Ultrasound Neuromodulation of the Spleen Has Time-Dependent Anti-Inflammatory Effect in a Pneumonia Model

Figures

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  Figure 1. S. Pneumonia dose titration and change in physiological markers. (A) Blood and lung cultures in sheep blood agar plates at 16 hours timepoint after bacterial inoculation of different doses from 10^5 to 10^7 CFU. The left column shows lung culture plates after saline instillation. The middle and right columns shows blood and lung culture plates after bacterial inoculation as different doses. (B–D; n=6) Body temperature, heart rate and lung culture after 16 hours of bacterial inoculation in sham and splenic ultrasound groups. (E, F; n=7) Change in breathing rate and body weight from the baseline (i.e., before bacterial inoculation) at 4 hours, 16 hours, and 48 hours post bacterial infection/instillation. (G) Grimace score for pain assessment at 4 hours, 16 hours, and 48 hours post bacterial infection/instillation. Grimace score reflects 0=normal, 1=moderate, and 2=obvious.

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  Figure 2. Whole blood cytokine response assay to saline and bacteria instillation. (A) TNF-α response to in-vitro LPS challenge test (10 ng/ml) in instillation naïve animals (left bars) and saline instilled animals (middle and right bars) before and after splenic ultrasound stimulation at multiple timepoints. (B) TNF-α response to in-vitro LPS challenge test in bacteria instilled animals before and after splenic ultrasound stimulation at 4 hours, 16 hours, and 48 hours post-bacterial infection. (C) Same as (B), but in sham animals (i.e, without active ultrasound stimulation). Asterisk indicates p<0.05 using non-paramteric Wilcoxon rank sum test. n=7 for all groups (except bacteria instillation + CAP activation which has n=13).

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  Figure 3. Plasma cytokine levels and blood cell counts after saline and bacterial instillation. (A) Plasma cytokines levels in pre- ultrasound/sham samples in control and bacteria instilled animals at 4 hours, 16 hours, and 48 hours timepoints. (B) Plasma cytokines levels in pre- and post- ultrasound samples in bacteria instilled animals at 16 hours timepoint. (C) Same as (B), but in saline instilled animals. (D) Plasma chemokine KC/GRO levels (a neutrophil chemokine) in control (no bacteria) and bacteria instilled animals at 4 hours, 16 hours, and 48 hours timepoints. (E) Plasma chemokine KC/GRO levels in bacteria instilled animals before and after ultrasound stimulation at 16 hours timepoint. (F) Same as (E), but in saline instilled animals. (Gi) Percentage of white blood cell differentials in saline and bacteria instilled animals before ultrasound or sham stimulation at 16 hours. (Gii) Same as (Gi), but after ultrasound or sham stimulation. Asterisk indicates p<0.05 using non-paramteric Wilcoxon rank sum test.

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  Figure 4. Receiver operating curves (ROC) for whole blood TNF-α. (A) ROC created for whole blood TNF-α pre-US samples (n=23 animals; Area Under the Curve=0.86; Mann Whitney U-test, p=0.003). (B) ROC created for whole blood TNF-α post-US samples (n=23 animals; Area Under the Curve=0.37; Mann Whitney U-test, p>0.05). (C) ROC created for change in whole blood TNF-α (post US – pre US) (n=23 animals; Area Under the Curve=0.97; Mann Whitney U-test, p<0.0001).