Mitochondrial cytochrome oxidase immunolabeling in aged human temporal bones.

Presbycusis, an age-related hearing loss, is accompanied by histopathological cochlear changes including variable amounts of degeneration of the auditory receptors, neurons and the stria vascularis. The causes of degeneration are unknown, although acoustic trauma and exposure to ototoxic agents are certainly contributors to the cellular degeneration. Acquired mitochondrial DNA defects are postulated as important determinants of aging in neuromuscular tissues. The cochlear neurons are highly metabolic and are, therefore, likely to be affected by mitochondrial DNA defects. Sequence analysis has demonstrated a significant number of acquired mutations in the cytochrome oxidase gene in the neurons from aged human cochleas. The current study used immunohistochemical labeling of cytochrome oxidase in the neuronal cell bodies in archival celloidin sections to evaluate relationships among label density, hearing loss, number of neurons and mitochondrial DNA changes within individual cochleas. Label density was less in many aged temporal bones, but not all. There was no relationship among any other variables. It is concluded that while there may be a decrease in the amount of cytochrome oxidase expression in aged spiral ganglion cell bodies, there are many other factors that contribute to hearing loss and cellular degeneration.

An immunohistochemical study of inducible nitric oxide synthase in the rat middle ear, with reference to tympanosclerosis.

Tympanosclerosis and myringosclerosis are well-known sequelae after acute and chronic otitis media and are also often seen after treatment of secretory otitis media with ventilation tubes. They sometimes cause serious hearing disability. There is no successful treatment for these conditions. There might be factors triggering an immunological or autoimmune chain reaction, which leads to tympanosclerosis. Intervention with the aim of abolishing this type of response might be possible if an interruption of the chain reaction can be found. Nitric oxide is a radical molecule with the ability to kill pathogens and is produced by the enzyme nitric oxide synthase. Expression of inducible nitric oxide synthase (iNOS) was analysed immunohistochemically in a rat model of acute otitis media. In rats sacrificed at days 3 and 6 after inoculation. iNOS was also strongly expressed in the middle ear mucosa and in the tympanic membrane as well as in the inner ear. In control specimens as well as in infected ones. iNOS was expressed in the tissue of the external ear canal. In rats sacrificed at day 10 and after 3 months, iNOS was expressed at the same locations, although less frequently. These data indicate that iNOS expression is induced during acute otitis media and suggest that nitric oxide may be important in the host defence against ear infections.

Glycosaminoglycan metabolism and cytokine release in normal and otosclerotic human bone cells interleukin-1 treated.

Glycosaminoglycans (GAGs), normal components of the extracellular matrix (ECM), and the glycosidases, that degrade them, play a key role in the bone remodelling process. The effects of interleukin-1 alpha (IL-1 alpha) on GAG metabolism in normal and otosclerotic human bone cells as well as its capacity to modulate IL-1 alpha, IL-1 beta and IL-6 secretion in both populations was analyzed. The amount of radiolabeled GAGs was lower in otosclerotic than in normal bone cells. IL-1 alpha reduced newly synthesized cellular and extracellular GAGs in normal cells, but only those of the cellular compartment in otosclerotic bone cells. It depressed heparan sulphate (HS) more in normal cells and chondroitin sulphate (CS) more in otosclerotic bone cells. The HA/total sulphated GAG ratio was shifted in favour of the latter in otosclerotic cells, whereas the opposite effect was seen after IL-1 alpha treatment. There was little difference in the beta-D-glucuronidase levels of the normal and pathological cells, while beta-N-acetyl-D-glucosaminidase was significantly increased in otosclerotic bone cells. As the activity of neither enzyme was modified by treatment with IL-1 alpha, the cytokine seems to exert its influences on GAG synthesis rather than on the degradation process. IL-1 alpha, IL-1 beta and IL-6 secretion was markedly higher in otosclerotic cells. IL-1 alpha modulated the secretion of each interleukin differently, thus resulting in a cytokine cascade that may act in autocrine/paracrine manner on target cells. The authors suggest that changes in the cytokine network may have a specific, yet still unknown, role during normal and pathological osteogenesis.

