用于口腔中性粒细胞病评价的鼠期牙周炎的强结合诱导模型
Summary
本文提出了建立涉及多个上颌骨的月牙病结扎诱导模型的协议，从而在涉及牙龈组织和骨骼的较大区域进行后续分析，并减少动物使用。还介绍了一种以类似于人类受试者的方式评估口服中性粒细胞的技术。
Abstract
利用鼠模型研究牙周病的病理生理学的主要优点是降低了动物的成本，一系列转基因菌株，大量的分析可以在收获的软组织和硬组织上进行。然而，其中许多制度受到程序上的批评。作为替代方案，可以采用牙周病的结扎诱导模型，由非生物性口腔微生物群的局部发展和保留驱动，这种模型是快速诱导的，相对可靠。不幸的是，结扎引起的鼠期牙周炎协议的变种被隔离到牙周的重点区域，并受到安装的连体过早的外泄。这最大限度地减少了可用于后续分析的组织数量，并增加了研究所需的动物数量。该协议描述了必要的精确操作，以放置扩展的摩尔结扎，改善保留和使用一种新的冲洗技术，以恢复小鼠的口服嗜中性粒细胞与另一种方法，以减轻上述技术挑战。
Introduction
牙周病（PD）是一种与重大宿主发病率和经济负担相关的骨解疾病，表现为牙龈炎症和软组织附件的丧失和对受影响的牙龈1、2、3、4的骨质支持。这个过程由宿主的口腔微生物群和先天免疫系统之间的相互作用所控制。它也与其他全身炎性疾病的恶化有关，包括糖尿病、心血管疾病和癌症5，6，7，8。从历史上看，据推测，PD发病机制依赖于大量的特定细菌，如波菲洛莫纳斯牙龈9。然而，最近的证据表明，PD的微生物成分是由牙科生物膜介导的。生物膜是一个有组织的，复杂的社区，由众多微生物组成，可以存在于健康的共生和破坏性的生物发育不良状态10，11。口服生物膜通常通过防止致病菌的建立来抵抗宿主，并通过调节宿主免疫反应12、13促进理想的牙龈组织结构和功能。口腔内共体生物体与宿主免疫系统之间平衡关系的扰动可能导致组织平衡的改变，导致细菌不及PD5、10、12、13、14的标记临床和放射外观的发展。
有趣的是，建立口腔不良细菌病，虽然需要启动PD，但不足以驱动PD在所有个体，逃避宿主免疫反应的能力，颠覆微生物群之间的共生和发育不良状态15过渡。这特别突出了PD影响先天免疫系统的主要特征之一，即多态核粒细胞（PMN），或中性粒细胞，从局部和系统的角度来看，从局部和系统的角度16，17。
在人类中，PMN在健康的牙周结缔组织中以+2 x 106细胞/小时的速度从循环中招募，它们是主要白细胞种群。在这里，它们随后被从牙龈硫酸盐中排出到口腔中，作为牙龈粘液的组成部分。在PD的存在，嗜中性粒细胞在循环和口腔中显现，其中这些效应细胞具有超炎表型，导致上述破坏的牙周17，18，19，20，21，22。因此，了解 PMN 在 PD 和其他全身炎症条件中的作用至关重要。
虽然人们普遍认为慢性病与PD有相互联系，但基本机制尚未得到阐明，造成这些病态和可能致命的系统疾病的管理困难。多个实验动物模型，每个都有独特的优点和缺点，已被利用来研究PD23，24的病理生理学。特别侧重于鼠模型，有各种协议，通过它促进对PD的研究;然而，他们有几个技术和生理缺陷25，26，27，28，29，30，31。
首先，口服腹膜小鼠模型需要大量人类牙周病原体的口服接种，以产生牙龈炎症和骨质流失。此外，它一般先有一段时间的抗生素治疗，以颠覆鼠群的口腔菌群25。这种模式通常需要专门的训练来安全地进行口腔结节，只使用一小部分来自更复杂的人类口腔微生物群的牙周病原病原体，并且需要几个月才能建立腹腔骨流失。
相反，化学诱导的鼠模型利用口服三硝基苯硫酸（TNBS）或硫酸钠（DSS），这种在几个月内建立结肠炎的鼠类模型时常用的制剂诱导牙周骨流失26。基于口腔内脓肿和外脓肿的模型可用，分别涉及地膜和钙化物的鼠切口和组织。在以前的脓肿模型中，多次注射细菌，产生多个牙龈脓肿和缺乏藻类骨质流失，限制了其在PD研究中的使用。后者脓肿模型明显更容易研究口腔外部位的细菌毒性、炎症和骨吸收，从而消除对牙周和口腔微生物群27、28、29、30、31的评价。
使用牙周炎的结结诱导模型，一个编织的丝线缝合线通常围绕第二摩尔周围安装。作为替代方案，缝合材料的单一线性段可以插入第一和第二摩尔32，33之间。结扎放置的目的是促进细菌积累和在牙龈硫化物内产生肌酸，导致牙周组织炎症和破坏组织组成牙周。最值得注意的是，这种模式能够产生显着更多的阿尔韦拉尔骨流失相比，更常用的口腔口盖模型34。使口服性食样模型的使用更加复杂的是，几种小鼠菌株（即C57BL/6）对发育的卵泡骨流失具有天然的抵抗力。这也是有问题的，因为这种菌株是最常用的基于鼠的动物研究35。
马尔切桑等人、安倍和哈吉森利斯所描述的现有程序旨在简化将连字33、36放置的技术操作。不幸的是，前一个协议需要专门的3D打印设备，并具有过早结扎丢失的可能性，从而增加动物的使用和与在手术室中花费的额外时间相关的成本。此外，这两种协议只产生可用于研究的患病牙周小区域。
