Effect of shock wave power spectrum on the inner ear pathophysiology in blast-induced hearing loss.

Blast exposure can induce various types of hearing impairment, including permanent hearing loss, tinnitus, and hyperacusis. Herein, we conducted a detailed investigation of the cochlear pathophysiology in blast-induced hearing loss in mice using two blasts with different characteristics: a low-frequency dominant blast generated by a shock tube and a high-frequency dominant shock wave generated by laser irradiation (laser-induced shock wave). The pattern of sensorineural hearing loss (SNHL) was low-frequency- and high-frequency-dominant in response to the low- and high-frequency blasts, respectively. Pathological examination revealed that cochlear synaptopathy was the most frequent cochlear pathology after blast exposure, which involved synapse loss in the inner hair cells without hair cell loss, depending on the power spectrum of the blast. This pathological change completely reflected the physiological analysis of wave I amplitude using auditory brainstem responses. Stereociliary bundle disruption in the outer hair cells was also dependent on the blast's power spectrum. Therefore, we demonstrated that the dominant frequency of the blast power spectrum was the principal factor determining the region of cochlear damage. We believe that the presenting models would be valuable both in blast research and the investigation of various types of hearing loss whose pathogenesis involves cochlear synaptopathy.

Establishment of a cochlear injury model using bone-conducted ultrasound irradiation in guinea pigs and investigation on peripheral coding and recognition of ultrasonic signals.

The cochlea of guinea pigs was irradiated with different frequencies of bone-conducted ultrasound (BCU) at a specific dose to induce cochlear hair cell-specific injuries, in order to establish frequency-related cochlear hair cell-specific injury models. Cochlear near-field potentials were then evoked using BCU of different frequencies and intensities to explore the peripheral coding and recognition of BCU by the cochlea. The inner ears of guinea pigs were irradiated by 30 kHz at 100 db and 80 kHz at100 db BCU for 6h to create frequency-related, ultrasound-specific cochlear injury models. Then, 30 kHz and 80 kHz BCU of different intensities were used to evoke auditory brainstem response (ABR) thresholds, compound action potential (CAP) thresholds, and action potential (AP) intensity-amplitude input-output curves in the normal control group and the ultrasonic cochlear injury group. This allowed us to explore the coding and recognition of BCU frequencies and intensities by cochlear hair cells. Immunofluorescence assay of outer hair cell (OHC) Prestin and inner hair cell (IHC) Otofelin was performed to verify the injury models. Irradiation of guinea pig inner ears by 30 kHz and 80 kHz BCU at a specific dose induced hair cell injuries at different sites. Irradiation with low frequency BCU mainly induced OHC injury, whereas irradiation with high frequency BCU induced IHC injury; moreover, IHC injury was more serious than OHC injury. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the normal control group. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the 80 kHz ultrasonic cochlear injury group. The difference in the 80 kHz-evoked ABR thresholds were not significant between the 30 kHz and 80 kHz ultrasonic cochlear injury groups. The click- and 30 kHz-evoked AP intensity-amplitude curves for the 30 kHz ultrasonic cochlear injury group indicate that the AP amplitude evoked at the same intensity was higher in the 30 kHz-evoked group than the click-evoked group. The spatial positions of cochlear hair cells in guinea pigs had a coding function for the frequencies of low-frequency ultrasound. OHCs have an amplification effect on the coding of low-frequency ultrasonic intensities. The peripheral perception of high frequency BCU may not require the participation of cochlear hair cells.

[The study on the targets of the optical evoked auditory brainstem response on the cochlea of guinea pig stimulating by infrared laser].

Objective: To identify the targets of the infrared laser stimulating on the cochlea of guinea pig which evoked auditory brainstem response (oABR), and explore the mechanisms of the infrared neurostimulation. Methods: A polished optical fiber with 200 µm diameter (NA=0.22) was planted into the scala tympani of guinea pigs to stimulate the cochlea of both the normal hearing and acute deafened guinea pigs. The direction of the fiber distal was changed to radiate different regions of the scala tympani, recording the oABR respectively. Differences of energy thresholds and amplitudes of oABR between normal hearing and acute deafened animals was concerned, and different responses were recorded as the optical path of laser fiber being changed to investigate the targets of the infrared laser stimulation. Immunofluorescence was used to detect the changes of inner and outer hair cells, and spiral ganglion neurons 7 days post-deafening, to looking for the probable association with the oABR changes at the same stimulus. SPSS 18.0 software was used to analyze the data. Results: Inner and outer hair cells were damaged in basal and middle turn, butresidual hair cells were observed in apical turn.Only when the optical fiber pointed to Rosenthal's canal stimulated the spiral ganglion region directly could the oABR be evoked. No response was recorded while the fiber pointed to other directions. Conclusion: Infrared laser stimulates cochlea evoked oABR generats from the response of spiral ganglion directly, the spiral ganglion neurons are the target of infrared stimulation.

