本文所述的工作得到了 RO1 CA166907、NIDCD RO1-DC 002396 和 RO3 DC011621 的支持。

雄性大鼠是按照国家卫生研究院的动物使用指南和由南伊利诺伊大学医学院实验室动物护理和使用委员会批准的协议处理的。在药物管理局麻醉前和72小时后, 对大鼠进行听觉脑干反应 (ABR) 检查, 以验证其对鼓膜药物传递的影响。
1. 听觉脑干反应 (ABR)
注意:通过听觉测试设备和软件采集 ABR 测量结果。ABR 代表诱发电位或高频波产生的第八颅神经 (波 I 和 II) 和其他高听觉脑干结构, 包括 anteroventral 耳蜗核 (波 III), 侧 lemniscus (波 IV), 和下位丘 (波浪 V)。这些波浪根据延迟被区分。ABR 测量是按照前面描述的12进行的。
<ol><li>麻醉大鼠, 混合90毫克/千克氯胺酮和17毫克/千克甲苯噻嗪通过腹腔注射。确认麻醉深度通过脚趾捏反射。对眼睛进行眼部润滑 (或矿物油滴), 防止眼部干燥, 避免麻醉时出现溃疡。</li>
	<li>把老鼠放在有控制的隔音亭内的加热垫 (37 °c) 的俯卧位置上。听力测试设备位于展台的外面和旁边。</li>
	<li>相应地插入不锈钢电极: 在后侧面的接地电极, 两个耳朵之间的正电极, 直接顶部的头骨, 和负电极在每耳耳廓下。</li>
	<li>在8、16和32赫上应用高频传感器作为5毫秒音爆发的声学刺激。确定刺激强度作为分贝声压水平 (dB SPL)。这开始在10分贝的 spl 和达到90分贝的 spl 与 10 db 的步骤大小。使用脉冲精密声电平计, 将声音强度校准为最大输出90分贝的 SPL。 注意:实验结果显示了周围听觉器官、耳蜗和上述听觉结构的完整性。波形是在15毫秒的刺激输入中产生的, 而波的潜伏期取决于传导时间, 这反过来又受到三个关键因素的调节: 大脑体积、强度和声音频率。利用制造商提供的软件记录听觉诱发电位。</li>
	<li>考虑最小强度, 可以唤起两个不同的波形 (II 和 III) 的0.5 µV 振幅作为阈值突出。 注意:阈值移位表示治疗后的阈值与治疗前获得的阈值的差异。</li>
</ol>2. 反式鼓膜注射
<ol><li>对药物进行 tympanically, 将大鼠置于左侧褥疮位置。<ol>
<li>将2.5 毫米的一次性耳镜面插入耳道。</li>
		<li>使用手术范围, 定位镜面, 使鼓膜可见。</li>
		<li>使用29克 X ½, 0.5 毫升胰岛素注射器, 绘制50µL 的 [r] n-苯基异丙腺苷 (r-PIA) 溶液 (1 µM), 8-环戊基-13-dipropylxanthine (DPCPX) 溶液 (3 µM), 或 siRNA 溶液 (0.9 µg) 注入 (5 单位)。</li>
		<li>使用镜面引导针到鼓膜的前下位区。</li>
		<li>用针在膜上戳一个洞, 并管理1.2.3 中提到的药物。允许老鼠在这个位置休息15分钟。 注意: 50 µL 应该是足够的体积, 以适应鼓膜后。用药后, 耳道不应有液体。</li>
		<li>将大鼠置于右侧压位位置, 并重复步骤1.2.1 通过 1.2.5, 以供另一耳管理。</li>
	</ol>
</li>
	<li>对于接受顺铂腹腔的大鼠, 在37摄氏度的加热垫上放置大鼠仰卧位。<ol>
<li>使用21克 X 3/4 蝶形针 (12 英寸长油管), 在无菌磷酸盐缓冲盐水中绘制顺铂 (11 毫克/千克, 1 毫克/毫升溶液)。</li>
		<li>使用注射器泵, 管理顺铂 (1 毫克/毫升)通过腹腔注射30分钟以上。 注意:对于一个250克大鼠, 体积将是2.75 毫升的速度约0.1 毫升每分钟。</li>
	</ol>
</li>
	<li>在这些程序中继续监测麻醉深度。一旦顺铂管理完成, 把老鼠放回笼子里的俯卧位置, 确保没有任何阻碍它的呼吸。</li>
	<li>监视老鼠直到完全恢复。</li>
</ol>3. 耳蜗解剖和脱钙
