Association of Prenatal Exposure to Maternal Drinking and Smoking With the Risk of Stillbirth.

Importance: Prenatal smoking is a known modifiable risk factor for stillbirth; however, the contribution of prenatal drinking or the combination of smoking and drinking is uncertain. Objective: To examine whether prenatal exposure to alcohol and tobacco cigarettes is associated with the risk of stillbirth. Design, Setting, and Participants: The Safe Passage Study was a longitudinal, prospective cohort study with data collection conducted between August 1, 2007, and January 31, 2015. Pregnant women from Cape Town, South Africa, and the Northern Plains region of the US were recruited and followed up throughout pregnancy. Data analysis was performed from November 1, 2018, to November 20, 2020. Exposure: Maternal consumption of alcohol and tobacco cigarettes in the prenatal period. Main Outcomes and Measures: The main outcomes were stillbirth, defined as fetal death at 20 or more weeks' gestation, and late stillbirth, defined as fetal death at 28 or more weeks' gestation. Self-reported alcohol and tobacco cigarette consumption was captured at the recruitment interview and up to 3 scheduled visits during pregnancy. Participants were followed up during pregnancy to obtain delivery outcome. Results: Of 11663 pregnancies (mean [SD] gestational age at enrollment, 18.6 [6.6] weeks) in 8506 women for whom the pregnancy outcome was known by 20 weeks' gestation or later and who did not terminate their pregnancies, there were 145 stillbirths (12.4 per 1000 pregnancies) and 82 late stillbirths (7.1 per 1000 pregnancies). A total of 59% of pregnancies were in women from South Africa, 59% were in multiracial women, 23% were in White women, 17% were in American Indian women, and 0.9% were in women of other races. A total of 8% were older than 35 years. In 51% of pregnancies, women reported no alcohol or tobacco cigarette exposure (risk of stillbirth, 4 per 1000 pregnancies). After the first trimester, 18% drank and smoked (risk of stillbirth, 15 per 1000 births), 9% drank only (risk of stillbirth, 10 per 1000 pregnancies), and 22% smoked only (risk of stillbirth, 8 per 1000 pregnancies). Compared with the reference group (pregnancies not prenatally exposed or without any exposure after the first trimester), the adjusted relative risk of late stillbirth was 2.78 (98.3% CI, 1.12-6.67) for pregnancies prenatally exposed to drinking and smoking, 2.22 (98.3% CI, 0.78-6.18) for pregnancies prenatally exposed to drinking only after the first trimester, and 1.60 (98.3% CI, 0.64-3.98) for pregnancies prenatally exposed to smoking only after the first trimester. The adjusted relative risk for all stillbirths was 1.75 (98.3% CI, 0.96-3.18) for dual exposure, 1.26 (98.3% CI, 0.58-2.74) for drinking only, and 1.27 (98.3% CI, 0.69-2.35) for smoking only compared with the reference group. Conclusions and Relevance: These results suggest that combined drinking and smoking after the first trimester of pregnancy, compared with no exposure or quitting before the end of the first trimester, may be associated with a significantly increased risk of late stillbirth.

Nicotinic Receptors in the Brainstem Ascending Arousal System in SIDS With Analysis of Pre-natal Exposures to Maternal Smoking and Alcohol in High-Risk Populations of the Safe Passage Study.

