Viviane I. Otto

Role of cerebral inflammation after traumatic brain injury : A revisited concept

ABSTRACT— Neuroinflammation occuring after traumatic brain injury (TBI) is a complex phenomenon comprising distinct cellular and molecular events involving the injured as well as the healthy cerebral tissue. Although immunoactivation only represents a one of the many cascades initiated in the pathophysiology of TBI, the exact function of each mediator, activated cell types or pathophysiological mechanism, needs to be further elucidated. It is widely accepted that inflammatory events display dual and opposing roles promoting, on the one hand, the repair of the injured tissue and, on the other hand, causing additional brain damage mediated by the numerous neurotoxic substances released. Most of the date supporting these hypotheses derive from experimental work based on both animal models and cultured neuronal cells. More recently, evidence has been provided that a complete elimination of selected inflammatory mediators is rather detrimental as shown by the attenuation of neurological recovery. However, there are conflicting results reported on this issue which strongly depend on the experimental setting used. The history of immunoactivation in neurotrauma is the subject of this review article, giving particular emphasis to the comparison of clinical versus experimental studies performed over the last 10 years. These results also are evaluated with respect to other neuropathologies, which are years ahead as compared to the research in TBI. The possible reciprocal influence of peripheral and intrathecal activation of the immune system will also be discussed. To conclude, the future directions of research in the field of neurotrauma is considered.

Experimental closed head injury: analysis of neurological outcome, blood-brain barrier dysfunction, intracranial neutrophil infiltration, and neuronal cell death in mice deficient in genes for pro-inflammatory cytokines.

Cytokines are important mediators of intracranial inflammation following traumatic brain injury (TBI). In the present study, the neurological impairment and mortality, blood-brain barrier (BBB) function, intracranial polymorphonuclear leukocyte (PMN) accumulation, and posttraumatic neuronal cell death were monitored in mice lacking the genes for tumor necrosis factor (TNF)/lymphotoxin-α (LT-α) (TNF/LT-α−/−) and interleukin-6 (IL-6) and in wild-type (WT) littermates subjected to experimental closed head injury (total n = 107). The posttraumatic mortality was significantly increased in TNF/LT-α−/− mice (40%; P < 0.02) compared with WT animals (10%). The IL-6−/− mice also showed a higher mortality (17%) than their WT littermates (5.6%), but the difference was not statistically significant (P > 0.05). The neurological severity score was similar among all groups from 1 to 72 hours after trauma, whereas at 7 days, the TNF/LT-α−/− mice showed a tendency toward better neurological recovery than their WT littermates. Interestingly, neither the degree of BBB dysfunction nor the number of infiltrating PMNs in the injured hemisphere was different between WT and cytokine-deficient mice. Furthermore, the analysis of brain sections by in situ DNA nick end labeling (TUNEL histochemistry) at 24 hours and 7 days after head injury revealed a similar extent of posttraumatic intracranial cell death in all animals. These results show that the pathophysiological sequelae of TBI are not significantly altered in mice lacking the genes for the proinflammatory cytokines TNF, LT-α, and IL-6. Nevertheless, the increased posttraumatic mortality in TNF/LT-α-deficient mice suggests a protective effect of these cytokines by mechanisms that have not been elucidated yet.

Elevated intracranial IL-18 in humans and mice after traumatic brain injury and evidence of neuroprotective effects of IL-18-binding protein after experimental closed head injury.

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18–binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP—treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.

sICAM-1 and TNF-? induce MIP-2 with distinct kinetics in astrocytes and brain microvascular endothelial cells