Glycosaminoglycan metabolism in otosclerotic bone cells.

Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.

Aberration of the tissue collagenase system in association with otosclerosis.

Studies of aural and other body tissues suggest that otosclerosis represents the local manifestation of a general disorder of connective tissue. In particular, collagen abnormalities have been described. We have undertaken a pilot study of the in vivo messenger RNA (mRNA) transcription for procollagenase (precursor of collagenase), as well as for stromelysin and tissue inhibitor of metalloprotease (TIMP), an activator and a specific inhibitor of tissue collagenase activity, respectively. Human skin from individuals with surgically confirmed otosclerosis was compared to skin from their family members (clinically positive and clinically negative) and from unrelated normal controls. Preliminary data indicate that on average there are significantly lower levels of mRNA production for stromelysin among individuals with otosclerosis as compared to all others tested. Similar trends were demonstrated for TIMP and procollagenase, although these did not achieve statistical significance. In addition to suggesting a pathogenetic mechanism for the development of the disease, these data could serve as the basis of possible confirmatory tests for early diagnosis of otosclerosis and as a method for evaluating the genotype of offspring of affected individuals prior to their age of clinical manifestation. This could translate into the application of prophylactic treatment regimens in the future. The proposed abnormalities also suggest candidate genes for otosclerosis.

Altered adenylate cyclase activity in human otosclerotic bone cell cultures.

Adenylate cyclase (AC) activity was studied in whole homogenates of normal and otosclerotic bone cell cultures. When Mn2+ or Ca2+ was added to the medium there was a similar increase in AC activity in both cell types. F- provoked a greater rise in normal than in pathological cells, whereas 0.01 mM guanosine triphosphate (GTP) significantly raised cAMP synthesis in otosclerotic cells only. Mn2+ + calcitonin (Ct) increased AC activity in both cell preparations. With Ca2+ as cofactor there was no significant rise in either normal or pathological cells. However, while the combination Ca2+ + Ct + GTP had little effect on normal cells, it markedly increased cAMP synthesis in the pathological cells. 1 microgram/ml of the beta-blocker propranolol inhibited the effect Ct exerts on AC in normal cells, but enhanced it in otosclerotic cells. It would, therefore, seem that the pathogenesis of otosclerosis could be associated with an alteration in the AC system associated with Ct receptors.

Etiology of otospongiotic sensorineural losses.

The etiology of otospongiotic-otosclerotic disease is enzymatic; the proteolytic enzymes released by the otospongiotic-otosclerotic foci damage the inner ear and are also the basis of the bony rebuilding of the OW niche leading to stapedial fixation. The trigger may be an autoimmune process due to the reaction of the enchondral otic capsule against the embryonic cartilaginous remnants, genetically determined to be located in the otic capsule and mainly in the fissula antefenestram.

Cathepsin D activity in otosclerotic bone and perilymph.

Otosclerotic bone and perilymph samples obtained during stapedectomy operations from patients suffering from pure conductive hearing loss of at least 40 dBs air-bone gap at 500 Hz. Control perilymph samples derived from patients suffering from round window membrane rupture or Meniere's disease. Enzyme activities were determined using a synthetic fluorogenic substrate by fluorescence spectrophotometry. No significant differences have been found between the protein content of otosclerotic and normal or non-otosclerotic perilymph samples. Cathepsin D activity was significantly higher (p less than 0.001) in the otosclerotic perilymph samples. Otosclerotic stapes footplate contained more activity than normal meatal cortical bone as well, though the difference was not significant. Biological significance of the elevated Cathepsin D activity must be considered.

Proteolytic enzymes in otosclerosis. Significance of proteolytic enzymes in otosclerotic bone remodelling.

The otosclerotic stapes footplate exhibits higher activities of cathepsin D and H and collagenase-like peptidase than those of normal cortical bone. The elevated enzyme activities of osteoblastic origin (Cl-peptidase and cathepsin D) emphasize the essential and probably primary role of the bone-forming cells, not only in bone formation, but in resorption as well. The highest activity of cathepsin D from among the measured enzymes highlights the adjuvant role of acidic glucosaminoglycans in the otosclerotic demineralization process. As the osteoblastic osteoid synthesis is known to be sharply reduced in otosclerotic bone remodelling, and the above data emphasize the role of proteolytic enzymes of osteoblastic origin too, indirectly, osteoblasts seem to be the otosclerosis signal-transducing cells.

