Effects of postirradiation carboxymethylglucan administration in mice.

The hemopoiesis-enhancing ability of a soluble glucan derivative, i.e. carboxymethylglucan (CMG), was investigated in gamma-irradiated mice. Attention was focused on the usefulness of its single or repeated postirradiation administration. CMG was administered i.p. at (a) single dose of 6 mg 2 h postirradiation, (b) four 6 mg doses in the first 4 days postirradiation, (c) four 1.5 mg doses at the same time intervals. Indices of granulopoiesis and inflammatory side effects (liver weight increase and hepatic granulomas) were investigated in mice irradiated with a sublethal dose of 7 Gy. All three CMG-treated groups of mice were found to exhibit enhanced hemopoietic recovery in comparison with the controls. Although the mice repeatedly given the 6 mg CMG doses showed the most rapid recoveries of all the evaluated parameters of granulopoiesis, the most pronounced hepatic side effects were found in these mice, too. When survival of mice was recorded in lethally (9 Gy) irradiated animals, the best protective response were obtained following the repeated administration of the 1.5 mg CMG dose, the survival by day 30 in this group being significantly higher not only in comparison with the controls but also with the mice repeatedly given the 6 mg dose of CMG. The results suggest that the postirradiation CMG administration can be useful for enhancing radiation suppressed hemopoiesis. However, repeated larger CMG doses may produce side effects which compromise the overall survival of irradiated mice.

Effects of sublethal irradiation with helium ions (300 MeV/nucleon) on basic hematological parameters of mice.

The aim of the experiment was to obtain new knowledge on the biological effectiveness of high-energy (300 MeV/nucleon) helium ions, which represent a part of the spectrum of cosmic rays. Male (CBA x C57BL)F1 mice, 4 months old, were exposed to a dose of 4 Gy helium ions (exposure rate 0.05 Gy/min). As a comparative standard irradiation the same dose of 4 Gy of 137Cs gamma-rays (exposure rate 0.07 Gy/min) was used. Material sampling was performed 6-8 h, 4 days and 9 days after irradiation for both experimental groups mentioned above. There were 7 animals in each group including the control group of non-irradiated mice. Eight basic hematological parameters of peripheral blood, bone marrow, spleen and thymus were studied. On day 4 after the irradiation with helium ions, the values of leukocyte counts in peripheral blood, bone marrow cellularity and spleen cellularity were reduced to about 10% of the respective control values while the decline after irradiation with gamma-rays amounted to about 50%. These and other results presented reflect a high relative biological effectiveness of 300 MeV/nucleon helium ions.

Hematopoietic recovery in repeatedly irradiated mice can be enhanced by a repeatedly administered combination of diclofenac and glucan.

A combination of diclofenac and glucan administered repeatedly in a protective regimen in the course of repeated gamma irradiation of mice (6 x 2 Gy during 3 weeks) enhanced granulopoiesis and other indices of hematopoietic recovery investigated from 3 to 7 days after the last radiation exposure. Repeated administration of diclofenac or glucan alone or treatment of the mice with the diclofenac-glucan combination given once before the first or the last radiation exposure did not induce such effects. The protective effect of the repeatedly administered combination of the drugs was realized despite the fact that the response of the serum colony-stimulating activity to the repeated combined drug administration was decreased at the end of the treatment regimen compared to that of mice given this drug combination only once. The combined treatment is supposed to act via increased proliferation of the hematopoietic stem or progenitor cells. Additivity or even synergism of the hematostimulatory action of glucan and of the strengthening of positive control of cell proliferation achieved by removing negatively acting prostaglandins (diclofenac action) may account for the radioprotective effects observed.

Hyporesponsiveness of murine myeloid progenitor cells to glucan following its repeated administration.

A moderate marrow granulocytic hyperplasia developed after 4 injections of glucan (soluble derivative carboxymethylglucan) administered to mice at 3-4-day intervals. However, when evaluating the response of the marrow granulocyte-macrophage colony-forming cells (GM-CFC) to the fifth glucan injection given in the repetitive treatment schedule, the development of hyporesponsiveness of these cells was found, contrary to the stimulatory action of a single glucan injection. In addition, the prompt increase in serum granulocyte-macrophage colony-stimulating activity occurring after a single glucan injection, and proposed to upregulate myelopoiesis, was absent in mice treated with glucan repeatedly. A joint administration of glucan with diclofenac, an inhibitor of prostaglandin synthesis, was able to enhance the GM-CFC response to a single glucan injection, probably due to the removal of the downregulation action of prostaglandins on these progenitor cells. However, a repeated administration of diclofenac with glucan did not reduce substantially the development of GM-CFC hyporesponsiveness. Thus, the results suggest that the GM-CFC hyporesponsiveness or tolerance to repeated glucan injections was induced by weakening the mechanisms of positive control mediated by the serum colony-stimulating activity.

