Chhavi Gupta

Molecular mechanisms involved in cochlear implantation trauma and the protection of hearing and auditory sensory cells by inhibition of c-jun-N-terminal kinase signaling

To investigate the molecular mechanisms involved in electrode insertion trauma (EIT) and to test the otoprotective effect of locally delivered AM‐111.

The Cochlear Implant: Historical Aspects and Future Prospects

The cochlear implant (CI) is the first effective treatment for deafness and severe losses in hearing. As such, the CI is now widely regarded as one of the great advances in modern medicine. This article reviews the key events and discoveries that led up to the current CI systems, and we review and present some among the many possibilities for further improvements in device design and performance. The past achievements include: (1) development of reliable devices that can be used over the lifetime of a patient; (2) development of arrays of implanted electrodes that can stimulate more than one site in the cochlea; and (3) progressive and large improvements in sound processing strategies for CIs. In addition, cooperation between research organizations and companies greatly accelerated the widespread availability and use of safe and effective devices. Possibilities for the future include: (1) use of otoprotective drugs; (2) further improvements in electrode designs and placements; (3) further improvements in sound processing strategies; (4) use of stem cells to replace lost sensory hair cells and neural structures in the cochlea; (5) gene therapy; (6) further reductions in the trauma caused by insertions of electrodes and other manipulations during implant surgeries; and (7) optical rather electrical stimulation of the auditory nerve. Each of these possibilities is the subject of active research. Although great progress has been made to date in the development of the CI, including the first substantial restoration of a human sense, much more progress seems likely and certainly would not be a surprise. Anat Rec, 2012.

Efficacy of three drugs for protecting against gentamicin-induced hair cell and hearing losses

BACKGROUND AND PURPOSE Exposure to an ototoxic level of an aminoglycoside can result in hearing loss. In this we study investigated the otoprotective efficacy of dexamethasone (DXM), melatonin (MLT) and tacrolimus (TCR) in gentamicin (GM)‐treated animals and cultures.

A Novel Organ of Corti Explant Model for the Study of Cochlear Implantation Trauma

This study presents a novel in vitro model of electrode insertion trauma‐induced hair cell (HC) damage and loss and its application for testing the efficacy of otoprotective drugs. In the cochlear implant (CI) procedure as a treatment for profound deafness, an electrode array is surgically inserted to provide electrical stimulation to the auditory nerve. Mechanical trauma from insertion of a CI electrode into the scala tympani can lead to inflammation and a high level of oxidative stress, which can initiate the apoptosis of auditory HCs and intracochlear fibrosis. HC apoptosis and intracochlear fibrosis are thought to be causes of poor CI functional outcomes. In order to gain insight into the molecular mechanisms that initiate HC apoptosis and scala tympani fibrosis following electrode insertion trauma (EIT), and the otoprotective effects of dexamethasone (DXM) observed in previous studies, an in vitro model of EIT was designed. Here we present and characterize a novel, reproducible in vitro model for the study of cellular and molecular events that occur following an EIT procedure. Cochleae from 3‐day‐old rats were subjected to a cochleostomy and were then divided into three groups: (1) control, (2) EIT, and (3) EIT + DXM (20 μg/mL). In Groups 2 and 3, a 0.28‐mm diameter monofilament fishing line was introduced through the small cochleostomy located next to the round window area, allowing for an insertion of between 110° and 150°. HC counts, gene expression for pro‐inflammatory cytokines (i.e., TNFα and IL‐1β), pro‐inflammatory inducible enzymes (i.e., iNOS and COX‐2) and growth factors (i.e., TGFβ1, TGFβ3 and CTGF), oxidative stress (i.e., CellROX), and analyses of apoptosis pathways (i.e., caspase‐3, apoptosis induced factor and Endonuclease G) were carried out on all explants at different time points. The results of this EIT in vitro model show the initiation of wound healing in which an inflammatory response is followed by a proliferative‐fibrosis phase. Moreover, DXM treatment of EIT explants inhibited the inflammatory response and promoted a nonscarring wound healing process. The novel in vitro model described here will improve our understanding of mechanisms underlying CI insertion trauma and protective strategies such as DXM treatment. Anat Rec, 2012.

Pulsed infrared radiation excites cultured neonatal spiral and vestibular ganglion neurons by modulating mitochondrial calcium cycling.

