U.S. patent application number 11/777958 was filed with the patent office on 2007-12-27 for device for inducing vascular injury and/or blockage in an animal model.
Invention is credited to Beth FRIEDMAN, David KLEINFELD, Patrick D. LYDEN, Nozomi NISHIMURA, Christopher B. SCHAFFER, Lee Frederick SCHROEDER, Philbert TSAI.
Application Number | 20070299331 11/777958 |
Document ID | / |
Family ID | 32507914 |
Filed Date | 2007-12-27 |
United States Patent
Application |
20070299331 |
Kind Code |
A1 |
FRIEDMAN; Beth ; et
al. |
December 27, 2007 |
DEVICE FOR INDUCING VASCULAR INJURY AND/OR BLOCKAGE IN AN ANIMAL
MODEL
Abstract
Ultrashort laser pulses are used to induce photodisruptive
breakdown in vasculature in an animal to controllably produce
hemorrhage, thrombosis or breach of blood-brain barrier in
individual, specifically targeted blood vessels. Damage is limited
to the targeted vessels such that neighboring vessels exhibit no
signs of vascular damage, including vessels directly above or below
the targeted vessel. Ultrashort laser pulses of lower energy are
also used to observe and quantify the baseline and altered states
of blood flow. Observation and measurement may be performed (1) by
TPLSM, OCT or other known techniques, providing a real-time, in
vivo model for the dynamics and effects of vascular injury.
Inventors: |
FRIEDMAN; Beth; (La Jolla,
CA) ; KLEINFELD; David; (La Jolla, CA) ;
LYDEN; Patrick D.; (San Diego, CA) ; NISHIMURA;
Nozomi; (La Jolla, CA) ; SCHAFFER; Christopher
B.; (La Jolla, CA) ; SCHROEDER; Lee Frederick;
(San Diego, CA) ; TSAI; Philbert; (La Jolla,
CA) |
Correspondence
Address: |
PROCOPIO, CORY, HARGREAVES & SAVITCH LLP
530 B STREET
SUITE 2100
SAN DIEGO
CA
92101
US
|
Family ID: |
32507914 |
Appl. No.: |
11/777958 |
Filed: |
July 13, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10538548 |
Jun 10, 2005 |
7258687 |
|
|
PCT/US03/39428 |
Dec 11, 2003 |
|
|
|
11777958 |
Jul 13, 2007 |
|
|
|
60432371 |
Dec 11, 2002 |
|
|
|
Current U.S.
Class: |
600/407 ;
119/712 |
Current CPC
Class: |
A61B 2018/00452
20130101; A61B 2017/00057 20130101; A61B 18/203 20130101; A61B
18/20 20130101 |
Class at
Publication: |
600/407 ;
119/712 |
International
Class: |
A61D 99/00 20060101
A61D099/00 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] Pursuant to 35 U.S.C. .sctn.202(c), it is hereby
acknowledged that the U.S. Government has certain rights in the
invention described herein, which was made in part with funds from
the National Institute of Neurological Disorders and Stroke, Grants
No. R01-NS41096 and No. RO1-NS043300-O1A1.
Claims
1.-48. (canceled)
49. A device for producing spatially-localized injury to
vasculature in an animal, comprising: an animal mount for holding
the animal in a fixed position; an optical source for producing a
photodisruption beam, wherein the photodisruption beam comprises a
plurality of ultrashort pulses adapted for driving a nonlinear
interaction within the target vasculature; and a microscope
objective for focusing the photodisruption beam onto target
vasculature in the animal; wherein the animal has a window formed
therein for providing optical access to the target vasculature.
50. The device of claim 49, wherein the optical source comprises an
optical oscillator and an optical pump.
51. The device of claim 49, wherein the optical source further
comprises an optical amplifier.
52. The device of claim 49, further comprising detectors for
detecting light produced in the animal by the ultrashort
pulses.
53. The device of claim 49, wherein an imaging beam is directed
through the microscope objective for imaging the animal.
54. The device of claim 49, wherein the microscope objective is
part of a two photon laser scanning microscope.
55. The device of claim 49, wherein the microscope objective is
part of an optical coherence tomography microscope.
56. The device of claim 49, wherein the animal mount comprises a
kinematic mount for the removal and repositioning of the
animal.
57. The device of claim 49, further comprising a measurement device
for observing blood flow in the animal.
58. The device of claim 49, wherein the ultrashort pulses have
pulsewidths in a range from 10 femtoseconds to 100 picoseconds.
59. A device for producing spatially-localized injury to
vasculature in a live animal, wherein the animal has an optically
transparent window disposed over an area comprising target
vasculature, the device comprising: an optical source for producing
a photodisruption beam, wherein the photodisruption beam comprises
a plurality of ultrashort pulses adapted for driving a nonlinear
interaction within the target vasculature; and a microscope
objective for focusing the photodisruption beam onto the target
vasculature in the animal; and an animal mount for stabilizing the
animal within an optical path of the photodisruption beam.
60. The device of claim 59, wherein the optical source comprises an
optical oscillator and an optical pump.
61. The device of claim 59, wherein the optical source further
comprises an optical amplifier.
62. The device of claim 59, further comprising detectors for
detecting light produced in the animal by the ultrashort
pulses.
63. The device of claim 59, wherein an imaging beam is directed
through the microscope objective for imaging the animal.
64. The device of claim 59, wherein the microscope objective is
part of a two photon laser scanning microscope.
65. The device of claim 59, wherein the microscope objective is
part of an optical coherence tomography microscope.
66. The device of claim 59, wherein the animal mount comprises a
kinematic mount for the removal and repositioning of the
animal.
67. The device of claim 59, further comprising a measurement device
for observing blood flow in the animal.
68. The device of claim 59, wherein the ultrashort pulses have
pulsewidths in a range from 10 femtoseconds to 100 picroseconds.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/538,548, filed, Jun. 10, 2005, which is a U.S. national
stage application of International Application No. PCT/US03/39428,
filed Dec. 11, 2003, which claims the benefit of priority of U.S.
Provisional Application Ser. No. 60/432,371 filed Dec. 11, 2002,
the disclosure of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a device and method for
inducing vascular injury and/or blockage in animal models for the
study of vascular disease, and more particularly an optical device
and method for producing laser-induced hemorrhage, thrombosis, and
breach of the blood-brain barrier in specifically targeted
individual blood vessels with micrometer precision.
BACKGROUND OF THE INVENTION
[0004] With the average lifespan and age of the population on the
increase, vascular diseases are guaranteed to strike growing
numbers within the population. Among such diseases are
neurovascular disorders, which encompass those conditions that
result in cerebrospinal ischemia, infarction, and hemorrhage. To
provide an example, every year, over 700,000 people in the United
States suffer a stroke, and roughly a quarter of those strokes are
fatal. Stroke is therefore the third leading cause of death in the
United States. In addition, a large segment of the elderly
population is debilitated by dementia. Recently, neuronal vascular
disorders, including microstrokes (lacunes), microbleeds, and
neurovascular disease have been linked with many forms of dementia,
such as Alzheimer's disease and vascular dementia. (Heye and
Cervos-Navarro 1996; del Zoppo and Mabuchi 2003; Wardlaw,
Sandercock et al. 2003) At present, options for treatment of stroke
remain few and of limited efficacy despite years of basic and
clinical research. Continued progress in stroke research depends
critically on animal models that allow stroke to be studied at
various stages, from initial changes in physiological parameters
(e.g., blood flow and blood oxygenation) to neuronal death,
behavioral impairment, and recovery. (del Zoppo 1998; Lipton 1999;
del Zoppo and Mabuchi 2003). Most ischemic stroke models developed
to date produce either large-scale injury, or a multitude of
small-scale injuries at uncontrolled sites. Most hemorrhagic stroke
models developed to date produce either large-scale hemorrhage or
systemic injury. These existing models do not allow the production
of small-scale, localized injury or blockage to specifically
targeted vessels at depth. Such a paradigm is particularly crucial
for the study of the effects of ischemic microstrokes and
microbleeds.
