这项工作得到了国家耳聋和其他传播障碍研究所（R21DC010806至CAM和JCS以及R01DC011285至CAM）的资助。

对于这个方案，在6和30个月龄时使用雄性Fischer 344-Brown Norway大鼠。大鼠从国家老龄鼠老鼠研究所获得。我们使用老鼠进行这项研究，因为大鼠喉的结构类似于人类的结构，功能上用于气道保护和物种特异性发声本研究是按照PHS对人类关心和使用实验动物的政策进行的， NIH"实验动物护理和使用指南"和"动物福利法"（7 USC等）;动物使用协议由肯塔基大学的机构动物护理和使用委员会（IACUC）批准。 大麻麻醉<ol><li>通过将盐酸氯胺酮（解离麻醉剂）和盐酸赛拉嗪（镇静和镇痛）混合在缓冲盐水中来制备麻醉药。最终溶液中氯胺酮和赛拉嗪的浓度为100mg / 8mg / kg体重分别为</li><li>通过使用25 G针的注射器通过腹膜内给药将麻醉剂注入大鼠。 </li><li>通过用镊子夹住脚趾或脚来确定大鼠是否充分麻醉。如果大鼠对痉挛没有反应，那么手术就可以开始了。如果大鼠对反射或肌肉收缩的脚趾反应，则等待1-2分钟，然后重复捏捏测试。如果大鼠再次反应，用新动物代替大鼠，并从步骤1.2开始重复操作。 </li><li>在大鼠不动之后，向大鼠的眼睛涂抹眼科软膏，以防止角膜变干。 </li></ol>渗透泵植入<ol><li>将大鼠腹侧置于无菌手术区域。管理美洛昔康作为美容前药。使用25 G针的注射器以1-4mg / kg体重腹膜内给药。 </li><li>使用剪刀去除一个近似值脖子后面有1"x 1"平方英尺的毛皮，肩膀之间的距离大约为1英尺，剃须尽可能靠近皮肤。 </li><li>用乙醇消毒（70％）将背部和颈部润湿。 </li><li>剃须后，用3次擦洗擦洗颈部背部碘酒精酒精。 </li><li>通过将其放置在设置在34℃的加热垫上来保持大鼠的体温。 </li><li> 用无菌制备的渗透泵用50μL的NTF4或盐水进行全身NTF4处理（ 图1 ）。 <ol><li>使用手术刀做一个大约2厘米宽的水平切口，通过皮肤，只是颅骨到肩胛骨之间的空间。用镊子用镊子提起切口的后边缘，同时插入止血钳的尖端，轻轻地推到切口的后方。 </li><li>止血垫的尖端约为2厘米c在切口处行走，打开止血钳上的手柄，将尖端扩张，在切口部位皮下形成一个空心的"口袋"。这将是泵的安置点。 </li></ol></li><li>在插入后首先定位泵输送入口端，以最小化NTF4与口腔切口部位愈合的任何相互作用。 </li><li>送出50μL的NTF4盐水7天14天。 7天组的NTF4达到6.72mg /天，总剂量为47.04mg。 14天组每天接受6.72mg /天的总剂量为94.08mg的NTF425。 </li><li>使用5-0尼龙缝合线，止血钳和镊子关闭用于泵放置的切口。 </li><li>从麻醉中恢复至少30分钟观察大鼠。完成监测的标准包括动物变得活跃，围绕笼子，饮水，开始其他正常的活动，如梳理。 </li><li>每天监视动物通过观察手术部位的愈合，正常饲料和耗水量以及尿/粪便的通过以及压力，疼痛或其他手术后并发症的任何异常行为迹象，第一周。 </li><li>如果大鼠似乎处于疼痛或痛苦状态，则每24小时给予大鼠5mg / kg皮下注射卡洛芬，持续5天以缓解疼痛。 </li><li>如果出现感染，请咨询兽医，以确保伤口愈合良好。 </li><li>根据大鼠在哪个实验组中，手术后7-10天取出5-0尼龙缝线，以防止线刺激。 </li></ol> 3.直接注射大鼠麻醉<ol><li>在手术前一天晚上从老鼠身上取食食物。这确保没有食物阻塞内窥镜或注射针。 </li><li>称重大鼠，并制备1-2mg / kg乙酰丙嗪的体重。肌内注射（IM位置是左侧甲状腺肌肉）。 </li><li>将大鼠放在感应盒中。在感应箱中诱导5％异氟烷和1 LO 2的麻醉。 </li><li>将大鼠移至2％异氟烷和600 mL O 2的鼻锥。 </li><li>通过用镊子夹住脚趾或脚来确定大鼠是否充分麻醉。如果大鼠对痉挛没有反应，则注射方案可以开始。如果大鼠对反射或肌肉收缩的脚趾反应，则等待1-2分钟，然后重复捏捏测试。如果大鼠再次反应，用新动物代替大鼠，并从步骤3.4开始重复操作。 </li></ol>直接注入和可视化<ol><li>将无菌制备的含有NTF4或盐水的50μL剂量在设定为25℃的H 2 O浴中注射30分钟。 </li><li>将大鼠放置在有机玻璃平台上的俯卧位置（ 图2 ）。野猪通过穿过平台顶部的导丝，从前额上切牙通过斜倚的姿势来摆放大鼠。 </li><li>将50毫米，30磅，100μL注射器连接到1.9毫米，30度鼻窦内窥镜（ 图3 ）。 注意:注射器组件通过将插管牢固地保持在内窥镜的外壁上的夹具连接。内窥镜允许在口腔内可视化的声带和注射器的引导。在每个动物之前调整套管尖端的位置，以确保通过内窥镜视图完全清晰地看到尖端（ 图4 ）。 </li><li>使用橡胶一对镊子延伸舌头并将其横向移动。之后，插入塑料窥器以保持口腔通畅。将5毫米塑料注射器筒中的勺子切成1.5至2厘米的长度，切割边缘去毛刺并抛光光滑。 </li><li>关掉车上的灯并将卤素光源附着到内窥镜。打开录像机以捕获该过程。 </li><li>将内窥镜的远端浸入温水中几秒钟，以便在插入大鼠口中时最大限度地减少玻璃尖端上凝结的发展。 </li><li>使用来自显示器的视觉反馈，仔细地将针引导到左声带的区域。 </li><li> 随着动物呼吸循环的吸气阶段注射溶液，以完全进入声带。在呼吸的吸气阶段，声带完全暴露。 <ol><li>一旦声带完全可见，将针插入左侧甲状腺激素，发现在声带的白色中间边缘的外侧。将针插入到位，通过注射器的凹陷输送注射器。 </li></ol></li><li>关闭内窥镜和视频播放器上的卤素光源，然后转回room灯。 </li><li>将老鼠回到家中的笼子上放在加热垫上。 </li><li>从加热垫取出前，让老鼠恢复。更换笼子里的食物和水。 </li><li>注射后监测大鼠7天，然后安乐死。取出喉部进行冷冻切片24 。 </li></ol> 5.大鼠安乐死<ol><li>用盐酸氯胺酮和盐酸赛拉嗪麻醉麻醉大鼠（100mg / 8mg / kg体重腹膜内注射）。 </li><li>通过内侧胸廓切开术安乐死。 </li></ol>

