Microglial activation and intracerebral hemorrhage.

INTRODUCTION: Microglia activate upon injury, migrate to the injury site, proliferate locally, undergo morphological and gene expression changes, and phagocytose injured and dying cells. Cytokines and proteases secreted by these cells contribute to the injury and edema formed. We studied the injury outcome after local elimination/paralysis of microglia. METHODS: Adult male mice were subjected to intracerebral hemorrhage (ICH) by intra-caudate injection of either collagenase or autologous blood. Mice survived for different periods of time, and were subsequently evaluated for neurological deficits, size of the hematoma, and microglia activation. Mice expressing an fms-GFP transgene or the CD11b-HSVTK transgene were also used. For elimination of monocytes/macrophages, CD11b-HSVTK mice were treated with ganciclovir prior to hemorrhage. Modifiers of microglial activation were also used. RESULTS: Induction of ICH resulted in robust microglia activation and recruitment of macrophages. Inactivation of these cells, genetically or pharmacologically, pointed to a critical role of the time of such inactivation, indicating that their role is distinct at different time points following injury. Edema formation is decreased when microglia activation is inhibited, and neurological outcomes are improved. CONCLUSIONS: Microglia, as immunomodulatory cells, have the ability to modify the final presentation of ICH.

Microglial activation and recruitment, but not proliferation, suffice to mediate neurodegeneration.

Microglial activation occurs during excitotoxin-induced neurodegeneration. We have reported that microglia can exhibit neurotoxic behaviors after injection of excitotoxins into the hippocampus. It is not known, however, whether microglial proliferation, which is part of the activation response, is required for neurodegeneration to be observed, or whether activation of the pre-existing resident microglia suffices. Using osteopetrotic (op/op) mice, in which injury-induced microglial proliferation does not take place, we demonstrate that only the microglia initially residing in the CNS are adequate to promote neurodegeneration. Our data suggest that there is a threshold at which a maximal microglial contribution to neurotoxicity is observed. This threshold appears to be sufficiently low, such that activation of just 40% of the microglia present in wild-type mice serves to trigger neurodegeneration. Furthermore, since the decrease in microglial numbers coincides with a decrease in tissue plasminogen activator's activity, we suggest that tissue plasminogen activator can be used as a marker for microglial proliferation.

Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system.

Tissue plasminogen activator mediates excitotoxin-induced neurodegeneration and microglial activation in the mouse hippocampus. Here we show that tissue plasminogen activator (tPA) acts in a protease-independent manner to modulate the activation of microglia, the cells of the central nervous system with macrophage properties. Cultured microglia from tPA-deficient mice can phagocytose as efficiently as wild-type microglia. However, tPA-deficient microglia in mixed cortical cultures exhibit attenuated activation in response to lipopolysaccharide, as judged by morphological changes, increased expression of the activation marker F4/80 and the release of the pro-inflammatory cytokine tumor necrosis factor-(&agr;). When tPA is added to tPA deficient cortical cultures prior to endotoxin stimulation, microglial activation is restored to levels comparable to that observed in wild-type cells. Proteolytically-inactive tPA can also restore activation of tPA-deficient microglia in culture and in vivo. However, this inactive enzyme does not restore susceptibility of tPA-deficient hippocampal neurons to excitotoxin-mediated cell death. These results dissociate two different functions of tPA: inactive enzyme can mediate microglial activation, whereas proteolytically-competent protein also promotes neuronal degeneration. Thus tPA is identified as a new cytokine in the central nervous system.

Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus.

BACKGROUND: Injury to the brain induces dramatic local changes in gene expression, cellular morphology and behavior. Activation of microglial cells occurs as an early event after central nervous system (CNS) injury, but it has not been determined whether such activation plays a causal role in neuronal death. We have investigated this question using an excitotoxin-mediated brain injury model system, in conjunction with an endogenous peptide factor (macrophage/microglial inhibiting factor, MIF) that ablates microglial contribution to the cascade. RESULTS: Using MIF, we inhibited the microglial activation that normally follows excitotoxic injury. In cell culture studies, we found that such inhibition blocked the rapid release of microglia-derived tissue plasminogen activator (tPA), an extracellular serine protease made by both neurons and microglia, which we had previously identified as mediating a critical step in excitotoxin-induced neuronal death. Finally, infusion of MIF into the mouse brain prior to excitotoxic insult resulted in the protection of neurons from cell death. CONCLUSIONS: Our results demonstrate that microglia undertake a neurotoxic role when excitotoxic injury occurs in the CNS. They also suggest that the tPA released from microglia has a critical role in triggering neurodegeneration.

An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus.

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.