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Hypothesis: Transforming growth factor beta-1 (TGFβ-1) and connective tissue growth factor (CTGF) are upregulated in the implanted human cochlea., Background: Cochlear implantation can lead to insertion trauma and intracochlear new tissue formation, which can detrimentally affect implant performance. TGFβ-1 and CTGF are profibrotic proteins implicated in various pathologic conditions, but little is known about their role in the cochlea. The present study aimed to characterize the expression of these proteins in the human implanted cochlea., Methods: Archival human temporal bones (HTB) acquired from 12 patients with previous CI and histopathological evidence of new tissue formation as well as surgical samples of human intracochlear scar tissue surrounding the explanted CI were used in this study. Histopathologic analysis of fibrosis and osteoneogenesis was conducted using H&E. Protein expression was characterized using immunofluorescence. RNA expression from surgical specimens of fibrotic tissue surrounding the CI was quantified using qRT-PCR., Results: TGFβ-1 and CTGF protein expressions were upregulated in the areas of fibrosis and osteoneogenesis surrounding the CI HTB. Similarly, surgical samples demonstrated upregulation of protein and mRNA expression of TGFβ-1 and mild upregulation of CTGF compared with control. TGFβ-1 was expressed diffusely within the fibrous capsule, whereas CTGF was expressed in the thickened portion toward the modiolus and the fibrosis-osteoneogensis junction., Conclusion: To our knowledge, this is the first study to demonstrate increased expression of TGFβ-1 and CTGF in the human implanted cochlea and may provide better understanding of the mechanism behind this pathogenic process to better develop future mitigating interventions., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

Background: Otosclerosis is a common ear disease that causes fixation of the stapes and conductive hearing impairment. However, the pathogenesis of otosclerosis is still unknown. Otosclerosis could be associated with the unique bony environment found in the otic capsule. Normal bone remodelling is almost completely absent around the inner ear after birth allowing degenerative changes and dead osteocytes to accumulate. High levels of inner ear anti resorptive osteoprotegerin (OPG) is most likely responsible for this capsular configuration. Studies have demonstrated how osteocyte lifespan variation creates occasional clusters of dead osteocytes, so-called cellular voids, at otosclerotic predilection sites in the human otic capsule. These cellular voids have been suggested as possible starting points of otosclerosis., Aim: To describe the cellular viability in otosclerotic lesions and compare it to that of cellular voids., Materials and Methods: The study was based on unbiased stereological quantifications in undecalcified human temporal bones with otosclerosis., Results: Osteocyte viability was found to vary within the otosclerotic lesions. Furthermore, the results presented here illustrate that inactive otosclerotic lesions consist of mainly dead interstitial bone, much like cellular voids., Conclusions and Significance: Focal degeneration in the otic capsule may play an important role in the pathogenesis of otosclerosis.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2 , the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

To investigate the possible association of pathogenic mutations of SLC26A4 and computerized tomography (CT) phenotypes of inner ear, and explore the feasibility of using the method of gene sequence analysis. A total of 155 patients with bilateral hearing loss carrying SLC26A4 gene mutations were further subjected to high-resolution temporal bone CT and thyroid B ultrasound tests. The potential relationship between the pathogenic mutations of gene and the CT phenotypes were analyzed. As a result, 65 patients (41.9%, 65/155) carried SLC26A4 gene mutations, and 27 cases were detected with pathogenic mutations of SLC26A4 where IVS7-2A>G (55.6%, 15/27) was the most common pathogenic mutation. Amongst them, 19 patients carrying bi-allelic SLC26A4 mutations were all confirmed to have inner ear malformation by CT scan including four cases of enlarged vestibular aqueduct (EVA) and 15 cases of Mondini dysplasia (MD). However, there was only one in eight cases of single allele pathogenic mutation who was confirmed to have EVA by CT scan. Further, only one patient with EVA was confirmed to be slightly higher of total T3 than normal by thyroid ultrasound scan and thyroid hormone assays. These findings suggested that CT detection and SLC26A4 gene detection are efficient methods to diagnose EVA, which can complement each other. Also, the bi-allelic pathogenic mutations of SLC26A4 are more likely to induce inner ear malformation than single allele pathogenic mutation., (© 2019 The Author(s).)

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 reconstruction is a persisting problem following middle ear cholesteatoma surgery. Seeking to advance the clinical transfer of stem cell therapy we attempted the reconstruction of temporal bone using a composite bioartificial graft based on a hydroxyapatite bone scaffold combined with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). The aim of this study was to evaluate the effect of the combined biomaterial on the healing of postoperative temporal bone defects and the preservation of physiological hearing functions in a guinea pig model. The treatment's effect could be observed at 1 and 2 months after implantation of the biomaterial, as opposed to the control group. The clinical evaluation of our results included animal survival, clinical signs of an inflammatory response, and exploration of the tympanic bulla. Osteogenesis, angiogenesis, and inflammation were evaluated by histopathological analyses, whereas hBM-MSCs survival was evaluated by immunofluorescence assays. Hearing capacity was evaluated by objective audiometric methods, i.e. auditory brainstem responses and otoacoustic emission. Our study shows that hBM-MSCs, in combination with hydroxyapatite scaffolds, improves the repair of bone defects providing a safe and effective alternative in their treatment following middle ear surgery due to cholesteatoma.

