This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.

注：所有动物的程序批准了美国约翰霍普金斯大学动物护理和使用委员会。麻醉机需要每年检查和校准是必要的。 1.麻醉和定位<ol><li>麻醉。 <ol><li>执行麻醉下用2-3％异氟烷所有的外科手术。确认麻醉通过脚趾捏和呼吸速率适当的水平。检查该大鼠不响应于脚趾捏退缩。 注：退缩表明麻醉是为时已晚，并且可能需要在开始之前再麻醉或更高浓度的异氟醚。的呼吸率小于1呼吸每2秒的速度太慢，指示麻醉太高，并且可能需要在异氟烷浓度的减轻。 </li><li>确保必要的动物，以避免在操作过程中的头部运动。将利多卡因对手术眼睛的下降。使用人工泪液，每10分钟，以防止干燥，而在麻醉下。给根据需要注射的0.01毫克/千克SC预可操作然后每6-8小时丁丙诺啡。 </li></ol></li><li>定位。 <ol><li>将老鼠变成一个立体框架和一个加热垫保暖。湿头皮毛皮用酒精小心地避免暴露在眼前。使用无菌的工具和无菌技术，以减少手术后感染的风险。 </li></ol></li></ol> 2.眼控<ol><li>放置4-0缝合线在横向和结膜有足够的缝合比分扳成允许牵引温柔。 </li></ol> 3.解剖<ol><li>初步解剖。 <ol><li>使一〜1英寸切口，在皮肤上覆使用大小为10解剖刀， 如图1中的轨道脊。缩回皮肤和下面的筋膜并小心剖析远离筋膜。为了防止出血过多进入手术，避免削减血管解剖时筋膜。使用PL的战略一杆进洞棉花提示，以提供止血。 </li></ol></li><li>更深层次的剖析。 注：随着对拉动结膜眼潦倒插槽温柔的牵引，眶上肌肉就会映入眼帘。为了揭露视神经，这种肌肉必须进行切割和眶脂肪去除。脂肪可以被丢弃，并应在注射后不能被替换。从这点来说，视神经筋膜应尽可能视神经本身束可见，随着包裹在硬脑膜血管（ 图1）。 <ol><li>让使用手术刀或31计斜面针头刺入做创伤小一小切口硬脑膜。 </li></ol></li></ol> 4.移取喷油器<ol><li>拉玻璃微量吸管到直径为50-100微米。以提供稳定性，装入微量上一个显微和附加到Hamilton注射器连接于输注泵。 </li><li>拉起珠（或干细胞）重组在0.5-1.0微升卷成随着甲基蓝溶液之前和之后的0.5微升体积逆行微量。 注意：一个输注率设定为每分钟0.5-2微升防止外伤视神经。 </li></ol> 5.注射<ol><li>降低微量的前端到视神经正上方的缺口硬脑膜。 注：在玻璃尖端的微小的，但轻快运动进入视神经导致损害最小。作为输液泵开始时，甲基蓝染料应突出注入视神经的面积。染料应视神经内不会泄漏出在蛛网膜下腔保持本地化。 </li><li>遵循甲基蓝的第二频带，以确定何时所述干细胞注射完成并打开输液泵关闭甲基蓝的第二剂量注入。保持微量仍然视神经内，2分钟，每1微升体积注入，以防止高在撤离的微压顶出。 注：另一种方法是将枪头连接到一个充有空气的注射器可手动操纵。无论哪种方式，避免用力过大，以减少损害视神经。我们建议注入不超过2微升体积的在每个视神经。 </li></ol> 6.跟进<ol><li>即时。 <ol><li>当注射完成后，取出微管以及用于提供止血棉的任何提示。缝合皮肤用3-0丝线和删除结膜缝合。 </li><li>保持温暖的老鼠在一个加热垫，直到他们从麻醉中出现。不要让动物无人值守，直到它已经恢复了意识足以维持胸骨斜卧。不返回已经历手术给其他动物的公司，直到完全恢复的动物。 </li><li>如果动物表现出疼痛的迹象，包括过度嗜睡，运动失调，或吃力的呼吸克，适当管理与镇痛丁丙诺啡肌肉注射（0.05毫克/千克），最多每日三次。 </li></ol></li><li>长期。 <ol><li>在接下来的24-48小时，观察大鼠进行手术的并发症，包括从伤口肿胀或分泌物或疼痛等体征如的发声，弯腰驼背的样子，不修饰，或不思饮食。考虑与兽医或伦理安乐死进行必要的磋商。 </li></ol></li></ol>

