长程施用抗癫痫药致婴鼠脑损伤的机理研究
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摘要
第一部分长程施用抗癫痫药致婴鼠脑损伤的组织病理学观察
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内组织病理学损伤的不同特征及恢复的可能性。
方法:健康SD大鼠144只,包括婴鼠用药组、长程用药再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组8只鼠。一日一次灌胃给药,连续给药28天,在各用药观察时间点结束后24小时记录体重、断头处死称取脑重后,取额叶及海马行HE和Nissl染色观察组织学损伤特征并进行神经细胞计数,用透射电镜观察脑组织超微结构。
结果:(1)婴鼠用药组中仅PB和CNP引起体重和脑重明显降低,其中脑重降低幅度分别达对照组的18.3%和19.4%,但停药4周后其体重和脑重与对照已无明显差异。这5种AEDs均未引起成年鼠体重脑重的明显改变。(2)HE和Nissl染色光镜检查发现使用PB和CNP后,婴鼠额叶皮层组织结构模糊,且在皮层及海马均见细胞明显肿胀、胞浆空泡形成,或Nissl小体消失,部分显示胞核变性等,额叶皮层和海马各区神经细胞计数均较正常对照组明显减少。与同龄对照比较,PB致额叶皮层神经细胞的减少幅度达15.06%(P<0.0001),海马DG、CA1和CA3区的减少幅度分别为16.59%、12.03%和11.52%(P<0.0001)。CNP引起的额叶皮层和海马各区神经细胞的减少幅度与PB较为相似,分别达14.37%、15.57%、12.30%和11.57%(P均<0.0001)。停药4周后,PB和CNP组的神经细胞分布基本均匀,细胞边界和核仁清晰,病变已不再明显,但额叶皮层和海马各区的神经元计数仍明显低于对照组。而婴鼠用药组中的CBZ、VPA和TPM组及所有的成年用药组神经细胞的镜下特征和计数与对照组相比无明显差异。(3)透射电镜发现婴鼠长程使用PB和CNP后染色质边集浓缩,线粒体肿胀和内质网扩张,但停药4周后这些超微结构的改变皆不再明显。
结论:(1)长程给予PB和CNP可致婴鼠脑重明显减轻,神经细胞变性及数量明显减少,电镜下超微结构显著异常。提示两药对幼鼠认知功能的持续影响是基于它们对未成熟脑的明显组织学损伤。(2)婴鼠长程给予PB和CNP并长时间停药后,大鼠的脑重、神经细胞损伤及其数量虽都有不同程度的恢复,但额叶皮层和海马各区神经元计数依然远低于同龄对照组。说明出生后婴儿早期使用PB或CNP所致的脑损伤难以完全康复,存在持久性脑病变可能性。(3)长程给予PB和CNP并不会对成年鼠造成明显的组织学损伤。而无论对于婴鼠或是成年鼠,长程给予CBZ、VPA和TPM均未引起明显的脑组织结构异常。
第二部分长程施用抗癫痫药致婴鼠脑内神经细胞过度凋亡
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞凋亡的不同影响。
方法:健康SD大鼠288只,包括婴鼠用药组、长程用药再停药4周婴鼠组,及成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组16只鼠。一日一次灌胃给药,连续给药28天。取与第一部分中HE染色紧邻脑片用原位末端标记法(TUNEL)检测脑内凋亡细胞,免疫组化法检测脑内凋亡相关基因Bcl-2、Bax蛋白表达。在各用药观察时间点结束后24小时,一半的大鼠用流式细胞仪检测额叶和海马神经细胞线粒体膜电位,另一半的大鼠用荧光分光光度仪检测额叶和海马神经细胞Caspase-3活性。
结果:(1)婴鼠用药组中仅PB和CNP引起大鼠额叶和海马TUNEL阳性细胞明显增高,与同龄对照比较,PB致额叶和海马神经TUNEL阳性细胞的增加达1.80倍和2.45倍(P<0.0001),而CNP达1.70倍和2.33倍(P<0.0001)。但在停药4周后较对照组已无明显差异。成年鼠长程使用这5种AEDs均没有出现TUNEL阳性细胞的显著增加。(2)婴鼠用药组中仅PB和CNP引起大鼠额叶和海马Bax蛋白表达明显增高(P<0.0001),而Bcl-2蛋白与对照无明显差异,进而导致Bax/Bcl-2比值均较对照显著性增高达1.57～2.26倍和1.74～2.29倍。停药4周后,PB和CNP所致的Bax蛋白和Bax/Bcl-2比值均与对照组无显著性差异。而成年鼠长程给予这5种AEDs均没有出现Bax蛋白和Bax/Bcl-2比值的异常。(3)长程给予PB和CNP还导致婴鼠脑内Caspase-3活性显著增高(P <0.01)和神经细胞线粒体膜电位显著降低(P <0.05),停药4周后与对照不再显示差异。所有的成年用药组均未显示与对照的差异。
