December 2007
Volume 48, Issue 12
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Retina  |   December 2007
Upregulation of VEGF in Murine Retina via Monocyte Recruitment after Retinal Scatter Laser Photocoagulation
Author Affiliations
  • Masahiro Itaya
    From the Department of Ophthalmology and Visual Science and the
  • Eiji Sakurai
    From the Department of Ophthalmology and Visual Science and the
  • Miho Nozaki
    From the Department of Ophthalmology and Visual Science and the
  • Kiyoshi Yamada
    From the Department of Ophthalmology and Visual Science and the
  • Satoshi Yamasaki
    From the Department of Ophthalmology and Visual Science and the
  • Kiyofumi Asai
    Department of Bioregulation, Research Institute for Molecular Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
  • Yuichiro Ogura
    From the Department of Ophthalmology and Visual Science and the
Investigative Ophthalmology & Visual Science December 2007, Vol.48, 5677-5683. doi:10.1167/iovs.07-0156
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      Masahiro Itaya, Eiji Sakurai, Miho Nozaki, Kiyoshi Yamada, Satoshi Yamasaki, Kiyofumi Asai, Yuichiro Ogura; Upregulation of VEGF in Murine Retina via Monocyte Recruitment after Retinal Scatter Laser Photocoagulation. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5677-5683. doi: 10.1167/iovs.07-0156.

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

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Abstract

purpose. This study was conducted to determine changes in the expression of vascular endothelial growth factor (VEGF) in murine retina after retinal scatter laser photocoagulation.

methods. Photocoagulation (PHC) was performed on wild-type C57BL/6J mice using a diode laser, and the eyes were enucleated 1, 2, 3, 4, 7, and 14 days after laser treatment. VEGF and monocyte chemoattractant protein (MCP)-1 levels in the sensory retina and retinal pigmented epithelial (RPE) cells in both tissues were measured by ELISA. The VEGF mRNA was measured by real-time RT-PCR. Leukocyte infiltration into the RPE-choroid was determined by flow cytometry. VEGF comparisons between mice subjected to PHC and those treated with monocyte recruitment inhibitor (anti-MCP-1) were performed and statistically analyzed. The expression of VEGF and MCP-1 in the retina was determined by immunohistochemistry.

results. VEGF protein levels significantly increased 1 day after PHC in both the RPE-choroid and the sensory retina. VEGF concentrations were maximum at day 3 after photocoagulation and stayed elevated until day 7. The number of choroid-infiltrating macrophages was markedly increased in mice with laser treatment compared with those without laser treatment. VEGF expression decreased after treatment with neutralized antibody to monocyte recruitment. We demonstrate that MCP-1 expression in the retina increased markedly after scatter laser photocoagulation by immunohistochemistry and ELISA.

conclusions. Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes.

