The brain beating and heart breathing: a unifying theory of the neuro- cardiac- respiratory control in infant and adult sudden unexpected deaths

Background: Sudden Infant Death Syndrome (SIDS) is characterized by the death of an infant that cannot be explained, despite a systematic case examination, including death scene investigation, autopsy and review of the clinical history. Nowadays, Sudden Unexpected Infant Death (SUID) is a wide-ranging concept used to describe any sudden and unexpected death, whether explained or unexplained, including SIDS, which occurs during the first year of life. Several differing and sometimes contradictory hypotheses of the underlying mechanisms of SIDS have been proposed. The most reliable seems to be the “triple risk hypothesis”. Based on this theory, unexpected infant deaths might arise as a consequence of the combination of three factors coming together: a vulnerable infant, a vulnerable phase of development and a final insult occurring in this window of vulnerability. Recently, a unified neuropathological theory contributes to describing SIDS. According to this, serotonergic neurons play a crucial homeostatic function in the cardiorespiratory brainstem centres. A high incidence of morphological abnormalities and biochemical defects of serotoninergic neurotransmission have been reported in the brainstem of SIDS victims. This brain region includes the main nuclei and structures that coordinate the vital activities, such as cardiovascular function and breathing, perinatal and after birth. Nevertheless, evidence suggests likely genomic complexity and a degree of overlap among SIDS, Sudden Intrauterine Death (SIUD), Sudden Cardiac Death (SCD) and Sudden Unexpected Death in Epilepsy (SUDEP). In SUDEP, which has clinical parallels with SIDS, alterations to medullary serotoninergic neural populations and autonomic dysregulation have been shown too. Molecular profiling of SUDEP cases and the investigation of genetic models have directed to the identification of putative SUDEP genes of which most are ion channel active along the neurocardiac, neuroautonomic, and neurorespiratory pathway. Concurrently, anomalous time- activation, transcription or regional expression of candidate neuro-cardiac-respiratory genes implicated for SUDEP, could be similarly involved in other unexpected sudden deaths. A small but significant proportion of infants who die suddenly and unexpectedly have been shown on postmortem genetic testing to have Developmental Serotonopathies, Cardiac Channelopathies and Autonomic Nervous System Dysregulation, with considerable implications for surviving and future family members. This has led to the demonstration that neuro-cardiac genes are expressed in both tissues (brain and heart) and recently in the respiratory system. Aim: Despite their decreasing incidence, SIDS and SUDEP are still important causes of death. There are many nuclei in the cardio and respiratory centres of the brain involved in unexpected and sudden deaths. Cardiac, sympathetic, and respiratory motor activities can be viewed as a unified rhythm controlled by brainstem neural circuits for effective and efficient gas exchange. We aim to describe abnormalities in these nuclei, in part because robust molecular or functional examination of these nuclei has not been carefully performed. We intend to perform detailed functional mapping of these brainstem nuclei. Specifically, the cardiorespiratory and cardioventilatory coupling can be understood as a unified vital rhythm controlled by brainstem neural circuits. By cardiorespiratory coupling, we mean the Respiratory Sinus Arrhythmia (RSA) that is characterized by a heart rate (HR) increasing during inspiration and an HR decrease during expiration. Conversely, Cardioventilatory coupling (CVC) is considered the influence of heartbeats and arterial pulse pressure on respiration with the tendency for the next inspiration to start at a preferred latency after the last heartbeat in expiration. We hypothesized that these two reflex systems are not separate, but constitute an integrated network. We defined this last concept as "unifying theory". By studying all the maps of the cardiorespiratory nuclei of the Literature, we integrated this concept into a reworking map of brainstem nuclei that could also explain the gasping and blocking cardiorespiratory of sudden deaths. The theory of a unique, unifying cardiorespiratory network, it has been recently demonstrated in some cases of arrhythmia, in some cases of SUDEP with striking systolic hypotensive changes and in some cases of SIDS too. Material and Methods: We investigated articles, reviews indexed in PubMed describing putative neuro-cardiac-respiratory genes and cardiorespiratory, and cardioventilatory coupling theories. Specifically, we evaluated cardiorespiratory brainstem nuclei and whole brains of fetal, infant and adult autopsies respectively to detect congenital errors in the cerebral development or malformations, but also to identify the “normal” or “dysplastic” brainstem centres. Results: Based on the Literature, we identified a brain-heart gene mapping and a scheme of cardiorespiratory brainstem nuclei network. Contemporary, we collected a large pool of fetal brain malformations and cardiorespiratory nuclei dysgenesis both in infants both in adult sudden deaths. We found dysgenesia, agenesia and hypoplasia of brainstem nuclei associated with SIDS cases, compared with post-mortem infant control cases. However, the arcuate nucleus showed insignificant inter-variations regarding adults autoptic cases. Discussion: Many intrinsic and extrinsic factors increase fetal, perinatal, infant, and adult sudden death susceptibility. The final common pathway for SIDS and SUDEP involves a failure to arouse and autoresuscitate in response to environmental challenge. The different risk factors, among these a prone position, can directly alter the function of cardiorespiratory nuclei and impair the ability of this network to coordinate cardiorespiratory–cardioventilatory coupling. Conclusions: Neuropathological analysis of the infant brainstem and neuro-cardiac-respiratory gene mapping represents a good tool to infer on the final events of SIDS and SUDEP, although nothing it is clear regarding the role of adult cardiorespiratory centres. An integrated study of postmortem neuropathology and molecular autopsies could help to understand the network of this beating-breathing-thinking unit

