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.

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.

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.

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.

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.

Enhancement of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced new bone formation by concurrent treatment with parathyroid hormone and a phosphodiesterase inhibitor, pentoxifylline.

We investigated the enhancement of new bone |formation elicited ectopically by recombinant human bone morphogenetic protein-2 (rhBMP-2), using parathyroid hormone (PTH) and a phosphodiesterase inhibitor (PDEi), pentoxifylline (PTX), in an animal model. Collagen sponge sheet discs containing rhBMP were implanted onto the back muscles of mice. PTX alone (200 mg/kg body weight [BW]), PTH(1-34) (10 microg/kg BW), PTX plus PTH (200 mg/kg BW and 10 microg/kg BW, respectively), or vehicle (control) were injected subcutaneously daily for 3 weeks after implantation. At the end of this period, rhBMP-2-induced ectopic ossicles were harvested from each group of animals. Ossicles from the PTX-treated group were significantly larger in size, with unchanged bone mineral density (BMD), as compared with the ossicles from the controls. In contrast, the ossicles from the PTH-treated group had significantly higher BMD, but showed no difference in size when compared with those from the control animals. The ossicles of the PTX + PTH treatment group were significantly larger than those of the control and PTH treatment groups. In addition, the BMD of the harvested tissues from the PTX + PTH treatment group was signifi-cantly higher than that of tissues from the control and PTX treatment groups. Although the calcium content of ossicles was significantly higher in the PTX-, PTH-, and PTX + PTH-treated groups than in the control group, the Ca content of ossicles from the PTH + PTX-treated group was highest (two times that of controls), followed by the PTH- and PTX-treated groups.

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.

Bone resorption in chronic otitis media: the role of the osteoclast.

Most of the pathology associated with cholesteatoma is the result of osteoclast-mediated bone resorption in the middle ear. Cytokines, prostaglandins, nitric oxide, neurotransmitters and growth factors are associated with chronic inflammation and have been implicated in cholesteatoma-induced bone resorption. Although many different factors are known to regulate osteoclast activation, there is a final common pathway of osteoclast differentiation and resorption. Recent advances in molecular techniques and the increasing availability of genetically altered mice have provided valuable insight into the molecular mechanism of osteoclastic bone resorption. This review focuses on osteoclast biology, lessons from genetically altered mice, and their contribution to our understanding of cholesteatoma-induced bone resorption.

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.

Ossicular chain reconstruction using a new tissue adhesive.

HYPOTHESIS: A new medical-grade cyanoacrylate tissue adhesive will improve the results of ossicular chain reconstruction in a rat model. BACKGROUND: An ideal tissue adhesive has long been awaited by otologists. Studies examining the older cyanoacrylates have demonstrated variable efficacy and toxicity. Octylcyanoacrylate is a new tissue adhesive that has many ideal properties for otologic surgery. METHODS: Thirteen female C-D rats were anesthetized, and preoperative auditory brainstem response (ABR) testing was performed. A left antrotomy was performed, and the incus was removed. In the adhesive group, the incus was dipped in octylcyanoacrylate and interposed between the tympanic membrane and the stapes; no adhesive was used in the control group. At 8 weeks, postoperative ABR was performed, the integrity of the ossicular chain inspected, and histopathologic analysis of the temporal bones performed. Statistical comparison of ABR results was performed with the Mann-Whitney test. RESULTS: Seven rats were randomized to the adhesive group and six to the control group, of which four survived. There were no histopathologic differences in the temporal bones of the animals other than the presence of mild foreign body reaction around the ossicular chain of the animals in the adhesive group. The ossicular chain was not intact in two of the four controls, whereas the rest were intact at 8 weeks. Postoperative air conduction ABR results (mean dB sound pressure level) (62.5 control versus 34.3 adhesive, p = 0.010) and air-bone gaps (47.5 control versus 18.9 adhesive, p = 0.008) were significantly better in the adhesive group. CONCLUSIONS: This new medical-grade tissue adhesive improves the hearing results of ossicular chain reconstruction, with no apparent histotoxicity in this animal model.

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.

Involvement of interleukin-1 in middle ear cholesteatoma.

PURPOSE: The histopathological characteristics of middle ear cholesteatoma are hyperproliferation and differentiation of the epithelium and accompanying destruction of the bones. We directed our attention to interleukin 1 alpha (IL-1 alpha) and demonstrated localization of IL-1 alpha in the epidermis of cholesteatoma. In addition, a comparative study was made of the relationship of IL-1 alpha with the developmental stage of cholesteatoma, the degree of destruction of the auditory ossicles, presence/absence of otorrhea, and the state of subepithelium. MATERIALS AND METHODS: Middle ear cholesteatoma tissues collected during operations were used as the experimental material, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and immunohistological techniques were used as methods. RESULTS: An anti-IL-1 alpha antibody-positive 17-kd band was observed in 2 of the 17 cases, whereas a 31-kd band was observed in 10 of the 17 cases. Neither the 17-kd band nor the 31-kd band was detected in normal skin tissues. The immunohistological staining showed the presence of IL-1 alpha in a region near the basal membrane of cholesteatoma epithelium. CONCLUSION: A correlation was observed between IL-1 alpha and patients with vigorous proliferation of granulation in the subepithelium. The presence of granulation in the reverse side of the eardrum plays an important role in the proliferation of cholesteatoma epithelium.

Distribution of hyaluronan in the middle and inner ear. A light microscopical study in the rat using a hyaluronan-binding protein as a specific probe.

The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analyzed in middle and inner ear tissues from rats by use of microwave-aided fixation, a hyaluronan-binding protein and the avidin-biotin/peroxidase staining procedure. In the middle ear HYA was mainly localized in the pars flaccida, the ossicles, the round window membrane and in the endomysium of the middle ear muscles. The subepithelial stroma of the Eustachian tube was strongly HYA-positive. In the inner ear, the spiral ligament of the cochlea and the connective tissue surrounding the nerve fibers emerging from the crista ampullaris stained intensely for HYA. Except for a weak staining of the basilar membrane, the Corti organ was devoid of HYA. The heterogenous distribution of HYA may indicate its specific involvement in middle and inner ear physiology and pathology.