The Developing Human Brain: Growth and Epidemiologic Neuropathology

E

CHAPTER 1

INTRODUCTION

F.H. Gilles

Publisher Summary

This chapter discusses the causes of neonatal mental dysfunction. Pathologists examining the brains of neonates with a wide variety of gestational ages need to be aware that some of the criteria of abnormality they use in the adult may be misleading. Lesions in the neonatal brain may range from simple loss of neural tissue to complex malformations resulting from various admixtures of developmental arrests, abnormalities of migration, and abortive attempts by the tissue to repair itself. However, assessment of delay in the developing nervous system presupposes that adequate standards controlled for site, systemic disease, etc., are available. At the end of gestation, the brain is growing at its greatest rate. By the second postnatal year, its weight will have increased to three times its weight at birth. The deposition of a large amount of myelin throughout the last weeks of gestation and over the first few months of postnatal life probably accounts for a large proportion of this weight gain. Thus, this transient special variety of tissue, myelinating white matter, might be susceptible to a unique class of insults, and evaluation of its degree of maturation is of the great potential value to the pathologist.

Background

Conceptual Problems

Hypothesis Generation

Pathologic Changes in Neonatal Brains

The Pathologic Material in the NINCDS Collaborative Perinatal Project

Observer Variability

The Criteria Used in This Study

The Assumptions of This Study

Goals

Confounding Factors

Range of Abnormalities

Preview

Project Directors

Pathologists

Acknowledgments

BACKGROUND

The physician is often frustrated in the traditional roles of diagnostician, healer, researcher, and teacher when trying to deal with the problems of children labelled as having retarded mentation, disabled learning ability, or cerebral palsy. First, it is difficult to make an etiologically and prognostically specific diagnosis based on the few neurologic signs and symptoms available to the examiner of the neonate or young child. This difficulty is compounded in older children when the physician has to deal with cerebral problems whose clinical manifestations lie entirely within the interaction between the child and his society (e.g., the child with retarded mentation or disabled learning ability). Second, the physician’s frustration heightens with his attempts to treat a paralyzed or malfunctioning part, or to prescribe within a discipline in which he is traditionally ill-prepared (e.g., the prescription of an educational plan for the child with an intellectual disability). The third role of the physician, that of the searcher for the significant antecedents to these problems, is even more difficult for a variety of reasons. Since these conditions do not terminate life, their analysis must wait for many years until brain specimens become available, many years during which the risk of incomplete data, losing records, or the death or dimming of memory of informants is very high indeed, making it unlikely that significant antecedents will be recognized. For these reasons it is understandable that the physician in his fourth role as teacher frequently has given short shrift to these conditions and their serious societal impact when instructing younger generations of physicians.

In this context, the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) undertook the prodigious task of prospectively collecting data throughout pregnancy and childhood for a large number of children in an attempt to identify those aspects of childhood cerebral dysfunction which might be accounted for by modifiable maternal, fetal, or perinatal events. The hope was that methods would be found to sort out the data and demonstrate potentially treatable or modifiable associations between events or sets of events and these conditions.

For our group to accomplish these ends for the study of brains of fetuses and infants who died, three conditions needed satisfaction: (1) Base lines or norms for many cerebral developmental events in populations of dead human infants had to be formulated as many were not available. (2) Reliable methods for measuring morphologic deviations from these norms and for measuring associations in populations of autopsied individuals between potential antecedents and morphologic abnormalities had to be developed. (3) Demonstrated associations between potential antecedents and morphologic abnormalities generate hypotheses applicable to the dead only. These hypotheses must then be recast in potentially negatable forms for testing in the living or in the laboratory before they can be used therapeutically. The data for the living and dead infants in this study were collected in identical fashion and the living were followed for many years, thus providing a substantial data base for the testing of hypotheses.

