Browsing by Subject "Brn3a"

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    7,8-Dihydroxiflavone maintains retinal functionality and protects various types of RGCs in adult rats with optic nerve transection
    (MDPI, 2021-10-30) Gallego Ortega, Alejandro; Vidal Villegas, Beatriz; Norte Muñoz, María; Salinas Navarro, Manuel Ángel; Avilés Trigueros, Marcelino; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    To analyze the neuroprotective effects of 7,8-Dihydroxyflavone (DHF) in vivo and ex vivo, adult albino Sprague-Dawley rats were given a left intraorbital optic nerve transection (IONT) and were divided in two groups: One was treated daily with intraperitoneal (ip) DHF (5 mg/kg) (n = 24) and the other (n = 18) received ip vehicle (1% DMSO in 0.9% NaCl) from one day before IONT until processing. At 5, 7, 10, 12, 14, and 21 days (d) after IONT, full field electroretinograms (ERG) were recorded from both experimental and one additional naïve-control group (n = 6). Treated rats were analyzed 7 (n = 14), 14 (n = 14) or 21 d (n = 14) after IONT, and the retinas immune stained against Brn3a, Osteopontin (OPN) and the T-box transcription factor T-brain 2 (Tbr2) to identify surviving retinal ganglion cells (RGCs) (Brn3a+), α-like (OPN+), α-OFF like (OPN+Brn3a+) or M4-like/α-ON sustained RGCs (OPN+Tbr+). Naïve and right treated retinas showed normal ERG recordings. Left vehicle-treated retinas showed decreased amplitudes of the scotopic threshold response (pSTR) (as early as 5 d), the rod b-wave, the mixed response and the cone response (as early as 10 d), which did not recover with time. In these retinas, by day 7 the total numbers of Brn3a+RGCs, OPN+RGCs and OPN+Tbr2+RGCs decreased to less than one half and OPN+Brn3a+RGCs decreased to approximately 0.5%, and Brn3a+RGCs showed a progressive loss with time, while OPN+RGCs and OPN+Tbr2+RGCs did not diminish after seven days. Compared to vehicle-treated, the left DHF-treated retinas showed significantly greater amplitudes of the pSTR, normal b-wave values and significantly greater numbers of OPN+RGCs and OPN+Tbr2+RGCs for up to 14 d and of Brn3a+RGCs for up to 21 days. DHF affords significant rescue of Brn3a+RGCs, OPN+RGCs and OPN+Tbr2+RGCs, but not OPN+Brn3a+RGCs, and preserves functional ERG responses after IONT.
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    A freely available semi-automated method for quantifying retinal ganglion cells in entire retinal flatmounts
    (Elsevier, 2016-04-20) Geeraerts, E. ; Dekeyster, E. ; Gaublomme, D. ; De Groef, L.; Moons, L.; Salinas Navarro, Manuel Ángel; Anatomía Humana y Psicobiología
    Glaucomatous optic neuropathies are characterized by progressive loss of retinal ganglion cells (RGCs), the neurons that connect the eye to the brain. Quantification of these RGCs is a cornerstone in experimental optic neuropathy research and commonly performed via manually quantifying parts of the retina. However, this is a time-consuming process subject to inter- and intra-observer variability. Here we present a freely available ImageJ script to semi-automatically quantify RGCs in entire retinal flatmounts after immunostaining for the RGC-specific transcription factor Brn3a. The blob-like signal of Brn3a-immunopositive RGCs is enhanced via eigenvalues of the Hessian matrix and the resulting local maxima are counted as RGCs. After the user has outlined the retinal flatmount area, the total RGC number and retinal area are reported and an isodensity map, showing the RGC density distribution across the retina, is created. The semi-automated quantification shows a very strong correlation (Pearson's r ≥ 0.99) with manual counts for both widefield and confocal images, thereby validating the data generated via the developed script. Moreover, application of this method in established glaucomatous optic neuropathy models such as N-methyl-D-aspartate-induced excitotoxicity, optic nerve crush and laser-induced ocular hypertension revealed RGC loss conform with literature. Compared to manual counting, the described automated quantification method is faster and shows user-independent consistency. Furthermore, as the script detects the RGC number in entire retinal flatmounts, the method allows detection of regional differences in RGC density. As such, it can help advance research investigating the degenerative mechanisms of glaucomatous optic neuropathies and the effectiveness of new neuroprotective treatments. Because the script is flexible and easy to optimize due to a low number of critical parameters, it can potentially be applied in combination with other tissues or alternative labeling protocols.
