作者想感谢美国糖尿病协会支持这项工作 (赠款 # #7-13-BS-180), 密歇根州立大学研究技术支持设施, 提供培训和访问的在体内成像系统和密歇根州立大学调查组织病理学实验室为组织病理学检查进行小鼠活检。

动物实验由密歇根州立大学动物保育和使用委员会批准. 
 1. 咬合敷料和硅胶垫片的制备 <ol> <li> 剪切透明的咬合敷料, 使正方形约 1 cm x 1 厘米与剪刀. </li>
	<li> 在0.5 毫米厚的硅胶片上用10毫米的活检拳切割10毫米圆圈. 和 #160; 中心5毫米活检冲床在 10 mm 圈的中间, 并按坚定地创建一个孔, 形成一个和 #34;d onut 和 #34;-像圆盘, 将被用作夹板. 和 #160; </li> </ol>
 2. 实验动物 <ol> <li> 使用8周老 (22-26 克) 雄性 SKH-1 小鼠。保持21和 #176 的标准条件下的小鼠; C 和12小时暗循环, 免费获得食物和水. </li>
	<li> 为了诱发糖尿病, 在5连续的天内, 用13毫克/毫升链脲佐菌素 (intraperitonially) solutionand 25% 葡萄糖 (250 和 #181; l/鼠标) 注射小鼠. 
	<ol> <li> 通过稀释65和 #181; 5 和 #181 中的脲佐菌素 g; 100 mM 柠檬酸, pH 4.5. </li>
		<li> 调整每只老鼠的注射量为每1公斤的老鼠体重65毫克的最终质量。100毫米柠檬酸溶液注射控制小鼠的 pH 值4.5 在同一天. </li>
	</ol> </li> <li> 在最后一次注射血糖后14天内通过血糖监测确认高血糖。糖尿病小鼠可能有尿, 所以他们的床上用品可能需要更频繁地改变, 以消除湿度和他们的体重应监测每周3次. </li>
</ol> 3。生物膜 <ol> <li> 生物膜制备 <ol> <li> 生长菌落生物膜 8 以给伤口接种。手术前两天开始一夜的生物发光的文化 铜绿假单胞菌 Xen 41 在胰的大豆肉汤中孵育在37和 #176; C 和震动 200 rpm 和消毒的聚碳酸酯膜过滤器与0.2 和 #181; m 孔径通过暴露在紫外光在一个生物安全罩15分钟每边. </li>
		<li> 一天前的手术离心机过夜培养 2万 x g 2 分钟和洗涤3次与1毫升的 Dulbelcco 和 #39; 磷酸缓冲盐水 (DPBS) 由移上下. </li>
		<li> 稀释悬浮在 DPBS 到0.05 在 600 nm 的吸光度. </li>
		<li> 吸管10和 #181; 在胰大豆琼脂 (TSA) 板上的每个膜上的稀释培养基的 L。在允许干燥后, 在35和 #176 孵育膜; C 为72小时生长生物膜, 每24小时向新的 TSA 板转移. </li>
	在实验开始之前, </ol> </li> <li> 标准曲线 <ol> <li> 将发光和细菌计数关联起来的标准曲线. </li>
		<li> 准备3.1 中描述的生物膜. </li>
		<li> 使生物膜的系列稀释从 #189; 到1/24 通过混合生物膜与 DPBS 和涡流, 直到产生一个视觉均匀的解决方案。吸管200和 #181; 在一个黑色96井板和图像的稀释溶液的 L 的 在体内 成像系统. </li>
		<li> 将板稀释到 TSA 板上并在35和 #176 上孵育; C 为 24 h. </li>
		<li> 在板上计数菌落形成单元 (CFU), 并创建标准曲线以关联生物发光和细菌计数. </li>
	</ol> </li> </ol> 4。伤口手术 <ol> <li> 在 95% oxygen/5%co 中使用异氟醚诱导全身麻醉 (以防止酮症酸中毒死亡), 流速为1升/分和 #160; 使用1-3% 异氟醚维持麻醉。在手术中保持动物的热垫. </li>
	<li> 确保通过用镊子捏住脚并将鼠标置于俯卧位置来抑制鼠标的深层踏板反射. </li>
	<li> 昔 (0.2 毫克/千克) 通过 sub-cutaneous 注射 (30 和 #956; l) 进行疼痛管理. </li>
	<li> 用10% 聚维酮碘三次和一个异丙醇垫擦拭背部的皮肤. </li>
	<li> 使用无菌 4 mm 活检穿孔, 为鼠标和 #39 的一侧的伤口勾勒一个圆形图案。用永久性标记勾勒图案. </li>
	<li> 使用锯齿钳在轮廓和虹膜剪刀的中部提起皮肤, 以创建一个全的伤口, 延伸到皮下组织, 包括 panniculus carnosus 和切除圆形的组织. </li>
	<li> 将医用防水皮肤粘合剂粘附在小鼠的皮肤上, 并将硅胶夹板施加轻度压力。用透明的咬合敷料盖住伤口。手术后, 将动物单独关在笼子里. </li>
</ol> 5。术后管理 <ol> <li> 每日通过 sub-cutaneous 注射昔 (0.2 毫克/千克), 用于术后2天内疼痛缓解. </li>
	<li> 每天监测动物的疼痛和体重减轻的表现。糖尿病动物需要胰岛素注射时, 他们已经失去了15% 或更多的体重. </li>
</ol> 6。生物膜接种的制备和感染 <ol> <li> 在手术后接种48小时的小鼠, 如以下步骤所述. </li>
	<li> 用无菌刮刀从膜上刮 72 h 生物膜, 将其放在离心管中, 并在 DPBS 中稀释1:2。混合通过短暂的移上下. </li>
	<li> 将板接种到 TSA 板上, 以计算总 CFU。为了确保计数是准确的, 打破了生物膜接种进一步由一系列的两个1分钟的涡流步骤插2分钟超声步骤在40赫在一个超声波清洗器. </li>
	<li> 删除敷料覆盖伤口和硅胶夹板, 并采取显微镜下的伤口与相机的摄像头使用的标尺, 以供参考. </li>
