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Dexamethasone Alters TNF-α Expression in Retinopathy
Molecular Genetics and Metabolism 72, 164 –167 (2001) doi:10.1006/mgme.2000.3124, available online at http://www.idealibrary.com on
Dexamethasone Alters TNF-␣ Expression in Retinopathy 1 Panitan Yossuck, Yun Yan Misrak Tadesse, and Rosemary D. Higgins 2 Department of Pediatrics, Division of Neonatology, Georgetown University Children’s Medical Center, Washington, DC 20007 Received August 16, 2000, and in revised form November 6, 2000; published online January 16, 2001
vide a potential site of action for therapeutic targets. © 2001 Academic Press
TNF-␣ has been found in the retina. Hyperoxia and hypoxia regulate TNF-␣ expression. TNF-␣ is an important factor in inflammation and angiogenesis. Dexamethasone inhibits TNF-␣ production. Changes in TNF-␣ expression in the retina may play an important role in the development of oxygen-induced retinopathy. Oxygen-induced retinopathy was produced in C57BL6 mice by exposure to 75% oxygen at Postnatal Day 7 (P7) for 5 days and the mice recovered in room air until Day 17 (P17). Dexamethasone was administered at 0.5 mg/kg/day once daily subcutaneously during the 5 days of oxygen exposure. TNF-␣ expression was evaluated at Day 7 prior to oxygen exposure, at Day 12 (P12) immediately upon removal from oxygen, and at Day 17, the time of maximal vasoproliferation by RT-PCR. TNF-␣ is developmentally regulated in the retinae of C57BL6 mice. From P7 to P12, there is a 3-fold increase in TNF-␣ expression and from P7 to P17 there is a 2.7-fold increase. There was 2.7-fold suppression in expression immediately following oxygen exposure at P12. The expression was dramatically increased at P17, the time of maximal vasoproliferation. Dexamethasone inhibited the expression of TNF-␣ at P17 by 6.4-fold. At this dose, it also suppressed the baseline TNF-␣ expression in the mouse model. In summary, TNF-␣ is altered in the development of oxygen-induced retinopathy in the mouse. It increased markedly during the vasoproliferative phase and was suppressed by dexamethasone. Modulation of TNF-␣ expression may pro-
Tumor necrosis factor alpha (TNF-␣), a monocyte/ macrophage-derived proinflammatory factor, is involved in the pathogenesis of proliferative retinopathy and ocular inflammation (1– 8). TNF-␣ has both proangiogenic and antiangiogenic effects on endothelial cells, depending on the biological context (9, 10). High-dose TNF-␣ was found to inhibit angiogenesis, in contrast to low doses that induced angiogenesis (11). The role of TNF-␣ and oxygen toxicity has been studied extensively (12–14). Hyperoxia in the lung induces the production and expression of TNF-␣ by alveolar macrophage and eventually leads to the development of pulmonary fibrosis (12). Hypoxia in several tissues was found to alter the production and expression of TNF-␣. Oxygen-induced retinopathy is the disease involved in both hyperoxia and hypoxia. TNF-␣ expression in the mouse model oxygen-induced retinopathy has not been reported. Dexamethasone is one of the most potent and widely used anti-inflammatory agents, although the mode of action is poorly understood. Administration of dexamethasone before (during Postnatal Days, 1–5) and during oxygen exposure (P7–11) decreases the severity of oxygen-induced retinopathy in the mouse model (15, 16). Dexamethasone has no effect when given after the oxygen exposure (during P12– 16) (16). It has been found that dexamethasone inhibits TNF-␣ production and expression in several tissues (17–21). The goals of this study are to explore the TNF-␣ expression in the retina of the mouse model during normal development and during the development of
1 Funded in part by Fight For Sight, Research Division of Prevent Blindness America. 2 To whom correspondence should be addressed at Department of Pediatrics, Division of Neonatology, Georgetown University Children’s Medical Center, 3800 Reservoir Road, NW Room M3400, Washington, DC 20007. Fax: (202) 784-4747. E-mail: [email protected].
