Investigation of cortical vascular functions and pericytes in mice with experimental autoimmune encephalomylities

Background & Objective: Multiple sclerosis (MS) is a major CNS inflammatory disorder that affects millions of people worldwide. It is known that inflammation, demyelination, and neurodegeneration are linked to the pathogenesis of MS. This disorder is thought to be of autoimmune origin and mediated by inflammatory cells that cross the blood-brain barrier to enter the central nervous system. These cells trigger inflammation, leading to lesion formation characterized by demyelination and neurodegeneration. Of note, neurodegeneration is not restricted to the lesions, and it is global. Although neurodegeneration is more prominent in later stages of MS, it is known to start since the beginning of disease. The exact mechanisms of neurodegeneration are still unclear. Precise regulation of the cerebrovascular function is critical for the brain health, and it is regulated by the neurovascular unit (NVU). Pericytes are important elements of the neurovascular unit (NVU) and BBB and play essential roles in cerebral blood flow regulation. The interaction between pericytes and endothelial cells enables rapid and efficient communication, regulating cerebral blood flow (CBF) through a process called neurovascular coupling (NVC) at the capillary level. NVU function and pericytes are adversely affected in Alzheimer's disease. However, the specific roles of NVC and pericytes and their connection to neurodegeneration in MS is not known. We hypothesize that cortical pericytes are affected globally through soluble factors released by inflammatory cells leading to NVU dysfunction and neurodegeneration. In this project, our aim is to reveal whether vascular dysfunction contributes to cortical neurodegeneration in the experimental autoimmune encephalomyelitis (EAE) model, which is widely accepted as an animal model of MS. Methods: Following chronic cranial window opening and habituation in C57BL6/J mice, we induced EAE model by injecting MOG peptides. Weight changes and clinical symptoms were tracked and scored for 26 days after induction. We measured cerebrovascular reactivity (CVR) by blowing air on the whisker pad and applying 5% hypercapnia while the animals were awake and their heads were fixed in three different times: pre-injection, preclinical stage and clinical peak period. We performed the measurements with both laser speckled contrast imaging (LSCI, Perimed) and a custom-made internal optical signal imaging (IOSI) system that was developed by us for that project. We also examined pericytes, vascular and neural cells by immunofluorescence staining and confocal imaging. Results: We detected by both LSCI (after 5% hypercapnia application) and IOSI (after whisker pad stimulation) that CVR was decreased at the peak time of inflammation in the EAE group compared to the controls (p<0.05 and p<0.01, respectively). In histological examinations, we found a significant increase in vascular coverage of pericytes in both cortex and hippocampus in the EAE group (p<0.001). In parallel, we detected multiple foci with loss of NeuN reactivity especially in the subpial and intracortical areas in the EAE group (p<0.05). We also found loss of NeuN reactivity in the ventral horns of the thoracic spinal cord section in the EAE group (p<0.05). Moreover, SMI-32 staining revealed neurite loss in the cortex and hippocampus of EAE mice (p<0.01). In addition, we observed IgG deposition on luminal side of some vessels and also in the parenchyma in both cortex (p<0.04) and hippocampus (p<0.04). We found that pericyte morphology was also impaired in these vessels. Furthermore, we detected decreased staining of three vascular markers, namely tLectin, CD31 and CD105, in cortex of EAE mice. Interestingly, there we did not detect any change in the hippocampus, or GM and WM of the spinal cord. Finally, we detected that microglia are activated diffusely throughout the cortex, some of which were microvessel-associated microglia. Conclusion and comment: Our findings show for the first time in the literature that cortical NVU function and pericytes are adversely affected during EAE. In parallel, neurons, microvessels and microglia were also diffusely affected throughout the cortex. As there is little or no lesion formation in the cortex of mice with EAE, these findings show that cortical structures are diffusely affected by the soluble factors released by inflammatory cells. Our findings bring novel insights into the mechanisms of global neurodegeneration seen in the so called "normal-appearing gray matter" in people with MS. In the future, the development of NVU-preserving treatments could be used to prevent neurodegeneration in MS.