神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞
Summary
This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.
Abstract
中风的实验动物模型是了解中风的病理和开发更有效的治疗策略非常宝贵的工具。 2周协议重复缺氧预处理（RHP）诱导长期保护，防止中枢神经系统（CNS）损伤的局灶性缺血性脑卒中的小鼠模型。 RHP由9随机暴露于缺氧该变化在两个持续时间（2或4小时）和强度（8％和11％的O 2）。 RHP减少梗死体积，血 – 脑屏障（BBB）的破坏，并为周后的最后暴露于缺氧的中风后的炎症反应，这表明了长期的感应的内源性的CNS保护表型。对于梗死体积和血脑屏障破坏的双重定量的方法是有效的评估小鼠RHP或其他神经保护剂推测神经血管的保护。成年雄性Swiss Webster小鼠被RHP或持续时间相当于曝光预处理到21％氧气<suB> 2（ 即室内空气）。 60分钟短暂的大脑中动脉闭塞（tMCAo）诱导后2周的最后低氧暴露。无论是缺血再灌注经颅激光多普勒血流仪证实。二十二个小时再灌注后，伊文思蓝（EB）进行静脉内，通过尾静脉注射给药。 2小时后，将动物用异氟烷过量和脑切片处死沾满2,3,5-氯化三苯基四唑（TTC）。梗死体积再量化。接着，EB从组织中提取超过48小时tMCAo后确定血脑屏障破坏。总之，RHP是一个简单的协议，它可以被复制，以最少的成本，以诱导中风损伤小鼠长期内源性神经血管保护，与平移潜力其它CNS基和全身促炎疾病状态。
Introduction
成人残疾和死亡的第四大病因的主要原因，中风是美国面临的成人人口中最衰弱的疾病之一。卒中1动物模型允许对减少缺血性损伤的新方法和实验研究改善中风后恢复。一个新的途径，例如翻译研究的预处理。预处理是有意使用的非破坏性的刺激，以减少从随后，和更严重的，损伤的破坏。2缺氧预处理已经显示出产生在大脑中的多效性的变化提供保护，防止中风在体内和体外研究3，当然 ，单次曝光缺氧只提供短期的神经保护作用，诱导少于72小时耐受缺血的成年小鼠的4连四个星期后的14小时日常暴露在低压缺氧，林等人。 FOUND的神经保护作用只持续了一个星期。5重复缺氧预处理（RHP）的特点是频率，持续时间和低氧暴露强度随机变化。在对比一个单一预处理挑战，RHP诱导脑保护表型，持续长达八周的小鼠。6 RHP减少梗死体积，血-脑屏障（BBB）的破坏，血管炎，和白细胞血细胞渗出数周的最后低氧暴露后。 RHP特别通过降低T细胞，单核细胞，和巨噬细胞群体，同时维持B细胞群体中的缺血性半球炎症减少了缺血性脑7实际上，RHP任何CNS损伤诱导小鼠免疫表型之前，包括中风。 RHP处理的B细胞从RHP处理健康小鼠中分离表现出独特的抗炎表型，具有两个抗原呈递和抗体产生的下调。该在促炎症性免疫机制的整体减少使得RHP极好方法，以诱导内源免疫抑制不仅CNS特异性炎性疾病，而且全身的损伤或疾病的模型，其中包括一个促炎性病理。
