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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.

Purpose The purpose of this study was to study the effect of minocycline and several neurotrophic factors, alone or in combination, on photoreceptor survival and macro/microglial reactivity in two rat models of retinal degeneration. Methods P23H-1 (rhodopsin mutation), Royal College of Surgeon (RCS, pigment epithelium malfunction), and age-matched control rats (Sprague-Dawley and Pievald Viro Glaxo, respectively) were divided into three groups that received at P10 for P23H-1 rats or P33 for RCS rats: (1) one intravitreal injection (IVI) of one of the following neurotrophic factors: ciliary neurotrophic factor (CNTF), pigment epithelium-derived factor (PEDF), or basic fibroblast growth factor (FGF2); (2) daily intraperitoneal administration of minocycline; or (3) a combination of IVI of FGF2 and intraperitoneal minocycline. All animals were processed 12 days after treatment initiation. Retinal microglial cells and cone photoreceptors were immunodetected and analyzed qualitatively in cross sections. The numbers of microglial cells in the different retinal layers and number of nuclei rows in the outer nuclear layer (ONL) were quantified. Results IVI of CNTF, PEDF, or FGF2 improved the morphology of the photoreceptors outer segment, but only FGF2 rescued a significant number of photoreceptors. None of the trophic factors had qualitative or quantitative effects on microglial cells. Minocycline treatment reduced activation and migration of microglia and produced a significant rescue of photoreceptors. Combined treatment with minocycline and FGF2 had higher neuroprotective effects than each of the treatments alone. Conclusions In two animal models of photoreceptor degeneration with different etiologies, minocycline reduces microglial activation and migration, and FGF2 and minocycline increase photoreceptor survival. The combination of FGF2 and minocycline show greater neuroprotective effects than their isolated effects.

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.

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a+RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m+RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a+RGCs, m+RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value.

Purpose To investigate retinal ganglion cell (RGC) survival and activation of caspase 3 after optic nerve crush (ONC) or transection (ONT) and treatment with brain-derived neurotrophic factor (BDNF) or Z-DEVD_fmk. Methods In albino Swiss mice, the left optic nerve was severed or crushed at 0.5 mm from the optic head and retinas were analyzed from 1 to 10 days. Additional groups were treated intravitreally with a single injection of BDNF (2.5 μg) or Z-DEVD_fmk (125 ng) right after injury, or with Z-DEVD_fmk at day 2, or with multiple injections of Z-DEVD_fmk. As controls intact or vehicle-treated retinas were used. In all retinas, Brn3a (RGCs) and cleaved-caspase 3 (c-casp3) were immunodetected and their numbers quantified. In an additional group, c-casp3 expression was assessed in RGCs retrogradely labeled before axotomy. Results The temporal loss of RGCs was the same after ONC or ONT and occurred in two phases with 65% loss during the first 7 days and an additional 4% loss from day 7 to 10. The appearance of c-casp3+RGCs is Gaussian, peaking at 4 days and declining thereafter. Brn3a down-regulates when RGCs start expressing c-casp3. Retinal ganglion cell rescue rate for BDNF or Z-DEVD_fmk is similar and both delay RGC loss by 1 day. Delayed treatment with Z-DEVD_fmk does not rescue RGCs, and several injections are not better than a single one at the time of the injury. Conclusions Brn3a down-regulation marks the beginning of RGC death, which after axotomy occurs by caspase-dependent apoptosis in at least half of the RGCs. These data should be considered when designing neuroprotective strategies.

The transcription factor Brn3a has been reported to be a good marker for adult rat retinal ganglion cells in control and injured retinas. However, it is still unclear if Brn3a expression declines progressively by the injury itself or otherwise its expression is maintained in retinal ganglion cells that, though being injured, are still alive, as might occur when assessing neuroprotective therapies. Therefore, we have automatically quantified the whole population of surviving Brn3a positive retinal ganglion cells in retinas subjected to intraorbital optic nerve transection and treated with either brain derived neurotrophic factor or vehicle. Brain derived neurotrophic factor is known to delay retinal ganglion cell death after axotomy. Thus, comparison of both groups would inform of the suitability of Brn3a as a retinal ganglion cell marker when testing neuroprotective molecules. As internal control, retinal ganglion cells were, as well, identified in all retinas by retrogradely tracing them with fluorogold. Our data show that at all the analyzed times post-lesion, the numbers of Brn3a positive retinal ganglion cells and of fluorogold positive retinal ganglion cells are significantly higher in the brain derived neurotrophic factor-treated retinas compared to the vehicle-treated ones. Moreover, detailed isodensity maps of the surviving Brn3a positive retinal ganglion cells show that a single injection of brain derived neurotrophic factor protects retinal ganglion cells throughout the entire retina. In conclusion, Brn3a is a reliable retinal ganglion cell marker that can be used to accurately measure the potential effect of a given neuroprotective therapy.