**6.3. Toxic gases**

262 Apoptosis and Medicine

BBB damage, as discussed below.

temporary CPA after insult. These observations suggest neuronal loss accompanied by active glial responses after severe burns regardless of CPA after insult. Lower glial GFAP immunopositivity in the white matter in prolonged deaths due to burns may be related to

Both *in vitro* and *in vivo*, bFGF has been well established as a neuroprotective and neurotrophic factor (Dietrich et al., 1996; Louis et al., 1993). Severe trauma, including burn injury, can result in whole body tissue damage, for which an important early sign is systemic inflammatory response syndrome (SIRS), which may lead to multiple organ dysfunction syndrome (MODS). The early appearance of inflammatory cytokines in the systemic circulation has been demonstrated following thermal injury both in humans (Cannon et al., 1992) and animals (Kataranovski et al., 1999). Systemic inflammatory responses also develop in the brain (Reyes et al., 2006), which may induce, enhance or accompany astrogliosis (Balasingam et al., 1994). Thus, the up-regulation of bFGF and GFAP as well as increased numbers of glial cells in the parietal cerebral cortex in prolonged death due to burns may reflect self-protective responses of the brain. In acute death, such glial responses may not be apparent due to the shorter survival time. Furthermore, bFGF may be involved in anti-apoptotic pathways; the exogenous application of bFGF prevented apoptosis in both *in vitro* and *in vivo* studies (Ay et al., 2001; Tamatani et al., 1998). High cerebral cortex glial bFGF immunopositivity accompanied by low neuronal ssDNA

expression suggests that endogenous bFGF is an anti-apoptosis factor in the brain.

2002); the damaged brain does not lose its self-protective capacity after severe burns.

To summarize, typical pathologies in the brain after fatal burns are: 1) in acute deaths, increased neuronal ssDNA immunopositivity in the cerebral cortex, irrespective of the severity of the burns and CO intoxication; 2) neuronal loss in prolonged death; 3) increase in glial cells in prolonged death, accompanied by higher glial bFGF immunopositivity in the cerebral cortex and white matter, higher and lower glial GFAP immunopositivity in the cortex and white matter, respectively, with low neuronal ssDNA immunopositivity (Table 3) (Wang et al., 2011a). These findings suggest that: 1) increased neuronal ssDNA positivity, together with other pathological findings, can be used as a vitality finding in acute fire death; 2) the brain retained self-protective response capacity in fire victims who died due to

The BBB between systemic circulation and the cerebral parenchyma is composed of interendothelial tight junctions, basal lamina and perivascular astrocytes, and may also be damaged by severe burns; thus, BBB permeability can be increased, causing advanced brain edema (Reyes et al., 2009). Low glial GFAP immunopositivity in the white matter in prolonged deaths due to severe burns suggests astrocyte damage related to BBB dysfunction. In addition, systemic inflammatory responses followed by hyperthermia may also induce BBB dysfunction, which is characterized by vasogenic brain edema (Sharma, 2006). Further investigation is needed to clarify the mechanism of BBB dysfunction in prolonged deaths due to severe burns. Meanwhile, high bFGF positivity in the white matter, which was detected in prolonged deaths due to severe burns, suggests that the selfprotective system involving bFGF is activated to maintain BBB function (Deguchi et al., A spectrum of toxic or asphyxiating gases produced by combustion, including CO, cyanide and carbon dioxide, can contribute to death in a fire. CO has histo-/cytotoxicity due to its high affinity to specific sites of the brain, including the globus pallidus and the midbrain substantia nigra, but cyanide did not have such neurotoxicity (Oehmichen et al., 2006). In fire fatality with a fatal level of blood COHb saturation (>60%), immunohistochemistry detected higher neuronal ssDNA immunopositivity in the pallidum than in cases of a lower COHb level (<60%), as described above, suggesting CO-specific neuronal damage (Michiue et al., 2008; Tofighi et al., 2006; Wang et al., 2011a). Cyanide did not appear to contribute to neuronal ssDNA immunopositivity.

In prolonged deaths, neuron and glial cell number was decreased in CO intoxication as well as asphyxiation, regardless of temporary CPA after insult, suggesting reduced glial reactivity due to CO intoxication and asphyxiation. Glial bFGF and GFAP immunopositivity was low at each site, but neuronal ssDNA immunopositivity was high in prolonged deaths due to CO intoxication (Fig. 2) (Wang et al., 2011a). These findings differed from those in prolonged death due to heart attack or mechanical asphyxiation involving simple cerebral ischemia or hypoxia regardless of temporary CPA after insult. These observations suggest neuronal loss and progressive apoptosis without glial responses after CO intoxication. When the crucial functions of glial bFGF and GFAP in the self-protective responses of the brain are considered, high neuronal ssDNA immunopositivity accompanied by low glial bFGF and GFAP expressions in prolonged deaths due to CO intoxication, as indicated above, suggests that the brain has failed to generate sufficient bFGF to prevent apoptosis, which may indicate serious damage to the brain due to CO intoxication; CO can exert direct damage on cells by inducing apoptosis (Tofighi et al., 2006). Low bFGF and GFAP positivity in the white matter in prolonged death due to CO intoxication suggests delayed effects of CO, characterized by bilateral, confluent lesions that reflect diffuse demyelination (Lo et al., 2007). Such injury may also be caused by slowly progressive cytotoxic edema related to the direct toxic effect of CO. These findings suggest persistent and irreversible damage to the brain white matter due to CO intoxication. Similar findings suggesting damage to the BBB in the white matter were partly seen in prolonged asphyxial deaths, but were milder in ischemic heart attack. Such white matter damage may be responsible for delayed CNS deterioration due to CO intoxication and asphyxiation (Lo et al., 2007; Strackx et al., 2008).

