视交叉前，单次注射自体血以诱导大鼠模型中实验性蛛网膜下腔出血
1Department of Neurosurgery,Rigshospitalet, 2Department of Clinical Experimental Research,Glostrup Research Institute, Rigshospitalet, 3Department of Clinical Medicine,University of Copenhagen, 4Department of Clinical Sciences, Division of Experimental Vascular Research,Lund University, 5Department of Clinical Neuroscience,Karolinska Institutet
Summary
蛛网膜下腔出血继续给人类带来很高的死亡率和发病率负担。为了便于进一步研究病情及其病理生理学，提出了一种视交叉前的单次注射模型。
Abstract
尽管过去几十年在治疗方面取得了进展，但蛛网膜下腔出血（SAH）仍然具有很高的发病率和死亡率负担，主要困扰着相当年轻的人群。已经开发了几种SAH动物模型来研究SAH背后的病理生理机制并测试药物干预。本文介绍的大鼠的视交叉前单次注射模型是具有预定血容量的SAH的实验模型。简而言之，对动物进行麻醉、插管并保持在机械通气下。温度通过加热垫调节。在尾动脉中放置导管，可以连续测量血压和进行血液采样。切开寰枕膜，并在大池中放置用于压力记录的导管，以测量脑内压。该导管也可用于鞘内治疗干预。将大鼠置于立体定位框架中，在前膛前钻一个毛刺孔，并通过毛刺孔插入导管并放置在视交叉前方。从尾导管抽出自体血 （0.3 mL） 并手动注射。这导致脑内压升高和脑血流量减少。将动物保持镇静30分钟，并给予皮下盐水和镇痛药。动物被拔管并返回笼子。前交叉模型具有很高的重现率，并且由于预先确定的血容量，动物之间的差异有限。它模仿人类的SAH，使其成为SAH研究的相关模型。
Introduction
非创伤性蛛网膜下腔出血（SAH）是卒中的一种形式，约占所有病例的5%。非创伤性 SAH 最常见的原因是动脉瘤 （aSAH） 突然破裂，占 SAH 的 85%。其他原因包括动脉静脉畸形破裂、凝血病和中脑周围出血的静脉破裂1。发病率为 9/100，000 人年，死亡率约为三分之一，另外三分之一需要在 SAH2，3 后维持日常生活。
初步稳定和确诊后，治疗取决于出血的严重程度。病情最严重的患者将在心室中插入心室外引流管以降低脑内压（ICP），并被收入神经重症监护室，在那里他们受到密切监测。患者将接受血管造影以识别（可能的）动脉瘤，然后将动脉瘤盘绕或夹住以防止再出血4.尽管进行了大量药物治疗试验，但只有尼莫地平（一种钙通道拮抗剂）已被证明可以改善结局5。目前正在进行多项临床试验。请参阅Daou及其同事的评论，以获取广泛的列表6。
动脉瘤的破裂被描述为有史以来最严重的头痛或霹雳性头痛的突然发作。破裂导致ICP急剧上升，随后脑血流量（CBF）减少。这种减少导致大脑的整体缺血，这可能导致意识丧失。这种更机械的途径，以及血液中外渗元素的启动分解，引起细胞因子释放和先天免疫系统的激活，导致无菌性神经炎症。此外，经常观察到血脑屏障的破坏，导致脑水肿和离子稳态紊乱。所有这些变化以及更多被称为早期脑损伤（EBI）的变化发生在头几天内，并导致神经元丢失和细胞凋亡7。
大约 1/3 的 aSAH 患者在第 4-14 天之间会出现迟发性脑缺血 （DCI）8。DCI被定义为局灶性神经损伤的首次出现，或者在格拉斯哥昏迷量表上至少下降两个点，持续至少1小时，当排除其他原因，包括癫痫发作和再出血时。DCI 与 aSAH9 后死亡风险增加和功能结局降低有关。脑血管痉挛（CVS）是脑动脉的狭窄，几十年来一直已知与DCI有关，以前被认为是DCI的唯一原因。此后已经表明，CVS可以在没有DCI发展的情况下发生，并且已经确定了更多因素，包括微血管血栓形成和收缩，皮质扩散抑制和EBI的炎症反应10，11，12。
由于EBI和DCI对疾病进程和患者结果的巨大影响，动物模型需要尽可能模仿这些，同时仍然可重复。研究人员在从小鼠到非人类灵长类动物的各种动物中采用了各种不同的模型来尝试模拟aSAH。Sprague-Dawley和Wistar野生型大鼠是目前最常用的实验动物，最常见的模型是血管内穿孔模型，cisterna-magna双注射模型，最后是视交叉前单次注射模型，本文将在本文中描述13。
前交叉单次注射模型最初由Prunell及其同事开发，以应对其他实验模型的一些缺点14。掌握手术后，具有高度可重复性，并最大限度地减少了动物之间的差异。该模型在多个点上模拟了人类的 SAH，包括注射血液后 ICP 突然升高，导致由于 CBF15，16 下降而导致短暂性整体缺血。它影响前循环，这是人类大多数aSAH发生的地方17。死亡率在 10%-33% 之间，具体取决于研究和注射的血液量14，18。延迟性细胞死亡和神经炎症可以在第2天和第7天检测到，从而为研究EBI和DCI19，20的后果提供变量。
该研究提供了大鼠交叉前单次注射模型的更新描述，以及如何利用ICP探针作为鞘内给药的端口的描述。
Protocol
