The Relative Importance Of The Melanocortin Receptors In Suppressing Cerebral Ischemia Reperfusion Induced Leukocyte Recruitment

Paul Holloway1, Stephen Getting2, Felicity Gavins1. 1Imperial College, London, UK, 2University of Westminster, London, UK

Cerebral ischemia reperfusion (I/R) has been shown to induce significant leukocyte recruitment which serves to exacerbate brain injury and amplify inflammatory responses. Potent anti-inflammatory and neuro-protective actions have been attributed to the melanocortin receptor system, thus targeting this system may provide a novel therapeutic strategy to treat stroke. Melanocortin receptor (MC) subtypes MC1 and MC3 have been shown to mediate MC anti-inflammatory actions, however their relative importance is poorly understood and may differ with the pathophysiological environment.

In the present study the bilateral common carotid artery occlusion (BCCAo) mouse model of global cerebral I/R has been used along with intravital microscopy to investigate the potential of melanocortin treatments in reducing cerebral I/R induced leukocyte recruitment. Investigations have utilised both MC selective compounds and receptor mutant mice. Briefly, male C57BL/6, recessive yellow (e/e) mice possessing a non-functional MC1 or MC3 -/- mice (18-30g) were anesthetised using sodium pentobarbital (100mg/kg i.p)and subjected to 5 min of BCCAo followed by either 40 min or 2 h of reperfusion. Melanocortin treatments were given (10μg/mouse i.p) at the start of reperfusion. Intravital microscopy was used at the end of the reperfusion time to quantify leukocyte recruitment in cerebral venules and was quantified in terms of rolling cell flux and adherent leukocytes, expressed as cells/min/mm2. C57BL/6 40 min reperfusion groups n = 6 mice/group, MC3 -/- and e/e groups n = 4. 2 h reperfusion groups n = 4. Serum cytokine levels were also assessed using ELISA and cytometric bead array and MC mRNA expression determined in both brain and blood using qRT-PCR. Statistical evaluation was performed using ANOVA with Bonferroni test post hoc analyses. P<0.05 was considered significant.

BCCAo was found to induced significant leukocyte recruitment by 40 min of reperfusion compared to sham operated animals, with leukocyte rolling increasing from 21.5 cells/mm2/min to 191.0 cells/mm2/min and adhesion from 42.1 cells/mm2/min to 282.3 cells/mm2/min. Extending the period of reperfusion to 2 h resulted in further increases in adhesion to 1500% of sham levels. Treatment with the non-selective melanocortin agonist, α-MSH, significantly reduced I/R induced leukocyte rolling (by 80%) and adhesion (by 68%) at 40 min reperfusion. Treatment remained effective in suppress I/R induced leukocyte recruitment when reperfusion was extended to 2 h. Selective pharmacological activation of MC1 with BMS-470,539 was found to provide similarly potent inhibition of leukocyte recruitment at 40 min furthermore, MC1 mutant e/e mice also displayed enhanced leukocyte rolling and serum TNF-α levels, while no inflammatory abnormalities were observed in MC3 -/- mice. However by 2 h the anti-inflammatory effects of BMS-470,539 subsided and e/e mice no longer showed elevated levels of leukocyte recruitment. On the other hand MC3 targeted compounds were highly effective at this later time point. These investigations reveal an important role for MC1 immediately following BCCAo with a later shift toward MC3 mediated processes by 2 h. qRT-PCR revealed no changes in receptor mRNA expression across these time points however by 2 h α-MSH treatment was found to reduce NF-κβ related gene expression but not at 40 min. It is thus possible that the apparent shift in receptor importance is due to the activation of distinct signalling pathways rather than change in receptor expression, with MC1 initiating rapidly acting anti-inflammatory signalling and MC3 allowing for a more delayed suppression of inflammatory processes through the inhibition of NF-KB signalling.