Diffusion Measurements To Understand Dynamics and Structuring in Solutions Involving a Homologous Series of Ionic Liquids.

The self-diffusion coefficients of each of the components in mixtures containing pyridine and each of the homologous series 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imides in acetonitrile were determined using NMR diffusometry (i. e., Pulsed Gradient Spin Echo). The nature of solvation was found to change significantly with the proportion of salt in the mixtures. Increased diffusion coefficients (when corrected for viscosity) for the molecular components were observed with increasing proportion of ionic liquid and with increasing alkyl chain length on the cation. Comparison of the molecular solvents suggests increased interactions in solution of the pyridine with other components of the mixture, consistent with the proposed interactions shown previously to drive changes in reaction kinetics. Discontinuities were seen in the diffusion data for each species in solution across different ionic liquids between the hexyl and octyl derivatives, suggesting a change in the structuring in solution as the alkyl chain on the cation changes and demonstrating the importance of such when considering homologous series.

Extracellular vesicles-Mediators of opioid use disorder?

Opioid use disorder (OUD) is a growing health emergency in the United States leading to an epidemic of overdose deaths. OUD is recognized as an addictive brain disorder resulting in psychological, cognitive and behavioural dysfunction. These observed clinical dysfunctions are a result of cellular changes that occur in the brain. Derangements in inflammation, neurogenesis and synaptic plasticity are observed in the brains of OUD patients. The mechanisms of these derangements are unclear; however, extracellular vesicles (EVs), membrane bound particles containing protein, nucleotides and lipids are currently being investigated as agents that invoke these cellular changes. The primary function of EVs is to facilitate intercellular communication by transfer of cargo (protein, nucleotides and lipids) between cells; however, changes in this cargo have been observed in models of OUD suggesting that EVs may be agents promoting the observed cellular derangements. This review summarizes evidence that altered cargo of EVs, specifically protein and miRNA, in models of OUD promote impairments in neurons, astrocytes and microglial cells. These findings support the premise that opioids alter EVs to detrimentally affect neuro-cellular function resulting in the observed addictive, psychological and neurocognitive deficits in OUD patients.

Effects of Ionic Liquids on the Nucleofugality of Chloride.

The nucleofugality of chloride has been measured in solvent mixtures containing ionic liquids for the first time, allowing reactivity in these solvents to be put in context with molecular solvents. Using well-described electrofuges, solvolysis rate constants were determined in mixtures containing different proportions of ethanol and the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide; the different solvent effects observed as the mixture changed could be explained using interactions of the ionic liquid with species along the reaction coordinate, determined using temperature dependent kinetic studies. The solvolysis data allowed determination of the nucleofugality of chloride in these mixtures, which varied with the proportion of salt in the reaction mixture, demonstrating quantitatively the importance of the amount of ionic liquid in the reaction mixture in determining reaction outcome. Nucleofugality data for chloride were determined in seven further ionic liquids, with the reactivity shown to vary over more than an order of magnitude. This outcome illustrates that the components of the ionic liquid are critical in determining reaction outcome. Overall, this work quantitatively extends the understanding of solvent effects in ionic liquids and demonstrates the potential for such information to be used to rationally select an ionic liquid to control reaction outcome.

Rapid relaxation NMR measurements to predict rate coefficients in ionic liquid mixtures. An examination of reaction outcome changes in a homologous series of ionic liquids.

A series of ionic liquids based on the 1-alkyl-3-methylimidazolium cations were examined as components of the solvent mixture for a bimolecular substitution process. The effects on both the rate coefficient of the process and the NMR spin-spin relaxation of the solvent components of changing either the alkyl chain length or the amount of ionic liquid in the reaction mixture were determined. At a constant mole fraction, a shorter alkyl chain length resulted in a greater rate coefficient enhancement and a longer relaxation time, with the opposite effects for a longer alkyl chain length. For a given ionic liquid, increasing the proportion of salt in the reaction mixture resulted in a greater rate coefficient and a shorter relaxation time. The microscopic origins of the rate coefficient enhancement were determined and a step change found in the activation parameters on increasing the alkyl chain length from hexyl to octyl, suggesting notable structuring in solution. Across a range of ionic liquids and solvent compositions, the relaxation time from NMR measurements was shown to relate to the reaction rate coefficient. The approach of using fast and simple NMR relaxation measurements to predict reaction outcomes was exemplified using a morpholinium-based ionic liquid.

