大鼠皮质发育障碍模型研究及γ-氨基丁酸检测
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摘要
目的：建立能模拟人类皮质发育障碍（disorders of cortical development, DCDs）的动物模型，探索DCDs的形成机制和与难治性癫痫(intractable epilepsy , IE)的关系，为临床诊治DCDs与IE提供理论依据。
方法：用两种方法制备DCDs模型：⑴采用剂量为2.0Gy的γ-射线照射妊娠15天Wistar大鼠造成胎鼠的损伤；⑵用低于-50℃的液氮钝性探针冰冻损伤出生第二天（P2）的乳鼠大脑皮质，制作出皮质发育障碍的动物模型。同时设正常对照组。
通过下列观察以确定DCDs模型的成功率和可靠性：①孕鼠后代及冰冻脑损伤鼠在出生第22天（P22）处死取脑，作巨检和病理切片观察大鼠脑皮质和海马神经元结构，确定DCDs的发生率和类型；②对大鼠日常行为表现进行观察，包括意识状态、生活能力、以及有无攻击行为和癫痫发作，并作脑电图记录；③利用热水浴诱导惊厥发作，记录发作的潜伏期；④采用Morris水迷宫方法监测大鼠学习能力和空间记忆能力；⑤用免疫组化方法观察γ-氨基丁酸（GABA）阳性神经元在大鼠大脑皮质中的表达。以上均与正常对照组比较。
结果： ①模型鼠平均脑重比正常对照鼠轻(P<0.05)。用剂量为
2.0Gy的γ-射线照射孕鼠组其后代全部（100%）产生了广泛的皮质发育畸形和皮质下神经元结节性异位。用低于-50℃液氮冰冻损伤P2乳鼠组全部（100%）产生多微脑回畸形，即在脑表面出现了类似于人类微脑回畸形的微沟。病理切片尼氏染色镜检可见大脑皮质变薄，层状结构紊乱。γ-射线模型鼠可见弥漫性皮质下和海马神经元成团状的结节状异位；冰冻损伤模型鼠具有多灶性、局限性的4层皮质，脑回变小等发育畸形；②模型鼠与正常对照鼠相比日常活动能力较差，但是没有观察到自发性癫痫发作；③模型组热水浴诱导惊厥发作的潜伏期较对照组明显缩短(P<0.05)；④模型组水迷宫实验中寻找水下平台的时间较对照组明显延长(P<0.05)；⑤模型鼠免疫组化可见GABA阳性神经元的表达减少P<0.05）。
结论：1. 用γ-射线照射妊娠15天Wistar大鼠及冰冻损伤P2幼鼠均能够成功地建立类似人类某些皮质发育障碍类型的动物模型。
2．模型组与正常对照组相比，体重和大脑重量均明显减轻，说明模型鼠的身体发育和大脑发育均受到影响。
3．射线照射鼠的后代100％出现广泛的皮质发育不良和神经元团状异位，类似人类神经元结节性移位。
4．冰冻P2乳鼠大脑皮质也100％出现类似人类多微脑回畸形的发育不良，且与冰冻损伤的程度相关。
5．皮质发育障碍模型鼠热性惊厥的发作阈值较正常对照鼠明显降低，即发作易感性增加，并伴有认知功能障碍。
6.通过免疫组化方法我们发现皮质发育障碍模型大鼠的皮质GABA阳性神经元的表达减少(P<0.05)，可能是皮质发育障碍所致癫痫发作和难治性癫痫的发病机制之一。
Objective: We developed two animal models of disorders of cortical development ( DCDs )which can mimic human to study the mechanisms underling epileptogenesis with DCDs and understand the relationship between DCDs and intractable epilepsy(IE). Both above may provide a theory base for prevention DCDs and IE .
Methods: Animal models of DCDs were made in two ways. ⑴ The first method was that pregnant Wistar rats were exposed toγ-ray using a single dose of 2.0 Gy on embryonic day 15 (E15) for 210 seconds. The brain of their offsprings might receive lesions through this method. ⑵ The other way was that we used a 2-mm-diameter copper cylinder cooled with liquid nitrogen that temperature below -50℃ to injure the cortical plate of neonatal rats (postnatal days 2, P2 ). The experimental little rats
were divided into model groups and normal control groups. We observed the following aspects to confirm the success ratio and the credibility in the animal models of DCDs : ① On postnatal days 22 , the freeze- lesion-rats and the offsprings of irrated-pregnat-rats were killed to be used to histomorphology and histopathologic studies. Neuronal changes in hippocampus and cerebral cortex were detected by Nissl stain. Through this way we can confirm the seizures ratio and styles of the DCDs. ② Before EEG recordings, rats were observed daily activities which included consciousness, living ability , attack behavior as well as spontaneous seizures. ③Rats were induced seizures by warm-water- immersion (45℃). ④ The memory capacity was tested by using a Morris-water-maze method. ⑤ The expressions of GABA in cortex were observed by immuno- hisochemistry.
