March 2015
Volume 56, Issue 3
Free
Glaucoma  |   March 2015
BDNF Rescues RGCs But Not Intrinsically Photosensitive RGCs in Ocular Hypertensive Albino Rat Retinas
Author Affiliations & Notes
  • Francisco J. Valiente-Soriano
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Francisco M. Nadal-Nicolás
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Manuel Salinas-Navarro
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Manuel Jiménez-López
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Jose M. Bernal-Garro
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Maria P. Villegas-Pérez
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Marta Agudo-Barriuso
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Manuel Vidal-Sanz
    Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, and Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain
  • Correspondence: Manuel Vidal-Sanz, Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, IMIB-Arrixaca, Campus de Espinardo, 30100 Murcia, Spain; manuel.vidal@um.es
  • Footnotes
     FJV-S and FMN-N are joint first authors.
Investigative Ophthalmology & Visual Science March 2015, Vol.56, 1924-1936. doi:10.1167/iovs.15-16454
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      Francisco J. Valiente-Soriano, Francisco M. Nadal-Nicolás, Manuel Salinas-Navarro, Manuel Jiménez-López, Jose M. Bernal-Garro, Maria P. Villegas-Pérez, Marta Agudo-Barriuso, Manuel Vidal-Sanz; BDNF Rescues RGCs But Not Intrinsically Photosensitive RGCs in Ocular Hypertensive Albino Rat Retinas. Invest. Ophthalmol. Vis. Sci. 2015;56(3):1924-1936. doi: 10.1167/iovs.15-16454.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Glaucoma is among the leading causes of blindness in developed countries.1 An elevated intraocular pressure (IOP) above normal levels is one of the most important risk factors and the only one that may be treated. Current therapy is devoted to controlling and/or diminishing ocular hypertension (OHT); however, in an important proportion of glaucoma patients, retinal ganglion cell (RGC) loss, the hallmark of glaucoma, keeps progressing with concomitant visual deficits that eventually lead to blindness. This has prompted investigators to look for alternative treatments that could prevent or slow cell death with neuroprotective drugs. 
To study the pathology of human glaucoma, a number of OHT models have been developed in rodents, taking advantage of the anatomy of the aqueous humor draining system.2 These include episcleral vein cauterization,3 hypertonic saline injection into episcleral radial veins,4 injections of microbeads or viscoelastics into the anterior chamber alone or in combination,5,6 and laser photocoagulation (LP) of the limbal tissues.7,8 Laser photocoagulation results in a number of features that make this model relevant to further our knowledge of the pathology of OHT-induced retinal degeneration and has been the method of choice in our laboratory to induce OHT in adult albino rats912 and in adult albino1318 or pigmented19,20 mice. Laser photocoagulation induces diffuse and sectorial loss of RGCs, early damage to RGC axons at the level of optic nerve head, and a protracted degeneration of RGC somas.10,13,2125 Moreover, LP of one eye results in important glial alterations in the contralateral retina,11,1517 and with time, OHT retinas develop alterations in the outer retina.12,13 There are, however, several issues that remain unresolved: (1) OHT-induced damage to the optic nerve head could explain the typical pie-shaped sectorial loss of RGCs, but whether the retinal vasculature also plays a role in this sectorial loss is presently unknown; (2) OHT-induced RGC degeneration in rodents can be prevented with the well-known neuroprotective agent brain-derived neurotrophic factor (BDNF),26 but whether injured but alive RGCs are amenable to neuroprotection is not known; and (3) most of the above-mentioned studies in rats report the responses of RGCs against OHT without making distinctions between different types of RGCs—thus it is of interest to study whether OHT elicits the same responses in all types of RGCs. 
Retinal ganglion cells can be protected against retinal injuries with neuroprotective agents,27,28 among which BDNF has been shown to be one of the most potent,2931 although its effect is transient and best observed during the first 2 weeks after injury.3234 Indeed, exogenous administration of BDNF alone26,3538 or in combination with lingo-1 antagonist39 has proven effective in preventing acute or chronic OHT-induced RGC loss. Here we further investigate the neuroprotective effects of BDNF on the survival of axonally intact and axonally nonintact RGCs, as well as on the general population of RGCs versus the population of intrinsically photosensitive RGCs. 
Non–image-forming visual behaviors depend on intrinsically photosensitive RGCs (ipRGCs), one class of RGC that contains the photopigment melanopsin (m+RGCs). Intrinsically photosensitive RGCs are responsible for the circadian photoentrainment, pupillary reflexes, and the regulation of pineal melatonin secretion.4044 In adult rodents, m+RGCs constitute approximately 2% to 3% of all RGCs (2.5% and 2.1% for pigmented and albino mice, respectively,44,45 and 2.5% and 2.7% for pigmented and albino rats, respectively46–48). There is evidence that in human glaucomatous patients, a number of non–visual-forming functions mediated by the ipRGC system are altered.4952 Several reports using experimental models of OHT have shown that the non–image-forming visual functions are affected, with important changes in the circadian timing system,5355 as well as in the locomotor activity and behavioral patterns,55 all of which are associated with dysfunctional ipRGCs. Moreover, there are some discrepancies regarding the survival of m+RGCs after OHT; while some studies have reported a better survival in rats55,56 and mice,57 others indicate the opposite for mice20 and rats.53,54,58 Furthermore, the effects of OHT on the topological distribution of m+RGCs and whether m+RGCs are responsive to neuroprotective strategies are presently unknown. 
In the present studies, using modern techniques to identify and map in the same retinal whole mounts the general population of RGCs (non–melanopsin-expressing RGCs, which can be immunodetected with Brn3a) and the subpopulation of RGCs expressing melanopsin (m+RGCs)4648 as well as to visualize the entire inner retinal arterial vasculature, we have compared for the first time the responses of non-m+RGCs to those of m+RGCs to OHT-induced retinal degeneration and neuroprotection afforded by BDNF. We further investigate the neuroprotective effects of BDNF on the survival of axonally intact and axonally nonintact RGCs. Finally, the inner retinal vasculature was examined to look for retinal vessel abnormalities that could account for the typical sectorial RGC loss that follows OHT. Our results indicate that the responses of m+RGCs against OHT-induced retinal degeneration and neuroprotection differ from those of the general population of non-m+RGCs (a short account of this work has been reported in Abstract form59). 
Materials and Methods
Animal Handling
All experiments were carried out following the Spanish and European Union regulations for the use of animals in research (Council Directive 86/609/EEC) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. This study was approved by the Ethics Committee for Animal Research of the University of Murcia (Spain). 
Adult female albino Sprague-Dawley (SD) rats (180–230 g) obtained from Charles River Laboratories (L'Arbresle, France) were housed at the University of Murcia animal facilities in temperature- and light-controlled rooms (12-hour light/dark cycle) with food and water ad libitum. 
All surgical manipulations were carried out under general anesthesia induced with an intraperitoneal (IP) injection of a mixture of ketamine (70 mg/kg, Ketolar; Parke-Davies, S.L., Barcelona, Spain) and xylazine (10 mg/kg, Rompun; Bayer, S.A., Barcelona, Spain), and all efforts were made to minimize suffering. Oral analgesia was provided from the first day to the day of euthanasia.60 During recovery from anesthesia, an ocular ointment (Tobrex; Alcon Cusí, S.A., Barcelona, Spain) was applied on the cornea to prevent corneal desiccation. Animals were euthanized with an IP injection of an overdose of pentobarbital (Dolethal Vetoquinol; Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain). 
Experimental Design
To investigate whether RGCs could be rescued from OHT-induced retinal degeneration, in one main group of rats, BDNF (n = 24) or vehicle (n = 21) was injected intravitreally and the retinas were examined 12 or 15 days later. These time intervals were chosen because BDNF has been shown to have maximal effects during the first 15 days after optic nerve injury.2931 The responses of m+RGCs were investigated in 16 retinas from this group, examined at 12 or 15 days after OHT. Because only a minute proportion (0.2%) of m+RGCs express Brn3a,46,48,61 in these retinas melanopsin and Brn3a were doubly immunodetected, an approach that allows one to study in parallel, but independently, the general RGC population (nonintrinsically photosensitive Brn3a+) and the population of ipRGCs (m+RGCs).20,4547 Finally, to investigate if OHT had an effect on the inner retinal vasculature that could be responsible for the geographical sectorial pattern of RGC loss, four retinas from the main group were examined 15 days after intravitreal administration of BDNF or vehicle and induction of OHT. The population of surviving RGCs was identified with Fluorogold and Brn3a, while the retinal vessels were identified with RECA1 antibodies. An additional group of untouched naïve retinas (n = 6) served as control for the normal RGC population retrogradely labeled with Fluorogold, or immunostained with Brn3a, and for the normal appearance of the inner retinal vessels. The experimental design is outlined in Table 1
Table 1
 
