The Effect Of New Generation Antiepiletic Drugs To Neonatal Rat Brain

PARTⅠTHE POSSIBILITY OF BRAIN DAMAGE INDUCED BY TRADITIONAL TREATMENT FOR NEONATAL SEIZURE IN RATSObjective: To investigate the influences of sigle clinical dose of traditional anticonvulsant drugs and PB in combind with others on brain development in neonatal rats.Methods: Dose of anticonvulsant drugs for neonatal rats were got by Meeh-Rubner mothod base on clinical dose of anticonvulsant drugs, Postnatal day 7 rats were divided into six treatment groups. Each group was administered with Phenobarbital (PB),Diazepam (DZP),Phenytoin (PHT),Phenobarbital in combination with Diazepam (PB+DZP), Phenobarbital in combination with Phenytoin (PB+PHT) and normal saline respectively by intraperitoneal injection (i.p). Investigate the body weight before and 24 hours after administration,and measure brain weight at 24 hours after administration, HE and Nissl staining for histological observation. The plasma neuron-specific enolase (NSE) was determined by ELISA at 24 hours after administration. Expression levels of apoptosis-related proteins Bax and Bcl-2 in neurocytes was detected by immunohistochemistry. Neurocyte apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and the caspase-3 activity was detected by spectrophotometric assay. The effects of anticonvulsant drugs on cognitive function were assessed by Morris water maze at postnatal day 23 rats. Using HE and Nissl staining to observe the pathological changes at postnatal day 30 rats, and counting neurons in cortex and hippocampus subiculum complex by Nissl staining.Results: (1) Postnatal day 7 rats in all treatment groups gain body weight less than control group, and the brain weight of all rats in PHT,PB+DZP,PB+PHT groups decreased significantly (P<0.01). Pathological changes such as karyopyknosis, cell shrinkage were found in hippocampus subiculum complex after HE staining, the more severe Pathological changes were found in combine groups than in single groups. Nissl bodies in some neurons in hippocampus subiculum complex dissolved and disappeared after Nissl staining, it is more severe in combine groups. (2) The concentration of plasma NSE exposed to PB,PHT,PB+DZP,PB+PHT significantly increased 52.48%,67.02%,152.48% and 175.53% respectively compared with controls [(0.282±0.1340)ng/L] (P<0.01), and the NSE exposed to PB+DZP and PB+PHT significantly increased 65.58% and 80.20% respectively compared with PB group (0.430±0.1345ng/L)(P<0.01). (3) The expression of Bax protein significantly elevated in all treatment groups compared with controls (P < 0.05). But the expression Bcl-2 protein just significantly decreased in the PB+PHT group compared to control (P<0.05). While the number of TUNEL positive cells in the subicular complex exposed to PB,DZP,PHT,PB+DZP and PB+PHT was significantly increased 4.74-fold,3.27-fold,5.44-fold,6.20-fold and 6.99-fold respectively compared with control group (P<0.01), moreover, PB+DZP and PB+PHT significantly increased 1.31-fold and 1.47-fold compared with PB group (P<0.01). The level of active Caspase-3 in frontal lobe and hippocampus increased significantly in all treatment groups compared with control (P < 0.05). (4) The escape latencies of rats treated with PB+DZP,PB+PHT were significantly longer than controls in Morris water maze (P<0.01). The rats treated with PHT(1.38±1.06),PB+DZP(1.38±2.00) and PB+PHT(1.12±1.126) had less platform crossing than control group (3.12±0.99) (P < 0.01). There was no significant difference between PB or DZP and control groups in Morris water maze test (P>0.05). (5) More red neurons degeneration in temporal cortex was found in combine groups after HE staining (23 days after administration, PD30). The number of hippocampal neurons (PD30) exposed to PHT,PB+DZP and PB+PHT significantly decreased 22.6%,23.8%,31.3% respectively compared with controls (82.8±14.44) after Nissl staining (P<0.01), there was no significant difference on the number of neurons in hippocampal between treatment groups and control groups in temporal cortex.Conclusion: (1) Even the neonatal rats treated one single therapeutic dose of PB,DZP or PHT will also lead to brain damage, and more severe damages will be caused by either PB+DZP or PB+PHT. (2) Treatment with PB+DZP or PB+PHT on PD7 rats only one time may cause persistent cognitive impairment and neurons decrease; Same Phenomenon were not found in sigle dose of anticonvulsant drugs. (3) Either single dose of PB, DZP, PHT or PB in combine with DZP or PHT may result in remarkable neuronal apoptosis and necrosis, especially in combined groups. PARTⅡTHE EFFECT OF NEW GENERATION ANTIEPILEPTIC DRUGS ON NEONATAL RAT BRAINObjective: To investigate the effect of new generation of antiepileptic drugs on brain development of newborn rats solely or in combination with PB administration under clinical doses.Methods: Dose of antiepileptic drugs for neonatal rats were got by Meeh-Rubner