Traumatic brain injury (TBI) is a leading cause of death and disability, contributing to one-third of all injury-related deaths in the United States, and a significant cause of loss of productivity [1]. It is estimated that 10-20% of returning veterans sustain TBI while deployed, making TBI the hallmark injury of current military casualties. In these cases, most TBI results from exposure to blast overpressure (BOP) from explosive devices, resulting in cell death and neuronal dysfunction. Furthermore, military casualties exposed to blast often experience delays in medical evacuation to higher echelons of care, with time intervals ranging from 1 hour to several days [2]. During this time frame, secondary molecular responses promote further neuronal injury and consequently, neurodegeneration. The development of secondary injury subsequent to the primary injury provides a window of opportunity for therapeutic intervention to prevent progressive tissue damage after traumatic brain injury (TBI) [3]. Neuroinflammation is a well-established secondary injury mechanism that largely contributes to damage observed after TBI [4].

Neuroinflammation following TBI is mediated by an early activation of the innate immune system [5]. The complement system, a key player in innate immunity, may participate as an important early mediator of neuroinflammation and neurodegeneration after TBI. Various studies in animal models and clinical studies have demonstrated elevated levels of complement components and complement activation fragments in serum, cerebrospinal fluid (CSF), and brain parenchyma after TBI, with broad parenchymal deposition of complement components [6–9]. In both clinical and experimental studies, severe secondary insults in TBI were observed in parallel to pronounced complement activation, specifically increased levels of the complement activation product, C5b-9 [10]. These secondary injuries include the recruitment of inflammatory cells into the intrathecal compartment, the induction of blood brain barrier (BBB) dysfunction by activated complement C3a and C5a, the induction of neuronal apoptosis through the neuronally expressed C5a receptor (C5aR), and homologous cell lysis mediated by C5b-9 [11]. Prior studies from our lab report that complement activation and C3a-C3aR interaction are associated with hypoxia-induced disruption of neuronal networks, loss of dendritic spines, and neuronal apoptosis [12].

Genetic and pharmacological manipulation of both complement levels and complement activation in mouse models of TBI are reported to be highly neuroprotective, suggesting the complement cascade is a critical target for managing post-TBI tissue damage. Mice deficient in the C3 gene [13, 14] or overexpressing complement regulatory proteins such as Crry or vaccinia virus complement control protein, exhibit significant neuroprotection, attenuated BBB disruption, and improved neurological outcome after injury [15, 16]. Inhibition or deficiency of other complement components have also been demonstrated to be effective in ameliorating injury, including inhibition of C1 by C1-inhibitor (C1-INH) [17], C4 deficiency or inhibition by C4 antibody [18], C5aR blockade [19], and factor B deficiency [20] or inhibition by factor B antibody [21]. In our most recent work, we found early complement activation was dramatically increased profound after blast injury. In addition, higher systemic and local levels of C5b-9 were detected as early as 3 h and persisting for up to 48 h were associated with BINT in rats exposed to blast overpressure (BOP) [22]. These results strongly suggest early administration of complement inhibitors as a novel and viable pharmaceutical strategy to mitigate BINT.

Administration of decay-accelerating factor (DAF), an inhibitor of alternative and classical complement activation pathways has been shown to protect neurons from hypoxia-induced disruption of neuronal networks, loss of dendritic spines, and neuronal apoptosis in cultured primary neuronal cells [12]. In the study presented here, we hypothesized that the complement system plays a critical role in the development of secondary injury and early administration of DAF would be neuroprotective in a rat model of blast-induced neurotrauma.

Previously, we have demonstrated that early complement activation and inflammatory response were associated with blast-induced neurotrauma in rats after a moderate BOP exposure (120 kPa) [22]. In this study, we evaluated the effects of complement inhibition on neuroprotection after blast injury. We found that: 1) administration of rhDAF 30 min after moderate blast exposure displays a protective effect on brain histological damage as well as BBB breakdown and brain edema during the first 24 and 48 h after BOP; 2) rhDAF treatment attenuates blast-triggered systemic complement activation at 3 h and local complement deposition during the first 48 h, as well as systemic and local cytokine release at 24 h after the injury; and 3) Treatment with rhDAF reduces blast-instigated C3a-C3aR interaction and tau phosphorylation during the first 24 h after the blast exposure. Taken together, the data presented here provides first line evidence of early complement inhibition as an effective therapeutic strategy for blast-induced neurotrauma and neuroinflammation.