164 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
future
TNF-␣, DEXAMETHASONE, AND RETINOPATHY
oxygen-induced retinopathy. Further, the effect of dexamethasone on TNF-␣ expression in oxygeninduced retinopathy is studied. MATERIALS AND METHODS
165
der an ultraviolet light and was photographed. The results were scanned for density using Quantiscan Program (Biosoft, Ferguson, MO). The results were normalized with the -actin expression from the same sample. The PCR product was also sequenced for confirmation.
Animal Model The study protocol was approved by Georgetown University Animal Use and Care Committee. C57BL6 mice were exposed to 75% oxygen from Postnatal Day 7 to 12 as previously described (22) and utilized in our laboratory (15, 16) to produce oxygen-induced retinopathy. Dexamethasone (0.5 mg/kg sc daily) was administered from Postnatal Day 7 for 5 days (15). The animals were sacrificed using lethal pentobarbital injections (120 mg/kg). The retinae were harvested on Postnatal Day 7 before oxygen exposure, Day 12 just after exposed to oxygen, and Day 17, the day of maximal neovascularization, and frozen immediately to ⫺70°C. Three batches of animals in each group were used in the study with total of at least 20 animals per group. TNF-␣ Expression Eight to 10 retinae were used per each pooled retinal RNA isolation from each group using TRIzol reagent (Life Technologies, Grand Island, NY) (the acid quanidinium thiocyanate-phenol-chloroform extraction technique of Chomczynski and Sacchi) (23). The concentration of RNA was measured using Beckman DU640B spectrophotometry (Beckman Instruments, Fullerton, CA). Ten micrograms of total RNA was used for firststrand cDNA synthesis using the SuperScript RTPCR kit (Life Technologies, Grand Island, NY). cDNA sample was used for PCR using sense primer (⫹412 to ⫹432; ⫹177; 5⬘-ATG AGC ACA GAA AGC ATG ATC-3⬘) and antisense primer (⫹412 to ⫹432; 5⬘-TAC AGG CTT GTC ACT CGA ATT-3⬘) (Life Technologies, Rockville, MD) as previously described (24). To verify that equal amounts of RNA were added in each PCR within each experiment and to verify a uniform amplification process, -actin mRNA was also transcribed and amplified for each sample as internal control. The RT-PCR step was repeated three times for each batch and each group of the animals. cDNA was loaded onto agarose gel and electrophoresis was performed. Each gel was analyzed un-
Data Analysis The data from cytokine expression dentisometry were expressed as means ⫾SEM in each group of the animals. Student’s t tests were used to determine statistical significance. RESULTS AND DISCUSSION During normal development of retina in the mouse, a developmental increase of TNF-␣ was demonstrated. There is a 3.0-fold increase from Postnatal Day 7 (n ⫽ 33) to Day 12 (n ⫽ 34) in control animals, and a 2.7-fold increase from Day 7 to Day 17 (n ⫽ 44). Immediately after hyperoxia exposure, at Postnatal Day 12, there was a 2.7-fold inhibition (P ⬍ 0.05) of TNF-␣ expression compared to control animals. At Day 17 (n ⫽ 44), the expression in the retina of the oxygen exposure group was induced 1.8-fold (P ⬍ 0.005) compared to that of the control at Postnatal Day 17 as shown in Fig. 1. The suppression of TNF-␣ immediately after oxygen exposure may be associated with the vasoconstriction phase of oxygen-induced retinopathy. It has been reported previously that retinal glial cells, Muller cells, and macrophages were responsible for the production of TNF-␣. It could be explained that during the process of vasoconstriction, these cells decreased their expression of TNF-␣. Thus TNF-␣ may be a factor which plays a role in the inflammation and angiogenesis during the development of oxygen-induced retinopathy. TNF-␣ is the important factor in the pathogenesis of several ocular inflammation (1– 8). Okata et al. (8) reported elevation of TNF-␣ in experimental autoimmune uveoretinitis in the rat. Limb et al. (4) demonstrated the association between serum TNF-␣ and development of proliferative diabetic retinopathy. He also postulated that local activation of TNF-␣ and enhanced expression of vascular cell adhesion molecules might play an important role in the development of the proliferative phase of diabetic retinopathy. Spranger et al. (3) reported an increased TNF-␣ in neovascular eye disease and proliferative diabetic retinopathy. The mechanism of action of
