Luigi Matturri

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.

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).

Association between Pulmonary Hypoplasia and Hypoplasia of Arcuate Nucleus in Stillbirth

OBJECTIVE: To investigate lung development and to correlate pulmonary hypoplasia with hypoplasia of the arcuate nucleus in stillbirths.STUDY DESIGN: We examined 26 stillbirths which occurred after 25 complete gestational weeks. The brainstem and the lung were the particular focus of this study. The brainstem was examined according to the protocol routinely followed in our Institute. As regards the lung examination, the development stage was evaluated on the basis of the correlation between lung and body weight (LW/BW), and according to microscopic parameters, that is, the presence of cartilaginous bronchi up to the distal level and the radial alveolar count (RAC). The normal reference values for the last 3 months of gestation correspond to >0.022 for LW/BW and from 2.2 to 4.4 for RAC.RESULTS: In 17 cases (65%) pulmonary hypoplasia was observed, characterized by a LW/BW value below 0.022 and RAC below 2.2. In nine cases (35%), microscopic examination of brainstem serial sections showed varying degrees of hypoplasia of the arcuate nucleus (ARCn). In eight cases (31%) the pulmonary hypoplasia was associated with hypoplasia/agenesis of the ARCn.CONCLUSIONS: This study demonstrated that in about a third of stillbirths there is a congenital hypodevelopment of both lung and arcuate nucleus. In these cases the ARCn hypoplasia would exert a negative effect on respiratory movements in utero and therefore on lung development. When the pulmonary hypoplasia is not accompanied by hypodevelopment of this nucleus the explanation could be a failure to block the inhibitory action of the Kölliker–Fuse nucleus.

Cytoarchitectural organization of the parabrachial/Kölliker-Fuse complex in man

While the parabrachial/Kölliker-Fuse complex has been described in a variety of animal species it has not been characterized in human brainstem. In the present study we investigated fetal and infant brainstems, focusing particularly on the dorsolateral part of the pontine tegmentum, with the aim of defining the precise cytoarchitecture of the medial parabrachial, lateral parabrachial, and Kölliker-Fuse nuclei in man, and analyzing the developmental stages of this complex. In serial sections of 28 human brainstems of subjects aged between 32 gestational weeks and 1 year we made a morphologic and morphometric analysis of the shape and size of the parabrachial/Kölliker-Fuse complex. We observed a homogeneous morphology in all cases, which enabled us to define the structure of the three nuclei. The features of the parabrachial nuclei are largely consistent with those reported in experimental studies. However, 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. The neuronal maturation of these nuclei was seen to occur between the 35th and the 36th gestational weeks.

Hypoplasia of the arcuate nucleus and maternal smoking during pregnancy in sudden unexplained perinatal and infant death

Maternal smoking during pregnancy is the most important risk factor for sudden perinatal and infant death in more industrialized countries. The frequent observation of hypoplasia of the arcuate nucleus in the brainstem of these victims prompted the verification of whether maternal cigarette smoking could be related to defective development of this nucleus during intrauterine life, by affecting the expression of specific genes involved in its developmental process. In serial sections of the brainstem of 54 cases of sudden and unexplained fetal and infant deaths (13 stillbirths, 7 neonatal deaths and 34 sudden infant death syndrome (SIDS) victims), morphological and morphometrical analysis was used to observe the different structural alterations of the arcuate nucleus (bilateral hypoplasia, monolateral hypoplasia, partial hypoplasia, delayed neuronal maturation and decreased neuronal density) detected in 24 cases (44%). Correlating this finding with smoking in pregnancy, a significantly increased incidence of cytoarchitectural alterations of the arcuate nucleus was found in stillborns and SIDS victims with smoker mothers compared to victims with non‐smoker mothers. Moreover, the observation of a wide range of developing morphological defects of the arcuate nucleus related to maternal smoking led to the hypothesis that the constituents of the gas phase in cigarette smoke could directly affect the expression of genes involved in the development of this nucleus, such as the homeobox En‐2 gene.

Maternal smoking and sudden infant death syndrome: epidemiological study related to pathology.

Various risk factors have been postulated to be related to sudden infant death syndrome (SIDS). Despite its reduction, thanks to the “Back to Sleep” campaign, SIDS is still a major cause of infant mortality in the first year of life. The purpose of this study was to correlate the different risk factors with the autopsy results and thus to determine if one or more of these variables is really specific for SIDS. We collected 128 sudden infant death victims with clinical diagnosis of SIDS and performed a complete autopsy with in-depth histology on serial sections, particularly of the brainstem, in accordance with our necropsy protocol. Histopathologic and immunohistochemical examination of the central autonomic nervous system revealed, in 78 cases of the SIDS group, the following anomalies: hypodevelopment of the arcuate nucleus, somatostatin positive hypoglossus nucleus, tyrosine hydroxylase negativity in the locus coeruleus, gliosis, and hypoplasia of the hypoglossus nucleus. A significant relation was found between maternal smoke and brainstem alterations.

