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Vulnerability of the developing brain Neuronal mechanisms Adnan T. Bhutta, MBBS, FAAP, K. J. S. Anand, MBBS, DPhil * University of Arkansas for Medical Sciences, Arkansas Children’s Hospital, 800 Marshall Street, Little Rock, AR 72202, USA

About 300,000 low birth weight neonates are born in the United States each year [1], and 60,000 of them are classified as very low birth weight (< 1500 g). An overwhelming majority of these children are born preterm, at a time when the brain’s architecture and vasculature have not developed completely. The percentage of low birth weight and very low birth weight babies has increased gradually in the last 20 years, with a concurrent decline in neonatal and infant mortality rates (Fig. 1). It has been suggested that this decline in mortality rates could be the result of improved obstetric and neonatal care [1]. Despite impressive achievements in the increasing survival of this population, parents and teachers may be dismayed to find a high prevalence of cognitive deficits, learning difficulties, and abnormal behaviors during their early childhood and primary school years. Multiple follow-up studies of ex-preterm neonates have reported a high prevalence of major neurodevelopmental deficits [2– 4], with lifetime needs for special assistance [5] and increasing burdens on the annual health care budget [6].

The neurobehavioral outcomes of ex-preterm children Some preterm neonates may suffer early neurological injury caused by intraventricular hemorrhage or white matter injury leading to periventricular leukomalacia, whereas others who are spared these lesions may also develop ‘‘hidden’’ areas of cognitive disability for unknown reasons [7,8]. A metaanalysis conducted by the authors to assess the cognitive and behavioral

* Corresponding author. E-mail address: [email protected] (K.S. Anand). 0095-5108/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 0 0 9 5 - 5 1 0 8 ( 0 2 ) 0 0 0 11 - 8

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Fig. 1. Trends in the infant mortality rate (IMR), neonatal mortality rate (NMR), and post-neonatal mortality rate (PNMR), when compared to the birth rates for low birth weight, very low birth weight, and preterm neonates in the United States between 1980 and 2000. IMP, infant deaths per 1000 live births; NMR, neonatal deaths per 1000 live births; PNMR, post-neonatal deaths per 1000 live births; LBW, percent low birth weight infants (< 2500 g birth weight); VLBW, percent very low birth weight infants (< 1500 g birth weight); PT, percent preterm neonates (< 37 week’s gestation). (From Hoyert DL, et al. Annual summary of vital statistics: 2000. Pediatrics 2001;108(6):1241 – 55; with permission.)

outcomes of ex-preterm children (cases) at school age compared with term-born controls shows that the mean IQ score of former preterm children is 10.9 points lower (95% confidence intervals: 9.2, 12.5, P < 0.001) than controls. The reduction in IQ scores was significantly correlated with the gestational age and birth weight of these children; that is, the lower their gestational age and birth weight, the greater the difference between the mean IQ scores of the cases and controls. The authors found that this difference does not vary with the age at evaluation, suggesting the persistence of these cognitive differences between ex-preterm and term-born children across their entire childhood [9]. Similarly, Hack et al [10] showed a 5-point difference between the IQ scores of 8-year-old ex-preterm and term-born children and found that this cognitive difference persisted until their follow-up at 20 years of age. Decreased cognitive function was associated with a higher degree of neurosensory impairment and with significantly lower educational achievements, even after adjusting for sociodemographic status [10].

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The authors’ meta-analysis further showed that ex-preterm school children have a 2.64-fold increased risk (95% confidence intervals: 1.8, 3.8, P < 0.001) for developing attention-deficit/hyperactivity disorder (diagnosed by the Diagnostic and Statistical Manual of Mental Disorders, Third Edition or Fourth Edition criteria) and frequently manifested externalizing or internalizing behaviors during school age [9]. Multiple studies report an increased prevalence of psychiatric disorders in ex-preterm children, which may contribute to increased parenting stress and maternal depression during early childhood. The authors propose that the cognitive differences reported in this meta-analysis will have a significant impact on the educational requirements for ex-preterm children and may determine their future socioeconomic potential. Despite their enormous clinical and societal significance, the biologic mechanisms underlying these neurodevelopmental deficits remain unclear and underinvestigated.

