| Bacterial meningitis is one of serious infectious diseases in central neural system inchildren.In recent years, although treatments with effective antibiotics, the disease stillhas high mortality and morbidity. So further study of pathological mechanism of bacterialmeningitis and looking for new means of intervention has great significance. Bacterialmeningitis can cause the disruption of blood-brain barrier, most severly it can result inbrain damage which effects prognosis.There are many studies that have confirmed thatnecrosis and apoptosis of brain neurons occur while brain damage is takingplace.Neuron-specific enolase(NSE) and S100b have been confirmed to be the specificmarkers of brain damage.Inducible nitric oxide synthase (iNOS) can biosynthesize NO,and it has been shown to play an important role in neurons’ apoptosis associated withbacterial meningitis.B7Homology3（B7-H3）is a new member of the B7family, whichplays an important role in the regulation of the immune response. In our previous study,wefound an abnormal high expression of soluble B7-H3protein in children with bacterialmeningitis. In a mouse model of Streptococcus pneumococcal(SP) meningitis,we alsofound B7-H3exacerbates the pathological injury of SP meningitis, enhanced inflammatorychemotaxis of this disease.Been based on those findings,In order to investigate the impactof B7-H3in brain damage caused in SP meningitis, to understand its role in theprogression of bacterial meningitis, in the present study,we further explored the effects ofB7-H3on the neurons necrosis and apoptosis and the mRNA expressions of NSE,S100b,and protein expression of NSE,S100b,iNOS in mice model of SP meningitis to providenew ideas and experimental evidences for underlying immune interventions in bacterialmeningitis.Methods：Part1. to detect the effects of B7-H3on the change of behavior score、the bodyweight.481.5-2months old healthy male BALB/C mice were randomly divided into4 groupss: normal saline control groups （CON groups）, B7-H3groups （B7-H3groups）,Streptococcus pneumonia groups （SP groups）, Streptococcus pneumoniae+B7-H3protein groups（SP+B7-H3groups）; each groups of12mice. Anesthesia were induced byintraperitoneal injection of pentobarbital in mice, and by lateral ventricular injection of10μ l of sterile normal saline+5μ l of Streptococcus pneumoniae suspension, bacterialmeningitis mouse model were established; CON groups mice were injected with15μ lsterile saline, and groups B7-H3with10μ lB7-H3(0.33μg.μ l-1)+5μ l sterile saline, SPgroups mice were injected with10μ l sterile saline+5μ lof Streptococcus pneumoniaesuspension, and SP+B7-H3groups mice with10μ lB7-H3protein (0.33μg.μl-1)+5μlStreptococcus pneumoniae suspension.Part2.18h、48h、72h after lateral ventricular injection, following steps wereprocessed:1） neurological score,2） change, recording of body mass(before and after theinjection of mice,we record the mice body weight at different time points and then analyzethe difference in weight).We detect the effects of B7-H3on the neurons necrosis andapoptosis in the brian of mice with SP meningitis and then evaluate brain injury.Mousemodel and groupss are the same to part1.In lateral ventricle injection of18h、48h、72h micein each groups:1)to assess neurons necrosis(Nissl staining),2)to assess neurons apoptosis(TUNEL staining)(Through the microscope,we observe the number of positive cells inmouse brain at different time points).Part3. to detect the effects of B7-H3on the relative expression of NSE、S100bmRNA and the expression of