The Study Of The Expression Of Multidrug Transporters In The PTZ-kindled Adenosine A1 Receptors Knock-out Mice And Correlation With COX-2

Part OneThe role of adenosine A1 receptors in seizures and histomorphology changes in mice after pentylenetetrazol-kindled epilepsyObjective:To observe the changes in seizures and histomorphology in adenosine A1 receptors knock-out mice after pentylenetetrazol(PTZ)-kindled epilepsy and investigate the role of adenosine A1 receptors in an experimental model of epilepsy.Methods:The animals were divided into wild type (WT) group (n=20) and adenosine A1 receptors knock-out (KO) group(n=20). The mortality rates, kindling rates, mean times to onsets, start times of seizures, durations of seizures, and the behavioral seizure scales of mice subjected to the pentylenetetrazol (PTZ)-induced kindling of seizures in two groups were measured and compared. HE staining and Nissl staining were adopted to observe the histomorphology of cortex and hippocampus of mice in two groups at 24 hours and 30 days post-kindling.Results:The rates of mortality and kindling in KO group were significantly higher than that in WT group (P<0.05). The mean times to onsets and start times of seizures in KO group were significantly shorter than that in WT group, while the durations of seizures in KO group were significantly longer than that in WT group (P<0.05). Mice in KO group showed a significant increase in behavioral seizure stage scores compared with those in WT group (P<0.05). Mice in KO group showed a more serious damage in brain tissues (reactivity of microglia, neuronal necrosis, astrocytosis, etc) than those in WT group.Conclusion:Adenosine A1 receptors knock-out mice have more susceptibility to seizures. Adenosine A1 receptors may play a neuroprotective role in seizures.Part TwoThe expression of PGP and MRP1 in adenosine A1 receptors knock-out mice after pentylenetetrazol-kindled epilepsyObjective:To observe the changes of expression of P-glycoprotein (PGP) and multidrug resistance-associated protein 1 (MRP1) in adenosine A1 receptors knock-out mice after pentylenetetrazol-kindled epilepsy and investigate the role of adenosine A1 receptors in an experimental model of epilepsy.Methods:The animals were divided into wild type (WT) group(n=40) and adenosine A1 receptors knock-out (KO) group(n=40). RT-PCR and immunohistofluorescence were adopted to observe the transcriptional level and protein expression of PGP and MRP1 respectively in cortex and hippocampus of mice subjected to the pentylenetetrazol (PTZ)-induced kindling of seizures at 24 hours,7 days and 30 days post-kindling or mormal ones in two groups.Results:Twenty-four hours post-kindling, there was no significant difference in the transcriptional level and protein expression of PGP and MRP1 between PTZ-kindled mice and normal ones in WT group (P>0.05), while the transcriptional level and protein expression of PGP and MRP1 in PTZ-kindled mice were significantly higher than that in normal ones in KO group (P<0.05). Seven days and 30 days post-kindling, the transcriptional level and protein expression of PGP and MRP1 in PTZ-kindled mice were significantly higher than that in normal ones in both WT and KO groups (P<0.05). The transcriptional level and protein expression of PGP and MRP1 in PTZ-kindled mice increased with the time went on and there were significant difference among 24 hours,7 days and 30 days post-kindling in both WT and KO groups (P<0.05). The transcriptional level and protein expression of PGP and MRP1 in PTZ-kindled mice in KO group were significantly higher than that i n WT group at the same ti me poi nts post-ki ndl i ng.Conclusion:The activation of adenosine A1 receptors decreased the transcriptional level and protein expression of PGP and M RP1 and local application of adenosine may be an effective treatment method for intractable epilepsy.Part ThreeThe effects of Celecoxib on the seizures, histomorphology and expression of PGP, MRP1 and COX-2 in adenosine A1 receptors knock-out mice after pentylenetetrazol-kindled epilepsyObjective:To observe the effects of Celecoxib on the seizures, histomorphology and expression of P-glycoprotein (PGP), multidrug resistance-associated protein 1 (MRP1) and cyclooxygenase-2 (COX-2) in adenosine A1 receptors knock-out mice after pentylenetetrazol(PTZ)-kindled epilepsy and investigate the role of COX-2 in an experimental model of epilepsy.Methods:Adenosine A1 receptors knock-out mice were randomly divided into treatment group (treated with Celecoxib, n=20) and non-treatment group (treated with normal saline solution, n=20). The mean times to onsets, start times of seizures, and durations of seizures of mice subjected to the pentylenetetrazol (PTZ)-induced kindling of seizures in two groups were measured and compared. HE staining was adopted to observe the histomorphology of cortex and hippocampus of mice in two groups at 24 hours and 30 days post-kindling. RT-PCR and immunohistofluorescence were adopted to observe the transcriptional level and protein expression of PGP, MRP1 and COX-2 respectively in cortex and hippocampus of mice subjected to the pentylenetetrazol (PTZ)-induced kindling of seizures i n two groups at 24 hours and 30 days post-kindling.Results:The mean times to onsets and start times of seizures in treatment group were significantly longer than that in non-treatment group, while the durations of seizures in treatment group were significantly shorter than that in non-treatment group (P<0.05). Mice in non-treatment group showed a more serious damage in brain tissues (cortex and hippocampus) than that in treatment group. Twenty-four hours post-kindling, there was no significant difference in the transcriptional level and protein expression of PGP, MRP1 and COX-2 between treatment group and control group (P>0.05), while the transcriptional level and protein expression of PGP, MRP1 and COX-2 in non-treatment group were significantly higher than that in control group (P＜0.05). Thirty days post-kindling, thetranscriptional level and protein expression of PGP, MRP1 and COX-2 in both treatment and non-treatment group were significantly higher than that in control groups(P<0.05). The transcriptional level and protein expression of PGP, MRP1 and COX-2 in both treatment and non-treatment group increased with the time went on. There were significant difference between 24 hours and 30 days post-kindling in both treatment and non-treatment groups (P<0.05). The transcriptional level and protein expression of PGP, MRP1 and COX-2 in treatment group were significantly lower than that in non-treatment group at the same time points post-kindling.Conclusion:COX-2 may have a close relationship with intractable epilepsy, in which adenosine A1 receptors may play an important role. Celecoxib, a selective inhibitor of COX-2, helps to reduce the generation of intractable epilepsy on the early stage of seizures. Intervention of both adenosine and COX-2 inflammatory reaction may be a promising treatment method for intractable epilepsy.