HES6 acts as a transcriptional repressor in myoblasts and can induce the myogenic differentiation program.

HES6 is a novel member of the family of basic helix-loop-helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box-containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box-containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6-VP16 activation domain fusion protein activated the N box-containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.

Coarsening of unstable thin films subject to gravity.

Thin films of viscous fluids coating hydrophobic substrates are unstable to dewetting instabilities, and long-time evolution leads to the formation of an array of near-equilibrium droplets connected by ultrathin fluid layers. In the absence of gravity, previous use of lubrication theory has shown that coarsening dynamics will ensue-the system will evolve by successively eliminating small drops to yield fewer larger drops. While gravity has only a weak influence on the initial thin film, we show that it has a significant influence on the later stages of the coarsening dynamics, dramatically slowing the rate of coarsening for large drops. Small drops are relatively unaffected, but as coarsening progresses, these aggregate into larger drops whose shape and dynamics are dominated by gravity. The change in the mean drop shape causes a corresponding gradual transition from power-law coarsening to a logarithmic behavior.

The effects of cyclic tensile and stress-relaxation tests on porcine skin.

When a living tissue is subjected to cyclic stretching, the stress-strain curves show a shift down with the increase in the number of cycles until stabilization. This phenomenon is referred to in the literature as a preconditioning and is performed to obtain repeatable and predictable measurements. Preconditioning has been routinely performed in skin tissue tests; however, its effects on the mechanical properties of the material such as viscoelastic response, tangent modulus, sensitivity to strain rate, the stress relaxation rate, etc….remain unclear. In addition, various physical interpretations of this phenomenon have been proposed and there is no general agreement on its origin at the microscopic or mesoscopic scales. The purpose of this study was to investigate the effect of the cyclical stretching and the stress-relaxation tests on the mechanical properties of the porcine skin. Cyclic uniaxial tensile tests at large and constant strain were performed on different skin samples. The change in the reaction force, and skin's tangent modulus as a function of the number of cycles, as well as the strain rate effect on the mechanical behavior of skin samples after cycling were investigated. Stress-relaxation tests were also performed on skin samples. The change in the reaction force as a function of relaxation time and the strain rate effect on the mechanical behavior of skin samples after the stress-relaxation were investigated. The mechanical behavior of a skin sample under stress-relaxation test was modeled using a combination of hyperelasticity and viscoelasticity. Overall, the results showed that the mechanical behavior of the skin was strongly influenced by cycling and stress relaxation tests. Indeed, it was observed that the skin's resistance decreased by about half for two hours of cycling; the tangent modulus degraded by nearly 30% and skin samples became insensitive to the strain rates and accumulated progressively an inelastic deformation over time during cycling. Finally, the hysteresis loops became very narrow at the end of cycling and after relaxation process.

Intravenous torsemide as adjunctive therapy in patients with acute pulmonary edema.

The safety and efficacy of intravenous (i.v.) torsemide as adjunctive therapy for acute cardiogenic pulmonary edema was evaluated. Thirteen patients were treated with i.v. torsemide and six patients, with i.v. furosemide, as a positive control. Doses of torsemide, 20 mg or 40 mg, and furosemide, 40 mg or 80 mg, were administered initially. The dose was titrated as necessary over the next 24 hours. In patients who received i.v. torsemide, median fractional sodium excretion significantly increased from 2.88% (0.04-10.1%) at baseline to 6.76% (0.71-11.6%) at peak (P = 0.0342). Hourly urine volume increased from 134 mL (25-400 mL) to 375 mL (145-790 mL) (P = 0.0034). Torsemide administration resulted in a significant improvement in both pulmonary rales and orthopnea. None of the patients experienced serious adverse events or required withdrawal from the study. These results suggest that i.v. torsemide is an effective and well-tolerated diuretic in patients with acute cardiogenic pulmonary edema.

Dose effectiveness and safety of butorphanol in acute migraine headache.

This study was undertaken to compare the effectiveness and safety of three dosage levels of butorphanol in 52 patients with acute, severe migraine headache. After baseline evaluation, patients were given a dose of butorphanol 1.0, 2.0, or 3.0 mg intramuscularly on a double-blind basis. Assessments of pain intensity and pain relief using 100 mm linear analog scales (LAS), vital signs, and medication side effects were made at 15, 30, 45, and 60 minutes after the dose. All three treatment groups were similar in baseline characteristics. Each dose of butorphanol demonstrated a significant decrease in pain intensity LAS compared to baseline and increase in pain relief LAS over the observation period. The majority of analgesic response was observed at the first (i.e., 15-min) assessment. Doses of 2.0 and 3.0 mg produced significantly greater analgesia than did 1.0 mg at all posttreatment evaluations. No significant difference was apparent between the 2.0- and 3.0-mg doses. Adverse cardiovascular and respiratory depressant effects were not observed. An analgesic response to butorphanol 2.0 and 3.0 mg is clearly and rapidly evident and near maximum 30-45 minutes after administration. We conclude that in these doses butorphanol provides effective and safe analgesia for patients with acute migraine headache.

