该出版物中报告的研究得到国家牙科和颅面研究研究所 (NIDCR) 和国立卫生研究院全国癌症研究所 (R56 DE025098、UG3 DE027695 和 F30 CA206296) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这项工作也得到了 NSF DMR 1206219 和 IADR 口腔护理奖 (2016) 创新的支持。
我们要感谢詹 Gavrity 在 IVIS 实验中的帮助。我们要感谢凯伦. 宾利在执行 EM 方面的投入和协助。我们要感谢裴翁对 IHC 的协助。我们要感谢马修·格尔斯在图准备方面的协助。我们要感谢伊莲 Smolock 博士和艾米莉. 吴对这篇手稿的批判性阅读。

下文概述的所有体内程序均由纽约罗切斯特大学动物资源大学委员会批准。
1. 准备
<ol><li>使用32G 颅内导管与线插入, 削减3厘米的油管形成一个斜角结束, 大约45°到长轴。确认导线的长度至少比油管长1厘米。</li>
	<li>将50µL PSMA 纳米粒子溶液 (图 1) 或其他注射材料加载到哈密尔顿注射器中。为减少注射过程中气压伤的几率, 请将导管管、鞘移除、注射器和排出死容积。</li>
	<li>检查注射液, 以确保纳米微粒完全溶解, 以防止导管梗阻后的管理。</li>
	<li>用0.1 毫克/毫升的方法制备阿托品溶液。 注: 由于阿托品是轻敏感和退化随着时间的推移, 这个解决方案应在注射日, 并保护从光, 直到管理。</li>
</ol>2. 进入和可视化导管入口点
<ol><li>用分析天平称 C57/BL6 小鼠。</li>
	<li>使用0.5 毫升注射器与 29G x ½ "针, 麻醉小鼠与腹腔注射无菌盐水溶液100毫克/千克氯胺酮和10毫克/千克甲苯噻嗪。当鼠标不再响应刺激时, 继续执行以下步骤, 通常在注入后5到10分钟内发生。 注: 此过程也可在异氟醚下执行, 但需要一个自定义的鼻锥, 允许进入口腔。</li>
	<li>为了防止在过程中的干燥, 应用润滑剂的眼睛, 并把鼠标放在一个容易的位置在一个自定义阶段。 注意: 为了保持口腔内程序的适当条件, 应在每次使用前对工具进行消毒或消毒。</li>
	<li>通过将上颌门牙固定在金属束上, 打开口腔, 并使用弹性带在下颌门牙后面施加下行张力 (图 2A)。</li>
	<li>在解剖显微镜下对齐鼠标, 使下颚的底座可视化。</li>
	<li>要扩大口腔, 使用一个定制的, 弯曲的钢牵引器, 以双边方式对颊粘膜施加张力。</li>
	<li>为了可视化颌下腺乳突, 用钝钳抓住并轻轻地从口中的舌上抬起舌头。 注: 乳突将出现在舌下的两个苍白突起 (图 2B)。</li>
	<li>为了缓解口腔内的可视化和进一步的操作, 在舌头和颊粘膜之间放置棉花。</li>
</ol>3. 导管插管和线路放置
<ol><li>采用细、弯钳, 用金属丝插入导管。为在插管过程中进行最佳手动控制, 请将油管与镊子的曲率对齐 (图 3A)。</li>
	<li>使用解剖显微镜, 将钳和导线移动到视野中。 注: 导线应从油管上凸出。</li>
	<li>轻轻地将压力压入一个下颌下腺的底部, 使用金属丝插入, 产生一个小的, 表面的, 粘膜穿刺 (0.076 毫米直径), 将方便以后进入导管管 (0.25 毫米直径)。如果遇到阻力, 用锋利的解剖剪刀在油管和导线的插入上切开新的斜面尖。</li>
	<li>进入后, 取出鞘, 并使用解剖显微镜, 确认穿刺部位是否存在唾液。避免对可能导致出血或损害导管完整性的鞘进行强力或突然的运动 (撤回或插入)。</li>
	<li>收回油管内的鞘 (图 3B)。</li>
	<li>为确保注射油管能适应沃顿商学院的管道开放, 插入含有鞘的油管作为先前穿刺的刚性指南 (图 2C)。 注: 如果不快速执行, 局部肿胀可能会阻止重新插入。</li>
	<li>为防止长期导管梗阻的背部压力, 请取出油管。检查, 以验证在显微镜下可见的开口, 可以看到在颌下腺的乳突。如果出现可见出血, 请将鞘和重试从3.2 步移到对立的颌下腺乳突上。</li>
	<li>不移动鼠标, 管理腹腔注射1毫克/千克阿托品溶液, 减少流涎过程中。等待 5-10 分钟。</li>
	<li>掌握注射器导管的末端, 并使用解剖显微镜 (图 3C) 插入孔内。如果遇到阻力, 切一个新的斜面端到油管和重试。</li>
	<li>一旦导管在颌下腺内, 慢慢地向前推进 3-5 毫米进入导管。从镊子上松开油管。</li>
	<li>为了改善导管与颌下腺之间的密封, 用纱布轻轻涂抹1分钟, 使界面干燥。</li>
	<li>检查以确认油管的位置在干燥过程中没有转移。</li>
</ol>4. 注塑
<ol><li>注射材料的速率为10µL/分. 检查以确认鼠标保持镇静, 并没有显示遇险迹象 (图 2D)。 注:15-50 µL 注射液耐受性好。注入较大的体积会导致气压伤。</li>
	<li>注射后, 保持注射器压力为5分钟, 以改善在沃顿商学院的管道和 SMG 的材料保留 (图 4)。定期检查下颌下腺, 以确保导管不出口导管口。</li>
	<li>使用细钳, 抓住并轻轻地从颌下腺的乳突中取出油管。 注意: 观察一些液体从乳突中出口是正常的。</li>
	<li>在将鼠标从舞台上移开之前, 从口腔中取出牵引器和棉。 注意: 动物不应被留在无人看管, 直到它恢复足够的意识, 以维持胸骨卧床。此外, 确保鼠标没有与其他老鼠住在一起, 直到完全恢复。</li>
</ol>5. 核查和分析
注意:体内成像系统 (IVIS) 可用于评估注射后荧光标记的纳米粒子的保留 (如在图 5中注射后显示1小时和24小时)。
<ol><li>为了更好地在 SMG 内通过皮肤可视化荧光信号, 可以通过剃须或使用化学脱毛去除 SMGs 上的腹毛。 注: 在安乐死后, SMG 组织也可以收获, 固定 (隔夜在4% 多聚甲醛), 并染色使用免疫组化, 以确认荧光的持久性标记 NP 一天后注射(图 6)。</li>
</ol>