Experimental Design
Table 1
 
Experimental Design
Intravitreal Administration of Brain-Derived Neurotrophic Factor
Right before lasering, 5 μL BDNF (Peprotech Laboratories, London, UK), diluted at 1 μg/μL in 1% bovine serum albumin-phosphate buffer saline (n = 24) or 5 μL vehicle (n = 21), was intravitreally injected into the left eyes as previously described in detail.27,30,3234 
Induction of OHT
To induce OHT, the left eyes were treated in a single session with diode laser burns (Viridis Ophthalmic Photocoagulator-532 nm laser; Quantel Medical, Clermont-Ferrand, France). The laser beam was delivered directly, without any lenses, aimed at the trabecular meshwork and the limbal and episcleral veins. The pupil of the treated eye was dilated with 1% tropicamide (Colircusi tropicamida 1%; Alcon-Cusí, S.A., Barcelona, Spain), and an average of 95 laser burns were administered per eye; the spot size, duration, and power were 50 to 100 μm, 0.5 seconds, and 0.4 W, respectively, as previously described in detail.12,13,24 Right eyes were kept as control. 
Measurement of the Intraocular Pressure
The IOP of both eyes was measured under anesthesia using a rat-adapted rebound tonometer (Tono-Lab; Tiolat, OY, Helsinki, Finland). The readings were obtained before LP and at 24 and 48 hours and 7 days after LP as described previously in detail.10,24 
Retrograde Labeling From the Superior Colliculi
To identify RGCs with a competent retrograde axonal transport in OHT retinas, Fluorogold (FG; Fluorochrome, Inc., Englewood, CO, USA) was applied to both superior colliculi (SCi) 1 week before euthanasia, following previously described methods that are standard in our laboratory.10,6264 In brief, after exposing the midbrain, a small piece of gelatin sponge (Espongostan Film; Ferrosan A/S, Soeborg, Denmark) soaked in saline containing 3% FG diluted in 10% dimethyl sulphoxide–saline was applied over the entire surface of both SCi. A similar procedure was used for the control group of naïve retinas to identify the RGC population. 
Tissue Processing: Retinal Whole Mounts
Rats were deeply anesthetized and perfused transcardially with saline and 4% paraformaldehyde in 0.1 M phosphate buffer. The eyes were enucleated, and both retinas were dissected and prepared as flattened whole mounts, maintaining retinal orientation by making four radial cuts (the deepest one signals the superior pole of the eye) as previously described.46,47,62,63 
Immunohistofluorescence
Immunodetection was carried out following previously described methods.20,34,4648,6568 In brief, after permeation, retinas were incubated overnight with the primary antibodies diluted in blocking buffer (PBS, 2% normal donkey serum, 2% Triton X-100). After washing in PBS, retinas were incubated at room temperature for 2 hours with the appropriate secondary antibodies diluted in PBS 0.5% Triton X-100. Finally, retinas were washed in PBS and mounted vitreal side up on slides and covered with antifading solution. 
The primary antibodies and dilutions used in this study were as follows: 
  1.  
    The general population of RGCs was detected using goat anti-Brn3a (1:500, C-20; Santa Cruz Biotechnologies, Heidelberg, Germany)46,47,68;
  2.  
    Melanopsin-expressing RGCs were detected using the rabbit anti-melanopsin PAI-780 (1:500; Thermo Scientific, Madrid, Spain)47,48 that detects the NH2 terminal of the melanopsin protein and thus both melanopsin isoforms (short and long46–48,69). Melanopsin immunodetection identifies the M1, M2, and M3 ipRGCs subtypes, because the M4 and M5 subtypes are not stained with anti-melanopsin antibodies70; and
  3.  
    Retinal vessels were identified with mouse anti-rat RECA1 (clone HIS52) that recognizes the rat endothelial cell antigen 1 (diluted 1:1000; MCA970GA Serotec, Bionova Scientific, Madrid, Spain).
As secondary antibodies we used donkey anti-goat DyLight 594, donkey anti-rabbit DyLight 488 (Jackson Immunoresearch, Suffolk, UK), and donkey anti-mouse Alexa Fluor 488 (Molecular Probes, ThermoFisher, Madrid, Spain). All were used at 1:500 dilution. 
Retinal Analysis
Retinal photographs were made to reconstruct retinal whole mounts following previously described procedures that are standard in our laboratory,45,48,62,68,71 using an epifluorescence microscope (Axioscop 2 Plus; Zeiss Mikroskopie, Jena, Germany) equipped with a computer-driven motorized stage (ProScan H128 Series; Prior Scientific Instruments, Cambridge, UK) controlled by image analysis software (Image-Pro Plus, IPP 5.1 for Windows; Media Cybernetics, Silver Spring, MD, USA). Each reconstructed whole mount was composed of 154 individual frames captured side by side with no gap or overlap between them with a ×10 objective (Plan-Neofluar, 20/0.50; Zeiss Mikroskopie). When required, images were further processed using a graphics editing program (Adobe Photoshop CS 8.0.1; Adobe Systems, Inc., San Jose, CA, USA). 
Automated Quantification and Spatial Distribution of RGCs
Fluorogold+RGCs and Brn3a+RGCs were counted automatically while m+RGCs were counted manually, and their topography was studied according to previously described methods.20,34,4548,62,68 
Retinal Vessel Analysis
The distribution of the inner retinal vessels was studied in naïve control as well as in OHT retinas to investigate whether OHT provoked anomalies in the retinal vasculature that could be responsible for the typical sectorial RGC loss found in these retinas. To facilitate the study of the inner retinal vessels, RECA1 signal was transformed in a black and white image, whereby the positive signal was detected as black and the negative signal (background) was detected as white. For that, an automatic IPP macro language routine was developed to highlight the vascular net enabling the qualitative evaluation of the retinas. In a first step, background variations were evened out by applying a Flatten Background correction filter; then the images were converted to 16-bit grayscale to discard color information. This step was followed by the application of the convolution filter HiGauss (7 × 7). The resultant image data were then filtered with a Sharpen (5 × 5) filter, which enhances fine detail using the unsharp masking technique. Finally, grayscale segmentation was performed, and a new black and white image of the highlighted vascular net was saved for further analysis. Blood vessel density was measured through a 3-mm-radius caliper centered on the optic nerve, and each interception in the perimeter of this caliper was automatically counted as a positive vessel. 
Statistics
All averaged data are presented as means with standard deviations (SD). Statistical analysis was done using SigmaStat 3.1 for Windows (SigmaStat for Windows Version 3.11; Systat Software, Inc., Richmond, CA, USA). Kruskal-Wallis was used when comparing more than two groups and Mann-Whitney test when comparing two groups only. Differences were considered significant when P < 0.05. 
Results
Induction of Ocular Hypertension
In Figure 1 is shown the evolution of the IOPs after a single session of LP in BDNF-treated (BDNF) and vehicle-treated (VEHI) groups of rats. Intraocular pressure peaked at 24 hours after LP, and the values kept statistically increasing up to 7 days compared to those of the right eyes, which remained basal during the entire study. There were no differences in the IOP values between the BDNF- and VEHI-treated groups. 
Figure 1
 
Intraocular pressure values. Graph showing the temporal evolution of the intraocular pressure in the left experimental eye (LE) and right control eye (RE) in BDNF and vehicle groups before laser photocoagulation (LP) and at 24 hours and 7 days after LP. n, number of animals analyzed.
Figure 1
 
Intraocular pressure values. Graph showing the temporal evolution of the intraocular pressure in the left experimental eye (LE) and right control eye (RE) in BDNF and vehicle groups before laser photocoagulation (LP) and at 24 hours and 7 days after LP. n, number of animals analyzed.
Effect of OHT With or Without BDNF Treatment on RGCs
In the right control retinas, FG+ and Brn3a+RGCs (Figs. 2A, 2B', 2a–d) showed normal distribution; they were denser in the medial and central retina and sparser in the retinal periphery, with higher densities in the visual streak located in the dorsal retina above the optic nerve, as previously reported.62,68,72 Moreover, the total numbers of FG+ or Brn3a+RGCs counted automatically were comparable to previously reported data from this laboratory.12,62,64 Melanopsin+RGCs, however, are preferentially found in the retinal periphery (Figs. 2C, 2C', 2e, 2f) with a topography complementary to that of the conventional population of RGCs that do not express melanopsin, identified here by Brn3a immunodetection, and this is in agreement with recent reports.48,64 The highest densities of m+RGCs were found in the superotemporal quadrant, and total numbers of m+RGCs were also within the range of previously published data.47,48 Both Brn3a+RGCs and m+RGCs were more abundant in the dorsal than in the ventral retina, as seen in the topological maps and in the magnifications shown in Figure 2
Figure 2
 
