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Venu Talla, Tsung-Han Chou, Vittorio Porciatti, John Guy; Kruppel-like factor 4 (KLF4) Knockout in Retinal Ganglion Cells Rescues Optic Neuritis in Experimental Autoimmune Encephalomyelitis (EAE) Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5735.
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© ARVO (1962-2015); The Authors (2016-present)
To rescue visual loss and optic neuropathy in the EAE mouse model using ssAAV2Cre-GFP mediated knockdown of floxed KLF4 (KLF4fl/fl) a transcriptional repressor of axon growth in RGCs and optic nerves (ON).
EAE was induced in floxed-KLF4 (n=6) and littermates (n=8) by sub-dermal injection of 0.1 ml homologous spinal cord emulsion in complete Freund's adjuvant at nuchal area. EAE sensitized floxed-KLF4 mice received an intravitreal injection of ssAAV-Cre-GFP into both eyes whereas the EAE littermates received scAAV-CMV-mCherry as an injection control. Un-sensitized littermates with (n=9) and without (n=7) scAAV-CMV-mCherry injections acted as additional controls. The visual function was assessed by recording the 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. All the mice were sacrificed after 9MPI. Retinas and ONs were dissected and Tuj1 labeled RGC numbers and ON axons were evaluated by immunofluorescence.
Expression: CSLO imaging at 5 and 9MPI in live EAE floxed-KLF4 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 corresponding PERG latencies were also delayed by 7%, 17% and 27% in EAE-mCherry at 1M, 4M (p<0.05) and 9MPI (p<0.05) respectively. Knockdown of KLF4 in EAE mice RGCs by AAV2-Cre-GFP injection rescued the amplitudes by 100% at all three time points tested (p<0.05), however the latencies were delayed significantly compared to control (p<0.001). OCT images of the EAE-mCherry showed a significant thinning in RNFL/RGC/inner plexiform layers while the OCT images of the KLF4 knockout EAE mice were comparable to control. Quantitative analysis of the Tuj1 positive RGCs and ONs revealed 32% fewer numbers (p<0.02) in EAE mice compared to KLF4 knockout mice.
KFL4 is involved in activation of inflammation in EAE and also has regenerative properties for RGCs and optic nerve axons that may be useful for the 8% of optic neuritis patients that experience permanent loss of vision.
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