Authors would like to acknowledge Indian Council of Medical Research (ICMR; ITR-2020-2882) for funding support to Dr. Nirmal J. We would also&#160;like to thank University Grant of Commission for providing Junior Research Fellowship to Manisha Malani and Central Analytical Laboratory Facility, BITS-Pilani, Hyderabad campus for providing infrastructural facility.

This protocol follows all the animal care guidelines provided by Institutional Animal Ethics Committee, BITS-Pilani, Hyderabad campus.
1. Retinal histology
<ol>
	<li>Enucleation and fixation of the eye
	<ol>
		<li>Euthanize a 2 to 3-month-old diabetic Wistar male rat along with the age-matched control (14 to 15 weeks old) using a high dose of pentobarbital (150 mg/kg) injected through the intraperitoneal route. No detectable heartbeat confirms the death within 2-5 min.</li>
		<li>Enucleate the eye by making incisions using a scalpel blade on the nasal and temporal regions of the eye. Then cut along its edges using forceps and micro scissors to remove the eye from the orbital socket. 
		NOTE: Before sacrificing rats, keep the fixative solutions ready. 
		CAUTION: Formaldehyde irritates the skin, eye, nose, and respiratory tract. It can also cause cancer as it is a potent skin sensitizer. Wear gloves and handle it under the hood21.</li>
		<li>Wash the eye thoroughly with 10 mL of phosphate-buffered saline (PBS) solution in a Petri dish to remove blood. Remove the excess fat surrounding the eye for easy penetration of the fixative solution.</li>
		<li>Immediately place the eye in 5 mL of fixative solution with the help of forceps, and make sure it is not stuck to walls of glass vials. Stir gently for a few seconds and replace the cap with proper labeling. Incubate eye in fixative solution for 24 to 48 h in dark conditions at room temperature.</li>
	</ol>
	</li>
	<li>Trimming of tissue
	<ol>
		<li>After fixation, remove the eye from the fixative solution, wash with 10 mL of PBS, and place it in a Petri dish containing 10 mL of PBS chilled at 4 &#176;C.</li>
		<li>Using micro forceps, hold the eye with the optic nerve and make a nick at pars plana with the help of micro scissors. Cut through the entire margin of the cornea to separate the anterior cup of an eye. Using forceps, pick the lens gently, discard it, and cut the optic nerve.</li>
		<li>Using curved micro scissors, make a longitudinal section of the posterior eyecup, passing through the optic disc dividing it into two halves. Place it in the base of the cassette and close the lid properly without any disturbance to the tissue. Label the cassettes with the tissue sample name and date.</li>
	</ol>
	</li>
	<li>Dehydration, clearing, and paraffin impregnation of tissue
	<ol>
		<li>Dehydrate the trimmed tissue by gradual transfer in 80 mL of 50%, 70%, 90%, and 100% ethanol. While transferring the cassettes from one concentration to another, make sure to dab them on clean tissue paper to minimize contamination. 
		NOTE: Allow the tissue to stand in each transfer for 30 min (twice). The total time consumed for this step is approximately 4 h.</li>
		<li>Upon dehydration, transfer the cassettes into 80 mL of xylene for 30 min (twice) to replace the ethanol with xylene. 
		NOTE: After incubation with xylene, the tissue becomes translucent. 
		CAUTION: Xylene is an aromatic compound with a benzene ring. It irritates the eye and mucous membrane and may also cause depressions.</li>
		<li>Finally, impregnate the tissue with pre-heated paraffin at 60 &#176;C to replace xylene in the tissue. Dab the cassettes several times on tissue paper to minimize the xylene content and place in 200 mL of paraffin (liquid at 60 &#176;C) for 2 h (1 h x 2). 
		&#8203;CAUTION: Avoid inhaling melted paraffin, as it produces tiny lipid droplets which may cause respiratory discomfort.</li>
	</ol>
	</li>
	<li>Paraffin embedding
	<ol>
		<li>Turn on the paraffin embedding machine at least 1 h before use to melt the paraffin and for the stations to reach desired temperatures. Fill a steel mold with melted paraffin wax by placing it on a hot surface, then remove one cassette at a time from the paraffin container.</li>
		<li>Move the steel mold from a hot to a cold surface (4 &#176;C). At this point, the wax in the mold will start to solidify. Before it solidifies completely, place the tissue in wax with the help of forceps and orient the tissue so that the optic disc portion of the posterior cup is facing the base of the mold. 
		NOTE: Ensure that the tissue does not move and keep its desired orientation. If not, put the mold back onto the warm work surface until the whole paraffin liquefies, then start again with step 1.4.2.</li>
