<div>This work was supported by&#160;the&#160;National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) DGE - 1644869 (to CCO), the&#160;National Eye Institute (NEI) 5T32EY013933 (to AMP)&#160;and the National Institute on Aging (NIA)&#160;R21AG063012 (to CMT).&#160;</div>

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
NOTE: All experiments used two-month-old male mice that weighed approximately 20 g.
1. Preparation and administration of tamoxifen for inducible genetic ablation of floxed genes
NOTE: Retinal vessel diameter can be affected by the weight of the animal. Make sure that all animals used for an experiment are of similar weights.
<ol>
	<li>Dilute tamoxifen in corn oil to a concentration of 20 mg/mL. 
	NOTE: Tamoxifen is a toxicant and is light-sensitive. Protect from light, e.g., with aluminum foil.</li>
	<li>Vortex the solution for a couple of seconds.</li>
	<li>Leave in the oven at 55 &#176;C for 15 min. 
	NOTE: Make sure that the tamoxifen has dissolved completely. Additional vortexing may be necessary.</li>
	<li>Store the solution at 4 &#176;C for up to 1 week.&#160;&#160;</li>
	<li>Use a 1 mL syringe fitted with a 26 G needle for tamoxifen injection. Clean the injection area with 70% ethanol.&#160; Administer 2 mg of tamoxifen (100 &#181;L of 20 mg/mL) intraperitoneally (IP) once daily&#160;for the established time according to the specific inducible Cre line.&#160;</li>
	<li>Allow two days of rest for the animals before starting the experiments.</li>
</ol>
2. Preparation of reagents for laser photocoagulation
<ol>
	<li>Rose bengal 
	NOTE: Rose bengal is light-sensitive. Store in the dark until usage and prepare fresh for best results.
	<ol>
		<li>Prepare rose bengal by diluting it to 5 mg/mL in sterile saline and filter it through a 0.2 &#181;m syringe filter.</li>
		<li>Prepare a 1 mL syringe fitted with a 26 G needle with rose bengal.</li>
	</ol>
	</li>
	<li>Ketamine/xylazine
	<ol>
		<li>Dilute ketamine and xylazine in sterile saline accordingly for the following concentrations: ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg).</li>
	</ol>
	</li>
	<li>Carprofen
	<ol>
		<li>Dilute carprofen to 1 mg/mL in sterile saline.</li>
		<li>Prepare a 1 mL syringe fitted with a 26 G needle with carprofen.</li>
	</ol>
	</li>
	<li>Sterile saline
	<ol>
		<li>Prepare a 5 mL syringe fitted with a 26 G needle with sterile saline.</li>
	</ol>
	</li>
</ol>
3. Laser setup
<ol>
	<li>Gently handle the fiber optic cable and connect it to the laser control box and the laser adapter of the retinal imaging microscope.</li>
	<li>Turn the retinal imaging microscope lamp box on.</li>
	<li>Turn the computer on and open the imaging program.</li>
	<li>Adjust the white balance by using a piece of white paper and putting it in front of the mouse eye piece and clicking on Adjust in the imaging program.</li>
	<li>Turn the laser control box on by turning the key and following the instructions on the screen of the laser control box. 
	NOTE: The laser used in this experiment is Class 3B and can cause eye damage. Wear protective goggles when operating the laser.</li>
	<li>Verify the baseline laser power.
	<ol>
		<li>Use a laser power meter.</li>
		<li>Adjust the screen of the laser control box to the following parameters: 50 mW and 2,000 ms.</li>
		<li>Turn the laser on and place the power meter in front of the eyepiece. 
		NOTE:&#160;Make sure microscope light is off while testing baseline laser power.</li>
		<li>Press the footswitch pedal to activate the laser.</li>
		<li>Aim for the laser power readout to be 13-15 mW. 
		NOTE: The laser power readout will determine the success rate for retinal vein occlusions. If the laser power readout is too low, adjustments can be made to the power and time of laser exposure. See Table 1 for recommendations.</li>
	</ol>
	</li>
	<li>Adjust the experimental laser power by setting up the screen of the laser control box for the following parameters: 100 mW, 1,000 ms.</li>
	<li>Turn off the laser. 
	&#8203;NOTE: For safety and to prevent overheating, it is best to keep the laser off between mice.</li>
</ol>
4. Mouse tail vein injection of rose bengal
<ol>
	<li>Pour 300 mL of water into a 500 mL beaker.</li>
	<li>Warm the beaker in a microwave oven for 1 min.</li>
	<li>Put gauze in the warm water in the beaker.</li>
	<li>Put the mouse in a restrainer.</li>
	<li>Press the gauze into the mouse tail gently and look for the dilated veins. Disinfect the injection site&#160;using an alcohol wipe after the warm water dilation.&#160;</li>