结论:(1)长程给予治疗剂量PB和CNP存在致未成熟脑内神经细胞广泛过度凋亡可能性,停药后此种异常凋亡将会逐渐消失。然而,PB和CNP却不引起成年鼠脑内凋亡增加。(2)PB和CNP导致的未成熟脑损伤主要通过上调Bax蛋白,导致胞内Bax/Bcl-2比值和线粒体膜电位增高,进而激活Caspase酶系这一内源性凋亡途径,导致不可逆性过度凋亡发生。(3)长程治疗剂量的CBZ、VPA和TPM,均未引起婴鼠或成年鼠脑内明显过度凋亡现象。
第三部分长程施用抗癫痫药致婴鼠脑内神经细胞线粒体功能损伤
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞线粒体内氧化-抗氧化体系、呼吸链酶活性和ATP酶活性的不同影响,探索线粒体功能受损在AEDs导致的过度凋亡启动中关键性作用。
方法:健康SD大鼠144只,包括婴鼠用药组、长程用药后再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组8只鼠。一日一次灌胃给药,连续给药28天。在各用药观察时间点结束后24小时处死,分离额叶皮层和海马并提取各自的线粒体,分光光度计测定线粒体内谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量,以及线粒体呼吸链酶复合物Ⅰ、Ⅳ与Na+K+-ATP酶活性。
结果:(1)除了TPM外,其余4种药均导致包括婴鼠和成年鼠额叶皮层和海马神经细胞线粒体抗氧化指标SOD活性和GSH含量不同程度的降低,脂质过氧化指标MDA不同程度的升高。婴鼠的改变程度明显较成年鼠严重,其中尤以PB和CNP组下降幅度最突出,较同龄对照降低42%～44%(SOD)、20%～23%(GSH)和升高63%～73%(MDA)(P均<0.01)。而PB、CNP、CBZ和VPA导致的成年鼠SOD、GSH降低及MDA升高均较婴鼠轻微且在各药间并无差异。停药4周后,这些氧化-过氧化指标的改变均明显恢复并与对照不再显著差异。(2)长程施用PB和CNP导致婴鼠额叶皮层和海马神经细胞线粒体呼吸链酶复合物Ⅰ和Ⅳ活性显著降低(P <0.0001)。与对照相比,PB对额叶皮层和海马复合物Ⅰ的降低分别为28%和37%,CNP为33%和35%;而PB对额叶皮层和海马复合物Ⅳ的降低分别为34%和33%,CNP为35%和32%。停药4周后,降低的复合物Ⅰ和Ⅳ活性均有所恢复,与对照组无显著差异。而婴鼠用药组中的CBZ、VPA和TPM组及所有的成年用药组两酶活性与对照组相比无明显差异。(3)长疗程施用PB和CNP还导致额叶皮层和海马细胞线粒体Na~+K~+-ATP酶活性的显著降低,降低幅度在17%～19%间(P<0.05)。停药4周后与对照不再显示差异。而所有的成年用药组脑线粒体内Na~+K~+-ATP酶活性与对照组相比无明显差异。
结论:(1)与CBZ、VPA和TPM不同,长程给予PB和CNP可致婴鼠脑细胞线粒体氧化-抗氧化持续且更为严重的失衡状态,主要表现为GSH和SOD等主要抗氧化物质活性明显降低,脂质过氧化水平显著增高,氧化磷酸化的呼吸传递链复合物I和IV活性下降导致能量枯竭,同时伴有线粒体内主要离子泵Na~+K~+-ATP酶活性低下,膜内外离子分布和电位失衡,及线粒体正常膜完整性丧失。(2)线粒体遭受的氧化应激性损伤和能量代谢损伤是PB和CNP诱发未成熟脑细胞过度凋亡的关键性启动环节。(3)长程给予PB和CNP导致的婴鼠脑内线粒体损伤在停药后可逐渐恢复正常,未显示持久不可逆性线粒体功能异常。(4)在成年鼠中长程给予PB和CNP,以及婴鼠和成年鼠中长程给予CBZ和VPA后也均发生程度较轻的线粒体抗氧化物质活性降低,和脂质过氧化增高,但呼吸酶复合物I和IV的活性,以及Na~+K~+-ATP酶活性均未发生改变,也未造成明显细胞凋亡和脑组织学损害,提示实验动物体内相关的代偿机制阻止了线粒体功能实质性破坏和过度凋亡发生。
第四部分长程施用抗癫痫药对婴鼠脑内Ca~(2+)和神经营养因子的影响
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞内及线粒体内Ca~(2+)超载的危险性与发生机理,及脑内神经营养因子的不同影响。
