| BackgroundMetalloproteinase (MMP) were found because of studying the cause of the body changes in frog development. In 1962, Gross define the unknown active material which can degrade tadpole tail as "collagenase", up to now, in this family there were more than 20 members which were validate.According to their function and target of substrate, they were divided into four kinds: collagenase, gelatinase, matrilysin and membrane-type metalloproteinase. Gelatinase was subdivided into metalloproteinase-2 and metalloproteinase-9 according to their structure and molecular weight.The synthesis and degradation of excetral cellular matrix (ECM) keep dynamic equilibrium. The dynamic equilibrium between MMPs and its tissue inhibitor of metalloproteinase (TIMP) is important in keeping integrity of ECM and regulating the relation between cell and enviroment. Four types of TIMP were found (TIMP-1, 2, 3, 4), they can bind with MMPs slectively to inhibit its activity. The selection of TIMP to MMPs is not specific. So people pay closed attention to man-made MMPs inhibitor. There are more than 10 kinds of man-made MMPs inhibitor reported at present,all of them were used in animal experiment only. GM6001 is one of artifical MMPs inhibitors which mainly inhibits the activity of MMP-2 and MMP-9.MMPs work in tissue development, wound healing, bone growth and vascularization physiologically . MMPs were excreted by neutrophis , glial cell, cerebellum star cell in developing process, neuron and blood vessel endothelium cell in central nervous system, MMPs were related with growth of neurite, encephalon blood vessel regeneration and cerebellum star cell . proliferation and migration. MMPs can also accelerate occurrece and progress of many diseases, such as the incursion and metastasis of tumor, atherosclerosis .multiple sclerosis, Alzheimer disease, malignancy glioma.and so on. Scientises gradually pay close attention to the role of MMP-2 and MMP-9 in braim injury. There is reported that MMP-2 and MMP-9 were significantly elevated after cerebral paralysis.Animal experiment also indicated that MMP-9 was activated early after local cerebral ischemia. The neuroprotective effects of MMPs inhibitor in traumatic brain injury was confirmed. The relation between MMP-2, MMP-9 and inflammatory brain injury and the role of MMPs inhibitor GM6001 in inflammatory brain injury, however, are not clear.On the other hand, bacterial meningitis is the most commom serious infection of the central nervous system. Though newer and more potent antibiotics'are continually developed and applied, and there are more and more advanced nurse, the mortality and permanent neurologic deficits still high occur following bacterial meningitis in children. Once bacteria have gained access to the central nervous system, their multiplication triggers a complex host response . Alteration of the cerebralvasculature, with disruption of the blood brain barrier and global and focal ischemia, ultimately lead to functional and structural brain damage, which leads to many sequelaes involving in epilepsia, mental retardation and acroparalysis in serious patients .We infer that there may be changes of MMP-2 and MMP-9 in bacterial meningitis, the changes of MMP-2 and MMP-9 may be correlated with cytokine, and MMPs inhibitor may have neuroprotective effect in bacterial meningitis. ObjectiveTo establish the immature rat model of pneumococcal meningitis and evaluate the pathogenesis of brain injury in the rat model of pneumococcal meningitis.To study the expression of matrix metalloproteinases(MMPs) in pneumococcal meningitis and the changes of mRNA for MMP-9 and TNF-a of brain tissue and the levels of MMP-9 and TNF- a of cerebrospinal fluid (CSF) in rat with pneumococcal meningitis at different time point and to evaluate the roles of GM6001. To investigate the neuroprotective effect of a new MMPs inhibitor, GM6001, on pneumococcal meningitis. Methods1. Wistar rats (21 days old;n=72) were infected intracisternally with 50 n 1 of streptococcus pneumoniae(serogroup 3; 3X104"8 colony forming unites/ml). After a single inoculation, rats were killed on the 1st, 2nd, 3rd, 10 th, 20 th and 30 th day separately. Illness was assessed clinically and the pathologically changes of brain tissue were analyzed.2. Immature rats(12-14 days old,n=36) were randomly divided into three groups .-controls, saline group and pneumococcal group. Rats infected intracisternally with 50u 1 of saline or streptococcus pneumoniae (3 X 104 colony formingunits). Twenty-four hours later,illness and pathological changes were observed. cerebrospinal fluid(CSF)was cultured.The expression of MMP-9 and MMP-2 in brain parenchyma were verified by immunohistochemical analysis and by reverse transcription-polymerase chain reaction(RT-PCR) analysis.The gelatinolytic activities of MMPs of CSF were performed by gelatin zymographic analysis.3. Sisty-four rats were randomly divided into the following groups:controls, meningitis group, GM6001-treated group(treatment with a hydroxamix acid-type MMP inhibitor:GM6001;65mg/kg intraperitoneally every 12h,beginning at the time of infection ),no-intervention group.The model of pneumococcal meningitis in rats was established by intracisternal injection of 50M-1 of saline containing 3104CFU Streptococcus pneumoniae(serogroup 3).At each time point, i. e. 2, 4, 8,16,24h after infection,the expression of MMP-9 and TNF- a in brain tissue was verified by reverse transcription- polymerase chain reaction analysis ;the gelatinolytic activities of MMP-9 of CSF were performed by gelatin zymographic analysis;the concentration of TNF-a in CSF were determined by radio-immunity.4. Wistar rats (21 days