Armando Faa

Hypoxia/reoxygenation-induced myocardial lesions in newborn piglets are related to interindividual variability and not to oxygen concentration

OBJECTIVE: Evaluation of myocardial histological changes in an experimental animal model of neonatal hypoxia-reoxygenation. METHODS: Normocapnic hypoxia was induced in 40 male Landrace/Large White piglets. Reoxygenation was initiated when the animals developed bradycardia (HR <60 beats/min) or severe hypotension (MAP <15 mmHg). The animals were divided into four groups based on the oxygen (O2) concentration used for reoxygenation; groups 1, 2, 3, and 4 received 18%, 21%, 40%, and 100% O2, respectively. The animals were further classified into five groups based on the time required for reoxygenation: A: fast recovery (<15 min); B: medium recovery (15-45 min); C: slow recovery (45-90 min); D: very slow recovery (>90 min), and E: nine deceased piglets. RESULTS: Histology revealed changes in all heart specimens. Interstitial edema, a wavy arrangement, hypereosinophilia and coagulative necrosis of cardiomyocytes were observed frequently. No differences in the incidence of changes were observed among groups 1-4, whereas marked differences regarding the frequency and the degree of changes were found among groups A-E. Coagulative necrosis was correlated with increased recovery time: this condition was detected post-asphyxia in 14%, 57%, and 100% of piglets with fast, medium, and slow or very slow recovery rates, respectively. CONCLUSIONS: The significant myocardial histological changes observed suggest that this experimental model might be a reliable model for investigating human neonatal cardiac hypoxia-related injury. No correlation was observed between the severity of histological changes and the fiO2 used during reoxygenation. Severe myocardial changes correlated strictly with recovery time, suggesting an unreported individual susceptibility of myocardiocytes to hypoxia, possibly leading to death after the typical time-sequence of events.

Neonatal asphyxia and forensic medicine

In the last decades, the scientific literature addressing neonatal encephalopathy has grown in a logarithmic way and malpractice claims in obstetrics and neonatology have become a major threat to the health service. At the moment, scientific evidence are insufficient to clearly identify in each single case whether the hypoxic insult has developed in the course of labor or in the first few hours after the birth or, otherwise, whether the damage has to recognize a remote and long-lasting cause acting during pregnancy. Several authors feel that this scientific uncertainty leads to a higher percentage of civil suit decisions prone to recognizing a guilty medical behavior, and they wish a more in-depth analysis of all these cases to clearly identify all the data either in favor or in contrary to the assumption of the existence of a causal correlation between neonatal encephalopathy and medical misbehavior. This article will focus on the medico-legal approach to a hypoxic–ischemic event in the perinatal period, addressing the relevant data to be collected in order to establish the medical and juridical cause of the neonatal damage.

Multiple organ failure syndrome in the newborn: morphological and immunohistochemical data

Multiple organ failure (MOF) syndrome, also known as multiple organ dysfunction syndrome (MODS) represents a common but complex problem in critically ill patients in neonatal intensive care unit (NICU) centers, and a major cause of morbidity and mortality in newborns. MOF is considered the result of an inappropriate generalized inflammatory response of the newborn to a variety of acute insults. This study was aimed at analyzing, at histology, multiple organ pathological changes in two newborns admitted to the NICU center of our University Hospital, who showed a progressive clinical picture of MOF, in order to verify the pathological changes of vascular structures and of endothelial cells in the different organs affected by MOF. All the samples obtained at autopsy for histological examination showed specific organ pathological changes, especially related to modifications in vascular structures and, in particular, in endothelial cells. The most interesting findings were found in the intestinal barrier, in the lower respiratory tract and in the endothelial barrier. The loss of the gut barrier could allow the passage into the blood of microbial factors that could trigger the production of tumor necrosis factor α (TNFα) leading to endothelial damage. Our preliminary study underlines the principal role probably played by intestinal and vascular changes in the origin of MOF in newborns.

WT1 expression in the human fetus during development

Wilms’ Tumor 1 (WT1) is a transcription factor involved in the development of the urogenital system. The purpose of this study was to analyze the immunoreactivity for WT1 protein in different tissues and organs in human fetuses in early phases of gestation. To this end, samples from multiple organs were obtained from 4 human fetuses, ranging from 7 up to 12 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained for WT1. Our data show that WT1 is involved in development of multiple human organs in a more vast series of cells types than previously reported. Immunostaining for WT1 was characterized by a predominant cytoplasmic reactivity in the vast majority of cell types. Mesenchimal progenitors in the fetal lung, ductal plate progenitors in fetal liver, cap mesenchimal cells in the developing kidney, fetal zone cells in adrenal glands, atrial and ventricular cardiomyocytes in the fetal heart, radial glial cells in the fetal cerebral cortex and skeletal muscle cell precursors showed the highest levels of WT1 immunoreactivity. Future studies will be needed to detect differences in the expression of WT1 in various organs at different gestational ages, in order to better evaluate the role of WT1 in cell proliferation and differentiation during intrauterine human development.

