Small Acetylcholinesterase-positive Cells Research Articles

Abnormal megakaryocytopoiesis in the Belgrade laboratory rat

The Belgrade laboratory (b/b) rat has a hereditary hypochromic microcytic anemia because of defective transmembrane iron transport into erythroblasts. The present study was prompted by our previous work in which we showed that the b/b rat has hypomegakaryocytic thrombocytopenia associated with increased megakaryocyte size. To define the basic mechanism underlying this abnormality in the b/b rat we have studied both megakaryocytopoiesis and granulopoiesis in anemic b/b rats, chronically transfused b/b rats, iron-treated b/b rats, and controls. We have found decreased concentrations of megakaryocyte and granulocyte progenitors in the marrow of b/b rats. Full correction of the severe anemia by chronic transfusion resulted in normalization of megakaryocyte progenitors, small acetylcholinesterase positive cells, megakaryocyte size, and platelet counts, along with granulocyte progenitors. In contrast, the partial correction of anemia obtained by iron treatment resulted in improvement, but not normalization, of these parameters. These findings indicate that abnormal megakaryocytopoiesis in the b/b rat can be best interpreted as a consequence of hypoxia because of the severe anemia. Because we have recently shown that the number of erythroid progenitors in b/b rats is also low, we propose that abnormal megakaryocytopoiesis in this animal is a reflection of an acquired stem cell disorder induced by the prolonged hypoxia resulting from the severe anemia.

Abnormal Megakaryocytopoiesis in the Belgrade Laboratory Rat

The Belgrade laboratory (b/b) rat has a hereditary hypochromic microcytic anemia because of defective transmembrane iron transport into erythroblasts. The present study was prompted by our previous work in which we showed that the b/b rat has hypomegakaryocytic thrombocytopenia associated with increased megakaryocyte size. To define the basic mechanism underlying this abnormality in the b/b rat we have studied both megakaryocytopoiesis and granulopoiesis in anemic b/b rats, chronically transfused b/b rats, iron-treated b/b rats, and controls. We have found decreased concentrations of megakaryocyte and granulocyte progenitors in the marrow of b/b rats. Full correction of the severe anemia by chronic transfusion resulted in normalization of megakaryocyte progenitors, small acetylcholinesterase positive cells, megakaryocyte size, and platelet counts, along with granulocyte progenitors. In contrast, the partial correction of anemia obtained by iron treatment resulted in improvement, but not normalization, of these parameters. These findings indicate that abnormal megakaryocytopoiesis in the b/b rat can be best interpreted as a consequence of hypoxia because of the severe anemia. Because we have recently shown that the number of erythroid progenitors in b/b rats is also low, we propose that abnormal megakaryocytopoiesis in this animal is a reflection of an acquired stem cell disorder induced by the prolonged hypoxia resulting from the severe anemia.

Effects of recombinant human erythropoietin on megakaryocytes and on platelet production in the rat

The contention that erythropoietin (Epo) affects platelet production was investigated in the rat with recombinant human Epo (rHuEpo). In normal rats, Epo caused a dose-dependent increase in both reticulocyte and platelet numbers, the reticulocyte response preceding that of platelets. Withdrawal of Epo resulted in reticulocytes and platelets returning to control levels. [75Se]-selenomethionine incorporation into platelets was also enhanced in response to Epo. Chronic daily administration of rHuEpo resulted in steady state erythrocyte levels after 12 to 14 days, which were elevated 20% above controls. Attainment of this steady state was associated with both reticulocytes and platelets returning to control levels despite continued administration of Epo, an effect not associated with a change in the half-life of circulating Epo. In polycythemic rats a platelet response was observed before an effect on reticulocytes. Erythropoietin caused a 2.4-fold increase in the frequency of small acetylcholinesterase-positive cells within 24 hours, and increased the mean megakaryocyte diameter within 48 hours. Furthermore, the [3H]-thymidine labeling index of megakaryocytes from rats treated for 24 hours with rHuEpo was increased for all stages of megakaryocyte maturation. These results support the proposal of an effect of Epo on rat megakaryocytes causing increased platelet production.

