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While such reorganization of the system and everyday practice of TBI care led to marked reduction in mortality and morbidity, the clinical care for the head injured has reached its limits (Vukic et al., 1999; Ghajar, 2000). Thereby, now basic scientists and/or pharmaceutical research should provide further ammunition for clinicians to fight the silent epidemic represented by TBI. The importance of translational studies in TBI is further highlighted by data from the field of health care management, indicating that effective treatment of TBI is considered one of the most cost-efficient medical interventions (Saltman and Findling, 1997).

Although detailed analysis of the pathobiolog-ical processes activated in TBI are beyond the scope of this work, the authors will provide a short theoretical background to each section addressing various approaches to rescue neurons and glia in TBI, while also referring to other chapters of this work for further relevant information.

Cell death routes

On noxious influences or agents three major forms of cell death (CD) may occur: autophagic, necrotic and apoptotic.

Autophagy is characterized by lysosomal sequestration and degradation of cellular components. This mode of cell death has its potential role in physiological processes as well as in neurode-generative disorders (for review, see Assuncao and Linden, 2004).

While primary brain injury leads to focal and diffuse alterations in brain parenchyma, which are predominantly necrotic by nature, secondary brain damage practically triggered and initiated at the moment of TBI is also associated with apopto-tic damage. In fact, as time elapses post-injury, apoptotic processes become more dominant and in late phases post-injury autophagy may also participate particularly in axonal demise (Lockshin and Zakeri, 2004; Ringger et al., 2004; Gallyas et al., 2006).

Necrosis is characterized with swelling and rupture of the cell (or its axon); membrane damage and activation of inflammatory reactions is a consistent feature of necrotic cell death. Such necrotic processes are also associated with energy depletion/negative energetic state of the cell (Cai et al., 1998; Arden and Betenbaugh, 2004). Recent observations proved that both in focal and diffuse forms of experimental TBI in injured soma as well as in axons, various proteolytic processes are triggered and are responsible for necrotic demise (Bartus, 1997; Yamashima, 2004; Farkas et al., 2006). These studies demonstrated that mechanic or enzymatic alterations of membrane integrity in conjunction with altered function of ionic pumps and channels should lead to intracellular calcium accumulation which in turn results in the activation of neutral proteases, primarily the cysteine-aspartate protease calpain (Bartus, 1997; Stys and Jiang, 2002). Besides direct proteolysis associated with calpain activation, indirect proteolytic consequences such as calpain-mediated lysosomal rupture and cathepsin activation (Yamashima, 2004) also contribute to cellular demise.

Although the association and feedback connection between permeability changes, activation of proteolytic processes, cellular demise and functional alterations following TBI is not entirely developed, preliminary data suggest that inhibition of necrotic enzymes may facilitate functional recovery (Saatman et al., 1996).

Regardless of the initiating factors and the route leading to altered intracellular ionic homeostasis, increased level of intracellular Ca++ represents a permissive environment for the activation of another form of CD that is apoptosis (Cai et al., 1998).

Apoptosis, frequently referred to as programmed cell death plays a pivotal role in embryogenesis and is frequently initiated by a non-lethal stimulus leading to cell death via the activation of proteolytic cascades. Characteristic features of apoptosis include DNA laddering (detectable by electrophoresis), DNA fragmentation (TUNEL/ terminal deoxynucleotidyl-transferase-mediated de-oxyuridine triphosphate-biotin nick-end labeling), sub-2N DNA (FACS), rounding/blebbing of the cell, fragmentation of nuclei with marginalization of chromatin, exteriorization of phosphatidylserine (annexin V binding) (for review, see Arden and Betenbaugh, 2004; Lockshin and Zakeri, 2004).

As apoptosis requires a relatively intact energetic state of the cell, most probably it will not be localized to the core of tissue laceration (focal damage) rather it will involve remote/diffuse ("penumbra") area of the central nervous system (CNS). In the last decade multiple lines of evidences pointed to the activation of apoptotic processes in various forms of TBI (Rink et al., 1995; Conti et al., 1998; Buki et al., 2000; Ginis et al., 2000).

