不同剂量托吡酯对新生大鼠缺氧缺血性脑损伤脑保护作用的实验研究
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摘要
研究背景
缺氧缺血性脑病(Hypoxic-ischemic encephalopathy,HIE)是新生儿期常见的疾病,是导致新生儿死亡和远期神经系统发育障碍的重要因素。由于其发病机制尚不明确,对其损伤后的治疗仍无有力措施。探讨发病机制并在其关键分子上进行有效干预,对于改善HIE预后,提高人口质量具有重要意义。
托吡酯(topiramate,TPM),是一种高效新型抗癫痫药,大量实验表明其在脑损伤后脑保护中发挥着重要作用。越来越多的研究表明托吡酯在保护缺氧缺血性脑损伤(Hypoxic-ischemic brain damage,HIBD)发挥着重要的作用,未来有可能用于临床治疗HIBD。但是关于托吡酯脑保护作用的具体机制、给药剂量及药物副作用等目前研究较少。
胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是TGF-β超家族的一个新亚族成员,发挥着多效能的神经营养作用,并且在改善和促进损伤后的神经元修复和再生中发挥着重要作用。脑缺氧缺血可能是脑内GDNF分泌增加的启动因素之一,本课题组前期通过对7日龄的Wistar大鼠研究发现,HIBD假手术组偶见GDNF表达;单纯缺氧缺血组GDNF于缺氧缺血后12h开始上升,第2d进一步升高,第3d达高峰,第5d有所回落。
有研究表明,星形胶质细胞除了表达Na~+和Ca~(2+)通道以外,GluR及GABAR在其细胞膜表明亦有表达,鉴于托吡酯具有抑制Na~+或/和Ca~(2+)通道激活以及调节脑内GluR及GABA平衡之功效。因此,我们推测托吡酯有可能通过作用于星形胶质细胞表达的相应受体,达到保护星形胶质细胞,促进GDNF的分泌,进而起到保护神经元的作用。本课题组的前期研究已证实这一点,我们的研究显示:不同时间托吡酯给药后观察组GDNF的分泌量具有差异性。
本课题组前期研究已证明托吡酯用于HIBD的治疗时间窗较短,为缺氧缺血后2h之内,且预防用药优于损伤后用药;托吡酯的脑保护作用与GDNF的分泌增加有直接关系。许多动物实验研究显示托吡酯对脑缺血有明显的治疗作用,剂量多在50mg/kg-200mg/kg之间,并未产生损伤边缘系统及影响骨骼发育等副作用。本课题通过建立HIBD动物模型,借助观察脑损伤后给予不同剂量托吡酯对脑组织病理学改变、GDNF动态表达水平等方面的影响,进一步探讨托吡酯脑保护作用的机制、用药剂量,为托吡酯临床治疗HIE提供强有力的理论和试验依据,为HIE的治疗开辟一条新的道路。
目的
1.观察新生大鼠各处理组相同时间点脑组织的病理学改变。
2.观察新生大鼠缺氧缺血性脑损伤后GDNF表达水平的动态变化及不同剂量托吡酯干预对GDNF表达的影响。
3.探讨托吡酯脑保护作用的机制。
方法
1.120只新生7日龄Wistar大鼠随机分为假手术组、单纯缺氧缺血组及托吡酯治疗组,托吡酯治疗组根据不同给药剂量分为50mg/kg、100mg/kg及150mg/kg治疗组。
2.建立HIBD动物模型。
3.给药及处理方法
(1)托吡酯治疗组:将托吡酯溶于注射用水配成2%溶液,采用小型灌胃器灌胃。不同剂量托吡酯治疗组分别于手术前30min给予1次、HIBD模型建成后即刻给予1次、之后每隔12h各一次,直至第5d。
(2)单纯缺氧缺血组和假手术组同一时间点给予等体积的生理盐水。
(3)各组均在缺氧缺血后12h、2d、3d、5d分别处死1/4的动物。
4.应用HE染色、免疫组织化学(Immunocytochemistry)及Western Blotting等试验方法,观察和测定研究指标。
结果
1.HE染色结果假手术组脑组织结构层次清晰,细胞轮廓清楚,核居中,尼氏体位于核周边;单纯缺氧缺血组在缺氧缺血后12h缺血侧皮质、纹状体出现轻微的病理变化,第2d病变最严重,表现为皮质、纹状体、海马、丘脑大片坏死区,第3d病变减轻,出现胶质细胞增生,第5d基本上未见破裂细胞,出现大片胶质瘢痕;50mg/kg、100mg/kg和150mg/kg托吡酯治疗组术后12h上述部位病变不明显,第2d出现轻微病变,表现为极少数细胞间隙增宽,细胞核浓缩,第3d、5d未见明显病灶;其中150mg/kg托吡酯治疗组脑损伤减轻程度较100mg/kg托吡酯治疗组明显,而100mg/kg托吡酯治疗组又优于50mg/kg托吡酯治疗组,50mg/kg托吡酯治疗组较单纯缺氧缺血组脑损伤程度减轻。
