Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Summary

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

Abstract

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

Introduction

Cerebral stroke is a leading cause of death and disability worldwide¹. The high mortality and morbidity of stroke result in immense public healthcare burden and serious socio-economic consequences. Although the rate of stroke incidence and mortality remain stable, the number of stroke patients and stroke-related deaths are increasing over decades^2,3. Strokes can be categorized as ischemic or hemorrhagic. The majority of stroke cases are ischemic strokes caused by the occlusion of a main cerebral artery⁴. To date, tissue plasminogen activator (tPA) is the only FDA-approved drug for ischemic stroke, but its application is highly limited by the narrow therapeutic window, complications, and contraindications^5,6,7. Thus, it is urgent to develop new treatment options with a wider therapeutic window to mitigate the effects of stroke.

Experimental animal models are useful tools to study the pathophysiology of ischemic stroke. In most human patients, ischemic stroke is caused by the blockage of the middle cerebral artery (MCA)⁸. Therefore, rodent models of middle cerebral artery occlusion (MCAO) have been developed to resemble human cerebral infarction. While there are several MCAO approaches that are used in small animal studies, the method most widely used is the intraluminal suture model, which involves inserting a nylon filament into the middle cerebral artery from either the external or internal carotid arteries, resulting in transient or permanent occlusion of the blood flow^4,9. This model leads to a large volume of cerebral infarction and allows examination of cell death signaling in the infarcted brain tissue with immunofluorescent techniques.

The area surrounding the infarcted core, referred to as penumbra, is the target for potential stroke therapies^10,11. Since blood flow in the penumbra is partially maintained, injured neurons and non-neuronal cells in this area may be salvaged by inhibiting the activation of cell death signaling. Targeting cell death pathways may be a promising strategy for neuroprotection after stroke¹². Therefore, evaluating cell death signaling is crucial for experimental stroke research. Recently, cathepsin-B, a lysosomal protease, has been shown to play a role in mediating programmed cell death after stroke, and it has gained substantial attention in the neurological field¹³. Cathepsin-B represents a potential target for neuroprotection that merits further investigation.

This article demonstrates how to induce MCAO using the intraluminal suture approach in rats. We also show how to perform 2,3,5-triphenyltetrazalium chloride (TTC) staining to determine infarct size and detect apoptotic cells using Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining.

Protocol

This protocol and the experiments reported here were approved by the Sichuan University Medical Ethics Committee. The care and use of animals were in compliance with local and international ethical criteria.

NOTE: Adult male Sprague-Dawley rats weighing 250-280 g were used in this study. The rats were housed in a standard environmental condition (20-22 °C temperature, 12 h light/dark cycle), fed with a standard rat formula diet and high-purity water. The surgical procedure was performed under sterile conditions.

