Stromal Cell-Derived Factor-1 Is Essential for Photoreceptor Cell Protection in Retinal Detachment
2010; Elsevier BV; Volume: 177; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.100134
ISSN1525-2191
AutoresHiroki Otsuka, Noboru Arimura, Shozo Sonoda, Makoto Nakamura, Teruto Hashiguchi, Ikuro Maruyama, Shintaro Nakao, Ali Hafezi‐Moghadam, Taiji Sakamoto,
Tópico(s)Retinopathy of Prematurity Studies
ResumoStromal cell-derived factor-1 (SDF-1) causes chemotaxis of CXCR4-expressing bone marrow-derived cells. SDF-1 is involved in the pathogenesis of various vascular diseases, including those of the eye. However, the role of SDF-1 in neuronal diseases is not completely understood. Here, we show higher SDF-1 levels in the vitreous humor of patients with retinal detachment (RD) compared with normal patients. SDF-1 correlated positively with the duration as well as the extent of RD. Furthermore, SDF-1 correlated significantly with levels of interleukin-6 and interleukin-8, but not with vascular endothelial growth factor. Western blot analysis results showed significant SDF-1 up-regulation in detached rat retinas compared with normal animals. Immunohistochemistry data showed that SDF-1 was co-localized with the glial cells of the detached retina. SDF-1 blockade with a neutralizing antibody increased photoreceptor cell loss and macrophage accumulation in the subretinal space. The retinal precursor cell line R28 expressed CXCR4. SDF-1 rescued serum starvation-induced apoptosis in R28 cells and enhanced their ability to participate in wound closure in a scratch assay. Our results indicate a surprising, protective role for SDF-1 in RD. This effect may be mediated directly or indirectly through other cell types. Stromal cell-derived factor-1 (SDF-1) causes chemotaxis of CXCR4-expressing bone marrow-derived cells. SDF-1 is involved in the pathogenesis of various vascular diseases, including those of the eye. However, the role of SDF-1 in neuronal diseases is not completely understood. Here, we show higher SDF-1 levels in the vitreous humor of patients with retinal detachment (RD) compared with normal patients. SDF-1 correlated positively with the duration as well as the extent of RD. Furthermore, SDF-1 correlated significantly with levels of interleukin-6 and interleukin-8, but not with vascular endothelial growth factor. Western blot analysis results showed significant SDF-1 up-regulation in detached rat retinas compared with normal animals. Immunohistochemistry data showed that SDF-1 was co-localized with the glial cells of the detached retina. SDF-1 blockade with a neutralizing antibody increased photoreceptor cell loss and macrophage accumulation in the subretinal space. The retinal precursor cell line R28 expressed CXCR4. SDF-1 rescued serum starvation-induced apoptosis in R28 cells and enhanced their ability to participate in wound closure in a scratch assay. Our results indicate a surprising, protective role for SDF-1 in RD. This effect may be mediated directly or indirectly through other cell types. Chemokines are a family of polypeptides that act as potent chemoattractants. 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115: 86-93Crossref PubMed Scopus (234) Google Scholar, 13Sonmez K Drenser KA Capone Jr, A Trese MT Vitreous levels of stromal cell-derived factor 1 and vascular endothelial growth factor in patients with retinopathy of prematurity.Ophthalmology. 2008; 115: 1065-1070Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Previously, we reported elevated vitreous levels of SDF-1 in patients with retinal vein occlusion.14Ki IY Arimura N Noda Y Yamakiri K Doi N Hashiguchi T Maruyama I Shimura M Sakamoto T Stromal-derived factor-1 and inflammatory cytokines in retinal vein occlusion.Curr Eye Res. 2007; 32: 1065-1072Crossref PubMed Scopus (19) Google Scholar In addition, SDF-1/CXCR4 potentially mediates ocular inflammation by recruiting CD4+ T-cells, and is potentially involved in the formation of proliferative membranes in eyes with proliferative vitreoretinopathy.15Curnow SJ Wloka K Faint JM Amft N Cheung CM Savant V Lord J Akbar AN Buckley CD Murray PI Salmon M Topical glucocorticoid therapy directly induces up-regulation of functional CXCR4 on primed T lymphocytes in the aqueous humor of patients with uveitis.J Immunol. 