Harnessing The Melanocortin Receptor System To Reduce Leukocyte Recruitment Following Stroke

Paul Holloway1, Stephen Getting0,2, Felicity Gavins1. 1Imperial college, London, UK, 2University of Westminster, London, UK

Introduction

Stroke is a leading cause of mortality worldwide and the largest single cause of adult disability; however, current pharmacological interventions remain extremely limited. A disproportionate inflammatory response exacerbates injury following stroke, thus, resolving inflammation may provide an effective treatment.

The G-protein coupled melanocortin receptors (MCs) have potent anti-inflammatory and neuro-protective activities, making them particularly attractive targets for stroke treatment. However, the relative contribution of each receptor subtype is not fully understood. The melanocortin receptors MC1, MC3 and MC4 have previously been shown to initiate anti-inflammatory processes in a number of different tissues and disease states. This project aims to determine the main receptor subtype/s involved in providing anti-inflammatory protection following cerebral ischemia reperfusion (I/R).

Methods

The bilateral common carotid artery occlusion (BCCAo) mouse model of global stroke was utilised in conjunction with intravital microscopy (IVM), to quantify neuro-inflammatory responses through real time visualisation of cerebral leukocyte recruitment. Investigations employed both pharmacological and genetic approaches using MC selective compounds and KO mice. Briefly; male WT (C57BL/6), recessive yellow (e/e) mice (possessing a non-functional MC1) or MC3KO mice (18-30g) were anesthetised (pentobarbital 100mg/kg i.p) and subjected to 5 minutes global brain ischemia followed by 40 minutes or 2 hours of reperfusion. Melanocortin treatments were given (10μg/mouse i.p) at the start of reperfusion. IVM was used to quantify leukocyte-endothelium interactions in terms of rolling cell flux and adherent leukocytes, expressed as cells/mm2. 40 minute reperfusion WT groups n=6 mice/group, MC3KO and e/e groups n=4. Two hour reperfusion groups n=3. P<0.05 was considered significant using ANOVA with Bonferroni test post hoc analyses.

Results

In comparison to sham-operated animals (rolling: 21.5±6.9 cells/mm2, adherence: 42.1±14.4 cells/mm2) BCCAo significantly increased leukocyte rolling (191.0 ±31.49 cells/mm2) and adherence (282.3 ± 49.4 cells/mm2) after 40 minutes of reperfusion in WT mice. Both MC3KO and e/e mice showed no significant differences in sham leukocyte-endothelial interactions and displayed I/R induced leukocyte rolling and adhesion at 40 minutes. However the I/R induced leukocyte rolling observed in e/e mice (539.28± 153.1 cells/mm2) was significantly greater than in WT mice, suggesting an enhanced inflammatory response and an important role for MC1.

WT mice treated with the pan receptor agonist, α-MSH. presented a significant reduction in I/R induced leukocyte rolling (58.1±10.4 cells/mm2) and adhesion (51.7±17.2 cells/mm2) at 40 minutes reperfusion. The MC3 selective agonist, Dtrp8-γ-MSH, similarly showed reduced leukocyte adherence, although did not significantly reduced rolling. Antagonism of MC3 and MC4 using SHU9119 did not significantly abrogate the anti-inflammatory actions of α-MSH.

Extending the reperfusion period to 2 hours further exacerbated leukocyte-endothelium interactions (rolling: 243.9 ±74.0 cells/mm2, adherence: 659.7 ±115.6 cells/mm2). At 2 hours reperfusion α-MSH mediated reduction in leukocyte recruitment remained significant however SHU9119 treatment blunted the anti-inflammatory actions of α-MSH and prevented Dtrp8-γ-MSH mediated reduction in rolling and adhesion.

Conclusions

These results demonstrate that the anti-inflammatory actions of MCs may prove useful in the context of stroke. Furthermore results MC1 activation may be of particular importance in the early stages of reperfusion. However as the inflammatory reaction progresses the role of MC3 may become more prominent.