Materials used to simulate physical properties of human skin.

BACKGROUND: For many applications in research, material development and testing, physical skin models are preferable to the use of human skin, because more reliable and reproducible results can be obtained. PURPOSE: This article gives an overview of materials applied to model physical properties of human skin to encourage multidisciplinary approaches for more realistic testing and improved understanding of skin-material interactions. METHODS: The literature databases Web of Science, PubMed and Google Scholar were searched using the terms 'skin model', 'skin phantom', 'skin equivalent', 'synthetic skin', 'skin substitute', 'artificial skin', 'skin replica', and 'skin model substrate.' Articles addressing material developments or measurements that include the replication of skin properties or behaviour were analysed. RESULTS: It was found that the most common materials used to simulate skin are liquid suspensions, gelatinous substances, elastomers, epoxy resins, metals and textiles. Nano- and micro-fillers can be incorporated in the skin models to tune their physical properties. CONCLUSION: While numerous physical skin models have been reported, most developments are research field-specific and based on trial-and-error methods. As the complexity of advanced measurement techniques increases, new interdisciplinary approaches are needed in future to achieve refined models which realistically simulate multiple properties of human skin.

CO2 and O2 concentrations in integral motorcycle helmets.

Inhaling air which contains excess CO2 and/or is oxygen-deficient is known to present health risks and to diminish human cognitive abilities. The average CO2 concentrations relevant to a motorcyclist wearing an integral helmet were measured 20 years ago and found to be alarmingly large. The purpose of the present study was to examine gas concentrations typically inhaled by a motorcyclist. Average concentrations of CO2 near the upper lip for persons (n = 4) wearing integral motorcycle helmets were measured in the laboratory and the field to facilitate comparison to previous work, and similarly high average concentrations were found: above 2% when stationary, well below 1% for speeds of 50 km/h or more. Very good agreement was obtained between laboratory and field measurements. Detailed measurements of the time-dependent CO2 concentrations passing through a mouthpiece for mouth-breathing showed inhaled levels slightly over half of the corresponding average concentrations, including 1.3+/-0.3% at standstill, though higher concentrations (4% or more) were inhaled at the beginning of each breath. Opening the visor at standstill had on average no effect. At a speed of 50 km/h the inhaled CO2 concentration resembles that for a person without a helmet in still air, at about 0.2%. The oxygen deficiency is generally equal to the CO2 concentration, and could also contribute negatively to a motorcyclist's cognitive abilities.

In-process cell counts during preparation of platelet concentrates from buffy coats.

To ensure the quality of platelet concentrates (PCs), we studied in-process recoveries of blood cell counts in pooled PCs derived from four or five buffy coats (BCs) from Biopack Compoflex Systems in Bern (PC-BC/4 or PC-BC/5) and from five BCs from Optipac (Baxter) in Zurich (PC-BC/5). BCs were pooled employing a sterile connecting device and flushing them with 300 mL of platelet additive solution. The pools were centrifuged for 12 min at 500 g at 20 degrees C and filtered with PALL's Auto-Stop BC-leukocyte removal filter. Automated platelet counting was performed on whole blood donation, on single BC, on pooled BC and in the final product. Four out of 10 PC-BC/4 (= 40%) and 29 out of 30 PC-BC/5 (= 97%) had a total platelet count of > 200 x 10(9) platelets. Average percentage recoveries in PC compared to the pre-centrifugation BC pools were similar with the Biopack Compoflex and the Optipac systems, 62% and 57% respectively, whereby the absolute platelet count per one donation was similar, i.e. 49.5 x 10(9), 55 x 10(9) and 53 x 10(9) in PC-BC/4 and PC-BC/5 from Bern and PC-BC/5 from Zurich. There was a significant positive correlation between the inital number of BCs taken for pooling and the final platelet counts in the PCs. In order to recover a minimal platelet content of 200 x 10(9) platelets per pooled unit, it is safer to start out with five rather than with four donations unless recoveries during the production steps can be improved.

Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve.

Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na+. Relief of TTX blockage was also observed by adding 110 mM chloride salts of Li+, hydrazine+, guanidine+ and K+ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.

The action of arginine-specific reagents on ionic and gating currents in frog myelinated nerve.