		<li>As the wax in the mold starts solidifying, immediately place the cassette base on top of the mold. Carefully fill the mold with paraffin above the upper edge of the cassette and slowly transfer it to a cold surface (near -20 &#176;C) for rapid solidification. Until it solidifies, repeat the process with other cassettes from step 1.4.2.</li>
		<li>Once all the molds are solidified, separate the steel mold from the cassette. If it is hard, wait a bit longer and try again (do not remove it forcefully). Separation becomes easier upon complete solidification. Now the cassette becomes the base of the paraffin block to be held during sectioning by microtome. 
		NOTE: This block can be stored at room temperature for further use. At this point, the process can be stopped and continued later (if required).</li>
	</ol>
	</li>
	<li>Sectioning
	<ol>
		<li>Install a disposable microtome blade in the blade holder. Remove excess paraffin wax around cassettes so that both the upper and lower portions of the blocks remain parallel to the knife. Fit the block on to cassette holder.</li>
		<li>Unlock the hand-wheel to advance it until the surface of the block is in contact with the edge of the knife. Trim the excess paraffin wax on the block until the tissue is visualized on the surface of the block. Set the section thickness to 5 &#181;m and cut a ribbon of five to six sections.</li>
		<li>Using forceps, gently transfer the ribbon onto the surface of the pre-warmed water bath (around 50 &#176;C) to unfold the ribbon. Collect sections on a glass slide coated with Mayer&#39;s albumin by holding the slide beneath the section. Gently lift the sections and allow the slides to dry horizontally overnight at 37 &#176;C. 
		&#8203;NOTE: Slides can be stored at room temperature in dry boxes for several months. At this step, the process can be stopped and continued later.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Heat the slides to be stained in a hot air oven at 60 &#176;C for 1 h before use for the paraffin to melt, and follow the steps as mentioned in Table 1. 
		CAUTION: Hematoxylin and Eosin stains are toxic when inhaled or ingested. They have been reported to be carcinogenic.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Standing Time</td>
			<td>Repetition (Number of times)</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>90% Ethanol</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>50% Ethanol</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>4 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Water wash</td>
		</tr>
		<tr>
			<td>1% Acid alcohol in 70% Ethanol</td>
			<td>30 s</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Water wash</td>
		</tr>
		<tr>
			<td>Scott&#39;s water</td>
			<td>1 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Water wash</td>
		</tr>
		<tr>
			<td>50% Ethanol</td>
			<td>1 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>1 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>0.25% Eosin</td>
			<td>5 s</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="3">Water wash</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>2 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>1 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>1 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>5 min</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="3">Mountant and coverslip</td>
		</tr>
	</tbody>
</table>
Table 1. Hematoxylin and Eosin staining procedure
2. Blood-brain barrier breakdown assay
<ol>
	<li>Blood and vitreous collection
	<ol>
		<li>Anesthetize a 2 to 3-month-old diabetic Wistar male rat along with the age-matched control (14 to 15 weeks) using ketamine (80 mg/kg) and xylazine (8 mg/kg). Confirm the anesthetic state by pedal withdraw reflex (toe pinch). Immediately collect 1 mL of blood by cardiac puncture into an ethylenediaminetetraacetic acid (EDTA) coated tube to separate plasma from whole blood.</li>
		<li>Euthanize the rat using a high dose of pentobarbital (150 mg/kg), injected through the intraperitoneal route. No detectable heartbeat confirms the death within 2-5 min. Enucleate the eye and immediately place it on dry ice.</li>
		<li>Place the eye in a clean Petri dish above dry ice (recommended) and start dissecting the eye as mentioned in step 1.2.2.</li>
		<li>Remove the lens, pull the vitreous carefully from the posterior cup using micro forceps and place it in a homogenization tube containing three to four glass beads (2-4 mm). 
		&#8203;NOTE: Dissection on dry ice is recommended as removing the lens and vitreous is easier under freezing conditions.</li>
