Name: culturing rat sympathetic neurons from embryonic superior cervical ganglia for morphological and proteomic analysis
Uploaded: 2020-09-27T14:00:00
Description: Watch this Scientific Journal Video about Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis at JoVE.com

这项工作得到了加州圣玛丽学院的教师发展基金和暑期研究计划赠款的支持。作者还要感谢加州大学戴维斯分校的帕梅拉·莱因博士和加州大学伯克利分校质谱设施的安东尼·伊瓦龙博士在开发这些协议期间提供的建议。作者还要感谢加州圣玛丽学院传播办公室的海莉·纳尔逊在视频制作和编辑方面的帮助。

加州圣玛丽学院的机构动物护理和使用委员会（IACUC）批准了有关动物研究的所有程序。圣玛丽学院的动物护理和使用指南是根据国家卫生研究所实验室动物福利办公室提供的准则（https://olaw.nih.gov/sites/default/files/PHSPolicyLabAnimals.pdf和https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf） https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf。
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<ol>
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本文介绍了从胚胎大鼠幼崽的上颈神经群中培养交感神经元的规程。使用这个模型系统的优点是，有可能获得一个均匀的神经元群，提供类似的反应生长因子，并且由于这些神经元的生长因子要求已经很好地特征化，有可能在无血清条件下在定义的介质中体外生长这些神经元。虽然该协议描述了从E21大鼠幼崽隔离SCG的过程，但该协议可用于从E17到E21的鼠幼幼解剖SCG，以及解剖胚?...

来自胚胎优越的宫颈神经群（SCG）的交感神经元已被广泛用作研究神经元发育的初级神经元培养系统，包括生长因子依赖、神经元靶向相互作用、神经递质信号、突克生长、树突发育和可塑性、突触发生和信号机制，其基础神经靶点/神经胶质相互作用,,,1,1、2、3、4、5、6、7、8、9。7,8,95,6,234尽管它们体积小（大约10000个神经元/神经），但有三个主要原因，发展和广泛使用这种文化系统是一）作为第一个神经群在同情链，他们更大，因此更容易隔离，比其余的同情神经10;ii） 与中央神经元不同，SCG 中的神经元相当均匀，所有神经元都来自神经峰，大小相似，依赖于神经生长因子，并且无肾上腺素。这使得它们成为形态学和基因组研究的宝贵模型10，11,11和iii）这些神经元可以保持在一个定义的无血清介质包含神经生长因子超过一个月10，12。10,12围产期SCG神经元已被广泛使用，用于研究树突2的启动和维持机制。这主要是因为，虽然SCG神经元在体内有广泛的树突状，但在缺乏血清的情况下，它们不在体外延伸树突，但在某些生长因子（如骨形态遗传蛋白,2、12、13）,12的存在下，可以诱导树突生长树突。13
本文介绍了胚胎大鼠SCG神经元的隔离和培养方案。在过去50年中，SCG的原发神经元培养主要用于形态学研究，研究大规模基因组或蛋白质组变化的研究数量有限。这主要是由于组织体积小，导致低量的DNA或蛋白质的分离，这使得很难对这些神经元进行基因组和蛋白质组分析。然而，近年来，检测敏感性的提高使得在树突生长发育过程中，在SCG神经元中检查基因组、miRNome和蛋白质组的方法的发展已经能够发展14、15、16、17。,15,16,17本文还将描述利用免疫细胞化学和获取神经蛋白提取物进行质谱分析的神经元形态分析方法。

