Fig. 4. Intra- and extracellular bacterial localization in the cranial marrow.

a, IVM of skull marrow from sham controls or mice 48 hrs after intracisternal injection of GFP⁺ Streptococcus pneumoniae JWV500 (n=3 sham, 4 meningitis from 2 independent experiments; scale: 50 μm, 25 μm). b, Experimental scheme for (d-j). c, Bacterial culture of pneumococcal growth in tibia versus skull. Skull sample contains pooled frontal, parietal and occipital bone. d, Quantitation of bacterial CFU (mean ± SD; n=10; P value represents a Mann-Whitney two-sided rank test). e, S. pneumoniae surface adhesion gene (psaA) expression in tibia versus skull normalized to sham (mean ± SD; n=9; P value represents a Mann-Whitney two-sided rank test). f, Gating strategy for GFP⁺ CD45⁺ leukocytes g, Histogram of GFP signal in CD45⁺ leukocytes obtained from mice with meningitis compared to CD45⁺ cells from sham controls. h, Quantitation of GFP⁺CD45⁺ cells (mean ± SD; n=5; P value represents an unpaired, two-tailed t-test) i, Transmission electron microscopy of S. pneumoniae in the skull marrow. Left, low-magnification view of calvarial marrow depicts a sinusoidal vessel lumen adjacent to a skull channel, the inner skull bone cortex and leukocytes. Right, insets illustrate multiple leukocytes containing S. pneumoniae (n=2 mice; scale: 10 μm and 600 nm). j, Flow cytometric analysis of extracellular GFP⁺ S. pneumoniae in supernatant fraction of tibia and skull (n=2 sham and 4 meningitis).