<ol>
	<li>Miranda-Rius, J., Brunet-Llobet, L., Lahor-Soler, E., Farre, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+Secretory+Disorders,+Inducing+Drugs,+and+Clinical+Management.">Salivary Secretory Disorders, Inducing Drugs, and Clinical Management.</a> International Journal Of Medical Sciences. 12 (10), 811-824 (2015).</li><li>Acauan, M. D., Figueiredo, M. A. Z., Cherubini, K., Gomes, A. P. N., Salum, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy-induced+salivary+dysfunction:+Structural+changes,+pathogenetic+mechanisms+and+therapies.">Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies.</a> Archives of Oral Biology. 60 (12), 1802-1810 (2015).</li><li>Dirix, P., Nuyts, S., Vander Poorten, V., Delaere, P., Van den Bogaert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+xerostomia+after+radiotherapy+on+quality+of+life.">The influence of xerostomia after radiotherapy on quality of life.</a> Supportive Care in Cancer. 16 (2), 171-179 (2008).</li><li>Vissink, A., 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=Clinical+management+of+salivary+gland+hypofunction+and+xerostomia+in+head-and-neck+cancer+patients:+successes+and+barriers.">Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers.</a> International Journal of Radiation Oncology Biology Physics. 78 (4), 983-991 (2010).</li><li>Delporte, 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=Increased+fluid+secretion+after+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+to+irradiated+rat+salivary+glands.">Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands.</a> Proceedings of the National Academy of Sciences. 94 (7), 3268-3273 (1997).</li><li>Samuni, Y., Baum, B. 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+delivery+in+salivary+glands:+from+the+bench+to+the+clinic.">Gene delivery in salivary glands: from the bench to the clinic.</a> Biochimica et Biophysica Acta. 1812 (11), 1515-1521 (2011).</li><li>Beahm, D. D., 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=Surgical+approaches+to+the+submandibular+gland:+A+review+of+literature.">Surgical approaches to the submandibular gland: A review of literature.</a> International Journal of Surgery. 7 (6), 503-509 (2009).</li><li>Zheng, C., Shinomiya, T., Goldsmith, C. M., Di Pasquale, G., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+and+reproducible+in+vivo+gene+transfer+to+mouse+parotid+glands.">Convenient and reproducible in vivo gene transfer to mouse parotid glands.</a> Oral diseases. 17 (1), 77-82 (2011).</li><li>Zheng, 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=Prevention+of+Radiation-Induced+Salivary+Hypofunction+Following+hKGF+Gene+Delivery+to+Murine+Submandibular+Glands.">Prevention of Radiation-Induced Salivary Hypofunction Following hKGF Gene Delivery to Murine Submandibular Glands.</a> Clinical Cancer Research. 17 (9), 2842-2851 (2011).</li><li>Okazaki, Y., 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=Acceleration+of+rat+salivary+gland+tissue+repair+by+basic+fibroblast+growth+factor.">Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor.</a> Archives of Oral Biology. 45 (10), 911-919 (2000).</li><li>Arany, S., Benoit, D. S., Dewhurst, S., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+gene+silencing+confers+radioprotection+to+salivary+glands+in+vivo.">Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo.</a> Molecular Therapy. 21 (6), 1182-1194 (2013).</li><li>Cotrim, A. P., Sowers, A., Mitchell, J. B., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+irradiation-induced+salivary+hypofunction+by+microvessel+protection+in+mouse+salivary+glands.">Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.</a> Molecular Therapy. 15 (12), 2101-2106 (2007).</li><li>Redman, R. S., Ball, W. D., Mezey, E., Key, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+donor+salivary+gland+cells+are+widely+distributed+in+the+recipient+gland+when+infused+up+the+ductal+tree.">Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.</a> Biotechnic &amp; Histochemistry. 84 (6), 253-260 (2009).</li><li>Grundmann, O., Fillinger, J. L., Victory, K. R., Burd, R., Limesand, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+radiation+therapy-induced+salivary+gland+dysfunction+in+mice+by+post+therapy+IGF-1+administration.">Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration.</a> BMC Cancer. 10, 417-417 (2010).</li><li>Limesand, K. H., 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=Insulin-Like+Growth+Factor-1+Preserves+Salivary+Gland+Function+After+Fractionated+Radiation.">Insulin-Like Growth Factor-1 Preserves Salivary Gland Function After Fractionated Radiation.