Salinas Navarro, Manuel Ángel

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Salinas Navarro, Manuel Ángel

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Salinas Navarro, Manuel Ángel

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Universidad de Murcia. Departamento de Anatomía Humana y Psicobiología

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Open Access
Displaced retinal ganglion cells in albino and pigmented rats
(Frontiers Media , 2014-10-06) Nadal-Nicolás, Francisco Manuel; Salinas Navarro, Manuel Ángel; Jiménez López, Manuel; Sobrado Calvo, Paloma; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
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.
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Retinal neurodegeneration in experimental glaucoma
(Elsevier, 2015-07-02) Vidal Sanz, Manuel; Valiente Soriano, Francisco Javier; Ortín Martínez, Arturo; Nadal-Nicolás, Francisco Manuel; Jiménez López, Manuel; Salinas Navarro, Manuel Ángel; García Ayuso, Diego; Avilés Trigueros, Marcelino; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Alarcón Martínez, Luis; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Bagetta, Giacinto; Nucci, Carlo; Facultades de la UMU::Facultad de Medicina
In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m(+)RGC), Brn3a immunodetection to identify most RGCs but not m(+)RGCs, RECA1 immunodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the GC layer. The outer retinal layers (ORLs) were examined in cross sections analyzed morphometrically or in wholemounts to study S- and L-cones. Innervation of the superior colliculi was examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B. By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG(+)RGCs or Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. Brn3a(+)RGCs were significantly greater than FG(+)RGCs, indicating the survival of large numbers of RGCs with their axonal transport impaired. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. m(+)RGCs decreased to approximately 50-51% in a diffuse loss across the retina. Cross sections showed focal areas of degeneration in the ORLs. RGC loss at 1m diminished to 20-25% and did not progress further with time, whereas the S- and L-cone populations diminished progressively up to 6m. The retinotectal projection was reduced by 10 days and did not progress further. LP-induced OHT results in retrograde degeneration of RGCs and m(+)RGCs, severe damage to the ORL, and loss of retinotectal terminals.
Open Access
Immediate upregulation of proteins belonging to different branches of the apoptotic cascade in the retina after optic nerve transection and optic nerve crush.
(Association for Research in Vision and Ophthalmology, 2009-01) Agudo, Marta; Pérez Marín, María C.; Sobrado Calvo, Paloma; Lönngren, Ulrika; Cánovas, Isabel; Miralles Imperial, Jaime; Hallböök, Finn; Vidal Sanz, Manuel; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Anatomía Humana y Psicobiología
Purpose. 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). methods. 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. results. 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. conclusions. 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.
Open Access
Characterizing microglia activation: a spatial statistics approach to maximize information extraction
(Nature Research, 2017) Salinas Navarro, Manuel Ángel; Cordeiro, M. Francesca; Moons, Lieve ; De Groef, Lies ; Davis, Benjamin M. ; Anatomía Humana y Psicobiología; Facultades de la UMU::Facultad de Medicina
Microglia play an important role in the pathology of CNS disorders, however, there remains significant uncertainty about the neuroprotective/degenerative role of these cells due to a lack of techniques to adequately assess their complex behaviour in response to injury. Advancing microscopy techniques, transgenic lines and well-characterized molecular markers, have made histological assessment of microglia populations more accessible. However, there is a distinct lack of tools to adequately extract information from these images to fully characterise microglia behaviour. This, combined with growing economic pressures and the ethical need to minimise the use of laboratory animals, led us to develop tools to maximise the amount of information obtained. This study describes a novel approach, combining image analysis with spatial statistical techniques. In addition to monitoring morphological parameters and global changes in microglia density, nearest neighbour distance, and regularity index, we used cluster analyses based on changes in soma size and roundness to yield novel insights into the behaviour of different microglia phenotypes in a murine optic nerve injury model. These methods should be considered a generic tool to quantitatively assess microglia activation, to profile phenotypic changes into microglia subpopulations, and to map spatial distributions in virtually every CNS region and disease state.
Open Access
Two methods to trace retinal ganglion cells with fluorogold: From the intact optic nerve or by stereotactic injection into the optic tract
(Elsevier, 2015-02-01) Salinas Navarro, Manuel Ángel; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Nadal-Nicolás, Francisco Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
Open Access
Retino-retinal projection in juvenile and young adult rats and mice
