Comparative determination of placental perfusion by magnetic resonance imaging and contrast-enhanced ultrasound in a murine model of intrauterine growth restriction.

INTRODUCTION: Exploration of placental perfusion is essential in screening for dysfunctions impairing fetal growth. The objective of this study was to assess the potential value of contrast-enhanced ultrasonography (CEUS) and magnetic resonance imaging (MRI) for examining placental perfusion in a murine model of intrauterine growth restriction (IUGR). We also studied the reproducibility of perfusion quantification by CEUS. METHODS: Pregnant Sprague Dawley rat models of IUGR were studied during the third trimester. Unilateral uterine artery ligation induced IUGR. Placental perfusion was evaluated by CEUS and perfusion MRI with gadolinium for both ligated and control fetoplacental units. The kinetic parameters of the two imaging modalities were then compared. RESULTS: The analysis included 20 rats. The study showed good reproducibility of the CEUS indicators. The CEUS perfusion index approximated the blood flow rate and was halved in the ligation group (27.9 [u.a] (±14.8)) versus 61 [u.a] (±22.3) on the control side (P = 0.0003). MRI with gadolinium injection showed a clear reduction in the blood flow rate to 51.2 mL/min/100 mL (IQR 34.9-54.9) in the ligated horn, compared with 90.9 mL/min/100 mL (IQR 85.1-95.7) for the control side (P < 0.0001). The semiquantitative indicators obtained from the kinetic curves for both CEUS and MRI showed similar trends. Nonetheless, values were more widely dispersed with CEUS than MRI. DISCUSSION: The similar results for the quantification of placental perfusion by MRI and CEUS reinforce the likelihood that CEUS can be used to identify IUGR in a murine model induced by uterine vessel ligation.

ATP-induced inhibition of gap junctional communication is enhanced by interleukin-1 beta treatment in cultured astrocytes.

Nucleotides are signaling molecules involved in variety of interactions between neurons, between glial cells as well as between neurons and glial cells. In addition, ATP and other nucleotides are massively released following brain insults, including inflammation, and may thereby be involved in mechanisms of cerebral injury. Recent concepts have shown that in astrocytes intercellular communication through gap junctions may play an important role in neuroprotection. Therefore, we have studied the effects of nucleotides on gap junction communication in astrocytes. Based on measurement of intercellular dye coupling and recording of junctional currents, the present study shows that ATP (10-100 microM) induces a rapid and a concentration-dependent inhibition of gap junction communication in cultured cortical astrocytes from newborn mice. Effects of agonists and antagonists of purinergic receptors indicate that the inhibition of gap junctional communication by ATP mainly involves the stimulation of metabotropic purinergic 1 (P2Y(1)) receptors. Pretreatment with the pro-inflammatory cytokine interleukin-1beta (10 ng/ml, 24 h), which has no effect by itself on gap junctional communication, increases the inhibitory effect of ATP and astrocytes become sensitive to uridine 5'-triphosphate (UTP). As indicated by the enhanced expression of P2Y(2) receptor mRNA, P2Y(2) receptors are responsible for the increased responses evoked by ATP and UTP in interleukin-1beta-treated cells. In addition, the effect of endothelin-1, a well-known inhibitor of gap junctional communication in astrocytes was also exacerbated following interleukin-1beta treatment. We conclude that ATP decreases intercellular communication through gap junctions in astrocytes and that the increased sensitivity of gap junction channels to nucleotides and endothelin-1 is a characteristic feature of astrocytes exposed to pro-inflammatory treatments.

Gap junctions and connexin expression in the normal and pathological central nervous system.

Gap junctions are widely expressed in the various cell types of the central nervous system. These specialized membrane intercellular junctions provide the morphological support for direct electrical and biochemical communication between adjacent cells. This intercellular coupling is controlled by neurotransmitters and other endogenous compounds produced and released in basal as well as in pathological situations. Changes in the expression and the function of connexins are associated with number of brain pathologies and lesions suggesting that they could contribute to the expansion of brain damages. The purpose of this review is to summarize data presently available concerning gap junctions and the expression and function of connexins in different cell types of the central nervous system and to present their physiopathological relevance in three major brain dysfunctions: inflammation, epilepsy and ischemia.

Low sodium inotropy is accompanied by diastolic Ca2+ gain and systolic loss in isolated guinea-pig ventricular myocytes.

We measured sarcolemmal Ca2+ fluxes responsible for the positive inotropic effects of solutions with reduced Na+ concentration in voltage-clamped guinea-pig ventricular myocytes; intracellular Ca2+ concentration ([Ca2+]i) was measured with Indo-1. Reduction of external Na+ concentration by 50 % (to 67 mM) produced an increase in systolic [Ca2+]i accompanied by a decrease in Ca2+ entry via the L-type Ca2+ current. With reduced Na+ concentration, there was an initial decrease in the Na+-Ca2+ exchange current on repolarization followed by an increase to greater than control. We attribute this initial decrease to a decrease in the Na+ gradient and the subsequent increase to a fall in intracellular Na+ concentration and increase in systolic [Ca2+]i. The decreased L-type Ca2+ current and increased Ca2+ efflux on Na+-Ca2+ exchange resulted in a calculated systolic loss of Ca2+. The calculated systolic loss of Ca2+ was accompanied by a measured increase in sarcoplasmic reticulum (SR) Ca2+ content. Reduction of the external Na+ concentration also produced an outward shift of holding current which was blocked by Ni2+. This is taken to represent Ca2+ influx via Na+-Ca2+ exchange. When diastolic influx is taken into account, the observed gain in SR Ca2+ content can be predicted. The measurements show that, in reduced Na+, much of the entry of Ca2+ into the cell occurs during diastole (via Na+-Ca2+ exchange) rather than in systole (via the L-type Ca2+ current).

