Wenquan Liu

Effects of cerebral ischemia on neuronal hemoglobin

This study examined whether neuronal hemoglobin (Hb) is present in rats. It then examined whether cerebral ischemia or ischemic preconditioning (IPC) affects neuronal Hb levels in vivo and in vitro. In vivo, male Sprague-Dawley rats were subjected to either 15 mins of transient middle cerebral artery occlusion (MCAO) with 24 h of reperfusion, an IPC stimulus, or 24 h of permanent MCAO (pMCAO), or IPC followed 3 days later by 24 h of pMCAO. In vitro, primary cultured neurons were exposed to 2 h of oxygen—glucose deprivation (OGD) with 22 h of reoxygenation. Results showed that Hb is widely expressed in rat cerebral neurons but not astrocytes. Hemoglobin expression was significantly upregulated in the ipsilateral caudate and the cortical core of the middle cerebral artery territory after IPC. Hemoglobin levels also increased more in the penumbral cortex and the contralateral hemisphere 24 h after pMCAO, but expressions in the ipsilateral caudate and the cortical core area were decreased. Ischemic preconditioning modified pMCAO-induced brain Hb changes. Neuronal Hb levels in vitro were increased by 2 h of OGD and 22 h of reoxygenation. These results indicate that Hb is synthesized in neurons and can be upregulated by ischemia.

Deferoxamine attenuates white matter injury in a piglet intracerebral hemorrhage model

Background and Purpose— Deferoxamine reduces neuronal death in a piglet model of intracerebral hemorrhage (ICH). This study examined the effect of deferoxamine on perihematomal white matter edema in piglets. Methods— ICH was induced by an injection of autologous blood into the right frontal lobe of piglets. In the first part of study, the time course of edema formation was determined. In the second part, the effects of deferoxamine on ICH-induced white matter edema, tumor necrosis factor &agr;, and receptor-interacting protein kinase 1 were examined. Results— ICH resulted in marked brain edema and increased tumor necrosis factor &agr; and receptor-interacting protein kinase 1 levels in white matter. Systemic treatment with deferoxamine markedly reduced white matter tumor necrosis factor &agr; and receptor-interacting protein kinase 1 levels and attenuated white matter edema after ICH. Conclusions— Deferoxamine reduces white matter edema, tumor necrosis factor &agr;, and receptor-interacting protein kinase 1 levels after ICH in piglets, suggesting deferoxamine is a potential effective therapeutic agent for patients with ICH.

Minocycline Attenuates Brain Edema, Brain Atrophy and Neurological Deficits After Intracerebral Hemorrhage

Evidence suggests that microglia activation contributes to brain injury after intracerebral hemorrhage (ICH). The present study aimed to determine if minocycline, an inhibitor of microglia activation, can reduce brain edema, brain atrophy and neurological deficits after ICH.Male Sprague-Dawley rats received an infusion of 100-microL autologous whole blood into the right basal ganglia. Rats received minocycline or vehicle treatment. There were two sets of experiments in this study. In the first set of experiments, the effects of minocycline on ICH-induced brain edema were examined at day 3. In the second set, behavioral tests were performed at days 1, 3, 7, 14 and 28. Rats were killed at day 28 for brain atrophy measurement (caudate and lateral ventricle size).Minocycline reduced perihematomal brain edema in the ipsilateral basal ganglia (78.8 +/- 0.4 vs. 80.9 +/- 1.1% in the vehicle-treated group, p < 0.01). Minocycline also improved functional outcome. In addition, minocycline reduced brain tissue loss in the ipsilateral caudate (p < 0.01) and ventricular enlargement (p < 0.05).In conclusion, minocycline attenuates ICH-induced brain edema formation, neurological deficits and brain atrophy in rats suggesting an important role of microglia in ICH-related brain injury.

Estrogen reduces iron-mediated brain edema and neuronal death.