Studies in more severe, focal forms of TBI have proven cytochrome c (cyto c) release and caspase-3 activation in the cerebral cortex 6-24 h post-injury (Sullivan et al., 2002). Caspase-12 activation was also demonstrated in cortex, hippocampus and cortical astrocytes in this model of TBI implicating endoplasmic reticulum-associated apoptotic processes (vide infra) in the pathogenesis of cell loss (Larner et al., 2004) These authors also demonstrated capsase-7 activation in the same model peaking at day five post-injury (Larner et al., 2005).

Knoblach et al. (2002), in the model of lateral fluid percussion TBI in accordance with other, previous reports (Yakovlev et al., 1997; Beer et al., 2000; Clark et al., 2000), demonstrated caspase-3 and -9 activation in the cerebral cortex and the hippocampus 1-12 h post-injury. Although these workers were not able to demonstrate considerable activation of caspase-8, in other models including cortical impact TBI caspase-8 (representing the induction of the extrinsic route of apoptosis) was also found activated in both neurons and glia (Beer et al., 2001).

Apoptotic pathways

Apoptosis could be initiated via three major pathways (Fig. 1), and in most of these pathways

Fig. 1. The three major routes of apoptosis. Note that different caspases participate at initiation and the execution of the cell death processes. Also note the crucial role of calcium and the mitochondrion (for further details, see text).

intracellular Ca+ + , mitochondria and the caspase enzyme family play a crucial role. The extrinsic receptor-mediated route utilizes specific cell surface receptors belonging to the tumor necrosis factor (TNF) super family where binding of the ''death signal'' (such as Fas by Fas ligand) triggers the activation of the caspase enzyme cascade (Chinnaiyan et al., 1995). Caspases are cysteine-aspartate proteases which are present in the cell in the inactive proenzyme (zymogen) form. Specifically, ligand-receptor interaction in the extrinsic route will activate pro-caspase-8, which, in turn activates pro-caspase-3.

Similarly to this pathway, the final executor of the second, intrinsic pathway is also caspase-3, however, this route is mediated by mitochondrial alterations (Cai et al., 1998; Kruman and Mattson, 1999; Adams and Cory, 2002; Kroemer, 2003).

Mitochondria play a key role in apoptosis. Several studies demonstrated both in vitro and in vivo that TBI leads to a perturbation of the energy homeostasis (Giza and Hovda, 2001). Partially due to excessive firing of neurons and/or ionic imbalance due to altered pump/channel function as well as mechanoporation in at least a subpopulation of cell bodies and axons, a clear metabolic dysfunction occurs, leading to a shift toward anaerobic glycolysis, lactate-production and the accumulation of reactive oxygen species (ROS) (Pettus et al., 1994; Stys and Jiang, 2002; Singleton and Povlishock, 2004; Farkas et al., 2006). N-methyl-D-aspartate (NMDA) receptor activation in neurons, failure of the Na-K-ATPase in axons and me-chanoporation in some soma and axons in conjunction with altered energy homeostasis leads to the accumulation of intracellular (intraaxonal) Ca++ (Siesjo et al., 1999; Stys and Jiang, 2002).

Mitochondria accumulate the excess amount of Ca++ leading to swelling and dissipation of their membrane potential, initiating the perm-ealization of their outer membrane for solutes under 1500 Da, widely explained by the opening of the mitochondrial permeability transition pore (MPTP) (Zoratti and Szabo, 1995; Susin et al., 1998; Kruman and Mattson, 1999). This purported pore is consisting of the adenine nuc-leotide translocase (ANT), voltage dependent anion channel (VDAC), cyclophilin D, peripheral benzodiazepine receptor, hexokinase, creatine kinase and Bcl-2 proteins (Susin et al., 1999) (some of these Bcl-2 proteins harbor anti-apoptotic potential, while others possess pro-apoptotic activity (Reed, 1998; Scorrano and Korsmeyer, 2003)). Opening of the MPTP leads to the release of pro-apoptotic substances — cyto c, second mitochondrial activator of caspases (SMAC)/direct inhibitor of apoptosis protein (IAP) binding protein with low pi (DIABLO) and apoptosis inducing factor (AIF). When released, cyto c together with the apoptosis activating factor 1 (Apaf-1), dATP and cytosolic pro-caspase-9 forms the "apo-ptosome'' resulting in the generation of active ca-spase-9 which, in turn triggers the transition of pro-caspase-3 to active caspase-3 (Cai et al., 1998; Adams and Cory, 2002).

