Human Otic Capsule Research Articles

Osteoprotegerin expression and sensitivity in otosclerosis with different histological activity

Otosclerosis is a complex bone dystrophy of the human otic capsule leading to conductive and sensorineural hearing loss. Since otosclerosis may, at least in part, be considered as an autoimmune-inflammatory disease, disturbed balance of TNF-alpha and osteoprotegerin (OPG) expression has been implicated in the pathological bone remodeling. It has been supposed that active otosclerosis is characterized by decreased or missing local OPG production with invariable OPG sensitivity of the otosclerotic foci. Ankylotic stapes footplates (n = 41) removed by stapedectomy were processed to histological examination, OPG-specific RT-PCR, tissue culturing and alkaline-phosphatase (AP) activity assessment, respectively. OPG concentration of serum specimens (n = 41) was measured by ELISA. Cortical bone fragments harvested from the external ear canal were used as negative controls of otosclerosis. Among 41 ankylotic stapes footplates, 22 active and 19 inactive otosclerosis cases were histologically diagnosed. OPG expression was significantly lower (p < 0.001) in active otosclerosis compared to inactive cases. Osteoclast cultures originated from active otosclerotic foci showed a considerable susceptibility against external OPG dosage, which resulted in a significant decrease of AP activity (p < 0.001). In contrast, OPG serum levels were in the normal range (5-100 ng/ml) indicating a non-systemic bone resorption. In conclusion, secondary decreased local OPG production might play an important role in the pathogenesis of otosclerotic bone remodeling disorder. As to previous and current results, decreased OPG sensitivity of lesion-forming cells should be excluded. These observations may indicate the potential role of recombinant OPG treatment in early stages of otosclerosis.

A method for MS E differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography–mass spectrometry (LC–MS) method for tissue sections embedded with celloidin. Q-TOF (Quadrupole-time of flight mass spectrometry) MS E (mass spectrometry where E represents collision energy) and Identity E™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins. Formalin-fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin de-cross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 μm sections of mouse inner ear samples, and FFCE 20 μm human inner ear and human otic capsule bone sections. Three of the five-tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified Liquid Tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Otosclerosis

Otosclerosis is an inflammatory bone remodeling disorder of the human otic capsule, which might be characterized by a disturbed balance between cell survival and apoptosis due to an increased expression of inflammatory cytokines, mainly tumor necrosis factor-[alpha] (TNF-[alpha]). Histologic features of otosclerosis have been well described; however, different histopathologic and clinical stages have not been attributed precisely to the molecular biology of the pathologically increased metabolism of bone-forming and bone-resorbing cells. Forty ankylotic stapes footplates (n = 40, males = 17, females = 23) removed by stapedectomy were histologically analyzed by conventional hematoxylin-eosin staining, and hCIAP1/2 (inhibitors of apoptosis) and granzyme-[beta] (apoptosis inducer) specific immunofluorescent assays were performed. Four normal stapes footplates obtained from cadavers with negative otologic history were used as negative controls. Active otosclerosis (n = 19) was featured by robust expression of apoptosis inhibitor proteins hCIAP1/2 and negligible expression of granzyme-[beta]. Inactive cases of otosclerosis (n = 8) were characterized by inverse reaction: granzyme-[beta] was highly expressed; however, hCIAP1/2 specific immunoreactions were absent. Nonotosclerotic and normal stapes specimens showed no considerable little granzyme-[beta] expression and moderate hCIAP1/2-specific immunoreactions. Expression pattern of apoptosis-associated proteins showed strong correlation with the histologic diagnosis and activity of otosclerosis (Yates-corrected [chi]2 test, p < 0.001). Detection of the inversely expressed apoptosis inhibitor and inducer proteins in active and inactive stages of otosclerosis demonstrates pathologic regulation of cell survival and apoptosis. These results may suggest active otosclerosis inactivation by TNF-[alpha] induced apoptosis. Anti-TNF-[alpha] biologics may serve as an option in the medical treatment of active otosclerosis.

Etiopathogenesis of otosclerosis

The objectives of our study was to review our current knowledge of the etiopathogenesis of otosclerotic bone remodeling including genetics, viral infection, autoimmunity and inflammation and to discuss disease pathogenesis with relevance to pharmacotherapy. Relevant publications on the etiopathogenesis, molecular biology, genetics and histopathology of otosclerosis from 1984 to 2009 were analyzed. Otosclerosis is a bone remodeling disorder of the human otic capsule; however, the etiopathogenesis remains unclear. Genetic predisposition, disturbed bone metabolism, persistent measles virus infection, autoimmunity, and hormonal and environmental factors also may play contributing roles in the pathogenesis of otosclerosis. Since diagnosis of otosclerosis is still based on histopathological examination of the removed stapes footplate, systemic prospective studies based on comprehensive histopathological and molecular biological analysis are necessary to obtain further information on the background of the disease.