Pre-natal exposures to nicotine and alcohol are known risk factors for sudden infant death syndrome (SIDS), the leading cause of post-neonatal infant mortality. Here, we present data on nicotinic receptor binding, as determined by 125I-epibatidine receptor autoradiography, in the brainstems of infants dying of SIDS and of other known causes of death collected from the Safe Passage Study, a prospective, multicenter study with clinical sites in Cape Town, South Africa and 5 United States sites, including 2 American Indian Reservations. We examined 15 pons and medulla regions related to cardiovascular control and arousal in infants dying of SIDS (n = 12) and infants dying from known causes (n = 20, 10 pre-discharge from time of birth, 10 post-discharge). Overall, there was a developmental decrease in 125I-epibatidine binding with increasing postconceptional age in 5 medullary sites [raphe obscurus, gigantocellularis, paragigantocellularis, centralis, and dorsal accessory olive (p = 0.0002-0.03)], three of which are nuclei containing serotonin cells. Comparing SIDS with post-discharge known cause of death (post-KCOD) controls, we found significant decreased binding in SIDS in the nucleus pontis oralis (p = 0.02), a critical component of the cholinergic ascending arousal system of the rostral pons (post-KCOD, 12.1 ± 0.9 fmol/mg and SIDS, 9.1 ± 0.78 fmol/mg). In addition, we found an effect of maternal smoking in SIDS (n = 11) combined with post-KCOD controls (n = 8) on the raphe obscurus (p = 0.01), gigantocellularis (p = 0.02), and the paragigantocellularis (p = 0.002), three medullary sites found in this study to have decreased binding with age and found in previous studies to have abnormal indices of serotonin neurotransmission in SIDS infants. At these sites, 125I-epibatidine binding increased with increasing cigarettes per week. We found no effect of maternal drinking on 125I-epibatidine binding at any site measured. Taken together, these data support changes in nicotinic receptor binding related to development, cause of death, and exposure to maternal cigarette smoking. These data present new evidence in a prospective study supporting the roles of developmental factors, as well as adverse exposure on nicotinic receptors, in serotonergic nuclei of the rostral medulla-a finding that highlights the interwoven and complex relationship between acetylcholine (via nicotinic receptors) and serotonergic neurotransmission in the medulla.

Medullary Serotonergic Binding Deficits and Hippocampal Abnormalities in Sudden Infant Death Syndrome: One or Two Entities?

Sudden infant death syndrome (SIDS) is understood as a syndrome that presents with the common phenotype of sudden death but involves heterogenous biological causes. Many pathological findings have been consistently reported in SIDS, notably in areas of the brain known to play a role in autonomic control and arousal. Our laboratory has reported abnormalities in SIDS cases in medullary serotonin (5-HT) receptor 1A and within the dentate gyrus of the hippocampus. Unknown, however, is whether the medullary and hippocampal abnormalities coexist in the same SIDS cases, supporting a biological relationship of one abnormality with the other. In this study, we begin with an analysis of medullary 5-HT1A binding, as determined by receptor ligand autoradiography, in a combined cohort of published and unpublished SIDS (n = 86) and control (n = 22) cases. We report 5-HT1A binding abnormalities consistent with previously reported data, including lower age-adjusted mean binding in SIDS and age vs. diagnosis interactions. Utilizing this combined cohort of cases, we identified 41 SIDS cases with overlapping medullary 5-HT1A binding data and hippocampal assessment and statistically addressed the relationship between abnormalities at each site. Within this SIDS analytic cohort, we defined abnormal (low) medullary 5-HT1A binding as within the lowest quartile of binding adjusted for age and we examined three specific hippocampal findings previously identified as significantly more prevalent in SIDS compared to controls (granular cell bilamination, clusters of immature cells in the subgranular layer, and single ectopic cells in the molecular layer of the dentate gyrus). Our data did not find a strong statistical relationship between low medullary 5-HT1A binding and the presence of any of the hippocampal abnormalities examined. It did, however, identify a subset of SIDS (~25%) with both low medullary 5-HT1A binding and hippocampal abnormalities. The subset of SIDS cases with both low medullary 5-HT1A binding and single ectopic cells in the molecular layer was associated with prenatal smoking (p = 0.02), suggesting a role for the exposure in development of the two abnormalities. Overall, our data present novel information on the relationship between neuropathogical abnormalities in SIDS and support the heterogenous nature and overall complexity of SIDS pathogenesis.

Concurrent prenatal drinking and smoking increases risk for SIDS: Safe Passage Study report.

