Abstract
Background
Neurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases.
Methods
To study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2’-azobis-2-methyl- propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (H2O2) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software.
Results
We optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count.
Conclusions
Following ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease.
Document Type
Article
Publication Date
2024
Digital Object Identifier (DOI)
https://doi.org/10.1186/s12987-024-00579-9
Funding Information
This project was supported by the University of Kentucky Neuroscience Research Priority Area Pilot Award. The studies were also supported by NIH NIGMS P20 GM148326 (WBH). This research was supported in part by IK2 BX004618 (WBH) from BLR&D of the Department of Veterans Affairs and award HT9425-24-1-0301 (WBH) from Department of Defense Congressionally Directed Medical Research Programs. This research was also supported in part by R01NS079507 (BB) from the NIH/NINDS and R01AG075583 (AMSH) from the NIH/NIA. The contents do not represent the views of the US Department of Veterans Affairs, the NIH/NINDS/NIA, or the United States government.
Repository Citation
Velmurugan, Gopal V.; Vekaria, Hemendra J.; Hartz, Anika M. S.; Bauer, Björn; and Hubbard, William Brad, "Oxidative stress alters mitochondrial homeostasis in isolated brain capillaries" (2024). Markey Cancer Center Faculty Publications. 267.
https://uknowledge.uky.edu/markey_facpub/267
Included in
Developmental Neuroscience Commons, Molecular and Cellular Neuroscience Commons, Neurology Commons, Oncology Commons
Notes/Citation Information
This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.