基质金属蛋白酶9在脑皮质发育障碍中的作用研究
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摘要
脑皮质发育障碍（MCDs）是一类以大脑皮层细胞构筑异常为表现的神经系统疾病，是临床上儿童药物难治性癫痫的重要病因之一。局灶性皮质发育不良（FCD）和结节性硬化（TSC）是MCDs常见的两种亚型，并且病理学表现非常类似。对FCD的病因研究认为，FCD是一类由胚胎发育期异常的神经细胞增殖、迁移和分化所致的发育性疾病。在FCD三种病理亚型中，IIb型FCD（FCDIIb）具有最典型的病理改变，且与其他类型相比具有临床癫痫发作较早、癫痫发作频率高、发作间期短等特点。TSC是一类有TSC1或者TSC2基因突变造成的多系统多器官常染色体显性遗传病。据统计，在高达70%的TSC患者中有癫痫发作，并且常常药物难以控制。尽管从病因学的角度出发，FCDIIb和TSC并不相同。但是，鉴于他们在组织病理学上共同的改变以及临床表现的相似性，我们自然而然地推测他们在癫痫发作的病理生理学机制上有着某种共性。此外，最近的遗传学研究也发现TSC1基因在FCDIIb的发育过程中可能起到重要作用，也在某种程度上支持了我们的猜想。在对FCDIIb及TSC致痫研究中发现，FCDIIb中局部皮质发育不良区以及TSC的皮层结节是癫痫放电灶，但是具体机制仍不明确。
迄今为止，神经递质受体及离子通道在癫痫发生中的作用吸引了大多数学者的眼球，很少有研究关注到细胞外蛋白酶在癫痫发生中的可能作用。基质金属蛋白酶（MMPs）是一类具有锌指结构的分泌性或者细胞膜结合性蛋白水解酶，在胚胎发育、血管生成、组织重塑等生理过程中发挥重要的作用。在中枢神经系统中（CNS），MMPs广泛地参与到肿瘤生成、炎症反应、缺血、脑创伤等病理过程。基质金属蛋白酶9（MMP9）是MMPs家族中最被大家所熟知的一员，正常情况下先以酶原（pro-MMP9）形式分泌到细胞外，然后再被分解成活性酶（act-MMP9）形式发挥生理功能。近来的研究发现MMP9在皮层发育、突触重塑和学习记忆等生理功能中发挥重要的作用。鉴于在癫痫发生的过程中伴随着神经网络组织重塑，最近一系列的动物实验证实MMP9参与癫痫的发生发展。然而,MMP9在MCDs中的具体作用仍有待研究。
为了阐明MMP9在MCDs中的作用及其可能的机制，首先，我们利用半定量RT-PCR、western blot、免疫组织化学技术观察了MMP9在MCDs亚型(FCDIIb和TSC)临床标本中的表达分布情况；其次，我们利用局部冰冻损伤新生大鼠脑皮质建立了一个模拟人类微脑回的MCDs大鼠模型，并且检测了MMP9蛋白及酶活性在不同发育期的表达情况；最后，为了直观的了解MMP9在MCDs中的作用，在课题组前期研究的基础上，我们进一步研究了MMP9对SVZ区来源的神经前体细胞（neural precursor cells,NPCs）向微脑回区迁移的影响。通过这些研究，我们得到以下结果：
一、MMP9在MCDs患者皮质病灶中的表达分布
1．FCDIIb及TSC皮层致痫灶中MP9的mRNA水平较正常对照皮层（CTX）明显升高。
2．FCDIIb，TSC及TLE组中MMP9蛋白的酶原表达水平较CTX明显增高，而活性酶表达量仅见FCDIIb及TSC较CTX明显增高，TLE与CTX无显著差异。
3．在正常皮层中可检测到MMP9表达，其表达模式与先前报道的一致：呈弱到中等强度表达于神经元及内皮细胞上，在灰质与白质中偶见胶质细胞表达。
4．FCDIIb病灶内呈现出强MMP9免疫反应。染色强度评分提示FCDIIb中MMP9蛋白表达较CTX明显升高。我们进一步对MMP9的细胞分布表达模式进行了研究，发现：80%±6.5%的BCs (n=352)强表达MMP9；89±3.3%的DNs (n=573)强表达MMP9；83±4.7%的HNs(n=420)强表达MMP9。同时，胶质细胞也呈现强MMP9免疫反应。免疫荧光双标结果证实MMP9分别共定位于vimentin（未成熟胶质细胞标志物，常见BCs表达）阳性的BCs(图1-3H)NF200（神经元标志物）阳性的DNs、HNs。进一步的研究发现，MMP9阳性（MMP9+）的胶质细胞是GFAP+的星形胶质细胞，而非Iba1+的小胶质细胞。
5．在TSC皮层结节中，MMP9IR呈现出与FCDIIb病灶类似的强反应。染色强度评分提示TSC中MMP9蛋白表达较CTX明显升高。进一步对MMP9的细胞分布表达模式研究发现，77%±5.3%的GCs (n=491)和85%±3.6%的DNs (n=627)呈中等到强MMP9IR。先前的研究发现，GCs是一类分化程度不一的细胞，我们免疫双标的结果也发现一些MMP9+GCs呈vimentin+，而另外一些则是NF200+。进一步的荧光双标实验发现，大多数MMP9+胶质细胞是GFAP+的星形胶质细胞，而非小胶质细胞。
二、MMP9在FCD大鼠模型微脑回中蛋白、酶活性及细胞定位研究
1．Western Blotting检测MMP9蛋白在FCD模型及对照组中可见，目的蛋白大小为92kD的MMP9蛋白在各个时相点均可被检测出，在P10和P20两个时相点，FCD组MMP9蛋白表达水平较对照组明显升高。
2．明胶酶谱法检测MMP9蛋白酶活性发现，在P10和P20两个时相点，FCD组MMP9蛋白酶活性较对照组明显升高。
3．P10正常大鼠皮层可观察到MMP9表达，且主要呈中等强度表达在锥体神经元上。P10FCD大鼠微脑回区可观察到MMP9表达，光密度值分析提示FCD微脑回区MMP9表达强于正常对照组。进一步的免疫荧光结果提示，微脑回区表达MMP9的细胞为NeuN阳性的神经元，偶见GFAP阳性的胶质细胞表达MMP9。
4．类似于P10的表达模式，我们观察到MMP9在P20正常大鼠皮层广泛表达，且主要呈中等强度表达在锥体神经元上。而在P20FCD大鼠微脑回区可观察到MMP9表达主要呈强表达于锥体神经元上。光密度值分析提示FCD微脑回区MMP9表达强于正常对照组。进一步的免疫荧光结果提示，微脑回区表达MMP9的细胞为NeuN阳性的神经元，未探及GFAP阳性的胶质细胞表达MMP9。
三、MMP9对SVZ区NPCs向微脑回区迁移的影响
