这项工作得到部分支持的 jsp KAKENHI 赠款号 24700349, 24659093, 25293239, JP18H02536 和 17K07086, 下个 KAKENHI 赠款号码25110737和 23110525, 阿曼德赠款号码 JP18ek0109311, SENSHIN 医学研究基金会和日本癫痫研究基金会。

所有实验程序均经东京都医学科学院动物护理和使用委员会批准。建议产后 8-16 周大鼠。任何近交菌株都是可以接受的实验。C57BL/6 小鼠对 ptz 更有抵抗力, 而 BALB/c 和瑞士白化小鼠对 ptz 更敏感。C57BL/6 在这项研究中被使用。对 PTZ 的脆弱性也取决于鼠标的年龄。与小鼠相比, 大鼠更难在 PTZ35。这种方法使用的动物数量可能会有所不同, 但每种情况下至少需要 6-10 只动物。
1. 云台的研制
<ol><li>溶解2毫克/毫升 PTZ 在无菌 0.9% (瓦特/v) 氯化钠。在使用日准备云台。</li>
</ol>2. 云台的注入
<ol><li>在上午9:00 至下午12:00 (中午) 之间进行所有实验。</li>
	<li>测量动物的体重。</li>
	<li>将动物放在观察室中以适应习惯。</li>
	<li>在适应期 (3 分钟) 内, 根据动物的体重和先前确定的注射剂量, 计算出该注射液的云台溶液体积。 注: 例如, 当使用35毫克/千克 ptz 时, 0.875 毫克的 ptz 是需要的鼠标重25克 (35 毫克/千克 x 0.025 公斤 = 0.875 毫克)。因此, 437.5 ul 的2毫克/毫升 PTZ 解决方案应注射 (0.875 毫克/2 毫克/毫升 = 0.4375 毫升)。云台的注射剂量取决于小鼠的基因型和应变。对于野生型 C57BL/6 小鼠, 建议第一次试验的剂量为30-35 毫克 PTZ/千克体重。对 PTZ 的灵敏度取决于使用的鼠标应变36,37。注射剂量根据实验目的而异。</li>
	<li>注射 PTZ 腹腔与一个1毫升注射器连接到一个27口径的针, 到左或右象限的动物腹部。避免在中线注射。<ol>
<li>避免重复注射在同一位置。</li>
	</ol>
</li>
	<li>观察云台管理后30分钟的动物行为 (图 1A), 并对异常行为进行分类和评分, 如下所示。如果可能的话, 保持观察动物24小时, 或至少增加 6-10 h 后注射, 特别是一旦癫痫严重评分达到3或以上。<ol>
<li>注意在30分钟的观察期以外的动物轻微的癫痫发作或行为改变。这一长期观察期可以解决一个特别重要和完全的影响, subconvulsive 剂量 PTZ 在产生慢性无端癫痫发作。</li>
		<li>此外, 测量每项观察到的癫痫发作持续时间, 因为扣押期间的变化与缉获严重程度有关。此外, 云台注射后第一次癫痫发作的潜伏期是另一项重要的收集措施。监测癫痫发作的频率、持续时间和潜伏期对于任何癫痫后的分子研究都是至关重要的。</li>
	</ol>
</li>
	<li>每隔一天注入 PTZ (图 1A)。注射的数量取决于实验的目的。有代表性的例子如下。<ol>
<li>要生成完全被点燃的动物, 一旦动物经历了癫痫发作, 分数为 5 (补挛癫痫), 在额外的三个行政部门内完成注射。在这种情况下, 增加注射剂量, 如果癫痫评分不增加三个连续的管理。每种动物可以接受不同数量和剂量的 PTZ 注射。</li>
		<li>确定每种情况下 ptz 注射的数量和剂量, 以评估对 ptz 的脆弱性, 包括对抗癫痫药物的评估和转基因小鼠表型的研究。给所有动物相同数量的 PTZ 注射;建议共注射8至12针。如果动物死亡, 癫痫评分应记为6。</li>
		<li>确定注射剂量, 以维持高癫痫评分 (4 或 5) 10 或更多的注射, 以研究发生癫痫发作的病理变化。在这种情况下, 总注入数应介于25到30之间。如果癫痫评分达到 5, 则减少注射剂量。如果癫痫评分降低到3或更低, 增加注射剂量。</li>
	</ol>
</li>
</ol>3. 扣押评分
<ol><li>观察动物行为, 记录分数。<ol>
