Macrophages in the Human Cochlea: Saviors or Predators-A Study Using Super-Resolution Immunohistochemistry.

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with "synapse-like" specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human's auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration.

Surgical access to the mammalian cochlea for cell-based therapies.

Cochlear implants are dependent on functionally viable spiral ganglion neurons (SGNs) - the primary auditory neurons of the inner ear. Cell-based therapies are being used experimentally in an attempt to rescue SGNs from deafness-induced degeneration or to generate new neurons. The success of these therapies will be dependent on the development of surgical techniques designed to ensure precise cell placement while minimizing surgical trauma, adverse tissue reaction and cell dispersal. Using 24 normal adult guinea pigs we assessed three surgical procedures for cell delivery into the cochlea: (i) a cochleostomy into the scala tympani (ST); (ii) direct access to Rosenthal's canal - the site of the SGN soma - via a localized fracture of the osseous spiral lamina (RC); and (iii) direct access to the auditory nerve via a translabyrinthine surgical approach (TL). Half the cohort had surgery alone while the other half had surgery combined with the delivery of biocompatible microspheres designed to model implanted cells. Following a four week survival period the inflammatory response and SGN survival were measured for each cohort and the location of microspheres were determined. We observed a wide variability across the three surgical approaches examined, including the extent of the inflammatory tissue response (TL>>RC> or =ST) and the survival of SGNs (ST>RC>>TL). Importantly, microspheres were effectively retained at the implant site after all three surgical approaches. Direct access to Rosenthal's canal offered the most promising surgical approach to the SGNs, although the technique must be further refined to reduce the localized trauma associated with the procedure.

Transfer of accelerated presbycusis by transplantation of bone marrow cells from senescence-accelerated mice.

Until now, there has been no effective therapy for chronic sensorineural hearing impairment. This study investigated the role of bone marrow cells (BMCs) in cochlear dysfunction. BALB/c mice (2 months of age), a non-presbycusis-prone mouse strain, were lethally irradiated and then transplanted with BMCs from SAMP1 mice (2 months of age), a presbycusis-prone mouse strain. Acceleration of age-related hearing loss, early degeneration of spiral ganglion cells (SGCs) and impairment of immune function were observed in the recipient mice as well as in the SAMP1 mice. However, no spiral ganglion cells of donor (SAMP1) origin were detected in the recipient mice. These results indicated that accelerated presbycusis, cochlear pathology, and immune dysfunction of SAMP1 mice can be transferred to BALB/c recipient mice using allogeneic bone marrow transplantation (BMT). However, although the BMCs themselves cannot differentiate into the spiral ganglion cells (SGCs), they indirectly cause the degeneration of the SGCs. Further studies into the relationship between the inner ear cells and BMCs are required.

Spiral ligament fibrocytes release chemokines in response to otitis media pathogens.

CONCLUSION: Spiral ligament fibrocytes (SLFs) may be involved in the innate immune response of the inner ear by producing chemoattractants for recruiting inflammatory cells such as neutrophils and monocytes. OBJECTIVE: The purpose of this study was to investigate the cellular responses of SLFs when challenged by inflammatory stimuli such as components of otitis media pathogens or proinflammatory cytokines. MATERIALS AND METHODS: To detect released inflammatory cytokines and chemokines, cells were treated for 48 h with whole lysates of nontypable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, or with interleukin 1 alpha (IL-1alpha). The culture medium was then collected and applied to protein arrays. To compare mRNA levels of chemokines, total RNA was extracted after 3 h of treatment with the above agents, and quantitative real-time PCR was performed. RESULTS: Protein array analysis showed that in response to NTHi or S. pneumoniae, rat SLFs released monocyte chemotactic protein 1, macrophage inflammatory protein 3 alpha, TNF-alpha, and cytokine-induced neutrophil chemoattractant 2 and 3. Treatment with IL-1alpha, on the other hand, resulted in release of MCP-1 but not the other molecules. Tissue inhibitor of metalloproteinase 1 and vascular endothelial growth factor were released regardless of the inflammatory stimulus used.

Localization of neurotensin immunoreactivity in neurons and organ of Corti of rat cochlea.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.

