Abstract
Purpose.:
The retina contains two distinct populations of monocyte-derived cells: perivascular macrophages, and microglia. The present study was undertaken to evaluate the presence and function in mouse and human retinas of a subtype of resident perivascular macrophages with scavenger function, different from microglia, in physiological conditions and during retinopathy.
Methods.:
Perivascular macrophages were characterized by means of confocal microscopy, electron microscopy, and flow cytometry analyses. Two murine models of blood–retinal barrier breakdown and photoreceptor degeneration were used to analyze the role of these macrophages during retinopathy.
Results.:
The macrophages analyzed constituted a small population of resident perivascular cells different from microglia, since they were Iba-1 negative. Although these cells expressed F4/80 and CD11b antigens in common with microglia, they also expressed BM8 and MOMA-2 epitopes, which are macrophagic markers not expressed by microglia. Perivascular macrophages emitted autofluorescence due to cytoplasmic inclusions containing protein-bound oxidized lipids. They constitutively expressed the scavenger receptor class A and moved along blood vessels, providing an additional coating to thinner areas of the basement membrane. Moreover, they accumulated blood-borne horseradish peroxidase and acetylated low-density lipoprotein in healthy retinas. In addition, during blood–retinal barrier breakdown and photoreceptor degeneration, these cells migrated to the lesion site.
Conclusions.:
All these morphologic and functional features are consistent with those described for brain Mato cells. Thus, this study showed the presence of autofluorescent perivascular macrophages, different from microglia, with a scavenger function that may contribute to the maintenance of the blood–retinal barrier in healthy conditions and that are also involved in retinopathy.
Autofluorescent perivascular macrophages different from microglia were described in the brain by Mato et al.
1 These cells contain autofluorescent inclusion bodies and are localized around blood vessels, between the blood vessel basement membrane and the astrocyte end feet.
1 They fulfill a major role in the scavenging of blood-borne proteins and lipids. In fact, the autofluorescent inclusion bodies have been categorized as lysosomes rich in hydrolytic enzymes that can efficiently concentrate and digest these substances.
1,2 Autofluorescent perivascular macrophages participate in blood–brain barrier (BBB) function, specifically in the exclusion of blood-borne molecules from the brain,
2 and are also implicated in hypertension and stroke.
3
The blood–retinal barrier (BRB) is a homologue of the BBB,
4 since the retina is an outgrowth of the brain and exclusion of blood-borne molecules is also required for normal retinal function. Perivascular cells have been described in normal retina.
5 A small population of macrophages and resident microglial cells both acting as antigen presenting cells have been found surrounding retinal blood vessels.
6 However, no perivascular cells with scavenger function have been described in healthy retina. Microglia only express scavenger receptors when activated in response to injury.
7,8 In this report, we describe and characterize a retinal autofluorescent perivascular macrophage (APM) population. These macrophages moved along retinal blood vessels, coating protein low-density areas in the basement membrane, scavenging and accumulating blood-borne molecules without BRB disruption. In mannitol-induced BRB breakdown and in experimental and human retinopathy, these perivascular cells migrated to the lesion site.
Materials and Methods
Animals
One- to six-month-old ICR mice were used. C57Bl/6 mice present loss of 2F8 immunoreactivity as a consequence of scavenger receptor class A polymorphism
9 and their inability for recognize 2F8 antibody led us to use ICR mice to study scavenger function in the APMs. The mice were fed ad libitum with a standard diet (Panlab SL, Barcelona, Spain). Animal care and experimental procedures were performed according to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research.
Human Retinas
Human retinas obtained from a 89-year-old female and an 83-year-old male donor were formalin fixed and used in the experiments.
Lipid Solubility Experiments
Six mouse retinas were treated with 70% methanol (1 hour) and ethyl ether (20 minutes) for lipid extraction.
10 Human retinas were stained with oil red O to demonstrate the presence of lipids in perivascular macrophage cytoplasmic inclusions.
