Morphological changes in spleen, bone marrow and blood of rats after continuous gamma irradiation and indomethacin treatment.

A possibility of modifying the radiation damage of haematopoiesis by indomethacin administered during continuous irradiation was followed. Indomethacin was given in drinking water (average dose of 740.7 micrograms/kg per day) within 7-days continuous irradiation with gamma rays (a daily dose rate of 2.055 Gy, total accumulated dose of 14.385 Gy). Under the given conditions no marked protective effect of indomethacin on haematopoiesis was found with the exception of mild stimulation of myelopoiesis and an increased release of developmentally younger cells of myeloid and erythroid series to the peripheral blood as well.     

Stem cell kinetics in spleen and bone marrow after single and fractionated irradiation of infant mice

Summary The number and type of stem cells in spleen and bone marrow of mice were determined after exposure to a single dose of 150 R on day 6, to a single dose of 500 R on day 6 or day 9 or to a fractionated dose of 150 R + 350 R on day 6 and 9. The stem cells were assayed on the basis of colony forming units (CFU) in spleen and of incorporation of iododeoxyuridine in spleen and bone marrow of lethally irradiated host mice. During the first month of life, the number of stem cells in non-irradiated mice increases markedly in bone marrow and slightly in spleen. Irradiation causes a long-lasting depression in stem cells, particularly in bone marrow and affecting preferentially erythropoietic precursor cells. Following a dose of only 150 R, the number of CFU in bone marrow is still below control levels 24 days later. An exposure to 500 R fractionated between day 6 and 9 has a markedly greater effect on stem cells in the spleen than the same dose given in a single application either at day 6 or 9.Supported by the Schutzkommission am Ministerium des Innern der BRD and contract B232-76-1BIOB of the Biology Division of the Commission of the European Community (Publikation No. 1727)     

Recovery of CFUc in rat bone marrow after prolonged fractionated irradiation with various total doses

The rate and the degree of recovery of committed precursors of granulocytes and monocytes (CFUc) following long-term fractionated irradiation were a function of a cumulative radiation dose. In rats exposed to doses of 9.7 and 19.4 Gy the number of CFUc of myelokaryocytes and granulocytes of blood reached the control values after 1-3 months. The increase in CFUc of animals exposed to a dose of 29.1 Gy was transient and did not provide the recovery of granulocytopoiesis.     

Changes in the transaldolase activity in the liver, spleen and bone marrow of rats after total X-ray irradiation

The paper deals with changes in the activity of transaldolase in tissues of the liver, spleen and bone marrow of rats 4.24 h and on the 3 d, 7th, 18th, 30th, 45th, and 60th day after total X-ray irradiation in a dose of 600 R. The ionizing irradiation causes a stable inhibition of the enzyme activity in All tissues. The observed changes are homogeneous in their direction but have their own peculiarities for each tissue. Differences in the degree and extent of the enzyme activity changes at various stages of the X-ray lesion development in the tissues under study may be explained by their different sensitivity to the penetrating radiation.     

Elevated cyclic GMP concentrations in rabbit bone marrow culture and mouse spleen following erythropoietic stimulation.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.     

Effect of spleen and bone marrow regeneration on the number of hematopoietic colonies in mouse spleen]

The spleen (2/3) was removed in CBA male mice (the 1st group); in the 2nd group the bone marrow from the right posterior shin was removed. The hemopoietic splenic colonies were counted on the 8th day after the lethal irradiation and injection of 1 X 10(-6) nucleated cells of the intact spleen. A significant increase of the number of colonies in comparison with their number in control intact mice was observed. The authors suppose that this increase could also be caused by the local influence of the regenerating stroma of the spleen and by some stimulating factor discharge by the regenerating hemopoietic tissue.     

Evaluation of cell regeneration of bone marrow after fractional irradiation of the mouse in toto]

We have studied the recovery for mice bone marrow cells after fractionated irradiation of the whole body. The additional dose (Dr) to obtain a given biological effect if the irradiation is split in two equal subfractions (2 Di) separated by a short interval of time (i) is 40 rad per day when the interval of time between the two irradiations is lengthened of one day.     

Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD₁ mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159–169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNP CE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscope conditions.     

Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation

Graft rejection presents a major obstacle for transplantation of T cell-depleted bone marrow in HLA-mismatched patients. In a primate model, after conditioning exactly as for leukemia patients, it was shown that over 99% of the residual host clonable T cells are concentrated in the spleen on day 5 after completion of cytoreduction. We have now corroborated these findings in a mouse model. After 9-Gy total body irradiation (TBI), the total number of Thy-1.2+ cells in the spleen reaches a peak between days 3 and 4 after TBI. The T cell population is composed of both L3T4 (helper) and Lyt-2 (suppressor) T cells, the former being the major subpopulation. Specific booster irradiation to the spleen (5 Gy twice) on days 2 and 4 after TBI greatly enhances production of donor-type chimera after transplantation of T cell-depleted allogeneic bone marrow. Similar enhancement can be achieved by splenectomy on day 3 or 4 after TBI but not if splenectomy is performed 1 day before TBI or 1 day after TBI, strengthening the hypothesis that, after lethal TBI in mice, the remaining host T cells migrate from the periphery to the spleen. These results suggest that a delayed booster irradiation to the spleen may be beneficial as an additional immunosuppressive agent in the conditioning of leukemia patients, in order to reduce the incidence of bone marrow allograft rejection.     

Hemopoiesis in spleen and bone marrow cultures

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.     

Ultrastructural and morphometric alterations in bone marrow stromal tissue after 7 Gy irradiation.

To evaluate the response of marrow stroma to 7 Gy irradiation, femoral bone marrow was fixed by vascular perfusion (so as to avoid the artificial destruction of sinus endothelia), and was examined using light and electron microscopy with morphometric methods. The radiation caused a marked decrease in hematopoietic cell number (NHC) within 3 days post-irradiation, followed by total recovery of hematopoiesis, which occurred gradually over 28 days. An increased number of fat cells was seen by 7 days. During the whole course of hypoplasia and recovery, the continuity of sinus wall, three-dimensional reticular mesh work in hematopoietic parenchyma, gap junctions (GJ) between stromal cells, the adventitial cell cover of sinus wall (ACC), and the stromal cell numbers of reticular cells (RC), sinus endothelia (SE), and macrophages (MP) were maintained. The cellularity of stromal components of RC, SE, and MP seemed passively increased in contrast to a reduction in numbers of NHC. A similar tendency was observed (1) between NHC and ACC and (2) between GJ and the cellularity of fat cells, which had a statistical significant correlation (p less than 0.05; t-test). The mechanism of radio resistance in bone marrow stroma and the possible functional adaptation and cellular coordination after irradiation are discussed.     

Endocrine dysfunction after total body irradiation and bone marrow transplantation

The present report describes our data regarding changes of endocrine parameters after total body irradiation (TBI) and bone marrow transplantation (BMT). Endocrine glands are usually resistant to irradiation under morphological aspects. But new methods of determination and sensitive tests were developed in the last few years. Now it is possible to detect already small functional changes. Endocrine studies in the course of the disease were followed serially at 16 patients with TBI and BMT. Pretransplant conditioning consisted of single-dose irradiation combined with a high-dose, short-term chemotherapy. Reactions of the endocrine system showed a defined temporary order. Changes of ACTH and cortisol were in the beginning. The pituitary-adrenal cortex system responds in a different way. The pituitary-thyroid system develop a short-term "low-T3-syndrome" reflecting the extreme stress of the organism. At the same time we obtained an increase of thyroxine. Testosterone and luteotropic hormone, the sexual steroids showed levels representing a primary gonadal insufficiency. The studies in the posttransplant period yielded a return to the normal range at most of the hormonal levels with the exception of the sexual steroids. Sterility is one of the late effects of TBI. A tendency towards hypothyroidism could be noticed in some cases being only subclinical forms. Reasons and possible therapy are discussed.     

Radiosensitivity of mouse bone marrow and spleen CFUdc during gamma-irradiation in different oxygenation conditions

After exposure in vitro and in situ CFUdc of CBA mouse bone marrow and spleen were characterized by the comparable parameters of radiosensitivity and oxygen-dependent modification: the values of D0 for bone marrow and spleen cells were 1.31 and 1.35 Gy (in vitro) and 1.36 and 1.37 Gy (in situ), and the values of the oxygen effect were 2.3 and 2.5, respectively.     

Mathematical model of the cytokinetics of erythropoiesis in mouse bone marrow and spleen]

The described model approximates the function of the erythropoietic system of the mouse to the function of a self-renewed cellular system, describable in the terms of cell population kinetics. The model is based on a number of experimentally proved ideas of contemporary haematology and arises from the assumption that there exists mutual negative influence between the cellular populations of the bone marrow and spleen. Considering the erythropoietic system in the mouse to be composed of two relatively independent parts - the bone marrow and spleen - the described model differs from the attempts so far made on the mathematical modelling of erythropoiesis.     

Antiviral immune responses of fully allogeneic irradiation bone marrow mouse chimeras

In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.