Cellular voids in the pathogenesis of otosclerosis.

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.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

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.

Study on the relationship between the pathogenic mutations of SLC26A4 and CT phenotypes of inner ear in patient with sensorineural hearing loss.

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.

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.

Clinical and molecular characterization of POU3F4 mutations in multiple DFNX2 Chinese families.

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.

Effects of calcium and estrogen on the development of the ceratohyal cartilage in zebrafish (Danio rerio) larvae upon embryo and maternal cadmium exposure.

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 Cd2+ 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 Ca2+ 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 (17ß-estradiol) contents were higher around 1.9 folds in the ovaries of females; CH angle were over 25°, and Cd2+ 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 ERß1 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 Cd2+ 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 Ca2+ contents upon Cd2+ exposure.

Connexin 26 Immunohistochemistry in Temporal Bones With Cochlear Otosclerosis.

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.

The Transplantation of hBM-MSCs Increases Bone Neo-Formation and Preserves Hearing Function in the Treatment of Temporal Bone Defects - on the Experience of Two Month Follow Up.

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.

Bone Metabolic Markers in the Clinical Assessment of Patients with Superior Semicircular Canal Dehiscence.

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 (rpb = -0.35, P = 0.001). Postoperative calcium supplementation negatively correlated with improvement in dizziness (rpb = -0.36, P = 0.01). The level of 25-hydroxyvitamin D correlated with preoperative hyperacusis (rpb = -0.98, P = 0.02) and postoperative autophony (rpb = 0.96, P = 0.04). Postoperative vitamin D supplementation positively correlated with hearing decline (rpb = 0.04, P = 0.04) The level of thyroid stimulating hormone correlated with preoperative autophony, amplification, and tinnitus (rpb = -0.71, rpb = -0.75, rpb = -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.

Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma.

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 tyr466) was investigated in temporal bone squamous cell carcinoma (TBSCC). MATERIALS AND METHODS: Immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr466) 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 tyr466) 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.

Immunohistological analysis of eotaxin and RANTES in the model animal of eosinophilic otitis media.

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.

Noise Induced DNA Damage Within the Auditory Nerve.

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.

Unilateral Opening of Rat Blood-Brain Barrier Assisted by Diagnostic Ultrasound Targeted Microbubbles Destruction.

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.

Racial Differences of Pigmentation in the Human Vestibular Organs.

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.

Anti CD163+, Iba1+, and CD68+ Cells in the Adult Human Inner Ear: Normal Distribution of an Unappreciated Class of Macrophages/Microglia and Implications for Inflammatory Otopathology in Humans.

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.

Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone.

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.

Fracturas de temporal graves en niños: presentación, complicaciones y secuelas observadas en los últimos 11 años / Severe temporal bone fractures in children: Clinical presentation, complications and sequelae observed in the last 11 years

OBJETIVO: Analizar las características de presentación, complicaciones y secuelas, de las fracturas de temporal ingresadas en UCIP.MATERIAL Y MÉTODOS: Análisis descriptivo retrospectivo de las historias clínicas y TC craneal. RESULTADOS: Veintisiete pacientes ingresados en UCIP presentaron fractura del temporal: 13 (48%) sin afectación de peñasco (grupo 1) y 14 (52%) con afectación de peñasco (grupo 2). El grupo 2 presentó mayor estancia en la UCIP: 4,5 días (RI: 2,75-22,25) vs. 2 (RI: 1-3) (p = 0,018), mayor frecuencia de secuelas (p = 0,04) y presencia de fístula del LCR (p < 0,02). Los scores de PRIMS III e índice de traumatismo pediátrico no mostraron diferencias significativas. El 31% presentó hipoacusia, 2 pacientes fallecieron y 4 (15%) presentaron secuelas permanentes. CONCLUSIONES: Las fracturas temporales pueden ser graves, especialmente si incluyen al peñasco, bien por las lesiones asociadas, que marcan el riesgo vital, como por las secuelas (déficit auditivo o fístula LCR). Los autores indican el seguimiento de estos pacientes a largo plazo por Otorrinolaringología

OBJECTIVES: To evaluate the clinical presentation, complications and sequelae in patients with temporal bone fracture in the last 11 years. MATERIAL AND METHODS: A total of 27 patient medical records were retrospectively analysed. RESULTS: Of the 27 patients who were admitted for temporal bone fracture from 2001 to 2012, 13 (48%) had no petrous involvement (Group 1), and 14 (52%) with petrous involvement (Group 2). Patients in Group 2 had a longer P-ICU stay: median 4.5 days (RI: 2.75-22.25 d) vs 2 (RI: 1-3 d) (P=.018); more days on mechanical ventilation support: median 3 days (RI: 1.50-17 d) vs 1 (RI: 1-1.25 d). This group also had a higher frequency in sequelae (P=.04 OR = 1.4 (95% CI: 1.05-1.95)) and a higher incidence in cerebrospinal fluid (CSF) fistula (P<.02; OR 2.33; 95% CI (1.27-4.27)). Severity scores (PRIMS III and PTI) showed no significant differences. Some degree of hearing loss was observed in 31% of the patients. Traffic accident was the main cause of trauma (33%), followed by falls (27%). There were 2 deaths and 4 (15%) had permanent sequelae. CONCLUSIONS: Isolated temporal bone fractures usually have a good outcome in children, but in some cases they can be fatal or have permanent sequelae. Long term follow up is recommendedby authors

Relaxin-2 expression in temporal bone carcinoma.

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.

Structure and Size-selective Permeability of the Synovial Membrane of the Temporomandibular Joint of the Mouse Measured by MR Imaging at 7T.

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.

Pou3f4-mediated regulation of ephrin-b2 controls temporal bone development in the mouse.

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.