CONCEPTUAL PROBLEMS

The problem of the diaphanous relationship between cause and effect has plagued the pathologist concerned with disease of the human (see Chap. 2, Epidemiologic Methods). Some relationships between antecedent and disease state are both sufficiently strong and clear that the physician has a good idea of the antecedent when he sees patients with some kinds of disease. However, even the celebrated man-in-the-street is well aware that although he may be exposed to a disease agent, he will not necessarily get the disease, or, if he gets the disease, he will not necessarily have to be hospitalized, or, if hospitalized, he will not necessarily die. The physician’s thinking has been too often colored by his hospital-based experience. He may find that all patients with a specific syndrome have a common antecedent (e.g., all patients with radiologic tuberculosis have tubercle bacilli demonstrable in a body fluid), and he may be then seduced into the untenable position that that antecedent is the only necessary antecedent and a sufficient cause (e.g., all people exposed to tubercle bacilli get tuberculosis).

The pathologist is in a similar position. If all patients autopsied with disease X have factor Y, he may be tempted to assume the validity of extrapolation to the living, that all people with factor Y have, or will have, disease X. What he has forgotten, of course, is the necessary corollary of only if they are autopsied, an event which he has no way of predicting about the living with accuracy from his data alone.

The detection of significant associations between neurologic damage and its antecedents in the neonate is on very shaky ground for several reasons. A complex interaction between two individuals (fetus and mother) is involved prenatally and postnatally. The living neonate, with its immature nervous system, sometimes stubbornly refuses to give up information of adequate diagnostic and prognostic value until long after the neonatal period. Even if neurologic abnormalities in the neonate could be ascertained with complete accuracy, the chance of correctly evaluating the potential antecedents of these abnormalities is lessened by the complex interaction between mother and fetus; certainly, with advanced postnatal age the chance of detecting significant neonatal or prenatal events is lessened. In addition, the chance that the analysis of a single case, with a small malformation within the cerebrum, for example, could give a direct clue to a specific etiologic relationship with some event in the past is very small indeed because very few abnormalities are specific to specific clinical antecedents. Further, since the plasticity and the repair capabilities of the human fetal nervous system are unknown, estimates of the timing of the abnormality would be, at best, speculative.

HYPOTHESIS GENERATION

Our primary goal in this volume is not to identify the elusive cause but to generate hypotheses about relationships between potential antecedents and morphologic events that can subsequently be tested in the living child using specific measures of neurologic or cerebral function. Thus, we feel that there is a strong advantage to us as autopsy pathologists in reviewing the material from the NINCDS Collaborative Perinatal Project in having all of the data collected prospectively in the same fashion in both the population of dead infants and in the population of clinical interest, namely, the living.

The NINCDS Collaborative Perinatal Project affords the neuropathologist and epidemiologist the power obtained from the use of populations (that of detecting trends not immediately obvious from inspection of antecedents alone or inspection of specimens alone) and the opportunity to do the heretofore impossible, namely, to extrapolate to the living derived from the same initial population as the dead. Thus, we have the opportunity to generate hypotheses about relationships between potential antecedents and morphologic events in the brains of the dead, to frame these hypotheses in negatable form, and to test these hypotheses in a population of living from which the perinatal data was collected in identical fashion, at the same institution at which the autopsies were done (i.e., from the same base population), and over the same limited period of time.

PATHOLOGIC CHANGES IN NEONATAL BRAINS

The pathologist faced with the necessity of examining the brains of neonates with a wide variety of gestational ages needs to be aware that some of the criteria of abnormality he uses in the adult may be potentially misleading. For instance, macrophages in moderately cellular leptomeninges, perivascular cuffs of small mononuclear cells near ventricular surfaces, or large numbers of microglia-like cells in telencephalic structures would be abnormal if encountered in the mature brain, but are usually normal in the brain of the infant.

Lesions in neonatal brain may range in apparent complexity from simple loss of neural tissue (e.g., necrosis or infarction) to complex malformations resulting from various admixtures of developmental arrests, abnormalities of migration, and abortive attempts of the tissue to repair itself. Another broad group of lesions is characterized merely by delay in acquisition of some component of the developing brain (e.g., myelin). The assessment of the latter lesion by the pathologist and the evaluation of its antecedents may have more long-term social significance than the more dramatic lesions listed above because of the greater number of children at risk of these antecedents (such as malnutrition). However, assessment of delay in the developing nervous system (in terms of weight, neuronal mass, complexity of dendritic tree and spine arborization, or degree of myelination, for instance) presupposes that adequate standards controlled for site, systemic disease, etc., are available. Unfortunately, such standards for the developing human nervous system are largely unsatisfactory since most are based on small samples or anecdotal evidence, even for such a rudimentary measure of growth as weight, or have been limited by excluding cases for ambiguous reasons. A similar limitation applies to the phenomenon of myelination in the human fetal brain; the available tables are constructed from relatively small numbers of cases or fail to estimate normal biologic variation within each tract as it myelinates, either for time of onset or rate of myelination.

