Biomedical subjects

E M Keithley

Publications and source records attributed to E M Keithley.

At least 19 recordsLinked to original sources

PubMed2001

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.

Aged↗

PubMed2001

Connection between the inner ear and the lymphatic system.

OBJECTIVE: The aim of this study was to identify the lymphatic drainage of the inner ear in guinea pigs. STUDY DESIGN: Prospective study. METHODS: The prospective study was performed in guinea pigs by injection of keyhole limpet hemocyanin (KLH) into either the right-side scala tympani or the middle ear cavity. The left side was not injected and served as a control. Fifteen minutes after injection, the animals were killed by intracardiac perfusion with paraformaldehyde and tissue specimens (right and left temporal bones, cervical lymph nodes, and the spleen) were collected. The presence of KLH in each specimen was determined by immunohistochemical assay of frozen sections using polyclonal mouse anti-KLH antibodies. RESULTS: After injection into the middle ear, labeled cells were identified in the parotid, superficial ventral, mandibular, and deep cranial cervical lymph nodes. However, after inner ear injections KLH was present in only the parotid and superficial ventral cervical nodes. The spleen contained KLH-positive cells following injection into either the middle or inner ear, but not all animals contained labeled spleen cells. CONCLUSIONS: The inner ear has a connection to the lymphatic drainage system. Because fewer lymph nodes contained labeled cells after inner ear injection than after middle ear injection, it is concluded that the inner ear does not simply drain to the middle ear and subsequently to the lymph nodes but seems likely to have its own connections.

Animals↗

PubMed2001

Autoimmune inner ear disorders.

There is considerable evidence to suggest that hearing and vestibular function can be influenced by autoimmune processes. A number of systemic autoimmune disorders include hearing loss and vertigo as part of their constellation of symptoms. Although classic evidence for a specific autoimmune etiology mediated by immune response directed solely at the inner ear is elusive, it appears that autoimmune damage can also exist as an entity confined to the labyrinth. Antigenic targets of autoimmunity within the labyrinth seem to be diverse. Partly because of this, the condition is difficult to diagnose. However, autoimmune disorders of the inner ear are of special interest since they are among the few forms of hearing loss that are amenable to medical treatment. Recent progress in understanding the etiology, diagnosis and treatment of autoimmune damage to the inner ear is reviewed.

Animals↗

PubMed2000

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.

Calcium↗

PubMed2000

Characterization of an experimentally induced inner ear immune response.

OBJECTIVE: To determine the effects of a sterile immune response on the structure and function of the cochlea. METHODS: An immune response was created in guinea pigs by systemically sensitizing the animals to keyhole limpet hemocyanin and subsequently challenging the inner ear with the protein. Animals were allowed to survive for 1 to 5 weeks, after which the cochlea was evaluated histologically. Hearing was measured by auditory brainstem response before the inner ear challenge, during the survival period, and prior to sacrifice. RESULTS: Inflammatory cells infiltrated the cochlea from the circulation. Surface preparations and plastic sections of the organ of Corti 1 and 2 weeks after the initiation of the inflammation demonstrated degeneration of the sensory and supporting cells in cochlear turns containing inflammatory cells. Good preservation of structures was seen in the more apical cochlear turns with little or no inflammatory cells. In cochleas from animals that survived 5 weeks, most of the infiltrated cells were cleared after undergoing apoptosis and the inflammatory matrix in the scala tympani began to calcify. Hearing loss was moderate to severe depending on the amount of inflammation. CONCLUSION: Although in general the immune response serves to protect an organism from infection, these results demonstrate that bystander injury associated with local immune responses in the cochlea, an organ incapable of regeneration, causes permanent cochlear destruction and hearing loss.

Adjuvants, Immunologic↗

PubMed2000

Intratympanic immunosuppressives for prevention of immune-mediated sensorineural hearing loss.

HYPOTHESIS: Round window membrane application of immunosuppressives reduces cochlear inflammation and hearing loss in a guinea pig model of sterile labyrinthitis. BACKGROUND: Systemic immunosuppressives are used to treat sensorineural hearing loss due to inflammation (autoimmune, bacterial, viral), which in animal models causes hearing loss. Transtympanic application of drugs is an attractive and possibly efficacious method of treatment that avoids systemic toxicities. METHODS: Sterile labyrinthitis was created using keyhole limpet hemocyanin (KLH). Dexamethasone (0.048 mg/day and 0.288 mg/day), cyclosporine (0.5%), prednisolone acetate (1%), fluorouracil, (5%), and FK506 (0.01%) were delivered to the round window membrane with one injection (filling middle ear space) or osmotic minipumps. Efficacy was evaluated by auditory brainstem response and histology. RESULTS: No drug was effective at reducing hearing loss or inflammation. CONCLUSION: Local application of immunosuppressives did not suppress inner ear inflammatory infiltrates and hearing loss in KLH-induced labyrinthitis in a guinea pig model.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Aging↗

PubMed1999

Application of labeling techniques to archival temporal bone sections.