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	<li>Dahlmann-Noor, A., Vijay, S., Jayaram, H., Limb, A., Khaw, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+and+future+prospects+for+stem+cell+rescue+and+regeneration+of+the+retina+and+optic+nerve.">Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve.</a> Canadian journal of ophthalmology Journal canadien d'ophtalmologie. 45 (4), 333-341 (2010).</li><li>Quigley, H. A., Iglesia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+to+replace+the+optic+nerve.">Stem cells to replace the optic nerve.</a> Eye. 18 (11), 1085-1088 (2004).</li><li>Ghaffarieh, A., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+disease+and+axon+pathophysiology.">Optic nerve disease and axon pathophysiology.</a> International review of neurobiology. 105, 1-17 (2012).</li><li>Fukui, Y., Hayasaka, S., Bedi, K. S., Ozaki, H. S., Takeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+the+development+of+the+optic+nerve+in+rats+reared+in+the+dark+during+early+postnatal+life.">Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.</a> Journal of anatomy. 174, 37-47 (1991).</li><li>Celestre, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+approaches+to+the+cervical+spine.">Minimally invasive approaches to the cervical spine.</a> The Orthopedic clinics of North America. 43 (1), 137-147 (2012).</li><li>Hallas, B. H., Wells, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Technique+for+Multiple+Injections+into+the+Mammalian+Optic+Nerve.">A Novel Technique for Multiple Injections into the Mammalian Optic Nerve.</a> Kopf Carrier. 54, (2001).</li><li>Harvey, A. R., Hellstrom, M., Rodger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+and+transplantation+in+the+retinofugal+pathway.">Gene therapy and transplantation in the retinofugal pathway.</a> Progress in brain research. 175, 151-161 (2009).</li><li>Adachi-Usami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+neuritis--from+diagnosis+to+optic+nerve+transplantation.">Optic neuritis--from diagnosis to optic nerve transplantation.</a> Nippon Ganka Gakkai zasshi. 104 (12), 841-857 (2000).</li><li>Slater, B. J., Vilson, F. L., Guo, Y., Weinreich, D., Hwang, S., Bernstein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+inflammation+and+demyelination+in+a+rodent+model+of+nonarteritic+anterior+ischemic+optic+neuropathy.">Optic nerve inflammation and demyelination in a rodent model of nonarteritic anterior ischemic optic neuropathy.</a> Investigative ophthalmology &amp; visual science. 54 (13), 7952-7961 (2013).</li><li>Zarbin, M. A., Arlow, T., Ritch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+nanomedicine+for+vision+restoration.">Regenerative nanomedicine for vision restoration.</a> Mayo Clinic proceedings. 88 (12), 1480-1490 (2013).</li></ol>

The authors have no meaningful disclosures to report.

Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery. 
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visua...

视神经为中枢神经系统（CNS）再生的研究，包括眼科疾病如视神经炎，青光眼和创伤的理想地点。的各种干细胞注射要么证明疗效或显示出希望在替换丢失髓磷脂，增加轴突计数和/或预防变性疾病。1,2- 人类视神经含有约120万平行轴突从视网膜到一个直径约为3.0-3.5毫米的视交叉行进。3为了模拟人类疾病在实验室中，老鼠被频繁使用。成年大鼠视神经包含大约100,000轴突直径大约0.5mm范围内。4之一在中枢神经系统的主要限制再生的研究是直接去骨访问。并发症和手术风险的动物都在颅骨或脊椎被删除更高。类似的好处微创方法在脊柱，5个直属视神经注射，无需打开颅骨报价减少并发症和更快的恢复。 该技术已在以前的研究。6在这个手稿和影随行，我们展示了微创方法，以干细胞注入到老鼠视神经。

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:151px;">Catalog Number</td>
			<td style="width:335px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lewis rat</td>
			<td style="width:151px;">Charles River</td>
			<td align="right" style="width:151px;">4</td>
			<td>Any rat strain will work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anesthesia machine</td>
			<td style="width:151px;">Surgivet</td>
			<td style="width:151px;">CDS9000</td>
			<td>CDS 9000 Small Animal Anesthesia Machine - Pole Mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infusion pump</td>
			<td style="width:151px;">Stoelting</td>
			<td align="right" style="width:151px;">53129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:151px;">National Optical</td>
			<td style="width:151px;">409-411-1105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiber-optic light source</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:151px;">12-562-21</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissection and Stereotaxic Instrument</td>
			<td style="width:151px;">Stoelting</td>
			<td align="right" style="width:151px;">51400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette Puller</td>
			<td style="width:151px;">Kopf</td>
			<td align="right" style="width:151px;">750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pipettes</td>
			<td style="width:151px;">World Precision Instruments</td>
			<td style="width:151px;">18150-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable scalpel blades</td>
			<td style="width:151px;">Harvard Apparatus</td>
			<td style="width:151px;">810-15-021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Iridectomy scissors</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">Uniband LA-4XF</td>
			<td></td>
		</tr>
	</tbody>
</table>

minimally-invasive technique for injection into rat optic nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

在实验结束时，处死大鼠，并灌注4％多聚甲醛。视神经被小心解剖和安装成用于低温恒温器切片。 图2示出了在低功率的鼠全视神经其中伊文思蓝染料以可视化的部位注入的一个例子。箭头识别注射的精确位置。此夹层被注射的几分钟内完成由下神经染料的弥散受限所指示的。在其它注射剂，我们观察到对在几个小时的过程中视交叉染料的缓慢扩散。 <p class="jove_content" fo:keep-togethe...

Watch this Scientific Journal Video about Minimally-invasive Technique for Injection into Rat Optic Nerve at JoVE.com

Minimally-invasive Technique for Injection into Rat Optic Nerve

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

微创技术注入到老鼠视神经

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

minimally-invasive-technique-for-injection-into-rat-optic-nerve

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Neuroscience

9.4K 次观看.  Johns Hopkins University. Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid. 