Effects of Recombinant Human Erythropoietin on Megakaryocytes and on Platelet Production in the Rat

Abstract The contention that erythropoietin (Epo) affects platelet production was investigated in the rat with recombinant human Epo (rHuEpo). In normal rats, Epo caused a dose-dependent increase in both reticulocyte and platelet numbers, the reticulocyte response preceding that of platelets. Withdrawal of Epo resulted in reticulocytes and platelets returning to control levels. [75Se]-selenomethionine incorporation into platelets was also enhanced in response to Epo. Chronic daily administration of rHuEpo resulted in steady state erythrocyte levels after 12 to 14 days, which were elevated 20% above controls. Attainment of this steady state was associated with both reticulocytes and platelets returning to control levels despite continued administration of Epo, an effect not associated with a change in the half-life of circulating Epo. In polycythemic rats a platelet response was observed before an effect on reticulocytes. Erythropoietin caused a 2.4-fold increase in the frequency of small acetylcholinesterase-positive cells within 24 hours, and increased the mean megakaryocyte diameter within 48 hours. Furthermore, the [3H]-thymidine labeling index of megakaryocytes from rats treated for 24 hours with rHuEpo was increased for all stages of megakaryocyte maturation. These results support the proposal of an effect of Epo on rat megakaryocytes causing increased platelet production.

Megakaryocy topoiesis: Incorporation of Tritiated Thymidine by Small Acetylcholinesterase-Positive Cells in Murine Bone Marrow During Antibody-Induced Thrombocytopenia

The ability of small acetylcholinesterase-positive (SACHE) cells to incorporate tritiated thymidine (3H-TdR) was studied in the bone marrow of mice made acutely thrombocytopenic by injection of guinea pig antimouse platelet serum (APS) and in control mice injected with normal guinea pig serum (NS). 3H-TdR was administered in vivo, and bone marrow was collected at various time points thereafter. The initial labeling index (LI) for both groups was about 30%. After four hours, the LI increased and reached peak values at 48 hours, but was lower in thrombocytopenic animals. The lower peak LI in APS-treated animals may be the result of both a faster rate of influx of unlabeled cells into the acetylcholinesterase (AChE) positive cell compartment and efflux of more heavily labeled, and probably polyploid, SACHE cells into the compartment of recognizable megakaryocytes. In both groups the increasing LI occurred concomitantly with a decreasing mean grain count. This may indicate cellular division by some fraction of SACHE cells. Heterogeneity in nuclear morphology was also demonstrated, and the frequency of individual morphologies was altered in APS-treated animals. In addition, cells that incorporated 3H-TdR were larger than those that did not. This group of small cells appears to represent a pivotal point in megakaryocytopoiesis in which the switch from mitosis to endomitosis is occurring.

Megakaryocytopoiesis: incorporation of tritiated thymidine by small acetylcholinesterase-positive cells in murine bone marrow during antibody-induced thrombocytopenia

The ability of small acetylcholinesterase-positive (SACHE) cells to incorporate tritiated thymidine (3H-TdR) was studied in the bone marrow of mice made acutely thrombocytopenic by injection of guinea pig antimouse platelet serum (APS) and in control mice injected with normal guinea pig serum (NS). 3H-TdR was administered in vivo, and bone marrow was collected at various time points thereafter. The initial labeling index (LI) for both groups was about 30%. After four hours, the LI increased and reached peak values at 48 hours, but was lower in thrombocytopenic animals. The lower peak LI in APS-treated animals may be the result of both a faster rate of influx of unlabeled cells into the acetylcholinesterase (AChE) positive cell compartment and efflux of more heavily labeled, and probably polyploid, SACHE cells into the compartment of recognizable megakaryocytes. In both groups the increasing LI occurred concomitantly with a decreasing mean grain count. This may indicate cellular division by some fraction of SACHE cells. Heterogeneity in nuclear morphology was also demonstrated, and the frequency of individual morphologies was altered in APS-treated animals. In addition, cells that incorporated 3H-TdR were larger than those that did not. This group of small cells appears to represent a pivotal point in megakaryocytopoiesis in which the switch from mitosis to endomitosis is occurring.

