Etiological Roles of p75NTR in a Mouse Model of Wet Age-Related Macular Degeneration

Choroidal neovascularization (CNV) is a pathological angiogenesis of the choroidal plexus of the retina and is a key feature in the wet form of age-related macular degeneration. Mononuclear phagocytic cells (MPCs) are known to accumulate in the subretinal space, generating a chronic inflammatory state that promotes the growth of the choroidal neovasculature. However, how the MPCs are recruited and activated to promote CNV pathology is not fully understood. Using genetic and pharmacological tools in a mouse model of laser-induced CNV, we demonstrate a role for the p75 neurotrophin receptor (p75NTR) in the recruitment of MPCs, in glial activation, and in vascular alterations. After laser injury, expression of p75NTR is increased in activated Muller glial cells near the CNV area in the retina and the retinal pigmented epithelium (RPE)-choroid. In p75NTR knockout mice (p75NTR KO) with CNV, there is significantly reduced recruitment of MPCs, reduced glial activation, reduced CNV area, and the retinal function is preserved, as compared to wild type mice with CNV. Notably, a single intravitreal injection of a pharmacological p75NTR antagonist in wild type mice with CNV phenocopied the results of the p75NTR KO mice. Our results demonstrate that p75NTR is etiological in the development of CNV.


Introduction
In industrialized countries, age-related macular degeneration (AMD) is one of the most frequent causes of visual impairment and blindness in adults over 60 years [1]. AMD is the deterioration of the macula, the region of the retina that generates the central images of the visual field, and it is associated with risk factors that include genetic background, environmental conditions, and aging [2].
The decrease in retinal function is associated with pathology of the retinal pigmented epithelium (RPE), a monolayer of pigmented cells that forms the external blood-retinal barrier and provides trophic and metabolic support. The RPE is the outermost layer of the retina, and it is sandwiched between photoreceptors that are located in the retina and the choriocapillaris outside the retina. The RPE is separated from the choroid by collagenous and elastic fibers called Bruch's membrane. The photoreceptors, RPE, Bruch's membrane, and choriocapillaris are ordered layers, and they constitute a "functional unit" whose organization is crucial for proper vision. This unit is impaired in AMD [3].

Immunofluorescent Detections in Retinal Cryosections
After mice sacrifice, eyes were enucleated with forceps and then fixed 2 h with 4% PFA during 2 h at room temperature. After that, eyes were incubated overnight in 10%, 20%, and 30% of sucrose in PBS at 4 • C to cryopreserve the tissues. For sections performance, eyes were embedded in optimum cutting temperature (OCT) (Tissue-TEK, Sakura) compound, and 10 µm-thick radial sections were obtained by using a cryostat. The obtained cryosections were stored at −20 • C under dry conditions. Immunostaining was performed as described previously [34]. Briefly, cryosections were washed with PBS, blocked with 2% of BSA in PBS containing 0.1% Tween-20 for 1 h, and then incubated ON at 4 • C with the following primary antibodies, respectively: rabbit polyclonal anti-GFAP (1/1000; Dako, Carpinteria, CA, USA), mouse β3 tubulin (1/500; ab78078, Abcam), and goat anti-p75 NTR (1/500; R&D system). Next, sections were washed with PBS 0.1% Tween-20 and incubated with secondary antibodies, including against goat, rabbit, or mouse IgG conjugated with Alexa Fluor 488 or 594 (1/250; Molecular Probes, Eugene, OR, USA), during 1 h at RT. The sections were also counterstained with Hoechst 33258 (1:3000; Molecular Probes) for 7 min. Cryosections were washed twice with PBS 0.1% Tween-20, mounted with Fluor Save (Calbiochem, La Jolla, CA, USA), and cover-slipped. The labeling was captured using a confocal laser-scanning microscope (Olympus Fluvial FV1200; Olympus Corp., New York, NY, USA). Finally, confocal microphotographs were processed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Negative controls without incubation with primary antibody were carried out for each inmmunodetection (data not shown).

