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Dorota Stankowska, Alena Minton, Raghu Krishnamoorthy; Brn3b mediated regeneration of the optic nerve in a rodent model of glaucoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6357.
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Glaucoma is an optic neuropathy, commonly associated with an increase in intraocular pressure (IOP). Studies using rodent models of glaucoma indicate the initial damage due to IOP occurs to optic nerve axons in the lamina-like region. The presence of intact somas following axonal damage provides a window of opportunity to attempt regeneration of the optic nerve. One good candidate to test for neuroregenerative ability is the class 4 POU domain transcription factor, Brn3b, which is selectively expressed in retinal ganglion cells. The purpose of this study was to determine if adeno-associated virus (AAV) mediated overexpression of transcription factor Brn3b in retinal ganglion cells could promote axonal regeneration after optic nerve injury in glaucomatous rats.
The Morrison’s rat model of glaucoma was used to elevate IOP in one eye of Brown Norway rats. One week after IOP elevation, rats were injected with either AAV-Brn3b or AAV-Empty viruses and maintained for an additional 3 weeks of IOP elevation. Retina sections through the optic nerve heads (ONHs) were obtained and subjected to immunohistochemical analysis of neuroregenerative marker proteins. Protein extracts were prepared from optic nerves and immunoblot analyses of GAP-43, neurofilament-M and abLIM were carried out. In separate set of experiments, an axonal tracer (CT-B) was intravitreally injected 48 h prior to sacrifice, to assess functionality of regenerated axons due to Brn3b overexpression following IOP elevation in rats.
An increased immunostaining of neuroregenerative markers GAP-43, abLIM and ac-Tuba was observed in both retinas and ONHs (posterior to the site of axonal injury) in AAV-Brn3b transduced, IOP-elevated rat eyes in comparison with IOP elevated, AAV-Empty virus injected rat eyes. Immunoblot analysis of rat optic nerves revealed prominent increase in proregenerative proteins: GAP-43, abLIM and neurofilament-M in IOP elevated AAV-Brn3b injected rats, compared to empty vector injected rats. An appreciable increase in CT-B tracer along the axonal tracts, posterior to the ONH was detected in IOP elevated AAV-Brn3b injected rats indicative of an increased regenerative response and improved axonal function, mediated by Brn3b overexpression.
The data indicates that administration of AAV-Brn3b virus has the potential to reverse optic nerve damage, possibly through regeneration of the damaged axons of the optic nerve.
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