NSE、S100b、iNOS protein in the brian of mice with SPmeningitis. Mouse model and groups are the same to part1.In lateral ventricle injection of18h、48h、72h mice in each groups:1) Real-time PCR method for detection of relativeexpression of mRNA of NSE、S100b in brain tissue,2）to detect the expression of NSE、S100b、iNOS protein in brain tissue with Immunohistochemical method.Results:Part1. The effects of B7-H3on clinical symptoms of mice with SP meningitis.1. Neuroethology evaluation:the score of each groups were recorded18h、48h、72hafter lateral ventricle injection,score of B7-H3groupss compared with CON groupss hadno significant difference,18h（5.0±0）vs（5.0±0）、48h（5.0±0）vs（5.0±0）、72h（5.0±0）vs（5.0±0），（P>0.05）; SP groupss were relative to the CON groupss decreased significantly at18h、48h、72h after induction of the meningitis, the difference hasstatistically significant18h（4.5±0.57）vs（5.0±0）、48h（3.5±0.57）vs（5.0±0）、72h（2.75±0.5）vs（5.0±0），（P <0.05）;SP+B7-H3groupss were relative to the CONgroupss decreased more significantly at18h、48h、72h after induction of the meningitis, thedifference has statistically significant,18h（4.0±0）vs（5.0±0）、48h（3.0±0）vs（5.0±0）、72h（1.75±0.5）vs（5.0±0），（P <0.05）;morever, the score of SP+B7-H3groupsswas lower than that of SP groupss at18h、48h、72h after induction of the meningitis，18h（4.0±0）vs（4.5±0.57）、48h（3.0±0）vs(3.5±0.57)、72h（1.75±0.5）vs(2.75±0.5),(P<0.05).2. The changes of body weight: before and after the injection of mice,we record themice body weight at different time points.Both of B7-H3groups compared with CONgroups at18h、48h、72hafter induction of the meningitis.18h（-0.01±0.24）vs（-0.05±0.42）、48h（-0.02±0.12）vs（-0.28±0.34）、72h（-0.02±0.13）vs（-0.07±0.4），had no significant difference（P>0.05）; after inoculation of bacteria,SP groupss had andecrease compared to CON groups in body weight at18h、48h、72h18h（0.8±0）vs（-0.05±0.42）、48h（1.48±0.05）vs（-0.28±0.34）、72h（2.53±0.07）vs（-0.07±0.4），the difference was statistically significant（P<0.05）；Both of SP+B7-H3groupss had andecrease compared to CON groupss in body weight after bacteria and B7-H3proteininjection18h（1.5±0.17）vs（-0.05±0.42）、48h（2.0±0.02）vs（-0.28±0.34）、72h（3.23±0.12）vs（-0.07±0.4）the difference was statistically significant（P<0.05）;butalso the score of SP+B7-H3groupss was lower than that of SP groupss at18h、48h、72hafter induction of the meningitis，18h（1.5±0.17）vs（0.8±0）、48h（2.0±0.02）vs（1.48±0.05）、72h（3.23±0.12）vs（2.53±0.07），the difference was statisticallysignificant （P<0.05）.Part2. The effects of B7-H3on the neurons necrosis and apoptosis in the brian ofmice with SP meningitis.1、Nissl staining:there are postive cells (neurons) in four groupss.In mice cerebralcortex,there was no difference about the number of neurons in B7-H3groupss and CONgroupss18h（113.33±5.77）vs（115.0±5.0）、48h（113.0±1.0）vs（113.0±2.65）、 72h（108.67±3.21）vs（107.67±3.21）,(P>0.05); SP groupss compared with CONgroupss,the number of neurons decreased18h（105.0±5.0）vs（115.0±5.0）、48h（94.33±4.04）vs（113.0±2.65）、72h（89.33±0.58）vs（107.67±3.21）,（P<0.05）, at eachtime point,the number of neurons in SP+B7-H3groupss compared with CON groupssdecreased further18h（90.0±1.0）vs（115.0±5.0），48h（82.0±2.65）vs（113.0±2.65）、72h（81.33±2.31）vs（107.67±3.21）（,P <0.01）; we also found that at three time points,the number of neurons in SP+B7-H3groupss decreased compared with the SP groupss（90.0±1.0）vs（105.0±5.0）、（82.0±2.65）vs（94.33±4.04）、72h（81.33±2.31）vs（89.33±0.58）,with statistical significance （P <0.05）.2.Tunel staining:in the mice cerebral cortex,the apoptosis cells of B7-H3groupss