**6.2. Burns**

Severe burns in a fire cause systemic disorders involving hypovolemic shock accompanied by hypoalbuminemia (burn shock), and hemolysis and skeletal muscle injury due to deep burns, followed by systemic inflammatory responses and hypoxia (Jeschke et al., 2008), which are usually detected by pathomorphology and biochemistry in postmortem investigation (Bohnert et al., 2010; Quan et al., 2009; Zhu et al., 2001b). Macro- and microscopic signs of vitality in fire death include soot deposits and thermal injury in the upper airways, but these findings may partly be sparse or even absent, especially in peracute deaths, making the diagnosis difficult (Bohnert et al., 2003). Recent immunohistochemical studies of the respiratory tract and lungs demonstrated intravital reactions in fatal burns (Boehm et al., 2010; Bohnert et al., 2010; Marschall et al., 2006); however, the pathophysiological process leading to death is still unclear. In particular, the influences of toxic gases usually do not leave significant pathology that is detectable after death, except that bilateral pallidum necrosis is occasionally seen in CO intoxication. With respect to this, previous studies detected specific neuronal damage in the pallidum due to CO intoxication by immunohistochemistry (Michiue et al., 2008; Piantadosi et al., 1997); immunohistochemical markers in the brain may be used to differentiate pathological conditions of the neurons and glial cells due to ischemic, metabolic, toxic and traumatic brain injury (Chen et al., 1997; Piantadosi et al., 1997; Zhang et al., 2010).

260 Apoptosis and Medicine

Very little knowledge is available with regard to the contribution of apoptosis to the neurotoxicity of other drugs and poisons at present; however, drug-related hyperthermia may induce neuronal apoptosis in a similar manner to that in heatstroke, described below. Various chemicals that trigger oxidative stress can induce neuronal apoptosis. Animal experiments showed that organophosphorus compounds caused acute necrosis of neurons in the brain at toxic doses, but induced apoptotic neuronal death at sublethal doses (Abou-

Fire fatality involves complex causes of death; major lethal factors involved in fire death are burns and inhalation of toxic gases, including CO and cyanide, which are produced by combustion, accompanied by smoke and ambient oxygen depletion (Stefanidou et al., 2008). Despite recent advances in clinical burn and CO intoxication care measures (Ipaktchi & Arbabi, 2006; Prockop & Chichkova, 2007), most fire victims are found dead, and in those found alive, severe burns or brain damage from CO intoxication can cause death despite intensive clinical care. In such cases, it is necessary to clarify the cause and process of death in a fire. In forensic casework, however, it may be difficult to determine the predominant cause of death due to fire or to exclude other causes of death, for which acute heart attack and asphyxiation are of particular interest, especially when clinical toxicological data are not available in cases of prolonged death without severe burns. In this respect, previous studies showed pulmonary pathology, and systemic hematological and biochemical disorders due to burns (Zhu et al., 2001a; Zhu et al., 2001b), while brain immunohistochemistry suggested specific findings of CO intoxication (Michiue et al., 2008). Thus, immunohistochemical markers in the brain that are involved in neuronal damage, apoptosis, degeneration and repair, including ssDNA, GFAP and bFGF, are useful to detect the specific neuropathology

of CO intoxication for differentiation from fatal burns as well as other fatal insults.

Severe burns in a fire cause systemic disorders involving hypovolemic shock accompanied by hypoalbuminemia (burn shock), and hemolysis and skeletal muscle injury due to deep burns, followed by systemic inflammatory responses and hypoxia (Jeschke et al., 2008), which are usually detected by pathomorphology and biochemistry in postmortem investigation (Bohnert et al., 2010; Quan et al., 2009; Zhu et al., 2001b). Macro- and microscopic signs of vitality in fire death include soot deposits and thermal injury in the upper airways, but these findings may partly be sparse or even absent, especially in peracute deaths, making the diagnosis difficult (Bohnert et al., 2003). Recent immunohistochemical studies of the respiratory tract and lungs demonstrated intravital reactions in fatal burns (Boehm et al., 2010; Bohnert et al., 2010; Marschall et al., 2006);

**5.4. Others** 

Donia, 2003).

**6.2. Burns** 

**6. Fire fatality** 

**6.1. General considerations** 

In acute fire fatality, immunohistochemistry demonstrated higher neuronal ssDNA immunopositivity in the parietal cortex than in acute cardiac and asphyxial deaths, suggesting the induction of neuronal apoptosis, irrespective of the blood COHb level; however, such findings were not seen in cases of postmortem burns (Wang et al., 2011a). These suggest that brain damage due to a fire is not simply caused by ischemia or hypoxia, but also involves cytotoxic factors, including massive thermal tissue injury and hemolysis, which can induce systemic oxidative stress involving the brain (Gatson et al., 2009). However, neuronal ssDNA immunopositivity in the pallidum was lower in cases of a low level of blood COHb saturation than in those with a fatal level of blood COHb saturation (>60%). Therefore, increased neuronal ssDNA immunopositivity in the parietal cortex and pallidum can be used as a vitality finding in acute fire deaths, with consideration of other pathological findings; these findings can be used to interpret death due to burns or CO intoxication in a fire. Furthermore, the topographical distribution of neuronal ssDNA immunopositivity in the brain may be helpful for determining the immediate cause of death in cases of other potentially fatal traumas or diseases, e.g. strangulation, drug abuse and acute cardiac attack. However, higher neuronal ssDNA immunopositivity was sporadically detected in other cases, suggesting the partial contribution of unspecific neuronal damage due to reperfusion, possibly involved with cardiopulmonary resuscitation measures (Li et al., 2010); this should be carefully considered when determining the cause of death, especially in cases where the vitality findings are sparse.

In prolonged deaths, the macro- and microscopic signs of vitality in fire death, described above, may become obscure, making the pathological diagnosis quite difficult. In immunohistochemical study of the brain, however, there were significant differences between fatal burns and CO intoxication in prolonged fire deaths under critical clinical care (Fig. 2). Neuronal loss was seen in those with burns and CO intoxication as well as in patients with a fatal ischemic heart attack and prolonged asphyxial deaths, while glial cells were increased in burns and heart attack; the glial cell number was larger in fatality due to burns than in CO intoxication and asphyxiation, regardless of temporary cardiopulmonary arrest (CPA) after insult, suggesting glial activation. The increase in glial cells in cases of fatal burns was accompanied by higher glial bFGF immunopositivity in the parietal cortex and white matter, and higher and lower glial GFAP immunopositivity in the cortex and white matter, respectively, showing low neuronal ssDNA immunopositivity. The abovementioned 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 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 BBB damage, as discussed below.

Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 263

severe burns. However, progressive systemic deterioration after severe burns, accompanied by preexisting disorders or physical predispositions, can cause fatality due to respiratory failure, hypoxic brain damage, hypovolemic shock and secondary infection involving sepsis, even under critical life support care (Barber et al., 2007; Williams et al., 2009). These death

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

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

processes should be assessed based on individual evidence.

**6.3. Toxic gases** 

neuronal ssDNA immunopositivity.

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.

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., 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 severe burns. However, progressive systemic deterioration after severe burns, accompanied by preexisting disorders or physical predispositions, can cause fatality due to respiratory failure, hypoxic brain damage, hypovolemic shock and secondary infection involving sepsis, even under critical life support care (Barber et al., 2007; Williams et al., 2009). These death processes should be assessed based on individual evidence.