Total numbers and spatial distribution of FG+RGCs, Brn3a+RGCs, and m+RGCs. (A) Photomontage of a naïve retina traced with FG from both superior colliculi and (B) inmunoreacted against Brn3a. Retinal ganglion cell topography is observed in the corresponding isodensity maps (A', B'). Melanopsin+RGCs are shown in (C) and their distribution is visualized with a neighbor map in (C'). Bottom left of each map: number of RGCs counted in that retina. Magnifications from the dorsal (a, c, e) or the ventral (b, d, f) retina showing FG, Brn3a, and melanopsin RGCs. Color scale for isodensity maps is shown in (B') and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C') and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V, ventral; T, temporal; N, nasal.
Figure 2
 
Total numbers and spatial distribution of FG+RGCs, Brn3a+RGCs, and m+RGCs. (A) Photomontage of a naïve retina traced with FG from both superior colliculi and (B) inmunoreacted against Brn3a. Retinal ganglion cell topography is observed in the corresponding isodensity maps (A', B'). Melanopsin+RGCs are shown in (C) and their distribution is visualized with a neighbor map in (C'). Bottom left of each map: number of RGCs counted in that retina. Magnifications from the dorsal (a, c, e) or the ventral (b, d, f) retina showing FG, Brn3a, and melanopsin RGCs. Color scale for isodensity maps is shown in (B') and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C') and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V, ventral; T, temporal; N, nasal.
Twelve and 15 days after LP of the left eyes, the numbers of FG+RGCs in the experimental retinas decreased significantly (Fig. 3) and showed the typical regions lacking FG+RGCs within pie-shaped sectors of the retinas with their base located in the periphery and their apex on the optic disc (Figs. 4, 5). This finding indicates that OHT causes an impairment of the retrograde axonal transport in a large proportion of the RGC population, as previously shown.10,12 Compared to their right contralateral retinas, the percentage of RGCs that had their retrograde axonal transport impaired was 79% or 78% and 77% or 84% at 12 and 15 days after BDNF or vehicle treatment, respectively. Thus, BDNF treatment did not ameliorate axonal damage triggered by OHT, because total numbers of FG+RGCs (i.e., those that maintained a competent retrograde axonal transport) were comparable among different groups (Kruskal-Wallis test, P > 0.05) (Fig. 3). 
Figure 3
 
Brain-derived neurotrophic factor delays OHT-induced RGC death. Histogram showing the mean ± standard deviation of the total number of FG+ and Brn3a+RGCs in control retinas and in experimental retinas treated with BDNF or vehicle and analyzed at 12 or 15 days after laser-induced ocular hypertension (OHT). In all groups, the number of FG+ and Brn3a+RGCs was significantly smaller in the experimental than in the control retinas (P < 0.05, Mann-Whitney test). The number of Brn3a+RGCs was significantly greater than FG+RGCs in all groups and time points analyzed (P < 0.003, Mann-Whitney test). The number of Brn3a+RGCs was greater in BDNF-treated than in vehicle-treated animals at 12 or 15 days after LP. After BDNF treatment the number of RGCs significantly decreased from 12 to 15 days. Statistical comparisons, Mann-Whitney test, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 for all groups. n, number of analyzed retinas.
Figure 3
 
Brain-derived neurotrophic factor delays OHT-induced RGC death. Histogram showing the mean ± standard deviation of the total number of FG+ and Brn3a+RGCs in control retinas and in experimental retinas treated with BDNF or vehicle and analyzed at 12 or 15 days after laser-induced ocular hypertension (OHT). In all groups, the number of FG+ and Brn3a+RGCs was significantly smaller in the experimental than in the control retinas (P < 0.05, Mann-Whitney test). The number of Brn3a+RGCs was significantly greater than FG+RGCs in all groups and time points analyzed (P < 0.003, Mann-Whitney test). The number of Brn3a+RGCs was greater in BDNF-treated than in vehicle-treated animals at 12 or 15 days after LP. After BDNF treatment the number of RGCs significantly decreased from 12 to 15 days. Statistical comparisons, Mann-Whitney test, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 for all groups. n, number of analyzed retinas.
Figure 4
 
Spatial distribution of FG+RGC, Brn3a+RGC, and m+RGC populations at 12 or 15 days after OHT with or without BDNF treatment. Isodensity maps of FG+RGCs (A, D, G, J) show that all retinas have a sectorial and/or diffuse damage. Their corresponding isodensity maps of Brn3a+RGCs (B, E, H, K) show that there are a large number of viable RGCs in the areas lacking FG+RGCs at the times analyzed, and that they are more abundant in the BDNF-treated retinas. The neighbor maps representing m+RGCs (C, F, I, L) show that their loss is the same in BNDF- or vehicle-treated retinas. Furthermore, m+RGC loss is diffuse although more severe in the dorsal retina. Bottom left of each map: number of RGCs counted in that retina. Color scale for isodensity maps is shown in (B) and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C) and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V,ventral; T, temporal; N, nasal.
Figure 4
 
Spatial distribution of FG+RGC, Brn3a+RGC, and m+RGC populations at 12 or 15 days after OHT with or without BDNF treatment. Isodensity maps of FG+RGCs (A, D, G, J) show that all retinas have a sectorial and/or diffuse damage. Their corresponding isodensity maps of Brn3a+RGCs (B, E, H, K) show that there are a large number of viable RGCs in the areas lacking FG+RGCs at the times analyzed, and that they are more abundant in the BDNF-treated retinas. The neighbor maps representing m+RGCs (C, F, I, L) show that their loss is the same in BNDF- or vehicle-treated retinas. Furthermore, m+RGC loss is diffuse although more severe in the dorsal retina. Bottom left of each map: number of RGCs counted in that retina. Color scale for isodensity maps is shown in (B) and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C) and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V,ventral; T, temporal; N, nasal.
Figure 5
 
