Post-ischaemic immunological response in the brain: targeting microglia in ischaemic stroke therapy

Microglia, the major endogenous immune cells of the central nervous system, mediate critical degenerative and regenerative responses in ischaemic stroke. Microglia become "activated", proliferating, and undergoing changes in morphology, gene and protein expression over days and weeks post-ischaemia, with deleterious and beneficial effects. Pro-inflammatory microglia (commonly referred to as M1) exacerbate secondary neuronal injury through the release of reactive oxygen species, cytokines and proteases. In contrast, microglia may facilitate neuronal recovery via tissue and vascular remodelling, through the secretion of anti-inflammatory cytokines and growth factors (a profile often termed M2). This M1/M2 nomenclature does not fully account for the microglial heterogeneity in the ischaemic brain, with some simultaneous expression of both M1 and M2 markers at the single-cell level. Understanding and regulating microglial activation status, reducing detrimental and promoting repair behaviours, present the potential for therapeutic intervention, and open a longer window of opportunity than offered by acute neuroprotective strategies. Pharmacological modulation of microglial activation status to promote anti-inflammatory gene expression can increase neurogenesis and improve functional recovery post-stroke, based on promising preclinical data. Cell-based therapies, using preconditioned microglia, are of interest as a method of therapeutic modulation of the post-ischaemic inflammatory response. Currently, there are no clinically-approved pharmacological options targeting post-ischaemic inflammation. A major developmental challenge for clinical translation will be the selective suppression of the deleterious effects of microglial activity after stroke whilst retaining (or enhancing) the neurovascular repair and remodelling responses of microglia

Proteomic analysis of age-related changes in ovine cerebrospinal fluid

Cerebrospinal fluid (CSF) circulates through the brain and has a unique composition reflecting the biological processes of the brain. Identifying ageing CSF biomarkers can aid in understanding the ageing process and interpreting CSF protein changes in neurodegenerative diseases. In this study, ovine CSF proteins from young (1-2 year old), middle aged (3-6 year old) and old (7-10 year old) sheep were systemically studied. CSF proteins were labelled with iTRAQ tagging reagents and fractionated by 2-dimensional high performance, liquid chromatography. Tryptic peptides were identified using MS/MS fragmentation ions for sequencing and quantified from iTRAQ reporter ion intensities at m/z 114, 115, 116 and 117. Two hundred thirty one peptides were detected, from which 143 proteins were identified. There were 52 proteins with >25% increase in concentrations in the old sheep compared to the young. 33 of them increased >25% but 50% but 1 fold [i.e. haptoglobin (Hp), haemoglobin, neuroendocrine protein 7B2, IgM, fibrous sheath interacting protein 1, vimentin]. There were 18 proteins with >25% decrease in concentrations in the old sheep compared to the young. 17 of them decreased >25% but <50%, and histone deacetylase 7 (HDAC7) was gradually decreased for over 80%. Glutathione S-transferase was decreased in middle aged CSF compared to both young and old CSF. The differential expressions of 3 proteins (Hp, neuroendocrine protein 7B2, IgM) were confirmed by immunoassays. These data expand our current knowledge regarding ovine CSF proteins, supply the necessary information to understand the ageing process in the brain and provide a basis for diagnosis of neurodegenerative diseases

Stem Cell Therapy for Ischaemic Stroke: Translation from Preclinical Studies to Clinical Treatment

No pharmacological intervention has been shown convincingly to improve neurological outcome in stroke patients after the brain tissue is infarcted. While conventional therapeutic strategies focus on preventing brain damage, stem cell treatment has the potential to repair the injured brain tissue. Stem cells not only produce a source of trophic molecules to minimize brain damage caused by ischaemia/reperfusion and promote recovery, but also potentially turn to new cells to replace those lost in ischaemic core. Although preclinical studies have shown promise, stem cell therapy for stroke treatment in human is still at an early stage and it is difficult to draw conclusions from current clinical trials about the efficacy of the different treatments used in humans. This article reviews the potential of various types of stem cells, from embryonic to adult to induced pluripotent stem cells, in stroke therapy, highlights new evidence from the ongoing clinical trials and discusses some of the problems associated with translating stem cell technology to a clinical therapy for stroke

Reactive Oxygen Species Formation in the Brain at Different Oxygen Levels: The Role of Hypoxia Inducible Factors

