This work was supported by research grants from the National Institutes of Health (EY022383 and EY022683; EJD) and Core grant (P30EY001765), Imaging and Microscopy Core Module.

Ethics Statement: All procedures were performed in accordance with the guidelines set forth by the Johns Hopkins University Institutional Animal Care and Use Committee.
Note: Mice used during filming are C57BL/6 mice from Jackson, although other rodent strains or species may also be used. When using other strains or species, be aware that anesthesia dosages and injury timeline may vary. It is important to adapt I/R conditions to accommodate strain, species, and experimental variations.
1. Prepare the Anesthesia Cocktail
<ol>
	<li>Combine 1.25 ml Ketamine, 0.625 ml Xylazine, 0.375 ml Acepromazine, and 22.75 ml phosphate buffered saline in a 50 ml centrifuge tube. 
	NOTE: For the remainder of the manuscript, this solution will be referred to as the cocktail.</li>
	<li>Filter the cocktail into a new sterile 50 ml centrifuge tube using a 60 ml syringe and a 0.20 &#181;m filter. Label and date this new tube.</li>
	<li>Fully wrap the cocktail tube in aluminum foil to prevent light-induced degradation of the anesthetic.&#160; 
	NOTE: The cocktail may be stored at room temperature and reused until the expiration date of its earliest-expiring ingredient.</li>
</ol>
2. Prepare the Anesthesia Booster
<ol>
	<li>In a 50 ml centrifuge tube, combine 4 ml Ketamine and 16 ml phosphate buffered saline. 
	NOTE: This solution will henceforth be referred to as the booster.</li>
	<li>Filter the booster into a new sterile 50 ml centrifuge tube using a 60 ml syringe and a 0.20 &#181;m filter. Label and date this new tube.&#160; 
	NOTE: The booster may be stored at room temperature and reused until the expiration date of its earliest-expiring ingredient.</li>
</ol>
3. Prepare the Surgical Suite
<ol>
	<li>Set the room temperature between 18 &#176;C and 21 &#176;C.</li>
	<li>Turn on the surgery table, and adjust its surface heat to the highest temperature.</li>
	<li>Cover all work surfaces with surgical underpads.</li>
	<li>Arrange an empty cage or other container on the surgical table to warm.</li>
	<li>Arrange a scale and an ear tagger on the worktop.</li>
</ol>
4. Prepare the Balanced Salt Solution with Heparin Sodium
<ol>
	<li>Add 0.5 ml of heparin sodium to a 500 ml IV bottle of balanced salt solution (HBSS).</li>
	<li>Insert the sharp end of the primary set prepierced Y-site tubing into the bottle of 0.1% heparin sodium.</li>
</ol>
5. Set up the IV Pole
<ol>
	<li>Hang the bottle of 0.1% heparin sodium from the IV pole extension, and snap open the air filter cap on the primary set prepierced Y-site tubing.</li>
	<li>Elevate the 0.1% heparin sodium bottle to 163 cm (120 mm Hg). Measure the height from the tabletop to the peak of the sodium heparin drip.</li>
	<li>Remove any air bubbles in the IV tubing by manually flicking the primary set prepierced Y-site tubing.</li>
</ol>
6. Set up the Sodium Heparin Drip
<ol>
	<li>Connect the primary set prepierced Y-site tubing to the five-valve manifold.</li>
	<li>Connect 30-gauge &#189; inch needles to the five-port manifold using Luer male to Luer male tube fittings.</li>
	<li>Insert each of the 30-gauge &#189; inch needles into its own 10-inch segment of 30-gauge tubing.</li>
	<li>Using hemostats, break the needle tips from new 30-gauge &#189; inch needles and insert their blunt ends into the 30-gauge tubing.&#160;Sterility or disinfection of the needle tips will be necessary for Step 8.3.</li>
	<li>Using tape, arrange the 30-gauge tubes with needles such that the tubes connected to the inner ports are positioned on top of the tubes connected to the outer ports. This arrangement will prevent tangling of tubing during anterior chamber cannulation.</li>
	<li>Turn on the 0.1% heparin sodium flow to the five-valve manifold and to each individual port.
