**3.3. Brain swelling and compression**

Brain swelling and compression, which cause brainstem dysfunction, are critically lifethreatening events in clinical trauma care. In patients with a brain compression sign, accompanied by increased brain weight, glial bFGF positivity in the parietal cerebral cortex was increased in acute death (survival time <0.5 h), followed by an increase of glial bFGF positivity in the parietal white matter in subacute death (survival time of 6–8 h). Such a finding was not detected in the hippocampus in acute–subacute deaths. The bFGF has been well documented as a neuroprotective and neurotrophic factor, both *in vitro* and *in vivo* (Bikfalvi et al., 1997; Dietrich et al., 1996; Louis et al., 1993); thus, the increase of glial bFGF positivity in these cases suggests a self-protective response to maintain BBB function in the early phase of brain swelling after trauma (Deguchi et al., 2002), which may start in the cerebral cortex and spread into the white matter, despite the fatal brain compression, as suggested by parahippocampal herniation. However, in some cases of acute death with or without the brain compression sign, the above-mentioned findings were not detected, suggesting other mechanisms of acute death, including rapid cardiorespiratory failure induced by SAH around the brainstem (Macmillan et al., 2002).

Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 255

important role in patients without a brain compression sign. Prolonged deaths with Duret hemorrhage, however, showed advanced glial and neuronal losses in the parietal cortex and hippocampus, accompanied by decreased GFAP positivity in the parietal white matter and hippocampus, overall low glial bFGF positivity, and high neuronal ssDNA positivity in the parietal cortex and hippocampus, which were more evident than in the early deaths with Duret hemorrhage and decompressive craniectomy described above. These findings suggested fatal CNS dysfunction due to posttraumatic progressive deterioration of whole brain involving the hippocampus, lacking glial bFGF and GFAP activation for

Of note, there were significantly different findings depending on the survival time in cases of brain compression, as mentioned above. Acute and subacute death cases (survival time <12 h) as well as early death cases without decompressive craniectomy had higher glial bFGF and GFAP, and lower neuronal ssDNA positivity without glial and neuronal losses, whereas glial and neuronal losses, accompanied by lower glial bFGF and GFAP, and higher neuronal ssDNA positivity, were evident in early death despite decompressive craniectomy and prolonged death, suggesting different pathologies and mechanisms of brain edema/swelling, depending on the time after brain injury (Wang et al., 2012b). In a classic concept, brain edema is divided into two types based on its pathogenesis (Klatzo, 1994; Unterberg et al., 2004): a) 'vasogenic (extracellular)' edema due to BBB disruption, resulting in extracellular water accumulation, and b) 'cytotoxic (intracellular)' edema due to sustained intracellular water collection. However, brain edema after mechanical brain injury is considered to be a mixed form; vasogenic edema may be predominant in the acute phase, followed by prolonged cytotoxic edema (Barzo et al., 1997). In acute and subacute phases, the mechanical/physical impact on the brain may injure blood vessels with subsequent disruption of endothelial membranes (Hellal et al., 2004) and minor damage to astrocytes, leading to BBB opening. Thereafter, in longer survival cases, increased intracranial pressure may be involved with diffuse cytotoxic brain edema (Marmarou et al., 2000; Unterberg et al., 2004), in which glial swelling is a major mediator (Kimelberg, 1995). The activation of glial bFGF in acute and subacute death cases and decreased glial GFAP positivity in longer survival cases with brain swelling suggest a self-protective response to maintain BBB function in the early phase after mechanical brain injury and the structural damage of astrocytes caused by cytotoxic edema, respectively. Furthermore, astrocyte damage can in turn deteriorate the extracellular microenvironment (e.g. persistent increase of extracellular glutamate levels), which causes

neuroprotection and repair, as a consequence of unimproved brain swelling.

both glial and neuronal damage (Barbeito et al., 2004; Matute et al., 2006).

To summarize, characteristic immunohistochemical findings were detected with regard to the influence of cerebral compression and survival time in mechanical brain injury (Table 1) (Wang et al., 2012b). Peracute deaths with severe open head injury without brain swelling presented with glial injury in the parietal white matter. Other fatalities without a brain compression sign did not show a significant loss of glial cells; however, glial injury in the parietal white matter was seen during a survival time of <12 h, while glial responses involving bFGF positivity were detected overall after 12 h–3-day survival, and delayed neuronal loss

