Paloma Sobrado - Academia.edu (original) (raw)

Papers by Paloma Sobrado

Research paper thumbnail of Effect of olfactory ensheathing glia clone TEG3 on neuroprotection, microglial activation and gene expression profile of allotransplanted retina with and without immunosuppression

Research paper thumbnail of Tnfr1 Death-Signalling Pathway Is Regulated in the Retina Both After Optic Nerve Transection and Optic Nerve Crush

Investigative Ophthalmology & Visual Science, 2009

Research paper thumbnail of Co-Relation Between Transcriptomic And Metabolomic Changes In The Axotomized Adult Rat Retina

Investigative Ophthalmology & Visual Science, 2012

Research paper thumbnail of Quantitative And Qualitative Analyses Of The Nerve Fiber Layer Degeneration After Optic Nerve Axotomy In Adult Mice

Investigative Ophthalmology & Visual Science, 2012

Research paper thumbnail of Corneal endothelial cell loss after trabeculectomy and phacoemulsification in one or two steps: a prospective study

Eye, 2021

Objective The objective of this study was to analyse the results of the surgical treatment of coe... more Objective The objective of this study was to analyse the results of the surgical treatment of coexisting cataract and glaucoma and its effects on corneal endothelial cell density (CECD). Methods We include two longitudinal prospective studies: one randomised that included 40 eyes with open angle glaucoma that received one- (n = 20) or two-step (n = 20) phacotrabeculectomy and another that included 20 eyes that received phacoemulsification. We assess the impact of surgery on different clinical variables and in particular in CECD using Confoscan 4™ confocal microscopy and semiautomatic counting methods. Results Phacoemulsification and phacotrabeculectomy, but not trabeculectomy, increase significantly best-corrected visual acuity and anterior chamber depth and trabeculectomy and one- or two-step phacotrabeculectomy decreased similarly the intraocular pressure. We document percentages of endothelial cell loss of 3.1%, 17.9%, 31.6% and 42.6% after trabeculectomy, phacoemulsification and...

Research paper thumbnail of Topical bromfenac transiently delays axotomy-induced retinal ganglion cell loss

Experimental Eye Research, 2019

Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on t... more Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on the survival of central nervous system neurons, the retinal ganglion cells (RGCs). Here we have analyzed the neuroprotective effect of topical bromfenac treatment, a nonsteroidal anti-inflammatory drug (NSAID) used in clinic to ameliorate post-operative inflammation, on axotomized rat RGCs. The left optic nerve of adult rats was subjected to optic nerve crush (ONC). Half of the rats were treated with a topical instillation of saline. On the other half, immediately after the surgery, 2 drops of bromfenac (0.09% Yellox; Bausch & Lomb) were instilled, and then every 12 h until analysis. Retinas in both groups were dissected 3, 5, 7, 9 and 14 days after ONC (n = 4-8/time point/group). Toxicity of bromfenac was assessed in intact retinas treated during 14 days (n = 6). Intact untreated retinas were used as control of the RGC population. RGCs were identified by Brn3a immunodetection and automatically quantified. Our results show that bromfenac does not cause RGC loss in intact retinas. In the injured groups, the number of RGCs at 7, 9 and 14 days after the lesion was significantly higher in treated vs. untreated retinas. To our knowledge this is the first report showing that a topical treatment with a NSAIDs delays axotomy-induced RGC loss and indicates that treatment with NSAIDs could be used as conjunctive therapy in diseases that proceed with optic nerve damage.

Research paper thumbnail of Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension

Frontiers in Neuroscience, 2017

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion ce... more Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a + RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m + RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a + RGCs, m + RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value.

