Leukocyte TNFR1 and TNFR2 Expression Contributes to the Peripheral Immune Response in Cases with Ischemic Stroke.

Tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2) have been found in brain parenchyma of stroke patients, and plasma levels are increased in the acute phase of stroke. We evaluated associations between TNFR1 and TNFR2 plasma levels and stroke severity, infarct size, and functional outcome. Furthermore, we examined cellular expression of TNFR1 and TNFR2 on leukocyte subpopulations to explore the origin of the increased receptor levels. Blood samples were taken from 33 acute ischemic stroke patients and 10 healthy controls. TNFR1 and TNFR2 plasma concentrations were measured and correlated against the Scandinavian Stroke Scale at admission, infarct volume, and the modified Rankin Scale score three months after stroke onset. Classical, intermediate, and non-classical monocytes as well as neutrophils were purified, and cellular expression of TNFR1 and TNFR2 was examined using flow cytometry. TNFR1 and TNFR2 plasma levels were both increased after ischemic stroke, but we found no correlation with patient outcome measurements. Compared to healthy controls, ischemic stroke patients had decreased non-classical monocyte and neutrophil populations expressing TNFR1 and increased neutrophils expressing TNFR2, and decreased non-classical populations co-expressing both TNFR1 and TNFR2. This study supports the hypothesis of an acute immunological response orchestrated by the peripheral immune system following an ischemic stroke. However, the origin of the increased TNFR1 and TNFR2 plasma levels could not be clearly linked to peripheral monocytes or neutrophils. Future studies are needed and will help clarify the potential role as treatment target.

Dopamine Binding and Analysis in Undiluted Human Serum and Blood by the RNA-Aptamer Electrode.

Specific analysis of such neurotransmitters as dopamine by the aptamer electrodes in biological fluids is detrimentally affected by nonspecific adsorption of media, particularly pronounced at positive charges of the electrode surface at which dopamine oxidizes. Here, we show that dopamine analysis at the RNA-aptamer/cysteamine-modified electrodes is strongly inhibited in undiluted human serum and blood due to nonspecific interfacial adsorption of serum and blood components. We demonstrate that nonspecific adsorption of serum proteins (but not of blood components) could be minimized when analysis is performed in a flow and injections of serum samples are followed by washing steps in a phosphate buffer solution (PBS) carrier. Under those conditions, the dopamine-aptamer binding affinity in whole human serum of (1.9 ± 0.3) × 104 M-1 s-1 was comparable to the (3.7 ± 0.3) × 104 M-1 s-1 found in PBS, and the dopamine oxidation signal linearly depended on the dopamine concentration, providing a sensitivity of analysis of 73 ± 3 nA µM-1 cm-2 and a LOD of 114 ± 8 nM. The flow-injection apatmer-electrode system was used for direct analysis of basal levels of dopamine in undiluted human serum samples, without using any physical separators (membranes) or filtration procedures. The results suggest a simple strategy for combatting biosurface fouling, otherwise most pronounced at positive electrode potentials used for dopamine detection, and assist in designing more efficient antifouling strategies for biomedical applications.

The Immunomodulatory Drug Glatiramer Acetate is Also an Effective Antimicrobial Agent that Kills Gram-negative Bacteria.

Classic drug development strategies have failed to meet the urgent clinical needs in treating infections with Gram-negative bacteria. Repurposing drugs can lead to timely availability of new antibiotics, accelerated by existing safety profiles. Glatiramer acetate (GA) is a widely used and safe formulation for treatment of multiple sclerosis. It contains a large diversity of essentially isomeric polypeptides with the cationic and amphiphilic character of many antimicrobial peptides (AMP). Here, we report that GA is antibacterial, targeting Gram-negative organisms with higher activity towards Pseudomonas aeruginosa than the naturally-occurring AMP LL-37 in human plasma. As judged from flow cytometric assays, bacterial killing by GA occurred within minutes. Laboratory strains of Escherichia coli and P. aeruginosa were killed by a process of condensing intracellular contents. Efficient killing by GA was also demonstrated in Acinetobacter baumannii clinical isolates and approximately 50% of clinical isolates of P. aeruginosa from chronic airway infection in CF patients. By contrast, the Gram-positive Staphylococcus aureus cells appeared to be protected from GA by an increased formation of nm-scale particulates. Our data identify GA as an attractive drug repurposing candidate to treat infections with Gram-negative bacteria.

Interleukin-6 is increased in plasma and cerebrospinal fluid of community-dwelling domestic dogs with acute ischaemic stroke.