Distribution of beta-tubulin in guinea pig inner ear.

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.

Localization of gamma-aminobutyric acid A receptor subunits in the rat spiral ganglion and organ of Corti.

Gamma-aminobutyric acid (GABA) is thought to be the major inhibitory neurotransmitter in the central nervous system. Although it is distributed in the olivo-cochlear bundles, which constitute the mammalian cochlear efferent system, its function in the cochlea is still obscure. In this study, we investigated the localization of GABAa receptor subunits (alpha1-6, beta1-3, gamma) in the rat cochlea in order to determine the role of GABA in the cochlea. Most spiral ganglion cells were intensely immunolabeled with all the anti-GABAa receptor subunit antibodies. In the organ of Corti, punctate immunoreactivities were observed in inner hair cell regions corresponding to the distribution of GABA. These data suggest that GABAa receptor was present in afferent nerve terminals in inner hair cell regions, and that GABA regulated afferent nerve transmission contacting efferent nerve endings by means of the axo-dendritic synapse function.

Induction of MHC class II antigens on cells of the inner ear.

Growing evidence supports the concept that immune reactions occur in the cochlea, where they can function either in protection or as a source of inflammation. Since immunity is generally initiated by antigen presentation of foreign substances to T cells, antigen-presenting cells expressing major histocompatibility complex (MHC) class II molecules are required. Under resting conditions, cochlear cells usually express no MHC class II. However, we show that exposure to -interferon in vitro induces an increase in MHC class II expression in neonatal cochlear cells of mice. In addition, MHC class II immunoreactivity was observed in the inner ear of adult mice after induction of sterile labyrinthitis in vivo. It is concluded that the induction of MHC class II molecules by inflammation may render cochlear cells competent to initiate and participate in immune reactions and may therefore contribute to both immunoprotective and immunopathological responses of the inner ear.

Growth of postoperative remnants of unilateral vestibular nerve schwannoma: role of the vestibular ganglion.

Histopathological examination of seven temporal bones from patients who underwent a removal of vestibular nerve schwannomas by the translabyrithine or middle fossa approaches has demonstrated small tumor remnants that failed to grow as long as 25 years after surgery. In spite of the high incidence of residual tumors, the clinical recurrence rate of tumors operated at our institution by the translabyrinthine or middle fossa approaches is low (0.3%). Immunohistochemical labeling of dividing cells demonstrated that segments of tumor adjacent to the vestibular nerve and ganglion contained more dividing cells than were present in areas of the tumor at a distance from them.

[Cochlear and retrocochlear deafness induced by immunity with different inner ear tissue antigens].

OBJECTIVES: To inquire into whether the different inner ear tissue antigens could cause different kind of hearing loss, and to find out the disorder positions of auditory system. METHODS: The basilar membrane (BM), spiral ligament (SL), and spiral ganglion (SG) of guinea pigs were removed for making antigens, respectively. Then, we used these antigens to immune guinea pigs. The special humour and cellular immune reaction, hearing function, and inner ear histopathological changes were observed. RESULTS: In BM-antigen and SL-antigen immune group, the various degrees of cochlear microphonic potential disturbance, recruitment, and immune pathological inflammation in cochlear duct and stria vascularis were found. In SG-antigen immune group, auditory nerve compound action potential changes were prominent, and the inner ear pathological damage mainly existed in cochlear axis vessels or surround areas, and SG. CONCLUSIONS: It was confirmed that the inner ear antigens come from different part of auditory system, which could cause cochlear or retrocochlear autoimmune disease.

Image analysis of neurofilament immunoreactivity in human spiral ganglion.

Computer-assisted image analysis was used to study the immunoreactivity to NF-L, NF-M and NF-H in human spiral ganglion cells. The concentrations, represented by the relative mean grey of NF-L, NF-M and NF-H, were calculated and compared. The cell-area mean-grey correlations for NF-L, NF-M and NF-H were analysed and calculated, showing that NF-M is more specific to the larger cells (type 1?) and NF-H is more specific for the smaller cells (type 2?), while NF-L is non-specific for cell size. These findings confirm several previous assumptions by providing a quantitative basis. We conclude that image analysis is a useful-even essential-tool for the analysis of immunostained temporal bone sections.

