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The American Journal of Pathology, Vol. 184, No. 6, June 2014
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Neuronal Cell Death in the Inner Retina and the Influence of Vascular Endothelial Growth Factor Inhibition in a Diabetic Rat Model Q10
Hae-Young Lopilly Park, Jie Hyun Kim, and Chan Kee Park From the Department of Ophthalmology and Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Accepted for publication February 19, 2014. Address correspondence to Chan Kee Park, M.D., Ph.D., Department of Ophthalmology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea. E-mail: [email protected].
Q3
To inhibit vascular changes in diabetic retinopathy, inhibiting vascular endothelial growth factor (VEGF) has become a mainstay of the treatment of diabetic retinopathy. However, its effects on neuronal cells remain to be elucidated. We aimed to evaluate the effect of VEGF inhibition on neuronal cells in a streptozotocin-induced diabetic rat retina. VEGF inhibition was performed by intravitreal VEGF-A antibody injection. After anti-VEGF treatment, apoptosis in retinal ganglion cells (RGCs) increased, and novel apoptosis in amacrine and bipolar cells of the inner nuclear layer was observed by TUNEL staining. Phosphorylated Akt expression was significantly higher in RGCs but was decreased in neuronal cells of the inner nuclear layer after anti-VEGF treatment by Western blot analysis and immunohistochemical staining. These results demonstrate that VEGF inhibition significantly increased RGC apoptosis and neuronal cell apoptosis in the inner nuclear layer of a diabetic retina, which seems to consist primarily of amacrine and bipolar cells. The phosphorylated Akt pathway, which plays a neuroprotective role via VEGF, was significantly affected by VEGF inhibition in the inner nuclear layer, suggesting that neurotrophic factor deprivation is the main mechanism for neuronal cell death after inhibiting VEGF. The results of this study show that inhibiting VEGF may have detrimental effects on the apoptosis of neuronal cells in the inner layers of the diabetic retina. (Am J Pathol 2014, 184: 1e11; http://dx.doi.org/10.1016/j.ajpath.2014.02.016)
Diabetic retinopathy is the most frequently occurring microvascular complication of diabetes mellitus (DM) and often leads to vision loss in diabetic patients. Vascular dysfunction and loss of perfusion in the retina remain hallmarks of diabetic retinopathy1,2; however, growing evidence indicates that the function of neuronal cells in the retina is also compromised, perhaps even before overt vessel changes.3 Changes in neuronal cells, such as apoptosis of retinal ganglion cells (RGCs), amacrine cells, and photoreceptors, have been identified in the diabetic retina.4e7 In diabetic retinopathy, the atrophic appearance and dysfunction of the neural retina continue to clinically worsen after vascular lesions become quiescent.8 Therefore, DM-induced retinal neural degeneration remains a major cause of final vision loss and may progress independently of vascular lesions. Current treatments for diabetic retinopathy are intended to regulate vascular changes mediated by the action of vascular endothelial growth factor (VEGF) via laser treatment or agents that inhibit VEGF.9,10 However, VEGF-A, which is
the target of anti-VEGF agents used in patients with diabetic retinopathy, is thought to play a neuroprotective role.11e13 Neurons express VEGF receptors and can respond to VEGF-A via phosphoinositide 3-kinase/Akt signaling.14 VEGF-A can protect neurons against various harmful conditions, such as hypoxia, glutamate excitotoxicity, and deprivation of serum.15e17 Therefore, an improved comprehension of the effects of inhibiting VEGF-A in the diabetic retina is critical to understanding the function of neuronal cells in this region. It is possible that compromised neuronal cells in the diabetic retina may be further affected by treatments for diabetic retinopathy that target vascular changes. Supported by a National Research Foundation of Korea grant funded by Q2 the Korean government (Ministry of Education, Science, and Technology; C.K.P.; NRF-2011-0007989). H.-Y.L.P. and J.H.K. contributed equally to this work. Disclosures: None declared.
Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.02.016
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Park et al The purpose of this study was to investigate changes in neuronal cells of the inner retina and to evaluate the effects of inhibiting VEGF in a streptozotocin (STZ)-induced diabetic rat model. Changes in phosphorylated Akt (phosphoAkt) expression were measured in the diabetic retina to determine the downstream regulation of VEGF receptor signaling, which mediates the antiapoptosis of neuronal cells. STZ is cytotoxic to pancreatic beta cells and is used to generate a type 1 DM model in animals. The STZ-induced DM model results in inner retinal dysfunction but is not associated with pathologic neovascularization in the retina, which, in the present study, has the advantage of investigating neuronal cells in the inner retina.
Materials and Methods Diabetic Rat Models
Q4
Adult male Sprague-Dawley rats (7 to 8 weeks old, 250 to 300 g) were used in this study. The diabetic and control groups comprised six animals for each experimental procedure; the total number of animals used was 96. All the animal experiments complied with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and animals were treated according to the regulations of the Catholic Ethics Committee of The Catholic University of Korea (Seoul, Korea) and the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 80-23, revised 1996; http://search.grants. nih.gov, accessed December 2013). All efforts were made to minimize suffering and the number of animals used in the study. DM was induced by a single i.p. injection of 60 ㎎/㎏ of STZ (Sigma-Aldrich, St. Louis, MO) in a citrate buffer solution (0.1 mol/L citric acid and 0.2 mol/L sodium phosphate, pH 4.5). Age-matched control rats received an equivalent volume of only the citrate buffer solution. Three days after STZ injection, blood glucose levels for each rat were measured using an automated Accu-Chek glucometer (Roche Diagnostics, Indianapolis, IN). Animals with a plasma glucose reading >350 mg/dL were considered diabetic and were used for further experimentation. Body weights and blood glucose levels were recorded once a week after the induction of DM.
Drug Administration All the rats were anesthetized via an i.p. injection of 50 mg/kg of tiletamine plus zolazepam (Zoletil; Virbac, Carros, France) and 15 mg/kg of xylazine hydrochloride (Rompun; Bayer, Leuverkeusen, Germany). For intravitreal injections, a small incision was made in the conjunctiva, and the eyeball was rotated by grasping the conjunctiva and gently pulling. In the left eye of diabetic rats, 4 mL of 25-mg/mL anti-VEGF antibody (Genentech Inc., San Francisco, CA) was injected intravitreally using a 30-gauge needle and a Hamilton syringe. The right eye of diabetic rats was injected
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with the same amount of goat IgG (IgG control). To reduce reflux after the injection, the needle remained in the eye for an additional 3 to 4 seconds and was then drawn back. The whole procedure was guided by a stereoscopic microscope, with care taken to avoid lens and retinal injury.
Fluorescein Dextran Angiography of the Retinal Blood Vessel A solution of fluorescein isothiocyanateedextran (2 106 molecular weight; Sigma-Aldrich) was prepared at a concentration of 10 mg/mL in PBS. After 8 weeks, three rats from each group were anesthetized with 50 mg/kg of ketamine and 5 mg/kg of xylazine. Fluorescein isothiocyanateedextran solution (0.5 mL) was injected into the tail vein of each rat, and a cover glass was placed on the cornea as a contact lens. Angiograms were established with a scan angle of 30 . Digital images at a magnification of 40 were captured using a charge-coupled device camera (DC500; Leica Microsystems, Heerbrugg, Switzerland) attached to a fluorescence stereomicroscope (MZ-III; Leica Microsytems, Wetzlar, Germany). Vascular abnormalities were scored from 0 to 5 in accordance with the following grading: grade 0, normal retinal vasculature, as observed in control fundus; þ1 point each for dilated or tortuous vessels, presence of microaneurysmal-like hyperfluorescent dots, and presence of dye-filling defects; and þ2 points for two or more of dilated or tortuous vessels, presence of microaneurysmal-like hyperfluorescent dots, and presence of dye-filling defects.18 Retinal vascular permeability was measured using the Evans blueealbumin leakage assay following an established protocol.19 Concentrations of Evans blue in the retina were normalized by total retinal protein concentrations and by Evans blue concentrations in the plasma.
Tissue Preparation At each time point, all the animals were deeply anesthetized with 50 mg/kg of ketamine and 5 mg/kg of xylazine for immunohistochemical analysis, the eyes were quickly enucleated and dissected, and the posterior eye cups were placed in chilled fixative (4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4). The isolated retinas were divided into small pieces and were immersion-fixed in the same fixative for 2 hours at 4 C. After washing several times, fixed retinas were cryoprotected in 30% sucrose containing 0.1 mol/L phosphate buffer for 6 hours at 4 C and then were stored in this buffer at 70 C. For Western blot analysis, retinal tissues were quickly dissected, frozen in liquid nitrogen, and stored at 70 C.
Immunohistochemical Analysis For fluorescence staining, samples were pre-embedded in 3% agar in deionized water. Fifty-micron vibratome sections were collected and washed several times in PBS. Sections
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Table 1
Body Weights and Serum Glucose Levels
Parameter
Control rats
STZ-induced diabetic rats
Body weight (g) Start Finish Serum glucose (mg/dL)
331.5 9.4 441.6 8.9 133.5 4.9
335.9 7.7 328.7 10.5 565.8 14.2
Data are expressed as means SD.
were incubated in 10% normal donkey serum in PBS for 1 hour at room temperature to block nonspecific binding activity, followed by incubation with the rabbit anti-VEGF (Abcam Plc, Cambridge, UK) and antieglial fibrillary acidic protein (GFAP) (Millipore, Billerica, MA) overnight at 4 C. The next day, after several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546 (Molecular Probes, Eugene, CA). For double-labeling studies, sections were incubated with mouse anti-NeuN (Millipore), anti-PKCa (Santa Cruz Biotechnology, Santa Cruz, CA), antiecleaved caspase-3 (Cell Signaling Technology Inc., Boston, MA), and anti-parvalbumin (Sigma-Aldrich) or in
0.1 mol/L PBS containing 0.5% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) overnight at 4 C. They were then rinsed for 30 minutes with 0.1 mol/L PBS and incubated with goat anti-mouse Alexa 488 (Molecular Probes) for 1 hour and 30 minutes at room temperature. After further washing in 0.1 mol/L phosphate buffer for 30 minutes, the sections were mounted using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Sections were washed, placed under cover glass, and examined by confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH, Jena, Germany).
