Volpe's View

September, 2024

INCONSISTENT PREDICTIVE VALUE OF MRI IN EARLY INFANCY AFTER NEONATAL HIE: DYSMATURATION MAY BE THE EXPLANATION

Introduction

Many studies have addressed the potential value of MRI in early infancy (beyond the neonatal period) for prediction of later neurodevelopmental outcome in term infants with neonatal hypoxic-ischemic encephalopathy (HIE) (see for reviews^1,2). Although severe lesions (especially those involving such deep nuclear structures as thalamus and basal ganglia) are associated clearly with unfavorable outcomes, such lesions involve the small minority of infants and are generally detectable in the neonatal period.^3,4 There is a larger proportion of infants who will later exhibit neurodevelopmental abnormality (particularly, cognitive impairments) but who do not show severe lesions in the neonatal period. Indeed, in a recent study of 451 term infants with HIE, infants with no injury on MRI at a median age of 5 days had similar cognitive, language and motor impairment scores as those infants with observable injury.³ A reasonable question is whether such infants with only mild or moderate or perhaps no observable neonatal injury might exhibit prognostically important structural abnormalities later, e.g., three months of age. Detection of structural abnormalities in early infancy, e.g., three months of age, is desirable for many reasons. The most important of these reasons is identification of those infants who should receive therapies to enhance neurodevelopmental potential (see later). The focus of this Commentary is this large proportion of infants with neonatal HIE who later exhibit neurodevelopmental impairment, but without severe neonatal brain injury.

Brain Abnormalities Identified on Late MRI Studies

Approximately 20 reports over the past 34 years have reported MRI data obtained in the months after neonatal HIE (see Table 1 in Sotelo et al.²). In general (see later for a few exceptions), compared to neonatal MRI data, later conventional MRIs do not consistently show more clearly useful information regarding prediction of later neurodevelopmental impairment. Severe neurological deficits involving motor (“cerebral palsy”) and cognitive functions are predicted by overt injury (usually necrosis) to deep nuclear structures (e.g., thalamus, putamen) or cerebral cortex or both. However, as just noted, these lesions generally are identified readily in the neonatal period and occur in only a small minority of current survivors of neonatal HIE.

The principal late MRI findings in the first postnatal months among the large majority of survivors without overt necrotic lesions have included signal abnormalities in cortex, deep nuclear structures and cerebral white matter, often with modest increases in lateral ventricular size or extracerebral space or both. The anatomic bases generally have been considered to be related to “Wallerian degeneration” and “atrophy.” Thus, in essence, the concept has been that the infant sustained a neonatal hypoxic-ischemic injury with cellular/tissue loss and that subsequent anatomic and neurodevelopmental findings are secondary to this loss. However, these modest later structural MRI findings do not show a robust relation to subsequent neurodevelopmental outcome.^1,2

In this Commentary, I propose that the reasons for this lack of a robust relation of later MRI studies to subsequent neurodevelopmental outcomes are that (1) the anatomic bases are not related simply to cellular/tissue loss, and (2) the previous later MRI approaches have not been adequate to capture the essence of the brain abnormalities.

Proposed Nature of the Brain Abnormalities in Neonatal HIE in Early Infancy

The hypothesis set forth in this Commentary is that the brain abnormalities in early infancy in those infants destined to have neurodevelopmental impairment are principally dysmaturational in nature, involve both gray and white matter structures, are persistent and dynamic, and potentially are modifiable. This dysmaturational concept^5,6 is accepted generally for premature infants with neonatal injury, and the manifestations are definable in such infants at term equivalent age by MRI studies of regional brain volumes, cerebral cortical gyrification, fiber tract development and connectivity (among other parameters).^6,7 I suggest that the dysmaturational concept is relevant also to the term infant in the months following neonatal hypoxic-ischemic injury. Indeed, vulnerable maturational events are very active in the term newborn brain and continue robustly over the months postnatal, as discussed next.

Principal Developmental Events at Term and in the First Postnatal Months

To place this dysmaturational idea in context, it is important to consider the principal developmental events at term and in the first postnatal months following term birth. These events involve key structures in cerebral white matter, thalamus and cerebral cortex.

Cerebral White Matter

The major developmental events in cerebral white matter at term and during the early post-term period include oligodendroglial maturation with ensheathment of axons (critical for such axonal growth as thalamo-cortical, cortico-callosal and cortico-cortical fibers and for early cerebral myelination).^5,6,8 Cerebral myelination begins during this period in the corona radiata (central cerebral white matter), the optic tract and chiasm, the auditory radiation, Heschl’s gyrus, thalamus and thalamic connections, among others.⁹

An additional important event in cerebral white matter involves migration of GABAergic neurons to cerebral cortex.¹⁰ This migration is especially active prior to term but continues until six postnatal months. Differentiation of these GABAergic neurons in cortex is also very active during this period (see later).

Thalamus

Thalamic development is very active at term and during the first months of life. The principal events involve axonal growth, myelination and connectivity.^8,9 This structure serves as a complex way station for axons ascending from brain stem, cerebellum and spinal cord and descending from cerebral cortex. Indeed, thalamic connections to the entirety of cerebral cortical and diencephalic neurons are crucial for a wide variety of complex motor, cognitive and behavioral functions. Thalamic neurons are highly sensitive to hypoxic-ischemic injury in the term infant.^11,12 Not surprisingly, a strong relation exists between overt hypoxic-ischemic thalamic/putaminal injury and severely impaired cognitive and motor outcomes.¹³ However, little is known about the relations of less obvious disturbance to thalamus during early development and a variety of subsequent neurodevelopmental impairments. Although not yet elucidated, in view of the very wide spectrum of thalamic connections, it seems reasonable to speculate that less obvious disturbances of thalamic developmental parameters could contribute importantly to later neurodevelopmental impairments.

Cerebral Cortex

The first several months after term birth is a remarkable period for cortical development. The key events are elaboration of the dendritic arbor and axonal ramifications, culminating in cortical synaptogenesis.^14-21 A detailed computerized analysis of Conel’s remarkable corpus of study of human cerebral cortical development in the first months and years of life showed that the first six months of life account for fully one-third of the developmental change in human cerebral cortex from term birth to 72 months of age.¹⁸

As noted earlier, GABAergic neuronal development is very active during the first postnatal months. Migration to upper cortical areas comes to a conclusion during this period. Differentiation of these neurons in upper cortical layers increases superficial cortical surface area and is important in provoking formation of gyri.²² During this time frame, GABAergic neurons differentiate from an initial, largely excitatory phenotype to their familiar inhibitory phenotype. These neurons play critical roles in development of cortical circuitry and determination of cortical critical periods.^23-25

Interaction of Cell Injury/Loss and Altered Development

Although not yet clearly accomplished for all parameters, it seems highly likely that the remarkable developmental events just described are highly vulnerable to such major perturbations as hypoxic-ischemic insults and especially to the subsequent persistent gliosis, involving both microglia and astrocytes (see later). This dysmaturational consequence to a destructive process would be entirely consistent with the phenomena shown for premature infants.^5,6

A particularly important brain area in this context of rapid developing structures likely to be affected by neonatal HIE is the hippocampus. Several seminal preclinical studies by Gunn and colleagues highlighted the particular vulnerability of hippocampal neurons.^26,27 The hippocampus is located deep in the medial temporal lobe, and contains a series of subfields of pyramidal cells that connect to multiple other structures, such as thalamus, hypothalamus, amygdala, and prefrontal cortex, among other structures (for review, see^28,29). The hippocampus later mediates key functions involved in memory, learning, emotional regulation and cognitive function. Hippocampal neurons, especially those of the CA1 and CA3 regions, are exquisitely vulnerable to injury by hypoxic-ischemic insults.^11,12

The loss of these hippocampal neurons during the acute period, as well as the deleterious effect of subsequent gliosis (see later), would be expected to disrupt the subsequent development of the hippocampus and its connections. The most active developmental processes occurring in hippocampus around the time of birth and in the first months of life are dendritic arborization, synaptogenesis and myelination. It is noteworthy in this context that later MRI studies of infants with HIE have shown evidence of decreased hippocampal volumes, parahippocampal white matter, and mammillary bodies.^{30 ,31,32} Decreased hippocampal volumes and mammillary body “atrophy” were associated with impaired cognition and memory at school age after neonatal HIE.³⁰ Notably, “atrophy” of the mammillary bodies has been shown as early as three postnatal months.³³ Experimental data suggest that the abnormalities of these hippocampal structures and connections may reflect impaired development rather than simply atrophy. Thus, Back and colleagues have shown in a preterm sheep model that hippocampal volume deficits identified four weeks after hypoxia-ischemia are not related to cell loss but rather to impaired development of basal and dendritic arborization.³⁴ These observations have important clinical implications (see later).

Interaction of cell loss, cell injury and impaired cerebral development likely involves other aspects of brain development active at term and in the first months of life, as outlined earlier. Disturbances of axonal growth, myelin elaboration, cortical and thalamic neuronal development and connectivity would be likely possibilities. A few earlier MRI studies in term infants months after HIE have noted impaired cerebral myelination³⁵ and reduced volumes.³⁶ However, more detailed information with state-of-the-art measures is lacking.

Likely Mechanisms Underlying Dysmaturation after Neonatal HIE

The mechanisms underlying potential dysmaturational events in the weeks and months after neonatal HIE likely relate largely to the persistent gliosis and related neuroinflammation characteristic of the so-called “tertiary phase” of perinatal hypoxic-ischemic injury (see^37,38). The key role of gliosis, involving both microglia and astrocytes, has been delineated best by Gressens and collaborators.^39,40 The mechanisms by which this gliosis leads to dysmaturation of differentiating oligodendroglial and neuronal-axonal structures are diverse. Pro-inflammatory (M1) microglia release reactive oxygen/nitrogen species and cytokines that then act on developing cells. Additionally, these pro-inflammatory microglia can induce formation of neurotoxic reactive astrocytes.^41,42 These astrocytes secrete cytokines and other molecular products important in impaired developmental events. Additionally, because microglia and astrocytes not in an active pro-inflammatory state are important in normal development of neurons, axons, and oligodendrocytes, vascularization, synaptic development, pruning and neural circuit formation,⁴³ diversion to the pro-inflammatory phenotypes could contribute importantly to disturbed maturational events subsequent to neonatal HIE.