<table>
	<tbody>
		<tr>
			<td>2D nanoACQUITY</td>
			<td>Waters Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-7</td>
			<td>R&#38;D Systems</td>
			<td>354-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Alumin</td>
			<td>Sigma-Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>CLS-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>4176</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar or Nunc Flat bottomed Cell culture plates</td>
			<td>Fisher Scientific</td>
			<td>07-200, 140675, 142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine- &#946;- D-arabinofuranoside</td>
			<td>Sigma-Aldrich</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>D-phosphate buffered saline (Calcium and magnesium free)</td>
			<td>ATCC</td>
			<td>30-2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Low glucose + Glutamine, + sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid Free BSA</td>
			<td>Calbiochem</td>
			<td>126609</td>
			<td>20 mg/mL stock in low glucose DMEM</td>
		</tr>
		<tr>
			<td>Fine forceps Dumont no.4 and no.5</td>
			<td>Ted Pella Inc</td>
			<td>5621, 5622</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps and Scissors for Dissection</td>
			<td>Ted Pella Inc</td>
			<td>1328, 1329, 5002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverlips - 12mm</td>
			<td>Neuvitro Corporation</td>
			<td>GG-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-Anti Mouse IgG Alexa 488 conjugated</td>
			<td>Thermo Fisher Scientific</td>
			<td>A32723</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>11765</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt soltion (Calcium and Magnesium free)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14185</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Selenium-Transferrin (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>A3221</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media for glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36931, P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse-anti- MAP2 antibody (SMI-52)</td>
			<td>BioLegend</td>
			<td>SMI 52</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve growth factor</td>
			<td>Envigo Bioproducts (formerly Harlan Bioproducts)</td>
			<td>BT5017</td>
			<td>Stock 125 &#956;g/mL in 0.2% Prionex in DMEM</td>
		</tr>
		<tr>
			<td>Paraformaldehye</td>
			<td>Spectrum Chemicals</td>
			<td>P1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P0899</td>
			<td></td>
		</tr>
		<tr>
			<td>Prionex</td>
			<td>Millipore</td>
			<td>529600</td>
			<td>10% solution, 100 mL</td>
		</tr>
		<tr>
			<td>RapiGest SF</td>
			<td>Waters Corporation</td>
			<td>186001861</td>
			<td>5 X 1 mg</td>
		</tr>
		<tr>
			<td>Synapt G2 High Definition Mass Spectrometry</td>
			<td>Waters Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoro acetic acid - Sequencing grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>28904</td>
			<td>10 X 1 mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega or NEB</td>
			<td>V511A, P8101S</td>
			<td>100 &#956;g or 5 X 20 mg</td>
		</tr>
		<tr>
			<td>Waters Total recovery vials</td>
			<td>Waters Corporation</td>
			<td>186000385c</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing rat sympathetic neurons from embryonic superior cervical ganglia for morphological and proteomic analysis

来自胚胎大鼠高级宫颈节（SCG）的交感神经元已被用作周围神经元的体外模型系统，用于研究共培养系统中的突触生长、斧质贩运、突触、树突生长、树突增合和神经靶相互作用。该协议描述了神经元与E21大鼠胚胎的优越宫颈神经分离，随后在无血清培养中制备和维持纯神经元培养。由于神经元不粘附于未涂覆的塑料，神经元将在 12 mm 玻璃盖玻片或涂有聚 D-lysine 的 6 孔板上培养。在用抗米理剂（Ara-C，细胞素β-D-阿拉伯富拉诺赛德）治疗后，该协议产生健康神经元培养，非神经元细胞少于5%，可以在体外维持一个多月。虽然胚胎大鼠SCG神经元是多极的，体内有5-8个树突;在无血清条件下，这些神经元在培养中只延伸单个轴突，并在培养期间继续是单极的。然而，这些神经元可以诱导扩大树突在地下膜提取物，骨形态遗传蛋白（BPS），或10%胎儿小牛血清的存在。这些同质神经元培养物可用于免疫细胞化学染色和生化研究。本文还介绍了这些神经元中微管相关蛋白-2（MAP-2）的免疫细胞化学染色的优化方案，以及用于质谱学的神经元提取物的制备。

隔离和维护胚胎SCG神经元的神经元培养 大鼠胚胎SCG分离的细胞被镀在多D-lysine涂层板或盖玻片中，并保留在含有b神经生长因子的血清自由培养基中。含有神经元和胶质细胞混合物的分离细胞在电镀时看起来是圆形的（图1A）。在电镀的24小时内，神经元扩展小轴突过程与胶质细胞扁平化，并在相位对比度显微镜下出现相暗（图1B）。在使?...

Watch this Scientific Journal Video about Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis at JoVE.com

