Cerebral c-jun expression mapping in sudden infant death syndrome

Despite a decline in the overall rate of sudden infant death syndrome (SIDS), it remains the leading cause of postneonatal infant mortality. Research into underlying mechanisms of SIDS has still not yielded a morphological, histopathological correlate explaining aetiology and pathophysiology of an infants sudden death. Of particular interest would be elucidating pathophysiological and molecular events immediately preceding the infants sudden death. C-jun is an immediate early gene with a physiological role in orchestrating cellular responses following injury including a role in cell death. It is also one of the earliest and most consistent markers for neurons undergoing stress, degeneration and survival. We investigated the immunohistochemical expression of the acute phase protein c-jun within 47 separate cerebral regions of interest in the brains of 23 SIDS and 7 control cases for evidence of acute neuronal injury immediately preceding sudden death and to assess the distribution of cellular damage suggestive of pathophysiologically sensitive or critical cerebral regions. Dividing SIDS-cases and controls into subgroups with (1) no neuronal expression of c-jun at all or (2) some c-jun expression, a statistically significant higher c-jun expression for SIDS compared with control cases was established for the inferior colliculi (P=0.008), the spinal trigeminal nuclei (P=0.046) and the premotoric cortex layers II and IV (P=0.029). The increased expression of c-jun in these areas might be directly related to the pathogenesis of sudden death, or may represent a secondary marker of neuronal injury with the primary event/injury being elsewhere in critical cardioventilatory circuits.     

Phrenic nerve maturation in the sudden infant death syndrome

It has been suggested that delayed development in either the peripheral or central nervous system could underlie the sudden infant death syndrome (SIDS). We studied the phrenic nerve in an attempt to find if maturation in this nerve was delayed in SIDS, and to see if fiber size differences could explain the paucity of type I muscle fibers described in SIDS diaphragms. Samples of phrenic nerves were taken at post mortem from 11 SIDS and 10 control infants. Myelinated fibers were quantitated in 1-μm silver-stained resin sections using a semi-automatic image analysis system. The numbers and sizes of all myelinated fibers in the right phrenic nerve were compared between SIDS and control groups. The proportion of small (less than 6-μm diameter) phrenic nerve fibers was similar in SIDS (53.1%, SD 12.8%) and control (53.6%, SD 14.0%) cases. The proportion of small fibers decreased with increasing age at an equivalent rate in both SIDS and control groups. Total numbers of myelinated fibers were similar in the right phrenic nerves of SIDS (3429, SD 440) and control (3457, SD 507) infants, but varied widely between cases and were not correlated with age. Maturation of the phrenic nerve, as judged by increasing size of myelinated fibers, is therefore similar in SIDS and control infants. This argues against a widespread developmental delay in SIDS. No changes in phrenic nerve fiber sizes were found to account for the finding of fewer type I fibers in SIDS diaphragms. Received: 27 July 1995 / Revised, accepted: 11 October 1995     

Severe hypoplasia of medullary arcuate nucleus: quantitative analysis in sudden infant death syndrome

The human arcuate nucleus (ARCn) is postulated to be homologous to ventral medullary cells involved in chemoreception, and respiratory and blood pressure responses. Abnormalities in central respiratory control may result from dysfunction of this anatomic ventral area. We evaluated the changes of the neuronal population of the medullary ARCn in infants victims of the sudden infant death syndrome (SIDS). In this study we tested the hypothesis that anatomical deficiency of the ARCn is associated with SIDS. The volume and neuronal density of the ARCn were morphometrically quantified with an image analyzer in 36 cases of SIDS and 12 age-matched controls. We found a marked hypoplasia in the SIDS ARCn compared to controls and, particularly, in 11 SIDS cases (30%) in which the ARCn exhibited a severe hypoplasia, being almost totally absent. Three-dimensional reconstructions and morphometric measurements of ARCn confirmed this marked hypoplasia in all the serial sections examined (P = 0.0001) and the reduced neuronal density (P = 0.0025) in relation to control cases. In conclusion these abnormalities observed in the ARCn are consistent with the idea that ARCn dysfunction plays an important role among the causative factors of sudden infant death. The hypoplasia of the ARCn represents the most frequent congenital abnormality in our experience, and can be a plausible morphological substrate for a subset of SIDS. Received: 11 May 1999 / Accepted: 6 August 1999     

Autonomic responses to sighs in healthy infants and in victims of sudden infant death

