In Situ Microwave Fixation to Define the Terminal Rodent Brain Metabolome

Abstract

The brain metabolome directly connects to brain physiology and neuronal function. Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. Therefore, challenges remain to capture an accurate snapshot of the physiological brain metabolome in healthy and diseased rodent models. To overcome this barrier, we employ a high-power focused microwave for the simultaneous euthanasia and fixation of mouse brain tissue to preserve metabolite pools prior to surgical removal and dissection of brain regions. We demonstrate exhaustion of glycogen and glucose and increase in lactate production during conventional rapid brain resection prior to preservation by liquid nitrogen that is not observed with microwave fixation. Next, microwave fixation was employed to define the impact of brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple regions of the mouse brain, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the TCA cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation to study terminal brain metabolism in rodent models.

Laforin targets malin to glycogen in Lafora progressive myoclonus epilepsy

“…Compared to brain, muscle is a glycogen-enriched tissue (0.1% of total brain weight versus 1-2% of muscle mass ( Brown, 2004 ; Nelson et al, 1968 ; Shulman et al, 1995 ). Additionally, adult mouse brain glycogen is known to be depleted as much as 46% within a minute of post-euthanasia by routine laboratory techniques such as cervical dislocation or decapitation before rapid freezing ( DiNuzzo et al, 2019 ; Hutchins and Rogers, 1970 ; Juras et al, 2022 preprint). Consequently, our attempt to track malin in brain HSP fraction failed.…”

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology

“…While this includes the study of brain-EVs from humans, rodent models, and non-human primates, postmortem analyses have caveats. The postmortem interval can alter RNA expression and integrity (81), shift brain metabolism (82), and accelerate protein degradation (83). Thus, postmortem tissue changes may affect brain-derived EVs by causing artifactual changes in the EV cargo that would be absent if isolated from a live brain.…”