BACKGROUND: Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality. Although the rate has plateaued, any unexpected death of an infant is a family tragedy thus finding causes and contributors to risk remains a major public health concern. The primary objective of this investigation was to determine patterns of drinking and smoking during pregnancy that increase risk of SIDS. METHODS: The Safe Passage Study was a prospective, multi-center, observational study with 10,088 women, 11,892 pregnancies, and 12,029 fetuses, followed to 1-year post delivery. Subjects were from two sites in Cape Town, South Africa and five United States sites, including two American Indian Reservations. Group-based trajectory modeling was utilized to categorize patterns of drinking and smoking exposure during pregnancy. FINDINGS: One-year outcome was ascertained in 94·2% infants, with 28 SIDS (2·43/1000) and 38 known causes of death (3·30/1000). The increase in relative risk for SIDS, adjusted for key demographic and clinical characteristics, was 11·79 (98·3% CI: 2·59-53·7, p < 0·001) in infants whose mothers reported both prenatal drinking and smoking beyond the first trimester, 3.95 (98·3% CI: 0·44-35·83, p = 0·14), for drinking only beyond the first trimester and 4·86 (95% CI: 0·97-24·27, p = 0·02) for smoking only beyond the first trimester as compared to those unexposed or reported quitting early in pregnancy. INTERPRETATION: Infants prenatally exposed to both alcohol and cigarettes continuing beyond the first trimester have a substantially higher risk for SIDS compared to those unexposed, exposed to alcohol or cigarettes alone, or when mother reported quitting early in pregnancy. Given that prenatal drinking and smoking are modifiable risk factors, these results address a major global public health problem. FUNDING: National Institute on Alcohol Abuse and Alcoholism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute on Deafness and Other Communication Disorders.

Mutations in NRXN1 and NRXN2 in a patient with early-onset epileptic encephalopathy and respiratory depression.

Early infantile epileptic encephalopathy (EIEE) is a severe disorder associated with epilepsy, developmental delay and intellectual disability, and in some cases premature mortality. We report the case of a female infant with EIEE and strikingly suppressed respiratory dysfunction that led to death. Postmortem research evaluation revealed hypoplasia of the arcuate nucleus of the medulla, a candidate region for respiratory regulation. Genetic evaluation revealed heterozygous variants in the related genes NRXN1 (c.2686C>T, p.Arg896Trp) and NRXN2 (c.3176G>A, p.Arg1059Gln), one inherited from the mother with family history of sudden infant death syndrome (SIDS) and one from the father with family history of febrile seizures. Although there are no previous reports with the digenic combination of NRXN1 and NRXN2 variants, patients with biallelic loss of NRXN1 in humans and double neurexin 1α/2α knockout mice have severe breathing abnormalities, corresponding to the respiratory phenotype of our patient. These observations and the known interaction between the NRXN1 and NRXN2 proteins lead us to hypothesize that digenic variants in NRXN1 and NRXN2 contributed to the phenotype of EIEE, arcuate nucleus hypoplasia, respiratory failure, and death.

Drinking and smoking patterns during pregnancy: Development of group-based trajectories in the Safe Passage Study.