Identification of Brain Dysmaturation in the First Months After Neonatal HIE

In view of the considerations just discussed, a reasonable goal in the term infant with HIE is to assess brain dysmaturation by approximately three months of age, principally to identify the nature and loci of the abnormalities and to institute interventions to improve outcomes. Three months of age obviously is a somewhat arbitrary time point, but the nature and loci of the dysmaturational events are likely to be apparent by this age and interventions should be instituted as early as possible.

To identify the nature of the likely abnormalities described earlier, state-of-the-art MRI approaches will be necessary. These approaches have been described in detail elsewhere (see, for example, Kelly et al.⁴⁴). It is beyond the scope of this Commentary to describe these techniques in detail. Suffice it to say here, such methods should include careful measures of volumes of cerebral cortical regions, including hippocampus, cerebral white matter, thalamus and other nuclear structures (basal ganglia, mammillary bodies), as well as measures of axonal growth and early myelination by diffusion based tractographic approaches (see later), and of intracortical and cortico-thalamic connectivity by functional MRI measures.

Detailed microstructural assessments will be critical in detecting early dysmaturational events.^44,45 For example, measures of cerebral cortical surface area, thickness and gyrification will provide insights into the cortical dysmaturational disturbances referred to earlier. In cerebral white matter, major white matter tracts can be interrogated by such advanced diffusion MRI analyses as fixel-based analysis⁴⁴ to provide insight into axonal growth and elaboration. Similarly, so-called NODDI imaging (Neurite Orientation Dispersion and Density Imaging) could provide major insight into axonal development in white matter tracts. Insight into disturbances of pre-oligodendroglial (pre-OL) ensheathment of axons and myelination in cerebral white matter, as well as dendritic and axonal development in cerebral cortex and such deep nuclear structures as thalamus and putamen, can be deduced by these approaches.

Clearly, all or most of these approaches are not available to the vast proportion of neonatal facilities. Moreover, the capacity to study a control population is limited and difficult. However, some research programs could pursue such a major investigative effort (with appropriate external funding). The aim then would be that some of the findings generated from such research could help direct use of specific MRI measures more generally available among neonatal facilities to target specific structures or regions of especial prognostic value. For example, could measures related to hippocampal structure and connections or to specific white matter maturational features be especially valuable for estimates of prognosis or for direction of areas of intervention?

Is There Value to Detect Dysmaturation by MRI at Three Months of Age?

Although understanding the neurobiology of dysmaturation by MRI at three months of age after HIE would be of major academic interest, could the information also lead to improved outcomes? I suggest that potentially it could lead to improved outcomes, for several reasons. First, neuro- restorative interventions could be instituted with force and direction. Interventions have been discussed in detail elsewhere^6,7 and will not be repeated here. In broad terms, the interventions include experiential factors (visual, auditory, pain, stress, early intervention programs, parenting-educational-social factors) and nutritional factors (breast-feeding; quality, source and components of milk, etc.).

Of potentially great importance, but not yet studied in human infants, are manipulations of microglia or astrocytes from a damaging pro-inflammatory to a neuroprotective/pro-repair, pro-development, anti-inflammatory phenotype.³⁷ This approach could minimize dysmaturational effects while restoring the important roles of these glial cells in promoting such normal developmental events as pre-OL development, axonal growth, synaptogenesis and synaptic pruning. In several experimental models, including hypoxic-ischemic injury, such agents as metformin, resveratrol, and pioglitazone have shown benefit for long-term outcomes via promotion of the M2 (anti-inflammatory) microglial phenotype.^46-49

A number of other agents show promise in this context. IGF-1 and EPO may have benefit for neuronal and pre-OL development.³⁷ Hyaluronidase inhibitors may be beneficial for pre-OL development.⁵⁰ A variety of stem cells has been shown beneficial for recovery from experimental stroke and related ischemic brain injuries in neonatal animals.^51,52 Intranasal delivery has been effective.⁵³ Exosomes derived from stem cells, also delivered intranasally, are similarly beneficial.⁵⁴ These issues are discussed in detail elsewhere (see, for examples,^37,40,55).

Conclusions

In this relatively brief Commentary, I have suggested (1) an explanation for the relatively disappointing predictive value of MRI in early infancy for prediction of long-term neurological outcome, (2) brain dysmaturation rather than simply tissue loss or atrophy is the principal mechanism for impaired outcomes, (3) highly advanced MRI techniques are capable of, and necessary for, identifying many of the dysmaturational deficits in the first months of life, and (4) early identification of such deficits could lead to both targeted and novel interventions to counteract or prevent such dysmaturation. The principal difficulty is that the needed highly advanced MRI techniques for in vivo detection are not available widely. In my view, carefully designed research that likely will require multiple institutions with the necessary MRI capabilities is needed. Supportive funding obviously is a major issue, and it is beyond the scope of this presentation to suggest the mechanisms. However, I hope that federal and nonfederal sources will accept this challenge.

