Patricia M. Boyer

Bone status in an animal model of chronic sub-optimal nutrition: a morphometric, densitometric and mechanical study.

In children, inappropriate eating habits can induce a disease known as nutritional dwarfing (ND). Due to the link between nutritional condition and bone growth, the effects induced by a 20 % reduction of food intake on bone competence were assessed in an animal model of ND. Bone status during catch-up growth was also analysed. Male Wistar rats were divided into control (C) and ND groups. C rats were fed ad libitum. ND received 80 % of the diet consumed by C for 4 weeks (T4); thereafter, they were fed ad libitum for 8 weeks. Results, expressed as mean (SEM) for ND v. C, were as follows. At T4, body weight (g) and length (cm) and femur weight (g) and length (mm) were 97.35 (SEM 5.89) v. 199.07 (SEM 9.24), 16.91 (SEM 0.41) v. 20.26 (SEM 0.31), 0.30 (SEM 0.01) v. 0.46 (SEM 0.01) and 23.09 (SEM 0.29) v. 26.98 (SEM 0.26), respectively (P<0.001); bone mineral content (g) and density (g/cm(2)) were 0.014 (SEM 0.002) v. 0.030 (SEM 0.002) and 0.061 (SEM 0.004) v. 0.080 (SEM 0.003), respectively (P<0.001); load-bearing capacity (N), yielding load (N) and elastic stiffness (N/mm) were 25.06 (SEM 1.24) v. 50.34 (SEM 2.94), 23.72 (SEM 1.02) v. 46.97 (SEM 1.75) and 65.98 (SEM 4.42) v. 115.07 (SEM 3.85), respectively (P<0.001); cross-sectional area (mm(2)) and moment of inertia (mm(4)) were 2.86 (SEM 0.19) v. 4.54 (SEM 0.17) and 1.27 (SEM 0.08) v. 3.03 (SEM 0.16), respectively (P<0.001). Significant effects were not evident in material properties. Parameters assessed normalized during re-feeding. These results suggest that the impaired mechanical femur competence in ND rats could be due to an altered bone mass and architectural distribution rather than to intrinsic quality. Re-feeding caused a reversal of the effects of food restriction on growth and bone parameters in ND rats.

Effect of Nutritional Stress on the Hypothalamo-Pituitary-Gonadal Axis in the Growing Male Rat

Background/Objective: Nutritional dwarfing (ND) consists of a decrease in weight and height gain and delayed onset of puberty. The aim of the present investigation was to study the modifications induced in male rats by the nutritional stress of a mere 20% reduction in food intake which, however, started immediately after weaning. Materials and Methods: At weaning, male Wistar rats were divided into two groups: Control (C) and ND. C rats were fed ad libitum with a balanced rodent diet. ND received 80% of the diet consumed by C for 4 weeks (T4); then they were fed ad libitum for another 4 (T8) and 8 weeks (T12). The rats were studied at T0, T4, T8 and T12 for the effects of nutritional stress and refeeding on nutritional status, body composition, hypothalamic-pituitary-gonadal axis, and sperm morphology and concentration. Results: ND body weight and length diminished vs. C (p < 0.001). ND body fat percentage decreased 40% (p < 0.001) without change in the percentage of body protein content. The hypothalamic content of LHRH did not change. However, FSH, LH and testosterone serum levels had significantly decreased (p < 0.001) at T4 in ND rats. A 48.4 % decrease in serum leptin in the ND group was observed at T4 (p < 0.05). The absolute testicular and seminal vesicle weight was significantly decreased by ND at T4 (p < 0.001). At T4 the percentage of anomalies of caudal spermatozoa increased in about 64% (p < 0.001) of ND vs. C rats, despite the unchanged sperm concentrations. All parameters normalized during refeeding. Conclusion: In this model, a decrease in leptin due to nutritional stress could be responsible, at least in part, for the inhibition of reproductive function. Refeeding normalized all parameters studied.

Growth deceleration and bone metabolism in nutritional dwarfing rats

Nutritional status as well as energy and protein intake are critical regulators of IGF1 and IGFBP-3 and contribute to the modulation of bone remodeling and formation. The purpose of this study was to investigate on an experimental model with nutritional dwarfing (ND), whether the alterations on body growth velocity, energy metabolism and body composition could affect serum concentrations of IGF-1 and IGFBP-3 and bone (tibiae and mandible) histology and histomorphometry. Twentyone male weanling Wistar rats (body weight = 38.20 ± 0.94 g) were randomized to three groups: seven of them were killed at day = 0 (CO, n = 7); control (C, n = 7); and experimental 80 (E80, n = 7). During 4 weeks, C was fed ad libitum with a 1: 1 carbohydrate to fat diet. E80 was being underfed with the same diet by 80% and the following parameters were measured: weight (Wt) for length (L) ratio z-score; oxygen consumption (VO 2); body composition (BC) by EM-SCAN SA 3000. At t = 28, E80 and C were killed. Serum IGF-1 and IGFBP-3 and bone histology and histomorphometry were performed on C0, E80 and C. E80 showed Wt for L z-score between lean and adequate, a decrease in VO 2 according to body proportions, a BC of a delayed puberty individual, IGF-1 and IGFBP-3 decreased by 56 and 53%, respectively. Tibiaes hematopoyetic and adipose bone marrow areas were combined, with sealing trabeculae on metaphyseal areas. This study suggests that there is a relationship among growth deceleration in ND rats and structural alterations on tibiae.