In early and prolonged death cases (survival time >12 h), parahippocampal hernia may not be identified because of brain softening (encephalomalacia) around the hippocampus, but Duret hemorrhage in the brainstem as a sign of advanced descending transtentorial herniation can be used as an indicator of fatally severe high intracranial pressure (Graham et al., 1987; Parizel et al., 2002). In early deaths (survival time of 12 h–3 days), cases without Duret hemorrhage, irrespective of craniotomy, as well as those with Duret hemorrhage without depression craniotomy had increased glial bFGF positivity in the parietal cortex and white matter as well as the hippocampus, without significant glial loss; however, GFAP positivity in parietal white matter began to decrease in cases with the sign of brain compression (Fig. 1) (Wang et al., 2012b). The up-regulation of bFGF in these cases may reflect the self-protective responses of the brain after brain injury. Furthermore, the bFGF may be involved in the anti-apoptosis pathways; exogenous application of bFGF could prevent apoptosis (Ay et al., 2001; Tamatani et al., 1998). In these early death cases, mostly involving subdural hemorrhage/hematoma (SDH), high glial bFGF positivity accompanied by low neuronal ssDNA expression is consistent with the function of endogenous bFGF as an anti-apoptosis factor in traumatized brains (Wang et al., 2011a); self-protective activity in the cerebrum is maintained despite a fatal outcome, even in patients with Duret hemorrhage as a sign of fatal brainstem compression. In patients without such a brain compression sign, death may be attributed to overall brain damage without brain swelling, accompanied by SDH (Graham et al., 1988).

Early deaths with Duret hemorrhage and decompressive craniectomy (survival time of 12– 60 h), often involving massive contusions, presented quite different findings, involving glial and neuronal losses in the parietal cortex and/or hippocampus, accompanied by decreased glial GFAP positivity in the parietal white matter and hippocampus, with overall low glial bFGF positivity and high neuronal ssDNA positivity (Fig. 1) (Wang et al., 2012b). These findings suggest that the brain failed to generate sufficient bFGF to prevent apoptosis as a consequence of serious brain damage involving uncontrollable progressive brain edema and swelling, which developed fatal brainstem compression and Duret hemorrhage.

In prolonged deaths (survival time >3 days), patients without Duret hemorrhage as a brain compression sign, irrespective of craniotomy, had increased glial bFGF positivity in the parietal cortex and white matter as well as the hippocampus without glial loss; however, neuronal loss without a significant increase of neuronal ssDNA positivity was detected in the parietal cortex, showing no neuronal loss in the hippocampus. This suggests gradual cortical neurodegeneration after trauma despite anti-apoptotic neuroprotective activity, as indicated by increased glial bFGF positivity, and different mechanisms of cell death involved in mechanical brain injury besides apoptosis (Castejon & Arismendi, 2006; Stoica & Faden, 2010). The fatal complication of secondary pneumonia was more frequent in these cases than in deaths with Duret hemorrhage; secondary complications may play an important role in patients without a brain compression sign. Prolonged deaths with Duret hemorrhage, however, showed advanced glial and neuronal losses in the parietal cortex and hippocampus, accompanied by decreased GFAP positivity in the parietal white matter and hippocampus, overall low glial bFGF positivity, and high neuronal ssDNA positivity in the parietal cortex and hippocampus, which were more evident than in the early deaths with Duret hemorrhage and decompressive craniectomy described above. These findings suggested fatal CNS dysfunction due to posttraumatic progressive deterioration of whole brain involving the hippocampus, lacking glial bFGF and GFAP activation for neuroprotection and repair, as a consequence of unimproved brain swelling.

254 Apoptosis and Medicine

suggested by parahippocampal herniation. However, in some cases of acute death with or without the brain compression sign, the above-mentioned findings were not detected, suggesting other mechanisms of acute death, including rapid cardiorespiratory failure

In early and prolonged death cases (survival time >12 h), parahippocampal hernia may not be identified because of brain softening (encephalomalacia) around the hippocampus, but Duret hemorrhage in the brainstem as a sign of advanced descending transtentorial herniation can be used as an indicator of fatally severe high intracranial pressure (Graham et al., 1987; Parizel et al., 2002). In early deaths (survival time of 12 h–3 days), cases without Duret hemorrhage, irrespective of craniotomy, as well as those with Duret hemorrhage without depression craniotomy had increased glial bFGF positivity in the parietal cortex and white matter as well as the hippocampus, without significant glial loss; however, GFAP positivity in parietal white matter began to decrease in cases with the sign of brain compression (Fig. 1) (Wang et al., 2012b). The up-regulation of bFGF in these cases may reflect the self-protective responses of the brain after brain injury. Furthermore, the bFGF may be involved in the anti-apoptosis pathways; exogenous application of bFGF could prevent apoptosis (Ay et al., 2001; Tamatani et al., 1998). In these early death cases, mostly involving subdural hemorrhage/hematoma (SDH), high glial bFGF positivity accompanied by low neuronal ssDNA expression is consistent with the function of endogenous bFGF as an anti-apoptosis factor in traumatized brains (Wang et al., 2011a); self-protective activity in the cerebrum is maintained despite a fatal outcome, even in patients with Duret hemorrhage as a sign of fatal brainstem compression. In patients without such a brain compression sign, death may be attributed to overall brain

induced by SAH around the brainstem (Macmillan et al., 2002).

damage without brain swelling, accompanied by SDH (Graham et al., 1988).

swelling, which developed fatal brainstem compression and Duret hemorrhage.

