| Recently, the incidence rate of traumatic brain injury (TBI)has increased year by year. Both in developed and developing countries, TBI has become one of the main causes of death for young people, especially those under 35 years. Therefore, clinicians and neuroscientists closely pay attention to the research on mechanism and treating measures of TBI.After traumatic brain injury, whether mechanical injury or ischemia, hypoxia, ionic disequilibrium and toxic effects of excitory amino acids appeared subsequently all could casuse neurons damage and death, further lead to secondary edema and dysfunction of brain. Hippocampus is an important target and highly sensitive to injury when traumatic brain injury occurrs. The function of hippocampus is closely related to learning and memory, which plays main roles in the process of acquiring, indexing, consolidating and storing spatial information. There are no effective measures at present to cure dysfunctions of learning and memeory because of hippocampus damage, which has become one of the important reasons resulting in badly treating effect and high physical disability after TBI. Therefore, the injured mechanism and treatment of hippocampal neurons after TBI have evoked considerable interests all over the world in recent years.It is indicated that glutamate is one of the primary neurotransmitters in synapse circuit of hippocampus. At the same time, the effects of glutamate and its receptors have been the main research content about molecular mechanism of TBI. Though many antagonists against glutamate receptor have been developed recent years, the results are not satisfying because of evident side effects. The reason of which is that low specificity of antagonists when the antagonist blocks calcium ions ,it also obstructs normal information transmission in synapses. So, it is very necessary to study the expression of Glu subunit of hippocampal neurons after TBI and the regulating process of transport deeply. It has not only theoretical significance to recognize the pathogenetic mechanism of TBI, but also clinical value to design more specific and less side effect drug against glutamate receptor.The Glutamate receptors (GluRs) are categorized into two classes, metabotropic ( namely G-proteins coupled receptor) and ionotropic receptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: N-methyl-D-aspartate (NMDA) receptor,a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA) and kainate receptors. It is demonstrated that the content of NMDA and AMPA receptors in CA1 region of hippocampus are more than those in other regions. In general, NMDA receptors located on membrane of CA1 neurons are characterized by a high permeability to Ca2+, while AMPA receptors show no permeability to Ca2+.The permeability to Ca2+ of AMPA receptors are related to GluR2 subunit, among which AMPA receptors with GluR2 subunit may prevent the channels from cellular influx of Ca2+ whereas AMPA receptors without GluR2 subunit show significant permeability to Ca2+. Extensive researches have been carried out about effects of NMDA receptor on TBI. Although blockage of NMDA receptor can protect hippocampal neurons from TBI, there was a distance to prospective effects. The biological effects of AMPA receptor in TBI attracted wide attention at present.It has been reported that in global ischemia of rat AMPA receptors with GluR2 located on postsynaptic membrane of hippocampal neurons diminished notably, whereas AMPA receptors without GluR2 increased, which obviously induced Ca2+ influx into cytoplasm and neuronal death. The mechanism of that remains unclear.AMPA receptors are located on the plasma membrane of neural cell. Although the function of receptors are regulated by many procedures outside or inside the cell, phosphorylation is the most important factor influencing the function of plasma receptors. Phosphatase and tensin homology deleted on chromosome ten (PTEN) was cloned as a novel tumor suppressor gene in 1997. PTEN can antagonize PI3K signaling pathway and inhibit the growth of cells by dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3).In addition, PTEN can interrupt cell cycle and induce apoptosis,further, suppresses migration, diffusion, aggregation and trafficking of cells. Recent studies implicated PTEN took part in the process of cerebral ischemia injury and was associated with biological function of excitory amino acid NMDA receptors. However the changes of PTEN expression and interaction with AMPA receptors in hippocampus damage by TBI has not been reported . Thus, we firstly established moderate traumatic brain injury model of rats by Impactor II weight-drop equipment; Secondly, the relationship between PTEN and changes in morphology and function of hippocampal neurons after TBI as well as the regulation on AMPA receptor were observed; Finally, the regulated effect of PTEN on hippocampal neurons injury after TBI was initially analyzed and the mechanism of which was discussed.Main techniques and methods1. Models of traumatic brain injury were established through strength inducing to temporal right side of the rat's cortex with Impactor-II weight-drop equipment modified by Wise Young.2. The