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This study investigated the effect of a soft diet, given to growing rats, on the biomechanical behaviour of the mandible. Female rats, 30 d of age, received an ordinary diet in the form of pellets (i.e. hard-diet group), and another group of female rats received the same diet, but ground and mixed with water, forming a paste (i.e. soft-diet group). The experiment lasted 8 wk. Body-weight and body-length gains were not affected by the consistency of the diet. No significant differences were found between groups concerning the length, height, and area of the right hemimandible. Mechanical properties of the right hemimandibles were determined using a three-point bending test, in which bones were stressed on a perpendicular line immediately posterior to the posterior face of the third molar. Structural properties (load at yielding, load at fracture, structural stiffness, and elastic energy absorption) and geometric properties of the fracture section (cross-sectional area, cortical area, and moment of inertia) were significantly lower in hemimandibles of rats of the soft-diet group than in those of rats of the hard-diet group. Material properties of the mandibular bone tissue (elastic modulus and maximal elastic stress), which were estimated through appropriate equations, did not differ between groups. It was concluded that the reduced physical consistency of the diet, possibly associated with a reduced masticatory load, diminished the skeletal load-bearing capacity of the mandible in growing rats. This observed reduction in the bone structural behaviour was attributed to changes occurring at the level of bone mass and its geometrical properties because intrinsic properties of the bone material tissue were unaffected.

BACKGROUND: Physical activity plays a tremendous role in determining bone mechanical behavior, which is superimposed to gravidity. OBJECTIVE: Compare the geometric and material responses of the rat femur to a high intensity treadmill running training of a relatively short duration, as assessed by 3-point mechanical test. METHODS: Mature male rats (180.0 ± 30 g) were assigned (7 rats/group) to no exercise (NE) or treadmill exercise (EX). After a preconditioning period, the running speed was set at 45 cm.seg−1 during 2 wks, frequency 5 d/wk, 2-hour sessions/day. Body weight and weight of the crural quadriceps were registered at euthanasia. The right femur was mechanically tested through 3-point bending. The left femur was ashed to estimate bone mineral content. Geometric and material bone properties were estimated directly or calculated by appropriate equations. RESULTS: 1) Final body weight was 14% reduced in EX rats, while the crural quadriceps was 47% increased. Yield and fracture loads, and structural stiffness were significantly higher in the EX rats, as were the apparent elastic modulus, the bone mineral content and the degree of mineralization. Geometric properties were not affected. CONCLUSIONS: High intensity treadmill running training increases bone strength and stiffness by increasing material stiffness and mineralization, without affecting geometric bone parameters. Fil: Bozzini, Clarisa. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina Fil: Viscor, Ginés. Universidad de Barcelona; España Fil: Kristjánsson, Juan C. Ríos. Universidad de Barcelona; España Fil: Picasso, Emilio. Universidad de Buenos Aires. Facultad de Ingeniería; Argentina Fil: Champin, Graciela Monica. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Alippi, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina Fil: Bozzini, Carlos Eduardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina

Both stiffness and strength of bones are thought to be controlled by the "bone mechanostat". Its natural stimuli would be the strains of bone tissue (sensed by osteocytes) that are induced by both gravitational forces (body weight) and contraction of regional muscles. Body weight and muscle mass increase with age. Biomechanical performance of load-bearing bones must adapt to these growth-induced changes. Hypophysectomy in the rat slows the rate of body growth. With time, a great difference in body size is established between a hypophysectomized rat and its age-matched control, which makes it difficult to establish the real effect of pituitary ablation on bone biomechanics. The purpose of the present investigation was to compare mid-shaft femoral mechanical properties between hypophysectomized and weight-matched normal rats, which will show similar sizes and thus will be exposed to similar habitual loads. Two groups of 10 female rats each (H and C) were established. H rats were 12-month-old that had been hypophysectomized 11 months before. C rats were 2.5-month-old normals. Right femur mechanical properties were tested in 3-point bending. Structural (load-bearing capacity and stiffness), geometric (cross-sectional area, cortical sectional area, and moment of inertia), and material (modulus of elasticity and maximum elastic stress) properties were evaluated. The left femur was ashed for calcium content. Comparisons between parameters were performed by the Student's t test. Average body weight, body length, femur weight, femur length, and gastrocnemius weight were not significantly different between H and C rats. Calcium content in ashes was significantly higher in H than in C rats. Cross-sectional area, medullary area, and cross-sectional moment of inertia were higher in C rats, whereas cortical area did not differ between groups. Structural properties (diaphyseal stiffness, elastic limit, and load at fracture) were about four times higher in hypophysectomized rats, as were the bone material stiffness or Young's modulus and the maximal elastic stress (about 7×). The femur obtained from a middle-aged H rat was stronger and stiffer than the femur obtained from a young-adult C rat, both specimens showing similar size and bone mass and almost equal geometric properties. The higher than normal structural properties shown by the hypophysectomized femur were entirely due to changes in the intrinsic properties of the bone; it was thus stronger at the tissue level. The change of the femoral bone tissue was associated with a high mineral content and an unusual high modulus of elasticity and was probably due to a diminished bone and collagen turnover.

The present study was designed to estimate the bone biomechanical repercussion of protein restriction imposed to rats between the 26th and the 135th days of age. Female rats were placed on either a 4% (P4) or 20% (P20) protein diets. Body weight at the end of the study was 350.5 g (SD 31.0 g) in P20 rats and 87.5 g (19.4 g) in P4 ones. Femur weight and length were significantly reduced. The diaphyseal cross-sectional area and the cross-sectional moment of inertia were also diminished. Femoral calcium concentrations in ashes were similar in both groups. However, the femoral mass (calcium mass) was significantly lower in P4 than in P20 rats. When the femoral bone mass was normalized per 100-g femur, no significant difference between groups was found. The right femur of each animal was subjected to a three-point bending test in order to obtain the load/deformation curve. A significant weakening of bone beams, shown by a correlative impairment of diaphyseal ultimate strength and stiffness, was observed in P4 rats. However, when their values were normalized for body weight, the differences disappeared. The bone material quality indicators, or pre-yield bending stiffness (elastic modulus) and yielding stress were similar in P4 and P20 rats. In summary, we have described a number of alterations in bone morphometrical and biomechanical variables in rat femur shafts resulting from protein restriction from weaning to early adulthood. The clear differences in strength and stiffness of bone beams 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.

