通过细胞内分娩诱导小鼠脑膜炎球菌性脑膜炎血清群C
Summary
在这里，我们描述了一种通过成年小鼠的间感染途径诱导脑膜炎球菌性脑膜炎的方法。提出了从接种制备到间感染的脑膜炎球菌感染的一步一步方案;然后记录动物的生存，并评估鼠组织中的细菌负荷。
Abstract
脑膜炎球菌（脑膜炎球菌）是一种窄宿主范围的微生物，全球公认的细菌性脑膜炎的主要原因。脑膜炎球菌是人类鼻咽的瞬时殖民者，约占健康受试者的10%。在特定情况下，它获得侵入能力，穿透粘膜屏障，并侵入血液导致败血症。在最近一个病例中，即使没有随后发展脑膜炎，也可能出现脓毒症。相反，细菌在血液中繁殖不良，穿过血脑屏障，到达中枢神经系统，导致脑膜炎。细菌性脑膜炎的鼠群模型是研究宿主-病原体相互作用和分析导致这种致命疾病的病理遗传机制的有用工具。虽然在过去几十年中，已经评估了几个实验模型系统，但其中没有一个能够重现脑膜炎球菌病的特征病理事件。在本实验协议中，我们描述了基于细菌内接种的小鼠模型中诱导脑膜炎球菌性脑膜炎的详细过程。通过评估临床参数（如温度、体重）、存活率评估、微生物分析和脑损伤组织学检查，在鼠宿主中记录了人类脑膜炎的奇特症状。当使用内囊虫（即cist.）接种时，meningococci完全传递到水凝管，导致脑组织中非常有效的脑膜炎球菌复制。在大约18小时内观察到细菌的活量增加了1000倍。此外，在受感染小鼠的脾脏和肝脏中也发现了脑膜炎球菌，这表明肝脏可能代表脑膜炎球菌复制的目标器官。
Introduction
奈瑟氏脑膜炎是一种限于人类宿主的革兰氏阴性β-蛋白酶，众所周知，它是全世界人群中脑膜炎和败血症的最常见原因之一。它殖民健康和无症状的携带者的上呼吸道（鼻子和喉咙），但细菌有时逃避各种宿主免疫防御，从血液传播到大脑，导致不受控制的局部炎症，称为脑膜炎球菌性脑膜炎。宿主和细菌因素的组合似乎有助于从共生体向侵入性行为1的过渡。
脑膜炎是专门用于人类殖民化和感染的。它的宿主范围很窄，因此，由于缺乏复制人类脑膜炎球菌病的合适动物模型，体内发病机制研究有限。因此，在理解脑膜炎球菌引起的败血症和脑膜炎的发病机制方面，它造成了根本的差距。在过去的几十年里，许多体外系统的发展使得几个脑膜炎球菌毒性因子2，3，4的识别。尽管这些有价值的研究为了解这些因素对成功脑膜炎球菌感染的作用提供了重要见解，但这些模型不允许评估细菌与体液和细胞相互作用的后果免疫系统，甚至更少与整个组织。体内动物感染模型对于评估疫苗制剂所赋予的保护程度也有很大的意义。作为人-热带病原体，脑膜炎球菌拥有成功感染所需的适当决定因素，如表面结构（即IV型皮利蛋白和不截面蛋白）和人体受体和运输蛋白的铁吸收系统（即转移蛋白和乳铁蛋白）5，6，7适当地坚持， 生存和入侵人类宿主.最后，病原体的遗传变异能力，以逃避和/或阻止人体免疫反应，进一步促进高物种对流性8，9。因此，缺乏特定的宿主因素，参与相互作用，可以阻碍病原体生命周期的步骤，在小型动物模型的发展中遇到重大困难，从而总结脑膜炎球菌的生命周期。
在过去的几十年中，已经开发出几种方法来增进我们对脑膜炎球菌感染周期的了解。两种动物模型，小鼠和大鼠的感染，无论是腹内（即）或内陆（即），被开发繁殖脑膜炎球菌病10，11，12，13，14 ，15，16，17.实验鼠可能是诱导实验性脑膜炎球菌感染的更通用的动物之一。
然而，i.p. 感染方式导致严重败血症的发展，虽然它不模仿感染的自然途径，而i.n. 感染途径是有用的评估脑膜炎球菌发病机制，即使它可能导致肺部感染在败血症10，11，12，13，14，15，16，17之前。
