<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
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<ol>
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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. 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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 ...

The protocol provides a model to investigate mechanisms of vascular injury and subsequent development of edema in a non-invasive manner. The main advantage of this technique is that you can follow the injury live over time without surgical intervention. This provides avenues to test treatments more efficiently.It can be challenging to learn how to perform proper tail veining and to accommodate the animal in the platform correctly. For both techniques I recommend being patient and taking things slowly. To begin, gently handle the fiber optic cable and connect it to the laser control box and the laser adaptor of the retinal imaging microscope, then turn on the retinal imaging microscope lamp box.Turn on the computer and open the imaging program. Place a piece of white paper in front of the microscope eyepiece and adjust the white balance by clicking on Adjust in the imaging program. Turn on the laser control box by turning the key and following the instructions on the screen of the laser control box.To verify the baseline laser power, set the laser control box to 50 milliwatts and 2, 000 milliseconds. Then turn on the laser and place the power meter in front of the eyepiece. Press the foot switch pedal to activate the laser, aiming for the laser power readout to be 13 to 15 milliwatts.Then, using the laser control box screen, adjust the experimental laser power to 100 milliwatts and 1, 000 milliseconds, and turn off the laser. Pour 300 milliliters of water into a 500-milliliter beaker and warm the beaker in a microwave oven for one minute. Then place a gauze in the warm water.Next place a two-month-old male mouse in a restrainer. Press the warm gauze into the mouse tail gently and look for the dilated veins. Using a 26-gauge needle, inject the appropriate amount of rose bengal according to the animal's weight and apply pressure on the injection site to prevent hematoma or bleeding before wiping the site.Then release the mouse from the restrainer and return it to the cage. Allow eight minutes for the rose bengal to circulate before the injection of anesthetics. Turn on the heated mouse platform.Then add one drop of phenylephrine and tropicamide in each eye of the mouse. After confirming anesthesia, add one drop of proparacaine hydrochloride per eye, then add eye ointment to both eyes. Next, inject 150 microliters of carprofen subcutaneously between the ears.Then accommodate the mouse on the platform and adjust the platform until the view of the retinal fundus is clear and focused. Count the retinal veins and take an image of the fundus. Next, turn on the laser and aim towards a retinal vein approximately 375 micrometers from the optic disc.Irradiate the vessel by pressing the foot switch and slightly moving the laser beam up to 100 micrometers. Repeat this step three times and move the laser beam after each pulse so that the irradiation is not focused in one spot. Repeat the irradiation on other major vessels to achieve two to three occlusions.After irradiating the vessels, turn off the lamp and wait for 10 minutes. Then turn the lamp back on, count the number of veins occluded, and take an image of the fundus. After image acquisition and saline injection, add lubricant eyedrops and gel ointment to both eyes.Watch the mouse as it recovers from anesthesia and return it to the cage with other animals only when fully recovered. The number of occlusions achieved immediately after laser irradiation was not different between animals that were lasered 10 and 20 minutes after rose bengal administration. The number of occlusions sustained up to one day after retinal vein occlusion significantly decreased in animals lasered 20 minutes after rose bengal administration independently of genotype.Without modifying the experimental power of 100 milliwatts, a low baseline laser power of 11.5 milliwatts resulted in no occlusions. In contrast, a baseline laser output of 13.5 milliwatts resulted in successful occlusions. Four main types of occlusions occur after laser photocoagulation:fully occluded vessels, partially occluded vessels, partially reperfused, and fully reperfused vessels.The irradiated vasculature of Tamoxifen-inducible endothelial cell Casp9 knockout mice spent more time in partially reperfused and partially occluded states than that of C57 Black 6 mice, which spent more time in fully occluded states. The occlusion state of the vessels changes rapidly within the first 10 minutes after laser photocoagulation and flame-shaped hemorrhages are observed 24 hours post-injury. Different ophthalmic pathologies occurred after retinal vein occlusion.The level of retinal edema after retinal vein occlusion can be quantified in the injured eyes using optical coherence tomography images. The state of neuronal layers can also be determined by assessing the disorganization of retinal inner layers. An example of an optical coherence tomography image with the corresponding labels for each layer is shown here.Measuring the baseline laser power is important. This readout will greatly impact the success of the occlusions and can be optimized. Additional methods such as OCT, ERG, and Optiger can be performed.This will help address the effects of neurovascular injury on retinal neuronal integrity and visual pathway function. This technique paves the way to interrogate the molecular pathways driving neurovascular disease and, by following individual animals over time, provides data which can be more readily translated to human disease.

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2.5K Views.  Columbia University. The protocol provides a model to investigate mechanisms of vascular injury and subsequent development of edema in a non-invasive manner. The main advantage of this technique is that you can follow the injury live over time without surgical intervention. This provides avenues to test treatments more efficiently.It can be challenging to learn how to perform proper tail veining and to accommodate the animal in the platform correctly. For both techniques I recommend being patient and takin...