2.免疫组织化学假手术组仅见较少的GDNF阳性细胞表达,着色较浅,稀疏地分布于整个大脑皮质、纹状体、海马,双侧对称,各时间点上检测未见明显的动态变化;单纯缺氧缺血组和托吡酯各治疗组非缺血侧GDNF阳性细胞染色的程度、数量及变化情况均类似于假手术组;单纯缺氧缺血组缺血侧皮质GDNF阳性细胞在损伤后12h开始表达,第2d进一步上升,第3d达高峰,第5d基本恢复正常,仅见极少量的GDNF表达;50mg/kg、100mg/kg和150mg/kg托吡酯治疗组缺血侧皮质在损伤后12h即可见较多GDNF表达,第2d进一步增加,第3d达高峰,第5d有所减少,但还可见较多的表达,且150mg/kg托吡酯治疗组GDNF的表达尤为明显,托吡酯治疗组在每个时间点上GDNF的表达均明显高于单纯缺氧缺血组(P＜0.05)。GDNF的表达增加与托吡酯成剂量正相关,有统计学意义(各时相点相比均P＜0.05)。
3.Western blotting单纯缺氧缺血组与托吡酯各处理组均有GDNF蛋白表达条带,150mg/kg托吡酯治疗组蛋白表达明显高于单纯缺氧缺血组,100mg/kg托吡酯治疗组稍弱,50mg/kg托吡酯治疗组较差。应用生物凝胶电泳图像分析系统(Alphaimager2200)扫描定量,通过计算出各组蛋白表达的相对积分值作出蛋白的相对含量,经过统计学处理发现:50mg/kg、100mg/kg和150mg/kg托吡酯治疗组各时间点上蛋白表达均高于单纯缺氧缺血组,有统计学意义(P＜0.05);GDNF的表达增加与托吡酯成剂量正相关,有统计学意义(各时相点相比P均＜0.05)。
结论
1.托吡酯可以明显减轻缺氧缺血导致的脑细胞水肿、液化、坏死,发挥较好的脑保护作用,托吡酯对神经元的保护作用呈剂量正相关。
2.托吡酯可使缺氧缺血性脑损伤后GDNF表达升高,其表达升高程度与托吡酯呈剂量正相关。
3.增加胶质细胞分泌GDNF可能是托吡酯脑保护作用机制之一。
Research background(Hypoxic-ischemic encephalopathy,HIE) was a newborn time common disease,which caused the newborn death and important attributed to the forward nervous system growth barrier.Its pathogenesis was not still clear about,therefore did not have the powerful treatment.To pay more attention to pathogenesis and effective intervention,would improve the prognosis and population quality of HIE.
Topiramate was a new kind of effective antiepileptic.A lot of experiments had indicated that topiramate played a vital role in the cerebellum protection after brain damage. More and more research indicated that topiramate played a vital role in protection of hypoxic-ischemic damage brain and would be used to treat HIBD clinically in future.But referring to the mechanism of protection,dosage and side effect,there was few studies.