Study of the retinal vessels in BDNF and vehicle-treated hypertensive retinas. (A, A') A naïve retina labeled with FG and its corresponding isodensity map. (B) The RECA1+ retinal vessels in black and white. (C, D) Magnifications of the retina in (A) taken from the dorsotemporal (C) and inferotemporal (D) quadrant showing FG+RGCs (white), Brn3a+RGCs (red), and RECA-1+ vessels (green). In the naïve retina, there is a viable RGC retrograde axonal transport (RAT), and the retinal vessels irrigate properly all the areas. In hypertensive retinas treated with vehicle (EF) or BDNF (IJ) and analyzed 15 days after LP, there is a sectorial loss of the axonal transport in the dorsal retina (E, E', I, I'). In the black and white representations (F, J), the retinal vessels appear normal and morphologically similar to those in the control naïve retina. This is observed clearly in the magnifications taken from areas with no RAT (G, K) and with RAT (H, L). Bottom in (A), (A'), (E), (E'), and (I), (I'): number of RGCs counted in that retina. D, dorsal; V, ventral; T, temporal; N, nasal.
Figure 5
 
Study of the retinal vessels in BDNF and vehicle-treated hypertensive retinas. (A, A') A naïve retina labeled with FG and its corresponding isodensity map. (B) The RECA1+ retinal vessels in black and white. (C, D) Magnifications of the retina in (A) taken from the dorsotemporal (C) and inferotemporal (D) quadrant showing FG+RGCs (white), Brn3a+RGCs (red), and RECA-1+ vessels (green). In the naïve retina, there is a viable RGC retrograde axonal transport (RAT), and the retinal vessels irrigate properly all the areas. In hypertensive retinas treated with vehicle (EF) or BDNF (IJ) and analyzed 15 days after LP, there is a sectorial loss of the axonal transport in the dorsal retina (E, E', I, I'). In the black and white representations (F, J), the retinal vessels appear normal and morphologically similar to those in the control naïve retina. This is observed clearly in the magnifications taken from areas with no RAT (G, K) and with RAT (H, L). Bottom in (A), (A'), (E), (E'), and (I), (I'): number of RGCs counted in that retina. D, dorsal; V, ventral; T, temporal; N, nasal.
When total numbers of Brn3a+RGCs in the left experimental retinas treated with vehicle were compared to their fellow eyes, there was a clear RGC loss of approximately 57% or 58% at 12 or 15 days after OHT, respectively. These data indicate that RGC death follows OHT. However, the numbers of Brn3a+RGCs were significantly greater than the numbers of FG+RGCs in both groups at both survival time intervals (Fig. 3); this indicates that at early times after OHT, large numbers of RGCs expressing Brn3a and thus alive, but with their retrograde axonal transport impaired, are still present in the retina (2-fold the number of traced RGCs in vehicle-treated retinas and 3- to 4-fold in BDNF-treated retinas) as previously reported.24,62 The loss of Brn3a+RGCs observed 12 or 15 days after lasering showed the typical pie-shaped sectors, but was also diffuse (Fig. 3). After BDNF treatment, the numbers of Brn3a+RGCs were significantly greater than in vehicle-treated retinas at both time points (Fig. 3). Additionally, there was a significant decrease of Brn3a+RGCs between 12 and 15 days after OHT in the retinas treated with BDNF (P = 0.045, Mann-Whitney test) (Fig. 3). Thus, overall these data demonstrate that a single BDNF injection delays OHT-induced RGC death. 
In Figure 4 are shown some representative examples of isodensity maps illustrating the distribution of FG+RGCs (Figs. 4A, 4D, 4G, 4J) and Brn3a+RGCs (Figs. 4B, 4E, 4H, 4K) in OHT retinas. Isodensity maps of FG+RGCs show that in all retinas there is well-defined sectorial damage, more severe in the dorsal retina (Figs. 4A, 4D, 4G, 4J) as previously reported.10,12,13,24 Isodensity maps of Brn3a+RGCs show that in these sectors of axonal damage (with a lack of FG+RGCs) there are still surviving RGCs. In agreement with the quantitative data, the isodensity maps illustrate that RGC survival in the areas lacking FG tracing is more obvious in the retinas treated with BDNF (Figs. 4B, 4E, 4H, 4K). 
Responses of m+RGCs to OHT and Treatment With BDNF or Vehicle
Following OHT, m+RGCs also diminish considerably when compared to the naïve group of control retinas (Table 2). Twelve or 15 days after LP+vehicle, the population of m+RGCs decreases approximately to 50% or 51%, respectively, of the naïve population. Interestingly, this survival was not significantly different from the survival of Brn3a+RGCs, since in these same retinas the percentages of Brn3a+RGCs were 56% and 43% (n = 4; Mann-Whitney test, P = 0.4 at 12 or P = 0.46 at 15 days). These data indicate that OHT causes the same response in the two RGC populations. There is, however, an important difference that relates to the topological distribution of the surviving m+RGCs; while the general population of RGCs (identified with Brn3a) exhibit both a diffuse and sectorial loss, the lack of m+RGCs is mainly diffuse in all retinas analyzed, with a more pronounced effect in the superotemporal quadrant (Figs. 4C, 4F, 4I, 4L). Another important difference in m+RGCs relates to their lack of response to intraocular BDNF. Indeed, while the number of Brn3a+RGCs was significantly greater for the BDNF-treated groups when compared to the vehicle-treated groups, within the same retinas the numbers of m+RGCs in the BDNF and vehicle groups were similar (Kruskal-Wallis test, P = 0.521) (Table 2), thus indicating that m+RGCs do not respond to BDNF administration. 
Table 2
 
Total Number of m+RGCs in Naïve or OHT Retinas Treated With BDNF or Vehicle
Table 2
 
