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Technical Advances |
From the Department of Chemistry and Biochemistry,*
W. M. Keck Center for Transgene Research and the Department of
Electrical Engineering,
University of Notre
Dame, Notre Dame, Indiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although mice provide an appropriate genetic model, their diminutive size presents a challenge in the study of hemostasis. Vascular injury models developed in larger mammals are sometimes difficult to miniaturize to apply to the mouse. Despite these difficulties, several murine vascular injury models have been developed. These include use of electrical21 and mechanical injuries,22-25 FeCl3,26,27 laser-induced injuries,28,29 and light-induced injuries using photoactivatable dyes.30-34 Most of these models involve surgical intervention that can lead to artifactual activation of the hemostatic system. The use of intravital microscopy to analyze photochemical injury to vessels of the ear of the hairless mouse addresses this concern.32,33 This latter model has been modified and used herein to induce injury with irradiation from an argon-ion laser. Specifically, laser radiation is introduced through an optical port of the microscope and focused through the objective on the target tissue. Because the laser energy is focused at the focal plane of the microscope, the intensity of the laser beam is adjusted so that injury is only induced at a particular depth in the tissue. It is thus possible to induce injury on the luminal surface of the vessel and monitor thrombogenesis of individual thrombi by intravital microscopy. This report describes the major characteristics of this injury model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice for the studies included C57BL6 and Swiss Webster wild-type mice. Housing and procedures involving experimental animals were approved by Institutional Animal Cure and Use Committee of the University of Notre Dame.
Laser-Induced Injury
The experimental arrangement of components to induce and record
vascular injury in real time in vivo is illustrated in
Figure 1
. The beam of an argon-ion laser
(Innova 90-5, Coherent, CA) was introduced into an optical port of a
TwinEpi attachment (Prairie Technologies, Madison, WI) to a Nikon
Eclipse 600 (Nikon, Tokyo, Japan) compound microscope and directed
through the objective to be focused on a sample. The laser was focused
on the target with a low intensity light (using power input to the
laser that does not induce lasing). An injury was induced with a short
high-intensity laser burst. After laser exposure, the image of the
injury generated by backlighting of the injured vessel was recorded
with a charge-coupled device camera (Optronics, Goleta, CA). The
recorded video image was digitized and then analyzed with Bioquant True
Color Windows software (Biometrics, Nashville, TN). The design of the
TwinEpi attachment also permitted illumination with a mercury lamp to
generate epifluorescent images of the thrombus.
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Video recordings of the injury were analyzed by capturing and digitizing the recorded image at prescribed intervals (1 or 2 seconds). The captured frames were analyzed using the BioQuant True Color Windows software package (Biometrics, Nashville TN). A region of interest (ROI) was defined for the target vessel that included a portion of the target vessel that is larger than the maximum injured area. Using software tools, thresholds were set to define the thrombus in the ROI. The software was programmed to review each frame, identify the thrombus in the ROI, and to measure several different parameters including area and intensity.
The increase in the integrated optical density (IOD) of the thrombus
was determined by first using the BioQuant software to adjust the
brightness of the video image such that the mean brightness of the
pixels in the ROI was at midrange (a brightness level of 128 in a scale
from 0 to 255). The background intensity was defined as the average
intensity in the ROI from a captured frame corresponding to the vessel
before injury. The IOD is defined as the 
-
(log[Ip/Ib] for each of
the pixels in the ROI where Ip is the light
intensity at the pth pixel and Ib is the
background intensity. The increase of the IOD is determined by
subtracting the average IOD of the first two captured frames
corresponding to the vessel before injury from the IOD of subsequent
frames.
Histological and Immunohistochemical Analyses
After the induction of injury to the vessels of the ear, the anesthetized animals were sacrificed and the animals perfused by pumping saline followed by buffered formalin into the left ventricle of the heart. Histological analyses were performed on paraffin-embedded serial sections of the injured ears. Sections at 80-µm intervals were stained with hematoxylin and eosin (H&E) for microscopic evaluation.