F-Actin is associated with a worsening qSOFA score and intensive care unit admission in emergency department patients at risk for sepsis.

Objective: We previously demonstrated that plasma levels of F-actin and Thymosin Beta 4 differs among patients with septic shock, non-infectious systemic inflammatory syndrome and healthy controls and may serve as biomarkers for the diagnosis of sepsis. The current study aims to determine if these proteins are associated with or predictive of illness severity in patients at risk for sepsis in the Emergency Department (ED).Methods: Prospective, biomarker study enrolling patients (>18 years) who met the Shock Precautions on Triage Sepsis rule placing them at-risk for sepsis.Results: In this study of 203 ED patients, F-actin plasma levels had a linear trend of increase when the quick Sequential Organ Failure Assessment (qSOFA) score increased. F-actin was also increased in patients who were admitted to the Intensive Care Unit (ICU) from the ED, and in those with positive urine cultures. Thymosin Beta 4 was not associated with or predictive of any significant outcome measures.Conclusion: Increased levels of plasma F-actin measured in the ED were associated with incremental illness severity as measured by the qSOFA score and need for ICU admission. F-actin may have utility in risk stratification of undifferentiated patients in the ED presenting with signs and symptoms of sepsis.

Controlling the outcome of SN2 reactions in ionic liquids: from rational data set design to predictive linear regression models.

Rate constants for a bimolecular nucleophilic substitution (SN2) process in a range of ionic liquids are correlated with calculated parameters associated with the charge localisation on the cation of the ionic liquid (including the molecular electrostatic potential). Simple linear regression models proved effective, though the interdependency of the descriptors needs to be taken into account when considering generality. A series of ionic liquids were then prepared and evaluated as solvents for the same process; this data set was rationally chosen to incorporate homologous series (to evaluate systematic variation) and functionalities not available in the original data set. These new data were used to evaluate and refine the original models, which were expanded to include simple artificial neural networks. Along with showing the importance of an appropriate data set and the perils of overfitting, the work demonstrates that such models can be used to reliably predict ionic liquid solvent effects on an organic process, within the limits of the data set.

Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.

Thymosin beta 4 (Tß4), a secreted 43 amino acid peptide, promotes oligodendrogenesis, and improves neurological outcome in rat models of neurologic injury. We demonstrated that exogenous Tß4 treatment up-regulated the expression of the miR-200a in vitro in rat brain progenitor cells and in vivo in the peri-infarct area of rats subjected to middle cerebral artery occlusion (MCAO). The up-regulation of miR-200a down-regulated the expression of the following targets in vitro and in vivo models: (i) growth factor receptor-bound protein 2 (Grb2), an adaptor protein involved in epidermal growth factor receptor (EGFR)/Grb2/Ras/MEK/ERK1/c-Jun signaling pathway, which negatively regulates the expression of myelin basic protein (MBP), a marker of mature oligodendrocyte; (ii) ERRFI-1/Mig-6, an endogenous potent kinase inhibitor of EGFR, which resulted in activation/phosphorylation of EGFR; (iii) friend of GATA 2, and phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which are potent inhibitors of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, and resulted in marked activation of AKT; and (iv) transcription factor, p53, which induces pro-apoptotic genes, and possibly reduced apoptosis of the progenitor cells subjected to oxygen glucose deprivation (OGD). Anti-miR-200a transfection reversed all the effects of Tß4 treatment in vitro. Thus, Tß4 up-regulated MBP synthesis, and inhibited OGD-induced apoptosis in a novel miR-200a dependent EGFR signaling pathway. Our findings of miR-200a-mediated protection of progenitor cells may provide a new therapeutic importance for the treatment of neurologic injury. Tß4-induced micro-RNA-200a (miR-200a) regulates EGFR signaling pathways for MBP synthesis and apoptosis: up-regulation of miR-200a after Tß4 treatment, increases MBP synthesis after targeting Grb2 and thereby inactivating c-Jun from inhibition of MBP synthesis; and also inhibits OGD-mediated apoptosis after targeting EGFR inhibitor (Mig-6), PI3K inhibitors (FOG2 and Pten) and an inducer (p53) of pro-apoptotic genes, for AKT activation and down-regulation of p53. These findings may contribute the therapeutic benefits for stroke and other neuronal diseases associated with demyelination disorders.