DAF, a ubiquitously expressed intrinsic complement regulatory protein, inhibits complement activation by preventing the assembly or accelerating the disassembly of the C3/C5 convertases in both the classic and alternative pathways thereby limiting the local C3a/C5a and C5b-9 production [28]. Human DAF has a structure similar to rat DAF and has displayed cross-species reactivity [29]. The selected dosage of rhDAF was in the titrated range used in the previous studies of hypoxia in rat primary neuronal cells [12], mouse ischemia-reperfusion [23, 28], swine hemorrhagic shock [30, 31] and rat hemorrhagic shock (unpublished data). The time window for rhDAF administration (30 min) after blast injury used in this study was based on previous findings that systemic complement activation after a moderate BOP exposure paralleled BBB breakdown as early as 3 h, persisting up to 48 h, and returning to control levels by 72 h after the injury [12, 22]. Intravenous administration of rhDAF accumulated in the brain cortex and hippocampus as early as 3 h and persisted up to 48 h following BOP (Figure 1). The distribution of rhDAF in the brain was associated with the cerebral endothelium, suggesting that it might bind to the damaged endothelium and enter into the brain through the breached BBB after the blast injury.

C3a-C3aR interaction is a common initiating signal for subsequent reactions that initiates an inflammatory response. C3aR, a G-protein coupled receptor, is constitutively expressed in the central nervous system, on both neurons and glia [32]. Significant up-regulation of C3aR in murine brain after ischemia has been observed [12, 33]. Interaction of C3a-C3aR leads to enhanced and maintained inflammatory responses such as leukocyte infiltration, vascular permeability, leukocyte activation, and inflammatory cytokine production [34]. Consistent with our previous findings [12], increased C3aR expression and interaction of C3a-C3aR were observed in rats exposed to BOP. Notably, C3a-C3aR engagement was markedly reduced in rhDAF-treated BOP injured animals, presumably through limiting local expression of C3aR and C3a and/or preventing the extravasation of systemic C3a through increased integrity of the BBB.

TBI results in brain microvasculature and BBB damage, leading to increasing BBB permeability. BBB integrity was assessed by immunoglobulin (IgG) staining. Staining for immunoglobulin (IgG) is an effective technique for assessing BBB integrity as the breach of the BBB to plasma protein, such as IgG, is a prominent feature of experimental brain injury [35]. The increased BBB permeability observed during the initial 48 h after blast (Figure 10) is congruent with previously reported increases in IgG staining at 3 and 24 h following blast [27]. In contrast, in rhDAF treated animals a significant reduction in BBB permeability was observed. BBB limited permeability with rhDAF treatment could be one way in which local cytokines and complement activity is down-regulated.

Our previous and current data demonstrated increased deposition of C3 and C5b-9 associated with cortical vasculatures at 3 and 48 h after BOP exposure, suggesting complement deposition could be playing pivotal role in BBB disruption post-injury. In agreement, early rhDAF treatment reduced local complement deposition (C3 and C5b-9) 3, 24 and 48 h post-injury and concurrently reduced BBB permeability (Figures 5, 6 and 10). The paralleled change between BBB dysfunction and complement deposition (C3 and C5b-9) indicates that systemic complement activation may contribute to complement protein accumulation in the brain cortex via crossing the damaged BBB following blast injury. Nevertheless, BOP exposure might have also resulted in elevation of C3 transcription in the cortex, suggesting the late complement protein accumulation could be, at least partially, produced locally through a C3a-C3aR interaction.

