Diane M. Prieskorn

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line‐derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad‐GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad‐GDNF or ES or both. Ad‐GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES‐treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad‐GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES‐treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF‐ and ES‐mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. J. Comp. Neurol. 454:350–360, 2002.

Deafferentiation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3‐week‐delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate‐ and delayed‐treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation‐induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin‐positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers. J. Comp. Neurol. 507:1602–1621, 2008.

Glutamatergic Neuronal Differentiation of Mouse Embryonic Stem Cells after Transient Expression of Neurogenin 1 and Treatment with BDNF and GDNF: In Vitro and In Vivo Studies

Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50–75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.

Technical report: Chronic and acute intracochlear infusion in rodents

As a follow-up to the Brown et al., 1993 technique, we have made several improvements to the micro-cannula, osmotic pump procedure, enabling chronic intracochlear infusions while maintaining hearing. In addition, acute bolus injection techniques are briefly described in guinea pig, rat and mouse.

Delayed neurotrophin treatment following deafness rescues spiral ganglion cells from death and promotes regrowth of auditory nerve peripheral processes: Effects of brain-derived neurotrophic factor and fibroblast growth factor

The extent to which neurotrophic factors are able to not only rescue the auditory nerve from deafferentation‐induced degeneration but also promote process regrowth is of basic and clinical interest, as regrowth may enhance the therapeutic efficacy of cochlear prostheses. The use of neurotrophic factors is also relevant to interventions to promote regrowth and repair at other sites of nerve trauma. Therefore, auditory nerve survival and peripheral process regrowth were assessed in the guinea pig cochlea following chronic infusion of BDNF + FGF1 into scala tympani, with treatment initiated 4 days, 3 weeks, or 6 weeks after deafferentation from deafening. Survival of auditory nerve somata (spiral ganglion neurons) was assessed from midmodiolar sections. Peripheral process regrowth was assessed using pan‐Trk immunostaining to selectively label afferent fibers. Significantly enhanced survival was seen in each of the treatment groups compared to controls receiving artificial perilymph. A large increase in peripheral processes was found with BDNF + FGF1 treatment after a 3‐week delay compared to the artificial perilymph controls and a smaller enhancement after a 6‐week delay. Neurotrophic factor treatment therefore has the potential to improve the benefits of cochlear implants by maintaining a larger excitable population of neurons and inducing neural regrowth.

Effects of hepatic arterial yttrium 90 glass microspheres in dogs

A 22‐μm glass microsphere called TheraSphere (Theragenics Corp., Atlanta, GA) has been developed in which yttrium 89 oxide is incorporated into the glass matrix and is activated by neutron bombardment to form the beta‐emitting isotope yttrium 90 (Y 90) before using the spheres as radiotherapeutic vehicles. The injection of up to 12 times (on a liver weight basis) the anticipated human dose of nonradioactive TheraSphere into the hepatic arteries of dogs was well tolerated and produced clinically silent alterations within centrolobular areas. The hepatic arterial (HA) injection of radioactive TheraSphere also produced portal changes similar to those observed in humans after external beam therapy. While the extent of damage increased with the delivered dose, radiation exposures in excess of 30,000 cGy did not cause total hepatic necrosis and were compatible with survival. No microspheres distributed to the bone marrow and absolutely no myelosuppression was encountered in any animal. Proposed hepatic exposures to humans of 5000 to 10,000 cGy by means of these microspheres, therefore, would appear to be feasible and tolerable. Radiotherapeutic microsphere administration preceded by regional infusion of a radiosensitizing agent and/or immediately following the redistribution of blood flow toward intrahepatic tumor by vasoactive agents can potentially yield a synergistic, highly selective attack on tumors confined to the liver.

KHRI-3 monoclonal antibody-induced damage to the inner ear : antibody staining of nascent scars

Intracochlear infusion of the KHRI-3 monoclonal antibody results in in vivo binding to guinea pig inner ear supporting cells, loss of hair cells and hearing loss. To further characterize the basis for KHRI-3-induced hearing loss, antibody was produced in a bioreactor in serum-free medium, affinity purified, and compared to conventionally prepared antibody by infusion into the scala tympani using mini-osmotic pumps. In vivo antibody binding was observed in 10 of 11 guinea pigs. A previously unreported pattern of KHRI-3 antibody binding to cells involved in scar formation was noted in five guinea pigs. All but one of the KHRI-3-infused animals demonstrated a hearing loss of > 10 dB in the treated ear. In five of 11 animals the threshold shift was 30 dB or more, and all had hair cell losses. In one guinea pig infused with 2 mg/ml of antibody, the organ of Corti was absent in the basal turn of the infused ear. This ear had a 45-50 dB threshold shift but, curiously, no detectable antibody binding in the residual organ of Corti. Organ of Corti tissue was fragile in antibody-infused ears. Breaks within the outer hair cell region occurred in 5/11 infused ears. The contralateral ears were normal except for one noise-exposed animal that demonstrated hair cell loss in the uninfused ear. Three animals were exposed to 6 kHz noise (108 dB) for 30 min on day 7. Antibody access to the organ of Corti may be increased in animals exposed to noise, since the strongest in vivo binding was observed in noise-exposed animals. Loss of integrity of the organ of Corti seems to be the primary mechanism of inner ear damage by KHRI-3 antibody. The binding of KHRI-3 antibody in new scars suggests a role of the antigen in scar formation. Antibodies with binding properties similar to KHRI-3 have been detected in 51% of patients diagnosed with autoimmune sensorineural hearing loss; thus, it seems likely that such autoantibodies also may have pathologic effects resulting in hearing loss in humans.