<li>分类和评分的癫痫行为如下38,39 (图 1B): 0: 正常行为, 无异常。 1: 固定, 躺在腹部。 2: 头点头, 面部, 前肢, 或后肢挛。 3: 连续的全身挛, 挛的抽搐, 尾巴僵硬地举行了。 4: 养育, 滋补发作, 跌倒在它的边。 5: 补药-挛发作, 跌倒在它的后面, 狂放的冲和跳跃。 6: 死亡。</li>
	</ol>
</li>
	<li>根据实验条件, 更改基于拉辛评分40的行为准则。</li>
</ol>4. 扣押后分析
<ol><li>Immunohistopathological 分析<ol>
<li>灌注固定<ol>
<li>制备<ol>
<li>溶解 4% (w/v) 多聚甲醛 (粉煤灰) 在0.1 米磷酸盐缓冲 (pH 7.4)。加热解答到大约70°c 以加法 1 m 氢氧化钠 (大约1µL 1 m 氢氧化钠为50毫升煤灰解答)。</li>
				<li>准备冰冷4% 粉煤灰和磷酸盐缓冲盐水 (PBS)。</li>
				<li>用管子和针头设置蠕动泵。通过管子运行大约20毫升的水来清除残留物。然后, 把管子的开端放在冰冷的 PBS 里。</li>
			</ol>
</li>
			<li>Transcardial 灌注与固定脑的制备<ol>
<li>用 50% isoflurane/50%ethanol 在一个小罐子里深深地麻醉老鼠。</li>
				<li>将老鼠的前肢和后肢的尖端固定在仰卧位。</li>
				<li>切开腹部腹中线, 露出隔膜。</li>
				<li>沿肋边切开隔膜。然后, 切开两侧的肋骨和身体。用锁钳捏剑突过程, 埃弗特肋骨笼。保持剑突过程的位置, 用针钉住它或用镊子捏紧它。暴露心脏。</li>
				<li>将针头插入左心室, 用剪刀在右心房切开。注意不要把针推到心脏太远, 因为它可以刺穿内壁。</li>
				<li>通过针开始一个稳定的冰的 PBS 流, 以洗涤血液 (大约 15-20 毫升/分钟)。</li>
				<li>当血液从体内清除后, 将管子移至4% 多聚甲醛溶液 (大约60毫升), 而不添加气泡。然后, 停止灌注。</li>
				<li>切开后颈部和脊柱, 剥去头部皮肤和头骨的背部部分用解剖剪刀。</li>
				<li>在眼球轨道之间切开前上颌骨骨, 并完全移除头骨的背部。</li>
				<li>在大脑和泪骨之间建立一个间隙, 并在大脑下方切开三叉神经和视神经交叉。小心地取出大脑, 放在含有4% 只粉煤灰的瓶子里。后固定的大脑在 4°c 12-16 小时。</li>
			</ol>
</li>
		</ol>
</li>
		<li>脑切片制备<ol>
<li>将固定的大脑转移到20% 的蔗糖/PBS, 直到大脑吸收到溶液中, 以允许抗冻。</li>
			<li>根据所需的切片方向和大脑需要的部分来切割固定的大脑。</li>
			<li>设置 cryomicrotome。设置刀片位置, 并保持在-20 摄氏度以下的温度切片阶段。</li>
			<li>将预切的大脑放在舞台上, 用20% 的蔗糖/PBS 将大脑涂上。蔗糖/PBS 溶液将逐渐冻结, 覆盖大脑的舞台上, 直到大脑完全嵌入冷冻20% 蔗糖/pbs。</li>
			<li>通过将刀片滑入组织, 切片大脑 (30 µm 厚度)。将切片转移到 PBS 并保持在4摄氏度。</li>
		</ol>
</li>
		<li>荧光免疫组化<ol>
<li>清洗必要的切片在0.1% 海卫 X-100/PBS (PBST) 10 分钟, 3 倍室温 (RT)。</li>
			<li>在 PBST 中用2% 山羊血清或5% 牛血清白蛋白 (BSA) 在 RT 中进行 1-2 小时的孵化, 阻断切片。</li>
			<li>以2% 山羊血清或 5% BSA 为1至7过夜, 在4摄氏度的 PBST 中, 以适当浓度的原抗体在抗体溶液中孵化切片。</li>
			<li>将 PBST 中的切片洗净10分钟, RT 3 次。</li>
			<li>在二次抗体溶液中孵育切片, 适当浓度为 1-2 小时的 PBST 中的二级抗体, 光保护。</li>
			<li>将切片安装到玻璃滑梯上, 并盖上安装介质和盖玻璃。</li>
			<li>用荧光显微镜观察切片。</li>
		</ol>
</li>
