[Show abstract][Hide abstract] ABSTRACT: 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.
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% 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.
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.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effect of retrograde tracing or axotomy on melanopsin mRNA expression and immunodetection in albino and pigmented rat retinas.
Groups were (1) intact-naïve retinas; (2) optic nerve crush (ONC) analyzed at 7 days (7d) or 2 months (2m); (3) Fluorogold (FG) tracing from the superior colliculi (SCi) analyzed at 7d or 2m; (4) tracing from the intact optic nerve (ON) with FG or hydroxystilbamidine methanesulfonate (OHSt), analyzed 3d later; and (5) sham tracing from the ON or sham surgery. Brn3a and melanopsin were double stained in whole mounts to quantify and assess the distribution of orthotopic and displaced Brn3a+ retinal ganglion cells (Brn3a+RGCs) and melanopsin+RGCs (m+RGCs). Freshly dissected retinas were used for melanopsin mRNA quantitative PCR.
Tracing from the SCi did not affect the number of Brn3a+RGCs or m+RGCs counted in pigmented rats. However, only 55% of m+RGCs were immunodetected in albinos at 7d, although by 2m the m+RGCs counts returned to normal. Optic nerve tracing had a more dramatic effect (38% or 77% of m+RGCs were immunodetected in albino or pigmented rats) that occurred irrespectively of the tracer (OHSt or FG). This effect was not observed in the sham groups. After ONC, Brn3a+RGCs decreased to 37% and 8% by 7d and 2m, respectively. Melanopsin +RGC counts diminished to 30% at 7d, but recovered to 49% of controls by 2m. Melanopsin mRNA was downregulated after ON tracing or 7d after ONC, but did not differ from intact values 2m after ONC.
Following ON injury or retrograde tracing there is a transient melanopsin downregulation that should be taken into account when assessing m+RGC survival.
[Show abstract][Hide abstract] ABSTRACT: 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.
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 μm 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.
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+ 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.
After IONT, RGC death is more severe and precedes thinning of the RNFL.
[Show abstract][Hide abstract] ABSTRACT: To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) extracted fresh to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (inmunolabeld). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a+RGCs but with large numbers of DAPI+nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.
Experimental Eye Research 01/2015; 14. DOI:10.1016/j.exer.2015.01.006 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.
PLoS ONE 12/2014; 9(12):e113798. DOI:10.1371/journal.pone.0113798 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied in parallel the population of displaced retinal ganglion cells (dRGCs) and normally placed (orthotopic RGCs, oRGCs) in albino and pigmented rats. Using retrograde tracing from the optic nerve, from both superior colliculi (SC) or from the ipsilateral SC in conjunction with Brn3 and melanopsin immunodetection, we report for the first time their total number and topography as well as the number and distribution of those dRGCs and oRGCs that project ipsi- or contralaterally and/or that express any of the three Brn3 isoforms or melanopsin. The total number of RGCs (oRGCs+dRGCs) is 84,706±1,249 in albino and 90,440±2,236 in pigmented, out of which 2,383 and 2,428 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 hypertension. Although the role of mammalian dRGCs remains to be determined, our data suggest that they are not misplaced by an ontogenic mistake.
Frontiers in Neuroanatomy 10/2014; 8:99. DOI:10.3389/fnana.2014.00099 · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We purpose here to analyze and compare the population and topography of cone photoreceptors in two mouse strains using automated routines, and to design a method of retinal sampling for their accurate manual quantification. In whole-mounted retinas from pigmented C57/BL6 and albino Swiss mice, the longwave-sensitive (L) and the shortwave-sensitive (S) opsins were immunodetected to analyze the population of each cone type. In another group of retinas both opsins were detected with the same fluorophore to quantify all cones. In a third set of retinas, L-opsin and Brn3a were immunodetected to determine whether L-opsin+cones and retinal ganglion cells (RGCs) have a parallel distribution. Cones and RGCs were automatically quantified and their topography illustrated with isodensity maps. Our results show that pigmented mice have a significantly higher number of total cones (all-cones) and of L-opsin+cones than albinos which, in turn, have a higher population of S-opsin+cones. In pigmented animals 40% of cones are dual (cones that express both opsins), 34% genuine-L (cones that only express the L-opsin), and 26% genuine-S (cones that only express the S-opsin). In albinos, 23% of cones are genuine-S and the proportion of dual cones increases to 76% at the expense of genuine-L cones. In both strains, L-opsin+cones are denser in the central than peripheral retina, and all-cones density increases dorso-ventrally. In pigmented animals S-opsin+cones are scarce in the dorsal retina and very numerous in the ventral retina, being densest in its nasal aspect. In albinos, S-opsin+cones are abundant in the dorsal retina, although their highest densities are also ventral. Based on the densities of each cone population, we propose a sampling method to manually quantify and infer their total population. In conclusion, these data provide the basis to study cone degeneration and its prevention in pathologic conditions.
