Effect of granulocyte colony-stimulating factor on the cochlear nuclei after creation of a partial nerve lesion: an experimental study in rats.

OBJECTIVE The risk of injury of the cochlear nerve during angle (CPA) surgery is high. Granulocyte colony-stimulating factor (G-CSF) has been found in various experimental models of peripheral and CNS injury to have a neuroprotective effect by inhibiting apoptosis and inflammation. However, to the authors' knowledge, the influence of G-CSF on cochlear nerve regeneration has not been reported. This study investigated the neuroprotective effect of G-CSF after a partial cochlear nerve lesion in rats. METHODS A lesion of the right cochlear nerve in adult male Sprague-Dawley rats was created using a water-jet dissector with a pressure of 8 bar. In the first group (G-CSF-post), G-CSF was administrated on Days 1, 3, and 5 after the surgery. The second group (G-CSF-pre/post) was treated with G-CSF 1 day before and 1, 3, and 5 days after applying the nerve injury. The control group received sodium chloride after nerve injury at the various time points. Brainstem auditory evoked potentials (BAEPs) were measured directly before and after nerve injury and on Days 1 and 7 to evaluate the acoustic function of the cochlear nerve. The animals were sacrificed 1 week after the operation, and their brains were fixed in formalin. Nissl staining of the cochlear nuclei was performed, and histological sections were analyzed with a light microscope and an image-processing program. The numbers of neurons in the cochlear nuclei were assessed. RESULTS The values for Waves 2 and 4 of the BAEPs decreased abruptly in all 3 groups in the direct postoperative measurement. Although the amplitude in the control group did not recover, it increased in both treatment groups. According to 2-way ANOVA, groups treated with G-CSF had a significant increase in BAEP Wave II amplitudes on the right side (p = 0.0401) after the applied cochlear nerve injury. With respect to Wave IV, a trend toward better recovery in the G-CSF groups was found, but this difference did not reach statistical significance. In the histological analysis, higher numbers of neurons were found in the G-CSF groups. In the statistical analysis, the difference in the numbers of neurons between the control and G-CSF-post groups reached significance (p = 0.0086). The difference in the numbers of neurons between the control and G-CSF-pre/post groups and between the G-CSF-post and G-CSF-pre/post groups did not reach statistical significance. CONCLUSIONS The use of G-CSF improved the function of the eighth cranial nerve and protected cochlear nucleus cells from destruction after a controlled partial injury of the nerve. These findings might be relevant for surgery that involves CPA tumors. The use of G-CSF in patients with a lesion in the CPA might improve postoperative outcomes.

EphB2 signaling regulates lesion-induced axon sprouting but not critical period length in the postnatal auditory brainstem.

BACKGROUND: Studies of developmental plasticity may provide insight into plasticity during adulthood, when neural circuitry is less responsive to losses or changes in input. In the mammalian auditory brainstem, globular bushy cell axons of the ventral cochlear nucleus (VCN) innervate the contralateral medial nucleus of the trapezoid body (MNTB) principal neurons. VCN axonal terminations in MNTB, known as calyces of Held, are very large and specialized for high-fidelity transmission of auditory information. Following unilateral deafferentation during postnatal development, VCN axons from the intact side form connections with novel targets, including the ipsilateral MNTB. EphB signaling has been shown to play a role in this process during the first postnatal week, but mechanisms involved in this reorganization during later developmental periods remain unknown. RESULTS: We found that EphB2 signaling reduces the number of induced ipsilateral projections to the MNTB after unilateral VCN removal at postnatal day seven (P7), but not after removal of the VCN on one side at P10, after the closure of the critical period for lesion-induced innervation of the ipsilateral MNTB. CONCLUSIONS: Results from this study indicate that molecular mechanisms involved in the development of circuitry may also play a part in rewiring after deafferentation during development, but do not appear to regulate the length of critical periods for plasticity.

Increased BrdU incorporation reflecting DNA repair, neuronal de-differentiation or possible neurogenesis in the adult cochlear nucleus following bilateral cochlear lesions in the rat.