model of moderate traumatic brain injury was produced by 50g.cm forece's strike. The animals were divided into three groups: normal controlled group, injured group; group treated with PTEN inhibitor bpv through intracerebroventricular injection. The expression of PTEN mRNA and protein at different intervals post injury was detected by immunohistochemistry, RT-PCR and western blot. The apoptosis and survival of neurons in hippocampus at different intervals post injury were observed by TUNEL and NF-200 immunofluorescent staining respectively.3. A stretch injury model was established with primary cultured hippocampal neurons in vitro. In this experiment , the cells were divided into normal controlled group, injured group and bpv treated group. Expression of GluR2 in neurons post-injury was detected by immunofluorescent staining. At the same time, the death of injured neurons was examined by PI staining.4. The expression of PTEN, p-PTEN and GluR2 in neurons after loaded stretch injury was estimated by western Blot and RT-PCR. The free Ca2+ in cytoplasm of neurons was labeled with Fura-3 and the changes of Ca2+ concentration were scanned with laser confocal microscope.Main Results:1. Mild,moderate,severe traumatic brain injury models were established through 20g.cm,50g.cm,100g.cm force's strike inducing to temporal right side of the rat's cortex respectively, with Impactor-II weight-drop equipment modified by Wise Young.2. Apoptotic neurons of hippocampus in injured side increased notably after moderate TBI, reaching peak on the 1st day, maintaining on the 3rd day and decreasing significantly on 7th day, which indicated that hippocampus damage caused by TBI was of regional selectivity and neurons in CA1 region were most sensitive to injury. The expression of PTENmRNA in injured hippocampal neurons increased remarkably 12 hours post-injury, so as the PTEN protein,which was prominent at 24 hours post-injury. All of the results implyed PTEN participated the pathophysiological procedure of TBI. The apoptosis and loss of hippocampal neurons after TBI can be diminished through intracerebroventricular injection of PTEN inhibitor bpv.3. PTENmRNA and protein in cultured hippocampal neurons both increased after stretched injury, accompanied with added dead neurons. The number of dead neurons post-injury was decreased by 100nM, 200nM and 500nM PTEN inhibitor pre-treatment, among which the protective effect of 200nM bpv was better than that of 100nM, but had no difference compared with 500nM group. This data demonstrated that to some extent the protective effect of PTEN inhibitor was of dose-dependent.4. It was firstly clarified by western blot text that level of PTEN and p-PTEN in injured group or bpv treated group were both more than that in normal controlled group at 12,24 and 72 hours after neurons stretched injury, but there was no difference between the injured group and bpv treated group. However, level of p-PTEN in bpv treated group was lower than that in the injured group at all time post-injury. It was indicated that bpv antagonized the phosphorylation of PTEN in injured hippocampal neurons.5. It was observed that expression of GluR2 on membrane of hippocampal neurons decreased by immunofluorescent staining and the permeability to Ca2+ increased after stretched injury. PTEN inhibitor could suppress GluR2 diminishing and Ca2+ influx. However total GluR2 in neurons didn't change post injury, which indicated PTEN inhibitor bpv antagonized phosphatase activity of PTEN, resulting in prevention GluR2-containing AMPA receptor located on membrane decreasing and relieving of neuron injury by Ca2+ influx, which may be the possible mechanism of PTEN inhibitor regulation of AMPA receptor and have protective effect on neurons.6. The changes of Fura-3 fluorescent intensity were detected by laser confocal microscope real-time scanning. It was observed that blockage of NMDA receptor partly prevented Ca2+ from influx into cytoplasm casused by Glu, at the same time , bpv combined with MK-801 could significantly decrease but not completely forbid Ca2+ influx stimulated by Glu. Ca2+ influx in bpv+ MK801+LY294002 pre-treated neurons stimualted by Glu was much more than those in bpv+ MK801 pre-treated group, which demonstrated that PTEN regulated AMPA receptor in the injured neurons through PI3K-Akt signaling pathway.Conclusions:1. The damage to brain tissue after TBI is of selectivity. Neurons in hippocampal CA1 region are most sensitive to injured factors. In this study we fisrtly observed that PTEN took part in the process of secondary injury to hippocampal neurons after TBI and was associated with apoptosis and survival of neurons.2. It was identified PTEN inhibitor bpv had protective effect on injured hippocampal neurons both in vivo and in vitro.3. It was firstly observed that PTEN inhibitor bpv antagonized phosphatase activity of PTEN, resulting in preventing GluR2-containing AMPA receptor located on membrane decreasing and relieving of neuron injury by Ca2+ influx.It may be the protective mechanism of bpv .4. It was well established that PTEN, via PI3K-Akt pathway, mediated GluR2 regulation.
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