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    Apoptotic retinal ganglion cell death after optic nerve transection or crush in mice: delayed RGC loss with BDNF or a Caspase 3 inhibitor
    (Association for Research in Vision and Ophthalmology, 2016-01) Sánchez-Migallón, María C.; Valiente Soriano, Francisco Javier; Nadal-Nicolás, Francisco Manuel; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
    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.
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    BDNF Rescues RGCs But Not Intrinsically Photosensitive RGCs in Ocular Hypertensive Albino Rat Retinas
    (Association for Research in Vision and Ophthalmology., 2015-02-26) Valiente Soriano, Francisco Javier; Nadal-Nicolás, Francisco Manuel; Salinas Navarro, Manuel Ángel; Jiménez López, Manuel; Bernal Garro, José M.; Villegas Pérez, Maria Paz; Agudo Barriuso, Marta; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    Purpose: To study the responses of the general population of retinal ganglion cells (Brn3a(+)RGCs) versus the intrinsically photosensitive RGCs (melanopsin-expressing RGCs [m(+)RGCs]) to ocular hypertension (OHT), the effects of brain-derived neurotrophic factor (BDNF) on the survival of axonally intact and axonally nonintact RGCs, and the correlation of vascular integrity with sectorial RGC loss. Methods: In Sprague-Dawley rats, 5 μg BDNF or vehicle was intravitreally injected into the left eye followed by laser photocoagulation of the limbal tissues. To identify RGCs with an active retrograde axonal transport, Fluorogold was applied to both superior colliculi 1 week before euthanasia (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: Ocular hypertension resulted at 12 to 15 days in sectorial loss of FG(+)RGCs (78%-84%, respectively) while Brn3a(+)RGCs were significantly greater, indicating that a substantial proportion (approximately 21%-26%) of RGCs with their retrograde axonal transport impaired survive in the retina. Brain-derived neurotrophic factor increased the survival of Brn3a(+)RGCs to 81% to 67% at 12 to 15 days, respectively. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. At 12 to 15 days, m(+)RGCs decreased to approximately 50% to 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.
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    Brain derived neurotrophic factor maintains Brn3a expression in axotomized rat retinal ganglion cells
    (Elsevier, 2009-08-16) Sánchez Migallón, María del Cielo; Nadal-Nicolás, Francisco Manuel; Jiménez López, Manuel; Sobrado Calvo, Paloma; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    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.
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    Comparison of retinal nerve fiber layer thinning and retinal ganglion cell loss after optic nerve transection in adult albino rats
    (Association for Research in Vision and Ophthalmology., 2015-07-15) Rovere, Giuseppe; Nadal-Nicolás, Francisco Manuel; Agudo Barriuso, Marta; Sobrado Calvo, Paloma; Nieto López, Leticia; Nucci, Carlo; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    PURPOSE. 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. METHODS. At 3, 7, 12, or 21 days, or 1, 2, or 4 months after ONT, the retinas were imaged with spectral-domain optical coherence tomography (SD-OCT) using the circular-peripapillary scan and volume scan raster pattern (61 horizontal sections equally spaced) both centered in the optic nerve. In all sections, the RNFL and retinal thickness were measured to obtain the total values of the peripapillary scan and the values of three concentric sectors (400, 1200, and 2400 lm in diameter) from the volume scan. After SD-OCT, retinas were dissected and immunoreacted for Brn3a and neurofilaments (pNFH) to identify RGCs and their intraretinal axons, respectively. Total numbers of RGCs were quantified. RESULTS. Thinning of the RNFL was first observed at 12 days in peripapillary scan (10% decrease) and progressed up to 4 months (72% decrease). The volume scan showed transient RNFL swelling in central and medial sectors at 3, 7, and 12 days followed by progressive significant thinning first observed at 21 days (central sector, 30%; medial sector, 40%) and 12 days (peripheral sector, 15%), respectively. Following IONT, Brn3aþ RGCs decreased to approximately 80%, 52%, 17%, 9%, 5%, 3%, and 2% at 3, 7, 12, 21 days, and at 1, 2, and 4 months, respectively. Retinal ganglion cell axon immunodetection decreased from 12 days onwards. CONCLUSIONS. After IONT, RGC death is more severe and precedes thinning of the RNFL.
