Acute administration of IV SDF-1 improves radiation-induced thrombocytopenia and is an additive with earlier TPO administration. (A) MK lineage kinetics at day 5 post-4 Gy TBI in mice treated with IV SDF-1 at day 4 (blue), 0.3 μg IP TPO (red) at 2 hours, both 2-hour TPO and day 4 SDF-1 (purple), or vehicle control (gray). IV SDF-1 increases platelet count following radiation injury. TPO administration at 2 hours post-TBI increases MK and MKP numbers, and IV SDF-1 at day 4 following initial TPO treatment results in an additive improvement in circulating platelets. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as a percentage of unirradiated control. Mean absolute numbers for 0 Gy controls: 10 939 MKP/femur, 50 595 MK/femur, 473 × 10³ platelets/μL. (B) Quantification of Gp1Bβ+ MKs in the vascular niche (physically associated with MECA32+ vessels) by femoral IHC prepared on day 5 post-4 Gy from irradiated vehicle control mice (gray), or mice treated with IV SDF-1 at day 4 (blue), 0.3 μg IP TPO (red) at 2 hours, or both 2-hour TPO and day 4 SDF-1 (purple). Percentage of MKs associated with vasculature (IHC) are normalized to per femur values (imaging flow cytometry). Mean 20 104 MK/femur associated with vasculature for 0 Gy controls. Sequential TPO and SDF-1 administration increases MKs in the vascular niche above either treatment alone. (C) Representative images of femoral marrow IHC for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) at day 5 post-4 Gy with hindlimbs isolated from vehicle controls (left panel), mice receiving IV SDF-1 at day 4 (middle panel), and mice receiving 2-hour TPO followed by day 4 SDF-1 (right panel) as described above. White arrows indicate examples of MKs physically associated with MECA32+ vessels. Images were acquired and processed as described in the “Immunohistochemistry” section. Error bars represent standard error of the mean of ≥3 independent experiments (n = 3-11 total mice per group). Statistical analyses comparing indicated platelet samples in panel A were performed by one-way analysis of variance with Tukey’s multiple comparisons test, and all other indicated comparisons were performed by a 2-tailed Student’s t test. Bar represents 100 μm (C). *P < .05; **P < .006.