这项工作得到了中国国家重点研究开发计划 (2017YFD0500102) 的资助;中国国家自然科学基金 [31572544];兽医病因生物学国家重点实验室 [SKLVEB2016KFKT005];上海农业应用技术发展项目, 中国 [G2016060201]。

小鼠感染实验由中国江苏省实验动物监测委员会批准, 并在南京农业大学实验动物中心 (许可证编号: SYXK (Su) 2017-0007) 上进行。
1. 细菌的制备
注:猪血清2型毒株 P1/7 是从患病的猪与脑膜炎19分离出来的。P1/7 菌株生长在托德-休伊特汤 (THB, 公式每公升 THB: 心脏输液, 3.1 克; neopeptone, 20.0 克; 葡萄糖, 2.0 克; 氯化钠, 2.0 克; 磷酸二钠, 0.4 克; 碳酸钠, 2.5 克) 和电镀在托德-威特特琼脂 (临屋区, 公式每公升心脏输液, 3.1 克;neopeptone, 20.0 克;葡萄糖, 2.0 克;氯化钠, 2.0 克;磷酸二钠, 0.4 克;碳酸钠, 2.5 克;琼脂, 15.0 克) 在37°c 和 5% CO2。
<ol><li>应变 P1/7 浮游细胞的采集<ol>
<li>收集5毫升浮游细胞从中对数阶段培养 (OD600= 0.6), 离心机为3分钟在 8000 x g, 然后洗涤3次与 PBS。</li>
		<li>并用重悬的细胞与5毫升25% 甘油在 THB, 整除成5管, 然后存储在-80 摄氏度。</li>
	</ol>
</li>
	<li>应变 P1/7 生物膜状态细胞的采集<ol>
<li>采取20毫升隔夜文化和增加180毫升新鲜的 THB;将稀释过的文化分成10圆的培养板, 然后把盘子放在37摄氏度的孵化器中, 5% CO2为 24 h。</li>
		<li>孵化后, 轻轻摇动盘子, 并用重悬不粘在盘子上的细菌, 然后用吸入法丢弃上清。 注意: 轻轻摇动盘子, 避免 resuspending 沉淀物。</li>
		<li>加入5毫升的 PBS, 以收获生物膜状态细胞, 完全并用重悬沉积物, 然后将样品转移到新管上。</li>
		<li>油脂实验生物膜状态细胞从最后一步以以下参量:60 W, 4 个周期, 5 s 在和十年代。</li>
		<li>离心机为3分钟在 8000 x g 和存放上清在-80 °c。 注: 上清可能含有生物膜成分, 可用于在感染前并用重悬生物膜细菌, 如动物实验所述 (步骤 3)。</li>
		<li>在 THB 中并用重悬10毫升25% 甘油的沉淀物, 然后将生物膜状态细胞储存在-80 摄氏度。</li>
	</ol>
</li>
</ol>2. 扫描电镜 (SEM) 分析
<ol><li>用4% 多聚甲醛 12-24 小时在4摄氏度固定收集的生物膜状态细胞和浮游细胞。</li>
	<li>用蒸馏水快速冲洗样品, 并以乙醇 (25%、50%、70%、90% 和 100%) 的浓度增加为30分钟, 在每个溶液中依次脱水样品。</li>
	<li>将干试样粘附在带有双面胶带的金属夹上, 最后将其涂在带有金和钯的蒸发器中。</li>
	<li>用扫描电子显微镜观察样品, 使用以下参数: EHT (超高压) = 10 伏;WD (工作距离) = 7.0 或8.0 毫米;放大 = 5000 x;信号 A = SE1。</li>
</ol>3. 动物实验
<ol><li>在本研究中使用 SPF 6 周的雌性 BALB/c 小鼠。通过颅内 subarachnoidal 感染的所有小鼠, 其注射部位位于 bregma 3.5 毫米延髓。Chiavolini 和al17清楚地描述了注射部位的立体定向坐标。<ol>
<li>病理学分析, 感染两组小鼠 (每组5只小鼠), 使用浮游细胞或生物膜状态细胞, 剂量为 3 x 107菌落形成单位 (CFU), 另有5只老鼠注射 PBS 作为控制。</li>
		<li>为检测 TLR2 和细胞因子在脑中的 mRNA 表达, 在 3 x 107 CFU 中使用浮游细胞或生物膜状态细胞感染两组小鼠 (每组6只小鼠)。</li>
		<li>通过电镀连续稀释来确定活菌的数量。</li>
		<li>弄死所有小鼠在12小时后感染后进行进一步的研究。 注: 在感染前必须清除在培养基中的甘油。从-80 °c 恢复的浮游细胞和生物膜状态细胞被洗涤3次与 PBS。然后, 浮游细胞悬浮与 PBS, 稀释到适当剂量的感染;生物膜状态细胞与储存的上清液悬浮 (从步 1.2.5) 和稀释到适当剂量的感染。感染量不应超过50µL。</li>
	</ol>
</li>
	<li>脑组织中 TLR2 和细胞因子 mRNA 表达的检测<ol>
