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in Activation & Migration of Microglia/Macrophages:
Implications in Neuroinflammation Elicited by Traumatic Brain Injury"
Molecular Biology, Genetics and Cancer Track
B.S. 2009, Shaheed Udham Singh College of Engineering and Technology, India
Thesis Advisor: Jorge E. Contreras, Ph.D.
Department of Pharmacology, Physiology and Neuroscience
Thursday, October 27, 2016
1:30 P.M., MSB Room H609b
The inflammatory based response during many neurodegenerative diseases, including Multiple Sclerosis and Traumatic Brain Injury (TBI), is mediated by glial cell activation, microglia and leukocyte recruitment towards the injury site, and up-regulation of inflammatory mediators. Activation of various immune cells releases various molecules such as ATP, pro-inflammatory cytokines (IL-1£], TNF-£) in an orchestrated sequence. The potential gates for the release of transmitters, such as ATP and glutamate, to the external milieu are recently attributed to the coordinated action of specialized channels called pannexin channels. Pannexin channels are large nonselective transmembrane channels that are permeable to ions and small molecules such as ATP in numerous cell types. In this study, we will focus on microglial/macrophage cells because of its importance in perpetuating the inflammatory responses in the brain. Despite the recognition of pannexin proteins as important channel pathways for ATP release during brain injury, the understanding of the neuroinflammatory component that will be mediated by pannexin channels, in particular, Panx1 in microglia/macrophage cells is still unknown. Our central hypothesis is that an increase in the activity of Panx1 channel potentiates neuroinflammation via microglia/macrophage activation and migration after injury and that in vivo delivery of pannexin channel blockers will improve the outcome after traumatic brain injury. As a first approach to examine the inflammatory response mediated Panx1 channels in activated microglia/macrophages in vitro, we used Carbenoxolone (CBX) - a general pannexin and connexin hemichannel and gap junction blocker, which has been shown to have anti-inflammatory actions under in vitro and in vivo conditions. Our results suggested that CBX significantly reduced the levels of pro-inflammatory cytokines (IL-1ƒÒ, TNF-ƒÑ, and IL-6), expression of iNOS and nitrite levels in LPS stimulated primary microglial and macrophage (RAW 264.7) cells. However, when more selective Panx1 channel blockers such as Brilliant Blue FCF (BBFCF), Trovafloxacin (TVX) and mimetic blocking peptides against Panx1 channels were used, neither the nitrite levels nor the pro-inflammatory cytokines were prevented in LPS stimulated primary microglial and RAW 264.7 cells. Similar results were found with specific Cx43 blockers. Altogether, our results indicated that CBX is a potent inhibitor of microglia/macrophage inflammatory response and suggest that some of the anti-inflammatory action of CBX could be mediated through a mechanism independent of Panx1 channel activity.
We found that LPS induced acute ATP release in microglial (BV-2) and macrophage (RAW 264.7) cells which was significantly attenuated in the presence of Panx1 blockers (Panx1 mimetic blocking peptides and pharmacological blockers such as BBFCF and TVX). As noted before, ATP is a pro-inflammatory and chemotactic molecule that has been implicated in promoting the migration and recruitment of various inflammatory cells upon injury. To examine whether blockade of Panx1 could reduce the migration of activated microglia/macrophages, we used another chemotactic stimulus, C5a that is a component of the complement system, activated upon tissue injury. Our results showed that C5a induced ATP release and migration which were drastically attenuated in the presence of Panx1 channel blockers.
Upon brain injury, ATP release and migration are two key features important in promoting the migration and recruitment of various immune cells. To evaluate the role of Panx1 in these processes in vivo, we used the Controlled Cortical Impact (CCI) model of traumatic brain injury (TBI) which is characterized by the infiltration of neutrophils and accumulation of microglia/macrophages at the injury site. In our CCI model of injury, immunofluorescence studies, western blot and RT-qPCR data showed that administration of Panx1 channel blocker - TVX, reduced the accumulation of microglial/macrophage cells (CD68, Iba-1) at 6 days post injury (dpi). We next tested whether the reduced accumulation of microglial/macrophage cells at the injury site was due to decreased inflammatory response. At 1dpi, RT-qPCR results indicated that TVX reduced the levels of pro-inflammatory cytokines (IL-1ƒÒ, IL-6 and TNF-a) and infiltrating neutrophils (MPO). In addition, mRNA and protein levels of activated astrocytes (GFAP) and the markers for tissue damage (ƒÑ-II-Spectrin and MMP-9) which peaked at 6 dpi in CCI injured mice were significantly attenuated by TVX treatment. Furthermore, TVX treatment also improved the behavioral outcome in injured mice beginning day 1 post injury. We next used homozygous Panx1 knockout mice (that has 70% reduced mRNA levels of Panx1) to examine the behavior in CCI injured wild type (WT) and homozygous Panx1 knockout (HMZ) mice. Our preliminary results indicated that Panx1 HMZ mice have a tendency to recover better than the WT injured mice. Further analysis to evaluate the anti-inflammatory effects in Panx1 HMZ mice are currently in progress. Taken together, our results suggest that Panx1 channels serve as a pharmacological target to reduce neuroinflammation and promote functional recovery upon brain injury.