ThicknessTool: automated ImageJ retinal layer thickness and profile in digital images

To develop an automated retina layer thickness measurement tool for the ImageJ platform, to quantitate nuclear layers following the retina contour. We developed the ThicknessTool (TT), an automated thickness measurement plugin for the ImageJ platform. To calibrate TT, we created a calibration dataset of mock binary skeletonized mask images with increasing thickness masks and different rotations. Following, we created a training dataset and performed an agreement analysis of thickness measurements between TT and two masked manual observers. Finally, we tested the performance of TT measurements in a validation dataset of retinal detachment images. In the calibration dataset, there were no differences in layer thickness between measured and known thickness masks, with an overall coefficient of variation of 0.00%. Training dataset measurements of immunofluorescence retina nuclear layers disclosed no significant differences between TT and any observer's average outer nuclear layer (ONL) (p = 0.998), inner nuclear layer (INL) (p = 0.807), and ONL/INL ratio (p = 0.944) measurements. Agreement analysis showed that bias between TT vs. observers' mean was lower than between any observers' mean against each other in the ONL (0.77 ± 0.34 µm vs 3.25 ± 0.33 µm) and INL (1.59 ± 0.28 µm vs 2.82 ± 0.36 µm). Validation dataset showed that TT can detect significant and true ONL thinning (p = 0.006), more sensitive than manual measurement capabilities (p = 0.069). ThicknessTool can measure retina nuclear layers thickness in a fast, accurate, and precise manner with multi-platform capabilities. In addition, the TT can be customized to user preferences and is freely available to download

Atorvastatin Promotes Phagocytosis and Attenuates Pro-Inflammatory Response in Human Retinal Pigment Epithelial Cells

Phagocytosis of daily shed photoreceptor outer segments is an important function of the retinal pigment epithelium (RPE) and it is essential for retinal homeostasis. RPE dysfunction, especially impairment of its phagocytic ability, plays an essential role in the pathogenesis of age-related macular degeneration (AMD). Statins, or HMG CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, are drugs with multiple properties that have been extensively used to treat hyperlipidemia. However, their effect on RPE cells has not been fully elucidated. Here we report that high dose atorvastatin increased the phagocytic function of ARPE-19 cells, as well as rescue the cells from the phagocytic dysfunction induced by cholesterol crystals and oxidized low-density lipoproteins (ox-LDL), potentially by increasing the cellular membrane fluidity. Similar effects were observed when evaluating two other hydrophobic statins, lovastatin and simvastatin. Furthermore, atorvastatin was able to block the induction of interleukins IL-6 and IL-8 triggered by pathologic stimuli relevant to AMD, such as cholesterol crystals and ox-LDL. Our study shows that statins, a well-tolerated class of drugs with rare serious adverse effects, help preserve the phagocytic function of the RPE while also exhibiting anti-inflammatory properties. Both characteristics make statins a potential effective medication for the prevention and treatment of AMD

Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration

Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis

Photoreceptor cell death and rescue in retinal detachment and degenerations

Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss

Treatment with cAMP analogue, cGMP analogue, and KT5720 in <i>ovl</i>.

<p>(A–E) Eye sections at 5 dpf <i>ovl</i> treated with different concentration of a cAMP analogue, 8-Bromo-cAMP. 8-Bromo-cAMP (B–E) or control water (A). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (Bar = 100 µm.) (F) Graph of survival rod photoreceptors of <i>ovl</i> mutants in control water (black dots) and cAMP analogue-treated water. cAMP analogue accelerated rod photoreceptor death. (Bars mean SD, * means p<0.05.) (G and H) Eye sections at 5 dpf <i>ovl</i> treated with an cGMP analogue, 8-Bromo-cGMP. (H) or control water (G). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (I) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and cGMP analogue-treated water. cGMP does not accelerate rod photoreceptor death. (Bars mean SD.) (J and K) Eye sections at 5 dpf <i>ovl</i> treated with KT5720 (K) or control water (J). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (L) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and KT5720 analogue-treated water. KT5720 suppresses rod photoreceptor death. (Bars mean SD, * means p<0.05.)</p

Mislocalized ADCY in rod outer segments induces photoreceptor cell death.

