Analbuminemic Nagase rats: blood pressure response to dietary salt challenge

BACKGROUND: The role of albumin on blood pressure response to different salt challenges is not known. Therefore, we studied the blood pressure response of analbuminemic Nagase rats (NAR) to different salt challenges. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme regulating the glucocorticoid access to the mineralocorticoid receptor, an enzyme that is decreased in humans with salt sensitive hypertension and other diseases with abnormal renal salt retention, was assessed during salt challenges. METHODS: Blood pressure was measured continuously by an intra-arterial catheter and a telemetry system in NAR (n = 8). NAR were set successively for 7 days on a normal (0.45% NaCl), high (8% NaCl), low (0.1% NaCl) and normal salt diet again, to assess salt related response in mean systolic (SBP) and diastolic blood pressure (DBP). 11beta-HSD2activity was assessed by measuring the urinary (THB + 5alpha-THB)/THA ratio with gas chromatography - mass spectrometry. RESULTS: Mean SBP and DBP increased with high salt intake (normal salt vs. high salt: SBP: 114 +/- 1 vs.119 +/- 3 mm Hg, p < 0.01; DBP: 84 +/- 1 vs. 88 +/- 3 mm Hg; n = 8; p < 0.01). Urinary (THB +5alpha-THB)/THA ratio increased during the high-salt period when compared to the normal-salt period (high salt vs. normal salt: 0.52 +/- 0.10 vs. 0.37 +/- 0.07; p = 0.05) indicating decreased 11beta-HSD2activity. CONCLUSION: Analbuminemic Nagase rats express increased blood pressure and reduced 11beta-HSD2 activity in response to a high-salt diet.

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.

Sodium thiosulfate prevents vascular calcifications in uremic rats

Accelerated vascular calcification is a severe complication of chronic kidney disease contributing to high morbidity and mortality in patients undergoing renal replacement therapy. Sodium thiosulfate is increasingly used for the treatment of soft tissue calcifications in calciphylaxis. Therefore, we determined whether it also prevents development of vascular calcifications in chronic kidney disease. We found that uremic rats treated by thiosulfate had no histological evidence of calcification in the aortic wall whereas almost three-fourths of untreated uremic rats showed aortic calcification. Urinary calcium excretion was elevated and the calcium content of aortic, heart, and renal tissue was significantly reduced in the thiosulfate-treated compared to non-treated animals. Sodium thiosulfate treatment transiently lowered plasma ionized calcium and induced metabolic acidosis. It also lowered bone strength in the treated animals compared to their normal controls. Hence, sodium thiosulfate prevented vascular calcifications in uremic rats, likely by enhancing acid- and/or chelation-induced urinary calcium loss. The negative impact on rat bone integrity necessitates a careful risk-benefit analysis before sodium thiosulfate can be used in individual human patients.

Prevention of adjuvant arthritis by cyclosporine in rats

The effect of cyclosporine A during the development phase of adjuvant arthritis was studied in 40 female rats. Five groups of eight animals each received oral cyclosporine, 2.5, 5, 10, 20, or 30 mg/kg daily for 30 days. Also, eight normal and eight diseased rats served as placebo controls. At the time of inoculation of the adjuvant suspension on day 0, measurement of disease parameters (paw swelling and vertebral density) was started concomitantly with beginning of therapy. On completion of the study, the animals were killed, and after measurement of total skeletal and segmental (hind legs and caudal spine plus two caudal vertebrae) calcium, the two assessed vertebrae and both femoral condyles were removed for histomorphometric evaluation (vertebrae) and for estimation of glycosaminoglycan (GAG) content of cartilage. Blood for osteocalcin determinations also was taken at term from control and untreated arthritic rats and from animals that had received 10 mg/kg cyclosporine. Treatment with 2.5 mg/kg was ineffective, but doses between 5 and 20 mg/kg prevented the development of articular and osseous lesions. The 20 mg/kg dose showed no better effect than 10 mg/kg. This was shown by the absence of inflammation and the presence of normal condylar GAG and total mineral content in the areas screened. Untreated animals showed marked reductions in all of these parameters. The 30 mg/kg dose was effective in blocking the GAG loss, but significant reductions in bone density and trabecular volume were seen. There was a close correlation between GAG and bone density values, suggesting a common causal relationship. Circulating osteocalcin was significantly elevated in the untreated animals with adjuvant arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mild vitamin a deficiency on lung maturation in newborn rats: a morphometric and morphologic study