Thus, one of our tasks in this volume is to point out the clues that can be gleaned by the pathologist from unmyelinated or myelinating brain, a tissue whose tactile and visual characteristics differ considerably from those of the homologue in the adult, a tissue whose normal constituents and reacting cells are still not in the adult form and have not reached mature capabilities.

At the end of gestation the brain is growing at its greatest rate. By the second postnatal year its weight will have increased to three times its weight at birth. The deposition of a large amount of myelin throughout the last weeks of gestation and over the first few months of postnatal life probably accounts for a large proportion of this weight gain. Thus, this transient special variety of tissue, myelinating white matter, might be susceptible to a unique class of insults, and evaluation of its degree of maturation is of great potential value to the pathologist. Similarly, another transient tissue, the tissue lining the ventricular system, may be subject to a unique set of insults.

THE PATHOLOGIC MATERIAL IN THE NINCDS COLLABORATIVE PERINATAL PROJECT

The population of brains in the NINCDS Collaborative Perinatal Project (NCPP) is derived largely from neonates who died a short time after birth; the number of children who died later in childhood and whose brains were available for examination was disappointingly small. The fact that the population we are dealing with died close to the time of birth raised another danger, namely, confusing changes in brains associated with death (e.g., ischemic change, neuronal sclerosis) with the changes likely to persist and underlie cerebral abnormality had the child lived. Another methodologic problem, devastating to concepts about antecedents, is that of selecting a group of patients with a common pathologic condition (e.g., hyaline membrane disease) and describing their neuropathology as though it were specific to their common pathologic condition without ascertaining whether this neuropathology differs from that found in appropriate controls. To describe the neuropathology of babies with hyaline membrane disease, for instance, one must show at a minimum a significant difference in the rates or proportions of cases with hemorrhages, necroses, etc., in a population of the same age dying with something other than hyaline membrane disease.

There are several distinct populations within the NINCDS Collaborative Perinatal Project. Approximately 1,100 brains were in a condition which allowed some degree of analysis. Of the approximately 640 brains collected during the first half of the study, 425 were selected by P. I. Yakovlev for the whole brain serial sectioning. The brains remaining in the first half and all of those in the second half were prepared by paraffin processing by L. Lipkin.

Various subpopulations were appropriate to the purposes of each of the studies in this volume. Several examples will show how we constructed these subpopulations: a) For the brain weight studies, all brains that had a fresh brain weight and a pathologist’s statement as to condition were used initially. For various aspects of the study, different subgroups were used; e.g., all non-stillbirth brain weights without gross hemorrhage, malformation, necrosis, or hydrocephalus were used for constructing the final Gompertz function, b) For the purpose of the selection of the group of brains for serial sectioning only minimal mechanical distortion was allowed, c) For the intracranial hemorrhage studies more distortion was allowed because of the nature of the distortion from rupture of large hemorrhage.

The use of multiple pathology departments located in many parts of the country in a collaborative study which lasted many years required a common, detailed autopsy protocol for the large number of pathologists who removed, first examined, and fixed the brains. Internal comparability of the final interpretation of cases was maximized by the use of a small number of neuropathologists responsible for the processing and handling of the fixed brains, and limiting the final preparation of the descriptive aspects of this study to members of one group.

Early in the study P. I. Yakovlev set aside some 425 brains for serial sectioning. Each of these brains was photographed in six planes resulting in some 2,400 black and white prints. The brains were embedded whole in celloidin and were serially sectioned and stained using standard techniques (Yakovlev, 1970). In excess of 100,000 slides were generated and are currently maintained at the Armed Forces Institute of Pathology.

