Foetal haemopoiesis during the hepatic period. III. Erythroid cell kinetics

Serial sections of haemopoietic liver from 12 to 17 days old mice embryos were studied. The percentual values of cycling erythroid cells, located at vascular level, show a continuous decay through the entire hepatic phase of erythropoiesis. Since the 15th day onwards no differences were found in the cellular blood composition between the general circulation and the vascular channels of the central area of the growing lobes. Nevertheless, other more peripheric areas of the liver have a slowed decrease of circulating young cells, when compared with the former. On the contrary, in the marginal zones, interstitial proerythroblasts increase their percentual values towards the 13th day, coinciding with the moment on which they predominate all over other more mature demes. This increment is not explainable by self renewal only, since their mitotic indexes undergo a decay, which is unexpected in a growing population. Interstitial non erythroid cells seem not to play a role in the increment of proerythroblasts in the proliferating areas.

Foetal haemopoiesis during the hepatic period. III. Erythroid cell kinetics.

Serial sections of haemopoietic liver from 12 to 17 days old mice embryos were studied. The percentual values of cycling erythroid cells, located at vascular level, show a continuous decay through the entire hepatic phase of erythropoiesis. Since the 15th day onwards no differences were found in the cellular blood composition between the general circulation and the vascular channels of the central area of the growing lobes. Nevertheless, other more peripheric areas of the liver have a slowed decrease of circulating young cells, when compared with the former. On the contrary, in the marginal zones, interstitial proerythroblasts increase their percentual values towards the 13th day, coinciding with the moment on which they predominate all over other more mature demes. This increment is not explainable by self renewal only, since their mitotic indexes undergo a decay, which is unexpected in a growing population. Interstitial non erythroid cells seem not to play a role in the increment of proerythroblasts in the proliferating areas.

Ultrastructural changes in somatotropic cells induced by anemic or hypoxic hypoxia.

Somatotropic cells from three months old male mice, made hypoxic by either bleeding or exposure to 0.5 atm. barometric pressure in a decompression chamber, showed important ultrastructural changes when compared with normoxic controls, during the 36 hours that follow initiation of hypoxia. These changes were found to be similar for both types of hypoxic animals. An equivalent number of dark cells, cells with a swollen rough endoplasmic reticulum (RER), extremely granulated and degranulated, were successively--and in this order--seen along the experiment. Most of the observed differences between experimental and control animals appears as a magnification of the normal circadian rhythms. The degranulation rate, which probably reflects growth hormone (GH) secretion rate, was found to be about three times greater in hypoxic than in control mice. These findings indicate that a decrease in the red cell mass or its consequence (hypoxia), induce important ultrastructural variations in somatotropic (STH) cells, which may or not be specific.

Ultrastructural changes in somatotropic cells induced by anemic or hypoxic hypoxia.

Las celulas somatotropas de ratones machos de 3 meses de edad mostraron variaciones ultraestructurales importantes tanto al provocar anemia como al someter a los animales a hipoxia hipobarica. Esos cambios, observados durante las primeras 36 horas de iniciado el tratamiento, fueron similares en las dos clases de animales hipoxicos. En forma sucesiva se pudo ver: un numero equivalente de celulas obscuras, celulas con el RER dilatado, extremadamente granuladas y desgranuladas, interpretandose la mayor parte de las diferencias entre animales tratados y controles como una magnificacion de los ritmos circadianos normales. La desgranulacion celular - reflejo de la secrecion de hormona de crecimiento - fue 3 veces mayor en los animales hipoxicos que en los testigos. Estos hallazgos indican que una disminucion real de la masa roja circulante o su consecuencia (hipoxia) induce variaciones ultraestructurales importantes en las celulas somatotropas de la adenohipofisis, que pueden (o no) ser especificas

Unmodified erythropoietic response to a beta adrenergic agonist in hypothyroid mice

