Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis  Adult hippocampal neurogenesis occurs in mammals, from mice to humans1246We performed an unbiased discovery proteomics screen in a stem cell model of human brain development to identify potential CSF biomarkers of neuronal differentiationSix family members, Gpc1-Gpc6, are all expressed in the developing nervous system, but only Gpc2 expression is exclusively restricted to the CNS9111213Using immunocompetitive capture coupled mass spectrometry, we showed that Gpc2 binds to Fibroblast Growth Factor 2 (FGF2) and its receptor. Gpc2 modulates neural progenitor cell proliferation by antagonizing the mitotic effect of FGF2. In rodents, Gpc2 expression is restricted to the neurogenic regions, the subgranular zone (SGZ) of the dentate gyrus (DG) and the subventricular zone-rostral migratory stream-olfactory bulb region (SVZ/RMS/OB), and can be detected in rodent and human CSF. We showed a marked reduction of Gpc2 in the CSF after genetic ablation of adult neurogenesis, thus addressing its specificity. In addition, known physiological modulators of adult neurogenesis, including running wheel and fluoxetine treatments, led to increased Gpc2 levels in CSF; conversely, aging led to a marked reduction in Gpc2 levels. Thus, CSF Gpc2 levels reflect the degree of adult hippocampal neurogenesis. Gpc2 was also detected in human CSF, and the initial experiments showed a moderate age-dependent reduction in Gpc2 levels in a small longitudinal cohort. Based on these results, Gpc2 may be a potential translational biomarker, justifying further investigations of adult human hippocampal neurogenesis and neurogenic activity in response to drug treatments.We performed a discovery proteomics screen of human embryonic stem cell-derived neural progenitor cells (hNPCs) at different developmental stages after the onset of neuronal differentiation to identify novel protein markers of neuronal differentiation. Protein lysates were collected at different days after the induction of neural differentiation and subjected to mass spectrometry analysis . Overallhttp://mouse.brain-map.org)We hypothesized that proteins displaying a dynamic expression pattern similar to known stage-specific marker proteins might be novel markers of immature neurons. Thus, we clustered the proteins according to their dynamic expression patterns during neural differentiation . BecauseGpcs are membrane-bound proteins belonging to the family of heparan sulfate proteoglycans (HSPGs)13Gpcs are attached to the plasma membrane via a GPI anchor but are proteolytically cleaved and released into the ECM18Thus, Gpc2 is dynamically and transiently expressed during the neural differentiation of human neural stem cells. It is released into the extracellular milieu, suggesting that Gpc2 has the potential to function as a CSF marker of neurogenesis.We performed immunocompetitive capture mass spectrometry (ICC-MS) to identify the proteins that bind Gpc2 and may explain the link between neurogenesis and Gpc2 20. Compe+ cells within the total population in situ hybridization (ISH) in neurogenic regions from adult mouse brains to corroborate these findings. The Gpc2 mRNA was predominantly located within the subgranular zone (SGZ)  of the dentate gyrus (DG) and the subventricular zone-rostral migratory stream-olfactory bulb regionne (SGZ) , the dorne (SGZ)  and withne (SGZ) , indicatne (SGZ) . We obsene (SGZ) .Hippocampal neurogenesis dramatically decreases during the initial postnatal period after birth23Proteins within cerebrospinal fluid are used as biomarkers for disease diagnosis and progression25262728We conclude that the CSF levels of Gpc2 protein during postnatal periods reflect the gradual decrease in neurogenesis, and we next tested whether Gpc2 has the potential as a CSF biomarker for adult neurogenesis.+ NSCs and also observed a significant reduction in the number of Dcx-positive cells in the DG  treatment is required for behavioral activity37383940adult DG  as well adult DG . This inadult DG . Thus, C+ progenitors233142Aging leads to a reduction in adult neurogenesis, as determined by loss of proliferating neural precursor cells and the reduction in total DcxBased on these results, the CSF levels of Gpc2 protein can be used as a CSF biomarker to detect changes in the extent of adult neurogenesis modulated by physical exercise, antidepressant treatment and aging.As an initial test to assess the feasibility of measuring Gpc2 protein levels in human CSF, we measured the levels of Gpc2 protein in commercially available CSF samples obtained from 83 individuals . Gpc2 waInter-subject variability might prohibit a correlation with age and potential technical sources of variability cannot be excluded in commercially available CSF samples. We therefore analyzed CSF Gpc2 levels in a small subset of human CSF samples for which longitudinal CSF sampling has been performed in order to reduce potential technical sources of variability and to better address intra-subject variability. When Gpc2 levels in three-year follow-up samples were compared to the baseline samples, we observed low intra-subject variability and a decrease in the CSF levels of Gpc2 in 9 out of 10 individuals .Our initial findings in human CSF support to the prospective use of Gpc2 as a potential CSF biomarker for human adult neurogenesis.post mortem tissue4414C-dating to date the nuclear content of these neurons has been used to show that neurogenesis exists in several regions of the adult human brain146Current standard