Inflammation & apoptosis in spinal cord injury

Inflammatory reactions after SCI

After spinal cord trauma, ruptured blood vessels disturb the blood-brain barrier and the injury site is rapidly infiltrated by blood-borne neutrophils. This process may contribute to the secondary damage that follows the initial primary injury. At 30-45 min post-SCI, tumour necrosis factor (TNF)-positive cells could be seen over the injured spinal cord segment and from 3 to 24 h, TNF-α and interleukin-6 (IL-6) were strongly upregulated around the contused area1011. The inflammatory cytokine mRNAs were shown to be induced as early as 15 min following contusion of rat spinal cord, with TNF-α increased first, followed by IL-6 mRNA1213. TNF-α could potentiate glutamate-mediated neuronal cell death in the rat spinal cord1415, while TNF antagonist reduced the development of inflammation and tissue injury events associated with SCI1617. Besides, IL-6 receptor monoclonal antibody treatment suppressed the astrocytic differentiation, decreased the number of inflammatory cells and the severity of connective tissue scar formation1819. In studies, hyper-IL-6 infusion induced a six-fold increase in the number of neutrophils, a two-fold increase in the areas of spinal tissue occupied by macrophages and activated microglia and a four-fold decrease in axonal growth at the lesion site2021.

Increased production of cytokines of the IL-1 family, such as IL-1α, is well documented, providing clear evidence for a pivotal role of this cytokine in triggering SCI-induced inflammatory processes22–24. IL-1α and IL-18 are potent mediators of inflammation and initiate and/or amplify a wide variety of effects associated with innate immunity, host responses to tissue injury, and microbial invasion. Moreover, it has been speculated that the inflammasome is kept in an inactive state in normal tissues by binding to a putative caspase-1 inhibitor, but the nature of this inhibitor has not been described25. The study showed that a molecular platform (NALP1 inflammasome) consisting of NALP1, adipose-derived stem cell (ASC), caspase-1, and caspase-11 was present in neurons of the normal rat spinal cord and formed a protein assembly with the inhibitor of apoptosis family member, X-linked inhibitor of apoptosis protein (XIAP). And SCI induced rapid processing of IL-1α and IL-18, activation of caspase-1, cleavage of XIAP, and promoted assembly of the NALP1 inflammasome. Further, neutralization of ASC reduced caspase-1 activation and XIAP cleavage and decreased processing of IL-1α and IL-18, leading to improved histopathological and functional outcomes after SCI26.

Central nervous system inflammatory responses that occur after SCI are initiated by peripherally derived immune cells, and activated glial cells that proliferate or migrate into the lesion site following injury27. T-cells are essential for activating macrophages and mounting a cellular or immune response. In rats, SCI activates myelin basic protein (MBP)-reactive T cells capable of causing neuron-inflammation and transient paralysis. In SCI, the frequency of MBP-reactive T cells increases, reaching levels that approximate those seen in multiple sclerosis (MS) patients. The pathogenic potential of SCI-activated B cells still remains to be directly tested, but early indications suggested that B cells also were pathological16. Data from other models also confirmed a direct link between primary CNS pathology and peripheral lymphocyte activation. Once lymphocytes gain access to the injury site, they persist indefinitely. Indeed, T and B cell numbers increase in the mouse SCI lesion through at least 9 wk post-injury1. Macrophages and neutrophils have also been proposed to participate in tissue destruction and enlargement of the lesion. Macrophages and microglia contribute to the secondary pathological and inflammatory response, in part through the release of cytokines, TNF, IL-1, IL-6, and IL-1028, interferon, and activation of interleukin receptors (IL-4R and IL-2R). Cytokines facilitate CNS inflammatory responses by inducing expression of additional cytokines, chemokines, nitric oxide (NO), and reactive oxygen. Based on the presence and position of the first cysteine residues, the chemokines have been divided into four subgroups, i.e., CC, XC, CX3C and CXC29.

