Survival of melanopsin expressing retinal ganglion cells long term after optic nerve trauma in mice.

In this study we have compared the response to optic nerve crush (ONC) and to optic nerve transection (ONT) of the general population of retinal ganglion cells in charge of the image-forming visual functions that express Brn3a (Brn3a+RGCs) with that of the sub-population of non-image forming RGCs that express melanopsin (m+RGCs). Intact animals were used as control. ONT and ONC were performed at 0.5 mm from the optic disk, and retinas dissected 3, 5, 7, 14, 30, 45 or 90 days later (n = 5/injury/time point). In all the retinas, Brn3a+RGCs and m+RGCs were identified and their survival analyzed quantitatively and topographically. There were no differences in the course of RGC loss between lesions. The decrease of RGCs was significant at short time points (3 or 5 days for Brn3a+ or m+ RGCs, respectively) and, up to 14 days, the course of loss of both RGC populations was similar, surviving at this time point between 20 and 22% of their original population. However, while the loss of Brn3a+RGCs continues steadily up to 90 days when only 5-6% of them still remain, the loss of m+RGCs stops at 14 days, and the proportion of surviving m+RGCs remains constant up to 90 days (26-30%). In conclusion, m+RGC do not respond to axotomy in the same way than the rest of RGCs, and so whilst image-forming RGCs die in two exponential phases a quick one and a slow protracted one, non-image forming RGCs die only during the first quick phase.

Nerve fibre layer degeneration and retinal ganglion cell loss long term after optic nerve crush or transection in adult mice.

We have investigated the long term effects of two different models of unilateral optic nerve (ON) lesion on retinal ganglion cells (RGCs) and their axons, in the injured and contralateral retinas of adult albino mice. Intact animals were used as controls. The left ON was intraorbitally crushed or transected at 0.5 mm from the optic disk and both retinas were analyzed at 2, 3, 5, 7, 14, 30, 45 or 90 days after injury. RGCs were immunoidentified with anti-Brn3a, and their axons with anti-highly phosphorylated axonal neurofilament subunit H (pNFH). After both lesions, RGC death in the injured retinas is first significant at day 3, and progresses quickly up to 7 days slowing down till 90 days. In the same retinas, the anatomical loss of RGC axons is not evident until day 30. However, by two days after both lesions there are changes in the expression pattern of pNFH: axonal beads, axonal club- or bulb-like formations, and pNFH+RGC somas. The number of pNFH+RGC somata peak at day 5 after either lesion and is significantly higher than in intact retinas at all time points. pNFH+RGC somata are distributed across the retina, in accordance with the pattern of RGC death which is diffuse and homogenous. In the contralateral retinas there is no RGC loss, but there are few pNFH+RGCs from day 2 to day 90. In conclusion, in albino mice, axotomy-induced RGC death precedes the loss of their intraretinal axons and occurs in two phases, a rapid and a slower, but steady, one. Injured retinas show similar changes in the pattern of pNFH expression and a comparable course of RGC loss.

Retino-retinal projection in juvenile and young adult rats and mice.

Identification of retino-retinal projecting RGCs (ret-ret RGCs) has been accomplished by tracing RGCs in one retina after intravitreal injection of different tracers in the other eye. In mammals, rabbit and rat, ret-ret RGCs are scarce and more abundant in newborn than in adult animals. To our knowledge, ret-ret RGCs have not been studied in mice. Here we purpose to revisit the presence of ret-ret RGCs in juvenile and young adult rats and mice by using retrograde tracers applied to the contralateral optic nerve instead of intravitreally. In P20 (juvenile) and P60 (young adult) animals, the left optic nerve was intraorbitally transected and Fluorogold (rats) or its analogue OHSt (mice) were applied onto its distal stump. P20 animals were sacrificed 3 (mice) or 5 (rats) days later and adult animals at 5 (mice) or 7 (rats) days. Right retinas were dissected as flat-mounts and double immunodetected for Brn3a and melanopsin. Ret-ret RGCs were those with tracer accumulation in their somas. Out of them some expressed Brn3a and/or melanopsin, while other were negative for both markers. In young adult rats, we found 2 ret-ret RGCs displaced to the inner nuclear layer. In both species, ret-ret RGCs are quite scarce and found predominantly in the nasal retina. In juvenile animals there are significantly more ret-ret RGCs (9 ± 3, rats, 13 ± 3 mice) than in young adult ones (5 ± 6 rats, 7 ± 3 mice). Finally, juvenile and young adult mice have more ret-ret RGCs than rats.