Imaging studies of ex-preterm and term-born children One possible explanation for these cognitive and behavioral differences arises from a study published by Peterson et al [11]. Using an advanced volumetric magnetic resonance imaging technique in 8-year-olds who were born preterm (n = 25) or at term (n = 39), Peterson et al [11] discovered that poor cognitive outcomes were correlated with reduced brain volumes in specific cortical and subcortical regions. From the 25 ex-preterm children studied, none had severe intraventricular hemorrhage (grade III or grade IV) and only 1 infant had periventricular leukomalacia. One child developed cerebral palsy and one developed a seizure disorder requiring medications at 8 years of age, suggesting a relatively low prevalence of neurologic morbidity in this group of ex-preterm children. Compared with term-born controls, however, their cognitive function and visualmotor integration were substantially impaired and they had a higher incidence of attention-deficit/hyperactivity disorder, separation anxiety disorder, and simple phobias. Perinatal risk factors (eg, hypoxia, hemorrhage, severity of illness) or demographic factors (eg, gender, minority status, maternal education) did not explain these findings, indicating subtler and perhaps more pervasive influences on brain development [11]. Further morphometric analyses revealed disproportionately smaller volumes of the sensorimotor, premotor, midtemporal, parieto-occipital, and subgenual cortical areas in the ex-preterm children (Fig. 2). Subcortical regions including the corpus callosum, amygdala, hippocampus, and basal ganglia were also significantly smaller compared with their term-born peers. IQs were positively correlated with the summed cortical volumes and, most consistently, with the volumes of the sensorimotor cortex, midtemporal cortex, and corpus callosum [11]. Although Peterson et al [11] were the first to correlate cognitive outcomes with regional neuroanatomic differences, they were unable to identify any physiologic (or pathologic) mechanisms responsible for the correlation between measured brain volumes and subsequent cognition or behavior. The same can be

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Fig. 2. Differences of regional brain volumes between ex-preterm and term-born children at 8 years of age. Bonferroni-corrected probability values from the multivariate analysis of covariance and post hoc univariate analyses are color coded and superimposed on stereotactically subdivided images of the brain and ventricular system. These analyses show disproportionately smaller volumes of various cortical (sensorimotor, premotor, midtemporal, parieto-occipital, subgenual) and subcortical areas (corpus callosum, amygdala, hippocampus, basal ganglia) in the ex-preterm children compared with their term-born peers. (From Peterson B, et al. Regional brain volume abnormalities and long-term cognitive outcome in preterm infants. JAMA 2000;284(15):1939 – 47; with permission.)

said for other recent magnetic resonance imaging findings in preterm neonates, which showed abnormal white matter signals within 48 hours after birth, leading to cerebral atrophy [12] or reduced cortical gray matter at term gestation [13]. When preterm neonates were studied in later childhood and adolescence, similar parenchymal and white matter lesions correlated with significant neurobehavioral abnormalities [14,15].

Mechanisms leading to neuroanatomic differences Significantly increased neuronal death and in the immature human brain may offer a plausible explanation for the widespread volumetric reductions in cortical and subcortical brain regions noted from these studies of ex-preterm children. The cellular mechanisms leading to these changes may be investigated in animal models and human studies that explore normal neurodevelopment in the fetus and neonate and study the long-term effects of pain, stress, or other adverse experiences during the neonatal period. The theoretical framework supported by

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these studies suggests that (1) immature neurons have an enhanced vulnerability to degenerative changes, and (2) repetitive pain or other elements of the extrauterine environment during neonatal intensive care may have a significant impact on neuronal survival.