Noradrenaline reduces cardiovascular effects of the combined dipyridamole and AMP administration but preserves radioprotective effects of these drugs on hematopoiesis in mice.

Recent results of the authors have demonstrated that the elevation of extracellular adenosine induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), a soluble adenosine prodrug, mediates radioprotective effects in mice. Furthermore, it has been shown that this action is induced by at least two mechanisms: (1) protection by hypoxia as a result of the effects of treatment on the cardiovascular system (bradycardia, vasodilation), and (2) an enhanced regeneration of the radiation-perturbed hematopoiesis. Here, it was ascertained that the joint use of an optimal dose of noradrenaline given with dipyridamole and AMP combination eliminates the hypothermic and hypoxic effects of the treatment, but preserves the radioprotective action of dipyridamole and AMP combination in terms of hematopoietic recovery and partially also survival enhancing effects of the drugs in gamma-irradiated mice. These findings might be of importance for attempts to obtain available and tolerable radioprotective pharmacological prescriptions for clinical use.

Enhancement of hematopoietic recovery in gamma-irradiated mice by the joint use of diclofenac, an inhibitor of prostaglandin production, and glucan, a macrophage activator.

The effects of diclofenac (inhibitor of prostaglandin production) and carboxymethylglucan (immunomodulator and an agent stimulating hematopoiesis), when given to mice 1 day before gamma-irradiation, were studied. Both of the agents were administered either alone or in combination. The investigations included the assessment of post-irradiation hematopoietic recovery in terms of bone marrow and spleen cellularity and endogenous spleen colony formation, as well as the determination of the survival of lethally irradiated mice. The results demonstrated at least additive radioprotective effects when mice were given diclofenac and carboxymethylglucan in combination. Experimental evidence provided by the increased 125iodo-deoxyuridine incorporation into the spleen and elevated hydroxyurea kill of endogenous spleen colony-forming units indicated that the beneficial action of the combined treatment could be a consequence of increased cell proliferation in the hematopoietic tissue. It is likely that the inhibition of prostaglandin production (diclofenac action) and the concomitant increased release of growth factors (glucan action) shift the regulatory balance towards the predominance of positive hematopoietic control.

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.

Effect of dipyridamole and adenosine monophosphate on cell proliferation in the hemopoietic tissue of normal and gamma-irradiated mice.

Combined treatment with dipyridamole and adenosine monophosphate enhances cell proliferation in the hemopoietic tissue of normal and gamma-irradiated mice. This effect can be explained by the elevation of extracellular adenosine, and the receptor-mediated activation of the cell adenylate cyclase system.

Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis.

An impairment of the survival of mice subjected to whole-body gamma-irradiation with a lethal dose of 10 Gy and treated with a repeated postirradiation administration of prostaglandin synthesis inhibitors (PGSIs), indomethacin or diclofenac, was observed. Morphological examination of the gastrointestinal tract and the estimation of blood loss into its lumen in animals treated with diclofenac did not show serious damage such as haemorrhages or perforation, but revealed structural injury to the intestinal mucosa indicating inflammatory processes. The lesions found are supposed to be connected with increased intestinal permeability which leads to endotoxin escape from the gut and a subsequent increased mortality rate of irradiated animals. It may be concluded that PGSIs are not suitable for the management of radiation sickness after an exposure to lethal doses of ionizing radiation.

Ultrastructure of erythroblast mitochondria in mice treated with mixtures of radioprotectors.

Unirradiated mice were given i.p. a mixture of mexamine (20 mg/kg body weight) and AET (150 mg/kg body weight) or mexamine (20 mg/kg body weight) and cystamine (150 mg/kg body weight). Acute effects of these mixtures of radioprotective agents on the ultrastructure of basophilic and polychromatic bone marrow erythroblast mitochondria were studied. One hour after injecting mice with above-mentioned mixtures, statistically significant differences in the size of mitochondria were found between control mice and mice given a mixture of mexamine and cystamine. The differences in the number of dilated cristae per micron 2 of the mitochondrion area between control mice and both experimental groups given mixtures of radioprotective agents were also statistically significant. Thus it was proved that also mixtures of radioprotectors induced acute ultrastructural changes of mitochondria which were so far described only after treating mice with only one radioprotective agent.

Hemopoiesis stimulating and radioprotective effects of carboxymethylglucan.

Carboxymethylglucan, a novel soluble derivative of beta-1,3-glucan, was found to enhance hemopoietic recovery in sublethally gamma-irradiated mice and to increase survival in lethally irradiated animals when given 24 hours prior to irradiation. Postirradiation treatment with carboxymethylglucan also induced favourable effects in terms of survival when used in combination with preirradiation cystamine administration.

The reaction of lymphatic component of hemopoiesis to fractionated irradiation and repeated bone marrow transplantation.