Changes in the immunoreactivity of protein gene product (PGP) 9.5 in the cochlea of spontaneously diabetic WBN/Kob rats.

The mechanism of hearing impairment due to diabetes mellitus was examined in relation to changes in the level of the immunoreaction for the protein gene product 9.5 in the cochlea of spontaneously diabetic WBN/Kob rats. At 7 months (33 weeks) of age, when half of the males of this strain manifest diabetes, male WBN/Kob rats were divided into two groups as follows: one group consisted of prediabetic animals showing slightly decreased tolerance to glucose with a normal plasma concentration of glucose, normal urinary excretion of glucose, and functional hearing impairment (assessed in terms of elevation of hearing threshold). The second group consisted of diabetic animals with glucose intolerance, high plasma glucose level, polyuria, urinary glucose excretion, and more apparent elevation of hearing threshold. According to morphometric analysis of the spiral ganglion, the number of ganglion cells was significantly smaller in both the prediabetic and the diabetic animals than in the age-matched control Wistar rats. The staining intensity for protein gene product 9.5 was increased in some spiral ganglion cells of diabetic animals, but decreased in others according to quantitative immunohistochemical analysis. On the other hand, the immunoreactivity for protein gene product 9.5 was similar in the prediabetic animals to that in the control Wistar rats. These results suggest that numerical and immunohistochemical changes in the spiral ganglion cells reflect the onset and degree of the diabetic hearing impairment.

Distribution of endothelin-1-like activity in the cochlea of normal guinea pigs.

Distribution of endothelin-1 (ET-1) in the cochlea of normal guinea pigs was determined by immunohistochemistry. ET-1 activity was identified with the mouse anti-human ET-1 IgG1 monoclonal antibody. ET-1 activity was distributed in the modiolus, spiral ligament, stria vascularis, spiral prominence. Reissner's membrane, supporting cells of the organ of Corti and spiral ganglion cells. These findings suggest that ET-1 may be involved in the regulation of fluid volume and ions of the cochlea.

The localization and specificity of guinea pig inner ear antigenic epitopes.

In this study, we investigated the relative localization of some antigenic epitopes in the inner ear. The inner ear protein antigens were extracted from various parts of the guinea pig inner ear. Brain, kidney, lung, heart and liver extracts were also obtained. We found by SDS-polyacrylamide gel electrophoresis that total inner ear extracts separated into three high concentration polypeptide bands with molecular weights of approximately 30, 42, 58 kd and three low density bands of 20, 25 and 35 kd. The 30 kd band was found mainly in the extract of the spiral ganglion and the acoustic nerve in the modiolus. The 42 and 58 kd bands were detected in the extract of the spiral ligament and the stria vascularis. The Organ of Corti and the basilar membrane extract gave rise to three bands of 30, 42 and 58 kd. Twenty-eight of the 75 sera from patients with inner ear disease reacted with the 30 and 58 kd bands of the inner ear protein extracts by immunoblotting. Sixteen of these 28 positive sera were then used to probe immunoblots of the brain, kidney, lung, heart and liver extracts. The 58 kd band was also found in protein extracts of the brain, the lung and the liver. This study suggests that the 30 kd antigenic epitope may be mainly related to the acoustic nerve and that the 58 kd antigenic epitope is not cochlear specific.

The significance of herpes viral latency in the spiral ganglia.

To better understand the pathogenesis of idiopathic sudden hearing loss (ISHL), the possibility of latent virus infection in the spiral ganglion cell was considered. Only few spiral ganglion cells showed positive viral antigen after systemic guinea pig-specific cytomegalovirus (GPCMV) inoculation indicating the absence of hearing loss but the possibility of a subsequent latent infection. By using a modern molecular biological technique we have detected the herpes simplex virus type-1 (HSV-1) DNA in human spiral ganglia. The concept of establishing viral latency in the spiral ganglion cells with periods of reactivation fits with the clinical picture seen in ISHL, even though the mechanism of reactivation still remains unclear.