肿瘤坏死因子-α对癫癎大鼠海马氨基酸递质的影响
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摘要
目的：肿瘤坏死因子-α作为一个重要的免疫介质参与了癫癇的发生与发展。我们旨在通过探讨肿瘤坏死因子-α对癇性发作程度和持续时间以及对谷氨酸(Glu)和γ-氨基丁酸(GABA)的影响，揭示它在癫癇发病中的具体作用机制。
方法：将24只成年雄性Wistar大鼠(180-230g)随机分为NC组、PTZ组和TNF组，PTZ组和TNF组腹腔注射阈下剂量的PTZ(35mg／kg)，每日一次，直至达到点燃标准，NC组腹腔注射等量生理盐水。点燃后，TNF组经腹腔注入TNF-α(5μg／kg)，动物在注射TNF后2h，再经腹腔内注射PTZ(同上述剂量)诱发癫癇发作；PTZ组经腹腔注入同TNF相同体积的生理盐水，动物在注射生理盐水后2h，再经腹腔内注射PTZ(同上述剂量)诱发癫癎发作。癫癎发作停止后行灌注固定、取材、切片。应用免疫组织化学的方法检测大鼠海马Glu能神经元和GABA能神经元的变化。
结果：1．应用TNF-α后，TNF组的发作潜伏期较PTZ组明显缩短，发作持续时问延长，发作强度增加(P＜0.05)。
2．大鼠海马谷氨酸能神经元平均阳性细胞记数和胞体平均光密度值的改变：PTZ组明显高于NC组(P＜0.01)，TNF组明显高于PTZ组(P＜0.01)和NC组(P＜0.01)。
3．大鼠海马GABA能神经元平均阳性细胞记数和胞体平均光密度值的改变：PTZ组明显低于NC组(P＜0.01)，TNF组明显低于PTZ组(P＜0.01)和NC组(P＜0.01)。
结论：1．TNF-α使癫癎的发作强度明显增加，发作持续时间延长，潜伏期缩短；它明显促进了癫癎的发生与发展。
2．TNF-α主要通过增强海马谷氨酸能神经元的活性和降低GABA能神经元的活性而促进癫癎的发生和发展。
Objectives: Tumor necrosis factor-α is an important immunity medium. It attachs itself to the occurrence and development of epilepsy. We had aimed to explore the effect of tumor necrosis factor-α on epileptic latency, degree, duration and glutamate acid-ergic and γ-aminobutyric acid-ergic neuron of hippocampus in seizures rats induced by PTZ and reveal the possible mechanism of tumor necrosis factor-α in seizure.
Methods: Twenty-four adults wistar rats(180-230g) were randomly divided into three groups, NC group, PTZ group and TNF group. A dose of PTZ(35mg/kg/day) was intraperitoneally injected into the rats of PTZ group and TNF group until the kindling criterion was reached. The rats of NC group were administered with the same dose of normal saline. After kindling, the rats of TNF group were administered intraperitoneally with TNF-α (5 u g/kg),after 2 hours, the above-mentioned same dose of PTZ was administered intraperitoneally and induced seizure; the rats of PTZ group were administered intraperitoneally with the same dose of normal saline, after 2 hours, the above-mentioned same dose of PTZ was administered intraperitoneally and induced seizure. The rats were killed after seizure terminating, and perfused, fixed, drawed the materials, sectioned. To study the influence of TNF-α on glutamate acid-ergic and Y -αminobutyric acid-ergic neuron immunoreactivity by using immunohistochemistry method.
Results: 1.After administering TNF-α .the epileptic latency of TNF group was shorter than PTZ group; the epileptic duration of TNF group was longer than PTZ group; the epileptic degree of TNF group was more reinforced than PTZ group(P<0.05).
2. The number and optical density of glutamate acid-ergic neuron in hippocampus: the PTZ group rats was higher than that of NC group rats (P<0.01); the TNF group rats was higher than that of PTZ group rats(P<0.01) and that of NC group rats (P<0.01).