(1) The effect of arginine-specific reagents on the sodium current (INa), potassium current (IK) and gating current (Igat) of myelinated nerve fibres was investigated. (2) Externally applied camphorquinone-10-sulfonic acid (Cqs-OH) had little effect, but 50 mM Cqs-OH applied to the cut ends of the fibre progressively reduced the amplitude of INa without significantly altering its time course. After 30 min INa was reduced to 52% (pH 9.0) or 66% (pH 6.75-7.6) of the control value. IK was decreased to a similar extent without changing its kinetics. Igat was less affected than the ionic currents. (3) Externally applied phenylglyoxal markedly reduced INa and Igat, but many fibres were lost during or shortly after the treatment. A few min treatment with 5 mM phenylglyoxal at pH 9 reduced INa to 20% and the on-response of Igat to 69.5%. The effect was to a large extent irreversible. (4) External nitrophenylglyoxal and hydroxyphenylglyoxal significantly reduced INa and were less damaging than phenylglyoxal. INa was decreased to 34.5% by 10 mM nitrophenylglyoxal and to 28.3% by 20 mM hydroxyphenylglyoxal. The effect of nitrophenylglyoxal was little reversible, but that of hydroxyphenylglyoxal to a large extent reversible. 20 mM hydroxyphenylglyoxal reduced the on-response of Igat to 62.5% of the control value, i.e. much less than INa. (5) 5 mM phenylglyoxal, 10 mM nitrophenylglyoxal and 20 mM hydroxyphenylglyoxal shifted the steady-state inactivation curve by 10-15 mV to more negative values of membrane potential but did not affect the descending branch of the INa(E) curve. (6) 20-30 mM glyoxal, 20 mM 1,2-cyclohexanedione and 10 mM 4-hydroxy-3-nitrophenylglyoxal had no effect on INa. (7) The results are compatible with the idea that arginine residues are principal components of the sodium channel macromolecule.

A comparison of sodium currents in rat and frog myelinated nerve: normal and modified sodium inactivation.

1. Sodium currents were measured under voltage-clamp conditions in Ranvier nodes of rat and frog nerve fibres at 20 degrees C. Voltage errors due to the resistance in series with the nodal membrane were minimized by reducing sodium currents with tetrodotoxin in the extracellular solutions. 2. The stationary and kinetic properties of sodium activation and inactivation were determined for a wide range of potentials (V) from -40 to 160 mV with respect to the initial holding level (V = 0 mV). 3. The curves m infinity(V) and h infinity(V) of stationary sodium activation and inactivation were not different in rat and frog fibres. 4. The time constants tau m, tau h of sodium activation and inactivation were normally larger in the rat than in the frog. At moderate depolarizations (0 less than or equal to V less than or equal to 80 mV) tau m in the rat was 15-50% larger; the ratio of the rat to the frog tau h values was usually smaller. Thus tau m/tau h = 0.116 for the rat and 0.0965 for the frog at V = 60 mV (potential with maximum peak sodium inward current). 5. Sodium inactivation in rat nerve was slowed and became incomplete by application of intra-axonal iodate or by treatment with external Anemonia toxin II (ATX II), chloramine-T or Ruthenium Red. Peak sodium currents were not increased by these substances. 6. Wash-out of ATX II from frog nerve was rapid and complete but partly irreversible in rat nerve. This suggests different properties or accessibilities of sodium channels in frog and rat nodes.

Comparison of the effects of Anemonia toxin II on sodium and gating currents in frog myelinated nerve.

Na+ and gating currents were measured in myelinated frog nerve fibres without and in the presence of 7 microM Anemonia toxin II in the extracellular solution. From the experiments, kinetic parameters of Na+ currents and of gating charge displacements during ('on' response) and after ('off' response) depolarizations were determined. The following parallel modifications of Na+ currents and charge displacements by Anemonia toxin II were observed: the toxin reduces the maximum Na+ permeability and the 'on' charge displacement; Na+ activation and 'on' charge displacement become faster; Na+ inactivation and the decline of the 'off' charge displacement with increasing pulse duration (charge immobilization) are prolonged; slow components of 'on' charge displacements are diminished. The observations support the notion that the fast 'on' charge displacement is connected with the process of Na+ activation, while Na+ inactivation is linked to charge immobilization. Our experiments suggest that slow 'on' charge displacements during longer depolarizations are correlated with the process of Na+ inactivation.

