这项工作得到了圣保罗研究基金会（FAPESP，拨款2017/00003-0）的支持。我们感谢提高高等教育人员的协调（CAPES）。我们感谢Anthony R. West博士，Heinz Steiner博士和Kuei Y. Tseng博士的支持和指导。

所有涉及动物的程序都遵循国家动物实验控制委员会（CONCEA）和阿鲁卡法（第11.794/2008号法律）的道德原则，并得到当地伦理委员会（CEUA-FFCLRP/USP（18.5.35.59.5）的批准。
1. 药物制备
<ol><li>用氯胺酮/西拉嗪麻醉 注意：氯胺酮的剂量为70mg / kg，木拉嗪的剂量为10mg / kg。<ol>
<li>要使用氯胺酮100mg / mL溶液和木拉嗪20mg / mL溶液制备1 mL麻醉剂，将0.35mL氯胺酮溶液，0.25mL木肼嗪溶液和0.4mL 0.9%无菌盐水溶液混合。以2 mL / kg的最终体积施用麻醉液。 注意：氯胺酮和木肼可以产生镇静60-80分钟。如果动物仍然有反射（例如，后腿俯仰和/或眨眼反射），则额外给予个体剂量的10%。</li>
	</ol>
</li>
	<li>丙咪嗪 注意：使用的丙咪嗪的个体剂量为20mg / kg。<ol>
<li>要制备1mL丙咪嗪20mg / mL溶液，将20mg丙咪嗪和1mL0.9%无菌盐水溶液混合。以1 mL / kg的最终体积施用丙咪嗪溶液。</li>
	</ol>
</li>
	<li>美洛昔康 注意：使用美洛昔康的个体剂量为1mg / kg。<ol>
<li>要制备1 mL美洛昔康1 mg / mL溶液，将0.05 mL美洛昔康2%和0.95mL0.9%无菌盐水溶液混合。以1 mL / kg的最终体积施用美洛昔康溶液，每天一次，持续两天。</li>
	</ol>
</li>
	<li>抗坏血酸0.1%<ol>
<li>要制备1 mL 0.1%抗坏血酸，将1mg抗坏血酸和1mL0.9%无菌盐水溶液混合。</li>
	</ol>
</li>
	<li>6-羟基多巴胺 注意：6-OHDA是一种神经毒素，用于选择性地破坏大脑中的多巴胺能和去甲肾上腺素能神经元。避免直接接触皮肤和眼睛、鼻子和嘴巴的粘膜。处理 6-OHDA 时，请戴上双丁腈手套、实验室外套、一次性防护服、护目镜、外科口罩或面罩。毒素的总输注体积为4μL/动物，个体量为10μg6-OHDA/动物。<ol>
<li>为了以2.5mg / mL的终浓度制备1mL 6-OHDA，混合2.5mg 6-OHDA和1mL含有0.1%抗坏血酸的0.9%盐水溶液（如上所述）。 注意：6-OHDA对光敏感，暴露在强光下时降解速度更快。它必须妥善处理并储存在避光的环境中。如果溶液的颜色为红色，请将其丢弃。</li>
	</ol>
</li>
	<li>盐酸利多卡因（2%）<ol>
<li>准备2%利多卡因溶液，用于动物局部施用。 注意：可以施加的最大剂量为7mg / kg。</li>
	</ol>
</li>
	<li>聚抗生素悬浮液 注意：含有链霉素和青霉素的聚抗生素悬浮液（见 材料表）必须在施用时用整个体积的稀释剂制备，其安瓿与粉末一起伴随小瓶。<ol>
<li>卸下橡胶塞上的金属制动盘。用酒精对橡胶塞进行消毒。</li>
		<li>使用针头为23 G的注射器，将稀释剂注入小瓶中。取出针头并用力摇晃小瓶，直到悬浮液完全均质化。将少量空气注入小瓶中，并抽出所需体积的悬浮液。</li>
		<li>进行深度肌内注射，在注射药物之前拉动柱塞，以确保没有到达血管。 注：最终应用的悬浮液体积为0.5 mL/kg。</li>
	</ol>
</li>
</ol>2. 准备材料
注意：处理化学品时，请始终遵循材料安全数据表中提供的说明。
<ol><li>立体定位装置<ol>
<li>将立体定位设备放在稳定干净的工作台上，并有适当的照明进行手术。用70%乙醇消毒设备。</li>
		<li>检查设备的耳朵和门牙条是否正确对齐。放置一条保暖毯，在手术期间放置动物，以便在手术过程中保持温暖。用精确的直肠探头监测动物的温度。 注意：热毯应处于37.5°C，以便动物保持37°C的身体温度。</li>
	</ol>
</li>
