这项工作得到北京自然科学基金 (7162124 号) 和北京中医药大学新奥基金会的支持。

所有的动物都是按照中国关于实验室动物的道德使用和护理的法律来维护的。
1. 预建模前准备
<ol><li>
实验室准备
	<ol>
<li>在实验前至少30分钟运行紫外线灯。</li>
		<li>控制实验室温度在 25 3 摄氏度, 相对湿度约30% 左右。</li>
		<li>打开实验室灯在6:00 和关闭在18:00 建立一个 12 h/12 h 光/暗循环。</li>
	</ol>
</li>
	<li>
多平台箱结构
	<ol>
<li>构造一个不透明的塑料罐, 不盖 110 x 60 x 40 厘米3。</li>
		<li>固定十五个圆形平台 (h = 8 cm, d = 6.5 cm) 在坦克的底部, 有序地分布在三行和五列。在每个平台之间留出足够的空间, 各列之间大约有10厘米, 行之间有13厘米。</li>
		<li>在水箱的侧面设置一个出水口, 并安装一个水龙头。</li>
		<li>用一个挂在上面的食物盒做一个铁网盖。</li>
	</ol>
</li>
	<li>
分组和住房大鼠 注: 实验中使用8周大的雄性大鼠, 重约 200-210 克。在建模过程中, 老鼠成群地活着。<ol>
<li>用记号笔数老鼠的尾巴根。</li>
		<li>对大鼠进行称重, 排除极轻或重的老鼠, 将其余的随机分为模型和对照组。</li>
		<li>把老鼠轻轻地放进干净的笼子里, 让他们适应实验室至少3天。提供充足的水和食物供应。</li>
	</ol>
</li>
</ol>2. 用 MMPM 建模
注: 该过程从18:00 开始, 第二天以8:00 为单位, 每天总计14小时, 超过21天。为了避免干扰因素, 同一个人需要进行整个实验, 同时穿着相同的实验室大衣。实验中使用了10只大鼠。
<ol><li>把油箱放在平坦的表面,例如地板上。然后用大约7厘米的温暖 (25 3 °c) 的水填满油箱, 在平台平坦的下面大约1厘米。</li>
	<li>准备足够的食物和饮料为所有的老鼠在坦克1天。把饲料和水放进食物盒里, 把它挂在盖子上。 注意: 一些聪明的老鼠学会在食物盒上休息。如果是的话, 把他们送回坦克。</li>
	<li>把模型组的老鼠从笼子里拿出来, 抓住它们的尾巴, 然后轻轻地放进油箱里。在水中发射所有的老鼠, 而不是平台来激发他们对水的恐惧。确保每只老鼠都有一个平台站立, 而控制组的老鼠留在他们原来的笼子里, 有足够的食物和水。</li>
	<li>把油箱盖上监视老鼠以避免意外伤害。如果一只老鼠在水中停留超过1小时而不爬上平台, 从油箱中取出, 然后从测试中取下。</li>
	<li>14小时后, 把模型鼠从水箱里拿出来, 用烘干机擦干头发。如果老鼠的尾巴褪色, 就重新标记。把老鼠送回原来的笼子里, 给他们提供足够的食物和水。</li>
	<li>冲洗油箱的每个角落。抬高油箱一侧, 打开水龙头流出污水。</li>
	<li>用75% 乙醇喷雾消毒油箱, 并将其暴露在紫外线照射下。</li>
</ol>3. 模型评估: 行为测试
注意: 所有的测试都是在行为实验室进行的。在测试期间不允许噪音和额外光线, 以避免干扰。如果可能, 请使用同一个人进行每次测试。图像处理中灰度识别需要深色外套和手套。执行经常第一, 因为它对老鼠的行为影响最小。
<ol><li>
经常
	<ol>
