Vestibular Aqueduct Morphology Correlates With Endolymphatic Sac Pathologies in Menière's Disease-A Correlative Histology and Computed Tomography Study.

HYPOTHESIS: The vestibular aqueduct (VA) in Menière's disease (MD) exhibits different angular trajectories depending on the presenting endolymphatic sac (ES) pathology, i.e., 1) ES hypoplasia or 2) ES degeneration. BACKGROUND: Hypoplasia or degeneration of the ES was consistently found in inner ears affected by MD. The two etiologically distinct ES pathologies presumably represent two disease "endotypes," which may be associated with different clinical traits ("phenotypes") of MD. Recognizing these endotypes in the clinical setting requires a diagnostic tool. METHODS: 1) Defining the angular trajectory of the VA (ATVA) in the axial plane. 2) Measuring age-dependent normative data for the ATVA in postmortem temporal bone histology material from normal adults and fetuses. 3) Validating ATVA measurements from normative CT imaging data. 4) Correlating the ATVA with different ES pathologies in histological materials and CT imaging data from MD patients. RESULTS: 1) The ATVA differed significantly between normal adults and MD cases with ES degeneration, as well as between fetuses and MD cases with ES hypoplasia; 2) a strong correlation between ATVA measurements in histological sections and CT imaging data was found; 3) a correlation between the ATVA, in particular its axial trajectory in the opercular region (angle αexit), with degenerative (αexit < 120°) and hypoplastic ES pathology (αexit > 140°) was demonstrated. CONCLUSION: We established the ATVA as a radiographic surrogate marker for ES pathologies. CT-imaging-based determination of the ATVA enables endotyping of MD patients according to ES pathology. Future studies will apply this method to investigate whether ES endotypes distinguish clinically meaningful subgroups of MD patients.

Clinical Imaging Findings of Vestibular Aqueduct Trauma in a Patient With Posttraumatic Meniere's Syndrome.

Posttraumatic Meniere's syndrome is a rare clinical entity. The pathomechanism by which temporal bone trauma leads to fluctuating audiovestibular symptoms, in some cases with a delay of onset many years after trauma, remains elusive. Here, a clinical case and the respective temporal bone imaging data were reviewed to investigate the underlying inner ear pathology. A 44-year-old patient presented with left-sided Meniere's syndrome 34 years after he suffered an ipsilateral temporal bone fracture caused by a car accident. Clinical imaging showed left cochleovestibular hydrops (gadolinium-enhanced MRI) and bony obliteration of the left VA (CT imaging), resulting in discontinuity of the ES. Our findings suggest that a temporal bone fracture with a "retrolabyrinthine" course, traversing the VA, caused intraaqueductal callus bone formation and progressive blockage of the VA. As a result, the extraosseous (distal) endolymphatic sac (eES) became separated from the cochleovestibular labyrinth, an event that presumably underlies endolymphatic hydrops formation and that precipitates the onset of clinical Meniere's symptoms in this case.

Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology.

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This "pressurized coverslipping" during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.

Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease.

Meniere's disease (MD), a syndromal inner ear disease, is commonly associated with a pathological accumulation of endolymphatic fluid in the inner ear, termed "idiopathic" endolymphatic hydrops (iEH). Although numerous precipitating/exacerbating factors have been proposed for MD, its etiology remains elusive. Here, using immunohistochemistry and in situ protein-protein interaction detection assays, we demonstrate mineralocorticoid-controlled sodium transport mechanisms in the epithelium of the extraosseous portion of the endolymphatic sac (eES) in the murine and human inner ears. Histological analysis of the eES in an extensive series of human temporal bones consistently revealed pathological changes in the eES in cases with iEH and a clinical history of MD, but no such changes were found in cases with "secondary" EH due to other otological diseases or in healthy controls. Notably, two etiologically different pathologies-degeneration and developmental hypoplasia-that selectively affect the eES in MD were distinguished. Clinical records from MD cases with degenerative and hypoplastic eES pathology revealed distinct intergroup differences in clinical disease presentation. Overall, we have identified for the first time two inner ear pathologies that are consistently present in MD and can be directly linked to the pathogenesis of EH, and which potentially affect the phenotypical presentation of MD.