Total Number of m+RGCs in Naïve or OHT Retinas Treated With BDNF or Vehicle
Inner Retinal Vasculature After OHT
The inner retinal vessels were examined in naïve and experimental OHT retinas to determine if the typical sectorial loss of RGCs was related to a defect in the retinal vasculature. To this end, retinas were immunostained with RECA1, which primarily labels the main retinal arteries and arterioles. Representative examples in black and white images of the typical inner arterial plexus in naïve as well as in OHT retinas are shown in Figure 5. The general appearance of the main plexus of the inner retinal vessels in naïve retinas is comparable to that found in OHT retinas treated with BDNF or vehicle and examined at 15 days. This was confirmed with the blood vessel density measurements showing no significant differences (Kruskal-Wallis test, P = 0.653) between the number of vessels per millimeter (v/mm) in naïve retinas (11.06 ± 0.45 v/mm, n = 6) compared with BDNF-treated retinas (11.30 ± 0.68 v/mm, n = 2) and vehicle-treated retinas (10.91 ± 0.55 v/mm, n = 2). Moreover, the areas of the OHT retinas containing RGCs were compared with areas lacking RGCs, and there were no major differences in the arterial blood supply. The vessels found within the areas with competent retrograde axonal transport (RAT, Figs. 5H, 5L) were compared to those in the areas with impaired retrograde axonal transport (no RAT, Figs. 5G, 5K); there were no obvious differences between RAT and no-RAT areas either between BDNF and vehicle retinas or if these were compared with the vasculature of naïve retinas (Figs. 5C, 5D). In conclusion, at these time points OHT does not change the morphology of the inner retinal vessels; thus, there appears to be no correlation between the retinal blood supply and the appearance of sectorial loss of RGCs induced by OHT. 
Discussion
The present studies rely on immunohistochemical identification of Brn3a, melanopsin, and RECA1. In rat, Brn3a is expressed by all RGCs except for m+RGCs and one-half of the ipsilaterally projecting RGCs46,47; all ipRGC subtypes except M5 express melanopsin,45,47 and RECA1 has been shown to identify retinal vessels both in control naïve and in injured retinas.65–67 Although injured RGCs may modify their level of protein expression,73,74 thus warranting a note of caution when interpreting RGC survival using immunodetection, both melanopsin and Brn3a have been shown to be expressed long after optic nerve injury.33,34,75 
IOP Elevations Are Comparable to Those in Previous Studies
Laser photocoagulation of the limbal plexus has resulted in consistent elevations of the IOP in several studies in our laboratory10,12,24 that are comparable to values obtained by others.8 In accordance, the levels of IOP obtained in the present experiments show the typical abrupt rise in IOP that is already measurable at 24 hours and is maintained for the first week, the latest time interval measured in this study. Neither BDNF nor vehicle injection modified the levels of IOP. 
OHT-Induced Loss of m+RGCs and Non-m+RGCs Is Comparable
The loss of non-m+RGCs (Brn3a+RGCs) found in this work is in agreement with previous studies in rats10,12,24 and mice13,14,20 from our laboratory and is also in the range of RGC loss reported by others7,8,53,54,76 using laser-induced OHT. Our results also indicate that m+RGCs degenerate as a consequence of OHT (Fig. 3; Table 2); we have found a loss of approximately 50% or 51% of the control population of m+RGCs at 12 or 15 days, respectively, which is comparable to the loss of the non-m+RGCs. These results are in agreement with recent findings from this laboratory in OHT-pigmented mice retinas20 and comparable to those in previous studies from other laboratories in adult rats53,54 and mice.58 Similarly, Jakobs and colleagues57 reported that in the DBA/2J mouse, a well-established model of inherited pigmentary glaucoma, the decrease in total numbers of m+RGCs is substantial, although proportionally less than the fall in total numbers of RGCs. Zhang and colleagues,55 using mice created by crossing DBA/2J mice with Thy1-CFP, reported that progressive increase in IOP was followed by a concomitant reduction in the numbers of RGCs and m+RGCs, which could have an impact on nonimage-forming visual behaviors. Another previous study in a chronic OHT model in adult albino rats reported no alterations in total numbers of m+RGCs or in their dendritic morphology, suggesting that m+RGCs are more resistant to injury.56 Possible explanations for the discrepancies between the latter studies and our own data may relate to the methodology employed to ascertain RGC survival, the time intervals analyzed after induction of OHT, and the types of m+RGCs analyzed in these studies; there are approximately six different types of m+RGCs.77 Certainly, m+RGCs have been shown to be more resistant than the rest of the RGCs to a number of retinal injuries77 including optic nerve axotomy,78 NMDA-induced RGC degeneration,79 and inherited mitochondrial optic neuropathies.80 
Topological Loss of Non-m+RGCs Is Different From That of m+RGCs and Unrelated to Inner Retinal Vessels