PLoS ONE 07/2014; 9(7):e102392. DOI:10.1371/journal.pone.0102392 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the cause of retinal ganglion cell (RGC) loss in dystrophic aged Royal College of Surgeons (RCS) rats.
RCS-p+ (dystrophic) female rats of postnatal times (P365, P450 and P540) and age-matched RCS-p1 rdy+ (non-dystrophic) rats were used. In whole-mounted retinas, RGCs were doubly labelled with Fluorogold (FG) retrogradely transported from the superior colliculi and Brn3a immunohistochemistry. RGC axons were labelled with anti-neurofilament antibodies. Automatic image analysis techniques allowed quantification of the total population of RGCs per retina and construction of isodensity maps to investigate RGC topology.
Dystrophic retinas showed at all times studied wedge-shaped sectors devoid of FG(+) and Brn3a(+) RGCs. These sectors were also devoid of neurofilament-labelled axons. The total number of FG(+)RGC and Brn3a(+)RGC per retina was significantly smaller in dystrophic rats at P540, revealing RGC death at this age. The total number of FG(+)RGCs was smaller than those of Brn3a(+)RGCs at P540, indicating a disturbance of the retrograde axonal transport at this age.
RGC double labelling documents that sectorial RGC loss in aged dystrophic RCS rats is mainly due to RGC death, although a deficit of the retrograde axonal transport exists also at the more advanced ages.
The British journal of ophthalmology 12/2013; 98(3). DOI:10.1136/bjophthalmol-2013-303958 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To identify metabolic pathways and metabolites affected by optic nerve crush that can act as predictors of the disease or therapeutic targets.
The left optic nerve of adult rats was intraorbitally crushed and retinas were dissected 24 hours or 14 days after the lesion (n = 10 per group). Metabolic profiling analysis was carried out by Metabolon, Inc. A total of 195 metabolites were unambiguously detected. Data were normalized and the regulated metabolites were identified after comparing the different conditions. Metabolite concentration changes were analyzed using single and multivariate statistical analysis to detect discriminatory metabolites. Functional clustering and meta-analysis of the regulated metabolites was run through the Metacore platform.
Comparison of 24 hours versus control, 14 days versus control samples, and 24 hours versus 14 days identified 9, 19, and 32 regulated metabolites, respectively. Single and multivariate analysis identified a total of 27 and 36 metabolites to discriminate between control and 14 days and between 24 hours and 14 days, respectively. Enrichment analysis showed alterations in the amino acid, carbohydrate, and lipid metabolism, which were further linked to translation, oxidative stress, energy (glucose and tricarboxylic acid cycle), and apoptosis through ceramide pathways.
Our analysis differentiates a set of metabolites that clearly discriminate control and early-injury samples from late-injury samples. These metabolites could have potential use as diagnostic molecules.
[Show abstract][Hide abstract] ABSTRACT: Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs -identified by their Brn3a expression- and the population of ipRGCs -identified by melanopsin immunodetection- thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard desviation of ipRGCs in the albino rat is 2,047±309. Their distribution in the retina seems to be complementary to that of Brn3a(+)RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.
Experimental Eye Research 01/2013; 108. DOI:10.1016/j.exer.2012.12.010 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory’s previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.
[Show abstract][Hide abstract] ABSTRACT: The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.
PLoS ONE 11/2012; 7(11):e49830. DOI:10.1371/journal.pone.0049830 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory's previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.
Progress in Retinal and Eye Research 09/2011; 31(1):1-27. DOI:10.1016/j.preteyeres.2011.08.001 · 8.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor Brn3a has been reported to be a good marker for adult rat retinal ganglion cells in control and injured retinas. However, it is still unclear if Brn3a expression declines progressively by the injury itself or otherwise its expression is maintained in retinal ganglion cells that, though being injured, are still alive, as might occur when assessing neuroprotective therapies. Therefore, we have automatically quantified the whole population of surviving Brn3a positive retinal ganglion cells in retinas subjected to intraorbital optic nerve transection and treated with either brain derived neurotrophic factor or vehicle. Brain derived neurotrophic factor is known to delay retinal ganglion cell death after axotomy. Thus, comparison of both groups would inform of the suitability of Brn3a as a retinal ganglion cell marker when testing neuroprotective molecules. As internal control, retinal ganglion cells were, as well, identified in all retinas by retrogradely tracing them with fluorogold. Our data show that at all the analyzed times post-lesion, the numbers of Brn3a positive retinal ganglion cells and of fluorogold positive retinal ganglion cells are significantly higher in the brain derived neurotrophic factor-treated retinas compared to the vehicle-treated ones. Moreover, detailed isodensity maps of the surviving Brn3a positive retinal ganglion cells show that a single injection of brain derived neurotrophic factor protects retinal ganglion cells throughout the entire retina. In conclusion, Brn3a is a reliable retinal ganglion cell marker that can be used to accurately measure the potential effect of a given neuroprotective therapy.
