Dynamics of recovery of morphometrical variables and pQCT-derived cortical bone properties after a short-term protein restriction in maturing rats.

Severe protein restriction during the post-weaning period in the rat markedly reduces femoral bone mass and produces a number of alterations in the shaft biomechanical properties. Body weight and femur length show an immediate and complete catch-up during nutritional rehabilitation. The aim of the present investigation was to assess whether the accelerated bone growth that occurs during protein rehabilitation is accompanied by recovery of cortical bone properties. The dynamics of the recovery of both material and geometric properties were thus evaluated on the femoral diaphyses in 45-day old female rats after a 10-day period of dietary protein restriction by peripheral quantitative computed tomography (pQCT). Protein starvation led to marked reduction of both body weight and femoral length (37% and 14% at day 10, respectively) which showed a complete catch-up after 30 d of protein refeeding. Protein restriction was associated with the interruption of the natural increase in cortical area (CtCSA), volumetric cortical bone mineral content (vCtBMC) and volumetric cortical bone mineral density (vCtBMD) which were 19.7, 25.8, and 14%, respectively, in malnourished than in control rats at the end of the protein starvation period. These parameters recovered completely during protein refeeding. Treatment also reduced by 30% both rectangular (xCSMI) and polar (pCSMI) moments of inertia. Although an improvement of these architectural indicators occurred with time, an approximately 20% deficit was still present at the end of the observation period (70 d), as was the bone strength index (BSI). It is concluded that protein restriction affected the adaptation of diaphyseal design which should reduce the mechanical competence of the femoral diaphysis because of an inadequate architectural distribution of cortical bone, and that the alteration did not show complete catch-up during the studied period.

Catch-up in mandibular growth after short-term dietary protein restriction in rats during the post-weaning period.

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.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80% suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone(AU)

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80

suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

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 observation 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 normocythemic mouse models subjected to different treatments. From calculations of the O2 carrying capacity of blood and body O2 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 x 3 weeks), phenylhydrazine administration (40 mg/kg at weekly intervals x 3 weeks) or repeated rh-EPO injections (1500 U/kg 3 times a week x 3 weeks) before transfusion were more than five times high 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 x 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.

[Effects of bisphosphonates on the mechanical efficiency of normal and osteopenic bones]. / Efectos de los bisfosfonatos sobre la eficiencia mecánica de esqueletos normales u osteopénicos.

Bone mechanical competence (stiffness, strength) at organ level is determined by mechanical quality (intrinsic stiffness) and spatial distribution (macro-architecture) of bone material in cortical tissue (in every bone) and trabecular network (in vertebral bodies). These properties are inter-related and controlled according to mechanical usage by a feed-back mechanism known as mechanostat. Therefore, the effects on bone fragility of any treatment should be evaluated concerning the way they may have affected bone material or geometric properties as well as the mechanostatical interactions between them. Standard densitometry does not provide the necessary data, but some alternative methodologies (as peripheral quantitative computed tomography, pQCT) are being developed to complement or even substitute SPA, DPA or DXA determinations. Bisphosphonate (BP) effects on bone biomechanics have been studied only in animal models. Many sources of variation of results (type of compound, dose, mode of administration, species, race, sex, age, age since menopause, type of bone, remodeling ability of the skeleton, endocrine-metabolic status, interactions with other treatments, etc.) have been reported. In general terms, BPs are beneficial concerning cortical bone strength in purely modeling species (rodents) and trabecular strength in remodeling mammals (dogs, baboons). This positive action at organ level depends on independent improvements in bone macro-architecture (mainly affected by bone modeling) and material stiffness (chiefly affected by bone composition and remodeling). On one hand, bone macro-architecture has been positively affected by BPs in normal (not in ovariectomy (OX), steroid- or disuse-induced osteopenic) animals. On the other, bone material quality has been improved in the latter but not in the former. Mechanostatic interrelationships have been differently affected according to the compound employed. Results reported by ours and other laboratories concerning the three derivatives available nowadays in Argentina were reviewed and summarized. Pamidronate improved small rodents' cortical bone strength and geometric properties at low doses but impaired mineralization, material properties and strength at toxic doses. In normal, remodeling animals it improved mechanical properties in vertebral bodies but not in long bones. It also prevented the negative impact of OX-, steroid- or disuse-induced osteopenia in rats by improving bone material properties without affecting normal mechanostatic interrelationships. Olpadronate exerted positive effects on long-bone strength at any dose in normal rats and mice by improving cross-sectional properties and preserving both mineralization and material properties. These effects were highly dependent upon bone deformability, body weight, and mechanical usage of the limb as an evidence of an anabolic interaction induced on bone modeling and mechanostatic interrelationships. This compound also prevented the OX- or disuse-induced impairment in rat cortical long-bone strength and recovered rat cortical bone when given since 3 months after OX by improving only bone material quality. No interaction with bone mechanostat was detected in these studies. Alendronate effects on bone biomechanics in normal rats and dogs were positive only in long treatments. They were highly dependent on body weight of the animals, hence a positive interaction with bone mechanostat should be hypothesized. It also prevented the negative impact of OX in rat femurs by improving cortical material quality with no effect on cross-sectional properties, i.e., exerting an anti-catabolic interaction with bone mechanostat. The effects of all the three compounds were found positive for bone health, yet their mechanisms of action varied with type of bone and subject condition. A striking dissociation between (positive) effects on bone strength and (variable) effects on bone stiffness was repeatedly observed in these studies. Also an enla

