Note: Descriptions are shown in the official language in which they were submitted.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
Medical Applications of Orthogonal Polarization
Spectral Imaging
Background of the Invention
1. Field of the Invention
The present invention relates to orthogonal polarization spectral (OPS)
imaging analysis. More particularly, the present invention relates to in vivo
medical and clinical uses of OPS imaging to directly, and in many cases non-
invasively, visualize, characterize, evaluate, monitor, and/or analyze a
subject's
microvascular and/or vascular system. The present invention also relates to in
vitro and in vivo applications of reflected spectral imaging analysis for
basic
research, clinical research, and as a teaching tool.
2. Related Art
Different disease states, including, e.g., diabetes, hypertension, numerous
opthalmological conditions, and coronary heart disease, produce distinctive
microvascular pathologies. To date, imaging of the human microcirculation for
diagnosis and/or treatment has been limited to vascular beds, where the
vessels are
visible and close to the surface (e.g., nailfold; conjunctiva).
For example, nailfold capillaroscopy has been used in the diagnosis and
treatment of peripheral vascular diseases, Raynaud's phenomenon, diabetes, and
hematological disorders (Forst, T., et al., Clinical Science 94:255-261
(1998);
Fagrell, B. & Bollinger, A., Clinical Capillaroscopy: A Guide to Its Use in
Clinical Research and Practice, Hogrefe & Huber, Seattle (1990); Fagrell, B. &
Intaglietta, M., J. Int. Med. 241:349-362 (1997)). These studies were done to
obtain experimental data regarding capillary density, capillary shape, and
blood
flow velocity, and were limited to gross physical measurements on capillaries.
No
spectral measurements, or individual cellular measurements, were made, and
Doppler techniques were used to assess velocity.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-2-
The use of the bulbar conjunctiva vessels for clinical applications in
ophthalmology has been restricted due to problems with movement (Davis, E. &
Landau, J., in Clinical Capillary Microscopy, Thomas, Springfield (1966);
Fenton, B.M., et al., Microvasc. Res. 18:153-166 (1979); Wolf, S., et al,
Hypertension 23:464-467 ( 1994)).
Other locations observed by intravital microscopy include the
microcirculation of the skin, lip, gingival tissue, and tongue (Davis, E. &
Landau,
J., supra). ,
Laser scanning confocal imaging is one new technique that does allow
reflected light imaging of the microcirculation in vivo (Bussau, L.J., et al.,
J. Anat
192 (Pt 2):187-194 (1998); Rajadhyaksha, M., et al., J. Invest. Dermatol.
104:946-952 ( 1995)). This method can distinguish between layers of skin and
can
image microcirculation in the skin. However, images obtained using laser
scanning confocal microscopy can only be collected at a fraction of normal
video
rate (up to 16 versus 30 frames/second) and the best microvascular images
using
this technique require fluorescent labels for contrast enhancement (Bussau,
L.J.,
et al., supra).
Direct observation, using conventional methods, of vascular beds of other
organs in humans has been prevented by the need for transillumination,
fluorescent
dyes for contrast enhancement, or by the size of instrumentation required to
acquire images, especially during surgery.
Several devices for in vivo analysis based on reflectance
spectrophotometry have been developed recently. However, these conventional
reflectance-based devices are less than optimal for several reasons.
One non-invasive device for in vivo analysis is disclosed in U.S. Patent
4,998,533 to James W. Winkelman (1991 ). This device uses image analysis and
reflectance spectrophotometry to measure individual cell parameters such as
cell
size. Measurements are taken only within small vessels, such as capillaries
where
individual cells can be visualized. Because this device takes measurements
only
in capillaries, measurements made will not accurately reflect measurements for
larger vessels. Consequently, the Winkelman device is not capable of measuring
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-3-
the central or true hematocrit, or the total hemoglobin concentration, which
depend upon the ratio of the volume of red blood cells to that of the whole
blood
in a large vessel such as a vein.
The Winkelman device measures the number of white blood cells relative
to the number of red blood cells by counting individual cells as they flow
through
a micro-capillary. The Winkelman device depends upon accumulating a
statistically reliable number of white blood cells in order to estimate the
concentration. The Winkelman device does not provide any means by which
platelets can be visualized and counted or a means by which the capillary
plasma
can be visualized, or the constituents ofthe capillary plasma quantified.
Also, this
device does not provide a means by which abnormal constituents of blood, such
as tumor cells, can be detected.
Other devices utilize light application means that focus an illumination
source directly onto a blood vessel in a detection region. As a result, these
devices are extremely sensitive to movements of the device with respect to the
patient. This increased sensitivity to device or patient movement can lead to
inconsistent results. To counteract this motion sensitivity, these devices
require
stabilizing and fixing means.
Other conventional devices have been developed based on a traditional
dark field illumination technique. As understood in traditional microscopy,
dark
field illumination is a method of illumination which illuminates a specimen
but does
not admit light directly to the objective. For example, a traditional dark
field
imaging approach is to illuminate an image plane such that the angular
distribution
of illumination and the angular distribution of light collected by an
objective for
imaging are mutually exclusive. The illumination is incident on the field of
view
of the detector, however, so in these devices scattering off optically active
tissue
in the image path can create an orientation dependent backscatter or image
glare
that reduces image contrast. Moreover, rotation of these devices causes a
change
in contrast.
To date, there is no non-invasive method to visualize and characterize the
microcirculation of the interior of the eye. Of major interest is the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-4-
microcirculation of the retina and optic disc. Also of interest is the
microcirculation of the external ocular structures and changes that occur
related
to disease processes such as benign and malignant tumors and circulatory
problems that occur, for example, the sludging and clumping of red blood cells
that occurs in various forms of sickle cell disease.
The retinal circulation is affected by many systemic diseases, such as
diabetes mellitus, sickle cell anemia, and macroglobulinemia. The various
types
of macular degenerations are also affected by circulatory changes. There are,
in
fact, many other ocular and systemic conditions which also affect the retinal
microcirculation. Medications also affect circulation. Glaucoma is a condition
of
the eye usually associated with a high eye pressure. There are, however, forms
of
glaucoma, often called low tension glaucoma, where the intraocular pressure is
not
found to be elevated when tested. In glaucoma, visual loss is associated with
loss
of function and death of the nerve fibers in the optic nerve which transmits
impulses to the brain where they are eventually interpreted as vision. There
are
changes in the microcirculation of the optic disc which contribute to the loss
of
function of some of the nerve fibers and death of some, thus affecting vision.
The
optic disc is easily visualized in the normal eye with the use of instruments
such
as various types of ophthalmoscopes or fundus cameras. Very little is now
known
about the microcirculation of the optic disc, the surrounding structures and
the
effects of medications on the micro-circulation in this area, as well as the
rest of
the intraocular structures.
With present techniques, microcirculation of various intraocular structures
are visualized only after various dyes are injected into the patient's
circulatory
system. Photographs are then taken. If treatment is necessary, such as laser
ablation of a leaking retinal vessel or capillary, this is done by comparing
the
photograph, which is usually placed on a screen next to the laser, with what
is
seen in the eye by the treating physician. Clearly, new methods are needed to
visualize and characterize the ocular microcirculation, as well as the
microcirculation of other human tissues and organs.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-5-
Orthogonal Polarization Spectral (OPS) Imaging
OPS imaging is a new method for visualizing and characterizing the
microcirculation using reflected light that allows imaging of the
microcirculation
noninvasively through mucus membranes, as well as on the surface of solid
organs.
OPS imaging has been described in Groner and Nadeau's U.S. Patent
5,983,120 (issued November 9, 1999), U.S. Patent 6,104,939 (issued August 15,
2000), and PCT publication WO 97/15229; all of which are incorporated by
reference in their entirety. In both the 5,983,120 and 6,104,939 patents and
the
WO 97/15229 publication, Groner and Nadeau used high resolution images of the
microcirculation to directly measure and compute key elements of the complete
blood count (CBC), including the white blood cell differential (CBC + Diff).
The
CBC + Diff is one of the most frequently requested diagnostic tests with about
two billion done in the United States per year. The combination of OPS
imaging,
image processing, reflectance spectroscopy, and algorithmic calculation was
used
to compute a rapid determination of hemoglobin concentration, hematocrit, red
and white blood cell count, and platelet count without drawing blood samples
from the body.
In addition, OPS imaging technology was used to non-invasively measure
other types of blood components, such as non-cellular constituents (e.g.,
blood
gases and bilirubin) present in the plasma component of blood. Rapidly and non-
invasively quantitatively measuring a variety of blood and vascular
characteristics
clearly eliminates the need to draw a venous blood sample to ascertain blood
characteristics, which may pose particular problems for newborns, elderly
patients,
burn patients, and patients in special care units. A device of this type also
eliminates the delay in waiting for the laboratory results in the evaluation
of the
patient. Such a device also has the advantage of added patient comfort, as
well
as obviating the risk of exposure to AIDS, viral hepatitis, and other blood-
borne
diseases. Noninvasive blood testing will have substantial utility in current
medical
practice.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-6-
An enhanced OPS imaging system for high contrast in vivo imaging of the
vascular system is described in copending U.S. Patent Application No.
09/401,859, filed September 22,1999, and is incorporated by reference in its
entirety.
Thus, OPS imaging provides an apparatus for complete non-invasive, in
vivo analysis of the vascular system with high-image quality. The apparatus
provides high resolution visualization and characterization of: blood cell
components (red blood cells, white blood cells, and platelets); blood
rheology; the
vessels in which blood travels; and vascularization throughout the vascular
system.
The apparatus further minimizes the glare and other deleterious artifacts
arising
in conventional reflectance spectrophotometric systems.
Although the above-referenced documents sufficiently describe OPS
imaging, a brief review of this technology follows.
In OPS imaging, the tissue is illuminated with linearly polarized light and
imaged through a polarizer oriented orthogonal to the plane of the
illuminating
light. Only depolarized photons scattered in the tissue contribute to the
image.
The optical response of OPS imaging is linear and can be used to perform
reflection spectrophotometry over the wide range of optical density typically
achieved by transmission spectrophotometry.
In OPS imaging, the subject medium is illuminated with light which has
been linearly polarized in one plane, while imaging the remitted light through
a
second polarizer (analyzer) oriented in a plane precisely orthogonal to that
of the
illumination. To form the image, the light is collected, passed through a
spectral
filter to isolate the wavelength region, and linearly polarized. The polarized
light
is then reflected toward the target by a beam sputter. An objective lens
focuses
the light onto a region of approximately 1 mm in diameter. The length of the
objective lens can vary; two exemplary OPS imaging probes have a 3 inch and an
8 inch objective lens. Light that is remitted from the target is collected by
the
same obj ective lens, which then forms an image of the illuminated region
within
the target upon an imaging detector such as a charge coupled device (CCD)
video
camera or a complementary metal oxide semiconductor (CMOS) video camera.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
The polarization analyzer is placed directly in front of the camera. A
polarizing
beam sputter can be chosen for maximum efficiency. That is, one orthogonal
polarization state is reflected while the other is transmitted.
In polarized light, the state of polarization of light is preserved in
ordinary
reflections as well as single scattering events. Typically, more than 10
scattering
events are required to effectively depolarize light (MacKintosh, F.C., et al.,
Phys.
Rev. B 40:9342 (1989); Schmitt, J.M., et al., Applied Optics 31:6535 (1992)).
Thus, the only remitted light from the subject that can pass through the
analyzer
results from multiple scattering occurring relatively deep (> 10 times the
single
scattering length) within the medium. This depolarized scattered light
effectively
back-illuminates any absorbing material in the foreground. If a wavelength
within
the hemoglobin absorption spectrum is chosen, the blood vessels of the
peripheral
microcirculation can be visualized using OPS imaging as in transilluminated
intravital microscopy. A wavelength region centered at an isobestic point of
oxy-
and deoxy-hemoglobin (548 nm) was chosen for optimal imaging of the
microcirculation. This wavelength region represented a compromise between
using an isobestic point in the Soret region (about 420 nm), where hemoglobin
absorption is maximum, but the scattering length is shorter, or one in the
near
infrared region (810 nm), where multiple scattering occurs deep in the tissue,
but
absorption for hemoglobin is insufficient to provide good contrast in smaller
vessels.
It is noted that the scattered light can be used to view subsurface cellular
structure irrespective ofthe absorption characteristics that allow
visualization and
characterization of blood cells.
Conventional reflectance spectrophotometry is carried out on an extended
diffuse reflecting surface to which the analyte has been applied. Typically,
reflectance spectrophotometry has a much more limited range of measurement of
optical density (OD) than does transmission spectrophotometry, which can
easily
measure changes from 0 to 3 OD. In reflected light spectrophotometry, the
apparent OD of the analyte is reduced by specular reflections and light
scattering.
The reflection and light scatter are due to physical characteristics rather
than the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-g-
chemical concentration of the analyte (Kortum, G., Reflectance Spectroscopy,
Springer-Verlag, New York (1969)).
OPS imaging was developed in part to eliminate some of the confounding
error inherent to reflectance spectroscopy that was due to reflection and
light
scatter. Studies have shown that OPS imaging techniques can be used to
accurately measure in reflection the wide range of OD typically achieved only
in
standard transmission spectrophotometry.
A theoretical explanation of OPS imaging begins by recalling that forming
an image in reflected light requires both scattering for illumination and
absorption
for contrast. In continuous media, these are typically considered the result
of a
pair of material constants (coefficients) (Star, W.M., et al., Phys Med Biol
33:437-454 (1988)). Both scattering and absorption depend linearly upon
penetration, and the remitted light intensity is given by the ratio of the
coefficients
(Kortum, G., Reflectance Spectroscopy, Springer-Verlag, New York (1969)).
High quality imaging is therefore generally not possible in turbid media due
to the
scattering which degrades the image resolution. However, in OPS imaging, the
remitted illumination is provided only by multiple scattering. This is a
distinctly
non-linear function of the penetration depth and thus is decoupled from the
absorption. Consequently, absorbing substances at shallow depth can be
visualized and characterized with both high contrast and good resolution.
Comparison of OPS Imaging With Intravital Microscopy
A comparison of fluorescence intravital microscopy (Harris, A.G., et al.,
Int. J. Microcirc.-Clin. Exp 17:322-327 (1997)) with OPS imaging in the
hamster
demonstrated equivalence in measured physiological parameters under controlled
conditions and after ischemic injury (Harris, A. G., et al., The study of the
microcirculation using orthogonal polarization spectral imaging, in Yearbook
of
Intensive Care and Emergency Medicine 2000, Ed. J.-L.Vincent, Springer-Verlag,
Berlin, pp 705-14 (2000); Harris, A. G., et al., Validation of OPS imaging, 21
st
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-9-
European Conference on Microcirculation, Ed. B. Fagrell, Bologna, Monduzzi
Editore, pp. 43-48 (2000)).
The ability of OPS imaging to provide quantitative measurements of
relevant physiological parameters and pathophysiological changes in the
microcirculation was shown by measurement of functional capillary density
(FCD)
in the dorsal skinfold chamber of the awake Syrian golden hamster. FCD is
defined as the length of red blood cell perfused capillaries per observation
area and
is given in cm/cm2. As the capillaries must contain flowing red cells to be
counted
in the measurement, FCD is a direct measure of nutritional tissue perfusion
and an
indirect measurement of oxygen delivery to tissue (Harris, A.G., et al., Am.
J.
Physiol. 271:H2388-2398 (1996)). The hamster dorsal skinfold model is
standardized and has been used extensively to study ischemia/reperfusion
injury
(Id.; Nolte, D., et al., Int. J. Microcirc.-Clin. Exp. 15:244-249 (1995)).
FCD was measured on the same capillary networks using standard
intravital fluorescence videomicroscopy (IVM) (Harris, A.G., et al., Int. J.
Microcirc.-Clin. Exp 17:322-327 (1997)) and OPS imaging at baseline, 0.5 hour,
and 2 hours after 4 hours of pressure ischemia (Nolte, D., et al.,
IntJ.Microcirc-
Clin Exp 15:9-16 (1995)). A total of six capillary networks per animal (n=10)
were imaged and recorded on videotape by both methods. The images from both
systems were analyzed during repetitive playback using a computer assisted
microcirculation analysis system (CapImageTM) (Klyscz, T., et al., Biomed Tech
(Berl) 42:168-175 (1997)) and FCD was determined using the images from both
methods. FCD was measured equally well by the two imaging methods.
Furthermore, Bland-Altman analysis (Bland, J.M. & Altman, D.G., Lancet 1:307-
310 (1986)) showed very good agreement between methods as well as no
systematic bias over the entire range of FCD measurements.
A direct comparison was made of the contrast between OPS imaging and
fluorescence on paired images of the hamster dorsal skinfold microcirculation.
There was no significant difference between the two methods in absolute
contrast
(p=0.43, paired t-test). Thus, contrast obtained by OPS imaging without the
use
of dyes was equivalent to that obtained with contrast enhancement by IVM.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 10-
In the following study, validation of the CYTOSCANTM A/R (a high
contrast OPS imaging device owned by Cytometrics, Inc.) under both
physiological and pathophysiological conditions was performed using standard
IVM.
S Methods: In the dorsal skin-fold chamber model in the awake Syrian
golden hamster measurements of vessel diameter, RBC velocity and functional
capillary density (FCD) were made before and after a 4 hour ischemia. In a
tumor
growing in the chamber, measurements were made 3, 6, and 9 days after
implantation of amelanomic melanoma tumor cells. In the rat liver, venular
diameter, RBC velocity and the number of perfused sinusoids were measured
before and after a 60 minute ischemia. The data were analyzed using Bland-
Altman plots to test the agreement between the two methods (IVM vs.
CYTOSCANTM A/R).
Results: There was excellent agreement between the two methods for the
1 S measurement of venular RBC velocity in all three models. There was no
bias, the
variance was constant over the entire range, the 95% confidence interval was
of
an acceptable range and 95% of all data points were within this range. For the
measurement of perfusion (FCD in the chamber, functional vessel density in the
tumor, and the number of perfused sinusoids in the liver) there was also good
agreement between the two methods. In the hamster chamber (muscle and
tumor), measurements of vessel diameter showed good agreement, although the
IFM demonstrated a bias of approximately 4 microns. This is actually to be
expected since the two methods measure two different diameters (from
endothelial
cell to endothelial cell (IFM) vs. RBC column (OPS imaging)) and since the
fluorescence scattering causes the vessels to appear larger. In the liver,
there was
good agreement between the two methods for the measurement of vessel
diameter. Similar results were obtained in the pancreas, intestine, brain,
skin-flap
and during wound healing.
Conclusions: The CYTOSCANTM A/R can be used to make accurate
microvascular measurements of vessel diameter, RBC velocity, and perfusion in
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-11-
a variety of organs in animal models. Similar measurements are therefore
possible
in humans.
Quantitative Determination of Optical Density In Vitro
Quantitative data regarding local hematocrit, which can be obtained with
S transillumination using the absorbance of blood hemoglobin, is not possible
using
epi-illumination methods without fluorescent labeling of the red cells.
Previous
studies to determine local hematocrit using intravital microscopy were carried
out
in thin tissue sections (such as the rat mesentery or cremaster muscle)
(Pittman,
R.N. & Duling, B.R., J. Appl. Physiol. 38:321-327 (1975)). The optical systems
used for those studies were evaluated and calibrated using glass capillaries
filled
with solutions of hemoglobin and/or whole blood (Pittman, R.N. & Duling, B.R.,
J. Appl. Physiol. 38:315-320 (1975); Lipowsky, H.H., et al., Microvasc Res
24:42-55 (1982)).
It has been demonstrated that the OPS imaging system produces images
of blood filled capillary tubes similar to those obtained by transillumination
(Groner, W., et al., Nat. Med. 5:1209-1213 (1999)). The optical densities
obtained from transillumination and OPS imaging were compared for a series of
measurements made on glass capillaries filled with diluted whole blood or
solutions of hemoglobin. The receiving optics were identical. The optical
densities were essentially identical (r2 = 0.984). Thus, OPS imaging can be
useful
in the quantitative determination of local hematocrit using the optical
density
approach in epi-illumination.
OPS imaging relies on the absorbance of hemoglobin to create contrast.
Thus, the vessel must contain RBCs to be visualized. For measurement, the
vessel
needs to be greater in diameter than the minimum resolution of the camera and
optics. For these particular images, the magnification at the camera was one
micrometer per pixel. Therefore, the resolving power ofthe system was
sufficient
to resolve a single RBC and individual capillaries of approximately 5 ~m in
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-12-
diameter. Finally, there must be sufficient contrast between RBCs and the
surrounding tissue.
Although OPS imaging technology has been described in U.S. Patents
5,983,120 and 6,104,939 to Groner and Nadeau, and an enhanced OPS imaging
system for high contrast in vivo imaging of the vascular system described in
copending U.S. Patent Application No. 09/401,859, filed September 22,1999, the
medical, clinical, and research applications of this exciting technology have
not yet
been fully realized.
Summary of the Invention
The present invention relates to medical applications of OPS imaging
technology for visualizing and characterizing the microcirculation. These new
medical applications may result in the development of new diagnostic tests for
human and/or veterinary microvascular pathologies.
Accordingly, the present invention provides a method of detecting a
circulation disturbance comprising: (a) obtaining a single captured image or a
sequence of images of the microcirculation of an individual afflicted with, or
suspected of being afflicted with a circulation disturbance, using an
orthogonal
polarization spectral ("OPS") imaging probe, comprising the steps of: (i)
illuminating a tissue in the microcirculatory system of said individual with
light
polarized in a first plane of polarization, and (ii) capturing at least one
image or
a sequence of images reflected from said tissue, wherein said reflected
images)
are passed through an analyzer having a plane of polarization substantially
orthogonal to said first plane of polarization to produce a raw reflected
image(s),
thereby obtaining the captured image(s); and (b) analyzing said captured
images) to identify characteristics of the microcirculation, thereby detecting
said
circulation disturbance.
In addition, the invention provides a method of monitoring the
microcirculation of an individual before, during, or after a medical procedure
comprising:(a) obtaining a single captured image or a sequence of images of
the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-13-
microcirculation of an individual before, during, or after a medical
procedure,
using an OPS imaging probe, comprising the steps of: (i) illuminating a tissue
in
the microcirculatory system of said individual with light polarized in a first
plane
of polarization, and (ii) capturing at least one image or a sequence of images
reflected from said tissue, wherein said reflected images) are passed through
an
analyzer having a plane of polarization substantially orthogonal to said first
plane
of polarization to produce a raw reflected image(s), thereby obtaining the
captured
image(s); and (b) analyzing said captured images) to monitor the
microcirculation
of said individual before, during, or after a medical procedure.