	</ol></li>
	<li>三室试验<ol>
<li>制备<ol>
<li>准备至少 2 2-6 月大的 129/Sv 小鼠相同的性别作为主题小鼠是 "陌生人老鼠"。习惯所有老鼠在笼子前进行分析。对于每个训练会话, 将鼠标放在笼子中, 并将鼠标留在15分钟。</li>
			<li>在每个适应阶段与主题老鼠, 清洗整个仪器和两个笼子通过擦拭表面与70% 乙醇。</li>
			<li>把空笼子放在3室器具的两个侧室里, 打开房间的门。</li>
			<li>要习惯主题鼠标, 请在中心腔内放置一个主题鼠标, 并允许鼠标在整个设备上自由移动, 直到鼠标对两个笼子进行了调查, 再加上5分钟. 可能是害怕笼子的主题动物不调查笼20分钟, 不应使用此分析。这样的动物避开笼子, 很少靠近笼子。</li>
			<li>动物进入中心腔后, 关闭车门, 让鼠标在中心房间内自由移动5分钟。在此期间, 把 129/Sv 老鼠的一个笼子里习惯到笼子里去。</li>
		</ol>
</li>
		<li>社会性分析<ol>
<li>在主题鼠标的适应期后立即进行分析。</li>
			<li>放置包含 129/Sv 鼠标的笼子, 称为 "陌生人1笼", 在其中一个侧面室和放置空笼, 称为 "对象笼", 在另一侧的房间。</li>
			<li>打开门并监视主题鼠标的行为。</li>
			<li>允许主题鼠标自由移动10分钟, 并记录以下行为参数。如果主题鼠标害怕在陌生人1笼中的鼠标, 如鼠标所示避免陌生人1笼和留在一个房间的角落里, 动物不应该用于分析。 a) 在每个会议厅、陌生人1室、对象室和中心室度过的时间。 b) 调查每个笼子、陌生人1笼和物体笼所花费的时间。(如果鼠标嗅探或增韧鼠标或笼子, 则将鼠标归类为调查笼子) c) 每个房间的入口数量 (可选) d) 旅行总距离 (可选)</li>
			<li>鼠标移动到中心房间后, 把门关上。</li>
		</ol>
</li>
		<li>社会新奇分析<ol>
<li>在社会性分析后立即进行分析。</li>
			<li>用70% 乙醇擦拭两个笼子。</li>
			<li>将另一只 129/Sv 鼠标放在其中一个笼子里, 称为 "陌生人2笼", 并将陌生人2笼放在其中一个房间里。将陌生人1只老鼠放在另一个笼子里, 称为 "陌生人1笼", 并将陌生人1笼放在另一个房间里。</li>
			<li>打开门并监视主题鼠标的行为。</li>
			<li>允许主题鼠标自由移动10分钟, 并测量相同的行为参数描述的社会性分析。 注: 陌生人1和陌生人2鼠标应随机选择。陌生人1房间和对象房间并且陌生人1个房间和陌生人2个房间应该被随机地确定。</li>
		</ol>
</li>
	</ol></li>
	<li>语境恐惧歧视<ol>
<li>调节<ol>
<li>每次调节试验前, 用70% 乙醇擦拭表面, 清洁实验装置。</li>
			<li>将鼠标放在具有特定条件的空调设备 (设备形状、墙壁颜色、地板材质、气味、照明和背景噪音音量应预先确定)。例如, 这里使用的空调设备是一个方形的设备, 具有清晰的有机玻璃墙, 金属网格地板, 乙醇气味, 100 勒克斯亮度和 65 dB 背景白噪声。</li>
			<li>在5分钟的时间内, 用 0.1 mA 电击的伪随机定时对小鼠进行足部冲击, 2 x 3 次。</li>
			<li>调节后将鼠标返回到主笼子。</li>
		</ol>
</li>
		<li>内存评估<ol>
<li>每次评估前, 用70% 乙醇擦拭表面, 清洁实验器具。</li>
			<li>第二天调理后, 将鼠标放到同一个装置中作为休克状态, 并在5分钟的过程中测量凝固时间。</li>
			<li>同一天晚些时候, 将鼠标放在一个新的仪器中, 在5分钟的评估中测量冰冻时间。在这里使用了一个三角形的设备, 白色有机玻璃墙, 平板地板, 没有气味, 30 莱克丝亮度和70分贝白噪音。</li>
			<li>比较了相同条件和新条件下的冻结时间。具有正常记忆能力的小鼠在相同的条件下会比在新的条件下冷冻更多。冰冻被定义为超过2秒的动物的静止。</li>
		</ol>
</li>
	</ol></li>
</ol>

