生酮饮食对幼鼠戊四氮诱导痫性发作的影响及海马区形态学观察
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摘要
前言
生酮饮食(ketogenic diet，KD)是一种高脂肪、低蛋白质和低碳水化合物的饮食，用于治疗难治性癫痫，特别是儿童难治性癫痫。有关KD的临床研究国外已有七十余年的历史，该方法比较安全、疗效确切。KD的动物实验研究，尤其是其作用机制的研究正成为国外癫痫治疗研究的热点之一，目前国内文献尚未检索到相关的临床或动物实验报道。本研究参照国外相关文献资料，通过对KD饲养的幼鼠采用戊四氮(Pentylenetetrazole，PTZ)诱导痫性发作阈值的测定及点燃后其海马病理改变的观察，探讨和验证KD对实验幼鼠的抗痫作用，同时观察KD对幼鼠痫性发作强度的影响。
材料和方法
健康生后25-30天Wistar幼鼠40只，体重90-120克，雌雄各20只，由沈阳市药品检验所实验动物中心提供。按随机表法则随机分为生酮饮食喂养实验组和常规饮食喂养对照组，每组20只。每只鼠放于一笼单独喂养，实验室温度控制在20℃左右。12小时昼夜亮暗周期。动物禁食12小时后，对照组和生酮饮食组分别饲以普通饲料和生酮饮食饲料。大鼠的普通饲料由沈阳市于洪
乡动物饲料厂提供，符合大鼠生长发育所需的各种营养成视准。
生酮饮食饲料的脂肪主要来源于动物，本实验参照Freeman等的
方案采用的是猪油，按4：二比例＊肪与碳水化合物及蛋白质的
重量比）混合于大鼠普通饮食中（由营养师协助配制人动物自由
饮用自来水。两组幼鼠分别在饲养10天后进行实验观察。通过
尾静脉输注pZuigm。公司生产）来进行化学诱导病性发作。将
300mg pZ溶于30Inl生理盐水中配成新鲜的 PIZ溶液，浓度为
10m4ml。IqZ溶液通过输液泵以恒定的速度即1．ondlnin输人
大鼠尾静脉。PIZ诱导痫性发作所需的阈剂量以大鼠产生双侧前
肢阵挛一强直所需时间为标准进行换算，此时停止输液，记录输注
时间及动物病性发作的行为评分。此后，用PIZ按亚致癌量问7．
sin才g）隔日上午分别给两组动物腹腔注射，直到达到点燃状态。
两组动物均达到点燃状态后，将动物常规麻醉灌流固定，断头取
脑，取海马标本制成光镜切片JE染色。光镜下观察比较海马结
构的细胞形态学改变。分别计数两组动物海马CA3区正常神经
细胞数。各组数据均取平均值，用均数上标准差如土8）表示，两
组间差异用t检验做差异显著性分析。
实验结果
1．动物及饮食情况摄取生酮饮食饲料组的动物在实验过程
中，进食良好，没有不健康迹象。对照组动物体重增长略高于实验
组，即至实验结束时普g饲料对照组体重平均增长约100克，生酮
饮食饲料喂养组平均增长约90克，差异无统计学意义。
2．生酮饮食对病性发作阈值及病性发作强度的影响生酮饮
食饲料喂养10天后，生酮饮食饲料喂养组动物的病性发作阈值明
显提高，两组间经统计学分析具有显著性差异u＜o．01人尽管
生酮饮食可以提高病性发作阈值，延迟PIZ诱导的病性发作产
·2·
生，但它对病性发作的强度没有明显影响。两组动物m诱导痫
性发作强度评分拥J为：实验组二．30。0．19；对照组二．37 S 0．2一
两组间差异无统计学意义瞩＞o．仍人
3．病理形态学观察光镜下生酮饮食喂养组和对照组大鼠的
海马CAI区人A3区及齿状回神经细胞发生明显变性坏死，主要
表现为锥体细胞的固缩性坏死，深染，呈三角形。胞浆染色深，空
泡形成，尼氏小体减少以至消失，细胞核浓缩，深染，核仁不明显。
一部分神经细胞肿胀，呈圆形。胶质细胞水肿。上述改变对照组
较KD组更为明显，程度更严重。两组动物海马CA3区正常锥体
细胞计数显示生酮饮食喂养组数目明显高于对照组，两组间差异
具有统计学意义u功．01人
讨 论
KD最早是由模仿饥饿时产生酮血症状态设计发展而来。传
统的KD（即本实验所采用的KDu肪主要来源于动物，脂肪与蛋
白质加碳水化合物的重量比为4：＊饮食中的脂肪消耗所致酮血
症是KD具有抗惊厥作用的基础。临床和动物实验显示，饲以KD
5—7天后实验对象即可产生稳定的酮血症水平并具有抗惊厥作
用和对痛性发作有保护作用。只要坚持这样的饮食，这种状态就
可以一直保持下去。需要特别提及的是，大量的临床实验证实：
m应用于儿童难治性癫痛显示了更显著更安全的疗效。这是由
于儿童较成人更易获得持久稳定的酮病状态，而大脑利用酮体的
能力随年龄的增加而下降。因此，本研究采用了生后30天大鼠，
饲以KD10天，也收到了较好的实验效果。
选择WZ尾静脉输注法为病性发作的诱导方法，是因其可获
得明确的、显而易见的痛性发作阈值。给予大鼠尾静脉输注册
致惊剂量，一般在给药后数分钟约97％的动物可产生痉挛性抽
·3·
动。p主要作用于脑干及大脑，使兴奋性突触的易化过程增加，
容易发生惊厥，其阈剂量是以引起动物头部及前肢抽动为标准。
本研究发现人D组大鼠痛性发作阈值明显高于对照组动物。
两组间统计学分析具有显著性差异，但两组动物的痛性发作强度
没有明显?
Introduction
The ketogenic diet(KD) is a kind of diet which contains high fat and low protein plus carbohydrate. The clinical studies on the treatment of intractable epilepsy, especially the intractable epilepsy in children by KD were reported 70 years ago. It was considered as a safty and effective method. The animal study on the mechanism of KDs action are becoming the popular topic of epilepsy treatment in the foreign countries.
In our study, in order to explore the anticonvulsant effect of KD on the experimental rats and observe the effect of KD on the seizure intensity, we designd to determinate the PTZ - induced seizure threshold of immatrure rats fed with KD and to observe their morphological changes in the hippocampus consulting the foreign relative document data.
