Glutamate Receptors, Cell death, & TBI

Following Central Nervous System (CNS) injury, the extracellular concentration of glutamate is increased and the expression and function of metabotropic glutamate receptors (mGluR) are also altered. We have been studying the effects of modulating group 1 mGluRs (mGluR1 and mGluR5) activity in models of CNS injury and neurodegeneration, and have demonstrated that mGluR1 activation exacerbates neuronal death mechanisms, particularly necrotic cell death. In contrast, activation of mGluR5 is neuroprotective and has anti-apoptotic properties in neuronal cell culture models and has strong anti-inflammatory effects in microglial cell culture models. In vivo studies confirm these findings and activation of mGluR5 by CHPG provides significant neuroprotection in models of focal cerebral ischemia, spinal cord injury (SCI), and traumatic brain injury (TBI).

Our recent studies demonstrate that selective activation of mGluR5 by the agonist, CHPG, reduces microglial activation and the associated release of pro-inflammatory mediators following SCI and TBI. Furthermore, the protective effects of mGluR5 activation are mediated, in part, via the inhibition of NADPH oxidase as CHPG treatment reduces NADPH oxidase activity levels, and siRNA knockdown or knockout (gp91phox-/-) of NADPH oxidase subunits in microglia reversed the neuroprotective effects of CHPG treatment. Notably, we have also demonstrated that delayed administration of CHPG at 1 month after TBI resulted in reduced expression of chronically activated microglia that expressed NADPH oxidase subunits, and these changes were associated with reduced neurodegeneration and improved functional recovery at 4 months post-injury. On-going studies in this research program include investigating the neuroprotective effects of novel mGluR5 positive allosteric modulators (PAMs) in models of neuronal cell death and microglial activation, as well as the therapeutic potential of these drugs when administered following TBI.

mGluR5 activation attenuates NFκB signaling in microglia and the anti-inflammatory effects of CHPG treatment are mediated by the inhibition of NADPH oxidase.

mGluR5 activation attenuates NFkB signaling
LPS stimulation resulted in a significant increase in NFκB activity in BV2 microglia (***p<0.001 versus control), and CHPG treatment significantly reduced NFκB activity (+++p<0.001 versus LPS) in a NF- κB-luciferase reporter assay.

Nitrite levels were measured in the media from the BV2 microglia transfected with the NF-κB-luciferase reporter plasmids. LPS stimulation resulted in a significant increase in nitrite levels (***p<0.001 versus control), and CHPG treatment significantly reduced LPS-stimulated nitrite production (+++p<0.001 versus LPS).

LPS stimulation resulted in a significant increase in intracellular ROS levels in gp91phox+/+ microglia (***p<0.001 versus control [gp91phox+/+]). CHPG treatment significantly reduced ROS levels in gp91phox+/+ microglia (+++p<0.001 versus LPS [gp91phox+/+]). In gp91phox-/- microglia LPS also resulted in a significant increase in intracellular ROS levels (###p<0.001 versus control [gp91phox-/-]). However, these levels were significantly reduced when compared with those in gp91phox+/+ microglia (^^^p<0.001 versus LPS [gp91phox+/+]). Notably, CHPG treatment failed to significantly reduce intracellular ROS levels in gp91phox-/- microglia. Analysis by one-way ANOVA, followed by post-hoc adjustments using a Student Newman-Keuls correction. Mean ± standard error of the mean (n=6/treatment).

From Loane et al., 2012

Delayed treatment with the mGluR5 agonist, CHPG, reduces lesion volume and increases white matter integrity after TBI.

Delayed treatment with the mGluR5 agonist
Lesion volume was assessed using T2-weighted MRI in TBI mice. One month post-injury, all TBI mice had significant and comparable lesions volumes and were subsequently randomized and treated with vehicle, CHPG or MTEP + CHPG. TBI-induced lesion volumes significantly increased over three months in the vehicle-treated TBI group (*P < 0.05 versus one month), whereas the lesions in the CHPG-treated TBI group did not expand and were significantly reduced at three months post-injury when compared to the vehicle-treated TBI group (+P < 0.05 versus vehicle). TBI mice that received MTEP co-administration with CHPG had lesion volumes that were not significantly different compared to vehicle treated TBI levels (n = 6 per group).

Representative MRI images show hyperintense lesion areas from the lesion epicenter and rostral/caudal regions at three months post-injury for each treatment group.

Ex vivo DTI was performed at four months post-injury. White matter tracts of interest were outlined in either color or FA images (Ci) on both the ipsilateral (pink) and contralateral (brown) sides. The ratio of the ipsilateral mean diffusivity (Cii) and FA (Ciii) to contralateral measurements demonstrate that CHPG treatment significantly reduced mean diffusivity and increased FA (*P < 0.05, n = 5).

White matter tracts were outlined (Di) to determine fiber volume (Diii). Three-dimensional visual representations are presented in (Dii). A naïve sample demonstrates the standard volume of white matter tracts in uninjured tissue. TBI resulted in a marked reduction in white matter volume. Fiber volume was significantly increased in CHPG-treated TBI tissue when compared to vehicle treated TBI tissue (*P < 0.05, n = 5). Statistical analysis was by two-way ANOVA with Bonferroni t-test post-hoc corrections in (A), Mann-Whitney U test in (Cii), and Student's t-test in (Ciii) and (Diii). Bars represent mean ± SEM.

From Byrnes et al., 2012