Microglia, the resident immune cells of the brain, become activated and mediate neuroinflammatory responses in response to traumatic brain injury (TBI). This neuroinflammation can be detrimental to the health of the brain; thus, inhibition of this natural response can benefit TBI patients. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a technological advancement that allow manipulation of specific cellular signaling pathways. The hM4D DREADD gene was inserted in a viral construct downstream of the CD68 promoter, which is markedly upregulated in activated microglia following TBI. This construct is activated with clozapine n-oxide (CNO) to downregulate secondary messengers in activated microglia and thus attenuates the inflammatory response by reducing microglial activation and proliferation after injury. After viral transfection to deliver the construct to cells, rats underwent a controlled cortical impact (CCI) - an experimental model of TBI - and were treated with a CNO injection at varied intervals after injury. After the rat brains were dissected and sectioned, we used immunohistochemistry techniques to label microglia with an anti-Iba1 antibody, proliferating cells with an anti-BrdU antibody, and cell nuclei with DAPI. This allowed for the visualization of microglia using fluorescence microscopy and microglia were quantified using stereological principles. The aim of this research project is to use pharmacological DREADD receptor-mediated inhibition of microglia at different intervals post-injury to quantify the proliferation of microglia following CCI. We hypothesize that increased duration between CCI and CNO injection leads to more pronounced microglial activation represented by increased numbers and morphological changes. We also hypothesize that microglial activation can be observed as a gradient with the greatest proliferation closest to the CCI epicenter.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic event confers neuronal and axonal protection against subsequent ischemic exposure. The cell types responsible for IPC in the brain are unknown. In a novel model of white matter (WM) IPC and ischemic injury, we identified specific innate immune signaling pathways in microglia as required for IPC-mediated axonal protection, leading us to suspect that microglia are required for IPC. The model of WM IPC involves exposing the mouse optic nerve (MON) to a brief ischemic event 72 hours before the MONs are isolated and exposed to oxygen-glucose deprivation. Animals were treated with PLX5622 - a colony stimulating factor-1 receptor (CSF1R) pharmacologic antagonist – to deplete microglia in the central nervous system, including the MON, to test our hypothesis. By recording axonal function, we determined that microglial depletion eliminated IPC-mediated axonal protection in the WM. This study examines the impacts of IPC and ischemia on the nodes of Ranvier, which we hypothesize are protected by preconditioned microglia. The length of the nodes of Ranvier affects conductance and action potential propagation through an axon, with recent publications suggesting elongation of the notes in disease or injury states. We hypothesize the nodes of Ranvier will be shorter in preconditioned MONs than non-preconditioned MONs, which would support IPC-mediated protection. Additionally, we expect the nodes of Ranvier in PLX5622-treated MONS will be of similar length as non-preconditioned control MONs, indicating loss of IPC-mediated protection in animals without microglia. We will use immunofluorescence and confocal microscopy to measure the nodes of Ranvier and correlate these anatomical findings to prior electrophysiology experiments. The results of this research would contextualize a novel understanding of how microglia and ischemia affect the WM in specific regions of the axon, which is vital for advancing the development of neurotherapeutics for stroke.