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 central vision³. Therefore, inhibition of retinal photoreceptor apoptosis is the pointcut for the prevention and treatment of RP.

Retinal cell apoptosis is a common feature of human RP and model animals⁴. Intraperitoneal injection of 60 mg/kg N-Methyl-N-Nitrosourea (MNU) in rats for 7 days can induce apoptosis and loss of retinal photoreceptor cells, and it is a commonly used animal model of RP^5,6. Analyzing the changes in pathological structure, specific cell apoptosis, and apoptosis-related protein expression of retinal tissue in model animals can provide effective experimental data and theoretical support in studying the pathogenesis of human RP and screening drugs^7,8. Therefore, the quality of retinal specimens determines the reliability of experimental data. However, due to the particularity of ocular tissue, there are very few reports on how to get the rat retina⁹.

This paper provides a simple, fast, standardized, and operable procedure for retinal sampling in rats to overcome the above shortcomings. Hematoxylin-eosin staining (HE), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) staining, and Western blot are used to analyze the pathological changes in retinal tissue damage and apoptosis in the rats. All the methods are derived from our research group's experience with the specific operational processes.

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 plates, inhalation anesthesia devices, embedded boxes, frozen storage tubes, disposable surgical gloves and masks, wide-mouth bottles, 4% paraformaldehyde, ice packs, ophthalmic scissors, ophthalmic tweezers, surgical blades and knife handles, 1.5 mL microcentrifuge tubes and sterilize them in advance (see Table of Materials).
2. Animal preparation
   1. Randomly divide 20 male SD rats into model group (n=10) and control group (n=10). Inject model rats intraperitoneally with 60 mg/kg MNU and inject control rats intraperitoneally with the same volume of saline.
   2. After 7 days of injection, fast the rats and allow them to drink freely the night before sampling.
   3. The next day, place the rat supine on the operating table and use a mask to administer anesthesia using 4% isoflurane and 2 L/min oxygen. Measure the depth of anesthesia by pinching the center of the foot to observe the rats' reactions.

2. Retinal sampling for protein factor detection

1. Fix the eye socket of the rat with the left hand and remove the eye with moderate force with the right hand using an ophthalmic bending forceps. Place the eyeball in a sagittal position in a cold glass dish.
2. With the left hand holding ophthalmic straight tweezers to fix the eyeball and the right hand holding the surgical blade, make a 2 mm longitudinal incision near the lens site.
3. Cut off the cornea with the ophthalmic scissors along the incision, and gently peel off the lens, vitreous, and expose eye cup.
4. Place the bottom of the eye cup at the tip of the 1.5 mL microcentrifuge tube, turn the eye cup over the tube, and expose the retina (the retina is faint yellow).
5. Remove the entire retina along the scleral wall of the eyeball with ophthalmic bending tweezers, then place it in a frozen tube and store it in a liquid nitrogen tank for future use (Figure 1).

3. Retinal sampling for pathological examination and specific cell staining

1. With the left hand, use the eye tweezers to lift the corner of the rat's eye. With the right hand, use a surgical blade to cut a 4 mm opening along the corner of the rat's eye.
2. Use the eye tweezers to clamp the edge of the eyeball at the opening in the left hand. Use ophthalmic scissors to cut off the surrounding tissues along the edge of the eyeball while paying attention to cleaning the surrounding tissues of the eyeball.
3. Separate the tissues around the optic nerve at the posterior pole of the eyeball with eye tweezers, cut the optic nerve, and pay attention to preserving it. Then, remove the eyeball.
4. Wash the removed eyeball in saline to remove any remaining blood, roll it on filter paper to absorb excess water, and place it on cold tin foil.
5. Fix the eye with ophthalmic tweezers. Make a 4 mm longitudinal incision along the junction of the cornea and retina with a surgical blade.
6. Remove the cornea with ophthalmic scissors, make a circular cut along the incision, and gently remove the lens and vitreous body.
7. Place the remaining eye cup with an optic nerve in an embedding box and fix it in 4% paraformaldehyde.
8. Embed paraformaldehyde-fixed samples in paraffin and cut into 4 µm slices for HE staining and TUNEL staining⁷.

