Objective: Recovery can occur after stroke in both human and animals, and this is attributed in part by rewiring of neural connections in areas adjacent to or remotely connected to the infarct. As stroke can cause brain-wide network changes, it is important to interrogate regenerative processes in the whole brain after stroke. In this study we use a high resolution 3D whole brain imaging technique called CLARITY to visualize the cellular and structural changes in stroke mice during recovery.
Hypothesis: We hypothesize that the CLARITY procedure will provide a more detailed and complete visualization of post-stroke regenerative processes in the whole brain.
Material/Methods: We used C57Bl6 WT mice with 10-12 weeks of age. Ischemic stroke was induced by transient middle cerebral artery occlusion using an intraluminal suture. Mice were sacrificed at different time points (Post-stroke day 1, 7, 14 and 28) and brains were processed with the CLARITY protocol. Brains were immunostained repeatedly with antibodies for neurons, glia, oligodendrocytes, microglia/macrophages and blood vessels.
Results: Stroke brains collected at different post-stroke timepoints became optically transparent after the CLARITY process. Interestingly, the ischemic area turned transparent for all timepoints except post-stroke day 7 and 28. At this time point the ischemic area remains opaque after CLARITY process, suggesting that the cellular composition at this timepoint is resistant to the CLARITY treatment. Immunostaining with MAP2 demonstrates labeling of neuronal processes in the whole brain. Current studies investigate other cell types and cellular processes, such as glial scarring (GFAP), microglia/macrophage accumulation (CD68) and angiogenesis (Collagen IV) to map the reorganization at the cellular level with high resolution in 3D whole brain.
Conclusion: Stroke brains can be made optically transparent after the CLARITY process. This allows high resolution whole brain 3D imaging to probe for the cellular and molecular mechanisms during stroke recovery.