Neonatal Cochlea Research Articles

Hes5 expression in the postnatal and adult mouse inner ear and the drug-damaged cochlea.

The Notch signaling pathway is known to have multiple roles during development of the inner ear. Notch signaling activates transcription of Hes5, a homologue of Drosophila hairy and enhancer of split, which encodes a basic helix-loop-helix transcriptional repressor. Previous studies have shown that Hes5 is expressed in the cochlea during embryonic development, and loss of Hes5 leads to overproduction of auditory and vestibular hair cells. However, due to technical limitations and inconsistency between previous reports, the precise spatial and temporal pattern of Hes5 expression in the postnatal and adult inner ear has remained unclear. In this study, we use Hes5-GFP transgenic mice and in situ hybridization to report the expression pattern of Hes5 in the inner ear. We find that Hes5 is expressed in the developing auditory epithelium of the cochlea beginning at embryonic day 14.5 (E14.5), becomes restricted to a particular subset of cochlear supporting cells, is downregulated in the postnatal cochlea, and is not present in adults. In the vestibular system, we detect Hes5 in developing supporting cells as early as E12.5 and find that Hes5 expression is maintained in some adult vestibular supporting cells. In order to determine the effect of hair cell damage on Notch signaling in the cochlea, we damaged cochlear hair cells of adult Hes5-GFP mice in vivo using injection of kanamycin and furosemide. Although outer hair cells were killed in treated animals and supporting cells were still present after damage, supporting cells did not upregulate Hes5-GFP in the damaged cochlea. Therefore, absence of Notch-Hes5 signaling in the normal and damaged adult cochlea is correlated with lack of regeneration potential, while its presence in the neonatal cochlea and adult vestibular epithelia is associated with greater capacity for plasticity or regeneration in these tissues; which suggests that this pathway may be involved in regulating regenerative potential.

Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features

Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.

Pharmacological inhibition of Notch signaling in the mature guinea pig cochlea

Recent studies using explant cultures have demonstrated that pharmacological inhibition of Notch signaling by gamma-secretase inhibitors generates supernumerary hair cells in embryonic or neonatal cochleae. The aim of this study was to examine the effects of such pharmacological inhibition on mature auditory epithelia in vivo. Normal adult guinea pig auditory epithelia exhibited weak or no immunoreactivity for Notch1 and Jagged1, whereas ototoxic treatment caused the upregulation of these molecules in damaged auditory epithelia. Local application of a gamma-secretase inhibitor in damaged cochleae generated ectopic hair cells in mature auditory epithelia. These findings indicate that pharmacological inhibition of Notch signaling is a possible strategy for hair cell regeneration in adult auditory epithelia.

Technical report: Laser microdissection and pressure catapulting is superior to conventional manual dissection for isolating pure spiral ganglion fractions from the cochlea

Isolating cells from the cochlea to perform molecular biology assessment presents a challenge, because it is not possible to dissect pure cell pools by conventional methods. Thus, we set out to demonstrate that laser microdissection and pressure catapulting (LMPC) is superior to conventional manual cochlea dissection for this purpose. Spiral ganglions (SG) were isolated from neonatal rat cochleae by manual dissection and LMPC. Also, modioli were manually dissected. Total RNA was isolated from all three cell pools. In order to demonstrate contamination of the dissected cell pool, we determined the expression of type II iodothyronine deiodinase (D2), claudin 11 (Cld-11), neurofilament light chain (NF-L) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts by RT-PCR. The results showed that LMPC is not only a suitable method for selectively dissecting cochlear tissues, but in addition the molecular markers confirmed pure spiral ganglion cell pools without indication for any contamination by other cells. This indicates that LMPC is capable of providing a pure SG cell pool in contrast to conventional manual dissection. Therefore, LMPC presents a new technique for cochlear tissue separation improving the validity of molecular biological studies of the inner ear.

Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.

Inhibition of Notch/RBP-J signaling induces hair cell formation in neonate mouse cochleas

Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation.

Distortion product otoacoustic emissions in term infants after hypoxia-ischaemia

Distortion product otoacoustic emissions (DPOAEs) were recorded in 46 term infants who suffered perinatal hypoxia-ischaemia to identify which frequencies in the cochlear audiogram are susceptible to perinatal hypoxia-ischaemia. On days 3-5 after birth, the pass rates across the frequencies of the f(2 )primary tone between 1 and 10 kHz, particularly 1-5 kHz, were all lower than those in normal term controls (X(2 )= 7.27-32.30, all P <0.01). Of the 92 ears, 15 (16.3%) failed the DPOAE test, which was significantly higher than in the controls (4.3%, X(2) = 5.81, P <0.05). At 1 month, 80 ears with a type A tympanogram were re-tested. The pass rates at most frequencies, mainly 1 and 2 kHz, were slightly further decreased. Thirteen ears (16.2%) failed the DPOAE test. These results suggest that the neonatal cochlea, mainly at the frequencies 1-5 kHz, is impaired shortly after perinatal hypoxia-ischaemia and the impairment remains at 1 month. Perinatal hypoxia-ischaemia impairs the neonatal cochlea mainly at the frequencies 1-5 kHz and the impairment detected on days 3-5 after birth is unlikely to improve in the later neonatal period. These findings may have implications for the management of hearing impairment in infants after perinatal hypoxia-ischaemia.

