Effects Of Intranasal EPO Application On NGB Expression, NOS Activity And NO Content In Cerebral Tissues Of MCAO Rats

Objective: In recent years, ischemic cerebrovascular disease has a gradually increasing incidence, a high fatal rate and disability rate, which severely affects the life quality of patients with ischemic cerebrovascular diseases. Erythropoietin (EPO) is a hemopoietic growth factor with tissue protective effects, and definite tissue protective effects are shown during cell culture and in various mammalian models with nervous system diseases. However, EPO is a macromolecular glucoprotein, with a low permeability rate through blood brain barrier. In various animal models, EPO inclines to protect the nerves, but it has multiple side effects, which limits the application of EPO in clinics. Neuroglobin (NGB) is a newly found globulin which can carry oxygen. It mainly expresses in nervous tissues, participating in cell oxygen supply, clearance of NO and oxyradical. Intranasal application can send EPO directly to cerebral tissues to avoid the obstruction of blood brain barrier, and it is a simple noninvasive drug administrating pathway. In this study, we apply EPO by using intranasal application and peritoneal injection, the NGB expression of different times points, NO level and NOS activity 24h post infarction are observed, thus to investigate the protective pathway of EPO on brain, and supply experimental evidence of intranasal EPO application in clinics.Methods: A hundred SD rats were randomly divided into four groups: intranasal application group, peritoneal injection group, sham operation group, normal saline control group. Three times points, 12h, 24h and 48h after construction of Middle cerebral artery occlusion (MCAO) were chosen, and rats at each time points were divided into three subgroups. MCAO rat model was constructed based on suture method as narrated by Longa et al [1]. No suture was applied in sham operation or control groups, 10 min after cerebral ischemia, 3000 U/kg EPO was injected into abdomen, and 20U/20μl recombinant human erythropoietin (rhEPO) was applied intranasally. Behavior score was conducted at corresponding time points, rats were decapitated after overdose anesthesia, and complete cerebral tissues were collected and observed after stained with hematoxylin-eosin, and NGB expression was detected by using immunohistochemistry staining. NO level in cerebral tissues was determined by using nitrate reductase method, and NOS activity was determined by using chemical colorimetry.Results: NGB expression was increased after cerebral ischemia and anoxemia, no NGB expression was observed in sham operation group, NGB positive cells were more than normal saline control group at three time points in rats with rhEPO intraperitoneally injected or intranasally applied, but no difference was observed in quantities of NGB positive cells in rats treated by two different methods at 12h and 24h. At 48h, NGB positive cells were more in peritoneal injection group than those in intranasal application group. No level and NOS activity were highly increased 24h after cerebral infarction, NO level in cerebral tissues around the infracted area could be decreased after EPO was applied by either method, and NOS activity could be depressed. NO level was lower in intranasal application group than in peritoneal injection group, but no significant difference was observed in NOS activities between the two drug groups.Conclusion: Intranasal EPO application is an effective drug application method, which can increase NGB expression in cerebral tissues, depress NOS activity, thus to decrease the toxic effects induced by NO.