	<li>Insert the needle into the injection site and&#160;pull on the syringe to ensure you are in&#160;the vein. Then,&#160;inject the mouse tail vein, administering the correct amount according to the weight of the animal (37.5 mg/kg). Apply pressure on the injection site to avoid hematoma or bleeding. Wipe the site.</li>
	<li>Release the mouse from the restrainer and return it to the cage.</li>
	<li>Allow 8 min for the rose bengal to circulate before the injection of anesthetics. 
	&#8203;NOTE: This will provide a total of 10 min between the rose bengal injection and laser irradiation.</li>
</ol>
5. Occlusion of major veins
<ol>
	<li>Turn on the heated mouse platform.</li>
	<li>Add one drop of phenylephrine and tropicamide in each eye.</li>
	<li>Inject 150 &#181;L of the anesthetics, ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg) IP. 
	NOTE: During this procedure, the mouse was given two IP injections. Hence, the sides were alternated. The IP injection for the anesthesia was given into&#160;the lower right abdominal quadrant, and the saline was injected intp&#160;the lower left abdominal quadrant. Pulling on the syringe before injecting is recommended to ensure that the needle is in the abdomen and not any organs.</li>
	<li>Toe-pinch the animal to determine the depth of anesthesia and wait until it is unresponsive.</li>
	<li>Add one drop of proparacaine hydrochloride per eye (analgesic).</li>
	<li>Add gel ointment to both eyes.</li>
	<li>Inject 150 &#181;L of carprofen subcutaneously between the ears.</li>
	<li>Accommodate the mouse on the platform.</li>
	<li>Adjust the platform until the view of the retinal fundus is clear and focused.</li>
	<li>Count the retinal veins and take an image of the fundus. 
	NOTE: Retinal veins are darker and broader than arteries. Veins and arteries alternate; however, sometimes there can be a branched artery close to the optic nerve, and therefore, two adjacent arteries.</li>
	<li>Turn the laser on and aim towards a retinal vein at approximately 375 &#181;m from the optic disc.</li>
	<li>Irradiate the vessel by pressing the footswitch and slightly moving the laser beam up to 100 &#181;m. Repeat this step three times and move the laser beam after each pulse so that the irradiation is not focused in one spot.</li>
	<li>Repeat irradiation on other major vessels to achieve 2-3 occlusions.</li>
</ol>
6. Establishing the number of veins occluded at day 0
<ol>
	<li>Turn off the lamp after irradiating the vessels and wait for 10 min. 
	NOTE: Light exposure can cause retinal damage and inflammation; turn off the lamp during the waiting time to minimize exposure7.</li>
	<li>Turn the lamp back on and count the number of veins occluded.</li>
	<li>Take an image of the fundus.</li>
</ol>
7. Aftercare
<ol>
	<li>Inject 1 mL of sterile saline IP. 
	NOTE: See IP injection details in section 5, step 3.</li>
	<li>Add lubricant eye drops to both eyes.</li>
	<li>Add gel ointment to both eyes.</li>
	<li>Watch the mouse as it recovers from anesthesia, and do not return it to the cage with the other animals until fully recovered. Carprofen (5 mg/kg) can be given daily up to 2 days post-procedure.&#160;If applied to humans, pain is not a symptom of RVO. 
	NOTE: &#160;Do not leave the animals unattended until they fully recover from anesthesia.&#160;&#160;</li>
</ol>
8. Assessment of retinal edema by optical coherence tomography (OCT)
NOTE: This step can be done at the investigator&#39;s time point of interest. The peak of retinal edema for a C57BL/6J mouse is 1 day after the RVO procedure. This time point might vary depending on the background of the mouse.
<ol>
	<li>Turn on the retinal imaging microscope light box, the OCT machine, and the heated mouse platform.</li>
	<li>The day after the occlusion, follow steps 5.2 to 5.7 to prepare the animal.</li>
	<li>Open the imaging and OCT software programs.</li>
	<li>In the OCT program, adjust the nudge to 5.</li>
	<li>Take OCT at 75 &#181;m distal from the burn or 4 clicks.</li>
	<li>Take OCT images at four quadrants of the retina.</li>
	<li>Analyze the OCT images using tracing software.</li>
	<li>Compare the retinal thickness of pre-irradiated measures to 1 day post RVO or at the time point of interest. 
	NOTE: When analyzing the data, take into consideration the number of veins irradiated as this can influence the development of retinal edema. Animals are then euthanized by administering anesthetic followed by perfusion non-survival surgery.</li>
</ol>

