Comparative Study On The Different Influence Of Antiepileptic Drugs At Therapeutic Level To Brain Maturation

PART I EFFECTS OF LONG TERM TREATMENT WITH ANTIEPILEPTIC DRUGS ON COGNITIVE FUNCTION OF IMMATURE BRAINObjective: To observe if cognitive function of immature brain would be impaired after long-term treatment with AEDs(antiepileptic drugs) at therapeutic level.Methods: The subjects selected for this study were 100 healthy infant (7 days) and adult Spraque-Dawley(SD) rats. Four kinds of AEDs commonly used in clinic to children were choosed, including phenobarbital (PB), clonazepam(CNP), valpric acid(VPA) and topiramate(TPM). The steady-state plasma concentrations of AEDs at the experimental dosage were coincided with the range of clinical therapeutic concentrations. Rats were divided into infant group and adult group by age. Each age group was administered with PB, CNP, VPA, TPM or CMC(carboxymethyl cellulose) respectively for persistent 5 weeks. 1, 14 and 30 days after AEDs withdrawed, effects of AEDs on cognitive function were assessed by Morris water maze and two-way shuttle box.Results: (1) The experimental doses at which the steady-state plasma concentrations of AEDs were coincided with the range of clinical therapeutic concentrations were as followed: PB 30mg/kg/d, VPA 250 mg/kg/d, CNP 6 mg/kg/d, TPM 40 mg/kg/d. (2) For immature rats, there were significant differences between AEDs-treated groups and control group in escape response latency (ERL) in the two-way shuttle box. ERLs were increased by 56.58%, 53.41%, 48.83% and 27.51% due to the use of CNP, PB, VPA and TPM respectively. 14 days after AEDs withdrawed, all groups but VPA-treated rats needed significant longer time to find the hidden platform than did controls(P<0.01). However, ERLs of immature rats receiving PB or CNP were still longer than that of untreated controls (P<0.01) after one month. ERLs were increased by 27.3% and 22.23% due to the use of CNP and PB respectively. As for adult groups, except PB group, there were significant differences between AEDs-treated groups and control group in ERL 1 day after drug withdrawed. ERLs were increased by 52.48%, 27.10% and 30.09% due to the use of CNP, VPA and TPM respectively. But these differences were all disappeared after two weeks. (3) For immature rats, except TPM group, escape latencies of AEDs-treated groups in the Morris water maze were all longer than that of controls 1 day after AEDs withdrawed (P<0.01). The same was also seen in all AEDs-treated adult rats. Howerer, there were no differences between AEDs-treated and controls in all of adult groups after two weeks. The same was seen in immature rats treated with VPA or TPM. Only immature rats treated with CNP or PB still performed worse than untreated controls even after one month(P<0.01). Escape latencies in PB and CNP immature rats during the Morris water maze test were increased by 2.27 times and 1.62 times compared with control .Conclusions: (1)The animal models were established successfully by long-term intragastric application of AEDs, and the steady-state plasma concentrations of AEDs were coincided with the range of clinical therapeutic concentrations. (2)The spatial learning and memory function of immature brain was impaired by the use of CNP or PB at therapeutic level for long term. This side effect induced by CNP or PB was severe and long lasting. While the impairment of spatial learning and memory function were transient in all of AEDs-treated adult rats, as well as VPA or TPM-treated immature rats. (3)Treatment with PB, CNP, VPA or TPM at therapeutic level for long term will impaire the conditional reflex reaction on learning and memory function of immature brain. The impairment resulted from TPM or VPA was transient and reversible, but persistent cognitive impairment would be seen in CNP or PB-treated immature rats. As for adult rats, transient impairment of conditional reflex reaction on learning and memory function can be seen in CNP, TPM or VPA treated groups except for PB-treated group.PART II EXPERIMENTAL STUDY ON PATHOLOGIC CHANGES OF IMMATURE BRAIN AFTER SHORT OR LONG TERM TREATMENT WITH ANTIEPILEPTIC DRUGS AT THERAPEUTIC LEVELObjective: To clarify the morphologic characteristics of brain after short or long term treatment with AEDs at therapeutic level in different age rats.Methods: 160 health Spraque-Dawley(SD) rats were divided into infant group and adult group by age. Each age group was administered PB, CNP, VPA, TPM or CMC respectively. Each group was further divided into long-term(5 weeks) treated group and short-term(2 weeks) treated group. Drug level in plasma corresponding to therapeutic range in clinic was determined by fluorescence polarization immunoassay. At the end of the therapeutic period, body and brain weight were got when the rats were sacrificed. Morphology in the frontal lobe and hippocampus were observed by HE staining and Nissl staining. And ultrastructural changes of the frontal lobe and hippocampus were observed by the tranmission electron microscopy. 