The small size of the optical probe will facilitate its use as a non-invasive
diagnostic tool in both experimental and clinical settings to evaluate and
monitor
the microvascular sequelae of conditions known to impact the microcirculation,
such as shock (hemorrhagic, septic), hypertension, high altitude sickness,
diabetes,
sickle cell anemia, and numerous other red blood cell or white blood cell
abnormalities. In addition to non-invasive applications, imaging and
quantitative
analysis of the microcirculation during surgery (transplant, cardiac,
vascular,
orthopedic, neurological, plastic, etc.), wound healing, tumor diagnosis and
therapy and intensive care medicine are also applications of OPS imaging
technology. The ability to obtain high contrast images of the human
microcirculation using reflected light will allow quantitative determination
of
parameters such as, capillary density, vessel (and microvessel) morphology,
vessel
density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics, such as
vasomotion, functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, at many
previously inaccessible sites. Preferably, two or more parameters will be
determined using OPS imaging technology. These measurements will be
instrumental in developing precise tools to evaluate perfusion during clinical
treatment of those diseases that impact tissue viability and microvascular
function.
Using this methodology, physicians may now be able to follow the progression
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-14-
and development of microvascular disease and directly monitor the effects of
treatment on the microcirculation.
The OPS image of the microcirculation may be a single captured OPS
image or a sequence of images, depending on the parameter to be determined.
Parameters which can be measured from a single captured OPS image
include: vessel diameter (vessel can include arteriole, capillary, and
venule); vessel
morphology; cell morphology; capillary density; vessel density; area-to-
perimeter
ratio; and vasospasm.
Parameters which are dynamic and can only be measured from a sequence
of images include: red blood cell velocity; functional capillary density;
functional
vessel density; blood flow; leukocyte-endothelial interactions (includes
rolling
leukocytes and sticking (or adherent) leukocytes); and vascular dynamics, such
as
vasomotion.
Thus, the present invention is directed to new medical applications of the
OPS imaging probe apparatus described in U.S. Patents 5,983,120 and 6,104,939
(also called herein, the "standard" probe), as well as the OPS imaging probe
described in copending U.S. Patent Application No. 09/401,859, filed September
22,1999 (also called herein, the "high contrast" probe). It is believed that
the
medical applications described herein will be applicable to both the standard
and
high contrast versions of the OPS imaging system. In many instances, however,
the high contrast OPS image will be preferred.
The OPS imaging technology described herein is useful for imaging human
subjects and animal subjects, as well as in vitro applications in research and
teaching.
The apparatus used includes a light source, an illumination system, and an
imaging system. The light source provides an illumination beam that propagates
along an illumination path between the light source and the plane in which the
object is located (the object plane). The illumination system transforms the
illumination beam into a high contrast illumination pattern and projects that
illumination pattern onto the sub-surface object. The illumination pattern has
a
high intensity portion and a low intensity portion. The imaging system
includes
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-15-
an image capturing device that detects an image of the sub-surface object,
such as
blood or tissue under the skin of a patient, as well as internal organs
(heart, brain,
colon) exposed during surgery.
Intravital imaging by transmission microscopy is not possible in humans or
animals for inaccessible sites or solid organs. The OPS imaging method employs
a small sized optical probe and produces clear images of the microcirculation
by
reflectance from the surface of solid organs and from sites such as the
sublingual
area in the awake human. OPS imaging could thus reveal differences between
normal and pathological microvascular structure and function non-invasively.
The
diagnosis and progression of disease, and the effectiveness of treatment could
be
monitored for disorders in which altered microvascular function has been
noted.
In one embodiment of the present invention, a standard or high contrast
OPS imaging probe is used for in vivo cancer diagnosis, prognosis, or as an
aid in
cancer surgery.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used for the direct diagnosis of epithelial or intraepithelial
neoplasms or
pre-cancerous conditions. Examples of epithelial or intraepithelial neoplasms
that
could be diagnosed by OPS imaging include cervical, dermal, esophageal,
bronchial, intestinal, or conjunctival neoplasms.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to assess tumor boundaries or tumor margins, prior to,
during, or following cancer therapy.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to diagnosis different types of tumors based on their
vascular structure.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to monitor the effects of cancer radiation therapy, such
as
the occurrence of telangiectasia or other microvascular or vascular effects on
irradiated tissue.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used for in vivo wound care and wound healing
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-16-
management. In this embodiment, the OPS imaging probe would be used in the
visualization, characterization, assessment and management of different types
of
wounds--venous ulcers, decubitis ulcers, traumatic wounds, non-healing
surgical
wounds, and burn wounds. Since venous ulcers are formed as a result of an
underlying circulatory problem, knowledge about the microcirculation would be
essential to effective treatment.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used to visualize and characterize microvessels in and around wounds.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to visualize, characterize, and quantify the perfused
microvessel density in venous stasis ulcers or diabetic ulcers.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to observe necrotic tissue and determine the degree of
debridement a wound would require.
1 S In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to assess the margins of a wound to determine the
likelihood it is going to heal.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to assess the viability of wound tissue to successfully
support a skin graft.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to more accurately and objectively determine the line of
amputation. In another aspect of this embodiment, a standard or high contrast
OPS imaging probe is used to measure and compare the revascularization and
healing of a wound using different wound healing therapies.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to monitor capillary budding during wound healing.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo during plastic surgery.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-17-
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to monitor blood flow (perfusion) during and following plastic,
reconstructive, reattachment, or microsurgery.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to determine healthy versus necrotic or dead tissue
around
a skin flap.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used after surgery to continuously monitor the
microcirculation
and identify any potential problems with reperfusion. Early indication of
reperfusion problems may help avoid the need for repeat surgery.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo in the field of cardiology and
cardiac
surgery.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to increase visualization and characterization of the cardiac
microcirculation, to confirm reperfusion during minimally invasive cardiac
surgical
procedures that avoid the heart/lung bypass machine, such as "keyhole"
surgeries
(i. e., Heartport) and thoracotomies.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used for monitoring and detecting changes in patient blood
flow
(the microcirculation) during open heart surgery while the patient is on the
heart-
lung machine. Thus, the OPS imaging probe could be used to monitor the
progress
of the patient on the heart-lung machine.
In another aspect of this embodiment, the OPS imaging probe also can be
used during coronary artery bypass graft (CABG) surgery to determine if the
graft
is getting good blood flow and providing the downstream vessels with good
blood
flow. That is, a cardiac surgeon could use the OPS imaging probe, directly
contacting the heart, to monitor the blood flow to the capillaries after
bypass
surgery.
In another aspect of this embodiment, the OPS imaging probe is used for
cardiac risk monitoring. The use of the OPS imaging probe to visualize and
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-18-
characterize the microcirculation may assist in non-invasively determining a
high
risk cardiac profile. The microvascular sequalae of hypertension could be
studied
and also be used in determining cardiac risk.
In another embodiment of the present invention, the OPS imaging probe
is used in the visualization, characterization, and assessment of lung tissue.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo prior to or during neurosurgery.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is applied directly to the brain during diagnostic or therapeutic
neurosurgery
to visualize and characterize the microcirculation, and measure parameters,
such
as, e.g., diameter, flow velocity, and functional capillary density.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to detect vasospasm following an aneurism or
subarachnoid
hemorrhage.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to detect boundaries of tumors in the brain.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used for the determination and typing of brain tumors based
on
the vascular structure and differences in the microcirculation of different
brain
tumors.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to directly visualize and characterize the vascular
consequences of neural trauma. The OPS imaging probe may also be used to
determine the extent of neural trauma.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo during organ transplantation.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used during transplant surgery to determine the amount of perfusion
after
the transplanted tissue/organ is connected.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-19-
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo during vascular grafting surgery,
such
as in Peripheral Arterial Occlusive Disease (PAOD).
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used during vascular graft surgery to determine the amount of
perfusion
after the graft is connected.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo during orthopedic surgery, as well
as
in the field of orthopedic medicine.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used during orthopedic surgery to identify and observe necrotic
tissue, for
surgical removal. The site of the probe would be on the area that has
undergone
trauma.
The probe can also be used to visualize bones, tendons, and ligaments.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to visualize and characterize the microcirculation
around
periosteum (bone). Differences in microcirculation were observed when the
image
was taken before or after a fracture.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo in the fields of gastroenterology
and
gastrointestinal (GI) or gastroesophageal surgery.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize and characterize the large intestine and diagnose
and
treat inflammatory bowel disease, ulcerative colitis, Crohn's disease, or
other
gastrointestinal disorders affecting the microcirculation. The probe can be
inserted
into the rectum to directly contact the wall of the laxge intestine.
In another aspect of this embodiment, the OPS imaging probe can be used
during gastrointestinal (GI) surgery to visualize and characterize the colon
during
bowel resection. Other GI organs may be visualized as well.
In another aspect of this embodiment, the OPS imaging probe can be used
to determine the boundaries of a cancerous GI tumor and to visualize and
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-20-
characterize necrotic tissue. Removal of the affected tissue from the stomach
and/or esophagus area can thus be more easily accomplished during surgery.
In another aspect of this embodiment, the OPS imaging probe can also be
used to visualize and characterize the rectal mucosal microcirculation, such
as, for
example, in patients with inflammatory bowel disease.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used in vivo in the field of opthamology.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize and characterize the ocular microcirculation. Such
a
visualization tool can be used for diagnostic purposes and treatment as well
as
providing information regarding the effectiveness of various types of
medications
on the circulatory system in the area examined.
In another aspect of this embodiment, OPS imaging technology can be
used to diagnose macular degeneration, retinal disorders (retinopathy), and
glaucoma.
In another aspect of this embodiment, OPS imaging technology can be
used to visualize and characterize the optic disk, retina, sclera,
conjunctiva, and
changes in the vitreous humor.
In another aspect of this embodiment, OPS imaging technology can be
used for early diagnosis and treatment of diabetes by looking at the ocular
microcirculation, especially changes or differences in the sclera and/or
aqueous
humor of the eye.
In another embodiment of the present invention, the OPS imaging probe
is used during normal or complicated pregnancy to monitor the woman's
microvascular function.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to detect or monitor women whose pregnancy is complicated by
preeclampsia (PE).
In another embodiment of the present invention, the OPS imaging probe
can be used in neonatology monitoring.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-21 -
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to quantitatively measure changes in the microcirculation of
neonates during different disease states like sepsis and meningitis, and
therefore
be used to diagnosis those conditions. Hemoglobin levels of the neonates can
be
monitored non-invasively.
In another embodiment of the present invention, OPS imaging can be used
in high altitude studies or to study space physiology.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to observe and evaluate changes in the microcirculation at high
altitudes to study, diagnose, and/or treat high altitude sickness.
In another embodiment of the present invention, the OPS imaging probe
can be used in vivo in critical care or intensive care medicine.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used as a sublingual measuring or monitoring device on critically ill
patients to diagnose, treat, or prevent sepsis and shock (hemorrhagic or
septic).
In another embodiment of the present invention, the OPS imaging probe
is used in the field of pharmaceutical development.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to study the effects of different pharmaceuticals on the
microcirculation, such as anti-angiogenesis drugs to determine if circulation
to a
tumor is cut off; or angiogenesis drugs to determine if vessel growth to an
organ
and thus circulation has improved; or anti-hypertensive agents to determine
mechanisms of action of new treatments or hypertension etiology at the
microvascular level.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to look at hemoglobin-based oxygen carriers (i. e., DCL
Hb,
a synthetic hemoglobin) to determine their effects on the microcirculation,
such
as, e.g., whether there is an increased flow of RBC's as a result of using the
product.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-22-
In another aspect of this embodiment, the OPS imaging probe can be used
to study the effect of ultrasound enhancers (i. e., inj ectable dyes) on the
microcirculation.
In another aspect of this embodiment, the OPS imaging probe can be used
to visualize and detect leakage of injectable dyes or other injectable
contrast-
S generating agents, from the blood vessels into tissues.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used to visualize and characterize capillary
beds in
the nailfold, as has been done before using standard capillaroscopy.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to study, diagnose, and evaluate patients with circulation
disturbances, such as, for example, Raynaud's phenomenon, osteoarthritis, or
systemic sclerosis.
In another embodiment of the present invention, the OPS imaging probe
is used in the area of anesthesiology.
' In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to monitor blood loss during surgery. For example, an OPS
imaging
probe can be used to non-invasively and continuously monitor the hemodynamic
parameters of an anesthetized patient, such as, e.g., hemoglobin concentration
and
hematocrit.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used on an anesthetized patient to monitor the clumping of
red
blood cells, and the early formation of microemboli during surgery.
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used to visualize, characterize, identify,
and/or
monitor disseminated intravascular coagulation (DIC) in a patient, which may
occur as a secondary complication of infection, obstetrics, malignancy, and
other
severe illnesses.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize, characterize, identify, and/or monitor DIC, due to
infection, and more particularly due to meningitis.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 23 -
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used to visualize, characterize, and monitor
changes in leukocyte-endothelial cell interactions, such as occurs during
inflammation or infection.
In another embodiment of the present invention, the OPS imaging probe
is used for in vitro or in vivo basic or clinical research in any or all of
the areas
mentioned above (i.e., cardiology, cardiac surgery, wound care, diabetes,
hypertension, opthalmology, neurosurgery, plastic surgery, transplantation,
anesthesiology, and pharmacology).
In another embodiment of the present invention, the OPS imaging probe
is used as a teaching tool for medical students and/or science students
studying,
for example, physiology, anatomy, pharmacology, the microcirculation, and
disease states affecting the microcirculation.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary and explanatory and are intended
to
provide further explanation of the invention as claimed.
Brief Description of the Figures
The accompanying figures, which are incorporated herein and form part
ofthe specification, illustrate embodiments ofthe present invention and,
together
with the description, further serve to explain the principles of the invention
and to
enable a person skilled in the pertinent art to make and use the invention. In
the
figures, like reference numbers indicate identical or functionally similar
elements.
Figure 1 shows a block diagram illustrating one particular embodiment of
an OPS imaging probe for non-invasive in vivo analysis of a subject's vascular
system. In this particular embodiment, the objective lens is 8 inches long.
This
embodiment would be particularly useful for imaging cervical tissue.
Figure 2 is an OPS image of arterioles (A) and venules (V) in normal brain
tissue after opening of the dura during neurosurgery. Bar indicates 100 Vim.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-24-
Figure 3 is an OPS image showing the site of interest in the brain
dominated by capillaries. Bar indicates 100~m.
Figure 4 is an OPS image of cortical microvessels in a patient with
subarachnoid hemorrhage. Extravasation of red blood cells is seen as black
dots
in the extravascular space. The arteriole (A) shows the pearl string sign of
multiple
segmental microvasospasm (arrows). No microvasospasm is seen in the venule
(V). Bar indicates 100~m.
Figure 5 is an OPS image ofthe opaque endothelial layer in several vessels
close to the area of brain tumor resection. The underlying pathophysiology is
unknown. The observation may represent endothelial swelling or a plasma layer
streaming along the endothelium. Bar indicates 100 Vim.
Figure 6 is an OPS image depicting tumor angiogenesis in a patient with
a glioblastoma multiforme WHO IV. Typical tortuous, irregular shaped
conglomerate of newly built tumor vessels. Bar indicates 100 Vim.
Figure 7 is a bar graph depicting functional capillary density before and
after tumor resection/clipping of aneurysm. Mean~SEM.
Figure 8 is a bar graph depicting the distribution of arteriolar and venular
diameters before and after tumor resection/clipping of aneurysm. The box and
bars
indicate the median and the 10'",25'", 75'n and 90'" percentiles.
Figure 9 is a bar graph depicting the frequency distribution of red blood
cell velocities in arterioles and venules before and after tumor
resection/clipping
of aneurysm given in percentages of total number of vessels. nm = no
measurement.
Figure 10 depicts changes of portal venous blood flow in Sham and
endotoxemic (ETX) animals during the investigation period. All data are
expressed as boxplots including median, 10'",25'", 75'" and 90'" percentiles,
as well
as the highest and the lowest value. # denotes significant difference versus
baseline, ~ indicates significant difference between the two groups.
Figure 11 depicts changes of the gastrointestinal mucosal-arterial pC02-
gap (r-aPC02) in Sham and endotoxemic animals during the investigation period.
All data are expressed as boxplots including median,10'", 25'", 75'" and 90'n
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 25 -
percentiles, as well as the highest and the lowest value. # denotes
significant
difference versus baseline, ~ indicates significant difference between the two
groups.
Figure 12 depicts changes in the numbers of perfused/heterogenously
perfused/unperfused villi in Sham and endotoxemic animals during the
investigation period.
Figure 13 depicts relative changes of perfused/heterogenously
perfused/unperfused villus count in Sham and Endotoxin animals during the
investigation period. Data are shown in % of counted villi.
Figure 14 is a schematic picture of the OPS imaging device and an image
of the capillaries of the nailfold.
Figures 15A-15B: Figure 15A depicts change in flux (%) during venous
occlusion and after arterial occlusion (peak flux) compared to flux at rest
measured at the palmer side. One subject of the preeclamptic group had an
increase in peak flux that was an increase of 178% compared to rest value.
Figure 15B depicts change in flux (%) during venous occlusion and after
arterial occlusion (peak flux) compared to flux at rest measured at the dorsal
side.
One subject of the control group had an increase in flux of 411% during venous
occlusion.
Figure 16 shows percentual change in velocity during venous occlusion
compared to velocity at rest.
Figure 17 depicts an OPS image of the sublingual area in a healthy
volunteer. Note the dense venular and capillary network.
Figure 18 is an OPS image of the sublingual area of a patient with septic
shock (mean arterial pressure 68 mmHg, lactate 3.8 mEq/L, dopamine 20
mcg/kg.min, norepinephrine 0.13 mcg/kg/min). Note the decrease in capillary
density with stop flow and transient flow in numerous capillaries.
Figure 19 is an OPS image of the sublingual area of a patient with severe
cardiogenic shock (mean arterial pressure 50 mmHg, lactate 10.5 mEq/L,
dobutamine 20 mcg/kg.min, dopamine 20 mcg/kg.min, norepinephrine 3
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-26-
mcg/kg.min). Note the decreased capillary density and the red blood cell
conglomerates in large vessels representative of stagnant flow.
Figure 20 is an OPS image of necrotic ileostomy. Note the decreased
number of gut mucosal capillaries, most of them not perfused.
Detailed Description of the Embodiments
The present invention relates to medical applications of OPS imaging
technology for visualizing and characterizing the microcirculation. Such
applications may result in the development of new diagnostic tests for human
microvascular pathologies.
Accordingly, the present invention provides a method of detecting a
circulation disturbance comprising: (a) obtaining a single captured image or a
sequence of images of the microcirculation of an individual afflicted with, or
suspected of being afflicted with a circulation disturbance, using an
orthogonal
polarization spectral ("OPS") imaging probe, comprising the steps of: (i)
illuminating a tissue in the microcirculatory system of said individual with
light
polarized in a first plane of polarization, and (ii) capturing at least one
image or
a sequence of images reflected from said tissue, wherein said reflected
images)
are passed through an analyzer having a plane of polarization substantially
orthogonal to said first plane of polarization to produce a raw reflected
image(s),
thereby obtaining the captured image(s); and (b) analyzing said captured
images) to identify characteristics ofthe microcirculation, thereby detecting
said
circulation disturbance.
In addition, the invention provides a method of monitoring the
microcirculation of an individual before, during, or after a medical procedure
comprising:(a) obtaining a single captured image or a sequence of images of
the
microcirculation of an individual before, during, or after a medical
procedure,
using an OPS imaging probe, comprising the steps of: (i) illuminating a tissue
in
the microcirculatory system of said individual with light polarized in a first
plane
of polarization, and (ii) capturing at least one image or a sequence of images
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-27-
reflected from said tissue, wherein said reflected images) are passed through
an
analyzer having a plane of polarization substantially orthogonal to said first
plane
of polarization to produce a raw reflected image(s), thereby obtaining the
captured
image(s); and (b) analyzing said captured images) to monitor the
microcirculation
of said individual before, during, or after a medical procedure. As used
herein, the
term "tissue" is intended to include blood.
The small size of the optical probe will facilitate its use as a non-invasive
diagnostic tool in both experimental and clinical settings to evaluate and
monitor
the microvascular sequelae of conditions known to impact the microcirculation,
such as shock (hemorrhagic, septic), hypertension, high altitude sickness,
diabetes,
sickle cell anemia, and numerous other red blood cell or white blood cell
abnormalities. In addition to non-invasive applications, imaging and
quantitative
analysis of the microcirculation during surgery (transplant, cardiac,
vascular,
orthopedic, neurological, plastic, etc.), wound healing, tumor diagnosis and
therapy and intensive care medicine are also applications of OPS imaging
technology.
The ability to obtain high contrast images of the human microcirculation
using reflected light will allow quantitative determination of one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel
density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, at many
previously inaccessible sites. Preferably, two or more parameters will be
determined using OPS imaging technology. These measurements will be
instrumental in developing precise tools to evaluate perfusion during clinical
treatment ofthose diseases that impact tissue viability and microvascular
function.
Using this methodology, physicians may now be able to follow the progression
and development of microvascular disease and directly monitor the effects of
treatment on the microcirculation.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-28-
As used herein, the term "capillary density"means the ratio of the length
of capillaries to the total area of observation, expressed as cm/cm2.
As used herein, the term "vessel (and/or microvessel) morphology" means
the physical or structural characteristics of a vessel (or microvessel). This
term
can refer to an individual vessel or to a network of vessels.
As used herein, the term "vessel density" means the area occupied by
vascular structures. Vessel density is expressed as a ratio of the area
occupied by
vessel structures to the total observation area. The number can be expressed
as
a percentage. Vessel density can also be referred to as "vascularization
index."
As used herein, the term "vasospasm" means prolonged abnormal
constriction of vessels, usually in response to trauma and/or the presence of
extracellular blood in the area adjacent to the vessel. For example,
vasospasms
are a well-known consequence of a sub-arachnoid hemorrhage in the brain.
As used herein, the term "red blood cell (RBC) velocity" means the
observed velocity of red blood cells within a blood vessel.
As used herein, the term "cell morphology" means the cell shape
characteristics of white blood cells, red blood cells, and/or epithelial
cells.
As used herein, the term "vessel diameter" means the distance between the
observable walls of a blood vessel.
As used herein, the term "leukocyte-endothelial cell interactions" means
any interaction between a leukocyte (white blood cell) and the inner surface
of a
blood vessel (the endothelial cell layer). These interactions can include
rolling
along the wall of the vessel at a rate slower than the RBC velocity, sticking
in one
place for a period of time, or migrating out through the vessel wall, perhaps
to a
site of inflammation.
As used herein, the term "vascular dynamics" means a temporal (time
dependent) change in vessel diameter. This may be a spontaneous change (e.g.,
vasomotion) or in response to a neuronal, hormonal, or pharmacological
stimulus.
In addition, the normal response of a vessel to a given stimulus may be
influenced
by other factors. These other factors would then be described as influencing
the
vascular dynamics.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-29-
As used herein, the term "functional vessel density" means a ratio of the
length of perfused vessels to the total observation area. A perfused vessel is
one
through which RBC's can be observed to flow.