TUNEL Assay Apoptotic cells were evaluated using TUNEL staining. The retinas were dissected from the choroid, and the central portion of the superior nasal quadrant, 1.5 mm from the optic disc, was trimmed into small pieces. Fifty-micron cryosections of the retina were embedded in 4% paraformaldehyde and were washed with PBS. The tissue was stained using the TUNEL method according to the manufacturer’s protocol (in situ cell
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VEGF Inhibition in Diabetic Rat Model Q1
Immunohistochemical staining of VEGF and NeuN, an RGC marker. Control retina (A, E, I, and M) and diabetic retina 1 week (B, F, J, and N), 4 Q7 weeks (C, G, K, and O), and 8 weeks (D, H, L, and P) after STZ injection. Basal VEGF expression in the control retina (A) increased gradually after DM induction through 8 weeks (BeD). There was no co-expression of VEGF (A) and NeuN (E) in the GCL of the control retina (I and M). VEGF and NeuN co-expression (arrowheads) appears in the GCL at week 1 (F, J, and N), week 4 (G, K, and O), and week 8 (H, L, and P) after STZ injection. n Z 5 for each time point and group. Scale bars: 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.
Figure 1
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death detection kit; Roche Applied Science, Indianapolis, IN). For double-labeling studies, sections were incubated with antiNeuN, anti-PKCa, and anti-parvalbumin solutions overnight at 4 C. The next day, after several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546, and after further washes in 0.1 mol/L phosphate buffer for 30 minutes they were mounted using VECTASHIELD mounting medium with DAPI. Sections were washed, put under cover glass, and examined using confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH). For quantitative analysis of the number of TUNEL-positive cells in the ganglion cell layer (GCL) and inner nuclear layer (INL), the cell bodies were counted in each layer. Three to four independent tests were repeated in different retinal sections by one investigator in a masked manner, not knowing the classified group of the retinal sections.
Western Blot Analysis Control and diabetic retinas were homogenized in radioimmunoprecipitation assay buffer (1% Triton X-100, 5% SDS, 5% deoxycholic acid, 0.5 mol/L Tris-HCl [pH 7.5], 10% glycerol, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mg/mL of aprotinin, 1 mg/mL of leupeptin, 1 mg/mL of pepstatin, 200 mmol/L sodium orthovanadate, and 200 mmol/L sodium fluoride). Tissue extracts were incubated for 10 minutes on ice and were clarified by centrifugation at 10,000 g for 25 minutes at 4 C. Total protein in the retinal extracts was measured using a standard bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). Retinal extracts containing 40 mg of total protein were resuspended in
5 sample buffer (60 mmol/L TriseHCl [pH 7.4], 25% glycerol, 2% SDS, 14.4 mmol/L 2-mercaptoethanol, and 0.1% bromophenol blue) at a 4:1 ratio, boiled for 5 minutes, and resolved by SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane, and blots were stained with Ponceau S (Sigma-Aldrich) to visualize the protein bands and ensure equal protein loading and uniform transfer. Blots were washed and blocked for 45 minutes with 5% nondried skim milk in Tris-buffered saline with Tween 20 buffer (20 mmol/L TrisHCl [pH 7.6], 137 mmol/L NaCl, and 0.1% Tween 20). Blots were then probed for 24 hours using antibodies against VEGF, phospho-Akt (Cell Signaling Technology Inc.), total Akt (Cell Signaling Technology Inc.), GFAP, brain-derived neurotrophic factor (BDNF) (Santa Cruz Biotechnology), and actin (Sigma-Aldrich). Blots were then probed with horseradish peroxidaseeconjugated goat anti-rabbit secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence system (Amersham, Piscataway, NJ) and X-ray film. Each immunoblot shown is representative of five independent experiments. Signal intensity was measured using an ImageMaster VDS system (Pharmacia Biotech, Piscataway, NJ). Signal quantities shown were standardized to background and normalized for loading. Controls were set as 1.0, and the fold changes were plotted in relation to the control value. Results are representative of five independent experiments. Data are expressed as means SD.
Statistical Analysis All the data are expressed as means SD. Comparisons between each time point were compared with the control
Figure 2 A: TUNEL staining showed increased apoptotic cells in the GCL after DM induction at weeks 4 and 8 compared with control retina. B: The number of TUNEL-positive cells in the GCL was significantly greater in the diabetic retina compared with the control retina at weeks 1, 4, and 8 after STZ injection. Data are expressed as means SD. n Z 5 for each time point and group. *P < 0.05 compared with controls. Scale bar Z 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.
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using a U-test. Results with P < 0.05 were considered statistically significant.
Results Body Weight and Blood Glucose Levels ½T1
The body weights and blood glucose levels of experimental rats were recorded (Table 1). At week 8 after STZ injection, body weight was lower and blood glucose levels were significantly higher in diabetic rats compared with control rats.
Inner Retinal Changes after DM Induction The immunostaining of VEGF revealed expression in all layers of the retina in control rats (Figure 1A). After STZ injection in experimental rats, this expression gradually increased in week 1 (Figure 1B), week 4 (Figure 1C), and week 8 (Figure 1D). At week 8 after STZ injection, VEGF was densely expressed in the GCL, inner plexiform layer, INL, outer plexiform layer, and outer nuclear layer in diabetic rats. Colabeling with VEGF and NeuN, an RGC marker, did not identify co-expression of VEGF and NeuN in the GCL in control retinas (Figure 1, E, I, and M). However, VEGF and NeuN co-expression was evident in the GCL at week 1 (Figure 1, F, J, and N), week 4 (Figure 1, G, K, and O), and week 8 (Figure 1, H, L, and P) in diabetic rats. A significant increase in apoptotic cells was identified in ½F2 the GCL at weeks 4 and 8 after STZ injection (Figure 2A). The number of TUNEL-positive cells in the GCL was significantly greater in the retinas of diabetic rats compared with those of control rats at weeks 1, 4, and 8 (Figure 2B). After colabeling with TUNEL staining and NeuN, apoptotic cells in the GCLs were found to be mostly RGCs ½F3 (Figure 3A), and the TUNEL-positive RGCs expressed VEGF in the cytoplasm (Figure 3B). GFAP immunostaining was performed to evaluate glial cell activation in response to retinal stress caused by DM. GFAP expression was restricted to astrocytes and the end feet of Muller cells in the inner limiting membrane in the ½F4 retinas of control rats (Figure 4A). In diabetic rats, however, GFAP expression was significantly greater at weeks 1, 4, and 8 after STZ injection (Figure 4, C, E, and G, respectively). The increased GFAP expression in astrocytes and the processes of Muller glia spanned the entire inner retina. These data demonstrate that VEGF levels are elevated throughout the retinas of STZ-induced diabetic rats and are especially up-regulated in the cytoplasm of RGCs after week 1 of STZ injection. VEGF up-regulated RGCs were shown to undergo apoptosis by TUNEL staining that gradually increased after STZ injection through week 8. Similarly, the activation of astrocytes and Muller glial cells also increased gradually after DM induction through week 8. ½F1
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559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 Figure 3 A: Colabeling with TUNEL staining and NeuN, a ganglion cell 596 marker, showed that TUNEL-positive cells in the ganglion cell layer (GCL) 597 are retinal ganglion cells undergoing apoptosis (arrows). B: Colabeling 598 with TUNEL staining and VEGF showed that these apoptotic RGCs express 599 VEGF (arrows). n Z 5 for each time point and group. Scale bar Z 50 mm. 600 INL, inner nuclear layer. 601 Effect of Inhibiting VEGF in the Diabetic Retina 602 603 There was no change in GFAP immunostaining in IgG 604 605 control retinas (Figure 4B). However, anti-VEGF antibody 606 administration resulted in an increase in GFAP expression 607 in diabetic retinas at weeks 1, 4, and 8 (Figure 4, D, F, and 608 H, respectively). 609 TUNEL staining revealed apoptotic cells in the GCL and 610 INL after intravitreal injection of the anti-VEGF antibody 611 (Figure 5A). The number of TUNEL-positive cells in the ½F5 612 GCL and INL significantly increased after anti-VEGF 613 antibody administration compared with control diabetic 614 retina (Table 2). TUNEL-positive cells were not identified ½T2 615 in the INL after DM induction or after intravitreal injection 616 617 of IgG in the diabetic retina. However, injection of the anti618 VEGF antibody resulted in additional apoptotic cells in the 619 INL of diabetic retinas. TUNEL-positive cells in the GCL 620
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Figure 4 GFAP immunostaining revealed an increase in glial cell activity in the diabetic retina at weeks 1 (C), 4 (E), and 8 (G) after STZ injection compared with the control retina (A). After antiVEGF antibody treatment, GFAP expression significantly increased in the diabetic retina at weeks 1 (D), 4 (F), and 8 (H) after STZ injection. However, the diabetic retina was not affected by IgG injection (B). n Z 5 for each time point and group. Scale bar Z 50 mm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
dilation or tortuosity, presence of microaneurysm, and dyefilling defects, diabetic retinas showed increased scores. However, after VEGF inhibition, these vascular features returned to normal levels (Figure 6D). Vascular permeability, representing vessel leakage, was also significantly increased in diabetic retinas, which returned to normal levels after VEGF inhibition (Figure 6E). These data demonstrate that inhibiting VEGF aggravates inner retinal cell death in the diabetic retina, although the vascular changes improve.
Differential Response of the GCL and INL in the Diabetic Retina after Inhibition of VEGF Western blot analyses revealed greater VEGF and GFAP expression in the diabetic retina at weeks 1, 4, and 8 after STZ injection (Figure 7A). Phospho-Akt levels gradually ½F7 decreased, despite no changes in total Akt levels, after DM induction at weeks 1, 4, and 8. The ratio of phospho-Akt to total Akt was significantly lower at weeks 4 and 8 in diabetic retinas compared with control retinas (Figure 7B).
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VEGF Inhibition in Diabetic Rat Model
After intravitreal injection of anti-VEGF antibody into diabetic rat eyes, VEGF was significantly decreased compared with IgG control eyes at weeks 1, 4, and 8 (Figure 7A). Likewise, GFAP was significantly greater after the injection of anti-VEGF antibody into diabetic eyes compared with control eyes at weeks 1, 4, and 8. Phospho-Akt levels were also decreased after anti-VEGF antibody injection into diabetic eyes at weeks 4 and 8. However, the ratio between phospho-Akt and total Akt did not show a difference after VEGF inhibition in diabetic retinas (Figure 7B). Immunostaining of phospho-Akt revealed baseline expression levels in the GCL and INL in control retinas (Figure 7C), which Table 2 Number of TUNEL-Positive Cells after Anti-VEGF Treatment in Diabetic Retinas Time after STZ injection Q9
1 week 4 weeks 8 weeks
Anti-VEGF treatment
GCL
þ þ þ
1.9 3.7 4.4 6.7 8.0 9.9
INL
0.7* 0.8*y 0.8* 1.1*y 1.3* 1.9*
1.3 1.0 30.8 4.4* 34.7 4.6*
n Z 3 to 4 for each time point and group. Data are expressed as means SD. *P < 0.05 compared with control retinas. y P < 0.05 compared with diabetic retinas without anti-VEGF treatment. -, no TUNEL-positive cells.
