Non-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF).t test, as applicable.Various inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, or Student’s Here, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells.Our study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS.The online version of this article (doi:10.1186/s12974-016-0698-0) contains supplementary material, which is available to authorized users. Etiopathogenesis of amyotrophic lateral sclerosis (ALS), the devastating and relentlessly progressing neurodegenerative disorder leading to muscular weakness, is poorly understood. Approximately 90 % of the total cases reported have unknown etiology and are categorized as sporadic ALS. However, the rest 10 % of the cases follow an autosomal dominant inheritance pattern , and only ~20 % of these may be mapped to the mutations in the SOD-1 gene, which forms the basis of the animal models of ALS .The activated astrocytes may adopt either a neuroprotective or a neurotoxic phenotype in a stimulus-dependent manner by a process termed as glial polarization; the end-results of which depend largely on the microenvironment experienced by the astrocytes , 6. For The plausible role of astrocytes in the initiation/progression of ALS has been studied employing chimeric mSOD1/TDP-43 models or human iPSC-derived astrocytes from ALS patients –13. SuchGlutamate-associated excitotoxicity following the selective loss of astroglial glutamate transporters, leading to reduced synaptic glutamate uptake, has been reported in the autopsy samples as well as the animal models of ALS , 15. HowTo address this, we have established and characterized a cellular/animal model for SALS, which involves studying the toxic effect of the cerebrospinal fluid from the sporadic ALS patients (ALS-CSF). Such a model recapitulates the SALS pathology effectively. Using behavioral, immunocytochemical, molecular, electron microscopy and electrophysiological approaches, we have earlier demonstrated the potential of ALS-CSF to induce morphological and functional changes in motor neurons and glia. This was seen in neurons in the form of necroptosis-mediated neuronal death, reduced ChAT, Nav1.6 and Kv1.6 expression, aberrant phosphorylation of neurofilaments; mitochondrial, Golgi apparatus, ER and lysosomal defects, and reduced expression of trophic factors –29. ConcMass spectrometric analysis of the ALS-CSF demonstrated a clear up-regulation of glial inflammatory proteins including chitotriosidase, osteopontin, and chitinases 3 like protein-1 and 2 in the CNS circulation of the ALS patients, thus emphasizing the role of glia in SALS pathology . We cond4 cells/ml to avoid contamination with the non-astroglial cells, including microglia [Enriched astrocyte cultures were obtained from Wistar rats (P0-P2) using a modified protocol of Kerstetter and Miller . Brieflyicroglia . The ast4 cells/ml in sterile 24-well plates and allowed to differentiate for 3–4 days in vitro, or until the cultures attained 70–80 % confluence. The cultures were then subjected to different experimental conditions as mentioned below.The NSC-34 motor neuronal cell line was procured , maintained, and propagated as per the published protocol . For expSix drug-naive patients diagnosed to have definite ALS and the age-matched neurological patients serving as disease control were selected for CSF collection. The samples were collected after obtaining informed consent as per the institutional human ethics committee guidelines. The diagnosis of ALS was based on the revised El Escorial criteria . The meaV/V CSF in DMEM, and the effects were studied in duplicates for all the six CSF samples discretely. Thus, depending on the experiments, a minimum of three and a maximum of six separate cultures were studied in replicates for each experimental group. The study consisted of three experimental groups, namely:Normal control (NC): cultures propagated in DMEMV/V in DMEM) other than neurodegenerative and neuroinflammatory disordersNALS (disease control): cultures exposed to CSF from the age-matched patients suffering from neurological diseases such as intracranial hypertension ALS: cultures exposed to CSF from ALS patients , and the standards were prepared according to the manufacturer’s instructions. For a temporal analysis, the cultures were exposed to CSF for 12, 24, and 48 h, respectively, and the media from the cultures was used for the estimation.6 cells were harvested from the astroglial cultures exposed to ALS-CSF or the normal controls for 48 h, and lysates were prepared. Simultaneously, the medium from the normal controls and each of the subsets exposed to ALS-CSF for 12, 24, and 48 h was collected and centrifuged at 14,000 rpm to remove the debris. The cytosolic and secreted glutamate levels were measured using the glutamate assay kit . The measurement was based on enzymatic conversion, and the absorbance was read at 450 nm using a colorimetric ELISA microplate plate reader .Approximately 10The lysates from the astroglial cultures exposed to ALS-CSF for 48 h or the normal controls were prepared as described above. Simultaneously, the medium from the normal cultures and those exposed to ALS-CSF was also collected. The level of nitrates was assayed in the samples using nitric oxide assay kit  at 540 nm using a colorimetric ELISA microplate reader .The ROS levels were measured in astroglial cultures exposed to ALS-CSF using dichlorofluorescin diacetate (DCFDA), a compound that reacts with the intracellular ROS to produce fluorescence. Briefly, the cultures were treated with DCFDA, harvested, and lysed, and the resultant fluorescence release was measured at 536 nm in an ELISA plate reader. Fluorescence/cell was calculated and statistically analyzed. For qualitative studies, the cells were plated onto 13-mm coverslips. After exposure, the cells were incubated with Locke’s solution and DCFDA and directly viewed  using confocal microscopy .Cell viability for NSC-34 cell line was assayed using MTT assay . Briefly4 cells/ml onto the 13-mm circular coverslips pre-coated with poly-l-lysine (0.1 mg/ml). These were exposed to the aforementioned experimental conditions for 48 h, after which the cells were fixed with 4 % paraformaldehyde (PFA) for 15 min at RT. Blocking was carried out with 3 % bovine serum albumin (BSA) and incubated with primary antibodies of interest for 24 h, followed by appropriate secondary antibodies tagged with FITC or CY3 for 2 h at RT. The specificity was ensured for both primary and secondary antibodies by adding relevant positive and/or negative controls to the experiments. For double immunofluorescence, the coverslips were re-equilibrated with 0.1 M PBS (pH 7.4) and blocked with 3 % BSA, following which the second set of primary and secondary antibodies were added. The list of antibodies, the dilution factor, incubation time, and the temperature are provided in Table Cellular localization, as well as the expression of inflammatory molecules and trophic factors, was studied using quantitative immunofluorescence. Briefly, astroglial cultures were plated at a density of 2.5 × 10Following 48 h of CSF exposure, the astroglial cells were lysed on ice using cell disruption buffer ). The lysates thus obtained were centrifuged at 12,000 rpm for 15 min at 4 °C. The suspension was carefully collected, and the total protein concentration was determined using Bradford’s reagent. Twenty micrograms of the protein sample was loaded for each sample. The proteins were separated on 10 % denaturing gel for COX2 and VEGF and 12 % denaturing gel for GDNF, in accordance with the predicted molecular weight. The proteins were then transferred to PVDF membrane. The membrane was then subjected to blocking with 7 % skimmed milk overnight at 4 °C. The blots were further incubated overnight at 4 °C with mouse monoclonal anti-GDNF , Rabbit polyclonal anti-VEGF , or Rabbit polyclonal anti-COX-2  depending on the experiments, followed by specific biotinylated anti-mouse for anti-GDNF  and anti-rabbit antibody for anti-VEGF and anti-COX-2  for 2 h. The blots were developed using Avidin-Biotin complex and developing solutions . The bands were visualized, and the intensity was quantified using the Gel Documentation System . β-Actin (Sigma-Aldrich) was used as the loading control, and the band intensity for the protein of interest was normalized to that of the β-actin protein.Upon exposure to CSF for 48 h, the astroglial cells were collected and the total RNA was isolated using RNeasy Plus Mini Kit  as per the manufacturer’s instructions. The messenger RNA (mRNA) was quantified using 1 μl of RNA in a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc). Approximately 10 ng of RNA was reverse transcribed (RT) using a high-capacity complementary DNA (cDNA) reverse transcription kit . Real-time PCR amplification was carried out in triplicates using specific primers and hydrolysis probes for the target genes listed in Table t test. GraphPad Prism was the software used to analyze the significance.The data for mRNA expression, quantitative immunofluorescence, semi-quantitative western blots, and MTT assay was statistically assessed for significance by one-way ANOVA followed by Tukey’s post hoc test. For ELISA, glutamate, NO, and ROS, the statistical analysis was carried out using Student’s p < 0.05 NC v/s ALS; Fig.p < 0.01 NC v/s ALS, 24 and 48 h; Fig. p < 0.05 NC v/s ALS, 24 h and **p < 0.01 NC v/s ALS, 48 h; Fig. p < 0.01 NC v/s ALS, 24 and 48 h; Fig. p < 0.05 NC and ##p < 0.01 NALS v/s ALS). However, the cellular level of IFN-γ was stable  and approximately fivefold in IFN-γ expression (**p < 0.01 NC and NALS v/s ALS) when compared to the NC subset. Surprisingly, IL-10 mRNA levels were up-regulated nearly 1.5fold, when compared to NC , but not at 12 or 24 h .Qualitative assessment of the cytosolic ROS by confocal microscopy also revealed enhanced ROS levels in cultures exposed to ALS-CSF for 48 h when compared to the normal control Fig. . Furtherp < 0.01 NC and #p < 0.05 NALS v/s ALS; Fig. p < 0.01 NC and NALS v/s ALS; Fig. p < 0.05 NC and ##p < 0.01 NALS v/s ALS; Fig. Further, to investigate the effect of ALS-CSF on the trophic factors secreted by astrocytes, we analyzed the mRNA and protein levels of GDNF and VEGF. ALS-CSF exposure resulted in near threefold down-regulation in the mRNA expression levels of GDNF in the astroglial cultures exposed to ALS-CSF for 48 h, when compared to the normal and the disease control .We also observed an approximately fourfold down-regulation of VEGF mRNA level as compared to NC and NALS subsets  or NALS-CSF (NALS-ACM) as disease control. For the normal control, NSC-34 cells were exposed to conditioned media from the astroglial cultures grown in normal medium (NC-ACM).Upon exposure, ALS-ACM induced morphological changes suggestive of degeneration in NSC-34 cells, while the cells appeared healthy in NC-ACM and NALS-ACM Fig. . ALS-ACMp < 0.001 NC-ACM and NALS-ACM v/s ALS-ACM; Fig. Further, there was a 25 % reduction in the cell viability in cultures exposed to ALS-ACM when compared to NALS-ACM and NC-ACM  in response to ALS-CSF . Here, wPro-inflammatory cytokines have gained notoriety for their role in triggering and propagating inflammation-mediated