Scatter laser photocoagulation is one of the most effective modalities for treating a variety of retinal diseases, especially ischemic retinal diseases such as diabetic retinopathy 1 and retinal vein occlusion. 2 However, inappropriate laser photocoagulation can induce some complications such as hemorrhage, 3 epiretinal membrane, 4 intraocular proliferation, 5 development of choroidal neovascularization, 6 and macular edema. 7 8 9 In particular, macular edema is sight-threatening, current therapy is ineffective, and the mechanism of action is still unknown. 9 10 11 12  
Vascular endothelial growth factor (VEGF) is a well-known potent angiogenic factor, and recruited macrophages can produce VEGF in response to laser injury. 13 14 15 16 17 VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells, an initial and essential step toward inflammation. 18 In addition, VEGF induces increased retinal vascular permeability. 19 20  
VEGF expression in laser induced choroidal neovascularization animal models has been studied 21 22 23 in recent years. In experimental models of laser photocoagulation, VEGF expression is upgraded as shown by the many cells (macrophages are predominant) that appear in the subretinal space after laser photocoagulation. 24  
In contrast, it has been demonstrated that intraocular levels of VEGF decline 19 25 when treatment with laser photocoagulation induces remission of retinal neovascularization for the treatment of diseases such as proliferative diabetic vitreoretinopathy, ischemic retinal vein occlusion, or retinopathy of prematurity where panretinal photocoagulation is used to prevent the formation of new blood vessels at the junction of vascularized tissue and the avascular retina. 
In this study, we observed changes in VEGF expression in the sensory retina and the retinal pigmented epithelial cells (RPE)-choroid after scatter laser photocoagulation in vivo. In addition, we compared this with VEGF expression after treatment with a series of specific monoclonal antibodies directed against monocyte chemoattractant protein (MCP)-1, which plays a role in the recruitment of macrophages and monocytes. 26  
Methods
Animals
Male wild-type C57BL/6 mice (Japan SLC, Shizuoka, Japan) between 6 and 8 weeks of age were used, to minimize variability. For all procedures, anesthesia was achieved by intramuscular injection of 50 mg/kg ketamine HCl (Sankyo, Tokyo, Japan) and 10 mg/kg xylazine (Bayer, Tokyo, Japan), and pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Santen, Osaka, Japan). All animal experiments were in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Induction of Scatter Laser Photocoagulation
Laser photocoagulation (Lumenis, Salt Lake City, UT) was performed (532 nm, 200 mW, 100 ms, 100 μm, 30 spots; Fig. 1 ) on both eyes of each mouse, and the eyeballs were enucleated 1, 2, 3, 7, and 14 day after laser photocoagulation. The fundus was observed using a slit lamp through a slide on the cornea with a 2% methylcellulose solution. 
Reagents
Reagents used in these studies were from the following sources. Anti-mouse CCL2/JE/MCP-1 antibody (R&D Systems, Minneapolis, MN); anti-goat IgG conjugated to Cy3 (Rockland, Gilbertsville, PA), rat anti-mouse F4/80 antibody (Serotec, Raleigh, NC), anti-IgG(H+L), goat rabbit-poly FITC (Vector Laboratories, Inc., Burlingame, CA), VEGF(147) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-rabbit IgG conjugated to FITC (Santa Cruz Biotechnology, Inc.). Antifade mounting medium for fluorescence with DAPI (4′,6′-diamino-2-phenylindole; Vectashield HardSet; Vector Laboratories); optimal cutting temperature (OCT) compound (Tissue Tek; Miles Laboratories, Elkhart, IN). 
Immunohistochemistry
Immunohistochemical methods were performed on cryosections for the detection of MCP-1, VEGF, and F4/80. After retinal scatter laser photocoagulation, the eyes were enucleated on days 2 and 3, and 5-μm sections were cut. Cryosections of eyes were fixed (Histochoice; Amresco, Inc., Solon, OH) for 15 minutes, washed with TBS and blocked with protein block (Dako, Inc., Carpinteria, CA) for 60 minutes. 
The primary antibodies against mouse MCP-1, VEGF, and F4/80 (1:100) were incubated for 1 hour at room temperature. After incubation with primary antibody (1:200), the slides were incubated with secondary antibody. Sections were then washed three times for 5 minutes in PBS and mounted in mounting medium with DAPI. Sections were viewed on a microscope (AX70; Olympus, Tokyo, Japan). 
Intravitreous Injection of Anti-MCP-1 Antibody
Immediately after laser photocoagulation, intravitreous injections of anti-MCP-1 antibody were performed by inserting a 33-gauge double-caliber needle (Ito Corp., Shizuoka, Japan) under an operating microscope. Animals received intravitreous injections of 1 μL sterile PBS containing 5, 1, or 0.2 ng of neutralization anti-mouse CCL2/JE/MCP-1 antibody (Clone 123616; R&D Systems). 
Enzyme-Linked Immunosorbent Assay for CCL2/MCP-1 and VEGF
