This work was supported by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) grant DGE - 1644869(to CKCO), the National Eye Institute (NEI) 5T32EY013933 (to AMP), the National Institute of Neurological Disorders and Stroke (RO1 NS081333, R03 NS099920 to CMT), and the Department of Defense Army/Air Force (DURIP to CMT).

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: Imaging was done on 2 month old C57BL/6J male mice that weighed approximately 23 g.
1. Preparation of reagents for retinal imaging
<ol>
	<li>Preparation of injectable fluorescein solution. 
	NOTE: Fluorescein is very light-sensitive. Protect from light and use it shortly after preparation.
	<ol>
		<li>Dilute fluorescein to a concentration of 1% in sterile saline.</li>
	</ol>
	</li>
	<li>Preparation of Ketamine/Xylazine
	<ol>
		<li>Dilute Ketamine and Xylazine in sterile saline accordingly for the following concentration: Ketamine (80-100 mg/kg) and Xylazine (5-10 mg/kg).</li>
	</ol>
	</li>
	<li>Sterile saline
	<ol>
		<li>Prepare a 5 mL syringe with a 26 G needle with sterile saline.</li>
	</ol>
	</li>
</ol>
2. OCT and fluorescein imaging
<ol>
	<li>Turn the retinal imaging microscope lightbox, the OCT machine, and the heated mouse platform ON.</li>
	<li>Turn the computer ON and open the imaging program.</li>
	<li>Add one drop of phenylephrine and tropicamide to each eye.</li>
	<li>Inject 150 &#181;L of anesthesia (Ketamine (80-100 mg/kg) and Xylazine (5-10 mg/kg)) intraperitoneally (IP). Determine the depth of anesthesia by toe pinch and wait until the animal is unresponsive. Apply ophthalmic ointment or artificial tears to both the eyes.</li>
	<li>Accommodate the mouse on the platform.</li>
	<li>Adjust the height and angle of the platform until the view of the retinal fundus is clear and focused. Take a picture of the fundus.</li>
	<li>Open the imaging and OCT software. In the OCT program, adjust nudge to 5.</li>
	<li>Take an OCT image at 75 &#181;m distal from the burn. Repeat for the other three quadrants of the retina.</li>
	<li>Inject 100 &#181;L of 1% fluorescein IP.</li>
	<li>Switch the camera to a 488 nm filter. Increase the camera gain to 5.</li>
	<li>Take a picture of the fundus at exactly 5 min after fluorescein injection. 
	&#8203;NOTE: Avoid prolonged exposure of the eye to the camera light at maximum setting, as fluorescein can exacerbate retinal photodamage. Keep light source off until the 5 min wait time has elapsed and the mouse is ready for imaging.</li>
</ol>
3. Aftercare
<ol>
	<li>Inject 1 mL of sterile saline IP. Apply lubricant eye drops to both the eyes. Apply ophthalmic ointment or artificial tears to both the eyes.</li>
	<li>Observe the mouse as it recovers from anesthesia. Return to the cage with other animals only when fully recovered, generally after around 40 min.</li>
</ol>
4. Assessment for exclusion criteria
<ol>
	<li>Open the fundus image taken at 24 h post-procedure to assess for exclusion criteria. Exclude the eye if any of the following criteria are identified.</li>
	<li>Assess whether the image has zero occlusions
	<ol>
		<li>Evaluate the image for the number of occluded vessels. 
		NOTE: A successful occlusion usually has some purple pigmentation on or around the burn, very thin or discontinuous vessel through the burn, faint or non-existent vessel appearance outside the burn area, and retinal discoloration from hypoxia. If the entire vessel can be seen through the white burn by the laser, the vessel failed to occlude. Sometimes the vessel will appear partially obstructed, but if it looks uninterrupted outside the burn, the vessel likely didn&#39;t occlude.</li>
		<li>For ambiguous cases, use FA imaging at the same time point to evaluate occlusions. In these images, an occlusion will appear as a break in the continuity of a vessel, often with a tapering of the surrounding vessel.</li>
		<li>If zero occlusions are identified, exclude the eye from analysis, as the RVO is considered ineffective. 
		NOTE: Occlusions typically resolve by 48-72 h post-RVO, and the presence of occlusions should no longer be used as an exclusion criterion at these time points.</li>
	</ol>
	</li>
	<li>Assess the fundus and OCT images for excessive retinal detachment 
	NOTE: Subretinal fluid accumulation is common after induction of RVO, and causes separation of neural retina from RPE. Exclusionary criteria for excessive retinal detachment are defined as follows: OCT will either be completely unviewable, or some layers will appear incredibly distorted. Image quality is poor, with a loss of resolution of outer plexiform and RPE layers. The separation between the neural retina and the choroid is greater than what the OCT field of view allows. On the fundus image, the retina tone will be nearly completely white, with some purple blotching. Part of the retina may appear distorted and out of focus. This is because it has detached and is at a different focal distance than the rest of the retina.
	<ol>
		<li>If the assessment of the images from an eye determines peripheral or complete detachment of the retina, exclude the eye from the analysis.</li>
	</ol>
	</li>
	<li>Exclude images with evidence of corneal cataract 
	NOTE: A corneal cataract appears as an opaque white dot on the mouse&#39;s cornea. Cataracts typically occur due to insufficient lubrication of the eyes while the animal is anesthetized and can be largely avoided by taking care to apply eye ointment generously. Cataracts can generally be identified before imaging by inspecting the animal. Mice that have developed cataracts should be excluded from the dataset without needing to undergo the imaging process. In imaging, cataracts will obscure the retina from the camera, and the OCT will appear warped.</li>
