12
The Encephalopathy of Prematurity: One Pediatric Neuropathologist’s Perspective Hannah C. Kinney, MD A major challenge in understanding brain injury in the premature brain is the establishment of the precise human neuropathology at the cellular and molecular levels, as such knowl- edge is the foundation upon which the elucidation of the cause(s), scientific experimenta- tion, and therapies in the field is by necessity based. In this essay, I provide my perspective as a pediatric neuropathologist upon pathologic studies in the developing human brain itself, including a review of past, present, and future aspects. My focus is upon the path that has brought us to the current recognition that preterm brain injury is a complex of white and gray matter damage that results in the modification of key developmental pathways during a critical period, which in turn defines the adverse clinical outcomes as important as the primary insult itself. The evolution of this recognition, as well as the introduction of the term “encephalopathy of prematurity” for the complex of gray and white matter damage because of acquired and developmental mechanisms, is discussed. Our enhanced understanding of the fundamental neuropathology of the human preterm brain should bring us closer to more effective therapy as the need to prevent and treat injury to developing oligodendrocytes and neurons in combination is appreciated. Semin Pediatr Neurol 16:179-190 © 2009 Published by Elsevier Inc. M y charge as a pediatric neuropathologist long interested in brain injury in the premature infant is to provide a perspective upon pathologic studies in the developing hu- man brain itself. At the request of Dr Adre DuPlessis, the editor of this series, I review here past, present, and future aspects of the neuropathology of the human premature brain in the context of its specific “challenges, advances, detours, and quantum leaps” (his words). This perspective, on the basis of personal insights gained over a two-decade career in the field, is hoped to benefit the readers. I am particularly fortunate in that my own research has been inspired and guided by 4 giants in the field who share in common a love of children, a fascination with the beauty of the developing nervous system, a passion to further our understanding of pediatric brain injury, and an unwavering commitment to mentor and with kindness: Drs Joseph J. Volpe, Miguel Ma- rine-Padilla, Dawna Armstrong, and Floyd Gilles. Below I have highlighted the lessons they have taught me individu- ally, as well as to the field as a whole. Significantly, the major challenge in understanding brain injury in the premature brain, in my opinion, is the establish- ment of the precise human neuropathology at the cellular and molecular levels, as such knowledge is the foundation upon which the elucidation of the cause(s), scientific exper- imentation, and therapies is by necessity based. To me, the major “quantum leap” in our understanding, in the last few years, has been the recognition that preterm brain injury is a complex of white and gray matter damage that results in the modification of key developmental pathways during a critical period, which in turn defines the adverse clinical outcomes as importantly as the primary insult itself. The concept of the devastating intersection between development and disease is not unique to any one investigator in perinatal brain injury; indeed, this intersection is the sine qua non of all pediatric disorders. Nevertheless, this concept as applied to premature brain injury is brilliantly synthesized and elegantly articu- lated by the world-renown “thinker” in neonatal neurology, Dr Volpe, 1 in his already classic review published but a few months ago. This quantum leap forward in our conceptual- ization of premature brain injury is best captured in the label “encephalopathy of prematurity” that was coined by Dr Volpe 2 to emphasize the combined acquired tissue loss and altered developmental trajectories in combined white and From the Department of Pathology, Children’s Hospital Boston and Harvard Medical School, Boston, MA. Supported by grants from the National Institute of Neurological Diseases and Stroke (PO1-NS38475), Hearst Foundation, March of Dimes, and National Institute of Child Health and Development (P30-HD18655) (Children’s Hospital Developmental Disabilities Research Center). Address reprint requests to Hannah C. Kinney, MD, Department of Pathol- ogy, Enders Building 1112, Children’s Hospital Boston, 300 Longwood Av, Boston, MA 02115. E-mail: [email protected] 179 1071-9091/09/$-see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.spen.2009.09.003

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he Encephalopathy of Prematurity: Oneediatric Neuropathologist’s Perspective

annah C. Kinney, MD

A major challenge in understanding brain injury in the premature brain is the establishmentof the precise human neuropathology at the cellular and molecular levels, as such knowl-edge is the foundation upon which the elucidation of the cause(s), scientific experimenta-tion, and therapies in the field is by necessity based. In this essay, I provide my perspectiveas a pediatric neuropathologist upon pathologic studies in the developing human brainitself, including a review of past, present, and future aspects. My focus is upon the path thathas brought us to the current recognition that preterm brain injury is a complex of white andgray matter damage that results in the modification of key developmental pathways duringa critical period, which in turn defines the adverse clinical outcomes as important as theprimary insult itself. The evolution of this recognition, as well as the introduction of the term“encephalopathy of prematurity” for the complex of gray and white matter damage becauseof acquired and developmental mechanisms, is discussed. Our enhanced understanding ofthe fundamental neuropathology of the human preterm brain should bring us closer to moreeffective therapy as the need to prevent and treat injury to developing oligodendrocytes andneurons in combination is appreciated.Semin Pediatr Neurol 16:179-190 © 2009 Published by Elsevier Inc.

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y charge as a pediatric neuropathologist long interestedin brain injury in the premature infant is to provide a

erspective upon pathologic studies in the developing hu-an brain itself. At the request of Dr Adre DuPlessis, the

ditor of this series, I review here past, present, and futurespects of the neuropathology of the human premature brainn the context of its specific “challenges, advances, detours,nd quantum leaps” (his words). This perspective, on theasis of personal insights gained over a two-decade career inhe field, is hoped to benefit the readers. I am particularlyortunate in that my own research has been inspired anduided by 4 giants in the field who share in common a love ofhildren, a fascination with the beauty of the developingervous system, a passion to further our understanding ofediatric brain injury, and an unwavering commitment toentor and with kindness: Drs Joseph J. Volpe, Miguel Ma-

ine-Padilla, Dawna Armstrong, and Floyd Gilles. Below I

rom the Department of Pathology, Children’s Hospital Boston and HarvardMedical School, Boston, MA.

upported by grants from the National Institute of Neurological Diseasesand Stroke (PO1-NS38475), Hearst Foundation, March of Dimes, andNational Institute of Child Health and Development (P30-HD18655)(Children’s Hospital Developmental Disabilities Research Center).

ddress reprint requests to Hannah C. Kinney, MD, Department of Pathol-ogy, Enders Building 1112, Children’s Hospital Boston, 300 Longwood

aAv, Boston, MA 02115. E-mail: [email protected]

071-9091/09/$-see front matter © 2009 Published by Elsevier Inc.oi:10.1016/j.spen.2009.09.003

ave highlighted the lessons they have taught me individu-lly, as well as to the field as a whole.

