Effects Of Inflammatory Reaction Induced By Lipopolysaccharide On Intracerebral Hemorrhage Injury

Stroke is called the second greatest killer to human beings. Intracerebral hemorrhage (ICH) accounts for 10 to 15% of all strokes, but results in a disproportionately high morbidity and mortality without effective therapy. It is expected that the incidence of ICH will increase as the overall population ages and the society prospers. The physiopathologic mechanism of ICH is very complicated not only involving in primary injury from space occupying effect of hematoma itself, but relating to secondary injury from hematoma enlargement and formation of perihematomal edema. Both hematoma enlargement and brain edema, especial the third phase of it, delayed edema lead to patients’ early neurological deterioration and poor prognosis, and there’s no valid controlling strategy up to now, so thorough study on their physiopathologic mechanisms is both necessary and imperative.Inflammation is a kind of defensive response of alive organisms with blood vascular systems to harmful stimuli. Despite it has been demonstrated that inflammatory response of brain perihematoma region contributes to edema formation, little investigation has been performed concerning its effects upon hematoma enlargement and delayed brain edema (DBE), which are just the aims of our present study. To induce the activation of inflammatory cells, lipopolysaccharide (LPS) was microinjected into rat corpus callosum 1 day prior to intrastriatal infusion of autologous whole blood. Three days after ICH, the effects of LPS pretreatment on brain injury was investigated according to hematoma size. Then the impact of heme oxygenase-1 (HO-1) on LPS pretreatment was further determined by DNA microarray, immunohistochemistry, western blot and lentiviral transfection, etc. Double immunofluorescence was also performed to assess HO-1 immunoexpression and cellular localization after ICH. The results showed hematoma enlargement at least partly resulted from overexpression of HO-1 in activated microglia/macrophage induced by LPS and autologous whole blood. This finding provides both new insights for pathologic mechanism of hematoma enlargement and new target for therapeutic intervention.DBE usually occurs 72 h after ICH and is probably due to the toxicity of hemoglobin and its degradation product, hemin released from lysed red cells. However, inflammatory response happens much earlier than hemin release. So neuronal or neuroglial cells were exposed to inflammatory environment prior to contact with hemin after ICH. Firstly, the dose-and time-effects of hemin on neuron, mixed glia, astrocyte and microglia were respectively evaluated by morphologic observation under microscope and kinetic lactate dehydrogenase (LDH) assay. Then the effects of LPS pretreatment on hemin toxicity in above-mentioned cells were further explored, by which we established a new in vitro model more close to pathologic mechanism of DBE. The results showed hemin-induced injury in these cells was dose-and time-dependent, and the order of vulnerability was microglia> neuron> astrocyte. In addition, LPS pretreatment increased the vulnerability of neuron and astrocyte to hemin injury, whereas decreased that of microglia. For the first time, we put forward that pathologic mechanism of DBE was implicated in exacerbated injury of neuron and astrocyte and increased tolerance of microglia to hemin injury by inflammatory stimulation.It has been demonstrated that microglia were rapidly activated possibly within minutes after the onset of ICH, while the activated condition persisted for 3 to 4 weeks by many in vivo experiments, which supported our previous data. To survive in a harmful hemorrhagic or post-hemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanisms to resist the toxicity of hemin.First, we examined the cell type-dependent protection of LPS pretreatment and compared the influence of different treatment methods of LPS upon hemin-mediated injury in microglia. Then nitric oxide (NO), induced by LPS pretreatment in microglia was focused on to further study its role in protective mechanism of LPS by propidium iodide (PI) staining or kinetic LDH assay. And the interactions between iNOS and HO-1 or mitogen-activated protein kinases (MAPKs) were respectively explored by western blot assay. Finally, the obtained data was further confirmed via in vivo experiments by immunno-histochemistry and-fluorescence. The results showed:1) LPS pretreatment attenuated hemin-povoked brain injury and the protective role was microglia-specific, whereas it disappeared by co-treatment with LPS and hemin.2) The preventive effects by LPS was significantly diminished by an iNOS inhibitor, whereas it was mimicked by a NO donor, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia.3) Hemin induced injury in microglia by time-dependent activation of JNK and p38 MAPKs pathways, whereas iNOS stimulated by LPS reduced hemin-mediated toxicity via inhibition of these two pathways.4) LPS-induced iNOS expressioin was the upstream regulator of HO-1, and there existed a negative feedback regulation of iNOS expression by HO-1 in microglia. Interactive regulations between NO and HO-1 may adjust the cellular level of each other within a physiologically endurable range to keep microglial homeostasis against pathological threats of cerebral hemorrhagic lesion.5) Silencing of HO-1 gene or inhibition of HO-1 activity attenuated the vulnerability of LPS-activated microglia to hemin-induced injury probably due to the recovery of NO release. In conclusion, for the first time, we investigated the mechanisms by which hemin induced injury in microglia at cellular levels and by which LPS pretreatment diminished the vulnerability of microglia to hemin toxicity as well as the interactions between iNOS and HO-1 in microglia after ICH.The present study focused on the effects of LPS-induced inflammatory response on hematoma enlargement and DBE. These findings not only shed new light upon physiopathologic mechanisms of secondary brain injury after ICH but provide promising therapeutic targets for ICH patients.