This work was supported by the National Institutes of Health [Grant no. R01NS069726] and the American Heart Association Grant in Aid Grant [Grant no. 13GRNT17020004] to SD.

Note: Mice (C57BL/6J, male) aged 10 &#8211; 12 weeks were used in this study. All the procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Missouri Institutional Animal Care and Use Committee (IACUC).&#160;
1. Pre-Surgery
<ol>
	<li>The day before surgery autoclave and sterilize all the surgical instruments. Wrap the instruments and autoclave at 121 oC at 15 psi for 30 min followed by 30 min of drying (121 oC, 15 psi, 30/30 cycle). Place the instruments in a clean and sterile environment until further use.</li>
	<li>Prepare fresh Rose Bengal (RB) solution (20 mg/ml in sterile saline) every time before the surgery. To completely dissolve RB, vortex the tube then followed by sonication for 5 min at 50/60 Hz with output power of 19 W. Wrap the tube in aluminum foil and protect it from light until further used during surgery.</li>
	<li>Prepare a mixture of ketamine/xylazine in sterile saline. Add 125 &#181;l of xylazine (stock concentration: 20 mg/ml) and 325 &#181;l of ketmaine (stock concentration: 100 mg/ml) and 550 &#181;l of sterile saline to make a final volume of 1 ml of anesthetic mixture.</li>
	<li>Pre-warm the homeothermic heating pad.</li>
	<li>Pre-warm the metal halide lamp (light source for FN1 epi-fluorescence microscope) for 30 min to stabilize the lamp power.</li>
	<li>Adjust the size of the illuminated region to a diameter of 1 mm using 10X objective and a reticle by adjusting the field diaphragm in the upright FN1 epi-fluorescence microscope.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Anesthetize the mouse with a dose of ketamine (130 mg/kg B. wt.) and xylazine (10 mg/kg B. wt.). Based on the cocktail in 1.3,&#160; 4 &#181;l per g of mouse B. wt. are needed. Sterilize the site of injection with an alcohol swab and administer the anesthetics through the intra-peritoneal (IP) route. Take care not to inject the anesthetics into the blood vessels or muscle as this would delay the induction and recovery of the animal.</li>
	<li>Apply artificial tear ointment to both eyes of the mouse to prevent drying and place the animal on the heating pad to prevent hypothermia.</li>
	<li>Preparation of animal
	<ol>
		<li>Check the animal for proper surgical level anesthesia by using the toe pinch response.</li>
		<li>Once the animal has reached surgical level of anesthesia, clip the hair on the dorsal surface around midline of the animal using an electric hair trimmer. Scrub the surgical site with 70% ethanol followed by betadine solution three times. Cover the site with a sterile surgical drape until next step.</li>
	</ol>
	</li>
	<li>Surgical procedure to thin the bone to expose the spinal cord
	<ol>
		<li>Place the mouse in a prone position over the homeothermic heating pad on the surgical platform (Figure 1A). Properly secure the mouse posture using a snout clamp to maintain an elongated neck region (Figure 1A, B).</li>
		<li>Make an incision (approximately 1 cm long) using surgical scissors along the dorsal mid line extending from the thoracic vertebrae T9 to T12. Move aside the skin to expose surgical area.</li>
		<li>Using a scalpel, carefully clear the muscle to expose the dorsal spines at T9 - T12 vertebrae. Stop bleeding in each step by applying gentle pressure with sterile cotton swab. Separate T10 - T12 vertebrae from the surrounding muscle and secure them using a vertebral clamp to stabilize and prevent any movement (Figure 1A, B).</li>
		<li>Using a high speed drill with bone polishing drill bit, carefully and gently thin the dorsal surface of T10 or T11 vertebra to visualize the posterior spinal vein and other small vessels on the dorsal surface of the SC (Figure 1C).</li>
		<li>To prevent thermal damage due to heat generated during the thinning procedure, apply a gentle and constant stream of normal saline along with constant suction to remove the debris.</li>
		<li>Using a scalpel carefully smooth the bone surface until the main vessel is clearly visible. Take care not to damage the spinal cord during this process.</li>
		<li>Once the blood vessel is visualized, administer RB at a dose of 30 mg/kg (body weight) through retro-orbital sinus route using an insulin syringe.</li>
