Spiral ganglion cell counts in an age-graded series of rat cochleas.

Spiral ganglion cells in the cochleas of Sprague-Dawley rats in various age groups were counted in order to assess the extent and location of cell degeneration with age. Cells in every tenth section of serially sectioned plastic embedded cochleas were counted in the light microscope. The median cell number for the 1- to 2-month-old animals was 15,800 cells. This number was first seen to be significantly reduced (-14%) in the 23-month-old animals. At 27 to 29 months the ganglion cell number was reduced by 20%, while at 33 to 34 months there was a 17% loss. Losses were found throughout the length of the ganglion with the greatest losses at the lower basal and apical ends. In the oldest group, these losses amounted to 28% and 33%, respectively. Type II ganglion cells first showed a significant decrease in number in the 27- to 29-month-old group, when a 32% loss was seen. The same loss was seen in the 33- to 34-month-old group. Unlike the type I cells which are lost throughout the length of the ganglion, type II cells were not significantly reduced in number at the basal end, but decreased by as much as 42% in the middle and apical regions.

Fibronectin-like immunoreactivity of the basilar membrane of young and aged rats.

Dysfunction of cochlear mechanics has been hypothesized to be a source of age-related hearing loss and the basilar membrane mass and stiffness contribute to normal cochlear mechanics. Fibronectin, a large, extracellular matrix protein and a major component of the basilar membrane, may contribute to both the mass and stiffness of the membrane. Mesothelial cells underlying the basilar membrane may produce the fibronectin and also contribute to the mass of the membrane. Changes in either the fibronectin or the mesothelial cells might, therefore, have an effect on cochlear mechanics. In order to assess basilar membrane changes in aged animals, young adult (2-4 months) and aged (24-26 months) Sprague-Dawley rats were evaluated for the presence of fibronectin-like protein and mesothelial cells. The basilar membrane in the young animals had strong fibronectin-like immunoreactivity throughout its length. The old animals, on the other hand, showed normal fibronectin immunoreactivity in the basilar membrane of the basal turn, but little or no reactivity in the apical cochlear turn. The number of mesothelial cells was reduced throughout the length of the membrane in aged animals, with the greatest loss in the basal turn (60% fewer cells). These two degenerative changes, which appear to be independent of each other, may contribute to the observed threshold shifts in aged cochleas.

Collaterals from lateral and medial olivocochlear efferent neurons innervate different regions of the cochlear nucleus and adjacent brainstem.

Two populations of superior olivary neurons which project to different sensory cell regions in the cochlea also give off collateral projections to the ventral cochlear nucleus (VCN) and adjacent brainstem. To determine whether these VCN projections also have different targets they were characterized by selective retrograde amino acid transport. Retrograde transport of 3H-d-aspartate (D-ASP) selectively labeled the unmyelinated fibers and neurons of the lateral olivocochlear (OC) system including a dense collateral projection to the central VCN. Retrograde transport of 3H-nipecotic acid (NIP) labeled the myelinated fibers and neurons of the medial OC system, including collateral projections to the peripheral VCN, subpeduncular granule cells, and nucleus Y. Medial and lateral OC efferent collaterals thus innervate different regions of the CN. Lateral system collaterals overlap extensively with Type I spiral ganglion cell afferent input. They are well positioned to play a role in modulating afferent input to the central auditory system, as is the primary projection of these efferents to the cochlea. The medial system collaterals project near the recently described afferent projections of Type II spiral ganglion cells. The medial system collaterals may therefore be related to the function of outer hair cells, as the medial system primary axons appear to be in the cochlea.

Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.

Immunopathology of the inner ear: an update.