1. Rat model of MCAO

1. Induce initial anesthesia using 4% isoflurane with oxygen at 1 L/min. Monitor depth of anesthesia by foot pinch. While anesthesia is achieved, administer an ocular ointment to prevent dryness. Inject rat with 1 mL of saline subcutaneously as volume replenishment and with 5 mg/kg carprofen and 0.1 mg/kg buprenorphine for preoperative analgesia. Reduce isoflurane to 3.5% initially and then to 2% gradually throughout the surgery.
2. Place the rat in a supine position and maintain body temperature at 36.5 ± 0.1 °C on a regulated heat mat with a rectal temperature monitor probe. Shave and disinfect the skin in the neck with an iodophor disinfecting solution.
3. Make a 2 cm midline incision in the neck and retract the soft tissues to expose the carotid vessels. Use forceps to isolate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA).
4. Use sutures to ligate the proximal right CCA and clamp the right ICA and ECA with a micro-clip at their origin.
5. Insert a nylon monofilament coated with a round silicone tip into the lumen of the CCA through a small incision and tie a knot in the CCA to prevent bleeding and dislodging of the nylon monofilament.
6. Open the right ICA micro-clip and insert the nylon monofilament into the right middle cerebral artery (MCA) through the ICA stump until the silicone tip occludes the origin of MCA (~18-20 mm).
7. Cut off the exposed part of the nylon monofilament and close the neck midline incision.
8. After 2 h occlusion of MCA, open the neck incision and untie the knot in the CCA. Then withdraw the nylon monofilament to reperfuse the MCA. Ligate the distal right CCA, and close the neck incision with either continuous or interrupted sutures.
9. Post-operatively, give buprenorphine by subcutaneous injection every 8 h, at a dose of 0.03 mg/kg. Observe rats throughout the recovery from the anesthesia in a heated cage with a temperature-regulated heating mat. To encourage eating, place a Petri dish with mashed chow in the cage. House one rat per cage after the surgery.
10. Subject the sham rats to the same procedure without the monofilament insertion.
    NOTE: A total of 9 animals are used in each group for measurement of infarct size (n = 3), immunofluorescence analysis (n = 3), and apoptosis detection(n = 3).
11. Evaluate neurological function at 2 h after MCAO and 22 h after reperfusion using Zea-Longa behavioral rating scale with a 0-5 point scale¹⁴. Exclude rates with a score of 0 or 4.

2. Measurement of infarct size (Figure 1)

1. Sacrifice rats under deep anesthesia (4% isoflurane) by guillotine and carefully isolate the whole brain from the skull.
2. Freeze the brain at -20 °C for 20 min and cut the frozen brain into six coronal slices with a 2 mm distance between slices. Stain the brain slices with 2% TTC for 15 min at 37 °C in the dark.
3. Fix brain slices with 4% paraformaldehyde for 24 h at room temperature (RT). Photograph the stained slices with a digital camera, with auto mode setting for close-up objects.
4. Transfer the pictures to a computer and measure the infarct size using ImageJ. Present the infarct size as the ratio of the sum of the infarct area in 6 slices to the sum of the whole brain area in the same slices. Express data as mean ± standard error of the mean (SEM).

3. Immunofluorescence analysis on brain cryosection (Figure 2)

1. Induce anesthesia with 4% isoflurane and 1 L/min oxygen. Incise the skin and subcutaneous tissue to expose the heart.
2. Remove the pericardium and make a small cut in the right atrium. Perfuse 200 mL of saline through the left ventricle, followed by 200 mL of 4% paraformaldehyde.
3. Open the skull and remove the intact brain.
4. Fix the brain in 4% paraformaldehyde at RT for over 24 h. Then, dehydrate it in a series of sucrose solution gradients of 10%, 15%, and 30% concentrations until the tissue sinks, approximately 6-8 hours for each concentration.
5. Embed the brain in optimal cutting temperature (OCT) compound and remove air bubbles if there are any. Then, snap-freeze the brain in liquid nitrogen and store it in the -80 °C freezer.
6. Section the brain using a cryostat at a thickness of 5 µm. Throughout sectioning, ensure the cabinet temperature remains between -18 °C and -20 °C. Label the sectioning location based on the model site and experimental conditions. Store the sections in a freezer.
7. Allow the frozen sections to equilibrate at RT for ~30 min to perform immunofluorescence staining. Then, soak the sections with sterile PBS 3x for 5 min each.
8. Outline the tissue with an immunohistochemistry pen. Incubate with 20 µg/mL Proteinase K (RNase-free) and 0.3% Triton X-100 at RT for 10 min.
9. Soak the sections with sterile PBS 3x for 5 min each. Apply 3% BSA to the outlined tissue and incubate at RT for 1 h for blocking.
10. After blocking, remove the solution, add the appropriate primary antibody (Rabbit recombinant monoclonal cathepsin-B antibody, 1:200), and incubate the sections overnight in the dark at 4 °C.
11. Remove the primary antibody and wash the sections with sterile PBS containing 0.1% Tween-20 3x for 5 min each.
12. Add the appropriate secondary antibody (Goat Anti-Rat IgG [H+L] [FITC conjugated], 1:200) with the required fluorophore, and incubate the sections in a humidified chamber at RT for 1 h, protecting them from light.
13. After secondary antibody incubation, wash the sections with sterile PBS containing 0.1% Tween-20 3x for 5 min each. Stain the sections with a mounting medium containing DAPI and cover them with a coverslip.
14. Capture images using a fluorescence microscope with the following settings: excitation wavelength of 488 nm, emission wavelength of 520 nm, and exposure time of 500 ms. Store the sections in the dark at 4 °C for long-term preservation.