neurotoxicity . The up-IL-10 is a beneficial anti-inflammatory cytokine. Vasoactive intestinal peptide (VIP) exerts its immune modulatory actions by regulating IL-10 as well as neurotrophic factors in an mSOD1 model of ALS . SimilarFurther, pro-inflammatory cytokines can regulate the glutamatergic transmission by directly inhibiting astroglial glutamate transporters, by inducing overexpression of glutamate receptors on synapses or by reducing glutamate uptake via NO-mediated mechanism , 49. Alt2+ influx, as reported in the case of acute viral encephalomyelitis and PD [The inflammatory role of COX-2 and PGE-2 has been demonstrated in ALS, while their role in conferring neuroprotection or toxicity in inflammation is debated . Consides and PD .Up-regulation of astroglial ROS, NO, and iNOS levels in response to ALS-CSF was another major finding. Elevated levels of ROS accentuate the disease pathology by causing oxidative stress, as well as reducing the glutathione (GSH) levels , 57. Sim2+ influx in normal glial cells, as well as neurons [In the present study, we also report the capability of the conditioned medium, here representing the secreted factors, to exert neurotoxicity comparable to that of our ALS-CSF model of pathology, thus validating the role of astrocytes in the disease progression. Recently, Rojas and colleagues demonstrated that the astroglial-conditioned media from mSOD1 model brought about mitochondrial dysfunction, ROS elevation, and Ca neurons . The abiConcomitant to the inflammatory and toxic changes observed in the astrocytes, we also observed the down-regulation of the neurotrophic factors, VEGF and GDNF. They may protector reverse the motor neuron degeneration since trophic factor supplementation has been reported to be beneficial , 61. MatIn conclusion, the up-regulation of pro-inflammatory cytokines and down-regulation of anti-inflammatory cytokine suggest a clear ALS-CSF-induced astroglial cytokine imbalance. The multifarious nature of astroglial involvement in ALS pathogenesis is implied by the increase in auxiliary pro-inflammatory factors like PGE-2, COX-2, NO, and glutamate. The depletion of endogenous astrocytic VEGF and GDNF provide the additional impounding effect. Such a plethora of responses encompassing the inflammatory process suggests a temporal hierarchy of the pathways and molecules involved. Based on our results, we would like to advocate the need for combinatorial therapies to combat the multidimensional glial pathology and its compounding effect on the degeneration of motor neurons.


The influence of isoform A of reticulon-4 (Nogo-A), also known as neurite outgrowth inhibitor, on primary brain tumor development was reported. Therefore the aim was the evaluation of Nogo-A concentrations in cerebrospinal fluid (CSF) and serum of brain tumor patients compared with non-tumoral individuals.3/μL, the mean cerebrospinal fluid Nogo-A concentration value decreases 1.12 times. In the model of multiple linear regression analysis predictor variables influencing cerebrospinal fluid Nogo-A concentrations included: diagnosis, sex, and sodium level. The mean cerebrospinal fluid Nogo-A concentration value was 1.9 times higher for women in comparison to men. In the astrocytic brain tumor group higher sodium level occurs with lower cerebrospinal fluid Nogo-A concentrations. We found the opposite situation in non-tumoral individuals.All serum results, except for two cases, obtained both in brain tumors and non-tumoral individuals, were below the lower limit of ELISA detection. Cerebrospinal fluid Nogo-A concentrations were significantly lower in primary brain tumor patients compared to non-tumoral individuals. The univariate linear regression analysis found that if white blood cell count increases by 1 × 10Univariate linear regression analysis revealed, that cerebrospinal fluid Nogo-A concentrations change in relation to white blood cell count. In the created model of multiple linear regression analysis we found, that within predictor variables influencing CSF Nogo-A concentrations were diagnosis, sex, and sodium level. Results may be relevant to the search for cerebrospinal fluid biomarkers and potential therapeutic targets in primary brain tumor patients.Nogo-A concentrations were tested by means of enzyme-linked immunosorbent assay (ELISA). Aside from its role in neurite growth isoform A of reticulon-4 (Nogo-A) has a variety of other functions, e.g. participation in neuronal stem cells differentiation, inhibition of angiogenesis, microglial activity .Kuhlman et al. found strong expression of Nogo-A in 71% of oligodendroglioma, but not in other brain tumors, such as: ependymomas WHO grade II, astrocytoma WHO grade I or II, dysembryoplastic neuroepithelial tumors, clear cell meningiomas, as well as metastases to the brain [in vitro. This effect may be mediated via the NgR (receptor for Nogo-66), as they revealed that treatment of U87MG cells with NgR antibodies resulted in lowering their potential for adhesion and migration [The role and expression of Nogo-A in brain tumors were studied by several authors –7. Kuhlmhe brain . The authe brain , 6. Morehe brain . Additiohe brain . Some auSo far Nogo-A concentrations have been analyzed only in patients serum; however these results were obtained in children with autism spectrum disorders and patients with traumatic brain injury –9. AccorIn the last few years great effort has been put into searching for diagnostic, prognostic, predictive, and therapeutic response biomarkers in primary brain tumors. Because the search for these kinds of biomarkers still represents an exciting research area, the aim of the current study was to evaluate Nogo-A concentrations in CSF and serum of primary brain tumor patients as compared to non-tumoral individuals.2 plasma, and cerebrospinal fluid (CSF) samples. All serum results, obtained both in brain tumors and non-tumoral individuals, were below the lower limit of detection, except in two cases . In EDTA-K2 plasma samples all results were below the lower limit of detection. We found Nogo-A results in all cerebrospinal fluid tumors as well as in non-tumoral samples.We tested Nogo-A concentrations in patients serum, EDTA-KP < 0.001 and P = 0.003, respectively)  Nogo-A was a little bit higher in patients with tumors of the meninges  compared to patients with astrocytic brain tumors ; however this difference was not significant. We also found that CSF Nogo-A concentrations were nearly 10-fold lower in the astrocytic brain tumor group and 7-fold lower in the group of tumors of the meninges compared to non-tumoral subjects  (P > 0.05).The analysis of the protein tested depending on the sex revealed that females had higher concentrations of Nogo-A  compared to males , but these differences were not statistically relevant , secondly in particular patients subgroups, and finally depending on patients sex.R = −0.407; P = 0.010). We did not find any correlation coefficient for Nogo-A in the astrocytic brain tumor subgroup as well as the group of patients with tumors of the meninges. In non-tumoral individuals Nogo-A was positively related to the duration of hospitalization after neurosurgery . We did not find any correlation coefficient for Nogo-A in the male or female group.We found that in the whole study group Nogo-A concentrations negatively correlated with white blood cell count . For patients with astrocytic brain tumors, the mean CSF Nogo-A concentration value decreases 8.4 times compared to non-tumoral patients . For patients with tumors of the meninges, the mean CSF Nogo-A concentration value decreases 8.5 times compared to non-tumoral patients  , if other model parameters are fixed. For patients with tumors of the meninges the mean CSF Nogo-A concentration value decreases 7.2 times compared to non-tumoral individuals , if other model parameters are fixed , if other model parameters are fixed. For non-tumoral individuals we found that if Na+ level increases by 1 mmol/L the mean CSF Nogo-A concentration value increases by 13% , if other model parameters are fixed  the mean CSF Nogo-A concentration value changes in relation to sodium (Na+) level and increases eβ1∙eβ5∙Na+ = e20.7∙e−0.16∙Na+ times , if other model parameters are fixed. In the astrocytic brain tumor group, higher sodium levels occur with lower CSF Nogo-A concentrations (R2 = −0.055). We found the opposite situation in non-tumoral individuals, where a higher sodium levels occur with higher CSF Nogo-A concentrations (R2 = 0.541)  Lumbar puncture is not routine in the diagnosis of patients with CNS tumors, thus the Local Bioethics Committee gave permission for CSF collection only within neurosurgery; 2) To compare CSF Nogo-A concentrations between tumoral and non-tumoral individuals, cerebrospinal fluid should be collected from the same CNS interspace; so our samples were relatively homogenous.However, control subjects are definitely non-tumoral individuals and there is a possibility that our choice was not appropriate, as it was reported that Nogo Δ-20, a second Nogo-A ligand domain, disorganizes F-actin filaments structures in multinucleated vascular endothelial cells (MVCEs) as well as MVCE's lamellipodia and filopodia retraction . Nevertha priori (prospective) power analysis, which is generally carried out in a big cooperative studies including hundreds of subjects, especially if a new diagnostic/therapeutic method is to be proposed in the conclusion of a study. Our purpose was far more modest—we only aimed at demonstrating that CSF concentration of Nogo-A is diminished in patients with primary brain tumors and to explore factors which might influence this phenomenon.The first study limitation is the small number of primary brain tumor patients included in the study, which resulted from the difficulties of the cerebrospinal fluid collection procedure . Also, CSF sampling in “healthy” control patients is rare nowadays in neuro departments, therefore the collection of these samples took us nearly 2 years. Due to the small number of cases which we were able to include in the study, we did not perform The second study limitation is a lack of correlation for Nogo-A concentrations in CSF with their expression in tumor samples, taking into consideration that no report on this is found in the available literature. As a matter of fact we have designed such a study, also including the expression a receptor for Nogo-A and we are currently in the process of applying for funds. Additionally we would like to analyze the expression for NgR, which is a receptor for Nogo-A. Nevertheless, we do not think it is good to wait indefinitely for these “full” results and to not attempt to publish our somewhat preliminary results.vs. non-tumoral subjects increases for women in comparison to men and changes in relation to sodium levels.To conclude, cerebrospinal fluid is the best material in which Nogo-A concentrations are detectable by means of ELISA. Neither serum, nor EDTA-K2 plasma are useful for the quantitative evaluation of Nogo-A. Univariate linear regression analysis revealed that cerebrospinal fluid Nogo-A concentrations change in relation to the white blood cell count. Median cerebrospinal Nogo-A concentration in females was higher compared