Zongjian Liu

Remote Ischemic Preconditioning-Mediated Neuroprotection against Stroke is Associated with Significant Alterations in Peripheral Immune Responses.

Remote ischemic preconditioning (RIPC) of a limb is a clinically feasible strategy to protect against ischemia–reperfusion injury after stroke. However, the mechanism underlying RIPC remains elusive.

Ethanol and Normobaric Oxygen: Novel Approach in Modulating Pyruvate Dehydrogenase Complex After Severe Transient and Permanent Ischemic Stroke

Background and Purpose— Ischemic stroke induces metabolic disarray. A central regulatory site, pyruvate dehydrogeanse complex (PDHC) sits at the cross-roads of 2 fundamental metabolic pathways: aerobic and anaerobic. In this study, we combined ethanol (EtOH) and normobaric oxygen (NBO) to develop a novel treatment to modulate PDHC and its regulatory proteins, namely pyruvate dehydrogenase phosphatase and pyruvate dehydrogenase kinase, leading to improved metabolism and reduced oxidative damage. Methods— Sprague–Dawley rats were subjected to transient (2, 3, or 4 hours) middle cerebral artery occlusion followed by 3- or 24-hour reperfusion, or permanent (28 hours) middle cerebral artery occlusion without reperfusion. At 2 hours after the onset of ischemia, rats received either an intraperitoneal injection of saline, 1 dose of EtOH (1.5 g/kg) for 2- and 3-hour middle cerebral artery occlusion, 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg in 2 hours) in 4 hours or permanent middle cerebral artery occlusion, and EtOH+95% NBO (at 2 hours after the onset of ischemia for 6 hours) in permanent stroke. Infarct volumes and neurological deficits were examined. Oxidative metabolism and stress were determined by measuring ADP/ATP ratio and reactive oxygen species levels. Protein levels of PDHC, pyruvate dehydrogenase kinase, and pyruvate dehydrogenase phosphatase were assessed. Results— EtOH induced dose-dependent neuroprotection in transient ischemia. Compared to EtOH or NBO alone, NBO+EtOH produced the best outcomes in permanent ischemia. These therapies improved brain oxidative metabolism by decreasing ADP/ATP ratios and reactive oxygen species levels, in association with significantly raised levels of PDHC and pyruvate dehydrogenase phosphatase, as well as decreased pyruvate dehydrogenase kinase. Conclusions— Both EtOH and EtOH+NBO treatments conferred neuroprotection in severe stroke by affecting brain metabolism. The treatment may modulate the damaging cascade of metabolic events by bringing the PDHC activity back to normal metabolic levels.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke.

Curcumin Protects against Ischemic Stroke by Titrating Microglia/Macrophage Polarization

Stroke is the most common type of cerebrovascular disease and is a leading cause of disability and death. Ischemic stroke accounts for approximately 80% of all strokes. The remaining 20% of strokes are hemorrhagic in nature. To date, therapeutic options for acute ischemic stroke are very limited. Recent research suggests that shifting microglial phenotype from the pro-inflammatory M1 state toward the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. The dietary phytochemical curcumin has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are unknown. Here we address these gaps by subjecting mice to distal middle cerebral artery occlusion (dMCAO) and administering curcumin intraperitoneally (150 mg/kg) immediately after ischemia and 24 h later. Histological studies revealed that curcumin post-treatment significantly reduced cerebral ischemic damage 3 days after dMCAO. Sensorimotor functions—as measured by the adhesive removal test and modified Garcia scores—were superior in curcumin-treated mice at 3, 5, 7 and 10 days after stroke. RT-PCR measurements revealed an elevation of M2 microglia/macrophage phenotypic markers and a reduction in M1 markers in curcumin-treated brains 3 days after dMCAO. Immunofluorescent staining further showed that curcumin treatment significantly increased the number of CD206+Iba1+ M2 microglia/macrophages and reduced the number of CD16+Iba1+ M1 cells 10 days after stroke. In vitro studies using the BV2 microglial cell line confirmed that curcumin inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced M1 polarization. Curcumin treatment concentration-dependently reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12p70, in the absence of any toxic effect on microglial cell survival. In conclusion, we demonstrate that curcumin has a profound regulatory effect on microglial responses, promoting M2 microglial polarization and inhibiting microglia-mediated pro-inflammatory responses. Curcumin post-treatment reduces ischemic stroke-induced brain damage and improves functional outcomes, providing new evidence that curcumin might be a promising therapeutic strategy for stroke.

Administration of human platelet-rich plasma reduces infarction volume and improves motor function in adult rats with focal ischemic stroke.