In the perinatal section at the NINCDS the remaining brains were prepared under the direction of L. Lipkin. After photographs of the external surfaces were obtained, the brains were cut in a standardized fashion at approximately three mm intervals; each slab was photographed, and multiple blocks were taken and processed in paraffin. In excess of 30,000 color transparencies of whole brains and slabs were prepared. Multiple stains on sections from each of the blocks were prepared. The vast majority of the resulting approximately 100,000 slides were prepared in a single laboratory. Thus, these two sets of brain material, each prepared by one of the traditional methods of neuropathology, potentially allow different kinds of questions to be answered.

OBSERVER VARIABILITY

To minimize observer variability in the ascertainment of the data collected for each of the morphologic studies in this volume, each photograph and each slide chosen for study was simultaneously reviewed either grossly or with a double-headed microscope by two observers (F. H. Gilles and E. C. Dooling), a technique thought to reduce observational variation (Gilles, Winston, Fulchiero, and Leviton, 1977). The data were recorded on standard check-off sheets abstracted from a matrix of approximately 700 possible combinations of site and disease process.

THE CRITERIA USED IN THIS STUDY

The criteria of morphologic abnormality used throughout this study satisfied three conditions: (1) they had to be simple or broad criteria which (2) were easily recognized and (3) were unlikely to have been modified by minor variations among institutions in handling and fixation. Thus we chose criteria like coagulative or cystic necrosis, hypertrophic astrocytes, mineralization, and hemorrhage rather than pyknosis, hyperchromia, or loosening of tissue. We chose neuronal phagocytes rather than satellitosis. Finally, we chose terms that were descriptive rather than interpretative for the primary collection of the data. Thus, attention was directed at each level of data collection toward methods of assuring maximal internal comparability of the data.

THE ASSUMPTIONS OF THIS STUDY

Any study is based on many assumptions and biases, some identified and many unrecognized. We recognized and tried to deal operationally with each of the following assumptions:

It was assumed for the purposes of the analyses in this study that all of the brains were abnormal. Most were derived from a group of infants sufficiently abnormal in some way to have aborted spontaneously or to have died relatively shortly after birth. The number of specimens obtained from induced abortions was very small. Thus, statements about delay in acquisition of a structure compare early and late in a population of abnormal infants. On one hand, this assumption may appear to exaggerate the contribution of a risk factor under some circumstances. But on the other hand, it may appear to diminish the contribution of a risk factor to a morphologic condition if the risk factor is common to all dead (therefore abnormal) children.

While assuming that all brains were abnormal, the suspicion cannot be stilled that some of the brains approached what is normal. Further, it is obvious that for judgments about developing human brain this is all we have now and all we will have in the future. We do not even have that figment of the imagination, the so-called normal brain obtained after a traumatic death.

Extrapolation of conclusions to the living human child from information about the dead or from experimental results derived from subhuman animals must be done with care. We may strongly suspect comparable processes in living children, but without additional evidence one must constrain this extrapolation. One new source of such evidence is the computer assisted tomography, which has shown intraventricular hemorrhage to be more common in living premature infants than previously suspected. Without this kind of data obtained in the living neonate we feel strongly that the above kind of direct extrapolation is not ethically justified. Thus, hypotheses can be generated about associations between clinical events and morphologic abnormalities in a population of the dead. These hypotheses are applicable to the dead only, until some additional information allows extrapolation to the living in whom a different manifestation can be expected (one can’t see a morphologic change within the brain of the living child, only the functional or radiologic abnormality). The NINCDS Collaborative Perinatal Project is unique in that it potentially allows testing of these hypotheses in a population of the living, with data that was collected prospectively in an identical fashion in both living and dead.

Operationally, for each of the studies in this volume, populations were chosen which seemed appropriate to the specific study. In each study the cases excluded and the reasons for exclusion are carefully delineated. For example, for the study of the development of gyri, cases with gross abnormality or cases in which the tissue had been grossly distorted were excluded. For the study of growth of the brain in weight, the operating procedure was sequentially modified so that all brain weights were included initially, and then selected subgroups (e.g., all stillbirths) were removed and the growth function and constants were again generated.