to males, but simple comparison analysis did not reveal a statistical difference. However, in the created model of multiple linear regression analysis we found that among the predictor variables influencing CSF Nogo-A concentrations were diagnosis, sex, and sodium level, as the mean CSF Nogo-A concentration in astrocytic brain tumor patients The results of our study may be relevant to the search for CSF biomarkers and/or future therapeutic targets in primary brain tumor patients, however these are only preliminary and should be treated as a foundation for future paths of investigation.The study group consisted of 28 patients with previously untreated primary central nervous system (CNS) tumors, divided into: “A” patients subgroup with astrocytic brain tumors  and “B” patients subgroup with meningeal tumors .Table The comparative group was composed of 11 non-tumoral subjects  with unruptured intracranial aneurysms, which is usually asymptomatic and only discovered incidentally . The excP > 0.05). The laboratory parameters on admission to the hospital of all subgroups studied are presented in Table Statistical analysis revealed, that patients subgroups were age-matched . All subjects included in the study gave their written informed consent.Procedures on patients were performed under a general anesthetic during neurosurgery at the Department of Neurosurgery at the Clinical Medical Hospital in Bialystok. Craniotomy was performed on all subjects included in the study. In patients undergoing tumor resection its size and localization was tailored according to location and size of the tumor, preferred access route and surrounding anatomy. Clipping of an aneurysm required an opening in the fronto-temporal region . Regardless of operation type or location, after placing the patient's head in a three-pin Mayfield headholder the surgical field was prepared in the standard fashion. Skin incision preceded lifting of bone flap and lancing of dura mater, which allowed for a visualization of arachnoid membrane and subarachnoid space. With the aid of an operating microscope the subarachnoid space was carefully opened and about 2–3 mL of CSF aspirated with a single-use, sterile syringe and soft venous catheter. The aforementioned steps were taken in the very beginning of each procedure, before any potential bleeding occurred. This routine allowed for keeping the CSF clean of blood and not mixed with warm saline solution used as an irrigation.Blood collected in tubes without anticoagulant, in EDTA-K2 tubes, and CSF samples were centrifuged for 20 minutes at 1000 × g. The obtained serum, plasma, and CSF supernatant were stored at –80° C until further analysis.Concentrations of Nogo-A were measured using Human RTN4 ELISA (enzyme-linked immunosorbent assay) kit  according the manufacturer's instruction. Samples were not diluted prior to analysis. According to the Vendor's instruction the range for the detection of Human RTN4 is between 78.125–5000 pg/mL; sensitivity of the assay kit is < 46.875 pg/mL. Between zero well (Standard dilution buffer) and the first standard (78.125 pg/mL) some optic density (OD) absorbance may be read and transformed into Nogo-A concentration. For example the Vendor of the assay kit referred to the value of 0.112 of optic density (OD) at 450 nm for the zero well and the value of 0.242 OD at 450 nm for the 78.125 pg/mL standard value. In our study for brain tumor cerebrospinal fluid samples we found three values, which were below 78 pg/mL  and one of them was below the sensitivity of the kit referred to by the Vendor (< 46.875 pg/mL).P < 0.05.The obtained results were statistically analyzed with the use of the STATISTICA 12.0 PL software  and STATA 12.1 (StataCorp LP). The concentrations of parameters tested did not follow a normal distribution in the preliminary statistical analysis (Shapiro-Wilk test), thus nonparametric statistical analysis was employed. The Mann–Whitney test was used in order to compare two independent samples, and the Kruskal-Wallis test was used for the comparison of three samples. Correlation coefficients were obtained by applying Spearman's rank method. If not otherwise stated, the values for each given measured variable are given as medians and interquartile ranges. Univariate linear regression analysis as well as multiple linear regression analysis models were created. Differences were considered statistically significant for


Here, we hypothesized, that treatment with granulocyte colony-stimulating factor (G-CSF) may improve bone healing by mobilization of CD34+ progenitor cells into the circulation, which in turn may facilitate vascularization at the defect site. In this pilot study, we aimed to characterize the different cell populations mobilized by G-CSF and investigate the influence of cell mobilization on the healing of a critical size femoral defect in rats. Cell mobilization was investigated by flow cytometry at different time points after five consecutive daily G-CSF injections. In a pilot study, bone healing of a 4.5-mm critical femoral defect in F344 rats was compared between a saline-treated control group and a G-CSF treatment group. In vivo microcomputed tomography and histology were applied to compare bone formation in both treatment groups. Our data revealed that leukocyte counts show a peak increase at the first day after the last G-CSF injection. In addition, we found that CD34+ progenitor cells, including EPCs, were significantly enriched at day 1, and further increased at day 5 and day 11. Upregulation of monocytes, granulocytes and macrophages peaked at day 1. G-CSF treatment significantly increased bone volume and bone density in the defect, which was confirmed by histology. Our data show that different cell populations are mobilized by G-CSF treatment in cell specific patterns. Although in this pilot study no bridging of the critical defect was observed, significantly improved bone formation by G-CSF treatment was clearly shown.Bone is an organ with high natural regenerative capacity and most fractures heal spontaneously when appropriate fracture fixation is provided. However, additional treatment is required for patients with large segmental defects exceeding the endogenous healing potential and for patients suffering from fracture non-unions. These cases are often associated with insufficient vascularization. Transplantation of CD34+ endothelial progenitor cells (EPCs) has been successfully applied to promote neovascularization of bone defects, however including extensive  While the majority of bone fractures and bone defects heal, nonunion is a common complication of a fracture; it indicates that fracture healing is not happening in a timely fashion. For example, nonunion occurs in about 10% of all tibia fractures  population in peripheral blood  is an important mediator of granulopoiesis. G-CSF-deficient mice suffer from neutropenia and impaired mobilization of neutrophils in the blood , G-CSF became of interest for the treatment of diseases involving impaired vascularization. G-CSF treatment can be applied to increase the frequency of EPCs in the circulation and by this to improve the yield of donor cells for transplantation approaches. For the treatment of critical limb ischemia, a phase I/IIa clinical trials has been performed to assess transplantation of G-CSF mobilized cells and suggested safety and feasibility of this approach  in an Association for Assessment and Accreditation of Laboratory Animal Care Internal  approved facility. Animals were group-housed and received standard diet  and water ad libidum. Adult specific pathogen free female F344 Fisher rats were obtained from Charles River (Germany). Animals were healthy based on health certificates provided by the breeder and clinical examination upon study entry.®, Streuli Pharma AG).Twenty-nine female rats  were used to assess the time course and composition of the G-CSF mobilized cell population and randomly assigned to five experimental groups Figure . Prior t®, Rechitt Benckiser). Under isoflurane anesthesia, the left femur was aseptically prepared and a skin incision was made. The sc fascia lata was incised and the M. quadriceps and M. biceps femoris were separated bluntly to expose the femur. The PEEK fixation plate (RIS.602.100) was fixed on the femur, with the center of the plate at the distal level of the lateral femoral crest using six self-cutting locking screws (RIS.402.120) after predrilling the screw holes (RIS.592.202). Facilitated by a jig (RIS.302.104), a 4.5 mm defect was created by two osteotomies using a Gigly saw . Finally, fascia and skin were closed in a continuous pattern using absorbable suture material . To prevent hypothermia, a heated mat during surgery and an infrared lamp during the recovery period (approx. 30 min) were used. All animals received 3 ml Ringer’s solution (Braun) intraperitoneally. Postoperative analgesia consisted of Buprenorphine  and Paracetamol . Animals recovered rapidly after surgery and started normal load-bearing within 30 min. All animals lost weight postoperatively (<5%) but recovered within 10 days. The animals were kept in the study for as long as bone was forming in the defect [according to microcomputed tomography (microCT)]. Once plateauing, the study was stopped (230 days).In a pilot study investigating the effect of G-CSF treatment on bone formation, 10 skeletally mature female rats  were randomly divided into two groups. Animals were injected subcutaneously once daily for 5 consecutive days after surgery with G-CSF (50 µg/kg) or saline, respectively . The animals were scanned under isoflurane anesthesia at the indicated time points. They were placed in a modified holder in lateral recumbency. The operated leg was positioned to align the femur with the scanner axis, then fixed with masking tape to minimize metal and motion artifacts. A 8 mm region, centered on the defect, was scanned with 200 ms integration time and 1,500 projections per rotation. The X-ray tube was operated at 70 kV tension and 114 µA current. The projections were then reconstructed across an image matrix of 2,048 × 2,048 pixels with an isotropic voxel size of 19 µm. Scans showing evidence of motion artifacts were repeated. The postoperative scans were rotated to align the femur longitudinally and the screws sagittally. The subsequent scans were registered to their respective postoperative scan using rigid registration .The region of interest (ROI) for analysis was defined as the defect volume on the postoperative scan. The ROI was propagated to the registered scans. Scans were Gaussian-filtered (sigma = 0.8 support = 1) to reduce noise, bone was segmented (threshold = 515 mgHA/ccm); bone volume and density were computed within the ROI using direct voxel counting methods. The gap size was then defined as the smallest distance found in the distance map  and stained with Giemsa/eosin as described before  was used according to the manufacturer’s protocol to determine the concentration of human G-CSF in rat serum collected upon euthanasia. Absorbance was measured using Viktor3 Plate Reader (Perkin Elmer).g, mononuclear cells (MNCs) were collected from the interphase and washed twice [PBS/5% fetal bovine serum ]. Cell counts were determined using