Table 1.

Morphine and TAT treatment in the context of S. pneumoniae infection in TLR 2, 4, and TLR2/4 knock-out mice

Groups	Treatment	ROS (% control)	Nitrite (mol/mg)	Caspase 3 (MFI)
Wild type	Placebo	175 ± 27	0.25 ± 0.03	121 ± 12
	Morphine	500 ± 35*	0.78 ± 0.07*	350 ± 35*
TLR2KO	Placebo	125 ± 18	0.18 ± 00.02	93 ± 10
	Morphine	196 ± 23	0.21 ± 0.019	143 ± 14
TLR4KO	Placebo	135 ± 31	0.16 ± 0.009	88 ± 9
	Morphine	182 ± 21	0.19 ± 0.02	132 ± 14
TLR2/4KO	Placebo	112 ± 29	0.12 ± 0.005	56 ± 6
	Morphine	105 ± 25	0.14 ± 0.006	52 ± 8

WT and TLR 2, 4 knock-out and TLR2/4 double knock-out mice were treated with either placebo or morphine pellet as described in Materials and Methods and infected with S. pneumoniae intranasally. Animals were killed for ROS and reactive nitrogen species measurement 24 h after infection and for caspase 3 activity 48 h after infection. ROS generation was measured using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate. Data is represented as percentage increase over vehicle control. NO levels were estimated by measuring the concentrations of nitrites. Supernatants free from cellular debris were mixed with Griess reagent [1 part 1% (w/v) sulfanilamide in 5% H₃PO₄, 1 part 0.1% (w/v) N-1-naphthylethylenediamine (v/v)] in 96-well tissue culture plates for 10 min at room temperature in the dark. The absorbance at 540 nm was determined using a microplate reader. Caspase 3 activity was determined using the CaspACE fluorometric activity assay according to the manufacturer's instruction. Protein levels of brain homogenates samples were determined using the bicinchoninic acid protein assay kit with an absorption band of 570 nm to normalize the protein levels between control and different treated groups. Sample size in each group was six animals. Data represents mean ± SD of three independent experiments.

*p < 0.01.