	<ol>
		<li>Ensure that the 0.1% heparin sodium is flowing strongly for each port. If a port is flowing weakly, replace the port or clear it with air from a sterile syringe.</li>
		<li>Allow the 0.1% heparin sodium to flow for 2 - 3 min to remove air bubbles from the 30-gauge tubes and 5-valve manifold.</li>
	</ol>
	</li>
	<li>Turn off all the ports on the 5-valve manifold.</li>
</ol>
7. Prepare the Mice for Surgery
<ol>
	<li>Bring the mice to the surgical suite. Supply water bottles for each cage in order to prevent animal dehydration during surgery.</li>
	<li>Record the weight of each mouse.</li>
	<li>Inject each mouse intraperitoneally&#160;with 0.02 ml cocktail per gram bodyweight.</li>
	<li>Tag and record the number of each mouse.</li>
	<li>Place all the mice into the empty container on the surgery table. Allow 5-10 min for all the mice to achieve deep anesthetization as confirmed by absence of the pedal withdrawal response to toe pinch.</li>
	<li>For each mouse, administer one drop of Tropicamide into each eye for pupil dilation and short-term lubrication.</li>
	<li>For each mouse, administer one drop of Proparacaine into each eye for local anesthesia and short-term lubrication.</li>
	<li>Arrange the mice in the order of anesthetization so that the first mouse to be anesthetized will be the first mouse to be cannulated.</li>
	<li>Allow approximately 2 min for the eye drops to take effect.</li>
	<li>Prepare straight 4-inch pieces of tape by pulling tightly on the tape. Set aside one piece of tape for each mouse.&#160;&#160;&#160;&#160; 
	NOTE: Failing to pull tightly on the tape will result in curling of the tape.</li>
</ol>
8. Cannulate the Anterior Chamber
<ol>
	<li>Arrange the first mouse under the surgical microscope, and focus the microscope on the preferred cornea.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Cannulation may be performed on either eye. Right-hand dominant surgeons may find it easiest to cannulate the left eye, while left-hand dominant surgeons may prefer the right.</li>
	<li>Turn on the first 0.1% heparin sodium port on the five-valve manifold.</li>
	<li>Under the surgical microscope, use a pair of forceps to gently proptose the eye. Insert the 30-gauge cannula needle into the anterior chamber approximately halfway between the zonule fibers and the apex of the cornea.
	<ol>
		<li>Take care to avoid scratching or puncturing the iris, lens, or inner corneal surface.</li>
		<li>Avoid penetrating the cornea a second time.</li>
		<li>Using a gentle twisting motion to overcome friction between the cannula and the cornea, insert the cannula deeply in the anterior chamber.</li>
	</ol>
	</li>
	<li>Use a strip of tape to secure the 30-gauge tubing to the table. To minimize movement of the inserted cannula, press the 30-gauge tubing against the tabletop while reaching for the tape.</li>
	<li>Record the start time of the surgery.</li>
	<li>Using the microscope, verify that no leakage is apparent. If leakage is present, the movement of fluid will be visible near the eye.</li>
	<li>Visually confirm ocular distention by observing that the I/R eye is larger than the contralateral eye. Together, the absence of leakage and the presence of ocular distention demonstrate a successful elevation of intraocular pressure.</li>
	<li>Apply hypromellose to both eyes. Hypromellose serves to lubricate the cornea and seal microleaks. Reapply hypromellose as needed (approximately every 30 min) for sustained lubrication.</li>
	<li>Repeat Step 8 until all animals have been cannulated.</li>
</ol>
9. Monitor Anesthesia
<ol>
	<li>Use the toe pinch, visual observation of the whiskers, or visual observation of the tail to verify that each mouse remains anesthetized. Should a mouse demonstrate a pedal withdrawal response, whisker twitching, or tail movement, proceed immediately to Step 9.2.</li>
	<li>If a mouse begins to awaken during surgery, lift the tail to inject 0.05 ml booster intraperitoneally into the lower abdomen from behind the mouse. Allow 1 - 2 min for the booster to take effect.</li>