We have reviewed the events of an inner-ear immune response. The perilymph contains antibody, presumably derived from the systemic circulation and CSF, which would allow for neutralization and help with opsonization and complement fixation. The endolymphatic sac contains immunocompetent cells capable of processing and presenting viral or bacterial antigen, potentiating the immune response, attacking the invaders directly or attacking infected cells, and developing immunoglobulin responses in situ. The early release of mediators such as IL-2 likely emanate from the endolymphatic sac and result in potentiation and regulation of the response and may assist in changes in the SMV, including expression of ICAM-1, which aid in the egress of immune cells from the systemic circulation. PMNs arrive first, followed by T cells and B cells, with secretion of specific antibody a relatively late event. Concomitant with the increase in cellular constituents is the formation of a dense extracellular matrix. The inner ear appears to have remarkable difficulty in clearing this matrix, ultimately resulting in ossification. The immune response is unfortunately deleterious to the inner ear, resulting in degeneration of the organ of Corti, stria vascularis, and spiral ganglion. Hearing loss is consistently seen following sterile and virally induced labyrinthitis. The inner ear also appears to be a target for autoimmune disease. While inner-ear damage has been described as part of non-organ-specific autoimmune disease, specific disease against the hearing apparatus is also likely. Experimental paradigms have allowed alterations of both the afferent and efferent limbs of this response; ultimately, with the hope that we can alter the course of the response and the subsequent damage in patients.

GDNF protects the cochlea against noise damage.

Glial-derived neurotrophic factor (GDNF) was tested for its ability to prevent hearing and sensory cell loss in guinea pigs exposed to acoustic trauma. Hearing was measured prior to any treatment. Animals were exposed to damaging levels of noise either before or after local application of GDNF to one ear. Four weeks later, hearing and sensory cell loss was greater in the control ear than in the ear receiving GDNF before acoustic trauma or 2 h after trauma, but not 4 or 6 h after trauma. The results indicate that GDNF treatment in vivo can prevent cochlear sensory cell damage and hearing loss if present during or shortly after acoustic trauma.

The spiral ganglion and hair cells of Bronx waltzer mice.

Inner hair cells and spiral ganglion cells were counted in a mutant mouse strain (Bronx waltzer) reported to have very few of these cells (6) in order to determine if the remaining ganglion cells would be predominantly type II cells. These cell counts indicate a 50% reduction of spiral ganglion cells in Bronx waltzer cochleas compared to normal mice. Averaged throughout the cochlea about 11% of the remaining cells are type II cells while in normal mice this percentage is 5%. In some regions however, as many as 20% of the remaining cells are type II cells. Counts of IHC in surface preparations reveal 37 normal looking IHC (about 5% of the normal population) in each of two Bronx waltzer mouse cochleas. There were also about 120 shrunken IHC in each cochlea, representing 17% of the normal cell population. While there appears to be an increased proportion of type II cells in the Bronx waltzer spiral ganglion there are also many more type I cells than might be expected from the small number of IHC.

Spiral ganglion cell endings in the cochlear nucleus of young and old rats.

The spiral ganglion cells (SGCs) forming the auditory nerve have been shown to degenerate with age in both human and animal models, presumably resulting in post-synaptic sites in the cochlear nucleus (CN) that have lost their inputs. The present study examined the morphological changes in the surviving SGC endings in the CN of aged animals. The auditory nerves of 2-3 MO and 25-26 MO male, Fisher 344 rats were anterogradely labeled with horseradish peroxidase and the CN prepared histochemically. This resulted in Golgi-like labeling of afferent fibers and their terminals. All endings within each section were drawn and the area and number of components per ending were measured. Young and old animals both had about the same proportion of "simple", "string" and "complex" endings within the ventral CN, with complex endings being predominant in both age groups. The area of many complex endings was greater in the old animals with some endings being twice as large as any seen in young animals. There was no evidence of smaller endings in the old animals, suggesting that endings are not shrinking with age. A comparison of the number of components per complex ending revealed significantly more complexity in the endings of aged animals. Following the degeneration of SGCs it seems likely that the remaining cells, by increasing the area and altering the shape of their central terminals, may cover some of the post-synaptic sites made available by degenerated endings in aged animals.

Hair cell counts in an age-graded series of rat cochleas.

