Carmela Fiore

Correlation between cardiac oxidative stress and myocardial pathology due to acute and chronic norepinephrine administration in rats

Background: To investigate the cardiotoxic role of reactive oxygen species (ROS) and of products derived from catecholamines auto‐oxidation, we studied: (1) the response of antioxidant cardiac cellular defence systems to oxidative stress induced by norepinephrine (NE) administration, (2) the effect of NE administration on cardiac β1‐adrenergic receptors by means of receptor binding assay, (3) the cellular morphological alterations related to the biologically cross‐talk between the NE administration and cytokines [tumor necrosis factor‐alpha (TNF‐α), monocyte chemotactic protein‐1 (MCP‐1), interleukins IL6, IL8, IL10]Methods and Results: A total of 195 male rats was used in the experiment. All animals underwent electrocardiogram (EKG) before being sacrificed. The results obtained show that NE administration influences the antioxidant cellular defence system significantly increasing glutathione peroxidase (GPx) activity, glutathione reductase (GR) and superoxide dismutase (SOD). The oxidized glutathione (GSH/GSSG) ratio significantly decreases and malondialdehyde (MDA) levels increase showing a state of lipoperoxidation of cardiac tissue. We describe a significant apoptotic process randomly sparse in the damaged myocardium and the effect of ROS on the NE‐mediated TNF‐α, MCP‐1, and IL6, IL8, IL10 production. Conclusions: Our results support the hypothesis that catecholamines may induce oxidative damage through reactive intermediates resulting from their auto‐oxidation, irrespective of their interaction with adrenergic receptors, thus representing an important factor in the pathogenesis of catecholamines‐induced cardiotoxicity. The rise of the cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium with respect to the cardiac dysfunction resulting from NE injection.

Side effects of cocaine abuse: multiorgan toxicity and pathological consequences.

Cocaine is a powerful stimulant of the sympathetic nervous system by inhibiting catecholamine reuptake, stimulating central sympathetic outflow, and increasing the sensitivity of adrenergic nerve endings to norepinephrine (NE). It is known, from numerous studies, that cocaine causes irreversible structural changes on the brain, heart, lung and other organs such as liver and kidney and there are many mechanisms involved in the genesis of these damages. Some effects are determined by the overstimulation of the adrenergic system. Most of the direct toxic effects are mediated by oxidative stress and by mitochondrial dysfunction produced during the metabolism of noradrenaline or during the metabolism of norcocaina, as in cocaine-induced hepathotoxicity. Cocaine is responsible for the coronary arteries vasoconstriction, atherosclerotic phenomena and thrombus formation. In this way, cocaine favors the myocardial infarction. While the arrhythmogenic effect of cocaine is mediated by the action on potassium channel (blocking), calcium channels (enhances the function) and inhibiting the flow of sodium during depolarization. Moreover chronic cocaine use is associated with myocarditis, ventricular hypertrophy, dilated cardiomyopathy and heart failure. A variety of respiratory problems temporally associated with crack inhalation have been reported. Cocaine may cause changes in the respiratory tract as a result of its pharmacologic effects exerted either locally or systemically, its method of administration (smoking, sniffing, injecting), or its alteration of central nervous system neuroregulation of pulmonary function. Renal failure resulting from cocaine abuse has been also well documented. A lot of studies demonstrated a high incidence of congenital cardiovascular and brain malformations in offspring born to mothers with a history of cocaine abuse.

Enzymatic–nonenzymatic cellular antioxidant defense systems response and immunohistochemical detection of MDMA, VMAT2, HSP70, and apoptosis as biomarkers for MDMA (Ecstasy) neurotoxicity

3,4‐Methylenedioxymethamphetamine (MDMA)‐induced neurotoxicity leads to the formation of quinone metabolities and hydroxyl radicals and then to the production of reactive oxygen species (ROS). We evaluated the effect of a single dose of MDMA (20 mg/kg, i.p.) on the enzymatic and nonenzymatic cellular antioxidant defense system in different areas of rat brain in the early hours (<6 hr) of the administration itself, and we identified the morphological expressions of neurotoxicity induced by MDMA on the vulnerable brain areas in the first 24 hr. The acute administration of MDMA produces a decrease of reduced and oxidized glutathione ratio, and antioxidant enzyme activities were significantly reduced after 3 hr and after 6 hr in frontal cortex. Ascorbic acid levels strongly increased in striatum, hippocampus, and frontal cortex after 3 and 6 hr. High levels of malonaldehyde with respect to control were measured in striatum after 3 and 6 hr and in hippocampus and frontal cortex after 6 hr. An immunohistochemical investigation on the frontal, thalamic, hypothalamic, and striatal areas was performed. A strong positive reaction to the antivesicular monoamine transporter 2 was observed in the frontal section, in the basal ganglia and thalamus. Cortical positivity, located in the most superficial layer was revealed only for heat shock protein 70 after 24 hr.

