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Phlebotomy-induced anemia reduces oxygen-induced retinopathy severity and dampens retinal developmental transcriptomic pathways in rats

Pediatric Research volume 97, pages 1237–1245 (2025)Cite this article

Abstract

Background

Phlebotomy-induced-anemia (PIA), which induces tissue hypoxia and angiogenesis, occurs universally among infants at risk for severe retinopathy of prematurity (ROP). We hypothesized that PIA exacerbates pathologic retinal neovascularization in ROP.

Methods

We induced PIA to a hematocrit of 18% among rats undergoing the established 50/10 oxygen-induced retinopathy (OIR) model. Rats were euthanized at P15 and P20, during the avascular and neovascular phases of OIR, respectively. Retinal vascular morphometry, cytokine/chemokine concentrations, transcriptomes, and mRNA expression of angiogenic and iron-deficiency markers were compared to non-PIA controls.

Results

In OIR, PIA decreased percent avascular area at P15 by 35%, percent neovascular area at P20 by 42%, and select pro-inflammatory cytokine/chemokine concentrations at both time points. At P20, PIA increased mRNA expression of angiopoietin 2/ vascular endothelial growth factor-A 2-fold and transferrin and transferrin receptor 5-fold. RNA sequencing showed dampened pathways of angiogenesis, inflammation, and neural development in anemic OIR females.

Conclusion

Contrary to our hypothesis, PIA decreased OIR severity and retinal cytokine and chemokine levels and dampened transcriptomic pathways central to retinal vascular and neural development in neonatal rats. These data suggest PIA provides a protective effect from OIR. Further investigation into the functional effect of these molecular changes is warranted.

Impact

  • This is the first preclinical study to investigate the impact of neonatal anemia on oxygen-induced retinopathy (OIR) outcomes.

  • This study adds to the literature that anemia decreases neovascularization, decreases cytokine and chemokine levels, and dampens angiogenic and neural transcriptomic pathways in the rat 50/10 OIR model.

  • The study identifies a sex-specific transcriptomic response to anemia in the 50/10 OIR model, with females primarily impacted.

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Fig. 1: Rat pup daily weight gain and hematocrits.
Fig. 2: Retinal vascular morphometric outcomes.
Fig. 3: Comparison of male versus female response to phlebotomy-induced anemia (PIA) in the OIR model, n = 4/ sex.
Fig. 4: Comparison of IPA-mapped gene pathways.
Fig. 5: Relative mRNA expression at P20 of Angiopoietin 2 (Ang2), Vascular endothelial growth factor-A (Vegf-A), Ang2/ Vegf-A ratio, Transferrin (Tf), and Transferrin receptor (TfR).
Fig. 6

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Data availability

The datasets generated during the current study are available in Gene Expression Omnibus: Accession number GSE272252.

References

  1. Meyer, M. P., O’Connor, K. L. & Meyer, J. H. Thresholds for blood transfusion in extremely preterm infants: A review of the latest evidence from two large clinical trials. Front Pediatr. 10, 957585, https://doi.org/10.3389/fped.2022.957585 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Strauss, R. G. Anaemia of prematurity: pathophysiology and treatment. Blood Rev. 24, 221–225, https://doi.org/10.1016/j.blre.2010.08.001 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yucel, O. E., Eraydin, B., Niyaz, L. & Terzi, O. Incidence and risk factors for retinopathy of prematurity in premature, extremely low birth weight and extremely low gestational age infants. BMC Ophthalmol. 22, 367, https://doi.org/10.1186/s12886-022-02591-9 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Widness, J. A. Pathophysiology of anemia during the neonatal period, including anemia of prematurity. Neoreviews 9, e520, https://doi.org/10.1542/neo.9-11-e520 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Özdemir, H. B. & Özdek, S. Late sequelae of retinopathy of prematurity in adolescence and adulthood. Saudi J. Ophthalmol. 36, 270–277, https://doi.org/10.4103/sjopt.sjopt_276_21 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Englert, J. A., Saunders, R. A., Purohit, D., Hulsey, T. C. & Ebeling, M. The effect of anemia on retinopathy of prematurity in extremely low birth weight infants. J. Perinatol. 21, 21–26 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Lundgren, P. et al. Duration of anaemia during the first week of life is an independent risk factor for retinopathy of prematurity. Acta Paediatr. 107, 759–766, https://doi.org/10.1111/apa.14187 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Lundgren, P. et al. Erythropoietin serum levels, versus anaemia as risk factors for severe retinopathy of prematurity. Pediatr. Res. https://doi.org/10.1038/s41390-018-0186-6 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Pheng, E., Lim, Z. D., Tai Li Min, E., Rostenberghe, H. V. & Shatriah, I. Haemoglobin levels in early life among infants with and without retinopathy of prematurity. Int J. Environ. Res Public Health 18, 7054, https://doi.org/10.3390/ijerph18137054 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Tandon, M., Ranjan, R., Muralidharan, U. & Kannan, A. Influence of anaemia on multifactorial disease retinopathy of prematurity: a prospective observational study. Cureus 14, e27877, https://doi.org/10.7759/cureus.27877 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Maria Cristina, C. Prevalence of retinopathy in patients with anemia or thrombocytopenia. Eur. J. Haematol. 67, 238–244 (2001).

