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>
...

<ol>
	<li>Jonas, J. B., Sabanayagam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+risk+factors+for+diabetic+retinopathy.">Epidemiology and risk factors for diabetic retinopathy.</a> Diabetic Retinopathy and Cardiovascular Disease. 27, 20-37 (2019).</li><li>Pandova, M. 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> Visual Impairment and Blindness. , (2019).</li><li>Mokdad, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+national,+and+subnational+big+data+to+inform+health+equity+research:+perspectives+from+the+Global+Burden+of+Disease+Study+2017.">Global, regional, national, and subnational big data to inform health equity research: perspectives from the Global Burden of Disease Study 2017.</a> Ethnicity &amp; Disease. 29, 159-172 (2019).</li><li>Barber, A. J., 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=Neural+apoptosis+in+the+retina+during+experimental+and+human+diabetes.+Early+onset+and+effect+of+insulin.">Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.</a> The Journal of Clinical Investigation. 102 (4), 783-791 (1998).</li><li>El-Asrar, A. M. A., Dralands, L., Missotten, L., Al-Jadaan, I. A., Geboes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+apoptosis+markers+in+the+retinas+of+human+subjects+with+diabetes.">Expression of apoptosis markers in the retinas of human subjects with diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 45 (8), 2760-2766 (2004).</li><li>Schr&#246;der, S., Palinski, W., Schmid-Sch&#246;nbein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+monocytes+and+granulocytes,+capillary+nonperfusion,+and+neovascularization+in+diabetic+retinopathy.">Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy.</a> The American Journal of Pathology. 139 (1), 81 (1991).</li><li>Miyamoto, 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=Prevention+of+leukostasis+and+vascular+leakage+in+streptozotocin-induced+diabetic+retinopathy+via+intercellular+adhesion+molecule-1+inhibition.">Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.</a> Proceedings of the National Academy of Sciences. 96 (19), 10836-10841 (1999).</li><li>Bhanushali, D., 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=Linking+retinal+microvasculature+features+with+severity+of+diabetic+retinopathy+using+optical+coherence+tomography+angiography.">Linking retinal microvasculature features with severity of diabetic retinopathy using optical coherence tomography angiography.</a> Investigative Ophthalmology &amp; Visual Science. 57 (9), 519-525 (2016).</li><li>Wang, W., Lo, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy:+pathophysiology+and+treatments.">Diabetic retinopathy: pathophysiology and treatments.</a> International Journal of Molecular Sciences. 19 (6), 1816 (2018).</li><li>Akbarzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+diabetes+by+streptozotocin+in+rats.">Induction of diabetes by streptozotocin in rats.</a> Indian Journal of Clinical Biochemistry. 22 (2), 60-64 (2007).</li><li>Weiss, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozocin:+a+review+of+its+pharmacology,+efficacy,+and+toxicity.">Streptozocin: a review of its pharmacology, efficacy, and toxicity.</a> Cancer Treatment Reports. 66 (3), 427-438 (1982).</li><li>Karunanayake, E. H., Hearse, D. J., Mellows, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolic+fate+and+elimination+of+streptozotocin.">The metabolic fate and elimination of streptozotocin.</a> Biochemical Society Transactions. 3 (3), 410-414 (1975).</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. , (1968).</li><li>Okunlola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+studies+on+the+retina+and+cerebellum+of+Wistar+rats+treated+with+Arteether.">Histological studies on the retina and cerebellum of Wistar rats treated with Arteether.</a> Journal of Morphological Sciences. 31 (01), 028-032 (2014).</li><li>Wallow, I., Engerman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+patency+of+retinal+blood+vessels+in+experimental+diabetes.">Permeability and patency of retinal blood vessels in experimental diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 16 (5), 447-461 (1977).</li><li>do Cartmo, A., Ramos, P., Reis, A., Proen&#231;a, R., Cunha-Vaz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakdown+of+the+inner+and+outer+blood+retinal+barrier+in+streptozotocin-induced+diabetes.">Breakdown of the inner and outer blood retinal barrier in streptozotocin-induced diabetes.</a> Experimental Eye Research. 67 (5), 569-575 (1998).</li><li>Shires, T., Faeth, J., Pulido, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+levels+in+the+vitreous+of+rats+with+streptozotocin-induced+diabetes+mellitus.">Protein levels in the vitreous of rats with streptozotocin-induced diabetes mellitus.