Anette Fransson

Morphological and functional preservation of the outer hair cells from noise trauma by sound conditioning

Guinea pigs were sound conditioned to a low-level, long-term pure tone stimulus (1 kHz, 81 dB SPL, 24 days) before exposure to a traumatic noise (1 kHz, 105 dB SPL, 72 h). Auditory brainstem response thresholds and distortion product otoacoustic emissions were obtained at selected frequencies before sound conditioning and at day 1, 5, 10, and 15 during sound conditioning as well as on the final 24th day. Auditory brainstem responses at 1 and 2 kHz were not affected at any time during sound conditioning. The amplitude of the distortion product otoacoustic emission showed minor alterations (below 10 dB) at selected frequencies only during the initial stages (day 1, 5, and 10) of sound conditioning in some, but not all the animals. Distortion product amplitudes were similar to control values on the 15th and 24th day of conditioning. Surface preparations of the organ of Corti did not reveal any significant hair cell loss induced by sound conditioning. The effect of a traumatic exposure (1 kHz, 105 dB SPL, 72 h) on a control group and a sound conditioned group was determined. The distortion product otoacoustic emission amplitude measured 4 weeks after the cessation of the traumatic exposure revealed significant differences. The amplitude of the distortion product otoacoustic emission for the control group was depressed at all tested frequencies and at lower frequencies (2.8, 2.1, and 1.75 kHz) the emissions did not show an increase in response to increases in intensity, of the primaries. The sound conditioned group showed increases in distortion product amplitude with increases in the intensity of the primaries for all tested frequencies and statistically significant reductions from the pre-exposure values were not found. Surface preparations from the control group indicated that the traumatic noise exposure affected nearly 100% of the outer hair cells around the 14 mm distance from the round window. The sound conditioned group showed a significantly less (50%) outer hair cell loss than the control group. The sound conditioned group illustrated an altered pattern of damage after subsequent noise trauma. There were two distinct regions of outer hair cell loss, one being around the 16 mm distance and the other around the 12 mm distance from the round window. These results imply that the intrinsic properties of the outer hair cells and/or the organ of Corti have been altered by sound conditioning.

Post-Treatment Effects of Local GDNF Administration to the Inner Ears of Deafened Guinea Pigs

For patients with profound hearing loss, a cochlear implant is the only treatment available today. The function of a cochlear implant depends in part on the function and survival of spiral ganglion neurons. Following deafferentation, glial cell-derived neurotrophic factor (GDNF) is known to affect spiral ganglion neuron survival. The purpose of this study was to assess delayed GDNF treatment after deafening, the effects of cessation of GDNF treatment, and the effects of subsequent antioxidants on responsiveness and survival of the spiral ganglion neurons. Three-week deafened (by local neomycin administration) guinea pigs were implanted in the scala tympani with a combined cochlear implant electrode and cannula. GDNF (1 μg/mL) or artificial perilymph was then delivered for 4 weeks, following which the animals received systemic ascorbic acid  +  Trolox or saline for an additional 4 weeks. Thresholds for electrically-evoked auditory brain stem responses (eABRs) were significantly elevated at 3 weeks with deafness, stabilized with GDNF, and showed no change with GDNF cessation and treatment with antioxidants or saline. The populations of spiral ganglion neurons were reduced with deafness (by 40% at 3 weeks and 70% at 11 weeks), and rescued from cell death by GDNF with no further reduction at 8 weeks following 4 weeks of cessation of GDNF treatment equally in both the antioxidant- and saline-treated groups. Local growth factor treatment of the deaf ear may prevent deterioration in electrical responsiveness and rescue auditory nerve cells from death; these effects outlast the period of treatment, and may enhance the benefits of cochlear implant therapy for the deaf.

Reducing noise damage by using a mid-frequency sound conditioning stimulus.

SOUND conditioning guinea pigs to a 6.3 kHz tone at 78 dB SPL for either 13 or 24 days provides significant physiological (auditory brain stem responses, ABR; and distortion product otoacoustic emissions, DPOAE) and morphological (cochleograms) protection against a subsequent traumatic exposure (6.3 kHz, 100 dB SPL for 24 h) delivered 2 h after sound conditioning. Threshold shifts (ABR, DPOAE) were significantly reduced and the degree of hair cell loss was minimal. When a 1 week pause was given between the end of the sound conditioning and the traumatic exposure, protection was still observed, but to a lesser degree. These findings demonstrate that mid-frequency sound conditioning protects against noise trauma and that the protective effect is maintained for at least 1 week.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.

Medial olivocochlear efferent terminals are protected by sound conditioning

Synaptophysin immunoreactivity was used as a marker for the olivocochlear efferent system that innervates the outer hair cells of the cochlea. An intense noise exposure at either 6.3 kHz or 1.0 kHz caused a significant reduction in anti-synaptophysin immunoreactivity within the 8-6 mm or 14-11 mm distance from the round window, respectively. In the region of the main lesion, the reduction in synaptophysin immunoreactivity for both the 6.3 and 1.0 kHz exposures correlated well with outer hair cell loss. In regions peripheral to the main lesion, some remnants of efferent nerve endings could remain even when their associated outer hair cells were missing. Pre-treatment with a low level sound conditioner (either at 6.3 tone or 1.0 kHz) effectively reduced the efferent and outer hair cell pathology induced by the 6.3 and 1.0 kHz intense noise exposures, respectively. The results demonstrate the feasibility of using anti-synaptophysin immunoreactivity as an effective means of quantifying pathological alterations to the medial cochlear efferent terminals throughout the cochlea. Furthermore, the results show that sound conditioning significantly reduces damage to the efferent terminals.