3. The number and optical density of GABA-ergic neuron in hippocampus:the PTZ group rats was lower than that of NC group rats(P<0.01); The TNF group rats was lower than that of PTZ group rats(P<0.01) and that of NC group rats(P<0.01).
Conclusion: 1. TNF-α reinforced the epileptic degree and shorted the epileptic latency, elongated the epileptic duration: It could obviously accelerate the occurrence and development of epilepsy.
2.TNF-α had obviously reinforced the activity and excitability of glutamate acid-ergic neuron in hippocampus and suppressed the activity and excitability of GABA-ergic neuron in hippocampus. The development and episode of epilepsy were reinforced.
方法：将24只成年雄性Wistar大鼠(180-230g)随机分为NC组、PTZ组和TNF组，PTZ组和TNF组腹腔注射阈下剂量的PTZ(35mg／kg)，每日一次，直至达到点燃标准，NC组腹腔注射等量生理盐水。点燃后，TNF组经腹腔注入TNF-α(5μg／kg)，动物在注射TNF后2h，再经腹腔内注射PTZ(同上述剂量)诱发癫癇发作；PTZ组经腹腔注入同TNF相同体积的生理盐水，动物在注射生理盐水后2h，再经腹腔内注射PTZ(同上述剂量)诱发癫癎发作。癫癎发作停止后行灌注固定、取材、切片。应用免疫组织化学的方法检测大鼠海马Glu能神经元和GABA能神经元的变化。
结果：1．应用TNF-α后，TNF组的发作潜伏期较PTZ组明显缩短，发作持续时问延长，发作强度增加(P＜0.05)。
2．大鼠海马谷氨酸能神经元平均阳性细胞记数和胞体平均光密度值的改变：PTZ组明显高于NC组(P＜0.01)，TNF组明显高于PTZ组(P＜0.01)和NC组(P＜0.01)。
3．大鼠海马GABA能神经元平均阳性细胞记数和胞体平均光密度值的改变：PTZ组明显低于NC组(P＜0.01)，TNF组明显低于PTZ组(P＜0.01)和NC组(P＜0.01)。
结论：1．TNF-α使癫癎的发作强度明显增加，发作持续时间延长，潜伏期缩短；它明显促进了癫癎的发生与发展。
2．TNF-α主要通过增强海马谷氨酸能神经元的活性和降低GABA能神经元的活性而促进癫癎的发生和发展。
Objectives: Tumor necrosis factor-α is an important immunity medium. It attachs itself to the occurrence and development of epilepsy. We had aimed to explore the effect of tumor necrosis factor-α on epileptic latency, degree, duration and glutamate acid-ergic and γ-aminobutyric acid-ergic neuron of hippocampus in seizures rats induced by PTZ and reveal the possible mechanism of tumor necrosis factor-α in seizure.
Methods: Twenty-four adults wistar rats(180-230g) were randomly divided into three groups, NC group, PTZ group and TNF group. A dose of PTZ(35mg/kg/day) was intraperitoneally injected into the rats of PTZ group and TNF group until the kindling criterion was reached. The rats of NC group were administered with the same dose of normal saline. After kindling, the rats of TNF group were administered intraperitoneally with TNF-α (5 u g/kg),after 2 hours, the above-mentioned same dose of PTZ was administered intraperitoneally and induced seizure; the rats of PTZ group were administered intraperitoneally with the same dose of normal saline, after 2 hours, the above-mentioned same dose of PTZ was administered intraperitoneally and induced seizure. The rats were killed after seizure terminating, and perfused, fixed, drawed the materials, sectioned. To study the influence of TNF-α on glutamate acid-ergic and Y -αminobutyric acid-ergic neuron immunoreactivity by using immunohistochemistry method.
Results: 1.After administering TNF-α .the epileptic latency of TNF group was shorter than PTZ group; the epileptic duration of TNF group was longer than PTZ group; the epileptic degree of TNF group was more reinforced than PTZ group(P<0.05).
2. The number and optical density of glutamate acid-ergic neuron in hippocampus: the PTZ group rats was higher than that of NC group rats (P<0.01); the TNF group rats was higher than that of PTZ group rats(P<0.01) and that of NC group rats (P<0.01).