2004; 172: 7154-7161PubMed Google Scholar, 16Abu El-Asrar AM Struyf S Kangave D Geboes K Van Damme J Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy.Eur Cytokine Netw. 2006; 17: 155-165PubMed Google Scholar Therefore, interest in understanding the role of SDF-1/CXCR4 in non-neovascular inflammatory or proliferative ocular diseases remains great. Retinal detachment (RD), the physical separation of the neural layer of the retina from the subjacent retinal pigment epithelium, results in photoreceptor cell death.17Cook B Lewis GP Fisher SK Adler R Apoptotic photoreceptor degeneration in experimental retinal detachment.Invest Ophthalmol Vis Sci. 1995; 36: 990-996PubMed Google Scholar, 18Yang L Bula D Arroyo JG Chen DF Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice.Invest Ophthalmol Vis Sci. 2004; 45: 648-654Crossref PubMed Scopus (89) Google Scholar Because of the irreversible nature of the damage, a long duration of RD can cause permanent vision loss.19Arroyo JG Yang L Bula D Chen DF Photoreceptor apoptosis in human retinal detachment.Am J Ophthalmol. 2005; 139: 605-610Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar Thus, new insights into the photoreceptor protection in RD would be of great clinical interest, as they could lead to new treatments. Because, the retina is an accessible part of the brain, it also offers a unique opportunity for studies of the central nervous system. Given that RD usually occurs without infectious inflammation or destructive ischemia, it provides a suitable context for investigating morphological changes in neural disorders and a local sterile inflammation. The CC chemokine monocyte chemotactic protein-1, erythropoietin, and interleukin (IL)-6 have recently been implicated in neuro protection.20Digicaylioglu M Lipton SA Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades.Nature. 2001; 412: 641-647Crossref PubMed Scopus (863) Google Scholar, 21Chong DY Boehlke CS Zheng QD Zhang L Han Y Zacks DN Interleukin-6 as a photoreceptor neuroprotectant in an experimental model of retinal detachment.Invest Ophthalmol Vis Sci. 2008; 49: 3193-3200Crossref PubMed Scopus (62) Google Scholar Monocyte chemotactic protein-1 is a critical mediator of RD-induced photoreceptor apoptosis.22Nakazawa T Hisatomi T Nakazawa C Noda K Maruyama K She H Matsubara A Miyahara S Nakao S Yin Y Benowitz L Hafezi-Moghadam A Miller JW Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.Proc Natl Acad Sci USA. 2007; 104: 2425-2430Crossref PubMed Scopus (234) Google Scholar This study elucidates the role of SDF-1 in RD by using human vitreous samples in vivo and in vitro. This study was approved by the institutional ethical committee at University of Kagoshima, and was performed in accordance with the Declaration of Helsinki. All surgeries were performed at Kagoshima University Hospital. All patients provided informed consent before receiving treatment. Undiluted vitreous fluid samples (0.5 to 0.7 ml) were obtained from the same sites of the anterior vitreous by pars plana vitrectomy. Vitreous humor was collected in sterile tubes, placed immediately on ice, centrifuged to remove cells and debris, and stored at −80°C until analysis. The clinical histories of all patients were obtained from their medical records. In patients with rhegmatogenous RD (RRD), preoperative data collection included time from onset of symptoms to surgery and extent of detached retina. Measurements of vitreous levels of SDF-1α, vascular endothelial growth factor (VEGF), and inflammatory cytokines/chemokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor-α) were measured as we described previously.23Arimura N Otsuka H Yamakiri K Sonoda Y Nakao S Noda Y Hashiguchi T Maruyama I Sakamoto T Vitreous mediators after intravitreal bevacizumab or triamcinolone acetonide in eyes with proliferative diabetic retinopathy.Ophthalmology. 2009; 116: 921-926Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar SDF-1α and VEGF were quantified by using commercial enzyme-linked immunosorbent assays (Human CXCL12/SDF-1 α Quantikine ELISA Kit, Human VEGF Quantikine ELISA Kit; R&D Systems, Minneapolis, MN). Six inflammatory cytokines/chemokines were quantified by using commercial multiplex Cytometric Bead Array systems: Human Inflammation Kit (BD Biosciences Pharmingen, San Diego, CA). All concentrations less than the detection level were assigned a value of 0 in the subsequent analysis. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and approval of our institutional animal care committee. Brown Norway rats (Kyudo, Fukuoka, Japan), postnatal 8 weeks, were studied as follows. RD was induced in the right eyes as we described previously.23Arimura N Otsuka H Yamakiri K Sonoda Y Nakao S Noda Y Hashiguchi T Maruyama I Sakamoto T Vitreous mediators after intravitreal bevacizumab or triamcinolone acetonide in eyes with proliferative diabetic retinopathy.Ophthalmology. 2009; 116: 921-926Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar The rats were anesthetized with an intramuscular injection of ketamine and xylazine, and their pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride. The retinas were detached by using a subretinal injection of 1% sodium hyaluronate (Opegan; Santen, Osaka, Japan) with an anterior chamber puncture to reduce intraocular pressure. Sclera was penetrated at the ocular nasal equator with a 30-Gauge needle. Then, sodium hyaluronate (0.05 ml) was gently injected through the sclera into the subretinal space to enlarge the RDs. These procedures were performed only in the right eye, with the left eye serving as a control. Eyes with lens injury, vitreous hemorrhage, infection, and spontaneous reattachment were excluded from the analysis. In some eyes, immediately after RD induction, 1 μg anti-SDF-1α antibody (MAB310; R&D Systems) or 1 μg isotype control IgG1 monoclonal antibody (MAB002; R&D Systems) was injected intravitreally from the temporal limbus of the same eye by using a 33-Gauge needle (Hamilton, Reno, NV) in a 10-μl volume. Doses were based on previous studies in mice.24Sasahara M Otani A Oishi A Kojima H Yodoi Y Kameda T Nakamura H Yoshimura N Activation of bone marrow-derived microglia promotes photoreceptor survival in inherited retinal degeneration.Am J Pathol. 2008; 172: 1693-1703Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar The rats were sacrificed on days 3 and 7 after treatment, and the eyes were harvested for study. SDF-1 expression in the detached retinas was examined by Western blot, as described previously.25Xie Z Wu X Qiu Q Gong Y Song Y Gu Q Li C Expression pattern of erythropoietin and erythropoietin receptor in experimental model of retinal detachment.Curr Eye Res. 2007; 32: 757-764Crossref PubMed Scopus (25) Google Scholar The rats were sacrificed 3 days after RD induction. The eyes were enucleated immediately, and the anterior segment and vitreous were removed. The detached portion of the neurosensory retina was carefully peeled and harvested. Retinas were resuspended in lysis buffer (30 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 1% Nonidet P-40, and 10% grycerol), and centrifuged for 10 minutes at 4°C. Protein concentration in the supernatant was calculated by using the micro bicinchoninic acid protein assay kit (Pierce, Rockford, IL). An aliquot of 50 μg total extract was mixed with protein loading buffer containing methoxyethanol, and boiled for 5 minutes before being loaded onto 15% SDS-polyacrylamide gels, then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked by incubation with blocking buffer (Tris-buffered saline [pH 7.5] with 5% nonfat dry milk and 0.1% Tween-20) for 1 hour at room temperature. The membrane was then incubated with rabbit polyclonal anti-SDF-1α antibody (1:1000; Abcam, Cambridge, UK) at 4°C overnight. The blots were subsequently probed with secondary anti-rabbit antibodies conjugated to horseradish peroxidase, and images were developed by using the enhanced chemiluminescence system plus (GE Health care, Tokyo, Japan). The eyes were fixed in 4% paraformaldehyde at 4°C overnight. The anterior segment and the lens were removed, and the remaining eye cup was cryoprotected with 10% to 30% sucrose in PBS. The eye cups were then frozen in an optimal cutting temperature compound (Sakura Finetech, Tokyo, Japan). Frozen sections (8 μm) were dried and blocked with blocking buffer for 1 hour. The antibodies used for staining were rabbit polyclonal anti-SDF-1α antibody, rabbit polyclonal anti-CXCR4 antibody (each 1:50; Abcam), monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (Sigma, St. Louis, MO), mouse anti-CD68 monoclonal antibody (ED1; 1:800; Serotec, Raleigh, NC), and mouse anti-CD31 monoclonal antibody (PECAM-1; 1:100; Abcam). Normal rabbit or mouse IgG was used instead of primary antibody as a negative control in each case. Secondary antibodies were Alexa-Fluor 488-conjugated goat anti-mouse IgG F(ab)2 fragment and Alexa-Fluor 594-conjugated goat anti-rabbit IgG F(ab)2 fragment (each 1:400; Molecular Probes, Carlsbad, CA). Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI), mounted with Shandon PermaFlour (Thermo Scientific, Waltham, MA), and viewed with a Olympus fluorescence microscope (Olympus, Tokyo, Japan). Images were captured by using the same exposure time for each comparative section. Confocal Laser Scanning Microscopy images were taken on an Olympus FV-500 confocal microscope (Olympus). For all experiments, at least three sections from each eye were evaluated. Immunofluorescent staining for CXCX4 was performed on R28 cells by using the same method. To evaluate the change of outer nuclear layer (ONL) thickness after RD, we compared the thickness of the ONL with the entire retina (defined as the distance between the internal limiting membrane to the external limiting membrane) in histological sections (three sections each point) stained with hematoxylin and eosin. Three separate measurements of retinal thickness were obtained from each retinal section by using image analysis software, Image J (National Institutes of Health, Bethesda, MD). To assess the number of ED1 positive macrophages infiltrating the subretinal space, we performed double immune-fluorescence staining with rabbit polyclonal anti-CXCR4 antibody and ED1. The number of ED1 positive cells was counted at three random fields in each eye in a masked fashion. The rat immortalized retinal precursor cell line R28, a gift from Dr. G. M. Siegel (The State University of New York, Buffalo), was cultured in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal bovine serum, 10 mmol/L nonessential amino acids, and 10 mg/ml gentamicin as described previously.26Neekhra A Luthra S Chwa M Seigel G Gramajo AL Kuppermann BD Kenney MC Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells.Invest Ophthalmol Vis Sci. 2007; 48: 1362-1367Crossref PubMed Scopus (25) Google Scholar Cells were incubated at 37°C in a 5% CO2 incubator and subcultured with 0.05% trypsin-EDTA. Subconfluent cultures were trypsinized and seeded for the following experiments. To induce cell death by serum starvation, R28 cells at 50% confluence in 24-well tissue culture plates were washed with PBS twice, and the culture medium was replaced with serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin. After 6 hours of synchronization, recombinant SDF-1α (PeproTech, London, UK) or PBS was added to the wells at the indicated concentrations. After incubation for 48 hours, cell survival was assessed by trypan blue dye exclusion assay in a masked fashion, as described previously.27Sareen D van Ginkel PR Takach JC Mohiuddin A Darjatmoko SR Albert DM Polans AS Mitochondria as the primary target of resveratrol-induced apoptosis in human retinoblastoma cells.Invest Ophthalmol Vis Sci. 2006; 47: 3708-3716Crossref PubMed Scopus (89) Google Scholar Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) procedure and quantification of TUNEL-positive cells were performed by using an ApopTag fluorescein direct in situ apoptosis detection kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. The number of TUNEL-positive cells was counted in a masked fashion. For scratch wound assay, R28 cells were grown to 90% confluence in 6-well tissue culture plates and serum starved for 6 hours before experiment. Then, R28 cells were scratched with a sterile 0.1- to 10-μl pipette tip (TipOne; USA Scientific, Ocala, FL) to remove cells with three parallel linear scrapes. The debris of damaged cells was removed by washing, and the cells were refed with serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin in the presence or absence of recombinant (rSDF-1) (100 ng/ml). The progression of wound healing was photographed immediately and 24 hours after wounding, at the same field near the marked point, using an inverted microscope (Olympus CKX41; Olympus) equipped with a digital camera. The extent of healing