Ischemic blood-brain barrier (BBB) disruption is a major contributor to tissue injury during stroke. Understanding the mechanisms that regulate this process will lead to new approaches to mitigate brain damage and lengthen the window for thrombolytic treatment. Pericytes are essential for development and maintenance of the BBB. However, little is known about their impact on BBB integrity during ischemic injury in the adult brain. Using in vivo two-photon microscopy, we have found that pericytes elicit punctate BBB disruptions specifically at their somata. Pericyte somata cover only 7% of the total capillary surface, while their fine and extensive processes cover the rest. However, harmful blood-borne molecules can diffuse far beyond the point of extravasation, strengthening our need to understand this uncharacterized leakage route. Our central hypothesis is that pericytes rapidly upregulate matrix metalloproteinase activity, leading to local disassembly of endothelial tight junctions (paracellular leakage). This hypothesis will be tested using state-of- the-art approaches.
In Aim 1, we will use in vivo two-photon microscopy to directly visualize MMP9 activity using a fluorescent gelatin probe, following stroke induction in pericyte-labeled mice. In vivo pharmacological and pericyte-specific deletion experiments will be performed to test putative signaling cascades that can lead to rapid MMP9 activation. Ex vivo biochemical studies will be performed to confirm the role of these signaling cascades.
In Aim 2, we will use 3-D serial block-face electron microscopy to examine the nature of endothelial disruption at pericyte somata. Our findings will be compared with neighboring capillary regions not covered by somata. The proposed research is significant because it is expected to define pericytes as inducers of BBB injury during ischemia, which contrasts their emerging role as nurturers of BBB integrity during development and normal brain function.
The proposed research will provide fundamental information on how capillary pericytes are involved in acute blood-brain barrier leakage during stroke. This work is relevant to public health because blood- brain barrier disruption causes brain swelling and limits the utility of thrombolytic treatment. The proposed experiments may reveal new strategies to manage this feature of stroke injury and thereby improve recovery.
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