Uncovering the secreted signals and transcription factors regulating the development of mammalian middle ear ossicles.

The mammalian middle ear comprises a chain of ossicles, the malleus, incus, and stapes that act as an impedance matching device during the transmission of sound from the tympanic membrane to the inner ear. These ossicles are derived from cranial neural crest cells that undergo endochondral ossification and subsequently differentiate into their final functional forms. Defects that occur during middle ear development can result in conductive hearing loss. In this review, we summarize studies describing the crucial roles played by signaling molecules such as sonic hedgehog, bone morphogenetic proteins, fibroblast growth factors, notch ligands, and chemokines during the differentiation of neural crest into the middle ear ossicles. In addition to these cell-extrinsic signals, we also discuss studies on the function of transcription factor genes such as Foxi3, Tbx1, Bapx1, Pou3f4, and Gsc in regulating the development and morphology of the middle ear ossicles.

Hox-A2 protein expression in mouse embryo middle ear ossicles.

The origin of the mammalian middle ear ossicles from mandibular and hyoid pharyngeal arches remains controversial and discussed. Two adverse theories are proposed. The first claims that malleus and incus derive from the Meckel's cartilage of the mandibular arch, and stapes from Reichert's cartilage of the hyoid arch. The second postulates that handle of malleus and long process of the incus are derived from the second arch as well as the stapes. Contradictory analyses support alternatively each theory without any experimental evidence. In order to bring new data, we analyzed by immunohistochemistry the expression of Hox-A2 protein in ossicular anlagen in E11 to 13 mouse embryos. HOXA2 gene is known to be expressed in second arch cells and to be absent from mandibular arch derivatives. Surprisingly, Hox-A2 protein was present in all ossicular primordia, as well in Reichert's cartilage. Meckel's cartilage was free of staining. Unlabeled cells were also present in ossicular blastemata. These results suggest that ossicular condensations could arise from mixed cell populations originated in both mandibular and hyoid pharyngeal arches. However, we cannot exclude that diffuse Hox-A2 immunoreactivity could correspond to a secondary expression in craniofacial mesenchyme independently from the branchial origin of cells.

The expression of tenascin-C and tenascin-W in human ossicles.

The ossicles of the middle ear (the malleus, incus and stapes) transmit forces resulting from vibrations of the tympanic membrane to the cochlea where they are coded as sound. Hearing loss can result from diseases such as rheumatoid arthritis (RA) that affect the joints between the ossicles or degenerative processes like otosclerosis that lead to ankylosis of the footplate of the stapes in the oval window of the cochlea. In this study, immunohistochemistry was used to determine if the extracellular matrix glycoproteins tenascin-C or tenascin-W are expressed in the incudomalleolar and incudostapedial joints of ossicles dissected from human cadavers. Tenascin-C, which is expressed during inflammatory conditions including RA, was seen in the articular cartilage of the incudomalleolar joints and the head of the stapes. Tenascin-W, in contrast, was enriched in the annular ligament that anchors the footplate of the stapes into the oval window of the cochlea.

The auditory ossicles as a DNA source for genetic identification of highly putrefied cadavers.

Genetic identification of putrefied bodies is a common task in forensic medicine. With advancing putrefaction, however, DNA integrity is rapidly decreasing and genetic typing of tissue might be impaired or impossible. Since DNA stability is generally higher in hard tissues, bones or teeth are frequently used as DNA source in such cases. However, isolation of DNA from hard tissues is usually very time-consuming and labor-intensive. This can be especially important in (forensic) cases where time is short and identification has to be carried out as fast as possible. Here, we present the identification of dead bodies by analyzing DNA from the auditory ossicles. These minuscule bones provided DNA of sufficient quality and quantity for identification purposes in all 40 investigated cases. Additionally, processing of the bones proved to be amazingly easy and fast, and a successful extraction is possible using a variety of different methods. We present a detailed protocol, results, and cases in which this new method has been successfully applied.

Fascin expression in cholesteatoma: correlation with destruction of the ossicular chain and extent of disease.