A standardized postmortem protocol to assess the real burden of sudden infant death syndrome

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Serotonin transporter in the temporal lobe, hippocampus and amygdala in SUDEP

Several lines of evidence link deficient serotonin function and SUDEP. Chronic treatment with serotonin reuptake inhibitors (SRIs) reduces ictal central apnoea, a risk factor for SUDEP. Reduced medullary serotonergic neurones, modulators of respiration in response to hypercapnia, were reported in a SUDEP post-mortem series. The amygdala and hippocampus have high serotonergic innervation and are functionally implicated in seizure-related respiratory dysregulation. We explored serotonergic networks in mesial temporal lobe structures in a surgical and post-mortem epilepsy series in relation to SUDEP risk. We stratified 75 temporal lobe epilepsy patients with hippocampal sclerosis (TLE/HS) into high (N = 16), medium (N = 11) and low risk (N = 48) groups for SUDEP based on generalised seizure frequency. We also included the amygdala in 35 post-mortem cases, including SUDEP (N = 17), epilepsy controls (N = 10) and non-epilepsy controls (N = 8). The immunohistochemistry labelling index (LI) and axonal length (AL) of serotonin transporter (SERT)-positive axons were quantified in 13 regions of interest with image analysis. SERT LI was highest in amygdala and subiculum regions. In the surgical series, higher SERT LI was observed in high risk than low risk cases in the dentate gyrus, CA1 and subiculum (p < 0.05). In the post-mortem cases higher SERT LI and AL was observed in the basal and accessory basal nuclei of the amygdala and peri-amygdala cortex in SUDEP compared to epilepsy controls (p < 0.05). Patients on SRI showed higher SERT in the dentate gyrus (p < 0.005) and CA4 (p < 0.05) but there was no difference in patients with or without a psychiatric history. Higher SERT in hippocampal subfields in TLE/HS cases with SUDEP risk factors and higher amygdala SERT in post-mortem SUDEP cases than epilepsy controls supports a role for altered serotonergic networks involving limbic regions in SUDEP. This may be of functional relevance through reduced 5-HT availability

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process

The Collaborative for the Research on Black Women and Girls

<p>Seven patients per group. **P<0.01 Mann-Whitney U test. Representative granule cell layer hippocampal sections from patients without granule cell pathology (B) or with type-2 GCP (C) exhibiting DAB-labeled ANTXR1-like immunoreactivity (LI). Omitting the primary antibody to estimate nonspecific signal yielded completely negative labeling (data not shown). Note a widespread increase in ANTXR1-LI in granule cells from patients with type-2 GCP (C).</p

The brain beating and heart breathing: a unifying theory of the neuro- cardiac- respiratory control in infant and adult sudden unexpected deaths