	<li>微量输液系统<ol>
<li>将汉密尔顿注射器（50μL或根据需要）填充（70-80%），连接到医用级聚乙烯微管和针头上，并用双蒸馏水（ddH 2 O）检查是否泄漏。</li>
		<li>将空气拉过系统，使单个气泡将注射器中的ddH2O与微管中的6-OHDA溶液分开。 注意：该程序避免了用6-OHDA污染汉密尔顿注射器，并允许在同一实验日使用几只大鼠。</li>
		<li>将 Hamilton 注射器放在输液泵上，使其牢固地连接，注射器的柱塞平行于将要移动以推动它的框架。将输液泵设置为0.5μL / min的速度，以便4μL6-OHDA的总施用持续8分钟。通过确认没有泄漏并且根据先前设定的时间和体积进行输液来测试输液系统。</li>
		<li>将附着在微管上的输液针连接到立体定位臂末端的装置上，并检查针头是否与表面成180°角。确保针是直的，而不是弯曲的。 注意：仔细检查所有描述的程序，因为如果输液系统中的任何项目不能正常工作，可能会危及手术的成功。</li>
	</ol>
</li>
	<li>缝合<ol>
<li>手术后使用无菌尼龙不可吸收缝合线和3/8圆针缝合切口。</li>
	</ol>
</li>
	<li>术后恢复站点<ol>
<li>放置一个干净且无菌的外壳盒，可以监测动物，直到完全恢复（对触摸和操作做出反应）。在盒子里放一个热毯进行体温调节。 注意：由于体温调节很重要，如有必要，请包括补充热源以保持体温。</li>
	</ol>
</li>
</ol>3. 外科手术
注意：在该协议中，成年雄性Sprague-Dawley大鼠（200-250g）被保持在温度（22±2°C），空气交换（15-20次交换/小时）和明暗循环（12小时/ 12小时）的受控条件下，与3或4只动物分组，可自由获得食物和水。
<ol><li>称量动物体重，以监测手术后几天内的体重变化。计算要施用的药物的剂量。</li>
	<li>术前30分钟（麻醉前约10-15分钟）腹膜内注射丙咪嗪，使用27G针头和1mL注射器。 注意：丙咪嗪会阻断去甲肾上腺素转运蛋白（NAT）并阻止去甲肾上腺素能神经元对6-OHDA的摄取，使病变对多巴胺能神经元更具选择性40。</li>
	<li>在给予丙咪嗪10-15分钟后，使用27G针头和1mL注射器进行腹膜内氯胺酮/木拉嗪麻醉。等到动物完全麻醉。验证当动物对后腿捏合没有反应并且没有表现出眨眼反射时，动物处于深度麻醉状态。</li>
	<li>剃掉头部区域的大鼠皮毛，在那里会发生切口。</li>
	<li>将大鼠置于立体定位装置中。<ol>
<li>将头部放在门牙杆上，并将门槛固定在耳际线以下3.3毫米处。</li>
		<li>将耳杆定位，一次一侧。放置门牙杆和耳杆，使颅骨的顶部笔直且平行于表面。</li>
		<li>调整鼻夹并测试头部是否牢固且不会向任何一侧移动。</li>
	</ol></li>
	<li>将无菌眼药膏涂抹在大鼠眼睛上，以防止角膜变干。</li>
	<li>将聚维酮碘涂抹在要切口的区域，以对部位进行消毒。</li>
	<li>局部利多卡因用于切口区域的镇痛;不要超过7毫克/公斤。</li>
	<li>使用27 G针头和1mL注射器皮下给药美洛昔康。 注意：美洛昔康是一种非甾体抗炎镇痛药，可帮助动物在手术后恢复。</li>
	<li>使用23 G针头和1mL注射器肌肉注射多抗生素悬浮液。 注意：多抗生素悬浮液作为预防性治疗给药，以避免术后恢复中可能的细菌感染。</li>
	<li>通过用镊子捏住后爪来检查动物是否处于深度麻醉状态，方法是检查眨眼反射或后肢反射。</li>
	<li>用手术刀在发生显微注射的区域做一个~1.5厘米的切口。 注意：无菌技术从此时开始应用，直到伤口闭合。</li>
	<li>用棉签和棉签清洁颅骨区域，直到可以看到Bregma和Lambda。用消毒的细笔标记Bregma和Lambda。</li>
	<li>检查Bregma和Lambda的背腹（DV）坐标是否相似。如果它们不同，则重新调整立体定位装置中的大鼠，因为大鼠的头部位置不正确。</li>
	<li>记下Bregma的前后（AP）和中外侧（ML）坐标。</li>