Cyclopiazonic acid and thapsigargin reduce Ca2+ influx in frog skeletal muscle fibres as a result of Ca2+ store depletion.

We have investigated the influence of the sarcoplasmic reticulum (SR) Ca2+ content on the retrograde control of skeletal muscle L-type Ca2+ channels activity by ryanodine receptors (RyR). The effects of cyclopiazonic acid (CPA) and thapsigargin (TG), two structurally unrelated inhibitors of SR Ca(2+)-adenosine triphosphatase (ATPase), were examined on the SR Ca2+ content, the calcium current and contraction in single frog semitendinosus fibres using the double mannitol-gap technique. At moderate concentrations that only partially inhibited Ca2+ sequestration by the SR, CPA (2-4 microM) induces a concentration dependent depression of contraction and Ca2+ current amplitudes. When Ba2+ is the charge carrier, the inward current is not changed by CPA suggesting that this Ca(2+)-pump inhibitor does not directly affect dihydropyridine Ca2+ channels. Similar effects were obtained with TG (1-5 microM). Changes in Ca2+ currents and contraction were accompanied by a reduced Ca2+ loading of the SR. We attribute the modulation of the Ca2+ current to the selective inhibition of the SR Ca2+ ATPase, resulting in a decreased Ca2+ release and thereby a reduced activation of calcium inward currents. This is therefore taken to represent a calcium release-dependent modulation of skeletal muscle L-type Ca2+ channels.

Na(+)-Ca2+ exchange induces low Na+ contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase.

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity.

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca2+-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1-10 microM) enhanced the amplitude and time-course of relaxation of 146 mM potassium contracture. In the presence of cyclopiazonic acid 0.5 microM, the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium.

Measurement of sarcomere length during fast contraction of muscle fibers by digital image analysis.

A low-cost, high-resolution (spatial and temporal) image analysis system was developed to measure sarcomere length (Sl) during fast twitch of isolated striated muscle fibers at different temperatures. Fiber images were examined during twitch with an imaging rate of 220 Hz. To increase temporal resolution beyond 220 Hz, consecutive temporally shifted image sequences (N sequences) were acquired. Individual or average Sl was directly measured from a horizontal profile without spatial-frequency assessment. Measurement precision (E) was determined and expressed as: E(%) = 100xPs/(IsxSl), where Ps is the pixel size and Is the involved sarcomere number. At 18 degrees C during isometric twitch, Sls were measured with 220 Hz temporal and 0.2% spatial resolutions. Sl shortened in the central region (0.21+/-0.12 microm) as tension developed, reaching a maximal shortening of 8.09 + 2.05% (at rest, Sl = 2.59+/-0.05 microm, n = 4) in 32.5+/-1.96 ms. At 30 degrees C, Sl variations were examined with 880 Hz temporal resolution, in which case maximal S1 shortening was reached in 15.74+/-1.99 ms, and then decreased to 5.19+/-1.97% (at rest, S1 = 2.6+/-0.06 microm). The twitch tension developed by the whole fiber was recorded and compared with sarcomere length behavior. Sarcomere length variations in the central region were representative of overall developed tensions at 18 and 30 degrees C.

Cyclopiazonic acid-induced changes in the contraction and Ca2+ transient of frog fast-twitch skeletal muscle.

The effects of cyclopiazonic acid (CPA) were investigated on isolated skeletal muscle fibers of frog semitendinosus muscle. CPA (0.5-10 microM) enhanced isometric twitch but produced little change in resting tension. At higher concentrations (10-50 microM), CPA depressed twitch and induced sustained contracture without affecting resting and action potentials. In Triton-skinned fibers, CPA had no significant effect on myofibrillar Ca2+ sensitivity but decreased maximal activated force at concentrations > 5 microM. In intact cells loaded with the Ca2+ fluorescence indicator indo 1, CPA (2 microM) induced an increase in Ca(2+)-transient amplitude (10 +/- 2.5%), which was associated with an increase in time to peak and in the time constant of decay. Consequently, peak force was increased by 35 +/- 4%, and both time to peak and the time constant of relaxation were prolonged. It is concluded that CPA effects, at a concentration of up to 2 microM, were associated with specific inhibition of sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase in intact skeletal muscle and that inhibition of the pump directly affected the handling of intracellular Ca2+ and force production.

Hypogravity increases cyclopiazonic acid sensitivity of rat soleus muscle.

The functional capacity of skeletal muscle sarcoplasmic reticulum was explored in slow rat soleus muscle after 21 days of hindlimb suspension. The sarcoplasmic reticulum function was assessed in intact and saponin-skinned fibers by using cyclopiazonic acid, a specific Ca(2+)-adenosinetriphosphatase inhibitor. After hindlimb unweighting, the sensitivity to cyclopiazonic acid of intact and skinned soleus fibers becomes similar to that found in fast-twitch muscles. This change could be related to the expression of fast Ca2(+)-adenosinetriphosphatase-pump protein in unloaded soleus muscles and agrees with a transformation of soleus muscle from slow- to fast-twitch type. These results also indicate that specific pharmacological tools, like cyclopiazonic acid, could be used to analyze subcellular functional changes due to hindlimb unweighting.