TBI leads to primary and secondary pathological events where various neuronal and non- neuronal cell types and proteins expressed by these cells are involved (Figure 1.2). These include myelin basic protein by oligodendrocytes (Thomas et al., 1978); interleukin-6 (Gebhard et al., 2000), glial fibrillary acidic protein and calcium binding protein B (Pelinka et al., 2004) by astrocytes; reactive microglia and macrophages (Ramlackhansingh et al., 2011); spectrin break down products (Pineda et al., 2007), neurofilament heavy, medium and light polypeptide chain (Petzold, 2005), microtubule-associated protein tau (Ost et al., 2006; Yang et al., 2017), ubiquitin C-terminal hydrolase-L1 (Papa et al., 2010; Papa et al., 2012) and amyloid precursor protein by neurons.
Figure 1.2: TBI biomarkers
Various proteins change in expression after brain injury expressed by neurons, microglia, macrophages and astrocytes.
Neurofilament is a dominant axonal cytoskeletal protein. The high molecular weight heavy neurofilament subunit (NFH) is phosphorylated and released by damaged axons in brain injury. In moderate to severe TBI rats, the NFH levels are detected in blood within 24 to 48 hr of injury and can be used as a blood biomarker (Anderson et al., 2008). The phosphorylated NFH in serum is high in severe TBI patients compared to mild to moderate
17 TBI cases observed at 24 hr and 72 hr post-trauma. The serum levels of elevated NFH following TBI correlate with the 6 months GOS and can be used as a predictive outcome after brain injury (Shibahashi et al., 2016). Neurofilament medium polypeptide concentration in serum and cerebrospinal fluid increase in 44% of severe TBI and polytrauma cases. The elevation of serum neurofilament medium polypeptide in polytrauma patients indicates the consequence of other injuries (Martinez-Morillo et al., 2015). Following brain injury, neurofilament light protein levels also elevated in serum of severe TBI patients and the levels predicted the poor 12-month outcome (Shahim et al., 2016).
The microtubule-associated intracellular tau protein plays an important role in neuronal development and axonal stabilization. The post-mortem analysis of cerebrospinal fluid and serum in severe TBI cases showed elevated levels of tau (Olczak et al., 2017). Different forms of tau have been reported to vary in expression under neuronal stress. The oligomeric and phosphorylated tau increases significantly in injured cortex from 4 hr to 2 weeks postinjury in rats, whereas no change in total tau and non-phosphorylated tau forms (Hawkins et al., 2013). The cis-form, but not trans-form of phosphorylated tau is increased in injured axons in the absence of tau oligomers or tau tangles in human TBI brains (Kondo et al., 2015; Albayram et al., 2017). The levels of cis phosphorylated tau in mice is robust in moderate to severe or blast induced TBI compared to transient expression in mild TBI (Kondo et al., 2015). The notable increase in cis phosphorylated tau is observed from 12 hr postinjury (Kondo et al., 2015). The cis phosphorylated tau following repeated mild TBI, spreads and causes neurotoxicity observed after 6 months of injury (Kondo et al., 2015). The increased levels of cis phosphorylated tau correlates with the poor brain injury outcome in TBI patients after 1 yr of injury (Albayram et al., 2017). In CTE patients, elevated cis phosphorylated tau staining correlates with the increased tau oligomerization, early tangles, astrogliosis and microgliosis in cortex and thalamus (Albayram et al., 2017). Treatment with cis monoclonal antibody prevented a range of pathological outcome in vivo and in vitro (Kondo et al., 2015; Albayram et al., 2017). The elevated levels of tau observed prior to the development of any macroscopic neuronal lesions in mild TBI (Olczak et al., 2017). Therefore, due to the rapid activation of tau to neuronal insult, it can be used as a diagnostic approach in TBI.