Materials and Methods
We divided 40 healthy 25-30days old Wistar rats into two groups randomly. They are experimental group(KD group) and control group (normal diet group). Every group has 20 naive rats. They were fed with ketogenic diet and normal diet respectively after 12 hours fast. The KD is the mixture of lard and the rodent chow by the ratio of 4:1 (fat:protein plus carbohydrate by weigh). Ten days later,seizures were chemically induced by tail-vein infusion of PTZ( concentration: 10mg/ml;infusion rate: 1.0ml/min). The threshold dose of PTZ( mg/ kg) was derived from the time required to produce the first bilateral myoclonic twitch of the forelimbs and the seizure behaviore were noted for scoring. Then the subepileptogenic dose of PTZ was administered intraperitoneally to the rats every other morning until to the kindling state. All rats were anesthetized and infusion fixing. The hippocampus slides were prepared for HE staining. The morphological changes in hippocampus formation were studied and the normal neuron cells in hippocampus CA3 area were counted.
Results
1. Animals Animals freely consumed the ketogenic diet did not show any signs of ill health. The increased animal body weight of control group was a little more than that of the experimental group.
2. Effects of the ketogenic diet on seizure threshold and the seizure severity Rats fed with the ketogenic diet showed an increasd resistance to seizure and a higher seizure threshold than the control
group animals(p<0.01). But there was no obvious difference of seizure severity between them(P>0.05)
3. Morphological study The neuron cells in hippocampus CA1 CA3area and dental gyrus of all animals showed degenerated and necrosis, and the severity of control group was more obvious than that of the KD group. The counts of normal neuron cells in hippocampus CA3 area of KD group are higher than that of the control group (P<0. 01).
Discussion
The ketogenic diet is designed to induce continuous systemic ke-tosis. It is based on very low carbohydrate and very high fat intake. The classic KD,which was used to control seizures, has a ratio of fat to protein plus carbohydrate of 4:1. The fat is derived from animal sources. Clinical and animal studies had showed that the experimental subjects could produce a steady ketonemia level and had the anticon-vulsant effect after 5-7days on KD. Ketonemia has been showed to result from consumption of high - fat diets. It was the basis of the seizure protection. We should emphasized that many clinical studies had suggested that we can gain more obvious and safty effect in the KD treatment of children intractable epilepsy. Because children had show to develop higher level of ketonemia. So in our study we choosed 30 days old rats and kept them on KD for 10 days.