Electroretinography (ERG)
Electroretinographic signals were measured as previously described [36]. Briefly, 7 days post laser, mice were adapted overnight (ON) in a dark room. The following day, mice were anesthetized intraperitoneally with a solution containing ketamine (90 mg/kg)/xilacine (8 mg/kg), under dim red illumination. The pupils were dilated with 1% tropicamide, and the cornea was lubricated with gel drops of 0.4% polyethyleneglycol 400 and 0.3% propylene glycol (Systane, Alcon, Buenos Aires, Argentina) to protect it from mechanical damage. Then, each mouse was placed in the ganzfeld and exposed to a light stimulus (10 cd·s/m 2 , 0.2 Hz) at a distance of 20 cm. A reference electrode was introduced on the back in the neck, a grounding electrode was placed on the tail, and a gold electrode was located in contact with the central cornea. Electroretinograms were simultaneously recorded from both eyes, and 10 responses to flashes of unattenuated white light from a photic stimulator set at maximum brightness were amplified, filtered (1.5-Hz low-pass filter, 1000 high-pass filter, notch activated), and averaged (Akonic BIO-PC, Buenos Aires, Argentina). The a-wave was determined as the difference in amplitude between the recording at onset and trough of the negative deflection, and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. The latencies of the a-and b-waves were measured from the time of flash stimulation to the trough of the a-wave or the peak of the b-wave, respectively. Responses were averaged across the two eyes for each mouse.

Fluorescence-Activated Cell Sorting (FACS) Analysis
Retinas and RPE-Choroid from both WT and p75 NTR KO mice, with or without laser, were obtained 4 days after the lesion. Under dissecting microscope, retinas and RPE-Choroid were collected in separate tubes and homogenized by gentle pipetting in FACS buffer (cold PBS with 2% FBS, 0.1% sodium azide). Cell suspensions were filtered through a 70 µm cell strainer and washed in FACS buffer. Viability of the cells was checked by Alexa Fluor 700 NHS ester dye (1/15.000; Molecular Probes, Eugene, OR, USA) [37]. Cells were incubated for 30 min at 4 • C with mouse anti f4/80 (1/50; Invitrogen), rabbit anti CX3CR1

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 5.0 software. A p-value < 0.05 was considered statistically significant. Parametric or nonparametric tests were used, according to variance homogeneity evaluated by F or Barlett's tests. Two-tailed unpaired t or Mann-Whitney tests were used in analysis of two groups. One-way or two-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison post-test or Kruskal-Wallis, followed by Dunn s multiple comparison post-test were used to determine statistical significance among more than two different groups. All the assays were performed in n ≥ 3 independent experiments, with n = 3 technical replicates in each assay, as indicated.

p75 NTR Expression Is Increased in the Choroidal Neovascularization Lesions
First, we quantified the expression of p75 NTR in RPE-Choroid tissue in the mouse model of wet AMD. Studies were carried out at 2 time points: 4 days post laser injury (P4 CNV, when the majority of infiltrating cells arrive to the RPE-Choroid) and 7 days post laser injury (P7 CNV, a time point when CNV is fully developed) [10,38].
Western blot analysis showed some expression of p75 NTR in healthy RPE-Choroid tissues and a significant increase 4 days after laser injury ( Figure 1A). However, the protein levels were non-significantly different between WT and CNV mice 7 days after laser injury (p = 0.2286; Figure 1B). The expression of p75 NTR in microglia and other mononuclear phagocytic cells was evaluated 4 days after the laser injury. Confocal images of the CNV lesion in choroidal whole mounts showed p75 NTR labelling in F4/80 positive cells ( Figure 1C) Cells 2023, 12, 297 6 of 18 and partially in Iba-1 and CX3CR1 positive microglial cells ( Figure 1D,E). For vascular and peri-vascular staining, we performed RPE-Choroid wholemounts 7 days after the laser injury. There was no detectable p75 NTR expression in the vasculature, as the p75 NTR label was not co-localized with markers of endothelial cells (isolectin IB4) ( Figure 1F) and pericytes (NG2) ( Figure 1G). laser injury (P7 CNV, a time point when CNV is fully developed) [10,38].
Western blot analysis showed some expression of p75 NTR in healthy RPE-Choro sues and a significant increase 4 days after laser injury ( Figure 1A). However, the p levels were non-significantly different between WT and CNV mice 7 days after laser (p = 0.2286; Figure 1B). The expression of p75 NTR in microglia and other mononuclear ocytic cells was evaluated 4 days after the laser injury. Confocal images of the CNV in choroidal whole mounts showed p75 NTR labelling in F4/80 positive cells ( Figure 1C partially in Iba-1 and CX3CR1 positive microglial cells ( Figure 1D,E). For vascula peri-vascular staining, we performed RPE-Choroid wholemounts 7 days after the injury. There was no detectable p75 NTR expression in the vasculature, as the p75 NT was not co-localized with markers of endothelial cells (isolectin IB4) ( Figure 1F) and cytes (NG2) ( Figure 1G). Representative Western blot of RPE-Choroid homogenates prepared from WT mice without CNV, or 4 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 5 mice/group. The asterisks show statistical differences respect to control. * p < 0.05. (B) Representative Western blot of RPE-Choroid homogenates prepared from WT mice without CNV, or 7 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. ns: non-significant. Bars denote the mean ± SD from triplicate experiments, n = at least 4 mice/group.