The third, most recently described route of apoptosis is initiated by endoplasmatic reticulum (ER)-stress leading to caspase-12 activation. This latter enzyme can act either as an executor caspase or via direct or indirect activation of caspase-3 (Oyadomari et al., 2002; Zong et al., 2003).

Crossroads of cell death

To develop rationally targeted therapeutic strategies beyond understanding these basic mechanisms of cell death, it is also mandatory to unravel those potential routes via the pathways initiated by noxious stimuli may communicate or interact.

Such connections or "detours" exist within the apoptotic machinery itself as well as between various major pathways of cell death that is necrosis, apoptosis and autophagy (Denecker et al., 2001; Lockshin and Zakeri, 2004). In addition to their role in the maintenance of the ionic and energy homeostasis and triggering the intrinsic pathway of apoptosis, mitochondria also serve as a crossroad or common pathway for all routes of apo-ptotic cell death (Cai and Jones, 1998; Saikumar et al., 1998) (Fig. 1).

To this end, besides pro-apoptotic members of the Bcl-2 family the mitochondrial membrane also contains other pro-apoptotic proteins such as Bax and Bak. These are responsible for the cross-talk between mitochondria (that is the intrinsic pathway of apoptosis) and the other two avenues of apoptotic cell death; the extrinsic route is converging to these pro-apoptotic proteins via the ca-spase-8 Bid-Bax/Bak interaction while the endoplasmatic reticulum stress also interacts with these proteins as well as via sending direct Ca+ + signals to the mitochondria (Jurgensmeier et al., 1998; Schendel et al., 1998; Ferri and Kroemer, 2001).

Not only the major intracellular "death-avenues'' but also the redundancy of the effector ca-spases provide considerable versatility for cell death; studies with knock-out mice as well as ca-spase-inhibitor studies have proven that an injured cell has the potential to select between various effector caspases and such decision is most probably influenced by various, physiologically active inhibitors of apoptosis (Assuncao and Linden, 2004).

Alternative, protease-mediated pathways may also contribute to cell death. Ubiquitination of some caspases and their subsequent destruction by the proteosome may participate in the regulation of apoptosis (Lockshin and Zakeri, 2002; Ditzel et al., 2003; Varshavsky, 2003). Recent observations have proven that disconnection of the cell from its neighboring structures specifically from the extracellular matrix should contribute to the initiation of cell death mechanisms. This process, primarily mediated by matrix metalloproteases is frequently described with the Greek word "an-oikis'' (homelessness) (Lockshin and Zakeri, 2002; Abe et al., 2004).

The above-detailed complexity of the cell death machinery provides multiple choices for a cell to react to a noxious stimulus. Apoptotic cell death requires energy and phagocytes that scavenge the dying cells. In cell cultures, in vitro in the absence of phagocytes the final mechanisms of cell death are always necrotic, despite of the initiating agent and the route the cell entered to die (Arden and Betenbaugh, 2004; Assuncao and Linden, 2004). Limited sources of ATP can easily halt the apo-ptotic processes and the cell may fail to display apoptotic morphology, however, this does not mean an arrest of cell death rather the initiation of autophagic or necrotic processes (Cai et al., 1998; Reed, 1998; Saikumar et al., 1998).