方法:健康SD大鼠288只,包括婴鼠用药组、长程用药再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组16只鼠。一日一次灌胃给药,连续给药28天。取与第一部分中HE染色紧邻脑片用免疫组化法检测脑内NMDA-R1、BDNF和NT-3的表达。取第三部分新鲜提取未经冻存的线粒体悬液行线粒体内Ca~(2+)浓度测定。在各用药观察时间点结束后24小时,一半的大鼠用于神经细胞胞浆内游离Ca~(2+)浓度检测,另一半的大鼠用于NMDA-R1、BDNF和NT-3 mRNA检测。
结果:(1)婴鼠用药组中仅PB和CNP引起皮层和海马细胞的胞内和线粒体内Ca~(2+)浓度显著增高,其中胞内Ca~(2+)浓度增幅为对照的48%～57%(P<0.0001),而线粒体Ca~(2+)浓度的增加更是高达85%～97%(P<0.0001)。停药4周后,PB和CNP所致婴鼠神经细胞内和线粒体增高的Ca~(2+)明显回落,较对照组已无显著性差异。而成年鼠长程施用这5种AEDs均未导致胞内或线粒体内Ca~(2+)浓度的明显变化。(2)无论从蛋白水平还是基因水平均未发现PB和CNP导致NMDA-R1改变。(3)使用这5种AEDs后,无论婴鼠还是成年鼠,脑内BDNF和NT-3蛋白与基因水平表达呈一致下调,但婴鼠组较成年鼠组下降明显,而婴鼠组中又以PB和CNP下降更突出。停药4周后BDNF和NT-3的表达均与对照组不再有显著性差异。
结论:(1)长程给予PB和CNP可致婴鼠脑内与过度凋亡相一致的胞内和线粒体内钙超载。(2)PB和CNP导致的未成熟脑内Ca~(2+)超载并没有通过增加NMDA-R1密度来加强NMDA-R介导的胞外Ca~(2+)内流,但与线粒体内ROS异常增多及促凋亡Bax蛋白增高有更大关系。(3)蛋白和基因水平的检测均一致发现,长程给予PB、CNP、CBZ、VPA和TPM后导致的婴鼠和成年鼠脑内BDNF和NT-3不同程度的下降。但与成年鼠相比,婴鼠的下降更明显,尤以使用PB和CNP后最突出,促进了凋亡的启动和加重。但在停药后会逐渐恢复正常。
第五部分长程施用抗癫痫药致婴鼠脑内神经发生的不良影响
目的:初步探索生后早期长程施用临床治疗血药浓度的PB、CNP、CBZ、VPA和TPM,对大鼠未成熟脑内海马新生细胞增殖、存活及神经发生的影响。
方法:健康生后7日龄SD大鼠192只,分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组32只鼠。一日一次灌胃给药,连续给药28天后,腹腔注射BrdU(150mg/kg×3)。其中一半大鼠于注射BrdU后24h处死进行BrdU、DCX、BrdU/DCX双标免疫组化检测及DCX免疫印迹检测,以反映用药后海马新生细胞增殖及新生神经元发生情况;另一半于注射BrdU后28天处死进行BrdU、NeuN和BrdU/NeuN、BrdU/GFAP双标免疫组化检测,及NeuN免疫印迹检测,以观察海马新生细胞存活及分化状态。
结果:(1)BrdU给药后24小时,PB和CNP组的BrdU~+细胞较对照下降了63%和59%(P<0.01),同时反映神经发生的DCX水平也较对照明显降低,但BrdU~+/DCX~+细胞比率却较对照组为高。未发现CBZ、VPA和TPM组对新生细胞增殖和神经发生有明显改变。(2)28天后,BrdU~+细胞较先前BrdU给药后24小时水平更明显降低,对照组有58%的新生细胞存活,而PB和CNP组的新生细胞存活率仅为45%和51%(P<0.01)。BrdU~+/NeuN~+和BrdU~+/GFAP~+细胞占总BrdU~+细胞的水平却与对照无明显差异,海马齿状回NeuN~+细胞和海马总体NeuN蛋白表达水平也较对照显著降低。仍未发现CBZ、VPA和TPM改变新生细胞存活和分化。
结论:(1)生后早期长程施用PB和CNP显著抑制未成熟脑内海马神经发生,主要表现为抑制新生细胞的增殖和存活。(2)长程施用PB和CNP对未成熟脑内海马新生细胞的分化过程无明显影响。(3)未发现长程施用CBZ、VPA和TPM对未成熟脑内海马神经发生的显著影响。
PARTⅠEXPERIMENTAL STUDY ON HISTOPATHOLOGY CHANGES OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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.
PARTⅡEXPERIMENTAL STUDY ON THE POSSIBILITY OF EXCESSIVE CELL APOPTOSIS IN INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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 PARTⅠwere 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.