The present study is to investigate the reason why the ceratohyal cartilage (CH) angle of zebrafish larvae were larger compared to the control group after their female parents were treated with cadmium (F-Cd). However, the CH angle was smaller compared to the control group when embryos were directly exposed to Cd 2+ for 72 h (D-Cd). Results showed that calcium contents of larvae were lower than the control, but the transporter isoforms trpv4 and trpv6 mRNA expressions were significantly increased upon D-Cd treatment. Furthermore, external Ca 2+ added during D-Cd treatment reveals that the CH angles of larvae did not appear significantly different compared to the control. On the other hand, E2 (17β-estradiol) contents were higher around 1.9 folds in the ovaries of females; CH angle were over 25°, and Cd 2+ contents were higher around 6 folds than the control group on larvae treated through F-Cd treatment; CH angles and E2 levels on larvae were higher than the control after the larvae were treated with 1.84 μM E2 (D-E2); Estradiol receptor (ER) isoforms ERβ1 and ERα mRNA expressions significantly increased when 0 hpf embryos were either treated with D-E2 or D-Cd. According to the results, we suggested that the CH angle of larvae become larger upon F-Cd treatment due to maternal Cd 2+ inducing E2 levels. However, the CH angle of larvae appeared to be smaller compared to the control upon D-Cd treatment. We suggested that the CH angle decreased due to the decrease of Ca 2+ contents upon Cd 2+ exposure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Many X-linked non-syndromic hearing loss (HL) cases are caused by various mutations in the POU domain class 3 transcription factor 4 (POU3F4) gene. This study aimed to identify allelic variants of this gene in two Chinese families displaying X-linked inheritance deafness-2 (DFNX2) and one sporadic case with indefinite inheritance pattern., Methods: Direct DNA sequencing of the POU3F4 gene was performed in these families and in 100 Chinese individuals with normal hearing., Results: There are characteristic imaging findings in DFNX2 Chinese families with POU3F4 mutations. The temporal bone computed tomography (CT) images of patients with DFNX2 are characterized by a thickened stapes footplate, hypoplasia of the cochlear base, absence of the bony modiolus, and dilated internal acoustic meatus (IAM) as well as by abnormally wide communication between the IAM and the basal turn of the cochlea. We identified three causative mutations in POU3F4 for three probands and their extended families. In family 1468, we observed a novel deletion mutation, c.973delT, which is predicted to result in a p.Trp325Gly amino acid frameshift. In family 2741, the mutation c.927delCTC was identified, which is predicted to result in the deletion of serine at position 310. In both families, the mutations were located in the POU homeodomain and are predicted to truncate the C-terminus of the POU domain. In the third family, a novel de novo transversion mutation (c.669 T > A) was identified in a 5-year-old boy that resulted in a nonsense mutation (p.Tyr223*). The mutation created a new stop codon and is predicted to result in a truncated POU3F4 protein., Conclusions: Based on characteristic radiological findings and clinical features, POU3F4 gene mutation analysis will increase the success rate of stapes operations and cochlear implantations, and improve molecular diagnosis, genetic counseling, and knowledge of the molecular epidemiology of HL among patients with DFNX2.

Hypothesis: Connexin-26 (Cx26) expression is diminished in the spiral ligament of subjects with hearing loss and cochlear otosclerosis (CO)., Background: Human temporal bone (HTB) studies have demonstrated that CO is associated with hyalinization of the spiral ligament. We hypothesize that hyalinization is associated with a loss of fibrocytes with a consequent decline in Cx26 expression. Cx26 and Connexin-30 (Cx30) encode gap junction proteins expressed in supporting cells of the organ of Corti, the spiral limbus, stria vascularis, and in fibrocytes of the spiral ligament. These gap junctions are critical for potassium recycling and maintenance of the endocochlear potential. Diminished expression of these proteins would likely be associated with hearing dysfunction., Methods: Histopathology and clinical characteristics of 45 HTB specimens with CO and spiral ligament hyalinization were reviewed. Those with sensorineural or mixed hearing loss but normal or near-normal hair cell counts were analyzed with light microscopy, and Cx26-immunoreactive (IR) signal was qualitatively assessed., Results: H&E staining demonstrated hyalinization in the spiral ligament and loss of type II and type III fibrocytes. Cx26-IR was diminished throughout the cochlea affected with CO compared with normal controls., Conclusions: Cx26-IR reduction in the spiral ligament of subjects with CO likely plays a role in hearing loss.

Background: Superior semicircular canal dehiscence (SSCD) is a bony defect in the osseous shell of the petrous temporal bone. The pathophysiological association between osteoporosis and SSCD remains poorly understood. We investigated the relationship between bone metabolic markers and symptoms in patients with SSCD., Methods: We collected patient demographics and clinical parameters for adult patients diagnosed with SSCD on high-resolution computed tomography scans. We used point-biserial correlation analysis to investigate the relationship between bone metabolic markers and symptoms in patients with SSCD. We compared clinical symptoms before and after surgical repair of SSCD through a middle fossa craniotomy using McNemar's test for paired comparisons of binary measures., Results: We included a total of 99 patients (64 females and 35 males; average age 52 years; 118 surgeries). The level of serum calcium correlated with the need for a second surgery (r pb  = -0.35, P = 0.001). Postoperative calcium supplementation negatively correlated with improvement in dizziness (r pb  = -0.36, P = 0.01). The level of 25-hydroxyvitamin D correlated with preoperative hyperacusis (r pb  = -0.98, P = 0.02) and postoperative autophony (r pb  = 0.96, P = 0.04). Postoperative vitamin D supplementation positively correlated with hearing decline (r pb  = 0.04, P = 0.04) The level of thyroid stimulating hormone correlated with preoperative autophony, amplification, and tinnitus (r pb  = -0.71, r pb  = -0.75, r pb  = -0.70, all P < 0.001)., Conclusions: Bone metabolic markers could be important in the clinical assessment of SSCD patients and could be potential targets for symptom management., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The most crucial clinical problem of Eosinophilic Otitis Media (EOM) is sensorineural hearing loss. A previous report revealed that repeated antigen stimulation of middle ear causes eosinophilic inflammation not only in the middle ear but also in the inner ear., Objective: The purpose of the present study was to elucidate the mechanism of eosinophil infiltration to the inner ear in the animal model of EOM., Methods: Continuous OVA stimulation to the middle ear of guinea pigs was performed for 7 days, 14 days, and 28 days. Histological observation was made for eosinophil infiltration and morphological change of the inner ear. Immunostaining for eotaxin and RANTES was performed to study immunoreactivity of those chemokines., Results: In the 7-day stimulation side, a few eosinophils were found in the scala tympani, without obvious morphological damage of the inner ear. Moreover, immunoreactivity of both eotaxin and RANTES was significantly higher in the OVA stimulation sides than control sides. For both eotaxin and RANTES, the number of immunopositive cells was significantly increased in the 14-day stimulation side over the 7-day stimulation side., Conclusions: Eotaxin and RANTES seem to play some important roles for the eosinophil infiltration in the middle and inner ear of model animal of EOM.