	<li>Assess the image for excessive hemorrhage 
	&#8203;NOTE: Excessive hemorrhage can be identified as amounts of red fluid in the image, usually obscuring retinal background, vessel, and burn. These areas of red fluid will be a brighter, opaquer red than the purple splotches that are normal in successful RVO. Hemorrhages show up at the ganglion cell layer on OCT imaging and interfere with the ability to visualize other retinal layers beneath the hemorrhage.
	<ol>
		<li>If the image is determined to have an excessive hemorrhage, exclude the eye from the analysis.</li>
	</ol>
	</li>
</ol>
5. Fluorescein image processing
<ol>
	<li>Open the fluorescein image in the image processing software.</li>
	<li>Duplicate the image</li>
	<li>Using a selection tool, carefully trace the major vessels.
	<ol>
		<li>The major vessels are the thicker veins and arteries radiating out from the optic disc. Ignore any vessels branching out from these vessels.</li>
		<li>If leakage prevents the outline of the vessel from being seen near the occlusion site, trace through the leakage in the approximate location of the vessel (maintain thickness, connect the last visible point to the next visible point).</li>
	</ol>
	</li>
	<li>In the first image, delete the selection, leaving only the background. Save this masked image.</li>
	<li>Move the selection to the second image, invert the selection and delete, isolating the vessels. Save this masked image.</li>
	<li>Open the two images in ImageJ. Open the background image and measure the integrated density.</li>
	<li>Open the vessel&#39;s image, select the outline of the vessels, and then measure the mean intensity.</li>
	<li>Divide the integrated density of the background by the mean intensity of the vessels, generating the leakage ratio for the eye.</li>
	<li>Record this leakage ratio for each eye in an experimental cohort.</li>
	<li>To further control for background, normalize experimental eyes to the mean leakage ratio of uninjured control eyes. 
	&#8203;NOTE: In order to create a standardized quantification of fluorescein leakage in the FA image, this calculation uses a ratio of the background density (where the leakage will be present) with the brightness of the major vessels to create results that control for the variation in brightness from image to image and can be reliably quantified. Eyes that are undamaged have no leakage and should theoretically have ratios of zero. The ratios calculated from these undamaged control eyes, therefore, represent background noise, and this value is used to further normalize experimental values.</li>
</ol>
6. Retinal layer thickness
<ol>
	<li>Open the OCT image in the image processing software.</li>
	<li>Trace the borders of the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, photoreceptor layer, and RPE layer. Measure the mean thickness of each layer.</li>
	<li>Repeat for OCT images from the other three quadrants of the retina. Average the mean layer thicknesses across the four quadrants to obtain the mean thickness of each retinal layer for the eye.</li>
	<li>Repeat for each eye in the experimental cohort.</li>
</ol>
7. Disorganization of retinal inner layers (DRIL)
<ol>
	<li>Open the OCT image in ImageJ.</li>
	<li>Using the line tool, measure the distance where the upper border of the outer plexiform layer is indistinct. 
	NOTE: It is important to differentiate between DRIL and areas of poor layer visibility caused by imaging artifacts. Poor OCT image quality may invalidate an eye for DRIL analysis if sufficient image resolution is not possible. Images with DRIL will typically have other regions or retinal layers that are clearly resolved and organized, which can be a good indicator of sufficient image quality.
	<ol>
		<li>Measure horizontally from the latitude where the disorganization begins to the latitude where the upper border of the outer plexiform layer becomes visible again, if at all. Even if the outer plexiform layer shifts upward or downward vertically, measure perfectly horizontally.</li>
		<li>There may be multiple areas of disorganization separated by areas with no disorganization. Measure these individually and calculate the sum of the distances.</li>
	</ol>
	</li>
	<li>Divide the length of disorganization by the total length of the retina visible in each OCT image to obtain the ratio of disorganization for the image.</li>
	<li>Repeat the measurement and calculation for OCT images from the other three quadrants of the retina.</li>
	<li>Take the mean of the ratios of disorganization from the four OCT images. This number represents the average disorganization for the whole retina. Repeat for each eye in the experimental cohort.</li>
</ol>

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The authors declare that they have no competing financial interests.

Noninvasive rodent retinal imaging presents an avenue to study pathology and develop interventions. Previous studies have developed and optimized a mouse model of RVO, limiting variability and allowing for reliable translation of common clinical pathologies in the murine retina5,7,13. Developments in ophthalmic imaging technology further allow for the use of clinical in vivo imaging techniques such as FA and OCT in expe...