an automated cell counter . For cell surface marker staining, 1 × 105 MNCs were washed with buffer (PBS/1% FBS) and incubated with CD32 antibody (BD) for 5 min on ice to reduce unspecific antibody binding. Afterward cells were stained for 30 min on ice with the following antibodies: CD45-PeCy5 (ThermoFisher Scientific), CD34-APC (Antibodies Online), CD11b-FITC (BD). Unstained and isotype controls (mouse IgG1-PeCy5 (ThermoFisher), rabbit IgG-Alexa647 (Bioconcept), mouse IgG2a, k-FITC (BD)) were performed. Antibodies were used according to the manufacturer’s recommendations. To investigate LDL uptake, 1 × 105 MNCs were washed and exposed to 10 µg/ml dil-labeled acLDL (ThermoFisher) for 4 h at 37°C. LDL which was not taken up was removed by washing. Cells were exposed to CD32 antibody, stained for CD11b-FITC, washed and analyzed using a FACS Aria III (BD). Leukocyte counts measured in blood from the tail vein were used to calculate results for the respective cell populations as cell number/μl blood.Anticoagulated blood was diluted in phosphate-buffered saline (PBS) and overlaid on Ficoll density medium . After centrifugation at 800 in vivo microCT data for bone volume, bone formation, gap size and gap closing speed were modeled using Bayesian generalized linear mixed modeling in R . At day 5, the serum G-CSF concentration was not significant different from untreated control animals indicating a fast pharmacokinetic. Next, we examined the increase of the blood leukocyte count over time .Serum levels of human G-CSF reached a peak value at day 1 after the last injection with G-CSF  in control animals  were detected at day 11. An upregulation was also detected for CD11b+leukocytes  compared to G-CSF-treated animals  but not in control animals  were identical between G-CSF and vehicle animals (data not shown). Furthermore, the bone formation outcomes from the defect site did not correlate with the contralateral bone parameters.The result of more efficient bone formation upon G-CSF treatment was confirmed by histology Figure . In bothDespite its fast pharmacokinetic, G-CSF induced an elevation of blood leukocytes. The short serum half-life of injected G-CSF is in agreement with an earlier study reporting a half-life of 1.8–2.4 h for Neupogen (recombinant G-CSF) , which might be explained by the different life span of the cell types. We also evaluated whether mature endothelial cells (CD11b−LDL+) would be targeted by G-CSF treatment. No mobilization of endothelial cells into the circulation was observed, but it is likely that G-CSF had a local effect on endothelial cells since various studies have reported a positive effect of G-CSF on proliferation and migration of endothelial cells  and performed in accordance with the Swiss Animal Protection Law in an Association for Assessment and Accreditation of Laboratory Animal Care Internal  approved facility.All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The authors contributed to the work as follows: MHe and VS: design and conception of work, acquisition, analysis and interpretation of data, drafting the article, revision; SZ: design and conception of work, acquisition of data, revision; UE: design of work, acquisition of data, revision; MHi, KC, and UM: acquisition and interpretation of data, revision; MA: design and conception of work, interpretation of data, revision; SV: design and conception of work, acquisition and interpretation of data, revision; VS: design and conception of work, acquisition, analysis and interpretation of data, drafting the paper, revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To systematically review the literature comparing the efficacy and safety of dexmedetomidine and midazolam when used for procedural sedation.We searched MEDLINE, EMBASE and COCHRANE for clinical trials comparing dexmedetomidine and midazolam for procedural sedation up to June 20, 2016. Inclusion criteria: clinical trial, human subjects, adult subjects (≥18 years), article written in English, German, French or Dutch, use of study medication for conscious sedation and at least one group receiving dexmedetomidine and one group receiving midazolam. Exclusion criteria: patients in intensive care, pediatric subjects and per protocol use of additional sedative medication other than rescue medication. Outcome measures for efficacy comparison were patient and clinician satisfaction scores and pain scores; outcome measures for safety comparison were hypotension, hypoxia, and circulatory and respiratory complications.We identified 89 papers, of which 12 satisfied the inclusion and exclusion criteria; 883 patients were included in these studies. Dexmedetomidine was associated with higher patient and operator satisfaction than midazolam. Patients receiving dexmedetomidine experienced less pain and had lower analgesic requirements. Respiratory and hemodynamic safety were similar.Dexmedetomidine is a promising alternative to midazolam for use in procedural sedation. Dexmedetomidine provides more comfort during the procedure for the patient and clinician. If carefully titrated, the safety profiles are similar. Procedural sedation can provide more comfort for the patient and an easier procedure for the clinician for painful or unpleasant diagnostic or therapeutic procedures. It may be preferred over general anesthesia due to physiological, financial and logistical considerations,.Midazolam is one of the classic sedatives for procedural sedation. While midazolam is thought to cause minimal hemodynamic effects, it does have the potential to cause loss of airway reflexes, respiratory depression, and even apnea . If an eDexmedetomidine  is a relatively new drug, which can also be used for procedural sedation. It has sedative and anxiolytic properties and is known for its analgesic potential owing to a reduction of sympathetic tone. Dexmedetomidine induces dose-dependent effects, ranging from minimal to deep sedation. Moreover, except at doses that cause very deep sedation or general anesthesia, the sedation is reversible. The patient can be easily roused to a lucent state, but when left undisturbed will fall back into a state very similar to natural sleep. These are unique properties among the sedative medications in common use. Dexmedetomidine does not impair the respiratory drive per se and seldom causes apnea. However, it has been shown to impair the respiratory responses to hypoxia and hypercapnia .Many studies have compared aspects of the safety and efficacy of midazolam and dexmedetomidine, but the results have not yet been systematically reviewed. Therefore, the aim of our systematic review was to systematically review the current literature on the relative efficacy and safety of dexmedetomidine and midazolam when used as monosedatives for conscious, procedural sedation. We included studies of all types of surgical or diagnostic procedures.The objective of this systematic review was to answer the following research question: does dexmedetomidine result in more efficacious and safer sedation compared to midazolam in the periprocedural period for adult patients undergoing procedural sedation?We searched the Cochrane, Pubmed and Embase databases to identify adult human clinical trials comparing the sedative efficacy and/or safety of dexmedetomidine versus midazolam. Studies that compared both drugs without routine use of other sedative medications—other than for rescue from inadequate sedation or analgesia—were eligible for inclusion.For the Pubmed search  we used the following search strategy: (("dexmedetomidine"[MeSH Terms] OR "dexmedetomidine"[All Fields]) AND ("midazolam"[MeSH Terms] OR "midazolam"[All Fields]) AND sedation[All Fields]) AND "humans"[MeSH Terms]). The Embase database was searched with a comparable search strategy as used for Pubmed : ('dexmedetomidine'/exp OR dexmedetomidine AND ('midazolam'/exp OR midazolam) AND ('conscious sedation'/exp OR 'conscious sedation')). The Cochrane library was searched for relevant reviews using the following search terms: dexmedetomidine AND midazolam AND sedation. There was no limit on the years considered.The papers thus identified were screened by two authors  by title and abstract for eligibility for inclusion . The resData extraction and assessment of risk of bias was done by the same two authors . Pilot forms were used to extract unfiltered data on all described outcome measures related to efficacy and safety. The outcome measures for the review were selected by means of team discussion . The selected outcome measures were placed onto forms for collecting data from the studies reviewed.Study selection was based on the following criteria:Participants: adults receiving procedural sedationType of intervention: dexmedetomidine use for procedural sedationType of comparison: midazolam use for procedural sedationStudy types: randomized controlled clinical trialsWe excluded studies where dexmedetomidine or midazolam was given as part of intensive or critical care and any studies including children. Also excluded were studies where additional medication with sedative properties was given other than as rescue medication.The Jadad score  was usedPatient and operator satisfaction scores and pain scores were used to compare efficacy. If these outcome measures were not expressed numerically in scores or ratings, we used the verbal descriptions provided in the studies.To compare the safety of dexmedetomidine and midazolam we recorded all reports of hypotension and hypoxia. When no explicit mention of these events was present, reports of hemodynamic and respiratory complications were discussed by three authors  to evaluate their eligibility for inclusion in the results. If the absence of complications was explicitly mentioned or if it was explicitly stated that there was no need for intervention, the incidence of hypotension or hypoxia was assumed to be zero.To compare the incidences of hypotension and respiratory adverse events, we isolated the studies and patient groups in which patients received either dexmedetomidine or midazolam for subgroup analysis. These pooled incidences of hypotension and respiratory adverse events were compared with the Chi-square test.The results of our search strategy according to the PRISMA method  are summTwelve publications were direct comparisons of dexmedetomidine with midazolam , Table 1Of the 12 included studies, 4 were given a final JADAD score of 2 or lower ,6,12,15 Eight studies n = 597 subjects) measured patient satisfaction by means of numerical rating scales or questionnaires with Likert scales . Four of subjectsEight studies n = 577) reported on the comparative analgesic effects of dexmedetomidine and midazolam . Two of 7 reporteIn two studies in which dexmedetomidine treatment resulted in higher patient satisfaction, this treatment was also associated with better analgetic properties compared to midazolam ,8. MidazNine studies n = 637) reported either clinician satisfaction or a measure of ease of performance of the procedure . Four of reportedKaya et al. reported that dexmedetomidine use, when compared to midazolam, was associated with more patients reaching the desired level of sedation . Muttu eIn 11 studies n = 767) both the number of patients included and the