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

本文描述了胚胎大鼠交感神经元从优越的宫颈神经的分离和培养。它还为免疫细胞化学染色和制备用于质谱分析的神经元提取物提供了详细的方案。

培养胚胎高级宫颈神经群中的大鼠交感神经元进行形态学和蛋白质学分析

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

在这段视频中，我们将展示胚胎大鼠的优质宫颈刚体用于形态学和蛋白质组分析的培养。在开始解剖之前，准备控制和解剖介质。控制介质含有F-12和低葡萄糖DMEM，辅以胰岛素-钚-转移素混合物、谷氨酰胺、神经生长因子和BSA。解剖介质包含 L-15，辅以笔链和 BSA。有关媒体准备的详细协议，请参阅手稿。培养神经元的板的准备。用无菌蒸馏水稀释每毫升1毫克多D-lysine，每毫升100微克。对于蛋白质组或基因组分析，解剖前一到两天，涂上六孔板约两毫升无菌100克多毫升多D-莱辛。这是确保细胞附着力到井中所必需的。用保鲜膜包裹盘子，在四摄氏度下隔夜存放板。解剖当天，在解剖开始前，从井中去除多D-lysine溶液，用无菌蒸馏水冲洗井五次，然后用低血糖DMEM冲洗一次。在乳心酶消化过程中，在电镀细胞前大约一小时，从板中吸出DMEM，用0.3毫升的控制介质代替。将板在 35 摄氏度以下的 5%CO2 中存放在加湿室中。在机罩中，设置以下项目进行解剖。四个50毫升无菌锥形管，每个管中约有20毫升解剖介质。四个无菌的35毫米菜，1.5毫升解剖介质。一个无菌的50毫升圆锥管，带20毫升解剖介质进行离心。一个15毫升的无菌锥形管，用于离心，一个无菌10毫升管收集分离的细胞。使用干珠消毒器对一对细钳进行消毒至少一分钟。加州圣玛丽学院的机构动物护理和使用委员会批准了有关动物研究的所有程序。加州圣玛丽学院的动物护理和使用指南是根据国家卫生研究院实验室动物福利办公室提供的指南制定。从怀孕大鼠中去除E21胚胎。请注意，如果对周围区域进行彻底消毒，可以在发动机罩外进行子宫角的切除。用二氧化碳吸入使怀孕大鼠安乐死。从腹部区域剪下毛皮，用 70% 酒精擦拭该区域的皮肤以消毒。使用一套新的无菌剪刀和钳子，切开皮肤，然后肌肉露出含有胚胎的子宫角。用胚胎取出子宫角，使用一套新的剪刀和钳子，注意不要在这个过程中损害肠道。将子宫角与胚胎一起转移到150毫米无菌培养皿中，然后将其转移到机罩中。使用一套新的钳子和剪刀，从子宫角取出胚胎，将胚胎从羊膜和胎盘分离。要对胚胎进行安乐死，请沿着右臂下的中线切割胚胎的脊髓。这将减少从胡萝卜动脉出血期间去除SCG。这些胚胎移植到准备好的50毫升锥形管中，含有解剖介质。确保胚胎浸入介质中。每个管最多可以容纳三个胚胎。将优越的宫颈从胚胎中分离。将一只小狗从解剖介质转移到无菌的 150 毫米培养皿上，该培养皿半填充固体基材，其后面在基材上。使用三根无菌的 23 针，将酒吧固定在盘子上，每根手臂下插一根针，第三根针穿过嘴，小心地超伸长颈部。用无菌细钳切开颈部区域的皮肤，以暴露下面的唾液腺。使用细钳去除这些腺体。分别找到锁骨和气管附近的胸腺肌和肌肽肌肉。首先，切割横向胸肌肌肉。然后用细钳小心地切割细小肌肉。一旦这些肌肉被移除，前端的胡萝卜动脉的分叉将在气管的两侧可见，而SCG位于胡萝卜状动脉的这个叉子下。使用闭合钳，轻轻抬起胡萝卜动脉以可视化 SCG。使用 SCG 两侧的一个钳子，拉出卡罗蒂德，将其转移到准备好的无菌 35 毫米盘。这种组织将最有可能包含SCG与胡萝卜动脉，迷走神经与结节，以及在该地区的其他部分的肌肉或脂肪。在另一侧重复解剖过程。在继续概述的其余解剖步骤之前，先从所有胚胎中取出 SCG。将分离的组织分配在 35 毫米的盘子中的两个之间，以便于组织样本的处理。SCG 的后期处理。要将SCG与解剖组织分离，首先使用细钳去除任何无关的组织，如肌肉或脂肪，注意避免在肉皮分叉附近的区域。一旦这些组织被移除，两个小将可见。当SCG是杏仁形时，结节更小，圆圆。轻轻拉上迷走神经，将迷走神经和结点神经与胡萝卜状神经分离，然后将SCG与胡萝卜动脉分离。使用精细钳子取出 SCG 周围的胶囊。将 SCG 转移到新的 35 毫米培养盘中。对所有解剖组织样本重复此过程。用解剖介质涂上经过消毒的棉质玻璃移液器，以防止组织粘附在移液器壁上。使用移液器将解剖介质替换为无菌的两毫升胶原酶II型，不含钙和无镁的HSSSII型，并在37摄氏度下孵育50分钟，以帮助分解组织。请注意，孵育时间可能需要针对不同批次的胶原酶/分离酶进行优化，通常从 40 分钟到 1 小时。孵育后，将 SCG 和胶原酶/分离转移到无菌的 15 毫升锥形管中。使用解剖介质冲洗板，将溶液转移到管中。添加足够的解剖介质，使体积达到约 10 毫升。