OBJECTIVE: Sigh, defined as an isolated breath with an increased tidal volume, can be associated with abrupt changes in heart rate (HR) or blood oxygenation. Sigh may be followed by a central apnea. As impairment of autonomic control was postulated in future SIDS victims, we hypothesized that their autonomic responses to sighs were different from those of healthy control infants. METHODS: Sighs followed by central apnea were studied in the sleep recordings of 18 infants who eventually died of SIDS and of 18 control infants. The infants of the two groups were matched for sex, gestational age, postnatal age, weight at birth and sleep position during sleep recording. HR autoregressive power spectral analysis was performed on RR intervals preceding and following sighs. RESULTS: In all infants, most sighs followed by an apnea were found in NREM sleep. Compared to the control infants, the future SIDS victims were characterized by a greater sympathovagal balance and a lower parasympathetic tonus before the sighs. Following the sighs, no more differences were found in NREM sleep. CONCLUSION: Based on the present findings, it can be postulated that sighs contribute to reset autonomic tonus during NREM sleep.     

Glial and neuronal alterations in the nucleus tractus solitarii of sudden infant death syndrome victims

The factors underlying the sudden infant death syndrome (SIDS) are still unknown, but in recent years much attention has been focused on the central cardiorespiratory control system. In the present work we analyzed the nucleus tractus solitarii (nTS) of 23 SIDS victims and 17 age-matched control cases. We studied the functional and morphological alterations of neurons and glial cells to evaluate the results of possible hypoxic-ischemic injury that could have led to sudden death. Morphometric and immunohistochemical analyses were performed on medullary sections. In the nTS of SIDS victims we observed modifications of both neuronal and glial cells. Brain injury triggers the activation of both astrocytes and microglia, which respond to neuronal damage by characteristic changes that could explain our observations in the nTS of SIDS victims. In our investigation of the nTS of SIDS victims we found a significant increase of reactive astrocytes density, a significantly higher percentage of necrotic cells, an increase of reactive microglial cells density, a significantly higher expression of substance P and the presence of NMDA receptors immunoreactivity. Our results support the hypothesis that there is injury of the nTS neurons in SIDS victims, even if the causes of this damage are still unknown. This neuronal damage may explain why adequate ventilation is often not maintained during hypoxia. Such histological findings have never been thought sufficient to explain SIDS, but the tissue findings could be an indication of the impairment of several pathophysiological mechanisms which may underlie brainstem dysfunction, affecting cardiorespiratory control.     

Delayed neuronal maturation of the medullary arcuate nucleus in sudden infant death syndrome

Recently, quantitative abnormalities in neuronal populations derived from the rhombic lip (inferior olive nucleus of the brain stem and external granular layer of the cerebellum) have been reported in victims of the sudden infant death syndrome (SIDS). In this study we examined the arcuate nucleus (ARCn) of 35 SIDS victims and 25 controls, to determine neuronal abnormalities involving this nucleus in SIDS. Computer-assisted cell evaluation was made on sections stained with hematoxylin and eosin to study the neuronal dimensions (nuclear and cytoplasmic area, nuclear/cytoplasmic ratio), the form factor and the density of reactive astrocytes. There was a significant reduction of the neuronal area (nuclear and cytoplasmic) in SIDS victims compared with controls. The neuronal populations of SIDS victims had a significantly higher form factor, index of immaturity. The SIDS victims were divided into two groups on the basis of ARCn development: 18 SIDS-A cases with a well-developed ARCn and 17 SIDS-B cases with severe bilateral hypoplasia. The results of our research indicate that the developmental defect is characterized by a reduction in size of the ARC neurons and by neuronal depletion. In SIDS the ARCn has the histomorphological features of neuronal immaturity, and there is a marked reduction of all quantitative cell parameters and lower astrocytes density with respect to controls. On the basis of the morphometric results of the arcuate neuronal populations, we hypothesize that infants whose neurons have failed to reach full maturity are at risk for SIDS because they are unable to develop appropriate cardioventilatory control.     