OBJECTIVE: Fascin is an actin-bundling protein found in cell membrane protrusions and increases cell motility. The expression of fascin in epithelial neoplasms has been described only recently. No data are available concerning the role of this protein in invasive cholesteatoma. Thus, we investigated the expression of fascin in cholesteatoma tissue and the relationship between fascin expression and intraoperative evaluation of the destruction of the ossicular chain and extent of disease. METHOD: Cholesteatoma specimens of 28 patients and external auditory canal (EAC) skin specimens of the same patients (as the control group) were collected from mastoidectomies. Immunohistochemical technique was used to investigate the fascin expression in all cholesteatoma tissues and EAC skin specimens. Immunohistochemical staining was assessed semiquantitatively based on the thickness of epithelium. SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA) was performed to statistically analyse the relationships between fascin expression and intraoperative evaluation destruction of ossicular chain and extent of the disease. RESULTS: Immunohistochemically, there was no or very low fascin expression observed in normal epithelial cells of EAC skin, while expressed in cholesteatoma tissue. Also, fascin expression in cholesteatoma tissues was significantly correlated with destruction of ossicular chain and extent of the disease. CONCLUSIONS: Fascin expression is usually found in cholesteatoma epithelium and is correlated with destruction of the ossicular chain and extent of disease. Considering all of the correlations between the clinical and histopathological findings, 'fascin immunoexpression scoring' may be used for histological grading of cholesteatoma.

The origin of the stapes and relationship to the otic capsule and oval window.

BACKGROUND: The stapes, an ossicle found within the middle ear, is involved in transmitting sound waves to the inner ear by means of the oval window. There are several developmental problems associated with this ossicle and the oval window, which cause hearing loss. The developmental origin of these tissues has not been fully elucidated. RESULTS: Using transgenic reporter mice, we have shown that the stapes is of dual origin with the stapedial footplate being composed of cells of both neural crest and mesodermal origin. Wnt1cre/Dicer mice fail to develop neural crest-derived cartilages, therefore, have no middle ear ossicles. We have shown in these mice the mesodermal stapedial footplate fails to form and the oval window is induced but underdeveloped. CONCLUSIONS: If the neural crest part of the stapes fails to form the mesodermal part does not develop, indicating that the two parts are interdependent. The stapes develops tightly associated with the otic capsule, however, it is not essential for the positioning of the oval window, suggesting that other tissues, perhaps within the inner ear are needed for oval window placement.

Identification of Id1 in acquired middle ear cholesteatoma.

OBJECTIVES: To determine (1) the relationship between chronic inflammatory changes in the ossicular chain area (OCA) and the formation of cholesteatoma and (2) the correlates between aberrant gene expression and abnormal proliferation of cholesteatoma. METHODS: Two hundred sixty-four ears with chronic otitis media that had undergone ear surgery were included in this study for statistical analysis of the relationship between abnormalities in the OCA and cholesteatoma. Fourteen middle ear cholesteatoma specimens were collected for immunohistochemical analysis of candidate molecules involved in the abnormal proliferation of keratinocytes. A cell model was used for verification of candidate molecule involvement. RESULTS: The formation of cholesteatoma was accompanied by chronic inflammatory changes in the OCA, including granulated tissue, adhesion, and stagnating effusion. The inhibitor of the DNA-binding (Id1) gene, which is involved in controlling cell cycle progression, was abundantly expressed in cholesteatoma epithelium. In vitro studies indicate that Id1 regulated the expression of nuclear factor kappaB, cyclin D1, proliferating cell nuclear antigen, and cell cycle progression of keratinocytes, CONCLUSIONS: Chronic inflammation in the OCA is closely related to the formation of cholesteatoma. The Id1/nuclear factor kappaB/cyclin D1/proliferating cell nuclear antigen signaling pathway is involved in the abnormal proliferation of keratinocytes in acquired cholesteatoma.

Regulated gene expression of hyaluronan synthases during Xenopus laevis development.

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.

Development of the chick columella: immunohistochemical studies with anti-collagen monoclonal antibodies.

Developmentally regulated changes in the extracellular matrices of the columella have been immunohistochemically analyzed with anti-collagen, type-specific monoclonal antibodies. In the 12-day chick embryo, the ossicle is entirely cartilagenous. By using immunohistochemical methods, we found that the 12-day columella contains type II collagen within the cartilagenous matrix and type I collagen in the surrounding perichondrium, but no type X collagen. Previous studies have shown that type X collagen is specific for hypertrophic cartilage (i.e., the site of future marrow cavity formation and ossification). By 16 days, hypertrophic cartilage is evident, type X collagen is present, and ossification has started medially adjacent to the oval window. These results both confirm and extend those of other chick endochondral bones that have been studied. Thus, the columella can serve as a model system for analysis of ossicular development and the associated temporal and spatial changes which occur within its extracellular matrices.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

Uptake and release of alcohol by homograft tissues in tympanoplasty.

