Identification of Cellular Voids in the Human Otic Capsule.

This study aimed to describe the spatial distribution of osteocyte-depleted areas, so-called cellular voids, in the human otic capsule and compare it with that of otosclerosis. Systematic histological studies of the bony otic capsule have revealed an osteoprotegerin (OPG)-mediated inhibition of normal bone remodeling around the inner ear. The resulting accumulation of bony degeneration and dead osteocytes has been thoroughly documented, and the spatial distribution of dead osteocytes and matrix microcracks resembles that of the human ear disease otosclerosis. Clusters of dead osteocytes that may interfere with osteocyte connectivity and thereby the OPG signaling pathway have been described in human temporal bones. It is possible that these cellular voids create disruptions in the antiresorptive OPG signal that may give rise to local pathological remodeling. Recently, a method of detecting cellular voids was developed. This study uses unbiased stereology to document the spatial distribution of cellular voids in bulk-stained undecalcified human temporal bone. Cellular voids accumulate around the inner ear and increase in number and size with age. Furthermore, cellular voids are more frequently found in the anterior and lateral regions of the otic capsule, which are known predilection sites of otosclerosis. This colocalization of cellular voids and otosclerosis suggests a causal relationship between focal degeneration and otosclerotic remodeling.

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.

Background The bony otic capsule is comprised of highly mineralized and dense compact bone. It is rarely remodelled and degenerative changes, therefore, accumulate around the inner ear. It is also a predilection site for the pathological remodelling seen in otosclerosis. Morphometric studies have documented increased numbers of dead osteocytes and microcracks in the human otic capsule. Microcracks may disrupt the lacuno-canalicular network and cause osteocyte apoptosis ultimately breaking up the perilabyrinthine bone signalling pathways and dynamics. This may be important to understand the pathogenesis of remodelling diseases like otosclerosis. Aims/Objectives This study describes the spatial and regional distribution of microcrack surface density in relation to the inner ear and compares it to that previously recorded for otosclerosis. Material and methods Forty-two temporal bones and five ribs were used. All samples were undecalcified, bulk stained in basic fuchsin and plastic embedded. Unbiased stereology was used to estimate the true surface density of microcracks (mm2/mm3) in perilabyrinthine bone. Results The surface density of microcracks accumulates around the inner ear spaces, particularly in the lateral window regions, and increases with age. Conclusions and significance This study documents the spatial and temporal association between microfractures and otosclerosis in the otic capsule.

Conclusions: Age-dependent microdamage (MDx) accumulates excessively in human perilabyrinthine bone, where the bone turnover is almost absent. This may have pathological implications for bone-specific disorders such as otosclerosis. The role of MDx accumulation is discussed from an osteodynamic perspective. Objectives: Bone remodelling is highly inhibited within the otic capsule compared with the rest of the skeleton. Consequently excessive accumulation of age-dependent capsular MDx is expected. This study describes the prevalence, size and topographical distribution of MDx in the human otic capsule. Methods: A total of 241 undecalcified human temporal bones were examined. Bulk staining and the cutting-grinding technique were used to separate in vivo MDx from microcrack artefacts induced post mortem by the milling procedure. Quantitative data were obtained by fluorescence microscopy by counting and measuring and by the use of stereology. Results: Microcracks accumulated continuously and extensively in the human otic capsule throughout life. Both the number and total length of MDx were higher close to the inner ear space as compared with the capsular periphery. The mean length of the MDx remained constant with age. There was no statistically significant sex difference.

There is no clinical imaging method to visualize the soft tissues of the human cochlea, which are crucial for sound transduction and are damaged in sensorineural hearing loss. Although optical coherence tomography (OCT) has been effective in small animal models, we show for the first time that it can image through the full thickness of the ex-vivo human otic capsule and resolve cochlear microstructures despite increased scattering. We aim to investigate whether OCT could image the cochlea through the otic capsule. We compared 1.7 and OCT to test if the reduced scattering at provided any appreciable advantage for imaging the cochleae. OCT interferometers were built for both 1.3 and wavelengths, using identical sample and reference arm optics in both systems. Imaging was performed on two fixed human temporal bones with intact cochleae. The interferometers were designed to allow seamless switching between 1.3 and OCT without disrupting the temporal bone during imaging. We took volumetric OCT images at the base, apex, and hook regions of fixed ex-vivo human cochleae and compared the images taken at with those taken at . At both wavelengths, we could see through the otic capsule and identify cochlear structures. In some cases, OCT resulted in clearer images of the lateral wall, interior scala, and fine cochlear structures due to reduced multiple scattering at depth compared with . We conclude that both and OCT can image through the human otic capsule, offering the potential for direct measurement of cochlear vibrometry or blood flow in living humans. Using light, we observed reduced multiple scattering in the otic capsule, leading to enhanced contrast of cochlear structures compared with . However, these improvements were marginal and came with trade-offs.

