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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Neurotrophin 4 | Pepro Tech | 450-04 | 200ng in 50μl |
| Alzet Osmotic Pump | DURECT Corporation | 2001D | |
| 30° endoscope | Stoltz | 61029D | |
| 50mm 30 gauge 100-μl syringe | Hamilton | 84850 and 201812 | |
| saline (sodium chloride solution) | Sigma-Aldrich | S8776 | |
| ketamine hydrochloride | Henry Schein | 56344 | |
| xylazine hydrochloride | Henry Schein | 33198 | |
| 25 G 5/8 needle | Becton-Dickinson | 305901 | |
| 1 ml syringe | Becton-Dickinson | 309659 | |
| ophthalmic ointment | Henry Schein | 8897 | |
| clippers | Oster | 44-018 | |
| ethanol | Decon | 2716 | |
| iodine (Betadine) | Purdue Pharma L.P. | 606404 | |
| heating pad | Sunbeam | 731-5 | |
| 5-0 nylon suture thread | AD Surgical | PMN-518R6 | |
| crile hemostat | Fine Science Tools | 13005-14 | |
| delicate suture tying forceps | Fine Science Tools | 11063-07 | |
| meloxicam | Henry Schein | 49756 | |
| carprofen | Merritt Veterinary Supplies | 148700 | |
| antibiotic ointment | Henry Schein | 57110 | |
| acepromizine Aceproject | Henry Schein | 3845 | |
| isoflurane Isothesia | Henry Schein | 50033 | |
| induction box (anesthetizing box) | Harvard Apparatus | 50-0116 | |
| oxygen compressed tank | Scott Gross | UN1072 | |
| plexiglas platform | Small Parts Inc (Amazon) | ||
| rubber tipped forceps | Fine science tools rubber | 11075-00 | |
| liquid rubber for forceps above | Lowe's | 42518 | |
| plastic spectula (BD syringe cut to length) | Becton-Dickinson | 309659 | |
| halogen light source rhino-laryngeal stroboscope | Kay-Pentax | RLS 9100 B | |
| video recorder | Kay-Pentax | ||
| sucrose | Sigma-Aldrich | S0389-500G | |
| phosphate buffered saline | Sigma-Aldrich | P4417-100TAB | |
| cryostat Mictotom HM525 | Thermo Scientific | HM 525 | |
| Gill 1 hematoxylin | VWR | 10143-142 | |
| Shandon eosin-Y alcoholic | Thermo Fisher Scientific | 6766007 | |
| anti-sodium channel Nav1.5 antibody produced in rabbit | Sigma-Aldrich | S0819 | |
| Texas red-X phalloidin | Sigma-Aldrich | T7471 | |
| alpha- bungarotoxin alexa fluor 488 conjugate | Thermo Fisher Scientific | B-13422 | |
| Small animal anaesthesia machine | Smiths Medical | CDS 9000 |
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Cite This Article
Andreatta, R. D., Stemple, J. C., Seward, T. S., McMullen, C. A. Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles. J. Vis. Exp. (124), e55837, doi:10.3791/55837 (2017).