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?...

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 ...

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

Research

JoVE Journal

Medicine

2.2K 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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

科研

医学

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

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

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

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

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)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

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

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

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

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

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

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  &amp;#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 ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

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

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

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

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

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

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

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

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 patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

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 ...

Methods Article

脊柱植入物感染的体内小鼠模型