Eotaxin/CCL11 levels correlate with myocardial fibrosis and mast cell density in native and transplanted rat hearts

Myocardial fibrosis contributes to hemodynamic and cardiac functional alterations commonly observed posttransplantation. Cardiac mast cells (MC) have been linked to fibrosis in posttransplantation hearts. Eotaxin, which has been shown to be involved in fibrogenesis, has been demonstrated to be increased in production in cardiac macrophages. The aim of our study was to correlate myocardial fibrosis during heart transplant rejection in the rat with eotaxin/chemokine [c-c motif] ligand 11 (CCL11) expression, and with various subtypes of infiltrating cardiac MC, namely connective-type MC (CTMC) and mucosa-type MC (MMC).

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.

Effects of local continuous release of brain derived neurotrophic factor (BDNF) on peripheral nerve regeneration in a rat model

The purpose of this study was to evaluate the effect of continuously released BDNF on peripheral nerve regeneration in a rat model. Initial in vitro evaluation of calcium alginate prolonged-release-capsules (PRC) proved a consistent release of BDNF for a minimum of 8 weeks. In vivo, a worst case scenario was created by surgical removal of a 20-mm section of the sciatic nerve of the rat. Twenty-four autologous fascia tubes were filled with calcium alginate spheres and sutured to the epineurium of both nerve ends. The animals were divided into 3 groups. In group 1, the fascial tube contained plain calcium alginate spheres. In groups 2 and 3, the fascial tube contained calcium alginate spheres with BDNF alone or BDNF stabilized with bovine serum albumin, respectively. The autocannibalization of the operated extremity was clinically assessed and documented in 12 additional rats. The regeneration was evaluated histologically at 4 weeks and 10 weeks in a blinded manner. The length of nerve fibers and the numbers of axons formed in the tube was measured. Over a 10-week period, axons have grown significantly faster in groups 2 and 3 with continuously released BDNF compared to the control. The rats treated with BDNF (groups 2 and 3) demonstrated significantly less autocannibalization than the control group (group 1). These results suggest that BDNF may not only stimulate faster peripheral nerve regeneration provided there is an ideal, biodegradable continuous delivery system but that it significantly reduces the neuropathic pain in the rat model.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Ventricular-arterial coupling in a rat model of reduced arterial compliance provoked by hypervitaminosis D and nicotine

The vitamin D(3) and nicotine (VDN) model is one of isolated systolic hypertension (ISH) in which arterial calcification raises arterial stiffness and vascular impedance. The effects of VDN treatment on arterial and cardiac hemodynamics have been investigated; however, a complete analysis of ventricular-arterial interaction is lacking. Wistar rats were treated with VDN (VDN group, n = 9), and a control group (n = 10) was included without the VDN. At week 8, invasive indexes of cardiac function were obtained using a conductance catheter. Simultaneously, aortic pressure and flow were measured to derive vascular impedance and characterize ventricular-vascular interaction. VDN caused significant increases in systolic (138 +/- 6 vs. 116 +/- 13 mmHg, P < 0.01) and pulse (42 +/- 10 vs. 26 +/- 4 mmHg, P < 0.01) pressures with respect to control. Total arterial compliance decreased (0.12 +/- 0.08 vs. 0.21 +/- 0.04 ml/mmHg in control, P < 0.05), and pulse wave velocity increased significantly (8.8 +/- 2.5 vs. 5.1 +/- 2.0 m/s in control, P < 0.05). The arterial elastance and end-systolic elastance rose significantly in the VDN group (P < 0.05). Wave reflection was augmented in the VDN group, as reflected by the increase in the wave reflection coefficient (0.63 +/- 0.06 vs. 0.52 +/- 0.05 in control, P < 0.05) and the amplitude of the reflected pressure wave (13.3 +/- 3.1 vs. 8.4 +/- 1.0 mmHg in control, P < 0.05). We studied ventricular-arterial coupling in a VDN-induced rat model of reduced arterial compliance. The VDN treatment led to development of ISH and provoked alterations in cardiac function, arterial impedance, arterial function, and ventricular-arterial interaction, which in many aspects are similar to effects of an aged and stiffened arterial tree.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.