V-Proportion: a method based on the Voronoi diagram to study spatial relations in neuronal mosaics of the retina

The visual system plays a predominant role in the human perception. Although all components of the eye are important to perceive visual information, the retina is a fundamental part of the visual system. In this work we study the spatial relations between neuronal mosaics in the retina. These relations have shown its importance to investigate possible constraints or connectivities between different spatially colocalized populations of neurons, and to explain how visual information spreads along the layers before being sent to the brain. We introduce the V-Proportion, a method based on the Voronoi diagram to study possible spatial interactions between two neuronal mosaics. Results in simulations as well as in real data demonstrate the effectiveness of this method to detect spatial relations between neurons in different layers

Design of a Trichromatic Cone Array

Cones with peak sensitivity to light at long (L), medium (M) and short (S) wavelengths are unequal in number on the human retina: S cones are rare (<10%) while increasing in fraction from center to periphery, and the L/M cone proportions are highly variable between individuals. What optical properties of the eye, and statistical properties of natural scenes, might drive this organization? We found that the spatial-chromatic structure of natural scenes was largely symmetric between the L, M and S sensitivity bands. Given this symmetry, short wavelength attenuation by ocular media gave L/M cones a modest signal-to-noise advantage, which was amplified, especially in the denser central retina, by long-wavelength accommodation of the lens. Meanwhile, total information represented by the cone mosaic remained relatively insensitive to L/M proportions. Thus, the observed cone array design along with a long-wavelength accommodated lens provides a selective advantage: it is maximally informative

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated approximately 100 kDa CX3CL1 form and a cleaved approximately 85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal approximately 95 kDa form and a decrease in the approximately 85-kDa form were observed. From P14, a approximately 95 kDa form became principal in wt retina, while in rd10 retinas a soluble approximately 85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1

Western blot analysis of fractalkine protein expression in mouse neural retina at P5 until P60 being representative of five independent experiments.

<p>Blots were re-probed with anti-GAPDH antibody. (A). Fractalkine membrane integrated (100-kDa) and soluble (85-kDa) protein forms are present in both control (wt) and rd10 retina lysates during development at P5. (B). Accumulation of presumably intracellular (95-kDa) protein form is seen in both wt (B) and rd10 (C) developing neural retina at P10. Remarkably, no cleaved (85-kDa) protein form was associated with membrane integrated (100-kDa) form in both rd10 and wt retina lysates at P10. Both wt and rd10 retinas show lower to beneath detection level of 100-kDa form and higher level of 95-kDa form at P14 through P60 (D-G). Increased level of 85-kDa form is clearly detectable in rd10 retinas at P45 and P60 (G). Histogram showing relative percent levels of each of the three CX3CL1 protein forms in wt retina (H) and degenerating rd10 retina (I). Data are expressed as percent of densitometric arbitrary units. Values are mean ± SEM, (n = 5). In rd10 retinas, positive correlation between the relative levels of a ∼100-kDa and corresponding 85-kDa bands was found at P45 and P60 (Pearson product-moment correlation coefficient r = 0.683, n = 12, p = 0.014 in RD10 retina at P45 and r = 0.882, n = 9, p = 0.0017 at P60) as well as for the relative levels of a 100-kDa and 95-kDa bands (r = 0.928, n = 11, p<0.0001 at P14, r = 0.861, n = 9, p = 0.0029 at P22, r = 0.892, n = 9, p = 0.0012 at P30, r = 0.966, n = 12, p<0.0001 at P45, and r = 0.628, n = 12, p = 0.0288 at P60 in rd10 retina). The degree of association between the levels of 95 kDa and 85 kDa protein band was r = 0.712, n = 12, p = 0.0094 at P45 and r = 0.907, n = 9, p = 0.0007 at P60. It was not, however, possible, to make similar analysis for the wild type retina samples, as the level of the ∼95-kDa protein form was the highest and the levels of cleaved and full-length forms were far below the limit of detection.</p

Localization of CX3CL1 mRNA expressing cells in the retina and brain of wt and rd10 mice.

<p>(A-D). Light micrographs of radial cryosections through mouse central retina probed with a DIG-labeled anti-sense CX3CL1 riboprobe. In both, wt (A) and rd10 (B) mouse retinas, CX3CL1 mRNA signal is present exclusively in the neurons of the ganglion cell layer (GCL) and the inner- and outermost areas of the inner nuclear layer (INL). The outer nuclear (photoreceptor) layer is devoid of CX3CL1 signal at the time points examined (P22 and P30). Enlargements showing juxtanuclear localization of mRNA in the cells of inner nuclear (C) and ganglion (D) cell layers in wt mouse retina. (E-G). Distribution of CX3CL1 mRNA expressing neurons in wt mouse hippocampus. Light micrographs of a paraffin section used as positive control. (E). Numerous CX3CL1 mRNA-positive neurons are located in all hippocampal subfields of the pyramidal (PCL) and in the dentate granule cell layers (GCL). (F). Higher magnification of the dentate gyrus tips and of hilus. (G). Enlargement showing high levels of CX3CL1 mRNA expression within the grey matter of the cerebral cortex. PCL, pyramidal cell layer; GCL, layer of ganglion cells in dentate granule cell layer; H, hilus; T, dentate gyrus tips. Cells expressing CX3CL1 mRNA are visualized as of brown product accumulations in cryosections and red-brown in paraffin sections. The different color of nuclei staining (blue in cryosections and purple in paraffin sections) is due to a hemalaun counterstaining. Scale bars represent: 50 µm (A, B), 35 µm (C, D), 250 µm (E), 125 µm (F) and 25 µm (G).</p

