Ex vivo determination of opiate antagonist binding at mu-opioid ([3H]-DAGO) receptors in hypothalamic micropunches from maturing female rats: comparison between SDZ 210-096 and nalmefene.

We have previously shown that the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effect following peripheral injection of naloxone and naltrexone. In the present work, we have used this ex vivo technique to compare receptor occupancy of a new opiate antagonist, SDZ 210-096 (SDZ), to that of nalmefene (NLM) in maturing female rats. Two doses (SDZ, 5.6 and 50 mg/kg; NLM, 2.5 and 50 mg/kg) were injected subcutaneously into 3 groups of rats (infantile, juvenile and peripubertal). Micropunches from hypothalamic coronal slices (300 microns) were removed at various times post-injection for quantification of mu-opioid receptors with [3H]-DAGO. Acute administration of the lower dose of SDZ inhibited ligand binding almost completely by 3 h but 50% recovery was observed in all age groups by 12 h. In contrast, SDZ 50 mg/kg provided 80-100% antagonism for at least 24 h. Age-related differences in the ability of SDZ to inhibit [3H]-DAGO binding were observed in that hypothalamic mu-opioid receptors were blocked for longer periods in younger rats. Determination of receptor occupation following NLM injection confirmed that it too has prolonged duration of action but a 24 h blockade is not achieved with either dose of this antagonist. Age-related and dose-related changes in receptor occupancy were minimal compared to SDZ. These studies clarify the interaction of these antagonists at hypothalamic mu-opioid receptors and provide information which allows a clearer interpretation of results in experiments involving opioid blockade.

Growth, free plasma and muscle amino-acids in uraemic rats fed various low-protein diets.

The nutritional effects of low-protein diets are difficult to assess in humans. Normal and uraemic growing rats were therefore fed: a moderately low-protein (12%) reference diet (diet R), two 5% casein diets, one supplemented with essential amino acids (AA) (diet A) and the other with their keto acids (diet K), and a 7% casein diet isonitrogenous with diet K (diet L). Appetite and growth of both uraemic and control rats were identical on diets R and A and were reduced on diets K and L. Stunting was prominent in rats fed diet L and more severe than in those on diet K. Diet K induced marked anorexia in controls. This effect was smaller in uraemic rats, which were all anorectic, regardless of the diet. Plasma essential AA were similar in rats on diets R and A but low in control rats fed diets L and K. In particular, diet K did not improve the branched-chain AA levels although it produced better growth than diet L. Plasma and muscle threonine were surprisingly elevated in rats on the semi-synthetic diets A and K, despite identical or lower consumptions. Regardless of the diet, uraemia resulted in unchanged or increased plasma essential AA, despite reduced appetite and stunting. Uraemia caused a marked rise in some non-essential AA. Muscle essential AA, except for threonine, were essentially unaltered and did not correlate with growth or uraemia.

Effects of prenatal exposure to methylazoxymethanol (MAM) on brain weight, hypothalamic cell number, pituitary structure, and postnatal growth in the rat.

Congenital brain damage syndromes typically are described in terms of behavioral symptoms. Many brain functions are not reflected in behavior, however, and prenatal injury to the developing nervous system could alter these functions, as well. To test the hypothesis that prenatal brain injury can result in postnatal endocrine malfunction, rats were exposed in utero to 20 mg/kg of methylazoxymethanol acetate, a potent neuroteratogen, at two stages of gestation when different sets of growth-controlling neurons of the hypothalamus are forming. The growth hormone releasing factor (GRF) neurons stimulate release of growth hormone from the somatotropes of the anterior pituitary, contributing to rapid growth in the period between weaning and puberty. The somatotropin release inhibiting factor (SRIF) neurons have the opposite effect on the pituitary and can inhibit the GRF cells directly. Growth of treated animals was monitored daily from birth to 40 days and compared to that of controls. Treatment on the 14th day of gestation produced a small number of dwarf animals characterized by normal weight at birth and a sudden decrease in growth rate at the beginning of the fourth postnatal week that led to a body weight about 50% of normal. Treatment on day 16 yielded an acceleration of postnatal growth (significant in males). In each group, most treated animals were like controls in adult size and pattern of growth. As adults, both treatment groups demonstrated massive reductions in brain weight which characterized all the subjects, whether or not they exhibited growth anomalies. The animals treated on day 14 were confirmed to have a significant, selective reduction in growth hormone releasing factor neurons. Reductions were greatest in the middle and posterior levels of the GRF cell distribution, the regions forming most actively at the time of exposure. Unexpectedly, the same group also had increased numbers of periventricular SRIF neurons. Neither type of neurons was significantly altered in the later treatment group. Examination of pituitary structure indicated that dwarfs had very small pituitaries, with an immature pattern of somatotrope distribution, and giants had very large pituitaries, with some hypertrophy of somatotropes. The results suggest that endocrine anomalies which manifest themselves long after birth may originate as birth defects of the nervous system.