The bone changes in hypothyroidism are characterized by a low bone turnover with a reduced osteoid apposition and bone mineralization rate, and a decreased osteoclastic resorption in cortical bone. These changes could affect the mechanical performance of bone. The evaluation of such changes was the object of the present investigation. Hypothyroidism was induced in female rats aged 21 days through administration of propylthiouracil in the drinking water for 70 days (HT group). Controls were untreated rats (C group). Right femur mechanical properties were tested in 3-point bending. Structural (load bearing capacity and stiffness), geometric (cross-sectional area and moment of inertia) and material (modulus of elasticity) properties were evaluated. The left femur was ashed for calcium content determination. Plasma T(4) concentration was significantly decreased in HT rats. Body and femur weight and length in HT rats were also reduced. Femoral calcium concentration in ash was higher in HT than in C rats. However, the femoral calcium mass was significantly lower in HT than in C rats because of the reduced femoral size seen in the former. The stiffness of bone material was higher in HT than in C rats, while the bone geometric properties were significantly lower. The "load capacity" was between 30 and 50% reduced in the HT group, although, the differences disappeared when the values were normalized per 100-g body weight. The lowered biomechanical ability observed in the femoral shafts of HT rats seems to be the expression of a diminished rate of growth. Qualitative alterations in the intrinsic mechanical properties of bone tissue were observed in HT rats, probably because the mineral content and the modulus of elasticity were positively affected. The cortical bone of the HT rat thus appears as a bone with a higher than normal strength and stiffness relative to body weight, probably due to improvement of bone material quality due to an increased matrix calcification.

Both undernutrition and hypoxia exert a negative influence on both growth pattern and bone mechanical properties in developing rats. The present study explored the effects of chronic food restriction on both variables in growing rats exposed to simulated high-altitude hypoxia. Male rats (n 80) aged 28 d were divided into normoxic (Nx) and hypoxic (Hx) groups. Hx rats were exposed to hypobaric air (380 mmHg) in decompression chambers. At T0, Nx and Hx rats were subdivided into four equal subgroups: normoxic control and hypoxic controls, and normoxic growth-restricted and hypoxic growth-restricted received 80 % of the amount of food consumed freely by their respective controls for a 4-week period. Half of these animals were studied at the end of this period (T4). The remaining rats in each group continued under the same environmental conditions, but food was offered ad libitum to explore the type of catch-up growth during 8 weeks. Structural bone properties (strength and stiffness) were evaluated in the right femur midshaft by the mechanical three-point bending test; geometric properties (length, cross-sectional area, cortical mass, bending cross-sectional moment of inertia) and intrinsic properties of the bone tissue (elastic modulus) were measured or derived from appropriate equations. Bone mineralisation was assessed by ash measurement of the left femur. These data indicate that the growth-retarded effects of diminished food intake, induced either by food restriction or hypoxia-related inhibition of appetite, generated the formation of corresponding smaller bones in which subnormal structural and geometric properties were observed. However, they seemed to be appropriate to the body mass of the animals and suggest, therefore, that the bones were not osteopenic. When food restriction was imposed in Hx rats, the combined effects of both variables were additive, inducing a further reduction of bone mass and bone load-carrying capacity. In all cases, the mechanical properties of the mineralised tissue were unaffected. This and the capacity of the treated bones to undergone complete catch-up growth with full restoration of the biomechanical properties suggest that undernutrition, under either Nx or Hx conditions, does not affect bone behaviour because it remains appropriate to its mechanical functions.

The growth of the body and bone mass and the mechanical properties of appendicular bone are impaired in immature rats exposed to different simulated high altitudes (SHA) (1850-5450 m) between the 32nd and the 74th days of postnatal life. Now, we report the effects of exposure to 4100 m on the above cited variables in female rats from infancy (age: 1 month) to adulthood (age: 8 months) to define the occurrence of catch up and to establish whether the effects of altitude are transient or permanent. The ex vivo right femur was mechanically tested in three-point bending. Body weight and length, and structural (loads at yielding and fracture, and stiffness) and architectural (diaphyseal cross-sectional area, cortical area, and cross-sectional moment of inertia) properties were measured at 2, 4, 6, and 8 months of exposure to SHA. The negative influence of hypoxia on all variables was similar at different ages or, in other words, the difference among ages was maintained at any extent of hypoxia. Hypoxia did not affect the elastic modulus, thus suggesting that the mechanical properties of the bone tissue were maintained. Catch up did not occur. The resulting osteopenic bone remained appropriate to its mechanical function during the entire exposure to SHA.

