Disruption of the brain-derived neurotrophic factor (BDNF) immunoreactivity in the human Kölliker-Fuse nucleus in victims of unexplained fetal and infant death.

Experimental studies have demonstrated that the neurotrophin brain-derived neutrophic factor (BDNF) is required for the appropriate development of the central respiratory network, a neuronal complex in the brainstem of vital importance to sustaining life. The pontine Kölliker-Fuse nucleus (KFN) is a fundamental component of this circuitry with strong implications in the pre- and postnatal breathing control. This study provides detailed account for the cytoarchitecture, the physiology and the BDNF behavior of the human KFN in perinatal age. We applied immunohistochemistry in formalin-fixed and paraffin-embedded brainstem samples (from 45 fetuses and newborns died of both known and unknown causes), to analyze BDNF, gliosis and apoptosis patterns of manifestation. The KFN showed clear signs of developmental immaturity, prevalently associated to BDNF altered expression, in high percentages of sudden intrauterine unexplained death syndrome (SIUDS) and sudden infant death syndrome (SIDS) victims. Our results indicate that BDNF pathway dysfunctions can derange the normal KFN development so preventing the breathing control in the sudden perinatal death. The data presented here are also relevant to a better understanding of how the BDNF expression in the KFN can be involved in several human respiratory pathologies such as the Rett's and the congenital central hypoventilation syndromes.

Possible role of the α7 nicotinic receptors in mediating nicotine's effect on developing lung - implications in unexplained human perinatal death.

BACKGROUND: It is well known that maternal smoking during pregnancy is very harmful to the fetus. Prenatal nicotine absorption, in particular, is associated with alterations in lung development and functions at birth and with respiratory disorders in infancy. Many of the pulmonary disorders are mediated by the interaction of nicotine with the nicotinic receptors (nAChRs), above all with the α7 nAChR subunits that are widely expressed in the developing lung. To determine whether the lung hypoplasia frequently observed in victims of sudden fetal and neonatal death with a smoker mother may result from nicotine interacting with lung nicotinic receptors, we investigated by immunohistochemistry the possible presence of the α7 nAChR subunit overexpression in these pathologies. METHODS: In lung histological sections from 45 subjects who died of sudden intrauterine unexplained death syndrome (SIUDS) and 15 subjects who died of sudden infant death syndrome (SIDS), we applied the radial alveolar count (RAC) to evaluate the degree of lung maturation, and the immunohistochemical technique for nAChRs, in particular for the α7 nAChR subunit identification. In the same cases, an in-depth study of the autonomic nervous system was performed to highlight possible developmental alterations of the main vital centers located in the brainstem. RESULTS: We diagnosed a "lung hypoplasia", on the basis of RAC values lower than the normal reference values, in 63% of SIUDS/SIDS cases and 8% of controls. In addition, we observed a significantly higher incidence of strong α7 nAChR immunostaining in lung epithelial cells and lung vessel walls in sudden fetal and infant death cases with a smoker mother than in age-matched controls. Hypoplasia of the raphe, the parafacial, the Kölliker-Fuse, the arcuate and the pre-Bötzinger nuclei was at the same time present in the brainstem of these victims. CONCLUSIONS: These findings demonstrate that when crossing the placenta, nicotine can interact with nicotinic receptors of both neuronal and non-neuronal cells, leading to lung and nervous system defective development, respectively. This work stresses the importance of implementing preventable measures to decrease the noxious potential of nicotine in pregnancy.

Cerebellar Purkinje cell vulnerability to prenatal nicotine exposure in sudden unexplained perinatal death.

The present study was aimed at supplementing our previous investigations on the morphological features of the Purkinje cells during the autonomic nervous system development, particularly in victims of sudden perinatal death (Sudden Intrauterine Unexplained Death Syndrome and Sudden Infant Death Syndrome), given their crucial role in determining connectivity patterns in the brain as well as in the control of autonomic functions. We highlighted in these pathologies, and precisely in 21 cases of sudden foetal death and 26 cases of sudden infant death, a high percentage of developmental defects of the Purkinje cells such as heterotopia, hypoplasia, hyperplasia, mitotic and/or shrunken features and abnormal neuronal nuclear antigen expression. These alterations can be interpreted as a result of a defective maturation and/or migration of Purkinje cells in foetal cerebellum, likely consequence of exposure to injuries, particularly to maternal cigarette smoke. Interestingly, we observed in sudden perinatal deaths an association with similar developmental defects of both the dentate and the inferior olivary nuclei. This suggests the existence of a Purkinje-Olivo-Dentate network playing a fundamental role in triggering a sudden death mechanism in perinatal life in the presence of specific risk factors.

Neuronal nuclear antigen (NeuN): a useful marker of neuronal immaturity in sudden unexplained perinatal death.

INTRODUCTION: In the developing brain neuronal differentiation is associated with permanent exit from the mitotic cycle. Neuronal nuclear antigen (NeuN) is a nuclear protein widely expressed in the mature postmitotic neurons. METHODS: We applied NeuN immunocytochemistry in 65 cases of perinatal death (16 victims of sudden intrauterine unexplained death syndrome/SIUDS, 19 of sudden infant death syndrome/SIDS and 30 controls) to test the physiological status of the brain neurons. In addition we applied both TUNEL and Caspase 3 immunohistochemical methods in order to highlight a possible relation between decreased NeuN expression and apoptotic outcome. We also attempted to see whether or not NeuN pathological changes can be related to cigarette smoke absorption in pregnancy. RESULTS: NeuN staining was considerably reduced or lost in SIUDS/SIDS compared to controls. However neurons with decreased NeuN-labeling showed no sign of apoptosis. A significant association was found between NeuN depletion and maternal smoking. CONCLUSION: Altered NeuN expression can be a marker of immature and/or suffering neurons. The exclusive presence of this pattern of expression in SIUDS/SIDS victims, leads us to recommend the NeuN immunohistochemistry as a routine method in neuropathological protocols to convalidate a diagnosis of sudden perinatal death.