TNF-α Receptor Expression Correlates With Histologic Activity of Otosclerosis

Otosclerosis is an inflammatory bone remodeling disorder of the human otic capsule, which might be characterized by variable levels and unique expression pattern of TNF-alpha receptors. Histologic characteristics of otosclerosis have been well described during the latest decades; however, the grading of different histopathologic and clinical stages has not been attributed precisely to the molecular biology of the pathologically increased metabolism of osteoclast-osteoblast axis. Forty otosclerotic- and 40 nonotosclerotic ankylotic stapes footplates (n = 80; men, 29; women, 51) were histologically analyzed: conventional hematoxylin-eosin staining and tumor necrosis factor-alpha receptor I and II (TNFRI/II)-specific immunofluorescent assay was performed. Active otosclerosis (Grades I-II; n = 24) was featured by increased expression of TNFRII and moderate expression of TNFRI; inactive cases (Grades III-IV) were characterized by permanent expression of TNFRI; however, TNFRII-specific immunoreaction was absent. Nonotosclerotic stapes specimens showed a negligible TNFR expression. Tumor necrosis factor receptor expression pattern showed a strong correlation with the histologic activity of otosclerosis (chi2 test; p < 0.001). Detection of elevated TNFR expression demonstrates activated osteoclast metabolism and inflammatory pathways in otosclerosis. Different etiopathogenesis of otosclerotic and nonotosclerotic stapes ankylosis should be distinguished. Administration of monoclonal anti-TNF-alpha antibody may be a reasonable option in the medical treatment of active stages of otosclerosis.

Restriction analysis of otosclerosis-associated CD46 splicing variants

Otosclerosis is a primary bone remodeling disorder of the human otic capsule and is associated with persistent measles virus infection. The human cellular receptor of measles virus is the membrane cofactor protein (MCP, CD46), which has 14 well-described splicing variants. Unique CD46 expression pattern of the otic capsule and the stapes footplate may determine the susceptibility for persistent measles virus infection. A total of 51 surgically removed ankylotic stapes footplates were analyzed by histopathological and molecular biological methods, respectively. Nucleic acids were extracted. Measles virus sequences were detected by nucleoprotein RNA-specific reverse transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNA of CD46 isoforms was amplified by RT-PCR; cDNA amplimers were separated by SDS poly-acrylamide gel electrophoresis and were purified from the gel. Complementary DNA of CD46 isoforms was restricted by endonuclease enzymes having CD46-specific recognition sites. The presence of viral RNA was associated exclusively with the histopathological diagnosis of otosclerosis; the stapes specimens with negative measles virus belonged to non-otosclerotic stapes fixations. All specimens (N = 51) were characterized by the consecutive expression of five CD46 variants (c, d, e, f and one shorter unidentified isoform). Histologically confirmed ostosclerotic specimens (N = 21) were characterized by increased expression levels of variant "f" and the unknown isoform. Increased expression levels of these isoforms and special CD46 expression pattern of the human otic capsule might produce modified or pathological intracellular signalization that could create the possibility of persistent measles virus infection.

Disease‐Associated Novel CD46 Splicing Variants and Pathologic Bone Remodeling in Otosclerosis

Otosclerotic bone is supposed to show unique CD46 expression pattern because otosclerosis is an organ-specific disease with viral etiology. Otosclerosis is a complex bone remodeling disorder of the human otic capsule, which is associated with persisting measles virus infection. The general cellular receptor of measles virus is the CD46, which has 14-known splicing isoforms. Nucleic acid was extracted from ankylotic stapes footplates (N = 99) removed during stapedectomies. Consecutive histological, CD46 specific immunohistologic analysis, and multiple polymerase chain reaction (PCR) amplifications were performed. Measles virus was detected by seminested reverse transcriptase-PCR. Splicing variants of CD46 were identified by nested reverse transcriptase-PCR and finally determined by mass sequencing of complementary DNA. Measles virus RNA was detectable only in histologically otosclerotic stapes footplates. Virus negative-fixed stapes represent degenerative disorders of variable histopathology. Otosclerosis is featured by an increased number of osteoclasts showing strong CD46 immunoreaction in contrast to nonotosclerotic stapes fixations. Normal and nonotosclerotic stapes footplates show consistent expression of "c," "d," "e," "f," and "l" CD46 splicing isoforms. In contrast, four novel CD46 splicing variants were additionally detected in otosclerosis: os1, os2, os3, and os4. Newly described CD46 isoforms have shorter or missing transmembrane domain and a rare cytoplasmic tail with pathological or uncommon signal transduction; however, virus binding ability remains equal and invariable. These changes may be responsible for the smooth virus replication. A special expression pattern and altered functions of CD46 could explain the organ-specific and virus-associated pathogenesis of otosclerosis.