(Elsevier, 2015-05-01) Nadal-Nicolás, Francisco Manuel; Valiente Soriano, Francisco Javier; Salinas Navarro, Manuel Ángel; Jiménez López, Manuel; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
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.
Open Access
Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naïve and optic nerve–injured retinas
(Association for Research in Vision and Ophthalmology, 2009-08) Jiménez López, Manuel; Sobrado Calvo, Paloma; Nieto López, Leticia; Cánovas Martínez, Isabel; Vidal Sanz, Manuel; Agudo, Marta; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Anatomía Humana y Psicobiología
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.
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Axotomy-induced retinal ganglion cell death in adult mice: quantitative and topographic time course analyses
(Elsevier, 2011-02-24) Galindo Romero, Caridad; Avilés Trigueros, Marcelino; Jiménez López, Manuel; Valiente Soriano, Francisco Javier; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Anatomía Humana y Psicobiología; Facultades de la UMU::Facultad de Medicina
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, tracedretinal ganglion cells were automatically quantified, using the previously reported method (SalinasNavarro 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) andofBRN3Apositive 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.
Open Access
Optogenetic Stimulation of the Superior Colliculus Confers Retinal Neuroprotection in a Mouse Glaucoma Model
(Society for Neuroscience, 2019-03-20) Geeraerts, Emiel ; Claes, Marie ; Dekeyster, Eline; Salinas Navarro, Manuel Ángel; De Groef, Lies ; Van den Haute, Chris ; Scheyltjens, Isabelle ; Baekelandt, Veerle ; Arckens, Lutgarde ; Moons, Lieve ; Anatomía Humana y Psicobiología; Facultades de la UMU::Facultad de Medicina
Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) in the eye, which ultimately results in visual impairment or even blindness. Because current therapies often fail to halt disease progression, there is an unmet need for novel neuroprotective therapies to support RGC survival. Various research lines suggest that visual target centers in the brain support RGC functioning and survival. Here, we explored whether increasing neuronal activity in one of these projection areas could improve survival of RGCs in a mouse glaucoma model. Prolonged activation of an important murine RGC target area, the superior colliculus (SC), was established via a novel optogenetic stimulation paradigm. By leveraging the unique channel kinetics of the stabilized step function opsin (SSFO), protracted stimulation of the SC was achieved with only a brief light pulse. SSFO-mediated collicular stimulation was confirmed by immunohistochemistry for the immediate-early gene c-Fos and behavioral tracking, which both demonstrated consistent neuronal activity upon repeated stimulation. Finally, the neuroprotective potential of optogenetic collicular stimulation was investigated in mice of either sex subjected to a glaucoma model and a 63% reduction in RGC loss was found. This work describes a new paradigm for optogenetic collicular stimulation and a first demonstration that increasing target neuron activity can increase survival of the projecting neurons.
Open Access
Retinal compensatory changes after light damage in albino mice
(Molecular Vision, 2012-03-24) Montalbán Soler, Luis; Jiménez López, Manuel ; Bezerra de Sá, Fabrízio; Salinas Navarro, Manuel Ángel; Alarcón Martínez, Luis; Galindo Romero, Caridad; García Ayuso, Diego; Avilés Trigueros, Marcelino; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Anatomía Humana y Psicobiología
Purpose: To investigate the anatomic and functional changes triggered by light exposure in the albino mouse retina and compare them with those observed in the albino rat. Methods: BALB/c albino mice were exposed to 3,000 lx of white light during 24 h and their retinas analyzed from 1 to 180 days after light exposure (ALE). Left pupil mydriasis was induced with topical atropine. Retinal function was analyzed by electroretinographic (ERG) recording. To assess retinal degeneration, hematoxylin and eosin staining, the TdT-mediated dUTP nick-end labeling (TUNEL) technique, and quantitative immunohistofluorescence for synaptophysin and protein kinase Cα (PKCα) were used in cross sections. Intravenous injection of horseradish peroxidase and Fluoro-Gold™ tracing were used in whole-mounted retinas to study the retinal vasculature and the retinal ganglion cell (RGC) population, respectively. Results: Light exposure caused apoptotic photoreceptor death in the central retina. This death was more severe in the dorsal than in the ventral retina, sparing the periphery. Neither retinal vascular leakage nor retinal ganglion cell death was observed ALE. The electroretinographic a-wave was permanently impaired, while the b-wave decreased but recovered gradually by 180 days ALE. The scotopic threshold responses, associated with the inner retinal function, diminished at first but recovered completely by 14 days ALE. This functional recovery was concomitant with the upregulation of protein kinase Cα and synaptophysin. Similar results were obtained in both eyes, irrespective of mydriasis. Conclusions: In albino mice, light exposure induces substantial retinal damage, but the surviving photoreceptors, together with compensatory morphological/molecular changes, allow an important restoration of the retinal function.