Significantly, the major challenge in understanding brainnjury in the premature brain, in my opinion, is the establish-

ent of the precise human neuropathology at the cellularnd molecular levels, as such knowledge is the foundationpon which the elucidation of the cause(s), scientific exper-

mentation, and therapies is by necessity based. To me, theajor “quantum leap” in our understanding, in the last few

ears, has been the recognition that preterm brain injury is aomplex of white and gray matter damage that results in theodification of key developmental pathways during a criticaleriod, which in turn defines the adverse clinical outcomes as

mportantly as the primary insult itself. The concept of theevastating intersection between development and disease isot unique to any one investigator in perinatal brain injury;

ndeed, this intersection is the sine qua non of all pediatricisorders. Nevertheless, this concept as applied to prematurerain injury is brilliantly synthesized and elegantly articu-

ated by the world-renown “thinker” in neonatal neurology,r Volpe,1 in his already classic review published but a fewonths ago. This quantum leap forward in our conceptual-

zation of premature brain injury is best captured in the labelencephalopathy of prematurity” that was coined by Drolpe2 to emphasize the combined acquired tissue loss and

ltered developmental trajectories in combined white and

179

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ray matter sites in the developing human brain. Moreover,he constellation of cognitive, motor, and emotional impair-ent in long-term survivors of prematurity reflects the par-

icular patterns of white and gray matter damage in combi-ation with arrested developmental programs—patterns thatepend upon the severity, timing, and chronicity of the in-

ury, as well as an individual confounding factors.1,3 Theeuropathology of the encephalopathy of prematurity, asell as its causes and pathogenesis, has been reviewed by Drolpe1 and by Dr Volpe in collaboration with me;3 it is alsoummarized by Dr Volpe elsewhere in this series. In the fol-owing perspective, I focus rather upon advances, detours,oadblocks, and future directions towards understandinghis entity in its entirety and ultimately abolishing it.

dvanceshe Concept of Preferential Whiteatter Vulnerability in the Fetal Brainmajor advance in our understanding of the preterm brain

njury was the early recognition (formally beginning in the9th century) that the developing cerebral white matter isarticularly vulnerable to insult in the human fetus. Histori-al reviews mention the key roles of Little, Virchow, Parrot,nd Schwartz in focusing attention upon brain damage in theremature infant and the predominance of white matter pa-hology in the perinatal period.4,5 Parrot’s studies at the endf the 19th century in particular emphasized yellow or chalkylaques, 5 to 6 mm in diameter, in the periventricular whiteatter and sparing of the gray matter.4 In their historical

eview, Banker and Larroche5 point out that Parrot ascribed aparticular vulnerability to the immature white matter, attrib-table to the fact that the affected zones are farthest from thelood supply”, because the lesions were centrally placed inhe white matter and the “cerebral cortex and basal gangliaere spared”, and that Parrot further suggested the causes of

he white matter damage were “circulatory and nutritionalisturbances” occurring in an “actively developing brain”.5

rom the outset, controversies abounded, including abouthe role of anoxia vs infection in its pathogenesis, the naturenormal or pathologic?) of diffuse fatty change or “metamor-hosis” of glial cells, and the significance of certain periven-ricular cells that were variously interpreted as inflammatoryr periventricular germinal rests.4,5 In 1962, the comprehen-ive study of “periventricular leukomalacia” (PVL) in 51 in-ants dying at the Children’s Hospital Boston was publishedy Banker and Larroche.5 In this landmark study, (1) theyeported that the clinical and pathologic features were corre-ated, leading to the conclusion that PVL is the basis of manynstances of mental retardation and spasticity; (2) the nameperiventricular leukomalacia” for this type of perinatal whiteatter damage was recommended; (3) the temporal se-

uences of the histopathology of PVL were delineated; (4) aonsistent topography of the necrotic lesions in the periven-ricular white matter was described (Fig 1); (5) axonal dam-ge (“retraction clubs”) in the focally necrotic lesions in the

eriventricular white matter was highlighted; and (6) a key G

ausative role for ischemia relative to border zones of theascular supply to the deep white matter and inadequatelood flow complicating systemic hypotension was empha-ized.5 Of note, astrogliosis in the white matter surroundingr distant from the necrotic foci was not described, nor wasiffuse microglial activation.The next conceptual advance in our understanding of the

europathology of the preterm brain, in my opinion, was theecognition of diffuse astrogliosis in association with the ne-rotic foci by Dr Gilles and colleagues6,7 in the 1970s and980s, leading them to introduce the terms “perinatal telen-ephalic leukoencephalopathy” (PTL) or “acquired perinataleukoencephalopathy”. Dr Gilles’ observations were based inarge part upon his monumental analysis of almost 200 fetalnd neonatal brains accrued under the auspice of the nationalollaborative perinatal project (NCPP) sponsored by the Na-ional Institute of Neurological and Communicative Disor-ers6,7 and during his time at Children’s Hospital Boston. Drilles considered 4 histologic features evidence of perinatalhite matter damage under the rubric of PTL, ie, necrotic foci

PVL), hypertrophic astrocytes (see below), perivascular am-hophilic globules, and cells described as “acutely damagedlia”.6 In a sample of newborns enrolled in the NCPP, ne-rotic foci were found in 10%, hypertrophic astrocytes in2%, amphophilic globules in 29%, and acutely damagedlia in 16%.6 Of note, Gilles postulated that the acutely dam-ged glia are comprised of “glial cells destined to becomeligodendroglia and to lay down and maintain myelin,” andhat they are “either destroyed or transformed by . . . in-ults”.6 He further suggested that “gliosis represents a trans-ormation of pluripotent glia into glia-fibril-producing cells,ather than into myelin-producing and -supporting oligo-endroglia”.6 Remarkably, these seminal observations by Dr

igure 1 Classic diagram from the seminal report of the neuropa-hology of periventricular leukomalacia by Drs Banker and Larrochen 1962,5 showing the distribution of the necrotic foci in theeriventricular leukomalacia. (Color version of figure is availablenline.)

illes were made before the introduction of immunocyto-

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Encephalopathy of prematurity 181

hemical markers for astrocytes (glial fibrillary acidic proteinGFAP]) and pre-myelinating OLs (pre-OLs) and/or matureLs [O4, O1, OLIG2, and myelin basic protein (MBP)]. Drilles’ other contributions to our understanding of pretermrain injury include (1) the application of large populationepidemiologic) analysis to determine the risk factors for theombined or separate features of PTL,6,7 (2) the recognitionased upon such risk factor analysis of the potential rolef endotoxin in the pathogenesis of perinatal white matternjury,6 and (3) the emphasis upon the testing of causativeypotheses generated from human pathology in develop-ental animal models, as exemplified by his analysis of en-otoxin toxicity to white matter in kittens.8