Stress erythropoiesis (SE) can be defined as a state of increased red cell production in response to an enhanced secretion of erythropoietin. Under normal conditions, erythropoiesis in the adult occurs in the erythropoietic tissue that is confined to the bone marrow within the skeleton. Hypertrophy of the erythropoietic marrow that occurs during SE must be accommodated in a larger space. The resulting expansion of the marrow space may induce bone resorption and could alter the biomechanical performance of bone. The present study was designed to estimate the effect of sustained SE on diaphyseal structure and biomechanics of rat femur by mechanically testing the diaphyseal stiffness and strength and calculating some indicators of bone material properties. Female Sprague–Dawley rats weighing 100.0 ± 5.2 g were divided in a control (C) and an experimental (E) group. E rats were biweekly injected with 60 mg/kg of phenylhydrazyne during 6 weeks to induce a haemolytic state. SE, in response to haemolysis, was estimated by reticulocyte count and erythrokinetic techniques, which were markedly increased. To assess bone mechanical properties, the right femur was tested in three-point bending test. Sections of the left femur were stained with haematoxylin–eosin. Body growth was not altered by treatment. Diaphyseal bone mass (CSA) was 13% lower in E than in C rats, while the cross-sectional bending moment of inertia (xCSMI) was significantly higher. The “load capacity” extrinsic properties of the femoral diaphysis were about 40% decreased in E rats when compared to C ones, while the bone material quality indicators (elastic modulus and yield stress of cortical bone tissue) were 54 and 38% lower, respectively. The histologic sections of E femora exhibited a marked thinning of cortical bone and the presence of woven bone in the medullary compartment. The results obtained in this study indicate that SE caused the impairment of the diaphyseal bone material properties (elastic modulus and elastic stress) but enhanced xCSMI over the control values. Neither this improvement in diaphyseal cross-sectional design nor the formation of woven bone could offset the impairment in the bone material stiffness. It is thus proposed that the biomechanical impact of SE upon the whole-bone stiffness should have been more determined by the negative effect on bone material quality than by the negative changes in the cortical area.

Erythropoietin-hypersecretory state (EPO-HS) has been defined as a condition elicited by inducers, which is easily observed in hypertransfused polycythemic rodents in which hypoxia-stimulated EPO secretion is higher than in non-treated controls at equal levels of polycythemia. Testosterone (T) is a potent inducer of EPO-HS. T has the potential of acting itself or of being metabolized to either 5α-dihydrotestosterone (5α-DHT) or 5β-dihydrotestosterone (5β-DHT). The former binds to the androgen receptor while the latter does not. However, both isomers can stimulate erythropoiesis, although through different mechanisms and possibly through different receptors. The objective of this investigation was to characterize the role of both receptors in mediating the EPO-HS induced by T. Male CF#1 mice were orchidectomized when aged 30 days. One month later, groups of 10 animals each received graded doses (0–3,200 μg) of T, 5α-DHT or 5β-DHT, subcutaneously, 3 times a week for 4 weeks. Mice were made polycythemic 4 days after the injection period and exposed to hypobaric air (506.5 mbar) for 6 h, 1 day later. Plasma EPO concentration (pEPO) was estimated by immunoassay at the end of the exposure period and taken as the expression of EPO production rates. Androgenic activity of the steroids was estimated by seminal vesicle weight, whereas the nephrotropic activity of the steroids was derived from the kidney weight. Orchidectomy significantly reduced the weights of both seminal vesicle and kidney. Dose-related increases of organ’s weights were elicited by both T and 5α-DHT; 5β-DHT being ineffective. The pEPO response to hypobaric hypoxia increased as a function of the dose of the androgenic steroids administered up to the 800 μg dose with no further increments beyond this dose. Administration of 5β-DHT had no effect on pEPO. The induced EPO-HS was associated with the androgenic and renotrophic actions of both testosterone and 5α-DHT; 5β-DHT being ineffective as EPO-HS inducer. Data indicate that the induction of an EPO-HS by testosterone requires the activation of the androgenic receptor. The possible activation by the steroid of the non-androgenic receptor, which appears to exert a direct erythropoiesis-stimulating effect on the bone marrow, does not induce an EPO-HS.

Long-term glucocorticoid administration to growing rats induces osteopenia and alterations in the biomechanical behavior of the bone. This study was performed to estimate the effects of dexamethasone (DTX), a synthetic steroid with predominant glucocorticoid activity, on the biomechanical properties of the mandible of rats during the growth phase, as assessed by bending test and peripheral quantitative computed tomographic (pQCT) analysis. The data obtained by the two methods will provide more precise information when analyzed together than separately. Female rats aged 23 d (n=7) received 500μg.kg-1 per day of DXT for 4 weeks. At the end of the treatment period, their body weight and body length were 51.3% and 20.6% lower, respectively, than controls. Hemimandible weight and area (an index of mandibular size) were 27.3% and 9.7% lower, respectively. The right hemimandible of each animal was subjected to a mechanical 3-point bending test. Significant weakening of the bone, as shown by a correlative impairment of strength and stiffness, was observed in experimental rats. Bone density and cross-sectional area were measured by pQCT. Cross-sectional, cortical and trabecular areas were reduced by 20% to 30% in the DTX group, as were other cortical parameters, including the bone density, mineral content and cross-sectional moment of inertia. The "bone strength index" (BSI, the product of the pQCT-assessed xCSMI and vCtBMD) was 56% lower in treated rats, which compares well with the 54% and 52% reduction observed in mandibular strength and stiffness determined through the bending test. Data suggest that the corticosteroid exerts a combined, negative action on bone geometry (mass and architecture) and volumetric bone mineral density of cortical bone, which would express independent effects on both cellular (material quality) and tissue (cross-sectional design) levels of biological organization of the skeleton in the species.