p75 NTR Expression Is Also Increased in the Injured Retina
In heathy retinas, p75 NTR is expressed at low levels. However, its expression pattern and protein levels may vary after an injury [24]. We evaluated expression of p75 NTR in the retinas of CNV mice 7 days after the laser injury, when the photoreceptor layer has been disorganized by neovessels. The RPE-Choroid tissues were excluded, as they were analysed above.
Western blot analysis showed increased expression of p75 NTR in retinas CNV mice 7 days after laser, compared to control mice (no laser retinas) (Figure 2A). To further determine which cells residing in the retina increased p75 NTR expression, immunostaining of p75 NTR was combined with a variety of cellular markers in double fluorescent immunolabelling in cryosections of retina from CNV mice 7 days after laser. There was increased expression of p75 NTR in activated Muller glial cells surrounding the lesioned area, identified by glial fibrillary acid protein (GFAP)-positivity and morphology ( Figure 2B, upper panel). However, no expression of p75 NTR was detected in neurons ( Figure 2B, lower panel). Moreover, there was no significant p75 NTR staining in pericytes of the superior vascular plexus in retinal whole mounts ( Figure 2C).
homogenates prepared from WT mice without CNV, or 4 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 5 mice/group. The asterisks show statistical differences respect to control. * p < 0.05. (B) Representative Western blot of RPE-Choroid homogenates prepared from WT mice without CNV, or 7 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. ns: non-significant. Bars denote the mean ± SD from triplicate experiments, n = at least 4 mice/group.

p75 NTR Expression Is Also Increased in the Injured Retina
In heathy retinas, p75 NTR is expressed at low levels. However, its expression pattern and protein levels may vary after an injury [24]. We evaluated expression of p75 NTR in the retinas of CNV mice 7 days after the laser injury, when the photoreceptor layer has been disorganized by neovessels. The RPE-Choroid tissues were excluded, as they were analysed above.
Western blot analysis showed increased expression of p75 NTR in retinas CNV mice 7 days after laser, compared to control mice (no laser retinas) (Figure 2A). To further determine which cells residing in the retina increased p75 NTR expression, immunostaining of p75 NTR was combined with a variety of cellular markers in double fluorescent immunolabelling in cryosections of retina from CNV mice 7 days after laser. There was increased expression of p75 NTR in activated Muller glial cells surrounding the lesioned area, identified by glial fibrillary acid protein (GFAP)-positivity and morphology ( Figure 2B, upper panel). However, no expression of p75 NTR was detected in neurons ( Figure 2B, lower panel). Moreover, there was no significant p75 NTR staining in pericytes of the superior vascular plexus in retinal whole mounts ( Figure 2C).  p75 NTR has been associated with immune response [39][40][41] and with playing a role in the activation [42] and migration [43,44] of monocytes and immune cells. Therefore, we evaluated if the genetic deletion of p75 NTR affected the arrival of MPCs cells to the injured area, using F4/80 flow cytometric analysis.
This quantitative approach allows estimating the percentage of MPCs related to the total CD45 positive cells (myeloid linage) that reach the lesioned area. In the RPE-Choroid suspended cells, we observed an increase in MPCs in WT CNV mice respect to control WT mice 4 days after laser ( Figure 3A, upper panels). Notably, p75 NTR KO CNV mice exhibited a significant decrease in MPCs population, as compared to WT CNV mice. A similar result was observed in retina tissues ( Figure 3A, lower panels). These results suggest that p75 NTR , which is expressed by MPCs, could have a role in the recruitment of these cells to the lesioned vascular area. There were no statistical differences in the basal percentage of MPCs between WT and p75 NTR KO mice control (no laser) in the retina or in RPE-Choroid. We did not detect F4/80 immunofluorescence differences in the RPE-Choroids between WT and p75 NTR KO mice 4 days after laser injury (F4/80 area p = 0.1106; F4/80 mean intensity p = 0.9833) ( Figure 3B). This result suggests that a MPCs subpopulation of F4/80 positive cells is recruited to the injured area.