	<li>Should a mouse&#160;require additional sedation, repeat Step 9 as necessary.</li>
</ol>
10. Remove the Cannula&#160;from the Anterior Chamber
<ol>
	<li>For each animal, after 90 min have elapsed, gently pull the cannula from the anterior chamber.</li>
	<li>Untape the mouse from the surgical table, taking care not to disturb the cannula tubing of adjacent animals.</li>
	<li>Lubricate both eyes with lubricating jelly.</li>
	<li>As subsequent cannulae are removed, arrange the mice in the empty container on the surgical table to recover from surgery. Do not turn off the heat of the surgical table.</li>
	<li>Allow, at minimum, 2 - 3 hr for recovery on the heated surgical table.&#160;Observe the mice frequently until they have fully recovered from the anesthesia.</li>
</ol>
11. Clean the Equipment
<ol>
	<li>Disinfect&#160;the cannulae using alcohol wipes.&#160; 
	NOTE: Other methods for disinfection or sterilization, such as autoclaving, may be substituted.</li>
	<li>Expel 0.1% heparin sodium from the 5-valve manifold, 30-gauge needles, 30-gauge tubing, and cannulae using a 60 ml syringe filled with air.</li>
	<li>Rinse the 5-valve manifold, 30-gauge needles, 30-gauge tubing, and cannulae using a 60 ml syringe filled with distilled water.</li>
	<li>Expel distilled water from the 5-port manifold, 30-gauge needles, 30-gauge tubing, and cannulae using a 60 ml syringe filled with air.</li>
	<li>After disinfecting&#160;the cannulae and rinsing the tubing apparatus, store this equipment for reuse.</li>
	<li>Store, discard, or turn off all other equipment. Leave the heat of the surgical table on.</li>
</ol>
12. Return All Mice to Their Home Cages
<ol>
	<li>After the mice awaken from surgery (after 2 - 3 hr), return each animal to its home cage. Provide gel food for each cage.&#160;Return all cages to their designated rooms.</li>
	<li>Turn off the heat of the surgical table. Discard all waste, and wipe down the surgical suite.</li>
</ol>
13. Perform Retinal Assessment
<ol>
	<li>As appropriate, collect retinas for histological analysis or dark adapt mice for electroretinogram recording.</li>
</ol>

<ol>
	<li>Osborne, N. N., 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=Retinal+ischemia:+mechanisms+of+damage+and+potential+therapeutic+strategies.">Retinal ischemia: mechanisms of damage and potential therapeutic strategies.</a> Prog Retin Eye Res. 23 (1), 91-147 (2004).</li><li>Bonne, C., Muller, A., Villain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+retinal+ischemia.">Free radicals in retinal ischemia.</a> Gen Pharmacol. 30 (3), 275-280 (1998).</li><li>Smith, G. G., Baird, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+time+of+retinal+cells+when+deprived+of+their+blood+supply+by+increased+intraocular+pressure.">Survival time of retinal cells when deprived of their blood supply by increased intraocular pressure.</a> Am J Ophthalmol. 35 (5:2), 133-136 (1952).</li><li>Buchi, E. R., Suivaizdis, I., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+retinal+ischemia+in+rats:+an+experimental+model+for+quantitative+study.">Pressure-induced retinal ischemia in rats: an experimental model for quantitative study.</a> Ophthalmologica. 203 (3), 138-147 (1991).</li><li>Block, F., Schwarz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+b-wave+of+the+electroretinogram+as+an+index+of+retinal+ischemia.">The b-wave of the electroretinogram as an index of retinal ischemia.</a> Gen Pharmacol. 30 (3), 281-287 (1998).</li><li>Katai, N., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+retinal+neuronal+death+by+ischemia-reperfusion+is+executed+by+two+distinct+caspase+family+proteases.">Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases.</a> Invest Ophthalmol Vis Sci. 40 (11), 2697-2705 (1999).