Cyclooxygenase-3 (Cox-3) is a recently identified splice variant of the Cox-1 gene (Chandrasekharan et al. 2002) in which part of intron 1 is retained. Cox-3 shows cyclooxygenase activity but is inhibited by paracetamol-related drugs rather than traditional non-steroidal anti-inflammatories. Cox-3 was originally identified in canine cortex; in this study the localisation of Cox-3 has been studied in rat brain.
Wistar rats (250-300g) were humanely killed by decapitation under halothane anaesthesia and brains were rapidly removed and frozen on dry ice. All animal procedures conformed to UK legislation. Brains were sectioned either horizontally or coronally at 10µm and thaw mounted on poly-L-lysine coated slides. Immunohistochemistry was performed using an antibody raised against a peptide corresponding to the first 13 amino acids of the predicted retained intron 1 sequence (Chandrasekharan et al. 2002). Sections were post-fixed in 4 % paraformaldhyde in phosphate buffer. Endogenous peroxidases were quenched by treatment with 0.5 % H2O2 in methanol and non-specific binding was blocked in normal goat serum diluted 1:50 in 0.1M PBS. Sections were incubated in affinity-purified anti-Cox-3 antibody (1:2000) overnight at 4°C. Sections were rinsed and incubated in biotinylated goat anti-rabbit secondary antibody (1:2000) for 30 mins. Following a further 30 mins incubation in Vectastain ABC reagent, the substrate, diaminobenzidine tetrahydrochloride was added for 2 min. As a negative control, the primary anti-serum was replaced with immune-depleted serum at the same dilution under exactly the same conditions.
Control sections of rat brain showed no positive staining at all. Brains incubated in anti-Cox-3 antibody showed specific staining in neurons, that in many cases seemed to be nuclear or peri-nuclear. Areas of the brain in which positive neurons were identified included the hippocampal formation, deep but not superficial cortical layers and the spinal trigeminal nuclei. Specific neuronal staining was also identified in spinal cord, particularly in motor neurons. No staining was seen in white matter areas such as the corpus callosum.
These data indicate that Cox-3 protein is expressed in neurons within the rat nervous system. Glial expression was not seen in these preliminary studies. Cox-3 has been shown to be potently inhibited by drugs such as paracetamol, at lower concentrations than those that inhibit either Cox-1 or Cox-2. The analgesic action of paracetamol is thought to be exerted centrally; these data support the hypothesis that Cox-3 may be the central target of paracetamol and related drugs.