Alterations of inner ear morphology in experimental hypercholesterolemia.

Previous clinical and experimental studies have indicated that auditory function may be compromised by hypercholesterolemia. In this investigation, inner ear tissue from chinchillas maintained on a cholesterol-supplemented diet for 3 months was examined for morphological alterations which might underlie the physiological changes observed with this condition in earlier studies. Ultrastructural analysis of cochleas from 16 hypercholesterolemic chinchillas revealed alterations in both the stria vascularis and outer hair cells. Strial marginal cells throughout the cochlea and outer hair cells of the apical turn, contained electron-lucent patches of an amorphous material. These patches had the morphological characteristics and histochemical properties of glycogen. Mild extracellular edema and increased numbers of lysosomes were also noted in the stria vascularis of experimental animals. These alterations suggest that chronic hypercholesterolemia metabolically stresses inner ear tissue. It is hypothesized that such changes could increase susceptibility of the cochlea to ototraumatic agents.

Alterations in microvasculature are associated with atrophy of the stria vascularis in quiet-aged gerbils.

Age-related changes in the integrity of the stria vascularis and its microvasculature were assessed in whole mount preparations of the gerbil cochlear lateral wall. Small focal regions containing few or no capillaries were present at the extreme ends of the stria vascularis in most 5-9 month-old gerbils. A few ears in this age range also contained small regions devoid of capillaries at the extreme basal end of the stria. These degenerate foci expanded in a systematic fashion toward the middle turn of the cochlea with increasing age. Gerbils aged 33 months or older exhibited a normal strial vasculature pattern only in portions of the middle and upper basal turns. The remainder of the stria in these older gerbils contained degenerate regions which showed both loss of capillaries and atrophy of strial marginal cells. Quantification via computer-aided image analysis confirmed that the areas of strial atrophy correlated well with the loss of strial capillaries at all ages. However, regions containing capillaries with decreased diameter were not necessarily accompanied by atrophic changes in marginal cells. The results suggest that degeneration of the stria vascularis begins prior to onset of auditory threshold shift and is preceded and possibly initiated by changes in the strial microvasculature.

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.

Expression patterns of ion transport enzymes in spiral ligament fibrocytes change in relation to strial atrophy in the aged gerbil cochlea.

Fibrocytes of the lateral wall function in conjunction with the stria vascularis (StV) to mediate cochlear ion homeostasis. Age-related changes in the expression patterns of ion transport enzymes in spiral ligament fibrocytes were investigated to ascertain their relation to metabolic presbyacusis in the gerbil. Immunoreactivity of fibrocytes for Na,K-ATPase (Na,K), carbonic anhydrase isozyme II (CA) and creatine kinase isozyme BB (CK) varied among and within cochleas from aged but not from young gerbils. The variable immunostaining was related to the extent and location of StV atrophy. Age-dependent degeneration and loss of Na,K in the StV occurred predominantly in the apex and lower base and hook of the cochlea, largely sparing more central regions. Immunostaining intensity for Na,K, CK, and CA in fibrocytes changed in relation to declines in strial marginal cell Na,K initially showing upregulation followed by downregulation. Spiral ligament fibrocytes in cochleas with more than two remaining normal turns often disclosed overexpression of CK in regions of strial atrophy. Conversely, CA in such cochleas was often increased in regions of normal StV adjacent to foci of atrophic StV. Senescent cochleas with two or fewer functional turns generally contained fibrocytes with diminished CK or CA immunoreactivity in regions of atrophic StV but in isolated instances exhibited fibrocytes with enhanced staining. Heightened staining for CK in type Ia fibrocytes underlying regions of complete or partial strial atrophy indicated an increased metabolic demand in fibrocytes in response to strial insufficiency. The findings provide further support for the role of spiral ligament fibrocytes in cochlear fluid and ion homeostasis.

Localization of actin in basal cells of stria vascularis.