Inflammatory cytokines are potential modulators of infarct progression in acute ischaemic stroke, and are therefore possible targets for future treatment strategies. Cytokine studies in animal models of surgically induced stroke may, however, be influenced by the fact that the surgical intervention itself contributes towards the cytokine response. Community-dwelling domestic dogs suffer from spontaneous ischaemic stroke, and therefore, offer the opportunity to study the cytokine response in a noninvasive set-up. The aims of this study were to investigate cytokine concentrations in plasma and cerebrospinal fluid (CSF) in dogs with acute ischaemic stroke and to search for correlations between infarct volume and cytokine concentrations. Blood and CSF were collected from dogs less than 72 h after a spontaneous ischaemic stroke. Infarct volumes were estimated on MRIs. Interleukin (IL)-2, IL-6, IL-8, IL-10 and tumour necrosis factor in the plasma, CSF and brain homogenates were measured using a canine-specific multiplex immunoassay. IL-6 was significantly increased in plasma (P=0.04) and CSF (P=0.04) in stroke dogs compared with healthy controls. The concentrations of other cytokines, such as tumour necrosis factor and IL-2, were unchanged. Plasma IL-8 levels correlated significantly with infarct volume (Spearman's r=0.8, P=0.013). The findings showed increased concentrations of IL-6 in the plasma and CSF of dogs with acute ischaemic stroke comparable to humans. We believe that dogs with spontaneous stroke offer a unique, noninvasive means of studying the inflammatory processes that accompany stroke while reducing confounds that are unavoidable in experimental models.

Uniform distributions of glucose oxidation and oxygen extraction in gray matter of normal human brain: No evidence of regional differences of aerobic glycolysis.

Regionally variable rates of aerobic glycolysis in brain networks identified by resting-state functional magnetic resonance imaging (R-fMRI) imply regionally variable adenosine triphosphate (ATP) regeneration. When regional glucose utilization is not matched to oxygen delivery, affected regions have correspondingly variable rates of ATP and lactate production. We tested the extent to which aerobic glycolysis and oxidative phosphorylation power R-fMRI networks by measuring quantitative differences between the oxygen to glucose index (OGI) and the oxygen extraction fraction (OEF) as measured by positron emission tomography (PET) in normal human brain (resting awake, eyes closed). Regionally uniform and correlated OEF and OGI estimates prevailed, with network values that matched the gray matter means, regardless of size, location, and origin. The spatial agreement between oxygen delivery (OEF≈0.4) and glucose oxidation (OGI ≈ 5.3) suggests that no specific regions have preferentially high aerobic glycolysis and low oxidative phosphorylation rates, with globally optimal maximum ATP turnover rates (VATP ≈ 9.4 µmol/g/min), in good agreement with (31)P and (13)C magnetic resonance spectroscopy measurements. These results imply that the intrinsic network activity in healthy human brain powers the entire gray matter with ubiquitously high rates of glucose oxidation. Reports of departures from normal brain-wide homogeny of oxygen extraction fraction and oxygen to glucose index may be due to normalization artefacts from relative PET measurements.

Effects of liraglutide on neurodegeneration, blood flow and cognition in Alzheimer´s disease - protocol for a controlled, randomized double-blinded trial.

INTRODUCTION: Type 2 diabetes (DM-2) increases the risk of developing Alzheimer´s disease (AD), and patients with AD are more likely to develop DM-2. DM-2 and AD share some pathophysiological features. In AD, amyloid-ß (Aß) is accumulated as extracellular plaques in the gray matter of the brain, while in DM-2 islet amyloid polypeptide (IAPP) is accumulated in the pancreas. Premature cellular degeneration is seen in both diseases. Glucagon-like peptide-1 (GLP-1) reduces the amount of Aß and improves cognition in animal studies. The present study tests the hypothesis that treatment with the long-acting GLP-1 receptor agonist liraglutide affects the accumulation of Aß in patients with AD. MATERIAL AND METHODS: This is a randomized, controlled, double-blinded intervention study with AD patients treated for six months with liraglutide (n = 20) or placebo (n = 20). The primary outcome is change in deposition of Aß in the central nervous system (CNS) by Pittsburgh compound B positron emission tomography (PET). The secondary outcome is evaluation of cognition using a neuro-psychological test battery, and examination of changes in glucose uptake in the CNS by 18F-fluoro-deoxy-glucose PET. Finally, a perfusion-weighted magnetic resonance imaging with contrast will be performed to evaluate blood flow. CONCLUSION: No registered drug affects the deposition of Aß in the brain of AD patients. Our goal is to find a new therapeutic agent that alters the pathophysiology in AD patients by decreasing the formation of Aß plaques and thereby presumably improves the cognitive function. FUNDING: The trial is investigator-initiated and investigator-driven and is supported by Novo Nordisk Scandinavia. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01469351.

Variable ATP yields and uncoupling of oxygen consumption in human brain.

