Rosa M. Alippi

Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible

Morphological and biomechanical features of the mandible are negatively affected by protein-energy malnutrition, whose effects are apparently dependent on the time of life of application. The aim here was to investigate, in neonatal rats nursed by dams put on a protein-free diet to depress milk production and thus create a state of protein-energy malnutrition in the offspring, subsequent growth and long-term effects by analyzing mandibular dimensions and bone quality in adulthood. Pregnant Wistar rats were fed a 20% protein diet (control) or a protein-free diet (malnourished) to obtain normal or subnormal milk production, respectively. After weaning, the offspring (males) were fed a 20% protein diet for 70 days. The dimensions of their excised mandibles were measured directly between anatomical points; the geometry and material quality of mandibular bone were assessed by peripheral quantitative computed tomography. Pups suckling from malnourished dams weighed 49.4% of those suckling from control dams at weaning; the actual difference between control and malnourished pups was 25.1g, which persisted until day 91 of age, indicating the absence of catch-up growth. As with body size, the mandibular base length, height and area (an index of mandibular size) were significantly smaller in malnourished than control rats at the end of the study. The mandibular cortical area, volumetric cortical bone mineral content and volumetric cortical bone mineral density assessed by peripheral quantitative computed tomography were similar in both groups of rats at the end of the observation period, but there was a significant reduction in the cortical axial moment of inertia in malnourished rats at this time of postnatal life. These findings suggest that catch-up growth was incomplete in rats malnourished during the suckling period and that the adaptation of mandibular bone architecture to body growth was apparently insufficient to attain normal values, thus not allowing complete compensation in mechanical competence at the end of the study because of an inadequate spatial distribution of resistive material through its cross-section rather than qualitative or quantitative impairment of cortical bone.

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.

Failure of Polycythemia-Induced Increase in Arterial Oxygen Content to Suppress the Anorexic Effect of Simulated High Altitude in the Adult Rat

The anorexic effect of exposure to high altitude may be related to the reduction in the arterial oxygen content (Ca(O2)) induced by hypoxemia and possibly the associated decreased convective oxygen transport (COT). This study was then performed to evaluate the effects of either transfusion-induced polycythemia or previous acclimation to hypobaria with endogenously induced polycythemia on the anorexic effect of simulated high altitude (SHA) in adult female rats. Food consumption, expressed in g/d/100 g body weight, was reduced by 40% in rats exposed to 506 mbar for 4 d, as compared to control rats maintained in room air. Transfusion polycythemia, which significantly increased hematocrit, hemoglobin concentration, Ca(O2), and COT, did not change the anorexic response to the exposure to hypobaric air. Depression of food intake during exposure to SHA also occurred in rats fasted during 31 h before exposure and allowed to eat ad libitum for 2 h during exposure. Body mass loss was similar in 48-h fasted rats that were either hypoxic or normoxic. Body mass loss was similar in normoxic and hypoxic rats, the former eating the amount of food freely eaten by the latter. Hypoxia-acclimated rats with endogenously induced polycythemia taken to SHA again had diminished food intake and lost body mass at rates that were very close to those found in nonacclimated ones. Exposure to SHA also led to a decrease in food consumption, body weight, and plasma leptin in adult female mice. Analysis of data suggest that body mass loss that accompanies SHA-induced hypoxia is due to hypophagia and that experimental manipulation of the blood oxygen transport capacity cannot ameliorate it. Leptin does not appear to be an inducer of the anorexic response to hypoxia, at least in mice and rats.

Effects of on/off anabolic hPTH and remodelling inhibitors on metaphyseal bone of immobilized rat femurs. Tomographical (pQCT) description and correlation with histomorphometric changes in tibial cancellous bone

An anabolic effect of hPTH(1-38) (s.c. doses of 200 micrograms/kg/d during 75 days) on trabecular and cortical bone mass is tomographically described in the metaphyseal region of immobilized rat femurs using pQCT technology, in agreement with previous histomorphometrical studies of the proximal tibial metaphyses. Correlations between pQCT and histomorphometrical data showed that this effect derived from a stimulation of endosteal and trabecular bone modeling that induced a transference from trabecular to cortical bone mass. Loss of effects after withdrawal, resulting from a stimulation of bone remodeling, could be total or partially prevented by subsequent s.c. injections of risedronate (5 micrograms/kg/2/wk), 17-B-estradiol (10 micrograms/kg/d) or calcitonin (10 micrograms/kg/d) given during 60 days, in this order of effectiveness. The preventive potency was proportionally related to the reduction induced in histomorphometric indices of bone resorption.