Enhanced Erythropoietin Response to Acute Hypoxemia in Mice with Pharmacological Depression of Erythropoiesis.

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).

Depression of stimulated erythropoietin production in mice with enhanced erythropoiesis.

BACKGROUND: The reports of lower plasma erythropoietin (EPO) in anemic patients with active erythropoiesis (hyperplastic) than in comparably anemic subjects with erythroid hypoplasia have generally been interpreted as the result of EPO utilization by the target cells of the hormone. An alternative explanation could be that there is a feedback mechanism through which EPO formation by EPO-producing cells is modulated by the erythroid activity of the erythropoietic organs. 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, the blood viscosity and the plasma EPO half-life are within normal values in spite of an intense stimulation of erythropoiesis. MATERIALS AND METHODS: Adult female mice of the CF1 strain with either normal or increased rates of erythropoiesis were used in this study. Erythropoiesis was stimulated by two injections of 10 units of rhEPO given 24 h apart. All experimental determinations were performed 24 h after the second EPO injection. Erythropoiesis was measured by the percent of a tracer dose of 59Fe incorporated into the spleen. Hypobaric hypoxemia was induced by exposing mice to atmospheric air maintained at 50% atmospheric pressure for 6 h. Plasma EPO concentration was determined by RIA. Plasma disappearance of radiolabeled rhEPO was determined by i.v. injection of the hormone and sampling by cardiac puncture every hour for 6 h. RESULTS: Administration of rhEPO to mice increased splenic 59Fe uptake significantly without affecting the hematocrit, the plasma EPO level or the plasma disappearance of radiolabeled EPO. Plasma EPO titer after 6 h of exposure to hypobaric air was about 70% lower in mice with EPO-induced stimulation of erythropoiesis than in mice with normal erythropoiesis. CONCLUSIONS: The results of this study suggest that there is an inverse relationship between the rate of stimulated EPO production and erythropoietic marrow activity. They also suggest that the variations in plasma EPO levels during periods of rapidly increasing erythropoiesis are the reflection of a decrease in the rate of production rather than an increase in the rate of utilization by a proliferating pool of erythroid cells.

Body weight loss during acute hypoxia: effects of increased convective oxygen transport or previous acclimation.