The glial cell line-derived neurotrophic factor(GDNF) was a new subtribe member of TGF-βsuper family,which could have nerve nutrition effect,improve and promote repairing and regeneration of the damage neuron.The brain,which shorted of blood and oxygen,would be a possibly start factors of GDNF,which could increased secretion.Based on the earlier research,we thought that there was few GDNF expression in HIBD sham-operation group in 7day Wistar rat and GDNF was started to rise at 12h,elevated at 2d,reached the peak at 3d,recedes at 5d,but the level wa still high in the simple oxygen deficit lacks blood group.
Some research found that,the astrocyte could express Na~+ and the Ca~(2+) channel,and also GluR and GABAR in its membrane,in view of the fact that holds the topiramate could suppresses the activation of Na~+ or/and in the Ca~(2+) channel and regulate the balance of GABA/Glu.Therefore,we guessed that topiramate would be effective through the corresponding acceptor,which expressed in the astrocyte.It also could protect astrocyte and promotes the GDNF secretion,which could protect the function of neuron.The earlier research of our team also confirmed this point.Given the topiramate at different times, we observated the different GDNF secretion.
This earlier period research had proven that,as topiramate used in HIBD,the treatment time window was short.Lacking the blood after the oxygen deficit in 2h,and also the prevention medication was better than after damage applies;The topiramate the brain protective function was related to the GDNF secretion directly.Many animal experiments study showed topiramate had the obvious treatment function to lack the blood to the brain.The effective dosage was between 50mg/kg-200mg/kg,and had no side effect on edge system and skeleton growth.This research was through established the HIBD animal model,the observation brain damage and topiramate treatment about the brain organization and pathology changes and GDNF dynamic expression.Further discuss the topiramate brain protective function,the mechanism,and the effective dosage, providing the powerful theory and the experimental basis and making a new HIE treatment way.
Objective
1.To observe the brain organization aspect and so on pathology change of various newborn mat treatment groups in the same time.
2.To observe the expression of GDNF,and dynamic changes of newborn rat after HIBD. With changes of the different dosage topiramate,the changes of the GDNF expression.
3.To discuss the mechanism of topiramate.
Methods
1.120 newborn on 7th the age Wistar rat were divided into the sham-operation group,the pure oxygen deficit stochastically lacks the blood group and topiramate treatment group, topiramate treatment group were weighted,and then given medicine 50mg/kg,100mg/kg and 150mg/kg especially.
2.Made the HIBD animal model.
3.Given the medicine and the methods
(1) Topiramate treatment group:Topiramate was dissolve into sterile water and maked into 2%solution.Using the small stomach fills into stomach.Each the different dosage topiramate treatment group separately 30min,then given 1 time.After the HIBD model completed,instantly given 1 time.12h after the surgery each one time,until 5d.
(2)The pure oxygen deficit stochastically lacks the blood group and the sham-operation group were given the equal volume physiological saline at identical time spot.
(3) Each group executes 1/4 animal separately at 12h,2d,3d,5d after HIBD.
4.Using HE dyeing,Immunochemistry and Western Blotting,we observed.
Results
1.HE dyeing findings
In sham-operation group,brain organizational structure level and the cell outline were clear and the nucleus was in the middle,Nepali body was peri-nucliar.In the pure oxygen deficit lacks the blood group,lacking the blood after the oxygen deficit 12h and also lacking the blood,side cerebral cortex and the corpus striatum appeared the slight pathological changes.The 2d pathological changes were most serious,the cerebral cortex, the corpus striatum,the seahorse and the cerebral ganglion displayed a great necrosis area. The 3d pathological changes were reduced,which appeared the spongiocyte proliferation. The 5d almostly did not see the breakage cells and could see big pieces of scar.After 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,the 12h above spot pathological change not to be obvious,2d appears the slight pathological change,displays increases the width for few intercellular space,the cell nucleus concentration,3d and 5d had not seen the obvious focus of infection.150mg/kg topiramate treatment,the group brain damage was prior to 100mg/kg topiramate treatment group,and 100mg/kg topiramate treatment group was prior to 50mg/kg topiramate treatment.