Precise identification of drinking and smoking patterns during pregnancy is crucial to better understand the risk to the fetus. The purpose of this manuscript is to describe the methodological approach used to define prenatal drinking and smoking trajectories from a large prospective pregnancy cohort, and to describe maternal characteristics associated with different exposure patterns. In the Safe Passage Study, detailed information regarding quantity, frequency, and timing of exposure was self-reported up to four times during pregnancy and at 1 month post-delivery. Exposure trajectories were developed using data from 11,692 pregnancies (9912 women) where pregnancy outcome was known. Women were from three diverse populations: white (23%) and American Indian (17%) in the Northern Plains, US, and mixed ancestry (59%) in South Africa (other/not specified [1%]). Group-based trajectory modeling was used to identify 5 unique drinking trajectories (1 none/minimal, 2 quitting groups, 2 continuous groups) and 7 smoking trajectories (1 none/minimal, 2 quitting groups, 4 continuous groups). Women with pregnancies assigned to the low- or high-continuous drinking groups were less likely to have completed high school and were more likely to have enrolled in the study in the third trimester, be of mixed ancestry, or be depressed than those assigned to the none/minimal or quit-drinking groups. Results were similar when comparing continuous smokers to none/minimal and quit-smoking groups. Further, women classified as high- or low-continuous drinkers were more likely to smoke at moderate-, high-, and very high-continuous levels, as compared to women classified as non-drinkers and quitters. This is the first study of this size to utilize group-based trajectory modeling to identify unique prenatal drinking and smoking trajectories. These trajectories will be used in future analyses to determine which specific exposure patterns subsequently manifest as poor peri- and postnatal outcomes.

The Lateral Temporal Lobe in Early Human Life.

Abnormalities of lateral temporal lobe development are associated with a spectrum of genetic and environmental pathologic processes, but more normative data are needed for a better understanding of gyrification in this brain region. Here, we begin to establish guidelines for the analysis of the lateral temporal lobe in humans in early life. We present quantitative methods for measuring gyrification at autopsy using photographs of the gross brain and simple computer-based quantitative tools in a cohort of 28 brains ranging in age from 27 to 70 postconceptional weeks (end of infancy). We provide normative ranges for different indices of gyrification and identify a constellation of qualitative features that should also be considered in these analyses. The ratio of the temporal area to the whole brain area increased dramatically in the second half of gestation, but then decelerated after birth before increasing linearly around 50 postconceptional weeks. Tertiary gyrification continued beyond birth in a linear process through infancy with considerable variation in patterns. Analysis of 2 brains with gyral disorders of the lateral temporal lobe demonstrated proof-of-principle that the proposed methods are of diagnostic value. These guidelines are proposed for assessments of temporal lobe pathology in pediatric brains in early life.

The Stillbirth Classification System for the Safe Passage Study.

Objective Describe the classification system for assigning the cause of stillbirth in the Safe Passage Study, an international, multi-institutional, prospective analysis conducted by the NIAAA/NICHD-funded Prenatal Alcohol in SIDS and Stillbirth (PASS) Research Network. The study mission is to determine the role of prenatal alcohol and/or cigarette smoke exposure in adverse pregnancy outcomes, including stillbirth, in a high-risk cohort of 12,000 maternal/fetal dyads. Methods The PASS Network classification system is based upon 5 "sites of origin" for cause of stillbirth, further subdivided into mechanism subcategories; both are employed to assign an ultimate cause of death. Each PASS stillbirth was assigned a cause of death and status of sporadic versus recurrent. Adjudication involved review of maternal and obstetrical records; fetal autopsy and placental findings; and required complete consensus in each case. Two published classification systems, ie, INCODE and ReCoDe, were used for comparison. Results Causes of stillbirth classified were fetal (26%), placental (53%), external (5%), and undetermined (16%). Nine cases (47%) had placental causes of death due to maternal disorders that carry recurrence risks. There was full agreement for cause of death across the 3 classification systems in 26% of cases and partial agreement among them in 42% of cases. Conclusions The proposed PASS schema employs a user-friendly classification that provides comparable information to previously published systems. Advantages include its simplicity, mechanistic formulations, tight clinicopathologic integration, provision for an undetermined category, and its wide applicability to perinatal mortality review boards with access to information routinely collected during clinicopathologic evaluations.

A Century of Germinal Matrix Intraventricular Hemorrhage in Autopsied Premature Infants: A Historical Account.

