| Part 1 The establishment of a model of 3-day-old neonatal rats with white matter damage.Objective: To establish a neonatal rat model of white matter damage(WMD)in premature infants,by giving intraperitoneal injection of lipopolysaccharide(LPS)and hypoxia-ischemia(HI)to 3-day-old neonatal rats.Method: A total of 96 3-day-old Sprague-Dawley(SD)neonatal rats were randomly divided into 4 groups,named as group NS(n=24),LPS(n=24),NS+HI(n=24),LPS+HI(n=24).Group NS were injected intraperitoneally with normal saline(0.05mg/kg)only,without any other treatment.Group LPS were injected intraperitoneally with lipopolysaccharide(0.05mg/kg)only,without hypoxic-ischemic treatment.Group NS+HI and LPS+HI were handled with hypoxia-ischemia treatment for 1 hour after intraperitoneal injection with normal saline or lipopolysaccharide(0.05mg/kg)3 hours respectively.Hypoxic-ischemic treatment is by ligating the right common carotid artery and being placed in a hypoxic environment(8%oxygen +92% nitrogen mixture)for 1 hour.Six rats of each group after treatment 0h,24 h,48h,72 h were infused with paraformaldehyde,taken out the periventricular white matter.Further experiments such as brain tissue HE staining and TUNEL detection of cell apoptosis were performed to identify the condition of neonatal white matter damage.Results:(1)Pathological changes of brain tissue.Through results of HE staining,no obvious cell damages were found in group NS、group NS+HI and group LPS.In LPS and HI group,the brain tissues display as periventricular loosened cortical structure,distorted neural arrangement,Nuclear condensation or fragmentation.(2)Cell apoptosis in brain tissue.From TUNEL staining results,no obvious apoptotic cells were found in group NS,group LPS and group NS+HI.In group LPS+HI,the cells’ apoptosis at 48 h was obvious,and reached the peak at 72 h.Conclusions:Individual treatment by intraperitoneal injection of low-dose lipopolysaccharide or short-term hypoxic ischemia failed to cause neonatal rat white matter damage,while combined both methods could successfully build a neonatal rat WMD model.Part 2 Impact of xenon on expression of microRNA-210 and HIF-1αin neonatal rats with white matter damageObjective: To reveal the molecular basis and neuroprotective mechanism of Xenon intervention in treating white matter damage by detecting the expression level of microRNA210(mi R-210)and hypoxia inducible factor 1α(HIF-1α)in brain tissues of 3-day-old neonatal rats after Xenon(Xe)intervention.Method: Atotal of 120 3-day-old Sprague-Dawley(SD)neonatal rats were randomly divided into 3 groups,named as group NS(n=24),LPS+HI(n=24),LPS+HI+Xe(n=72).Group NS and LPS+HI were prepared as described previously.Group LPS+HI+Xe was given Xenon gas(50% xenon +30% oxygen +20% nitrogen)inhalation for three hours after treatment of LPS injection and hypoxia-ischemia cultivation at 0h,2h,and 5h,and divided into three groups randomly,named as group LPS+HI+Xe1(n=24,at 0h);group LPS+HI+Xe2(n=24,at 2h);group LPS+HI+Xe3(n=24,at 5h).Six rats of each group after treatment 0h,24 h,48h,72 h were infuesd with paraformaldehyde,taken out the periventricular white matter tissue.Further experiments including brain tissue HE staining and TUNEL detection of cell apoptosis,RT-PCR of microRNA-210 and Western blot of HIF-1a were performed to find out the modulation mechanism of Xenon intervention in treating white matter damage.Results:(1)Pathological changes of brain tissue.Through results of HE staining,no obvious cell damages were found in group NS.In LPS and HI group,the brain tissues display as periventricular loosened cortical structure,distorted neural arrangement,Nuclear condensation or fragmentation,while less necrosis or structure distortion were found in Xenon treated groups.(2)Cell apoptosis in brain tissue.From TUNEL staining results,no obvious apoptotic cells were found in group NS.In group LPS+HI,the cells’ apoptosis at48 h was obvious,and reached the peak at 72 h.In comparison,the quantity of cell necrosis showed statistically significant decrease in Xenon treated groups(P<0.05).(3)Detection of mi R-210 level by RT-PCR.Compared with in NS group,the expression level of mi R-210 in neonatal rats’ periventricular tissue increased significantly at all time points in LPS+HI group(p<0.05).While the expression level of mi R-210 in brain tissues of group LPS+HI was significantly lower at 48 h and 72 h than that of group LPS+HI+Xe1(p<0.05),but made no difference with that of group LPS+HI+Xe2 or group LPS+HI+Xe3(p>0.05).Compared with group LPS+HI+Xe1,the expression of mi R-210 in brain tissues of group LPS+HI+Xe2decreased significantly at 24 h and 72h(p<0.05),and group LPS+HI+Xe3 decreased significantly at 0h,24 h,48h and 72h(p<0.05).And the expression of mi R-210 at each group increased firstly and then decreased,and reached the peak at 48 hours.(4)Detection of HIF-1α protein by Western blot.The expression level of HIF-1α protein increased firstly and then decreased in each group,reaching peak at 24 h,and was statistically differentiated with each other at every time point(p<0.05).The level of HIF-1α protein in group LPS+HI brain tissues was significantly higher than that of group NS at each time point,while significantly lower than that of group LPS+HI+Xe1 and LPS+HI+Xe2 and LPS+HI+Xe3 at 0h,24 h and72h(p<0.05).Compared with group LPS+HI+Xe1 and group LPS+HI+Xe2,the expression of HIF-1α in the brain tissue of group LPS+HI+Xe3 decreased significantly at 24h(p<0.05).Conclusions: We successfully build a neonatal rat white matter damage model by intraperitoneal injection of lipopolysaccharide combined with hypoxic ischemia.In WMD rats,the expression of HIF-1α and mi R-210 increased in periventricular tissues and Xenon could relieve the white matter damage by up-regulating the expression of HIF-1α and its target gene mi R-210.The Xenon therapeutic time window was within 5 hours after intervention,the sooner the better. |
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