</a> International Journal of Radiation Oncology Biology Physics. 78 (2), 579-586 (2010).</li><li>Marmary, Y., 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=Radiation-induced+loss+of+salivary+gland+function+is+driven+by+cellular+senescence+and+prevented+by+IL-6+modulation.">Radiation-induced loss of salivary gland function is driven by cellular senescence and prevented by IL-6 modulation.</a> Cancer Research. , (2016).</li><li>Baum, B. J., 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=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (47), 19403-19407 (2012).</li><li>Arany, S., 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=Pro-apoptotic+gene+knockdown+mediated+by+nanocomplexed+siRNA+reduces+radiation+damage+in+primary+salivary+gland+cultures.">Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.</a> Journal of Cellular Biochemistry. 113 (6), 1955-1965 (2012).</li><li>Benoit, D. S. W., Henry, S. M., Shubin, A. D., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-responsive+polymeric+siRNA+carriers+sensitize+multidrug+resistant+ovarian+cancer+cells+to+doxorubicin+via+knockdown+of+polo-like+kinase+1.">pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.</a> Molecular pharmaceutics. 7 (2), 442-455 (2010).</li><li>Malcolm, D. W., Varghese, J. J., Sorrells, J. E., Ovitt, C. E., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Biological+Fluids+on+Colloidal+Stability+and+siRNA+Delivery+of+a+pH-Responsive+Micellar+Nanoparticle+Delivery+System.">The Effects of Biological Fluids on Colloidal Stability and siRNA Delivery of a pH-Responsive Micellar Nanoparticle Delivery System.</a> ACS Nano. , (2017).</li><li>Baranello, M. P., Bauer, L., Benoit, 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=Poly(styrene-alt-maleic+anhydride)-based+diblock+copolymer+micelles+exhibit+versatile+hydrophobic+drug+loading,+drug-dependent+release,+and+internalization+by+multidrug+resistant+ovarian+cancer+cells.">Poly(styrene-alt-maleic anhydride)-based diblock copolymer micelles exhibit versatile hydrophobic drug loading, drug-dependent release, and internalization by multidrug resistant ovarian cancer cells.</a> Biomacromolecules. 15 (7), 2629-2641 (2014).</li><li>Wang, Y., 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=Fracture-Targeted+Delivery+of+&#946;-Catenin+Agonists+via+Peptide-Functionalized+Nanoparticles+Augments+Fracture+Healing.">Fracture-Targeted Delivery of &#946;-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing.</a> ACS Nano. 11 (9), 9445-9458 (2017).</li><li>Baranello, M. P., Bauer, L., Jordan, C. T., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micelle+Delivery+of+Parthenolide+to+Acute+Myeloid+Leukemia+Cells.">Micelle Delivery of Parthenolide to Acute Myeloid Leukemia Cells.</a> Cellular and Molecular Bioengineering. 8 (3), 455-470 (2015).</li><li>Kuriki, Y., 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=Cannulation+of+the+Mouse+Submandibular+Salivary+Gland+via+the+Wharton's+Duct.">Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct.</a> Journal of Visualized Experiments. (51), e3074 (2011).</li><li>Nair, R. P., Zheng, C., Sunavala-Dossabhoy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroductal+Submandibular+Gland+Instillation+and+Localized+Fractionated+Irradiation+in+a+Rat+Model+of+Salivary+Hypofunction.">Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction.</a> Journal of Visualized Experiments. (110), (2016).</li><li>Wang, Y., Malcolm, D. W., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+and+sustained+delivery+of+siRNA/NPs+from+hydrogels+expedites+bone+fracture+healing.">Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone fracture healing.</a> Biomaterials. 139 (Supplement C), 127-138 (2017).</li><li>Hoffman, M. D., Van Hove, A. H., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradable+hydrogels+for+spatiotemporal+control+of+mesenchymal+stem+cells+localized+at+decellularized+bone+allografts.">Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts.</a> Acta Biomaterialia. 10 (8), 3431-3441 (2014).</li><li>Nguyen, C. Q., Yin, H., Lee, B. H., Chiorini, J. A., Peck, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL17:+potential+therapeutic+target+in+Sjogren's+syndrome+using+adenovirus-mediated+gene+transfer.">IL17: potential therapeutic target in Sjogren's syndrome using adenovirus-mediated gene transfer.</a> Laboratory Investigation. 91 (1), 54-62 (2011).</li></ol>