Bone is continuously remodeled to repair and strengthen degenerative bone with accumulating dead osteocytes and microfractures. Inner ear osteoprotegerin (OPG)-mediated inhibition of otic capsular bone remodeling causes excessive perilabyrinthine bone degeneration. Consequently, microcracks accumulate around the inner ear. Microcracks cause osteocyte apoptosis and may disrupt the canalicular network connecting osteocytes. Despite their linear microscopic appearance, microcracks are three-dimensional disruption planes and represent surface areas inside a tissue space. With an elevated microcrack burden the number of disconnected osteocytes is expected to increase. This may prove relevant to ongoing research in otic focal pathologies like otosclerosis. Therefore, an unbiased quantification of the microcrack surface density (mm2 /mm3 ) in the human otic capsule is essential. In this study unbiased stereology was applied to undecalcified bulk stained human temporal bones to demonstrate its feasibility in describing the three-dimensional reality behind two dimensional observations of microcracks. A total of 28 human temporal bones and five ribs were bulk stained in basic fuchsin, serially sectioned and hand-ground to a thickness of 80-120 μm. Both horizontal and vertical sections were produced and compared. This study showed that surface density of microcracks was significantly higher around the inner ear compared to ribs. Furthermore, no significant difference in microcrack surface density between horizontal and vertical sections in the temporal bone was demonstrated.

Conclusions: 1. The principle of bilateral symmetry depends on the chordal cartilage that is the keystone in cranial base ossification in rats and humans, due to its anatomical situation and for the production of the chordin protein that regulates the bone morphogenetic protein BMP-7. 2. In humans and in rats, foramen lacerum closure follows a line of intramembranous ossification that depends on BMP-7, regulated by the first branchial pouch. 3. The cranial base ossification patterns and centres are similar in humans and in rats, except in the otic capsule, palate and the lateral pterygoid plate. 4. The neural crest may induce cranial ossification through the cranial nerves. Objectives: To study the patterns of cranial base ossification in humans and in rats, considering the chordal cartilage, and the otic, nasal and orbit capsules, as well as the participation of the branchial arches and pouches. Methods: This was a light microscopy study of human fetal specimens obtained from spontaneous abortions with the following crown-rump-lengths (crl) 45, 74, 90, 134, 145 and 270 mm, and a 1-day-old neonate (360 mm crl), who had died of sudden death syndrome. We also examined Webster albino rat embryos of 16, 18 and 20 days of gestation and a postnatal series of rats 8 h and 1, 3, 4, 6, 7, 10 and 13 days old, as well as adult animals. Results: In the 45 mm human fetus, the chordal cartilage with the nasal, otic and orbit capsules initiates cranial base ossification. Foramen lacerum closure begins in the 16-day-old rat embryo, following a line of membranous ossification between the external pterygoid process and the lateral alisphenoidal wing at ovalis foramen level. This is not a timing symmetrical process, which may persist until the 10th postnatal day in the rat. In the human fetus of 74 mm, the foramen lacerum space is closed by a membranous fusion ossification between the chordal cartilage and otic capsule, finishing at the 270 mm specimen. Endochondral ossification of the human otic capsule first appeared in the 145 mm (18 weeks) fetal specimen with four ossifying centres. The rat otic cartilaginous capsule showed rapid endochondral ossification, in the third and fourth postnatal day specimens.

The otic capsule, when compared with other bones in the body, is unique in that it undergoes no significant remodeling of bone after development. We previously demonstrated that osteoprotegerin (OPG), which inhibits formation and function of osteoclasts, is produced at high levels in the inner ear of normal mice and secreted into the perilymph from where it diffuses into the surrounding otic capsule bone through a lacunocanalicular system. To test our hypothesis that the high level of OPG may be important in the inhibition of otic capsule remodeling, we studied the light microscopic histology of the otic capsule in OPG knockout mice for evidence of abnormal remodeling of bone. We also tested the hearing in OPG knockout mice to determine whether OPG and its influence on surrounding bone is important for auditory function. Temporal bone histopathology and pathophysiology were compared in homozygous OPG knockout mice and C57BL/6 (B6) mice, the background strain for the knockouts. Auditory function in age-matched animals from each group was evaluated at approximately 4-week intervals from 8 to 21 weeks using frequency-specific auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). After each of the last three evaluations, the cochleae from one mouse of each group were harvested, processed, and examined by light microscopy. Osteoprotegerin knockout mice demonstrated abnormal remodeling of bone within the otic capsule with multiple foci showing osteoclastic bone resorption and formation of new bone. Such changes were not seen in the age-matched B6 controls. The active bone remodeling process in the knockout animals showed many similarities to otosclerosis seen in human temporal bones. Over the time period that we monitored, auditory function was significantly and progressively compromised in the knockout animals relative to B6 controls. At the earliest age of test (8 wk), the loss was apparent as a mild, high-frequency reduction in sensitivity by ABR. In contrast, DPOAE losses in the knockouts were substantial even at 8 weeks, and by 21 weeks, these losses exceeded our equipment limits. Results of ABR testing showed hearing sensitivity changes in the animals of the background strain were confined largely to the high frequencies, whereas OPG knockouts demonstrated substantial low-frequency shifts in addition to those at high frequencies. The histopathological and pathophysiological findings in OPG knockout mice support the hypothesis that OPG is important in the inhibition of bone remodeling within the otic capsule and the maintenance of normal auditory function. This mouse may provide a valuable animal model of human otosclerosis.