	<li>移动到右侧 MFB 的 AP 和 ML 坐标，依次为 41：AP：-4.3 毫米，ML：距 Bregma 1.6 毫米。</li>
	<li>用无菌的细笔标记钻孔区域。</li>
	<li>用消毒的钻头，慢慢刺穿动物的头骨，注意不要伤害硬脑膜。</li>
	<li>将显微注射针放在硬脑膜上，并记下DV坐标。取一根细针，轻轻地将硬脑膜破裂。将针头插入MFB的DV坐标（8.3mm腹侧），在那里将进行显微注射。</li>
	<li>操作显微注射泵，将 6-OHDA 溶液释放到 MFB 中。显微注射完成后，检查汉密尔顿注射器，看看是否注射了4μL的6-OHDA。 注意：显微注射应持续8分钟。</li>
	<li>施用6-OHDA后，等待10分钟，然后取出针头，以避免药物回流。从动物的大脑中慢慢取出显微注射针头。</li>
	<li>再次用聚维酮碘消毒切口区域。</li>
	<li>用约3-4个手术结缝合切口区域。 注意：结不应太强或太松。</li>
	<li>将大鼠从立体定位装置中取出，并将其放在干净的盒子中，以便在热毯上进行恢复，直到动物从麻醉中完全恢复。每15分钟观察一次动物，直到它从麻醉中完全清醒。</li>
</ol>4. 术后程序
<ol><li>在手术后四天内监测动物的体重。术后每天一次皮下注射美洛昔康，持续两天，调整每天体重的剂量。 注意：所有动物应在手术后第三天评估是否需要镇痛药。</li>
	<li>每天检查切口至少四天，以确保它们没有被感染。寻找热，肿胀，疼痛，分泌物和发红，直到切口愈合。</li>
	<li>通过监测动物的体重来检查食欲和水的消耗。给予湿饲料，以鼓励动物进食。手术后至少四天每天观察一般身体状况，态度和活动能力。手术后7-10天取下缝线。 注意：如果达到道德程序中定义的终点，则应对动物实施安乐死。</li>
</ol>5. 步进测试
<ol><li>训练 注意：动物应在测试前训练三天。根据下面描述的协议，训练应每天进行两次，一次在早上，一次在下午，或者两次训练之间间隔至少2小时。使用计时器跟踪时间。<ol>
<li>第 1 天<ol>
<li>在第一次会议中，通过戴上手套处理大鼠约1-2分钟，让大鼠熟悉处理者/实验者。</li>
			<li>在第二个会话中，交替将大鼠保持20秒并将其放在协议表上20秒。重复此训练步骤3分钟，以使大鼠熟悉步进测试的实验设置。</li>
		</ol>
</li>
		<li>第 2 天<ol>
<li>在第一个会话中，将大鼠的两个前爪放在协议表上，用一只手握住它的后爪和背部。将大鼠头朝下倾斜，角度为45°，与实验表的平坦表面成45°。在桌子上从头到尾水平移动，让老鼠用两只爪子踩在桌子上（在4秒内覆盖90厘米）。用手套将老鼠抱10秒，让它休息;重复此模式3分钟。</li>
			<li>在第二个会话中，将大鼠的一个前爪放在协议表上，用一只手握住另一个前爪，用另一只手握住大鼠的背部和后爪（见步骤5.1.2.1）。在4秒内水平移动桌子上的头到尾，让老鼠用它自由的爪子踩踏。将老鼠戴上手套10秒，让它休息，然后用另一个前爪重复，然后是休息时间。重复此模式，在两个前爪之间交替，休息3分钟。</li>
			<li>重复训练步骤3次，每次1分钟。</li>
		</ol>
</li>
		<li>第 3 天<ol>
<li>在第一个会话中，按照步骤 5.1.2.2 中描述的过程进行一次前爪。用另一个前爪重复上述步骤，然后是休息时间。重复此模式，在两个前爪之间交替，休息3分钟。</li>
			<li>在第二个会话中，按照步骤 5.1.2.2 中描述的过程进行操作。</li>
		</ol>
</li>
	</ol>
</li>
	<li>测试 注意：踏步试验在手术前，立体定位手术后2周和4周进行，以评估对侧前肢的运动障碍和6-OHDA可能引起的损伤。<ol>
<li>将大鼠保持在与表面45°的角度，使其后肢固定，并且如上所述，在训练的第3天，只允许其中一个前肢在平台上休息。</li>
		<li>在4秒内将老鼠向前拖动90厘米的距离，右爪或左爪放在表面上。</li>
		<li>做笔记并量化每个爪子在每个方向上采取的正手调整步骤的数量。</li>
	</ol>
</li>
</ol>