10
Analysis of Autofluorescence
Autofluorescence fingerprints of 10 wholemount, unstained retinas and their corresponding retinal pigment epithelium attached to the inner surface of choroids were analyzed. The lambda mode of a laser scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany) with an excitation laser of 488 nm was used.
Immunohistochemistry
The retinas were fixed in 10% neutral buffered formalin for 1 hour at room temperature. Each one of the antibodies was used for the six different mouse retinas. After they were washed in 0.01-M phosphate-buffered saline (PBS), wholemount retinas or paraffin-embedded sections were incubated over night at 4°C with the following markers: rabbit anti-mouse collagen IV (Chemicon- Millipore Iberica SAU, Madrid, Spain) at 1:200 dilution; rabbit anti-mouse glial fibrillary acidic protein (GFAP; Dako, Barcelona, Spain) at 1:500 dilution; rabbit anti-mouse Iba-1 (Wako, Neuss, Germany) at 1:3000 dilution; rat anti-mouse CD11b and rat anti-mouse anti-2F8 (Serotec, Raleigh, NC) at 1:100 and 1:50 dilutions, respectively; rat anti-mouse F4/80 (CI:A3-I; BMA Biomedicals, Augst, Switzerland) at 1:100 dilution; rat anti-mouse BM8 (eBioscience, San Diego, CA) at 1:50 dilution; rat anti-mouse MOMA-2 (Abcam, Cambridge, UK) at 1:50 dilution and also with the biotinylated Lycopersicon esculentum lectin (20 μg/mL; Vector Laboratories, Burlingame, CA). After they were washed in PBS, the retinas were incubated at 4°C over night with specific secondary antibodies: biotinylated anti-rabbit (1:100) and anti-rat mouse absorbed (1:100) IgGs (Vector Laboratories). After the retinas were washed in PBS, an infrared fluorochrome Cy5 (GE Healthcare Espana SA, Alcobendas, Spain) at 1:500 dilution was used to avoid interference with yellow autofluorescence capture; the incubation was made at 4°C overnight. Nuclear counterstaining with Hoechst stain solution (Sigma-Aldrich, Química, Spain) and iodide (To-Pro-3; Invitrogen SA-Molecular Probes, Barcelona, Spain) was performed for microscopic analysis with the laser scanning confocal microscope (TCS SP2; Leica Microsystems).
Transmission Electron Microscopic Analysis
Retinal fragments from five different mice were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, dehydrated, and embedded in epoxy resin. Ultrathin sections (70 nm) were stained using lead citrate and examined by a transmission electron microscope (H-7000; Hitachi, Tokyo, Japan).
Time-Lapse Imaging of Perivascular Cell Movement
Four retinas were cultured in fresh Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich) penicillin and streptomycin, in a CO2/air (5% / 95%) humidified atmosphere at 37°C. Time-lapse imaging was performed in a laser-scanning confocal microscope (Leica; TCS SP2). Retinal cell viability was determined by neutral red staining (Sigma-Aldrich).
Flow Cytometry
Each one of the antibodies was tested in 36 retinas in three different experiments. Each experiment was performed on 12 retinas to obtain enough cells for flow cytometry analysis. After cell suspension, the retinas were pooled to obtain three samples of four retinas each, to repeat the analysis. The retinas were mechanically disaggregated and further digested with collagenase B (Roche, Madrid, Spain), followed by filtration with 30-μm preseparation filters (Miltenyl Biotec, Madrid, Spain) to obtain a single cell suspension. For PBMC purification, blood was diluted 1:1 with PBS, layered over a density gradient (Histopaque-1.083; Sigma-Aldrich), and centrifuged. Mononuclear cells were collected from the interface. APC-conjugated anti-CD11b (BD Pharmingen, Madird, Spain), biotinylated anti-BM8 (eBioscience), and biotinylated anti-2F8 (Serotec) antibodies and Cy5-conjugated streptavidin (GE Healthcare) were used. Isotyped matched antibody served as the control. Samples were analyzed with a flow cytometer (FACScanto; BD Pharmigen). Autofluorescence was detected in the FLH2 channel.