When we consider, how to inhibit apoptosis, we also have to answer, whether inhibition of apoptosis is useful. Theoretically, in a philosophical approach, we may conclude that a lethal or even non-lethal stimulus may ignite any of the above-detailed pathways. Due to the cross-talk between these avenues and the internal diversity of the individual pathways it is quite possible that pharmacological intervention to halt a death process may just shift the cell death mechanism to another pathway of cellular demise. Inhibition of apoptotic cell death can lead to autophagy instead of resulting in cell survival (Lang-Rollin et al., 2003). Similarly, although the application of ca-spase inhibitors is a popular way to interfere with apoptosis, more upstream targets should be advocated for pharmacological intervention. Specifically, as caspase-9 and -3 represent the post-mitochondrial phase of cell death their activation requires the release of cyto c and other pro-apo-ptotic substances. Such excessive mitochondrial damage also points to a considerable disruption of the electron transport chain leading to mass generation of free radicals as well as to altered energy homeostasis both facilitating the activation of ne-crotic processes in the cell. In traumatic axonal injury (TAI) the activation of caspases leads to irreversible digestion of the membrane skeleton and other cytoskeletal structures indicative of imminent, irreversible axonal demise (Wang et al., 1998; Buki et al., 2000) (Fig. 2). The relevance of such co-activation of the "necrotic" and "apopto-tic'' machinery was also demonstrated in an elegant study by Liu et al. (2006) in hippocampal lysates from rats which have undergone controlled cortical impact injury, where degradome-studies identified various common targets of calpain-2 and caspase-3 including beta-II-spectrin, synaptotag-min-1 and striatin. Similar "collaboration" between calpain and caspase-3 was also demonstrated by Warren et al. (2005) in metamphetamine induced experimental neurotoxicity.

Inhibition of apoptosis

Several studies with caspase-inhibitors corroborate — at least from some aspects — the above-described

Fig. 2. Images of damaged axonal profiles from the medial lemniscus of rat exposed to impact acceleration brain injury (60 min post-injury). Cytochrome c release from damaged mitochondria (immunofluorescent label on A) and caspase-3 activation (immunofluorescent label on B) are co-localised in the same damaged axons (digital overlay on C) pointing to the contribution of calcium-induced mitochondrial damage and caspase activation to the pathogenesis of traumatically induced diffuse brain injury (axonal injury).

Fig. 2. Images of damaged axonal profiles from the medial lemniscus of rat exposed to impact acceleration brain injury (60 min post-injury). Cytochrome c release from damaged mitochondria (immunofluorescent label on A) and caspase-3 activation (immunofluorescent label on B) are co-localised in the same damaged axons (digital overlay on C) pointing to the contribution of calcium-induced mitochondrial damage and caspase activation to the pathogenesis of traumatically induced diffuse brain injury (axonal injury).

critical attitude to the usefulness of inhibiting the effector-phase of apoptosis alone. Abrahamson et al. (2006) demonstrated that a pan-caspase inhibitor (BAF) significantly reduced the formation of caspase-cleaved amyloid precursor protein fragments also leading to a reduced volume of hippo-campal cell loss, however, in their experiment performed in mice applying the cortical impact model of injury significant necrotic areas where still noted in the cortex and the hippocampus. In another work, the pan-caspase inhibitor FK-011 was not able to achieve reduction in cortical lesion size measured 7 days post-injury (Sullivan et al., 2002). Although Knoblach et al.

(2004) were able to prove significant improvement of motor and neurological function using the pan-caspase inhibitor z-VAD-fmk, they also stress that this beneficial effect was purportedly also attributable to the inhibition of calpain that is co-inhibition of necrosis in the dose they applied. In their review Lockshin and Zakeri (2004) also stress that co-inhibition of lysosomal proteases may lead to overestimation of the efficacy of caspase-inhibition.

Keeping these warnings in mind one should conclude that inhibition of apoptosis alone might be "too little, too late''. Thus, the most promising way to interfere with the progression of the patho-biological processes evoked by/operant in TBI must either be the application of therapeutic agents with multiple targets like the above mentioned pan-caspase inhibitors or targeting pre-mitochondrial/mitochondrial events in the course of cellular demise.

As far as multitargetic therapeutic approaches are considered, sex steroids should be included to our list; several studies suggested that steroids could have beneficial effects both on neurons and glia. In ischemic and TBI sex steroids have been proven to inhibit necrotic as well as apoptotic cell death (Roof and Hall, 2000). Although the exact mechanism of their action still requires further investigation, sex steroids are thought to inhibit lipid peroxydation and the formation of ROS while also exerting anti-apoptotic properties facilitating the expression of anti-apoptotic proteins including bcl-2 and Bax (Soustiel et al., 2005). Similarly, estrogen treatment has a positive effect on oligoden-droglia function (Curry and Heim, 1966) and astrocytic scar formation (Garcia-Estrada et al., 1999). Testosterone can influence the expression of the astrocytic water channel protein aquaporin-4 indicating that sex steroids may also exert their action on the formation of brain edema — a major player in secondary brain injury (Gu et al., 2003).