Experimental Eye Research 02/2011; 92(4):260-7. DOI:10.1016/j.exer.2011.02.001 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fate of retinal ganglion cells after optic nerve injury has been thoroughly described in rat, but not in mice, despite the fact that this species is amply used as a model to study different experimental paradigms that affect retinal ganglion cell population. Here we have analyzed, quantitatively and topographically, the course of mice retinal ganglion cells loss induced by intraorbital nerve transection. To do this, we have doubly identified retinal ganglion cells in all retinas by tracing them from their main retinorecipient area, the superior colliculi, and by their expression of BRN3A (product of Pou4f1 gene). In rat, this transcription factor is expressed by a majority of retinal ganglion cells; however in mice it is not known how many out of the whole population of these neurons express it. Thus, in this work we have assessed, as well, the total population of BRN3A positive retinal ganglion cells. These were automatically quantified in all whole-mounted retinas using a newly developed routine. In control retinas, traced-retinal ganglion cells were automatically quantified, using the previously reported method (Salinas-Navarro et al., 2009b). After optic nerve injury, though, traced-retinal ganglion cells had to be manually quantified by retinal sampling and their total population was afterwards inferred. In naïve whole-mounts, the mean (±standard deviation) total number of traced-retinal ganglion cells was 40,437(±3196) and of BRN3A positive ones was 34,697(±1821). Retinal ganglion cell loss was first significant for both markers 5 days post-axotomy and by day 21, the last time point analyzed, only 15% or 12% of traced or BRN3A positive retinal ganglion cells respectively, survived. Isodensity maps showed that, in control retinas, BRN3A and traced-retinal ganglion cells were distributed similarly, being densest in the dorsal retina along the naso-temporal axis. After axotomy the progressive loss of BRN3A positive retinal ganglion cells was diffuse and affected the entire retina. In conclusion, this is the first study assessing the values, in terms of total number and density, of the retinal ganglion cells surviving axotomy from 2 till 21 days post-lesion. Besides, we have demonstrated that BRN3A is expressed by 85.6% of the total retinal ganglion cell population, and because BRN3A positive retinal ganglion cells show the same spatial distribution and temporal course of degeneration than traced ones, BRN3A is a reliable marker to identify, quantify and assess, ex-vivo, retinal ganglion cell loss in this species.
Experimental Eye Research 02/2011; 92(5):377-87. DOI:10.1016/j.exer.2011.02.008 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE. To quantify the whole population of S-and L-cones in the albino (Sprague-Dawley, SD) and pigmented (Piebald Virol Glaxo, PVG) rats and to study their topographical distribution within the retina. METHODS. Retinal radial sections and whole-mounted retinas were double immunodetected with antibodies against UV-sen-sitive and L-opsins to detect the S-and L-cones, respectively. Two automated routines were developed to quantify the whole population of S-and L-cones. Detailed isodensity maps of each cone type were generated. In both strains, the presence of dual cones was detected, these were semiautomatically quantified and their distribution determined. The matching distribution of retinal ganglion cells (RGCs) and L-cones was attained by dou-ble immunodetection of Brn3a and L-opsin, respectively RESULTS. The mean number Ϯ SEM of L-or S-cones in SD and PVG retinas was 231,736 Ϯ 14,517 and 239,939 Ϯ 6,494 or 41,028 Ϯ 5,074, and 27,316 Ϯ 2,235, respectively. There was an increasing gradient of S-cone density along the inferonasal quadrant, although the highest densities were found in the retinal rims. The distribution of L-cones seemed to be comple-mentary to the S-cones. The highest densities were observed in the superior nasotemporal axis, paralleling the distribution of Brn3a-positive RGCs. CONCLUSIONS. These data establish, for the first time, the total number and the topographical distribution of S-and L-cones in two rat strains and demonstrate the correlation of L-cones and RGC spatial distribution. (Invest Ophthalmol Vis Sci. 2010;51: 3171–3183) DOI:10.1167/iovs.09-4861 I n the mammalian retina, cone photoreceptors are responsi-ble for daylight (photopic) vision and color discrimination. Color discrimination in nonprimate mammals is achieved by two types of cones: S and M/L. These cones are distinguished mainly by the portion of light spectrum to which each is more sensitive: short wave lengths stimulate S-cones and medium to long wave lengths stimulate M/L-cones. This spectral sensitivity is conferred by the protein part of the visual pigments, the opsin. Thus, opsins are slightly different, depending on their sensitivity and color preference. Each cone type carries a specific opsin. In rodents, S-cones express the ultraviolet-sen-sitive or SWS1 opsin, 1,2 and M/L-cones express the LWS opsin, 3