The care of premature infants in the 20th century is remarkable for technical advances that have dramatically improved survival, but little is known about temporal changes in the neuropathology of the premature infant over this time frame. We hypothesize that the autopsy rate of germinal matrix hemorrhage changed in the 20th century relative to combined influences of clinical interventions that were both harmful and helpful. We examined germinal matrix hemorrhage with intraventricular hemorrhage (GMH-IVH) in 345 premature infants (gestational age 25-36 weeks) autopsied at Boston Children's Hospital from 1914 to 2015. There was a median of 19 cases/decade (range 7-68). Over the course of the study median gestational age decreased from 33 to 27 gestational weeks (P<0.001), and median postnatal survival increased from 2 to 26 days (P=0.02). The incidence of GMH-IVH increased from 4.7% before 1960 to 50.0% from 1975 to 1980, and then decreased to 12.5% after 2005 (P<0.001). The incidence of GMH-IVH increased >3-fold around the time of the introduction of positive pressure ventilation into premature intensive care in the mid-1960s. The increased incidence of GMH-IVH in the 1970s-1980s likely reflects respiratory and hemodynamic imbalances complicating mechanical ventilation. We speculate that the subsequent decreased incidence of GMH-IVH likely reflects stabilization of respiratory function with improvements in ventilators and in ventilator management beginning in the 1970s and the use of surfactant and antenatal steroids in the 1980s.

Prenatal nicotine exposure selectively affects nicotinic receptor expression in primary and associative visual cortices of the fetal baboon.

Exposure to nicotine during pregnancy via maternal cigarette smoking is associated with visual deficits in children. This is possibly due to the activation of nicotinic acetylcholine receptors (nAChRs) in the occipital cortex, which are important in the development of visual mapping. Using a baboon model, we explored the effects of prenatal nicotine on parameters in the primary and associated visual cortices. Pregnant baboons were infused with nicotine (0.5 mg/h, intravenous) or saline from 86 days gestation. At 161 days gestation, fetal brains were collected (n = 5 per group) and the occipital lobe assessed for nAChRs and markers of the serotonergic and catecholaminergic systems using tissue autoradiography and/or high-performance liquid chromatography. Neuronal nAChRs and serotonergic markers were expressed in a region- and subunit-dependent manner. Prenatal nicotine exposure was associated with increased binding for (3) H-epibatidine sensitive nAChRs in the primary visual cortex [Brodmann areas (BA) 17] and BA 18, but not BA 19, of the associative visual cortex (P < 0.05). Markers of the serotonergic or catecholaminergic systems were not significantly altered. Thus, prenatal nicotine exposure is associated with alterations in the cholinergic system in the occipital lobe, which may aid in the explanation of the appearance of visual deficits in children from mothers who smoke during pregnancy.

Neuropathologic studies of the encephalopathy of prematurity in the late preterm infant.

It has been widely suggested that brain damage in survivors of late preterm deliveries is similar to that in early preterm infants, only less severe. This report addresses this concept through reanalysis of published neuropathologic data obtained according to late preterm in comparison with early preterm ages. Findings suggest that the spectrum of brain injury in the late preterm infant, as determined in an autopsy population, is similar to that found in early preterm infants, with potential differential susceptibility for different neuronal, glial, and vascular indices. Further research is needed to more clearly define developmental cellular susceptibilities in preterm populations.

Whole genome sequencing identifies SCN2A mutation in monozygotic twins with Ohtahara syndrome and unique neuropathologic findings.

Mutations in SCN2A gene cause a variety of epilepsy syndromes. We report a novel SCN2A-associated epilepsy phenotype in monozygotic twins with tonic seizures soon after birth and a suppression-burst electroencephalography (EEG) pattern. We reviewed the medical records, EEG tracings, magnetic resonance imaging (MRI), and neuropathologic findings, and performed whole genome sequencing (WGS) on Twin B's DNA and Sanger sequencing (SS) on candidate gene mutations. Extensive neurometabolic evaluation and early neuroimaging studies were normal. Twin A died of an iatrogenic cause at 2 weeks of life. His neuropathologic examination was remarkable for dentate-olivary dysplasia and granule cell dispersion of the dentate gyrus. Twin B became seizure free at 8 months and was off antiepileptic drugs by 2 years. His brain MRI, normal at 2 months, revealed evolving brainstem and basal ganglia abnormalities at 8 and 15 months that resolved by 20 months. At 2.5 years, Twin B demonstrated significant developmental delay. Twin B's WGS revealed a heterozygous variant c.788C>T predicted to cause p.Ala263Val change in SCN2A and confirmed to be de novo in both twins by SS. In conclusion, we have identified a de novo SCN2A mutation as the etiology for Ohtahara syndrome in monozygotic twins associated with a unique dentate-olivary dysplasia in the deceased twin.

