Role of Free Radical Formation in Murine Cytomegalovirus-Induced Hearing Loss.

OBJECTIVES: The goal of the study was to determine whether reactive oxygen species (ROS) mediates cytomegalovirus (CMV)-induced labyrinthitis. STUDY DESIGN: Murine model of CMV infection. SETTING: University of Utah laboratory. SUBJECTS AND METHODS: Nrf2 knockout mice were inoculated with murine CMV. Auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) were then performed on these and uninfected controls. BALB/c mice were inoculated with murine CMV to determine whether a marker for ROS production, dihydroethidium (DHE), is expressed 7 days after inoculation. Finally, 2 antioxidants-D-methionine and ACE-Mg (vitamins A, C, and E with magnesium)-were administered 1 hour before and after infection in inoculated mice for 14 days. Temporal bones were harvested at postnatal day 10 for DHE detection. ABR and DPOAE testing was done at postnatal day 30. Scanning electron microscopy was also performed at postnatal day 30 to evaluate outer hair cell integrity. RESULTS: Nrf2-infected mice had worse hearing than uninfected mice (P < .001). A statistically significant increase in DHE fluorescence was detected in BALB/c-infected mice as compared with uninfected mice 7 days after inoculation. D-methionine- and ACE-Mg-treated mice demonstrated an attenuation of the DHE fluorescence and a significant improvement in ABR and DPOAE thresholds when compared with untreated infected controls (P < .0001). Scanning electron microscopy demonstrated less outer hair cell loss in the treated versus untreated infected controls. CONCLUSION: These results demonstrate for the first time that excessive ROS mediates CMV-induced hearing loss in a mouse model.

Nitrogen and oxygen molecules in meningitis-associated labyrinthitis and hearing impairment.

Pneumococcal meningitis remains a serious disease with a case fatality rate of 15%-25%. Furthermore, long-term residues affect up to 50% of survivors. One of the most frequent sequelae is sensorineural hearing loss, which occurs in 26% of survivors of pneumococcal meningitis. Unfortunately, sufficient treatment regimens are still missing. New insights into the pathology and pathophysiology of meningitis-associated hearing loss have come from animal models of bacterial meningitis. Most likely, bacteria reach the cochlea through the cochlear aquaeduct. Once arrived in the perilymphatic spaces, they induce a severe suppurative labyrinthitis. The blood-labyrinth barrier breaks, hair cells are damaged, and neurons in the spiral ganglion undergo cell death, leading to meningitis-associated hearing loss. Reactive oxygen and nitrogen species, in particular peroxynitrite, seem to be among the crucial mediators of cochlear damage and hearing loss during meningitis. In our rat model of pneumococcal meningitis, adjunctive therapy with the antioxidants and peroxynitrite scavengers Mn(III)tetrakis(4-bencoic acid)-porphyrin (MnTBAP) and N-Acetyl-L-Cystein (NAC) significantly attenuated acute and long-term hearing loss. In several other animal studies of pneumococcal meningitis, adjunctive antioxidant therapy also protected infected animals from intracranial complications. Therefore, the use of antioxidants seems to be a promising future treatment option in pneumococcal meningitis.

Transforming growth factor beta expression during an inner ear immune response.

OBJECTIVES: The involvement of transforming growth factor beta (TGF-beta), a strong mediator of fibrogenesis, during cochlear immune responses was investigated. METHODS: An inner ear adaptive immune response to antigen was created in mice that were painlessly sacrificed 3 to 48 hours and 7 days after initiation of the immune response. The cochleas were assayed by immunocytochemistry for TGF-beta and latency-associated peptide (LAP). RESULTS: We found LAP expressed in normal cochleas and the endolymphatic sac, in the small round cells in the cochlear scalae and the mesothelial cells under the basilar membrane, and in the endolymphatic sac perisaccular area. We found TGF-beta expressed in infiltrated, inflammatory cells in the scalae and the endolymphatic sac lumen 3 hours after cochlear antigen challenge. At this time, LAP immunoreactivity was decreased. This rapid shift in immunoreactivity provides evidence for activation of TGF-beta during an immune response. This reversal of expression persisted for 48 hours, but conditions reverted to normal after 7 days. Surgical controls did not show TGF-beta expression. CONCLUSIONS: We conclude that TGF-beta activation occurs in the early phase of a cochlear adaptive immune response and is down-regulated as the response resolves. This finding suggests that the process of cochlear fibrosis starts early and that proper treatment could prevent cochlear fibrosis.