In the last two decades several studies suggested that poly(ADP-ribose)polymerase-1 (PARP-1, EC 2.4.2.30) inhibition might be beneficial in the inhibition of CNS injury of various origin (Burkle, 2001; Besson et al., 2003, 2005). This enzyme was originally associated with DNA-repair function: stress induced strand DNA breaks activate PARP

that will transfer ADP-ribose units to nuclear proteins from NAD. This energy consuming process was recognized to lead to NAD depletion and loss of ATP leading to a collapse in cellular energy homeostasis and cell death (Burkle, 2001). While long-term inhibition of PARP could theoretically lead to mutagenesis and carcinogenesis, in the acute phase such intervention would be beneficial in terms of restoring/preserving the cellular energy pool. As it has recently been stressed by Komjati et al. (2005) PARP inhibition is not only interfering with ROS-generated necrosis. Beyond representing a potential pathway in initiating and/or modulating necrosis via AIF, PARP has a substantial role in the induction of apoptosis. Via the NF-kappaB-pathway PARP is also capable of influencing the expression of inflammatory cytokines and mediators (Komjati et al., 2005).

In the last few years several PARP inhibitors have been proven to inhibit TBI. In the lateral fluid percussion head injury model LaPlaca et al. (2001) have demonstrated that the PARP inhibitor GPI 1650 significantly reduced the lesion volume while not influencing the number of TUNEL positive cells.

Other experiments proved that PARP inhibitors were able to improve the neurological score of injured animals without reducing the extent of the cellular lesion (Besson et al., 2005). These data in conjunction with those results indicating that PARP inhibitors reduce the extent of TAI assessed by beta amyloid precursor protein immuno-reactivity implicate the inhibition of TAI in the neuroprotective effects of PARP inhibitors.

Fluid percussion TBI is not the only model where PARP inhibitors proved their neuroprotec-tive effect: 3-aminobenzanide significantly reduced the lesion size in cold injury (Hortobagyi et al., 2003) and proved beneficial functional effects (PJ34) as well as improved effectiveness of neural stem cell transportation (Lacza et al., 2003).

In the last decades, several studies implicated the cytosolic neutral cysteine protease calpain (EC 3.4.22.17) in the pathogenesis of both diffuse and focal TBI (Bartus, 1997; Kampfl et al., 1997; Buki et al., 1999b). This Ca++-induced enzyme is capable of reversibly cleaving spectrin and other constituents of the cytoskeleton (Wang et al.,

1998). While classic thought appreciated calpain as an agent contributing to necrotic processes several lines of evidences demonstrated that calpain can participate in the induction of the mitochondrial (intrinsic) pathway of apoptosis. This communication between the classic, Ca++-induced necrotic processes and the apoptotic enzyme system is partially executed that way that calcium accumulation leads to axolemmal permeability alteration through proteolytic modification of the subaxo-lemmal network (cortical cytoskeleton) which in turn at least in some neurons and in axons contributes to further membrane-permeability changes (Buki et al., 1999b). As it has been described in the above passages, excessive influx of Ca++ induces mitochondrial damage via MPTP-opening. In addition to this pathway, calpain itself is capable — as it has been demonstrated in liver — to open the MPTP via its direct proteolytic modification (Aguilar et al., 1996). Once MPTP is open and the intrinsic route, thus caspase-3 is activated, it can cleave calpastatin, the major inhibitor of calpain, contributing to further uncontrolled activation of calpain. Taking into consideration such interactions and the temporal/ causative relationship between calpain and caspase activation a logical way to influence the release of apoptotic enzymes should be the inhibition of calcium-mediated proteolytic alterations that is the inhibition of calpain.