Neuron deficit in the white matter and subplate in periventricular leukomalacia.

OBJECTIVE: The cellular basis of cognitive abnormalities in preterm infants with periventricular leukomalacia (PVL) is uncertain. One important possibility is that damage to white matter and subplate neurons that are critical to the formation of the cerebral cortex occurs in conjunction with oligodendrocyte and axonal injury in PVL. We tested the hypothesis that the overall density of neurons in the white matter and subplate region is significantly lower in PVL cases compared to non-PVL controls. METHODS: We used a computer-based method for the determination of the density of microtubule-associated protein 2-immunolabeled neurons in the ventricular/subventricular region, periventricular white matter, central white matter, and subplate region in PVL cases and controls. RESULTS: There were 5 subtypes of subcortical neurons: granular, unipolar, bipolar, inverted pyramidal, and multipolar. The neuronal density of the granular neurons in each of the 4 regions was 54 to 80% lower (p≤0.01) in the PVL cases (n=15) compared to controls adjusted for age and postmortem interval (n=10). The overall densities of unipolar, bipolar, multipolar, and inverted pyramidal neurons did not differ significantly between the PVL cases and controls. No granular neurons expressed markers of neuronal and glial immaturity (Tuj1, doublecortin, or NG2). INTERPRETATION: These data suggest that quantitative deficits in susceptible granular neurons occur in the white matter distant from periventricular foci, including the subplate region, in PVL, and may contribute to abnormal cortical formation and cognitive dysfunction in preterm survivors.

Risk factor changes for sudden infant death syndrome after initiation of Back-to-Sleep campaign.

OBJECTIVE: To test the hypothesis that the profile of sudden infant death syndrome (SIDS) changed after the Back-to-Sleep (BTS) campaign initiation, document prevalence and patterns of multiple risks, and determine the age profile of risk factors. METHODS: The San Diego SIDS/Sudden Unexplained Death in Childhood Research Project recorded risk factors for 568 SIDS deaths from 1991 to 2008 based upon standardized death scene investigations and autopsies. Risks were divided into intrinsic (eg, male gender) and extrinsic (eg, prone sleep). RESULTS: Between 1991-1993 and 1996-2008, the percentage of SIDS infants found prone decreased from 84.0% to 48.5% (P < .001), bed-sharing increased from 19.2% to 37.9% (P < .001), especially among infants <2 months (29.0% vs 63.8%), prematurity rate increased from 20.0% to 29.0% (P = .05), whereas symptoms of upper respiratory tract infection decreased from 46.6% to 24.8% (P < .001). Ninety-nine percent of SIDS infants had at least 1 risk factor, 57% had at least 2 extrinsic and 1 intrinsic risk factor, and only 5% had no extrinsic risk. The average number of risks per SIDS infant did not change after initiation of the BTS campaign. CONCLUSIONS: SIDS infants in the BTS era show more variation in risk factors. There was a consistently high prevalence of both intrinsic and especially extrinsic risks both before and during the Back-to-Sleep era. Risk reduction campaigns emphasizing the importance of avoiding multiple and simultaneous SIDS risks are essential to prevent SIDS, including among infants who may already be vulnerable.

Aberrant upregulation of astroglial ceramide potentiates oligodendrocyte injury.

