**2. Brain neuronal apoptosis in human death**

Apoptosis is programmed cell death, regulated by specific 'death genes.' The process involves active protein synthesis, initiated by changes in the microenvironment and impaired metabolic and tropic supply (Alison & Sarraf, 1992), with the participation of immediate early gene transcription factors (e.g. c-jun, jun-B, jun-D, c-fos, AP-1, ATF and nuclear factor (NF)-κB), proteases (e.g. calpains and caspases), and glutamate-mediate toxicity, including free radicals, protein kinases, Ca2+ homeostasis, and second messenger systems (Vaux & Strasser, 1996). It is known that microglial cells have an anti-apoptotic function to protect neurons from apoptotic death (Polazzi et al., 2001). Mechanical brain injury is accompanied by the apoptosis of neurons and glial cells surrounding the site of contusion and hemorrhage, which undergoes cell degeneration and necrosis (Oehmichen et al., 2006). Apoptosis begins hours after a traumatic event, and remains demonstrable for about 3 days (Yakovlev & Faden, 2001). These survival time-dependent changes are useful for timing brain contusions and hemorrhages in forensic pathology (Hausmann et al., 2004); however, apoptosis has been detected in the white matter as long as 1 year after injury (Williams et al., 2001). Apoptosis is also induced by other insults, including cerebral ischemia and hypoxia/asphyxia (Rosenblum, 1997), carbon monoxide (CO) intoxication (Piantadosi et al., 1997) and drug toxicity (Cadet & Krasnova, 2009). It is of particular importance that apoptosis may be involved in delayed neuronal loss (Becker & Bonni, 2004), which may contribute to delayed death or posttraumatic neurological disorders and sequelae.

Neuronal apoptosis is usually detected by *in situ* labeling of DNA fragments, e.g. terminal deoxynucleotidyl-transferase-mediated dUDP nick end-labeling (TUNEL) or *in situ* nick translation (ISNT) (Clark et al., 2001; Gavrieli et al., 1992; Rink et al., 1995). However, experimental studies have shown that single-stranded DNA (ssDNA) degradation precedes DNA double-strand breaks (DNAdsb) during a delayed neuronal death process caused by reperfusion after transient brain ischemia or intracerebral hemorrhage, possibly due to oxidative stress (Chen et al., 1997; Gong et al., 2001; Love, 1999; Nakamura et al., 2005). Thus, ssDNA can be used as an earlier marker of apoptosis and programmed cell death, which causes neuronal loss (Chen et al., 1997; Frankfurt et al., 1996; Michiue, 2008). This marker may contribute to the investigation of neuronal damage in acute death and also the timing of brain injury in the early phase (Chen et al., 1997; Hausmann et al., 2004).

248 Apoptosis and Medicine

sequelae.

PCR), and immunohisto-/immunocytochemistry combined with biochemistry, respectively. These observations will also contribute to understanding life-threatening events after

In forensic and clinical medicine, head injury is a major trauma, and primary or secondary brain damage, e.g. due to ischemic, hypoxic and toxic insults, is involved in most fatal traumas and diseases; thus, the investigation of brain damage after such insults is essential to assess the etiology and evaluate the severity of brain impairment relevant to central nervous system (CNS) dysfunction (Oehmichen et al., 2006). Necrosis and apoptosis are involved in morphological deterioration of the brain, involving cell and tissue decay (Fawthrop et al., 1991). Neuronal apoptosis is involved in both early and delayed responses after insults; however, this type of neuronal degeneration and cell death is of greater importance in connection with delayed or intermittent CNS dysfunction (Martin et al., 1998). This chapter reviews neuronal apoptosis and related pathologies in the brain after fatal traumas and diseases as demonstrated in forensic autopsy casework, summarizing previous observations (Michiue et al., 2008; Wang et al., 2011a; Wang et al., 2012a; Wang et al., 2012b).