<li>在打开的字段框上检查录音机, 确保它正确地连接到工作站上, 并覆盖盒子的每个角落。调整照明以消除框中的阴影。</li>
		<li>把老鼠搬进他们原来的笼子里的行为实验室。允许他们在测试前至少适应1小时。</li>
		<li>用75% 乙醇清洁和消毒盒子, 以确保以前的实验没有留下任何粪便或气味。</li>
		<li>从笼子里取出一只老鼠的背部, 轻轻地放进盒子的中央区域。迅速撤退的武器从箱子, 以免阻止射击。</li>
		<li>输入老鼠的号码, 开始录音。计数和记录大鼠垂直活动的频率, 包括饲养和攀登。</li>
		<li>5分钟后, 停止录音, 把老鼠从盒子里拿出来, 把它还给笼子。</li>
		<li>重复步骤 3.1.3 3.1.6 直到所有的老鼠都完成了测试。</li>
	</ol>
</li>
	<li>
EPM
	<ol>
<li>对经常 (步骤 3.1.1 3.1.2) 执行预检和驯化步骤。</li>
		<li>把老鼠从笼子里拿回来, 轻轻地放到两只胳膊的接合处。把老鼠朝左张开的手臂, 并迅速离开, 以免阻止射击。</li>
		<li>输入老鼠的号码, 开始录音。计数和记录不同臂入口的频率。如果老鼠在测试中掉下迷宫, 把它捡起来送回迷宫。记录数据分析的详细信息。</li>
		<li>5分钟后, 停止录音, 把老鼠拿出来, 把它还给笼子。</li>
		<li>去除粪便, 用75% 乙醇擦拭迷宫, 消除前鼠的气味。</li>
		<li>重复步骤 3.2.2 3.2.5 直到所有的老鼠都完成了测试。</li>
	</ol>
</li>
	<li>
ES 试验
	<ol>
<li>填充泳池 (70 x 30 x 110 厘米3) 与 80 cm 温暖 (25 @ 3 °c) 水。 注: 如果水箱中有恒温器, 水温应设定在37摄氏度左右, 这与大鼠的体温相似。如果没有, 将其设置为室温以保持其恒定。</li>
		<li>给每只有针束的老鼠装上重物, 然后轻轻地把它绑在尾巴根部。负荷重于大鼠体重的10%。</li>
		<li>抓住一只老鼠的尾巴, 把它扔进泳池。如果老鼠蜷缩或附着在墙上, 把它们分开, 把它们驱回水中。</li>
		<li>开始计时的时候, 当老鼠被放进水中, 停止时间时, 它的疲惫, 这是证明是失败的水与嘴和鼻子在水下超过十年代。 注意: 有时, 疲惫和溺水突然发生。一定要有足够的实验者记录和保存的动物在同一时间。</li>
		<li>把筋疲力尽的老鼠从水中取出, 不要打断别人。干他们的头发, 重新标记他们的数字, 并把他们送回笼子。</li>
		<li>一组完成后, 更换水箱中的水。在所有的老鼠都完成后, 清空泳池, 用乙醇和紫外光清洁和消毒。</li>
	</ol>
</li>
</ol>4. 模型评估: 中枢神经递质检测
<ol><li>麻醉大鼠腹腔内注射10% 水合氯醛 (3 毫升/千克) 直到昏迷。</li>
	<li>斩首老鼠。</li>
	<li>沿后内侧线进行纵切口, 打开颅骨两侧, 露出大脑。把头骨翻过来, 把大脑移开, 把大脑放在冰袋上。</li>
	<li>分离并除去下丘脑, 它是位于大脑基底中央部分的菱形区域, 与周围组织有明显的边界。把它放在不育的管子里, 用液氮冷冻。把所有的样品都存放在-80 摄氏度的冰箱里。</li>
	<li>用高效液相色谱法 (HPLC)8测定下丘脑中 DA 和 5 HT 的含量。</li>
</ol>