</li><li>Toriu, N., 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=Lomerizine,+a+Ca2++channel+blocker,+reduces+glutamate-induced+neurotoxicity+and+ischemia/reperfusion+damage+in+rat+retina.">Lomerizine, a Ca2+ channel blocker, reduces glutamate-induced neurotoxicity and ischemia/reperfusion damage in rat retina.</a> Exp Eye Res. 70 (4), 475-484 (2000).</li><li>Kawai, S. I., 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=Modeling+of+risk+factors+for+the+degeneration+of+retinal+ganglion+cells+after+ischemia/reperfusion+in+rats:+effects+of+age,+caloric+restriction,+diabetes,+pigmentation,+and+glaucoma.">Modeling of risk factors for the degeneration of retinal ganglion cells after ischemia/reperfusion in rats: effects of age, caloric restriction, diabetes, pigmentation, and glaucoma.</a> FASEB J. 15 (7), 1285-1287 (2001).</li><li>Chidlow, G., Schmidt, K. G., Wood, J. P., Melena, J., Osborne, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-lipoic+acid+protects+the+retina+against+ischemia-reperfusion.">Alpha-lipoic acid protects the retina against ischemia-reperfusion.</a> Neuropharmacology. 43 (6), 1015-1025 (2002).</li><li>Fei, F., 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=Upregulation+of+Homer1a+Promoted+Retinal+Ganglion+Cell+Survival+After+Retinal+Ischemia+and+Reperfusion+via+Interacting+with+Erk+Pathway.">Upregulation of Homer1a Promoted Retinal Ganglion Cell Survival After Retinal Ischemia and Reperfusion via Interacting with Erk Pathway.</a> Cell Mol Neurobiol. , (2015).</li><li>Xu, 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=Neuroprotective+role+of+Nrf2+for+retinal+ganglion+cells+in+ischemia-reperfusion.">Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.</a> J Neurochem. 133 (2), 233-241 (2015).</li><li>Kim, B. J., Braun, T. A., Wordinger, R. J., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+morphological+changes+and+impaired+retinal+function+associated+with+temporal+regulation+of+gene+expression+after+retinal+ischemia/reperfusion+injury+in+mice.">Progressive morphological changes and impaired retinal function associated with temporal regulation of gene expression after retinal ischemia/reperfusion injury in mice.</a> Mol Neurodegener. 8 (21), (2013).</li><li>Portillo, J. 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=CD40+mediates+retinal+inflammation+and+neurovascular+degeneration.">CD40 mediates retinal inflammation and neurovascular degeneration.</a> J Immunol. 181 (12), 8719-8726 (2008).</li><li>Wei, 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=Nrf2+has+a+protective+role+against+neuronal+and+capillary+degeneration+in+retinal+ischemia-reperfusion+injury.">Nrf2 has a protective role against neuronal and capillary degeneration in retinal ischemia-reperfusion injury.</a> Free Radic Biol Med. 51 (1), 216-224 (2011).</li><li>Abcouwer, S. F., 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=Effects+of+ischemic+preconditioning+and+bevacizumab+on+apoptosis+and+vascular+permeability+following+retinal+ischemia-reperfusion+injury.">Effects of ischemic preconditioning and bevacizumab on apoptosis and vascular permeability following retinal ischemia-reperfusion injury.</a> Invest Ophthalmol Vis Sci. 51 (11), 5920-5933 (2010).</li><li>Abcouwer, S. F., 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=Minocycline+prevents+retinal+inflammation+and+vascular+permeability+following+ischemia-reperfusion+injury.">Minocycline prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury.</a> J Neuroinflammation. 10 (149), (2013).</li><li>Zheng, L., Gong, B., Hatala, D. A., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ischemia+and+reperfusion+causes+capillary+degeneration:+similarities+to+diabetes.">Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes.</a> Invest Ophthalmol Vis Sci. 48 (1), 361-367 (2007).</li><li>Weyker, P. D., Webb, C. A., Kiamanesh, D., Flynn, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+ischemia+reperfusion+injury:+a+bench-to-bedside+review.">Lung ischemia reperfusion injury: a bench-to-bedside review.</a> Semin Cardiothorac Vasc Anesth. 