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(Investigative Ophthalmology and Visual Science. 2000;41:3095-3099.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Dexamethasone and Critical Effect of Timing on Retinopathy

Panitan Yossuck, Yun Yan, Misrak Tadesse and Rosemary D. Higgins

From the Department of Pediatrics, Division of Neonatology, Georgetown University Medical Center, Washington DC.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. Administration of corticosteroids soon after birth has been reported to have deleterious, protective, and no effect on retinopathy of prematurity. Conflicting results may be due to timing of corticosteroid administration. The goal of this study was to determine effects of pretreatment and late dexamethasone on retinopathy in a mouse model.

METHODS. The C57BL6 mouse model of oxygen-induced retinopathy (by placing animals in 75% oxygen from postnatal days 7 through 12) was used to create retinal neovascularization. Dexamethasone at 0.5 mg/kg per day was administered from day 1 through day 5 in the pretreatment group. The late-treatment group received 5 days of dexamethasone at the same dose beginning on day 12. Mice were killed at days 17 through 20, and retinal vasculature was assessed by a retinal scoring system of wholemount preparation after high-molecular-weight fluorescein-labeled dextran perfusion. In addition, retinal neovascularization was assessed by quantification of extraretinal neovascular nuclei in retinal sections. Statistical significance was defined as P < 0.05 and was determined by the Kruskal–Wallis test, Mann–Whitney test, and Student’s t-test.

RESULTS. Oxygen-exposed animals that received treatment with dexamethasone before oxygen exposure had an improvement in retinopathy, with a median score of 6 (5,7; 25th,75th quartiles) compared with 10 (8,11) in the untreated oxygen-exposed (P < 0.05). The group treated late (after oxygen exposure) with dexamethasone had a median score of 10 (9,11). Pretreatment reduced extraretinal vascularization, when assessed by quantification of neovascular nuclei, to a mean ± SEM of 19 ± 9, significantly less than in the untreated oxygen-exposed group (55 ± 12; P < 0.05). No difference was observed in the late-treatment group when compared with the untreated oxygen-exposed group. Significant growth retardation, indicated by body weight, was observed in the pretreatment (P < 0.01) and late-treatment (P < 0.05) groups when compared with the control group.

CONCLUSIONS. Timing of dexamethasone administration was critical to the inhibition of development of retinopathy in the mouse model. Degree of growth retardation, measured by body weight, also appeared to be time dependent. These data may explain the different results of clinical observations with respect to corticosteroid treatment, timing, and development of retinopathy.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Birth weight, gestational age, and duration of supplemental oxygen have been found to be the leading risk factors for retinopathy of prematurity (ROP).1 2 3 4 5 Corticosteroid treatment has been used with increasing frequency in neonatology. Antenatal corticosteroid administration is now a recommended treatment to promote lung maturity in premature delivery at gestational ages of 24 to 34 weeks.6 Higgins et al.,7 Kennedy,8 and the Italian ROP study,9 reported decreasing severity of ROP in infants born to mothers who had received antenatal steroid treatment. Postnatal administration of corticosteroids has also been studied extensively with the goal of decreasing the incidence of chronic lung disease, duration of mechanical ventilation, and supplemental oxygen requirement.10 There are still no clear-cut guidelines for postnatal administration of steroids. With some, treatment begins as early as 24 hours after birth and with others, after 2 to 3 weeks of life. Results are controversial. ROP has also been evaluated after postnatal corticosteroid therapy, because it has anti-inflammatory and angiostatic effects. At least four studies11 12 13 14 have focused on the association between corticosteroid treatment and ROP, with contradictory results. Wright and Wright11 reported that there is no association, Sobel and Philip12 demonstrated that prolonged use of steroids beginning on a mean of day 23 after birth reduces the need for cryotherapy, and Batton et al.13 and Ramanathan et al.14 both reported that postnatal steroid therapy is associated with severe ROP and requirement for cryotherapy. Regression analyses to control for degree of systemic illness of preterm infants were performed and confirmed no association with ROP11 12 and, in contrast, a significant increase in risk13 for ROP in infants treated with dexamethasone.

Timing and underlying systemic disease may explain the different observations. Rotchild et al.15 reported the protective effect of dexamethasone administered concurrently with oxygen exposure in a mouse model. We designed this study to assess the effect of timing of dexamethasone, before and after oxygen exposure, in a mouse model of oxygen-induced retinopathy.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animal Model and Dexamethasone Administration
The protocol was approved by Georgetown University Animal Care and Use Committee. C57BL6 mice were obtained from Taconic Farms (Germantown, NY). Mice were placed with their nursing mothers in an infant incubator (Ohmeda, Columbia, MD) with 75% oxygen from postnatal day (P)7 through P12, as previously described16 and used in our laboratory.17 Oxygen concentration was measured using an oxygen analyzer (Hudson Ventronics, Temecula, CA) and was checked at least twice daily during the period of oxygen exposure. Animals were returned to room air on P12.

