Francisco M Nadal-Nicolás

University of Murcia, Murcia, Murcia, Spain

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Publications (20)69.93 Total impact

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    ABSTRACT: 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. Copyright © 2015. Published by Elsevier Ltd.
    Experimental Eye Research 03/2015; 591. DOI:10.1016/j.exer.2015.03.015 · 3.02 Impact Factor
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    ABSTRACT: Purpose: To study: the effects of ocular hypertension (OHT) on the general population of retinal ganglion cells (Brn3a+RGCs) versus the intrinsically-photosensitive (melanopsin-expressing-RGCs m+RGCs); the effects of BDNF on the survival of axonally intact and axonally non-intact RGCs, and; the correlation of vascular integrity with sectorial RGC loss. Methods: In Sprague-Dawley rats, 5µg BDNF or Vehicle was intravitreally injected in the left eye followed by laser photocoagulation of the limbar tissues. To identify RGCs with an active retrograde axonal transport, Fluorogold was applied to both superior colliculi one week before sacrifice (FG+RGCs). Retinas were dissected 12 or 15 days after lasering and immunoreacted against Brn3a (to identify all RGCs except m+RGCs), melanopsin or RECA1 (inner retinal vasculature). Results: OHT resulted at 12-15d in sectorial loss of FG+RGCs (78-84%, respectively), and Brn3a+RGCs were significantly greater, indicating that a substantial proportion (≈21-26%) of RGCs with their retrograde axonal transport impaired survive in the retina. BDNF increased the survival of Brn3a+RGCs to 81-67% at 12-15 days, respectively. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. At 12-15d, m+RGCs decreased to approximately 50-51%, but this loss was diffuse across the retina and was not prevented by BDNF. Conclusions: The responses of m+RGCs against OHT-induced retinal degeneration and neuroprotection differ from those of Brn3a+RGCs; while OHT induces similar loss of Brn3a+RGCs and m+RGCs, Brn3a+RGCs are lost in sectors and can be rescued with BDNF but m+RGCs do not respond to BDNF and their loss is diffuse. Copyright © 2015 by Association for Research in Vision and Ophthalmology.
    Investigative Ophthalmology &amp Visual Science 02/2015; 56(3). DOI:10.1167/iovs.15-16454 · 3.66 Impact Factor
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    ABSTRACT: To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) extracted fresh to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (inmunolabeld). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a+RGCs but with large numbers of DAPI+nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.
    Experimental Eye Research 01/2015; 14. DOI:10.1016/j.exer.2015.01.006 · 3.02 Impact Factor
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    Experimental Eye Research 12/2014; 131. DOI:10.1016/j.exer.2014.12.005 · 3.02 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 12/2014; 9(12):e113798. DOI:10.1371/journal.pone.0113798 · 3.53 Impact Factor
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    ABSTRACT: We have studied in parallel the population of displaced retinal ganglion cells (dRGCs) and normally placed (orthotopic RGCs, oRGCs) in albino and pigmented rats. Using retrograde tracing from the optic nerve, from both superior colliculi (SC) or from the ipsilateral SC in conjunction with Brn3 and melanopsin immunodetection, we report for the first time their total number and topography as well as the number and distribution of those dRGCs and oRGCs that project ipsi- or contralaterally and/or that express any of the three Brn3 isoforms or melanopsin. The total number of RGCs (oRGCs+dRGCs) is 84,706 ± 1249 in albino and 90,440 ± 2236 in pigmented, out of which 2383 and 2428 are melanopsin positive (m-RGCs), respectively. Regarding dRGCs: i/ albino rats have a significantly lower number of dRGCs than pigmented animals (0.5% of the total number of RGCs vs. 2.5%, respectively), ii/ dRGCs project massively to the contralateral SC, iii/ the percentage of ipsilaterality is higher for dRGCs than for oRGCs, iv/ a higher proportion of ipsilateral dRGCs is observed in albino than pigmented animals, v/ dRGC topography is very specific, they predominate in the equatorial temporal retina, being densest where the oRGCs are densest, vi/ Brn3a detects all dRGCs except half of the ipsilateral ones and those that express melanopsin, vii/ the proportion of dRGCs that express Brn3b or Brn3c is slightly lower than in the oRGC population, viii/ a higher percentage of dRGCs (13% albino, 9% pigmented) than oRGCs (2.6%) express melanopsin, ix/ few m-RGCs (displaced and orthotopic) project to the ipsilateral SC, x/ the topography of m-dRGCs does not resemble the general distribution of dRGCs, xi/ The soma size in m-oRGCs ranges from 10 to 21 μm and in m-dRGCs from 8 to 15 μm, xii/ oRGCs and dRGCs have the same susceptibility to axonal injury and ocular hypertension. Although the role of mammalian dRGCs remains to be determined, our data suggest that they are not misplaced by an ontogenic mistake.
