Electrical Impedance Spectroscopic Studies on Broiler Chicken Tissue Suitable for the Development of Practical Phantoms in Multifrequency EIT

Phantoms are essential for assessing the system performance in Electrical Impedance Tomography (EIT). Saline phantoms with insulator inhomogeneity fail to mimic the physiological structure of real body tissue in several aspects. Saline or any other salt solution is purely resistive and hence studying multifrequency EIT systems cannot be assessed with saline phantoms because the response of the purely resistive materials do not change over frequency. Animal tissues show a variable response over a wide band of signal frequency due to their complex physiological and physiochemical structures and hence they can be suitably used as bathing medium and inhomogeneity in the phantoms of multifrequency EIT systems. An efficient assessment of a multifrequency EIT system with a real tissue phantom needs a prior knowledge of the impedance profile of the bathing medium as well as the inhomogeneity. In this direction Electrical Impedance Spectroscopy (EIS) studies on broiler chicken muscle tissue paste, muscle tissue blocks and fat tissue blocks are conducted over a wide range of signal frequency using impedance analyzers, and their impedance profiles are analyzed. Results show that the chicken muscle tissue paste is less resistive than the fat tissue and hence it can be used successfully as the bathing medium of the phantoms for impedance imaging in multifrequency EIT. Fat tissue is found more resistive than the muscle tissue which makes it more suitable for the inhomogeneity in phantoms of impedance imaging study. Moreover, as there is a large difference between the resistivities of muscle tissue and fat tissue they can be used as either inhomogeneity or background medium. EIS studies also show that the variations in the impedance parameters of a muscle tissue block are greater than in the tissue paste as the cell membrane structures are destroyed in tissue paste. Results also show that the α and β dispersions are visible in all the parameters of both the tissue samples, but both the dispersions are larger in the muscle tissue block. The Nyquist plot obtained for the muscle tissue block demonstrates that the equivalent electric model of the tissue sample contains Warburg impedance and a constant phase element.

Water and ion transport across the eversible vesicles in the collophore of a springtail Orchesella cincta

ABSTRACTSpringtails (Collembola) are ancient close relatives of the insects. The eversible vesicles are their unique paired transporting organs, which consist of an epithelium located inside a tube-like structure on the first abdominal segment called the collophore. The vesicles can be protruded out of the collophore and several lines of evidence indicate that they have a vital function in water uptake and ion balance. However, the amount of water absorbed by the vesicles and which other ions apart from sodium are transported remain unknown. Using Orchesella cincta as a model, we developed protocols for two assays that enabled us to study water and ion movement across the eversible vesicles in whole living springtails. Using an inverse Ramsay assay we demonstrate that the eversible vesicles absorb water from a droplet applied onto their surface. Using the scanning ion-selective electrode technique (SIET) we show that the vesicles absorb Na+ and Cl− from the bathing medium, secrete NH4+, and both absorb and secrete K+, H+ is secreted at a low level in the anterior part and absorbed at the posterior. We did not detect transport of Ca2+ at significant levels. The highest flux was the absorption of Cl−, and the magnitude of ion fluxes were significantly lower in fully hydrated springtails. Our data demonstrate that the eversible vesicles are a transporting epithelium functioning in osmo- and ionoregulation, nitrogenous waste excretion and likely acid-base balance.

Microvascular dilation evoked by chemical stimulation of C-fibers in rats

Activation of pulmonary C-fibers can reflexively decrease heart rate, blood pressure, and peripheral vascular resistance. However, the effects of these afferents on microvascular tone remain incompletely understood. In this study, we examined the effects of these afferents on microvascular tone in a striated muscle vascular bed. The right cremaster muscle in pentobarbital-anesthetized rats with intact circulation and innervation was suspended in a tissue bath, and diameters of small arterioles were measured by intravital video microscopy. Stimulation of pulmonary C-fibers by injecting capsaicin (5 μg/kg) or phenylbiguanide (20 μg/kg) into the right atrium dilated small arterioles and decreased blood pressure and heart rate. The effects persisted when the cervical vagus nerves were cooled to 5 to 7°C (blocking myelinated fibers), but were prevented by cooling to 0°C (blocking C-fibers and myelinated fibers), by cutting the genital femoral nerve (GFN) supplying the cremaster to block the nerve supply to the muscle, or by adding 6-hydroxydopamine to the bathing medium to selectively block sympathetic effects by depleting norepinephrine from adrenergic nerve terminals. Our results show that stimulation of pulmonary C-fibers reflexively dilates small arterioles in striated muscle by a mechanism that could involve withdrawal of sympathetic adrenergic tone. In conclusion, pulmonary C-fibers can exert an inhibitory influence on neural tone of the microcirculation at an important site where microvascular resistance and tissue blood flow are regulated.