Twenty-nine litters (n = 118 animals) were assigned to either the room air–reared group or the oxygen-reared group. Within individual litters, animals were randomly assigned to receive no treatment, sham injection of normal saline (sham-treatment group), pretreatment with dexamethasone (pretreatment group), or late treatment with dexamethasone (late-treatment group). Pretreatment with dexamethasone from P1 through P5 was selected to expose animals to dexamethasone before oxygen-induced injury to the retinal vessels to attempt to simulate antenatal corticosteroid administration or very early postnatal administration.

Late dexamethasone treatment was used to simulate administration of dexamethasone, because it is commonly used in neonatal intensive care nurseries to facilitate weaning from mechanical ventilation. A single dose of 0.5 mg/kg per day for 5 days was selected based on a previous study15 and based on doses used clinically.11 12 13 14 Dexamethasone-treated animals were divided into two groups: pretreatment and late-treatment groups. Animals assigned to the pretreatment group were given a single daily dose of 0.5 mg/kg per day of dexamethasone (American Regent Laboratories, Shirley, NY) subcutaneously in the nape of the neck for 5 days from P1 to P5 before exposure to oxygen. The late-treatment group was given the same dose of dexamethasone for 5 days, beginning on P12, after the mice were removed from oxygen and returned to room air. The animals were killed by lethal intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL) at P17 through P20. P17 through P20 was chosen as the time of death, because maximal retinal neovascularization has been reported at these time points.16 17 A sham group received normal saline injection at the same volume and for the same periods as the dexamethasone treatment groups and was also divided into groups according to exposure and nonexposure to oxygen. The weights of the animals were recorded at P1, P7, P12, and on the day killed (P17 through P20).

Fluorescein Dextran Perfusion of the Retinal Blood Vessels
To study the retinal vascular pattern, systemic perfusion was performed18 using high-molecular-weight (MW = 2,000,000) fluorescein-conjugated dextran (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS; Gibco, Grand Island, NY). Briefly, animals were given a lethal dose of sodium pentobarbital, and a median sternotomy was performed. The left ventricle of the heart was identified and perfused with 1 ml fluorescein-conjugated dextran (50 mg/ml in 4% PBS) using a 1-milliliter tuberculin syringe with a 27-gauge needle. Eyes were then enucleated and placed in 4% paraformaldehyde (Sigma) in PBS for 4 to 24 hours. Under a dissecting microscope, the retina was removed, and a flatmount was prepared by making radial cuts. A coverslip was applied over the retinas after placement of a drop of 2% gelatin (Sigma). The edge of the coverslip was sealed with transparent nail polish. The scoring of retinal wholemounts was performed using fluorescence microscopy. Each retina was scored by two investigators working independently in a masked fashion and using the retinopathy scoring system17 shown in Table 1 , and the average retinopathy score (average of two eyes and two investigators) for each animal was used for the statistical analysis.


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  		Table 1. Retinopathy Scoring System

 
PAS Stain of Retinal Sections
Mice were killed as indicated. The eyes were enucleated, placed immediately in optimal cutting temperature (OCT) embedding compound (Sakura Fine Tek, Torrance, CA), and frozen at -70°C. Serial sections (7–9 µm thick) over a minimum of 450 µm were cut in a sagittal plane through the cornea, parallel to the optic disc. Tissue sections were stained with periodic acid–Schiff (PAS) reagent and hematoxylin.19 Multiple sections from individual eyes were scored in a masked fashion under light microscopy by counting all nuclei extending beyond the inner limiting membrane into the vitreous, as previously described.16 A minimum of six sections at least 50 µm apart were evaluated and counted per eye and averaged. The mean number of neovascular nuclei per section for each eye was used in the statistical analyses.

Statistical Analyses
Analysis of variance using the Kruskal–Wallis test was performed to test for differences in retinopathy score among the various treatment groups. Mann–Whitney tests were used to compare the total retinopathy scores between individual groups of animals. Student’s t-tests were used to compare the mean number of neovascular nuclei on retinal sections between individual groups. Student’s t-tests were also used to compare animal weight between the room air–reared group and the various other groups at the various time points. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Total Retinopathy Scores
Pretreatment with dexamethasone (n = 9, from seven litters) before oxygen exposure significantly decreased the total retinopathy score to 6 (5,7; 25th,75th quartiles) compared with the nontreated oxygen-exposed group (n = 14, from seven litters) with a score of 10, (8,11; P < 0.05). Late dexamethasone treatment (n = 12, from six litters) did not have a significant effect on the total retinopathy score, with a median score of 10 (9,11) when compared with the nontreated oxygen-exposed group. Sham-treated animals had similar scores to animals reared in oxygen alone: for P1 through P5 (n = 2 from one litter) 9.5 (8.25,10.25) and for P12 through 16 (n = 4 from 3 litters) 10.5 (7.75, 11.25). Figure 1 shows representative fluorescein-conjugated dextran–perfused retinal wholemounts.



