Mechanism Of Neurovascular Unit Injury During Hypoxia/Ischemia

Objectives1. To clarify the hypothesis that aquaporin-4 (AQP4) may play different roles in ischemic brain edema based on different cellular elements of the neurovascular unit.2. To verify the hypothesis that AQP4 may be a key protein in adult neurogenesis after hypoxia/ischemia.3. To validate the hypothesis that caveolin-1 may regulate matrix metalloproteinases (MMPs) expression in brain microvascular endothelium cell (BMEC) after hypoxia/ischemia.4. To verify the hypothesis that caveolin-1 may be a critical protein associated with the nitric oxide (NO)-induced MMPs activation, degradation of tight junction (TJ) proteins and subsequent BBB disruption in cerebral ischemia/reperfusion injury.Methods1. Western blot analysis of AQP4 expression in bEnd.3 cell line; Western blot analysis of AQP4 expression in isolated brain microvessels at ischemia/reperfusion 6 h,24 h and 48 h; Western blotting of AQP4 expression in cultured astrocyte after oxygen and glucose deprivation (OGD).2. Western blot analysis of AQP4 expression in cultured neural stem cells (NSCs) under hypoxic and normoxic conditions; Western blot analysis of AQP4 and neuronal class Ⅲ β-tubulin (Tuj1) expression in NSCs after using siRNA against AQP4.3. Western blotting of caveolin-1, MMP-2/-9 expression in isolated brain microvessels during ischemia/reperfusion; Immuno-fluorescence assay for cavolin-1 expression; In situ gelatin zymography for MMP-2/-9 activities; In vitro gelatin zymography for MMP-2 activity after using siRNA against cavolin-1 in primary culture of rat brain endothelial cells.4. The rats were divided into the following groups:sham control; 2 h of ischemia followed by 24 h of reperfusion (I/R); I/R+3 mg/kg N’-nitro-L-arginine methylester (L-NAME); I/R+25 mg/kg 7-nitroindazole (7-NI). All of the NOS inhibitors were intraperitoneally administrated 15 min before the onset of ischemia (pre-ischemia). The same amounts of normal saline were also administered in the sham control rats. Assessment of blood-brain barrier permeability by the measurement of Evans blue dye. Western blotting of caveolin-1 expression in isolated brain microvessels; immuno-fluorescence assay for 3-nitrotyrosine, caveolin-1, and zona occulden-1(ZO-1) expression; and in situ gelatin zymography for MMPs activity.Results1. AQP4 expression was observed in a mouse BMEC strain:bEnd.3. Compared with sham group, level of AQP4 expression increased in isolated BMECs at 6 h,24 h and 48 h of ischemia/reperfusion groups. Compared with control group, AQP4 expression in cultured rat astrocytes declined in OGD group.2. AQP4 expression gradually increased for 14 days under normoxia and hypoxia conditions in cultured NSCs. Compared with control group, knocking down the expression of AQP4 down-regulated Tuj-1 expression.3. In isolated BMECs, MMP-2 expression increased sharply at 6 h,24 h and 48 h, while MMP-9 obviously increased at 48 h of ischemia/reperfusion insults. Compared with sham operation group, MMPs activity obviously increased after ischemia/reperfusion. The expression of caveolin-1 protein decreased at 6 h after ischemia/reperfusion in isolated BMECs. The number of caveolin-1 positive microvessels decreased in ischemia/reperfusion insult groups. In In vitro gelatin zymography, MMP2 activity increased in BMECs after caveolin-1 knockdown.4. L-NAME and 7-NI decreased BBB permeability respectively. Compared with saline group, caveolin-1 was rescued after treatment with L-NAME in isolated BMECs. Compared with sham control, nitration was completely blocked by treatment with L-NAME in ischemia/reperfusion 24 h group. Compared with saline group, L-NAME treatment could rescue caveolin-1 positive microvessels, decrease MMPs activity, and restore ZO-1 positive vessels to normal conditions.Conelusions1. AQP4 in brain microvessels may facilitate early brain edema after ischemic stroke and early temporary loss of glial AQP4 may have a protective role against postischemic edema formation.2. AQP4 may play a specific functional role in regulating the adult neurogenesis after ischemic stroke.3. Caveolin-1 may be a critical protein associated with MMPs activation and BBB disruption in cerebral ischemia/reperfusion injury.4. Caveolin-1 may control BBB permeability via protecting TJ-associated proteins from degradation by NO-induced MMPs activation in ischemic/reperfused BMECs and brain tissues.