As a neuropathology fellow in training with Dr Gilles, Iame to appreciate the full extent of astrogliosis in pretermrain injury in the daily sign-out of autopsy cases with him.hese teachings were supplemented by the neuroimagingbservations emphasized by my other mentor, Dr Volpe,9 inhich periventricular necrotic lesions are accompanied byiffuse signal intensity in the surrounding white matter. Theombined neuroimaging and autopsy teachings convincede of the validity of the formulation that white matter dam-

ge in the preterm infant is comprised of 2 key components,hich are defined as (1) the “focal” component of periven-

ricular necrosis, and (2) the “diffuse” component of gliosis inhe surrounding white matter.3,10 The identification of diffusestrogliosis in the white matter in preterm brain injurytermed PVL) is important because it suggests a penumbra ofiffuse (reversible) injury to pre-OLs in the surroundinghite matter that is more amenable to intervention, repair,

nd recovery than the global injury to tissue in the coreirreversible) infarcts of the periventricular regions3—an ideaor testing in animal models.

Advances in our understanding of preterm brain injuryeflect in large part the application of immunocytochemistry,

Figure 2 (A) A key feature of the white matter damagemyelin basic protein (MBP) staining in the perikarya of oto MBP using immunocytochemistry.12 (B) The diagrtrafficking in the mature (or developing) OL contribuabnormality in OL-axonal interaction (red lines). (Colo

omputer-based quantitation and graphics, western blotting, i

n situ hybridization, and other modern methods directly tohe postmortem human brain. Under the visionary leader-hip of Dr Volpe in a NINDS-funded program beginning in000, our group in neuropathology at Children’s Hospitaloston began concentrated efforts to define systematicallyhe cellular basis of preterm injury directly in the humanrain at autopsy.11-22 The application of immunomarkers forL cell lineage in conjunction with computer-based quanti-

ation by our group revealed that: (1) the number and densityf pre-OLs are not reduced in the subacute and chronictages of the diffuse component of PVL; (2) rather, they arencreased immediately adjacent to the periventricular ne-rotic foci, suggesting the possibility of attempted regenera-ion and repair; and (3) dysfunctional myelin synthesis inreserved pre-OLs likely accounts in large part for subse-uent impairment in myelination (Fig 2).12 We observed, fornatance, abortive attempts at myelin formation around theecrotic foci, as well as intense MPB immunostaining in OLerikarya, suggestive of a “hang-up” in MPB trafficking from

ts site of synthesis in the cytoplasm to its deposition in pro-esses involved in axonal ensheathment (Fig 2). During thisame period, Dr Stephen Back et al23 reported reduced num-ers of pre-OLs in the white matter in PVL cases with “acute”

esions in a smaller sample size, raising the possibility thatre-OLs may undergo cell death immediately after injuryut, in conjunction with our findings, be “replaced” in theubacute and chronic stages. This possibility is supported byn earlier report by our group of striking (qualitative) pre-OLepletion in 2 PVL cases compared with controls.21 Of note,ida et al24 reported previously a loss of OLs with the non-pecific marker ferritin in PVL cases in which there was as-ociated myelin-staining in tissue sections. Taken together,hese human data suggest that the number of pre-OLs onalance may depend upon the stage at which the histopathol-gy is captured at the time of death, and that the “snapshot”

encephalopathy of prematurity is the accumulation ofndrocytes (OL) (arrows), as indicated with an antibodyicates schematically that the putative arrest of MBPmpaired ensheathment of the axons and a functionaln of figure is available online.)

in theligodeam indtes to i

s taken relative to the timing of the acute insult, progression

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f tissue damage, and extent and type of OL regeneration andepair. The application by us of CD68 antibody as an immu-omarker for the microglial/macrophagocytic lineage re-ealed diffuse activation of the microglia in the diffuse com-onent of PVL,21 of major importance given that a role foricroglia in the pathogenesis of PVL was previously not de-

cribed (see above), as well as the known roles of microglia inhe inflammatory response and cytokine and free radical tox-city to pre-OLs.3,25-27

The application by us of the fraction antibody as an immu-omarker for axonal damage revealed a striking degree ofxonal injury in the diffuse component of PVL, of majormportance in potentially redefining our concept of pretermhite matter injury as a disorder not exclusively of develop-

ng OLs but also of developing axons, and raising the possi-ility that this white matter injury even reflects a primaryxonapathy1,3,13 (Fig 3). The application by us of immuno-arkers of oxidative and nitrative stress indicated extensive

ree radical injury in the diffuse component of PVL, includingarticularly to pre-OLs, which demonstrate coexpression ofhese markers by double-labeling techniques.21 These obser-

Figure 3 Diffuse axonal injury in the encephalopathy of pment (C). (A) In this diagram of a representative sectioncallosum) in a preterm infant with the encephalopathy owhite matter surrounding the microcystic foci (microcysof involvement of the intrahemispheric crossing fibers.strated with an antibody to fraction, marker of apoptoticpeak period of the encephalopathy of prematurity and thmatter, as indicated using the growth associated protein-protein-43 levels are expressed as the percentage of an adof prematurity may reflect: (1) secondary degeneration to(antegrade degeneration) that is located in the thalamucoursing through the white matter to and from the co(dying-back or retrograde degeneration). (Color version

ations concerning free radical injury in PVL was supported c

y those of Dr Back et al23 in PVL cases compared withontrols with tissue measurement of F(2)-isoprostane.