    Article  Google Scholar 

  12. Widness, J. A. Treatment and prevention of neonatal anemia. Neoreviews 9, 526–533 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Widness, J. A. et al. Reduction in red blood cell transfusions among preterm infants: results of a randomized trial with an in-line blood gas and chemistry monitor. Pediatrics 115, 1299–1306, https://doi.org/10.1542/peds.2004-1680 (2005).

    Article  PubMed  Google Scholar 

  14. Moreno-Fernandez, J., Ochoa, J. J., Latunde-Dada, G. O. & Diaz-Castro, J. Iron deficiency and iron homeostasis in low birth weight preterm infants: a systematic review. Nutrients 11, 1090, https://doi.org/10.3390/nu11051090 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hartnett, M. E. Pathophysiology and mechanisms of severe retinopathy of prematurity. Ophthalmology 122, 200–210, https://doi.org/10.1016/j.ophtha.2014.07.050 (2015).

    Article  PubMed  Google Scholar 

  16. Penn, J. S., Henry, M. M. & Tolman, B. L. Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat. Pediatr. Res. 36, 724–731, https://doi.org/10.1203/00006450-199412000-00007 (1994).

    Article  CAS  PubMed  Google Scholar 

  17. Penn, J. S., Henry, M. M., Wall, P. T. & Tolman, B. L. The range of PaO2 variation determines the severity of oxygen-induced retinopathy in newborn rats. Invest. Ophthalmol. Vis. Sci. 36, 2063–2070 (1995).

    CAS  PubMed  Google Scholar 

  18. Penn, J. “Penn Model Tips”. Personal communication. (ed E. Ingolfsland) (2016).

  19. Lee, H. et al. Differences in oxygen-induced retinopathy susceptibility between two Sprague Dawley rat vendors: a comparison of retinal transcriptomes. Curr. Eye Res., 1–12, https://doi.org/10.1080/02713683.2023.2297346 (2023).

  20. Molomjamts, M. & Ingolfsland, E. C. Identification of reference genes for the normalization of retinal mRNA expression by RT-qPCR in oxygen induced retinopathy, anemia, and erythropoietin administration. PLoS One 18, e0284764 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Holmes, J. M. & Duffner, L. A. The effect of postnatal growth retardation on abnormal neovascularization in the oxygen exposed neonatal rat. Curr. Eye Res. 15, 403–409 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Hartnett, M. E. & Penn, J. S. Mechanisms and management of retinopathy of prematurity. N. Engl. J. Med. 367, 2515–2526, https://doi.org/10.1056/NEJMra1208129 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rao, R. et al. Iron supplementation dose for perinatal iron deficiency differentially alters the neurochemistry of the frontal cortex and hippocampus in adult rats. Pediatr. Res. 73, 31–37, https://doi.org/10.1038/pr.2012.143 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Wallin, D. J. et al. Phlebotomy-induced anemia alters hippocampal neurochemistry in neonatal mice. Pediatr. Res. 77, 765–771, https://doi.org/10.1038/pr.2015.41 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Singh, N. K. & Rao, G. N. Emerging role of 12/15-Lipoxygenase (ALOX15) in human pathologies. Prog. Lipid Res. 73, 28–45, https://doi.org/10.1016/j.plipres.2018.11.001 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Oshima, Y. et al. Angiopoietin 2 (Ang2) Increases or Decreases Neovascularization (NV) depending upon the setting. IOVS 44 (2003).

  27. Oshima, Y. et al. Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive. FASEB J. 19, 963–965, https://doi.org/10.1096/fj.04-2209fje (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Idzerda, R. L., Huebers, H., Finch, C. A. & McKnight, G. S. Rat transferrin gene expression: tissue-specific regulation by iron deficiency. Proc. Natl. Acad. Sci. USA 83, 3723–3727, https://doi.org/10.1073/pnas.83.11.3723 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dupic, F. et al. Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice. Gut 51, 648–653, https://doi.org/10.1136/gut.51.5.648 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schoephoerster, J. et al. Identification of clinical factors associated with timing and duration of spontaneous regression of retinopathy of prematurity not requiring treatment. J. Perinatol. 43, 702–708, https://doi.org/10.1038/s41372-023-01649-w (2023).