Heterogeneity of external surface charges near sodium channels in the nodal membrane of frog nerve.

The conductance gamma and the number of No of Na channels in the nodal membrane of frog nerve fibres were determined from ensemble average values of the Na current and the variance of Na current fluctuations. Replacement of extracellular Cl- by NO3- shifts the voltage dependencies of all Na gating parameters towards more negative voltages, reduces gamma by a factor of 0.84 and hardly changes the number No of channels not blocked by 8 nM TTX. Adding 0.1 mM LaCl3 to the extracellular solution shifts the voltage dependencies of all Na gating parameters towards more positive voltages, reduces gamma by a factor of 0.75 and hardly changes the number No of channels not blocked by 8 nM TTX. It is concluded that changes of the external surface potential induced by Cl-, NO3- replacement do not alter the local Na+ concentration in the outer mouth of the Na channel and hardly affect the TTX binding to toxin receptors. Surface potential changes by addition of LaCl3 also have no clear effect on TTX binding. The reduction of gamma in 0.1 mM LaCl3 is probably due to a direct interaction of La3+ with Na channels. Our results suggest a heterogeneous distribution of external fixed surface charges in the outer mouth of the Na channel, at the TTX binding site and near the Na channel gates.

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin.

(1) Na+ currents and Na+-current fluctuations were measured in myelinated frog nerve fibres at 15 degrees C during 7.7 ms depolarizations to V = 40, 60 and 80 mV. (2) The conductance gamma of a single Na+ channel and the number No of channels per node were calculated from ensemble average values of the mean Na+ current and the variance of Na+-current fluctuations. (3) For a hyperpolarizing holding potential of VH = -28 mV the mean values of the channel conductance and number were gamma = 9.8 pS and No = 74000. (4) After changing the holding potential to the resting potential (VH = 0) the conductance gamma increased by a factor of 1.37 whereas the number No decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of VH = -28 mV increased gamma by a factor of 1.55 and reduced No by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na+ channels in the nodal membrane.

Sodium currents and sodium-current fluctuations in rat myelinated nerve fibres.

1. Sodium currents and fluctuations of sodium currents were measured in myelinated fibres of rat sciatic nerve under voltage clamp at 20 degrees C.2. Relaxations of sodium currents during various test potentials were recorded in the presence of 6 nM-TTX in the extracellular solution. The activation of sodium currents at low depolarizations could be described with the m(2) formulation. At increasing potentials higher powers of m up to 4 were required. The mid-point of the P(Na) (E) curve was located near E = -32 mV. Sodium inactivation during various depolarizations developed in two phases.3. The resistance in series with the nodal membrane was calculated from peak sodium currents without and with 6 nM-TTX in the extracellular solution. The resistance varied between different fibres and ranged between 190 and 620 kOmega.4. From peak sodium currents at the same mambrane potential without and in the presence of TTX an apparent equilibrium dissociation constant of 1.6 nM was calculated for TTX binding to sodium channels.5. The conductance gamma and the number N(0) (corrected for series-resistance effects) of sodium channels were evaluated from ensemble average values of the mean sodium current and the variance of sodium-current fluctuations at the beginning of a test pulse. The mean values were gamma = 14.5 pS, N(0) = 21,000 per node.6. The spectral density of stationary sodium-current fluctuations exhibited two relaxation components whose time constants were comparable to those of sodium activation and inactivation. At low depolarizations the variance produced by inactivation fluctuations was larger than predicted by the m(3). h formulation.7. It is concluded that individual sodium channels of rat and frog nerve have similar gating properties. In mammalian nodes the number of sodium channels is lower and the single-channel conductance higher than in amphibian nodes.

Fluctuation analysis of Na+ channels modified by batrachotoxin in myelinated nerve.

(1) Single myelinated nerve fibers of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na+ currents and Na+-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses the fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na+-current fluctuations was fitted by the sum of a 1/f component and a Lorentzian function. The time constant tau c = 1/(2 pi fc) obtained from the corner frequency fc of the Lorentzian function approximately agreed with the activation time constant tau m of the macroscopic currents. (3) The conductance gamma of a single Na+ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na+ current. At the resting potential V = 0 we obtained gamma - 1.6 pS, higher gamma-values of 3.2 and 3.45 pS were found at V = --8 and --16 mV, respectively. (4) The conductance of a modified Na+ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na+ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an 'all-or-none' manner on Na+ channels and creates a distinct population of modified channels.