The application of cytokine immunomarkers by us16 andr Kadhim et al29 indicated the marked expression of inter-

eukins, tumor necrosis factor-�, interferon-�, and other cy-okines in astrocytes and macrophages in both the diffusend focal components of PVL, suggesting an important roleor them in the pathogenesis of the white matter injury,hich must be accounted for in the development of experi-ental models and that may prove useful in discovery of

herapeutic agents. Moreover, the demonstration of receptorsor interferon-� on pre-OLs indicated that the vulnerabilityf these cells to cytokine toxicity reflects at least in part re-eptor-mediated interactions.16,30 The application of immu-omarkers for the glutamate transporter GLT1 indicated itsxpression in the reactive astrocytes and macrophages of thenflammatory response of PVL, suggesting that these cellslay, an as yet to be defined, role in preventing excitotoxicamage to pre-OLs.17 Finally, the application of immuno-arkers for stem and progenitor cells by us10 and others31 is

pening major new avenues of research by indicating that the

urity (A,B) in the context of normative axonal develop-posterior frontal lobe (level of the body of the corpusturity, there is axonal fragmentation (small lines) in theote the fragmentation in the corpus callosum indicativexonal damage throughout the white matter is demon-, with immunocytochemical methods.13 (C) During theinfancy, axonal elongation occurs in the cerebral white

rker with western blot analysis.28 The growth associatedndard.28 (D) The axonal damage in the encephalopathyon because of a primary insult to the neuronal cell bodyerebral cortex; and/or (2) primary insult to the axonsith secondary degeneration to the neuronal cell bodyre is available online.)

rematof the

f premat).13 N(B) Ainjuryrough43 mault stathe axs or crtex w

erebral white matter in human premature infant has the

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Encephalopathy of prematurity 183

nnate capacity for regeneration and repair, and that thera-eutic strategies to augment this capacity may prove im-ensely effective in the prevention of long-term neurologicalisabilities. Of note, our autopsy analysis of brain injury inhe early preterm period compared with the late pretermeriod reveals that the late preterm infant is at risk for theame types of damage that occurs in early preterm infants, ie,he encephalopathy of prematurity.18,19 Moreover, it suggestshat the underlying neuropathologic substrate of the subtleognitive and emotional problems increasingly recognized inhe late preterm infant32 are likely the result of milder degreesf injury than the severe injury that results in major clinicalroblems in low birth weight infants.In addition to pathologic insights, the application of modern

echniques to the human fetal brain at autopsy has revealedultiple developmental factors that contribute to the biological

usceptibility to PVL. Of particular interest are the factors thatncrease vulnerability to cerebral ischemia in the setting of theick premature infant with pulmonary immaturity, respiratoryistress syndrome, and impaired cerebral vascular autoregula-ion.1,3 Work by our group indicates that these factors in thehite matter in the last half of gestation, ie, the peak time framef PVL, include (1) the predominance of pre-OLs in the whiteatter at the peak age for PVL, which are known to be especially

ulnerable to excitoxic and free radical injury compared withature OLs,33,34 (2) the developmental delay in the matura-

ion of the superoxide dismutases,35 (3) the transient expres-ion of the glutamate transporter GLT1 in the cerebral whiteatter,36 (4) transient expression of AMPA receptors, with

Figure 4 Classic Golgi drawing by Dr Marin-Padilla41 ofwhite matter necrosis in a human infant. Some neurons aothers show alterations suggestive of centripetal dendricontact with Layer I with progressive distal absorption. Texplanation of the figure.41

he lack of the relative expression of GluR2,37 (5) the tran- t

ient elevation in the density of ameboid microglia,38 and (6)mmaturity of axonal projections and cellular structure (neu-ofilaments).28 The work of Dr Takashima and Tanaka40 alsondicates the morphologic immaturity of the arterial vascula-ure supplying white matter over this same period, therebyncreasing the white matter’s vulnerability to ischemia, whichomplicates deleterious hemodynamic changes in the sickremature infant.39 Thus, the basis of the vulnerability of theetal white matter to ischemic injury alone is complex andnvolves multiple factors that collide in time and space toorm the “perfect storm” of susceptibility.

orphological Evidence for the Interactionetween Injury and Development

n the Encephalopathy of Prematurityerhaps the best illustration of this principle is the demon-tration by Dr Marin-Padilla41 of the consequences of whiteatter damage upon the subsequent development of the ce-

ebral cortex (Fig 4). In his landmark Golgi study of theerebral cortex in long-term survivors of severe white matterecrosis, Dr Marin-Padilla41 showed secondary neuronal ab-ormalities, including in Layer V (Fig 4). He found that theeveloping cortex was deprived of afferent terminals due toheir severance from the cortex by the extensive white matterecrosis.41 Moreover, some of the cortical neurons failed toeach their subcortical targets because their axons also wereestroyed by the white matter lesion.41 Of major interest wasis stunning observation that the axotomized pyramidal cells

al abnormalities in the cerebral cortex overlying severesformed into small stellate neurons post injury, whereassorption. Some pyramidal cells also seem to have loster is referred to the original publication for the in-depth

neuronre trantic reabhe read

ransformed from long-projecting into local-circuit neu-

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ons41 (Fig 4). On the basis of the observations by Golgi,arine-Padilla41 proposed that the neurological sequelae of

erinatal white matter lesions are a direct consequence ofostinjury, gray matter “transformations”. Dr Marin-Padillaersonally taught me that there is no substitute for carefulbservation with the microscope in which the mind contin-ously works to correlate structure with function in 3 dimen-ions as the eye sees two-dimensional cells on the slides inonderment.

etourshe major detour, in my opinion, in our understanding of theeuropathology of the preterm brain was the historical focuspon white matter injury to the almost total exclusion of grayatter injury. Indeed, the conventional teaching became that

ray matter structures, particularly the cerebral cortex, arepared in preterm brain injury and are developmentally re-istant to hypoxic-ischemic damage in particular in the fetaleriod. Although gray matter injury in the preterm brain was

ndeed recognized,3-5,10,42 it was downplayed, and the con-entional wisdom emerged, as evidenced in 3 representativeuotations:

1. “The human fetal brain seems to be extremely vulner-able to injury and the patterns of injury are distinctfrom those seen in adults and older children. In partic-ular, the cerebral white matter is unusually vulnerableto injury whereas the gray matter is less frequently af-fected”.42

2. “Hypoxic-ischemic injury during the prenatal periodtends to lead to white matter injury and subsequent lossin gray matter during maturation. On the other hand,hypoxia-ischemia in the term infant, ie, neonatal orpostnatal exposure, leads preferentially to damage inthe deep gray matter and parasagittal regions of theCNS”.43

3. “Though these differences are not absolute, the pretermbrain exhibits a higher degree of susceptibility of thewhite matter whereas the term brain exhibits primarilygray matter injury”.44