Molecules acting as anti-inflammatory agents

It has been indicated that in various types of injuries, some molecules act as anti-inflammatory agents and regulate invasive migration of immune cells to the site of damage. Many of anti-inflammatory agents could have potential also in the elimination of the secondary damage after SCI6. Based on the observation that the protective effects of gluococorticoids were independent on their receptors, a new group of steroid analogues, lazaroids, were developed. These analogues inhibit lipid peroxidation without glucocorticoid/mineralocorticoid activity and in such a way avoid the complications of steroid therapy. Accordingly, aminosteroid lazaroid U-74389G reduced the production of systemic and spinal IL-8 (neutrophile attractant and activator) as well as systemic interleukin-1 receptor antagonist (IL-1ra) after SCI induced by aortic cross-clamping. This report indicates that lazaroid may attenuate ischaemic endothelial cell injury or activation of leukocytes effect30. Moreover, neutrophil infiltration to the site of injured spinal cord could be eliminated by specific and potent neutrophil elastase inhibitor ONO-504631. It was shown that spinal cord compression increased CINC-1 mRNA expression and protein synthesis. CINC-1 is a neutrophil chemoattractant and acute-phase protein induced by focal brain injury causing leukocyte mobilization and liver injury. This increase correlates with neurologic damage in injured rats. March et al32 showed that ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production. In addition, sphingosine-1-phosphate (Sph-1-P) could act as a specific and an effective motility regulator of human neutrophils. It was demonstrated that Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into human umbilical vein endothelial cells (HUVEC)-covered collagen layers, while no effect on their adhesion to HUVECs was observed. Although the mechanism of its action is not yet fully understood, this result indicates that Sph-1-P has the potential to be used as anti-inflammatory agent regulating invasive migration of neutrophils through endothelial layers at injured vascular site22.

A novel phosphoprotein, proliferation related acidic leucine-rich protein (PAL31) has been identified in the nervous system, and its expression gradually declines with the developmental process and is rarely expressed in the adult nervous system, including the spinal cord. In addition to the function in proliferation, PAL31 could act as a caspase-3 inhibitor, which might negatively regulate the expression of macrophage chemoattractant protein 1 (MCP-1) and signal transducer and activator of transcription-1 (STAT-1) and rescue macrophages from apoptosis during an inflammatory response33. Besides, alleviation of this damage-induced signal in the repair-model SCI rat showed a good correlation with better recovery of damage spinal cords, and PAL31 might behave like an inflammatory modulator in response to the regeneration process in SCI rats34. Most interestingly, knockdown of PAL31 in macrophages triggered apoptosis in cells stimulated with interferon (IFN-γ) or lipopolysaccharide (LPS), which suggested that PAL31 might play an important role in maintaining the survival of macrophage in the presence of inflammatory stress9.

Mediators of cellular apoptosis

SCI pathology results from complex interactions between different cell types and secreted molecules in a time-dependent manner. SCI leads to increased expression of death receptors and their ligands as well as activation of caspases and calpain.

Oxidants have, and continue to receive much attention as triggers of apoptosis. Studies have focused on the mechanisms by which H₂O₂ modulates the apoptotic pathway given the pivotal role that H₂O₂ plays in ischaemia/reperfusion injury to cerebral microvasculature and neuronal cells52. An integrated model of H₂O₂-mediated cellular apoptosis is unresolved although existing evidence implicates H₂O₂ in apoptosis initiation in both the mitochondrial and the death receptor signaling pathways. The more popular paradigm supports H₂O₂ as a mediator of mitochondrial membrane potential collapse that leads to the release of cytochrome c and the activation of caspase-9. Mitochondrial as well as extramitochondrial systems, such as cytoplasmic cytochrome P-450 and membrane bound NADPH oxidase are examples of physiologically relevant H₂O₂ sources52.

The glutathione/glutathione disulphide (GSH/GSSG) redox system is a major contributor to the maintenance of the cellular thiol redox status. Evidence showed that decrease in cell GSH was associated with enhanced cellular apoptosis while increases in GSH were associated with expression of the anti-apoptotic protein, Bcl-253. In more recent studies, they showed that it was the change in cellular GSH-to-GSSG ratio rather than changes in GSH per se that specifically mediated cell apoptosis and that this redox imbalance induced apoptosis was preceded by caspase-3 activation54. The two identified targets for redox control in apoptotic signaling are the mitochondrial permeability transition and caspases35.

Current evidence shows TNFα, a proinflammatory cytokine which is best known for its role in immune and vascular responses, can induce apoptosis in non-immune tissues via the death domain of its cell surface receptor, TNF-R1. However, there are conflicting reports as to the role of cell death in SCI that probably reflect the known capacity of TNF to be both pro- and anti-apoptotic54–56.

Fas-mediated neuronal and oligodendroglial apoptosis through the mitochondrial signaling pathway could be an important event that might ultimately contribute to demyelination, axonal degeneration and neurological dysfunction after SCI57. Preventing the activation of Fas-mediated cell death using neutralization of endogenous FasL is, therefore, a highly relevant neuroprotective approach, and warrants further investigation. Yu et al58 showed that Fas-mediated apoptosis could be amplified by the intrinsic mitochondrial pathway after SCI.