Apoptosis is programmed cell death, regulated by specific 'death genes.' The process involves active protein synthesis, initiated by changes in the microenvironment and impaired metabolic and tropic supply (Alison & Sarraf, 1992), with the participation of immediate early gene transcription factors (e.g. c-jun, jun-B, jun-D, c-fos, AP-1, ATF and nuclear factor (NF)-κB), proteases (e.g. calpains and caspases), and glutamate-mediate toxicity, including free radicals, protein kinases, Ca2+ homeostasis, and second messenger systems (Vaux & Strasser, 1996). It is known that microglial cells have an anti-apoptotic function to protect neurons from apoptotic death (Polazzi et al., 2001). Mechanical brain injury is accompanied by the apoptosis of neurons and glial cells surrounding the site of contusion and hemorrhage, which undergoes cell degeneration and necrosis (Oehmichen et al., 2006). Apoptosis begins hours after a traumatic event, and remains demonstrable for about 3 days (Yakovlev & Faden, 2001). These survival time-dependent changes are useful for timing brain contusions and hemorrhages in forensic pathology (Hausmann et al., 2004); however, apoptosis has been detected in the white matter as long as 1 year after injury (Williams et al., 2001). Apoptosis is also induced by other insults, including cerebral ischemia and hypoxia/asphyxia (Rosenblum, 1997), carbon monoxide (CO) intoxication (Piantadosi et al., 1997) and drug toxicity (Cadet & Krasnova, 2009). It is of particular importance that apoptosis may be involved in delayed neuronal loss (Becker & Bonni, 2004), which may contribute to delayed death or posttraumatic neurological disorders and

Neuronal apoptosis is usually detected by *in situ* labeling of DNA fragments, e.g. terminal deoxynucleotidyl-transferase-mediated dUDP nick end-labeling (TUNEL) or *in situ* nick translation (ISNT) (Clark et al., 2001; Gavrieli et al., 1992; Rink et al., 1995). However, experimental studies have shown that single-stranded DNA (ssDNA) degradation precedes

traumas in the clinical management of critical patients.

**2. Brain neuronal apoptosis in human death** 

Animal experimentation has shown that ssDNA positivity could be detected after as little as 1 min of reperfusion following transient brain ischemia, showing a progressive increase, and exclusively in neurons exhibiting normal nuclear morphology within the first hour of reperfusion before the appearance of DNAdsb, whereas DNAdsb was first detected after 1 h of reperfusion. Thereafter, at 16–72 h of reperfusion, both ssDNA and DNAdsb positivity were found in many neurons and astrocytes, showing morphological changes consistent with apoptosis (Chen et al., 1997). Alternatively, ssDNA-positive neurons may be decreased after several hours of reperfusion, possibly due to active DNA repair. These findings suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA single-strand breaks due to oxidative stress may contribute to delayed ischemic neuronal death by triggering apoptosis. Other experimental studies have suggested that oxidative stress contributes to DNA damage and brain injury after intracerebral hemorrhage (Gong et al., 2001; Nakamura et al., 2005). These observations indicate that neuronal ssDNA positivity can be a marker of early brain damage, possibly within the first hour after an insult involving oxidative stress, including reperfusion and hemorrhage (Michiue et al., 2008). The detection of neuronal ssDNA may depend on the cause of death and survival time after a fatal insult. Brain reperfusion during cardiopulmonary resuscitation (CPR) may also contribute to positivity.