作者没有利益冲突要声明。

本文描述了一种在MFB中对6-OHDA进行单侧微输注手术的方案，能够在黑纹状体通路的神经元中引起强大的病变并在动物中产生运动障碍。还描述了用于执行步进测试的方案，这是一种易于应用且无创的测试，可用于证明病变的成功并评估前肢运动障碍。正如代表性结果所示，接受6-OHDA的动物显示出与损伤对侧调整步骤的次数减少，这意味着6-OHDA受伤的动物在输液手术后2周内表现出强烈的运动障碍。...

PD 的主要神经病理学特征是黑质 （SNc） 中多巴胺能神经元的慢性进行性神经变性以及存在含有α-突触核蛋白的路易体1。当 SNc 多巴胺能神经元通过纹状体通路将其轴突投射到纹状体中时，SNc 中神经元的神经变性导致纹状体中多巴胺能缺陷2。纹状体中多巴胺的缺失导致直接和间接运动控制途径的活动失衡，这是PD的主要运动症状的原因：运动障碍（运动缓慢），运动迟缓（开始运动困难），肌肉僵硬和休息时的震颤3，4，5。
由于参与PD发作的分子和生理机制仍未完全了解，目前可用的主要治疗方法旨在通过药物治疗，深部脑刺激6，7，遗传疗法8和细胞移植来缓解运动症状9。因此，临床前研究对于阐明PD发病的机制以及发现早期诊断的新方法和新疗法以防止或阻止受PD10影响的神经元变性至关重要。
动物模型已被广泛用于在实验室中模拟人类病理学，为医学和科学的进步做出了贡献11，12，13，14。然而，必须强调的是，动物模型的正确选择是研究成功的基础。因此，动物模型必须在三个主要方面进行验证：i）面部有效性，其中动物模型必须具有人类病理学的特征;ii）建设性有效性，其中动物模型必须具有坚实的理论基础;iii）预测有效性，其中动物模型必须以与临床治疗类似的方式对治疗做出反应。
目前，几种动物被用作PD的动物模型。主要群体包括哺乳动物，如啮齿动物，灵长类动物，迷你猪，狗和猫，以及其他群体，如果蝇和斑马鱼。啮齿动物是PD最经典的动物模型，由于其易于处理和维护而使用最多。此外，PD的解剖学和分子，细胞和药理学机制在啮齿动物和人类中是相似的15。
Kin及其同事在2019年发表的一篇综述分析了2000年代用于PD的主要动物模型方法，发现最常用的动物模型涉及神经毒素，如6-羟基多巴胺（6-OHDA）和1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）。这两种神经毒素都会导致黑纹状体通路中多巴胺能神经元的线粒体失调，导致细胞死亡16。另一种广泛使用的模型涉及通过参与PD发作的特定基因的突变进行遗传操作，从而导致线粒体失调17。神经毒素模型通常用于评估和比较治疗方法，而遗传模型用于研究预防性疗法和特发性PD15的发展。
神经毒素MPTP在20世纪80年代中期被发现会导致帕金森综合征，此前有七名患者使用该物质并表现出严重的PD症状。除了症状之外，患者对L-DOPA的治疗也有反应，这使得研究人员将分子直接与PD联系起来。该病例于1986年发表后，一些研究人员开始在临床前PD研究中使用MPTP18。研究人员发现，作为一种亲脂性分子，MPTP可以穿过血脑屏障（BBB）并转化为MPP + 19。这种有毒物质积聚在神经元内，对线粒体呼吸链的复合物1造成损害，导致多巴胺能神经元死亡20。
6-OHDA神经毒素模型于196821年首次用于诱导黑纹状体通路单胺神经元的变性。6-OHDA模型通常用于引起黑纹状体通路中的神经变性，因为它是一种多巴胺类似物，对含儿茶酚胺的细胞有毒。6-OHDA进入大脑后，它可能被多巴胺能神经元中的多巴胺转运蛋白（DAT）吸收，导致黑纹状体通路变性22。由于6-OHDA不穿透BBB，因此必须直接通过脑内立体定向注射给药23。去甲肾上腺素再摄取抑制剂通常与 6-OHDA 显微注射相结合，以保留去甲肾上腺素能纤维，并提供更具选择性的多巴胺能神经元变性24。
DAT吸收6-OHDA后，它会积聚在神经元的细胞质基质中，产生活性氧（ROS）并导致细胞死亡15。经常使用6-OHDA的三种不同病变模型：i）SNc25，26的病变;ii）纹状体病变27，28;iii）MFB29，30的病变。纹状体引起的病变导致SNpc中多巴胺能神经元的缓慢和逆行变性。相反，在SNpc和MFB中引起的病变导致神经元的快速和完全变性，导致更晚期的帕金森症状31。
单侧或双侧注射 6-OHDA 可导致多巴胺能神经元的神经变性。6-OHDA并不总是对神经元造成严重损害;有时，注射会导致部分损坏，这也用于模拟PD32的早期阶段。单侧注射更常用，因为该模型能够评估动物的运动缺陷，并通过安非他明/阿扑吗啡诱导的旋转和步进测试等测试预测细胞损失29。双侧注射最常用于评估空间记忆和识别33。
苯丙胺/阿扑吗啡诱导的旋转试验是一种行为试验，通常用于预测黑纹状体通路中的细胞损失。它被定义为重复施用多巴胺激动剂导致6-OHDA病变动物旋转行为加剧的过程34。旋转行为包括量化单侧病变啮齿动物中苯丙胺诱导的同侧旋转或阿扑吗啡诱导的对侧旋转。药物诱导的旋转行为受到批评，因为旋转与人类的PD症状不对应，并且可能受到耐受性，致敏和“启动”等变量的影响35。
启动是这些行为测试中最关键的因素之一。据报道，一些病例中，单剂量的左旋多巴导致旋转行为失败36。此外，与苯丙胺诱导试验和阿扑吗啡诱导试验联合应用并行使用相关的另一个关键因素是，由于作用机制不同，它们测量不同的终点，反映了不同信号传导机制和途径的失活。此外，苯丙胺诱导的试验更准确地测量高于50-60%的黑质纹状体病变，而阿扑吗啡诱导的试验对于80%以上的病变更准确37。
步进测试已成为一种行为测试，表明与多巴胺能神经元变性和治疗效果相关的缺陷。它能够分析由多巴胺能神经元中的6-OHDA病变引起的运动障碍，而无需药物诱导的程序。此外，自1995年以来，该测试已经得到很好的建立和广泛使用，当时Olsson等人首次对其进行了描述。1999年，Chang等人还分析 并比较了大鼠在步进试验中的表现与6-OHDA引起的变性水平，发现在步进试验中表现较差的动物也具有更显着的多巴胺能神经元变性。