<li>使用 rna 提取试剂盒从脑组织中提取总 RNA。<ol>
<li>对于每只老鼠, 使用全脑组织提取 RNA。把整个脑组织分成5管。服用多达100毫克的脑组织, 并添加1毫升的裂解液, 每管含有株溶藻基质提供的试剂盒。</li>
			<li>在6.0 的设置中, 在一个均质机上处理管, 然后离心管在 12000 x g 和4°c 为5分钟。</li>
			<li>离心后, 将上相转移到一个新的离心管。</li>
			<li>在室温下孵化转移样品5分钟;添加300µL 的氯仿, 漩涡十年代, 然后孵化5分钟室温。</li>
			<li>离心管在 12000 x g 和4°c 5 分钟转移上部阶段到新的管子。</li>
			<li>将500µL 的冷绝对乙醇添加到管中, 然后将其反转5次, 然后将样品贮存在-20 摄氏度, 至少为1小时。</li>
			<li>离心管在 12000 x g 和4°c 为15分钟和去除上清。用500µL 冷75% 乙醇在 RNase 的 H2O 上清洗颗粒。</li>
			<li>除去乙醇, 空气干燥的颗粒在室温下5分钟, 并并用重悬 RNA 在100µL RNase 无2O。</li>
			<li>在室温下孵化 rna 5 分钟, 用 rna bioanalyzer 测定 rna 浓度。</li>
		</ol>
</li>
		<li>进行 cDNA 合成, 包括消除基因组 DNA (gDNA), 使用 thermocycler 与反向转录 (RT) 试剂盒。<ol>
<li>将1µg 的 RNA 添加到含有2µL 5x gDNA 消除缓冲液的管中, 1 µL gDNA 消除酶和 RNase 的 H2O, 直到体积达到10µL. 在2摄氏度孵育42分钟。</li>
			<li>添加10µL 的主混合 (1 µL 的 rt 酶混合 I, 1 µL 的 rt 底漆混合, 4 µL 5x 缓冲器 2, 4 µL RNase 免费 H2O) 到反应解决方案从最后一步, 然后轻轻混合。立即进行 RT 反应:37 °c 为15分钟, 然后85°c 5 s。</li>
			<li>使用 SYBR qPCR 试剂盒实时 pcr 机进行定量实时 pcr (RT qPCR) 分析。添加10µL 的2x 酶, 0.8 µL 的前底漆, 0.8 µL 的反向底漆, 0.4 µL 50x 火箭参考染料 II, 2 µL 的 cDNA 模板, 和 RNase 免费 H2O, 直到总容量20µL。</li>
			<li>使用以下热参数运行每个样本: 三十年代在94°c, 跟随40个周期 5 s 在95°c 和三十四年代在60°c, 以十五年代的最后阶段在95°c, 1 分钟在60°c, 十五年代在95摄氏度。qPCR 的底漆列于表 1。管家基因B2m和18s rRNA被用作内部控制。</li>
		</ol>
</li>
		<li>根据 2-ΔΔCt方法计算相对褶皱变化。</li>
	</ol>
</li>
	<li>
组织学切片观察
	<ol>
<li>24 h 固定10% 福尔马林后, 将固定的脑组织重建成适当大小的组织块, 在 2-3 毫米。</li>
		<li>把纸巾放在嵌入的卡带里, 浸泡在一个新的固定液中, 用自动真空脱水脱水。</li>
		<li>取出固定组织, 并在每种溶液中依次脱水以增加乙醇 (70%、80%、95%、95% 和 100%) 的浓度。</li>
		<li>将该样品与二甲苯 Transparentize 15 分钟, 将其浸在液体石蜡中60°c 90 分钟, 然后用嵌入机嵌入。</li>
		<li>将石蜡块嵌入组织切成3µm 片, 在45摄氏度的水上平铺切片, 然后在空气中晾干。</li>
		<li>在60摄氏度孵育3小时的切片, 用 haematoxylin 和伊红 (他) 方法染色, 并用中性胶封住。</li>
		<li>用光学显微镜观察大脑的组织病理学变化。采用四点分级系统评价组织学变化。使用未配对的t检验进行统计分析。 充血/出血:缺席, 评分 0;小面积的局灶充血/出血, 评分 1;大面积的局灶充血/出血, 或多处小面积的局灶充血/出血, 评分 2;多达三个大焦距充血/出血部位, 得分 3;弥漫性充血/出血, 评分4。 坏死:缺席, 评分 0;病灶坏死, 评分 1;弥漫性发生 1-10 坏死细胞, 评分 2;汇合坏死灶, 评分 3;广泛的汇合坏死, 评分4。 炎症细胞浸润:缺席, 评分 0;少数和焦点渗透, 得分 1;多焦浸润或渗透形成聚集, 得分 2;最多三集, 分数 3;超过四集, 分数4。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