number of respiratory adverse events or complications was reported ,7–16. We67 both tExcept for two studies ,6, all sThe data from the eight good quality studies was pooled, showing a similar incidence of hypotension: 10 events of hypotension among 281 dexmedetomidine-treated patients compared to 7 events among 280 midazolam-treated patients (p = 0.80) ,13,14,16Finally, two studies ,14 reporFor adult patients undergoing procedural sedation, dexmedetomidine results in more efficacious sedation than midazolam in the periprocedural period. The safety profile of both drugs appears to be similar. We found that dexmedetomidine has potential benefits over midazolam when used for procedural sedation. No studies reported that patients or clinicians were more satisfied with the result of midazolam sedation, whereas several studies found dexmedetomidine use to be associated with greater patient and clinician satisfaction and greater analgetic potential. The safety of both drugs seems to be similar with respect to respiratory or hemodynamic complications.Patients expect procedural sedation to provide them with comfort during an otherwise stressful or painful period. Alleviation of pain and discomfort is an important determinant for patient satisfaction scores. This may explain the higher patient satisfaction scores for dexmedetomidine in several studies ,8. DexmeAll studies, except for the volunteer studies of Frölich et al. ,6, used Midazolam also provides amnesia. Although this may be preferred by some patients, for instance those with dentophobia, amnesia could also be considered undesirable for this group of patients. These patients aim to alleviate their fear through gradual exposure, and amnesia prevents this learning effect.Like patients, clinicians in the selected studies tended to have a preference for dexmedetomidine. Based on their additional comments in these studies, this preference might be explained by the improved cooperation of patients treated with dexmedetomidine and the absence of paradoxical reactions in this group ,11,15. TParadoxical reactions are a rare but well known side effect of benzodiazepines. The occurrence of restlessness, agitation and even aggression is very disturbing to the patient as well as to the clinician. The etiology is unclear but children and the elderly are more prone. Although the reported incidence is <1% these reactions can prevent clinicians from completing the procedure. Moreover, the outright hostility seen in some patients exhibiting a paradoxical reaction to midazolam presents a danger to both patient and clinician . DexmedeAll of the above findings can be explained from the pharmacodynamics of both drugs. Midazolam produces its effects through the GABAa receptors and inhibits the excitatory reaction of the brain to stimuli . DexmedeIn the pooled results dexmedetomidine and midazolam did not differ in terms of respiratory safety. This was surprising because midazolam is well known for causing respiratory depression , and we Dexmedetomidine has a reputation of causing hypotension, which is sometimes preceded paradoxically by hypertension. In contrast, midazolam is known for its hemodynamic stability. However, the hypotensive effect of dexmedetomidine can be mitigated by preventing rapid infusion and by not using bolus dosing. High peak plasma levels are responsible for the complex hemodynamic effects of dexmedetomidine . In all Careful dosing, preferably by titration, is the key to procedural sedation. Within the confines of carefully protocollized studies, dexmedetomidine (when used with slow loading dosages) and midazolam (in a careful infusion regimen) would appear to have similar safety profiles. For general practice, this would require intravenous access and infusion pumps for titration, and appropriate monitoring would still be needed. When patients are sedated with dexmedetomidine, they remain rousable. However, it should be remembered that like midazolam, dexmedetomidine has moderately slow pharmacokinetics, so that during the recovery period, the patient may be sleepy when not stimulated. As with midazolam, close observation is necessary for a period of time before discharge commensurate with the pharmacokinetics of the agent. Dexmedetomidine, however, seems to yield better results with respect to patient and clinician satisfaction. And the possibility of using this drug safely with intranasal administration may be very useful for office-based procedural sedation. The safe use of dexmedetomidine in the general population, and more specifically the frail or the elderly should be the subject of further investigation before this treatment can be used in the office-based or nursing home-based care setting.The included studies varied widely with respect to dosing regimens, procedures and outcome measures. Also, not all studies reported in enough detail on these outcome measures. This prevented us from performing more formal meta-analyses.We excluded studies where analgesics with an additional sedative effect were given other than as rescue medication and placebo-controlled studies where midazolam served as rescue medication. This led to the exclusion of many papers studying dexmedetomidine versus midazolam for conscious sedation. However, the exclusion of these studies allowed for a more precise comparison of the effects of both drugs without accounting for numerous (unpredictable) pharmacological interactions.We included four studies of moderate to low quality,6,12,15.Midazolam causes profound amnesia. The amnesic effect appears at lower dosages and is more apparent than the sedative effect. This amnesia could have confounded results when patient questionnaires are used after midazolam treatment.The frail and elderly are an underrepresented group in these studies. With only one study including some ASA 3 patients or a substantial number of patients aged over 60, the safety and efficacy of dexmedetomidine compared to midazolam for use in procedural sedation in frail patients has not been well assessed . Su et aWe have shown that dexmedetomidine has advantages over midazolam in terms of reliability, analgesia and patients’ and clinicians’ satisfaction. Moreover, within the scope of this review, dexmedetomidine and midazolam appear to have a similar cardio-respiratory safety profile when both are carefully titrated. Combined with the use of local anesthesia, dexmedetomidine provides a good alternative for midazolam for procedural sedation.S1 PRISMA Checklist(DOC)Click here for additional data file.


A fragment of continental crust has been postulated to underlie the young plume-related lavas of the Indian Ocean island of Mauritius based on the recovery of Proterozoic zircons from basaltic beach sands. Here we document the first U–Pb zircon ages recovered directly from 5.7 Ma Mauritian trachytic rocks. We identified concordant Archaean xenocrystic zircons ranging in age between 2.5 and 3.0 Ga within a trachyte plug that crosscuts Older Series plume-related basalts of Mauritius. Our results demonstrate the existence of ancient continental crust beneath Mauritius; based on the entire spectrum of U–Pb ages for old Mauritian zircons, we demonstrate that this ancient crust is of central-east Madagascar affinity, which is presently located ∼700 km west of Mauritius. This makes possible a detailed reconstruction of Mauritius and other Mauritian continental fragments, which once formed part of the ancient nucleus of Madagascar and southern India. There is growing evidence for the presence of continental fragments within the young oceanic basins, but this is still based on limited geological data. Here, the authors use zircon isotope geochronology to demonstrate the presence of Archaean continental crust beneath the young hotspot volcanoes of Mauritius. It has been noted that other compositions, including older Proterozoic or even Archaean components, can also be plausibly modelledEvidence is accumulating that old continental crust may occur beneath some young ocean-island volcanoes, contributing contaminating components to their chemical and isotopic compositions. For example, the Sr, Pb and Nd isotopic compositions of basalts to rhyolites in the Öræfajökull volcano of southeastern Iceland were modelled to have incorporated 2–6% of Precambrian continental crust−1); extreme care was taken during sample processing to avoid any risk of contamination. Thirteen zircon grains were recovered, from which we report 68 individual point analyses acquired using the Cameca 1280-HR SIMS (secondary ion mass spectrometer) instrument at GFZ Potsdam; details of the sample processing and analytical methods are provided in the Methods section. Three zircon grains show uniquely mid- to late-Archaean U–Pb systematics, with no evidence for Phanerozoic components. Cathodoluminescence (CL) and backscattered electron (BSE) images  images  show tha52±11 Ma . We inte206Pb/238U age of 5.7±0.2 Ma . In Grains 3 and 8, there is a tendency for spots with lower Th/U to occur on grain margins. These features suggest that the Archaean zircons were derived from source rocks that experienced a complex history of magmatic and metamorphic events. The young (Late Miocene) zircon grains all show much higher Th/U ratios of 0.7–2.6  typical of magmatic zircons in alkaline rocks10U and Th concentrations of all measured individual spots in the zircon grains are given in 91118OSMOW values between 5.5‰ (Grain 3) and 9.7–9.9‰ (Grain 8). The lower value is comparable to the average δ18OSMOW value of 5.5±0.4‰ for Archaean zircons in tonalite–trondhjemite–granodiorite rocks in the Superior Province of Canada18OSMOW for magmatic zircons18OSMOW>8.5‰ have been reported in Precambrian granitoids from Fennoscandia18OSMOW value of 4.59±0.20‰ (1 s.d.)  . These v3 km2) and smaller  continental fragments as potential correlatives of the sub-Mauritius continental crust. Granitoid rocks of the Seychelles222424262 area in east-central Madagascar, as illustrated in Can the spectrum of U–Pb ages for old Mauritian zircons be correlated with exposed Precambrian terranes in nearby continental entities? We considered major continental masses like India, as well as large  were reported in young lavas and beach sands from the Galapagos, but so far only mentioned in a conference abstractMauritia acted as a buffer zone between the western Indian subcontinent and eastern Madagascar, and was fragmented by numerous tectonic and volcanic events that occurred in that region since the early Cretaceous. Due to the early opening between India and East Antarctica in the Enderby Basin, a sinistral shear zone must have acted between India and Madagascar, with India displaced northward relative to Madagascar. That caused convergence and perhaps subduction between northwestern India and Somalia/Arabiain situ from outcrop located at 20.32840° S; 57.71312° E , yielding an unweighted mean 206Pb/238U age of 1062.3±6.6 Ma (1 s.d.). We confirmed the accuracy of the U–Pb fractionation correction by measuring N=21 determinations on the Temora2 zircon Quality Control Material. Here we determined a mean, unweighted 206Pb/238U age of 421.1±2.8 Ma (1 s.d.), which is in reasonable agreement with the published age of 416.50 Ma (ref. 204Pb/206Pb ratio (older grains) or 207Pb/206Pb ratio (younger grains), in conjunction with a recent common Pb composition.Our initial analyses employed a 1–3 nA  Ma ref. . A total Ma ref. , confirm16O2− primary beam employing a Gaussian beam distribution was focused to a ∼2 μm diameter at the sample surface; this lower primary beam current resulted in a much lower ion collection rate. In order to produce a flat-bottomed sputtering cater, a 5 × 5 μm raster was applied to the primary beam, and this was compensated for using the dynamic transfer capability of the 1280-HR's secondary ion optics. All other aspects of the second data acquisition series were the same as those for earlier 2 nA analyses. Measurements of the Temora2 Quality Control Material gave an unweighted 206Pb/238U age of 410±5.3 Ma ; despite the low primary ion current used, this is in reasonable agreement with the assigned 416.50 Ma ageOur initial survey of U–Pb ages identified three grains with Archaean ages. We decided to survey the two larger of these grains Results of all SIMS spot analyses of zircons in trachyte sample MAU-8 are given in 18OSMOW value of 9.86±0.11‰, based on gas source mass spectrometry16O from the SIMS primary beam, there were only three locations on the Archean grains that could be analyzed. These results, along with the results from 29 locations on Miocene zircons, are given in With the intent of further characterizing the nature of our suite of Mauritian zircons, we undertook additional oxygen isotope determinations by SIMS. Our analytical technique closely followed that described for the Potsdam SIMS laboratoryBackscattered electron images show the presence of numerous mineral inclusions in 2 of the 3 Archaean grains , whereasThe authors declare that all relevant data are available within the article and its How to cite this article: Ashwal, L. D. et al. Archaean zircons in Miocene oceanic hotspot rocks establish ancient continental crust beneath Mauritius. Nat. Commun.8, 14086 doi: 10.1038/ncomms14086 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.U-Pb isotopic data acquired by SIMS for zircons recovered from Mauritius trachyte sample MAU-8Oxygen isotope data acquired by SIMS for zircons recovered from Mauritius trachyte sample MAU-8


Gesture recognition in non-intrusive muscle-computer interfaces is usually based on windowed descriptive and discriminatory surface electromyography (sEMG) features because the recorded amplitude of a myoelectric signal may rapidly fluctuate between voltages above and below zero. Here, we present that the patterns inside the instantaneous values of high-density sEMG enables gesture recognition to be performed merely with sEMG signals at a specific instant. We introduce the concept of an sEMG image spatially composed from high-density sEMG and verify our findings from a computational perspective with experiments on gesture recognition based on sEMG images with a classification scheme of a deep convolutional network. Without any windowed features, the resultant recognition accuracy of an 8-gesture within-subject test reached 89.3% on a single frame of sEMG image and reached 99.0% using simple majority voting over 40 frames with a 1,000 Hz sampling rate. Experiments on the recognition of 52 gestures of NinaPro database and 27 gestures of CSL-HDEMG database also validated that our approach outperforms state-of-the-arts methods. Our findings are a starting point for the development of more fluid and natural muscle-computer interfaces with very little observational latency. For example, active prostheses and exoskeletons based on high-density electrodes could be controlled with instantaneous responses. A muscle-computer interface (MCI) is a communication system that transforms myoelectrical signals from mere reflections of muscle activities into interaction commands that convey the intent of the user s movement. A muscle is composed of many motor units (MU), and the “discharge” or “firing” of each MU activation generates a “motor unit action potential” (MUAP), which is the sum of the contributions from the individual fibres that compose the MU. Surface electromyography (sEMG) records a muscle’s electrical activity from the surface of the skin and thus reflects the generation and propagation of MUAPs. sEMG-based gesture recognition is the technical core of non-intrusive muscle-computer interfaces, which are often directed at controlling active prosthesesetc.) on sEMG feature space, i.e., the sequence of myoelectric signals of each channel often need to be transformed into a set of descriptive and discriminatory features extracted using a window of EMG data (or segment)56Gesture recognition based on sEMG can be naturally framed as a pattern classification problem in which a classifier is usually trained through supervised learning. It has generally been accepted that feeding the instantaneous value of myoelectric signals directly to a classifier is impractical and useless for pattern recognition techniques5788Existing gesture recognition approaches can be broadly divided into two categories: (1) methods based on sparse multi-channel sEMG15161718202123The collected HD-sEMG data are composed of myoelectric signals that characterize the spatiotemporal distribution of myoelectric activity over the muscles that reside within the electrode pick-up area. The collected HD-sEMG data also provide a global view of the varying states of electric fields on the surface of the involved muscles sampled via arrayed electrodes within the covered region, i.e., the instantaneous values of HD-sEMG present a relative global measure of the physiological processes underlying muscle activities at a specific time. However, whether the instantaneous HD-sEMG exhibits spatial patterns or is random like individual sEMG channels remains unclear.We have determined that there are patterns inside the instantaneous HD-sEMG that are reproducible across trials of the same gesture and discriminative among different gestures. To verify this hypothesis computationally, we introduced the concept of an sEMG image and designed a series of experiments using three public databases employing an image-classification framework to recognize gestures from HD-sEMG.An acquired HD-sEMG describes a potential distribution in space . The resulting recognition accuracy was 84.6% on a single difference image, and 99.4% using simple majority voting over 149 difference images. By simple majority voting over 150 instantaneous sEMG images together with their 149 difference images, we obtained a recognition accuracy of 99.6%. Such a high recognition accuracy on the difference sEMG images further verify that there are patterns in the instantaneous HD-sEMG signals from a temporal perspective.Finally, we developed a prototype MCI based on real-time gesture recognition shown in , which rMotivated by the very high accuracy of the HD-sEMG-based gesture recognition via deep learning, in the second experiment, we replaced ConvNet with conventional classifiers. The dataset was identical to that in experiment 1, DB-a, where instantaneous values of sEMG signals were directly formed as a feature vector (denoted as sEMG vector). This procedure ensured that the conventional classifier did not utilize any additional information or manually specified features of myoelectric activity residing within the electrode pick-up area. We evaluated five classical classifiers: MLP (multilayer perceptron), KNN (K-Nearest Neighbours), SVM (Support Vector Machine), Random Forests and LDA . The resulting evaluation of the DB-a dataset demonstrated that patterns can be observed from gesture recognition with MLP and Random Forests. Thus, patterns can be captured by certain classical recognizers and not merely by the deep-learning framework.et al.We evaluated our ConvNet-based gesture recognition on the CSL-HDEMG databaseet al.We also evaluated the sEMG map proposed by Rojas In experiment 4, we performed the tests of 8 figure gestures on the NinaPro databaseMoreover, we evaluated the ConvNet-based recognition of all 52 hand gestures of NinaPro DB1 using the aforementioned classification procedure. As shown in In this study, we performed a series of experiments to verify our assumptions on the patterns inside instantaneous sEMG images and demonstrate that the hand gestures of a specific subject will be effectively recognized directly on the instantaneous sEMG images with an image classifier trained on the samples of hand gestures from the same subject. We achieved state-of-the-art performance for sEMG-based gesture recognition on three public databases  ReLU non-linearityOur ConvNet had eight layers. The input to the ConvNet consisted of a 8 × 16 image for CapgMyo, a 7 × 24 image for CSL-HDEMGWe used stochastic gradient descent (SGD)For MLP, we used a single hidden layer of 1,024 units with ReLU non-linearity and applied the same training scheme as that of the ConvNet case. For KNN, SVM, Random Forests and LDA, we used the implementation and default hyper-parameters of Scikit-learn 0.17.0The hyper-parameters of the entire recognition model included 1) a method for detecting malfunctioning channels; 2) a method for the removal of power-line interference; 3) a method for rectification; 4) a method for low-pass filtering; 5) a method for amplitude normalization; 6) a method for cross-talk removal; 7) a colour scheme for instantaneous sEMG images; and 8) a method for training data augmentation.The hyper-parameters were selected by comparing the recognition accuracy of a specific configuration with that of a baseline model on DB-a .