Results: ① The weight of the brains in model groups rats were lighter than the control groups (P﹤0.05 ). All of the irrated-pregnat-rats′ offsprings ( 100% ) were caused brain lesions resulting in neuronal migration disorders. All of the neonatal little rats ( 100% ) received liquid nitrogen freeze lesions had cortical malformation mimics human microgyria. Model rat shows a thin cortical plate and distinct clusters of neuronal elements that represent heterotopias. In the control rat cortex, the typical six-layered lamination pattern is apparent, whereas the model rat
cortex shows laminar disorganization and thinning. In addition, there are clusters of large neurons in both superficial and deep layers in the model rat cortex. Nissl stains showed the presence of multiple cortical areas of neuronal clustering and a focal four-layered cortex, moreover pyramidal cell dispersion was seen in the hippocampal formations. ② Animal model groups have less activities than the normal control groups. We did not observed the spontaneous seizures. ③ The latent period that warm- water- immerse induced the seizures was significantly shorter than the control (P﹤0.05). ④ The searching platform latency of model rats was significantly longer than that of the normal controls (P﹤0.05) . ⑤ The GABA positive cells in the cortex of model rats could be detectived loss by immuno- hisochemistry.
Conclusions:
1. we can succeed in developing the animal models of DCDs , The first method was that pregnant rats were exposed toγ-ray on embryonic day 15 (E15) , The other method was that we used a copper cylinder cooled with liquid nitrogen that temperature below -50℃ to injure the cortical plate of neonatal rats (postnatal days 2, P2 ).
2. The weight of the brain and body in model groups rats were lighter than those of the control groups. DCDs was a handicap to the development of the body and brain.
3. All of the irrated-pregnat-rats′offsprings ( 100% ) were caused brain lesions resulting in cortical dysplasia and neuronal migration disorders which similar to human neuronal heterotopia malformation.
4. All of the neonatal rats ( 100% ) received liquid nitrogen freeze lesions had cortical malformation mimics human microgyria.
5. Opposite to the control groups, models rats have decreasing seizure threshold and increased seizure susceptibility as well as cognitive handicap
方法：用两种方法制备DCDs模型：⑴采用剂量为2.0Gy的γ-射线照射妊娠15天Wistar大鼠造成胎鼠的损伤；⑵用低于-50℃的液氮钝性探针冰冻损伤出生第二天（P2）的乳鼠大脑皮质，制作出皮质发育障碍的动物模型。同时设正常对照组。
通过下列观察以确定DCDs模型的成功率和可靠性：①孕鼠后代及冰冻脑损伤鼠在出生第22天（P22）处死取脑，作巨检和病理切片观察大鼠脑皮质和海马神经元结构，确定DCDs的发生率和类型；②对大鼠日常行为表现进行观察，包括意识状态、生活能力、以及有无攻击行为和癫痫发作，并作脑电图记录；③利用热水浴诱导惊厥发作，记录发作的潜伏期；④采用Morris水迷宫方法监测大鼠学习能力和空间记忆能力；⑤用免疫组化方法观察γ-氨基丁酸（GABA）阳性神经元在大鼠大脑皮质中的表达。以上均与正常对照组比较。
结果： ①模型鼠平均脑重比正常对照鼠轻(P<0.05)。用剂量为
2.0Gy的γ-射线照射孕鼠组其后代全部（100%）产生了广泛的皮质发育畸形和皮质下神经元结节性异位。用低于-50℃液氮冰冻损伤P2乳鼠组全部（100%）产生多微脑回畸形，即在脑表面出现了类似于人类微脑回畸形的微沟。病理切片尼氏染色镜检可见大脑皮质变薄，层状结构紊乱。γ-射线模型鼠可见弥漫性皮质下和海马神经元成团状的结节状异位；冰冻损伤模型鼠具有多灶性、局限性的4层皮质，脑回变小等发育畸形；②模型鼠与正常对照鼠相比日常活动能力较差，但是没有观察到自发性癫痫发作；③模型组热水浴诱导惊厥发作的潜伏期较对照组明显缩短(P<0.05)；④模型组水迷宫实验中寻找水下平台的时间较对照组明显延长(P<0.05)；⑤模型鼠免疫组化可见GABA阳性神经元的表达减少P<0.05）。
结论：1. 用γ-射线照射妊娠15天Wistar大鼠及冰冻损伤P2幼鼠均能够成功地建立类似人类某些皮质发育障碍类型的动物模型。
2．模型组与正常对照组相比，体重和大脑重量均明显减轻，说明模型鼠的身体发育和大脑发育均受到影响。
3．射线照射鼠的后代100％出现广泛的皮质发育不良和神经元团状异位，类似人类神经元结节性移位。
4．冰冻P2乳鼠大脑皮质也100％出现类似人类多微脑回畸形的发育不良，且与冰冻损伤的程度相关。
5．皮质发育障碍模型鼠热性惊厥的发作阈值较正常对照鼠明显降低，即发作易感性增加，并伴有认知功能障碍。
6.通过免疫组化方法我们发现皮质发育障碍模型大鼠的皮质GABA阳性神经元的表达减少(P<0.05)，可能是皮质发育障碍所致癫痫发作和难治性癫痫的发病机制之一。