Hypoxia inducible factor (HIF) is the master oxygen sensor within cells and is central to the regulation of cell responses to varying oxygen levels. HIF activation during hypoxia ensures optimum ATP production and cell integrity, and is associated both directly and indirectly with reactive oxygen species (ROS) formation. HIF activation can either reduce ROS formation by suppressing the function of mitochondrial tricarboxylic acid cycle (TCA cycle), or increase ROS formation via NADPH oxidase (NOX), a target gene of HIF pathway. ROS is an unavoidable consequence of aerobic metabolism. In normal conditions (i.e., physioxia), ROS is produced at minimal levels and acts as a signaling molecule subject to the dedicated balance between ROS production and scavenging. Changes in oxygen concentrations affect ROS formation. When ROS levels exceed defense mechanisms, ROS causes oxidative stress. Increased ROS levels can also be a contributing factor to HIF stabilization during hypoxia and reoxygenation. In this review, we systemically review HIF activation and ROS formation in the brain during hypoxia and hypoxia/reoxygenation. We will then explore the literature describing how changes in HIF levels might provide pharmacological targets for effective ischaemic stroke treatment. HIF accumulation in the brain via HIF prolyl hydroxylase (PHD) inhibition is proposed as an effective therapy for ischaemia stroke due to its antioxidation and anti-inflammatory properties in addition to HIF pro-survival signaling. PHD is a key regulator of HIF levels in cells. Pharmacological inhibition of PHD increases HIF levels in normoxia (i.e., at 20.9% O2 level). Preconditioning with HIF PHD inhibitors show a neuroprotective effect in both in vitro and in vivo ischaemia stroke models, but post-stroke treatment with PHD inhibitors remains debatable. HIF PHD inhibition during reperfusion can reduce ROS formation and activate a number of cellular survival pathways. Given agents targeting individual molecules in the ischaemic cascade (e.g., antioxidants) fail to be translated in the clinic setting, thus far, HIF pathway targeting and thereby impacting entire physiological networks is a promising drug target for reducing the adverse effects of ischaemic stroke

Toward highly potent cancer agents by modulating the c-2 group of the arylthioindole class of tubulin polymerization inhibitors

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC50 = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes

New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway

Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture

Cells respond to reduced oxygen availability predominately by activation of the hypoxia-inducible factor (HIF) pathway. HIF activation upregulates hundreds of genes that help cells survive in the reduced oxygen environment. The aim of this study is to determine whether chemical-induced HIF accumulation mimics all aspects of the hypoxic response of cells. We compared the effects of dimethyloxalylglycine (DMOG) (a HIF stabiliser) on PC12 cells cultured in air oxygen (20.9% O2, AO) with those cultured in either intermittent 20.9% O2 to 2% O2 (IH) or constant 2% O2 (CN). Cell viability, cell cycle, HIF accumulation, reactive oxygen species (ROS) formation, mitochondrial function and differentiation were used to characterise the PC12 cells and evaluate the impact of DMOG. IH and CN culture reduced the increase in cell numbers after 72 and 96 h and MTT activity after 48 h compared to AO culture. Further, DMOG supplementation in AO induced a dose-dependent reduction in the increase in PC12 cell numbers and MTT activity. IH-cultured PC12 cells displayed increased and sustained HIF-1 expression over 96 h. This was accompanied by increased ROS and mitochondrial burden. PC12 cells in CN displayed little changes in HIF-1 expression or ROS levels. DMOG (0.1 mM) supplementation resulted in an IH-like HIF-1 profile. The mitochondrial burden and action potential of DMOG-supplemented PC12 cells did not mirror those seen in other conditions. DMOG significantly increased S phase cell populations after 72 and 96 h. No significant effect on PC12 cell differentiation was noted with IH and CN culture without induction by nerve growth factor (NGF), while DMOG significantly increased PC12 cell differentiation with and without NGF. In conclusion, DMOG and reduced oxygen levels stabilise HIF and affect mitochondrial activity and cell behaviour. However, DMOG does not provide an accurate replication of the reduced oxygen environments

Editorial (Current and Emerging Pharmacological Therapies of Ischaemic Stroke)

Stroke is a form of cardiovascular disease affecting blood supply to the brain. About 87% of strokes are ischemic, and the remainder are haemorrhagic. Ischaemic stroke is caused by the obstruction of blood flow in a brain artery leading to the lack of oxygen and glucose supply to the brain region, resulting in the collapse of oxidative metabolism, decreased ATP synthesis, increased free radical production, unregulated influx of calcium/sodium and membrane depolarization, and calcium excitotoxicity. Eventually, cerebral ischaemia leads to formation of an “unviable” core and damaged but “amenable” penumbral regions

The Duration of Oxygen and Glucose Deprivation (OGD) Determines the Effects of Subsequent Reperfusion on Rat Pheochromocytoma (PC12) Cells and Primary Cortical Neurons

Reperfusion is the fundamental treatment for ischaemic stroke; however, many ischaemic stroke patients cannot undergo reperfusion treatment. Furthermore, reperfusion can cause ischaemic reperfusion injuries. This study aimed to determine the effects of reperfusion in an in vitro ischaemic stroke model—oxygen and glucose deprivation (OGD) (0.3% O2)—with rat pheochromocytoma (PC12) cells and cortical neurons. In PC12 cells, OGD resulted in a time-dependent increase in cytotoxicity and apoptosis, and reduction in MTT activity from 2 h onwards. Reperfusion following shorter periods (4 and 6 h) of OGD recovered apoptotic PC12 cells, whereas after 12 h, OGD increased LDH release. In primary neurons, 6 h OGD led to significant increase in cytotoxicity, reduction in MTT activity and dendritic MAP2 staining. Reperfusion following 6 h OGD increased the cytotoxicity. HIF-1a was stabilised by 4 and 6 h OGD in PC12 cells and 2 h OGD onwards in primary neurons. A panel of hypoxic genes were upregulated by the OGD treatments depending on the duration. In conclusion, the duration of OGD determines the mitochondrial activity, cell viability, HIF-1a stabilization, and hypoxic gene expression in both cell types. Reperfusion following OGD of short duration is neuroprotective, whereas OGD of long duration is cytotoxic