**4.3. Cerebral hypoxia – Asphyxia**

256 Apoptosis and Medicine

without an increase of neuronal ssDNA positivity was seen after 3 days at the time of death, mostly due to complications. Fatalities with signs of brain swelling and compression showed gradual losses of glial cells and neurons with an early increase of glial bFGF positivity in the parietal cerebral cortex, which was followed by an increase of glial bFGF positivity in the parietal white matter and hippocampus, and final decreases of glial bFGF and GFAP positivity with increased neuronal ssDNA positivity in the parietal lobe and hippocampus, suggesting the involvement of neuronal apoptosis in progressive brain damage after injury. Such findings were detected earlier in death despite decompression craniotomy. These observations suggested different mechanisms of whole brain damage in the death process, depending on the severity of brain compression. ssDNA, bFGF and GFAP immunohistochemistry is useful to investigate such different death processes after brain injury with regard to the survival time. These findings may also contribute to wound timing when the pathology of the primary injury

Ischemia implies a local loss of blood supply due to arterial occlusion/disruption or vasoconstriction, or as part of systemic circulatory insufficiency or blood loss, resulting in a lack of oxygen (ischemic hypoxia), while other causes are also involved in hypoxia (oxygen deficiency), for which ischemic hypoxia is the simplest model (Table 2). Brain ischemia and hypoxia are common consequences of trauma or disease involving severe cardiac and peripheral vascular injury; the brain is more susceptible to ischemia/hypoxia than other viscera. Asphyxia in the forensic context implies systemic hypoxia associated with carbon dioxide retention due to a mechanical insult, causing acidosis, which aggravates tissue damage involving the brain; however, a lack of atmospheric oxygen (suffocation) is also included. Susceptibility of neurons in the brain to oxygen deficiency depends on the vasculature and the vulnerability of individual neurons; ischemic hypoxia first affects the watershed/arterial border zone of the frontal gyri, the globus pallidus, the Ammon horn (hippocampus), and the cerebral cortex (Oehmichen et al., 2006). These sitedependent susceptibilities of the brain to ischemia/hypoxia present with various pathologies of neurons and glial cells following cardiac arrest and asphyxia, depending

It is known that transient cerebral ischemia induces neuronal apoptosis (Chan, 2004); however, the usual feature of global ischemia involves neuronal necrosis in the cerebral cortex (watershed/arterial border zone of the frontal gyri), the globus pallidus, the hippocampus and the celebellar Purkinje cells. Immunohistochemistry detected no evident changes of the brain in sudden death due to acute heart attack (simple cerebral ischemia); however, prolonged deaths under intensive medical care, possibly involving reperfusion,

involving brain contusion and hematoma is considered.

**4. Cerebral ischemia and hypoxia/asphyxia** 

**4.1. General considerations** 

on the survival time.

**4.2. Cerebral ischemia** 

The classification of asphyxia in the forensic context is not uniform (Byard, 2011; Sauvageau & Boghossian, 2010). From a practical point of view, however, the causes of mechanical asphyxia can grossly be divided into types with and without neck compression; the former (strangulation) involves lethal factors including brain ischemia/congestion due to closure of the blood vessels and/or air passages of the neck, whereas the latter (choking and smothering) causes hypoxia due to obstruction of the air passages. In addition, neurogenic cardiac suppression may be involved in both types (Oehmichen et al., 2006). The diagnosis of mechanical asphyxia as a cause of death is one of the most difficult tasks in forensic pathology, especially in cases lacking significant pathological evidence, even when 'classic signs of asphyxia' are apparent; for example, a very careful examination is needed to discriminate between smothering and sudden cardiac attack in cases without bruises or abrasions around the nose and mouth. The diagnosis of choking may also be obstructed when a foreign body has been removed in resuscitation measures. Furthermore, it is difficult to determine whether a food bolus in the air passages was the cause of death or a result of agonal or postmortem spillage; therefore, various procedures have been developed to detect and explain the pathophysiology of asphyxial death (Ishida et al., 2002; Zhu et al., 2000). In prolonged death cases, however, it is difficult to differentiate asphyxia from heart attack. With respect to this, immunohistochemistry of the brain detected no specific findings in acute asphyxial death, compared with sudden cardiac death; however, prolonged asphyxial death showed lower parietal glial GFAP positivity and neuronal loss with increased ssDNA positivity as a sign of apoptosis following advanced brain hypoxia, which was usually not detected in cardiac death (Table 3) (Wang et al., 2011a).


Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 259

Numerous chemical substances are involved in accidental, suicidal and even homicidal intoxication; it is a very difficult task to screen and identify individual intoxication in forensic and clinical routine work, especially in cases where anamnesis or circumstantial evidence is obscure or absent, since intoxication often presents with non-specific signs and symptoms, or poor morphological findings. It is also important for forensic pathologists to discriminate other insults as the cause of death or contributory factor even when drugs or poisons are detected. Previous studies showed systemic deterioration in fatal intoxication, involving CNS, using biochemical markers (Maeda et al., 2011). Some drugs and poisons primarily affect the nervous system, and secondary brain damage is almost inevitable in any kind of intoxication; however, CO intoxication and drug abuse are most frequent in forensic

The histo-/cytotoxicity of CO is due to its high affinity to iron-containing structures such as hemoglobin and myoglobin, as well as to specific sites of the brain, including the globus pallidus and the midbrain substantia nigra, which is different from cyanide, sulfide and azide (Knight & Saukko, 2004; Oehmichen et al., 2006). CO also depresses myocardial function, resulting in severe hypotension and subsequent global cerebral ischemia and hypoxia. Bilateral necrosis of the globus pallidus and the pars reticulata of the midbrain substatia nigra are known as non-specific alterations (Oehmichen et al., 2006); however, neuronal apoptosis in the pallidum has been suggested as an early change due to CO intoxication in an animal experiment (Piantadosi et al., 1997). With respect to this, immunohistochemistry of ssDNA demonstrated high positivity as a sign of apoptosis in the pallidum of the human brain in fatal CO intoxication (Table 3) (Michiue et al., 2008; Wang et al., 2011a). Similar findings were detected in acute and delayed fire fatalities having a fatal level of blood carboxyhemoglobin (COHb) saturation, different from those with lower COHb level, as described below. These

A variety of psychostimulants, narcotics and hallucinogens are involved in drug abuse, which results in brain damage and functional impairment. Among these drugs, animal experiments have shown that amphetamine and its derivatives, such as methamphetamine and ecstasy, induce apoptosis of cortical and striatal neurons, and cerebellar granular cells via various pathways (Cunha-Oliveira et al., 2008). Neuronal apoptosis has also been suggested with cocaine and opiates (Cunha-Oliveira et al., 2008). In forensic autopsy materials, immunohistochemistry also detected high neuronal ssDNA positivity as a sign of apoptosis in the cerebral cortex, pallidum and substantia nigra in fatal abuse of sedative hypnotics as well as methamphetamine, suggesting selective neuronal damage (Table 3) (Michiue et al., 2008).

findings indicate the specific neurotoxicity of CO to the pallidum.

**5. Intoxication** 

routine work.

**5.3. Drug abuse** 

**5.2. Carbon monoxide** 

**5.1. General considerations** 

CO, carbon monoxide; ssDNA, single-stranded DNA, bFGF, basic fibroblast growth factor; GFAP, glial fibrillary acidic protein

**Table 3.** Immunohistochemical findings of apoptosis-related biomarkers in non-injury traumas with regard to the survival time