Inhibitors of apoptosis

To control aberrant caspase activation, which can kill the cell, additional molecules inhibit caspase-mediated pathways. Among these are proteins known as inhibitors of apoptosis. These inhibitors interact directly with modulators of cell death. For example, the X-linked inhibitor of apoptosis and the neuronal inhibitor of apoptosis are proteins in neurons that directly inhibit caspase-3 activity and protect neurons from ischaemic injury3959.

The inhibitor of apoptosis protein (IAP) family of anti-apoptotic proteins, which are conserved across evolution with homologues found in vertebrate and invertebrate species, have a key function in the negative regulation of programmed cell death in a variety of organisms. Several mammalian homologues (XIAP, cIAP-1, cIAP-2, NAIP, Bruce, Survivin, and pIAP) have been identified, most of which have been demonstrated to inhibit cell death. Although the biochemical mechanism by which IAP-family proteins suppress apoptosis is controversial, at least some of the human IAPs (XIAP, cIAP-1, and cIAP-2) have been reported to directly bind and inhibit certain caspases, including caspases-3, -7 and -9. Thus, IAPs can inhibit caspases within both the death receptor and mitochondrial pathways. During apoptosis induced by the TNF family member Fas, XIAP is cleaved, separating the BIR1-2 domains from the BIR3-Ring domain. The BIR1-2 fragment is capable of inhibiting active caspases-3 and -7, but it is turned over rapidly in cells. Thus, cleavage of XIAP may be a mechanism for lowering the threshold of caspase activity necessary for inducing apoptosis60.

Anti-inflammation strategies

Because multiple harmful substances are involved in the secondary SCI, it is unlikely that blocking one substance or mechanism would significantly prevent the course of secondary injury. Recently, several laboratories have shown remarkable protection and recovery of function in models of spinal cord injury using treatments that target components of the CNS inflammatory response.

A candidate molecule that could serve as a common or converging mediator for the secondary SCI is phospholipase A2 (PLA2)67, which could be induced by multiple harmful substances including inflammatory cytokines, free radicals68, and excitatory amino acids69–71, and its metabolic products are involved in multiple injury processes. Meanwhile, increased levels of PLA2 and their metabolites may also induce inflammation, oxidation, and neuron toxicity72, which could further exacerbate the injury.

Many of the pharmacologic approaches to SCI have been aimed at cell death caused by excitatory amino acids (EAAs). However, for a variety of possible reasons, clinical trials of EAA antagonists have not been efficacious73. The recently reported studies illustrate new therapeutic approaches that might be effective, at least in part, by interfering with the acute CNS inflammatory cascade. It was shown that injury induced the immune receptor CD95 and that blockade of this receptor produced a better recovery after experimental SCI in mice74. Meanwhile, an antibiotic tetracycline derivative, minocyline, that has anti-inflammatory and anti-apoptotic actions provided substantial sparing of both neurons and glial cells and also resulted in better neurological outcomes in two different SCI models in rats75–77. Another recent paper reports a novel effective treatment that reduces the NO release that is associated with acute inflammation after SCI78. The effects on apoptosis were manifest hours to days later, but there were also effects on lesion size, which were perhaps due to reductions in acute necrotic cell death79. The results of each study indicated a reduction of axonal ‘dieback’ and hinted at enhanced regeneration.

Lipid peroxidation (LP) is one of the most harmful mechanisms developed after SCI. Several strategies have been explored in order to control this phenomenon. Protective autoimmunity (PA) is in fact a new concept that refers to an innovative approach where autoreactive mechanisms are modulated in order to promote neuroprotection. In light of this concept, immunization with neural-derived antigens has shown to modulate this autoreactive response and to render protective and restorative effects after SC injury80–82. For instance, a study demonstrated that immunization with neural-derived peptides induces a better motor recovery compared to control animals83. The same study showed that this strategy improves neuronal survival and myelin preservation in SC-injured rats8485.

The use of minocycline, an antibiotic that reduces microglial activation, antibody blockade of the CD95 (FAS) ligand and the blockade of glycosphingolipid-induced iNOS (inducible nitric oxide synthase) has recently been shown to reduce neuronal and glial apoptosis with concomitant improvement in neurological function, and to enhance the efficacy of cell transplantation strategies79. Dexmedetomidine is a highly selective and potent adrenergic agonist that is increasingly being used as an adjunct for general anaesthesia8687. Use of dexmedetomidine as an anaesthetic adjunct does not change somatosensory or motor-evoked potential responses during complex spine surgery by any clinically significant amount88. Dexmedetomidine is found to be safe and effective in various neuraxial and regional anaesthetics in humans8990. Methylprednisolone is often used in the setting of acute SCI with anti-inflammatory properties that were thought to reduce spinal cord oedema91.