Protein Intravenous Administration Experiments
Single Injection.
HRP type II (Sigma-Aldrich), 15 mg/mL, and DiI acetylated-low-density lipoprotein (DiI-ac-LDL; Molecular Probes Europe BV, Leiden, The Netherlands) 200 μg/mL, in physiological saline solution (PSS), were injected in two different groups of four animals. Six hours after HRP injection and 2 hours after DiI-ac-LDL injection, the retinas were fixed in formalin. HRP was revealed with 3′3′-diaminobenzidine (Sigma-Aldrich). Red fluorescence emitted by DiI showed the localization of DiI-ac-LDL. Images were acquired with an epifluorescence microscope (Eclipse E-800; Nikon Corp., Tokyo, Japan) with a digital camera (DXM 1200F; Nikon Corp.).
Combination of Bis-benzimide and HRP Injections.
Ten minutes after tail injection of 250 μL of a 0.5% solution of Bis-benzimide (Hoechst stain, H33258) (Calbiochem, San Diego, CA) dissolved in PSS, a second injection of 250 μL of HRP type II (Sigma-Aldrich), 15 mg/mL in PSS, was performed in four mice. One hour after HRP injection, the mice were euthanatized and the retinas were formalin fixed at room temperature for 30 minutes. HRP was revealed with 3′3′-diaminobenzidine and nuclear counterstaining was performed with iodide (To-Pro-3; Molecular Probes).
Combination of Mannitol, Bis-benzimide, and HRP Injections.
Four mice were intravenously injected with 250 μL of a 25% solution of mannitol (Sigma-Aldrich) dissolved in PSS. Ten minutes later, a combination of bis-benzimide and HRP injections was performed as previously described. The retinas were obtained 10 minutes later and HRP was revealed with 3′3′-diaminobenzidine. Nuclear counterstaining was performed with iodide (To-Pro-3; Molecular Probes).
Images in these experiments were acquired with an epifluorescence microscope (Eclipse E-800; Nikon Corp.) with a digital camera (DXM 1200F; Nikon Corp.) and a laser scanning confocal microscope (TCS SP2; Leica).
Mouse Retinopathy Model Experiments
Four groups of eight mice each were intraperitoneally injected with 100 mg/kg of sodium iodate (NaIO3; Sigma-Aldrich) in PSS and euthanatized immediately after injection or 1, 2, or 3 days after treatment. A group injected only with PSS was used as the control.
Statistical Analysis
The number of autofluorescent cells present in the inner retina (ganglion cell layer+inner plexiform layer+inner nuclear layer) and the outer retina (outer plexiform layer+external nuclear layer+inner and outer photoreceptor segments) after sodium iodate injection was counted. Three wholemount retinas per group, half sectioned, and each half, with the inner or the outer retina facing up, were counterstained with iodide, to visualize the nuclei, and were analyzed by laser confocal microscopy, using the same settings used to reveal autofluorescence. Ten random fields at 63×, corresponding to a retinal area of 56,691 μm2 each were counted immediately after injection and 24 and 48 hours after injection. The results are expressed as the mean ± SEM.
Results
Autofluorescent Perivascular Cells in Mouse Retina
Confocal microscopy analysis revealed the presence of autofluorescence in cells surrounding retinal blood vessels (
Fig. 1). Autofluorescence was detected in granular inclusions close to the cellular nuclei (
Figs. 1A,
1B,
1C). Oxidized lipids are known to produce fluorescence.
1 To determine whether perivascular cell autofluorescent inclusions contained protein-bound oxidized lipids, we treated the retinas with lipid solvents. Methanol and ethyl ether remove lipids from cells, whereas protein-bound lipids are insoluble to lipid solvents.
