作者要感谢北美小儿骨科学会 Biomet 脊柱补助金和美国国立卫生研究院临床与转化科学研究所 KL2 补助金以及 HH Lee 外科研究补助金作为这些实验的主要资金来源。

所有动物均严格按照《动物福利法》（AWA）、1996 年《实验动物护理和使用指南》、《PHS 实验动物人道护理和使用政策》以及《动物护理和使用培训手册》中规定的机构政策和程序中规定的良好动物规范进行处理， 所有动物工作均获得加州大学洛杉矶分校校长动物研究委员会（ARC）的批准。
1. 金黄色葡萄球 菌生物发光菌株选择
<ol><li>使用生物发光 金黄色葡萄球菌菌 株 Xen36 作为感兴趣的接种物。 注意:该菌株来源于 亲本金黄色葡萄球菌 ATCC-49525，它是来自脓毒症患者的临床分离物。 金黄色葡萄球菌 Xen36 独特地利用 了 luxABCDE 操纵子，该操纵子经过优化并集成到宿主的天然质粒中。19 因此，Xen36 菌株能够产生峰值波长发射为 490 nm 的蓝绿色生物发光光。这种发射信号仅由具有代谢活性的活细菌生物产生。</li>
</ol>2. S. 的制备接种用金黄色葡萄球菌
<ol><li>将 200 μg/mL 卡那霉素加入 Luria 肉汤中，加上 1.5% 琼脂，利用与 lux 操纵子19 相连的卡那霉素抗性基因，从潜在污染物中分离金黄色葡萄球菌 Xen36。</li>
	<li>将 金黄色葡萄球 菌Xen36细菌条纹到胰蛋白酶大豆琼脂平板（胰蛋白酶大豆肉汤[TSB]加1.5%琼脂）上，并在37°C下孵育12-16小时。</li>
	<li>分离 金黄色葡萄球 菌Xen36的单个菌落，并在37°C下在振荡培养箱（200rpm）中在TSB中单独培养12-16小时。</li>
	<li>以 1:50 的比例稀释所得培养物。</li>
	<li>在37°C下再培养2小时以分离中对数期细菌。</li>
	<li>在磷酸盐缓冲盐水 （PBS） 中沉淀、重悬和洗涤细菌 3 次。</li>
	<li>测量600nm处的吸光度。理想的 OD600 介于 0.700 和 0.750 之间，相当于 4x105 CFU/mL。进行连续稀释以获得所需的细菌接种物（1 x 103 CFU / 2μL）。 注:发现用于建立慢性感染的Xen36的最佳浓度为1 x 103 CFU。较低剂量的细菌被宿主免疫系统清除，较高剂量导致伤口破裂。伤口破损不区分深部植入物感染和浅表伤口感染，因此在此模型中避免了（图 1）20。</li>
</ol>3. 老鼠
<ol><li>使用12周龄雄性C57BL / 6J野生型小鼠。</li>
	<li>将老鼠饲养在笼子里，一次最多4只。</li>
	<li>随时保持水的供应。保持 12 小时的明/暗循环，不要在循环的暗阶段进行实验。</li>
	<li>使用不含苜蓿的食物进行喂养，因为可能会干扰荧光信号。</li>
	<li>让研究人员或兽医人员每天评估小鼠，以确保动物在整个实验过程中的健康。</li>
</ol>4. 小鼠外科手术
<ol><li>通过将小鼠置于异氟醚（2%）腔室中约5分钟来诱导麻醉。通过监测呼吸以保持有节奏且比清醒时慢，并且不会因有害刺激（例如，手术操作、脚趾捏）而改变，从而确认适当的麻醉深度。</li>
	<li>将麻醉的小鼠转移到准备站，并用啮齿动物剪将毛发从骶骨去除到上胸椎。</li>
	<li>用交替的倍他丁溶液和异丙醇进行三次洗涤清洁和消毒。</li>
	<li>将俯卧位的麻醉和灭菌小鼠转移到无菌手术床上，通过鼻锥给予吸入异氟醚（2%）维持麻醉。</li>
	<li>最大限度地弯曲臀部并确定膝盖在脊柱水平的位置，以接近腰椎 4 椎体。</li>
