托吡酯对硝酸甘油致大鼠偏头痛模型血浆和脑组织中水通道蛋白1表达的影响
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摘要
目的
研究硝酸甘油致大鼠偏头痛模型血浆和脑组织中水通道蛋白1(AQP1)的表达,并研究托吡酯(TPM)对此表达的影响。
方法
SD大鼠50只,随机分成3组,对照组10只,模型组25只,治疗组15只。对照组皮下注射生理盐水,4小时后灌胃生理盐水2ml/kg;其余大鼠均皮下注射硝酸甘油注射剂10mg/kg,建立实验性偏头痛模型,造模成功后随机分为两组,模型组每日相同时间皮下注射硝酸甘油注射剂,4小时后灌胃生理盐水,共7d；治疗组每日相同时间皮下注射硝酸甘油注射剂,4小时后灌胃TPM 7d。各组末次处理4h后,将动物处死,取血和脑组织备用。实验结束时统计每组大鼠的存活数,应用酶联免疫吸附测定(ELISA)检测血浆中的AQP1浓度,脑组织切片进行HE染色及AQP1免疫组化染色检测,免疫组化染色切片经NIS-ELEMENTS F系统采集图像,MetaMorph offline软件系统测量平均积分光密度(IOD)值。
结果
1、三组存活数：对照组存活10只,模型组25只存活10只,治疗组15只存活10只。模型组与治疗组的存活数比较有统计学差异(p<0.05),
2、ELISA检测结果：对照组AQP1的浓度明显低于模型组和治疗组(p<0.05),模型组与治疗组的AQP1浓度无明显差异(p>0.05),
3、HE染色：三组未见异常病变,也未见明显差异,
4、AQP1免疫组化检测：阳性细胞为在细胞膜上和细胞浆中有棕黄色表达,通过测量平均IOD值比较：模型组和治疗组脑组织的AQP1的免疫组化IOD值高于对照组,有统计学差异(p<0.05),模型组脑组织中的AQP1平均IOD值稍高于治疗组,但统计学上无明显差异(p>0.05)。
结论
AQP1在硝酸甘油致偏头痛大鼠模型血浆和脑组织中的表达增加,提示其在偏头痛的发病机制中可能发挥着重要的作用,而TPM是否通过影响AQP1的表达来治疗偏头痛不能确定。
Objective
To investigate the expression of aquaporin 1 (AQP1) in blood plasma and cerebral tissue from rat model of migraine with nitroglycerin and effect of topiramate (TPM) on the expression.
Method
50 SD rats were selected and randomly divided into 3 groups:control group:10 rats, modelset group:25 rats, treatment group:15rats. From the first day, rats of control group were given Normal Saline(2ml/kg) by hypodermic injection and given Normal Saline(2ml/kg) by lavage after 4 hour (to eliminate the effect to rats of hypodermic injection and lavage) from 1st day to 7th day; rats of modelset group were given nitroglycerin injection (10mg/kg) by hypodermic injection and were given Normal saline(2ml/kg) by lavage after 4 hour from 1st day to 7th day; rats of treatment group were given nitroglycerin injection (10mg/kg) by hypodermic injection and then were given topiramate (50mg/kg) by lavage after 4 hour from 1st day to 7th day. At 7th day, rats of each group were narcotized by 10%chloral hydrate at 4 hours after the last respective operation,then get blood and cerebral tissue, to measure concentration in blood and to observe histological change by light microscope and to measure values of average Integrated Optical Density (IOD) for AQP1 by immunohistochemical stains.
Result
1、survival of the rats:10 rats of control group survived,10 rats in 25 of modelset group survived,10 rats in 15 of treatment group survived, the difference of survival between modelset group and treatment group has statistical difference (P<0.05).
2、results of AQP1 from ELISA:modelset group and treatment group show no statistical difference (P>0.05), control group and modelset group or treatment group both show statistical difference (P<0.05).
3、results from HE stains:there were no abnormal changes obviously, and no obvious difference among groups.