In OHT retinas the surviving non-m+RGC population adopts the typical distribution in pie-shaped sectors, with their base located on the retinal periphery and their vertex on the optic nerve head. This is in contrast to the topology of surviving m+RGCs, which showed a diffuse loss throughout the retina without clear sectors. The observation of an apparent normal inner retinal vasculature, together with the preservation of the displaced amacrine cell population,12,20,57,8183 further supports the idea that sectorial loss is related to an axotomy-like injury to bundles of axons somewhere near the optic nerve head where retinal axons maintain their highest retinotopic arrangement.22,84,85 Why m+RGCs do not show the sectorial loss that is observed for conventional non-m+RGCs, including the small population of displaced RGCs,47 is not completely understood.20 One possible explanation could be the absence of retinotopic arrangement in m+RGCS. Nonimage-forming visual retinorecipient nuclei in the brain are thought to lack retinotopy; however, recent studies have shown that m+RGCs also project to image-forming visual retinorecipient nuclei,45,86,87 and these nuclei receive visual information with a very precise retinotopic arrangement.88 
BDNF Protects Non-m+RGCs But Not m+RGCs Against OHT-Induced Retinal Degeneration
We found a significant rescue of the general non-m+RGC population (Brn3a+RGCs) against OHT-induced RGC loss in the BDNF-treated groups analyzed 12 or 15 days after LP, in agreement with previous reports26,3539 showing that BDNF and other neurotrophic factors such as neurotrophin 4/5, insulin-like growth factor, or glial-derived neurotrophic factor provide temporary protection against injury-induced RGC loss.27,3034,8991 Perhaps a longer effect would require the establishment of permanent synaptic connections with appropriate target regions in the brain, as has been shown for adult rat RGCs.9296 It is possible that the presence of intraretinal axonal collaterals97,98 could provide m+RGCs with alternative trophic support, other than that originating from their target territories in the brain. This latter possibility, however, does not fully explain why there is still an important overall diminution in the m+RGC population of up to 50% by 15 days following OHT. Nevertheless, there was no significant rescue in the numbers of m+RGCs in the BDNF-treated groups when compared to the vehicle-treated groups; such a finding was surprising because in the same retinas BDNF induced a substantial rescue of non-m+RGCs (Figs. 3, 4; Table 2). An explanation for the neuroprotective effect of BDNF on injured RGCs has been that these neurons express the tropomyosin receptor kinase B (TrkB),99,100 which is needed for the activation of intracellular signaling pathways and whose expression is not significantly altered shortly after injury.101103 However, which proportion of the conventional RGC population and whether all types of m+RGCs express TrkB receptor is not known. 
Axonal transport after optic nerve104 or retinal105 injury has been shown to be altered. Following OHT, several studies in adult rats22,23,76 and mice23,57,106,107 have shown retrograde axonal transport impairment to precede RGCs loss. Indeed, surviving RGCs in adult albino mice13 or rat10 retinas outnumbered RGCs with an active and competent retrograde axonal transport.24 In these studies, impaired retrograde axonal transport was observed as early as 1 to 2 weeks after LP and did not progress further up to 6 months12,24; RGC loss was observed as early as 1 week after LP, the earliest time interval analyzed, and progressed up to 32 or 35 days in rats or mice, respectively.24 It is interesting to note that in the present studies, the neuroprotective effects of BDNF were evident for the population of RGCs with their retrograde axonal transport impaired (axonally nonintact) but not for the population of RGCs with their retrograde axonal transport maintained (axonally intact), thus indicating that compromised RGC somas are amenable to neuroprotection. Further studies are required to determine if such neuroprotection is maintained for longer periods of time. 
In conclusion, in the present study we have compared for the first time the responses of non-m+RGCs to those of the m+RGCs to OHT-induced retinal degeneration and neuroprotection afforded by intravitreal administration of BDNF. In addition, the inner retinal vasculature was examined, and our data indicate that OHT does not induce retinal vessel abnormalities that could account for the typical sectorial loss of RGCS that follows OHT. Our data further document that in adult albino rats 12 or 15 days after laser-induced OHT there is loss of approximately 57% or 58%, respectively, of the general RGC population. Intravitreal administration of BDNF prior to lasering results in significant rescue of approximately 39% or 26% more RGCs than in vehicle-treated controls at 12 or 15 days, respectively. While the proportion of loss of m+RGCs and non-m+RGCs after OHT is comparable, m+RGCs differ from non-m+RGCs in two aspects: m+RGC topological loss is not sectorial but diffuse, and m+RGCs appear to be insensitive to BDNF neuroprotection. Thus, our present study does not support that m+RGCs are more resistant to injury than non-m+RGCs but shows important differences in their response to BDNF rescue and topology of loss when compared to non-m+RGCs. 
Acknowledgments
Supported by the Spanish Ministry of Economy and Competitiveness: SAF-2012-38328, Instituto de Salud Carlos III (ISCIII) - Fondos Europeos de Desarrollo Regional (FEDER) “Una manera de hacer Europa,” PI13/01266, PI13/00643, and Red Temática de Investigación Cooperativa en Salud (RETICS): RD12/0034/0014. 
Disclosure: F.J. Valiente-Soriano, None; F.M. Nadal-Nicolás, None; M. Salinas-Navarro, None; M. Jiménez-López, None; J.M. Bernal-Garro, None; M.P. Villegas-Pérez, None; M. Agudo-Barriuso, None; M. Vidal-Sanz, None 
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Figure 1
 