OBJECTIVE To evaluate the effects of a 60% vitamin A deficiency (VAD) on the two postnatal stages of lung development: alveolarization and microvascular maturation. Lungs from deficient rats were compared to age-matched controls. STUDY DESIGN Starting at 3 weeks before mating, female rats were maintained under a diet lacking vitamin A. Due to the slow depletion of the vitamin A liver stores the pregnant rats carried to term and delivered pups under mild VAD conditions. Mothers and offspring were then kept under the same diet what resulted in a mean reduction of vitamin A plasma concentration of about 60% vs. controls during the whole experimental period. Pups were sacrificed on days 4, 10 and 21 and their lungs fixed and analyzed by means of a combined morphologic and morphometric investigation at light and electron microscopic levels. RESULTS During the whole experiment, body weights of VAD animals were lower than controls with a significant decrease on day 10. On days 4, 10 and 21 the pulmonary structure was in a comparable gross morphologic state in both groups. Despite this morphologic normality, quantitative alterations in some functional parameters could be detected. On day 4, lung volume and the volume and surface area of air spaces were decreased, while the arithmetic mean barrier thickness and type 2 pneumocyte volume were increased in the VAD group. On day 21, some changes were again manifest mainly consisting in an augmentation of the vascularization and a decrease in interstitial volume in deficient animals. CONCLUSIONS Mild VAD causes no gross disturbances in the postnatal phases of lung development in rats. However, a body weight-related transient retardation of lung maturation was detectable in the first postnatal week. At 3 weeks, the VAD lungs showed a more mature vascular system substantiated by an increase in volume of both capillary volume and the large non-parenchymal vessels. In view of these quantitative alterations, we suspect that mild VAD deregulates the normal phases of body and lung growth, but does not induce serious functional impairments.

Geometric properties of the lung parenchyma after postnatal glucocorticoid treatment in rats

While glucocorticoid (GC) administration appears to be beneficial during the acute phase of treatment of neonates at risk of developing chronic lung disease, it is still not clear whether steroid application has an adverse long-term effect on the lung maturation. Thus, the goal of the present work was to analyze GC effects on the pulmonary structure in a rat model where dosage and timing of drug administration were adapted to the therapeutic situation in human neonatology. The animals received daily a maximum of 0.1 mg dexamethasone phosphate per kilogram body weight during the first 4 postnatal days. Investigations were performed at the light microscopic level by means of a digital image analysis system. While there were no differences in the lung architecture between experimental animals and controls on day 4, the earliest time point of observation, we found a widening of airspaces with a concomitant decrease in the alveolar surface area density, representing a loss of parenchymal complexity, on days 10 and 21 in treated rats. On days 36 and 60, however, no alterations in the pulmonary parenchyma could be detected in experimental animals. We conclude from these findings that the GC-induced initial inhibition of development (days 10 and 21) was completely reversed, so that a normal parenchymal architecture and also a normal alveolar surface area density were found in adult rats (days 36 and 60). From the results obtained using the regimen of GC administration described, mimicking more closely the steroid treatment in human neonatology, we conclude that the observed short-term adverse effects on lung development can be fully compensated until adult age.

Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain

PURPOSE Synchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. METHODS Tissue responses to MRT (two orthogonal arrays (2 × 400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. RESULTS In tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. CONCLUSION We provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.

Early gene expression analysis in 9L orthotopic tumor-bearing rats identifies immune modulation in molecular response to synchrotron microbeam radiation therapy