17 (1), 28-43 (2013).</li><li>Raedschelders, K., Ansley, D. M., Chen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+and+molecular+origin+of+reactive+oxygen+species+generation+during+myocardial+ischemia+and+reperfusion.">The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion.</a> Pharmacol Ther. 133 (2), 230-255 (2012).</li><li>Di, 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=MicroRNAs+expression+and+function+in+cerebral+ischemia+reperfusion+injury.">MicroRNAs expression and function in cerebral ischemia reperfusion injury.</a> J Mol Neurosci. 53 (2), 242-250 (2014).</li><li>Saidi, R. F., Kenari, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+ischemia/reperfusion+injury:+an+overview.">Liver ischemia/reperfusion injury: an overview.</a> J Invest Surg. 27 (6), 366-379 (2014).</li><li>Malek, M., Nematbakhsh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+ischemia/reperfusion+injury;+from+pathophysiology+to+treatment.">Renal ischemia/reperfusion injury; from pathophysiology to treatment.</a> J Renal Inj Prev. 4 (2), 20-27 (2015).</li><li>Mallick, I. H., Yang, W., Winslet, M. C., Seifalian, 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=Ischemia-reperfusion+injury+of+the+intestine+and+protective+strategies+against+injury.">Ischemia-reperfusion injury of the intestine and protective strategies against injury.</a> Dig Dis Sci. 49 (9), 1359-1377 (2004).</li><li>Hesketh, E. E., 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=Renal+ischaemia+reperfusion+injury:+a+mouse+model+of+injury+and+regeneration.">Renal ischaemia reperfusion injury: a mouse model of injury and regeneration.</a> J Vis Exp. (88), (2014).</li><li>Stefansson, E., Wilson, C. A., Schoen, T., Kuwabara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+ischemia+induces+cell+mitosis+in+the+adult+rat+retina.">Experimental ischemia induces cell mitosis in the adult rat retina.</a> Invest Ophthalmol Vis Sci. 29 (7), 1050-1055 (1988).</li><li>Honjo, 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=Statin+inhibits+leukocyte-endothelial+interaction+and+prevents+neuronal+death+induced+by+ischemia-reperfusion+injury+in+the+rat+retina.">Statin inhibits leukocyte-endothelial interaction and prevents neuronal death induced by ischemia-reperfusion injury in the rat retina.</a> Arch Ophthalmol. 120 (12), 1707-1713 (2002).</li><li>Otori, 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=Expression+of+c-fos+and+c-jun+mRNA+following+transient+retinal+ischemia:+an+approach+using+ligation+of+the+retinal+central+artery+in+the+rat.">Expression of c-fos and c-jun mRNA following transient retinal ischemia: an approach using ligation of the retinal central artery in the rat.</a> Surv Ophthalmol. 42, 96-104 (1997).</li><li>Liu, J., 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=Epac2-deficiency+leads+to+more+severe+retinal+swelling,+glial+reactivity+and+oxidative+stress+in+transient+middle+cerebral+artery+occlusion+induced+ischemic+retinopathy.">Epac2-deficiency leads to more severe retinal swelling, glial reactivity and oxidative stress in transient middle cerebral artery occlusion induced ischemic retinopathy.</a> Sci China Life Sci. 58 (6), 521-530 (2015).</li><li>Zhang, Y., Zhang, Z., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simvastatin+inhibits+ischemia/reperfusion+injury-induced+apoptosis+of+retinal+cells+via+downregulation+of+the+tumor+necrosis+factor-alpha/nuclear+factor-kappaB+pathway.">Simvastatin inhibits ischemia/reperfusion injury-induced apoptosis of retinal cells via downregulation of the tumor necrosis factor-alpha/nuclear factor-kappaB pathway.</a> Int J Mol Med. , (2015).</li><li>Li, B., Pang, I. H., Barnes, G., McLaughlin, M., Holt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+and+device+to+induce+transient+retinal+ischemia+in+the+rat.">A new method and device to induce transient retinal ischemia in the rat.</a> Curr Eye Res. 24 (6), 458-464 (2002).</li></ol>

The authors have no disclosures.

