Stimulation of dental pulp with simultaneous recording of the jaw opening reflex in the rat.

The processing of nociceptive information in the central nervous system has been analysed in most studies by activation of peripheral nerves. However, the limitation of this method is the simultaneous activation of noxious and inocuous fibers. Nevertheless, the stimulation of the tooth pulp is believed to activate mainly nociceptive fibers which could be the method of choice. On the other hand, the response to nociceptive activation of the dental pulp is easily quantified by the amplitude of the jaw opening reflex, a nocifensive flexion withdrawal reflex. In this protocol we describe a technique to manufacture and implant electrodes in lower incisors of the rat and a method to prepare and insert stainless steel twisted bipolar electrodes to record the electromyographic activity of both digastric muscles, in response to nociceptive dental pulp stimulation. This approach was applied in the study of the analgesic effects of the rat's striatal stimulation.

Striatal modulation of the jaw opening reflex.

The effect of striatal electrical and chemical conditioning stimulation (L-glutamate 80-160 nmoles/0.5 microl) on the jaw opening reflex (JOR) was studied in Sprague-Dawley male rats anesthetized with urethane. The JOR was evoked by stimulation of the tooth pulp of lower incisors. This response was suppressed by transection of the dental root, which indicates according with the bibliography, a specific activation of the pulp nerves. Three type of responses were obtained on the evoked JOR by conditioning stimulation of the striatum; being the main one the suppression of the reflex elicited by tooth pulp activation. A second type of response was an increase of the tooth-JOR amplitude. This effect was observed more frequently with glutamate stimulation rather than with electrical activation of the striatum. A third response was observed with chemical stimulation but not by electrical stimulation of the striatum. This was a triphasic response which consisted in an increase followed by an inhibition and a late increase of the tooth-JOR amplitude. A biphasic effect, an increase prior to a decrease of the JOR amplitude, was also recorded with a minor frequency. The distribution of effective sites for electrical and chemical stimulation within the striatum are mainly similar located in the rostral aspect of the nucleus, with the inhibitory sites in the middle of the nucleus and intermingled with the excitatory ones. The complex responses (tri/biphasic) were observed ventrally and caudally in the nucleus. On the basis of the results mentioned above, one could assume that the striatum is related to the modulation of the JOR evoked probably by nociceptive stimulation. However, activation of other type of fibers could not be ruled out.

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(AU)

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 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.

Unmodified erythropoietic response to a beta adrenergic agonist in hypothyroid mice.

beta-adrenergic agonists are able to increase erythropoiesis in the polycythemic mouse model by possibly increasing erythropoietin secretion. Since a great deal of evidence indicates that the actions of thyroid hormones and catecholamines are intimately interrelated, the present study was designed to estimate the erythropoietic response to isoproterenol, a very well-known beta-adrenergic agonist, in hypothyroid mice. Adult male CF-1 mice, maintained on a standard rodent chow and water (euthyroid) or 0.1% propylthiouracil (PTU) solution (hypothyroid) ad libitum during 37 days. Plasma T4 concentration was 1.75 +/- 0.25 micrograms/ml in euthyroid and < 1.0 microgram/ml in hypothyroid mice at this time. Mice were transfused with 1.0 ml of packed homologous red cells and the erythropoietic effect of graded doses (50, 500 and 5000 micrograms/kg) were tested by the RBC-59Fe incorporation method. No statistically significant differences (unpaired t test) were found between euthyroid and hypothyroid mice. Hypothyroidism, therefore, does not affect beta-adrenergic agonist-induced erythropoietin secretion in the present experimental conditions.

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)

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 dÝas. 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.

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.

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.

Impaired response of polycythemic mice to erythropoietin induced by protein starvation imposed after hormone administration.

The present study was performed to determine the stage of the erythropoietic pathway which is affected by starvation or protein deprivation and whose manifestation is a depressed response to exogenous erythropoietin (EPO). The response to recombinant human EPO was measured in post-hypoxic polycythemic mice by determination of 59Fe uptake into red cells, spleen and femur and/or erythroid colony forming units (CFU-E) and erythroid precursor cell concentrations in femoral marrow. Experimental mice were either starved or fed one of seven different diets whose protein (casein) content ranged from 0 to 20%. All diets were isocaloric. The response of mice maintained on the standard diet (Purina Lab chow) was taken as the normal one. Starvation during the 48-hour period immediately before EPO injection had no effect on the response to the hormone. Starvation, and protein deprivation to a lesser extent, during the 48-hour period following EPO, on the other hand, significantly reduced the response. There was a progressive increase in the response as the casein content of the diet was increased. A normal response was observed when dietary casein concentration was 10%. These findings indicate that nutritional deprivation or dietary protein alterations during the period immediately following EPO injection in polycythemic mice can have detrimental effects on the erythroid response in a model in which nutritional deprivation was relatively short and acute. They also indicate that the subnormal response is not due to a decreased size of the erythroid progenitor pool available for differentiation but to deficient rates of differentiation of erythropoietic units.