Evidence for the enhanced vulnerability of immature neurons Toward the end of human gestation, the relatively mature fetus faces a critical period of brain development just before and after birth, which corresponds to the neurologic maturity of 6- to10-day-old rat pups. This critical window is defined by peak rates of brain growth [16], exuberant synaptogenesis [17], and the developmental regulation of specific receptor populations. Among the developing populations of neuronal receptors, the commonly distributed N-methyl-D-aspartate (NMDA) receptors, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, and metabotropic glutamate receptors as sites for binding the excitatory amino acid glutamate play a key role in neuronal proliferation [18], migration [19], synaptogenesis [20], and synaptic plasticity in the developing brain [21]. NMDA receptors allow Ca++ entry into the cell, which leads to phosphorylation of second messengers and alteration in gene regulation. NMDA receptors reach a peak density at birth [22,23] and are coupled with an increased magnitude of ligand-gated Ca++ currents [24] in newborn rats. They are abundantly expressed in the human fetal brain as well [25]. Immature neurons appear to have an enhanced vulnerability to excitotoxic damage [26], which may be due to altered molecular mechanisms for Ca++ signaling [27]. This enhanced vulnerability of the immature nervous system, in the setting of increased stimulation by pathologic stressors, may lead to an excessive amount of Ca++ entry into the cell, which can initiate excitotoxic cell death (Fig. 3). Prolonged blockade of NMDA receptors or the activation of neuronal cytokine receptors (eg, the tumor necrosis factor a receptor) may also trigger apoptosis in developing neurons [28] directly or indirectly through the sequential activation of initiator (eg, caspase-8, caspase-9) and effector caspases (eg, caspase-3) [29 – 31]. This critical period is also characterized by enhanced degrees of naturally occurring neuronal death (or physiologic cell death) via apoptotic mechanisms [32,33]. Such neuronal cell death follows a developmental pattern, affecting partcular brain regions during specific developmental phases, such as the brain stem in the perinatal period [34], thalamus and other subcortical areas soon after birth [35,36], and cortical areas in the first 2 postnatal weeks [36 – 38]. The regional expression of Bcl-2 and caspase-3 appear to mediate this susceptibility to neuronal apoptosis and this phase is terminated by the reduced cellular expression of caspase-3 [39]. In situ hybridization revealed a profound developmental regulation of caspase-3, the main effector enzyme for neuronal apoptosis, with a high abundance of caspase-3 mRNA observed in fetal and neonatal neurons and decreased expression in adult neurons [40]. Rabinowicz

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Fig. 3. Mechanisms of neuronal excitotoxicity mediated via activation of glutamate receptors and receptors for other excitatory amino acids. NMDA receptors are ligand-gated ion channels that allow the entry of cations such as CA2+ and NA+; increased calcium entry activates the CA2+-calmodulin complex leading to altered gene expression, with the induction of immediate early genes (IEG) and heat shock proteins (HSP). Lysosomal degranulation results in necrosis of the neuron and excitotoxic damage to adjacent neurons. The authors hypothesize that signal transduction via opioid receptors can lead to phosphorylation of the NMDA receptor complex, resulting in stabilization of the magnesium (Mg2+) block and diminished CA2+ entry. m-OR, m-opioid receptor; Gi/Go, inhibitory G-proteins, PKA, cAMP-dependent protein kinase, PKC, protein kinase C. (From Anand KJS, Scalzo FM. Can adverse neonatal experiences alter brain development and subsequent behavior? Biol Neonate 2000;77:69 – 82; with permission.)

et al [32] calculated that large numbers of cortical neurons undergo apoptosis after 28 weeks of human gestation, with neuronal numbers decreasing by > 50% to achieve a stable number of neurons at birth. This vulnerability is not only limited to neurons but also extends to other cells of the nervous system. In a series of experiments performed on an early differentiating lineage of oligodendroglia isolated by cell culture, Volpe and colleagues [41] demonstrated that oligodendroglial cells that are present in premature human infants are exquisitely sensitive to free radical injury. The predominant mechanism of oligodendroglial cell death occurs by apoptosis. This sensitivity to free