Effects of acarbose on the development of diabetes in BB/Wor rats.

This study was designed to determine whether the addition of acarbose (a dissacharidase inhibitor) to the diet can prevent or delay the development of diabetes in the BB/Wor rat. Acarbose reduces postprandial glucose and insulin levels in normal and diabetic rats by delaying the absorption of carbohydrates. Data by others suggested that reduced activity of beta cells renders them less prone to immunological attack. It was speculated that feeding acarbose to diabetes susceptible BB/Wor rats may similarly reduce islet immunogenicity by reducing the burden on the beta cells, and decrease the incidence of disease. Acarbose was added to regular chow and chow containing 15% sucrose. The rationale for using chow containing sucrose was to increase the effectiveness of acarbose, since sucrose is one of the primary carbohydrates whose digestion is blocked by acarbose. Diabetes-prone BB/Wor rats were fed either regular chow (A;N = 30), regular chow plus acarbose at 40 mg/100 g chow (B;N = 30), 15% sucrose-containing chow (C;N = 30), or 15% sucrose-containing chow plus acarbose (D;N = 30). Oral chow tolerance tests, performed using 500 mg of chow, confirmed a reduction in plasma glucose and insulin values in acarbose-fed animals. The mean incidence of diabetes in groups A through D was 87%, 73%, 87%, and 93%, respectively (not significant). The mean (+/- SEM) ages (days) of onset of diabetes in animals fed diets A through D were 88 +/- 2.1, 90 +/- 3.2, 86 +/- 1.9, and 91 +/- 2.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Desenvolvimento pós-natal de crias de ratas tratadas com decocto de sucuuba Himathantus sucuuba (Spr.) wood / Post natal development of rat pups from sucuuba (Spr.) wood decoction treated mothers

O trabalho foi planejado para avaliar o desenvolvimento pós-natal de crias de ratas, tratadas com decocto de sucuuba (40 mg/dia/rata/via oral) do 6§ ao 15§ dia de gravidez. Os animais foram colocados em gaiolas individuais, no 20§ dia de gestaçäo, de modo que se pudesse acompanhar, em cada rata, o desenvolvimento da ninhada. Foram estabelecidos os seguintes parâmetros: tamanho de cria, peso ao nascimento, aos 4, 10 e 30 dias de vida, índice de crescimento e de mortalidade neonatal e sinais de desenvolvimento físico. Apesar de se ter observado atraso de um dia na abertura dos olhos, erupçäo de incisivo inferior e aparecimento de lanugo, tais alteraçöes näo parecem ter significado biológico importante, levando a que se considere que o decocto de sucuuba, administrado a ratas grávidas näo altera o desenvolvimento pós-natal da cria.

The toxicity of p-aminophenol in the Sprague-Dawley rat: effects on growth, reproduction and foetal development.

p-Aminophenol (p-AP) was fed in the diet to groups of 40 male and 45 female Sprague-Dawley rats at levels of 0.07, 0.2 or 0.7% for up to 6 months. Methaemoglobin levels were determined after 6 wk. During wk 12, urine was collected from ten rats/sex/group for evaluation of mutagenicity in the Ames test. Clinical chemistry, haematology and histopathology studies were performed in subgroups after 13 and 27 wk. In addition, after 13 wk, 25 females/group were mated to untreated males in a teratology study. After 20 wk, 20 males/group were removed from the test diets and mated to untreated virgin females in a dominant lethal mutagenicity study. These males remained untreated until they were killed at 27 wk. Rats that had been maintained on the test diets throughout the study were also killed at wk 27. The high dose level of 0.7% p-AP resulted in a significant (10-15%) reduction in body-weight gain in both sexes. There was no increase in the level of methaemoglobin and, other than slight reductions in total erythrocytes and haemoglobin in female rats at 13 wk, there were no toxicologically important differences between groups in haematology or clinical chemistry values at any time during the 27 wk of treatment. Dose-related nephrosis was seen in both sexes after 13 and 27 wk and in the high-dose males that were removed from the test diet for a 7-wk recovery period. The compound was not teratogenic, but an increase in developmental variations associated with maternal toxicity was noted at the mid- and high-dose levels. In the dominant lethal study, an increase in the total number of resorptions (but not litters with resorptions) was observed in the high-dose group in the first of two matings but this observation was not confirmed in a follow-up study. Mutagenic activity was not detected in the urine of rats fed p-AP.

