Figure 4. Quantitative analysis of the numbers of NOS-immunopositive cells and the total NOS-immunopositive area. We used image analysis software to count immunopositive cell numbers and to measure the immunopositive area in the unit area (200 µm x 200 µm). In comparison with the normal group, the numbers of eNOS- and iNOS-immunopositive cells (A,B) significantly decreased in the hypofunction group, and they increased to close-to-normal levels after the recovery period. eNOS cell numbers: normal, 17.41 ± 3.11 cells/unit; hypofunction, 9.05 ± 2.17 cells/unit; and recovery, 15.75 ± 2.92 cells/unit. iNOS cell numbers: normal, 74.75 ± 5.47 cells/unit; hypofunction, 30.58 ± 4.18 cells/unit; and recovery, 70.58 ± 6.11 cells/unit. Immunopositive area (%) means the percentage of immunopositive cells in the unit area. The pattern changes in the immunopositive area were similar to those in the cell number (C,D). The change in the NOS-immunopositive area was greater than the NOS-immunopositive cell number in both eNOS and iNOS. eNOS-immunopositive area: normal, 1.71 ± 0.29%; hypofunction, 0.72 ± 0.19%; and recovery, 1.62 ± 0.19%. iNOS-immunopositive area: normal, 6.00 ± 0.56%; hypofunction, 1.81 ± 0.32%; and recovery, 5.64 ± 0.57%. The data are means ± SD. N = 12 for each group. *p < 0.05. **p < 0.01.