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....

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 u.......

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