New Step in Understanding the Pathogenetic Mechanism of Sudden Infant Death Syndrome: Involvement of the Pontine Reticular Gigantocellular Nucleus.

This study aimed to investigate, for the first time, the potential role of the gigantocellular nucleus, a component of the reticular formation, in the pathogenetic mechanism of Sudden Infant Death Syndrome (SIDS), an event frequently ascribed to failure to arouse from sleep. This research was motivated by previous experimental studies demonstrating the gigantocellular nucleus involvement in regulating the sleep-wake cycle. We analyzed the brains of 48 infants who died suddenly within the first 7 months of life, including 28 SIDS cases and 20 controls. All brains underwent a thorough histological and immunohistochemical examination, focusing specifically on the gigantocellular nucleus. This examination aimed to characterize its developmental cytoarchitecture and tyrosine hydroxylase expression, with particular attention to potential associations with SIDS risk factors. In 68% of SIDS cases, but never in controls, we observed hypoplasia of the pontine portion of the gigantocellular nucleus. Alterations in the catecholaminergic system were present in 61% of SIDS cases but only in 10% of controls. A strong correlation was observed between these findings and maternal smoking in SIDS cases when compared with controls. In conclusion we believe that this study sheds new light on the pathogenetic processes underlying SIDS, particularly in cases associated with maternal smoking during pregnancy.

Mitochondrial DNA content: a new potential biomarker for Sudden Infant Death Syndrome.

BACKGROUND: Sudden Infant Death Syndrome (SIDS) occurs in apparently healthy infants and is unpredictable and unexplained despite thorough investigations and enormous research efforts. The hypothesis tested in this case-control study concerns mitochondrial involvement in SIDS occurrence. METHODS: Mitochondrial DNA content (MtDNAcn) was measured in 24 SIDS cerebral cortex samples and 18 controls using real-time PCR. RESULTS: The median (interquartile range) mtDNAcn in SIDS and controls was 2578 (2224-3838) and 1452 (724-2517) copies per nuclear DNA, respectively (P = 0.0001). MtDNAcn values were higher in SIDS victims born to non-smoking parents (n = 7) 4984 (2832-6908) compared to the controls (n = 5) 2020 (478-2386) (P = 0.006). Increased levels of mtDNAcn have been observed in the SIDS cases with mild defects in nuclei not essential for life compared to those found in SIDS cases with severe alterations of respiratory function (P = 0.034) 3571 (2568-5053) (n = 14) 2356 (1909-3132) (n = 8), respectively. CONCLUSIONS: Our study revealed for the first time higher mtDNAcn in the cerebral cortex of the SIDS cases than the controls, indicating metabolic alterations. MtDNAcn plays an important role in compensatory mechanisms against environmental factors affecting human health. Despite the small sample size, mtDNA may prove to be a potential forensic biomarker for autopsied SIDS victims for gaining new insights into the etiology of SIDS. IMPACT: Mitochondrial DNA content evaluated in cerebral cortex samples is higher in SIDS victims than controls. These results represent a novel line of investigation for the etiology of SIDS and could have a significant role in the compensatory mechanism due to environmental factors affecting human health. These findings suggest that the mitochondria are involved in SIDS: mtDNA content may represent a biomarker of this syndrome.

Determination of selected endocrine disrupting compounds in human fetal and newborn tissues by GC-MS.

