Figure 4. Age-related reduction in nitrergic neurons in the MP of mouse GI tract. Whole-mount preparations stained with NADPH-d histochemistry showed the variations in number of nitrergic neurons in stomach (A), jejunum (B) and colon (C) which appeared at 16, 20 and 20 mo, respectively. In higher magnification of 2-mo-old stomach (D), more typical NOS(+) neurons were oval in shape and intensely stained, with unstained nuclei. Some lighter cell bodies of NOS(+) neurons (left and right figures, arrows) were also seen which suggested that the activity of NOS were lower. Few cells with vaguely remained cellular outlines (right figure, arrowhead) shown in ganglia were confirmed to be other types of neurons. Statistical analysis showed that the decrease of NOS(+) neuronal numbers was started from 16 mo in stomach, 20 mo in jejunum and colon (E). Western blotting indicated that the trend of nNOS expression was coincident with the number of NOS(+) neurons in all three organs (F-H), and densitometric analysis of protein expressions normalized to β-actin. Statistical analysis was performed using one-way analysis of variance and data were represented as mean ± SD, statistical significance is: (E) # P < 0.05, ## P < 0.01 compared with previous stomach group; *** P < 0.001 compared with previous jejunum group; ††† P < 0.001 compared with previous colon group; (F-H) # P < 0.05, ### P < 0.001 compared with 2-mo-old group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with previous group; n=5 mice per group. Abbreviation: Sto, stomach; Jej, jejunum; Co, colon.