4、expression of AQP1 by immunohistochemical stains in cerebral tissue:average IOD value of control group show lower than that of modelset group or treatment group and both show statistical difference (P<0.05), modelset group show slightly stronger than treatment group,but show no statistical difference (P>0.05).
Conclusion
In the attack of rat model of migraine with nitroglycerin, AQP1 may partake in the attack of migraine, but it cannot conclude that TPM maybe has effect on the expression of AQP1 as to be one of mechanisms of treatment for topiramate to migraine.
研究硝酸甘油致大鼠偏头痛模型血浆和脑组织中水通道蛋白1(AQP1)的表达,并研究托吡酯(TPM)对此表达的影响。
方法
SD大鼠50只,随机分成3组,对照组10只,模型组25只,治疗组15只。对照组皮下注射生理盐水,4小时后灌胃生理盐水2ml/kg;其余大鼠均皮下注射硝酸甘油注射剂10mg/kg,建立实验性偏头痛模型,造模成功后随机分为两组,模型组每日相同时间皮下注射硝酸甘油注射剂,4小时后灌胃生理盐水,共7d；治疗组每日相同时间皮下注射硝酸甘油注射剂,4小时后灌胃TPM 7d。各组末次处理4h后,将动物处死,取血和脑组织备用。实验结束时统计每组大鼠的存活数,应用酶联免疫吸附测定(ELISA)检测血浆中的AQP1浓度,脑组织切片进行HE染色及AQP1免疫组化染色检测,免疫组化染色切片经NIS-ELEMENTS F系统采集图像,MetaMorph offline软件系统测量平均积分光密度(IOD)值。
结果
1、三组存活数：对照组存活10只,模型组25只存活10只,治疗组15只存活10只。模型组与治疗组的存活数比较有统计学差异(p<0.05),
2、ELISA检测结果：对照组AQP1的浓度明显低于模型组和治疗组(p<0.05),模型组与治疗组的AQP1浓度无明显差异(p>0.05),
3、HE染色：三组未见异常病变,也未见明显差异,
4、AQP1免疫组化检测：阳性细胞为在细胞膜上和细胞浆中有棕黄色表达,通过测量平均IOD值比较：模型组和治疗组脑组织的AQP1的免疫组化IOD值高于对照组,有统计学差异(p<0.05),模型组脑组织中的AQP1平均IOD值稍高于治疗组,但统计学上无明显差异(p>0.05)。
结论
AQP1在硝酸甘油致偏头痛大鼠模型血浆和脑组织中的表达增加,提示其在偏头痛的发病机制中可能发挥着重要的作用,而TPM是否通过影响AQP1的表达来治疗偏头痛不能确定。
Objective
To investigate the expression of aquaporin 1 (AQP1) in blood plasma and cerebral tissue from rat model of migraine with nitroglycerin and effect of topiramate (TPM) on the expression.
Method
50 SD rats were selected and randomly divided into 3 groups:control group:10 rats, modelset group:25 rats, treatment group:15rats. From the first day, rats of control group were given Normal Saline(2ml/kg) by hypodermic injection and given Normal Saline(2ml/kg) by lavage after 4 hour (to eliminate the effect to rats of hypodermic injection and lavage) from 1st day to 7th day; rats of modelset group were given nitroglycerin injection (10mg/kg) by hypodermic injection and were given Normal saline(2ml/kg) by lavage after 4 hour from 1st day to 7th day; rats of treatment group were given nitroglycerin injection (10mg/kg) by hypodermic injection and then were given topiramate (50mg/kg) by lavage after 4 hour from 1st day to 7th day. At 7th day, rats of each group were narcotized by 10%chloral hydrate at 4 hours after the last respective operation,then get blood and cerebral tissue, to measure concentration in blood and to observe histological change by light microscope and to measure values of average Integrated Optical Density (IOD) for AQP1 by immunohistochemical stains.
Result
1、survival of the rats:10 rats of control group survived,10 rats in 25 of modelset group survived,10 rats in 15 of treatment group survived, the difference of survival between modelset group and treatment group has statistical difference (P<0.05).
