Based on our spinal cord findings that β1 integrin inhibition greatly increases hypoxia-induced vascular disruption almost exclusively in WM, we next extended our analysis to the brain to see if our proposed model also holds true there. Here we examined two well demarcated WM tracts; the WM tracts of the cerebellum and the corpus callosum, and compared them with the surrounding GM areas of the cerebellum and cerebral cortex, respectively. First, we compared the angiogenic response in WM and GM regions by quantifying the density of proliferating endothelial cells, using CD31/Ki67 dual-IF. This showed that in the cerebellum, almost all the proliferating endothelial cells were in the WM tract, with very few in the neighboring GM (Fig.
5A). This was confirmed using fluoromyelin/Ki67 dual-IF (Fig.
5B). Quantification of CD31
+/Ki67
+ cells in the cerebellum revealed a WM:GM ratio of 43:1 (Fig.
5C). In a similar manner, in the forebrain, the WM corpus callosum tract contained a much higher density of proliferating endothelial cells than the neighboring cerebral cortex (Fig.
5D), which was confirmed by fluoromyelin/Ki67 dual-IF (Fig.
5E). Quantification of CD31
+/Ki67
+ cells in the WM corpus callosum and the GM cerebral cortex revealed a WM:GM ratio of 5:1, (Fig.
5F). Thus, consistent with our observations in spinal cord WM [
17], in both these brain regions, the WM contained a much greater number of proliferating endothelial cells. Next, we compared the relative distribution of BBB disruption in the WM and GM regions of the cerebellum and the forebrain (cerebral cortex and corpus callosum). Similar to spinal cord, this revealed that under normoxic conditions, no BBB disruption was detected in mice receiving isotype control antibody, while β1 integrin blockade triggered a very small number of vascular leaks (Fig.
6). However, under hypoxic conditions, while a small number of leaks occurred in control antibody treated mice, β1 integrin inhibition greatly enhanced the number of vascular leaks both in the cerebellum and the forebrain (
\( \sim \)75-fold and 13-fold respectively compared to isotype control conditions). Most strikingly, these vascular leaks occurred almost exclusively in the WM, both in the cerebellum (Fig.
6A) and the corpus callosum of the forebrain (Fig.
6C). In the cerebellum, almost all vascular leaks occurred in the WM tract, with very few in the surrounding GM (WM:GM ratio of 41:1, Fig.
6E), while the corpus callosum WM tract contained many vascular leaks with just a few in the surrounding cerebral cortex GM (WM:GM ratio of 12, Fig.
6F). Fluoromyelin/fibrinogen dual-IF confirmed that almost all vascular leaks identified in these brain regions occurred in the myelinated WM regions (Fig.
6B and D). In addition, in both the cerebellum and forebrain, fluoromyelin/fibrinogen dual-IF demonstrated a strong spatial association between vascular disruption and myelin degradation like that found in the spinal cord (note the motheaten appearance of myelin in Fig.
6B and D). These observations support the concept that the preferential tendency for WM (in both brain and spinal cord) to show vascular disruption in response to CMH in the presence of β1 integrin blockade, is a result of lower vascularity triggering stronger angiogenic remodelling, allowing the β1 integrin blocking antibody more opportunity to prevent the stabilization of newly formed blood vessels in these regions.