Osteoprotegrin knockout mice demonstrate abnormal remodeling of the otic capsule and progressive hearing loss

Abstract

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Schematic diagram showing the relation between osteoblasts and osteoclasts. RANKL expressed by osteoblasts stimulates its specific receptor RANK on preosteoclasts and osteoclasts to initiate the differentiation, activation, and survival of osteoclasts. Osteoprotegerin acts as soluble neutralizing factor and blocks the effect of RANKL.

Fig. 2

All examples are from 13-week-old mice. (A) Lower basal turn of normal C57 control mouse. Open arrow indicates the squamous middle ear epithelium. Filled arrow indicates the otic capsule separating the stapedial artery and the underlying spiral ligament. (B) OPG −/− basal turn. The middle ear mucosa (open arrow) is thickened. The bony shell between the stapedial artery and spiral ligament (closed arrow) has been eroded and the bone superior to the artery is thickened and filled with cells (arrowhead). (C) Higher magnification of the highly cellular otic capsule indicated by the boxed area in B. The arrow indicates a multinucleated osteoclast. (D and E) Closed arrows indicate places where the otic capsule of the upper turn of the cochlea has been eroded in a −/− animal to create a large opening between the spiral ligament and the interior of the capsule. (F and G) Open arrows show a comparison of the normal (F) and OPG −/− (G) at the incudomalleal joint (H) The footplate of the stapes of an OPG −/− case showing only connective tissue (arrow) at one point along the eroded bone. (I) The otic capsule beneath the basal turn of a control mouse with few cells and few open spaces. (J) The corresponding region of an OPG −/− animal showing hypercellularity and numerous large vascular channels (arrows).

Fig. 3

Auditory function in osteoprotegerin knockout and control (B6) groups of mice at 8 weeks and 21 weeks, as determined by auditory brainstem response and distortion product otoacoustic emissions testing. Data shown are mean ± standard error.