Research paper thumbnail of Topical Treatment With Bromfenac Reduces Retinal Gliosis and Inflammation After Optic Nerve Crush

Investigative Opthalmology & Visual Science, 2016

PURPOSE. To study the effect of topical administration of bromfenac, a nonsteroidal antiinflammat... more PURPOSE. To study the effect of topical administration of bromfenac, a nonsteroidal antiinflammatory drug (NSAID), on retinal gliosis and levels of prostaglandin E 2 (PGE 2) after complete optic nerve crush (ONC). METHODS. Adult albino rats were divided into the following groups (n ¼ 8 retinas/group): (1) intact, (2) intact and bromfenac treatment (twice a day during 7 days), (3) ONC (7 days), and (4) ONC (7 days) þ bromfenac treatment (twice a day during 7 days). Animals from groups 3 and 4 were imaged in vivo with spectral-domain optical coherence tomography (SD-OCT) before the procedure and 15 minutes, 3, 5, or 7 days later. Retinas from all groups were analyzed by immunodetection, Western blotting, or enzyme-linked immunoabsorbent assay (ELISA). RESULTS. Quantification of Brn3a (brain-specific homeobox/POU domain protein 3A) þ RGCs (retinal ganglion cells) in cross sections showed that bromfenac treatment does not accelerate ONC-induced degeneration. Cellular retinaldehyde binding protein 1 regulation indicated that bromfenac improves retinal homeostasis in injured retinas. Spectral-domain OCT showed that the thickness of the retina and the retinal nerve fiber layer at 7 days post ONC was significantly reduced in bromfenac-treated animals when compared to untreated animals. In agreement with these data, hypertrophy of astrocytes and Müller cells and expression of glial fibrillary acidic protein and vimentin were greatly diminished by bromfenac treatment. While no changes in cyclooxygenase (COX) enzyme COX1 and COX2 expression were observed, there was a significant increase of PGE 2 after ONC that was controlled by bromfenac treatment. CONCLUSIONS. Topical administration of bromfenac is an efficient and noninvasive treatment to control the retinal gliosis and release of proinflammatory mediators that follow a massive insult to the RGC population.

Research paper thumbnail of Ketorolac Administration Attenuates Retinal Ganglion Cell Death After Axonal Injury

Investigative Opthalmology & Visual Science, 2016

PURPOSE. To assess the neuroprotective effects of ketorolac administration, in solution or delive... more PURPOSE. To assess the neuroprotective effects of ketorolac administration, in solution or delivered from biodegradable microspheres, on the survival of axotomized retinal ganglion cells (RGCs). METHODS. Retinas were treated intravitreally with a single injection of tromethamine ketorolac solution and/or with ketorolac-loaded poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Ketorolac treatments were administered either 1 week before optic nerve crush (pre-ONC) or right after the ONC (simultaneous). In all cases, animals were euthanized 7 days after the ONC. As control, nonloaded microspheres or vehicle (balanced salt solution, BSS) were administered in parallel groups. All retinas were dissected as flat mounts; RGCs were immunodetected with brain-specific homeobox/POU domain protein 3A (Brn3a), and their number was automatically quantified. RESULTS. The percentage of Brn3a þ RGCs was 36% to 41% in all control groups (ONC with or without BSS or nonloaded microparticles). Ketorolac solution administered pre-ONC resulted in 63% survival of RGCs, while simultaneous administration promoted a 53% survival. Ketorolac-loaded microspheres were not as efficient as ketorolac solution (43% and 42% of RGC survival pre-ONC or simultaneous, respectively). The combination of ketorolac solution and ketorolac-loaded microspheres did not have an additive effect (54% and 55% survival pre-ONC and simultaneous delivery, respectively). CONCLUSIONS. Treatment with the nonsteroidal anti-inflammatory drug ketorolac delays RGC death triggered by a traumatic axonal insult. Pretreatment seems to elicit a better output than simultaneous administration of ketorolac solution. This may be taken into account when performing procedures resulting in RGC axonal injury.

Research paper thumbnail of Long-Term Effect of Optic Nerve Axotomy on the Retinal Ganglion Cell Layer

Investigative ophthalmology & visual science, 2015

To analyze the long-term effect of optic nerve injury on retinal ganglion cells (RGCs) and melano... more 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. 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. Orthotopic and displaced RGCs behave similarly within the RGC and m+RGC populations. Both lesions cause an exponential loss of RGCs (4%-1%...

