No abstract

Reactive oxygen and nitrogen species (RO/NS) such as nitric oxide (NO), hydroxyl radical (OH·), and superoxide anion (O2−) are generated in a variety of neuropathological processes and damage neurons. In the present study, we investigated the neuroprotective effects of rat astrocytes against RO/NS‐induced damage using neuron–glia cocultures, and the effects were compared to those of microglial cells. Sodium nitroprusside (SNP), 3‐morpholinosydnonimine (SIN‐1), and FeSO4 were used to generate NO, O2− and NO, and OH·, respectively. Solely cultured neurons, which were transiently exposed to these agents, degenerated, possibly through apoptotic mechanisms as revealed by in situ detection of DNA fragmentation, whereas neurons cocultured with either astrocytes or microglial cells were viable even after exposure to RO/NS. In contrast, most neurons cocultured with meningeal fibroblasts degenerated. Astrocyte‐conditioned medium partially attenuated RO/NS‐induced neuronal damage. When neurons were cultured on astrocyte‐derived extracellular matrix (AsECM), neuronal death induced by SNP and FeSO4 was almost completely inhibited. AsECM contained significant amounts of laminin and fibronectin, and pure fibronectin and laminin also protected neurons against RO/NS‐induced damage in the same manner as AsECM. These results suggest that astrocytes can protect neurons against RO/NS‐induced damage by secreting soluble and insoluble factors. GLIA 28:85–96, 1999. © 1999 Wiley‐Liss, Inc.

The apolipoprotein E4 isoform (apoE4) was initially identified as a susceptibility gene for the development of Alzheimer's disease, and has also recently been associated with poor outcome after acute traumatic and ischemic brain injury. One mechanism by which apoE may influence outcome in acute and chronic neurological disease is by downregulating glial activation and the neuroinflammatory response. Because it does not readily cross the blood-brain barrier (BBB), the apoE holoprotein has limited therapeutic potential. However, smaller peptides derived from the receptor binding region of apoE have been developed that mimic the functional anti-inflammatory and neuroprotective effects of the intact apoE protein. These apoE-derived therapeutic peptides cross the BBB and have been demonstrated to improve functional and histological outcomes in murine models of brain injury. Thus, the development of apoE-derived peptides represent a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

Microglia transform from ameboid to ramified cells during development and display an ameboid appearance again under certain pathological conditions. Some cytokines produced by astrocytes may be responsible for the microglial transformation. In the present study, we compared the effects of cytokines, granulocyte/macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor (M‐CSF), and interleukin‐3 (IL‐3) on the morphology of rat cultured microglia. For quantitative evaluation, we employed “transformation index” as calculated by (perimeter of cell)2/4 π (cell area). GM‐CSF facilitated the ramification of cultured rat microglia, which was effectively induced in a serum‐free medium. However, M‐CSF and IL‐3 did not induce the ramification. A certain serum adhesion protein (possibly vitronectin) as well as other high molecular weight substances in fetal calf serum inhibited the GM‐CSF‐induced microglial ramification. Among ordinary supplements for a chemically defined medium, progesterone, insulin, and a high concentration of glucose suppressed the ramification. These findings suggest that GM‐CSF may be involved in microglial ramification and that many kinds of supplements that are added to culture media profoundly affect the morphology of microglial cells. © 1996 Wiley‐Liss, Inc.

Apoptotic neuronal death is known to occur in the developing brain and in the mature brain of patients with ischemic and degenerative disorders. Although microglial cells are known to become activated in specific conditions, it has not been elucidated whether they enhance or prevent neuronal apoptosis. The present study was intended to observe how microglial cells are involved in neuronal death. When rat primary cortical neurons were incubated with a nitric oxide (NO) donor sodium nitroprusside (SNP; 300 microM) for 10 min, neuronal death occurred 12-16 hr later. The NO-induced neuronal death was inhibited by cycloheximide, and the SNP-treated neurons were characterized by nuclear fragmentation and intact cell membrane under electron microscopy. Agarose gel electrophoresis demonstrated DNA fragmentation of the SNP-treated neurons. Thus, the NO-induced neuronal death appeared to be apoptosis. When neurons were cocultured with rat primary microglial cells, the SNP treatment failed to induce the neuronal death. Because microglia-conditioned medium also prevented apoptotic neuronal death, microglial cells were considered to secrete antiapoptotic factors. The microglia-conditioned medium rescued neurons even when they were added to neuronal cultures after the SNP treatment, implying that the factors acted on neurons in a manner other than scavenging NO. Interleukin-3, interleukin-6, macrophage colony-stimulating factor, and basic fibroblast growth factor, which are known to be secreted by microglial cells, were not effective in preventing NO-induced neuronal death. Among microglia-derived substances, tumor necrosis factor alpha and plasminogen, which are heat-labile proteins, inhibited neuronal apoptosis. The neuroprotective action of the microglia-conditioned medium, however, still remained, even after it was heated. These findings suggest that microglial cells protect neurons against NO-induced lethal damage by secreting heat-labile and heat-stable neuroprotective factors in vitro.