	<li>用 15 刀片手术刀在皮肤上做一个 2 厘米的纵向切口。</li>
	<li>触诊棘突以确认中线，并继续切口直至骨骼。</li>
	<li>在L4棘突右侧的骨膜下解剖，向外侧延伸至横突。</li>
	<li>将 5-0 头侧和尾部大小的可吸收编织缝合线通过筋膜传递到 L4 体并保持开放状态，为将来的闭合做准备。</li>
	<li>使用 25 G 脊髓针，使用 25 G 脊髓针铰接 L4 的棘突，并沿椎板插入直径为 0.1 毫米、长 1 厘米的"L 形"手术级不锈钢植入物，长臂铺设头侧。</li>
	<li>用 1 x 103 CFU/2 μL 生物发光 金黄色葡萄球 菌 Xen36 接种植入物，注意确保所有溶液都接触植入物。</li>
	<li>在接种后将先前通过的可吸收缝合线绑扎起来约10秒钟，以确保接种物包含在植入物上。</li>
	<li>用可吸收的缝合线以跑步的方式紧贴皮肤。</li>
	<li>立即皮下注射丁丙诺啡 （0.1 mg/kg），然后每 12 小时注射一次止痛药，持续 3 天。</li>
	<li>在加热垫上恢复小鼠并监测恢复正常活动。</li>
	<li>获取术后 X 光片以确认植入物的正确放置。</li>
</ol>5. 纵向活体生物发光成像测量细菌负荷
<ol><li>用吸入异氟醚（2%）麻醉小鼠。通过监测呼吸以保持有节奏且比清醒时慢，并且不会因有害刺激（例如，手术操作、脚趾捏）而改变，从而确认适当的麻醉深度。</li>
	<li>用啮齿动物剪去除从骶骨到上胸椎的毛发。</li>
	<li>将小鼠加载到生物发光成像平台的视野中，以进行体内生物发光成像（BLI）19。</li>
	<li>在 5 分钟的采集时间内捕获生物发光信号。利用具有 15 厘米视野 （B） 的大型像素合并设置。</li>
	<li>在术后第 0、1、3、5、7、10、14、18、21、25、28 和 35 天（或基于特定实验设计的其他天数）重复步骤 5.1-5.4 以监测细菌负荷。</li>
	<li>通过色阶和叠加在灰度照片上呈现BLI数据。使用 BLI 软件分离标准卵形感兴趣区域 （ROI），以量化总通量（光子/秒/秒）或平均最大通量（光子/秒/厘米2/球面度）的 BLI。</li>
</ol>6. 量化粘附在植入物和周围组织的细菌
<ol><li>根据 AVMA 指南，在 POD 35 或选择的替代术后日期暴露于二氧化碳的情况下对小鼠实施安乐死。确认安乐死伴继发性颈椎脱位。</li>
	<li>根据步骤4.3对背部皮肤进行消毒，并将小鼠俯卧在无菌手术区域上。</li>
	<li>使用 15 刀片手术刀锐利切开先前的切口。</li>
	<li>使用无菌剪刀钝性解剖 L4 棘突并识别手术植入物。</li>
	<li>使用针头轻轻扭转植入物并将其从 L4 棘突中的位置移开。</li>
	<li>使用无菌镊子和剪刀，收获紧邻手术植入物周围的约 0.1 g 棘突骨和软组织，并将 1 mL PBS 放入带有 4 个锋利均质珠的小锥形犀牛管中。</li>
	<li>通过称量锥形管在收获前后记录软组织的重量。</li>
	<li>将植入物置于 TSB 中的 0.5 mL 0.3% 吐温-80 中并超声处理 15 分钟。</li>
	<li>涡旋所得植入物悬浮液2分钟，培养过夜12-16小时。</li>
	<li>使用均质器将先前放置在植入物周围的1ml PBS中的软组织和棘突均质化。</li>
	<li>涡旋所得软组织悬浮液5分钟，培养过夜12-16小时。</li>
	<li>过夜培养后，分别从植入物和周围组织中计数CFU。表示为软组织的总 CFU/g 和超声植入物的总 CFU/mL。</li>
</ol>

