C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法
Summary
本报告概述了一个简单的方法, 成功诱导实验性自身免疫神经炎 (一) 使用髓磷脂蛋白零 (P0)180-199肽结合弗氏的完整佐剂和百日咳毒素。我们提出了一个复杂的范例, 能够准确地评估在这一过程中出现的功能性赤字和神经病的程度。
Abstract
实验性自身免疫神经炎 (欢迎) 是自身免疫性外周脱髓鞘疾病的实验模型。通过免疫小鼠的神经源性多肽诱导炎症, 使其对周围神经系统的成分有直接的攻击作用。最近的进展使得在相对耐药的 C57BL/6 小鼠线使用髓鞘蛋白零 (P0)106-125或 P0 的180-199多肽在佐剂和注射百日咳毒素的结合。在 C57BL/6 菌株中诱导药物的能力允许使用在这个老鼠背景上存在的众多的基因工具, 从而允许对疾病的发病机制和对新疗法的机械作用的审讯的复杂研究与转基因方法相结合。在本研究中, 我们展示了一个简单的方法, 成功地诱导使用 P0180-199肽的 C57BL/6 小鼠。我们还概述了在这个模型中出现的功能缺陷评估的一个协议, 同时还有一系列的病理特征。因此, 该模型是研究人外周脱髓鞘神经病的一个强有力的实验模型, 并确定了旨在促进髓鞘修复和预防自身免疫性神经损伤的潜在疗法的疗效。炎.
Introduction
外周神经病可以是遗传的起源或后天, 与后天神经病有代谢, 缺血性, 炎症, 或毒性沉淀。这些疾病也可以被归类为轴突或鞘的来源。最常见的后天性脱髓鞘外周神经病是急性炎症性脱髓鞘神经病 (AIDP, 也称为格林-巴利综合征, GBS) 和慢性炎症性脱髓鞘神经病 (CIDP)1,2,3,4;两者都是 pathogenetically 的特点是对髓鞘的自身免疫反应, 导致鞘周围神经。在这些疾病中, 活化的 T 细胞穿过血神经屏障, 在三七皂苷内产生免疫反应。激活的巨噬细胞, 然后导致鞘要么直接通过吞噬的攻击或间接通过分泌炎症介质, 导致临床残疾, 如麻痹和感觉障碍5。虽然鞘轴突保留 remyelinated 跟随鞘的能力, 髓通常是延迟或不完整的, 导致裸轴突的易感性到不可逆转的损害, 这是永久临床的主要原因残疾.目前, 最有效的治疗方法是免疫调节, 但尽管他们的疗效, 在许多情况下, 恢复往往是缓慢的和〜25% 患者将体验到剩余的功能赤字, 大大降低他们的生活质量6, 7。
是一种广泛使用的脱髓鞘周围神经病的动物模型, 它提供了有价值的洞察机制和评估新的治疗药物的方法4。该模型可诱导不同种类的家兔、大鼠、小鼠和豚鼠, 并由神经源性抗原免疫诱发。然而, 最终成功的细胞诱导依赖于适当的免疫反应的疾病发生。鉴于免疫功能的种类 (和间/应变) 的变化, 多种抗原和佐剂的组合已被开发成功地诱导了。在小鼠遗传工具方面, C57BL/6 是最广泛使用的;然而, 传统的 P2 蛋白肽 57-81 (P257-81), 导致在易感 SJL 小鼠菌株的疾病8是不能非法发病导致功能性赤字的 C57BL/6 菌株。幸运的是, 使用 P0106-125或 P0180-199多肽的敏化范例, 连同注射百日咳毒素的佐剂一起提供, 可以克服这一障碍, 使复杂的遗传工具能够用于小鼠模型。
本文介绍了一种简单的 C57BL/6 小鼠的诱导方法。此外, 还提供了一个综合详细的方法来评估与疾病相关的功能和病理的缺陷。P0180–199多肽9首选于 P057-81备选10。与 P057-81备选10相比, P0180–199模型已被描述为产生较不严重的临床症状, 因此可能会承受引入潜在有害的遗传摄动, 从外科手术中恢复 (如渗透泵植入), 并经得起跑步机的步态功能测试4。然而, 在 P0 的57-81诱导变异中研究这种疾病时, 可以很容易地使用在这里描述的跑步机步态功能测试和组织学协议。
Protocol
Representative Results
Discussion
本报告概述了一个简单的方法, 诱导使用 P0180-199多肽在 C57BL/6 小鼠, 使量化的关键病理和功能缺陷的小鼠的免疫诱导。不同的是, 这里所描述的归纳协议是使用麻醉, 而执行免疫注射。使用异氟醚麻醉, 大大提高了能力, 以确保总体积的接种被注入皮下在理想的位置, 最小的错误和压力的动物。
小鼠将开始显示疾病的临床症状从 6 dpi4使用这种方法的诱?…
Declarações
The authors have nothing to disclose.