Oligodendroglial injury is a pathological hallmark of many human white matter diseases, including multiple sclerosis (MS) and periventricular leukomalacia (PVL). Critical regulatory mechanisms of oligodendroglia destruction, however, remain incompletely understood. Ceramide, a bioactive sphingolipid pivotal to sphingolipid metabolism pathways, regulates cell death in response to diverse stimuli and has been implicated in neurodegenerative disorders. We report here that ceramide accumulates in reactive astrocytes in active lesions of MS and PVL, as well as in animal models of demyelination. Serine palmitoyltransferase, the rate-limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination. Mass spectrometry confirmed the upregulation of specific ceramides during demyelination, and revealed a concomitant increase of sphingosine and a suppression of sphingosine-1-phosphate, a potent signaling molecule with key roles in cell survival and mitogenesis. Importantly, this altered sphingolipid metabolism during demyelination was restored upon active remyelination. In culture, ceramide acted synergistically with tumor necrosis factor, leading to apoptotic death of oligodendroglia in an astrocyte-dependent manner. Taken together, our findings implicate that disturbed sphingolipid pathways in reactive astrocytes may indirectly contribute to oligodendroglial injury in cerebral white matter disorders.

The developing oligodendrocyte: key cellular target in brain injury in the premature infant.

Brain injury in the premature infant, a problem of enormous importance, is associated with a high risk of neurodevelopmental disability. The major type of injury involves cerebral white matter and the principal cellular target is the developing oligodendrocyte. The specific phase of the oligodendroglial lineage affected has been defined from study of both human brain and experimental models. This premyelinating cell (pre-OL) is vulnerable because of a series of maturation-dependent events. The pathogenesis of pre-OL injury relates to operation of two upstream mechanisms, hypoxia-ischemia and systemic infection/inflammation, both of which are common occurrences in premature infants. The focus of this review and of our research over the past 15-20 years has been the cellular and molecular bases for the maturation-dependent vulnerability of the pre-OL to the action of the two upstream mechanisms. Three downstream mechanisms have been identified, i.e., microglial activation, excitotoxicity and free radical attack. The work in both experimental models and human brain has identified a remarkable confluence of maturation-dependent factors that render the pre-OL so exquisitely vulnerable to these downstream mechanisms. Most importantly, elucidation of these factors has led to delineation of a series of potential therapeutic interventions, which in experimental models show marked protective properties. The critical next step, i.e., clinical trials in the living infant, is now on the horizon.

Potential neuronal repair in cerebral white matter injury in the human neonate.

Periventricular leukomalacia (PVL) in the premature infant represents the major substrate underlying cognitive deficits and cerebral palsy and is characterized as focal periventricular necrosis and diffuse gliosis in the immature cerebral white matter. We have recently shown a significant decrease in the density of neurons in PVL relative to controls throughout the white matter, including the subventricular, periventricular, and subcortical regions. These neurons are likely to be remnants of the subplate and/or GABAergic neurons in late migration to the cerebral cortex, both of which are important for proper cortical circuitry in development and throughout adulthood. Here, we tested the hypothesis that intrinsic repair occurs in PVL to attempt to compensate for the deficits in white matter neurons. By using doublecortin (DCX) immunopositivity as a marker of postmitotic migrating neurons, we found significantly increased densities (p < 0.05) of DCX-immunopositive cells in PVL cases (n = 9) compared with controls (n = 7) in the subventricular zone (their presumed site of origin), necrotic foci, and subcortical white matter in the perinatal time-window, i.e. 35-42 postconceptional weeks. These data provide the first evidence suggestive of an attempt at neuronal repair or regeneration in human neonatal white matter injury.

The cerebral cortex overlying periventricular leukomalacia: analysis of pyramidal neurons.

