Activation Of Metabotropic Glutamate Receptor 1a And Protective Effect Of Its Antagonist AIDA In Mechanically Neuronal Injury In Vitro And In Vivo

Traumatic brain injury is a major cause of death and disability. The pathophysiology of trauma is believed to consist of two major phases. The primary phase refers to the neuronal damage at the moment of insult by direct mechanical impact on the brain. A secondary or delayed phase refers to the biochemical and cellular events that occur at the time of insult but do not have clinical manifestation until hours or days after injury, as shown by sudden death after clinical regain of consciousness for the patient. At present, the specific mechanisms involved in the secondary injury are not clear. A major mechanism mediating neuronal damage during trauma is the massive release of endogenous glutamate/aspartate and over-stimulation of excitatory amino acid receptors following trauma. This excitotoxic neuronal injury appears to be mediated by ionotropic receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Activation of iGluRs causes influx of Ca²⁺ and Na⁺, leading to excitatory neuronal damage. Recent evidence suggests that activation of mGluRs, which are coupled to second messenger cascades through G proteins, are implicated in the neuronal lost.The mGluRs is a distinct group of G protein-coupled receptor superfamily with at least eight genes encoding for the 8 subtypes of mGluRs (mGluR1-8). These 8 subtypes have been classified into three groups on the basis of sequence homology and coupling of the second-messenger systems. Group I includes mGluR1 and mGluR5, which are coupled with phosphoinositide (PI) hydrolysis. The hydrolysis of PI leads to the generation of inositol triphosphate (IP3) and diacylglycerol, with the subsequent release of intracellular Ca²⁺. Recent studies indicate that acute activation of group I mGluRs contribute to the pathogenesis of trauma.For studying the traumatic brain injury, a model with fine reproducibility and controllability is often indispensable. Applicable injury models include experimental animal models and cell injury models cultured in vitro. Recently, researchers have attached great importance to the in vitro models because they allow for the precisecontrol of the extracellular environment, easy and perhaps repeated access to the cells, and lower associated costs. In this study, we described a novel in vitro traumatic brain injury model in which mechanical injury is delivered by the use of a specially designed device. We hypothesized that blockade of mGluR1a may be neuroprotective in traumatic brain injury. Using both in vitro and in vivo traumatic brain models, we explored the role of mGluR1a in post-traumatic neuronal death and the effects of mGluR1 antagonist AIDA.Part I Establishment of a mechanical injury model of rat cortical neuron in vitroObjective: To introduce an easy culturing method of rat cortical neuron and establish a novel, practical, and reproducible mechanical injury model of the cortical neurons in vitro.Methods: Cortical neurons of neonatal Sprague-Dawly (SD) rats were cultured in vitro. Mechanical injuries were delivered to the neurons by sliding of a syringe needle over cell surface. The control uninjured cultures was treated identically exceptfor the injury procedure. Neuronal damage was assessed by measuring the LDH activity in the cultrre medium and cellular viability at different time points after the injury.Results: Cultured neurons grow well and kept the normal structure. Degeneration, degradation and necrosis of the cultured neurons were observed after injury. The injuried neuronal uptake of Trypan blue increased and cellular viability of injuried neuron was significantly lower than that of the control group at different time points after injury (P<0.05). Compared with that of the control group, LDH activity in injured groups increased significantly from 30 min after the injury (P<0.05). Furthermore, the neuronal uptake of Trypan blue and LDH activity were positively correlated with the degree of injury.Conclusions: This in vitro model of cortical neurons injury can be easily repeated and can simulate the damage mechanism of traumatic brain injury. Graded mechanical injury also can be induced in vitro. This model can be used in the further research oftraumatic brain injury.Part II Expression of mGluRla in in vitro mechanical injury model of rat cortical neuron and the effect of its competive antagonist AIDAObjective: To