RHP降低了梗死体积和血脑屏障破坏下一个暂时性大脑中动脉闭塞（tMCAo）。中风的动物模型，如常用的tMCAo，显着地提高中风的病理生理学的了解，以及更有效的neurotherapeutics设计。首先由小泉等人开发的。，1986年，8 tMCAo过程是诱导中风在啮齿类动物和优选的方法为以下灌注调查炎症一个的广泛使用的方法。作为方法tMCAo演进，更多的近期使用的有机硅涂覆的长丝进一步减少相对于其他模型9,10 <蛛网膜下腔出血的风险/ SUP>，提高了可靠性，但不幸的tMCAo往往产生于梗死体积变化很大。11-13这些研究大多描绘的冠状脑切片梗死区与2,3,5-三苯基氯化（TTC）染色，被认为是金标准梗死定量，因为它是产生生动的，可复制的结果的简单和廉价的方式。 TTC用作存在于线粒体脱氢酶的底物。当脑切片暴露在TTC溶液中，TTC选择性考虑活细胞，其中其非水溶性还原产物，甲，沉淀到深红色存活线粒体。因为在缺血组织线粒体功能障碍的，这个组织保持白色，使受损和健康组织的分化。14
RHP也减少了在缺血半球血脑屏障破坏。6因此，血脑屏障完整性的双重定量同一B内部降雨作为TTC基梗塞体积测定15将提供大约源性保护的充分发挥药效的有用信息，并在未处理的和处理的动物血脑屏障破坏和梗死之间潜在的因果关系。外周血通过具有破坏血脑屏障，继发于中风，潮增加白细胞群，促炎性细胞因子，氧化应激，血管性水肿，和出血性转化中的缺血性半球，最终增加了感染和死亡的发生率在缺血性脑卒中患者16,17测量血脑屏障损伤的动物模型中的常用方法是通过定量的伊文思蓝（EB）的染料泄漏入脑。15,18-21 EB选择性地结合血清白蛋白，球状蛋白质（MW = 65 kDa）的不穿过BBB在未受伤的动物。22继缺血性中风，EB渗入脑和荧光在620nm，允许光密度withi的测量n中的灌注损伤实质22的光密度成正比血脑屏障的时EB已经洗出的验尸皮质脉 管由transcardiac灌注的渗透性。随着动物EB管理TTC染色大脑的即时处理，既梗死体积和血脑屏障破坏，可以有效地量化。应当指出，然而，神经元损伤和血脑屏障破坏不是在中风后大脑伴随工序，23,24这样的牺牲的时间的选择是一个重要的考虑因素。
接下来的协议细节的RHP方法，该tMCAo方法用于诱导临时动脉闭塞该模型中脑动脉闭塞在人类患者，和双组织学方法，用于确定神经和血管中风损伤端点。 TTC测量细胞死亡和累积的组织损伤，从而允许整体梗塞体积的量化乌梅，而电子束提供的血脑屏障损伤的半球定量。
Protocol
注：此协议被批准的机构动物护理和使用委员会（IACUC）在得克萨斯大学西南医学中心这为健康（NIH）的政策实验动物的使用遵循全国学院。 1.重复缺氧预处理定制设计上的气体调节器4流量计和附加到标准15升感应腔与PVC管，以允许压缩气体从氧气（O 2）的罐，以经由入口端口流入所述腔室。看装备和材料有关自定义设计的更多细节。 小鼠分成2组：重?…
Representative Results
这项研究包括25男瑞士韦伯斯特小鼠为10周龄时随机开始进入RHP（N = 10）或21％O 2（N = 15）组。两周后的最终RHP曝光，外科手术进行，以盲基和天之间平衡。继tMCAo，1个鼠标术后恢复过程中死亡和1个鼠标被排除在研究，因为它不符合再灌注CBF标准。排除两种小鼠在21％O 2组。按照ARRIVE准则，28表2示出的外科参数。间接的梗死体积，半球肿胀（ 即 ，水肿），和伊文思?…
Discussion
单次暴露于全身缺氧（ 即人力资源，2 11％O 2）在小鼠“瞬时”免受tMCAo，29大脑意义缺氧预处理挑战后生反应是持续时间短，基线表型内恢复天。缺氧预处理刺激重复演示大大延长神经保护型的持续时间。6，许多研究表明，频率，幅度和持续时间的反复刺激训练都是这种反应的关键因素。例如，简单地重复同样的强度和缺氧的持续时间（2小时的11％的氧气）…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).