		<li>Measure blood flow using a Laser Doppler flowmeter after 3 min following RB injection if necessary (Figure 2A, B). Maintain asepsis during entire procedure.</li>
	</ol>
	</li>
</ol>
3. Induction of PT
<ol>
	<li>Place the animal on an X-Y position adjustable stage over a Lab-Jack which can adjust the height. Adjust the position of the mouse so the exposed region of T11 spinal cord is directly under the 10x objective of the FN1 epi-fluorescence microscope (Figure 3A).</li>
	<li>Set the power of the light source at 12% and irradiate the T11 region with a diameter of 0.75 mm in the middle of thinned spinal cord (Note: this region includes the posterior spinal vein and other capillaries) with a green light (wavelength 540 - 580 nm, which is achieved by the filter cube in the microscope) through the 10X objective for 2 min. Take images at the beginning and end of irradiation (Figure 3B, C) and record the time of the experiment at this point.</li>
	<li>Measure blood flow again for 10 min if necessary by placing the laser Doppler probe to the same position above the spinal cord as in 2.4.8 (Figure 2A, B).</li>
	<li>After irradiation check for any hemorrhage and if none found proceed to suturing the animal. Suture the superficial fascia along with the muscles on the either side of spinal cord using an absorbable suture or&#160;4-0 size silk&#160;suture. Take care not to damage the exposed SC. Suture the skin with 4-0 silk suture. Apply Betadine or iodine to the edges of the skin after suturing.</li>
</ol>
4. Post-surgery Care
<ol>
	<li>After suturing, place the animal on the heating pad for recovery. After recovery check the animals for signs of neurological deficits by observing the movement of both hind-limbs. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Transfer the animals to the home cage. Do not return the animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Check the animals at regular intervals. In case of severe neurological deficits, provide proper care like evacuation of bladder, administration of analgesics (buprenorphine, 0.05 - 0.1 mg/kg). Check for dehydration and administer normal saline subcutaneously in severe case.&#160;Usually, buprenorphine ( 0.1 mg/kg) will be administered after suturing to relieve pain in the surgical site.</li>
	<li>If animals are not immediately euthanized after surgery, we will put high water content diet on the cage floor, so the animals can reach the food easily.</li>
</ol>
5. Transcardial Perfusion, Nissl staining and Immunostaining
<ol>
	<li>Transcardially perfuse the animal as described previously17-20.
	<ol>
		<li>Anesthetize the animal as described earlier in the protocol and transcardially perfuse with phosphate buffer saline (PBS, pH 7.4), followed by ice-cold 4% paraformaldehyde (PFA) in PBS.</li>
		<li>After perfusion, remove the spinal cord (SC) and post-fix it in 4% PFA in PBS at 4 &#176;C O/N. Transfer the fixed SC into PBS with 30% sucrose and keep it for 2 - 3 days until it sinks to the bottom of the tube.</li>
		<li>Using a cryostat cut the spinal cord into 30 &#181;m thick sections and place them serially on a gelatin-coated glass slides or in a 48-well plate with 0.01 M PBS.</li>
	</ol>
	</li>
	<li>Nissl staining: To inspect the injury caused by PT perform a Nissl staining on spinal cord sections as previously described17-20.
	<ol>
		<li>Briefly, collect every fifth spinal cord slice on the glass slides and stain with 0.25% cresyl violet. Take images of the stained sections (Figure 4).</li>
	</ol>
	</li>
	<li>Immunostaining: As described previously using a floating section method17,18,20.
	<ol>
		<li>Briefly, stain the spinal cord sections by incubating O/N at 4 &#176;C with rabbit anti-glia fibrillary acidic protein (GFAP) polyclonal antibody (1:300), rabbit anti-NeuN antibody (1:300), and rabbit anti-Iba1 antibody (1:500) followed by donkey anti rabbit Alexa 568-conjugated IgG (1:400) secondary antibodies for 4 h at RT. Take images with a fluorescent microscope (Figure 5).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cadotte, D. W., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+a+systematic+review+of+current+treatment+options.">Spinal cord injury: a systematic review of current treatment options.</a> Clin Orthop Relat Res. 469 (3), 732-741 (2011).