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作者没有什么可以披露的。

喉部肌肉易受老化不利影响。以前的研究已经证明了老化喉部肌肉的变化，包括纤维尺寸变化，纤维总数，再生能力，NMJ大小和数量变化，以及收缩功能和肌球蛋白异构体转变的变化4,11,27,30,31 。老化喉肌可以通过应用神经营养蛋白来改变。可以容易地测量这些变化。因此，大鼠喉部肌肉提供了一个有用的模型来研究衰老和声音疾病/失调的影响。这些肌肉的研究也可能有助于在老龄化人群中发展?...

喉部肌肉迅速，一致地收缩，容易受到衰老的不利影响。这种不断的活动被认为有助于在65岁以上的人，1,2,3,4,5,6,7人群中观察到的语音问题或吞咽困难。几种分子和病理生理机制有助于这种年龄相关的功能障碍。这些机制可能包括喉粘膜的重塑，肌肉纤维的萎缩或丧失，缺乏肌纤维再生或萎缩，导致声带弯曲和声门关闭不能8,9,10,11。目前还没有经过证实的药物治疗彻底预防或恢复这些年龄相关的这些肌肉变化。 调节神经肌肉传播的有效性可以极大地影响神经运动的表现。神经营养蛋白家族包括神经生长因子（NGF），脑源性神经生长因子（BDNF），神经营养因子3（NTF3）和NTF4 12,13 。神经营养因子已经显示出调节突触功效1,4 。肝细胞生长因子，转化生长因子β和成纤维细胞生长因子最近已被用于人类治疗声带皱纹瘢痕15-17 。 NTF4也调节NMJ的有效性;缺乏NTF4的小鼠显示了拆卸的NMJ 11,18,19。这些研究导致治疗有希望的效果t老化喉部肌肉疾病和去神经与生长因子。 对人体组织的直接注射治疗不是人类前所未有的。例如，局部注射肉毒杆菌毒素目前用作影响喉部肌肉的神经运动障碍的有效治疗方法，如痉挛性发音障碍和双侧复发性喉神经麻痹20,21 。透明质酸水凝胶是另一种可注射的，用于治疗声带褶皱和声门功能不全22,23 。注射喉成形术可用于治疗各种通气障碍24 。这些直接注射方法有望改善老年人群的声音功能和吞咽。

<table><tbody><tr><td>Neurotrophin 4</td><td>Pepro Tech</td><td>450-04</td><td>200 ng in 50 &amp;mu;L</td></tr><tr><td>Alzet Osmotic Pump</td><td>DURECT Corporation</td><td>2001D</td><td></td></tr><tr><td>30 &amp;deg; endoscope</td><td>Stoltz</td><td>61029D</td><td></td></tr><tr><td>50 mm 30 gauge 100-&amp;mu;L syringe</td><td>Hamilton</td><td>84850 and 201812</td><td></td></tr><tr><td>saline (sodium chloride solution)</td><td>Sigma-Aldrich</td><td>S8776</td><td></td></tr><tr><td>ketamine hydrochloride</td><td>Henry Schein</td><td>56344</td><td></td></tr><tr><td>xylazine hydrochloride</td><td>Henry Schein</td><td>33198</td><td></td></tr><tr><td>25 G 5/8 needle</td><td>Becton-Dickinson</td><td>305901</td><td></td></tr><tr><td>1 mL syringe</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>ophthalmic ointment</td><td>Henry Schein</td><td>8897</td><td></td></tr><tr><td>clippers</td><td>Oster</td><td>44-018</td><td></td></tr><tr><td>ethanol</td><td>Decon</td><td>2716</td><td></td></tr><tr><td>iodine (Betadine)</td><td>Purdue Pharma L.P.</td><td>606404</td><td></td></tr><tr><td>heating pad</td><td>Sunbeam</td><td>731-5</td><td></td></tr><tr><td>5-0 nylon suture thread</td><td>AD Surgical</td><td>PMN-518R6</td><td></td></tr><tr><td>crile hemostat</td><td>Fine Science Tools</td><td>13005-14</td><td></td></tr><tr><td>delicate suture tying forceps</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>meloxicam</td><td>Henry Schein</td><td>49756</td><td></td></tr><tr><td>carprofen</td><td>Merritt Veterinary Supplies</td><td>148700</td><td></td></tr><tr><td>antibiotic ointment</td><td>Henry Schein</td><td>57110</td><td></td></tr><tr><td>acepromizine Aceproject</td><td>Henry Schein</td><td>3845</td><td></td></tr><tr><td>isoflurane Isothesia</td><td>Henry Schein</td><td>50033</td><td></td></tr><tr><td>induction box (anesthetizing box)</td><td>Harvard Apparatus</td><td>50-0116</td><td></td></tr><tr><td>oxygen compressed tank</td><td>Scott Gross</td><td>UN1072</td><td></td></tr><tr><td>plexiglas platform</td><td>Small Parts Inc (Amazon)</td><td></td><td></td></tr><tr><td>rubber tipped forceps</td><td>Fine science tools rubber</td><td>11075-00</td><td></td></tr><tr><td>liquid rubber for forceps above</td><td>Lowe&#039;s</td><td>42518</td><td></td></tr><tr><td>plastic spectula (BD syringe cut to length)</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>halogen light source rhino-laryngeal stroboscope</td><td>Kay-Pentax</td><td>RLS 9100 B</td><td></td></tr><tr><td>video recorder</td><td>Kay-Pentax</td><td></td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389-500G</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P4417-100TAB</td><td></td></tr><tr><td>cryostat Mictotom HM525</td><td>Thermo Scientific</td><td>HM 525</td><td></td></tr><tr><td>Gill 1 hematoxylin</td><td>VWR</td><td>10143-142</td><td></td></tr><tr><td>Shandon eosin-Y alcoholic</td><td>Thermo Fisher Scientific</td><td>6766007</td><td></td></tr><tr><td>anti-sodium channel Nav1.5 antibody produced in rabbit</td><td>Sigma-Aldrich</td><td>S0819</td><td></td></tr><tr><td>Texas red-X phalloidin</td><td>Sigma-Aldrich</td><td>T7471</td><td></td></tr><tr><td>alpha- bungarotoxin alexa fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>B-13422</td><td></td></tr><tr><td>Small animal anaesthesia machine</td><td>Smiths Medical</td><td>CDS 9000</td><td></td></tr></tbody></table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