作者声明没有利益冲突。

在这里, 我们提出了一个广泛的可访问的协议, 建立一个药理动物模型的癫痫。云台介导的化学点火具有悠久的历史, 是一种普遍接受的模型, 研究的病理学和细胞病理学的癫痫41。Suzdak 和詹森对癫痫的化学点燃模型进行了回顾, 199542。药物癫痫诱导, 特别是与 PTZ, 是一种简单和简单的方法来唤起严重癫痫发作。注射剂量的变化与癫痫严重程度相关。因此, 通过改?...

癫痫是一种慢性神经系统疾病, 其特点是反复发作, 影响大约1% 的人。在临床研究中不能充分阐明癫痫患者负反馈和癫痫产生的机制。因此, 对癫痫1的研究需要一个适宜的动物模型。
各种动物模型的癫痫已被用来调查癫痫的生理和识别抗癫痫药物2,3。在这些模型中, 药物癫痫诱导是一种常见的方法, 用于生成动物模型, 以调查4的癫痫病理。这种方法既便宜又简单。电极介导的点燃也是一种常用的方法, 但这一过程的成本较高, 该方法需要手术和电气技能, 以诱发重复性发作5。
药理诱导也有利, 因为发作的时间和数量容易控制。在癫痫的研究中也使用了表现为自发癫痫的基因小鼠模型。然而, 预测在这些基因模型中癫痫发作的时间和频率可能是不可能的6。为了观察基因修饰小鼠6的癫痫行为, 需要一个监测系统。
海仁酸、匹戊四氮 (PTZ) 被广泛用作癫痫诱导药物7。海仁酸是谷氨酸受体的促激动剂, 而匹属激活胆碱受体。PTZ 是一种伽玛丁酸 (GABA)-受体拮抗剂8。PTZ 抑制抑制性突触的功能, 导致神经元活动增加。这个章程导致普遍的缉获在动物9。单一注射海仁酸和匹特能诱发急性癫痫发作, 特别是癫痫持续状态 (SE)10,11和海仁酸-或匹特式介导硒促进慢性自发性和复发性癫痫12,13. 脑电图 (EEG) 录音和行为分析表明, 在一次注射12、13后一个月内, 自然复发性癫痫发作。单一注射的抽搐剂量的 PTZ 也诱发急性发作。然而, 单一注射 PTZ 后的慢性自发性癫痫很难推广。需要对 PTZ 进行慢性管理, 以诱发重复性癫痫14。在这两种方法中, 重复性癫痫的产生都能诱发一种与人类癫痫相似的病理, 而非急性癫痫的发生。在 PTZ 的情况下, 每次注射都能引起癫痫发作, 每次注射时, 检出的严重性都会逐步加重。最后, 单低剂量 PTZ 注射诱发严重的挛癫痫发作。在这个阶段, 每次注射都能唤起严重的癫痫发作。此外, 在注射过程中, 癫痫潜伏期和持续时间也会发生变化。在点燃15的后阶段, 补剂发作的潜伏期往往会缩短。此外, 癫痫发作加重还伴有长期发作期16。研究控制癫痫发作严重性、潜伏期和持续时间的分子机制对于筛查抗癫痫药物17、18、19有很重要的意义。
癫痫发作通常是由一个单一的系统管理的 PTZ, 恢复速度非常快, 在30分钟4,5。因此, 在云台点火模型中, 癫痫发作的次数更能控制。然而, 脑电图监测表明, 在云台介导的癫痫发作20后, 一般峰值可达12小时。因此, 动物最好保持观察24小时后, 挛或补药发作21 , 以更精确的分析点燃机制。
抗癫痫药物, 如 ethosuximide, 丙戊酸, 苯巴比妥, 氨乙烯酸, 和 retigabine3, 在 PTZ 注射之前或之后, 降低发作严重性3,22的恶化, 23。同样, 没有基因发作加重的击倒小鼠, 如基质 metalloproteinase-924, FGF-2225和 neuritin26, 已被证明在多台 PTZ 注射后, 检出严重程度降低。此外, 通过这种方法可以观察癫痫发作后的组织病理改变。颞叶癫痫患者有典型的组织学改变, 如苔藓纤维发芽27,28, 异常颗粒神经元迁移29, astrogliosis30, 神经细胞死亡在海马31,32和海马硬化症33。癫痫模型动物也观察到类似的变化。在现有的方法中, 云台介导的化学点火是一种很好的, 重现性和廉价的方法来产生一种动物模型的癫痫。在一个由匹凡达介导的 se 模型中, 癫痫控制是困难的, 许多小鼠死亡或无法发展 se34。相比之下, 在 PTZ 模型中, 死亡率和癫痫严重程度更可控制。此外, PTZ 比海仁酸便宜, 而且在小鼠脑外科的技能是不需要药物管理。