As used herein, the term "functional capillary density" means a ratio of the
S length (or number) of perfused capillaries to the total observation area. A
perfused capillary is one through which RBC's can be observed to flow. This is
expressed as cm/cmz (or number/cm2).
As used herein, the term "blood flow" means the observed movement of
red blood cells through a vessel.
As used herein, the term "area-to-perimeter ratio" means a measure of the
clumping or heterogeneity of red blood cells in a vessel. The ratio is defined
as
the ratio of the area of red blood cell conglomerates detected within a vessel
to the
length of the perimeter or outline of the red blood cell conglomerates. A
fully
perfused vessel without plasma spaces leads to a higher area-to-perimeter
ratio.
Plasma spaces or partially filled vessels will result in a smaller area-to-
perimeter
ratio value. This parameter might be used to assess stagnant flow or clumped
cells, for example.
As used herein, the term "hemoglobin (or Hb) concentration" means the
concentration of the iron-containing protein pigment (hemoglobin) found in red
blood cells. Hemoglobin, the main component of the red blood cell, is a
conjugated protein that serves as a vehicle for the transportation of oxygen
and
CO2, throughout the body. When fully saturated, each gram of hemoglobin holds
1.34 ml of oxygen. The red cell mass of the adult contains approximately 600 g
of hemoglobin, capable of carrying 800 ml of oxygen. The main function of
hemoglobin is to transport oxygen from the lungs, where oxygen tension is
high,
to the tissues, where it is lower.
As used herein, the term "hematocrit" means the ratio of the volume of
erythrocytes (red blood cells) in a sample of blood to that of the whole
blood. It
is expressed as a percentage or, preferably, as a decimal fraction.
Thus, the present invention is directed to new medical applications of the
OPS imaging probe apparatus described in U. S. Patents 5,983,120 and 6,104,939
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-30-
(also called herein, the "standard" probe), as well as the OPS imaging probe
described in copending U.S. Patent Application No. 09/401,859, filed September
22,1999 (also called herein, the "high contrast" . probe). It is believed that
the
medical applications described herein will be applicable to both the standard
and
high contrast versions of the OPS imaging system. In certain medical
applications,
however, the high contrast OPS image will be preferred.
The OPS imaging technology described herein is useful for imaging human
subjects and animal subjects, as well as in vitro applications in research and
teaching.
The apparatus used includes a light source, an illumination system, and an
imaging system. The light source provides an illumination beam that propagates
along an illumination path between the light source and the plane in which the
object is located (the object plane). The illumination system transforms the
illumination beam into a high contrast illumination pattern and projects that
illumination pattern onto the sub-surface object. The illumination pattern has
a
high intensity portion and a low intensity portion. The imaging system
includes
an image capturing device that detects an image of the sub-surface obj ect,
such as
blood and/or tissue under the skin of a patient, from sublingual sites, as
well as
from the surface of solid organs (heart, brain, colon, lungs) exposed during
surgery, or epithelial or intraepithelial tissue (i.e., cervix). Intravital
imaging by
transmission microscopy is not possible in humans or animals for inaccessible
sites
or solid organs. The OPS imaging method employs a small sized optical probe
and produces clear images of the microcirculation by reflectance from the
surface
of solid organs and from sites such as the sublingual area in the awake human.
OPS imaging could thus reveal differences between normal and pathological
microvascular structure and function non-invasively. The diagnosis and
progression of disease, and the effectiveness of treatment could be monitored
for
disorders in which altered microvascular function has been noted.
The image of the microcirculation may be a single captured OPS image or
a sequence of images, depending on the parameter to be determined.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-31 -
Parameters which can be measured from a single captured OPS image
include: vessel diameter (vessel can include arteriole, capillary, and
venule); vessel
morphology; cell morphology, capillary density; vessel density; area-to-
perimeter
ratio; and vasospasm.
Parameters which are dynamic and can only be measured from a sequence
of images include: red blood cell velocity; functional capillary density;
functional
vessel density; blood flow; leukocyte-endothelial interactions (includes
rolling
leukocytes and sticking (or adherent) leukocytes); and vascular dynamics, such
as
vasomotion.
The present invention is illustrated by numerous examples and
experimental data presented below. This information is provided to aid in the
understanding and enablement of the present invention, but is not to be
construed
as a limitation thereof.
Cancer Applications
In this embodiment of the present invention, a high contrast OPS imaging
probe is used for in vivo cancer diagnosis, prognosis, or as an aid in cancer
surgery.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used for the direct diagnosis of epithelial or intraepithelial
neoplasms or
pre-cancerous conditions. Examples of epithelial or intraepithelial neoplasms
that
could be diagnosed by OPS imaging include cervical, dermal, esophageal,
bronchial, intestinal, or conjunctival neoplasms.
Regarding cervical intraepithelial neoplasms (CIN), although there has
been a significant decline in the incidence and mortality of invasive cervical
carcinoma over the last 50 years, there has been an increase in both the
reported
and actual incidence of CIN. As a result, it has been estimated that the
mortality
of cervical carcinoma may rise by 20% in the years 2000-2004 unless screening
techniques for CIN are improved.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-32-
Present screening for CIN and cervical cancer is relatively inexpensive but
labor intensive because it initially relies on the results of a Pap smear; a
false
negative error rate of 20-30% is associated with insufficient cell sampling
and/or
inexpert reading of Pap smears. On the other hand, false positive results on a
Pap
S smear, such as occurs in 70-80% of women with the Pap smear classification
"ASCUS" (which stands for Atypical Cells of Undetermined Significance under
the newer "Bethesda" classification system), may lead to unnecessary, time-
consuming, and costly follow-up testing (such as colposcopyand/or biopsy) and
needless worry for the patient.
Thus, a more reliable method to classify cervical tissue as normal or
abnormal, and in the latter case to distinguish inflammation or benign HPV
infection from CIN, is needed.
It was noted that the excellent images of the microcirculation obtained
through OPS imaging technology were overlain by a "mosaic" of tesserae that
were epithelial cell-shaped. They were rhomboidal, penta, hexa, septahedronal
and quite flat and regular. Their diameters were approximately 10-40 microns
based on a comparison with the RBCs and arterioles and venules in the same
fields. They were clearly overlaying the vascular structures. Their cell
margins
were illuminated (refractile). The cells were quite "flat," suggesting that
they were
superficial rather than deeper epithelium, and definitely not basal layer
cells which
are more cuboidal. In addition, the size and shape of cell nuclei could also
be
distinguished. These characteristics are particularly important in an
assessment of
premalignant and malignant states.
With the information that the focal "plane" of the system as configured was
~ 150-200 microns from the surface of the optical probe, which corresponds to
the
depth at which the vasculature exists, it was theorized that the depiction of
superficial epithelium must arise from a mechanism of light scattering and
back
illumination, or other effects. It was then realized that the optics of the
OPS
imaging probe could be optimized to make the epithelium the main feature of
the
image. Thus, by using the OPS imaging probe to bring out the epithelial image
at
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 33 -
different depths, then the direct diagnosis of intraepithelial lesions,
including
carcinoma-in-situ or early invasive carcinoma can be achieved.
In gynecological oncology, recognizing and treating carcinoma-in-situ is
challenging. Current practice usually begins with a Pap smear test, sometimes
associated with papilloma virus detection, followed by colposcopy with biopsy
and
then a surgical removal. This is time-consuming, involving multiple visits by
the
patient for pelvic examination, cytologic and wet laboratory (for papilloma
virus)
testing and pathological examination of biopsy specimens. This is also quite
expensive.
In this embodiment of the invention, OPS imaging technology, or
derivatives of it, can be employed to directly visualize, characterize, and
also
quantify pre-malignant and malignant epithelial lesions. For example, in the
case
of carcinoma-in-situ of the cervix, a gynecological examination by eye could
be
immediately followed by employing an OPS imaging probe that reveals epithelial
cell layers. The lesions mentioned above are defined as disruptions of the
regular
pattern, by cells with distinguishable characteristics that include abnormal
size,
shape and cellular features such as nuclear: cytoplasmic ratios. They produce
an
abnormal "architecture," in histopathological terms, that has heretofore
required
examination by a pathologist of biopsy material. Distinguishing the abnormal
disruptive architecture of pre-malignant and malignant epithelial lesions from
normal cells could be done by reconstructing 3-dimensional images of the
tissue
from OPS images gathered while focusing through the tissue.
Using an OPS imaging probe, a direct diagnosis could be made by
comparing uninvolved, normal, portions of the cervix with "suspicious"
regions.
Image analysis of the mosaic could lead to characterizable differences in the
regularity of the mosaic. Cell dimensions, shapes and other morphological
characteristics could be quantified; alterations in blood flow and velocity
between
normal and suspicious areas, or even between different tumor types may also be
observed and quantified. This has never been done before and pathological
diagnosis is still impressionistic and subjective rather than statistical.
Direct
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-34-
diagnosis using OPS imaging technology, of course, does not preclude
conventional biopsy.
The approach to intraepithelial diagnosis might well require optimization
of the OPS imaging probe. For example, image analysis of mosaic patterns,
different magnifications, different and perhaps continuously variable focus,
different dimensions of light source, masking of the aperture for dark field
and
perhaps epi-illumination (perhaps also at different angles) to accentuate the
epithelial cell boundaries and interior structure, image quantification of the
normal
vs. inspected abnormal region, etc, may need to be optimized by those skilled
in
the art.
FIG. 1 shows a block diagram of an OPS imaging probe 200, having an
elongated objective 214 (approximately 8 inch), that could be used for the
screening and/or diagnosis of cervical neoplasms or cervical pre-cancerous
lesions.
Probe 200 includes a light source 202, collection lenses 204, relay lenses
208, a
detector 260, and an objective 217. One or more band pass filters 206 may be
placed in the light path to enhance the quality of the image obtained. The
type of
filter used is a function of the object being imaged, as is well-known to
skilled
workers in the relevant arts.
Light source 202 illuminates a tissue region of a subject (shown generally
at 224). Although one light source is shown in FIG. 1, it is to be understood
that
the present invention is not limited to the use of one light source, and more
than
one light source can be used. In an embodiment where more than one light
source
is used, each light source can be monochromatic or polychromatic. Light source
202 can be a light capable of being pulsed, a non-pulsed light source
providing
continuous light, or one capable of either type of operation. Light source
202, can
include, for example, a pulsed xenon arc light or lamp, a mercury arc light or
lamp,
a halogen light or lamp, a tungsten light or lamp, a laser, a laser diode, or
a light
emitting diode (LED). Light source 202 can be a source for coherent light, or
a
source for incoherent light.
A folding mirror or beam splitter 218 is used to form a light path between
light source 202 and subject 224. According to one embodiment of the present
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-35-
invention, beam sputter 218 is a coated plate having 50% reflection of
illumination
beam 209. Other embodiments of beam sputter 218 are well known to persons
skilled in the relevant arts.
In a preferred embodiment, a first polarizer 210 is placed between light
source 202 and subject 224. First polarizer 210 polarizes light from light
source
202. A second polarizer or analyzer 220 is placed between object 224 and image
capturing means 260 along image path 207. Polarizers 210 and 220 preferably
have planes of polarization oriented 90 ° relative to each other.
Polarizers, such
as polarizers 210 and 220, having planes of polarization oriented 90 °
relative to
each other are referred to herein as "crossed-polarizers."
Preferably, the image from object 224 emanates from a depth less than a
multiple scattering length and travels along image path 207 to image capturing
means 260. However, the imaging system of the present invention can also
capture images formed from a depth greater than a multiple scattering length.
Objective 217 is used to magnify the image of object 224 onto image capturing
means 260. Objective 217 is placed co-axially in illumination path 209 and
image
path 207. Image capturing means 260 is located in a magnified image plane of
objective 217. Objective 217 can comprise one or more optical elements or
lenses, depending on the space and imaging requirements of apparatus 200, as
will
be apparent to one of skill in the art based on the present description.
Suitable image capturing means 260 include those devices capable of
capturing a high resolution image as defined above. The image capturing means
captures all or part of an image for purpose of analysis. Suitable image
capturing
means include, but are not limited to, a camera, a film medium, a
photosensitive
detector, a photocell, a photodiode, a photodetector, or a CCD or CMOS camera.
Image capturing means 260 can be coupled to an image correcting and
analyzing means (not shown) for carrying out image correction and analysis.
The
resolution required for the image capturing means can depend upon the type of
measurement and analysis being performed by the in vivo apparatus.
Preferably, objective 217 can be one or more lenses that are selected with
the lowest magnification level required to visualize the illuminated object.
The
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-36-
magnification required is a function of the size of the object in the
illuminated
tissue to be visualized, along with the size of the pixels used for the image.
According to a preferred embodiment, illumination path 209 and image
path 207 share a common axis. This coaxial nature allows for objective 217 to
be
utilized for more than one purpose. First, objective 217 acts as the objective
for
image capturing means 260. In other words, it collects the image beam
emanating
from object 224 onto image capturing means 260. Second, objective 217 acts to
focus the high contrast illumination pattern onto the object plane. The high
intensity portion of illumination beam 209 is directed outside the field of
view
(FOV) of the image capturing means 260.
The combination of the optical characteristics of objective 217 and image
capturing means 260 determine the FOV of device 200. The FOV of the image
capturing means can be limited by many parameters including the numerical
aperture of its objective (here objective 217), entrance pupils, exit pupils,
and the
area of the detector comprising image capturing means 260.
While the cervix is a particularly important target because of the frequency
of disease and the regularity of examination for carcinoma, and also because
the
cervical epithelium is thick, flat based and regular, other intraepithelial
lesions and
immediate sub-epithelial extensions of epithelial malignancies through the
basal
layer and basal membrane are also good targets for this approach to diagnosis.
For example, the difference between a benign activated nevus and a malignant
melanoma presents a serious diagnostic dilemma that usually results in a
biopsy.
With the OPS imaging technique of this invention, such skin lesions, or their
precancerous precursors could be diagnosed, and distinguished, directly. An
"inventory" of pigmented lesions could be recorded by sight on an individual's
body and archived images and quantitative measures be employed as "baseline."
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to assess tumor boundaries or tumor margins, prior to, during, or
following
cancer therapy. For example, OPS imaging can be used to visualize and
characterize true skin cancer margins that may not be clinically visible to
the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-37-
dermatologist with the unaided eye. This may reduce the need for additional
surgery.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to diagnosis different types of tumors based on their vascular
structure.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to monitor the microvascular effects of cancer radiation therapy, such
as
the occurrence of telangiectasia (the dilation of smooth blood vessels). A
high
contrast OPS imaging probe could also be used to study other microvascular or
vascular effects on irradiated tissue, such as changes in perfused microvessel
density following varying doses of radiation.
Applications in Wound Care and Wound Healing Management
In this embodiment, the OPS imaging probe is used in the visualization,
characterization, assessment, and management of different types of wounds--
venous ulcers (caused by chronic venous insufficiency or diabetes); decubitis
ulcers (also known as pressure sores, which form when chronic pressure
inhibits
blood flow, cutting off oxygen and nutrients and leading to tissue ulceration
and
death); traumatic wounds (wounds sustained during an accident or violent
episode); non-healing surgical wounds (incisions made during a surgical
procedure that do not heal within the expected timeframe) and burn wounds.
Since
venous ulcers are formed as a result of an underlying circulatory problem,
knowledge about the microcirculation would be essential to effective
treatment.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-38-
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize and characterize microvessels in and around wounds.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to visualize, characterize, and quantify the perfused
microvessel density in venous stasis ulcers or diabetic ulcers. Using OPS
imaging
on chronic wounds in diabetic patients, a relative lack of microvessels in the
wound bed or in the adjacent tissue has been observed compared to uninvolved
skin.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to observe necrotic tissue and determine the degree of
debridement a wound would require.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to assess the margins of a wound to determine the
likelihood it is going to heal.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to assess the viability of wound tissue to successfully
support a skin graft.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to more accurately and objectively determine the line of
amputation.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to measure and compare the revascularization and healing
of a wound using different wound healing therapies.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to monitor capillary "budding" (i.e., the creation of
new
capillaries) during wound healing.
In the following study, the microcirculation of the skin was visualized and
characterized using OPS imaging. The aim of this study was to validate the use
of OPS imaging (Groner, W., et al., Nat. Med. 5:1209-1213 (1999)) for making
microvascular measurements in the skin against standard fluorescent
videomicroscopy, under normal conditions and in a disease state (in this case
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-39-
during the process of wound healing). Clearly, the assessment of cutaneous
microcirculation would have important clinical implications since several
dermatological pathological states are associated with changes in the
microvasculature. Through quantitative analysis of skin microcirculation,
vascular
pathologies, angiogenesis during wound healing and impaired blood flow after
skin flap creation could be identified.
Materials and Methods: Experiments were carried out on ears of hairless
(SKH-1 hr) mice (n=9). A circular wound was created according to Bondar and
coworkers (Bondar, L, et al., Res. Ex. Med. 191:379-388 (1991). The OPS
imaging device (CYTOSCANTM A/R) was attached to the intravital microscope
to take advantage of the computer controlled XY-plate. Observations were made
using both the intravital fluorescence microscopy (IVM) and OPS imaging in the
exact same regions of interest at baseline as well as 4, 7, 10 and 15 days
after
wound creation. For the measurements with IVM, 0.02 ml of a 3% solution of
FITC-Dextran i.v. was used as a plasma marker. The images obtained from both
instruments were analyzed during playback of the videotapes using a computer
assisted analysis system (CapImageTM) (Klyscz, T., et al., Biomed. Tech.
(Berl)
42:168-175 (1997)). Measurements of arteriolar diameter, venular diameter, red
blood cell velocity and functional capillary density (FCD) were made under
baseline conditions, as well as during wound healing.
Conclusions: OPS imaging produced high quality images of skin
microcirculation with optical contrast comparable to that achieved with IVM.
Further, using OPS imaging, it was possible to make accurate quantitative
measurements of vessel diameter, venular RBC velocity, and functional
capillary
density during the physiological conditions and during the process of wound
healing. The small size and portability of the instrument, as well as the
quality of
the images obtained without the need of a fluorescent dye, indicate that OPS
imaging offers a great potential to be used for diagnostic measurements in
human
skin.
In another study, wound induced angiogenesis was observed using OPS
imaging.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-40-
Introduction: The trauma associated with wounding creates the first
signals for the tissue to begin to repair itself. This initial trauma
initiates
inflammation, which leads to a cascade of molecular signals, encouraging
angiogenesis. Conheim was one of the first in modern history to describe the
initial changes in the vasculature that occur following wounding (Conheim, J.,
"The pathology of the circulation," Section 1 in Lectures in General
Pathology:
A Handbook for Practitioners and Students, traps. by A.B. McKee, ed.., New
Sydenham Society, London. V. 126, 129, 133, pp. xvii-528, 1889-1890 (1889)).
More recently, Clark and Clark suggested that angiogenesis was an important
step
in the initiation of the healing of wounds (Clark, E.R. and Clark, E.L., Am
JAnat
64:251-299 (1939)).
One of the experimental models that have been widely used to study
wound healing is the window chamber. Hashomoto and Prewitt placed a chamber
in the rabbit ear and documented the changes that occur in this model of a
healing
wound (Hashimoto, H., et al., Int. J. Microcirc: Clip. Exp. 5:303-310 (1987)).
Numerous investigators, such as Dewhirst et al. (Dewhirst et al., Rad Res.
112:581-591 (1987)), have placed a window chamber in the dorsal skin fold of
rats and indicated that this is an excellent model for the study of early
angiogenesis. However, such a chamber is not feasible for clinical use in
humans.
This study utilized a similar principle of using a wound to stimulate
angiogenesis.
A reproducible model for the assessment of angiogenesis would be of
value in a variety of clinical situations, such as testing the efficacy of a
particular
therapy for inhibiting angiogenesis in cancer patients. Auerbach et al.,
Pharmacol
Therapeutics 51:1-11 (1991) indicated in a recent review that understanding of
angiogenesis has been important in the increased knowledge of wound healing.
Similarly, Folkman proposed that understanding angiogenesis would permit the
development of therapies to inhibit normal angiogenesis (Folkman, Adv. Cancer
Res.19:351-358 (1974)). The goal of this study was to characterize the
development of microvessels surrounding full thickness wounds in humans.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-41 -
Materials and Methods: Subjects received local anesthesia on the volar
aspect of their forearm, followed by a full thickness 4 mm diameter cutaneous
punch biopsy extending down to fat or fascia. The wound was dressed with
antibiotic ointment and dressed. Every other day, the wound was examined with
a variety of optical devices, including an operating microscope with video
camera
and a CYTOSCANTM A/R. On each day of observation, the dressing was
removed and the cutaneous tissue adjacent to the wound was recorded for
several
seconds in each position, moving around the biopsy margin in a clock-wise
motion. Approximately five minutes were needed to complete one cycle of the
wound margin. The video images were stored in either analog or digital form.
For analysis, a semi-quantitative scale with four grades was established.
Grade 0 was characterized by a total lack of vascular elements and color.
Grade
1 was characterized by a diffuse pink or red color with very few clearly
visible
microvessels in the tissue adjacent to the wound, with no identifiable
organization
or pattern. Grade 2 was typified by short, vertical microvessel loops seen on
end
that were poorly organized with no preferential orientation and little
branching.
Grade 3 was characterized by longer microvessels that were generally curved
but
were beginning to orient radially extending out from the edge of the wound.
Some minimal branching was also visible. Grade 4 consisted of long
microvessels
that were aligned radially like the rays of the sun with some interconnected
loops
or anastomoses. Some limited branching was also observed. Once the semi-
quantitative scale had been defined, blinded observers viewed the images and
scored them using these detailed criteria.
Results: The grading of the vascular pattern observed was reproducible
based on the well-defined criteria. The coefficient of variation was
approximately
10% or less. It took only a few days for the vascular grade to increase to an
average of 1.5. A score of 2.0 was achieved in less than five days following
wounding. The change from an average grade of 2.0 to a grade of 2.5 was
slower, requiring approximately 10 days after wounding. Then, the newly formed
vessels appeared to quickly elongate and begin to align radially, achieving a
grade
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-42-
of 3.0 less than one full day later. The score of 1.0 was achieved soon after
initial
wounding.
Microvascular architecture was also observed in patients with diabetic or
venous ulcers. The CYTOSCANTM A/R was used to perform video microscopy
of each patient prior to treatment and during several weeks of conservative
non-
surgical therapy. A pattern of vascular grades was observed. The most
difficult
wounds showed a general lack of vascularity adjacent to the wounds (as in
grade
1). The diabetic patients, in particular, showed a very low number of visible
microvessels in the superficial skin immediately adj acent to the wound.