P75 NTR Deletion Reduces Choroidal Neovessels Formation, Lesion Size, and Neuronal Alterations in CNV Mice
As p75 NTR expression after laser CNV is incremented in the RPE-Choroid ( Figure 1) and in the retina (Figure 2), we investigated a possible role of p75 NTR in the formation of choroidal neovessels and the subsequent retinal neurodegeneration in this model. We performed laser lesions in both wild type (WT) and p75 NTR knockout (KO) mice to evaluate vascular, glial, and neuronal changes that may occur after laser-induced CNV. Similar lesions (assessed by the actin-labelled structure of the RPE-Choroid cells) were detected in the RPE-Choroid for WT and p75 NTR KO mice 1 day after the laser ( Figure 4A), suggesting that genotypes do not interfere with the initial injury by the laser (area of lesion p = 0.7519; perimeter of lesion p = 0.6713). However, as disease progresses, 7 days after laser, there was a significant reduction in the choroidal neovessel area and perimeter in the p75 NTR KO mice, as compared to WT mice ( Figure 4B). This result indicates that p75 NTR participates in the promotion of CNV development.
Upon damage, macroglial cells in the retina set up a protective response to assure neuronal well-being. The up-regulation of intermediate filaments, hypertrophy, proliferation, and migration of glial cells are key events of reactive gliosis [45]. To evaluate if p75 NTR impacts the gliotic response, we measured glial activation by evaluation of GFAP protein levels. Western blot and immunostaining showed an increase in GFAP protein in the WT retina 7 days after laser injury ( Figure 4C,D), but no increase in GFAP in the p75 NTR KO CNV mice was found, suggesting decreased astrogliosis. Cells 2023, 12, x FOR PEER REVIEW 9 of 18

P75 NTR Deletion Reduces Choroidal Neovessels Formation, Lesion Size, and Neuronal Alterations in CNV Mice
As p75 NTR expression after laser CNV is incremented in the RPE-Choroid (Figure 1) and in the retina (Figure 2), we investigated a possible role of p75 NTR in the formation of Figure 3. Reduced inflammatory phenotype in the RPE-Choroids and retinas of p75 NTR knockout mice, after laser injury. Mononuclear phagocytic cell recruitment is significantly reduced in retinas and RPE-Choroids of p75 NTR KO mice after CNV. (A) Representative flow cytometry pseudocolor plots from WT and p75 NTR KO mice without CNV, or 4 days after laser injury. Cells in the gate were quantified and the number of cells in the gate/total cells ratio is represented in the bar graph expressed as units relative to WT no laser control. Graphs denote the mean ± SD from triplicate experiments, n = 6 mice/group. The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative confocal images of RPE-Choroid flat-mounts of WT and p75 NTR KO mice 4 days after laser, showing immunofluorescence staining with Isolectin IB-4 (grey) and F4/80 (green). Scale bar: 200 µm. F4/80 fluorescence intensity and area were quantified with ImageJ FIJI software Version 1.53t, and represented in the bar graph expressed as units relative to CNV WT control. Bars denote the mean ± SD from triplicate experiments, n = at least 5 mice/group. ns: non-significant. Similar lesions (assessed by the actin-labelled structure of the RPE-Choroid cells) were detected in the RPE-Choroid for WT and p75 NTR KO mice 1 day after the laser ( Figure  4A), suggesting that genotypes do not interfere with the initial injury by the laser (area of lesion p = 0.7519; perimeter of lesion p = 0.6713). However, as disease progresses, 7 days after laser, there was a significant reduction in the choroidal