Murillo-Cuesta Silvia, Vallecillo Néstor, Cediel Rafael, Celaya Adelaida M, Lassaletta Luis, Varela-Nieto Isabel, Contreras Julio
Instituto de Investigaciones Biomédicas (IIBm) Alberto Sols CSIC-UAM; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII); Instituto de Investigación Sanitaria La Paz (IdiPAZ);
Instituto de Investigaciones Biomédicas (IIBm) Alberto Sols CSIC-UAM.
J Vis Exp. 2017 Mar 8(121):54951. doi: 10.3791/54951.
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´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 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.
我们介绍了两种在啮齿动物中进行的微创显微手术技术，用于将特定药物递送至中耳，以便药物能够到达内耳。第一种手术包括鼓泡穿孔，称为鼓泡造口术；第二种是经鼓膜注射。这两种技术都模仿了人类临床鼓室内手术。壳聚糖-甘油磷酸酯（CGP）和乳酸林格氏缓冲液（RL）被用作局部药物递送的生物相容性载体。CGP是一种无毒的可生物降解聚合物，广泛应用于制药领域。它在室温下是一种粘性液体，但在体温下会凝结成半固体状态。RL是一种用于人类静脉给药的等渗溶液。通过鼓泡造口术将少量这种载体精确地放置在圆窗（RW）龛上。经鼓膜注射可充满中耳，但控制较少，但对内耳的进入范围更广。通过功能和形态学测试研究并比较了这两种技术的安全性。在显微手术前和手术后多次记录听觉脑干反应（ABR）来评估听力。通过常规组织学技术在多聚甲醛固定和脱钙的耳蜗样本中研究耳蜗结构的细胞结构和保存水平。同时，采集未固定的耳蜗样本并立即冷冻，通过定量逆转录聚合酶链反应（qRT-PCR）分析炎症标志物的基因表达谱。这两种方法都适合作为向小鼠中耳递送药物的方法，尽管经鼓膜注射与鼓泡造口术相比侵入性较小。