	</ol>
	</li>
	<li>Preparation of samples
	<ol>
		<li>Homogenize vitreous humor in a bead homogenizer at medium speed for 10 s.</li>
		<li>Transfer the blood (1 mL) and vitreous samples (10-20 &#181;L) into the microcentrifuge tubes and centrifuge both the samples at 5200 x g for 10 min at 4 &#176;C. 
		&#8203;NOTE: In the case of successful homogenization, vitreous has uniform consistency after centrifugation. However, in the case of non-uniform consistency of vitreous, repeat from step 2.2.1 with increased homogenization time.</li>
		<li>Collect supernatant from both samples, and dilute vitreous humor and plasma samples in 1:10 and 1:20 ratio, respectively, with PBS.</li>
	</ol>
	</li>
	<li>Protein quantification and vitreous-plasma protein ratio
	<ol>
		<li>To quantitate protein concentration in vitreous humor and plasma samples, add 5 &#181;L of diluted samples to 250 &#181;L of Bradford reagent, mix well, and read the absorbance at 590 nm within 40 min. 
		NOTE: If the absorption value of the samples is above 1.0, dilute the samples further and repeat the procedure from step 2.2.3.</li>
		<li>Normalize the vitreous protein level to plasma protein level from the same rat and measure the fold difference between healthy vs. diabetic rats.</li>
	</ol>
	</li>
</ol>
3. Fluorescence angiography
<ol>
	<li>FITC-dextran70000 dye injection
	<ol>
		<li>Anesthetize a 2 to 3-month-old diabetic Wistar male rat along with the age-matched control (14 to 15 weeks) using 3% isoflurane and confirm the pedal withdrawal reflex (toe pinch). Dip the tail in a beaker containing warm water (37-40 &#176;C). Clean the tail with 70% ethanol before injecting the dye. Locate the tail&#39;s lateral vein and mark the injection position in the lower portion of the tail. 
		NOTE: If the insertion of the needle fails, try to insert in another location above the current position (tip to the base of tail). However, it is advised to inject once as repeated pricking may lead to stress development in the rat, which could cause vasoconstriction.</li>
		<li>Inject 1 mL of 50 mg/mL FITC-dextran70000 dye solution through the tail vein and allow the dye to circulate for 5 min. Euthanize the rat with a high dose of pentobarbital (150mg/kg), injected through the intraperitoneal route. No detectable heartbeat confirms the death within 2-5 min. Enucleate the eye as mentioned in step 1.1.1. 
		NOTE: If required, the eye can be fixed in 4% formaldehyde solution, for no more than 30 min.</li>
	</ol>
	</li>
	<li>Flat mount preparation
	<ol>
		<li>For preparing flat mounts, keep the dissecting tools ready along with the slides and coverslips. Place the eye in a Petri dish filled with chilled PBS and start dissecting the eye as mentioned in step 1.2.2.</li>
		<li>Now place the tip of a pointed forcep between the sclera and retina. Gently move it all along the rim of the cup, making sure the retina is not attached to the sclera at any point. If there is any obstruction, slowly cut that portion along the rim using curved micro scissors.</li>
		<li>Once it is confirmed that the retina is not attached to the sclera from any side, cut near the optic disc, making a small hole such that the retina completely detaches from the sclera. Slowly push the retina into PBS solution and place it on a clean flat slide with the help of a spatula or forceps.</li>
		<li>Using micro scissors or scalpel blades, make minor cuts in the retinal tissue, dividing it into four quadrants. Make sure that the cuts are at least 2 mm away from the hole left by the optic disc at the center, as shown in Figure 1.</li>
		<li>Remove excess PBS from the sides of tissue using 0.2 mL tips without disturbing the flat mount.</li>
		<li>Place a drop of anti-fading mounting medium (50 &#181;L to 100 &#181;L) on a flat mount, cover with a coverslip, and visualize immediately under a confocal microscope. 
		NOTE: Maintain minimum light during the entire procedure.</li>
	</ol>
	</li>
	<li>Visualization of flat mount
	<ol>
		<li>Visualize the flat mount under 10x objective of a confocal microscope. Perform tile scanning to capture the entire flat mount as a single image. The number of tiles depends upon the size of the retinal flat mount. Also, perform Z-stacking to visualize the veins and arteries in different foci (preferred size for Z-stack is 5 &#181;m, depending on which, the number of Z-stack steps varies).</li>
		<li>Use PMT and HyD detector at % gain as 700-900 and 100-150, respectively. Choose emission range between 510-530 nm for FITC-dextran dye.</li>
	</ol>
	</li>
</ol>