The dysfunction of the blood‐brain barrier (BBB) occurring after traumatic brain injury (TBI) is mediated by intracerebral neutrophil accumulation, chemokine release (e.g., interleukin (IL)‐8) and upregulation of adhesion molecules (e.g., intercellular adhesion molecule (ICAM)‐1). In patients with severe TBI, we previously found that elevated cerebrospinal fluid (CSF) IL‐8 and soluble (s)ICAM‐1 correlate with BBB dysfunction, and this prompted us to concomitantly monitor IL‐8, sICAM‐1 and their stimulator tumor necrosis factor (TNF)‐α in CSF. Potential mechanisms for upregulation of the IL‐8 analogue, murine macrophage inflammatory protein (MIP)‐2, and sICAM‐1 at the BBB were studied using cultured mouse astrocytes and brain microvascular endothelial cells (MVEC). In CSF of seven patients, IL‐8 and sICAM‐1 were elevated for 19 days after severe TBI, whereas TNF‐α exceeded normal values on 9 days. Stimulation of MVEC and astrocytes with TNF‐α simultaneously induced the release of MIP‐2 reaching saturation by 4–8 hr and of sICAM‐1 increasing continuously from 2–4 hr to 12 hr. Augmented sICAM‐1 production correlated with enhanced membrane‐bound (m)ICAM‐1 expression in both cell types (rs = 0.96 and 0.90, P < 0.0001), but was markedly higher in astrocytes. The release of sICAM‐1 was not influenced by IL‐8 or MIP‐2, although astrocytes and MVEC expressed the IL‐8/MIP‐2 receptor (CXCR‐2) as determined by FACS analysis. Instead, we found that sICAM‐1 strongly induced MIP‐2 secretion by both cell types with kinetics differing from those evoked by TNF‐α. If added together, sICAM‐1 and TNF‐α synergistically induced MIP‐2 production suggesting the involvement of two different pathways for MIP‐2 regulation. J. Neurosci. Res. 60:733–742, 2000.

Upregulation of ICAM-1 and MCP-1 but not of MIP-2 and sensorimotor deficit in response to traumatic axonal injury in rats.

The pathophysiology of traumatic axonal injury (TAI) is only partially understood. In this study, we investigated the inflammatory response as well as the extent of neurological deficit in a rat model of traumatic brain injury (TBI). Forty‐two adult rats were subjected to moderate impact‐acceleration brain injury and their brains were analyzed immunohistochemically for ICAM‐1 expression and neutrophil infiltration from 1 hr up to 14 days after trauma. In addition, the chemotactic factors MIP‐2 and MCP‐1 were measured in brain homogenates by ELISA. For evaluating the neurological deficit, three sensorimotor tests were applied for the first time in this model. In the first 24 hr after trauma, the number of ICAM‐1 positive vessels increased up to 4‐fold in cortical and subcortical regions compared with sham operated controls (P < 0.05). Maximal ICAM‐1 expression (up to 8‐fold increase) was detected after 4 days (P < 0.001 vs. 24 hr), returning to control levels in all brain regions by 7 days after trauma. MCP‐1 was elevated between 4 hr and 16 hr post‐injury as compared with controls. In contrast, neither neutrophil infiltration nor elevation of MIP‐2, both events relevant in focal brain injury, could be detected. In all neurological tests, a significant deficit was observed in traumatized rats as compared with sham operated animals from Day 1 post‐injury (grasping reflex of the hindpaws: P < 0.001, vibrissae‐evoked forelimb placing: P = 0.002, lateral stepping: P = 0.037). In conclusion, after moderate impact acceleration brain injury ICAM‐1 upregulation has been demonstrated in the absence of neutrophil infiltration and is paralleled by a selective induction of chemokines, pointing out that individual and distinct inflammatory events occur after diffuse vs. focal TBI. J. Neurosci. Res. 63:438–446, 2001.

Interleukin-6 and its soluble receptor in serum and cerebrospinal fluid after cerebral trauma.

Interleukin-6 (IL-6) and its soluble receptor (sIL-6-R) were measured in cerebrospinal fluid (CSF) and serum of 11 severely head injured patients for up to 3 weeks following trauma. IL-6 increased immediately after injury displaying much higher concentrations in CSF than in serum (n = 11). Differently, median levels of sIL-6-R remained in the normal ranges being 10 times higher in serum than in CSF. However, increased amounts over control levels were found in CSF (n = 7) and intrathecal release of sIL-6-R was also suggested (n = 7). Although no correlation with the extent of cerebral lesion or with clinical outcome was evident, elevation of sIL-6-R in CSF supports a pivotal role for IL-6/sIL-6-R complex in the injured brain.

Regulation of chemokines and chemokine receptors after experimental closed head injury.