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作者没有要披露的利益冲突。

脊柱植入物相关感染预示着患者1、2、3、4、5 的不良预后。与身体的许多其他部位不同，脊柱中受感染的硬件通常无法切除，因为存在不稳定和神经系统受损的风险。在对全身抗生素治疗耐药的生物膜细菌的情况下，这种独特的挑战需要新的治疗方法12.先前对SII新疗法的研究受到昂贵，低效的动物模型的限制。为了更好地研究这些感染并有效评估潜在治疗的疗效，我们使用体内生物发光成像开发了一种无创的SII纵向小鼠模型。
先前的器械性脊柱感染动物模型需要体外组织分析来评估结果，需要大型队列，并且无法随着时间的推移监测感染13,14,26。相比之下，本文中提出的SII小鼠模型利用BLI可靠地监测细菌负荷随时间的变化16,17,18。这种新方法使研究人员能够评估细菌和宿主对抗生素、涂层或免疫调节的反应。
方案中的关键步骤包括:适当制备 Xen36 金黄色葡萄球菌 和估计单次接种物 1 x 103 CFU/2 μL;精确的手术植入、接种和封堵;体内生物发光成像;并使用 CFU 计数确认细菌负荷。
对模型的修改可能包括替代的生物发光细菌菌株、更长的时间点，或使用其他类型的基因工程小鼠的整合，例如在骨髓细胞中表达绿色荧光蛋白的小鼠 （Lys-EGFP） 以同时测量中性粒细胞浸润与体内荧光成像20.可以使用其他方法来补充方案中描述的方法，以解决椎骨溶解、椎间盘退行性变、软组织感染和植入物生物膜感染等过程。在这些过程中提供定量结果的技术可能包括但不限于:显微计算机断层扫描、磁共振成像、实时定量 PCR、血清学、组织学、免疫组化和/或可变压力扫描电子显微镜。
此模型有几个限制。首先，与临床实践相比，脊柱手术的模式是一种粗略的简化。与高危脊柱手术患者典型的广泛减压和多层次融合手术相比，模型手术涉及使用单个不锈钢植入物进行最小的骨切除术。由于临床脊柱植入物具有多种不同的材料，因此这些材料对细菌感染和生物膜形成的敏感性可能不同。此外，与所有动物模型一样，宿主对小鼠感染的反应与人类不同。
将来，该模型可用于评估使用其他抗生素递送方式（包括万古霉素粉、抗生素负载珠或涂层植入物）对 SII 的治疗。此外，该模型可用于研究宿主对SII反应的机制基础。

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

该方法的目的是描述一种新的脊柱植入物感染（SII）小鼠模型。该模型旨在提供一种廉价且准确的工具，以灵活评估宿主、病原体和/或植入物变量在体内的影响。在该模型中测试脊柱植入物感染的潜在疗法和治疗策略旨在指导在应用于大型动物模型和临床试验之前的研究开发。
脊柱手术后植入物相关感染是一种毁灭性的并发症，不幸的是，大约 3-8% 的择期脊柱手术患者1、2、3、4、5 和高达 65% 的接受多节段或翻修手术的患者6。脊柱植入物感染的治疗通常需要多次住院、多次手术和长期抗生素治疗。SII 预示着不良的患者预后，包括神经功能受损、残疾和死亡风险增加。SII 的管理非常昂贵，每位患者的费用超过 900,000 美元7。
金黄色葡萄球菌是 SII8、9、10、11 最常见的致病病原体。细菌可以在手术过程中直接播种硬件，在术后期间通过伤口播种，或者以后通过血行播散。在金属植入物存在的情况下，金黄色葡萄球菌形成生物膜，保护细菌免受抗生素治疗和免疫细胞的侵害。虽然移除受感染的硬件可能有助于有效根除感染，但这在脊柱中通常不可行，否则会导致不稳定并有神经系统损害的风险12.
在没有移植受感染的硬件的情况下，需要新的方法来预防、检测和治疗 SII。从历史上看，SII的动物模型有限，无法有效评估新疗法的安全性和有效性。以前的SII动物模型需要大量的动物和需要安乐死的数据点的收集，包括菌落计数，组织学和培养13,14,15。由于缺乏纵向体内监测，这些模型仅为每只动物提供一个数据点，因此成本高昂且效率低下。
先前研究膝关节置换术感染小鼠模型的工作确定了无创活体光学成像在纵向监测感染负担方面的价值和准确性16。生物发光的检测允许在单个动物的纵向时间过程中以人道、准确和高效的方式量化细菌负荷。此外，先前的研究表明，体内生物发光与粘附在植入物上的CFUs之间存在高度相关性17。随着时间的推移跟踪感染的能力，使人们对植入物相关感染有了更细致入微的理解。此外，以这种方式监测纵向感染，可以准确评估抗生素治疗和新型抗菌剂的有效性16,17,18。
利用这些工具，我们开发并验证了术后脊柱植入物感染的模型。在所提出的方法中，我们利用生物发光金黄色葡萄球菌 Xen36 的接种物来建立 SII 的体内小鼠模型，以纵向监测细菌负荷16、17、18。这种新颖的模型提供了一种有价值的工具，可以在将其应用于大型动物模型和临床试验之前有效地测试SII的潜在检测、预防和治疗策略。