Research paper thumbnail of Comparison of Retinal Nerve Fiber Layer Thinning and Retinal Ganglion Cell Loss After Optic Nerve Transection in Adult Albino Rats

Investigative Opthalmology & Visual Science, 2015

PURPOSE. We compared the time-course and magnitude of retinal nerve fiber layer (RNFL) thinning w... more 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, Brn3a þ 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.

Research paper thumbnail of Temporal response of the phagocytic microglia in the axotomized rat retina: optic nerve crush vs. transection

Acta Ophthalmologica, 2014

Purpose To analyze the appearance of phagocytic microglial cells (PMC) in rat retinas after optic... more Purpose To analyze the appearance of phagocytic microglial cells (PMC) in rat retinas after optic nerve crush or transection.Methods Retinal ganglion cells (RGCs) from adult Sprague Dawley rats were traced with fluorogold applied to both superior colliculi. One week later, the left optic nerve was either crushed (ONC, n=32) or transected (ONT, n=32). Rats were euthanized at 2, 5, 9 and 14 days after the axotomy (n=8/group). Retinas were dissected as flatmounts and Brn3a immunodetected to identify RGCs. Retinas were photographed for each signal. Brn3a+RGCs were automatically quantified and their distribution visualized by isodensity maps. The position of each PMC found in the ganglion cell layer (identified by their transcellularly labelling with fluorogold) was dotted on the retinal photomontages. These dots were counted and graphically represented with neighbour maps.Results PMCs were observed at 2 days(43±8, ONT; 11±5, ONC). Their number increased quicker after ONT than after ONC ...

Research paper thumbnail of Measurement of retinal injury in the rat after optic nerve transection: an RT-PCR study

Molecular vision, Jan 2, 2005

In the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to p... more In the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to provide information on retinal ganglion cell (RGC) injury and survival after optic nerve transection (ONT). The levels of mRNAs synthesized by RGCs and glial components were initially measured at defined time points after ONT. Subsequently, a comparison was made between the levels of these mRNAs in the ONT retinas of rats treated with the neuroprotectant BDNF and in rats which received vehicle. Wistar rats received an ONT in one eye, while the fellow eye served as a control. ONT was performed 1-2 mm from the optic disc without damaging the retinal blood supply. In the first experiment, rats were killed at 1, 3, 5, 7, and 21 days after ONT. In the second experiment, brain derived neurotrophic factor (BDNF; 5 microg) or vehicle was injected intravitreally at the same time as the ONT and animals were killed after 7 days. After ONT, mRNA levels of RGC markers (NF-L and Thy-1) decreased substa...

Research paper thumbnail of Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death

Investigative ophthalmology & visual science, 2001

To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-... more To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA re...

Research paper thumbnail of Neuroprotective Effects of a2Selective Adrenergic Agonists against Ischemia-Induced Retinal Ganglion Cell Death

determined by the duration of the period of ischemia.18,19 The a2-adrenergic receptors are G prot... more determined by the duration of the period of ischemia.18,19 The a2-adrenergic receptors are G protein- coupled recep- tors localized in the retina within the RGC layer, inner nuclear layer, and inner segments of the photoreceptors.20 -22 Recent evidence indicates that retinal a2-adrenergic receptors mediate neuroprotective responses in the retina. a2-Selective adrener- gic agonists (a2-SAs) mediate the expression of bFGF mRNA

Research paper thumbnail of Displaced retinal ganglion cells in albino and pigmented rats

Frontiers in Neuroanatomy, 2014

Research paper thumbnail of Whole number and spacial distribution of the POU4f family of transcription factors in the adult rat retina

Research paper thumbnail of Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: A comparative study

Vision Research, 2009

We examined qualitatively and quantitatively in adult rat retinas the temporal degeneration of th... more 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.

Research paper thumbnail of Whole Number, Distribution and Co-Expression of Brn3 Transcription Factors in Retinal Ganglion Cells of Adult Albino and Pigmented Rats

PLoS ONE, 2012

The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Br... more 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.