The role of the cerebral cortex in the cognitive deficits in preterm survivors is poorly understood. Periventricular leukomalacia (PVL), the key feature of encephalopathy of prematurity, is characterized by periventricular necrotic foci and diffuse gliosis in the surrounding cerebral white matter. Here, we tested the hypothesis that reductions in the density of layer I neurons and/or pyramidal neurons in layers III and/or V are associated with PVL, indicating cortical pathology potentially associated with cognitive deficits in long-term survivors. In controls (23 gestational weeks to 18 postnatal months) (n = 15), a lack of significant differences in pyramidal density among incipient Brodmann areas suggested that cytoarchitectonic differences across functional areas are not fully mature in the fetal and infant periods. There was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases (n = 17) compared to controls (n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six-layer cortex is visually distinct (P < 0.024). This may reflect a dying-back loss of somata complicating transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the potential role of secondary cortical injury in the encephalopathy of prematurity.

Prenatal nicotine-exposure alters fetal autonomic activity and medullary neurotransmitter receptors: implications for sudden infant death syndrome.

During pregnancy, exposure to nicotine and other compounds in cigarette smoke increases the risk of the sudden infant death syndrome (SIDS) two- to fivefold. Serotonergic (5-HT) abnormalities are found, in infants who die of SIDS, in regions of the medulla oblongata known to modulate cardiorespiratory function. Using a baboon model, we tested the hypothesis that prenatal exposure to nicotine alters 5-HT receptor and/or transporter binding in the fetal medullary 5-HT system in association with cardiorespiratory dysfunction. At 87 (mean) days gestation (dg), mothers were continuously infused with saline (n = 5) or nicotine (n = 5) at 0.5 mg/h. Fetuses were surgically instrumented at 129 dg for cardiorespiratory monitoring. Cesarean section delivery and retrieval of fetal medulla were performed at 161 (mean) dg for autoradiographic analyses of nicotinic and 5-HT receptor and transporter binding. In nicotine-exposed fetuses, high-frequency heart rate variability was increased 55%, possibly reflecting increases in the parasympathetic control of heart rate. This effect was more pronounced with greater levels of fetal breathing and age. These changes in heart rate variability were associated with increased 5-HT(1A) receptor binding in the raphé obscurus (P = 0.04) and increased nicotinic receptor binding in the raphé obscurus and vagal complex (P < 0.05) in the nicotine-exposed animals compared with controls (n = 6). The shift in autonomic balance in the fetal primate toward parasympathetic predominance with chronic exposure to nicotine may be related, in part, to abnormal 5-HT-nicotine alterations in the raphé obscurus. Thus increased risk for SIDS due to maternal smoking may be partly related to the effects of nicotine on 5-HT and/or nicotinic receptors.

Nitrosative stress and inducible nitric oxide synthase expression in periventricular leukomalacia.

Periventricular leukomalacia (PVL) is a lesion of the immature cerebral white matter in the perinatal period and associated predominantly with prematurity and cerebral ischemia/reperfusion as well as inflammation due to maternofetal infection. It consists of focal necrosis in the periventricular region and diffuse gliosis with microglial activation and premyelinating oligodendrocyte (pre-OL) injury in the surrounding white matter. We previously showed nitrotyrosine in pre-OLs in PVL, suggesting involvement of nitrosative stress in this disorder. Here we hypothesize that inducible nitric oxide synthase (iNOS) expression is increased in PVL relative to controls. Using immunocytochemistry in human archival tissue, the density of iNOS-expressing cells was determined in the cerebral white matter of 15 PVL cases [29-51 postconceptional (PC) weeks] and 16 control cases (20-144 PC weeks). Using a standardization score of 0-3, the density of iNOS-positive cells was significantly increased in the diffuse component of PVL (score of 1.8 +/- 0.3) cases compared to controls (score of 0.7 +/- 0.3) (P = 0.01). Intense iNOS expression occurred in reactive astrocytes in acute through chronic stages and in activated microglia primarily in the acute stage, suggesting an early role for microglial iNOS in PVL's pathogenesis. This study supports an important role for iNOS-induced nitrosative stress in the reactive/inflammatory component of PVL.