<ol><li>继最后 ABR (72 小时后), 麻醉大鼠与90毫克/千克氯胺酮的混合物和17毫克/千克甲苯噻嗪通过腹腔注射。用脚趾捏的反射来确认麻醉。弄死老鼠的通过斩首。</li>
	<li>解剖出的颞骨, 如前所述13。</li>
	<li>在一个7毫升的玻璃闪烁瓶 (完全覆盖耳蜗) 的 1x PBS 溶液中放置耳蜗4% 多聚甲醛。隔夜冷藏4摄氏度。在室温下取出多聚甲醛并用 1x PBS 清洗。</li>
	<li>去除 PBS, 完全用120毫米 EDTA 溶液 (pH 7.3) 填满管子。在室温下放置在转子上, 允许脱钙2至3周, 每天更换 EDTA 溶液。</li>
</ol>4. Cryosectioning
<ol><li>在完成脱钙后, 完全浸入以下溶液中的耳蜗 (每7毫升) 24 小时4摄氏度: 10% 蔗糖, 20% 蔗糖, 1:1 混合物20% 蔗糖和最佳切削温度 (OCT) 嵌入化合物。</li>
	<li>在15毫米 x 15 毫米 x 5 毫米一次性嵌入模具中, 将新鲜的 OCT 填充并完全覆盖耳蜗。将耳蜗定向到其侧面, 使其与嵌入模具的底部平行, 如 Whitlon et所述。14
</li>
	<li>立即将模具放在干冰上固化 OCT, 并在一夜之间存放在-80 摄氏度。</li>
	<li>0.01% 聚 l-赖氨酸在室温下浸泡30分钟. 取出幻灯片, 不冲洗, 并允许夜间干燥。将这些幻灯片用于 cryosections。</li>
	<li>将 OCT 中的耳蜗从-80 摄氏度冷藏库中取出, 放在干冰上。使用锋利的切片刀片 (L x W:80 毫米 x 8 毫米, 厚度0.25 毫米, 切削角度34°), 部分 OCT 块在10µm 使用恒温器在-30 °c。每张幻灯片放置两个部分。</li>
	<li>完成后, 将幻灯片冷藏4摄氏度。</li>
</ol>5. 免疫组化
<ol><li>将幻灯片放在幻灯片架上的玻璃显微镜滑动染色盘中。用350毫升的 1x PBS 填充这道菜, 在室温下将幻灯片冲洗5分钟三次。</li>
	<li>将幻灯片从盘子中取出, 用干擦拭的方法将组织周围的区域晾干, 确保不使组织干燥。<ol>
<li>使用液体阻断笔, 在组织切片周围画一个圆圈。</li>
	</ol>
</li>
	<li>在室温下, 在 1x PBS 中加入150µL, 其中含有10% 正常 (驴) 血清、1% 非离子洗涤剂和 1% BSA, 以1小时的温度阻断组织。</li>
	<li>在 1x PBS 中, 用150µL 10% 正常 (驴) 血清、0.1% 非离子洗涤剂和主要抗体 (见下文注), 在4摄氏度的湿润室中, 利用多余的阻断溶液, 在一夜之间孵化组织。 注意:对于下列抗体, 使用了这些稀释: 对于图 2和3, p-STAT1 系列727 1:300。对于图 4, p-STAT1 系列727、1:100 和 TRPV1 1:100。</li>
	<li>将幻灯片放在幻灯片架上的玻璃显微镜滑动染色盘中。用350毫升的 1x PBS 填充盘子, 在室温下清洗5分钟三倍的幻灯片。</li>
	<li>在一个含有0.01% 非离子洗涤剂和10% 正常 (驴) 血清的溶液中, 在黑暗中的一个加湿室中, 在室温下进行2到3小时的二次抗体的孵育。 注意:对于以下二次抗体, 使用了这些稀释: 对于图 2和3, 驴抗兔 IgG 1:600。对于图 4, 罗丹明 (TRITC) 驴抗兔 igg 1:500 和驴抗山羊 igg 1:500。</li>
	<li>将幻灯片放在幻灯片架上的玻璃显微镜滑动染色盘中。用350毫升的 1x PBS 填充盘子, 在室温下清洗5分钟三倍的幻灯片。</li>
	<li>从幻灯片上轻拍多余的液体, 用干燥的纸巾擦干幻灯片。通过将安装代理的一滴 DAPI 直接添加到组织中来装载幻灯片。慢慢地将盖玻片放在顶部, 确保没有气泡在下面形成, 并允许幻灯片在室温下夜间在黑暗中治愈。将幻灯片存放在4摄氏度。</li>
	<li>图像幻灯片使用共焦显微镜。使用激光成像如下: DAPI 紫外线激光器, 488 nm 抗兔 IgG 激光, 543 nm 激光为罗丹明 TRITC。</li>
</ol>