We had found in our study that the seizure threshold of KD group are higher than that of control group. The difference are stastically significant. But the seizure severity showed no obvious difference. This indicated that KD can elevate the PTZ - induced seizure threshold, but
donot change the seizure severity. Thease findings were also confirmed by Kristophe. J et al. However, some researchers had suggested in their studies that KD had a proconvu
生酮饮食(ketogenic diet，KD)是一种高脂肪、低蛋白质和低碳水化合物的饮食，用于治疗难治性癫痫，特别是儿童难治性癫痫。有关KD的临床研究国外已有七十余年的历史，该方法比较安全、疗效确切。KD的动物实验研究，尤其是其作用机制的研究正成为国外癫痫治疗研究的热点之一，目前国内文献尚未检索到相关的临床或动物实验报道。本研究参照国外相关文献资料，通过对KD饲养的幼鼠采用戊四氮(Pentylenetetrazole，PTZ)诱导痫性发作阈值的测定及点燃后其海马病理改变的观察，探讨和验证KD对实验幼鼠的抗痫作用，同时观察KD对幼鼠痫性发作强度的影响。
材料和方法
健康生后25-30天Wistar幼鼠40只，体重90-120克，雌雄各20只，由沈阳市药品检验所实验动物中心提供。按随机表法则随机分为生酮饮食喂养实验组和常规饮食喂养对照组，每组20只。每只鼠放于一笼单独喂养，实验室温度控制在20℃左右。12小时昼夜亮暗周期。动物禁食12小时后，对照组和生酮饮食组分别饲以普通饲料和生酮饮食饲料。大鼠的普通饲料由沈阳市于洪
乡动物饲料厂提供，符合大鼠生长发育所需的各种营养成视准。
生酮饮食饲料的脂肪主要来源于动物，本实验参照Freeman等的
方案采用的是猪油，按4：二比例＊肪与碳水化合物及蛋白质的
重量比）混合于大鼠普通饮食中（由营养师协助配制人动物自由
饮用自来水。两组幼鼠分别在饲养10天后进行实验观察。通过
尾静脉输注pZuigm。公司生产）来进行化学诱导病性发作。将
300mg pZ溶于30Inl生理盐水中配成新鲜的 PIZ溶液，浓度为
10m4ml。IqZ溶液通过输液泵以恒定的速度即1．ondlnin输人
大鼠尾静脉。PIZ诱导痫性发作所需的阈剂量以大鼠产生双侧前
肢阵挛一强直所需时间为标准进行换算，此时停止输液，记录输注
时间及动物病性发作的行为评分。此后，用PIZ按亚致癌量问7．
sin才g）隔日上午分别给两组动物腹腔注射，直到达到点燃状态。
两组动物均达到点燃状态后，将动物常规麻醉灌流固定，断头取
脑，取海马标本制成光镜切片JE染色。光镜下观察比较海马结
构的细胞形态学改变。分别计数两组动物海马CA3区正常神经
细胞数。各组数据均取平均值，用均数上标准差如土8）表示，两
组间差异用t检验做差异显著性分析。
实验结果
1．动物及饮食情况摄取生酮饮食饲料组的动物在实验过程
中，进食良好，没有不健康迹象。对照组动物体重增长略高于实验
组，即至实验结束时普g饲料对照组体重平均增长约100克，生酮
饮食饲料喂养组平均增长约90克，差异无统计学意义。
2．生酮饮食对病性发作阈值及病性发作强度的影响生酮饮
食饲料喂养10天后，生酮饮食饲料喂养组动物的病性发作阈值明
显提高，两组间经统计学分析具有显著性差异u＜o．01人尽管
生酮饮食可以提高病性发作阈值，延迟PIZ诱导的病性发作产
·2·
生，但它对病性发作的强度没有明显影响。两组动物m诱导痫
性发作强度评分拥J为：实验组二．30。0．19；对照组二．37 S 0．2一
两组间差异无统计学意义瞩＞o．仍人
3．病理形态学观察光镜下生酮饮食喂养组和对照组大鼠的
海马CAI区人A3区及齿状回神经细胞发生明显变性坏死，主要
表现为锥体细胞的固缩性坏死，深染，呈三角形。胞浆染色深，空
泡形成，尼氏小体减少以至消失，细胞核浓缩，深染，核仁不明显。
一部分神经细胞肿胀，呈圆形。胶质细胞水肿。上述改变对照组
较KD组更为明显，程度更严重。两组动物海马CA3区正常锥体
细胞计数显示生酮饮食喂养组数目明显高于对照组，两组间差异
具有统计学意义u功．01人
讨 论
KD最早是由模仿饥饿时产生酮血症状态设计发展而来。传
统的KD（即本实验所采用的KDu肪主要来源于动物，脂肪与蛋
白质加碳水化合物的重量比为4：＊饮食中的脂肪消耗所致酮血
症是KD具有抗惊厥作用的基础。临床和动物实验显示，饲以KD
5—7天后实验对象即可产生稳定的酮血症水平并具有抗惊厥作
用和对痛性发作有保护作用。只要坚持这样的饮食，这种状态就
可以一直保持下去。需要特别提及的是，大量的临床实验证实：
m应用于儿童难治性癫痛显示了更显著更安全的疗效。这是由
于儿童较成人更易获得持久稳定的酮病状态，而大脑利用酮体的
能力随年龄的增加而下降。因此，本研究采用了生后30天大鼠，
饲以KD10天，也收到了较好的实验效果。
选择WZ尾静脉输注法为病性发作的诱导方法，是因其可获
得明确的、显而易见的痛性发作阈值。给予大鼠尾静脉输注册
致惊剂量，一般在给药后数分钟约97％的动物可产生痉挛性抽
·3·
动。p主要作用于脑干及大脑，使兴奋性突触的易化过程增加，
容易发生惊厥，其阈剂量是以引起动物头部及前肢抽动为标准。
本研究发现人D组大鼠痛性发作阈值明显高于对照组动物。
两组间统计学分析具有显著性差异，但两组动物的痛性发作强度
没有明显?
Introduction