methods for monitoring adult neurogenesis are based on a retrospective analysis of Currently, efforts to identify biomarkers that reflect the generation of newborn neurons in living humans have been hampered by the correlative nature, sensitivity or reproducibility of the results73048The detection of proteins in CSF shows promise as a reflection of the physiological and pathophysiological changes in the central nervous system. The beta-amyloid and Tau proteins have proven to be valuable diagnostic biomarkers for Alzheimer’s disease504852In preliminary experiments, we show that Gpc2 is present in human CSF but its levels are highly variable . Little To this end, we tested Gpc2 levels in a small cohort of non-demented aged individuals . CSF sam56Our data highlights the novelty and the advantage of Gpc2 compared to DCX as a CSF biomarker of the process of adult neurogenesis. Unlike DCX, Gpc2 expression is not restricted to early postnatal stages of development, which enabled us to directly assess the effect of altered adult neurogenesis on Gpc2 CSF levels in animal models . Gpc2 leOur first experiments using human CSF samples indicate that while CSF Gpc2 levels show high variability between subjects, the intra-subject variability is low. Further longitudinal experiments with samples from large, well-characterized cohorts of individuals with and without central nervous system diseases or under controlled conditions that modulate neurogenesis are required to validate this initial finding and to assess utility of CSF Gpc2 analysis in the clinic.Notably, another member of the Glypican family, Gpc3, may serve as a diagnostic serum marker of hepatocellular carcinomasFrom a biological perspective, we provide insights as to how a specific HSPG may control the availability of growth factors in the neurogenic niche. Cellular and molecular components provide a specific local microenvironment in the adult SGZ that provides the necessary signals to maintain the stem cell population and to support neural differentiation and maturation. Microglia, astrocytes, neurons and the local vasculature provide direct cell-to-cell contacts, neurotrophic support, neurotransmitters and growth factors to control individual steps in the process of neurogenesis6061in vivoHSPGs constitute part of the local environment in the adult DG and have also been shown to play a critical role in the modulation of key signaling pathways for secreted factors, such as Wnt, Shh, BMP and FGF during mammalian developmentin-situ-hybridization, (b) its correlation with expression changes in DCX as a marker of neural blast and immature neurons, and (c) its reduced CSF protein levels after ablation of neurogenesis in different transgenic models. Due to technical limitations, it was not possible to perform a stage specific cellular analysis of Gpc2 protein expression. Since a previous study reported that Gpc2 is a Sox11 target gene, it is likely to be restricted to Sox11+ neuronal precursors and immature neurons, but this will require further confirmationWe hypothesize that Gpc2 functions as a niche factor that regulates the availability of FGF2 and thus modulates progenitor cell proliferation. The binding of Gpc2 to FGF2 may reduce the local concentrations within the niche, potentially resulting in a negative feedback loop that reduces the net amount of proliferation. As shown in our study, the presence of high CSF levels of Gpc2 and abundant expression of membrane-bound HSPGs in the brainIn summary, we have identified a CNS-specific protein, Glypican-2, that may modulate proliferation by sequestering FGF2 in the neurogenic niche and may be used as a CSF biomarker for adult neurogenesis. Further studies focusing on characterization of the CSF Gpc2 signal and stability, as well as analyses in diseases in which neurogenesis is affected, are required to validate Gpc2 as a biomarker for neurogenesis. We are tempted to speculate that changes in CSF Gpc2 levels may be observed in depression and Alzheimer’s disease, human diseases in which a reduction in adult neurogenesis has been described692 and maintained in basic medium supplemented with 20 ng/ml BDNF (Peprotech) to induce differentiation. Alternatively, differentiation was induced by replating NPCs at a density of 12,000 cells/cm2 and culturing in basal medium supplemented with 200 ng/ml Shh (Peprotech), 100 ng/ml FGF8 (Peprotech) and 100 μM ascorbic acid (Sigma) for 7 days, with subsequent replating in basal medium supplemented with 20 ng/ml BDNF (Peprotech), 10 ng/ml GDNF (Peprotech), 0.5 mM cAMP (BIOLOG Life Science) and 100 μM ascorbic acid (Sigma) at a density of 45,000 cells/cm2.The conditions used for the neural progenitor cultures have been previously describedNeural progenitor cells were cultured in basic medium composed of equal volumes of DMEM:F12 Glutamax medium and Neurobasal medium  supplemented with 2% B27 ; 1% N2 ; 0.1% beta-mercaptoethanol ; 0, 1 or 5 ng/ml FGF2 (Peprotech); and 0, 0.1, 1, 10, 100 or 500 nM recombinant human Gpc2 (R&D Systems) to analyze the effect of Gpc2 on FGF2-mediated proliferation.6 cells per condition) were harvested after t = 0, 1, 2, 4, 7, 14, 21 or 28 days. After lysis by sonication, a crude membrane fraction was obtained by centrifugation, washed in high salt/high pH to deplete the soluble proteins and further fractionated by SDS-PAGE. Each lane was divided into 12 bands that were subjected to in-gel trypsinization and individually analyzed on a nanoLC-MS/MS using a Thermo LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific).In