<ol>
	<li>McKeage, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+adverse+effect+profiles+of+platinum+drugs.">Comparative adverse effect profiles of platinum drugs.</a> Drug Saf. 13 (4), 228-244 (1995).</li><li>Boulikas, T., Vougiouka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+and+platinum+drugs+at+the+molecular+level.">Cisplatin and platinum drugs at the molecular level.</a> Oncol Rep. 10 (6), 1663-1682 (2003).</li><li>Fouladi, M., 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=Phase+II+study+of+oxaliplatin+in+children+with+recurrent+or+refractory+medulloblastoma,+supratentorial+primitive+neuroectodermal+tumors,+and+atypical+teratoid+rhabdoid+tumors:+a+pediatric+brain+tumor+consortium+study.">Phase II study of oxaliplatin in children with recurrent or refractory medulloblastoma, supratentorial primitive neuroectodermal tumors, and atypical teratoid rhabdoid tumors: a pediatric brain tumor consortium study.</a> Cancer. 107 (9), 2291-2297 (2006).</li><li>Pasetto, L. M., D'Andrea, M. R., Rossi, E., Monfardini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-related+neurotoxicity:+how+and+why.">Oxaliplatin-related neurotoxicity: how and why.</a> Crit Rev Oncol Hematol. 59 (2), 159-168 (2006).</li><li>Ardizzoni, 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=Cisplatin-+versus+carboplatin-based+chemotherapy+in+first-line+treatment+of+advanced+non-small-cell+lung+cancer:+an+individual+patient+data+meta-analysis.">Cisplatin- versus carboplatin-based chemotherapy in first-line treatment of advanced non-small-cell lung cancer: an individual patient data meta-analysis.</a> J Natl Cancer Inst. 99 (11), 847-857 (2007).</li><li>Banfi, B., Malgrange, B., Knisz, J., Steger, K., Dubois-Dauphin, M., Krause, 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=NOX3,+a+superoxide-generating+NADPH+oxidase+of+the+inner+ear.">NOX3, a superoxide-generating NADPH oxidase of the inner ear.</a> J Biol Chem. 279 (44), 46065-46072 (2004).</li><li>Mukherjea, D., Whitworth, C. A., Nandish, S., Dunaway, G. A., Rybak, L. P., Ramkumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+kidney+injury+molecule+1+in+the+rat+cochlea+and+induction+by+cisplatin.">Expression of the kidney injury molecule 1 in the rat cochlea and induction by cisplatin.</a> Neuroscience. 139 (2), 733-740 (2006).</li><li>Rybak, L. P., Husain, K., Morris, C., Whitworth, C., Somani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+protective+agents+against+cisplatin+ototoxicity.">Effect of protective agents against cisplatin ototoxicity.</a> Am J Otol. 21 (4), 513-520 (2000).</li><li>Lee, J. E., 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=Role+of+reactive+radicals+in+degeneration+of+the+auditory+system+of+mice+following+cisplatin+treatment.">Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment.</a> Acta Otolaryngol. 124 (10), 1131-1135 (2004).</li><li>Lee, J. E., 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=Mechanisms+of+apoptosis+induced+by+cisplatin+in+marginal+cells+in+mouse+stria+vascularis.">Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis.</a> ORL J Otorhinolaryngol Relat Spec. 66 (3), 111-118 (2004).</li><li>Lawenda, B. D., Kelly, K. M., Ladas, E. J., Sagar, S. M., Vickers, A., Blumberg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+supplemental+antioxidant+administration+be+avoided+during+chemotherapy+and+radiation+therapy.">Should supplemental antioxidant administration be avoided during chemotherapy and radiation therapy.</a> J Natl Cancer Inst. 100 (11), 773-783 (2008).</li><li>Akil, O., Oursler, A. E., Fan, K., Lustig, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+auditory+brainstem+response+testing.">Mouse auditory brainstem response testing.</a> Bio Protoc. 6 (6), 1768 (2016).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Mount+Dissection+and+Immunofluorescence+of+the+Adult+Mouse+Cochlea.">Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.</a> J. Vis. Exp. (107), e53561 (2016).</li><li>Whitlon, D. S., Szakaly, R., Greiner, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoembedding+and+sectioning+of+cochleas+for+immunocytochemistry+and+in+situ+hybridization.">Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.</a> Brain Res Brain Res Protoc. 6 (3), 159-166 (2001).</li><li>Londos, C., Cooper, D. M., Wolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclasses+of+external+adenosine+receptors.">Subclasses of external adenosine receptors.</a> Proc Natl Acad Sci. 77 (5), 2551-2554 (1980).</li><li>Lohse, M. J., Klotz, K. N., Lindenborn-Fotinos, J., Reddington, M., Schwabe, U., Olsson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-Cyclopentyl-1,3-dipropylxanthine+(DPCPX)--a+selective+high+affinity+antagonist+radioligand+for+A1+adenosine+receptors.">8-Cyclopentyl-1,3-dipropylxanthine (DPCPX)--a selective high affinity antagonist radioligand for A1 adenosine receptors.</a> Naunyn Schmiedebergs Arch Pharmacol. 336 (2), 204-210 (1987).</li><li>Rybak, L. P., Whitworth, C., Scott, V., Weberg, A. D., Bhardwaj, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+as+a+potential+model+for+hearing+loss+in+biotinidase+deficiency.">Rat as a potential model for hearing loss in biotinidase deficiency.</a> Ann Otol Rhinol Laryngol. 100 (4), 294-300 (1991).</li><li>Mukherjea, 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=NOX3+NADPH+oxidase+couples+transient+receptor+potential+vanilloid+1+to+signal+transducer+and+activator+of+transcription+1-mediated+inflammation+and+hearing+loss.">NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss.</a> Antioxid Redox Signal. 14 (6), 999-1010 (2011).</li><li>Kaur, T., 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=Adenosine+A1+receptor+protects+against+cisplatin+ototoxicity+by+suppressing+the+NOX3/STAT1+inflammatory+pathway+in+the+cochlea.">Adenosine A1 receptor protects against cisplatin ototoxicity by suppressing the NOX3/STAT1 inflammatory pathway in the cochlea.</a> J Neurosci. 36 (14), 3962-3977 (2016).</li><li>Kaur, T., Mukherjea, D., Sheehan, K., Jajoo, S., Rybak, L. P., Ramkumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+interfering+RNA+against+STAT1+attenuates+cisplatin-induced+ototoxicity+in+the+rat+by+suppressing+inflammation.">Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation.</a> Cell Death Dis. 2 (180), (2011).</li></ol>