Neurogenesis is known to occur in response to injury in the brain, for example, as a result of neurodegenerative diseases. However, there have been few investigations into how the brain responds to damage to peripheral sensory nerves, in other areas such as the brainstem. Here, we report that bilateral surgical lesions of the cochlea result in increased incorporation of the DNA replication marker, bromodeoxyuridine (BrdU), in cells of the brainstem cochlear nucleus (CN) of the adult rat, suggesting either cell proliferation or DNA repair. Some of the BrdU-labelled cells colabelled for the mature neuron marker, NeuN and the GABAergic enzyme GAD-65, suggesting the possibility that neurogenesis might have occurred and resulted in the generation of new neurons with a GABAergic phenotype. However, some of the mature neurons also re-expressed immature neuronal intermediate filament and microtuble-associated proteins, without apoptotic neuronal death, which suggests that the colabelling of BrdU with NeuN and GAD-65 may not be a true reflection of neurogenesis, but injury-stimulated neuronal dedifferentiation. These results suggest the possibility that DNA repair, neuronal de-differentiation or possible neurogenesis occurs in the cochlear nucleus, in response to damage to the peripheral auditory system.

Short-term pathophysiologic changes and histopathologic findings of the auditory pathway after closed head injury, using a rabbit model.

Hearing impairment is a well-known consequence of closed head injury (CHI). The aim of this study was to elucidate the pathogenesis of CHI-induced hearing loss, using a rabbit model. Twelve New Zealand white rabbits were divided into two groups of 6. In the first group, CHI was induced mechanically, whereas the rabbits of the second group served as controls. Baseline distortion product otoacoustic emissions (DPOAEs), contralateral suppression (CS) of the DPOAEs and auditory brainstem response (ABR) were obtained. The same measurements were performed in the first group after CHI. Three hours later, the animals were sacrificed and their brain was excised and subjected to histopathologic examination. Mean I-III ABR latencies were increased and DPOAE amplitudes and CS values were reduced in the trauma group after CHI, at a statistically significant level. Histopathologic examination of the temporal lobe and brainstem showed multiple hemorrhagic and necrotic areas, with edema in the surrounding region. The vestibulocochlear nerve was severely damaged at its emerging site at the brainstem. In conclusion, both peripheral and central involvement of the auditory pathway was found after CHI. Otoacoustic emissions in conjunction with ABR may provide significant information on both peripheral and central auditory function.

Modulation of inhibitory and excitatory synaptic transmission in rat inferior colliculus after unilateral cochleectomy: an in situ and immunofluorescence study.

We investigated whether inhibitory synaptic transmission mediated through glycinergic receptor, GABAA receptors, glutamic acid decarboxylase, the enzyme synthesizing GABA, and excitatory synaptic transmission through alpha-amino-3-hydroxi-5-methylisoxazole-4-propionic acid receptors and N-methyl-D-aspartate receptors are affected in the inferior colliculus by unilateral surgical cochleectomy. In situ hybridization and immunohistofluorescence studies were performed in normal and lesioned adult rats at various times following the lesion (1-150 days). Unilateral auditory deprivation decreased glycine receptor alpha1 and glutamic acid decarboxylase 67 expression in the contralateral central nucleus of the inferior colliculus. This decrease began one day after cochleectomy, and continued until day 8; thereafter expression was consistently low until day 150. The glycine receptor alpha1 subunit decrease did not occur if a second contralateral cochleectomy was performed either on day 8 or 150 after the first cochleectomy. Bilateral cochleectomy caused also a bilateral inferior colliculus diminution of glutamic acid decarboxylase 67 mRNA at post-lesion day 8 but there were no changes in glycine receptor alpha1 compared with controls. In contrast, the abundance of other alpha2-3, and beta glycine receptor, gephyrin, the anchoring protein of glycine receptor, the alpha1, beta2 and gamma2 subunits of GABAA receptors, GluR2, R3 subunits of alpha-amino-3-hydroxi-5-methylisoxazole-4-propionic acid receptors, and NR1 and NR2A transcripts of N-methyl-D-aspartate receptors was unaffected during the first week following the lesion. Thus, unilateral cochlear removal resulted in a selective and long-term decrease in the amount of the glycine receptor alpha1 subunit and of glutamic acid decarboxylase 67 in the contralateral central nucleus of the inferior colliculus. These changes most probably result from the induced asymmetry of excitatory auditory inputs into the central nucleus of the inferior colliculus and may be one of the mechanisms involved in the tinnitus frequently encountered in patients suffering from a sudden hearing loss.

Effects of exposing DBA/2J mice to a high-frequency augmented acoustic environment on the cochlea and anteroventral cochlear nucleus.