4. HE staining of rat retina

1. Dewaxing and hydration: Place the slices in xylene for 10 min, repeat the process for another 10 min, followed by absolute ethanol for 5 min, repeat ethanol treatment for another 5 min, followed by treatment in 95% alcohol for 5 min, then in 85% alcohol for 5 min, and finally in 75% alcohol for 5 min.
2. Hematoxylin staining: Rinse the slice with deionized water for 5 min. Use 100 µL of hematoxylin staining solution and stain for 5 min, rinse with deionized water for 5-10 s. Add 100 µL of 1% hydrochloric acid ethanol for 30 s, rinse with deionized water for 20 min, and add 100 µL of 0.2% ammonia water for 1 min. Rinse with deionized water for 5 min.
3. Eosin staining: Use 100 µL of eosin staining solution to stain for 5 min and rinse with deionized water for 30 s.
4. Dehydration and seal: Immerse the slices in 70% alcohol for 5 min, then 85% alcohol for 5 min, followed by 90% alcohol for 5 min, and absolute ethanol for 5 min. Repeat the absolute ethanol treatment for another 5 min, then immerse in xylene for 5 min, and repeat the process for another 5 min. Finally, seal by adding approximately 100 µL of neutral resin to the surface of the slices and then cover it with a cover glass.
5. Imaging the slides: Position the stained slices on the microscope, adjust the microscope's focus until the image is clearly visible, set the magnification at 400x, and capture the image.
6. Measure the total retinal thickness and outer retinal thickness: Measure the total retinal thickness and outer retinal thickness (thickness of the outer nuclear layer and photoreceptor layer), and calculate the outer retinal thickness percentage
   Outer retinal thickness (%) = (outer retinal thickness/ total retinal thickness) x 100.

5. Determination of apoptosis rate of retinal photoreceptor cells with TUNEL method

1. Dewaxing and hydration: Place the slices in xylene for 10 min, repeat the process for another 10 min, then place in absolute ethanol for 5 min, and repeat this step for another 5 min. Perform dehydration by placing in 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and rinse with deionized water for 5 min.
2. Rinse with 0.85% sodium chloride solution for 5 min and PBS for 5 min. Fix with paraformaldehyde using 4% paraformaldehyde for 15 min. Rinse with PBS for 5 min, and repeat the process for another 5 min.
3. Incubation and equilibration⁷: Incubate with 20 µg/mL proteinase K solution for digestion for 15 min, equilibrate in the equilibrium buffer for 10 min, and incubate with recombinant terminal deoxynucleotidyltransferase (rTdT) buffer at 37 °C for 60 min.
4. Stopping reaction: Add 100 µL the standard citrate solution (2x SSC solution) for 15 min. Rinse with PBS for 5 min, and repeat the process for another 5 min.
5. Nuclear dye: Add 100 µL 4 ′, 6-diamidine-2-phenindl (DAPI) reagent for 5 min. Wash with deionized water for 5 min, and repeat the process for another 5 min.
6. Seal: Wipe excess xylene around the slices, and seal by adding approximately 100 µL of neutral resin to the surface of the slices, and then cover it with a cover glass.
7. Microscopy: Place the stained slices on the microscope, adjust the microscope's focus for clear visibility, set the magnification to 400x, and capture the image. Observe the green fluorescence at 520 ± 20 nm using a standard fluorescence filter. Observe the blue DAPI at 460 nm.
8. Calculate the apoptosis rate: measure the green fluorescence values and blue fluorescence values of the outer retinal nucleus layer. Calculate the apoptosis rate as
   Apoptosis cell rate = green fluorescence value/blue fluorescence value x 100.

6. Western blot analysis ¹⁰

1. Lysis buffer treatment: Mince and homogenize one frozen retina with 50 µL of cold lysis buffer (1 mL of lysis buffer containing 5 µL of phosphatase inhibitor, 1 µL of protease inhibitor, and 5 µL of 100 mM PMSF).
2. Subject to a 28 kHz sonication for 5 s on an ice bath; repeat 3x. Centrifuge at 4 °C for 5 min at 10,000 x g to remove cellular debris. Keep the supernatant.
3. em>6.3Quantification of protein: Determine the protein concentration using a BCA protein assay reagent kit.
4. em>6.4Electrophoresis: Load 50 µg protein sample and separate on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
5. Transfer to membrane: Electrophoretically transfer to PVDF membranes using an electrophoretic transfer system.
6. Subdivide membranes: Subdivide the membranes, and analyze each protein of interest, β-actin and GAPDH from a single transfer.
7. Blocking: Block the membranes for 1 h in TBST containing 5% non-fat milk at room temperature.
8. Incubate primary antibody: Incubate with primary antibody of cleaved caspase-9 (1:200 dilution), cleaved caspase-3 (1:200 dilution), cleaved caspase-7 (1:200 dilution), and β-actin (1:1000 dilution) overnight at 4 °C. Wash the membranes with TBST for 5 min, repeat 3x.
9. Incubate second antibody: Incubate with horseradish peroxidase-conjugated secondary antibodies (1:2000) for 2 h at room temperature. Wash the membranes with TBST for 5 min, repeat 3x.
10. Exposure: Visualize the labeled proteins using Western blot substrate, and finally, expose the membranes to visualize the protein bands in an automatic exposure mode.
11. Calculate the protein content⁷ by measuring the protein band density.

7. Statistical analysis

1. Analyze the data using the SPSS 19.0 software and present it as the mean ± standard deviation. Perform statistical analyses of observed values using one-way analysis of variance. Take p< 0.05 as statistically significant.

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

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