Developmental alterations of the respiratory human retrotrapezoid nucleus in sudden unexplained fetal and infant death.

The study aims were twofold: 1) identify the localization and the cytoarchitecture of the retrotrapezoid nucleus (RTN) in the human fetus and infant and 2) ascertain if the RTN, given its essential role in animal studies for the maintenance of breathing and chemoreception, showed abnormalities in victims of sudden perinatal and infant death (sudden intrauterine unexplained death/SIUD - and sudden infant death syndrome/SIDS). We examined SIDS and SIUD cases and Controls (n=58) from 34 gestational weeks to 8 months of postnatal age by complete autopsy, in-depth autonomic nervous system histological examination, and immunohistochemical analysis of the PHOX2B gene, a transcriptional factor involved in Congenital Central Hypoventilation Syndrome that has been defined as a marker of rat RTN neurons. We identified a group of PHOX2B-immunopositive neurons within the caudal pons, contiguous to the facial/parafacial complex, in 90% of Controls, likely the homologous human RTN (hRTN). We observed structural and/or PHOX2B-expression abnormalities of the hRTN in 71% of SIUD/SIDS cases vs 10% of Controls (p<0.05). In conclusion we suggest that developmental abnormalities of the hRTN may seriously compromise chemoreception control, playing a critical role in the pathogenesis of both SIUD and SIDS.

Brain iron accumulation in unexplained fetal and infant death victims with smoker mothers--the possible involvement of maternal methemoglobinemia.

BACKGROUND: Iron is involved in important vital functions as an essential component of the oxygen-transporting heme mechanism. In this study we aimed to evaluate whether oxidative metabolites from maternal cigarette smoke could affect iron homeostasis in the brain of victims of sudden unexplained fetal and infant death, maybe through the induction of maternal hemoglobin damage, such as in case of methemoglobinemia. METHODS: Histochemical investigations by Prussian blue reaction were made on brain nonheme ferric iron deposits, gaining detailed data on their localization in the brainstem and cerebellum of victims of sudden death and controls. The Gless and Marsland's modification of Bielschowsky's was used to identify neuronal cell bodies and neurofilaments. RESULTS: Our approach highlighted accumulations of blue granulations, indicative of iron positive reactions, in the brainstem and cerebellum of 33% of victims of sudden death and in none of the control group. The modified Bielschowsky's method confirmed that the cells with iron accumulations were neuronal cells. CONCLUSIONS: We propose that the free iron deposition in the brain of sudden fetal and infant death victims could be a catabolic product of maternal methemoglobinemia, a biomarker of oxidative stress likely due to nicotine absorption.

Ependymal alterations in sudden intrauterine unexplained death and sudden infant death syndrome: possible primary consequence of prenatal exposure to cigarette smoking.

BACKGROUND: The ependyma, the lining providing a protective barrier and filtration system separating brain parenchyma from cerebrospinal fluid, is still inadequately understood in humans. In this study we aimed to define, by morphological and immunohistochemical methods, the sequence of developmental steps of the human ependyma in the brainstem (ventricular ependyma) and thoracic spinal cord (central canal ependyma) of a large sample of fetal and infant death victims, aged from 17 gestational weeks to 8 postnatal months. Additionally, we investigated a possible link between alterations of this structure, sudden unexplained fetal and infant death and maternal smoking. RESULTS: Our results demonstrate that in early fetal life the human ependyma shows a pseudostratified cytoarchitecture including many tanycytes and ciliated cells together with numerous apoptotic and reactive astrocytes in the subependymal layer. The ependyma is fully differentiated, with a monolayer of uniform cells, after 32 to 34 gestational weeks. We observed a wide spectrum of ependymal pathological changes in sudden death victims, such as desquamation, clusters of ependymal cells in the subventricular zone, radial glial cells, and the unusual presence of neurons within and over the ependymal lining. These alterations were significantly related to maternal smoking in pregnancy. CONCLUSIONS: We conclude that in smoking mothers, nicotine and its derivatives easily reach the cerebrospinal fluid in the fetus, immediately causing ependymal damage. Consequently, we suggest that the ependyma should be examined in-depth first in victims of sudden fetal or infant death with mothers who smoke.

Neuropathology of the intermediolateral nucleus of the spinal cord in sudden unexplained perinatal and infant death.

Experimental studies have demonstrated that breathing activity in rats is generated early in embryonic stages in rostral spinal cord, precisely in the intermediolateral nucleus, then establishing a spinal cord-brainstem network. In this study we aimed to individuate and to define the developmental steps of the intermediolateral nucleus, still inadequately known in humans, in the thoracic spinal cord of a large series of perinatal and infant death victims, aged from 17 gestational weeks to 10 months of life. Besides we investigated a possible link between alterations of this nucleus and sudden unexplained perinatal and infant death. The normal developmental pattern of the human intermediolateral nucleus consists of a progressive maturation of its neurons, that change from a round to a polygonal shape with long axons and significantly decrease in number. Various degrees of intermediolateral nucleus hypodevelopment (neuronal immaturity in a normal structure/hypoplasia/agenesis) were found almost exclusively in unexplained fetal and infant death victims. Besides, a significant correlation was found between maternal smoking in pregnancy and the neuropathological results. In conclusion this work underlines the negative effects of prenatal nicotine exposure on the development of autonomic nervous centers checking the vital functions, already in early gestational stages, when the integrity of the intermediolateral nucleus is indispensable for the first breathing bursts.