该程序是根据欧盟关于保护用于科学目的的动物的指令2010/63 / EU完成的，并由丹麦动物实验检查局批准（许可证号2016-15-0201-00940）。尽可能使用无菌技术进行手术，包括无菌器械、手套、导管和缝合线。该研究使用体重230-350g的雄性和雌性Sprague-Dawley大鼠，组饲养12小时光照/黑暗循环，恒温22°C（±2°C），湿度为55%（±10%）。为动物提供标准的食物和随意的水。手术后将动物饲养在单笼中，但当ICP?…
Representative Results
与男性相比，女性患 aSAH 的风险更高。尽管如此，雄性啮齿动物主要用于实验，因为雌性发情周期的异质性可能存在偏差。这里介绍的代表性结果来自最近比较雌性和雄性大鼠的出版物，证实该模型在雌性动物中产生的结果与雄性21相似。该研究包括34只雌性Sprague-Dawley大鼠（18只SAH和16只shams）。Shams没有将脊髓针下降到视交叉或注射血液。所有其他程序均在与SAH相同的Shams上进行…
Discussion
SAH的视交叉前单次注射模型模拟了人类SAH的几个重要元素，包括ICP峰值，CBF降低，短暂性全面缺血，神经炎症标志物上调和CVS14，15，16，18，19，20。ICP探针也用作鞘内给药的端口（图5）。此外，研究表明，该模型在雄性和雌?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了灵北基金会和灵北卓越奖（编号R59-A5404）的支持。资助者在手稿的任何部分都没有角色。
Materials
| 16 G peripheral vein catheter | BD Venflon | 393229 | Needle shortened, distal 1 cm curved. Wings removed |
| Anesthesia bell/ chamber | Unknown | ||
| Blood gas analyzer | Radiometer | ABL80 | |
| Blood pressure (BP) monitor | Adinstruments | ML117 | Connects to Powerlab |
| Curved forceps, 12 cm x 3 | F.S.T | 11001-12 | For anesthesia |
| Cylindrical pillow, 28 cm x 4 cm | Homemade | Made from surgical towels | |
| Data acquisition hardware | Adinstruments | ML870 Powerlab | |
| Data acquistion software | Adinstruments | LabChart 6.0 | |
| Drill | KMD | 1189 | |
| Drill controller | Silfradent | 300 IN | |
| Flexible light | Schott | KL200 | |
| Heating pad | Minco | 1135 | |
| Hypodermic needle, 20 G | KD Medical | 301300 | Connects to stereotaxic frame |
| ICP monitor | Adinstruments | ML117 | Connects to Powerlab |
| Isoflurane vaporizer | Ohmeda | TEC3 | |
| Laptop | Lenovo | T410 | |
| Laser doppler monitor | Adinstruments | ML191 | |
| Laser doppler probe | Oxford Optronics | MSF100XP | Connects to laser doppler monitor |
| Needle holder, 13 cm | F.S.T | 12001-13 | For anesthesia |
| Precision syringe, 0.025 mL | Hamilton | 547407 | |
| Stereotaxic frame | Kopf Instruments | M900 | |
| Surgical microscope | Carl Zeiss | F170 | |
| Suture needle | Allgaier | 1245 | For anesthesia |
| Temperaure controller | CWE,INC. | TC-1000 | |
| Transducer x 2 | Adinstruments | MLT0699 | Connects to BP and ICP monitor |
| Ventilator | Ugo Basile | 7025 | |
| Veterinary clipper | Aesculap | GT421 | |
| 3-pronged Blair retractor, 13.5 cm | Agnthos | 17022–13 | |
| Blunt Alm retractor | F.S.T | 17008-07 | |
| Curved forceps, 12 cm x 2 | F.S.T | 11001-12 | |