<ol>
	<li>Song, P., Xu, Y., Zha, M., Zhang, Y., Rudan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epidemiology+of+retinal+vein+occlusion:+a+systematic+review+and+meta-analysis+of+prevalence,+incidence,+and+risk+factors.">Global epidemiology of retinal vein occlusion: a systematic review and meta-analysis of prevalence, incidence, and risk factors.</a> Journal of Global Health. 9 (1), 010427 (2019).</li><li>Ehlers, J. P., Fekrat, 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+vein+occlusion:+beyond+the+acute+event.">Retinal vein occlusion: beyond the acute event.</a> Survey of Ophthalmology. 56 (4), 281-299 (2011).</li><li>Iftikhar, 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=Loss+of+peak+vision+in+retinal+vein+occlusion+patients+treated+for+macular+edema.">Loss of peak vision in retinal vein occlusion patients treated for macular edema.</a> American Journal of Ophthalmology. 205, 17-26 (2019).</li><li>Zhang, 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=Development+of+a+new+mouse+model+of+branch+retinal+vein+occlusion+and+retinal+neovascularization.">Development of a new mouse model of branch retinal vein occlusion and retinal neovascularization.</a> Japanese Journal of Ophthalmology. 51 (4), 251-257 (2007).</li><li>Ebneter, A., Agca, C., Dysli, C., Zinkernagel, 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=Investigation+of+retinal+morphology+alterations+using+spectral+domain+optical+coherence+tomography+in+a+mouse+model+of+retinal+branch+and+central+retinal+vein+occlusion.">Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.</a> PLoS One. 10 (3), 0119046 (2015).</li><li>Fuma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacological+approach+in+newly+established+retinal+vein+occlusion+model.">A pharmacological approach in newly established retinal vein occlusion model.</a> Scientific Reports. 7, 43509 (2017).</li><li>Zhang, C., 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=Activation+of+microglia+and+chemokines+in+light-induced+retinal+degeneration.">Activation of microglia and chemokines in light-induced retinal degeneration.</a> Molecular Vision. 11, 887-895 (2005).</li><li>Avrutsky, M. 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=Endothelial+activation+of+caspase-9+promotes+neurovascular+injury+in+retinal+vein+occlusion.">Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion.</a> Nature Communications. 11 (1), 3173 (2020).</li><li>Nicholson, 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=Diagnostic+accuracy+of+disorganization+of+the+retinal+inner+layers+in+detecting+macular+capillary+non-perfusion+in+diabetic+retinopathy.">Diagnostic accuracy of disorganization of the retinal inner layers in detecting macular capillary non-perfusion in diabetic retinopathy.</a> Clinical &amp; Experimental Ophthalmology. 43 (8), 735-741 (2015).</li><li>Moein, H. R., 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=Optical+coherence+tomography+angiography+to+detect+macular+capillary+ischemia+in+patients+with+inner+retinal+changes+after+resolved+diabetic+macular+edema.">Optical coherence tomography angiography to detect macular capillary ischemia in patients with inner retinal changes after resolved diabetic macular edema.</a> Retina. 38 (12), 2277-2284 (2018).</li><li>Hirabayashi, 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=Development+of+a+novel+model+of+central+retinal+vascular+occlusion+and+the+therapeutic+potential+of+the+adrenomedullin-receptor+activity-modifying+protein+2+system.">Development of a novel model of central retinal vascular occlusion and the therapeutic potential of the adrenomedullin-receptor activity-modifying protein 2 system.</a> American Journal of Pathology. 189 (2), 449-466 (2019).</li><li>Martin, G., Conrad, D., Cakir, B., Schlunck, G., Agostini, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+in+a+mouse+model+of+retinal+vein+occlusion+induced+by+laser+treatment+reveals+a+predominant+inflammatory+and+tissue+damage+response.">Gene expression profiling in a mouse model of retinal vein occlusion induced by laser treatment reveals a predominant inflammatory and tissue damage response.</a> PLoS One. 13 (3), 0191338 (2018).