    Frontiers in Neuroanatomy 10/2014; 8:99. DOI:10.3389/fnana.2014.00099 · 4.18 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 07/2014; 9(7):e102392. DOI:10.1371/journal.pone.0102392 · 3.53 Impact Factor
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    ABSTRACT: To investigate the cause of retinal ganglion cell (RGC) loss in dystrophic aged Royal College of Surgeons (RCS) rats. RCS-p+ (dystrophic) female rats of postnatal times (P365, P450 and P540) and age-matched RCS-p1 rdy+ (non-dystrophic) rats were used. In whole-mounted retinas, RGCs were doubly labelled with Fluorogold (FG) retrogradely transported from the superior colliculi and Brn3a immunohistochemistry. RGC axons were labelled with anti-neurofilament antibodies. Automatic image analysis techniques allowed quantification of the total population of RGCs per retina and construction of isodensity maps to investigate RGC topology. Dystrophic retinas showed at all times studied wedge-shaped sectors devoid of FG(+) and Brn3a(+) RGCs. These sectors were also devoid of neurofilament-labelled axons. The total number of FG(+)RGC and Brn3a(+)RGC per retina was significantly smaller in dystrophic rats at P540, revealing RGC death at this age. The total number of FG(+)RGCs was smaller than those of Brn3a(+)RGCs at P540, indicating a disturbance of the retrograde axonal transport at this age. RGC double labelling documents that sectorial RGC loss in aged dystrophic RCS rats is mainly due to RGC death, although a deficit of the retrograde axonal transport exists also at the more advanced ages.
    The British journal of ophthalmology 12/2013; 98(3). DOI:10.1136/bjophthalmol-2013-303958 · 2.81 Impact Factor
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    ABSTRACT: PURPOSE: To identify metabolic pathways and metabolites affected by optic nerve crush that can act as predictors of the disease or therapeutic targets. METHODS: The left optic nerve of adult rats was intraorbitally crushed and retinas were dissected 24 hours (h) or 14 days (d) after the lesion (n=10 per group). Metabolic profiling analysis was carried out by Metabolon (Inc). A total of 195 metabolites were unambiguously detected. Data were normalized and the regulated metabolites were identified after comparing the different conditions. Metabolites concentration changes were analyzed using single and multivariate statistical analysis to detect discriminatory metabolites. Functional clustering and meta-analysis of the regulated metabolites was run through the Metacore (Thomson Reuters) platform. RESULTS: Comparison between 24h vs. control, 14d vs. control samples and 24h vs. 14d identified 9, 19 and 32 regulated metabolites, respectively. Single and multivariate analysis identified a total of 27 and 36 metabolites to discriminate between control and 14d and between 24h and 14d, respectively. Enrichment analysis showed alterations in the aminoacid, carbohydrate and lipid metabolism which were further linked to translation, oxidative stress, energy (glucose and tricarboxilic acid cycle) and apoptosis through ceramide pathways. CONCLUSIONS: Our analysis differentiates a set of metabolites that clearly discriminate control and early injury samples from late injury samples. These metabolites could have potential use as diagnostic molecules.