| Needle holder, 13 cm | F.S.T | 12001-13 | |
| Straight Dumont forceps, 11 cm | F.S.T | 11252-00 | |
| Straight Halsted-Mosquito hemostat x 2 | F.S.T | 13008-12 | |
| Straight Iris scissor, 9 cm | F.S.T | 14090-09 | |
| Straight Vannas scissor, 10.5 cm | F.S.T | 15018-10 | |
| Absorpable swabs | Kettenbach | 31603 | |
| Black silk thread, 4-0, 5 x 15 cm | Vömel | 14757 | |
| Bone wax | Aesculap | 1029754 | |
| Carbomer eye gel 2 mg/g | Paranova | ||
| Cotton swab | Heinz Herenz | WA-1 | |
| Cotton tipped applicator x 4 | Selefa | 120788 | |
| Hypodermic needle, 23 G x2 | KD Medical | 900284 | Connects to stopcock. Remove distal end |
| Hypodermic needle, 23 G x3 | KD Medical | 900284 | Remove distal end. 2 connects to stopcock, 1 to syringe |
| ICP probe: | Homemade | Made of the following: | |
| Polythene tubing, 20 mm | Smiths medical | 800/100/200 | Inner diameter (ID): 0.58 mm, Outer diameter (OD): 0.96 mm. |
| Silicone tubing, 10 mm | Fisher | 15202710 | ID: 0.76 mm, OD: 2.4 mm. |
| Silicone tubing, 2 mm | Fisher | 11716513 | ID: 1.0 mm, OD: 3.0 mm. |
| Micro hematocrit tubes | Brand | 7493 11 | |
| OP-towel, 45 cm x75 cm | Mölnlycke | 800430 | |
| PinPort adapter, 22 G | Instech | PNP3F22 | |
| PinPort injector | Instech | PNP3M | |
| Polythene tubing, 2 x 20 cm | Smiths medical | 800/100/200 | Connects to syringe. ID: 0.58 mm, OD: 0.96 mm. |
| Rubberband | Unknown | ||
| Scalpel, 10 blade | Kiato | 23110 | |
| Spinalneedle, 25 G x 3.5'' | Braun | 5405905-01 | |
| Stopcock system, Discofix x 2 | Braun | 16494C | Connects to transducer |
| Suture, 4-0, monofil, non-resorbable x 3 | Ethicon | EH7145H | |
| Syringe, 1 mL | BD Plastipak | 1710023 | |
| Syringe, luer-lock, 10 mL x 4 | BD Plastipak | 305959 | Connects to transducer |
| Tissue adhesive glue | 3M | 1469SB | |
| 0.5% Chlorhexidine spirit | Faaborg Pharma | 210918 | |
| Carprofen 50 mg/mL | ScanVet | 43715 | Diluted 1:10 |
| Isoflurane | Baxter | ||
| Isotonic saline | Amgros | 16404 | |
| Lidocaine-Adrenaline 10 mg/5 µg/mL | Amgros | 16318 |
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Citazione di questo articolo
Bömers, J. P., Johansson, S. E., Edvinsson, L., Mathiesen, T. I., Haanes, K. A. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. Exp. (172), e62567, doi:10.3791/62567 (2021).