Distribution of melanopsin positive neurons in pigmented and albino mice: evidence for melanopsin interneurons in the mouse retina.

Here we have studied the population of intrinsically photosensitive retinal ganglion cells (ipRGCs) in adult pigmented and albino mice. Our data show that although pigmented (C57Bl/6) and albino (Swiss) mice have a similar total number of ipRGCs, their distribution is slightly different: while in pigmented mice ipRGCs are more abundant in the temporal retina, in albinos the ipRGCs are more abundant in superior retina. In both strains, ipRGCs are located in the retinal periphery, in the areas of lower Brn3a(+)RGC density. Both strains also contain displaced ipRGCs (d-ipRGCs) in the inner nuclear layer (INL) that account for 14% of total ipRGCs in pigmented mice and 5% in albinos. Tracing from both superior colliculli shows that 98% (pigmented) and 97% (albino) of the total ipRGCs, become retrogradely labeled, while double immunodetection of melanopsin and Brn3a confirms that few ipRGCs express this transcription factor in mice. Rather surprisingly, application of a retrograde tracer to the optic nerve (ON) labels all ipRGCs, except for a sub-population of the d-ipRGCs (14% in pigmented and 28% in albino, respectively) and melanopsin positive cells residing in the ciliary marginal zone (CMZ) of the retina. In the CMZ, between 20% (pigmented) and 24% (albino) of the melanopsin positive cells are unlabeled by the tracer and we suggest that this may be because they fail to send an axon into the ON. As such, this study provides the first evidence for a population of melanopsin interneurons in the mammalian retina

Comparison of retinal nerve fiber layer thinning and retinal ganglion cell loss after optic nerve transection in adult albino rats

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

Inherited photoreceptor degeneration causes the death of melanopsin-positive retinal ganglion cells and increases their coexpression of brn3a

Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a

Topical Treatment With Bromfenac Reduces Retinal Gliosis and Inflammation After Optic Nerve Crush.

Purpose To study the effect of topical administration of bromfenac, a nonsteroidal anti-inflammatory drug (NSAID), on retinal gliosis and levels of prostaglandin E2 (PGE2) after complete optic nerve crush (ONC). Methods Adult albino rats were divided into the following groups (n = 8 retinas/group): (1) intact, (2) intact and bromfenac treatment (twice a day during 7 days), (3) ONC (7 days), and (4) ONC (7 days) + bromfenac treatment (twice a day during 7 days). Animals from groups 3 and 4 were imaged in vivo with spectral-domain optical coherence tomography (SD-OCT) before the procedure and 15 minutes, 3, 5, or 7 days later. Retinas from all groups were analyzed by immunodetection, Western blotting, or enzyme-linked immunoabsorbent assay (ELISA). Results Quantification of Brn3a (brain-specific homeobox/POU domain protein 3A) +RGCs (retinal ganglion cells) in cross sections showed that bromfenac treatment does not accelerate ONC-induced degeneration. Cellular retinaldehyde binding protein 1 regulation indicated that bromfenac improves retinal homeostasis in injured retinas. Spectral-domain OCT showed that the thickness of the retina and the retinal nerve fiber layer at 7 days post ONC was significantly reduced in bromfenac-treated animals when compared to untreated animals. In agreement with these data, hypertrophy of astrocytes and Muller cells and expression of glial fibrillary acidic protein and vimentin were greatly diminished by bromfenac treatment. While no changes in cyclooxygenase (COX) enzyme COX1 and COX2 expression were observed, there was a significant increase of PGE2 after ONC that was controlled by bromfenac treatment. Conclusions Topical administration of bromfenac is an efficient and noninvasive treatment to control the retinal gliosis and release of proinflammatory mediators that follow a massive insult to the RGC population

Tracing the retina to analyze the integrity and phagocytic capacity of the retinal pigment epithelium

We have developed a new technique to study the integrity, morphology and functionality of the retinal neurons and the retinal pigment epithelium (RPE). Young and old control albino (Sprague-Dawley) and pigmented (Piebald Virol Glaxo) rats, and dystrophic albino (P23H-1) and pigmented (Royal College of Surgeons) rats received a single intravitreal injection of 3% Fluorogold (FG) and their retinas were analyzed from 5 minutes to 30 days later. Retinas were imaged in vivo with SD-OCT and ex vivo in flat-mounts and in cross-sections. Fifteen minutes and 24 hours after intravitreal administration of FG retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the RPE of the RCS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. In both dystrophic strains, the RPE cells were pleomorphic and polymegathic

Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a+ , bIII-tubulin+ and Islet-1+ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG+ and Brn3a+ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin+ RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3astained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage

Pleural cancer mortality in Spain: time-trends and updating of predictions up to 2020

Background A total of 2,514,346 metric tons (Mt) of asbestos were imported into Spain from 1906 until the ban on asbestos in 2002. Our objective was to study pleural cancer mortality trends as an indicator of mesothelioma mortality and update mortality predictions for the periods 2011–2015 and 2016–2020 in Spain.Methods Log-linear Poisson models were fitted to study the effect of age, period of death and birth cohort (APC) on mortality trends. Change points in cohort- and period-effect curvatures were assessed using segmented regression. Fractional power-link APC models were used to predict mortality until 2020. In addition, an alternative model based on national asbestos consumption figures was also used to perform long-term predictions.Results Pleural cancer deaths increased across the study period, rising from 491 in 1976–1980 to 1,249 in 2006–2010. Predictions for the five-year period 2016–2020 indicated a total of 1,319 pleural cancer deaths (264 deaths/year). Forecasts up to 2020 indicated that this increase would continue, though the age-adjusted rates showed a levelling-off in male mortality from 2001 to 2005, corresponding to the lower risk in post-1960 generations. Among women, rates were lower and the mortality trend was also different, indicating that occupational exposure was possibly the single factor having most influence on pleural cancer mortality.Conclusion The cancer mortality-related consequences of human exposure to asbestos are set to persist and remain in evidence until the last surviving members of the exposed cohorts have disappeared. It can thus be assumed that occupationally-related deaths due to pleural mesothelioma will continue to occur in Spain until at least 2040.The study was partially supported by a research grant from the Spanish Health Research Fund (FIS PI11/00871) and the HAR2009-07543 project of the Ministry of Science and Innovation. The Department of Labour of the Government of Catalonia provided the asbestos consumption data

Microglial dynamics after axotomy-induced retinal ganglion cell death

Abstract Background Microglial cells (MCs) are the sentries of the central nervous system. In health, they are known as surveying MCs because they examine the tissue to maintain the homeostasis. In disease, they activate and, among other functions, become phagocytic to clean the cellular debris. In this work, we have studied the behavior of rat retinal MCs in two models of unilateral complete intraorbital optic nerve axotomy which elicit a different time course of retinal ganglion cell (RGC) loss. Methods Albino Sprague-Dawley rats were divided into these groups: (a) intact (no surgery), (b) fluorogold (FG) tracing from the superior colliculi, and (c) FG tracing + crush or transection of the left optic nerve. The retinas were dissected from 2 days to 2 months after the lesions (n = 4–12 group/lesion and time point) and then were subjected to Brn3a and Iba1 double immunodetection. In each intact retina, the total number of Brn3a+RGCs and Iba+MCs was quantified. In each traced retina (b and c groups), FG-traced RGCs and phagocytic microglial cells (PMCs, FG+Iba+) were also quantified. Topographical distribution was assessed by neighbor maps. Results In intact retinas, surveying MCs are homogenously distributed in the ganglion cell layer and the inner plexiform layer. Independently of the axotomy model, RGC death occurs in two phases, one quick and one protracted, and there is a lineal and topographical correlation between the appearance of PMCs and the loss of traced RGCs. Furthermore, the clearance of FG+RGCs by PMCs occurs 3 days after the actual loss of Brn3a expression that marks RGC death. In addition, almost 50% of MCs from the inner plexiform layer migrate to the ganglion cell layer during the quick phase of RGC loss, returning to the inner plexiform layer during the slow degeneration phase. Finally, in contrast to what happens in mice, in rats, there is no microglial phagocytosis in the contralateral uninjured retina. Conclusions Axotomy-induced RGC death occurs earlier than RGC clearance and there is an inverse correlation between RGC loss and PMC appearance, both numerically and topographically, suggesting that phagocytosis occurs as a direct response to RGC death rather than to axonal damage