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作者没有什么可透露的。

MMPM 最初设计为睡眠剥夺9。老鼠被发射到水箱里, 平台固定在底部。由于对水的本能的恐惧, 老鼠仍然站在平台上, 没有睡眠发生。研究表明, 不同时间的睡眠剥夺导致大鼠行为和情绪的变化, 包括认知障碍10, 消极情绪11, 中心疲劳。一些研究人员证明, 采用单平台法 (SPM) 的慢性睡眠剥夺可诱发中枢性疲劳, 并有认知障碍和社会障碍12。其他研究表明, 连续几天间歇性剥夺会导致情绪紊乱和中枢性疲劳, 这可以用内啡肽13治疗。我们以前的研究证明, 与5天14天相比, 21 天的剥夺导致更多的中枢性疲劳, 而不是与压力相关的情绪障碍14。MMPM 的许多因素可能导致中枢性疲劳, 包括长时间站立、狭小的遮蔽空间、单调乏味和重复的环境以及缺乏睡眠。睡眠剥夺与中枢性疲劳之间的潜在相关性可能与下丘脑-垂体-肾上腺 (HPA) 轴在不同的水平, 其中单胺类神经递质的变化可能发挥关键作用。
在本协议中, 我们开发了一个使用 MMPM 的中心疲劳模型, 并通过行为测试和神经递质检测对其进行评估。首先, 我们在实验室创建一个 12 h/12 h 光/暗循环 (6:00-18:00) 来模拟大鼠的自然昼夜节律, 其平均睡眠时间为12.6 小时, 夜间约 2.4-4.2 小时, 白天 8.2-9.6 小时.然后将模型鼠放入多个平台盒中, 每天连续21天站立14小时 (18:00-8:00), 对照组设置为对比。实验结束时, 模型组中的大鼠表现出明显的疲劳状态, 包括钝毛、微弱的尾部颜色、暗淡的眼睛以及笼中活动的减少。
行为测试的结果显示身体和情绪方面的变化。经常被广泛应用于啮齿动物模型评估中, 以评估勘探行为和自愿活动16。啮齿类动物有 thigmotaxis 的本能, 即一旦它们被放进一个开阔的领域, 它们就会迅速地移动到墙附近的外环。同时, 他们对新的环境感到好奇, 并且渴望通过垂直饲养和横向运动来探索中心区域。两种动机的冲突反映了焦虑情绪17。结果表明, 模型大鼠的自愿活动下降的平均速度下降的基础上。此外, 模型大鼠的饲养频率与控制大鼠相比明显降低, 这可能意味着焦虑情绪。此外, 模型大鼠在进入外环时往往花更多的时间, 在探索中没有明显的偏好, 提示模型大鼠的空间认知障碍。该测验是评估焦虑的经典测试。焦虑的大鼠倾向于保持密切的手臂安全, 而不是探索开放的手臂18。结果表明, 模型大鼠与对照组相比, 在闭合臂上花费的时间较多, 张开臂的时间较短, 与不同臂的进入频率相似, 总体上验证了模型大鼠的焦虑情绪。在 ES 试验中, 模型大鼠的游泳时间比控制大鼠短得多, 提示疲劳导致的肌肉表现不佳。结果表明, 模型大鼠焦虑、认知障碍、肌肉表现不佳、自愿活动有限, 均表现为中枢性疲劳。
对于中枢神经系统, 中枢神经递质的所有变化都表明中枢疲劳。我们发现大容量的显著下降和下丘脑的 5 HT 的增加。5-HT 是由氨基酸色氨酸 (跨国激进党) 合成的单胺类神经递质。剧烈的活动会增加5多羟的世代, 释放更多的自由激进党进入血液;累积的 5 HT 作为回报, 抑制了电机系统的中央控制, 导致较差的肌肉验19。DA 是一种易激动的神经递质, 在运动神经活动开始时增加, 并通过疲劳20的出现而下降。DA 和 5-HT 关联并相互作用作为一个励磁抑制系统, 影响到电机系统的中心控制21。因此, DA 与 5-HT 比值的下降是中枢疲劳的一个重要指标。
《议定书》中有一些对成功至关重要的说明。首先, 对雄性大鼠的模型持续时间和条件进行了测试。温度偏好和睡眠持续时间在菌株和性别之间差异14。其次, 大鼠应该在一个笼子里生活, 最多有6只老鼠, 在整个实验中提供足够的食物和水。在建模的头两个星期里, 老鼠很恼火, 可能在坦克和笼子里打架。继续监视他们, 防止受伤的老鼠死亡。另外, 记得在从水箱中取出后, 把老鼠的头发晒干, 尤其是在冬天, 以防寒冷的情况。
虽然该模型是为中心疲劳设计的, 但增加复杂因素以扩大其使用是可行的。例如, 我们安装振动马达和弹簧到平台上模拟海浪和改变剥夺模式, 试图建立一个导航疲劳模型。通过调整模型持续时间, 可以在许多其他字段中使用。作为动物模型研究, 该研究有其局限性。首先, 模型的预测有效性没有证据。在今后的研究中, 我们应该对大鼠进行抗疲劳治疗, 并对其恢复进行评估, 以证明模型的有效性。此外, 目前对该模型的评价更侧重于消极情绪和中枢神经系统的变化;然而, 中央疲劳也体现为学习困难和社会回避12。行为测试如莫里斯水迷宫和社会互动测试可以进行, 以获得更全面的了解疾病。我们希望在这里引入的中心疲劳模型可以帮助探讨中枢疲劳的病理机制。

