By using an animal model in which inflammatory cytokines are induced in lipopolysaccharide (LPS)-injected rat brain, we investigated the induction of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and IL-6. Immunoblotting and immunohistochemistry revealed that all three cytokines were transiently induced in the cerebral cortex at about 12 h after LPS injection. To clarify which glial cell type induced the cytokines, we examined the respective abilities of astrocytes and microglia in vitro. Primary microglia largely induced TNFα, IL-1β and IL-6 in response to LPS, but primary astrocytes induced only limited levels of TNFα. Thus, we used specific inhibitors to focus on microglia in surveying signaling molecules involved in the induction of TNFα, IL-1β, and IL-6. The experiments using mitogen-activated protein kinases (MAPK) inhibitors revealed that c-Jun N-terminal kinase (JNK)/p38, external signal regulated kinase (ERK)/JNK, and ERK/JNK/p38 are necessary for the induction of TNFα, IL-1β, and IL-6, respectively. The experiments using protein kinase C (PKC) inhibitor clarified that PKCα is required for the induction of all these cytokines in LPS-stimulated microglia. Furthermore, LPS-dependent IL-1β/IL-6 induction was suppressed by pretreatment with a nitric oxide (NO) scavenger, suggesting that NO is involved in the signaling cascade of IL-1β/IL-6 induction. Thus, an inducible NO synthase induced in the LPS-injected cerebral cortex might be related to the induction of IL-1β/IL-6 through the production of NO in vivo. Taken together, these results demonstrated that microglia induce different kinds of inflammatory cytokine through specific combinations of MAPKs and by the presence or absence of NO.

No abstract

Transection of the rat facial nerve leads to a variety of alterations not only in motoneurons, but also in glial cells and inhibitory neurons in the ipsilateral facial nucleus. In injured motoneurons, the levels of energy metabolism-related molecules are elevated, while those of neurofunction-related molecules are decreased. In tandem with these motoneuron changes, microglia are activated and start to proliferate around injured motoneurons, and astrocytes become activated for a long period without mitosis. Inhibitory GABAergic neurons reduce the levels of neurofunction-related molecules. These facts indicate that injured motoneurons somehow closely interact with glial cells and inhibitory neurons. At the same time, these events allow us to predict the occurrence of tissue remodeling in the axotomized facial nucleus. This review summarizes the events occurring in the axotomized facial nucleus and the cellular and molecular mechanisms associated with each event.

Axotomy of the rat facial nerve causes downregulation of motoneuron-specific molecules, including choline acetyltransferase and the vesicular acetylcholine transporter, in surviving motoneurons. Subsequently, resident microglia are activated and proliferate. These cellular responses are thought to promote the survival, repair and regeneration of motoneurons. However, it is still unclear which signaling molecules are involved in these responses. In this study, we investigated the changes and localizations of several signaling molecules, including immediate early genes (IEGs) such as c-Jun and c-Fos, transcription factors such as cAMP responsive element binding protein (CREB) and activating transcription factor 2 (ATF2), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38. Immunoblotting and immunohistochemical analyses revealed the following. Among the IEGs, c-Jun was increased in injured motoneurons, but c-Fos did not respond to neuronal injury. Among the CREB/ATF family members, phosphorylated CREB (p-CREB) was significantly decreased in injured motoneurons. The levels of p-CREB/CREB and ATF2 were immunohistochemically increased in microglia. Among MAPKs, p-ERK1/2 and p-JNK1 were decreased in injured motoneurons at the late stage. p-p38 and p38 were markedly increased in microglia. In vitro experiments revealed that p38 and CREB were activated in proliferating microglia. These results strongly suggested that c-Jun is involved in the survival, repair and regeneration of motoneurons, but p-CREB/CREB, p-ERK/ERK and p-JNK/JNK are associated with the downregulation of motoneuron-specific molecules. On the other hand, p-p38/p38 and p-CREB/CREB were suggested to be closely involved in the activation/proliferation of microglia.