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  		Figure 1. Representative fluorescein-conjugated dextran-perfused retinal wholemount preparations. (A) Room air–reared control animal (score = 0). (B ) Noninjected 75% oxygen-exposed animal (score = 12) demonstrates significant loss of central blood vessels, tortuosity of the vessels, and tuft formation of neovascularization. (C) Animal exposed to 75% oxygen and pretreated with dexamethasone (score = 5) showing less tortuosity of the vessels and less tuft formation. There is also less central loss of vessels compared with (B). (D) Animal exposed to 75% oxygen treated late with dexamethasone (score = 12) showing no significant change compared with no treatment in (B).

 
All room air control groups had median retinopathy scores of 0 (0,0 or 0,1), whether untreated (n = 12, nine litters), sham-treated (n = 6 from four litters), pretreated with dexamethasone (n = 6 from four litters), or treated late with dexamethasone (n = 8 from four litters).

The day of death (i.e., P17 to P20) did not affect the results. Specifically, animals exposed to oxygen (n = 2) and killed on P17 had median retinopathy scores of 9 (7.5,10.25), pretreatment scores (n = 4) of 6 (5,6), and late-treatment scores (n = 4) of 10 (9,10). For P18, oxygen-exposed (n = 7) scores were 9 (9,13), and pretreatment scores (n = 3) scores were 7.5 (6.25,8). For P19, oxygen-exposed (n = 4) scores were 10 (8,11.5), pretreatment (n = 5) scores were 6 (5,7), and late-treatment scores (n = 7) were 10 (8,11). One late dexamethasone–treated animal was killed on P20 and had a score of 12.

Retinal Sections
To corroborate the finding of extraretinal neovascularization in the retinal flatmounts, retinal sections were examined. Extraretinal nuclei count also decreased significantly in the pretreatment group (18.9 ± 8.9) compared with the nontreated oxygen-exposed group (55.0 ± 12.1; P = 0 0.04). There was no significant change in nuclei count between the late-treatment group (30.8 ± 0.79) compared with the nontreated oxygen-exposed group (Fig. 2) . All room air–reared animals had nuclei counts similar to those shown in Figure 2 .



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  		Figure 2. Extraretinal nuclei counts from retinal sections. The open bars represent the mean number of extraretinal neovascular nuclei in the 75% oxygen-exposed animals. The solid bars represent the room air–reared animals. Error bars: SEM. *Represents P < 0.05 when compared with the oxygen-exposed group by Student’s t-test. In the control group (n = 9 animals, five litters) five were killed on P17, one on P18, two on P19, and one on P20. In the oxygen-exposed group (n = 7 animals, four litters), six were killed on P18 and one on P19. In the room air pretreatment group (n = 6, three litters), two were killed on P17 and four on P18. In the oxygen-exposed dexamethasone pretreatment group (n = 7, four litters), six were killed on P18 and one on P19. In the room air–reared late-treatment dexamethasone group (n = 10 animals, five litters), four were killed on P17 and six on P18. In the oxygen-exposed late dexamethasone treatment group (n = 6, four litters) two were killed on P17, two on P18, and two on P19.

 
Growth Suppression
Dexamethasone significantly decreased the growth as indicated by body weight of the animals (Table 2) . The maximum effect of growth suppression was found in the pretreatment group.


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  		Table 2. Animal Weight

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Timing of treatment with dexamethasone is a critical factor in this mouse model of oxygen-induced retinopathy. Pretreatment with dexamethasone before oxygen exposure significantly reduced total retinopathy score and the number of extraretinal neovascular nuclei on PAS-stained retinal sections compared with the nontreated oxygen-exposed group. Late dexamethasone treatment did not show any effect on the severity of retinopathy, according to both retinopathy score and the number of neovascular nuclei. Although a beneficial effect was observed in the development of retinopathy in the mice, dexamethasone treatment caused significant growth retardation, measured by body weight. The effect of growth suppression was more prominent in the pretreatment group when compared with the late-treatment group. Rotschild et al.15 reported a protective effect of dexamethasone at 0.5 mg/kg per day when administered concurrently with oxygen exposure beginning on P7 and continuing for 5 days.

Dexamethasone reduced both the retinopathy score and neovascular nuclei in that study. Barks et al.20 demonstrated that timing and dose of dexamethasone were important with modulation of injury: pretreatment 24 hours before unilateral cerebral hypoxia–ischemia prevented infarction. Higgins et al.,7 Kennedy,8 and the Italian ROP study9 reported decreased severity of ROP associated with antenatal dexamethasone treatment.

