这项工作得到CIHR神经肌肉研究合作伙伴（JNM - 90959;到JAEB）的补助。

使用这个模型之前，研究者必须接受手术动物从他们的机构的理事机构使用协议的批准。该模型是研究伦理委员会，汉密尔顿健康科学公司，麦克马斯特大学（AUP＃10-04-24）批准，并严格按照加拿大动物保护协会的建议进行。 1。鼠标准备<ol><li>称量鼠标。异氟醚或5％，2％氟烷麻醉。应确保有足够的麻醉清除保护外科医生使用的电路。 2-3分钟后，动物的呼吸会减缓。确保眨眼反射消失，并在爪子捏叉空间，以确认手术麻醉（ 即通过鼠标无响应）。眼科润滑剂应用的眼球，在手术过程中防止角膜干燥。 </li><li>剃须大腿外侧和臀部坐骨神经缺口膝盖和消毒proviodine。剃须将免费保持切口的头发的部位，以确保有足够的可视化手术野，以尽量减少干扰与神经剥离和横断。可以被识别，这是优越，后方股骨，坐骨切迹通过触诊。 </li></ol> 2。手术过程<ol><li>减少吸入异氟醚2％（1％氟烷），将鼠标放在其侧面（侧用于手术朝上），经营或解剖显微镜下。另外可以进行手术治疗与手术放大镜为成人（20-25克）鼠标自3.5X放大倍率是令人满意的。 </li><li>戴上无菌手套。通过触诊识别坐骨切迹。使用手术刀，切割皮肤从坐骨切迹大腿外侧膝（约1厘米）。 </li><li>轻轻涂抹皮肤。识别股二头肌，这是平坦的表面肌肉大腿外侧紧接下方的皮肤。用细剪刀，拆股二头肌沿着肌纤维，并举行公开的弹簧拉钩暴露坐骨神经及其分支。 </li><li>确定坐骨神经紧接深至股二头肌。都可以识别通过其特性有光泽的白色，直径约0.8毫米。它运行从坐骨切迹到膝盖，分支到胫骨，腓骨和腓肠神经的胭窝上面的水平。 </li><li>轻轻分开胫，腓神经和腓肠神经分支与超细钳和弹簧microdissecting的剪刀。胫神经是最大的分支，通常是中央的。重要的是不要的神经，而分离的树枝粉碎。控股超细镊子外adventicial层的神经，使神经松弛（不教），将避免神经损伤和牵拉损伤。 </li><li>完整和持久denervati的，切开胫骨神经尽量远侧microdissecting剪刀，小心地避免腘血管。另外，对于暂时​​神经预期在2-4周内完整的神经再支配，可以简单地被粉碎的胫骨神经与超细钳为15秒，而不是横断。 （周围神经损伤后再生将reinnervate目标肌肉。） </li><li>是必需的，如果完全去神经的横断胫神经缝合结束股二头肌的前表面用5-0薇乔以10-0尼龙和重近似股二头肌，以防止异常神经支配腓肠肌和比目鱼肌。 </li><li>关闭与正在运行的5-0薇乔缝合皮肤。 </li></ol> 3。术后护理<ol><li>关闭吸入麻醉剂，但维持氧气流量。管理buprinorphine（或代替）镇痛皮下注射。 </li><li>没有鼠标转移到一个干净的笼从麻醉中醒来时，床上用品。请毯上一个笼子内，直接观察下，直到走动。 </li><li>软底笼（线）的转让及房屋充足的软床上用品。 </li><li>在每天检查执行肢体状况的发展褥疮的鞋跟溃疡或咀嚼的证据的外科伤以及脚。如proviodine外用抗生素或防腐剂，可管理的小问题。规定动物安乐死的终点指标是减肥，恶劣的自我护理的证据（竖起毛皮），弯腰驼背的姿势。此外，主要伤口的干扰或溃疡不愈合在1-2周内局部抗生素或出现有疼痛的动物应该被牺牲掉。 </li></ol> 4。失神经支配腓肠肌和比目鱼肌收获<ol><li>在手术后所需的时间点，权衡与CO 2过量的鼠标和牺牲。 </li><li>剃须双方的内侧方面手术和对侧控制的腿和用酒精清洁。将鼠标操作或解剖显微镜下，或者使用手术观赏放大镜放大。 </li><li>手术肢体的小腿内侧皮肤切开，用解剖刀从脚踝到膝关节和踝关节周围的圆周上。轻轻拉扯皮肤，肌肉和大腿近端向钳断。这暴露了所有的腿部肌肉。确定，这是从膝盖后方的腿的脚踝上，紧靠下面的皮肤的小腿肌肉的腓肠肌。识别远端插入股二头肌，腓肠肌近端上的内侧面的膝盖。股二头肌出现在其远端插入薄薄膜和覆盖在腓肠肌的最基端部。使用剪刀钝端剥离，轻轻地从气体中分离的远端插入股二头肌肌肉trocnemius。 </li><li>在其远端插入到跟骨，腓肠肌逐渐变细到跟腱。确定白色和强壮有力的跟腱，这似乎。保持用钳子跟腱，照顾不持有或粉碎腓肠肌，把跟腱跟骨插入用剪刀。 </li><li>仍持有肌腱，轻轻抬起腓肠肌（淡红色）关闭深比目鱼（更深的红色），从远端插入朝着它的起源在膝盖（比目鱼可以收获另发）。 </li><li>解剖腓肠肌关闭腿，除以用剪刀从内侧和外侧的股骨chondyles的起源腓肠肌。很温柔的对肌肉的牵引促进这一进程。小心不要压碎肌肉。 </li><li>比目鱼现在清晰可见，立刻根据网站的腓肠肌。电梯比目鱼其插入跟腱或其小腿后部igin。如果不经意间提出的腓肠肌，比目鱼轻轻分开，从收获的标本。 （淡红色）腓肠肌和比目鱼（暗红色）在收获的标本由于其颜色差异仍然很容易识别。 </li><li>精密的规模分别称量肌肉。 </li><li>垂直拆分肌肉，半金鱼草冷冻在液氮中（随后的蛋白质和/或RNA的提取），组织学（ 即形态学评估，免疫组化）的一半固定在10％福尔马林或异戊烷中冷冻固定用液氮冷却，然后随意。 </li><li>重复上述步骤4.3上的控制，取消操作，一边收获控制腓肠肌和比目鱼肌。 </li></ol>