Adenohypophyseal hormones are important for normal growth and maintenance of skeletal mass. From a mechanical point of view, the skeleton can be viewed as a biomechanically regulated structure, the strength of which depends on the intrinsic stiffness (material properties) and the spatial distribution (architectural properties) of the mineralized tissue. Both material and architectural properties of bone change during ontogeny. Both muscle strength and body weight, the main two types of load producing strain, remain practically unchanged in a young rat after hypophysectomy (HX). Therefore, this study was performed to analyse the effect of HX on bone development in a model in which the main loads on bone are almost constant from infancy to adulthood. Female rats were HX at 30 days of age. A “basal control group” (BC) was sacrificed on the same day. An “age-matched control group” (CON) was also included. HX and CON rats were sacrificed 5 months later. Mechanical properties of the right femur were determined using a three-point bending mechanical test. HX rats failed to gain body weight and body and femur length. Femur weight was about 1.09 times increased and femoral calcium content (left femur) was 2.25 times greater in HX than in BC rats. The “load-capacity” extrinsic properties (stiffness, elastic limit, and ultimate load) were 3.47, 3.42, and 3.11 times increased, respectively, in HX rats in relation to BC ones. Focussing on the intrinsic material properties, the cortical elastic modulus (E) was 10.73 and 2.54 times higher in HX rats than in BC and CON rats, respectively. In summary, HX in the rat induced the transformation of the juvenile femur to a type of adult bone characterised by its high mineral density and its unusual large modulus of elasticity in spite of cessation of lineal growth. These effects are probably due to secondary hypothyroidism induced by TSH deficiency and associated to increased collagen maturation and cross-linking, and degree of bone calcification.

The erythropoietin-hypersecretory state (EPO-HS) is a condition elicited by several inducers that is easily observed in hypertransfused-polycythaemic rats and mice, in which hypoxia-stimulated erythropoietin (EPO) secretion is higher than in polycythaemic controls. Steroids with different androgenic/anabolic ratios have proved to be potent inducers of EPO-HS. However, both steroid activities do not appear to have similar importance. The purpose of the present study was to assess the ability of oxymetholone, a synthetic derivate of testosterone that shows the highest anabolic/androgenic ratio, to elicit an EPO-HS. Mice were orchidectomised at 30 days of age. One month later, groups of animals were injected with graded doses of oxymetholone for 4 weeks. All orchidectomised mice were hypertransfused 4 days after the end of the injection period. On the next day, they were exposed to air maintained at 506.5 mbar for 6 h to stimulate EPO production. Plasma EPO titre was determined by immuno-analysis and taken as a reflection of the EPO production rate. Kidney, seminal vesicle and levator ani muscle weights were registered as index of renotrophic, androgenic and anabolic effects, respectively. The steroid brought out a significant anabolic, and a poor androgenic, response. Any of the doses of oxymetholone tested increased EPO production in orchidectomised polycythaemic mice and, therefore, an EPO-HS was not induced. This finding, coupled with previously reported data, suggests that only steroids showing a certain degree of androgenic action have the capacity to evoke an EPO-HS.

SummaryChemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to −26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.

Summary. Mice myocardia, in which plasma erythropoietin (EPO) concentrations were modified in response to different experimental conditions, were studied to evaluate contractility (dF/dt). CF1 mice were randomly separated into four main groups: group I, normocythaemic normoxic; group II-a, normocythaemic intermittently exposed to hypobaria for 72 h; group II-b, normocythaemic intermittently exposed to hypobaria for 3 weeks; group III, hypertransfused polycythaemic exposed to 72 h hypobaria; and group IV, hypertransfused polycythaemic maintained in normobaric air. Plasma EPO, contractile studies and binding assays were performed. The dF/dt was significantly higher in group II-a than in group I and group II-b; but in groups III and IV, the dF/dt was reduced. The toxic action of ouabain was reduced and delayed in its onset, accompanied by increased numbers of 3H-ouabain binding sites in group II-a. Contractility was positively correlated with plasma EPO (pEPO) in the different groups. Treating group I with recombinant human (rHu)-EPO enhanced contractility while treating group II-a with a monoclonal anti-EPO decreased the dF/dt. The inhibition of enzymatic pathway(s) known to participate in the cytokines signal transduction, decreased the basal dF/dt values on atria from group II-a and on group I atria treated with rHu-EPO. The results demonstrated: (1) a cardiac non-haematopoietic effect of EPO; (2) that mice in which the pEPO concentration increased showed improvement in contractility and in the therapeutic action of ouabain; and (3) it is possible that EPO may act as a cardioprotective agent by modulating the cardiac Na+–K+ pump.

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.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Catch-up growth has been defined as growth with a velocity above the statistical limits of normality for age during a defined period of time which follows a period of impaired growth. Since no data are available on catch-up in mandibular growth, the present study was designed to estimate the dynamics of the mandibular size after short-term dietary protein restriction in rats during the post-weaning period. Weanling male rats, 22 d of age, were divided into two groups, control (C) and experimental (E). E rats were fed a protein-free diet during the first 10 d; from this time on, they were placed on a 20% protein diet, as were C rats during the entire experimental period, which lasted 70 d. Five rats from both groups were randomly selected every 10 d and sacrificed. Mandibular growth was estimated directly on the right mandible by measuring several dimensions (mandibular area, base length, mandibular height, mandibular length, alveolar length and incisor alveolar process length). Alveolar and incisor alveolar process lengths did not change with age or dietary protein. All other dimensions increased with age and were thus negatively affected by protein restriction. After growth restriction ceased, the rate of increase of all affected dimensions was above normal values and deficits were swiftly eliminated. Since age-independent dimensions compose roughly the anterior portion of the mandible, this portion of the bone was not affected by protein restriction. It was, thus, the posterior part of the mandible which stopped growth during the nutritional insult and showed catch-up during nutritional rehabilitation. In summary, the rat mandible has a high potential for catch-up during the post-weaning period, showing the ability to achieve complete catch-up in about 30 d.

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 (t 1/2) 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.