is defined as the ratio of the area difference between the original wound and the remaining wound 24 hours after injury compared with the original wound.28Xu KP Yu FS Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing.Invest Ophthalmol Vis Sci. 2007; 48: 2242-2248Crossref PubMed Scopus (77) Google Scholar The wound area was determined by the number of pixels in histogram (Photoshop CS3; Adobe, San Jose, CA). R28 cells (5 × 105) were subcultured on 6-cm tissue culture dishes. The cells were serum-starved for 6 hours and then stimulated with or without rSDF-1 (100 ng/ml) for 24 hours.29Murakami Y Ikeda Y Yonemitsu Y Onimaru M Nakagawa K Kohno R Miyazaki M Hisatomi T Nakamura M Yabe T Hasegawa M Ishibashi T Sueishi K Inhibition of nuclear translocation of apoptosis-inducing factor is an essential mechanism of the neuroprotective activity of pigment epithelium-derived factor in a rat model of retinal degeneration.Am J Pathol. 2008; 173: 1326-1338Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar For inhibition studies, U0126 (Promega, Madison, WI) was added 1 hour before SDF-1 treatment. In brief, whole cells were lysed with SDS sample buffer and a 80-μg volume of protein extracts was loaded onto 15% SDS-polyacrylamide gels, then transferred onto a polyvinylidene difluoride membrane. After blocking, the membrane was reacted with anti-Bcl-2 antibody (1:1000; Cell Signaling Technology, Beverly, MA) at 4°C overnight. The blots were subsequently probed with secondary antibodies and images were developed. To analyze activation of ERK-1/2, the membrane was reacted with phospho-ERK-1/2 (1:1000; Cell Signaling Technology). After detection of phospho-ERK-1/2, the blot was stripped and re-probed with an antibody against total ERK-1/2 (1:1000; Cell Signaling Technology), as described previously.30Arimura N Ki-i Y Hashiguchi T Kawahara K Biswas KK Nakamura M Sonoda Y Yamakiri K Okubo A Sakamoto T Maruyama I Intraocular expression and release of high-mobility group box 1 protein in retinal detachment.Lab Invest. 2009; 89: 278-289Crossref PubMed Scopus (47) Google Scholar The vitreous inflammatory cytokine/chemokine concentrations in each group were compared by using the Mann-Whitney U-test. The correlation between inflammatory cytokines/chemokines in RD samples was analyzed by using a simple linear regression analysis and Spearman's rank correlation coefficient. All in vitro and vivo data are presented as mean ± SEM, and the significance of differences between groups was determined by Student's t-test. P values less than 0.05 were considered significant. To investigate the role of SDF-1 in non-neovascular ocular diseases, we quantified the levels of human vitreous SDF-1 in RRD, epiretinal membrane (ERM), and macular hole (MH). PDR, a neovascular disease with high levels of SDF-1 in the vitreous, was used as a positive control.31Brooks Jr, HL Caballero Jr, S Newell CK Steinmetz RL Watson D Segal MS Harrison JK Scott EW Grant MB Vitreous levels of vascular endothelial growth factor and stromal-derived factor 1 in patients with diabetic retinopathy and cystoid macular edema before and after intraocular injection of triamcinolone.Arch Ophthalmol. 2004; 122: 1801-1807Crossref PubMed Scopus (194) Google Scholar Samples were harvested from 30 eyes with PDR, 44 eyes with RRD, 11 eyes with ERM, and 18 eyes with MH. There were no statistically significant differences in age and sex of patients (Table 1).Table 1Patient CharacteristicsCharacteristicsPDRRRDERMMHPSubjects, no.30441118Age (yr)62 (31–80)59 (44–86)69 (50–75)68 (49–78)0.184*Kruskal-Wallis variance analysis.Female sex no. (%)15 (50)18 (41)6 (55)11 (61)0.849†χ2 test.Values are expressed as the median (range) or number (%).* Kruskal-Wallis variance analysis.