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

Summary

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.

Abstract

Retinopathy can be observed in most ocular diseases and the late complications of diabetes mellitus. The specific pigment epithelial cells and optic cells' damage and degeneration are the main features of retinopathy. Many 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'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 changes. 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.

Introduction

Retinitis pigmentosa (RP) is a hereditary and blinding retinal disease¹. The incidence of RP is between 1/3500 and 1/5000, affecting the visual function of about 2.5 million people in the world. It is one of the most well-known diseases leading to visual impairment in human beings, imposing a significant burden on the whole society². The disease is characterized by the gradual loss of retinal pigment epithelial cell function and the progressive apoptosis of photoreceptors. In the early stage, patients experience night blindness, which manifests as peripheral visual field defects and eventually leads to the loss of centra....

Protocol

All protocols and surgical procedures were approved by the Ethics Committee of Ningxia Medical University (Ethics number: NO. 2020-Q066). SPF Sprauge-Dawley (SD) male rats, aged 7-8 weeks and weighing 200-220 g, were purchased from Ningxia Medical University with the animal license number SCXK (Ning) 2020-0001. All the animals were raised in the Laboratory Animal Center of Ningxia Medical University. The temperature and humidity were suitable, the day-night light cycle was maintained, and the food and water were free and sufficient.

1. Preoperative preparation

1. Preparation of materials
   1. Prepare rat pla....

Results

After HE staining, all retinal layers of rats in the control group had clear tissue structure, orderly cell arrangement, and uniform staining, while the retinal structure of rats in the model group was significantly damaged, the outer retinal layer was thinner, the outer nuclear layer was almost completely integrated with the inner core layer, the arrangement of cells in the layer was extremely disordered, the number of cell layers was significantly reduced, and the thickness of the photosensitive cell layer and outer nu.......

Discussion

RP is a common retinal disease in clinics. The onset, severity, and progression of RP are related to genes and genetic modes and are affected by the environment^8,11. RP includes familial RP and occasional RP, of which familial RP accounts for about 60% of the patients. Through tracing the family genetic history, 83 genes related to RP have been identified so far, while occasional RP accounts for about 40%, which means this kind of patient lacks a family history a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amersham Imager	GE	680
Ammonium persulfate	Boster Biological Technology Co.,Ltd	A8090
Analytical Balance	Mettler Toledo	ME104E
BCA protein quantization kit	Ken Gen Biotech. Co. Ltd	KGP902
cleaved caspase-3 antibody	Cell Signaling Technology	#9664
cleaved caspase-7 antibody	Cell Signaling Technology	#8438
cleaved caspase-9 antibody	Cell Signaling Technology	#9507
DeadEnd Fluorometric TUNEL System	Promega	G3250
Glycine	Boster Biological Technology Co.,Ltd	AR1200
Goat Anti-Rabbit IgG H&L (HRP)	Bioss Antibodies	bs-80295G-HRP
Goat serum	Biosharp	BL210A
High speed  crusher	Thermo Fisher Scientific	AG22331
Immobilon-P SQ Transfer membranes	Merck Millipore. Ltd	ISEQ00010
Isoflurane	RWD Life Science	R510-22-10
Methanol	Chengdu Kelong Chemical Co., Ltd	20230108
Microplate Reader	Thermo Fisher Scientific	1510
Microscope	Olympus	IX73
Microscope slide	Citotest Labware Manufacturing Co., Ltd	7105P-G
Mini-PROTEAN Tetra	BIO-RAD	1658001
N-Methyl-N-Nitrosourea	sigma-Aldrich	N4766–100G
Oven	Shanghai Yuejin Medical Equipment Co., Ltd	DHG-8145
Page Pre-solution (30% )	Doublehelix Biology Science and Technology Co.,Ltd	L3202A
PageRuler Prestained Protein Ladder	Thermo Fisher Scientific	26617
PBS buffer	Biosharp	G4202
SDS-PAGE Protein loading buffer (5×)	Beyotime Biotechnology	P0015
Skim milk powder	BioFroxx	1172GR500
Sprague Dawley rats	Ningxia Medical University	SCXK (Ning) 2020-0001
TEMED	Boster Biological Technology Co.,Ltd	AR1165
Total protein extraction kit	Ken Gen Biotech. Co. Ltd	KGP2100
Trans-Blot Module	BIO-RAD	1703935
Tris base	Boster Biological Technology Co.,Ltd	AR1162
Tweezer	Changde BKMAM Biotechnology Co., Ltd	130302027
β-actin	Cell Signaling Technology	#4970