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Introduction. The radicular groove is a developmental groove which is usually found on the palatal or lateral aspects of the maxillary incisor teeth. The present case is a maxillary lateral incisor with a small second root and a deep radicular groove. The developmental groove caused a combined periodontal-endodontic lesion. Methods. Case was managed using a combined treatment procedure involving nonsurgical root canal therapy and surgical periodontal treatment. After completion of root canal treatment, guided tissue regeneration (GTR) was carried out using decalcified freeze dried bone allograft (DFDBA) and a bioabsorbable collagenous membrane. Tooth also was splinted for two months. Results. After 12 months the tooth was asymptomatic. The periapical radiolucency disappeared and probing depth did not exceed 3 mm. Conclusion. Combined treatment procedure involving nonsurgical root canal therapy and surgical periodontal regenerative treatment can be a predictable technique in treating combined endodontic-periodontal lesions caused by radicular groove. The radicular groove is a developmental groove which is usually found on the palatal or lateral aspects of the maxillary incisor teeth. It is a linear depression that sometimes reaches the apex . The groRadicular grooves have been classified into three groups based on their severity. Type I, the groove is limited to the coronal third of the root. Type II, the groove is extended beyond the coronal third of the root but it is shallow and pulp is not exposed. Type III, the groove is long and deep, it is extended beyond the coronal third of the root, and root canal system is involved .It has been stated that the radicular groove is analogous to the pathogenesis of dens invaginatus because it happens due to a slight in-folding of the enamel organ and Hertwig's epithelial root sheath cells during odontogenesis .Radicular groove can be an appropriate habitat for microorganisms and plaque accumulation. Therefore, a local inflammation and deep periodontal pocket are general findings. A deep pocket that reaches to the root apex can affect the pulp vitality and causes a combined periodontal-endodontic lesion .In order to treat a radicular groove the following procedures have been suggested: curettage of the affected periodontal tissues , sealingAlthough most anatomical studies have shown that maxillary incisors always have a single root, there are case reports suggesting lateral incisor with two , 12 and The aim of the present paper is to report a case involving a maxillary lateral incisor with a small second root and a Type III deep radicular groove which caused a combined periodontal-endodontic lesion. Therefore, a combined treatment procedure involving nonsurgical root canal therapy and surgical periodontal treatment was required.A 32-year-old female patient presented to endodontic department of Qazvin School of Dentistry with a chief complaint of mobility of maxillary left lateral incisor. Clinical examination was performed. A 7 mm localized pocket was detected on the palatal of tooth #10 . ProbingConsidering patient's history, clinical and radiographic examination, the lesion was provisionally diagnosed as necrotic pulp and localized periodontitis secondary to the radicular groove.Treatment options were presented to the patient including a combined procedure involving endodontic and periodontal regenerative treatments. She was informed that due to length and depth of the radicular groove, long-term prognosis of tooth #10 is questionable. A written informed consent was obtained and the patient was scheduled for endodontic treatment.At the patient's return, local anesthesia was administered using local infiltration  technique with a cartridge of lidocaine ; after proper isolation, access cavity on tooth #10 was prepared. Canal preparation was performed using the ProTaper system  according to the manufacturer's instructions. An attempt was made to negotiate the second root canal, but no canal opening was found for the supernumerary root. Sodium hypochlorite 2.5%  was used as irrigating solution. Canal was obturated using lateral compaction technique with gutta-percha  and AH26 sealer . The access cavity was sealed with Cavit  .μm  and a 20 × 25 mm (0.4 to 0.6 mm thickness) bioabsorbable collagenous membrane  . Sulcular incisions were placed on the labial side from #9 to #11; to increase the access a releasing incision was also made on the distal of #11. A full thickness mucoperiosteal flap was raised on labial and palatal aspects to access the radicular groove . Granulad, Iran) . The flaThe histologic examination results revealed that the structure of supernumerary root has no abnormality, no dysplastic cell is detected, and the surrounding tissue consists of connective tissue and inflammatory cells .Twelve months' follow-up revealed absence of signs and symptoms, and probing depth did not exceed 3 mm and radiographic examination indicated disappearance of the radiolucency around the tooth #10 due to bone grafting and simultaneous bone regeneration .Combined endodontic-periodontal lesions are real clinical dilemmas due to difficulty of making a differential diagnosis and deciding a prognosis. One of the serious factors that causes this kind of combined lesions is radicular groove. These developmental grooves act as “plaque trap” and initiating factor in localized gingivitis and periodontitis . In caseThe key factor to achieve success in management of this type of anomalies is accurate diagnosis . TherefoSurgical removal of granulation tissue and irritantsGingivectomy and apically positioned flapSurgical exposure and flattening of the groove by grinding, with or without application of guided tissue regeneration techniquesPlacing amalgam restoration in the grooveOrthodontic extrusion of the toothAlthough shallow grooves which are located entirely on the crown can be corrected by odontoplasty and curettage of granulation tissue, more complicated radicular grooves that are associated with severe periodontal breakdown and extensive periapical lesion need surgical intervention [Kerezoudis et al.  suggestervention .In our case, due to presence of deep periodontal pocket and severe attachment loss, surgical procedure was performed. After flattering and odontoplasty of the groove, the anatomy of the root was favorable. That is why no restorative material was used to restore the groove.Regenerative periodontal therapy aims to predictably restore the periodontal tissue and leads to formation of a new cementum with inserting periodontal ligament fibers .Therefore, to enhance the periodontal attachment of the tooth in the present case, DFDBA was used. It has been stated that DFDBA is a scaffold for bone formation and is able to be converted into bone . Also, tThe applied treatment regimen was successful as it can be observed on the follow-up radiography. After 12 months the subjected tooth was asymptomatic, and a 3 mm healthy gingival sulcus was restored in relation to the radicular groove.Combined treatment procedure involving nonsurgical root canal therapy and surgical periodontal regenerative treatment can be a predictable technique in treating combined endodontic-periodontal lesions caused by radicular groove.


The PI was positively correlated with SAH volume (p = 0.025). For patients with lower SAH volume, patients with favorable outcome had a higher PI than those with poor outcome . Using multivariable logistic regression analysis, the PI was a positive determinant for favorable outcome  that more proliferation-promoting factors in CSF was associated with better functional outcome in SAH patients.Neurogenesis from endogenous neural stem cells (NSCs) might contribute to functional recovery after stroke based on animal studies; however, the relationship between neurogenesis and post-stroke outcome has rarely been demonstrated in humans. We prospectively collected cerebrospinal fluid (CSF) from 36 patients with subarachnoid hemorrhage (SAH). The CSF was added to the culture medium of the rat NSCs to test the effects on proliferation . We correlated the PI with functional outcome based on the modified Rankin Scale at 3 months post-SAH. Treatment with the CSF samples collected from SAH patients showed a higher PI compared with those collected from patients with normal pressure hydrocephalus and untreated controls (20.3 ± 8.8  Many recent studies have focused on regenerative therapies for stroke3. Endogenous neural stem cells (NSCs), which are located mainly in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus, may also provide another attractive source for cell therapy4. Recently, many studies have shown that various kinds of brain injury, including stroke, may enhance neurogenesis at the SVZ and SGZ regions6. Stroke-induced neurogenesis may play an important role in functional recovery after stroke7. This concept has been demonstrated by conditional ablation of NSCs in adult mice diminishing poststroke motor and cognitive functional improvement and reduced synaptic connectivity9. However, the beneficial effects of neurogenesis have not been demonstrated in human studies, primarily because there is no available method to evaluate the neurogensis capacity in the brain of stroke patients.Stroke is one of the major causes of death and disability all over the world10. In rodent brain, increased proliferation capacity of NSCs has been reported after SAH12. In human brain, SAH also induced the expression of NSC proliferation markers, indicating enhancement of neurogenesis13. To investigate the relationship between neurogenesis and SAH provides a chance not only to delineate the pathogenesis of the disease, but also to develop a potential treatment strategy, especially since there is still no effective therapy for SAH patients14.Subarachnoid hemorrhage (SAH) is a subtype of stroke, which carries significant morbidity and mortality, with one-third of survivors suffering long-term physical and neurocognitive impairments15. Since the SVZ and SGZ areas are close to cerebral ventricle without the blood–brain barrier, the above factors may also appear in cerebrospinal fluid (CSF). Investigating CSF is thus a potential way to understand the relationships between neurogenesis and features of SAH. We have demonstrated that the NSC proliferation capacity is increased at the SVZ regions in a rat model of SAH at post-SAH 5–7 days11. Notably, the CSF collected from SAH rats during post-SAH 5–7 days can enhance the proliferating potentials of cultured rodent NSCs11. These findings imply the existence of proliferation-promoting factors in CSF of SAH rats. In this study, we tried to obtain CSF from SAH patients, measure the proliferation-promoting capacity of the CSF samples, and correlate this capacity to clinical features of SAH patients. We showed that the proliferation capacity of cultured NSCs promoted by CSF collected from SAH patients correlates well with disease severity and functional outcome.In pathological conditions, injured brain may release some factors that diffuse to the SVZ and SGZ regions and stimulate neurogenesis16.We prospectively collected CSF samples from SAH patients Fig. . The CSF11. Therefore, we first tested the effects of human CSF samples collected from three SAH patients on post-SAH day 5 at different concentrations. The PI was higher in the SAH group than both normal pressure hydrocephalus (NPH) and untreated control groups in a dose-dependent manner (p < 0.05)  . We thus collected the CSF samples on day 5 post-SAH in all other patients. We also used BrdU method and cell viability assay kit  to analyze the NSC proliferation, and found similar results that NSCs treated with CSF samples (n = 6) collected at 5 days post-SAH showed higher proliferation capacity as compared to those treated with CSF samples obtained from NPH patients and those without treatment   in six SAH patients. Only NSCs treated with CSF samples collected at 5 days post-SAH showed a higher PI as compared to those treated with CSF samples obtained from NPH patients and those without treatment (18.5 ± 9.5 05) Fig. . The PI The demographic data and clinical features of 36 SAH patients  are summarized in Supplementary Table vs. 8.2 ± 5.1 and 7.8 ± 3.0, P < 0.001)  Fig. . The PI 01) Fig. . On the 01) Fig. .Figure 3The overall correlation between the PI and mRS at post-SAH 3 months was not significant   and a higher PI  than those with poor outcome. Age, gender, SAH volume, aneurysm location, and presence of IVH or vasospasm were not significantly different between the groups. In the high SAH volume group, patients with favorable outcome had a higher proportion of males  than those with poor outcome, while other parameters including the PI were not significantly different between the groups.We further compared the features of patients with favorable and poor outcome Table . In the Using