† χ2 test. Open table in a new tab Values are expressed as the median (range) or number (%). SDF-1 concentration of RRD (58.5 pg/ml) was significantly higher than that of ERM (21.2 pg/ml, P = 0.012 and P < 0.016 after Bonferroni correction). Significant differences were not found between RRD and MH (10.0 pg/ml), or between MH and ERM (Figure 1A). The vitreous VEGF level in PDR was highest and comparable to that of SDF-1. In contrast, there was surprisingly no VEGF detectable in RRD, ERM, and MH (Figure 1B). Next, we examined whether there is a relation between vitreous SDF-1 concentration and pathological condition in eyes with RRD, in terms of duration of disease (range, 3 to 120 days) and extent of detached retina (range, 0.5 to 4 quadrants; Table 2). The vitreous SDF-1 level in RRD was positively correlated with duration of disease and extent of detached retina in RRD by a simple linear regression (r = 0.501, P < 0.001, r = 0.339, P = 0.024, respectively) and by a Spearman's rank correlation coefficient (r = 0.347, P = 0.025, r = 0.323, P = 0.040, respectively; Figure 1, C and D).Table 2Clinical State of Rhegmatogenous Retinal DetachmentDuration of disease (days)15 (3–120)Extent of detached retina (quadrants)1.5 (0.5–4)Values are expressed as the median (range). Open table in a new tab Values are expressed as the median (range). Furthermore, vitreous SDF-1 showed a positive correlation with IL-6 (r = 0.484, P = 0.002) and IL-8 (r = 0.400, P = 0.009) by Spearman's rank correlation coefficient (Figure 1, E and F), but not by simple linear regression. The vitreous IL-6 in RRD positively correlated with the extent of detached retina by a Spearman's rank correlation coefficient (r = 0.342, P = 0.027; data not shown); however, it did not correlate with disease duration. To investigate SDF-1 and CXCR4 expression in the retina after RD, 3 days after detachment was induced in Brown Norway rats, neurosensory retina was harvested and expression of these proteins was analyzed by Western blotting. The SDF-1 protein level in the detached retina was substantially higher (P < 0.01), 3 days after RD, compared with the untreated control (Figure 2, A and B). Immunofluorescent staining revealed sparse SDF-1 expression in the inner border and the outer plexiform layer of the retina in the control. In contrast, up-regulation of the expression of SDF-1 occurred with RD (Figure 3, A and B), which seemed to be colocalized with the activated GFAP-positive astrocytes or Müller cells (Figure 3, C–F). In the normal retina, CXCR4 staining mainly was present in the ganglion cell layer and the inner nuclear layer of the retina. Additionally, CXCR4 was strongly positive in the outer nuclear layer and photoreceptor inner segments of the detached retina (Figure 4, A–D). Double immunofluorescent staining of CXCR4 showed strong staining in some ED-1-positive macrophages infiltrating into the subretinal space (Figure 5, A–C).Figure 5CXCR4 is expressed in the macrophages infiltrating into subretinal space. The retinal sections were derived at 3 days after detachment. A: CXCR4 staining was positive in the macrophages infiltrating into subretinal space. B: ED-1 stained macrophages in subretinal space. C: Merged image of CXCR4 (red), ED1 (green), and DAPI (blue). ONL, Outer nuclear layer; IS, inner segment. Original magnifications, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further define the role of SDF-1 in RD, after sodium hyaluronate injection into the subretinal space, some rats were intravitreally injected with anti-SDF-1 antibody or nonbinding control antibody. Three days after RD, there were no apparent morphological differences between the Ab-injected animals and normal controls. Seven days after RD, there was thinning of the outer nuclear layer due to photoreceptor loss and deconstruction of the inner and outer segments of the photoreceptors. The ratio of the thickness of the ONL to the entire retina differed significantly between the noninjected (0.278 ± 0.015) and anti-SDF-1 Ab group (0.228 ± 0.011, P < 0.05), or between anti-SDF-1 Ab and control Ab group (0.294 ± 0.018, P < 0.01), respectively. No significant difference was found between noninjected and control Ab group (Figure 6, A and B). In normal eyes without detachment, SDF-1 blockade did not cause apparent retinal morphological change (data not shown). This suggests that SDF-1 blockade results in photoreceptor cell loss only at the site of detachment. TUNEL staining of rat eyes 3 days after detachment revealed significantly higher percentages of TUNEL-positive cells in the ONL of retinas treated with anti-SDF-1 Ab compared with those treated with control Ab (P < 0.05) or untreated control (P < 0.05; Figure 6, C and D). To assess the impact of SDF-1 blockade on the recruitme
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