没有宣布利益冲突。

通过鼓膜管理途径, 可以将药物和其他药物的局部运送到耳蜗, 如果系统地进行管理, 可能会产生明显的系统性副作用。这种药物管理方法允许药物迅速进入行动地点, 其剂量要比通过系统途径所能达到的要高得多。在这里和发表的结果表明, 通过鼓膜管理的 [R] n-苯基异丙腺苷 (r-PIA) 保护耳蜗从顺铂诱导的外毛细胞损失 (图 1)19和诱导炎症的表现,...

外周听觉系统非常敏感的药物, 如顺铂和氨基糖苷类抗生素。顺铂是一种广泛使用的化疗剂, 用于治疗各种实体肿瘤, 如卵巢, 睾丸, 头颈癌。使用这种药物的耳毒性是剂量限制和相当常见的, 影响75-100% 的患者治疗1。其他药物, 如卡铂和奥沙利铂, 已成为替代顺铂2,3,4,5, 但它们的用处仅限于少数癌症。
早期研究表明活性氧 (ROS) 对顺铂和氨基糖苷类产生的耳毒性有重要作用。随后的研究表明, NOX3 异构体氧化酶是耳蜗中 ROS 的主要来源, 并由顺铂6,7激活。ROS 的产生危及细胞的抗氧化缓冲能力, 导致细胞膜脂质过氧化增加8。此外, 顺铂增加了羟基自由基产生剧毒醛 4-hydroxynonenal (4-他), 一个引发细胞死亡9,10。根据这些发现, 对治疗顺铂耳毒性的几种抗氧化剂进行了研究。这些包括 n-乙酰半胱氨酸 (NAC), 硫代硫酸钠 (STS), 汀和 d-蛋氨酸。然而, 抗氧化剂治疗的一个主要关注是, 这些抗氧化剂可以降低顺铂化疗的功效时, 系统地管理11通过与硫醇组的相互作用的抗氧化剂分子。
鉴于这些问题的抗氧化剂治疗, 本研究的目的是检查传递抗氧化剂和其他药物的耳蜗, 以减少听力损失的跨鼓膜路线。下面描述的药物和短干涉 (si) RNA 的跨鼓膜路线似乎特别有希望。