Endocrine disrupting compounds (EDCs) include organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), carbamate pesticides, and plasticizers, such as bisphenol A (BPA). They persist in the environment because of their degradation resistance and bioaccumulate in the body tissues of humans and other mammals. Many studies are focused on the possible correlation between in utero exposure to EDCs and adverse health hazards in fetuses and newborns. In the last decade, environmental pollution has been considered a possible trigger for Sudden Infant Death Syndrome (SIDS) and Sudden Intrauterine Unexplained Death Syndrome (SIUDS), the most important death-causing syndromes in fetuses and newborns in developed countries. In this work, a rapid and sensitive analytical method was developed to determine the level of OCPs and OPPs, carbamates, and phenols in human fetal and newborn tissues (liver and brain) and to unveil the possible presence of non-targeted compounds. The target analytes where selected on the basis of their documented presence in the Trentino-Alto Adige region, an intensive agricultural area in northern Italy. A liquid-solid extraction procedure was applied on human and animal tissues and the extracts, after a solid phase extraction (SPE) clean-up procedure, were analyzed by gas chromatography coupled to a quadrupole mass spectrometric detector (GC-qMS). A GC-TOFMS (time-of-flight) instrument, because of its higher full-scan sensitivity, was used for a parallel detection of non-targeted compounds. Method validation included accuracy, precision, detection, and quantification limits (LODs; LOQs), and linearity response using swine liver and lamb brain spiked at different concentrations in the range of 0.4-8000.0 ng/g. The method gave good repeatability and extraction efficiency. Method LOQs ranged from 0.4-4.0 ng/g in the selected matrices. Good linearity was obtained over four orders of magnitude starting from LOQs. Isotopically labeled internal standards were used for quantitative calculations. The method was then successfully applied to the analysis of liver and brain tissues from SIUDS and SIDS victims coming from the above mentioned region.

Investigation of 5-HTT expression using quantitative real-time PCR in the human brain in SIDS Italian cases.

The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant death, being defined as the sudden death of an infant under one year of age that remains unexplained after a complete clinical review, autopsy and death scene investigation. The neurotransmitter serotonin (5-HT) is involved in the regulation of a broad array of behavioral and biological functions. By controlling the reuptake of 5-HT from the extracellular space, the serotonin transporter (5-HTT) regulates the duration and strength of the interactions between 5-HT and its receptors. It has been shown that the activity of the human 5-HTT gene promoter is regulated by polymorphic repetitive elements, resulting in differences in the efficacy of 5-HTT reuptake among the allelic variants: the short (S) allele is associated with lower transcriptional efficiency of the promoter compared with the long (L) allele. Using qRT-PCR we studied the gene expression of 5-HTT in ten SIDS cases, previously analyzed at a molecular level and which showed the genetic S/S profile. In nine cases we observed 5-HTT expression levels comparable to those seen in the control case, while in one case there was a remarkable reduction in the expression of the gene. It is presumable that, despite the presence of the same S/S genotype, the different genetic background could influence the transcript stability and that the polimorphic variant of the 5-HTT gene could respond differently to the external environmental stimuli.

Obstructive sleep apnea syndrome (OSAS) in children with Class III malocclusion: involvement of the PHOX2B gene.

PURPOSE: The aim of this study is to provide new molecular approaches to the children with obstructive sleep apnea syndrome by evaluating the possible involvement of the PHOX2B gene, notoriously associated to congenital central hypoventilation syndrome (CCHS), in Class III malocclusion. METHODS: Fifty subjects with Class III malocclusion, aged from 8 to 14 years, and with history of sleep apneic episodes, and 20 age-matched controls were submitted to genomic DNA examination from oral cells to specifically analyze the PHOX2B genotype. RESULTS: Point "silent" mutations affecting different nucleotides of the PHOX2B gene were observed in 32 % of patients with Class III malocclusion and never in controls (0 %). CONCLUSION: The genetic data obtained in this study in children with Class III malocclusion and sleep-related breathing disorders provide new information useful to the genetic characterization of this pathology. The PHOX2B gene silent mutations can lead to structural and functional modification of their product providing to a group of children with Class III malocclusion similar features to those of CCHS (sleep apnea episodes and craniofacial malformations).

Altered Development of Mesencephalic Dopaminergic Neurons in SIDS: New Insights into Understanding Sudden Infant Death Pathogenesis.