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807 808 809 810 811 812 813 814 815 Figure 5 A: VEGF inhibition of the STZ-induced 816 diabetic eye resulted in the apoptosis of cells in the GCL and INL as observed by TUNEL staining. B: 817 TUNEL-positive cells in the GCL were RGCs shown 818 by colabeling with NeuN (arrows). Of TUNEL819 positive cells in the INL, some were bipolar cells 820 shown by colabeling with PCKa (C; arrows), and 821 others were amacrine cells shown by colabeling 822 with parvalbumin (D; arrows). Few cells in the GCL 823 are colabeled with parvalbumin (arrowhead), 824 showing some apoptosis of displaced amacrine 825 cells in the GCL. n Z 6 for each time point and 826 group. Scale bar Z 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nu827 clear layer; OPL, outer plexiform layer. 828 829 830 831 832 833 834 835 836 837 838 did not change in IgG control retinas (Figure 7D). Subse839 quent to anti-VEGF antibody injection into diabetic eyes, 840 phospho-Akt expression was significantly greater in the 841 Q5 GCL but was decreased in the INL (Figure 7E). 842 RGC death in the GCL was increased after diabetes in843 duction but decreased after anti-VEGF injection in the 844 diabetic retina. Immunostaining of cleaved caspase-3 in the 845 RGC shows decreased RGC death after inhibition of VEGF 846 (Figure 8A). Western blot analysis revealed increased ½F8 847 848 BDNF levels after inhibition of VEGF in diabetic retinas 849 (Figure 8B). Downstream of BDNF, phospho-Akt immu850 nostaining was increased in the RGCs (Figure 8A). 851 852 853 Table 3 Number of Cells in Each Retinal Layer after Anti-VEGF 854 Treatment in Diabetic Retinas 855 Time after Anti-VEGF 856 STZ injection treatment GCL INL 857 1 week 16.3 1.6* 119.9 8.7 858 þ 17.9 1.7* 120.1 9.8 859 4 weeks 14.1 1.8* 113.0 8.9 860 þ 14.9 1.3* 119.6 8.1y 861 8 weeks 11.0 1.5* 121.4 9.8 862 þ 14.0 1.4* 115.3 8.1y 863 864 n Z 3 to 4 for each time point and group. Data are expressed as 865 means SD. 866 *P < 0.05 compared with control retinas. y 867 P < 0.05 compared with diabetic retinas without anti-VEGF treatment. 868
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Figure 6 Representative fluorescein angiograms from control retina (A), diabetic retina (B), and diabetic retina with anti-VFGF treatment (C) 8 weeks after STZ injection. B and C: Dye-filling defects (arrows), dilated or tourtous vessels (white arrowheads), and microaneurysmal-like hyperfluorescent dots (gray arrowheads) were present in the diabetic retina. C: VEGF inhibition improved the microvascular pathologic changes of diabetic retinas. D: Grading showed significantly increased vascular changes in diabetic retinas, which returned to near normal levels after VEGF inhibition. E: The permeability assay, indicating fluorescence leakage, showed increased leakage from vessels in diabetic retinas, which also returned to near normal after VEGF inhibition. Data are expressed as means SD. n Z 3 for each time point and group. Scale bar Z 200 mm.
Discussion DM causes systemic microvascular complications, including retinopathy.20e22 Vascular and ischemic changes of the retina caused by high glucose levels result in up-regulation of VEGF in the diabetic retina.23 Up-regulation of VEGF could be a compensatory mechanism for the retina to withstand the vascular and ischemic changes by DM; however, this causes devastating effects to the retina, such as compromised bloodretinal barrier, vascular leakage, and neovascularization.24 Treatment with an anti-VEGF agent is reported to be effective in eliminating the abnormal vessels and retinal edema of diabetic retinopathy. However, although vascular changes caused by DM are improved after inhibiting VEGF, neuronal cell death increases in the inner retina, as shown in the present results. Neuronal cells, such as bipolar and amacrine cells, may use VEGF for survival, and inhibiting VEGF had a harmful effect on these cells, with decreased phospho-Akt expression in the INL, which is downstream of the growth factor pathway. Phospho-Akt levels were increased in RGCs and may be mediated by increased BDNF levels after diabetic induction, and inhibiting VEGF had less effect on these cells. Several studies have indicated that not only vascular abnormalities but also neuronal abnormalities are associated with early diabetic retinopathy.4e7 However, the precise mechanism of neuronal cell death in early diabetic retinopathy
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is not well established. In the human retina, VEGF is found in all major classes of retinal neurons and is especially prominent in amacrine cells and RGCs.25 Retinal neurons normally provide continuous trophic support for their own survival, and VEGF expression in these neurons is increased in an ischemic retina.25,26 VEGF-A exerts its neuroprotective effects and directly prevents neuronal cell apoptosis via phosphoinositide 3-kinase/Akt signaling.8,14 In this study, the expression of phospho-Akt was primarily centered in the GCL and INL of control retinas and is in agreement with prominent locations of VEGF expression found in previous studies.25 However, although VEGF protein levels increased after DM induction, phospho-Akt protein levels decreased at weeks 4 and 8 after STZ injection. Immunostaining did not reveal specific changes in phospho-Akt expression in the GCL or INL of the diabetic retina. Likewise, the total amount of phospho-Akt protein did not exhibit any changes subsequent to VEGF inhibition. However, the pattern of phosphoAkt expression did change after anti-VEGF treatment in that expression significantly increased in RGCs in the GCL but decreased in neuronal cells of the INL. A decrease in phospho-Akt expression in the INL explains the presence of apoptotic cells subsequent to anti-VEGF treatment because the neuroprotective effects of VEGF via phospho-Akt signaling were down-regulated by VEGF inhibition. The inhibition of VEGF significantly increases apoptosis Q6 in amacrine and bipolar cells in the INL, but the increase in
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Figure 7 Representative results of Western blot analysis (A), quantitative analysis of the ratio of phospho-Akt to total Akt (B), and immunohistochemical staining of phospho-Akt (CeE). A: The proteins of VEGF and GFAP increased significantly after diabetes induction. However, VEGF protein levels decreased and GFAP protein levels increased after VEGF inhibition. B: The phospho-Akt/total Akt ratio decreased significantly at weeks 4 and 8 after STZ injection. VEGF inhibition decreased the phospho-Akt/total Akt ratio compared with IgG control 4 and 8 weeks after STZ injection; however, it is not statistically significant. Phospho-Akt immunoreactions (arrows) were found in the GCL and INL after diabetes induction (C), which is not changed in the IgG control retina (D). E: After Q8 VEGF inhibition, phospho-Akt expression increased in the GCL (arrows) and decreased in the inner nuclear layer. Data are expressed as means SD. n Z 6 for each time point and group. *P < 0.05 compared with controls. Scale bar Z 50 mm.
RGC apoptosis after VEGF inhibition is relatively less prominent. This finding may suggest that neuronal cells use VEGF differently in the diabetic retina and that the neuroprotective effects of VEGF may be different between RGCs and inner nuclear cells, including amacrine and bipolar cells. However, BDNF is the most potent survival factor for injured RGCs,27e29 and these cells may be less affected by VEGF inhibition.
Anti-VEGF agents are widely used for the treatment of age-related macular degeneration to reduce angiogenesis associated with choroidal neovascularization and are currently Food and Drug Administrationeapproved for the treatment of age-related macular degeneration and retinal vessel occlusion. Furthermore, bevacizumab is extensively used as an off-label treatment for diabetic macular edema, proliferative diabetic retinopathy, and neovascular glaucoma.
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Representative results of immunohistochemical staining of phospho-Akt and cleaved caspase-3 (A) and Western blot analysis (B). A: PhosphoAkt immunoreactions increased in the RGC, colabeled with NeuN (arrowheads), in the ganglion cell layer (GCL) after VEGF inhibition compared with the IgG control retina. Cleaved caspase-3 showed decreased RGC death in the GCL after VEGF inhibition. B: BDNF levels increased after VEGF inhibition up to 4 weeks after STZ injection. n Z 6 for each time point and group. Scale bar Z 50 mm.
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Park et al Accumulating clinical evidence indicates the safety of using anti-VEGF agents in age-related macular degeneration because there are no changes in the retinal nerve fiber layer (where the axons of RGCs pass through) or the optic disc after multiple injections of anti-VEGF agents.30e32 However, the diabetic retina is different from the retina with age-related macular degeneration. The function of neuronal cells in the diabetic retina is compromised, and the presence of apoptosis in RGCs and retinal stress, as evidenced by the elevated expression of GFAP, before overt neovascularization is clear, as shown in the present study. Previously, we reported that the retinal nerve fiber layer gets thinner as diabetic retinopathy progresses and that retinal nerve fiber layer thinning was present in patients with DM without diabetic retinopathy.33 These findings together indicate that neuronal stress and RGC damage are clinically apparent in diabetic retinas before evident neovascularization. In the adult retina, VEGF is expressed in the absence of active neovascularization and is implicated in the maintenance and function of adult retinal neuronal cells.34 It is possible that the inhibition of VEGF in the diabetic retina may further compromise the function of neuronal cells and influence apoptosis. However, the effect of anti-VEGF agents on neuronal cells in the inner diabetic retina has not been fully investigated, and there is a lack of long-term clinical data showing that anti-VEGF therapy is safe for diabetic retinopathy.35 Neither the in vivo intravitreal injection nor the direct application of various VEGF inhibitors to RGCs in vitro show toxic effects on RGCs in previous reports.36e38 Likewise, VEGF inhibition did not have a toxic effect on RGCs under stressful conditions induced by N-methyl-D-aspartate or oxidative stress,37,39 and Muller glial cells were not affected in vivo or in vitro by VEGF inhibition.40 However, anti-VEGF agents neutralize the protective effect of VEGF when applied together under stressful conditions.38 RGC apoptosis has been observed after multiple injections of an anti-VEGF agent,41 including under conditions in which RGCs require VEGF for survival or in which repeated treatment with anti-VEGF agents may interfere with the neuroprotective action of VEGF.38 The present study demonstrates that VEGF expression is increased in RGCs after DM induction and that these VEGFexpressing RGCs undergo apoptosis. After VEGF inhibition to the diabetic eye, inner retinal stress is increased by GFAP expression, apoptosis of RGCs is increased, and novel apoptosis of amacrine and bipolar cells in the INL is present. These results show that under diabetic conditions, neuronal cells in the inner retina use VEGF for survival, which is interfered by VEGF inhibition, after which they undergo apoptosis. This neuronal cell death was independent of the changes in the retinal vessels after VEGF inhibition, as shown in the angiographic results. In conclusion, RGC apoptosis is increased after DM induction in an STZ injection model and is further increased after VEGF inhibition in the diabetic retina. More important, VEGF inhibition significantly increases neuronal cell apoptosis in the INL in the diabetic retina and seems to primarily include
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amacrine and bipolar cells. The phospho-Akt pathway, which is integral to the neuroprotective role of VEGF, is significantly affected by VEGF inhibition, and its pattern is different between RGCs and neuronal cells in the INL. This study provides useful information concerning the use of antiVEGF agents in the diabetic retina as the treatment of vascular changes due to diabetic retinopathy via the inhibition of VEGF may be crucial for the preservation of vision. However, note that this treatment may have detrimental effects on apoptosis in neuronal cells of the inner retina. Further strategies for the management of vascular changes and the preservation of inner neuronal cells are needed to save the vision of diabetic patients.