    Article  PubMed  Google Scholar 

  31. Zhou, Z. et al. Distinguished functions of microglia in the two stages of oxygen-induced retinopathy: a novel target in the treatment of ischemic retinopathy. Life 12, 1676, https://doi.org/10.3390/life12101676 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rivera, J. C. et al. Retinopathy of prematurity: inflammation, choroidal degeneration, and novel promising therapeutic strategies. J. Neuroinflamm. 14, 165, https://doi.org/10.1186/s12974-017-0943-1 (2017).

    Article  CAS  Google Scholar 

  33. Deliyanti, D. et al. Early depletion of neutrophils reduces retinal inflammation and neovascularization in mice with oxygen-induced retinopathy. Int J. Mol. Sci. 24, 15680, https://doi.org/10.3390/ijms242115680 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Perelli, R. M., O’Sullivan, M. L., Zarnick, S. & Kay, J. N. Environmental oxygen regulates astrocyte proliferation to guide angiogenesis during retinal development. Development 148, dev199418, https://doi.org/10.1242/dev.199418 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wu, P. Y. et al. Systemic cytokines in retinopathy of prematurity. J. Pers. Med 13, 291, https://doi.org/10.3390/jpm13020291 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Arthur, C. M. et al. Anemia induces gut inflammation and injury in an animal model of preterm infants. Transfusion 59, 1233–1245, https://doi.org/10.1111/trf.15254 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bastian, T. W. et al. Fetal and neonatal iron deficiency but not copper deficiency increases vascular complexity in the developing rat brain. Nutr. Neurosci. 18, 365–375, https://doi.org/10.1179/1476830515Y.0000000037 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Woodman, A. G. et al. Modest and severe maternal iron deficiency in pregnancy are associated with fetal anaemia and organ-specific hypoxia in rats. Sci. Rep. 7, 46573, https://doi.org/10.1038/srep46573 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Erber, L., Liu, S., Gong, Y., Tran, P. & Chen, Y. Quantitative Proteome and Transcriptome dynamics analysis reveals iron deficiency response networks and signature in neuronal cells. Molecules 27, 484, https://doi.org/10.3390/molecules27020484 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Toblli, J. E., Cao, G., Oliveri, L. & Angerosa, M. Effects of iron deficiency anemia and its treatment with iron polymaltose complex in pregnant rats, their fetuses and placentas: oxidative stress markers and pregnancy outcome. Placenta 33, 81–87, https://doi.org/10.1016/j.placenta.2011.11.017 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Shirley Ding, S. L. et al. Revisiting the role of erythropoietin for treatment of ocular disorders. Eye 30, 1293–1309, https://doi.org/10.1038/eye.2016.94 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Park, A. M., Sanders, T. A. & Maltepe, E. Hypoxia-inducible factor (HIF) and HIF-stabilizing agents in neonatal care. Semin Fetal Neonatal Med 15, 196–202, https://doi.org/10.1016/j.siny.2010.05.006 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Becker, S. et al. Protective effect of maternal uteroplacental insufficiency on oxygen-induced retinopathy in offspring: removing bias of premature birth. Sci. Rep. 7, 42301, https://doi.org/10.1038/srep42301 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bretz, C. A., Ramshekar, A., Kunz, E., Wang, H. & Hartnett, M. E. Signaling through the erythropoietin receptor affects angiogenesis in retinovascular disease. Invest. Ophthalmol. Vis. Sci. 61, 23, https://doi.org/10.1167/iovs.61.10.23 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bretz, C. A. et al. Erythropoietin receptor signaling supports retinal function after vascular injury. Am. J. Pathol. 190, 630–641, https://doi.org/10.1016/j.ajpath.2019.11.009 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Fahim, N. M. et al. Endogenous erythropoietin concentrations and association with retinopathy of prematurity and brain injury in preterm infants. PLoS One 16, e0252655, https://doi.org/10.1371/journal.pone.0252655 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bórquez, D. A., Castro, F., Núñez, M. T. & Urrutia, P. J. New players in neuronal iron homeostasis: Insights from CRISPRi studies. Antioxidants 11, 1807, https://doi.org/10.3390/antiox11091807 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bastian, T. W., von Hohenberg, W. C., Mickelson, D. J., Lanier, L. M. & Georgieff, M. K. Iron deficiency impairs developing hippocampal neuron gene expression, energy metabolism, and dendrite complexity. Dev. Neurosci. 38, 264–276, https://doi.org/10.1159/000448514 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Bastian, T. W., Rao, R., Tran, P. V. & Georgieff, M. K. The effects of early-life iron deficiency on brain energy metabolism. Neurosci. Insights 15, 2633105520935104, https://doi.org/10.1177/2633105520935104 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Vessey, K. A., Wilkinson-Berka, J. L. & Fletcher, E. L. Characterization of retinal function and glial cell response in a mouse model of oxygen-induced retinopathy. J. Comp. Neurol. 519, 506–527, https://doi.org/10.1002/cne.22530 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. Irani, Y. D. et al. Sex differences in corneal neovascularization in response to superficial corneal cautery in the rat. PLoS One 14, e0221566, https://doi.org/10.1371/journal.pone.0221566 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Al Mamun, A., Yu, H., Romana, S. & Liu, F. Inflammatory responses are sex specific in chronic hypoxic-ischemic Encephalopathy. Cell Transpl. 27, 1328–1339, https://doi.org/10.1177/0963689718766362 (2018).