120 Frequency-Specific Hearing Impairment in Neonates After Perinatal Hypoxia-Ischaemia

Background/Aim: Perinatal hypoxia-ischaemia (HI) is one of the major risk factors of acquired sensorineural hearing impairment in infants. So far, little is known about which frequencies on the audiogram of the cochlea in newborn infants are highly susceptible to perinatal HI. By measuring distortion product otoacoustic emissions (DPOAEs), this study aimed to identify the frequencies that are impaired in neonates after HI.Methods: The subjects were 46 term neonates who suffered perinatal HI (5 min Apgar score ≥7 and clinical evidence of HI). Normal controls were 35 healthy term neonates without problems. DPOAE was elicited by two pure tones (f 1 and f 2; f 2/ f 1 = 1.22) presented simultaneously, with the lower frequency primary tone (f 1) at 65 dB SPL and the higher frequency primary tone (f 2) at 55 dB SPL. The f 2 primary tone was presented at 10 frequencies between 0.5 and 10 kHz. In individual subjects, a testing result that 6 or more out of the 10 frequencies passed the criteria was classified as a total pass. Both the left and right ears were tested. DPOAE was measured on day 3–5 after birth. One month later, all neonates after HI who had normal acoustic impedance (n = 41, 82 ears), and the normal controls who failed in the first DPOA testing were re-tested.Results: On day 3–5 after birth, the pass rates across the frequencies, mainly 1–5 kHz, in the neonates after HI were all lower than in the controls. The total pass rate was 77.2% (71/92 ears), compared with 95.7% (67/70 ears) in the controls. The patterns of 2 f 1− f 2 DPOAE amplitudes at different frequencies in both the neonates after HI and the controls were generally similar to those of the pass rates, with a ‘dip' at the frequencies 750 Hz and 1 kHz. The difference in the patterns between the two groups was also similar to that in the pass rates. One month later, all the 3 ears which did not pass the first DPOAE testing in the controls passed the re-test. Of the 92 ears in neonates after HI, 80 had Type A tympanogram, and the remaining had Type B (n= 2) or Type As/C (n= 10), suggesting middle ear disorders. The DPOAE pass rates in the 80 neonates with Type A tympanogram tended to decrease slightly further at all frequencies. The total pass rate also decreased further to 62.5%.Conclusions: The neonatal cochlea, mainly at the frequency range of 1–5 kHz, was impaired after perinatal HI. One month later, the impairment did not show any improvement. The findings may have important implication to early intervention. Follow-up study is needed to detect any permanent frequency-specific impairment.

DPOAE suppression tuning: cochlear immaturity in premature neonates or auditory aging in normal-hearing adults?

Previous work has shown that distortion product otoacoustic emission (DPOAE) suppression tuning curves (STCs) recorded from premature neonates are narrower than adult STCs at both low and high frequencies. This has been interpreted to indicate an immaturity in cochlear function prior to term birth. However, an alternative explanation for this finding is that adult DPOAE STCs are broadened and reflect cochlear hair cell loss in normal-hearing adults due to aging, and natural exposure to noise and ototoxins. This alternative hypothesis can be tested by studying suppression tuning in normal-hearing school-aged children. If normal-hearing children, who have not aged significantly or been exposed to noise/ototoxins, have DPOAE suppression tuning similar to adults, the auditory aging hypothesis can be ruled out. However, if children have tuning similar to premature neonates and dissimilar from adults, it implicates aging or other factors intrinsic to the adult cochlea. DPOAE STCs were recorded at 1500, 3000, and 6000 Hz using optimal parameters in normal-hearing children and adults. DPOAE STCs collected previously from premature neonates were used for age comparisons. In general, results indicate that tuning curves from children are comparable to adult STCs and significantly different from neonatal STCS at 1500 and 6000 Hz. Only the growth of suppression was not adultlike in children and only at 6000 Hz. These findings do not strongly support the auditory aging hypothesis as a primary explanation for previously observed neonatal-adult differences in DPOAE suppression tuning. It suggests that these age differences are most likely due to immaturities in the neonatal cochlea. However, nonadultlike suppression growth observed in children at 6000 Hz warrants further attention and may be indicative of subtle alternations in the adult cochlea at high frequencies.