Temporal bone histopathology in a case of sensorineural hearing loss caused by superficial siderosis of the central nervous system and treated by cochlear implantation.

OBJECTIVE: To evaluate the histopathology of the temporal bones of a patient with documented superficial siderosis of the central nervous system who underwent right cochlear implantation six years before death. BACKGROUND: Superficial siderosis of the central nervous system is due to chronic or repeated subarachnoid hemorrhage and results in sensorineural deafness in 95% of affected individuals in addition to other neurologic findings. The deposition of hemosiderin in the meninges and around cranial nerves is thought to be causative. There have been no previous reports of temporal bone pathology in this disorder.This 57 year old man developed progressive, bilateral hearing loss starting in his 30's with loss of pure tone thresholds and word recognition. He underwent a right cochlear implant at age 51 with full insertion of the device. METHODS: The temporal bones and brainstem were fixed in formalin and prepared for histologic study by standard techniques. Special stains, including Gomori stain for iron were performed on sections of the temporal bones and cochlear nucleus. RESULTS: There was severe bilateral degeneration of the organ of Corti, spiral ligament, stria vascularis, and spiral ganglion cells. Gomori stain revealed iron deposits within the spiral ligament, stria vascularis and in the subepithelial mesenchymal tissue of the maculae of the vestibular system. Evaluation of the cochlear nucleus revealed iron deposits within glial cells and larger cells, probably macrophages, near the CSF surface. On the right side, the track created by the cochlear implant entered the scala tympani and continued to mm17, as measured from the round window. DISCUSSION AND CONCLUSION: This is the first known case of superficial siderosis with documented temporal bone histopathology. Hearing loss was likely caused by severe degeneration of spiral ganglion cells in both ears, despite the presence of remaining hair cells in the middle and apical turns. This was consistent with cochlear neuronal degeneration and retrograde degeneration of spiral ganglion cells within the inner ear, or alternatively, consistent with primary degeneration of hair cells and neural structures within the cochlea. Despite the presence of neural degeneration, the patient achieved a word recognition score of 28% six months following implantation.

Extralabyrinthine manifestations of DFNA9.

DFNA9 is an autosomal dominant cause of non-syndromic adult-onset sensorineural hearing loss with associated variable vestibular dysfunction caused by mutations in the COCH gene. DFNA9 has previously been characterized by the presence of unique histopathologic features limited to the cochlear and vestibular labyrinth. This report describes newly discovered extralabyrinthine findings within the middle ear in DFNA9 and discusses their implications. The histopathologic anatomy of extralabyrinthine structures was reviewed in 12 temporal bones from seven individuals with DFNA9 and compared with age-matched controls. All temporal bones with DFNA9 had abnormal deposits within the tympanic membrane, incudomalleal joint, and incudostapedial joint. Hematoxylin and eosin stain and Movat's pentachrome stain both revealed different staining patterns of the extralabyrinthine deposits compared with the intralabyrinthine deposits suggesting that the composition of the deposits varies with location. The deposits within the tympanic membrane resembled cartilage morphologically and stained positively for aggrecan, an extracellular matrix protein found in cartilage. However, the cellular component of the tympanic membrane deposits did not stain with immunomarkers for chondrocytes (s100 and connective tissue growth factor). These novel findings in DFNA9 have implications for the phenotypic expression of the disorder and the clinical workup of adult-onset sensorineural hearing loss.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Techniques of celloidin removal from temporal bone sections.

OBJECTIVES: We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. METHODS: We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. RESULTS: Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol-sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. CONCLUSIONS: Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested.

Wolfram syndrome: a clinicopathologic correlation.

Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) is a neurodegenerative disorder characterized by diabetes mellitus and optic atrophy as well as diabetes insipidus and deafness in many cases. We report the post-mortem neuropathologic findings of a patient with Wolfram syndrome and correlate them with his clinical presentation. In the hypothalamus, neurons in the paraventricular and supraoptic nuclei were markedly decreased and minimal neurohypophyseal tissue remained in the pituitary. The pontine base and inferior olivary nucleus showed gross shrinkage and neuron loss, while the cerebellum was relatively unaffected. The visual system had moderate to marked loss of retinal ganglion neurons, commensurate loss of myelinated axons in the optic nerve, chiasm and tract, and neuron loss in the lateral geniculate nucleus but preservation of the primary visual cortex. The patient's inner ear showed loss of the organ of Corti in the basal turn of the cochleae and mild focal atrophy of the stria vascularis. These findings correlated well with the patient's high-frequency hearing loss. The pathologic findings correlated closely with the patient's clinical symptoms and further support the concept of Wolfram syndrome as a neurodegenerative disorder. Our findings extend prior neuropathologic reports of Wolfram syndrome by providing contributions to our understanding of eye, inner ear and olivopontine pathology in this disease.

Immunocytochemical traits of type IV fibrocytes and their possible relations to cochlear function and pathology.

One of the more consistent and least understood changes in the aging human cochlea is the progressive loss of fibrocytes within the spiral ligament. This report presents an animal model for type IV fibrocyte loss, along with immunocytochemical evidence that noise-induced loss of these cells may account for previously unexplained hearing losses. The remarkably low threshold for noise-induced loss of type IV fibrocytes, approximately 24 dB less than the threshold for adjacent hair cell destruction, may account for the prevalence of missing fibrocytes in humans. In mice, changes in the spectrum of traumatizing noise had little effect upon the site of loss of the fibrocytes, suggesting that the primary site of damage that induced the loss was the basal-most cochlear turn, a site expected to be damaged by all three noise bands. Type IV fibrocytes were found to immunostain for connective tissue growth factor (CTGF) and for transforming growth factor beta receptor 3, a receptor that is known to activate CTGF expression. Type IV fibrocytes lack immunostaining for adenosine triphosphatase and connexins that are key players in potassium ion uptake and transmission, which suggests that they play little, if any, role in potassium recycling from perilymphatic space to the endolymphatic space. Consequently, their loss probably does not directly reduce this process. Immunostaining for a receptor for CTGF, low-density-lipoprotein-related protein 1, indicated that CTGF acts as an autocrine and a paracrine agent within the cochlea. The lack of CTGF paracrine effects following noise-induced loss of type IV fibrocytes may account for previously unexplained hearing losses.

CARMA3 mediates lysophosphatidic acid-stimulated cytokine secretion by bronchial epithelial cells.

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.

Effects of fixative and embedding medium on morphology and immunostaining of the cochlea.

The localization of proteins by immunostaining is a powerful method to investigate otologic disorders. However, the use of fixatives and embedding media (necessary for the preservation of morphology) can obscure antigens, making it difficult to perform immunoassays. We performed a systematic investigation of the effects of fixative and embedding medium on morphology and immunostaining of the mouse cochlea. Three different fixative solutions [4% formaldehyde (F), 4% formaldehyde + 1% acetic acid (FA), and 4% formaldehyde + 1% acetic acid + 0.1% glutaraldehyde (FGA)] and 3 different embedding media (paraffin, polyester wax, and celloidin) were used. Morphology was assessed using light microscopy. Immunostaining was studied using a panel of 6 antibodies (to prostaglandin D synthase, aquaporin 1, connective tissue growth factor, 200-kDa neurofilament, tubulin and Na(+),K(+)-ATPase). Preservation of morphology was suboptimal with paraffin, adequate with polyester wax and superb with celloidin. Immunostaining was successful using all 6 antibodies in all 3 fixatives and all 3 embedding media. While there were differences in strength of signal and localization of antigen between the 3 fixatives, overall, FA and FGA gave the most uniform results. For a given fixative and antibody, there was surprisingly little difference in the quality of immunostaining between celloidin and paraffin, while results in polyester wax were not as good in some cases. These results suggest that celloidin may be the embedding medium of choice for both morphological and pathological studies, including immunostaining when morphology must be optimized.