Round window membrane and labyrinthine pathological changes: an overview.

The round window membrane is considered the most likely pathway from the middle to the inner ear. Various substances placed in the middle ear have been seen to pass through the round window membrane. Once toxic substances or inflammatory mediators such as cytokines and nitric oxide enter the inner ear, various inner ear sequelae such as labyrinthitis, endolymphatic hydrops, sensorineural hearing loss or more insidious diseases can occur.

Location and timing of initial osteoid deposition in postmeningitic labyrinthitis ossificans determined by multiple fluorescent labels.

OBJECTIVES/HYPOTHESIS: Variable amounts of fibrosis and neo-ossification fill the cochlea following bacterial meningitis. The purpose of the study was to delineate the timing and location of initial ossification following pneumococcal meningitis, as well as subsequent remodeling and resorption, over the 3-month period after infection. STUDY DESIGN: Randomized, double-blind study. METHODS: Fluorochromes are compounds that specifically incorporate into ossifying bone. Sequential addition of different colored fluorochromes during osteoneogenesis define the timing and location of osteoid deposition and mineralization. Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3, and control gerbils received saline. Both groups were injected with calcein on postoperative day 3, followed by xylenol orange, oxytetracycline, and alizarin red on days 7, 14, and 28 respectively. Ten experimental gerbils were killed 24 hours after each label, and an additional group at 84 days after infection. Two groups of 10 control gerbils were killed at 29 and 84 days after treatment. The temporal bones and tibias were harvested, embedded in plastic, and sliced with a diamond saw. Wafers at a thickness of 200 microm were mounted in sequence and examined. RESULTS: Sixteen of 49 experimental animals (33%) were positive for at least one of the fluorescent labels. Fluorescent labeled osteoid was present at all sampling times. Label extended from the endosteal wall into the lumen of the scala tympani between the vestibule and the round window membrane. Discrete sites of fluorescence varied among specimens and were associated with the opening of the cochlear aqueduct, the scala tympani, organ of Corti, and the stria vascularis and spiral ligament in all turns from base to apex. CONCLUSION: The results indicate that osteoid is deposited and begins mineralization by day 3 after infection, at least, and continues, at least, through the first 28 days after infection. There was no apparent resorption of new bone and remodeling by 84 days after infection.

Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation.

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3-48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1beta, TNFalpha, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1beta and TNFalpha between 3 and 12 h after KLH injection. On the other hand, TNFalpha, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFalpha was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFalpha, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

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.

Experimental autoimmune labyrinthitis: assessment of molecular size of autoantigens in fractions of inner ear proteins eluted on the Mini Whole Gel Eluter.

In order to determine the molecular size of the causative autoantigens of experimental autoimmune labyrinthitis, crude inner ear antigen was separated into 14 fractions and the constituent molecules were identified by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Mini Whole Gel Eluter. The mean total protein recovery was approximately 66%. Sensitization with 55-65 kDa proteins induced the highest number of infiltrating cells among the fractions and sensitization with 38-45 kDa proteins induced the second highest number of inflammatory cells. These results suggest that 38-45 kDa and 55-65 kDa proteins are the causative autoantigens of experimental autoimmune labyrinthitis.

Inflammatory signals increase Fas ligand expression by inner ear cells.