DBA/2J (D2) mice, which exhibit very early progressive sensorineural hearing loss, were treated for 12h nightly with an augmented acoustic environment (AAE) initiated before the onset of hearing. The AAE consisted of repetitive bursts of a 70 dB sound pressure level, half-octave noise band centered at 20 kHz (i.e. low frequencies were excluded). At 55 days of age, AAE-treated mice, compared to control mice, exhibited less elevation of auditory brainstem response thresholds for tone frequencies from 16 to 32 kHz and fewer missing outer hair cells in the high-frequency tonotopic region of the cochlea. The dorsal region of their anteroventral cochlear nucleus (most strongly stimulated by the AAE) was larger, had more surviving neurons, and larger neurons than those of untreated control mice. These and previous findings using an AAE band containing lower frequencies indicate that AAE treatment effects are frequency-related. The findings provide support for the hypothesis that the beneficial effects of AAE treatment on the cochlea are associated with increased physiological activity evoked by the AAE, and the central AAE effects result from increased AAE-evoked neural activity and a healthier cochlea providing the auditory input.

Developmental regulation and adult maintenance of potassium channel proteins (Kv 1.1 and Kv 1.2) in the cochlear nucleus of the rat.

The development and maintenance of the adult expression and distribution of Kv 1.1 and Kv 1.2, two voltage-dependent potassium channel subunits, were investigated in the anteroventral cochlear nucleus (AVCN) of the rat. Both Kv 1.1 and Kv 1.2 were found in AVCN neuronal cell bodies at birth, as detected by in situ hybridization and immunocytochemistry. However, Kv 1.1 and Kv 1.2 were not seen in axons until the end of the third postnatal week. From postnatal day 21 through adulthood, labeling for both potassium channels was in axonal processes, whereas the number of cell bodies labeled for Kv 1.1 decreased and there were no cell bodies labeled for Kv 1.2. Therefore, these two potassium channel proteins are targeted to their final subcellular destinations in axons well after hearing onset. Once the adult distribution pattern of Kv 1.1 and Kv 1.2 is attained, its maintenance does not depend on signals from auditory nerve synapses. Eliminating auditory nerve input to the cochlear nucleus by means of bilateral cochleotomy did not change Kv 1.1 or Kv 1.2 expression or distribution, as seen by in situ hybridization, immunocytochemistry and Western blot. Thus, normal excitatory synaptic input in adult animals is not a requirement to regulate the expression and cellular and subcellular distribution of these potassium channel proteins.

Medial olivocochlear reflex interneurons are located in the posteroventral cochlear nucleus: a kainic acid lesion study in guinea pigs.

The medial olivocochlear (MOC) reflex arc is probably a three-neuron pathway consisting of type I spiral ganglion neurons, reflex interneurons in the cochlear nucleus, and MOC neurons that project to the outer hair cells of the cochlea. We investigated the identity of MOC reflex interneurons in the cochlear nucleus by assaying their regional distribution using focal injections of kainic acid. Our reflex metric was the amount of change in the distortion product otoacoustic emission (at 2f(1)-f(2)) just after onset of the primary tones. This metric for MOC reflex strength has been shown to depend on an intact reflex pathway. Lesions involving the posteroventral cochlear nucleus (PVCN), but not the other subdivisions, produced long-term decreases in MOC reflex strength. The degree of cell loss within the dorsal part of the PVCN was a predictor of whether the lesion affected MOC reflex strength. We suggest that multipolar cells within the PVCN have the distribution and response characteristics appropriate to be the MOC reflex interneurons.

ERK and SAPK signaling in auditory brainstem neurons after unilateral cochlear ablation.

Unilateral cochlear ablation (UCA) in adults deafferented one cochlear nucleus (CN) and induced several plasticities in central auditory pathways. To assess whether signal transduction could contribute to these changes, we determined if UCA induced activity in the extracellular signal-regulated kinase (ERK) and the stress-activated protein kinase (SAPK) signal transduction pathways. Using Western blots, we measured phosphorylated ERK1 (ERK1-P), ERK2 (ERK2-P), p46 and p54 SAPK (SAPK-P) and c-Jun (c-Jun-P) levels in the major subdivisions of the CN, the principal nuclei of the superior olivary complex (SOC) and the central nucleus of the inferior colliculus (ICc) for up to 145 days postablation. ERK1-P and ERK2-P were typically elevated at 7 and 145 days but depressed at 30 days, 60 days, or both. In addition, ERK1-P and ERK2-P were elevated at 3 days in the anteroventral (AVCN) and posteroventral CN (PVCN). Immunohistochemical labeling indicated that after 5 days, most ERK1/2-P in the CN was in neuronal nuclei. Only minor changes were evident in total ERK1 and ERK2 levels. Several correlations were evident between the postablation plasticities observed previously and altered ERK1-P and ERK2-P levels. Few changes were found in SAPK-P and c-Jun-P levels. Concomitant elevations of SAPK-P and c-Jun-P were not evident, except in the superficial dorsal CN (DCN) at postablation day 3, consistent with a cell-stress response. These findings suggest that signals induced as a consequence of UCA are transduced mainly through the neuronal ERK pathway. This activity probably influenced gene expression and cytoplasmic regulatory mechanisms that contributed to the plasticities induced by UCA.