Recent observations indicated that systemic administration of calpain inhibitors reduced the overall degree of the cerebral ischemia (Bartus, 1997). Further, Saatman et al. (1996) demonstrated that the use of calpain inhibitors resulted in improved behavioral outcome in a contusional model of TBI. Intriguingly enough, in an extension of this work Saatman et al. failed to detect any significant change in the size of contusion, concluding that the beneficial therapeutic effects of AK-295 could be explained by inhibiting TAI in remote areas (Saatman et al., 2000). The correctness of this assumption was further supported by the finding that in a rodent model of TAI the cell permeable peptidyl-aldehyde calpain inhibitor MDL-28170 (carbobenzylzoxy-Val-Phe-H) proved to prevent axonal injury in the brainstem fiber tracts (Buki et al., 2003). Most recent observations indicate that this agent is also potent in terms of reducing the length of axonal segments displaying traumatically induced axolemmal permeability changes frequently associated with the activation of the Ca++-induced protease calpain coupled in some axons to the initial, strain-induced axolem-mal mechanoporation (Buki et al., unpublished) (vide supra). Although physical and chemical properties of MDL-28170 do not favor its application in the clinical care, the family of such cell-permeable calpain-inhibitors that readily cross the blood brain barrier and exert their beneficial effects in a relatively wide therapeutic window (Bartus, 1997; Markgraf et al., 1998) should be considered candidates for further pharmacological modifications and studies.

Calpain inhibitors are of particular interest as recent reports demonstrated that besides decreasing the formation of cleavage products of this cysteine protease primarily participating in necro-tic processes, they are capable of inhibiting the formation of caspase-cleaved byproducts, thereby apoptosis too (Kawamura et al., 2005).

In the line of therapeutic approaches aiming to inhibit the mitochondrial/premitochondrial phase of apoptosis one of the most promising interventions is the use of the inhibitors of mPTP opening. Among these interventions the immunophilin lig-and cyclosporine A (CsA) has been used in a set of experiments targeting TAI in the rodent impact acceleration model of TBI. First, immuno-electron microscopic studies by Okonkwo and Povlishock (1999) demonstrated that CsA significantly attenuated mitochondrial swelling and rupture, translating into reduced numbers of damaged, disconnected axonal segments displaying immonu-reactivity for the transport protein beta amyloid precursor protein. Subsequent studies also proved that both pre- and post-injury administration of CsA significantly reduced the extent of TAI detected by immunohistochemical markers of calpain-mediated spectrin proteolysis, axonal disconnection and cytoskeletal alterations — neurofilament compaction (NFC). Dose response curves for the beneficial effects of CsA in the experimental model of TBI have also been established (Okonkwo et al., 2003). More recent studies also demonstrated that CsA limits/improves some aspects of functional outcome following TBI (Riess et al., 2001). Preliminary data regarding the use of CsA in head injury in man indicate that this compound is safe and potentially beneficial (Alves et al., 2003).

Another immunophilin ligand FK506, a substance that has no known effect on MPT and exerts its action via inhibition of calcineurin activity (Liu et al., 1991; Kuroda et al., 1999) has also displayed beneficial effects on TAI in a pre-injury administration paradigm (Singleton et al., 2001) and reduced the complications associated with rapid re-warming following hypothermic intervention for TAI (Suehiro et al., 2001).

Inhibition of the Ca++-induced phosphatase calcineurin is supposed to be beneficial in axonal injury by preventing dephosphorylization of the neurofilament side arms that should decrease the repelling forces between the neurofilaments thereby permitting NFC leading to impairment of axoplasmatic transport (Povlishock et al., 1997; Okonkwo et al., 1998). Such dephosphorylated neurofilaments would be more susceptible for pro-teolytic degradation by calpain and caspase (Pant, 1988). It is of note that calcineurine might also participate in the signaling pathways regulating proliferative responses of astrocytes and its inhibition by CsA as well as FK-506 diminishes as-trocytic reactions (Pyrzynska et al., 2001). Calcineurin also contributes to the control of synaptic activity by inactivating dynamin I and synapsin I, proteins participating in neurotrans-mitter release (Cousin and Robinson, 2001) and by dephosporylation-mediated induction of nitric oxide synthase leading to increased nitric oxide level and neurotransmitter release (Dawson et al., 1993).

Other important aspects of TBI might also be influenced by immunophyllin ligands. Specifically, CsA inhibited ischemia-induced edema in vitro, suggesting that such a beneficial effect should also be worth investigating in secondary brain injury where hypoperfusion-hypoxya-induced edema and tissue damage is an issue, too (MacGregor et al., 2003).

