Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

Summary

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Abstract

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

Introduction

One of the most common causes of morbidity and mortality worldwide is stroke. Globally, ischemic stroke represents 68% of all stroke cases, while in the United States ischemic stroke accounts for 87% of stroke cases^1,2. It is estimated that the economic burden of stroke reaches $34 billion in the United States² and €45 billion in the European Union³. Animal models of stroke are necessary to study its pathophysiology, develop new methods for evaluation, and propose new therapeutic options⁴.

Ischemic stroke ....

Protocol

The following procedures were conducted according to the recommendations of the Declaration of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.

1. Preparing rats for the experimental procedure

1. Select adult male Sprague-Dawley rats without overt pathology, each weighing between 300 and 350 g.
2. Maintain all rats at room temperature at 22 °C, with 12 hours of light and dark cycles before experiment.
3. Ensure that food and water are available ad libitum.<....

Results

Infarct zone measurement

An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated a significant increase in brain infarct volume compared to the 16 sham-operated rats (MCAO=7.49% ± 3.57 vs. Sham = 0.31% ± 1.9, t(28.49) = 7.56, p < 0.01 (see Figure 2A)). The data is expressed as a mean percentage of the contralateral hemisphere ± SD.

Brain.......

Discussion

The principal goal of the present protocol was to demonstrate consistent measurements of three main parameters of ischemic injury: IZ, BE and BBB permeability. Previous studies in this field have demonstrated the possibility of performing one or two of these parameters together in the same sample. Besides the cost reduction that this three-part method offers, it also provides a more desirable bioethical model that limits the number of animals that must be operated on and subsequently euthanized. As in all histological te.......

Materials

Name	Company	Catalog Number	Comments
2 mL Syringe	Braun	4606027V
2% chlorhexidine in 70% alcohol solution	Sigma-Aldrich	500 cc	Provides general antisepsis of the skin in the operatory field
27 G Needle with Syringe	Braun	305620
3-0 Silk sutures	Henry Schein	1007842
4-0 Nylon suture	4-00
Brain & Tissue Matrices	Sigma-Aldrich	15013
Cannula Venflon 22 G	KD-FIX	183603985447
Centrifuge Sigma 2-16P	Sigma-Aldrich	Sigma 2-16P
Compact Analytical Balances	Sigma-Aldrich	HR-AZ/HR-A
Digital weighing scale	Sigma-Aldrich	Rs 4,000
Dissecting scissors	Sigma-Aldrich	Z265969
Eppendorf pipette	Sigma-Aldrich	Z683884
Eppendorf tube	Sigma-Aldrich	EP0030119460
Fluorescence detector	Tecan, Männedorf Switzerland	Model: Infinite 200 PRO multimode reader	Optional.
Fluorescence detector	Molecular Devices LLC	VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument	Optional.
Gauze sponges	Fisher	22-362-178
Heater with thermometer	Heatingpad-1	Model: HEATINGPAD-1/2
Hemostatic microclips	Sigma-Aldrich
Horizon-XL	Mennen Medical Ltd
Infusion cuff	ABN	IC-500
Micro forceps	Sigma-Aldrich
Micro scissors	Sigma-Aldrich
Multiset	Teva Medical	998702
Olympus BX 40 microscope	Olympus
Operating forceps	Sigma-Aldrich
Operating scissors	Sigma-Aldrich
Optical scanner	Canon	Cano Scan 4200F	Resolution 3200 x 6400 dpi
Petri dishes	Sigma-Aldrich	P5606
Purina Chow	Purina	5001	Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.
Rat cages	Techniplast	2000P	Conventional housing for rodents. Cages were used for housing rats throughout the experiment
Scalpel blades #11	Sigma-Aldrich	S2771
Software
Adobe Photoshop CS2 for Windows	Adobe
ImageJ 1.37v	NIH		The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA
Office 365 ProPlus	Microsoft	-	Microsoft Office Excel
Windows 10	Microsoft
Reagents
2,3,5-Triphenyltetrazolium chloride	Sigma-Aldrich	298-96-4
50% trichloroacetic acid	Sigma-Aldrich	76-03-9
Ethanol 96 %	Romical		Flammable liquid
Evans blue 2%	Sigma-Aldrich	314-13-6
Isoflurane, USP 100%	Piramamal Critical Care, Inc	NDC 66794-017