166
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effect of dexamethasone on oxygen-induced retinopathy in the mouse has been previously described (15, 16). Rotschild et al. (15) demonstrated a protective effect of dexamethasone given during the oxygen exposure. The total retinopathy score and extraretinal neovascularization were both improved. Yossuck et al. (16) reported a critical effect of timing of dexamethasone in the mouse model of oxygen-induced retinopathy. A protective effect of dexamethasone could be demonstrated if given before oxygen exposure but not after. Glucocorticoids have inhibitory effects on TNF-␣. Wagge and Bakke (17) and Joyce et al. (18) both reported that dexamethasone suppresses the production of TNF by LPS-stimulated human monocytes. Brenner et al. (19) demonstrated stimulation of TNF-␣ production by mycoplasma and inhibition by dexamethasone in cultured astrocytes. Behur et al. (19) found inhibitory effects of dexamethasone on TNF-␣ expression in the retina of
FIG. 1. (A) Representative gel of TNF-␣ expression. Lane 1 shows a DNA (100-bp ladder); lane 2, P7 retinal sample; lane 3, P12 room-air sample; lane 4, P17 room-air sample; lane 5, oxygen-reared P12 sample; and lane 6, oxygen-reared P17 sample. (B) Expression of TNF-␣ in retinal tissue at P7, P12, and P17 in room-air and oxygen-treated animals.
TNF-␣ may be through increasing permeability of the blood-retinal barrier, leading to an inflammatory response (25, 26). An increase in TNF-␣ expression was observed at the time of maximal retinal neovascularization (P17) in the mouse model. Administration of dexamethasone suppressed the normal developmental expression of TNF-␣ in the room-air-reared animals. There was 1.5-fold and 4.7-fold reduction of expression at Postnatal Day 12 (n ⫽ 20) and Day 17 (n ⫽ 32) accordingly in the room-air-reared animals who received dexamethasone during P7–P11. In the oxygen-exposed animals, dexamethasone inhibited the suppression of TNF-␣ expression at Postnatal Day 12 (n ⫽ 32) immediately after oxygen exposure. The expression of TNF-␣ was suppressed 2.3-fold (P ⬍ 0.05) by dexamethasone at Postnatal Day 17, the time of maximal neovascularization, in the oxygen-exposed animals (n ⫽ 32) as shown in Fig. 2. Reaction products were sequenced and base pairs were confirmed. Dexamethasone is one of the most often used corticosteroids in perinatal and neonatal medicine. The
FIG. 2. (A) Representative gel of TNF-␣ expression. Lane 1 shows a DNA (100-bp ladder); lane 2, P7 retinal sample; lane 3, P12 room-air sample; lane 4, P17 room-air sample; lane 5, oxygen-reared P12 sample; lane 6, oxygen-reared P17 sample; lane 7, P12 room-air-reared dexamethasone-treated sample; lane 8, P17 room-air-reared dexamethasone-treated sample; lane 9, P12 oxygen-exposed dexamethasone-treated sample; and lane 10, P17 oxygen-exposed dexamethasone-treated sample. (B) Expression of TNF-␣ in retinal tissue at P7, P12, and P17 in room-air and oxygen-treated animals with exposure to dexamethasone.
TNF-␣, DEXAMETHASONE, AND RETINOPATHY
endotoxin-induced uveitis using a local delivery system in a rat model. From this study we demonstrated a dramatic decrease in TNF-␣ expression after dexamethasone treatment during oxygen exposure. TNF-␣ may be responsible for the protective effect of dexamethasone on this oxygen-induced retinopathy as previously reported (15, 16). In summary, we postulate that TNF-␣ is one of the important factors in the pathophysiology of oxygeninduced retinopathy. Modulation of TNF-␣ expression by dexamethasone provides a protective mechanism of action against the development of severe retinopathy.