[0005] Existing in vivo animal models of stroke fall into one of
five broad categories: 1) occlusion of large vessels by ligation or
filament insertion; 2) occlusion of a multitude of microvessels by
injection of embolus into the bloodstream; 3) hemorrhagic damage
(vessel rupture) by injection of a tissue-degrading substance; 4)
model of hemorrhage by injection of whole or fractionated blood;
and 5) optically-induced thrombosis of blood vessels by linear
adsorption of light. There is no reported technique that is capable
of producing both thrombotic and hemorrhagic stroke to specific
individual vessels deep within the same preparation.
[0006] In the case of mechanical occlusion, current techniques
involve the blockage of blood flow by a variety of methods. These
methods include ligation of large arteries (e.g., carotid artery)
(McBean and Kelly 1998)], ligation of smaller arteries (Wei,
Rovainen et al. 1995; Wei, Erinjeri et al. 2001), and insertion of
a filament into a large artery for the occlusion of a main arterial
branch, (e.g., the middle cerebral artery) (Tamura, Graham et al.
1981; Chen, Hsu et al. 1986; Busch, Kruger et al. 1998). Artery
ligation results in neuronal injury to large, millimeter or larger
sized regions of the rodent brain and is a model for major
infarcts.
[0007] As a model for microstrokes, microspheres (Lyden and Hedges
1992; Lyden, Zivin et al. 1992; Lyden, Lonzo et al. 1997) or
preformed clots (Kudo, Aoyama et al. 1982; Overgaard 1994; Krueger
and Busch 2002) can be injected into an artery, leading to
occlusion of smaller vessels downstream from the injection site,
but without allowing specific individual vessels to be targeted. As
a result, physiological changes cannot be correlated to specific
local disruptions.
[0008] Hemorrhages can be induced by systemic or local injections
of agents such as collagenase (Rosenberg, Mun-Bryce et al. 1990],
or tissue plasminogen activator (tPA) (Dijkhuizen, Asahi et al.
2002) to weaken vessels or disrupt the blood-brain barrier. Using
such models to evaluate potential treatments is difficult because
the effects of these agents can be spread over large, uncontrolled
volumes. In addition, the effects of the agent cannot be isolated
to the vasculature alone, because the agent can directly affect the
surrounding tissue.
[0009] Alternatively, direct injection of whole or fractionated
blood into the extracellular space has been reported as a model for
hemorrhagic stroke (Deinsberger, Vogel et al. 1996; Hickenbottom,
Grotta et al. 1999). The spatial localization is limited by
diffusion of the injected materials. Additionally, this model is
deficient in other aspects of natural hemorrhagic stroke, including
the vascular and endothelial response. Current models of hemorrhage
cannot be used as models of small hemorrhage, which are necessary
for studies of vascular dementia.
[0010] For the case of optically-induced thrombosis, previous work
utilized green light to excite an intravenously injected
photosensitizer. When excited by exposure to light,
photosensitizers generate single oxygen (Pooler and Valenzeno
1981), which attacks the membranes of the vessel walls (Herrmann,
1983). Damage to the vessel walls then starts a natural cascade of
activation that results in the formation of a clot in all exposed
vessels (Watson, Dietrich et al. 1985; Krammer 2001). In earlier
work, transcranial illumination with diffuse green light exposed
blood vessels over a wide lateral and axial extent, 1-3 millimeters
in diameter (Watson, Dietrich et al. 1985; Dietrich, Ginsberg et
al. 1986; Dietrich, Ginsberg et al. 1986).
[0011] More recently, work has been done using green light that is
tightly focused through a microscope objective, constraining the
lateral dimension of exposure at the focal plane to approximately
one micrometer (Schaffer, Ebner et al. 2003; Schaffer, Ebner et al.
2003; Schaffer, Tsai et al. 2003), allowing individual vessels to
be clotted. While very powerful, this focal photothrombotic stroke
model has one major drawback: localized clotting can be achieved
only in surface vessels. This limitation is due to the
single-photon excitation of the photosensitizer molecule. When
focused on a deep-lying vessel to induce a clot, all vasculature
lying above that vessel is also clotted, preventing the use of this
model for studying the effect of localized thrombosis in individual
vessels at depth.
[0012] Alternatively, highly absorbed wavelengths of light (e.g.,
10.2 microns from a CO.sub.2 laser) are used extensively in
neurosurgery to simultaneously remove neuronal and vascular tissue
while concurrently cauterizing the remaining portions of the
removed blood vessels. The mechanism of damage relies on the linear
adsorption of the laser light by water and other tissue
constituents and, therefore, does not require the presence of an
exogenous photosensitizer. The high absorption coefficient of the
tissue at these wavelengths results in substantial energy
absorption and thermal buildup within the targeted tissue. The
concurrent thermal diffusion out of the targeted volume results in
an extended region of collateral thermal damage.
[0013] Current medical treatment for stroke requires therapeutic
intervention within hours of the stroke to be optimally effective.
Full understanding of the mechanisms and efficacy of these
interventions therefore requires real-time visualization of stroke
with high spatial and temporal resolution. Previously, real-time
visualization and quantification of the effects of vascular damage
on blood flow and blood vessel morphology have been performed using
technologies, such as laser Doppler flowmetry (Dirnagl, Kaplan et
al. 1989; Nakase, Kakizaki et al. 1995), magnetic resonance imaging
(MRI) (Hoehn-Berlage, Norris et al. 1995; Busch, Kruger et al.
1998), positron emission tomography (PET) (Marchal, Young et al.
1999), computer-aided tomography (CAT), fluorescent video
microscopy (Wei, Rovainen et al. 1995; Wei, Erinjeri et al. 2001;
Ishikawa, Sekizuka et al. 2002), or confocal laser scanning
microscopy (Seylaz, Charbonne et al. 1999; Pinard, Nallet et al.
2002). With the exception of the light microscopy, these techniques
are limited to determining average blood flow over
100-1000-micrometer-sized areas. While such averages may be
relevant for determining the degree of ischemia or hemorrhage, they
provide no input on changes in flow and morphology in individual
vessels, save for the largest branches of the cerebral vasculature.
Fluorescent video microscopy allows individual vessels to be
studied, but is limited to the observation of surface vessels only,
while confocal microscopy allows vessels up to approximately 50 um
beneath the surface to be visualized. These observation techniques
have allowed quantitative characterization of changes in blood flow
velocity and blood vessel dilation as a result of large-scale
ischemia produced by surgical occlusion of arteries and arterioles
(Wei, Rovainen et al. 1995; Wei, Craven et al. 1998; Seylaz,
Charbonne et al. 1999; Wei, Erinjeri et al. 2001; Pinard, Nallet et
al. 2002). These studies could not, however, address local changes
in blood flow and vessels near an isolated occlusion. Recently,
fluorescent video microscopy was used to study vessel dilation
after photochemically-induced clots in individual arterioles, but
the results were limited to surface vessels and blood flow could
not be resolved (Ishikawa, Sekizuka et al. 2002).
[0014] Another modality of analysis for current models of induced
stroke is based on the observation of behavior deficits in the
subject and post-mortem histology of the targeted and collateral
tissue regions. These widely utilized methods are performed hours
to days after the onset of damage and, therefore, are unable to
elucidate the dynamics and mechanisms involved in the propagation
of injury due to vascular damage.