Sudden infant death syndrome (SIDS) is defined as the unexpected sudden death of an infant under 1 year of age that remains unexplained after a thorough case investigation. The SIDS pathogenesis is still unknown; however, abnormalities in brain centers that control breathing and arousal from sleep, including dramatic changes in neurotransmitter levels, have been supposed in these deaths. This is the first study focusing on mesencephalic dopaminergic neurons, so far extensively studied only in animals and human neurological diseases, in SIDS. Dopaminergic structures in midbrain sections of a large series of sudden infant deaths (36 SIDS and 26 controls) were identified using polyclonal rabbit antibodies against tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, and the dopamine transporter, a membrane protein specifically expressed in dopaminergic cells. Dopamine-immunolabeled neurons were observed concentrated in two specific structures: the pars compacta of the substantia nigra and in the subnucleus medialis of the periaqueductal gray matter. Anatomical and functional degenerations of dopaminergic neurons in these regions were observed in most SIDS cases but never in controls. These results indicate that dopamine depletion, which is already known to be linked especially to Parkinson's disease, is strongly involved even in SIDS pathogenesis.

Neuropathological explanation of minimal COVID-19 infection rate in newborns, infants and children - a mystery so far. New insight into the role of Substance P.

Sars-Cov-2 or Novel coronavirus infection (COVID-19) has become a global challenge, affecting elderly population at large, causing a burden on hospitals. It has been affecting the world from a health and economic perspective after its emergence since October 2019 at Wuhan province of China. Later on it became a pandemic, with aged people most affected. Surprisingly, the infants and children were not severely infected and mortality among them was reported infrequently. If they died it was due to some comorbidity or congenital heart problems. Why the rate of infection varies in different age groups around the world and what is the protective mechanism in children remains a mystery. Based on our neuropathological experience at the "Lino Rossi Research Center for the study and prevention of the unexpected perinatal death and Sudden Infant Death Syndrome (SIDS)" of the University of Milan, Italy, we hypothesize that the decreased severity of the disease in infants compared to the elderly may be due to alteration at neurotransmitter levels especially of the Substance P (SP) and of the spinal trigeminal nucleus in the brainstem that is responsible for its secretion. This neurotransmitter may be directly related to the respiratory illness as is in COVID-19 infection. It is responsible for the increased inflammation and the characteristic symptoms associated with this disease. It is the main switch that must be urgently turned off using the NK-1R antagonist which is the receptor of SP and responsible for its functionality, especially in the elderly.

Silver nanoparticles in the fetal brain: new perspectives in understanding the pathogenesis of unexplained stillbirths.

We report, for the first time, the surprising presence of toxic nanoparticles, especially silver, in the brain of a fetus, who died unexpectedly at the end of a regular pregnancy. After an accurate autopsy, including the examination of the fetal annexes, an in-depth anatomopathological study of the nervous system and a search by scanning electron microscopy of nanoparticles in the brain, we highlighted the sequence of events that may have led to this fetal death, triggered primarily by the transition of nanosized xenobiotics from the mother to the fetal bloodstream. From this report emerges the importance of considering the search of nanosubstances in the brain during routine investigations following unexpected and unexplained fetal and infant deaths.

Optimisation of postmortem tissue preservation and alternative protocol for serotonin transporter gene polymorphisms amplification in SIDS and SIUD cases.

The major obstacle to genetic research in SIUD (sudden intrauterine unexplained death) and SIDS (sudden infant death syndrome) cases is the complex characteristics of the human anatomic samples available. In fact, in Italy autopsies are performed at least 24 h post-mortem and tissues can be left in formalin for long fixation times (>4/5 days), thus compromising nucleic acids integrity. In this study we compared the quality of DNA and RNA extracted from tissues differently preserved. As expected, the DNA and RNA from formalin-fixed and paraffin-embedded tissues, formalin-acetic acid-alcohol tissues and ethanol tissues were of poor quality and not adequate for subsequent molecular analysis. The best results were obtained with RNAlater preserved tissues: this buffer was equivalent, if not superior, to freezing method for preservation of postmortem DNA and RNA. In addition, we introduce a new protocol for the amplification of the serotonin transporter gene promoter region (5-HTT) ideal to obtain the increase of specific product, avoiding artifacts formation.

Association of dopamine transporter and monoamine oxidase molecular polymorphisms with sudden infant death syndrome and stillbirth: new insights into the serotonin hypothesis.