4. Detection of apoptosis by TUNEL staining (Figure 3)

NOTE: A commercial TUNEL assay kit is used for TUNEL staining. This TUNEL kit includes a DNase I Buffer working solution, deoxynucleotidyl transferase (TdT) equilibration buffer, labeling working solution, and DAPI working solution.

1. Perfuse and isolate the brain using the same procedure as above (steps 3.2-3.3). Immerse the brain in 4% paraformaldehyde for 3-4 h for fixation.
2. Cut the brain into coronal slices approximately 2 mm thick, starting from the rostral end to the caudal end, and immerse in 4% paraformaldehyde for an additional 12 h. Following fixation, wash the brain slices in running water for 1 h.
3. Immerse the brain slices in ethanol solutions of 50%, 70%, 80%, 90%, 95%, and 100% concentrations sequentially for 35-50 min each for dehydration.
4. Treat the brain slices with TO-type transparent biological agent I for 35-50 min, followed by clearing agent II for 35-50 min, until the tissue is fully transparent.
5. Immerse the brain slices sequentially in embedding medium I for 60 min, embedding medium II for 60 min, embedding medium III for 60 min, and embedding medium IV for 60 min.
6. Place the brain slices into an embedding cassette. Seal the cassette, label it clearly, and place it in a cold storage room for solidification.
7. Set the microtome to an initial thickness of 10 µm to trim the tissue block. Then, adjust the thickness to 5 µm for slicing.
8. Heat the water bath to 45 °C, float the tissue sections on the water surface, and pick them up onto slides. After approximately 30 s, remove the slides, dry them at RT, and store them in a drying box.
9. Deparaffinize the stored brain sections by immersing them in a clearing agent for 2 cycles, 10 min each.
10. To rehydrate the sections, soak them in anhydrous ethanol for 5 min, repeating 3 times. Transfer the sections to 90% ethanol for 3 min. Further, soak the sections in 80% ethanol for 3 min. Finally, immerse the sections in 70% ethanol for 3 min.
11. Rinse the samples in PBS 3x for 5 min each. Gently remove excess moisture using filter paper.
12. Administer 100 µL of 1x proteinase K working solution to each sample and incubate at 37 °C for 20 min. Following permeabilization, rinse the samples in PBS 3x for 5 min each.
13. Prepare samples for positive control.
    1. Add 100 µL of 1x DNase I Buffer working solution (TUNEL assay kit) to the permeabilized sample and allow it to equilibrate at RT for 5 min. Carefully remove any excess liquid from the sample using absorbent paper.
    2. Add 100 µL of diluted DNase I working solution (200 U/mL) to the sample and incubate at 37 °C for 10-30 min. Rinse the samples in PBS 3x for 5 min each.
14. Prepare samples for negative control.
    1. Apply 100 µL of 1x DNase I Buffer working solution to the permeabilized sample and equilibrate at RT for 5 min. Incubate the negative control sample with DNase I Buffer at 37 °C for 10-30 min, ensuring no DNase I enzyme is added. Rinse the samples in PBS 3x for 5 min each.
15. Apply 100 µL of terminal deoxynucleotidyl transferase (TdT) equilibration buffer (TUNEL assay kit) to each sample and incubate in a humidified chamber at 37 °C for 10-30 min. Then, remove the TdT Equilibration Buffer with absorbent paper.
16. Add 50 µL of labeling working solution (TUNEL assay kit) to each sample, then incubate them at 37 °C in a humidified, dark chamber for 60 min. If the signal intensity is low, consider extending the DNA labeling reaction time. Then, rinse the samples in PBS 3x for 5 min each.
17. After blotting excess moisture, apply the DAPI working solution (TUNEL assay kit) and incubate at RT in the dark for 5 min to counterstain the nuclei. Then, rinse the samples in PBS 4x for 5 min each. Remove excess liquid with absorbent paper, and seal the slides using an anti-fade mounting medium.
18. Scan the stained slides using the Vectra Polaris multispectral imaging system and analyze data with the system software.