the initial discovery proteomics step, biological triplicates of NSCs . Anti-FGF2 from Abcam (ab126861) was used for both IP and blot detection. The Gpc2 immunocompetitive capture experiment was performed as previously describedT2 RBPjkfl/fl model were conducted under SPF conditions according to institutional regulations under license number 2537 . All testing of the corticosterone model was conducted in compliance with the laboratory animal care guidelines and with protocols approved by the Institutional Animal Care and Use Committee .All experiments involving mice and rats were performed by authorized investigators following national and European ethical guidelines. Young adult mice  between 8 and 10 weeks of age were used for the experiments, unless stated otherwise. Mice were maintained on a 12-h day/night cycle with adequate food and water, according to the guidelines from local veterinary authorities under license numbers 2012–0016/2584 . Experiments using the Hes5::CreERRat tissues and cerebrospinal fluid (Sprague-Dawley) obtained at different postnatal developmental stages were purchased from QPS Austria GmbH.An agreement (license L-O 15-2015/0) between the NIH and the Université Paris-Sud provides UMRS 1178 with the use of transgenic mice that express herpes simplex virus thymidine kinase (TK) under control of the glial fibrillary acidic protein (GFAP) promoter (GFAP-TK mice), as previously describedGanciclovir  was administered to the treatment group in the chow for a period of 4 weeks using the method reported in previous studies34ad libitum. CORT-treated water was changed every 3 days to prevent possible degradation. Fluoxetine hydrochloride  was purchased from Anawa Trading  and dissolved in a 0.45% β-CD/corticosterone solution. Thereafter, during the administration of β-CD or corticosterone, mice were treated with vehicle (0.45% β-CD) or fluoxetine in the drinking water, as previously describedThe hypothalamic-pituitary-adrenal (HPA) axis is often dysregulated in clinical depression. The glucocorticoid levels are exogenously increased in this model. This chronic elevation of corticosterone (CORT) levels results in dysregulation of the HPA axis. For example, a blunting of the HPA axis in response to stress is observed in CORT-treated mice, as shown by the marked attenuation of stress-induced CORT levels3838Hes5::CreERT2 and floxed RBPjk mice have been previously described33Stock solutions of tamoxifen (Sigma) were prepared at a concentration of 20 mg/ml in corn oil (Sigma). Adult mice were i.p. injected with 2 mg/day tamoxifen once per day for 5 consecutive days to induce recombination and were euthanized for analysis 21 or 100 days after the last injection. The brains were prepared for further processing as described below.The running experiment has been previously described35For CSF collection, the mice were deeply anesthetized by an intraperitoneal (i.p.) injection of pentobarbital solution (150 mg/kg body weight). CSF was isolated by puncturing the cisterna magna with a narrow microcapillary needle (Hirschmann). The skin and musculature overlaying the cisterna magna were carefully removed to expose the arachnoid membrane covering the cistern. The surrounding area was gently cleaned to remove the residual blood and interstitial fluid. Upon puncture of the membrane, CSF was readily collected by capillary forces. In cases with strong blood contamination, the sample was excluded from further analysis. CSF was snap-frozen on dry ice until further analysis. Immunoassays for Gpc2 were performed using the AlphaLISA bead-based technology (Perkin Elmer) according to the manufacturer’s protocol. Briefly, AlphaLISA acceptor beads were conjugated with a novel monoclonal antibody against mouse Gpc2, mAb4/7, to obtain a final concentration of 5 mg/ml of conjugated antibody in PBS with 0.05% Proclin300 (Sigma). Biotinylated mAb4/15, a second novel monoclonal antibody against mouse Gpc2, was used to detect Gpc2. The rodent CSF samples were diluted in AlphaLISA HiBlock buffer (Perkin Elmer) in the presence of 1% normal human serum (Sigma) to analyze Gpc2 levels. Acceptor-bead coupled mAb4/7 (10 μg/ml) and 1 nM biotinylated mAb4/15 were incubated with the corresponding sample for 3 h at RT. Streptavidin-conjugated donor beads were added to a final concentration of 40 μg/ml and incubated for an additional 30 min at RT in the dark. The plates were analyzed in a 2104 EnVision Multilabel Reader (Perkin Elmer). Recombinant mouse Glypican-2 (R&D Systems) was used as a positive control. Selectivity for Glypican-2 was shown by the absence of immunoreactivity for recombinant Glypican-1, Glypican-3 and Glypican-4.Biotinylated AF2304 (R&D Systems) was used at a final concentration of 0.2 nM in combination with a final concentration of 10 μg/ml novel mAb2/32 against human Gpc2 coupled to acceptor beads to detect human Glypican-2. The assay was performed in AlphaLISA HiBlock buffer without additional supplements to detect human Gpc2.Human CSF samples were purchased from Bioreclamation IVT, NY, USA. Recombinant human Glypican-2 (R&D Systems) was used as a positive control. In addition, subjects of both sexes aged 40 years or above were recruited from two different clinical centers in France and Sweden after the protocol was approved by the ethics committee of the corresponding countries: CCPPRB Alsace I Strasbourg, University Clinic Huddinge and Uppsala. All procedures were conducted according to the principles expressed in the Declaration of Helsinki, and