The Effects Of Sodium Valproate On Spatial Cognition Of Rats With Convulsive Status Epilepticus And Its Possible Mechanisms At Effective Steady State Plasma Concentration

PART ONE THE EFFECTS OF SODIUM VALPROATE ON SPATIAL LEARNING AND MEMORY OF RATS WITH CONVULSIVE STATUS EPILEPTICUS AT EFFECTIVE STEADY STATE PLASMA CONCENTRATIONSTUDY ONE ESTABLISHMENT OF DOSAGE REGIMEN TO MAINTAIN SODIUM VALPROATE AND PHENOBARBITAL TO BE AT EFFECTIVE STEADY STATE PLASMA CONCENTRATIONObjective: To establish the dosage regimen to maintain sodium valproate (VPA) and phenobarbital (PB) to be at effective steady state plasma concentration in Wistar rats of different ages in different gender.Method: The subjects selected for this study were postnatal 15days age (P15) and postage 35days age (P35) Wistar rats. All rats were divided into many groups with differrent dosage regimen and VPA and PB drugs level in plasma were determined by fluorescence polarization immunoassay after 2days and 5days therapeutic period.Results: (1) The dosage regimen to maintain VPA and PB to be at effective steady state plasma concentration in P15 male rats were 75 mg/kg (12/day), 10 mg/kg (2/day) and the dosage regimen in P35 male rats were 350 mg/kg (6/day),35 mg/kg (2/day); (2) With 200 mg/kg (6/day) dosage regimen, the VPA plasma concentration in P15 and P35 male rats were 150.00±0.00μg/ml,34.83±8.47μg/ml, however with 30 mg/kg (2/day) dosage regimen, the PB plasma concentration in P15 and P35 male rats were 42.15±6.34μg/ml, 13.00±3.47μg/ml; (3) With dosage regimen of 200 mg/kg, 300 mg/kg and 400 mg/kg (6/day), the VPA plasma concentration were higher in male rats than in female rats, but with dosage regimen of 45 mg/kg, 60 mg/kg (2/day) the PB plasma concentration were higher in female rats than in male rats; (4) The effective dose and lethal dose of VPA were closely 2400 mg/kg/day, 2700 mg/kg/day.Conclusions: (1) The dosage regimen to maintain VPA to be at effective steady state plasma concentration in P15 and P35 male rats were75 mg/kg (12/day), 350 mg/kg (6/day), and the dosage regimen of PB were 10 mg/kg (2/day), 35 mg/kg (2/day); (2) The rate of VPA and PB metabolism were age-dependent, which were faster in P35 rats than P15 rats; (3) The rate of VPA and PB metabolism were gender-dependent. The rate of VPA metabolism were faster in female rats than male rats while the rate of PB metabolism were faster in male rats than female rats and female rats were not suitable for studies wich require VPA plasma concentration to be at effective steady state.STUDY TWO THE EFFECTS OF VPA ON SPATIAL LEARNING AND MEMORY OF RATS WITH CONVULSIVE STATUS EPILEPTICUS AT EFFECTIVE STEADY STATE PLASMA CONCENTRATIONObjective: To study the effects of sodium valproate on spatial learning and memory of rats with convulsive status epilepticus (CSE) at effective steady state plasma concentration compared with PB treatment.Method: CSE was induced in P15 andP35 Wistar rats injected with lithium and pilocarpine intraperitoneally and controlled 60 minutes later. The effects of VPA and PB on spatial reference memory(SRM) and spatial working memory(SWM) were assessed by Morris water maze.Results: (1) The spatial cognition after CSE in P35 rats was more serious than in P15 rats, and with VPA treatment SRM and SWM impairment could be eased; (2) The VPA treatment could induce SRM and SWM impairment in P15 and P35 rats; (3) Within effective steady state plasma concentration the spatial cognition impairment in VPA-treated P15 and P35 rats were similar with PB-treated rats.Conclusions: VPA treatment could improve the spatial cognition after CSE in P35 rats, but with normal rats VPA treatment could induce the spatial cognition impairment in P15 and P35 rats. Within effective steady state plasma concentration there were no differences in spatial cognition impairment in CSE and non-CSE rats compared with PB-treated rats.PART TWO THE EFFECTS OF VPA ON LONG-TERM POTENTIATION IN CA1 AREA OF HIPPOCAMPAL SLICES PREPARED FROM RATS WITH CSE WITH EFFECTIVE CONCENTRATIONSTUDY ONE THE EFFECTS OF VPA ON LONG-TERM POTENTIATION IN CA1 AREA OF HIPPOCAMPAL SLICES WITH INTERVENTION IN THE BODY. Objective: To explore the effects of VPA on long-term potentiation (LTP) in CA1 area of hippocampal slices prepared from rats with CSE at effective steady state concentration and to study the possible underline mechanisms.Method: Hippocampal slices were prepared from male Wistar rats on postnatal 35 days. The effects of VPA on