神经发育复检新生幼鼠
Summary
行为测试是确定脑损伤后的结果的黄金标准，并可以识别发育障碍的婴儿和儿童的存在。神经反射是这些异常的早期指标。在新生儿啮齿动物很容易地完成发育反射测试的主机进行了开发和说明。
Abstract
神经发育反射测试是在临床实践中通常使用的，以评估中枢神经系统的成熟。神经反射也被称为原始反射。他们是敏感的，并与后来的结果是一致的。反射异常被描述为不存在，持久性，再现，或延迟反射，其是处于对神经发育障碍的高风险的婴儿的预测指标。神经发育障碍，如脑瘫的动物模型，通常表现出发育异常反射，如将人类婴儿进行观察。所描述的技术评估各种新生大鼠神经发育反射的。神经反射测试提供了研究者的测试方法，是不是在如此年轻的动物，否则可用。这里介绍的方法，目的是协助调查人员在审查新生大鼠的发育里程碑检测早发BR的方法AIN损伤和/或确定治疗性干预的有效性。这里介绍的方法，目的是为研究者提供的一般准则。
Introduction
神经反射，或发展的里程碑，是对人类新生儿和婴儿最早使用的评估项目之一。神经反射是展示脑干和脊髓反射不由自主的和重复的动作。更高皮质网络，其特征在于演进迁移，髓鞘形成，和突触的成熟促进自愿控制和皮质抑制。在中枢神经系统演进的正常进展的改变可以破坏大脑发育，产生异常皮质布线，功能，和髓鞘形成，神经发育造成反射延迟或缺席。在神经发育残疾的高风险与人婴儿通常表现出异常的早期反射。反射异常可呈现在采集，不存在延迟，延长的存在，或再现以后的生活中，并且预测发育障碍的。 1，2因此，它模仿神经发育障碍的实验模型反射延迟是很重要的。
鼠害通常用作实验模型。出生的时候幼鼠是altricial，因此太不成熟承担具体的或复杂的运动，感觉和/或认知行为的任务。在这方面，他们的发育不成熟同时涉及他们的身体和器官的发育。老鼠是天生无毛为不能thermoregulate，是盲目的，无法行走。参照大脑发育，皮质相当成熟出生后发生。新生大鼠幼崽（出生当天称为1日龄; PD1）已经被建议以达到脑成熟水平是类似于23早产人脑 – 妊娠28周，而PD7-10幼仔相当于近长期人类的大脑。 3，4，5，6这种相关性是基于大体解剖分析，但是，如髓鞘形成和振幅整合脑电图脑成熟的其他措施也已经被描述。 5,7例如，预少突胶质细胞是主要的细胞在发展人胎脑从23 – 宫内 32周，这的成熟阶段对应于PD1-3啮齿动物。 5，8，9，10此外，髓鞘形成开始于宫内在人类中，而在大鼠幼仔它出现在周围PD7-10前脑;在新生啮齿动物脑在很大程度上仍然未髓。 11，12 Tucker 等。发现，P1大鼠的振幅整合脑电图图案为类似于一个23周gesta重刑人类胎儿，而PD7和PD10小狗是类似于一个30 – 52周和足月儿，分别。 7出于这些原因，在新生幼鼠新生儿反射测试提供了捕获个体发育和/或大脑发育受阻的机会。
下面描述反射的电池适于从由姆·福克斯和A. Lubics 13,14 WM福克斯相对于在小鼠反射的个体发育的最早研究者之一的研究。 13个这些反射包括，但不限于，四肢抓握和放置，悬崖回避，扶正，加速扶正，步态，听觉惊跳，姿势，和眼图张度。两个前肢及后肢把握（分别被称为人类掌跖把握，）由脊髓反射和从非主运动区皮质抑制变得容易。 15,16后肢放置（跖反射）反映皮质脊髓束的成熟。 16，17，18崖避免（保护反应），扶正（迷宫），以及加速扶正涉及感觉输入和电动机输出之间的集成和通信（例如，那些涉及触须和前庭系统）。 19，20，21步态体现运动。 14听觉惊跳评估声刺激和巨神经元的突触连接在细胞核脑桥网状尾。 21姿势涉及适当皮质脊髓/脊柱皮质突起，肌肉力量，和神经肌肉的神经支配。 22，23马伽马氨基丁酸受体turation可与眼图张度相关。 24重要的是要记住的是，反射反映一个更复杂的网络，在这里提供的是一般的关系是非常重要的。此外，这些反射提供在非常年轻的时候在那里更复杂的行为测试是不可行的评价神经发育的快速简便的方法。
本文的目的是提供一种可以很容易地纳入实验新生大鼠研究神经发育复检的一般准则。描述的方法是在长埃文斯新生幼鼠进行和结果的定量是基于外观的第一天。该反射测试当天被启动，并且使用的设备可以被修改以更好地适应不同的实验模型（例如用于不同菌株和物种）。通过建立ref的正常生理进展在一个特定的动物模型成熟法，调查员可以评估外部应激物，内源性的操作，和/或在新生大鼠的神经发育的治疗性干预的效果。总体而言，使用反射的脑成熟的确定是在预测围产期脑损伤有利的，并且是后面反射神经发育结局的。
Protocol
动物护理和使用委员会，健康科学阿尔伯塔大学批准了所有动物研究。 注意：虽然此协议可以适用于其他物种和品系，该协议是为Long-Evans大鼠写的。这些大鼠已经显示出具有相对于其他啮齿动物品系优越马达性能和视觉敏锐度。 25，26，用于定时怀孕，膳食补充，产妇炎症，和神经发育反射的协议如下。 <p class="jove_tit…
Representative Results
本实验设计的时间轴示于图2。 30种的方法和结果之前已公布。 30本研究的目的是评估膳食补充妊娠期间花椰菜芽和断奶前周期是否保护， 在子宫内暴露于LPS诱导的神经发育由延迟的后代。定时怀孕的大鼠给予生理盐水（对照组，100μL）或LPS（200微克/千克在100μL无菌生理盐水稀释）上E19和E20每12小?…
Discussion
神经反射测试是不正常的皮质发育和成熟的预测指标，这可能是情况下，其中明显的神经病理学并不明显意义。在神经发育测试，关键是要确保幼崽在同一时间每天检查。鼠是夜间活动，因此，如果测试是在一天中的不同时间执行他们的昼夜节律可能会改变性能。 34测试应在安静的房间内完成为大声喧哗，可以加应力的幼崽。 35只幼仔需要至少1个小时，以适应…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢我们的资助机构，其中包括NeuroDevNet（卓越的国家中心），将ALVA基金会，妇女和儿童健康研究所和艾伯塔大学。