Our previous studies found that 17-beta estradiol attenuates edema formation after intracerebral hemorrhage (ICH). As brain iron overload occurs after ICH and contributes to ICH-induced brain injury, the present study examined the effects of estrogen on iron-induced brain injury in vivo and in vitro.There were two sets of experiments in this study. In the first set, male Sprague-Dawley rats were pretreated with 17-beta estradiol or vehicle prior to an intracerebral injection of ferrous iron. Ferrous iron was injected into the right caudate and the rats were killed 24 h later for brain edema measurement. In the second set, primary cultured neurons were pretreated with different doses of 17-beta estradiol or vehicle for 24 h. The cells were then exposed to ferrous iron for 48 h when culture medium was collected for lactate dehydrogenase measurement. Neuronal death was also assessed by live/dead cell assay.Estrogen pretreatment reduced brain water content (p < 0.01) 24 h after iron injection. Estrogen also protected against iron-induced cell death in cultured neurons. Estrogen reduces iron-induced brain edema in vivo and neuronal death in vitro suggesting estrogen could be a potential therapeutic agent for ICH.

Hyperbaric oxygen preconditioning activates ribosomal protein S6 kinases and reduces brain swelling after intracerebral hemorrhage

BACKGROUND New protein synthesis is key to ischemic tolerance induced by preconditioning and ribosomal protein S6 kinases (p70 S6 K) are important enzymes in protein synthesis. Hyperbaric oxygen preconditioning (HBOP) reduces ischemic brain damage. This study investigated if HBOP can activate p70 S6 K and increase new protein synthesis and if HBOP induces brain tolerance against brain swelling after intracerebral hemorrhage (ICH). METHODS There were two parts of the studies. 1) Rats received five consecutive sessions of HBOP. Twenty-four hours after HBOP, the rats had an ICH and were sacrificed one or three days later for brain edema measurement. 2) Rats received five sessions of HBOP or control pretreatment and were sacrificed for Western blot analysis and immunohistochemistry of activated p70 S6 K and heme oxygenase-1 (HO-1). FINDINGS Five sessions of HBOP significantly reduced brain edema in the ipsilateral basal ganglia after ICH. Western blot analysis showed that HBOP activated p70 S6 K and increased HO-1 levels in the basal ganglia. Strong activated p70 S6 K immunoreactivity was also found in the basal ganglia. CONCLUSIONS Our results suggest activation of p70 S6 K may have a role in heat shock protein synthesis after HBOP and may contribute to HBOP-induced brain protection.

Hemoglobin Expression in Neurons and Glia After Intracerebral Hemorrhage

The purpose of this study was to examine the expression of hemoglobin (Hb) in the brain after intracerebral hemorrhage (ICH) and the effects of hemin and iron on neuronal Hb. For the in vivo studies, male Sprague-Dawley rats received either a sham operation or an ICH. The rats were killed 1, 4, 24 or 72 h later, and brains were used for real-time polymerase chain reaction (PCR) and immunohistochemistry. For the in vitro study, primary cultured neurons were exposed to either hemin or vehicle. Some neurons also received treatment with deferoxamine, an iron chelator. Neurons were collected 24 h later for real-time PCR. We found that α-globin (HbA) and β-globin (HbB) mRNA levels in the ipsilateral basal ganglia are significantly increased after ICH, and Hb is localized in neurons and glia cells. Exposure of neurons to hemin also upregulated HbA and HbB mRNA levels. Hemin-induced HbA and HbB expression in cultured neurons was reduced by deferoxamine treatment. These results indicate that ICH increases HbA and HbB expression in neurons and glia cells, and that heme and iron may be important factors in inducing endogenous Hb expression after ICH.

Thrombin-induced neuronal protection: Role of the mitogen activated protein kinase/ribosomal protein S6 kinase pathway

Our previous studies have found that intracerebral pretreatment with a low dose of thrombin (thrombin preconditioning, TPC) reduces infarct volume and attenuates brain edema after focal cerebral ischemia. In this study, we examined whether TPC protects against the neuronal death induced by oxygen glucose deprivation (OGD), and whether the protection is through thrombin receptors and the p44/42 mitogen activated protein kinases (MAPK)/ribosomal protein S6 kinases (p70 S6K) pathway. Expression of protease-activated receptors (PARs) mRNA was detected in cultured primary rat neurons and thrombin upregulated PAR-1 and PAR-4 mRNA expression. TPC reduced OGD-induced neuronal death (e.g. dead cells: 52.5 ± 5.4% vs. 72.3 ± 7.2% in the control group, n=6, p<0.01). Agonists of PAR-1 and PAR-4 mimicked the effects of thrombin and reduced OGD-induced neuronal death. Pretreatment with thrombin or PAR agonists induced the upregulation of activated p44/42 MAPK and p70S6K (Thr 421/Ser 424). PD98059, an inhibitor of p44/42 MAPK kinase, blocked thrombin-induced upregulation of activated p44/42 MAPK and p70S6K. It also reduced TPC-induced neuronal protection (e.g. dead cells: 68.2 ± 5.2% vs. 56.9 ± 4.6% in vehicle+TPC group, n=6, p<0.05). These results suggest that TPC-induced ischemic tolerance is through activation of thrombin receptors and the p44/42 MAPK/p70S6K pathway.