all subjects provided written informed consent. In cases where the patients were considered not to have the capacity to consent, informed consent was provided by their relatives.in situ hybridization, coronal sections of equivalent regions of the caudal and rostral hippocampus were embedded in paraffin. Blocks containing the caudal hippocampus were prepared, and four series of slides per block were produced with a gap of 10 slides. IHC was performed on consecutive slides with an anti-DCX  primary antibody on Ventana Discovery® XT autostainer, and ISH was performed with an anti-Gpc2 probe  on Ventana Discovery® Ultra autostainer.After CSF collection, the brains were removed from the skull and split into the two hemispheres. One hemisphere was fixed by a 24-h immersion in 10% neutral-buffered formalin for immunohistochemistry. The other hemisphere was further micro-dissected to prepare the dentate gyrus or respective subanatomical brain regions. Tissues were snap-frozen on dry ice for subsequent RNA analyses. For histology and Total RNA was isolated from snap-frozen brain tissues using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol, and the RNA concentration was quantified using spectrophotometry. Four nanograms of total RNA were used for RT-PCR in a 384-well plate on a Roche Light Cycler LC480. The following Taqman assays (ABI) were used: Mm00549653 ; Mm00438401 ; Mm00727586 ; Mm99999915 ; Rn00593367 ; Rn00584505 ; Rn99999916 . Relative quantification was based on expression of the GAPDH gene.®. Semi-automated image analysis of immunostaining was performed with Definiens Tissue Studio™ software based on manually annotated regions of interest comprising the dentate gyrus.All slides were scanned with Aperio ScanScopeP values were obtained from moderated t tests and adjusted for multiple tests according to the methodology described by Augustin et al.t-test. Differences were considered statistically significant at *p < 0.05, **p < 0.01 and ***p < 0.001. The data are presented as the mean ± SEM. RNA expression data are presented as the mean ± CI.The data processing and statistical analyses of the ICC-MS experiments have been thoroughly described elsewhere+ cells divided by the number of Ki67+ cells (proliferation index). Statistical comparisons were conducted with a two-tailed unpaired Student’s t-test. Differences were considered statistically significant at *p < 0.05, **p < 0.01 and ***p < 0.001. The data are presented as the mean ± SEM. For the CORT and GFAP-TK experiments, the data were analyzed using GraphPad Prism v6.0 f. For the CORT experiments, one-way ANOVA was applied to the data as appropriate. Significant main effects and/or interactions were analyzed using Fisher’s protected least significant difference (PLSD) post hoc tests or unpaired t-tests.Immunofluorescent staining with DAPI and Ki67 was manually traced using a 20x objective and analyzed with ImageJ software. Cell densities were expressed as the number of DAPIHow to cite this article: Lugert, S. et al. Glypican-2 level in cerebrospinal fluid predicts the status of adult hippocampal neurogenesis. Sci. Rep.7, 46543; doi: 10.1038/srep46543 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Midazolam is commonly used as a pre-anesthesia anxiolytic. It`s elimination may not be fast enough for short procedures. In orally premedicated patients we obtained midazolam plasma concentrations at the end of surgical procedures and compared those to concentrations at anesthesia induction.p <0.05).The study was conducted prospectively with consent of the local ethics committee  and carried out with written informed consent of each patient. Female patients aged 20 to 60 years undergoing elective procedures with general anesthesia were included, and were divided in two groups according to the planned surgical time: group S (<30 min) and group L (90–120 min), respectively. All patients received 7.5 mg Midazolam po as premedication. Blood samples were drawn at anesthesia induction, and at the end of surgery. Data were compared with t-test . None of the patients from group L (n = 24), but five patients in group S (n = 22) did have a higher plasma level of Midazolam at the end of the case compared to the beginning.Twenty-five patients per group were included. Four patients were excluded from analysis, since midazolam was not detectable in any samples. Time of premedication to the 1st blood sample was not statistically different between groups, neither were Midazolam plasma levels at this time point ,  In our institution, for many years Midazolam has been the premedication drug of choice for anxiolysis in patients undergoing general anesthetics.Generally, the standard dosage for Midazolam used as an oral premedication before general anesthesia is 7.5–15 mg in adults [This may be an issue because of the rather long elimination half-life of 90–150 min that possibly could result in a comparatively long sedative effect of midazolam. Further, in daily routine, it is not clear, when the peak of absorption actually occurs in relation to the anesthetic process. This can potentially contribute to relatively high plasma levels of midazolam at the end of short procedures and therefore lead to delayed and prolonged emergence from the anesthetic.In the present study we determined midazolam plasma levels in an everyday clinical setting at the time of induction of anesthesia and at the end of the surgical procedure in procedures of different durations. The study attempted to validate the hypothesis that some patients