Axonal Injury
FIGURE 8-1
Categories of mechanisms proposed to be involved in the evolution of secondary damage after severe TBI in infants and chil-dren. Three major categories for these secondary mechanisms include (i) ischemia, excitotoxicity, energy failure, and cell death cascades; (ii) cerebral swelling; and (iii) axonal injury. A fourth category, inflammation and regeneration, contributes to each of these cascades. K potassium; EAA excitatory amino acids; Ca calcium; O2 superoxide; AA arachidonic acid;
BBB blood brain barrier; ICP intracranial pressure; CBV cerebral blood volume
TBI: PATHOBIOLOGY 83
extra-cerebral insults (i.e., hypotension, hypoxemia) from the injury scene through the intensive care unit (ICU).
Studies in models of TBI have begun to unravel the mechanisms producing secondary damage. Four categories of mechanisms can be defined (Figure 8-1), those associ-ated with (i) ischemia, excitotoxicity, energy failure, and resultant cell death cascades; (ii) secondary cerebral swelling; (iii) axonal injury; and (iv) inflammation and regeneration. Within each category, a constellation of medi-ators of secondary damage, endogenous neuroprotection repair, and regeneration are involved. The quantitative con-tribution of each mediator to outcome and the interplay between these mediators remains poorly defined.
A variety of methods have been used to study the evolution of secondary damage in human head injury including (i) the analysis of brain biochemistry and mol-ecular biology via of ventricular cerebrospinal fluid (CSF) drained in the treatment of intracranial hypertension, (ii) assessment of brain interstitial fluid by cerebral micro-dialysis, (iii) imaging techniques linked to assessment of cerebral blood flow (CBF) and cerebral metabolism, and (iv) the assessment of molecular markers in brain tissue obtained from patients treated with surgical decompres-sion for refractory intracranial hypertendecompres-sion. We discuss these studies and cite the clinical evidence supporting pro-posed mechanisms of secondary damage. It is impossible to address all of the mediators that may be involved;
however, key mechanisms will be considered.
POST-TRAUMATIC ISCHEMIA
Clinical studies in adults have indicated that soon after severe TBI, CBF is reduced and suggest that early post-traumatic ischemia might represent a therapeutic target (10,11). Clinical studies applying the stable xenon com-puted tomographic (CT) method of CBF assessment in the initial hours after severe TBI have been the most important in this regard. Early hypoperfusion or ischemia after severe TBI appears to represent a finding that is seen in most cases and is associated with poor outcome. The devastating consequences of secondary extra-cerebral insults early after injury (i.e., hypotension, hypoxemia) early post-trauma are also consistent with this possibility—
because a hypoperfused brain is at high risk and may be incapable of mounting an appropriate vasodilatory response during these added insults (12). This is not to sug-gest that secondary insults are limited to the field or emer-gency department. Secondary ischemic insults can also occur in the ICU. This was best described in the classic report of Gopinath et al., (13) who used a jugular venous catheter to identify episodes of jugular venous desat-uration (SjvO2 50 percent for more than 10 minutes) in the ICU in 116 patients with severe TBI. In that study, 46 of the 116 patients had at least one episode of
desaturation—suggesting ischemia. The causes of these episodes were either systemic such as hypotension or cere-bral such as refractory intracranial hypertension.
Episodes of desaturation were strongly associated with a poor neurological outcome. Just a single desaturation increased the incidence of poor outcome from 55 percent to 74 percent.
Numerous mechanisms may underlie the early post-traumatic hypoperfusion. Armstead reported reductions in the vasodilatory response to nitric oxide (NO), cGMP, cAMP, and prostanoids after experimental TBI in pigs, along with the release of superoxide anion (14,15). Also, greater injury-induced release of the potent vasoconstric-tor peptide endothelin-1 in the newborn versus the juvenile pig was posed to mediate the hypoperfusion (16). Others have suggested a loss of either endothelial NO production, or reduced responsivity to NO as mediating hypoperfusion.
Treatment with L-arginine (the substrate for NO production) improved CBF after TBI in rats (17). Similarly, treatment with L-arginine improved CBF and reduced con-tusion volume after TBI in rats (18). L-arginine is being tested in a clinical TBI trial in adults (personal communi-cation, C. Robertson, MD). Loss of vasodilators and elab-oration of vasoconstrictors, or other mechanisms, might be involved in producing early post-traumatic hypoperfusion (Figure 8-2).
Increases in metabolic demands, related to uptake of glutamate, as reflected by increases in brain tissue and CSF lactate, early after TBI have been reported in both models (19) and humans (20–22). Thus, reduced meta-bolic demands with a coupled CBF reduction in severely injured brain regions, early after injury, is an unlikely explanation for the hypoperfusion.
