**3.1. General considerations**

Mechanical brain injury is a major trauma in both forensic and clinical medicine and is caused by various insults, resulting in various types of brain damage, often accompanied by secondary brain dysfunction, involving brain edema, swelling and compression; these are subdivided into focal and diffuse brain injury (Greenfield & Ellison, 2008; Knight & Saukko, 2004; Oehmichen et al., 2006). Classic concepts of CNS dysfunction due to mechanical brain injury comprise the disruption of brain structures by laceration and contusion, subarachnoid hemorrhage (SAH), compression by space-occupying intracranial hematoma or increased intracranial pressure due to edema, axonal injury, ischemic brain damage and primary acute brain swelling, especially in infancy. Brain compression or swelling accompanied by increased intracranial pressure is critical for survival in the early phase after brain injury in most cases. Previous studies have made great strides in investigating the morphology and causal mechanism of brain injury and dysfunction; forensic neuropathological case studies have demonstrated findings useful for establishing practical investigation procedures. Estimation of the age of brain injury and hematomas at the site or in the area adjacent to the injury has especially important criminological implications (Bratzke, 2004; Dressler et al., 2007; Hausmann & Betz, 2001; Hausmann et al., 1999; Hausmann et al., 2000; Oehmichen et al., 2003; Takamiya et al., 2007); however, some patients may survive for months or years after severe brain injury, while it may be difficult to explain the causal relationship between a focal brain injury and death in some fatalities. Cerebral edema/swelling alone may be a distinct finding of brain injury at autopsy, with mild or even no other structural lesions, to explain the cause of death, involving increased intracranial pressure affecting vital centers in 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 related to a fatal outcome, causing brainstem dysfunction.

250 Apoptosis and Medicine

**3. Brain injury** 

**3.1. General considerations** 

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

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

Mechanical brain injury is a major trauma in both forensic and clinical medicine and is caused by various insults, resulting in various types of brain damage, often accompanied by secondary brain dysfunction, involving brain edema, swelling and compression; these are subdivided into focal and diffuse brain injury (Greenfield & Ellison, 2008; Knight & Saukko, 2004; Oehmichen et al., 2006). Classic concepts of CNS dysfunction due to mechanical brain injury comprise the disruption of brain structures by laceration and contusion, subarachnoid hemorrhage (SAH), compression by space-occupying intracranial hematoma or increased intracranial pressure due to edema, axonal injury, ischemic brain damage and primary acute brain swelling, especially in infancy. Brain compression or swelling accompanied by increased intracranial pressure is critical for survival in the early phase after brain injury in most cases. Previous studies have made great strides in investigating the morphology and causal mechanism of brain injury and dysfunction; forensic neuropathological case studies have demonstrated findings useful for establishing practical investigation procedures. Estimation of the age of brain injury and hematomas at the site or in the area adjacent to the injury has especially important criminological implications (Bratzke, 2004; Dressler et al., 2007; Hausmann & Betz, 2001; Hausmann et al., 1999; Hausmann et al., 2000; Oehmichen et al., 2003; Takamiya et al., 2007); however, some patients may survive for months or years after severe brain injury, while it may be difficult to explain the causal relationship between a focal brain injury and death in some fatalities. Cerebral edema/swelling alone may be a distinct finding of brain injury at autopsy, with mild or even no other structural lesions, to explain the cause of death, involving increased intracranial pressure affecting vital centers in

can be used to monitor the self-protective capacity of the brain after injury.

et al., 2012a; Wang et al., 2012b). Details are described below.

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

**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


Immunohistochemistry of Neuronal Apoptosis in Fatal Traumas: The Contribution of Forensic Molecular Pathology in Medical Science 253

Diffuse mechanical brain injury clinically involves primary and secondary CNS dysfunction, which may result in permanent disability or fatal outcome. This type of mechanical brain injury is morphologically associated with specific white matter injury, usually termed diffuse axonal injury (DAI); however, other non-specific factors, including disrupted BBB, ischemia and vascular injury, also contribute to posttraumatic CNS dysfunction (Oehmichen et al., 2006). The macropathology may present with brain swelling and hemorrhages in the deep part of white matter, for which histology often involves focal edema and demyelination, accompanied by axonal injury, along the junction of gray and white matter, but these findings cannot be detected in very short survival cases. In such cases of peracute or instantaneous death within minutes, involving severe open head injury and apparently fatal structural brain damage, immunohistochemistry detected decreased glial GFAP positivity in the parietal white matter without glial or neuronal loss; however, this finding was not evident in the cerebral cortex (Wang et al., 2012b). GFAP as a marker of fibrous astrocytes in the white matter and molecular layer of the cerebral cortex is usually not detectable in protoplasmatic astrocytes or neurons in the cerebral cortex by routine immunohistochemistry (Li et al., 2009b). An increase in GFAP immunoreactivity in the cerebral cortex may be detected in classic astrocytic activation or astrogliosis; however, GFAP immunopositivity in the cerebral cortex showed no difference among all mechanical brain injury and control groups, irrespective of survival times, indicating a morphologically intact cerebral cortex. In peracute death, however, a significant decrease of white matter GFAP immunopositivity indicated the immediate, diffuse disruption of brain white matter; such findings were not detected in the hippocampus. Similar findings were detected in acute and subacute deaths (survival time <12 h) due to closed head injury without parahippocampal hernia as a brain compression sign, irrespective of the type of brain injury. This glial change in the parietal white matter may represent damage to the whole brain white matter immediately due to mechanical brain injury, suggesting fatal CNS dysfunction

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

**3.2. Diffuse brain injury** 

without brain swelling (Graham et al., 1988).

**3.3. Brain swelling and compression** 

ST, survival time; SAH, subarachnoid hemorrhage; SDH, subdural hemorrhage or hematoma; ssDNA, single-stranded DNA, bFGF, basic fibroblast growth factor; GFAP, glial fibrillary acidic protein

**Table 1.** Immunohistochemical findings of apoptosis-related biomarkers in mechanical brain injury with regard to the survival time
