Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Note: All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center San Antonio and were consistent with the ARRIVE guidelines.
1.&#160;Anesthetizing for Cortical Preparation
<ol>
	<li>Place the mouse in an induction chamber with 2-3% isofluorane mixed with oxygen to induce anesthesia. Observe the respiration rate decrease as the mouse is induced. Pinch the paw of the mouse to determine whether the mouse is ready to move to the nose cone. Note: Anesthesia level is a critical step in any in vivo preparation and care should be taken not to induce a level that will cause global ischemia.</li>
	<li>Once the mouse is sufficiently anesthetized, transfer the animal to the surgery/imaging platform and place the mouse&#8217;s nose in the nose cone and apply 1-1.2% isofluorane to maintain an anesthetized state. Ensure that the mouse is lying on a temperature controlled heating pad to maintain the body temperature (37&#176;C +/- 0.5&#176;C) throughout the remaining procedures. Place vet ointment over the eyes to prevent dryness while under anesthesia.</li>
	<li>Monitor the physiology of the mouse using a pulse oximetry system using the tail or foot clip provided with the system. Check that the respiratory rate is maintained between 50-65 breaths/min. Check that the heart rate remains between 300 to 450 bpm and oxygen saturation is maintained between 97-98% to ensure long-term survival of the animal.</li>
	<li>When the mouse is adequately anesthetized, shave the hair over the cranium using electric clippers, remove residual hair and clean with betadine, followed by an ethanol swab. Repeat this procedure up to three times to ensure a sterile surgical environment.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>With the scalp fully cleaned and shaven, make a 5 mm incision in the scalp of the mouse to reveal the cranial fissures and to locate bregma.</li>
	<li>Use a sterile cotton applicator to remove any remaining fascia overlying the cranium.</li>
	<li>Glue a custom made stainless steel ring (Figure 1) with tissue adhesive to the bone overlying the parietal cortex using the stereotaxic coordinates of Bregma: -1 to -3 mm and lateral: 2-4 mm. Note: The glue typically sets within 2 min&#160;after the placement of the ring onto the bone.</li>
	<li>Affix the ring to a stereotaxic holder (Figure 2) to stabilize the mouse and to prevent movement during imaging.</li>
	<li>Under a surgical grade dissecting microscope, slowly drill a 1-2 mm section in the cranium using a speed controlled dremel-like tool (Meisinger 3.9 mm drill bit) making sure to keep the area level as it is drilled. Achieve this using a zig-zag pattern. To avoid heat build up, set the drill speed to low and take frequent breaks.</li>
	<li>When the cranial skull becomes shinny in appearance, continue the thinning of the skull using a scalpel blade utilizing the same zig-zag pattern to keep the thinned surface level to facilitate smooth removal of thin layers of cranial skull. Using the tip of the scalpel blade make small linear strokes with light pressure to remove thin layers of bone at a time. Continue this until the vasculature is clearly visible through the dissecting microscope.</li>
	<li>If the experimenter punctures through or break the skull during the thinning process, euthanize the animal due to likely damage to the underlying cortex. 