PARTⅢEXPERIMENTAL STUDY ON CELL MITOCHONDRIUM DYSFUNCTION OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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 complexⅠ,Ⅳand 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 complexⅠandⅣconcentration in frontal lobe and hippocampus of infant brain (P<0.0001). In frontal lobe and hippocampus, the complexⅠlevel was reduced respectively by 28% and 37% after PB administration and by 33% and 35% after CNP administration compared with control, and the complexⅣlevel was reduced respectively by 34% and 33% after PB administration, and 35% and 32% after CNP administration. But the decreased level of complexⅠandⅣweren’t found when drugs withdrawal. In both infant and adult rat administration group, there was no significant decrease of complexⅠandⅣafter 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 complexⅠ,Ⅳand 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 complexⅠ,Ⅳand Na~+K~+-ATP enzyme. So the compensation mechanism in body prevents the further mitochondria damage and the excessive apoptosis.
PARTⅥTHE 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 PARTⅢwere used to assay calcium concentration in mitochondria by spectrophotometer. Sections from PARTⅠwere 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 Ca~(2+) concentration. Compared with control, intracellular Ca~(2+) concentration was increased by 48%～57% (P<0.0001), and mitochondria Ca~(2+) concentration was increased by 85%～97% (P<0.0001). Four weeks after withdrawal, the high level of Ca~(2+) concentration didn’t detected anymore. No significant Ca~(2+) 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.
PARTⅤEXPERIMENTAL STUDY ON THE ADVERSE EFFECT ON NEUROGENESIS INDUCED BY LONG-TERM ANTIEPILPTIC DRUGS ADMINISTRATION
Objective: 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.