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Fig. 4. Evidence and mechanisms suggesting an increased vulnerability of cultured oligodendrogliocyte precursors (Pre Ols) to free radicals. (A) Free radical attack is produced by 24 hour growth in cystine-deprived medium (Cys ), which results in glutathione depletion and a greater loss of Pre Ols, showing that free radicals are more toxic to Pre Ols than to mature oligodendrocytes (Ols). (B) Cystine deprivation causes a greater accumulation of free radicals in Pre Ols than in mature Ols. Free radicals were determined by a fluorescence technique. (C) Free radical metabolism with ischemia-reperfusion. The superoxide anion is generated and undergoes conversion to hydrogen peroxide (H2O2) by the action of superoxide dismutase (SOD). H2O2 is detoxified by catalase and glutathione (GSH) peroxidase. If this detoxification step fails or H2O2 accumulates, and if Fe++ is available, the Fenton reaction can produce the deadly hydroxyl radical (OH ). (D) A summary scheme for pathogenesis of Ol death under conditions of ischemia-reperfusion. The central role of free radical attack and the basis of the vulnerability of Pre Ols (impaired antioxidant defenses and acquisition of Fe++) are shown. (From Volpe JJ. Neurobiology of periventricular leukomalacia in the premature infant. Pediatr Res 2001;50(5):553 – 62; with permission.)

radical injury is maturation dependent because mature oligodendroglia survive in much greater numbers when exposed to free radicals (Fig. 4) [42]. Effects of the extrauterine environment and neonatal intensive care The developmental regulation of excitotoxic and apoptotic mechanisms, as noted above, heightens the susceptibility of the immature nervous system to the adverse experiences or metabolic insults (eg, hypoxia, hypoglycemia, and sepsis) that occur commonly in preterm neonates. Accordingly, models of hypoxicischemic injury in neonatal rats show increased neuronal necrosis in the cerebral cortex, striatum, thalamus, and hippocampus [43]. Viral infections of the neonatal mouse brain can cause increased cortical and hippocampal apoptosis [44], whereas remote stressors such as neonatal peritonitis also lead to neuronal and

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astrocytic injury, associated with impaired integrity of the blood-brain barrier in the frontal cortex [45]. Survival experiments further report a hyper-responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis, correlated with permanent changes in the median eminence and hippocampus of adult rats following exposure to neonatal endotoxemia [46]. Although neonatal intensive care aggressively treats hypoxia, hypoglycemia, or sepsis, other factors contributing to neuronal damage, such as repetitive pain or maternal separation, have received little therapeutic attention until recently [47]. When the 8-year-old children studied by Peterson et al [11] were receiving neonatal intensive care, it was customary to ignore the effects of invasive procedures (eg, heel lancing, venous catheterization, chest tube placement, etc) or aversive environmental stimuli (eg, loud noises, bright lights). Recent clinical and experimental observations suggest that the repetitive pain caused by invasive procedures and maternal separation leading to a lack of social (tactile, kinesthetic, and verbal) stimulation may have independent and perhaps inter-related [48] effects on the developmental vulnerability of immature neurons.

Effects of maternal contact With regard to maternal separation, rodent models have elegantly demonstrated the long-term effects of neonatal manipulation on the cognitive, behavioral, and neuroendocrine responses of adult rats [49 – 51]. These studies noted life-long increases in anxiety and defensive withdrawal behavior, accentuated HPA-axis stress responses, as well as deficits in memory and learning correlated with progressive damage to hippocampal neurons. In contrast, several lines of evidence underscore the importance of maternal care-giving practices on neonatal physiology and brain development, with similar results reported in a variety of species [52]. Models associated with increased handling or maternal grooming behavior resulted in decreased HPA-axis responses, enhanced neuronal survival and synaptogenesis in the hippocampus (mediated by brain-derived neurotrophic factor), and improved performance in cognitive tests (eg, the Morris water-maze test) [53]. Beneficial effects of on weight gain and neurodevelopment were noted from the tactile-kinesthetic stimulation of infant rats [54] and preterm neonates [55]. These results have helped to formulate the principles of ‘‘kangaroo care’’ (skin-to-skin contact of preterm neonates and their mothers), which may provide additional physiologic and neurodevelopmental benefits in preterm neonates [56,57].