Intraocular pressure values. Graph showing the temporal evolution of the intraocular pressure in the left experimental eye (LE) and right control eye (RE) in BDNF and vehicle groups before laser photocoagulation (LP) and at 24 hours and 7 days after LP. n, number of animals analyzed.
Figure 1
 
Intraocular pressure values. Graph showing the temporal evolution of the intraocular pressure in the left experimental eye (LE) and right control eye (RE) in BDNF and vehicle groups before laser photocoagulation (LP) and at 24 hours and 7 days after LP. n, number of animals analyzed.
Figure 2
 
Total numbers and spatial distribution of FG+RGCs, Brn3a+RGCs, and m+RGCs. (A) Photomontage of a naïve retina traced with FG from both superior colliculi and (B) inmunoreacted against Brn3a. Retinal ganglion cell topography is observed in the corresponding isodensity maps (A', B'). Melanopsin+RGCs are shown in (C) and their distribution is visualized with a neighbor map in (C'). Bottom left of each map: number of RGCs counted in that retina. Magnifications from the dorsal (a, c, e) or the ventral (b, d, f) retina showing FG, Brn3a, and melanopsin RGCs. Color scale for isodensity maps is shown in (B') and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C') and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V, ventral; T, temporal; N, nasal.
Figure 2
 
Total numbers and spatial distribution of FG+RGCs, Brn3a+RGCs, and m+RGCs. (A) Photomontage of a naïve retina traced with FG from both superior colliculi and (B) inmunoreacted against Brn3a. Retinal ganglion cell topography is observed in the corresponding isodensity maps (A', B'). Melanopsin+RGCs are shown in (C) and their distribution is visualized with a neighbor map in (C'). Bottom left of each map: number of RGCs counted in that retina. Magnifications from the dorsal (a, c, e) or the ventral (b, d, f) retina showing FG, Brn3a, and melanopsin RGCs. Color scale for isodensity maps is shown in (B') and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C') and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V, ventral; T, temporal; N, nasal.
Figure 3
 