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

Fixed negative charge and the Donnan effect: a description of the driving forces associated with brain tissue swelling and oedema

Cerebral oedema or brain tissue swelling is a significant complication following traumatic brain injury or stroke that can increase the intracranial pressure (ICP) and impair blood flow. Here, we have identified a potential driver of oedema: the negatively charged molecules fixed within cells. This fixed charge density (FCD), once exposed, could increase ICP through the Donnan effect. We have shown that metabolic processes and membrane integrity are required for concealing this FCD as slices of rat cortex swelled immediately (within 30 min) following dissection if treated with 2 deoxyglucose + cyanide (2DG+CN) or Triton X-100. Slices given ample oxygen and glucose, however, did not swell significantly. We also found that dead brain tissue swells and shrinks in response to changes in ionic strength of the bathing medium, which suggests that the Donnan effect is capable of pressurizing and swelling brain tissue. As predicted, a non-ionic osmolyte, 1,2 propanediol, elicited no volume change at 2000×10 −3 osmoles l −1 (Osm). Swelling data were well described by triphasic mixture theory with the calculated reference state FCD similar to that measured with a 1,9 dimethylmethylene blue assay. Taken together, these data suggest that intracellular fixed charges may contribute to the driving forces responsible for brain swelling.

Direct observation of failing fibers in muscles of dystrophic mice provides mechanistic insight into muscular dystrophy

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin. Dystrophin's function is not known, but its cellular location and associations with both the force-generating contractile core and membrane-spanning entities suggest a role in mechanically coupling force from its intracellular origins to the fiber membrane and beyond. We report here the presence of destructive contractile activity in lumbrical muscles from dystrophin-deficient ( mdx) mice during nominally quiescent periods following exposure to mechanical stress. The ectopic activity, which was observable microscopically, resulted in longitudinal separation and clotting of fiber myoplasm and was absent when calcium (Ca2+) was removed from the bathing medium. Separation and clotting of myoplasm were also produced in dystrophin-deficient muscles by local application of a Ca2+ ionophore to create membrane breaches in the absence of mechanical stress, whereas muscles from control mice tolerated ionophore-induced entry of Ca2+ without damage. These observations suggest a failure cascade in dystrophin-deficient fibers that 1) is initiated by a stress-induced influx of extracellular Ca2+, causing localized activation to continue after cessation of stimulation, and 2) proceeds as the persistent local activation, combined with reduced lateral mechanical coupling between the contractile core and the extracellular matrix, results in longitudinal separation of myoplasm in nonactivated regions of the fiber. This mechanism invokes both the membrane stabilization and the mechanical coupling functions frequently proposed for dystrophin and suggests that, whereas the absence of either function alone is not sufficient to cause fiber failure, their combined absence is catastrophic.

Serum Lipoproteins Promote Efficient Presentation of the Malaria Virulence Protein PfEMP1 at the Erythrocyte Surface

ABSTRACT The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.

Low-voltage activated calcium currents in ganglion cells of the tiger salamander retina: Experiment and simulation

We examined the functional properties of a low-voltage-activated (LVA) calcium current in ganglion cells of the neotenous tiger salamander (Ambystoma tigrinum) retina. Our analysis was based on whole-cell recordings from acutely dissociated ganglion cell bodies identified by retrograde dye injections. Using a continuously perfused cell preparation, the LVA current was isolated with the use of potassium channel blocking agents added to the bathing medium and the pipette solution, while tetrodotoxin was added to the bathing medium to block Na+channels. Approximately 70% of ganglion cells had an easily identified LVA current. The LVA current activated at membrane potentials more positive than −90 mV, and inactivated rapidly. It was relatively insensitive to nickel (IC50 > 500 μM) and amiloride (IC50 > 750 μM). Voltage- and current-clamp studies allowed us to generate a model of this current using the NEURON simulation program. Studies were also carried out to measure the LVA Ca2+current in ganglion cells with dendrites to confirm that it had a significant dendritic representation. Physiological mechanisms that may depend on LVA Ca2+currents are discussed with an emphasis on the role that dendrites play in ganglion cell function.

Angiotensin II Induces Calcium-Dependent Rhythmic Activity in a Subpopulation of Rat Hypothalamic Median Preoptic Nucleus Neurons

Whole cell patch-clamp recordings revealed a subpopulation (16%, n = 18/112) of rat median preoptic nucleus (MnPO) neurons responded to bath-applied angiotensin II (Ang II; 100 nM to 5 μM; 30–90 s) with a prolonged TTX-resistant membrane depolarization and rhythmic bursting activity. At rest, cells characteristically displayed relatively low input resistance and negative resting potentials. Ang-II-induced responses featured increased input resistance, a reversal potential of −95 ± 2 mV, an increase in action potential duration from 2.9 ± 0.5 to 4.3 ± 0.8 ms, and the appearance of a rebound excitation at the offset of membrane responses to hyperpolarizing current injection. The latter was sensitive to Ni2+ (0.5–1 mM; n = 5), insensitive to extracellular Cs+ (1 mM, n = 7), and intracellular QX-314 (4 mM, n = 5), consistent with activation of a T-type Ca2+ conductance. Coincident with the Ang-II-induced depolarization was the appearance of rhythmic depolarizing shifts at a frequency of 0.14 ± 0.09 Hz with superimposed bursts of 4–22 action potentials interspersed with silent periods persisting for >1 h after washout. These TTX-resistant depolarizing shifts increased in amplitude and decreased in frequency with membrane hyperpolarization with activity ceasing beyond approximately –80mV, and were abolished in low-Ca2+/high-Mg2+ bathing medium ( n = 6), Co2+ (1 mM; n = 6), or Ni2+ (0.5–1 mM; n = 8). Thus in a subpopulation of MnPO neurons, Ang II induces “pacemaker-like” activity by reducing a K+-dependent leak conductance that contributes to resting membrane potential and promoting of Ca2+-dependent regenerative auto-excitation mediated, in part, by a T-type Ca2+ conductance.