2-chloro-5-hydroxyphenylglycine and Brain-Injuries

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.

Topics: Allosteric Regulation; Animals; Brain Injuries; Cell Count; Cerebral Cortex; Disease Models, Animal; Glycine; Hippocampus; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Microglia; Motor Activity; NADPH Oxidase 2; NADPH Oxidases; Neuroprotective Agents; Nitric Oxide Synthase Type II; Phenylacetates; Pyridines; Receptor, Metabotropic Glutamate 5; Recovery of Function; Thiazoles

Abstract Traumatic brain injury (TBI) induces microglial activation, which can contribute to secondary tissue loss. Activation of mGluR5 reduces microglial activation and inhibits microglial-mediated neurodegeneration in vitro, and is neuroprotective in experimental models of CNS injury. In vitro studies also suggest that the beneficial effects of mGluR5 activation involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition in activated microglia. We hypothesized that activation of mGluR5 by the selective agonist CHPG after TBI in mice is neuroprotective and that its therapeutic actions are mediated by NADPH oxidase inhibition. Vehicle, CHPG, or CHPG plus the mGluR5 antagonist (MPEP), were administered centrally, 30 minutes post-TBI, and functional recovery and lesion volume was assessed. CHPG significantly attenuated post-traumatic sensorimotor and cognitive deficits, and reduced lesion volumes; these effects were blocked by MPEP, thereby indicating neuroprotection involved selective activation of mGluR5. CHPG treatment also reduced NFκB activity and nitrite production in lipopolysaccharide-stimulated microglia and the protective effects of CHPG treatment were abrogated in NADPH oxidase deficient microglial cultures (gp91(phox-/-)). To address whether the neuroprotective effects of CHPG are mediated via the inhibition of NADPH oxidase, we administered the NADPH oxidase inhibitor apocynin with or without CHPG treatment after TBI. Both apocynin or CHPG treatment alone improved sensorimotor deficits and reduced lesion volumes when compared with vehicle-treated mice; however, the combined CHPG + apocynin treatment was not superior to CHPG alone. These data suggest that the neuroprotective effects of activating mGluR5 receptors after TBI are mediated, in part, via the inhibition of NADPH oxidase.

Topics: Animals; Blotting, Western; Brain Injuries; Enzyme Inhibitors; Glycine; Male; Mice; Mice, Inbred C57BL; NADPH Oxidases; Neuroprotective Agents; Phenylacetates; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Recovery of Function

A wealth of evidence has shown that microglia-associated neuro-inflammation is involved in the secondary brain injury contributed to the poor outcome after traumatic brain injury (TBI). In vitro studies were reported that activation of metabotropic glutamate receptor 5 (mGluR5) could inhibit the microglia-associated inflammation in response to lipopolysaccharide and our previous study indicated that mGluR5 was expressed in activated microglia following TBI. However, there is little known about whether mGluR5 activation can provide neuro-protection and reduce microglia-associated neuro-inflammation in rats after TBI. The goal of the present study was to investigate the effects of mGluR5 activation with selective agonist CHPG, on cerebral edema, neuronal degeneration, microglia activation and the releasing of pro-inflammatory cytokines, in a rat model of TBI. Rats were randomly distributed into various subgroups undergoing the sham surgery or TBI procedures, and 250 nmol of CHPG or equal volume vehicle was given through intracerebroventricular injection at 30 min post-TBI. All rats were sacrificed at 24 h after TBI for the further measurements. Our data indicated that post-TBI treatment with CHPG could significantly reduce the secondary brain injury characterized by the cerebral edema and neuronal degeneration, lead to the inhibition of microglia activation and decrease the expression of pro-inflammatory cytokines in both mRNA transcription and protein synthesis. These results provide the substantial evidence that activation of mGluR5 reduces the secondary brain injury after TBI, in part, through modulating microglia-associated neuro-inflammation.

Topics: Animals; Brain Edema; Brain Injuries; Excitatory Amino Acid Agonists; Glycine; Male; Microglia; Nerve Degeneration; Phenylacetates; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

Group I metabotropic glutamate receptors (mGluRs) have been implicated in the pathophysiology of central nervous system injury, but the role of mGluR5 in traumatic brain injury (TBI) remains unclear. In the present study, we investigated the neuroprotective potency of (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG), a selective mGluR5 agonist, for protecting against TBI in both in vitro and in vivo models. Primary cortical neurons were treated with 1 mM CHPG in an in vitro preparation 30 min before TBI, and 250 nM CHPG was injected into the right lateral ventricle of rats 30 min before TBI was induced in in vivo studies. The results showed that CHPG significantly attenuated lactate dehydrogenase (LDH) release and neuronal apoptosis and reduced lesion volume. Compared to the control or vehicle group, the phosphorylation levels of extracellular signal-regulated kinase (ERK) and Akt were increased in the presence of CHPG, even following the induction of TBI. Furthermore, treatment with either the ERK inhibitor PD98059 or Akt inhibitor LY294002 partially reversed the CHPG's neuroprotective effects. These data suggest that CHPG minimizes brain damage after induction of TBI both in vitro and in vivo, and that these protective effects were possibly mediated by activation of the ERK and Akt signaling pathways. Thus, potentiating mGluR5 activity with selective agonists such as CHPG may be useful for the treatment of traumatic brain injury.