The distribution of actin in the lateral wall of the cochlear duct was investigated in the gerbil, rat, mouse and hamster. A monoclonal antibody specific for muscle alpha and gamma actins, and a polyclonal antiserum reactive with smooth muscle gamma and non-muscle beta actins yielded strong immunostaining of basal cells in the stria vascularis and of smooth muscle cells in lateral wall blood vessels. Both cell types stained in all four genera. Diffuse cytosolic staining was observed along the full-length of the basal cell layer including the blunt cell processes which they extend toward strial marginal cells. Immunoreactive basal cells appeared continuous with morphologically similar cells investing vessels penetrating the stria from the spiral ligament. The basal cells failed to bind antibodies to smooth muscle alpha actin and sarcomeric actin. By electron microscopic immunocytochemistry, gold labeling for actin was observed on dense, fine fibrils in the cytoplasm of the basal cells. In paraffin sections adjacent to those stained for actin, antibody to vimentin stained intermediate and basal cells in the stria vascularis whereas antibody to isoform 1 of the facilitated glucose transporter protein family (GLUT1) labeled only the non-overlapping apical and basal plasmalemma of basal cells. Content of vimentin, GLUT1 and muscle gamma actin supports the derivation of basal cells from mesoderm. The presence of stress fibers containing muscle gamma actin points to a possible contractile activity of basal cells which conceivably could be related to transport of K+ to and within the intrastrial compartment or regulation of blood flow in the stria vascularis.

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.

Characterization and development of an inner ear type I fibrocyte cell culture.

A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils. The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types. Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes. The cells showed little evidence of intercellular contact even when confluent. The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase. The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament. These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics. Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis. The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis.

Quantification of the stria vascularis and strial capillary areas in quiet-reared young and aged gerbils.

The area of the stria vascularis (StV) and of StV capillaries was measured in radial sections from regions corresponding to 0.5, 2, 4, 10, 20 and 40 kHz. In young gerbils, StV area ranged from 3700 to 8500 microm2 and that of individual StV capillaries from 70 to 110 microm2. The maximal StV area as well as the largest number of capillaries occurred at the 20 kHz region. In quiet-aged gerbils, the StV area also varied with frequency and was 28-67% smaller than corresponding measures in young gerbils. The decrease in StV area was statistically significant at all but the 2 and 4 kHz regions. The area of individual StV capillaries declined also (8-29%) with age even when the StV area remained near normal. Reductions in capillary area were statistically significant at the 2, 20 and 40 kHz regions. The large variance in StV radial area among aged gerbils reflects the patchy nature of strial degeneration previously observed in this species. The data agree with those of our previous studies and indicate alterations in StV capillaries are a primary cause of presbyacusis in the gerbil.

Ca2+-dependence and nifedipine-sensitivity of vascular tone and contractility in the isolated superfused spiral modiolar artery in vitro.

The regulation of the vascular diameter of the spiral modiolar artery may play a major role in the regulation of cochlear blood flow and tissue oxygenation since the spiral modiolar artery provides the main blood supply to the cochlea. The goal of the present study was to determine whether vascular tone and contractility of the spiral modiolar artery depend on the presence of extracellular Ca2+ and involves nifedipine-sensitive Ca2+ channels. The spiral modiolar artery was isolated and superfused in vitro and the diameter was measured continuously by video microscopy. Isolated segments of the spiral modiolar artery had an outer diameter of 61 +/- 3 microm (n = 59) and displayed vasomotion characterized by 5-15 clearly distinguishable constrictions per min. Removal of Ca2+ from the superfusion medium caused a reversible relaxation and cessation of vasomotion and was used to determine the magnitude of basal vascular tone. The basal vascular tone consisted of a sustained reduction of the vascular diameter to 95.1 +/- 0.3% (n = 51) of the maximal diameter in Ca2+-free medium. Nifedipine reduced the basal vascular tone with an IC50 of (1.1 +/- 0.3) x 10(-9)) M although 22% of the basal vascular tone was insensitive to nifedipine. Elevation of the K+ concentration from 3.6 to 150 mM caused a transient vasoconstriction which was dependent on the presence of extracellular Ca2+. Nifedipine fully inhibited K+-induced vasoconstriction with an IC50 of (2.0 +/- 0.7) x 10(-9) M. Norepinephrine (10(-4) M) caused a transient vasoconstriction and an increase of vasomotion at branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction was fully inhibited in the absence of Ca2+ and partially inhibited by 10(-7) M nifedipine. These observations suggest that the spiral modiolar artery contains voltage-dependent nifedipine-sensitive Ca2+ channels which are involved in the maintenance of basal vascular tone as well as in the mediation of K+- and norepinephrine-induced contractility. Further, the data suggest that cytosolic Ca2+ stores, if present in the spiral modiolar artery, are of limited capacity compared to other vessels.

Interaction of cisplatin and noise on the peripheral auditory system.

The potentiation of cisplatin ototoxicity by noise was explored in the chinchilla. The effects of exposure to cisplatin alone, noise alone or concurrent exposure to both agents were compared in terms of the threshold shift of the auditory evoked potential and the amount of hair cell loss. The combination of cisplatin plus noise produced significantly more hair cell loss and hearing loss at the high frequencies than did either the noise or cisplatin alone when the noise level was 85 dB SPL or higher; no interaction was seen when the noise level was 70 dB SPL. The amount of the interaction, when present, was constant regardless of the noise level. These results indicate that moderate to high levels of noise can exacerbate cisplatin ototoxicity.