neovessel area and perimeter in the p75 NTR KO mice, as compared to WT mice ( Figure 4B). This result indicates that p75 NTR participates in the promotion of CNV development. represented in the bar graph expressed as units relative to CNV WT control. Bars denote the mean ± SD from triplicate experiments, n = at least 6 mice/group. The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Representative Western blot of total retinal homogenates prepared from WT and p75 NTR KO mice without CNV, or 7 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and GFAP/tubulin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 3 mice/group. (D) Representative immunofluorescence analysis of GFAP (green) in retinal cryosections from WT and p75 NTR KO mice without injury or 7 days after laser injury. Scale bar: 50 µm. Cell nuclei counterstained with Hoechst are also shown (blue). Abbreviations: GCL (ganglion cells layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer). (E) Amplitudes and implicit times of a-and b-waves from scotopic electroretinograms recorded in WT and p75 NTR KO mice without injury or 7 days after laser injury. The data show averages of responses of both eyes. Graphs denote the mean ± SD from triplicate experiments, n = at least 6 mice/group. The symbol correspond: WT no laser (filled circle), WT CNV (filled square), p75 NTR KO no laser (filled triangle up), p75 NTR KO CNV (filled triangle down). The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
Based on the reduced choroidal neovessel area and the reduced GFAP glial activation of the p75 NTR KO after laser injury, we anticipated a healthier neuro-retinal functional unit and improved visual function. This was quantified by scotopic electroretinography studies 7 days after laser injury ( Figure 4E). In WT CNV mice, we observed a decreased amplitude of the a-and b-waves, as compared to the control WT control (no laser mice). Remarkably, p75 NTR KO CNV mice showed preserved a-and b-waves amplitude, as compared to control p75 NTR KO healthy mice. No differences were observed in the a-and b-waves latencies between the experimental groups.
Confocal images of RPE-Choroid flat mounts labelled with isolectin IB-4 showed in the THX-B-treated eyes a significant reduction in CNV area and perimeter 7 days after laser injury ( Figure 5A,B).
In functional studies, scotopic electroretinography showed that THX-B injection prevents the decrease in the a-wave amplitude ( Figure 5C). However, the injection of THX-B did not prevent the reduction in the b-wave observed in CNV-vehicle mice ( Figure 5C). No variations were detected in the a-and b-latency in any of the conditions ( Figure 5C).
We further conducted RPE-Choroids cytometric analysis, which showed that in laserinjured mice, THX-B treatment decreased the percentage of MPCs cells, as compared to vehicle treatment. The decreased in MPCs cells in the mice that received THX-B was quantified in the RPE-Choroids ( Figure 5D, upper panels) and was quantified also in retinas ( Figure 5D, lower panels).
Lastly, THX-B administration did not alter the MPCs fluorescence area and intensity in the RPE-Choroid 4 days after laser injury, as compared with CNV mice with vehicle (F4/80 area p = 0.4418; F4/80 mean intensity p = 0.1492) ( Figure 5E).
Collectively, these p75 NTR pharmacological antagonist results are a phenocopy of the results using p75 NTR knockout mice. Confocal images of RPE-Choroid flat mounts labelled with isolectin IB-4 showed in the THX-B-treated eyes a significant reduction in CNV area and perimeter 7 days after laser injury ( Figure 5A,B).