Materials
| Breeding | |||
| Transfer pipettes | Fisherbrand | 12-711-9AM | Used for vaginal flushes. |
| Sterile Saline | Hospira | 7983254 | The solution used to collect cells during vaginal flushes. |
| 400 µl Microcentrifuge tubes | Fisherbrand | 05-408-120 | Used to hold the saline solution. |
| Light microscope | Leica | Leica ATC 2000 | For observation of the saline solution. Can be any light microscope used in the lab. |
| Slides | Fisherbrand | 12-552-5 | The saline solution is placed on the slide. Can be any slides used in the lab. |
| Coverslips | Fisherbrand | 12-545-F | To coverslip the slides. Can use any coverslips used in the lab. |
| Dietary Supplementation | |||
| Broccoli Sprouts seeds | Mumm's Sprouting Seeds | Broccoli sprouts seeds are ordered and grown in the lab. | |
| Countertop Seed Sprouter Box | Mumm's Sprouting Seeds | A box is used to germinate and grow the seeds prior to harvest. | |
| 250 mL beaker | The beaker is used to soak the seed. Any size beaker that would fit can be used. | ||
| Maternal Inflammation | |||
| Lipoplysaccharide (LPS) | Sigma | L3129 | The endotoxin used to mimic maternal inflammation. |
| 1 mL Syringe | BD Syringe | 309659 | Used to inject the pregnant rat. |
| Gauge (30G X 1/2) | BD PrecisionGlide Needle | 305106 | Use the smallest needle to avoid pain and discomfort. |
| Sterile Saline (0.9% Sodium Chloride, USP) | Hospira | Saline is used to dissolve LPS. | |
| Weights | |||
| Scale | Denver Instrument | For recording the weights. Can be any scale with 2 decimal places used in the lab. | |
| Neurodevelopmental Reflexes | |||
| Thin blunt rod | Can be a paperclip or toothpick. This is for forelimb and hindlimb grasping. | ||
| Round filter paper | Whatman | 1001 150 | 15 cm diameter paper used for gait analysis. |
| Timer | Fisher Scientific | 06-662-51 | For timing the time allocated to righting and gait. |
| Blunt surface | Can be an edge of a table. This is for hindlimb placing and cliff avoidance. | ||
| Foam landing | For when the pups perform accelerated righting. | ||
| Video recorder | Sony | VCT-D580RM | To record all reflexes tested. Must be able to record at 1/1000 fps |
| Bell | For auditory startle. | ||
| Heat lamp or pad | To maintain the body temperature of the pups underoing examination. |
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Cite This Article
Nguyen, A. T., Armstrong, E. A., Yager, J. Y. Neurodevelopmental Reflex Testing in Neonatal Rat Pups. J. Vis. Exp. (122), e55261, doi:10.3791/55261 (2017).