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E., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+of+whole+eyes:+the+role+of+fixative+osmolarity+in+the+production+of+tissue+artifact.">Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 233 (6), 366-370 (1995).</li><li>Tokuda, 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=Optimization+of+fixative+solution+for+retinal+morphology:+a+comparison+with+Davidson's+fixative+and+other+fixation+solutions.">Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.</a> Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).</li><li>Luna, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third edition. , (1968).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+mouse+vitreous+and+retina+for+proteomic+analyses.">Evisceration of mouse vitreous and retina for proteomic analyses.</a> Journal of Visualized Experiments. (50), e2795 (2011).</li><li>D'Amato, R., Wesolowski, E., Smith, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+visualization+of+the+retina+by+angiography+with+high-molecular-weight+fluorescein-labeled+dextrans+in+the+mouse.">Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse.</a> Microvascular Research. 46 (2), 135-142 (1993).</li><li>Atkinson, E. G., Jones, S., Ellis, B. A., Dumonde, D. C., Graham, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+size+of+retinal+vascular+leakage+determined+by+FITC-dextran+angiography+in+patients+with+posterior+uveitis.">Molecular size of retinal vascular leakage determined by FITC-dextran angiography in patients with posterior uveitis.</a> Eye (Lond). 5, 440-446 (1991).</li></ol>