Discussion
In this work, we found that p75 NTR pharmacological or genetic ablation prevented the neovascularization and the consequent functional deficits of the retina in a murine model of wet age-related macular degeneration (AMD). Moreover, our results suggest that at least part of the p75 NTR actions in this model are related to the recruitment of mononuclear phagocytic cells, which are known to promote the angiogenic environment. These results are relevant to understanding the wet AMD pathogenic process and to potentially propose p75 NTR as a relevant therapeutic target.
The most widely described effects mediated by p75 NTR include neuronal death, longterm depression, and neuronal cytoarchitecture rearrangements [46][47][48][49]. However, the expression of p75 NTR and its roles in ocular vasculopathies are less studied. In a recent work, p75 NTR KO mice showed a significant reduction in occluded capillaries and a major number of pericytes in the retinas of diabetic mice induced by streptozotocine [50]. In another report, the deletion of p75 NTR suppressed the pro-angiogenic factors induced by hypoxia in the RPE [51]. A variety of mechanisms were described to explain these p75 NTR effects, ranging from the activation of the transcription factor NF-κB to the cytoplasmic stabilization of HIF-1α by the intracellular fragment of p75 NTR generated after proteolytic cleavage [31,52]. In models of retinal vascular disease, increased expression of p75 NTR was predominantly in Müller glia, a macroglial cell that is known to secrete pro-angiogenic proteins and to participate in the settlement of the inflammatory response [24], although p75 NTR may also be expressed in leucocytes and in infiltrating monocytes after a traumatic injury [53].
Here, in a CNV mouse model, we report expression of p75 NTR in MPCs identified as F4/80 positive cells and partially in Iba-1-or CX3CR1-positive cells (Figure 2), suggesting that this receptor is mainly present in MPCs derived from myeloid progenitors. The major expression of the neurotrophin receptor was detected 4 days after the laser injury, coincidental with the arrival of MPCs to the tissue. These protein levels tend to decrease after P4 CNV, with no significant differences between no laser and CNV at P7. Moreover, in the retina, p75 NTR expression was restricted to Müller glial cells after the laser-induced lesion. The increased expression of p75 NTR in Müller glial cells in the retina and in MPCs in the RPE-Choroid suggest a role of p75 NTR in the inflammatory response (Figures 1 and 2). The absence of p75 NTR in pericytes and endothelial cells indicates that this receptor is not mediating a direct effect on vascular cells.
We asked whether and how is p75 NTR involved in AMD. We focused on MPCs, which we determined are a population expressing the receptor abundantly after the laser. The MPCs population is heterogenous, and they can roughly be classified as tissue-resident or bone-derived, meaning that at least part of them can be recruited from the circulatory system [54]. Classical immunostaining techniques were not sensitive enough to detect differences in MPCs. However, cytometric analysis showed that at least a subpopulation of MPCs were significantly reduced in the injured area of p75 NTR KO CNV mice, as compared to WT CNV animals ( Figure 3). The difference in the MPCs population resulted in a different inflammatory environment, a key modulator of the angiogenic response. MPC proliferation, activation, and/or recruitment to the lesion area may be directly (as they express p75 NTR ) or indirectly regulated by p75 NTR . However, we cannot discard that other cells expressing p75 NTR may also participate in the settlement and progression of CNV.
The development of neovessels encompasses a strong angiogenic response, mediated by several proteins, including growth factors and lectins, among others. Under different insults, such as hypoxia, hyperglycemia, or chronic inflammation, certain cells residing in both RPE-Choroid and the retina are able to express and secrete pro-angiogenic proteins. However, in many animal models, the blooming of the vascular changes is reached with the arrival of MPCs due to the fact that the inflammation potentiates the response, favoring the secretion of pro-angiogenic molecules [55,56]. In the mouse model of laser-induced CNV, the angiogenic response is known to be regulated by the RPE together with MPCs [17]. Two strategies were employed to determine the participation of p75 NTR in CNV: genetic ablation of the receptor utilizing a p75 NTR KO mice and pharmacological inhibition by intraocular injection of the p75 NTR small-molecule antagonist THX-B.
To reach the RPE-Choroid, the laser beam must go through all the anterior segment, the vitreous, and the retina. We verified that the initial laser lesion areas and perimeters were similar in both mice genotypes, WT and p75 NTR KO (estimated with F-actin labelling), suggesting that the initial injury is comparable for both genotypes. Although the initial laser lesion was indistinguishable, vascular outgrowth was reduced in p75 NTR KO mice 7 days after the laser, indicating that the receptor is involved in the development of CNV. The fact that p75 NTR only alters the response of a subgroup of MPCs would explain why there is a partial reduction in the CNV area and perimeter in p75 NTR KO CNV mice. GFAP expression also provided evidence of reduced retinal damage in p75 NTR KO CNV mice, compared to WT CNV mice, 7 days after the laser (Figure 3).
In different retinopathies, such as glaucoma, diabetic retinopathy, optic nerve atrophy, and retinitis pigmentosa, p75 NTR is mainly expressed in Muller glial cells [24]. In this regard, it has been reported that the interaction of proNGF with p75 NTR in Muller cells increases the production of inflammatory cytokines as tumor necrosis factor α and other proteins as alpha-2 macroglobulin to induce neurodegeneration [21,24,57]. Further experiments should be performed to unravel the role of p75 NTR in Muller cells and to unmask the potential ligand involved in its actions in the CNV model.
Electrophysiological studies in CNV animals show that the damage alters retinal functionality, at least in part due to the loss of trophic support provided by the RPE to photoreceptors, as well as the disruption of the retinal architecture by proliferating vessels [58]. Thus, our results suggest that the preserved retinal functionality observed in p75 NTR KO CNV mice is a consequence of the reduced neovascular area (Figure 4).
For treatment of neovascular retinopathies, novel therapeutic strategies are needed, especially for patients who become refractory to the classical anti-VEGF therapy. Antiinflammatory strategies, such as corticosteroid treatment, are one approach [59,60]. Here, the intra-ocular treatment with THX-B demonstrated that p75 NTR inhibition reduces the neovessels coverage and the MPCs recruitment, and, importantly, preserved the retinal function ( Figure 5). This result suggests that cytokines and other molecules secreted by MPCs regulate the vascular growth and contribute significantly to the development of CNV.
Overall, in this work, we evidenced that p75 NTR , a receptor frequently linked to neurobiological effects, participates in neovascularization of the choroidal vessels in a clinically relevant model of macular degeneration.