[0015] The study of microstrokes and microhemorrhages requires
microscopic resolution, coupled with the ability to either
precisely target or locate the microscopic vascular disturbance
within the brain volume. Using nonlinear microscopy, local changes
in blood flow due to isolated occlusions can be studied and
quantified in real-time.
[0016] The use of nonlinear optical effects to provide contrast for
image formation has revolutionized microscopy over the past decade.
Many nonlinear effects are now used for imaging, including second-
and third-harmonic generation, Coherent Anti-Stokes Raman
scattering, the Kerr effect, and multi-photon excited
fluorescence.
[0017] One non-linear technique is two-photon laser scanning
microscopy, or "TPLSM" (Denk, Strickler et al. 1990; Denk 1994),
which allows fluorescence imaging with intrinsic optical sectioning
deep inside scattering specimens with diffraction-limited
resolution. Briefly, an ultrashort laser pulse is tightly focused
inside a specimen tagged with a fluorescent molecule that does not
linearly absorb at the wavelength of the ultrashort laser. At the
laser focus, the laser intensity can become high enough to induce
two-photon excitation of the fluorescent molecule. Because the
excitation is nonlinear, this fluorescence is only produced in the
focal volume where the laser intensity is high. The fluorescence
intensity is then recorded as the position of the laser focus is
scanned throughout the specimen forming a three-dimensional image.
In addition, because photoexcitation occurs only at the laser
focus, there is significantly reduced bleaching of fluorescent dyes
and photodamage to the sample as compared to linear imaging
techniques.
[0018] TPLSM is especially well suited to in vivo imaging deep into
highly scattering specimens, such as brain. In widefield or
confocal fluorescence microscopy, the fluorescence must be imaged
to a camera or to a pinhole, respectively. Scattering of the
fluorescence leads to an unwanted background in widefield
microscopy and to decreased signal strength in confocal microscopy.
In TPLSM, however, because all the fluorescence originates from the
focal volume, it need only be detected in order to contribute to
the signal, not imaged. Thus fluorescence that is scattered on the
way to the detector still contributes to image formation, and does
not produce unwanted background. This immunity to scattering of the
fluorescence allows imaging deep into scattering samples. The
imaging depth is ultimately limited by scattering of the ultrashort
laser beam. In practice, one can image up to 500 micrometer beneath
the cortical surface in rat (providing access to layers 1-4 of the
cortex), without loss of image resolution. For neuronal tissue with
labeling throughout the tissue, a theoretical limit for imaging
depth is approximately 1 millimeter.
[0019] TPLSM further provides means for measuring and quantifying
the velocity, i.e., direction and speed, and flux of red blood cell
(RBC) movement and plasma flow in vivo under acute as well as
chronic conditions. These measurements make use of either
fluorescently labeled plasma, in which case cells in the blood,
such as RBCs and leukocytes, appear as dark objects on a bright
background, or the use of fluorescently labeled RBCs.
[0020] The above-described technologies provide means for forming
and observing strokes. However, these techniques for induction of
stroke are incapable of producing hemorrhage, thrombosis, and
breach of the blood-brain barrier targeted to specific individual
blood vessels. Further, no technique is currently available for the
production of surface or subsurface vascular injury localized with
micrometer precision, thereby permitting the disruption of the
smallest vessels, i.e., capillaries. Accordingly, the need remains
for a device and method with such capabilities.
SUMMARY OF INVENTION
[0021] The present invention provides a device and method for
optically inducing precision vascular injury and/or blockage deep
in the tissue of an animal. No exogenous agents are required to
facilitate the injury, as the laser light interacts directly with
the endogenous tissue and fluids. In an exemplary embodiment, a
tightly focused beam of high intensity, ultrashort, laser pulses
drives nonlinear interactions between the laser light and the
tissue at the focus of the beam, producing photodisruption at the
targeted vasculature. These nonlinear interactions result in both
direct photoionization of the tissue at the focus and thermoelastic
damage to the local surrounding tissue. As a result of these damage
mechanisms, a localized hemorrhage, thrombosis, or breach of the
blood-brain barrier is formed within a single targeted vessel,
providing an in vivo animal model for vascular injury or blockage.
Because of the nonlinear nature of the laser-tissue interaction,
photodisruption can be targeted deep to the surface of the tissue
without extensive collateral damage to tissue surrounding the
targeted vessel.
[0022] According to the inventive method for production and
observation of localized photodisruption, optical access to
vasculature of the peripheral and central nervous system is
obtained through a window. For the case of brain vasculature, an
optically transparent cranial window consisting of either a
coverslip-sealed craniotomy, a thinned-skull preparation, or an
intrinsically thin skull is utilized. In the preferred embodiment,
an intravenous injection of fluorescent water-soluble tracer is
used to visualize target vessels and quantify the blood flow
through the cranial window. In the preferred embodiment, two-photon
laser scanning microscopy (TPLSM) is used, however, other
observation procedures may also be used.
[0023] According to the present invention, ultrashort laser pulses
are used to induce photodisruptive breakdown in vasculature thereby
controllably producing hemorrhage, thrombosis, as well as breach of
the blood-brain barrier in individual, specifically targeted blood
vessels. Ultrashort laser pulses of lower energy are also used to
image and quantify the baseline and altered states of blood flow.
Such analysis provide a real-time, in vivo model for the dynamics
and effects of vascular injury, such as occurs in stroke. Breakdown
is produced in individually targeted vessels with approximately 100
to 10000 nanojoule pulses. Induced thrombi are found to be stable
past 6 hours. Thrombosis has been demonstrated in vessels ranging
in diameter from 5 to 50 micrometers. Hemorrhagic damage has been
demonstrated in vessels ranging in diameter from 5 to 1000
micrometers. Three-dimensional localization of damage has been
demonstrated by optical sections that were taken before and after
the vascular damage has been induced, and from histological
sections of post-mortem tissue. In both cases the sections spans
the entire three-dimensional volume of interest. Blood vessels
neighboring the targeted vessel showed no signs of vascular damage,
including vessels directly above and directly below the targeted
vessel.
[0024] The inventive device and method are not limited to
applications for modeling of stroke or other vascular injury of the
brain, but can be used for the study of vascular disease in other
organs, including, but not limited to heart, liver and kidney. In
general, any disease involving disruption of normal vascular
function can be modeled and studied using the device and method of
the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The invention, both as to its organization and manner of
operation may be further understood by reference to the following
description taken in conjunction with the following drawings
wherein:
[0026] FIG. 1 is a schematic diagram of an exemplary arrangement
for the optics and optomechanics of the inventive device, including
scan optics, detectors, and sample stage;
[0027] FIG. 2 is a schematic diagram of laser sources for a first
embodiment of the device, wherein two coupled laser sources are
used for imaging and photodisruption;
[0028] FIG. 3 is a schematic diagram of laser sources for a second
embodiment of the device, wherein two separate laser sources are
used for imaging and photodisruption;
[0029] FIGS. 4a and 4b are diagrammatic views of a cross-section of
tissue before and during exposure to photodisruption, respectively.
FIGS. 4c, 4d, and 4e are diagrammatic views of the tissue showing a
hemorrhage, a breach of the blood-brain barrier, and a thrombosis,
respectively, induced by photodisruption.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0030] The following detailed description provides examples of
application of the device and method of the invention to modeling
of stroke or other vascular injury in the brain. These examples are
not intended to be limiting. Applications of the invention go
beyond those relating to simulation and study of stroke, extending
to the study of vascular disease in other organs, including, but
not limited to, heart, liver and kidney, and other areas of the
body, for conditions such as peripheral vascular disease.