疲劳是威胁人类健康的主要因素之一1。在过去的几十年里, 各种研究已经证明疲劳是外围触发的, 但集中驱动, 总是伴随着情感和认知障碍。意大利生理学家 a. Mosso 首先提出了 "中心疲劳2" 这个词。它一般被定义为有限的自愿活动和认知功能障碍, 由于中枢神经系统的冲动传输功能障碍 (CNS)3。与周围肌肉疲劳相比, 中枢性疲劳强调中枢神经系统的变化, 以及由此产生的情绪/行为紊乱, 包括抑郁、焦虑、认知障碍和记忆力减退。一项研究表明, 许多因素会导致中枢性疲劳, 其中过度的体力活动和精神压力是相当不可缺少的4。至于发病机制, 如色氨酸-犬尿氨路径假说5解释某些途径的变化;然而, 还需要更深入的研究来揭示中枢性疲劳的中枢-周边相关性。
由于中心疲劳的基本机制尚不清楚, 有效的动物模型对进一步的研究具有十分重要的意义。现有的疲劳模型大多是由过度运动引起的, 如跑步机6和负重游泳7, 很少关注心理因素。为了更好地模拟中心疲劳的发展, 我们小组开发了 MMPM 的大鼠模型。在建模过程中, 老鼠仍然站在狭窄的平台上, 在多个平台盒长时间, 包括部分睡眠时间。与过度运动模型不同, MMPM 模型采用部分睡眠剥夺作为心理因素, 考虑到复杂的中枢性疲劳发病机制。
对于模型评价, 我们使用经常和测验来确定焦虑情绪和自愿活动。采用 ES 试验对周围肌肉的性能进行测量。此外, 我们采取大鼠的大脑和检测多巴胺 (DA)/血清素 (5 HT) 的内容, 在两个 hypothalamuses 观察中枢神经递质的差异。
下面提出的协议旨在模拟在人类生活中常见的情况下, 反复的体力活动和缺乏睡眠导致的中枢疲劳。然而, 通过调整模型的持续时间, 它可以用于许多其他领域, 如睡眠观察和应力研究。在今后的研究中, 我们希望这一模型能帮助发现更多的中枢神经系统变化及其与周边体系的联系, 揭示中枢性疲劳的发病机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