To summarize, typical pathologies in the brain of fire fatality with a fatal level of blood COHb saturation are: 1) in acute deaths, increased neuronal ssDNA immunopositivity in the cerebral cortex, irrespective of the severity of CO intoxication, and higher neuronal ssDNA positivity in the pallidum due to CO intoxication; 2) neuronal loss in prolonged death; 3) overall low glial bFGF and GFAP immunopositivities with high neuronal ssDNA immunopositivity in prolonged death due to CO intoxication (Table 3) (Wang et al., 2011a). These findings suggest neuronal loss and progressive apoptosis without glial responses after CO intoxication; the brain sustained serious damage involving the loss of selfprotective capacity in CO intoxication, thus causing delayed death.

Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 265

Saukko, 2010; Madea et al., 2010); previous studies have suggested that postmortem biochemistry, immunohistochemistry and molecular biology can detect systemic functional alterations in these fatalities (Fineschi et al., 2005; Ishikawa et al., 2008; Jakubeniene et al., 2009; Maeda et al., 2011; Yoshida et al., 2011). Immunohistochemistry of the brain using ssDNA, bFGF, GFAP and S100 can also demonstrate functional alterations in fatalities due to extreme ambient temperature, involving glial responses and neuronal apoptosis (Wang et

When the human body cannot compensate for heat loss in an extremely cold environment, the body temperature decreases progressively, resulting in cerebral and cardiorespiratory dysfunction, and finally fatal arrhythmia and asystole. Metabolic deterioration involves dehydration, acidosis, azotemia and enhanced fat metabolism with ketonemia/ketouria, but

In immunohistochemcal investigation of the brain, hypothermia cases showed higher glial bFGF immunopositivity in the cerebral cortex and white matter, and higher S100β immunopositivity in the cerebral cortex with a lower CSF S100β concentration, without glial or neuronal loss (Fig. 3 and Table 3) (Wang et al., 2012a). The up-regulation of glial bFGF and S100β in the cerebral cortex suggests the self-protective responses of the brain and possible neurotrophic properties, respectively (Gomide & Chadi, 1999). Furthermore, since bFGF may be involved in the anti-apoptotic pathways (Ay et al., 2001; Tamatani et al., 1998), high glial bFGF immunopositivity accompanied by low neuronal ssDNA expression in hypothermia cases can indicate the activation of endogenous bFGF as an anti-apoptosis factor in the brain, which is similar to previous findings in prolonged fire fatality due to burns (Wang et al., 2011a). As above, the brain may retain self-protective response capacity without marked glial or neuronal damage in fatal hypothermia. The mechanism of death may mainly involve cardiac dysfunction, including ventricular fibrillation or asystole, resulting from myocardial ischemia, hypoxia, electrolyte abnormalities and elevated catecholamine levels (Turk, 2010), although there have been few postmortem investigations (Ishikawa et al., 2010; Wang et al., 2011b). To summarize, fatal hypothermia cases showed neuroprotective glial responses without marked neuronal or glial damage, which can serve as a condition for possible recovery and survival by means of adequate resuscitation and

A high ambient temperature in combination with predisposing factors and individual susceptibility ultimately impairs thermoregulation, and the body temperature rises precipitously; the main pathophysiology of heatstroke consists of hyperpyrexia involving impaired thermoregulation, accompanied by dehydration and profound systemic hypoxia, which is followed by further complications of pulmonary edema, renal tubular necrosis, adrenal hemorrhage, hepatic necrosis, myocardial necrosis, rhabdomyolysis,

myocardial and brain tissue damage are usually mild (Maeda et al., 2011).

al., 2012a).

**7.2. Hypothermia (Cold exposure)** 

life-supporting measures.

**7.3. Hyperthermia (Heatstroke)** 

**Figure 2.** Immunohistochemistry of single-stranded DNA (ssDNA), basic fibroblast growth factor (bFGF) and glial fibrillary acidic protein (GFAP) in the parietal cortex of prolonged fire death cases: 1) a case of fatal burns and low blood cardoxyhemoglobin (COHb) saturation (72 h survival), showing low ssDNA (a), and high bFGF (b) and GFAP (c) positivity; 2) a case of a fatal level of blood COHb saturation (48 h survival), showing high ssDNA (d), and low bFGF (e) and GFAP (f) positivity