Serotoninergic receptor 1A in the sudden infant death syndrome brainstem medulla and associations with clinical risk factors

The immunoreactivity of the serotoninergic receptor subtype 1A (5HT_1AR) was quantitatively analyzed in the human infant brainstem medulla (caudal and rostral levels). We hypothesized that immunoreactivity of 5HT_1AR would be reduced in infants diagnosed with sudden infant death syndrome (SIDS). In particular that those infants with known clinical risk factors (including cigarette smoke exposure, bed sharing and sleep position) would have greater changes than those without clinical risks. Comparing SIDS (n = 67) to infants who died suddenly with another diagnosis (non-SIDS, n = 25), we found decreased 5HT_1AR immunoreactivity in the majority of the nuclei studied at the rostral medulla level including dorsal motor nucleus of the vagus (DMNV), nucleus of the solitary tract, vestibular, and inferior olivary nucleus (ION). There was a significant relationship with all risk factors for 5HT_1AR, especially for DMNV, suggesting that 5HT_1ARs are highly vulnerable to various insults within the SIDS DMNV. This study not only provides further evidence of abnormalities within the brainstem serotoninergic system of SIDS infants, but also shows that these changes may be associated with exposure to clinical risk factors.     

Cytological investigations on the cerebellar cortex of sudden infant death victims

Summary The study was based on the hypothesis that cerebellar hypoxia may play a role in sudden infant death syndrome resulting in morphological changes of the cerebellar cortex, especially with respect to Purkinje cell density. In the morphological evaluation of the Purkinje cell layer, special consideration was additionally given to secondary alterations (i.e., macrophage and/or astrocyte reaction). A total of 12 sudden infant death syndrome cases were compared with an age- and sex-matched control group. The Purkinje cell density was evaluated by determining the number of these cells per suface unit on parasagittal sections in both hemispheres. The myelomonocytic and glial reaction was demonstrated by immunohistochemical methods using lysozyme as leukocyte and macrophage markers and glial fibrillary acidic protein as an astrocyte marker. Qualitatively, no alterations resembling a macrophage or glial cell reaction were detected in sudden infant death syndrome. No differences between the right and left cerebellar hemisphere could be established in the victims of sudden infant death syndrome nor in the controls. The number of Purkinje cells per 0.352 mm² cortex was higher in the younger victims of sudden infant death (younger than 45 weeks of gestation) than in all matched controls. A statistically significant difference in Purkinje cell density, however, could not be established, and, especially, no indications of hypoxia were observed in the cerebellar cortex.     

Astrocytes in the hypoglossal nuclei of sudden infant death syndrome (SIDS) infants: a quantitative study

It has been suggested that brain stem hypoxia or ischaemia underlies the sudden infant death syndrome (SIDS), but previous reports of astrocytosis in the brain stems of SIDS infants have been contradictory. A volumetric quantitative technique was, therefore, developed to compare astrocyte numbers and sizes in the hypoglossal nuclei of SIDS and control infancts. In 12 SIDS and eight control infants, serial sagittal sections were taken through the hypoglossal nucleus and every tenth section through the h ypoglossal nucleus and every tenth section was stained for glial fibrillary acidic protein. Astrocytes were counted in the central 4% of a grid stepped throughout the hypoglossal nucleus, and the heights of 100 astrocyte nuclei were measured with a mocrocator. Astrocyte number, corrected for section thickness and nuclear height, was divided by the volume of the hypoglossal nucleus to calculate astrocyte density. Numbers of astrocytes did not differ significantly between SIDS (mean number 44 729, SD 12 096) and control (mean number 46 562, SD 11 060) infants. Astorcyte nuclear height did not differ significantly between groups (SIDS: mean height 3.98μm, SD 0.31). Astrocyte density was similar in SIDS (mean density 24 378 astrocyte/mm³, SD 6155) and control (mean density 23 978 astrocytes/mm³, SD 4031) infants. No quantitative evidence of astrocytosis was found in the hypoglossal nuclei of SIDS infants. This implies that SIDS infants die without previous episodes of hypoxia/ischaemia severe enough to damage the brain stem.     

PrP<Superscript>Sc</Superscript>-like prion protein conformer in sudden infant death syndrome brain

Nothing is known about the pathophysiology of sudden infant death syndrome (SIDS). Here we show the presence of misfolded prion protein (PrP^Sc-like) in extracts of various sections of the brains of two SIDS victims. DNA sequence information for one of these (death at 12 days) revealed two nucleotide variants in the protein coding region of the PrP gene. This may be a key finding in the understanding of SIDS pathology, and may suggest ways for identifying risk factors for SIDS in newborn infants.     

Polysomnographic study of the autonomic nervous system in potential victims of sudden infant death syndrome

To determine the influence of sleep stages and night-time distribution on cardiac autonomic activity, the polysomnographic recordings of 18 victims of sudden infant death syndrome (SIDS) and of 36 control infants were studied. Autoregressive spectral analyses of heart rate (HR), using both short-term and whole-night methods, were evaluated as a function of sleep stages. The two main peaks of normalized LF and HF were computed during the night and the sympathovagal balance (LF/HF) was determined.In both NREM and REM sleep, SIDS infants were characterized by significantly lower normalized HF powers and higher LF/HF ratios. This finding was observed in both short-term and whole-night HR spectral analyses. In addition, SIDS victims were characterized by the presence of a high desynchronized peak of sympathetic tonus in the late hours of the night, a finding not seen in the control subjects.This report adds further indirect evidence for a possible sleep-related impairment of autonomic controls in some infants who died of SIDS.     

