Erythropoietin and the HEAL Study: Timing Issues

January 2024

Introduction

The recently reported studies1-3 derived from the High-Dose Erythropoietin (EPO) For Asphyxia and Encephalopathy (HEAL) study raise two important questions relevant to timing. The first of these questions concerns the time of occurrence of the hypoxic-ischemic injury leading to hypoxic-ischemic encephalopathy (HIE), and the second, the optimal duration of treatment with EPO in the context of HIE. The HEAL study was a multicenter, double-blind, randomized, placebo-controlled trial involving 501 infants born at gestational ages of 36 weeks or more with moderate or severe HIE, to receive EPO or placebo in conjunction with standard therapeutic hypothermia (begun within 6 hours after birth and continued for 72 hours). EPO was administered before 26 hours of age and at 2, 3, 4 and 7 days of age. The primary outcome was death or neurodevelopmental impairment of any severity at 22 to 36 months of age. The incidence of death or neurodevelopmental impairment was similar in both groups (52% in the EPO group, 49% in the Placebo group). 

Time of Occurrence of Hypoxic-Ischemic Injury

Concerning the time of occurrence of the hypoxic-ischemic insults leading to HIE, in the HEAL study MRI findings at a median age of approximately 5 days (4.9 days) were interpreted to indicate that brain injury was “acute only” in 23%, “subacute only” in 22%, and “acute and subacute” in 21% (chronic injury was identified in only 2%).3 Acute lesions were defined as foci of restricted diffusion (i.e., reduced ADC) with or without corresponding signal abnormalities on T1w and T2w MRI. Subacute lesions were defined as signal abnormalities on T1w and/or T2w MRI without corresponding diffusion restriction. Acute and subacute lesions were defined by MRI features of both types. The surprising finding of subacute injury in 43% of infants deserves further consideration.

The relatively high proportion of subacute injury reported in the HEAL study should be viewed in the context of (a) previous neuropathological data and (b) the MRI findings utilized to conclude injury was subacute. In general, my view is that the gold standard for assessment of timing of neonatal injury is the neuropathology, although in this era of rare postmortem studies, we must rely on less direct measures of pathology, such as neuroimaging.

Concerning neuropathological data, in the largest reported series of infants with HIE studied postmortem, all 45 infants showed cellular evidence of acutely evolving lesions thought to be of “hypoxic-ischemic origin.”4 (The principal neuronal changes following an acute, injurious, hypoxic-ischemic insult are, after 24 to 36 hours, marked eosinophilia of neuronal cytoplasm [“red dead neuron”], condensation [pyknosis] or fragmentation [karyorrhexis] of nuclei and cellular edema, followed in the next 72 hours by the appearance of macrophages/activated microglia and still later by hypertrophic astrocytes.5) In a later series, utilizing staining for different stages of activated microglia/macrophages, a similar preponderance of acute injury was observed (only two of 23 cases showed changes consistent with onset of the hypoxic-ischemic event “2-3 days before birth”).6

Concerning the MRI data to identify “subacute” injury in the HEAL study, diffusion measures were of central importance.3 MRI was performed at a median age of 4.9 days. Acute injury was determined by the MRI findings noted earlier. Previous work by Bednarek et al. in their landmark study of the time course of diffusion changes in HIE had shown that in infants with HIE treated with hypothermia, “pseudonormalization” occurs by approximately 10 days,7 as opposed to normothermic infants with HIE in whom pseudonormalization occurs by approximately 6 days.8 Utilizing data for hypothermic infants, as noted earlier, Wisnowski et al. found overall that in the HEAL population 23% exhibited only acute, 22% only subacute, and 21% both acute and subacute injuries.3 However, considering only the subacute injuries, it is noteworthy that of the 93 examples 83 had moderate HIE and only 10, severe HIE. This preponderance of moderate HIE in the subacute group may be important, because as reported by Bednarek et al.7, at five days of age, the differences between the mean diffusivity (MD) ratio of 1.0 (“pseudonormalization”) and the values obtained in the moderate HIE group are relatively small (see Fig. 2B in Bednarek et al.).7 The small differences raise the possibility that “subacute” injury in the HEAL study could be overestimated. Confirmation of the MRI findings reported by Wisnowski et al.3, especially in infants with moderate HIE, will be important.