Hair cells of Sprague-Dawley rats aged 2-33 months were counted in order to assess the magnitude, location and time course of cell degeneration. The mean number of hair cell places (hair cells plus phalangeal scars) was approximately 4700: 960 inner hair cell places and 3470 outer hair cell places. These numbers do not vary systematically with age. Hair cell degeneration was observed in all animals. At 31-33 months of age, animals had inner hair cell losses ranging from 1.6 to 4.2% and outer hair cell losses ranging from 2.1 to 23.3%. The loss of hair cells was greatest in the upper apex, where the 31-33-month-old animals had 3.1-9.2% inner hair cell losses and 7.4-46.8% outer hair cell losses. Outer hair cell losses were also large in the basal end, where inner hair cell losses were small. In the older animals, hair cell losses were consistently most prominent in the third row of outer hair cells. Following examination of the hair cell population, the ganglion cells in the apical region were evaluated in a number of cochleas. No significant correlation was found between the magnitude of inner hair cell and ganglion cell losses.

Cochlear spiral ganglion cell degeneration in wild-caught mice as a function of age.

Presbyacusis in humans is an age-related bilateral sensorineural hearing impairment generally associated with degeneration of cochlear hair cells and spiral ganglion cells (SGC) predominantly in the basal turn but present in the apical turn. Investigations of cochleas of aged rats and gerbils reveal a large loss of SGCs in the apical as well as the basal turns. Genetically inbred aged mice, on the other hand, seem to have variable amounts of SGC loss beginning in some strains very early in the life span of the animals and greatest in the basal turn. Three age groups of wild-caught, then laboratory-bred, mice were investigated to determine the pattern of SGC degeneration. In 18-19-month-old animals the main loss of SGCs occurred in the basal turn (49% loss compared to 2-3 months) followed by the apical turn (31%). The greatest SGC losses in the 28-31-month-old animals were in both the apical (76%) and basal turns (74%). Thus, this strain of mice is similar to other rodents in that both ends of the ganglion are affected by SGC degeneration associated with aging.

2-Deoxyglucose uptake patterns in response to pure tone stimuli in the aged rat inferior colliculus.

The tonotopic map of the inferior colliculus (IC) of aged rats (25 months old) was examined to determine whether age-related changes known to occur in the cochlea are reflected in the 2-deoxyglucose (2-DG) uptake pattern of the IC. Because aged animals have hearing losses, auditory brainstem response thresholds were measured. Animals with threshold shifts of no greater than 30 dB relative to young animals were used. Animals were injected with radiolabeled 2-DG and stimulated with continuous pure tones presented at 70 dB above the behavioral thresholds for young animals at either 1, 4 or 32 kHz for one hour in a sound attenuated booth. The stimulus sound pressure levels were chosen to achieve comparable sensation levels between the young and aged animals. The tonotopic map of the IC in aged rats was different from that reported previously for young animals (Ryan et al., 1988), in that, the regions stimulated by 1 and 4 kHz were shifted towards the higher frequencies and the uptake areas were twice as broad for the aged animals as for the young animals. The observed 2-DG uptake patterns are consistent with an activation pattern of a high intensity stimulus and a loss of responsive elements in the cochlear apex. Similar broad and shifted bands of activated tissue may contribute to difficulties in auditory perception in aged humans with increased thresholds and sound amplification.

Spiral ganglion cell density in young and old gerbils.

The Mongolian gerbil, like other mammalian species, has a decreased number of spiral ganglion cells as a function of age. This loss of cells was first seen in 24- to 30-month old animals in the basal end of the ganglion. In the oldest individuals the apical end of the ganglion was also affected. There were approximately 15-25% fewer cells in the affected areas in the 36- to 42-month old animals. In the oldest animals degeneration of the stria vascularis was seen in the apical turn and some degenerative changes in the organ of Corti were seen throughout the length of the cochlear duct. The aging pattern in the gerbil cochlea, is similar to that described for other species. Vacuoles, previously described in the gerbil cochlear nucleus, were also seen in the auditory nerve within the modiolus, but central to the Schwann-glial border in all animals. Vacuoles were not present within the spiral ganglion or the peripheral processes of the ganglion cells. Because the ganglion cell axons should be similar on either side of the Schwann-glial border, but the vacuoles were confined to the central nervous system, it is concluded that the degenerative process affects glial cells as opposed to neurons.

Immunohistochemistry and microwave decalcification of human temporal bones.