Joseph J. Volpe, MD

Department of Neurology, Boston Children’s Hospital

Bronson Crothers Professor of Neurology, Emeritus, Harvard Medical School

Boston MA

References

1. Parmentier CEJ, Kropman T, Groenendaal F, et al.: Cranial MRI beyond the Neonatal Period and Neurodevelopmental Outcomes in Neonatal Encephalopathy Due to Perinatal Asphyxia: A Systematic Review. J Clin Med 12, 2023. DOI: 10.3390/jcm12247526
2. Sotelo E, Sharon D, Gagoski B, et al.: Insights from serial magnetic resonance imaging in neonatal encephalopathy in term infants. Pediatr Res, 2024. DOI: 10.1038/s41390-024-03258-5
3. Wu YW, Monsell SE, Glass HC, et al.: How well does neonatal neuroimaging correlate with neurodevelopmental outcomes in infants with hypoxic-ischemic encephalopathy? Pediatr Res 94:1018-25, 2023. DOI: 10.1038/s41390-023-02510-8
4. Parmentier CEJ, Lequin MH, Alderliesten T, et al.: Additional Value of 3-Month Cranial Magnetic Resonance Imaging in Infants with Neonatal Encephalopathy following Perinatal Asphyxia. J Pediatr 258:113402, 2023. DOI: 10.1016/j.jpeds.2023.113402
5. Volpe JJ: Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances. Lancet Neurol 8:110-24, 2009. DOI: 10.1016/S1474-4422(08)70294-1
6. 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
7. Inder TE, Volpe JJ, Anderson PJ: Defining the Neurologic Consequences of Preterm Birth. N Engl J Med 389:441-53, 2023. DOI: 10.1056/NEJMra2303347
8. Haynes RL, Borenstein NS, Desilva TM, et al.: Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 484:156-67, 2005. DOI: 10.1002/cne.20453
9. Brody BA, Kinney HC, Kloman AS, Gilles FH: Sequence of central nervous system myelination in human infancy. I. An autopsy study of myelination. J Neuropathol Exp Neurol 46:283-301, 1987. DOI: 10.1097/00005072-198705000-00005
10. Xu G, Broadbelt KG, Haynes RL, et al.: Late development of the GABAergic system in the human cerebral cortex and white matter. J Neuropathol Exp Neurol 70:841-58, 2011. DOI: 10.1097/NEN.0b013e31822f471c
11. Del Bigio M. Supratentorial Gray Matter and Disruptions. Chapter 32. In: Lechpammer M, Del Bigio M, Folkerth RD, editors. Perinatal Neuropathology. Cambridge UK: Cambridge University Press; 2021. p. 165-77.
12. Pierson CR, Volpe JJ. Hypoxic-Ischemic Encephalopathy in the Term Infant: Neuropathology. Chapter 22. In: Volpe JJ, Inder T, Darras BT, de Vries LS, du Plessis AJ, Ferriero DM, Perlman JM, editors. Volpe’s Neurology of the Newborn. 7th ed. Philadelphia PA: Elsevier; 2024. p. 619-31.
13. Martinez-Biarge M, Diez-Sebastian J, Rutherford MA, Cowan FM: Outcomes after central grey matter injury in term perinatal hypoxic-ischaemic encephalopathy. Early Hum Dev 86:675-82, 2010. DOI: 10.1016/j.earlhumdev.2010.08.013
14. Conel JL. Postnatal Development of the Human Cerebral Cortex. Vol I: The Cortex of the Newborn. Cambridge MA: Harvard University Press; 1939.
15. Conel JL. Postnatal Development of the Human Cerebral Cortex. Vol II: The Cortex of the One-Month Infant. Cambridge MA: Harvard University Press; 1941.
16. Conel JL. Postnatal Development of the Human Cerebral Cortex. Vol III: The Cortex of the Three-Month Infant. Cambridge MA: Harvard University Press; 1947.
17. Conel JL. The Postnatal Development of the Human Cerebral Cortex. Vol IV: The Cortex of the Six-Month Infant. Cambridge MA: Harvard University Press; 1951.
18. Shankle WR, Romney AK, Landing BH, Hara J: Developmental patterns in the cytoarchitecture of the human cerebral cortex from birth to 6 years examined by correspondence analysis. Proc Natl Acad Sci U S A 95:4023-8, 1998. DOI: 10.1073/pnas.95.7.4023
19. Huttenlocher PR: Morphometric study of human cerebral cortex development. Neuropsychologia 28:517-27, 1990. DOI: 10.1016/0028-3932(90)90031-i
20. Rakic P, Bourgeois JP, Goldman-Rakic PS: Synaptic development of the cerebral cortex: implications for learning, memory, and mental illness. Prog Brain Res 102:227-43, 1994. DOI: 10.1016/S0079-6123(08)60543-9
21. Huttenlocher PR, Dabholkar AS: Regional differences in synaptogenesis in human cerebral cortex. J Comp Neurol 387:167-78, 1997. DOI: 10.1002/(sici)1096-9861(19971020)387:2<167::aid-cne1>3.0.co;2-z
22. D’Gama AM, Poduri AH, Volpe JJ. Neuronal Migration. Chapter 6. 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; 2024. p. 142-65.
23. Wang DD, Kriegstein AR: Defining the role of GABA in cortical development. J Physiol 587:1873-9, 2009. DOI: 10.1113/jphysiol.2008.167635
24. Schwartz ML, Meinecke DL: Early expression of GABA-containing neurons in the prefrontal and visual cortices of rhesus monkeys. Cereb Cortex 2:16-37, 1992. DOI: 10.1093/cercor/2.1.16
25. Hensch TK: Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6:877-88, 2005. DOI: 10.1038/nrn1787
26. Mallard EC, Gunn AJ, Williams CE, et al.: Transient umbilical cord occlusion causes hippocampal damage in the fetal sheep. Am J Obstet Gynecol 167:1423-30, 1992. DOI: 10.1016/s0002-9378(11)91728-1
27. Gunn AJ, Parer JT, Mallard EC, et al.: Cerebral histologic and electrocorticographic changes after asphyxia in fetal sheep. Pediatr Res 31:486-91, 1992. DOI: 10.1203/00006450-199205000-00016
28. White TA, Miller SL, Sutherland AE, et al.: Perinatal compromise affects development, form, and function of the hippocampus part two; preclinical studies. Pediatr Res 95:1709-19, 2024. DOI: 10.1038/s41390-024-03144-0
29. White TA, Miller SL, Sutherland AE, et al.: Perinatal compromise affects development, form, and function of the hippocampus part one; clinical studies. Pediatr Res 95:1698-708, 2024. DOI: 10.1038/s41390-024-03105-7
30. Annink KV, de Vries LS, Groenendaal F, et al.: Mammillary body atrophy and other MRI correlates of school-age outcome following neonatal hypoxic-ischemic encephalopathy. Sci Rep 11:5017, 2021. DOI: 10.1038/s41598-021-83982-8
31. Annink KV, de Vries LS, Groenendaal F, et al.: The long-term effect of perinatal asphyxia on hippocampal volumes. Pediatr Res 85:43-9, 2019. DOI: 10.1038/s41390-018-0115-8
32. Pfister KM, Stoyell SM, Miller ZR, et al.: Reduced Hippocampal Volumes in Children with History of Hypoxic Ischemic Encephalopathy after Therapeutic Hypothermia. Children (Basel) 10, 2023. DOI: 10.3390/children10061005
33. Molavi M, Vann SD, de Vries LS, et al.: Signal Change in the Mammillary Bodies after Perinatal Asphyxia. AJNR Am J Neuroradiol 40:1829-34, 2019. DOI: 10.3174/ajnr.A6232
34. McClendon E, Wang K, Degener-O’Brien K, et al.: Transient Hypoxemia Disrupts Anatomical and Functional Maturation of Preterm Fetal Ovine CA1 Pyramidal Neurons. J Neurosci 39:7853-71, 2019. DOI: 10.1523/JNEUROSCI.1364-19.2019
35. Byrne P, Welch R, Johnson MA, et al.: Serial magnetic resonance imaging in neonatal hypoxic-ischemic encephalopathy. J Pediatr 117:694-700, 1990. DOI: 10.1016/s0022-3476(05)83323-2
36. Mulkey SB, Yap VL, Swearingen CJ, et al.: Quantitative cranial magnetic resonance imaging in neonatal hypoxic-ischemic encephalopathy. Pediatr Neurol 47:101-8, 2012. DOI: 10.1016/j.pediatrneurol.2012.05.009
37. 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
38. Gonzalez FF, Ferriero DM, Volpe JJ. Hypoxic-Ischemic Injury in the Term Infant: Pathophysiology. Chapter 23. 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; 2024. p. 632-42.
39. Fleiss B, Gressens P: Tertiary mechanisms of brain damage: a new hope for treatment of cerebral palsy? Lancet Neurol 11:556-66, 2012. DOI: 10.1016/S1474-4422(12)70058-3
40. Fleiss B, Van Steenwinckel J, Bokobza C, et al.: Microglia-Mediated Neurodegeneration in Perinatal Brain Injuries. Biomolecules 11, 2021. DOI: 10.3390/biom11010099
41. Liddelow SA, Barres BA: Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity 46:957-67, 2017. DOI: 10.1016/j.immuni.2017.06.006
42. Liddelow SA, Guttenplan KA, Clarke LE, et al.: Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541:481-7, 2017. DOI: 10.1038/nature21029
43. Hammond TR, Robinton D, Stevens B: Microglia and the Brain: Complementary Partners in Development and Disease. Annu Rev Cell Dev Biol 34:523-44, 2018. DOI: 10.1146/annurev-cellbio-100616-060509
44. Kelly CE, Thompson DK, Adamson CL, et al.: Cortical growth from infancy to adolescence in preterm and term-born children. Brain 147:1526-38, 2024. DOI: 10.1093/brain/awad348
45. Kelly CE, Shaul M, Thompson DK, et al.: Long-lasting effects of very preterm birth on brain structure in adulthood: A systematic review and meta-analysis. Neurosci Biobehav Rev 147:105082, 2023. DOI: 10.1016/j.neubiorev.2023.105082
46. Zhao Q, Wang Q, Wang J, et al.: Maternal immune activation-induced PPARgamma-dependent dysfunction of microglia associated with neurogenic impairment and aberrant postnatal behaviors in offspring. Neurobiol Dis 125:1-13, 2019. DOI: 10.1016/j.nbd.2019.01.005
47. Jin Q, Cheng J, Liu Y, et al.: Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain Behav Immun 40:131-42, 2014. DOI: 10.1016/j.bbi.2014.03.003
48. Mandrekar-Colucci S, Karlo JC, Landreth GE: Mechanisms underlying the rapid peroxisome proliferator-activated receptor-gamma-mediated amyloid clearance and reversal of cognitive deficits in a murine model of Alzheimer’s disease. J Neurosci 32:10117-28, 2012. DOI: 10.1523/JNEUROSCI.5268-11.2012
49. Karalis F, Soubasi V, Georgiou T, et al.: Resveratrol ameliorates hypoxia/ischemia-induced behavioral deficits and brain injury in the neonatal rat brain. Brain Res 1425:98-110, 2011. DOI: 10.1016/j.brainres.2011.09.044
50. Buser JR, Maire J, Riddle A, et al.: Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants. Ann Neurol 71:93-109, 2012. DOI: 10.1002/ana.22627
51. van Velthoven CT, Sheldon RA, Kavelaars A, et al.: Mesenchymal stem cell transplantation attenuates brain injury after neonatal stroke. Stroke 44:1426-32, 2013. DOI: 10.1161/STROKEAHA.111.000326
52. van Velthoven CT, Gonzalez F, Vexler ZS, Ferriero DM: Stem cells for neonatal stroke- the future is here. Front Cell Neurosci 8:207, 2014. DOI: 10.3389/fncel.2014.00207
53. Oppliger B, Joerger-Messerli M, Mueller M, et al.: Intranasal Delivery of Umbilical Cord-Derived Mesenchymal Stem Cells Preserves Myelination in Perinatal Brain Damage. Stem Cells Dev 25:1234-42, 2016. DOI: 10.1089/scd.2016.0027
54. Thomi G, Joerger-Messerli M, Haesler V, et al.: Intranasally Administered Exosomes from Umbilical Cord Stem Cells Have Preventive Neuroprotective Effects and Contribute to Functional Recovery after Perinatal Brain Injury. Cells 8, 2019. DOI: 10.3390/cells8080855
55. Gressens P. Inflammation and the Newborn Brain. 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; 2024. p. 494-505.

July, 2024

BRAIN DYSMATURATION AFTER VERY PRETERM BIRTH: A PROGRESSIVE AND LONG-TERM CHALLENGE

Introduction

This Commentary was stimulated by a recent report published in April in Brain.¹ The report addresses cerebral cortical growth in very preterm (VPT) and term born (TB) children followed to adolescence and studied by advanced MRI techniques. A major strength of the study is its standing as the first longitudinal study of the same population of VPT and TB individuals from birth to adolescence. The work is notable for insights into normal cortical development in TB infants, disturbances of this development in VPT infants, and the course of these disturbances through childhood and adolescence. The data raise important questions concerning the cellular and mechanistic bases for the cortical disturbances and, critically, concerning the long-term neurological implications for survivors of VPT birth.

Cerebral Cortical Development from Birth to Adolescence in VPT and TB Infants

This unique study (the Victorian Infant Brain Study) concerned infants born between 2001-2004 and included 201 infants born VPT (<30 weeks gestation or <1250 g birth weight) and 66 TB infants. The VPT infants had “no major neonatal brain injuries.” Advanced MRI methodologies allowed assessments of cerebral cortical volume, area and thickness for 62 cortical regions at term, 7 years, and 13 years.

Cerebral Cortical Development in TB Infants

In TB infants, from term to 7 years, developmental changes in 62 cortical regions consisted, generally, of large increases in cortical volume, area and thickness (375%, 147%, and 78%, respectively). However, notably, from 7 years to 13 years, generally only minor increases or slight decreases were found. It is beyond the scope of this Commentary to discuss regional differences in detail, but the general theme of pronounced increases to mid childhood and subsequent stabilization or slight decreases to adolescence is apparent.

