Abstract
Purpose: :
In response to central nervous system injury connexin43 (Cx43) gap junction channels are upregulated in the vascular bed and the blood-brain barrier is compromised, contributing to lesion spread and neuron loss. We used systemically delivered Cx43 mimetic peptides to block gap junction hemichannel opening following rat retinal ischemia-reperfusion in order to investigate the possibility of reducing leak and inflammation, and so protect retinal ganglion cells (RGCs).
Methods: :
Raised intraocular pressure was used to induce one hour of hypoxia in a rat retina ischemia-reperfusion model. Vessel leak was determined using Evans Blue dye injection. Cx43 changes, vascular integrity (Isolectin-B4 label), astrocytosis (GFAP levels) and RGC loss (Brn3A label) was analysed in wholemounts at 4, 8 and 24 hours, or 7 and 21 days post-ischemia. Cx43 mimetic peptide (or scrambled control peptide) was delivered by a single intraperitoneal injection immediately following reperfusion and effects on all parameters investigated.
Results: :
Cx43 was significantly upregulated in endothelial cells after reperfusion, peaking within 4 hours and remaining high until 24 hours (p<0.05). Vessel leak resulting from hemichannel mediated endothelial cell rupture peaked at 4 hours post reperfusion, but remained significant at 8 and 24 hours (p<0.05). Astrocytosis was not initially universal, but correlated with regions of vascular leak. Within 21 days of ischemia 35% of RGCs were lost. A single injection of Cx43 mimetic peptide reduced vascular leak and associated astrocytosis. Leak at 4 hours was 5% of that in the control (then at its highest level), and peaked at 8 hours post perfusion when it reached 20% of hypoxia only controls at that time point. The mimetic peptide treatment significantly reduced RGC loss by two thirds to 9% - 14 % (p<0.05).
Conclusions: :
Cx43 upregulation and hemichannel opening mediates vascular leak which is associated with astrocytosis and subsequent RGC loss following retinal ischemia. Reduction in vascular leak was brought about by systemic delivery of Cx43 mimetic peptide at a level expected to block hemichannels, but not to uncouple gap junctions. Modulation of Cx43 after retinal ischemia-reperfusion injury alleviates vascular leakage, reduces inflammation and provides significant neuron sparing.
Keywords: ganglion cells • gap junctions/coupling • ischemia