title: Permeabilization of brain tissue in situ enables multiregion analysis of mitochondrial function in a single mouse brain.
abstract: CONCLUSIONS
  Mitochondrial function in the brain is traditionally assessed through analysing respiration in isolated mitochondria, a technique that possesses significant tissue and time requirements while also disrupting the cooperative mitochondrial reticulum. We permeabilized brain tissue in situ to permit analysis of mitochondrial respiration with the native mitochondrial morphology intact, removing the need for isolation time and minimizing tissue requirements to ∼2 mg wet weight. The permeabilized brain technique was validated against the traditional method of isolated mitochondria and was then further applied to assess regional variation in the mouse brain with ischaemia-reperfusion injuries. A transgenic mouse model overexpressing catalase within mitochondria was applied to show the contribution of mitochondrial reactive oxygen species to ischaemia-reperfusion injuries in different brain regions. This technique enhances the accessibility of addressing physiological questions in small brain regions and in applying transgenic mouse models to assess mechanisms regulating mitochondrial function in health and disease.
  Mitochondria function as the core energy providers in the brain and symptoms of neurodegenerative diseases are often attributed to their dysregulation. Assessing mitochondrial function is classically performed in isolated mitochondria; however, this process requires significant isolation time, demand for abundant tissue and disruption of the cooperative mitochondrial reticulum, all of which reduce reliability when attempting to assess in vivo mitochondrial bioenergetics. Here we introduce a method that advances the assessment of mitochondrial respiration in the brain by permeabilizing existing brain tissue to grant direct access to the mitochondrial reticulum in situ. The permeabilized brain preparation allows for instant analysis of mitochondrial function with unaltered mitochondrial morphology using significantly small sample sizes (∼2 mg), which permits the analysis of mitochondrial function in multiple subregions within a single mouse brain. Here this technique was applied to assess regional variation in brain mitochondrial function with acute ischaemia-reperfusion injuries and to determine the role of reactive oxygen species in exacerbating dysfunction through the application of a transgenic mouse model overexpressing catalase within mitochondria. Through creating accessibility to small regions for the investigation of mitochondrial function, the permeabilized brain preparation enhances the capacity for examining regional differences in mitochondrial regulation within the brain, as the majority of genetic models used for unique approaches exist in the mouse model.
authors.affiliation: Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
authors.forename: Eric A F
authors.initials: EA
authors.lastname: Herbst
authors.forename: Graham P
authors.initials: GP
authors.lastname: Holloway
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism / Q000502:physiology*; D002374:Catalase / Q000235:genetics; D019069:Cell Respiration / Q000502:physiology; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008928:Mitochondria / Q000502:physiology*; D010539:Permeability; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000503:physiopathology*
chemical_list: D017382:Reactive Oxygen Species; D002374:Catalase