NeuN area measurements and statistical analysis
To measure the NeuN+ areas, six sections from the three representative animals in each of the main time groups, i.e., 10, 35, and 90 days were selected. The stained sections were digitized and the areas covered by DG and CA1 were outlined using a cursor. The same criteria for threshold detection were used for all 36 sections (9 dorsal 2VO, 9 ventral 2VO, 9 dorsal sham and 9 ventral sham). The area measurements were obtained using Image J software (Http://rsb.info.nih.gov/ij/). Mean values of these areas are presented in Table 1, giving reliable, comparative estimates of the neuronal populations in CA1 and DG of 2VO and sham animals.
Table 1.  Summary of NeuN+ area measurements indicating regional changes in hippocampal cell layers at different time points after ischemia.
10 days  35 days  90 days
Area  Sham  2VO  %Δ  Sham  2VO  %Δ  Sham  2VO  %Δ
DG Dorsal  230,000 ± 19,920  214,100 ± 4,500  −7%  213,200 ± 30,410  255,600 ± 29,830  ±20%  230,800 ± 24,240  288,800* ± 50,390  ±25%
DG Ventral  193,100 ± 9,840  191,100 ± 27,360  −1.4%  178,800 ± 12,290  200,300 ± 11,870  −12%  200,800 ± 19,320  202,700 ± 7,410  ±1%
CM Dorsal  131,200 ± 20,160  8,200* ± 725  −94%  124,500 ± 9,410  1,500* ± 1,120  −99%  124,700 ± 7,240  8,900* ± 12,950  −93%
CM Ventral  56,900 ± 7,640  45,600* ± 13,790  −20%  83,700 ± 12,760  38,600* ± 5,340  −54%  68,900 ± 6,570  39,800* ± 26,900  −42%
Mean areas (μm2) and standard deviations are shown for each group. *indicates statistically significant difference between sham and 2VO groups. 2 way ANOVA with n = 3 in each group taking total time and treatment as variables. Area measurements (μm2) were done using Image J software as detailed in methods. Statistical analysis was performed with SigmaStat software. Data sets from control and 2VO groups were compared using 2-way ANOVA with time and treatment as the two variables and post-hoc pairwise comparisons with Holm-Sidak method at P < 0.05. In some cases where only two groups were compared at a single time point a t-test or equivalent non-parametric test were used.