Synchrotron Microbeam Radiation Therapy (MRT) relies on the spatial fractionation of the synchrotron photon beam into parallel micro-beams applying several hundred of grays in their paths. Several works have reported the therapeutic interest of the radiotherapy modality at preclinical level, but biological mechanisms responsible for the described efficacy are not fully understood to date. The aim of this study was to identify the early transcriptomic responses of normal brain and glioma tissue in rats after MRT irradiation (400Gy). The transcriptomic analysis of similarly irradiated normal brain and tumor tissues was performed 6 hours after irradiation of 9 L orthotopically tumor-bearing rats. Pangenomic analysis revealed 1012 overexpressed and 497 repressed genes in the irradiated contralateral normal tissue and 344 induced and 210 repressed genes in tumor tissue. These genes were grouped in a total of 135 canonical pathways. More than half were common to both tissues with a predominance for immunity or inflammation (64 and 67% of genes for normal and tumor tissues, respectively). Several pathways involving HMGB1, toll-like receptors, C-type lectins and CD36 may serve as a link between biochemical changes triggered by irradiation and inflammation and immunological challenge. Most immune cell populations were involved: macrophages, dendritic cells, natural killer, T and B lymphocytes. Among them, our results highlighted the involvement of Th17 cell population, recently described in tumor. The immune response was regulated by a large network of mediators comprising growth factors, cytokines, lymphokines. In conclusion, early response to MRT is mainly based on inflammation and immunity which appear therefore as major contributors to MRT efficacy.

Expression of neuromuscular junction messenger RNAs and protein in aged rats

The loss of skeletal muscle mass is believed to be the dominant reason for reduced strength in aging humans. The purpose of this investigation was to gain some information as to why skeletal muscles lose mass as we age. Since nervous system innervation is essential for skeletal muscle fiber viability, incomplete regional reinnervation during normal synaptic junction turnover has been hypothesized to result in selective muscle fiber loss. Examined here was the age-related association in skeletal muscle between atrophy and the expression of mRNAs encoding the γ- and ϵ-subunits of the nicotinic acetylcholine receptor, myogenin, and muscle specific receptor kinase (MuSK). Gastrocnemius and biceps brachii muscles were collected from young (2 month), adult (18 month), and old (31 month) Fischer 344 cross brown Norway F 1 male rats. In the gastrocnemius, muscles of old vs. young and adult rats, lower muscle mass was accompanied by significantly elevated acetylcholine receptor γ-subunit, myogenin, and MuSK mRNA levels. In contrast, the biceps brachii muscle in the same animals exhibited neither atrophy nor a change in acetylcholine receptor γ-subunit, myogenin, or MuSK mRNA levels. Expression of the acetylcholine receptor ϵ-subunit mRNA did not change with age in either gastrocnemius or biceps brachii muscles. Since acetylcholine receptor γ-subunit, myogenin, and MuSK mRNA levels are upregulated in surgically denervated skeletal muscles of young rats while expression of the acetylcholine receptor ϵ-subunit does not change, the findings of the current investigation suggest that a select fiber population within atrophied skeletal muscles of old rats may be in a denervated-like state. I speculate that increases in γ-subunit, myogenin, and MuSK mRNA levels in atrophied muscles of old rats are compensatory responses to nerve terminal retraction. Indeed, a prolongation of denervation in these muscle fibers would subsequently result in their atrophy and death, ultimately leading to a decline in the number of force generating elements present in the muscle. ^

Inhalation anesthesia of rats: influence of the fraction of inspired oxygen on limb ischemia/reperfusion injury

Inhalation anesthesia with isoflurane is a well-established and safe method used in small laboratory animals. In most cases oxygen is used as a carrier gas for isoflurane, but room air or mixtures of oxygen with air or nitrous oxide are also being used. Anesthesia is therefore administered using different fractions of inspired oxygen (FiO2), and this may have consequences for the outcome of experiments. The aim of the present study was to investigate the influence of FiO2 on rat hind limb ischemia/reperfusion injury and to refine the used inhalation anesthesia. Male Wistar rats were subjected to 3.5 h of ischemia and 2 h of reperfusion, and divided into three groups according to FiO2 in the O2/air/isoflurane anesthesia gas mixture: 40%, 60%, and 100% O2. Normal, healthy rats were used as controls. Muscle edema and creatine kinase MM, a marker for myocyte necrosis, were significantly increased with 40% FiO2 as compared with 100% FiO2 (P<0.05). Partial pressure of oxygen, oxygen saturation, and oxyhemoglobin were significantly higher in the 100% O2 group as compared with 40% O2. No significant differences were detected for other parameters, such as the oxidative stress markers malondialdehyde and superoxide dismutase. We conclude that a refined inhalation anesthesia setting using 40% FiO2, reflecting more or less the clinical situation, leads to a more severe and more physiologically relevant reperfusion injury than higher FiO2. Oxidative stress did not correlate with FiO2 and seemed to have no influence on reperfusion injury.

Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes

Sterol regulatory element binding proteins (SREBPs) enhance transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis and uptake. In the current experiments, we observed a decline in the mRNA encoding one SREBP isoform, SREBP-1c, in the livers of rats that were rendered diabetic by treatment with streptozotocin. There was no change in the mRNA encoding SREBP-1a, which is derived from the same gene as SREBP-1c but uses a different promoter. The ratio of SREBP-1c:1a transcripts fell 25-fold from 5:1 in control rats to 0.2:1 in the diabetic animals. The SREBP-1c mRNA rose nearly to normal, and the 1c:1a ratio increased 17-fold when the diabetic rats were treated for 6 h with insulin. These treatments produced no change in the mRNA for SREBP-2, which is encoded by a separate gene. The SREBP-1c mRNA also fell selectively in freshly isolated rat hepatocytes and rose when the cells were treated with insulin. Considered together with recent data on hepatocytes [Foretz, M., Pacot, C., Dugal, I., et al. (1999) Mol. Cell. Biol. 19, 3760–3768], the current in vivo studies suggest that insulin may stimulate lipid synthesis in the liver by selectively inducing transcription of the SREBP-1c gene.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Lipotoxic heart disease in obese rats: Implications for human obesity

To determine the mechanism of the cardiac dilatation and reduced contractility of obese Zucker Diabetic Fatty rats, myocardial triacylglycerol (TG) was assayed chemically and morphologically. TG was high because of underexpression of fatty acid oxidative enzymes and their transcription factor, peroxisome proliferator-activated receptor-α. Levels of ceramide, a mediator of apoptosis, were 2–3 times those of controls and inducible nitric oxide synthase levels were 4 times greater than normal. Myocardial DNA laddering, an index of apoptosis, reached 20 times the normal level. Troglitazone therapy lowered myocardial TG and ceramide and completely prevented DNA laddering and loss of cardiac function. In this paper, we conclude that cardiac dysfunction in obesity is caused by lipoapoptosis and is prevented by reducing cardiac lipids.

Temporal distribution of the ganglion cell volleys in the normal rat optic nerve

We describe experiments on behaving rats with electrodes implanted on the cornea, in the optic chiasm, and on the visual cortex; in addition, two red light-emitting diodes (LED) are permanently attached to the skull over the left eye. Recordings timelocked to the LED flashes reveal both the local events at each electrode site and the orderly transfer of visual information from retina to cortex. The major finding is that every stimulus, regardless of its luminance, duration, or the state of retinal light adaptation, elicits an optic nerve volley with a latency of about 10 ms and a duration of about 300 ms. This phenomenon has not been reported previously, so far as we are aware. We conclude that the retina, which originates from the forebrain of the developing embryo, behaves like a typical brain structure: it translates, within a few hundred milliseconds, the chemical information in each pattern of bleached photoreceptors into a corresponding pattern of ganglion cell neuronal information that leaves via the optic nerve. The attributes of each rat ganglion cell appear to include whether the retinal neuropile calls on it to leave after a stimulus and, if so when, within a 300-ms poststimulus epoch. The resulting retinal analysis of the scene, on arrival at the cortical level, is presumed to participate importantly in the creation of visual perceptual experiences.

Sleep modifies retinal ganglion cell responses in the normal rat

Recordings were obtained from the visual system of rats as they cycled normally between waking (W), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Responses to flashes delivered by a light-emitting diode attached permanently to the skull were recorded through electrodes implanted on the cornea, in the chiasm, and on the cortex. The chiasm response reveals the temporal order in which the activated ganglion cell population exits the eyeball; as reported, this triphasic event is invariably short in latency (5–10 ms) and around 300 ms in duration, called the histogram. Here we describe the differences in the histograms recorded during W, SWS, and REM. SWS histograms are always larger than W histograms, and an REM histogram can resemble either. In other words, the optic nerve response to a given stimulus is labile; its configuration depends on whether the rat is asleep or awake. We link this physiological information with the anatomical fact that the brain dorsal raphe region, which is known to have a sleep regulatory role, sends fibers to the rat retina and receives fibers from it. At the cortical electrode, the visual cortical response amplitudes also vary, being largest during SWS. This well known phenomenon often is explained by changes taking place at the thalamic level. However, in the rat, the labile cortical response covaries with the labile optic nerve response, which suggests the cortical response enhancement during SWS is determined more by what happens in the retina than by what happens in the thalamus.