Ultrastructural observations on nodes of Ranvier from isolated single frog peripheral nerve fibres.

A preparative procedure is described by which well-preserved nodes of Ranvier from isolated frog peripheral nerve fibres may be obtained. The following steps are crucial: careful and gentle dissection and isolation of a single nerve fibre, mounting in a chamber limiting lateral movements of the fibre during the fluid exchange, simultaneous glutaraldehyde-OsO4 fixation and embedding in the same chamber used for fixation. Serial sectioning of individual nodes from both motor and sensory fibres made it possible to reconstitute three-dimensional models of several nodes and to study their morphology extensively. In addition to well-known ultrastructural features of the nodal and paranodal architecture, evaginations of a nodal membrane containing mitochondria and outpouchings of the paranodal axoplasm containing vesicles are described.

Block of Na channels in the membrane of myelinated nerve by benzocaine.

The actions of the neutral local anesthetic benzocaine on Na channels were studied in voltage-clamp experiments on single myelinated nerve fibres of the frog by measurements of sodium currents, asymmetry currents, and sodium current fluctuations. 2. 1 mM benzocaine reduced the peak Na currents during various depolarizations V between 20 and 120 nV to 63% of their control values but did not change the time constant of Na activation. 3. 1 mM benzocaine altered asymmetry currents during 1 ms pulses V between 20 and 120 mV in the same was as the early Na currents: It reduced the amplitude to 64% but did not affect the kinetics of the currents. 4. The charge displacement of the asymmetry current during the pulse (Qon) was compared with the charge displacement after the pulse (Qoff). Without benzocaine the relative charge Qoff/Qon Declined to a constant level (0.42 at V = 40mV, 0.25 at V = 100 mV) with increasing pulse durations. In the presence of 1 mM benzocaine the charges Qoff after pulses to V = 40 or 100 mV are almost independent of pulse duration and approximately equal to the control Qoff values after 5.6 ms pulses. Thus, the immobilizations caused by Na inactivation and benzocaine are not additive. 5. Na currents and Na-current fluctuations were recorded during depolarizations V between 24 and 48 mV in the presence of 0.1 mM benzocaine and 7 microM Anemonia toxin II. A lower limit of 8.6 pS was derived for the conductance of a single Na channel. The value agrees with other estimates of the conductance of Na channels which had not been treated by local anesthetics. This suggests an "all-or-none blocking" of Na channels by benzocaine.

Potassium ion accumulation at the external surface of the nodal membrane in frog myelinated fibers.

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in myelinated fibers and analyzed in terms of two models-three-compartment and diffusion in an unstirred layer. In the myelinated fibers, as in squid giant axons, the three-compartment model satisfactorily describes potassium accumulation. Within this framework the average space thickness, theta, in frog was 5,900 +/- 700 A, while the permeability coefficient of the external barrier, PK, was (1.5 +/- 0.1) X 10(-2) cm/s. The model of ionic diffusion in an unstirred aqueous layer adjacent to the axolemma, as an alternative explanation for ion accumulation, was also consistent with the experimental data, provided that D, the diffusion constant, was (1.8 +/- 0.2) X 10(-6) cm/s and l, the unstirred layer thickness, was 1.4 +/- 0.1 micron, i.e., similar to the depth of the nodal gap. An empirical equation relating the extent of potassium accumulation to the amplitude and duration of depolarization is given.

Potassium currents and conductance. Comparison between motor and sensory myelinated fibers.

The potassium conductance system of sensory and motor fibers from the frog Rana esculenta were studied and compared by means of the voltage clamp. The potassium ion accumulation was first estimated from the currents and reversal potentials within the framework of both a three-compartment model and diffusion-in-an-unstirred-layer model. The potassium conductance parameters were then computed using the measured currents and corrected ionic driving forces. It was found that the potassium accumulation is faster and more pronounced in sensory fibers, the voltage dependency of the potassium conductance is steeper in sensory fibers, the maximal potassium conductance, corrected for accumulation, is approximately 1.1 S/cm2 in sensory and 0.55 S/cm2 in motor fibers, and that the conductance time constants, tau n, are smaller in sensory than in motor fibers. These differences, which increase progressively with depolarization, are not detectable for depolarization of 50 mV or smaller. The interpretation of these findings in terms of different types of potassium channels as well as their implications with regard to the differences between the excitability phenomena in motor and sensory fibers are discussed.