Fibroblast growth factors (FGFs) in the cochlear nucleus of the adult mouse following acoustic overstimulation.

To see if fibroblast growth factors (FGFs) might function in the central changes following auditory overstimulation we tracked immunostaining in the cochlear nucleus of adult mice with monoclonal antibodies to FGFs (FGF-1, FGF-2) and FGF receptor. After exposure nearly all outer hair cells died, while inner hair cell and fiber loss were restricted to a region midway along the cochlear spiral. FGFs staining in the cochlear nucleus appeared in hypertrophied astrocytes in the regions of nerve fiber degeneration only. For normal-sized astrocytes there was an increase in the number stained and the intensity of staining across all frequency domains, but not in neurons. The increases were modest at 3-7 days, pronounced at 14 days, modest again by 30 days, and back to control levels by 60 days. FGF receptor staining of neurons occurred equally in all mice, exposed or not. The findings suggest that astrocytes play a role in the central responses to acoustic overstimulation and cochlear damage, involving FGFs, possibly regulating the activity of intrinsic neurons or signaling axonal growth. Not limited to regions of cochlear nerve fiber and inner hair cell loss, the changes in FGFs may represent a reaction to outer hair cell damage which spreads broadly across the central pathways.

Effects of acoustic trauma on dorsal cochlear nucleus neuron activity in slices.

Previous studies found increased multi-unit spontaneous activity in the dorsal cochlear nucleus (DCN) of animals that had been exposed to intense sound. Such activity may be related to tinnitus. Our study examined effects of previous exposure to intense sound on single neurons in the DCN, by measuring spontaneous activities and sensitivities to acetylcholine, an important neurotransmitter of centrifugal pathways to the cochlear nucleus, in brain slices. Spontaneous discharges were recorded extracellularly in the DCN portion of brain slices from control and intense-tone-exposed rats. Slices from exposed rats showed increased prevalence of bursting and decreased regular spontaneous activity. Since regular neurons include fusiform cells, and bursting neurons include cartwheel cells, intense tone exposure may lead to increased activity of DCN cartwheel cells and decreased activity of fusiform cells. Alternatively, the activity of some fusiform cells might change to bursting. Intense tone exposure also appeared to increase bursting neuron sensitivity to carbachol. This suggests that changes in DCN cartwheel cell spontaneous activity may reflect changes in effects of cholinergic centrifugal pathways following intense tone exposure. We conclude that acoustic trauma may lead to changes in the physiology and pharmacology of DCN neurons. These changes may be related to underlying mechanisms of central tinnitus.

Apoptosis in auditory brainstem neurons after a severe noise trauma of the organ of Corti: intracochlear GDNF treatment reduces the number of apoptotic cells.

We have studied the morphological and cellular changes in the cochlear nucleus (CN) after cochlear nerve degeneration and whether these changes can be prevented by rescuing the primary cochlear neurons from degeneration with local glial cell line derived neurotrophic factor (GDNF) treatment. Degeneration of spiral ganglion neurons was seen to lead to a reduction of the volume of the anteroventral cochlear nucleus (AVCN); the size of the cell nuclei in the AVCN also was reduced. No differences were observed in cell density. After intrascalar GDNF treatment the volume of the AVCN was significantly larger when compared to the untreated side, and the size of the cell nuclei in the AVCN was significantly larger on the treated side. After degeneration of spiral ganglion neurons, an increased number of apoptotic cell nuclei were seen in ipsilateral CN and superior olivary complex. This increase was significantly smaller after intrascalar GDNF treatment. Degeneration of primary cochlear neurons seems to lead to an increase in the number of CN neurons undergoing apoptotic cell death. This can be prevented partially by rescuing primary cochlear neurons from degeneration with local GDNF treatment.