<table><tbody><tr><td>Ketathesia (100 mg/ml) 10 ml</td><td>Henry Schein</td><td>56344</td><td>Controlled substance&amp;nbsp;</td></tr><tr><td>AnaSed Injection/Xylazine (20 mg/ml) 20 ml</td><td>Henry Schein</td><td>33197</td><td></td></tr><tr><td>2.5 mm disposable ear specula</td><td>Welch Allyn</td><td>52432</td><td></td></tr><tr><td>Surgical Scope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>29 G X 1/2 insulin syringe</td><td>Fisher Scientific</td><td>14-841-32&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>cis-Diammineplatinum(II) dichloride</td><td>Sigma Aldrich</td><td>P4394</td><td>TOXIC - wear proper PPE</td></tr><tr><td>Harvard 50-7103 Homeothermic Blanket Control Unit</td><td>Harvard Apparatus</td><td>Series 863</td><td></td></tr><tr><td>Excel International 21 G X 3/4 butterfly needle</td><td>Fisher</td><td>14-840-34&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>BSP Single Speed Syringe Pump</td><td>Brain Tree Sci, Inc</td><td>BSP-99</td><td></td></tr><tr><td>Pulse Sound Measurement System</td><td>Bruel &amp;amp; Kjaer</td><td>Pulse 13 software</td><td></td></tr><tr><td>High-Frequency Module</td><td>Bruel &amp;amp; Kjaer</td><td>3560C</td><td></td></tr><tr><td>1/8&amp;Prime; Pressure-field Microphone &amp;mdash;-Type 4138</td><td>Bruel &amp;amp; Kjaer</td><td>bp2030</td><td></td></tr><tr><td>High Frequency Transducer</td><td>Intelligent Hearing System</td><td>M014600</td><td></td></tr><tr><td>Opti-Amp Power Transmitter</td><td>Intelligent Hearing System</td><td>M013010P</td><td></td></tr><tr><td>SmartEP ABR System</td><td>Intelligent Hearing System</td><td>M011110</td><td></td></tr><tr><td>Disposable Subdermal EEG Electrodes</td><td>CareFusion</td><td>019-409700</td><td></td></tr><tr><td>16% Formaldehyde, Methanol-free</td><td>Fisher Scientific</td><td>28908</td><td>TOXIC - wear proper PPE&amp;nbsp;</td></tr><tr><td>7 mL Borosilicate Glass Scintillation Vial</td><td>Fisher Scientific</td><td>03-337-26</td><td>Can be purchased through other vendors</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP118-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Tissue Plus OCT Compound</td><td>Fisher Scientific</td><td>4585</td><td></td></tr><tr><td>CryoMolds (15 mm x 15 mm x 5mm)</td><td>Fisher Scientific</td><td>22-363-553</td><td>Can be purchased through other vendors</td></tr><tr><td>Microscope Slides (25mm x 75mm)</td><td>MidSci</td><td>1354W</td><td>Can be purchased through other vendors</td></tr><tr><td>Coverslips (22 x 22 x 1)</td><td>Fisher Scientific</td><td>12-542-B</td><td>Can be purchased through other