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. 2011 Apr;3(4):235–245. doi: 10.1002/emmm.201100126

Figure 2. Phenotype assessment of CLDN5 AAV-2/9.

Figure 2

  1. An AAV-2/9 with an eGFP reporter gene was injected sub-retinally in an adult C57/Bl6 mouse and 3 weeks post-injection, a retinal whole-mount showed the pattern of transduction to be widespread (green).
  2. Although transduction was observed throughout the retina, it was apparent that this transduction was heavily localized just to the neural retina (30–50% transduction) at the site of injection (arrow and high magnification inset), with the entire RPE being transduced. There was no transduction of AAV-2/9 observed along the optic nerve.
  3. High-magnification microscopy of retinal cryosections injected sub-retinally with the eGFP AAV-2/9 (green) in the region of neural retina transduction and subsequent staining with a griffonia-simplicifolia isolectin-Alexa-568 (endothelial cell staining—red), showed the transduction efficiency of the AAV-2/9 serotype for endothelial cells.
  4. Contrast-enhanced MRI showed extravasation of the MRI contrasting agent Gd-DTPA (MW 742 Da) in mice injected in the right eye with CLDN5 AAV-2/9, but not in the left eye, injected with NT AAV-2/9, when animals were supplemented with the inducing agent doxycycline (2 mg/ml with 5% sucrose in their drinking water) for 3 weeks post-injection. The inverted (LUT) image highlights the localized extravasation of Gd-DTPA in the right eye compared to the contra-lateral control (arrows).
  5. Both horizontal and sagittal MRI show the pattern of extravasation of Gd-DTPA localized to the site of sub-retinal inoculation but that Gd-DTPA permeates the entire retina of the CLDN5 AAV-2/9-injected eye.
  6. This extravasation was a consistent observation (***p = 0.0001) in all mice analysed (n = 9), with the background pixel intensity from the NT AAV-2/9-injected eye being subtracted from the CLDN5 AAV-2/9-injected eye.
  7. Retinal cryosections of CLDN5 AAV-2/9-injected mice perfused with microperoxidase (1881 Da) showed this tracer molecule to be confined within the retinal microvasculature, without signs of extravasation (n = 4).
  8. Rod and cone isolated ERGs showed typical ERG tracings expected 3 weeks post-sub-retinal injection and no differences between the NT AAV-2/9-injected eyes and the CLDN5 AAV-2/9-injected eyes.
  9. T-2-weighted coronal MRI analysis revealed no signs of retinal oedema in either NT AAV-2/9 or CLDN5 AAV-2/9 injected eyes (n = 9).