Recent findings demonstrated the role of neurotransmitters in the aetiopathogenesis of sudden unexpected deaths in infancy. Although genes involved in serotonin metabolism have been proposed as risk factors for sudden infant death syndrome (SIDS), the contribution of additional neurotransmitters and genes different from the serotonin transporter (SLC6A4, 5-HTT) has not been investigated. Considering the common metabolic pathway and synergism between dopamine and serotonin, the role of dopamine transporter (SLC6A3, DAT) and monoamine oxidase A (MAOA) genes in SIDS and stillbirth (sudden intrauterine unexplained death, SIUD) was investigated. Genotypes and allelic frequencies of DAT and MAOA were determined in 20 SIDS and five stillbirth cases and compared with 150 controls. No association was found between DAT polymorphisms and SIDS either at genotype (P = 0.64) or allelic (P = 0.86) level; however, a highly significant association was found between MAOA genotypes (P = 0.047) and alleles (P = 0.002) regulating different expression patterns (3R/3R vs 3.5R/3.5R + 4R/4R) in SIDS + SIUD and controls. Analysis of combined 5-HTTLPR (serotonin transporter linked polymorphic region)/MAOA genotypes revealed that frequency of L/L-4R/4R genotype combination was eightfold higher in SIDS + SIUD than in controls (P < 0.001). Findings are discussed considering the metabolic association among DAT, 5-HTT and MAOA with special emphasis on the linked action of 5-HTT/MAOA in regulating serotonin metabolism of SIDS and SIUD infants.

Genes regulating the serotonin metabolic pathway in the brain stem and their role in the etiopathogenesis of the sudden infant death syndrome.

Genotypes and allelic frequencies of TPH2, 5-HTTLPR, the 5-HTT (SLC6A4) intron 2 variable-number tandem repeat (VNTR) region, and the MAOA VNTR region were determined in brain-stem samples of 20 "genuine" SIDS cases and compared with results obtained from 150 healthy controls. The SNP G1463A responsible for 80% functionality loss of TPH2 (tryptophan hydroxylase 2) was not detected, neither in SIDS infants nor in the controls. In contrast, a strict relation was found between the 5-HTTLPR genotype and its allelic frequencies with SIDS cases. The L/L genotype and the long allele (L) of the promoter region of the serotonin transporter were significantly associated (likelihood ratio (LR) test, p<0.001) with the syndrome (L/L, 60% SIDS vs 14% controls; L, 80% SIDS vs 42.6% controls). Polymorphisms of the intron 2 VNTR of the same gene showed a trend for significant differences between genotypes 10/10 and 12/12 (LR test, p=0.068), with the L-12 haplotype being almost twofold in SIDS (44.5%) with respect to controls (23.4%). Differences were even higher considering the genotype combination L/L-12/12 (20% SIDS vs 2.6%), and variations among categories were statistically highly significant (p<0.001). Although additional differences were observed in the frequency of the MAOA (monoamine oxidase A) VNTR genotype 3R/3R between SIDS and controls (respectively 15% vs 26%), the results were not supported by statistical significance. Molecular polymorphisms are discussed considering their functional role in regulating serotonin synthesis (TPH2), neuronal reuptake (5-HTTLPR and 5-HTT intron 2), and catabolism (MAOA) in the nervous system of Italian SIDS infants. Comparisons are made with previous data obtained in different ethnic groups.

Guidelines for neuropathologic diagnostics of perinatal unexpected loss and sudden infant death syndrome (SIDS): a technical protocol.

In light of the growing information on the pathophysiology and clinical aspects of unexpected perinatal loss and sudden infant death syndrome (SIDS), a novel approach to the inherent problems by pathologists has become necessary. Herein, we propose an up-to-date protocol for accurate examination of the central autonomic nervous system and of the cardiac conduction system, which can encompass morphological and/or functional abnormalities of reliable epicritical value in unexplained perinatal loss and SIDS, particularly in those cases (still quite numerous) lacking adequate clinical documentation. Anatomo-pathologic examination of the central autonomic nervous system includes an in-depth study on histological serial sections of the brainstem, cerebellum, and spinal cord, where the main structures participating in control of the vital functions are located. For the histological study of the cardiac conductions system, serial sections were obtained from two blocks, including the sino-atrial node and the atrio-ventricular system, respectively. This type of updated investigation is yielding important arguments for a broader discussion of the pathogenesis of unexpected stillbirth, early neonatal death, and SIDS, besides allowing a more complete forensic-medical documentation of individual cases.