The second assumption was that, in evaluating the development of an organ with the complexity of the human brain, growth may be measured in many ways. There is no single correct way. Growth is generally a continuous process; however, we are unable to sample a single growing fetus repeatedly during gestation. This limits us to providing best estimates of growth from fetuses dead at different times in development, thinking of the fetus as moving from one stage to the next. Since each way of measuring growth has its own strengths in contributing to our understanding, each is valid. Although we use the traditional strategy of measuring growth against the independent variable of estimated gestational age, we also "use other parameters as the independent variable in some of the studies.

One traditional independent variable, estimated gestational age, may be in error by several weeks. Further, estimates of gestational age adjusted for correctness of body length or weight may be improved but suffer seriously because the bias of selective loss of data (for instance, how do you then deal with the excluded case?) and because of contamination of the external independent variable (gestational age) by factors which may, in fact, be dependent (vide infra). The major advantage of the use of gestational age is that this independent variable is regularly sequenced throughout gestation and, once determined, is free of events occurring within the mother or baby.

Body weight and crown-rump length are two variables frequently used as abscissae. While they are easily ascertained at birth or autopsy, they suffer from serious limitations for the purposes of this study. First, changes in body length or in weight of the body or brain are unequal throughout gestation and tend to decrease near term (Chap. 6) where greatest discrimination might be desired (in the comparison with the degree of myelination, for instance). Thus, one would recognize only coarse discrepancies, probably even with the use of ratios such as body/brain weight. Second, factors which might inhibit or fail to support some component of brain growth could be expected to also affect body weight or length. Consequently, if weight or length are used as the independent variable, these factors could conceivably be overlooked. Third, the allometric relationship between the simultaneous growth of the brain and some other part of the fetus precludes, to some extent, meaningful analysis of the processes of growth itself (Laird, Barton, and Tyler, 1968).

The following assumptions are related. We assumed that recognizable structural abnormalities underlie some of the functional cerebral deficits classified as mental retardation, cerebral palsy, learning disability, etc. Some of these structural aberrations appear during gestation and may have their origin in events that alter the environment of the fetus and neonate. Morphologic expressions of these alterations of the normal course of human cerebral development constitute natural experiments which can be used in the investigation of disease etiology. This last point is important because appropriate experiments on the human infant are inconceivable and the direct extrapolation from experiments on subhuman primates is fraught with conceptual and ethical problems. It is hoped that maternal and perinatal factors that contribute to perinatal cerebral morbidity may be directly susceptible to modification (even though the exact etiologic mechanism may not be appreciated). A related assumption was that these structural abnormalities can be separated from morphologic abnormalities of the nervous system found in any fetus, neonate, or infant as a component of dying. The significance of the last assumptions seems self-evident; the significance of the next may not be immediately obvious.

In spite of the societal definition, the process of death is rarely instantaneous. Even when cardiac action ceases, some cellular metabolic activities may continue. Prior to final cardiostasis one may assume that there is considerable discord in the internal milieu from instability of cardiovascular, respiratory, and metabolic control. Thus, a wide variety of changes in the central nervous system (e.g., vacuolization of neuropil, cellular shrinkage, neuronal hyperchromia) potentially may reflect merely those events accompanying the termination of life. While the acute terminal morphologic changes in the brain may in fact be similar to those changes which take place during an insult that is survived, their similarity has never been adequately demonstrated in the human. Further, one must assume that there are most likely quantitative and qualitative differences between those insults which damage developing brain, but that allow survival, and those insults which accompany death. The fact that termination of life may indeed be associated in some brains with abnormalities traditionally called ischemic or hypoxic neuronal change, as well as with relative lack of oxygen, supports, but does not prove, a relationship between the two. Consequently, the observation that relative hypoxia and brain damage both appear to accompany difficult delivery hardly proves a casual relationship, and, even more important, may preclude adequate search for other potentially treatable antecedents coincident upon difficult delivery. For our purposes, we found karyorrhexis, coagulative necrosis, macrophages, glial scars, intramural vascular deposits, frank neuronal depletion, malformation, etc., of greater interest than acute changes in neurons.