    Investigative ophthalmology & visual science 05/2013; 54(6). DOI:10.1167/iovs.12-11451 · 3.66 Impact Factor
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    ABSTRACT: Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs -identified by their Brn3a expression- and the population of ipRGCs -identified by melanopsin immunodetection- thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard desviation of ipRGCs in the albino rat is 2,047±309. Their distribution in the retina seems to be complementary to that of Brn3a(+)RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.
    Experimental Eye Research 01/2013; DOI:10.1016/j.exer.2012.12.010 · 3.02 Impact Factor
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    ABSTRACT: Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory’s previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.
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    ABSTRACT: The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.
    PLoS ONE 11/2012; 7(11):e49830. DOI:10.1371/journal.pone.0049830 · 3.53 Impact Factor
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    ABSTRACT: Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory's previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.
    Progress in Retinal and Eye Research 09/2011; 31(1):1-27. DOI:10.1016/j.preteyeres.2011.08.001 · 9.90 Impact Factor
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    ABSTRACT: 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.
    Experimental Eye Research 02/2011; 92(4):260-7. DOI:10.1016/j.exer.2011.02.001 · 3.02 Impact Factor
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    ABSTRACT: 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.
    Experimental Eye Research 02/2011; 92(5):377-87. DOI:10.1016/j.exer.2011.02.008 · 3.02 Impact Factor
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    ABSTRACT: PURPOSE. To quantify the whole population of S-and L-cones in the albino (Sprague-Dawley, SD) and pigmented (Piebald Virol Glaxo, PVG) rats and to study their topographical distribution within the retina. METHODS. Retinal radial sections and whole-mounted retinas were double immunodetected with antibodies against UV-sen-sitive and L-opsins to detect the S-and L-cones, respectively. Two automated routines were developed to quantify the whole population of S-and L-cones. Detailed isodensity maps of each cone type were generated. In both strains, the presence of dual cones was detected, these were semiautomatically quantified and their distribution determined. The matching distribution of retinal ganglion cells (RGCs) and L-cones was attained by dou-ble immunodetection of Brn3a and L-opsin, respectively RESULTS. The mean number Ϯ SEM of L-or S-cones in SD and PVG retinas was 231,736 Ϯ 14,517 and 239,939 Ϯ 6,494 or 41,028 Ϯ 5,074, and 27,316 Ϯ 2,235, respectively. There was an increasing gradient of S-cone density along the inferonasal quadrant, although the highest densities were found in the retinal rims. The distribution of L-cones seemed to be comple-mentary to the S-cones. The highest densities were observed in the superior nasotemporal axis, paralleling the distribution of Brn3a-positive RGCs. CONCLUSIONS. These data establish, for the first time, the total number and the topographical distribution of S-and L-cones in two rat strains and demonstrate the correlation of L-cones and RGC spatial distribution. (Invest Ophthalmol Vis Sci. 2010;51: 3171–3183) DOI:10.1167/iovs.09-4861 I n the mammalian retina, cone photoreceptors are responsi-ble for daylight (photopic) vision and color discrimination. Color discrimination in nonprimate mammals is achieved by two types of cones: S and M/L. These cones are distinguished mainly by the portion of light spectrum to which each is more sensitive: short wave lengths stimulate S-cones and medium to long wave lengths stimulate M/L-cones. This spectral sensitivity is conferred by the protein part of the visual pigments, the opsin. Thus, opsins are slightly different, depending on their sensitivity and color preference. Each cone type carries a specific opsin. In rodents, S-cones express the ultraviolet-sen-sitive or SWS1 opsin, 1,2 and M/L-cones express the LWS opsin, 3
    Investigative ophthalmology & visual science 06/2010; · 3.66 Impact Factor
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    ABSTRACT: To quantify the whole population of S- and L-cones in the albino (Sprague-Dawley, SD) and pigmented (Piebald Virol Glaxo, PVG) rats and to study their topographical distribution within the retina. Retinal radial sections and whole-mounted retinas were double immunodetected with antibodies against UV-sensitive and L-opsins to detect the S- and L-cones, respectively. Two automated routines were developed to quantify the whole population of S- and L-cones. Detailed isodensity maps of each cone type were generated. In both strains, the presence of dual cones was detected, these were semiautomatically quantified and their distribution determined. The matching distribution of retinal ganglion cells (RGCs) and L-cones was attained by double immunodetection of Brn3a and L-opsin, respectively. The mean number +/- SEM of L- or S-cones in SD and PVG retinas was 231,736 +/- 14,517 and 239,939 +/- 6,494 or 41,028 +/- 5,074, and 27,316 +/- 2,235, respectively. There was an increasing gradient of S-cone density along the inferonasal quadrant, although the highest densities were found in the retinal rims. The distribution of L-cones seemed to be complementary to the S-cones. The highest densities were observed in the superior nasotemporal axis, paralleling the distribution of Brn3a-positive RGCs. These data establish, for the first time, the total number and the topographical distribution of S- and L-cones in two rat strains and demonstrate the correlation of L-cones and RGC spatial distribution.