Acknowledgements
DGG 是一个澳洲彼得多尔蒂和多发性硬化研究澳大利亚 (微软亚洲研究院) 早期职业研究员。JLF 得到微软亚洲研究院博士后奖学金的支持。这项工作得到澳大利亚国家卫生和医学研究理事会 (澳洲) 项目赠款 #APP1058647 JX。
Materials
| C57BL/6, male, 6-8 weeks old | Australian Bioresources Cenre, WA, Australia | ||
| Pertussis toxin | List Biological Laboratories, Inc., CA, USA | #181 | |
| 0.1M mouse-isotonic phosphated buffered salined (MT-PBS) | Laboratories will have their own protocol. | ||
| Isoflurane | Pharmachem, QLD, Australia | Laboratories will have their own protocol for administration. | |
| P0180–199 peptide | Wuxi Nordisk Biotech Co. Lt. SHG, CHN | P0180–199, sequence S–S–K–R–G–R– Q–T–P–V–L–Y–A–M–L–D–H–S–R–S | |
| Heat killed Mycobacterium tuberculosis (strain H37RA) | Difco, MI, USA | #231141 | |
| Freund's complete adjuvant (FCA) | Difco, MI, USA | #263910 | |
| 16% Paraformaldehyde (PFA) | Electron Microscopy Services | #15710 | Dilute to 4% PFA day of tissue collection. |
| 25% glutaraldheyde | ProSciTech Pty Ltd, QLD, Australia | #11-30-8 | Dilute to 2.5% glutaraldehyde day of fixation. |
| Sodium azide | Chem-Supply Pty Ltd, SA, Australia | SL189 | Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v). |
| Sucrose | Chem-Supply Pty Ltd, SA, Australia | SA030 | Use at 30% (w/v). |
| Optimum cutting temperature (OCT) medium | Sakura Finetek, CA, USA | #4583 | |
| Normal donkey serum | Merck Millipore, MA, USA | #S30-100 | Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory. |
| Triton-X 100 | Sigma Aldrich, MI, USA | #90o2-31-1 | Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory. |
| rabbit anti-amyloid precursor protein (APP) | Invitrogen (Life Technologies), CA, USA | S12700 | Used at 1:400 or titrate in own lab. |
| rabbit anti-contactin-associated protein-1 (Caspr) | Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel | Used at 1:500 or titrate in own lab. | |
| Appropriate Alexa Fluor conjugated secondary antibodies | Molecular Probes (Life Technologies), OR, USA | Various | Use at 1:200 or titrate in own lab. Choice of species the antibody was raised in and Alexa Fluor chosen is at the discretion of each laboratory. |
| Aqueous mounting solution | Dako (Agilent), CA, USA | #S3023 | Each laboratory will have their own preference. |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| 0.5 mL syringe with 301/2 g needles | BD | #326105 | |
| 23 g needles | BD | #305143 | |
| Red ink pad | Any red ink pad or red food dye could be used to mark the animals' feet. | ||
| DigiGate apparatus (includes treadmill) | eMouse Specifics Inc. Framingham, MA | ||
| DigiGate Imaging System | eMouse Specifics Inc. Framingham, MA | ||
| Stopwatch | Any timer may be used. | ||
| DigiGait 8 Software | eMouse Specifics Inc. Framingham, MA | ||
| Dissecting microscope | Zeiss | Any appropriate dissecting microscope may be used. | |
| Charged slides | Superfrost Plus, Lomb Scientific Pty Ltd | SF41296SP | |
| Cyrostat | Leica | Any suitable cyrostat may be used. | |
| Perfusion equipment and dissecting instruments | Labs will have their own perfusion protoctols. | ||
| Opaque humified chamber | Labs may produce their own using an opaque plastic container. | ||
| PAP pene | GeneTex (USA) | Wax pencil, or surface tension may also be used to create a well around the tissue section. | |
| Confocal microscope | Zeis LSM780 | Any confocal microscope with appropriate laser lines may be used. | |
| FIJI/Image J | National Institues of Health | Available from www.fiji.sc |
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