Browsing by Subject "Fluorogold"

Now showing 1 - 5 of 5
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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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    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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    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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    Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration
    (2010-01) Valiente Soriano, Francisco J.; Jiménez López, Manuel; Mayor Torroglosa, Sergio; Avilés Trigueros, Marcelino; Villegas Pérez, María Paz; Vidal Sanz, Manuel; Salinas Navarro, Manuel Ángel; Alarcón Martínez, Luis; Anatomía Humana y Psicobiología
    Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG+ or DTMR+RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR+RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR+RGCs and FG+RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.
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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.