In vivo binding and hearing loss after intracochlear infusion of KHRI-3 antibody

The IgG1 mouse monoclonal antibody (MAb) KHRI-3, binds to an antigen of 65-68 kDa expressed on inner ear supporting cells in guinea pigs. We previously showed [Nair et al. (1995) Monoclonal antibody induced hearing loss. Hear. Res. 83, 101-113] that mice carrying the KHRI-3 hybridoma develop high frequency hearing loss and loss of hair cells in the basal turn suggesting that this MAb causes immune-mediated sensorineural hearing loss. To evaluate the specificity of this effect, sterile KHRI-3 and control IgG1 preparations were infused directly into the guinea pig cochlea using Alzet mini-osmotic pumps. Assessments included: (1) hearing, measured by click auditory brain stem responses (ABRs); (2) in vivo antibody binding; and (3) the structural integrity of the organ of Corti. Nine animals were infused with KHRI-3 preparations and 5 controls were infused with control IgG1. Four guinea pigs given KHRI-3 developed 25-55 dB hearing loss. Control animals showed no difference from baseline. In vivo binding of KHRI-3 was detected in the organ of Corti in 6 of the 9 animals, including all 4 that had hearing loss. No staining was observed with control antibody. Confocal microscopy revealed that the in vivo KHRI-3 antibody binding pattern was identical to that obtained by incubating fixed tissue in vitro with KHRI-3. Histologic examination revealed an increased frequency of hair cell loss in KHRI-3 treated ears when compared to either the contralateral ears of the same guinea pigs or the IgG1 treated ears of control animals. The lesions in the infused ears of guinea pigs were scattered throughout the cochlea from base to apex. These experiments demonstrate the following points: (1) Antibodies can be chronically infused directly into the cochlea of living animals. (2) The KHRI-3 antibody binds to live supporting cells within the organ of Corti. (3) Infusion of an inner ear specific antibody affects auditory function. (4) The infusion of irrelevant antibody had no effect on the structure or function of the ear. This system provides an animal model for further studies of antibody-induced sensorineural hearing loss.

Functional evaluation of a cell replacement therapy in the inner ear

Hypothesis: Cell replacement therapy in the inner ear will contribute to the functional recovery of hearing loss. Background: Cell replacement therapy is a potentially powerful approach to replace degenerated or severely damaged spiral ganglion neurons. This study aimed at stimulating the neurite outgrowth of the implanted neurons and enhancing the potential therapeutic of inner ear cell implants. Methods: Chronic electrical stimulation (CES) and exogenous neurotrophic growth factor (NGF) were applied to 46 guinea pigs transplanted with embryonic dorsal root ganglion (DRG) neurons 4 days postdeafening. The animals were evaluated with the electrically evoked auditory brainstem responses (EABRs) at experimental Days 7, 11, 17, 24, and 31. The animals were euthanized at Day 31, and the inner ears were dissected for immunohistochemistry investigation. Results: Implanted DRG cells, identified by enhanced green fluorescent protein fluorescence and a neuronal marker, were found close to Rosenthal canal in the adult inner ear for up to 4 weeks after transplantation. Extensive neurite projections clearly, greater than in nontreated animals, were observed to penetrate the bony modiolus and reach the spiral ganglion region in animals supplied with CES and/or NGF. There was, however, no significant difference in the thresholds of EABRs between DRG-transplanted animals supplied with CES and/or NGF and DRG-transplanted animals without CES or NGF supplement. Conclusion: The results suggest that CES and/or NGF can stimulate neurite outgrowth from implanted neurons, although based on EABR measurement, these interventions did not induce functional connections to the central auditory pathway. Additional time or novel approaches may enhance functional responsiveness of implanted cells in the adult cochlea.

Mechanism of electrical stimulation-induced neuroprotection: effects of verapamil on protection of primary auditory afferents☆

In order to assess the role of L-type voltage-gated calcium channels in electrical stimulation-mediated neuroprotection in vivo, we assessed survival of primary auditory afferents (spiral ganglion cells) in systemically deafened guinea pigs following chronic electrical stimulation with or without intracochlear infusion of verapamil, an L-type voltage-gated calcium channel antagonist. Continuous intracochlear drug delivery (0.5 microl/h) was provided using a delivery system developed previously in our laboratory using Alzet mini-osmotic pumps. In the absence of chronic stimulation, spiral ganglion cell survival was relatively symmetric in animals treated unilaterally with either artificial perilymph or verapamil (50 microg/ml). In the presence of unilateral chronic electrical stimulation, spiral ganglion cell survival was significantly greater in stimulated, perilymph-infused ears, relative to the contralateral ear. In contrast, spiral ganglion cell survival was bilaterally symmetric in chronically stimulated, verapamil-infused animals. The difference in symmetry of spiral ganglion cell survival between the two groups was statistically significant. In vitro, passive depolarization has been demonstrated to enhance survival of cultured neurons via activation of L-type calcium channels. The results of this study indicate that, as suggested by in vitro depolarization models, in vivo electrical stimulation-mediated neuroprotection requires the activation of L-type voltage-gated calcium channels. Chronic electrical stimulation of the deaf ear is an ideal preparation for further studies in which to extrapolate findings from in vitro depolarization models.