The authors declare that they have no competing financial interests.

Histology 
Retinal histology is performed to visualize the morphological changes of retinal cells and layers. Various steps, including choice of fixative solution, fixation duration, dehydration, and paraffin impregnation, need to be optimized. The tissue size should not exceed 3 mm, as the fixative penetration becomes slow. The commonly used 4% paraformaldehyde leads to retinal detachment even in the healthy eye due to the relatively high osmolarity of the solution compared to aqueous humor and vit...

Diabetic retinopathy (DR) is one of the most complex secondary complications of diabetes mellitus. It is also the leading cause of preventable blindness in the working-age population worldwide. In a recent meta-analysis of 32.4 million blind people, 830,000 (2.6%) people were blind due to DR1. The proportion of vision loss attributed to diabetes ranked seventh in 2015 at 1.06% (0.15-2.38) globally2,3.
Diabetic retinopathy is diagnosed by vascular abnormalities in the posterior ocular tissues. Clinically, it is divided into two stages - Non-Proliferative DR (NPDR) and Proliferative DR (PDR), based on the vascularization in the retina. Hyperglycemia is considered the potent regulator of DR as it implicates several pathways involved in neurodegeneration4,5, inflammation6,7, and microvasculature8 in the retina. Multiple metabolic complications induced due to hyperglycemia include the accumulation of advanced glycation end products (AGEs), polyol pathway, hexosamine pathway, and protein kinase-C pathway. These pathways are responsible for cell proliferation (endothelial cells), migration (pericytes), and apoptosis (neural retinal cells, pericytes, and endothelial cells) based on different stages of diabetic retinopathy. These metabolic alterations can lead to physiological changes such as retinal detachment, loss of retinal cells, breakdown of the blood-retinal barrier (BRB), aneurysms, and angiogenesis9.
Streptozotocin (STZ) induced type-1 diabetes is a well-established and well-accepted practice in rats for evaluating diabetes pathogenesis and its complications. Diabetogenic effects of STZ are due to selective destruction of pancreatic islet &#946;-cells10. As a result, the animals will undergo insulin deficiency, hyperglycemia, polydipsia, and polyuria, all of which are characteristic of human type-1 diabetes mellitus11. For severe diabetes induction, STZ is administered at 40-65 mg/kg body weight intravenously or intraperitoneally during adulthood. After approximately 72 h, these animals present blood glucose levels greater than 250 mg/dL10,12.
To understand the physiological alterations of the retina due to neurodegeneration, inflammation, and angiogenesis, different techniques should be optimized in experimental animal models. Structural and functional changes in retinal cells and retinal vessels can be studied by various techniques such as histology, BRB breakdown assay, and fluorescence angiography.
Histology involves the study of the anatomy of cells, tissues, and organs at a microscopic level. It establishes a correlation between the structure and function of cells/tissue. Several steps are performed to visualize and identify the microscopic alterations in tissue structure, thereby comparing healthy and diseased counterparts13. Hence, it is essential to standardize each step of histology meticulously. Various steps involved in retinal histology are fixation of the specimen, trimming the specimen, dehydration, clearing, impregnation with paraffin, paraffin embedding, sectioning, and staining (Hematoxylin and Eosin staining)13,14.
In a healthy retina, the transport of molecules across the retina is controlled by BRB, composed of endothelial cells and pericytes on the inner side, and retinal pigment epithelial cells on the outer side. However, inner BRB endothelial cells and pericytes start degenerating during the diseased condition, and BRB is also compromised15. Due to this BRB breakdown, many low molecular weight molecules leak into vitreous and retinal tissue16. As the disease progresses, many other protein molecules (low and high molecular weight) also leak into vitreous and retinal tissue due to homeostasis disturbance17. It leads to various other complications and ultimately macular edema and blindness. Hence, quantifying the protein levels in the vitreous and comparing healthy and diabetic states measures compromised BRB.
Fluorescence angiography is a technique used to study blood circulation of the retina and choroid using fluorescent dye. It is used to visualize vasculature of the retina and choroid by injecting fluorescein dye via intravenous route or cardiac injection18. Once the dye is injected, it first reaches the retinal arteries, followed by retinal veins. This circulation of dye is usually completed within 5 to 10 min from the injection of dye19. It is an important technique to diagnose various posterior segment ocular diseases, including diabetic retinopathy and choroidal neovascularization20. It helps to detect major and minor vasculature changes in normal and diseased conditions.