</li><li>Cheriyan, T., 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=Spinal+cord+injury+models:+a+review.">Spinal cord injury models: a review.</a> Spinal Cord. 52 (8), 588-595 (2014).</li><li>Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+injury+mechanisms+in+acute+spinal+cord+injury.">Secondary injury mechanisms in acute spinal cord injury.</a> J Emerg Med. 11, 13-22 (1993).</li><li>Crowe, M. J., Bresnahan, J. C., Shuman, S. L., Masters, J. N., Beattie, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+delayed+degeneration+after+spinal+cord+injury+in+rats+and+monkeys.">Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys.</a> Nat Med. 3 (1), 73-76 (1997).</li><li>Soubeyrand, M., 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=Effect+of+norepinephrine+on+spinal+cord+blood+flow+and+parenchymal+hemorrhage+size+in+acute-phase+experimental+spinal+cord+injury.">Effect of norepinephrine on spinal cord blood flow and parenchymal hemorrhage size in acute-phase experimental spinal cord injury.</a> Eur Spine J. 23 (3), 658-665 (2014).</li><li>Soubeyrand, M., 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=Real-time+and+spatial+quantification+using+contrast-enhanced+ultrasonography+of+spinal+cord+perfusion+during+experimental+spinal+cord+injury.">Real-time and spatial quantification using contrast-enhanced ultrasonography of spinal cord perfusion during experimental spinal cord injury.</a> Spine (Phila Pa 1976). 37 (22), E1376-E1382 (2012).</li><li>Mautes, A. E., Weinzierl, M. R., Donovan, F., Noble, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+events+after+spinal+cord+injury:+contribution+to+secondary+pathogenesis).">Vascular events after spinal cord injury: contribution to secondary pathogenesis).</a> Phys Ther. 80 (7), 673-687 (2000).</li><li>Liu, X. Z., 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=Neuronal+and+glial+apoptosis+after+traumatic+spinal+cord+injury.">Neuronal and glial apoptosis after traumatic spinal cord injury.</a> J Neurosci. 17 (14), 5395-5406 (1997).</li><li>Liu, L., 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=An+experimental+study+of+cell+apoptosis+and+correlative+gene+expression+after+tractive+spinal+cord+injury+in+rats.">An experimental study of cell apoptosis and correlative gene expression after tractive spinal cord injury in rats.</a> Zhonghua Wai Ke Za Zhi. 42 (23), 1434-1437 (2004).</li><li>Hirose, K., 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=Activated+protein+C+reduces+the+ischemia/reperfusion-induced+spinal+cord+injury+in+rats+by+inhibiting+neutrophil+activation.">Activated protein C reduces the ischemia/reperfusion-induced spinal cord injury in rats by inhibiting neutrophil activation.</a> Ann Surg. 232 (2), 272-280 (2000).</li><li>Oyinbo, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+injury+mechanisms+in+traumatic+spinal+cord+injury:+a+nugget+of+this+multiply+cascade.">Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade.</a> Acta Neurobiol Exp (Wars). 71 (2), 281-299 (2011).</li><li>Guerit, J. M., Dion, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-of-the-art+of+neuromonitoring+for+prevention+of+immediate+and+delayed+paraplegia+in+thoracic+and+thoracoabdominal+aorta+surgery).">State-of-the-art of neuromonitoring for prevention of immediate and delayed paraplegia in thoracic and thoracoabdominal aorta surgery).</a> Ann Thorac Surg. 74 (5), S1867-S1869 (2002).</li><li>Schepens, M. A., Heijmen, R. H., Ranschaert, W., Sonker, U., Morshuis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thoracoabdominal+aortic+aneurysm+repair:+results+of+conventional+open+surgery.">Thoracoabdominal aortic aneurysm repair: results of conventional open surgery.</a> Eur J Vasc Endovasc Surg. 37 (6), 640-645 (2009).</li><li>Braeuninger, S., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia:+procedural+pitfalls+and+translational+problems.">Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems.</a> Exp Transl Stroke Med. 1, 8 (2009).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2 (3), 396-409 (2005).</li><li>Dietrich, W. D., Ginsberg, M. D., Busto, R., Watson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cortical+infarction+in+the+rat.+1.+Time+course+of+hemodynamic+consequences.">Photochemically induced cortical infarction in the rat. 1. Time course of hemodynamic consequences.