Cellular generators of the binaural difference potential in cat.

In humans, lateralization and fusion of binaurally presented clicks are correlated with the latency and amplitude of the binaural difference potential (BDP) (e.g., Furst et al., 1985). The BDP is derived by subtracting the brainstem auditory evoked potential (BAEP) for binaural stimulation from the sum of the BAEPs for left and right monaural stimulation. Our aim in this work was to determine the cellular generators of the BDP and thus identify cells that may be crucial for specific types of binaural sound processing. To this end, we injected kainic acid into the superior olivary complex (SOC) or the cochlear nucleus (CN) in cats and examined the effects of the resulting lesions on the click-evoked BDP. Lesions confined to the anterior anteroventral CN (AVCNa) substantially reduced the BDP, while lesions primarily involving more posterior parts of the CN had little or no effect. BDP reductions occurred for lesions involving either high (> 10 kHz) or lower (< 10 kHz) characteristic frequency (CF) regions of the AVCNa (as well as the posterior CN). Lesions involving the SOC reduced the BDP and, in one case, eliminated the high-pass filtered (270 Hz cutoff) BDP. Combining these results with published information about the physiology and anatomy of auditory brainstem cells, we conclude that: (1) spherical cells in the AVCNa are essential for BDP production, (2) the earliest part of the BDP is generated by medial superior olive (MSO) principal cells which receive spherical cell inputs, (3) a later part is probably generated by the cellular targets of MSO principal cells and, (4) the cells involved in BDP generation have CFs above, as well as below, 10 kHz. Since humans, like cats, have a well-developed MSO, we suggest that the MSO may also be essential for BDP production in humans. Thus, perceptual correlates of the BDP, binaural fusion and click lateralization, apparently involve the MSO.

Exposure to low frequency noise during rearing induces spongiform lesions in gerbil cochlear nucleus: high frequency exposure does not.

Spongiform lesions of the gerbil cochlear nucleus are reduced in number and extent by rearing in acoustic isolation compared with rearing while exposed to normal colony low-frequency background noise. This study tested whether rearing under exposure to noise bands of moderate intensity would increase the number and extent of cochlear nucleus spongiform lesions. Gerbils were reared from weaning to young adulthood in acoustic isolation chambers while continually exposed to moderately intense bands of either high frequency or low frequency noise. Exposure to low frequency noise resulted in lesion number and area densities that were more than twice those seen in gerbils exposed to high frequency noise. Lesion extent in the low frequency group was similar to that in colony-reared gerbils; lesion extent in the high frequency group was similar to gerbils reared in acoustic isolation. Comparisons within the posterior ventral cochlear nucleus revealed that the differences in lesion extent were most pronounced in the middle and dorsal-medial portions, the regions that are most responsive to middle and high frequencies. These finding suggest that the regional restriction of spongiform lesions within the cochlear nucleus does not have a tonotopic basis.

Stimulus parameters affecting tissue injury during microstimulation in the cochlear nucleus of the cat.

We investigated the effects of continuous microstimulation in the cats' posteroventral cochlear nucleus, using chronically implanted activated iridium microelectrodes. We examined 51 electrode sites (39 pulsed sites, and 12 unpulsed sites). Seven hours of continuous stimulation at 500 Hz often produced tissue injury near the tips of the pulsed microelectrodes. The damage took the form of a region of vacuolated tissue extending 200 microns or more from the site of the electrode tip. Electron microscope studies showed the vacuoles to be severely edematous segments of myelinated axons. The statistical correlation between the amount of damaged tissue and the charge per phase was large and highly significant (P < 0.0001). When the electrodes were pulsed for 7 h at 500 Hz with charge-balanced biphasic pulse pairs, the threshold for the damage was approximately 3 nC/phase. The damage threshold was not appreciably lower than the stimulation protocol was extended to 35 h (7 h/day for 5 days). In contrast, the threshold for exciting neurons near the microelectrode is approximately 1 nC/phase, as determined by the evoked response recorded in the inferior colliculus. There was little correlation between the severity of the tissue damage and the geometric charge density at the surface of the electrodes, between the damage and amplitude of the cathodic phase of the voltage transient induced across the stimulating electrodes by the stimulus current pulses, or between the damage and the stimulus pulse duration.