Histological and biological developmental characterization of the human cerebellar cortex

The aim of this study was to investigate the histological and biological features of the human cerebellar cortex development and differentiation. We analyzed 52 brains of fetal and infant death victims, aged from 17 gestational weeks to 12th postnatal month. In particular, in the cerebellar cortex at different ages we evaluated, besides the structural aspects, the expression of several biomarkers implicated in proliferative processes (c‐fos, PCNA and apoptosis). We observed morphological patterns progressively evolving every month, from the indefinite structure of the second gestational trimester to the four‐layered structure (external granular layer, molecular layer, Purkinje cell layer, internal granular layer) of the late fetal cortex and subsequently to the three‐layered postnatal definitive morphology, due to involution of the external granular layer. The evaluation of the biological features of the cerebellar cortex showed high proliferative activity mainly confined to the transient external granular layer in prenatal life, and high apoptotic index after birth. Thus, the histological examination, better with the support of biomarker investigations, allows with accuracy to describe the dynamic sequence of steps that occur in human cerebellar cortex development and to establish in each case the age, namely the pre‐ or postnatal month of life. Consequently, we can diagnose delayed or altered processes of differentiation during the development of the human cerebellar cortex.

Hypoplasia of the Parafacial/Facial Complex: A Very Frequent Finding in Sudden Unexplained Fetal Death

AIMS - To define firstly in man the localization and the anatomical boundaries of the parafacial respiratory group in the brainstem. Thereafter, to determine whether this center, given its essential role in the respiratory rhythm- generating circuit, showed abnormalities in sudden unexplained perinatal and infant deaths, like other nuclei and/or struc- tures of the brainstem and cerebellum checking vital functions, that we have previously reported. METHODS - In 67 brains collected from 29 stillbirths, 9 newborns and 29 infants, died of both known and unknown cause, an in-depth histological examination of the autonomic nervous system was made, according to the protocol rou- tinely followed by the Institute of Pathology, University of Milan. In particular we analyzed the parafacial and facial nu- clei in serial sections of caudal pons. RESULTS - We firstly identified and defined the normal structure of the parafacial/facial complex in control cases. Be- sides we diagnosed the hypoplasia of these nuclei in 75% of sudden unexplained fetal deaths and never after birth. CONCLUSIONS - We formulate the hypothesis that the hypoplasia of the parafacial/facial complex is a specific marker of unexplained stillbirth, and that the normal development of this complex is essential for extra-uterine life.

Histopathology of the cardiac conduction system in sudden intrauterine unexplained death

BACKGROUNDnSudden intrauterine unexplained death (SIUD) is one of the most heartbreaking tragedies that any parent can experience. It remains poorly understood and incompletely examined both morphologically and functionally. The aim of this work is to examine the likely role of cardiac conduction system in relation to sudden and unexplained fetal death.nnnMETHODSnWe analyzed and compared the autopsy results in 15 cases of SIUD (6 males and 9 females, ranging in age from 35 to 40 weeks) and 11 cases of intrauterine explained death (IED). A complete autopsy was performed, focusing on the examination of the cardiac conduction system on serial sections.nnnRESULTSnThe following findings were observed: resorptive degeneration (33% of SIUD, 36% of IED), dispersion or septation of the atrioventricular (AV) junction (60% of SIUD, 64% of IED), islands of the conduction system in the central fibrous body (80% of SIUD, 73% of IED), Mahaim fibers (20% of SIUD), cartilaginous metahyperplasia (20% of SIUD, 18% of IED), an AV node (AVN) tongue (13% of SIUD), hemorrhage of the cardiac conduction system (7% of SIUD, 9% of IED), left-sided bifurcation (7% of SIUD), an intramural right bundle (7% of SIUD), central fibrous body hypoplasia (7% of SIUD), and thickening of the conduction system arteries (13% of SIUD).nnnCONCLUSIONSnMost of the abnormal cardiac conduction findings were detected only in SIUD and were absent in controls, i.e., Mahaim fibers, AVN tongue, left-sided bifurcation, intramural right bundle, and central fibrous body hypoplasia. We are convinced that these cardiac conduction abnormalities, in association with altered neurovegetative stimuli, could underlie potentially malignant arrhythmias.

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.