Sudden unexpected death in young athletes.

A 13-year-old white boy died suddenly and unexpectedly while playing soccer. This case acquires a unique interest because of the coincidence of sudden unexpected death in a 13-year-old boy, anomalous origin of the left coronary artery from the right aortic sinus of Valsalva, anomalous location of the right coronary ostium within proper aortic sinus of Valsalva, hyperacute myocardial infarction, and myocardial fibrosis. The authors are convinced that the cardiovascular evaluation of young athletes needs to be focused on the identification of individuals at high risk of sudden cardiac arrest, paying attention to suggestive symptoms and to a family history of sudden death due to cardiac arrest, particularly at an early age. In addition, enquiry should be made into the concomitant presence of a smoking habit or of passive smoke exposure.

Sudden unexpected death related to medullary brain lesions.

The sudden unexpected death of a person believed healthy has occasionally been followed by a detailed postmortem examination that revealed no cause of death except for the unexpected presence of a medullary brain lesion. Our review of all available cases of sudden unexpected death related to medullary brain lesions (SUD-MBL) revealed the absence of any specific constellation of ante-mortem disease characteristics, together with the finding that major motor and sensory pathways were grossly preserved in most cases. The wide variety in ages of the victims, and in specific types of tissue pathology affecting the medulla, makes this illness extremely difficult to anticipate when the medullary lesions are not otherwise known to exist during life. SUD-MBL may be a specific clinico-neuropathologic disease entity, having significant importance for forensic investigators trying to establish the cause of sudden unexpected death in a victim of any age. Because victims often harbor their medullary lesions for days or weeks before SUD-MBL, clinical physicians as well need to consider the possibility of medullary brain involvement by any disease process, neurologic or systemic, while managing their patients.

Toxic Effect of Cigarette Smoke on Brainstem Nicotinic Receptor Expression: Primary Cause of Sudden Unexplained Perinatal Death.

Among the neurotoxicants contained in tobacco smoke, if absorbed during pregnancy, nicotine significantly affects α7-nicotinic acetylcholine receptors, which play essential roles in the development of the brainstem regions receiving cholinergic projections in perinatal life. Immunohistochemical procedures for analysing formalin-fixed and paraffin-embedded brainstem samples from 68 fetuses and early newborns, with smoking and non-smoking mothers, who died of known and unknown causes, were carried out in order to determine if nicotine had activated the α7-nicotinic acetylcholine receptors. High α7-nicotinic acetylcholine receptor expression levels were only observed in the victims with smoking mothers. Frequently, these findings were associated with the hypoplasia of the brainstem structures controlling vital functions. The results of this study indicate that the exposition to nicotine in pregnancy exerts a strong direct effect on α7-nicotinic acetylcholine receptor activity especially in perinatal life and may be one of the primary risk factors leading to the sudden unexplained death of fetuses and newborns.

Variability of the medullary arcuate nucleus in humans.

INTRODUCTION: The arcuate nucleus is a component of the ventral medullary surface involved in chemoreception and breathing control. The hypoplasia of this nucleus is a very frequent finding in victims of sudden unexplained fetal and infant death (from the last weeks of pregnancy to the first year of life). On the contrary, this developmental alteration is rarely present in age-matched controls who died of defined causes. These observations lead to hypothesize that a well-developed and functional arcuate nucleus is generally required to sustain life. The aim of this study was to investigate whether the arcuate nucleus maintains the same supposed function throughout life. METHODS: We carried out neuropathological examinations of brainstems obtained from 25 adult subjects, 18 males and 7 females, aged between 34 and 89 years, who died from various causes. RESULTS: For almost half of the cases (44%) microscopic examinations of serial histological sections of medulla oblongata showed a normal cytoarchitecture of the arcuate nucleus, extending along the pyramids. For the remaining 56% of cases, various degrees of hypodevelopment of this nucleus were observed, validated through the application of quantitative morphometric investigations, from decreased area, neuron number and volume, to full aplasia. CONCLUSIONS: These unexpected findings indicate that the involvement of the arcuate nucleus in chemoreception in adulthood is questionable, given the possibility of living until late age without this nucleus. This opens new perspectives for researchers on the role and function of the arcuate nucleus in humans from birth to old age.

