Abstract
Purpose :
Connexin 43 (Cx43) hemichannels(HC) are known to be involved in promoting central nerve system inflammation and secondary injury. In this study, we demonstrated that inhibition of Cx43 hemichannels function by antibodies improved retinal function and reduced ganglion cell damages.
Methods :
Cx43 HC is specifically inhibited by Cx43(E2) and Cx43(M1) antibodies for in vitro and in vivo studies. For vitro study, conditioned medium (CM) collected from primary astrocytes treated with IL-1β and TNF-α for 24 h in the absence or presence of E2 was used to treat differentiated RGC-like R28 cells for 48 h. Ethidium bromide (EB) was used to assess HC opening. The apoptosis, live cells and axon damage were quantified by AnnexinV/ Propidium Iodide (PI), Typan blue and phalloidin. ATP and glutamate levels in CM were detected by kits. Optic nerve compression injury (ONC) was applied to C57BL/6J wild-type (WT) and Cx43 heterozygous (+/-) mice. M1 was administrated through intravitreal injection 30 mins after ONC and the expression of neuroinflammation markers (CD68, inducible nitric oxide synthase: iNOS) and reactive gliosis markers (glial fibrillary acidic protein: GFAP) was assessed by immunofluorescence and western blotting. Optical coherence tomography (OCT), electroretinogram (ERG) and immunofluorescence (BRN3A and SMI-32) were performed to evaluate injuries.Apoptosis of RGC was determined by TUNEL.
Results :
IL-1β and TNF-α treatment increased opening of Cx43 HC and increased ATP and glutamate release in primary astrocytes. The R28 apoptosis was increased significantly associated with decreased number of live cells after culturing in CM collected from IL-1β and TNF-α-treated astrocytes. However, CM from Cx43(E2)-treated astrocytes showed reduced ATP and glutamate levels, apoptotic cells and axonal damage. ONC promoted neuroinflammation and gliosis, which increase retinal injury and RGC apoptosis. Haplosufficiency of Cx43 improved retinal function and reduced retinal injury after ONC. In addition, inhibition of Cx43 HC by M1 reduced microglia and neuroinflammation, reactive gliosis and apoptosis, leading to preservation of RGCs and visual function after ONC.
Conclusions :
The results suggest that opening of Cx43 HC in response to optical nerve injury and neuroinflammation promotes RGC loss and impedes retinal function, while inhibition of Cx43 HC protects RGC and retinal function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.