Damage of tracer erythropoietin results in erroneous estimation of concentration in mouse submaxillary gland.

It has been previously reported that 1) plasma erythropoietin (Epo) titer during exposure to hypobaria is lower in nephrectomized rats and mice whose submaxillary glands (SMG) were either ablated or atrophied than in nephrectomized controls whose SMG were intact and 2) that the gland shows one of the highest levels of immunoreactive Epo (iEpo) in the body. The latter observation, however, was questioned recently when it was observed that SMG extracts degrade labeled Epo used as tracer antigen in the radioimmunoassay (RIA), thus giving invalid estimates of Epo. Since this interpretation was in turn questioned, the present study was conducted to obtain more information on the subject and make these conflicting points clear. Investigation of the reported/possible degradation of Epo by SMG homogenates was conducted via polyacrylamide gel electrophoresis followed by radioautography or by a RIA in solid phase in which there was no simultaneous incubation of the tracer antigen with the SMG homogenates. It was observed that 125I-labeled rhEpo was degraded when incubated with SMG homogenates. Degradation was rapid, being evident when incubation lasted 30 minutes, and occurred in the presence of a protease inhibitor. It showed a high degree of specificity since it did not occur when Epo was incubated with kidney homogenate or normal mouse serum. SMG homogenate did not degrade labeled thyrotrophic hormone and degraded alpha interferon (IFN-alpha) only partially. When estimates of iEpo in SMG homogenate were performed in conditions of simultaneous (SI-RIA) or nonsimultaneous (NSI-RIA) incubation of the homogenate with tracer Epo, it was observed that while estimates of Epo in plasma were similar in both types of RIA and somewhat higher in kidney homogenate in the SI-RIA than in the NSI-RIA, estimates of Epo in SMG were about 60 times higher in the former than in the latter. Therefore, it could be concluded that most of the Epo detected by standard RIA in SMG homogenate does not represent true Epo because of damage of tracer Epo which determines loss of the integrity of the RIA system.

Higher erythropoietin secretion in response to cobaltous chloride in post-hypoxic than in hypertransfused polycythemic mice.

BACKGROUND: We have shown previously that both erythrocyte production rate (EPR) and plasma erythropoietin (EPO) levels in response to hypoxia or to compounds able to stimulate EPO secretion are very much higher in post-hypoxic (PH) than in hypertransfused (HT) polycythemic mice with similar levels of hematocrit. Since it has been demonstrated that cobalt (Co) treatment rises renal EPO-mRNA and increases plasma EPO levels, the present study was conducted to determine whether there is a difference between PH and HT mice in relation to the erythropoietic response to Co and whether the stimulatory effect of Co on EPO secretion can be blunted by polycythemia. METHODS: Adult female mice of the CF-1 strain were made polycythemic by either exposing them to 270 h of discontinuous hypoxia (18 h/d) in a hypobaric chamber maintained at 456 hPA (PH mice) or by injecting them with 0.8 ml of washed packed red cells on two consecutive days (HT mice). Measurement of the erythrocyte production rate (EPR) was made by RBC-59Fe uptake. Plasma EPO concentration was determined by RIA. Cobalt chloride (CoC12) was dissolved in saline and injected in doses of 4 and 8 umoles/mouse. Recombinant human EPO (HEMAX 4000, Bio Sidus SA, Argentina) was dissolved in PBS + albumin to the desired concentration. RESULTS: By comparison with the corresponding dose-regression line for rHu-EPO, it was estimated that the responses (EPR) (measured as RBC-59Fe incorporation) of PH mice to sc injections of 4 and 8 umoles of CoC12 were equivalent to 95 and 145 mU of rHu-EPO, respectively. The response of HT mice to 4 umoles of the drug was not detectable. At the upper dose level, the response was equivalent to 52 mU of rHuEPO. Plasma immunoreactive EPO (iEPO) titers 12 h after COC12 (8 umoles) were not significantly different between normocythemic and PH mice. The observed values were significantly higher than those found in HT mice. DISCUSSION: These findings demonstrate that EPO production in response to COC12 is depressed by polycythemia when induced by transfusion but not when induced by chronic exposure to hypobaric hypoxia. They also confirm, but not explain the nature of the conditioning effect of exposure to hypoxia which makes the mechanism controlling EPO secretion either more sensitive to EPO-secreting stimuli or unable to recognize the polycythemic state.

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.

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.

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.