Wounds
that were progressing toward healing showed some increased number of
microvessels with no obvious orientation (as in grade 2). Microvessel
lengthening
and the emergence of a preferential microvessel orientation (as in grades 3
and 4)
paralleled a clinical improvement in the wound.
Discussion: The semi-quantitative assessment of angiogenesis induced by
wounds appears to be possible using instruments permitting intravital
microscopy,
such as the CYTOSCANTM A/R. For hundreds of years, numerous investigators
have observed microvessels associated with various types of wounds. However,
clinical studies in patients were extremely difficult to execute due to the
extensive
and cumbersome equipment required. Even in experimental animals, intravital
microscopy has been difficult. OPS imaging greatly facilitates the acquisition
of
microscopic images ofthe living microvasculature. Minimal training ofpersonnel
was required before they were able to accurately conduct the video microscopy
protocols for these clinical studies.
In the following case report, OPS imaging was used to assess the
microcirculation in a burn wound.
Introduction: The treatment of choice of deep second and third degree
burns is toward early excision and grafting. As a prerequisite for such an
aggressive surgical treatment, an accurate diagnosis of the burn lesion is
required.
This underscores the importance of accurate assessment of the severity of a
burn
wound as the key in early decision making. Clinical examination, based on
wound
appearance, remains the unchallenged method for such procedures, even though
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 43 -
it has been reported that clinical assessment of burns performed by clinically
experienced burn surgeons is not always satisfying (Robson, M.C., et al.,
Clin.Plast.Surg. 19:663-671 (1992); Heimbach, D.M., etal., J. Trauma. 24:373-
378 (1984)). Thus, adjunct diagnostic techniques might help surgeons to
accurately analyze the severity of a burn and therefore might help for rapid
decision making at bedside. However, despite all technical innovations an
ideal
device for such an application has not been established for routine use as
yet.
Recently, a novel microscopic technique, orthogonal polarization spectral
(OPS) imaging, has been introduced (Groner, W., et al., Nat.Med. 5:1209-1212
(1999); Messmer, K., ed., Orthogonal Polarization Spectral imaging: aNew Tool
for the Observation and Measurement of the Human Microcirculation, Prog. Appl.
Microcirc., Karger, Basel (2000), pp 1-117). OPS imaging provides a method
which can be used to directly visualize the organ microcirculation in animals
and
in human beings. OPS imaging uses polarized reflected light at a wavelength of
548 nm to visualize hemoglobin carrying microvessels without the use of
fluorescent dyes, thereby avoiding phototoxic effects (Saetzler, R.K., et al.,
J.Histochem.Cytochem. 45:505-513 (1997)). The OPS imaging technique has
been validated for the measurement of functional capillary density against the
standard method for such measurements, the fluorescence intravital microscopy
(Harris, A., et al., J. vast. Res. (In Press) (2000)). Functional capillary
density is
a parameter reflecting capillary tissue perfusion and is given as the length
of the
red cell perfused capillaries per observation area (Harris, A.G., Am. J.
Physiol.
271:H2388-H2398 (1996)).
Tissue blood flow in human burns had been the subject of several studies
and it was hypothesized that the degree of reduction of dermal blood flow in
the
thermally injured skin correlates with the level of its destruction (Micheels,
J., et
al., Stand. J. Plast. Reconstr. Surg. 18:65-73 ( 1984); Alsbjorn, B., et al.,
Stand.
J. Plast. Reconstr. Surg. 18:75-79 (1984); O'Reilly, T.J., et al., J.Burn.Care
Rehabil. 10:1-6 (1989)). However, a direct visualization ofthe
microcirculation
in a human burn, was not yet possible due to methodological difficulties. In
this
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-44-
case study, OPS imaging was introduced as a novel technique for the assessment
of the microcirculation in a burn wound.
Patient: The patient was a 26 year old male patient with a burn injury on
his left hand. The burn was inflicted by boiling oil during cooking and was
immediately rinsed with cold water prior to the emergency room admission. A
second degree burn was diagnosed by clinical observation after initial
debridement
of devitalized tissue. Blisters were opened but left intact. The burn was
managed
conservatively using topical 1 % silversulfadizine in a semi-solid oil in
water
emulsion and the fingers were wrapped separately in a soft gauze dressing. The
hand was elevated for the first 48 hours after the burn and analgesics were
given
orally for pain control.
Study Protocol: The OPS imaging technique has been incorporated into
a small, easy-to-use device called the CYTOSCANTM A/R (Cytometrics Inc.,
Philadelphia, PA). After obtaining informed consent, observations of the
microcirculation were performed with OPS imaging starting day 3 following the
injury. Measurements were carried out at constant room temperature
(27°C) in
our outpatient clinic. Approximately 10 minutes after cleaning of the wound by
irrigation with sterile saline solution to remove the topical agent, the OPS
imaging
probe was applied to 6 different areas of interest at the dorsal surface of
the hand
including the fingers. Images of the microcirculation were recorded on
super-VHS videotapes (Sony, Cologne, Germany). The working distance of the
OPS imaging probe, which was covered with a disposable sterile plastic cap,
was
approximately 2 mm. Sterile ultrasound gel was applied in between the probe
and
the tissue, and helped to improve index matching. The patient never showed
signs
of pain or discomfort during the application of the OPS imaging probe which
required approximately 5 minutes of duration. A custom made holder helped to
prevent the probe from moving during the observations, as well as from
compressing the capillary circulation. Subsequent measurements of the
microcirculation were performed at days 6, 12, 20, 23, 26, and 30 after the
burn,
always returning to the same areas within the affected site. Quantitative
analysis
of the microcirculation was performed off line during play back of the
videotapes
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-45-
using the CapImageTM computer program (Klyscz, T., et al., Biomed. Tech.
(Berl.)
42:168-175 (1997)). Functional capillary density was assessed from single
images
which were digitalized from the running video sequence. Capillaries which were
in focus and could be clearly identified as red blood cell perfused were
included
into the evaluation. Data is given as the number of perfused capillaries per
observation area [n/cm2] as described earlier in studies of clinical
capillaroscopy
(Bollinger, A. and Fagrell, B., Clinical Capillaroscopy-A Guide to its Use in
Clinical Research and Practice, Toronto, Hofgrefe & Huber Publishers, pp. l-
166
( 1990).
Results: OPS imaging produced high quality images ofthe microcirculation
in a burn wound using a total on-screen magnification of 254x. Capillary
morphology was clearly identified and from these images the quantitative
analysis
of the number of red blood cell perfused capillaries was feasible. The
functional
capillary density (FCD) which was measured at day 3 following the injury was
11.2 ~ 4.6 n/mmz (mean ~ SEM). During the initial phase of healing,
microcirculatory changes were characterized by a moderate but steady increase
of
FCD, and showed marked increase beginning from day 12 following the burn
( 16.6 ~ 6.9). Maximal FCD measured at day 23 (48.2 ~ 19.7) decreased from
this
point in time to finally reach 25.2 ~ 10.3 n/mm2 at the end of observation.
The
patient tolerated the measurements very well, and was enthusiastic about
seeing
his own capillaries in the healing wound. The wound, which presented a surface
area of 122 em2 at the day of injury, calculated by digital planimetry, showed
an
uncomplicated healing within 3 weeks without leaving residual scars.
Discussion: The ability to determine precisely and as early as possible the
severity of a thermal injury is prerequisite for planning surgical treatment
thus to
optimize patients functional and cosmetic results (Kao, C.C. and Garner W.L.,
Plast. Reconstr. Surg. 105:2482-2492 (2000); Heimbach, D., et al., World J.
Surg. 16:10-15 (1992); Germann, G., et al., Pediatr. Surg. Int. 12:321-326
(1997)). Early burn wound excision and closure by grafted skin confers several
advantages (Heimbach, D., etal., WorldJ. Surg. 16:10-15 (1992); Engrav, L.H.,
etal., J. Trauma. 23:1001-1004 (1983); Gray, D.T., etal., Am.J.Surg. 144:76-80
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-46-
(1982); Schiller, W.R., et al., J. Trauma. 43:35-39 (1997)), however clinical
evaluation is often inaccurate and estimated not to be better than 64% even
for
experienced burn surgeons (Heimbach, D., et al., supra). Due to the
limitations
of clinical observation, it is not surprising, that for decades interest has
been
focused on devices and methods that could support clinical observation,
particularly to distinguish between burns which heal without complications and
those which need surgery. Numerous modalities such as vital dyes, fluorescein
fluorometry and nuclear magnetic resonance imaging are available to classify
burns
(Heimbach, D., et al., supra). However, none of these techniques has been
accepted for widespread clinical application so far. One reason for that is
the
additional injury some of these invasive approaches inflict, however, the main
reason is that, except for the laser doppler (LD) technique, they have not
proven
to give reliable results. For a review, see, Heimbach, D., et al., World J.
Surg.
16:10-15 (1992) and Shakespeare, P.G., Burns 18:287-295 (1992).
The LD technique was first used for assessing the extent of skin
destruction in human burns by Micheels and coworkers (Micheels, J., et al.,
Scand. J. Plast. Reconstr. Surg. 18:65-73 ( 1984)). The principle of the use
of LD
for such investigations is the hypothesis that the amount of skin blood flow
correlates with the level of its destruction (O'Reilly, T.J., et al., ,I Burn.
Care
Rehabil. 10:1-6 (1989)). As described by Braverman, the nutritive components
of
the skin are formed by the dermal papillary loops which are situated 1-2 mm
below
the dermal surface (Braverman, LM., Microcirculation 4:329-340 (1997)). Thus,
if the papillary loops are destroyed (as is the case in deep dermal burns), a
standstill, or at least a reduction of the microcirculatory blood flow must be
present.
Given this, LD measurements might be useful to obtain data of the amount
of blood flow from which a predictive statement concerning the severity of the
burn lesion can be made. This was already done in clinical investigations, and
a
significant relationship between burn blood flow and clinical ultimate fate
has been
demonstrated in adult patients (Alsbjorn, B., et al., Scand. J. Plast.
Reconstr.
Surg. 18:75-79 (1984); O'Reilly, T.J., et al., J.Burn. Care Rehabil. 10:1-6
(1989);
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-47-
Schiller, W.R., etal., J. Trauma. 43:35-39 (1997); Green, M., etal., J. Burn.
Care
Rehabil. 9:57-62 (1988); Niazi, Z.B., et al., Burns. 19:485-489 (1993); Yeong,
E.K., et al., J. Trauma.40:956-961 (1996)) as well as in children (Miles, L.,
et al.,
J. Burn. Care Rehabil. 16:596-601 (1995)). However, despite these encouraging
results, the use of LD is limited to unexcited and cooperative patients, since
movements or agitation make the measurements difficult to interpret or even
useless (Micheels, J., Scand. J. Plast. Reconstr. Surg. 18:65-73 (1984); Park,
D.H., et al., Plast.Reconstr.Surg. 101:1516-1523 (1998)). In addition, since
the
measurements are reported to be relatively time-consuming for the patient and
the
research staff (Micheels, J., Scand. J. Plast. Reconstr. Surg. 18:65-73 (
1984)), not
all of the patients are suitable for such kind of examinations. Importantly,
additional staff is required, unquestionably raising costs of patient care.
Further
limitations of the LD technique are the pressure exerted by the probe on the
skin,
meaning that the measurement itself influences the parameter which is under
study
(Obeid, A.N., etal., J.Med.Eng.Technol. 14:178-181 (1990)).
In clinical investigations using LD on wounds which ultimately healed, a
steady increase of the average perfusion level was found during the first post
burn
days. In deeper burn wounds, on the other hand, such a clear pattern of
recovery
of blood flow has not been detected (O'Reilly, T.J., et al., J. Burn. Care
Rehabil.
10:1-6 (1989); Green, M., etal., J. Burn. CareRehabil. 9:57-62 (1988); Atiles,
L.,
et al., J. Burn. Care Rehabil. 16:596-601 (1995)). From these investigations,
it
became evident that one limiting factor in the healing of a burn is the amount
of
damage that occurred to the microvessels of the skin.
The rheologic changes in the microvascular network following a scald burn
have also been studied in an animal model using intravital fluorescent
microscopy
and a fluorescent agent for contrast enhancement (Boykin, J.V., et al.,
Plast.Reconstr.Surg. 66:191-198 (1980)). These studies allowed for a close
insight into the dynamic alterations ofthe microcirculation following aburn
injury,
however, this kind of investigation is limited to the laboratory so far.
In this case report, the microcirculation of a human burn wound was
assessed for the first time by means of OPS imaging. This technique allowed
for
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-48-
direct and noninvasive visualization of the cutaneous and dermal
microcirculation
repeatedly during the healing process. The observations were performed within
minutes and the patient was spared to undergo a period of adaptation to
specific
environmental conditions as needed for investigations using LD (Micheels, J.,
et
al., supra). There was no need for surface contact of the OPS imaging probe
thus
excluding pressure artifacts. In contrast to LD measurements, using OPS
imaging,
the individual capillaries in the burn wound were visualized in order to study
their
morphology, and to quantitatively assess their dynamics during the process of
healing from the video recordings.
Microcirculatory data obtained from OPS imaging showed that there was
a similar recovery of FCD in the healing process as reported in previous
studies
for perfusion data using LD (O'Reilly, T.J., et al., J. Burn. Care Rehabil.
10:1-6
(1989); Heimbach, D., et al., World J. Surg. 16:10-15 (1992); Green, M., et
al.,
J. Burn. Care Rehabil. 9:57-62 (1988)). Given that, from LD measurements, a
correlation between perfusion and the burn wound's ability to heal was made,
it
is likely that FCD might also be a useful parameter to predict whether a burn
lesion will heal or not.
The results obtained in the patient reported here have shown that OPS
imaging should provide a reliable tool for quantitative estimation of the
functional
capillary density in burn wounds. The device is convenient to operate,
portable,
and does not require special skills. Moreover, the probe can be wrapped in
sterile
plastic foil also allowing for measurements during surgery. These results on
the
use of OPS imaging to assess the microcirculation in burns appear promising,
and
it is anticipated that this technique will permit our knowledge of the
dynamics of
the microcirculation in the pathophysiology of thermal injury to grow. Beyond
this, OPS imaging, in conjunction with the clinical observation, seems to be
an
encouraging diagnostic tool for the assessment of the severity of burn
lesions.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-49-
Applications iu Plastic Surgery
In this embodiment of the present invention, a high contrast OPS imaging
probe is used in vivo during plastic surgery.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, area-to-
perimeter ratio, blood flow, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a high contrast OPS imaging probe is
used to monitor blood flow (perfusion) during and following plastic,
reconstructive, reattachment, or microsurgery.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to determine healthy versus necrotic or dead tissue around a skin
flap.
During skin flap reconstruction, one's own tissue is used to reconstruct or
replace
tissue lost during surgical tumor removal or trauma. It is important for the
plastic
surgeon to monitor blood flow (perfusion) to the flap both intraoperatively,
and
especially postoperatively to insure that there is good perfusion in the
tissue. This
could be accomplished using functional capillary density measurements, RBC
velocity, and diameter.
In the following study, skin flap perfusion was monitored using OPS
imaging. Despite recent advances in technology, there is currently no
monitoring
system for a reliable detection of perfusion failure in human skin flaps.
There is,
however, a large need for an obj ective method that identifies perfusion
inadequacy
within transferred tissues. OPS imaging allows for the direct visualization
and
characterization of the microcirculation using polarized light. The aim ofthis
study
was to validate OPS imaging for microvascular measurements in skin flaps. The
validation was performed against the standard technique for quantitative
microcirculatory measurements, intravital fluorescence microscopy (IFM).
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-50-
Material and Methods: An established skin flap model of male hairless
mice (hr/hr) was used (Galla, T.J., et al., Br. J. Plastic Surg. 45:578-585
(1992).
After flap creation (n=9), examinations of the skin microcirculation were
performed at 1 hour, 6 hours and 24 hours following flap creation with both
OPS
imaging and IFM. Application of FITC-Dextran (Smg/kgBW) was a prerequisite
for IFM measurements, but not for OPS imaging. For direct comparison of the
two techniques the identical regions of interest were sequentially monitored.
The
flaps were scanned from the distal part to their base. A total of 60 regions
of
interest were captured on videotape of each time point. Functional capillary
density (FCD) was measured off line using the CapImageTM computer program.
All procedures were performed under general isoflurane anesthesia.
Results: Using OPS imaging it was possible to visualize and characterize
the skin flap microcirculation without the use of tracers (FITC-Dextran). From
these images quantitative analysis of FCD was feasible. FCD was significantly
lower in the distal part of the flap compared to the base ( 171.834.7 vs
62.025.6,
mean~SD; 1 h data). Comparison of OPS imaging and IFM revealed a significant
correlation of FCD values (p<0.001, Spearman rank sum test) at all time
points.
Bland-Altman plots revealed a good agreement between the two methods.
Discussion: OPS imaging allows for quantitative analysis of skin flap
perfusion. Given the success of this validation study on mouse skin flaps,
further
investigations have to certify that OPS imaging can also successfully be used
in
humans. Implementation of this novel technique in reconstructive surgery will
improve our knowledge of the function of skin flap microcirculation and
provide
a novel tool for inter- and postoperative monitoring of skin flap perfusion.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to visualize and characterize the microcirculation in
cutaneous and myocutaneous free flaps, as well as free flaps that develop
complications. For example, in questionable free flaps, the direct observation
of
red blood cell flow in capillaries, by using OPS imaging, gave indication that
the
flap was viable.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- S1 -
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used after plastic or microsurgery to continuously monitor
the
microcirculation and identify any potential problems with reperfusion. Early
indication of reperfusion problems may help avoid the need for repeat surgery.
Cardiac Applications
In this embodiment of the present invention, a high contrast OPS imaging
probe is used in vivo in the field of cardiology and/or cardiac surgery.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In the following study, OPS imaging was applied to a beating pig heart
using an epicardial stabilization device, to see if it could be used to
detect,
visualize, and characterize changes in the epicardial microcirculation during
regional induced ischemia.
Methods: Eight pigs (70 kg body weight) underwent median sternotomy
under general anesthesia. After exposure of the heart, an epicardial suction
device
(Octopus, Medtronic Inc.) was applied. The two fork-like extensions of the
device
were placed on top of the left anterior descending (LAD) artery, and the
myocardial area was stabilized by applying 400mmHg of suction. The OPS
imaging probe was placed in this stablized region to visualize the epicardial
microvessels. Regional ischemia of this area was achieved with a monofilament
tourniquet suture around the proximal LAD.
Results: It was possible to obtain microvascular images of the epicardium
in all 8 animals. Semiquantitative analysis showed a decrease in microvascular
blood flow on occlusion of the LAD.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-52-
Conclusions: OPS imaging in conjunction with regional myocardial wall
immobilization device allows the visualization and characterization of the
microcirculation of the epicardium. From the images obtained, it is possible
to
quantify changes in epicardial blood flow and allow a unique assessment of the
S microcirculatory changes during regional ischemia. The ability to detect and
visualize changes in the epicardial microcirculation on the beating heart may
be of
particular interest and a valuable tool during coronary revascularization.
In one aspect of this embodiment, a high contrast OPS imaging probe is
used to increase visualization and characterization of the cardiac
microcirculation
to confirm reperfusion during minimally invasive cardiac surgical procedures
that
avoid the heart-lung bypass machine, such as "keyhole" surgeries (i. e. ,
Heartport)
and thoracotomies.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used for monitoring and detecting changes in patient blood flow (the
microcirculation) during open heart surgery while the patient is on the heart-
lung
machine. The OPS imaging probe would be in the patient's mouth during
monitoring. Differences in blood flow were observed after the patient was
placed
on the heart-lung machine and after coming off of it, when compared to prior
to
the start of surgery. An increased incidence of vessels were also observed
where
leukocytes can be observed. In particular, the longer the patient is on the
heart-
lung machine the worse the flow in the microcirculation is. Thus, the OPS
imaging probe could be used to monitor the progress of the patient while on
the
heart-lung machine, as well as when "off' the machine.
In the following study, OPS imaging was used to directly visualize and
characterize microvascular changes in human patients undergoing cardiac
surgery.
Methods: OPS imaging was used on 12 male patients (mean age 61.1
years) undergoing cardiopulmonary bypass (CPB) surgery to examine the changes
in microvascular perfusion during CPB. Leukocyte-endothelial cell interaction
was also examined. Microvascular diameter (DIA [~cm]), red cell velocity (VEL
[mm/s]), as well as functional capillary density (FCD [cm/cm2]) were measured
in
images taken from the sublingual mucosa, immediately after induction of
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-53-
anaesthesia (T1), in the early phase of CPB (T2), the late phase of CPB (T3),
and
one hour after reperfusion (T4).
Results: DIA was significantly increased at T3 and T4. VEL was
decreased at T2 and increased at T3. FCD did not change significantly (Table 1
).
S Table 1
T1 T2 T3 T4
VEL (mm/s)0.55 0.49* 0.66* 0.59
(0.37-0.81)(0.33-0.69)0.45-1.09) (0.40-0.79)
DIA ( Vim)20.8 23.0 23.8* 25.6*
( 15.5-28.9)( 15.4-32.2)( 18.2-32.0)( 19.0-34.0)
FCD (cm/cmZ)152 135 126 139
I 1123-1751 lyX-1641 1100-1541 1109-17
I I I l
median (25%-75%); *p<0.05 vs. T1
Conclusions: This data provides evidence for microcirculatory changes
during CPB. However, nutritive blood flow seems to be well-maintained during
uncomplicated CPB, since FCD only showed a non-significant decrease.
In another study, described below, OPS imaging was used to visualize and
characterize the changes in epicardial microcirculation during cardiopulmonary
bypass in humans.
Methods: In a pilot study, OPS imaging was used in 3 humans who
underwent median sternotomy under general anesthesia and were placed on
cardiopulmonary bypass for different cardiac procedures. The area perfused by
LAD was visualized in all patients during each period of cardioplegic
perfusion
with blood cardioplegia. Microvascular diameter (DIA [mm]) and red cell
velocity
(VEL [mm/s]) were measured in images taken directly from the heart surface.
Results: Using OPS imaging, microvascular images of the epicardium
were obtained in all patients. The diameters of arterioles and venules ranged
between 10 and 70m. The red cell velocities reached a maximum of 0.6 mm/sec
in these vessels during the application of the cardioplegic solution. Evidence
of
vasomotion was also found since arteries and arterioles were contracting and
dilating. Frequently, areas with no apparent flow of erythrocytes were
identified,
which may suggest that the cardioplegic solution had not reached this part of
the
myocardium.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-54-
Conclusions: This data provides the first intravital microscopic images of
the surface of the human heart during the application of cardioplegic solution
in
humans. The semiquantitative analysis done so far suggests remarkable
differences
in microvascular blood flow. OPS imaging may become of great importance
during cardiac surgery, since it allows direct assessment of microvascular
changes
in the human heart.