At more delayed times after injury (several hours to days), oxidative metabolism has been noted to be reduced to levels of ~50 percent of baseline for the
FIGURE 8-2
Schematic outlining putative mediators involved in the pro-duction of early post-traumatic hypoperfusion and/or ischemia after severe TBI. NOS NO synthase. (See text for details.)
majority of the ICU course (20). The complex issue of issue alterations in metabolic demands after severe TBI is discussed in greater detail in the section on brain swelling later in this chapter.
EXCITOTOXICITY
Excitoxicity describes the process by which glutamate and other excitatory amino acids (EAAs) cause neuronal damage. Lucas and Newhouse (23) first described the tox-icity of glutamate. Olney (24) subsequently reported that intraperitoneal administration of glutamate produces brain injury. Although glutamate is the most abundant neurotransmitter in the brain, exposure to toxic levels produces neuronal death (25).
Glutamate exposure produces neuronal injury in two phases. Minutes after exposure sodium-dependent neu-ronal swelling occurs (26). This is followed by delayed,
calcium-dependent degeneration. These effects are medi-ated through both ionophore-linked receptors, labeled according to specific agonists (N-methyl-D-aspartate [NMDA], kainate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA]), and receptors linked to second messenger systems, called metabotropic recep-tors. Activation of these receptors leads to calcium influx through receptor-gated or voltage-gated channels, or through the release of intracellular calcium stores.
Increased intracellular calcium concentration is the trigger for a number of processes that can lead to cellular injury or death (Figure 8-3). One mechanism involves activation of constitutive NO synthase, leading to NO production, peroxynitrite formation and resultant DNA damage.
Poly(ADP-ribose) polymerase (PARP) is an enzyme oper-ative in DNA repair, and in the face of DNA damage, PARP activation leads to ATP depletion, metabolic failure and cell death (27). This may be important since PARP knock-out mice exhibit improved knock-outcome versus controls (28).
FIGURE 8-3
Mechanisms involved in excitotoxicity. Glutamate causes an increase in intracellular calcium concentration through stimula-tion of (i) the NMDA receptor with opening of the receptor-linked calcium ionophore, (ii) the AMPA receptor with opening of the voltage-gated calcium channels, and (iii) the metabotropic receptor, with the release of intracellular calcium stores via the second messengers inositol triphosphate and diacylglycerol. Increased intracellular calcium concentration leads to activation of proteases, lipases and endonucleases, along with neuronal NOS stimulation and production of oxygen radicals. This results in peroxynitrite formation, mitochondrial damage and DNA injury with subsequent cellular injury and death.
Abbreviations: GLY: glycine coagonist site; NMDA: N-methyl-D-aspartate receptor; AMPA: a-amino-3-hydroxy-5-methylisoazole-4-proprionic acid receptor; METAB: glutamate metabotropic receptor; PIP2: phosphoinositide; IP3: inositol triphosphate;
DG: diacylglycerol
TBI: PATHOBIOLOGY 85
Recent evidence suggests that activation of PARP within mitochondria may contribute importantly to their failure (29).
Faden et al. (30) first reported an increase in intersti-tial EAAs to neurotoxic levels after experimental TBI. Anti-excitotoxic therapies improve outcome after experimental TBI. Pretreatment with NMDA antagonists (phencyclidine, MK-801) attenuate behavioral deficits after TBI in rats (31, 32). Other therapies that modify the glutamate-NMDA receptor interaction and improve outcome following exper-imental TBI are magnesium (33), glycine site antagonists (34), hypothermia (35), and pentobarbital (36).
Palmer et al. (37) first demonstrated increased con-centrations of EAAs in ventricular CSF from adult patients with TBI. Glutamate concentrations were about 5-fold greater than in control patients (up to 7 M)—
levels sufficient to cause neuronal death in cell culture (38). However, CSF glutamate concentrations do not cor-relate to outcome after TBI in adults (39). Bullock et al.
(40) characterized patterns of glutamate release by mea-suring EAAs by microdialysis after adult TBI. Patients with a normal head CT and no secondary ischemic events had interstitial concentrations of glutamate that were increased early in their course, then returned to normal, similar to the pattern seen in most experimental models.
A second group of patients had an intermediate increase in glutamate concentration (5–20 M) that declined over time, but remained higher than normal. Most of these patients had ischemic events or intracranial hypertension.
A third group of patients had markedly increased con-centrations of glutamate (over 20 M). All patients with a progressively rising level of glutamate died.
Despite these findings, clinical trials with anti-excitotoxic therapies have been unsuccessful. This may be due to the fact that most therapies have been applied to all patients with TBI rather than those with excitotoxicity (41). Also, treatment may have been initiated too late. Inhi-bition of plasticity by anti-excitotoxic therapies may limit their efficacy—especially at the interfact between the acute and subacute periods after injury (42).
