Felipe Santos

Identification of Genetic and Chemical Modulators of Zebrafish Mechanosensory Hair Cell Death

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.

Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio)

Developmental differences in hair cell susceptibility to aminoglycoside-induced cell death has been observed in multiple species. Increased sensitivity to aminoglycosides has been temporally correlated with the onset of mechanotransduction-dependent activity. We have used in vivo fluorescent vital dye markers to further investigate the determinants of aminoglycoside induced hair cell death in the lateral line of zebrafish (Danio rerio). Labeling hair cells of the lateral line in vivo with the dyes FM 1-43, To-Pro-3, and Yo-Pro-1 served as reliable indicators of hair cell viability. Results indicate that hair cell maturation is a determinant of developmental differences in susceptibility. The age dependent differences in susceptibility to aminoglycosides are independent of the onset of mechanotransduction-dependent activity as measured by FM 1-43 uptake and independent of hair cell ability to take up fluorescently conjugated aminoglycosides.

Drug screening for hearing loss: Using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss

Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause and prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. The identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection.

Implantation of the Semicircular Canals with Preservation of Hearing and Rotational Sensitivity: a vestibular neurostimulator suitable for clinical research

Hypothesis It is possible to implant a stimulating electrode array in the semicircular canals without damaging rotational sensitivity or hearing. The electrodes will evoke robust and precisely controlled eye movements. Background A number of groups are attempting to develop a neural prosthesis to ameliorate abnormal vestibular function. Animal studies demonstrate that electrodes near the canal ampullae can produce electrically evoked eye movements. The target condition of these studies is typically bilateral vestibular hypofunction. Such a device could potentially be more widely useful clinically and would have a simpler roadmap to regulatory approval if it produced minimal or no damage to the native vestibular and auditory systems. Methods An electrode array was designed for insertion into the bony semicircular canal adjacent to the membranous canal. It was designed to be sufficiently narrow so as to not compress the membranous canal. The arrays were manufactured by Cochlear, Ltd., and linked to a Nucleus Freedom receiver/stimulator. Seven behaviorally trained rhesus macaques had arrays placed in 2 semicircular canals using a transmastoid approach and “soft surgical” procedures borrowed from Hybrid cochlear implant surgery. Postoperative vestibulo-ocular reflex was measured in a rotary chair. Click-evoked auditory brainstem responses were also measured in the 7 animals using the contralateral ear as a control. Results All animals had minimal postoperative vestibular signs and were eating within hours of surgery. Of 6 animals tested, all had normal postoperative sinusoidal gain. Of 7 animals, 6 had symmetric postoperative velocity step responses toward and away from the implanted ear. The 1 animal with significantly asymmetric velocity step responses also had a significant sensorineural hearing loss. One control animal that underwent canal plugging had substantial loss of the velocity step response toward the canal-plugged ear. In 5 animals, intraoperative electrically evoked vestibular compound action potential recordings facilitated electrode placement. Postoperatively, electrically evoked eye movements were obtained from electrodes associated with an electrically evoked vestibular compound action potential wave form. Hearing was largely preserved in 6 animals and lost in 1 animal. Conclusion It is possible to implant the vestibular system with prosthetic stimulating electrodes without loss of rotational sensitivity or hearing. Because electrically evoked eye movements can be reliably obtained with the assistance of intraoperative electrophysiology, it is appropriate to consider treatment of a variety of vestibular disorders using prosthetic electrical stimulation. Based on these findings, and others, a feasibility study for the treatment of human subjects with disabling Ménière’s disease has begun.

Supraclavicular Artery Island Flap for Reconstruction of Complex Parotidectomy, Lateral Skull Base, and Total Auriculectomy Defects

IMPORTANCE There are limited data on the use of the supraclavicular artery island flap (SCAIF) for parotid and lateral skull base (LSB) surgery. This flap can be an important reconstructive tool for these procedures. OBJECTIVE To describe the use of the SCAIF for parotid and LSB surgery and its success, as well as important technique modifications for successful use of the flap in this setting. DESIGN, SETTING, AND PARTICIPANTS Retrospective single-institution review from July 1, 2011, to September 30, 2013, of patients in a tertiary care referral center. A prospectively collected institutional database was reviewed to identify patients who received SCAIF reconstruction for parotid and/or LSB surgery. Forty-six SCAIF reconstructions were identified; 16 were performed for the indication of parotidectomy or LSB surgery. INTERVENTIONS The SCAIF reconstruction for parotid and/or LSB surgery. MAIN OUTCOMES AND MEASURES Indication for reconstruction, flap viability, flap size, reconstruction site complication, and donor site complication. RESULTS Resection was performed for advanced cutaneous malignant tumor in 10 patients, primary salivary gland malignant tumor in 4 patients, and chronic infection and mastoid cutaneous fistula in 2 patients. All defects were complex, involving multiple subsites; 5 patients underwent facial nerve resection and 4 had previous radiation therapy. No complete flap loss occurred. One partial flap loss occurred. The average flap island size was 7 × 10 cm. No major complications occurred. Two minor reconstruction site complications and 3 donor site seromas occurred. CONCLUSIONS AND RELEVANCE The SCAIF can be successfully and reliably used for complex defects following parotid and LSB surgery. There are 3 important technique modifications to help facilitate rotation and coverage of this region.

Side-to-end hypoglossal to facial anastomosis with transposition of the intratemporal facial nerve.