The Concentration of Dietary Casein Required for Normal Mandibular Growth in the Rat

To determine a suitable casein concentration for normal, undeformed mandibular growth, we placed weanling male rats on diets containing graded levels of casein between 0% and 30% for 19 days. Some weanlings were killed so that initial values could be established. Ten linear dimensions corresponding to the six skeletal units of the mandible were evaluated so that their growth rates at the end of the experimental period could be established. Other dimensions were also evaluated for study of the growth rate of the bone as a whole. The macroscopic growth of the mandible showed a sigmoidal relationship with dietary casein concentration, most of the measurements reaching a plateau at 20% casein. Within the skeletal units, four dimensions corresponding to the alveolar and symphyseal regions did not change with age and were not affected by the casein content of the diet. The remaining six dimensions-corresponding to condylar, coronoid, angular, and basal regions of the mandible-increased with age and were related positively to dietary casein concentration. Their growth patterns were not uniform, although all of them reached maximal values when the diet contained 20% casein. Therefore, deformation of the mandible appears to occur in rats fed diets with a casein concentration lower than 20%. It appears that a dietary casein concentration of 20% is required for normal, undeformed mandibular growth.

TOMOGRAPHIC (pQCT) AND BIOMECHANICAL EFFECTS OF hPTH(1-38) ON CHRONICALLY IMMOBILIZED OR OVERLOADED RAT FEMURS

Six-month old rats chronically submitted to right hindlimb immobilization (IM) with mechanical overload (OL) of the left leg were treated 1 month later with 200 micrograms/kg/d of hPTH(1-38) for 15 or 75 days. Peripheral quantitative computed tomography (pQCT) scans and bending tests showed that hPTH increased cortical mass and volumetric BMD (vCtBMD) in both legs. However, elastic modulus of cortical bone and diaphyseal load-bearing capacity were improved only in OL bones. Improvement of diaphyseal strength was attributable to that of cortical bone quality, yet a stronger mechanostatic response of cortical modeling to bone material quality was also observed in treated OL bones. Data support hPTH(1-38) use for improving cortical bone mass and strength and point out a physical activity interaction with therapeutic results.

Growth-dependent effects of dietary protein concentration and quality on the biomechanical properties of the diaphyseal rat femur☆

OBJECTIVES This study compares the effects of feeding growing rats with increasing concentrations of casein (C) and wheat gluten (G), proteins that show different biological qualities, on the morphometrical and biomechanical properties of the femoral diaphysis. MATERIALS AND METHODS Female rats were fed with one of ten diets containing different concentrations (5-30%) of C and G between the 30th and 90th days of life (Control=C-20%). Biomechanical structural properties of the right femur middiaphysis were estimated using a 3-point bending mechanical test with calculation of some indicators of bone material properties. RESULTS Body weight and length were affected by treatments, values being highest in rats fed the C-20% diet. G diets affected negatively both parameters. Changes in cross-sectional geometry (mid-diaphyseal cross-sectional and cortical areas, femoral volume, and rectangular moment of inertia) were positively related to the C content of the diet, while they were severely and negatively affected by G diets. Similar behaviors were observed in the bone structural properties (fracture load, yielding load, diaphyseal stiffness and elastic energy absorption). When values of strength and stiffness were normalized for body weight, the differences disappeared. The bone material quality indicators (elastic modulus, yielding stress, elastic energy absorption/volume) did not differ significantly among all studied groups. Femoral calcium concentration in ashes was not significantly different among groups. CONCLUSION The clear differences in strength and stiffness of bone beams induced by dietary protein concentration and quality seemed to be the result of an induced subnormal gain in bone structural properties as a consequence of a correlative subnormal gain in bone growth and mass, yet not in bone material properties.