Acknowledgments We thank Jun-Sub Choi, Ph.D., (The Catholic University of Korea) for advice and support in experiments of retinal angiography. C.K.P. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
References 1. Kern TS, Engerman RL: Vascular lesions in diabetes are distributed non-uniformly within the retina. Exp Eye Res 1995, 60:545e549 2. Ljubimov AV, Burgeson RE, Butkowski RJ, Couchman JR, Zardi L, Ninomiya Y, Sado Y, Huang ZS, Nesburn AB, Kenney MC: Basement membrane abnormalities in human eyes with diabetic retinopathy. J Histochem Cytochem 1996, 44:1469e1479 3. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, Jefferson LS, Kester M, Kimball SR, Krady JK, LaNoue KF, Norbury CC, Quinn PG, Sandirasegarane L, Simpson IA; Group JDRC: Diabetic retinopathy: seeing beyond glucose-induced microvascular disease. Diabetes 2006, 55:2401e2411 4. Barber AJ, Lieth E, Khin SA, Antonetti DA, Buchanan AG, Gardner TW: Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. J Clin Invest 1998, 102:783e791 5. Gastinger MJ, Singh RS, Barber AJ: Loss of cholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akitadiabetic mouse retinas. Invest Ophthalmol Vis Sci 2006, 47: 3143e3150 6. Martin PM, Roon P, Van Ells TK, Ganapathy V, Smith SB: Death of retinal neurons in streptozotocin-induced diabetic mice. Invest Ophthalmol Vis Sci 2004, 45:3330e3336 7. Park SH, Park JW, Park SJ, Kim KY, Chung JW, Chun MH, Oh SJ: Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia 2003, 46:1260e1268 8. Foxton RH, Finkelstein A, Vijay S, Dahlmann-Noor A, Khaw PT, Morgan JE, Shima DT, Ng YS: VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma. Am J Pathol 2013, 182:1379e1390 9. Kim LA, D’Amore PA: A brief history of anti-VEGF for the treatment of ocular angiogenesis. Am J Pathol 2012, 181:376e379 10. Ozaki H, Seo MS, Ozaki K, Yamada H, Yamada E, Okamoto N, Hofmann F, Wood JM, Campochiaro PA: Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to
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11. 12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24. 25.
26.
completely prevent retinal neovascularization. Am J Pathol 2000, 156: 697e707 D’Amore PA: Vascular endothelial cell growth factor-A: not just for endothelial cells anymore. Am J Pathol 2007, 171:14e18 Beazley-Long N, Hua J, Jehle T, Hulse RP, Dersch R, Lehrling C, Bevan H, Qiu Y, Lagreze WA, Wynick D, Churchill AJ, Kehoe P, Harper SJ, Bates DO, Donaldson LF: VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro. Am J Pathol 2013, 183:918e929 Miguel NC, Matsuda M, Portes AL, Allodi S, Mendez-Otero R, Puntar T, Sholl-Franco A, Krempel PG, Monteiro ML: In vitro effects of bevacizumab treatment on newborn rat retinal cell proliferation, death, and differentiation. Invest Ophthalmol Vis Sci 2012, 53:7904e7911 Kim I, Ryan AM, Rohan R, Amano S, Agular S, Miller JW, Adamis AP: Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes. Invest Ophthalmol Vis Sci 1999, 40:2115e2121 Jin KL, Mao XO, Greenberg DA: Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc Natl Acad Sci U S A 2000, 97:10242e10247 Gomes E, Papa L, Hao T, Rockwell P: The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells. Mol Cell Biochem 2007, 305:179e190 Tolosa L, Mir M, Asensio VJ, Olmos G, Llado J: Vascular endothelial growth factor protects spinal cord motoneurons against glutamateinduced excitotoxicity via phosphatidylinositol 3-kinase. J Neurochem 2008, 105:1080e1090 Kowalczuk L, Touchard E, Omri S, Jonet L, Klein C, Valamanes F, Berdugo M, Bigey P, Massin P, Jeanny JC, Behar-Cohen F: Placental growth factor contributes to micro-vascular abnormalization and bloodretinal barrier breakdown in diabetic retinopathy. PLoS One 2011, 6:e17462 Xu Q, Qaum T, Adamis AP: Sensitive blood-retinal barrier breakdown quantitation using Evans blue. Invest Ophthalmol Vis Sci 2001, 42: 789e794 Kohner EM, Patel V, Rassam SM: Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy. Diabetes 1995, 44:603e607 Gardiner TA, Archer DB, Curtis TM, Stitt AW: Arteriolar involvement in the microvascular lesions of diabetic retinopathy: implications for pathogenesis. Microcirculation 2007, 14:25e38 Mizutani M, Kern TS, Lorenzi M: Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest 1996, 97:2883e2890 Qaum T, Xu Q, Joussen AM, Clemens MW, Qin W, Miyamoto K, Hassessian H, Wiegand SJ, Rudge J, Yancopoulos GD, Adamis AP: VEGF-initiated blood-retinal barrier breakdown in early diabetes. Invest Ophthalmol Vis Sci 2001, 42:2408e2413 Stitt AW, Lois N, Medina RJ, Adamson P, Curtis TM: Advances in our understanding of diabetic retinopathy. Clin Sci 2013, 125:1e17 Famiglietti EV, Stopa EG, McGookin ED, Song P, LeBlanc V, Streeten BW: Immunocytochemical localization of vascular endothelial growth factor in neurons and glial cells of human retina. Brain Res 2003, 969:195e204 Shima DT, Gougos A, Miller JW, Tolentino M, Robinson G, Adamis AP, D’Amore PA: Cloning and mRNA expression of vascular
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27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40. 41.
endothelial growth factor in ischemic retinas of Macaca fascicularis. Invest Ophthalmol Vis Sci 1996, 37:1334e1340 Jelsma TN, Friedman HH, Berkelaar M, Bray GM, Aguayo AJ: Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve. J Neurobiol 1993, 24:1207e1214 Mansour-Robaey S, Clarke DB, Wang YC, Bray GM, Aguayo AJ: Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci U S A 1994, 91:1632e1636 Perez MT, Caminos E: Expression of brain-derived neurotrophic factor and of its functional receptor in neonatal and adult rat retina. Neurosci Lett 1995, 183:96e99 Martinez-de-la-Casa JM, Ruiz-Calvo A, Saenz-Frances F, RecheFrutos J, Calvo-Gonzalez C, Donate-Lopez J, Garcia-Feijoo J: Retinal nerve fiber layer thickness changes in patients with age-related macular degeneration treated with intravitreal ranibizumab. Invest Ophthalmol Vis Sci 2012, 53:6214e6218 Seth RK, Salim S, Shields MB, Adelman RA: Assessment of optic nerve cup-to-disk ratio changes in patients receiving multiple intravitreal injections of antivascular endothelial growth factor agents. Retina 2009, 29:956e959 Horsley MB, Mandava N, Maycotte MA, Kahook MY: Retinal nerve fiber layer thickness in patients receiving chronic anti-vascular endothelial growth factor therapy. Am J Ophthalmol 2010, 150:558e561.e1 Park HY, Kim IT, Park CK: Early diabetic changes in the nerve fibre layer at the macula detected by spectral domain optical coherence tomography. Br J Ophthalmol 2011, 95:1223e1228 Wang Y, Mao XO, Xie L, Banwait S, Marti HH, Greenberg DA, Jin K: Vascular endothelial growth factor overexpression delays neurodegeneration and prolongs survival in amyotrophic lateral sclerosis mice. J Neurosci 2007, 27:304e307 Al-Latayfeh M, Silva PS, Sun JK, Aiello LP: Antiangiogenic therapy for ischemic retinopathies. Cold Spring Harb Perspect Med 2012, 2: a006411 Iriyama A, Chen YN, Tamaki Y, Yanagi Y: Effect of anti-VEGF antibody on retinal ganglion cells in rats. Br J Ophthalmol 2007, 91: 1230e1233 Thaler S, Fiedorowicz M, Choragiewicz TJ, Bolz S, Tura A, HenkeFahle S, Yoeruek E, Zrenner E, Bartz-Schmidt KU, Ziemssen F, Schuettauf F: Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage. Acta Ophthalmol 2010, 88:e170ee176 Brar VS, Sharma RK, Murthy RK, Chalam KV: Bevacizumab neutralizes the protective effect of vascular endothelial growth factor on retinal ganglion cells. Mol Vis 2010, 16:1848e1853 Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D’Amore PA: Endogenous VEGF is required for visual function: evidence for a survival role on muller cells and photoreceptors. PLoS One 2008, 3:e3554 Guo B, Wang Y, Hui Y, Yang X, Fan Q: Effects of anti-VEGF agents on rat retinal Muller glial cells. Mol Vis 2010, 16:793e799 Romano MR, Biagioni F, Besozzi G, Carrizzo A, Vecchione C, Fornai F, Lograno MD: Effects of bevacizumab on neuronal viability of retinal ganglion cells in rats. Brain Res 2012, 1478:55e63
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Neuronal Cell Death in the Inner Retina and the Influence of Vascular Endothelial Growth Factor Inhibition in a Diabetic Rat Model Q10
Hae-Young Lopilly Park, Jie Hyun Kim, and Chan Kee Park From the Department of Ophthalmology and Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Accepted for publication February 19, 2014. Address correspondence to Chan Kee Park, M.D., Ph.D., Department of Ophthalmology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea. E-mail: [email protected].