1．我们成功地将增强型绿色荧光蛋白腺病毒表达载体（Ad-eGFP）成功地转染到了SVZ区细胞中。并且观察到，FCD皮层冰冻损伤病灶内可见大量散在分布的eGFP+细胞，并且分布趋势类似从喙侧神经干细胞迁移到嗅球的细胞迁移流，称之为SFMS。
2．免疫荧光双标结果发现，eGFP+细胞与神经干细胞标志物nestin、SVZ区来源的具有迁移能力神经前体细胞标志物DCX共标，提示SFMS中eGFP+细胞是由SVZ区迁移而来。
3．P10期，运用蛋白酶抑制剂FN-439抑制MMP9活性后，我们发现，FN-439组迁移流（SFMS）中eGFP+细胞数量较对照组（saline）明显下降。酶谱检测也证实，FN-439组微脑回区MMP9活性较saline组确实显著降低。
在P20时间点，抑制MMP9活性后，观察到类似的结果：FN-439组SFMS中eGFP+细胞数量较saline组明显下降。同时，酶谱检测也证实，FN-439组微脑回区MMP9活性较saline组确实显著降低。
综上所述，本研究结果表明：第一、人MCDs组织中MMP9表达显著上调，且特征性地高表达于与癫痫放电活性相关的异形细胞（如异形神经元、肥大神经元、巨细胞、气球样细胞）上；第二、我们建立了一个模拟人类微脑回的MCDs大鼠模型，并且证实在P10和P20时刻微脑回区MMP9蛋白表达及酶活性较对照皮层显著上调。第三、与课题组前期研究一致，我们观察到eGFP+SVZ区来源的NPCs不断性微脑回区迁移，形成脑室下区至FCD脑区迁移流。抑制MMP9活性之后，迁移流中eGFP+细胞数量明显减少。总之，我们的结果提示MMP9可能与MCDs的病理发生密切相关。
Malformations of cortical development (MCDs), which are characterized by abnormalcytoarchitecture of the cerebral cortex, are important causes of drug-resistant epilepsy inchildren. Two common subtypes of MCDs, focal cortical dysplasia (FCD) and tuberoussclerosis complex (TSC), have many similar histological features. The pathogenesis of FCD isthought to be mainly caused by embryonic developmental insults that result in the formationof dysplastic lesions with abnormal neuronal proliferation, migration and differentiation.Among the three types of FCD, type IIb (FCDIIb) results in the most typical histologicalchanges and has a younger age of seizure onset, a shorter epilepsy duration, and an increasedseizure frequency compared with the other variants. TSC is an autosomal dominantmultisystem neurocutaneous syndrome that results from mutations of the TSC1or TSC2genes. Seizures occur in more than70to80%of the patients with TSC and are oftenrefractory to treatment. Despite the different etiologies of FCDIIb and TSC, they do share anumber of characteristic cellular and histological abnormalities as well as common clinicalfeatures, which suggest that they could have common pathomechanisms of epileptogenesis.Moreover, human genetic studies linked the TSC1gene to the development of FCDIIb.Recent evidence suggests that seizures originate within the dysplastic cortical lesions inFCDIIb and the cortical tubers in TSC. However, understanding epilepsy in FCDIIb and TSCremains a challenge.
Until now, most of our interest has focused on the role of neurotransmitter receptors andion channels in the epileptogenesis of MCDs; little attention has been paid to the potentialrole of extracellular proteinases. Matrix metalloproteinases (MMPs) are a large family ofstructurally related zinc-dependent secreted or cellular membrane-bound proteinases. MMPsare essential for various normal biological processes such as embryonic development,angiogenesis and tissue