Figure 10.

Morphine + TAT + S. pneumoniae (luciferase tagged) induced synergistic increase in bacterial dissemination and proinflammatory cytokines are significantly attenuated in TLR 2, 4 knock-outs and TLR2/4 double knock-out mice. A, A significant decrease in bacterial dissemination into the CNS of TLR 2, 4 knock-outs and TLR2/4 double knock-out mice following morphine, TAT, and S. pneumoniae lysate treatment. B, Quantification of bacterial dissemination in the brain tissue of WT, TLR2KO, and TLR4KO mice. Data represents mean ± SEM of three independent experiments. *p < 0.01, ***p < 0.001. C, To determine apoptosis, animals (6 per group) were treated as described in Figure 4A and killed at 72 h. Brains were removed and snap frozen in liquid nitrogen. Cryostat sections (5 μm) were used to evaluate apoptosis using TUNEL staining (Intergen) according to the manufacturer's instruction. DAPI staining shows the nuclei of cells. Apoptosis is significantly lower in the TLR knock-out animals compared with wild-type following treatment with morphine, TAT, and S. pneumoniae (S.p.) lysate. Scale bars, 10 μm. D, Synergistic increase in proinflammatory cytokines in WT mice following morphine, TAT, and S. pneumoniae lysate treatment is significantly attenuated in brain homogenate harvested from TLR 2, 4 knock-outs and TLR2/4 double knock-out mice. Data represents mean ± SEM of three independent experiments. *p < 0.01. E, Survival curve of WT and TLR 2, 4 knock-outs and TLR2/4 double knock-out mice treated with morphine + TAT + S. pneumoniae. Animals were treated as described in Figure 4A and survival followed for 5 d. Data represents mean ± SEM of three independent experiments.