Postnatal weight gain modifies severity and functional outcome of oxygen-induced proliferative retinopathy

Abstract

In clinical studies, postnatal weight gain is strongly associated with retinopathy of prematurity (ROP). However, animal studies are needed to investigate the pathophysiological mechanisms of how postnatal weight gain affects the severity of ROP. In the present study, we identify nutritional supply as one potent parameter that affects the extent of retinopathy in mice with identical birth weights and the same genetic background. Wild-type pups with poor postnatal nutrition and poor weight gain (PWG) exhibit a remarkably prolonged phase of retinopathy compared to medium weight gain or extensive weight gain pups. A high (r(2) = 0.83) parabolic association between postnatal weight gain and oxygen-induced retinopathy severity is observed, as is a significantly prolonged phase of proliferative retinopathy in PWG pups (20 days) compared with extensive weight gain pups (6 days). The extended retinopathy is concomitant with prolonged overexpression of retinal vascular endothelial growth factor in PWG pups. Importantly, PWG pups show low serum levels of nonfasting glucose, insulin, and insulin-like growth factor-1 as well as high levels of ghrelin in the early postoxygen-induced retinopathy phase, a combination indicative of poor metabolic supply. These differences translate into visual deficits in adult PWG mice, as demonstrated by impaired bipolar and proximal neuronal function. Together, these results provide evidence for a pathophysiological correlation between poor postnatal nutritional supply, slow weight gain, prolonged retinal vascular endothelial growth factor overexpression, protracted retinopathy, and reduced final visual outcome.

Figure 1

Postnatal weight gain modifies course and severity of OIR. A: Neovascularization (NV) in mice with oxygen-induced retinopathy shows a parabolic correlation with body weight at P17 (r² = 0.83). Examplary lectin-stained retinal flat-mounts illustrate the highest amount of NV (black areas) in medium weight pups (middle) compared with high weight pups (top) and low weight pups (bottom); n = 15 litters. B: Longitudinal development of postnatal weight measured in 226 C57BL/6 wild-type OIR pups. Mice were exposed to hyperoxia (75% O₂) from P7–P12, followed by relative hypoxia at room air (21% O₂) from P12 onwards. Three groups were defined based on weights at P17: PWG (P17 weight < 5 g), MWG (P17 weight 5–7.5 g), and EWG (P17 weight >7.5g). The dashed line represents normoxic control mice. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 for significant differences from MWG group (ANOVA with Tukey-Kramer honestly significant difference); n = 10–98 mice/group and time point. C and D: Retinal NV and VO from all three weight groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; *****P < 10⁻⁵ for difference from MWG group (ANOVA with Tukey-Kramer honestly significant difference). n = 3–42 retinas/group and time point. E: P17 weight data for three independent litters that were split at P1. One-half of each litter was raised with additional surrogate mother (black bar), while the other half of each litter was raised without additional surrogate (w/o surrogate; gray bar). Weights from surrogated pups resemble EWG values at P17, while littermates without surrogate resemble MWG values at P17. F and G: Retinal NV is lower in surrogated pups at P17 compared with nonsurrogated littermates. NV values in surrogated pups resemble EWG values. NV values in nonsurrogated pups resemble MWG values. VO does not differ between the two groups; **P < 0.01.

Figure 2

Retinal VEGF mRNA up-regulation mirrors duration and severity of proliferative retinopathy. A: In EWG pups, retinal up-regulation of VEGF mRNA (bars) peaks at P17, coinciding with the spike in retinal neovascularization (NV; lines). Representative lectin-stained retinal flat-mounts illustrate relatively mild proliferative retinopathy with almost complete resolution by P21. B: MWG pups show a similar pattern of VEGF up-regulation and course of retinopathy. Peak VEGF expression as well as maximal disease severity, however, are higher compared with EWG pups. Representative images on the right illustrate the more severe course of retinopathy. C: PWG pups exhibit a strikingly different pattern of VEGF up-regulation and retinopathy with peak VEGF expression between P19 and P21, coinciding with prolonged maximal NV formation. Representative retinal flat-mounts illustrate the extended course of retinopathy in PWG pups with active NV and remaining avascular retinal areas until P29.

Figure 3

Postnatal weight gain groups differ in serum markers of metabolic supply and long-term functional outcome. A–D: Non-fasting serum levels for glucose (A), insulin (B), ghrelin (C), and IGF-1 (D) differ between the three weight groups. Significantly lower levels for nonfasting glucose and IGF-1 combined with higher serum ghrelin levels indicate low nutritional supply in PWG pups. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA with Tukey-Kramer honestly significant difference); n = 3–10 retinas/group. E and F: Surrogated pups from the litter-matched experiment show similar nonfasting blood glucose levels but higher insulin serum levels compared with their nonsurrogated littermates; *P < 0.05 (Student’s t-test). G: H&E-stained retinal cross-sections from adult PWG mice (right) show no morphological difference to EWG mice (left). H: Vascularization of the inner retina shows no difference between adult EWG and PWG mice. Exemplary flat-mounts from P70 EWG (top) and PWG (bottom) mice are shown on the right. I: At P70, PWG pups retain a significant difference in body weight compared to EWG pups (19.46 ± 0.5 versus 23.05 ± 0.75 g). J: Functional retinal analysis using ERG shows significant deficits in PWG mice at P70 compared with EWG pups. PIII = photoreceptor function with RmP₃ being the saturating amplitude of the rod photoreceptor response and S the sensitivity of the rod photoreceptor response. PII = bipolar cell function with RmP₂ being the saturating amplitude of the bipolar cell component and Logk_P2 the sensitivity of the bipolar cell response. OPs = oscillatory potentials with Em being the maximal energy in the oscillatory potential. Flicker = retinal response to intermittent light stimulus with R₈ being the response amplitude at 8 Hz.