remodeling. In the central nervous system (CNS), they are involved inseveral pathologies, including tumorigenesis, inflammation, ischemia, trauma and epilepsy.MMP9, one of the best characterized members in MMP family, is synthesized and secreted into the extracellular space as the inactive zymogen pro-MMP9, then converted to the activeform, act-MMP9. Recent studies have suggested that MMP9plays important roles in corticaldevelopment, synaptic plasticity, learning and memory. As epileptogenic plasticity involvesextensive nervous tissue remodeling, a series of experiments with animal models haveindicated that MMP9is intimately involved in epileptogenesis. However, little is known aboutthe role of MMP9in MCDs.
To address the above concerns: First, we identified the expression and distribution ofMMP9in epileptic lesions of FCDIIb and TSC patients by means of RT-PCR, westernblotting and immunohistochemistry. Second, we tested the expression, activity anddistribution of MMP9in a FCD rat model. Third, to reveal the role of MMP9in thepathogenesis of MCDs, we further investigated the effect of MMP9on the migration ofSVZ-derived neural precursor cells (NPCs) to the microgyrus. The main results are asfollows:
1．Expression and cellular distribution of MMP9in cortical lesions of patiens withMCDs
1.1RT-PCR analysis showed the expression of MMP9mRNA in total homogenates ofFCDIIb and TSC lesions was dramatically higher than that in CTX samples.
1.2Western blotting analysis indicated that the pro-MMP9protein levels weresignificantly higher in the epilepsy groups (including FCDIIb, TSC and TLE) than in the CTXsamples. However, only in FCDIIb and TSC, the act-MMP9protein levels were significantlyelevated compared with CTX.
1.3As described in the previous studies, weak to moderate MMP9IR was observed inneurons and endothelial cells, and MMP9IR-positive glial cells were occasionally seen in theGM and WM of the CTX tissues
1.4A strong MMP9IR was observed in the FCDIIb cortical lesions. The intensity scoresindicated a higher expression of MMP9in the FCDIIb samples compared with the CTXsamples. Moreover, the MMP9IR showed a specific cellular distribution pattern: i.e. in allFCDIIb tissues (n=9) there was a strong staining of MMP9in80%±6.5%of the BCs (n=352)and in89±3.3%of the DNs (n=573) and83±4.7%of the HNs (n=420), and also glial cells.Double labeling experiments confirmed that the MMP9IR was colocalized with vimentin （amarker of less mature glial cells commonly expressed by BCs）and NF200(a neuronalmarker). Further immunofluorescent studies demonstrated that most of the MMP9+glial cells were theGFAP+astrocytes and not the microglia.