In another aspect of this embodiment, the OPS imaging probe also can be
used during coronary artery bypass graft (CABG) surgery to determine if the
area
supplied by the graft is getting good blood flow. That is, a cardiac surgeon
could
use the OPS imaging probe, directly contacting the heart, to monitor the blood
flow to the capillaries after bypass surgery.
In another aspect of this embodiment, the OPS imaging probe can be used
to monitor a patient's microcirculation during cardiac surgery to repair
congenital
heart defects. For example, the OPS imaging probe has been used during
pediatric
cardiac surgery to image the surface of the heart of two infants during
ventricular
septal defect (VSD) repair. The OPS imaging probe, which has the size of a
large
pen, was applied to the surface of the heart. The probe was hand-held and
focused
manually in response to the images seen on a high resolution monitor. Images
were recorded during the application of cardioplegic solution (Brettschneider
solution), which is used to protect the heart during the ischemia associated
with
open heart surgery. Initially, the heart was still beating with < 20
beats/minute
followed by a period of cardioplegia. The well-established CapImageTM analysis
program was used for quantification of the intravital microscopic images.
Using OPS imaging, it was possible to obtain images from the surface of
the heart, and calculate the diameters of the microvessels observed. Despite
the
fact that the Brettschneider solution does not contain erythrocytes, it was
possible
to visualize the vessels due to the remaining erythrocytes within the vessel.
In
general, the cardioplegic solution reached most ofthe microvessels, however
some
microvessels were not reached as they still contained columns of erythrocytes
with
no evident erythrocyte flow. Thus, certain areas of the heart that did not
receive
the cardioplegic solution, remained unprotected from the surgery induced
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-55-
ischemia. Moreover, profound vasomotion in arterioles was observed during the
application of the cardioplegic solution with a diameter change from 70 ~cm to
32
Vim.
Thus, in view of these findings, as well as the prior report, in yet another
aspect of this embodiment, OPS imaging can be used to make both qualitative
and
quantitative assessments of the effectiveness of cardioplegic solution
perfusion
when applied during any cardiac surgery procedure. Further, microvascular
phenomena like vasomotion can be studied with OPS imaging technology.
In another aspect of this embodiment, the OPS imaging probe can be used
to monitor a patient's microcirculation during any type of cardiac surgery.
Examples include surgery for heart valve replacement or heart valve repair.
In another aspect of this embodiment, the OPS imaging probe can be used
for cardiac risk monitoring. The use of the OPS imaging probe to visualize and
characterize the microcirculation may assist in non-invasively determining a
high
risk cardiac profile. The microvascular sequalae of hypertension could be
studied
and also be used in determining cardiac risk.
Pulmonary Medicine Applications
In this embodiment, the OPS imaging probe is used in the visualization,
characterization and assessment of lung tissue.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
The lung from a pig was visualized using the CYTOSCANTM A/R with
two objectives; one objective having an optical magnification of Sx, the other
objective having an optical magnification of 1 Ox.. Using the A/R 1 Ox, much
detail
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-56-
was observed, such as the corner vessels (the vessels that surround the air
sac),
and also the capillaries providing gas exchange within the alveolus. The
plural
vessels were also visualized through the thin membrane overlying the lung
proper.
With the A/R Sx, the air sacs were visualized much more clearly, but the
"corner"
vessels were now almost too small to quantitate (~10-15 microns). The greater
definition seen at Sx vs. l Ox may be due to the different depth of field
between the
two objectives.
Neurosurgical Applications
In this embodiment of the present invention, a high contrast OPS imaging
probe is used in vivo prior to or during neurosurgery.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a high contrast OPS imaging probe is
applied directly to the brain during diagnostic or therapeutic neurosurgery to
view
the cortical or pial microcirculation, and measure parameters such as vessel
diameter, flow velocity, and functional capillary density.
Using a specially prepared OPS imaging device, the pial microcirculation
was imaged in anesthetized humans just prior to neurosurgery. To allow the
surgeon to position the light guide on the brain surface and to hold the image
guide in place for stable video recordings, a stainless steel surgical arm was
developed having three pivot points. The arm could be made rigid by twisting a
number of securing knobs. Before surgery, the arm with the OPS imager was
secured to the rails of the operating table. The arm was covered by sterile
foil
used for covering endoscopes, which in turn was attached to the sterile Teflon
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-57-
sleeve covering the light guide. Following craniotomy, the imager was gently
positioned on the surface of the pial mater. The surgeon was able to observe
the
microcirculation of the brain on the TV monitor.
Although difficult to see in a static image, during video playback, red
blood cells could be observed flowing in single file through the capillaries.
Vessels
could be readily identified as arterioles, capillaries, orvenules. Video
images were
of sufficient quality to allow quantification of red cell flux using
commercially
available image processing software originally developed for analysis of video
images obtained from intravital microscopy (Klyscz, T., et al., Biomed. Tech.
(Berl.) 42:168-175 (1997)). Equally revealing images of other human
microcirculatory beds in the esophagus and the stomach have also been
obtained.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to detect vasospasm following an aneurism or subarachnoid hemorrhage.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to detect boundaries of tumors in the brain. Brain tumors typically do
not
lie at the surface, so they cannot be seen. In another neurosurgical
application of
OPS imaging, the OPS imaging probe can be used during surgery to detect
changes in functional capillary density in "normal" tissue resting above the
tumor,
and therefore can be used to assess brain tumor margins.
Microcirculatory abnormalities in human brain tumors have been observed
using OPS imaging technology. Thus, in another aspect of this embodiment, a
high
contrast OPS imaging probe is used for the determination and typing of brain
tumors based on the vascular structure and differences in the microcirculation
of
different brain tumors.
The microcirculation of human tumors is a highly researched area due to
the importance of tumor hypoxia and angiogenesis. In the following study, OPS
imaging was used to visualize and characterize the microcirculation of human
brain tumors in 11 patients (55.9 ~ 5.17 years old; mean ~ SEM) during
surgery.
These patients were selected because of the superficial position of the tumor
in the
brain. In this way, the microcirculation of the tumor could be observed with
minimal manipulation of the tumor and surrounding tissue. Comparisons between
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-58-
the microcirculation of the patients' healthy cortex and their tumor were made
by
visualizing different random areas.
Three types of brain tumors were examined: benign meningioma (n = 5),
glioblastoma multiforme (n = 4), and metastasis (n = 2). The meningiomas had a
dark background compared to normal, almost no blood flow, chaotic and dilated
vascular pattern. The glioblastoma had a background similar to normal, low
blood
flow, and few vessels compared to normal. The metastases had a very dark
background compared to normal, almost no blood flow, and a chaotic vascular
pattern. This study showed the feasibility of in situ identification of
pathologic
microcirculation in brain tumors during surgery using OPS imaging.
Evaluation of microcirculatory parameters was performed using a
computer asssisted microcirculatory analsysis system (CapImageTM, Dr. Zeintl
Ingenieurbiiro, Heidelberg, Germany). Significant differences in
microcirculatory
parameters (such as red blood cell velocity (RBCV) and functional vessel
density
1 S (total vessel length per area)) were found between the different brain
tumors and
healthy cortex. The RBCV in the glioblastoma was 0.40 ~ 0.10 mm/s compared
to 1.59 ~ 0.32 in the arterioles and 0.64 ~ 0.09 in the venules. Differences
(p<0.05) in vessel length per area (vessel density) were found between the
healthy
cortex and the different tumor types: Meningioma (control; tumor): 68.10 t
5.65
cm/cmz; 41.75 ~ 6.39; Glioblastoma multiforme (control; tumor): 88.47 ~ 5.70;
50.02 ~ 6.91; Metastasis (control; tumor): 72.96 ~ 17.04; 25.04 ~ 6.02). This
study showed the first recording of human brain tumor microcirculation. It is
expected that OPS imaging will provide more insight into the pathogenesis and
possibly treatment of brain tumors.
The following study reports on intraoperative observation of human
cerebral microcirculation using OPS imaging.
Our knowledge of human brain microcirculation is mainly derived from
histological studies (Hunziker, O., J. Geront. 34:345-50 (1979); Craigie,
E.H.,
Biol. Rev. 20:133-146 (1945)), or conclusions drawn from in vivo observations
in animals (Uhl, E., et al., Stroke 30:873-879 (1999)). With the
implementation
of the OPS imaging method into a small hand held device (CYTOSCANTM A/R),
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-59-
it is now possible to observe human brain microcirculation during
neurosurgical
procedures (Groner, W., et al., Nature Medicine 5:1209-1213 (1999)). The
technique is based on the illumination of the tissue with linearly polarized
light,
while the reflected light is imaged using a orthogonally polarized analyzer.
The
application of fluorescent dyes is not required. The aim of the current study
was
to test the feasibility of OPS imaging during neurosurgical procedures and to
obtain basic quantitative data of human cortical microcirculation under normal
and
pathological conditions.
Patients and Methods: The study was approved by the local ethical
committee and informed consent was obtained of each patient prior to surgery.
So
far, 12 patients (6 male, 6 female patients) have been entered in the study.
Four
patients were operated on an incidental intracerebral aneurysm and served as
control group. Three patients had suffered from a subarachnoid hemorrhage
(SAH) due to a ruptured intracranial aneurysm and underwent surgery for
clipping
of the aneurysm. Five patients were operated on a brain tumor. The mean age
was
47.39.7 years. None of the patients had been operated on before. The
operations
were performed under general anesthesia using a standard regimen with
sufentanil
(lgg/kg b.w. for induction of anesthesia), propofol (400-700mg/h) and
remifentanil (0.25-O.S~g/kg/min). In addition, all patients received 8mg of
dexamethasone and 250m1 of 20% mannitol before trephination. Patients with
SAH also received a continous infusion of nimodipine (0.5-2mg/h). Systemic
parameters were routinely measured during the operation.
Intraoperative Measurements: Intravital microscopy was performed with
the CYTOSCANTM A/R equipped with a lens of Sx magnification and a CCD
camera. The technical details of the system have been described elsewhere
(Groner, W., et al., Nature Medicine 5:1209-1213 (1999)). The device, which
was covered with a sterile plastic cap (CYTOLENSTM, Cytometrics Inc.,
Philadelpha, PA) and a sterile plastic foil, was adapted to a Leyla-retractor
which
allowed stable positioning of the probe on the brain surface during the
measurements. Online observations of the cortical microvessels were performed
right after opening the dura, before clipping the aneurysm or resection of the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-60-
tumor, respectively. A second measurement was performed at the end of the
operation before closing the dura. The time between the first and the second
measurement differed depending on the type and difficulty of the surgical
approach. In each patient, at least five sites of interest (SOI) on the
cortical
surface were randomly selected. Each SOI was scanned for 30 seconds and the
images stored on video tape for off line evaluation. Due to the intraoperative
shift
and resection of brain tissue it was not possible to evaluate identical vessel
segments. Evaluation of microcirculatory parameters was performed using a
computer asssisted microcirculatory analysis system (CapImageTM, Dr. Zeintl
Ingenieurbiiro, Heidelberg, Germany) at a final magnification of x260 (Klyscz,
T.,
et al., Biomed. Tech (Berl) 42:168-175 (1997)). Vessel diameters [pm], red
blood cell velocity [mm/s] in arterioles and venules as well as functional
capillary
density [cm'] were measured. The latter is defined as the length of all
capillaries
per cm2, which are perfused with red blood cells at the time of observation.
The results were as follows:
Morphological Aspects of Cortical Vessels: With the OPS system,
arterioles with their typical structure, capillaries, and draining venules
were able
to be clearly distinguished (FIGS. 2 and 3). Capillaries were found to have an
irregular, however mostly circular shape. The number of capillaries can vary
within
a certain area and decreases in the vicinity ofthe junction of venules and
arterioles.
In SAH extravasation, red blood cells in the subarachnoid space were observed
(FIG. 4). In these patients, segmental vasospams in arterioles were detected
leading to a reduction of the vascular diameter of up to 50%. In some
arterioles
this vasospasm was limited to one segment only, in others multiple spasms led
to
a pearl string like appearance of the vessel (FIG. 4). In addition, changes of
the
vascular endothelium were observed initially in patients with SAH as well as
in
patients with brain tumor at the border of tumor resection (FIG. S). In these
vessels, an opaque layer was observed along the luminal vessel surface which
led
to a reduction of the intraluminal space resulting in a reduced flow of red
blood
cells.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-61 -
In cases of the tumor reaching the surface, angiogenetic tumor vessels
could be distinguished from normal brain microcirculation. The tumor vessels
had
an irregular and tortuous shape with sometimes sinusoidal aspect, forming a
conglomerate of vessels at certain areas with normal microcirculation in
between
(FIG. 6). These tumor vessels had close contact to venules whereas there was
no
obvious relation to the arterioles in this area.
Functional Capillary Density: The inital functional capillary density in
control patients was 94.7 ~ 9.lcm-~ (mean~SEM), in tumor patients 79.1 ~ 5.7.
In patients with SAH the initial value was lower than in the other patients
reaching
a mean value of 61.7 ~ 12.5. In all patients the functional capillary density
had
increased at the end of the operation as compared to the first measurement
(FIG. 7) with the most pronounced increase observed after aneurysm surgery in
patients with SAH.
Vessel diameter: The diameters of the observed vessels ranged between
10 and 150 ~m in arterioles/small arteries, and 10 to 210 ~m in venules/small
veins. Since the vessels were randomly chosen it was not possible to measure
the
identical vessel segments at the beginning and the end ofthe operation.
Therefore,
actual changes of specific vessels could not be derived from our observations.
Thus, the distribution of the data is presented rather than the mean (FIG. 8).
Except segmental microvasospasm in patients with SAH no dramatic change of
the distribution of the vessel diameters during the course of the operation
was
observed.
Red blood cell velocity: With the line-shift diagram incorporated in the
CapImageTM system it is possible to measure RBC-velocities up to 2mm/s. In
many cortical vessels, especially in arterioles, RBC-velocity exceeds this
value and
the exact value cannot be obtained. Therefore RBC-velocities were categorized
in 6 classes and the percentage of the vessel classes with a certain RBC-
velocity
is given for each time point (FIG. 9). Vessels with RBC-velocities, that were
too
high to be measured, were comprised in the class of velocities higher than
2mm/s.
Our data show that in all 3 groups of patients, the number of vessels with a
RBC-
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-62-
velocity higher than 2mm/s had increased at the end of the operation with the
most
pronounced changes found in aneurysm surgery following SAH.
Discussion: This experience with the OPS imaging system shows that
CYTOSCANTM A/R is a suitable device for intraoperative observation of human
cortical microcirculation. Brain capillaries, arterioles and venules were
visualized,
but also a quantitative analysis of microcirculatory parameters was obtained.
In
some patients at high magnification, however, brain movement, which mainly
depends on respiration rather than on transmission of the cardiac pulse, did
impair
postoperative off line quantification of the microcirculatory parameters
despite
excellent quality of the images. Although the brain with the translucent and
thin
pia mater is an ideal organ to be studied with a system that works on light
reflectance the quality of the images strongly depends on the underlying
pathology. Whereas in patients with incidental aneurysms and tumors the
quality
was good or excellent, visualization was impaired in patients with
subarachnoid
hemorrhage due to the extravasation of red blood cells and brain swelling. In
addition to extravasated red blood cells we could observe distinct
microvasospasm
in arterioles (10-95pm) and small arteries (100-150pm) as described above.
These
microvasospams were observed in patients with normal transcranial Doppler
values and without clinical signs of vasospasm at the time of the operation.
Whether this intraoperative finding can be of prognostic value for the
development
of clinically relevant vasospasm later in the course of the disease should be
evaluated in the future.
Furthermore, an opaque layer associated with the microvascular
endothelium was observed in patients with SAH as well as at the border of
resection in patients with brain tumor. The underlying pathology of this
observation is not known. It may be speculated that this finding represents
either
endothelial swelling or a dense layer of plasma proteins along the inner
surface of
the endothelium leading to a reduced intraluminal space. The result is a
reduced
flow of red blood cells in the specific vessel.
In addition to the intraoperative visualisation of brain capillaries,
arterioles
and venules, a quantitative analysis of microcirculatory parameters was also
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-63-
obtained. In all patients, FCD was found to be higher at the end of the
operation,
which is probably due to the reduction of intracranial pressure either by
drainage
of cerebrospinal fluid in patients with an aneurysm or by debulking of the
tumor
mass in patients with brain tumor. The resulting relaxation of brain tissue
may also
be the underlying reason for the higher number of microvessels, especially
venules,
with a RBC-velocity exceeding 2mm/s. In contrast to the mean vessel diameters,
which essentially depend on the selection of SOIs by the observer, functional
capillary density and red blood cell-velocity seem to be mainly independent
and
remain as the quanitative parameters of choice.
It is expected that OPS imaging will be a helpful tool in brain tumor
surgery.
In another aspect of this embodiment, a high contrast OPS imaging probe
is used to directly visualize and characterize the vascular consequences of
neural
trauma, and to determine the extent of neural trauma.
In the following study, it was shown that cortical hypoperfusion precedes
hyperperfusion following controlled cortical impact injury in rats.
Impaired cerebral perfusion contributes to tissue damage following
traumatic brain injury. In this longitudinal study, persistence of reduced
cortical
perfusion employing laser doppler flowmetry and intravital microscopy using
OPS
imaging (CYTOSCANTM A/R) were investigated following controlled cortical
impact injury (CCII).
Methods: Before, 30 minutes, 4, 24, and 48 hours after CCII, perfusion
in pericontusional and non-traumatized cortex were determined by moving a
laser
doppler probe in 50 x 0.2 mm steps over the traumatized hemisphere in 6 rats.
Diameter and flow velocity in arterioles and venules were assessed using
orthogonal polarization spectral imaging in the same rats.
Results: At 4 hours after CCII, cortical perfusion was significantly
diminished by 33% (p< 0.05) compared to pre-trauma levels. Despite normal
paCOz values (mean: 42.1 +/- 1.0 mmHg) cortical perfusion was significantly
increased by 43 and 107% (p< 0.005), respectively at 24 and 48 hours following
CCII. Intravital microscopy revealed corresponding alterations. In the early
phase
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-64-
after trauma, vessel diameter was reduced in arterioles by 24% while the
diameter
in venules remained unchanged. Alterations in flow velocity were mostly
sustained
in venules as it was decreased by 39%. In the late phase, vessel diameter was
significantly increased in arterioles (+ 39%) and venules (+ 75%) (p< 0.005).
In
S venules flow velocity exceeded measurable values, being similar to
velocities
determined in arterioles at all time points.
Conclusions: Cortical hypoperfusion found within the early phase
following CCII seems reversible as it precedes a long-lasting phase of
hyperperfusion. Changes in tissue mediators (acidosis, NO, serotonin) could
account for these findings.
Organ Transplantation Applications
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used in vivo during organ transplantation.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used during transplant surgery to determine the amount of perfusion
after
the transplanted tissue/organ is connected. Any transplanted organ can be
imaged,
including, e.g., liver, lung, pancreas, bowel, kidney, and heart. This would
be
especially useful during liver transplantation surgery, as perfusion would be
difficult to assess otherwise (unlike kidney transplantation, for example). In
liver,
the number of perfused sinusoids could be measured. The OPS probe can be
placed directly on the transplanted part of the organ.
One or more parameters, such as, capillary density, vessel (and
microvessel) morphology, vessel density, vasospasm, red blood cell (RBC)
velocity, cell morphology, vessel diameter, leukocyte-endothelial cell
interactions,
vascular dynamics (such as vasomotion), functional vessel density, functional
capillary density, blood flow, area-to-perimeter ratio, hemoglobin
concentration,
and hematocrit, may be quantitatively determined. Preferably, two or more
parameters are determined.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-65-
Vascular Grafting
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used in vivo during vascular grafting, such as in
Peripheral
Arterial Occlusive Disease (PAOD).
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used during vascular graft surgery to determine the amount of
perfusion
after the vascular graft is connected.
Orthopedic Surgery Applications
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used in vivo during orthopedic surgery, as well as in the
field of orthopedic medicine.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used during orthopedic surgery to identify and observe necrotic
tissue, for
surgical removal. The site of the probe would be on the area that has
undergone
trauma.
The probe can also be used to visualize bones, tendons, and ligaments.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to visualize and characterize the microcirculation
around
periosteum (bone). Differences in microcirculation were observed when the
image
was taken before or after a fracture.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-66-
One or more parameters, such as, capillary density, vessel (and
microvessel) morphology, vessel density, vasospasm, red blood cell (RBC)
velocity, cell morphology, vessel diameter, leukocyte-endothelial cell
interactions,
vascular dynamics (such as vasomotion), functional vessel density, functional
capillary density, blood flow, area-to-perimeter ratio, hemoglobin
concentration,
and hematocrit, may be quantitatively determined. Preferably, two or more
parameters are determined.
Gastrointestinal Applications
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used in vivo in the fields of gastroenterology and
gastrointestinal (GI) or gastroesophageal surgery.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize and characterize the large intestine and diagnose
and
treat inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease,
or
other gastrointestinal disorders affecting the microcirculation. OPS imaging
technology can be used to distinguish Crohn's disease and ulcerative colitis,
and
therefore serve as an aid in accurate diagnoses. The probe can be inserted
into the
rectum to directly contact the wall of the large intestine.
In the following study, OPS imaging (CYTOSCANTM A/R) was compared
to intravital fluorescence microscopy for the visualization and
characterization of
colon microcirculation in a mouse model. In this study, the colon
microcirculation
of Balb/c mice under control conditions and after induction of DS S-induced
colitis
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-67-
was investigated (n=7 for each group studied). As microcirculatory parameters,
postcapillary venular diameter, venular red blood cell velocity and functional
capillary density were analyzed on the outer wall of the colon
(serosa/muscularis),
as well as on the luminal wall (mucosa). To examine the agreement between both
methods, linear regression, Spearman's correlation coefficient and Bland-
Altman-
plots were analyzed.
All measured parameters correlated significantly between the two methods
in both the control and colitis groups. It has been demonstrated that OPS
imaging
can be used to visualize and characterize the colon microcirculation without
the
use of fluorescent dyes, and allows quantitative measurement of relevant
microcirculatory parameters of the mouse colon under physiological and
pathophysiological conditions. The results obtained showed a significant
correlation with those obtained with IVM. OPS images were of superior quality
and sharpness compared to those obtained from IVM.
In the following study, OPS imaging detected microcirculatory shunting
in the pig ileum after supra-mesenteric aortic cross-clamping.
Changes in the microcirculation due to ischemia and reperfusion injury are
thought to cause "no-reflow" phenomenon and organ failure after aortic cross-
clamping (AoX). Until now, in vivo observations of the microcirculation were
only possible in rodents by the use of intravital microscopy. In this study,
dynamic
changes in the serosal microcirculation of the pig ileum after AoX were
observed
by using OPS imaging.