Q3
To inhibit vascular changes in diabetic retinopathy, inhibiting vascular endothelial growth factor (VEGF) has become a mainstay of the treatment of diabetic retinopathy. However, its effects on neuronal cells remain to be elucidated. We aimed to evaluate the effect of VEGF inhibition on neuronal cells in a streptozotocin-induced diabetic rat retina. VEGF inhibition was performed by intravitreal VEGF-A antibody injection. After anti-VEGF treatment, apoptosis in retinal ganglion cells (RGCs) increased, and novel apoptosis in amacrine and bipolar cells of the inner nuclear layer was observed by TUNEL staining. Phosphorylated Akt expression was significantly higher in RGCs but was decreased in neuronal cells of the inner nuclear layer after anti-VEGF treatment by Western blot analysis and immunohistochemical staining. These results demonstrate that VEGF inhibition significantly increased RGC apoptosis and neuronal cell apoptosis in the inner nuclear layer of a diabetic retina, which seems to consist primarily of amacrine and bipolar cells. The phosphorylated Akt pathway, which plays a neuroprotective role via VEGF, was significantly affected by VEGF inhibition in the inner nuclear layer, suggesting that neurotrophic factor deprivation is the main mechanism for neuronal cell death after inhibiting VEGF. The results of this study show that inhibiting VEGF may have detrimental effects on the apoptosis of neuronal cells in the inner layers of the diabetic retina. (Am J Pathol 2014, 184: 1e11; http://dx.doi.org/10.1016/j.ajpath.2014.02.016)
Diabetic retinopathy is the most frequently occurring microvascular complication of diabetes mellitus (DM) and often leads to vision loss in diabetic patients. Vascular dysfunction and loss of perfusion in the retina remain hallmarks of diabetic retinopathy1,2; however, growing evidence indicates that the function of neuronal cells in the retina is also compromised, perhaps even before overt vessel changes.3 Changes in neuronal cells, such as apoptosis of retinal ganglion cells (RGCs), amacrine cells, and photoreceptors, have been identified in the diabetic retina.4e7 In diabetic retinopathy, the atrophic appearance and dysfunction of the neural retina continue to clinically worsen after vascular lesions become quiescent.8 Therefore, DM-induced retinal neural degeneration remains a major cause of final vision loss and may progress independently of vascular lesions. Current treatments for diabetic retinopathy are intended to regulate vascular changes mediated by the action of vascular endothelial growth factor (VEGF) via laser treatment or agents that inhibit VEGF.9,10 However, VEGF-A, which is
the target of anti-VEGF agents used in patients with diabetic retinopathy, is thought to play a neuroprotective role.11e13 Neurons express VEGF receptors and can respond to VEGF-A via phosphoinositide 3-kinase/Akt signaling.14 VEGF-A can protect neurons against various harmful conditions, such as hypoxia, glutamate excitotoxicity, and deprivation of serum.15e17 Therefore, an improved comprehension of the effects of inhibiting VEGF-A in the diabetic retina is critical to understanding the function of neuronal cells in this region. It is possible that compromised neuronal cells in the diabetic retina may be further affected by treatments for diabetic retinopathy that target vascular changes. Supported by a National Research Foundation of Korea grant funded by Q2 the Korean government (Ministry of Education, Science, and Technology; C.K.P.; NRF-2011-0007989). H.-Y.L.P. and J.H.K. contributed equally to this work. Disclosures: None declared.
Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.02.016
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Park et al The purpose of this study was to investigate changes in neuronal cells of the inner retina and to evaluate the effects of inhibiting VEGF in a streptozotocin (STZ)-induced diabetic rat model. Changes in phosphorylated Akt (phosphoAkt) expression were measured in the diabetic retina to determine the downstream regulation of VEGF receptor signaling, which mediates the antiapoptosis of neuronal cells. STZ is cytotoxic to pancreatic beta cells and is used to generate a type 1 DM model in animals. The STZ-induced DM model results in inner retinal dysfunction but is not associated with pathologic neovascularization in the retina, which, in the present study, has the advantage of investigating neuronal cells in the inner retina.
Materials and Methods Diabetic Rat Models
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Adult male Sprague-Dawley rats (7 to 8 weeks old, 250 to 300 g) were used in this study. The diabetic and control groups comprised six animals for each experimental procedure; the total number of animals used was 96. All the animal experiments complied with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and animals were treated according to the regulations of the Catholic Ethics Committee of The Catholic University of Korea (Seoul, Korea) and the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 80-23, revised 1996; http://search.grants. nih.gov, accessed December 2013). All efforts were made to minimize suffering and the number of animals used in the study. DM was induced by a single i.p. injection of 60 ㎎/㎏ of STZ (Sigma-Aldrich, St. Louis, MO) in a citrate buffer solution (0.1 mol/L citric acid and 0.2 mol/L sodium phosphate, pH 4.5). Age-matched control rats received an equivalent volume of only the citrate buffer solution. Three days after STZ injection, blood glucose levels for each rat were measured using an automated Accu-Chek glucometer (Roche Diagnostics, Indianapolis, IN). Animals with a plasma glucose reading >350 mg/dL were considered diabetic and were used for further experimentation. Body weights and blood glucose levels were recorded once a week after the induction of DM.
Drug Administration All the rats were anesthetized via an i.p. injection of 50 mg/kg of tiletamine plus zolazepam (Zoletil; Virbac, Carros, France) and 15 mg/kg of xylazine hydrochloride (Rompun; Bayer, Leuverkeusen, Germany). For intravitreal injections, a small incision was made in the conjunctiva, and the eyeball was rotated by grasping the conjunctiva and gently pulling. In the left eye of diabetic rats, 4 mL of 25-mg/mL anti-VEGF antibody (Genentech Inc., San Francisco, CA) was injected intravitreally using a 30-gauge needle and a Hamilton syringe. The right eye of diabetic rats was injected
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with the same amount of goat IgG (IgG control). To reduce reflux after the injection, the needle remained in the eye for an additional 3 to 4 seconds and was then drawn back. The whole procedure was guided by a stereoscopic microscope, with care taken to avoid lens and retinal injury.
Fluorescein Dextran Angiography of the Retinal Blood Vessel A solution of fluorescein isothiocyanateedextran (2 106 molecular weight; Sigma-Aldrich) was prepared at a concentration of 10 mg/mL in PBS. After 8 weeks, three rats from each group were anesthetized with 50 mg/kg of ketamine and 5 mg/kg of xylazine. Fluorescein isothiocyanateedextran solution (0.5 mL) was injected into the tail vein of each rat, and a cover glass was placed on the cornea as a contact lens. Angiograms were established with a scan angle of 30 . Digital images at a magnification of 40 were captured using a charge-coupled device camera (DC500; Leica Microsystems, Heerbrugg, Switzerland) attached to a fluorescence stereomicroscope (MZ-III; Leica Microsytems, Wetzlar, Germany). Vascular abnormalities were scored from 0 to 5 in accordance with the following grading: grade 0, normal retinal vasculature, as observed in control fundus; þ1 point each for dilated or tortuous vessels, presence of microaneurysmal-like hyperfluorescent dots, and presence of dye-filling defects; and þ2 points for two or more of dilated or tortuous vessels, presence of microaneurysmal-like hyperfluorescent dots, and presence of dye-filling defects.18 Retinal vascular permeability was measured using the Evans blueealbumin leakage assay following an established protocol.19 Concentrations of Evans blue in the retina were normalized by total retinal protein concentrations and by Evans blue concentrations in the plasma.
Tissue Preparation At each time point, all the animals were deeply anesthetized with 50 mg/kg of ketamine and 5 mg/kg of xylazine for immunohistochemical analysis, the eyes were quickly enucleated and dissected, and the posterior eye cups were placed in chilled fixative (4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4). The isolated retinas were divided into small pieces and were immersion-fixed in the same fixative for 2 hours at 4 C. After washing several times, fixed retinas were cryoprotected in 30% sucrose containing 0.1 mol/L phosphate buffer for 6 hours at 4 C and then were stored in this buffer at 70 C. For Western blot analysis, retinal tissues were quickly dissected, frozen in liquid nitrogen, and stored at 70 C.
Immunohistochemical Analysis For fluorescence staining, samples were pre-embedded in 3% agar in deionized water. Fifty-micron vibratome sections were collected and washed several times in PBS. Sections
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Table 1
Body Weights and Serum Glucose Levels
Parameter
Control rats
STZ-induced diabetic rats
Body weight (g) Start Finish Serum glucose (mg/dL)
331.5 9.4 441.6 8.9 133.5 4.9
335.9 7.7 328.7 10.5 565.8 14.2
Data are expressed as means SD.
were incubated in 10% normal donkey serum in PBS for 1 hour at room temperature to block nonspecific binding activity, followed by incubation with the rabbit anti-VEGF (Abcam Plc, Cambridge, UK) and antieglial fibrillary acidic protein (GFAP) (Millipore, Billerica, MA) overnight at 4 C. The next day, after several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546 (Molecular Probes, Eugene, CA). For double-labeling studies, sections were incubated with mouse anti-NeuN (Millipore), anti-PKCa (Santa Cruz Biotechnology, Santa Cruz, CA), antiecleaved caspase-3 (Cell Signaling Technology Inc., Boston, MA), and anti-parvalbumin (Sigma-Aldrich) or in
0.1 mol/L PBS containing 0.5% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) overnight at 4 C. They were then rinsed for 30 minutes with 0.1 mol/L PBS and incubated with goat anti-mouse Alexa 488 (Molecular Probes) for 1 hour and 30 minutes at room temperature. After further washing in 0.1 mol/L phosphate buffer for 30 minutes, the sections were mounted using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Sections were washed, placed under cover glass, and examined by confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH, Jena, Germany).