The anatomical correlates of these cortical developmental changes are not entirely known, but for the increases during later infancy and early childhood, available data indicate importance for pronounced axonal input to cortex, cortical dendritic development, synaptogenesis, and intracortical (and likely immediate subcortical) myelination.^2-6 A detailed study of human parietal cerebrum with markers of axonal growth and elongation (GAP-43) and axonal maturity (Anti-SMI31) indicates rapid axonal growth in white matter during the preterm period, and in cerebral cortex in the first two years of life.² Presumably this axonal input into cortex leads to activity-dependent development of cortical dendritic arbors.^3,7,8 The classic studies of Huttenlocher and Rakic show that this axonal-dendritic development is followed in cortex by synaptogenesis, with an excess of synapses apparent in mid-childhood in multiple cortical areas.^9-12 Later in childhood and into adolescence, synapse elimination is active, and this pruning may be important in the plateau (and the modest decline) in cortical volume and thickness observed by MRI by Kelly et al.¹ However, anatomical studies show that myelination of the cortical neuropil and subcortical association areas also becomes prominent in late infancy and early childhood and continues beyond adolescence.^13-16 This process likely contributes to the increase in both cortical volume and thickness observed by MRI into childhood.¹

Cerebral Cortical Development in VPT Infants

            As previously described,^17,18 at term-equivalent age VPT infants already exhibited abnormalities of cerebral cortical development, especially lower cortical volume and area.¹ From 0-7 years, the volume differences between VPT and TB infants became more pronounced, especially in higher-order frontal, temporal and parietal regions. Earlier work by the same group, utilizing a voxel-based (rather than surface-based) approach, had shown that the lower brain volume in VPT infants becomes more exaggerated between ages 0-7 years, particularly in temporal regions.^19,20 The current approach, which allowed fractionation of cortical volume, provided the new insight that the increasingly lower volume in temporal regions in the childhood years is related principally to increasingly lower cortical thickness. Accompanying the latter was persistently reduced cortical area. By the age 13 measurements, values in VPT infants for volumes, area and thickness had nearly plateaued and were persistently lower than in TB infants. No evidence of catch-up was apparent, and indeed pronounced differences between VPT and TB cortical regions persisted. These findings may have major implications for longer-term follow-up (see later).

            The anatomical correlates and cellular mechanisms underlying the apparent impairment in cortical development in survivors of VPT birth remain to be elucidated. However, a superb earlier study by the same group provides excellent clues.²¹ Assessing the same cohort as in the current cortical study, Kelly et al. utilized advanced diffusion MRI analyses, i.e., fixel-based analysis (FBA), to evaluate the long-term development of white matter microstructure. The principal findings were that at ages 7 and 13 VPT children, when compared to TB children, exhibited axonal reductions in many white matter fiber tracts and slower axonal growth in several areas. Degenerative axonal changes have been documented in VP infants with cerebral white matter injury,² and in the study of Kelly et al., white matter abnormalities were modestly associated with the axonal deficits.²¹ Notably, however, impairments of axonal development and function have been shown to occur with even milder white matter abnormalities, not readily visible by early-life MRI.¹⁷ Such impairment is likely related to the principal cellular abnormality in cerebral white matter injury, i.e., impaired pre-oligodendrocyte (pre-OL) differentiation with failure of oligodendroglial ensheathment of axons.¹⁷ This failure would be expected to lead to impaired axonal function, and thereby impaired activity-dependent cerebral cortical differentiation, as described in the previous section. Similarly, impaired intracortical myelination, a process that occurs over many years in childhood, adolescence and adulthood,¹³ would be affected.

            The possibility that impaired cortical neuronal development, as delineated in the study of Kelly et al.,¹ represents a primary neuronal dysmaturation, rather than a dysmaturation secondary to altered axonal and oligodendroglial-myelin development as just outlined, needs consideration. An excellent clinical study, involving 95 VPT infants studied by diffusion-based MRI at two time points (32 and 40 weeks post-conceptional age), provided evidence for delayed microstructural development of cerebral cortex (in association with impaired somatic growth), but cerebral white matter appeared unaffected.²² Similarly consistent with primary neuronal dysmaturation, two careful experimental studies, utilizing a well characterized fetal sheep model, showed disturbances in cortex, in dendritic development and synapse formation, and in subplate neurons, in dendritic arborization and synaptic activity, four weeks after a hypoxic-ischemic insult.^23,24 Available data in the population studied by Kelly et al.¹ suggest that such insults were not likely in their population. A later investigation in the same animal model showed that even a brief hypoxic episode led four weeks later to disturbances of basal and apical dendritic arborization of hippocampal CA1 neurons and impaired connectivity.²⁵ (Such brief hypoxic events, common in VPT infants, likely would not have been detected or quantitated in the study of Kelly et al.¹) Importantly, in the animal model the neurophysiological correlate of memory formation, a hallmark of hippocampal function, was impaired in hippocampus.²⁵ Notably, disturbances of working memory have been documented in survivors of VPT birth.²⁶

Long-Term Implications of Disturbed Cerebral Cortical Development in VPT Infants

The current study raises the question of the impact of the magnitude and evolution of the cortical structural deficits found by age 13 in VPT infants by Kelly et al.¹ on later neurological outcomes. Clearly the latter study will be at the vanguard of this quest. Current information raises interesting and concerning questions.

Impact of Cerebral Cortical Disturbance on Childhood Neurological Outcomes

As noted in the study of Kelly et al.,¹ alterations in frontal and temporal regions are prominent in the VPT infants at childhood age. Previous work has shown that morphological alterations in these regions in VPT children are associated with lower IQ and deficits in language and executive function.^27-29 Notably, the hippocampus, an important component of temporal cortex, has been shown to be smaller, straighter and with less infolding in adolescents and young adults after VPT birth.^29-31 The findings were related to memory deficits.

Impact of Cerebral Cortical Disturbance on Adult Neurological Outcomes

The possible relation of the cortical disturbances in survivors of VPT birth, especially involving temporal cortex, to still later neurological outcomes should be considered. Thus, Heinonen et al.³² studied 919 Finnish men and women born between 1934 and 1944 as late preterms (34 to 36-6/7 weeks gestational age) and evaluated at 68.1 years of age. Among those who had attained a basic or upper secondary education, late preterm birth was associated with lower scores on multiple cognitive sub-tests and a 2.70 times higher risk of mild cognitive impairment. Among those with tertiary levels of education, late preterm birth was not associated with such deficits. The authors concluded that late preterm birth should be added as “a novel risk factor to the list of neurocognitive impairment in late adulthood.” They also raise the possibility that “lifetime education may mitigate aging-related neurocognitive impairment among those born late preterm.” Although a number of questions could be raised about the study, the findings are noteworthy.

A later MRI study³³ of 260 VPT and 229 TB individuals at age 26 years showed MRI features of increased “brain age” in the VPT individuals. Details are beyond the scope of this Commentary, but the morphological difference is interesting. Additionally noteworthy, in older individuals a relationship between increased brain age and dementia has been observed. One potential importance of this observation in this context is that aggregation of tau protein in neurons, a key feature in Alzheimer’s disease, begins in peripheral dendrites and thereby might reach the cell body more rapidly in underdeveloped dendrites, as described earlier in the studies of VPT infants.³³

Consistent with these reports, a study of 70 VPT and 67 TB adults identified aberrant connectivity between thalamus and temporal and prefrontal cortices.³⁴ Impairments in verbal cognitive abilities were also observed in the PT adults.

Another study focused on the claustrum, a structure critical in a variety of cognitive functions, in 70 VPT adults and 87 TB adults and found increased diffusivity in the former individuals. Reduced IQ also was observed in the VPT adults.³⁵

Finally, a large literature has focused on the potential relationship between early life stresses and risk of Alzheimer’s disease (see, for review,³⁶). VPT birth is not discussed separately, but many of the “stresses” considered important are characteristic of the premature period and subsequent infancy-childhood. Of particular note, as noted earlier, the cortical areas particularly characterized by aberrant development in VPT infants, children and adolescents, i.e., temporal cortex, hippocampus, are the areas initially and particularly affected by the neuropathology of Alzheimer’s dementia.

Potentially Additive or Potentiating Factors in Long-Term Neurological Outcome in VPT Infants

            Although the previous sections have emphasized the long-term implications of the disturbances in cerebral cortical development in VPT children and adolescents, certain non-neurological factors may enhance the possibility of subsequent impairments in neurological outcome. These factors concern cardiac function and cerebrovascular disease. A recent review has focused on factors associated with preterm birth, such as reduced cardiac reserve, smaller left and right ventricular volumes, decreased vascularity, increased vascular stiffness and higher pressure of both the pulmonary and systemic vasculatures.³⁷ In a nationwide Swedish study that included 73,489 adults who were born <37 weeks gestation,³⁸ among those born before 32 weeks gestation, a nearly two-fold increased risk of cerebrovascular disease was observed compared to TB individuals. In a more recent report, also from Sweden, among individuals at ages 18-43 years, when compared with TB infants, the adjusted hazard ratio for stroke associated with preterm birth (<37 weeks gestation) was 1.26 (95% C.I., 1.12-1.43, P<0.001) and when further stratified, was 1.42 (1.11-1.85, P=0.005) for early preterm (22-33 weeks) and 1.22 (1.06-1.40, P=0.004) for late preterm (34-36 weeks).³⁹

Conclusions

The recent report of Kelly et al.¹ establishes broad and important points concerning cerebral cortical development in survivors of VPT birth. First, relative to TB infants, cortical development is abnormal, not only at term (as previously reported many times), but progressively so subsequently, at least into late childhood and adolescence, and perhaps beyond. Second, the cellular mechanisms underlying the apparent progression of dysmaturation, although not fully known, appear to reflect a cascade of developmental disturbances, with abnormalities of white matter axons and immature oligodendrocytes likely important at the onset. Additionally, data from other studies suggest that the cortical dysmaturational events may render the brain of the VPT infant more susceptible to later neurodegenerative disorders. Finally, abnormalities of cerebrovascular development may aggravate or render the VPT brain vulnerable to later degenerative or destructive effects.