Detoxification genes polymorphisms in SIDS exposed to tobacco smoke.

The best hypothesis to explain Sudden Infant Death Syndrome (SIDS) pathogenesis is offered by the "triple risk model", which suggests that an interaction of different variables related to exogenous stressors and infant vulnerability may lead to the syndrome. Environmental factors are triggers that act during a particular sensible period, modulated by intrinsic genetic characteristics. Although literature data show that one of the major SIDS risk factors is smoking exposure, a specific involvement of molecular components has never been highlighted. Starting from these observations and considering the role of GSTT1 and GSTM1 genes functional polymorphisms in the detoxification process, we analyzed GSTM1 and GSTT1 null genotype frequencies in 47 SIDS exposed to tobacco smoke and 75 healthy individuals. A significant association (p < .0001) between the GSTM1 null genotype and SIDS exposed to smoke was found. On the contrary, no association between GSTT1 polymorphism and SIDS was determined. Results indicated the contribution of the GSTM1 -/- genotype resulting in null detoxification activity in SIDS cases, and led to a better comprehension of the triple risk model, highlighting smoking exposure as a real SIDS risk factor on a biochemical basis.

Ontogenesis of human cerebellar cortex and biopathological characterization in sudden unexplained fetal and infant death.

The aims of this study were to investigate in the human cerebellar cortex the structural and biological ontogenetic features, the possible presence of alterations in cases of sudden unexplained fetal and infant death, and the involvement of the maternal cigarette smoking in developmental abnormalities. We analyzed 52 brains of fetal and infant death victims, aged from the second gestational trimester to 12th postnatal month. In the cerebellar cortex we evaluated, besides the morphological aspects, the expression of several biomarkers implicated in proliferative processes (c-fos, proliferating cell nuclear antigen, and apoptosis) as well as the presence of the neurotransmitter somatostatin, which is strongly implicated in central nervous system differentiation, and of EN2 gene. The observed features of the cerebellar cortex, mainly confined to the transient external granular layer, were high proliferative activity and high expression of both somatostatin and EN2 gene in prenatal life and high apoptotic index after birth. In 41% of the sudden unexplained death victims, in the greater part with smoking mothers, we observed different biopathological alterations of the cerebellar cortex. Maternal smoking is increasingly being demonstrated to be one of the main contributors to developmental neurological alterations in the offspring.

Biopathology of the dentate-olivary complex in sudden unexplained perinatal death and sudden infant death syndrome related to maternal cigarette smoking.

OBJECTIVES: The present study was aimed to evaluate the possible presence of cytohistologic and/or biologic modifications of the human dentate-olivary complex in sudden unexplained perinatal and infant deaths. METHODS: We investigated the histologic morphology of the dentate and inferior olivary nuclei, the glial index, the c-fos and apoptotic immunopositivity, as well as the possible effects elicited by maternal cigarette smoking, in 44 cases of perinatal and infant death victims, aged from the 26th gestational week to 10 months of life. RESULTS: We observed subtle alterations of both the medullary inferior olivary nucleus and of the cerebellar dentate nucleus, represented by a significant increase in the reactive astrocyte density and in the neuronal c-fos and apoptotic expression in unexplained death victims, compared with age-matched controls. These alterations were closely related to a maternal cigarette smoking habit. DISCUSSION: We postulate that maternal smoking, besides inducing the previously demonstrated morpho-functional alterations of the autonomic central nervous system, could also exert an adverse influence on the dentate-olivary complex, leading to sudden death in vulnerable periods of perinatal development or early infancy.