2.Immunochemistry findings
sham-operation group only showed the few GDNF masculine cell expression,the coloration was shallow,distribution sparsely in the entire cerebral cortex,the corpus striatum,the seahorse,the two-sided symmetry and did not see the obvious dynamic change in various time light the examination;The pure oxygen deficit lacks the blood group,after 12h of damage,there were to GDNF expression in side cerebral cortex,2d further rised,3d reached the peak,5d was descented to normal and only extremely few GDNF expression; 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,after lacking the blood side cerebral cortex 12h,it could see many GDNF expression,2d further increased,3d reached the peak,5d reduced,but also obviously many expressions,and 150mg/kg topiramate treatment group,the expression of GDNF was obvious,in each time the expression of GDNF was obviously higher than the pure oxygen deficit to lack the blood group (P＜0.05).The GDNF expression was related to the dosage of topiramate,which was statistics significance(P＜0.05).
3.Western blotting findings
The pure oxygen deficit lacks the blood group and with topiramate treatment groups all had the GDNF protein expression banding.150mg/kg topiramate treatment GDNF protein expression was higher than the pure oxygen deficit to lack the blood group obviously, 100mg/kg topiramate treatment group was slightly weak,50mg/kg topiramate treatment group was worst.The application biology gel electrophoresis image analysis system (Alphaimager 2200) scans the quota,through calculated various histone expression,and the relative integral valve to make the protein the comparative content,discovered after statistics processes:50mg/kg,100mg/kg and 150mg/kg topiramate treatment group at various time to light the protein expression to be higher than the pure oxygen deficit to lack the blood group,had statistics significance(P＜0.05);The GDNF expression increased with holds the topiramate ester to become the dosage related,had statistics significance(P＜0.05).
Conclusion
1.Topiramate could obviously reduce the brain cell dropsy,the liquefication and necrosis, which was caused by oxygen deficit and short of blood.It was also showed the good brain protective function and the dosage dependent.
2.Topiramate would be increased the GDNF expression after HIBD,and its expression degree was related to the dosage of topiramate.
3.Topiramate's brain protective mechanisms may be related to GDNF increased,which were secreted by spongiocyte.
缺氧缺血性脑病(Hypoxic-ischemic encephalopathy,HIE)是新生儿期常见的疾病,是导致新生儿死亡和远期神经系统发育障碍的重要因素。由于其发病机制尚不明确,对其损伤后的治疗仍无有力措施。探讨发病机制并在其关键分子上进行有效干预,对于改善HIE预后,提高人口质量具有重要意义。
托吡酯(topiramate,TPM),是一种高效新型抗癫痫药,大量实验表明其在脑损伤后脑保护中发挥着重要作用。越来越多的研究表明托吡酯在保护缺氧缺血性脑损伤(Hypoxic-ischemic brain damage,HIBD)发挥着重要的作用,未来有可能用于临床治疗HIBD。但是关于托吡酯脑保护作用的具体机制、给药剂量及药物副作用等目前研究较少。
胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是TGF-β超家族的一个新亚族成员,发挥着多效能的神经营养作用,并且在改善和促进损伤后的神经元修复和再生中发挥着重要作用。脑缺氧缺血可能是脑内GDNF分泌增加的启动因素之一,本课题组前期通过对7日龄的Wistar大鼠研究发现,HIBD假手术组偶见GDNF表达;单纯缺氧缺血组GDNF于缺氧缺血后12h开始上升,第2d进一步升高,第3d达高峰,第5d有所回落。
有研究表明,星形胶质细胞除了表达Na~+和Ca~(2+)通道以外,GluR及GABAR在其细胞膜表明亦有表达,鉴于托吡酯具有抑制Na~+或/和Ca~(2+)通道激活以及调节脑内GluR及GABA平衡之功效。因此,我们推测托吡酯有可能通过作用于星形胶质细胞表达的相应受体,达到保护星形胶质细胞,促进GDNF的分泌,进而起到保护神经元的作用。本课题组的前期研究已证实这一点,我们的研究显示:不同时间托吡酯给药后观察组GDNF的分泌量具有差异性。
本课题组前期研究已证明托吡酯用于HIBD的治疗时间窗较短,为缺氧缺血后2h之内,且预防用药优于损伤后用药;托吡酯的脑保护作用与GDNF的分泌增加有直接关系。许多动物实验研究显示托吡酯对脑缺血有明显的治疗作用,剂量多在50mg/kg-200mg/kg之间,并未产生损伤边缘系统及影响骨骼发育等副作用。本课题通过建立HIBD动物模型,借助观察脑损伤后给予不同剂量托吡酯对脑组织病理学改变、GDNF动态表达水平等方面的影响,进一步探讨托吡酯脑保护作用的机制、用药剂量,为托吡酯临床治疗HIE提供强有力的理论和试验依据,为HIE的治疗开辟一条新的道路。
目的
1.观察新生大鼠各处理组相同时间点脑组织的病理学改变。
2.观察新生大鼠缺氧缺血性脑损伤后GDNF表达水平的动态变化及不同剂量托吡酯干预对GDNF表达的影响。
3.探讨托吡酯脑保护作用的机制。
方法
1.120只新生7日龄Wistar大鼠随机分为假手术组、单纯缺氧缺血组及托吡酯治疗组,托吡酯治疗组根据不同给药剂量分为50mg/kg、100mg/kg及150mg/kg治疗组。
2.建立HIBD动物模型。
3.给药及处理方法
(1)托吡酯治疗组:将托吡酯溶于注射用水配成2%溶液,采用小型灌胃器灌胃。不同剂量托吡酯治疗组分别于手术前30min给予1次、HIBD模型建成后即刻给予1次、之后每隔12h各一次,直至第5d。
(2)单纯缺氧缺血组和假手术组同一时间点给予等体积的生理盐水。
(3)各组均在缺氧缺血后12h、2d、3d、5d分别处死1/4的动物。
4.应用HE染色、免疫组织化学(Immunocytochemistry)及Western Blotting等试验方法,观察和测定研究指标。
结果
1.HE染色结果假手术组脑组织结构层次清晰,细胞轮廓清楚,核居中,尼氏体位于核周边;单纯缺氧缺血组在缺氧缺血后12h缺血侧皮质、纹状体出现轻微的病理变化,第2d病变最严重,表现为皮质、纹状体、海马、丘脑大片坏死区,第3d病变减轻,出现胶质细胞增生,第5d基本上未见破裂细胞,出现大片胶质瘢痕;50mg/kg、100mg/kg和150mg/kg托吡酯治疗组术后12h上述部位病变不明显,第2d出现轻微病变,表现为极少数细胞间隙增宽,细胞核浓缩,第3d、5d未见明显病灶;其中150mg/kg托吡酯治疗组脑损伤减轻程度较100mg/kg托吡酯治疗组明显,而100mg/kg托吡酯治疗组又优于50mg/kg托吡酯治疗组,50mg/kg托吡酯治疗组较单纯缺氧缺血组脑损伤程度减轻。
2.免疫组织化学假手术组仅见较少的GDNF阳性细胞表达,着色较浅,稀疏地分布于整个大脑皮质、纹状体、海马,双侧对称,各时间点上检测未见明显的动态变化;单纯缺氧缺血组和托吡酯各治疗组非缺血侧GDNF阳性细胞染色的程度、数量及变化情况均类似于假手术组;单纯缺氧缺血组缺血侧皮质GDNF阳性细胞在损伤后12h开始表达,第2d进一步上升,第3d达高峰,第5d基本恢复正常,仅见极少量的GDNF表达;50mg/kg、100mg/kg和150mg/kg托吡酯治疗组缺血侧皮质在损伤后12h即可见较多GDNF表达,第2d进一步增加,第3d达高峰,第5d有所减少,但还可见较多的表达,且150mg/kg托吡酯治疗组GDNF的表达尤为明显,托吡酯治疗组在每个时间点上GDNF的表达均明显高于单纯缺氧缺血组(P＜0.05)。GDNF的表达增加与托吡酯成剂量正相关,有统计学意义(各时相点相比均P＜0.05)。
3.Western blotting单纯缺氧缺血组与托吡酯各处理组均有GDNF蛋白表达条带,150mg/kg托吡酯治疗组蛋白表达明显高于单纯缺氧缺血组,100mg/kg托吡酯治疗组稍弱,50mg/kg托吡酯治疗组较差。应用生物凝胶电泳图像分析系统(Alphaimager2200)扫描定量,通过计算出各组蛋白表达的相对积分值作出蛋白的相对含量,经过统计学处理发现:50mg/kg、100mg/kg和150mg/kg托吡酯治疗组各时间点上蛋白表达均高于单纯缺氧缺血组,有统计学意义(P＜0.05);GDNF的表达增加与托吡酯成剂量正相关,有统计学意义(各时相点相比P均＜0.05)。
结论
1.托吡酯可以明显减轻缺氧缺血导致的脑细胞水肿、液化、坏死,发挥较好的脑保护作用,托吡酯对神经元的保护作用呈剂量正相关。
2.托吡酯可使缺氧缺血性脑损伤后GDNF表达升高,其表达升高程度与托吡酯呈剂量正相关。
3.增加胶质细胞分泌GDNF可能是托吡酯脑保护作用机制之一。
Research background(Hypoxic-ischemic encephalopathy,HIE) was a newborn time common disease,which caused the newborn death and important attributed to the forward nervous system growth barrier.Its pathogenesis was not still clear about,therefore did not have the powerful treatment.To pay more attention to pathogenesis and effective intervention,would improve the prognosis and population quality of HIE.
