Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a ‘stringent’ one [60min MCAO/48h reperfusion] and a ‘mild’ one [30min MCAO/72h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the ‘mild’ model at 3d after MCAO. Furthermore, MT- II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long- term studies with different species and larger sampling sizes are required before a clinical use can be envisaged

Оптимизация работы установок электроцентробежных насосов в процессе добычи нефти на Снежном нефтегазоконденсатном месторождении (Томская область)

Объектом исследования является Снежное нефтегазоконденсатное месторождение. Цель работы – изучить оптимизацию работы установок электроцентробежных насосов в процессе добычи нефти на Снежном нефтегазоконденсатном месторождении. В процессе работы были рассмотрены причины отказов в работе установок электроцентробежных насосов, выполнен статистический анализ отказов в работе скважин, оборудованных УЭЦН, показавший, что большая доля причин проведения подземных ремонтов (до 15%) связана с рассогласованием гидравлических характеристик центробежного насоса и пласта.The object of research is the Snezhnoye oil and gas condensate field. The purpose of the work is to study the optimization of the operation of electric centrifugal pump installations in the process of oil production at the Snezhnoye oil and gas condensate field. During the work, the reasons for failures in the operation of electric centrifugal pump installations were considered, a statistical analysis of failures in the operation of wells equipped with ESP was performed, which showed that a large share of the reasons for underground repairs (up to 15%) is associated with a mismatch in the hydraulic characteristics of the centrifugal pump and the formation

Mendelian adult-onset leukodystrophy genes in Alzheimer´s disease. Critical influence of CSF1R and NOTCH3

Mendelian adult-onset leukodystrophies are a spectrum of rare inherited progressive neurodegenerative disorders affecting the white matter of the central nervous system. Among these, Cerebral Autosomal Dominant and Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL and CARASIL), Cerebroretinal vasculopathy (CRV), Metachromatic leukodystrophy (MLD), Hereditary diffuse Leukoencephalopathy with spheroids (HDLS), Vanishing white matter disease (VWM) present with rapidly progressive dementia as dominant feature and are caused by mutations in NOTCH3, HTRA1, TREX1, ARSA, CSF1R, EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5, respectively. Given the rare incidence of these disorders and the lack of unequivocally diagnostic features, leukodystrophies are frequently misdiagnosed with common sporadic dementing diseases such as Alzheimer’s disease (AD), raising the question of whether these overlapping phenotypes may be explained by shared genetic risk factors. To investigate this intriguing hypothesis, we have combined gene expression analysis 1) in 6 different AD mouse strains (APPPS1, HOTASTPM, HETASTPM, TPM, TAS10 and TAU), at 5 different developmental stages (Embryo [E15], 2 months, 4 months, 8 months and 18 months), 2) in APPPS1 primary cortical neurons under stress conditions (oxygen-glucose deprivation) and single-variant and single-gene (c-alpha and SKAT tests) based genetic screening in a cohort composed of 332 Caucasian late-onset AD patients and 676 Caucasian elderly controls. Csf1r was significantly overexpressed (Log2FC>1, adj. p-val<0.05) in the cortex and hippocampus of aged HOTASTPM mice with extensive Aβ core dense plaque pathology. We identified 3 likely pathogenic mutations in CSF1R TK domain (p.L868R, p.Q691H and p.H703Y) in our discovery and validation cohort, composed of 465 AD and MCI Caucasian patients from the UK. Moreover, NOTCH3 was a significant hit in the c-alpha test (adj p-val = 0.01). Adult onset Mendelian leukodystrophy genes are not common factors implicated in AD. Nevertheless, our study suggests a potential pathogenic link between NOTCH3, CSF1R and sporadic LOAD, that warrants further investigation

Effect of metallothionein-II <i>i</i>.<i>p</i>. treatment <i>in vivo</i> 48h after MCAO for 60 min.

<p>Infarct volumes of vehicle (0.9% NaCl) (n = 7) or MT-II (<i>i</i>.<i>p</i>.) treated (n = 8) male adult wildtype C57BL/6N mice after 60min MCAO and 48h reperfusion. <b>A</b>: direct infarct volumes. <b>B</b>: inflammatory cell count in the ischemic hemisphere as determined by counting Iba1-positive cells in whole ischemic brain hemispheres of mice at 48h of reperfusion after 60min MCAO. Inflammatory cell accumulation (macrophages and activated microglia) was determined by counting Iba1-positive cells at interaural position No.III (distance to bregma 3.9mm) in the whole ischemic/ipsilateral hemisphere. <b>C</b>: Brain swelling, as calculated by the difference between direct and indirect infarct volumes. Whereas indirect infarct volumes were calculated as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral/ischemic hemisphere (p-values between groups were >0.05 as calculated by Mann Whitney U-Test, one-tailed). <b>D:</b> neurological deficit of mice at 48h after induction of MCAO as determined by a modified Bederson score [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144035#pone.0144035.ref041" target="_blank">41</a>]: 0 represents no deficits, 1 represents an extension deficit in the contralateral leg, 2 a hemiparesis with circling, 3 loss of postural reflexes, and 4 death.</p

Gene expression analysis of pro-inflammatory proteins with real-time PCR.