Six pigs were anesthetized and fully hemodynamically monitored. An
ultrasonic flow probe was placed around the superior mesenteric artery (SMA).
Following 60 minutes of stabilization, AoX was performed above the SMA for 45
minutes. The deal serosal microcirculation was observed using OPS imaging
during baseline, 5 minutes after AoX, and after 2 hours reperfusion. Video
images
were computer analysed by using CapImageTM software. The number of perfused
vessels per imaged microcirculatory area (3 per pig) were determined. RBC
velocity was calculated and graded in comparison to baseline flow. The number
of perfused capillaries did not change from baseline 9 X1.79 to 5 minutes
after
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-68-
unclamping 10.5 X1.378, although the flow in the SMA nearly doubled. Two
hours after reperfusion, the number of perfused vessels fell significantly to
5 X2.1
(p< 0.05). The RBC velocity 5 minutes after AoX and after 120 minutes of
reperfusion was significant lower (p< 0.05) than at baseline. These results
indicate
a decrease in capillary density and RBC velocity in the microcirculation of
the
ileum after AoX despite an increase in superior mesenteric artery flow. These
results suggest the presence of shunting pathways during "no-reflow"
phenomenon.
In the following study, it was shown by using OPS imaging, that
endotoxin-induced deal mucosal acidosis was associated with impaired villus
microcirculation in pigs.
The following abbreviations are used: PA-cath. = pulmonary artery
catheter for the measurement of mean pulmonary artery pressure and cardiac
output; ECG = electrocardiogram to monitor heart rate; Flow V.portae =
regional
blood flow over the V. porta measured by ultrasonic flow probes; A.fem.-cath.
_
Arterial line in the A. femoralis to measure blood pressure and to take blood
samples; PCOZ-Sensor = fiberoptic pC02 sensor for the continuous measurement
of regional pC02 of the gut mucosa wall within an ileostoma; CYTOSCANTM
A/R= OPS imaging instrument to visualize and characterize the microcirculation
of the gastrointestinal mucosa within an ileostoma.
Introduction: The tonometric determination of the gastrointestinal
mucosal-arterial pC02-gap (0r-aPC02) is used to monitor adequacy of the
gastrointestinal perfusion and can indicate mucosal acidosis (Fiddian-Green,
R.G.,
Br. J. Anaesth. 74:591-606 (1995); Brinkmann, A., et al., Intensive Care Med
24:542-556 (1998)). Several different pathophysiological conditions, however,
can influence regional pC02 homoeostasis such as changes of regional blood
flow,
oxygen delivery and consumption, COZ production and disturbencies of cellular
energy metabolism (Schlichtig, R., et al., J. Crit. Care 11: 51-56 (1996);
Vandermeer, T.J., et al., Crit. Care Med 23:1217-1226 (1995)). The role of the
villus microcirculation for the development of an increased Dr-aPC02 and deal
mucosal acidosis, respectively, has never been investigated in longterm animal
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-69-
models of larger species as no suitable intruments to visualize the gut
mucosal
microcirculation in situ had been available. The CYTOSCANTM A/R, a new
noninvasive method based on orthogonal polarization spectral (OPS) imaging
enabled the visualization, characterization, and recording of the
microcirculation
at this previously inaccessible site.
Thus, the aim of this study was to analyze the influence of villi
microcirculation on the development of mucosal gut acidosis in a hyperdynamic
porcine endotoxic shock model and to compare this to the regional blood flow.
In addition, the applicability and feasibility of the CYTOSCANTM A/R for the
analysis of the gastrointestinal microcirculation is discussed.
Material and Methods:
(1) Animal Model: The endotoxic pig model has been previously
described in detail (Santak, B., et al., Br. J. Pharmacol 124:1689-1697
(1998)).
In the present experiment, 16 pigs (mean body weight 48 kg) were investigated.
1 S All pigs were anesthesized and mechanically ventilated. A pulmonary artery
catheter was inserted through the right jugular vein for the determination of
mean
pulmonary arterial pressure and cardiac output by the thermodilution
principle. In
one femoral artery a catheter was placed for continuous blood pressure
recording
and blood sampling. Ringer's lactate solution (10 ml kg-'h-') was infused
intravenously to maintain fluid balance.
A midline laparotomy was performed and a precalibrated Doppler-
ultrasound flow probe (Transonic Systems, Ithaca, NY) was placed around the
portal vein. The flow was continuously recorded by a T206 flow meter
(Transonic
Systems). An ileostomy was performed for the insertion of a fiberoptic COZ
sensor
(Multiparameter Intravascular Sensor; Pfizer, Karlsruhe) connected to a
monitor
(Paratrend 7; Pfizer), and intravital video records of the ileum
microcirculation by
the CYTOSCANTM A/R.
After instrumentation, a stabilization period of 8 hours was allowed before
baseline measurements.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-70-
(2) Protocol: The animals were randomly assigned to two groups:
Endotoxin (ETX) (n=10) and Sham (n=6). After recording baseline measurements
continuous i.v. endotoxin (Escherichia coli lipopolysaccharide B O 111:B4
[Difco
Laboratories], 20 mg L-' in 5% dextrose) or saline was started. The endotoxin
infusion rate was incrementally increased until mean pulmonary arterial
pressure
(MPAP) reached SO mmHg and then subsequently adjusted to result in moderate
pulmonary hypertension with MPAP 35-40 mmHg. Hydroxyethylstarch was
administered to stabilize hemodynamics and keep mean arterial pressure (MAP)
above 60 mmHg. Further hemodynamic and 0r-aPC02 measurements as well as
CYTOSCANT"' A/R recordings of the deal mucosal microcirculation were
obtained at 12 and 24 hours after the start of the endotoxin or saline
infusion,
respectively. At the end of the investigation, the animals were killed by KCl
inj ection.
The Or-aPC02 was calculated by the difference of deal mucosal and
arterial pCOz. At each measurement time point, 6 video sequences of 1 minute
duration each of the villus microcirculation from randomly chosen locations of
the
ileum mucosa were recorded using the CYTOSCANTM A/R. All villi were counted
and semiquantitatively classified as perfused, heterogenously perfused (i. e.
existence of both perfused and unperfused capillaries within the same villus)
and
unperfused. Due to technical difficulties the microcirculation of two animals
in the
Sham-group were not recorded.
(3) Statistical analysis: All values shown are median and interquartile
range unless otherwise stated. Intragroup differences were tested using a
Friedman repeated measures analysis of variance on ranks and a subsequent
Student-Newman-Keuls test for multiple comparisons. P<0.05 was regarded as
significant. Intergroup differences were analyzed using the Mann-Whitney-Rank-
Sum-Test for unpaired samples.
Results: Endotoxin caused a significant progressive fall of the mean arterial
blood pressure from baseline to 12 and 24 hours of endotoxemia concomitant
with
a significant sustained increase in cardiac output, whereas there was no
effect in
the Sham animals (Table 2).
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-71 -
Table 2
Baseline 12h shoc 24h shock
MAP ETX 102(93;107) 83(74;95)# 79(61;84)#
mmH
Sham 95(91;97) 97(94;99) 108(103;108)
CO ETX 107(97;120) 160( 148;170)#162( 131;200)#
ml/minxk
Sham 115 106;124 107 103;115 116 110;130
# p< 0.05 vs. baseline; ~ p < 0.05 ETX alone vs. Sham
Table 2. Time-dependent variations of cardiac output (CO) and mean arterial
pressure (MAP) in sham-operated and endotoxemic pigs. ETX means endotoxin
group (n=10): animals which received an endotoxin infusion after recording
ofthe
baseline data; Sham means Sham-operated group (n=6): animals which were
prepared like the above mentioned, but received saline instead of endotoxin.
Portal venous blood flow (FIG. 10) remained unchanged in both groups
without intergroup difference. The (0r-aPC02) increased significantly from
baseline to 12 and 24 hours in the endotoxin group, while remained
uninfluenced
1 S in the Sham-group (FIG. 11 ).
FIGS. 12 and 13 show the microcirculatory changes in the ileum mucosa
during the investigation period: At baseline, all villi were perfused while 12
hours
of endotoxin infusion lead to considerable heterogeneity of the
microcirculation
in the endotoxin-group: half of the villi were not or heterogenously perfused,
whereas in the sham group, 5 % only of the classified villi were unperfused (p
<
0.05). Virtually the same pattern was observed at 24 hours of endotoxemia.
Discussion: The aim of this study was to compare the influence of the
macro- and microcirculation on the development of ileum mucosal acidosis
during
longterm hyperdynamic porcine endotoxemia mimicking the clinical features of
septic shock in humans.
Earlier studies showed an deal mucosal acidosis during endotoxemia in
pigs although regional blood flow maintained, and thus, microcirculatory
changes
were expected. Unfortunately, up to now, there were no instruments available
to
visualize and characterize microcirculatory changes at the small bowel mucosa
in
situ which could be integrated in an experimental setting. Thus, the
CYTOSCANTM A/R was an optimal addition, as in comparison to capillary
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-72-
microscopy, it is cheaper, easier to use, and it was not necessary to isolate
the
ileum from the animal. Using this techniques care should be taken not to press
the
probe too hard on the tissue, because this can compromise capillary perfusion.
The key finding was the marked heterogeniety of the villus
microcirculatory status at 12 and 24 hours of endotoxemia, respectively, with
about half of the counted villi being unperfused. For this finding it was
favorable
to use a lower magnification to identify as many villi as possible in one
field of
vision. Moreover, a reduced capillary network within the villus during
endotoxemia was observed, which has also been found by other authors in
smaller
animal septic shock models and described as decreased capillary density
(Farquhar
L, et al., J. Surg. Res. 61:190-196 (1996)). Unfortunately, there exists no
simple
and time-sparing analytical device to quantitate these changes. Measurements
of
flow velocity within the villus capillaries were not possible, because of the
gut
peristaltic, which could not be corrected by movement control functions in the
1 S analytical software.
Besides this, microcirculatory changes during endotoxemia were so
obvious, that perfused vs. not perfused villi was the focus of the evaluation.
These changes were accompanied by a significantly increased pC02-gap
in the endotoxin group indicating intramucosal acidosis (Leverve, X.M.,
Intensive
Care Med. 25:890- 892 (1999)). Taking into consideration the unchanged portal
venous blood flow, i. e. the well-maintained macrocirculatory oxygen
availability
the progressive increase in Or-aPC02 is mainly due to the heterogeniety of
capillary villus perfusion. This finding is in striking contrast to data
recently
published by Knichwitz et al., Crit. Care Med. 26:1550-1557(1998)) stating
that
a reduction of the arterial blood supply of at least 60-70 % is necessary to
result
in mucosal acidosis with increased pCOz-gap.
In conclusion, it was demonstrated, by using the OPS imaging
incorporated into the CYTOSCANTM AR, that the development of ileum mucosal
acidosis in a longterm hyperdynamic porcine endotoxic shock model was
associated with marked alterations of the villus microcirculation, even when
both
portal venous blood flow of the gut wall were well-preserved.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-73-
In another aspect of this embodiment, the OPS imaging probe can be used
during gastrointestinal (GI) surgery to visualize and characterize the colon
during
bowel resection.
In another aspect of this embodiment, the OPS imaging probe can also be
used to determine the boundaries of a cancerous GI tumor and to visualize and
characterize the necrotic tissue. Removal of the affected tissue from the
stomach
and/or esophagus area can thus be more easily accomplished during surgery.
In another aspect of this embodiment, the OPS imaging probe can also be
used to visualize and characterize the rectal mucosal microcirculation, such
as, for
example, in patients with inflammatory bowel disease (IBD).
There is strong evidence that the microcirculation is involved in IBD. OPS
imaging was used to visualize the microcirculation of the rectal mucosa of 11
healthy volunteers and 25 patients with IBD. Of these, a few patients (3
healthy;
6 IBD) were selected to apply a well-known image analysis algorithm in order
to
quantify the images. Macroscopic images were also made using conventional
endoscopy, during which biopsies were taken. OPS imaging allowed detailed
visualization of the rectal mucosa to be made and was well-tolerated by
patients.
Erythrocyte movement could be observed in capillaries and venules. Mucosal
crypts, from which the mucosa is renewed and where mucus is formed, could be
clearly identified. The normal rectal mucosa is characterized by a very
distinct
vascular pattern of the elevated crypts surrounded by hexagonal capillary
rings.
Marked differences were found between the rectal microcirculation of healthy
volunteers and that of IBD patients. The distinct mucosal and capillary
structures
were completely distorted to the extent that the crypts were not identifiable
anymore, and the capillaries were dilated and more tortuous than normal. Image
analysis using a polyhedron-recognition algorithm (expressed in a Euler-
number)
showed a marked difference in results between normal (15.7), severe (-434) and
mild (-36.3) IBD.
In another aspect of this embodiment, the OPS imaging probe can be used
to assess and characterize the microcirculation of solid organs, such as the
liver
and pancreas. Other organs within the gut can be visualized as well using OPS
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-74-
imaging, such as, kidney, small intestine, gall bladder, mesentery, bladder,
diaphragm, stomach, and esophagus. In addition, the OPS imaging probe can be
used to visualize the villi and microcirculation of the villi in the ileum,
through an
ileostomy.
The aim of the following study was to validate OPS imaging against
standard intravital fluorescence microscopy (IFM) under normal and
pathophysiological conditions in the rat liver.
Background: Quantitative analysis of the liver microcirculation using IFM
in animals has increased our knowledge about ischemia/reperfusion injury.
However, because of the size of the instrumentation and the necessity of
fluochromes for contrast enhancement the human liver microcirculation cannot
be
observed. OPS imaging is a recently introduced technique which can be used to
visualize the microcirculation without the need for fluorescent dyes. It is a
small,
hand held device and could potentially be used to study the microcirculation
of the
human liver in a clinical setting. However, before implementation into
clinical use
its ability to quantitatively measure microcirculatory parameters must be
validated.
Methods: The livers of Sprague-Dawley rats (n=9) were exteriorized and
images were obtained using OPS imaging and IFM of the identical microvascular
regions prior to and after the induction of a 20 minute warm lobar ischemia.
Images were videotaped for later computer assisted off line analysis.
Results: OPS imaging can be used to accurately quantify the sinusoidal
perfusion rate, vessel diameter and venular red blood cell velocity.
Correlation
parameters were significant and Bland-Altman analyses showed good agreement
for data obtained from the two methods at baseline as well as during
reperfusion.
Conclusion: OPS imaging can be used to visualize the hepatic
microcirculation and quantitatively measure microcirculatory parameters in the
rat
liver under both physiological and pathophysiological conditions. Thus, OPS
imaging has the potential to be used to make quantitative measurements of the
microcirculation in the human liver. As it can be seen from the correlation
parameters and the Bland-Altman analyses, there is a statistically significant
agreement of the data obtained from OPS imaging and the standard method for
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-75-
such measurements, the intravital fluorescent microcope. In contrast to the
fluorescent method, OPS imaging can be used to visualize the microcirculation
in
humans since it does not require fluorescent dyes for contrast enhancement.
Furthermore, the OPS imaging device is small and can easily be used in a
clinical
setting, e.g., during surgery or transplantation.
One of the most frequent complications following liver transplantation is
the dysfunction of the graft which results from severe deterioration of
hepatic
microcirculation. Therefore, the ability to assess blood flow in the human
liver is
of great clinical importance.
The following validation study has also been done using OPS imaging to
image the rat pancreas. The quantification of capillary perfusion in human
pancreas during transplantation and surgery could correlate the results of
laboratory experiments and provide a new insight into the pathophysiology of
acute pancreatitis and pancreatic ischemia/reperfusion damage.
Materials and Methods: Eight rats (n=8) were anaesthetized and
monitored as described previously. Following transverse laparotomy, the short
gastric arteries were ligated and dissected. For access to the pancreas the
greater
omentum was than removed from the greater curvature of the stomach. The
microsurgical technique performed has been described previously in detail
(Hoffmann et al., Res. Exp. Med. (Berl.) 195:125-144 (1995)). After
mobilization, the pancreas and the attached spleen were gently exteriorized
onto
an adjustable stage and covered by a thin plastic foil to protect it from room
air
and drying. For visualization of the capillary networks 0.3 ml 5 % bovine
serum
albumin labeled with the plasmamarker FITC (fluorescein-isothiocyanate, Sigma-
Aldrich Chemie, Deisenhofen, Germany) were injected intravenously. For
comparison of the capillary perfusion using the two techniques the identical
setup
as for the liver was used. In each animal 6 identical microvascular fields
were
assessed using IVM and OPS imaging. Functional capillary density, defined as
the
length of red blood cell perfused capillaries per observation area [cm/cm2],
was
analyzed by offline computer evaluation of the recorded images using the
CapImageTM computer program.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-76-
Results and discussion: The typical honeycomb-like capillary network of
the pancreas can be visualized with the CYTOSCANTM A/R. The quantitative
analysis of the OPS images showed a mean functional capillary density of 385.4
X45 cm/cm2 which is in agreement with previous studies using intravital
microscopy. As it can be seen in the Bland-Altman analysis, these values are
on
average only 2.2 % (8.7 cm/cm2) less those obtained from IVM images.
In addition, just two data points are outside of the 95 % confidence
interval and the regression line coincides with the mean. Thus, it becomes
clear
that there is a very good agreement between the two methods (IVM and OPS
imaging) for the measurement of FCD in the rat pancreas under baseline
conditions. The contrast and quality of the images obtained using IVM and the
CYTOSCANTM A/R are qualitatively the same, and therefore the time required for
the analysis of images was comparable for both techniques.
Thus, OPS imaging is a suitable tool for quantitative analysis of pancreatic
capillary perfusion during baseline conditions. The CYTOSCANTM A/R would
be a useful tool for the scientific and clinical evaluation of the
microcirculation of
the pancreas during surgery and transplantation in humans. Since pancreatic
perfusion failure is an indication of the degree of postischemic damage to the
organ (Hoffmann et al., Res. Exp. Med. (Berl.) 195:125-44 (1995); Hoffmann et
al., Microsc. Res. Tech. 37:557-571 (1997)) the measurement of the FCD would
be a useful diagnostic parameter for monitoring the condition of the pancreas.
Opthalmological Applications
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used in vivo in the field of opthalmology.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
_77_
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a high contrast OPS imaging probe is
used to visualize and characterize the microcirculation of the interior of the
eye
(especially the retinal microcirculation). In one embodiment, the OPS imaging
probe can be utilized in a non-contact method. In this embodiment, the optics
are
set up so that the relaxed eye (focusing near infinity) focuses and images the
OPS
light onto the retina; alternatively, the imaging probe could be utilized in a
direct
contact manner, such as, by the use of a special diagnostic type of corneal
contact
lens. Such a visualization tool can be used for diagnostic purposes and
treatment.
In a preferred embodiment, a fundus photographic unit , a laser delivering
light of suitable wavelength to treat specific intraocular lesions, and the
OPS
imaging probe is functionally integrated. This type of instrument could be
used
for diagnostic purposes and treatment, as well as providing information
regarding
the effectiveness of various types of medications on the circulatory system in
the
area examined.
In order to be accurate, the exact area (spot) to be determined and treated
or examined and re-examined in the eye must be easily found again. Therefore,
a
centering, tracking and grid device may be incorporated into the system. The
tracking device is important to allow the area being evaluated and treated to
always be in the same position relative to the instrumentation, even though
there
may be fine eye movements.
Also important in the optimization of such instrumentation is the
incorporation of a recording device indicating the size of the vessels,
lesions, etc.,
irrespective of magnification. At present, the field visualized by the
instrument
when used as a direct contact device externally is about O.Smm. There will be
some variation in the size of the intraocular field, depending upon the
dioptic
power of the eye being examined and the power of a corneal contact lens, if
one
is being used.
Image intensification can be incorporated so that minimal light of the
desired wavelength is used to define the images. Ideally, the image will be in
color
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
_78_
and in real time. The instrument will allow for immediate diagnosis of
pathologic
micro-circulatory problems. Real time video or photographs can be done for
permanent records. Immediate laser or other types of treatment such as
photodynamic therapy of the pathology can be performed in real time.
Optimization of this device will include some or all of the following
features: a centering device; a method to visualize the same spot or area; a
device
to track eye movements and always be in the same position; a recording device
indicating the size of vessels irrespective of magnification, i. e.,
calibration device;
an image intensifying device; and a magnification means to expand the size of
the
image. Color images would be optional.
In another aspect of this embodiment, OPS imaging technology can be
used to diagnose macular degeneration; retinal disorders (retinopathy), and
glaucoma.
In another aspect of this embodiment, OPS imaging technology can be
used to visualize the optic disk, retina, sclera, and changes in the vitreous
humor.
In another aspect of this embodiment, OPS imaging technology can be
used for early diagnosis and treatment of diabetes by looking at the ocular
microcirculation, especially changes or differences in the sclera and/or
aqueous
humor of the eye.
In another aspect of this embodiment, OPS imaging was used to analyze
the ocular microcirculation during operations on the internal cartoid artery
(ICA).
The aim of the following study was the intraoperative visualization and
quantitative analysis of the microcirculation using OPS imaging in the flow
area
of the internal (ICA) and external (ECA) cartoid artery during reconstruction
of
stenosis of the cartoid artery.
Materials and Methods: Interoperatively, in 15 patients, the microvascular
perfusion of the front part of the eye (sclera) was visualized and quantified
using
a CYTOSCANTM A/R. The FCD, capillary diameter, and blood flow velocity
were measured in the microvascular network of the ICA stenosis area in the
ipsi
and contralateral eye under control conditions (I); during ECA ischemia (II);
during ICA ischemia (III); during shunt perfusion (IV); during shunt removal
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-79-
(ischemia), selective after reperfusion of the ECA (V); and ICA (VI); 5
minutes
after reperfusion (VII); and 20 minutes after reperfusion (VIII).
Results: The temporary ischemia during the implantation of the shunt
caused a significant reduction in the nutritive ocular capillary perfusion of
approximately 50% in comparison to the baseline values before ischemia.
Furthermore, the short ischemia period was also associated with a significant
decrease in the capillary diameter. The changes were even more pronounced
following clamping of the ICA and the ECA when compared to a simple short
ECA ischemia. In contrast, the post-ischemic shunt perfusion of the ICA was
characterized by a significant increase (p<0.05, paired t-test) of the FCD,
the
capillary diameter and the RBC velocity when compared to I and III. Renewed
temporary ischemia during the shunt removal led to similar perfusion deficits
like
during shunt implantation. In contrast, the immediate post-ischemic
reperfusion
(VI) was associated with a significant increase (P<0.05) in the FCD, the
capillary
diameter, and the RBC velocity. The increases in perfusion could also be
observed both during shunt perfusion during the reperfusion phase and in the
contralateral eye and 20 minutes post reperfusion less evidently.