A role for the outer retina in development of the intrinsic pupillary light reflex in mice.

Mice do not require the brain in order to maintain constricted pupils. However, little is known about this intrinsic pupillary light reflex (iPLR) beyond a requirement for melanopsin in the iris and an intact retinal ciliary marginal zone (CMZ). Here, we study the mouse iPLR in vitro and examine a potential role for outer retina (rods and cones) in this response. In wild-type mice the iPLR was absent at postnatal day 17 (P17), developing progressively from P21-P49. However, the iPLR only achieved ∼ 30% of the wild-type constriction in adult mice with severe outer retinal degeneration (rd and rdcl). Paradoxically, the iPLR increased significantly in retinal degenerate mice >1.5 years of age. This was accompanied by an increase in baseline pupil tone in the dark to levels indistinguishable from those in adult wild types. This rejuvenated iPLR response was slowed by atropine application, suggesting the involvement of cholinergic neurotransmission. We could find no evidence of an increase in melanopsin expression by quantitative PCR in the iris and ciliary body of aged retinal degenerates and a detailed anatomical analysis revealed a significant decline in melanopsin-positive intrinsically photosensitive retinal ganglion cells (ipRGCs) in rdcl mice >1.5 years. Adult mice lacking rod function (Gnat1(-/-)) also had a weak iPLR, while mice lacking functional cones (Cpfl5) maintained a robust response. We also identify an important role for pigmentation in the development of the mouse iPLR, with only a weak and transient response present in albino animals. Our results show that the iPLR in mice develops unexpectedly late and are consistent with a role for rods and pigmentation in the development of this response in mice. The enhancement of the iPLR in aged degenerate mice was extremely surprising but may have relevance to behavioral observations in mice and patients with retinitis pigmentosa.

Axotomy-induced retinal ganglion cell death in adult mice: quantitative and topographic time course analyses.

The fate of retinal ganglion cells after optic nerve injury has been thoroughly described in rat, but not in mice, despite the fact that this species is amply used as a model to study different experimental paradigms that affect retinal ganglion cell population. Here we have analyzed, quantitatively and topographically, the course of mice retinal ganglion cells loss induced by intraorbital nerve transection. To do this, we have doubly identified retinal ganglion cells in all retinas by tracing them from their main retinorecipient area, the superior colliculi, and by their expression of BRN3A (product of Pou4f1 gene). In rat, this transcription factor is expressed by a majority of retinal ganglion cells; however in mice it is not known how many out of the whole population of these neurons express it. Thus, in this work we have assessed, as well, the total population of BRN3A positive retinal ganglion cells. These were automatically quantified in all whole-mounted retinas using a newly developed routine. In control retinas, traced-retinal ganglion cells were automatically quantified, using the previously reported method (Salinas-Navarro et al., 2009b). After optic nerve injury, though, traced-retinal ganglion cells had to be manually quantified by retinal sampling and their total population was afterwards inferred. In naïve whole-mounts, the mean (±standard deviation) total number of traced-retinal ganglion cells was 40,437(±3196) and of BRN3A positive ones was 34,697(±1821). Retinal ganglion cell loss was first significant for both markers 5 days post-axotomy and by day 21, the last time point analyzed, only 15% or 12% of traced or BRN3A positive retinal ganglion cells respectively, survived. Isodensity maps showed that, in control retinas, BRN3A and traced-retinal ganglion cells were distributed similarly, being densest in the dorsal retina along the naso-temporal axis. After axotomy the progressive loss of BRN3A positive retinal ganglion cells was diffuse and affected the entire retina. In conclusion, this is the first study assessing the values, in terms of total number and density, of the retinal ganglion cells surviving axotomy from 2 till 21 days post-lesion. Besides, we have demonstrated that BRN3A is expressed by 85.6% of the total retinal ganglion cell population, and because BRN3A positive retinal ganglion cells show the same spatial distribution and temporal course of degeneration than traced ones, BRN3A is a reliable marker to identify, quantify and assess, ex-vivo, retinal ganglion cell loss in this species.

Retinal ganglion cell population in adult albino and pigmented mice: a computerized analysis of the entire population and its spatial distribution.

UNLABELLED: In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus((R)) (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina. RESULTS: The mean total numbers of RGCs labeled from the ON, in Swiss (49,493+/-3936; n=18) or C57 mice (42,658+/-1540; n=10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733+/-3954; n=43) or C57 mice (41,192+/-2821; n=42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.