Several studies focused on postnatal corticosteroids and their effect on ROP.11 12 13 14 Ehrenkranz21 commented on some of these data12 13 and speculated that the difference in association between corticosteroids and ROP may be due to the difference in age at initiation, dosage, length of treatment, and indications for treatment with corticosteroids. An animal model, as used in this study, would be able to demonstrate the effect of intervention without confounding factors present in clinical investigation. Dexamethasone, when administered before oxygen exposure, improves retinopathy during oxygen exposure,15 but has no effect when administered after oxygen exposure.

The obvious side effect of dexamethasone was growth retardation in the mouse, which was also found by Rotschild et al.15 Growth retardation is a known side effect of corticosteroids in clinical practice. It is currently premature to plan a clinical trial based on the current data, because of the significant growth retardation caused by dexamethasone. The dosage of dexamethasone should be determined to minimize the side effect of poor weight gain and maximize the protective effect against retinopathy.

The timing of dexamethasone administration is critical as we demonstrated in this study. These animal model data may help to explain the controversy among observations of effects of corticosteroids on ROP in clinical practice.


   Footnotes
 
Supported in part by Fight For Sight, Research Division of Prevent Blindness America (RDH).

Submitted for publication January 14, 2000; revised March 28 and May 15, 2000; accepted May 22, 2000.

Commercial relationships policy: N.

Corresponding author: Rosemary D. Higgins, Department of Pediatrics, Division of Neonatology, Georgetown University Medical Center, 3800 Reservoir Road, NW M3400, Washington, DC 20007. higginsr1{at}gunet.georgetown.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kinsey, VE, Arnold, HJ, Kalina, RE, et al (1977) PaO2 levels and retrolental fibroplasia: a report of the cooperative study Pediatrics 60,655-668[Abstract/Free Full Text]
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  3. Shahinian, L, Malachowski, N. (1978) Retrolental fibroplasia: a new analysis of risk factors based on recent cases Arch Ophthalmol 96,70-74[Abstract/Free Full Text]
  4. Flynn, JT, Bancalari, E, Bachynski, B, et al (1987) Retinopathy of prematurity; diagnosis, severity, and natural history Ophthalmology 94,620-629[Medline][Order article via Infotrieve]
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  6. . NIH Consensus Statement (1994) Effect of corticosteroids for fetal maturation on perinatal outcomes 12,1-24
  7. Higgins, RD, Mendelsohn, AL, DeFeo, MJ, Ucsel, R, Hendricks–Munoz, K. (1998) Antenatal dexamethasone and decreased severity of retinopathy of prematurity Arch Ophthalmol 116,601-605[Abstract/Free Full Text]
  8. Kennedy, JE (1997) Premature birth and retinopathy of prematurity Progress in Retinopathy of Prematurity ,25-29 Kugler Amsterdam/New York.
  9. . The Italian ROP Study Group (1997) Italian multicenter study on retinopathy of prematurity Eur J Pediatr 156,939-943[Medline][Order article via Infotrieve]
  10. Halliday, HL (1999) Clinical trials of postnatal corticosteroids: inhaled and systemic Biol Neonate 76(suppl),29-40
  11. Wright, K, Wright, SP (1994) Lack of association of glucocorticoid therapy and retinopathy of prematurity Arch Pediatr Adolesc Med 148,848-852[Abstract/Free Full Text]
  12. Sobel, DB, Philip, AGS (1992) Prolonged dexamethasone therapy reduces the incidence of cryotherapy for retinopathy of prematurity in infants of less than 1 kilogram birth weight with bronchopulmonary dysplasia Pediatrics 90,529-533[Abstract/Free Full Text]
  13. Batton, DG, Roberts, C, Trese, M, Maisels, MJ (1992) Severe retinopathy of prematurity and steroid exposure Pediatrics 90,534-536[Abstract/Free Full Text]
  14. Ramanathan, R, Siassi, B, DeLemos, RA (1995) Severe retinopathy of prematurity on extremely low birth weight infants after short-term dexamethasone therapy J Perinatol 15,178-182[Medline][Order article via Infotrieve]
  15. Rotschild, T, Nandgaonkar, BN, Yu, K, Higgins, RD (1999) Dexamethasone reduces oxygen induced retinopathy in the mouse model Pediatr Res 46,94-100[Medline][Order article via Infotrieve]
  16. Smith, LEH, Wesolowski, E, McLellan, A, et al (1994) Oxygen-induced retinopathy in the mouse Invest Ophthalmol Vis Sci 35,101-111[Abstract/Free Full Text]
  17. Higgins, RD, Yu, K, Sander, RJ, Nandgaonkar, BN, Rothchild, T, Rifkin, DB (1999) Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy Curr Eye Res 18,20-27[Medline][Order article via Infotrieve]
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