</a> Brain Research Bulletin. 30 (1-2), 85-90 (1993).</li><li>D'amato, R., Wesolowski, E., Smith, L. H. <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>Gupta, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+angiography+refresher+course:+Here's+how+to+interpret+the+findings+of+this+useful+diagnostic+tool.">Fluorescein angiography refresher course: Here's how to interpret the findings of this useful diagnostic tool.</a> Review of Optometry. 138 (11), 60-65 (2001).</li><li>Edelman, J. L., Castro, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+image+analysis+of+laser-induced+choroidal+neovascularization+in+rat.">Quantitative image analysis of laser-induced choroidal neovascularization in rat.</a> Experimental Eye Research. 71 (5), 523-533 (2000).</li><li>Szab&#243;, 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=Histological+evaluation+of+diabetic+neurodegeneration+in+the+retina+of+Zucker+diabetic+fatty+(ZDF)+rats.">Histological evaluation of diabetic neurodegeneration in the retina of Zucker diabetic fatty (ZDF) rats.</a> Scientific Reports. 7 (1), 1-17 (2017).</li><li>Margo, C. 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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</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&#39;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>Equipments</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>Blood Retinal Barrier breakdown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</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>Equipments</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>Fluorescence Angiography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</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>Equipments</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 ...

The overall goal of these techniques is to evaluate the retinal pathophysiology in streptozotocin-induced diabetic retinopathy rat model. The diabetic retinopathy is one of the leading cause of blindness. The continuous search is going on to discover pharmacological agents with better efficacy and longer action to treat diabetic retinopathy.The process of discovering pharmacological agents in pre-clinical animal models plays a vital role. These animal models helps to understand the structural and functional alterations of the posterior segment tissues of the eye. Therefore, we'll be demonstrating three different techniques to study these alterations in the posterior segment issues.We are demonstrating histology to study morphological changes of retinal cells in layers, BRB breakdown assay to study compromised BRB by quantifying leaked protein in the vitreous from plasma, and also fluoresce angiography to visualize retinal vasculature using FITC-dextran dye. Euthanize rat using high dose of pentobarbital and confirm its death with no detectable heartbeat. Enucleate the eye by making incisions using a scalpel blade on the nasal and temporal regions of the eye.Then cut along its edges to remove the eye from the orbital socket. Remove the excess fat surrounding the eye and place it in 5 mL of fixative solution. Incubate the eye in fixative solution for 24 to 48 hours.After fixation, remove the eye from the fixative solution and transfer to Petri dish filled with 10 mL PBS. Using micro forceps, hold the eye with 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 and posterior cup of eye.Remove the lens and cut the optic nerve. Cut the posterior cup into two halves, passing through the optic nerve. Transfer each half into the separate cassettes.Dehydrate the tissue by gradually increasing the concentration of ethanol from 50%to 100%ethanol. And finally, clear the ethanol with xylene. Impregnate the tissue with pre-heated paraffin at 60 degrees Celsius to replace xylene in the tissue.Prepare the paraffin blocks using a steel mold to hold the tissue and cassette as its base. While preparing the block, the optic disc portion of the eye should face the bottom of the steel mold. Fill the cassette with paraffin and transfer it to a cool surface.Mount the blade and cassette onto the respective holders on microtome. Using a microtome, trim the excess paraffin wax and cut a ribbon of five to six sections with a thickness of 5 micrometer. Transfer the ribbon onto the surface of pre-warmed water bath to unfold the ribbon.Collect the sections on a glass slide coated with albumin. Allow the slides to dry overnight at 37 degrees Celsius. To perform Hematoxylin and Eosin staining, preheat the slides at 60 degrees Celsius for 1 hour to melt the paraffin.Follow the staining procedure as given in manuscript. Begin with the rehydration of tissue, and then stain with Hematoxylin followed by Eosin. Now, dehydrate the tissue and add mounting medium on the sections.Place the cover slip on sections and visualize