Name: in vivo mouse model of spinal implant infection
Uploaded: 2020-06-23T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

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?...

<ol>
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S., 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=Management+of+postprocedural+discitis.">Management of postprocedural discitis.</a> Spine Journal. 2 (4), 279-287 (2002).</li><li>Pappou, I. P., Papadopoulos, E. C., Sama, A. A., Girardi, F. P., Cammisa, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+infections+in+interbody+fusion+for+degenerative+spinal+disease.">Postoperative infections in interbody fusion for degenerative spinal disease.</a> Clinical Orthopaedics and Related Research. 444, 120-128 (2006).</li><li>Sampedro, M. F., 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=A+biofilm+approach+to+detect+bacteria+on+removed+spinal+implants.">A biofilm approach to detect bacteria on removed spinal implants.</a> Spine. 35 (12), 1218-1224 (2010).</li><li>Pull ter Gunne, A. F., Mohamed, A. S., Skolasky, R. L., van Laarhoven, C. J., Cohen, 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=The+presentation,+incidence,+etiology,+and+treatment+of+surgical+site+infections+after+spinal+surgery.">The presentation, incidence, etiology, and treatment of surgical site infections after spinal surgery.</a> Spine. 35 (13), 1323-1328 (2010).</li><li>Olsen, M. 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A rabbit model.</a> Spine. 23 (6), 653-656 (1998).</li><li>Stavrakis, A. I., 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=Current+Animal+Models+of+Postoperative+Spine+Infection+and+Potential+Future+Advances.">Current Animal Models of Postoperative Spine Infection and Potential Future Advances.</a> Frontiers in Medicine (Lausanne). 2, 34 (2015).</li><li>Pribaz, J. R., 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=Mouse+model+of+chronic+post-arthroplasty+infection:+noninvasive+in+vivo+bioluminescence+imaging+to+monitor+bacterial+burden+for+long-term+study.">Mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study.</a> Journal of Orthopaedic Research. 30 (3), 335-340 (2012).</li><li>Bernthal, N. 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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?...

An erratum was issued for:&#160;<a href="https://www.jove.com/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/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&#8482; minimizes sample heat up. Uses 1.5ml screw-cap RINO&#174; tubes or snap-cap Eppendorf&#174; Safe-lock&#8482; 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 
			used exclusively for research purposes. The Germinator 500 should not be used as a substitute for 
			traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine 
			sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been 
			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&#8212;and answers&#8212;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...

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 ...

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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

Combined In vivo Optical and Ãƒâ€šÃ‚ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

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 ...

Controlled Cortical Impact Model for Traumatic Brain Injury

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Neonatal Mouse Spinal Cord Compression Injury Model

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...