Perinatal Heart Programming: Long-term Consequences

OBJECTIVE evaluate the relationship between impaired growth during intrauterine life and adult risk of cardiovascular disease and death. MATERIALS review of the most important contributions to the relationship between intrauterine fetal life and heart disease insurgence in childhood and adulthood, starting with a schematic representation of the principal steps in human heart development, discussion of the new theory on the relevance of the number of cardiomyocytes that every heart shows at birth. RESULTS intrauterine environment defines the epigenetic profile of newborns, with implications for the risk of developing diseases later in adult life. This means that the programming of cardiovascular risk and other pathologies, such as obesity, in adulthood takes place starting from intrauterine life. CONCLUSIONS it can be hypothesized that by preventing and eventually treating cardiovascular diseases in the pediatric age, if these are already present in their early and/or in light forms, the long-term management of complications could be approached differently and more effectively than by postponing the treatment to adulthood. The future challenge in this fascinating field of clinical research is the discovery of the molecular mechanisms underlying the association between intrauterine growth restriction and fetal onset of adult cardiac disease, so as to make a dream come true by applying primary prevention of adult heart disease in the womb.

Hypoxia-induced endothelial damage and microthrombosis in myocardial vessels of newborn landrace/large white piglets.

Objective. Evaluating the presence of endothelial changes in myocardial vessels in an experimental model of hypoxia and resuscitation in newborn piglets. Methods. Fifty male Landrace/Large White neonatal piglets were studied: ten of them were allocated in group A (control group, SHAM-operated). In group B (forty animals, experimental group) normocapnic hypoxia was induced by decreasing inspired concentration of O2 to 6%–8%. When the animals developed bradycardia or severe hypotension, reoxygenation was initiated. The animals of group B were allocated in 4 subgroups of 10, according to the concentration of O2 they were resuscitated with (groups 1, 2, 3, and 4 received 18%, 21%, 40%, and 100% O2, resp.). Results. Control group animals did not show any significant endothelial lesions. Contrarily, endothelial lesions were detected in all experimental group cases. When these lesions were analyzed in the different heart zones, no significant difference in their incidence was observed; analyzing the frequency in the animals of the 4 subgroups, only microthrombosis showed a higher frequency in animals in groups 4 and 3. Conclusions. Endothelial damage represents a diffuse pathological feature in the myocardial vessels of piglets subjected to normocapnic hypoxia and resuscitation suggesting a possible role of hyperoxygenation in aggravating endothelial damage.

Low vascularization of the nephrogenic zone of the fetal kidney suggests a major role for hypoxia in human nephrogenesis

CD31 reactivity is generally utilized as a marker of endothelial cells. CD31 immunoreactivity in the developing human kidney revealed that fetal glomerular capillary endothelial cells change their immunohistochemical phenotype during maturation. The aim of this study was to analyze CD31 reactivity in the fetal human kidney in the different stages of intrauterine development: We observed different distribution of CD31-reactive vascular progenitors in the different areas of the developing kidney. In particular, the nephrogenic zone and the renal capsule were characterized by a scarcity of CD31-reactive cells at all gestational ages. These data suggest the hypothesis that nephrogenesis does not need high oxygen levels and confirms a major role of hypoxia in nephrogenesis.

S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker?

OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death. METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody. RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes. CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.

Stem cell markers in the heart of the human newborn

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. Several recent studies now show that the different cell types that characterize the adult human heart arise from a common ancestor. Human cardiac stem cells differentiate into cardiomyocytes, and, in lesser extent, into smooth muscle and endothelial cells. The characterization of human cardiac stem cells (CSCs) has important clinical implications. In recent years, CD117 (c-kit) has been reported to mark a subtype of stem/progenitor cells in the human heart, with stem cell-like properties, including the ability to self-renewal and clonogenicity multipotentiality. Proceedings of the 2 nd International Course on Perinatal Pathology (part of the 11 th International Workshop on Neonatology · October 26 th -31 st , 2015) · Cagliari (Italy) · October 31 st , 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

S100B immunoreactivity: a new marker of hypoxia-related cardiac damage in newborn piglets.

Abstract Objective: The evaluation of the expression of S100B protein, in the swine heart in an experimental model of hypoxia - reoxygenation. Methods: Normocapnic hypoxia was induced in 40 male Landrace/Large White neonatal piglets by decreasing the inspired concentration of oxygen to 6–8%. When animals developed bradycardia or severe hypotension, reoxygenation was initiated. Piglets were allocated in four groups of 10, according to the oxygen concentration they were reoxygenated with: Group 1, 2, 3 and 4 resuscitated with 18%, 21%, 40% and 100% oxygen, respectively. The animals were further classified into 4 groups according with the time required for reoxygenation: group A (<15 min); group B (16–60 min); group C (>60 min); group D (deceased animals). Results: Immunostaining for S100B protein was detected in 14 out of the 40 heart samples (35%), both inside the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. Significant differences were observed among groups 1–4 regarding S100B expression. Reactivity for S100B in cardiac cells was detected in 50%, 50%, 10% and 33% of animals in groups 1 and 2, 3 and 4, respectively. Marked differences were also observed among groups A–D: 75%, 33%, 12% and 22% of the animals in group 1, 2, 3 and 4, respectively, showed reactivity for S100B in the heart. Conclusions: Expression of S100B protein occurred in the heart of some of newborn piglets following severe hypoxia. S100B storage in cardiomyocytes correlates with the different oxygen concentration used during reoxygenation, being higher in piglets reoxygenated with 18% and 21%, and lower in animals reoxygenated with 40% oxygen. Intermediate levels of S100B expression were found in 100% O2-treated animals. The finding of a higher percentage of S100B-immunoreactive hearts in piglets with a fast recovery and the detection of a decreased reactivity in animals with a slow and a very slow recovery clearly indicates S100B protein as an early protective factor with a positive prognostic value in asphyxiated newborn piglets.