The distribution of brain oxidative metabolism values among healthy humans is astoundingly wide for a measure that reflects normal brain function and is known to change very little with most changes of brain function. It is possible that the part of the oxygen consumption rate that is coupled to ATP turnover is the same in all healthy human brains, with different degrees of uncoupling explaining the variability of total oxygen consumption among people. To test the hypothesis that about 75% of the average total oxygen consumption of human brains is common to all individuals, we determined the variability in a large group of normal healthy adults. To establish the degree of variability in different regions of the brain, we measured the regional cerebral metabolic rate for oxygen in 50 healthy volunteers aged 21-66 and projected the values to a common age of 25.Within each subject and region, we normalized the metabolic rate to the population average of that region. Coefficients of variation ranged from 10 to 15% in the different regions of the human brain and the normalized regional metabolic rates ranged from 70% to 140% of the population average for each region, equal to a two-fold variation. Thus the hypothetical threshold of oxygen metabolism coupled to ATP turnover in all subjects is no more than 70% of the average oxygen consumption of that population.

Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools.

BACKGROUND: We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats (SHRs) and Sprague-Dawley rats (SDs) one week after tMCAo; (2) to present the practical use of the optical fractionator and 2D nucleator in stereological brain tissue analyses; and (3) to report our experiences with an intraluminal tMCAo model where the occluding filament is advanced 22 mm beyond the carotid bifurcation and the common carotid artery is clamped during tMCAo. METHODS: Twenty-three SDs and twenty SHRs were randomized into four groups subjected to 90 minutes tMCAo or sham. BrdU (50 mg/kg) was administered intraperitoneally twice daily on Day 4 to 7 after surgery. On Day 8 all animals were euthanized. NeuN-stained tissue sections were used for brain and infarct volume estimation with the 2D nucleator and Cavalieri principle. Brains were studied for the presence of activated microglia (ED-1) and hippocampal BrdU incorporation using the optical fractionator. RESULTS: We found no significant difference or increase in post-ischemic NPC proliferation between the two strains. However, the response to remote ischemia may differ between SDs and SHRs. In three animals increased post-stroke NPC proliferation was associated with hippocampal ischemic injury. The mean infarct volume was 89.2 +/- 76.1 mm3 in SHRs and 16.9 +/- 22.7 mm3 in SDs (p < 0.005). Eight out of eleven SHRs had ischemic neocortical damage in contrast to only one out of 12 SDs. We observed involvement of the anterior choroidal and hypothalamic arteries in several animals from both strains and the anterior cerebral artery in two SHRs. CONCLUSIONS: We found no evidence of an early hippocampal NPC proliferation one week after tMCAo in both strains. Infarction within the anterior choroidal artery could induce hippocampal ischemia and increase NPC proliferation profoundly. NPC proliferation was not aggravated by the presence of activated microglia. Intraluminal tMCAo in SHRs gave a more reliable infarct with neocortical involvement, but affected territories supplied by the anterior cerebral, anterior choroidal and hypothalamic arteries.

Serotonin depletion results in a decrease of the neuronal activation caused by rivastigmine in the rat hippocampus.

Interactions between the serotonergic and cholinergic systems are known to occur and are believed to play a role in the mechanism underlying both major depression and Alzheimer's disease. On a molecular level, studies suggest that acetylcholine (ACh) increases serotonin (5-HT) release through nicotinic receptors located at nerve terminals. The aim of the present study was to determine in which areas and to what extent 5-HT mediates the neuronal response to ACh release. For this purpose, neuronal activity was measured in rats with rivastigmine-induced elevated ACh levels after a 95% 5-HT depletion obtained by dosing p-chlorophenylalanine followed by D,L-fenfluramine. Neuronal activation was quantified by stereological measurements of c-Fos immunoreactivity. The brain areas examined were medial prefrontal cortex, septum, dorsal hippocampus, and dorsal raphe nucleus. Rivastigmine significantly increased c-Fos immunoreactivity in medial prefrontal cortex and the hippocampus, but not in the septum and dorsal raphe nucleus. 5-HT depletion decreased ACh-induced c-Fos immunoreactivity in the dentate gyrus. By contrast, 5-HT depletion had no effect on the ACh-induced activity in the other brain areas examined. It is concluded that 5-HT mediates part of the ACh-induced hippocampal neuronal activation, possibly mediated via locally released 5-HT.

Increased in vitro and in vivo transgene expression levels mediated through cis-acting elements.

BACKGROUND: Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. METHODS: Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. RESULTS: Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. CONCLUSION: The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

NS 1231, a novel compound with neurotrophic-like effects in vitro and in vivo.

NS 1231 [5-(4-chlorophenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3.2-h]naphthalene-2,3-dione-3-oxime] belongs to a chemical series of compounds, which exhibit neurotrophic-like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)-differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF-induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF-induced signalling events, such as TrkA phosphorylation at the Shc-binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA-induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.