<div>This work was supported by National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) grant DGE &#8211; 1644869 (to CKCO), the National Eye Institute (NEI) 5T32EY013933 (to AMP), the National Institute on Aging (NIA) R21AG063012 (to CMT) and by the Department of Defense Army Research Office Defense University Research Instrumentation Program (DURIP) (to CMT).</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>

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<li>Drechsler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+intravitreal+anti-vascular+endothelial+growth+factor+treatment+on+the+retinal+gene+expression+in+acute+experimental+central+retinal+vein+occlusion%5BTitle%5D)+AND+%22Ophthalmic+Research%22%5BJournal%5D)">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>
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<li>Nguyen, V. P., Li, Y., Zhang, W., Wang, X., Paulus, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+multimodal+photoacoustic+microscopy+and+optical+coherence+tomography+image-guided+laser+induced+branch+retinal+vein+occlusion+in+living+rabbits%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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>
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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><tbody><tr><td>Carprofen</td><td>Rimadyl</td><td>NADA #141-199</td><td>keep at 4 &amp;deg;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,&amp;nbsp; 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 &amp;deg;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...

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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 ...

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2.6K 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

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Medicine

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Summary

Explore More Videos

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

A Mouse Model for Laser-induced Choroidal Neovascularization

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images