Topics: Animals; Brain Injuries; Cells, Cultured; Chromones; Glycine; L-Lactate Dehydrogenase; Male; Mitogen-Activated Protein Kinases; Morpholines; Neuroprotective Agents; Phenylacetates; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Signal Transduction

Several previous studies utilizing selective pharmacological antagonists have demonstrated that type 5 metabotropic glutamate receptors (mGluR5) are potential therapeutic targets for the treatment of numerous disorders of the central nervous system, but the role of mGluR5 activation in traumatic brain injury (TBI) is not fully understood. Here in an in vitro TBI model, the mGluR5 agonist (RS)-2-chloro-5- hydroxyphenylglycine (CHPG) and the positive allosteric modulators 3-cyano-N-(1,3- diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) were used to investigate the neuroprotective potency of mGluR5 activation. Data showed that CHPG and CDPPB suppressed the increase of LDH release and caspase-3 activation induced by traumatic neuronal injury in a dose-dependent manner, and the salutary effects were also present when these compounds were added 1 h after injury. Western blot was used to examine the activation of three members of mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase (p38). CHPG and CDPPB enhanced the activation of ERK after traumatic neuronal injury, and PD98059 and U0126, two selective MEK/ERK inhibitors, partly revised the protective effects. Furthermore, we also investigated the role of protein kinase C (PKC) in CHPG and CDPPB-induced neuroprotection. With the pretreatment of chelerythrine chloride, a PKC inhibitor, the surpressing effects of CHPG and CDPPB on traumatic injury-evoked LDH release and caspase-3 activation were blocked. All of these findings extended the protective role of mGluR5 activation in an in vitro model of TBI and suggested that these protective effects might be mediated by the PKC-dependent activation of MEK/ERK pathway. These results may have important implications for the development of mGluR5 modulators to treat TBI.

Topics: Animals; Benzamides; Blotting, Western; Brain Injuries; Cells, Cultured; Enzyme Activation; Excitatory Amino Acid Agonists; Female; Glycine; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neuroprotective Agents; Phenylacetates; Phosphorylation; Pregnancy; Protein Kinase C; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate

Traumatic brain injury initiates biochemical processes that lead to secondary neurodegeneration. Imaging studies suggest that tissue loss may continue for months or years after traumatic brain injury in association with chronic microglial activation. Recently we found that metabotropic glutamate receptor 5 (mGluR5) activation by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) decreases microglial activation and release of associated pro-inflammatory factors in vitro, which is mediated in part through inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Here we examined whether delayed CHPG administration reduces chronic neuroinflammation and associated neurodegeneration after experimental traumatic brain injury in mice.. One month after controlled cortical impact traumatic brain injury, C57Bl/6 mice were randomly assigned to treatment with single dose intracerebroventricular CHPG, vehicle or CHPG plus a selective mGluR5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine. Lesion volume, white matter tract integrity and neurological recovery were assessed over the following three months.. Traumatic brain injury resulted in mGluR5 expression in reactive microglia of the cortex and hippocampus at one month post-injury. Delayed CHPG treatment reduced expression of reactive microglia expressing NADPH oxidase subunits; decreased hippocampal neuronal loss; limited lesion progression, as measured by repeated T2-weighted magnetic resonance imaging (at one, two and three months) and white matter loss, as measured by high field ex vivo diffusion tensor imaging at four months; and significantly improved motor and cognitive recovery in comparison to the other treatment groups.. Markedly delayed, single dose treatment with CHPG significantly improves functional recovery and limits lesion progression after experimental traumatic brain injury, likely in part through actions at mGluR5 receptors that modulate neuroinflammation.

Topics: Animals; Brain Injuries; Calcium-Binding Proteins; Cognition Disorders; Diffusion Tensor Imaging; Disease Models, Animal; Ectodysplasins; Encephalitis; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Glycine; Hippocampus; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Movement Disorders; Neurodegenerative Diseases; Neurons; Phenylacetates; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Immunologic; Receptors, Metabotropic Glutamate; Recovery of Function; Statistics, Nonparametric; Thiazoles; Time Factors

Traumatic brain injury (TBI)-released excessive glutamate resulted in the activation of glutamate receptors including the metabotropic glutamate receptor 5 (mGluR5). To investigate the expression and cell distribution of mGluR5 in the rat cortex following TBI, western blot and quantitative real-time PCR were used to study the protein and mRNA level of mGluR5 respectively while immunohistochemistry analysis and double immunofluorescence with neural cell marker were used to define the cell distribution of mGluR5. Furthermore, we examined the effects of post-TBI administration of (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG), a selective mGluR5 agonist, on the neuronal degeneration in the cortex. In the present study, we found that the protein level of mGluR5 was up-regulated by traumatic brain injury, while TBI-induced mGluR5 mRNA expression displayed biphasic changes with up-regulation in the early time and down-regulation in the late time after TBI. And neuron, astrocyte and microglia in the cortex after TBI all expressed mGluR5. Moreover, CHPG treatment significantly reduced the number of degenerating neurons detected by Fluoro-Jade C staining. These findings demonstrate that expression of mGluR5 differentially changes both spatially and temporally after TBI and may be related to the neuroprotection after TBI. Therefore, understanding the expression and cell distribution of mGluR5 after TBI may give insight into pathophysiology after TBI and provide a new target for the therapy of TBI.

Topics: Animals; Brain Injuries; Cerebral Cortex; Excitatory Amino Acid Agonists; Glycine; Male; Nerve Degeneration; Neurons; Phenylacetates; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Tissue Distribution