Effects of microbubble size on ultrasound-mediated gene transfection in auditory cells.

Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 µm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 µm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

Short-wavelength near infrared stimulation of the inner ear hair cells.

To explore whether the short wavelength near infrared laser can stimulate the functional hair cells, pulsed laser with wavelength of 808-nm was used to stimulate guinea pigs cochlea. Compound action potential (CAP) and auditory brainstem responses (ABR) were recorded during the experiments. We successfully recorded photomechanical responses from normal hearing animals and demonstrated the responses were not induced by optical acoustic events. Furthermore, we studied the effect of different stimulation parameters on neural response. The results show that cochlear activation can be modulated with different optical parameters.

[Effects of laser-assisted cochleostomy on inner hair cell ribbon synapse in rats].

OBJECTIVE: To investigate the effects on ribbon synapse of inner hair cells after superpulsed CO2 laser-assisted cochleostomy in SD rats. METHODS: Eighteen SD rats were randomly divided into laser-assisted surgery groups (2 W group and 5 W group), sham-operated group and control group. Ten of those were performed a cochleostomy using superpulsed CO2 laser with a corresponding power. Auditory brainstem responses (ABR) were measured pre-and postoperatively. The ribbon synapses at apical and middle cochlear turns were observed under laser scanning confocal microscope and then were quantified with 3ds Max software. RESULTS: The postoperative ABR thresholds of the 2 W and 5 W groups were larger than the preoperative case (t = -5.65, P < 0.01; t = -4.97, P < 0.01). The synapse number at the middle turn decreased significantly in 5 W group (F = 17.15, P < 0.01), while no significant changes were noted at the apical turn (P > 0.05). There was no statistical difference in 2 W group (P > 0.05). CONCLUSIONS: The superpulsed CO2 laser-assisted cochleostomy with high-power is accompanied by a synaptic injury, while no obvious effects after the low-power laser surgery, which might be a safe strategy to preform cochleostomy.

New fin-line devices for radiofrequency exposure of small biological samples in vitro allowing whole-cell patch clamp recordings.

The development and analysis of three waveguides for the exposure of small biological in vitro samples to mobile communication signals at 900 MHz (GSM, Global System for Mobile Communications), 1.8 GHz (GSM), and 2 GHz (UMTS, Universal Mobile Telecommunications System) is presented. The waveguides were based on a fin-line concept and the chamber containing the samples bathed in extracellular solution was placed onto two fins with a slot in between, where the exposure field concentrates. Measures were taken to allow for patch clamp recordings during radiofrequency (RF) exposure. The necessary power for the achievement of the maximum desired specific absorption rate (SAR) of 20 W/kg (average over the mass of the solution) was approximately P(in) = 50 mW, P(in) = 19 mW, and P(in) = 18 mW for the 900 MHz, 1800 MHz, and 2 GHz devices, respectively. At 20 W/kg, a slight RF-induced temperature elevation in the solution of no more than 0.3 °C was detected, while no thermal offsets due to the electromagnetic exposure could be detected at the lower SAR settings (2, 0.2, and 0.02 W/kg). A deviation of 10% from the intended solution volume yielded a calculated SAR deviation of 8% from the desired value. A maximum ±10% variation in the local SAR could occur when the position of the patch clamp electrode was altered within the area where the cells to be investigated were located.

Tonotopic variation in the calcium dependence of neurotransmitter release and vesicle pool replenishment at mammalian auditory ribbon synapses.

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (DeltaC(m)) in low-frequency (apical region, approximately 300 Hz) and high-frequency (basal, approximately 30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca(2+) and body temperature). With maturation, the Ca(2+) efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca(2+) cooperativity of exocytosis despite the smaller DeltaC(m) in apical cells. After maturation, DeltaC(m) in high-frequency IHCs increased linearly with the Ca(2+) current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca(2+) sensor otoferlin or the spatial coupling between Ca(2+) channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

Persistence of Ca(v)1.3 Ca2+ channels in mature outer hair cells supports outer hair cell afferent signaling.