The expression of the chemokines macrophage inflammatory protein (MIP)-2 and MIP-1α and of their receptors CXCR2 and CCR5 was assessed in wild type (WT) and TNF/lymphotoxin-α knockout (TNF/LT-α−/−) mice subjected to closed head injury (CHI). At 4 h after trauma intracerebral MIP-2 and MIP-1α levels were increased in both groups with MIP-2 concentrations being significantly higher in WT than in TNF/LT-α−/− animals (p < 0.05). Thereafter, MIP-2 production declined rapidly, whereas MIP-1α remained elevated for 7 days. Expression of CXCR2 was confined to astrocytes and increased dramatically within 24 h in both mouse types. Contrarily, CCR5 expression remained constitutively low and was mainly localized to microglia. These results show that after CHI, chemokines and their receptors are regulated differentially and with independent kinetics.

The role of inflammation in neurologic disease

The central nervous system (CNS) requires an intact and peculiar environment in order to function properly. This homeostasis is maintained by the blood-brain barrier, which separates the CNS from the peripheral circulation. The ability of the CNS tissue to counteract the pathogenic effect of infectious agents is poor, because immune cells and humoral factors have no free access into the brain due to the formation of tight junctions by cerebral endothelial cells. In addition, under normal conditions the low expression of histocompatibility antigens, adhesion molecules, and immune mediators renders the CNS refractory to immune responses compared with peripheral organs. In addition, once inflammation within the nervous system occurs, it is difficult to control. Although inflammatory events are aimed at defending the CNS from pathogens and repairing lesioned tissue, they can also be deleterious by contributing to tissue damage and impairment of neurologic functions. Abundant research activity in this field has revealed that resident cells of the nervous system actively participate in intracranial immune defense by releasing inflammatory mediators able to regulate, neuronal functions in addition to immunological functions. The dichotomy of neuroinflammation is discussed in this review article, which reports diverse examples of chronic and acute diseases of the CNS in humans as well as in animal models.

Specific determination of germ cell alkaline phosphatase for early diagnosis and monitoring of seminoma: performance and limitations of different analytical techniques

Two-dimensional electrophoresis, ion-exchange chromatography and immunoassay were evaluated in order to improve the diagnostic specificity of the germ cell specific isoenzyme of alkaline phosphatase (GCAP) for the detection of seminoma. Assessment of GCAP is hampered by its structural heterogeneity and low serum concentration. The structural heterogeneity of GCAP from seminoma tissue could be clearly visualized by two-dimensional electrophoresis. We inferred that it depended on allelic amino acid substitutions, varying sialylation and differential cleavage of the membrane anchor. The allelic variability of GCAP affects the accuracy of immunological measurements. However, immunoassay was found to be the only technique sensitive enough to assess GCAP in serum. The elevated GCAP levels in 15% of healthy blood donors were shown to be correlated with smoking. Further studies clarifying how to interpret the values measured in smokers are prerequisite for the introduction of GCAP as a serum marker for seminoma. In the future, GCAP might be utilized for the detection of carcinoma in situ (CIS) cells in ejaculate. Assessment of the enhanced expression of cellular GCAP by CIS cells exfoliated into ejaculate could be a means for noninvasive, early diagnosis that presumably will not be hampered by the patients smoking habits.

Inflammatory Response to Brain Injury

Historically, it was believed that the central nervous system (CNS) fails to mount an inflammatory response due to its protection from the systemic immune system mediated by the blood-brain barrier (BBB). However, in the last decades, participation of neuronal and glial cells in inflammatory events occurring in infectious, autoimmune and degenerative neuropathologies became increasingly evident (Morganti-Kossmann et al. 2000). With regard to traumatic brain injury (TBI), it has been reported in clinical (Bell et al. 1997; Morganti-Kossmann et al. 1997; Holmin et al. 1998) as well as experimental studies (Taupin et al. 1993; Shohami et al. 1994; Fan et al. 1995; Fan et al. 1996; Carlos et al. 1997; Hans et al. 1999; Csuka et al. 2000) that, despite the presence of the BBB, a profound inflammatory response occurs within the brain immediately following injury. It is nowadays accepted that 1) immune surveillance is present in the CNS, 2) an immune response can be induced within the brain, 3) most of the molecules and mechanisms modulating the inflammatory response are present and active within the brain and 4) bi-directional communication exists between the nervous and the immune systems.