i.p. 小鼠模型有助于评估对脑膜炎球菌挑战10、11、12的保护。基于i.n.感染途径的脑膜炎球菌殖民化小鼠模型已经开发给幼鼠，因为它们更容易感染脑膜炎球菌，以繁殖模仿人类脑膜炎球菌病的侵入性感染13，14，15，16，17。此外，为了促进脑膜炎球菌在鼠宿主的复制，越来越多的技术策略也被应用，包括给动物施用铁，以改善感染，使用高细菌接种，通过小鼠的细菌菌株，以及雇用婴儿或免疫功能低下的动物宿主10，13，15，18，19。表达特定的人为因素，如CD4620或转移性药物21增加了小鼠对这种人-热带细菌的易感性;人类皮肤异种移植模型的感染也有助于评估脑膜炎球菌对人内皮的粘附能力22，23。总体而言，人化转基因小鼠的最近发展提高了对脑膜炎球菌发病机制及其宿主相互作用的理解。
此前，我们开发了脑膜炎球菌性脑膜炎的鼠模型，其中细菌的接种被执行到成年小鼠的水箱中，这些老鼠带有老鼠通过的细菌24。受感染小鼠的临床参数和存活率表明，脑膜炎的建立具有与人类宿主相似的特点，以及大脑的微生物学和组织学分析。从这些受感染的小鼠中，细菌也从血液、肝脏和脾脏中恢复，而周围器官的细菌负荷也与传染性剂量相关。特别是，该模型用于评估在L-谷氨酸运输机GltT24中有缺陷的异源突变菌株的毒性。最近，使用我们的小鼠模型脑膜炎球菌性脑膜炎基于i.cist。血清组 C 菌株 93/42862，24和异体突变缺陷在cssA基因编码为 UDP-N-乙酰葡萄糖胺 2-表皮酶25，我们分析了暴露的西沙酸在建立中的作用在老鼠的疾病。
在本协议中，我们描述了一种基于i.cist诱导实验性脑膜炎球菌性脑膜炎的直截了当的方法。巴尔布/c成年小鼠的感染途径。该方法对于鼠类宿主脑膜炎球菌感染的表征，以及野生型参考菌株和异种突变体之间的毒性评估特别有用。水池内感染途径确保将脑膜炎直接输送到凝乳中，从而促进脑脊液 （CSF） 中的细菌复制，并诱导脑膜炎具有模仿这些特征存在于人类2，24，25，26。
Protocol
根据欧洲共同体理事会1986年11月24日指令（86/609/EEC），执行这项议定书是为了尽量减少动物痛苦和减少小鼠数量。本研究中报告的体内实验得到了伦理动物护理和使用委员会（2012年12月14日第2号）和意大利卫生部（Prot. 编号 0000094-A-03/01/2013）的批准。所有程序应在 BSL2 房间的生物安全机柜 2 （BSC2） 内执行，潜在受感染的废物应放在专用容器中。 1. 小鼠感染N. 脑膜炎血?…
Representative Results
感染N.脑膜炎野生型和异体突变菌株的小鼠存活。这些代表性结果中使用的奈瑟利亚脑膜炎菌株是血清群 C 参考菌株 93/4286 （ET-37） 及其同源突变体 93/4286+cssA通过插入性失活cssA基因获得，编码为UDP-N-乙酰葡萄糖胺2-表皮酶，该图在胶囊合成位点25。为了评估目前鼠模型中cssA缺陷菌株的毒性程度，评估了能够确定50%受感染动物死亡?…
Discussion
在这项研究中，我们描述了一种实验方案，通过i.cist诱导成年小鼠的脑膜炎球菌性脑膜炎。脑膜炎球菌的接种。据我们所知，在感染i.cist的实验鼠中，还没有开发出其他脑膜炎球菌性脑膜炎模型。路线;过去，人们一直在探索这种方法，为大鼠31和兔子32提供脑膜炎球菌性脑膜炎的模型。众所周知，脑膜炎球菌病发病率最高的是幼儿、青少年和33…
Divulgations
The authors have nothing to disclose.