Biomechanical properties of the mandible, as assessed by bending test, in rats fed a low-quality protein

OBJECTIVE The present study describes the effects of feeding growing rats with diets containing increasing concentrations of wheat gluten (a low quality protein, G) on both the morphometrical and the biomechanical properties of the mandible. DESIGN Female rats were fed one of six diets containing different concentrations (5-30%) of G between the 30th and 90th days of life. Control rats were fed a diet containing 20% casein (C), which allows a normal growth and development of the bone. Mandibular growth was estimated directly on excised and cleaned bones by taking measurements between anatomical points. Mechanical properties of the right hemimandibles were determined by using a three-point bending mechanical test to obtain a load/deformation curve and estimate the structural properties of the bone. Bone material properties were calculated from structural and geometric properties. The left hemimandibles were ashed and the ash weight obtained. Calcium content was determined by atomic energy absorption. Results were summarised as means±SEM. Comparisons between parameters were performed by ANOVA and post-test. RESULTS None of the G-fed groups could achieve a normal growth performance as compared to the C-fed control group. Like body size, age-related increments in mandibular weight, length, height and area (index of mandibular size) were negatively affected by the G diets, as was the posterior part of the bone (posterior to molar III). The cross-sectional geometry of the mandible (cross-sectional area and rectangular moment of inertia) as well as its structural properties (yielding load, fracture load, and stiffness) were also severely affected by the G diets. However, material properties (Youngs modulus and maximum elastic stress) and calcium concentration in ashes and the degree of mineralisation were unaffected. CONCLUSIONS The differences in strength and stiffness between treated and control rats seemed to be the result of an induced loss of gain in bone growth and mass, in the absence of changes in the quality of the bone mineralised material.

Androgens and Erythropoiesis. Induction of Erythropoietin- Hypersecretory State and Effect of Finasteride on Erythropoietin Secretion

The major activity of androgens in promoting erythropoiesis is mediated by the ability of these steroids to increase erythropoietin (EPO) production. Testosterone exerts a nephrotrophic action which is evidenced by an increased renal mass following chronic administration of this steroid. The increased renal mass, in turn, may be associated with an increased capacity for production of EPO by the organ. The present study was thus designed to test the hypothesis that chronic administration of testosterone induces an EPO-hypersecretory state (EPO-HS) defined as a condition of higher than normal EPO synthesis and secretion in response to well-known erythropoietic stimuli, such as hypoxia, anaemia or cobalt. In addition, since 5α-dehydrotestosterone (5α-DHT) was found to be more active than testosterone in stimulating EPO production, the effect of inhibition of the 5α-reductase by finasteride on both constitutive and stimulated EPO secretion was also estimated. Female adult mice receiving 2 mg testosterone propionate three times a week for 3.5 weeks increased their erythrocyte production rates at day 4 and at the time of each sampling points thereafter during the injection period. Average renal weight was about 1.8 times higher in testosterone-treated than in vehicle-treated mice. When mice were hypertransfused at the end of the injection period and exposed to hypobaric air, both RBC-59Fe uptake and plasma EPO concentration (pEPO) were several times greater in androgen-treated than in vehicle-injected mice. No changes were observed in both parameters under normobaric conditions Testosterone treatment altered neither the plasma disappearance (t1/2) of radiolabelled rh-EPO nor the erythropoietic response to exogenously administered rh-EPO. pEPO was similar into normoxic male and female mice and neither orchidectomy nor finasteride (10 mg/kg/day for 12 days) modified these levels. Significant differences were seen in pEPO levels between male and female mice under hypoxic conditions. Orchidectomy and finasteride both lowered the hypoxia-dependent increment in pEPO in male mice to levels that were not significantly different from those of hypoxic female mice. In summary, an EPO-HS followed chronic administration of testosterone in mice, which may or may not be related to the nephrotrophic effect of this androgen. Since finasteride adversely affected stimulated-EPO production in mice, it is suggested that the testosterone effect on stimulated-EPO secretion is mediated by DHT. In contrast, the findings of unchanged baseline pEPO in either orchidectomised or finasteride-treated mice suggest that constitutional EPO production is not influenced by testosterone.

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.