3. The number and optical density of GABA-ergic neuron in hippocampus:the PTZ group rats was lower than that of NC group rats(P<0.01); The TNF group rats was lower than that of PTZ group rats(P<0.01) and that of NC group rats(P<0.01).
Conclusion: 1. TNF-α reinforced the epileptic degree and shorted the epileptic latency, elongated the epileptic duration: It could obviously accelerate the occurrence and development of epilepsy.
2.TNF-α had obviously reinforced the activity and excitability of glutamate acid-ergic neuron in hippocampus and suppressed the activity and excitability of GABA-ergic neuron in hippocampus. The development and episode of epilepsy were reinforced.
引文
1. Bostantjopoulou S,Hatzizisi O,Argyopoulou O,et al. Immunological parameters in patients with epilepsy. Funct Neurol, 1994,9(1): 11-15.
2. Vezzani A,Conti M,De Luigi M,et al. Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures. J Neurosci, 1999, 19(12):5054-5065.
3. Vezzani A,Moneta D,Conti M,et al. Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice. Proc Natl Acad Sci USA, 2000, 97(21):11534-11539.
4. Liu ZS,Wang QW, Wang FL,et al.Serum cytokine levels are altered in patients with West syndrome. Brain Dev, 2001, 23(7):548-551
5. Bartolomei F, Boucraut J,Barrie M,et al. Cryptogenic partial epilepsies with anti-GM1 antibodies: a new form of immune-mediated epilepsy? Epilepsia, 1996,37(10):922-926;
6. Dambinova SA,Izykenova GA,Burov SV, et al. The presence of autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptor in the blood serum of patients with epilepsy.J Neurol Sci, 1997,152(1):93-97;
7. Dambinova SA,Ganstrem OK,Tourov A,et al. Monitoring of brain spiking activity and autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptors in blood serum of rats with cobalt-induced epilepsy. J Neurochem, 1998,71 (5):2088-2093.
8. Aarli JA. Epilepsy and the immune system. Arch Neurology, 2000, 57(12): 1689-1692
9. F Villani GA.The use of immunoglobulins in the treatment of human epilepsy.Neurol Sci.2002,(23):S33-S37.
10. Watkins, Maier SF, Goehler LE, et al. Cytokin-brain communication: a review and analysis of alternative mechanisms. [J] Life sci. 1995,57:1011-1026.
11. Fleshner M, Goehler LE, Schwwartz BA, et al. Thermogenic and corticosterone responses to intravenous IL-1 β and TNF-α are attenuated by subdiaphragamatic vagotomy. [J] J Neuroimmunol. 1998,86:134-141.
12．成祥林，张晓琴．肿瘤坏死因子-a与癫癎．国外医学·神经病学神经外科学分册，2003，30(3)：278-280．
13. Mittelman A,Puccio C,Cafney E,et al.A phase Ⅰ pharmacokinetic study of recombinant human tumor necrosis factor administered by a 5-day continuous infusion.Invest New Drugs.1992,10:183-190.
14．毕国荣，卢丽萍，丁雁，等．癫癎患者血浆及脑脊液中肿瘤坏死因子含量测定及其意义．临床神经病学杂志．1999，6：350-351．
15．杜静，方敏，龚非力，等．癫痫患者脑组织TNF-α mRNA，IL-6mRNA以及HLA-Ⅱ抗原表达的改变．同济医科大学学报．2000，2：111-113．
16. Shandra A.A, Godlevshy LS, Vastyanov KS,et al. The role of TNF-α in amygdala kindled rats. [J] Neurosci Res. 2002,42:147-153.
17. Probert L, Akassoglou K, Pasparakis M,et al. Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor α. [J] Proc Nat Acad Sci U.S.A. 1995,92:11294-11298.
18. Plata-Salaman CR, Ilyin SE, Turrin NP, et al. Kindling modulates the IL-1beta system, TNF-alpha, TGF-betal , and neuropeptide mRNA in specific brain regions. [J] Brain Res Mol Brain Res. 2000,75:248-258.