[0031] Nonlinear optical induction of vascular injury and/or
blockage is performed using high intensity, ultrashort laser
pulses. Nonlinear microscopy is used to monitor and quantify
changes of physiological parameters (e.g., blood flow, or blood
oxygenation) in real-time.
[0032] Photodisruption relies on nonlinear interaction between the
laser light and endogenous tissue constituents to generate either
thrombosis, breach of the blood-brain barrier resulting in
extravasation of blood plasma but not red blood cells, or
hemorrhage resulting in extravasation of both blood plasma and red
blood cells. This method does not require the presence of an
exogenous photosensitizer. Use of laser wavelengths that are
neither highly scattered nor highly absorbed by the tissue allow
for both imaging and photodisruption to be targeted deep to the
tissue surface, localized in three dimensions, and performed with
negligible thermal damage to the surrounding tissue.
Photodisruption is instigated by multiphoton and avalanche
ionization. The resulting damage is from either direct
vaporization, if the tissue is located in the focal volume where
the laser energy is nonlinearly absorbed, or mechanical disruption
by either a shock wave or cavitation bubble. By locating the laser
focus at different positions inside or on the vessel wall, a
variety of vascular injuries can be produced. When the laser pulse
is focused on the wall of a vessel, the cells comprising the wall
are vaporized, producing a hemorrhage. When the laser pulse is
focused into the lumen of the vessel, the vessel wall is most
likely damaged by the shock wave, potentially triggering the
clotting cascade and formation of a thrombus, and/or leading to
hemorrhage or breach of the blood-brain barrier.
[0033] In the preferred embodiment, targeting of vessels, as well
as imaging and quantification of physiological parameters are
performed by TPLSM. Other techniques for observation of the effects
of photodisruption, which may be used in lieu of or in combination
with TPLSM include optical coherence tomography (OCT), magnetic
resonance imaging (MRI), functional magnetic resonance imaging,
multi-spectral intrinsic imaging, positron emission tomography
(PET), time resolved light scattering, Doppler flowmetry, and
surface imaging of blood vessels with wide-field videography.
[0034] The major optics and optomechanics of the modified two
photon laser scanning microscope are illustrated in FIG. 1. As
illustrated, the device broadly includes multiple laser
source/electro-optics assemblies 10 and 12, a modified two-photon
laser scanning microscope 20, and an animal preparation mount 30
attached to a translation stage 40. The laser sources for the two
beams of laser pulses have pulse parameters appropriate for
nonlinear imaging and photodisruption, respectively. In the
preferred embodiment, the first source assembly 10 is capable of
producing roughly 100 femtosecond, 720 to 900 nanometer laser
pulses with energies of up to approximately 10 nanojoules at a
repetition rate of 76 megahertz for the purpose of nonlinear
microscopy. A second source assembly 12 is capable of producing
roughly 100 femtosecond, 800 nanometer laser pulses with energies
of up to approximately 1 millijoule at a repetition rate of 1
kilohertz. Alternatively, the laser sources can be any other laser
systems or combinations of systems appropriate for nonlinear
microscopy and photodisruption, respectively. The two-photon laser
scanning microscope 20 is adapted for concurrent delivery of a
second beam line for photodisruption. The animal preparation mount
30 is designed to stably hold the animal preparation 32 for optical
access to the vasculature. A translation stage 30 and kinematic
mount allow positioning to micrometer accuracy. Auxiliary equipment
can be included for monitoring and maintaining homeostatic
conditions for the animal preparation.
[0035] Referred to FIG. 2, in a first embodiment, the laser source
for TPLSM and the laser source for photodisruption are separate but
coupled laser sources. In this first exemplary embodiment, laser
oscillator 110 is a Titanium:sapphire (Ti:sapphire) laser with a
pulse width of approximately 100 femtoseconds. For purposes of the
present invention, pulse widths may fall in the range of around 10
femtoseconds to the 100 picoseconds. Appropriate lasers are
commercially available, for example, under the trademark Mirao 900
(Coherent, Inc., Santa Clara, Calif.). Laser oscillator 110 is used
both as the imaging source for a modified two-photon laser scanning
microscope, and as a seed for a multi-pass optical amplifier 140
through a electrooptic pulse picker 130.
[0036] An example of an appropriate amplifier for use in the
inventive device is that of Kapteyn and Murnane (Backus, Bartels et
al. 2001). Such systems, producing pulses with a duration of
approximately 100 femtoseconds and energies up to approximately 1
millijoule at a repetition rate of approximately 1 kilohertz, are
commercially available, for example, under the trademark Hurricane
(Positive Light, Inc., Los Gatos, Calif.). The amplified beam from
the multi-pass optical amplifier serves as a photodisruption beam
for vascular injury. The pump laser 100 for the laser oscillator is
a continuous wave (CW) solid state laser, such as the 1OW Verdi-V10
laser available from Coherent, Inc. The pump laser 160 for the
optical amplifier 140 is a pulsed solid state laser. Beam
diagnostics 122,142 include a power meter, spectrometer, and
autocorrelator, which receive light from the optical oscillator or
amplifier via beamsplitters 120,144. Intensity controls 132,146 and
mechanical shutters 134,148 are provided independently for each
beam path 136,152. A half-wave plate 150 is placed in the
photodisruption beam path 152 to rotate the polarization of the
beam for effective polarization mixing with the oscillator
(imaging) beam 152 in the modified microscope.
[0037] In a second embodiment, a separate laser source assembly can
be used for each of TPLSM and photodisruption of subsurface
vessels, as illustrated in FIG. 3. In this alternative embodiment,
the first laser/electrooptics assembly 10 includes a
photodisruption laser 210 pumped by a separate pump laser 200 to
produce the ablation beam 222 of high energy pulses with or without
the use of an optical amplifier. As in the first embodiment,
beamsplitter 212 diverts a portion of the beam 222 to beam
diagnostics 214, which includes a power meter, spectrometer, and
autocorrelator. Intensity control 216 and mechanical shutter 218
are provided in the beam path 222. A half-wave plate 220 is placed
in the beam path 152 to rotate the polarization of the beam for
effective polarization mixing with the imaging beam 270 in the
modified microscope.
[0038] In the separate, second laser/electrooptics assembly 12,
imaging beam 270 is produced in a similar manner to that of
oscillating beam 136 of the first embodiment. Imaging laser 240 is
pumped by laser 230. Beam diagnostics 252 receives light from the
imaging laser 240 via beamsplitter 250. Intensity control 262 and
mechanical shutter 264 are provided in beam path 270. A half-wave
plate 150 is placed in the amplified beam path 152 to rotate the
polarization of the beam for effective polarization mixing with the
oscillator (imaging) beam 152 in the modified microscope.
[0039] Referring again to FIG. 1, the modified two photon laser
scanning microscope includes a pair of scan mirrors 14 together
with a scan lens 16, a tube lens 18, and an objective 22, which
together serve to raster the oscillator beam across the animal
preparation 32 for imaging. The objective 22 has a high numerical
aperture (NA) in the range from 0.1 to 1.3 NA, which is typically
available with standard water-immersion objectives. The choice of
the numerical aperture is based on considerations that tie the NA
of the microscope objective 22 to the working distance, laser
penetration depth and resolution at a given location of tissue. The
amplified ultrashort pulses for photodisruption (from source 12)
are combined into a common optical path with the imaging beam by
polarizing beamsplitter 36.
[0040] The detection optics comprise a dichroic mirror 24, a
mixture of colored glass and interference filters 28, collection
lens 26, and detectors 34, all receiving photons from the animal
preparation 32 through the objective lens 22. A digital image
acquisition and storage system 70 is provided to store sections in
the form of digital images. Such a system 70 comprises a computer
system and suitable acquisition software and imaging software to
visualize and quantify the blood flow in the animal preparation.