Retinal I/R&#160;injury by elevated IOP has proven its utility in modeling cellular damage and dysfunction, particularly neurodegeneration, in the rodent retinal neurovascular unit. This procedure provides a robust control tissue and is easily accessible in terms of technical sophistication. It has been noted in this and other I/R injury models that increasing the pressure and duration of ischemia may increase injury severity24. For this reason, some practitioners have elected to use ischemic pressures and durations differing from those presented here4,6-10,12. Therefore retinal I/R injury by elevated IOP offers an advantage over alternative retinal I/R techniques in that it allows one to adjust surgical parameters to accommodate one&#39;s particular experimental goals.
Nonetheless, alternative techniques have been employed for inducing retinal I/R injury in rodents. Ligation of the optic nerve bundle25,26 or the central retinal artery alone27 has been used to arrest retinal blood flow temporarily. Similar strategies for systemic conditions have required ligation of the cerebral artery28 or the cephalic artery29 to reduce blood flow without fully obstructing it. One rarer methodology involves circumferentially compressing the retinal globe using a thread that is weighted on both ends30. While such strategies have successfully contributed to an understanding of hypoxia-induced neurovascular changes in the retina, the technique described herein offers several advantages over these alternatives. Necessitating minimal non-retinal damage only to the cornea, I/R by elevated IOP provides a more specifically targeted retinal injury than is afforded by ligation techniques and so may be more useful for researchers interested in retina-specific disease. Also, elevated IOP methods are more tractable than ligation or compression models, such that the IOP method allows quick attainment of retinal ischemia as well as subsequent&#160;reperfusion. Finally, elevated IOP protocols require minimal surgical and technological sophistication and so may be more widely accessible than their alternatives.
Retinal I/R injury by elevated IOP is not without its challenges. Cannulation of the anterior chamber requires manual dexterity, and care must be taken to preserve the integrity of the iris, lens, and cornea. Caution is also advised after insertion of the cannula, as the cannula may be pulled from the anterior chamber while the 30-gauge tubing is secured with tape.
Other critical processes in this protocol include maintaining a warm body temperature for anesthetized animals, administering booster anesthesia in a timely manner, and sustaining lubrication of the cornea using hypromellose. It is also important to note mouse stress or sickness behaviors (for example, lack of grooming, hunching, etc.) prior to surgery, as these extra-surgical variables may influence drug potency and mortality. By attending to these issues, one may achieve a highly regulated and reproducible model for retinal I/R injury.
It should also be acknowledged that retinal I/R injury by elevated IOP is only a model, and caution is advised when extrapolating findings to specific diseases, particularly chronic conditions. While differing from these diseases in time frame and etiological origin, however, retinal I/R injury by elevated IOP may nonetheless provide a sound platform for evaluating mechanisms of retinal degeneration and recovery.
The retina is composed of multiform cells and signaling processes, and a comprehensive story of retinal dysregulation remains to be elucidated. The current literature demonstrates the utility of retinal I/R injury by elevated IOP not only in examining retinal degenerative processes6-8,10,12, but also in identifying targets for therapeutic prevention and intervention7,9,11,12,14. In addition, there is growing evidence to support the utility of retinal I/R injury by elevated IOP in non-neuronal endpoints such as retinal inflammation, vascular degeneration, and leakage14,15,17. Given its anatomical specificity, experimental tractability, and technical accessibility, retinal I/R injury by elevated IOP promises to maintain a leading role in pursuing these inquiries.

Retinal ischemia-reperfusion (I/R) characterizes many human retinal pathologies, including glaucoma, diabetic retinopathy, and retinal vascular occlusions1. In retinal I/R, reduced blood flow (ischemia) in the retinal vasculature creates a state of retinal hypersensitivity to oxygen and other nutrients, precipitating severe oxidative and inflammatory damage when circulation is subsequently reinstated (reperfusion)2. The neural retina appears particularly vulnerable to these changes, with retinal neurodegeneration being perhaps the most salient feature of I/R-induced damage. Presented here is a protocol for modeling retinal I/R injury in the mouse. This technique enables researchers to examine potential mechanisms and treatment strategies for human diseases of the retinal neurovascular unit.