Purpose: Cortactin is a multidomain protein engaged in several cellular mechanisms involving actin assembly and cytoskeletal arrangement. Cortactin overexpression in several malignancies has been associated with increased cell migration, invasion, and metastatic potential. Cortactin needs to be activated by tyrosine or serine/threonine phosphorylation. The role of cortactin and phosphorylated cortactin (residue tyr 466 ) was investigated in temporal bone squamous cell carcinoma (TBSCC)., Materials and Methods: Immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr 466 ) was assessed in 27 consecutively-operated TBSCCs., Results: Several clinicopathological variables correlated with recurrence (pT stage, dura mater involvement), and disease-free survival (DFS) (cT stage, pT stage, pN status, dura mater involvement). Twenty-three of 24 immunohistochemically evaluable TBSCCs were cortactin-positive. Median cortactin expression was 75.0%. Cortactin reaction in the cytoplasm was more intense in carcinoma cells than in normal adjacent tissue. Recurrence and DFS rates did not correlate with cortactin and phosphorylated cortactin (residue tyr 466 ) expression in TBSCC specimens., Conclusions: Cortactin upregulation in TBSCC supports the conviction that inhibiting cortactin functions could have selective effects on this malignancy. Multi-institutional studies should further investigate the role of cortactin and phosphorylated cortactin in TBSCC, and their potential clinical application in integrated treatment modalities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An understanding of the molecular pathology that underlies noise induced neurotoxicity is a prerequisite to the design of targeted therapies. The objective of the current experiment was to determine whether or not DNA damage is part of the pathophysiologic sequela of noise induced neurotoxicity. The experiment consisted of 41 hooded Long-Evans rats (2 month old males) that were randomized into control and noise exposed groups. Both the control and the noise group followed the same time schedule and therefore started and ended the experiment together. The noise dose consisted of a 6000 Hz noise band at 105 dB SPL. Temporal bones from both groups were harvested, and immunohistochemistry was used to identify neurons with DNA damage. Quantitative morphometric analyses was then employed to determine the level of DNA damage. Neural action potentials were recorded to assess the functional impact of noise induced DNA damage. Immunohistochemical reactions revealed that the noise exposure precipitated DNA damage within the nucleus of auditory neurons. Quantitative morphometry confirmed the noise induced increase in DNA damage levels and the precipitation of DNA damage was associated with a significant loss of nerve sensitivity. Therefore, DNA damage is part of the molecular pathology that drives noise induced neurotoxicity. Anat Rec, 300:520-526, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objectives: Melanin pigmentation is present in the human inner ear. In this study, we quantify the melanin pigmentation in the vestibular system and examine racial differences of vestibular melanin pigmentation using human cadaveric temporal bone specimens., Study Design: Basic research., Setting: Laboratory., Subjects and Methods: Light microscopy was used to examine specimens from 40 left temporal bones from the Johns Hopkins Human Temporal Bone Collection. Color images of (1) ampulla of the horizontal canal, (2) utricular wall, (3) endolymphatic duct, and (4) posterior ampullary nerve as it enters the posterior canal were acquired with a digital camera attached to the microscope and image acquisition software. Acquired images of each anatomic area of interest were processed offline through ImageJ. Melanin content was then compared according to ethnicity, age, sex, and location., Results: Fifteen African American and 25 Caucasian specimens were analyzed. Mean age was 68.8 years. African American specimens had a significantly greater amount of pigment at all 4 sampled locations as compared with Caucasian specimens (P < .01). Between sexes, there was a statistically significant difference (P < .05) at the posterior ampullary nerve, with males having more than females. Melanin content was not associated with age., Conclusions: There is greater melanin pigmentation within the vestibular system of African Americans than in Caucasians, similar to what has been described in the cochlea. Racial differences in vestibular physiologic function have been observed in the literature and may be explained by differences in melanin pigmentation., (© American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.)

Hypothesis: Identification, characterization, and location of cells involved in the innate immune defense system of the human inner ear may lead to a better understanding of many otologic diseases and new treatments for hearing and balance-related disorders., Background: Many otologic disorders are thought to have, as part of their disease process, an immune component. Although resident macrophages are known to exist in the mouse inner ear, the innate immune cells in the human inner ear are, to date, unknown., Methods: Primary antibodies against CD163, Iba1, and CD68 (markers known to be specific for macrophages/microglia) were used to immunohistochemically stain celloidin embedded archival temporal bone tissue of normal individuals with no known otologic disorders other than changes associated with age., Results: Cells were positively stained throughout the temporal bone within the connective tissue and supporting cells with all three markers. They were often associated with neurons and on occasion entered the sensory cell areas of the auditory and vestibular epithelium., Conclusions: We have immunohistochemically identified an unappreciated class of cells in the normal adult inner ear consistent in staining characteristics and morphology with macrophages/microglia. As in other organ systems, it is likely these cells play an essential role in organ homeostasis that has not yet been elucidated within the ear.

Objective. Blood-brain barrier (BBB) is a key obstacle that prevents the medication from blood to the brain. Microbubble-enhanced cavitation by focused ultrasound can open the BBB and proves to be valuable in the brain drug delivery. The study aimed to explore the feasibility, efficacy, and safety of unilateral opening of BBB using diagnostic ultrasound targeted microbubbles destruction in rats. Methods. A transtemporal bone irradiation of diagnostic ultrasound and intravenous injection of lipid-coated microbubbles were performed at unilateral hemisphere. Pathological changes were monitored. Evans Blue extravasation grades, extraction from brain tissue, and fluorescence optical density were quantified. Lanthanum nitrate was traced by transmission electron microscopy. Results. After diagnostic ultrasound mediated microbubbles destruction, Evans Blue extravasation and fluorescence integrated optical density were significantly higher in the irradiated hemisphere than the contralateral side (all p < 0.01). Erythrocytes extravasations were demonstrated in the ultrasound-exposed hemisphere (4 ± 1, grade 2) while being invisible in the control side. Lanthanum nitrate tracers leaked through interendothelial cleft and spread to the nerve fiber existed in the irradiation side. Conclusions. Transtemporal bone irradiation under DUS mediated microbubble destruction provides us with a more accessible, safer, and higher selective BBB opening approach in rats, which is advantageous in brain targeted drugs delivery.