    Article  Google Scholar 

  53. Mirza, M. A., Ritzel, R., Xu, Y., McCullough, L. D. & Liu, F. Sexually dimorphic outcomes and inflammatory responses in hypoxic-ischemic encephalopathy. J. Neuroinflamm. 12, 32, https://doi.org/10.1186/s12974-015-0251-6 (2015).

    Article  CAS  Google Scholar 

  54. Singh, G. et al. Dose- and sex-dependent effects of phlebotomy-induced anemia on the neonatal mouse hippocampal transcriptome. Pediatr. Res. 92, 712–720, https://doi.org/10.1038/s41390-021-01832-9 (2022).

    Article  CAS  PubMed  Google Scholar 

  55. Lundgren, P. et al. WINROP identifies severe retinopathy of prematurity at an early stage in a nation-based cohort of extremely preterm infants. PLoS One 8, e73256, https://doi.org/10.1371/journal.pone.0073256 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Al-Mousawi, A. M. et al. Impact of anesthesia, analgesia, and euthanasia technique on the inflammatory cytokine profile in a rodent model of severe burn injury. Shock 34, 261–268, https://doi.org/10.1097/shk.0b013e3181d8e2a6 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Boivin, G. P., Hickman, D. L., Creamer-Hente, M. A., Pritchett-Corning, K. R. & Bratcher, N. A. Review of CO2 as a Euthanasia agent for laboratory rats and mice. J. Am. Assoc. Lab Anim. Sci. 56, 491–499 (2017).

    PubMed  PubMed Central  Google Scholar 

  58. van der Schrier, R. et al. Carbon dioxide tolerability and toxicity in rat and man: A translational study. Front Toxicol. 4, 1001709, https://doi.org/10.3389/ftox.2022.1001709 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to acknowledge Juan E. Abrahante Lloréns, PhD of the University of Minnesota Informatics Institute who performed the pairwise comparisons generating the differentially expressed genes from the RNA sequencing data. We would also like to acknowledge Dr. John Penn and his laboratory team at Vanderbilt University who trained EI in the OIR model.

Funding

Funding This research was funded by the American Academy of Pediatrics (Marshall Klaus award to E.I., no award number) and the Knights Templar Eye Foundation (Career Starter grant to E.I., no award number).

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Authors and Affiliations

  1. Department of Pediatrics, Division of Neonatology, University of Minnesota Medical School, Minneapolis, MN, USA

    Ellen C. Ingolfsland, Mandkhai Molomjamts, Ann Foster, Haeyeon Lee, Amelia Morikuni, Husaam Qureishy, Phu V. Tran & Michael K. Georgieff

  2. Department of Ophthalmology and Visual Neurosciences, University of Minnesota Medical School, Minneapolis, MN, USA

    Heidi Roehrich & Linda K. McLoon

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  1. Ellen C. Ingolfsland
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  2. Mandkhai Molomjamts
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  3. Ann Foster
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  10. Michael K. Georgieff
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Contributions

Each author contributed substantially, as follows: Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data: E.I., M.M., A.F., H.L., H.R., A.M., H.Q., P.T., L.M., M.G. Drafting the article or revising it critically for important intellectual content: E.I., H.R., P.T., L.M., M.G. Final approval of the version to be published: E.I., M.M., A.F., H.L., H.R., A.M., H.Q., P.T., L.M., M.G.

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Correspondence to Ellen C. Ingolfsland.

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Ingolfsland, E.C., Molomjamts, M., Foster, A. et al. Phlebotomy-induced anemia reduces oxygen-induced retinopathy severity and dampens retinal developmental transcriptomic pathways in rats. Pediatr Res 97, 1237–1245 (2025). https://doi.org/10.1038/s41390-024-03477-w

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