under light microscope. To perform blood-retinal barrier breakdown assay, anesthetize the rat using ketamine and xylazine and confirm the anesthetic state by toe pinch. Locate the heart and insert 1 mL syringe with 24-gauge needle to draw the blood by cardiac puncture.Once the sufficient blood is collected, transfer it to EDTA-coated tube. Mix it gently to prevent hemolysis. Enucleate the eye and immediately transfer it to dry ice.Under frozen conditions, begin the dissection of eye by removal of cornea and lens. Pull the vitreous from posterior segment of eye using forceps without any contamination from retina. Transfer it to homogenizing tube with three to four beads.Homogenize the samples using bead homogenizer at medium speed for 10 seconds. Transfer blood and vitreous humor samples into microcentrifuge tubes to process further. Centrifuge both the samples at 5, 200 g for 10 minutes at 4 degrees Celsius.Using a multichannel pipette, add 250 microliters of Bradford reagent into wells. Dilute vitreous samples 10 times and plasma samples 20 times using PBS. Add 5 microliters of diluted samples to Bradford reagent and mix well.Incubate the samples for 20 to 30 minutes, and then measure the absorbance at 590 nanometer using a 96-well plate reader. Intensity of sample is directly proportional to the concentration of protein. To perform fluorescence angiography, anesthetize the rat using 3%isoflurane and confirm the anesthetic state by toe punch.Dip the tail in warm water before injecting the dye. Inject 1 mL of 50 mg per mL FITC-dextran solution into lateral tail vein. Allow the dye to circulate for 5 to 10 minutes.Euthanize the rat using pentobarbital and confirm its death by no detectable heartbeat. Enucleate the eye and proceed for flat mount preparation. To prepare retinal flat mount, place the eye on a fiber-free Kimwipe and cut the anterior portion of eye.Slowly pull the lens, along with the vitreous humor, from the posterior segment of eye. Transfer the posterior segment into Petri dish filled with PBS and cut the optic nerve. Now, place the tip of the pointed forcep between sclera and retina.Gently move it all along the rim of the cup to make sure that retina is not attached to sclera at any point. Cut near the optic disc, such that retina completely detaches from the sclera. Once it is confirmed that retina is not attached to sclera on any side, push it slowly into the PBS solution.With the help of a forcep, transfer retina onto a clean glass slide without causing any damage to it. Using micro scissor or scalpel blade, make four cuts on the retina, as shown, to divide it into four quadrants. Remove excess PBS from retinal surroundings and add anti-fading mounting medium on the retinal flat mount.Cover it with a coverslip and visualize the flat mount under confocal microscope. It is important to note here that the entire process of fluorescence angiography is performed under minimum light to avoid the quenching of fluorescence from FITC-dextran dye. In diabetic rat, the retinal cells undergo degeneration, which leads to further complications.These structural alterations can be visualized by histology technique to determine the cell number and thickness of retina. In case of diabetic rats, the thickness of retinal layers increases due to edema. Hence, this technique helps to screen various potential pharmacological agents to treat diabetic retinopathy.Metabolic changes in retina due to diabetes causes loss of pericytes and breakdown of blood-retinal barrier. As there is leakage of protein and other biomolecules into retina and vitreous, the levels of total protein increase in the vitreous of diabetic rats. Preventing the breakdown of blood-retinal barrier could significantly hamper the progress of diabetic retinopathy.In diabetic retinopathy, expression of vascular endothelial growth factor increases that lead to formation of new vessels. These vessels disturb the normal retinal structure, and therefore its innervation is of primary concern to treat diabetic retinopathy. Fluorescence angiography technique is utilized to visualize new blood vessels and also leakage of blood vessels, as highlighted here.However, angiogenesis is prominent only after six to 12 months of diabetes induction. This technique helps us to quantify the effect of drug treatment on innervation of angiogenesis and vascular leakage. The future treatment of diabetic retinopathy needs more efficient pharmacological agents, but the future treatments can only be achieved through better understanding of the disease pathogenesis.Mastering these techniques could help researchers to have a better understanding about pathogenesis of diabetic retinopathy, which could lead to a discovery of better pharmacological interventions.

Retinal Pathophysiological Evaluation in a Rat Model

Abstract