The use of modern neuroimaging to study preterm brainnjury and its consequences was the crucial event that refo-used, in my opinion, the neuropathologist’s attention uponhe gray matter. Volumetric MRI studies in living infants overperiod have reproducibly demonstrated deficits in multipleortical regions, thalamus, basal ganglia, hippocampus, anderebellum at term equivalent and into childhood and ado-escence.1,45-50 Moreover, these studies have shown that theseeuroimaging deficits correlate with multiple cognitive, mem-ry, or emotional problems upon follow-up evaluation.1,46,50

ognizant of the overwhelming evidence for widespread (andndeed profound) gray matter injury in the preterm brain byeuroimaging, our group undertook a “re-look” at the entirepectrum of the histopathology of prematurity through the sys-ematic survey of all gray and white matter brain regions in 41remature infants dying between 1997 and 1999, ie, during

he modern era of the premature intensive care nursery.14 We s

ivided the cases into 3 groups according to white matteristopathology, ie, PVL (n � 17), diffuse white matter gliosisDWMG) without focal periventricular necrosis (n � 17),nd neither PVL nor DWMG (n � 7). We applied the mostmportant tool of all neuropathology to this study, ie, the eyehrough the microscope. We found that over one-third of theVL cases also demonstrated gray matter lesions which wereharacterized by neuronal loss and/or gliosis41 (Fig 5). Neu-onal loss of any degree of severity by visual assessment oc-urred exclusively in the PVL cases compared with non-PVLases; its incidence in the PVL cases was 38% in the thalamus,3% in the globus pallidus and hippocampus, and 29% inhe dentate nucleus.41 Gliosis without obvious neuronal lossas more common than neuronal loss and gliosis combined

n the thalamus (56% of cases), globus pallidus (60%), hip-ocampus (47%), basis pontis (100%), inferior olive (92%),nd brainstem tegmentum (43%). Not unexpected were thendings of obvious neuronal loss in only 13% of the cerebralortex (frontal only) and gliosis alone (31%) in the PVL cases,einforcing the long-time observation that the cerebral cortexbut not the thalamus or other gray matter sites) seems his-ologically spared relative to the white matter. In a survey ofhe frequency of histopathologic features of brain injury inow birth weight infants (n � 67) by Dr Gilles and col-eagues,51 “neuronal loss” (not further defined) was foundnfrequently compared with white matter abnormalities; the

igure 5 Summary diagram of the topography of lesions in the en-ephalopathy of prematurity as determined in a cohort of 41remature infants.14 The incidence of neuronal loss or gliosis inach brain region is indicated. The large red circle represents theeriventricular foci of necrosis; the small red dots represent reactivestrocytes in the diffusely gliotic component of the white matterathology. (Color version of figure is available online.)

pecific gray matter sites were not reported.

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Encephalopathy of prematurity 185

Our neuropathologic survey of the entire preterm brainndicated to us that neuronal loss and/or gliosis are the his-opathologic hallmarks of gray matter injury in PVL and isidespread, affecting virtually all gray matter sites, albeit inariable combinations and degrees.3 Moreover, it reinforceshe finding of the neuroimaging studies that gray matter in-ury is common in PVL, occurring in our series collectively inver one-third of the cases.14 This survey has formed the basisf our current strategy, to pursue with computer-based quan-itation and immunocytochemistry in detail the neuropatho-ogic findings in all the major gray matter sites affected, be-inning with the thalamus15 and the axons coursing throughhe white matter.13 In-depth analysis by us of the thalamus,or instance, in 22 PVL cases and 16 non-PVL cases revealedhat 59% of PVL cases demonstrated thalamic damage com-ared with 16% of non-PVL cases (P � .01). Moreover, thisamage occurred in 4 different patterns, ie, diffuse gliosisith or without neuronal loss, microinfarcts with focal neu-

onal loss, macro-infarcts in the distribution of the posteriorerebral artery, and status marmoratus.15 These heteroge-eous patterns likely reflect different mechanisms, includingiffuse hypoxia-ischemia and focal embolism,15 that requireursuit each in their right. Although the damage involved allhalamic nuclei, it was noteworthy in the mediodorsal nu-leus because of key role in working memory and the reportf deficits in this function in preterm survivors, as well as inhe reticular nucleus because of key role in attention androusal and the report of deficits in these functions in suchurvivors.15

The question arises: how did we in preterm brain injuryet side-tracked to focus upon white matter injury almost tohe exclusion of gray matter injury? The answer lies, I believe,n part in the subtlety of the gray matter lesions, ie, micro-copic neuronal loss and gliosis, which are “overshadowed”y the striking cystic cavities of PVL obvious to the naked eyet brain cutting. Dr Volpe has also suggested to me the pos-ibility that early ultrasound imaging demonstrated largeeriventricular cysts; once the clinical incidence of these ra-iographic cysts substantially decreased over the last 2 de-ades,52 we were able to “see” the gray matter damage, noonger distracted by cystic PVL recognition (Dr Volpe, per-onal communication); the fortuitous introduction of volu-etric MRI over this same period also helped. In addition,istorically we did not have specific tools to “enhance” theetection of subtle neuronal loss and gliosis, such as computer-ased methods to determine gray matter volumes and neuro-al number and density or immunocytochemical methodsith the GFAP antibody to highlight reactive astrocytes, in

issue sections. It is also likely that the discernment of subtlenjury is behind the capabilities of current tools, particularlyn autopsy tissue with lengthy postmortem intervals. Detec-ion of astrocytes (comprised of subtypes with protoplasmicorphology in the cerebral cortex and fibrillary morphology

n the cerebral white matter) illustrate this issue well.53 Re-ently, a study of the adult human cortex found that proto-lasmic astrocytes could be detected only in freshly resectedpecimens obtained at surgery, for instance, temporal lobe

esections, which were fixed in paraformaldehyde, and that r

hey could not be detected in autopsy specimens fixed inormalin.53 Consequently, the concept of the preferential in-olvement of white matter over cortex in preterm brain injuryay “simply” reflect an inability to immunostain cortical pro-

oplasmic astrocytes, as opposed to white matter fibrillarystrocytes, for unknown biological or technical reasons inostmortem tissue. The failure to detect gray matter injury inhe preterm brain may also reflect the involvement of subcel-ular structures, such as dendrites, axonal terminals, andpines in neuropil that require for their elucidation certainools that are not conventionally applied at autopsy. Marine-adilla’s findings of dendritic and axonal pathology in theerebral cortex associated with white matter injury (seebove) illustrates this point well, as this pathology was de-ected only with the application of the labor-intensive Golgi’stain that is not standard in clinical neuropathology.41 Rec-gnizing the potential role of subcellular pathology in grayatter, Dr Gilles wrote in his review of acquired perinatal

eukoencephalopathy with Dr Leviton in 1984:

“Disorders with gray matter damage are not a focus ofthis review because there is little epidemiological knowl-edge about them. To a large extent this might reflect alack of data from routine postmortem examinationabout disorders of synaptogenesis, which probably con-tribute more to functionally important deficits than doesfrank neuron destruction”.6

The detection of “disorders of synaptogenesis” in the pre-erm brain will require the application of immunocytochem-stry and western blotting to assess synaptic proteins, as wells of Golgi and other special stains to visualize dendrites andpines, the major synaptic sites.