The ketogenic diet(KD) is a kind of diet which contains high fat and low protein plus carbohydrate. The clinical studies on the treatment of intractable epilepsy, especially the intractable epilepsy in children by KD were reported 70 years ago. It was considered as a safty and effective method. The animal study on the mechanism of KDs action are becoming the popular topic of epilepsy treatment in the foreign countries.
In our study, in order to explore the anticonvulsant effect of KD on the experimental rats and observe the effect of KD on the seizure intensity, we designd to determinate the PTZ - induced seizure threshold of immatrure rats fed with KD and to observe their morphological changes in the hippocampus consulting the foreign relative document data.
Materials and Methods
We divided 40 healthy 25-30days old Wistar rats into two groups randomly. They are experimental group(KD group) and control group (normal diet group). Every group has 20 naive rats. They were fed with ketogenic diet and normal diet respectively after 12 hours fast. The KD is the mixture of lard and the rodent chow by the ratio of 4:1 (fat:protein plus carbohydrate by weigh). Ten days later,seizures were chemically induced by tail-vein infusion of PTZ( concentration: 10mg/ml;infusion rate: 1.0ml/min). The threshold dose of PTZ( mg/ kg) was derived from the time required to produce the first bilateral myoclonic twitch of the forelimbs and the seizure behaviore were noted for scoring. Then the subepileptogenic dose of PTZ was administered intraperitoneally to the rats every other morning until to the kindling state. All rats were anesthetized and infusion fixing. The hippocampus slides were prepared for HE staining. The morphological changes in hippocampus formation were studied and the normal neuron cells in hippocampus CA3 area were counted.
Results
1. Animals Animals freely consumed the ketogenic diet did not show any signs of ill health. The increased animal body weight of control group was a little more than that of the experimental group.
2. Effects of the ketogenic diet on seizure threshold and the seizure severity Rats fed with the ketogenic diet showed an increasd resistance to seizure and a higher seizure threshold than the control
group animals(p<0.01). But there was no obvious difference of seizure severity between them(P>0.05)
3. Morphological study The neuron cells in hippocampus CA1 CA3area and dental gyrus of all animals showed degenerated and necrosis, and the severity of control group was more obvious than that of the KD group. The counts of normal neuron cells in hippocampus CA3 area of KD group are higher than that of the control group (P<0. 01).