Spatial statistics of S-cones In primate/human retinal periphery suggests a correlation with domains of vascular subtypes

Purpose:In central primate retina the multitiered vasculature leads to nonuniform gradients of oxygen along radial retinal profiles (Yu &Cringle,2001). In the far periphery, vasculature is reduced and spread out into a non-overlapping monolayered plexus with alternating cascades of arterioles, venules and a mediating capillary zones. This compartmentalization may create microenvironments with different metabolic profiles. We therefore developed a procedure for studying possible effects of such vascular domains on the spatial distribution of retinal cell classes. In particular, it is applied to detect eventual biases in the overlying short wavelength sensitive (S-) cone mosaic. Methods:A statistical framework has been designed and implemented in Matlab. In a set of iterations applied to binary maps of vascular elements (collagen IV labeled) and S-cones (S-opsin labeled) in peripheral sectors from an Orang Utan (6yrs,f) and a Human (76 yrs,f) retina, we obtained the percentage of cones located within increasing domains along a vessel network and compare the results with data from Monte Carlo (MC) simulations. Results:The comparisons suggest differential positioning of S-cones with respect to the type of underlying vasculature (Fig.). For 90 of S-cones a significant positive correlation to retinal capillaries (c) was found. For arterioles (a), a small circumferential "aura" (~2 x 115 μm) of lower than expected S-cone densities was detected. No significant correlation was found along underlying venules (v) for S-cones within 300 µm domains. The pattern of peripheral S-cone mosaic itself is random (Chi-Square, 95). Conclusions:Peripheral S-cone positioning appears influenced by vicinitiy to underling vessels types, although the timing and specific catalysts for the shaping of these correlation are yet unclear. The inferred tiling of peripheral retina could also demarcate sites, predisposed for initial manifestation of progressive pathologic conditions as from retinitis pigmentosa, diabetes or from aging

Ultrahigh resolution optical coherence tomography of the monkey fovea. Identification of retinal sublayers by correlation with semithin histology sections

Optical coherence tomography (OCT) has become an established diagnostic tool for the clinical assessment of retinal pathology but correlation of acquired signals with retinal substructures has often been ambiguous. In the monkey retina we have now obtained ultrahigh resolution (UHR) OCT images with 1·4 μm axial×3 μm transverse resolution from perfusion-fixed eye cups of Macaca fascicularis and optimized the identification of retinal anatomy by correction of spatial artefacts in correlated histology. After resin embedding, serial semithin sections were obtained that corresponded to OCT transects. The direct overlay of features identified in histological sections with corresponding OCT locations was limited by non-linear tissue shrinkage due to dehydration and sectioning stress. In the present study, these misalignments were further corrected by using polygonal spline morphing based on corresponding unequivocal landmarks. The geometric normalization then allowed detailed comparison of both profiles including delicate sublayers of photoreceptor inner- and outer segments. Such correlation will facilitate the extraction of structural information from in vivo ultrahigh resolution OCT images in clinical and experimental applications

Enhanced visualization of choroidal vessels using ultrahigh resolution ophthalmic OCT at 1050nm

In this article the ability of ultrahigh resolution ophthalmic optical coherence tomography (OCT) to image small choroidal blood vessels below the highly reflective and absorbing retinal pigment epithelium is demonstrated for the first time. A new light source (?c= 1050 nm, ??? = 165 nm, Pout= 10 mW), based on a photonic crystal fiber pumped by a compact, self-starting Ti:Al2O3 laser has therefore been developed. Ex-vivo ultrahigh resolution OCT images of freshly excised pig retinas acquired with this light source demonstrate enhanced penetration into the choroid and better visualization of choroidal vessels as compared to tomograms acquired with a state-of-the art Ti:Al2O3 laser (Femtolasers Compact Pro, lc= 780 nm, ???= 160 nm, Pout= 400 mW), normally used in clinical studies for in vivo ultrahigh resolution ophthalmic OCT imaging. These results were also compared with retinal tomograms acquired with a novel, spectrally broadened fiber laser (MenloSystems, ?c= 1350 nm, ???= 470 nm, Pout = 4 mW) permitting even greater penetration in the choroid. Due to high water absorption at longer wavelengths retinal OCT imaging at ~1300 nm may find applications in animal ophthalmic studies. Detection and follow-up of choroidal neovascularization improves early diagnosis of many retinal pathologies, e.g. age-related macular degeneration or diabetic retinopathy and can aid development of novel therapy approaches