The Expression Of Tyrosine Phosphatase SHP2 In Experimental Pneumococcal Meningitis And Sodium Orthovanadate Aggravates Brain Damage In Bacterial Meningitis

Background:Bacterial meningitis,aslo named purulent meningitis,is an important infectious disease that threatens lives of children and adults. It is an infectious disease of central nervous system(CNS) that caused by purulent bacteria and has the main characteristics of meningitis in pathology.It often affects infants,nearly 75% of patients are less than 2 years old.The peak age is from 6 to 12 months.Its main clinical characteristics are fever,headache, vomit, convulsion, conscious disturbance,positive in meningeal irritation sign and purulent changes in cerebrospinal fluid (CSF). With the widely use of antibiotics and glucocorticoid, the attack rate of bacterial meningitis decreased remarkably. But the mortality and permanent neurologic deficits remain high. Up to 30% of the patients die and half of the survivors continue suffering from permanent neurological sequelae. To explore the pathophysiology of bacterial meningitis, the protective and detrimental factors in it and their mechanisms are helpful to increase the protective factor,decrease the detrimental factor and consequently increase the outcome of the disease. Animal models of bacterial meningitis give people more convenience to explore the pathophysiology of the disease.In the intracellular signal transduction, I-kB kinase NF-kB pathways and 3 mitogen-activated protein kinase (MAPK) pathways (i.e.Erk,Jnk and p38) have been considered to be activated by lipopolysaccharide (LPS) and peptidoglycan of the germ. As a result of activation of these signaling pathways, a variety of transcription factors are activated: NF-kB (p50/p65) and activator protein-1 (cFos/cJun), which coordinate the induction of many genes encoding a variety of inflammatory mediators. Protein tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases) are responsible for phosphorylation and dephosphorylation of tyrosine residues of different signaling proteins respectively. There has been growing evidence that protein tyrosyl phosphorylation control many important processes, including cellular proliferation and differentiation. SHP2, together with SHP1,are the two known mammalian PTPases that contain dual SH2 domains at their amino-terminal end and one phosphatase domain at the carboxyl- terminal end. The SH2 domain directs the specific protein-protein interaction. Despite high homology between SHP2 and SHP1, their functions might be distinct. In the cell signaling,SHP1 generally acts as a negative signal transducer and inhibits cell proliferation and differentiation. The ultimate effect of SHP2 in most signaling pathways is to enhance the signal transduction and promote cell proliferation and differentiation. SHP2 appears to be involved in a variety of signal transduction processes, such as the Ras-Raf-MAP kinase.There may be a balance and antagonism between the opposite activities of SHP1 and SHP2. The change in the balance will influence cell proliferation and differentiation and signal transduction. In experimental ischemic brain injury, both SHP1 and SHP2 were induced to express in reactive astrocytes. SHP2 was thought to control the switch of mature committed astrocytes to reactive astrocytes, while SHP1 appeared to limit injury-induced astrocyte proliferation.As both bacterial meningitis and SHP2 are implicated in the same signal transduction, i.e.MAPK, we deduce that SHP2 might be involved in the inflammatory reaction. In the gene map of cute stage of pneumococcal meningitis, SHP1 has been shown to be upregulated. Therefore,we deduce that SHP2 will be upregulated in bacterial meningitis.To verify this hypothesis, we established a juvenile rat pneumococcal meningitis model, observed the expression of SHP2 at both mRNA and protein levels and its association with tumor necrosis factor-alpha (TNF-alpha) and white blood cell(WBC) count in CSF. To explore the role of SHP2 and PTPases in bacterial meningitis, we tested the effects of sodium orthovanadate (SOV), a potent inhibitor of PTPases, on brain damage in experimental pneumococcal meningitis.Materials and Methods:â‘ Animal: A total of 101 3-week-old Wistar rats without consideration sex were used. Their weight range was 60-80g. All animal studies were conducted in accordance with institutional guidelines of Shandong University for the care and use of laboratory animals. Fifty-eight rats were randomly divided into meningitis group(n = 48) and control group(n = 10). The meningitis group were subdivided into four groups, i.e.day 1,3,7,14 group.Forty-three rats with pneumococcal meningitis were were randomly divided into SOV-treated group and saline-treated group.Among these,27 rats were used for histopathologic examination, 16 rats for the Morris water maze.