Discussion
The ketogenic diet is designed to induce continuous systemic ke-tosis. It is based on very low carbohydrate and very high fat intake. The classic KD,which was used to control seizures, has a ratio of fat to protein plus carbohydrate of 4:1. The fat is derived from animal sources. Clinical and animal studies had showed that the experimental subjects could produce a steady ketonemia level and had the anticon-vulsant effect after 5-7days on KD. Ketonemia has been showed to result from consumption of high - fat diets. It was the basis of the seizure protection. We should emphasized that many clinical studies had suggested that we can gain more obvious and safty effect in the KD treatment of children intractable epilepsy. Because children had show to develop higher level of ketonemia. So in our study we choosed 30 days old rats and kept them on KD for 10 days.
We had found in our study that the seizure threshold of KD group are higher than that of control group. The difference are stastically significant. But the seizure severity showed no obvious difference. This indicated that KD can elevate the PTZ - induced seizure threshold, but
donot change the seizure severity. Thease findings were also confirmed by Kristophe. J et al. However, some researchers had suggested in their studies that KD had a proconvu
引文
1. Wilder RM. The effects of ketonemia on the course of epilepsy. Mayo Clin Pro 1921,2:307-8
2. Freeman JM. Kelly MT. Freeman JB. The epilepsy diet treatment: an introduction to the ketogenic diet. New York: Demos Vermande, 1996.
3. Bough and Douglas A. Eagles. A ketogenic diet increase the resistance to pentylenetetrazole- induced seizures in the rats. Epilepsia, 1999, Vol(40) 138-142
4. Foote F, Gale K. Proconvulsant effect of morphine on seizures induced by pentylenetrazole in the rat. Eur J Pharm. 1984,105:179-84
5．包新民，舒斯云。大鼠脑立体定位图谱。北京：人民卫生出版社，1991：41-47
6. Huttenlocher P. R, A.J. Wilbourn and J. M. Signore. Medium -chain triglycerides as a therapy for intractable childhood epilepsy. Neurology, 1997,21 (1) :1097-1103.
7. Freeman J. M. , E. P. G. Vining, D. J. Pillas, et al. The efficacy of the ketogenic diet - 1998: A prospective evaluation of intervention in 150 children. Pediatrics, 1998, 102(6):1358-1363.
8. Sirven J. , Whedon B, Caplan D, et al. The ketogenic diet for intractable epilepsy in adults: preliminary results. Epilepsi, 1999, Dec ;40(12): 172-6
9．解学孔。癫痫病学。北京：人民卫生出版社。199，234
10. Patrick J. Matthews, Kristophen J. Bough, Douglas A. A ketogenie diet markedly exacerbate seizure severity but elevates sei-
zure threshold in the same animals. Epilepsia, 1998, Vol, 39 (suppl. 6)
11. Sergei S. Likhodii, musa K, Mendoca A. et al. Dietary fat, ketosis and seizure resistance in rats on the ketogenic diet. Epilepsia, 2000, 4(1) :1400-1410.
12. Thavendiranathan P, Mandonca A, Dell C et al. The MCT ketogenic diet :Effects on animal seizure models. Experimental Neurology, 2000 ,Feb 161,696-703.
13. Uhlemann, E. R. A. H. Neims. Anticonvulsant properties of the ketogenic diet in mice. Pharmacol. Exp. ther, 1982,180: 231-238.
14. Nakazawa. M,S. Kodama,and J. Matsuo. Effects of ketogenic diet on electroconvulsive threshold and brain contents of adenosine nucleotides. Brain Dev, 1983,5 (4): 375-380.
15. Millichap, J. G, J. D. Jones. and B. P. Rudis Mechanism of anticonvulsant avtion of ketogenic diet. Am. J. Dis. Child, 1984 , 107: 593-6O4
16. Appleton, D. B. and D. C. Devivo. An animal mode of the ketogenic diet. Epilepsia , 1974, 15:211-227
17．高旭光，崔莹，胡刚等。海马硬化与癫痫的因果关系探讨。中华神经科杂志，1996，29(4)：214-216
18. Douglas R. Nordli J, and Darryl C. et al. The ketogenic diet revisited:back to the future. Epilepsia, 1997,38(7):743-749.
19. Pan SW, Bebin EM, CHu WJ, et al. Related articles ketosis and epilepsy: 31P spectroscopic imaging at 4. 1T. Epilepsia, 1999,Tun ;40 (6) :703-7.