Spatial and temporal patterns of oligodendrocyte differentiation in rat cerebrum and cerebellum.

Oligodendrocytes are largely generated postnatally during mammalian CNS development. We have used a variety of antibodies to label immature neuroectodermal cells and developing oligodendrocytes in several areas of the rat CNS. Antibodies included those to GD3 ganglioside, a characteristic glycolipid of immature cells; carbonic anhydrase (CA), contained primarily in oligodendrocytes; and galactocerebroside and myelin basic protein, myelin components. Several aspects of oligodendrocyte development were examined: changes in shapes of immature cells with respect to time and to location within the brain, the sequential acquisition of the various markers, and possible sites of origin and pathways of precursor cell migration. Our observations suggest that oligodendrocytes in the forebrain and cerebellum arise from cells of the subventricular zone (SVZ) adjacent to the ventricles and migrate into and through nearby white and gray matter. During maturation, there are distinct patterns of morphological changes that correlate with time, locations of the cells in the brain, and acquisition of specific markers.

Effects of calcium deprivation on n-6 fatty acid metabolism in growing rats.

Two separate experiments examining the effects of calcium deficiency on plasma and liver fatty acids in rats were conducted. In Experiment I, weanling male Sprague-Dawley rats were fed a calcium-deficient diet with or without the supplementation of 5 or 20 g/kg calcium for 22 days. There were no significant differences in plasma and liver fatty acid distribution between the two calcium-supplemented groups. However, calcium deficiency significantly elevated the levels of 18:3n-6 in plasma and liver cholesteryl esters and liver phospholipids, while it reduced the levels of 20:3n-6 in plasma cholesteryl esters. In Experiment II, weanling rats were fed a calcium-deficient diet supplemented with 5 g/kg calcium for 22 days. After overnight fast, animals were given by intragastric feeding a dose of 4 g/kg body wt gamma-linolenic acid concentrate (containing 92% 18:3n-6 ethyl ester), and were killed 22 hr later. The levels of 18:3n-6 were significantly higher, whereas the levels of 20:3n-6 were either not changed or lower than those in calcium-supplemented group. In both experiments, the ratios of (20:3n-6 + 20:4n-6)/18:3n-6 in plasma and liver lipids were significantly reduced in calcium-deficient rats. These results suggest that calcium may play an important and specific role in the process of elongation of 18:3n-6 to 20:3n-6.

Neurotransmitters and neuropeptides in blood pressure regulation in the spontaneously hypertensive rat.

Studies of the roles played by neurotransmitters in the development of hypertension in the spontaneously hypertensive (SHR) rat are complicated by the presence of genetic differences between SHR and normotensive control rats, which are not related to differences in blood pressure. One approach that may be used in an attempt to overcome this difficulty is to study the manner in which neurotransmitter and metabolite levels change with age, and to relate these changes to alterations in blood pressure with ageing. Noradrenaline (NA) levels in the brainstem and spinal cord of SHR and Wistar Kyoto rats fell with age, while 3,4-dihydroxyphenylethyleneglycol (DHPG) levels (a neuronal metabolite of noradrenaline) remained constant. Similar changes were seen when NA and DHPG levels were measured in the discrete brainstem A1, A2, and C2 region, and when adrenaline, NA, and DHPG levels were examined in the C1 region. Differences in age-related changes of neuropeptide Y (NPY) levels were also found in the ventromedial nucleus of the hypothalamus and the locus coeruleus, and of beta-endorphin in the anterior hypothalamic nucleus, the paragigantocellular nucleus of the brainstem, and the locus coeruleus. These changes may indicate either a progressive increase in the activity of neurons in the sympathoexcitatory C1 region or a progressive reduction in the activity of vasodepressor A1, A2, and C2 regions with ageing, or both. However, changes in catecholamines and metabolites with age were similar in both strains and therefore cannot readily explain the more rapid rise in blood pressure with ageing in SHR rats.

Postnatal development of TRH and TSH rhythms in the rat.

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.

Valorization of rapeseed meal. 5. Effects of sinapine and other phenolic compounds on food intake and nutrient utilization in growing rats.