Postnatal hypoxia blunts body mass growth. It is also known that the quality of the fetal environment can influence the subsequent adult phenotype. The main purpose of the study was to determine whether gestational hypoxia and early postnatal hypoxia are able to blunt growth when the offspring is raised under normoxia. Hypobaric hypoxia was induced in simulated high altitude (SHA) chambers in which air was maintained at 380 mmHg (5450 m). Mature Sprague-Dawley rats of both sexes were divided in normoxic (NX) and hypoxic (HX) groups and, in the case of the HX group, maintained for 1 month at 5450 m. Mating was then allowed under NX or HX conditions. Offspring were NX-NX, NX-HX, HX-HX, or HX-NX: the first term indicates NX or HX during both gestation and the first 30 days of life; the second term indicates NX or HX during postnatal life between days 30 and 133. Body mass (g) was measured periodically and body mass growth rate (BMGR, g/d) was estimated between days 33 and 65 of postnatal life. Results can be summarized as follows: 1) BM was significantly higher in NX than in HX rats at weaning; 2) BMGR was not significantly different between NX-NX and HX-NX rats, and between HX-HX and NX-HX animals; and 3) BMGR was significantly higher in rats living under NX conditions than in those living under HX conditions during postnatal life. Data suggest that that hypobaric hypoxia during gestational and early postnatal development of rats does not alter the regulation of body mass growth in rats when compared to that seen under sea-level conditions. Fil: Bozzini, Carlos Eduardo Jose. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Champin, Graciela Monica. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bozzini, Clarisa. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina Fil: Alippi, Rosa Maria. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Bozzini, Clarisa, Graciela M. Champin, Rosa M. Alippi, and Carlos E. Bozzini. Static biomechanics in bone from growing rats exposed chronically to simulated high altitudes. High Alt Med Biol 14:367–374, 2013.—Biomechanical behavior of bone is related to the amount (bone mass) and its architectural distribution, as well as the mechanical quality of bone material. This investigation reports the effects of exposure to different simulated high altitudes (SHA) (1850, 2900, 4100, and 5450 m) on femur biomechanical properties in female growing rats exposed to SHA (22–23 h/d) between the 32° and the 74° days of life. The ex vivo right femur was mechanically tested in three-point bending. The left femur was ashed at 600°C and the ash weight obtained. Final body weight and structural (loads at yielding and fracture, stiffness, and elastic energy absorption) and architectural (diaphyseal cross-sectional area, cortical area, and cross-sectional moment of inertia) were negatively affected in the animals exposed to the two highest SHA. Material properties of the mineralized tissue (Young's modulus and limit elastic stress) and the degree of mineralization were unaffected. In conclusion, hypoxic bone is weaker than normoxic one because of its smaller bone mass, which appear to have been negatively influenced by SHA in relation to its effects on overall body mass.

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 (Young?s 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. Fil: Bozzini, Carlos Eduardo Jose. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Champin, Graciela Monica. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alippi, Rosa Maria. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bozzini, Clarisa. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina

The recent report of a depression of stimulated production of erythropoietin (EPO) in mice with enhanced erythropoiesis suggests that unknown mechanism (s) other than hypoxia may be involved in the regulation of EPO formation. The present study was thus designed to investigate EPO production during acute hypoxemia in a mouse model in which the oxygen-carrying capacity of blood, the plasma EPO level, and the plasma EPO half-life were within normal values in spite of a marked depression of the red cell production rate (RCPR) induced by cyclophosphamide (CP) administration. Injection of 100 mg/Kg of the drug into adult female CF-1 mice that previously received 0.4 ml of packed red cells depressed markedly the 24-hour RBC 59Fe uptake without affecting the plasma immunoreactive EPO level and the plasma disappearance of 1251-labeled recombinant human EPO. The EPO production rate, calculated from the change in plasma EPO levels and the estimated EPO clearance rate, after 4 h of exposure to hypobaric air was about 2.8 times higher in mice with CP-induced inhibition of the RCPR than in mice with normal RCPR. The results support the hypothesis that the EPO production rate in mammals is not only related to the oxygen supply to the tissues relative to their oxygen needs (main stimulus) but also to the erythroid activity of the marrow (modulatory action).

In previous studies with cortisol, betamethasone and oxazacort we attributed glucocorticoid effects on bone biomechanics to changes in bone mass and geometry rather than to an action on bone material properties. In this experiment, groups of 7 rats each received subcutaneous doses of 15.6, 31.2, 62.5, 125, 250, 500 or 1000 micrograms/kg per day of dexamethasone (DMS) and an additional 14 animals were controlled untreated for 4 weeks. Their fresh femurs were then scanned by peripheral quantitative computerized tomography (pQCT; XCT-960, Stratec, Germany) at the midshaft and submitted to three-point bending tests. In consonance with our earlier investigations, a significant, log-dose-related reduction in bone load-bearing capacity was observed, associated with an impairment in bone geometric properties (cross-sectional area and moment of inertia) and in body weight gain. However, the pQCT-assessed volumetric mineral density of cortical bone (vCtBMD; regarded as a material quality indicator in terms of mineralization) was significantly reduced by DMS following a dose-response relationship. Furthermore, a direct association was detected between vCtBMD and diaphyseal load-bearing capacity and stiffness. In contrast with our previous approach, data suggests that, apart from changes in bone geometric properties, glucocorticoid effects on bone material quality--as assessed by vCtBMD changes in this study--seem also to play a significant role in the determination of their biomechanical consequences.