Conclusions: OPS imaging makes it possible for the first time to directly
visualize and quantify the ocular microcirculation. It allows immediate and
reliable
intraoperative monitoring of ischemia (perfusion deficit) and reperfusion
caused
changes in the cerebral microcirculation during reconstruction of the carotid
artery. The ocular interruption of the microcirculation such as capillary
stasis and
constriction, as well as oscillating flow which occur immediately after even
short
ICA and ECA ischemia, indicate that there is a rapid manifestation of ischemia
induced microvascular dysfunction. The maintenance of an adequate perfusion in
the ICA perfusion area during the reconstruction leads to a significant
improvement and protection of the ipsi and contralateral ocular
microcirculation.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-80-
Applications During Pregnancy
In this embodiment of the present invention, the OPS imaging probe is
used during normal or complicated pregnancy to monitor the woman's
microvascular function.
S In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used to detect or monitor women whose pregnancy is complicated by
preeclampsia (PE). As-used herein, "preeclampsia" is a complication of a
current
or recent pregnancy, characterized by hypertension with proteinuria and/or
edema.
Preeclampsia is a systemic disease with an endothelial cell dysfunction. To
investigate the way in which PE effects the microcirculation of the skin,
microvascular function was studied with OPS imaging.
The aim of the following study was to investigate in vivo microcirculatory
function in pregnancy and pregnancy complicated with preeclampsia.
Summary: In 10 preeclamptic and 10 normotensive pregnant women, the
microcirculation of the skin was studied. The red blood cell velocity at rest
(rCBV) and the local sympathetic veno-arteriolar reflex (VAR) during venous
occlusion was studied in the nailfold using OPS imaging. Laser-Doppler
fluxmetry
was used to assess skin microcirculatory function during venous occlusion,
arterial
occlusion, and during rest.
Laser-Doppler fluxmetry showed no difference between the normotensive
and the preeclamptic group. Although no differences were found in the absolute
velocities during rest and during venous occlusion between the two groups as a
whole, OPS imaging, showed a decrease in red blood cell velocity following
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-81-
venous occlusion (58% (40-94) (median and range)) in the group with
preeclampsia, compared to 84% (74-89) in the control group (P = 0.003).
This study shows that by using OPS imaging, there is an impaired local
veno-arteriolar reflex in preeclampsia, not detectable by laser-Doppler
fluxmetry
measurements. OPS imaging may also permit the study of the effects of
preeclampsia in other organ beds.
Introduction: The maternal cardiovascular system reacts to pregnancy
with a decrease in peripheral vascular resistance and systemic blood pressure,
accompanied by an increase in blood volume and cardiac output resulting in a
normotensive condition (NT). These adaptations are necessary to meet the
demands of normal pregnancy. This response, however, is impaired in
pregnancies
complicated with preeclampsia (PE). Pathophysiologic findings in PE are high
blood pressure, proteinuria, decreased plasma volume, increased peripheral
vascular resistance, vasoconstriction, and reduced organ perfusion (Roberts,
J.M.,
and Redman, C.W.G., Lancet 341:1447-1451 (1993); Bosio, P.M., et al., Obstet.
Gynacol. 94:978-984 (1999)). The coagulation cascade is activated and there is
an imbalance between prostacylin/thromboxane ratio causing platelet
aggregation
and a further increase in vasoconstriction (Walsh, S.W., Am. J. Obstet.
Gynocol.
152:355-340 (1985)). Accumulating evidence suggests ageneralized endothelial
cell dysfunction in PE (Roberts, J.M., and Redman, C.W.G., Lancet 341:1447-
1451 (1993)). The cause of the endothelial dysfunction is still elusive, but
it is
suggested that it is caused by blood borne products from a poorly perfused
placenta (Roberts, J.M., and Redman, C.W.G., Lancet 341:1447-1451 (1993)).
In PE, a vascular dysfunction was found in isolated arteries from several
organs (Aalkjaer, C., et al., Clin. Sci. 69:477-482 (1985); McCarthy, A.L., et
al.,
Am. J. Obstet. Gynacol. 168:1323-1330 (1993); Vedernikov, Y., et al., Semin.
Perinatol. 23:34-44 (1999)). There is an impaired endothelium-dependent
dilatation in PE compared to NT (McCarthy, A.L., et al., Am. J. Obstet.
Gynacol.
168:1323-1330 (1993); Knock, G.A., and Poston, L., Am. J. Obstet. Gynacol.
175:1668-1674 (1996)), and vascular smooth muscle cells of women with PE
showed an increased sensitivity to vasopressors (Gant, N.F., et al., J. Clin.
Invest.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-82-
52:2682-2689 (1973)). Also, an increased sympathetic activity was found in
blood vessels of skeletal muscle in vivo in PE (Schobel, H.P., et al., N.
Engl. J.
Med. 335:1480-1485 (1996)). These findings suggest that not only endothelial
dysfunction is responsible for changes in vascular reactivity in PE.
However, most of these studies have been performed in vitro and
assessment of vascular function in vivo has been done less frequently because
of
the difficulties in studying vascular function in vivo. An in vivo study with
laser-
Doppler fluxmetry (LDF), using ionophoresis of vascoactive agents, showed no
altered vascular function in the microcirculation of the skin in women with PE
(Eneroth-Grimfors, H., etal., Brit. J. Obstet. Gynacol. 100:469-471 (1993)).
The
reaction after arterial occlusion (the hyperemic reaction, an endothelial-
dependent
vasodilation) studied with LDF was increased in the skin of the hand in PE
compared to NT, while in the skin of the arm no difference was seen (Beinder,
E.,
and Lang, N., Geburtsh a Frauenheilk 54:268-272 (1994)). Vascular reactivity
in local cooling, causing vasoconstriction, was significantly greater in both
areas
in PE (Beinder, E., and Lang, N., Geburtsh a Frauenheilk 54:268-272 (1994)).
However, with capillaroscopy of the skin in the nailfold, no difference was
found
in the hyperemic reaction in women with PE (Rosen, L., et al., Int. J.
Microcirc.
Clin. Exp. 8:237-244 (1989)), while the brachial arteries of women with PE
showed an altered hyperemic reaction studied with high-resolution
ultrasonography and Doppler ultrasonography compared to NT (Yoshida, A., et
al., Hypertension 31:1200 (1998); Veille, J.C., et al., J. Soc. Gynacol.
Invest.
5:3 8-43 ( 1998)).
Microcirculatory dysfunction was demonstrated by Rosen, L., et al., Int.
J. Microcirc. Clin. Exp. 9:257-266 (1990), who showed that the reaction to
venous occlusion, causing a veno-arteriolar reflex (a local sympathetic reflex
leading to an endothelium-independent vasoconstriction reaction), was
depressed
in the skin in PE studied with capillaroscopy. This study, however, was never
repeated and the cause of an impaired veno-arteriolar reflex in combination
with
3 0 an increased sympathetic activity remains uncertain. That is the reason
the present
study was undertaken. Since the use of different methods and various vascular
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-83-
beds resulted in contradictory results concerning vascular reaction to
arterial
occlusion in PE, the present study also studied the hyperemic reaction.
To observe vascular function of the cutaneous microcirculation in PE in
vivo in a simple and non-invasive way, OPS imaging was used. Implanted in a
small and hand-held device, this technique allows intravital observation not
only
of the microcirculation of the skin but also of internal human organs at
bedside and
during surgery (Groner, W., et al., Nature in Medicine 5:1209-1212 (1999). The
technique has been validated by comparison to conventional capillaroscopy. To
compare the results with another method that measures in vivo microvascular
function also, laser-Doppler fluxmetry (LDF) was used. The control of vascular
function by the sympathetic system was studied by assessment of the veno
arteriolar reflex (VAR) (Henriksen, O., Acta Physiol. Scand. 143:33-39 (1991))
using both devices. The control of endothelium-dependent vasodilation was
studied with the hyperemic reaction (Ostergren, J., and Fagrell, B., Int. J.
Clin.
Microcirc. Clin. Exp. 5:37-51 (1986)) with LDF.
Material and Methods:
(I) Subjects: Ten women with PE and 10 women with a normotensive
pregnancy (control group) were enrolled after written informed consent. PE was
defined as a diastolic blood pressur >90 mmHg developed after a gestational
age
of 20 weeks and proteinuria >300 mg/24 hours or dipstick ++/+++ (National High
Blood Pressure Education Program Working Group Report on High Blood
Pressure in Pregnancy, Am. J. Obstet. Gynacol. 163:1689-1712 (1990)). The
Karotkoff V was used to determine diastolic blood pressure. Of the women with
PE, six used medication of which three used infedipine (Adalat, 3-4 x 10 mg)
and
three methyldopa (Aldomet, 3 x 250 or 500 mg). Women from the control group
were recruited by advertisements in the midwifery outpatient clinic of the
hospital.
None of them used medication. Women with diabetes mellitus, hypertension, and
Raynaud's phenomenon diagnosed before pregnancy, or with fever, were not
included in this study. The characteristics of the subjects are listed in
Table 3.
(2) Methods: Laser Doppler Fluxmetry (LDF) (Perifleux 4001, PF 408
standard probe time constant 0.2 sec, Perimed, Sweden) was used to assess
total
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-84-
cutaneous blood flow (Stern, M.D., et al., Am. J. Physiol. 232:H441-H448
( 1977)). Two probes were attached to the pulp of the finger using a probe
holder
(Perimed, Sweden) with double-sided adhesive tape. Laser light with a
wavelength of 780 nm was conducted through optical fibers to the skin where it
penetrated the skin to a depth of 1-1.5 mm and was partly reflected. The
light,
when backscattered by moving obj ects (erythrocytes), undergoes a frequency
shift
proportional to the velocity and number of moving objects and is expressed in
arbitrary units (volts) and referred to as flux. The data were recorded and
analyzed off line (AcqKnowledge III and MP 100 WSW, Biopac System Inc.,
Santa Barbara, California).
The microcirculation of the nailfold was studied with OPS imaging
(version E2, Cytometrics Inc., Philadelphia, PA). This technique makes use of
green polarized light passed through a light guide and is placed above the
organ
bed to be measured. By cross-polarization of the reflected light all surface
reflection is eliminated and remarkably sharp images can be recorded (FIG.
14).
A 75 W halogen precision lamp was used as light source with light of a
wavelength of 540 nm. A removable Sx objective was used with a final onscreen
magnification of 325x for analysis. All images were recorded on a digital
video
recorder (Sony DSR-20P), off line analyses ofthe images were accomplished with
a software program (CapImageTM, Dr. Zeintl Software engineering, Heidelberg,
Germany) (Klyscz, T., et al., Biomed Tech (Berl) 42:168-175 (1997)).
(3) Measurements: With LDF, the mean flux at rest (RF) during 5
minutes, and the mean flux during 2 minutes of venous occlusion after 1 minute
occlusion (VOF), to determine the VAR, were measured. Also, the highest flux
after 1 minute of arterial occlusion (PF) and time until PF were measured. The
decrease or increase in flux normalized to rest values was calculated as
((RF-VOF)/RF) and ((PF-RF)/RF), and expressed as percentages. During arterial
occlusion, the residual LDF (biological zero) (Caspary, L., et al., Int. J.
Microcirc.
Clin. Exp. 7:367-371 (1988)) was determined, which was subsequently subtracted
from all other measured LDF values. LDF measurements could be performed in
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-85-
all but two women from the control group because of the unavailability of the
laser-Doppler device on those occasions.
With OPS, the capillary blood cell velocity at rest (rCBV) was measured
as the mean velocity during 30 seconds and the VAR was determined as the mean
velocity during 30 seconds after 30 seconds of venous occlusion (roCBV). Both
velocities were measured by use of the line-shift diagram method (Klyscz, T.,
et
al., Biomed Tech (Berl) 42:168-175 (1997)). The decrease in red blood cell
velocity during venous occlusion normalized to rest value was calculated and
expressed in percentages ((rCBV-voCBV)/rCBV). From every subject, three
capillaries were studied and mean values used for comparisons.
(4) Protocol: All subjects were asked to refrain from drinking caffeine-
containing drinks from 2 hours before the investigation. The investigation
took
place in an air conditioned room with a temperature between 23 and 25
°C after
an acclimatization period of 15 minutes. The subjects lay down in lateral
position
on their dominating side, with their arm slightly bent at heart level on a
pillow. A
cuff was placed around their upper arm. At first, the capillaries of the 4th
finger
of the non-dominating hand were studied with OPS imaging, the device placed
slightly above the surface of the nailfold. The fingers were imbedded in a
mass of
clay to stabilize the hand and a drop of paraffin oil on the nailfold was used
to
make the skin more transparent. Skin temperature was measured continuously
with a digital thermometer (Keithley 871 A) taped on the skin proximal to the
nailfold. From the capillaries of the nailfold, three well visualized
capillaries were
chosen for measurements. Each capillary was recorded for 2 minutes to measure
rCBV, after which the cuff was inflated to 50 mmHg for 2 minutes to measure
voCBV.
After these recordings, the measurements with LDF took place. The two
probes were attached to the middle phalanx of the same finger, one at the
palmer
side and one at the dorsal side. After a period of rest for at least 5 minutes
to
determine RF, the cuff was inflated to 50 mmHg to measure VOF during 3
minutes. The cuff was then inflated to 200 mmHg for arterial occlusion to
determine the biological zero, and deflated after 1 minute. For 2 more minutes
the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-86-
flux was recorded for PF and time to peak. The blood pressure was measured at
the end of the investigation.
(5) Statistics: Values were expressed as median with range unless stated
otherwise and p-values were calculated with the Mann-Whitney U test and the
Wilcoxon signal rank test. A p-value <0.05 was considered statistically
significant.
Results: There were no differences in patient characteristics between both
groups except for blood pressure (Table 3). The results of the laser-Doppler
fluxmetry are presented in Table 4 and FIGS. 15A and 15B. There was no
significant difference in flux or percentual change between patients with PE
and
the control group at rest, during venous occlusion or after arterial
occlusion, at
palmer and dorsal side of the finger. In the control group, there was even no
significant change in flux during venous occlusion at dorsal side, while in
the
patient group, venous occlusion at palmer side did not provoke a significant
reaction.
Analysis of capillary red blood cell kinetics obtained from OPS images
revealed no difference between the rCBV and voCBV between the two groups.
However, the percentual decrease in the blood cell velocity during venous
occlusion in the women with PE, was significantly smaller than the decrease in
the
control group, 58% (40-94) vs. 84% (74-89) (P = 0.003, see FIG. 16).
Comment: The present study confirms an impaired VAR in PE identified
using OPS imaging. This impaired reflex was also found by Rosen et al., supra,
with capillaroscopy. The VAR is one of the reflexes causing arteriolar
constriction for maintaining arterial blood pressure in an upright position.
It is a
local sympathetic axon reflex (Henriksen, O., Acta Physiol. Scand. 143:33-39
(1991); Vissing, S.F., et al., Acta Physiol. Scand. 159:131-138 (1997)), the
role
of the endothelium in the VAR is still unknown (Henriksen, O., Acta Physiol.
Scand. 143:33-39 (1991)). The VAR is elicited by venous distension causing
arteriolar constriction resulting in a decrease in blood flow. Increasing
transmural
3 0 pressure of more than 25 mmHg by inflating a cuff around the arm causing
venous
occlusion can also provoke this reflex.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
_87_
An impaired reflex could not be detected with LDF. This discrepancy may
be explained by the fact that in LDF, not only the blood flow in the nutritive
capillaries of the skin is measured like with OPS imaging, but also the flow
in the
subpapillary (thermoregulatory) plexus at a greater depth. It has been
suggested
that the VAR in these vessels is less obvious (Tooke, J.E., et al., Int. J.
Microcirc.
Clin. Exp. 2:277-284 (1983)). This could explain why no differences were found
in the VAR with LDF in contrast to the results with OPS imaging.
Furthermore, we found no defect in hyperemia response following arterial
occlusion between PE and NT. The hyperemic reaction is a largely endothelium-
dependent vasodilation response to arterial occlusion. After the release of
the
cuff, the blood flow starts again and increases until it reaches a peak
velocity after
a few seconds and returns to rest value.
Previous investigators found an impaired hyperemic reaction of the
brachial artery in women with PE compared to NT (Yoshida, A., et al.,
Hypertension 31:1200 (1998); Veille, J.C., etal., J. Soc. Gynacol. Invest.
5:38-43
(1998)). These results could not be confirmed in the cutaneous
microcirculation
using LDF. LDF is widely used, often to evaluate the microcirculation in
vascular
diseases and diabetes mellitus (Schabauer, A.M.A., nad Rooke, T. W., Mayo
Clin.
Proc. 69:564-574 (1994)). However, there is a great physiologic variability in
flux, which causes poor reproducibility (Schabauer, A.M.A., nad Rooke, T.W.,
Mayo Clin. Proc. 69:564-574 (1994)). Several measures, like venous occlusion
to provoke the VAR, and reactive hyperemia, by which the subjects are their
own
control, are used to reduce this variability.
In PE, a sympathetic overactivity has been found in the blood vessels of
skeletal muscle. This could cause an increase in sympathetic vasoconstrictor
activity and could play a role in the increase in peripheral vascular
resistance
(Schobel, H.P., et al., N. Engl. J. Med. 335:1480-1485 (1996)). Sympathetic
overactivity may thus explain the impaired reaction to venous occlusion. An
increased arteriolar constriction in PE would impair further vasoconstriction
by
sympathetic stimulation. Methyldopa, a centrally acting drug reducing
sympathetic outflow, is first choice medication for long term blood pressure
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
_88_
control in PE, indeed suggesting involvement of the sympathetic nervous
system.
An alternative hypothesis could be that because of the sympathetic
overactivity,
the receptors of the smooth muscle cells are down-regulated, causing less
vasoconstriction when stimulated. There is a down-regulation of the a,- and
az-adenoreceptors of the myocardium in heart failure, a condition associated
with
elevated sympathetic activity (Brodde, O.C., Pharmacol. Rev. 43:203-242
(1991)). It is not known what happens with receptors of the circulation of the
skin during sympathetic overactivity. However, it cannot be excluded that the
impaired VAR is a consequence of functional or structural changes of the
microcirculation in PE, which are independent of the sympathetic nervous
system
and contribute to impaired vascular function.
In this study, the microcirculation of the skin with OPS imaging and LDF
was investigated. In conclusion, OPS provided a more sensitive technique to
evaluate vascular function in pregnancy than LDF. Using OPS imaging, an
impaired veno-arteriolar reflex in women with PE was detected, that could not
be detected with LDF. This result suggests impaired sympathetic
vasoconstriction
in the microcirculation of the skin. The ability to use OPS imaging for
observation
in other organ beds could help to clarify vascular dysfunction in PE.
Table 3:
Characteristics of Subjects
Control (n=10) Preeclampsia
(n=10)
Age (year) 33.0 (22.5-33.6)29.7 (22.8-32.4)
Gestational age (weeks)31.3 (22.1-37.1)32.9 (21.4-40.3)
Diastolic blood pressure68 (50-78) 100* (75-112)
(mmHg)
Systolic blood pressure108 (90-110) 144* (120-172)
(mmHg)
Hb (mmol/1) 7.5 (6.4-8.1) 7.9 (6.3-8.7)
Skin temperature (C) 34.5 (32.9-35.6)34.4 (31.9-35.5)
Results given as median and range. P < 0.001 for comparison with control
group,
Mann-Whitney U test.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-89-
Table 4:
Laser Doppler Fluxmetry Values in Volts
Control (n=8) Preeclampsia
(n=10)
almar dorsal almar dorsal
1.40 0.39 0.71 0.31
(0.35-3.23) (0.22-0.63)(0.13-2.28)(0.22-0.99)
0.62* 0.29 0.47 0.25*
VOF (0.25-3.31) (0.20-1.14)(0.03-2.94)(0.13-0.56)
pF 2.28* 1.16* 1.63* 0.71*
(0.78-5.44) (1.00-1.83)(0.53-3.58)(1.39-2.60)
Time 4.50 5.85 5.65 5.90
to
peak (3.4-7.1) (3.7-8.3) (3.1-7.9) (2.8-8
(sec) 7
.)
Results given as median and range.
* Values significantly different compared to the RF
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to monitor intact placental microvasculature in
preeclampsia/ fetal growth retardation.
It is well-known that fetal nutrition and oxygenation are dependent on
well-developed and capillarised terminal villi of the placenta. In
preeclampsia, fetal
growth retardation is a common symptom. Using scanning electron microscopy,
it has been shown that hypocapillarisation exists in the terminal villi
(Habashi S.
et al, Placenta 4:41-56 (1983)).
OPS imaging was used to examine, ex vivo, the surface of the maternal site
of the placenta from 10 normotensive women with neonates appropriate for
gestational age, and from 10 preeclamptic women with neonates small for
gestational age. The aim of this study was to determine if images could be
obtained of the microvasculature, and if the terminal villi of the latter
group were
hypocapillarised, compared with the first.
Within 10 minutes after delivery ofthe placenta, umbilical arteries and vein
were cannulated and perfused with a mixture of 50 mL Ringers glucose, 5 mL
Heparin and 50 mL Custodiol. Subsequently, the arteries were perfused with 5
mL
Indian ink each and 10-13 mL synthetic baryta. It was clear that OPS images of
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-90-
the capillaries in the terminal villi compare with images obtained by SEM
showing
microcirculatory units that are hypocapillarized, normo-capillarized, or
' hypercapillorized. OPS imaging thus adds a new and versatile facility for
the
postnatal study of the nature of fetal growth retardation (i.e., due to
genetics vs.
due to placental deficiency, such as in pre-eclampsia).
Neonatology Monitoring
In this embodiment of the present invention, the OPS imaging probe is
used in neonatology monitoring.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used to quantitatively measure changes in the microcirculation of
neonates during different disease states like sepsis and meningitis, and
therefore
be used for diagnosis of these conditions. Hemoglobin levels of the neonates
can
be monitored non-invasively.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to measure changes in the neonate microcirculation prior
to, during, or following shock (hemorrhagic or septic).
The above applications would also be useful in monitoring premature
infants.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-91-
High Altitude StudieslSpace Physiology
In this embodiment of the present invention, the OPS imaging probe can
be used in high altitude studies or to study space physiology.
One or more parameters, such as, capillary density, vessel (and
microvessel) morphology, vessel density, vasospasm, red blood cell (RBC)
velocity, cell morphology, vessel diameter, leukocyte-endothelial cell
interactions,
vascular dynamics (such as vasomotion), functional vessel density, functional
capillary density, blood flow, area-to-perimeter ratio, hemoglobin
concentration,
and hematocrit, may be quantitatively determined. Preferably, two or more
parameters are determined.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to observe and evaluate changes in the microcirculation at high
altitudes to study, diagnose, and/or treat high altitude sickness. Changes in
the
microcirculation include an increase in the number of vessels, an increase in
the
number of WBCs that can be seen in those vessels, as well as an increase in
leukocyte-endothelial cell interactions. Changes in Hb could also be
determined
as a result of high altitude.