Demonstration of intravital microfissures in undecalcified plastic-embedded temporal bones with the prestaining technique.

Microfissures in the human otic capsule have been observed since the start of the century, but it was Otto Mayer, in 1930, who first realized that some of them were of intravital origin and not just processing artifacts. Since then, a small number of publications, based on decalcified temporal bones, have mostly confirmed his findings. With the introduction by Frost in the late 1950s of the undecalcified bone technique and the bulk staining technique for peripheral bones, a method was developed and refined for identifying even very small intravital microfissures (microdamage). Bulk staining of undecalcified otic capsules has not yet been used to verify the findings from the previous decalcified specimens. The present report presents our experience with the pertinent techniques, and suggests modifications and shortcuts pertinent to temporal bone research. Both large and tiny microfissures of intravital genesis are demonstrable within bulk-stained undecalcified human otic capsules. The importance of microfissures in the petrous bone in the causation of otosclerosis and perilymphatic leakage has long been discussed, and the present techniques may advance our understanding of these pathological conditions

Recognition of basic fuchsin prestained microfissures of intravital origin with fluorescence microscopy: validation of a shortcut

For 70 years it has been suspected that not all microfissures in histological bone sections are artifacts, but that some are provoked in vivo through repetitive stress. The development of undecalcified bone techniques and of the bulk staining technique has established a method for demonstrating the existence of intravital cracks and enhanced the discrimination towards artifactual microfissures in the load-bearing skeleton. Recently the presence of intravital microfissures has also been ascertained in temporal bones by these techniques. Due to the fluorescent properties of basic fuchsin it is possible to use epifluorescence microscopy for analysis of microfissures after bulk staining with basic fuchsin. This provides a more steady microscopic background and a sharper delineation of surface level structures since no projection from lower levels interfere. Artifactual cracks, which in transmitted light microscopy may look like darkly stained intravital microfissures due to refraction phenomena, become invisible or colorless. Epifluorescence microscopy enhances the detection of both smaller and larger prestained intravital microfissures, and leaves only a minor part of the cracks without certain categorization. The epifluorescence mode of analysis has the further advantage of being independent of slice thickness, making feasible whole-specimen analysis by serial stepwise grinding. The present study shows that the number and the length of microfissures in the human otic capsule, counted and measured under the epifluorescence microscope, equals numerically the findings in light microscopy, enabling the routine use of this mode of analysis. This may prove to be of particular value in the research into the etiology and pathogenesis of otosclerosis as well as perilymphatic fistulae.

Ultrastructure of otic capsule sclerosis in Palmerston North autoimmune mice

Purpose: Numerous temporal bone studies have reported a correlation between systemic autoimmune disease and osteogenic lesions within the inner ear. However, little is known of the cellular mechanisms that relate these two disease processes. The Palmerston North (PN) autoimmune strain mouse exhibits both spontaneous systemic autoimmune disease and otic capsule sclerotic lesions that are similar in many ways to those reported in humans. This suggests the PN mouse is a potential model in which to study the cellular events responsible for immune-related otic capsule lesions. Therefore, an evaluation of the fine structure of the PN modiolus was conducted to better understand these matrix changes of the inner ear. Materlals and Methods: Inner ears were collected from 15 PN mice at ages from 17 to 24 months and prepared for electron microscopy. The ears were ultrastructurally evaluated to characterize the lesions and their associated cytoarchitecture. Results: The sclerotic lesions consisted of an electron-dense mass that appeared lobulated or layered, usually adjacent to the modiolar bone and blood vessels. Immediately surrounding the lesions were activated fibroblasts and fine fibrillar material in the extracellular space between them. The sclerotic foci often were apposed to normal modiolar bone that never appeared degraded. Conclusions: The similarities between these bony lesions and known human otic capsule diseases suggests parallel processes are involved. Thus, further study of the PN inner ear may provide insight into the cellular events that underlie otic capsule and other temporal bone alterations in systemic autoimmune diseases.