The present investigation was performed to assess the biomechanical repercussion of protein malnutrition imposed on rats between the 26th and 135th days of postnatal life on the mandible, which is not a weight-bearing bone but supports the loads related to the masticatory activity. Female Wistar rats aged 26 d (n = 14) were placed on either a 4%-protein diet (ICN 960254, P4 group) or a 20%-protein diet (ICN 960260, P20 group) and killed 111 d later. Both body weight and length were recorded regularly. The mandibles were dissected and cleaned of adhering soft tissue. Mandibular growth was estimated directly by taking measurements between anatomical points. Areal Bone Mineral Density (BMD) was estimated using a bone densitometer (LUNAR DPX-L). Mechanical properties of the right hemimandible were determined using a three-point bending mechanical test to obtain the load/deformation curve and estimate the structural properties of the bone. Results were summarized as means +/- SD. Comparisons between parameters were performed by Student's t test. A 75% reduction in body weight and a 32% reduction in body length were observed in P4 rats. Like body size, mandibular weight, length, height and area (index of mandibular size) were negatively affected by P4 diet, as was the posterior part of the bone (posterior to molar III). The anterior part (alveolar and incisor alveolar process) was not affected by age or diet. The "load capacity" extrinsic properties of the mandible (load fracture, stiffness, yielding point) were between 43% and 64% of control value in protein restricted rats. BMD was similar in both groups of animals.1) Chronic protein malnutrition imposed on rats from infancy to early adulthood reduces the growth of the posterior part of the mandible without inducing changes in the anterior part, which produces some deformation of the bone in relation to age-matched rats; and 2) the significant reduction of strength and stiffness of the mandible seem to be the result of an induced loss of gain in bone structural properties as a consequence of a correlative loss of gain in both growth and mass, yet not in bone material properties.

Objectives This study compares the effects of feeding growing rats with increasing concentra-tions 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 con-centrations (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 geome-try (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.

Summary Hypoxia leads to an increase in erythropoiesis, which induces hypertrophy of the erythropoietic marrow and may induce bone resorption. This study investigates the effect of chronic hypobaric hypoxia (simulated high altitude, SHA) on the biomechanics of rat femurs by mechanical tests of diaphyseal stiffness and strength and calculation of some indicators of bone material properties. Adult female rats were exposed to SHA (5500 m, 23.5 h/d for 60d=HX rats; NX=normoxic). This treatment induced reticulocytosis and polycythemia. Bone mineral content did not differ between NX and HX rats at the end of the study. To determine cortical bone mechanical properties, the right femur was assessed using a 3-point bending test. Endosteum and periosteum moved away from the central axis in HX rats as deduced by an increase in the cross-sectional moment of inertia. The two indicators of bone material properties, the elastic modulus and stress at the yielding point, were significantly reduced. However, the strength of the femur as an intact organ (“load capacity” parameters) was not compromised by exposure to SHA. It is thus proposed that the negative effect of SHA on bone material quality was completely offset by an improvement in diaphyseal cross-sectional design, thus allowing a normal biomechanical response to bending of the femur as a whole.

It has been previously reported that several doses of cyclophosphamide (CPA) reduce body weight gain, diaphyseal torsional strength and longitudinal femoral growth in the growing rat. The present study was thus designed to estimate both the initial and the possible long-term effects of CPA treatment, by analyzing mandibular dimensions and biomechanical performance of the bone in adulthood in rats treated with the drug around weaning. Female Sprague–Dawley rats (N = 20), 26 d of age, received 100 mg/kg of CPA by the intraperitoneal route during days 0, 7 and 21 of the experimental period. Controls (C) received saline. Groups of rats were sacrificed at day 28 to estimate initial changes induced by the drug and on day 126 in order to determine long-term effects. The dimensions of the excised mandibles were measured directly between anatomical points; the geometry and material biomechanical quality of mandibular bone were assessed using a three-point bending mechanical test in an Instron Universal Testing Machine model 4442. CPA reduced body weight, body length and mandibular size (posterior part of the bone) significantly, when the parameters were measured at day 28. They did not recover with time, which means that catch-up growth did not occur and that the overall growth of the body was permanently affected by the drug. CPA treatment was also associated with a marked depression of the natural increase in the mandibular bone mass (cross-sectional area). The bending cross-sectional moment of inertia of the fracture sections (xCSMI) was also negatively affected by treatment. Significant decreases of both ultimate load and stiffness were also observed. The above structural parameters did not recover enough with time to attain control values at the end of the study. The intrinsic stiffness (E) of the mandibular bone was not affected by treatment. These findings suggest that CPA treatment during early postnatal life causes permanent changes in mandibular morphology and affects the adaptation of mandibular bone architecture to body growth, thus not allowing complete compensation at the end of the study because of an inadequate distribution of the resistive material through its cross-section rather than a qualitative impairment of cortical bone.

The depression of body growth rate and the reduction of body mass for chronological age and gender in growing experimental animals exposed to hypobaric air (simulated high altitude = SHA) have been associated with hypophagia because of reduced appetite. Catch-up growth during protein recovery after a short period of protein restriction only occurs if food intake becomes super-normal, which should not be possible under hypoxic conditions if the set-point for appetite is adjusted by the level of SHA. The present investigation was designed to test the hypothesis that growth retardation during exposure to SHA is due to an alteration of the neural mechanism for setting body mass size rather than a primary alteration of the central set-point for appetite. One group of female rats aged 35 d were exposed to SHA (5460m) in a SHA chamber for 27 d (HX rats). Other group was maintained under local barometric pressure conditions (NX rats). One half of both NX and HX rats were fed a protein-free diet for the initial 9 d of the experimental period. From this time on, they were fed a diet containing 20% protein, as were the remaining rats of both groups during the entire experimental period. The growth rates of both mass and length of the body were significantly depressed in well-nourished rats exposed to SHA during the entire observation period when compared to normoxic ones. At its end, body mass and body length were 24% and 21% less in HX than in NX rats. Growth rates were negatively affected by protein restriction in both NX and HX rats. During protein recovery, they reached supernormal values in response to supernormal levels of energy intake that allowed a complete catch-up of both body mass and length. The finding that energy intake during the period of protein rehabilitation in HX rats previously stunted by protein restriction was markedly higher than in HX control ones at equal levels of hypoxia demonstrates that the degree of hypoxia does not determine directly the degree of appetite and energy intake. Furthermore, the finding that catch-up growth in the stunted HX rats returns the animal only to the stunted size appropriate for the hypoxic animal supports the hypothesis that hypoxia lowers the set-point for body mass size, which is reached by inhibition of appetite. Confirmation of the hypothesis was done by assessment of the set-point of body mass by the behavioral method of the weight threshold to hoard food. It was lowered by 17.0% in HX rats.

