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.

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.

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.

Public-access defibrillation: where do we place the AEDs?

BACKGROUND: Many prehospital cardiac arrests occur in public places. Even the best EMS systems have a finite response time. Therefore, it has been recommended that automated external defibrillators (AEDs) be placed in public areas for immediate access by trained members of the general public. OBJECTIVE: To determine the locations of multiple cardiac arrests in order to plan for placement of public-access AEDs. METHODS: Retrospective review of all primary cardiac arrests in calendar year 1997. Cardiac arrests in which resuscitation was not attempted (DOA), traumatic cases, pediatric cases, and those due to "other" causes were excluded. Location of the cardiac arrest was obtained from the ambulance run ticket. The EMS system is an urban, Midwestern, all-ALS, public-utility model system with fire department first responders that transports approximately 58,000 patients annually. RESULTS: There was scene response to 922 cardiac arrests. 377 DOAs and 219 nonprimary cardiac arrests were excluded. There were 326 primary cardiac arrests. Sixteen locations had more than one cardiac arrest: 11 locations had two cardiac arrests, four locations had three cardiac arrests, and one location had four cardiac arrests. The airport, an airline overhaul facility, a casino, and two hotels each had two cardiac arrests; the other locations of multiple cardiac arrests were in nursing homes. The professional sports stadiums had no cardiac arrests. CONCLUSIONS: Since very few locations had more than one cardiac arrest, it may be difficult to identify high-yield public places in which to place an AED. Nursing homes may want to consider AED availability.

Effects of nimodipine on noise-induced hearing loss.

The effects of nimodipine, a calcium channel blocker, on noise-induced hearing loss were examined in gerbils. Animals were implanted subcutaneously with a timed-release pellet containing either nimodipine (approximately 10 mg/kg/day) or placebo and exposed to either 102 or 107 dBA noise. Serum levels were tested in two subjects and were in the range known to protect humans from cerebral artery vasospasm and ischemia-related neurologic deficits. Nimodipine and control groups had similar amounts of noise-induced (a) permanent threshold shift; (b) reductions in distortion product otoacoustic emissions; (c) reductions in tuning and suppression of the compound action potential; and (d) loss of outer hair cells. The results suggest that nimodipine, at a dose which results in clinically relevant serum levels, does not provide protection from the effects of moderately intense noise exposures.

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.

Effectiveness of intermittent and continuous acoustic stimulation in preventing noise-induced hearing and hair cell loss.

Resistance to noise-induced hearing loss (NIHL) was studied in gerbils exposed either to intermittent or continuous low-level noise prior to an intense noise. Auditory-evoked brainstem response (ABR) thresholds, distortion product otoacoustic emissions (DPOAEs), Q10dB values from compound action potential (CAP) tuning curves, and outer hair cell (OHC) loss were measured for each group. Subjects were exposed to A-weighted noise (octave band noise centered at 2 kHz) on an intermittent (80 dB, 6 h/day) or continuous schedule (74 dB, 24 h/day) for 10 days, allowed to rest in quiet for 2 days, then exposed to intense A-weighted noise (107 dB, 24 h/day) for 2 days. A "noise-only" group was exposed only to the intense noise. Gerbils exposed in both the "intermittent" and "continuous" groups had less (15-30 dB) temporary threshold shift (TTS) than those in the noise-only group. In addition, the continuous group had less (10-15 dB) permanent threshold shift (PTS) than the other groups. These data suggest that resistance to NIHL is evident in both the intermittent and continuous groups when TTS is measured, but resistance to PTS is afforded only by the continuous paradigm. Both paradigms decreased OHC loss as compared to the noise-only group, with the continuous paradigm being most effective. However, neither paradigm conserved DPOAE amplitudes or tuning curve Q10dB values relative to the noise-only group.

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.

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.

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.

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.

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.

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.

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.

Na,K-ATPase activity decreases in the cochlear lateral wall of quiet-aged gerbils.

Alterations in the distribution and activity of Na,K-ATPase have been implicated in declining cell function with age. However, the location, size and anatomical complexity of the cochlea have limited study of this essential enzyme. Here we describe a micro-colorimetric assay which measures Na,K-ATPase activity in subregions of individual cochleae. Na,K-ATPase activity was determined in lateral wall and organ of Corti tissues by measuring liberation of inorganic phosphate (Pi) from ATP against a standard phosphate curve. Na,K-ATPase specific activity, expressed as mu mol Pi liberated/mg protein/h, was calculated as the difference between total Pi liberated versus Pi liberated in the presence of ouabain. Na,K-ATPase specific activity and total protein content in the lateral wall significantly exceeded those of the organ of Corti. Although lateral wall protein content remained constant with age, Na,K-ATPase specific activity declined in some older gerbils, suggesting a basis for age-related reductions in magnitude of the endocochlear potential and confirming previous histochemical results. This microassay offers a sensitive, reliable means to assay enzyme activity in subregions or single turns of the cochlea that unlike other methods does not rely on use of radioisotopes, enzymatic cycling or sample pooling.