Background: Sudden Infant Death Syndrome (SIDS) is characterized by the death of an infant that cannot be explained, despite a systematic case examination, including death scene investigation, autopsy and review of the clinical history. Nowadays, Sudden Unexpected Infant Death (SUID) is a wide-ranging concept used to describe any sudden and unexpected death, whether explained or unexplained, including SIDS, which occurs during the first year of life. Several differing and sometimes contradictory hypotheses of the underlying mechanisms of SIDS have been proposed. The most reliable seems to be the “triple risk hypothesis”. Based on this theory, unexpected infant deaths might arise as a consequence of the combination of three factors coming together: a vulnerable infant, a vulnerable phase of development and a final insult occurring in this window of vulnerability. Recently, a unified neuropathological theory contributes to describing SIDS. According to this, serotonergic neurons play a crucial homeostatic function in the cardiorespiratory brainstem centres. A high incidence of morphological abnormalities and biochemical defects of serotoninergic neurotransmission have been reported in the brainstem of SIDS victims. This brain region includes the main nuclei and structures that coordinate the vital activities, such as cardiovascular function and breathing, perinatal and after birth. Nevertheless, evidence suggests likely genomic complexity and a degree of overlap among SIDS, Sudden Intrauterine Death (SIUD), Sudden Cardiac Death (SCD) and Sudden Unexpected Death in Epilepsy (SUDEP). In SUDEP, which has clinical parallels with SIDS, alterations to medullary serotoninergic neural populations and autonomic dysregulation have been shown too. Molecular profiling of SUDEP cases and the investigation of genetic models have directed to the identification of putative SUDEP genes of which most are ion channel active along the neurocardiac, neuroautonomic, and neurorespiratory pathway. Concurrently, anomalous time- activation, transcription or regional expression of candidate neuro-cardiac-respiratory genes implicated for SUDEP, could be similarly involved in other unexpected sudden deaths. A small but significant proportion of infants who die suddenly and unexpectedly have been shown on postmortem genetic testing to have Developmental Serotonopathies, Cardiac Channelopathies and Autonomic Nervous System Dysregulation, with considerable implications for surviving and future family members. This has led to the demonstration that neuro-cardiac genes are expressed in both tissues (brain and heart) and recently in the respiratory system. Aim: Despite their decreasing incidence, SIDS and SUDEP are still important causes of death. There are many nuclei in the cardio and respiratory centres of the brain involved in unexpected and sudden deaths. Cardiac, sympathetic, and respiratory motor activities can be viewed as a unified rhythm controlled by brainstem neural circuits for effective and efficient gas exchange. We aim to describe abnormalities in these nuclei, in part because robust molecular or functional examination of these nuclei has not been carefully performed. We intend to perform detailed functional mapping of these brainstem nuclei. Specifically, the cardiorespiratory and cardioventilatory coupling can be understood as a unified vital rhythm controlled by brainstem neural circuits. By cardiorespiratory coupling, we mean the Respiratory Sinus Arrhythmia (RSA) that is characterized by a heart rate (HR) increasing during inspiration and an HR decrease during expiration. Conversely, Cardioventilatory coupling (CVC) is considered the influence of heartbeats and arterial pulse pressure on respiration with the tendency for the next inspiration to start at a preferred latency after the last heartbeat in expiration. We hypothesized that these two reflex systems are not separate, but constitute an integrated network. We defined this last concept as "unifying theory". By studying all the maps of the cardiorespiratory nuclei of the Literature, we integrated this concept into a reworking map of brainstem nuclei that could also explain the gasping and blocking cardiorespiratory of sudden deaths. The theory of a unique, unifying cardiorespiratory network, it has been recently demonstrated in some cases of arrhythmia, in some cases of SUDEP with striking systolic hypotensive changes and in some cases of SIDS too. Material and Methods: We investigated articles, reviews indexed in PubMed describing putative neuro-cardiac-respiratory genes and cardiorespiratory, and cardioventilatory coupling theories. Specifically, we evaluated cardiorespiratory brainstem nuclei and whole brains of fetal, infant and adult autopsies respectively to detect congenital errors in the cerebral development or malformations, but also to identify the “normal” or “dysplastic” brainstem centres. Results: Based on the Literature, we identified a brain-heart gene mapping and a scheme of cardiorespiratory brainstem nuclei network. Contemporary, we collected a large pool of fetal brain malformations and cardiorespiratory nuclei dysgenesis both in infants both in adult sudden deaths. We found dysgenesia, agenesia and hypoplasia of brainstem nuclei associated with SIDS cases, compared with post-mortem infant control cases. However, the arcuate nucleus showed insignificant inter-variations regarding adults autoptic cases. Discussion: Many intrinsic and extrinsic factors increase fetal, perinatal, infant, and adult sudden death susceptibility. The final common pathway for SIDS and SUDEP involves a failure to arouse and autoresuscitate in response to environmental challenge. The different risk factors, among these a prone position, can directly alter the function of cardiorespiratory nuclei and impair the ability of this network to coordinate cardiorespiratory–cardioventilatory coupling. Conclusions: Neuropathological analysis of the infant brainstem and neuro-cardiac-respiratory gene mapping represents a good tool to infer on the final events of SIDS and SUDEP, although nothing it is clear regarding the role of adult cardiorespiratory centres. An integrated study of postmortem neuropathology and molecular autopsies could help to understand the network of this beating-breathing-thinking unit