Objective To describe results in a large series of patients using a recent variation of hypoglossal-facial nerve anastomosis (HFA) in which the intratemporal facial nerve segment is used, obviating the need for a sensory nerve “jump graft.” Study Design Retrospective chart review. Setting Tertiary neurotologic referral center. Patients Nineteen patients (12 female/7 male subjects) with facial paralysis because of posterior fossa surgery for tumor (n = 15), Bell’s palsy (n = 1), facial neuroma (n = 1), hemangioma (n = 1), and trauma (n = 1) who underwent HFA from 1997 to 2011, with at least 1-year follow-up. Mean age at surgery is 47.4 years (range, 11.2–83 yr). Mean follow-up is 4.0 years. Intervention Side-to-end hypoglossal to facial anastomosis with transposition of the intratemporal facial nerve (swingdown HFA). Main Outcome Measure House-Brackmann (H-B) facial nerve grade. Results Seven patients (36.8%) achieved an H-B Grade III, 9 patients (47.4%) a grade IV, and 3 patients (15.8%) a grade V at last follow-up. No patients complained of dysphagia, dysarthria, or had evidence of oral incompetence. One patient complained of mild tongue weakness. Age at time of HFA (p ⩽ 0.049, III younger than V) and time from facial nerve injury to HFA (p ⩽ 0.02, III<IV and V) were significant factors for ultimate facial nerve outcome. All patients with an H-B III result had HFA within 6 months of injury. Other factors were not significant. Conclusion The HFA swingdown technique is a safe and effective method to restore facial nerve function in patients with facial paralysis and obviates the need for an interposition jump graft.

Otopathology in Congenital Toxoplasmosis

Objective To describe the temporal bone histopathology in children with congenital toxoplasmosis. Background Toxoplasmosis is a parasitic infection caused by Toxoplasma gondii. If fetal infection occurs early in gestation, severe inflammation and necrosis can cause brain lesions, chorioretinitis, and hearing loss. Hearing loss in congenital toxoplasmosis may be preventable with early diagnosis and treatment. Materials and Methods The temporal bones of 9 subjects with congenital toxoplasmosis were removed at autopsy and studied under light microscopy. Cytocochleograms were constructed for hair cells, the stria vascularis, and cochlear neuronal cells. Results Three (33%) of 9 subjects were found to have parasites in the temporal bone. The organism was identified in the internal auditory canal, the spiral ligament, stria vascularis, and saccular macula. The cystic form of the parasite was not associated with the inflammatory response seen in the active tachyzoite form. Conclusion We infer that the hearing loss of toxoplasmosis is likely the result of a postnatal inflammatory response to the tachyzoite form of T. gondii. Our findings have implications for the early identification and management of Toxoplasmosis.

Otopathology in Osteogenesis Imperfecta

Background Osteogenesis Imperfecta (OI) is a genetic disorder of connective tissue matrix. OI is caused by mutations that affect type I collagen. The hearing loss in OI is characterized by onset in early adulthood and can be conductive, sensorineural, or mixed. Objectives To describe the temporal bone histopathology in 9 individuals with OI. Materials and Methods Four adult, 1 pediatric, and 4 infant specimens were identified. Temporal bones were removed at autopsy and studied using light microscopy. Results All adults and 1 pediatric specimen showed otosclerotic lesions. The findings included examples of clinical, histologic, and cochlear otosclerosis. The temporal bones of infants showed delayed ossification of the endochondral layer of bone and of the ossicles. There were no infant specimens with otosclerotic lesions. Conclusion Hearing loss in OI may be the result of clinical or cochlear otosclerosis. Fracture or atrophy of the ossicles may also be present in OI. A third unidentified mechanism of hearing loss may lead to cochlear degeneration. The described findings of otosclerotic lesions have implications for the observed heterogeneity of hearing loss patterns and for the surgical management of hearing loss in OI.

Otologic manifestations of Fanconi anemia

Objectives To identify and define the otologic manifestations in patients with Fanconi anemia. Design Retrospective case series. Setting Tertiary referral center. Patients Sixty-nine patients in all age groups who received a diagnosis of Fanconi anemia with or without otologic presentation. Methods Medical records, audiograms, and tympanograms of all patients were reviewed. Data were analyzed and compared with the literature. Results Of 69 patients with Fanconi anemia, many had morphologic anomalies affecting the structures of the ear and the head and neck. Conductive hearing loss, external auditory canal stenosis, and auricular malformation were among the most common. Conclusions Fanconi anemia is a clinically heterogenous disorder. Conductive hearing loss, external auditory canal stenosis, and auricular malformations are the most common otologic presentations but are not present in the majority of patients.

Endolymphatic Sac Amputation Without Hydrops

The endolymphatic sac has been proposed as the site of endolymph resorption. Blockage of the duct or sac resulting in endolymphatic hydrops has been observed in animal studies (1). Human examples of endolymphatic obstruction with hydrops have also been observed. In syphilis, microgummata obstructing the endolymphatic duct and sac have been observed in patients with concurrent hydrops and dilation of the inner ear membranes (2). Blockage in the endolymphatic duct was also seen in 8 of 46 temporal bones from patients with a clinical history of Ménière_s disease (3). This case presents a complete transection of the endolymphatic sac during resection of a large vestibular schwannoma. The patient died 2 months later without histopathologic evidence of endolymphatic hydrops, suggesting that endolymph is absorbed by the endolymphatic duct independent of the sac.