<table>
	<tbody>
		<tr>
			<td>Pentylenetetrazole</td>
			<td>Sigma-Aldrich</td>
			<td>P6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>MANAC</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>CLEA Japan</td>
			<td></td>
			<td>C57Bl/6NJcl, postnatal 8 week, male</td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle(27G x 3/4&#34;) (0.40 x 19 mm)</td>
			<td>Terumo</td>
			<td>NN-2719S</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing scale</td>
			<td>Mettler</td>
			<td>PE2000</td>
			<td>This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&#38;D company.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>nacalai tesque</td>
			<td>31511-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristatic pump</td>
			<td>ATTO</td>
			<td>SJ1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>nacalai tesque</td>
			<td>30404-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Yamato</td>
			<td>REM-700</td>
			<td>This item is a discontinued product. Almost equivalent to REM-710</td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Feather</td>
			<td>S35</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-synaptoporin antibody</td>
			<td>Synaptic systems</td>
			<td>102 002</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ZnT3 antibody</td>
			<td>Synaptic systems</td>
			<td>197 002</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-doublecortin</td>
			<td>Santa Cruz</td>
			<td>sc-8066</td>
			<td>This item is a discontinued product. We did not test equivalent product (sc-271390).</td>
		</tr>
		<tr>
			<td>Contextual fear discrimination test apparatus</td>
			<td>O&#39;hara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Three chamber test apparatus</td>
			<td>Muromachi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pentylenetetrazole-induced kindling mouse model

戊四氮 (PTZ) 是 GABA-受体拮抗剂。腹腔内注射 PTZ 进入动物诱导急性, 严重癫痫的高剂量, 而顺序注射 subconvulsive 剂量已用于发展化学点燃, 癫痫模型。单次低剂量的 PTZ 诱导轻度发作, 不抽搐。然而, 反复低剂量的 PTZ 注射降低了诱发痉挛性癫痫发作的门槛。最后, 云台连续低剂量管理诱发严重的挛癫痫发作。这种方法简单, 广泛适用于研究癫痫的病理生理学, 它被定义为一种涉及重复性癫痫的慢性疾病。这种化学点燃协议导致动物反复发作。利用这种方法, 估计易受云台介导的癫痫发作或癫痫发作加重的程度。这些优势导致了使用这种方法筛选抗癫痫药物和癫痫相关的基因。此外, 该方法已被用来调查癫痫发作后的神经细胞损伤, 因为在癫痫患者的大脑中观察到的组织学改变也出现在化学点燃动物的大脑中。因此, 该协议对于方便地制作癫痫动物模型是有益的。

反复注射 PTZ 诱发癫痫发作的严重性增加。对六 C57BL/6 小鼠进行了 PTZ 治疗, 另有6只小鼠以生理盐水作为对照组治疗。PTZ 剂量为35毫克/千克, 10 次注射。随着 PTZ 注射, 癫痫评分逐渐增加, 而生理盐水注射没有诱发癫痫发作或异常行为 (图 2)。方差分析后的 Bonferroni 试验显示, 云台治疗组与生理盐水治疗组有显著性差异。
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Watch this Scientific Journal Video about Pentylenetetrazole-Induced Kindling Mouse Model at JoVE.com

Pentylenetetrazole-Induced Kindling Mouse Model

本协议描述了一种戊四氮化学点燃方法, 并提供了一种小鼠癫痫模型。该协议还可用于调查小鼠癫痫发作后癫痫诱导和发病机制的脆弱性。

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

pentylenetetrazole-induced-kindling-mouse-model

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Unit 1

Pharmacotherapy of the Epilepsies

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32.5K 次观看.  Tokyo Metropolitan Institute of Medical Science. 本协议描述了一种戊四氮化学点燃方法, 并提供了一种小鼠癫痫模型。该协议还可用于调查小鼠癫痫发作后癫痫诱导和发病机制的脆弱性。

视频: Pentylenetetrazole-Induced Kindling Mouse Model

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量:血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

在多发性硬化症的小鼠模型测量进行性神经功能残疾

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

小鼠后脑作为研究胚胎神经再生的模型

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

电点燃VGAT-CRE小鼠电极的制备和植入，以生成颞叶癫痫模型

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...