Figure 1.
Intrajugular injection of SE in mice less than 24 h old.
A) Depiction of murine anatomy including the ventral muscles and blood vessels of the neck, highlighting the location of the external jugular vein (image used with permission of Nature Publishing Group). B) Positioning of mouse and exposure of neck for injection into right external jugular vein. C) Insertion of needle into external jugular vein. D) Injection of inoculum into external jugular vein. E) Removal of needle showing blood return following vessel puncture.
Figure 2.
Induction of bacteremia is proportional to injection scoring criteria.
Newborn mice less than 24 h old were injected with 108 CFU of SE. Mice were euthanized, organs harvested, and CFUs measured. CFU data were stratified by injection score, determined as per Table 2. Prospective injection scores of 3 or higher correlated with substantially greater CFUs in mouse spleen and liver (N = 5–8, ** p<0.01, *** p<0.001, Mann-Whitney t-test).
Table 1.
Technique diagram for intra-jugular injection of newborn mice.
Table 2.
Injection scoring criteria.
Figure 3.
SE-induced inoculum-dependent infection of solid organs and blood.
Bacterial load in A) spleen, B) liver, and C) blood at 2 h following intra-jugular injection of SE at 106, 107, and 108 CFU showing inoculum-dependent increase in bacterial counts. Graph represents individual data points with median values indicated by a horizontal line. Only mice with injection scores of 3–5 were used in analysis. Groups were compared using the Mann-Whitney t-test (N = 7–31, * p<0.05, ** p<0.01, *** p<0.001).
Figure 4.
Neonatal mice clear SE infection within 48 h post-injection.
Effect of time on bacterial load in A) spleen, B) liver, and C) blood following intra-jugular injection of SE at 106, 107, and 108 CFU shows significant clearance of bacteria by 48 h when compared to inoculation at 2 h (N = 5–31, ** p24 h<0.01, *** p24 h<0.001, ++p48 h<0.01, +++p48 h<0.001, Mann-Whitney t-test).
Figure 5.
SE inoculum-dependent transcription of TLR signaling pathway genes.
Neonatal mice were injected IV with SE at A) 106, B) 107, and C) 108 CFU and euthanized at 2 h. Liver tissue was harvested, RNA isolated and transcription of TLR-signaling pathway genes assessed. Log10 normalized gene expression levels are shown for both saline (x-axis) and inoculum SE (y-axis) injected mice. Inoculum-dependent up-regulation of TLR-gene transcription is evident, with mRNA transcripts that are significantly up- or down-regulated shown in red.
Figure 6.
SE induces selective mRNA expression of TLR2 and MyD88.
Liver samples were collected 2 h post-SE injection and analyzed for mRNA expression of TLR2, TLR4, MyD88, and MD-2. Relative fold change in mRNA transcripts, each normalized to housekeeping genes as described in methods, was expressed as a ratio of each transcript in the SE injected animals to that in saline-injected animals. SE induced significant inoculum-dependent increases in mRNAs encoding TLR2 and MyD88 (* p<0.05, Student t-test). By contrast, TLR4 and MD-2 were not up-regulated over saline-injected mice.
Table 3.
Changes in liver mRNA regulation 2 h following injection with 108 SE relative to control.
Figure 7.
SE-induced systemic IL-6 and TNF-α production.
IL-6 and TNF-α were both up-regulated in response to intra-jugular injection with SE showing a high ratio of IL-6 to TNF-α at 2 h post-injection. IL-6 was induced in an inoculum-dependent manner following injection with 106, 107 and 108 CFUs. TNF-α was also induced, but at substantially lower concentrations, with significance only at the highest inoculum when compared to saline injections (N = 4–9, * p<0.05, ** p<0.01, *** p<0.001, Mann-Whitney t-test).
Figure 8.
SE induced systemic cytokine production.
Radar plot representation of Milliplex cytokine data that highlights the fold change in cytokine induction demonstrating up-regulation of both IL-6 and TNF-α in mice injected with 108 CFU of SE at 2 h. Results represent 3 independent experiments.
Figure 9.
SE inoculum-dependent impairment of neonatal weight gain.
Newborn pups were weighed at birth (birth weight = BW) and at 24 and 48 h following injection with saline or the indicated inocula of SE. There was significant impairment of weight gain noted in mice injected with 108 CFU of SE. This pattern of growth was observed at both 24 and 48 h (N = 5–13, * p<0.05, *** p<0.001, Mann-Whitney t-test).