Mice were repeatedly subjected to individual doses of 60Co-gamma rays at intervals of four days up to total doses of 3, 6, 9, 12, 15, 18, 21 and 24 Gy. Under these conditions, signs of partial adaptation of the lymphatic component of hemopoiesis to conditions of repeated irradiations were found in mice which were given transplants of 10(6) nuclear cells of syngenetic bone marrow after each irradiation. Systematic findings of statistically significant differences in the values of the lymphatic hemopoietic component parameters studied between bone marrow recipients and animals subjected only to repeated irradiations were recorded after 24th day of experiment in mice which received total doses of 21 and 24 Gy. A positive influence of repeated transplantations in bone marrow recipients became manifested in these experimental groups already upon withdrawals made on day 4 after the last irradiation. A quite pronounced increase in the values of the bone marrow recipients' lymphatic hemopoietic component studied was then recorded upon withdrawals made on day 9 after the last irradiation.

Changes in hemopoiesis of dying and surviving mice after fractionated irradiation and repeated bone marrow transplantation.

Mice received doses of 3 Gy of 60Co-gamma rays total body irradiation at four-day intervals up to a total dose of 24 Gy. After each dose per fraction half of the animals were injected with 10(6) bone marrow cells. At four- and nine-day intervals evaluations were made of the blood count, bone marrow and spleen cellularities, and spleen mass. In animals subjected only to irradiation the damage of hemopoietic organs was becoming deeper until the end of observation; the majority of these mice died by nine days after the irradiation with the last dose per fraction (by 37 days of the experiment). The authors consider anemia as the main cause of their death. All of the mice that were given bone marrow injections survived; nine days after the last dose of irradiation the mean cellularities of their bone marrows and spleens were 76.8% and 112.3% of the unirradiated controls respectively. In general, regeneration of erythropoiesis was quite successful, the number of thrombocytes was positively influenced, and the number of leukocytes nearly unchanged in bone marrow recipients when compared with the only irradiated mice. We observed two periods of maximum and one of minimum bone marrow and spleen regeneration, which were not synchronized. These results deny an unrepairable damage to the hemopoietic microenvironment in conditions of our experiment. This paper follows up with our preceding work [10] describing results of an experiment which ended on day 24.

Haemopoiesis in murine bone marrow and spleen after fractionated irradiation and repeated bone marrow transplantation. I. Erythropoiesis.

Erythropoiesis was studied in mice repeatedly subjected to individual doses of 3 Gy of 60Co gamma-rays at 4-day intervals up to a total dose of 24 Gy on the basis of total bone marrow and spleen cellularity follow-up and analysis of myelograms and splenograms. Half of the mice received 10(6) nuclear cells of syngeneic bone marrow after each fractional radiation dose. It was mainly the spleen which was involved in the adaptation and regeneration of erythropoiesis, its contribution to total erythropoiesis in bone marrow recipients having been as much as 73.9% (day 20 of experiment, total dose 15 Gy). In mice only irradiated, the number of nuclear cells of erythroid lineage decreased to zero values sooner in the spleen (day 16 of experiment, total dose 12 Gy) when compared to the bone marrow (day 24 of experiment, total dose 18 Gy). Analysis of the results of collections made on day 9 after the last irradiation, however, revealed that the haemopoietic microenvironment of the spleen and haemopoietic cells capable of differentiation in the erythroid direction are so resistant to irradiation in only irradiated mice that erythropoiesis in their spleens exhibits signs of regeneration even after the highest total dose of 24 Gy.

Haemopoiesis in murine bone marrow and spleen after fractionated irradiation and repeated bone marrow transplantation. II. Granulopoiesis.

Granulopoiesis was studied in mice repeatedly subjected to individual doses of 3 Gy of 60Co gamma-rays at 4-day intervals up to a total dose of 24 Gy on the basis of total bone marrow cellularity follow-up and analysis of myelograms and splenograms. Half of the mice received 10(6) nuclear cells of syngeneic bone marrow after each fractional radiation dose. After an initial steep decrease, the number of granuloid cells in the spleen increased about 30-fold between days 12 and 16 of the experiment (total dose 9 and 12 Gy, respectively). This increase was temporary and between days 20 and 24 (total dose 15 and 18 Gy, respectively) a steep decrease again occurred. At a low level (below 10% of the control value) remained the granuloid cells in the spleens of bone marrow recipients until the end of the experiment (day 37, total dose 24 Gy). The behaviour of the granuloid compartment of haemopoiesis thus contrasts with findings in the erythroid compartment (see Hofer et al., 1989b) when high numbers of erythroid nuclear cells remained in the spleens of bone marrow recipients until the end of the experiment. On the whole, the influence of repeated bone marrow transplantation on granulopoiesis in the bone marrow and spleen is positive. Of the 22 comparisons made between bone marrow recipients and mice only irradiated, 14 differences are statistically significant, always in favour of bone marrow recipients.