In human Alzheimer’s brain tissue, reactive microglia colocalizes with tau, and extracellular tau internalizes to microglia indicating the downstream pathological events (Bolos et al., 2016). Some studies show that microglial activation precedes tau phosphorylation during neuronal damage and reactive microglia induces tau hyperphosphorylation in vivo (Bhaskar
18 et al., 2010; Maphis et al., 2015). The microglial activation correlates with the tau pathology
in vitro and in vivo (Maphis et al., 2015). The soluble factors derived from microglia induce
tau phosphorylation via p38 mitogen-activated protein kinase pathway observed in mouse primary cortical cultures and is inhibited by treatment with interleukin-1 receptor antagonist (Bhaskar et al., 2010). In addition, toll-like receptor-4 knockout mice shows decrease tau phosphorylation in neuronal injury (Bhaskar et al., 2010). This suggests that interleukin-1 and toll-like receptor-4 play an important role in microglial activation which further results in tau phosphorylation in neuronal insult.
Astrocytes in response to brain injury play a role in axonal injury, neuroinflammation and immune response. The immune response induced by pro-inflammatory cytokines precedes activation of astrocytes and microglia under the neuronal stress (Norden et al., 2016). The microglial (Ionized calcium binding adaptor molecule-1, Iba1) and astrocyte (Glial fibrillary acid protein, GFAP) reactivity was detected after 24 hr of injury whereas pro-inflammatory cytokine expression in microglia precedes astrocyte cytokine expression observed within 2 hr of injury (Norden et al., 2016). A similar outcome was observed in mice subjected to moderate to severe brain injury showing rapid changes in cytokine expression precedes Iba1 and GFAP reactivity (Villapol et al., 2017).
Among these proteins, amyloid precursor protein (APP) is known to play a direct role in modulating brain injury. The expression of APP increases significantly following brain injury observed in both animal models and TBI patients (McKenzie et al., 1996). The extent of neuronal damage is initially identified by axonal swelling observed within 15 min of injury in the corpus callosum and fornix following mild or severe head injury (Blumbergs et al., 1995; Greer et al., 2013). The upregulated APP levels in axons stay elevated at 90 days following brain injury (Itoh et al., 2009). The elevated APP positive neurites correlate with increased expression of activated microglia in TBI cases (Griffin et al., 1994) where APP positive damaged axons are later phagocytosed by microglia or macrophages (Itoh et al., 2009). APP expression also increases significantly in astrocytes following injury (Siman et al., 1989; Otsuka et al., 1991). In excitotoxic injury, APP is mainly produced by reactive astrocytes rather than reactive microglia at injury area (Topper et al., 1995). Along with APP, the inflammatory cytokine, interleukin-1β expression also increased rapidly in cortex and hippocampus observed within 6 hr of controlled cortical impact injury in rat (Ciallella et al., 2002). This suggests the role of APP in primary and secondary effects of brain injury.
19 The morphology of the axonal injury varies with the injury severity and time (McKenzie et al., 1996; Hayashi et al., 2009). Mice subjected to mild TBI show axonal swelling within 3 to 4 min of injury and then major axonal swelling has expanded in size as time progress (Greer et al., 2013). Majority of the injured axons show multifocal axonal swelling and disconnections, a limited number of axons demonstrate continuity (Greer et al., 2013). The majority of APP accumulates in swollen axons where further disconnections are happening (Greer et al., 2013).
The axonal damage and bulb formation observed in TBI patients within 2 hr of injury and increases with the survival time observed up to 24 hr (McKenzie et al., 1996). The post- mortem analysis of severe TBI cases survived less than 2 hr show neurofilament stained beaded axons and density or thickness increases with the survival time. Initial APP staining shows weaker immunoreactivity within 2 hr of injury compared to neurofilament but increases significantly by 24 hr of injury indicating the widespread axonal damage (Romero Tirado et al., 2018). In TBI cases survived more than 24 hr show loss of beaded axons and white matter cellular density stained with neurofilament whereas APP immunoreactivity increases until 26 days of injury (Romero Tirado et al., 2018).
Overall, the expression of APP by neurons and glia, rapid onset and long-lasting APP expression changes to neuronal injury has made APP as a gold standard measure of axonal injury (Topper et al., 1995; Itoh et al., 2009; Ryu et al., 2014). APP is also found to be neuroprotective in brain injury (Thornton et al., 2006; Corrigan et al., 2011; Corrigan et al., 2012c, b, a). The dual role of APP in neuroprotection and neurotoxicity is due to the difference in proteolytic processing of APP, detailed in section 1.2.1 (APP proteolytic processing, figure 1.4).
20