11.
12.
13.
14.
15.
REFERENCES 1.
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Palexas GN, Sussman G, Welsh NH. Ocular and systemic determination of IL-1␣ and tumour necrosis factor in a patient with ocular inflammation. Scan J Immunol Suppl 11:173–175, 1992. Planck SR, Huang XN, Robertson JE, Rosenbaum JT. Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest Ophthalmol Vis Sci 35:924 –930, 1994. Spranger J, Meyer-Schwickerath R, Klein M, Schatz H, Pfeiffer A. TNF-alpha level in the vitreous body. Increase in neovascular eye diseases and proliferative diabetic retinopathy. Med Clin 90:134 –137, 1995. Limb GA, Chignell AH, Green W, LeRoy F, Dumonde DC. Distribution of TNF-␣ and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy. Br J Ophthalmol 80:168 –173, 1996. Dick AD, McMenamin PG, Korner H, Scallon BJ, Ghrayeb J, Forrester JV, Sedgwick JD. Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina. Eur J Immunol 26: 1018 –1025, 1996. De Kozak Y, Cotinet A, Goureau O, Hicks D, Thillaye-Goldenberg B. Tumor necrosis factor and nitric oxide production by resident retinal glial cell from rats presenting hereditary retinal degeneration. Ocul Immunol Inflamm 5:85–94, 1997. Armstrong D, Augustin AJ, Spengler R, Al-Jada A, Nickola T, Grus F, Koch F. Detection of vascular endothelial growth factor and tumor necrosis factor alpha in epiretinal membranes of proliferative diabetic retinopathy, proliferative vitreoretinopathy and macular pucker. Ophthalmologica 212: 410 – 414, 1998. Okada AA, Sakai J, Usui M, Mizuguchi J. Intraocular cytokine quantification of experimental autoimmune uveoretinitis in rats. Ocul Immunol Inflamm 6:111–120, 1998. Klagsbrun M, D’Amore PA. Regulators of angiogenesis. Ann Rev Physiol 53:217–239, 1991. Sunderkotter C, Goebeler M, Schulze-Osthoff K, Bhardwaj R, Sorg C. Macrophage-derived angiogenesis factors. Pharmacol Ther 51:195–216, 1991.
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Fajardo LF, Kwan HH, Kowalski J, Prionas SD, Allison AC. Dual role of tumor necrosis factor-alpha in angiogenesis. Am J Pathol 140:539 –544, 1992. Jensen JC, Pogrebniak HW, Pass HI, Buresh C, Merino MJ, Kauffman D, Venzon D, Langstein HN, Norton JA. Role of tumor necrosis factor in oxygen toxicity. J Appl Physiol 72:1902–1907, 1992. O’Brien-Ladner AR, Nelson ME, Cowley BD, Bailey K, Wesselius LJ. Hyperoxia amplifies TNF-␣ production in LPSstimulated human alveolar macrophages. Am J Respir Cell Mol Biol 12:275–279, 1995. Shea LM, Beehler C, Schwartz M, Shenkar R, Tuder R, Abraham E. Hyperoxia activates NF-B and increases TNF-␣ and IFN-␥ gene expression in mouse pulmonary lymphocytes. J Immunol 157:3902–3908, 1996. Rotschild T, Nandgaonkar BN, Yu K, Higgins RD. Dexamethasone reduces oxygen induced retinopathy in the mouse model. Pediatr Res 46:94 –100, 1999. Yossuck P, Yun Y, Tadesse M, Higgins RD. Dexamethasone and critical effect of timing on retinopathy. Invest Ophthalmol Vis Sci 41:3095–3099, 2000. Wagge A, Bakke O. Glucocorticoids suppress the production of tumor necrosis factor by lipopolysaccharide-stimulated human monocyte. Immunology 63:303–311, 1988. Joyce DA, Steer JH, Kloda A. Dexamethasone antagonizes IL-4 and IL-10-induced release of IL-1RA by monocytes but augments IL-4-, IL-10-, and TGF--induced suppression of TNF-␣ release. J Interferon Cytokine Res 16:511–517, 1996. Brenner T, Yamin A, Abramsky O, Gallily R. Stimulation of tumor necrosis factor-␣ production by mycoplasmas and inhibition by dexamethasone in cultured astrocytes. Brain Res 608:273–279, 1993. Behar-Cohen F, Parel JM, Pouliquen Y, Thillaye-Goldenberg B, Goureau O, Heydolph S, Courtois Y, De Kozak Y. Iontophoresis of dexamethasone in the treatment of endotoxin-induced-uveitis in rats. Exp Eye Res 65:533–545, 1997. Joyce DA, Kloda A, Steer JH. Dexamethasone suppresses release of soluble TNF receptors by human monocytes concurrently with TNF-␣ suppression. Immunol Cell Biol 75: 345–350, 1997. Smith LEH, Wesolowski E, McLellan A, Kostyk S, D’Amato R, Sullivan R, D’Amore PA. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101–111, 1994. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156, 1987. Macica CM, Escalante BA, Conners MS, Ferreri NR. TNF production by the medullary thick ascending limb of Henle’s loop. Kidney Int. 46:113–121, 1994. Bamforth SD, Lightman S, Greenwood J. The effect of TNF-␣ and IL-6 on the permeability of the rat blood-retinal barrier in vivo. Acta Neuropathol 91:624 – 632, 1996. Luna JD, Chan C, Derevjanik NL, Mahlow J, Chiu C, Peng B, Tobe T, Campochiaro PA, Vinores SA. Blood-retinal barrier (BRB) breakdown in experimental autoimmune uveoretinitis: Comparison with vascular endothelial growth factor, tumor necrosis factor ␣, and interleukin-1-mediated breakdown. J Neurosci Res 49:268 –280, 1997.
Dexamethasone Alters TNF-␣ Expression in Retinopathy 1 Panitan Yossuck, Yun Yan Misrak Tadesse, and Rosemary D. Higgins 2 Department of Pediatrics, Division of Neonatology, Georgetown University Children’s Medical Center, Washington, DC 20007 Received August 16, 2000, and in revised form November 6, 2000; published online January 16, 2001
vide a potential site of action for therapeutic targets. © 2001 Academic Press
TNF-␣ has been found in the retina. Hyperoxia and hypoxia regulate TNF-␣ expression. TNF-␣ is an important factor in inflammation and angiogenesis. Dexamethasone inhibits TNF-␣ production. Changes in TNF-␣ expression in the retina may play an important role in the development of oxygen-induced retinopathy. Oxygen-induced retinopathy was produced in C57BL6 mice by exposure to 75% oxygen at Postnatal Day 7 (P7) for 5 days and the mice recovered in room air until Day 17 (P17). Dexamethasone was administered at 0.5 mg/kg/day once daily subcutaneously during the 5 days of oxygen exposure. TNF-␣ expression was evaluated at Day 7 prior to oxygen exposure, at Day 12 (P12) immediately upon removal from oxygen, and at Day 17, the time of maximal vasoproliferation by RT-PCR. TNF-␣ is developmentally regulated in the retinae of C57BL6 mice. From P7 to P12, there is a 3-fold increase in TNF-␣ expression and from P7 to P17 there is a 2.7-fold increase. There was 2.7-fold suppression in expression immediately following oxygen exposure at P12. The expression was dramatically increased at P17, the time of maximal vasoproliferation. Dexamethasone inhibited the expression of TNF-␣ at P17 by 6.4-fold. At this dose, it also suppressed the baseline TNF-␣ expression in the mouse model. In summary, TNF-␣ is altered in the development of oxygen-induced retinopathy in the mouse. It increased markedly during the vasoproliferative phase and was suppressed by dexamethasone. Modulation of TNF-␣ expression may pro-