Sudden deafness: is it viral?

A number of theories have been proposed to explain the etiopathogenesis of idiopathic sudden sensorineural hearing loss (ISSHL), including viral infection, vascular occlusion, breaks of labyrinthine membranes, immune-mediated mechanisms and abnormal cellular stress responses within the cochlea. In the present paper, we provide a critical review of the viral hypothesis of ISSHL. The evidence reviewed includes published reports of epidemiological and serological studies, clinical observations and results of antiviral therapy, morphological and histopathological studies, as well as results of animal experiments. The published evidence does not satisfy the majority of the Henle-Koch postulates for viral causation of an infectious disease. Possible explanations as to why these postulates remain unfulfilled are reviewed, and future studies that may provide more insight are described. We also discuss other mechanisms that have been postulated to explain ISSHL. Our review indicates that vascular occlusion, labyrinthine membrane breaks and immune-mediated mechanisms are unlikely to be common causes of ISSHL. Finally, we review our recently proposed theory that abnormal cellular stress responses within the cochlea may be responsible for ISSHL.

Cooperative function of Tbx1 and Brn4 in the periotic mesenchyme is necessary for cochlea formation.

The T-box transcription factor TBX1 has been identified as the major gene responsible for the etiology of velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS). Conductive hearing loss occurs in a majority of patients with this syndrome, while sensorineural deafness has also been reported in some cases. Mutations in POU3F4/BRN4, a POU domain transcription factor, cause DFN3, an X-linked nonsyndromic form of deafness characterized by mixed conductive and sensorineural hearing loss. Inactivation of the murine orthologues of these genes causes similar defects to those seen in humans and has provided excellent models for the study of inner ear development. Tbx1 and Brn4 are expressed in the mesenchymal cells surrounding the otic vesicle and have been shown to play roles in cochlear outgrowth. Furthermore, expression of Brn4 is reduced in Tbx1 null mutants, suggesting a possible genetic interaction between these genes. To test whether Tbx1 and Brn4 function in a common pathway, mice mutant for both genes were generated and analyzed for inner ear defects. Brn4-;Tbx1+/- mutants displayed a significant reduction in the number of turns of the cochlea compared to Brn4- or Tbx1+/- mice. In addition, Brn4-;Tbx1+/- mice displayed structural defects in the apical cochlea indicative of Mondini dysplasia found in patients with either VCFS/DGS or DFN3. These data establish a genetic interaction between Tbx1 and Brn4 relevant to human disease and indicate a function of these genes in signaling from the periotic mesenchyme to the otic vesicle to direct proper coiling of the cochlear duct.

Studies of otic capsule morphology and gene expression in the Mov13 mouse--an animal model of type I osteogenesis imperfecta.

Type I osteogenesis imperfecta (OI) is a disorder of skeletal bones characterized by bone fragility and blue sclera, which can result from mutations in genes encoding for type I collagen--the COL1A1 and COL1A2 genes. Fifty percent of patients with type I OI develop hearing loss and associated histopathological changes in the otic capsule that are indistinguishable from otosclerosis, a major cause of acquired hearing loss. In an attempt to elucidate molecular and cellular mechanisms of hearing loss in type I OI, we have studied the Mov13 mouse, which has served as an animal model of type I OI by virtue of exhibiting variable transcriptional block of the COL1A1 gene. We studied the morphometry of the Mov13 otic capsule and compared expression levels of 60 genes in the otic capsule with those in the tibia and parietal bone of the Mov13 and wild-type mice. The degree of transcriptional block of the COL1A1 gene and its downstream effects differed significantly between the bones examined. We found that expression levels of bone morphogenetic protein 3 and nuclear factor kappa-B1 best distinguished Mov13 otic capsule from wild-type otic capsule, and that osteoprotegerin, caspase recruitment domain containing protein 1, and partitioning defective protein 3 best distinguished Mov13 otic capsule from Mov13 tibia and parietal bone. Although the Mov13 mouse did not demonstrate evidence of active abnormal otic capsule remodeling as seen in type I OI and otosclerosis, studying gene expression in the Mov13 mouse has provided evidence that osteocytes of the otic capsule differ from osteocytes in other bones.