本文采用改进的多平台法 (MMPM), 介绍了一种大鼠中心疲劳模型。多平台箱设计成一个水箱, 底部有狭窄的平台。模型鼠被投入了坦克并且在平台上站立了14小时 (18:00-8:00) 每天连续21天, 与空白控制小组设置为对比。建模结束时, 模型组大鼠表现出明显的疲劳状态。为了评估模型, 我们进行了几个行为测试, 包括开放场测试 (常), 高附加迷宫 (吸盘) 测试, 和详尽的游泳 (ES) 测试。结果表明: 模型大鼠焦虑、空间认知功能障碍、肌肉表现不佳、自愿活动减少, 证实了中枢性疲劳的诊断。中枢神经递质的变化也验证了结果。最后, 该模型成功地模拟了中心疲劳, 未来的研究与模型可以帮助揭示疾病的病理机制。

我们用 MMPM 描述了一种大鼠中心疲劳模型。24只大鼠随机分为对照组和模型组, 每组12只大鼠。该模型装置设计成一个水箱, 底部有狭窄的平台 (图 1)。模型鼠每天在平台上站立14小时, 包括部分睡眠时间, 21 天 (图 2)。
行为测试是在建模后进行的, 以评估大鼠的情绪和物理变化。通常的结果 (图 3) 表明, 与对照组相比 (n = 10), 在模型大鼠 (n = 10) 的饲养运动和自愿活动的平均速度 (p < 0.05, p < 0.01) 显著下降, 和外环入口的延迟明显增加 (p < 0.01)。该测试 (图 4) 显示, 21 天的建模减少了张开臂输入和持续时间的频率与对照组相比 (n = 10) (p < 0.05, p < 0.01), 同时增加了接近臂进入和持续时间的频率 (p < 0.05)。ES 试验 (图 5) 的结果表明, 模型组 (n = 10) 的游泳持续时间明显少于对照组 (n = 10) (p < 0.001)。
接下来, 我们检测到两个 hypothalamuses 中的 DA 和 5 HT 含量, 观察中枢神经递质的差异。结果 (图 6) 显示, 与对照组相比 (n = 10) (p < 0.05, p < 0.01), 下丘脑的 da 和 da 到5的比值显著降低 (n = 10), 而 5 HT 含量增加显著 (p < 0.05)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57362/57362fig1.jpg"> 图 1: 多平台框的示意图.(A) 前视图。(B) 最高视图。多平台盒是一个 coverless 塑料罐 (110 x 60 x 40 cm3) 与十五个丙烯酸平台固定在底部和一个水龙头的侧面。每个平台由一根柱子和一个圆形平面 (d = 6.5 cm) 平台组成, 大于柱子顶部。平台 (h = 8 厘米) 分布在三行和五列中。相邻的平台在列中相隔10厘米, 行中为13厘米。该罐最多可容纳15只老鼠。<a href="/files/ftp_upload/57362/57362fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57362/57362fig2.jpg"> 图 2: 建模的照片.站在站台上的老鼠是建模的第十五天。它的干头发和暗淡的眼睛表明一个明显的疲劳状态。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57362/57362fig3.jpg"> 图 3: 经常分析.(A) 垂直活动频率的比较。数据显示为均值电子扫描电镜 (n = 10)。以不等方差 (F = 9.877, p = 0.006 < 0.05), 意义由独立样品 t 测试, t = 2.226, p = 0.049 < 0.05 决定。模型大鼠 (n = 10) 与对照组相比, 垂直活动 (饲养) 的频率下降 (n = 10)。(B) 对自愿活动的平均速度进行比较。数据显示为中 IQR (n = 10)。意义是由曼-惠特尼 U 测试, z =-2.685, p = 0.007 < 0.01 决定。模型大鼠自愿活动的平均速度与对照组相比下降。(C) 对外环入口的延迟进行比较。数据显示为均值电子扫描电镜 (n = 10)。以不等方差 (F = 5.748, p = 0.028 < 0.05), 重要性由 t 测试, t =-3.724, p = 0.03 < 0.01 决定。模型大鼠的外环入口潜伏期增加, 这意味着它们比控制大鼠花更多的时间进入出环。注: p< 0.05 (*);p <0.01 (**);p < 0.001 (**)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57362/57362fig4.jpg"> 图 4: 对吸盘测试的分析.(A) 对张开臂输入频率的比较。数据显示为均值电子扫描电镜 (n = 10)。具有等方差 (F = 0.982, p = 0.348 > 0.05), 意义由 t 检验, t = 2.710, p = 0.014 < 0.05 决定。模型大鼠 (n = 10) 的张开臂输入频率与对照鼠 (n = 10) 相比下降。(B) 在张开臂上的持续时间比较。数据显示为均值电子扫描电镜 (n = 10)。