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内组织病理学损伤的不同特征及恢复的可能性。
方法:健康SD大鼠144只,包括婴鼠用药组、长程用药再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组8只鼠。一日一次灌胃给药,连续给药28天,在各用药观察时间点结束后24小时记录体重、断头处死称取脑重后,取额叶及海马行HE和Nissl染色观察组织学损伤特征并进行神经细胞计数,用透射电镜观察脑组织超微结构。
结果:(1)婴鼠用药组中仅PB和CNP引起体重和脑重明显降低,其中脑重降低幅度分别达对照组的18.3%和19.4%,但停药4周后其体重和脑重与对照已无明显差异。这5种AEDs均未引起成年鼠体重脑重的明显改变。(2)HE和Nissl染色光镜检查发现使用PB和CNP后,婴鼠额叶皮层组织结构模糊,且在皮层及海马均见细胞明显肿胀、胞浆空泡形成,或Nissl小体消失,部分显示胞核变性等,额叶皮层和海马各区神经细胞计数均较正常对照组明显减少。与同龄对照比较,PB致额叶皮层神经细胞的减少幅度达15.06%(P<0.0001),海马DG、CA1和CA3区的减少幅度分别为16.59%、12.03%和11.52%(P<0.0001)。CNP引起的额叶皮层和海马各区神经细胞的减少幅度与PB较为相似,分别达14.37%、15.57%、12.30%和11.57%(P均<0.0001)。停药4周后,PB和CNP组的神经细胞分布基本均匀,细胞边界和核仁清晰,病变已不再明显,但额叶皮层和海马各区的神经元计数仍明显低于对照组。而婴鼠用药组中的CBZ、VPA和TPM组及所有的成年用药组神经细胞的镜下特征和计数与对照组相比无明显差异。(3)透射电镜发现婴鼠长程使用PB和CNP后染色质边集浓缩,线粒体肿胀和内质网扩张,但停药4周后这些超微结构的改变皆不再明显。
结论:(1)长程给予PB和CNP可致婴鼠脑重明显减轻,神经细胞变性及数量明显减少,电镜下超微结构显著异常。提示两药对幼鼠认知功能的持续影响是基于它们对未成熟脑的明显组织学损伤。(2)婴鼠长程给予PB和CNP并长时间停药后,大鼠的脑重、神经细胞损伤及其数量虽都有不同程度的恢复,但额叶皮层和海马各区神经元计数依然远低于同龄对照组。说明出生后婴儿早期使用PB或CNP所致的脑损伤难以完全康复,存在持久性脑病变可能性。(3)长程给予PB和CNP并不会对成年鼠造成明显的组织学损伤。而无论对于婴鼠或是成年鼠,长程给予CBZ、VPA和TPM均未引起明显的脑组织结构异常。
第二部分长程施用抗癫痫药致婴鼠脑内神经细胞过度凋亡
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞凋亡的不同影响。
方法:健康SD大鼠288只,包括婴鼠用药组、长程用药再停药4周婴鼠组,及成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组16只鼠。一日一次灌胃给药,连续给药28天。取与第一部分中HE染色紧邻脑片用原位末端标记法(TUNEL)检测脑内凋亡细胞,免疫组化法检测脑内凋亡相关基因Bcl-2、Bax蛋白表达。在各用药观察时间点结束后24小时,一半的大鼠用流式细胞仪检测额叶和海马神经细胞线粒体膜电位,另一半的大鼠用荧光分光光度仪检测额叶和海马神经细胞Caspase-3活性。
结果:(1)婴鼠用药组中仅PB和CNP引起大鼠额叶和海马TUNEL阳性细胞明显增高,与同龄对照比较,PB致额叶和海马神经TUNEL阳性细胞的增加达1.80倍和2.45倍(P<0.0001),而CNP达1.70倍和2.33倍(P<0.0001)。但在停药4周后较对照组已无明显差异。成年鼠长程使用这5种AEDs均没有出现TUNEL阳性细胞的显著增加。(2)婴鼠用药组中仅PB和CNP引起大鼠额叶和海马Bax蛋白表达明显增高(P<0.0001),而Bcl-2蛋白与对照无明显差异,进而导致Bax/Bcl-2比值均较对照显著性增高达1.57～2.26倍和1.74～2.29倍。停药4周后,PB和CNP所致的Bax蛋白和Bax/Bcl-2比值均与对照组无显著性差异。而成年鼠长程给予这5种AEDs均没有出现Bax蛋白和Bax/Bcl-2比值的异常。(3)长程给予PB和CNP还导致婴鼠脑内Caspase-3活性显著增高(P <0.01)和神经细胞线粒体膜电位显著降低(P <0.05),停药4周后与对照不再显示差异。所有的成年用药组均未显示与对照的差异。