The present study provides baseline data for a number of mandibular growth dimensions, specially on bone mass and bone strength, that were collected between the 21st and the 180th days of postnatal life, which are intended as a reference for researchers designing experimental studies, specially on mandibular catch-up growth, and as an aid for clinicians who must evaluate results from published animal studies for validity and potential extrapolation to the human clinical situation. Fifty weanling female Wistar (Hsd:Wi) rats were fed ad libitum a diet previously shown to allow normal, undeformed mandibular growth. Five of them were randomly selected at different times between 21 and 180 d of life. Mandibular growth was estimated directly on the right hemimandible by taking measurements between anatomical points; mandibular bone mass (calcium mass) was estimated from the mg of calcium, determined by atomic absorption spectrophotometry, present in the ashes of the left hemimandible; and mechanical properties of the right hemimandible were determined using three-point bending mechanical test. Dimensions, bone calcium mass and bone strength of the female rat mandible increased linearly from day 21 to approximately day 90. Bone growth, as expected, was more than twice when assessed from bone weight than when derived from mandibular area, length or height when the parameters were expressed as the relative increase from the mean infant condition. The growth rate of the posterior part of the mandible (behind the third molar) was almost five times greater than that of the anterior part. The rates of growth of the studied parameters showed a marked decline after day 90. ANOVA indicated that no statistical differences were found between day 90 and day 120 values. It could be concluded that the female rat mandible attains its adult size, peak bone calcium mass and bone structural mechanical properties at some point between 90 and 120 d of postnatal life. Because of the extremely high positive correlation between mandibular bone calcium mass and both mandibular area and mandibular weight, it was possible to calculate the mandibular peak bone mass from the relations 7.69 mgCa/cm2 and 0.19 mgCa/mg bone.

Current evidence suggests that a modulatory action on O(2)-dependent EPO secretion is exerted by the erythroid/precursor cell population in the erythropoietic organs through a negative feedback system. The hypothesis is based on studies of stimulated-EPO secretion performed in mice in whom the erythropoietic rates were either enhanced or depressed in the presence of normal plasma EPO half-lives. Since erythropoietic depression was elicited by cyclophosphamide administration, which could have altered EPO production directly, the aim of the present investigation was to estimate hypoxia-stimulated EPO secretion in a mouse model of functional depressed erythropoiesis induced by exposure to normobaric hyperoxia. Females CF#1 mice aged 70 d were divided into control (C) and experimental (E) groups. The former was maintained in plastic cages in a normal environment, while the latter was placed in an environment of 60% O(2)/40% N(2) in an 85-dm(3) atmospheric chamber with air flow of 1 L/min. Erythropoiesis was evaluated by either 24-h RBC-(59)Fe uptake or iron kinetics performed 3 h after IV injection of a tracer dose of (59)Fe. Both indexes of the red cell production rate were significantly depressed in E mice. Plasma disappearance of exogenous EPO in C mice, as well as in E mice exposed to hyperoxia for 4 d, was estimated by injecting (125)I-rHuEPO intravenously. Linear regression analysis indicated that neither the differences between the slopes of both curves nor the Y-intercepts were significant. Hypobaric hypoxemia was used as stimulus for EPO production. Plasma immuno-EPO titer after a 4-h exposure to hypobaric air was 73% higher in mice with hyperoxia-induced hypoerythropoiesis than in control mice with normal erythropoiesis. Data support the concept that the rate of erythropoiesis, perhaps through the number of the erythroid progenitor/precursor cell population, modulates O(2)-dependent EPO secretion.

Mice myocardia, in which plasma erythropoietin (EPO) concentrations were modified in response to different experimental conditions, were studied to evaluate contractility (dF/dt). CF1 mice were randomly separated into four main groups: group I, normocythaemic normoxic; group II-a, normocythaemic intermittently exposed to hypobaria for 72 h; group II-b, normocythaemic intermittently exposed to hypobaria for 3 weeks; group III, hypertransfused polycythaemic exposed to 72 h hypobaria; and group IV, hypertransfused polycythaemic maintained in normobaric air. Plasma EPO, contractile studies and binding assays were performed. The dF/dt was significantly higher in group II-a than in group I and group II-b; but in groups III and IV, the dF/dt was reduced. The toxic action of ouabain was reduced and delayed in its onset, accompanied by increased numbers of 3H-ouabain binding sites in group II-a. Contractility was positively correlated with plasma EPO (pEPO) in the different groups. Treating group I with recombinant human (rHu)-EPO enhanced contractility while treating group II-a with a monoclonal anti-EPO decreased the dF/dt. The inhibition of enzymatic pathway(s) known to participate in the cytokines signal transduction, decreased the basal dF/dt values on atria from group II-a and on group I atria treated with rHu-EPO. The results demonstrated: (1) a cardiac non-haematopoietic effect of EPO; (2) that mice in which the pEPO concentration increased showed improvement in contractility and in the therapeutic action of ouabain; and (3) it is possible that EPO may act as a cardioprotective agent by modulating the cardiac Na+-K+ pump.

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.