TUNEL Assay Apoptotic cells were evaluated using TUNEL staining. The retinas were dissected from the choroid, and the central portion of the superior nasal quadrant, 1.5 mm from the optic disc, was trimmed into small pieces. Fifty-micron cryosections of the retina were embedded in 4% paraformaldehyde and were washed with PBS. The tissue was stained using the TUNEL method according to the manufacturer’s protocol (in situ cell
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VEGF Inhibition in Diabetic Rat Model Q1
Immunohistochemical staining of VEGF and NeuN, an RGC marker. Control retina (A, E, I, and M) and diabetic retina 1 week (B, F, J, and N), 4 Q7 weeks (C, G, K, and O), and 8 weeks (D, H, L, and P) after STZ injection. Basal VEGF expression in the control retina (A) increased gradually after DM induction through 8 weeks (BeD). There was no co-expression of VEGF (A) and NeuN (E) in the GCL of the control retina (I and M). VEGF and NeuN co-expression (arrowheads) appears in the GCL at week 1 (F, J, and N), week 4 (G, K, and O), and week 8 (H, L, and P) after STZ injection. n Z 5 for each time point and group. Scale bars: 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.
Figure 1
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death detection kit; Roche Applied Science, Indianapolis, IN). For double-labeling studies, sections were incubated with antiNeuN, anti-PKCa, and anti-parvalbumin solutions overnight at 4 C. The next day, after several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546, and after further washes in 0.1 mol/L phosphate buffer for 30 minutes they were mounted using VECTASHIELD mounting medium with DAPI. Sections were washed, put under cover glass, and examined using confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH). For quantitative analysis of the number of TUNEL-positive cells in the ganglion cell layer (GCL) and inner nuclear layer (INL), the cell bodies were counted in each layer. Three to four independent tests were repeated in different retinal sections by one investigator in a masked manner, not knowing the classified group of the retinal sections.
Western Blot Analysis Control and diabetic retinas were homogenized in radioimmunoprecipitation assay buffer (1% Triton X-100, 5% SDS, 5% deoxycholic acid, 0.5 mol/L Tris-HCl [pH 7.5], 10% glycerol, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mg/mL of aprotinin, 1 mg/mL of leupeptin, 1 mg/mL of pepstatin, 200 mmol/L sodium orthovanadate, and 200 mmol/L sodium fluoride). Tissue extracts were incubated for 10 minutes on ice and were clarified by centrifugation at 10,000 g for 25 minutes at 4 C. Total protein in the retinal extracts was measured using a standard bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). Retinal extracts containing 40 mg of total protein were resuspended in
5 sample buffer (60 mmol/L TriseHCl [pH 7.4], 25% glycerol, 2% SDS, 14.4 mmol/L 2-mercaptoethanol, and 0.1% bromophenol blue) at a 4:1 ratio, boiled for 5 minutes, and resolved by SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane, and blots were stained with Ponceau S (Sigma-Aldrich) to visualize the protein bands and ensure equal protein loading and uniform transfer. Blots were washed and blocked for 45 minutes with 5% nondried skim milk in Tris-buffered saline with Tween 20 buffer (20 mmol/L TrisHCl [pH 7.6], 137 mmol/L NaCl, and 0.1% Tween 20). Blots were then probed for 24 hours using antibodies against VEGF, phospho-Akt (Cell Signaling Technology Inc.), total Akt (Cell Signaling Technology Inc.), GFAP, brain-derived neurotrophic factor (BDNF) (Santa Cruz Biotechnology), and actin (Sigma-Aldrich). Blots were then probed with horseradish peroxidaseeconjugated goat anti-rabbit secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence system (Amersham, Piscataway, NJ) and X-ray film. Each immunoblot shown is representative of five independent experiments. Signal intensity was measured using an ImageMaster VDS system (Pharmacia Biotech, Piscataway, NJ). Signal quantities shown were standardized to background and normalized for loading. Controls were set as 1.0, and the fold changes were plotted in relation to the control value. Results are representative of five independent experiments. Data are expressed as means SD.
Statistical Analysis All the data are expressed as means SD. Comparisons between each time point were compared with the control
Figure 2 A: TUNEL staining showed increased apoptotic cells in the GCL after DM induction at weeks 4 and 8 compared with control retina. B: The number of TUNEL-positive cells in the GCL was significantly greater in the diabetic retina compared with the control retina at weeks 1, 4, and 8 after STZ injection. Data are expressed as means SD. n Z 5 for each time point and group. *P < 0.05 compared with controls. Scale bar Z 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.
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using a U-test. Results with P < 0.05 were considered statistically significant.
Results Body Weight and Blood Glucose Levels ½T1
The body weights and blood glucose levels of experimental rats were recorded (Table 1). At week 8 after STZ injection, body weight was lower and blood glucose levels were significantly higher in diabetic rats compared with control rats.
Inner Retinal Changes after DM Induction The immunostaining of VEGF revealed expression in all layers of the retina in control rats (Figure 1A). After STZ injection in experimental rats, this expression gradually increased in week 1 (Figure 1B), week 4 (Figure 1C), and week 8 (Figure 1D). At week 8 after STZ injection, VEGF was densely expressed in the GCL, inner plexiform layer, INL, outer plexiform layer, and outer nuclear layer in diabetic rats. Colabeling with VEGF and NeuN, an RGC marker, did not identify co-expression of VEGF and NeuN in the GCL in control retinas (Figure 1, E, I, and M). However, VEGF and NeuN co-expression was evident in the GCL at week 1 (Figure 1, F, J, and N), week 4 (Figure 1, G, K, and O), and week 8 (Figure 1, H, L, and P) in diabetic rats. A significant increase in apoptotic cells was identified in ½F2 the GCL at weeks 4 and 8 after STZ injection (Figure 2A). The number of TUNEL-positive cells in the GCL was significantly greater in the retinas of diabetic rats compared with those of control rats at weeks 1, 4, and 8 (Figure 2B). After colabeling with TUNEL staining and NeuN, apoptotic cells in the GCLs were found to be mostly RGCs ½F3 (Figure 3A), and the TUNEL-positive RGCs expressed VEGF in the cytoplasm (Figure 3B). GFAP immunostaining was performed to evaluate glial cell activation in response to retinal stress caused by DM. GFAP expression was restricted to astrocytes and the end feet of Muller cells in the inner limiting membrane in the ½F4 retinas of control rats (Figure 4A). In diabetic rats, however, GFAP expression was significantly greater at weeks 1, 4, and 8 after STZ injection (Figure 4, C, E, and G, respectively). The increased GFAP expression in astrocytes and the processes of Muller glia spanned the entire inner retina. These data demonstrate that VEGF levels are elevated throughout the retinas of STZ-induced diabetic rats and are especially up-regulated in the cytoplasm of RGCs after week 1 of STZ injection. VEGF up-regulated RGCs were shown to undergo apoptosis by TUNEL staining that gradually increased after STZ injection through week 8. Similarly, the activation of astrocytes and Muller glial cells also increased gradually after DM induction through week 8. ½F1
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559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 Figure 3 A: Colabeling with TUNEL staining and NeuN, a ganglion cell 596 marker, showed that TUNEL-positive cells in the ganglion cell layer (GCL) 597 are retinal ganglion cells undergoing apoptosis (arrows). B: Colabeling 598 with TUNEL staining and VEGF showed that these apoptotic RGCs express 599 VEGF (arrows). n Z 5 for each time point and group. Scale bar Z 50 mm. 600 INL, inner nuclear layer. 601 Effect of Inhibiting VEGF in the Diabetic Retina 602 603 There was no change in GFAP immunostaining in IgG 604 605 control retinas (Figure 4B). However, anti-VEGF antibody 606 administration resulted in an increase in GFAP expression 607 in diabetic retinas at weeks 1, 4, and 8 (Figure 4, D, F, and 608 H, respectively). 609 TUNEL staining revealed apoptotic cells in the GCL and 610 INL after intravitreal injection of the anti-VEGF antibody 611 (Figure 5A). The number of TUNEL-positive cells in the ½F5 612 GCL and INL significantly increased after anti-VEGF 613 antibody administration compared with control diabetic 614 retina (Table 2). TUNEL-positive cells were not identified ½T2 615 in the INL after DM induction or after intravitreal injection 616 617 of IgG in the diabetic retina. However, injection of the anti618 VEGF antibody resulted in additional apoptotic cells in the 619 INL of diabetic retinas. TUNEL-positive cells in the GCL 620
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Figure 4 GFAP immunostaining revealed an increase in glial cell activity in the diabetic retina at weeks 1 (C), 4 (E), and 8 (G) after STZ injection compared with the control retina (A). After antiVEGF antibody treatment, GFAP expression significantly increased in the diabetic retina at weeks 1 (D), 4 (F), and 8 (H) after STZ injection. However, the diabetic retina was not affected by IgG injection (B). n Z 5 for each time point and group. Scale bar Z 50 mm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
dilation or tortuosity, presence of microaneurysm, and dyefilling defects, diabetic retinas showed increased scores. However, after VEGF inhibition, these vascular features returned to normal levels (Figure 6D). Vascular permeability, representing vessel leakage, was also significantly increased in diabetic retinas, which returned to normal levels after VEGF inhibition (Figure 6E). These data demonstrate that inhibiting VEGF aggravates inner retinal cell death in the diabetic retina, although the vascular changes improve.
Differential Response of the GCL and INL in the Diabetic Retina after Inhibition of VEGF Western blot analyses revealed greater VEGF and GFAP expression in the diabetic retina at weeks 1, 4, and 8 after STZ injection (Figure 7A). Phospho-Akt levels gradually ½F7 decreased, despite no changes in total Akt levels, after DM induction at weeks 1, 4, and 8. The ratio of phospho-Akt to total Akt was significantly lower at weeks 4 and 8 in diabetic retinas compared with control retinas (Figure 7B).
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VEGF Inhibition in Diabetic Rat Model
After intravitreal injection of anti-VEGF antibody into diabetic rat eyes, VEGF was significantly decreased compared with IgG control eyes at weeks 1, 4, and 8 (Figure 7A). Likewise, GFAP was significantly greater after the injection of anti-VEGF antibody into diabetic eyes compared with control eyes at weeks 1, 4, and 8. Phospho-Akt levels were also decreased after anti-VEGF antibody injection into diabetic eyes at weeks 4 and 8. However, the ratio between phospho-Akt and total Akt did not show a difference after VEGF inhibition in diabetic retinas (Figure 7B). Immunostaining of phospho-Akt revealed baseline expression levels in the GCL and INL in control retinas (Figure 7C), which Table 2 Number of TUNEL-Positive Cells after Anti-VEGF Treatment in Diabetic Retinas Time after STZ injection Q9
1 week 4 weeks 8 weeks
Anti-VEGF treatment
GCL
þ þ þ
1.9 3.7 4.4 6.7 8.0 9.9
INL
0.7* 0.8*y 0.8* 1.1*y 1.3* 1.9*
1.3 1.0 30.8 4.4* 34.7 4.6*
n Z 3 to 4 for each time point and group. Data are expressed as means SD. *P < 0.05 compared with control retinas. y P < 0.05 compared with diabetic retinas without anti-VEGF treatment. -, no TUNEL-positive cells.