Temporal bone squamous cell carcinoma (TBSCC) is an uncommon, aggressive malignancy with a significant local recurrence rate even in patients with postoperative pathology reports of free surgical margins. This raises the question of how "free" negative margins should be to be oncologically safe, especially in bone tissue. A potential role for relaxin-2 hormone in tumor-driven osteolysis has recently been reported. The aim of this study was to assess the prognostic role of relaxin-2 expression in TBSCC tissue specimens and pathologically negative bone margins. Relaxin-2 immunohistochemical expression was assessed in 25 consecutively operated TBSCC patients. Several pathological variables correlated with recurrence rate (pT stage, dura mater involvement), disease-free survival (DFS) (pT stage, pN status, grade, and dura mater involvement), and disease-specific survival (DSS) (pT stage, pN status, grade, and dura mater involvement). The recurrence rate, DFS, and DSS did not correlate with relaxin-2 expression in TBSCC specimens or pathologically negative bone margins. Although local recurrence in TBSCC could relate to neoplastic bone invasion not apparent on conventional pathological investigations, the present preliminary findings seem to rule out any role of relaxin-2 in mediating this local aggressiveness. Molecular mechanisms of TBSCC recurrence after curative treatment should be further investigated.

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.

Purpose: We analyzed the anatomical structure of the temporomandibular joint (TMJ) and molecular weight dependency of synovial membrane permeability in mice using 7-tesla magnetic resonance (MR) imaging., Methods: We obtained 3-dimensional (3D) T1-weighted gradient echo (3D-T1W) and 3D T2-weighted rapid acquisition with relaxation enhancement (3D-T2W RARE) MR images of the TMJ of male C57BL6 mice with voxel resolution of 65 µm. Two-dimensional (2D) T1w images were measured every 45 s before and after bolus intravenous (IV) injection of contrast reagents: gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA; 0.5 kDa); oligomer-based contrast agent (CH3-DTPA-Gd; 2.1 kDa); gadolinium-labeled polylysine (Gd-polylysine; 10 kDa); and gadolinium-labeled albumin (Gd-albumin; 74 kDa)., Results: T1W images depicted the temporal bone and mandibular condyle as regions with lower signal intensity and the disc as a region of intermediate intensity. In the Gd-DTPA-enhanced T1W and T2W images, the articular disc could be identified as a region with lower signal intensity than that of the upper and lower joint cavities. After IV injection of Gd-DTPA or CH3-DTPA-Gd, the signal intensity of the joint cavities increased within 10 min, but this increase was not shown with Gd-polylysine and Gd-albumin., Conclusion: The structural findings obtained by MR imaging agreed with those obtained by hematoxylin-eosin staining under light microscopy. Contrast-enhanced MR imaging suggested that smaller (<2.1 kDa) but not larger (>10 kDa) molecules can permeate the synovial membrane. Our results suggest the utility of MR imaging for analyzing the structure of the TMJ as well as permeability of the synovial membrane.

Opioid peptides and their receptors have been localized to the inner ear of the rat and guinea pig mammalian models. The expression of mu opioid receptor (MOR) in the human and mouse cochlea is not yet known. We present MOR protein localization by immunohistochemistry and mRNA expression by in situ hybridization in the human and mouse spiral ganglia (SG) and organ of Corti. In the human most of the (SG) neurons were immunoreactive; a subset was non-immunoreactive. In situ hybridization revealed a similar labeling pattern across the neurons of the SG. A similar distribution MOR pattern was demonstrated in the mouse SG. In the mouse organ of Corti MOR was expressed in inner and outer hair cells. Fibers underneath the inner hair cells were also MOR immunoreactive. These results are consistent with a role of MOR in neuromodulation of the auditory periphery. The present results show that the expression of MORs is well-conserved across multiple mammalian species, indicative of an important role in auditory processing., (Copyright © 2014. Published by Elsevier B.V.)

The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.

Abnormal elongation of the styloid process, or Eagle syndrome, can be painful, and is associated with differential diagnoses including cranio-facial malformations and vasculo-neurological disturbances. The precise molecular mechanism leading to styloid process elongation is unknown. In this study, elongated styloid processes with periosteal fibrous ligament tissue were obtained from three patients with Eagle syndrome and examined by immunohistochemical methods using different antisera. In all cases, marked bony deposition was found at the apex of the styloid process. The osteogenetic proteins, such as osteonectin, osteocalcin, BMP-2, BMP-4, and RANKL were strongly positive by immunohistochemistry in both the ligament fibers and the periosteal membrane attached to the styloid process apex. Staining for protective proteins, HO-1, HSP-70, and HSP-90 was also positive. These results suggest that styloid process elongation is related to increased expression of osteogenetic and protective proteins. Therefore, we propose that Eagle syndrome results from a protective response to increased tensile stress in the ligament attached to the styloid process, which could also signal osteogenetic protein expression in the periosteal fibrous tissue., (Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. All rights reserved.)

Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10(+) cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10(+) marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10(+) cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10(+) cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10(+) cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.

