Abstract:Complete examination of the brainstem involves transverse serial 5-m sections made throughout the entire brainstem. The number of serial sections varies from 360 in sudden intrauterine unexplained death (SIUD) to 600 in term fetuses to over 1400 sections in sudden infant death syndrome (SIDS) victims. The procedure is not applicable in all histopathological laboratories, owing to the need for additional technical personnel. The simplified procedure allows a remarkable reduction of the number of sections. The brainstem is divided into 3 blocks. The first, cranial block, extends from the border between the medulla oblongata and pons up to the upper pole of the olivary nucleus. The second, intermediate block, corresponding to the submedian area of the inferior olivary nucleus, has as reference point the obex and extends 2 to 3 mm above and below the obex itself. The third, caudal block, includes the lower pole of the inferior olivary nucleus and the lower adjacent area of the medulla oblongata. Examinations of the brainstems from 106 SIDS victims, 30 controls, and 51 stillborns underlined a remarkable variability, particularly of the arcuate nucleus. The simplified examination of the brainstem makes it possible to evaluate the structures, examining 3 specific levels, defined by morphologic reference points.

The developmental defects found in the locus coeruleus complex in victims of sudden unexplained fetal and infant death imply alterations of the vital activities related to the widespread brain connections arising from this neuronal center, including coordination of the sleep-waking cycle and control of the cardio-respiratory system.

BackgroundIt 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.MethodsIn 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.ResultsWe 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.ConclusionsThese 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.

BackgroundIron 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.MethodsHistochemical 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.ResultsOur 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.ConclusionsWe 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.

AimsThe nucleolus is an important cellular component involved in the biogenesis of the ribosome. This study was performed in order to validate the introduction of the argyrophilic nucleolar organiser region (AgNOR) stain technique, specific for the nucleoli detection, in neuropathological studies on sudden fetal and infant death.MethodsIn a wide set of fetuses and infants, aged from 27 gestational weeks to eight postnatal months and dead from both known and unknown causes, an in-depth neuropathological study usually applied at the Lino Rossi Research Center of the Milan University was implemented by the AgNOR method.ResultsPeculiar abnormalities of the nucleoli, as partial or total disruption above all in Purkinje cells (PCs), were exclusively found in victims of sudden fetal and infant death, and not in controls. The observed nucleolar alterations were frequently related to nicotine absorption in pregnancy.ConclusionsWe conclude that these findings represent early hallmarks of PC degeneration, contributing to the pathophysiology of sudden perinatal death.

Background - The purpose of this study was to research possible morphological and functional alterations of the substantia nigra in SIDS. Methods - Brainstems of 46 victims of sudden infant death, aged from 1 to 7 months, were investigated. Twenty-six of these cases were diagnosed as SIDS, due to the lack of any pathological finding, while the remaining 20 cases, in which the cause of death was determined at autopsy, served as controls. Maternal smoking was reported in 10% of controls and 77% of SIDS. Histopathological examination of the substantia nigra was done on midbrain 5-µm-thick sections stained with hematoxylin-eosin and Klüver-Barrera. Densitometry, immunohistochemistry and histochemistry were applied to highlight the neuronal concentration, the expression of tyrosine hydroxylase and neuromelanin, respectively. Results - Hypoplasia of the pars compacta of the substantia nigra was observed in SIDS but not in controls. Tyrosine hydroxylase expression in the substantia nigra was significantly higher in controls than in SIDS. Neuromelanin was observed as dark granules only in 4 infants of the control group but never in SIDS. A significant correlation was found between hypoplasia/low neuronal density, low tyrosine hydroxylase expression in the substantia nigra pars compacta and maternal smoking. Conclusion - The substantia nigra pars compacta, that is the main dopamine center in the brain, is involved in the control of many functions, including the sleep-arousal phase, and hence, its structural and/or functional alterations may explain the pathogenesis of SIDS, that often occurs during sleep. This study also indicates that maternal smoking can may have strongly influenced the SN altered development. Trial registration – not applicable for this study

Brain-derived neurotrophic factor (BDNF), a neurotrophin of the central nervous system, is able to regulate neuronal differentiation and modulate synaptic plasticity, being particularly involved in the development of the cerebellar cortical structure. The main aim of this study was to delineate, by immunohistochemistry, the BDNF expression in human cerebellar cortex of victims of fetal and infant death. The study was performed on a total of 45 cases, aged between 25 gestational weeks and 6 postnatal months, including 29 victims of sudden fetal and infant death and 16 age-matched subjects who died of known causes (Controls). We observed, in sudden death groups compared with Controls, a significantly higher incidence of defective BDNF expression in granule layers of the cerebellar cortex, which was particularly evident in the posterior lobule, a region that participates in respiratory control. These results were related to maternal smoking, allowing to speculate that nicotine, in addition to the well-known damages, can exert adverse effects during cerebellar cortex development, in particular in hindering the BDNF expression in the posterior lobule. This implies modifications of synaptic transmission in the respiratory circuits, with obvious deleterious consequences on survival.

The purpose of this study was to research possible developmental alterations of the substantia nigra (SN) in sudden infant death syndrome (SIDS), a syndrome frequently attributed to arousal failure from sleep. Brain stems of 46 victims of sudden infant death, aged from 1 to about 7 months (4 to 30 postnatal weeks), were investigated. Twenty-six of these cases were diagnosed as SIDS, due to the lack of any pathological finding, while the remaining 20 cases in which the cause of death was determined at autopsy served as controls. Maternal smoking was reported in 77% of SIDS and 10% of controls. Histopathological examination of the SN was done on 5-µm-thick sections of caudal midbrain stained with both hematoxylin-eosin and Klüver-Barrera. Densitometry, immunohistochemistry and histochemistry were applied to highlight the neuronal concentration, the tyrosine hydroxylase (TH) expression, and the presence of neuromelanin (NM) in this structure. Hypoplasia of the pars compacta portion of the SN was observed in 69% of SIDS but never in controls; TH expression was significantly higher in controls than in SIDS; and NM was observed only in 4 infants of the control group but not in SIDS. A significant correlation was found between SIDS, hypoplasia/low neuronal density, low TH expression in the pars compacta, and maternal smoking. Because the SN pars compacta, being the major dopamine brain center, controls many functions, including the sleep-arousal phase, its alterations, especially concurrently with smoking exposure, may contribute to explain the pathogenesis of SIDS that occur in the great part of cases at awakening from sleep.