Type II collagen-induced otospongiosis-like lesions in rats.

Otospongiosis-like lesions were induced in rats by immunizing them with type II collagen. After seven months' immunization, the rats were killed and processed for histologic study. We found otospongiotic lesions in the bony cochlea, vestibule, semicircular canal, and in the regions near the oval window and round window. The spongiotic lesions in the otic capsules were similar to human otospongiosis and were characterized by the following types of microscopic appearances. 1) The classic type showed enlarged vascular spaces with congestion, macrophages, fibroblasts, and sometimes osteoclasts. 2) The fibrotic type showed vascular spaces filled with fibrous tissues. 3) The osteoporotic type had a porous appearance and was devoid of content. 4) The sclerotic type showed bone spaces partially or entirely being replaced by new bone with blue mantles and a mosaic appearance. Some spongiotic lesions showed a mixture of the above types. The findings suggest that this animal model may provide important information to help understand the process of human otosclerosis.

Inhibition of myosin light-chain kinase activity in the organ of Corti by 0.3-5 kilodalton substances of the otosclerotic perilymph.

The guinea pig organ of Corti contains myosin light-chain kinase (MLCK) activity. The upper and lower most parts of the cochlea do not show significantly different activities of the enzyme, which is Ca2+ and calmodulin-dependent. Short-term noise exposure does not cause a significant change. 0.3-5 Kilodalton substances of the otosclerotic perilymph, separated by SG-25 column chromatography, inhibit the MLCK activity in in vitro organ of Corti preparations. This inhibitory action of the perilymph substances can also be observed with the purified MLCK of turkey gizzard. The activity of the enzyme can be specifically inhibited by cyclic AMP-dependent protein kinase.

Vitamin D deficiency and otosclerosis.

A prospective study of 47 patients with otosclerosis was undertaken to investigate the possible etiologic role of vitamin D undernutrition. The population comprised 27 women and 20 men, with a mean age of 46.4 years (range 21 to 79). The disease was bilateral in 43 patients, and cochlear involvement was present in 84.4%. The mean duration of symptoms was 17.1 years. Vitamin D status was evaluated by measuring the plasma 25-hydroxy vitamin D3 (25-OHD), which is the main storage metabolite. Abnormally low 25-OHD levels were found in 10 patients (21.7%) and borderline low levels in another two. Raised serum alkaline phosphatase levels were present in 32.6%, calcium in 6.5%, and inorganic phosphate in 4.3%. Calcium and vitamin D replacement therapy resulted in significant hearing improvement in 3 of 16 patients; these data support a causal correlation. Vitamin D deficiency is probably a factor in the etiology of some cases of otosclerosis and is important, since the deafness resulting from cochlear involvement may be reversible.

Otospongiosis as a genetic disease. Early detection, medical management, and prevention.

Extensive research into the inmost mechanism of otospongiotic disease and an extended study of otosclerotic patients' ancestry have led the authors to a plan for prevention of this disease, which appears to be a genetic deafness with autosomal dominant inheritance and about 40 percent penetrance of genes. Progress in impedance audiometry has permitted early detection of stapedial fixation by means of systematic audiometric investigations, particularly the elicitation of stapedius reflex showing a very special pattern called the "on-off effect." The evidence supporting an enzymatic origin of the sensoringeural component of hearing loss in otosclerotic families has led us to treat otospongiotic children with very low doses of sodium fluoride, with no risk of stunting growt. We have been applying this procedure for four years to the families of stapedectomized otosclerotic patients. We believe it would be advisable to extend this type of prevention to schoolchildren by means of systematic audiometric investigations, including the elicitation of stapedius reflex to detect a possible on-off effect. We also studied sodium fluoride therapy, which derives from the enzymatic origin of the otospongiotic disease. This treatment, based on enzymogenesis inhibitors, should be given to young otospongiotic/otosclerotic patients detected by systematic audiometric investigations. Sodium fluoride could even be prescribed for otospongiotic mothers to prevent disease in their newborn children, exactly as in dental prevention. Otospongiotic/otosclerotic disease can be easily controlled by medical treatment, combined with surgery if needed. This treatment is effective thanks to early detection.