2、results of AQP1 from ELISA:modelset group and treatment group show no statistical difference (P>0.05), control group and modelset group or treatment group both show statistical difference (P<0.05).
3、results from HE stains:there were no abnormal changes obviously, and no obvious difference among groups.
4、expression of AQP1 by immunohistochemical stains in cerebral tissue:average IOD value of control group show lower than that of modelset group or treatment group and both show statistical difference (P<0.05), modelset group show slightly stronger than treatment group,but show no statistical difference (P>0.05).
Conclusion
In the attack of rat model of migraine with nitroglycerin, AQP1 may partake in the attack of migraine, but it cannot conclude that TPM maybe has effect on the expression of AQP1 as to be one of mechanisms of treatment for topiramate to migraine.
引文
1 杜艳芬,王纪佐.偏头痛发病机制研究进展.中国临床神经科学,2002；10(3)：314-317.
2 Read SJ, Manning P, McNeil, et al. Effects of sumatriptan on nitrie oxide and superoxide balance during glyceryl trinitrate infusion in the rat:Implications for antimigraine mechanismsn. Brain Research,1999; 847(1):1-8.
3 程波,蒋静.水通道蛋白亚型AQP1的研究进展.实用医学杂志,2008；24(2)：317-318.
4 Oshio K, Watanabe H, Yan D, et al. Impaired pain sensation in mice lacking Aquaporin-1 water channels. Headache,2007; 47(10):1457-8.
5 Adelman J, Freitag FG, Lainez M, et al. Analysis of safety and tolerability data obtained from over 1,500 patients receiving topiramate for migraine prevention in controlled trials. Pain Med, 2008; 9(2):175-185.
6 周永红,王新陆,胡怀强等.硝酸甘油型实验性偏头痛大鼠模型建立与评价.中国神经免疫学和神经病杂志,2005；12(2)：116-117.
7 Zhou S, Sun X, Liu L, et al. Increased expression of aquaporin-1 in the anterior temporal neocortex of patients with intractable epilepsy. Neurol Res,2008; 30(4):400-5.
8 Shields SD, Mazario J, Skinner K, et al. Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons. Pain.2007; 131:8-20.
9 Wakayama Y, Inoue M, Takahashi J. Aquaporin 1 may be involved in the pathophysiology of migraine:a hypothesis. Headache.2007 Nov-Dec; 47(10):1457-8.
10 Silberstein S D, Neto W, Schmitt J, et al. Topiramate in migraine prevention, results of a large controlled trial [J]. A rch Neurol,2004,61 (4):490-495.
11 Brandes J L, Saper J R, Diamond M, et al. Topiramate for migraine prevention:a randomized controlled trial[J]. JAMA,2004,291 (8):965-973.
12 Trojnar MK, Malek R, Chroscinska M, et al. Neuroprotective efects of anticpileptic drugs, Pol J Pharmacol,2002,54(6):557-566.
13 Ma T,Yang B,Verkman AS. Cloning of a novel water and urea permeable aquaporin f rom mouse expressed st rongly in colon, placenta, liver, and heart [J]. Biochem Biophys Res Commun,1997,240 (2):324-328
1 Magni F, Chinello C, Raimondo F. AQP1 expression analysis in human diseases:implications for proteomic characterization. Expert Rev Proteomics.2008 Feb; 5(1):29-43.
2 Yool AJ. Aquaporins:multiple roles in the central nervous system Neuroscientist.2007 Oct; 13(5):470-85.
3 Oshio K, Watanabe H, Yan D, et al. Impaired pain sensation in mice lacking Aquaporin-1 water channels. Manley GT Headache.2007 Nov-Dec; 47(10):1457-8.
4 Dolman D, Drndarski S, Abbott NJ, et al. Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture. Neurochem. 2005 May; 93(4):825-33.
5 Yool AJ. Functional domains of aquaporin-1:keys to physiology, and targets for drug discovery. Curr Pharm Des.2007; 13(31):3212-21.