Additional software and/or hardware can be included to provide
positioning control and coordination of the translation stage 40,
allowing precise positioning and assignment of reference
coordinates to the stored images. Additional optics can be easily
inserted for simultaneous detection and discrimination in multiple
wavelength bands.
[0041] In the preferred embodiment, targeting of specific vessels
and visualization of blood flow can be performed by two-photon
laser scanning microscopy at multiple adjacent fields of view.
Then, the animal can be removed and precisely repositioned in the
apparatus using a kinematic mount for observation using, in one
preferred embodiment, optical coherence tomography (OCT), a
technique developed in the early 1990s which enables non-invasive,
high resolution in vivo imaging in turbid biological tissue (see,
e.g., Fujimoto, 2003). Additional or alternative observation and
vessel targeting modalities may be used, including magnetic
resonance imaging (MRI), functional magnetic resonance imaging,
multi-spectral intrinsic imaging, positron emission tomography,
time resolved light scattering, Doppler flowmetry, or surface
imaging of blood vessels with wide-field videography. As an
alternative to repositioning, the animal can remain in a fixed
position and the instrumentation moved into position viewing. In
this latter embodiment, the TPLSM assembly would be mounted on an
appropriate translation stage or platform in a system that includes
one or more additional types of observation instrumentation. The
second instrument, also mounted on a stable translation stage, can
be positioned for observing the animal.
[0042] The head-fixed mount 30 is constructed from a metal plate 62
which is directly attached to the animal preparation 32. The metal
plate 62 is mounted onto metal rods 64 which attach to a kinematic
baseplate 66 that can be removed and replaced with high precision.
The kinematic base plate 66 attaches to a translation stage 40 that
can be connected to system controller 70 to provide computer
control to deliver micrometer position accuracy.
[0043] Additional details of the components of the TPLSM and
examples of commercial sources for the components of the TPLSM are
provided in Chapter 6 ("Principles, Design and Construction of a
Two-Photon Laser-Scanning Microscope for In Vitro and In Vivo Brain
Imaging", by P. S. Tsai, et al.) of In Vivo Optical Imaging of
Brain Function, ed. Ron D. Frostig, 2002, CRC Press, pp. 113-171,
which is incorporated herein by reference.
[0044] For practicing the method of the present invention,
mode-locked laser 10 produces a train of ultrashort laser pulses
capable of being focused to peak intensities exceeding
approximately 10.sup.10 W/cm.sup.2 appropriate for nonlinear
microscopy. This train of pulses is directed to laser scanning
microscope 20 and focused at the animal preparation 32 for the
purpose of monitoring physiological parameters, such as blood flow,
blood oxygenation or cellular physiology. A subset of the pulses is
diverted by pulse picker 130 to optical amplifier 140 to produce
ultrashort laser pulses capable of being focused to peak
intensities exceeding approximately 10.sup.13 W/cm.sup.2 as
appropriate for photodisruption. This beam of amplified pulses is
also focused at the animal preparation 32 for the purpose of
producing photodisruption by nonlinear interaction with the
endogenous tissue constituents. Due to the high-order dependence of
these nonlinear interactions on laser intensity, the probability of
interaction is negligible everywhere except in the immediate
vicinity of the focus of the laser beam. Sub-femtoliter focal
volumes can be achieved, resulting in localization of vascular
damage down to a single specific blood vessel, and imaging with
sub-micrometer resolution.
[0045] Three categories of vascular injury-thrombosis, breach of
the blood-brain and hemorrhage--can be selectively produced by
optimization of three parameters: the pulse energy, the number of
pulses applied, and the targeting location within the vessel.
Targeting the pulses to the vessel wall results in direct
photoionization of the cells comprising the vasculature. Targeting
the pulses to the vessel lumen results in photoionization of the
fluid within the lumen, leading to a cavitation bubble and a shock
wave which propagates to the vessel wall, and causes injury. The
injury resulting from these mechanisms may be severe enough to
degrade the blood-brain barrier, trigger a natural clotting
cascade, or rupture the vessel. Because the amplitude of produced
shock waves falls off rapidly with propagation distance, collateral
damages to surrounding tissue is minimal.
[0046] An animal is prepared for optical access to neuronal
vasculature by performing a craniotomy and sealing the opening with
a coverslip and 1% agarose in artificial cerebral spinal fluid
(ACSF). Small openings were left around one or more edges of the
coverslip to permit insertion of small electrical probes to contact
or penetrate the brain tissue for the purpose of electrical
stimulation and/or recording (Svoboda, Denk et al. 1997). The
animal was the placed into a head-fixed mount 30 at the base of the
apparatus. Alternatively, optical tracking (e.g., using fiber laser
delivery), can be used for animal preparations that are not fixed
to a stationary mount. Auxiliary equipment may be provided at the
animal preparation to monitor and maintain homeostatic conditions,
as well as provide sensory stimulation.
[0047] Fluorescent labeling of the blood plasma allows targeting of
the vessels as well as observation of blood flow using TPLSM. The
blood plasma is labeled by intravenous injection of a water-soluble
fluorescent tracer. Blood flow is visualized by monitoring the
motion of erythrocytes or other blood stream constituents, which
appear as dark objects moving against a fluorescent blood plasma
background. Imaging of the neuronal blood flow is performed and
maps of the vascular connectivity are generated with micrometer
resolution.
[0048] Quantified maps of blood flow are generated by analysis of
the collected images. Quantitative blood flow analysis consists of
calculating the streak angle of contrast-generating objects in the
bloodstream visualized by TPLSM. Alternatively, two-point
correlation of intensity changes along the length of the imaged
vessel can be used to quantify the blood flow.
[0049] Vascular damage is induced in a subpopulation of the mapped
blood vessels by tightly focusing a controlled number of
photodisruptive laser pulses in those blood vessels. Either
hemorrhage, thrombosis, or breach of the blood-brain barrier can be
produced without requiring the presence of an exogenous
photosensitizer in the blood stream. Additionally, multiple types
of vascular injury can be induced to different targets within the
same animal preparation. After vascular injury has been induced,
more imaging or neuronal blood flow is performed, and changes in
blood flow are quantified. Further, any other physiological
parameter amenable to fluorescence microscopy can also be observed
with TPLSM, e.g., intracellular Ca.sup.2+ concentration, Reduced
Nicotinamide Adenine Dinucleotide/Nicotinamide Adenine Dinucleotide
(NADH/NAD.sup.+) ratio, or the transmembrane voltage.
Post-operative observations are used to correlate the induced
vascular injury and real-time observed physiological changes to
behavioral deficits or post-mortem histology.
[0050] FIGS. 4a and 4b provide simulated images of a cross-section
of tissue 300 before and during exposure to the photodisruptive
pulses, respectively. Microscope objective lens 22 is positioned
over the desired target area for irradiation on the animal by
movement of one or both of the kinematic mount 66 and translation
stage 40. In FIG. 4b, the photodisruption beam 222 is focused by
objective lens 22 onto target vasculature 400. FIGS. 4c, 4d, and 4e
are images of the tissue showing a hemorrhage 402, a breach of the
blood-brain barrier 404, and a thrombosis 406, respectively, all of
which were induced by photodisruption.