Objective: Temporal bone squamous cell carcinoma (SCC) accounts for less than 0.2% of all head and neck tumors. Although some progress has been made in treating this aggressive tumor, the prognosis in advanced cases remains poor. More effective therapeutic strategies need to be considered, including receptor-mediated carcinoma-targeted therapy. Phosphorylated STAT3 (pSTAT3) regulates many genes that are necessarily expressed in cancer initiation, development, and progression, being involved in proliferation, anti-apoptosis, invasion, angiogenesis, and immune surveillance evasion. The aim of the present study was to preliminarily investigate the potential prognostic role of pSTAT3 expression in temporal bone SCC., Study Design: Retrospective clinicopathologic investigation., Setting: Tertiary referral centers., Patients: Twenty-five consecutively operated patients with primary temporal bone SCC., Intervention: pSTAT3 immunohistochemical expression in primary temporal bone SCCs was assessed with the aid of computer-based image analysis., Main Outcome Measures: Conventional clinicopathologic parameters and pSTAT3 expression were correlated with SCC prognosis., Results: pT, stage, and surgical margin status were significantly related with recurrence rate (p = 0.002, p = 0.01, and p = 0.047, respectively) and disease-free survival (DFS) (p = 0.0049, p = 0.031, and p = 0.035, respectively). pT classification was also related with disease-specific survival (DSS) (p = 0.035). The SCC recurrence rate did not correlate with pSTAT3 expression. Statistical analyses ruled out any significant difference in DFS or DSS when patients were stratified by pSTAT3 expression (>80.0% or ≤80.0%)., Conclusion: Despite our preliminary results, the role of pSTAT3 in temporal bone SCC warrants further investigation in larger series because there is increasing evidence in preclinical models to indicate that inhibiting STAT3 phosphorylation can be a useful addition to different anticancer strategies.

Glutamate plays an important role in the central nervous system as an excitatory neurotransmitter. However, its abundance can lead to excitotoxicity which necessitates the proper function of active glutamate transporters. The glutamate-aspartate transporter (GLAST) has been shown to exist and function within non-human cochlear specimens regulating the inner ear glutamate concentration. In this study, we examined human cochleas from formalin-fixed celloidin-embedded temporal bone specimens of three different types of patients (Meniere's disease, normal controls, and other otopathologic conditions) and examined the differential expression of GLAST in the spiral ligament of the basal, middle, and apical turns of the cochlea. Immunohistochemical staining was performed with polyclonal antibodies against GLAST and image analysis was carried out with the Image J analysis software. In contrast to other studies with non-human specimens, GLAST was expressed in the spiral ligament fibrocytes but was not detected in the satellite cells of the spiral ganglia or supporting cells of the Organ of Corti in the human cochlea. Our data also showed that GLAST expression significantly differs in the basal and apical turns of the cochlea. Lastly, post-hoc analysis showed a difference in the GLAST immunoreactive area of patients with Meniere's disease when compared to that of patients with other otopathologic conditions-such as presbycusis or ototoxicity. These results may potentially lead to further understanding of different disease states that affect hearing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection., Methods: Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone., Results: COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases., Conclusion: We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: There is ongoing debate on the pathogenic mechanisms of cholesterol granuloma formation in the temporal bone. The purpose of this report is to provide evidence in support of the exposed marrow hypothesis in explaining the pathogenesis of petrous apex cholesterol granuloma., Study Design: Retrospective single case study., Main Outcome Measurement: The primary outcome evaluated was the diagnosis of a new cholesterol granuloma in a petrous apex that previously demonstrated radiologic evidence of bone marrow exposed to petrous apex air cells., Results: A patient with a unilateral petrous apex cholesterol granuloma develops a new, contralateral cholesterol granuloma in a hyperpneumatized temporal bone petrous apex shown previously to have medullary bone exposed to air cells., Conclusion: This report implicates the medullary-air cell interface in a hyperaerated temporal bone petrous apex in the development and growth of a petrous apex cholesterol granuloma.

This article presents a unique approach for the delivery of gene therapy vectors into the cochlea of the laboratory rat. Mice and guinea pigs are established in vivo models for cochlear gene therapy each of which has distinct advantages and disadvantages. The rat has some of the molecular advantages of a mouse model combined with size advantages for surgical approaches. Vector delivery via cochleostomy or injection through the round window causes concomitant sensorineural hearing loss and is therefore not suitable for studies where the change in hearing is being followed. Compared to the mouse, the rat does not demonstrate easily recognizable landmarks that allow for use of the semicircular canal as an approach to the inner ear. We analyzed sagittal and coronal temporal bone sections of Long Evans rats and identified the bony entrance of the facial nerve as a crucial landmark for canalostomy. Auditory brainstem response and distortion product otoacustic emission measurements revealed minimal differences in the hearing threshold after adenovirus vector application when large volumes of vector were infused to the inner ear. Canalostomy and infusion of adenoviral vectors also resulted in temporary balance disturbance in the rat. Immunohistochemical assessment after delivery of a green fluorescent protein expressing vector showed significant GFP expression in the cochlea., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: Temporal bone squamous cell carcinoma (SCC) accounts for less than 2% of all head and neck tumors. Its biologic parameters should be investigated because clinicopathologic factors are often inaccurate for the purposes of its prognosis. CD105 is a proliferation-associated protein expressed in angiogenic endothelial cells and a potential prognostic indicator for several solid malignancies. The present study is the first to investigate the prognostic role of CD105 expression in temporal bone SCC., Study Design: Retrospective clinicopathologic investigation., Setting: Tertiary referral centers., Patients: Twenty consecutive operable patients with temporal bone SCC., Intervention: CD105 immunohistochemical expression in primary temporal bone SCCs was assessed using image analysis., Main Outcome Measures: CD105 expression was correlated with conventional clinicopathologic and prognostic parameters., Results: Using the revised Pittsburgh staging system, T and stage correlated with local recurrence rate (p = 0.0001 and p = 0.0001, respectively) and disease-free survival (p = 0.043 and p = 0.018, respectively). The recurrence rate was significantly higher (p = 0.038) and the disease-free survival shorter in patients with CD105 expression of 9.44% or higher (p = 0.038) than in cases where it was less than 9.44%. The crude carcinoma recurrence risk ratio of was 5.9 times higher for patients whose CD105 expression was 9.44% or higher., Conclusion: CD105 expression in activated endothelial cells of temporal bone SCC can be considered potentially useful for detecting patients at a higher risk of local disease recurrence after treatment. Further investigations are needed to ascertain the feasibility of incorporating targeted anti-CD105 therapy in multimodality or multitarget strategies for temporal bone SCC treatment.

