Correlation between venous tortuosity and concentration of VEGF in the aqueous humor.

<p>The degree of venous tortuosity is significantly correlated with the VEGF concentration in the aqueous. (r = 0.49, <i>P</i> = 0.004)</p

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for <em>In Vivo</em> Rescue of Developmental Photoreceptor Disorders

<div><h3>Background</h3><p>Adeno-associated virus (AAV) is well established as a vehicle for <em>in vivo</em> gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for <em>in vivo</em> functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.</p> <h3>Methodology/Principal Findings</h3><p>We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the <em>Cone-rod homeobox</em> gene knockout (<em>Crx</em> KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing <em>Crx</em> under the control of the <em>Crx 2kb</em> promoter into the neonatal <em>Crx</em> KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the <em>Crx</em> KO retina.</p> <h3>Conclusion/Significance</h3><p>In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of <em>Crx</em>.</p> </div

CRVO tortuosity Data English

CRVO tortuosity Data Englis

Quantitative analysis of ERG amplitudes in control and AAV-Crx treated eyes.

<p>Quantitative analysis of ERG amplitudes in control and AAV-Crx treated eyes.</p

Correlation between venous tortuosity index and foveal thickness.

<p>The degree of venous tortuosity is significantly correlated with the foveal thickness. (r = 0.40, <i>P</i> = 0.02)</p

Clinical Characteristics and SD-OCT findings of Patients with AZOOR-Complex.

<p>Abbreviations: AZOOR, acute zonal occult outer retinopathy; MEWDS, multiple evanescent white dot syndrome; AIBSE, acute idiopathic blind spot enlargement; BCVA, best-corrected visual acuity; ELM, external limiting membrane; EZ, ellipsoid zone; IDZ, interdigitation zone; I, continuous; II, discontinuous; III, absent.</p><p>Clinical Characteristics and SD-OCT findings of Patients with AZOOR-Complex.</p

Gene expression analysis of the <i>Crx</i> KO retina treated with AAV-Crx.

<p>(A) Schematic diagram of the AAV2/5-Crx2kb-Flag-Crx construct. (B) Expression analysis of <i>Crx</i> by RT-qPCR using RNA isolated from control and AAV-treated <i>Crx</i> KO retinas (Control <i>Crx</i> KO retinas: n = 3 from three different mice and AAV-treated <i>Crx</i> KO retinas: n = 4 from four different mice). (C) Western blot analysis of FLAG-CRX using control and AAV-treated <i>Crx</i> KO retinas from three different mice respectively. An anti-FLAG antibody was used to detect FLAG-CRX. ACTB (β-actin) was used as a loading control. (D–F) Immunostaining of the <i>Crx</i> KO retinas treated with AAV-Crx or PBS using an anti-FLAG antibody. The distribution of FLAG-CRX expression in control and AAV-Crx treated <i>Crx</i> KO retinas (E, F). Enlarged images in white boxes (a-c and d-f) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054146#pone-0054146-g003" target="_blank">Figure 3E and F</a>, respectively. Scale bar represents 50 µm (D) and 1 mm (E, F). (G–Q) Expression analyses of eleven genes related to human retinal diseases three weeks after treatment (Control <i>Crx</i> KO retinas: n = 3 from three different mice and AAV-treated <i>Crx</i> KO retinas: n = 4 from four different mice). <i>Rhodopsin</i> (G), <i>Gnat1</i> (H), <i>S-opsin</i> (I), <i>M-opsin</i> (J), <i>Pde6g</i> (K), <i>Slc24a1</i> (L), <i>Rdh12</i> (M), <i>Rpgrip1</i> (N), <i>Nrl</i> (O), <i>Cabp4</i> (P), and <i>Fscn2</i> (Q). Control retinas were injected with PBS (Vehicle). <i>Rpl4</i> was used for normalization. Primers for qPCR were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054146#pone.0054146.s001" target="_blank">Table S1</a>. Error bar represents the SD from the means of three control retinas and four treated retinas. ^**<i>p</i><0.01, ^*<i>p</i><0.05. ITR: inverted terminal repeat, RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer.</p

Static visual field and spectral-domain optical coherence tomography (SD-OCT) results of the right eye of Case 1 at the initial visit (A–C) and six months after the initial visit (D–F).

<p>A: Deviation plot obtained with the Humphrey 30-2 program at the initial visit. B: Horizontal SD-OCT image through the fovea at the initial visit. C: Magnified view of the area outlined by dashed yellow line box in B. D: Deviation plot obtained with the Humphrey 30-2 program at six months after the initial visit. E: Horizontal SD-OCT image through the fovea at six months after the initial visit. F: Magnified view of the area outlined by dashed yellow line box in the image of B. ELM, external limiting membrane. EZ, ellipsoid zone. IDZ, interdigitation zone. Several column-shaped highly reflective materials are seen at the outer retinal area of visual field defect at the initial visit (red arrows).</p

Calculation of venous tortuosity index.

<p>Measurements of superior and inferior venous arcades were obtained starting from the optic disc margin to the crossing point of a circle (A) whose diameter is the distance from the center of optic disc to the fovea. The course of the veins were traced using Photoshop (Adobe Systems, Inc. Ca, USA). NIH ImageJ software was used to measure the lengths of the vein (A) and chord (B and C) of the vessels. The venous tortuosity index was calculated by dividing the length of the retinal veins by the chord length of the same segment (B/ A and C/A). The average of the venous tortuosity ((B/A + C/A)/2) was calculated to obtain the venous tortuosity index.</p

Characteristics of patients with CRVO.

<p>*Data are expressed as mean ± SD (range)</p><p>Characteristics of patients with CRVO.</p