Electronmicroscopic Analysis
After laser illumination, mice were perfused with saline Karnovsky’s solution (1% paraformaldahyde/2.5% glutaraldehyde, in 0.1 mol/L Na-cacodylate buffer, in which the osmolarity was adjusted with NaCl and sucrose). The injured section of ear was excised, and incubated in Karnovsky’s solution for 16 hours. The samples were rinsed twice in 0.1 mol/L Na-cacodylate buffer for 30 minutes each, and postfixed in 4% osmium tetroxide for 1 hour, and rinsed again in the same buffer. After dehydration through progressive alcohol incubations, the samples were embedded in epoxy resin. Semithin sections (0.5 µm) were stained in toluidine blue and examined with a compound microscope to identify regions of interest. Once the thrombus was identified, thin sections (90 nm) were placed onto copper grids and stained with Reynold’s lead citrate and (2%) uranyl acetate for transmission electron microscopy analysis.35
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experimental set-up illustrated in Figure 1
permits
photo-induced injury at specific sites within the vasculature of the
ear of a mouse. The hair on the ear of an anesthetized mouse is removed
and the animal positioned in a plastic restraint on a microscope stage.
The restraint is designed so one ear can be taped to a glass platform
so that blood flow through the vasculature of the ear can be visualized
by transillumination. Although circulation in both arteries and veins
is easily visualized, this report presents a characterization of the
induction of venous injuries. Although the architecture of the
vasculature of the ear varies among mice, generally there are either
one or two primary veins proximal to the head with a diameter of 
200
to 350 µm. This primary vein(s) is fed by secondary veins of 150
to 250 µm, which in turn are fed by tertiary vessels of 100 to 200
µm. The primary, secondary, and tertiary veins are targeted in these
studies because the increased pigmentation in more distal segments of
the ear introduces experimental complications reducing the
reproducibility of the model.
Using the microscope, laser, and imaging system described in Figure 1
,
injury can be induced in the vessel by two distinct methods. The first
involves direct laser injury of the wall of the vessel by focusing a
high-energy laser pulse at the luminal surface of the vessel wall. The
second involves intravenous injection of the photoactivatable dye, Rose
Bengal. In this latter case, subsequent laser illumination of the
target vessel results in photochemical injury at the site of
illumination. Both models are minimally invasive, generate reproducible
injuries, and can produce multiple injuries in a single animal.
Although the two methods for inducing injuries are similar, thrombus
development by the two procedures exhibits different sensitivities to
anticoagulants and platelet antagonists.
Direct Laser Injury
After positioning the anesthetized animal in a restraint on the
microscope platform, the laser was focused on the target vessel.
Because the laser beam focused through the microscope objective is
confocal with the image generated by transmitted light, the laser
energy is concentrated to a small volume around the focal plane.
Initial studies established parameters to generate subocclusive
thrombi. Laser illumination of larger venules (primary to tertiary
venules in the ear) with the 517-nm line at 100 mW for 1 second
consistently induced a subocclusive thrombus that grew in size and
intensity during the course of several minutes (Figure 2A)
. After reaching its maximum size
(25 to 75% the diameter of the vessel), the thrombus began to fade
and then usually to embolize. Several minutes after the laser pulse,
the image of the vessel stabilizes and a detectable mural thrombus
remains.
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Effects of Antithrombotics
To determine the nature of the thrombi generated in this model,
the effects of coagulation inhibitors on thrombus development were
examined. Anesthetized animals were administered the anti-GPIIb/IIIa
inhibitor, G4120,36
in normal saline at a dose of 1 µg/g
body weight by intravenous tail vein injection. The panels in Figure 2
illustrate the progression of two injuries induced in a C57BL6 mouse in
the absence (Figure 2
, top) or presence (Figure 2
, bottom) of the
inhibitor. In Figure 3
, the areas of
thrombi generated after multiple injuries as determined with the
BioQuant imaging system in the presence or absence of G4120 are
displayed graphically. These images (Figure 2)
and analyses (Figure 3)
show that thrombus development is inhibited in the presence of the
antiplatelet compound.
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Because measuring the area of the thrombus (as plotted in Figure 3
)
does not take into consideration the intensity or brightness of the
clot, the increase of the IOD of the developing thrombus was
determined. The IOD calculates the log of the ratio of the intensity at
each pixel to the background intensity before injury and sums that
value for each pixel in the ROI. The ROI is drawn to include a region
of the target vessel that extends beyond the maximum extent of the
clot. The increase in the IOD of the clot can be calculated by
subtracting the value of the IOD from images of the vessel before
injury from the value of the IOD after injury. Pixels in the ROI that
do not change in intensity during the course of the injury
(corresponding to uninjured sections of the vessel in the ROI) thus do
not contribute to the increase in IOD.