Topiramate was a new kind of effective antiepileptic.A lot of experiments had indicated that topiramate played a vital role in the cerebellum protection after brain damage. More and more research indicated that topiramate played a vital role in protection of hypoxic-ischemic damage brain and would be used to treat HIBD clinically in future.But referring to the mechanism of protection,dosage and side effect,there was few studies.
The glial cell line-derived neurotrophic factor(GDNF) was a new subtribe member of TGF-βsuper family,which could have nerve nutrition effect,improve and promote repairing and regeneration of the damage neuron.The brain,which shorted of blood and oxygen,would be a possibly start factors of GDNF,which could increased secretion.Based on the earlier research,we thought that there was few GDNF expression in HIBD sham-operation group in 7day Wistar rat and GDNF was started to rise at 12h,elevated at 2d,reached the peak at 3d,recedes at 5d,but the level wa still high in the simple oxygen deficit lacks blood group.
Some research found that,the astrocyte could express Na~+ and the Ca~(2+) channel,and also GluR and GABAR in its membrane,in view of the fact that holds the topiramate could suppresses the activation of Na~+ or/and in the Ca~(2+) channel and regulate the balance of GABA/Glu.Therefore,we guessed that topiramate would be effective through the corresponding acceptor,which expressed in the astrocyte.It also could protect astrocyte and promotes the GDNF secretion,which could protect the function of neuron.The earlier research of our team also confirmed this point.Given the topiramate at different times, we observated the different GDNF secretion.
This earlier period research had proven that,as topiramate used in HIBD,the treatment time window was short.Lacking the blood after the oxygen deficit in 2h,and also the prevention medication was better than after damage applies;The topiramate the brain protective function was related to the GDNF secretion directly.Many animal experiments study showed topiramate had the obvious treatment function to lack the blood to the brain.The effective dosage was between 50mg/kg-200mg/kg,and had no side effect on edge system and skeleton growth.This research was through established the HIBD animal model,the observation brain damage and topiramate treatment about the brain organization and pathology changes and GDNF dynamic expression.Further discuss the topiramate brain protective function,the mechanism,and the effective dosage, providing the powerful theory and the experimental basis and making a new HIE treatment way.
Objective
1.To observe the brain organization aspect and so on pathology change of various newborn mat treatment groups in the same time.
2.To observe the expression of GDNF,and dynamic changes of newborn rat after HIBD. With changes of the different dosage topiramate,the changes of the GDNF expression.
3.To discuss the mechanism of topiramate.