Another assumption was based upon a continuum of causality hypothesis. It is that the morphologic abnormalities commonly found in the brain of the dead newborn are comparable in nature, site, and rate to those found in the brains of people afflicted with mental retardation, cerebral palsy, and learning disability. This is such a difficult assumption to resolve because of the lack of adequate data that, for the purposes of this study, we have assumed comparability at least at some level. There are two corollaries to this assumption. While there is little representation in the NINCDS Collaborative Perinatal Project of nervous system storage disease, tumors, progressive degenerative diseases, etc., it is clear that the great mass of patients afflicted with mental retardation, learning disabilities, cerebral palsy, etc., do not have storage, degenerative, or neoplastic diseases. Thus, the population encompassed by the brains derived from the NINCDS Collaborative Perinatal Project may be quite appropriate in this respect. The second obvious corollary is that the abnormalities in the brains of humans so severely afflicted by their cerebral condition to be institutionalized for life may or may not be the same qualitatively or quantitatively as the abnormalities in the brains of individuals partially socially disabled but not institutionalized.

GOALS

The goals of the contributors to this volume are easily delineated. The first is to determine the developmental sequence of selected maturational events. The second is to identify gestational, perinatal, or neonatal experiences which delay, inhibit, or prevent the orderly expression of sequential maturational events. Of course, those experiences identified as risk factors in the studies which follow apply only to this population; namely, a population collected largely before the advent of modern respiratory therapy and neonatal intensive care units. However, we can construct hypotheses about the contribution of these risk factors to mental retardation, neurologic deficit, and learning disability that can be tested in living children for whom comparable data was collected under identical conditions. The third goal is that of refining, where necessary, our current concepts of reactivity, repair, and plasticity, insofar as possible on the basis of the central nervous system material in the NINCDS Collaborative Perinatal Project.

CONFOUNDING FACTORS

In each of the studies in this volume we have tried to keep these goals in mind during the data analysis and simultaneously to consider several confounding factors. The first of these factors is that there normally may be a large biologic variation in an organ as complex as the developing human brain. The second is the difficulty of drawing firm conclusions when multiple mutually interdependent variables simultaneously contribute to a morphologic abnormality. For instance, there is a multitude of factors which increase the risk of necrosis. The third is the contribution of observer variability.

RANGE OF ABNORMALITIES

The range of cerebral abnormalities encompassed in the NCPP neuropathology sample was similar to that encountered in the neonatal neuropathology service at the Children’s Hospital Medical Center in Boston. There were relatively few congenital malformations of the brain. In many ways this reflects what is found in populations of brains derived from institutions concerned with the care of the neurologically damaged. It would appear that the bulk of dead neonates, as well as those older institutionalized children, do not die with congenital malformations of the brain. Some infants or children in both populations have no apparent morphologic abnormality. A large proportion of these infants and children had what appears to be brain tissue which had been normally formed until the time of the insult. Thus, this volume will reflect these observations in a general sense, and will be largely concerned with topics such as delay in myelination, intracranial hemorrhage, and the telencephalic leucoencephalopathies rather than with malformations.

PREVIEW

This volume is organized along the following lines: Section 1 (Chaps. 2-5) introduces the reader to the epidemiologic methods we have used, to the limitations of autopsy data in general, to the selection biases inherent in the creation of the NINCDS Collaborative Perinatal Project neuropathology sample, and to an overall description of the morphologic sample. Section 2 looks at growth of the fetal human brain from several viewpoints. In Chap. 6 a growth function is derived and presented as a model for the growth in weight of the human fetal brain. In Chap. 7 the beginning of telencephalic growth is briefly reviewed and the developing cerebra are considered as olfacto-cerebral outpouchings. The presence of the primordia of the fornix, hippocampus, striatum, and amygdala at the time of the telencephalic outpouching seems to diminish the need for the ideas of secondary cleavage, rotation of the cerebral hemisphere on its stalk, or secondary fusion between the telencephalon and the diencephalon. The evaluation of growth is then continued in Chap. 8, which deals with changes in surface area of the cerebral hemispheres and volume of the subventricular zones of the ganglionic eminence. Chap. 9 depicts growth as changes in sulcal patterns. The section on growth is continued with two more chapters; one deals with the mesodiencephalic junction and the transient melanin pigment in the pineal, and the other deals with changes in the ependymal lining of the lateral ventricles with advancing fetal age. In Chap. 12 regional myelination is used to explore growth further. The degree of myelination ascertained at 53 sites is used to examine sequence and synchrony among central nervous system tracts myelinating at different gestational times. Information on the timing of myelination is contained in charts of the age-specific rates for each degree of myelination at each site. An easily used method of evaluating the overall degree of myelination in a new fetal brain is presented.