Cathepsin-B activity in otosclerosis.

Cathepsin-B activity was determined fluorimetrically in the otosclerotic stapes footplate, the stapes superstructure, normal temporal cortical bone, and os frontale osteoma. Measurements with a synthetic substrate made determinations in individual samples possible. The cathepsin-B activity in the otosclerotic stapes footplate was one order of magnitude higher than that of the superstructure, which was not affected by the disease. The cortical bone and the superstructure displayed similar activities, as did os frontale osteoma and otosclerosis. The high lysosomal proteinase activity appears to be closely connected to the otosclerotic bone resorption process.

The enzymatic mechanism of the otospongiotic disease and NaF action on the enzymatic balance.

Extensive research on enzymes led to the discovery of the innermost mechanism of the otospongiotic disease. The authors present the results of multiple correlations made from February 1976 to September 1980 on 648 samples of perilymph taken during stapedectomies performed on otosclerotic patients. We studied microdosages of three selected enzymes--trypsin, alpha 1 antitrypsin, and alpha 2 macroglobulin--in each of the samples and their relationship with cochlear deterioration expressed in dBs of bone conduction decrease in pure-tone audiometry testing. This study allowed us a better knowledge of the enzymatic mechanism of the otospongiotic disease, based on the previously reported trypsin-alpha 1 antitrypsin balance, but in which alpha 2 macroglobulin appears to play a role as essential as that of alpha 1 antitrypsin. This enzymatic mechanism explains NaF efficiency, which is due in fact to a double action: not only direct trypsin inhibition, but also an overall reduction in enzymatic values in the perilymph of otospongiotic/otosclerotic patients. Current studies could lead to the possibility of future NaF replacement by proteinase inhibitors either of microbial origin, under study by Japanese researchers, or even of synthetic origin, which should be investigated. In fact, the role of NaF therapy could already be taken over by diphosphonates currently under study.

Bone resorption in otospongiosis.

"Considerable interest has been raised in recent years concerning the basic mechanisms involved in bone destruction and rebuilding in the otospongiotic focus. Under general bone resorption the osteoclasts play a decisive role, but in otospongiotic tissue electron-microscopic and cytochemical studies have shown that osteoclasts alone are not responsible for the bone resorption. Mononuclear histiocytes found in the marrow spaces and in the surrounding bone of an otospongiotic focus together with osteocytes take active part in the resorption. Cytochemical studies of acid phosphatase activity have shown that hydrolases in the lysosomes are expelled into the surrounding tissue, resulting in its resorption.

[Enzymatic mechanism of otosclerosis. Action of NaF]. / Méchanisme enzymatique de l'otospongiose. Action du NaF.

The authors present the conclusions they reached after more than four years of multiple correlations made from february 1976 to september 1980. 648 perilymph samples were selected from a total number of 811 samples taken during stapedectomies on otosclerotic patients. These multiple correlations were based on micro-dosages of three selected enzymes (trypsin-alpha 1 antitrypsin and alpha 2 macroglobulin, the fourth cathepsin B having not been found, even in perilymph pools) in each of the selected samples, and on their relationship with the cochlear deterioration expressed in dBs of B.C. decrease and in audiometric stages corrected with reference to the patients' age. This study leads to an enzymatic mechanism, based on the previously reported trypsin-alpha 1 antitrypsin balance, but in which alpha 2 macroglobulin appears to play as essential a role as that of alpha 1 A. This enzymatic mechanism explains NaF efficiency, due in fact to a double action, not only to direct trypsin inhibition, but also to an overall reduction in enzymatic levels in the perilymph of otosclerotic patients. The authors conclude by suggesting the possibility of future NaF replacement by proteinase inhibitors, either of microbial origin currently under study by Japanese researchers, or even of synthetic origin.