Acknowledgements
研究部分得到了PRIN 2012 [授权编号 2012WJSX8K]的支持：”粘膜感染中的宿主-微生物相互作用模型：新治疗策略的开发”，以及 PRIN 2017 [2017SFBFER]：”解决粘膜感染之间的相互作用的综合方法具有挑战性的病原体的适应、压力条件和抗微生物药物耐药性”。
Materials
| 1,8 Skirted Cryovial With external thread | Starlab | E3090-6222 | |
| 50ml Polypropylene Conical Tube | Falcon | 352070 | 30 x 115mm |
| Adson Forceps | F.S.T. | 11006-12 | Stainless Steel |
| Alarm-Thermometer | TESTO | 9000530 | |
| BactoTM Proteose Peptone | BD | 211693 | |
| BD Micro Fine syringe | BD | 320837 | U-100 Insulin |
| BD Plastipak syringe 1ml 25GA 5/8in | BD | 300014 | 05x16mm |
| BD Plastipak syringe 5ml | BD | 308062 | 07 x 30mm |
| BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET | Bio Air s.c.r.l. | Aura B3 | |
| BioPhotometer | Eppendorf | Model #6131 | |
| Bottle D | Tecniplast | D | Graduated up to:400ml, Total Volume 450ml, 72x72x122mm |
| C150 CO2 Incubator | Binder | 9040-0078 | |
| Cage Body Eurostandard Type II | Tecniplast | 1264C | 267x207x140mm, Floor area 370cm2 |
| Cell Culture Petri Dish With Lid | Thermo Scientific | 150288 | Working Volume: 5mL |
| Centrifuge | Eppendorf | Microcentrifuge 5415R | |
| Cuvetta semi-micro L. Form | Kartell S.p.A. | 01938-00 | |
| di-Potassium hydrogen phosphate trihydrate | Carlo erba | 471767 | |
| di-Sodium hydrogen phosphate anhydrous ACS-for analysis | Carlo Erba | 480141 | g1000 |
| Diete Standard Certificate | Mucedola s.r.l. | 4RF21 | Food pellet for animal |
| Dumont Hp Tweezers 5 Stainless Steel | F.S.T. by DUMONT | AGT5034 | 0,10 x 0,06 mm tip |
| Electronic Balance | Gibertini | EU-C1200 | Max 1200g, d=0,01g, T=-1200g |
| Eppendorf Microcentrifuge tube safe-lock | Eppendorf | T3545-1000EA | |
| Erythromycin | Sigma-Aldrich | E-6376 | 25g |
| Extra Fine Bonn Scissors | F.S.T. | 14084-08 | Stainless Steel |
| Filter Top (mini- Isolator), H-Temp with lock clamps | Tecniplast | 1264C400SUC | |
| GC agar base | OXOID | CM0367 | |
| Gillies Forceps 1 x2 teeth | F.S.T. | 11028-15 | Stainless Steel |
| Glicerin RPE | Carlo Erba | 453752 | 1L |
| Graefe Forceps | F.S.T. | 11052-10 | Serrated Tip Width: 0.8mm |
| Inner lid | Tecniplast | 1264C116 | |
| Iron dextran solution | Sigma-Aldrich | D8517-25ML | |
| Ketamine | Intervet | ||
| Microbiological Safety Cabinet BH-EN and BHG Class II | Faster | BH-EN 2004 | |
| Microcentrifuge tubes 1.5ml | BRAND | PP780751 | screw cap PP, grad |
| Mouse Handling Forceps | F.S.T. | 11035-20 | Serrated rubber; Gripping surface:15 x 20 mm |
| Mucotit-F2000 | MERZ | 61846 | 2000ml |
| Natural Latex Gloves | Medica | M101 | |
| New Brunswick Classic C24 Incubator Shaker | PBI international | C-24 Classic Benchtop Incubator Shaker | |
| Petri PS Dishes | VWR | 391-0453 | 90X14.2MM |
| Pipetman Classic P20 | Gilson | F123600 | 2-20microL |
| Pipetman Classic P200 | Gilson | F123601 | 20-200microL |
| Pipetman Classim P1000 | Gilson | F123602 | 200-1000microL |
| Polyvitox | OXOID | SR0090A | |
| Potassium Chloride | J.T. Baker Chemicals B.V. | 0208 | 250g |
| Potassium Dihydrogen Phosphate | J.T. Baker Chemicals B.V. | 0240 | 1Kg |
| PS Disposible forceps | VWR | 232-0191 | |
| Removable Divider | Tecniplast | 1264C812 | |
| Round-Bottom Polypropylene Tubes | Falcon | 352063 | 5ml |
| Sodium Chloride | MOLEKULA | 41272436 | |
| SS retainer and Polyester FilterSheet | Tecniplast | 1264C | |
| Standard Pattern Forceps | F.S.T. | 11000-12 | Stainless |
| Stevens Tenotomy Scissors | F.S.T. | 14066-11 | Stainless Steel |
| Surgical Scissor – ToughCut | F.S.T. | 14130-17 | Stainless |
| Touch N Tuff disposible nitrile gloves | Ansell | 92-500 | |
| Ultra Low Temperature (ULT) Freezer | Haier | DW-86L288 | Volume= 288L |
| Wagner Scissors | F.S.T. | 14070-12 | Stainless Steel |
| Xylazine | Intervet |
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