Role of cerebral inflammation after traumatic brain injury: a revisited concept.

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 data 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.

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

The expression of the chemokines macrophage inflammatory protein (MIP)-2 and MIP-1alpha and of their receptors CXCR2 and CCR5 was assessed in wild type (WT) and TNF/lymphotoxin-alpha knockout (TNF/LT-alpha-/-) mice subjected to closed head injury (CHI). At 4 h after trauma intracerebral MIP-2 and MIP-1alpha levels were increased in both groups with MIP-2 concentrations being significantly higher in WT than in TNF/LT-alpha-/- animals (p < 0.05). Thereafter, MIP-2 production declined rapidly, whereas MIP-1alpha 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.

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.

sICAM-1 and TNF-alpha 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)-alpha 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-alpha exceeded normal values on 9 days. Stimulation of MVEC and astrocytes with TNF-alpha 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 (r(s) = 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-alpha. If added together, sICAM-1 and TNF-alpha synergistically induced MIP-2 production suggesting the involvement of two different pathways for MIP-2 regulation.

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-alpha (LT-alpha) (TNF/LT-alpha-/-) 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-alpha-/- 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-alpha-/- 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-alpha, and IL-6. Nevertheless, the increased posttraumatic mortality in TNF/LT-alpha-deficient mice suggests a protective effect of these cytokines by mechanisms that have not been elucidated yet.

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 patient's smoking habits.

Simultaneous detection of all four alkaline phosphatase isoenzymes in human germ cell tumors using reverse transcription-PCR.

We have developed a reverse transcription-PCR method that clearly distinguishes between the RNA transcripts of all four alkaline phosphatase (AP) genes. If compared to the methods used up to the present, the main advantages of the reverse transcription-PCR method presented are its specificity and high sensitivity. The germ cell AP and the placental AP, which are the two most closely related AP isoenzymes (98% homology), can clearly be distinguished without any interference by other AP isoenzymes. An enhanced expression of AP isoenzymes has been reported for various tumors. The examination of the pattern of AP isoenzyme expression in a specific tumor and the corresponding tissue of origin enables discrimination between eutopically and ectopically expressed isoenzymes and thus represents an important tool in the elucidation of AP isoenzymes as potential tumor markers. The pattern of AP expression in 15 germ cell tumors, 2 germinal epithelia adjacent to seminoma, 2 cell lines of germ cell tumor origin (Tera-1 and BeWo), and 5 normal testes was studied. In comparison to normal testes, in all seminomatous germ cell tumors eutopic expression of germ cell AP and ectopic expression of tissue-nonspecific AP were demonstrated. In both samples of pure embryonal carcinoma and in the embryonal carcinoma cell line, the transcription of all four mRNAs was shown. These results indicate that the expression of the isoenzymes depends on the degree of differentiation of a tumor and that a simultaneous up-regulation of all AP isoenzymes in all types of germ cell tumors does not exist.

Separation of the two most closely related isoenzymes of alkaline phosphatase by two-dimensional electrophoresis.

Seminoma is the most frequent testicular germ cell tumor. While effective curative treatment of the disease is available today, there is to date no tumor marker suited for the diagnosis and follow-up. Several authors have suggested that the germ-cell-specific isoenzyme of alkaline phosphatase (GCAP) might be valuable for this purpose. GCAP shows 98% sequence homology with the placental isoenzyme of alkaline phosphatase (PLAP). Both display a high degree of phenotypic heterogeneity. Until now all attempts to raise an antibody reacting specifically with GCAP have failed. Consequently there is no immunological assay that allows the measurement of GCAP in the presence of PLAP. Two-dimensional electrophoresis with a sigmoid immobilized pH-gradient of 3-10 for the first dimension makes it possible to differentiate clearly between these two closely related isoenzymes. Additionally, it resolves their many phenotypic variants. This is of special interest, since malignant transformation affects the glycosylation patterns of many glycoproteins. For the detection of GCAP and PLAP in two-dimensional electrophoresis it is essential to purify the raw tissue extracts thoroughly. A chromatographic method suited for this purpose is presented.