Susceptibility to intracerebral hemorrhage-induced brain injury segregates with low aerobic capacity in rats

Although low exercise capacity is a risk factor for stroke, the exact mechanisms that underlie this connection are not known. As a model system for exploring the association between aerobic capacity and disease risks we applied two-way artificial selection over numerous generations in rats to produce low capacity runners (LCR) and high capacity runners (HCR). Here we compared intracerebral hemorrhage (ICH)-induced brain injury in both genders of these rat lines. HCR and LCR rats had 100μl blood injected into the right caudate and were killed at days 1, 3, 7 and 28 for brain water content determination, immunohistochemistry, histology, Western blot, and behavioral tests. Compared to male HCRs, male LCRs had more severe ICH-induced brain injury including worse brain edema, necroptosis, brain atrophy, and neurological deficits, but not increased numbers of Fluoro-Jade C positive cells or elevated cleaved caspase-3 levels. This was associated with greater microglial activation, and heme oxygenase-1 and protease activated receptor (PAR)-1 upregulation. In females, edema was also greater in LCRs than in HCRs, although it was less severe in females than in males for both LCRs and HCRs. Thus, ICH-induced brain injury was more severe in LCRs, a model of low exercise capacity, than in HCRs. Increased activation of microglia and PAR-1 may participate mechanistically in increased ICH-susceptibility. Females were protected against ICH-induced brain edema formation in both HCRs and LCRs.

Induction of autophagy in rat hippocampus and cultured neurons by iron.

Autophagy occurs in the brain after intracerebral hemorrhage (ICH). Iron is an important factor causing neuronal death and brain atrophy after ICH. In this study, we examined whether iron can induce autophagy in the hippocampus and in cultured neurons. For in vivo studies, rats received an infusion of either saline or ferrous iron into the right hippocampus and were killed 1, 3, or 7 days later for Western blot analysis of microtubule-associated protein light chain-3 (LC3). For in vitro studies, primary cultured cortex neurons from rat embryos were exposed to ferrous iron. Cells were used for Western blot analysis of LC3 and monodansylcadaverine (MDC) staining 24h later. Intrahippocampal injection of ferrous iron resulted in an increased conversion of LC3-I to LC3-II. Exposure of primary cultured neurons to ferrous iron also induced an enhanced conversion of LC3-I to LC3-II. MDC labeling showed an accumulation of MDC in cultured neurons exposed to ferrous iron. These results indicate that autophagy is induced by iron in neurons and that iron-induced autophagy may contribute to brain injury after ICH.

Red Blood Cell Lysis and Brain Tissue-Type Transglutaminase Upregulation in a Hippocampal Model of Intracerebral Hemorrhage

Red blood cell (RBC) lysis and iron release contribute to intracerebral hemorrhage (ICH)-induced brain injury. Tissue-type transglutaminase (tTG), which has a role in neurodegeneration, is upregulated after ICH. The current study investigated the effect of RBC lysis and iron release on brain tTG levels and neuronal death in a rat model of ICH. This study had three parts: (1) Male Sprague-Dawley rats received an intrahippocampal injection of 10 μL of either packed RBCs or lysed RBCs; (2) rats had a 10 μL injection of either saline, hemoglobin or FeCl2; (3) rats received a 10 μL injection of hemoglobin and were treated with an iron chelator, deferoxamine or vehicle. All rats were killed 24 h later, and the brains were sectioned for tTG and Fluoro-Jade C staining. Lysed but not packed RBCs caused marked tTG upregulation (p<0.05) and neuronal death (p<0.05) in the ipsilateral hippocampus CA-1 region. Both hemoglobin and iron mimicked the effects of lysed RBCs, resulting in tTG expression and neuronal death (p<0.05). Hemoglobin-induced tTG upreglution and neuronal death were reduced by deferoxamine (p<0.05). These results indicate that RBC lysis and iron toxicity contribute to neurodegeneration after ICH.