Research paper thumbnail of Death and neuroprotection of retinal ganglion cells after different types of injury

Neurotoxicity Research, 2000

Research paper thumbnail of Effect of olfactory ensheathing glia clone TEG3 on neuroprotection, microglial activation and gene expression profile of allotransplanted retina with and without immunosuppression

Research paper thumbnail of Tnfr1 Death-Signalling Pathway Is Regulated in the Retina Both After Optic Nerve Transection and Optic Nerve Crush

Investigative Ophthalmology & Visual Science, 2009

Research paper thumbnail of Co-Relation Between Transcriptomic And Metabolomic Changes In The Axotomized Adult Rat Retina

Investigative Ophthalmology & Visual Science, 2012

Research paper thumbnail of Quantitative And Qualitative Analyses Of The Nerve Fiber Layer Degeneration After Optic Nerve Axotomy In Adult Mice

Investigative Ophthalmology & Visual Science, 2012

Research paper thumbnail of Corneal endothelial cell loss after trabeculectomy and phacoemulsification in one or two steps: a prospective study

Eye, 2021

Objective The objective of this study was to analyse the results of the surgical treatment of coe... more Objective The objective of this study was to analyse the results of the surgical treatment of coexisting cataract and glaucoma and its effects on corneal endothelial cell density (CECD). Methods We include two longitudinal prospective studies: one randomised that included 40 eyes with open angle glaucoma that received one- (n = 20) or two-step (n = 20) phacotrabeculectomy and another that included 20 eyes that received phacoemulsification. We assess the impact of surgery on different clinical variables and in particular in CECD using Confoscan 4™ confocal microscopy and semiautomatic counting methods. Results Phacoemulsification and phacotrabeculectomy, but not trabeculectomy, increase significantly best-corrected visual acuity and anterior chamber depth and trabeculectomy and one- or two-step phacotrabeculectomy decreased similarly the intraocular pressure. We document percentages of endothelial cell loss of 3.1%, 17.9%, 31.6% and 42.6% after trabeculectomy, phacoemulsification and...

Research paper thumbnail of Topical bromfenac transiently delays axotomy-induced retinal ganglion cell loss

Experimental Eye Research, 2019

Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on t... more Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on the survival of central nervous system neurons, the retinal ganglion cells (RGCs). Here we have analyzed the neuroprotective effect of topical bromfenac treatment, a nonsteroidal anti-inflammatory drug (NSAID) used in clinic to ameliorate post-operative inflammation, on axotomized rat RGCs. The left optic nerve of adult rats was subjected to optic nerve crush (ONC). Half of the rats were treated with a topical instillation of saline. On the other half, immediately after the surgery, 2 drops of bromfenac (0.09% Yellox; Bausch & Lomb) were instilled, and then every 12 h until analysis. Retinas in both groups were dissected 3, 5, 7, 9 and 14 days after ONC (n = 4-8/time point/group). Toxicity of bromfenac was assessed in intact retinas treated during 14 days (n = 6). Intact untreated retinas were used as control of the RGC population. RGCs were identified by Brn3a immunodetection and automatically quantified. Our results show that bromfenac does not cause RGC loss in intact retinas. In the injured groups, the number of RGCs at 7, 9 and 14 days after the lesion was significantly higher in treated vs. untreated retinas. To our knowledge this is the first report showing that a topical treatment with a NSAIDs delays axotomy-induced RGC loss and indicates that treatment with NSAIDs could be used as conjunctive therapy in diseases that proceed with optic nerve damage.

Research paper thumbnail of Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension

Frontiers in Neuroscience, 2017

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion ce... more Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a + RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m + RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a + RGCs, m + RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value.

