In situ generation of RBC aggregates, slow blood cell flow speed, and increased cell apoptosis are pathologic features of SCD BM. (A) Thirteen- to 15-week-old AA or SS mice were subjected to transcardial perfusion with phosphate-buffered saline, immediately after being euthanized with CO₂, to flush nonadherent RBCs in the vasculatures, followed by fixation. Frozen thin sections (5 μm thickness) of SCD mouse femurs were stained with antibodies against Ter119 and laminin, followed by DAPI staining. Confocal microscopy (Leica SP8X) images were processed to show Ter119⁺ erythrocyte aggregates. Arrows indicate intra- and peri-sinusoidal sickle RBC aggregates. Arrowheads indicate sinusoids identified by vascular marker laminin. Scale bars, 10 μm. (B) Rabbit antibody against CD71 (transferrin receptor) was used to identify immature erythroid cells (Ter119⁺ CD71⁺ DAPI⁺) in confocal analysis (Leica SP8X) after immunofluorescence staining of frozen sections (5 μm thickness) of SCD mouse femurs. Arrowheads indicate aggregates of immature sickle RBC in sinusoids. Scale bars, 50 μm. (C) Gr-1/CD11b⁺ myeloid cells aggregated with sickle RBCs perisinusoidally in the femoral BM sections of SCD mice were analyzed by LaSC. Arrowheads denote sinusoids. Scale bars, 50 μm. (D) Gr-1⁺ cells, aggregated with sickle RBCs intrasinusoidally in frozen sections (5 μm thickness) of femoral BM of SCD mice, were analyzed by confocal microscopy (Leica SP8X). Arrowheads denote an intrasinusoidal Gr-1⁺ myeloid cell. Scale bar, 20 μm. (E) Frozen sections of femurs were stained with a Click-It Plus TUNEL Assay (Life Technologies). TUNEL⁺ apoptotic cells and DAPI⁺ BM cells were quantitated by LaSC. Bar graphs are plotted as mean + standard error of the mean (SEM); n = 4 mice per group (13-15 weeks old; 2 female and 2 male mice). Scale bars, 50 μm. **P < .01, Student t test. (F and G) For the flow speed measurement, Rhodamine B-dextran (70 kDa) was injected retro-orbitally, followed by calvarial vasculature recording using confocal intravital microscopy and calculation of endogenous blood cell flow speed (displacement over time) by negative contrast approach using ImageJ, as described previously (see also supplemental Figure 2A).^80,81  (F) Flow speed value per cell was plotted as scatter dot plots for each individual mouse, with a mean of individual mouse and pooled total shown as thinner and wider horizontal lines, respectively, by Prism 8 software (GraphPad). Each circle represents an individual RBC. The numbers of cells plotted from each mouse (total 4 mice) were 14, 18, 19, 13 (AA)/ 21, 20, 29, 6 (SS) for arterioles and 15, 26, 13, 19 (AA)/ 14, 19, 11, 6 (SS) for sinusoids. The P values were analyzed by a generalized linear model with a random effect of mouse nested within genotype (n = 4 mice). (G) The average RBC flow speed of each mouse was calculated as 1 data point and represented as dots. Bar graphs depict the mean of mean flow speeds of individual mouse as mean + SEM, n = 4 mice (12-15 weeks old; 2 female mice and 2 male mice). *P < .05, Student t test with Bonferroni correction for multiple comparisons.