receiving oral midazolam premedication will show higher plasma levels at the end of short procedures (<30 min) compared to the levels determined just before induction of anesthesia. In contrast, patients undergoing longer procedures (> 1.5 h) will always have lower midazolam levels at the end of anesthesia compared to levels measured just before induction of anesthesia.www.drks.de; DRKS00005429). All study participants were informed beforehand and gave their written consent.This study was conducted prospectively with the approval of the local ethics committee  and after registration with the German Clinical Trials Register  before they were transported to the operating room. The order was called to the ward from the operating room by anesthesia providers that were not part of the study group. In accordance to the standard practice for patients receiving general anesthesia at our institution, the premedication with midazolam po ideally would take place 30 to 45 min before the patient came to the operating room. For the study patients, the time when the po midazolam was given on the ward was recorded.A total of 50 patients (25 each with a planned operating time of ≤ 30 min (group S), and 90–120 min (Group L), respectively) were given 7.5 mg midazolam po  . The OAAFor the general anesthetic, we placed the standard monitoring first , an initial set of values was recorded, before moving on and establishing peripheral venous access on the back of the hand or forearm. While placing the venous cannula, 5 ml of blood were collected in a serum tube. The time of blood collection was recorded (time point 1).The further course of anesthesia mainly was a Propofol based anesthesia, supplemented with fentanyl and remifentanil. Anesthesia conduct was left to the responsible anesthetist, according to the standard of our institution. At the end of the surgical procedure, before emerging from the anesthesia, a second blood sample  was drawn from the opposite arm, and the time was noted (time point 2).http://www.usz.ch) where the plasma levels of midazolam were determined by liquid chromatography-mass spectrometry (LC-MS). After addition of stable-isotope labelled internal standards, samples were centrifuged. Twenty microliters of the clear supernatant was submitted to the analysis using a turbulent flow online extraction system. As extraction column, a Cyclone column  was used, as analytical column, a Uptisphere C18 (125 × 2 mm). The mobile phases consisted of 10 mM ammonium acetate in water + 0.1 % formic acid and 10 mM ammonium acetate in methanol/acetonitrile 50/50 (v/v) + 0.1 % formic acid. Calibration was done using an in-house prepared six point calibration curve. The method is validated and has an imprecision of less than 5.3 %. The quantification limit is at 0.01 mcmol/l.Both serum tubes were labeled and properly stored in the laboratory of our hospital . After completion of the entire study all the samples were sent to the Institute of Clinical Chemistry of the University Hospital Zurich .All data (midazolam plasma levels etc.) are presented descriptively as mean ± standard deviation, BIS values and sedation scores are presented as median (minimum - maximum).Comparisons between group S and group L were made using the t test for independent samples, and using the Mann-Whitney U-test for BIS values and sedation scores.The statistical evaluations were performed using Microsoft Excel 2010 , and StatView 5.0.1 .As there are no data about midazolam plasma levels at the end of anesthetics and the main outcome of our study was the number of patients with higher midazolam plasma levels at the end of anesthesia compared to anesthesia induction, there was no formal power analysis performed.Fifty patients were enrolled in this study (25 for each group). Four patients (three in group S and one in group L) were excluded from the statistical analysis, because neither midazolam, nor its metabolite 1-OH-midazolam were detectable in any blood samples, and we assumed that the patients have not taken the medication.The demographic data of the patients are summarized in Table p = 0.12). In accordance with the study protocol, the time between premedication and the second blood sample was significantly shorter in group S (112.2 ± 35.1 min) than in Group L .The timespan between taking the midazolam po and the 1st blood sample was 63.9 ± 31.4 min in group S and 52.6 ± 18.1 min in group L (p = 0.81) as did the first measured BIS values .The O/AAS values upon arrival in the operating room differed very little between group S (4.2 ± 0.7) and Group L . In the second blood sample at the end of the surgical procedure, the midazolam plasma levels in group S (0.06 ± 0.04 mcmol/l) were significantly higher than in group L .In the first blood sample the midazolam plasma levels were similar in both groups  was the midazolam plasma level higher at the end of surgery than before induction of anesthesia, whereas five patients of the group S (n = 22) did have an increased plasma level at the end of the case compared to the pre procedure value .Excluding the five patients with higher midazolam plasma levels at the end of surgery from group S, plasma levels in group S remained significantly higher than in group L .The satisfaction VAS score did not differ significantly between the two groups , a substantial percentage had higher midazolam plasma levels at the end of the operation compared to the time of anesthesia induction. In the group of patients with a longer duration of surgery (90–120 min), all patients