mothod base on clinical dose of antiepileptic drugs, Postnatal day 7 rats were divided into eight treatment groups. Each group was administered with small dose of oxcarbazepine (OXC30), emergency dose of Oxcarbazepine (OXC60), topiramate (TPM), levetiracetam (LEV), Phenobarbital in combination with small dose of oxcarbazepine (PB+OXC30), Phenobarbital in combination with topiramate (PB+TPM) or Phenobarbital in combination with levetiracetam (PB+LEV) and normal saline respectively by intraperitoneal injection (i.p) or intragastric administration (i.g). Investigate the body weight before and 24 hours after administration,and measure brain weight at 24 hours after administration, HE and Nissl staining for histological observation. The plasma neuron-specific enolase (NSE) was determined by ELISA at 24 hours after administration. Expression levels of apoptosis-related proteins Bax and Bcl-2 in neurocytes was detected by immunohistochemistry. Neurocyte apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and the caspase-3 activity was detected by spectrophotometric assay. The effects of antiepileptic drugs on cognitive function were assessed by Morris water maze at postnatal day 23 rats. Using HE and Nissl staining to observe the pathological changes at postnatal day 30 rats, and counting neurons in cortex and hippocampus subiculum complex by Nissl staining.Results: (1) Postnatal day 7 rats in OXC60 group and combined groups gain body weight less than control group, and the brain weight of all rats in OXC60 and PB+OXC30 groups decreased significantly (P<0.01). Pathological changes such as karyopyknosis, cell shrinkage were found in hippocampus subiculum complex of OXC60 group and combined groups after HE staining, the more severe Pathological changes were found in PB+OXC30 group than in PB group, and Nissl bodies in some neurons in hippocampus subiculum complex dissolved and disappeared after Nissl staining, it is more severe in PB+OXC30 group. But rats in OXC30, TPM, and LEV groups gain their body weight normally compared with control group (P>0.05) and the brain weight was also normal (P>0.05). Moreover, there was no obvious difference between PB+TPM or PB+LEV group and PB group on pathological changes. (2) The concentration of plasma NSE exposed to OXC60,PB+OXC30,PB+TPM and PB+LEV significantly increased 43.62%,108.87%,45.74% and 74.82% respectively compared with controls [(0.282±0.1340)ng/L] (P<0.01), and the NSE exposed to PB+OXC30 significantly increased 36.98% compared with PB group (0.430±0.1345 ng/L) (P<0.05). There was no significant difference between OXC30,TPM or LEV and control groups on the concentration of plasma NSE (P>0.05). (3) The expression of Bax protein significantly elevated in OXC60 and combined groups compared with control (P < 0.05). But the expression Bcl-2 protein just significantly decreased in the PB+OXC30 group compared to control (P<0.05). While the number of TUNEL positive cells in the subicular complex exposed to OXC60,PB+OXC30,PB+TPM and PB+LEV was significantly increased 4.02-fold,7.49-fold,3.73-fold and 4.62-fold respectively compared with control group (P<0.01); Moreover, PB+OXC30 group significantly increased 1.58-fold compared with PB group (P<0.01); There was no significant difference between OXC30,TPM or LEV and control groups as well. The level of active Caspase-3 in frontal lobe and hippocampus increased significantly in OXC60 and combined groups compared with control (P < 0.05); Additionally, the level of active Caspase-3 also increased significantly in PB+OXC30 group compared with PB group (P<0.01). (4) The escape latencies of rats treated with PB+OXC30 were significantly longer than controls in Morris water maze (P<0.01). The rats treated with OXC60 (1.38±1.19) and PB+OXC30 (1.12±0.99) had less platform crossing than control group (3.12±0.99) (P<0.01). There was no significant difference between OXC30,TPM,LEV,PB+TPM or PB+LEV and control groups in Morris water maze test (P>0.05). (5) More red neurons degeneration in temporal cortex was found in PB+OXC30 group after HE staining (23 days after administration, PD30). The number of hippocampal neurons (23 days after administration) exposed to PB+OXC30 (63.1±10.12) decreased significantly compared with controls (82.8±14.44) after Nissl staining (P<0.01), there was no significant difference on the number of neurons in hippocampal between treatment groups and control groups in temporal cortex (P>0.05).Conclusion: (1) High dose of OXC can cause PD7 rat acute brain damage and slightly cognitive decline. (2) Low dose of OXC will not affect immature brain, but, in combine with PB, cause obvious brain damage and persistent cognitive impairment. (3) Similar to PB,DZP and PHT, OXC can damage immature brain by excessive neuronal apoptosis and necrosis However, no pathological damage has been found after 3 weeks (PD30). (4) TPM and LEV neither cause brain damage nor aggravate PB induced brain damage.