6 Boassa D, Stamer WD, Yool AJ. Ion channel function of aquaporin-1 natively expressed in choroid plexus. Biochem Biophys Res Commun.2006 Mar 24; 341(4):1022-8. Epub 2006 Jan 25.
7 Zhou S, Sun X, Liu L, Wang X et al. Increased expression of aquaporin-1 in the anterior temporal neocortex of patients with intractable epilepsy. Neurol Res.2008 May; 30(4): 400-5.
8 Rodriguez A, Perez-Gracia E, Espinosa JC, et al. Increased expression of water channel aquaporin 1 and aquaporin 4 in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP transgenic mice, Acta Neuropathol.2006 Nov; 112(5): 573-85.
9 Costa C, Tortosa R, Rodriguez A, et al. Aquaporin 1 and aquaporin 4 overexpression in bovine spongiform encephalopathy in a transgenic murine model and in cattle field cases. Pumarola M. Brain Res.2007 Oct 17; 1175:96-106. Epub 2007 Jul 27.
10 Ma T, Yang B, Verkman AS. Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun, 1997,240 (2):324-328.
11 Shields SD, Mazario J, Skinner K, et al. Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons. Pain.2007; 131:8-20.
12刘军综述.水通道蛋白与脑水肿,国际检验医学杂志2006,27(11)：999-1000.
13 Wakayama Y, Inoue M, Takahashi J. Aquaporin 1 may be involved in the pathophysiology of migraine:a hypothesis. Headache.2007 Nov-Dec; 47(10):1457-8.
14 Lewis D, Ashwal S, Hershey A, Hirtz D, et al. Practice parameter:pharmacological treatment of migraine headache in children and adolescents:report of the American Academy of Neurology Quality Standards Subcommittee and the Practice Committee of the Child Neurology Society. Neurology,2004,63(12):2215-2224.
15 Silberstein S D, Neto W, Schmitt J, et al. Topiramate in migraine prevention, results of a large controlled trial. A rch Neurol,2004,61 (4):490-495.
16 Brandes J L, Saper J R, Diamond M, et al. Topiramate for migraine prevention:a randomized controlled trial. JAMA,2004,291 (8):965-973.
17 Evers S, Afra J, Frese A, et al. EFNS guideline on the drug treatment of migraine-report of an EFNS task force. Eur J Neurol,2006,13 (6):560-572.
18 Arvio M, Sillanpaa M. Topiramate in long-term treatment of epilepsy in the intellectually disabled. J Intellect Disabil Res,2005,49(Pt 3):183-189.
19 Campistol J, Campos J, Casas C, et al. Topiramate in the prophylactic treatmemt of migraine in children. J child Neurol,2005,20(3)251-253.
20 肖波,张进,章蓓.新型抗癫痫药妥泰的研究近况.国外医学·神经病学神经外科学分册,2000,27(6)：296-299.
21 Gibbs JW, Sombati S, DeLorenzo RJ, et al. Cellular actions of topiramate:blockade of kainate evoked inward currents in cultured hippocmnpal neurons. Epilepsia.2000,41(Suppl1): S10-S16.
22 Angehagen M, Ben Menachem E, Ronnback L, et al. Topiramate protects against glutamate-and kainate-induced neurotoxicity in primary neumnalastrecial cultures. Epilepsy Res.2003,54(1):63-71.
23 Maragakis NJ, Jackson M, Ganel R, et al. Topiramate protects against motor neuron degeneration in organotypic spinal cord cultures but not in G93A SOD1 transgenic mice. Neumsei Lett,2003,338(2):107-110.
24陈春富综述,托吡酯神经保护作用研究进展,国际神经病学神经外科学杂志2006,33(2)：149-152.
25 Kubera M, Budziszewska B, Jaworska Feil L, et al. Effect of topiramate on the kainate-induced status epilepticus, lipid peroxidation and immunoreactivity of rats. Pol J Pharm acol.2004,56(5):553-561.
26 Leniger T, Thane J, Wiemann M. Topiramate modulates pH of hippocampal CA3 neurons by combined effects on carbonic anhydrase and C1-/HCO3-exchange. Br J Pharmacol,2004, 142(5):831-842.