The increase in IOD for multiple laser-induced thrombi generated in
three animals is shown in Figure 4
. The
general shape of the tracings for each thrombus is very similar. The
IOD increases rapidly within the first 30 seconds after injury and then
is maintained or increases slightly throughout the next several minutes
of observation. In addition, the maximal increase in the IOD
(IODmax) for each thrombus was determined.
The average values of the (IODmax) for thrombi
generated in each of three animals are displayed in Table 1
. Interestingly, the average increase in
the IOD for injuries made in each animal is very reproducible; the
average (IODmax) calculated for different animals
in this series of injuries was 184 ± 20%.
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To analyze the nature of the laser-induced injury histologically
after injury, animals were perfused by ventricular puncture first with
saline, then with buffered formalin. The region of the ear surrounding
the injury was excised and embedded in paraffin. The target vein was
followed through serial sections enabling identification of sections of
the target vessel with the induced thrombus. The panels in Figure 4
represent histological characterization of sections through a single
clot induced by direct laser injury. As expected, sections preceding
the injury were free of detectable clot, consistent with the local
nature of the injury recorded by intravital microscopy (Figure 5A)
. The periphery of the thrombus
includes less condensed fibrous material (Figure 5B)
whereas the main
body of the thrombus contains condensed material attached to the vessel
wall. Histochemical analysis of other laser-induced thrombi indicates
the less condensed fibrous material stains with antibodies recognizing
fibrin(ogen) (data not shown). Unfortunately, the antibody recognizes
both fibrinogen and fibrin, so it is not possible to tell whether
fibrin generation and polymerization are required for thrombus
generation. The thrombus also contains P-selectin antigen (data not
shown) suggesting the presence of activated platelets within the clot.
This result is consistent with the sensitivity of thrombus development
to the anti-GPIIb/IIIa inhibitor G4120 (Figures 2 and 3)
. Sections
stained for von Willebrand factor (data not shown), which serves
as a marker for the vascular endothelium, indicate that the lumen of
the vein is ringed with a layer of endothelial cells. At the resolution
of the micrographs for immunohistochemical analysis, there does not
seem to be major disruption of the endothelial lining of the vein. In
addition, it was noted in these figures that there was no apparent
damage to the tissue surrounding the target vessel. This observation
supports the contention that the focusing of the laser induces injury
in a small volume within the target rather than burning a vertical
column through the ear.
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Ultrastructural analysis has been initiated to further
characterize the thrombus generated by laser-induced injury. Figure 6a
presents a transmission electron
micrograph of a thrombus induced by direct laser injury. The figure
shows polymorphonuclear cells (PMN) attached to damaged, highly
vacuolated endothelium. There are numerous platelets at various stages
of activation. Although the endothelium is injured, it is still
attached to the substratum of the vessel wall. However, there is one
site at which the subendothelial matrix is exposed and a projection
from a neutrophil is shown to make contact with the subendothelial
matrix (Figure 6a
, insert). From this, it seems that thrombus formation
does not require major denudation of the endothelium and exposure of
platelets or coagulation factors to the subendothelial matrix. The
micrographs also confirm the local nature of the injury. Sections
100-µm proximal or distal to the thrombus are free of injury. In
addition, composite micrographs that allow tracing along the luminal
surface indicate that while the endothelium at the site of injury is
damaged, the lining on the opposite surface of the vessel appears
normal (data not shown).
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In addition to the induction of direct laser injury with a
relatively high-energy pulse for a short duration, the experimental
apparatus shown in Figure 1
can also be used to induce photochemical
injury. In the photochemical injury mode, mice were administered 40
µg/g body weight by tail vein injection of a 10 mg/ml solution of
Rose Bengal dye dissolved in normal saline. Subsequent laser
illumination induced mural clots in the target vessels near the site of
illumination. Presumably, photoactivation of the Rose Bengal dye
results in the generation of singlet oxygen, which induces
chemical damage to the endothelium.
