Effect of daily systemic injections of donepezil (0, 0.5, 1, 1.5 mg/kg, ip) on morphine-induced apoptosis in rat lumbar spinal cord. A. Tissue sections from rat lumbar spinal cord were prepared and assayed with an In Situ Cell Death Detection Kit, POD. Slides were counterstained with toluidine blue. Representative photos illustrate the subpopulation of apoptotic cells, which are scattered throughout the tissue section and were intensely stained (brown) by the TUNEL treatment. Slides were analyzed with a light microscope (40× objective). a) Normal saline (1 mL/kg/d ip, for 14 days). b) Morphine (10 mg/kg/d ip, for 14 days) + donepezil (0 mg/kg, ip for 14 days). c) Morphine + Donepezil (0.5 mg/kg ip, for 14 days). d) Morphine + Donepezil (1 mg/kg ip, for 14 days). e) Morphine + Donepezil (1.5 mg/kg ip, for 14 days). f) Donepezil (1.5 mg/kg ip, for 14 days). g) Morphine + MK801(0.1 mg/kg ip, for 14 days). B. Quantification of apoptotic cells in rat lumbar spinal cords. The data represent the mean ± sem number of apoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] positive) cells in 30 fields, which were counted at a magnification of 40 × with a light microscope. A one-way analysis of variance (ANOVA) followed by Tukey’s test was used to analyze the statistical significances. The Scale bar represents a length of 20 μm. A P value of <0.05 was considered significant for all analyses. ***P < 0.001 in comparison with the corresponding control group. ^^^P < 0.001 in comparison with the corresponding saline group. ^^P < 0.01 in comparison with the corresponding saline group. DPZ = Donepezil, M = Morphine.