Accumulation of macrophage-like cells expressing NG2 proteoglycan and Iba1 in ischemic core of rat brain after transient middle cerebral artery occlusion.

Although neurons and glia inevitably undergo degeneration in the core of ischemic lesions, many cells, particularly immune cells, infiltrate the core and survive in it. Such infiltrating cells may play certain roles in the regeneration and repair of damaged brain tissues. In this study, we characterized macrophage-like cells that accumulated in the ischemic core of a rat brain whose right middle cerebral artery was transiently occluded for 90 mins. Many of the accumulated macrophage-like cells expressed Iba1, a marker of macrophages/microglia, as well as NG2 chondroitin sulfate proteoglycan (NG2), which has been recognized as a marker of oligodendrocyte progenitor cells. Such macrophage-like cells were termed BINCs (brain Iba1(+)/NG2(+) cells) to distinguish them from NG2(-)/Iba1(+) or NG2(+)/Iba1(-) cells that were also present in the perilesion and the contralateral hemisphere. Electron microscopy showed the localization of NG2 along the plasma membrane of cells that had many phagosomes and irregular-shaped or reniform heterochromatin-rich nuclei, which are characteristics of monocytes/macrophages. Brain Iba1(+)/NG2(+) cells were highly proliferative and their number peaked at 7 days post-reperfusion. An immunoblot analysis of NG2 revealed the presence of two NG2s: one expressed by BINCs with a molecular weight of 300 kDa, and the other found in the contralateral hemisphere with a molecular weight of 290 kDa. Taken the various functions of NG2, BINCs may be involved in not only phagocytosis of degenerated cells but also the healing and regeneration of lesion cores.

Continuous intrathecal infusion of SB203580, a selective inhibitor of p38 mitogen-activated protein kinase, reduces the damage of hind-limb function after thoracic spinal cord injury in rat.

P38 mitogen-activated protein kinase (MAPK) is one of the key enzymes in apoptosis induction pathways. We tested continuous intrathecal infusion of SB203580, a selective inhibitor of p38-MAPK, after spinal cord compression injury by a 20 g weight for 40 min at the 11th vertebra level-thoracic spinal cord. SB203580 (1 microg/day) was infused for 1 week after the compression. Hind-limb function was evaluated by measuring the frequency of 'standing' posture; raising fore limbs and sustaining body weight with hind-limbs. One-week after the compression, frequency of standing spinal cord injured rat was decreased to about half of that in sham operated animals which underwent laminectomy without compression. The frequency of standing in rats infused SB203580 recovered 2-3 weeks after the spinal cord injury, on the other hand, vehicle animals infused with saline did not recover. Myelin staining by Luxol fast blue showed severe myelin degradation in vehicle animals in lateral and dorsal funiculi. Apoptotic cells, detected by TUNEL staining, appeared in lateral funiculi of spinal cord injured rats. The application of SB203580 decreased the number of apoptotic cells. The SB203580-treated animals showed no significant degeneration of myelin structure. These results suggest that inhibition of p38-MAPK is one candidate for therapeutic agents against neurological deficits after spinal cord injury.

Adenosine triphosphate inhibits cytokine release from lipopolysaccharide-activated microglia via P2y receptors.

Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10-300 microM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P(2)y receptor agonists (ADPbetaS and 2-methylthio-ATP) on LPS-induced TNF-alpha release. The rank order of inhibitory potency of endogenous purines on TNF-alpha release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 microM 2-methylthio-ATP showed a potency similar to 100 microM ATP. The addition of ADPbetaS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P(2)y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.

Prostaglandin E1 analog inhibits the microglia function: suppression of lipopolysaccharide-induced nitric oxide and TNF-alpha release.

Release of nitric oxide and TNF-alpha, a toxic cytokine, have been reported to accelerate neuronal damage under several pathological conditions, such as trauma or ischemia in the central nervous system. In the present study, we tested the effect of alprostadil alfadex, a prostaglandin E1 analog, on cultured microglia from the rat spinal cord. The cultured microglia were exposed to lipopolysaccharide (LPS) (100 ng/ml), an endotoxin, for 24 h, then the released nitric oxide and TNF-alpha in the culture media was analyzed. The released nitric oxide was detected by the Griess reaction and released TNF-alpha was measured using ELISA method. The LPS-induced nitric oxide release was inhibited by the simultaneous addition of alprostadil alfadex in a dose-dependent manner (0.1-100 microM). The LPS-induced TNF-alpha release was also inhibited by alprostadil alfadex addition (0.1-100 microM). The IC50 values of alprostadil alfadex on nitric oxide and TNF-alpha release were about 1 and 10 microM, respectively. These results suggest that prostaglandin E1 possibly protects spinal cord neurons from several types of neurodegenerative damage, not only via increased blood supply, but also via inhibition of pathological immunoreactions of activated microglia.