At 1, 2, 3, 7, and 14 days after laser photocoagulation, eyes were enucleated, and the sensory retina and RPE-choroid complex were carefully isolated, placed in 150 μL of lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl2, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA with protease inhibitor; Sigma-Aldrich) and sonicated on ice for 15 seconds. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the CCL2/MCP-1 or VEGF levels in the supernatant were determined with a mouse CCL2/MCP-1 or VEGF ELISA kit (threshold of detection 3 pg/mL; Quantikine; R&D Systems) at 450 to 570 nm, with an absorption spectrophotometer (SpectraMax 34000A ROM, ver. 2.04; Bio-Rad, Hercules, CA), and normalized to total protein, according to the manufacturer’s protocol. A standard curve was plotted from measurements of diluted standard solutions (7.8–500 pg/mL) and the concentration of CCL2/MCP-1 or VEGF in each sample was determined in comparison with this curve. 
Quantitative Real-Time RT-PCR for VEGF
Real-time RT-PCR was performed in 96-well plates on a sequence-detection system (Prism 7700; Applied Biosystems, Inc. [ABI], Foster City, CA). Total RNA was extracted from the RPE-choroid (RNAqueous-4PCR Kit; Ambion, Inc., Austin, TX), and the cDNAs were reverse transcribed from total RNA samples (P/N 4322171, High-Capacity cDNA Archive Kit; ABI), as described by the manufacturer. RT-PCR was performed in 50-μL volumes containing 22.5 μL cDNA samples diluted with RNase-free water, 25 μL master mix (P/N 4324018; TaqMan Universal PCR Master Mix; ABI), and 2.5 μL random hexamers from gene expression assays (P/N 4331182; ABI). Reverse transcription included an incubation period of 2 minutes, holding at 50°C, 10 minutes hold at 95°C, followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. The assay number and target genes were MN_00441242 for mouse VEGF. The housekeeping gene (P/N 4308313; TaqMan Rodent GAPDH; ABI) was used as an endogenous control to normalize the expression data for each gene. The method used for obtaining quantitative data of relative gene expression, the comparative Ct (ΔΔCt) method, was as described by the manufacturer (ABI). 
Flow Cytometry
To determine the number of macrophages in the neurosensory retina or RPE-choroid, 3 days after laser injury by 30 laser spots, the eyes of each group were enucleated, and the neurosensory retina or RPE-choroid was isolated. Neurosensory retina and RPE-choroid were homogenized with a needle and treated with collagenase. Then they were filtered, and the single-cell suspensions isolated from mouse RPE-choroid via collagenase D (20 U/mL; Worthington, Lakewood, NJ) treatment were incubated in Fc block (0.5 mg/mL; BD Biosciences, Franklin Lakes, NJ) for 15 minutes on ice, and stained with Cy5-rat antibody against mouse F4/80 27 (1:30; Serotec, Cergy Saint-Christophe, France). Live cells were detected by gating on forward versus side scatter, followed by analysis of F4/80 in the fluorescence channel (FACSCalibur; BD Biosciences). At least 100,000 viable cells were analyzed per condition. Data were analyzed with the system software (Cellquest software; BD Biosciences). 
Statistics
VEGF data and results of flow cytometry were analyzed with commercial software (Excel; Microsoft, Redmond, WA). Results were considered significant at P < 0.05. 
Results
ELISA for VEGF
VEGF protein level was increased compared with control mice after laser treatment (Fig. 2) . VEGF concentrations in the treated mice increased in the RPE-choroid complex and the peak expression of VEGF protein was found on day 3 (11.2 ± 0.42 pg/mL; P < 0.001) after laser treatment. These levels had decreased on day 7 but remained high and decreased further on day 14 after laser treatment. 
Quantitative RT-PCR for VEGF
VEGF mRNA obtained from the RPE-choroid at 0 (no laser) days and at 1, 2, and 3 days after scatter laser photocoagulation were analyzed. The mRNA levels were increased from day 1 to day 3 after laser photocoagulation compared with untreated control animals, and the highest level of VEGF was a threefold increase at day 2 (Fig. 3 , P < 0.05). 
MCP-1 Expression after Retinal Scatter Laser Photocoagulation
MCP-1 was not detected in the control normal retina by immunohistochemistry and ELISA, and was strongly expressed mainly in the RPE layer of the laser injury site on day 2 by immunohistochemistry (Fig. 4) . MCP-1 levels in the neurosensory retina and RPE-choroid on day 0 were not detected using ELISA. Then the MCP-1 levels increased dramatically within 1 day after laser injury (neurosensory retina: 18.98 ± 1.234, RPE-choroid: 41.12 ± 8.312) and the peak expression of MCP-1 protein was found on day 2 (neurosensory retina: 28.61 ± 11.102, RPE-choroid: 48.19 ± 1.849) after scatter laser treatment. These levels decreased by day 7 (neurosensory retina: 0.73 ± 1.249, RPE-choroid: 1.86 ± 1.109; Fig. 5 ). 
Immunohistochemical Increase in VEGF in the Retina
Strong VEGF-positive immunoreactivity was detected in the photocoagulated site of the retinal pigmented epithelial layer. VEGF localized in infiltrated macrophages (Fig. 6)
Effect of Intravitreous Injection of Anti-MCP-1 Antibody
VEGF levels in the RPE-choroid complex on day 3 after intravitreous injection of the anti-MCP-1 antibody were comparably low and dependent on the administered anti-MCP-1 antibody concentration (Fig. 7) . VEGF levels of the group with intravitreous injections of 5 ng anti-MCP-1 antibody were significantly low (2.70 ± 1.37 pg/mL; P < 0.01) in comparison with the 0.2-ng injection (4.59 ± 0.90 pg/mL; P < 0.01) and compared with the non–drug-treated control subjects. 
Flow Cytometry
