Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

BACKGROUND AND PURPOSE: Microglia are resident immunocompetent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat. METHODS: Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR. RESULTS: HMO6 human microglial cells transplantation group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group. CONCLUSIONS: Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin

Effect of the triaminopyridine flupirtine on calcium uptake, membrane potential and ATP synthesis in rat heart mitochondria

1. Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular. 2. The findings show that flupirtine increases Ca(2+) uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg(−1) mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg(−1) mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria. 3. Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg(−1) mitochondrial protein and a return to control values at 20 nmol mg(−1) protein. 4. The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume. 5. Calcium at 80 and 160 nmol mg(−1) mitochondrial protein decreased ATP synthesis by 20–25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg(−1) protein was added before the addition of calcium. 6. Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg(−1) mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level

Influence of flupirtine on a G-protein coupled inwardly rectifying potassium current in hippocampal neurones

1. Previous studies have shown that flupirtine, a centrally acting, non-opioid analgesic agent, also exhibits neuroprotective activity in focal cerebral ischaemia in mice and reduces apoptosis induced by NMDA, gp 120 of HIV, prior protein fragment or lead acetate as well as necrosis induced by glutamate or NMDA in cell culture. To study the potential mechanism of the neuroprotective action of flupirtine, we investigated whether flupirtine is able to modulate potassium or NMDA-induced currents in rat cultured hippocampal neurones by use of the whole-cell configuration of the patch-clamp technique. 2. We demonstrated that 1 μM flupirtine activated an inwardly rectifying potassium current (K(ir)) in hippocampal neurones (ΔI=−39±18 pA at −130 mV; n=10). This effect was dose-dependent (EC(50)=0.6 μM). The reversal potential for K(ir) was in agreement with the potassium equilibrium potential predicted from the Nernst equation showing that K(ir) was predominantly carried by K(+). Furthermore, the induced current was blocked completely by Ba(2+) (1 mM), an effect typical for K(ir). 3. The activation of K(ir) by flupirtine was largely prevented by pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX-sensitive G-protein in the transduction mechanism (ΔI=−3±6 pA at −130 mV; n=8). Inclusion of cyclic AMP in the intracellular solution completely abolished the activation of K(ir) (n=7). 4. The selective α(2)-adrenoceptor antagonist SKF-86466 (10 μM), the selective 5-HT(1A) antagonist NAN 190 as well as the selective GABA(B) antagonist 2-hydroxysaclofen (10 μM) failed to block the flupirtine effect on the inward rectifier. 5. Flupirtine (1 μM) could not change the current induced by 50 μM NMDA. 6. These results show that in cultured hippocampal neurones flupirtine activates an inwardly rectifying potassium current and that a PTX-sensitive G-protein is involved in the transduction mechanism