The Open Pediatric Medicine Journal, 2007, 1, 1-8 1 Pathology of the Central Autonomic Nervous System in Stillbirth Luigi Matturri* and Anna Maria Lavezzi “Lino Rossi” Research Center, Institute of Pathology, University of Milan, Italy Abstract: The aim of this study was to identify in stillbirth a possible involvement of morphological and/or physiological alterations of structures of the central autonomic nervous system in the mechanism of death. The study, including the in- depth histological examination of brainstem and cerebellum, was performed on 42 stillbirths, aged from 22 to 40 gesta- tional weeks, 12 of which were explained and 30 were unexplained deaths. In the sudden unexplained stillbirths a variety of morphological and/or biological abnormalities of different structures and nuclei was found, above all the hypoplasia of the parafacial complex, frequently associated with hypoplasia of the arcuate and pre-Bötzinger nuclei, and with thyrosine-hydroxylase immunonegativity in the locus coeruleus. A significant correla- tion was also observed between the neuropathologic findings and mother’s smoking habit. Keywords: Stillbirth, central autonomic nervous system, brainstem, neuropathology, developmental alterations. INTRODUCTION applied immunohistochemical methods to analyze the ex- pression of neurotransmitters (namely catecholamines and While society displays an increased awareness of sudden somatostatin) regulating important physiological functions infant death syndrome (SIDS), less attention has been fo- already from the first weeks of gestation [7-10] and gliosis, a cused on another form of unexplained death that occurs with process of astrocytic activation, indicative of a response to a greater frequency in late pregnancy, namely “stillbirth”. In central nervous system injury [11,12]. fact, despite the high number of unexplained fetal deaths worldwide, very little research has been devoted to the issue MATERIALS AND METHODOLOGY of stillbirths, particularly in the anatomopathologic field. We studied 42 fresh stillbirths, 28 males and 14 females, More frequently than is currently admitted, the lethal aged from 22 to 40 gestational weeks. The gestational age event seems to be primarily triggered by a stroke of anatomic peaked in incidence at 36 to 40 weeks (62%). The weights of “lightning”, sufficient to impair the vital functions. victims ranged from 352 to 3500 grams. Accordingly, the vital centers of the central autonomic Our collected cases were subjected to complete autopsy, nervous system need to be given a close pathologic investi- including examination of the placental disk, umbilical cord gation, which may seem a simplistic approach to such an and membranes and an in-depth histological examination of extremely complex problem, yet it can enable a structural the autonomic nervous system, according to the protocol control of the reflexogenic neural network. issued by the “Lino Rossi” Research Center for the study and Stillbirth or fetal death is defined as death prior to com- prevention of the unexpected perinatal death and SIDS of the plete expulsion or extraction of a product of conception, irre- University of Milan and available on the web site: spective of the duration of the pregnancy. The perinatal pe- http://users.unimi.it/~pathol/sids_e.html [13-16]. riod commences at 22 completed weeks (154 days) of gesta- A diagnosis of “unexplained stillbirth” was established tion and ends seven completed days after birth [1]. Statistics for 30 fetuses who had died suddenly, with no defined cause and research on stillborn infants have been largely neglected of death. [2-5]. In the remaining 12 victims a precise cause of death was Calls for a routine stillbirth autopsy and for a more stan- diagnosed at autopsy and they were classified as "explained dardized stillbirth autopsy protocol have been made by the stillbirths” and considered as control cases. International Stillbirth Alliance (ISA), a recent organization Intrauterine growth retardation, with a weight below the devoted to understanding the causes and prevention of Still- birth [6]. corresponding reference value, was more frequent in the unexplained stillbirth group compared with the control Herein we illustrate the results of a neuropathologic group. study on the autonomic central nervous system in 42 fresh For every case a complete clinical history, particularly stillbirths, 12 of which were explained and 30 were unex- referred to mother, was collected. None of the mothers had plained deaths. Besides the histopathologic examination of the nuclei and structures of the brainstem, cerebellum and any significant pathology. None of the mothers used drugs, high doses of caffeine, sedatives or alcohol. With reference upper thoracic spinal cord in serial histological sections, we to smoking habit, 16 mothers (38%) declared that they were active smokers (all of more than 3 cigarettes/day and all be- *Address correspondence to this author at the Institute of Pathology, Uni- fore becoming pregnant) and 26 (62%) were non smokers. versity of Milan, Via della Commenda, 19, 20122 Milan, Italy; Tel: +39-02- Table 1 summarizes the case profiles in this study, with 50320800; Fax: +39-02-50320823; E-mail: [email protected] their relative death diagnosis and mother's smoking status. 