结论:(1)长程给予治疗剂量PB和CNP存在致未成熟脑内神经细胞广泛过度凋亡可能性,停药后此种异常凋亡将会逐渐消失。然而,PB和CNP却不引起成年鼠脑内凋亡增加。(2)PB和CNP导致的未成熟脑损伤主要通过上调Bax蛋白,导致胞内Bax/Bcl-2比值和线粒体膜电位增高,进而激活Caspase酶系这一内源性凋亡途径,导致不可逆性过度凋亡发生。(3)长程治疗剂量的CBZ、VPA和TPM,均未引起婴鼠或成年鼠脑内明显过度凋亡现象。
第三部分长程施用抗癫痫药致婴鼠脑内神经细胞线粒体功能损伤
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞线粒体内氧化-抗氧化体系、呼吸链酶活性和ATP酶活性的不同影响,探索线粒体功能受损在AEDs导致的过度凋亡启动中关键性作用。
方法:健康SD大鼠144只,包括婴鼠用药组、长程用药后再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组8只鼠。一日一次灌胃给药,连续给药28天。在各用药观察时间点结束后24小时处死,分离额叶皮层和海马并提取各自的线粒体,分光光度计测定线粒体内谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量,以及线粒体呼吸链酶复合物Ⅰ、Ⅳ与Na+K+-ATP酶活性。
结果:(1)除了TPM外,其余4种药均导致包括婴鼠和成年鼠额叶皮层和海马神经细胞线粒体抗氧化指标SOD活性和GSH含量不同程度的降低,脂质过氧化指标MDA不同程度的升高。婴鼠的改变程度明显较成年鼠严重,其中尤以PB和CNP组下降幅度最突出,较同龄对照降低42%～44%(SOD)、20%～23%(GSH)和升高63%～73%(MDA)(P均<0.01)。而PB、CNP、CBZ和VPA导致的成年鼠SOD、GSH降低及MDA升高均较婴鼠轻微且在各药间并无差异。停药4周后,这些氧化-过氧化指标的改变均明显恢复并与对照不再显著差异。(2)长程施用PB和CNP导致婴鼠额叶皮层和海马神经细胞线粒体呼吸链酶复合物Ⅰ和Ⅳ活性显著降低(P <0.0001)。与对照相比,PB对额叶皮层和海马复合物Ⅰ的降低分别为28%和37%,CNP为33%和35%;而PB对额叶皮层和海马复合物Ⅳ的降低分别为34%和33%,CNP为35%和32%。停药4周后,降低的复合物Ⅰ和Ⅳ活性均有所恢复,与对照组无显著差异。而婴鼠用药组中的CBZ、VPA和TPM组及所有的成年用药组两酶活性与对照组相比无明显差异。(3)长疗程施用PB和CNP还导致额叶皮层和海马细胞线粒体Na~+K~+-ATP酶活性的显著降低,降低幅度在17%～19%间(P<0.05)。停药4周后与对照不再显示差异。而所有的成年用药组脑线粒体内Na~+K~+-ATP酶活性与对照组相比无明显差异。
结论:(1)与CBZ、VPA和TPM不同,长程给予PB和CNP可致婴鼠脑细胞线粒体氧化-抗氧化持续且更为严重的失衡状态,主要表现为GSH和SOD等主要抗氧化物质活性明显降低,脂质过氧化水平显著增高,氧化磷酸化的呼吸传递链复合物I和IV活性下降导致能量枯竭,同时伴有线粒体内主要离子泵Na~+K~+-ATP酶活性低下,膜内外离子分布和电位失衡,及线粒体正常膜完整性丧失。(2)线粒体遭受的氧化应激性损伤和能量代谢损伤是PB和CNP诱发未成熟脑细胞过度凋亡的关键性启动环节。(3)长程给予PB和CNP导致的婴鼠脑内线粒体损伤在停药后可逐渐恢复正常,未显示持久不可逆性线粒体功能异常。(4)在成年鼠中长程给予PB和CNP,以及婴鼠和成年鼠中长程给予CBZ和VPA后也均发生程度较轻的线粒体抗氧化物质活性降低,和脂质过氧化增高,但呼吸酶复合物I和IV的活性,以及Na~+K~+-ATP酶活性均未发生改变,也未造成明显细胞凋亡和脑组织学损害,提示实验动物体内相关的代偿机制阻止了线粒体功能实质性破坏和过度凋亡发生。
第四部分长程施用抗癫痫药对婴鼠脑内Ca~(2+)和神经营养因子的影响
目的:对比性观察长程施用与临床治疗浓度相匹配的抗癫痫药PB、CNP、CBZ、VPA和TPM,对健康婴鼠和成年大鼠脑内神经细胞内及线粒体内Ca~(2+)超载的危险性与发生机理,及脑内神经营养因子的不同影响。
方法:健康SD大鼠288只,包括婴鼠用药组、长程用药再停药4周婴鼠组和成年鼠用药组三个观察组,各组再分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组16只鼠。一日一次灌胃给药,连续给药28天。取与第一部分中HE染色紧邻脑片用免疫组化法检测脑内NMDA-R1、BDNF和NT-3的表达。取第三部分新鲜提取未经冻存的线粒体悬液行线粒体内Ca~(2+)浓度测定。在各用药观察时间点结束后24小时,一半的大鼠用于神经细胞胞浆内游离Ca~(2+)浓度检测,另一半的大鼠用于NMDA-R1、BDNF和NT-3 mRNA检测。