<p>(A) Expression analysis of adenylyl cyclases in wild-type retina by RT-PCR. (B) RT-PCR analysis of recombinant adenylyl cyclase 2B from wild-type (lane1), ADCY RHO tail (−) (lane2) and ADCY RHO tail (+) (lane3). Lanes 4 to 6 are B-actin expression of each group. Ectopic expressions were confirmed. (C) Immunohistochemistry (IHC) section of retina of wild-type. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY did not expressed at OS. (D) Schematic diagrams of over-expression constructs. ADCY RHO tail (−) and (+) are downstream of zebrafish RH1 promoter between tol2 arms. (E and F) IHC sections of retina of ADCY RHO tail (−) fish (E) and ADCY RHO tail (+) fish (F) at 14 dpf. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). Arrows indicate outer segments. IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY is mis-localized at OS in only tail(+) animals. (G and H) Eye sections of ADCY RHO tail (−) and (+) animals at 14 dpf. Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) The number of rod photoreceptors was significantly decreased in tail (+) animals. (I) Graph of the number of rod photoreceptor of ADCY RHO tail (−) (black dots) and (+) (red dots). (Bars mean SD, ** means p<0.01.) (J and K) TUNEL (green) assay of sections in ADCY RHO tail (−) (J) and (+) (K) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). The signals of outer nuclear layer were observed only in tail (+) animals. (L) Magnification of the white square in (K). INL: inner uclear layer, ONL: outer nuclear layer. (M) Graph of the number of TUNEL assay positive cells, comparing ADCY RHO tail (−) (black dots) and (+) (red dots) animals. (Bars mean SD, ** means p<0.01.)</p

Effects on photoreceptor cell death in Q344X fish.

<p>(A and B) Retina sections of eyes from rhodopsin Q344X transgenic at 5 dpf. Animals were reared in constant darkness (A) or in constant light (B). Light exposure reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) Light accelerated the rod cell death. (C) Graph of the number of rod photoreceptors in rhodopsin Q344X transgenic fish at 5 dpf. Darkness and light exposure are compared. (Bars mean SD, ** means p<0.01.) (D and E) Eye sections of eyes treated by anti-transducin morpholinos (E) and control MO (D) in Q344X at 5 dpf. Suppression of transducin α expression enhances the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.). (F) Graph of the number of rods in Q344X, control morpholino-treated and anti-transducin morpholinos. (Bars mean SD, * means p<0.05.) (G and H) Eye sections of eyes treated by anti-phosphodiesterase 6β morpholinos (H) and control MO (G) in Q344X at 5 dpf. Suppression of phosphodiesterase expression reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized with EGFP (Bar = 100 µm.). (I) Graph of the number of rods in Q344X, control morpholino-treated and anti-phosphodiesterase 6β morpholinos. (Bars mean SD, ** means p<0.01.) (J and K) Eye ections of Q344X transgenic fish bred in SQ22536-treated water (K) and normal control water (J) at 5 dpf. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) ADCY antagonist rescued rod photoreceptor cell death. (L) Graph of the number of rod photoreceptor cells in Q344X 5 dpf. Black dots indicate control and red dots indicate SQ22536-treated (10, 20 and 100 mM) water. (Bars mean SD, * means p<0.05.)</p

Inhibitor of ADCY suppresses photoreceptor cell death.

<p>(A–D) Sections of <i>ovl</i> mutants bred in SQ22536-treated water (B–D) and control water (A). (E) The number of surviving rod photoreceptors from <i>ovl</i> mutants in control water (black dots) and SQ22536-treated water. SQ22536 increased survival rod photoreceptors in concentrations of 1 and 10 mM. (Bars mean SD, * means p<0.05.)</p