Improved bone status by the β-blocker propranolol in an animal model of nutritional growth retardation

The aim of the present research was to study if the beta-blocker propranolol, which is known to increase bone mass, could reverse the adverse skeletal effects of mild chronic food restriction in weanling rats. Male Wistar rats were divided into four groups: control, control+propranolol (CP), nutritional growth retardation (NGR) and nutritional growth retardation+propranolol (NGRP). Control and CP rats were fed freely with the standard diet. NGR and NGRP rats received, for 4 weeks, 80 % of the amount of food consumed by the control and CP rats, respectively. Results were expressed as mean values and sem. Food restriction induced detrimental effects on body and femur weight and length (P < 0.05) and bone structural and geometrical properties (P < 0.001), confirming results previously shown in our laboratory. However, the beta-blocker overcame the deleterious effect of nutritional stress on load-bearing capacity, yielding load, bone stiffness, cross-sectional cortical bone area and second moment of inertia of the cross-section in relation to the horizontal axis without affecting anthropometric, histomorphometric and bone morphometric parameters. The results suggest that propranolol administration to mildly chronically undernourished rats markedly attenuates the impaired bone status in this animal model of growth retardation.

Atherogenic cholesterol-rich diet and periodontal disease

OBJECTIVE This study investigated the effect of an atherogenic cholesterol-rich diet (AT) on the alveolar bone loss in rats with ligature-induced experimental periodontitis (EP). METHODS Female Wistar adult rats were assigned either a control (Co) or an AT diet fed for 9 weeks. The AT diet was high in saturated fat, cholesterol and energy. At week 2, animals were subjected to a unilateral ligature (L) around the left first molar (Co+L and AT+L). The contra lateral first right molar (not ligated) of both groups (Co and AT) were used as untreated controls. At week 9, blood was drawn, rats were euthanized, hemi-mandibles removed and stained digital photographs (buccal and lingual surfaces) and radiographs were obtained for quantification of alveolar bone loss (ABL). The ABL was determined by distance and area methods (mm(2)) and X-rays were used for periodontal bone support (PBS), (%). RESULTS Rats in the AT group exhibited a 17% increase in energy intake, gained significant body weight and showed the highest serum total-cholesterol (T-C) and non-high density lipoprotein-cholesterol (HDL-C) levels (p<0.001). The amount of lost periodontal bone was the greatest in AT+L rats. AT feedings significantly increased the buccal area and distance of bone loss when compared with the unligated-teeth (p<0.001). The rats in the AT+L group also achieved the lowest percentage of PBS (p<0.001). The AT and Co+L rats showed similar PBS. This method more clearly elucidated the effect of the cholesterol-rich AT, with and without the influence of molar ligature, compared to the morphometric analysis. CONCLUSION The alveolar bone loss of EP was magnified by ingestion of an atherogenic diet high in saturated fatty acids and cholesterol.

Hypothalamic Noradrenergic Hyperactivity and Detrimental Bone Status in an Animal Model of Nutritional Growth Retardation

We have studied hypothalamic noradrenergic activity in relation with bone status in a nutritional growth retar- dation model (ND). Control rats (C) were fed ad libitum. ND received 80% of the diet consumed by C for 4 weeks and later refed ad libitum for 8 weeks. Food restriction induced detrimental effects on body and femur weight and length (P<0.05) and bone biomechanical properties (P<0.001). Thickness of proliferative and hypertrophic zone (�m) of growth plate cartilage and bone volume (%, mean±SE) were 225.96±5.70 v. 280.70±12.52, 95.16±5.81 v. 134.60±9.30, 17.64±3.23 v. 26.80±2.03, respectively (P<0.05); anterior and posterior hypothalamus norepinephrine uptake and release and tyrosine hydroxylase activity (% of control) were 79.05±3.56, 67.00±10.00, 164.26±16.58 and 80.65±5.92, 147.00±1.00, 152.42±9.30, respectively (P<0.05). Thus, impaired biomechanical bone performance in ND could be due, in part, to the increased hypothalamic noradrenergic activity in response to restriction. Normalization of parameters with refeeding suggests no long-term side-effects in undernourished rats.