EXAMPLE 1
[0051] Sprague-Dawley rats, 100-300 grams in weight, were prepared
in order to provide optical access to neuronal blood flow. The
animals were anesthetized with urethane and craniotomies, roughly 4
millimeters by 4 millimeters in extent, were performed over
parietal cortex to create a cranial window. The dura was removed
and a metal frame 62 was glued to the skull. The exposed brain
surface was covered with 1.0 to 1.5% low gelling temperature
agarose (W/V) in artificial cerebral spinal fluid (ACSF). A glass
coverslip was clipped in place on top of the agarose to maintain
pressure over the brain and to provide optical access. Water
soluble fluorescein-isothiocyanate dextran was injected
intravenously to label the blood plasma for the purpose of
targeting and imaging vessels. Alternatively, a thinned-skull
preparation can be performed to gain optical access to the neuronal
blood flow. The metal frame is then mounted to a kinematic mount
and computer-controlled translation stage at the base of the
apparatus via metal rods. Homeostatic conditions are monitored and
maintained by auxiliary equipment at the base of the apparatus,
including, thermometer, heat blanket, oxygenated gas flow,
electrocardiogram, and blood pressure monitor. Additional auxiliary
equipment (e.g., mechanical whisker stimulator) at the base allows
for sensory stimulation during the course of the experiments.
[0052] TPLSM was used to visualize vessels and select a target for
photodisruption. Alternative means for labeling include the
injection of fluorescently-labeled erythrocytes, microscopic
exogenous fluorescent probes or addition of a stain to the blood
stream that localizes to endogenous cellular components. For
observation of the vessel walls, a lipophilic stain can be used for
labeling.
[0053] Once a target has been selected, a stack of TPLSM optical
sections was taken, spanning the area around the target from the
surface to roughly 300 micrometers below the surface to visualize
the three-dimensional organization of regional blood vessels.
Multiple adjacent image stacks can be taken to extend the field of
observation. A rapid time series of line scans were taken of a
target blood vessel as well as surrounding blood vessels for
quantification (measurement of velocity, flux and mass flux) of
blood flow, as discussed further below.
[0054] To initiate vascular disruption, a 1-kilohertz train of
laser pulses of roughly 100-femtosecond pulsewidth with a
wavelength of 800-nanometers were focused into the lumen of the
vessel or at the surface of the vessel while continuously
monitoring with TPLSM. After a train of approximately 10 laser
pulses, the irradiated area was observed for any vascular changes.
The laser pulse energy started at .about.50 nanojoules. If no
vascular changes were observed, the laser power was increased and
irradiation repeated. Vascular disruption occurred at energies of
around .about.0.3 microjoules.
[0055] The morphology of vessels can be monitored at a frame rate
of up of several hertz, while the flow velocity in vessels can be
monitored at kHz rates by scanning only a single line along the
length of the vessel. In some cases, velocity of RBCs was measured
in the target and surrounding vessels. To measure RBC velocity in a
single vessel with TPLSM, the imaging laser is scanned repeatedly
along the length of a vessel. This line-scan results in a
space-time image, in which the motion of the RBCs through the
vessel is recorded as dark streaks. Using the time between
successive line-scans and length of each line-scan, the velocity of
the RBC can be computed by an automated process.
[0056] At the end of the imaging experiments (5 to 6 hours
post-clot formation), the animal is administered an overdose of
Nembutal and transcardially perfused with phosphate buffered saline
(PBS) followed by 4% paraformaldehyde (W/V) in PBS. The brain is
removed and equilibrated with solution of 30% sucrose (W/V) in PBS.
The brain is sectioned (50 micrometer thick sections) on a
freezing, sliding microtome and serially collected in PBS with 0.2%
azide (W/V).
[0057] The expression of cFOS by immunolocalization has been linked
to cell stress and damage associated with ischemia and also with
exposure to ultraviolet laser radiation. Control experiments were
performed to eliminate the possibility of cFOS upregulation in
response to the surgical preparation or two-photon imaging process.
In these control experiments, cFOS immunostaining was only
scattered and sparse. Next, the induction of cFOS expression was
used as an indicator of cell stress in response to
photodisruptively induced vessel injury or blockage. In contrast to
the control experiments, cFOS upregulation is induced by local
photodisruption of blood flow in the vicinity of targeted vessels.
This provides corroborative evidence for downstream ischemic
cellular pathology following photodisruption-induced injury to
blood vessels.
[0058] Additionally, immunodetection of infused fluorescein reveals
sites of blood-brain barrier breach. The large molecular size of
the intravenously injected dye (2 megadalton
fluorescein-isothiocyanate dextran) resulted in exclusion of dye
from most of the brain parenchyma. Sections stained with
anti-fluorescein antibody demonstrated only restricted sites with
marked parenchymal labeling within several hundred micrometers of
the cortical surface. This staining outlined neurons and in some
cases appeared to also be incorporated where plasma extravasation
was visualized in vivo during photodisruptive production of
hemorrhage or breach of the blood-brain barrier.
EXAMPLE 2
[0059] Using the experimental set-up (animal preparation, system
configuration and measurement) described in Example 1,
photodisruption was performed on vessels in the cortex ranging in
depth from the surface to--175 micrometers in depth. The resulting
vascular disruption was found to divide into three categories,
which are listed below in order of the approximate severity and
size of the damage to the microvessel:
[0060] Thrombosis--Near the threshold energy for vascular
disruption and with a limited number of pulses, photodisruption
results in extremely limited extravasation of plasma. TPSLM images
were taken at various time points before, during and after vascular
photodisruption. In an exemplary experiment, the vessel is intact
before irradiation. After irradiation with 10 pulses of 0.3
microjoules, a small amount of extravasated plasma could be
visualized as fluorescence outside the vessel walls, however, the
vessel lumen remained unobstructed. Extravasated fluorescence
continued to spread for several seconds after irradiation, but
remained spatially confined to within 5 micrometers of the vessel.
After a second irradiation with 10 pulses at 0.3 microjoules,
thrombosis began within several seconds. In the targeted vessel,
unmoving, dark areas indicated the coalescence of RBCs and perhaps
platelets. Bright stationary areas indicate plasma within the
vessel that may be stagnant and without RBCs. A clot that was
formed was observed to be stable for the entire period of
observation (2 hours).
[0061] Breach of the blood-brain barrier--Following
photodisruption, a weakening of the blood-brain barrier can allow
the extravasation of fluorescein-labeled plasma to fill a volume
around the targeted vessel. Penetration of the plasma into the
extravascular space was not necessarily limited to regions
immediately adjacent to the target vessel. In exemplary
experiments, fluorescent dye penetrates the parenchyma up to--30
micrometers radially from the target vessel. In some instances,
leakage through the blood-brain barrier was accompanied by the
formation of a thombotic clot within the vessel. In other cases,
blood flow remained unobstructed within the target vessel
throughout irradiation, extravasation and subsequent observation.
RBC velocity remained unchanged by the irradiation and subsequent
vessel leakage.
[0062] Hemorrhage--Greater laser energies and/or increased numbers
of pulses, lead to a larger disruption of the target vessel. In an
exemplary experiment, a microvessel 125 micrometers below the
cortical surface was observed before and after irradiation with 10
pulses of 1 microjoule energy. Initial fluorescein leakage was
rapid, reaching a diameter of 60 micrometer within 1 second. The
plasma continued to expand, stabilizing to a volume of about 0.002
mm.sup.3. In addition to fluorescently labeled plasma, RBCs were
pushed into the parenchyma and were visualized with white light
microscopy immediately after photodisruption.
[0063] Ultrashort laser-induced photodisruption comprises
electron-ion plasma, shock wave, and cavitation bubble formation,
as described above. Vascular disruption can be caused by any one of
these optically-triggered events. It is believed that when the
laser is focused directly on the vessel wall, ionization removes
portions of the endothelial cell. When the laser is focused into
the vessel lumen, ionization occurs in blood plasma, or perhaps in
a passing red blood cell. In this case, the vessel walls are likely
not directly affected by the ionization because the ionization
volume is small relative to the vessel lumen, and the products of
ionization are swept downstream by the flowing blood. However, the
shock wave and the cavitation bubble that follow optical breakdown
may locally disrupt endothelial cells. The size and strength of the
shock wave and cavitation bubble depend on the total amount of
laser energy, so that the extend of injury to the vasculature and
the tissue can be modulated by the laser power and number of
applied pulses.
