Leticia Nieto-Lopez

Brn3a as a Marker of Retinal Ganglion Cells: Qualitative and Quantitative Time Course Studies in Naïve and Optic Nerve-Injured Retinas

PURPOSE To characterize Brn3a expression in adult albino rat retinal ganglion cells (RGCs) in naïve animals and in animals subjected to complete intraorbital optic nerve transection (IONT) or crush (IONC). METHODS Rats were divided into three groups, naïve, IONT, and IONC. Two-, 5-, 9-, or 14-day postlesion (dpl) retinas were examined for immunoreactivity for Brn3a. Before the injury, the RGCs were labeled with Fluorogold (FG; Fluorochrome, Corp. Denver, CO). Brn3a retinal expression was also determined by Western blot analysis. The proportion of RGCs double labeled with Brn3a and FG was determined in radial sections. The temporal course of reduction in Brn3a(+) RGCs and FG(+) RGCs induced by IONC or IONT was assessed by quantifying, in the same wholemounts, the number of surviving FG-labeled RGCs and Brn3a(+)RGCs at the mentioned time points. The total number of FG(+)RGCs was automatically counted in naïve and injured retinas (2 and 5 dpl) or estimated by manual quantification in retinas processed at 9 and 14 dpl. All Brn3a immunopositive RGCs were counted using an automatic routine specifically developed for this purpose. This protocol allowed, as well, the investigation of the spatial distribution of these neurons. RESULTS Brn3a(+) cells were only present in the ganglion cell layer and showed a spatial distribution comparable to that of FG(+) cells. In the naïve retinal wholemounts the mean (mean +/- SEM; n = 14) total number of FG(+)RGCs and Brn3a(+)RGCs was 80,251 +/- 2,210 and 83,449 +/- 4,541, respectively. Whereas in the radial sections, 92.2% of the FG(+)RGCs were also Brn3a(+), 4.4% of the RGCs were Brn3a(+)FG(-) and 3.4% were FG(+)Brn3a(-). Brn3a expression pattern was maintained in injured RGCs. The temporal course of Brn3a(+)RGC and FG(+)RGC loss induced by IONC or IONT followed a similar trend, but Brn3a(+)RGCs loss was detected earlier than that of FG(+)RGCs. Independent of the marker used to detect the RGCs, it was observed that their loss was quicker and more severe after IONT than after IONC. CONCLUSIONS Brn3a can be used as a reliable, efficient ex vivo marker to identify and quantify RGCs in control and optic nerve-injured retinas.

Number and Distribution of Mouse Retinal Cone Photoreceptors: Differences between an Albino (Swiss) and a Pigmented (C57/BL6) Strain

We purpose here to analyze and compare the population and topography of cone photoreceptors in two mouse strains using automated routines, and to design a method of retinal sampling for their accurate manual quantification. In whole-mounted retinas from pigmented C57/BL6 and albino Swiss mice, the longwave-sensitive (L) and the shortwave-sensitive (S) opsins were immunodetected to analyze the population of each cone type. In another group of retinas both opsins were detected with the same fluorophore to quantify all cones. In a third set of retinas, L-opsin and Brn3a were immunodetected to determine whether L-opsin+cones and retinal ganglion cells (RGCs) have a parallel distribution. Cones and RGCs were automatically quantified and their topography illustrated with isodensity maps. Our results show that pigmented mice have a significantly higher number of total cones (all-cones) and of L-opsin+cones than albinos which, in turn, have a higher population of S-opsin+cones. In pigmented animals 40% of cones are dual (cones that express both opsins), 34% genuine-L (cones that only express the L-opsin), and 26% genuine-S (cones that only express the S-opsin). In albinos, 23% of cones are genuine-S and the proportion of dual cones increases to 76% at the expense of genuine-L cones. In both strains, L-opsin+cones are denser in the central than peripheral retina, and all-cones density increases dorso-ventrally. In pigmented animals S-opsin+cones are scarce in the dorsal retina and very numerous in the ventral retina, being densest in its nasal aspect. In albinos, S-opsin+cones are abundant in the dorsal retina, although their highest densities are also ventral. Based on the densities of each cone population, we propose a sampling method to manually quantify and infer their total population. In conclusion, these data provide the basis to study cone degeneration and its prevention in pathologic conditions.

A Novel In Vivo Model of Focal Light Emitting Diode-Induced Cone-Photoreceptor Phototoxicity: Neuroprotection Afforded by Brimonidine, BDNF, PEDF or bFGF

We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.

Comparison of Retinal Nerve Fiber Layer Thinning and Retinal Ganglion Cell Loss After Optic Nerve Transection in Adult Albino Rats.

PURPOSE We compared the time-course and magnitude of retinal nerve fiber layer (RNFL) thinning with that of retinal ganglion cell (RGC) loss after intraorbital optic nerve transection (IONT) in adult rats. METHODS At 3, 7, 12, or 21 days, or 1, 2, or 4 months after ONT, the retinas were imaged with spectral-domain optical coherence tomography (SD-OCT) using the circular-peripapillary scan and volume scan raster pattern (61 horizontal sections equally spaced) both centered in the optic nerve. In all sections, the RNFL and retinal thickness were measured to obtain the total values of the peripapillary scan and the values of three concentric sectors (400, 1200, and 2400 μm in diameter) from the volume scan. After SD-OCT, retinas were dissected and immunoreacted for Brn3a and neurofilaments (pNFH) to identify RGCs and their intraretinal axons, respectively. Total numbers of RGCs were quantified. RESULTS Thinning of the RNFL was first observed at 12 days in peripapillary scan (10% decrease) and progressed up to 4 months (72% decrease). The volume scan showed transient RNFL swelling in central and medial sectors at 3, 7, and 12 days followed by progressive significant thinning first observed at 21 days (central sector, 30%; medial sector, 40%) and 12 days (peripheral sector, 15%), respectively. Following IONT, Brn3a+ RGCs decreased to approximately 80%, 52%, 17%, 9%, 5%, 3%, and 2% at 3, 7, 12, 21 days, and at 1, 2, and 4 months, respectively. Retinal ganglion cell axon immunodetection decreased from 12 days onwards. CONCLUSIONS After IONT, RGC death is more severe and precedes thinning of the RNFL.