19. De Simoni MG, Perego C, Ravizza T,et al. Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus. [J] Eur J Neurosci. 2000,12(7):2623-2633.
20. De Bock F, Dornand J, Rondouin G,et al. Release of TNF-alpha in the rat hippocampus following epileptic seizures and excitotoxic neuronal damage. [J] Neuroreport. 1996,7:1125-1129.
21. Bruce AJ, Boling W, Kindy MS,et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. [J] Nature. 1996,2:788-794.
22. Diehl RG, Smialowski A, Gotwo T. Development and persistence of kindled seizures after repeated injections of pentylenetetrazol in rats and guinea pigs. Epilepsia, 1984,25(4): 506-5.10.
23. Yuhas Y. Shulman L, Weizman A,et al. Involvement of tumor necrosis factor alpha and interleukin-1 in enhancement of pentylenetetrazole induced seizures caused by Shigella dysenteriae. [J] Infect Immun. 1999,67(3):1455-1460.
24. Sorkin LS, Xiao WH, Wanger R,et al.Tumor necrosis factor alpha induces ctopic activity in nocieptove primary afferent fibers.Neuroscience. 1997,81 ;255.
25. Sloviter RS.The functional organization of the hippocampsl dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy.Ann Neurol. 1994,35(6):640-54.
26. Bradford HF. Glutamate, GABA and epilepsy. Progress in Neurobio, 1995,47:477-511.
27. Chao-CC,Hu S. Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures. Deve Neurosci. 1994,16(3-4): 172-179.
28. Liao SL, Chen C . Differential effects of cytokines and redox potential on glutamate uptake in rat cortical glial cultures. Neurosci Lett. 2001,299(1-2): 113-116.
29. Hu S,Sheng WS,Ehrlich LC,et al. Cytokine effects on glutamate uptake by human astrocytes. [J] Neuroimmunomodulation, 2000, 7(3): 153-9.
30．赵珠峰、刘庆莹、朱长庚．TNFα激活的星形胶质细胞在慢性癫癎复发中的作用．解剖学报．2003，34(1)：21-26．
31. Bureau M,Laschet J,Minier F, et al.Intervention of GABAergic neurotransmission in partial epilepsies.Rev Neurol(Paris). 1997,153 suppl 1 :S46-54.
32. De Laurentiis A,Pisera D,Lasaga M,et al. Effect of interleukin-6 and tumor necrosis factor-alpha on GABA release from mediobasal hypothalamus and posterior pituitary. Neuroimmunomodulation. 2000, 7(2): 77-83.
2. Vezzani A,Conti M,De Luigi M,et al. Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures. J Neurosci, 1999, 19(12):5054-5065.
3. Vezzani A,Moneta D,Conti M,et al. Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice. Proc Natl Acad Sci USA, 2000, 97(21):11534-11539.
4. Liu ZS,Wang QW, Wang FL,et al.Serum cytokine levels are altered in patients with West syndrome. Brain Dev, 2001, 23(7):548-551
5. Bartolomei F, Boucraut J,Barrie M,et al. Cryptogenic partial epilepsies with anti-GM1 antibodies: a new form of immune-mediated epilepsy? Epilepsia, 1996,37(10):922-926;
6. Dambinova SA,Izykenova GA,Burov SV, et al. The presence of autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptor in the blood serum of patients with epilepsy.J Neurol Sci, 1997,152(1):93-97;
7. Dambinova SA,Ganstrem OK,Tourov A,et al. Monitoring of brain spiking activity and autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptors in blood serum of rats with cobalt-induced epilepsy. J Neurochem, 1998,71 (5):2088-2093.
8. Aarli JA. Epilepsy and the immune system. Arch Neurology, 2000, 57(12): 1689-1692
9. F Villani GA.The use of immunoglobulins in the treatment of human epilepsy.Neurol Sci.2002,(23):S33-S37.
10. Watkins, Maier SF, Goehler LE, et al. Cytokin-brain communication: a review and analysis of alternative mechanisms. [J] Life sci. 1995,57:1011-1026.
