Fig. 7. Histopathological analysis of hCOX-2 and nontransgenic age-matched littermates. A, Levels of GFAP were compared with an assay for astrocytic activation, proliferation, or both in aged hCOX-2 mice. Quantitative Western blot analysis (50 μg of protein/lane) of GFAP and actin was performed at 20–24 months (n = 7 for each COX-2 and nontransgenic group). A representative set of four pairs of COX-2 and nontransgenic (NTg) controls demonstrates increases in GFAP in aged hCOX-2 brain compared with nontransgenic controls in three of four pairs, with the fourth pair displaying an equal amount of GFAP staining. B, Standardized GFAP of 20- to 24-month-old hCOX-2 and nontransgenic mice demonstrates a significant increase in GFAP in hCOX-2 mice compared with nontransgenic mice at 20–24 months of age. *Significant between-genotype difference as a result of ANOVA. Data are mean ± SEM. C, TUNEL analysis of 8-, 14-, and 22-month-old hCOX-2 and age-matched control littermates. Two-way (age × genotype) ANOVA with the square root transformation of the number of apoptotic cells in cerebral cortex per section showed significance for both effects (age,F(2,335) = 16.34; p< 0.0001; genotype, F(1,335) = 5.87;p < 0.02). Both groups of mice demonstrated a higher number of apoptotic cells at 14 and 22 months. However, the age-associated increase in apoptosis was more pronounced in hCOX-2 mice compared with nontransgenic littermates. #,##Significant age-related increase in the number of stained cells as a result ofpost hoc tests; #p < 0.01; ##p < 0.0001. *p < 0.01 for post hoc tests applied to the effect of genotype within each age group. Data are mean ± SEM. D–F, Determination of cellular phenotype of TUNEL-positive cells using double immunofluorescent staining. After TUNEL labeling, selected sections were doubly stained with Neu N monoclonal and anti-GFAP polyclonal antibodies to identify astrocytes and neurons, respectively. Apoptotic cells uniformly stained for Neu N and did not stain for GFAP, indicating that the TUNEL-positive cells are neurons. D, Light micrograph 630× magnification of a TUNEL-positive cell; note nuclear condensation and margination. E, Immunofluorescent staining of the same region with anti-Neu N antibody demonstrating staining of the nuclear compartment of a TUNEL-positive cell (vertical arrow); horizontal arrows in all panels demonstrate nonapoptotic neurons also stained with Neu N.F, Immunofluroscent staining with anti-GFAP antibody demonstrating the absence of GFAP staining of the TUNEL-positive cell; note GFAP-stained astrocyte (asterisks in all three panels).