To detect the origin of VEGF, we measured the number of monocytes-macrophages in the neurosensory retina and RPE-choroid. In the group without laser injury, absolutely no macrophages were detected in the neurosensory retina and RPE-choroid. In the laser photocoagulation group without anti-MCP-1 antibody treatment, macrophages infiltrated the laser injury within 1 day, with peak response on day 3, followed by rapid disappearance by day 5 (data not shown). At day 3 after laser photocoagulation, macrophages significantly increased compared with the number in the control animals without laser treatment and were remarkably reduced by intravitreous injections of anti-MCP-1 antibody (Fig. 8)
Discussion
In the present study, we were able to show that VEGF is expressed in the sensory retina and RPE-choroid in increased intensities within a week after retinal scatter photocoagulation by ELISA, RT-PCR, and immunohistochemistry. In a preliminary experiment with flow cytometry, macrophages infiltrated the region of laser injury within 1 day, with peak response on day 3, followed by rapid disappearance by day 5. These changes in mRNA preceded macrophage infiltration. The VEGF mRNA level increased dramatically within 1 day after laser injury, and peak mRNA of VEGF was recorded on day 2. We measured VEGF protein levels in wild-type mice on days 0 through 5. VEGF protein levels began to increase 1 day after laser injury, and peaked on day 3, coinciding with peak macrophage infiltration, followed by a rapid decrease from day 3 to day 5. The levels of VEGF protein correlated with the mRNA levels. These data correlated highly with the number of macrophages after laser injury. 
Monocyte–macrophage recruitment is an early step in the initiation of inflammatory and angiogenic processes, and MCP-1, a CC chemoattractant protein, plays an important role in monocyte recruitment. 28 In our study, MCP-1 was strongly expressed, mainly in the RPE layer of the laser injury site on day 2. In addition, we examined VEGF expression and macrophage recruitment in the RPE-choroid with MCP-1 suppression by intravitreous injection of the neutral antibody. VEGF expression and macrophage recruitment had decreased in the anti-MCP-1 antibody treatment group, suggesting that the source of VEGF is the macrophage. 
The major sites of retinal damage after laser photocoagulation are in the RPE and outer retina. Ogata et al. 29 described the expression of cytokines including VEGF in photocoagulated RPE in vitro. In that report, the upregulation of VEGF was observed early (6 hours) after photocoagulation, and by 72 hours after photocoagulation, the expression of VEGF had decreased to the levels before photocoagulation. In contrast, in our in vivo results, the peak VEGF level was recorded on day 3. We speculate that the difference in the peak period may perhaps be because recruited macrophages contribute more toward upregulation of VEGF than does RPE or because RPE secretion of VEGF may be induced, in part, by macrophage-RPE interaction. Through their own VEGF release, macrophages can amplify the local VEGF response. 30 In addition, macrophage-derived cytokines can stimulate VEGF production in RPE cells. 23  
Scatter laser photocoagulation is one of the most effective modalities for treating a variety of retinal diseases, especially ischemic retinal diseases such as diabetic retinopathy and central retinal vein occlusion. 1 2 However, inappropriate laser photocoagulation can induce some complications such as hemorrhage, epiretinal membrane, intraocular proliferation, development of choroidal neovascularization, and macular edema. In particular, macular edema is sight threatening, and current therapy is ineffective; however, the pathologic mechanisms of macular edema and other ocular disorders are still poorly understood. 
VEGF is an endothelial cell mitogen and vasopermeability factor. 31 It increases vascular permeability and is a major factor in blood–retinal barrier breakdown in diabetic retinopathy. 19 The VEGF concentration is elevated in both the vitreous fluid and the aqueous humor of patients with active proliferative diabetic retinopathy 32 , whereas it has been reported that VEGF expression can be detected in excised subfoveal hard exudates from patients with macular edema. 33  
Clinically, it has been found that the vitreous levels of both interleukin-6 and VEGF are correlated with the severity of diabetic macular edema and show a strong correlation with each other. 34  
Scatter laser photocoagulation induces regression of active diabetic neovascularization. Immunostaining for VEGF is reduced in diabetic retinas that have no overt preretinal neovascularization after laser therapy. This finding is in close agreement with the results reported by Aiello et al., 19 who found decreased vitreous levels of VEGF in patients after laser therapy. It is possible that a reduction in retinal ischemia after laser treatment reduces the production of VEGF suppressing neovascularization and leading to regression and quiescence. 
The mechanisms by which laser photocoagulation achieves its results are not fully understood. It has recently been reported that TGF-b or PEDF may influence the regression of neovascularization after laser photocoagulation. 35 36  
Although the mechanisms of macular edema after scatter laser photocoagulation are still unclear, the potential usefulness of anti-MCP-1 antibody therapy is suggested. 
 