Enhanced evoked response amplitudes in the inferior colliculus of the chinchilla following acoustic trauma.

Evoked response amplitude-level functions were measured from electrodes in the inferior colliculus of the chinchilla before and after exposure to a 2 kHz pure tone of 105 dB SPL. The exposure produced approximately 20-30 dB of permanent threshold shift from 2 to 8 kHz, but little or no hearing loss at higher or lower frequencies. Generally less than 60% of the outer hair cells were missing in the region of hearing loss. The amplitude-level functions measured at 4 and 8 kHz generally showed a loss in sensitivity at low sound levels, a reduction in the maximum amplitude and sometimes steeper than normal slopes. The amplitude-level functions measured at 2 kHz also showed a loss in sensitivity; however, the maximum amplitude was often greater than normal. Even though there was no loss in sensitivity at 0.5 kHz, the amplitude-level function was steeper than normal and the maximum amplitude of the evoked response was almost always substantially larger than normal. The enhancement of the evoked response amplitude from the inferior colliculus does not appear to originate in the cochlea, but may reflect a reorganization of neural activity in the central auditory pathway.

Age-related thickening of basement membrane in stria vascularis capillaries.

Ultrastructural examination was undertaken to investigate the pathogenesis of age-related atrophy of the stria vascularis (StV). Basement membrane (BM) thickness was increased in 65-85% of strial capillaries in gerbils aged 33 months or older and often exceeded by several-fold that observed in young controls. In an early stage of thickening the BM expanded slightly around the full capillary profile, after which nodular expansions of BM encircling slender cell processes were often observed at or near one or both poles of the elliptical vessel profile. As widening progressed, the BM consisted of 2-3 layers separated by cell processes in the nodules but fewer strata elsewhere. Association of slender processes of both endothelial cells and pericytes with focal thickening outside the process suggested their participation in genesis of the capillary lesion. In later stages of atrophy, pericytes degenerated and disappeared, while endothelial cells remained intact. Eventually, thick multilayered BM devoid of endothelial cells surrounded a narrow lumen occluded by debris. The age-related change in BM in the inner ear was confined to StV capillaries. Degenerative changes in StV epithelial cells occurred apparently as a secondary consequence of the capillary lesion. The pathologic alterations in marginal cells included extrusion of blebs from the luminal surface, separation and loss of basolateral interfoldings, alteration and depletion of mitochondria and nuclear pyknosis. At the end-stage of degeneration, the StV consisted of a simple or multiple layer of squamous cells lining the scala media.

Decline in the endocochlear potential corresponds to decreased Na,K-ATPase activity in the lateral wall of quiet-aged gerbils.

The ion transport-mediating enzyme, Na,K-ATPase, is abundantly present in the cochlear lateral wall. This enzyme is essential for the generation and maintenance of the endocochlear potential. Diminished enzyme activity has been observed previously in the lateral wall of quiet-aged gerbils. The present study was designed to investigate the impact of the age-related decline in Na,K-ATPase specific activity upon auditory function. Measures of the resting endocochlear potential value and the level of Na,K-ATPase specific activity were made in cochleae obtained from gerbils aged in quiet conditions. Analysis revealed a high degree of correspondence between the level of lateral wall Na,K-ATPase specific activity and the value of the endocochlear potential measured in the round window/turn 1 region of the cochlea. Nonlinear regression models showed a strong relationship between the age-related reductions in enzyme activity and the magnitude of the endocochlear potential. The data suggest that during metabolic presbyacusis a decrease in Na,K-ATPase specific activity can explain most, but not all, of the decline in the endocochlear potential.

Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea.

Alterations in the enzymatic activity and antigenicity of Na,K-ATPase as well as changes in cochlear morphology were assessed in gerbil inner ears harvested at selected time intervals up to 18 h postmortem. Na,K-ATPase activity was assayed biochemically in one cochlea from each animal and the other cochlea was fixed and embedded in paraffin for evaluation by light microscopy. Na,K-ATPase antigenicity was assessed by immunostaining with a broad-spectrum antiserum reactive with all known isoforms of the enzyme, and structural preservation was evaluated on adjacent sections stained with hematoxylin and eosin. The results showed a downward trend in enzymatic activity of Na,K-ATPase in lateral wall tissues within 1 h of death. In contrast, Na,K-ATPase immunoreactivity was fairly well preserved with postmortem fixation delays up to 12 h, despite the considerable structural degradation of cochlear tissues which began 2-3 h postmortem. It is concluded that under controlled environmental conditions, cochleas collected up to 4 h postmortem are suitable for morphological and immunohistochemical study of Na,K-ATPase by light microscopy. Cochleas collected more than 5 h postmortem were useful only for relatively gross immunohistochemical studies. It is suggested that cochleas intended for biochemical assays of Na,K-ATPase and probably most other enzymes should be collected within 1 h of death.