Histological Evaluation of Megakaryocytes and Angiogenic Signaling Molecules of Bone Marrow in Experimentally Induced Iron Deficiency Anemia

Iron deficiency Anemia (IDA) induced reactive thrombocytosis occurs in children. The mechanisms involved in this phenomenon are indeterminate. Traditional cytokines involved in megakaryopoiesis such as Thrombopoietin (TPO), IL-6, and IL-11 have not been shown to be associated with this IDA induced thrombocytosis. Recent studies suggest that growth factors and signaling molecules involved in angiogenesis influence the proliferation and/or differentiation of megakaryocytes. A recent study observed that VEGFR1-mediated pathway up-regulates CXCR4 on megakaryocytes, leading to enhanced platelet production via distribution of megakaryocytes. We previously reported a statistically increased serum/plasma levels of FLT-3 and PDGF, but did not find an increase in plasma levels of TPO, VEGF and CXCR4 in an experimentally induced IDA in rats, when compared to control rats. We now present the histological evaluation of megakaryocytes and the expression of angiogenic signaling molecules, VEGF and CXCR4, in bone marrows of control and IDA rats.

Six week old male Sprague-Dawley rats with jugular vein cannulas were obtained. Diet for control rats (N=9) and iron deficient diet rats (N=18) had 50 ppm and 7-8 ppm iron in Purina chow respectively. CBC, Iron Panel, and cytokines were drawn at baseline and five weeks later. On day 0, 1.5 mL of blood was drawn from iron deficient diet rats to further induce anemia. Rats were euthanized by CO₂ asphyxiation and cardiac puncture. Femurs were collected, decalcified, and embedded in paraffin. Thin sliced sections were obtained to make slides. The slides were stained with hematoxylin and eosin (H&E), and with peroxidase linked anti factor VIII, VEGF, and CXCR4 according to manufacturer's instructions. The slides were evaluated under 40x microscopy. An area of 0.1 mm² was selected and the numbers of megakaryocytes in the selected area were visually quantitated. Immunoperoxidase stained slides were analyzed using Image J software.

When reviewing H&E stained bone marrow slides per 0.1 mm², control rats contained 4 megakaryocytes, while those from IDA rats contained 11 megakaryocytes (P=0.0001). In Factor VIII stained slides, quantitative analysis of peroxidase stained megakaryocytes in control group contained 49,271 pixels, while staining in the IDA rats was 185,076 pixels (P=0.00002). When the analysis was carried out looking at vessel staining, there was a significant difference between controls (3.6) and IDA (8.5) per 0.1 mm² (P=0.00001). In the VEGF stained slides, visual analysis of peroxidase stain showed increased intensity of staining per cell in the IDA rats. In the CXCR4 stained slides, visual inspection of the control bone marrows showed a rare small round cell weakly stained while these cells were more frequent and strongly stained in IDA rats.

We successfully induced IDA in an animal model with coexisting thrombocytosis. Bone marrow slides in IDA rats documented the expected increase in number of megakaryocytes. In addition, we documented a marked increase in vascular structures of IDA rats. Contrary to our previously reported plasma levels, VEGF intensity of stain was greater within IDA rat megakaryocytes when compared to control rat megakaryocytes. We also documented an increase of CXCR4 in the bone marrows of IDA rats. However, this increase was limited to early stage megakaryocyte development cells suggesting a role during the differentiation process of megakaryocytes. Both our previous report on circulating angiogenic signaling molecules and the current histological data suggest an important role for angiogenesis in the development of IDA induced thrombocytosis.