Mechanism Study On Brain Injury Of Infant Rat Induced By Long-Term Antiepileptic Drugs Administration

PARTâ… EXPERIMENTAL STUDY ON HISTOPATHOLOGY CHANGES OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATIONObjective: To observe the histopathology changes and the recovery extents of brain in infant and adult rats after long-term administration of the five antiepileptic drugs (AEDs) - PB, CNP, CBZ, VPA and TPM- at therapy level.Methods: 144 healthy SD rats were divided into three groups including infant rat administration group, four weeks withdrawal after long-term administration infant rats group and adult rat administration group. Each group was further divided into six subgroups and given PB, CNP, CBZ, VPA, TPM or distilled water respectively (n=8), once a day for 28 consecutive days. At the end of observation period, body and brain weight were recorded. The morphology of frontal lobe and hippocampus was observed by HE and Nissl staining, meanwhile nerve cells number was counted. And ultrastructural changes of the nerve cells were observed by transmission electron microscopy.Results: (1) In infant rat administration group, only PB and CNP decreased body and brain weight. Brain weight was decreased by 18.3% and 19.4% after PB and CNP administration respectively. But four weeks after withdrawal, brain weight was returned to normal level. No significant body and brain weight changes were observed in all adult rat administration groups. (2) HE and Nissl staining revealed nerve cell swelling and vacuolization, Nissl body degradation, or nuclear fragmentation in frontal lobe and hippocampus of infant rats after PB and CNP administration, and nerve cells number was significantly decreased. Compared with control, nerve cells number was reduced by 15.06%, 16.59%, 12.03% and 11.52% respectively in frontal lobe, DG, CA1 and CA3 region of hippocampus after PB administration (P <0.0001), while that was 14.37%, 15.57%, 12.30% and 11.57% after CNP administration (P <0.0001). Four weeks after withdrawal, the morphology changes weren't detected anymore, but nerve cells number remained decrease. There were no significant changes of nerve cell morphological characteristics and number in infant rats after long-term CBZ, VPA and TPM exposure and all adult rats after the five AEDs exposure. (3) Electron microscope showed chromatin condensation, mitochondria swelling and endocytoplasmic reticulum dilatation in infant rats after PB and CNP administration, while four weeks after withdrawl, none pathologic changes detected anymore.Conclusions: (1) Long-term administration of PB and CNP to infant rats can decrease brain weight, induce nerve cells degeneration and loss, and ultrastructural changes, all of which are suggested the persistent cognition interference induced by PB and CNP due to potential histological impairment. (2) A long time after withdrawl, brain damage induced by PB and CNP recover to different extent, but the nerve cell number of frontal lobe and hippocampus remained lower than control, which suggested the brain injury caused by long-term administration of PB and CNP during postnatal early life are hardly recovery and may persisted for long time. (3) Long-term administration of PB and CNP to adult rats and long-term administration of CBZ, VPA and TPM to both infant and adult rats doesn't cause significant morphological changes of brain.PARTâ…¡EXPERIMENTAL STUDY ON THE POSSIBILITY OF EXCESSIVE CELL APOPTOSIS IN INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATIONObjective: To compare the different effect on cell apoptosis in the brain of infant and adult rats after long-term administration of the five antiepileptic drugs (AEDs)-PB, CNP, CBZ, VPA and TPM- at therapy level.Methods: 288 healthy SD rats were divided into three groups including infant rat administration group, four weeks withdrawal after long-term administration infant rats group and adult rat administration group. Each group was further divided into six subgroups and given PB, CNP, CBZ, VPA, TPM or distilled water respectively (n=16), once a day for 28 consecutive days. Sections from PARTâ… were used to detect nerve cells apoptosis by TUNEL and apoptosis-related proteins Bax and Bcl-2 expression by immunohistochemistry. At the end of observation period, half rats in each group were sacrificed to detect cell mitochondria membrane potential in frontal lobe and hippocampus by flow cytometry, and another half of rats were sacrificed to detect Caspase 3 activity by spectrophotometric assay.Results: (1) In infant rat administration group, only PB and CNP increased the TUNEL positive cells in frontal lobe and hippocampus. Four weeks after withdrawal, the increased brain TUNEL positive cells induced by the two drugs didn't detected anymore. No significant TUNEL positive cells changes were observed in all adult rat administration groups. (2) In infant rat administration group, only PB and CNP significant increased the expression of Bax protein in frontal lobe and hippocampus, while the expression of Bcl-2 protein didn't have difference. And then the ratio of Bax to Bcl-2 was increased respectively by 1.57～2.26 times and 1.74～2.29 times in the two groups. Four weeks after withdrawal, the expression of Bax and the ratio of