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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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) globally2,3.

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.
    2. .......

Representative Results

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.......

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.......

Acknowledgements

Authors would like to acknowledge Indian Council of Medical Research (ICMR; ITR-2020-2882) for funding support to Dr. Nirmal J. We would also 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.

....

Materials

NameCompanyCatalog NumberComments
Histology
Reagents
IsofluraneAbbottAnesthesia agent
Ketamine hydrochlorideTroikaa PharmaceuticalsAnesthesia agent
XylazineIndian Immunologicals LimitedAnesthesia agent
Pentobarbital sodiumZora PharmaEuthanesia agent
Fixative solution (1 % formaldehyde, 1.25 % GlutaraldehydeHiMedia, AvraMB059, ASG2529Prepared in-house
EthanolHaymanF204325Dehydration
XyleneHiMediaMB-180Clearing of ethanol or paraffin
Paraffin waxHiMediaGRM10702used for embedding tissue
GlycerolHiMediaTC503To prepare albumin coated slides. Glycerol and egg albumin is mixed in 1:1 ratio to coat on slides
Hydrochloric acidSisco Research laboratories Pvt. Ltd.65955For preparation of 1 % acid alcohol
Acetic acidHiMediaAS119For preparation of eosin
Scotts waterLeica3802900Bluing reagent
Papanicolaou's solution 1b Hematoxylin solutionSigma1.09254.0500Staining of nuclei
EosinHiMediaGRM115Staining of cytoplasm, 0.25 % solution was prepared in-house
DPX Mountant mediaSigma6522Visualization and protection of retinal sections
Equipments
GlasswareBorosil
Corneal forcepStephens InstrumentsS5-1200Dissection
Colibri forcepStephens InstrumentsS5-1135Dissection
Curved micro scissorStephens InstrumentsS7-1311Dissection
Vannas scissorStephens InstrumentsS7-1387Dissection
Iris scissorStephens InstrumentsS7-1015Dissection
CassettesHiMediaPW1292To hold tissue during histology processing
Water bathGT SonicGT Sonic-D9Temperature maintenance
Paraffin embedding stationMyrEC 350Preparation of paraffin blocks
MicrotomeZhengzhou Nanbei Instrument Equipment Co., Ltd.YD-335ASectioning
BladesLeicaLeica 818Sectioning
SlidesHiMediaBG005Holding paraffin-tissue sections
CoverslipsHiMediaBG014CTo cover tissue after adding mounting media
Blood Retinal Barrier breakdown
Reagents
IsofluraneAbbottB506Anesthesia
Dry iceNot applicableNot applicableDissection
Bradford reagentSigmaB6916Protein quantification
Equipments
Corneal forcepStephens InstrumentsS5-1200Dissection
Colibri forcepStephens InstrumentsS5-1135Dissection
Curved micro scissorStephens InstrumentsS7-1311Dissection
Vannas scissorStephens InstrumentsS7-1387Dissection
Iris scissorStephens InstrumentsS7-1015Dissection
GlasswareBorosilNot applicable
EDTA coated tubesJ.K DiagnosticsNot applicableSeparate plasma from whole blood
Homogenization tubesMP BiomedicalsSKU: 115076200-CFHomogenization of vitreous
Homogenization capsMP BiomedicalsSKU: 115063002-CFHomogenization of vitreous
Glass beadsMP BiomedicalsSKU: 116914801Homogenization of vitreous
HomogeniserBertin InstrumentsP000673-MLYS0-AHomogenization of vitreous
96-well plate - TransparentGrenierGN655101Protein quantification
Plate readerMolecular devicesSpectrMax M4Absorbance measurement
CentrifugeREMICPR240 PlusCentrifugation
Fluorescence Angiography
Reagents
IsofluraneAbbottB506Anesthesia
FITC-dextran 70 kD (FITC, Dextran, Dibutylin dilaurate, DMSOFITC, Dextran and Dibutylin dilaurate from Sigma; DMSO from HiMediaFITC-F3651,Dextran-31390,Dibutylin dilaurate -29123, DMSO-TC185Prepared in-house
FluoroshiedSigmaF6182Anti-fading mounting medium
Equipments
Corneal forcepStephens InstrumentsS5-1200Dissection
Colibri forcepStephens InstrumentsS5-1135Dissection
Curved micro scissorStephens InstrumentsS7-1311Dissection
Vannas scissorStephens InstrumentsS7-1387Dissection
Iris scissorStephens InstrumentsS7-1015Dissection
GlasswareBorosilNot applicable
SlidesHiMediaBG005Flatmount preparation
CoverslipsHiMediaBG014CTo cover tissue after adding mounting media
Confocal microscopeLeicaDMi8Visualization of flatmount

References

  1. Jonas, J. B., Sabanayagam, C. Epidemiology and risk factors for diabetic retinopathy. Diabetic Retinopathy and Cardiovascular Disease. 27, 20-37 (2019).
  2. Pandova, M. G. . Visual Impairment and Blindness. , (2019).
  3. Mokdad, A. H., et al.

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