had lower midazolam levels at the end of the procedure.Midazolam serves as a very common choice to achieve anxiolysis in children, adolescents and adults before anesthesia , 3, 4. WThe optimal time point for the administration and dosing of midazolam preoperatively remains unclear and appears to be individually different. According to the study by Lim the sedative effects started to wear off after 45–60 min from the time point when midazolam was given as a tablet [In our study it took 48 min from the time of application of the drug to the first pre-operative blood sample. For some individual patients the peak sedative effect could have already happened before they actually came to the operating room.In addition, the interindividual metabolism of midazolam can differ greatly. After oral administration of 15 mg po in young, healthy people its bioavailability varies between 30 and 50 %, and the elimination half-life is between 1.5 and 2.5 h . A compahttp://www.compendium.ch), 7.5 to 15 mg midazolam PO is given as the standard dosing for anesthesia premedication. The laboratory of our institution regards 0.3 to 1 micromol/l as therapeutic plasma level, however without differentiating for which indication. Accordingly, both the subjectively and objectively detectable sedative effect preoperatively was only moderate, but at the same time the subjective satisfaction was rather good. OAA/S scores and BIS values at arrival in the operating room showed only a narrow variation, meaning the effect of the premedication being fairly predictable.The patients in our study were consistently medicated with 7.5 mg midazolam po. As this study was designed to assess clinical routine, this fixed dose was chosen, instead of dosing in relation to body weigth. This resulted in moderate midazolam plasma levels. The reported mean of 0.11 (± 0.06) micromol/l is corresponding to about 30 ng/ml. In the Swiss Drugs registry  compared to the induction of anesthesia when anxiolysis and sedation were most desirable.All the above mentioned points add to our main finding that under clinical routine conditions some patients having been orally premedicated with DormicumBrosius and Bannister were not able to show an interaction of midazolam with general anesthetics (Sevoflurane-based) in their setting and the recovery period after a standardized anesthetic in their midazolam group compared with a placebo group was not significantly longer , 8. HoweOur study setup had some limitations. The actual intake of the midazolam tablet on the ward happened largely unsupervised. The fact that four patients had no detectable plasma levels for neither midazolam nor its metabolites could be the result from the patients not having actually taken the tablet. It is also conceivable that some patients may have ingested only fractions of the actual tablet. Furthermore, it would have been very interesting to correlate the midazolam plasma levels at the end of the procedure with the time it took to emerge from anesthesia. In order to do so, the anesthetic management would have needed standardization with a protocol, which was not the case in our study. Also, if correlating midazolam plasma levels to clinical effect, it might be necessary to consider levels of α-hydroxy-midazolam, a metabolite with intrinsic action. However, the midazolam plasma levels in our study were quite low, and therefore most probably not significantly influencing emerging times after anesthesia.© tablets.Finally, dosing in relation to body weight might lead to more reliable plasma concentrations, but is not practicable with standard DormicumThe results of this study might lead the anesthesiologist to reconsider the use of standard, rather long acting midazolam premedication in patients undergoing surgery with a planned duration of less than 30 min. Non-pharmacological anxiolysis or new, shorter acting substances may be the solution to this problem.In summary, the most pertinent finding in this study was the fact that midazolam po as an anxiolytic before general anesthesia in short interventions can result in higher plasma levels at the end of the procedure.


CSF). G‐CSF‐producing esophageal squamous cell carcinoma is an extremely aggressive carcinoma. Leukocyte counts, neutrophil counts, and serum C‐reactive protein levels may be markers of its progression.There are very few reports of esophageal carcinoma producing granulocyte colony‐stimulating factor (G‐ Patients with malignant neoplasms may show various levels of leukocytosis because of accompanying bacterial infections, such as aspiration pneumonia in esophageal cancer and obstructive colitis in advanced colon cancer μL, and his neutrophil count was also elevated at 20,600 cells/μL. This unreasonable elevation of his blood counts prompted the analysis of his serum G‐CSF level, which was elevated , and an immunohistochemical examination of the biopsy specimens also revealed that the cancer cells were G‐CSF‐positive  of the diagnostic criteria.Granulocyte colony‐stimulating factor is a hematopoietic growth factor that regulates the production of granulocytes and macrophages We were able to diagnose G‐CSF‐producing squamous cell carcinoma using a small block of biopsied specimens. First, the histopathological findings of the biopsied specimens showed atypical findings, such as remarkable neutrophil infiltration surrounding the malignant cells. With suspicion of G‐CSF production, we performed an immunohistochemical analysis, which revealed numerous G‐CSF‐positive malignant cells. In