Concerning the implications of “subacute injury” for timing of the injurious hypoxic-ischemic insult(s) operative in the infants with HIE, Wisnowski et al.3 “speculate that the onset of injury was likely incurred days before birth.” Although I remain uncertain about the magnitude of subacute injury in HIE, the data raise the question of what role placental factors might play in pathogenesis of such injury. In the HEAL study, a complete placental pathological examination was available for 321 of the 500 participants (64%).1 Acute abnormalities were noted in 20%, chronic abnormalities in 21%, and both acute and chronic abnormalities in 43%. Thus, fully 84% of placentas exhibited abnormalities. Lesions considered high risk were observed in 21% of placentas, and most of these were chronic abnormalities, such as maternal vascular malperfusion, fetal vascular malperfusion, and chronic villitis. Similar lesions have been reported in smaller series of HIE.9-13  Although histological chorioamnionitis was relatively common (39%), only 2% were accompanied by a high-grade fetal inflammatory response and therefore considered high risk.

The prominence of chronic placental abnormalities in the HEAL population is notable, particularly because such disturbances may prime the brain to sustain hypoxic-ischemic brain injury in the peripartum period.12,14 It is noteworthy, in this regard, that the frequency of high-risk chronic abnormalities (approximately 20%) is similar to the frequency of “subacute injury” (22%) defined by the MRI studies discussed earlier.3 It would be of great interest if an analysis of the relations between specific placental findings and MRI analyses could be carried out in individual patients in the HEAL population. 

Also consistent with the notion that placental factors can be important in pathogenesis of some cases of HIE and, thereby, the response to postnatal EPO is the result of an earlier EPO trial by Wu and colleagues.13 In those cases with available placental pathology reports (n=35), EPO was associated with less brain injury than in nontreated infants only in those whose placentas exhibited no chronic histologic abnormalities.13 Thus, it is possible that in the EPO-unresponsive infants overt placental disturbance led to the hypoxic-ischemic brain injury that occurred prior to the onset of EPO therapy or that was set in motion prior to the onset of EPO therapy. More data are needed concerning placental structure and physiology in pregnancy, especially in relation to neonatal HIE. Recent advances in the application of sophisticated MRI methods for such study suggest that important insights may be gained in vivo in the near future.15,16

Timing of Treatment with EPO

The second question relating to timing from the results of the HEAL study concerns the duration of treatment with EPO. Is seven days of treatment sufficient to determine whether EPO has benefit for management of HIE? The study’s conclusion is that this duration of treatment does not lead to clear benefit re: death or neurodevelopmental impairment at 22 to 36 months of age. Should this well-designed, large study be the final word on any value of EPO in management of infants with HIE? This issue, including the risk/benefits of longer treatment, is addressed carefully by Wu et al. in the Discussion section of the article in the New England Journal of Medicine describing the trial.2 (In addition to the apparent lack of benefit on outcomes, the mean number of serious adverse effects per child was higher in the EPO group than in the placebo group.) In experimental models, EPO is well-established to have benefit in neuroprotection in primary, latent, and secondary phases of hypoxic-ischemic injury occurring over the first several days after the insult.17 This neuroprotection includes antiexcitotoxic, antioxidant, and antiapoptotic effects (among others).18 Many of these beneficial effects of EPO are shared by hypothermia, and as suggested by Wisnowski et al.3, it is possible that a substantial proportion of hypoxic-ischemic lesions are treated successfully by hypothermia, thereby shifting the distribution of residual lesions apparent after hypothermia to include a higher proportion of less responsive lesions.