<table><tbody><tr><td>Analytical Balance ME104</td><td>Mettler Toledo</td><td>30029067</td><td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td></tr><tr><td>BD Bacto Tryptic Soy Broth</td><td>Becton Dickinson (BD)</td><td>BD 211825</td><td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td></tr><tr><td>Biomate 3S UV-VIS Spectrophotometer</td><td>Thermo Scientific</td><td>840-208300</td><td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td></tr><tr><td>Bioshield 720+ swinging bucket rotor</td><td>Thermo Scientific</td><td>75003183</td><td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td></tr><tr><td>Branson Ultrasonics 2510R-MTH (Sonicator)</td><td>Branson Ultrasonics</td><td>CPX952217R</td><td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td></tr><tr><td>Bullet Blender Storm Homogenizer</td><td>Next Advance</td><td>BBY24M</td><td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&amp;trade; minimizes sample heat up. Uses 1.5ml screw-cap RINO&amp;reg; tubes or snap-cap Eppendorf&amp;reg; Safe-lock&amp;trade; tubes.</td></tr><tr><td>Germinator 500</td><td>Electron Microscopy Sciences</td><td>66118-10</td><td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be&lt;br/&gt; used exclusively for research purposes. The Germinator 500 should not be used as a substitute for&lt;br/&gt; traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine&lt;br/&gt; sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been&lt;br/&gt; designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td></tr><tr><td>Heracell 150i CO2 Incubator</td><td>Thermo Scientific</td><td>51026282</td><td>Single 150L</td></tr><tr><td>IVIS Lumina X5 Imaging System</td><td>Perkin Elmer</td><td>CLS148590</td><td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&amp;mdash;and answers&amp;mdash;on anatomical and molecular aspects of disease.</td></tr><tr><td>MAXQ 4450 Digtial Incubating Bench Shaker</td><td>Thermo Scientific</td><td>SHKE4450</td><td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td></tr><tr><td>PBS, Phosphate Buffered Saline</td><td>Fisher Bioreagents</td><td>BP24384</td><td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td></tr><tr><td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td><td>Thermo Scientific</td><td>75002436</td><td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td></tr><tr><td>SORVALL LEGEND X1R 120V Centrifuge</td><td>Thermo Scientific</td><td>75004261</td><td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td></tr><tr><td>Staphylococcus aureus - Xen36</td><td>Perkin Elmer</td><td>119243</td><td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td></tr><tr><td>TUTTNAUER AUTOCLAVE 2540E 120V</td><td>Heidolph Tuttnauer</td><td>23210401</td><td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td></tr><tr><td>Tween 80</td><td>Fisher Bioreagents</td><td>BP338-500</td><td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td></tr><tr><td>Vortex mixer VX-200</td><td>Labnet Internation</td><td>S0200</td><td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td></tr><tr><td>0.9% Sodium Chloride</td><td>Pfizer Injectables/Hospira</td><td>00409-4888-10</td><td>0.9% Sodium Chloride Injection, USP</td></tr></tbody></table>