20. Carl E. Statstrom. Ketogenic diet reduces CA1 excitability in hippocampal slices from kainic acid -treated rats. Epilesia, 1998,
Vil (39). Suppl. 634.
21. Schwartzkroin PA. Related articles Mechanisms underlying the anti - epileptic efficacy of the ketogenic diet. Epilepsy Res, 1999, Dec ;37(3): 171-80. Review.
22. Ordway RW, Singer JJ, Walsh JVJ. Direct regulation of inon channels by fatty acids. Trends Neurosci, 1991,14:96-100.
2. Freeman JM. Kelly MT. Freeman JB. The epilepsy diet treatment: an introduction to the ketogenic diet. New York: Demos Vermande, 1996.
3. Bough and Douglas A. Eagles. A ketogenic diet increase the resistance to pentylenetetrazole- induced seizures in the rats. Epilepsia, 1999, Vol(40) 138-142
4. Foote F, Gale K. Proconvulsant effect of morphine on seizures induced by pentylenetrazole in the rat. Eur J Pharm. 1984,105:179-84
5．包新民，舒斯云。大鼠脑立体定位图谱。北京：人民卫生出版社，1991：41-47
6. Huttenlocher P. R, A.J. Wilbourn and J. M. Signore. Medium -chain triglycerides as a therapy for intractable childhood epilepsy. Neurology, 1997,21 (1) :1097-1103.
7. Freeman J. M. , E. P. G. Vining, D. J. Pillas, et al. The efficacy of the ketogenic diet - 1998: A prospective evaluation of intervention in 150 children. Pediatrics, 1998, 102(6):1358-1363.
8. Sirven J. , Whedon B, Caplan D, et al. The ketogenic diet for intractable epilepsy in adults: preliminary results. Epilepsi, 1999, Dec ;40(12): 172-6
9．解学孔。癫痫病学。北京：人民卫生出版社。199，234
10. Patrick J. Matthews, Kristophen J. Bough, Douglas A. A ketogenie diet markedly exacerbate seizure severity but elevates sei-
zure threshold in the same animals. Epilepsia, 1998, Vol, 39 (suppl. 6)
11. Sergei S. Likhodii, musa K, Mendoca A. et al. Dietary fat, ketosis and seizure resistance in rats on the ketogenic diet. Epilepsia, 2000, 4(1) :1400-1410.
12. Thavendiranathan P, Mandonca A, Dell C et al. The MCT ketogenic diet :Effects on animal seizure models. Experimental Neurology, 2000 ,Feb 161,696-703.
13. Uhlemann, E. R. A. H. Neims. Anticonvulsant properties of the ketogenic diet in mice. Pharmacol. Exp. ther, 1982,180: 231-238.
14. Nakazawa. M,S. Kodama,and J. Matsuo. Effects of ketogenic diet on electroconvulsive threshold and brain contents of adenosine nucleotides. Brain Dev, 1983,5 (4): 375-380.
15. Millichap, J. G, J. D. Jones. and B. P. Rudis Mechanism of anticonvulsant avtion of ketogenic diet. Am. J. Dis. Child, 1984 , 107: 593-6O4
16. Appleton, D. B. and D. C. Devivo. An animal mode of the ketogenic diet. Epilepsia , 1974, 15:211-227
17．高旭光，崔莹，胡刚等。海马硬化与癫痫的因果关系探讨。中华神经科杂志，1996，29(4)：214-216
18. Douglas R. Nordli J, and Darryl C. et al. The ketogenic diet revisited:back to the future. Epilepsia, 1997,38(7):743-749.
19. Pan SW, Bebin EM, CHu WJ, et al. Related articles ketosis and epilepsy: 31P spectroscopic imaging at 4. 1T. Epilepsia, 1999,Tun ;40 (6) :703-7.
20. Carl E. Statstrom. Ketogenic diet reduces CA1 excitability in hippocampal slices from kainic acid -treated rats. Epilesia, 1998,
Vil (39). Suppl. 634.
21. Schwartzkroin PA. Related articles Mechanisms underlying the anti - epileptic efficacy of the ketogenic diet. Epilepsy Res, 1999, Dec ;37(3): 171-80. Review.
22. Ordway RW, Singer JJ, Walsh JVJ. Direct regulation of inon channels by fatty acids. Trends Neurosci, 1991,14:96-100.