	<li> 切割200和 #181 的提示; l 吸管提示和吸管10和 #181; 生物膜的 l 接种在每个伤口上. </li>
	<li> 图像鼠标使用 在体内 成像系统使用自动设置: 曝光时间5-300 秒, 与中等分, 1 f/停止和开放过滤器, 和视野 C (12.9 cm x 12.9 cm). </li>
	<li> 用新敷料覆盖伤口. </li>
</ol> 7。伤口测量和成像 <ol> <li> 每日评估动物临床症状 9 。
	<li> 根据需要提供食物、水和更换笼子. </li>
	<li> 每天检查敷料的完整性。当敷料是存在的, 只有生物发光可以测量, 由于闭塞的伤口。在8天, 敷料被删除, 并没有取代, 允许测量伤口闭合. </li>
	<li> 每隔一天称一次动物. </li>
	<li> 所有天数, 将小鼠单独放置在隔离室中使用 活体 成像系统每天或每隔一天都配备一个高效的过滤器和图像, 直到发光值降到背景水平以下. </li>
	<li> 8 天后, 在伤口愈合前, 用一把尺子, 用一把相机, 用一把显微, 用显微镜将伤口全身麻醉. </li>
</ol> 8。组织学分析 <ol> <li> 在安乐死室和 #160 中安乐2升/分钟的 CO 2 的小鼠; 发光后降到背景水平以下, 伤口完全愈合。确认宫颈脱位死亡是第二种安乐死方法. </li>
	<li> 使用虹膜剪刀创建一个宽, 完整的切除周围和伤口面积 (约1厘米直径), 并保存在4% 甲醛组织病理分析. </li>
	<li> 其他分析: 细胞因子检测 <ol> <li> 使用毛细管玻璃管从动物体内收集血液轨道, 并将其转移到 EDTA 处理过的管中。
		<li> 离心机的血液在 2000 rpm 20 分钟, 在4和 #176; C 和冷冻血浆细胞因子检测. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>James, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+in+chronic+wounds.">Biofilms in chronic wounds.</a> Wound Repair Regen. 16 (1), 37-44 (2008).</li><li>Gordois, A., Scuffham, P., Shearer, A., Oglesby, A., Tobian, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+care+costs+of+diabetic+peripheral+neuropathy+in+the+US.">The health care costs of diabetic peripheral neuropathy in the US.</a> Diabetes Care. 26 (6), 1790-1795 (2003).</li><li>Reiber, G. E., McDonell, M. B., Schleyer, A. M., Fihn, S. D., Reda, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+system+for+quality+improvement+in+ambulatory+care:+assessing+the+quality+of+diabetes+care.">A comprehensive system for quality improvement in ambulatory care: assessing the quality of diabetes care.</a> Patient Educ Couns. 26 (1-3), 337-341 (1995).</li><li>Driver, V. R., Fabbi, M., Lavery, L. A., Gibbons, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+of+diabetic+foot:+The+economic+case+for+the+limb+salvage+team.">The costs of diabetic foot: The economic case for the limb salvage team.</a> J Vasc Surg. 52 (Suppl 3), 17S-22S (2010).</li><li>Contag, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18 (4), 593-603 (1995).</li><li>Rocchetta, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+noninvasive,+real-time+imaging+technology+using+bioluminescent+Escherichia+coli+in+the+neutropenic+mouse+thigh+model+of+infection.">Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.</a> Antimicrob Agents Chemother. 45 (1), 129-137 (2001).