	Note: The mouse skull is approximately 300 &#181;m in thickness and is comprised of two thin layers of compact bone (one external and one internal layer) and a layer of spongy bone sandwiched between the two layers of compact bone. The external layer of compact bone and most of the spongy bone are removed within the 5 mm drilling area resulting in an approximate 50 &#181;m layer of compact bone remaining (see Figure 2B). Visualization of the vasculature will ensure that the final intact thinned skull is approximately 50 &#181;m in thickness. The skull is therefore still present when thinned to this thickness.</li>
</ol>
3. Microscope Set-up
<ol>
	<li>Use an inverted microscope system (conventional, confocal or two-photon systems) that has an objective inverter. Note: It is also possible to utilize a standard upright microscope. The limiting factor will be the space between the stage and the objectives. Modifications to the stage may be necessary to accomplish this setup.</li>
	<li>Secure the surgical/imaging platform to a custom made stage that is located aside the base of the microscope. Note: The platform is made using a laboratory jack to allow vertical movement of the surgical/imaging platform under the objective. The laboratory jack is mounted to a plate affixed to four cylindrical poles. (See Figure 2).</li>
	<li>Position the objective inverter containing a 20X objective over the cranial window. Use an external light source to find the cranial window by looking through the eyepieces of the microscope and position the objective in the imaging area. Note: The imaging area will be denoted by the presence of the vasculature.</li>
	<li>For water-based objectives, use artificial cerebral spinal fluid (aCSF) (130 mM NaCl; 30 mM KCl; 12 mM KH2PO4; 200 mM NaHCO3; 30 mM HEPES; and 100 mM glucose) as the medium due to potential leakage into the cranial cavity during imaging (Figure 3).</li>
</ol>
4. Rose Bengal Dye Preparation, Administration and Induction of Stroke
<ol>
	<li>Prepare a fresh 20 mg/ml solution of Rose Bengal in artificial cerebral spinal fluid (aCSF); filter and sterilize before administration. Do not reuse or store the Rose Bengal once it has been mixed. Make a fresh solution for each experiment.</li>
	<li>Give a 0.1 ml tail vein injection of Rose Bengal while scanning the cranial window with a 561 nm laser to ensure adequate injection of the solution. Note: Rose Bengal will be visualized within 5 sec after injection in the vasculature of the brain. The entire vessel should be filled with Rose Bengal.</li>
	<li>Following adequate injection of Rose Bengal dye choose an appropriate vessel for thrombosis based on vessel diameter (40-80 &#181;m) to ensure reproducibility of a particular lesion volume. Differentiate between arteries and veins by looking at the direction of blood flow: arteries will move from larger diameter to smaller diameter vessels, veins move from smaller to larger diameter vessels. This is easily accomplished by visualization once Rose Bengal is injected.</li>
	<li>Change the microscope setting as follows:
	<ol>
		<li>Increase the dwell time. Note: This will vary depending on the microscope system being utilized.</li>
		<li>Increase the laser power to 100%.</li>
		<li>Collect time sequence images at 1 frame/sec&#160;using the maximum scan speed.</li>
	</ol>
	</li>
	<li>Scan the mouse until a stable clot is formed within the vessel. Note: This typically is achieved within 5 min&#160;of continuous scanning (See Figure 4).</li>
	<li>Following the induction of clot formation using Rose Bengal, remove the mouse from the imaging area back to the dissecting microscope. Carefully remove the stainless steel ring from the cranial skull. Examine the cranial window for any bleeding. If bleeding occurs, terminate the experiment.</li>
	<li>Use 6.0 monofilament suture to close the incision over the skull. Place antibiotic ointment along the suture line to prevent infection. Inject Buprenex (0.05 mg/kg) subcutaneously every 12 hr for three days for pain management.</li>
	<li>Return the mouse to a recovery chamber following removal from the anesthetic until fully awake and freely moving.</li>
	<li>Return the mouse to a clean cage for further investigation at a later time.</li>
</ol>
5. Longitudinal Imaging on Subsequent Days
<ol>
	<li>Employ the following methods to perform longitudinal imaging on subsequent days post-photothrombosis.
