Stefania Goncalves

Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma

Loss of auditory sensory hair cells (HCs) is the most common cause of hearing loss. This review addresses the signaling pathways that are involved in the programmed and necrotic cell death of auditory HCs that occur in response to ototoxic and traumatic stressor events. The roles of inflammatory processes, oxidative stress, mitochondrial damage, cell death receptors, members of the mitogen-activated protein kinase (MAPK) signal pathway and pro- and anti-cell death members of the Bcl-2 family are explored. The molecular interaction of these signal pathways that initiates the loss of auditory HCs following acoustic trauma is covered and possible therapeutic interventions that may protect these sensory HCs from loss via apoptotic or non-apoptotic cell death are explored.

Spiral ganglion cells and macrophages initiate neuro-inflammation and scarring following cochlear implantation

Conservation of a patients residual hearing and prevention of fibrous tissue/new bone formation around an electrode array are some of the major challenges in cochlear implant (CI) surgery. Although it is well-known that fibrotic tissue formation around the electrode array can interfere with hearing performance in implanted patients, and that associated intracochlear inflammation can initiate loss of residual hearing, little is known about the molecular and cellular mechanisms that promote this response in the cochlea. In vitro studies in neonatal rats and in vivo studies in adult mice were performed to gain insight into the pro-inflammatory, proliferative, and remodeling phases of pathological wound healing that occur in the cochlea following an electrode analog insertion. Resident Schwann cells (SC), macrophages, and fibroblasts had a prominent role in the inflammatory process in the cochlea. Leukocytes were recruited to the cochlea following insertion of a nylon filament in adult mice, where contributed to the inflammatory response. The reparative stages in wound healing are characterized by persistent neuro-inflammation of spiral ganglion neurons (SGN) and expression of regenerative monocytes/macrophages in the cochlea. Accordingly, genes involved in extracellular matrix (ECM) deposition and remodeling were up-regulated in implanted cochleae. Maturation of scar tissue occurs in the remodeling phase of wound healing in the cochlea. Similar to other damaged peripheral nerves, M2 macrophages and de-differentiated SC were observed in damaged cochleae and may play a role in cell survival and axonal regeneration. In conclusion, the insertion of an electrode analog into the cochlea is associated with robust early and chronic inflammatory responses characterized by recruitment of leukocytes and expression of pro-inflammatory cytokines that promote intracochlear fibrosis and loss of the auditory hair cells (HC) and SGN important for hearing after CI surgery.

Effects of Cell-Based Therapy for Treating Tympanic Membrane Perforations in Mice

Objective To investigate the effectiveness of scaffold-embedded mesenchymal stem cells (MSCs) as a topical treatment for healing tympanic membrane perforations (TMPs) in a mouse model. Study Design Prospective animal study. Setting Experimental. Subjects and Methods In vitro: under sterile conditions, porcine-derived (Gelita-Spon [GS]), hyaluronate-derived (EpiDisc [ED]), and polyvinyl alcohol (PVA) scaffolds were cut into small pieces and cocultured with murine bone marrow–derived MSCs (BM-MSCs) expressing green fluorescent protein (GFP) for 72 hours. The cultures were either analyzed by confocal microscopy or used for subsequent in vivo experiments. In vivo: 26 mice were divided into 3 groups (ie, control [n = 9], GS [n = 8], ED [n = 9]). Under general anesthesia, TMPs of equal sizes were performed bilaterally using a sterile 27-gauge needle under a surgical microscope. The BM-MSCs embedded within GS or ED scaffolds were soaked in phosphate-buffered saline and then topically applied on right TMPs, and scaffolds alone were applied on left TMPs 6 to 8 hours after injury. Control mice did not receive treatment. On day 7, animals were euthanized and bullae were harvested for histological analysis. Results In vitro: BM-MSCs grew well on both GS (P = .0012) and ED (P = .0001) scaffolds compared with PVA. In vivo: 100% of untreated (control) TMPs remained open after 7 days. Animals treated with MSC-embedded ED scaffolds had a higher percentage of TMP closure (P = .016) and a thicker neotympanum (P = .0033) than control animals. The experimentally applied BM-MSCs engrafted and differentiated into epithelial cells suggested by the colocalized expression of cytokeratin-19 and GFP. Conclusions The topical application of bone marrow–derived MSCs enhances the healing of TMPs in this animal model and is a promising alternative to tympanoplasty.

Atypical radiographic features of skull base cholesterol granuloma

Cholesterol granulomas (CGs) are the most common benign lesions of the petrous apex (PA) and have distinct computed tomography (CT) and magnetic resonance imaging (MRI) characteristics. On CT, CGs of the PA (PACG) present as expansile lesions with erosion of bony trabeculae. MRI shows a hyperintense lesion on T1-and T2-weighted images and do not enhance with gadolinium. The objective is to describe the radiographic features of CGs of the skull base that do not arise from the PA. This study is a retrospective review. Three patients were operated on for suspected recurrent endolymphatic sac tumor, intracranial cholesteatoma, and recurrent sphenoid wing meningioma based on CT and MRI findings. Pathology results were consistent with CG in all three cases. All patients had bone erosion on CT. These skull base CGs did not demonstrate similar MRI features. These lesions were hyperintense, iso-to-hyperintense, and hypointense on T1-weighted MRI, respectively. These CGs were hyperintense in two cases and iso-to-hyperintense in one case on T2-weighted MRI. These lesions either demonstrated central or rim enhancement after gadolinium administration. Skull base CGs that do not arise from the PA demonstrate a broad spectrum of radiographic characteristics on MRI that are not typical of PACG.