10 Autofluorescence was not released from perivascular cell inclusions treated with methanol and ethyl ether (
Figs. 1B,
1C). These results suggest that protein-bound oxidized lipids are present in the autofluorescent inclusions. Fluorescence produced by lipofuscin accumulation has been described in perivascular microglia in aging mice.
11 Lipofuscin fluorescence is faded by lipid solvents
12 ; therefore, autofluorescence in retinal perivascular cells was probably not due to lipofuscin. In addition, the emission spectrum of both perivascular cell autofluorescent inclusions and retinal pigment epithelium lipofuscin granules was evaluated. The fluorescence fingerprint of autofluorescent inclusions (
Fig. 1D) was different from that of lipofuscin (
Fig. 1E), which was consistent with the fact that perivascular cell autofluorescence was due to protein-bound oxidized lipids and not to lipofuscin.
Topography of Autofluorescent Perivascular Cells in the Retina
Retinas immunohistochemically labeled for collagen IV, a basement membrane marker,
13 showed that autofluorescent perivascular cells were located externally to blood vessel basement membrane (
Figs. 1F,
1H). Thus, these cells were not pericytes or smooth muscle cells, since these latter are components of the vascular wall and are completely surrounded by the basement membrane.
14 Retinas immunohistochemically labeled for glial fibrillary acidic protein (GFAP), a marker of neuroglial cells,
15 revealed that glial end feet did not separate autofluorescent perivascular cells from the blood vessel basement membrane (
Figs. 1G,
1H). Transmission electron microscopic (TEM) analysis confirmed the localization of autofluorescent perivascular cells in the retinal perivascular space (
Fig. 2). The location of retinal autofluorescent perivascular cells was frequently associated with endothelial cell tight junctions (
Fig. 2B). Osmium tetroxide, used during postfixation in TEM studies, binds to lipids leading to electron density of protein-bound oxidized lipids.
10 Similarly to brain Mato cells, retinal autofluorescent perivascular cells presented lipidic electron-dense inclusion bodies (
Figs. 2B,
2C,
2D). A schematic representation of retinal autofluorescent perivascular cell topography is presented in
Figure 2E.
Cell Type of Retinal Autofluorescent Perivascular Cells
Immunocytochemistry and histochemistry revealed that retinal autofluorescent perivascular cells were not microglia, since they were Iba-1
− (
Fig. 3A) and only faintly marked by tomato lectin (
Fig. 3B). Iba-1 is a calcium-binding protein of microglial cells present in rodents,
16 and recently has been considered the most reliable marker of microglia in mouse retina.
17 The tomato lectin, a lectin obtained from
Lycopersicon esculentum, with affinity for poly-
N-acetyl lactosamine sugar residues, has also been reported to mark microglia in mouse retina.
17 The tomato lectin labeling of autofluorescent perivascular cells was substantially different from that of microglial cells, which exhibit a characteristic ramified or ameboid cytoplasmic labeling with this marker (
Fig. 3B). Immunocytochemistry also revealed that retinal autofluorescent perivascular cells were retinal macrophages since they expressed CD11b (
Fig. 3C) and F4/80 antigens (
Fig. 3D), which are both markers for murine macrophagic cells.
18,19 Moreover, retinal autofluorescent perivascular cells were not microglia since they also expressed the BM8 antigen (
Fig. 3E), a 125-kDa extracellular membrane protein sensitive to 2-mercaptoethanol, which is not expressed by microglial cells.
20,21 Flow cytometry analysis showed that autofluorescent CD11b- and BM8-positive cells constituted approximately 20% of retinal macrophages and that they were not found in blood (
Figs. 3F,
3G), indicating that retinal autofluorescent perivascular cells were resident macrophages. Overall these findings suggest that retinal autofluorescent perivascular cells are a subset of the macrophagic cell lineage and do not constitute microglial cells.
Movement of Retinal Autofluorescent Perivascular Cells
Perivascular cells migrate along brain blood vessels.