Plasma levels of F-actin and F:G-actin ratio as potential new biomarkers in patients with septic shock.

OBJECTIVE: To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls. RESULTS: F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml). CONCLUSIONS: F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.

Thymosin ß4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.

Thymosin ß4 (Tß4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tß4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tß4 on the TLR pathway. Cells were grown in the presence of Tß4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tß4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tß4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tß4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tß4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tß4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.

Small-Angle Neutron Scattering Insights into 2-Ethylhexyl Laurate: A Remarkable Bioester.

Commercial (protiated) samples of the "green" and biodegradable bioester 2-ethylhexyl laurate (2-EHL) were mixed with D-2-EHL synthesized by hydrothermal deuteration, with the mixtures demonstrating bulk structuring in small-angle neutron scattering measurements. Analysis in a polymer scattering framework yielded a radius of gyration (Rg) of 6.5 Å and a Kuhn length (alternatively described as the persistence length or average segment length) of 11.2 Å. Samples of 2-EHL dispersed in acetonitrile formed self-assembled structures exceeding the molecular dimensions of the 2-EHL, with a mean aggregation number (Nagg) of 3.5 ± 0.2 molecules across the tested concentrations. We therefore present structural evidence that this ester can function as a nonionic (co)surfactant. The available surfactant-like conformations appear to enable performance beyond the low calculated hydrophilic-lipophilic balance value of 2.9. Overall, our data offer an explanation for 2-EHL's interfacial adsorption properties via self-assembly, resulting in strong emolliency and lubricity for this sustainable ester-based bio-oil.

CLINICAL, MOLECULAR, AND EXOSOMAL MECHANISMS OF CARDIAC AND BRAIN DYSFUNCTION IN SEPSIS.

ABSTRACT: Sepsis is a complex disease resulting from a dysregulated inflammatory response to an infection. Initiation of sepsis occurs from a localized infection that disseminates to the bloodstream placing all organ systems at risk. Septic shock is classically observed to manifest itself as systemic hypotension with hyporesponsiveness to vasopressor agents. Myocardial dysfunction occurs resulting in an inability to perfuse major organ systems throughout the body. Most importantly, the brain is hypoperfused creating an ischemic and inflammatory state resulting in the clinical observation of acute mental status changes and cognitive dysfunction commonly known as sepsis-associated encephalopathy. This short review describes the inflammatory molecular mechanisms of myocardial dysfunction, discusses the evidence of the dual roles of the microglia resulting in blood-brain barrier disruption, and suggests that septic-derived exosomes, endosome-derived lipid bilayer spheroids released from living cells, influence cardiac and neurological cellular function.

Thymosin ß 4 mediates oligodendrocyte differentiation by upregulating p38 MAPK.

Thymosin beta 4 (Tß4), a G-actin sequestering peptide, increases oligodendrogenesis and improves functional outcome in models of neurological injury. The molecular mechanisms of Tß4 mediated oligodendrogenesis are unclear. The p38 mitogen-activated protein kinase (p38MAPK) regulates oligodendrocyte (OL) differentiation and myelin gene expression in other models. Therefore, we investigated p38MAPK signaling pathways. We used primary rat neural progenitor cells (NPCs) and a mouse oligodendrocyte progenitor cell (OPC) line (N20.1 cells) to investigate the molecular mechanisms of Tß4-enhanced oligodendrogenesis. NPCs were isolated from rat subventricular zone (SVZ) of the lateral ventricles (n = 12). Primary NPCs and N20.1 cells were grown in the presence of 0, 25, and 50 ng/mL of Tß4 (RegeneRx Biopharmaceuticals Inc, Rockville, MD) for 14 days. Quantitative real-time PCR and Western blot data showed significant induction of both expression and phosphorylation of p38MAPK with simultaneous inhibition of phosphorylation of extracellular signal regulated kinase (ERK1), c-Jun N-terminal kinase 1 (JNK1), leading to reduction of phosphorylation of c-Jun, a potent negative regulator of transcription of myelin genes. These effects were reversed with transfection of Tß4siRNA. Our data indicate that Tß4 treatment induces OL differentiation by inducing p38MAPK with parallel inactivation of ERK1 and JNK1, thus preventing the accumulation of phosphorylated c-Jun.