</a> J Cereb Blood Flow Metab. 6 (2), 184-194 (1986).</li><li>Zhang, 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=Neuronal+protective+role+of+PBEF+in+a+mouse+model+of+cerebral+ischemia.">Neuronal protective role of PBEF in a mouse model of cerebral ischemia.</a> J Cereb Blood Flow Metab. 30 (12), 1962-1971 (2010).</li><li>Li, H., Zhang, N., Sun, G., 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=Inhibition+of+the+group+I+mGluRs+reduces+acute+brain+damage+and+improves+long-term+histological+outcomes+after+photothrombosis-induced+ischaemia.">Inhibition of the group I mGluRs reduces acute brain damage and improves long-term histological outcomes after photothrombosis-induced ischaemia.</a> ASN Neuro. 5 (3), 195-207 (2013).</li><li>Li, H., 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=Histological,+cellular+and+behavioral+assessments+of+stroke+outcomes+after+photothrombosis-induced+ischemia+in+adult+mice.">Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice.</a> BMC Neurosci. 15, 58 (2014).</li><li>Wang, T., Cui, W., 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 (1), 33-42 (2010).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J Neurosci Methods. 117 (1), 43-49 (2002).</li><li>Boquillon, M., Boquillon, J. P., Bralet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced,+graded+cerebral+infarction+in+the+mouse+by+laser+irradiation+evolution+of+brain+edema.">Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema.</a> J Pharmacol Toxicol Methods. 27 (1), 1-6 (1992).</li><li>Kim, G. W., Lewen, A., Copin, J., Watson, B. D., Chan, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytosolic+antioxidant,+copper/zinc+superoxide+dismutase,+attenuates+blood-brain+barrier+disruption+and+oxidative+cellular+injury+after+photothrombotic+cortical+ischemia+in+mice.">The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood-brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice.</a> Neuroscience. 105 (4), 1007-1018 (2001).</li><li>Schmidt, A., 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=Photochemically+induced+ischemic+stroke+in+rats.">Photochemically induced ischemic stroke in rats.</a> Exp Transl Stroke Med. 4 (1), 13 (2012).</li><li>Lang-Lazdunski, L., 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=Spinal+cord+ischemia.+Development+of+a+model+in+the+mouse.">Spinal cord ischemia. Development of a model in the mouse.</a> Stroke. 31 (1), 208-213 (2000).</li><li>Wang, Z., 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=Development+of+a+simplified+spinal+cord+ischemia+model+in+mice.">Development of a simplified spinal cord ischemia model in mice.</a> J Neurosci Methods. 189 (2), 246-251 (2010).</li><li>Labat-gest, V., Tomasi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+ischemia:+a+minimally+invasive+and+reproducible+photochemical+cortical+lesion+model+for+mouse+stroke+studies.">Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.</a> J Vis Exp. (76), (2013).</li><li>Lu, H., 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=Induction+and+imaging+of+photothrombotic+stroke+in+conscious+and+freely+moving+rats.">Induction and imaging of photothrombotic stroke in conscious and freely moving rats.</a> J Biomed Opt. 19 (9), 96013 (2014).</li><li>Seto, A., 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=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Front Neuroenergetics. 6 (1), (2014).</li><li>Bell, M. T., 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=Toll-like+receptor+4-dependent+microglial+activation+mediates+spinal+cord+ischemia-reperfusion+injury.">Toll-like receptor 4-dependent microglial activation mediates spinal cord ischemia-reperfusion injury.</a> Circulation. 128 (11 Suppl 1), S152-S156 (2013).</li><li>Smith, P. D., 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=The+evolution+of+chemokine+release+supports+a+bimodal+mechanism+of+spinal+cord+ischemia+and+reperfusion+injury.">The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury.</a> Circulation. 126 (11 Suppl 1), S110-S117 (2012).</li><li>Jia, Z., 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=Oxidative+stress+in+spinal+cord+injury+and+antioxidant-based+intervention.">Oxidative stress in spinal cord injury and antioxidant-based intervention.</a> Spinal Cord. 50 (4), 264-274 (2012).</li></ol>

