Cerebral Expression Of 5-lipoxygenase And Its Possible Roles In A Rat Model Of Surgical Brain Injury

Objective:To investigate the spatio-temporal expression of 5-Lipoxygenase(5-LOX)and its possible roles and underlying mechanisms in the pathogenesis of surgically induced brain injury(SBI)with a rat model of SBI.Methods:1.SBI rat model was established by partial removal of the prefrontal cortex.2.The changes of neurobehavioral function were measured with modified Garcia and balance beam tests.3.The brain water content(BWC)after SBI in different groups were calculated according to wet-dry weight formula.4.The pathological changes of brain tissue in rats after SBI were observed by Hematoxylin &Eosi Staining.5.The distribution and cellular location of 5-LOX were detected by immunofluorescent staining.6.The activity of SOD and the amount of MDA in the injured brain tissue were determined using biochemistry method.7.Western Blotting were used to investigate the expression of 5-LOX,p-5-LOX(Ser271)and NF-?B in the damaged brain tissue in order to explore their possible roles.Results:1.Neurobehavioural tests: Compared with the control group,obvious functional deficits were observed in the rats of SBI-1d group and SBI-3d group.The Garcia Scores in 1d,3d and 7d group after SBI were significantly decreased compared with the Sham group(P<0.01),there was a significant difference between 1d group and 3d group after SBI(P<0.05);compared with 7d group post SBI,there was a significant difference in 1d and 3d group post SBI.The beam balance scores of 1d,3d and 7d group after SBI were evidently decreased compared with the sham group(P<0.01),but there was no significant difference among the 1d,3d and7 d groups after SBI.2.Brain water content(BWC)measurement: BWC of injured side(right frontal lobe)increased in group day 1 and day3 groups and peaked on day 3 after SBI,but tended to recover on day 7 post SBI.Compared with the Sham and SBI-7d group,BWC increased significantly in SBI-1d and SBI-3d group(P<0.05).There was no significant difference between SBI-3d group and SBI-1d group with higher BWC in SBI-3d group.All other brain regions remained similar in BWC among groups.3.H&E staining: In Sham group,the cellular structure was intact and there was no edema signs.In SBI group,the general arrangement of brain cells appeared in disorder and the intercelluar space was increased.Around the injured area,neuronal deformation and necrosis were found in accompany with the inflitration of proliferative astrocytes,polymorphonuclear cells and macrophages.In necrotic neurons,the eosinophilic cytoplasm wrapped the nucleus and the tissue gap is broadened.Vacuolar formation was observed in each time point especially obvious in SBI-3d group.The lesion signs can be observed till day 7 post surgery,but cell edema and leukocyte infiltration decreased.4.Immunofluorencent staining: There were almost no expression of 5-LOX discovered in the sham group.5-LOX was increased in the brains of SBI-1d and SBI-3d groups.In SBI-7d group,the expression of 5-LOX was reduced but still higher than that in the Sham group.Compared with normal brain and contralateral counterpart,the expression of 5-LOX was significantly increased in the ipsilateral injured area.For celluar localization,5-LOX was found to distribute around the lesions and primarily localize in neurons,followed by glial cells and microglia.The invaded leucocytes are also 5-LOX-positive.5.Determination of oxidative stress marks: Compared with the control group,the activity of SOD decreased in SBI-1d group and SBI-3d group(P<0.05)with significance,and tended to rebound on day 7 post-surgery.The amount of MDA increased in SBI-1d and SBI-3d groups significantly(P<0.05),and returned to normal level in SBI-7d group.6.Western blotting:The levels of 5-LOX,p-5-LOX(Ser 271)and NF-?B in brain tissue were significantly higher in SBI-1d and SBI-3d groups compared with sham group(P<0.05),with the peak on day1 after operation.The expressions of 5-LOX,p-5-LOX(Ser271)and NF-?B were decreased in the SBI-7d group compared with SBI-1d and SBI-3d groups with statistical significance(P<0.05).Conclusion:5-LOX expression is mainly located in the cytoplasm of neurons in injured brain area and reachs peak on day 1 post surgery.The activation of 5-LOX after brain injury initiates the inflammatory cascade by it enzymatic and non-enzymatic actions,it is also possible that it participates in SBI pathogenesis by oxidative stress mechanism.In order to improve the outcome of brain injury,it may be an effective strategy to inhibit 5-LOX expression by preconditioning prior to operation or administrating inhibitors immediately after operation.