</li><li>Kadurugamuwa, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+direct+method+for+monitoring+antibiotics+in+a+mouse+model+of+bacterial+biofilm+infection.">Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection.</a> Antimicrob Agents Chemother. 47 (0066-4804), 3130-3137 (2003).</li><li>Anderl, J. N., Franklin, M. J., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+antibiotic+penetration+limitation+in+Klebsiella+pneumoniae+biofilm+resistance+to+ampicillin+and+ciprofloxacin.">Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin.</a> Antimicrob Agents Chemother. 44 (7), 1818-1824 (2000).</li><li>Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+for+establishing+humane+endpoints.">A systematic approach for establishing humane endpoints.</a> ILAR J. 41 (2), 80-86 (2000).</li><li>Dunn, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+wound+healing.">Murine model of wound healing.</a> J Vis Exp. (75), e50265 (2013).</li><li>Zhao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+wound+healing+in+diabetic+(db/db)+mice+with+Pseudomonas+aeruginosa+biofilm+challenge+-+a+model+for+the+study+of+chronic+wounds.">Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge - a model for the study of chronic wounds.</a> Wound Repair Regen. 18 (5), 467-477 (2010).</li><li>Holley, A. K., Xu, Y., Noel, T., Bakthavatchalu, V., Batinic-Haberle, I., St. Clair, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+superoxide+dismutase-mediated+inside-out+signaling+in+HaCaT+human+keratinocytes+and+SKH-1+mouse+skin.">Manganese superoxide dismutase-mediated inside-out signaling in HaCaT human keratinocytes and SKH-1 mouse skin.</a> Antioxid Redox Signal. 20 (15), 2347-2360 (2014).</li><li>Abbas, S., Alam, S., Pal, A., Kumar, M., Singh, D., Ansari, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UVB+exposure+enhanced+benzanthrone-induced+inflammatory+responses+in+SKH-1+mouse+skin+by+activating+the+expression+of+COX-2+and+iNOS+through+MAP+kinases/NF-&#312;B/AP-1+signalling+pathways.">UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-&#312;B/AP-1 signalling pathways.</a> Food Chem Toxicol. 96, 183-190 (2016).</li><li>Watters, C., Everett, J. A., Haley, C., Clinton, A., Rumbaugh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+treatment+modulates+the+host+immune+system+to+enhance+Pseudomonas+aeruginosa+wound+biofilms.">Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.</a> Infect Immun. 82 (1), 92-100 (2014).</li></ol>