1.5A strong MMP9IR was present in the TSC tubers (n=14), which was similar to thatin the FCDIIb lesions. The intensity scores of MMP9IR in the TSC tubers were dramaticallyhigher than those in the CTX samples. For the cellular MMP9IR, there was a moderate tostrong IR in77%±5.3%of the GCs (n=491) and in85%±3.6%of the DNs (n=627). Inagreement with the abnormal differentiation of the GCs, our double labeling experimentsindicated that some of MMP9+GCs were vimentin+, and that some are NF200+. Furtherimmunofluorescent studies revealed that, as in the FCDIIb lesions, most MMP9+glial cells inthe TSC tubers were the GFAP+reactive astrocytes and not microglia.
2．Expression, activity and cellular distribution of MMP9in FCD rat model
2.1Western blotting analysis indicated that the MMP9protein levels were significantlyhigher in the FCD groups than in the control rats at postnatal10(P10) and postnatal20(P20).
2.2Zymography assay indicated that the activity of MMP9was dramatically higher inmicrogyrus of FCD rats than in the control cortices at P10and P20.
2.3Immunohistochemical studies suggested a stronger MMP9IR was observed inmicrogyrus of FCD rats than in the control cortices at P10. Further immunofluorescent studiesdemonstrated NeuN+neurons are the major sources of MMP9. MMP9IR-positive GFAP+glial cells were occasionally seen in the microgyrus.
2.4Similar to the observation of P10, a stronger MMP9IR was observed in microgyrusof FCD rats than in the control cortices. Double labeling experiments indicated that MMP9colabelled with NeuN, and not GFAP.
3．Effect of MMP9on the migration of SVZ-derived NPCs to the microgyrus
3.1We successfully transfected the Ad-eGFP into the cells in the SVZ, and observed theSFMS as our previous research.
3.2Immunofluorescent studies demonstrated the eGFP+cells colabelled with nestin (aneural stem cell marker) and DCX (a cytoskeletal protein expressed by the migrating NPCsfrom SVZ), suggesting that the eGFP+cells in the SFMS were derived from SVZ.
3.3Inhibition of MMP9by FN-439significantly attenuated the number of eGFP+cellsin the SFMS at P10and P20.
In summary, in the present study, we found an obvious increase of MMP9mRNA and protein expression in MCDs specimens compared with the CTX samples. The IHC studiesdetected MMP9at characteristically high levels in misshapen cells (e.g. DNs, HNs, GCs andBCs), which may be the intrinsic “pacemakers” that initiate and drive the epileptiform activityin MCDs. Further, we established a rat model of microgyral malformation and confirmed thatthe protein levels and activity of MMP9were dramatically higher in microgyrus than in thecontrol cortices at P10and P20. Consistent with our previous researches, we observed themigration of eGFP+SVZ-derived NPCs to the microgyrus, and inhibition of MMP9significantly attenuated the number of eGFP+cells in the SFMS at P10and P20. Takentogether, our findings suggest that MMP9may be linked with the pathogenesis of MCDs.
