The Role of Temperature in the Preparation and Viability of Rodent Brain Slices, and Propensity to Evoke Spreading Depolarization
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Authors
Wolkoff, Sophie Rose
Date
2024-09-19
Type
thesis
Language
eng
Keyword
Spreading depolarization , Rodent brain slicing , Temperature , Hypothermia , Cold-SD , Ischemia , Stroke , Neocortex , Hippocampus , Cytotoxic edema , Dendritic beading , Light transmittance imaging
Alternative Title
Abstract
Following an ischemic stroke, recurrent spreading depolarization (SD) propagates across the higher grey matter of the brain and is the primary contributor to acute neuronal injury. Within minutes of the brain being deprived of oxygen and glucose (OGD), Na+/K+ pumps on neuronal membranes fail and membrane potential is lost, resulting in SD. Experimentally, SD can be evoked in rodent brain slices using several methods. In fact, evidence in the Andrew Lab has shown that upon cooling to 2-6 °C, the brain will undergo a bout of SD induced by the cold.
Temperature also plays a role in the preparation of rodent brain slices. Brain slicing is a fundamental method used in cellular neuroscience research, and it is common practice to submerge the brain in ice-cold artificial cerebrospinal fluid (aCSF) throughout the dissection and slicing process. Slicing in cold temperatures lowers metabolism and facilitates firmer slicing. However, knowing that cold-SD occurs at temperatures approaching freezing, this led to the question of whether rodent brain slices prepared in warmer conditions may be of better quality than those prepared in ice-cold conditions.
In this thesis, hippocampal slices of male CD-1 mice prepared in aCSF at 2 °C, 10 °C, 22 °C or 35 °C, were compared, to determine the optimal temperature for producing healthy tissue, and study the effects of cold-SD. Warm slicing proved to be extremely challenging, due to the brain’s lack of rigidity at higher temperatures. To assess any differences in slice viability, SD was induced in the neocortex and hippocampus of slices by exposing them to OGD-aCSF, and this was observed via light transmittance (LT) imaging. Results showed that neocortical tissue viability was not affected by the temperature held during slice preparation, with surprisingly little variation among groups in this brain region. On the other hand, viability of the hippocampus slightly improved with cooling during preparation, as SD was generated more frequently and propagated at faster speeds in those slices. Therefore, we determined that using ice-cold conditions are marginally better for preparing healthy hippocampal slices from mice, rather than using warmer preparatory temperatures. Finally, despite our finding that slices prepared below 6 °C undergo a cold-SD, we concluded here that it does not affect tissue quality.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial-ShareAlike 4.0 International