Cochlear implants are currently the most effective solution for profound sensorineural hearing loss, and vestibular prostheses are under development to treat bilateral vestibulopathies. Electrical current spread in these neuroprostheses limits channel independence and, in some cases, may impair their performance. In comparison, optical stimuli that are spatially confined may result in a significant functional improvement. Pulsed infrared radiation (IR) has previously been shown to elicit responses in neurons. This study analyzes the response of neonatal rat spiral and vestibular ganglion neurons in vitro to IR (wavelength = 1,863 nm) using Ca(2+) imaging. Both types of neurons responded consistently with robust intracellular Ca(2+) ([Ca(2+)]i) transients that matched the low-frequency IR pulses applied (4 ms, 0.25-1 pps). Radiant exposures of ∼637 mJ/cm(2) resulted in continual neuronal activation. Temperature or [Ca(2+)] variations in the media did not alter the IR-evoked transients, ruling out extracellular Ca(2+) involvement or primary mediation by thermal effects on the plasma membrane. While blockage of Na(+), K(+), and Ca(2+) plasma membrane channels did not alter the IR-evoked response, blocking of mitochondrial Ca(2+) cycling with CGP-37157 or ruthenium red reversibly inhibited the IR-evoked [Ca(2+)]i transients. Additionally, the magnitude of the IR-evoked transients was dependent on ryanodine and cyclopiazonic acid-dependent Ca(2+) release. These results suggest that IR modulation of intracellular calcium cycling contributes to stimulation of spiral and vestibular ganglion neurons. As a whole, the results suggest selective excitation of neurons in the IR beam path and the potential of IR stimulation in future auditory and vestibular prostheses.

Biomedical Engineering Principles of Modern Cochlear Implants and Recent Surgical Innovations

This review covers the most recent clinical and surgical advances made in the development and application of cochlear implants (CIs). In recent years, dramatic progress has been made in both clinical and basic science aspect of cochlear implantation. Todays modern CI uses multi‐channel electrodes with highly miniaturized powerful digital processing chips. This review article describes the function of various components of the modern multi‐channel CIs. A selection of the most recent clinical and surgical innovations is presented. This includes the preliminary results with electro‐acoustic stimulation or hybrid devices and ongoing basic science research that is focused on the preservation of residual hearing post‐implantation. The result of an original device that uses a binaural stimulation mode with a single implanted receiver/stimulator is also presented. The benefit and surgical design of a temporalis pocket technique for the implants receiver stimulator is discussed. Advances in biomedical engineering and surgical innovations that lead to an increasingly favorable clinical outcome and to an expansion of the indication of CI surgery are presented and discussed. Anat Rec, 2012.

Mechanisms of programmed cell death signaling in hair cells and support cells post-electrode insertion trauma.

Abstract Conclusion: Programmed cell death (PCD) initially starts in the support cells (SCs) after electrode insertion trauma (EIT), followed by PCD in hair cells (HCs). Activation of caspase-3 was observed only in SCs. Protecting both SCs and HCs with selective otoprotective drugs at an early stage post implantation may help to preserve residual hearing. Objectives: Cochlear implant EIT can initiate sensory cell losses via necrosis and PCD within the organ of Corti, which can lead to a loss of residual hearing. PCD appears to be a major factor in HC loss post-EIT. The current study aimed to: (1) determine the onset of PCD in both SCs and HCs within the traumatized organ of Corti; and (2) identify the molecular mechanisms active within the HCs and SCs that are undergoing PCD. Methods: Adult guinea pigs were assigned to one of two groups: (1) EIT and (2) unoperated contralateral ears as controls. Immunostaining of dissected organ of Corti surface preparations for phosphorylated-Jun, cleaved caspase-3, and 4-hydroxy-2,3-nonenal (HNE) were performed at 6, 12, and 24 h post-EIT and for contralateral control ears. Results: At 6 h post-EIT the SCs immunolabeled for the presence of phosphorylated-Jun and activated caspase-3. Phosphorylated p-Jun labeling was observed at 12 h in both the HCs and SCs of middle and basal cochlear turns. Cleaved caspase-3 was not observed in HCs of any cochlear turn at up to 24 h post-EIT. Lipid peroxidation (HNE immunostaining) was first observed at 12 h post-EIT in both the HCs and SCs of the basal turn, and reached the apical turn by 24 h post-EIT.

Otoprotective properties of mannitol against gentamicin induced hair cell loss.

