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Updated: Dec 23, 2025
Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation
Published on: January 12, 2016
Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy
Ibolya Rutkai1,2, Wesley R Evans1,2, Nikita Bess1
1Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana.
Abstract:
Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice.NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.
Insights
Researchers developed a new method for real-time, long-term in vivo imaging of mitochondria in mouse cerebral vasculature. This technique allows detailed study of mitochondrial function in endothelial cells, advancing our understanding of brain blood vessel health and disease.
Area of Science:
- Neuroscience
- Cell Biology
- Biomedical Imaging
Background:
- Mitochondria are crucial for cerebral vascular function but studying them in vivo has been challenging due to methodological limitations.
- Understanding mitochondrial roles in the brain's vasculature is vital for addressing neurological diseases.
Purpose of the Study:
- To develop and validate a novel in vivo imaging technique for real-time, longitudinal observation of mitochondria in the cerebral endothelium.
- To enable detailed analysis of mitochondrial dynamics and their relationship with vascular parameters in the brain's microcirculation.
Main Methods:
- Generation of mice with mitochondria-specific fluorescent protein (mito-Dendra2) expression in the cerebral vascular endothelium using a Cre/Lox system.
- Cranial window implantation and two-photon microscopy for in vivo imaging of mito-Dendra2 labeled mitochondria.
- Simultaneous assessment of mitochondrial density, vessel dimensions, and mitochondria-to-vessel ratio using mito-Dendra2 and fluorescent dextran.
Main Results:
- Successfully established stable, long-term in vivo imaging of mitochondria within the cerebral vascular endothelium in live mice.
- Confirmed the specificity of mito-Dendra2 labeling to mitochondria in endothelial cells through immunohistochemistry and in vitro studies.
- Demonstrated the stability of the mito-Dendra2 signal over time while observing dynamic movement of individual mitochondria within endothelial cells.
Conclusions:
- The developed mito-Dendra2 imaging approach is suitable for real-time and longitudinal examination of mitochondria in the mouse cerebral vasculature.
- This innovative technique provides a powerful tool for investigating the role of mitochondria in endothelial cells and other cell types within the cerebral circulation.
- The method facilitates a deeper understanding of mitochondrial contributions to cerebrovascular health and disease.