vendors</td></tr><tr><td>Poly-L-Lysine Solution (0.01%)</td><td>EMD Millipore</td><td>A-005-C</td><td>Can be purchased through other vendors</td></tr><tr><td>HM525 NX Cryostat</td><td>Thermo Fischer Scientific</td><td>956640</td><td></td></tr><tr><td>MX35 Premier Disposable Low-Profile Microtome Blades</td><td>Thermo Fischer Scientific</td><td>3052835</td><td></td></tr><tr><td>Wheaton&amp;trade;&amp;nbsp;Glass 20-Slide Staining Dish with Removable Rack</td><td>Fisher Scientific</td><td>08-812</td><td></td></tr><tr><td>Super Pap Pen Liquid Blocker</td><td>Ted Pella, Inc.</td><td>22309</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-121</td><td>Can be purchased through other vendors</td></tr><tr><td>TritonX-100</td><td>Acros</td><td>21568</td><td>Can be purchased through other vendors</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>Can be purchased through other vendors</td></tr><tr><td>Phospho-Stat1 (Ser727) antibody</td><td>Cell Signaling</td><td>9177</td><td></td></tr><tr><td>VR1 Antibody (C-15)</td><td>Santa Cruz</td><td>sc-12503</td><td></td></tr><tr><td>DyLight 488 Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-485-152</td><td>Discontinued</td></tr><tr><td>DyLight 488 Donkey anti Goat</td><td>Jackson Immuno Research</td><td>705-485-003</td><td>Discontinued</td></tr><tr><td>Rhodamine (TRTIC) Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-025-152</td><td>Discontinued</td></tr><tr><td>ProLong&amp;reg; Diamond Antifade Mountant w/ DAPI</td><td>Thermo Fisher</td><td>P36971</td><td></td></tr><tr><td>(&amp;minus;)-N6-(2-Phenylisopropyl)adenosine</td><td>Sigma Aldrich</td><td>P4532</td><td></td></tr><tr><td>8-Cyclopentyl-1,3-dipropylxanthine</td><td>Sigma Aldrich</td><td>C101</td><td></td></tr><tr><td>siRNA pSTAT1</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>siRNA NOX3</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Scrambled Negative Control siRNA</td><td>Qiagen</td><td>1022076</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr></tbody></table>

trans-tympanic drug delivery for the treatment of ototoxicity

保护剂治疗药物性耳毒性的系统管理受到限制, 因为这些保护剂可能会干扰初级药物的化疗效果。这种情况尤其适用于药物顺铂, 其抗癌作用因抗氧化剂而减弱, 为听力丧失提供了足够的保护。其他当前或潜在的 otoprotective 代理可能会造成类似的问题, 如果系统管理。将各种生物制品或保护剂直接应用于耳蜗, 可以使这些药物的高水平在局部具有有限的系统性副作用。在本报告中, 我们展示了一种跨鼓膜的方法, 提供各种药物或生物试剂的耳蜗, 这应该加强对耳蜗的基础科学研究, 并提供一个简单的方法指导使用 otoprotective 剂在诊所。本报告详细介绍了一种跨鼓膜药物传递方法, 并举例说明了该技术如何成功地应用于实验动物治疗顺铂耳毒性。

在顺铂治疗后三天大鼠的 ABR 反应显示阈值显著升高。通过鼓膜 (r) n-phenylisopropyladenosine (r-PIA), 腺苷 A1受体激动剂15, 顺铂前, 这些阈值的升高明显减少。R-PIA 在腺苷 A1受体上的作用的特异性表明, 它是由 8-环戊基-13-dipropylxanthine (DPCPX) 对抗,一个1 受体特定拮抗剂16。这种药物增强顺铂诱发的 ABR ?...

Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

本文提出了一种通过鼓膜入耳蜗进行药物局部化管理的技术。通过这条途径运送药物不会影响化疗药物如顺铂的抗癌功效。

经鼓膜药物治疗耳毒性的临床研究

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

trans-tympanic-drug-delivery-for-the-treatment-of-ototoxicity

Research

JoVE Journal

Medicine

9.3K 次观看.  Southern Illinois University School of Medicine. 本文提出了一种通过鼓膜入耳蜗进行药物局部化管理的技术。通过这条途径运送药物不会影响化疗药物如顺铂的抗癌功效。

视频: Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

热消融治疗腹部肿瘤

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

科研

医学

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

遗传学

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

神经科学

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Endaural Endoscopic Atticoantrotomy (Retrograde Mastoidectomy) using a Constant Suction Bone-drilling Technique

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the main technical challenge of endoscopic middle ear surgery. The accompanying video describes the detailed protocol of a constant-suction bone-drilling technique and the procedure of endaural exclusive endoscopic atticoantrotomy (retrograde mastoidectomy) using this technique. The main components of this bone-drilling technique include a soft and flexible suction tube, which is placed into the tympanic cavity to provide constant suction, and a soft sleeve, which is wrapped around the drill shaft to prevent the high-speed rotating shaft from damaging the lens of the endoscope. With these simple modifications, the traditional otological electrodrill can be used for drilling a tiny endaural incision in endoscopic middle ear surgery. Based on this bone-drilling technique, endaural endoscopic atticoantrotomy (retrograde mastoidectomy) can be successfully established for the removal of various amounts of bone, depending on the extent of the lesion. The short-term postoperative outcome seems promising.

Endaural Endoscopic Atticoantrotomy Retrograde Mastoidectomy using a Constant Suction Bone-drilling Technique

In this paper, we present the protocol and short-term outcomes of endaural exclusive endoscopic atticoantrotomy using a constant suction bone-drilling technique.

使用恒定抽吸骨术的内窥镜下盂骨切开术（逆行切除术）

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

针对鼠标内耳给药方法的比较研究:Bullostomy<em&gt;对战</em&gt;鼓室注射

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

生物学

<meta charset="utf8"></head><body>电声刺激中长电极的听力保护

Enhanced Cochlear Coverage and Hearing Preservation in High-Frequency Hearing Loss via Electric Acoustic Stimulation with Longer Electrode

Electric-acoustic stimulation (EAS) is a promising treatment to improve hearing ability in patients with high-frequency hearing loss (HL). In EAS surgeries, shorter electrodes have been preferred to avoid the presence of an electrode covering the residual hearing region. However, our earlier studies showed that EAS with longer electrodes (28 mm) could preserve acoustic hearing. Additionally, we reported that the hearing preservation (HP) scores were independent of the length of the inserted electrodes, consistent with the systematic review. As most EAS patients gradually lose residual hearing over time due to the natural course of HL, in these cases, providing broader cochlear coverage using longer electrodes was beneficial toward better place-pitch matching. In addition to preparing for the deterioration in&#160;hearing in&#160;the future, EAS with longer electrodes could offer various types of map strategies. Herein, we show the pre-, intra-, and post-procedures for EAS surgery. Appropriate preoperative evaluation, less invasive surgery, flexible lateral-wall electrodes, and steroid administration resulted in good HP following EAS with longer electrodes.

<meta charset="utf8"></head><body>作者聚焦:使用长电极优化 EAS 以增强人工耳蜗覆盖率和听力保护

Electric acoustic stimulation (EAS) with longer electrodes can offer broader cochlear coverage and various types of maps in cases of high-frequency hearing loss. Combining less invasive surgery, flexible lateral-wall electrodes, and steroid administration permits deeper insertion with little or no surgical trauma, resulting in good preservation of hearing.