Neuropathology of Early Sudden Infant Death Syndrome-Hypoplasia of the Pontine Kolliker-Fuse Nucleus: A Possible Marker of Unexpected Collapse during Skin-to-Skin Care.

OBJECTIVE: \u2003To find a possible pathogenetic mechanism of the early sudden infant death occurring in newborns during the skin-to-skin care (SSC), through the examination of neuronal centers regulating the vital activities. STUDY DESIGN: \u2003This is an in-depth examination of the brain stem in 22 healthy term newborns, suddenly died in the first hour of life without the identification of a cause at autopsy (early sudden infant death syndrome [eSIDS]), 12 of them concomitantly with SSC, and 10 with age-matched controls died of known pathology. RESULTS: \u2003Developmental alterations of neuronal structures of the brain stem were highlighted in 19 of the 22 eSIDS, but not in control. The hypoplasia of the pontine K\uf6lliker-Fuse nucleus (KFN), an important respiratory center, was diagnosed at the histological examination, validated by morphometric quantifications, in 11 of the 12 eSIDS while they were placed on the mother's chest and in 2 of the 10 SSC unrelated neonatal deaths. CONCLUSION: \u2003The delayed development of the KFN could represent a specific finding of eSIDS occurring during SSC. Therefore, it is necessary to point out that the SSC represents a further risk factor that must be added to others already known for sudden infant death syndrome. Then this practice needs appropriate monitoring strategies of the infant's conditions

From fix to fit into the autoptic human brains

Formalin-fixed, paraffin embedded (FFPE) human brain tissues are very often stored in formalin for long time. Formalin fixation reduces immunostaining, and the DNA/RNA extraction from FFPE brain tissue becomes suboptimal. At present, there are different protocols of fixation and several procedures and kits to extract DNA/RNA from paraffin embedding tissue, but a gold standard protocol remains distant. In this study, we analysed four types of fixation systems and compared histo- and immunostaining. Based on our results, we propose a modified method of combined fixation in formalin and formic acid for the autoptic adult brain to obtain easy, fast, safe and efficient immunolabelling of long-stored FFPE tissue. In particular, we have achieved an improved preservation of cellular morphology and obtained success in post-mortem immunostaining for NeuN. This nuclear antigen is an important marker for mapping neurons, for example, to evaluate the histopathology of temporal lobe epilepsy or to draw the topography of cardiorespiratory brainstem nuclei in sudden infant death syndrome (SIDS). However, NeuN staining is frequently faint or lost in post-mortem human brain tissues. In addition, we attained Fluoro Jade C staining, a marker of neurodegeneration, and immunofluorescent staining for stem cell antigens in the postnatal human brain, utilizing custom fit fixation procedures

Calsequestrin in Purkinje cells of mammalian cerebellum

: Cerebellum is devoted to motor coordination and cognitive functions. Endoplasmic reticulum is the largest intracellular calcium store involved in all neuronal functions. Intralumenal calcium binding proteins play a pivotal role in calcium storage and contribute to both calcium release and uptake. Calsequestrin, a key calcium binding protein of sarco-endoplasmic reticulum in skeletal and cardiac muscles, was identified in chicken and fish cerebellum Purkinje cells, but its expression in mammals and human counterpart has not been studied in depth. Aim of the present paper was to investigate expression and localization of Calsequestrin in mammalian cerebellum. Calsequestrin was found to be expressed at low level in cerebellum, but specifically concentrated in Calbindin D28- and zebrin- immunopositive-Purkinje cells. Two additional fundamental calcium store markers, sarco-endoplasmic calcium pump isoform 2, SERCA2, and Inositol-trisphosphate receptor isoform 1, IP3R1, were found to be co-expressed in the region, with some localization peculiarities. In conclusion, a new marker was identified for Purkinje cells in adult mammals, including humans. Such a marker might help in staminal neuronal cells specification and in dissection of still unknown neurodegeneration and physio-pathological effects of dysregulated calcium homeostasis