具有等方差 (F = 0.100, p = 0.755 > 0.05), 意义由 t 检验, t = 3.304, p = 0.004 < 0.01 决定。模型大鼠的张开臂持续时间与控制大鼠相比下降, 这意味着模型大鼠在张开臂上的时间较少。(C) 接近臂输入频率的比较。数据显示为均值电子扫描电镜 (n = 10)。具有等方差 (F = 0.141, p = 0.712 > 0.05), 意义由 t 检验, t =-2.466, p = 0.024 < 0.05 决定。模型大鼠闭合臂项的频率随对照组的增大而增加。(D) 密切武装的持续时间比较。数据显示为均值电子扫描电镜 (n = 10)。以不等方差 (F = 4.796, p = 0.042 < 0.05), 重要性由 t 测试, t =-2.736, p = 0.0016 < 0.05 决定。模型大鼠的闭合臂持续时间与控制大鼠相比增加, 这意味着模型大鼠在闭合臂上花费的时间更多。注: p < 0.05 (*);p <0.01 (**);p < 0.001 (**)。<a href="/files/ftp_upload/57362/57362fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57362/57362fig5.jpg"> 图 5: 详尽的游泳试验分析.数据显示为中 IQR (n = 10), 并报告为p < 0.05 (*), p < 0.01 (**), p < 0.001 (**)。意义是由曼-惠特尼 U 测试, z =-3.326, p = 0.001 确定。模型大鼠的游泳时间 (n = 10) 比对照鼠明显短 (n = 10)。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57362/57362fig6.jpg"> 图 6: 中央神经递质含量分析.(A) 对 DA 内容的比较。数据显示为均值电子扫描电镜 (n = 10)。具有等方差 (F = 0.088, p = 0.771 > 0.05), 意义由 t 检验, t = 3.717, p = 0.002 < 0.01 决定。模型大鼠 (n = 10) 与对照鼠 (n = 10) 相比, 两 hypothalamuses 的 DA 含量均呈下降趋势。(B) 5 HT 含量的比较。数据显示为均值电子扫描电镜 (n = 10)。以不等方差 (F = 5.282, p = 0.034 < 0.05), 重要性由 t 测试, t =-2.997, p = 0.012 < 0.05 决定。在模型大鼠中, hypothalamuses 的 5 HT 含量与对照大鼠相比均有下降趋势。比率比较。数据显示为中 IQR (n = 10)。意义是由曼-惠特尼 U 测试, z =-3.175, p = 0.001 确定。模型大鼠与对照大鼠相比, DA-5-HT 的比值显著降低。注: p < 0.05 (*);p <0.01 (**);p < 0.001 (**)。

Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

在这里, 我们提出了一个使用改进的多平台方法 (MMPM) 来引入大鼠中心疲劳模型的协议。

基于改进多平台法的大鼠中心疲劳模型

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

a-rat-model-central-fatigue-using-modified-multiple-platform

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Beijing University of Chinese Medicine. 在这里, 我们提出了一个使用改进的多平台方法 (MMPM) 来引入大鼠中心疲劳模型的协议。

视频: A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

科研

神经科学

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

区域选择性基因枪瞄准的器官脑片用改进的基因枪

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

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 ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

高接触密度的制作，平面接口神经电极用于记录和刺激的应用

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

动态定量感觉测试表征中央疼痛处理

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

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 ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...