22 To investigate the retinal autofluorescent perivascular cells' dynamic behavior, we performed time-lapse laser scanning confocal imaging. Autofluorescent perivascular cells were identified by their characteristic autofluorescence, and blood vessels were identified in transmission mode after gelatinized India ink injection. Time-lapse sequences showed that retinal autofluorescent perivascular cells moved in an oscillatory manner along the external surface of the vascular wall at rates up to 15 μm/h (
Fig. 4A). Retinal venules presented thinner areas in the basement membrane as a consequence of lower expression of collagen IV (
Fig. 4B), similarly to what occurs in cremasteric venules.
23 Retinal autofluorescent perivascular cells were observed in close contact with collagen IV low-density areas in the basement membrane (
Fig. 4B).
Expression of Scavenger Receptor Class A and Accumulation of Blood-Borne Molecules by APMs in Healthy Conditions
In the brain, APMs are considered scavenger cells.
2 To determine whether retinal APMs expressed scavenger receptors, the anti-2F8 antibody, a specific marker for macrophage scavenger receptor class A (SR-A),
24 was used. Confocal microscopy and flow cytometry showed that retinal APMs expressed SR-A (
Figs. 5A,
5B). These scavenger receptors appeared surrounding autofluorescent inclusions, suggesting that SR-A were localized in the lysosomal membrane. The percentage of autofluorescent 2F8-positive cells in retina was similar to that of autofluorescent BM8-positive cells (
Fig. 3G), suggesting they are the same cellular population. Microglia only expresses scavenger receptors when activated in response to injury.
7,8 Therefore, these findings suggest that retinal autofluorescent perivascular cells are a perivascular macrophagic cell type with scavenger function in healthy conditions.
In the brain APMs accumulate blood-borne proteins under physiological conditions.
1,2 HRP is a tracer used to assess The BRB's integrity.
25,26 To investigate whether retinal autofluorescent perivascular cells accumulate blood-borne proteins without BRB disruption, HRP was intravenously injected in healthy mice. As expected, no HRP leakage from blood vessels was found in healthy retinas, even when a threefold higher concentration of HRP than that used in previous studies of BRB integrity was used.
27 However, 6 hours after intravenous injection of HRP, perivascular cells, in a position and distribution compatible with that of APMs, presented multiple HRP brownish inclusions in their cytoplasm (
Fig. 6A). To examine whether retinal autofluorescent perivascular cells accumulate blood-borne low-density lipoproteins (LDLs), DiI-ac-LDL was intravenously injected in healthy mice. Two hours after injection, DiI cytoplasmic red granular fluorescence was observed in cells in a position and distribution compatible with that of APMs (
Fig. 6B).
To rule out the possibility that APMs that take up HRP molecules in healthy retina are circulating monocytes/macrophages that, after taking up plasma HRP, migrate through the vessel wall, we performed an experiment with the combination of Bis-benzimide and HRP tail vein injections. Bis-benzimide interacts with DNA and stains nuclei of all cell types. In the retina, as this stain does not cross the BRB because the tight junctions between endothelial cells prevent access of the stain to extravascular elements, no nuclear staining is expected in the retinal parenchyma.
28 However, when the BRB is disrupted, the dye can access the retinal parenchyma labeling neurons and glia.
29 In our experiment Bis-benzimide was injected 10 minutes before HRP. This situation allows us to mark all intravascular monocytes/macrophages before the HRP injection. Thus, any HRP-loaded monocyte/macrophage that migrates through the vessel wall will be detected by both nuclear bright blue Bis-benzimide staining and HRP brownish cytoplasmic content.
The results obtained showed that, as expected, only the perivascular macrophages contained HRP (
Fig. 6C), and, in addition, no Bis-benzimide nuclear staining of HRP loaded APMs was observed (
Figs. 6D,
6E). This result suggests that APMs are not circulating cells that, after taking up plasma HRP, migrate through the vessel wall. Taken together, all these results suggest that retinal autofluorescent perivascular cells scavenge and accumulate blood-borne proteins in healthy retinas.
