Introduction: Post-stroke inflammation has been shown to amplify neuronal damage and impair long-term functional recovery. However, leukocyte navigation of dural lymphatic channels and interactions with neurovascular units after stroke remain unknown. Current methods to assess neuroinflammation either have limited ability for continuous monitoring over time, like ex-vivo flow cytometry, or require extra processing steps, such as exogenous labeling for fluorescent imaging. Here, we developed a novel, non-invasive, and label-free approach to directly visualize mouse brain compartments (sinuses, brain parenchyma, dura) in-vivo and simultaneously quantify temporal and spatial immune subpopulation recruitment during neuroinflammation.
Methods: After placing a chronic cranial window over the mouse sagittal sinus, we used oblique back-illumination microscopy (OBM) with sub-cellular resolution to image different compartments at baseline. Then, bacterial lipopolysaccharides were injected at three dosage levels into the cisterna magna to induce varying extents of meningitis. The same locations were revisited at multiple time points (6, 12, 24, and 48 hours) to assess the dynamics of immune response. Finally, we analyzed OBM videos to quantify and differentiate leukocytes participating in immune recruitment following meningitis
Results: We were able to visualize intravital immune cell recruitment at different stages in real-time, including margination, rolling, adhesion within flow, extravasation, and migration into brain compartments. We have imaged 26 mice (14 females, 12 males) at both pre-/post-neuroinflammation and compared the video immune cell counts with flow cytometry and CBCs of peripheral blood samples. Both microscopic quantifications and flow cytometry showed a significant increase in immune cell recruitment post-meningitis (p=0.000045), whereas CBCs demonstrated an opposite decreasing trend in WBC counts (p=0.000015).
Conclusion : Intravital OBM allows us to noninvasively quantify and monitor neuroinflammation in extensive brain compartments at various time points without exogenous labeling. This represents an important step towards gaining more insight into post-stroke inflammation, which will eventually help develop new therapeutic approaches.
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