Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro

Abstract

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.

Plasminogen inhibits TNFα-induced apoptosis in monocytes

“…67 Patients with severe type I plasminogen deficiency (plasminogen activity and antigen both extremely low) exhibit the neurologic developmental defect of symmetric internal hydrocephalus with a Dandy-Walker malformation, hypoplasia of the cerebellum, and a hypoplastic corpus callosum. 68 Because development of neurologic pathways requires apoptosis, the presence of this defect is consistent with defective apoptosis in these patients and mice.…”

Loss of Neurons in the Hippocampus and Cerebral Cortex of AMSH-Deficient Mice

“…Neuronal cells isolated from the hippocampus or cerebellar cortex were cultured as described above in the absence or presence of natural murine nerve growth factor (NGF) (Life Technologies, Inc.), recombinant human brain-derived neurotrophic factor (BDNF; PeproTech, Inc.), recombinant human transforming growth factor ␤1 (TGF-␤1; PeproTech, Inc.), and recombinant murine tumor necrosis factor alpha (TNF-␣; PeproTech, Inc.). To estimate the cell survival, Alamar blue fluorescent dye (Alamar Biosciences, Sacramento, Calif.), which is a redox indicator to assess viability and mitochondrial activity of the cells (40,41), was used by following a protocol manual for Fluoroskan Ascent (Labsystems). The culture method that we used induces the differentiation of neurons, during which mitochondrial numbers and activity in the individual neuronal cells increase.…”

Loss of Hippocampal CA3 Pyramidal Neurons in Mice Lacking STAM1

“…In this context, we demonstrated that STAM1 Ϫ/Ϫ primary hippocampal neurons were more sensitive to death induced by kainic acid and an NO donor in vitro than were wild-type neurons. Since both kainic acid (3,29) and NO donors (29,36,37) are known to induce neuronal cell death, it can be assumed that STAM1 may have a role in protection against these stresses in hippocampal neurons. The NO donor-induced neuronal apoptosis could be inhibited by antiapoptotic proteins, such as Bcl-2 and Bcl-X (36); however, when we investigated the expression of Bcl-2 and Bcl-X in STAM1…”

“…The cultured neurons were resuspended in Neurobasal medium plus B27 supplement but minus AO (Life Technologies) supplement, because B27 supplement contains some antioxidants. They were exposed to 150 M kainic acid or 50 M sodium nitroprusside (SNP) for 24 h and stained for 2 h with 10% Alamar blue dye (Alamar Biosciences, Sacramento, Calif.), which is a redox indicator, to assess the viability and metabolic activity of the cells (37,42). The light absorbance of the reduced form of the dye was measured at 540 nm, whereas the oxidized form was measured at 620 nm.…”