Six groups of 10 growing rats each were fed ad libitum for 15 days one of six diets: diet A, rapeseed (3.80 g of sinapine/kg DM); diet B, ethanol/water-extracted rapeseed (0.48 g of sinapine); diet C, control diet; diet G, control diet + 3.74 g of extracted sinapine; diet H, control diet + 3.72 g of sinapine + other phenolic compounds; or diet I, control diet + the hydrolysis products of sinapine and other phenolic compounds. During the first 8 days, the dry matter intake and live weight gain of the rats were significantly reduced by the intake of sinapine and other phenolic compounds. However, after this adaptation period their performances were similar to those of the control group. Dry matter, energy and nitrogen digestibility and protein utilization were not altered by sinapine intake but were slightly reduced with diets H and I.

Valorization of rapeseed meal. 4. Effects of iodine, copper and ferrous salt supplementation in growing rats.

Ten groups of 10 growing male rats (65 g) were fed ad libitum either a control diet or rapeseed diets (15 mM glucosinolates/kg DM) supplemented either with l- alone, l- and Cu++ (2 levels), l- + Cu++ and methionine or l- and Fe++ (2 levels) (table 1) for 35 days. When the rapeseed diets were given as powder or as mash, food intake decreased 24 and 32%, respectively; live weight gain was reduced by 36 and 46%, liver weight increased 20 and 33%, thyroïd gland was 3 and 4 times as big, and a 30 and 50% decrease in the T4 plasma level was noted (table 2). l- supplementation did not prevent the deleterious effects of rapeseed meal feeding. l- and Fe++ supplementation had detrimental effects on food intake, live weight gain and kidney weight, but beneficial effects on liver and thyroïd weights and on plasma thyroïd hormone levels. The low level of l- and Cu++ supplementation suppressed the depressive effects of rapeseed meal on food intake and live weight gain and reduced the deleterious effects on kidney, liver and thyroïd weights and on plasma thyroïd hormone levels, whereas the higher level almost suppressed them. The results are discussed considering the likelihood of a bivalent cation effect on glucosinolates.

Immunoassayable adrenocorticotropin in peripheral organs: concentrations during early development.

Although many have identified immunoassayable adrenocorticotropin (ACTH) in sites outside the pituitary (brain, gastrointestinal tract), there is relatively little information regarding immunoassayable ACTH in other major peripheral organs. Several major peripheral organs (pancreas, liver, kidney, heart) of rats were found to contain variable amounts of immunoreactive (IR-) ACTH which appeared to be authentic IR-ACTH on the basis of parallelism to ACTH1-39 upon serial dilution of extracts and gel filtration chromatography. Concentrations of IR-ACTH in peripheral organs were also studied to determine if changes occur during early development. Concentrations of IR-ACTH did not show significant changes in liver and heart at various ages between 10 and 80 days, but IR-ACTH in pancreas and kidney (day 10 vs. 80) did show significant decrements with aging.

Developmental changes in the phosphaturic response to parathyroid hormone in the rat.

The need for young, immature rats to maintain positive phosphate balance for growth is well recognized. However, whether this process is associated with a resistance to the phosphaturic effect of parathyroid hormone (PTH) is not clear. In these experiments we examined the effect of PTH on urinary phosphate and cAMP excretion in rats at 3, 6, 12, and 20 wk of age. Clearance experiments were performed in acutely thyroparathyroidectomized (TPTX) rats fed a normal phosphate diet (0.86%). Basal fractional excretion of phosphate (FEPi) was low in all TPTX rats (less than 1%). The phosphaturic response to a high dose of PTH (1 U X kg-1 X min-1) increased with development (from 4 to 29%). The responses to increasing doses of PTH demonstrated a decrease in sensitivity to PTH in 6- compared with 20-wk-old rats. Urinary cAMP excretion (either per milliliter glomerular filtrate or per gram kidney weight) following PTH was not different among 6-, 12-, and 20-wk-old rats, thus demonstrating a dissociation between the increase in phosphate excretion and cAMP excretion. These results indicate that the phosphaturic response to PTH is blunted in immature, acutely TPTX rats and that the phosphaturia increases progressively with development.

The effect of protein levels on the response of weanling rats to dietary pectin.

We have previously proposed that the inhibiting effects of pectin on feed consumption and growth might be due in part to the reduction it causes in protein digestibility. The present work was intended to test this hypothesis by feeding higher levels of protein in order to compensate for the lower digestibility. We fed diets containing 10, 20 and 30% casein (initial levels) and 0, 4.8, 16.7 and 28.6% pectin (by diet dilution). Despite the higher casein levels, pectin strongly inhibited the animals' feed intake, growth, protein efficiency ratio (PER), net protein ratio (NPR) and net protein utilization (NPU) as well as protein and energy digestibilities. Apart from determining the base (zero pectin) level of the various parameters measured, the dietary casein level in itself did not influence the effect of pectin on feed intake, digestible energy intake, protein digestibility, body weight growth or parameters of protein utilization such as PER, NPR and NPU. We thus cannot conclude that these effects of pectin were to any major extent secondary to the reduction in protein digestibility observed in these animals.