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    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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    Inherited photoreceptor degeneration causes the death of melanopsin-positive retinal ganglion cells and increases their coexpression of Brn3a
    (Association for Research in Vision and Ophthalmology, 2015-07-20) García Ayuso, Diego; Di Pierdomenico, Johnny; Esquiva, Gema; Nadal-Nicolás, Francisco Manuel; Pinilla, Isabel; Cuenca, Nicolás; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    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.
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    Long-term effect of optic nerve axotomy on the retinal ganglion cell layer
    (Association for Research in Vision and Ophthalmology., 2015-09-01) Nadal-Nicolás, Francisco Manuel; Sobrado Calvo, Paloma; Jiménez López, Manuel; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    Purpose: To analyze the long-term effect of optic nerve injury on retinal ganglion cells (RGCs) and melanopsin+RGCs orthotopic and displaced, and on the rest of the ganglion cell layer (GCL) cells. Methods: In adult albino rats, the left optic nerve was crushed (ONC) or transected (ONT). Injured and contralateral retinas were analyzed at increasing survival intervals (up to 15 months). To study all GCL cells and RGCs, retinas were immunodetected with Brn3a and melanopsin to identify the general RGC population (Brn3a+) and m+RGCs, and counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Brn3a+RGCs and m+RGCs displaced to the inner nuclear layer were analyzed as well. In additional retinas, glial cells in the GCL were identified with glial fibrillary acidic protein (GFAP) or Iba1, and in some retinas, Brn3a, calretinin, and γ-synuclein were immunodetected. Results: Orthotopic and displaced RGCs behave similarly within the RGC and m+RGC populations. Both lesions cause an exponential loss of RGCs (4%–1% survival at 6 months after ONC or ONT), but not of m+RGCs, whose number remains stable from 1 to 15 months (34%–44% of the initial population). γ-synuclein is expressed by RGCs and displaced amacrine cells (dACs), allowing us to confirm that axotomy does not affect the latter, and to determine that out of the approximately 217,406 cells that compose the GCL (excluding endothelia), 10% are glial cells, 50% dACs, and the remaining 40% are RGCs. Conclusions: In the GCL, only RGCs are lost after axotomy, and there are important differences in the course of loss and rate of survival between melanopsin+RGCs and the rest of RGCs.
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    Melanopsin-containing or non-melanopsin–containing retinal ganglion cells response to acute ocular hypertension with or without brain-derived neurotrophic factor neuroprotection
    (Association for Research in Vision and Ophthalmology, 2016-12) Rovere, Giuseppe ; Nadal-Nicolás, Francisco Manuel; Wang, Jiawei; Bernal-Garro, José M.; García-Carrillo, Nuria; Villegas Pérez, Maria Paz; Agudo Barriuso, Marta; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
    PURPOSE. To analyze the responses of different retinal ganglion cell (RGC) types to acute ocular hypertension (AOH) and intravitreal administration of brain-derived neurotrophic factor (BDNF). METHODS. In adult albino rats, the anterior chamber of the left eye was cannulated with a needle connected to a saline container elevated 1½ meters above the eye for 75 minutes. Rats received 12 hours before a 5 ul intravitreal injection containing 5 ug BDNF in 1% albumin PBS or vehicle and were analyzed 3, 7, 14, or 45 days later. Both retinas were dissected as wholemounts and immunolabeled for melanopsin (to identify intrinsically photosensitive RGCs) or Brn3a (to identify all RGCs except melanopsin +RGCs). RESULTS. During AOH there is ischemic damage and mechanical eye-globe deformation. Acute ocular hypertension results in a progressive loss of Brn3a+RGCs in the vehicle-treated retinas (39%, 35%, 25%, and 13% of the original value, at 3, 7, 14, or 45 days, respectively), whereas BDNF increases their survival to 81%, 73%, 59%, or 57% at the same time periods. In vehicletreated retinas, 37% or 39% of m+RGCs survive at 14 or 45 days, respectively, whereas BDNF treatment increases their survival to 40% or 78% at the same time points. CONCLUSIONS. Different types of RGCs respond differently to AOH because Brn3a+RGCs die progressively, but m+RGCs do not. After a transient downregulation of melanopsin expression, their number remains constant and their survival is proportionally higher than that of Brn3a+RGCs. BDNF affords a permanent protection up to 45 days after AOH injury inboth types of RGCs.
