Anna Maria Lavezzi

Adverse effects of prenatal tobacco smoke exposure on biological parameters of the developing brainstem.

We aimed to study the consequences of chronic exposure to tobacco smoke in utero on the morphological and functional maturation of the brainstem by comparing stillbirths of smoker mothers versus nonsmoker mothers. A total of 42 stillbirths, aged 25-40 gestational weeks, underwent autopsy according to our guidelines (). The brainstem was studied on serial sections and by immunohistochemistry to assay the expression of the EN2 gene, somatostatin (SS) and the tyrosine hydroxylase enzyme (TH). We observed a significant correlation between maternal smoking and sudden intrauterine unexplained death (SIUD), hypoplasia of the ArcN, no immunostaining of the EN2 in the arcuate nucleus (ArcN), and of TH in the locus coeruleus (LC) (P < 0.05). An increased incidence of maternal smoking was also observed in fetuses with SS negativity in the hypoglossus nucleus (HypoglN). Exposure in utero to maternal smoking may strongly interfere with brain biological parameters, giving rise not only to structural developmental abnormalities of the arcuate nucleus, but also to a decrease of noradrenergic activity in the LC, of EN2 gene expression in the ArcN and of SS in the HypoglN.

Functional neuroanatomy of the human pre-Botzinger complex with particular reference to sudden unexplained perinatal and infant death

The authors are the first to identify in man the pre‐Bötzinger complex, a structure of the brainstem critical for respiratory rhythmogenesis, previously investigated only in rats. The evaluation of the neurokinin 1 receptors and somatostatin immunoreactivity in a total of 63 brains from 25 fetuses, nine newborns and 29 infants, allowed to delineate the anatomic structure and the boundaries of this human neural center in a restricted area of the ventrolateral medulla at the obex level, ventral to the semicompact ambiguus nucleus. The neurons of the pre‐Bötzinger complex were roundish in fetuses before 30 gestational weeks and lengthened after birth, embedded in a dendritic system belonging to the reticular formation. Besides, structural and/or functional alterations of the pre‐Bötzinger complex were present in a high percentage of sudden deaths (47%), prevalent in late fetal deaths. In particular, different developmental defects (hypoplasia with a decreased neuronal number and/or dendritic hypodevelopment of the reticular formation, abnormal neuronal morphology, immunonegativity of neurotransmitters, and agenesis) were found. The authors suggest that the pre‐Bötzinger complex contains a variety of neurons not only involved in respiratory rhythm generation, but more extensively, essential to the control of all vital functions. Sudden death and in particular sudden unexpected fetal death could therefore be ascribed to a selective process when developmental alterations of the pre‐Bötzinger complex arise.

Techniques and criteria in pathologic and forensic-medical diagnostics in sudden unexpected infant and perinatal death.

For each case of sudden infant and perinatal death, a full review of clinical and epidemiologic data and a complete necropsy study were performed according to the necropsy protocol devised by the Institute of Pathology, University of Milan, Milan, Italy (available at: http://users.unimi.it/~pathol/sids_e.html). Histopathologic examination of unexpected late fetal and neonatal death and SIDS cases allowed us to identify frequent alterations, mainly congenital, of the autonomic nervous system, modulating respiratory, cardiovascular, arousal, and upper digestive activities. The data and arguments presented herein provide a brief survey tending to open, rather than conclude, a far-reaching subject and to motivate medicolegal specialists and pathologists to perform more in-depth study.

Study of the Brainstem, Particularly the Arcuate Nucleus, in Sudden Infant Death Syndrome (SIDS) and Sudden Intrauterine Unexplained Death (SIUD)

Complete examination of the brainstem involves transverse serial 5-&mgr;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.

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.

Preliminary Study on the Cytoarchitecture of the Human Parabrachial/Kölliker-Fuse Complex, with Reference to Sudden Infant Death Syndrome and Sudden Intrauterine Unexplained Death

The parabrachial/Kölliker-Fuse complex has been defined, in different animal species, to lie in the dorsolateral part of the pontine tegmentum and to be subdivided into three well-defined regions: the medial parabrachial nucleus, the lateral parabrachial nucleus, and the Kölliker-Fuse nucleus. Experimental studies have shown that the parabrachial/Kölliker-Fuse complex is involved in a variety of functional activities and above all plays an important role in respiratory modulation. In human brainstem, the cytoarchitecture and physiology of this complex have not yet been fully characterized. The aim of the present study was to examine fetal and infant human brainstems in order to define the precise morphology of the three nuclei of the parabrachial/Kölliker-Fuse complex, and to determine whether this nervous center shows morphologic alterations in sudden infant death syndrome (SIDS) and in sudden intrauterine unexplained death (SIUD). In serial sections of 31 brainstems of subjects aged from 32 gestational wk to 10 months of life, we studied, by morphologic and morphometric analyses, the cytoarchitecture and the extension of the three nuclei of the parabrachial/Kölliker-Fuse complex. All the morphometric parameters were very similar in SIUD and SIDS cases to those of the respective control group, as shown by the absence of significant statistical differences between the two fetus and infant groups. We observed that the features of both the lateral and the medial parabrachial nuclei are largely consistent with those reported in experimental studies. In contrast, the Kölliker-Fuse nucleus appears to be more developed in human beings than in other animal species, showing a greater extension and a more complex structure, as well as subdivision into two subnuclei (compactus and dissipatus).