这里描述的颅内 subarachnoidal 感染途径与其他感染途径相比有明显的优势。它允许调查人员研究宿主-细菌相互作用和细菌成分对宿主免疫应答的影响直接在大脑, 这模仿细菌进入中枢神经系统。因此, 这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。
为了获得良好的结果, 使用这个模型, 以下关键步骤是明确的。生物膜...

猪链球菌(猪) 是全世界猪的主要细菌病原体, 造成严重疾病, 包括脑膜炎、肺炎、败血症、心内膜炎和关节炎1。它也是一个新兴的人畜共患病的特工。到目前为止, 据报道, 九型血清型可能导致人类感染, 包括血清型 2, 4, 5, 9, 14, 16, 21, 24, 312,3,4。在人类和猪中, 脑膜炎是感染猪的主要临床征象之一。在越南和泰国,美国猪是5成人脑膜炎的主要病因。微生物生物膜是粘在一起的微生物, 并集中在界面上;它们对细菌的毒力、不同环境下的生存和耐药性5至关重要。生物膜通常被一个细胞外基质所包围, 通常含有多糖、蛋白质和 DNA6。后者能够诱发宿主炎症反应和细胞因子的产生7。在以前的研究中, 生物膜的形成已被用于链球菌脑膜炎。生物膜有助于罗非鱼鱼模型中的链球菌脑膜炎链球菌, 生物膜的形成已经通过腹腔内接种8在体内的脑组织和脑膜表面周围显露出来。在脑膜炎期间,肺炎链球菌处于生物膜状状态, 而这种生物膜状态下的细菌更有效地诱发了老鼠感染模型9中的脑膜炎。此外, 在我们以前的研究中, 在小鼠脑中与猪的相关的生物膜状态通过生存分析导致细菌毒力10。然而, 直接证据的生物膜参与的猪链球菌脑膜炎需要进一步的调查。
采用腹腔 (ip)11、鼻腔 (i.n.)12、静脉注射 (静脉注射)13和 intracisternal (中) 感染14条的方法, 研制出了猪鼠感染动物模型.15,16. 然而, 感染的 ip、i.n. 和静脉注射途径不适合于直接在脑部研究在脑膜炎中猪的表面成分的作用。这些包括生物膜胞外基质。虽然在美国猪感染中使用了内源接种, 但是这些文件中没有描述精确的注射部位。与此相反, 颅内 subarachnoidal 接种的注射部位的立体定向坐标在前一项研究中已经明确描述过17。这允许容易地承认接种点和更加简单的实验性协议。此外, 颅内 subarachnoidal 感染的途径, 模拟细菌进入中枢神经系统从鼻窦或中耳17, 中耳和脑膜炎的关系, 由猪.已经证明了马德森et al18。此外, 通过应用颅内 subarachnoidal 感染的小鼠, 我们已经证明了猪的小 RNA rss04 导致脑膜炎在我们以前的研究10。
在本研究中, 采用颅内 subarachnoidal 感染途径对小鼠进行了研究, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过这种感染途径, 小鼠感染了浮游细胞或猪的生物膜状态细胞。生物膜对小鼠脑组织中 TLR2 和细胞因子表达的组织病理学分析清楚地表明,猪的生物膜对脑膜炎有促进作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
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			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
			<td style="width:115px;">Z00QSVCUS</td>
			<td style="width:304px;">Without Dnase/ Rnase</td>
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		<tr height="21">
			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="20">
			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="24">
			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="42">
			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
			<td style="width:304px;"></td>
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			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
			<td style="width:115px;">DS-11+</td>