Of greatest importance in this context, however, EPO has been shown in experimental models to have important neurorestorative effects and, critically, such beneficial effects can be demonstrated with delayed EPO treatment. The neurorestorative potential of EPO in developing animals subjected to hypoxia-ischemia (stroke model) was shown initially by Gonzalez et al.19,20 Treatment was begun in the days following the production of stroke. The mechanisms of benefit included stimulation of neurogenesis and diminution of reactive astrocytosis. Subsequent work with the stroke model, which included immediate and delayed EPO treatment, showed enhanced neurogenesis and oligodendrogliosis. Other studies in hypoxic-ischemic models in developing animals have shown that EPO leads to decreased microglial activation, diminished oligodendroglial injury, and improved myelination.21 Beneficial effects on angiogenesis have also been shown. The first demonstration of the benefit of only delayed EPO therapy involved a stroke model (P10 rats), in which EPO therapy was instituted at P17, P20 and P23, a long delay when comparing rat and human brain development.22 In neonatal brain injury, neuronal development, axonal outgrowth, and oligodendroglial development can be impaired by the prolonged action of reactive astrocytes and activated microglia.23 At least in experimental models, EPO appears to have the potential to interrupt these effects when administered long-term, after the acute periods of injury. Clearly, because in human brain these developmental processes are occurring over many weeks to months, prolonged therapy with EPO could be needed and potentially could be beneficial. However, serious safety and risk-benefit issues require very careful consideration before controlled study of such an approach could be undertaken.

Conclusion

To conclude, the HEAL study has raised very important issues relating to timing of hypoxic-ischemic injury and timing of EPO therapy. My view is that the EPO story concerning the infant with HIE, hopefully, hasn’t finished and will add other important chapters.