27 Artemowicz B, Sobaniec W. Ncuroprotection possibilities in epieptic children. Rocz Akad Med Bialymst,2005,50(Suppl1):s91-s95.
28 Kudin AP, Debska-Viehaber G, Vielhaker S, et al. The mechanism of neuroprotection by topiramate in an animal model of epilepsy. Epilepsia.2004 Dec; 45(12):1478-87.
2 Read SJ, Manning P, McNeil, et al. Effects of sumatriptan on nitrie oxide and superoxide balance during glyceryl trinitrate infusion in the rat:Implications for antimigraine mechanismsn. Brain Research,1999; 847(1):1-8.
3 程波,蒋静.水通道蛋白亚型AQP1的研究进展.实用医学杂志,2008；24(2)：317-318.
4 Oshio K, Watanabe H, Yan D, et al. Impaired pain sensation in mice lacking Aquaporin-1 water channels. Headache,2007; 47(10):1457-8.
5 Adelman J, Freitag FG, Lainez M, et al. Analysis of safety and tolerability data obtained from over 1,500 patients receiving topiramate for migraine prevention in controlled trials. Pain Med, 2008; 9(2):175-185.
6 周永红,王新陆,胡怀强等.硝酸甘油型实验性偏头痛大鼠模型建立与评价.中国神经免疫学和神经病杂志,2005；12(2)：116-117.
7 Zhou S, Sun X, Liu L, et al. Increased expression of aquaporin-1 in the anterior temporal neocortex of patients with intractable epilepsy. Neurol Res,2008; 30(4):400-5.
8 Shields SD, Mazario J, Skinner K, et al. Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons. Pain.2007; 131:8-20.
9 Wakayama Y, Inoue M, Takahashi J. Aquaporin 1 may be involved in the pathophysiology of migraine:a hypothesis. Headache.2007 Nov-Dec; 47(10):1457-8.
10 Silberstein S D, Neto W, Schmitt J, et al. Topiramate in migraine prevention, results of a large controlled trial [J]. A rch Neurol,2004,61 (4):490-495.
11 Brandes J L, Saper J R, Diamond M, et al. Topiramate for migraine prevention:a randomized controlled trial[J]. JAMA,2004,291 (8):965-973.
12 Trojnar MK, Malek R, Chroscinska M, et al. Neuroprotective efects of anticpileptic drugs, Pol J Pharmacol,2002,54(6):557-566.
13 Ma T,Yang B,Verkman AS. Cloning of a novel water and urea permeable aquaporin f rom mouse expressed st rongly in colon, placenta, liver, and heart [J]. Biochem Biophys Res Commun,1997,240 (2):324-328
1 Magni F, Chinello C, Raimondo F. AQP1 expression analysis in human diseases:implications for proteomic characterization. Expert Rev Proteomics.2008 Feb; 5(1):29-43.
2 Yool AJ. Aquaporins:multiple roles in the central nervous system Neuroscientist.2007 Oct; 13(5):470-85.
3 Oshio K, Watanabe H, Yan D, et al. Impaired pain sensation in mice lacking Aquaporin-1 water channels. Manley GT Headache.2007 Nov-Dec; 47(10):1457-8.
4 Dolman D, Drndarski S, Abbott NJ, et al. Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture. Neurochem. 2005 May; 93(4):825-33.
5 Yool AJ. Functional domains of aquaporin-1:keys to physiology, and targets for drug discovery. Curr Pharm Des.2007; 13(31):3212-21.
6 Boassa D, Stamer WD, Yool AJ. Ion channel function of aquaporin-1 natively expressed in choroid plexus. Biochem Biophys Res Commun.2006 Mar 24; 341(4):1022-8. Epub 2006 Jan 25.
7 Zhou S, Sun X, Liu L, Wang X et al. Increased expression of aquaporin-1 in the anterior temporal neocortex of patients with intractable epilepsy. Neurol Res.2008 May; 30(4): 400-5.