Proteomic Profiles of Exosomes of Septic Patients Presenting to the Emergency Department Compared to Healthy Controls.

BACKGROUND: Septic Emergency Department (ED) patients provide a unique opportunity to investigate early sepsis. Recent work focuses on exosomes, nanoparticle-sized lipid vesicles (30-130 nm) that are released into the bloodstream to transfer its contents (RNA, miRNA, DNA, protein) to other cells. Little is known about how early changes related to exosomes may contribute to the dysregulated inflammatory septic response that leads to multi-organ dysfunction. We aimed to evaluate proteomic profiles of plasma derived exosomes obtained from septic ED patients and healthy controls. METHODS: This is a prospective observational pilot study evaluating a plasma proteomic exosome profile at an urban tertiary care hospital ED using a single venipuncture blood draw, collecting 40 cc Ethylenediaminetetraacetic acid (EDTA) blood. MEASUREMENTS: We recruited seven patients in the ED within 6 h of their presentation and five healthy controls. Plasma exosomes were isolated using the Invitrogen Total Exosome Isolation Kit. Exosome proteomic profiles were analyzed using fusion mass spectroscopy and Proteome Discoverer. Principal component analysis (PCA) and differential expression analysis (DEA) for sepsis versus control was performed. RESULTS: PCA of 261 proteins demonstrated septic patients and healthy controls were distributed in two groups. DEA revealed that 62 (23.8%) proteins differed between the exosomes of septic patients and healthy controls, p-value < 0.05. Adjustments using the False Discovery Rate (FDR) showed 23 proteins remained significantly different (FDR < 0.05) between sepsis and controls. Septic patients and controls were classified into two distinct groups by hierarchical clustering using the 62 nominally DE proteins. After adjustment multiple comparisons, three acute phase proteins remained significantly different between patients and controls: Serum amyloid A-1, C-reactive protein and Serum Amyloid A-2. Inflammatory response proteins immunoglobulin heavy constant Δ and Fc-fragment of IgG binding protein were increased. CONCLUSION: Exosome proteomic profiles of septic ED patients differ from their healthy counterparts with regard to acute phase response and inflammation.

Sildenafil treatment of subacute ischemic stroke: a safety study at 25-mg daily for 2 weeks.

BACKGROUND: In several animal studies of young and aged rats with ischemic stroke, treatment with sildenafil improved functional outcomes compared with placebo. We conducted a safety study of sildenafil (25 mg daily for 2 weeks) shortly after ischemic stroke onset. METHODS: We recruited patients aged 18 to 80 years with ischemic stroke, National Institutes of Health stroke scale (NIHSS) score 2 to 21, between days 2 and 9 after symptom onset. Patients were treated with sildenafil for 2 weeks (25 mg daily). The primary outcome measure was the adverse occurrence of any of the following during the treatment period: stroke worsening, new stroke, myocardial infarction, vision loss, hearing loss, or death from any cause. Secondary outcome measures were NIHSS score, Barthel indices, and modified Rankin score at 90 days. RESULTS: Twelve patients were recruited. Mean age was 57 years, 5 were female, and median NIHSS score at entry was 9.5 (range 2-20). The primary outcome measure occurred in one patient (sudden death). Another patient committed suicide 2 months after study entry (and 6 weeks after treatment with sildenafil had been completed). Among the 10 survivors, at 90 days, median NIHSS score was 2 (range 0-12), median Barthel index was 95 (range 15-100), and median modified Rankin score was 1.5 (range 0-5). CONCLUSIONS: Sildenafil (25 mg daily for 2 weeks) appeared to be safe in this group of patients with mild to moderately severe stroke. Further studies of higher doses will be tested.

Blood pressure declines and less favorable outcomes in the NINDS tPA stroke study.