Effects of repetitive pain Despite the recent recognition of the benefits of maternal contact, the acceptance that repetitive or prolonged pain can insidiously hinder cognitive development has, as yet, been largely ignored. For example, a similar pattern of long-term behavioral changes was noted from adult rats that were exposed to

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repetitive acute pain during the first postnatal week. Rats exposed to neonatal pain had lower pain thresholds during infancy, with increased alcohol preference, defensive withdrawal behavior, and hypervigilance noted during adulthood [58]. Neonatal rats subjected to inflammatory pain (induced by injection of formalin, complete Freund’s adjuvant, or carrageenan) also manifest robust behavioral changes during adulthood. Following complete Freund’s adjuvant injection in the neonatal period, adult rats were hyper-responsive to subsequent painful stimuli (pinch, formalin injection) [59]. Following exposure to repeated formalin injections, adult rats showed longer latencies to the hot plate, decreased alcohol preference and diminished locomotor activity [60]. It is likely that these longterm behavioral changes may result from developmental alterations of the immature pain system at the peripheral, spinal, and supraspinal levels [47]. At the peripheral and spinal level, these changes include increased sprouting of peripheral cutaneous nerves and their primary afferent connections with the dorsal horn of the spinal cord, corresponding to the area of tissue injury [59,61]. Somatotopically related dorsal horn neurons showed a marked hyperexcitability, both at rest and following noxious stimulation, as well as decreases in their receptive field size [59,62]. In the cortical areas associated with pain processing, it appears that repetitive inflammatory pain in neonatal rats leads to a significant accentuation of naturally occurring neuronal cell death [63]. Specific regions, particularly in areas of the piriform, temporal, and occipital cortex show twice as many neurons dying in 1-day-old and 7-day-old rat pups subjected to inflammatory pain compared with age-matched controls, but this vulnerability was not evident in 14-day-old rat pups [63]. Mechanisms leading to these long-term changes may include neuronal excitotoxicity (mediated via activation of NMDA or other excitatory receptors) or apoptosis (mediated via inflammatory cytokine receptors or mitochondrial injury). NMDA-dependent mechanisms not only mediate the spinal transmission of pain but also the long-term effects of pain such as hyperalgesia, allodynia, windup, and central sensitization [64] involved in the pathogenesis of chronic pain states [65,66]. Accumulating data suggest that exposure to neonatal pain promotes an increased susceptibility to chronic pain states mediated by NMDAdependent neuroplasticity [67,68]. If neonatal pain or localized inflammation truly produces these long-term changes, then analgesia or anti-inflammatory treatment should prevent or ameliorate the expression of the reported cellular and behavioral changes. A paucity of published data, however, does not allow any firm conclusions in this regard. One recent experiment showed that pre-emptive analgesia with morphine in neonatal rats exposed to inflammatory pain reduced some but not all the long-term behavioral changes noted in adult rats [60]. Preliminary evidence for the beneficial effects of pre-emptive morphine analgesia in preterm neonates comes from a blinded and placebo-controlled randomized clinical trial, which suggests a reduced incidence of early neurologic injury in the morphine-treated neonates [69]. The cognitive and neurobehavioral outcomes from a larger clinical trial (currently underway) may answer the question of whether the outcomes reported by Peterson

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et al [11] are altered by opioid analgesia, thus supporting the possibility that these changes resulted from pain-induced neuronal or white matter damage [70].