			<td style="width:304px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
			<td style="width:115px;">JY96-IIN</td>
			<td style="width:304px;"></td>
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			<td height="21" style="height:21px;width:184px;">Centrifuge</td>
			<td style="width:225px;">Hitachi Koki Co., Ltd</td>
			<td style="width:115px;">CT15RE</td>
			<td style="width:304px;"></td>
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			<td height="21" style="height:21px;width:184px;">Refrigerator</td>
			<td style="width:225px;">Aucma Co., Ltd</td>
			<td style="width:115px;">DW-86L500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
			<td style="width:304px;"></td>
		</tr>
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			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
			<td style="width:225px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">4485689</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR820A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">PrimeScript RT reagent kit with gDNA Eraser</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
			<td style="width:304px;"></td>
		</tr>
	</tbody>
</table>

intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

猪链球菌不仅是全球猪的主要细菌病原体, 而且是一种新兴的人畜共患病剂。在人类和猪中, 脑膜炎是感染猪的主要表现。适当的感染模型是了解病原体致病机制的基本工具。研究了小鼠感染的几种途径, 探讨了猪感染的发病机制。然而, 腹腔, 鼻腔和静脉感染的途径不适合研究猪的表面成分在脑膜炎直接在脑中的作用, 如生物膜胞外基质。虽然 intracisternal 接种已被用于猪的感染, 但精确注射部位尚未描述。本文用小鼠模型描述了颅内 subarachnoidal 感染的途径, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过位于 bregma 3.5 毫米延髓的注射部位, 将猪的浮游细胞或生物膜状态细胞直接注入小鼠蛛网膜下腔。用生物膜状态细胞注射小鼠脑组织中 TLR2 和细胞因子的病理学分析和 mRNA 表达明显表明,猪的生物膜在猪的脑膜炎中起着决定性的作用。这种感染途径明显优于其他感染途径, 允许研究宿主-细菌相互作用。此外, 它允许细菌成分对宿主免疫应答的影响直接在大脑被评估, 并模仿细菌进入中枢神经系统。这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。

在实验条件下对生物膜的形成进行了 SEM 分析。如图 1所示, 浮游细胞 (图 1a) 与生物膜状态细胞之间的生物膜形成有显著差异 (图 1B)。SEM 分析表明, 生物膜细菌在团簇和多层, 它们被包裹在细胞外基质, 而浮游细菌的密度更低, 主要分散单独。
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Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

本文描述了小鼠颅内 subarachnoidal 感染的途径, 研究了生物膜在猪链球菌脑膜炎中的作用。这种感染模型也适用于研究其他细菌性脑膜炎的发病机制和新药对细菌性脑膜炎的疗效。

