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

The Role of Mislocalized Phototransduction in Photoreceptor Cell Death of Retinitis Pigmentosa

Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy

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

Rod photoreceptor cell death in rhodopsin Q344X transgenic fish.

<p>(A) RT-PCR analysis of expression of ectopic rhodopsin Q344X transgene. (B) Sequence analysis of transgene in Q344X animal at 5 dpf. (C–H) Sections of normal rhodopsin fish at 3 dpf (C), 5 dpf (E), 7 dpf (G) and rhodopsin Q344X transgenic fish at 3 dpf (D), 5 dpf (F), 7 dpf (H). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) (I) Graph of the number of rod photoreceptor of normal rhodopsin and rhodopsin Q344X mutant at 3, 5 and 7 dpf. (Bars mean SD, * means p<0.05, ** means p<0.01.) Rod photoreceptors decreased by 5 dpf. (J and K) Immunohistochemistry sections of retina of wild-type (J) and Q344X (K) animal. F-actin is visualized with phalloidin (red), rod opsin with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) Cell localization of rhodopsin is abnormal in Q344X. (L and M) TUNEL (green) assay of sections of normal rhodopsin (L) and rhodopsin Q344X transgenic (M) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). Arrows indicate TUNEL positive photoreceptor cells. TUNEL staining in ONL was observed only in Q344X. (N) Graph of the number of TUNEL assay positive cells, comparing normal rhodopsin (black dots) and rhodopsin Q344X (red dots) transgenic animals. (Bars mean SD, * means p<0.05.)</p

SQ22536 treatment of <i>rd10</i> mice.

<p>(A and B) HE (Hematoxilin-Eosin) stained sections from eyes of <i>rd10</i> mice at P28. Control PBS treated eye (A) and SQ22536 treated eye (B). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.). (C) Graph of the thickness of INL (outlined bar) and ONL (solid bar) in <i>rd10</i> mice at P28. Control group (black) and SQ treated group (red) are compared. (Bars mean SD, * means p<0.05.) (D) Graph of the ONL/INL ratio of SQ22536 treated control (black bar) and untreated retina (red bar) in <i>rd10</i> mice. (E) Schematic illustration of adenylyl cyclase and apoptosis in rod photoreceptors. OS: outer segment, CC: connecting cilium, IS: inner segment, R: rhodopsin, T: transducin, AC: adenylyl cyclase.</p

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