| Objective: To explore the temporal expression of Neuroligin-1(NLG-1)and Postsynaptic density protein 95(PSD-95)in the injured cortex of mice after a moderate traumatic brain injury.The cognitive and motor function were also studied at different time points after the injury.Methods: In the present study,we built a mouse model of a moderate traumatic brain injury(TBI)using the free fall impact method.Mice were randomly divided into Sham operation group(SHAM group)and different time points post TBI,including 12 h group,24 h group,3 d group,7 d group,14 d group and 21 d group.The neurological function score,learning and memory ability and limb motor function were also tested at these time points.We used HE staining to observe the morphological changes in the cortex of injured mice.Immunofluorescence staining,immunohistochemical staining and Western blotting were used to detect the expression of NLG-1 and PSD-95 protein.The grayscale value and integrated density were analyzed by Image J,and the above results were statistically analyzed by Graph Pad Prism 7.0.Finally,we analyzed the ratio of NLG-1 to PSD-95 and investigate the relationship between the ratio and the timing of injuries.Results: 1.In the grip test,the limb motor ability of mice after TBI was generally lower than that in SHAM group,especially at 12 h group after TBI(p < 0.05).2.In the new object recognition(NOR)test,the index of NOR in mice decreased significantly at 12 h and 24 h after TBI(p < 0.05).3.HE staining showed that in the SHAM group,the neurons in the brain tissue were arranged orderly and evenly distributed,with regular shape,no obvious neuronal swelling,clear oval cell outline,blue staining in the nucleus,symmetrical red staining in the cytoplasm,without widening of vascular space in the interstitium,dilatation of blood vessels and inflammatory cell infiltration.The group after TBI : the brain tissue of the injury and its surrounding area showed obvious edema and part of the nuclear boundary was unclear,the vacuolar degeneration and atrophy of neurons,the nucleus was deeply stained,the intercellular space was widened,the eosinophilia of the cytoplasm was weakened,the nucleus was deeply stained,and inflammatory cells infiltrated.4.The results of immunohistochemical staining showed that NLG-1 and PSD-95 were mainly expressed on the membrane of nerve cells.Combined with Western blotting results,the expression of NLG-1 from 12 h to 14 d in the injured cortex was significantly lower than that in the SHAM group(p < 0.05),and there was no significant difference between the SHAM group and the 21 d after injury.The expression of PSD-95 decreased significantly at 12 h,24 h and 7 d after injury(p <0.05),but there was no significant difference between 14 d ～ 21 d and SHAM group at3 d after injury.There was no significant difference between 14 d ～ 21 d group and SHAM group.During 12 h ～ 3 d after injury,the ratio of NLG-1 to PSD-95 showed a downward-rising-downward-fluctuating trend and the expression of NLG-1 and PSD-95 were gradually increased,but it was still lower than that of the SHAM group.Thus,the ratio of the two-protein expression was decreased from 0.47 to 0.25.During 3 d ～ 7 d after injury,because of the gradually increasing expression of NLG-1 and rapid descent of PSD-95,the ratio was increased from 0.25 to 0.62.During 7 d ～ 14 d after injury,the expression of PSD-95 was gradually stable and the ratio was decreased rapidly from0.62 to 0.36.During 14 d ～ 21 d after injury,the expression of both proteins tended to be stable and the ratio was maintained around 0.4.Conclusion: 1.The learning and memory and the motor ability of limbs were significantly impaired after injury during 12 h ～ 24 h.2.The expression of NLG-1 in ipsilateral cortex between 12 h and 14 d was decreased and returned to the normal level on 21 d after a moderate TBI.The expression of PSD-95 in ipsilateral cortex between12 h ～ 24 h and 3 d ～ 7 d were decreased and returned to the normal level during 14 d ～21 d.3.The expression of NLG-1 and PSD-95 in the ipsilateral cortex showed temporal expression patterns after a moderate TBI,and the ratio of them is expected to be an index to infer the injury time after TBI. |
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