Calcium oxalate crystals in acute ethylene glycol poisoning: A confocal laser scanning microscope study in a fatal case

Introduction. The severity of ethylene glycol toxicity is related to the metabolic acidosis resulting from the biotransformation of ethylene glycol into toxic metabolites. Glycolic acid causes severe acidosis and oxalate precipitates as calcium oxalate in the kidneys and other tissues. Case Report. An adult male was taken to the local hospital by the team rescue and was apparently unconscious; severe metabolic acidosis and renal failure led to death a few hours after the arrival. Confocal laser scanning microscopy demonstrated oxalate crystals deposition within the tubular epithelial cells and widespread necrosis of the tubular epithelium in the proximal tubules. Toxicological examinations revealed ethylene glycol; the blood level was 250 mg/L and in urine the concentration was 0.3%. Discussion. In cases of ethylene glycol poisoning, calcium oxalate may be excreted not only as dihydrate crystals, but also as monohydrate crystals. Direct toxicity, cortical edema, and inhibition of mitochondrial activity, as evidenced by decreased succinate dehydrogenase activity, are possible mechanisms of crystal damage. Since calcium oxalate monohydrate crystals are transported intracellularly by kidney cells, the renal toxicity of ethylene glycol may result from inhibition of mitochondrial respiratory function in proximal tubular cells by calcium oxalate monohydrate crystals. Conclusions. The histologic diagnosis of acute renal failure secondary to ethylene glycol poisoning depends on the recognitions of the changes of acute tubular damage in association with calcium oxalate crystals deposition within the tubular epithelial cells and the widespread necrosis of the tubular epithelium in the proximal tubules.

Neurotoxicity by Synthetic Androgen Steroids: Oxidative Stress, Apoptosis, and Neuropathology: A Review

Anabolic-androgenic steroids (AAS) are synthetic substances derived from testosterone that are largely employed due to their trophic effect on muscle tissue of athletes at all levels. Since a great number of organs and systems are a target of AAS, their adverse effects are primarily on the following systems: reproductive, hepatic, musculoskeletal, endocrine, renal, immunological, infectious, cardiovascular, cerebrovascular, and hematological. Neuropsychiatric and behavioral effects as a result of AAS abuse are well known and described in the literature. Mounting evidence exists suggesting that in addition to psychiatric and behavioral effects, non-medical use of AAS carries neurodegenerative potential. Although, the nature of this association remains largely unexplored, recent animal studies have shown the recurrence of this AAS effect, ranging from neurotrophin unbalance to increased neuronal susceptibility to apoptotic stimuli. Experimental and animal studies strongly suggest that apoptotic mechanisms are at least in part involved in AAS-induced neurotoxicity. Furthermore, a great body of evidence is emerging suggesting that increased susceptibility to cellular oxidative stress could play a pivotal role in the pathogenesis of many neurodegenerative disorders and cognitive impairment. As in other drug-evoked encephalopathies, the key mechanisms involved in AAS – induced neuropathology could represent a target for future neuroprotective strategies. Progress in the understanding of these mechanisms will provide important insights into the complex pathophysiology of AAS-induced neurodegeneration, and will pave the way for forthcoming studies. Supplementary to abandoning the drug abuse that represents the first step in reducing the possibility of irreversible brain damage in AAS abusers, neuroprotective strategies have to be developed and implemented in future.

Myocardial expression of TNF-alpha, IL-1beta, IL-6, IL-8, IL-10 and MCP-1 after a single MDMA dose administered in a rat model.

Indirect effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and metabolites on the cardiac cells are well-known, the mechanism(s) underlying direct MDMA-induced cardiotoxicity remaining to be clarified. To better understand the immuno-inflammatory phenomena accompanying the cardiac alterations during MDMA administration, we conducted a study in an in vivo animal model to evaluate the cellular morphological alterations related to the biological response between MDMA administration and inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, 8, 10, and monocyte chemotactic protein-1). A total of 25 male rats were used. The effects were evaluated at 6, 16 and 24 hours after a single dose MDMA administered (20 mg/kg i.p.). We found high levels of the cardioinhibitory cytokines in rat heart after 3 and 6 hs from MDMA administration. Strongest reaction was observed at 24 hs for TNF-alpha, IL-1beta, IL-6, 8, 10 and for MCP-1. Furthermore, we still determined the presence of MDMA and MDA in the plasma of rats treated with MDMA intra-peritoneal single injection; it was present as early at 6 hs and still present 24 hs after treatment. Western blot analysis in cardiac samples demonstrated the IL-1beta and IL-6 reactions in rats died spontaneously at fourth hour. The rise of the selective cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium to the cardiac dysfunction resulting from MDMA injection.

