2009, 2010), and (4) markers reflecting changes in astrocytic and microglial responses in spinal cord and DRG. Existing evidence shows that AM1241 acts as an agonist at the cannabinoid 2 receptor (CB2R) that results in suppression of nociceptive responses (Rahn et al. 2010), and prevents neuropathic and inflammatory pain (Nackley et al. 2004; Beltramo et al. 2006; Rahn et al. 2008), with selectivity demonstrated Inhibitors,research,lifescience,medical not only by utilizing pharmacological CB2R antagonists, but also by examining AM1241 analgesic efficacy in CB2R knockout mice (Ibrahim et al. 2003, 2006). CB2Rs are characterized on microglia

and macrophages in cell culture (Walter et al. 2003; Cabral and Marciano–Cabral 2005; Ehrhart et al. 2005) and in the rodent spinal cord following peripheral nerve damage (Zhang et al. 2003; Romero-Sandoval et al. 2008a) or in transgenic Inhibitors,research,lifescience,medical mice overexpressing the CB2R (Racz et al. 2008b), as well as in the human central nervous system (CNS) under inflammatory diseased conditions (Nunez et al. 2008). Given that AM1241 can act on CB2Rs expressed on spinal Inhibitors,research,lifescience,medical microglia, the potential additional (1) cytokine and (2) p-p38MAPK involvement in AM1241s efficacy in producing spinal anti-inflammatory actions concurrent

with anti-allodynia were examined in these studies. Related to these goals, we examined two methods to analyze immunofluorescent images of spinal cord tissue sections to identify Inhibitors,research,lifescience,medical the most sensitive procedure for detecting and quantifying differences in specific immunoreactive protein markers. In this context, an alternative method that utilizes spectral analysis procedures, demonstrated here, can be advantageous over conventional methods of image analysis. Materials

and Methods Animals A total of 52 pathogen-free adult male Sprague Dawley rats (300–400 g; Harlan Labs, Madison, WI) were used in all experiments. Rats were double housed in a temperature- and light-controlled (12 h light/dark; lights on at 6:00 AM) environment, with standard rodent chow and water available ad libitum. All procedures Inhibitors,research,lifescience,medical were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of New Mexico Health Sciences Center. Drugs The CB2R agonist used in these experiments was (R,S)-(2-iodo-5-nitrophenyl)-(1-[(1-lifescience methylpiperidin-2-yl)methyl]-1H-indol-3yl)-methanone ADP ribosylation factor (AM1241) from the aminoalkyndole classification (Yao et al. 2006). Water-soluble hydrochloride salt of racemic AM1241 was generously gifted (A. Makriyannis, Center for Drug Discovery, Northeastern University). Initial doses of AM1241 were based on those previously reported for i.v. injection (Beltramo et al. 2006) and pilot studies. A 1000-fold dose range of AM1241, dissolved in sterile saline (Hospira Inc, Lake Forest, IL) was tested (10–0.01 μg in 10 μl) or equivolume sterile saline as vehicle.