Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Cocaine-related deaths: a comparative study on immunohistochemistry as an innovative diagnostic tool

Cocaine abuse remains a major global health concern, with mortality rates showing a progressive rise over the past decades. An estimated 25 million people used cocaine in 2023, representing 0.47% of the global population aged between 15 and 64. According to the EUDA data, cocaine is the second most frequently reported illicit drug. The mortality rate due to overdoses in the European Union in 2022 is estimated at 22.5 deaths per million population aged 15 to 64. Cocaine is a tropane alkaloid derived from the leaves of Erythroxylon coca, and its pharmacological effects depend on both its chemical form (hydrochloride salt or free base) and the route of administration. Cocaine exhibits multiple pharmacodynamic actions: its stimulant and psychoactive effects are explained by its action on presynaptic monoamine transporters; by blocking the reuptake of dopamine, serotonin, and noradrenaline, it prolongs neurotransmitter activity in the synaptic cleft. Pathologically, cocaine abuse has been linked to a wide spectrum of organ damage. Chronic patterns of use are often associated with progressive neuropathology, vascular impairment, premature cerebral ageing, and mood disorders. In contrast, acute intoxication - even after a single high dose - can lead to serious complications including tachycardia, elevated blood pressure, hyperthermia, excessive sweating, tremors, seizures, pupil dilation, headaches, gastrointestinal discomfort, skeletal muscle overactivity, hemorrhagic stroke, and multi-organ dysfunction. Several cocaine metabolites can cross the blood–brain barrier, contributing both to the psychoactive properties of the drug and to its toxicological profile. These complications can be life-threatening and may arise in young and healthy individuals with no pre-existing risk factors. Remarkably, there is no identifiable set of conditions that reliably predict their occurrence. Severe outcomes have been reported following small quantities of cocaine and even after first-time use. Although higher doses and frequent consumption generally increase the probability of adverse effects, the threshold at which such complications develop varies greatly between individuals. Consequently, while cocaine toxicity is broadly dose-related, it cannot be considered dose-specific. The forensic interpretation of cocaine concentrations in postmortem cases remains a major challenge. Measured drug concentrations after death do not necessarily reflect ante-mortem levels because of postmortem redistribution (PMR) - the movement of drugs from tissues into blood and other fluids after death. For cocaine, interpretation is complicated by its pharmacokinetics and metabolism. Wide interindividual variability in tolerance results in overlap between postmortem cocaine concentrations from cocaine-related fatalities, non-cocaine-related deaths, and even living individuals. In light of the complexity of toxic-related deaths (crime scene investigation, autopsy, histology and toxicological findings), post-mortem diagnosis of cocaine-related death could be difficult. Moreover, substance blood levels alone often cannot explain the fatal event. On the other hand, it is well known that cocaine related deaths are mostly linked to the cardiovascular effects of such substance, making myocardial changes pivotal in the understanding of its mechanism of action. The aim of this study is to identify a correlation between cocaine administration and the heart distributive response in cocaine-related deaths, investigating the relationship between classics toxicological analysis and the immunohistochemistry heart features through the use of an anti-cocaine antibody, analyzing its potential use as a new or complementary diagnostic marker. 7 cases of cocaine-related death were selected (four men, three women, mean age 36,57 years). Only cases with toxicological data positive for cocaine and negative for any other drug (ethanol included) were selected. Post-mortem examination confirmed the diagnosis of cocaine-related death in all the cases. Toxicological analyses were performed on peripheral blood, urine and bile. The control group was composed of cases that died from car accidents with no clinical history of drug abuse and negative toxicological analysis for drugs. Such deaths were characterized by their rapidity. A Mouse anti Cocaine Monoclonal Antibody was selected; in the literature, there were no indications on the best method to be used in immunohistochemistry tests with this antibody; lacking other similar experimental studies, we had to establish from scratch an adequate method and the appropriate dilution of the antibody. Routine microscopic histopathological studies were performed using formalin-fixed paraffin-embedded tissue sectioned at 4 μm and stained with haematoxylin-eosin (H & E). The specimens were examined under transmitted bright field illumination and phase contrast mode using a light microscope connected to a photo camera computer system. The immunohistochemical findings and the gradation of the immunohistochemical reaction have been described with an ordinary scale. The immunostaining was clear and evident in all of the heart samples examined, obtained from the seven cocaine-related deaths. The mean semi-quantitative score was 2.83 (moderate to strong expression) in lateral left ventricular wall and 2.29 (moderate expression) in interventricular septum samples. In the lateral left ventricular wall samples, the reaction showed a predominantly nuclear and myofibrillar staining, distributed in widespread foci; a mainly nuclear staining with isolated to scattered foci was observed in the interventricular septum samples. The observation of heart samples from cocaine-related deaths revealed a wide range of cardiomyocyte alterations, highlithed by the positive staining. An observed difference was the timing of the immunostaining expression: longer post-mortem intervals (PMI) are associated with decreased staining intensity in cardiomyocytes myofibers. On the other hand, the detectable level of nuclear immunohistochemical staining was not influenced by the duration of post-mortem interval. In our study, all of the tested cases showed the presence of a positive immunostaining in hypercontracted myocardial cells with rhexis of the myofibrillar apparatus into cross-fiber, anomalous, and irregular bands (contraction band necrosis, CBN). The results of our immunohistochemical study, although conducted on a limited population, may be of interest to better understand the timing of cocaine metabolism and mechanism of action: the nuclear immunostaining may reflect the cocaine-mediated oxygen DNA damage. Furthermore, our results may support the use of immunohistochemistry, using the anti-cocaine antibody, as a valuable aid for the post-mortem diagnosis of cocaine-related deaths, in light of the scarce reliability of toxicological data when dealing with cocaine. A great limitation of our study is the small sample size: this factor is partially linked to precise and strict inclusion criteria, when selecting the subjects. The scarcity of single cocaine-positivity cases suitable for this research, may reflect the ongoing rising in polydrug consumption, including ethanol. Also, detailed immunohistochemical anti-cocaine studies are not available, which excluded a comparison of our work with others. As a result, the present research should be regarded as preliminary, and the conclusions drawn cannot yet be considered definitive or conclusive. Although limited by the small sample size, this study provides novel insights into the mechanisms underlying cocaine-induced cardiotoxicity. In light of the growing evidence of oxidative/nitrosative stress and oxidative DNA damage in cocaine-induced cardiotoxicity, the observed differential post-mortem immunohistochemical patterns in nuclear and myofibrillar compartments, support the hypothesis that redox imbalance plays a pivotal role in the acute cardiac effects of cocaine. Future studies involving larger cohorts and molecular-level analyses are necessary to confirm these findings and to elucidate the mechanisms linking oxidative stress, mitochondrial dysfunction, and myocardial injury in cocaine-related cardiac pathology.