In our previous study, we reported that the levels of m2 muscarinic acetylcholine receptor and gammaaminobutyric acid receptor α1 in motoneurons were downregulated in the axotomized adult facial nucleus. These results led us to speculate that the phenomenon was attributable to an interruption of the retrograde supply of glial cell line-derived neurotrophic factor (GDNF). To investigate this possibility, in this study we determined the levels of GDNF in the adult rat axotomized facial nucleus. Western blotting revealed that the amounts of GDNF in the transected facial nucleus significantly decreased from 6 h to 7 d post-insult and returned to control levels at 3-5 weeks post-insult. Immunohistochemical study of the facial nucleus at 3 d post-insult indicated that many cells were positively stained by anti-GDNF antibody in the control facial nucleus, but the degree of staining was lower in the axotomized facial nucleus. The anti-GDNF antibody-stained cells in the facial nucleus were almost coincident with the anti-NMDA receptor 3B subunit (NR3B) antibody-positive cells, suggesting that the former were motoneurons. Together, these results demonstrated that GDNF levels in injured facial motoneurons transiently decreased from 6 h to 7 d post-insult but subsequently returned to the control levels.

Although microglia exist as a minor glial cell type in the normal state of the brain, they increase in number in response to various disorders and insults. However, it remains unclear whether microglia proliferate in the affected area, and the mechanism of the proliferation has long attracted the attention of researchers. We analyzed microglial mitosis using a facial nerve transection model in which the blood–brain barrier is left unimpaired when the nerves are axotomized. Our results showed that the levels of macrophage colony-stimulating factor (M-CSF), cFms (the receptor for M-CSF), cyclin A/D, and proliferating cell nuclear antigen (PCNA) were increased in microglia in the axotomized facial nucleus (axotFN). In vitro experiments revealed that M-CSF induced cFms, cyclin A/D, and PCNA in microglia, suggesting that microglia proliferate in response to M-CSF in vivo. In addition, M-CSF caused the activation of c-Jun N-terminal kinase (JNK) and p38, and the specific inhibitors of JNK and p38 arrested the microglial mitosis. JNK and p38 were shown to play roles in the induction of cyclins/PCNA and cFms, respectively. cFms was suggested to be induced through a signaling cascade of p38-mitogen- and stress-activated kinase-1 (MSK1)-cAMP-responsive element binding protein (CREB) and/or p38-activating transcription factor 2 (ATF2). Microglia proliferating in the axotFN are anticipated to serve as neuroprotective cells by supplying neurotrophic factors and/or scavenging excite toxins and reactive oxygen radicals.

No abstract

Previously we reported that endotoxin-dependent induction of tumor necrosis factor alpha (TNF expression in microglia was significantly suppressed by the superoxide anion scavenger N-acetyl cysteine (NAC), and that microglia induced TNF in response to a superoxide anion donor 3-(4-morpholinyl)sydnonimine (SIN-1) in vitro. Those findings strongly suggested that superoxide anion is associated with the induction of TNF in microglia. However, whether TNF is actually induced in microglia in vivo remains to be determined. In the present study, we confirmed the ability of microglia to induce TNF in vitro and examined the effects of SIN-1 on microglial induction of TNF in vivo. The accumulation of SIN-1 solution in rat cerebral cortex led to the induction of TNF on the ipsilateral, but not the contralateral side. The levels of TNF in the ipsilateral cortex peaked at 6-12 h post-accumulation. Immunohistochemical study revealed that anti-TNF antibody-positive cells in the SIN-1-injected region were mainly antiionized Ca 2+ binding adapter molecule-1 (Iba-1) antibody-positive, suggesting that microglia are a major cell type for inducing TNF. On the other hand, interleukin 1beta (IL-1) and IL-6 were not detected in the SIN-1-injected cortex. Together, these results indicate that microglia induced TNF in vivo in response to superoxide anion.