How to prevent or ameliorate this cascade of dysmaturation and disability? It is beyond the scope of this Commentary to address this key question. However, the potential for beneficial intervention is great, especially in early childhood years, before pronounced deficits are established. For lack of a better word, I have termed these “neurorestorative interventions.”¹⁷ Many can be established readily in the neonatal period, such as minimization of pain and stress, provision of optimal nutrition (breast feeding, optimal quality and source of milk, etc.), attention to experiential factors (relating to auditory, verbal and visual input), parenting, family-based interventions, socioeconomic factors and later, educational opportunities.²⁹

Finally, in view of the life-long course of neurologic and related challenges, interventions need to be continued through adult years. With the increasing recognition of the importance of lifestyle changes and perhaps specific drugs to prevent later neurodegenerative disorders,^40,41 caregivers beyond the neonatal and pediatric years should be made aware of the long-term challenges potentially faced by survivors of preterm birth. In my view, it behooves those of us who focus on newborn brain disease to alert pediatricians and later caregivers concerning the potential health hazards that survivors of VPT birth may face.

Joseph J. Volpe, MD

Department of Neurology, Boston Children’s Hospital

Bronson Crothers Professor of Neurology, Emeritus, Harvard Medical School

Boston MA

References

1. Kelly CE, Thompson DK, Adamson CL, et al.: Cortical growth from infancy to adolescence in preterm and term-born children. Brain 147:1526-38, 2024. DOI: 10.1093/brain/awad348
2. Haynes RL, Borenstein NS, Desilva TM, et al.: Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 484:156-67, 2005. DOI: 10.1002/cne.20453
3. Haynes RL, Kinney HC, Volpe JJ. Organizational events. Chapter 7. 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.
4. Marin-Padilla M: Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: a unifying theory. J Comp Neurol 321:223-40, 1992. DOI: 10.1002/cne.903210205
5. Marin-Padilla M: Prenatal and early postnatal ontogenesis of the human motor cortex: a golgi study. I. The sequential development of the cortical layers. Brain Res 23:167-83, 1970. DOI: 10.1016/0006-8993(70)90037-5
6. Haynes RL, Kinney HC, Volpe JJ. Myelination events. Chapter 8. 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.
7. Yap EL, Greenberg ME: Activity-Regulated Transcription: Bridging the Gap between Neural Activity and Behavior. Neuron 100:330-48, 2018. DOI: 10.1016/j.neuron.2018.10.013
8. Goodman CS, Shatz CJ: Developmental mechanisms that generate precise patterns of neuronal connectivity. Cell 72 Suppl:77-98, 1993. DOI: 10.1016/s0092-8674(05)80030-3
9. Huttenlocher PR: Synaptic density in human frontal cortex – developmental changes and effects of aging. Brain Res 163:195-205, 1979. DOI: 10.1016/0006-8993(79)90349-4
10. Rakic P, Goldman-Rakic PS: The development and modifiability of the cerebral cortex. Overview. Neurosci Res Program Bull 20:433-8, 1982.
11. Bourgeois JP, Goldman-Rakic PS, Rakic P: Synaptogenesis in the prefrontal cortex of rhesus monkeys. Cereb Cortex 4:78-96, 1994. DOI: 10.1093/cercor/4.1.78
12. Huttenlocher PR: Morphometric study of human cerebral cortex development. Neuropsychologia 28:517-27, 1990. DOI: 10.1016/0028-3932(90)90031-i
13. Yakovlev PI, Lecours AR. The myelogenetic cycles of regional maturation of the brain. In: Minkowski A, editor. Regional Development of the Brain in Early Life. Oxford: Blackwell Scientific; 1967. p. 3-70.
14. Brody BA, Kinney HC, Kloman AS, Gilles FH: Sequence of central nervous system myelination in human infancy. I. An autopsy study of myelination. J Neuropathol Exp Neurol 46:283-301, 1987. DOI: 10.1097/00005072-198705000-00005
15. Kinney HC, Brody BA, Kloman AS, Gilles FH: Sequence of central nervous system myelination in human infancy. II. Patterns of myelination in autopsied infants. J Neuropathol Exp Neurol 47:217-34, 1988. DOI: 10.1097/00005072-198805000-00003
16. Miller DJ, Duka T, Stimpson CD, et al.: Prolonged myelination in human neocortical evolution. Proc Natl Acad Sci U S A 109:16480-5, 2012. DOI: 10.1073/pnas.1117943109
17. 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
18. 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.
19. Monson BB, Anderson PJ, Matthews LG, et al.: Examination of the Pattern of Growth of Cerebral Tissue Volumes From Hospital Discharge to Early Childhood in Very Preterm Infants. JAMA Pediatr 170:772-9, 2016. DOI: 10.1001/jamapediatrics.2016.0781
20. Thompson DK, Matthews LG, Alexander B, et al.: Tracking regional brain growth up to age 13 in children born term and very preterm. Nat Commun 11:696, 2020. DOI: 10.1038/s41467-020-14334-9
21. Kelly CE, Thompson DK, Genc S, et al.: Long-term development of white matter fibre density and morphology up to 13 years after preterm birth: A fixel-based analysis. Neuroimage 220:117068, 2020. DOI: 10.1016/j.neuroimage.2020.117068
22. Vinall J, Grunau RE, Brant R, et al.: Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns. Sci Transl Med 5:168ra8, 2013. DOI: 10.1126/scitranslmed.3004666
23. Dean JM, McClendon E, Hansen K, et al.: Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization. Sci Transl Med 5:168ra7, 2013. DOI: 10.1126/scitranslmed.3004669
24. McClendon E, Shaver DC, Degener-O’Brien K, et al.: Transient Hypoxemia Chronically Disrupts Maturation of Preterm Fetal Ovine Subplate Neuron Arborization and Activity. J Neurosci 37:11912-29, 2017. DOI: 10.1523/JNEUROSCI.2396-17.2017
25. McClendon E, Wang K, Degener-O’Brien K, et al.: Transient Hypoxemia Disrupts Anatomical and Functional Maturation of Preterm Fetal Ovine CA1 Pyramidal Neurons. J Neurosci 39:7853-71, 2019. DOI: 10.1523/JNEUROSCI.1364-19.2019
26. Nosarti C, Froudist-Walsh S: Alterations in development of hippocampal and cortical memory mechanisms following very preterm birth. Dev Med Child Neurol 58 Suppl 4:35-45, 2016. DOI: 10.1111/dmcn.13042
27. Nosarti C, Nam KW, Walshe M, et al.: Preterm birth and structural brain alterations in early adulthood. Neuroimage Clin 6:180-91, 2014. DOI: 10.1016/j.nicl.2014.08.005
28. Thompson DK, Omizzolo C, Adamson C, et al.: Longitudinal growth and morphology of the hippocampus through childhood: Impact of prematurity and implications for memory and learning. Hum Brain Mapp 35:4129-39, 2014. DOI: 10.1002/hbm.22464
29. Inder TE, Volpe JJ, Anderson PJ: Defining the Neurologic Consequences of Preterm Birth. N Engl J Med 389:441-53, 2023. DOI: 10.1056/NEJMra2303347
30. Aanes S, Bjuland KJ, Skranes J, Lohaugen GC: Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults. Neuroimage 105:76-83, 2015. DOI: 10.1016/j.neuroimage.2014.10.023
31. Gimenez M, Junque C, Narberhaus A, et al.: Hippocampal gray matter reduction associates with memory deficits in adolescents with history of prematurity. Neuroimage 23:869-77, 2004. DOI: 10.1016/j.neuroimage.2004.07.029
32. Heinonen K, Eriksson JG, Lahti J, et al.: Late preterm birth and neurocognitive performance in late adulthood: a birth cohort study. Pediatrics 135:e818-25, 2015. DOI: 10.1542/peds.2014-3556
33. Hedderich DM, Menegaux A, Schmitz-Koep B, et al.: Increased Brain Age Gap Estimate (BrainAGE) in Young Adults After Premature Birth. Front Aging Neurosci 13:653365, 2021. DOI: 10.3389/fnagi.2021.653365
34. Menegaux A, Meng C, Bauml JG, et al.: Aberrant cortico-thalamic structural connectivity in premature-born adults. Cortex 141:347-62, 2021. DOI: 10.1016/j.cortex.2021.04.009
35. Hedderich DM, Menegaux A, Li H, et al.: Aberrant Claustrum Microstructure in Humans after Premature Birth. Cereb Cortex 31:5549-59, 2021. DOI: 10.1093/cercor/bhab178
36. Hoeijmakers L, Lesuis SL, Krugers H, et al.: A preclinical perspective on the enhanced vulnerability to Alzheimer’s disease after early-life stress. Neurobiol Stress 8:172-85, 2018. DOI: 10.1016/j.ynstr.2018.02.003
37. Lewandowski AJ, Levy PT, Bates ML, et al.: Impact of the Vulnerable Preterm Heart and Circulation on Adult Cardiovascular Disease Risk. Hypertension 76:1028-37, 2020. DOI: 10.1161/HYPERTENSIONAHA.120.15574
38. Ueda P, Cnattingius S, Stephansson O, et al.: Cerebrovascular and ischemic heart disease in young adults born preterm: a population-based Swedish cohort study. Eur J Epidemiol 29:253-60, 2014. DOI: 10.1007/s10654-014-9892-5
39. Crump C, Sundquist J, Sundquist K: Stroke Risks in Adult Survivors of Preterm Birth: National Cohort and Cosibling Study. Stroke 52:2609-17, 2021. DOI: 10.1161/STROKEAHA.120.033797
40. Gonzalez-Madrid A, Calfio C, Gonzalez A, et al.: Toward Prevention and Reduction of Alzheimer’s Disease. J Alzheimers Dis 96:439-57, 2023. DOI: 10.3233/JAD-230454
41. Sperling RA, Donohue MC, Raman R, et al.: Trial of Solanezumab in Preclinical Alzheimer’s Disease. N Engl J Med 389:1096-107, 2023. DOI: 10.1056/NEJMoa2305032