迄今为止，神经递质受体及离子通道在癫痫发生中的作用吸引了大多数学者的眼球，很少有研究关注到细胞外蛋白酶在癫痫发生中的可能作用。基质金属蛋白酶（MMPs）是一类具有锌指结构的分泌性或者细胞膜结合性蛋白水解酶，在胚胎发育、血管生成、组织重塑等生理过程中发挥重要的作用。在中枢神经系统中（CNS），MMPs广泛地参与到肿瘤生成、炎症反应、缺血、脑创伤等病理过程。基质金属蛋白酶9（MMP9）是MMPs家族中最被大家所熟知的一员，正常情况下先以酶原（pro-MMP9）形式分泌到细胞外，然后再被分解成活性酶（act-MMP9）形式发挥生理功能。近来的研究发现MMP9在皮层发育、突触重塑和学习记忆等生理功能中发挥重要的作用。鉴于在癫痫发生的过程中伴随着神经网络组织重塑，最近一系列的动物实验证实MMP9参与癫痫的发生发展。然而,MMP9在MCDs中的具体作用仍有待研究。
为了阐明MMP9在MCDs中的作用及其可能的机制，首先，我们利用半定量RT-PCR、western blot、免疫组织化学技术观察了MMP9在MCDs亚型(FCDIIb和TSC)临床标本中的表达分布情况；其次，我们利用局部冰冻损伤新生大鼠脑皮质建立了一个模拟人类微脑回的MCDs大鼠模型，并且检测了MMP9蛋白及酶活性在不同发育期的表达情况；最后，为了直观的了解MMP9在MCDs中的作用，在课题组前期研究的基础上，我们进一步研究了MMP9对SVZ区来源的神经前体细胞（neural precursor cells,NPCs）向微脑回区迁移的影响。通过这些研究，我们得到以下结果：
一、MMP9在MCDs患者皮质病灶中的表达分布
1．FCDIIb及TSC皮层致痫灶中MP9的mRNA水平较正常对照皮层（CTX）明显升高。
2．FCDIIb，TSC及TLE组中MMP9蛋白的酶原表达水平较CTX明显增高，而活性酶表达量仅见FCDIIb及TSC较CTX明显增高，TLE与CTX无显著差异。
3．在正常皮层中可检测到MMP9表达，其表达模式与先前报道的一致：呈弱到中等强度表达于神经元及内皮细胞上，在灰质与白质中偶见胶质细胞表达。
4．FCDIIb病灶内呈现出强MMP9免疫反应。染色强度评分提示FCDIIb中MMP9蛋白表达较CTX明显升高。我们进一步对MMP9的细胞分布表达模式进行了研究，发现：80%±6.5%的BCs (n=352)强表达MMP9；89±3.3%的DNs (n=573)强表达MMP9；83±4.7%的HNs(n=420)强表达MMP9。同时，胶质细胞也呈现强MMP9免疫反应。免疫荧光双标结果证实MMP9分别共定位于vimentin（未成熟胶质细胞标志物，常见BCs表达）阳性的BCs(图1-3H)NF200（神经元标志物）阳性的DNs、HNs。进一步的研究发现，MMP9阳性（MMP9+）的胶质细胞是GFAP+的星形胶质细胞，而非Iba1+的小胶质细胞。
5．在TSC皮层结节中，MMP9IR呈现出与FCDIIb病灶类似的强反应。染色强度评分提示TSC中MMP9蛋白表达较CTX明显升高。进一步对MMP9的细胞分布表达模式研究发现，77%±5.3%的GCs (n=491)和85%±3.6%的DNs (n=627)呈中等到强MMP9IR。先前的研究发现，GCs是一类分化程度不一的细胞，我们免疫双标的结果也发现一些MMP9+GCs呈vimentin+，而另外一些则是NF200+。进一步的荧光双标实验发现，大多数MMP9+胶质细胞是GFAP+的星形胶质细胞，而非小胶质细胞。
二、MMP9在FCD大鼠模型微脑回中蛋白、酶活性及细胞定位研究
1．Western Blotting检测MMP9蛋白在FCD模型及对照组中可见，目的蛋白大小为92kD的MMP9蛋白在各个时相点均可被检测出，在P10和P20两个时相点，FCD组MMP9蛋白表达水平较对照组明显升高。
2．明胶酶谱法检测MMP9蛋白酶活性发现，在P10和P20两个时相点，FCD组MMP9蛋白酶活性较对照组明显升高。
3．P10正常大鼠皮层可观察到MMP9表达，且主要呈中等强度表达在锥体神经元上。P10FCD大鼠微脑回区可观察到MMP9表达，光密度值分析提示FCD微脑回区MMP9表达强于正常对照组。进一步的免疫荧光结果提示，微脑回区表达MMP9的细胞为NeuN阳性的神经元，偶见GFAP阳性的胶质细胞表达MMP9。
4．类似于P10的表达模式，我们观察到MMP9在P20正常大鼠皮层广泛表达，且主要呈中等强度表达在锥体神经元上。而在P20FCD大鼠微脑回区可观察到MMP9表达主要呈强表达于锥体神经元上。光密度值分析提示FCD微脑回区MMP9表达强于正常对照组。进一步的免疫荧光结果提示，微脑回区表达MMP9的细胞为NeuN阳性的神经元，未探及GFAP阳性的胶质细胞表达MMP9。
三、MMP9对SVZ区NPCs向微脑回区迁移的影响
1．我们成功地将增强型绿色荧光蛋白腺病毒表达载体（Ad-eGFP）成功地转染到了SVZ区细胞中。并且观察到，FCD皮层冰冻损伤病灶内可见大量散在分布的eGFP+细胞，并且分布趋势类似从喙侧神经干细胞迁移到嗅球的细胞迁移流，称之为SFMS。
2．免疫荧光双标结果发现，eGFP+细胞与神经干细胞标志物nestin、SVZ区来源的具有迁移能力神经前体细胞标志物DCX共标，提示SFMS中eGFP+细胞是由SVZ区迁移而来。
3．P10期，运用蛋白酶抑制剂FN-439抑制MMP9活性后，我们发现，FN-439组迁移流（SFMS）中eGFP+细胞数量较对照组（saline）明显下降。酶谱检测也证实，FN-439组微脑回区MMP9活性较saline组确实显著降低。
在P20时间点，抑制MMP9活性后，观察到类似的结果：FN-439组SFMS中eGFP+细胞数量较saline组明显下降。同时，酶谱检测也证实，FN-439组微脑回区MMP9活性较saline组确实显著降低。
综上所述，本研究结果表明：第一、人MCDs组织中MMP9表达显著上调，且特征性地高表达于与癫痫放电活性相关的异形细胞（如异形神经元、肥大神经元、巨细胞、气球样细胞）上；第二、我们建立了一个模拟人类微脑回的MCDs大鼠模型，并且证实在P10和P20时刻微脑回区MMP9蛋白表达及酶活性较对照皮层显著上调。第三、与课题组前期研究一致，我们观察到eGFP+SVZ区来源的NPCs不断性微脑回区迁移，形成脑室下区至FCD脑区迁移流。抑制MMP9活性之后，迁移流中eGFP+细胞数量明显减少。总之，我们的结果提示MMP9可能与MCDs的病理发生密切相关。