Processing of human temporal bones is a long, expensive process and the resulting celloidin sections are difficult to use for immunohistochemistry. We tested the ability of immunohistochemical assays to work in human temporal bones that were decalcified using a microwave oven. Tissue was trimmed to an approximate cube (1.5-2 cm/side) containing only the cochlea and immersed in fresh EDTA with paraformaldehyde every 6 h. This sized block required 190-400 h to decalcify. The decalcified tissue was embedded in paraffin and sectioned. Sections were immunoassayed with anti-cytochrome c oxidase, anti-neurofilament or anti-peripherin. All three antibodies labeled the appropriate structures. This procedure may stimulate advancement in the understanding of human inner ear pathology.

The spatial representation of frequency in the rat dorsal cochlear nucleus and inferior colliculus.

The spatial distribution of neural activity produced by tones was assessed in the rat dorsal cochlear nucleus (DCN) and inferior colliculus (IC), using the 2-deoxyglucose (2-DG) technique. Eight pure tones, spanning the range of reported single unit characteristic frequencies in the rat, were presented at 40 dB above behavioral threshold. The relationship between frequency of stimulation and location of neural activity within each nucleus was evaluated quantitatively. Based on the 2-DG uptake pattern across animals, a tonotopic axis in the transverse plane was defined for each nucleus. This axis transected the centers of regions of evoked 2-DG uptake for each frequency. There was an orderly relationship between stimulus frequency and the location of evoked neural activity along the axis. Each pure tone stimulus activated an approximately equal proportion of this axis, for all frequencies tested, in both the DCN and IC. This suggests the existence of equal 'spatial bandwidths, in rat central auditory structures, across its entire frequency range. Equal spatial bandwidths could facilitate signal analysis strategies which require interaction between neurons with closely-related CFs. In the horizontal plane, however, the proportion of stimulated tissue was not equal across frequency. High-frequency (greater than 8 kHz) tones produced increased neural activity along a much greater extent of the anterior-to-posterior axis of the IC than did low-frequency tones.(ABSTRACT TRUNCATED AT 250 WORDS)

Cochlear degeneration in aged rats of four strains.

Animals with various degrees of inbreeding, some of which are albino, are frequently used in biological research. Albinos do not produce melanin and it is therefore absent from the cochlea. While the function of melanin is unknown, it has been hypothesized that it is involved in cochlear homeostasis. It is possible then, that age-related degeneration may be affected by the presence or absence of melanin. We therefore evaluated young (2-6 months old) and aged (24-36 months old) cochleas in 4 different rat strains: albino Fischer 344 and Lewis rats and pigmented Lewis-Brown Norway F1 rats and Brown Norway rats. Cochlear morphology was the same across all strains of young adult animals with the exception that the pigmented animals had small, darkly stained granules in the stria vascularis. The aged pigmented animals all had large granules as well as small ones. Degeneration of spiral ganglion cells in the apical region of the ganglion had occurred in the old animals of all strains. Strial degeneration at the apex was also present in aged animals. There was no correlation between the presence or absence of melanin and the magnitude of cochlear degenerative changes in the aged animals. The presence or absence of melanin therefore, appears to have no effect on cochlear degeneration in the aged rat cochlea.

Mitochondrial cytochrome oxidase immunolabeling in aged human temporal bones.

Presbycusis, an age-related hearing loss, is accompanied by histopathological cochlear changes including variable amounts of degeneration of the auditory receptors, neurons and the stria vascularis. The causes of degeneration are unknown, although acoustic trauma and exposure to ototoxic agents are certainly contributors to the cellular degeneration. Acquired mitochondrial DNA defects are postulated as important determinants of aging in neuromuscular tissues. The cochlear neurons are highly metabolic and are, therefore, likely to be affected by mitochondrial DNA defects. Sequence analysis has demonstrated a significant number of acquired mutations in the cytochrome oxidase gene in the neurons from aged human cochleas. The current study used immunohistochemical labeling of cytochrome oxidase in the neuronal cell bodies in archival celloidin sections to evaluate relationships among label density, hearing loss, number of neurons and mitochondrial DNA changes within individual cochleas. Label density was less in many aged temporal bones, but not all. There was no relationship among any other variables. It is concluded that while there may be a decrease in the amount of cytochrome oxidase expression in aged spiral ganglion cell bodies, there are many other factors that contribute to hearing loss and cellular degeneration.