11. Fleshner M, Goehler LE, Schwwartz BA, et al. Thermogenic and corticosterone responses to intravenous IL-1 β and TNF-α are attenuated by subdiaphragamatic vagotomy. [J] J Neuroimmunol. 1998,86:134-141.
12．成祥林，张晓琴．肿瘤坏死因子-a与癫癎．国外医学·神经病学神经外科学分册，2003，30(3)：278-280．
13. Mittelman A,Puccio C,Cafney E,et al.A phase Ⅰ pharmacokinetic study of recombinant human tumor necrosis factor administered by a 5-day continuous infusion.Invest New Drugs.1992,10:183-190.
14．毕国荣，卢丽萍，丁雁，等．癫癎患者血浆及脑脊液中肿瘤坏死因子含量测定及其意义．临床神经病学杂志．1999，6：350-351．
15．杜静，方敏，龚非力，等．癫痫患者脑组织TNF-α mRNA，IL-6mRNA以及HLA-Ⅱ抗原表达的改变．同济医科大学学报．2000，2：111-113．
16. Shandra A.A, Godlevshy LS, Vastyanov KS,et al. The role of TNF-α in amygdala kindled rats. [J] Neurosci Res. 2002,42:147-153.
17. Probert L, Akassoglou K, Pasparakis M,et al. Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor α. [J] Proc Nat Acad Sci U.S.A. 1995,92:11294-11298.
18. Plata-Salaman CR, Ilyin SE, Turrin NP, et al. Kindling modulates the IL-1beta system, TNF-alpha, TGF-betal , and neuropeptide mRNA in specific brain regions. [J] Brain Res Mol Brain Res. 2000,75:248-258.
19. De Simoni MG, Perego C, Ravizza T,et al. Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus. [J] Eur J Neurosci. 2000,12(7):2623-2633.
20. De Bock F, Dornand J, Rondouin G,et al. Release of TNF-alpha in the rat hippocampus following epileptic seizures and excitotoxic neuronal damage. [J] Neuroreport. 1996,7:1125-1129.
21. Bruce AJ, Boling W, Kindy MS,et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. [J] Nature. 1996,2:788-794.
22. Diehl RG, Smialowski A, Gotwo T. Development and persistence of kindled seizures after repeated injections of pentylenetetrazol in rats and guinea pigs. Epilepsia, 1984,25(4): 506-5.10.
23. Yuhas Y. Shulman L, Weizman A,et al. Involvement of tumor necrosis factor alpha and interleukin-1 in enhancement of pentylenetetrazole induced seizures caused by Shigella dysenteriae. [J] Infect Immun. 1999,67(3):1455-1460.
24. Sorkin LS, Xiao WH, Wanger R,et al.Tumor necrosis factor alpha induces ctopic activity in nocieptove primary afferent fibers.Neuroscience. 1997,81 ;255.
25. Sloviter RS.The functional organization of the hippocampsl dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy.Ann Neurol. 1994,35(6):640-54.
26. Bradford HF. Glutamate, GABA and epilepsy. Progress in Neurobio, 1995,47:477-511.
27. Chao-CC,Hu S. Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures. Deve Neurosci. 1994,16(3-4): 172-179.
28. Liao SL, Chen C . Differential effects of cytokines and redox potential on glutamate uptake in rat cortical glial cultures. Neurosci Lett. 2001,299(1-2): 113-116.
29. Hu S,Sheng WS,Ehrlich LC,et al. Cytokine effects on glutamate uptake by human astrocytes. [J] Neuroimmunomodulation, 2000, 7(3): 153-9.
30．赵珠峰、刘庆莹、朱长庚．TNFα激活的星形胶质细胞在慢性癫癎复发中的作用．解剖学报．2003，34(1)：21-26．
31. Bureau M,Laschet J,Minier F, et al.Intervention of GABAergic neurotransmission in partial epilepsies.Rev Neurol(Paris). 1997,153 suppl 1 :S46-54.
32. De Laurentiis A,Pisera D,Lasaga M,et al. Effect of interleukin-6 and tumor necrosis factor-alpha on GABA release from mediobasal hypothalamus and posterior pituitary. Neuroimmunomodulation. 2000, 7(2): 77-83.