investigate the expression of mGluR1a in in vitro mechanical injury model of rat cortical neuron and the effect of its antagonist AIDAMethods: Cortical neurons of SD rats were cultured in vitro for 1 week. Mechanical injuries were delivered to the neurons by syringe needle sliding. The control uninjured cultures were treated likewise except for the injury procedure. Level of mGluR1a mRNA was analyzed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). To confirm result obtained by qualitative RT-PCR and to demonstrate the presence of mGluR1a protein in neuronal culture, immunoblot analysis was also performed with a specific anti-mGluR1a antibody. Immunohistochemical examination was made at different time points after the injury. Cell damage was also assessed by measuring LDH activity in the culture medium at different time points after injury. In AIDA-treated group, cell was treated with 100ul AIDA 30mins before the injury procedure. Neuronal Ca²⁺ level was detected with fura-2/AM fluoresce method.Results: There was weak expresion of mGluR1a in the neurons of the control group. RT-PCR perfromed with primers specific for mGluR1a produced a band of the expected size (467bp) in RNA sample extracted from neuronal culture. mGluR1a protein presented as a band at 140kDa in neuronal culture. In the injury groups, widespread expression of mGluR1a positive neurons was seen and the positive staining particles mainly distributed in the cytoplasm around the nucleus after injury (P<0.05). RT-PCR demonstrated that the expression of mGluR1a mRNA increased significantly within 12 hours after injury (P<0.05). Treatment with mGluR1a antagonists AIDA attenuated trauma-induced LDH release from 12 hours after the injury (P<0.05). Ca²⁺ level of injured groups increased significant compared with the control (P<0.05). AIDAtreatment significantly decreased the Ca²⁺ level compared with injury alone (P<0.05). Conclusions: mGluR1a positive neurons increased significantly in in vitro mechanical injury model of the neuron. Its competitive antagonist AIDA can block the Ca²⁺ influx triggered by mGluR1a. AIDA has evident neuroprotective effects after mechanical neuronal injury.Part III mGluR1a expressional pattern and efficacy of its competive antagonist AIDA after traumatic brain injuryObjective: To study the changes of mGluR1a expression in an in vivo model of diffuse brain injury (DBI) and the effect of its competive antagonist AIDA.Methods: SD rats were randomized into the following four groups: sham-injury control group; DBI group; normal saline (NS) treated DBI group; and AIDA treated DBI group. After induction of Marmarouou's rodent DBI model by free drop impact, rats were decapitated and mGluR1a positive neurons were examined by immunohistochemistry at different time points after injury. The water content of brain was also counted. RT-PCR was performed to investigate mGluR1a mRNA level after injury. The sham-injury group underwent the same treatment expect for the injury procedure. AIDA (100nmol) or saline was injected into the right lateral cerebral ventricle (-1.0 mm bregma, 1.5 mm lateral, and 3.6 mm depth) 30 minutes before injury. A total volume of 10ul was injected over a 5 min interval. The water content of brain was counted at diferent time points after injury. Neurological scoring was performed at 24 h and 1 and 2 weeks after TBIResults: In the sham-injury group, the mGluR1a positive neurons remained low at any given time points with normal structure. The water content of brain also remains normal. In DBI group, the water content of brain increased significantly 1 h after injury (P<0.05). Compared with the sham group, the number of mGluR1a -positive cells increased significantly within 1 week and reached its peak value at about 24 hours after injury (P<0.05). RT-PCR demonstrated of the similar mGluR1a mRNA changing pattern as the protein.Compared with NS treatment DBI group, administration of AIDA 30 minutes before DBI significantly decreased the water content of brain 12 h after injury (P<0.05). Neurological scoring demonstrated that administration of AIDA improved neurological behaviors at 2 weeks as compared with NS treated group (P<0.05).Conclusions: The increased mGluR1a expresson after DBI suggested that the activation of mGluR1a may take part in the neuronal death after traumatic brain injury. The marked neuroprotection produced by AIDA against in vivo DBI indicates that mGluR1 agonists may prove to be beneficial in clinical head injury management.