作者声明他们没有竞争的金融利益。

在这里, 我们描述了一个新的老鼠模型的研究糖尿病慢性伤口的生物膜有很多优势, 创造一个可复制的, 可翻译的, 灵活的模型。
第一项创新是使用无毛小鼠。其他的小鼠模型已经开发, 以研究糖尿病慢性伤口愈合10,11, 但所有这些都依赖于头发小鼠的使用, 需要脱毛的过程, 包括打蜡或头发修剪结合脱毛霜这一步不仅费时和凌乱, 但可?...

生物膜是嵌在高分子物质基质中的复杂的微生物群落, 被强调为导致慢性伤口的不良解决的一个因素1。对这些高度有组织的, 持久性微生物种群的研究对于糖尿病患者来说尤为重要, 因为在肢体上的循环不良和周围感觉机制的改变会导致未被察觉的病变2。在美国, 据估计, 15% 的糖尿病患者在生命过程中至少会产生一种溃疡。这转化为经济支出约280亿美元的治疗3,4, 更不用说 immensurable 的情感和社会负担。了解允许微生物群落在创面中持续存在的因素, 以及这些生物膜在愈合过程中所产生的影响, 对于更好地照顾受影响的患者和推动新的治疗方法的发展是势在必行的。因此, 建立可重复和可翻译的在体内模型, 以探索细菌与宿主的相互作用是最重要的。
小鼠模型已经成功地研究了生物膜对慢性伤口的影响。然而, 这些模型经常利用头发的物种, 并通过平板计数来评估生物膜清除量, 为被牺牲的动物切除组织的活细菌细胞, 使它们耗费大量时间和成本。
Contag et al首次提出了一种 biophotonic 的动物在评估感染时的终点取样的替代方法。(1995)5,谁开发了一种方法来捕获发光从组成生物发光的沙门氏菌沙门菌测量抗生素治疗的功效。其它利用生物发光细菌的研究也随之而来。例如, Rochetta et al。(2001)6通过使用增强的电荷耦合装置和后来的 Kadurugamuwa et al来验证感染模型, 以研究大肠杆菌小鼠的大腿感染。(2003)7利用了一株被工程化的金黄色葡萄球菌的光子发射特性, 研究了几种抗生素在小鼠导管伤口模型中的有效性。
该方法的特点是在无毛小鼠中诱导糖尿病的直接协议, 预先生物荧光生物膜产生和接种伤口, 并对感染的铜绿假单胞菌进行 biophotonic 监测。使用在体内成像系统。它提供了一个直接, 快速,原位, 非侵入性和定量的过程, 以评估慢性伤口的生物膜, 此外, 允许额外的分析, 如显微成像愈合伤口, 间歇血液收集细胞因子的测量, 和组织学的晚期组织收集。

<table>
	<tbody>
		<tr>
			<td>Opsite</td>
			<td>Smith &#38; Nephew</td>
			<td>Model 66000041</td>
			<td>Smith &#38; Nephew Flexfix Opsite Transparent Adhesive Film Roll 4&#34; x 11yards</td>
		</tr>
		<tr>
			<td>SKH-1 mice Crl:SKH1-Hrhr</td>
			<td>Charles River Breeding Laboratories</td>
			<td>SKH1</td>
			<td>Hairless mice, 8 weeks old</td>
		</tr>
		<tr>
			<td>Streptozotocin (STZ)</td>
			<td>Sigma Aldrich</td>
			<td>S0130-1G</td>
			<td>Streptozocin powder, 1g</td>
		</tr>
		<tr>
			<td>AccuChek glucometer</td>
			<td>Accu-Chek Roche</td>
			<td>Art No. 05046025001</td>
			<td>ACCU-CHEK CompactPlus Diabetes Monitoring Care Kit</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa Xen 41</td>
			<td>Perkin Elmer</td>
			<td>119229</td>
			<td>Bioluminescent Pseudomonas aeruginosa</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane filters</td>
			<td>Sigma Aldrich</td>
			<td>P9199</td>
			<td>Millipore polycarbonate membrane filters with 0.2 &#956;m pore size</td>
		</tr>
		<tr>
			<td>Dulbelcco phosphate buffer saline (DPBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Tryptic soy agar</td>
			<td>Sigma Aldrich</td>
			<td>22091</td>
			<td>Culture agar</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Henry Schein Animal Health</td>
			<td>49755</td>
			<td>Eloxiject (Meloxicam) 5mg/mL, solution for injection</td>
		</tr>
		<tr>
			<td>10% povidone-iodine (Betadine)</td>
			<td>Purdue Products LP</td>
			<td>301879-OA</td>
			<td>Swabstick, Betadine Solution. Antiseptic. Individ. Wrapped, 200/case</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>AAJ61899AK</td>
			<td>Alfa Aesar Paraformaldehyde, 4% in PBS</td>
		</tr>
		<tr>
			<td>Capillary glass tube</td>
			<td>Fisher Scientific</td>
			<td>22-362-566</td>
			<td>Heparinized Micro-Hematocrit Capillary Tubes</td>
		</tr>
		<tr>
			<td>Silicone to make splints</td>
			<td>Invitrogen Life Technologies Corp</td>
			<td>P-18178</td>
			<td>Press-to-Seal Silicone Sheet, 13cm x 18cm, 0.5mm thick, set of 5 sheets</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>Sigma Aldrich</td>
			<td>22092</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>IVIS Spectrum Isolation chamber</td>
			<td>Perkin Elmer</td>
			<td>123997</td>
			<td>XIC-3 animal isolation chamber</td>
		</tr>
		<tr>
			<td>HEPA filter</td>
			<td>Teleflex</td>
			<td>28022</td>
			<td>Gibeck ISO-Gard HEPA Light number 28022</td>
		</tr>
		<tr>
			<td>Biopsy punches</td>
			<td>VWR International Inc</td>
			<td>21909-142</td>
			<td>Disposable Biopsy Punch, 5mm, Sterile, pack of 50.</td>
		</tr>
		<tr>
			<td>Biopsy punches</td>
			<td>VWR International Inc</td>
			<td>21909-140</td>
			<td>Disposable Biopsy Punch, 4mm, Sterile, pack of 50.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>J.T.Baker</td>
			<td>1916-01</td>
			<td>Dextrose, Anhydrous, Powder</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma Aldrich</td>
			<td>C2404-100G</td>
			<td>Citric Acid</td>
		</tr>
		<tr>
			<td>Mastisol</td>
			<td>Eloquest Healthcare</td>
			<td>HRI 0496-0523-48</td>
			<td>Mastisol Medical Liquid Adhesive 2/3 mL vial, box of 48</td>
		</tr>
		<tr>
			<td>Corning 96-well black plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-567</td>
			<td>96-well clear bottom black polysterene microplates</td>
		</tr>
		<tr>
			<td>25 gauge 5/8 inch needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>Regular bevel needle</td>
		</tr>
		<tr>
			<td>Bransonic M Ultrasonic Cleaning Bath</td>
			<td>Branson Ultrasonics</td>
			<td>N/A</td>
			<td>Ultrasonic Cleaner</td>
		</tr>
	</tbody>
</table>