Body weight loss and growth retardation occur in rats exposed to simulated high altitude, which may be related to the hypoxemia-induced reduction in the convective oxygen transport (COT). The present study was thus performed to determine whether transfusion polycythemia, increased affinity of hemoglobin for oxygen, or previous acclimation to hypobaria (factors that increase COT) are able to counteract its effect on body weight during the early period of exposure, which appears to be a suitable parameter to test the effectiveness of acclimatization. Polycythemia was induced in weanling rats by two ip injections of 2.5 ml/100 g b.wt of packed homologous red cells. The rise in hemoglobin O2 affinity was brought about in adult rats by giving them 0.5 g/dl sodium cyanate in the drinking water for 3 weeks. A lower body weight loss during the early period of exposure to hypobaria was seen in treated rats than in controls. However, body weight loss was still important, which would indicate that compensation was probably not complete. When growing rats were acclimated to simulated altitude, a sudden increase in body weight was observed when they were brought back to ground levels. When animals were taken to altitude again, they lost weight at a rate not significantly different to that found in non-acclimated ones. The results obtained indicate that treatments do not prevent the studied effect of hypoxia and suggest that hypophagia and the resultant initial body weight loss and secondary depression of body growth could be considered as protective mechanisms against the environmental challenge, although further investigation will be necessary to confirm the hypothesis.

Body weight loss during acute hypoxia: effects of increased convective oxygen transport or previous acclimation

La pérdida de peso corporal y el retardo del crecimiento corporal que se observan en ratas expuestas a condiciones de altura simulada podrían estar relacionados con la reducción del transporte convectivo de O2 (COT) inducida por hipoxemia. El presente estudio fue realizado para determinar si la policitemia transfusional, el incremento de la afinidad de la hemoglobina por el O2, o la aclimatación previa a hipobaria (factores que incrementan (COT) son capaces de contrarrestar los efectos señalados sobre el peso corporal durante el "período inicial" de la exposición, el que puede se considerado como um parámetro útil para comprobar la efectividad de la aclimatación. La policitemia fue inducida en ratas jóvenes mediante dos inyecciones ip de 2,5 ml/100g de suspensión de eritrócitos homólogos al 80 por ciento. La disminución de la P50 fue inducida en ratas adultas mediante la administración de o.5g/dl de cianato de sodio en el agua de bebida durante 3 semanas. Ambos tratamientos indujeron una menor pérdida de peso lo que sugeriría que la compensación fue probablemente insuficiente. Cuando ratas jóvenes fueron aclimatadas a condiciones de altura simulada, se observó un marcado incremento del peso corporal cuando perdieron peso en la misma proporción que la observada en animales controles no aclimatados previamente. Los resultados obtenidos indican que el incremento del COT no previene los efectos estudiados de la exposición a hipobária y sugieren que la hipofagia y la pérdida inicial de peso, así como la depressión secundaria del crecimiento corporal, podrían ser consideradas como un mecanismo protector contra la hipoxia resultante

Body weight loss during acute hypoxia: effects of increased convective oxygen transport or previous acclimation

La pérdida de peso corporal y el retardo del crecimiento corporal que se observan en ratas expuestas a condiciones de altura simulada podrían estar relacionados con la reducción del transporte convectivo de O2 (COT) inducida por hipoxemia. El presente estudio fue realizado para determinar si la policitemia transfusional, el incremento de la afinidad de la hemoglobina por el O2, o la aclimatación previa a hipobaria (factores que incrementan (COT) son capaces de contrarrestar los efectos señalados sobre el peso corporal durante el "período inicial" de la exposición, el que puede se considerado como um parámetro útil para comprobar la efectividad de la aclimatación. La policitemia fue inducida en ratas jóvenes mediante dos inyecciones ip de 2,5 ml/100g de suspensión de eritrócitos homólogos al 80 por ciento. La disminución de la P50 fue inducida en ratas adultas mediante la administración de o.5g/dl de cianato de sodio en el agua de bebida durante 3 semanas. Ambos tratamientos indujeron una menor pérdida de peso lo que sugeriría que la compensación fue probablemente insuficiente. Cuando ratas jóvenes fueron aclimatadas a condiciones de altura simulada, se observó un marcado incremento del peso corporal cuando perdieron peso en la misma proporción que la observada en animales controles no aclimatados previamente. Los resultados obtenidos indican que el incremento del COT no previene los efectos estudiados de la exposición a hipobária y sugieren que la hipofagia y la pérdida inicial de peso, así como la depressión secundaria del crecimiento corporal, podrían ser consideradas como un mecanismo protector contra la hipoxia resultante (AU)

Body weight loss during acute hypoxia: effects of increased convective oxygen transport or previous acclimation.