BACKGROUND AND PURPOSE: Hypertension is the most important modifiable risk factor for secondary stroke prevention but the immediate management of blood pressure after stroke is uncertain. We evaluated outcomes in the NINDS tPA stroke study in relation to blood pressure declines during the first 24 h after randomization. METHODS: Declines in blood pressure compared to baseline and preceding time points were analyzed in relationship to favorable outcomes (by a global test), poor outcomes (Rankin scale >3) and death at 3 months. RESULTS: 551 patients did not receive immediate pre-randomization anti-hypertensive treatment and had available blood pressures. Multivariate analysis showed significantly and progressively reducing likelihoods of a favorable outcome with each 10 mmHg decline in systolic blood pressure (SBP) >50 mmHg compared to any preceding measurement. Poor outcomes were significantly more likely in patients with >50 mmHg SBP reduction (or >30 mmHg compared to any immediately preceding measurement). There was an increased risk of death with blood pressure declines >60 mmHg. tPA treatment still produced favorable outcomes compared with placebo even with blood pressure declines. The median largest SBP reduction from baseline in patients treated with tPA was 35 mmHg compared to 30 mmHg in placebo-treated patients (p<0.01). CONCLUSIONS: In this post hoc analysis, progressively reducing likelihoods of a favorable outcome were seen with increasing declines in SBP. Despite a greater likelihood of favorable outcomes, tPA treatment was associated with a greater reduction in blood pressure than placebo. Randomized trials of blood pressure management are needed.

Thymosin ß4 for the treatment of acute stroke: neurorestorative or neuroprotective?

INTRODUCTION: Thymosin ß4 (Tß4) is a 5K peptide which influences cellular migration by inhibiting organization of the actin-cytoskeleton. Treatment of acute stroke presently involves use of rt-PA and/or endovascular treatment with thrombectomy, both of which have time limitations. Therefore, development of a treatment beyond these times is necessary as most stroke patients present beyond these time limits. A drug which could be administered within 24 h from symptom onset would provide substantial benefit. AREAS COVERED: This review summarizes the data and results of two in-vivo studies testing Tß4 in an embolic stroke model of young and aged rats. In addition, we describe in-vitro investigations of the neurorestorative and neuroprotective properties of Tß4 in a variety of neuroprogenitor and oligoprogenitor cell models. EXPERT OPINION: Tß4 acts as a neurorestorative agent when employed in a young male rat model of embolic stroke while in an aged model it acts a neuroprotectant. However evaluation of Tß4 as a treatment of stroke requires further preclinical evaluation in females and in males and females with comorbidities such as, hypertension and diabetes in models of embolic stroke to further define the mechanism of action and potential as a treatment of stroke in humans.

Thymosin beta 4 regulation of actin in sepsis.

INTRODUCTION: Sepsis is the dysregulated host response to an infection resulting in life-threatening organ damage. Thymosin Beta 4 is an actin binding protein that inhibits the polymerization of G-actin into F-actin and improves mortality when administered intravenously to septic rats. Thymosin Beta 4 decreases inflammatory mediators, lowers reactive oxygen species, up-regulates anti-oxidative enzymes, anti-inflammatory genes, and anti-apoptotic enzymes making it an interesting protein to study in sepsis. AREAS COVERED: The authors summarize the current knowledge of actin and Thymosin Beta 4 as it relates to sepsis via a comprehensive literature search. EXPERT OPINION: Sepsis results in measurable levels of F-actin in the circulation as well as a decreased concentration of Thymosin Beta 4. It is speculated that F-actinemia contributes to microcirculatory perturbations present in patients with sepsis by disturbing laminar flow. Given that Thymosin Beta 4 inhibits the polymerization of F-actin, it is possible that Thymosin Beta 4 decreases mortality in sepsis via the regulation of actin as well as its other anti-inflammatory properties and should be further pursued as a clinical trial in humans with sepsis.

Stroke induces gene profile changes associated with neurogenesis and angiogenesis in adult subventricular zone progenitor cells.

Neural progenitor cells in the subventricular zone (SVZ) of the lateral ventricular wall give rise to new neurons throughout rodent life. Ischemic stroke induces angiogenesis and neurogenesis. Using laser capture microdissection (LCM) in combination with microarrays containing approximately 400 known genes associated with stem cells and angiogenesis, we investigated gene profiles of SVZ cells in the adult mouse subjected to middle cerebral artery occlusion. Our data revealed that nonstroke SVZ cells expressed sets of genes that are important for neural progenitor cell proliferation, differentiation, and migration. In addition, stroke SVZ cells expressed many genes involved in neurogenesis during embryonic development but were not detected in nonstroke SVZ cells. Stroke upregulated genes were verified by real-time reverse transcriptase-polymerase chain reaction and immunostaining. These data indicate that adult SVZ cells recapture embryonic molecular signals after stroke and provide insight into the molecular mechanisms, which regulate the biological function of neural progenitor cells in the SVZ of adult rodent brain under physiological and stroke conditions.