Research paper thumbnail of Topical Treatment With Bromfenac Reduces Retinal Gliosis and Inflammation After Optic Nerve Crush

Investigative Opthalmology & Visual Science, 2016

PURPOSE. To study the effect of topical administration of bromfenac, a nonsteroidal antiinflammat... more PURPOSE. To study the effect of topical administration of bromfenac, a nonsteroidal antiinflammatory drug (NSAID), on retinal gliosis and levels of prostaglandin E 2 (PGE 2) after complete optic nerve crush (ONC). METHODS. Adult albino rats were divided into the following groups (n ¼ 8 retinas/group): (1) intact, (2) intact and bromfenac treatment (twice a day during 7 days), (3) ONC (7 days), and (4) ONC (7 days) þ bromfenac treatment (twice a day during 7 days). Animals from groups 3 and 4 were imaged in vivo with spectral-domain optical coherence tomography (SD-OCT) before the procedure and 15 minutes, 3, 5, or 7 days later. Retinas from all groups were analyzed by immunodetection, Western blotting, or enzyme-linked immunoabsorbent assay (ELISA). RESULTS. Quantification of Brn3a (brain-specific homeobox/POU domain protein 3A) þ RGCs (retinal ganglion cells) in cross sections showed that bromfenac treatment does not accelerate ONC-induced degeneration. Cellular retinaldehyde binding protein 1 regulation indicated that bromfenac improves retinal homeostasis in injured retinas. Spectral-domain OCT showed that the thickness of the retina and the retinal nerve fiber layer at 7 days post ONC was significantly reduced in bromfenac-treated animals when compared to untreated animals. In agreement with these data, hypertrophy of astrocytes and Müller cells and expression of glial fibrillary acidic protein and vimentin were greatly diminished by bromfenac treatment. While no changes in cyclooxygenase (COX) enzyme COX1 and COX2 expression were observed, there was a significant increase of PGE 2 after ONC that was controlled by bromfenac treatment. CONCLUSIONS. Topical administration of bromfenac is an efficient and noninvasive treatment to control the retinal gliosis and release of proinflammatory mediators that follow a massive insult to the RGC population.

Research paper thumbnail of Ketorolac Administration Attenuates Retinal Ganglion Cell Death After Axonal Injury

Investigative Opthalmology & Visual Science, 2016

PURPOSE. To assess the neuroprotective effects of ketorolac administration, in solution or delive... more PURPOSE. To assess the neuroprotective effects of ketorolac administration, in solution or delivered from biodegradable microspheres, on the survival of axotomized retinal ganglion cells (RGCs). METHODS. Retinas were treated intravitreally with a single injection of tromethamine ketorolac solution and/or with ketorolac-loaded poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Ketorolac treatments were administered either 1 week before optic nerve crush (pre-ONC) or right after the ONC (simultaneous). In all cases, animals were euthanized 7 days after the ONC. As control, nonloaded microspheres or vehicle (balanced salt solution, BSS) were administered in parallel groups. All retinas were dissected as flat mounts; RGCs were immunodetected with brain-specific homeobox/POU domain protein 3A (Brn3a), and their number was automatically quantified. RESULTS. The percentage of Brn3a þ RGCs was 36% to 41% in all control groups (ONC with or without BSS or nonloaded microparticles). Ketorolac solution administered pre-ONC resulted in 63% survival of RGCs, while simultaneous administration promoted a 53% survival. Ketorolac-loaded microspheres were not as efficient as ketorolac solution (43% and 42% of RGC survival pre-ONC or simultaneous, respectively). The combination of ketorolac solution and ketorolac-loaded microspheres did not have an additive effect (54% and 55% survival pre-ONC and simultaneous delivery, respectively). CONCLUSIONS. Treatment with the nonsteroidal anti-inflammatory drug ketorolac delays RGC death triggered by a traumatic axonal insult. Pretreatment seems to elicit a better output than simultaneous administration of ketorolac solution. This may be taken into account when performing procedures resulting in RGC axonal injury.

Research paper thumbnail of Long-Term Effect of Optic Nerve Axotomy on the Retinal Ganglion Cell Layer

Investigative ophthalmology & visual science, 2015

To analyze the long-term effect of optic nerve injury on retinal ganglion cells (RGCs) and melano... more 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. 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. Orthotopic and displaced RGCs behave similarly within the RGC and m+RGC populations. Both lesions cause an exponential loss of RGCs (4%-1%...

