Fig. 2

Effectiveness of the expression cassette with regard to coupling to GFAP transcription and Müller cell specificity in vivo. A Experimental set up for in vivo studies: eyes of C57BL/6 J wildtype mice were exposed to hypoxic conditions by elevated intraocular pressure (IOP), resulting in ischemic tissue responses. Subsequently, AAV vectors carrying expression cassettes for regulatory truncated FH variants and an EGFP-only control were injected. Tissue samples were collected for evaluation at intervals of 3 and 14 days post injections (dpi). B Scheme delineating the viral constructs used in the present study. C Cryosections of the central retina showed that EGFP was present in Müller cells identifiable by their unique morphology spanning the whole tissue from the ganglion cell layer (GCL) to the outer border of the outer nuclear layer (ONL). This indicated a cell type-specific expression of the AAV construct mainly in Müller cells. D Immunohistochemistry (IHC) analysis of peripheral retinal flat mounts at 14 dpi at the level of the nerve fiber layer after ischemic injury and AAV application was performed to assess whether also astrocytes, that even in the healthy retina express high levels of GFAP, were transduced by the AAV. EGFP-positive cells co-expressed the Müller cell marker glutamine synthetase (Glul), but not high levels of GFAP. In contrast, highly GFAP-positive astrocytes were not highlighted by EGFP-labeling (also see Additional file 1: Fig. S2). E Quantitative real-time PCR of isolated Müller cells showed a decrease in Glul expression in ischemic Müller cells at 3 dpi (left panel). This reduction is attenuated by 14 dpi. In contrast, Gfap expression increased by 3 dpi and remained elevated until 14 dpi (right panel). Each data point per biological replicate (n = 3–4) is represented by a dot in the graph. F EGFP expression levels showed correlation to Gfap expression in all three treatment groups as determined by qPCR (14 dpi). Pearson’s correlation coefficients of Gfap (-)dCT vs EGFP (-)dCT were as follows: ρ_{(AAV_EGFP)} = 0.8561, ρ_{(AAV_FH1-4^19-20)} = 0.8561 and ρ_{(AAV_FH1-7^19-20)} = 0.8534. Each data point per biological replicate (n = 3–4) is represented by a dot in the graph. G Representative microscopic images showing GFAP immunostaining on sections at 14 dpi (left panel). Quantitative analysis of mean fluorescence intensity encompassing the retina from the outer limiting membrane to the inner plexiform layer (right panel). The ganglion and nerve fiber layers were excluded from the analysis to eliminate signals from astrocytic GFAP expression. Each data point per biological replicate (n = 3–4) is represented by a dot in the graph