    Investigative ophthalmology & visual science 06/2010; 51(6):3171-83. DOI:10.1167/iovs.09-4861 · 3.66 Impact Factor
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    ABSTRACT: We examined qualitatively and quantitatively in adult rat retinas the temporal degeneration of the nerve fibre layer after intra-orbital optic nerve transection (IONT) or crush (IONC). Retinal ganglion cell (RGC) axons were identified by their heavy neurofilament subunit phosphorylated isoform (pNFH) expression. Optic nerve injury induces a progressive axonal degeneration which after IONT proceeds mainly with abnormal pNFH-accumulations in RCG axons and after IONC in RGCs somas and dendrites. Importantly, this aberrant pNFH-expression pattern starts earlier and is more dramatic after IONT than after IONC, highlighting the importance that the type of injury has on the time-course of RGC degeneration.
    Vision research 09/2009; 49(23):2808-25. DOI:10.1016/j.visres.2009.08.020 · 2.38 Impact Factor
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    ABSTRACT: 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). 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. 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. Brn3a can be used as a reliable, efficient ex vivo marker to identify and quantify RGCs in control and optic nerve-injured retinas.
    Investigative ophthalmology & visual science 04/2009; 50(8):3860-8. DOI:10.1167/iovs.08-3267 · 3.66 Impact Factor
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    ABSTRACT: To further investigate the molecular signals underlying optic nerve (ON) injury, the authors analyzed in adult control, ON-transected, and ON-crushed retinas the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, caspase 11 (inflammation and death), cathepsins C and B (lysosomal death pathway), calpain 1 (endoplasmic reticulum stress), calreticulin (apoptosis marker), Jun (early response), and aryl hydrocarbon receptor (cell cycle arrest). Adult female rats were subjected to intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON-injured adult rat retinas was extracted at different times postlesion, and Western blotting experiments were performed. For immunohistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradely identified with fluorogold applied to the superior colliculi 1 week before injury. Western blotting analyses revealed upregulation of all the analyzed proteins as early as 12 hours postlesion (hpl), peaking at 48 hpl, in agreement with our previous RNA study findings. Furthermore, immunohistofluorescence to radial sections showed that all but Stat 1 were expressed by the primarily injured neurons, the RGCs, as seen by colocalization with fluorogold. All analyzed proteins were upregulated in the retina after IONT or IONC as early as 12 hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.
    Investigative ophthalmology & visual science 10/2008; 50(1):424-31. DOI:10.1167/iovs.08-2404 · 3.66 Impact Factor

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398 Citations
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Institutions

  • 2008–2015
    • University of Murcia
      • • Department of Ophthalmology, Optometry, Otolaryngology and Pathological Anatomy
      • • Facultad de Medicina
      Murcia, Murcia, Spain
  • 2009–2013
    • Hospital Universitario Virgen de la Arrixaca
      Murcia, Murcia, Spain