Figure 1.
 
Fundus photograph of mouse eye immediately after retinal scatter laser photocoagulation.
Figure 1.
 
Fundus photograph of mouse eye immediately after retinal scatter laser photocoagulation.
Figure 2.
 
VEGF protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured with the ELISA technique. VEGF level in the RPE-choroid on day 0 was 0.68 ± 0.119, and the peak expression of VEGF protein was found on day 3 (11.2 ± 0.42 pg/mL, P < 0.001) after laser treatment. These levels decreased by day 7 (3.92 ± 2.73, P < 0.0001) but remained high, and decreased on day 14 after laser treatment (n = 8; *P < 0.001; Ω P = 0.014; ε P = 0.075).
Figure 2.
 
VEGF protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured with the ELISA technique. VEGF level in the RPE-choroid on day 0 was 0.68 ± 0.119, and the peak expression of VEGF protein was found on day 3 (11.2 ± 0.42 pg/mL, P < 0.001) after laser treatment. These levels decreased by day 7 (3.92 ± 2.73, P < 0.0001) but remained high, and decreased on day 14 after laser treatment (n = 8; *P < 0.001; Ω P = 0.014; ε P = 0.075).
Figure 3.
 
Levels of VEGF mRNA were detected by RT-PCR at 1, 2, and 3 days after laser photocoagulation. VEGF signals were corrected for rodent Gapdh. The each mRNA levels were increased compared with untreated control subjects on day 0 and the highest level of VEGF mRNA was a threefold increase at day 2 (n = 4; *P < 0.05).
Figure 3.
 
Levels of VEGF mRNA were detected by RT-PCR at 1, 2, and 3 days after laser photocoagulation. VEGF signals were corrected for rodent Gapdh. The each mRNA levels were increased compared with untreated control subjects on day 0 and the highest level of VEGF mRNA was a threefold increase at day 2 (n = 4; *P < 0.05).
Figure 4.
 
Fluorescence microscopic detection of MCP-1 in retinas after retinal scatter photocoagulation on day 3 (A), with omission of primary antibody (second antibody only) (B) and control nonlasered retina. (C) Bright red fluorescent signal (arrows) indicates MCP-1 expression. Positive staining was localized in the photocoagulated site of the RPE epithelial layer (Nomarski image). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Arrowhead: photocoagulated site. MCP-1 was not expressed in the normal control retina. And omission of primary antibody has undetectable fluorescence. Scale bar: 100 μm.
Figure 4.
 
Fluorescence microscopic detection of MCP-1 in retinas after retinal scatter photocoagulation on day 3 (A), with omission of primary antibody (second antibody only) (B) and control nonlasered retina. (C) Bright red fluorescent signal (arrows) indicates MCP-1 expression. Positive staining was localized in the photocoagulated site of the RPE epithelial layer (Nomarski image). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Arrowhead: photocoagulated site. MCP-1 was not expressed in the normal control retina. And omission of primary antibody has undetectable fluorescence. Scale bar: 100 μm.
Figure 5.
 