There is considerable evidence that hearing and vestibular function can be influenced by immune processes. The inner ear has evolved mechanisms, such as the blood-labyrinthine barrier that limit immune responses and autoimmune processes to reduce the potential for damage to cochlear cells. Recently, expression of Fas ligand (FasL) in some non-lymphoid tissue, as in the anterior chamber of the eye, has been hypothesized to play a role in protection of sensitive organs from activated T-cells. We show that under resting conditions, cochlear cells express little or no FasL. However, after exposure to interferon-gamma in vitro, FasL is induced in many neonatal cochlear cells. In addition, we show that FasL is upregulated in adult cochlear cells after induction of a sterile labyrinthitis in vivo. The induction of FasL by inflammation may serve to limit cochlear immune responses, and to protect sensorineural tissue from immune and autoimmune damage.

Th1: mediator lymphocytes in experimental autoimmune labyrinthitis.

A previous study on experimental autoimmune labyrinthitis (EAL) consistently demonstrated transient infiltration of lymphocytes only into the inner ear of mice. To clarify the profile of lymphocytes in the initiation of EAL, the present study investigated cell surface antigens, as well as cytokines, from Day 4 to Day 35, using immunohistochemical techniques. Many CD4+ cells mainly infiltrated the endolymphatic sac as early as Day 4 and gradually spread to the rest of the inner ear. Infiltration peaked on Day 12 and persisted in most animals until Day 35, although the number of cells gradually decreased. In contrast, very few CD8+ cells were found to have appeared in the inner ear of all animals on Day 10, and the number of cells rapidly decreased. Many cells positive for IFN-gamma and IL-2 were identified in the endolymphatic sac on Day 4. These results suggest that helper T1 lymphocytes, rather than cytotoxic T lymphocytes, may play a central role in the initiation of EAL.

Possible involvement of free radicals in lipopolysaccharide-induced labyrinthitis in the guinea pig: a morphological and functional investigation.

Intratympanic injection of bacterial lipopolysaccharide impaired caloric responses and caused severe and widespread morphological damage to vestibular end organs and the endolymphatic sac in the guinea pig. These effects could be blocked with N-nitro-L-arginine methylester, a competitive inhibitor of nitric oxide synthase, with superoxide dismutase, an O2 scavenger, with dexamethasone, and with ebselen, a scavenger of peroxynitrite. These observations indicate that enhanced nitric oxide and superoxide production, resulting in formation of peroxynitrite, is probably an important factor responsible for the pathological damage to vestibuli. If this is so, we may have found a way to study vestibular pathogenesis in inner ear disease.

Expression of intercellular adhesion molecule-1 during inner ear inflammation.

This study was designed to investigate the expression of intercellular adhesion molecule-1 (ICAM-1) on the spiral modiolar vein (SMV) with its collecting venules (CVs) and the venules of the endolymphatic sac during inner ear inflammation. These data will further elucidate the role of adhesion molecules in extravasation of inflammatory cells from blood vessels during an inner ear immune response. Labyrinthitis was induced in rats by inoculation of keyhole limpet hemocyanin into the scala tympani of animals who had been systemically sensitized to it. Expression of ICAM-1 was examined with a mouse monoclonal antibody to rat ICAM-1 by immunohistochemistry. ICAM-1 was found weakly on the epithelium of SMVs and CVs as early as 6 hours postchallenge, reaching a maximum by day 2 and then fading away gradually. The maximum influx of immunocompetent cells into the cochlea was seen between days 3 and 7. Staining for ICAM-1 was observed on the epithelium of the endolymphatic sac and perisaccular region at 12 and 24 hours, respectively, and this was associated with infiltration of cells into these areas 3 days postchallenge. By day 28, the inner ear had developed endolymphatic hydrops, but at this time it showed almost no significant staining with anti-ICAM-1. The molecule was also expressed in the mesothelium of perilymph, the perineurium of cochlear nerves, the spiral ligament, and the basal cells of the stria vascularis following immunization. Our data provide evidence that endothelial cells of the SMV and its CVs, as well as other inner sites, have the potential to express ICAM-1. This expression precedes the influx of immune cells; therefore, it is possible that this ligand plays a pivotal role in the onset of inflammation in the inner ear. This study also confirmed that the immune response results in endolymphatic hydrops as a long-term consequence.