Early deaths with Duret hemorrhage and decompressive craniectomy (survival time of 12– 60 h), often involving massive contusions, presented quite different findings, involving glial and neuronal losses in the parietal cortex and/or hippocampus, accompanied by decreased glial GFAP positivity in the parietal white matter and hippocampus, with overall low glial bFGF positivity and high neuronal ssDNA positivity (Fig. 1) (Wang et al., 2012b). These findings suggest that the brain failed to generate sufficient bFGF to prevent apoptosis as a consequence of serious brain damage involving uncontrollable progressive brain edema and

In prolonged deaths (survival time >3 days), patients without Duret hemorrhage as a brain compression sign, irrespective of craniotomy, had increased glial bFGF positivity in the parietal cortex and white matter as well as the hippocampus without glial loss; however, neuronal loss without a significant increase of neuronal ssDNA positivity was detected in the parietal cortex, showing no neuronal loss in the hippocampus. This suggests gradual cortical neurodegeneration after trauma despite anti-apoptotic neuroprotective activity, as indicated by increased glial bFGF positivity, and different mechanisms of cell death involved in mechanical brain injury besides apoptosis (Castejon & Arismendi, 2006; Stoica & Faden, 2010). The fatal complication of secondary pneumonia was more frequent in these cases than in deaths with Duret hemorrhage; secondary complications may play an Of note, there were significantly different findings depending on the survival time in cases of brain compression, as mentioned above. Acute and subacute death cases (survival time <12 h) as well as early death cases without decompressive craniectomy had higher glial bFGF and GFAP, and lower neuronal ssDNA positivity without glial and neuronal losses, whereas glial and neuronal losses, accompanied by lower glial bFGF and GFAP, and higher neuronal ssDNA positivity, were evident in early death despite decompressive craniectomy and prolonged death, suggesting different pathologies and mechanisms of brain edema/swelling, depending on the time after brain injury (Wang et al., 2012b). In a classic concept, brain edema is divided into two types based on its pathogenesis (Klatzo, 1994; Unterberg et al., 2004): a) 'vasogenic (extracellular)' edema due to BBB disruption, resulting in extracellular water accumulation, and b) 'cytotoxic (intracellular)' edema due to sustained intracellular water collection. However, brain edema after mechanical brain injury is considered to be a mixed form; vasogenic edema may be predominant in the acute phase, followed by prolonged cytotoxic edema (Barzo et al., 1997). In acute and subacute phases, the mechanical/physical impact on the brain may injure blood vessels with subsequent disruption of endothelial membranes (Hellal et al., 2004) and minor damage to astrocytes, leading to BBB opening. Thereafter, in longer survival cases, increased intracranial pressure may be involved with diffuse cytotoxic brain edema (Marmarou et al., 2000; Unterberg et al., 2004), in which glial swelling is a major mediator (Kimelberg, 1995). The activation of glial bFGF in acute and subacute death cases and decreased glial GFAP positivity in longer survival cases with brain swelling suggest a self-protective response to maintain BBB function in the early phase after mechanical brain injury and the structural damage of astrocytes caused by cytotoxic edema, respectively. Furthermore, astrocyte damage can in turn deteriorate the extracellular microenvironment (e.g. persistent increase of extracellular glutamate levels), which causes both glial and neuronal damage (Barbeito et al., 2004; Matute et al., 2006).

To summarize, characteristic immunohistochemical findings were detected with regard to the influence of cerebral compression and survival time in mechanical brain injury (Table 1) (Wang et al., 2012b). Peracute deaths with severe open head injury without brain swelling presented with glial injury in the parietal white matter. Other fatalities without a brain compression sign did not show a significant loss of glial cells; however, glial injury in the parietal white matter was seen during a survival time of <12 h, while glial responses involving bFGF positivity were detected overall after 12 h–3-day survival, and delayed neuronal loss 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 involving brain contusion and hematoma is considered.

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showed higher parietal glial bFGF positivity and neuronal loss with low ssDNA positivity, indicating incomplete necrosis or selective neuronal necrosis without positive evidence of

2. Hypoxic hypoxia – reduced blood oxygenation in the lung

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

II Asphyxia: 1. Neck compression – hanging and ligature/manual strangulation

3. Choking – foreign body in the airway

**Table 2.** Major causes of hypoxia and asphyxia in the forensic context

usually not detected in cardiac death (Table 3) (Wang et al., 2011a).

4. Suffocation – lack of atmospheric oxygen

3. Others: e.g. anemic, stagnant, oxygen affinity and histotoxic hypoxia

2. Smothering – obstruction of the airway orifices (nose and mouth)

apoptosis (Table 3) (Wang et al., 2011a).

**4.3. Cerebral hypoxia – Asphyxia** 

I Hypoxia: 1. Ischemic hypoxia – diminished blood supply
