Characteristic Features of Deep Brain Lymphatic Vessels and Their Regulation by Chronic Stress

Studies have demonstrated that a functional network of meningeal lymphatic vessels exists in the brain. However, it is unknown whether lymphatic vessels could also extend deep into the brain parenchyma and whether the vessels could be regulated by stressful life events. We used tissue clearing techniques, immunostaining, light-sheet whole-brain imaging, confocal imaging in thick brain sections and flow cytometry to demonstrate the existence of lymphatic vessels deep in the brain parenchyma. Chronic unpredictable mild stress or chronic corticosterone treatment was used to examine the regulation of brain lymphatic vessels by stressful events. Western blotting and coimmunoprecipitation were used to provide mechanistic insights. We demonstrated the existence of lymphatic vessels deep in the brain parenchyma and characterized their features in the cortex, cerebellum, hippocampus, midbrain, and brainstem. Furthermore, we showed that deep brain lymphatic vessels can be regulated by stressful life events. Chronic stress reduced the length and areas of lymphatic vessels in the hippocampus and thalamus but increased the diameter of lymphatic vessels in the amygdala. No changes were observed in prefrontal cortex, lateral habenula, or dorsal raphe nucleus. Chronic corticosterone treatment reduced lymphatic endothelial cell markers in the hippocampus. Mechanistically, chronic stress might reduce hippocampal lymphatic vessels by down-regulating vascular endothelial growth factor C receptors and up-regulating vascular endothelial growth factor C neutralization mechanisms. Our results provide new insights into the characteristic features of deep brain lymphatic vessels, as well as their regulation by stressful life events.