title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.
abstract: Connexin43 gap junction protein is expressed in astrocytes and the vascular endothelium in the central nervous system. It is upregulated following central nervous system injury and is recognized as playing an important role in modulating the extent of damage. Studies that have transiently blocked connexin43 in spinal cord injury and central nervous system epileptic models have reported neuronal rescue. The purpose of this study was to investigate neuronal rescue following retinal ischaemia-reperfusion by transiently blocking connexin43 activity using a connexin43 mimetic peptide. A further aim was to evaluate the effect of transiently blocking connexin43 on vascular permeability as this is known to increase following central nervous system ischaemia. Adult male Wistar rats were exposed to 60 min of retinal ischaemia. Treatment groups consisted of no treatment, connexin43 mimetic peptide and scrambled peptide. Retinas were then evaluated at 1-2, 4, 8 and 24 h, and 7 and 21 days post-ischaemia. Evans blue dye leak from retinal blood vessels was used to assess vascular leakage. Blood vessel integrity was examined using isolectin-B4 labelling. Connexin43 levels and astrocyte activation (glial fibrillary acidic protein) were assessed using immunohistochemistry and western blot analysis. Retinal whole mounts and retinal ganglion cell counts were used to quantify neurodegeneration. An in vitro cell culture model of endothelial cell ischaemia was used to assess the effect of connexin43 mimetic peptide on endothelial cell survival and connexin43 hemichannel opening using propidium iodide dye uptake. We found that retinal ischaemia-reperfusion induced significant vascular leakage and disruption at 1-2, 4 and 24 h following injury with a peak at 4 h. Connexin43 immunoreactivity was significantly increased at 1-2, 4, 8 and 24 h post ischaemia-reperfusion injury co-localizing with activated astrocytes, Muller cells and vascular endothelial cells. Connexin43 mimetic peptide significantly reduced dye leak at 4 and 24 h. In vitro studies on endothelial cells demonstrate that endothelial cell death following hypoxia can be mediated directly by opening of connexin43 hemichannels in endothelial cells. Blocking connexin43 mediated vascular leakage using a connexin43 mimetic peptide led to increased retinal ganglion cell survival at 7 and 21 days to levels of uninjured retinas. Treatment with scrambled peptide did not result in retinal ganglion cell rescue. Pharmacological targeting of connexin43 gap junction protein by transiently blocking gap junction hemichannels following injury provides new opportunities for treatment of central nervous system ischaemia.
authors.affiliation: Department of Ophthalmology, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand. h.daneshmeyer@auckland.ac.nz
authors.forename: Helen V
authors.initials: HV
authors.lastname: Danesh-Meyer
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Kerr
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Elizabeth K
authors.initials: EK
authors.lastname: Eady
authors.forename: Simon J
authors.initials: SJ
authors.lastname: O'Carroll
authors.forename: Louise F B
authors.initials: LF
authors.lastname: Nicholson
authors.forename: Cameron S
authors.initials: CS
authors.lastname: Johnson
authors.forename: Colin R
authors.initials: CR
authors.lastname: Green
mesh_terms: D000818:Animals; D018031:Connexin 43 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007511:Ischemia / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D009842:Oligopeptides / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012164:Retinal Diseases / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
chemical_list: D018031:Connexin 43; D009842:Oligopeptides; C571669:valyl-aspartyl-cysteinyl-phenylalanyl-leucyl-seryl-arginyl-prolyl-threonyl-glutamyl-lysyl-threonine