作者没有什么可透露的。

Retroductal 注射液对涎腺的局部药物传递至关重要。该技术在筛选治疗剂的条件, 包括干燥综合征和 RIH9,10,28的应用。通过 retroductal 注射液直接向 SMG 提供药物, 在降低靶向效应 (包括免疫激活11) 的潜在效果方面比系统性管理具有重要优势。在不积累周围组织的情况下, 最大限度地提高当地药物的输送能力, 也能使治疗试验在更广泛的剂量范围内进行, 而不是系统地实现。
我们提出这个协议, 与故障排除和质量检查步骤, 作为一个详细的和更新的方法, 通过沃顿商学院的管道提供聚合物纳米材料的小鼠 SMG20。例如, 正确使用导线指南有助于套管放置。 此外, 通过使用干印迹代替氰胶水在注射过程中保持套管到位, 减少了粘膜损伤的风险。此方法可用于处理具有一系列化合物的小鼠, 并且可以用相同的鼠标在多天内执行, 以评估重复管理11的 timecourse。
正常腺体分泌物将提供一个简单而直接的清除机制, 为过剩的有效载荷, 但这一策略应该通过仔细选择注射物质和阿托品剂量滴定来优化不同的应用。在这种情况下, NPs 在 SMG 中坚持至少24小时。通过使用能够进行药物加载的 NPs 或类似的纳米材料, 今后这项工作的应用包括克服溶解度限制, 否则将防止使用 retroductal 注入20,21测试疏水性剂。