Malformations of cortical development (MCDs), which are characterized by abnormalcytoarchitecture of the cerebral cortex, are important causes of drug-resistant epilepsy inchildren. Two common subtypes of MCDs, focal cortical dysplasia (FCD) and tuberoussclerosis complex (TSC), have many similar histological features. The pathogenesis of FCD isthought to be mainly caused by embryonic developmental insults that result in the formationof dysplastic lesions with abnormal neuronal proliferation, migration and differentiation.Among the three types of FCD, type IIb (FCDIIb) results in the most typical histologicalchanges and has a younger age of seizure onset, a shorter epilepsy duration, and an increasedseizure frequency compared with the other variants. TSC is an autosomal dominantmultisystem neurocutaneous syndrome that results from mutations of the TSC1or TSC2genes. Seizures occur in more than70to80%of the patients with TSC and are oftenrefractory to treatment. Despite the different etiologies of FCDIIb and TSC, they do share anumber of characteristic cellular and histological abnormalities as well as common clinicalfeatures, which suggest that they could have common pathomechanisms of epileptogenesis.Moreover, human genetic studies linked the TSC1gene to the development of FCDIIb.Recent evidence suggests that seizures originate within the dysplastic cortical lesions inFCDIIb and the cortical tubers in TSC. However, understanding epilepsy in FCDIIb and TSCremains a challenge.
Until now, most of our interest has focused on the role of neurotransmitter receptors andion channels in the epileptogenesis of MCDs; little attention has been paid to the potentialrole of extracellular proteinases. Matrix metalloproteinases (MMPs) are a large family ofstructurally related zinc-dependent secreted or cellular membrane-bound proteinases. MMPsare essential for various normal biological processes such as embryonic development,angiogenesis and tissue remodeling. In the central nervous system (CNS), they are involved inseveral pathologies, including tumorigenesis, inflammation, ischemia, trauma and epilepsy.MMP9, one of the best characterized members in MMP family, is synthesized and secreted into the extracellular space as the inactive zymogen pro-MMP9, then converted to the activeform, act-MMP9. Recent studies have suggested that MMP9plays important roles in corticaldevelopment, synaptic plasticity, learning and memory. As epileptogenic plasticity involvesextensive nervous tissue remodeling, a series of experiments with animal models haveindicated that MMP9is intimately involved in epileptogenesis. However, little is known aboutthe role of MMP9in MCDs.
To address the above concerns: First, we identified the expression and distribution ofMMP9in epileptic lesions of FCDIIb and TSC patients by means of RT-PCR, westernblotting and immunohistochemistry. Second, we tested the expression, activity anddistribution of MMP9in a FCD rat model. Third, to reveal the role of MMP9in thepathogenesis of MCDs, we further investigated the effect of MMP9on the migration ofSVZ-derived neural precursor cells (NPCs) to the microgyrus. The main results are asfollows:
1．Expression and cellular distribution of MMP9in cortical lesions of patiens withMCDs
1.1RT-PCR analysis showed the expression of MMP9mRNA in total homogenates ofFCDIIb and TSC lesions was dramatically higher than that in CTX samples.