come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model

在慢性创伤中, 特别是在糖尿病患者中, 细菌的存在是一种结构化的生物膜, 被认为可以防止伤口的愈合和解决。慢性小鼠伤口模型已经被用来理解微生物和宿主之间的潜在相互作用。该模型的发展至今依赖于头发动物的使用和伤口组织的终端收集, 以确定可行的细菌。虽然这些模型已经获得了很大的洞察力, 但这个实验过程需要大量的动物, 取样是费时的。我们开发了一种新的小鼠模型, 它结合了几种最佳的创新方法来评估慢性伤口中生物膜的进展: a) 它利用无毛老鼠, 消除了脱毛的需要;b) 将预先生物膜应用于伤口, 以便立即评估这些社区对宿主的持久性和影响;c) 监测生物膜的进展, 通过量化由基因工程生物荧光菌株的铜绿假单胞菌的光生产, 使 real-time 监测的感染, 从而减少了每项研究所需的动物数量。在该模型中, 在脲佐菌素诱导的糖尿病无毛小鼠的背上产生单全创面, 并在其上接种了铜绿绿脓杆菌生物发光菌株41的生物膜。从伤口的光输出每天记录在一个体内成像系统, 允许在体内和原位快速生物膜可视化和局部化生物膜细菌在伤口。这种新的方法是灵活的, 因为它可以用来研究其他微生物, 包括基因工程物种和多生物膜, 并可能具有特殊价值的测试 anti-biofilm 战略, 包括抗菌闭塞敷料。

在这个新模型的开发中, 我们观察到了利用无毛 SKH-1 C57BL/6J 小鼠的许多优点, 这是我们过去使用过的。在糖尿病的发病过程中, 受注射脲佐菌素的动物通常会逐渐减轻体重;然而, 在先前由我们的实验室进行的伤口愈合实验中, 复制了邓恩. et al提出的模型。(2012)9使用 C57BL/6J, 观察到了剧烈的失重 (图 1)。与此相反, 当使用 SKH-1 小?...

Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

在这里, 我们描述了一个新的糖尿病小鼠模型利用无毛 real-time, 无创, 监测生物膜伤口感染生物发光的铜绿假单胞菌。该方法可用于评价其他细菌种类和转基因微生物的感染, 包括多生物膜, 并对 antibiofilm 策略的有效性进行测试。

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

come-to-light-side-vivo-monitoring-pseudomonas-aeruginosa-biofilm

Research

JoVE Journal

Immunology and Infection

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

13.1K 次观看.  Michigan State University. 在这里, 我们描述了一个新的糖尿病小鼠模型利用无毛 real-time, 无创, 监测生物膜伤口感染生物发光的铜绿假单胞菌。该方法可用于评价其他细菌种类和转基因微生物的感染, 包括多生物膜, 并对 antibiofilm 策略的有效性进行测试。

视频: Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

科研

免疫与感染

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

摘要

探索更多视频

此视频中的章节

铜绿假单胞菌和酿酒酵母流细胞生物膜

RNA的分离绿脓杆菌殖民小鼠胃肠道

移植物抗宿主病的诱导和

使用人工痰介质测试抗生素药效试验

长期慢性

绿脓杆菌引起的肺损伤模型

研究微生物群落

人胎盘及蜕膜器官培养研究在母胎界面感染

可视化痰生物膜发展影响使用腔室盖玻片型号

慢性假单位病伤口感染的延迟接种模型