Activation Of Anterior Cingulate Cortex Extracellular Signal-Regulated Kinase-1and-2(ERK1/2)Regulates Acetic Acid-Induced, Pain-Related Anxiety In Adult Female Mice

Objective To define the role of ACC extracellular signal-regulated (ERK)-1and-2(ERK2) activity in the development of pain-related anxiety/depression and the nocifensive response in acetic acid (AA)-elicited visceral pain.Methods The study was carried out on female Kunming mice. The model of visceral pain was established by intraperitoneal(ip) injection of AA(0.6%,10ml/kg), the same amount of saline was administered to another cohort of mice as the vehicle control group. The expression of pERK1/2in the ACC of mice was tested by immunohistochemistry and Western Blot. To reveal neurochemical phenotypes of activated ACC neurons following AA injection, double immunofluorescence labeling was performed. In order to explore the role of ERK1/2activity in the development of pain-related anxiety/depression and the nocifensive response in acetic acid (AA)-elicited visceral pain, thirty min before AA injection, the mice were injected subcutaneously with SL327(30mg/kg) to inhibited ERK1/2activation. Visceral pain behavior was assessed blindly by counting abdominal contractions for60min after AA injection, and the pain-related anxiety behavior was tested by Elevated plus-maze and Open field test2hr after AA injection.Results Biphasic ERK1/2activation in the ACC Following AA injection:We determined that0.6%AA injection induces abdominal contractions in female Kunming mice (p<0.0001, versus control). We found that ERK1/2was rapidly activated (phosphorylated) after AA injection. Immunohistochemistry and Western Blot revealed that there was a significant increase in the number of p-ERK1/2-labeled cells compared with those of the control group10min after AA injection (p<0.001). However, this increase was transient and returned to baseline levels30min after AA injection (p>0.05). Interestingly, p-ERK1/2expression increased again1hr after AA injection (p<0.05), but returned to baseline levels2hr after AA injection (p>0.05). Western blotting further confirmed that no significant changes in total ERK1/2levels were found between groups at any of the time points examined(p>0.05).Colocalization of p-ERKl/2and GAD following AA injection in ACC:To reveal neurochemical phenotypes of activated ACC neurons following AA injection, p-ERK1/2and GAD67(a marker of GABAergic-neurons) double immunofluorescence labeling was performed. Result shown that there was no co-localization of p-ERK1/2and GAD67was found in ACC neurons following AA injection.SL327reduces AA-induced anxiety-like behavior but not nociceptive pain:Thirty min before AA injection, the mice injected sc with SL327(30mg/kg) could inhibited ERK1/2activation, the positive cells of ERK1/2in ACC was significant reduced (p<0.001). We also found that blocking ERK1/2activation has no effect on AA-induced abdominal contractions (p>0.05). Two hours after AA injection, the Elevated plus-maze tests showed that the time spent in the open arm was significantly longer in SL327-treated mice compared with control animals (p<0.001), and the Open field tests revealed that the percentage of time in the inner area were significantly longer in SL327-treated mice compared with control animals (p<0.05)Conclusion The present study demonstrates that the activation of p-ERK1/2in the ACC, the brain area in control of mood disorders and pain processing, is responsible for acute visceral pain-related anxiety behaviors. Attenuation of the overactivity of ACC p-ERK1/2represents a potential, valuable therapeutic strategy for relief of pain-related anxiety.