1874-3099/07 2007 Bentham Science Publishers Ltd. 2 The Open Pediatric Medicine Journal, 2007, Volume 1 Matturri and Lavezzi Table 1. Case Profiles of the Study Sex Gestational Weeks Weight (g) SGA Maternal Smoking Habit Death Diagnosis M F Range Mean ± DS Range Mean ± DS n (%) n (%) n (%) Smokers Non Smokers unexplained stillbirth (n = 30) 13 (31) 17 (40) 25-40 34.5±5.2 830-3930 2100±99 26 (62) 15 (36) 15 (36) explained stillbirth* (n = 12) 7 (17) 5 (12) 22-40 33.4±5.3 352-3940 2550±128 4 (9) 2 (5) 10 (24) *The death causes were: placental pathologies (4 cases); congenital heart diseases: dilated cardiomyopathy (1 case), Ebstein’s anomaly (2 cases); pneumonia (1 case); severe renal dysplasia (1 case); septicemia (1 case); Down’s syndrome (1 case); Potter’s syndrome (1 case). SGA = small for gestational age. Multiple samples of all organs were fixed in 10% phos- 40x lens) in order to estimate the number of alveoli tran- phate-buffered formalin, processed and embedded in paraf- sected by a perpendicular line drawn from the center of the fin. Sections of 4 μm were stained with hematoxylin-eosin most peripheral bronchiole (recognizable by not being com- and Heidenhaim-azan. pletely covered by epithelium) to the pleura or the nearest Target of this study was the in-depth histological exami- interlobular septum. To carry out this examination, we used samples of fetal lung sectioned parallel to the frontal plane nation of the central autonomic nervous system. The vital and passing through the hilus. The normal reference values centers, where afferents from peripheral receptors converge for the last three months of gestation correspond to > 0.022 on neural circuits processing the efferent reflexogenic re- for LW/BW and range from 2.2 to 4.4 [17] for the RAC. sponses, are located in the brainstem, linked with the cere- bellum and the sympathetic intermediolateral nucleus in the Immunohistochemical Methods thoracic (T1-T5) cord. In order to analyze the immunoexpression of soma- After fixation in 10% phosphate-buffered formalin, the tostatin and tyrosine-hydroxylase and to reveal the presence brainstem, cerebellum and upper spinal cord were processed of reactive astrocytes by the glial fibrillary acidic protein and embedded in paraffin [12-14]. Transverse serial sections (GFAP) method, we used specific primary antibodies after were made at intervals of 30 μm. For each level, twelve 5 the application of the avidin-biotin-peroxidase technique, in μm sections were obtained, two of which were routinely conformity with conventional immunohistochemical proce- stained for histological examination using alternately hema- dures. toxylin-eosin and Klüver-Barrera stains. Three additional sections at each level were subjected to immunohistochemis- A detailed description of the immunohistochemical try for the study of a) the neurotransmitter somatostatin; b) methods, including the immunopositivity evaluation, applied tyrosine-hydroxylase (TH), an enzyme involved in catecho- in this study is available in our previous works [9,10,18]. lamine (adrenalin, noradrenalin and dopamine) synthesis and Morphometric Evaluation c) gliosis. The remaining sections were saved and stained as deemed necessary for further investigations. The morphologic examination of the central autonomic nervous system was supplemented by the morphometric As regards the brainstem, identification in serial sections analysis of the main brainstem nuclei, performed with an and analysis were made of the main nuclei, namely the locus image analyzer. The following parameters were evaluated coeruleus and the parabrachial/Kölliker-Fuse complex in the and indicated as mean values and standard deviation: nucleus pons and mesencephalon, the hypoglossus, the dorsal motor area (expressed in mm2), neuronal density (expressed as vagal, the tractus solitarius, the ambiguus, the pre-Bötzinger, number of neurons per mm2) and neuronal size (cell body the inferior olivary and the arcuate nuclei in the medulla ob- area, expressed in μm2). The in-depth description of the longata. In the upper thoracic spinal cord the intermedio- methodology and of the related results can be found in our lateral nucleus was examined. Fig. (1) shows a diagram of previous works [10,19,20].