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干预或保护过程。 我们的全身或直接应用NTF4的方法在老化大鼠喉中产生重塑反应肌肉。这表明神经营养蛋白可能对衰老相关的肌肉功能障碍具有治疗潜力。神经营养因子的直接应用可以通过注射载体很容易地转化为人类老龄化模型。 使用注射方案时要考虑很多因素。首先，考虑到内窥镜的尖端具有尖锐的针，必须注意防止在喉前庭周围的软组织的划伤和损伤。其次，在动物呼吸周期的吸气阶段期间，针尖进入喉部的运动是准确的，准确地注射时间。第三，通过预先在计算机显示器上目视检查设备，确保针尖在尝试过程前清晰可见。第四，在热水中加热内窥镜的尖端几秒是确定内镜雾化的重要一步避免了镜头。最后，在任何范围界定程序之前，晚上将鼠标放在同一位置也是很重要的。如果没有食用食物，它们很可能在咽中仍然有食物残留，这使得注射程序实际上是不可能执行的。每天应检查大鼠的脱水迹象并进行称重，以确保没有明显的体重减轻（大于体重的10％）。 方案开发中的两个关键步骤是注射器与内窥镜的连接以及口腔窥器的使用。注射器组件的牢固锚定是必要的，以便于单手操作以进行示范性插入和注射。考虑到研究的目的，没有直接的可视化来指导注射声带，实验就不可能实现。此外，创建口腔窥器被认为是重要的，以防止舌头移动和维持会议开放整个程序。 视觉引导的注射方法进行了几轮修改，主要涉及找到将注射器和针牢固地固定在内窥镜上的最佳方式。在测试了几种不同形式的胶带粘合剂之后，发现市售的弹性运动胶带是将注射器固定在内窥镜主体上的最佳方式。 视觉引导注射方法的最大限制与仪器无关，而与专利咽和气道的需要无关。虽然在手术前24小时限制食物解决了大部分问题，但老鼠会在笼子里摄入任何东西，包括床上用品和粪便。发生这种情况时，有两种解决办法:（1）推迟注射，直到动物自然清除咽部，或（2）用镊子手动取出堵塞物。这是我们的经历e，前者是更好的选择，因为它减少了对动物咽部区域的潜在伤害的风险。 鉴于该方案的新颖性以及需要直接将化合物注入声带，所以没有其他可靠的方法存在。鉴于动物喉部系统的体积小，通过口腔和咽部区域的视觉指导注射，以确保化合物在活体动物中的正确和一致的放置。直接注射声带的唯一其他手段是尝试从外部位置通过喉部的皮肤和软骨进行。虽然这种方法在经过肉毒杆菌毒素治疗的人中成功地进行以缓解喉部肌张力障碍，但是经皮注射方法在小动物中是不可行的。 该技术是强大的，可用于不仅注射声带，而且可用于注射部位咽和口腔地区。另外，该方法可以通过去除注射器来适应于简单的视觉监测动物咽和喉部区域。 总之，这种注射方法是研究与人类衰老相关的声音功能障碍治疗相关的生物学机制的新手段。这种方法也有可能应用于影响人的声音，声音功能，沟通和吞咽的其他疾病模型。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurotrophin 4</td>
			<td>Pepro Tech</td>
			<td>450-04</td>
			<td>200ng in 50&#956;l</td>
		</tr>
		<tr>
			<td>Alzet Osmotic Pump</td>
			<td>DURECT Corporation</td>
			<td>2001D</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176; endoscope</td>
			<td>Stoltz</td>
			<td>61029D</td>
			<td></td>
		</tr>
		<tr>
			<td>50mm 30 gauge 100-&#956;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&#39;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 ）。治疗后也定性观察到较少的纤维化。去神经纤维的百分比随老化大鼠全身和直接应用NTF4而降低（ 图6 ）。 NMJ的数量也增加（ 图7 ）。这种增长的意义<html>取决于治疗或治疗途径的长短。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55837/55837fig1.jpg" /> 渗透泵的代表性图像。在用NTF4填充泵后，将流量调节剂插入主体以密封泵。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55837/55837fig2.jpg" /> 图2:注射程序平台。将大鼠放置在平台上的仰卧姿势中，并通过安装在平台顶部的引导线由前上切牙悬挂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55837/55837fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> / etc / ftp_upload / 55837 / 55837fig3.jpg"/&gt; 图3:注射器的代表性图像。用NTF4和盐水混合物填充注射器。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55837/55837fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55837/55837fig4.jpg" /> 图4:注射程序。 （ A ）注射器连接到19.9mm 30°内窥镜（如图所示）; （B）检查光线以确保该程序有光线; （ C ）实时视频确保针头被引导到正确的位置。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55837/55837fig5.jpg" /> 图5:纤维尺寸和形态的变化。代表他在30日龄26,27,28天，7天治疗（右）和未治疗（左）甲状腺肌肉的全身NTF4的酪蛋白和伊红染色切片。从控制到处理的动物的纤维尺寸有所增加。用NTF4治疗可将30 mo纤维尺寸改为6月龄较小的动物。 * p &lt;0.05与30个月（照片在40倍放大倍数捕获;比例尺=25μm，P &lt;0.001）。顶部图片中的箭头指向纤维化区域。治疗动物纤维化也有定性的降低。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55837/55837fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55837/55837fig6.jpg" /> 图6:内在变化使用NTF4治疗。代表性Nav1.5染色切片，全身NTF4 14天处理（右）和未处理（左）甲状腺肌肉30个月龄（图片以40倍放大倍数拍摄;比例尺= 25微米） 29 。 Na v 1.5标记（绿色）有年龄减少的去神经支配。左图是代表性的Na v 1.5（绿色）和鬼笔环肽，表示来自未经治疗的甲状腺样肌肉的纤维（红色）染色切片，右图为处理肌肉。绿色插入物是单独的Na v 1.5染色。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55837/55837fig7.jpg" /> 图7:NTF4治疗NMJ的变化。来自全身NTF4的NMJ的代表性荧光显微镜图像用α-银环蛇毒素（绿色）标记的不同动物的14天处理的甲状腺素肌肉切片和显示NMJ值在30个月时NTF4治疗（比例尺=25μm），（P &lt;0.001） 11的鬼笔环肽（红色）。

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

Nanyang Technological University

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

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...