Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats.

Our aim was to observe the effects of psychological stress on the structure of the temporomandibular joint (TMJ), and to evaluate the expression of matrix metallopeptidase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in condylar chondrocytes in rats. The rats were divided into 3 groups of 12 according to the duration of psychological stress: 3 weeks or 6 weeks, and 6 weeks of recovery. A fourth group of 12 rats was used as controls. Each rat was evaluated by the open-field test and the weight measured. The results confirmed psychological stress in 24 of the 36 rats (67%). The tissues of the TMJ were stained with haematoxylin and eosin and pathological changes were studied under a light microscope. MMP-3 and TIMP-3 expression was investigated using the SP kit. The experimental groups showed thinning of articular cartilage, shedding of collagen fibres, cracks in the articular discs, and other structural changes that were aggravated with time, from three weeks to six weeks. The 6-week recovery group showed an improvement in these changes, which indicated the initiation of joint repair. The MMP-3 expression rate correlated with the degree of joint lesion, while the TIMP-3 rate showed an opposite trend and was highest in the 6-week recovery group. Our findings clearly indicate that psychological stress may play an important part in the development of TMJ diseases in rats; further studies should be made to extrapolate the results to other models before clinical use.

Calcitonin gene-related Peptide and choline acetyltransferase colocalization in the human vestibular periphery.

Within the vestibular system, calcitonin gene-related peptide (CGRP) has been localized in the efferent terminals and their brainstem neuronal cell bodies in several animal models. Presently, very few studies have verified these findings in the vestibular system in adult primates or humans. CGRP immunoreactivity (CGRPi) and its colocalization with choline acetyltransferase immunoreactivity (ChATi) in human vestibular end organs and Scarpa's ganglion were studied using polyclonal antibodies against CGRP and ChAT, at the light-microscopic level. The CGRPi axons ramified to produce numerous CGRPi terminals throughout the neurosensory epithelium of the maculae and cristae, primarily in the basal and midbasal areas. Numerous CGRPi efferent terminals made contact with both type II vestibular hair cells and the afferent chalices surrounding type I vestibular hair cells. All CGRP immunoreactive fibers also exhibited ChATi. As in the animal models, no CGRPi was found within Scarpa's ganglion. This study provides evidence for CGRPi in the human vestibular periphery and validates the biomedical relevance of the current animal models.

Toxic effects of potassium bromate and thioglycolate on vestibuloocular reflex systems of Guinea pigs and humans.

Potassium bromate (KBrO(3)) and thioglycolate are two components of hair curling solution. The neurotoxic effects of KBrO(3) and thioglycolate on the vestibuloocular reflex (VOR) system have not been elucidated. In this paper, we report the adverse effects of KBrO(3) and thioglycolate on the VOR system of Hartley-strain guinea pigs. The function of the VOR system was evaluated by caloric test coupled with the electronystagmographic recordings after subcutaneous injection of 20 or 50 mg/kg KBrO(3) or 15 mg/kg thioglycolate, either alone or in combination once daily for 14 consecutive days. The results showed that KBrO(3) produced abnormal caloric responses in a concentration-dependent manner and thioglycolate enhanced this abnormality. Our clinical patients, 10 female hairdressers exposed to the hair curling solution for 10-30 years revealed a similar dysfunction in the caloric test. The possible mechanism of this adverse effect was studied: the cerebellar-regulated functions such as motor equilibrium performance and spontaneous locomotor activity of guinea pigs were reduced, the enzymatic Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities of cerebellar tissues were significantly decreased, and the loss of Purkinje cells as well as the derangement of the granular cell layer of the cerebellar cortex was revealed after treatment with KBrO(3) plus thioglycolate. These findings imply that KBrO(3) plus thioglycolate is toxic to the VOR system, mediated by, at least in part, the dysfunction of a higher cerebellar regulatory mechanism. We suggest that the caloric test is a noninvasive method for monitoring the consequences of hazardous exposure of hair curling solution in humans. Our clinical findings together with the animal study imply that clinicians should be alert to the risk of bromate exposure in hairdressers, especially those with vertigo, tinnitus, or hearing loss.

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.

Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction.

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-alpha). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal.

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs.

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct (n = 7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls (n = 6) as well as of sham-operated animals (n = 3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.

Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs.