in vivo mouse model of spinal implant infection

脊柱植入物感染预示着不良结果，因为诊断具有挑战性，并且手术根除与机械脊柱稳定性不一致。该方法的目的是描述一种新型的脊柱植入物感染 （SII） 小鼠模型，该模型旨在提供一种廉价、快速和准确的体内工具来测试脊柱植入物感染的潜在治疗方法和治疗策略。
在这种方法中，我们提出了一种后入路脊柱手术模型，其中将不锈钢 k 线固定到 12 周龄 C57BL/6J 野生型小鼠的 L4 棘突中，并接种 1 x 103 CFU 金黄色葡萄球菌 Xen36 细菌的生物发光菌株。然后在术后第0,1,3,5,7,10,14,18,21,25,28和35天纵向成像小鼠体内生物发光。对来自标准化视野的生物发光成像 （BLI） 信号进行量化，以测量体内细菌负荷。
为了量化粘附在植入物和植入物周围组织的细菌，将小鼠安乐死并收获植入物和周围的软组织。通过超声处理将细菌从植入物中分离出来，培养过夜，然后计数菌落形成单位（CFU）。从该方法获得的结果包括通过体内 金黄色葡萄球 菌生物发光（平均最大通量）测量的纵向细菌计数和安乐死后的 CFU 计数。
虽然先前的器械性脊柱感染动物模型涉及侵入性、离体组织分析，但本文提出的 SII 小鼠模型利用生物发光细菌的非侵入性实时体内光学成像来取代静态组织研究。该模型的应用范围很广，可能包括利用替代的生物发光细菌菌株，结合其他类型的基因工程小鼠来同时研究宿主免疫反应，以及评估当前或研究新的诊断和治疗方式，如抗生素或植入物涂层。

本文介绍的程序用于评估抗生素方案在SII体内小鼠模型中的疗效。具体而言，将万古霉素和利福平联合抗生素治疗与万古霉素单药治疗和未经治疗的感染对照组的疗效进行比较。
手术前，小鼠被随机分配到联合治疗组、单药治疗组或感染对照组。进行统计功效分析以计算样本量。平均最大通量 1 x 10 5 ± 3.2 x 10 4 和1.4 x 105 的预期平均值用于确定样本量，每组计算为 N=10。小鼠接受手术植入，接种金黄色葡萄球菌 Xen36，并在 POD 0、1、3、5、7、10、14、18、21、25、28 和 35 上测量体内金黄色葡萄球菌生物发光。在 POD 35 上，处死小鼠并定量植入物粘附和周围组织细菌的 CFU。
单药治疗组的小鼠接受治疗剂量的万古霉素（110mg / kg，每日两次）皮下给药。选择该剂量是为了近似于万古霉素21、22、23 典型人体暴露的曲线下面积。联合治疗组的小鼠接受治疗剂量的万古霉素（110 mg/kg，每日两次）和利福平（25 mg/kg，每日）24。感染对照组的小鼠接受假注射无菌生理盐水。所有组的治疗从术后第 7 天到第 14 天进行。
抗生素治疗对BLI的影响 受感染的对照小鼠的BLI信号在POD 10上达到峰值，该信号保持在1.0 x 105光子/ s / cm 2 / sr以上，直到处死，成功地模拟了慢性SII（图2）。与感染对照组相比，接受万古霉素单药治疗的小鼠的BLI信号显著降低，POD 10-21减少了2倍（p<0.03）。POD 21 后，单药治疗组和感染对照组的 BLI 差异无统计学意义。用万古霉素-利福平联合治疗的小鼠具有更低的BLI信号，比POD 10上的感染对照低20倍。显著减少一直持续到POD 28（p<0.01）。POD 28后，两组间BLI差异无统计学意义。在POD35的最终成像中，三组中的任何一组之间的BLI均无显著差异。
来自植入物和周围组织的 CFU 在POD 35上处死小鼠。收获植入物和周围组织并处理CFU计数（图3）。感染对照组、单药治疗组或联合治疗组之间在CFU中未观察到显著差异。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60560/60560fig1v2.jpg"> 图 1:高剂量 金黄色葡萄球菌 Xen36 接种物中的伤口破损。在脊柱植入物感染期间接种 金黄色葡萄球菌 Xen36的小鼠背部皮肤的图像。（A） 接种 1 x 103 CFU 的小鼠，背部皮肤完整。（B） 接种 1 x 104 CFU 的小鼠，并有明显伤口破裂的证据。图经Dworsky et al.许可改编和转载25<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60560/60560fig1v2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60560/60560fig2v2.jpg"> 图 2:使用体内生物发光测量细菌负荷。 在不锈钢植入物存在下，将具有稳定质粒（Xen36）中生物发光结构的1 x 103 CFU金黄色葡萄球菌接种到小鼠（每组n = 10只小鼠）的L4棘突中。（A） 通过体内金黄色葡萄球菌生物发光测量的细菌计数（平均最大通量 [光子/s/cm2/sr] ± sem [对数刻度]），实验方案的流程图如下。在 POD 7 上，抗生素给药开始于万古霉素、万古霉素和利福平的组合或无菌生理盐水对照。POD 14 停止抗生素给药。在 POD 35 上，处死小鼠并测量来自植入物和周围组织的 CFU。（B）在小鼠灰度图像上叠加的色标上的代表性体内金黄色葡萄球菌生物发光。图经 Hu et al.25 许可改编和转载 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60560/60560fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60560/60560fig03.jpg"> 图 3:使用 CFU 计数确认细菌负荷。 在 POD 35 处死小鼠，对针进行超声处理，使组织匀浆，培养和计数细菌。图经 Hu 等人许可改编和转载25。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60560/60560fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