涎腺功能障碍有许多病因, 包括 Sjögren 综合征, 自身免疫介导的功能性分泌组织丧失, 以及辐射诱发的 hyposalivation (RIH), 共同 sequella 头颈肿瘤放疗1。由于任一条件而导致的唾液功能丧失会诱发个人口腔和全身感染、蛀牙、消化和吞咽功能障碍、言语障碍和主要抑郁症1,2,3。因此, 生活质量显著受损, 干预措施仅限于缓解症状, 而不是治疗4。为了研究新的治疗方法在体内, 直接对涎腺进行生物活性化合物是有好处的。
Retroductal 注射液是一种很有价值的方法, 可以直接向唾液腺提供生物活性化合物, 并检测疾病、损伤或正常组织稳态下的疗效。三个主要唾液腺是腮腺 (PG), 颌下腺 (SMG) 和舌下 (SLG), 所有这些都空入口腔通过排泄导管。该小鼠的解剖允许通过插管的沃顿商学院的管道直接进入, 位于舌下的口底部5。插管后, 溶解药物可以直接管理到 SMG。在 retroductal 分娩后, 附加腺的扩散受周围组织胶囊的限制, 该囊调节与周围结构的材料交换6。smg 和它的导管是相似的结构在人, 并且经常被访问在 SMG 手术期间和 sialoendoscopy7。在人类和小鼠, PG 同样可以通过 Stensen 的导管在颊粘膜8。
在 RIH 的小鼠模型中, SMG retroductal 注射液用于提供治疗方法, 包括生长因子、原代细胞、腺病毒载体、细胞因子和抗氧化化合物, 以调节细胞对损伤的反应, 并减少由此产生的组织损伤5,9,10,11,12,13,14,15,16。retroductal 注射液的临床成功最显著的是对腺病毒载体的管理, 以直接表达的水道 (水通道蛋白 1;AQP1) 治疗头颈癌放疗后的患者17。
以前, 我们已经开发并显示了 retroductally 注射聚合物纳米 siRNA 系统的功效, 以保护唾液腺功能从 RIH11,18,19,20。作为我们过去工作的延伸, 在这里, 我们展示了我们的协议 retroductal SMG 注射使用一个荧光标记纳米粒子 (NP) 能够加载和交付否则不太可溶性药物21,22,23。
我们已经合成了 NP 从两嵌段共聚物组成的聚 (苯乙烯-alt-马来酸酐)-b 聚 (苯乙烯) (PSMA) 通过可逆添加链碎片 (筏) 聚合, 如前所述21。通过溶剂交换, 这些聚合物自发地自组装成胶束 NP 结构, 具有疏水性内部和亲水性外部的21。NPs 被标记与得克萨斯红色荧光允许验证 NP 交付入腺体, 不用牺牲动物。活体动物成像和 SMG 免疫组化显示在注射后1小时和1天。
此更新和可重现的插管协议应使其他人能够实现 retroductal 注射。我们预计, 这种精制技术将成为体内研究和治疗发展的关键24,25。

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			<td>Syringe for intraperitoneal injection</td>
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retroductal nanoparticle injection to the murine submandibular gland

涎腺治疗的两个共同目标是在自身免疫或辐射损伤后组织功能障碍的预防和治疗。通过局部向涎腺提供生物活性化合物, 可以安全地实现更大的组织浓度, 而不是系统性的管理。此外, 离体超腺堆积物的靶组织效应可以显著降低。在这方面, retroductal 注射液是一种广泛应用的方法, 以调查涎腺生物学和病理生理学。Retroductal 对生长因子、原代细胞、腺病毒载体和小分子药物的管理在损伤的设置中支持腺体功能。我们以前已经证明了 retroductally 注射纳米粒子-siRNA 策略在辐照后维持腺体功能的有效性。在这里, 一个高效和可重复的方法管理纳米材料的小鼠颌下腺通过沃顿的管道是详细 (图 1)。我们描述了进入口腔, 并概述了必要的步骤 cannulate 沃顿的管道, 并进一步的意见, 作为质量检查整个过程。