May, 2024

MUSIC EXPOSURE IN PRETERM INFANTS LEADS TO ENHANCED CEREBRAL CORTICAL AND WHITE MATTER DEVELOPMENT

Introduction

This Commentary was stimulated by a recent report in Developmental Cognitive Neuroscience, entitled “Music impacts brain cortical microstructural maturation in very preterm infants: A longitudinal diffusion MR imaging study.”¹ The work provides important insight into (a) normal development of the brain in the very preterm infant from 33 to 40 weeks postconceptional age, and (b) the effect of a music intervention during that period on this development. The findings suggest particular value for music exposure in very preterm infants. Although this Commentary focuses on the current report, the study is the most recent of a superb series from the same group focused on the effect of a music intervention in very preterm infants on subsequent structural and functional (connectivity) brain development and behavioral outcomes.^2-5

Music Intervention—Study Design

The sample of very preterm infants (mean gestational age 29 weeks) with no overt brain lesions was allocated randomly to a control group (n=19) (no music intervention) or a music intervention group (n=21). MRI (3.0T scanner) was performed during the 33^rd week gestational age (GA) and at term equivalent age (TEA). The MRI assessments involved advanced diffusion-based methodologies, i.e., fixel-based analysis (FBA) and neurite orientation dispersion and density imagery (NODDI). FBA provides insight into micro- and macrostructural changes, including such measures as fiber cross-section (FC) and fiber density (FD). NODDI provides microstructural information and, as the name implies, data relevant to neurite density and orientation dispersion.¹

The music intervention was carried out two times a day from 33 weeks GA to TEA. The infants were exposed to the music through headphones. (Infants in the no music intervention group were exposed similarly to headphones.) The music was composed of “a calming background, bells, harp, and punji (charming snake flute) interactively creating a melody” of 8 minutes duration. Three different music tracks were available for use, and the track utilized for each exposure was chosen “by the nurse according to the state of wakefulness of the child (waking up, falling asleep, being alert).”¹ More detailed assessment of sleep state was not carried out.

The large corpus of data obtained in this report is reviewed best according to insights into brain development during the study period and the effects of the music intervention on this development. To maintain the relative brevity of the Commentary, I will review each of these concisely next.

Brain Developmental Changes

The advanced MRI measures provided important insight into normal development of multiple structures from the 33^rd week GA to TEA. For the sake of convenience, I will divide the structures broadly into cerebral white matter and cerebral cortical gray matter. The imaging features will be correlated with current structural concepts based on direct anatomical studies, the gold standard for assessments of brain development.

Cerebral White Matter

In cerebral white matter, from the 33^rd week GA to TEA, prominent increases in FC and FD were observed and are likely indicative of increases in axonal fiber organization, fiber bundle cross-section, axonal coherence and axonal number.¹ These indicators of axonal growth and development are consistent with anatomical studies, including studies utilizing GAP-43, a marker of growing axons, and other markers.^6-10 Thus, Haynes et al.¹⁰ showed abundant expression of GAP-43 in cerebral white matter during this period, as well as expression in cortex, presumably marking the influx of growing axons into overlying cortex at this time.

The major white matter fibers defined by Sa de Almeida et al. included thalamic, commissural, association, and projection fibers.¹ From 33 weeks GA to TEA, the increase in FC and FD observed in thalamic regions is likely a correlate of the well-established growth and elaboration of thalamo-cortical axons^7,8,11 occurring particularly in cerebral white matter during this period, as the subplate layer gradually decreases. Also at this time, anatomical studies demonstrate that commissural (callosal), commissural-cortical and corticocortical (association fibers) axons depart the transient subplate and enter cerebral cortex.^7,8 Finally, the finding that the most pronounced increases in FD and FC involve central projection fibers (e.g., corona radiata) is consistent with the later myelination order of Kinney (see later) of projection fibers before association fibers.^12,13

As will be discussed later, it is possible that the onset of axonal ensheathment by pre-myelinating oligodendrocytes (OLs) accounts for some of the MRI findings. The O4+, O1+ immature OLs that begin this ensheathment are abundant in cerebral white matter as term approaches.¹⁴ Indeed, MBP staining of myelin sheaths was demonstrated in periventricular white matter by Back et al.¹⁴ as early as 30 weeks GA. However, microscopic myelin, stained by conventional markers (e.g., Luxol Fast Blue), is not detected in cerebral white matter tracts until many weeks or months post-term.^12,15 Exceptions to this statement are the central corona radiata and the posterior limb of the internal capsule.

Cerebral Cortical Gray Matter

Important developmental changes were identified by MRI in cortical gray matter.¹ FC increased, as it did in white matter, but FD decreased. The findings are most consistent with a decline in the “simple” radial orientation of earlier cortex (created by radially orientated neurons with limited dendritic arbors, and to a lesser extent, by radial glia) and an increase in complexity related to dendritic growth and arborization, influx of axonal ramifications, loss of radial glial fibers with conversion to astrocytes, among other events.⁷ The findings are reminiscent of the decrease in fractional anisotropy previously shown by DTI.¹⁶ The NODDI findings support and expand the conclusion that the changes likely are related to dendritic arborization and disruption of the radial glia scaffold.¹ Taken together, the two MRI approaches, FBA and NODDI, provided considerable insight into cortical development from 33-40 weeks GA.

Additionally, Sa de Almeida et al. also consider the cortical gray matter changes as indicative of “intra-cortical myelination” (italics mine).¹ This conclusion is the only one in this fine study that I consider problematic. Thus, exhaustive studies of myelination in the human fetus and term infant do not show intracortical myelin at 40 weeks post-conceptional age and overall indicate that myelination within cortex occurs principally over the ensuing months and years post-term.^12,17 However, such traditional myelin stains require 7-10 lamellae for detection.^13,15,17,18 It could be argued that the MRI data might reflect early ensheathment of axons in cortex by O4+, O1+ immature (pre-myelinating) OLs (which would not stain with traditional myelin stains). Although these cells constitute 50% of the oligodendroglial lineage in periventricular white matter at TEA, they are not present, at least in parietal cortex, at this time.^14,19 Thus, sufficient ensheathment by immature Ols to be detected by the myelin stains used by Kinney et al.^12,13 and Yakovlev and Lecours¹⁷ would not be expected. Moreover, as noted earlier, the sensitive means of detection of myelin, i.e., MBP staining, shows such staining in periventricular white matter and, to a lesser extent, more superficial white matter in human cerebrum at TEA,¹⁴ but not in parietal or frontal cortex. Perhaps part of the difficulty in interpreting “myelin” indicators by diffusion-based MRI relates to the very shallow depth of cortex at this time (i.e., only approximately 1.5 mm).²⁰ Is it possible that the MRI findings in the study of Sa de Almeida et al.¹ are related to early ensheathment of axons in subcortical association fibers? However, again, I am unaware of clear anatomical evidence for such an occurrence. Thus, at present, the conclusion that “intracortical myelination” was enhanced does not seem plausible to me. This conclusion does not detract from the predominance of new and important findings in this report.

Effects of Early Music Intervention from 33 weeks GA on Brain Maturation Assessed at TEA

The sophisticated MRI assessments at TEA indicate that the music intervention leads to enhanced maturation of both white matter and gray matter structures. Thus, this study and previous work from this group showed enhanced development of several white matter tracts² consistent with enhanced axonal growth/organization, and, potentially, oligodendroglial differentiation. This finding is consistent with the well-established relation between axonal activity and oligodendroglial differentiation.^6,21 These findings likely underlie, at least in part, the enhanced functional connectivity previously shown with the music intervention.³

The current study indicates that the music intervention also enhanced the maturation of several cortical gray matter regions, especially those involved in auditory, cognitive and socio-emotional processing.¹ Coupled with the findings from the NODDI data, the data are consistent with increased dendritic arborization and remodeling of axonal connections leading to enhancement of the overall complexity of the cortical neuropil. Sa de Almeida et al.¹ use the term cortical “complexification.” As just noted, the cortical changes are especially apparent in regions (e.g., insulo-orbito-temporopolar complex) which have been related to later socio-emotional deficits commonly observed in follow-up of very preterm infants.¹ Notably, earlier work from this group showed that a music intervention during the NICU period was associated with improved “fear-reactivity scores at 12 months and anger-reactivity scores at 24 months,” suggesting a beneficial effect on later socio-emotional development.⁵

Also relevant to the enhancement of insular-orbito-temporal connections in these preterm infants are earlier findings in adults that music listening has been shown to activate these cortical regions,²² that auditory cortex is functionally connected to these regions when musical stimuli elicit emotions,^1,23 and that music training may lead to re-organization of insular-based networks, potentially enhancing high-level and cognitive and affection functions.^1,24 If these beneficial effects could be produced in the developing cerebrum of the preterm infant, the benefits would be appreciable.

Taken together, the role of music intervention in the context of the preterm NICU stay could be substantial over the longer term. Thus, subsequent work assessing the effect of music intervention on a broad range of later neurodevelopmental parameters will be of great interest.

Conclusion

The current work, showing the impact of a music intervention on parameters of cerebral white and gray matter development, sets the stage for future studies assessing a broad range of potential functional effects. The intervention is not complex and should be readily adaptable to current NICUs. As with any intervention in the NICU, however, careful attention to the timing of the activity is important. Disturbance of beneficial sleep states²⁵ would not be desirable. Nonetheless, my view is that the findings are of exceptional interest and could lead to an effective, readily applied neonatal intervention. Future work from this superb group will be of great interest.