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807 808 809 810 811 812 813 814 815 Figure 5 A: VEGF inhibition of the STZ-induced 816 diabetic eye resulted in the apoptosis of cells in the GCL and INL as observed by TUNEL staining. B: 817 TUNEL-positive cells in the GCL were RGCs shown 818 by colabeling with NeuN (arrows). Of TUNEL819 positive cells in the INL, some were bipolar cells 820 shown by colabeling with PCKa (C; arrows), and 821 others were amacrine cells shown by colabeling 822 with parvalbumin (D; arrows). Few cells in the GCL 823 are colabeled with parvalbumin (arrowhead), 824 showing some apoptosis of displaced amacrine 825 cells in the GCL. n Z 6 for each time point and 826 group. Scale bar Z 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nu827 clear layer; OPL, outer plexiform layer. 828 829 830 831 832 833 834 835 836 837 838 did not change in IgG control retinas (Figure 7D). Subse839 quent to anti-VEGF antibody injection into diabetic eyes, 840 phospho-Akt expression was significantly greater in the 841 Q5 GCL but was decreased in the INL (Figure 7E). 842 RGC death in the GCL was increased after diabetes in843 duction but decreased after anti-VEGF injection in the 844 diabetic retina. Immunostaining of cleaved caspase-3 in the 845 RGC shows decreased RGC death after inhibition of VEGF 846 (Figure 8A). Western blot analysis revealed increased ½F8 847 848 BDNF levels after inhibition of VEGF in diabetic retinas 849 (Figure 8B). Downstream of BDNF, phospho-Akt immu850 nostaining was increased in the RGCs (Figure 8A). 851 852 853 Table 3 Number of Cells in Each Retinal Layer after Anti-VEGF 854 Treatment in Diabetic Retinas 855 Time after Anti-VEGF 856 STZ injection treatment GCL INL 857 1 week 16.3 1.6* 119.9 8.7 858 þ 17.9 1.7* 120.1 9.8 859 4 weeks 14.1 1.8* 113.0 8.9 860 þ 14.9 1.3* 119.6 8.1y 861 8 weeks 11.0 1.5* 121.4 9.8 862 þ 14.0 1.4* 115.3 8.1y 863 864 n Z 3 to 4 for each time point and group. Data are expressed as 865 means SD. 866 *P < 0.05 compared with control retinas. y 867 P < 0.05 compared with diabetic retinas without anti-VEGF treatment. 868
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Figure 6 Representative fluorescein angiograms from control retina (A), diabetic retina (B), and diabetic retina with anti-VFGF treatment (C) 8 weeks after STZ injection. B and C: Dye-filling defects (arrows), dilated or tourtous vessels (white arrowheads), and microaneurysmal-like hyperfluorescent dots (gray arrowheads) were present in the diabetic retina. C: VEGF inhibition improved the microvascular pathologic changes of diabetic retinas. D: Grading showed significantly increased vascular changes in diabetic retinas, which returned to near normal levels after VEGF inhibition. E: The permeability assay, indicating fluorescence leakage, showed increased leakage from vessels in diabetic retinas, which also returned to near normal after VEGF inhibition. Data are expressed as means SD. n Z 3 for each time point and group. Scale bar Z 200 mm.
Discussion DM causes systemic microvascular complications, including retinopathy.20e22 Vascular and ischemic changes of the retina caused by high glucose levels result in up-regulation of VEGF in the diabetic retina.23 Up-regulation of VEGF could be a compensatory mechanism for the retina to withstand the vascular and ischemic changes by DM; however, this causes devastating effects to the retina, such as compromised bloodretinal barrier, vascular leakage, and neovascularization.24 Treatment with an anti-VEGF agent is reported to be effective in eliminating the abnormal vessels and retinal edema of diabetic retinopathy. However, although vascular changes caused by DM are improved after inhibiting VEGF, neuronal cell death increases in the inner retina, as shown in the present results. Neuronal cells, such as bipolar and amacrine cells, may use VEGF for survival, and inhibiting VEGF had a harmful effect on these cells, with decreased phospho-Akt expression in the INL, which is downstream of the growth factor pathway. Phospho-Akt levels were increased in RGCs and may be mediated by increased BDNF levels after diabetic induction, and inhibiting VEGF had less effect on these cells. Several studies have indicated that not only vascular abnormalities but also neuronal abnormalities are associated with early diabetic retinopathy.4e7 However, the precise mechanism of neuronal cell death in early diabetic retinopathy
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is not well established. In the human retina, VEGF is found in all major classes of retinal neurons and is especially prominent in amacrine cells and RGCs.25 Retinal neurons normally provide continuous trophic support for their own survival, and VEGF expression in these neurons is increased in an ischemic retina.25,26 VEGF-A exerts its neuroprotective effects and directly prevents neuronal cell apoptosis via phosphoinositide 3-kinase/Akt signaling.8,14 In this study, the expression of phospho-Akt was primarily centered in the GCL and INL of control retinas and is in agreement with prominent locations of VEGF expression found in previous studies.25 However, although VEGF protein levels increased after DM induction, phospho-Akt protein levels decreased at weeks 4 and 8 after STZ injection. Immunostaining did not reveal specific changes in phospho-Akt expression in the GCL or INL of the diabetic retina. Likewise, the total amount of phospho-Akt protein did not exhibit any changes subsequent to VEGF inhibition. However, the pattern of phosphoAkt expression did change after anti-VEGF treatment in that expression significantly increased in RGCs in the GCL but decreased in neuronal cells of the INL. A decrease in phospho-Akt expression in the INL explains the presence of apoptotic cells subsequent to anti-VEGF treatment because the neuroprotective effects of VEGF via phospho-Akt signaling were down-regulated by VEGF inhibition. The inhibition of VEGF significantly increases apoptosis Q6 in amacrine and bipolar cells in the INL, but the increase in
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Figure 7 Representative results of Western blot analysis (A), quantitative analysis of the ratio of phospho-Akt to total Akt (B), and immunohistochemical staining of phospho-Akt (CeE). A: The proteins of VEGF and GFAP increased significantly after diabetes induction. However, VEGF protein levels decreased and GFAP protein levels increased after VEGF inhibition. B: The phospho-Akt/total Akt ratio decreased significantly at weeks 4 and 8 after STZ injection. VEGF inhibition decreased the phospho-Akt/total Akt ratio compared with IgG control 4 and 8 weeks after STZ injection; however, it is not statistically significant. Phospho-Akt immunoreactions (arrows) were found in the GCL and INL after diabetes induction (C), which is not changed in the IgG control retina (D). E: After Q8 VEGF inhibition, phospho-Akt expression increased in the GCL (arrows) and decreased in the inner nuclear layer. Data are expressed as means SD. n Z 6 for each time point and group. *P < 0.05 compared with controls. Scale bar Z 50 mm.
RGC apoptosis after VEGF inhibition is relatively less prominent. This finding may suggest that neuronal cells use VEGF differently in the diabetic retina and that the neuroprotective effects of VEGF may be different between RGCs and inner nuclear cells, including amacrine and bipolar cells. However, BDNF is the most potent survival factor for injured RGCs,27e29 and these cells may be less affected by VEGF inhibition.
Anti-VEGF agents are widely used for the treatment of age-related macular degeneration to reduce angiogenesis associated with choroidal neovascularization and are currently Food and Drug Administrationeapproved for the treatment of age-related macular degeneration and retinal vessel occlusion. Furthermore, bevacizumab is extensively used as an off-label treatment for diabetic macular edema, proliferative diabetic retinopathy, and neovascular glaucoma.
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VEGF Inhibition in Diabetic Rat Model
Figure 8
Representative results of immunohistochemical staining of phospho-Akt and cleaved caspase-3 (A) and Western blot analysis (B). A: PhosphoAkt immunoreactions increased in the RGC, colabeled with NeuN (arrowheads), in the ganglion cell layer (GCL) after VEGF inhibition compared with the IgG control retina. Cleaved caspase-3 showed decreased RGC death in the GCL after VEGF inhibition. B: BDNF levels increased after VEGF inhibition up to 4 weeks after STZ injection. n Z 6 for each time point and group. Scale bar Z 50 mm.
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Park et al Accumulating clinical evidence indicates the safety of using anti-VEGF agents in age-related macular degeneration because there are no changes in the retinal nerve fiber layer (where the axons of RGCs pass through) or the optic disc after multiple injections of anti-VEGF agents.30e32 However, the diabetic retina is different from the retina with age-related macular degeneration. The function of neuronal cells in the diabetic retina is compromised, and the presence of apoptosis in RGCs and retinal stress, as evidenced by the elevated expression of GFAP, before overt neovascularization is clear, as shown in the present study. Previously, we reported that the retinal nerve fiber layer gets thinner as diabetic retinopathy progresses and that retinal nerve fiber layer thinning was present in patients with DM without diabetic retinopathy.33 These findings together indicate that neuronal stress and RGC damage are clinically apparent in diabetic retinas before evident neovascularization. In the adult retina, VEGF is expressed in the absence of active neovascularization and is implicated in the maintenance and function of adult retinal neuronal cells.34 It is possible that the inhibition of VEGF in the diabetic retina may further compromise the function of neuronal cells and influence apoptosis. However, the effect of anti-VEGF agents on neuronal cells in the inner diabetic retina has not been fully investigated, and there is a lack of long-term clinical data showing that anti-VEGF therapy is safe for diabetic retinopathy.35 Neither the in vivo intravitreal injection nor the direct application of various VEGF inhibitors to RGCs in vitro show toxic effects on RGCs in previous reports.36e38 Likewise, VEGF inhibition did not have a toxic effect on RGCs under stressful conditions induced by N-methyl-D-aspartate or oxidative stress,37,39 and Muller glial cells were not affected in vivo or in vitro by VEGF inhibition.40 However, anti-VEGF agents neutralize the protective effect of VEGF when applied together under stressful conditions.38 RGC apoptosis has been observed after multiple injections of an anti-VEGF agent,41 including under conditions in which RGCs require VEGF for survival or in which repeated treatment with anti-VEGF agents may interfere with the neuroprotective action of VEGF.38 The present study demonstrates that VEGF expression is increased in RGCs after DM induction and that these VEGFexpressing RGCs undergo apoptosis. After VEGF inhibition to the diabetic eye, inner retinal stress is increased by GFAP expression, apoptosis of RGCs is increased, and novel apoptosis of amacrine and bipolar cells in the INL is present. These results show that under diabetic conditions, neuronal cells in the inner retina use VEGF for survival, which is interfered by VEGF inhibition, after which they undergo apoptosis. This neuronal cell death was independent of the changes in the retinal vessels after VEGF inhibition, as shown in the angiographic results. In conclusion, RGC apoptosis is increased after DM induction in an STZ injection model and is further increased after VEGF inhibition in the diabetic retina. More important, VEGF inhibition significantly increases neuronal cell apoptosis in the INL in the diabetic retina and seems to primarily include
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amacrine and bipolar cells. The phospho-Akt pathway, which is integral to the neuroprotective role of VEGF, is significantly affected by VEGF inhibition, and its pattern is different between RGCs and neuronal cells in the INL. This study provides useful information concerning the use of antiVEGF agents in the diabetic retina as the treatment of vascular changes due to diabetic retinopathy via the inhibition of VEGF may be crucial for the preservation of vision. However, note that this treatment may have detrimental effects on apoptosis in neuronal cells of the inner retina. Further strategies for the management of vascular changes and the preservation of inner neuronal cells are needed to save the vision of diabetic patients.