Migration of Retinal APMs during BRB Breakdown
To better understand the scavenging function of APMs, we performed an experiment in which the BRB was disrupted with mannitol. Mannitol induces an osmotic opening of the BRB, with widening of the interendothelial tight junctions to an estimated radius of 200 Å.
30 Plasma HRP was seen to leak from the blood vessels to the retinal parenchyma after mannitol-induced BRB breakdown (
Fig. 7A). As expected, cellular nuclei in the leakage area were marked with Bis-benzimide (
Fig. 7B), confirming that the BRB was broken. The analysis of autofluorescence by laser confocal microscopy showed that autofluorescent cells quickly (30 minutes) migrate to the leakage area (
Fig. 7C). Some of the migrating autofluorescent cells in the leakage area were not marked by Bis-benzimide (
Fig. 7C), suggesting that these autofluorescent cells migrate from non-BRB–disrupted areas to the lesion site. These results reinforce the scavenging function of APMs during BRB breakdown.
Migration of Retinal APMs in a Photoreceptor Degeneration Mouse Model
In the brain, APMs are implicated in encephalopathies. After cerebral injury, blood plasma infiltrating perivascular spaces is taken up by APMs that are the first cell type activated during encephalopathy.
3,31 We used a murine model of photoreceptor degeneration, to determine the role of APMs in retinopathy. Forty-eight hours after sodium iodate injection, alteration of photoreceptors and gliosis were observed (
Fig. 8A), indicating that retinopathy was well established. In the altered retinas, autofluorescent cells positive for BM8 (
Fig. 8B), MOMA-2 (
Fig. 8C), and 2F8 (
Fig. 8D) were detected within the outer nuclear layer, suggesting that retinal APMs migrate to lesion sites.
To provide quantitative data on the number of APMs cells through the retinal layers after sodium iodate injection, we counted the number of autofluorescent cells present in the inner retina (ganglion cell layer+inner plexiform layer+inner nuclear layer) and the outer retina (outer plexiform layer+external nuclear layer+inner and outer photoreceptor segments;
Fig. 9). After injection, 2.2 ± 0.06 autofluorescent perivascular cells per field in the inner retina were observed. However, no autofluorescent cells were detected in the outer retina (
Figs. 9A,
9B). Twenty-four hours after injection, the perivascular autofluorescent cells leave the inner retina and only 0.9 ± 0.13 cells per field were detected, whereas in the outer retina 3.3 ± 0.71 autofluorescent cells per field were counted (
Fig. 9A). Finally, at 48 hours after injection autofluorescent perivascular cells nearly disappeared (0.2 ± 0.01) and a big population of autofluorescent cells (50.6 ± 7.25, per field) were observed in the outer retina (
Figs. 9A,
9C). From the data it is clear that the population of autofluorescent cells that migrate to the outer retina do not represent only the APMs, and it is likely that activated microglial cells also migrate to the lesion site. To clarify this point, the Iba-1 microglial marker was used. As expected, many autofluorescent cells in the outer retina were microglial cells (
Fig. 9C).
Autofluorescent Perivascular Cells in Human Retinas
APMs in the brain were described not only in animals but also in humans.
3 To determine whether human retinas may also contain autofluorescent perivascular cells, wholemount retinas were analyzed. Similar to what was observed in mouse, perinuclear autofluorescent granular inclusions were detected in cells around the blood vessels (
Figs. 10A–C). These perivascular cells accumulated lipids marked with oil red O (
Fig. 10D). All these findings suggest that autofluorescent perivascular cells exist in human retina. Furthermore, in contrast with healthy retinas (
Fig. 10E), autofluorescent cells were detected at lesions in a donor with marked gliosis (
Fig. 10F) as occurred in the murine model of retinopathy induced by sodium iodate.