Yet, a major consideration of the reasons that substantialray matter injury was not historically recognized in the pre-erm brain was because it was not there. In my view, this haso be a consideration given the impressive skills of the pub-ishing investigators to recognize the whole spectrum of hu-

an developmental neuropathology. Perhaps gray matter in-ury has become more pronounced in the era of modernntensive premature care—an era which coincides with thatf modern neuroimaging and neuropathology—due to themergence of new diseases in association with the prolongedurvival rates of premature infants, for example, opportunis-ic infections, or to complications of the new technologieshemselves, for example, oxygen toxicity secondary to thenadvertent use of nonphysiologic levels of oxygen with me-hanical ventilation. To take but one example, thalamic dam-ge is not mentioned at all by Banker and Larroche5 in theiromprehensive study of perinatal brain injury in 1962 com-ared with multiple reports of substantial volume loss andathology in this structure in perinatal brains over a per-

od.1,3,45-50 Indeed, neuronal loss and gliosis are nonspecificonsequences of cell injury and result from oxygen toxicity,ilirubin toxicity, hypoglycemia, and infection,3,4,10 in addi-ion to hypoxia-ischemia. Perhaps the substantial thalamicnjury in the preterm brain reported by us in 2008—almost

half century following the report of Banker and Lar-

oche—is a manifestation of other metabolic/infectious in-
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ults in the modern day nursery in isolation or in combina-ion with hypoxia-ischemia. Indeed, we have already seenecular trends in the neuropathology of preterm brain injuryver the last 2 to 3 decades in the decline of cystic (as op-osed to noncystic) PVL as detected by neuroimaging in liv-

ng infants.2 We can only speculate that this decline reflectshe temporal introduction of more effective treatments forypotension and hypoxia, among other possibilities. Ourroup at Children’s Hospital Boston needs to go back to therchives in the Department of Pathology and reexamine theame microscopic slides reviewed by Banker and Larroche toest the hypothesis that the severity and type of gray matternjury we (the current neuropathologists at this venerableospital) observe was indeed not present in their day, partic-larly in the thalamus as a case in point. In the ever-wiseords of Dr Volpe (elsewhere in this series), we need to go

back to the future”.“What’s in a name?” My answer, Juliet, is “Everything”—as

itnessed by the subtle effect of the name “periventriculareukomalacia”, in my opinion, upon research in pretermrain injury. In their seminal report, Banker and Larroche did

ndeed describe gray matter damage in association with thehite matter damage, reporting that most of their cases

howed “minor changes” in the gray matter that was charac-erized by mild neuronal loss and gliosis, as well as neuronalaryorrhexis in the acute stages.5 The affected sites listed byhem were the cerebral cortex (lower layers), hippocampusSommer’s Sector), subiculum, griseum pontis, dentate nu-leus, and cerebellar Purkinje cells; germinal matrix hemor-hages were also present in 5 premature infants.5 In theirummary, Banker and Larroche stated: “Attention is drawn tounique disease of the cerebral white matter that we have

ncountered in 19% of all infants who died under 1 month ofge . . . In addition, there was a diffuse loss of nerve cells inhe cerebral cortex”.5 Thus, although they recognized thessociation of cortical injury, they recommended the termeriventricular “leukomalacia”, ie, “white matter softening”,hereby emphasizing the pathology of the white matter to theeglect of that in the gray matter. Consequently, brain injury

n the preterm infant became synonymous, I believe, withVL although Banker and Larroche were emphasizing theredominate and not exclusive feature of perinatal brain in-

ury in their experience. One major exception to the histori-al focus upon white matter injury was an insightful report in987 by my mentor, the pediatric neuropathologist Dr Arm-trong.54 In this report, Dr Armstrong observed that intraven-ricular hemorrhage in 20 infants who survived more than 1ostnatal week did not occur in isolation but rather wasssociated with choroid plexus hemorrhages in 46% of theases and additional brain findings in 92% of the cases, in-luding PVL, brainstem necrosis, hydrocephalus, and cere-ellar necrosis.54 Thus, she pointed out that PVL occurredith germinal matrix hemorrhages and other brain abnor-alities. The recognition of a complex of hemorrhagic andonhemorrhagic lesions in the white and gray in perinatalrain injury was the basis of her visionary advice that thentire spectrum of injury be considered in attempts to pre-

ent and treat any one of the lesions, and that their combined P

resence should be “suspected during clinical assessment ofurvivors” (Armstrong, 1987). Indeed, Dr Armstrong alwaysaught us to step back from the 1 slide under the microscopend “observe” the whole picture, a gift she herself so greatlyossessed.On the basis primarily of neuroimaging studies, Dr Volpe

n 2005 first introduced the term “encephalopathy of prema-urity” to emphasize the constellation of abnormalities in thereterm brain, including PVL, germinal matrix hemorrhages,ydrocephalus, and neuronal/axonal pathology.2 The spe-ific neuropathologic features of the neuronal/axonal abnor-alities in particular were substantiated by systematic stud-

es of gray matter sites and axons by our group under hisnspiration.13-15,19 The value of the use of the term “enceph-lopathy of prematurity” cannot, in my opinion, be underes-imated for it conveys the real complexity of preterm brainnjury without focus upon one specific feature (white matterathology) over another (gray matter pathology), andhereby crystallizes awareness of this complexity for the con-eptualization of comprehensive strategies to address causa-ion, treatment, and prevention. In short, treatment of whiteatter injury without the simultaneous treatment of grayatter injury will likely not prevent the complex outcomes ofreterm survivors; both treatments are needed for the “en-ephalopathy of prematurity”. Of note, Dr Volpe and I haveebated the proper term for brain injury in early life, partic-larly because the constellation of gray and white matteramage is not unique to premature infants but is also ob-erved in full-term infants, particularly those with congenitaleart disease.3,55 Indeed, the term “perinatal encephalopa-hy” may ultimately prove more accurate, with the applica-ion of “perinatal encephalopathy in premature infants” ineference particularly to premature infants.3 One additionalote on nomenclature: Dr Volpe and I have often discussedhe best abbreviation for the tongue-tying appellation “en-ephalopathy of prematurity”; we suggest the abbreviationEP”.