Methods
1.120 newborn on 7th the age Wistar rat were divided into the sham-operation group,the pure oxygen deficit stochastically lacks the blood group and topiramate treatment group, topiramate treatment group were weighted,and then given medicine 50mg/kg,100mg/kg and 150mg/kg especially.
2.Made the HIBD animal model.
3.Given the medicine and the methods
(1) Topiramate treatment group:Topiramate was dissolve into sterile water and maked into 2%solution.Using the small stomach fills into stomach.Each the different dosage topiramate treatment group separately 30min,then given 1 time.After the HIBD model completed,instantly given 1 time.12h after the surgery each one time,until 5d.
(2)The pure oxygen deficit stochastically lacks the blood group and the sham-operation group were given the equal volume physiological saline at identical time spot.
(3) Each group executes 1/4 animal separately at 12h,2d,3d,5d after HIBD.
4.Using HE dyeing,Immunochemistry and Western Blotting,we observed.
Results
1.HE dyeing findings
In sham-operation group,brain organizational structure level and the cell outline were clear and the nucleus was in the middle,Nepali body was peri-nucliar.In the pure oxygen deficit lacks the blood group,lacking the blood after the oxygen deficit 12h and also lacking the blood,side cerebral cortex and the corpus striatum appeared the slight pathological changes.The 2d pathological changes were most serious,the cerebral cortex, the corpus striatum,the seahorse and the cerebral ganglion displayed a great necrosis area. The 3d pathological changes were reduced,which appeared the spongiocyte proliferation. The 5d almostly did not see the breakage cells and could see big pieces of scar.After 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,the 12h above spot pathological change not to be obvious,2d appears the slight pathological change,displays increases the width for few intercellular space,the cell nucleus concentration,3d and 5d had not seen the obvious focus of infection.150mg/kg topiramate treatment,the group brain damage was prior to 100mg/kg topiramate treatment group,and 100mg/kg topiramate treatment group was prior to 50mg/kg topiramate treatment.
2.Immunochemistry findings
sham-operation group only showed the few GDNF masculine cell expression,the coloration was shallow,distribution sparsely in the entire cerebral cortex,the corpus striatum,the seahorse,the two-sided symmetry and did not see the obvious dynamic change in various time light the examination;The pure oxygen deficit lacks the blood group,after 12h of damage,there were to GDNF expression in side cerebral cortex,2d further rised,3d reached the peak,5d was descented to normal and only extremely few GDNF expression; 50mg/kg,100mg/kg and 150mg/kg topiramate treatment group,after lacking the blood side cerebral cortex 12h,it could see many GDNF expression,2d further increased,3d reached the peak,5d reduced,but also obviously many expressions,and 150mg/kg topiramate treatment group,the expression of GDNF was obvious,in each time the expression of GDNF was obviously higher than the pure oxygen deficit to lack the blood group (P＜0.05).The GDNF expression was related to the dosage of topiramate,which was statistics significance(P＜0.05).
3.Western blotting findings
The pure oxygen deficit lacks the blood group and with topiramate treatment groups all had the GDNF protein expression banding.150mg/kg topiramate treatment GDNF protein expression was higher than the pure oxygen deficit to lack the blood group obviously, 100mg/kg topiramate treatment group was slightly weak,50mg/kg topiramate treatment group was worst.The application biology gel electrophoresis image analysis system (Alphaimager 2200) scans the quota,through calculated various histone expression,and the relative integral valve to make the protein the comparative content,discovered after statistics processes:50mg/kg,100mg/kg and 150mg/kg topiramate treatment group at various time to light the protein expression to be higher than the pure oxygen deficit to lack the blood group,had statistics significance(P＜0.05);The GDNF expression increased with holds the topiramate ester to become the dosage related,had statistics significance(P＜0.05).
Conclusion
1.Topiramate could obviously reduce the brain cell dropsy,the liquefication and necrosis, which was caused by oxygen deficit and short of blood.It was also showed the good brain protective function and the dosage dependent.
2.Topiramate would be increased the GDNF expression after HIBD,and its expression degree was related to the dosage of topiramate.
3.Topiramate's brain protective mechanisms may be related to GDNF increased,which were secreted by spongiocyte.
引文
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