Neurovascular disease is a major healthcare problem responsible for ischemic strokes, a leading cause of mortality and morbidity, and retinal vascular diseases that lead to vision loss1,2. To model neurovascular disease, we employ a mouse model of retinal vein occlusion (RVO). This model is noninvasive and utilizes similar in vivo imaging techniques to those used to examine people with retinal vascular disease in a clinical setting. The use of this model thus increases the translational potential of studies utilizing this model. As with all mouse models, it is critical to maximize reproducibility of the model.
Retinal vascular diseases are a major cause of vision loss in people under the age of 70. RVO is the second most common retinal vascular disease after diabetic retinopathy3. Clinical features characteristic of RVO include ischemic injury, retinal edema, and vision loss as a consequence of neuronal loss3,4. Mouse models of RVO using laser photocoagulation of major vessels have been developed and refined to replicate key clinical pathologies observed in human RVO5,6,7. Advancements in ophthalmic imaging also allow for replication of noninvasive diagnostic tools used in humans, namely, fluorescein angiography (FA) and optical coherence tomography (OCT)6. Fluorescein Angiography allows for the observation of leakage due to the breakdown of the blood-retinal barrier (BRB) as well as blood flow dynamics in the retina, including sites of occlusion, using the injection of fluorescein, a small fluorescent dye8,9. OCT imaging allows for the acquisition of high-resolution cross-sectional images of the retina and the study of the thickness and organization of retinal layers10. Analysis of FA images has historically been largely qualitative, which limits the potential for direct and reproducible comparison between studies. Recently, a number of methods have been developed for the quantification of layer thickness in OCT imaging, though there is currently no standardized analysis protocol and the site of OCT image acquisition varies11. In order to properly leverage these tools, standardized, quantitative, and replicable data analysis methodology are needed. In this paper, we present three such vascular readouts used to evaluate pathological damage in a mouse model of RVO-fluorescein leakage, OCT layer thickness, and disorganization of retinal layers.

<table><tbody><tr><td>AK-Fluor 10%</td><td>Akorn</td><td>NDC: 17478-253-10</td><td>light-sensitive</td></tr><tr><td>Carprofen</td><td>Rimadyl</td><td>NADA #141-199</td><td>keep at 4 &amp;deg;C</td></tr><tr><td>GenTeal</td><td>Alcon</td><td>00658 06401</td><td></td></tr><tr><td>Image J</td><td>NIH</td><td></td><td></td></tr><tr><td>InSight 2D</td><td>Phoenix Technology Group</td><td></td><td>OCT analysis software</td></tr><tr><td>Ketamine Hydrochloride</td><td>Henry Schein</td><td>NDC: 11695-0702-1</td><td></td></tr><tr><td>Phenylephrine</td><td>Akorn</td><td>NDCL174478-201-15</td><td></td></tr><tr><td>Phoenix Micron IV</td><td>Phoenix Technology Group</td><td></td><td>Retinal imaging microscope</td></tr><tr><td>Phoenix Micron Meridian Module</td><td>Phoenix Technology Group</td><td></td><td>Laser photocoagulator software</td></tr><tr><td>Phoenix Micron Optical Coherence Tomography Module</td><td>Phoenix Technology Group</td><td></td><td>OCT imaging software</td></tr><tr><td>Phoenix Micron StreamPix Module</td><td>Phoenix Technology Group</td><td></td><td>Fundus imaging and acquisition targeting</td></tr><tr><td>Photoshop</td><td>Adobe</td><td></td><td></td></tr><tr><td>Refresh</td><td>Allergan</td><td>94170</td><td></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>

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.

These analysis methods allow for the quantification of retinal pathology captured by FA and OCT imaging. The experiments from which the representative data is extracted used C57BL/6J male mice who either served as uninjured controls or underwent the RVO procedure and received either Pen1-XBir3 treatment eyedrops or Pen1-Saline vehicle eyedrops. The RVO injury model involved the laser irradiation (532 nm) of the major veins in each eye of an anesthetized mouse following a tail-vein injection of rose bengal, a photoactivat...

Watch this Scientific Journal Video about In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility at JoVE.com

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).

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 ...

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JoVE Journal

Neuroscience

2.1K Views.  Columbia University. Dit protocol stelt de gebruiker in staat om klinisch analoge markers van schade in retinale beeldvorming van muizen kwantitatief te meten, wat de vertaalbaarheid van latere bevindingen versterkt. Deze methoden stellen ons in staat om de analyse te stroomlijnen en geven ons de mogelijkheid om op betrouwbare wijze beeldvorming tussen proefdieren te vergelijken. Hoewel deze methoden zijn ontwikkeld om een overzicht van muismodellen te bestuderen, kunnen ze zich gemakkelijk uitbreiden tot onderzo...

Video: In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Medicine

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

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). ...

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 ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

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.

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 ...

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, ...

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. ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

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