univariate logistic regression analysis, only initial GCS  was a positive determinant for favorable outcome Table .Table 2U17. However, the relationship between neurogenesis and post-stroke outcome has rarely been demonstrated in humans. After stroke, injured brain may release some factors to stimulate endogenous neurogenesis and these factors may also appear in CSF15. Therefore, investigating CSF is a potential way to understand the relationships between neurogenesis and features of stroke. We chose a stroke subtype, SAH, for this pilot study because lumbar drain is a routine management procedure for our SAH patients and therefore we could easily obtain the CSF samples in clinical practice. Here, we showed that the CSF from SAH patients enhanced the proliferation capacity of cultured rat NSCs in a severity-dependent manner, which indicates the existence of proliferation-promoting factors in CSF after SAH. The proliferation capacity positively correlated with clinical outcome, which may imply that more proliferation-promoting factors in CSF is associated with better functional outcome in SAH patients. Therefore, this study for the first time demonstrated the importance of NSC proliferation on the functional outcome of stroke patients, focusing on SAH.Endogenous neurogenesis might contribute to the repair process following brain damage after stroke based on studies in mice which show ablation of neurogenesis impedes motor function recovery after cerebral ischemia12. Since fresh blood containing red blood cells (RBCs) and plasma would appear in CSF of patients with SAH, we also treated the cultured NSCs with human RBCs and plasma to rule out their contributions to NSC proliferation. The PI was not increased in treatment with lytic or non-lytic RBCs  for further treatment that patients with low PI values and relatively lower SAH volume might be a good candidate to receive certain management to enhance the endogenous NSC proliferation.Using multivariable logistic regression with control of the variables including age, infarct volume, initial GCS, and presence of IVH or vasospasm, the proliferation promoting capacity of post-SAH CSF positively independently correlated with clinical outcome, which indicates that more proliferation promoting factors in CSF is associated with better functional outcome in SAH patients. It is reasonable that with the higher post-SAH production of proliferation promoting factors in CSF, the endogenous NSCs at the SVZ and SGZ of post-SAH brain would receive stronger stimulation to undergo cell proliferation, as we observed in the culture system. Post-stroke neurogenesis can promote tissue remodeling and neural repair, which have been demonstrated in the rodent model of cerebral infarct1. Future studies should try to identify the proliferation-promoting factors in CSF and extend the investigation to other types of stroke. Third, in vitro NSC proliferation and actual in vivo neurogenesis may be different. However, in our previous rodent study, we found that the neurogenesis at the SVZ region is enhanced in a rat model of SAH on days 5–7 post-SAH; the CSF collected from SAH rats during days 5–7 post-SAH can promote NSC proliferation11. The result may suggest that in vitro NSC proliferation is likely associated with in vivo post-SAH neurogenesis.This study had some limitations. First, we could not obtain CSF from healthy volunteers and therefore used CSF from NPH patients as a control instead. However, we cannot guarantee that the results obtained with CSF samples from NPH patients are totally normal. Second, although we clearly demonstrated the existence of proliferation promoting factors in CSF after SAH, we did not identify the specific factors in this study. In addition, we only enrolled patients with SAH but not cerebral infarct or intracerebral hemorrhage, which are more common subtypes of strokeIn summary, we provided a novel method using human CSF to study the relationship between NSC proliferation capacity and functional outcome in patients with SAH. The CSF from SAH patients enhanced the proliferation capacity of cultured NSCs, indicating the presence of neurogenesis promoting factors in CSF after SAH. The proliferation capacity positively correlated with clinical outcome, which implies that more proliferation-promoting factors in CSF is associated with better functional outcome in SAH patients. This study thus supports the rationale to enhance the proliferation capacity of NSCs as a reasonable therapeutic strategy for stroke treatment, especially for SAH.26. The patients were all monitored in the neurointensive care unit and treated with the standard protocol which consisted of resuscitation, early surgical or endovascular obliteration of the aneurysm, standard management of intracranial pressure, neurointensive care, and aggressive medical or endovascular therapy for vasospasm if present14. All patients received nimodipine for prevention of vasospasm.We prospectively recruited 36 adult patients with SAH from National Taiwan University Hospital who received lumbar drainage for CSF diversion. Initial head computed tomography (CT) was performed all within 1 day except for one patient (within 2 days) after the onset of SAH. We only included SAH patients with modified Fisher grading score of 3 and 4, which are defined as thick SAH (>1 mm in depth) without and with IVH, respectivelyThe CSF was collected via lumbar drain on day 5 after SAH. We obtained CSF on days 3, 5, and 7 after SAH in the first six patients for initial analysis of the optimal time to collect the CSF. The CSF samples were immediately centrifuged at 900 g at 4 °C for 20 min before being divided into suitable aliquots and snap-frozen at −80 °C within 30 min. We also collected CSF from six patients with NPH to use as a control. The research was approved by the National Taiwan University Hospital Committee of Human Research and conducted in accordance with human ethics regulations (No. 201605042RINB). Written informed consent was obtained from the patients or from the next of kin of patients who had decreased consciousness levels.27. First, embryos were removed from the rat, and the embryonic cerebral cortices were dissected out, washed, triturated, and cultured in the complete media containing Dulbecco’s Modified Eagle Media (DMEM)/F-12  supplemented with 1% N2 supplement (Gibco), 20 ng/ml basic fibroblast growth factor (bFGF), and 1% antibiotic solution absence of serum. Cultures were incubated at 37 °C in a humidified atmosphere and 5% CO2 for 6 days by which time primary neurospheres would form. Neurospheres were then cultured on 24-welled tissue culture polystyrene  at 200 ± 20 neurospheres/cm2 in the DMEM/F12 medium supplemented with 1% N2 supplemented, 1% antibiotic-antimycotic solution (Gibco), and different concentration  of CSF for 3 days without changing the culture medium. In addition to immunocytochemistry study, the Cell Counting Kit-8 (CCK-8)  was used to assess the NSC proliferation capacity. Equal amount of NSC was cultured with or without 0.5% CSF. After 3 days, CCK-8 solution was added to culture medium and NSCs were incubated at 37 °C for additional 4 hours. Optical density (OD) was then determined at a wave-length of 450 nm.NSCs were obtained from pregnant Wistar rats at the gestational age of 15 days according to a protocol previously describedThe animal experimental procedures were approved by the National Taiwan University Institutional Laboratory Animal Care committee and the Utilization Committee (No. 20160109). All procedures met the requirements of the Animal Welfare Protection Act of the Department of Agriculture, Executive Yuan, Taiwan.In immunocytochemical characterization, cells were fixed with ice-cold methanol for 20 min, blocked with 1% bovine serum albumin for 30 minutes at room temperature, and then incubated with primary antibodies overnight at 4 °C. For BrdU labeling, NSCs were pretreated with BrdU  at 10 μM for 3 days, fixed with methanol, and then treated with 4% Formaldehyde/1% Triton X100 for 10 min and 1 M HCl for 10 min before blocked. The primary antibodies used in this study were rabbit anti-Ki67 polyclonal antibody , mouse anti-BrdU monoclonal antibody , and mouse anti-nestin monoclonal antibody . After wash, cells were incubated with secondary antibodies and Hoechst 33258 (diluted in PBS) for 2 hours at room temperature. The secondary antibodies used in the experiment were Alexa Fluor 488-conjugated goat anti-ribbit IgG and CyTM3-conjugated donkey anti-mouse IgG . The immunostained cells were visualized by confocal LSM880 microscope  with the optical thickness of 2 μm. For each condition, we analyzed at least three neurospheres with at least three planes for each neurosphere. The sub-PI of each plane was calculated as the areas with Ki-67 (or BudU) immunoreactive cells divided by the areas with Hoechst 33258-stained cells; the areas were determined automatically using MetaMorph software . All the sub-PI values were then averaged to get a final PI for each condition.28. The researcher who measured the PI was blinded to the clinical condition of SAH patients and the researchers who analyzed the SAH volume or accessed the mRS were blinded to the PI values.The severity of SAH was determined by the volume of blood that appeared in the subarachnoid cisterns  on initial axial head CT . The area of hemorrhage on each film was measured manually using software of ImageJ. The median SAH volume in the subarachnoid cistern of 36 patients was 15.2 ml; therefore, we set the cutoff value for high and low SAH volume at 15 ml. The location of aneurysm is stratified into anterior circulation  and posterior circulation . A follow-up head CT was performed around 1 week post-SAH in all patients to detect potential new intracerebral hypodense lesions, which indicate post-SAH cerebral infarction. An additional head CT with CT angiography was performed if neurological deterioration developed or when vasospasm was suspected during post-SAH care within 2 weeks. The existence of vasospasm was defined as the presence of post-SAH cerebral infarct detected with head CT or vasospasm detected with CT angiography. The functional outcome was evaluated using the mRS at 3 months after onset of SAH. An mRS score less than 3 (able to look after own affairs without assistance) was defined as a favorable functional outcomeThe results are reported as mean ± standard deviation (SD). To compare the PI between groups, the Kruskal-Wallis test with the Mann-Whitney test as post-hoc analysis was used. We correlated the PI value with clinical data, including age, gender, initial GCS, SAH volume, IVH, vasospasm, location of aneurysm, and mRS using linear regression analysis. To analyze the outcome, the Mann-Whitney test and Fisher’s exact test were used to determine the differences between groups for continuous data and categorical data, respectively. A logistic regression model was used to analyze the determinants of favorable outcome. The model included SAH volume, initial GCS, PI value, age, presence of IVH, and presence of vasospasm. A p-value of less than 0.05 was considered statistically significant. The SPSS statistical software  was used for the statistical analyses.Supplementary Information