Enhanced reactivity of Alz-50 antibody in brains of sudden infant death syndrome victims versus brains with lethal hypoxic/ischemic injury

Alz-50 antibody is immunoreactive with brain tissue of subjects with Alzheimer’s disease and can also be demonstrated by immunocytochemistry in neurons of vibratome-prepared brain tissue of victims of sudden infant death syndrome (SIDS). The application of a slightly modified ImmunoMax method enabled us to demonstrate Alz-50 immunoreactivity in paraffin-embedded material. The Alz-50 epitope was detected in the hippocampus region and in nuclei of the medulla oblongata at the level of the inferior olivary protuberance in three diagnostic groups: victims of SIDS (n = 10), infants dying of subacute hypoxia/ischemia with subsequent (re-)perfusion (n=9), and infants dying of acute ischemia without (re-) perfusion (n = 7). Quantitative evaluation of the hippocampal cortex and the nucleus olivaris inferior disclosed a significantly (P < 0.05) higher percentage of Alz-50-reactive neurons in SIDS cases than in the control groups (hippocampal cortex and nucleus olivaris; SIDS victims: median = 100%; subacute hypoxia/ischemia: median = 33.6– 81%; acute ischemia: median = 89.2–99%). Semiquantitative analysis revealed an equally pronounced preponderance of Alz-50-reactive neurons in SIDS victims versus the control groups. This greater expression in SIDS victims may be due to an ongoing hypoxia/ischemia during agony, but the present paucity of knowledge prohibits definitive elucidation. Nevertheless, the method described here appears to offer the realistic possibility of distinguishing SIDS cases from cases of sudden death in infants due to other causes, i.e., it offers for the first time a positive criterion for the diagnosis of SIDS. Received: 17 April 1997 / Revised: 7 August 1997 / Revised, accepted: 30 September 1997     

Brainstem immaturity in sudden infant death syndrome: A quantitative rapid Golgi study of dendritic spines in 95 infants

Quantitative analysis of reticular dendritic spines was performed on rapid Golgi impregnated neurons in 7 brainstem areas from 61 sudden infant death syndrome (SIDS) and 34 control infants. Throughout the first postnatal year, mean spine density in SIDS was significantly greater than the mean density in controls (P less than 0.0001). There were significantly higher values of spine density in SIDS compared to controls (P less than 0.0001) in both term and preterm infants. Within the SIDS brainstem itself, the density of dendritic spines was significantly different (P less than 0.05) between two medullary regions and between reticular and non-reticular formation areas. Among these brainstem areas in controls, there was no significant difference. Our findings indicate an immature developmental pattern of increased dendritic spine density in the SIDS brainstem which may be responsible for abnormal central respiratory and arousal control. These significant quantitative differences in spine density are considered in the present study to represent an anatomical substrate of brainstem immaturity in the multifactorial pathogenesis of SIDS.     

Vulnerability of fourth ventricle choroid plexus in sudden unexplained fetal and infant death syndromes related to smoking mothers

The human choroid plexuses in the ventricular system represent the main source of cerebrospinal fluid secretion and constitute a major barrier interface that controls the brain's environment. The present study focused on the choroid plexus of the fourth ventricle, the main cavity of the brainstem containing important nuclei and/or structures mediating autonomic vital functions.     

Brain stem nuclei in sudden infant death syndrome (SIDS): volumes, neuronal numbers and positions

It has been suggested that the defect underlying the sudden infant death syndrome (SIDS) lies in brain stem nuclei involved in cardiac and respiratory function. However, most studies have not used rigorous quantitative techniques to assess brain stem nuclear volumes and neuronal numbers. We have measured the volume, neuronal numbers and position of brain stem nuclei in 11 SIDS and 11 aged-matched control infants. Using serial sagittal sections, nuclei involved in maintaining airway patency (hypoglossal, ambiguus and retro-ambiguus), heart rate (dorsal vagal) and generation of respiratory rhythm (ambiguus and dorsal vagal) were studied. No significant differences were found in nuclear volume increase with age, total neuronal number or nuclear position between SIDS and control cases. These findings support the hypothesis that the nervous system in SIDS may be normal until the final event that kills these infants.     