Unlabelled: Hearing loss is a very significant health problem. The methods currently available for inner ear drug delivery are limited and a noninvasive cell-specific drug delivery strategy needs to be found., Aim: In this study we investigated the ability of polymersomes, lipid core nanocapsules and hyperbranched poly-L-lysine to cross the round window membrane., Materials & Methods: Nanoparticles (NPs) used in this study have different size and chemical compositions. Freshly frozen human temporal bones were used for this investigation. Intact human round window membrane within the freshly frozen human temporal bone served as an excellent model to test the membrane permeation and distribution within the tissues., Results: In this investigation we were able to visualize the NPs across the round window membrane. The NPs were subsequently found to be distributed in the sensory hair cells, nerve fibers and to other cells of the cochlea., Conclusion: This finding raises hope in terms of future multifunctional NP-based drug delivery strategy to the human inner ear.

The evolution of the cetacean skeleton followed a path that differentiated this group from other terrestrial mammals about 50 million years ago [1], and debate is still going on about the relationships between Cetacea and Artiodactyla [2], [3], [4]. Some skeletal traits of the basilosaurids (the more advanced forms of Archaeocetes), such as the expansion of the peribullary air sinuses, dental modification and vertebral size uniformity [5] are maintained and further emphasized also in contemporary odontocetes and mysticetes. Using Dual-Energy X-Ray Absorptiometry here we report that the deposition of bone mineral in fetal and newborn specimens of the fin whale Balaenoptera physalus is remarkably higher in the bulla tympanica than in the adjacent basal skull or in the rest of the skeleton. Ossification of the tympanic bulla in fetal Artiodactyla (bovine, hippopotamus) is minimal, becomes sensible after birth and then progresses during growth, contrarily to the precocious mineralization that we observed in fin whales. Given the importance of the ear bones for the precise identification of phylogenetic relationship in therian evolution [6], this feature may indicate a specific evolutionary trait of fin whales and possibly other cetacean species or families. Early mineralization of the tympanic bulla allows immediate sound conduction in the aquatic medium and consequently holds potential importance for mother-calf relationship and postnatal survival.

Objective: To determine the expression patterns of B ephrins and Ephs in the cochlea and identify functional consequences of disruption of ephrin B/Eph B interactions in both cultured spiral ganglion neurons and in the cochlea of live animals., Study Design: The expression patterns of various B ephrins and Ephs were determined in mice with Lac-Z mutation. Mice with null function of individual B ephrin and Eph proteins and those with multiple knockouts were studied for cochlear innervation patterns., Methods: Mice with B ephrins and Ephs disrupted with the β-galactosidase gene were sacrificed at P6, and their cochleae isolated and processed for Lac-Z staining to determine expression of these proteins in cochlear tissue. Spiral ganglion cells from wild-type as well as ephrin B1 knockout mice were isolated and cocultured with Eph B2 expressing Cos1 cells and neurite lengths were determined. Fluorescent lipophillic dyes were used to label spiral ganglion cell nerve fibers to determine cochlear innervation patterns in wild-type and knockout mice., Results: Eph B1, B2, and ephrin B2 but not B3 was expressed in the cochlea. Eph B2 inhibited outgrowth of spiral ganglion cell axons from wild-type mice, but not from ephrin B1 knockout mice in culture. Knockout mice with null function of ephrin B1 alone or Eph B1, Eph B2, Eph B3 in combination demonstrated abnormal innervation patterns in the organ of Corti., Conclusions: Disruption of B ephrins and Ephs results in functional consequences in spiral ganglion cells, suggesting that these proteins play a role in establishing normal innervation patterns in the cochlea., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

Objectives: The authors recently demonstrated that extensive bone erosion correlated with a worse prognosis of the squamous cell carcinoma (SCC) of the temporal bone. The present objective was to investigate the expressions and the roles of epithelial-mesenchymal transition (EMT) in SCC of the temporal bone., Study Design: Retrospective patient file review and immunohistochemical study of tissues of patients with SCC of the temporal bone., Patients: The authors examined the expressions of vimentin in 16 patients with SCC of the temporal bone by immunohistochemical analysis., Results: As expected, vimentin was expressed on SCC of the temporal bone. We regard the patient who has vimentin-positive cells as EMT-positive patient because gain of vimentin is a hallmark in the EMT reported in several types of cancer. EMT positive tumors significantly correlated with extensive bone erosion, and extensive bone erosion correlated with a worse prognosis of the SCC of the temporal bone., Conclusion: The study results suggest that EMT in tumor cells becomes an important indicator of poor prognosis in SCC of the temporal bone., (© 2011, Otology & Neurotology, Inc.)

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.

Objective: To evaluate cellular hypertrophic activities in the mandibular condylar cartilage (MCC) and the glenoid fossa (GF) during mandibular advancement in the temporomandibular joint (TMJ) of Sprague-Dawley rats, as evidenced by fibroblast growth factor 8 (FGF8)., Methods and Materials: Fifty-five female 24-day-old Sprague-Dawley rats were randomly divided into four experimental and control groups, with a mandibular advancement appliance on the experimental rats' lower incisors. The rats were euthanized on days 3, 14, 21, and 30 of the study, and their TMJ was prepared for a immunohistochemical staining procedure to detect FGF8., Results: FGF8 expression was significantly higher among the experimental rats (P  =  .002). Patterns of ascension and descension of FGF8 expression were similar in experimental and control samples. The results show an overall enhanced osteogenic transition occurring in both the MCC and the GF in experimental rats in comparison with controls. The level of cellular changes in the MCC is remarkably higher than in the GF., Conclusion: In the MCC and the GF, cellular morphologic and hypertrophic differentiations increase significantly during mandibular advancement. It is also concluded that endochondral ossification in the MCC and intramembranous ossification in the GF occur during adaptive remodeling.

Objectives: To histologically and immunohistochemically assess the pattern of expression of bone morphogenic proteins 2 and 4 (BMP2/4) and proliferating cell nuclear antigen (PCNA) in response to bite jumping appliances in the condylar cartilage and the glenoid fossa., Materials and Methods: Fifty-five 4-week-old female Sprague-Dawley rats were randomly divided into four experimental and four control groups. Bite-jumping appliances were fitted to the experimental animals. The rats were sacrificed at 3, 14, 21, and 30 days, and the temporomandibular structures were analyzed histologically and immunohistochemically., Results: The expression of BMP2/4 in response to bite-jumping appliances was statistically significant in the condylar cartilage and the glenoid fossa. Cell proliferation was not significant., Conclusion: BMP2/4 plays an important role in bone formation in response to mandibular advancement by accelerating and enhancing the differentiation of mesenchymal cells into bone-forming cells.

