Quantitative evaluation of altered hepatic spaces and membrane transport in fibrotic rat liver

Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl4-treated group (phenobarbital with CCl4 treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of H-3-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl4-treated rats were found to have the smallest vascular space, extravascular albumin space, H-3-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of H-3-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses.

The role of Doppler left ventricular filling indexes and Doppler tissue echocardiography in the assessment of cardiac involvement in hereditary hemochromatosis

Although cardiac dysfunction in hereditary hemochromatosis (HHC) can be evaluated by conventional echocardiography, findings are often not specific. To test the hypothesis that the assessment of (1) conventional Doppler left ventricular filling indexes and (2) intrinsic elastic properties of the myocardium by Doppler tissue echocardiography can both enhance the accuracy of echocardiographic diagnosis of cardiac involvement in HHC, a group of 18 patients with HHC (mean age 50+/-7 years) and 22 age-matched healthy subjects were studied. The following indexes were characteristic for HHC: (1) the duration of atrial reversal measured from pulmonary venous flow (ms) was longer(118+/-20 vs 90+/-16; P

Influence of wall stress and left ventricular geometry on the accuracy of Dobutamine stress Echocardiography

OBJECTIVES The goal of this study was to determine whether wall stress at rest and during stress could explain the influence of left ventricular (LV) morphology on the accuracy of dobutamine stress echocardiography (DSE). BACKGROUND The sensitivity of DSE appears to be reduced in patients with concentric remodeling, but the cause of this finding is unclear. METHODS We studied 161 patients without resting wall motion abnormalities who underwent DSE and coronary angiography. Patients were classified into four groups according to relative wan thickness (normal

Influence of left ventricular size and hemodynamics on the systolic longitudinal myocardial Doppler velocity response to stress

Background Systolic myocardial Doppler velocity accurately identifies coronary artery disease. However, these velocities may be affected by age, hemodynamic responses to stress, and left ventricular cavity size. We sought to examine the influences of these variables on myocardial velocity during dobutamine stress in patients with normal wall motion. Methods One hundred seventy-nine consecutive patients with normal dobutamine echocardiograms were studied. Color myocardial tissue Doppler data were obtained at rest and peak stress, and peak systolic myocardial velocity (PSV) was measured in all basal and midventricular segments. Velocities at rest and peak stress were compared with left ventricular diastolic and systolic volumes, blood pressure, heart rate, and age by Pearson correlation and interdecile analysis by use of analysis of variance. Results The only clinical variable correlating with velocity was age; PSV showed only mild correlation with age at rest (r(2) = 0.01, P = .001) and peak stress (r(2) = 0.02, P = .001), but the normal peak velocity was significantly different between the extremes of age (<44 years and >74 years). There was very weak correlation of PSV with systolic and diastolic blood pressure (r(2) < 0.01), heart rate (r(2) < 0.01), systemic vascular resistance (r(2) = 0.08), and left ventricular volumes (r(2) < 0.01). Conclusions Peak systolic velocity during dobutamine stress is relatively independent of hemodynamic factors and left ventricular cavity size. The extremes of age may influence peak systolic Doppler velocities. These results suggest that peak systolic velocity may be a robust quantitative measure during dobutamine echocardiography across most patient subgroups.

Use of cyclic variation of integrated backscatter to assess contractile reserve and myocardial viability in chronic ischemic left ventricular dysfunction

The detection of viable myocardium has important implications for management, but use of stress echocardiography to detect this is subjective and requires exposure to dobutamine. We investigated whether cyclic variation (CV) of integrated backscatter (IB) from the apical views could provide a resting study for detection of contractile reserve (CR) and prediction of myocardial viability in 27 patients with chronic ischemic left ventricular (LV) dysfunction. Repeat echocardiography was performed after 6.7 +/- 3.8 months of follow-up; 14 patients underwent revascularization and 13 were treated medically. Using a standardized dobutamine echocardiography (DbE) protocol, images from three apical views were acquired at 80-120 frames/sec at rest and during stress. CR was identified if improvement of wall motion was observed at low dose (5 or 10 mug/kg/min) DbE. Myocardial viability was characterized by improvement at follow-up echocardiography in patients with revascularization. CVIB at rest and low dose dobutamine were assessed in 194 segments with resting asynergy (severe hypokinesis or akinesis), of which 88 (45%) were in patients who underwent revascularization. Of these, CVIB could be measured in 190 (98%) segments at rest and 185 (95%) at low dose dobutamine. Sixty-two (33%) segments had CR during low dose DbE and 50 (57%) segments showed wall-motion recovery (myocardial viability) at follow-up echocardiography. Segments with CR had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P = 0.005) than segments without CR. Using optimal thresholds of CVIB (> 8.2 dB) at rest, the accuracy of CVIB for detecting CR was 70%. Compared with nonviable segments, viable segments had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P < 0.001). Using optimal thresholds of CVIB (> 5.3 dB) at rest, the accuracy of CVIB for detecting myocardial viability was 85%, which was higher than that in conventional DbE (62%, P < 0.01). Thus, assessment of CV.TB from the apical views is a feasible and accurate tool for detecting CR and predicting myocardial viability in chronic LV dysfunction.

Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain

Abnormal left ventricular (LV) filling is common, but not universal, in hypertensive LV hypertrophy (LVH). We sought to elucidate the relative contributions of myocardial structural changes, loading and hypertrophy to LV dysfunction in 113 patients: 85 with hypertensive LVH and 28 controls without LVH and with normal filling. Patients with normal dobutamine stress echocardiography and no history of coronary artery disease were selected, in order to exclude a contribution from ischaemia or scar. Abnormal LV filling was identified in 65 LVH patients, based on Doppler measurement of transmitral filling and annular velocities. All patients underwent grey-scale and colour tissue Doppler imaging from three apical views, which were stored and analysed off line. Integrated backscatter (113) and strain rate imaging were used to detect changes in structure and function; average cyclic variation of 113, strain rate and peak systolic strain were calculated by averaging each segment. Calibrated 113 intensity, corrected for pericardial 113 intensity, was measured in the septum and posterior wall from the parasternal long-axis view. Patients with LVH differed significantly from controls with respect to all backscatter and strain parameters, irrespective of the presence or absence of abnormal LV filling. LVH patients with and without abnormal LV filling differed with regard to age, LV mass and incidence of diabetes mellitus, but also showed significant differences in cyclic variation (P < 0.01), calibrated 113 in the posterior wall (P < 0.05) and strain rate (P < 0.01), although blood pressure, heart rate and LV systolic function were similar. Multivariate logistic regression analysis demonstrated that age, LV mass index and calibrated IB in the posterior wall were independent determinants of abnormal LV filling in patients with LVH. Thus structural and functional abnormalities can be detected in hypertensive patients with LVH with and without abnormal LV filling. In addition to age and LVH, structural (not functional) abnormalities are likely to contribute to abnormal LV filling, and may be an early sign of LV damage. 113 is useful for the detection of myocardial abnormalities in patients with hypertensive LVH.

Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid. (C) 2002 Elsevier Science B.V. All rights reserved.

Ultrastructure of the mature spermatozoon of the musky rat-kangaroo, Hypsiprymnodon moschatus (Potoroidae : Marsupialia)

It is currently accepted that Hypsiprymnodon moschatus is a basal macropod, retaining several primitive features from the ancestral phalangeroid that gave rise both to modern possums and macropods. Sperm ultrastructure is frequently found to provide informative characters for phylogenetic analysis as these features are not strongly selected for and are thus unlikely to be confounded by effects such as convergence. Caudal epididymal biopsies were taken from two male H. moschatus and prepared for transmission and scanning electron microscopy in order to study mature spermatozoan ultrastructure. Within the diprotodont group, several features were found to be unique to H. moschatus. These were an unusual acrosome covering nearly 100% of the dorsal nuclear surface, a midpiece fibre network which is loose, indistinct and extends to the anterior-most aspect of the midpiece, a nucleus that is very streamlined, while the principal piece is comparatively short, and a mitochondrial helix and annulus which are similar to those of dasyurids. Also reported is the presence of a fibrous network in die connecting piece, not previously reported for any marsupial.

Distribution kinetics of solutes in the isolated in-situ perfused rat head using the multiple indicator dilution technique and a physiological two-barrier model

The purpose of this study was to determine the pharmacokinetics of [C-14]diclofenac, [C-14]salicylate and [H-3]clonidine using a single pass rat head perfusion preparation. The head was perfused with 3-[N-morpholino] propane-sulfonic acid-buffered Ringer's solution. Tc-99m-red blood cells and a drug were injected in a bolus into the internal carotid artery and collected from the posterior facial vein over 28 min. A two-barrier stochastic organ model was used to estimate the statistical moments of the solutes. Plasma, interstitial and cellular distribution volumes for the solutes ranged from 1.0 mL (diclofenac) to 1.6 mL (salicylate), 2.0 mL (diclofenac) to 4.2 mL (water) and 3.9 mL (salicylate) to 20.9 mL (diclofenac), respectively. A comparison of these volumes to water indicated some exclusion of the drugs from the interstitial space and salicylate from the cellular space. Permeability-surface area (PS) products calculated from plasma to interstitial fluid permeation clearances (CLPI) (range 0.02-0.40 mL s(-1)) and fractions of solute unbound in the perfusate were in the order: diclofenac>salicylate >clonidine>sucrose (from 41.8 to 0.10 mL s(-1)). The slow efflux of diclofenac, compared with clonidine and salicylate, may be related to its low average unbound fraction in the cells. This work accounts for the tail of disposition curves in describing pharmacokinetics in the head.

Cyclosporin A pretreatment in a rat model of warm ischaemia/reperfusion injury

Background/Aims: These studies investigated the role of apoptosis following ischaemia/reperfusion (I/R) injury to the liver and the effect of pretreatment with Cyclosporin A. Methods: Male Sprague-Dawley rats received 30 min of warm ischaemia followed by a period of reperfusion of 6 h. Rats were given olive oil or Cyclosporin A (30 mg/kg p.o.) the day before surgery. Neutrophil numbers were assessed in haematoxylin-eosin-stained sections of liver. In situ staining of sections using TdT-mediated dUTP-fluoreseein nick-end labelling was carried out to determine the extent of apoptosis, followed by electron microscopy. Semi-quantitative polymerase chain reaction (PCR) analysis of the transcript for Fas antigen was performed. Results and Conclusions: High levels of apoptosis were observed in I/R injury, which were greatly ameliorated in Cyclosporin A-pretreated groups. PCR analysis indicated a reduction in the level of expression of Fas transcript in Cyclosporin A-treated rats. Histological analysis showed a significant increase in the number of neutrophils infiltrating I/R-injured tissue (62 +/- 10.69, it = 16), which was markedly reduced by Cyclosporin A pretreatment (16 +/- 7, n = 6, P < 0.05). These results indicate a role of parenchymal apoptosis in the pathogenesis of I/R injury, which occurs in association with neutrophil infiltration, both of which can be significantly reduced by Cyclosporin A pretreatment. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

A prostaglandin F2a Analog induces suppressors of the cytokine signalling-3 expression n the corpus luteum of the pregnant rat: A potential new mechanism in luteolysis

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F-2alpha (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.