Joseph J. Volpe, MD

Department of Neurology, Boston Children’s Hospital

Bronson Crothers Professor of Neurology, Emeritus, Harvard Medical School

Boston MA

References

1. Sa de Almeida J, Baud O, Fau S, et al.: Music impacts brain cortical microstructural maturation in very preterm infants: A longitudinal diffusion MR imaging study. Dev Cogn Neurosci 61:101254, 2023. DOI: 10.1016/j.dcn.2023.101254
2. Sa de Almeida J, Lordier L, Zollinger B, et al.: Music enhances structural maturation of emotional processing neural pathways in very preterm infants. Neuroimage 207:116391, 2020. DOI: 10.1016/j.neuroimage.2019.116391
3. Lordier L, Meskaldji DE, Grouiller F, et al.: Music in premature infants enhances high-level cognitive brain networks. Proc Natl Acad Sci U S A 116:12103-8, 2019. DOI: 10.1073/pnas.1817536116
4. Filippa M, Lordier L, Sa de Almeida J, et al.: Early vocal contact and music in the NICU: new insights into preventive interventions. Pediatr Res 87:249-64, 2020. DOI: 10.1038/s41390-019-0490-9
5. Lejeune F, Lordier L, Pittet MP, et al.: Effects of an Early Postnatal Music Intervention on Cognitive and Emotional Development in Preterm Children at 12 and 24 Months: Preliminary Findings. Front Psychol 10:494, 2019. DOI: 10.3389/fpsyg.2019.00494
6. Haynes RL, Kinney HC, Volpe JJ. Organizational events. Chapter 7. 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.
7. 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
8. Kostovic I, Jovanov-Milosevic N: The development of cerebral connections during the first 20-45 weeks’ gestation. Semin Fetal Neonatal Med 11:415-22, 2006. DOI: 10.1016/j.siny.2006.07.001
9. Kostovic I, Jovanov-Milosevic N, Rados M, et al.: Perinatal and early postnatal reorganization of the subplate and related cellular compartments in the human cerebral wall as revealed by histological and MRI approaches. Brain Struct Funct 219:231-53, 2014. DOI: 10.1007/s00429-012-0496-0
10. Haynes RL, Borenstein NS, Desilva TM, et al.: Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 484:156-67, 2005. DOI: 10.1002/cne.20453
11. Kostovic I, Judas M: The development of the subplate and thalamocortical connections in the human foetal brain. Acta Paediatr 99:1119-27, 2010. DOI: 10.1111/j.1651-2227.2010.01811.x
12. Kinney HC, Brody BA, Kloman AS, Gilles FH: Sequence of central nervous system myelination in human infancy. II. Patterns of myelination in autopsied infants. J Neuropathol Exp Neurol 47:217-34, 1988. DOI: 10.1097/00005072-198805000-00003
13. Kinney HC, Volpe JJ. Myelination events. Chapter 8. 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. 176-88.
14. Back SA, Luo NL, Borenstein NS, et al.: Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury. J Neurosci 21:1302-12, 2001. DOI: 10.1523/JNEUROSCI.21-04-01302.2001
15. Brody BA, Kinney HC, Kloman AS, Gilles FH: Sequence of central nervous system myelination in human infancy. I. An autopsy study of myelination. J Neuropathol Exp Neurol 46:283-301, 1987. DOI: 10.1097/00005072-198705000-00005
16. Neil JJ, Volpe JJ. Specialized neurological studies. Chapter 10. 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; 2015. p. 222-54.
17. Yakovlev PI, Lecours AR. The myelogenetic cycles of regional maturation of the brain. In: Minkowski A, editor. Regional Development of the Brain in Early Life. Oxford: Blackwell Scientific; 1967. p. 3-70.
18. Haynes RL, Kinney HC, Volpe JJ. Myelination events. Chapter 8. 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.
19. Dean JM, Moravec MD, Grafe M, et al.: Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human. Dev Neurosci 33:251-60, 2011. DOI: 10.1159/000327242
20. Marin-Padilla M: Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: a unifying theory. J Comp Neurol 321:223-40, 1992. DOI: 10.1002/cne.903210205
21. Gibson EM, Purger D, Mount CW, et al.: Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. Science 344:1252304, 2014. DOI: 10.1126/science.1252304
22. Koelsch S: Brain correlates of music-evoked emotions. Nat Rev Neurosci 15:170-80, 2014. DOI: 10.1038/nrn3666
23. Koelsch S, Skouras S, Lohmann G: The auditory cortex hosts network nodes influential for emotion processing: An fMRI study on music-evoked fear and joy. PLoS One 13:e0190057, 2018. DOI: 10.1371/journal.pone.0190057
24. Zamorano AM, Cifre I, Montoya P, et al.: Insula-based networks in professional musicians: Evidence for increased functional connectivity during resting state fMRI. Hum Brain Mapp 38:4834-49, 2017. DOI: 10.1002/hbm.23682
25. Volpe JJ. Preterm Infants Need their Sleep, Especially Active (REM) Sleep. Volpe’s View [Internet]. March 2024. Available from: newbornbrainsociety.org.

March, 2024

PRETERM INFANTS NEED THEIR SLEEP, ESPECIALLY ACTIVE (REM) SLEEP

Introduction

A recent report¹ from a distinguished group in the field of sleep in infancy concludes that active sleep is related beneficially to cerebral white matter development in preterm infants. The findings suggest potential value of incorporating sleep monitoring into routine caregiving practices in NICU settings (see later). Recall that in the neonatal period sleep is divided generally into two main periods, quiet sleep, a precursor to non-rapid eye movement sleep, and active sleep, a precursor to rapid eye movement (REM) sleep.² In general, preterm infants, when uninterrupted, spend approximately 80-90% of the time asleep (see later), and the majority of this time is in active sleep.³

Sleep and Brain Development—Animal Studies

The possibility that altered sleep behaviors in infants could be related to impaired brain development has been raised by a variety of animal studies.^3-6 In particular, deprivation of active sleep during development has been followed by smaller brain size, reduced brain growth and subsequent alterations of brain function. At the microstructural level, depending on the model, active sleep has been associated with synaptogenesis, maintenance of dendritic spines, and strengthening of synapses.³  At the electrophysiological level, REM sleep in early development has been associated with a number of effects indicative of functional maturation of neural circuits. As summarized by Knoop et al., “During early maturation REM sleep seems to provide the stimulation needed for preliminary development and survival of sensorimotor neural networks. It does this by driving the generation of endogenous, intense and generalized neural activity across sensorimotor systems.”³ This phenomenon is reminiscent of activity-dependent differentiation of neuronal circuits in a variety of sensory systems during brain development.⁷ (Although beyond the scope of this discussion, animal studies support the notion that non-REM sleep is more important for regulating synaptic homeostasis and later brain maturation.²)

Several concerns could be raised about the animal studies. First, the models generally involved animals that are more mature than human infants.^4,5,8 Additionally, experimental paradigms often decreased active sleep by pharmacological methods and did not control for such factors as stress (as in experiments utilizing ocular deprivation). Moreover, study of brain maturation in the animal models has often not been sufficiently detailed, especially at the regional and cellular levels. Nevertheless, the findings are compelling.

Sleep and Brain Development—Human Studies

The few relevant studies in human preterm infants have been of interest but have been limited by methodological challenges. Several reports describe an association of decreased active sleep during the preterm or term period to later cognitive impairments.^9-12 Correlation with imaging markers of brain development was not reported. Moreover, the most prevalent methodologies utilized for sleep assessment, i.e., behavioral observation or polysomnography, limit daily sleep assessments to only a few minutes to hours.^13,14

Current Study

Study Design. The study that stimulated this Commentary is entitled: “Machine learning-derived active sleep as an early predictor of white matter development in preterm infants,” by Wang et al., and as noted earlier, was published in the Journal of Neuroscience (January 2024). (The senior author, Jeroen Dudink, is a well-established leader in the field.) Stimulated by the experimental and human studies reviewed earlier, this study “aimed to determine the potential of sleep as an early predictor for subsequent white matter development in preterm infants.” To overcome the deficiencies of conventional sleep studies, after an initial study of infants for multiple consecutive hours by visual sleep scoring by trained sleep observers, the group was able to utilize heart and respiratory rates as routinely monitored in the NICU to develop a machine learning-based automated sleep stage classifier. This automated classifier subsequently was applied to a cohort of 58 preterm infants to extract sleep percentage over 5-7 consecutive days from 29-32 weeks of postmenstrual age. Excluded from the cohort were infants with major congenital malformations and such overt brain injury as grade III IVH or cystic periventricular leukomalacia. Also excluded were infants on mechanical ventilation. The study infants then had an MRI scan at term equivalent age on a clinical 3-Tesla scanner to assess volumetric tissue segmentation.

Principal Finding. The results were very interesting. The infants spent most of their time asleep (mean percentage of 90% per 24 hours), and on average, time in active sleep and in quiet sleep were approximately equal.¹ Notably, higher active sleep percentage was significantly associated with larger white matter volume at term after adjusting for potential confounding covariates (standardized regression coefficient, 0.31; 95% CI, 0.09, 0.53; adjusted P-value 0.021).

Data Implications. The data raise the critical question concerning the basis for increased cerebral white matter volume in the infants with higher active sleep percentages. Cerebral white matter volume during the last 10 weeks of brain development in the premature infant increases nearly 80%, and approximately 80% of this increase is unmyelinated white matter.^15,16 The latter is composed principally of rapidly developing axons and premyelinating oligodendrocytes. Indeed, the period of observation of the sleep parameters in the study of Wang et al.,¹ 29 to 32 weeks postmenstrual age, is the time of onset of ensheathment of developing axons by immature oligodendrocytes in human cerebral white matter.¹⁷ This ensheathment is important for the subsequent development of the abundant growing axons in cerebral white matter at this time.⁷ In turn, the resulting axonal activity stimulates the further development of these ensheathing immature oligodendroglia.¹⁸ Analysis of developing cerebral white matter during this period with GAP-43, a marker of growing axons, shows marked expression in the third trimester.^19,20 Anatomical studies have elucidated the details of thalamacortical, commissural cortical and cortico-cerebral axonal development during this period.^7,21,22 Similarly, state-of-the-art diffusion-based MRI study of human fetal brain in utero shows changes consistent with exuberant axonal development during the last trimester of gestation, although the regional changes vary in magnitude and timing.²³

Notably, during the developmental period just described, accompanying these changes in cerebral white matter are increases in cerebral cortical surface area and gyrification.²⁴ Anatomical and diffusion MRI studies are indicative of marked cortical dendritic and synaptic development also during this time.⁷ Additionally, functional MRI studies show the onset of cortical neuronal activation and connectivity after visual, auditory and somatosensory stimulation at this time.²⁵ Assessment of these individual events in future studies of the role of active sleep in premature infants in subsequent brain development will be of great interest.

