Experimental Research Of The Effects Of Melatonin On The Secondary Brain Injury After Intracerebral Hemorrhage In Rats

Objective: This study was designed to investigate the therapeutic effect and mechanism of melatonin on secondary brain injury(SBI)in a rat model of intracerebral hemorrhage(ICH).Methods:An in vivo ICH model was induced via autologous whole blood injection into the right basal ganglia in Sprague-Dawley(SD)rats.The SD rats were then divided into the following 4 groups: sham group,ICH group,ICH + vehicle group and ICH + melatonin group(all n = 12).Melatonin(5mg/kg)and an equal volume of vehicle were injected into the rats intraperitoneally at 1,24 and 48 hours post-ICH.After 72 hours,each group we chose 6 rats and took the brain tissue 1.5-2mm area around the hematoma for western blot analysis(albumin,?-H2 AX,XRCC1,MMP-9,NOX-1,NOX-2,HO-1,NQO1,c-caspase 3,c-PARP,BAX and Bcl-2)and assay of oxidative stress indicators.The brain cortex of six rats was taken for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)method,immunofluorescence analysis,and nissl staining.The blood samples were collected from the heart for serum and cerebrospinal fluid was collected from the foramen magnum to measure the levels of TNF-? and IL-1?by enzyme linked immunosorbent assay(enzyme-linked immunosorbent assay,ELISA)and brain edema index was measured.To generate an in vitro ICH model,harvested primary rat cortical neurons were treated with oxygen hemoglobin(OxyHb).The primary rat cortical neurons were divided into the following 4 groups: control group,OxyHb group,OxyHb + vehicle group and OxyHb + melatonin group(all n = 6).Flow cytometry was used to detect neuronal apoptosis and JC-1 and TMRM were used to determine the mitochondrial permeability transition pore opening.Results:The results of the in vivo study showed that melatonin can alleviate severe brain edema and behavior disorders induced by ICH.Indicators of blood-brain barrier(BBB)integrity,DNA damage,inflammation,oxidative-stress,apoptosis,mitochondriadamage showed increased expression following ICH induction,while melatonin increases the expression levels of HO-1 and NQO1 and reduces the others' expression levels(p < 0.05).However,Bcl-2 expression and the ratio of Bcl-2/BAX was reduced following ICH induction,with expression increased following melatonin treatment.Microscopically,TUNEL and Nissl staining showed that melatonin can decrease the numbers of ICH-induced apoptotic cells.Inflammatory and DNA-damage indicators exhibited an identical pattern compared to those above(p< 0.05).Adttionally,the in vitro study demonstrated that melatonin treatment can reduce the ratio of apoptotic neurons induced by OxyHb and thus offers a protective effect to the mitochondrial membrane potential.Conclusion: Melatonin treatment exhibited the benefit of alleviating SBI,which includes apoptosis,inflammation,oxidative stress,DNA damage,brain edema,BBB integrity damage,induced by ICH and may play a mitochondria protective role by reducing mitochondrial membrane permeability transition pore(MPTP)opening.