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没有利益冲突的声明。

去神经引起的骨骼肌萎缩胫神经横断模型大鼠模型是一种常用的和很好的验证。我们已经适应了这个模型在小鼠中的使用，这使得研究者利用遗传工程小鼠的存在，并在体内研究的过程中，肌肉萎缩，肌肉质量7,8的调节至关重要的蛋白质在没有。腓肠肌和比目鱼肌，神经在这个模型中，能够容易且迅速，解剖以最小的处理能力，从而提供优良的品质mRNA和蛋白用于后续的分子分析。?...

骨骼肌失神经支配，由于外伤性周围神经损伤，疾病或药物干预，肌肉自愿收缩功能在眼前亏的结果。伴随肌肉开始萎缩，这种萎缩是可逆的，如果及时，优质的神经支配发生1,2。在没有神经支配，肌纤维萎缩的进展，在肌肉与发生不可逆的生物变化肌肉纤维化及肌纤维死亡。在这里，我们展示了胫神经横断模型，模型引起的失神经支配骨骼肌萎缩和纤维化，在小鼠体内。这种模式使科学家研究背后体内肌肉萎缩，腓肠肌和比目鱼肌的生理，细胞和分子生物学机制。虽然历史上主要用于大鼠，更近的应用，让这个模型，基因敲除和转基因小鼠系专门调查，以评估它们的作用特定的蛋白质（S）的感应，开发和维护，或者决议，肌肉萎缩及纤维化体内的兴趣。 胫神经是一个混合的运动感觉的啮齿类动物的后肢，外周神经和是三个终端坐骨神经分支之一。胫神经横断denervates腓肠肌，比目鱼肌和跖肌（三个小深屈肌的脚，包括胫骨后肌，趾长屈肌和屈hallicus的长肌），是一个标准化和验证模型大鼠3,4 。序列的时间点，可以很容易地解剖腓肠肌和比目鱼肌交的胫骨神经切断术，评估肌肉组织学和肌纤维形态测量或闪存冻结肌肉RNA和蛋白质的，目的是研究提取固定和处理，例如，调节肌肉萎缩的细胞信号网络。遗传算法strocnemius肌肉的混合纤维型肌（I型和II型，虽然主要是II型）和比目鱼肌I型纤维组成的相当大的比例，从而提供了快速和慢速肌5,6评估。胫神经横断模型适用于研究失神经性肌肉萎缩的过程中，短期（日）7和长期（周月）4,8。 在坐骨神经横断模型（失神经性肌肉萎缩的第二个模型常用于啮齿动物），胫骨神经切断术诱导少在动物发病率，使其成为一个更具吸引力的模型。坐骨神经横断denervates腿部（膝盖以下）和脚的肌肉，损害动物的走动能力2，而胫神经横断离开坐骨神经腓神经和腓肠神经分支不变，从而保持他们的目标肌肉和感官领土的。鼠标是无法跖屈或脚倒置，但能够轻易走动，体重承担同样的两后肢，从而大大减少发病率的模型。步态分析研究，评估步行模式已在胫骨和坐骨神经损伤的大鼠进行胫骨受伤9,10证明足迹和负重保存较好。此外，在胫骨神经横断模型，腓总神经可以在稍后的时间点被动员，迟发神经再生和转移作为源的研究设计，如果需要3。相比之下，延迟神经再生坐骨神经横断模型需要使用的神经移植到坐骨神经赤字，模型的技术难度非常显着增加，并限制其使用，熟练的外科医生。 虽然胫神经横断模型Requires熟悉无菌手术技术在动物手术操作，无论是胫神经和支配小腿肌肉很容易访问和操纵识别，从而使个人谁是不是外科医生，或具有丰富的经验与动物外科手术，可以很容易地掌握这个模型。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>10-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>2850G</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Vicryl suture</td>
			<td>Ethicon</td>
			<td>J553G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Spring microdissecting scissors</td>
			<td>Fine Surgical Tools</td>
			<td>15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Fine Surgical Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Non locking micro needle holder (driver)</td>
			<td>Fine Surgical Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring retractor</td>
			<td>Fine Surgical Tools</td>
			<td>17000-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice

胫神经横断模型在啮齿类动物中去神经引起的骨骼肌萎缩是一种耐受性良好，验证和可重复的模型。虽然最初的开发中广泛使用，由于其较大规模的大鼠，小鼠的胫神经，留下完好的坐骨神经腓神经和腓肠神经分支，从而保持足够大，它可以很容易被操纵，无论是挤压或横断他们的目标肌肉。因此，这种模式提供的优势诱导坐骨神经横断模型少的发病率和障碍比下地，还允许调查研究生理，细胞和分子生物学机制的调节肌肉萎缩的过程中，在遗传工程小鼠。胫神经建筑材料的腓肠肌，比目鱼肌和跖肌，所以它的横断允许失神经骨骼肌快肌II型纤维和/或慢肌型组成的研究，我纤维。在这里，我们展示了胫神经横断模型在C57Black6鼠标。我们评估腓肠肌的萎缩，作为一个有代表性的肌肉，在1，2，和4周后去神经通过测量肌肉重量和纤维类型特定的横截面面积，对石蜡包埋的组织切片进行免疫染色快肌的肌球蛋白。

<html>胫神经的横断denervates的小腿腓肠肌，比目鱼肌和跖肌。在这里，我们评估腓肠肌萎缩的发展，作为一个代表性的肌肉。从2-3个月大的程琳，程雪，闵连秋，中国保健CHINESE HEALTH CARE 2007年第12期C57black 6小鼠（杰克逊实验室），1，2，或4周神经腓肠肌收获。肌肉的重量逐渐减小（ 图1），II型快肌纤维（ 图2），随着时间的推移的横截面的面积。腓肠肌是混纤型肌（I型和I...

Watch this Scientific Journal Video about Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice at JoVE.com

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

胫神经横断模型骨骼肌萎缩是一种耐受性良好，验证和可重复的模型。中描述和演示C57Black6小鼠模型手术协议。

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

tibial-nerve-transection-standardized-model-for-denervation-induced

Research

JoVE Journal

Medicine

24.2K 次观看.  St Michaels Hospital. 胫神经横断模型骨骼肌萎缩是一种耐受性良好，验证和可重复的模型。中描述和演示C57Black6小鼠模型手术协议。 

视频: Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

科研

医学

Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P&#60;0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split&#160;hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split&#160;hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.

Dissociated atrophy of intrinsic hand muscles, termed the split hand, appears to be a specific feature of amyotrophic lateral sclerosis (ALS).&#160;Consequently, a novel neurodiagnostic test, termed the split hand&#160;index, was developed to quantify the clinical phenomenon of the split hand, which differentiated ALS from mimic disorders.

在肌萎缩侧索硬化症的诊断游离本质手部肌肉萎缩实用工具

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

神经病理性疼痛小鼠坐骨神经掴型号

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

面神经干切断小鼠：模型，研究运动神经元损伤反应

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

小鼠骨骼肌形态及纤维型成分的半自动化分析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...