Background Gentamicin is a widely used antibiotic, which causes hearing loss because of destruction of auditory hair cells. Mannitol has been shown to have cytoprotective properties in the cochlea both in vitro and in vivo. Mannitol has been shown to be safe in concentrations up to 100 mM in organ of Corti explants. It is proposed as an otoprotective agent against gentamicin ototoxicity. Methods Organ of Corti were dissected from P-3 rat pups and cultured under the following conditions for 96 hours: 1) control, 2) gentamicin (10 &mgr;M for all hair cell count experiments), 3) gentamicin + mannitol 10 mM, 4) gentamicin + mannitol 50 mM, and 5) gentamicin + mannitol 100 mM. The tissues were then fixed and stained, and hair cells were counted for segments of the apex, middle, and basal turns. Quantitative RT-PCR (qRT-PCR) was performed on organ of Corti explant extracted RNA after 24 hours in vitro: 1) control; 2) gentamicin (100 &mgr;M for all gene expression and CellRox experiments); 3) gentamicin +mannitol 100 mM; and 4) mannitol 100 mM for tumor necrosis factor–alpha (TNF-&agr;), TNF-&agr; receptor (TNFR1A), interleukin-1 beta (IL-1&bgr;) and cyclooxygenase-2 (COX-2). In vitro examination of oxidative stress was performed for the same test groups at 24 hours using CellRox Deep Red assay. Results Gentamicin induced loss of both inner hair cells and outer hair cells with increasing severity from apex to middle to basal segments (Pearson r = −0.999 for inner hair cells and −0.972 for outer hair cells). Mannitol demonstrated dose-dependent otoprotection of IHCs and outer hair cells (p < 0.001 for mannitol at 100 mM). CellRox demonstrated increased oxidative stress induced by gentamicin exposure, and this effect was attenuated by treatment of gentamicin-exposed explants with mannitol (p < 0.05). TNF-&agr;, IL-1&bgr; TNFR1A, and COX-2 mRNA levels were upregulated by gentamicin (p < 0.05). Mannitol treatment of gentamicin explants downregulated the gene expression of the proinflammatory cytokines, but this difference did not achieve significance. Interestingly, in gentamicin-challenged organ of Corti explants, Mannitol upregulated the expression of TNFR1A, but this increase did not achieve significance (p > 0.05). Conclusion Gentamicin ototoxicity is increasingly severe from the apex to basal turn of the cochlea. Treatment with mannitol prevents gentamicin-induced hair cell loss in a dose-dependent manner, protecting both IHCs and outer hair cells. Mannitol appears to act as a free radical scavenger to reduce the cytotoxic effects of gentamicin by reducing the level of oxidative stress.

Mannitol protects hair cells against tumor necrosis factor α-induced loss.

Hypothesis Mannitol has otoprotective effects against tumor necrosis factor (TNF) &agr;–induced auditory hair cell (HC) loss. Background Mannitol has been demonstrated to possess cytoprotective effects in several organ systems. Its protective effect on postischemic hearing loss has also been shown. Mannitol’s otoprotective mechanism and site of action are at present unknown. Materials and Methods Organ of Corti (OC) explants were dissected from 3 day-old rat pups. The safety (nonototoxicity) of mannitol was assessed at 4 different concentrations (1–100 mM). Three experimental arms were designed including: a control group, TNF&agr; group, and TNF&agr; + mannitol group. Cell viability was determined by counts of fluorescein isothiocyanate (FITC) phalloidin stained HC. Immunofluorescence assay of phospho-c-Jun and the proapoptotic mediators, cleaved caspase-3, apoptosis inducing factor (AIF), and endonuclease G (Endo G) were performed. Results Analysis of HC density confirmed the safety of mannitol at concentration ranges of 1 to 100 mM. The ototoxic effect of TNF&agr; was demonstrated (p < 0.05). The otoprotective effect of 100 mM mannitol in TNF&agr;-challenged OC explants was also demonstrated (p < 0.001). Mannitol treatment reduced the high levels of phospho-c-Jun observed in the TNF&agr;-challenged group. AIF cluster formation and EndoG translocation into the nuclei of HCs were also reduced by mannitol treatment. Conclusion Mannitol significantly reduces the ototoxic effects of TNF&agr; against auditory HC’s potentially by inhibiting c-Jun N terminal kinase (JNK) activation pathway and AIF, EndoG nuclear translocation. This local otoprotective effect may have therapeutic implications in inner ear surgery, for example, cochlear implants, protection of residual hearing, as well as implications for postischemic inner ear insults.

Hypoxia and kinase activity regulate lung epithelial cell glutathione

ABSTRACT The authors investigated the mechanisms by which hypoxia regulates glutathione (GSH) in lung epithelial cells, and specifically whether the mitogen-activated protein kinase (MAPK) system is involved in the response to hypoxia. Hypoxia decreased cellular GSH content and appeared to decrease the effect of N-acetylcysteine on repletion of GSH after hypoxia. Hypoxia decreased 2 key enzyme activities that regulate GSH synthesis, glutamate cysteine ligase (GCL) (E.C. 6.3.2.2) and glutathione synthase (GS) (E.C. 6.3.2.3). No hypoxia-dependent change occurred in GCL or GS protein expression on Western blots. When epithelial cells were transfected with an adenoviral vector that caused over expression of human catalase protein (Ad.Cat or Ad.mCat), GCL and GS activities did not decrease in hypoxia. Inhibition of p38MAPK (using SB203580) or extracellular signal-regulated kinase (ERK; PD98059) prevented the hypoxia-dependent decrease in GCL and GS activity. To seek in vivo correlation, the authors assayed total glutathione in lungs and livers from MK2−/− (homozygous knockout) mice. MK2−/− mice are presumably unable to phosphorylate heat shock protein 27 (Hsp27) normally, because of absent kinase (MK2) activity. Liver GSH content (expressed per mg protein) was 20%% less in MK2−/− mice than in nontransgenic Black 6 controls. Down-regulation of lung GSH content in hypoxia depends on peroxide tone of the cell and the p38MAPK system.