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The rapid pupillary constriction to an abrupt light stimulus is signaled through an oligosynaptic neural pathway that dominates over other supranuclear influences on pupillary movement. A pupillometric recording of the pupil light reflex shows the steep change in pupil size from baseline to maximal constriction. However, when the pupil is recorded in darkness in the phase after light stimulation or in response to non-light stimuli like a sudden noise or cognitive activity, pupil size changes are small and slow. In such cases, pre-processing of pupil recordings to reduce the noise due to intrusion of various artifactual and non-evoked pupillary movements is particularly important but may be time-consuming. To address the paucity of automated tools for pupil light reflex analysis in pupillometry, we aimed to develop a software for automated, user-guided pupillometric data analysis. We identified two types of commonly observed artifacts on pupil recordings. We designed a software, called PupilMetrics, which imports and displays raw pupil data, detects and removes these two types of artifacts, and quantifies outcome measures like pupil size, response time, maximal contraction amplitude and PIPR. The right pupil of 29 healthy adults was recorded using a Neurolight pupillometer (IDMed, Marseilles) in response to 9 different light stimuli. Data analysis of the total 261 pupil responses were performed manually or automatically using PupilMetrics. High correlation was observed between PupilMetrics and manual analysis outcome measures across all stimuli (average R2 = 0.9891 and p < 0.0001) with a near 1-to-1 correspondence (Beta = 0.9940). PupilMetrics reduced the total analysis time from 30 h to under 1 h. PupilMetrics offers a time-efficient alternative to manual processing and delivers comparable results. Such software can facilitate standardization of pupillometry for clinical and research uses. [ABSTRACT FROM AUTHOR]

Rods, cones and melanopsin contribute in various proportions, depending on the stimulus light, to the pupil light response. This study used a first derivative analysis to focus on the quantification of the dynamics of pupillary dilation that immediately follows light-induced pupilloconstriction in order to identify novel parameters that reflect rod and cone activity. In 18 healthy adults, the pupil response to a 1 s blue light stimulus ranging from − 6.0 to 2.65 log cd/m2 in dark-adapted conditions and to a 1 s blue light stimulus (2.65 log cd/m2) in light-adapted conditions was recorded on a customized pupillometer. Three derivative parameters which describe the 2.75 s following the light onset were quantified: dAMP (maximal amplitude of the positive peak), dLAT (latency of the positive peak), dAUC (area under the curve of the positive peak). We found that dAMP and dAUC but not dLAT have graded responses over a range of light intensities. The maximal positive value of dAMP, representing maximal rate of change of early pupillary dilation phase, occurs at − 1.0 log cd/m2 and this stimulus intensity appears useful for activating rods and cones. From − 0.5 log cd/m2 to brighter intensities dAMP and dAUC progressively decrease, reaching negligible values at 2.65 log cd/m2 indicative of a melanopsin-driven pupil response that masks the contribution from rods and cones to the early phase of pupillary dilation. [ABSTRACT FROM AUTHOR]

FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161atm1b/tm1b, lacking the major exon #3 which was replaced by a construct that include LacZ under the expression of the Fam161a promoter. LacZ staining was evident in ganglion cells, inner and outer nuclear layers and inner and outer-segments of photoreceptors in KO mice. No immunofluorescence staining of Fam161a was evident in the KO retina. Visual acuity and electroretinographic (ERG) responses showed a gradual decrease between the ages of 1 and 8 months. Optical coherence tomography (OCT) showed thinning of the whole retina. Hypoautofluorescence and hyperautofluorescence pigments was observed in retinas of older mice. Histological analysis revealed a progressive degeneration of photoreceptors along time and high-resolution transmission electron microscopy (TEM) analysis showed that photoreceptor outer segment disks were disorganized in a perpendicular orientation and outer segment base was wider and shorter than in WT mice. Molecular degenerative markers, such as microglia and CALPAIN-2, appear already in a 1-month old KO retina. These results indicate that a homozygous Fam161a frameshift mutation affects retinal function and causes retinal degeneration. This model will be used for gene therapy treatment in the future. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level. [ABSTRACT FROM AUTHOR]

The EB-1 cell line is a stable transfectant of EB, a p53 null colon carcinoma cell line, with an inducible promoter controlling expression of a wild type p53 cDNA. The induced p53 is transcriptionally active and gives rise to apoptosis in these cells. Using this cellular model for presence or absence of the transcription factor p53 and transactivated genes, the Suppression Subtractive Hybridization (SSH) technique permitted the isolation of 17 mRNA candidates (GIPs-Genes induced by p53), whose expression appears to be p53-dependent. Identity has been established for nine of the 17 isolated candidates. These are HGFL/MSP, Zap-70, APOBEC2, Ponsin/SH3P12/CAP/FLAF2, CDCrel2b/H5/Pnutl2, IgG, lats 2, cytokeratin 15 and PIG-3 (quinone oxidoreductase). The latter gene is the only GIP previously demonstrated to be p53 regulated. Of the eight remaining GIPs, six correspond to Unigene clusters. One candidate, GIP #1, is significantly homologous (72% identity) to a chicken zinc finger protein, CTCF, which binds to insulator elements and thus attenuates enhancer cross-talk between physically adjacent promoters. The p53-dependent expression of GIPs was confirmed by dependence of expression upon induction of wt p53 expression in the EB-1 cellular model and by up-regulation following activation of an endogenous wt p53 by treatment with adriamycin. Oncogene (2000) 19, 3978–3987 [ABSTRACT FROM AUTHOR]