步进试验是预测6-OHDA病变大鼠严重多巴胺能黑质纹状体损伤的极好方法。有证据表明，当 SNc 中的多巴胺能损失程度为 >90%39 时，在步进试验期间，6-OHDA 输注的对侧前肢会出现运动功能障碍。本文介绍了用于进行立体定向手术的方案，方法和材料，用于将6-OHDA单侧输注到大鼠的MFB中，以及如何通过步进测试预测由毒素引起的多巴胺能病变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-OHDA</td>
			<td>Sigma Aldrich</td>
			<td>H4381</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt&#38;region=BR&#38;cm_sp=Insite-_-caSrpResults_srpRecs_srpModel 
			_6-ohda-_-srpRecs3-1</td>
		</tr>
		<tr>
			<td>70% Alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>795437</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sial/795437?lang=pt&#38;region=BR&#38;gclid= 
			Cj0KCQjw4cOEBhDMARIsAA3XD 
			RipyOnxOxkKAm3J1PxvIsvw09 
			_kfaS2jYcD9E5OyuHYr4n89kO 
			6yicaAot6EALw_wcB</td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drill or tap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 50 uL</td>
			<td>Hamilton</td>
			<td>80539</td>
			<td>https://www.hamiltoncompany.com/laboratory-products/syringes/80539</td>
		</tr>
		<tr>
			<td>Imipramine</td>
			<td>Alfa Aeser</td>
			<td>J63723</td>
			<td>https://www.alfa.com/pt/catalog/J63723/</td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>Insight</td>
			<td>EFF-311</td>
			<td>https://insightltda.com.br/produto/eff-311-bomba-de-infusao-2-seringas/</td>
		</tr>
		<tr>
			<td>Ketamine (Dopalen)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/DOPALEN</td>
		</tr>
		<tr>
			<td>Machine for trichotomy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Maxicam 2%&#160; Ourofino)</td>
			<td>Ourofino</td>
			<td></td>
			<td>https://terrazoo.com.br/produto/maxicam-injetavel-2-50ml-ouro-fino/</td>
		</tr>
		<tr>
			<td>Metal Disposal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentabiotic</td>
			<td>Zoetis</td>
			<td></td>
			<td>https://www.zoetis.com.br/pentabiotico-veterinario.aspx</td>
		</tr>
		<tr>
			<td>Plastic waste garbage can</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-antibiotic</td>
			<td>Pentabiotic (Wealth)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel and blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scraper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Insight</td>
			<td>EFF-331</td>
			<td>https://insightltda.com.br/produto/eff-331-estereotaxico-1-torre/</td>
		</tr>
		<tr>
			<td>Sterile saline solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Anasedan)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/ANASEDAN</td>
		</tr>
	</tbody>
</table>