Ultrastructural pathology in the stria vascularis of the MRL-Fasl(lpr) mouse.

The MRL-Fas(lpr) mouse, a model of multisystemic, organ nonspecific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. A preliminary study employing light microscopy indicated that it develops cochlear pathology that appeared most striking in the stria vascularis, where cells underwent edema and degeneration. However, other structures, including the inner and outer hair cells and the supporting cells, also appeared to display pathology. The current study analyzed cochlear ultrastructure using transmission electron microscopy to better delineate the cochlear lesions found in these animals. MRL-Fas(lpr) animals were allowed to develop systemic disease (20 weeks old) and then had auditory brainstem response (ABR) thresholds determined. Animals were then killed and their cochleas prepared for electron microscopy. Age-matched MRL-+/+ and BALB/c mice served as controls. Results indicated that MRL-Fas(lpr) mice demonstrated elevated ABR thresholds. In contrast to a preliminary report, the cochlear pathology was observed exclusively in the stria vascularis, where cells demonstrated hydropic degeneration. Strial capillary structure was normal as were the rest of the cellular cochlear constituents. No inflammatory infiltrate was noted. These studies confirm that the MRL-Fas(lpr) mouse develops cochlear abnormalities focused in the stria vascularis. Whether the mechanism of the cellular degeneration involves autoimmune, genetic, or uremic processes has yet to be determined.

Effects of a hair cell transcription factor, Brn-3.1, gene deletion on homozygous and heterozygous mouse cochleas in adulthood and aging.

The transcription factor Brn-3.1, is expressed in the inner ear hair cells throughout life and is necessary for the development of these cells. Mutant mice in which the Brn-3.1 encoding region has been deleted have no identifiable hair cells, greatly reduced numbers of spiral ganglion cells and are deaf. A mutation in the human homologue of this gene has been shown to be related to adult onset, sensorineural hearing loss (Vahava et al., 1998). The question whether haploinsufficiency in the mutant Brn-3.1 mouse with a mixed C57BL6/129Sv genetic background could affect the adult or aged cochlea was tested, therefore, by measuring the auditory brainstem responses and examining the cochlea's histologically at 2, 18 and 24 months of age. The heterozygotes had a comparable hearing to the wild-type animals and similar patterns of cochlear degeneration. Both groups showed an about 30 dB hearing loss beginning at 18 months of age, outer hair cell degeneration and loss of spiral ganglion neurons in the basal turn. There appeared to be no effect of Brn-3.1 haploinsufficiency on the mouse cochlea, implying that one intact copy of the gene is sufficient to maintain a normal cochlea.

Late sequelae of cochlear infection.

Inflammatory reactions within the inner ear are deleterious to cochlear function and can result in server hearing loss that does not recover. This study investigated a guinea pig model of long-term cytomegalovirus infection. At high doses active inflammation was still present after 35 days. At lower doses some ears showed partial resolution while others were still inflamed. Hearing was totally lost in all cases of persistent inflammation. There was some residual hearing in the cases that had resolved. Cochlear structures including the organ of Corti, stria vascularis, and spiral ganglion were partially degenerated. Fibrotic matrix within scala tympani was ossified in many cases. These changes are consistent with those described for human cochleas following putative viral infections.

Reduction of endotoxin-induced inflammation of the middle ear by polymyxin B.

Endotoxin (ET) is an aggregate of lipo-oligosaccharide and protein found in the cell wall of gram-negative bacteria. A potent mediator of inflammatory responses, ET has been detected in middle ear effusions from patients with otitis media with effusion and chronic suppurative otitis media and used to induce inflammation of the middle ear mucosa and disruption of mucociliary transport in experimental animals. Polymyxin B, a polypeptide antibiotic, has been shown to bond to and inactivate the ET molecule. This study investigated the efficacy of polymyxin B as a modulator of the inflammatory response to endotoxin in the middle ear. In a guinea pig model, cellular infiltrate, effusion volume, and mucosal edema in response to ET were reduced in the presence of polymyxin B. These results suggest a potential role for the use of polymyxin B in the management of middle ear effusion.