在 200 x g、1，000 至 1，200 rpm 下在室温下离心 5 分钟，以对样品进行颗粒。吸上一道，注意不要将颗粒分离。用10毫升解剖介质重新补充颗粒，重复离心并丢弃上经剂。用一到两毫升的培养介质代替。使用窄孔、弯尖无菌巴斯德移液器，用培养介质预涂，通过轻轻三角 5 到 6 次，机械地分离团块。让较大的团块沉淀约一分钟，并转移上清细胞悬浮液到新的10毫升管。每次重复此过程三次，每次增加三角化力，以确保几乎完全分离 SCG。添加足够的培养介质，使体积达到 8 到 10 毫升。轻轻混合细胞悬浮液，用血细胞计量化细胞密度。将细胞悬浮液以适当的细胞密度分配到孔中，用于实验。在电镀过程中连续混合细胞悬浮液，以确保细胞均匀分布到井中。对于形态分析，在24井的盘子里，每井约有8000个细胞。对于基因组和蛋白质细胞协议，每井的细胞板高达3万个细胞。将板转移到底部有无菌水的玻璃干燥器，以形成加湿室。在密封之前，注入足够的 CO2（约 120 毫升）以在干燥器中获得 5% 的 CO2 环境。将板保持 35.5 摄氏度。这在协议中称为第 0 天。这些板也可以保持在一个正常的5%CO2培养箱。上述方法可最大限度降低温度和pH值变化，还有助于防止交叉污染。维持培养的SCG神经元和治疗。这些图片显示了第0天、第1天和第5天分离的优越宫颈神经节细胞。天零图像显示电镀时的细胞。在零日镀的细胞包含神经元和非神经元细胞的混合物。第一天，电镀24小时后，小心地取出一半的培养基，代之以两个微摩尔阿拉-C，细胞素D-arabinofuranoside，一种抗粒细胞剂。将治疗留在细胞上48小时。通常，这种治疗时间足以消除培养中的非神经元细胞。第三天，轻轻吸气一半的介质，代之以控制介质。第四天，细胞准备进行实验治疗。每隔一天用适当的媒介喂养文化。用新鲜的介质轻轻更换井中介质的一半。第五天的图片显示了广泛的斧弓生长，没有观察到树突生长。为了诱导树突生长，神经元可以在Matrigel上生长，或用每毫升骨形态遗传蛋白-7的10%血清或50毫微克进行治疗。图二显示了使用BMP-7治疗后E21大鼠SCG神经元神经元形态的变化。10 X 放大率的代表性相位对比显微图显示面板 A 中控制神经元中的圆神经元细胞体，以及面板 B.培养 SCG 神经元的箭头使用 B. 培养 SCG 神经元显示的 BMP-7 处理时具有多个短树突状过程的神经元，这些神经元可用于使用免疫细胞化学染色进行形态分析，用于基因组分析和生化研究，例如用于蛋白质学分析。有关免疫污处理和蛋白质组分析的样品制备的详细方案，请参阅手稿。图三显示了从 MAP-2 蛋白在培养胚胎大鼠交感神经元中的免疫抑制。面板 A 到 D 显示使用控制介质和面板 E 治疗的培养 SCG 神经元，通过 H 显示使用 BMP-7 治疗的神经元，每毫升 50 毫微克，为期五天。具有代表性的显微图显示具有C和G中微管相关蛋白-2的神经元的免疫细胞化学染色，D和H中的核染色，B和F中具有相对比显微图，以及来自微管相关蛋白-2的三个通道出现， 存在于细胞体中，在面板C中控制神经元的近端轴突中存在，并且从细胞体中的 MAP-2 蛋白中染色， BMP-7 治疗神经元中的树突位于面板 G. 这里是使用控制介质治疗的优越宫颈神经群的样本运行，该样本检测出 1，100 个蛋白质。使用黑豹数据库的基因本体学发现，大多数蛋白质是细胞质的。然而，在样本中，细胞结点有膜结合的蛋白质和蛋白质。这一分析还表明，在样本中检测到涉及各种神经元信号通路中的蛋白质。总之，看完这段视频后，你应该能够分离和培养大鼠胚胎优越的宫颈结节神经元进行形态学和蛋白质学分析。这项工作由加州圣玛丽学院的教师发展基金和暑期研究计划资助。

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JoVE Core

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Anatomy and Physiology

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Unit 1

The Autonomic Nervous System

SCIENTISTS IN ACTION

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

结状神经节注射液只SD大鼠的方法

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Diffusion Imaging in the Rat Cervical Spinal Cord

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...