Section 3 begins with a study of the risk factors of delayed myelination. Chapters follow about intracranial hemorrhages and their risk factors in the fetus. The next group of chapters begins with an overview of the kinds of damage which can be recognized in the fetal human nervous system (Chap. 17). This is followed by eight chapters dealing with the epidemiology of the telencephalic leucoencephalopathies. The final two chapters deal with the importance of the end of the second trimester to the growing brain and provide a summary. While we have sampled the literature freely, we have made no attempt to review it completely.

PROJECT DIRECTORS

This volume depended upon multiple individuals for clinical data collection and case follow-up, and for the preparation of neuropathology material. The project directors at the 14 collaborating institutions were as follows: Harold Abramson, M.D., John Adriani, M.D., John A. Anderson, M.D., Virginia Apgar, M.D., Richard Baetz, M.D., A. A. Baker, M.D., Daniel F. Baker, M.D., Harry M. Beirne, M.D., Ralph C. Benson, M.D., Heinz W. Berendes, M.D., Edward H. Bishop, M.D., Thomas Boggs, M.D., John Bornhofen, M.D., A. M. Bongiovanni, M.D., Glidden L. Brooks, M.D., Sidney Carter, M.D., William M. Clark, Jr., M.D., Stewart H. Clifford, M.D., Conrad Collins, M.D., Robert E. Cooke, M.D., Jean A. Cortner, M.Q., A. Reynolds Crane, M.D., Edward C. Curnen, Jr., M.D., Brigitte de la Burd6, M.D., D. Anthony D’Esopo, M.D., Arthur G. De Voe, M.D., James E. Drorbaugh, M.D., Charles Dunlap, M.D., Donal Dunphy, M.D., Rudolf Engel, M.D., Robert O. Fisch, M.D., Edmund P. Fowler, M.D., Richard Fowler, M.D., Marguerite J. Gates, M.D., Luke Gillespie, M.D., Marvin Green, M.D., Janet B. Hardy, M.D., Ruth Hase, M.D., Alan J. Hill, M.D. (deceased), James G. Hughes, M.D., S. Leon Israel, M.D., Charles A. Janeway, M.D., Charles Kennedy, M.D., Donald W. King, M.D., Lawrence J. Kolb, M.D., Sheldon B. Korones, M.D., Robert Kugel, M.D., William E. Laupus, M.D., Lewis P. Lipsitt, Ph.D., Milton McCall, M.D. (deceased), Rustin Mcintosh, M.D., Donald G. McKay, M.D., John McKelvey, M.D., Gilbert Mellin, M.D., H. Houston Merritt, M.D. Abe Mickal, M.D., Agnes Milan, M.D., J. George Moore, M.D., Craig Muckle, M.D., James G. Mulé M.D., W. T. Newsom, M.D., Kenneth R. Niswander, M.D., Richard W. Olmsted, M.D., Richard Pattison, M.D., Richmond Paine, M.D. (deceased), Emanuel M. Papper, M.D., R. V. Platon, M.D. (deceased), L. Paul Rasmussen, M.D., Sheldon Reed, Ph.D., Duncan E. Reid, M.D., John A. Rose, M.D., Mitchell I. Rubin, M.D., Robert Sappenfield, M.D., Phil C. Schreier, M.D., T. F. McNair Scott, M.D., Adolph H. Sellman, M.D., Richard L. Sleeter, M.D., Lawrence B. Slobody, M.D., Charles Steer, M.D., Joseph Stokes, M.D., Martin L. Stone, M.D., Howard C. Taylor, Jr., M.D, H. Hudnall Ware, Jr., M.D, Edward Wasserman, M.D, Milton C. Westphal, M.D, and Anthony Zangara, M.D.