Acknowledgments We thank Jun-Sub Choi, Ph.D., (The Catholic University of Korea) for advice and support in experiments of retinal angiography. C.K.P. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
References 1. Kern TS, Engerman RL: Vascular lesions in diabetes are distributed non-uniformly within the retina. Exp Eye Res 1995, 60:545e549 2. Ljubimov AV, Burgeson RE, Butkowski RJ, Couchman JR, Zardi L, Ninomiya Y, Sado Y, Huang ZS, Nesburn AB, Kenney MC: Basement membrane abnormalities in human eyes with diabetic retinopathy. J Histochem Cytochem 1996, 44:1469e1479 3. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, Jefferson LS, Kester M, Kimball SR, Krady JK, LaNoue KF, Norbury CC, Quinn PG, Sandirasegarane L, Simpson IA; Group JDRC: Diabetic retinopathy: seeing beyond glucose-induced microvascular disease. Diabetes 2006, 55:2401e2411 4. Barber AJ, Lieth E, Khin SA, Antonetti DA, Buchanan AG, Gardner TW: Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. J Clin Invest 1998, 102:783e791 5. Gastinger MJ, Singh RS, Barber AJ: Loss of cholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akitadiabetic mouse retinas. Invest Ophthalmol Vis Sci 2006, 47: 3143e3150 6. Martin PM, Roon P, Van Ells TK, Ganapathy V, Smith SB: Death of retinal neurons in streptozotocin-induced diabetic mice. Invest Ophthalmol Vis Sci 2004, 45:3330e3336 7. Park SH, Park JW, Park SJ, Kim KY, Chung JW, Chun MH, Oh SJ: Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia 2003, 46:1260e1268 8. Foxton RH, Finkelstein A, Vijay S, Dahlmann-Noor A, Khaw PT, Morgan JE, Shima DT, Ng YS: VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma. Am J Pathol 2013, 182:1379e1390 9. Kim LA, D’Amore PA: A brief history of anti-VEGF for the treatment of ocular angiogenesis. Am J Pathol 2012, 181:376e379 10. Ozaki H, Seo MS, Ozaki K, Yamada H, Yamada E, Okamoto N, Hofmann F, Wood JM, Campochiaro PA: Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to
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1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240
1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294
VEGF Inhibition in Diabetic Rat Model
11. 12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24. 25.
26.
completely prevent retinal neovascularization. Am J Pathol 2000, 156: 697e707 D’Amore PA: Vascular endothelial cell growth factor-A: not just for endothelial cells anymore. Am J Pathol 2007, 171:14e18 Beazley-Long N, Hua J, Jehle T, Hulse RP, Dersch R, Lehrling C, Bevan H, Qiu Y, Lagreze WA, Wynick D, Churchill AJ, Kehoe P, Harper SJ, Bates DO, Donaldson LF: VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro. Am J Pathol 2013, 183:918e929 Miguel NC, Matsuda M, Portes AL, Allodi S, Mendez-Otero R, Puntar T, Sholl-Franco A, Krempel PG, Monteiro ML: In vitro effects of bevacizumab treatment on newborn rat retinal cell proliferation, death, and differentiation. Invest Ophthalmol Vis Sci 2012, 53:7904e7911 Kim I, Ryan AM, Rohan R, Amano S, Agular S, Miller JW, Adamis AP: Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes. Invest Ophthalmol Vis Sci 1999, 40:2115e2121 Jin KL, Mao XO, Greenberg DA: Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc Natl Acad Sci U S A 2000, 97:10242e10247 Gomes E, Papa L, Hao T, Rockwell P: The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells. Mol Cell Biochem 2007, 305:179e190 Tolosa L, Mir M, Asensio VJ, Olmos G, Llado J: Vascular endothelial growth factor protects spinal cord motoneurons against glutamateinduced excitotoxicity via phosphatidylinositol 3-kinase. J Neurochem 2008, 105:1080e1090 Kowalczuk L, Touchard E, Omri S, Jonet L, Klein C, Valamanes F, Berdugo M, Bigey P, Massin P, Jeanny JC, Behar-Cohen F: Placental growth factor contributes to micro-vascular abnormalization and bloodretinal barrier breakdown in diabetic retinopathy. PLoS One 2011, 6:e17462 Xu Q, Qaum T, Adamis AP: Sensitive blood-retinal barrier breakdown quantitation using Evans blue. Invest Ophthalmol Vis Sci 2001, 42: 789e794 Kohner EM, Patel V, Rassam SM: Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy. Diabetes 1995, 44:603e607 Gardiner TA, Archer DB, Curtis TM, Stitt AW: Arteriolar involvement in the microvascular lesions of diabetic retinopathy: implications for pathogenesis. Microcirculation 2007, 14:25e38 Mizutani M, Kern TS, Lorenzi M: Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest 1996, 97:2883e2890 Qaum T, Xu Q, Joussen AM, Clemens MW, Qin W, Miyamoto K, Hassessian H, Wiegand SJ, Rudge J, Yancopoulos GD, Adamis AP: VEGF-initiated blood-retinal barrier breakdown in early diabetes. Invest Ophthalmol Vis Sci 2001, 42:2408e2413 Stitt AW, Lois N, Medina RJ, Adamson P, Curtis TM: Advances in our understanding of diabetic retinopathy. Clin Sci 2013, 125:1e17 Famiglietti EV, Stopa EG, McGookin ED, Song P, LeBlanc V, Streeten BW: Immunocytochemical localization of vascular endothelial growth factor in neurons and glial cells of human retina. Brain Res 2003, 969:195e204 Shima DT, Gougos A, Miller JW, Tolentino M, Robinson G, Adamis AP, D’Amore PA: Cloning and mRNA expression of vascular
The American Journal of Pathology
-
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40. 41.
endothelial growth factor in ischemic retinas of Macaca fascicularis. Invest Ophthalmol Vis Sci 1996, 37:1334e1340 Jelsma TN, Friedman HH, Berkelaar M, Bray GM, Aguayo AJ: Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve. J Neurobiol 1993, 24:1207e1214 Mansour-Robaey S, Clarke DB, Wang YC, Bray GM, Aguayo AJ: Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci U S A 1994, 91:1632e1636 Perez MT, Caminos E: Expression of brain-derived neurotrophic factor and of its functional receptor in neonatal and adult rat retina. Neurosci Lett 1995, 183:96e99 Martinez-de-la-Casa JM, Ruiz-Calvo A, Saenz-Frances F, RecheFrutos J, Calvo-Gonzalez C, Donate-Lopez J, Garcia-Feijoo J: Retinal nerve fiber layer thickness changes in patients with age-related macular degeneration treated with intravitreal ranibizumab. Invest Ophthalmol Vis Sci 2012, 53:6214e6218 Seth RK, Salim S, Shields MB, Adelman RA: Assessment of optic nerve cup-to-disk ratio changes in patients receiving multiple intravitreal injections of antivascular endothelial growth factor agents. Retina 2009, 29:956e959 Horsley MB, Mandava N, Maycotte MA, Kahook MY: Retinal nerve fiber layer thickness in patients receiving chronic anti-vascular endothelial growth factor therapy. Am J Ophthalmol 2010, 150:558e561.e1 Park HY, Kim IT, Park CK: Early diabetic changes in the nerve fibre layer at the macula detected by spectral domain optical coherence tomography. Br J Ophthalmol 2011, 95:1223e1228 Wang Y, Mao XO, Xie L, Banwait S, Marti HH, Greenberg DA, Jin K: Vascular endothelial growth factor overexpression delays neurodegeneration and prolongs survival in amyotrophic lateral sclerosis mice. J Neurosci 2007, 27:304e307 Al-Latayfeh M, Silva PS, Sun JK, Aiello LP: Antiangiogenic therapy for ischemic retinopathies. Cold Spring Harb Perspect Med 2012, 2: a006411 Iriyama A, Chen YN, Tamaki Y, Yanagi Y: Effect of anti-VEGF antibody on retinal ganglion cells in rats. Br J Ophthalmol 2007, 91: 1230e1233 Thaler S, Fiedorowicz M, Choragiewicz TJ, Bolz S, Tura A, HenkeFahle S, Yoeruek E, Zrenner E, Bartz-Schmidt KU, Ziemssen F, Schuettauf F: Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage. Acta Ophthalmol 2010, 88:e170ee176 Brar VS, Sharma RK, Murthy RK, Chalam KV: Bevacizumab neutralizes the protective effect of vascular endothelial growth factor on retinal ganglion cells. Mol Vis 2010, 16:1848e1853 Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D’Amore PA: Endogenous VEGF is required for visual function: evidence for a survival role on muller cells and photoreceptors. PLoS One 2008, 3:e3554 Guo B, Wang Y, Hui Y, Yang X, Fan Q: Effects of anti-VEGF agents on rat retinal Muller glial cells. Mol Vis 2010, 16:793e799 Romano MR, Biagioni F, Besozzi G, Carrizzo A, Vecchione C, Fornai F, Lograno MD: Effects of bevacizumab on neuronal viability of retinal ganglion cells in rats. Brain Res 2012, 1478:55e63
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