Los agonistas beta-adrenérgicos poseen la propriedad de estimular la eritropoyesis en el modelo del ratón policitémico mediante inducción de la secreción de eritropoyetina. Considerando la existencia de un cúmulo de evidencias que indica que las hormonas tiroideas y las catecolaminas están íntimamente relacionadas, el objetivo del presente trabajo fue estimar la potencia eritropoyética del isoproterenol, un agonista beta-adrenérgico bien conocido, en ratones hipotiroideos. Animales adultos de la cepa CF-1 fueron alimentados con dieta estandard para roedores y agua (eutiroideos) o solución al 0.1 por ciento de propiltiouracilo durante 37 dYas. La concentración de T4 en plasma, medida por RIA, fue 1.75 mug/ml y < 1.0 mug/ml en ratones eutiroideos e hipotiroideos, respectivamente. Los animales fueron transfundidos con 1.0 ml de eritrocitos homólogos y el efecto eritropoyético de 50, 500 o 5000 pg/kg de isoproterenol estimado mediante el método de incorporación de (59)Fe al eritrón. No se observaron diferencias estadísticamente significativas (P<0.05, test de t no apareado) entre animales eutiroideos e hipotiroideos. El hipotiroidismo, por lo tanto, no afecta la secreción de eritropoyetina inducida por isoproterenol en las presentes condiciones experimentales. (AU)

Suppressive effect of acetylsalicylic acid on erythropoietin-responsive cells in mice.

The time-response curve for RBC-59Fe uptake following i.p. injections of 3 doses of 5 mg of acetylsalicylic acid (ASA) at 4 hour interval into normal, nonpolycythemic mice, shows a maximal depression (35

of normal) at 3 days after ASA with return to almost normal values by 7 days. The effect is dose-related, showing a plateau with doses of ASA above 5 mg/4 hr. The shape of the time-response curve indicates that the more mature cells in the erythron are not affected by ASA and that the major effect of the drug must be on earlier erythroid cells. Administration of ASA prior to administration of erythropoietin (Epo) into post-hypoxic polycythemic mice depresses the incorporation of 59Fe into erythrocytes. The depression of radioiron uptake is similar when ASA is given prior to or simultaneously with Epo. When ASA is given 24 hr after injection of Epo, suppression is less marked. These results suggest a suppressive effect of the drug on the erythropoietin-responsive cells (ERC).

Immunoreactive erythropoietin in rodent submaxillary gland. Autonomic control of exocrine secretion and factors influencing its concentration

The SMG of mice and rats contain a heterologous group of biologically active factors. Some are well known, can be obtained at high purity and are well-characterized. There is strong evidence for the presence of others although they have not been purified. Finally, some of them are questionable and/or have not yet been characterized. EPO would be one of the factors whose presence in the SMG is strongly suspected, although its biological activity has not been demonstrated yet. Its presence in the gland, therefore, is only supported by radioimmunoassay data and immunocytochemical methods. Immunoreactive EPO is undetectable in the mouse SMG until the 30th day of postnatal life, increasing thereafter at a uniform rate and reaching adult levels by 50-60 days of age. The parallelism between its concentration in extracts of the gland, the size and relative proportion of GCT cells, could be accepted as indirect evidence for its localization in these cells. The rise in iEPO concentration in SMGs after androgen treatment, its fall following orchiectomy, and its reduction after duct ligation in proportion to the degree of degranulation of GCT cells lend support to the above hypothesis. Salivary secretions induced by either NE or ISO contain high levels of iEPO. A significant depletion of gland content is also observed. These two sets of data indicate that SMG exocrine iEPO secretion occurs and that this secretion is mediated by adrenergic receptors. The question whether the SMG also functions as an endocrine organ in relation to EPO can not be answered at present.

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.

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)

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)

Immunoreactive erythropoietin in rodent submaxillary gland. Autonomic control of exocrine secretion and factors influencing its concentration.

The SMG of mice and rats contain a heterologous group of biologically active factors. Some are well known, can be obtained at high purity and are well-characterized. There is strong evidence for the presence of others although they have not been purified. Finally, some of them are questionable and/or have not yet been characterized. EPO would be one of the factors whose presence in the SMG is strongly suspected, although its biological activity has not been demonstrated yet. Its presence in the gland, therefore, is only supported by radioimmunoassay data and immunocytochemical methods. Immunoreactive EPO is undetectable in the mouse SMG until the 30th day of postnatal life, increasing thereafter at a uniform rate and reaching adult levels by 50-60 days of age. The parallelism between its concentration in extracts of the gland, the size and relative proportion of GCT cells, could be accepted as indirect evidence for its localization in these cells. The rise in iEPO concentration in SMGs after androgen treatment, its fall following orchiectomy, and its reduction after duct ligation in proportion to the degree of degranulation of GCT cells lend support to the above hypothesis. Salivary secretions induced by either NE or ISO contain high levels of iEPO. A significant depletion of gland content is also observed. These two sets of data indicate that SMG exocrine iEPO secretion occurs and that this secretion is mediated by adrenergic receptors. The question whether the SMG also functions as an endocrine organ in relation to EPO can not be answered at present.