[Effect of diets supplemented with pork lard and vitamin C on cholesterol and triglyceride levels in young rats]. / Efecto de las dietas suplementadas con grasa de cerdo y vitamina C sobre los niveles de colesterol y triglicéridos en ratas jóvenes.

Plasma cholesterol and triglycerides in young female rats fed for 5 weeks on three experimental diets containing vitamin C and/or lard in comparison with the commercial pellet diet, were studied. The rats fed vitamin C showed no change of body weight whilst the animals fed lard gained in body weight. The variations in comparison with control are statistically significant. Plasma levels, cholesterol and triglycerides increased significantly in rats fed lard supplements. The cholesterol and triglycerides content did not change significatively with supplementary ascorbic acid.

Muscle protein turnover in rats fed on diets containing different levels of Vicia faba L. and casein as source of protein.

The effects of intake of Vicia faba L. and casein as the sources of protein on protein synthesis and breakdown were investigated. The results showed that the protein deposition as a percentage of body weight as well as the muscle and myofibrillar nitrogen content are not altered by the experimental diets. The values of myofibrillar gain (mg N2/day) were higher in the animals fed on casein diets than those fed on legume protein at both levels (12 and 18%) while no differences were found in the myofibrillar protein breakdown assessed by the urinary excretion of 3-methylhistidine. Therefore, it is suggested that the stunting of growth is not due to an increased protein degradation, but an impaired protein synthesis.

[Nutritional correction of an excess of unsaturated fatty acids in the body lipids of the growing rat]. / Correction par voie nutritionnelle d'un excès d'acides gras insaturés dans les lipides corporels du rat en croissance.

The use of hydrogenated coconut oil as a putative substance for correcting an excess of unsaturated fatty acids in reserve lipids was tested in growing rats. Young rats with a live weight of about 70 g were given diet A including 4% of sunflower oil which very rapidly created a triglyceride store containing 25 to 30% of linoleic acid (18:2 n-6). At different weights (200, 300 and 350 g), diet A was replaced by diet B containing 15% of hydrogenated coconut oil so that there was the same number of animals in each group. All the rats were killed at 400 g, and diet-A and B were compared as to final fatty acid composition of body lipids (i.e. values taken all during growth from 70 to 400 g). Hydrogenated coconut oil was a very effective substance for rapidly decreasing levels of reserve lipid linoleic acid and for increasing triglyceride melting-point. The impact on the decrease in total unsaturated fatty acid concentrations was more marked the earlier the diet was replaced. However, the latest replacements (at 300 and 350 g) still permitted 70 and 50%, respectively, of the maximal effect observed in rats eating diet B from 70 g. The present experiment shows that when coconut oil was introduced in the diet over a period corresponding to the last one-eight of total body growth in rat, the final concentrations of unsaturated fatty acids could still be considerably reduced, particularly that of linoleic acid. In the same way, there was increasing esterification of myristic acid (14:0), and especially of lauric acid (12:0), which appeared preferentially in sn-1 and sn-3 positions in triglycerides. This esterification of medium-chain fatty acids led to a modification in the mode of other fatty acid distribution. Analysis of liver phospholipids showed that when diet A was replaced above 200 g by diet B, there was no biochemical evidence of any deficiency of essential fatty acids of the n-6 series.

Effect of excess L-lysine on rat growth and on plasma and tissue concentrations of copper, iron and zinc.

A 28-day feeding study was conducted to test the effect of excess dietary lysine on rat growth and the concentration of copper, iron and zinc in plasma and liver. Young male Sprague-Dawley rats were fed a 10% protein casein diet with or without excess lysine. There were no significant differences in body weight gain, food intake or plasma proteins among the dietary treatment groups. Supplementation of the basal diet with 2.1% L-lysine caused a 53% reduction in hepatic copper and a significant reduction in hepatic iron. The addition of 0.7% or 2.1% lysine to the basal diet caused significant reductions in levels of plasma copper. The 2.1% level of lysine tended to lower the concentration of zinc in plasma. The data suggest that lysine may interfere with the availability of selected minerals by reducing tissue utilization or promoting excretion, or both.