andCON groupss had no significant difference18h（0.67±0.57）vs（1.33±0.57）、48h（0.68±0.57）vs（0.67±0.58）、72h（1.0±1.0）vs（0.33±0.58）,(P>0.05）; SP groupss comparedwith CON groupss,the apoptosis cells increased18h（11.33±1.53）vs（1.33±0.57）、48h（19.0±1.0）vs（0.67±0.58）、72h（26.32±1.54）vs（0.33±0.58）,（P<0.01）;at eachtime point,the apoptosis cells in SP+B7-H3groupss compared with CON groupssincreased obviously18h（17.67±2.08）vs（1.33±0.57），48h（26.33±1.53）vs（0.67±0.58）、72h（38.0±2.65）vs（0.33±0.58）,（P <0.01）; moreever at all time pointsSP+B7-H3groupss, the apoptosis cells also increased compared with the SPgroupss18h(17.67±2.08)vs（11.33±1.53），48h（26.33±1.53）vs（19.0±1.0）、72h（38.00±2.65）vs（26.32±1.54）,with statistical significance (P <0.05).Part3. The effects of B7-H3on the brain damage marker and iNOS in the brian ofmice with SP meningitis.1.Brain tissue homogenate Real-time PCR detection of NSE, S100b mRNA relativeexpression in bacteria meningitis: after injection of18h,48h,72h, the relative expression ofNSE mRNA in B7-H3groupss compared with CON groupss had no significant difference（-0.01±0.24）vs（-0.05±0.42）、（-0.02±0.12）vs（-0.28±0.34）、(-0.02±0.13）v（s-0.07±0.4）（,P>0.05）;at18h, NSE mRNA relative expression in SP groupss comparedwith CON groupss increased obviously（0.095±0.03）vs（0.028±0.01）, the differencewas statistically significant （P<0.05）; the relative expression of NSE mRNA inSP+B7-H3groupss compared with SP groupss increased obviously（0.124±0.049）vs （0.095±0.03）, the difference was statistically significant （P<0.05）;48h, NSE mRNArelative expression in SP groupss compared with the volume of CON groupss increasedobviously（0.177±0.021）vs（0.028±0.003）, the difference was statistically significant（P<0.05）; the relative expression of NSE mRNA in SP+B7-H3groups compared with SPgroups increased obviously（0.33±0.088）vs（0.177±0.021）, the difference wasstatistically significant （P<0.05）; after72h injection, SP groupss NSE mRNA relativeexpression increased than CON groups（0.369±0.035）vs（0.028±0.001）, the differencewas statistically significant （P<0.05）, the relative expression of SP+B7-H3groups NSEmRNA compared with that of SP groupss increased obviously（0.529±0.02）vs（0.369±0.035）, the difference also has statistically significant （P<0.05）.After bacteria injection, the relative expression of S100b mRNA in B7-H3groupsscompared with CON groupss had no significant difference18h(0.04±0.018)vs(0.03±0.006)、48h(0.029±0.003)vs(0.029±0.002)、72h(0.048±0.001)vs(0.046±0.002),(P>0.05);at18h, S100b mRNA relative expression in SP groupss compared withCON groupss increased obviously(0.061±.006)vs(0.03±0.006), the difference wasstatistically significant (P<0.05); the relative expression of S100b mRNA in SP+B7-H3groupss compared with SP groupss increased obviously（0.148±0.052）vs（0.061±.006）,the difference was statistically significant (P<0.05);48h, S100b mRNA relative expressionin SP groupss compared with the volume of CON groupss increased obviously（0.185±0.012）vs（0.029±0.002）, the difference was statistically significant （P<0.05）; therelative expression of S100b mRNA in SP+B7-H3groups compared with SP groupsincreased obviously（0.194±0.018）vs（0.185±0.012）, the difference was statisticallysignificant （P<0.05）; after72h injection, SP groupss S100b mRNA relative expressionincreased than CON groups（0.284±0.008）vs（0.046±0.002）, the difference wasstatistically significant （P<0.05）, the relative expression of SP+B7-H3groups S100bmRNA compared with that of SP groupss increased（1.62±0.118）vs（0.284±0.008）, thedifference also has