the 6-hydroxydopamine rat model of parkinson's disease

帕金森病 （PD） 的运动症状 - 运动迟缓、运动障碍和休息时震颤 - 是黑质 （SNc） 中多巴胺能神经元神经变性和多巴胺能纹状体缺陷的结果。动物模型已被广泛用于在实验室中模拟人类病理学。啮齿动物是PD最常用的动物模型，因为它们易于处理和维护。此外，PD的解剖学和分子，细胞和药理学机制在啮齿动物和人类中是相似的。将神经毒素6-羟基多巴胺（6-OHDA）输注到大鼠的内侧前脑束（MFB）中再现多巴胺能神经元的严重破坏并模拟PD症状。该协议演示了如何在PD的大鼠模型中对MFB中6-OHDA进行单侧显微注射，并通过步进测试显示了6-OHDA诱导的运动缺陷和预测的多巴胺能病变。6-OHDA导致对侧前肢进行的步数显着受损。

多巴胺能病变评估 步进试验能够评估病变前肢对侧的运动障碍，并选择可能由6-OHDA输注诱导的黑纹状体通路病变的动物（图1）。对侧前肢步进试验术前和术后2周和4周的表现的比较揭示了时间（术前，术后2 周和4周）和治疗（假手术和6-OHDA病变）之间的相互作用（F2，74 = 93.63;p <0.0001;双向重复测量方差分析）。Bonferroni的 事后 测试显示，与手术...

Watch this Scientific Journal Video about The 6-hydroxydopamine Rat Model of Parkinson's Disease at JoVE.com

The 6-hydroxydopamine Rat Model of Parkinson&#039;s Disease

6-羟基多巴胺（6-OHDA）模型已经使用了几十年，以推进对帕金森病的理解。在该协议中，我们展示了如何通过在内侧前脑束中注射6-OHDA来执行大鼠的单侧黑质纹状体病变，评估运动缺陷并使用步进测试预测病变。