Unexpected hypoxia-dependent erythropoietin secretion during experimental conditions not affecting tissue oxygen supply/demand ratio. Although a great deal of evidence supports the hypothesis that plasma erythropoietin (EPO) levels of mammals are related to the oxygen supply to the tissues relative to their oxygen needs, several observations millitate against its inherent simplicity. This study presents our results obtained from in vivo experiments that suggest that hypoxia-dependent EPO production can be altered by conditions which apparently do not modify the tissue oxygen supply/demand ratio. Hypoxia-dependent EPO production rate (EPO-PR), derived from plasma EPO titers and plasma EPO half-lives, were estimated in both transfused-polycythemic and normocy-themic mouse models subjected to different treatments. From calculations of the o 2 carrying capacity of blood and body o 2 consumption, it was assumed that the tissue supply/demand ratios were similar in both experimental and control mice of the same model at the time of induction of EPO production. The following observations were worth noting: (1) EPO-PRs in transfused polycythemic mice whose erythropoietic rates were stimulated by intermittent exposure to hypobaria (0·5 atm, 18 hr/day × 3 weeks), phenylhydrazine administration (40 mg/kg at weekly intervals × 3 weeks) or repeated rh-EPO injections (1500 U/kg 3 times a week × 3 weeks) before transfusion were more than five times higher than in comparabily polycythemic mice whose erythropoietic rates were not stimulated previously; and (2) EPO-PR in response to hypobaric hypoxia was 2·08 times normal in normocythemic mice with cyclophosphamide (100 mg/kg) induced depression of erythropoiesis, and 0·33 times normal in normocythemic mice with rh-EPO (400 U/kg × 2) induced enhancement of erythropoiesis. Although the results obtained in polycythemic mice are difficult to explain, those from normocythemic mice suggest the existence of a feedback mechanism between EPO-responsive cells and EPO-producing cells. Both demonstrate the existence of experimental conditions in which modulation of the hypoxia-dependent expression of the EPO gene appears to occur. This modulation would be dependent on factors other than oxygen.

Both body weight and somatic muscle forces are the main "mechanical factors" in the determination of bone strength in the "weight-bearing bones". However, other "non-mechanical factors", such as dietary proteins, also exist, which modulate bone physiology. This study was designed to explore the mechanical behavior of the femur in post-weaning female rats stunted by feeding on cornstarch. Forty female rats aged 30 days were fed freely with one of the two diets: control (CD) and experimental (ED). CD was the standard rat laboratory diet, whereas ED was cornstarch supplemented with vitamins and minerals. Control (C) and experimental (E) animals were divided into 4 groups: C40 and E40 rats were given CD and ED, respectively, for 40 days; C105 were fed the CD for 105 days; and E40-105 were given the ED for 40 days and then the CD for the remaining experimental period (65 days). Growth of rats was assessed following Parks' model. The biomechanical structural properties of the right femur middiaphysis were estimated using a 3-point bending test. The geometric properties of both the entire bone and the cross-section were determined. The left femur was ashed and both the Ca mass and the Ca concentration were determined. Rats fed the ED failed to achieve normal weight gain. Complete catch-up was observed at the end of a 65 day period of nutritional rehabilitation. The femoral weight and length were negatively affected by the ED, as were the mid-diaphyseal cross-sectional area, the mineralized cortical area, and the cross-sectional moment of inertia. All of these parameters showed incomplete catch-up. The structural bone mechanical properties indicative of strength and stiffness were seriously negatively affected. Intrinsic material bone properties, as assessed by the modulus of elasticity and the maximal elastic stress, were within normal values. In summary, the experimental bone was weaker than the control and structurally incompetent. The considered bone was smaller than the control one, showing a significant reduction in the cross-sectional area and the moment of inertia. However, material properties as well as the ash fraction and Ca concentration were similar in E and C bones. Therefore, E bone is weaker than the C one because of its smaller bone mass, which appears to have been negatively influenced by the ED in relation to its effects on overall body mass.

Male rats weighing 400 g were hypophysectomized, and erythropoietic marrow distribution, determination of number of erythroid cells in marrow and spleen, and iron kinetic studies were done 75-85 days after hypophysectomy. Rats of the same age served as normal controls. Positron camera pictures of the distribution of erythropoietic marrow within the skeleton using 52Fe showed the erythroid marrow of the hypophysectomized rats to be concentrated in the thoracic and lumbar spine, with less erythropoietic marrow in the extremities and proximal caudal vertebrae than in the controls. Total number of nucleated erythroid cells in marrow plus spleen, red cell mass, and rate of hemoglobin synthesis per 100 g of body weight in hypophysectomized rats were approximately 55% of the values in normal rats. The estimated mean erythron life span and mean effective erythron hemoglobinization time were similar in the 2 groups studied. It is concluded that erythropoiesis is decreased in the long-term hypophysectomized rat bec...

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.

The present study was undertaken to determine plasma EPO levels in response to acute exposure to hypobaria in transfused-polycythemic mice previously exposed to different forms of erythropoietic stimulation.

Cultures of the hepatic bud, under different experimental conditions, show a direct relation between foetal age at the time of dissection and the further organogenesis of the explant. In cultures of the septum transversum plus hepatic bud—with or without splanchnic mesenchyme—obtained from embryos of four to 25 somites, the capacity of endodermic cells to differentiate into hepatocytes appears only in a small number of samples; whereas, in the hepatic bud from older embryos (26 to 40 somites), this differentiation occurs in all cases. The amount of time cultures were allowed to grow was important for hepatic organogenesis, as measured by cord-like organization of hepatocytes plus their storage capacity for glycogen. The possibility of the explants showing complete haemopoiesis was also a condition of foetal age at the time of explant. Haemopoiesis was not found in cultures from embryos of less than 25 somites. On the other hand, development of haemopoiesis did not show a direct relation to days of culture, since the peak of this activity was observed towards the third day in vitro. Most explants showed a generalized haemopoiesis (both interstitial and vascular) with a decay towards the sixth day. Endodermal cells of the hepatic bud were capable of both proliferation and differentiation into hepatocytes, even in those mesenchymes considered inadequate such as limb mesenchyme. In the latter case we were unable to find haemopoiesis at any time. The septum transversum, when cultured alone, did not contain haemopoietic cells.