5. Statistical analysis

1. Present the experimental results as means ± SEM and analyze data with a Student's t-test for two group comparisons using GraphPad Prism 10 software (GraphPad Software, San Diego, CA).
2. Set a p-value less than 0.05 as a statistical difference.

Results

Rats are subjected to 120 min ischemia during MCAO followed by 22 h reperfusion. The mortality rate was minimal during MCAO surgery and was around 25% within the period of reperfusion. Significant brain damage was observed in the MCAO group, whereas no infarct was observed in the cerebrum of the Sham group (24.87 ± 1.21% vs. 0% sum of infarct area to the whole brain area; MCAO [n = 3] vs. Sham [n = 3], P < 0.001; Figure 1A,B). In the MCAO group, immunoreact...

Discussion

Occlusion of a cerebral artery leads to deprivation of oxygen and nutrition, followed by the activation of reactive oxygen species, intracellular calcium overload, glutamate release, and induction of inflammatory responses¹⁵. This series of events results in cell death and irreversible tissue damage in the infarcted brain. The penumbra is potentially salvageable through medical intervention within an appropriate therapeutic window¹⁶. The only approved treatment for ischemic...

Materials

Name	Company	Catalog Number	Comments
Akoya Vectra Polaris	Akoya Biosciences	N/A	Vectra Polaris multispectral imaging system
Anhydrous ethanol	Chengdu Jinshan Chemical Reagent Co.	N/A
Buprenorphine	West China Hospital	N/A
Carprofen	West China Hospital	N/A
Cathepsin-B antibody	Abcam	Ab214428
Cryostat	LEICA	LEICA CM 1950
Digital camera	OLYMPUS	TG-7
Goat Anti-Rat IgG (H+L) (FITC conjugated)	Elabscience	E-AB-1021
Goat serum	Solarbio	SL038
GraphPad Prism 10 software	N/A	https://www.graphpad-prism.cn/?c=i&a=prismdownload_cn
ImageJ	N/A	https://imagej.net/ij/
Immunohistochemistry pen	Biosharp	http://www.biosharp.cn/index/product/details/language/en/product_id/2828.html
InForm software	Akoya Biosciences	Version 2.6.0	system software for the multispectral imaging system
Iodophor disinfecting solution	Huatian Technology Industry Co.	N/A
Isoflurane	RWD Life Science Co.	https://www.rwdstco.com/inhalation-anesthesia-solutions/
Mounting medium containing DAPI	Solarbio	S2110
Nylon monofilament	RWD Life Science Co.	https://www.rwdstco.com/
Optimal cutting temperature (OCT) compound	Epredia	N/A
Paraformaldehyde	Biosharp	N/A
Phosphate buffer solution (PBS)	Solarbio	N/A
Prism	GraphPad	Version 10
Proteinase K	Tiangen	RT403-02
Saline	Kelun Co.	N/A
Sprague-Dawley rats	Huafukang Co.	N/A
Sucrose	BioFroxx	1245GR500
Surgical instruments	RWD Life Science Co.	N/A
TO-type transparent biological agent	Beijing Solarbio Science & Tecnology Co., Ltd.	http://www.solarbio.net/
Triphenyltetrazolium chloride (TTC) solution	Solarbio	G3005
Triton X-100	BioFroxx	1139ML100
TUNEL kit	Elabscience	E-CK-A321