Alcohol is known to produce severe cochlear damage and it is possible that deafness as a complication of tympanoplasty can be caused by alcohol which remains in homograft materials after inadequate washing. An investigation was made in which the quantity of alcohol taken up by ossicles, cartilage, temporalis fascia, dura and ear-drum was estimated. The rate of release of alcohol from such materials into saline during washing was also measured. The amount absorbed was found to depend upon the nature of the material, its weight and its surface area. The rate of release of alcohol from these materials differed significantly for each material.

Effect of flavonoid on PGE2-induced alterations in percentage collagen synthesis in ossicle organ cultures.

In adult guinea-pig stapes organ cultures, 3H (2,3)-proline incorporation into the collagenase-digestible protein (CDP) and non-collagen protein (NCP) fractions of the ossicles was measured and the percentage of collagen synthesis (PCS) was calculated. Prostaglandin E2 (PGE2), a potent stimulator of bone resorption, inhibited the PCS in low concentrations (5 and 25 microM), whereas it stimulated it in pharmacological concentrations (50 and 100 microM). Ipriflavon, an isoflavone derivative of therapeutical potential in otosclerosis, also reduced the PCS in 1, 10 and 50 microM concentrations. 50 microM Ipriflavon stimulated the PCS inhibited by 5 microM PGE2, but decreased the PGE2-induced PCS enhancement in vitro.

Incorporation of radioactive calcium into otolithic membranes and middle ear ossicles of the gerbil.

45CaCl2 was injected into gerbils in single or multiple doses, and the resulting radioactivity in serum, otoconial CaCO3, bone samples, and selected labyrinthine epithelium was determined by liquid scintillation spectrometry. Incorporation into both utricular and saccular otoconia occurred at the rate of 0.06-0.07 nmole Ca++ per day, corresponding to a fractional rate of uptake of 0.1%. The retention of radioactivity had a half-life of approximately 11 days. The rate of incorporation of calcium for the middle ear ossicles was 5-7 times that for otoconia and was similar to that for otic capsule and skeletal bone. The level of 45Ca++ was higher in the pigmented regions of the utricular membranous wall than in the non-pigmented areas of the utricular and ampullary wall and in the stria vascularis.

Effects of cadmium on the hearing system.

The functional resemblance between kidney proximal tubular and inner ear epithelial cells which has often been pointed out in the literature led us to hypothesize that nephrotoxic agents that cause renal tubular injury might also impair the function of inner ear cells. As one of the most toxic environmental nephrotoxic agents is cadmium, we aimed to study its effects on hearing experimentally in rats. In this study, increased blood and renal cortical cadmium levels were associated with high cadmium accumulation in ear ossicles and labyrinth in rats exposed to cadmium. The changes in auditory brainstem response (ABR) and otoacoustic emission in 2-month-old male rats exposed to drinking water containing 5 and 15 ppm CdCl2 for 30 days showed that cadmium-induced nephrotoxicity was associated with signs of defective hearing at a concentration of 15 ppm CdCl2 but that 5 ppm CdCl2 caused hearing loss without affecting kidney function. The mean latency of ABR wave 1, which indicates the function of the cochlea, was 1.335 +/- 0.31 ms in the control group and 1.641 +/- 0.052 and 1.74 +/- 0.88 ms in the rats subjected to 5 and 15 ppm CdCl2, respectively (p < 0.001). In the cadmium-treated groups short interpeak wave I-III latencies (p < 0.01) indicated cochlear dysfunction and this was also supported by the distortion product otoacoustic emission results (p < 0.001). Non-significant changes in wave III and V latencies were accepted as evidence of unaltered function of the other parts of the auditory system. These results suggest that hair cells are more sensitive to cadmium than kidney tubule cells and that the cochlear component of hearing is more vulnerable to cadmium toxicity than other parts of the auditory system.

Age-independent constancy of mineral contents in human vertebra and auditory ossicle.

To elucidate age-related changes of mineral contents in human bones, element contents of human vertebrae and auditory ossicles were determined by inductively coupled plasma atomic-emission spectrometry. The cervical, thoracic, and lumbar vertebrae were removed from 12 vertebral columns. The mallei of auditory ossicle were removed from 27 cadavers. It was found that average relative contents (RCs) of calcium and phosphorus in cervical, thoracic, and lumbar vertebrae remained almost constant within ages ranging from 46 to 99 y. In addition, it was found that the RCs of calcium and phosphorus in men's and women's mallei remained constant within ages ranging from 40 to 98 yr. These results support the view that there is no significant age-dependent change of mineral contents in human bones.