	<ol>
		<li>Anesthetize the mouse as described in Section 1 of the methods.</li>
		<li>Upon adequate anesthesia re-open the scalp by removing any residual sutures to reopen the skin overlying the previously drilled imaging field.</li>
		<li>Use a sterile cotton applicator to remove any remaining fascia overlying the cranium.</li>
		<li>Glue a custom made stainless steel ring (Figure 1) with tissue adhesive to the bone overlying the previous imaging field.</li>
		<li>Affix the ring to a stereotaxic holder (Figure 2) to stabilize the mouse and to prevent movement during imaging.</li>
		<li>Locate the vasculature underlying the previously thinned skull. Use tail vein injection of FITC-Dextran to verify the presence of the previously induced clot.</li>
		<li>Use 6.0 monofilament suture to close the incision over the skull. Place antibiotic ointment along the suture line to prevent infection. Inject Buprenex (0.05mg/kg) subcutaneously every 12 hr for three days for pain management.</li>
		<li>Return the mouse to a recovery chamber following removal from the anesthetic until fully awake and freely moving.</li>
	</ol>
	</li>
</ol>
6. Verification of Stroke Induction (Post-mortem)
<ol>
	<li>At the conclusion of a study, verify stroke volume using 2,3,5-triphenyltetrazolium chloride (TTC) staining as shown in Figure 5. The full method can be found in12.</li>
</ol>

<ol>
	<li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Annals of Neurology. 17, 497-504 (1985).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circulation Research. 40, 320-328 (1977).</li><li>Owens, A. P., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+tissue+factor+that+contribute+to+thrombosis+after+rupture+of+an+atherosclerotic+plaque.">Sources of tissue factor that contribute to thrombosis after rupture of an atherosclerotic plaque.</a> Thrombosis Research. 129, S30-S33 (2012).</li><li>Carmichael, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion+by+an+intraluminal+thread+compromises+collateral+blood+flow.">Rat middle cerebral artery occlusion by an intraluminal thread compromises collateral blood flow.</a> Brain Research. 791, 223-228 (1998).</li><li>Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J., Pulsinelli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+brain+ischemia+in+the+rat:+methods+for+reproducible+neocortical+infarction+using+tandem+occlusion+of+the+distal+middle+cerebral+and+ipsilateral+common+carotid+arteries.">Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.</a> Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 8, 474-485 (1988).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptor+stimulation+reduces+cytotoxic+edema+and+brain+infarcts+in+mouse+induced+by+photothrombosis+by+energizing+glial+mitochondria.">Purinergic receptor stimulation reduces cytotoxic edema and brain infarcts in mouse induced by photothrombosis by energizing glial mitochondria.</a> PloS One. 5, e14401 (2010).</li><li>Zheng, D. M., Wewer, J., Lechleiter, J. P. 2. Y. 1. R. -. i. n. i. t. i. a. t. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IP3R-dependent+stimulation+of+astrocyte+mitochondrial+metabolism+reduces+and+partially+reverses+ischemic+neuronal+damage+in+mouse.">IP3R-dependent stimulation of astrocyte mitochondrial metabolism reduces and partially reverses ischemic neuronal damage in mouse.</a> Journal of Cerebral Blood Flow and Metabolism. 33, 600-611 (2013).</li><li>Witte, O. 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Y., Rafie, C., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+basis+for+the+robust+distribution+of+blood+to+rodent+neocortex.">Topological basis for the robust distribution of blood to rodent neocortex.</a> Proceedings of the National Academy of Sciences. 107, 12670-12675 (2010).</li><li>Nishimura, N., Rosidi, N. L., Iadecola, C., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+collateral+flow+after+occlusion+of+a+single+cortical+penetrating+arteriole.">Limitations of collateral flow after occlusion of a single cortical penetrating arteriole.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30, 1914-1927 (2010).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proceedings of the National Academy of Sciences. 104, 365 (2007).</li><li>Blum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+after+silent+stroke:+Hippocampus+and+infarcts+both+matter.">Memory after silent stroke: Hippocampus and infarcts both matter.</a> Neurology. 78, 38-46 (2012).</li><li>Heinsius, T., Bogousslavsky, J., Van Melle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+infarcts+in+the+middle+cerebral+artery+territory+Etiology+and+outcome+patterns.">Large infarcts in the middle cerebral artery territory Etiology and outcome patterns.