Intimal preatherosclerotic thickening of the coronary arteries in human fetuses of smoker mothers

Summary.  Background: Many studies have described the development of preatherosclerotic coronary artery lesions in infancy. The observations reported in the literature regarding the fetal origin of coronary artery lesions are rare and controversial. Objectives: To identify the features of preatherosclerotic coronary artery lesions in late fetal stillborns and the possible atherogenic role of maternal cigarette smoking. Methods: We examined 22 stillborns (13 males and nine females), all of whom had died sine causa after the 32nd week of gestation. All underwent autopsy. Twelve of the mothers smoked over five cigarettes per day before and during the pregnancy. The four major epicardial coronary arteries were isolated along their whole length, embedded in paraffin and serially cut for histologic examination and immunohistochemical studies, particularly searching for the proliferating cell nuclear antigen and c‐Fos expression. Alterations of chromosome 7 were also investigated by the fluorescence in situ hybridization technique. Results: In over 50% of the fetuses, almost all from smoker mothers, multifocal structural alterations of coronary walls were evident. The smooth muscle cells (SMCs) presented loss of polarity, forming columns perpendicular to the axis of the media and infiltrating the subendothelial connective tissue. Increased amounts of mucoid ground substance were also observed in the subendothelial connective tissue. In all the cases with coronary alterations, study of the biological markers showed intense c‐Fos positivity of the SMCs. Conclusions: Preatherosclerotic intimal alterations of the coronary arteries are already detectable in the prenatal period and are significantly associated with maternal cigarette smoking.

Sudden infant death triggered by dive reflex

The dive reflex is the reflex mechanism most frequently considered in the aetiopathogenesis of sudden infant death syndrome (SIDS). This seems to persist in human beings as an inheritance from diver birds and amphibians. It has been reported that washing the face with cold water or plunging into cold water can provoke cardiac deceleration through the intervention of the ambiguus and the vagal dorsal nuclei. This report describes a case of SIDS that offers a unique insight into the role of the dive reflex in determining a lethal outcome. Examination of the brainstem on serial sections revealed severe bilateral hypoplasia of the arcuate nucleus and gliosis of the other cardiorespiratory medullary nuclei. The coronary and cardiac conduction arteries presented early atherosclerotic lesions. The possible role of parental cigarette smoking in the pathogenesis of arcuate nucleus hypoplasia and early coronary atherosclerotic lesions is also discussed.

Chromosomal alterations in atherosclerotic plaques

Alterations of chromosomes 7 and 11 have been involved in the progression of atherosclerosis. Twenty-three carotid endarterectomy specimens were studied for the presence of alterations in chromosomes 7 and 11, and fibroblastic growth factor-3 (FGF-3) gene amplification. Besides classic histological stainings, immunophenotyping of cellular and vascular components and fluorescence in situ hybridization (FISH) were performed. At the caps, unstable plaques (n=18) showed inflammatory infiltration of macrophages, smooth muscle cells, and T-lymphocytes. Specifically in these regions, the FISH showed varying percentages of trisomy (15/18) and tetrasomy (8/15) of chromosome 7. In four cases polisomy 7 was noted in some nuclei. Monosomy of chromosome 11 and gene amplification of FGF-3 gene was observed. The FISH of the five stable plaques and normal arterial walls showed no chromosome alterations; furthermore, chromosome 3, which is not involved in atherosclerotic progression, presented a normal ploidy of smooth muscle cells in stable and unstable plaques and normal arterial walls. In conclusion, chromosome 7 and 11 alterations and FGF-3 gene amplification are components of unstable plaques, and might contribute to the evolution of stable plaques into complicated plaques.

Alterations of biological features of the cerebellum in sudden perinatal and infant death.

This article intends to show how the cerebellum, a structure ordinarily not considered in mediating breathing or cardiovascular control, may play a critical role in compensatory responses particularly to hypoxic insults occurring pre and/or postnatally and thus may be involved in the sudden unexplained perinatal and infant death. Besides the ontogenesis of the cerebellar cortex in man, we reported alterations of biopathological features (neuronal immaturity, altered apoptotic programs, negative expression of somatostatin and EN2 gene, intense c-fos expression positivity, astrogliosis) in the cortex and in the dentate nucleus of the 63% of sudden deaths, and only in 10% of the controls. The correlation of these results with the mothers smoking habit was highly significant. Therefore, we support the hypothesis, already expressed in previous studies on brainstem, of a close relation between maternal cigarette smoking and a wide range of morpho-physiological defects of the brain, leading to unexplained sudden death in stillbirths, newborns, and Sudden Infant Death Syndrome (SIDS) victims.