Laser-Doppler Measurements and Electrocochleography during Ischemia of the Guinea Pig Cochlea: Implications for Hearing Preservation in Acoustic Neuroma Surgery

Interruption of cochlear blood flow has been implicated as one of the causes of the sensorineural hearing loss that may occur during acoustic neuroma surgery. With the guinea pig as an animal model for cerebellopontine angle surgery, laser-Doppler measurements were used to estimate the cochlear blood flow changes caused by compression of the eighth nerve complex. With compression, the laser-Doppler measurements decreased abruptly; somewhat later, the electrocochleographic potentials declined. When compression was released, laser-Doppler measurements usually returned immediately, followed later by return of the electrical potentials. Some of these potentials, including the compound action potential of the auditory nerve, often became transiently larger than their precompression values. Interposing bone between the laser-Doppler probe and the otic capsule, so that the total bone thickness approximated the thickness of the human otic capsule, decreased the laser-Doppler measurement, but changes caused by compression were still apparent. Thus, although the human otic capsule is much thicker than the guinea pig capsule, it may still be possible to make laser-Doppler estimates of human cochlear blood flow. Laser-Doppler monitoring during acoustic neuroma surgery may be beneficial, because it could give earlier warning of ischemia than is currently available from electrocochleographic monitoring, thereby enabling earlier corrective action. Electrocochleography complements laser-Doppler measurements by indicating the physiologic state of the cochlea.

Early Bone Formation in the Human Fetal Otic Capsule

The present study was concentrated on the use of energy-dispersive X-ray analysis and backscattered electron imaging as practical tools for advanced autopsy of human fetuses when diagnostic evaluation of ear pathology is required. These methods were used to revisit the primary calcification front of the fetal otic capsule between 18 and 36 weeks' gestational age. Energy-dispersive X-ray analysis indicates an equal Ca/P ratio in all the three layers of the otic capsule. These results are discussed in view of calcium homeostasis and inner ear function.

Early ossification within the human fetal otic capsule: morphological and microanalytical findings.

Besides the use of conventional techniques such as light and polarization microscopy, the present paper proposes the combined use of transmission electron microscopy, secondary and backscattered electron imaging, energy dispersive X-ray analysis and computed tomography for the diagnostic evaluation of ear pathology in the human fetus. These methods were used to revisit the primary calcification front of the fetal otic capsule between 16 and 23 weeks gestational age. Ultramicroscopic evaluation demonstrates similar fetal bone formation to that found in other bones of the human fetus. The formation of the endosteal and periosteal layers is a typical example of early intra-membranous ossification. The enchondral layer is made up of fibrillar bone, laid down around the calcified cartilage remnants. Microchemical analysis indicates a significantly higher Ca/P ratio in the endochondral layer with respect to the endosteum and periosteum. The consequences of a lower Ca/P ratio in the endosteal layer are discussed in view of calcium homeostasis and inner ear function.

Articular and internal remodeling in the human otic capsule.

AbstractA comprehensive histologic study of the human otic capsule is presented demonstrating the interrelations between cartilage, bone, blood vessels and soft tissue throughout life. Remodeling occurs through continual degeneration of chondrocytes within the uncalcified articular surfaces of the stapediovestibular joint and through continual degeneration of cartilage about the cochlea and semicircular canals. Degenerated chondrocytes are removed by macrophages and new endochondral bone forms from adjacent osteogenically active blood vessels. The cartilage surfaces of the footplate and oval window are constantly replenished by new cartilage formed by mesenchymal cells of the annular ligament. Within the otic capsule, new cartilage forms from mesenchymal cells lining the labyrinth. Cartilage foci continually undergo partial replacement by endochondral bone with remnants of uncalcified cartilage matrix remaining, forming globuli ossei and interglobular spaces. In addition to continual endochondral bone formation partially replacing the continually forming and degenerating cartilage, chondroid bone forms by direct transformation of some cartilage to bone. These chondroid bone areas are cellular in young age groups, but become pale and relatively acellular with age. These processes occur throughout life, regardless of age or sex.

Fissula ante fenestram and the adjacent tissue in the human otic capsule

(1935). Fissula ante fenestram and the adjacent tissue in the human otic capsule. Acta Oto-Laryngologica: Vol. 22, No. 3, pp. 382-392.