Brain-derived neurotrophic factor delays OHT-induced RGC death. Histogram showing the mean ± standard deviation of the total number of FG+ and Brn3a+RGCs in control retinas and in experimental retinas treated with BDNF or vehicle and analyzed at 12 or 15 days after laser-induced ocular hypertension (OHT). In all groups, the number of FG+ and Brn3a+RGCs was significantly smaller in the experimental than in the control retinas (P < 0.05, Mann-Whitney test). The number of Brn3a+RGCs was significantly greater than FG+RGCs in all groups and time points analyzed (P < 0.003, Mann-Whitney test). The number of Brn3a+RGCs was greater in BDNF-treated than in vehicle-treated animals at 12 or 15 days after LP. After BDNF treatment the number of RGCs significantly decreased from 12 to 15 days. Statistical comparisons, Mann-Whitney test, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 for all groups. n, number of analyzed retinas.
Figure 3
 
Brain-derived neurotrophic factor delays OHT-induced RGC death. Histogram showing the mean ± standard deviation of the total number of FG+ and Brn3a+RGCs in control retinas and in experimental retinas treated with BDNF or vehicle and analyzed at 12 or 15 days after laser-induced ocular hypertension (OHT). In all groups, the number of FG+ and Brn3a+RGCs was significantly smaller in the experimental than in the control retinas (P < 0.05, Mann-Whitney test). The number of Brn3a+RGCs was significantly greater than FG+RGCs in all groups and time points analyzed (P < 0.003, Mann-Whitney test). The number of Brn3a+RGCs was greater in BDNF-treated than in vehicle-treated animals at 12 or 15 days after LP. After BDNF treatment the number of RGCs significantly decreased from 12 to 15 days. Statistical comparisons, Mann-Whitney test, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 for all groups. n, number of analyzed retinas.
Figure 4
 
Spatial distribution of FG+RGC, Brn3a+RGC, and m+RGC populations at 12 or 15 days after OHT with or without BDNF treatment. Isodensity maps of FG+RGCs (A, D, G, J) show that all retinas have a sectorial and/or diffuse damage. Their corresponding isodensity maps of Brn3a+RGCs (B, E, H, K) show that there are a large number of viable RGCs in the areas lacking FG+RGCs at the times analyzed, and that they are more abundant in the BDNF-treated retinas. The neighbor maps representing m+RGCs (C, F, I, L) show that their loss is the same in BNDF- or vehicle-treated retinas. Furthermore, m+RGC loss is diffuse although more severe in the dorsal retina. Bottom left of each map: number of RGCs counted in that retina. Color scale for isodensity maps is shown in (B) and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C) and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V,ventral; T, temporal; N, nasal.
Figure 4
 
Spatial distribution of FG+RGC, Brn3a+RGC, and m+RGC populations at 12 or 15 days after OHT with or without BDNF treatment. Isodensity maps of FG+RGCs (A, D, G, J) show that all retinas have a sectorial and/or diffuse damage. Their corresponding isodensity maps of Brn3a+RGCs (B, E, H, K) show that there are a large number of viable RGCs in the areas lacking FG+RGCs at the times analyzed, and that they are more abundant in the BDNF-treated retinas. The neighbor maps representing m+RGCs (C, F, I, L) show that their loss is the same in BNDF- or vehicle-treated retinas. Furthermore, m+RGC loss is diffuse although more severe in the dorsal retina. Bottom left of each map: number of RGCs counted in that retina. Color scale for isodensity maps is shown in (B) and goes from 0 (purple) to ≥3500 (red) RGCs/mm2. Color scale for the neighbor map is shown in (C) and goes from 0 to 2 (purple) to ≥21 to 23 (red) neighbors in a radius of 0.22 mm. D, dorsal; V,ventral; T, temporal; N, nasal.
Figure 5
 
Study of the retinal vessels in BDNF and vehicle-treated hypertensive retinas. (A, A') A naïve retina labeled with FG and its corresponding isodensity map. (B) The RECA1+ retinal vessels in black and white. (C, D) Magnifications of the retina in (A) taken from the dorsotemporal (C) and inferotemporal (D) quadrant showing FG+RGCs (white), Brn3a+RGCs (red), and RECA-1+ vessels (green). In the naïve retina, there is a viable RGC retrograde axonal transport (RAT), and the retinal vessels irrigate properly all the areas. In hypertensive retinas treated with vehicle (EF) or BDNF (IJ) and analyzed 15 days after LP, there is a sectorial loss of the axonal transport in the dorsal retina (E, E', I, I'). In the black and white representations (F, J), the retinal vessels appear normal and morphologically similar to those in the control naïve retina. This is observed clearly in the magnifications taken from areas with no RAT (G, K) and with RAT (H, L). Bottom in (A), (A'), (E), (E'), and (I), (I'): number of RGCs counted in that retina. D, dorsal; V, ventral; T, temporal; N, nasal.
Figure 5
 
Study of the retinal vessels in BDNF and vehicle-treated hypertensive retinas. (A, A') A naïve retina labeled with FG and its corresponding isodensity map. (B) The RECA1+ retinal vessels in black and white. (C, D) Magnifications of the retina in (A) taken from the dorsotemporal (C) and inferotemporal (D) quadrant showing FG+RGCs (white), Brn3a+RGCs (red), and RECA-1+ vessels (green). In the naïve retina, there is a viable RGC retrograde axonal transport (RAT), and the retinal vessels irrigate properly all the areas. In hypertensive retinas treated with vehicle (EF) or BDNF (IJ) and analyzed 15 days after LP, there is a sectorial loss of the axonal transport in the dorsal retina (E, E', I, I'). In the black and white representations (F, J), the retinal vessels appear normal and morphologically similar to those in the control naïve retina. This is observed clearly in the magnifications taken from areas with no RAT (G, K) and with RAT (H, L). Bottom in (A), (A'), (E), (E'), and (I), (I'): number of RGCs counted in that retina. D, dorsal; V, ventral; T, temporal; N, nasal.
Table 1
 
Experimental Design
Table 1
 
Experimental Design
Table 2
 
Total Number of m+RGCs in Naïve or OHT Retinas Treated With BDNF or Vehicle
Table 2
 
Total Number of m+RGCs in Naïve or OHT Retinas Treated With BDNF or Vehicle
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