Depressed plasma erythropoietin levels in rats with hemodynamically-mediated acute renal failure.

Adult female Wistar rats were injected with 125 mg/kg b.w. of human methemoglobin (M-Hb) in order to induce a first episode of hemodynamically-mediated acute renal failure (HMARF). Eleven days after the injection of M-Hb, other groups of rats received another equal dose of the drug in order to induce a second episode of HMARF. Evaluation of renal function, histopathology studies, and determinations of plasma and kidney erythropoietin (Epo) titers by radioimmunoassay in normoxic and hypoxic conditions were performed 1, 2, 3, 5 and 10 days after M-Hb administration. Treatment induced transient increases in plasma urea concentration, fractional sodium excretion, and urine volume, and significant depression in urine osmolality. In every case, the maximal effect of the first injection of M-Hb on the individual parameters was always greater than that of the second injection, and observed on the 5th post-injection day. Histologic sections showed interstitial cellular infiltration, desquamation of the proximal tubular epithelium and collapse or dilation of the tubular lumen. Treatment with M-Hb depressed Epo titers in both kidney homogenates and plasma in normoxic as well as hypoxic rats. Here again, the effect of the first injection of the drug was higher than that of the second one. These observations indicate that there is a negative correlation between kidney tubule injury and Epo production in normoxic and hypoxic conditions. The findings give support to the concept that Epo production is related to proximal tubular function.

Additive effects of dietary protein and energy deficiencies on diaphysis and bone tissue of rat femurs as determined by bending tests.

The present study was designed to study the effects of diets moderately restricted in protein and/or carbohydrate derived calories on morphometric parameters (geometric properties) and mechanical performance of both diaphysis (structural properties) and cortical bone tissue (material properties) from growing rat femurs, as determined by bending tests at low strain rates. Male rats aged 30 days were divided in four groups, namely NN = normal protein and energy, NPLE = normal protein and low energy, LP = low protein and normal energy, and LPLE = low protein and low energy. Each group was fed on a corresponding diet for 20 days. Both body weight and femoral length were greater in NN and lower in LL than in LE and LP groups. Geometrical and structural variables (with the exception of wall/lumen ratio) grossly paralleled changes in body weight, while material properties showed independent and less significant changes. Therefore, the assayed levels of restriction of either plastic or energetic nutrients seemed to alter bone biomechanics proportionally to the way it affected bone growth.

Growth retardation in the early developing rat exposed to continuous hypobaric hypoxia.

Weanling male rats weighing 48.5 +/- 1.4 g were divided into two groups, hypoxic and normoxic. The former was placed into an altitude chamber and maintained at a pressure equivalent to 0.45 atm. (6 100 m) over a period of 23 days. The normoxic group was maintained at sea level conditions. Food intake, body weight, body length and tail length were recorded every day. Body weight gain in hypoxic rats was 35

of that seen in normoxic controls at the end of the experimental period. Body length gain was 55

and tail length gain was 59

of normal at the same time. The amount of food eaten by the hypoxic animals during the entire exposure period was 55

of that consumed by normoxic ones. The average daily caloric intake related to metabolic body weight (appetite quotient) of hypoxic rats was 60

of the normoxic control value. Efficiency of protein utilization was not significantly different between both groups of rats. These results indicate that exposure to hypobaric hypoxia induces growth retardation in the rat, which appears to be the result of a diminution in food intake because of a decreased appetite.

Effect of acute exposure to reduced atmospheric pressures on body weight, food intake and body composition of growing rats.

Growing male rats were exposed to simulated altitudes of 1850, 2900, 4100, 5450 or 7100 m in a hypobaric chamber to determine the effects of altitude on body weight gain and food intake as function of time of exposure. Female rats were exposed to a simulated altitude of 7100 m for 24 h to determine the effect of altitude on body composition. The results obtained indicate that in growing rats exposed to acute simulated altitude the initial body weight loss and the depressed growth rate, on one hand, and the reduced food intake, on the other hand, are related to the degree of the altitude; the parameters are not affected at altitudes below 1 850 m; the initial weight loss is not solely due to reduction in food intake, the additional loss being attributed to the added stress of hypoxia; the body weight loss occurs without marked alterations in body composition, although a tendency to dehydration exists; and the body compositional changes are the reflection of the altitude-induced hypophagia.

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.

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.

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.