The authors have nothing to disclose.

In this study, we described a photothrombotic model of SC ischemia. Due to advances in genetic engineering there has been a surge in commercially available transgenic mice which has made it possible to study the impact of specific genes involved in the ischemic pathophysiology in the SC. The aim of the study was to develop a reproducible mouse model of spinal cord ischemia. Here we adapted a cortical PT model to induce SCI in mice. Following surgery the posterior spinal vein and capillaries on the dorsal...

Traumatic spinal cord injury (SCI) is a devastating clinical condition affecting the sensorimotor and autonomic functions of the SC. Patients surviving SCI are often left with debilitating paraplegia that significantly affects their daily activities and quality of life1. Experimental SCI models have been an indispensable tool in the scientific investigation to understand the pathophysiology of SCI and associated neural repair processes. These models have also been used to test the preclinical efficacy of various experimental neuroprotective interventions that are aimed at functional recovery. Currently, majority of the SCI models in practice employ the use of physical blunt force to mechanically disrupt and injure the SC. These methods include contusion, compression, dislocation and transection of the SC2. It has been suggested that after the primary mechanical insult a secondary injury in the form of ischemia sets in in the injured SC3,4. The etiology of secondary ischemia includes extensive tissue degeneration, parenchymal hemorrhage and sometimes by obstruction of blood vessels by tissue edema5-7. As a result of the secondary injury the integrity of SC is further affected, neurons and glial cells are severely impaired in function and viability and undergo apoptosis which leads to infarct growth during the chronic stage of injury, analogous to the growth of ischemic penumbra following stroke8,9. Several mechanisms like excitotoxicity, free radical production, and inflammation have been reported to be responsible for the ischemic cell death following SCI10,11. In addition, SC ischemia is a serious complication of thoraco-abdominal aortic aneurysm repair surgeries which often lead to paraplegia in the patients12,13. In spite of such high clinical impact very few models of spinal cord ischemia with high reproducibility are currently available.
Photothrombosis (PT) is a commonly used method for the induction of focal ischemia in the brain14-20. The technique is fairly non-invasive, highly reproducible and produces a precise focal ischemic lesion in the exposed area of the brain17-21. This is achieved by systemic administration of photoactive dyes like Rose Bengal (RB)16-20,22 or erythrosine B23 followed by localized irradiation of blood vessels with proper light source. Photoactivation of the dye causes the generation of free radicals which disrupt the integrity of the smooth vascular endothelium, and cause the platelets to accumulate, which subsequently forms a thrombus. The obstruction of blood flow by the thrombus results in an infarct in the region supplied by the vessel24. Due to ease of control on the intensity and duration of irradiation this procedure yields a highly uniform and reproducible infarct. Furthermore, this method can be employed to induce an infarct at various anatomical locations enabling spatial (e.g., grey matter vs. white matter) understanding of the effect of ischemia.
The aim of the current study is to develop an easy and highly reproducible model of SC ischemia in mice. We described the procedure of a PT model of SC ischemia in mice. Results from histology and immunostaining demonstrated that PT can effectively induce SC infarction, neuronal loss and reactive gliosis.