â‘¡Establish experimental pneumococcal meningitis: Based on a previously described model of bacterial meningitis by Liu and Leib, experimental meningitis was induced by direct intracisternal injection of 50μl of saline containg 3-9×10⁵ CFU/ml Streptococcus pneumoniae (type 3) after removing 50μl CSF. Uninfected control animals were mock-infected with sterile saline. At 18h later, small amounts of CSF were obtained to be cultured on blood agar plates to confirm meningitis.â‘¢Tissue collection: Before the animal was sacrificed, CSF tap was performed for the determination of WBC level and concentration of TNF-alpha. After perfused via the left cardiac ventricle with 100ml of ice-cold phosphate-buffered saline (PBS) for assessment of mRNA and protein in brain homogenates or with 4% paraformaldehyde in PBS for immunohistochemical and histopathological evaluation, cortices and hippocampi were dissected from rat brains for assessment of mRNA and protein levels and stored in -80℃refrigerator. Rat brains for immunohistochemical and histopathological evaluation were stored in 4% paraformaldehyde for 1-3days, transferred to 30% sucrose for 1-3 days until the brain sank. Cryostat sections were cut in the coronal plane and stored at -20℃.â‘£Measurement methods: The SHP2 mRNA level was assessed by RT-PCR technique. mRNA was isolated by using the TRNzolRNA Purification Reagent and converted to cDNA by using the Universal RiboClone System.After PCR, the levels of SHP2 mRNA were measured by comparison to the mRNA levels of the beta-actin housekeeping gene. The SHP2 protein level was assessed by Western blot technique. Briefly, protein extracts were subjected to 15% SDS-polyacrylamide gelelectrophoresis, electrophoretically transferred to nitrocellulose membranes. After blocking, the membranes were incubated with the primary antibodies. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG and visualized using DAB kit. The levels of SHP2 protein were measured by comparison to the levels of the beta-actin housekeeping protein.Immunohistochemical localization of SHP2 in rat brain was detected by three-step immunohistochemical staining system (SP method). Briefly, after antigen repairation and endogenous H₂O₂ enzyme elimination, sections were preincubated with blocking serum(10% normal goat serum), then incubated with a primary antibody diluted in 1% PBS overnight at 4℃. Sections were then incubated with the secondary antibody. After further washes in PBS, sections were exposed to the avidin-biotin-HRP complex. In the end, the sections were counterstained with hematoxylin. The concentration of TNF-alpha in CSF of rats was measured by sensitive radioimmunoassays kit (no.R0701PR-B; Beijing Chemclin Biotech Co.,Ltd, Beijing, China). The assays were strictly complied to the instruction and were performed by professional staff.⑤Twenty-seven rats with bacterial meningitis were randomly divided into SOV treated group(n = 12) and saline-treated group(n = 15). Treatment with SOV(at 10 mg/ kg/day i.p.qd) was initiated from the time of infection and continued for 7 days. SOV was dissolved in 0.9% saline and injected at a pH of 7.4 and concentration of 27mM. The untreated controls were intraperitoneally (i.p.) injected with 2ml/kg sterile saline.We investigated the difference of clinical outcome, WBC count in CSF and blood, TNF-alpha concentration in CSF and histological brain damage scores between the two groups.â‘¥Learning function 3 weeks after infection was assessed by the Morris water maze. Forty-two-day-old survivors of meningitis (SOV-treated,n = 8; Nacl-treated, n = 8) performed for 4 days. The time to reach the invisible platform was documented for each day.