<table><tbody><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td></td><td>Anesthesia agent</td></tr><tr><td>Ketamine hydrochloride</td><td>Troikaa Pharmaceuticals</td><td></td><td>Anesthesia agent</td></tr><tr><td>Xylazine</td><td>Indian Immunologicals Limited</td><td></td><td>Anesthesia agent</td></tr><tr><td>Pentobarbital sodium</td><td>Zora Pharma</td><td></td><td>Euthanesia agent</td></tr><tr><td>Fixative solution (1 % formaldehyde, 1.25 % Glutaraldehyde</td><td>HiMedia, Avra</td><td>MB059, ASG2529</td><td>Prepared in-house</td></tr><tr><td>Ethanol</td><td>Hayman</td><td>F204325</td><td>Dehydration</td></tr><tr><td>Xylene</td><td>HiMedia</td><td>MB-180</td><td>Clearing of ethanol or paraffin</td></tr><tr><td>Paraffin wax</td><td>HiMedia</td><td>GRM10702</td><td>used for embedding tissue</td></tr><tr><td>Glycerol</td><td>HiMedia</td><td>TC503</td><td>To prepare albumin coated slides. Glycerol and egg albumin is mixed in 1:1 ratio to coat on slides</td></tr><tr><td>Hydrochloric acid</td><td>Sisco Research laboratories Pvt. Ltd.</td><td>65955</td><td>For preparation of 1 % acid alcohol</td></tr><tr><td>Acetic acid</td><td>HiMedia</td><td>AS119</td><td>For preparation of eosin</td></tr><tr><td>Scotts water</td><td>Leica</td><td>3802900</td><td>Bluing reagent</td></tr><tr><td>Papanicolaou&#039;s solution 1b Hematoxylin solution</td><td>Sigma</td><td>1.09254.0500</td><td>Staining of nuclei</td></tr><tr><td>Eosin</td><td>HiMedia</td><td>GRM115</td><td>Staining of cytoplasm, 0.25 % solution was prepared in-house</td></tr><tr><td>DPX Mountant media</td><td>Sigma</td><td>6522</td><td>Visualization and protection of retinal sections</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glassware</td><td>Borosil</td><td></td><td></td></tr><tr><td>Corneal forcep</td><td>Stephens Instruments</td><td>S5-1200</td><td>Dissection</td></tr><tr><td>Colibri forcep</td><td>Stephens Instruments</td><td>S5-1135</td><td>Dissection</td></tr><tr><td>Curved micro scissor</td><td>Stephens Instruments</td><td>S7-1311</td><td>Dissection</td></tr><tr><td>Vannas scissor</td><td>Stephens Instruments</td><td>S7-1387</td><td>Dissection</td></tr><tr><td>Iris scissor</td><td>Stephens Instruments</td><td>S7-1015</td><td>Dissection</td></tr><tr><td>Cassettes</td><td>HiMedia</td><td>PW1292</td><td>To hold tissue during histology processing</td></tr><tr><td>Water bath</td><td>GT Sonic</td><td>GT Sonic-D9</td><td>Temperature maintenance</td></tr><tr><td>Paraffin embedding station</td><td>Myr</td><td>EC 350</td><td>Preparation of paraffin blocks</td></tr><tr><td>Microtome</td><td>Zhengzhou Nanbei Instrument Equipment Co., Ltd.</td><td>YD-335A</td><td>Sectioning</td></tr><tr><td>Blades</td><td>Leica</td><td>Leica 818</td><td>Sectioning</td></tr><tr><td>Slides</td><td>HiMedia</td><td>BG005</td><td>Holding paraffin-tissue sections</td></tr><tr><td>Coverslips</td><td>HiMedia</td><td>BG014C</td><td>To cover tissue after adding mounting media</td></tr><tr><td>&lt;strong&gt;Blood Retinal Barrier breakdown&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>Anesthesia</td></tr><tr><td>Dry ice</td><td>Not applicable</td><td>Not applicable</td><td>Dissection</td></tr><tr><td>Bradford reagent</td><td>Sigma</td><td>B6916</td><td>Protein quantification</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Corneal forcep</td><td>Stephens Instruments</td><td>S5-1200</td><td>Dissection</td></tr><tr><td>Colibri forcep</td><td>Stephens Instruments</td><td>S5-1135</td><td>Dissection</td></tr><tr><td>Curved micro scissor</td><td>Stephens Instruments</td><td>S7-1311</td><td>Dissection</td></tr><tr><td>Vannas scissor</td><td>Stephens Instruments</td><td>S7-1387</td><td>Dissection</td></tr><tr><td>Iris scissor</td><td>Stephens Instruments</td><td>S7-1015</td><td>Dissection</td></tr><tr><td>Glassware</td><td>Borosil</td><td>Not applicable</td><td></td></tr><tr><td>EDTA coated tubes</td><td>J.K Diagnostics</td><td>Not applicable</td><td>Separate plasma from whole blood</td></tr><tr><td>Homogenization tubes</td><td>MP Biomedicals</td><td>SKU: 115076200-CF</td><td>Homogenization of vitreous</td></tr><tr><td>Homogenization caps</td><td>MP Biomedicals</td><td>SKU: 115063002-CF</td><td>Homogenization of vitreous</td></tr><tr><td>Glass beads</td><td>MP Biomedicals</td><td>SKU: 116914801</td><td>Homogenization of vitreous</td></tr><tr><td>Homogeniser</td><td>Bertin Instruments</td><td>P000673-MLYS0-A</td><td>Homogenization of vitreous</td></tr><tr><td>96-well plate - Transparent</td><td>Grenier</td><td>GN655101</td><td>Protein quantification</td></tr><tr><td>Plate reader</td><td>Molecular devices</td><td>SpectrMax M4</td><td>Absorbance measurement</td></tr><tr><td>Centrifuge</td><td>REMI</td><td>CPR240 Plus</td><td>Centrifugation</td></tr><tr><td>&lt;strong&gt;Fluorescence Angiography&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>Anesthesia</td></tr><tr><td>FITC-dextran 70 kD (FITC, Dextran, Dibutylin dilaurate, DMSO</td><td>FITC, Dextran and Dibutylin dilaurate from Sigma; DMSO from HiMedia</td><td>FITC-F3651,Dextran-31390,Dibutylin dilaurate -29123, DMSO-TC185</td><td>Prepared in-house</td></tr><tr><td>Fluoroshied</td><td>Sigma</td><td>F6182</td><td>Anti-fading mounting medium</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Corneal forcep</td><td>Stephens Instruments</td><td>S5-1200</td><td>Dissection</td></tr><tr><td>Colibri forcep</td><td>Stephens Instruments</td><td>S5-1135</td><td>Dissection</td></tr><tr><td>Curved micro scissor</td><td>Stephens Instruments</td><td>S7-1311</td><td>Dissection</td></tr><tr><td>Vannas scissor</td><td>Stephens Instruments</td><td>S7-1387</td><td>Dissection</td></tr><tr><td>Iris scissor</td><td>Stephens Instruments</td><td>S7-1015</td><td>Dissection</td></tr><tr><td>Glassware</td><td>Borosil</td><td>Not applicable</td><td></td></tr><tr><td>Slides</td><td>HiMedia</td><td>BG005</td><td>Flatmount preparation</td></tr><tr><td>Coverslips</td><td>HiMedia</td><td>BG014C</td><td>To cover tissue after adding mounting media</td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>DMi8</td><td>Visualization of flatmount</td></tr></tbody></table>