Interestingly, treatment with rhDAF decreased systemic and local cytokine levels at 24 h but not at 3 h after blast. The delayed effect of rhDAF on systemic cytokine release suggests an indirect effect of DAF on cytokine production presumably through the interruption of the C3a-C3aR and/or C5a-C5a receptor (C5aR) interaction and subsequent attenuation of cytokine synthesis via down-regulation of inflammatory gene transcriptional activity [36–38]. However, the effect of rhDAF on local cytokine attenuation in the cortex could be due to both the inhibition of cytokine synthesis via the interruption of C3a-C3aR engagement and the decrease of extravasation of systemic cytokines through the enhancement of BBB integrity.

AQP4, the principal member of the aquaporin protein family in the central nervous system, is expressed in astrocyte processes of the glia limitants externa and in perivascular astrocyte foot processes. AQP4 plays a key role in cerebral cytotoxic edema formation [26]. We observed an increased expression of AQP4 in the brain during the first 24 h which correlated with early cytotoxic edema. AQP4 expression largely normalized 48 h after the blast injury, indicating that cytotoxic edema is an early event in blast-induced brain injury. It is interesting to note, complement system activation has been reported to enhance AQP4 expression [22] and the observed increase in complement activation after blast exposure may play an important role in brain cytotoxic edema induced by AQP4. In agreement, our data shows that rhDAF exerted a protective effect on cytotoxic edema, and normalized blast-induced overexpression of AQP4. In addition, pro-inflammatory cytokines have been reported to induce the expression of AQP4 in rat astrocytes [39, 40]. This regulatory effect of DAF on AQP4 expression could be at least partially explained by its role in attenuating the expression of IL-1β and IL-18, which has been shown to down-regulate AQP4 expression after TBI.

Increasing evidence suggests that TBI is associated with tauopathy, characterized by neurofibrillary tangles and neuropil threads composed of hyperphosphorylated tau [41]. Recent studies in mice with genetic deficiency of CD59 and Cr-related protein Y have demonstrated that the complement system has an active role in the development of tau pathology and neurodegeneration [42, 43]. In the present study, hyperphosphorylated tau developed during the first 48 h after blast injury. These results are consistent with other reports which showed tauopathy in military personnel exposed to explosive blast as well as in blast-exposed mice [25]. Intriguingly, rhDAF treatment reduced early tau phosphorylation at 3 and 24 h, but not at 48 h after the blast. One possible explanation could be early tau phosphorylation, but not late tau phosphorylation, is complement activation-dependent. Another possibility is rhDAF, although present in the brain tissues 48 h after administration may not be functional to down regulate tau phosphorylation. Further studies will be needed to confirm these speculations.

Increased expression of C5aR was reported to be associated with enhanced phosphorylated tau in the brain of Alzheimer’s patients [44]. Moreover, treatment with a C5aR antagonist reduced the amyloid deposits and tau phosphorylation with enhanced neuronal functions in a murine model of Alzheimer’s disease [45]. Although increased expression of C5aR and interaction of C5a-C5aR was not observed in this study (data not shown), it is very likely that early tau phosphorylation is regulated by the enhanced C3a-C3aR signaling axis in our blast TBI model. Tau is also reported as a potent, antibody-independent activator of the classical complement pathway in chronic neurodegenerative disease [46, 47]. Thus hyperphosphorylated tau, in turn, might lead to and/or maintain local cerebral complement activation after blast injury. Blast exposure has been considered to increase the risk for late development of chronic traumatic encephalopathy (CTE), a tau protein-linked neurodegenerative disorder, which is associated with post-traumatic stress disorder (PTSD) [20]. Consequently, the ability of tau to activate classical complement pathway could provide a mechanism for initiating and sustaining a chronic, low-level cerebral inflammatory response that may cumulate over the disease course and contribute to PTSD after blast injury. Future studies will be necessary to look into the relationship between complement and tauopathy at subacute and chronic phases of blast injury, and to determine whether complement inhibition has beneficial effects on blast-induced CTE.

DAF protects against BINT by suppressing the systemic and local inflammatory response, reducing tau phosphorylation, improving BBB integrity, and decreasing cytotoxic edema. Modulation of the complement system after TBI is a novel and promising therapeutic tool aimed at manipulating secondary injury processes after BINT.