Immunohistochemical characterization of Fas (CD95) and Fas Ligand (FasL/CD95L) expression in the injured brain: relationship with neuronal cell death and inflammatory mediators.

Traumatic brain injury causes progressive tissue atrophy and consequent neurological dysfunction, resulting from neuronal cell death in both animal models and patients. Fas (CD95) and Fas ligand (FasL/CD95L) are important mediators of apoptosis. However, little is known about the relationship between Fas and FasL and neuronal cell death in mice lacking the genes for inflammatory cytokines. In the present study, double tumor necrosis factor/lymphotoxin-alpha knockout (-/-) and interleukin-6-/- mice were subjected to closed head injury (CHI) and sacrificed at 24 hours or 7 days post-injury. Consecutive brain sections were evaluated for Fas and FasL expression, in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; TUNEL), morphologic characteristics of apoptotic cell death and leukocyte infiltration. A peak incidence of TUNEL positive cells was found in the injured cortex at 24 hours which remained slightly elevated at 7 days and coincided with maximum Fas expression. FasL was only moderately increased at 24 hours and showed maximum expression at 7 days. A few TUNEL positive cells were also found in the ipsilateral hippocampus at 24 hours. Apoptotic, TUNEL positive cells mostly co-localized with neurons and Fas and FasL immunoreactivity. The amount of accumulated polymorphonuclear leukocytes and CD11b positive cells was maximal in the injured hemispheres at 24 hours. We show strong evidence that Fas and FasL might be involved in neuronal apoptosis after CHI. Furthermore, Fas and FasL upregulation seems to be independent of neuroinflammation since no differences were found between cytokine-/- and wild-type mice.

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.