Influence of dietary calcium concentration on body size and bone composition in rats during recovery from malnutrition

OBJECTIVE The purpose of our study was to assess the influence of different levels of calcium (Ca) in a diet containing 30% protein on the rehabilitated of the body size from protein-energy malnutrition (PEM) and to establish the optimal Ca/protein ratio for attaining a normal body composition. METHODS Weanling female Wistar rats were fed with protein-free diet up to a weight deficit of 20 +/- 1%. Then they were arranged in groups (TO) and fed diets with 30% protein and 0.0, 0.2, 0.4, 0.6, 0.9 or 1.2% Ca for 28 days (T28). Food and deionized water were given ad libitum. Body weight and length were recorded every 3 days. At T28, the animals were sacrificed to determine femur composition. RESULTS At T13, weight-for-age (W/A) was within the normal range for rats consuming > or = 0.6% Ca. At T28 all groups showed adequate W/A. Although length-for-age was adequate during rehabilitated period, rate of weight gain improved when Ca was > or = 0.6%. Femur length did not show significant difference between groups. Total femur Ca content and mg Ca/g of dry-weight tissue increased with increments in dietary Ca concentration and tended to plateau with 0.4% Ca. Ca/P ratio reached the highest value with 0.9% Ca. CONCLUSIONS Our findings indicate that at a dietary protein level of 30% the Ca/protein ratio is a limiting factor in attaining of normal body size; this is achievable when Ca concentration is 1.2% and the Ca/protein ratio is 0.04.

Hypophagia-induced hypometabolism during experimental anaemia in rats

Several investigators have reported a drop in oxygen (O2) consumption (VO2) and body temperature in laboratory animals during normobaric or hypobaric hypoxia. Hypophagia, with normal efficiency of protein utilisation for growth, was also observed. It has recently also been observed that hypometabolism is present during anaemic hypoxia. The present study was designed to test the experimental hypothesis that anaemic hypoxia induces hypometabolism secondary to hypophagia. Episodes of anaemia were created in adult male rats by either blood withdrawal through cardiac puncture (haemorrhagic anaemia) or phenylhydrazine administration (haemolytic anaemia). Haematrocrit, VO2, and food consumption, as indirect estimations of the level of anaemia, energy production, and appetite, respectively, were serially measured in all animals during 7 days (acute experiments) or 17 days (chronic experiments). Positive correlations were found between the three parameters during development of and recovery from anaemia during each anaemic episode. When the amount of food offered to non-anaemic rats was equalised to that freely eaten by anaemic rats, VO2 dropped in the former to almost the level found in the latter. Body composition changed during chronic anaemia because of a decrease in the lipid fraction of the body. The results confirmed the working hypothesis that hypometabolism, which has been considered as an immediate, emergency-type response to both hypoxic and anaemic hypoxia, can be considered as a response secondary to hypophagia because of depressed appetite. How appetite is adapted to the mechanisms which control O2 convection and O2 availability is not known at present.

Efficacy of phytosterols and fish-oil supplemented high-oleic-sunflower oil rich diets in hypercholesterolemic growing rats.

Abstract Phytosterols (P) and fish-oil (F) efficacy on high-oleic-sunflower oil (HOSO) diets were assessed in hypercholesterolemic growing rats. Controls (C) received a standard diet for 8 weeks; experimental rats were fed an atherogenic diet (AT) for 3 weeks, thereafter were divided into four groups fed for 5 weeks a monounsaturated fatty acid diet (MUFA) containing either: extra virgin olive oil (OO), HOSO or HOSO supplemented with P or F. The diets did not alter body weight or growth. HOSO-P and HOSO-F rats showed reduced total cholesterol (T-chol), non-high-density lipoprotein-cholesterol (non-HDL-chol) and triglycerides and increased HDL-chol levels, comparably to the OO rats. Total body fat (%) was similar among all rats; but HOSO-F showed the lowest intestinal, epididymal and perirenal fat. However, bone mineral content and density, and bone yield stress and modulus of elasticity were unchanged. Growing hypercholesterolemic rats fed HOSO with P or F improved serum lipids and fat distribution, but did not influence material bone quality.

Monounsaturated fatty acids-rich diets in hypercholesterolemic-growing rats.

Abstract The effects of replacing dietary saturated fat by different monounsaturated fatty acid (ω-9MUFA) sources on serum lipids, body fat and bone in growing hypercholesterolemic rats were studied. Rats received one of the six different diets: AIN-93G (control, C); extra virgin olive oil (OO) + C; high-oleic sunflower oil (HOSO) + C or atherogenic diet (AT) for 8 weeks; the remaining two groups received AT for 3 weeks and then, the saturated fat was replaced by an oil mixture of soybean oil added with OO or HOSO for 5 weeks. Rats consuming MUFA-rich diets showed the highest body fat, hepatic index and epididymal, intestinal and perirenal fat, and triglycerides. T-chol and non-HDL-chol were increased in HOSO rats but decreased in OO rats. Bone mineral content and density were higher in both OO and HOSO groups than in AT rats. This study casts caution to the generalization of the benefits of MUFA for the treatment of hypercholesterolemia.