Joseph J. Volpe, MD

Department of Neurology, Boston Children’s Hospital

Bronson Crothers Professor of Neurology, Emeritus, Harvard Medical School

Boston MA

References

1. Chalak L, Redline RW, Goodman AM, et al.: Acute and Chronic Placental Abnormalities in a Multicenter Cohort of Newborn Infants with Hypoxic-Ischemic Encephalopathy. J Pediatr 237:190-6, 2021. DOI: 10.1016/j.jpeds.2021.06.023
2. Wu YW, Comstock BA, Gonzalez FF, et al.: Trial of Erythropoietin for Hypoxic-Ischemic Encephalopathy in Newborns. N Engl J Med 387:148-59, 2022. DOI: 10.1056/NEJMoa2119660
3. Wisnowski JL, Monsell SE, Bluml S, et al.: Brain Injury Outcomes after Adjuvant Erythropoietin Neuroprotection for Moderate or Severe Neonatal Hypoxic-Ischemic Encephalopathy: A Report from the HEAL Trial. Dev Neurosci, 2023. DOI: 10.1159/000534618
4. Cowan F, Rutherford M, Groenendaal F, et al.: Origin and timing of brain lesions in term infants with neonatal encephalopathy. Lancet 361:736-42, 2003. DOI: 10.1016/S0140-6736(03)12658-X
5. Kinney HC, Volpe JJ. Hypoxic-ischemic injury in the term infant: neuropathology. Chapter 18. In: Volpe JJ, inder TE, Darras BT, de Vries LS, du Plessis AJ, Neil JJ, Perlman JM, editors. Volpe’s Neurology of the Newborn. 6th ed. Philadelphia PA: Elsevier; 2018. p. 484-99.
6. Alderliesten T, Nikkels PG, Benders MJ, et al.: Antemortem cranial MRI compared with postmortem histopathologic examination of the brain in term infants with neonatal encephalopathy following perinatal asphyxia. Arch Dis Child Fetal Neonatal Ed 98:F304-9, 2013. DOI: 10.1136/archdischild-2012-301768
7. Bednarek N, Mathur A, Inder T, et al.: Impact of therapeutic hypothermia on MRI diffusion changes in neonatal encephalopathy. Neurology 78:1420-7, 2012. DOI: 10.1212/WNL.0b013e318253d589
8. McKinstry RC, Miller JH, Snyder AZ, et al.: A prospective, longitudinal diffusion tensor imaging study of brain injury in newborns. Neurology 59:824-33, 2002. DOI: 10.1212/wnl.59.6.824
9. Vik T, Redline R, Nelson KB, et al.: The Placenta in Neonatal Encephalopathy: A Case-Control Study. J Pediatr 202:77-85 e3, 2018. DOI: 10.1016/j.jpeds.2018.06.005
10. Harteman JC, Nikkels PG, Benders MJ, et al.: Placental pathology in full-term infants with hypoxic-ischemic neonatal encephalopathy and association with magnetic resonance imaging pattern of brain injury. J Pediatr 163:968-95 e2, 2013. DOI: 10.1016/j.jpeds.2013.06.010
11. Mir IN, Johnson-Welch SF, Nelson DB, et al.: Placental pathology is associated with severity of neonatal encephalopathy and adverse developmental outcomes following hypothermia. Am J Obstet Gynecol 213:849 e1-7, 2015. DOI: 10.1016/j.ajog.2015.09.072
12. Volpe JJ: Placental assessment provides insight into mechanisms and timing of neonatal hypoxic-ischemic encephalopathy. J Neonatal Perinatal Med 12:113-6, 2019. DOI: 10.3233/NPM-190270
13. Wu YW, Goodman AM, Chang T, et al.: Placental pathology and neonatal brain MRI in a randomized trial of erythropoietin for hypoxic-ischemic encephalopathy. Pediatr Res 87:879-84, 2020. DOI: 10.1038/s41390-019-0493-6
14. Inder TE, Volpe JJ. Pathophysiology: general principles. Chapter 13. In: Volpe JJ, Inder TE, Darras BT, de Vries LS, du Plessis AJ, Neil JJ, Perlman JM, editors. Volpe’s Neurology of the Newborn. 6th ed. Philadelphia PA: Elsevier; 2018. p. 325-88.
15. Hutter J, Slator PJ, Jackson L, et al.: Multi-modal functional MRI to explore placental function over gestation. Magn Reson Med 81:1191-204, 2019. DOI: 10.1002/mrm.27447
16. du Plessis AJ, Volpe JJ. Placental conditions with consequences for the fetal brain. Chapter 10. In: Volpe JJ, Inder TE, Darras BT, De Vries LS, du Plessis AJ, Ferriero DM, Perlman JM, editors. Volpe’s Neurology of the Newborn. 7th ed. Philadelphia PA: Elsevier; in press.
17. Davidson JO, Gonzalez F, Gressens P, et al.: Update on mechanisms of the pathophysiology of neonatal encephalopathy. Semin Fetal Neonatal Med 26:101267, 2021. DOI: 10.1016/j.siny.2021.101267
18. Inder TE, Volpe JJ. Hypoxic-ischemic injury in the term infant: clinical-neurological features, diagnosis, imaging, prognosis, therapy. Chapter 20. In: Volpe JJ, Inder TE, Darras BT, de Vries LS, du Plessis AJ, Neil JJ, Perlman JM, editors. Volpe’s Neurology of the Newborn. 6th ed. Philadelphia PA: Elsevier; 2018. p. 510-63.
19. Gonzalez FF, McQuillen P, Mu D, et al.: Erythropoietin enhances long-term neuroprotection and neurogenesis in neonatal stroke. Dev Neurosci 29:321-30, 2007. DOI: 10.1159/000105473
20. Gonzalez FF, Larpthaveesarp A, McQuillen P, et al.: Erythropoietin increases neurogenesis and oligodendrogliosis of subventricular zone precursor cells after neonatal stroke. Stroke 44:753-8, 2013. DOI: 10.1161/STROKEAHA.111.000104
21. Liu W, Shen Y, Plane JM, et al.: Neuroprotective potential of erythropoietin and its derivative carbamylated erythropoietin in periventricular leukomalacia. Exp Neurol 230:227-39, 2011. DOI: 10.1016/j.expneurol.2011.04.021
22. Larpthaveesarp A, Georgevits M, Ferriero DM, Gonzalez FF: Delayed erythropoietin therapy improves histological and behavioral outcomes after transient neonatal stroke. Neurobiol Dis 93:57-63, 2016. DOI: 10.1016/j.nbd.2016.04.006
23. Volpe JJ: Dysmaturation of Premature Brain: Importance, Cellular Mechanisms, and Potential Interventions. Pediatr Neurol 95:42-66, 2019. DOI: 10.1016/j.pediatrneurol.2019.02.016