A Pilot Study On The Dynamic Alteration Of MMP-2and MMP-9and Correlation With The Blood-brain Barrier Disruption During Acute Phase Of Seizures

Part Ⅰ Dynamic alteration of serum levels of MMP-2and MMP-9during acute phase of seizuresObjective:Matrix metalloproteinases (MMPs) are degrading enzymes which plays the important role in blood-brain-barrier (BBB) disruption in central neuvous system diseases. To get more insight into the role of MMPs in occurrence and progression of epilepsy, we investigate the serum levels of MMP-2and MMP-9of patients in acute phase of seizures.Methods:Observational study comprised of36patients in acute phase of seizures and40Healthy controls were performed to evaluate the serum levels of MMP-2and MMP-9by enzyme linked immunosorbent assay (ELISA) at4h,24h,60h and7d after the seizure attacks, as well as40normal controls. It was performed to evaluate the severity of the seizures (with National Hospital Seizure Severity Scale (NHS3)). duration and relapse.Results:Observational study included36patients in acute phase of seizures, of which20were primary,16secondary, and28generalized. The levels of MMP-2and MMP-9were high regulated during the acute phase of seizures, which altered dynamically.At4h after seizures, serum MMP-2level (82.53±17.16ng/ml), compared with healthy controls (68.85±11.64ng/ml) was significantly higher (P<0.01), while24h(78.52±18.68ng/ml)、60h(76.44±18.17ng/ml) group gradually decreased, but still higher than the control group (P<0.01, P<0.05). After7d of the onset, serum MMP-2concentration (64.34±13.34ng/ml) was no significant difference with the control. At4h after seizures, compared with normal controls (10.65±1.92ng/ml), serum MMP-9level (18.26±9.12ng/ml) significantly high (P<0.01), while24h group (16.54±10.54ng/ml) decreased, but still higher than the control (P<0.01). After60h (13.43±9.26ng/ml) and7d (13.18±5.59ng/ml) of the onset, serum MMP-9concentration was no significant difference with the control. At4h and24h after seizures, the serum level of MMP-9was more distinguished in the cases with longer-lasting time of seizures(>2minutes)(P<0.05, P<0.05). In addition, MMP-2and MMP-9were correlated with the NHS3scores (P<0.05).Conclusion:This study suggested that MMP-2and MMP-9were altered dynamically in acute phase of seizures. At4h and24h after seizures, the serum level of MMP-9was more distinguished in the cases with longer-lasting time of seizures. In addition, MMP-2and MMP-9were correlated with the NHS3scores. It revealed that MMP-2and MMP-9were associated with the seizure severity.Part II The pilot study on the serum levels of MMP-2and MMP-9and the correlation with expression of the tight junction associated protein, claudin-5and occluding, during acute phase of seizures of the lithium chloride-pilocarpine induced epilepsy ratsObjective:To observe behavior changes of the lithium chloride-pilocarpine induced temporal lobe epilepsy mod el, assess degree of blood-brain barrier damage, and detect of serum levels of MMP-2and MMP-9. Then we analysed of the tight junction associated protein express ion to explore the mechanism of lood-brain barrier damage in the acute exacerbation of epilepsy.Methods:Lithium chloride-pilocarpine-induced epilepsy model were made. Brain weighing method and Evans Blue injection were used for assessing the blood-brain barrier damage, while ELISA assaying serum levels of MMP-2, MMP-9. Serum MMP-2, MMP-9were contented and animal behavior, blood-brain barrier damage changes were observed after the intervention of Rho kinase inhibitor. Tight junction associated proteins in brain tissue, claudin-5and occludin, were detected,and analysed of the relationship between serum levels of MMP-2,MMP-9and the expression of claudin-5, occludin.Results:A total of45rats were injected intraperitoneally with lithium chloride-pilocarpine, including four deaths, two episodes of less than Racine IV level, with the modeling success rate of86.7%. Compared to control group, the epileptogenic group had higher brain water content and intracerebral EB penetration at24h after induced seizures (P<0.01, P<0.05). The levels of MMP-2and MMP-9were high regulated during the acute phase of seizures, which altered dynamically. Serum MMP-2reached a peak at24hours after the model and72hours after decreased, while MMP-9reached a peak in the attack instantly and decline at24hours. After the intervention of the Rho kinase inhibitor, the intervention group was showed significantly lower MMP-2, MMP-9levels (P<0.05, P<0.01), decreased spontaneous seizures (P<0.05), and shorter duration of attack (P<0.05). And blood-brain barrier damage is reduced (P<0.05, P<0.05). Brain tissue claudin-5of acute exacerbation with epileptic rats decreased; After the intervention of Rho kinase inhibitors, the brain tissue claudin-5of epileptogenic group increased (P<0.05).Conclusion:The blood-brain barrier damage was presented in acute exacerbation of lithium chloride-pilocarpine-induced temporal lobe epilepsy model; The levels of MMP-2and MMP-9were high regulated during the acute phase of seizures, which altered dynamically, which were related to the severity and the blood-brain barrier damage; After the intervention of Rho kinase inhibitors, MMP-2, MMP-9levels decreased, and blood-brain barrier damage is reduced, while the brain tissue claudin-5of epileptogenic group increased,which demonstrated MMPs may be involved in the mechanism of the destruction of the blood-brain barrier in seizures.Conclusion1. At4h after seizures, serum MMP-2level was significantly high, while it was no significant difference with the control after7d o f the onset. At4h after seizures, serum MMP-9level was significantly high, while it was no significant difference with the control after60h of the onset;2. At4h and24h after seizures, the serum level of MMP-9was more distinguished in the cases with longer-lasting time of seizures. In addition, MMP-2and MMP-9were correlated with the NHS3scores. It revealed that MMP-2and MMP-9were associated with the seizure severity;3. The blood-brain barrier damage was presented in acute exacerbation of lithium chloride-pilocarpine-induced temporal lobe epilepsy model;4. After the intervention of Rho kinase inhibitors, serum MMP-2, MMP-9levels decreased, while the severity of seizure and blood-brain barrier damage is reduced, and the brain tissue claudin-5of epileptogenic group increased. It demonstrated MMPs may be involved in the mechanism of the destruction of the blood-brain barrier in seizures.