Megakaryoblast precursors in rodent bone marrow: specificity of acetylcholinesterase staining.

Small cells staining diffusely for acetylcholinesterase, corresponding closely to the description of Jackson (1973, 1974), have been quantitated in the bone marrow of mouse and rat. These cells fulfil the requirements of megakaryoblast precursors, showing elevation in numbers prior to megakaryocytes in response to thrombocytopenia, and carrying platelet-specific antigens demonstrated by immunofluorescence. Some show a clearly defined nucleus whilst in others the nucleus is obscured; the femoral content of both types follows the same time course after acute thrombocytopenia and the latter can be distinguished from megakaryocyte cytoplasmic fragments. Other small cells present in rodent bone marrow contain granular positivity after acetylcholinesterase staining but also granules staining with Luxol-Fast-Blue, suggesting that these cells are eosinophils. These cells are greatly increased in number with stimulation of eosinophilia by parasite infestation but show no response to acute thrombocytopenia. Peripheral blood eosinophils exhibit both acetylcholinesterase and Luxol-Fast-Blue staining. A number of properties ascribed to granular-staining small acetylcholinesterase-positive cells previously presumed to be megakaryoblast precursors will need to be reassessed.

Thrombocytosis-induced suppression of small acetylcholinesterase- positive cells in bone marrow of rats

Transfusion of platelet concentrates was used to establish a thrombocytosis of approximately three times normal platelet levels in male rats. This thrombocytosis resulted in a rebound thrombocytopenia to 60% of normal counts. Examination of the small acetylcholinesterase (ACh-E) positive cells of the marrow at this time showed a reduction to 50% of normal levels without significant changes in control animals. A second group of experiments indicated that this suppression developed as early as the third day posttransfusion, persisted until day 7, and returned to baseline levels by day 9. Incorporation of 75SeM indicated that the reduction in platelet count was due to decreased platelet production. Little or no changes were observed in the hematocrit or WBC. This evidence supports the hypothesis that these cells are early cells in the megakaryocytic series. They are the earliest cells of the series seen to be affected by thrombocytosis. Feedback control by platelets or platelet extracts of this cell population may represent one level of regulation of megakaryopoiesis.

Thrombocytosis-Induced Suppression of Small Acetylcholinesterase-Positive Cells in Bone Marrow of Rats

Transfusion of platelet concentrates was used to establish a thrombocytosis of approximately three times normal platelet levels in male rats. This thrombocytosis resulted in a rebound thrombocytopenia to 60% of normal counts. Examination of the small acetylcholinesterase (ACh-E) positive cells of the marrow at this time showed a reduction to 50% of normal levels without significant changes in control animals. A second group of experiments indicated that this suppression developed as early as the third day posttransfusion, persisted until day 7, and returned to baseline levels by day 9. Incorporation of 75SeM indicated that the reduction in platelet count was due to decreased platelet production. Little or no changes were observed in the hematocrit or WBC. This evidence supports the hypothesis that these cells are early cells in the megakaryocytic series. They are the earliest cells of the series seen to be affected by thrombocytosis. Feedback control by platelets or platelet extracts of this cell population may represent one level of regulation of megakaryopoiesis.

Cholinesterase as a Possible Marker for Early Cells of the Megakaryocytic Series

Abstract Among marrow cells of rats only megkaryocytes stain appreciably for acetylcholinesterase. An investigation was therefore undertaken to determine whether this staining characteristic might be used to identify precursors of recognizable megakaryocytes. Marrow cells smaller than recognizable megakaryocytes were positive for the enzyme. The number of these cells increased after induction of severe thrombocytopenia by injection of antiplatelet antiserum. Subsequently, the number of recognizable megakaryocytes increased, suggesting their derivation from the small acetylcholinesterase-positive cells. These results suggest that acetylcholinesterase activity can be used as a marker for megakaryocytic cells that cannot be detected with conventional staining methods.