该协议描述了一种新型的脊柱植入物感染体内小鼠模型，其中不锈钢 k 线植入物感染生物发光 金黄色葡萄球 菌 Xen36。通过生物发光成像纵向监测细菌负荷，并在安乐死后通过菌落形成单位计数确认。

脊柱植入物感染的体内小鼠模型

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

in-vivo-mouse-model-of-spinal-implant-infection

Universidad San Sebastian

Research

JoVE Journal

Medicine

2.3K 次观看.  University of California Los Angeles. 该协议描述了一种新型的脊柱植入物感染体内小鼠模型，其中不锈钢 k 线植入物感染生物发光 金黄色葡萄球 菌 Xen36。通过生物发光成像纵向监测细菌负荷，并在安乐死后通过菌落形成单位计数确认。

视频: In Vivo Mouse Model of Spinal Implant Infection

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神经入侵的体内小鼠坐骨神经模型

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

科研

癌症研究

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

神经科学

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

医学

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

<meta charset="utf8"></head><body>开发高效、精确的脊髓神经胶质瘤小鼠模型

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

<meta charset="utf8"></head><body>肿瘤细胞手术移植到小鼠脊髓中

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

肿瘤细胞手术移植到小鼠脊髓中

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

免疫与感染

建立 白色念珠菌 生物膜相关 PJI 的小鼠模型

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

小鼠假体周围关节 白色念珠菌 感染模型

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

脊柱植入物感染的体内小鼠模型

摘要

探索更多视频

神经入侵的体内小鼠坐骨神经模型

在体内脊髓的鼠标线使用双光子显微镜成像

结合

一个程序，植入脊柱商会纵

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

小鼠假体周围关节白色念珠菌感染模型

小鼠假体周围关节白色念珠菌感染模型

脊柱植入物感染的体内小鼠模型

摘要

探索更多视频

神经入侵的体内小鼠坐骨神经模型

在体内脊髓的鼠标线使用双光子显微镜成像

结合

一个程序，植入脊柱商会纵

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

小鼠假体周围关节 白色念珠菌 感染模型

小鼠假体周围关节 白色念珠菌 感染模型

小鼠假体周围关节白色念珠菌感染模型

小鼠假体周围关节白色念珠菌感染模型