1.2Western blotting analysis indicated that the pro-MMP9protein levels weresignificantly higher in the epilepsy groups (including FCDIIb, TSC and TLE) than in the CTXsamples. However, only in FCDIIb and TSC, the act-MMP9protein levels were significantlyelevated compared with CTX.
1.3As described in the previous studies, weak to moderate MMP9IR was observed inneurons and endothelial cells, and MMP9IR-positive glial cells were occasionally seen in theGM and WM of the CTX tissues
1.4A strong MMP9IR was observed in the FCDIIb cortical lesions. The intensity scoresindicated a higher expression of MMP9in the FCDIIb samples compared with the CTXsamples. Moreover, the MMP9IR showed a specific cellular distribution pattern: i.e. in allFCDIIb tissues (n=9) there was a strong staining of MMP9in80%±6.5%of the BCs (n=352)and in89±3.3%of the DNs (n=573) and83±4.7%of the HNs (n=420), and also glial cells.Double labeling experiments confirmed that the MMP9IR was colocalized with vimentin （amarker of less mature glial cells commonly expressed by BCs）and NF200(a neuronalmarker). Further immunofluorescent studies demonstrated that most of the MMP9+glial cells were theGFAP+astrocytes and not the microglia.
1.5A strong MMP9IR was present in the TSC tubers (n=14), which was similar to thatin the FCDIIb lesions. The intensity scores of MMP9IR in the TSC tubers were dramaticallyhigher than those in the CTX samples. For the cellular MMP9IR, there was a moderate tostrong IR in77%±5.3%of the GCs (n=491) and in85%±3.6%of the DNs (n=627). Inagreement with the abnormal differentiation of the GCs, our double labeling experimentsindicated that some of MMP9+GCs were vimentin+, and that some are NF200+. Furtherimmunofluorescent studies revealed that, as in the FCDIIb lesions, most MMP9+glial cells inthe TSC tubers were the GFAP+reactive astrocytes and not microglia.
2．Expression, activity and cellular distribution of MMP9in FCD rat model
2.1Western blotting analysis indicated that the MMP9protein levels were significantlyhigher in the FCD groups than in the control rats at postnatal10(P10) and postnatal20(P20).
2.2Zymography assay indicated that the activity of MMP9was dramatically higher inmicrogyrus of FCD rats than in the control cortices at P10and P20.
2.3Immunohistochemical studies suggested a stronger MMP9IR was observed inmicrogyrus of FCD rats than in the control cortices at P10. Further immunofluorescent studiesdemonstrated NeuN+neurons are the major sources of MMP9. MMP9IR-positive GFAP+glial cells were occasionally seen in the microgyrus.
2.4Similar to the observation of P10, a stronger MMP9IR was observed in microgyrusof FCD rats than in the control cortices. Double labeling experiments indicated that MMP9colabelled with NeuN, and not GFAP.
3．Effect of MMP9on the migration of SVZ-derived NPCs to the microgyrus
3.1We successfully transfected the Ad-eGFP into the cells in the SVZ, and observed theSFMS as our previous research.
3.2Immunofluorescent studies demonstrated the eGFP+cells colabelled with nestin (aneural stem cell marker) and DCX (a cytoskeletal protein expressed by the migrating NPCsfrom SVZ), suggesting that the eGFP+cells in the SFMS were derived from SVZ.
3.3Inhibition of MMP9by FN-439significantly attenuated the number of eGFP+cellsin the SFMS at P10and P20.
In summary, in the present study, we found an obvious increase of MMP9mRNA and protein expression in MCDs specimens compared with the CTX samples. The IHC studiesdetected MMP9at characteristically high levels in misshapen cells (e.g. DNs, HNs, GCs andBCs), which may be the intrinsic “pacemakers” that initiate and drive the epileptiform activityin MCDs. Further, we established a rat model of microgyral malformation and confirmed thatthe protein levels and activity of MMP9were dramatically higher in microgyrus than in thecontrol cortices at P10and P20. Consistent with our previous researches, we observed themigration of eGFP+SVZ-derived NPCs to the microgyrus, and inhibition of MMP9significantly attenuated the number of eGFP+cells in the SFMS at P10and P20. Takentogether, our findings suggest that MMP9may be linked with the pathogenesis of MCDs.
引文
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