[0064] At low energies, near the threshold for vascular disruption,
a weak shockwave and small cavitation bubble transiently injure the
endothelial cells. The injury may be severe enough to degrade the
blood-brain barrier, allowing the observed extravasation of
fluorescein-labeled plasma, but the leakage is transient. The
injury to the endothelial cells may be sufficiently mild that the
cells recover, or the endogenous clotting cascade may seal the
breach quickly. In some cases, the injury can also trigger
thrombosis that completely blocks the vessel, but in other cases,
the lumen remains unobstructed.
[0065] At higher laser energies, the shockwave may induce
sufficient damage to the endothelial cells to disrupt the
blood-brain barrier for longer times and over larger areas,
allowing bodies such as RBCs to invade the parenchyma. At even
higher energies, the shockwave is sufficiently strong that it can
completely rupture the vessel and possibly induce direct damage to
the tissue surrounding the vessels. These larger vascular
disruptions result in a hemorrhage that develops into an
intra-parenchymal hematoma, a clotted mass that includes RBCs. It
may be noted that even these larger vascular hemorrhages are still
three-dimensionally localized, as tissue surrounding the hemorrhage
is not disrupted. This tissue immediately bordering the vascular
injuries will be at the greatest risk for infarction and
consequently, the most interesting to study.
[0066] Extravascular tissue remains relatively unaffected by the
photodisruption in the vessel for several reasons. Because only a
small amount of total energy is delivered by the laser, collateral
damage by thermal mechanisms is insignificant. The ionization
plasma is confined to volume less than 1 micrometer in diameter.
The pressure induced by the shockwaves falls off with increasing
distance, thereby limiting the total volume affected by the shock.
With appropriate selection of energy, this volume is comprised
mostly of the vessel lumen and the endothelial cells. Vascular
cells and tissue which wrap completely around the target vessel,
e.g., endothelial cells and basement membrane, are preferentially
affected by the photodisruption when compared to cells which simply
abut the vessel. Because vascular cells wrap around the source of
the shockwave, the pressure wave translates into tensile stress
which can rupture cells.
[0067] The above-described device and method of the invention
provides means for producing injury to single, selected
microvessels in the depth of the cortex using ultrashort
laser-induced photodisruption. This model produces three types of
vascular damage: thrombosis, breach of the blood-brain barrier, and
hemorrhage. Thrombosis of single vessels may be a good model of
local blockages of the microvasculature that lead to small infarcts
in the brain. Previous models of lacunes and microinfarcts involved
the systemic injection of small clotting agents such as
microspheres or other emboli. The locations of the occlusions are
random and unpredictable, and infarcts must be located post-mortem
by the tedious inspection of the entire brain after histological
sectioning. Using the inventive method and model, single
microvessel occlusions can be placed in a predictable manner in the
cortex, allowing the subsequent cellular and physiological events
to be systematically studied. Further, the inventive method permits
real-time monitoring of physiological parameters amenable to
fluorescence microscopy (e.g. blood flow, intracellular Ca.sup.2+
concentration, Reduced Nicotinamide Adenine
Dinucleotide/Nicotinamide Adenine Dinucleotide (NADH/NAD.sup.+)
ratio, or the transmembrane voltage). Similarly, vessel ruptures
can be investigated in controlled experiments. Microvessels can be
ruptured throughout the cortex to model the nature of hemorrhages
that are detected in human brains by MRI. A third modality of
vascular injury produced by the inventive device and method is the
transient disruption of the blood-brain barrier leaving an intact
vessel. The present invention can be used to study the effects of
leaking blood plasma and its constituents into the neuronal
parenchyma with and without ischemia.
[0068] By coupling real-time TPLSM with ultrashort laser
photodisruption, the occlusions and hemorrhages in vessels can be
monitored as they are formed. In addition, RBC velocities in the
intact vessels surrounding an occluded microvessel can be
measured.
[0069] The device and method of the present invention provide novel
means for observing in real time and furthering the understanding
of mechanisms and treatment of stroke and vascular dementia in the
brain, and of vascular disease in other parts of the body, using
animal models. The ability to induce and study vascular disorders
in real time will provide means for evaluating treatments which can
prevent, limit and/or reverse the damage caused by stroke and
similar vascular insults in the brain and other organs within the
body.
[0070] Other embodiments and modifications of the present invention
will occur readily to those of ordinary skill in the art in view of
these teachings. Therefore, this invention is to be limited only by
the following claims which include all such other embodiments and
modifications when viewed in conjunction with the above
specification and accompanying drawings.
REFERENCES (Incorporated Herein by Reference)
[0071] 1. N. Heye and J. Cervos-Navarro, "Microthromboemboli in
acute infarcts: analysis of 40 autopsy cases," Stroke 27(3), 431-4.
(1996). [0072] 2. G. J. del Zoppo and T. Mabuchi, "Cerebral
microvessel responses to focal ischemia," J Cereb Blood Flow Metab
23(8), 879-94. (2003). [0073] 3. J. M. Wardlaw, P. A. Sandercock,
et al., "Is breakdown of the blood-brain barrier responsible for
lacunar stroke, leukoaraiosis, and dementia?," Stroke 34(3),
806-12. (2003). [0074] 4. G. J. del Zoppo, "Clinical trials in
acute stroke: Why have they not been successful?," Neurology 51,
S59-61. (1998). [0075] 5. P. Lipton, "Ischemic cell death in brain
neurons," Physiol Rev 79(4), 1431-568. (1999). [0076] 6. D. E.
McBean and P. A. Kelly, "Rodent models of global cerebral ischemia:
a comparison of two-vessel occlusion and four-vessel occlusion,"
Gen Pharmacol 30(4), 431-4. (1998). [0077] 7. L. Wei, C. M.
Rovainen, et al., "Ministrokes in rat barrel cortex," Stroke 26,
1459-1462. (1995). [0078] 8. L. Wei, J. P. Erinjeri, et al.,
"Collateral growth and angiogenesis around cortical stroke," Stroke
32(9), 2179-84. (2001). [0079] 9. A. Tamura, D. I. Graham, et al.,
"Focal cerebral ischaemia in the rat: 1. Description of technique
and early neuropathological consequences following middle cerebral
artery occlusion," J Cereb Blood Flow Metab 1 (1), 53-60. (1981).
[0080] 10. S. T. Chen, C. Y. Hsu, et al., "A model of focal
ischemic stroke in the rat: reproducible extensive cortical
infarction," Stroke 17(4), 738-43. (1986). [0081] 11. E. Busch, K.
Kruger, et al., "Reperfusion after thrombolytic therapy of embolic
stroke in the rat: Magnetic resonance and biochemical imaging,"
Journal of Cerebral Blood Flow and Metabolism 18(4), 407-418.
(1998) [0082] 12. P. D. Lyden and B. Hedges, "Protective effect of
synaptic inhibition during cerebral ischemia in rats and rabbits,"
Stroke 23, 1463-1469. (1992). [0083] 13. P. D. Lyden, J. A. Zivin,
et al., "Quantitative effects of cerebral infarction on spatial
learning in rats," Experimental Neurology 116(2), 122-132. (1992).