Effect on cochlea function of guinea pig after controlled release recombinant human bone morphogenetic protein 2.

BACKGROUND: The recombinant human bone morphogenetic protein 2 (rhBMP-2) has been used to induce osteogenesis in animals' middle ear and this technique is possible to be used to reconstruct the defects of ossicles. The side effects of the rhBMP-2 in middle ear should be observed before using in clinic. Thus we prepared the controlled release rhBMP-2 and implanted it into the acoustic bulla of guinea pigs. The effect on the cochlea was observed. METHODS: We prepared the acellular cancellous bone, accompanied with rhBMP-2. The material accompanied with rhBMP-2 was implanted into one acoustic bulla of the animal and the opposite side of the acoustic bulla was implanted with acellular cancellous bone without rhBMP-2. Totally 20 guinea pigs were undergone this procedure. After the operation, the auditory brainstem response (ABR) of the animals was tested according to the time sequence. Three months after the operation, the animals were sacrificed. The osteogenesis induced by rhBMP-2, the acoustic bulla and cochlea affected by rhBMP-2 were observed. The structures of hair cells were observed after silver nitrate staining. RESULTS: The animals were recovered soon after surgery. The hearing thresholds of the animals were declined slightly just after the surgery and come back completely after 3 months. Also, the bulla and cochlea were normal in shape. The osteogenesis occurred in the pore of the acellular cancellous bone with rhBMP-2. There was not any abnormal hyperplasia of bone in the bulla and cochlea. The articulation between the stapes and oval window was not merged. The shapes of the hair cells were normal and there was no obvious deletion of the hair cells compared with control group. CONCLUSIONS: The controlled release rhBMP-2 transplanted into the middle ear could induce osteogenesis in the bulla of the animals. It did not affect the shape of the bulla and the hearing threshold of the animal, and did not induce the abnormal hyperplasia of bone in the bulla and might be used to reconstruct the defects of ossicles.

A study on reconstruction of ossicular chain by an in situ bone tissue engineering technique.

CONCLUSION: The acellular cancellous bone with recombinant bone morphogenetic proteins 2 (rhBMP-2) could induce osteogenesis in the acoustic vesicle; this material might be used to reconstruct defects of the ossicular chain. OBJECTIVE: In recent years, the in situ tissue engineering technique has improved rapidly, especially in bone regeneration. The aim of this study was to make an ossicular prosthesis using columnar acellular cancellous bone combined with rhBMP-2, implant this prosthesis into the acoustic vesicle of rabbits, and then observe the osteogenesis in situ. MATERIALS AND METHODS: We prepared the acellular cancellous bone combined with rhBMP-2 as an ossicular prosthesis, while the same material without rhBMP-2 was used in the control group. From the retroauricular approach, we made a hole in the posterolateral bone wall of the acoustic vesicle, and the prepared materials were implanted. After 3 months, we observed the osteogenesis of the prosthesis by macroscopic anatomic and histologic methods. RESULTS: The rabbits recovered soon after surgery. The implanted acellular cancellous bones were connected tightly with the bone of the acoustic vesicle wall. The surfaces of all materials were covered with mucosa, and osteogenesis was observed in the material with rhBMP-2.

Mice with vestibular deficiency display hyperactivity, disorientation, and signs of anxiety.

Previous studies revealed that vestibular cues are crucial for exploration in the absence of visual cues. The working hypothesis of this study was, accordingly, that mice with vestibular dysfunction would become disoriented or unable to globally explore an unfamiliar environment. In 2- and 3-month-old mutant headbanger (Hdb) mice, stereocilia of hair cells are abnormally elongated, yet maintain partial staircase arrangement, suggesting some spared vestibular function at these ages. Here we tested a group of 3-month-old mutant Hdb and a group of non-mutant mice obtained from the same litters (Wt mice). Each individual mouse was introduced into a dark 120 cm x 120 cm arena and its behavior was followed for 10 min. Hdb mice were hyperactive and appeared to engage in local exploration, traveling in a restricted zone for a while and then shifting to travel in another zone. In contrast, Wt mice traveled across zones incessantly with fewer visits to recently entered zones. Thus, Hdb seemed to display local compared with the global exploration of Wt mice, indicating that they were less oriented in the global environment. In addition, Hdb exhibited numerous stretch-attends, which is suggested as a sign of elevated anxiety. Altogether, the three comorbidities of hyperactivity, anxiety, and disorientation can be presented as a syndrome associated with vestibular deficiency in this animal model, and serve in studying vestibular deficiency in humans.