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a β-galactosidase (β-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of β-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of β-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of β-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express β-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.

On the spectral periodicity of transient-evoked otoacoustic emissions from normal and damaged cochleas.

The spectral quasi-periodicity of transient-evoked otoacoustic emissions (TEOAE) is well acknowledged since Zwicker described a preferred spacing of 0.4 bark between consecutive peaks in the spectrum of otoacoustic emissions from normal ears. While there is scarce evidence of any anatomical reason for this regularity, several functional models of the cochlea have predicted that the structure of emission spectra reflects important characteristics of cochlear filters. In an attempt to check such predictions, the average regularity of TEOAE spectra was studied in three groups of human subjects, normally hearing adults, healthy neonates, and adults suffering from noise-induced hearing loss. Significant differences in emission periodicities were found. Around 1 kHz, the preferred spacing was close to 130 Hz in normally hearing adult ears and neonates. In contrast, no clear periodicity was found in the group of damaged ears, even though they had clinically normal pure-tone audiometry below 2 kHz. Around 4 kHz, the preferred spacing was close to 240 Hz in normal adults and neonates, whereas TEOAEs were absent in many impaired ears. A phenomenological model assuming that TEOAEs stem from the responses of a slightly disarrayed bank of highly tuned filters predicts that the filter width would be the same in healthy young adults and neonates. In contrast, ears suffering from high-frequency hearing loss could exhibit early damaged filters. The proposed method might provide an objective assessment of parameters otherwise difficult to evaluate, especially in neonatal cochleas.

An Earful of AChR α9

Even before hearing becomes active, there is a transient cholinergic innervation in the developing mammalian inner ear. The physiological significance of these synapses has never been demonstrated. Glowatzki and Fuchs show that the novel neuronal nicotinic acetylcholine receptor α9 is functionally present in neonatal cochlear inner hair cells and behaves like a true acetylcholine receptor. Its stimulation results in the activation of a fast, calcium-dependent potassium channel. Acetylcholine thus inhibits afferent activity and acts to impose rhythmicity onto the immature auditory pathway. Glowatzki, E., and Fuchs, P.A. (2000) Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea. Science 288 : 2366-2368. [Abstract] [Full Text]

Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea.

Efferent feedback onto sensory organs provides a means to modulate input to the central nervous system. In the developing mammalian cochlea, inner hair cells are transiently innervated by efferent fibers, even before sensory function begins. Here, we show that neonatal inner hair cells are inhibited by cholinergic synaptic input before the onset of hearing. The synaptic currents, as well as the inner hair cell's response to acetylcholine, are mediated by a nicotinic (alpha9-containing) receptor and result in the activation of small-conductance calcium-dependent potassium channels.

Effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian cochlea in vitro

The aim of this study was to test the possible regenerative potential of several molecules and growth factors such as retinoic acid (RA), insulin, epidermal growth factor (EGF) and transforming growth factors alpha (TGFα) and beta (TGFβ) on the neonatal cochlea in vitro after neomycin intoxication. Our studies show that cochlear sensory epithelium behaves differently while maintained in various culture conditions, although we did not observe regeneration whatever the molecules or growth factors tested. The ototoxic action of neomycin in vitro produced a specific death of hair cells, except in the apical region. Organ of Corti of rats 3 days after birth always presented two regions that responded differently to the antibiotic: a widespread scar region extending from the basal cochlea up to the beginning of the apical turn, where most hair cells had disappeared, and a second region called the resistance region localized in the apex, and which was more or less developed depending on culture conditions. The length of the resistance region was modulated by molecules or growth factors added to the feeding solution suggesting that some of them could produce a protective action on hair cells against neomycin. Slight protection effects may be found with RA and insulin, however, the most definite protection results from the combination of insulin with TGFα as shown by the large increase in the length of the resistance region compared to organ of Corti treated with antibiotic alone. The tested molecules and growth factors did not promote cochlear hair cell regeneration in vitro after neomycin treatment, however some of them may offer a protective action against ototoxicity.

Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse.