</a> Neurology. 50, 341-350 (1998).</li><li>Wardlaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+causes+lacunar+stroke.">What causes lacunar stroke.</a> Journal of Neurology, Neurosurgery &amp; Psychiatry. 76, 617-619 (2005).</li><li>Inoue, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+stroke+under+anticoagulant+therapy].+Rinsho+shinkeigaku.">Ischemic stroke under anticoagulant therapy]. Rinsho shinkeigaku.</a> Clinical Neurology. 50, 455-460 (2010).</li><li>Tiannan Wang, W. C., Xie, Y., Zhang, W., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Volume+of+the+Focal+Cerebral+Ischemic+Lesion+through+Photothrombosis.">Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis.</a> American Journal of Biomedical Sciences. 2, 33-42 (2009).</li><li>Head, B. P., Patel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+brain+protection.">Anesthetics and brain protection.</a> Current Opinion in Anaesthesiology. 20, 395-399 (2007).</li><li>Kirsch, J. R., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+cerebroprotection:+experimental+aspects.">Anesthetics and cerebroprotection: experimental aspects.</a> International Anesthesiology Clinics. 34, 73-93 (1996).</li><li>Koerner, I. P., Brambrink, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+protection+by+anesthetic+agents.">Brain protection by anesthetic agents.</a> Current Opinion in Anaesthesiology. 19, 481-486 (2006).</li><li>Gelb, A. W., Bayona, N. A., Wilson, J. X., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+anesthesia+compared+to+awake+reduces+infarct+size+in+rats.">Propofol anesthesia compared to awake reduces infarct size in rats.</a> Anesthesiology. 96, 1183-1190 (2002).</li><li>Bhardwaj, A., Castro, I. A., Alkayed, N. J., Hurn, P. D., Kirsch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+choice+of+halothane+versus+propofol:+impact+on+experimental+perioperative+stroke.">Anesthetic choice of halothane versus propofol: impact on experimental perioperative stroke.</a> Stroke; A Journal Of Cerebral Circulation. 32, 1920-1925 (2001).</li><li>Barretto, R. P., Messerschmidt, B., Schnitzer, M. 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The authors declare that they have no competing financial interests.

The ability to translate experimental stroke pathophysiology from animal to human application has been plagued with failure. However, the use of animal models, such as the photothrombosis model, allows for improved understanding of stroke pathophysiology and the exploration of new therapeutic approaches to provide neuroprotection following a stroke. Small cortical strokes and microinfarctions produced by the photothrombotic model are clinically relevant to subclinical or &#8220;silent&#8221; stroke13-15, which...

The technique described permits visualization of in vivo cellular responses immediately following induction of Rose Bengal photothrombosis in an intact mouse. Rose Bengal (4,5,6,7-tetrachloro-2&#39;,4&#39;,5&#39;,7&#39;-tetraiodofluorescein) is a photosensitive dye used to induce ischemic stroke in animal models (mouse and rat). Following a bolus injection of RB through the tail vein and subsequent illumination through a thinned skull with a 564 nm laser light, a thrombus is induced causing a physiologic stroke1. The method was originally described by Rosenblum and El-Sabban in 1977, and was later adapted by Watson in the mid 1980s1,2. In brief, Rose Bengal is irradiated with green excitation light (561 nm laser in our case), which generates the production of reactive oxygen species, which subsequently activates tissue factor, an initiator of the coagulation cascade. The induction of the coagulation cascade produces an ischemic lesion that is pathologically relevant to clinical stroke3.
Stroke has a complex pathophysiology due to the interplay of many different cells types including neurons, glia, endothelium and the immune system. Choosing the best technique to study a particular cellular process requires multiple considerations. Experimental techniques fall broadly into one of three categories: in vitro, in vivo and in silico with each having advantages and disadvantages. In vitro studies have the primary disadvantage of removing cells from their natural environment and therefore may not reproduce effects seen in an intact, living animal. In vivo techniques provide for enhanced experimental replication of disease states with increased translational significance. In silico generally refers to computer modeling of a disease or cellular process, and while increasingly utilized to study potential drug interactions for example, any information gleaned must still be tested in living cells or tissue.