CsA also proved neuroprotective in the hands of Gabbita et al. (2005) in the controlled cortical impact model where it inhibited the TBI-induced increase of cleaved tau (cytoskeletal protein) level in the ipsilateral hippocampus and its accumulation in the CSF of injured rodents. Van Den et al. (2004) have demonstrated another beneficial effect of CsA in TBI proving that 30min post-injury administration of CsA reduced APP mRNA at 2 and 6 h post-injury and likewise reduced APP accumulation in injured cell bodies. Nevertheless, they did not prove axonal preservation in terms of APP-immunoreactivity in their experiments. While these results might reflect the complexity in the pathogenesis of TBI, it is of note that several studies have proved that APP immunoreactivity alone roughly underestimates the extent of TBI and, in some axon-populations other mechanisms than impaired axoplasmatic transport and axonal swelling should be held accountable for axonal demise (Singleton and Povlishock, 2004; Farkas et al., 2006).

While, without doubt, a continuous development of various anti-apoptotic or, even more promising, anti-apoptotic, anti-necrotic drugs should be a major goal of pharmaceutical research, neuroprotective substances physiologically produced in the brain itself should also be taken into account. To this end, a bulk of recently collected evidences point to the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP).

This member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family (Miyata et al., 1989) was discovered as a hypothalamic peptide on its potential of increasing adenylate cyclase activity in the pituitary gland and proved to exert various effects in the central and peripheral nervous systems including neurotrophic and neuropro-tective ones (Arimura, 1998; Vaudry et al., 2000). In the extradural static weight compression model of spinal cord injury in rat, post-injury PACAP treatment significantly reduced the number of apoptotic cells assessed by TUNEL in the spinal cord (Katahira et al., 2003).

Further, PACAP has been shown to eliminate the increase of TNF after spinal cord transection (Kim et al., 2000) and in experimental TBI postinjury induction of PACAP-mRNA and its upreg-ulation paralleled the decrease in the number of apoptotic cells (Skoglosa et al., 1999). These studies indicate that PACAP is a promising therapeutic agent in traumatic brain and spinal cord injuries, which is further supported by our observations.

Unfortunately, the exact mechanism underlying the in vivo neuroprotective effect of PACAP has not been clarified so far. In vitro studies assign both anti-apoptotic and anti-inflammatory actions to PACAP. In cerebellar granule cells, PACAP significantly inhibited the activation of caspase-3 (Vaudry et al., 2004). PACAP was proven to be a potent inactivator of induced microglial release of pro-inflammatory cytokines and nitric oxide (Kong et al., 1999; Kim et al., 2000; Delgado et al., 2003). It is also believed to influence mi-tochondrial integrity as PACAP inhibited the mi-tochondrial Ca++ uptake induced inactivation of aconitase, a key mitochondrial enzyme influencing the viability of neurons (Tabuchi et al., 2003).

In the last few years our laboratories extensively investigated the purported neuroprotective role of PACAP in a diffuse model of TBI. Intriguingly enough, we were not able to reproduce neuropro-tective effects in terms of preserving axonal integrity assessed by APP-immunoreactivity (vide supra) with the administration paradigm (125 mg pre-injury, iv) that had worked in a middle cerebral artery occlusion model of stroke (Reglodi et al., 2002). Nevertheless, we were able to establish the dose-response curve for intracerebroventricu-lar (icv) administration of PACAP, proving that 100 mg PACAP significantly reduced the density of damaged, immunoreactive axons in the cortico-spinal tract (Farkas et al., 2004). Our most recent observations proved that a considerable therapeutic window exists for post-injury PACAP treatment demonstrating significant axonal protection in an icv administration paradigm 2h following impact acceleration TBI (Tamas et al., 2006).

A new avenue of pharmacotherapy targeting apoptosis in TBI is represented by the inhibition of cell cycle proteins. Di Giovanni et al. (2005) demonstrated that the cell cycle inhibitor flavopir-idol was not only capable of inhibiting etoposide-induced, capase-mediated cell death in cultured primary cortical neurons but also inhibited the proliferation of astrocytes and microglia and decreased the extension of TBI-induced tissue lesion and improved functional recovery in the lateral fluid percussion rodent model.