ENDOGENOUS NEUROPROTECTANTS Ischemia, excitotoxicity, or their combination is a key facet of secondary injury. These mechanisms are linked to calcium overload, oxidative stress, and mitochondrial failure. Studies have begun to define, in infants and chil-dren with severe TBI, the endogenous retaliatory response to these ischemic and excitotoxic insults. Space limita-tions have directed us to focus on two examples of this cascade—namely, adenosine and heat shock protein 70 (HSP 70). Endogenous neuroprotective responses related to the apoptosis, cell signaling, and inflammatory cas-cades are discussed later.
Adenosine is an endogenous neuroprotectant pro-duced in response to both ischemia and excitotoxicity.
Adenosine antagonizes a number of events thought to mediate neuronal death (43). Breakdown of adenosine triphosphate (ATP) leads to formation of adenosine, a purine nucleoside that decreases neuronal metabolism and increases CBF, among other mechanisms. Adenosine binding to A1 receptors decreases metabolism by increas-ing Kand Cland decreasing Caconductances in the neuronal membrane. A1 receptors are located on neurons in brain regions that are susceptible to injury (i.e., hip-pocampus) and are spatially associated with NMDA receptors (44). Thus, released adenosine minimizes exci-totoxicity. Binding of adenosine to A2 receptors (on cere-brovascular smooth muscle) causes vasodilation, although binding to A2a receptors on neurons may be detrimental. Brain interstitial levels of adenosine are increased early after TBI in rats (45–47). In experimen-tal TBI, brain interstitial adenosine increases immediately after injury to levels 50- to 100-fold greater than base-line (47). In clinical studies, marked increases in brain interstitial levels of adenosine in adults with TBI, were seen during episodes of jugular venous desaturation (secondary insults), supporting a role of adenosine as a
“retaliatory” defense metabolite (48).
Another putative endogenous neuroprotectant that plays a role after severe TBI is HSP 70. This protein is induced as part of the classic preconditioning response in brain and has recently been shown to be increased in both CSF and brain tissue after severe TBI in humans (49–51). HSP 70 is believed to play an important role in optimizing protein folding as a molecular chaperone. It also inhibits pro-inflammatory signaling (52). Thus, the brain mounts an important endogenous defense response to TBI. Therapies designed to augment these pathways have not been examined adequately.
APOPTOSIS CASCADES
It is now increasingly clear from experimental models and human data that cells dying after TBI can be categorized on a morphological continuum ranging from necrosis to apoptosis (53,54). Apoptosis is a morphological descrip-tion of cell death defined by cell shrinkage and nuclear condensation, internucleosomal DNA fragmentation, and the formation of apoptotic bodies (55). In contrast, cells dying of necrosis display cellular and nuclear swelling with dissolution of membranes. Apoptosis requires a cas-cade of intracellular events for completion of cell death;
thus, “programmed-cell death” is the currently accepted term for the process of cell death that leads to apoptosis (56). In diseases with complex and multiple mechanisms, such as TBI, it is typically difficult to distinguish clinical apoptotic vs. necrotic cell death as classically defined (57).
Some cells may display DNA fragmentation and activa-tion of proteases involved in programmed-cell death, despite having nuclear and cellular swelling. Dying cells with mixed phenotypes may represent particularly diffi-cult therapeutic targets after TBI.
In mature tissues, programmed-cell death requires ini-tiation via either intracellular or extracellular signals (see Figure 8-4). These signals have now been well character-ized in vitro, and are becoming better charactercharacter-ized in vivo.
Intracellular signaling appears to be initiated in mitochon-dria, triggered by disturbances in cellular homeostasis such as ATP depletion, oxidative stress, or calcium fluxes (58).
Mitochondrial dysfunction leads to egress of cytochrome c from the inner mitochondrial membrane into the cytosol.
Cytochrome c release can be blocked by anti-apoptotic members of the bcl-2 family (e.g., bcl-2, bcl-xL, bcl-w, and Mcl-1), and promoted by pro-apoptotic members of the bcl-2 family (e.g., bax, bcl-xS, bad, and bid) (59).
Cytochrome c in the presence of dATP and a specific apoptotic-protease activating factor (Apaf-1) in cytosol activates the initiator cysteine protease caspase-9 (60). Cas-pase-9 then activates the effector cysteine protease caspase-3, a key apoptosis effector that cleaves cytoskeletal proteins, DNA repair proteins, and activators of endonucleases (61).
An additional intracellular cascade of programmed cell death linked to mitochondrial injury is the apoptosis-inducing factor (AIF) pathway (62–66). This caspase-independent pathway is activated by mitochondrial permeability transition and results in the release of AIF from the mitochondrial membrane. AIF release leads to large-scale DNA fragmentation (50–700 kilo-base-pair in size). Recently, Zhang et al. (67) reported that the AIF pathway is activated in experimental TBI. To date, spe-cific pharmacologic inhibitors of this pathway are lack-ing, however, this alternative form of delayed neuronal death may represent an important therapeutic target.