</li><li>Drechsler, 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=Effect+of+intravitreal+anti-vascular+endothelial+growth+factor+treatment+on+the+retinal+gene+expression+in+acute+experimental+central+retinal+vein+occlusion.">Effect of intravitreal anti-vascular endothelial growth factor treatment on the retinal gene expression in acute experimental central retinal vein occlusion.</a> Ophthalmic Research. 47 (3), 157-162 (2012).</li><li>Genevois, O., 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=Microvascular+remodeling+after+occlusion-recanalization+of+a+branch+retinal+vein+in+rats.">Microvascular remodeling after occlusion-recanalization of a branch retinal vein in rats.</a> Investigative Ophthalmology &amp; Visual Science. 45 (2), 594-600 (2004).</li><li>Khayat, M., Lois, N., Williams, M., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+retinal+vein+occlusion.">Animal models of retinal vein occlusion.</a> Investigative Ophthalmology &amp; Visual Science. 58 (14), 6175-6192 (2017).</li><li>Nguyen, V. P., Li, Y., Zhang, W., Wang, X., Paulus, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+multimodal+photoacoustic+microscopy+and+optical+coherence+tomography+image-guided+laser+induced+branch+retinal+vein+occlusion+in+living+rabbits.">High-resolution multimodal photoacoustic microscopy and optical coherence tomography image-guided laser induced branch retinal vein occlusion in living rabbits.</a> Scientific Reports. 9 (1), 10560 (2019).</li><li>Sayyed, S. A. A. R., Beedri, N. I., Kadam, V. S., Pathan, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rose+Bengal+sensitized+bilayered+photoanode+of+nano-crystalline+TiO2-CeO2+for+dye-sensitized+solar+cell+application.">Rose Bengal sensitized bilayered photoanode of nano-crystalline TiO2-CeO2 for dye-sensitized solar cell application.</a> Applied Nanoscience. 6 (6), 875-881 (2015).</li><li>Emmart, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+absorption+spectra+of+fluorescein,+fluorescein+derivatives+and+conjugates.">Observations on the absorption spectra of fluorescein, fluorescein derivatives and conjugates.</a> Archives of Biochemistry and Biophysics. 73 (1), 1-8 (1958).</li><li>Yu, L., Liu, Z., Liu, S., Hu, X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+spectrophotometric+method+for+the+determination+of+polyvinylpyrrolidone+with+eosin+Y.">Fading spectrophotometric method for the determination of polyvinylpyrrolidone with eosin Y.</a> Chinese Journal of Chemistry. 27 (8), 1505-1509 (2009).</li><li>MacDonald, 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+ABCs+of+RVO:+a+review+of+retinal+venous+occlusion.">The ABCs of RVO: a review of retinal venous occlusion.</a> Clinical &amp; Experimental Optometry. 97 (4), 311-323 (2014).</li><li>Stahl, 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=Postnatal+weight+gain+modifies+severity+and+functional+outcome+of+oxygen-induced+proliferative+retinopathy.">Postnatal weight gain modifies severity and functional outcome of oxygen-induced proliferative retinopathy.</a> American Journal of Pathology. 177 (6), 2715-2723 (2010).</li><li>LaVail, M. M., Gorrin, G. M., Repaci, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+in+sensitivity+to+light-induced+photoreceptor+degeneration+in+albino+mice.">Strain differences in sensitivity to light-induced photoreceptor degeneration in albino mice.</a> Current Eye Research. 6 (6), 825-834 (1987).</li><li>Jeffery, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+albino+retina:+an+abnormality+that+provides+insight+into+normal+retinal+development.">The albino retina: an abnormality that provides insight into normal retinal development.</a> Trends in Neurosciences. 20 (4), 165-169 (1997).</li><li>Kinnear, P. E., Jay, B., Witkop, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Albinism.">Albinism.</a> Survey of Ophthalmology. 30 (2), 75-101 (1985).</li><li>Stahl, 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=Postnatal+weight+gain+modifies+severity+and+functional+outcome+of+oxygen-induced+proliferative+retinopathy.">Postnatal weight gain modifies severity and functional outcome of oxygen-induced proliferative retinopathy.</a> American Journal of Pathology. 177 (6), 2715-2723 (2010).</li></ol>