field excitatory postsynaptic potentials (fEPSPs) after high-frequency stimulation (HFS) were examined with patch clamp technique. The expression of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and phosphorylated CaMKⅡ(p-CaMKⅡ) were examined by Western Blot analysis.Results: (1) CSE in P35 rats significantly reduced the successful induction rate and maintenance but no influence in P15 rats, and VPA treatment could improve the successful induction rate and maintenance in P35 rats; (2) The expression of p-CaMKⅡand CaMKⅡhad no changes in P15 and P35 rats after CSE and VPA teatment could reduce p-CaMKⅡand CaMKⅡexpression in P15 rats. However in P35 rats VPA teatment only reduced p-CaMKⅡexpression but CaMKⅡexpression.Conclusions: (1) The influence of CSE on LTP was age-dependent which was gental in P15 rats than in P35 rats, and there were no relationship between the negative influence and p-CaMKⅡand CaMKⅡexpression; (2) The spatial cognition improvement of CSE rats after VPA treatment maybe due to the change of LTP induction and maintenance; (3) The contradiction between inhibition of p-CaMKⅡand CaMKⅡexpression and no-changes in LTP maybe due to the washing-out effect.STUDY TWO THE EFFECTS OF VPA ON LONG-TERM POTENTIATION IN CA1 AREA OF HIPPOCAMPAL SLICES WITH ISOLATED INTERVENTIONObjective: To explore the effects of VPA on LTP in CA1 area of hippocampal slices and the possible underline mechanisms.Method: Hippocampal slices were prepared from male Wistar rats on postnatal 35 days. The effects of VPA on field excitatory postsynaptic potentials (fEPSPs) after high-frequency stimulation (HFS) or double-pulse stimulation were examined with patch clamp technique. The expression of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and phosphorylated CaMKⅡ(p-CaMKⅡ) were examined by Western Blot analysis.Results: (1) VPA inhibited fEPSPs without modifying paired-pulse facilitation (PPF) which solely occured presynaptically as a simple form of synaptic plasticity; (2) pretreatment with VPA before HFS decreased the fEPSPs slope; (3) no significant effects of VPA on fEPSPs slope were found in two maintenance phase of LTP; (4) expression of p-CaMKⅡbut CaMKⅡdecreased in VPA pre-treatment group, and however no differences were detected of p-CaMKⅡand CaMKⅡexpression in VPA early-treatment group and VPA late- treatment group.Conclusions: VPA inhibited the induction of LTP postsynaptically through inhibiting the expression of p-CaMKⅡwithout modifying presynaptic neurotransmitter release and VPA had no significant influence on the two maintenance phase of LTP with no effects on the expression of CaMKⅡand p-CaMKⅡ.STUDY THREE THE EFFECTS OF VPA ON CA2+ CONCENTRATION IN HIPPOCAMPAL NEURONS WITH NMDA APPLICATIONObjective: To explore the effects of VPA on Ca2+ concentration in hippocampal neurons with NMDA application.Method: Neonatal rat hippocampal neurons were isolated and cultured in the serum-free B27 supplemented culture media. Primary hippocampal neurons cultured for 7 days were randomly divided into 3 groups: control group, VPA-treated group and negetive control group. All groups were interrupted with NMDA and the Ca2+ concentration in cultured hippocampal neurons were detected with furo-2.Results: VPA at effective concentration signicantly reduced ratio of fluorescence at 340nm, 380nm.Conclusions: VPA could reduce Ca2+ concentration in hippocampal neurons after NMDA application.PART THREE THE INFLUENCE OF VPA ON NEUROGENESIS AND MIGRATION OF NEWBORN NEURONS AFTER CSEObjective: To explore the influence of VPA at effective steady state plasma concentration on neurogenesis and migration of newborn neurons after CSE.Method: Rats were induced by chemoconvulsants lithium-pilocarpine and all rats received injections of bromodeoxyuridine (BrdU). Animals were perfused and brains were processed for immunocytochemistry antibodies against BrdU and Doublecortin (DCX). Results: The amount of BrdU positive cells and DCX positive neurons increased in dentate gyrus granule cell layer after CSE and some appeared in hilus of dentate gyrus. There were BrdU positive cells and DCX positive neurons in CA1 area. VPA treatment could decrease the amount of BrdU positive cells and DCX positive neurons in dentate gyrus and CA1 area. Inadition there were more and longer axons in the DCX positive neurons and VPA treatment could inhibit the abnormal phenomenon.Conclusions: VPA treatment could inhibit the neurogenesis and abnormal migration of newborn neurons.