Study On Expression Alteration Of NR2B Subunit Of NMDA Receptor And Preliminary Mechanism In Hypothalamus Of Rats After Severe Traumatic Injury Stress

The activation of hypothalamic-pituitary-adrenal cortex axis(HPA axis) exerts protectiveeffects on our body through glucocorticoid secretion and regulation of immunologic andinflammatory response and metabolism after severe traumatic injury. However,excessive andlong-duration HPA axis activation may cause severe secondary injury.As it reported by manyreseachers,the long-term rise of serum glucocorticoid afer severe traumatic and bury injury orin patients in intensive unit care,has a negative close correlation withprognosis.Therefore,how to prevent and manage the secondary injury afer excessive HPA axisactivation is the hot and key problem planned to be solved of clinically curing severe tramaticinjury.Glutamic acid and its Ionotropic receptor--N-methyl-D-aspartic acid receptor(NMDAreceptor) are involved in many central nervous system process of physiology andpathology,such as plasticity of synapse,learning and memory,cerebral ischemic disease andeplipsy.Some authors reported that NMDA receptor,especially NR2B subunit,is highlyexpressed in paraventricular nucleus(PVN) and Supraoptic nucleus in hypothalamus.With thein-depth study,the functional diversity of NR2B and its role in excitability regulation ofneuroendocrine and PVN neurons draw much attention to researcher.At the same time,the dramatic increasing expression of corticotropin releasing hormone(CRH) and its receptor corticotropin releasing hormone receptor(CRHR) afer server tramaticinjury may play a important role in serum glucocorticoid rise.Furthermore,an animalexperiment suggests that afer acute stress reponse,the NMDA subunit in PVN neuronschanged,and CRH may have a key role in this expression alteration.However,there is stillunclear that the hypothalamus NMDA subunit expression pattern and its role in HPA activityregulation and there is also unknown that whether the key hormone of stress response—CRHhas a regulatory fucnction in NR2B expression afer severe burn injury. The current traumatic injury animal models are mainly including gunshotinjury,explosive injury,collision injury and burn injury. In this experiment,we established30％total body surface area burn of rats as the severe tramatic animal model,because of theinjury conditions, injury parameters,injury degree and lesion location of gunshotinjury,explosive injury and collision injury are difficult to precise control and are notconducive to study stress response after tramatic injury.We were using serum cortisolconcentration as an indicators of HPA axis activity.To observe the expression pattern of NR1and NR2B in hypothalamus,we used immunochemistry and western-blotting techniques totest protein expression afer burn injury.In addition,we established intracerebroventricularcannulated rat model using unique cannula system and the agent is administered through theimplanted tube system.To futher verify the regulatory role of CRH in NR2B subunitexpreesion,the reverse transcription polymerase chain reaction(RT-PCR) andimmunochemistry techniques are adopted to test RNA and protein expression level.Main results and conclusions:1.After severe burn injury,the serum cotisol concentration was significantincrease,especially noted at6hours afer burn(P<0.05),and mantaied a high levelconcentration to post-burn48hours.Besides,the antagonist of CRH receptor1and NMDAreceptor can partly reverse this effects.Compared to post-burn6hours group,injection ofCP154,526and MK-801groups serum cortisol concentration are notably decreased(P<0.05).To testify cortisol concentration alteration,we established intracerebroventricular cannulatedrat model.Compare to saline injection group (44.5±4.6) ng/ml,the lower CRH concentrationinjection group’s serum concentration was significant increased (199.5±18.4) ng/ml(P<0.01)and the increase of CRH injection concentration was accompanied by serum corisolconcentration rise. The results suggest that the signaling pathway of CRH and NMDA mayplay a role in high excitability of HPA afer severe burn injury.2.The immunochemistry staining and western-blotting testing showed that NR1subunitexpression was enhanced,in particular at post-burn24hours(P<0.05),and was not increased inCP154,526injection group.However,NR2B expression had a opposite results.There was anotably reduction of NR2B subunit expression in6hours after burn injury and injection ofCP154,526can antagognize this effects,causing NR2B increase(P<0.05).These resultssuggested that CRH may play,at least, a partial role in NR2B decrease after severe burn injury.This results further suggest that downregulation of NR2B by CRH may be onemechanism of HPA high excitability.3.Using RT-PCR technique to examine NR1and NR2B expressions after agentinjection,the results showed that NR2B subunit expression was not changed in CP154,526andCRH plus CP154,526injection groups and was decreased in the lower CRH (1ug/6ul) andhiger CRH(5ug/6ul)concentration injection groups.Furthermore,the immunochemistrystaining obtained the similar results.Besides,To observe the effects of CRH on NR1subunitexpression,we also used RT-PCR technique to testify NR1expression inhypothalamus.Compared to saline injection group,there was no significant difference in CRHinjection rats.The futher immunochemistry staining results suggest that CRH may play animportant role in NR2B expression regulation.In summary,NMDA receptor subunits NR1and NR2B was obviously changed afersevere burn injury and further experiment through establishing intracerebroventricularcannulated rat model suggested that CRH may have a negative regulatory role in NR2B andhave no regulatory funcntion on NR1.Moreover,these results implied that regulation of NR2Bin hypothalamus by CRH may be one of important underlying mechanisms of centralinitiation and mantainence of stress response.To inhibit or antagonize CRH receptor may havea important clinical significance in excessive stress response prevention and treatment.