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

Dmitry Frank; Benjamin  F. Gruenbaum; Julia Grinshpun; Israel Melamed; Olena Severynovska; Ruslan Kuts; Michael Semyonov; Evgeni Brotfain; Alexander Zlotnik; Matthew Boyko

doi:10.3791/61309

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single ...

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Neuroscience

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JoVE Journal Neuroscience

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

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04:32 min

October 23, 2020

DOI: 10.3791/61309-v

Dmitry Frank*¹, Benjamin F. Gruenbaum*², Julia Grinshpun¹, Israel Melamed³, Olena Severynovska⁴, Ruslan Kuts¹, Michael Semyonov¹, Evgeni Brotfain¹, Alexander Zlotnik¹, Matthew Boyko¹

¹Department of Anesthesiology and Critical Care, Soroka Medical Center,Ben-Gurion University of the Negev, ²Department of Anesthesiology,Yale University School of Medicine, ³Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences,Ben-Gurion University of the Negev, ⁴Department of Physiology, Faculty of Biology, Ecology and Medicine,Dnepropetrovsk State University

Overview

This protocol details a modified middle cerebral artery occlusion technique to assess ischemic brain injury by evaluating infarct zones, brain edema, and blood-brain barrier (BBB) permeability in the same rodent brain samples. Utilizing a single brain sample enhances ethical and economic efficiency while allowing comprehensive examination of ischemic stroke pathophysiology.

Key Study Components

Area of Science

Neuroscience
Ischemic brain injury
Rodent models

Background

Ischemic stroke is a significant cause of morbidity and mortality.
Evaluating multiple parameters of brain injury can enhance understanding of stroke mechanisms.
Current methods often require multiple samples, creating ethical and resource constraints.
This study introduces a streamlined approach to measure various damage indicators using one sample.

Purpose of Study

To develop a technique for measuring infarct volume, edema, and BBB disruption in rodent brains.
To improve resource efficiency and reduce reliance on multiple animal samples.
To facilitate a more comprehensive understanding of ischemic brain injury and recovery.

Methods Used

The method involves using rat brain samples after inducing ischemia via the middle cerebral artery occlusion.
Brain sections are prepared and analyzed for various injury parameters including edema and BBB integrity.
Key steps include image processing, pixel analysis for infarct volume calculations, and spectrophotometry for BBB disruption assessment.
Data is quantified and compared between ischemic and control groups using statistical analysis.

Main Results

The protocol allowed simultaneous measurement of infarct volume, edema, and BBB integrity in rat models.
Significant increases in these parameters were observed in rats with induced ischemia compared to controls.
Findings highlight the effectiveness of this technique in assessing ischemic damage while reducing animal use.

Conclusions

This study demonstrates a novel method for the simultaneous assessment of key parameters of ischemic brain injury.
The approach enhances understanding of stroke pathology and facilitates more ethical research practices.
Implications extend to potential therapeutic developments and understanding of neuroprotective strategies.

Frequently Asked Questions

What are the advantages of this method?

The technique allows for assessing multiple parameters of ischemic brain injury using a single brain sample, enhancing ethical considerations and reducing costs.

How is the ischemic injury induced in the model?

Ischemic injury is induced using a modified middle cerebral artery occlusion technique in rodent models.

What types of data are obtained from this study?

The study provides data on infarct volume, brain edema, and blood-brain barrier integrity, allowing comprehensive analysis of ischemic damage.

Can this method be adapted for other types of injuries?

While primarily designed for ischemic brain injury, the methodological framework could be adapted to study other central nervous system injuries.

What are the key limitations of this protocol?

Potential limitations include the need for precise imaging and analysis techniques to ensure accurate measurement of parameters across samples.

How does this study contribute to our understanding of ischemic stroke?

The study enhances understanding of the pathophysiology of ischemic stroke by enabling the assessment of multiple injury parameters in a single sample.

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.

In the present protocol, we present a modified middle cerebral artery occlusion technique that measures ischemic brain injury to determinate an infarct zone, brain edema, and BBB permeability in the same set of rodent brains. In terms of ethical and economic costs, the main advantage of this technique is that only one brain sample is required for all three measurements. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate pathophysiology of ischemic stroke.

To begin, remove the rat brain, and cut 2 millimeter thick horizontal sections with a 0.009-inch stainless steel, uncoated, single edge razor blade. Place the sections in 0.005%TTC and incubate them for 30 minutes at 37 degrees Celsius, then place the brain tissue on the microscope slides and perform optical scanning of the six brain slices with a resolution of 1600 x 1600 DPI. Use a photo editor to add a blue filter to the images.

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