结果:(1)婴鼠用药组中仅PB和CNP引起皮层和海马细胞的胞内和线粒体内Ca~(2+)浓度显著增高,其中胞内Ca~(2+)浓度增幅为对照的48%～57%(P<0.0001),而线粒体Ca~(2+)浓度的增加更是高达85%～97%(P<0.0001)。停药4周后,PB和CNP所致婴鼠神经细胞内和线粒体增高的Ca~(2+)明显回落,较对照组已无显著性差异。而成年鼠长程施用这5种AEDs均未导致胞内或线粒体内Ca~(2+)浓度的明显变化。(2)无论从蛋白水平还是基因水平均未发现PB和CNP导致NMDA-R1改变。(3)使用这5种AEDs后,无论婴鼠还是成年鼠,脑内BDNF和NT-3蛋白与基因水平表达呈一致下调,但婴鼠组较成年鼠组下降明显,而婴鼠组中又以PB和CNP下降更突出。停药4周后BDNF和NT-3的表达均与对照组不再有显著性差异。
结论:(1)长程给予PB和CNP可致婴鼠脑内与过度凋亡相一致的胞内和线粒体内钙超载。(2)PB和CNP导致的未成熟脑内Ca~(2+)超载并没有通过增加NMDA-R1密度来加强NMDA-R介导的胞外Ca~(2+)内流,但与线粒体内ROS异常增多及促凋亡Bax蛋白增高有更大关系。(3)蛋白和基因水平的检测均一致发现,长程给予PB、CNP、CBZ、VPA和TPM后导致的婴鼠和成年鼠脑内BDNF和NT-3不同程度的下降。但与成年鼠相比,婴鼠的下降更明显,尤以使用PB和CNP后最突出,促进了凋亡的启动和加重。但在停药后会逐渐恢复正常。
第五部分长程施用抗癫痫药致婴鼠脑内神经发生的不良影响
目的:初步探索生后早期长程施用临床治疗血药浓度的PB、CNP、CBZ、VPA和TPM,对大鼠未成熟脑内海马新生细胞增殖、存活及神经发生的影响。
方法:健康生后7日龄SD大鼠192只,分为6小组,分别给予PB、CNP、CBZ、VPA、TPM和蒸馏水(对照),每组32只鼠。一日一次灌胃给药,连续给药28天后,腹腔注射BrdU(150mg/kg×3)。其中一半大鼠于注射BrdU后24h处死进行BrdU、DCX、BrdU/DCX双标免疫组化检测及DCX免疫印迹检测,以反映用药后海马新生细胞增殖及新生神经元发生情况;另一半于注射BrdU后28天处死进行BrdU、NeuN和BrdU/NeuN、BrdU/GFAP双标免疫组化检测,及NeuN免疫印迹检测,以观察海马新生细胞存活及分化状态。
结果:(1)BrdU给药后24小时,PB和CNP组的BrdU~+细胞较对照下降了63%和59%(P<0.01),同时反映神经发生的DCX水平也较对照明显降低,但BrdU~+/DCX~+细胞比率却较对照组为高。未发现CBZ、VPA和TPM组对新生细胞增殖和神经发生有明显改变。(2)28天后,BrdU~+细胞较先前BrdU给药后24小时水平更明显降低,对照组有58%的新生细胞存活,而PB和CNP组的新生细胞存活率仅为45%和51%(P<0.01)。BrdU~+/NeuN~+和BrdU~+/GFAP~+细胞占总BrdU~+细胞的水平却与对照无明显差异,海马齿状回NeuN~+细胞和海马总体NeuN蛋白表达水平也较对照显著降低。仍未发现CBZ、VPA和TPM改变新生细胞存活和分化。
结论:(1)生后早期长程施用PB和CNP显著抑制未成熟脑内海马神经发生,主要表现为抑制新生细胞的增殖和存活。(2)长程施用PB和CNP对未成熟脑内海马新生细胞的分化过程无明显影响。(3)未发现长程施用CBZ、VPA和TPM对未成熟脑内海马神经发生的显著影响。
PARTⅠEXPERIMENTAL STUDY ON HISTOPATHOLOGY CHANGES OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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.
PARTⅡEXPERIMENTAL STUDY ON THE POSSIBILITY OF EXCESSIVE CELL APOPTOSIS IN INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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 PARTⅠwere 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.