The question of fetal pain Interestingly, there is a growing body of literature shows that the human fetus, from midgestation onward, starts responding to painful stimulation by mounting a stress response. This could be another vulnerable population subjected to repetitive or prolonged pain because fetal surgery or other invasive procedures [71] are usually performed under local or regional anesthesia without consideration for the analgesic requirements of the fetus [72,73]. Despite the epochal developmental changes occurring in the immature brain, exposure of the fetus to unanesthetized surgery or repeated invasive procedures may or may not carry the same long-term neurodevelopmental consequences associated with prolonged or repetitive pain in the term or preterm neonate [58,59,74,75]. Physiologically active nociceptive pathways and reflexes in the human fetus cannot be equated with a ‘‘conscious experience’’ of pain, and the question of fetal consciousness is fraught with intense controversy. Does consciousness appear at birth or does it exist in utero, distinct from the mother’s consciousness? A British Commission of Inquiry into Fetal Sentience [76] declared that fetuses may be conscious from 6 weeks of gestation, whereas the Royal College of Obstetrics and Gynaecology countered that fetuses cannot be considered sentient before 26 weeks of gestation [77]. Afferent inputs can alter the activity of neurons in the neocortical alange by 20 weeks of gestation, when thalamocortical and cholinergic afferents form synapses with the upper subplate neurons [78], whereas noradrenergic and dopaminergic fibers start to penetrate the subplate zone by 13 weeks’ gestation and reach the cortical plate by 16 weeks [79]. Thalamocortical axons penetrate the primary somatosensory cortex by 24 weeks of gestation [80], providing the final anatomic link for the developing somatosensory system. Thus, somatosensory-evoked potentials were recorded from the sensory cortex of 25-week preterm neonates [81]. From about 20 weeks’ gestation, electroencephalographic recordings and ultrasound studies can differentiate sleep states and wakefulness [82,83], as well as responses to touch [84] and sound [85]. Experimental paradigms investigating the prenatal acquisition of memories further support the concept of fetal consciousness [86]. Thus, multiple lines of evidence suggest fetal consciousness from about 20 to 22 weeks of gestation. Fisk et al [87 – 91] and others [92] have reported robust hormonal (cortisol, b-endorphin) stress responses to the pain associated with needling of the intrahepatic vein in fetuses of 22 to 35 weeks’ gestation undergoing intrauterine transfusions. In addition, the pulsatility index of the middle cerebral artery decreased within 70 seconds after painful stimulation in fetuses from as early as 16 weeks’ gestation [93]. Although hormonal or circulatory responses do not guarantee conscious pain perception, their absence would be more likely if

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human fetuses were impervious to pain and if the sensory stimuli from invasive procedures were not reaching regulatory areas for the HPA axis or the sympathoadrenergic system, such as the prefrontal cortex and hypothalamus. In a recent comparative study, fentanyl analgesia (12.5 mg/kg of estimated fetal weight) was given directly to fetuses after intrahepatic vein cannulation and found to attenuate their stress hormone responses. Despite the possibility of direct cardiovascular or hormonal effects [94 –96], the most prominent effect of intravenous fentanyl is analgesia/sedation, and other physiologic changes in the fetus are consequent to its analgesic effect [97]. The responses of fetuses given fentanyl in this study were comparable with the physiologic responses of preterm neonates receiving fentanyl for analgesia or sedation [98,99], despite a lower gestational age (22 – 32 weeks). Therefore, these data provide preliminary evidence that the human fetus responds to pain that can be treated by opioid analgesia.

Summary Despite the improved survival of tiny preterm neonates, their neurodevelopmental outcomes remain a cause for grave concern. The authors propose two primary mechanisms leading to enhanced neuronal cell death in the immature brain: (1) NMDA-mediated excitotoxicity resulting from repetitive or prolonged pain, and (2) enhanced naturally occurring neuronal apoptosis during early development due to multiple metabolic stresses or lack of social stimulation. The pattern and magnitude of abnormalities will depend on genetic variability as well as the timing, intensity, and duration of adverse environmental experiences. Thus, cumulative brain damage during infancy will finally lead to reductions in brain volume, abnormal behavioral and neuroendocrine regulation, and poor cognitive outcomes during childhood and adolescence. The public health and economic importance of preventing or ameliorating the subtle brain damage caused by these mechanisms cannot be overestimated. This certainly justifies concerted efforts by neuroscientists and clinicians to investigate the mechanisms underlying early neuronal injury, to minimize the impact of adverse experiences and environmental factors in neonates, and to develop novel therapeutic strategies for improving the cognitive and behavioral outcomes of ex-preterm neonates.

Acknowledgments The authors gratefully acknowledge grant support from the National Institute for Child Health & Human Development and the Blowitz-Ridgeway Foundation.

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