The Effect Of Thalidomide On The Expression Of P-glycoprotein And Cyclooxygenase-2on Hippocampus Microvascular In Temporal Lobe Epileptic Model Rats

OBJECTIVESThe aim of this study is to test the effect of thalidomide on the expression of P-glycoprotein (P-gp) and cyclooxygenase-2(COX-2) on hippocampus microvascular in temporal lobe epileptic model rats, in order to provide a basis for investigation of thalidomide chemosensitizing and synergistic effect on antiepileptic drugs later.METHODS1. Development of temporal lobe epilepsy (TLE) model in ratAs a TLE model, the spontaneous recurrent seizures (SRS) was induced by lithium chloride-pilocarpine in Sprague Dawley male rats. Briefly, after one week’s adaptive feeding, the rats were injected intraperitoneally with lithium chloride.20hours later, pilocarpine was given by intraperitoneal injection. If the onset of ongoing generalized convulsive seizures (status epilepticus, SE) was not induced within30min, additional administration of half-dose pilocarpine was given by intraperitoneal injection every30min until the onset of it. After lasting60min, SE was terminated by intraperitoneal injection of diazepam and repeated every3min if seizure activity continued. The healthy control rats received responding vehicle instead of drug every time. Then the activity of rats was monitored everyday. The TLE model succeed when a relative stable frequency of SRS was observed for3consecutive weeks. Only the TLE model rats were used for further study.2. The treatment of thalidomideAfter83days of SE, the SRS activity (frequency and stage) of rats was monitored from9:00a.m. to5:00p.m. for7consecutive days. Then the TLE model rats are separated randomly into two groups, the model control and the thalidomide treatment group. Then thalidomide (10mg/kg/day) was injected intraperitoneally into treatment group rats at9:00a.m. everyday for7days. The healthy control and model control rats received responding vehicle instead of drug. The SRS activity of treatment group rats was monitored everyday within these7days.3. Sample collection and analysisAfter one week of thalidomide treatment, all rats were decapitated and brains were immediately removed. Then the hippocampus was isolated, cleaned and stored in formalin solution in4℃until measurement. The expression of P-gp and COX-2on brain micro vascular in hilus and CA3region of hippocampus was measured by immunohistochemistry and stereology and image analysis (SIA).4. Data processingThe data was analysised by Excel and statistical program for social sciences (SPSS)13.0software. The P-gp surface density, COX-2number density and hippocampus weight of each group was tested by one-factor analysis of variance, and diference between every two groups was tested by leaet significant difference (LSD)-t test. The difference of body weight between healthy control and TLE model group was tested by independent-samples t test. The difference of SRS frequency and stage pre-and during treatment was tested by paired-samples t test. P<0.05is considered significant.RESULTS1. Development of TLE model in ratThe TLE model was successfully induced in25.86%(15/58) rats, and a mean dosage required of pilocarpine is (28.75±7.86) mg/kg. The mortality rate is19.44%(14/72), and a mean latency to first SRS is (47.9±20.6) days. SRS frequency per7days is (12.0±4.1) per rat. After SE, the body weight of TLE model rats increased slowly, then stopped increasing, and finally dropped gradually compared to healthy control rats, and there is a significant difference after60days of SE till the end of study (P<0.01).2. The effect of thalidomide on weight of rat body and hippocampusThe difference of body weight between model control and treatment group after thalidomide (10mg/kg/day) treatment for7days is not significant. The hippocampus weight of model control and treatment group rats tended to drop to some extent compared to that of healthy control group, and there is no significant difference between treatment and model control group.3. Antiepileptic effect of thalidomideIn thalidomide (10mg/kg/day) treatment group, the mean SRS frequency was decreased significantly from (11.9±4.5) per rat before treatment to (8.0±3.5) per rat during treatment (P<0.01). Also, the mean SRS behavioral stage was retarded from (4.9±0.1) to (4.5±0.1), and there is a significant difference between two periods (P<0.05).4. P-gp expressionThe expression of P-gp on hippocampus microvascular in model control group rats is up-regulated significantly compared to that of healthy control group (P<0.05), and thalidomide (10mg/kg/day) treatment for7days tended to reverse the up-regulation of P-gp in model control group rats closing to the level of healthy control group, though there is no significant difference (P=0.06).5. COX-2expressionThe expression of COX-2on hippocampus microvascular in model control group rats tended to up-regulate to some extent compared to that of healthy control group (P=0.17), but the expression of it was significantly decreased by thalidomide (10mg/kg/day) treatment for7days (P<0.05). CONCLUSIONS1. Thalidomide (10mg/kg/day) can reduce the frequency and stage of SRS in TLE model rats significantly during the treatment period.2. SRS tends to up-regulate the expression of COX-2on brain microvascular of hippocampus in TLE model rats to some extent, and thalidomide (10mg/kg/day) treatment for7days can decrease the expression of COX-2in TLE model rats significantly.3. SRS can up-regulate the expression of P-gp on brain microvascular of hippocampus in TLE model rats significantly, and thalidomide (10mg/kg/day) treatment for7days can partially reverse this up-regulation closing to the level of healthy control group, and it is associated with the inhibition of COX-2expression on hippocampus microvascular in TLE model rats.