To assess tumor hypoxia after treatment, LH
BETAT
AG mice were injected intraperitoneally with a 0.16 mL suspension of pimonidazole (a drug used to detect hypoxia that penetrates all tissues, including the brain). This suspension consisted of 10 mg of pimonidazole hydrochloride (Chemicon, Temecula, CA) in 1 mL saline. Pimonidazole is known to bind to thiol-containing proteins in cells under low O
2 tension.
17 These adducts can be detected with specific antibodies and stained using immunohistochemical techniques. Animals were euthanized 2 hours after pimonidazole injection, and eyes were harvested and sectioned for histopathologic examination. Eyes were fixed with cold methanol for 10 minutes and immunostained with a directly labeled antibody recognizing pimonidazole adducts (Hypoxyprobe 1-Mab-1-FITC, clone 4.3.11.3; Chemicon) or the same concentration of a directly labeled isotype control antibody (mouse IgG1-FITC; Caltag, Burlingame, CA). Background signal intensities were minimal. All samples were normalized to intensities from isotype controls.
Both the density and the spatial distribution of hypoxic regions were analyzed independently. The density of hypoxia was measured by calculating the ratio of the amount of pixels stained with pimonidazole (i.e., marker for hypoxia) over the amount of pixels for total tumor area (100 × HPF; Adobe Photoshop CS5; Adobe, San Jose, CA). The spatial distribution of hypoxia in each tumor was performed by doing a grading analysis of different size hypoxic areas (i.e., small, medium, large, and very large) present in each intratumoral region (i.e., apex, center, lateral, and base; 100 × HPF). The different size hypoxic areas were labeled based on a cell count in the following manner: small size hypoxic areas were represented by ≤20 hypoxic cells and were assigned a value of 1; medium size hypoxic areas were represented by 21 to 40 hypoxic cells and were assigned a value of 2; large size hypoxic areas were represented by 41 to 80 hypoxic cells and were assigned a value of 4; and very large size hypoxic areas were represented by ≥81 hypoxic cells and were assigned a value of 8. A final value for each intratumoral region (e.g., apex) in all the tumors was obtained by adding all the values assigned for the different size hypoxic areas.