Tumor necrosis factor alpha (TNF-␣), a monocyte/ macrophage-derived proinflammatory factor, is involved in the pathogenesis of proliferative retinopathy and ocular inflammation (1– 8). TNF-␣ has both proangiogenic and antiangiogenic effects on endothelial cells, depending on the biological context (9, 10). High-dose TNF-␣ was found to inhibit angiogenesis, in contrast to low doses that induced angiogenesis (11). The role of TNF-␣ and oxygen toxicity has been studied extensively (12–14). Hyperoxia in the lung induces the production and expression of TNF-␣ by alveolar macrophage and eventually leads to the development of pulmonary fibrosis (12). Hypoxia in several tissues was found to alter the production and expression of TNF-␣. Oxygen-induced retinopathy is the disease involved in both hyperoxia and hypoxia. TNF-␣ expression in the mouse model oxygen-induced retinopathy has not been reported. Dexamethasone is one of the most potent and widely used anti-inflammatory agents, although the mode of action is poorly understood. Administration of dexamethasone before (during Postnatal Days, 1–5) and during oxygen exposure (P7–11) decreases the severity of oxygen-induced retinopathy in the mouse model (15, 16). Dexamethasone has no effect when given after the oxygen exposure (during P12– 16) (16). It has been found that dexamethasone inhibits TNF-␣ production and expression in several tissues (17–21). The goals of this study are to explore the TNF-␣ expression in the retina of the mouse model during normal development and during the development of
1 Funded in part by Fight For Sight, Research Division of Prevent Blindness America. 2 To whom correspondence should be addressed at Department of Pediatrics, Division of Neonatology, Georgetown University Children’s Medical Center, 3800 Reservoir Road, NW Room M3400, Washington, DC 20007. Fax: (202) 784-4747. E-mail: [email protected].
164 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
future
TNF-␣, DEXAMETHASONE, AND RETINOPATHY
oxygen-induced retinopathy. Further, the effect of dexamethasone on TNF-␣ expression in oxygeninduced retinopathy is studied. MATERIALS AND METHODS
165
der an ultraviolet light and was photographed. The results were scanned for density using Quantiscan Program (Biosoft, Ferguson, MO). The results were normalized with the -actin expression from the same sample. The PCR product was also sequenced for confirmation.
Animal Model The study protocol was approved by Georgetown University Animal Use and Care Committee. C57BL6 mice were exposed to 75% oxygen from Postnatal Day 7 to 12 as previously described (22) and utilized in our laboratory (15, 16) to produce oxygen-induced retinopathy. Dexamethasone (0.5 mg/kg sc daily) was administered from Postnatal Day 7 for 5 days (15). The animals were sacrificed using lethal pentobarbital injections (120 mg/kg). The retinae were harvested on Postnatal Day 7 before oxygen exposure, Day 12 just after exposed to oxygen, and Day 17, the day of maximal neovascularization, and frozen immediately to ⫺70°C. Three batches of animals in each group were used in the study with total of at least 20 animals per group. TNF-␣ Expression Eight to 10 retinae were used per each pooled retinal RNA isolation from each group using TRIzol reagent (Life Technologies, Grand Island, NY) (the acid quanidinium thiocyanate-phenol-chloroform extraction technique of Chomczynski and Sacchi) (23). The concentration of RNA was measured using Beckman DU640B spectrophotometry (Beckman Instruments, Fullerton, CA). Ten micrograms of total RNA was used for firststrand cDNA synthesis using the SuperScript RTPCR kit (Life Technologies, Grand Island, NY). cDNA sample was used for PCR using sense primer (⫹412 to ⫹432; ⫹177; 5⬘-ATG AGC ACA GAA AGC ATG ATC-3⬘) and antisense primer (⫹412 to ⫹432; 5⬘-TAC AGG CTT GTC ACT CGA ATT-3⬘) (Life Technologies, Rockville, MD) as previously described (24). To verify that equal amounts of RNA were added in each PCR within each experiment and to verify a uniform amplification process, -actin mRNA was also transcribed and amplified for each sample as internal control. The RT-PCR step was repeated three times for each batch and each group of the animals. cDNA was loaded onto agarose gel and electrophoresis was performed. Each gel was analyzed un-