Research paper thumbnail of Comparison of Retinal Nerve Fiber Layer Thinning and Retinal Ganglion Cell Loss After Optic Nerve Transection in Adult Albino Rats

Investigative Opthalmology & Visual Science, 2015

PURPOSE. We compared the time-course and magnitude of retinal nerve fiber layer (RNFL) thinning w... more 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, Brn3a þ 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.

Research paper thumbnail of Temporal response of the phagocytic microglia in the axotomized rat retina: optic nerve crush vs. transection

Acta Ophthalmologica, 2014

Purpose To analyze the appearance of phagocytic microglial cells (PMC) in rat retinas after optic... more Purpose To analyze the appearance of phagocytic microglial cells (PMC) in rat retinas after optic nerve crush or transection.Methods Retinal ganglion cells (RGCs) from adult Sprague Dawley rats were traced with fluorogold applied to both superior colliculi. One week later, the left optic nerve was either crushed (ONC, n=32) or transected (ONT, n=32). Rats were euthanized at 2, 5, 9 and 14 days after the axotomy (n=8/group). Retinas were dissected as flatmounts and Brn3a immunodetected to identify RGCs. Retinas were photographed for each signal. Brn3a+RGCs were automatically quantified and their distribution visualized by isodensity maps. The position of each PMC found in the ganglion cell layer (identified by their transcellularly labelling with fluorogold) was dotted on the retinal photomontages. These dots were counted and graphically represented with neighbour maps.Results PMCs were observed at 2 days(43±8, ONT; 11±5, ONC). Their number increased quicker after ONT than after ONC ...

Research paper thumbnail of Measurement of retinal injury in the rat after optic nerve transection: an RT-PCR study

Molecular vision, Jan 2, 2005

In the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to p... more In the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to provide information on retinal ganglion cell (RGC) injury and survival after optic nerve transection (ONT). The levels of mRNAs synthesized by RGCs and glial components were initially measured at defined time points after ONT. Subsequently, a comparison was made between the levels of these mRNAs in the ONT retinas of rats treated with the neuroprotectant BDNF and in rats which received vehicle. Wistar rats received an ONT in one eye, while the fellow eye served as a control. ONT was performed 1-2 mm from the optic disc without damaging the retinal blood supply. In the first experiment, rats were killed at 1, 3, 5, 7, and 21 days after ONT. In the second experiment, brain derived neurotrophic factor (BDNF; 5 microg) or vehicle was injected intravitreally at the same time as the ONT and animals were killed after 7 days. After ONT, mRNA levels of RGC markers (NF-L and Thy-1) decreased substa...

Research paper thumbnail of Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death

Investigative ophthalmology & visual science, 2001

To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-... more To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA re...

Research paper thumbnail of Neuroprotective Effects of a2Selective Adrenergic Agonists against Ischemia-Induced Retinal Ganglion Cell Death

determined by the duration of the period of ischemia.18,19 The a2-adrenergic receptors are G prot... more determined by the duration of the period of ischemia.18,19 The a2-adrenergic receptors are G protein- coupled recep- tors localized in the retina within the RGC layer, inner nuclear layer, and inner segments of the photoreceptors.20 -22 Recent evidence indicates that retinal a2-adrenergic receptors mediate neuroprotective responses in the retina. a2-Selective adrener- gic agonists (a2-SAs) mediate the expression of bFGF mRNA

Research paper thumbnail of Displaced retinal ganglion cells in albino and pigmented rats

Frontiers in Neuroanatomy, 2014

Research paper thumbnail of Whole number and spacial distribution of the POU4f family of transcription factors in the adult rat retina

Research paper thumbnail of Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: A comparative study

Vision Research, 2009

We examined qualitatively and quantitatively in adult rat retinas the temporal degeneration of th... more 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.

Research paper thumbnail of Whole Number, Distribution and Co-Expression of Brn3 Transcription Factors in Retinal Ganglion Cells of Adult Albino and Pigmented Rats

PLoS ONE, 2012

The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Br... more 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.

Research paper thumbnail of Death and neuroprotection of retinal ganglion cells after different types of injury

Neurotoxicity Research, 2000