Materials
| Material/ Equipment | Company | Catalog Number | Comments/Description |
| Flowmeters, regulators | VetEquip, Inc | Specialty order | Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5" x 1" x 1" metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench. |
| 21% O2 tank | AirGas | OX USP200 | |
| 11% O2 tank | AirGas | Specialty order | |
| 8% O2 tank | AirGas | Specialty order | |
| 15L induction chambers | VetEquip | 941454 | |
| Moor Laber Dopper Flow | Moor Instruments | moorVMS-LDF1-HP | 0.8mm diameter probe |
| High Intensity Illuminator | Nikon | NI-150 | |
| Zoom Stereo Microscope | NIkon | SMZ800 | Other surgical microscopes may be used. |
| Kent Scientific Right Temperature CODA | Kent Scientific Corporation | Discontinued | Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT). |
| Hovabator Incubator | Stromberg's | 2362-E | Our model is the 2362N. 2362E is a later model and includes an electronic thermostat. |
| V010 Anesthesia system | VetEquip | 901807 | Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters |
| 250 mL isoflurane | Butler Schein | NDC-11695 | |
| D-6 Vet Trim Animal Cordless Trimmer | Andis | #23905 | Replacement blades are available from Andis (#23995) |
| Betadine | Fisher Scientific | 19-898-867 | |
| Q-tips | Multiple sellers | Catalog number not available | |
| Gauze Pads | Fisher Scientific | 67622 | |
| Surgical drape | Fisher Scientific | GM300 | |
| Silk Sutures | Look/Div Surgical Specialties | SP115 | |
| Nylon Sutures | Look/Div Surgical Specialties | SP185 | |
| Durmont #5 forceps (2) | Fine Science Tools | 11251-35 | Angled 45° |
| Surgical Scissors | Fine Science Tools | 14028-10 | |
| 3mm Vannas | Kent Scientific Corporation | INS600177 | Straight blade |
| Hartman Hemostats | Fine Scientific Tools | 13002-10 | |
| Occluding filaments | Washington University | Specialty order | Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee. |
| Evans Blue | Sigma Aldritch | E2129-10G | |
| Filter Paper | Sigma Aldritch | WHA1001150 | 150 mm, circles, Grade 1 |
| Weigh Boats | Fisher Scientific | 02-202-101 | 2.5" diameter |
| 0.9% Sodium Chloride Injection USP | Baxter Pharmaceutics | 2B1321 | |
| 0.3cc insulin syringe with 29 g needle | Becton Dickinson Labware | 309301 | |
| Flat bottom restrainer | Braintree Scientific | FB M | 2.0" diameter |
| TTC | Sigma | T8877 | |
| 10X PBS, pH 7.4 | Fisher Scientific | BP399-20 | |
| Water Bath | Multiple sellers | Catalog number not available | Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath. |
| Styrofoam board | Multiple sellers | Catalog number not available | |
| Large Syringe Kit | PumpSystems Inc | P-SYRKIT-LG | |
| Perfusion Pump | PumpSystems Inc | NE-300 | |
| 60 cc syringe | Fisher Scientific | NC9203256 | |
| 27g winged infusion set | Kawasumi Laboratories, Inc | D3K1-25G 1 | |
| 20 ml scintillation vial | Fisher Scientific | 50-367-126 | |
| Stainless steel spatula | Fisher Scientific | 14-373-25A | |
| Alto acrylic 1.0 mm mouse brain, coronal | CellPoint Scientific | Catalog number not available | |
| 0.21 mm stainless steel blades, 25 pk | CellPoint Scientific | Catalog number not available | Reusable cryostat blades are an inexpensive alternative. |
| 4% paraformaldehyde | Santa Cruz Biotechnology | SC-281692 | |
| Superfrost microscope slides | Fisher Scientific | 12-550-15 | |
| HP Scanjet G4050 | Multiple sellers | Catalog number not available | Other commercial scanners are suitable for this step in the protocol. |
| ImageJ | National Institute of Health | Catalog number not available | |
| Analytical Balance | Mettler Toledo | XSE 205U | |
| Precision Compact Oven | Thermo Scientific | PR305225M | |
| 1.7 mL microcentrifuge tubes (Eppendorfs) | Denville Scientific | C2170 | |
| Formamide | Fisher Scientific | BP228-100 | |
| 96-well plates | Fisher Scientific | 07-200-9 | |
| Epoch Microplate Spectrophotometer | BioTek | Catalog number not available |
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Citazione di questo articolo
Poinsatte, K., Selvaraj, U. M., Ortega, S. B., Plautz, E. J., Kong, X., Gidday, J. M., Stowe, A. M. Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice. J. Vis. Exp. (99), e52675, doi:10.3791/52675 (2015).