该技术的优点在于同时研究口腔发育不良和免疫学，这些研究控制着牙周，利用具有不同遗传背景的低成本动物，以及简单的住房和饲养方法。因此，目标应该是尽量扩大病组织的数量，并为了实践减少动物研究的原则，将动物消费减少到尽可能低的水平。这需要确保所有动物都能被纳入实验分析37。然而，应该指出的是，无论使用哪种牙周病的动物模型，没有一个单一的模型，包括人类PD病理生理学的每一个元素。
这项新协议使用大多数实验室中的仪器和材料，在多个超颌摩尔牙齿周围放置结扎。它允许足够的时间轻松而自信地安装不太可能过早发生的连字。最后，当PMN协调PD中牙周牙的销毁时，还提出了一种以类似人类的方式恢复口服中性粒细胞的新方法。
Protocol
Representative Results
Discussion
与使用月冠引起的牙周炎模型相关的最关键的因素是围绕保留结扎，直到牺牲或故意去除的时间。安装的生物膜保持结扎能够导致在6天内导致阿尔韦拉尔骨高度的显著损失，在11-16天期间39之间稳定下来。决定在骨质流失的最长时期之前牺牲动物受试者，使这个结扎引起的牙周炎模型更短，被选择进一步减少过早结扎的发生率，定义为在牺牲前失去结扎，使动物无法诊断。
<p …Divulgations
The authors have nothing to disclose.
Acknowledgements
J. W. C. 得到加拿大卫生研究所（CIHR）的支持。作者要感谢孙春祥博士协助进行试青蓝染色。
Materials
| Anti-mouse F4/80 Antibody | BioLegend | 123131 | BV421, Clone BM8 |
| Anti-mouse Ly6G Antibody | BD | 560602 | PerCP-Cy5.5, Clone 1A8 |
| C57BL/6 Male Mice | Charles River | 8 to 12 weeks old | |
| Conical Centrifuge Tube | FroggaBio | TB15-500 | 15 mL |
| Conical Centrifuge Tube | FroggaBio | TB50-500 | 50 mL |
| FACS Buffer | Multiple | 1% BSA (BioShop), 2mM EDTA (Merck), 1x HBSS-/- (Gibco) | |
| FACSDiva | BD | v8.0.1 | |
| Fibre-Lite | Dolan-Jenner | Model 180 | |
| FlowJo | Tree Star | v10.0.8r1 | |
| Heat Therapy Pump | Hallowell | HTP-1500 | |
| Hot Glass Bead Sterilizer | Electron Microscopy Sciences | 66118-10 | Germinator 500 |
| Iris Scissors | Almedic | 7602-A8-684 | Straight |
| Ketamine | Vetoquinol | 100mg/mL | |
| LSRFortessa | BD | X-20 | |
| Mouse Serum | Sigma | M5905-5ML | |
| Nylon Mesh Filter | Fisher Scientific | 22-363-547 | 40 µm |
| Paraformaldehyde | Fisher Scientific | 28908 | 16% (w/v), Methanol Free |
| Phosphate-buffered Saline | Sigma | D1408-500ML | Without CaCl2 and MgCl2, 10x |
| Plastic Disposable Syringes | BD | 309659 | 1 mL |
| Rat Serum | Sigma | R9759-5ML | |
| Silk Suture | Covidien | SS652 | C13 USP 5-0 |
| Splinter Forceps | Almedic | 7726-A10-700 | #1 |
| Splinter Forceps | Almedic | 7727-A10-704 | #5 |
| Stereo Dissecting Microscope | Carl Zeiss | 28865 | Photo-Zusatz |
| Sterile Hypodemic Needle | BD | 305111 | 26G X 1/2" |
| Syringe | BD | 309659 | 1 mL |
| Xylazine | Rompun | 20mg/mL |
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