The Bronx Waltzer (vb) mutation in the mouse results in the degeneration of most but not all of the primary auditory receptors, the inner hair cells, and their afferent neurons. We analyzed the ultrastructure of 94 inner hair cells in the intact postnatal mutant mouse and in neonatal cochleas in culture to understand the pathogenesis of hair cell death and to detect factors that may prevent it. The vb spiral neurons of the Bronx Waltzer display two distinctive features: some of them continue to divide mitotically for at least seven postnatal days, and the type I radial fibers that innervate inner hair cells display a deficiency in immunoexpression of GAD. The growing endings of spiral neurons converge around the inner hair cells or, in their absence, invade the outer hair cell region. Their profuse sprouting among inner spiral sulcus cells contributes to the characteristic ultrastructural picture of the bv cochlea. During the first three days after birth, 40% of the inner hair cells appear normal and innervated, 40% are mostly denervated and degenerating, and 20% are immature, with minimal or no neuronal appositions. However, in mutants 6 days and older only a few inner hair cells survive, and these show either normal or superfluous afferent innervation and axosomatic GABAergic efferent innervation. Degeneration of inner hair cells begins with a distention of the nuclear envelope and the ribosomal endoplasmic reticulum. The outer nuclear membrane eventually breaks, and exudate fills the cell interior. The cellular edema leads to cell death. We propose that success or failure in synaptic acquisition is a decisive factor in the survival or decline of the mutant inner hair cells. We also suggest that the developmental delay in maturation of the spiral ganglion neurons (type I) and the failure in their synaptogenesis may be caused by an impairment in neurotrophin (NT3/BDNF) synthesis by their mutant receptor cells.

Expression of mRNA encoding vasopressin V1a, vasopressin V2, and ANP-B receptors in the rat cochlea

The expression of mRNAs encoding vasopressin V1a, V2, and ANP-B receptors in the rat cochlea was examined by PCR and in situ hybridization. After reverse-transcription of rat cochlear RNA, cDNA was amplified by PCR using pairs of primers specific to these receptors. After subcloning of the PCR products, clones with sequences identical to those cloned previously from the rat liver (V1a receptor), kidney (V2 receptor) and brain (ANP-B receptor) were obtained. The localization of expression of those receptors in the developing and adult rat cochlea was examined by in situ hybridization using 35S-labeled cRNA probes. The V1a and V2 receptors were expressed throughout the whole of the neonatal rat cochlea, while no expression was detected in the adult cochlea. The ANP-B receptor was expressed throughout the whole of the neonatal cochlea. In the adult cochlea, expression was observed in the spiral ganglion and the spiral ligament. These results suggest that vasopressin may play a role in the development of the cochlea, and that natriuretic peptide may play a role in the function of the spiral ganglion and the spiral ligament.

Factors modulating supernumerary hair cell production in the postnatal rat cochlea in vitro

It has been shown in the past that extra hair cells or supernumerary cells can be produced when neonatal cochleae are maintained in vitro. In this report, we investigated the effects of the culture methods, molecules and growth factors that are thought to be involved in cell proliferation. Quantitative studies of supernumerary hair cells were made by measuring the cell density over the entire spiral lamina at two postnatal stages: birth and 3 days after birth. With a standard feeding solution without serum, a difference in cell density was observed between the two methods of culture. Cochlear explants in a standard feeding solution supplemented with serum showed an increase of cell density only when the explantation is made at birth. Retinoic acid added to the standard feeding solution did not increase the hair cell density, while insulin induced an increase, especially at 5 μg/ml. Several growth factors were tested. Epidermal growth factor (EGF) presented a dose dependent effect with an increase of up to 30% of hair cell density that was observed in the basal region when the explantation was made at birth. Transforming growth factor-a did not induce an increase of cell density, whereas transforming growth factor-β presented an effect on hair cell density, with a dose dependent effect reaching 37.4% for the basal inner hair cells. Interpretation of these results is limited because of the lack of data concerning the presence of specific membrane receptors. One possibility is that insulin stimulates hair cell differentiation from existing undifferentiated cells. Another hypothesis may be related to the EGF and transforming growth factor-β, where these molecules might induce transdifferentiation of cells by acting on the transmembrane molecules and the extracellular matrix.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons from part of a later, secondary innervation to the cochlea.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons from part of a later, secondary innervation to the cochlea.

Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice

This study examined the potential for hair cell regeneration in embryonic and neonatal mouse organs of Corti maintained in vitro. Small numbers of hair cells were killed by laser microbeam irradiation and the subsequent recovery processes were monitored by differential interference contrast (DIC) microscopy combined with continuous time-lapse video recordings. Replacement hair cells were observed to develop in lesion sites in embryonic cochleae and on rare occasions in neonatal cochleae. In embryonic cochleae, replacement hair cells did not arise through renewed proliferation, but instead developed from preexisting cells that changed from their normal developmental fates in response to the loss of adjacent hair cells. In cochleae established from neonates, lost hair cells usually were not replaced, but 11 apparently regenerated hair cells and a single hair cell labeled by 3H-thymidine were observed as rare responses to the creation of hair cell lesions in these organs. The results indicate that the organ of Corti can replace lost hair cells during embryonic and on rare occasions during early neonatal development. The ability of preexisting cells to change their developmental fates in response to hair cell death is consistent with the hypothesis that during embryonic development hair cells may inhibit neighboring cells from specializing as hair cells. In neonatal cultures, the rare occurrence of apparently regenerated hair cells indicates that some cells in the postembryonic organ of Corti retain response mechanisms that can lead to self-repair.