Acute plasmalemma permeability and protracted clearance of injured cells after controlled cortical impact in mice.

Cell death after traumatic brain injury (TBI) evolves over days to weeks. Despite advances in understanding biochemical mechanisms that contribute to posttraumatic brain cell death, the time course of cell injury, death, and removal remains incompletely characterized in experimental TBI models. In a mouse controlled cortical impact (CCI) model, plasmalemma permeability to propidium iodide (PI) was an early and persistent feature of posttraumatic cellular injury in cortex and hippocampus. In cortical and hippocampal brain regions known to be vulnerable to traumatic cell death, the number of PI+ cells peaked early after CCI, and increased with increasing injury severity in hippocampus but not cortex (P<0.05). Propidium iodide labeling correlated strongly with hematoxylin and eosin staining in injured cells (r=0.99, P<0.001), suggesting that plasmalemma damage portends fatal cellular injury. Using PI pulse labeling to identify and follow the fate of a cohort of injured cells, we found that many PI+ cells recovered plasmalemma integrity by 24 h and were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling negative, but nonetheless disappeared from injured brain by 7 days. Propidium iodide-positive cells in dentate gyrus showed significant ultrastructural damage, including plasmalemma and nuclear membrane damage or overt membrane loss, in all cells when examined by laser capture microdissection and transmission electron microscopy 1 to 24 h after CCI. The data suggest that plasmalemma damage is a fundamental marker of cellular injury after CCI; some injured cells might have an extended window for potential rescue by neuroprotective strategies.

Administration of interferon-alpha in mice provokes peripheral and central modulation of immune cells, accompanied by behavioral effects.

BACKGROUND: Interferon-alpha (IFN-alpha) is used in the treatment of many viral and malignant diseases. Although IFN-alpha administration is highly efficacious, treatment is often complicated by psychiatric side effects such as depression, which may require discontinuation of the therapy. Unfortunately, the mechanisms underlying IFN-alpha-induced depression are still not well understood. METHODS: In this study, we explored behavioral and immune effects of IFN-alpha administration in mice. BALB/c mice received daily intraperitoneal injections of 60,000 U/kg murine IFN-alpha for 8 days. Behavioral and immunological analysis was performed at least 15 h after injection to avoid any acute IFN-alpha effect. We monitored depression and anxiety-like behavior in mice using the Forced Swimming Test (FST), Tail Suspension Test (TST), and Elevated Plus Maze (EPM). Moreover, we studied the expression of adhesion molecules on peripheral blood leukocytes and analyzed the recruitment of lymphocyte subsets into the brain. RESULTS: IFN-alpha administration resulted in increased immobility of mice in the late phase of FST, without significant effects in TST and EPM. Increased percentages of natural killer cells and lymphocytes expressing LFA-1 or Mac-1 were observed in peripheral blood. The percentages of CD4+ and CD8+ lymphocytes as well as the percentages of LFA-1-expressing CD4+ and CD8+ lymphocytes were increased in the brains of IFN-alpha-treated mice. CONCLUSION: Our data suggest that IFN-alpha administration leads to an increase in peripheral blood cells with migratory potential, accompanied by an increased number of lymphocytes in brain, whilst the detectable modulation of the behavior was rather modest.

Pneumolysin causes neuronal cell death through mitochondrial damage.

Bacterial toxins such as pneumolysin are key mediators of cytotoxicity in infections. Pneumolysin is a pore-forming toxin released by Streptococcus pneumoniae, the major cause of bacterial meningitis. We found that pneumolysin is the pneumococcal factor that accounts for the cell death pathways induced by live bacteria in primary neurons. The pore-forming activity of pneumolysin is essential for the induction of mitochondrial damage and apoptosis. Pneumolysin colocalized with mitochondrial membranes, altered the mitochondrial membrane potential, and caused the release of apoptosis-inducing factor and cell death. Pneumolysin induced neuronal apoptosis without activating caspase-1, -3, or -8. Wild-type pneumococci also induced apoptosis without activation of caspase-3, whereas pneumolysin-negative pneumococci activated caspase-3 through the release of bacterial hydrogen peroxide. Pneumolysin caused upregulation of X-chromosome-linked inhibitor of apoptosis protein and inhibited staurosporine-induced caspase activation, suggesting the presence of actively suppressive mechanisms on caspases. In conclusion, our results indicate additional functions of pneumolysin as a mitochondrial toxin and as a determinant of caspase-independent apoptosis. Considering this, blocking of pneumolysin may be a promising cytoprotective strategy in pneumococcal meningitis and other infections.

