Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome.

Importance: Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed. Objective: To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection. Design, Setting, and Participants: In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes. Exposures: There were no interventions or exposures. Main Outcomes and Measures: CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue. Results: A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels. Conclusions and Relevance: Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Dysregulation of platelet serotonin, 14-3-3, and GPIX in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant mortality, but the underlying cause(s) are unclear. A subset of SIDS infants has abnormalities in the neurotransmitter, serotonin (5-hydroxytryptamine [5-HT]) and the adaptor molecule, 14-3-3 pathways in regions of the brain involved in gasping, response to hypoxia, and arousal. To evaluate our hypothesis that SIDS is, at least in part, a multi-organ dysregulation of 5-HT, we examined whether blood platelets, which have 5-HT and 14-3-3 signaling pathways similar to brain neurons, are abnormal in SIDS. We also studied platelet surface glycoprotein IX (GPIX), a cell adhesion receptor which is physically linked to 14-3-3. In infants dying of SIDS compared to infants dying of known causes, we found significantly higher intra-platelet 5-HT and 14-3-3 and lower platelet surface GPIX. Serum and plasma 5-HT were also elevated in SIDS compared to controls. The presence in SIDS of both platelet and brainstem 5-HT and 14-3-3 abnormalities suggests a global dysregulation of these pathways and the potential for platelets to be used as a model system to study 5-HT and 14-3-3 interactions in SIDS. Platelet and serum biomarkers may aid in the forensic determination of SIDS and have the potential to be predictive of SIDS risk in living infants.

Altered 5-HT2A/C receptor binding in the medulla oblongata in the sudden infant death syndrome (SIDS): Part II. Age-associated alterations in serotonin receptor binding profiles within medullary nuclei supporting cardiorespiratory homeostasis.

The failure of chemoreflexes, arousal, and/or autoresuscitation to asphyxia may underlie some sudden infant death syndrome (SIDS) cases. In Part I, we showed that some SIDS infants had altered 5-hydroxytryptamine (5-HT)2A/C receptor binding in medullary nuclei supporting chemoreflexes, arousal, and autoresuscitation. Here, using the same dataset, we tested the hypotheses that the prevalence of low 5-HT1A and/or 5-HT2A/C receptor binding (defined as levels below the 95% confidence interval of controls-a new approach), and the percentages of nuclei affected are greater in SIDS versus controls, and that the distribution of low binding varied with age of death. The prevalence and percentage of nuclei with low 5-HT1A and 5-HT2A/C binding in SIDS were twice that of controls. The percentage of nuclei with low 5-HT2A/C binding was greater in older SIDS infants. In >80% of older SIDS infants, low 5-HT2A/C binding characterized the hypoglossal nucleus, vagal dorsal nucleus, nucleus of solitary tract, and nuclei of the olivocerebellar subnetwork (important for blood pressure regulation). Together, our findings from SIDS infants and from animal models of serotonergic dysfunction suggest that some SIDS cases represent a serotonopathy. We present new hypotheses, yet to be tested, about how defects within serotonergic subnetworks may lead to SIDS.

Multimodal MRI reveals brainstem connections that sustain wakefulness in human consciousness.

Consciousness is composed of arousal (i.e., wakefulness) and awareness. Substantial progress has been made in mapping the cortical networks that underlie awareness in the human brain, but knowledge about the subcortical networks that sustain arousal in humans is incomplete. Here, we aimed to map the connectivity of a proposed subcortical arousal network that sustains wakefulness in the human brain, analogous to the cortical default mode network (DMN) that has been shown to contribute to awareness. We integrated data from ex vivo diffusion magnetic resonance imaging (MRI) of three human brains, obtained at autopsy from neurologically normal individuals, with immunohistochemical staining of subcortical brain sections. We identified nodes of the proposed default ascending arousal network (dAAN) in the brainstem, hypothalamus, thalamus, and basal forebrain. Deterministic and probabilistic tractography analyses of the ex vivo diffusion MRI data revealed projection, association, and commissural pathways linking dAAN nodes with one another and with DMN nodes. Complementary analyses of in vivo 7-tesla resting-state functional MRI data from the Human Connectome Project identified the dopaminergic ventral tegmental area in the midbrain as a widely connected hub node at the nexus of the subcortical arousal and cortical awareness networks. Our network-based autopsy methods and connectivity data provide a putative neuroanatomic architecture for the integration of arousal and awareness in human consciousness.