MCP-1 protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured by using the ELISA technique. MCP-1 levels in the neurosensory retina and RPE-choroid were not detected on day 0. Then the MCP-1 level increased dramatically within 1 day after laser injury (neurosensory retina: 18.98 ± 1.234, RPE-choroid: 41.12 ± 8.312) and the peak expression of MCP-1 protein was found on day 2 after scatter laser treatment (neurosensory retina: 28.61 ± 11.102, RPE-choroid: 48.19 ± 1.849). These levels decreased on day 7 (neurosensory retina: 0.73 ± 1.249, RPE-choroid: 1.86 ± 1.109; n = 4).
Figure 5.
 
MCP-1 protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured by using the ELISA technique. MCP-1 levels in the neurosensory retina and RPE-choroid were not detected on day 0. Then the MCP-1 level increased dramatically within 1 day after laser injury (neurosensory retina: 18.98 ± 1.234, RPE-choroid: 41.12 ± 8.312) and the peak expression of MCP-1 protein was found on day 2 after scatter laser treatment (neurosensory retina: 28.61 ± 11.102, RPE-choroid: 48.19 ± 1.849). These levels decreased on day 7 (neurosensory retina: 0.73 ± 1.249, RPE-choroid: 1.86 ± 1.109; n = 4).
Figure 6.
 
Immunohistochemical studies after scatter laser photocoagulation. (A) Fluorescent signal in the nuclei of all cells stained with DAPI (blue), (B) VEGF (green), and (C) F4/80 (red) in the retina 3 days after retinal scatter photocoagulation. (D) Localization of VEGF (green) and F4/80 (red). Merged picture shows yellow costaining. With omission of primary antibody (second antibody only) has undetectable fluorescence (E). VEGF-positive staining was localized in the photocoagulated site of the RPE layer. VEGF localized in infiltrated macrophages. Scale bar, 100 μm.
Figure 6.
 
Immunohistochemical studies after scatter laser photocoagulation. (A) Fluorescent signal in the nuclei of all cells stained with DAPI (blue), (B) VEGF (green), and (C) F4/80 (red) in the retina 3 days after retinal scatter photocoagulation. (D) Localization of VEGF (green) and F4/80 (red). Merged picture shows yellow costaining. With omission of primary antibody (second antibody only) has undetectable fluorescence (E). VEGF-positive staining was localized in the photocoagulated site of the RPE layer. VEGF localized in infiltrated macrophages. Scale bar, 100 μm.
Figure 7.
 
Neutralizing anti-mouse MCP-1 antibody (5 ng, 1 ng, and 0.2 ng) was injected after laser photocoagulation and VEGF levels were detected by ELISA in the RPE-choroid at day 3 after that treatment. Each VEGF level was suppressed against uninjected control (11.2 ± 0.42 pg/mL, P < 0.001) at 0.2 ng (4.59 ± 0.896, P = 0.0038), 1 ng (3.41 ± 1.566, P = 0.0003), and 5 ng (2.70 ± 1.366, P = 0.001) (n = 4; *P < 0.001; Ω P < 0.005 vs. laser only group).
Figure 7.
 
Neutralizing anti-mouse MCP-1 antibody (5 ng, 1 ng, and 0.2 ng) was injected after laser photocoagulation and VEGF levels were detected by ELISA in the RPE-choroid at day 3 after that treatment. Each VEGF level was suppressed against uninjected control (11.2 ± 0.42 pg/mL, P < 0.001) at 0.2 ng (4.59 ± 0.896, P = 0.0038), 1 ng (3.41 ± 1.566, P = 0.0003), and 5 ng (2.70 ± 1.366, P = 0.001) (n = 4; *P < 0.001; Ω P < 0.005 vs. laser only group).
Figure 8.
 
Top: flow cytometric analysis data with F4/80 staining of neurosensory retina or RPE-choroid 3 days after retinal scatter photocoagulation. At day 3 after laser photocoagulation, the number of macrophages was detected by flow cytometry (bottom). These significantly increased compared with no laser photocoagulation controls and were remarkably reduced by intravitreous injections of 5 ng anti-MCP-1 antibody. Anti-MCP-1 Ab inhibited monocyte–macrophage recruitment and VEGF expression after retinal scatter photocoagulation (*P < 0.001 vs. laser only group; n = 4).
Figure 8.
 