Data Analysis The data from cytokine expression dentisometry were expressed as means ⫾SEM in each group of the animals. Student’s t tests were used to determine statistical significance. RESULTS AND DISCUSSION During normal development of retina in the mouse, a developmental increase of TNF-␣ was demonstrated. There is a 3.0-fold increase from Postnatal Day 7 (n ⫽ 33) to Day 12 (n ⫽ 34) in control animals, and a 2.7-fold increase from Day 7 to Day 17 (n ⫽ 44). Immediately after hyperoxia exposure, at Postnatal Day 12, there was a 2.7-fold inhibition (P ⬍ 0.05) of TNF-␣ expression compared to control animals. At Day 17 (n ⫽ 44), the expression in the retina of the oxygen exposure group was induced 1.8-fold (P ⬍ 0.005) compared to that of the control at Postnatal Day 17 as shown in Fig. 1. The suppression of TNF-␣ immediately after oxygen exposure may be associated with the vasoconstriction phase of oxygen-induced retinopathy. It has been reported previously that retinal glial cells, Muller cells, and macrophages were responsible for the production of TNF-␣. It could be explained that during the process of vasoconstriction, these cells decreased their expression of TNF-␣. Thus TNF-␣ may be a factor which plays a role in the inflammation and angiogenesis during the development of oxygen-induced retinopathy. TNF-␣ is the important factor in the pathogenesis of several ocular inflammation (1– 8). Okata et al. (8) reported elevation of TNF-␣ in experimental autoimmune uveoretinitis in the rat. Limb et al. (4) demonstrated the association between serum TNF-␣ and development of proliferative diabetic retinopathy. He also postulated that local activation of TNF-␣ and enhanced expression of vascular cell adhesion molecules might play an important role in the development of the proliferative phase of diabetic retinopathy. Spranger et al. (3) reported an increased TNF-␣ in neovascular eye disease and proliferative diabetic retinopathy. The mechanism of action of
166
YOSSUCK ET AL.
effect of dexamethasone on oxygen-induced retinopathy in the mouse has been previously described (15, 16). Rotschild et al. (15) demonstrated a protective effect of dexamethasone given during the oxygen exposure. The total retinopathy score and extraretinal neovascularization were both improved. Yossuck et al. (16) reported a critical effect of timing of dexamethasone in the mouse model of oxygen-induced retinopathy. A protective effect of dexamethasone could be demonstrated if given before oxygen exposure but not after. Glucocorticoids have inhibitory effects on TNF-␣. Wagge and Bakke (17) and Joyce et al. (18) both reported that dexamethasone suppresses the production of TNF by LPS-stimulated human monocytes. Brenner et al. (19) demonstrated stimulation of TNF-␣ production by mycoplasma and inhibition by dexamethasone in cultured astrocytes. Behur et al. (19) found inhibitory effects of dexamethasone on TNF-␣ expression in the retina of
FIG. 1. (A) Representative gel of TNF-␣ expression. Lane 1 shows a DNA (100-bp ladder); lane 2, P7 retinal sample; lane 3, P12 room-air sample; lane 4, P17 room-air sample; lane 5, oxygen-reared P12 sample; and lane 6, oxygen-reared P17 sample. (B) Expression of TNF-␣ in retinal tissue at P7, P12, and P17 in room-air and oxygen-treated animals.