Objective: We developed two animal models of disorders of cortical development ( DCDs )which can mimic human to study the mechanisms underling epileptogenesis with DCDs and understand the relationship between DCDs and intractable epilepsy(IE). Both above may provide a theory base for prevention DCDs and IE .
Methods: Animal models of DCDs were made in two ways. ⑴ The first method was that pregnant Wistar rats were exposed toγ-ray using a single dose of 2.0 Gy on embryonic day 15 (E15) for 210 seconds. The brain of their offsprings might receive lesions through this method. ⑵ The other way was that we used a 2-mm-diameter copper cylinder cooled with liquid nitrogen that temperature below -50℃ to injure the cortical plate of neonatal rats (postnatal days 2, P2 ). The experimental little rats
were divided into model groups and normal control groups. We observed the following aspects to confirm the success ratio and the credibility in the animal models of DCDs : ① On postnatal days 22 , the freeze- lesion-rats and the offsprings of irrated-pregnat-rats were killed to be used to histomorphology and histopathologic studies. Neuronal changes in hippocampus and cerebral cortex were detected by Nissl stain. Through this way we can confirm the seizures ratio and styles of the DCDs. ② Before EEG recordings, rats were observed daily activities which included consciousness, living ability , attack behavior as well as spontaneous seizures. ③Rats were induced seizures by warm-water- immersion (45℃). ④ The memory capacity was tested by using a Morris-water-maze method. ⑤ The expressions of GABA in cortex were observed by immuno- hisochemistry.
Results: ① The weight of the brains in model groups rats were lighter than the control groups (P﹤0.05 ). All of the irrated-pregnat-rats′ offsprings ( 100% ) were caused brain lesions resulting in neuronal migration disorders. All of the neonatal little rats ( 100% ) received liquid nitrogen freeze lesions had cortical malformation mimics human microgyria. Model rat shows a thin cortical plate and distinct clusters of neuronal elements that represent heterotopias. In the control rat cortex, the typical six-layered lamination pattern is apparent, whereas the model rat
cortex shows laminar disorganization and thinning. In addition, there are clusters of large neurons in both superficial and deep layers in the model rat cortex. Nissl stains showed the presence of multiple cortical areas of neuronal clustering and a focal four-layered cortex, moreover pyramidal cell dispersion was seen in the hippocampal formations. ② Animal model groups have less activities than the normal control groups. We did not observed the spontaneous seizures. ③ The latent period that warm- water- immerse induced the seizures was significantly shorter than the control (P﹤0.05). ④ The searching platform latency of model rats was significantly longer than that of the normal controls (P﹤0.05) . ⑤ The GABA positive cells in the cortex of model rats could be detectived loss by immuno- hisochemistry.
Conclusions:
1. we can succeed in developing the animal models of DCDs , The first method was that pregnant rats were exposed toγ-ray on embryonic day 15 (E15) , The other method was that we used a copper cylinder cooled with liquid nitrogen that temperature below -50℃ to injure the cortical plate of neonatal rats (postnatal days 2, P2 ).
2. The weight of the brain and body in model groups rats were lighter than those of the control groups. DCDs was a handicap to the development of the body and brain.
