创伤性脑损伤后评估成年大鼠空间工作记忆的指标测试
1Department of Anesthesiology and Critical Care,Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology and Perioperative Medicine,Mayo Clinic, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences,Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine,Dnepropetrovsk State University
Summary
创伤性脑损伤 （TBI） 通常与记忆障碍相关。在此，我们提出一个协议，通过公制任务评估 TBI 之后的空间工作内存。公制测试是研究 TBI 后空间工作记忆障碍的有用工具。
Abstract
感觉、短期和长期记忆损伤是创伤性脑损伤 （TBI） 后常见的副作用。由于人类研究的伦理局限性，动物模型为测试治疗方法和研究该病的机理和相关并发症提供了合适的替代方案。实验啮齿动物模型历来是使用最广泛的，因为它的可访问性，低成本，可重复性和经过验证的方法。公制测试是一种技术，可以测试在TBI之后研究空间工作记忆 （SWM） 损伤的技术，它测试了在彼此之间不同距离和角度上回忆两个物体放置的能力。公制任务的主要优点包括动态观察的可能性、低成本、可重复性、相对易于实施和低应力环境。在这里，我们提出了一个公制测试协议，以测量TBI后成年大鼠SWM的损伤。该测试为更有效地评估大脑功能的生理学和病理生理学提供了可行的方法。
Introduction
中度创伤性脑损伤（TBI）后，注意力、执行功能和某些记忆缺陷等神经缺陷的患病率超过50%，1、2、3、4、5、6、7、8。TBI 可导致空间短期、长期和工作记忆9的严重损伤。这些记忆障碍在TBI的啮齿动物模型中已经观察到。Rodent 模型使测试记忆的技术得以发展，从而能够更深入地检查 TBI 对神经记忆系统内存处理的影响。
两项测试分别与拓扑和公制空间信息处理有关，有助于测量空间工作记忆 （SWM）。拓扑测试取决于改变环境空间的大小或相关空间的连接或围绕一个物体，而公制测试评估在角度或距离对象10，11之间的变化。古德里奇-亨萨克等人首先对大鼠10号进行了人体拓扑测试，并应用了指标任务，将腹膜皮层（PC）和背海马在空间信息处理中的作用分离。同样，古尔科夫和他的同事评估了横向液体打击伤害9后的指标、拓扑和时间顺序记忆任务。大脑某些区域的损伤与公制或拓扑记忆的损伤之间存在相关性。有人提出，公制记忆损伤与海马的双侧牙周收缩陀螺和角膜氨基（CA）亚区域CA3的病变有关，拓扑记忆损伤与双侧皮质病变10、12有关。
此协议的目的是通过公制任务评估大鼠群体的空间记忆缺陷。该方法是脑损伤后SWM研究机制的合适替代方案，其优点包括实施相对容易、灵敏度高、可重复性低、动态观察的可能性大、应力环境低等。与其他行为任务，如巴恩斯迷宫13，14，莫里斯水导航任务15，16，17，或空间迷宫任务18，19，这个指标测试是不那么复杂。由于其易于实施，指标测试需要更短和压力较小的训练时间，只进行2天9：1天的习惯和1天的任务。此外，我们建议的测试比其他低压力测试更容易执行，如新颖的对象识别 （NOR） 任务，并且不需要额外的一天习惯20。
本文为脑损伤后SWM的评价提供了一个简单的模型。这种对TBI后SWM的评估可能有助于对其病理生理学进行更全面的调查。
Protocol
这些实验是根据《赫尔辛基宣言》和《东京宣言》以及《欧洲共同体实验动物使用准则》的建议进行的。这些实验得到了内盖夫本-古里安大学动物护理委员会的批准。协议时间表在 图1中说明。 1. 外科手术和液体打击TBI 选择雄性及雌性成年斯普拉格-道利大鼠，其室温为22±1°C，湿度为40%-60%，光黑周期为12-12小时。 提供食物作为周和水 <em…
Representative Results
使用曼-惠特尼测试确定组之间的比较的重要性。研究结果的统计意义在P<0.05，而统计高度相关性在P<0.01。 结果显示，在干预前和TBI之后的28天，所有群体在NSS上没有差异。每组由12只雌性或12只雄性大鼠组成。在 TBI出现在表1中后，NSS分数获得48小时。TBI组的老鼠在受伤后第28天表现出明显的神经缺陷，被排除在实验之外。数据按计数进行测量，并显示为中位数±范…
Discussion
通过专门针对公制空间信息过程，此指标测试为了解 TBI 之后的记忆缺陷提供了必要的工具。本文提出的协议是对先前描述的行为任务11的修改。先前描述的一个指标任务使用了两种不同的范式，每个范式包括三个习惯会话和一个测试会话。第一个范式包括习惯后将熟悉的物体移近在一起，第二个范式将物体移得更远。
与巴恩斯迷宫相比，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢奥莱娜·塞韦里诺夫斯卡教授;玛丽娜·库舍里亚瓦 M.Sc;马克西姆·克里沃诺索夫 M.Sc：达里娜·亚库门科 M.Sc：叶夫根尼亚·贡查里克 M.Sc：和奥尔哈·沙波瓦尔，生理学系，生物，生态学和医学系的博士候选人，奥尔斯·洪查尔·德尼普罗大学，第尼普罗，乌克兰的支持和有用的贡献。这些数据是作为德米特里·弗兰克博士论文的一部分获得的。
Materials
| 2% chlorhexidine in 70% alcohol solution | SIGMA – ALDRICH | 500 cc | For general antisepsis of the skin in the operatory field |
| Bupivacaine 0.1 % | |||
| 4 boards of different thicknesses (1.5cm, 2.5cm, 5cm and 8.5cm) | This is to evaluate neurological defect | ||
| 4-0 Nylon suture | 4-00 | ||
| Bottles | Techniplast | ACBT0262SU | 150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution |
| Bottlses (four) for topological an metric tasks | For objects used two little bottles, first round (height 13.5 cm) and second faceted (height 20 cm) shape and two big faceted bottles, first 9×6 cm (height 21 cm) and second 7×7 cm (height 21 cm). | ||
| Diamond Hole Saw Drill 3mm diameter | Glass Hole Saw Kit | Optional. | |
| Digital Weighing Scale | SIGMA – ALDRICH | Rs 4,000 | |
| Dissecting scissors | SIGMA – ALDRICH | Z265969 | |
| Ethanol 99.9 % | Pharmacy | 5%-10% solution used to clean equipment and remove odors | |
| EthoVision XT (Video software) | Noldus, Wageningen, Netherlands | Optional | |
| Fluid-percussion device | custom-made at the university workshop | No specific brand is recommended. | |
| Gauze Sponges | Fisher | 22-362-178 | |
| Gloves (thin laboratory gloves) | Optional. | ||
| Heater with thermometer | Heatingpad-1 | Model: HEATINGPAD-1/2 | No specific brand is recommended. |
| Horizon-XL | Mennen Medical Ltd | ||
| Isofluran, USP 100% | Piramamal Critical Care, Inc | NDC 66794-017 | Anesthetic liquid for inhalation |
| Office 365 ProPlus | Microsoft | – | Microsoft Office Excel |
| Olympus BX 40 microscope | Olympus | ||
| Operating forceps | SIGMA – ALDRICH | ||
| Operating Scissors | SIGMA – ALDRICH | ||
| PC Computer for USV recording and data analyses | Intel | Intel® core i5-6500 CPU @ 3.2GHz, 16 GB RAM, 64-bit operating system | |
| Plexiglass boxes linked by a narrow passage | Two transparent 30 cm × 20 cm × 20 cm plexiglass boxes linked by a narrow 15 cm × 15 cm × 60 cm passage | ||
| Purina Chow | Purina | 5001 | Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical researc for over 5 |
| Rat cages (rat home cage or another enclosure) | Techniplast | 2000P | No specific brand is recommended |
| Scalpel blades 11 | SIGMA – ALDRICH | S2771 | |
| SPSS | SPSS Inc., Chicago, IL, USA | 20 package | |
| Stereotaxic Instrument | custom-made at the university workshop | No specific brand is recommended | |
| Timing device | Interval Timer:Timing for recording USV's | Optional. Any timer will do, although it is convenient to use an interval timer if you are tickling multiple rats | |
| Topological and metric tasks device | Self made in Ben Gurion University of Negev | White circular platform 200 cm in diameter and 1 cm thick on table | |
| Video camera | Logitech | C920 HD PRO WEBCAM | Digital video camera for high definition recording of rat behavior under plus maze test |
| Windows 10 | Microsoft | ||
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Cite This Article
Frank, D., Gruenbaum, B. F., Melamed, I., Grinshpun, J., Benjamin, Y., Vzhetson, I., Kravchenko, N., Dubilet, M., Boyko, M., Zlotnik, A. A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury. J. Vis. Exp. (171), e62291, doi:10.3791/62291 (2021).