contrast, Shimomura reported that it was difficult to detect G‐CSF in an immunohistological examination because G‐CSF is rapidly secreted from cancer cells after production in vitro To the best of our knowledge, there have been only 10 reported cases of G‐CSF‐producing esophageal squamous cell carcinomas Granulocyte colony‐stimulating factor is a key regulator of neutrophil production. In addition, the correlation between G‐CSF and CRP has been reported in several conditions The opportunity to administer radical treatment is often lost in patients with G‐CSF‐producing esophageal cancer due to the rapid progression of the disease. Similarly, to ordinary esophageal cancer, surgical resection with neoadjuvant therapy is considered to be the mainstay radical treatment for G‐CSF‐producing esophageal cancer. In Japan, the standard treatment strategy includes radical esophagectomy through a thoracotomy or a thoracoscopic approach with neoadjuvant chemotherapy using 5‐FU and CDDP Several studies have reported that the autocrine and paracrine secretions of G‐CSF via the JAK2/STAT3 pathway confer more proliferative, invasive, and chemoresistant features to malignant cells In conclusion, we report a case of aggressive G‐CSF‐producing esophageal squamous cell carcinoma. WBC, neutrophils, and serum CRP levels may be markers of its progression. There is currently no established therapeutic strategy for this disease, and a multidisciplinary approach will be required to overcome G‐CSF‐producing esophageal squamous cell carcinoma in the future.All of the authors have read and approved the manuscript. SY and SK: collected, analyzed, and interpreted the patient disease data and edited the manuscript. SY and SK: contributed equally to the manuscript. KK: supervised the patients’ treatments and the research project. YN, FF, and MT: participated in the discussion. NK and JF: are pathologists and participated in the diagnosis of G‐CSF‐producing esophageal cancer. SM: also collected the data and participated in the case presentation. SE: approved the final submission of this manuscript.Informed consent for this report was obtained from the patient and his family.The publication of the present study was in accordance with the ethical standards of our institution.The authors declare no conflicts of interest in association with the present study.


The management of posttraumatic cerebrospinal fluid (CSF) rhinorrhoea remains a clinical challenge. Cerebrospinal fistula is a dural defect responsible for possible CSF leakage into the contiguous air-filled cavities located at the skull base. The risk of central nervous system infection in these conditions is severe and can be life threatening. Consequently, a specific CSF biomarker might be used in case of difficult diagnosis of CSF rhinorrhoea. CSF Tau protein is a neuronal protein, commonly assessed for diagnosis of Alzheimer Disease (AD). The aim of this study was to determine whether the Tau protein could be a relevant marker of CSF leakage.Tau protein measurement was performed by enzyme-linked immunosorbent assay in 13 patients with CSF leakage (CSF rhinorrhoea group), and 8 patients with spontaneous aqueous rhinorrhoea (non-CSF leakage group). The serum concentration of Tau protein was measured by ELISA in both CSF rhinorrhoea group and non-CSF leakage group.In patients with CSF leakage, CSF Tau protein median concentration was 479 ng/L (197 - 2325 ng/L). On the other hand, the Tau protein concentration was below the lower limit of quantification (LLoQ) (< 87 ng/L) in non-CSF leakage group. Serum Tau protein concentration by ELISA was also below LLoQ (< 87 ng/L) for all subjects.ELISA measurement of Tau protein in rhinorrhoea fluid may be a reliable and relevant marker for detecting the presence of CSF in the nasal discharge and sign the existence of a CSF leakage. The diagnosis and management of posttraumatic cerebrospinal fluid (CSF) rhinorrhoea remain a clinical challenge  when additional radiological or interventional examinations do not provide evidence of diagnosis; (ii) when further examination cannot be performed; (iii) in case of discrepancies between clinical features and radiological examinations.A specific CSF biomarker should be used for difficult diagnosis of CSF rhinorrhoea. Tau protein is commonly assessed in CSF for diagnosis of AD and should be used as a possible specific biomarker of CSF. The aim of this study was to evaluate the diagnostic value of Tau protein as a marker of CF in patients with rhinorrhoea.This study was performed on samples collected after medical prescription of Tau protein assay in the Reims University Hospital . The patients were hospitalized in the Otorhinolaryngology or Neurosurgery department. Patients were included in this study from October 2011 to December 2016. Three groups of patients were assessed. The CSF rhinorrhoea group included 13 patients with rhinorrhoea due to CF leakage ; 8 post chirurgical CF leakages and 5 posttraumatic CF leakages). The diagnosis of CSF rhinorrhoea was confirmed after surgery exploration, associated in some cases with sodium-fluorescein detection. The non-CSF leakage group included 8 patients with spontaneous aqueous rhinorrhoea due to viral or allergic rhinopathy ). These patients never presented any clinical feature of dural defect, head trauma, meningitis, or meningeal weakness. The sera group included the sera of ten patients from CSF rhinorrhoea group, three patients of non-CSF leakage group and three other healthy control patients ). It is therefore important