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

This method can help answer key questions about the roles of streptococcus suis'components in many sites, such as the extracellular matrix from its biofilms. The main advantage of this technique is that it enables investigations of bacterial components that affect how the immune responses directly in a brain and mimic bacteria entrance into the central nervous system. Demonstrating the procedure will be Mister Shouming Zhang, a graduate student from my laboratory.To collect strain P1/7 planktonic cells, first collect five milliliters of the cells from mid-lock phase culture. Centrifuge for three minutes at 8000 times G before washing the cells three times in PBS. Re-suspend the cells with five milliliters of 25%glycerol in THB and aliquot into five tubes.Alternatively, to collect strain P1/7 biofilm state cells, take 20 milliliters of an overnight culture and add to 180 milliliters of fresh THB. Equally divide the diluted culture into 10 round culture plates, then incubate the plates at 37 degrees celsius in 5%carbon dioxide for 24 hours. Following incubation, shake the plate gently to re-suspend the bacteria that have not adhered to the plate and avoid re-suspending the sediment.Then, discard the supernatant by aspiration. Add five milliliters of PBS to harvest biofilm state cells. Re-suspend the sediment completely and then transfer the sample to a new tube.Sonicate the re-suspended biofilm state cells as listed in the text protocol. Next, centrifuge the sonicated cells for three minutes at 8000 times G and store the supernatant at minus 80 degrees celsius. The supernatant may contain biofilm components and it can be used to re-suspend the biofilm bacteria before infection.Re-suspend the sediment with 10 milliliters of 25%glycerol in THB before observing the samples by scanning electron microscopy as described in the text protocol. Infect all mice using planktonic cells or biofilm state cells and perform euthanasia as described in the text protocol. Next, extract the total RNA from brain tissue using an RNA extraction kit.For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into five tubes. Take up to 100 milligrams of brain tissue and add one milliliter of lysis solution to each tube containing the lysis matrix provided by the kit.Extract the RNA following the steps listed in the text protocol. Next, perform CDMA synthesis, including elimination of genomic DNA, using a thermocycler with a reverse transcription reagent kit. Add one microgram of RNA to a tube containing two microliters of 5X genomic DNA elimination buffer and one microliter of geneomic DNA elimination enzyme.Add RNAse free water until the volume reaches 10 microliters. Incubate for two minutes at 42 degrees celsius. Add 10 microliters of master mix to the reaction solution and then mix gently.Proceed immediately with the reverse transcription reaction by placing the tube at 37 degrees celsius for 15 minutes, then transfer the reaction to 85 degrees celsius for five seconds. Perform the quantitative real-time PCR analysis using a real-time PCR machine with a cyber quantitative real-time PCR kit. Following the kit instructions, combine the enzyme, primers, 50X ROX reference dye two, and CDMA template, then add RNAse free water until a total volume of 20 microliters is reached.Run each sample in triplicate using the thermal parameters listed in the text protocol and calculate the relative fold-change based on the two minus delta delta CT method. Also perform the observation and scoring of histological sections of the infected brain tissue as detailed in the text protocol. The effect of biofilms on inflammatory response in mirroring brain tissue is shown here.At 12 hours post-infection, the expression of CCL2, IL6, TNF-alpha, and TLR2 from brains of infected mice was significantly higher for biofilm state cells as compared to planktonic cells. Far more severe pathological changes were observed in the meninges and cerebral cortex of mice infected with the biofilm bacteria as compared to mice infected with the planktonic bacteria. These changes included large areas of necrosis and intense inflammatory cell infiltration.However, much less inflammatory cell infiltration was observed in the meninges from mice infected with the planktonic bacteria. Furthermore, there was far more intense inflammatory cell infiltration observed in ventricles from mice infected with biofilm bacteria as compared to mice infected with the planktonic bacteria. As shown here, much less inflammatory cell infiltration was observed in ventricles from mice infected with the planktonic bacteria.While attempting this procedure, it is important to keep in mind that different strains may have different infective doses, so a preliminary experiment is strongly recommended to determine the appropriate infection dose. After watching this video, you should have a good understanding of how to collect biofilm cells and perform intercranial subretinal direct infection in mice.

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Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

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