The ideal model of stroke in the laboratory setting should demonstrate similar pathological features to those seen in the human population. While there are common physiologic characteristics of stroke in the human population, there are also many differences depending on the type of injury experienced. Stroke in the human population occurs as small or large vessel occlusions, hemorrhagic lesions, and artery-to artery or cardio-embolisms that result in varied infarct volumes as well as differences in mechanisms related to each pathology. The advantage of utilizing animal stroke models is the generation of reproducible infarcts that mimic characteristics of human stroke. The most common animal stroke models include artery occlusion using: middle cerebral artery occlusion (embolic or endovascular filament methods) which models distal MCAO and the photothrombosis model. The advantages and disadvantages of each model have been reviewed elsewhere (see 4 and 5). Global ischemic models (MCAO), while relatively easy to perform are less relevant to human stroke than are focal stroke models. In addition, these methods are highly variable in inducing reproducible brain infarct lesions. The photothrombosis model is highly reproducible as long as the experimenter controls their experiments well, providing a clear advantage over MCAO models. However, due to microvasculature insult the model has been described to display a minimal ischemic penumbra, the area where cells are thought to be salvageable 6,7. Additionally, vasogenic edema and cytotoxic edema formation may also be induced following irradiation of the imaging area. Despite these limitations the technique has provided new insight into many physiological processes following stroke8,9,10,11.

<table border="0" cellpadding="0" cellspacing="0">
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	</colgroup>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Anesthetic</td>
			<td>MWI Veterinary Supply</td>
			<td>088-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>aCSF&#160;</td>
			<td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Bioindustrial Products</td>
			<td>500-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors 14 cm</td>
			<td>Bioindustrial Products</td>
			<td>12-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont High Tech #5 style, straight</td>
			<td>Bioindustrial Products</td>
			<td>TWZ-301.22</td>
			<td></td>
		</tr>
		<tr>
			<td>LabJack 132X80</td>
			<td>Optosigma Co</td>
			<td>123-6670</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform for Labjack 8X 8</td>
			<td>Optosigma Co</td>
			<td>145-1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispomed Labvent Rodent anesthesia machine</td>
			<td>DRE, Inc.</td>
			<td>15001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tech IV Isoflurane vaporizer</td>
			<td>DRE, Inc.</td>
			<td>34001</td>
			<td></td>
		</tr>
		<tr>
			<td>F Air Canister</td>
			<td>DRE, Inc</td>
			<td>80120</td>
			<td></td>
		</tr>
		<tr>
			<td>Bain circuit breathing tube</td>
			<td>DRE, Inc</td>
			<td>86111B</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 regulator for oxygen tanks</td>
			<td>DRE, Inc</td>
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		<tr>
			<td>Rodent induction chamber</td>
			<td>DRE, Inc</td>
			<td>15004C</td>
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		</tr>
		<tr>
			<td>Ethicon Silk 6-0; 18 in with P-3 needle</td>
			<td>Suture Express</td>
			<td>1639G</td>
			<td></td>
		</tr>
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			<td>Objective inverter Optical Adapter</td>
			<td>LSM technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foredom drill Dual voltage 110/120</td>
			<td>Foredom</td>
			<td>134.53</td>
			<td></td>
		</tr>
	</tbody>
</table>

rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

The aim of this method was to induce an ischemic stroke in animal models (mouse and rat) following a bolus injection of RB through the tail vein and subsequent illumination of a thinned skull with a 561 nm laser light. The images in Figure 4 demonstrate the progression of clot formation within a single vessel following irradiation of the area at 0, 1, 1.5 and 2 min. Prior to clot formation the entire vessel is white due to free flowing Rose Bengal. Following the induction of irradiation of the vessel the...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

14.9K Views.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

Video: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...