Top: flow cytometric analysis data with F4/80 staining of neurosensory retina or RPE-choroid 3 days after retinal scatter photocoagulation. At day 3 after laser photocoagulation, the number of macrophages was detected by flow cytometry (bottom). These significantly increased compared with no laser photocoagulation controls and were remarkably reduced by intravitreous injections of 5 ng anti-MCP-1 antibody. Anti-MCP-1 Ab inhibited monocyte–macrophage recruitment and VEGF expression after retinal scatter photocoagulation (*P < 0.001 vs. laser only group; n = 4).
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Figure 1.
 
Fundus photograph of mouse eye immediately after retinal scatter laser photocoagulation.
Figure 1.
 
Fundus photograph of mouse eye immediately after retinal scatter laser photocoagulation.
Figure 2.
 
VEGF protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured with the ELISA technique. VEGF level in the RPE-choroid on day 0 was 0.68 ± 0.119, and the peak expression of VEGF protein was found on day 3 (11.2 ± 0.42 pg/mL, P < 0.001) after laser treatment. These levels decreased by day 7 (3.92 ± 2.73, P < 0.0001) but remained high, and decreased on day 14 after laser treatment (n = 8; *P < 0.001; Ω P = 0.014; ε P = 0.075).
Figure 2.
 
VEGF protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured with the ELISA technique. VEGF level in the RPE-choroid on day 0 was 0.68 ± 0.119, and the peak expression of VEGF protein was found on day 3 (11.2 ± 0.42 pg/mL, P < 0.001) after laser treatment. These levels decreased by day 7 (3.92 ± 2.73, P < 0.0001) but remained high, and decreased on day 14 after laser treatment (n = 8; *P < 0.001; Ω P = 0.014; ε P = 0.075).
Figure 3.
 
Levels of VEGF mRNA were detected by RT-PCR at 1, 2, and 3 days after laser photocoagulation. VEGF signals were corrected for rodent Gapdh. The each mRNA levels were increased compared with untreated control subjects on day 0 and the highest level of VEGF mRNA was a threefold increase at day 2 (n = 4; *P < 0.05).
Figure 3.
 
Levels of VEGF mRNA were detected by RT-PCR at 1, 2, and 3 days after laser photocoagulation. VEGF signals were corrected for rodent Gapdh. The each mRNA levels were increased compared with untreated control subjects on day 0 and the highest level of VEGF mRNA was a threefold increase at day 2 (n = 4; *P < 0.05).
Figure 4.
 
Fluorescence microscopic detection of MCP-1 in retinas after retinal scatter photocoagulation on day 3 (A), with omission of primary antibody (second antibody only) (B) and control nonlasered retina. (C) Bright red fluorescent signal (arrows) indicates MCP-1 expression. Positive staining was localized in the photocoagulated site of the RPE epithelial layer (Nomarski image). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Arrowhead: photocoagulated site. MCP-1 was not expressed in the normal control retina. And omission of primary antibody has undetectable fluorescence. Scale bar: 100 μm.
Figure 4.
 
Fluorescence microscopic detection of MCP-1 in retinas after retinal scatter photocoagulation on day 3 (A), with omission of primary antibody (second antibody only) (B) and control nonlasered retina. (C) Bright red fluorescent signal (arrows) indicates MCP-1 expression. Positive staining was localized in the photocoagulated site of the RPE epithelial layer (Nomarski image). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Arrowhead: photocoagulated site. MCP-1 was not expressed in the normal control retina. And omission of primary antibody has undetectable fluorescence. Scale bar: 100 μm.
Figure 5.
 
MCP-1 protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured by using the ELISA technique. MCP-1 levels in the neurosensory retina and RPE-choroid were not detected on day 0. Then the MCP-1 level increased dramatically within 1 day after laser injury (neurosensory retina: 18.98 ± 1.234, RPE-choroid: 41.12 ± 8.312) and the peak expression of MCP-1 protein was found on day 2 after scatter laser treatment (neurosensory retina: 28.61 ± 11.102, RPE-choroid: 48.19 ± 1.849). These levels decreased on day 7 (neurosensory retina: 0.73 ± 1.249, RPE-choroid: 1.86 ± 1.109; n = 4).
Figure 5.
 