Morphology of interactions of the haematopoietic microenvironment with the haematopoietic cells in erythroid spleen colonies.

The morphology of the interactions of the cells of the haematopoietic microenvironment with haematopoietic cells was studied in exogenous erythroid spleen colonies formed in mice, on the fifth and eighth day after their irradiation with a lethal dose of gamma rays and bone marrow transplantation. The characteristic type of stromal cell interacting with less mature cells of the erythroid series was a dark, branching reticular cell. The typical structural interaction of the reticular cells with erythroblasts was the formation of very long, fine cytoplasmic processes by the reticular cells. The processes were in close contact with the erythroblasts and formed a three-dimensional network stretching long distances from the nucleus of the reticular cells. Other cells of the haematopoietic microenvironment, in contact chiefly with poly- and ortochromic erythroblasts, were macrophages and the two together formed typical erythroblastic islands. In places, the macrophages and erythroblasts formed close, firm contacts by means of their cytoplasmic membranes. These morphological observations support the conception that close functional cooperation exists between the cells of the haematopoietic microenvironment and the haematopoietic cells in differentiation and proliferation processes in the haematopoietic tissue of spleen colonies.

A contribution to the study of damage and regeneration of hemopoiesis during fractionated irradiation and repeated bone marrow transplantation.

The experiment was aimed at studying two contradictory actions: damage to hemopoietic organs during application of radiation doses per fraction and regenerative efforts of the organism supported by repeated bone marrow transplantation. The mice received doses of 3 Gy of 60Co-gamma rays total body irradiation at four-day intervals up to a total dose of 18 Gy. After each dose per fraction half of the animals were injected with 10(6) bone marrow cells. At four-day intervals evaluations were made of the blood count, bone marrow and spleen cellularities, and spleen mass. In animals subjected only to irradiation the damage to hemopoietic organs was becoming deeper until the end of observation. In bone marrow recipients the decrease in bone marrow cellularity and the number of leukocytes stopped after day 16, and there was a steep growth of splenic cellularity and mass; the decrease in erythrocyte count stopped on day 20. Obviously a certain recovery of hemopoiesis occurred after the total dose of 12 Gy between days 12 and 16. The lodging of injected hemopoietic stem cells and their proliferation had to be preceded by a regeneration of the hemopoietic microenvironment.

Interstrain differences in the responsiveness of mice to glucan with respect to hematological effects and manifestations of late damage.

A study was made of the effect of a single s.c. injection of 4 mg glucan in the mouse strains C57BL/10, C3H and a hybrid population of mice. In mice of the strain C57BL/10 high responsiveness was demonstrated, manifested in a long-term increase in the granulocyte counts in the peripheral blood. These effects were accompanied by a fall in the mean red cell volume and the mean corpuscular hemoglobin, a fall in body weight, and mortality. 10 weeks after administration of glucan histology revealed necrotic foci in the livers of animals of the C57BL/10 strain, and an increased amount of the fibrous connective component in the pulmonary interstitium. The strain C3H exhibited a low responsiveness in respect of the hematological indices, while the hybrids did not show any significant response.

The kinetics of endogenous erythroid haemopoietic spleen colonies in mice between 4 and 10 days after irradiation with doses of 5, 7, or 9 Gy.

The kinetics of post-radiation haemopoietic tissue recovery was studied by evaluating quantitatively the results from histologic studies of the kinetics of endogenous erythroid spleen colony counts in subserial sections of spleens from mice irradiated with 5, 7, or 9 Gy of 60Co gamma-rays at intervals of 4, 6, 8, and 10 days following irradiation. Emphasis was put on individual types of colony-forming cells and on time intervals at which these cells enter into action. Large numbers of rapidly maturing microscopic erythroid colonies appeared between 8 and 10 days after irradiation. From this finding we conclude that in addition to colony-forming cells giving rise to macroscopic colonies, large numbers of already differentiated progenitor cells--the products of the first regeneration wave with a peak approximately 6 days following irradiation--participate in the second wave of regeneration which is typically manifested from day 10 after irradiation.

Effect of indomethacin on the proliferative and differentiation activity in the haemopoietic system of lethally irradiated mice after syngeneic bone marrow transplantation.

The effects of indomethacin treatment on the proliferation and differentiation of haemopoietic stem cells of bone marrow grafts in lethally irradiated mice were investigated. Indomethacin was given subcutaneously, on days 3 to 5 after irradiation, in 6 doses of 0.05 mg per mouse. On day 6 after irradiation, an increased self-renewal of the settled colony-forming cells and an increased amount of differentiated cells were observed in the spleens of the mice treated. Simultaneously, a decreased maturation of the erythroid cells was demonstrated. The effects observed may be explained by the indomethacin-induced inhibition of prostaglandin biosynthesis.