8 Rodriguez A, Perez-Gracia E, Espinosa JC, et al. Increased expression of water channel aquaporin 1 and aquaporin 4 in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP transgenic mice, Acta Neuropathol.2006 Nov; 112(5): 573-85.
9 Costa C, Tortosa R, Rodriguez A, et al. Aquaporin 1 and aquaporin 4 overexpression in bovine spongiform encephalopathy in a transgenic murine model and in cattle field cases. Pumarola M. Brain Res.2007 Oct 17; 1175:96-106. Epub 2007 Jul 27.
10 Ma T, Yang B, Verkman AS. Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun, 1997,240 (2):324-328.
11 Shields SD, Mazario J, Skinner K, et al. Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons. Pain.2007; 131:8-20.
12刘军综述.水通道蛋白与脑水肿,国际检验医学杂志2006,27(11)：999-1000.
13 Wakayama Y, Inoue M, Takahashi J. Aquaporin 1 may be involved in the pathophysiology of migraine:a hypothesis. Headache.2007 Nov-Dec; 47(10):1457-8.
14 Lewis D, Ashwal S, Hershey A, Hirtz D, et al. Practice parameter:pharmacological treatment of migraine headache in children and adolescents:report of the American Academy of Neurology Quality Standards Subcommittee and the Practice Committee of the Child Neurology Society. Neurology,2004,63(12):2215-2224.
15 Silberstein S D, Neto W, Schmitt J, et al. Topiramate in migraine prevention, results of a large controlled trial. A rch Neurol,2004,61 (4):490-495.
16 Brandes J L, Saper J R, Diamond M, et al. Topiramate for migraine prevention:a randomized controlled trial. JAMA,2004,291 (8):965-973.
17 Evers S, Afra J, Frese A, et al. EFNS guideline on the drug treatment of migraine-report of an EFNS task force. Eur J Neurol,2006,13 (6):560-572.
18 Arvio M, Sillanpaa M. Topiramate in long-term treatment of epilepsy in the intellectually disabled. J Intellect Disabil Res,2005,49(Pt 3):183-189.
19 Campistol J, Campos J, Casas C, et al. Topiramate in the prophylactic treatmemt of migraine in children. J child Neurol,2005,20(3)251-253.
20 肖波,张进,章蓓.新型抗癫痫药妥泰的研究近况.国外医学·神经病学神经外科学分册,2000,27(6)：296-299.
21 Gibbs JW, Sombati S, DeLorenzo RJ, et al. Cellular actions of topiramate:blockade of kainate evoked inward currents in cultured hippocmnpal neurons. Epilepsia.2000,41(Suppl1): S10-S16.
22 Angehagen M, Ben Menachem E, Ronnback L, et al. Topiramate protects against glutamate-and kainate-induced neurotoxicity in primary neumnalastrecial cultures. Epilepsy Res.2003,54(1):63-71.
23 Maragakis NJ, Jackson M, Ganel R, et al. Topiramate protects against motor neuron degeneration in organotypic spinal cord cultures but not in G93A SOD1 transgenic mice. Neumsei Lett,2003,338(2):107-110.
24陈春富综述,托吡酯神经保护作用研究进展,国际神经病学神经外科学杂志2006,33(2)：149-152.
25 Kubera M, Budziszewska B, Jaworska Feil L, et al. Effect of topiramate on the kainate-induced status epilepticus, lipid peroxidation and immunoreactivity of rats. Pol J Pharm acol.2004,56(5):553-561.
26 Leniger T, Thane J, Wiemann M. Topiramate modulates pH of hippocampal CA3 neurons by combined effects on carbonic anhydrase and C1-/HCO3-exchange. Br J Pharmacol,2004, 142(5):831-842.
27 Artemowicz B, Sobaniec W. Ncuroprotection possibilities in epieptic children. Rocz Akad Med Bialymst,2005,50(Suppl1):s91-s95.
28 Kudin AP, Debska-Viehaber G, Vielhaker S, et al. The mechanism of neuroprotection by topiramate in an animal model of epilepsy. Epilepsia.2004 Dec; 45(12):1478-87.