⑦Normally distributed variables were presented as mean±standard deviation, and differences between two groups were analyzed by an unpaired t test. Multiple comparison used Student-Newman-Keuls(SNK).Variables that were not normally distributed were compared by the Kruskal-Wallis test or the Wilcoxon test. Proportions between different groups were compared by Fisher's exact test. The association between variables was assessed by using the Pearson correlation coefficient. A P value of <0.05 was considered significant.Results1 The same streptococcus pneumoniae were cultured in CSF of meningitis group and had high bacterial titers.Negative results were found in control group.2 Rats with bacterial meningitis had the clinical outcome from normal to obtusion and lethargy.Some animals manifested motor sequelae as ambulatory circling. The histopathological changes in the subarachnoid space ,ventricles and cerebral pia mater were leukocyte and fibrin exudation.The main cortical damage type were neutrophil accumulation and neuronal necrosis in the cortex. Cerebral cortical damage was focused on ventral part of the brain without apparent damage on dorsal part of the brain. Control animals had no cortical damage detected by histopathology.3. In cortices, bacterial meningitis led to a significant upregulation of mRNA encoding SHP2 (P< 0.01), reaching a maximal increase of more than fivefold at 3 days after infection(0.200±0.049 vs. 0.031±0.028, P< 0.05) and remained at high levels at 7 days(0.129±0.032 vs. 0.031±0.028, P< 0.05).4. Western blot analysis of SHP2 demonstrated that pneumococcal meningitis led to an increased expression of SHP2 protein in cortices(P< 0.01).Compared to control group,the levels of SHP2 protein began to increase at 3 days after infection(0.678±0.041 vs. 0.119±0.020,P< 0.05),reaching a maximal increase of nearly ninefold at 7 days(1.181±0.042 vs. 0.119±0.020, P< 0.01) and remained at high levels at 14 days(0.556±0.071 vs. 0.119±0.020,P< 0.01).5. Immunohistochemical staining demonstrated that immunoreactive cells were widely expressed in the cerebral cortex of the infected and control animals. The increase in SHP2 immunostaining was evident around the third ventricle in animals with bacterial meningitis in contrast to uninfected controls.Judged from the profile,the increased immunoreactive cells were mainly glial cells.6. The concentration of TNF-alpha and WBC count in CSF were significantly increased in meningitis group compared to control group(t = 3.29 and 3.08,respectively, P<0.005). SHP2 protein levels in the cortex and WBC count in CSF were significantly correlated (r =0.77,P<0.001).There was no significant correlation between levels of SHP2 protein and TNF-alpha concentrations in CSF.7. Treatment of animals with meningitis with SOV(n = 12) increased the concentration of TNF-alpha in CSF significantly compared to the concentration in saline-treated animals(n = 15) with meningitis(4.51±1.22ng/ml vs. 1.99±0.31ng/ml, P<0.001). The brain tissue injury scores were significantly higher in SOV treatment group than in saline-treated group(medium[range] 42[36-48] vs.18[12-24], P<0.02). For the water maze procedure, treatment with SOV, compared with treatment with saline,led to a significantly inferior performance (23.8±3.6 vs.19.5±4.2s,P<0.05).Conclusion1 Intracisternal injection of saline containg 3-9×10⁵ CFU/ml Streptococcus pneumoniae (type 3) can establish pneumococcal meningitis successfully.2 Protein-tyrosine phosphatase SHP2 was induced to express at both mRNA and protein level in pneumococcal meningitis.3 The levels of SHP2 protein in the cortex and WBC count in CSF were positively correlated. However, there was no significant correlation between the levels of SHP2 protein and TNF-alpha concentrations in CSF. These findings do not support a essential role of SHP2 in the pathogenesis of experimental pneumoniae meningitis, but it is possible that SHP2 protein expression may be used as a marker of disease activity. 4 SOV, which inhibited the activity of SHP2 and PTPases,significantly increased TNF-alpha concentration and brain damage scores in experimental pneumococcal meningitis, led to further decrease in learning function of survivors in the Morris water maze.These results suggest that PTPases in CNS,especially SHP2 may exert protective effect in bacterial meningitis.