30 days after AEDs withdrawed, electron-microscopic changes of the frontal lobe and hippocampus of immature rats exposed to CNP or PB were investigated again.Results: (1)Compared with untreated group, there were no differences between immature and adult rats in body weight, brain weight and the ratio of brain /body weight at the end of short therapeutic period. As for long-term treated rats, reduction of body weight was only observed in TPM-treated adult rats and CNP-treated immature rats compared with control rats. And there were no differences in brain weight among all adults groups. While remarkable reduction of brain weight was observed in immature rats exposed to CNP or PB compared with that of VPA group(P<0.05) and control group(P<0.01). Brain weights were reduced by 4.73% and 5.36% when immature rats exposed to CNP and PB for 5 weeks respectively. And the ratio of brain /body weight was also reduced by 6.67% in PB-treated immature rats (P<0.01). (2)No significant morphologic changes of brain were observed in all adult rats and immature rats exposed to VPA or TPM only. But significant neurodegeneration and neuronal necrosis can be observed by HE staining in immature rats exposed to CNP or PB. (3)Nissl staining detected the loss of neurons, degradation of nissle body and disorganization of frontal lobe in immature rats exposed to CNP or PB. The number of neurons was reduced by 29.6% and 33.5% because of administration of CNP and PB, respectively. (4)The neurons of immature rats receiving CNP for long term had numerous chromatin clumps, mild swelling of rough endoplasmic reticulum and mitochondria. However 30 days after CNP withdrawed, the neurons had a normal appearance basically. Ultrastructural changes of neurons in PB-treated immature rats were chromatin clump, prominent swelling of endoplasmic reticulum and mitochondria, as well unclear mitochondria cristae. Even after one month, swelling of neurons and fragmenting of caryoplasm still could be seen.Conclusions: (1)No significant morphologic changes of brain can be seen in different age groups exposed to PB,CNP,VPA or TPM for short term. As for long-term treated groups, there will be no significant changs in mature brain. But the reduction of brain weight, degeneration and necrosis of neurons, loss of neurons and ultrastructure changs of brain can be seen in the immature rats exposed to CNP or PB for long term. It suggest that the influence of AEDs on cognitive function may be based on the significant morphologic changes of brain. (2)Even 30 days after PB withdrawed, the abnormal ultrastructure changs of brain still can be seen in immature rats exposed to PB for long term. It suggest that brain damge of PB at therapeutic level to immature brain may be irreversible. (3)The differences of brain damage between CNP and PB are as follows: firstly, the brain damage to immature brain induced by CNP may be reversible because 30 days after drug withdrawed, the ultrastructure changs of brain were come to normal; Secondly, in contrast with PB, the damage to brain development induced by CNP could not only result from its direct side effect to immature brain but also reduce their body weight and metabolism process.PART III THE MECHANISM OF INFLUENCE OF ANTIEPILEPTIC DRUGS AT THERAPEUTIC LEVEL TO BRAIN MATURATIONObjective: To study the mechanism of brain damage induced by AEDs at therapeutic level to brain maturation.Methods: Animal grouping and drug level detection were as above. At the end of the therapeutic period, blood and brain were taken when rats were sacrificed. plasma NSE was quantitated by ELISA. Expression of apoptosis-related proteins Bcl-2 and Bax was detected by immunohistochemistry. Neuronal apoptosis was detected by TUNEL.Results: (1)As for immature rats, concentration of plasma NSE was increased by 41.10% after short term treatment with PB compared with control group. And it was increased by 34.18% and 35.49% after long term treatment with PB and CNP, respectively. But there were no differences of concentrations of NSE between AEDs-treated adult rats and control rats.(2) The expression of Bcl-2 and Bax protein in mature brain did not change either short or long term treatment with AEDs at therapeutic level. In contrast, expression of Bax protein in the frontal lobe was increased by 2.49 times and 2.57 times in immature rats receiving CNP and PB for long term, and also the ratio of Bax / Bcl-2 was increased by 2.28 times and 2.10 times respectively compared with control. (3)No significant TUNEL -positive cells were detected in short-term treated adult rats and immature rats receiving VPA or TPM for long term compared to the control rats. Howerer the number of TUNEL positive cells in immature rats exposed to CNP or PB for long term was obviously more than that in the control and the fluorescent intensity of TUNEL positive cells was significantly increased too.Conclusions: (1)AEDs at therapeutic level will not induce neuronal apoptosis and necrosis in mature brain no matter short or long therapeutic period. (2)Long term treatment with PB or CNP at therapeutic level can induce neuronal apoptosis and necrosis in immature brain significantly. (3)Even short term treatment with PB at therapeutic level may induce neuronal necrosis in immature brain. It suggest that immature brain may be more sensitive to be damaged on PB use...