<table><tbody><tr><td>Rose Bengal</td><td>Sigma-Aldrich</td><td>330000</td><td>20 mg/ml in sterile saline</td></tr><tr><td>C57BL/6J</td><td>Jackson lab</td><td>664</td><td>22 - 25 g</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-996-06</td><td>100 mg/ml</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-234-11</td><td>100 mg/ml</td></tr><tr><td>Betadine solution</td><td>Purdue</td><td>NDC-67618-150-01</td><td>10% povidone iodine topical solution</td></tr><tr><td>Normal saline</td><td>Abott Laboratories</td><td>04930-04-10</td><td>For diluting RB, anaesthesia and for preventing tissue from drying</td></tr><tr><td>Artificial tears ointment&amp;nbsp;</td><td>Rugby</td><td>NDC-0536-6550-91</td><td>83% white petrolatum</td></tr><tr><td>Ethanol</td><td>Decon labs.Inc</td><td>2716</td><td>70% ethanol for disinfection</td></tr><tr><td>Metal halide lamp</td><td>EXFO, Canada</td><td>X-Cite 120 PC&amp;nbsp;</td><td>Set power at 12%</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>for minor dissection</td></tr><tr><td>Scissors (angled to side)</td><td>Fine Science Tool</td><td>14063-011</td><td>No. 3 handle</td></tr><tr><td>Standard scalpel</td><td>Fine Science Tool</td><td>10003-12</td><td>for removing muscle</td></tr><tr><td>Scalpel blade</td><td>Feather</td><td>2976</td><td>No. 10</td></tr><tr><td>Forceps (curved)</td><td>Fine Science Tool</td><td>11150-10</td><td>for holding tissue</td></tr><tr><td>Forceps (straight)</td><td>Fine Science Tool</td><td>11151-10</td><td>for holding tissue</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Science Tool</td><td>12002-12</td><td>for suturing</td></tr><tr><td>Tissue adhesive glue</td><td>3M Vetbond</td><td>1469SB</td><td>to adhere to edges of the cut skin</td></tr><tr><td>Monofilament polypropylene&amp;nbsp;</td><td>USSC Sutures</td><td>VP-521</td><td>Size = 4-0 (for fascia)</td></tr><tr><td>Perma-hand silk</td><td>Ethicon</td><td>683G</td><td>Size = 4-0 (for skin)</td></tr><tr><td>Micro drill</td><td>Roboz Surgical Instrument Co. Inc.</td><td>RS-6300</td><td>with bone polishing drill bit</td></tr><tr><td>Laser doppler flowmeter</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td>for monitoring change in blood flow</td></tr><tr><td>Heating pad</td><td>Fine Science Tool</td><td>21052-00</td><td>to prevent hypothermia</td></tr><tr><td>Lab-Jack</td><td>Fisher scientific&amp;nbsp;</td><td>14-673-50</td><td>4 x 4 in plate to adjust the height of the animal</td></tr><tr><td>X-Y gliding stage&amp;nbsp;</td><td>Amscope</td><td>GT100</td><td>for positioning the animal under microscope&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

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.

The aim of this study was to produce spinal cord ischemia in mice using a PT model. After the desired region of bone above the spinal cord (T10 - T12) was thinned, Rose Bengal was injected through retro-orbital sinus route, and ischemia was induced by PT. Figure 1A, B show the mouse positioned in a custom-made surgical platform during surgery. The mouse was held in place by a snout clamp and two adjustable vertebrate clamps to stabilize the spinal cord. Figure 1C show a thinned window ab...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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

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) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

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13.3K Views.  University of Missouri. 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. 

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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Neuroscience

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www.jove.com/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Modelowanie udaru fotozakrzepowego w modelu mysim: technika oświetlenia laserowego przez czaszkę myszy po podaniu barwnika światłoczułego w celu wywołania fotozakrzepicy

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

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Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app.jove.com/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.