Severe postictal laryngospasm as a potential mechanism for sudden unexpected death in epilepsy: a near-miss in an EMU

A 42-year-old man with refractory epilepsy experienced a 1-min generalized tonic-clonic seizure followed by persistent inspiratory stridor and cyanosis while being monitored in our epilepsy monitoring unit (EMU). Although his cardiac parameters remained stable throughout the event, the patient's respiratory status rapidly declined, despite the urgent administration of oxygen via bag-valve-mask. He was subsequently intubated by the emergency code team, who noted severe laryngospasm while trying to insert the endotracheal tube. The patient was successfully resuscitated. This monitored case demonstrates that postictal laryngospasm may represent another potential cause of sudden unexpected death in epilepsy (SUDEP).     

Neuronal cell death in the arcuate nucleus of the medulla oblongata in stillbirth

The hypothesis that unexplained stillbirth arises in a similar manner as the sudden infant death syndrome (SIDS) is based in part on shared neuropathologic features between the two entities, including hypoxic-ischemic lesions such as white matter and brainstem gliosis, as well as aplasia or hypoplasia of the arcuate nucleus on the ventral surface of the medulla. The arcuate nucleus is the putative homologue of the respiratory chemosensory region at the ventral medullary surface in animals that is involved in central chemosensitivity. To determine arcuate nucleus pathology in stillbirth, and its co-occurrence with evidence of hypoxia-ischemia, we reviewed brain specimens from the archives of our hospitals from 22 consecutive stillbirths from 22 to 41 gestational weeks. Explained causes of death (n=17) included nuchal cord, acute chorioamnionitis, placental abruption, and fetal glomerulosclerosis; 5 cases were unexplained. In 12 brains, we observed nuclear karyorrhexis and/or pyknosis with cytoplasmic hypereosinophilia in neurons in the arcuate nucleus in both explained (n=8) and unexplained (n=4) cases (54.5% of total cases). Three additional cases had arcuate aplasia (n=1) or hypoplasia (n=2) (13.6% of total cases); one of the latter cases also had neuronal necrosis in the hypoplastic arcuate. The degree of gliosis in the region of the arcuate nucleus was variable across all cases, without statistically significant differences between groups with and without arcuate nucleus necrosis. Other lesions in association with (n=14) and without (n=8) arcuate nucleus abnormalities were diffuse cerebral white matter gliosis, periventricular leukomalacia (PVL), and neuronal necrosis in the hippocampus, basal ganglia, thalamus, basis pontis, and brainstem tegmentum. In 16/20 (80.0%) cases (with or without histologic necrosis of the arcuate), immunostaining with caspase-3 demonstrated positive neurons. Our findings suggest that neuronal pathology in the arcuate nucleus may be both developmental (13.6%) and acquired (54.5%). The association of neuronal necrosis and apoptosis in the arcuate nucleus with systemic entities involving fetal ischemia, and with other brain lesions consistent with ischemia, e.g., cerebral white matter gliosis, suggests that ischemia plays a role in the arcuate nucleus damage as well. Thus, the underpopulation of arcuate neurons detected postnatally in some SIDS infants may be secondary to an acquired insult in mid- or late gestation, and in other cases, a primary developmental lesion in early gestation, or both. The role of arcuate nucleus pathology in the pathogenesis of fetal demise remains to be determined.     

Post-mortem analyses of neuropeptides in brains from sudden infant death victims

The causative factors underlying SIDS are still unknown, but in recent years much interest has been focused on the central ventilatory control system. In this study, peptides which are known to affect respiration were examined in brains from SIDS victims and controls. The levels of Met-enkephalin and substance P were measured in cortex, medulla oblongata, pons and hypothalamus. Substance P1-7, substance P C-terminal fragments, Met-enkephalin-Lys6 and neuropeptide Y (NPY) were estimated in medulla oblongata. The substance P levels in the medulla oblongata from the SIDS victims were significantly elevated compared with the controls. No change, however, was observed in the Met-enkephalin levels, but a tendency to higher levels in the youngest infants was noticed. As substance P and enkephalins have opposite effects on respiration, their relative concentrations were calculated in each individual sample. The ratio was significantly higher in the medulla oblongata from the SIDS victims. The levels of NPY, substance P1-7, C-terminal fragments of substance P and Met-enkephalin-Lys6 were similar in both groups. A significant correlation between the NPY levels and age was observed, however.