Objective: To elaborate whether rAAV2 can be used for future TMJ gene therapy, we examined the infection efficiencies of rAAV2 in vitro, and the transgene expression pattern mediated by rAAV2 in glenoid fossa, TMJ disc and condylar cartilage in vivo., Materials and Methods: Different dosages of rAAV2-eGFP (MOI: 5 x 10(4), 1 x 10(4), 5 x 10(3)) were applied to primary cultured condylar chondrocytes of rats. Infection efficiencies were analysed by FACSCalitur at different time points. Vastatin, a molecule not naturally expressed in TMJ, was used as a reporter for detection of rAAV2 mediated transgene expression in vivo. Thirty SD rats were injected with either rAAV2-sec-Vastatin (experimental group) or rAAV2-eGFP (control group) into both sides of TMJ. They were sacrificed at the indicated time (7, 14, 21, 30 and 60 days of injection) and the TMJ samples were collected for RT-PCR and immunostaining analysis., Results: High dosage (MOI 5 x 10(4)) of rAAV2-eGFP can achieve desirable transduction efficiencies in vitro after 5 days. Transgene expression of rAAV-sec-Vastatin persisted for about 21 days in glenoid fossa, around 7 days in TMJ disc and at least 60 days in condylar cartilage in vivo. In condylar cartilage, transgene expression was found in the proliferative layer and chondroblast layer (day 7), chondrocyte layer (day 14), pre-hypertrophic and hypertrophic layer (day 21), hypertrophic layer and deep hypertrophic layer (day 30 and 60)., Conclusion: Recombinant AAV2 could be considered as a promising vector for gene therapy in TMJ which can mediate therapeutic gene expression in glenoid fossa, articular disc and condylar cartilage in vivo.

Pre- and perinatal asphyxia is known to be an important risk factor in the development of neonatal hearing impairment. This study aims to evaluate the role of apoptosis, which is known to play an essential role in the development of the inner ear structures, in the development of neonatal hearing loss caused by pre- and perinatal asphyxia. Eight temporal bones of six different newborns were included. We performed a morphologic analysis by both light microscopy, and transmission electron microscopy, as well as immunohistochemical staining to detect the cleaved form of caspase 3 as apoptosis marker and Bcl 2 as anti-apoptotic marker. Early and late phases of apoptosis were evidenced by condensation of chromatin (electron-dense, black structure along nuclear membrane) and fragmentation of the nucleus, respectively. Changes in nuclear morphology during apoptosis correlate with cleavage by caspase 3 located downstream of Bcl 2 action. The immunohistochemistry for cleaved caspase 3 showed a particular predilection for the inner and outer hair cells, spiral ganglion cells and the marginal cells of the stria vascularis. The brain of all examined cases did not show signs of apoptosis. In summary, this investigation suggests that apoptosis takes place before brain tissue apoptosis and is probably an earlier event than thought. Apoptosis of the cochlea is known to play an essential role in the development of the inner ear. Additionally, this study shows that apoptosis may play an important role in the development of hearing impairment, caused by pre- and perinatal asphyxia.

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., (2008 S. Karger AG, Basel.)

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this study, we describe a technique for isolating cochlear structures and individual spiral ganglion cells from archival celloidin embedded human temporal bone sections. The specimens isolated are suitable for quantifying the mitochondrial DNA (mtDNA) common deletion (CD) within these tissues using a real time polymerase chain reaction (PCR) assay. The results presented in this manuscript demonstrate the feasibility of using this LMD technique to study the accumulation of mtDNA deletions in diseases of the ear. To our knowledge, this approach to analyzing archival human temporal bone tissues has not been previously reported.

The temporomandibular joint (TMJ) is a unique synovial joint whose development differs from the formation of other synovial joints. Mutations have been associated with the developmental defects of the TMJ only in a few genes. In this study, we report the expression of the homeobox gene Shox2 in the cranial neural crest derived mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and undifferentiated chondrocytes of the condyle as well as the glenoid fossa of the developing TMJ. A conditional inactivation of Shox2 in the cranial neural crest-derived cells causes developmental abnormalities in the TMJ, including dysplasia of the condyle and glenoid fossa. The articulating disc forms but fuses with the fibrous layers of the condyle and glenoid fossa, clinically known as TMJ ankylosis. Histological examination indicates a delay in development in the mutant TMJ, accompanied by a significantly reduced rate of cell proliferation. In situ hybridization further demonstrates an altered expression of several key osteogenic genes and a delayed expression of the osteogenic differentiation markers. Shox2 appears to regulate the expression of osteogenic genes and is essential for the development and function of the TMJ. The Shox2 conditional mutant thus provides a unique animal model of TMJ ankylosis.

Studies of transtympanic gentamicin have focused on clinical use and outcomes. This study presents evidence of bilateral uptake and retention of gentamicin in certain inner ear cells and structures following transtympanic gentamicin application. Middle ear application of gentamicin was performed by either minipump (Alza model, 2002) or transtympanic injection in a chinchilla model. Histological sections of decalcified temporal bones were stained to identify the distribution of gentamicin. Using both anti-gentamicin immunohistochemistry and autoradiography of tracer amounts of tritiated gentamicin, Scarpa's and spiral ganglion cells, stria vascularis, and vestibular dark cells of the injected ear were found to have higher levels of gentamicin and retain it within cell bodies while staining levels fell to background levels in the rest of the injected ear over the course of 14 days. There was no evidence of an apical to basal gradient of anti-gentamicin staining within the spiral ganglion. Contralateral inner ear cells showed light anti-gentamicin staining. Cell bodies in the ipsilateral dorsal cochlear nucleus bordering the cochlear aqueduct (CA) showed a lateral to medial gradient of gentamicin staining, suggesting the CA as a potential site of transfer of gentamicin to the contralateral ear. Direct effects of aminoglycosides on ganglion cells may have implications on both the success of cochlear implantation in patients deafened following systemic aminoglycoside therapy and on the advisability of clinical practices of transtympanic gentamicin therapy and ototopic aminoglycoside treatment.