帕金森病的6-羟基多巴胺大鼠模型

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

该方案展示了如何通过在内侧前脑束中注射6-羟基多巴胺来在大鼠中进行单侧黑质纹状体病变。该协议还演示了如何使用步进测试评估运动缺陷和预测病变。该模型再现了多巴胺能神经元的严重破坏，并模拟了大鼠的帕金森病症状。这种大鼠模型方案使得在实验室中研究帕金森病成为可能，涵盖了几个感兴趣的领域，如生理学，解剖学和疾病的病理机制。该测试使用两个前肢进行，一次一个，如预测试协议中所述。执行为期三天的培训课程。在第一天，在第一次会议中，保持大鼠1到2分钟，让大鼠熟悉处理者或实验者。在第二个会话中，为了使大鼠熟悉步进测试的实验设置，将大鼠保持20秒，然后将其放在桌子上20秒。重复此训练步骤 3 分钟。在第二天和第三天，在第一次训练中，用一只手握住后爪和背部，将大鼠的两个前爪放在协议表上。将大鼠向下倾斜，头部优先，角度为45

the-6-hydroxydopamine-rat-model-of-parkinson-s-disease

Research

JoVE Journal

Neuroscience

The 6-hydroxydopamine Rat Model of Parkinson's Disease

9.6K 次观看.  University of São Paulo. 该方案展示了如何通过在内侧前脑束中注射6-羟基多巴胺来在大鼠中进行单侧黑质纹状体病变。该协议还演示了如何使用步进测试评估运动缺陷和预测病变。该模型再现了多巴胺能神经元的严重破坏，并模拟了大鼠的帕金森病症状。这种大鼠模型方案使得在实验室中研究帕金森病成为可能，涵盖了几个感兴趣的领域，如生理学，解剖学和疾病的病理机制。该测试使用两个前肢...

视频: The 6-hydroxydopamine Rat Model of Parkinson's Disease

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.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

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 ...

科研

神经科学

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

方法来描述自发和惊吓引起的步态中的鱼藤酮诱导的帕金森病模型<em&gt;果蝇</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Characterizing the Relationship Between Eye Movement Parameters and Cognitive Functions in Non-demented Parkinson's Disease Patients with Eye Tracking

Cognitive impairment is a common phenomenon in Parkinson&#8217;s disease that has implications on the prognosis. A simple, noninvasive and objective proxy measurement of cognitive function in Parkinson&#8217;s disease will be helpful in detecting early cognitive decline. As a physiological metric, eye movement parameter is not confounded by the subject&#39;s attributes and intelligence and can function as a proxy marker if it correlates with cognitive functions. To this end, this study explored the relationship between the eye movement parameters and performance in cognitive tests in multiple domains. In the experiment, a visual search task with eye tracking was set up, where subjects were asked to look for a number embedded in an array of alphabets scattered randomly on a computer screen. The differentiation between the number and the alphabet is an overlearned task such that the confounding effect of cognitive ability on the eye movement parameters is minimized. The average saccadic amplitude and fixation duration were captured and calculated during the visual search task. The cognitive assessment battery covered domains of frontal-executive functions, attention, verbal and visual memory. It was found that prolonged fixation duration was associated with poorer performance in verbal fluency, visual and verbal memory, allowing further exploration on the use of eye movement parameters as proxy markers for cognitive function in Parkinson&#8217;s disease patients. The experimental paradigm has been found to be highly tolerable in our group of Parkinson&#39;s disease patients and could be applied transdiagnostically to other disease entities for similar research questions.

Characterizing the Relationship Between Eye Movement Parameters and Cognitive Functions in Non-demented Parkinson&#039;s Disease Patients with Eye Tracking

Here, we present a protocol to study the relationship between the eye movement parameters and cognitive functions in non-demented Parkinson&#39;s disease patients. The experiment used an eye tracker to measure the saccadic amplitude and fixation duration in a visual search task. The correlation with performance in multi-domain cognitive tasks was subsequently measured.

描述眼动参数与无视力帕金森病患者眼动功能的关系

Cognitive impairment is a common phenomenon in Parkinson&#8217;s disease that has implications on the prognosis. A simple, noninvasive and objective proxy ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson&#039;s Disease

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

将RatWalker系统应用于帕金森病遗传大鼠模型中的步态分析

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

通过螺纹栓塞方法建立高级射手座-鼻塞的鼠模型

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

左旋多巴诱导的帕金森病大鼠模型中运动障碍的诱导与评估

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

Analyzing the Parkinson&#039;s Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &amp;#945;-Synuclein

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.