Discussion
Several terms are used to describe macrophages around retinal blood vessels, including perivascular macrophages and perivascular and paravascular microglia, which reflects the heterogeneity within this population.
5 Fluorescence, topography, immunophenotype, and scavenger activity, observed in the autofluorescent perivascular retinal cell population analyzed, are consistent with those described by Mato et al.
1 for brain APMs.
Autofluorescent perivascular cells found in the retina were different from perivascular retinal microglia due to the following features: (1) They expressed the BM8 and MOMA-2 antigens which are never expressed by microglial cells
20,21 ; (2) they do not express Iba-1 protein, one of the most reliable markers of microglia
17 ; (3) they constitutively expressed scavenger receptors class A; microglia express these scavenger receptors only when activated
7,8 ; and (4) they emitted autofluorescence different from that of microglia.
11 In addition, autofluorescence in perivascular microglia was observed only in aging mice, whereas retinal autofluorescent perivascular cells presented autofluorescent granules even in the first month of life.
Table 1 summarizes the immunophenotype of the APMs in comparison with the previously published data for microglia.
| Iba-1 | TL | CD11b | F4/80 | BM8 | MOMA-2 | 2F8 |
| APM | − | + | +++ | +++ | +++ | +++ | +++ |
| Microglia | +++17 | +++17 | +++18 | +++19 | −20,21 | −21 | −7,8,24 |
Normal retinal function requires a strict control of the extracellular environment. The BRB regulates the extracellular environment by excluding blood-borne proteins from the retina.
32 This barrier is composed of the retinal vascular endothelium and the retinal pigment epithelium.
32 However, the data presented herein may suggest that small amounts of blood-borne proteins pass through the vascular endothelium in healthy retinas without BRB pathologic disruption. Autofluorescent perivascular cells found in mouse retina may act as scavenger macrophagic cells phagocytosing and accumulating blood-borne proteins (HRP and ac-LDL). This is also observed in BBB, where APMs phagocytose and accumulate these leakage proteins, thereby helping to prevent brain malfunction.
2,33 Therefore, retinal APMs, by their scavenger activity, may contribute to the BRB function in healthy conditions. Moreover, APMs migrate to the leakage areas during BRB breakdown.
APMs in the brain constitute the first type of macrophage activated during encephalopathy.
2,3 In experimental retinopathy, macrophagic 5D4-negative cells migrate early to the lesion site, whereas 5D4-positive microglial cells appear only in the late phase of photoreceptor degeneration,
34,35 suggesting that macrophages different from microglial cells are involved earlier in retinopathy. Similarly, our results indicated that APMs different from microglia appeared early at the lesion site in a murine model of experimental retinopathy.
In summary, this study describes a macrophagic, nonmicroglial, autofluorescent perivascular cell type in healthy retinas that is similar to the APMs found in brain. Retinal autofluorescent perivascular cells may contribute to the BRB function, scavenging and accumulating blood-borne proteins. Furthermore, their migration to the lesion site in BRB breakdown and in experimental and human retinopathy may suggest a key role of retinal APMs during retinopathy.
Supported by two grants from Instituto de Salud Carlos III (FIS PI061837 and CIBER de Diabetes y Enfermedades Metabólicas Asociadas) and the Interdisciplinary Centre of Research in Animal Health (CIISA) from the Faculty of Veterinary Medicine, Lisbon, Portugal (72.Retina).
Disclosure:
L. Mendes-Jorge, None;
D. Ramos, None;
M. Luppo, None;
C. Llombart, None;
G. Alexandre-Pires, None;
V. Nacher, None;
V. Melgarejo, None;
M. Correia, None;
M. Navarro, None;
A. Carretero, None;
S. Tafuro, None;
A. Rodriguez-Baeza, None;
J.A. Esperança-Pina, None;
F. Bosch, None;
J. Ruberte, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Carlos Andrade Fontes for critical revision of the manuscript and Pedro Otaegui and Lorena Noya for technical assistance.
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