Cytokines, chaperones and neuroinflammatory responses in heroin-related death: what can we learn from different patterns of cellular expression?

Heroin (3,6-diacetylmorphine) has various effects on the central nervous system with several neuropathological alterations including hypoxic-ischemic brain damage from respiratory depressing effects and neuroinflammatory response. Both of these mechanisms induce the release of cytokines, chemokines and other inflammatory mediators by the activation of many cell types such as leucocytes and endothelial and glial cells, especially microglia, the predominant immunocompetent cell type within the central nervous system. The aim of this study is to clarify the correlation between intravenous heroin administration in heroin related death and the neuroinflammatory response. We selected 45 cases among autopsies executed for heroin-related death (358 total cases); immunohistochemical studies and Western blotting analyses were used to investigate the expression of brain markers such as tumor necrosis factor-α, oxygen-regulated protein 150, (interleukins) IL-1β, IL-6, IL-8, IL-10, IL-15, cyclooxygenase-2, heat shock protein 70, and CD68 (MAC387). Findings demonstrated that morphine induces inflammatory response and cytokine release. In particular, oxygen-regulated protein 150, cyclooxygenase-2, heat shock protein 70, IL-6 and IL-15 cytokines were over-expressed with different patterns of cellular expression.

Reconstruction of the Weapon in a Case of Homicidal Decapitation

Decapitation as homicidal mode of death is relatively rare. In most cases of decapitation, the differentiation between the modes of death might be difficult to some extent, particularly in cases where essential investigative elements, like the decedents head and the weapon, are unavailable. Our report concerns a case of homicide by decapitation without any further mutilation of the victim, where only the combination of autopsy results, histologic findings, and engineering technical reconstruction allowed us to identify with certainty the mode of death as vital decapitation. The technical reconstruction of the alleged weapon allowed the identification and the discovery of the hand saw used by the murderer to decapitate the victim.

Confocal Laser Scanning Microscope, Raman Microscopy and Western Blotting to Evaluate Inflammatory Response after Myocardial Infarction

Cardiac muscle necrosis is associated with inflammatory cascade that clears the infarct from dead cells and matrix debris, and then replaces the damaged tissue with scar, through three overlapping phases: the inflammatory phase, the proliferative phase and the maturation phase. Western blotting, laser confocal microscopy, Raman microscopy are valuable tools for studying the inflammatory response following myocardial infarction both humoral and cellular phase, allowing the identification and semiquantitative analysis of proteins produced during the inflammatory cascade activation and the topographical distribution and expression of proteins and cells involved in myocardial inflammation. Confocal laser scanning microscopy (CLSM) is a relatively new technique for microscopic imaging, that allows greater resolution, optical sectioning of the sample and three-dimensional reconstruction of the same sample. Western blotting used to detect the presence of a specific protein with antibody-antigen interaction in the midst of a complex protein mixture extracted from cells, produced semi-quantitative data quite easy to interpret. Confocal Raman microscopy combines the three-dimensional optical resolution of confocal microscopy and the sensitivity to molecular vibrations, which characterizes Raman spectroscopy. The combined use of western blotting and confocal microscope allows detecting the presence of proteins in the sample and trying to observe the exact location within the tissue, or the topographical distribution of the same. Once demonstrated the presence of proteins (cytokines, chemokines, etc.) is important to know the topographical distribution, obtaining in this way additional information regarding the extension of the inflammatory process in function of the time stayed from the time of myocardial infarction. These methods may be useful to study and define the expression of a wide range of inflammatory mediators at several different timepoints providing a more detailed analysis of the time course of the infarct.

Decomposing Human Blood: Canine Detection Odor Signature and Volatile Organic Compounds

The admissibility of human “odor mortis” discrimination in courts depends on the lack of comprehension of volatile organic compounds (VOCs) during the human decay process and of the lack in standardized procedures in training cadaver dogs. Blood was collected from four young people who died from traffic accidents and analyzed using HS‐SPME/GC‐MS at different decompositional stages. Two dogs, professionally trained, were tested to exactly locate blood samples, for each time point of the experiment. We found a long list of VOCs which varied from fresh to decomposed blood samples, showing differences in specific compounds. Dog performance showed a positive predictive value between 98.96% and 100% for DOG A, and between 99.47% and 100% for DOG B. Our findings demonstrated that decomposing human blood is a good source of VOCs and a good target for canine training.