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

Experimental erbium laser surgery in the guinea pig cochlea: its use in the study of afferent cochlear neurotransmitters.

Microiontophoretic techniques were used to examine the changes in activity of inner half cell afferents in the guinea pig following circumscribed penetration of cochlear bone with erbium:YSGG laser pulses. Neuronal responses to the application of the glutamate agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) before laser use and after return to normal spontaneous activity were equivalent, implying total reversibility of the changes occurring. Suppression of laser-induced hair cell activity was possible with NMDA and AMPA receptor antagonists and lasted 10-15 min. These findings suggest a transmitter-related increase of neuronal activity. Our results show that use of the erbium laser in inner ear microsurgery might be possible with low risk if the amount of energy applied is kept under a safe limit of 10 J/cm2.

The influence of prenatal gamma irradiation on the ageing of the cochlea.

Pregnant CBA/Ca mice were subjected to whole-body irradiation on the 12th, 13th or 16th day of gestation with 0.5, 1 or 2 Gy, respectively. Auditory brainstem response (ABR) thresholds in the offspring at one month of age were significantly elevated. The irradiated mice were raised to an age of between 25 and 36 months. Before the animals were killed, the ABR thresholds were again determined and compared with those in age-matched controls. The most pronounced ABR threshold loss at a high age occurred in those animals irradiated on gestation day 12. There was a significant (up to p less than 0.001) potentiation of the age-related loss of ABR threshold in the old irradiated mice. In the scanning electron microscope, both inner and outer hair cells were missing in large numbers in exposed animals, irrespective of irradiation dose. Remaining hair cells and also the pillar cells showed signs of degeneration. In particular, damage observed in the mid-cochlear region was significantly more pronounced in the old irradiated mice than in the old control mice. It is concluded 1) that elevation of ABR thresholds in ageing irradiated animals is due to damage to cells and nerve endings in the peripheral receptor organ, and 2) that prenatal irradiation significantly potentiates age-related hearing loss. Exposure to gamma rays seems to function as a sensitizer causing premature ageing.

Morphological and electrophysiological study of the inner ear and the central auditory pathways following whole body fetal irradiation.

Pregnant CBA/CBA mice were whole body irradiated with 2 Gy on the 13th or 16th day of gestation, respectively. The exposed fetuses were raised to an age of 21 postnatal days. Auditory brainstem recordings of threshold levels showed a considerable elevation independent of if irradiation had been performed on either the 13th gestational day or the 16th gestational day. In exposed animals a latency difference occurs in the peaks that increases from peak 1 to peak 5, measuring in peak 5 up to 1.16 ms. Also the peak-to-peak length of waves 1-5 increases in irradiated animals. Scanning electron microscopy of the cochleae showed varying degrees of stereociliary derangement of both outer and inner hair cells, particularly in cochleae where irradiation had been performed on the 13th gestational day, but not loss of hair cells. Light microscopic analysis of auditory brainstem nuclei revealed normal conditions except that in inner ears exposed on the 16th gestational day the flocculus was fused to the lateral surface of the anterior ventral cochlear nucleus. It is concluded that the elevated threshold levels in irradiated animals are most likely due to pathological changes in the peripheral receptor organ whereas the increased latencies and the increased peak-to-peak length likewise reflect functional changes in the brainstem auditory nuclei.

Interaural correlations in normal and traumatized cochleas: length and sensory cell loss.

Sizable intraspecies variations have been found in both the length of the organ of Corti (OC) and the amount of damage resulting from exposure to a particular ototraumatic agent. These variations have made it difficult to address certain research questions such as the susceptibility of the previously injured ear to further damage. If intra-animal correlation is high, the variability problem could be circumvented by using the two ears from a given animal for different aspects of the same study. Therefore, correlation coefficients were calculated for OC length and for percentage of missing inner (IHCs) and outer hair cells (OHCs) in a large sample of chinchillas which included controls and animals which had been exposed to noise or treated with ionizing radiation. The correlation coefficients were +0.96 for OC length, +0.93 for IHC loss, and +0.97 for OHC loss.