3. All of the irrated-pregnat-rats′offsprings ( 100% ) were caused brain lesions resulting in cortical dysplasia and neuronal migration disorders which similar to human neuronal heterotopia malformation.
4. All of the neonatal rats ( 100% ) received liquid nitrogen freeze lesions had cortical malformation mimics human microgyria.
5. Opposite to the control groups, models rats have decreasing seizure threshold and increased seizure susceptibility as well as cognitive handicap
引文
晏勇. 皮质发育障碍与癫痫发作. 重庆医学 2001；30（5）：464-466.
Chevassus-au-Louis N, Baraban SC, Gaiarsa JL ,et al. Cortical malformations and epilepsy: New insights from animal models. Epilepsia 1999；40(7):812-815.
Morrell F. Secondary epileptogenic lesion. Epilepsia 1959/1960,；1:538-560.
Dvorak K, Feit J. Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats: contribution to the problems of morphological development and developmental period of cerebral microgyria. Acta Neuropathol 1977：38: 302-312.
Dvorak K, Feit J, Jurankova Z. Experimentally induced focal microgyria and status verucosus deformis in rats: pathogenesis and interrelation histological and autoradiographical study. Acta Neuropathol 1978；44:121-129.
Taylor D C, Falconer M A, Bruton C J,et al. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiat 1971；34：369-387.
Meencke H J. Pathology of childhood epilepsies. Clev Clin J Med 1988； 56： S111-S120.
Raymond A A, Fish D R, Sisodiya S M, et al. Abnormalities of gyration, heterotopias, Tuberose sclerosis, focal cortical dysplasia, microdysgenesis, dysembryoplastic neuroepithelial tumour and dysgenesis of the archicortex. Brain 1995；118: 629-665.
王学峰，沈鼎烈，曾可斌，等. 难治性癫痫的基础. 见：王学峰，主编. 难治性癫痫. 第1版. 上海: 上海科技出版社 2002. 1-40.
Palmini A, Andermann F, Olivier A, et al. Focal neuronal migration disorders and intractable partial epilepsy: results of surgical treatment. Ann Neurol 1991; 30: 750-757.
Guerrini R, Andermann E, Avoli M, et al. Cortical dysplasias, genetics, and
epileptogenesis. Adv Neurol 1999；79:95-121.
Guerrini R, Sicca F, Parmeggiani L. Epilepsy and malformations of the cerebral cortex. Epileptic Disord 2003；5 (Suppl 2):9-26.
北京市科学技术委员会. 北京市实验动物管理暂行实施细则. 北京实验动物科学 1993；10（4）：3-6.
北京市科学技术委员会. 北京市实验动物管理条例. 实验动物科学与管理1997；14（1）：1-3.
Babb T L, Zhong Y, Mikuni N, et al. Brain plasticity and cellular mechanisms of epileptogenesis in human and experimental cortical dysplasia. Epilepsia 2000； 41(Suppl.6): 76-81.
Schwarz P, Stichel C C, Luhmann H J. Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons? Epilepsia 2000；41(7):781-787.
Racine R J. Modification of seizure activity by electrical activity Ⅱ: motor seizure, electro encephalogy [J]. Clin Neurophysiol 1972；32:281-294.
Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neuro Sci Methods 1984；11(1): 47-60.
kondo S, Najm I, Kunieda T, et al. Electroencephalographic characterization of an adult rat model of radiation-induced cortical dysplasia. Epilepsia 2001； 42(10): 1221-1223.
Kaufmann W E, and Galaburda A M. Cerebrocortical microdysgenesis in neurologically normal subjects: a histopathologic study. Neurology 1989； 39: 238-244
Humphreys P, Kaufmann W E, and Galaburda A M. Developmental dyslexia in women: neuropathological findings in three patients. Ann Neurol 1990； 28: 727-738
Jacobs K M, Hwang B J, Prince D A. Focal Epileptogenesis in a Rat Model of Polymicrogyria . The Journal of Neurophysiology 1999； 81 (1)：159-173
Gabel L A and LoTurco J J . Layer I Ectopias and IncreasedExcitability in Murine Neocortex. J. Neurophysiol 2002； 87: 2471-2479.
Luhmann H J, Karpuk N, Qu M, et al. Characterization of neuronal migration disorders in neocortical structures. II. Intracellular in vitro recordings. J Neurophysiol 1998；80: 92-102,
Jiang W, Duong TM, Delanerolle NC. Neuropathology of hyperthermic seizures in the rat. Epilepsia 1999; 40(1):5-19.