PARTⅢEXPERIMENTAL STUDY ON CELL MITOCHONDRIUM DYSFUNCTION OF INFANT RAT BRAIN INDUCED BY LONG-TERM ANTIEPILEPTIC DRUGS ADMINISTRATION
Objective: 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 complexⅠ,Ⅳand 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 complexⅠandⅣconcentration in frontal lobe and hippocampus of infant brain (P<0.0001). In frontal lobe and hippocampus, the complexⅠlevel was reduced respectively by 28% and 37% after PB administration and by 33% and 35% after CNP administration compared with control, and the complexⅣlevel was reduced respectively by 34% and 33% after PB administration, and 35% and 32% after CNP administration. But the decreased level of complexⅠandⅣweren’t found when drugs withdrawal. In both infant and adult rat administration group, there was no significant decrease of complexⅠandⅣafter 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 complexⅠ,Ⅳand 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 complexⅠ,Ⅳand Na~+K~+-ATP enzyme. So the compensation mechanism in body prevents the further mitochondria damage and the excessive apoptosis.
PARTⅥTHE 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 PARTⅢwere used to assay calcium concentration in mitochondria by spectrophotometer. Sections from PARTⅠwere 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 Ca~(2+) concentration. Compared with control, intracellular Ca~(2+) concentration was increased by 48%～57% (P<0.0001), and mitochondria Ca~(2+) concentration was increased by 85%～97% (P<0.0001). Four weeks after withdrawal, the high level of Ca~(2+) concentration didn’t detected anymore. No significant Ca~(2+) 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.
PARTⅤEXPERIMENTAL STUDY ON THE ADVERSE EFFECT ON NEUROGENESIS INDUCED BY LONG-TERM ANTIEPILPTIC DRUGS ADMINISTRATION
Objective: 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.
引文
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