As proteolytic processes are key players in TBI-evoked apoptosis, the list of potential therapeutic measures would not be complete without mentioning the neuroprotective capacity of therapeutic hypothermia. This intervention is purported to act via general inhibition or slowing of the proteolytic processes which could provide an extended therapeutic window for further, specific therapeutic approaches while also enabling the neurons to keep their mitochondria up and running and their local energy homeostasis intact (Buki et al., 1999a). Although the use of controlled hypothermia in the treatment of human TBI remains controversial, on the basis of multiple lines of evidence this therapeutic modality might be worth further investigations and probably further clinical studies planned on the basis of past experience could resolve the controversy concerning the role of hypothermia in the treatment of the severely head injured (Clifton, 2004). Specifically, on the basis of our current knowledge on its action, hypothermia should be introduced relatively earlier in the care of TBI paying special attention to gradual re-warming in conjunction with introduction of other treatment strategies such as FK-506 that has already proved its potential to inhibit diffuse brain injury associated with post-hypothermic rewarm-ing (Suehiro et al., 2001).

Concluding remarks

There are several controversies in the development of novel pharmacotherpeutic agents aimed at the inhibition of pathobiological processes evoked by TBI. While everybody in the field considers the treatment of TBI an extremely cost efficient medical procedure, we still lack any novel, efficient therapeutic agent. Despite of the promising results achieved with various pre-clinical studies all candidates failed at the clinical phase and so far none of them proved useful for the everyday practice. While detailed analysis of such failure is well beyond the scope of this review, the authors feel appropriate to note that more detailed understanding and appreciation of cell death mechanisms and experimental models applied should be a prerequisite for successful development of neuroprotec-tive agents. As far as study design is considered we should appreciate that the rather complex and versatile nature of TBI occurring in human is hard to copy in any experimental model existing currently in our hands. In the overwhelming majority of cases human TBI is a mixture of focal and diffuse injuries complicated with secondary brain injury primarily caused by hypoxia and hypoper-fusion. The most frequently used models like controlled cortical impact or moderate/severe forms of fluid percussion brain injury produce mass tissue destruction where strain and shearing-force distribution leads to excessive influx and release of Ca++, and excitatory amino acids, activation and generation of ROS. Although such models may mimic the situation in the most severe forms of human TBI, they ignite such a bulk of various pathways that could hardly be controlled with a drug exerting its effect on a single target.

While diffuse brain injury models seem to provide a better environment to study more subtle forms of TBI and theoretically provide a more convenient field to investigate the effect of pharmaceutical agents, recent observations shed light on the extremely complex and heterogenous nature of both diffuse axonal and neuronal injury. Specifically, in different fiber populations considerably different markers of axonal damage may appear representing heterogeneity in the pathobiology as well as in therapeutic efficacy (Stone et al., 2004). Similarly, acceleration-deceleration injury evokes various forms of neuronal-cellular damage indicating a likewise complex field to target therapeu-tically (Singleton and Povlishock, 2004; Farkas et al., 2006).

In light of the above passages and the diversity and interactivity of cell death pathways, a fatalistic approach would be to conclude that when a cell is committed to die due to an extrinsic or intrinsic death signal it will do so regardless of whatever therapeutic measures have been taken; inhibition of apoptosis will ignite autophagic or necrotic cell death, ultimately leading to cellular demise. Nevertheless, the authors still feel appropriate to address this very issue from a different point of view with the believe that combined therapeutic approaches or ''polyhparmacia'' might successfully target intermingled death pathways with resulting improved morphological as well as functional outcome. To this end one can envision a scenario where ultra-early (pre-hospital) application of therapeutic measures aimed to slow down proteolytic processes (such as hypothermia treatment) and preserving energy homeostasis (PARP-inhibition, CsA-treatment) is followed or joined by caspase and calpain inhibitors, or PACAP treatment.

As far as the title of the present article is considered, to date it is hard to predict, whether inhibition of apoptosis is useful; one can only conclude that it is most probably beneficial in conjunction with other measures taken to inhibit various different pathways of traumatically induced neural demise.

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