Parameters to Generate Subocclusive Thrombi
Initial experiments were designed to determine parameters necessary to generate subocclusive thrombi in the target vessels in 8- to 12-week-old Swiss-Webster mice. Irradiation at 10 mW failed to generate thrombi in the presence or absence of Rose Bengal, even after 4 minutes of continuous illumination. Irradiation at 20 mW for 30 seconds in the presence of Rose Bengal at 40 µg/g body weight occasionally induced a faint thrombus. However, irradiation at 30 mW for 20 to 30 seconds consistently generated subocclusive thrombi in target veins. In contrast, more intense irradiation (75 mW) for 20 seconds at the same Rose Bengal concentration consistently generated occlusive clots near the site of illumination. These results indicate that the extent of thrombus development in the primary, secondary, or tertiary veins of the ear is a function of the intensity of irradiation. Similarly, the extent of injury is also affected by the duration of irradiation and concentration of Rose Bengal. Furthermore, it is possible to identify irradiation parameters to consistently generate subocclusive thrombi in the particular target vessel. In particular, subocclusive clots in secondary veins are consistently generated by 30 seconds of irradiation at 30 mW in Swiss Webster mice injected with Rose Bengal with a dose of 40 µg/g body weight. As might be expected, the parameters required to induce an injury of a particular intensity vary with the genetic background of the mouse. For instance, irradiation at 20 mW for 30 seconds induces extensive subocclusive thrombi in 129/C57BL6 mice, parameters that only induce a faint thrombus in Swiss-Webster mice.
Effects of Coagulation Inhibitors
To explore the role of the coagulation system on thrombus
development after photochemical injury, injuries were made in the
presence or absence of the thrombin inhibitor PPACK. Anesthetized mice
were administered Rose Bengal either with or without PPACK by
intravenous injection into the tail vein. Figure 7
illustrates the effect of the thrombin
inhibitor PPACK on photochemical injury. Figure 7
, top, shows
photochemical injury in the absence of PPACK. The blue light in panel A
indicates the target site for the laser, whereas panel B shows the
vessel before laser illumination. Panels C and D show the vessel 30 and
180 seconds after laser injury and indicate the growth and development
of the thrombus. Panels E through H represent similar images after
irradiation in an animal injected with PPACK and Rose Bengal. In this
latter case, no detectable thrombus was detected near the site of
illumination. These results indicate that thrombin activity is required
for thrombus development after laser-induced photochemical injury.
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Transmission electron micrographs of a thrombus generated by
laser-induced photoactivation of Rose Bengal (Figure 6b)
show the
aggregation of activated platelets at the site of injury. Although
the endothelium is intact and there is no detectable exposure of the
subendothelial matrix. The micrographs show the endothelial cells
are damaged and highly vacuolated. Examination of composite micrographs
that trace around the entire vessel indicate that the damage to the
endothelium extends to a larger area, and is not limited to the region
of illumination as was the case with the direct laser injury (data not
shown). This is consistent with phototoxins being generated at the site
of illumination, but damaging cells in a somewhat wider area.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The goal of this study was to develop a vascular injury model incorporating some of the advantages described in earlier models. In particular, a noninvasive photochemical injury model for the vasculature of the ear has been described that involved illuminating the ear with a mercury lamp after injection with fluorescein isothiocyanate-labeled dextran.32 In addition, there have been several reports of laser-induced photo-injuries to the rodent microvasculature. It is possible to focus the laser beam through the compound objective of a microscope to concentrate the laser radiation at the focal plane of the objective.27,28 If the laser is aimed at the inside surface of the vessel wall and the focal plane is adjusted so blood flowing in the vein is in sharp focus, then it is possible to induce injury consistently to the luminal surface of the vessel. Furthermore, the use of intravital microscopy to monitor thrombus development by transillumination in real time has been described.22 Relevant features of these models have been incorporated to noninvasively generate and monitor in real time laser-induced injury to the vasculature of the ear.
The models described in the present study offer a number of advantages over those previously described. They are noninvasive and require minimal animal handling and preparation. Because the injuries that are generated are small, there is no major perturbation of the coagulation system and no significant consumption of coagulation factors. Therefore, multiple injuries can be induced and recorded in a single animal. In addition, the model permits monitoring of thrombogenesis after individual injuries. Furthermore, parameters have been established that allow subocclusive injuries to be generated reproducibly. Therefore, the effects of genetic or pharmacological manipulations that shift the hemostatic balance in either thrombotic or hemorrhagic directions can be detected.
Using the experimental set-up described in Figure 1
, two types of
laser-induced injuries can be induced. In the first model of direct
laser injury, illumination in the range of 75 to 200 mW by the argon
laser for 1 to 2 seconds induces reproducible injuries in
veins of the ear. The focusing of the laser beam by the objective of
the microscope results in concentration of the energy of the laser beam
at the focal point. If the time and power during irradiation are
adjusted appropriately, tissue damage is limited to the focal point of
the targeted laser beam. By focusing the laser to the luminal surface
of the vein, it is possible to make reliable injuries inside the
vessel. Because the energy density of the beam is too low both above
and below the focal plane, no injury is induced above and below the
vessel. Therefore, injuries inside the vein can be generated in a
noninvasive manner.