Pathology of the human inner ear has traditionally been studied in celloidin-embedded, hematoxylin and eosin-stained sections of the temporal bone. Although the traditional histologic approach has yielded valuable information, it is now possible to extend these studies to include analysis of molecules using immunohistochemical and histochemical staining techniques. Fourteen antibodies and 6 lectins have been applied to 420 archival, celloidin-embedded human temporal bone sections. Tissues had been fixed in 10% formalin, embedded in celloidin, sectioned, and stored for as many as 40 years. The staining intensities varied among sections, so they were ranked from 'no label" to "dense label." To investigate the relationships between the extent of postmortem changes (PMCs), storage time, and staining intensity for each antibody, the sections were graded according to their PMCs, which ranged from good preservation of the temporal bone histologic structure to severe postmortem autolysis. Although statistical analysis indicated that both extent of PMCs and storage time in general decrease the staining intensity, both poorly fixed tissue and sections stored for a long time can yield good immunostaining results with some antibodies.

Antibodies↗

PubMed1998

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.

Animals↗

PubMed1998

Clinical diagnoses associated with histologic findings of fibrotic tissue and new bone in the inner ear.

Fibrotic tissue or new bone occurs following inner ear inflammation, fracture, or surgery. The prevalence is unknown and was investigated using the National Temporal Bone, Hearing and Balance Pathology Resource Registry database. A search yielded 264 temporal bones with diagnoses of otosclerosis, tumor, Meniere's disease, meningitis, labyrinthitis, chronic otitis media, autoimmune disease, temporal bone fracture, or sensorineural hearing loss. All autoimmune cases contained some new bone, whereas only 20% to 30% of the labyrinthitis/meningitis cases were reported to contain new bone. Otosclerosis, Meniere's disease, and otitis media had relatively few cases containing new bone. Although new bone may derive from surgical trauma, it is also likely to be a result of the disease process. It seems that all these disease processes may contain a common feature that acts as a stimulus to induce fibrosis or bone growth in the inner ear.

Autoimmune Diseases↗

PubMed1998

Immunohistochemical analysis of proliferating cells in a sterile labyrinthitis animal model.

Inflammatory reactions within the cochlea lead to the formation of fibrotic tissue and bone. To determine which cells are involved in the proliferation of the inflammatory response within the cochlea, sterile labyrinthitis was created by inoculating keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized animals. Cellular proliferation was assessed immunohistochemically using the monoclonal antibody Ki-67. Proliferating cells were identified among inflammatory cells and fibroblasts within the matrix, as well as in endosteal cells lining the scala tympani. Inflammatory and potential osteoprogenitor cells were labeled as late as 6 weeks after inoculation, suggesting the absence of a strong immunosuppressive mechanism. Endosteal cells may proliferate and secrete the extracellular matrix used by the inflammatory cells to move within the cochlear scalae. They may also participate in the ossification of the inflammatory matrix.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Comparison of anti-heat shock protein 70 (anti-hsp70) and anti-68-kDa inner ear protein in the sera of patients with Meniere's disease.

The 68-kDa antigen detected in the sera of patients with autoimmune inner ear disease is known to represent the highly inducible heat shock protein 70 (hsp70). To evaluate the existence of anti-hsp70 in the sera of patients with Meniere's disease and to develop a more reliable method to detect this antibody, the sera of patients and controls were examined. Bovine kidney (MDBK) cells were cultured and some of them were heat shocked. Proteins in the cells were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sera were reacted simultaneously with the blots of non-heat-shocked cells and heat-shocked cells. The serum was considered positive if the band in the 70-kDa location was denser in the lane with heat-shocked cells relative to non-heat-shocked cells. Presence of the antibody against the 68-kDa protein was compared with the result of immunoblotting with MDBK cells. In immunoblotting with MDBK cells, 33.3% of patients with Meniere's disease had anti-hsp70, while in the control group, only 5% had this antibody. Of the 60 cases, 13 were positive against both hsp70 and the 68-kDa protein, whereas 7 were positive only against hsp70 and 6 only against the 68-kDa protein. These differences appeared to result from the greater sensitivity of the differential anti-hsp assay and from difficulties in interpreting the results in blots with bovine inner ear extracts because of faint, broad, or overlapping multiple bands. Quite a number of patients with Meniere's disease have anti-hsp70, and it may be indicative of an immune etiology of the disease. The Western blot using heat-shocked and non-heat-shocked cells could be a reliable method to detect this antibody.

Adult↗

PubMed1996

Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.

The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn-3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

Animals↗

PubMed1996

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.

Aging↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Animals↗