To elucidate factors that may be responsible for the inhibition of remodeling of bone within the otic capsule. Expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK), and RANK ligand (RANKL) were assayed in samples of bone obtained from the otic capsule, calvarium, and femur, and from the soft tissue within the cochlea using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in mice. Immunostaining was used for histologic localization of the gene products. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of OPG within perilymph, serum, and cerebrospinal fluid. The micro-anatomy of the interface between the otic capsule and the fluid spaces of the cochlea was investigated by brightfield and phase-contrast microscopy and by three-dimensional reconstruction in the mouse and human. OPG, a powerful inhibitor of bone remodeling, was expressed at extremely high levels within the soft tissue of the cochlea and was present in the perilymph at very high concentrations. The OPG produced within the inner ear may diffuse into the surrounding otic capsule, where it may be responsible for inhibition of bone turnover. Our anatomic studies revealed an extensive system of interconnected canaliculi within the otic capsule that had direct openings into the fluid spaces of the inner ear, thus providing a possible anatomic route for the diffusion of OPG from the inner ear into the surrounding bone. OPG, a potent inhibitor of osteoclast formation and function, is expressed at high levels within the inner ear and is secreted into the perilymph and the surrounding bone and may serve to inhibit active bone remodeling within the otic capsule, especially immediately adjacent to the cochlea. By this means, the cochlear soft tissue may control the nature of the surrounding petrous bone.

The CaV1.2 L-type calcium channel regulates bone homeostasis in the middle and inner ear.

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

A number of bone-related genes may be responsible for the unique suppression of perilabyrinthine bone remodeling. Bone remodeling is highly inhibited around the inner ear space most likely because of osteoprotegerin (OPG), which is a well-known potent inhibitor of osteoclast formation and function. However, other signaling molecules may also be responsible for the inhibition of bone remodeling within the otic capsule. Microarray technology was used to determine bone-related genes differentially expressed between the lining tissues of the otic capsule (spiral ligament and stria vascularis) and the lining tissues from the middle ear of the rat. Data was analyzed with statistical bioinformatics tools. Gene expression levels of selected genes were validated using quantitative polymerase chain reaction. A total of 413 genes were identified when young inner bulla (growing) were compared with young otic capsule and 358 genes were identified when adult inner bulla (quiescent) were compared with adult otic capsule. Fourteen genes were involved in bone metabolism of which four genes have been previously discussed in the literature of perilabyrinthine bone biology. The gene expression of the otic capsule was significantly different from that of the middle ear. This study identified a number of differentially expressed bone-related mRNAs of potential significance and confirmed the OPG/receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) pathway as the key signaling system for the unique behavior of bone cells within the otic capsule. No differentially expressed up- or downstream messengers in the OPG/RANK/RANKL pathway were found.

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.

A 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.

Local delivery of bisphosphonates results in superior localization of these compounds for the treatment of cochlear otosclerosis, without ototoxicity. Otosclerosis is a common disorder of abnormal bone remodeling within the human otic capsule. It is a frequent cause of conductive hearing loss from stapes fixation. Large lesions that penetrate the cochlear endosteum and injure the spiral ligament result in sensorineural hearing loss. Nitrogen-containing bisphosphonates (e.g., zoledronate) are potent inhibitors of bone remodeling with proven efficacy in the treatment of metabolic bone diseases, including otosclerosis. Local delivery to the cochlea may allow for improved drug targeting, higher local concentrations, and the avoidance of systemic complications. In this study, we use a fluorescently labeled bisphosphonate compound (6-FAM-ZOL) to determine drug localization and concentration within the otic capsule. Various methods for delivery are compared. Ototoxicity is evaluated by auditory brainstem responses and distortion product otoacoustic emissions. 6-FAM-ZOL was administered to guinea pigs via intraperitoneal injection, placement of alginate beads onto the round window membrane, or microfluidic pump infusion via a cochleostomy. Hearing was evaluated. Specimens were embedded into resin blocks, ground to a mid-modiolar section, and quantitatively imaged using fluorescence microscopy. There was a dose-dependent increase in fluorescent signal after systemic 6-FAM-ZOL treatment. Local delivery via the round window membrane or a cochleostomy increased delivery efficiency. No significant ototoxicity was observed after either systemic or local 6-FAM-ZOL delivery. These findings establish important preclinical parameters for the treatment of cochlear otosclerosis in humans.