statistically significant （P<0.05）.2.Brain tissue immunohistochemistry analysis2.1The expression of NSE in brain:NSE positive cells are visible in cerebral cortexof four groupss.At18h,48h,72h, there was no difference about expression of NSE in B7-H3groupss and CON groupss18h(100.0±4.58)vs(99.67±0.58)、48h（100.33±0.58）vs（99.66±2.08）、72h（99.67±2.31）vs（100.0±2.0）,（P>0.05）; SP groupss comparedwith CON groups,the NSE expression decreased18h（96.67±1.53）vs（99.67±0.58）、48h（82.0±2.65）vs（99.66±2.08）、72h（75.33±0.58）vs（100.0±2.0）,（P<0.05）,at each time point SP+B7-H3groupss compared with CON groups decreased furthe（r86.67±2.89）vs（99.67±0.58）、（66.0±5.29）vs（99.66±2.08）、（55.0±5.0）vs（100.0±2.0）（P <0.01）; we also found that at all time points SP+B7-H3intervention groups, theexpression of NSE decreased compared with the SP groupss,（86.67±2.89）vs（96.67±1.53）、（66.0±5.29）vs（82.0±2.65）、（55.0±5.0）vs（75.33±0.58）,with statisticalsignificance （P <0.05）.2.2The expression of S100b in brain: there are S100b postive cells expression in allfour groupss.At18h,48h,72h,although S100b positive cells are found in B7-H3groupssand CON groupss,there was no difference18h(23.45±1.98） vs （23.54±2.3）、48h（23.67±2.32）vs（23.53±2.12）、72h（23.78±2.34）vs（23.76±1.32）,(P>0.05); SP groupscompared with CON groups,the S100b expression all increased18h （30.1±3.21） vs（23.54±2.3）、48h （38.23±3.23） vs （23.53±2.12）、72h （43.12±1.33） vs（23.76±1.32）,(P<0.05）, at each time point, the S100b expression of SP+B7-H3comparedwith CON groups increased significantly18h （40.6±1.45） vs （23.54±2.3）、48h（55.34±1.53）vs（23.53±2.12）、72h（68.12±2.89）vs（23.76±1.32）,（P <0.01）; wealso found that at three time points SP+B7-H3intervention groups, the level of S100bexpression was higher than SP groupss18h(40.6±1.45)vs(30.1±3.21)、48h(55.34±1.53)vs(38.23±3.23)、72h(68.12±2.89)vs(43.12±1.33)with statistical significance (P <0.05).2.3The expression of iNOS in brain:in B7-H3groupss and CON groupss, iNOSpositive cells are rarely visible at18h,48h,72h after injection. SP groups compared withCON groups,the iNOS expression all increased18h（29.33±1.15）vs（0.0±0.0）、48h（45.0±5.0）vs（1.0±0.0）、72h（71.67±2.89）vs（0.0±0.0）,(P<0.05）, at each time point,the iNOS expression of SP+B7-H3compared with CON groups increased obviously18h（35.33±0.58）vs（0.0±0.0）、48h（56.33±1.53）vs（1.0±0.0）、72h（81.67±2.89）vs （0.0±0.0）,（P <0.01）; morever,at all time points,the level of S100b expression inSP+B7-H3groupss was higher than SP groupss18h（35.33±0.58）vs（29.33±1.15）、48h（56.33±1.53）v（s45.0±5.0）、72h（81.67±2.89）v（s71.67±2.89）,with statistical significance(P <0.05）.Conclusions:1.By intraventricular injection of Streptococcus pneumoniae suspension, wesuccessflly established a mouse model of pneumococcal meningitis.2. Our results confirm that in mouse model of SP meningitis,the neuroethology scoredecreased,the mice body weight lost.In the cerebral cortex,the Nissl positive cellsdecreased,tunnel stained positive cells increased.The expression of NSE,S100b mRNA andS100b,iNOS protein were increased, while the expression of NSE protein decreased.Theseresults suggest that brain damages exists in SP meningitis.3. Collaborative signal molecule B7-H3can aggravate the clinical symptoms of SPmeningitis,boost the necrosis and apoptosis of brain neurons and increase the mRNAexpressions of NSE、S100b and the protein expressions of iNOS and S100b,while theNSE positive cells decreased furtherly.These results indicate that B7-H3aggravates thebrain injury of mice with SP meningitis. |
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