老年人的喉功能障碍是残疾的主要原因，从语音障碍到吞咽困难和气道保护性反射丧失。存在少量针对年龄相关性喉部肌肉功能障碍的治疗方法。神经营养因子参与神经肌肉接头（NMJ）的肌肉神经支配和分化。据认为神经营养蛋白通过增加神经递质释放来增强神经肌肉传递。神经肌肉接头（NMJ）在衰老的大鼠喉部肌肉中变得越来越小，具有功能去神经支配的证据。我们探讨了NTF4对未来临床应用的作用，作为改善老年人类喉部肌肉功能的治疗剂。在这里，我们提供了系统应用和直接注射NTF4的详细方案，以调查老化大鼠喉肌肉响应NTF4应用重建的能力。在这种方法中，大鼠通过 o系统地接受NTF4雾状泵或直接注射通过声带。然后解剖喉部肌肉，用于组织学检查形态学和年龄相关的去神经支配。

大鼠在渗透泵输注2周后直接注射NTF4后1周安乐死。收获喉，置于冷冻保护剂（30％蔗糖和70％磷酸盐缓冲盐水）中，然后用低温恒温器以10μm的宽度连续切片。老化的喉部肌肉受NTF4 25的施用影响。除了老年和老鼠之外，我们比较了甲状腺素肌肉的注射和非注射侧。通常，我们看到纤维尺寸随年龄的变化，其根据NTF4的施用途径而变化（ 图5</s...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

在这里，我们提出一个协议来描述神经营养因子4（NTF4）的系统和直接用于改造大鼠老化的喉部肌肉。

使用渗透泵或直接肌肉注射的皮下神经营养因子4输液增强老化大鼠喉肌肉

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.3K 次观看.  University of Kentucky. 在这里，我们提出一个协议来描述神经营养因子4（NTF4）的系统和直接用于改造大鼠老化的喉部肌肉。 

视频: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

椎管内针对肌萎缩性脊髓侧索硬化症和外伤性脊髓损伤颈腹角细胞移植

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

科研

医学

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

神经科学

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

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

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. ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

神经刺激器引导注射自体干细胞近马左喉返神经

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

慢性活体电成像的植入系统

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

用于神经肌肉分析的大鼠声带的制备

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

<meta charset="utf8"></head><body>小鼠耳长提肌电穿孔和神经损伤

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

<meta charset="utf8"></head><body>作者聚焦：肌肉蛋白在神经损伤后神经肌肉接头的再生作用

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

颅提肌耳长骨骼肌的体内电穿孔和短期神经再支配相结合

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...