In vivo infusion of UTP and uridine to the deafened guinea pig inner ear: effects on response thresholds and neural survival.

Nucleotides and nucleosides are known to function as neurotransmitters and neuromodulators but have recently been shown to have a trophic effect on neurons. It has previously been shown, in an animal model for cochlear implants, that local infusion of neurotrophic factors intervenes with the degenerative processes occurring after deafening and protects the auditory spiral ganglion neurons so that electrical responsiveness is maintained. Here we test the hypothesis that nucleosides and nucleotides have a similar effect on the acutely damaged inner ear. Pigmented guinea pigs received a cochlear implant electrode for measuring electrically evoked auditory brainstem responses and a miniosmotic pump for delivering drugs directly to the cochlea. The animals were deafened by a 48‐hr infusion with 10% neomycin, followed by 23 days of treatment with primarily UTP, uridine nucleotides, or as control artificial perilymph. Electrically evoked responses were measured weekly, and at the end of the experiment the cochleae were collected and processed for morphological analysis and spiral ganglion neuron counting. Both UTP‐ and uridine‐treated groups showed significantly better response after 23 days of treatment compared with the control group. The densities of spiral ganglion neuron were significantly higher for both treated groups compared with the control group treated with artificial perilymph. The results demonstrate that UTP and uridine rescue auditory neurons and suggest that drugs acting on purinoceptors could be of clinical importance.

Dendrogenin A and B two new steroidal alkaloids increasing neural responsiveness in the deafened guinea pig.

Aim: To investigate the therapeutic potential for treating inner ear damage of two new steroidal alkaloid compounds, Dendrogenin A and Dendrogenin B, previously shown to be potent inductors of cell differentiation. Methods: Guinea pigs, unilaterally deafened by neomycin infusion, received a cochlear implant followed by immediate or a 2-week delayed treatment with Dendrogenin A, Dendrogenin B, and, as comparison artificial perilymph and glial cell-line derived neurotrophic factor. After a 4-week treatment period the animals were sacrificed and the cochleae processed for morphological analysis. Electrically-evoked auditory brainstem responses (eABRs) were measured weekly throughout the experiment. Results: Following immediate or delayed Dendrogenin treatment the electrical responsiveness was significantly maintained, in a similar extent as has been shown using neurotrophic factors. Histological analysis showed that the spiral ganglion neurons density was only slightly higher than the untreated group. Conclusions: Our results suggest that Dendrogenins constitute a new class of drugs with strong potential to improve cochlear implant efficacy and to treat neuropathy/synaptopathy related hearing loss. That electrical responsiveness was maintained despite a significantly reduced neural population suggests that the efficacy of cochlear implants is more related to the functional state of the spiral ganglion neurons than merely their number.

Structural changes in the inner ear over time studied in the experimentally deafened guinea pig.

Today a cochlear implant (CI) may significantly restore auditory function, even for people with a profound hearing loss. Because the efficacy of a CI is believed to depend mainly on the remaining population of spiral ganglion neurons (SGNs), it is important to understand the timeline of the degenerative process of the auditory neurons following deafness. Guinea pigs were transtympanically deafened with neomycin, verified by recording auditory brainstem responses (ABRs), and then sacrificed at different time points. Loss of SGNs as well as changes in cell body and nuclear volume were estimated. To study the effect of delayed treatment, a group of animals that had been deaf for 12 weeks was implanted with a stimulus electrode mimicking a CI, after which they received a 4‐week treatment with glial cell‐derived neurotrophic factor (GDNF). The electrical responsiveness of the SGNs was measured by recording electrically evoked ABRs. There was a rapid degeneration during the first 7 weeks, shown as a significant reduction of the SGN population. The degenerative process then slowed, and there was no difference in the amount of remaining neurons between weeks 7 and 18.

The feasibility of an encapsulated cell approach in an animal deafness model

Abstract For patients with profound hearing loss a cochlear implant (CI) is the only treatment today. The function of a CI depends in part of the function and survival of the remaining spiral ganglion neurons (SGN). It is well known from animal models that inner ear infusion of neurotrophic factors prevents SGN degeneration and maintains electrical responsiveness in deafened animals. The purpose with this study was to investigate the effects of a novel encapsulated cell (EC) device releasing neurotrophic factors in the deafened guinea pig. The results showed that an EC device releasing glial cell line‐derived neurotrophic factor (GDNF) or brain‐derived neurotrophic factor (BDNF) implanted for four weeks in deafened guinea pigs significantly preserved the SGNs and maintained their electrical responsiveness. There was a significant difference between BDNF and GDNF in favour of GDNF. This study, demonstrating positive structural and functional effects in the deafened inner ear, suggests that an implanted EC device releasing biologically protective substances offers a feasible approach for treating progressive hearing impairment. Graphical abstract Figure. No Caption available.

Identification of endothelin-converting enzyme-2 as an autoantigen in autoimmune polyendocrine syndrome type 1

Abstract Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.