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Venu Talla, Vince Chiodo, Vittorio Porciatti, Sanford L Boye, William W Hauswirth, John Guy; Phosphatase and Tensin Homolog (PTEN) Knockout in Retinal Ganglion Cells (RGCs) Rescues Optic Neuritis in Experimental Autoimmune Encephalomyelitis (EAE) Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5531.
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© ARVO (1962-2015); The Authors (2016-present)
To rescue visual loss and optic neuropathy in EAE mouse model using ssAAV2Cre-GFP mediated knockout (KO) of floxed PTEN (PTEN fl/fl), a negative regulator of mTOR (mammalian target of rapamycin) pathway involved in optic nerve axonal regeneration in RGCs.
EAE was induced in floxed-PTEN mice (n=11) and littermates (n=8) by subdermal injection of 0.1 ml homologous spinal cord emulsion in complete Freunds adjuvant in the nuchal area. EAE sensitized floxed-PTEN mice received an intravitreal injection of ssAAV-Cre-GFP into both eyes whereas EAE littermates received scAAV-mCherry as an injection control. Unsensitized littermates with or without scAAV-mCherry injections acted as additional controls (n=16). Visual function was assessed by recording pattern electroretinograms (PERG). Spectral domain OCT evaluated the thickness of the inner plexiform layer to the nerve fiber layer at 1, 4 and 9 months post injection (MPI). Expression of the Cre-GFP in the RGCs and ONs were evaluated in live mice by confocal scanning laser ophthalmoscope (CSLO) imaging at 5 and 9 MPI. Retina and ON tissues were dissected and Tuj1 labeled RGCs and ON axons were evaluated by immunofluorescence.
Expression: CSLO imaging at 5 and 9MPI in live EAE floxed-PTEN mice revealed expression of Cre-GFP in RGC layer. Rescue: PERG analysis at 1M, 4M and 9MPI showed a 25%, 37% and 41% reduction in amplitude of EAE-mCherry mice compared to mCherry/control mice (p<0.005). The PERG latencies were also delayed by 7%, 17% and 27% in EAE-mCherry at 1M, 4M (p<0.05) and 9MPI (p<0.05). Interestingly, knockout of PTEN in EAE mice RGCs by AAV2-Cre-GFP injection rescued the amplitudes by 51, 54 and 77% (p<0.005) at 1, 4 & 9MPI. However, the latencies were not significantly rescued compared to EAE-mcherry. OCT images of the EAE-mCherry mice showed a significant thinning (p<0.005) in RNFL/RGC/inner plexiform layers while the OCT images of the PTEN (KO) EAE mice were 150% (p<0.0001) & 122% (p<0.0001) thicker compared to control at 5 & 9 MPI. Quantitative analysis of Tuj1 positive cells revealed 57% rescue of RGCs in PTEN (KO) mice compared to EAE mice (p<0.05).
PTEN is involved in intrinsic axonal growth arrest and knocking out the PTEN in RGCs is a strategy that may be useful for treating the 8% of optic neuritis patients experiencing permanent loss of vision.
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