Astrocytes are essential for the structural integrity of neurons and also for maintaining their physiological environment, involving electrolyte and water homeostasis, pH and osmotic regulation, and elimination of transmitter amino acids and plasma proteins, as well as the control of vascular tone and intercellular transport of molecules from the vessel to the neuron, supporting the blood-brain barrier (BBB) (Nag, 2011). In forensic neuropathology, glial fibrillary acidic protein (GFAP) and S100β, as specific markers of differentiated astrocytes in the brain, are used to detect their morphological and functional alterations involved in brain damage (Liedtke et al., 1996; Stroick et al., 2006). GFAP is normally detected in fibrous astrocytes in the white matter and molecular layer of the cerebral cortex, but is usually not detectable in protoplasmatic astrocytes in the cerebral cortex by a routine immunohistochemical procedure (Li et al., 2009b; Oehmichen et al., 2006). GFAP is essential for fibrous astrocyte functions, including maintenance of the integrity of CNS white matter and the blood-brain barrier (Liedtke et al., 1996), and can therefore be used to detect the morphological and functional alterations of astrocytes due to brain damage; the decrease of white matter GFAP immunopositivity indicates the disruption of astrocytes, while reactive astrogliosis involves an increase in the gray matter (Wang et al., 2011a; Wang et al., 2012a). S100β is a calcium-binding peptide and is used as a clinical parameter of glial activation and/or death in a spectrum of CNS disorders (Stroick et al., 2006); S100β levels in serum and cerebrospinal fluid (CSF) can be used as a marker of brain damage in clinical and postmortem investigations (Korfias et al., 2006; Li et al., 2006a; Li et al., 2009a). Basic fibroblast growth factor (bFGF) is closely involved in neuronal protection and repair after ischemic, metabolic or traumatic brain injury, and has emerged as a central player in acute brain damage (Bikfalvi et al., 1997); the increase of glial bFGF positivity indicates a selfprotective response (Wang et al., 2011a; Wang et al., 2012a; Wang et al., 2012b). Thus, bFGF can be used to monitor the self-protective capacity of the brain after injury.

Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 251

the brainstem. Animal experiments have demonstrated the rapid onset of brain edema following injury (Byard et al., 2009). Considering the anatomical and metabolic species differences, however, it is necessary to investigate human materials. Moreover, human brain injury is rarely as simple as in experimental models; thus, the changes to the whole human brain after injury should be clarified to establish the relationship to death. Brain damage to a part distant from primary lesions may provide more significant information about the whole brain condition. In particular, the evaluation of human brain damage with regard to parahippocampal herniation or secondary brainstem hemorrhage of Duret as a macroscopic sign of brain swelling and compression is important since they are believed to be closely

Immunohistochemical investigation of the expressions of bFGF and GFAP in glial cells as well as ssDNA positivity in the neurons as a sign of neuronal apoptosis at sites distant from the primary injury to detect survival time-dependent changes in forensic autopsy cases of fatal mechanical brain injury demonstrated characteristic posttraumatic glial and neuronal changes in regions that were not involved in the primary injury, with regard to the influence of brain swelling and compression (Fig. 1 and Table 1). These changes involved early glial changes in peracute to subacute death with survival time within 12 h and neuronal loss in prolonged death after 3 days, which depended on brain swelling and compression,

**Figure 1.** Immunohistochemistry of single-stranded DNA (ssDNA), basic fibroblast growth factor (bFGF) and glial fibrillary acidic protein (GFAP) in the parietal cortex of mechanical brain injury cases: 1) early death without Duret hemorrhage (2-day survival), showing low ssDNA (a) and high bFGF (b) positivity with unaffected GFAP positivity (c); 2) prolonged death with Duret hemorrhage (9 days

survival), showing high ssDNA (d), and low bFGF (e) and GFAP (f) positivity

related to a fatal outcome, causing brainstem dysfunction.

irrespective of the type of primary brain injury, as follows.

Previous studies of neuronal apoptosis in forensic pathology have mostly focused on the healing process at the site of brain injury for wound timing in the forensic context (Hausmann et al., 2004; Tao et al., 2006); however, it is of great forensic and clinical importance to investigate overall brain damage to evaluate the severity of insults. Immunohistochemistry of neuronal apoptosis and related molecular pathology using biological markers, including ssDNA, bFGF, GFAP and S100β, demonstrated various type of brain damage due to head injury, ischemia/hypoxia or asphyxia, intoxication, burns, and extreme ambient temperatures (hyperthermia and hypothermia) (Wang et al., 2011a; Wang et al., 2012a; Wang et al., 2012b). Details are described below.