In the underlying mechanism(s) of the encephalopathy ofrematurity, the pathogenesis of white matter abnormalities

s likely to be the same in the gray matter, with the keyifferences that the targeted cell in the gray matter is theeveloping neuron, as opposed to the pre-OLs in the whiteatter. Thus, we propose that the white and gray matter

njury triggered simultaneously by hypoxia-ischemia occursn the sick premature infant with respiratory compromisend systemic hypotension, leading to glutamate, free radical,nd cytokine toxicity to developing OLs and neurons, withifferent topographic patterns of injury on the basis of theirevelopmental and genetic susceptibilities.3 The search for aingle or even primary cause of the encephalopathy of pre-aturity seems outmoded in light of the evidence for multi-le metabolic and infectious insults bombarding simulta-eously sick premature infants in multiple organ systems andultiple time points.3 In addition, a growing body of multi-isciplinary evidence suggests that infection/inflammationnd hypoxia-ischemia potentiate each other to produce

VL.3 Targeting the shared pathways to necrosis and apopto-
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Encephalopathy of prematurity 187

is in developing OLs and neurons is an important strategy torevent all cell death.3

oadblockshe road blocks to advancement in our understanding of theeuropathology of the preterm brain are unfortunately mul-iple and daunting. Below I have highlighted the major roadlocks that effect every day upon human research in preterm

njury in the human brain.

ncomplete Knowledge oformal Human Brain Development

n 1973, Sidman and Rakic56 published their landmark chap-er in the textbook Cytology and Cellular Neuropathologyedited by RD Adams and W Haymaker) about the develop-ent of the human brain, a chapter that provided the foun-ation, in my opinion, for all subsequent studies of humanrain development and developmental pathology. In thisomprehensive chapter, they laid out the major milestonesnd sequences of the morphologic development of the cere-ral cortex, thalamus, hippocampus, basal ganglia, cerebel-

um, brainstem, and spinal cord.56 In a way, all subsequenttudies of human brain development have been but a “fill-ng-in” of the biochemical, cellular, and molecular details ofhis beautifully delineated, structural ground plan. The ap-lication of modern immunocytochemical, western blotting,

n situ hybridization, and autoradiographic tools to humanrain development has been instrumental in supplementinglassical Golgi, myelin, and cell stains in this endeavor. Al-hough we have made tremendous strides in understandinguman brain development, including in regards to the cellu-

ar factors underlying white matter susceptibility to hypoxia-

Figure 6 The hypothesis that the hypertrophic astrocyteillustrated. (A) There is a peak in the density of astrocyprotein, the major astrocytic marker, in the cerebral whperiod between Stage 1 (OL growth, migration, and pmyelination. This peak coincides with a peak in the dmarkers of oxidative [4-hydroxynonenal (HNE)] and nitare involved in physiological processes related to free raThe changing presence of HNE-immunostaining astrocy2 of myelination, compared with their absence in the fullthe specificity of the HNE antibody used in this study.4

schemia (see above), we still do not know much about even t

uch basic events as when in the fetal period neuronal migra-ion to the cerebral cortex is complete.

A prime example of how the lack of normative informationinders insights into developmental neuropathology con-erns astrocytes. The presence of “hypertrophic astrocytes”ithout associated periventricular necrosis has long been

ecognized in the cerebral white matter of premature infants,nd Dr Gilles and his colleagues emphasized their potentialathologic significance under the PTL rubric.6,7 Indeed, hy-oxic-ischemic white matter injury may follow a continuumf damage from mild gliosis (hypertrophic astrocytes) aloneo severe (periventricular necrosis combined with gliosis).3

et, the possibility exists that astrocytes may normally un-ergo hypertrophy in the late fetal and perinatal white matters an obligatory developmental change, potentially due to thephysiological oxidative stress” of active myelin sheath syn-hesis, and thus “hypertrophic astrocytes” may not be aarker of pathology at all57 (Fig 6). On the basis of brain

nalysis in the NCPP, Dr Gilles and colleagues reported thatypertrophic astrocytes were present in the white matter of1.4% of fetuses at 20 to 27 gestational weeks and increasedo 67.7% at 36 to 44 weeks with a progressive increase fromidgestation to term.6 This observation suggests that theormative morphology of astrocytes changes with age andecomes increasingly “hypertrophic”, as opposed to the con-entionally held alternative interpretation that fetal whiteatter at term is more susceptible to injury than at midges-

ation.6 In our survey of the neuropathology of prematurity,e found gliosis (without associated focal necrosis) in thehite matter of 25% of cases at 23- to 29 weeks, 86% at 30-

o 36 weeks, and 100% at term,14 again indicating a progres-ive increased incidence with age; the attainment of “gliosis”n all the term brains raises further the interesting possibility

uman fetal brain is normative rather than pathologic ish pronounced immunostaining of glial fibrillary acidictter around the time of birth and during the transitiontion] and Stage 2 (active myelin sheath synthesis) ofof immunostained morphologic astrocytes expressinginducible nitric oxide ), suggesting that these astrocytesjury potentially relevant to myelin synthesis.21,47 (B)dicated in the human white matter in Stage 1 and Stage

inated adult white matter.47 A negative control indicatesr version of figure is available online.)

in the htes witite maroliferaensityrative (dical intes is iny-myel

hat the finding is not pathologic but rather “normal” because

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ll cases share it, perhaps as myelin synthesis and its concom-tant physiological oxidative stress triggering astrocytic hy-ertrophy is increasing57 (Fig 6). Further studies are neededo examine the significance of astrocytic hypertrophy in de-elopmental neuropathology. This challenge is heightenedy the unavoidable fact that live-born infants dying duringhe last half of gestation are not “normal” but rather die inntensive care units with multiple complications of prematu-ity that are known to adversely affect the brain (see below).