<p>Relative mRNA expression of selected pro-inflammatory proteins was analysed in real-time PCR with gene-specific primers in whole mouse brain hemispheres of adult male wild-type C57BL/6N mice treated with either MT-II <i>i</i>.<i>p</i>. only without MCAO (MT-II-treated; n = 3), in mice at 72h after 30min MCAO that were treated with MT-II <i>i</i>.<i>p</i>. (n = 4), and in mice at 72h after 30min MCAO that were treated with vehicle only (n = 6). cDNA was separately isolated from each hemisphere. Relative Expression is presented as fold changes, calculated with the ΔΔCt-Method, normalized to HPRT mRNA expression and related to the non-ischemic/contralateral hemispheres as references. TNFα induction after MCAO is significantly reduced in mice that were treated with MT-II <i>i</i>.<i>p</i> when compared to vehicle-only treated mice (*p < 0.05, students one-side t-test). Data are shown as relative mRNA expression of each gene and is calculated from two technical replications of n = 3 to 6 mice for each condition (values are given as mean ± SEM).</p

Effect of metallothionein-II <i>i</i>.<i>p</i>. treatment <i>in vivo</i> 72h after MCAO for 30 min.

<p>Infarct volumes of vehicle (0.9% NaCl) (n = 8) or MT-II <i>i</i>.<i>p</i>. (n = 9) treated male adult wildtype C57BL/6N mice after 60min MCAO and 48h reperfusion. <b>A</b>: Direct infarct volumes (*<i>p</i>-value < 0.05 as calculated by Mann Whitney U-Test, one-tailed). <b>B</b>: Inflammatory cell count in the ischemic hemisphere as determined by counting Iba1-positive cells in whole ischemic brain hemispheres of mice at 72h of reperfusion after 30min MCAO. Inflammatory cell accumulation (macrophages and activated microglia) was determined by counting Iba1-positive cells at interaural position No.III (distance to bregma 3.9mm) in the whole ischemic/ipsilateral hemisphere <b>C</b>: Brain swelling, as calculated by the difference between direct and indirect infarct volumes. Whereas indirect infarct volumes were calculated as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral/ischemic hemisphere. (NaCl: vehicle-treated mice, MT-II: MT-II <i>i</i>.<i>p</i>.-treated wild-type mice). <b>D:</b> Neurological deficit of mice at 72h after induction of MCAO as determined by a modified Bederson score [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144035#pone.0144035.ref041" target="_blank">41</a>]: 0 represents no deficits, 1 represents an extension deficit in the contralateral leg, 2 a hemiparesis with circling, 3 loss of postural reflexes, and 4 death (*<i>p</i> < 0.05).</p

Blocking TLR2 in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy

Effect of <i>i</i>.<i>v</i>. metallothionein-II treatment <i>in vivo</i> evaluated at 48h after MCAO for 45 min.

<p>Infarct volumes of vehicle (0.9% NaCl) (n = 8), MT-II (<i>i</i>.<i>v</i>.) (n = 12), rtPA (<i>i</i>.<i>v</i>.) (n = 11), or rtPA and MT-II co-application (<i>i</i>.<i>v</i>.) (n = 11) injected wildtype C57BL6/N male mice after 45min MCAO and 48h (<b>A</b>), Kaplan-Meier Analysis of animal survival (<b>B</b>), and brain swelling (<b>C</b>; as calculated by the difference of direct and indirect infarct volumes), and neurological deficit (<b>D</b>) in a standard model of cerebral ischemia (45min MCAO and 48h reperfusion) after intravenous treatment with metallothionein-II or vehicle only in combination with or without co-application of rtPA (<i>*p</i> = 0.0187; as calculated by log rank Mantel Cox test). Data <b>A</b> presented as scatter dot blots in combination with mean ± SEM. For comparison of infarct volumes and neurological deficit, ANOVA with Kruskal-Wallis test was used.</p

Primary neuronal cells treated with metallothionein-II (MT-II) are protected against oxygen glucose deprivation (OGD) <i>in vitro</i>.

<p>Shown is the relative LDH release of primary neuronal cells after OGD (shown as “cell death (%LDH release)”). Cells are treated with various different concentrations of MT-II before induction of oxygen and glucose deprivation (<b>MT-II</b>: primary neuronal cells pretreated with metallothionein-II 12 h before OGD; <b>co</b>: control cells without OGD [BSS₂₀]). (*p = 0.0198, as calculated by Mann Whitney test; one-tailed).</p

Improving quality of preclinical academic research through auditing:

How much can we rely on whether what was reported in a study was actually done? Systematic and independent examination of records, documents and processes through audits are a central element of quality management systems. In the context of current concerns about the robustness and reproducibility of experimental biomedical research audits have been suggested as a remedy a number of times. However, audits are resource intense and time consuming, and due to their very nature may be perceived as inquisition. Consequently, there is very little experience or literature on auditing and assessments in the complex preclinical biomedical research environment. To gain some insight into which audit approaches might best suit biomedical research in academia, in this study we have applied a number of them in a typical academic neuroscience environment consisting of twelve research groups with about 100 researchers, students and technicians, utilizing the full gamut of state of the art methodology. Several types of assessments and internal as well as external audits (including the novel format of a peer audit) were systematically explored by a team of quality management specialists. An experimental design template was developed (and is provided here) that takes into account and mitigates difficulties, risks and systematic errors that may occur during the course of a study. All audits were performed according to a pre-defined workflow developed by us. Outcomes were assessed qualitatively. We asked for feedback from participating employees in every final discussion of an audit and documented this in the audit reports. Based on these reports follow-up audits were improved. We conclude that several realistic options for auditing exist which have the potential to improve preclinical biomedical research in academia, and have listed specific recommendations regarding their benefits and provided practical resources for their implementation (e.g. study design and audit templates, audit workflow)