Hypothesis: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones., Background: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway., Methods: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase., Results: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods., Conclusion: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Conclusion: Current neurotransmission models based on animal studies on the mammalian inner ear not always reflect the situation in human. Rodents and primates show significant differences in characteristics of efferent innervation as well as the distribution of neuroactive substances., Objective: Immunohistochemistry demonstrates the mammalian efferent system as neurochemically complex and diverse: several neuroactive substances may co-exist within the same efferent terminal. Using light and electron microscopic immunohistochemistry, this study presents a comparative overview of the distribution patterns of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, GABA, CGRP, and enkephalins within the peripheral nerve fiber systems of the human inner ear., Materials and Methods: Human temporal bones were obtained post mortem and prepared according to a pre-embedding immunohistochemical technique to detect immunoreactivities to ChAT, GABA, CGRP, leu- and met-enkephalins at the electron microscopic level., Results: Immunoreactivities of all the antigens were present within both the lateral and medial efferent systems of the cochlea, whereas only ChAT, GABA, and CGRP were detected in efferent pathways of the vestibular end organs.

Seven missense mutations and one in-frame deletion mutation have been reported in the coagulation factor C homology (COCH) gene, causing the adult-onset, progressive sensorineural hearing loss and vestibular disorder at the DFNA9 locus. Prevalence of COCH mutations worldwide is unknown, as there is no systematic screening effort for late-onset hearing disorders; however, to date, COCH mutations have been found on four continents and the possibility of COCH playing an important role in presbycusis and disorders of imbalance has been considered. Cochlin (encoded by COCH) has also been shown as a major target antigen for autoimmune sensorineural hearing loss. In this report, we present histopathology, immunohistochemistry and proteomic analyses of inner ear tissues from post-mortem DFNA9 temporal bone samples of an individual from a large Dutch kindred segregating the P51S mutation and adult human unaffected controls, and wild-type (+/+) and Coch null (-/-) knock-out mice. DFNA9 is an inner ear disorder with a unique histopathology showing loss of cellularity and aggregation of abundant homogeneous acellular eosinophilic deposits in the cochlear and vestibular labyrinths, similar to protein aggregation in well-known neurodegenerative disorders. By immunohistochemistry on the DFNA9 temporal bone sections, we have shown cochlin staining of the characteristic cochlear and vestibular deposits, indicating aggregation of cochlin in the same structures in which it is normally expressed. Proteomic analysis identified cochlin as the most abundant protein in mouse and human cochleae. The high-level expression and stability of cochlin in the inner ear, even in the absence and severe atrophy of the fibrocytes that normally express COCH, are shown through these studies and further elucidate the pathobiologic events occurring in DFNA9 leading to hearing loss and vestibular dysfunction.

In this paper, the influence of collagen fibres in the behaviour of the temporomandibular joint disc is studied. A three-dimensional finite element model of the joint is developed from a set of medical images. The model comprises the mandible, part of the cranium and both temporomandibular joints. Joints have been considered to be composed of the articular discs and the temporomandibular ligaments. A fibre-reinforced porohyperelastic model was used to study the response under clenching of the fibrocartilage that composes the articular disc. This was divided in an intermediate zone, and two bands, an anterior and other posterior, in order to define the orientation of collagen fibres. The study demonstrates that the introduction of collagen fibres in the biphasic behaviour of the articular disc implies for a prescribed displacement not only an increase of the pressurization in the tissue, but also higher stresses in the anterior and posterior bands, as well as in the lateral zone of the disc. Thus, modelling the disc as an isotropic solid matrix leads in this case to an overestimation of the stresses in the intermediate zone, an underestimation of the pore pressure in this area, and an underestimation of the stresses in the rest of the disc.

The objective of this paper was to review temporal bone chondroblastomas in regard to their presentation, radiographic findings, histopathology, and treatment. A case report of a 38-year-old man who presented with the left-sided hearing impairment and temporal swelling was reviewed. A CT scan revealed an osteolytic lobulated expansile mass. MRI depicted two cystic components with fluid-fluid level and enhanced solid mass. Immunohistochemical study of S-100 was performed using avidinbiotin-complex method. The tumor was totally removed, with eroded squamous bone and temporal muscle, via the left zygomatic-extended middle fossa approach. The pathology of the tumor showed that the tumor cell was spindle-shaped, along with multinucleated giant cells. These cells had oval to polygonal nuclei; some cells showed grooved nuclei. Intercelluar calcification and hemorrhagic components were also observed in the tumor. Tumor cells were strongly positive for S-100 protein. Temporal bone chondroblastomas are extremely rare osseous tumors with only 45 cases previously reported in the published literature. They may be confused with more common lesions seen in the temporal bone. Diagnostic radiology, including CT and/or MRI, as well as immunohistochemical staining with S-100 protein, may assist in making the diagnosis. Treatment is complete surgical excision with preservation of vital neurovascular structures.

In this study, the deposits of basophilic material on the cupula of the semicircular canals in temporal bones from patients who had aminoglycoside administration within six months prior to death were compared with normal temporal bones. Subjects were divided into two groups. Group I included 24 normal control temporal bones age-matched to group II patients. Group II consisted of 23 temporal bones that had received aminoglycosides within six months prior to death. All temporal bones were examined under light microscopy. One (4.2 per cent) of 24 temporal bones in group I (normal) showed basophilic deposits. In group II, deposits were observed in 8 (34.8 per cent) of 23 temporal bones. The prevalence of basophilic deposits in group II was significantly higher than group I. This study demonstrates that within six months after aminoglycoside administration there is an increased prevalence of basophilic deposits on the surface of the cupula. Such changes may be related to the benign paroxysmal positional vertigo (BPPV) seen in some patients who have had aminoglycoside administration.