TNF-␣ may be through increasing permeability of the blood-retinal barrier, leading to an inflammatory response (25, 26). An increase in TNF-␣ expression was observed at the time of maximal retinal neovascularization (P17) in the mouse model. Administration of dexamethasone suppressed the normal developmental expression of TNF-␣ in the room-air-reared animals. There was 1.5-fold and 4.7-fold reduction of expression at Postnatal Day 12 (n ⫽ 20) and Day 17 (n ⫽ 32) accordingly in the room-air-reared animals who received dexamethasone during P7–P11. In the oxygen-exposed animals, dexamethasone inhibited the suppression of TNF-␣ expression at Postnatal Day 12 (n ⫽ 32) immediately after oxygen exposure. The expression of TNF-␣ was suppressed 2.3-fold (P ⬍ 0.05) by dexamethasone at Postnatal Day 17, the time of maximal neovascularization, in the oxygen-exposed animals (n ⫽ 32) as shown in Fig. 2. Reaction products were sequenced and base pairs were confirmed. Dexamethasone is one of the most often used corticosteroids in perinatal and neonatal medicine. The
FIG. 2. (A) Representative gel of TNF-␣ expression. Lane 1 shows a DNA (100-bp ladder); lane 2, P7 retinal sample; lane 3, P12 room-air sample; lane 4, P17 room-air sample; lane 5, oxygen-reared P12 sample; lane 6, oxygen-reared P17 sample; lane 7, P12 room-air-reared dexamethasone-treated sample; lane 8, P17 room-air-reared dexamethasone-treated sample; lane 9, P12 oxygen-exposed dexamethasone-treated sample; and lane 10, P17 oxygen-exposed dexamethasone-treated sample. (B) Expression of TNF-␣ in retinal tissue at P7, P12, and P17 in room-air and oxygen-treated animals with exposure to dexamethasone.
TNF-␣, DEXAMETHASONE, AND RETINOPATHY
endotoxin-induced uveitis using a local delivery system in a rat model. From this study we demonstrated a dramatic decrease in TNF-␣ expression after dexamethasone treatment during oxygen exposure. TNF-␣ may be responsible for the protective effect of dexamethasone on this oxygen-induced retinopathy as previously reported (15, 16). In summary, we postulate that TNF-␣ is one of the important factors in the pathophysiology of oxygeninduced retinopathy. Modulation of TNF-␣ expression by dexamethasone provides a protective mechanism of action against the development of severe retinopathy.
11.
12.
13.
14.
15.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9. 10.
Palexas GN, Sussman G, Welsh NH. Ocular and systemic determination of IL-1␣ and tumour necrosis factor in a patient with ocular inflammation. Scan J Immunol Suppl 11:173–175, 1992. Planck SR, Huang XN, Robertson JE, Rosenbaum JT. Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest Ophthalmol Vis Sci 35:924 –930, 1994. Spranger J, Meyer-Schwickerath R, Klein M, Schatz H, Pfeiffer A. TNF-alpha level in the vitreous body. Increase in neovascular eye diseases and proliferative diabetic retinopathy. Med Clin 90:134 –137, 1995. Limb GA, Chignell AH, Green W, LeRoy F, Dumonde DC. Distribution of TNF-␣ and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy. Br J Ophthalmol 80:168 –173, 1996. Dick AD, McMenamin PG, Korner H, Scallon BJ, Ghrayeb J, Forrester JV, Sedgwick JD. Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina. Eur J Immunol 26: 1018 –1025, 1996. De Kozak Y, Cotinet A, Goureau O, Hicks D, Thillaye-Goldenberg B. Tumor necrosis factor and nitric oxide production by resident retinal glial cell from rats presenting hereditary retinal degeneration. Ocul Immunol Inflamm 5:85–94, 1997. Armstrong D, Augustin AJ, Spengler R, Al-Jada A, Nickola T, Grus F, Koch F. Detection of vascular endothelial growth factor and tumor necrosis factor alpha in epiretinal membranes of proliferative diabetic retinopathy, proliferative vitreoretinopathy and macular pucker. Ophthalmologica 212: 410 – 414, 1998. Okada AA, Sakai J, Usui M, Mizuguchi J. Intraocular cytokine quantification of experimental autoimmune uveoretinitis in rats. Ocul Immunol Inflamm 6:111–120, 1998. Klagsbrun M, D’Amore PA. Regulators of angiogenesis. Ann Rev Physiol 53:217–239, 1991. Sunderkotter C, Goebeler M, Schulze-Osthoff K, Bhardwaj R, Sorg C. Macrophage-derived angiogenesis factors. Pharmacol Ther 51:195–216, 1991.
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