[0084] 14. P. D. Lyden, L. M. Lonzo, et al., "Effect of ischemic
cerebral volume changes on behavior," Behavioral Brain Research 87,
59-67. (1997). [0085] 15. M. Kudo, A. Aoyama, et al., "An animal
model of cerebral infarction. Homologous blood clot emboli in
rats," Stroke 13(4), 505-8. (1982). [0086] 16. K. Overgaard,
"Thrombolytic therapy in experimental embolic stroke," Cerebrovasc.
Brain Metab. Rev 6(3), 257-86. (1994). [0087] 17. K. Krueger and E.
Busch, "Protocol of a thromboembolic stroke model in the rat:
review of the experimental procedure and comparison of models,"
Invest. Radiol 37(11), 600-8. (2002). [0088] 18. G. A. Rosenberg,
S. Mun-Bryce, et al., "Collagenase-induced intracerebral hemorrhage
in rats," Stroke 21(5), 801-7. (1990). [0089] 19. R. M. Dijkhuizen,
M. Asahi, et al., "Rapid breakdown of microvascular barriers and
subsequent hemorrhagic transformation after delayed recombinant
tissue plasminogen activator treatment in a rat embolic stroke
model," Stroke 33(8), 2100-4. (2002). [0090] 20. W. Deinsberger, J.
Vogel, et al., "Experimental intracerebral hemorrhage: description
of a double injection model in rats," Neurol Res 18(5), 475-7.
(1996). [0091] 21. S. L. Hickenbottom, J. C. Grotta, et al.,
"Nuclear factor-kappaB and cell death after experimental
intracerebral hemorrhage in rats," Stroke 30 (11), 2472-7;
discussion 2477-8. (1999). [0092] 22. J. P. Pooler and D. P.
Valenzeno, "Dye-sensitized photodynamic inactivation of cells," Med
Phys 8(5), 614-28. (1981). [0093] 23. K. S. Hermann, "Platelet
aggregation induced in the hamster cheek pouch by a photochemical
process with excited fluorescein isothiocyanate-dextran,"
MicrovascRes 26(2), 238-49. (1983). [0094] 24. B. D. Watson, W. D.
Dietrich, et al. "Induction of reproducible brain infarction by
photochemically initiated thrombosis," Annals of Neurology 17,
497-504. (1985) [0095] 25. B. Krammer, "Vascular effects of
photodynamic therapy," Anticancer Res 21(6B), 4271-7. (2001) [0096]
26. W. D. Dietrich, M.D. Ginsberg, et al., "Photochemically induced
cortical infarction in the rat. 1. Time course of hemodynamic
consequences," J Cereb Blood Flow Metab 6(2), 184-94. (1986) [0097]
27. W. D. Dietrich, M. D. Ginsberg, et al., "Photochemically
induced cortical infarction in the rat. 2. Acute and subacute
alterations in local glucose utilization," J Cereb Blood Flow Metab
6(2), 195-202. (1986) [0098] 28. C. B Schaffer, F. F. Ebner, et
al., "Two-photon fluorescence microscopy of collateral blood flow
following photothrombotic stroke in rat neocortex", Optical Society
of America, Tuscon, Ariz. (2003) [0099] 29. C. B. Schaffer, F. F.
Ebner, et al., "Arteriole blood flow reverses direction at the
first branch that lies downstream from a localized photothrombotic
clot", Society of Neuroscience, New Orleans, La. (2003) [0100] 30.
C. B. Schaffer, P. S. Tsai, et al., "All optical thrombotic stroke
model for near-surface blood vessels in rat: Focal illumination of
exogenous photosensitizers combined with real-time two-photon
imaging", Commercial and Biomedical Applications of Ultrafast
Lasers III, San Jose, SPIE-International Society of Optical
Engineering (2003). [0101] 31. U. Dirnagl, B. Kaplan, et al.,
"Continuous measurement of cerebral cortical blood flow by
laser-doppler flowmetry in a rat stroke model," J Cereb Blood Flow
Metab 9, 589-596. (1989) [0102] 32. H. Nakase, T. Kakizaki, et al.,
"Use of local cerebral blood flow monitoring to predict brain
damage after disturbance to the venous circulation: cortical vein
occlusion model by photochemical dye," Neurosurgery 37(2), 280-5;
discussion 285-6. (1995) [0103] 33. M. Hoehn-Berlage, D. G. Norris,
et al., "Evolution of regional changes in apparent diffusion
coefficient during focal ischemia of rat brain: the relationship of
quantitative diffusion NMR imaging to reduction in cerebral blood
flow and metabolic disturbances," J Cereb Blood Flow Metab 15(66),
1002-1011. (1995) [0104] 34. G. Marchal, A. R. Young, et al.,
"Early postischemic hyperperfusion: pathophysiologic insights from
positron emission tomography," J Cereb Blood Flow Metab 19(5),
467-82. (1999) [0105] 35. M. Ishikawa, E. Sekizuka, et al.,
"Platelet adhesion and arteriolar dilation in the photothrombosis:
observation with the rat closed cranial and spinal windows," J
Neurol Sci 194(1), 59-69. (2002) [0106] 36. J. Seylaz, R.
Charbonne, et al., "Dynamic in vivo measurement of erythrocyte
velocity and flow in capillaries and of microvessel diameter in the
rat brain by confocal laser microscopy," J Cereb Blood Flow Metab
19(8), 863-70. (1999) [0107] 37. E. Pinard, Nallet, et al.,
"Penumbral microcirculatory changes associated with peri-infarct
depolarizations in the rat," Stroke 33, 606-612. (2002) [0108] 38.
L. Wei, K. Craven, et al., "Local cerebral blood flow during the
first hour following acute ligation of multiple arterioles in rat
whisker barrel cortex," Neurobiol Dis 5(3), 142-50. (1998) [0109]
39. W. Denk, J. H. Strickler, et al., "Two-photon laser scanning
fluorescence microscopy," Science 248, 73-76. (1990) [0110] 40. W.
Denk, "Two-photon scanning photochemical microscopy: Mapping
ligand-gated ion channel distributions," Proceedings of the
National Academy of Sciences USA 91, 6629-6633. (1994) [0111] 41.
D. Kleinfeld, P. P. Mitra, et al., "Fluctuations and
stimulus-induced changes in blood flow observed in individual
capillaries in layers 2 through 4 of rat neocortex," Proceedings of
the National Academy of Sciences USA 95, 15741-15746. (1998) [0112]
42. D. Kleinfeld and W. Denk (2000). Two-photon imaging of
neocortical microcirculation. Imaging Neurons. A Laboratory Manual.
R. Yuste, F. Lanni and A. Konnerth, Cold Spring Harbor, Cold Spring
Harbor Laboratory Press: 23, 1-23. 15 [0113] 43. N. Suhm, M. H.
Gotz, et al., "Ablation of neural tissue by short-pulsed lasers--A
technical report," Acta Neurochirurgica 138, 346-349. (1996) [0114]
44. F. H. Loesel, J. P. Fischer, et al., "Non-thermal ablation of
neural tissue with femtosecond laser pulses," Applied Physics B 66,
121-128. (1998) [0115] 45. P. S. Tsai, B. Friedman, et al.,
"All-optical histology using ultrashort laser pulses," Neuron 39,
27-41. (2003) [0116] 46. S. Backus, R. Bartels, et al.,
"High-efficiency, single-stage 7-kHz high-average-power ultrafast
laser system" Optics Letters 26, 465-467. (2001) [0117] 47. K.
Svoboda, W. Denk, et al., "In vivo dendritic calcium dynamics in
neocortical pyramidal neurons." Nature 385: 161-165. (1997) [0118]
48. J. G. Fujimoto, "Optical coherence tomography for ultrahigh
resolution in vivo imaging", Nature Biotechnology, 21: 11 pp.
1361-1367 (2003).
* * * * *