Dexamethasone Protects Against Apoptotic Cell Death of Cisplatin-exposed Auditory Hair Cells In Vitro.

Hypothesis: Dexamethasone (DXM) protects against cisplatin-induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants in vitro by reducing levels of oxidative stress and NADPH-Oxidase-3 (NOX-3). Background: Intratympanic DXM has demonstrated protective effects against cisplatin-induced hearing loss in a few animal studies and one clinical trial. However, levels of protection with intratympanic DXM vary significantly between studies, which may not be a result of the intrinsic properties of DXM but rather reflect the diffusion of DXM into the cochlea. The molecular mechanisms and degree of DXM protection against cisplatin ototoxicity are currently unknown. Methods: OC explants from 3-day-old rats were cultured with no treatment or various concentrations of cisplatin (2, 5, or 10 &mgr;M) and DXM (75, 150, or 300 &mgr;g/mL) in vitro. HC viability and TUNEL assay were performed after 72 hours in vitro and levels of oxidative stress and NOX-3 were evaluated with confocal microscopy after 48 hours in vitro. Analysis of variance with Tukeys post hoc testing was performed. Results: Cisplatin initiated dose-dependent losses of outer HCs (OHCs) in the basal turns of exposed explants (p < 0.001). DXM protected against cisplatin (2 &mgr;M)-induced OHC loss in a dose-dependent manner with complete protection at 300 &mgr;g/mL of DXM (p < 0.001). DXM (150 &mgr;g/mL) significantly reduced levels of oxidative stress, NOX-3, and apoptosis in the basal turn of explants exposed to cisplatin (2 &mgr;M). Conclusions: DXM protects against cisplatin-induced loss of OHCs in the basal turn of rat OC explants as demonstrated by reductions in oxidative stress and NOX-3 production and decreased levels of apoptotic cell death.

Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr-/- mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.

Predicting depth of electrode insertion by cochlear measurements on computed tomography scans

To evaluate the effectiveness of cochlear measures obtained by high‐resolution computed tomography (HRCT) scan in predicting depth of cochlear implant insertion.

Pathophysiology of Olfactory Disorders and Potential Treatment Strategies.

Olfactory disorders have been regarded in the past with a sense of therapeutic nihilism. However, there have been remarkable advances in chemosensory research over the past several years. The clinical importance of olfactory disorders is well established, and entities such as presbyosmia have gained considerable broad attention. Powerful basic science experimental approaches have revealed aspects of olfactory neuron physiology, olfactory tissue maintenance, and regeneration that provide new potential therapeutic targets for certain forms of olfactory dysfunction. Although many recent advances remain in pre-clinical stages, there is considerable reason for optimism regarding future approaches for treatment of patients with olfactory loss.

Dexamethasone Protects Against Radiation-induced Loss of Auditory Hair Cells In Vitro.

Hypothesis: Dexamethasone (DXM) protects against radiation-induced loss of auditory hair cells (HCs) in rat organ of Corti (OC) explants by reducing levels of oxidative stress and apoptosis. Background: Radiation-induced sensorineural hearing loss (HL) is progressive, dose-dependent, and irreversible. Currently, there are no preventative therapeutic modalities for radiation-induced HL. DXM is a synthetic steroid that can potentially target many of the pathways involved in radiation-induced ototoxicity. Methods: Whole OC explants were dissected from 3-day-old rat cochleae exposed to specific dosages of single-fraction radiation (0, 2, 5, 10, or 20 Gy), were either untreated or treated with DXM (75, 150, 300 &mgr;g/mL), and then cultured for 48 or 96 hours. Confocal microscopy for oxidative stress (CellRox, 48 h) and apoptosis (TUNEL assay, 96 h) and fluorescent microscopy for viable HC counts (fluorescein isothiocyanate-phalloidin, 96 h) were performed. Analysis of variance and Tukey post hoc testing were used for statistical analysis. Results: Radiation exposure initiated dose-dependent losses of inner and outer HCs, predominantly in the basal turns of the OC explants. DXM protected against radiation-induced HC losses in a dose-dependent manner. DXM significantly reduced levels of oxidative stress and apoptosis in radiation-injured OC explants (p < 0.001). Conclusions: Radiation-initiated HC losses were dose-dependent in OC explants. DXM treatment protected explant HCs against radiation-initiated losses by decreasing the levels of oxidative stress and apoptosis. DXM may potentially be a therapeutic modality for preventing radiation-induced HL; further in vivo studies are necessary.

Trauma, Inflammation, Cochlear Implantation Induced Hearing Loss and Otoprotective Strategies to Limit Hair Cell Death and Hearing Loss

Hair cells are highly sensitive units that in response to trauma, inflammation, and cochlear implantation activate different signaling pathways leading to hair cell death and hearing impairment. In this chapter we discuss the most recent literature regarding signaling pathways of hair cell loss, mechanisms and inflammatory responses after noise exposure and electrode insertion, and otoprotective strategies that can limit hair cell death and hearing loss.