Body weight loss and growth retardation occur in rats exposed to simulated high altitude, which may be related to the hypoxemia-induced reduction in the convective oxygen transport (COT). The present study was thus performed to determine whether transfusion polycythemia, increased affinity of hemoglobin for oxygen, or previous acclimation to hypobaria (factors that increase COT) are able to counteract its effect on body weight during the early period of exposure, which appears to be a suitable parameter to test the effectiveness of acclimatization. Polycythemia was induced in weanling rats by two ip injections of 2.5 ml/100 g b.wt of packed homologous red cells. The rise in hemoglobin O2 affinity was brought about in adult rats by giving them 0.5 g/dl sodium cyanate in the drinking water for 3 weeks. A lower body weight loss during the early period of exposure to hypobaria was seen in treated rats than in controls. However, body weight loss was still important, which would indicate that compensation was probably not complete. When growing rats were acclimated to simulated altitude, a sudden increase in body weight was observed when they were brought back to ground levels. When animals were taken to altitude again, they lost weight at a rate not significantly different to that found in non-acclimated ones. The results obtained indicate that treatments do not prevent the studied effect of hypoxia and suggest that hypophagia and the resultant initial body weight loss and secondary depression of body growth could be considered as protective mechanisms against the environmental challenge, although further investigation will be necessary to confirm the hypothesis.

Inhibitory effect of an alpha 1-adrenergic antagonist on erythropoiesis in normoxic or hypoxic mice.

The present study was undertaken to assess the effect of prazosin, a selective postsynaptic alpha 1-adrenergic receptor blocking agent, on normoxic and hypoxic mice, in order to evaluate experimentally its use in the treatment of the excessive erythrocytosis that characterizes chronic mountain sickness. The drug, injected intraperitoneally to adult mice at a dose of 400 micrograms/kg per day, induced a significant depression of the rate or erythropoiesis, as measured by red blood cell 59iron uptake, with a decrease in the hematocrit from the 3rd day. The drug also inhibited the oxygen-dependent secretion of erythropoietin (estimated by the plasma immunoreactive hormone concentration) in hypoxemic mice when injected between 0 and 2 h after initiation of the hypoxic stimulation. When injected daily into mice exposed to intermittent hypobaric hypoxia, prazosin limited the degree of polycythemia or induced a sustained decrease in the hematocrit when polycythemia was already present due to previous exposure. It is postulated that the drug, by reducing the peripheral vascular resistance seen during hypoxia, could increase renal blood flow, thus improving the renal oxygen supply and partially restoring the imbalance between gas supply and demand, which drives erythropoietin formation.

Additive effects of dietary protein and energy deficiencies on mandibular growth in the weanling rat.

Dietary protein restriction adversely affects mandibular growth in the weanling rat. Protein deficiency is usually accompanied by reduced food intake which, in turn, induces energy deficiency. The present study was thus designed to dissociate the effects of dietary protein and energy deficiencies on the growth of the mandible in rapidly growing rats. Four groups of Sprague-Dawley rats aged 30 days were fed a normal diet, a low-energy diet, a low protein diet, and a low-protein and low-energy diet for 20 days. Rats were sacrificed at the end of experimental period and body weight and mandibular dimensions were recorded to evaluate body growth and mandibular growth. The growth of the mandible was affected almost in the same order of magnitude by both protein and energy restrictions. When both were applied together, mandibular growth was even more severely affected. Two way analysis of variance revealed the absence of synergism between variables, indicating that the negative effects of dietary protein and energy restrictions on mandibular growth could be considered to be additive.