3p-- syndrome defines a hearing loss locus in 3p25.3.

Deletions affecting the terminal end of chromosome 3p result in a characteristic set of clinical features termed 3p-- syndrome. Bilateral, sensorineural hearing loss (SNHL) has been found in some but not all cases, suggesting the possibility that it is due to loss of a critical gene in band 3p25. To date, no genetic locus in this region has been shown to cause human hearing loss. However, the ATP2B2 gene is located in 3p25.3, and haploinsufficiency of the mouse homolog results in SNHL with similar severity. We compared auditory test results with fine deletion mapping in seven previously unreported 3p-- syndrome patients and identified a 1.38Mb region in 3p25.3 in which deletions were associated with moderate to severe, bilateral SNHL. This novel hearing loss locus contains 18 genes, including ATP2B2. ATP2B2 encodes the plasma membrane calcium pump PMCA2. We used immunohistochemistry in human cochlear sections to show that PMCA2 is located in the stereocilia of hair cells, suggesting its function in the auditory system is conserved between humans and mice. Although other genes in this region remain candidates, we conclude that haploinsufficiency of ATP2B2 is the most likely cause of SNHL in 3p-- syndrome.

Cochleosaccular dysplasia associated with a connexin 26 mutation in keratitis-ichthyosis-deafness syndrome.

OBJECTIVE: The objective of this study was to characterize the temporal bone phenotype associated with a mutation of GJB2 (encoding connexin 26). STUDY DESIGN: The authors conducted correlative clinical, molecular genetic, and postmortem histopathologic analysis. METHODS: The study subject was a male infant with keratitis-ichthyosis-deafness (KID) syndrome. We performed a nucleotide sequence analysis of GJB2 and a histopathologic analysis of the temporal bones. RESULTS: The subject was heterozygous for G45E, a previously reported KID syndrome mutation of GJB2. The primary inner ear abnormality was dysplasia of the cochlear and saccular neuroepithelium. CONCLUSIONS: GJB2 mutations can cause deafness in KID syndrome, and possibly in other GJB2 mutant phenotypes, by disrupting cochlear differentiation.

Tissue-specific roles of Tbx1 in the development of the outer, middle and inner ear, defective in 22q11DS patients.

Most 22q11.2 deletion syndrome (22q11DS) patients have middle and outer ear anomalies, whereas some have inner ear malformations. Tbx1, a gene hemizygously deleted in 22q11DS patients and required for ear development, is expressed in multiple tissues during embryogenesis. To determine the role of Tbx1 in the first pharyngeal pouch (PPI) in forming outer and middle ears, we tissue-specifically inactivated the gene using Foxg1-Cre. In the conditional mutants, PPI failed to outgrow, preventing the middle ear bone condensations from forming. Tbx1 was also inactivated in the otic vesicle (OV), resulting in the failure of inner ear sensory organ formation, and in duplication of the cochleovestibular ganglion (CVG). Consistent with the anatomical defects, the sensory genes, Otx1 and Bmp4 were downregulated, whereas the CVG genes, Fgf3 and NeuroD, were upregulated. To delineate Tbx1 cell-autonomous roles, a more selective ablation, exclusively in the OV, was performed using Pax2-Cre. In contrast to the Foxg1-Cre mutants, Pax2-Cre conditional mutant mice survived to adulthood and had normal outer and middle ears but had the same inner ear defects as the Tbx1 null mice, with the same gene expression changes. These results demonstrate that Tbx1 has non-cell autonomous roles in PPI in the formation of outer and middle ears and cell-autonomous roles in the OV. Periotic mesenchymal markers, Prx2 and Brn4 were normal in both conditional mutants, whereas they were diminished in Tbx1-/- embryos. Thus, Tbx1 in the surrounding mesenchyme in both sets of conditional mutants cannot suppress the defects in the OV that occur in the null mutants.