The authors declare that they have no competing financial interests.

The mouse RVO model provides an avenue to further understand RVO pathology and to test potential therapeutics. While the mouse RVO model is widely used in the field, there is a need for a current detailed protocol of the model that addresses its variability and describes the optimization of the model. Here, we provide a guide with examples from experience on what can be altered to get the most consistent results across a cohort of experimental animals and provide reliable data.
The two most es...

Retinal vein occlusion (RVO) is a common retinal vascular disease that affected approximately 28 million people worldwide in 20151. RVO leads to vision decline and loss in working aged adults and elders, representing an ongoing sight-threatening disease estimated to increase over the proximate decade. Some of the distinct pathologies of RVO include hypoxic-ischemic injury, retinal edema, inflammation, and neuronal loss2. Currently, the first line of treatment for this disorder is through the administration of vascular endothelial growth factor (VEGF) inhibitors. While anti-VEGF treatment has helped ameliorate retinal edema, many patients still face vision decline3. To further understand the pathophysiology of this disease and to test potential new lines of treatment, there is a need to constitute a functional and detailed RVO mouse model protocol for different mouse strains.
Mouse models have been developed implementing the same laser device used in human patients, paired with an imaging system scaled to the correct size for a mouse. This mouse model of RVO was first reported in 20074 and further established by Ebneter and others4,5. Eventually, the model was optimized by Fuma et al. to replicate key clinical manifestations of RVO such as retinal edema6. Since the model was first reported, many studies have employed it using the administration of a photosensitizer dye followed by photocoagulation of major retinal veins with a laser. However, the amount and type of the dye that is administered, laser power, and time of exposure vary significantly across studies that have used this method. These differences can often lead to increased variability in the model, making it difficult to replicate. To date, there are no published studies with specific details about potential avenues for its optimization.
This report presents a detailed methodology of the RVO mouse model in the C57BL/6J strain and a tamoxifen-inducible endothelial caspase-9 knockout (iEC Casp9KO) strain with a C57BL/6J background and of relevance to RVO pathology as a reference strain for a genetically modified mouse. A previous study had shown that non-apoptotic activation of endothelial caspase-9 instigates retinal edema and promotes neuronal death8. Experience using this strain helped determine and provide insight towards potential modifications to tailor the RVO mouse model, which can be applicable to other genetically modified strains.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>NADA #141-199</td>
			<td>keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Corn Oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Patch Cable</td>
			<td>Thor Labs</td>
			<td>M14L02</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal</td>
			<td>Alcon</td>
			<td>00658 06401</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lasercheck</td>
			<td>Coherent</td>
			<td>1098293</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Akorn</td>
			<td>NDCL174478-201-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoneix Micron IV with Meridian,&#160; StreamPix, and OCT modules</td>
			<td>Phoenix Technology Group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride</td>
			<td>Akorn</td>
			<td>NDC: 17478-263-12</td>
			<td>keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Refresh</td>
			<td>Allergan</td>
			<td>94170</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma-Aldrich</td>
			<td>330000-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-5G</td>
			<td>light-sensitive</td>
		</tr>
		<tr>
			<td>Tropicamide</td>
			<td>Akorn</td>
			<td>NDC: 174478-102-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of the retinal vein occlusion mouse model to limit variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

The RVO mouse model aims to successfully achieve occlusions in the retinal veins, leading to hypoxic-ischemic injury, breakdown of the blood retinal barrier, neuronal death, and retinal edema8. Figure 1 shows a timeline of steps to ensure reproducibility, a schematic of the experimental design, and outlines steps that can be further optimized depending on the experimental questions. The three main steps that can be modified are the waiting time after rose bengal admin...

Watch this Scientific Journal Video about Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability at JoVE.com

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

optimization-retinal-vein-occlusion-mouse-model-to-limit

Research

JoVE Journal

Neuroscience

2.5K Views.  Columbia University. Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains. 

Video: Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...