Pioneered in 1952 by surgeons seeking to understand the neurodegenerative consequences of surgical anemia3, rodent retinal I/R by elevated intraocular pressure (IOP) was reestablished in 1991 for the purpose of standardizing neurodegenerative endpoints after ischemic insult4. Using the drip of a saline reservoir to raise IOP above systolic blood pressure, these studies demonstrated that pressurized ocular cannulation was sufficient to suspend the retinal circulation and thereby initiate neuronal degeneration. More recent efforts using retinal I/R by elevated IOP have begun to elaborate the mechanisms underlying I/R-induced retinal neurodegeneration5-12. Multiple groups have reported additional pathologic changes including inflammation13,14, vascular permeability15,16, and capillary degeneration14,17. Taken together, these studies have established retinal I/R injury by elevated IOP as a model of retinal neurovascular disease more generally.
Characterizing the mechanisms of I/R injury is essential for the study of vascular disease. Retinal I/R injury by elevated IOP is one of many hypoxia-induced injury models, including I/R injuries in lung18, heart19, brain20, liver21, kidney22, and intestine23. These models have been paramount in advancing our understanding of vascular illness and its clinical remedies. By extending the investigation of I/R processes to ocular tissues, retinal I/R injury by elevated IOP helps to paint a more comprehensive picture of these related conditions.
Corresponding closely with clinical neurodegenerative conditions in retina, retinal I/R injury by elevated IOP presents a valuable tool for researchers interested in exploring ischemic pathogenesis. The protocol described herein is targeted, tractable, and accessible. It is complemented well by endpoints in neuronal degeneration, such as quantification of retinal neurons, measurement of retinal thickness, and electrophysiological recording of retinal neuron function. This model has proven its utility in advancing neurovascular inquiry, and it shows promise in earning status as a foundational protocol in visual medicine research.

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a mouse model of retinal ischemia-reperfusion injury through elevation of intraocular pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

The neurodegenerative effects of retinal I/R by elevated IOP are commonly evaluated using two standard approaches. NeuN immunolabeling of neuronal nuclei has revealed significant neuronal cell loss following I/R insult (Figure 1). Briefly, eyes enucleated 7 days after I/R were fixed in paraformaldehyde, labeled with the neuronal cell marker NeuN, and whole-mounted. Images were captured using confocal microscopy, and cells labeled with NeuN were quantified by counting11. Decreases in ganglion cell layer neuron counts indicate I/R-induced cell death.
Impairments in retinal neuron function have been documented using electroretinography (Figure 2). Briefly, seven days after I/R, scotopic electroretinograms were recorded at multiple flash intensities. Amplitudes of the a- and b-waves were quantified using image analysis software12. Here, lower a- and b-wave amplitudes signify I/R-induced impairments in retinal neuronal function. In these and other I/R endpoints, the non-I/R eye serves as a robust negative control for I/R-induced damage.
<img alt="Figure 1" src="/files/ftp_upload/54065/54065fig1.jpg" /> 
Figure 1. Retinal I/R Induces Neuronal Cell Death in the Ganglion Cell Layer (GCL).&#160;Representative retinal images from control and I/R eyes are shown with scale bars denoting 100 &#181;m. (A) The number of NeuN-positive cells in the GCL is significantly reduced in I/R eyes as compared to controls (B). n = 9, error bar: standard error, ***p &#60; 0.0001 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54065/54065fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54065/54065fig2.jpg" /> 
Figure 2. Retinal I/R Impairs Retinal Neuron Function. Decreased a- and b-wave amplitudes indicate impaired membrane physiology in neuronal cells following I/R. n = 6, error bar: standard error, *p &#60; 0.05 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54065/54065fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure at JoVE.com

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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14.8K Views.  Johns Hopkins University. This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Video: A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...