Bax to Bcl-2 returned to normal level. No significant changes of Bax expression and Bax to Bcl-2 ratio were observed in all adult rat administration groups. (3) Long-term administration of PB and CNP also increased the activity of Caspase 3 (P<0.01) and mitochondria membrane potential (P<0.05), which were didn't observed at four weeks after withdrawal. No significant changes were found in all adult rat administration groups.Conclusions: (1) Long-term administration of PB and CNP may cause excessive cell apoptosis in immature brain, which aren't seen in adult brain. And the excessive cell apoptosis in immature brain may disappear with drug withdrawal. (2) PB and CNP up-regulate Bax protein expression resulted in enhanced ratio of Bax to Bcl-2 and mitochondria membrane potential, and then activate Caspase enzymes. At last lead to irreversible excessive apoptosis. (3) No matter infant or adult rats, long-term administration of CBZ, VPA and TPM don't induce excessive cell apoptosis in brain.PARTâ…¢EXPERIMENTAL STUDY ON CELL MITOCHONDRIUM DYSFUNCTION OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATIONObjective: To compare the different effect on cell mitochondria oxidative- antioxidative level, respiratory chain enzymes activity, ATP enzyme activity in brain of infant and adult rats after long-term administration of the five antiepileptic drugs (AEDs)-PB, CNP, CBZ, VPA and TPM- at therapy level, and to explore the key role of mitochondria dysfunction in apoptosis initiation.Methods: 144 healthy SD rats were divided into three groups including infant rat administration group, four weeks withdrawal after long-term administration infant rats group and adult rat administration group. Each group was further divided into six subgroups and given PB, CNP, CBZ, VPA, TPM or distilled water respectively (n=8), once a day for 28 consecutive days. At the end of observation period, rats were sacrificed by decapitation and brains were removed. Frontal lobe and hippocampus were separated, then extracted and purified mitochondria. Concentration of GSH, SOD, MDA, respiratory chain complexâ… ,â…£and Na⁺K⁺-ATP enzyme in mitochondria was all determined by spectrophotometer. All procedures completely complied with the kits instructions.Results: (1) Except for TPM, the other four AEDs decreased antioxidant level indicated by SOD and GSH, and increased peroxidant level indicated by MDA in both infant and adult rats. The injury in infant rats was much more serious than that in adult rats, special after PB and CNP administration. SOD and GSH were reduced by 42%～44% (P<0.01) and 20%～23% (P<0.01) respectively, and MDA was increased by 63%～73% (P<0.01) in infant rats after PB and CNP administration. In adult rat administration group, there were no significant differences among the change of SOD, GSH and MDA induced by the four AEDs. All of these changes weren't exist when drugs withdrawal. (2) Long-term PB and CNP administration significantly decreased the respiratory chain complexâ… andâ…£concentration in frontal lobe and hippocampus of infant brain (P<0.0001). In frontal lobe and hippocampus, the complexâ… level was reduced respectively by 28% and 37% after PB administration and by 33% and 35% after CNP administration compared with control, and the complexâ…£level was reduced respectively by 34% and 33% after PB administration, and 35% and 32% after CNP administration. But the decreased level of complexâ… andâ…£weren't found when drugs withdrawal. In both infant and adult rat administration group, there was no significant decrease of complexâ… andâ…£after long-term CBZ, VPA and TPM administration. (3) Long-term PB and CNP administration also significantly decreased the level of Na⁺K⁺-ATP enzyme in the frontal lobe and hippocampus of infant brain between 17%～19% (P<0.05). But the level of Na⁺K⁺-ATP enzyme was returned to normal level four weeks after withdrawl. No significant decrease was detected in adult rat administration group.Conclusions: (1) Different from CBZ, VPA and TPM, long-term PB and CNP administration can induce persistent and significant imbalance between oxidation and antioxidation in mitochondria, the lower level of antioxidation and the higher level of lipid peroxidation. They can also decrease the activity of mitochondrial respiratory chain complexâ… ,â…£and Na⁺K⁺-ATP enzyme which result in lower energy and mitochondrial membrane potential level, and the loss of membrane integrity of mitochondrial. (2) Both the oxidative stress and energy metabolism disorder occurred to mitochondria may play a key role in the process of cell apoptosis in immature brain after PB and CNP administration. (3) The mitochondria dysfunction can returned to normal level when drugs withdrawl, which indicate the reversibility of the damage induced by PB and CNP in immature brain. (4) Long-term PB and CNP administration to adult rats and long-term CBZ and VPA administration to both infant and adult rats can result in mild changes of SOD, GSH and MDA level, while they didn't change the activity of complexâ… ,â…£and Na⁺K⁺-ATP enzyme. So the compensation mechanism in body prevents the further mitochondria damage and the excessive apoptosis.PARTâ…¥THE EFFECT ON CALCIUM CONCENTRATION AND NEUROTROPHIC FACTORS OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION Objective: To clarify whether intracellular or mitochondria calcium overload is happened in brain of infant and adult rats after long-term administration of the five antiepileptic drugs (AEDs) - PB, CNP, CBZ, VPA and TPM- at therapy level and to explore the underlined mechanism. In addition, the expression of neurotrophic factors was tested.Methods: 288 healthy SD rats were divided into three groups including infant rat administration group, four weeks withdrawal after long-term administration infant rats group and adult rat administration group. Each group was further divided into six subgroups and respectively was given PB, CNP, CBZ, VPA, TPM or distilled water (n=16), once a day for 28 consecutive days. At the end of observation period, half rats were sacrificed to detect calcium concentration by spectrophotometer, and another half rats were sacrificed to detect the level of BDNF mRNA and NT-3mRNA by reverse transcriptase polymerase chain reaction. Samples from PARTâ…¢were used to assay calcium concentration in mitochondria by spectrophotometer. Sections from PARTâ… were used to assay the expression of NMDA-R1, BDNF and NT-3 by immunohistochemistry.Results: (1) In infant rat administration group, only PB and CNP increased both the intracellular and mitochondria Ca²⁺ concentration. Compared with control, intracellular Ca²⁺ concentration was increased by 48%～57% (P<0.0001), and mitochondria Ca²⁺ concentration was increased by 85%～97% (P<0.0001). Four weeks after withdrawal, the high level of Ca²⁺ concentration didn't detected anymore. No significant Ca²⁺ concentration changes were observed in all adult rat administration groups. (2) No matter at protein level or gene level, NMDA-R1 expression in infant and adult brain weren't changed after long-term PB and CNP administration. (3) The five AEDs decreased BDNF and NT-3 expression both at protein level and gene level, and the decrease in infant rats was more obvious than that in adult rats, special after PB and CNP administration. But four weeks after withdrawl, BDNF and NT-3 level was returned to normal.Conclusions: (1) Long-term PB and CNP administration can induce significant intracellular and mitochondria calcium overload coincided with the process of excessive apoptosis. (2) Calcium overload in immature brain is greatly related with the abnormal increased ROS in mitochondria and the high level of pro-apoptotic protein Bax, and no evidence of increased NMDA-R1 density is related with the increased calcium influx induced by PB and CNP. (3) Long-term PB, CNP, CBZ, VPA and TPM administration can decrease BDNF and NT-3 expression, no matter in infant and adult rat brain. While the decrease is more significant in infant brain than that in adult brain, special after PB and CNP administration, which promote and aggravate the process of apoptosis. All of these abnormal changes return to normal level when drugs withdrawal. PARTâ…¤EXPERIMENTAL STUDY ON THE ADVERSE EFFECT ON NEUROGENESIS INDUCED BY LONG-TERM ANTIEPILPTIC DRUGS ADMINISTRATIONObjective: To explore the effect of PB, CNP, CBZ, VPA and TPM at therapeutic level on cells perliferation, survival and neurogenesis in hippocampus of infant rat.Methods: 192 healthy P7 SD rats were divided into six subgroups and given PB, CNP, CBZ, VPA, TPM or distilled water respectively (n=32), once a day for 28 consecutive days. At the end of therapeutic period, BrdU was injected intraperitoneally three times to label newly generated cells. Half rats of each group were killed 24 h later to examine cell proliferation and neurogenesis by detecting expression of BrdU and DCX, co-expression of BrdU and DCX. Another half of rats were sacrificed 28 days later to examine cell survival and cell phenotype by detecting expression of BrdU and NeuN, co-expression of BrdU and NeuN or GFAP.Results: (1) 24 hours after last BrdU injection, the number of BrdU⁺ cell was significantly decreased by 63% and 59% in PB- and CNP-exposed rats (P<0.01), and the DCX expression was also significantly decreased. But the percentage of BrdU⁺/DCX⁺ cells in the two groups was higher than that in the vehicle group. There were no changes of cell proliferation and neurogensis in rats exposed to CBZ, VPA or TPM. (2) 28 days after last BrdU injection, the number of BrdU⁺ cell was sustained decrease compared with that at 24 hours. In the vehicle group, 58% of newly born cells had survived, whereas in the PB and CNP groups the survival was only 45% and 51% respectively (P < 0.01). There was no difference in the relative proportions of BrdU⁺/NeuN⁺ or BrdU⁺/GFAP⁺ cells in the two groups. The number of NeuN⁺ cell was significant decreased in all region of hippocampus (P < 0.01). There were no changes of cell survival and differentiation in rats exposed to CBZ, VPA or TPM.Conclusions: (1) Long-term PB and CNP administration during early life inhibits hippocampal neurogenesis in the developing brain, mainly in decreasing proliferation and survival of newly cells. (2) There are no effect on cell differentiation toward neuron and glial cells after PB and CNP administration during early life. (3) There are no evidence that of CBZ, VPA and TPM cause significant neurogenesis changes in immature brain of rats.