TNF alpha and Fas mediate tissue damage and functional outcome after traumatic brain injury in mice.

Tumor necrosis factor-alpha (TNFalpha) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFalpha, Fas, or both (TNFalpha/Fas-/-), we hypothesized that TNFalpha and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFalpha/Fas-/- mice had improved motor performance from 1 to 4 days (P<0.05), improved spatial memory acquisition at 8 to 14 days (P<0.05), and decreased brain lesion size at 2 and 6 weeks after CCI (P<0.05). Protection in TNFalpha/Fas-/- mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFalpha before CCI, and TNFalpha-/- mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice (P<0.05). Tumor necrosis factor-alpha/Fas-/- mice had decreased the numbers of cortical cells with plasmalemma damage at 6 h (P<0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFalpha and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P<0.05 versus WT), suggesting that the beneficial effects of TNFalpha/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFalpha and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFalpha/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.

Traumatic brain injury in mice deficient in Bid: effects on histopathology and functional outcome.

Bid is a proapoptotic member of the Bcl-2 family that mediates cell death by caspase-dependent and -independent pathways. We tested mice genetically deficient in Bid in a controlled cortical impact (CCI) model to examine the hypothesis that Bid contributes to cell death and functional outcome after traumatic brain injury. After CCI, truncated Bid (15 kDa) was robustly detected in cortical brain homogenates of wild-type mice. Bid-/- mice had decreased numbers of cortical cells with acute plasmalemma injury at 6 h (wild type (WT), 1721+/-124; Bid-/-, 1173+/-129 cells/ x 200 field; P<0.01), decreased numbers of cells expressing cleaved caspase-3 in the dentate gyrus at 48 h (WT, 113+/-15; Bid-/-, 65+/-9 cells/ x 200 field; P<0.05), and reduced lesion volume at 12 days (Bid-/-, 5.9+/-0.4 mm(3); WT, 8.4+/-0.4 mm(3); P<0.001), but did not differ from WT mice at later times after injury regarding lesion size (30 days) or brain tissue atrophy (40 days). Compared with naïve mice, injured mice in both groups performed significantly worse on motor and Morris water maze (MWM) tests; however, mice deficient in Bid did not differ from WT in postinjury motor and MWM performance. The data show that Bid deficiency decreases early posttraumatic brain cell death and tissue damage, but does not reduce functional outcome deficits after CCI in mice.

Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways.

Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H2O2 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.

Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex.

Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain-specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24-72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI.

Cortical spreading depression activates and upregulates MMP-9.

Cortical spreading depression (CSD) is a propagating wave of neuronal and glial depolarization and has been implicated in disorders of neurovascular regulation such as stroke, head trauma, and migraine. In this study, we found that CSD alters blood-brain barrier (BBB) permeability by activating brain MMPs. Beginning at 3-6 hours, MMP-9 levels increased within cortex ipsilateral to the CSD, reaching a maximum at 24 hours and persisting for at least 48 hours. Gelatinolytic activity was detected earliest within the matrix of cortical blood vessels and later within neurons and pia arachnoid (> or =3 hours), particularly within piriform cortex; this activity was suppressed by injection of the metalloprotease inhibitor GM6001 or in vitro by the addition of a zinc chelator (1,10-phenanthroline). At 3-24 hours, immunoreactive laminin, endothelial barrier antigen, and zona occludens-1 diminished in the ipsilateral cortex, suggesting that CSD altered proteins critical to the integrity of the BBB. At 3 hours after CSD, plasma protein leakage and brain edema developed contemporaneously. Albumin leakage was suppressed by the administration of GM6001. Protein leakage was not detected in MMP-9-null mice, implicating the MMP-9 isoform in barrier disruption. We conclude that intense neuronal and glial depolarization initiates a cascade that disrupts the BBB via an MMP-9-dependent mechanism.