Applications in Clinical Blood Rheology
In this embodiment of the present invention, the OPS imaging probe can
be 'used to examine rheology of the blood.
One or more parameters, such as, capillary density, vessel (and
microvessel) morphology, vessel density, vasospasm, red blood cell (RBC)
velocity, cell morphology, vessel diameter, leukocyte-endothelial cell
interactions,
vascular dynamics (such as vasomotion), functional vessel density, functional
capillary density, blood flow, area-to-perimeter ratio, hemoglobin
concentration,
and hematocrit, may be quantitatively determined. Preferably, two or more
parameters are determined.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-92-
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to observe and evaluate changes in a patient's blood rheology,
that
may be due to numerous clinical disorders, including, for example, sickle cell
anemia, or excessive blood clotting that may occur in patients infected with
malaria. This information could be used to study diseases which alter blood
clotting, red blood cell aggregation, or alter blood rheology.
Applications in Critical Care or Intensive Care Medicine
In this embodiment of the present invention, the OPS imaging probe is
used in vivo in critical care or intensive care medicine.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used as a sublingual monitoring device on critically ill patients to
diagnose, treat, or prevent sepsis and/or shock (hemorrhagic or septic). It
has
been found that in cases of sepsis and/or shock, there is either a drastic
reduction
of flow or no flow of the microcirculation. OPS imaging technology could be
used to prevent the two conditions in critically ill patients or to try some
therapies
that would change the outcome. OPS imaging could also be used to monitor the
efficacy of therapy.
In this patient population, one or more parameters, such as, capillary
density, vessel (and microvessel) morphology, vessel density, vasospasm, red
blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte-
endothelial
cell interactions, vascular dynamics (such as vasomotion), functional vessel
density, functional capillary density, blood flow, area-to-perimeter ratio,
hemoglobin concentration, and hematocrit, may be quantitatively determined
using
OPS imaging. Preferably, two or more parameters are determined.
In the following study, OPS imaging was used on intensive care patients.
Introduction: Whatever the cause, circulatory shock is one of the major
challenges in intensive care medicine. Multiple organ failure often develops
despite
improvement in the profound cardiovascular alterations. Even when global
hemodynamic alterations appear to be stabilized with fluids and vasoactive
agents,
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 93 -
significant alterations in the microcirculation may persist and participate in
the
development ofmultiple organ failure (Garrison, R.N., etal., Ann. Surg.
227:851-
860 (1998)).
The contribution of altered microvascular blood supply is quite
straightforward in shock states associated with critical reductions in blood
supply
as in severe heart failure or hypovolemia. The alterations are more complex
and
also more controversial in septic shock. Using intravital microscopy in
experimental conditions, various authors (Lam, C.J., e1 al., J. Clin. Invest.
94:2077-2083 (1994); Piper, R.D., etal., Am. .I Respir. Cril. Care Med.
157:129-
134 (1998); Farquhar, L, et al., J. Surg. Res. 61:190-196 (1996); Wang, P.,
Am.
J. Physiol. 263:638-643 (1992); Madorin, W.S., et al., Crit Care Med 27:394-
400 (1999); Pannen, B.H.J., et al., Am. J. Physiol. 272:H2736-H2745 (1997))
observed that blood flow heterogeneity was increased after endotoxin
administration. The number of transiently non perfused capillaries is
increased in
sepsis and this is probably due to the activation of various cellular elements
with
aggregation of leukocytes and platelets, and increased red blood cell
stiffness
(McCuskey, R.S., et al., Cardiovasc. Res. 32:752-763 (1996); Piper, R.D., et
al.,
Am. J. Respir. Crit. Care. Med 154:931-937 (1996)). Various authors have
demonstrated the link between blood flow heterogeneity and decreased oxygen
extraction capabilities in sepsis (Walley, K.R., J. Appl. Physiol. 81:885-894
(1996); Humer, M.F., etal., J. Appl. Physiol. 81:895-904 (1996); Drazenovic,
R.,
et al., J. Appl. Physiol. 72:259-265 ( 1992)). Also transient flow may lead to
focal
ischemia/ reperfusion injury in areas vascularized by these capillaries.
Inpatients,
the evidence for involvement of microcirculatory disturbances is still
lacking.
Access to the human microcirculation has for a long time been limited to the
nailfold. Indeed, the size of intravital microscopes and the depth of the
capillaries
precluded their use in other sites. OPS imaging, which is based on reflection
spectroscopy, in the clinical area recently allowed the investigation of
mucosal
sites. In critically ill patients, the usual sites available for non-invasive
microcirculation visualization are the lip, the sublingual area, and sometimes
the
enterostomy site. Although technically feasible, the investigation of the
rectal and
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-94-
vaginal mucosa is more difficult. The OPS imaging technique had been
incorporated in the CYTOSCANTM A/R (Cytometrics, Philadelphia, PA). In this
study, results using the CYTOSCANTM A/R in the intensive care setting are
reported.
PARA--Sublingual area: Although access to the sublingual area is very
easy, an important question is whether this area is representative of
microcirculatory alterations in circulatory failure. There is indeed some
evidence
to support an early involvement of the sublingual area in shock states. Weil
and
coworkers (Nakagawa Y, et al., Am JRespir Crit Care Med.157(6 Pt 1):1838-
1843 (1998)) recently reported that sublingual PCOZ (PslCOz), which represents
the balance between oxygen supply and demand in this area, is markedly
increased
in shock states. Furthermore, PslCOz is inversely related to blood pressure
and
directly related to arterial lactate levels, a marker of tissue hypoxia.
Ps1C02 was
also very sensitive to therapeutic interventions.
Imaging the sublingual microcirculation is very promising. Contrary to
animal preparations in which a quantitative measurement of the
microcirculatory
flow is easily available, the quantitative estimation is actually not feasible
in
critically ill patients because of the movements artifacts. Hence, a semi-
quantitative analysis was developed in which the capillary index was
calculated by
the number of vessels crossing 3 vertical and 3 horizontal lines that were
traced
on the screen. These vessels were separated into large vessels (>20 Vim,
mainly
venules) and small vessels (<20 ~cm, mainly capillaries). Blood flow velocity
was
estimated in a 20 second video sequence by eye, and classified into 4 groups:
normal or high flow, low flow, intermittent flow, and no flow. In 3 healthy
volunteers, sublingual vascularization consisted of large and small vessels in
a ratio
40/60% (FIG.17). Almost all these vessels were well perfused (95%), although
intermittent or absent flow could be observed in a very limited number of
capillaries (5%).
In 21 patients with septic shock, a very different pattern was observed. The
number of perfused vessels was markedly reduced (78%, p<0.05), and especially
in the small vessels (39%, p<0.01). In these vessels, an increased number of
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-95-
capillaries where flow was transient or absent was observed. A typical example
is
reported in FIG. 18. In 8 patients with low flow due to cardiogenic shock,
capillary index was decreased and red blood cell conglomerates could be
visualized in venules (FIG. 19). In these patients also, a decrease in the
number of
perfused vessels (75%, p<0.05) was observed, especially in the small vessels.
These preliminary results suggest that severe alterations in the
microcirculation can be observed in various shock states. It is likely that
these
alterations may be responsible for the increase in PslCOz that was reported by
other groups (Nakagawa Y. et al., supra), but further studies are required to
confirm this hypothesis.
Other sites of interest: The inner side of the mouth, including the lip and
cheek can also be investigated. In many cases, microcirculation in the lip is
better
preserved, even in severe shock states. In 11 patients with septic shock and
in
cardiogenic shock, it was observed that 95% of the small vessels and all the
large
vessels were perfused in the lip area, while only 32% of the small vessels and
all
the large vessels were perfused in the sublingual area (p<0.05). Hence, the
study
of the sublingual microcirculation is probably more relevant than lip
microcirculation in shock states.
Probably more promising is the study of enterostomies, since the
application of OPS imaging to the bowel mucosa allows direct visualization and
characterization of gut mucosal blood flow. In FIG. 20, the OPS image of a
patient with recurrent bowel necrosis is shown. In this patient, a very
limited
number of capillaries was observed in the gut mucosa and blood flow was almost
completely stopped in these capillaries. This report supports the value of the
use
of OPS imaging in mesenteric ischemia and infarction. One use of OPS imaging
is the early detection of ischemia, the quantification of the lesions, and the
delineation of ischemic zones with some potential for recovery.
Conclusions: The introduction of OPS imaging through the
CYTOSCANTM A/R in the intensive care opens a new area of investigation with
direct and non- invasive visualization of the microcirculation at the bedside.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-96-
Pharmaceutical Applications
In this embodiment of the present invention, the OPS imaging probe is
used in the field of pharmaceutical development.
In one aspect of this embodiment, a standard or high contrast OPS imaging
S probe is used to study the effects of different pharmaceuticals on the
microcirculation, such as tumor anti-angiogenesis drugs to determine if
circulation
to a tumor is cut off; cardiac angiogenesis drugs to determine if vessel
growth and
thus circulation (to the heart, for example) has improved; or anti-
hypertension
agents to determine the mechanisms of action of new treatments or hypertension
etiology at the microvascular or cellular level. This could be measured under
the
tongue or directly on the tissue. Real time serial images and measurements
could
be obtained.
One or more parameters, such as, capillary density, vessel (and
microvessel) morphology, vessel density, vasospasm, red blood cell (RBC)
velocity, cell morphology; vessel diameter, leukocyte-endothelial cell
interactions,
vascular dynamics (such as vasomotion), functional vessel density, functional
capillary density, blood flow, area-to-perimeter ratio, hemoglobin
concentration,
and hematocrit, may be quantitatively determined. Preferably, two or more
parameters are determined.
Tumor angiogenesis plays a key role in tumor growth, formation of
metastasis, detection and treatment of malignant tumors. Recent investigations
provided increasing evidence that quantitative analysis of tumor angiogenesis
is
an indispensable prerequisite for developing novel treatment strategies such
as
antiangiogenic and antivascular treatment options. OPS imaging has been
validated for non-invasive quantitative imaging of tumor angiogenesis in vivo
and
used to assess antiangiogenic tumor treatment in vivo.
Experiments were performed in amelanotic melanoma A-MEL 3 implanted
in a transparent dorsal skinfold chamber of the hamster. Starting at day 0
after
tumor cell implantation, animals were treated daily with the antiangiogenic
compound SU5416 (25 mg/kg/bw) or vehicle (control) only. Functional vessel
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-97-
density (fvd), diameter of microvessels (d) and red blood cell velocity (VRB~)
were
visualized by both OPS imaging and fluorescence microscopy and analyzed using
a digital image system.
The results were as follows: the morphological and functional properties
of the tumor microvasculature could be clearly identified by OPS imaging. Data
for fvd correlated excellently with data obtained by fluroescence microscopy
(y =
0.99x + 0.48, r2 = 0.97, RS = 0.98, precision: 8.22 cm-' and bias: -0.32 cm-
').
Correlation parameters d and VRB~ were similar (r2 = 0.97, RS = 0.99 and r2 =
0.93, RS = 0.94 for d and VR,~c, respectively). Treatment with SU5416 reduced
tumor angiogenesis. At day 3 and 6 after tumor cell implantation,
respectively, fvd
was 4.82.1 cm-' and 87.2110.2 cm-' compared to values of control animals of
66.6+10.1 cm-' and 147.413.2 cm-'. In addition to the inhibition of tumor
angiogenesis, tumor growth and the development of metastasis was strongly
reduced in SU5416 treated animals.
1 S OPS imaging enabled noninvasive, repeated, and quantitative assessment
of tumor angiogenesis and the effects of antiangiogenic treatment on tumor
vasculature. Tumor angiogenesis can be used to more accurately classify and
monitor tumor biologic characteristics and to explore aggressiveness of tumors
in
vivo.
In a related aspect of this embodiment, an OPS imaging probe is used to
look at the effect of pharmaceuticals that are thought to improve perfusion.
This
includes, for example, the vasoactive class of drugs, which includes, e.g.,
naftidrofuryl, pentoxifylline, and buflomedil. That is, pharmaceuticals used
to
combat perfusion problems can be tested in animal models or humans more
accurately using OPS imaging, since the OPS imaging probe can be used to
observe and quantify perfusion following administration of the drug. Further,
OPS
imaging could actually assist in determining the mechanisms) by which these
drugs exert their effects in humans.
In another aspect of this embodiment, a standard or high contrast OPS
imaging probe is used to look at hemoglobin-based oxygen carriers (i. e. , DCL
Hb,
a synthetic hemoglobin) to determine their effects on the microcirculation,
such
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-98-
as, e.g., whether there is an increased flow of RBC's as a result of using the
product.
In another aspect of this embodiment, the OPS imaging probe can be used
to study the effect of ultrasound enhancers (i.e., injectable dyes) on the
microcirculation.
In another aspect of this embodiment, the OPS imaging probe can be used
to visualize and detect leakage of injectable dyes or other injectable
contrast-
generating agents, from the blood vessels into tissues.
Capillariscope Comparison
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used to visualize and characterize capillary beds in the
nailfold, as has been done before using standard capillaroscopy.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used to study, diagnose, and evaluate patients with circulation
disturbances, such as, for example, Raynaud's phenomenon, osteoarthritis, or
systemic sclerosis.
The OPS imaging probe produces better results than the standard
capillariscope used to visualize and characterize capillaries in the skin.
Comparisons have show that the method is comparable (and in most cases better)
when looking at healthy and disease state individuals. The site ofthe OPS
imaging
probe is on the nailfold. The measurements obtained were RBC velocity and
diameter. Other parameters, such as capillary density, vessel (and
microvessel)
morphology, cell morphology, vessel density, vasospasm, vessel diameter,
leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion),
functional vessel density, functional capillary density, area-to-perimeter
ratio,
blood flow, hemoglobin concentration, and hematocrit, may be quantitatively
determined. Preferably, two or more parameters are determined. I n t h a
following study, OPS imaging was validated with conventional capillaroscopy.
To
this end, capillaroscopy (CAP) was compared to OPS imaging in the
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-99-
microcirculation of the nailfold. Three capillaries of 10 healthy male
volunteers,
age 23.5 ~ 0.8 years (mean ~ SD), were visualized with CAP and OPS and stored
on videotape for offline analyses. The red blood cell velocity at rest (rCBV)
was
measured with CapImageTM. To assess the respective quality of the images a
contrast ratio was calculated for both devices. The rCBV was not significantly
different between OPS and CAP, P=0.32, Wilcoxon matched pairs test. The rCBV
measured by OPS was 0.737 t 0.237 mm/sec and with CAP 0.782 ~ 0.196
mm/sec, mean difference between the single measurements: 0.071 ~ 0.130
mm/sec. A significant higher contrast ratio was found for the images of OPS
(OPS
0.34 ~ 0.08, CAP 0.17 t 0.06 P= 0.0039). It can be concluded that OPS imaging
provides recordings of the microcirculation of the nailfold with the same, if
not
better, accuracy and resolution as CAP. Additionally, advantages of OPS
imaging
include its ease of use and being applicable in many other organ beds.
Anesthesiology
In this embodiment of the present invention, the OPS imaging probe is
used in the area of anesthesiology.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect ofthis embodiment, a standard or high contrast OPS imaging
probe is used to monitor blood loss during surgery. For example, an OPS
imaging
probe can be used to non-invasively and continuously monitor the hemodynamic
parameters of an anesthetized patient, such as, e.g., hemoglobin concentration
and
hematocrit.
In another aspect of this embodiment, a standard or high contrast OPS
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 100 -
imaging probe is used on an anesthetized patient to monitor the clumping of
red
blood cells, and the early formation of microemboli during a surgical
procedure.
A high contrast OPS imaging probe is used to monitor and detect air or fat
bubbles in the blood that may arise due to any number of clinical situations,
including, for example, the use of bubble oxygenators during cardiac bypass
surgery, the introduction of air from a dialysis circuit, or decompression
during
laparoscopy. The ability to directly detect bubbles in the blood may foster
the
early discovery of a lung or brain embolism, that would prove fatal if not
treated.
OPS imaging has been used to study ventilation with Positive End-
Expiratory Pressure (PEEP), which reduces splanchnic blood flow and could
thereby contribute to gut-derived sepsis. PEEP effects on the capillary level
are
largely unknown. Research into this issue in a marine model could provide
insight
into molecular mechanisms in PEEP as genetically modified mice and marine
antibody-markers are widely available. Therefore, a marine model was
established
using OPS-imaging to visualize the intestinal microcirculation. Anesthetized
mice
(n=14, c57b16, male) were mechanically ventilated. After laparotomy, the
distal
2cm of the ileum was exposed and continuously superfused with Tyrode solution.
An OPS-probe was positioned over the exposed intestinal serosa. Mice were
randomly assigned to PEEP or control groups. In the latter, recordings were
made
at three stages (steady state); at baseline, at PEEP of 3 mmHg and at PEEP of
7mmHg. In controls, PEEP was not applied and recordings were made at
corresponding times. Functional Capillary Density (FCD) was measured as the
number of visible capillaries per area (CapImageTM-software). In the PEEP-
group, FCD decreased significantly (p<0.05 vs. baseline and controls) to 56~
18%
(PEEP3 mmHg, mean t SD in % of baseline) and again significantly (p<0.05 vs.
PEEP3 and controls) to 3914% (PEEP=7 mmHg), whereas controls remained
stable (9623% and 8023%). This study demonstrated that PEEP reduces
intestinal capillary perfusion. Furthermore, a marine model for studying the
effects of mechanical ventilation was successfully established.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 101 -
Disseminated Intravascular Coagulation (DIC)
As used herein,"DIC" is the generation of fibrin in the blood and the
consumption of procoagulants and platelets occurring in complications of
obstetrics, (e.g., abruptio placenta), infection (especially gram-negative),
malignancy and other severe illnesses. Some other causes of DIC include
intravascular hemolysis, vascular disorders, thrombosis, snake bite, massive
tissue
injury, trauma (especially head trauma in children), hypoxia, liver disease,
infant
and adult respiratory distress syndrome (RDS), Purpura fulminans, and thermal
injury.
The most serious clinical form of DIC is shown in extensive consumption
of coagulation proteins, significant deposition of fibrin, and bleeding. In
mild
forms of DIC, there are endogenous markers of thrombin generation with little
or
no obvious coagulation problems.
In this embodiment of the present invention, a standard or high contrast
OPS imaging probe is used to visualize, characterize, identify, and/or monitor
DIC
in a patient.
In each of the following aspects of this embodiment, one or more
parameters, such as, capillary density, vessel (and microvessel) morphology,
vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
vessel
diameter, leukocyte-endothelial cell interactions, vascular dynamics (such as
vasomotion), functional vessel density, functional capillary density, blood
flow,
area-to-perimeter ratio, hemoglobin concentration, and hematocrit, may be
quantitatively determined. Preferably, two or more parameters are determined.
In one aspect of this embodiment, a standard or high contrast OPS imaging
probe is used to visualize, identify, and/or monitor DIC, due to infection,
and
more particularly due to meningitis.
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 102 -
Leukocyte KineticslLeukocyte-Endothelial Cell Interactions
In another embodiment of the present invention, a standard or high
contrast OPS imaging probe is used to visualize, characterize, and monitor
changes in leukocyte kinetics, such as occurs during inflammation and
infection,
or any other disease or therapy that effects leukocytes. This may assist the
medical practitioner in determining the source of "fever of unknown origin."
Leukocyte-endothelial cell interactions (such as, for example, leukocyte
adhesion
or leukocyte rolling) could also be visualized, characterized, and monitored.
As used herein, the term "leukocyte adhesion" means a Type of leukocyte-
endothelial cell interaction whereby the leukocytes are sticking in one place
for a
period of time.
As used herein, the term "leukocyte rolling" means a type of leukocyte-
endothelial cell interaction whereby the leukocytes are rolling along the wall
of the
vessel at a rate slower than the RBC velocity.
The effects of TNF-a on leukocyte rolling in iNOS Deficient Mice were
studied using OPS Imaging.
Nitric oxide (NO) is an important endogenous modulator of leukocyte-
endothelial cell interactions. The aim of this study was to determine the
manner
in which NO affects TNF-a induced leukocyte rolling and adhesion.
Postcapillary
venules were examined with OPS imaging under baseline conditions with tyrode
superfusion including 96% NZ and 5% COz and following TNF-a (100ng/ml)
superfusion for 3 hours on the hind leg muscle of iNOS knockout mice and their
wild-types. Leukocyte rolling and adhesion were quantified off line from the
video recordings at 30 minute intervals.
No difference was found in leukocyte rolling or adherence both in iNOS
deficient and wild type mice between baseline and 3 hours tyrode superfusion.
When TNF-a was included in the superfusate, the total number of leukocytes
increased in both groups. The number of rolling leukocytes however was less in
iNOS deficient compared to wild-type mice, due to the increased number of
adhering leukocytes in the iNOS knockout mice. In some venules of the iNOS
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
-103-
knockout mice, leukocyte accumulation was so abundant, that plugging of the
vessels occurred. In the wild-type mice, adherence was less and more
leukocytes
exhibited rolling behavior. This in vivo study was supported by post-mortem
histologic analyses.
Basic and Clinical Research Applications
In this embodiment of the present invention, the OPS imaging probe is
used for in vitro or in vivo basic or clinical research in any or all of the
areas
mentioned above (i.e., cardiology, cardiac surgery, wound care, diabetes,
hypertension, opthalmology, neurosurgery, plastic/reconstructive surgery,
transplantation, anesthesiology, and pharmacology, especially for evaluating
agents that inhibit or promote angiogenesis, or anti-hypertension agents).
Experimental animal models of numerous diseases with microvascular
pathologies (i. e., diabetes, hypertension, Raynaud's) could be developed and
OPS
imaging technology used to study disease etiology, improved or earlier methods
of diagnosis, disease progession, and new therapies.
Teaching Tool
In a final embodiment of the present invention, the OPS imaging probe
can be used as a teaching tool for medical students, and/or science students
studying, for example, physiology, anatomy, pharmacology, the
microcirculation,
and disease states affecting the microcirculation.
While various embodiments of the present invention have been described
above, it should be understood that they have been presented by way of example
only, and not limitation. The illumination techniques of the present invention
can
be used in any analytical, in vivo, or in vitro medical application (clinical
or
research) that requires optically measuring or visually observing
characteristics of
an object. The spectral absorption and scattering features of the object can
also
be measured with OPS imaging. Thus, the breadth and scope of the present
CA 02385849 2002-03-22
WO 01/22741 PCT/US00/26106
- 104 -
invention should not be limited by any of the above-described exemplary
embodiments, but is intended to cover all changes and modifications that are
within the spirit and scope of the invention as defined by the following
claims and
their equivalents.
All publications and patents mentioned in this specification are indicative
of the level of skill of those skilled in the art to which this invention
pertains. All
publications and patents are herein incorporated by reference to the same
extent
as if each individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