Wnt expression in the adult rat subventricular zone after stroke.

Neurogenesis occurs in adult brain neural progenitor cells (NPCs) located in the subventricular zone (SVZ) of the lateral ventricle and the subgrandular zone of the hippocampal dentate gyrus. After ischemic stroke, NPCs in the SVZ proliferate and migrate towards the ischemic boundary region to replenish damaged neurons. During development, the Wnt pathways contribute to stem cell maintenance and promote neurogenesis. We hypothesized that stroke up regulates Wnt family genes in SVZ cells. Non-ischemic and ischemic cultured SVZ cells and a single population of non-ischemic and ischemic SVZ cells isolated by laser capture microdissection (LCM) were analyzed for Wnt pathway expression using real-time RT-PCR and immunostaining. The number of neurospheres increased significantly (p<0.05) in SVZ cells derived from ischemic (32+/-4.7/rat) compared with the number in non-ischemic SVZ cells (18+/-3/rat). Wnt family gene mRNA levels were detected in SVZ cells isolated from both cultured and LCM SVZ cells, however there was no up regulation between non-ischemic and ischemic SVZ cells. Immunostaining on brain sections also demonstrated no up regulation of Wnt pathway protein between ischemic and non-ischemic SVZ cells. Expression of the Wnt family genes in SVZ cells support the hypothesis that the Wnt pathway may be involved in neurogenesis in the adult brain. However, ischemia does not up regulate Wnt family gene expression.

Thymosin ß4 for the treatment of acute stroke in aged rats.

Thymosin ß4 (Tß4) is a 5K peptide which influences cellular migration by inhibiting organization of the actin-cytoskeleton. Tß4 has neurorestorative properties and is a potential candidate for the treatment of sub-acute stroke. Previous research demonstrated that Tß4 improved neurological outcome in a young (3 months) rat model of embolic stroke. We hypothesized that Tß4 would improve neurological outcome in an aged rat model of embolic stroke when administered 24h after embolic stroke. Aged Male Wistar rats (Charles River, France 18-21 months) were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were randomized to receive Tß4 (12mg/kg, RegeneRx Biopharmaceuticals, Inc.) or control 24h after MCAo and then every 3days for 4 additional doses. The dose of 12mg/kg was the maximal dose of Tß4 that showed functional improvement in a young rat model of embolic stroke. Functional tests (adhesive-removal test (ART), foot fault test (FFT) and the modified Neurological Severity Score (mNSS)) were performed weekly. The rats were sacrificed 56days after MCAo and lesion volumes were measured. Immunohistochemical analysis for oligodendrogenesis, myelination and gliosis was also performed. Twenty-three rats were included in the study: control group (n=12) and Tß4 group (n=11). After randomization, there were three deaths in both the control and Tß4 groups. The Tß4 treatment reduced infarct volume by more than 50% (12.8%±9.3%, mean±SE, p<0.05) compared to the control group (26.0%±4.3%). However, Tß4 did not show improvement in functional outcome compared to control. There was no significant increase in oligodendrogenesis, myelination and gliosis between control and treatment with Tß4, however, we unexpectedly observed that overall (control and Tß4 groups) astrocytic gliosis as measured by GFAP immunoreactivity was significantly inversely correlated with neurological outcome measured using the modified Neurological Severity Score (mNSS) (p<0.01), suggesting that greater gliosis may be related to improvement of neurological outcome in aged rats. In summary, Tß4 treatment of stroke aged rats significantly reduces infarct volume compared to vehicle treated stroke, however, Tß4 treatment did not show improvement in functional outcome, myelination or gliosis when compared to control. GFAP staining was significantly inversely correlated to improvement in the mNSS, suggesting that gliosis in the aged rat may be of benefit in improvement of functional outcome.