PATHOLOGISTS

The responsible pathologists: George W. Anderson, M.D., Lester Belter, M.D., William A. Blanc, M.D., Barbara Burke, M.D., Shirley Driscoll, M.D., Federico G. Fuste, M.D., Warren W. Johnson, M.D., Harry Kim, M.D., Gordon Madge, M.D., and Alexander Sedlis, M.D.

Perinatal Research Branch pathologists: Lewis E. Lipkin, M.D., Luz A. Froehlich, M.D., Toshio Fujikura, M.D., Jack H. Carleton, M.D., Gerald M. Fenichel, M.D., and Carl M. Leventhal, M.D.

ACKNOWLEDGMENTS

The authors acknowledge their responsibility to these individuals:

To Elaine Burke for her editorial work and unfailing enthusiasm; to Jean Kanski for her assistance with the medical art; to Ellen Nash for the preparation of the references; and to Terry Wrightson and his group who were responsible for the photographic work. Multiple publishers and the Armed Forces Institute of Pathology generously allowed use of their material. Alan R. Liss, Inc., Publisher, Journal of Comparative Neurology for Figs. 7-1, 7-7, 7-8, 7-10, and 7-15; Franz Deuticke for Figs. 7-2, 7-3, 7-11, 7-12, 7-13, 7-14, and 7-19; Lippincott for Fig. 7-4; Armed Forces Institute of Pathology (Yakovlev Collection) for Figs. 7-5, 7-16, 7-17, 9-2, 9-3, 10-2, and 10-3; Cambridge University Press for Figs. 7-6 and 7-9; Journal für Hirnforschung for Fig. 7-18; Little, Brown and Co., Publisher, Annals of Neurology for Fig. 9-1 and 11-1; and the Archives of Neurology for Fig. 9-4.

Finally, I find it necessary to mention the not inconsiderable awe the NINCDS Collaborative Perinatal Project evokes when one considers the magnitude of joint human endeavor which it represents. First, of course, was the prolonged patience and understanding of a large number of enlightened parents and their children to complete their individual contributions to the study. Second, was the gigantic administrative task of the Perinatal Research Branch and the Office of Biometry and Epidemiology of the National Institute of Neurological and Communicative Disorders and Stroke and the faculties and administrations of the 14 collaborating institutions towards accomplishing a goal established over 20 years ago by an enlightened group of physicians, administrators, and legislators. Individual acknowledgements to many of these dedicated people were made in the first major monograph from the project (see THE WOMEN AND THEIR PREGNANCIES, 1972, K. R. Niswander and M. Gordon, editors, Appendix A). Special thanks are also expressed to the many individuals who served on the Perinatal Research Committee, ad hoc advisory committees, task forces, and as individual consultants to the project. Dr. Stanley M. Aronson, chairman of the Pathology Task Force, deserves special recognition. I wish to express my thanks to the Directors of the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS), who, over the many years of the project, have supported and promoted its development: Pearce Bailey, M.D., Director, 1951 to 1959; Richard L. Masland, M.D., Director, 1959 to 1968; Edward F. MacNichol, Jr., Ph.D., Director, 1968 to 1972; Donald B. Tower, M.D., Director, 1972 to 1981; and Dr. Murray Goldstein, Acting Director, 1981 to the present. I also wish to acknowledge the contributions to the project by Dr. Heinz Berendes, Chief, Perinatal Research Branch, NINCDS, 1960 to 1971, Dr. Joseph S. Drage, M.D., Chief of the Developmental Neurology Branch (formerly the Perinatal Research Branch), and William Weiss, Chief, Office of Biometry and Field Studies, NINCDS. I hope that we, in turn, have done justice to this effort which has given physicians interested in the morphology of the developing human brain a unique opportunity to examine, record observations, and provide interpretations of this material, to generate hypotheses about relationships between antecedents and morphologic events, and to test these hypotheses in a living population whose perinatal data was collected in the same prospective fashion as that of the subgroup of the children who failed to