Abstract
Purpose: :
Ischaemic optic neuropathy (ION) is a devastating ocular disease that can result in partial or complete blindness. We believe that gap junctions play a role in the spread of damage in the optic nerve, and undertook this study to 1) Establish in vitro models to study the disease, 2) Identify the role of connexins in the spread of the lesion, and 3) Evaluate the use of connexin43 specific antisense oligodeoxynucleotides (AsODN) in blocking lesion spread.
Methods: :
Optic nerve segments from p21–25 wistar rats were cultured using an in vitro interphase method. ION was simulated using oxygen–glucose deprivation for two hours before adding connexin43 specific AsODN in Pluronic F–127 gel. Controls were treated with gel vehicle alone or no treatment. The nerves were processed for immunohistochemistry (including Cx43) and quantitatively analysed using cell death, tissue morphology, vascular defects and inflammation (astrocyte numbers / microglial cell phenotype change) as indicators of neuropathy.
Results: :
Cx43 was upregulated in control ION nerves. Treatment with Cx43 AsODN arrested this up–regulation. Tissue swelling associated with ischaemia was over 25% in controls but constrained to 10% with AsODN. Cell death was also much higher in untreated controls than AsODN treated tissue at 6 hours. Analysis of blood vessel integrity showed that mean vessel length remained constant in AsODN treated nerves but was reduced by 30% in untreated nerves over a 6 day period. Astrocyte numbers were less in AsODN treated optic nerves and microglial cells retained their normal star shaped phenotype, whereas, in control nerves microglial cells changed to an activated macrophage state by day 3.
Conclusions: :
We have established an in vitro model of ION. Treatment of ION with Cx43 specific AsODN significantly reduced the effects of ischaemia and signs of inflammation compared to control optic nerves.
Keywords: neuro-ophthalmology: optic nerve • ischemia • gap junctions/coupling