Retinal Pathophysiological Evaluation in a Rat Model

Summary

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.

Abstract

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.

Introduction

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) globally^2,3.

Diabetic retinopathy is diagnosed by vascular abnormalities in the posterior ocular tissues. Clinically, it is divided into two stages - Non-Proliferative DR (N....

Protocol

This protocol follows all the animal care guidelines provided by Institutional Animal Ethics Committee, BITS-Pilani, Hyderabad campus.

1. Retinal histology

1. Enucleation and fixation of the eye
   1. 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.
   .......

Discussion

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.......

Materials

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