The exact distribution of nitric oxide-synthases (NOS) in the vestibular system has not been described satisfying yet. Immunostaining, using specific antibodies to the three known NOS-isoforms, to cyclic guanosine monophosphate (cGMP) and soluble guanylyl-cyclase (sGC), the second messenger system of nitric oxide (NO), was performed on paraffin sections of temporal bone from guinea pigs. eNOS could be detected in vestibular ganglion cells and in nerve fibres, including the calyces, surrounding the type 1 hair cells (HC). bNOS was found in the sensory epithelium, ganglion cells and in bone, while iNOS could not be found. NOS-detection was accompanied by reactivity to sGC and to cGMP. This finding implies that b- and eNOS-generated NO is involved in regulative processes in neurotransmission and regulation of blood flow.

Na,K-ATPase in the cochlear lateral wall of human temporal bones with endolymphatic hydrops.

Meniere's disease has traditionally been thought to arise from a disruption in longitudinal endolymphatic flow. This view has been brought into question by recent experimental studies that have focused attention on derangements of cochlear fluid and electrolyte homeostatic mechanisms in Meniere's disease, including abnormalities in Na,K-ATPase enzymes found in the cochlear lateral wall. The current study examined the immunohistochemical labeling pattern of the major ion-transporting enzyme of the stria vascularis, Na,K-ATPase, in archival sections of hydropic and nonhydropic human temporal bones for increased density of label that could indicate overproduction of fluid. The results showed good labeling of the stria vascularis in the celloidin sections. The hydropic ears tended to have darker label, but the difference was not statistically significant. The findings are consistent with normal functioning of the stria vascularis in cases of Meniere's disease.

Collagenase activity in cholesteatoma.

Collagenase activity of cholesteatoma epithelium, temporal bone and ear canal skin was investigated. The enzyme extracted from these tissues was proved to be collagenase by disc electrophoresis. Collagenase activity was measured directly in homogenates of tissues. Activity was greatest in the skin. The activity in cholesteatomas was either higher than that of the bone, or the activity was higher in bone than in the cholesteatoma. These findings suggest that some variation of collagenase activity may be due to the stage of cholesteatoma, active or inactive in collagen metabolism of bone destruction, and that the ear canal skin plays a great role in bone destruction, this supporting the immigration theory.

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.

Bone resorption in chronic otitis media.

Bone resorption is an important aspect of chronic otitis media contributing to many complications of this disease. It is postulated that the mechanism of this localized destructive process is chemical in origin. Collagenase, lysosomal enzymes, prostaglandins, and other cell mediators are thought to induce bone resorption, but the site of action and cellular origin of these substances remains unclear. In this report, we demonstrate the location and attempt to delineate the cellular origin of two enzymes, collagenase and the lysosomal enzyme acid phosphatase in guinea pig temporal bones and human ossicles from ears containing chronic otitis media. Tissue localization of these enzymes identifies sites of active bone resorption and demonstrates the cells initiating this process. Using immunohistochemical and immunocytochemical techniques, collagenase was seen surrounding mononuclear inflammatory cells of granulation tissue at bone resorbing margins and at the periphery of osteocyte lacunae adjacent to resorbing areas. Electron microscopic data suggests that collagenase is an extracellular enzyme foun at the periphery of osteocytes. In addition, abundant acid phosphatase activity was seen in the same cells that exhibited collagenase staining, lending credence to the destructive function of these cells. The chronic inflammatory reaction found in chronic otitis media appears to activate bone destruction through the dynamic activity of mononuclear inflammatory cells and stimulates bone cells to increase their destructive biochemical functions.

Histochemistry of normal middle ear mucosa.

Mucosal biopsy specimens were taken from the middle ear in 19 patients with otosclerosis and stained by Alcan blue and PAS. They were also stained for hydrolytic and oxidative enzymes. In additon, 11 temporal bones from patients without any previous ear disease were serially sectioned for mucopolysaccharides. In otosclerosis, both acid and neutral mucopolysaccharides were present in the mucosa, but not to the same extent as in the temporal bone specimens. Acid phosphatase, lactate malate dehydrogenases, and nonspecific esterase were demonstrated even in very thin epithelium, providing a potential basis for the appearance of these enzymes in large amounts in various inflammatory middle ear diseases.