FIGURE 8-4
Simplified schematic depicting intracellular and extracellular pathways for programmed-cell death. Mitochondrial dysfunction caused by injurious stimuli such as oxidative stress or calcium fluxes can trigger release of cytochrome c or apoptosis inducing factor (AIF). Cytochrome c in the cytosol along with other enzymes and cofactors initiates activation of a cascade of caspases, culminating in apoptosis, with nucleosomal DNA cleavage. AIF triggers caspase-independent large-scale DNA fragmentation (see text for details). Programmed cell death can also be initiated by cell death receptors on the cell surface. Fas-ligand (Fas-L), either presented by an effector cell or in soluble form, binding to Fas-receptor (Fas-Rc), or TNF binding to TNF-receptor (TNF-Rc), can also initiate a cascade of caspases via intracellular death domains
TBI: PATHOBIOLOGY 87
Extracellular signaling of apoptosis occurs through the TNF superfamily of cell surface death receptors which include TNFR1 and Fas/Apo1/CD95 (68). Receptor-ligand binding of TNFR1-TNF or Fas-FasL promotes formation of a trimeric complex of TNF- or Fas-associated death domains, respectively. These death domains contain caspase recruitment domains. The proximity of multiple caspases, in this case caspase-8, allows for activation of the effector cysteine protease followed by activation of caspase-3, where the mitochondrial- and cell death receptor-pathways converge. The cell death receptor path-way can also be regulated by soluble receptors and ligands that prevent and promote apoptosis, respectively, and by receptors lacking death domains. Finally, there is cross-talk between mitochondrial- and cell death receptor-pathways (69).
Bcl-2 is an important endogenous inhibitor of programmed-cell death in vitro (70). It is induced after experimental TBI (71) and reduces cortical tissue loss (72).
Bcl-2 is increased in injured brain after severe TBI in humans (54). CSF levels of bcl-2 were increased ~4-fold in TBI compared with control patients. Moreover, CSF bcl-2 was associated with patient survival (73).
Clearly there is now substantial evidence, even in the clinical setting, for an important role for delayed neuronal death by apoptosis or mixed “apo-necrotic” phenotypes after severe TBI. This may represent a valuable opportu-nity for the development of new therapeutic approaches in the future.
CELL SIGNALING ABNORMALITIES IN NEURONAL DEATH
Neuronal death occurs after both experimental and clini-cal TBI. In addition to regions of brain directly contused, the hippocampus appears particularly vulnerable to TBI (74–78). As previously discussed, cell death execution path-ways are activated by a sufficient severity of TBI involving mitochondrial injury, cytochrome C release with caspase activation, AIF release, and receptor-coupled pro-death pathways. Neurotransmitters, neurotrophins, cytokines, other growth factors, and oxidative stress activate multiple upstream signaling pathways linked to either pro-survival or pro-death activities (79). These receptors couple to sig-nal transduction pathways involving interactions and cross-talk between multiple serine/threonine and tyrosine protein kinase cascades.
Many kinases involved in cell death process are ser-ine/threonine protein kinases. Important participants in the cell death cascades include the mitogen activated protein kinases (MAPK). MAPKs cascades are complex and are mediated by successive protein kinases that sequentially activate each other by phosphorylation.
They are importantly linked to two key components of
the cell death cascade, jun kinase (JNK) and P38 MAPK (Figure 8-5). JNK and p38 MAPK pathways activate cas-pase-3 (80–82). Activation of JNK leads to induction of pro-death genes including FasL (81–83). JNK increases p53 and Bax levels which increase cell death. JNK and p38 function in different stress signaling pathways and both target similar nuclear transcription factors that can be activated by pro death stimuli such as oxidative stress (84). Studies in various TBI models have docu-mented significant changes in both JNK, and p38 MAPKs that may be related to cell death and functional impair-ment after injury (85–87). MAPKs are also linked to sur-vival signals through the ERK pathway, highlighting the complex cross-talk between these cascades (Figure 8-5).
Several protein kinase cascades play a major survival role. Phosphoinositide 3-kinase (PI3-K), protein kinase B (PKB), and protein kinase A (PKA) pathways are proto-type examples (Figure 8-6). PKB is also called akt; the complex nomenclature of these kinases has evolved across many disease processes. PKB is activated upstream by PI3K in response to survival signals, and have numerous pro-survival, growth, differentiation and synaptic plas-ticity actions (88, 89). PKB affects survival by a number of mechanisms including the phosphorylation and inac-tivation of several pro-death mediators such as Bad. Bad, a member of the Bcl-2 family, is phosphorylated by PKB at ser136 resulting in Bad dissociation from Bcl-xL and binding to 14-3-3 proteins inhibiting cell death