Retroductal 注射液可用于管理 NPs 到小鼠 SMG (图 1)。在这里, 我们提供了50µg PSMA NPs 标记与德州红荧光。
正确放置鼠标可以方便地访问和可视化口底 (图 2A-b)。颌下腺的乳突被确定为舌下的两个肉质突起。在插管 (图 2C) 和阿托品注射液之后, 注射器导管可以放入颌下腺乳突 (图 2D)。
为便于插管, 在下颌下腺的一小口穿刺首先使用导管管内的导线鞘 (图 3A)。完成此操作后, 鞘应在油管内缩回, 以作为刚性导轨, 同时进行较大的开口 (图 3B)。鞘的直径为0.076 毫米, 而导管管的外径为0.25 毫米。在这个更大的开口的创建之后, 附加到注射注射器上的预加载导管油管可以引导进入导管孔 (图 3C)。
注射后, 建议将注射器固定, 并保持注射压力。如果压力不被应用, 交付将是成功的, 虽然效率和重现性较低。这表明, 通过注射50µL 1% 甲苯胺蓝染料双边和观察微弱的染色在腺体没有保持压力后, 注射 (图 4)。
为了验证 NP 传递, IVIS 可以用于检测鼠标中的荧光信号, 这是一侧到注入区域 1 h post 管理 (图 5)。此方法可在不 euthanizing 鼠标的情况下进行确认, 并且可以纵向持续, 直到信号不再被检测到26、27。
为了确认在 24 h 以下注射后的 NP 持久性, 腺体可以切片, 并通过荧光成像查看。Aqp5 和 Krt5 IHC 分别标记了 SMG 的分泌和导管细胞, 并在两个车厢中显示 NPs (图 6)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57521/57521fig1.jpg"> 图 1.逆行注射示意图.导管插管和注射器放置后, 50 µL 1 毫克/毫升聚合 NP 溶液注入到 SMG。具有代表性的透射电子显微图像 (TEM) 显示单分散 (多分散性指数 = 0.2) NP 人口。<a href="/files/ftp_upload/57521/57521fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57521/57521fig2.jpg"> 图 2.逆行注射步骤.(A)通过将上颌和下颌门牙分开来访问口腔。(B)可视化口腔底部舌下的乳突 (盒装), 这标志着沃顿商学院管道的位置。(C)使用带插入线的导管, 轻轻地 cannulate 下颌下腺的基底。(D)插管后, 导管管可与注射器油管交换<a href="/files/ftp_upload/57521/57521fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57521/57521fig3.jpg"> 图 3.鞘导管插管的有效定位.(A)将油管与镊子的曲率对齐, 并在油管和导线上切割一个斜面末端, 最初刺穿舌下的乳突。(B)收回油管内的鞘, 使其在舌下的乳突内插入导管的刚性指南。(C)在先前制作的孔内插入导管导管 (鞘取出), 加入注射注射器。<a href="/files/ftp_upload/57521/57521fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57521/57521fig4.jpg"> 图 4.注射后保持注射器压力可以提高材料的保留率.在注射50µL 甲苯胺蓝后, 注射器压力要么维持5分钟 (右 retroductal 第一次注射), 要么注射器被立即撤回 (左 smg 第二次注射)。<a href="/files/ftp_upload/57521/57521fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57521/57521fig5.jpg"> 图 5.retroductal NP 后注入的确认.(A)体内成像系统 (IVIS) 显示了红色荧光信号的侧向性 (左) 侧的小鼠 1 h 后注射。(B) NP IVIS 信号在24小时内显著降低。<a href="/files/ftp_upload/57521/57521fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57521/57521fig6.jpg"> 图 6.确认 retroductal NP 持久性 24 h 后注入.A、C。Uninjected 控制 Aqp5 和 Krt5 染色, 分别标记分泌腺泡和导管细胞。B、D。在 retroductal NP 注入的 SMG, Aqp5 和 Krt5 污渍显示正常腺体形态和 NPs 占去了腺泡细胞和管道 (鳞片酒吧:75 µm)。<a href="/files/ftp_upload/57521/57521fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

局部药物向颌下腺的传递对了解涎腺生物学和新疗法的发展有很浓厚的兴趣。我们提出了一个更新和详细的 retroductal 注射协议, 旨在提高交付准确性和实验再现性。本文提出的应用是高分子纳米粒子的传递。

Retroductal 纳米微粒注射液对小鼠颌下腺的治疗

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.9K 次观看.  University of Rochester. 局部药物向颌下腺的传递对了解涎腺生物学和新疗法的发展有很浓厚的兴趣。我们提出了一个更新和详细的 retroductal 注射协议, 旨在提高交付准确性和实验再现性。本文提出的应用是高分子纳米粒子的传递。

视频: Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

导管内送货到兔乳腺

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...