Although it is possible to define parameters for making reproducible subocclusive injuries in the vein of the ear, the elaboration of conditions to provoke reproducible injuries in the artery has been problematic. Conceivably, the thicker wall of the artery and smaller diameter of the vessel disrupts the optical path of the laser beam and prevents reliable focusing of the beam to the luminal surface of the artery.
Because photo-injury models have been described to induce vascular
damage in a number of different animal systems, the possibility of
using the experimental system described in Figure 1
was explored for
inducing photochemical damage. If animals are administered Rose Bengal
dye, subsequent illumination of vessels with light of a wavelength of
540 nm leads to singlet oxygen production, chemical damage to
endothelial cells, and thrombus development near the site of
illumination. Although initial reports describe the use of xenon lamps
to provide the illumination, the 514-nm band of the argon laser has
been used to induce photochemical injury in rat spinal cord and
middle cerebral arteries.39,40
Therefore, the possibility of focusing the laser through the microscope to induce photochemical injury in vessels of the ear was explored. Using mice injected with a Rose Bengal solution, parameters were identified to generate reproducible subocclusive thrombi in secondary veins of the ear. Similarly, conditions have been developed to induce reproducible thrombi in ear arteries.
Both direct laser-induced injury and photochemical injury lead to the development of platelet-rich thrombi. Platelets were detected histochemically by staining with anti-P-selectin antibodies and by transmission electron microscopy. Although anti-murine fibrin antibodies detect antigen in the thrombi, distinction cannot be made as to whether the antigen is fibrin or fibrinogen. Initial ultrastructural analyses using transmission of thrombi generated by these models fail to detect significant cross-linked fibrin in the clot. Thus, both models appear to generate platelet-rich thrombi that are more characteristic of arterial injuries even though they are induced in a vein.
The sensitivity of the thrombi to various anti-thrombotic compounds has been explored. The anti-GPIIb/IIIa compound, G4120, inhibits thrombus formation in the direct laser injury model, consistent with the platelet-rich nature of the developing thrombi. However, tests with anticoagulants were less consistent. The anti-thrombin compound, PPACK, and coagulation inhibitor, NapC2 (which targets the FXa/FVIIa/TF complex), did not consistently inhibit thrombogenesis. Because thrombin is thought to be a physiologically important trigger of platelet activation in vivo, it was somewhat surprising that coagulation inhibitors failed to block thrombogenesis. However, both coagulation inhibitors act after the appropriate zymogens have been activated. Therefore, small but sufficient concentrations of thrombin (in the case of PPACK) or FVIIa and FXa (in the case of NapC2) could be generated locally at the site of injury and result in significant platelet activation.
In contrast to the direct laser injury, the development of Rose Bengal-induced thrombi are inhibited by PPACK. Conceivably, the slower rate at which these clots develop might prevent the accumulation of local concentrations of thrombin at the site of injury to promote platelet aggregation. Determining the components required for thrombogenesis in these models is under further investigation.
Ultrastructural analyses indicate that both injuries do not lead to denudation of the endothelium lining the lumen of the vessel. The endothelial cells at the site of injury are more highly vacuolated than undamaged endothelial cells, but the cell membrane is intact and the cells are attached to subendothelial matrix. Thus, thrombogenesis does not require exposure of blood components to subendothelial matrix.
In summary, minimally invasive photo-injury models of vessels in the ear have been described. Using the same experimental system in which a laser beam is focused through the objective of a compound microscope, injuries can be induced by direct laser illumination or by photochemical damage after the administration of photoactivatable dyes. These systems offer significant advantages including that the procedures are minimally invasive; no surgical procedures that could activate or otherwise modulate the hemostatic system are involved other than anesthetization of the animal. Additionally, multiple injuries can be made on a single animal. Furthermore, variation in the development of multiple thrombi in a single animal is similar to the variation in development of injuries between animals. Finally, the system enables monitoring and recording of individual thrombus development in real time. This system will be valuable for the investigation of the role of specific components of the hemostatic system in thrombogenesis in vivo.
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Footnotes |
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Supported by National Institutes of Health grants HL-13423 and HL-19982 (to F. J. C.), a grant from the W. M. Keck Foundation, by the Kleiderer/Pezold Family Endowed Professorship (to F. J. C.)., and a grant from the American Heart Association Midwest Affiliate (to E. D. R.).
Accepted for publication January 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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