Which of the developmental processes just reviewed could underlie the increase in cerebral white matter volume related to time in active sleep in the study of Wang et al.?¹ Based on the likelihood that the increase in cerebral white matter volume during the 30-40 week gestational period is related largely to axonal development, an important possibility is that active sleep in some way leads to enhanced axonal development. One critical driving force for axonal development during this period from 30-40 weeks is the development of synaptic connections in cerebral cortex by terminals of axons coursing through cerebral white matter from thalamus, subplate, and other cortical sites via commissural-cortical and cortico-cortical connections (see earlier). These events are especially prominent in the period from 30-40 weeks’ gestation.⁷ Studies in animal models (see earlier) indicate that active sleep is important in synaptic development and maintenance. Taken together, the findings suggest that cerebral white matter volume may increase with active sleep because of an increase in axonal development stimulated in an activity-dependent manner by the synaptic effects of this sleep state. As noted earlier, the period of the study, 29-32 weeks postmenstrual age, is the onset of pre-oligodendroglial ensheathment of axons, a process critical for axonal function.⁷ Future studies of cerebral connectivity in the context of active sleep will be important.

Clinical Implications

The authors of the interesting report by Wang et al.¹ conclude that the discovery of a positive association between the percentage of machine learning-derived active sleep during the preterm period and white matter volume at term-equivalent age: (1) “provides new insights into the role of active sleep in early human brain development, confirming and extending findings from animal research,” and (2) “further indicates the potential benefit of integrating automated sleep monitoring into routine caregiving practices in NICU settings.”¹ I agree with both of these conclusions.

Concerning caregiving practices, a review of studies assessing interventions to promote “sleep” in NICU settings identified fourteen, of which 10 were RCT.²⁶ Approaches studied include kangaroo care, gentle human touch, different sleep surfaces, sound, cycled light, and LED light. None of the studies showed marked benefit, although methodological concerns, including the means of assessment of sleep state, render comparisons difficult. One study utilizing an instrument that creates a sound effect similar to the sound of moving fluids did show an increase in time spent in active sleep.²⁷ The authors of this review concluded that there is insufficient evidence to recommend any new intervention to promote neonatal sleep in the NICU.²⁶ However, with the recent work of Wang et al.¹ an important breakthrough may be apparent. Automated sleep monitoring in the NICU could allow caregivers to carry out various daily interventions in such a way as to safeguard active sleep and, presumably, to promote growth of cerebral white matter and the likely anatomical correlates describe earlier.

Currently, rational changes in caregiving would be difficult if sleep assessment is carried out by observation by individuals not highly trained in such assessment. REM sleep in the premature infant often can be difficult to detect because of its close resemblance to an awake state, a resemblance that results from the spontaneous activity generated during the sleep state (Jereon Dudink, personal communication). Thus, as communicated to me by Dr. Dudink, caretakers “will more likely disturb the infants during this sleep state than during quiet sleep. … Our extended monitoring indicates that sleep is disrupted many times in an NICU and that in a less intrusive environment, the duration of active sleep in preterm infants increases, underscoring the profound impact of the NICU setting on their sleep architecture.” Thus, the value for the continuous assessment of sleep state afforded by the approach described by Wang et al.¹ is particularly apparent.

A final caveat—it should be noted that the role of non-REM sleep during the preterm period in promoting brain development is unclear. Available data suggest that non-REM sleep is related more to later brain development, rather than to that occurring during the NICU period for preterm infants.³ More research is needed on the issue of non-REM sleep in preterm infants and brain development.

Conclusion

The methodological advances concerning sleep monitoring and the correlation with enhanced cerebral white matter development, as shown by MRI described here,¹ could lead to caregiving practices that enhance specific sleep behavior in the preterm infant, promote cerebral white matter development, and perhaps most importantly, improve neurological outcome. My view is that such improved sleep management, carefully devised, could prove to be a safe, readily applied, and beneficial management approach. Future research that includes assessment of cerebral cortical and white matter microstructure by advanced diffusion-based MRI methods^28,29 and of subsequent neurodevelopmental outcomes will be of particular interest.

Joseph J. Volpe, MD

Department of Neurology, Boston Children’s Hospital

Bronson Crothers Professor of Neurology, Emeritus, Harvard Medical School

Boston MA

References

1. Wang X, de Groot ER, Tataranno ML, et al.: Machine Learning-Derived Active Sleep as an Early Predictor of White Matter Development in Preterm Infants. J Neurosci 44, 2024. DOI: 10.1523/JNEUROSCI.1024-23.2023
2. Lokhandwala S, Spencer RMC: Relations between sleep patterns early in life and brain development: A review. Dev Cogn Neurosci 56:101130, 2022. DOI: 10.1016/j.dcn.2022.101130
3. Knoop MS, de Groot ER, Dudink J: Current ideas about the roles of rapid eye movement and non-rapid eye movement sleep in brain development. Acta Paediatr 110:36-44, 2021. DOI: 10.1111/apa.15485
4. Frank MG, Issa NP, Stryker MP: Sleep enhances plasticity in the developing visual cortex. Neuron 30:275-87, 2001. DOI: 10.1016/s0896-6273(01)00279-3
5. Blumberg MS, Dooley JC, Tiriac A: Sleep, plasticity, and sensory neurodevelopment. Neuron 110:3230-42, 2022. DOI: 10.1016/j.neuron.2022.08.005
6. Dang-Vu TT, Desseilles M, Peigneux P, Maquet P: A role for sleep in brain plasticity. Pediatr Rehabil 9:98-118, 2006. DOI: 10.1080/13638490500138702
7. 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
8. Del Rio-Bermudez C, Blumberg MS: Sleep as a window on the sensorimotor foundations of the developing hippocampus. Hippocampus 32:89-97, 2022. DOI: 10.1002/hipo.23334
9. Arditi-Babchuk H, Feldman R, Eidelman AI: Rapid eye movement (REM) in premature neonates and developmental outcome at 6 months. Infant Behav Dev 32:27-32, 2009. DOI: 10.1016/j.infbeh.2008.09.001
10. Bennet L, Walker DW, Horne RSC: Waking up too early – the consequences of preterm birth on sleep development. J Physiol 596:5687-708, 2018. DOI: 10.1113/JP274950
11. Freudigman KA, Thoman EB: Infant sleep during the first postnatal day: an opportunity for assessment of vulnerability. Pediatrics 92:373-9, 1993.
12. Shellhaas RA, Burns JW, Hassan F, et al.: Neonatal Sleep-Wake Analyses Predict 18-month Neurodevelopmental Outcomes. Sleep 40, 2017. DOI: 10.1093/sleep/zsx144
13. Werth J, Atallah L, Andriessen P, et al.: Unobtrusive sleep state measurements in preterm infants – A review. Sleep Med Rev 32:109-22, 2017. DOI: 10.1016/j.smrv.2016.03.005
14. Georgoulas A, Jones L, Laudiano-Dray MP, et al.: Sleep-wake regulation in preterm and term infants. Sleep 44, 2021. DOI: 10.1093/sleep/zsaa148
15. Huppi PS, Warfield S, Kikinis R, et al.: Quantitative magnetic resonance imaging of brain development in premature and mature newborns. Ann Neurol 43:224-35, 1998. DOI: 10.1002/ana.410430213
16. de Vries LS, Volpe JJ. Specialized neurological studies. Chapter 13. 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. Back SA, Luo NL, Borenstein NS, et al.: Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury. J Neurosci 21:1302-12, 2001. DOI: 10.1523/JNEUROSCI.21-04-01302.2001
18. Gibson EM, Purger D, Mount CW, et al.: Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. Science 344:1252304, 2014. DOI: 10.1126/science.1252304
19. Haynes RL, Borenstein NS, Desilva TM, et al.: Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 484:156-67, 2005. DOI: 10.1002/cne.20453
20. Haynes RL, Volpe JJ. Organizational events. Chapter 7. 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.
21. Kostovic I, Judas M, Rados M, Hrabac P: Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging. Cereb Cortex 12:536-44, 2002. DOI: 10.1093/cercor/12.5.536
22. Kostovic I, Jovanov-Milosevic N: The development of cerebral connections during the first 20-45 weeks’ gestation. Semin Fetal Neonatal Med 11:415-22, 2006. DOI: 10.1016/j.siny.2006.07.001
23. Wilson S, Pietsch M, Cordero-Grande L, et al.: Development of human white matter pathways in utero over the second and third trimester. Proc Natl Acad Sci U S A 118, 2021. DOI: 10.1073/pnas.2023598118
24. Kapellou O, Counsell SJ, Kennea N, et al.: Abnormal cortical development after premature birth shown by altered allometric scaling of brain growth. PLoS Med 3:e265, 2006. DOI: 10.1371/journal.pmed.0030265
25. Neil JJ, Volpe JJ. Specialized neurological studies. Chapter 10. 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; 2015. p. 222-54.
26. van den Hoogen A, Teunis CJ, Shellhaas RA, et al.: How to improve sleep in a neonatal intensive care unit: A systematic review. Early Hum Dev 113:78-86, 2017. DOI: 10.1016/j.earlhumdev.2017.07.002
27. Loewy J, Stewart K, Dassler AM, et al.: The effects of music therapy on vital signs, feeding, and sleep in premature infants. Pediatrics 131:902-18, 2013. DOI: 10.1542/peds.2012-1367
28. Raffelt DA, Tournier JD, Smith RE, et al.: Investigating white matter fibre density and morphology using fixel-based analysis. Neuroimage 144:58-73, 2017. DOI: 10.1016/j.neuroimage.2016.09.029
29. Batalle D, O’Muircheartaigh J, Makropoulos A, et al.: Different patterns of cortical maturation before and after 38 weeks gestational age demonstrated by diffusion MRI in vivo. Neuroimage 185:764-75, 2019. DOI: 10.1016/j.neuroimage.2018.05.046

January, 2024

ERYTHROPOIETIN AND THE HEAL STUDY: TIMING ISSUES

Introduction

The recently reported studies^1-3 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