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    Microglial dynamics after axotomy-induced retinal ganglion cell death
    (BioMed Central, 2017-11-09) Nadal-Nicolás, Francisco Manuel; Jiménez-López, Manuel; Salinas Navarro, Manuel Ángel; Sobrado Calvo, Paloma; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
    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.
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    Nerve fibre layer degeneration and retinal ganglion cell loss long term after optic nerve crush or transection in adult mice
    (Elsevier, 2018-02-13) Sánchez-Migallón Carreras, María del Cielo; Valiente Soriano, Francisco Javier; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Jiménez-López, M.; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
    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
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    Number and spatial distribution of intrinsically photosensitive retinal ganglion cells in the adult albino rat
    (Elsevier, 2013-01-04) Galindo Romero, Caridad; Jiménez López, Manuel; García Ayuso, Diego; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Avilés Trigueros, Marcelino; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    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 deviation of ipRGCs in the albino rat is 2047 ± 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.
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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.
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    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.
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    Systemic treatment with 7,8-Dihydroxiflavone activates TtkB and affords protection of two different retinal ganglion cell populations against axotomy in adult rats
    (Elsevier, 2021-07-08) Vidal-Villegas, Beatriz; Di Pierdomenico, Johnny; Gallego Ortega, Alejandro; Galindo Romero, Caridad; Martínez-de-la-Casa, José M.; García-Feijoo, Julián; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Óptica y Optometría
    Purpose: To analyze responses of different RGC populations to left intraorbital optic nerve transection (IONT) and intraperitoneal (i.p.) treatment with 7,8-Dihydroxyflavone (DHF), a potent selective TrkB agonist. Methods: Adult albino Sprague-Dawley rats received, following IONT, daily i.p. injections of vehicle (1%DMSO in 0.9%NaCl) or DHF. Group-1 (n = 58) assessed at 7days (d) the optimal DHF amount (1–25 mg/kg). Group-2, using freshly dissected naïve or treated retinas (n = 28), investigated if DHF treatment was associated with TrkB activation using Western-blotting at 1, 3 or 7d. Group-3 (n = 98) explored persistence of protection and was analyzed at survival intervals from 7 to 60d after IONT. Groups 2–3 received daily i.p. vehicle or DHF (5 mg/kg). Retinal wholemounts were immunolabelled for Brn3a and melanopsin to identify Brn3a+RGCs and m+RGCs, respectively. Results: Optimal neuroprotection was achieved with 5 mg/kg DHF and resulted in TrkB phosphorylation. The percentage of surviving Brn3a+RGCs in vehicle treated rats was 60, 28, 18, 13, 12 or 8% of the original value at 7, 10, 14, 21, 30 or 60d, respectively, while in DHF treated retinas was 94, 70, 64, 17, 10 or 9% at the same time intervals. The percentages of m+RGCs diminished by 7d–13%, and recovered by 14d–38% in vehicle-treated and to 48% in DHF-treated retinas, without further variations. Conclusions: DHF neuroprotects Brn3a + RGCs and m + RGCs; its protective effects for Brn3a+RGCs are maximal at 7 days but still significant at 21d, whereas for m+RGCs neuroprotection was significant at 14d and permanent.
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    The aging rat retina: from function to anatomy
    (Elsevier, 2018-01-01) Nadal-Nicolás, Francisco Manuel; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
    In healthy beings, age is the ultimate reason of cellular malfunction and death. In the rat retina, age causes a functional decline and loss of specific neuronal populations. In this regard, controversial conclusions have been reported for the innermost retina. Here, we have studied the albino and pigmented retina for the duration of the rat life-span. Independent of age (21 dayse22 months), the electroretinographic recordings and the volume of the retina and its layers are smaller in albinos. Functionally, aging causes in both strains a loss of cone- and rod-mediated responses. Anatomically, cell density decreases with age because the retina grows linearly with time; no cell loss is observed in the ganglion cell layer; and only in the pigmented rat, there is a decrease in cone photoreceptors. In old animals of both strains, there is gliosis in the superior colliculi and a diminution of the area innervated by retinal ganglion cells. In conclusion, this work provides the basis for further studies linking senescence to neurodegenerative retinal diseases.
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    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