retinal pathophysiological evaluation in a rat model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Retinal histology 
In the diabetic retina, retinal cells undergo degeneration. In addition, the thickness of the retinal layers increase due to edema22. The images obtained after Hematoxylin and Eosin staining can be used for cell count and measurement of the thickness of different layers, as shown in Figure 2 using ImageJ.
Blood-retinal barrier breakdown assay 
As the BRB is compromised in diabet...

Watch this Scientific Journal Video about Retinal Pathophysiological Evaluation in a Rat Model at JoVE.com

Retinal Pathophysiological Evaluation in a Rat Model

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

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4.4K Views.  Birla Institute of Technology & Science — Pilani, Hyderabad Campus. Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Video: Retinal Pathophysiological Evaluation in a Rat Model

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Bioengineering

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

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

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

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

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

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

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 ...

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 ...

Neuroscience

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Sampling of N-methyl-N-nitrosourea-induced Rat's Retina Damage and Multi-index Evaluation of Pathological Changes

Retinopathy can be observed in most ocular diseases and the late complications of diabetes mellitus. The specific pigment epithelial cells and optic cells&#39; damage and degeneration are the main features of retinopathy. Many&#160;traditional medicines have shown substantial clinical efficacy in treating retinopathy. How to obtain a retina quickly and completely is a key step in traditional medicine research for the treatment of retinopathy. In this study, we aim to provide a standardized and exercisable procedure for sampling of N-methyl-N-nitrosourea (MNU)-induced rat&#39;s retina damage and multi-index evaluation of pathological changes. Rats were injected intraperitoneally with 60 mg/kg MNU once to induce retina damage, and retina samples were obtained after 7 days. Additionally, we performed hematoxylin-eosin staining to assess retinal pathological&#160;changes.&#160;Determination of the apoptosis rate and apoptosis protein by TUNEL and Western blot. These standardized protocols for retinal sampling and evaluation of pathological changes are helpful in promoting the exploration of the mechanism of retinopathy and the discovery of novel and effective traditional herbs.

This protocol describes a procedure for rat retinal sampling and utilizing hematoxylin-eosin staining, TUNEL assay, and Western blot to detect pathological changes and apoptosis in the retina after intraperitoneal injection of N-Methyl-N-Nitrosourea.

Retinopathy can be observed in most ocular diseases and the late complications of diabetes mellitus. The specific pigment epithelial cells and optic ...

Retinal Pathophysiological Evaluation in a Rat Model

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Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

Sampling of N-methyl-N-nitrosourea-induced Rat's Retina Damage and Multi-index Evaluation of Pathological Changes