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Guerrini R, Andermann E, Avoli M, et al. Cortical dysplasias, genetics, and epileptogenesis. Adv Neurol 1999； 79: 95-121.
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interical spikes after intrahippocampal kainate in developing rats. Epilepsy Res 1996; 26:219-231.
De Lanerrolle, Kim JH, Robbins RJ, et al. Hippocampal interneuron loss and plastity in human temporal lobe epilepsy. Brain Res 1989; 495:387-395.
Holmes GL, Sarkisian M, Ben-Ari Y, et al. Mossy fiber sprouting following recurrent seizures during early development in rats. J. Comp Neurol 1999; 404: 537-553.
Kupfer WR, Pretorius JK. Recurrent excitatory circuits by “Sprouted” mossy fibers into the fascia dentate of human hippocampal epilepsy. Epilepsy 1988; 29: 674-681.
Chevassus-au-Louis N, Baraban SC, Gaiarsa JL ,et al. Cortical malformations and epilepsy: New insights from animal models. Epilepsia 1999；40(7):812-815.
Morrell F. Secondary epileptogenic lesion. Epilepsia 1959/1960,；1:538-560.
Dvorak K, Feit J. Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats: contribution to the problems of morphological development and developmental period of cerebral microgyria. Acta Neuropathol 1977：38: 302-312.
Dvorak K, Feit J, Jurankova Z. Experimentally induced focal microgyria and status verucosus deformis in rats: pathogenesis and interrelation histological and autoradiographical study. Acta Neuropathol 1978；44:121-129.
Taylor D C, Falconer M A, Bruton C J,et al. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiat 1971；34：369-387.
Meencke H J. Pathology of childhood epilepsies. Clev Clin J Med 1988； 56： S111-S120.
Raymond A A, Fish D R, Sisodiya S M, et al. Abnormalities of gyration, heterotopias, Tuberose sclerosis, focal cortical dysplasia, microdysgenesis, dysembryoplastic neuroepithelial tumour and dysgenesis of the archicortex. Brain 1995；118: 629-665.
王学峰，沈鼎烈，曾可斌，等. 难治性癫痫的基础. 见：王学峰，主编. 难治性癫痫. 第1版. 上海: 上海科技出版社 2002. 1-40.
Palmini A, Andermann F, Olivier A, et al. Focal neuronal migration disorders and intractable partial epilepsy: results of surgical treatment. Ann Neurol 1991; 30: 750-757.
Guerrini R, Andermann E, Avoli M, et al. Cortical dysplasias, genetics, and
epileptogenesis. Adv Neurol 1999；79:95-121.
Guerrini R, Sicca F, Parmeggiani L. Epilepsy and malformations of the cerebral cortex. Epileptic Disord 2003；5 (Suppl 2):9-26.
北京市科学技术委员会. 北京市实验动物管理暂行实施细则. 北京实验动物科学 1993；10（4）：3-6.
北京市科学技术委员会. 北京市实验动物管理条例. 实验动物科学与管理1997；14（1）：1-3.
Babb T L, Zhong Y, Mikuni N, et al. Brain plasticity and cellular mechanisms of epileptogenesis in human and experimental cortical dysplasia. Epilepsia 2000； 41(Suppl.6): 76-81.
Schwarz P, Stichel C C, Luhmann H J. Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons? Epilepsia 2000；41(7):781-787.
Racine R J. Modification of seizure activity by electrical activity Ⅱ: motor seizure, electro encephalogy [J]. Clin Neurophysiol 1972；32:281-294.
Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neuro Sci Methods 1984；11(1): 47-60.
kondo S, Najm I, Kunieda T, et al. Electroencephalographic characterization of an adult rat model of radiation-induced cortical dysplasia. Epilepsia 2001； 42(10): 1221-1223.
Kaufmann W E, and Galaburda A M. Cerebrocortical microdysgenesis in neurologically normal subjects: a histopathologic study. Neurology 1989； 39: 238-244
Humphreys P, Kaufmann W E, and Galaburda A M. Developmental dyslexia in women: neuropathological findings in three patients. Ann Neurol 1990； 28: 727-738
Jacobs K M, Hwang B J, Prince D A. Focal Epileptogenesis in a Rat Model of Polymicrogyria . The Journal of Neurophysiology 1999； 81 (1)：159-173
Gabel L A and LoTurco J J . Layer I Ectopias and IncreasedExcitability in Murine Neocortex. J. Neurophysiol 2002； 87: 2471-2479.
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