old;n=32) were infected intracisternally with 50 u 1 of streptococcus pneumoniae (serogroup 3;3X 104cfu/ml). Thirty-two rats were randomly divided into the following groups:short-term treated group with GM6001[i.p.65mg/(kg.12h)] for one day,control group,long-term treated group with GM6001[i.p. 65mg/(kg. 12h)] for three days, control group. Illness score and cerebral spinal fluid(CSF) titers were determined.The number of cortical and hippocampal neurons were then calculated with HE staining. The Dpecial memorywas examined by Morris water maze in rats. Results1. By 24h after infection, meningitis was fully developed in all infected animals, independent of the initial inoculum. Pneumococcal meningitis was characterized chinically by severe obtundation and seizures, and histopathologically by acute inflammation in the subarachnoid space and ventricles, a vasculopathy characterized by vascular engorgement,and focal neuronal necrosis in the cortex. Incidence of seizures,vasculopathy and neuronal injury correlated with the inoculum size(p<0. 01). Thirty days post-infection, brain weights of infected animals treated with antibiotics were decreased compared to uninfected controls(1. 38 ± 0.18g vs 1.65 ± 0. 12g;p<0.05).2. By 24h after infection, the same streptococcus pneumoniae were cultured in CSF of pneumococcal group and there was acute inflammation in subarachnoid spac,there were no such changes in saline group and controls. The Mrna expression of MMP-9 in pneumococcal group(l. 04 + 0. 34) was significantly increased than that in controls (0. '28 + 0. 12, P<0. 01). The protein expression of MMP-9 in pneumococcal groupQl. 5 ± 2. 4) was significantly increased than that in controls (3. 9 + 0. 3) as well (K0. 01). The relative gelatinolytic activities of MMP-9 in pneumococcal group(1554. 2 + 264. 5) was also significantly higher than that in controls (203. 4 + 57. 3, K0. 01). No difference in the expression and relative gelatinolytic of MMP-2 between pneumococcal group and controls (all P>0.05). The expression and relative gelatinolytic activities of MMP-9 and MMP-2 in saline group were similar to that of-controls(all P>0. 05).3. As compared to controls,the Mrna for MMP-9 and TNF-a of brain tissue in rat with pneumococcal meningitis was significantly upregulated 4h after infection(0. 72±O. 03, P<0. 05; 0.30 ± 0.03,P<0.05;),reaching maximum levels 8h after infection(1.02 ± 0. 03, P<0. 01; 0.78 ± 0. 07, P<0. 01;), persisting until 24h after infection. In parallel, there was a marked increase as early as 4h (877. 7 + 163. 5, P<0. 05; 656± 197, P<0. 05) and peak concentrations were reached at 16h (1557. 8+145. 4, P<0. 001; 3347 + 1513, P<0. 001 ) .persisting until 24h after infection.The concentrations of MMP-9 and TNF- a in CSF were significantly correlate(r=0. 83, P<0. 01).Treatment with GM6001 lowered the MMP-9 and TNF-a levels- in CSF ( 305. 8 + 129. 8, P<0. 001 ; 288 + 54ng/ml, P<0.001) .4. Treatment with GM6001, compared with control group, led to a reduction in the illness score (3. 3 + 1. 1 for GM6001 versus 3. 8 + 0. 5 for control group, P<0. 03) , whereas bacterial titers of CSF[ (1.7 + 0.5) X102cfu/ml for GM6001 versus (1.7 + 0.5) X 102cfu/ml for control group, p>0. 05] were not affected. The number of cortical and hippocampal neurons in GM6001 treated group( 23. 93 ±1.12/urn2, 53. 42 + 4. 30/ y m2) were all significantly higher compared to that in the control group (16. 23 + 0. 78/ u m\. 39.56 + 8. 25/p m2, P<0. 01, respectively). Brain water contents in GM6001 treated group (103. 45 + 12. 30%) was significantly lower than that in control group (179.15 + 22.15%, P<0.01 ) .Learning ability assessed by Morris water maze was significantly better in GM6001 treated group in comparison with control group (P<0.01) . Conclusions1. neumococcal meningitis in rats caused cortical neuronal injury resembling meningitis in humans, the focal disturbances of cerebral blood flow resulted from inflammatory involvement of cerebral vasculature maybe the pathogenesis ofbrain injury.2. The findings suggested that there was up regulation of MMP-9 in pneumococcal meningitis which may contributed to the pathogenesis of pneumococcal meningitis.3. The enhancement of MMP-9 and TNF-a may be involved in the process of inflammation of pneumococcal meningitis .The concentrations of MMP-9 and TNF- a in CSF showed a close correlation. GM6001 may reduced the concentrations of of MMP-9 and TNF-a in CSF.4. GM6001 can ameliorate cerebral edema and lessen neuronal death, thus GM6001 may be neuroprotective for rat with pneumococcal meningitis.SignificanceIn our model, meningitis is induced quickly and reliably by the intracisternal injection of the infecting organism. This study documents a role for MMPs in the pathogenesis of neuronal injury in a model of immature pneumococcal meningitis. In the cerebral cortex of animals with established meningitis , we found an increase in the levels of mRNAs encoding MMP-9 and TNF-a . In cerebrospinal fluid (CSF), concentrations of MMP-9 and TNF-a were upregulated and showed a close correlation. However, MMP-2 was not changed during bacterial meningitis. Inhibition of MMPs with GM6001 reduced the concentrations of MMP-9 and TNF-a in CSF and prevent brain edema and attenuated the extent of cortical brain damage. The present study provides strong evidence that MMP-9-mediated processes are critically involved in the pathogenesis of brain injury in bacterial meningitis and demonstrate a significant beneficial effect of an MMP inhibitor GM6001 on the neuropathologic outcome in bacterial meningitis. This study provides theory basis for the pathogenesiss therapyN...
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