Abstract
Background: Thrombocytopenia is a frequent problem in sick neonates. Neonatal megakaryocytes (Mks) are smaller and of lower ploidy than adult Mks, and it has been hypothesized that this may represent a limitation in the ability of neonates to respond to thrombocytopenia. However, the study of megakaryopoiesis in sick thrombocytopenic neonates is intrinsically difficult, and has been further hampered by the lack of animal models.
Objective: We generated a murine model of immune neonatal thrombocytopenia to test the hypothesis that neonates have a developmental deficiency in the ability to increase platelet production.
Design/Methods: Neonatal thrombocytopenia was induced by injecting a commercially available anti-platelet antibody (MWReg30) into pregnant C57BL/6 mice. The antibody was administered daily by tail vein injection, starting on gestation day 12.5. Healthy non-pregnant adults were similarly treated to establish adult responses. On the first day of life, pups were bled by intracardiac puncture and complete blood counts were obtained on an automated cell counter. Livers, spleens and femoral bones were isolated, fixed, and immunohistochemically stained with anti-vWF antibody to highlight the Mks. Mks were then quantified and their diameters measured using an eyepiece reticle and an eyepiece micrometer, respectively.
Results: Results are displayed as mean±SEM in Table 1. As shown, both newborn and adult thrombocytopenic mice had platelet counts approximately 1/3 of the normal mean for age. The main site of megakaryopoiesis was the liver (Liv) in newborn vs. the bone marrow (BM) in adult mice. As in human neonates, Mks in newborn pups were significantly smaller than in adult mice. The response to thrombocytopenia was different in newborn compared to adult mice: While the adults doubled the number of Mks in BM and spleen (Spl), newborn mice only exhibited a small increase in the diameter of their hepatic Mks, which did not reach normal adult size. To account for the combined contribution of changes in size and number, these measurements were multiplied to give an estimate of Mk "mass", expressed in arbitrary units. Overall, pups did not change their Mk mass in response to thrombocytopenia, while adult mice approximately doubled it.
Conclusions: Newborn mice do not significantly increase their Mk mass in response to immune thrombocytopenia, suggesting that neonates have a developmental limitation in their ability to increase platelet production.
Table 1. Megakaryocyte number, size, and mass Newborn mice Adult mice Control(n=25) Thrombo.(n=19) Control(n=14) Thrombo.(n=12) Plt, platelet; #, number; D., diameter; †, mean of 8 normal and 10 thrombocytopenic newborn samples; * p<0.05 vs. age-matched controls; **p<0.0001 vs. age-matched controls. Plt. count(×109/L) 711.4±23.2 292.0±18.6** 1298.2±46.0 418.1±71.6** Mk#(BM, /250 μ2)† 0.25±0.05 0.25±0.03 4.57±0.26 8.12±0.46** Mk#(Liv, /250 μ2) 1.70±0.13 1.88±0.12 0 0 Mk#(Spl, /250 μ2) 0.72±0.11 0.68±0.07 0.63±0.15 1.35±0.33* Mk D.(BM, μ) 14.06±1.08 15.59±0.43 20.80±0.41 20.53±0.40 Mk D.(Liv, μ) 15.76±0.12 17.55±0.28** Mk D.(Spl, μ) 15.04±0.38 15.92±0.37 21.71±0.47 22.55±0.67 Mk mass(BM) 3.41±0.78 3.59±0.52 97.48±4.31 167.43±10.73** Mk mass(Liv) 26.88±2.09 29.06±2.74 Mk mass(Spl) 10.78±1.95 10.49±1.32 14.15±3.63 32.32±6.07*
Neonatal thrombocytopenia: A review. II. Non-immune thrombocytopenia; platelet transfusion