Conductance fluctuations from the inactivation process of sodium channels in myelinated nerve fibres.

1. Na currents and fluctuations of Na currents were studied under voltage clamp in the same myelinated nerve fibres of Rana esculenta at 13 degrees C. The results were used to test several kinetic models for the gating process of Na channels.2. Long voltage pulses, depolarizing the membrane by 16-48 mV from a hyperpolarizing holding level of - 28 mV, were applied in 4 sec intervals. The d.c. and a.c. components of the membrane current were recorded during the last 328 msec of the 473 msec pulses. For each depolarization, ninety-six trials were made with the node in Ringer solution and, again, after adding 300 nm-tetrodotoxin (TTX) in that solution.3. The TTX-sensitive d.c. component declined during the 328 msec records by 14-51% of its time average. The a.c. component was corrected for this trend by subtracting the first from the second of each pair of subsequent records. The TTX-sensitive part of its variance declined, on the average, in parallel to the current, as if the open probability rather than the conductance of the individual Na channels was reduced by a slow process.4. Single-channel conductances, gamma, were calculated on the assumption that Na channels have only one non-zero conductance and were corrected for the limited band width (5 kHz) of the a.c. records. Values of gamma increased slightly (< 30% from 16 to 40 mV), and averaged 8.85 +/- 0.7 pS (s.e. of mean, seventeen measurements on ten fibres). This small degree of change in gamma suggests that deviations from the all-or-none gating are very small.5. Power spectral densities of the fluctuations between 3 Hz and 5 kHz were calculated from the trend-free a.c. records and corrected for the TTX-insensitive noise component. Control calculations showed that the only effect of the nonstationarity in the Na current was to enhance the low-frequency points of such spectra by less than 10%. The spectra revealed at least two Lorentzian components with cut-off frequencies in the range expected from the activation and inactivation kinetics. The low-frequency component became dominant as depolarization was increased.6. Na currents recorded during brief (< 40 msec) depolarizations were analysed in terms of various all-or-none gating models, in which inactivation either was independent of activation (Hodgkin-Huxley (HH) model) or could occur only from the partly or fully activated states (coupled models). The transient Na currents were reproduced by all models.7. With the parameters from such fits, the fluctuation spectra expected for each model were calculated. The predictions differed in the fraction, r(h), of the variance contributed by the slow (inactivation) fluctuations; r(h) was larger in the coupled models than in the HH model.8. The experimental spectra were divided into two spectral components to yield empirical values for r(h). We used as templates the spectral curves derived for the fast and for the slow fluctuations of the HH model. The empirical r(h) values were one (48 mV) to four (16 mV) times larger than those expected for the HH model. They were also larger than the theoretical r(h) of the coupled models at the small depolarizations, but became equal or smaller than those at the largest depolarization. Direct comparison of the measured and theoretical spectra revealed the same discrepancies.9. We conclude that all of the simple gating models considered in this paper are inconsistent with the fluctuation measurements, the coupled models giving slightly smaller deviations than the model with independent activation and inactivation.

Increased charge displacement in the membrane of myelinated nerve at reduced extracellular pH.

Asymmetry currents were measured in nodes of myelinated nerve fibers from Rana esculenta at extracellular pH values of 5.2, 7.0, and 8.1 by averaging the currents during and after 1-ms depolarizing and hyperpolarizing voltage pulses. The charge displacement in the nodal membrane was obtained by numerical integration of the asymmetry currents. Lowering the pH from 7.0 to 5.2 significantly slows down the kinetics of the fast charge displacement during depolarization but hardly affects the kinetics after repolarization. The pH reduction increases the maximum charge displacement during depolarization by 46%. No differences between asymmetry currents were found between pH 7.0 and 8.1. It is concluded that protonation by extracellular H+ ions may increase the net charge or the transition range of mobile subunits in the nerve membrane.