Cardiac changes following overdose Faa A et al. coronary vasoconstriction due to the stimulation of coronary a-adrenergic receptors; and third, it leads to an increase in platelet aggregability. Furthermore, the mismatch between myocardial oxygen supply and the demand from cocaine- induced vasoconstriction and increased myocardial work- load are often invoked as the major mechanisms by which cocaine might induce myocardial ischemia. In contrast, there is limited information available on the acute effects of heroin on the heart; to the best of our knowledge, only a few cases of AMI after heroin injections have been reported (18). Experimental studies have demon- strated that heroin may cause systemic and direct effects on the heart (19,20), most likely mediated by a local coronary spasm or inflammation (21). Heroin could act directly on the vasomotor center by increasing parasympathetic activity, producing bradycardia and hypotension and stimulating the release of histamine from mast cells (22,23). Heroin- induced coronary vasospasm after heroin sniffing has also been reported (24-26). Taken together, these reports indicate that heroin may have a direct toxic effect on coronary arteries and induce acute coronary occlusion. The S100 protein family comprises 16 low-molecular- weight (21 KDa) calcium-binding proteins that contain two calcium-binding sites. S100B, one of the two founding members of this family, is concentrated in oligodendrocytes, astrocytes, ependymocytes, Schwann cells and neurons (27). In clinical practice, it is a reliable biochemical marker of cerebral damage and is also utilized in postmortem diagnosis in medico-legal autopsy cases (28). Because S100B protein plasma levels have been shown to increase during ischemic stroke, S100 protein serum levels are used in clinical practice to monitor neurovascular status and functional outcome in subjects affected by cerebral damage (29) or subarachnoid hemorrhage (30,31). Elevated serum S100B levels have been reported in patients affected by obstructive sleep apnea syndrome (32). Moreover, S100B urine levels have been proposed as a useful tool to identify newborns that are at a higher risk of neonatal death (33) or full-term infants at risk of hypoxic-ischemic encephalopathy (34). S100B is not restricted to the nervous system; chondro- cytes, adipocytes, melanocytes, and Langerhans cells may also release S100B (35,36). In addition, recent investigations showed that elevated S100B serum levels may also be detected in patients without head injury, suggesting that it may originate from other damaged tissues (37,38), such as skeletal myofibers, muscle satellite cells and myoblasts. Isolated rat heart cells were shown to release S100B (39), and the myocardium was indicated as the putative main source for the increase in S100B serum levels in patients affected by dilated cardiomyopathy (40). Recently, S100B protein expression was shown to be induced in the heart of human subjects after myocardial infarction (41), and it has been proposed as a marker of poor prognosis in patients undergoing cardiac surgery (42). Based on these data, the current study was aimed at verifying, with immunohistochemical data, whether the S100B protein is expressed in the hearts of subjects who underwent sudden cardiac death, including drug addicts and patients without a history of drug abuse. CLINICS 2012;67(7):821-826 study. The subjects tested were subdivided into two main groups: the overdose group (15 subjects; cocaine, heroin or polypharmacological admixtures, including ecstasy) and the non-overdose group (59 subjects; traumatic and non-trau- matic deaths). The traumatic group was subdivided into three subgroups (sidearms, firearms, and drowning). The victims ranged in age from 9 to 85 years old. In each case, a full autopsy and toxicological analysis were performed within 24-72 hours after death. No macroscopic sign of congenital cardiomyopathy was found in any case. The macroscopic examination of the heart did not show any sign of severe coronary atherosclerosis. Multiple tissue samples collected from the left ventricular wall were fixed in 10% formalin, routinely processed and paraffin-embedded. The initial block was cut into 6-7 blocks approximately 2-3 mm wide. At least two histological sections were prepared from each block. The tissue sections were stained with hematox- ylin-eosin or immunostained with a polyclonal rabbit anti- S100B antibody (DakoCytomation A/S, Glostrup, diluted 1/ 2000) followed by incubation for three hours at 37˚C in with universal streptavidin/biotin immunoperoxidase detection reagents. Endogenous peroxidase was inactivated by incu- bation with 3% hydrogen peroxide for 5 min. To confirm the specificity of immunostaining, phosphate-buffered saline was substituted for the primary antibody in control sections. The peripheral nerves present in each section served as a positive internal control. Stained sections were read by two pathologists blinded to the clinical diagnosis. RESULTS Immunoreactivity for the S100B protein was mainly detected in the overdose group; less reactivity was detected in the 59 subjects in the non-overdose-related group (Figure 1). The pattern of S100B reactivity varied between cases, with different localization and different intensities. S100B staining was mainly observed inside the cytoplasm of scattered cardiac cells (Figure 2). The signals appeared as differently sized globular deposits and granules, often located near the nucleus. Similar globular deposits were also found in the interstitial spaces and were unevenly distributed throughout the cardiac tissue (Figure 3). Immunoreactive signals were also occasionally found in the perivascular spaces in the cytoplasm of stromal cells surrounding the wall of intra- cardiac arteries. No significant differences were found among the different subgroups in which the overdose patients were subdivided: S100B protein deposits were found in the heart in cocaine-, heroin- and other drug-related deaths. We observed the highest levels of immunoreactivity for S100B in 3 patients with a clinical history of combined cocaine and heroin abuse (Figure 4). In these cases, a large number of cardiomyocytes showed very large globular deposits of the protein. No reactivity for the S100B protein was detected in the subpericardial fatty tissue or other soft tissues, with the exclusion of nerve fibers, which represented the internal positive control. Finally, hematoxylin-eosin staining was performed to investigate the pathological status of these hearts; edema, and a wavy arrangement of cardiomyocytes were observed in all S100B-positive cases (Figure 5). MATERIALS AND METHODS DISCUSSION Seventy-four serial autopsies conducted from 2007 to 2010 (40 males and 34 females) were included in the present The current study investigated whether the S100B protein is expressed and immunodetectable in the hearts of patients 822