MCP-1 protein concentration in the neurosensory retina and RPE-choroid were quantitatively measured by using the ELISA technique. MCP-1 levels in the neurosensory retina and RPE-choroid were not detected on day 0. Then the MCP-1 level increased dramatically within 1 day after laser injury (neurosensory retina: 18.98 ± 1.234, RPE-choroid: 41.12 ± 8.312) and the peak expression of MCP-1 protein was found on day 2 after scatter laser treatment (neurosensory retina: 28.61 ± 11.102, RPE-choroid: 48.19 ± 1.849). These levels decreased on day 7 (neurosensory retina: 0.73 ± 1.249, RPE-choroid: 1.86 ± 1.109; n = 4).
Figure 6.
 
Immunohistochemical studies after scatter laser photocoagulation. (A) Fluorescent signal in the nuclei of all cells stained with DAPI (blue), (B) VEGF (green), and (C) F4/80 (red) in the retina 3 days after retinal scatter photocoagulation. (D) Localization of VEGF (green) and F4/80 (red). Merged picture shows yellow costaining. With omission of primary antibody (second antibody only) has undetectable fluorescence (E). VEGF-positive staining was localized in the photocoagulated site of the RPE layer. VEGF localized in infiltrated macrophages. Scale bar, 100 μm.
Figure 6.
 
Immunohistochemical studies after scatter laser photocoagulation. (A) Fluorescent signal in the nuclei of all cells stained with DAPI (blue), (B) VEGF (green), and (C) F4/80 (red) in the retina 3 days after retinal scatter photocoagulation. (D) Localization of VEGF (green) and F4/80 (red). Merged picture shows yellow costaining. With omission of primary antibody (second antibody only) has undetectable fluorescence (E). VEGF-positive staining was localized in the photocoagulated site of the RPE layer. VEGF localized in infiltrated macrophages. Scale bar, 100 μm.
Figure 7.
 
Neutralizing anti-mouse MCP-1 antibody (5 ng, 1 ng, and 0.2 ng) was injected after laser photocoagulation and VEGF levels were detected by ELISA in the RPE-choroid at day 3 after that treatment. Each VEGF level was suppressed against uninjected control (11.2 ± 0.42 pg/mL, P < 0.001) at 0.2 ng (4.59 ± 0.896, P = 0.0038), 1 ng (3.41 ± 1.566, P = 0.0003), and 5 ng (2.70 ± 1.366, P = 0.001) (n = 4; *P < 0.001; Ω P < 0.005 vs. laser only group).
Figure 7.
 
Neutralizing anti-mouse MCP-1 antibody (5 ng, 1 ng, and 0.2 ng) was injected after laser photocoagulation and VEGF levels were detected by ELISA in the RPE-choroid at day 3 after that treatment. Each VEGF level was suppressed against uninjected control (11.2 ± 0.42 pg/mL, P < 0.001) at 0.2 ng (4.59 ± 0.896, P = 0.0038), 1 ng (3.41 ± 1.566, P = 0.0003), and 5 ng (2.70 ± 1.366, P = 0.001) (n = 4; *P < 0.001; Ω P < 0.005 vs. laser only group).
Figure 8.
 
Top: flow cytometric analysis data with F4/80 staining of neurosensory retina or RPE-choroid 3 days after retinal scatter photocoagulation. At day 3 after laser photocoagulation, the number of macrophages was detected by flow cytometry (bottom). These significantly increased compared with no laser photocoagulation controls and were remarkably reduced by intravitreous injections of 5 ng anti-MCP-1 antibody. Anti-MCP-1 Ab inhibited monocyte–macrophage recruitment and VEGF expression after retinal scatter photocoagulation (*P < 0.001 vs. laser only group; n = 4).
Figure 8.
 
Top: flow cytometric analysis data with F4/80 staining of neurosensory retina or RPE-choroid 3 days after retinal scatter photocoagulation. At day 3 after laser photocoagulation, the number of macrophages was detected by flow cytometry (bottom). These significantly increased compared with no laser photocoagulation controls and were remarkably reduced by intravitreous injections of 5 ng anti-MCP-1 antibody. Anti-MCP-1 Ab inhibited monocyte–macrophage recruitment and VEGF expression after retinal scatter photocoagulation (*P < 0.001 vs. laser only group; n = 4).
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