Platelet-rich plasma (PRP) is a milieu of bioactive factors, including platelet derived growth factor, transforming growth factor beta, among many others. Despite accumulating evidence on PRPs safety and efficacy for treating musculoskeletal injuries, limited studies have been performed using PRP in brain disorders. This study aimed to explore the potential benefits of administration of human PRP lysate after ischemic stroke in rats. An ischemic stroke model was generated by occlusion of the right middle cerebral artery, then 90 min later, stroke rats were randomly assigned to receive local infusion to the ischemic area of human PRP lysate, human albumin solution (HSA), saline or no treatment at all. An additional group of stroke rats received systemic infusion of human PRP lysate to further assess the therapeutic effects of this treatment. Results showed that while local infusion of HSA or saline, and systemic administration of human PRP lysate, compared to no treatment significantly reduced infarct volume (37.4%, 40.1%, and 39.9% vs 49.7%) and neurological deficit score (2.2, 2.6, and 2.8 vs 3.7), the greatest neuroprotection (31.0% infarct volume and 1.6 neurological deficit score) was found in stroke animals that received local intra-arterial infusion of human PRP lysate (ps<0.05). In conclusion, administration of human PRP attenuates brain injury after focal ischemia. Our results suggest PRP should be investigated further as a potential point-of-care biomaterial following stroke.

Frequencies of circulating B- and T-lymphocytes as indicators for stroke outcomes

Background Stroke has high mortality and morbidity. Biomarkers are required for to predict stroke outcomes, which could help clinicians to provide rationale approaches for patient management. The dynamic changes in circulating immune cells have been reported in stroke patients and animal models of stroke. Aim The aim of this study was to explore biomarkers to predict stroke outcomes by investigating the relationship between the frequencies of circulating immune cells and stroke outcomes. Methods In all, 50 acute ischemic stroke (AIS) patients were enrolled. Their blood samples were collected upon hospital admission and on day 1 and day 7 after stroke, and the leukocyte subsets were analyzed by flow cytometry. The dynamic changes in some types of immune cells in the AIS course and their correlation with clinical parameters were analyzed. Blood samples from 123 age- and gender-matched healthy subjects were used as controls. Results The proportions of T-lymphocytes and NK cells in stroke patients were significantly lower than in healthy controls. The frequencies of B- and T-lymphocytes were negatively correlated with stroke severity at onset, including neurological deficits as assessed by National Institutes of Health Stroke Scale (NIHSS), and infarct volume as measured by the diffusion-weighted images (DWIs) of magnetic resonance (MR). Logistic regression analysis showed that modified Rankin scale (mRs) scores, a score system for the long-term neurological dysfunctions, were negatively correlated with the percentage of B-lymphocytes at stroke onset. Conclusion The proportions of B- and T-lymphocytes are associated with neurological outcomes of stroke patients and might be used as an indicator for severity and prognosis of ischemic stroke.

Weight loss: indication of brain damage and effect of combined normobaric oxygen and ethanol therapy after stroke

Abstract Background and Purpose: Weight loss is commonly seen after stroke. However, there is paucity of data that objectively examines the relationship between weight loss and infarction. We have used two unique models of middle cerebral artery (MCA) occlusion in rats to determine if weight loss can be used as indicator for severity of brain damage and for beneficial effects of stroke therapy. Methods: Sprague-Dawley rats underwent MCA occlusion using the rat intraluminal filament or auto-thrombus stroke models. Reperfusion was established at different intervals by removing the intraluminal filament or injecting r-tPA, followed by treatment with either ethanol (EtOH), normobaric oxygen (NBO), NBO plus EtOH or no treatment. The extent of brain injury was determined using infarct volume and motor performance. Results: The intraluminal filament ischaemic model demonstrated a significant positive correlation between weight loss and infarct volume size after acute stroke, as well as compared to infarct volumes post-treatment with NBO, EtOH and NBO plus EtOH. There was also a positive significant correlation between weight loss and infarct volume size in the thromboembolism ischaemic model with or without treatment. Furthermore a positive correlation was observed between weight loss and deficit score in both ischaemic models. Discussion: Degree of weight loss after stroke is largely associated with severity of infarction as well as damage reduction after treatment administration.

Splenectomy Fails to Provide Long-Term Protection Against Ischemic Stroke

Splenectomy before or immediately after stroke provides early brain protection. This study aims to explore the effect of splenectomy on long-term neurological recovery after stroke, which is currently lacking in the field. Adult male rats were randomized into splenectomy or sham groups and then subjected to 90 min of middle cerebral artery occlusion (MCAO). Spleen was removed right upon reperfusion or 3d after MCAO. Infarct volume, neurological functions, and peripheral immune cell populations were assessed up to 28d after stroke. The results show that delayed removal of spleen did not reduce brain tissue loss and showed no effect on sensorimotor function (Rotarod, beam balance, forelimb placing, grid walk, and adhesive removal tests) or cognitive function (Morris water maze). Spleen removal immediately upon reperfusion, although significantly reduced the infarct size and immune cell infiltration 3d after MCAO, also failed to promote long-term recovery. Flow cytometry analysis demonstrated that immediate splenectomy after MCAO resulted in a prolonged decrease in the percentage of CD3+CD4+ and CD3+CD8+ T cells in total lymphocytes as compared to non-splenectomy MCAO rats. In contrast, the percentage of CD3-CD45RA+ B cells was significantly elevated after splenectomy. As a result, the ratio of T/B cells was significantly reduced in stroke rats with splenectomy. In conclusion, delayed splenectomy failed to provide long-term protection to the ischemic brain or improve functional recovery. The acute neuroprotective effect achieved by early splenectomy after stroke cannot last for long term. This loss of neuroprotection might be related to the prolonged disturbance in the T cell to B cell ratio.