ack of the “Perfect Control”he selection of controls for neuropathologic analysis in pre-

erm brains is problematic given that all infants who die in theate fetal and neonatal period, ie, the period of risk for PVL,ie in extremis such that most have received some form ofechanical ventilation. The control brain without any injury

n this time frame is simply a myth. Given that all autopsiedreterm infants experience some degree of hypoxia-ischemiand other metabolic derangements at birth or during theirospital course, their brains may not be truly representativef those of “normal” living infants. Pathologic changes inreterm brains must be interpreted in light of potential (mi-or) abnormalities in the “control” brains. Given this caveat,e have found robust differences between PVL and controlrains in markers, for example, of thalamic and axonal dam-ge, oxidative and nitrative stress, and cytokine expressionhat suggest disease-specific phenomenon.11-22 In addition,odern neuroimaging techniques have the novel potential to

erify neuropathologic findings directly in living infants withass spectrometry, tractography, and diffusion tensor imag-

ng for myelin ensheathment abnormalities.45 In addition,hese neuroimaging techniques are able to verify sequences oformative development that were determined in autopsyrains, as illustrated for the sequences of myelination.58,59

ecline in Autopsyates in Fetal and Infant Caseshese declining rates are a major road block that is not fullyppreciated, in my opinion, by the neonatology community.t Children’s Hospital Boston, for example, the overall au-

opsy rate has plummeted in half, to 30%, between 1976 and007 (Dr HPW Kozakewich, personal communication).oreover, the autopsy rate in infants dying in our neonatal

ntensive care nursery in 2007, the last year in which suchata were finalized, was only 22%, representing 5 of 23eaths (Dr HPW Kozakewich, personal communication).rom this statistic alone, one can appreciate the difficulties inccruing a large sample size of both case and control brainsor comparative statistical analysis. The reasons for the de-line in autopsy rates include (1) fear of “upsetting” parentsurther at the time of great loss; (2) fear of litigation related tootential significant findings “missed” clinically; (3) delega-ion of autopsy consent to the most junior (inexperienced)hysicians; and (4) lack of training in physician communica-ion around autopsy-related issues, including about specificutopsy procedures.60 In addition, societal changes over the

ast 2 decades has led to an almost total lack of availability of a

mbryonic and early fetal (� 20 gestational weeks) tissues fortudy that were previously attained form therapeutic abor-ions. In short, there is a serious crisis in the capability tottain human brain tissues in the embryonic, fetal, and neo-atal periods for research—made all the more distressing byhe potential missed opportunity to study these brains withodern tissue techniques. This crisis needs to be systemati-

ally addressed, and in order not to jeopardize the progress inur understanding of preterm brain injury, which is the criticalrst step in developing strategies to prevent or ameliorate it.

ack of Realistic Quantitativeools in Autopsied Brain Tissueshe most valuable resource we have for obtaining tissue sam-les for analysis of preterm injury in the human brain is therchives of pathology departments throughout the country;ndeed, these archives at pediatric hospitals in particular arenational treasures”. To use this resource to its fullest poten-ial, it is essential to devise quantitative tools that are valid inrchival (paraffin-embedded) tissue sections. Stereology, forxample, is argued to be the most reliable method to counteurons,61 but it depends upon the accrual of fresh tissue that

s subsequently fixed and stained according to methods thatre not standard in pathology departments. Indeed, stereol-gy necessitates the prospective collection of the brains ofare cases and controls, which is logistically difficult in theaily operation of the autopsy room. Thus, we need to bepen, albeit without compromise for validity, to two-dimen-ional cell counting approaches.62 It is argued, however thathree-dimensional counting provides “unbiased” counts ofeurons, whereas two-dimensional approaches are “assump-ion-based” and therefore potentially yield inaccurate re-ults.62 Yet, all approaches are assumption-based and involvenherent biases, and thus the selection of two-vs three-di-

ensional approaches for a particular study is based uponelative strengths and weaknesses.62 Due to our dependencen archival tissue in which stereology is not an option, cou-led with the validity of two-dimensional methods in gene-al,62 the use of a two-dimensional approach is reasonablend valid.

uture Directionso advance further our understanding of the neuropathologyf brain injury in the premature infant, several future direc-ions are recommended. First and foremost, there needs to beore crosstalk between basic scientists and neuropatholo-

ists to ensure that the spectacular insights made in develop-ental neuroscience are applied to human preterm pathol-

gy. These insights relate, for example, to the relevant issuesf glutamate, free radical, and cytokine toxicity; neuronal,ligodendrocyte, and astrocytic cell lineage; synaptogenesis;xonal path finding; subplate neurons; neuronal migration;nd stem cell biology. Moreover, such crosstalk is needed tonsure hypotheses generated from human studies are testedn animal models that precise mechanisms for the cellular

bnormalities in the human brain are determined. The obser-
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Encephalopathy of prematurity 189

ation of diffuse microglial activation in the white matterurrounding focal periventricular necrosis in the encepha-opathy of prematurity, for example, focuses attention of ba-ic research upon the means to ameliorate such activation torevent pre-OL injury.63 The human findings need to guidehe experimental studies to ensure the relevance of the lattero the former.

Second, there needs to be more crosstalk between neuro-adiologists and neuropathologists to ensure that the spectac-lar insights made by modern neuroimaging guide futureeuropathological studies, and vice-versa. The autopsy find-

ng of diffuse axonal injury in the encephalopathy of prema-urity, for example, needs to be correlated with axonal pa-hology by modern tractography to translate into clinicalsefulness in diagnosis and treatment.64 Imaging of autopsyrains, although logistically demanding, is needed to corre-

ate directly pathology with neuroimaging.Third, more studies of normative human brain develop-ent at the cellular and molecular levels are needed, partic-larly in regards to the arterial vasculature, subplate neurons,

ate migrating GABAergic neurons, astrocytes, stem cells, andynapses.

Fourth, modern tissue techniques, for example, immuno-ytochemistry, receptor autoradiography, western blot anal-sis, high-performance liquid chromatography, computer-ased quantitation, and in situ hybridization, need to be

ncreasingly applied but nevertheless in creative combina-ions with classical histologic techniques that are the basis ofll neuropathological insights—witness the contributions ofrs Gilles, Marian-Padilla, and Armstrong.Fifth, we must train and mentor the pediatric neuropa-

hologists of the future with passion, as I have luckily beenentored, to bridge the gap between bedside and bench.Sixth, the decline in the autopsy rate must be reversed.Finally, the neuropathological issues so beautifully pre-

ented by Dr Volpe in the Lancet article1 and reiterated in thiseries need to be systematically addressed. We must studyn-depth the cerebral cortex, white matter, thalamus, basalanglia, amygdale, basal forebrain, hippocampus, cerebel-um, and brainstem—one by one—to characterize fully theeuropathologic substrate of the encephalopathy of prema-urity.

At the end I wonder: what do Banker and Larroche think,nd what will we think a half-century from now? Let theuture be for us as truly amazing as today must be for them.

cknowledgmentsam extremely grateful for the wonderful collaborations overhe years with Rebecca D. Folkerth, Robin L. Haynes, Ashokanigrahy, Saraid S. Billiards, Sarah Andiman, Christopherierson, Tara DeSilva, Natalia Borenstein, Stephen A. Back,nd Felicia L. Trachtenberg in neuropathologic studies of theuman preterm brain at Children’s Hospital Boston. I thankr Haynes and Mr Richard A. Belliveau for assistance in the

reparation of this manuscript.

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