to assay in parallel Tau protein in the serum and in the rhinorrhoea fluid using the same technique. For each patient, rhinorrhoea fluid and serum Tau protein concentrations must be assessed in the same way to avoid any influence of blood contamination of rhinorrhoea sample.All nasal discharge (400 μL) were collected, stored and processed in 5 mL polypropylene tubes without additive . These tubes do not adsorb the Tau protein and thus do not affect results (Concentration of Tau protein was measured in the three groups by a sandwich Enzyme Linked ImmunoSorbent Assay (ELISA)  according to manufacturer’s instructions. This assay was able to quantify total Tau protein, including its six isoforms. For each kit, we used the lower limit of quantification (LLoQ) according to the manufacturer’s instruction. LLoQ may vary between 34 to 87 ng/L depending on the kit used in the study. In case of concentrations higher than the limit of quantification (about 1000 ng/L) the samples were diluted. According to the manufacturer recommendations, the reference values for the Tau protein in the cerebrospinal fluid measured by ELISA are as follows: 21 to 51 years: 136 ± 89 ng/L, 51 to 70 years: 243 ± 127 ng/L, > 70 years: 341 ± 171 ng/L.The analysis was blind to clinical data and performed when required by the clinician. Tau protein measurement was assessed after medical prescription and performed routinely in our laboratory. Our laboratory is involved in external quality control program from Alzheimer’s association. No additional examinations were carried out and there were no risk or constraint for the patient. Approbation by an institutional/national ethics committee is not necessary in this case.The results obtained were analysed using StatEL software . Comparison between groups was done with Mann-Whitney nonparametric test and P values less than 0.05 were considered statistically significant. Cut-off for the best sensibility and specificity of the marker was determined with a ROC Curve.Tau protein concentration was detected in all patients in CSF rhinorrhoea group. Its mean concentration was 711 ng/L ranging from 197 ng/L to 2325 ng/L (median concentration 479 ng/L). By contrast, Tau protein was below the LLoQ of the ELISA assay (< 87 ng/L) in all samples from non-CSF leakage group. In all cases, the serum concentration of the Tau protein was also below the LLoQ (< 87 ng/L). The concentration of Tau protein was significantly higher in the CSF rhinorrhoea group than in non-CSF leakage group (P < 0.001).vs 0% in the non-CSF leakage group.According to these results, a cut-off of 87 ng/L was proposed to discriminate the CSF rhinorrhoea group and non-CSF leakage group. With this cut-off, sensitivity (100%) and specificity (100%) of Tau protein measurement by ELISA were excellent since Tau protein was present at detectable concentration in 100% of CSF rhinorrhoea In this study, we demonstrate that Tau protein measurement in the rhinorrhoea fluid may be a relevant new marker for the diagnosis of CSF leakage with a good sensitivity (100%) and good specificity (100%). Tau protein was higher in the CSF rhinorrhoea group compared to non-CSF leakage group.Diagnosis of skull base defects, either traumatic or not, is sometimes difficult. Laboratory tests are then of great interest. Several biological methods have been proposed to differentiate CSF rhinorrhoea from other spontaneous rhinorrhoea .Biological investigation of a suspected rhinorrhoea consisted for a long time in glucose content determination, using glucose oxidase strips. This screening test has the advantage of being easily done in an emergency context and at the bedside. It is fast, inexpensive and widely available. However, it has a poor sensitivity and specificity with high potential of misdiagnosis  have also been proposed for the diagnosis of CF , a member of the family of lipocalins, is the second most abundant protein in CSF after albumin . Tau protein must be assessed in both serum and rhinorrhoea fluid, using the same technique. According to the ELISA assay used in this study, the serum concentration of Tau protein was below the LLoQ of the assay, which demonstrate that blood contamination did not influence the rhinorrhoea of Tau protein. ELISA has also the advantage to require a low sample volume (50 μL). This is an important point, given the low volumes available for these types of samples. The main disadvantages of the ELISA assay is the high number of patients needed to completely fill the plate (cost of the test) and the duration of the assay (first incubation lasts overnight). Moreover, for each run, duplicate wells for calibrators, controls, and samples are needed. Thus, a major limitation of ELISA is the fact that it is not suitable as a rapid and emergency assay. The development of automated assay could overcome this limitation.By contrast, Tau protein is lower in case of normal pressure hydrocephalus, which could lead to false negative results (In conclusion, determination of Tau protein by ELISA uses well standardized steps or the sample handling procedure, which permits to obtain reproducible results. It is widely used in the diagnosis of AD although internationally recognized reference material is not yet available. This pilot study is encouraging and suggests that, in the future, it could be easily performed routinely and allows reliable detection of CSF in the case of rhinorrhoea related to a CSF fistula. Contamination by blood does not confound the results since serum Tau protein was below the LLoQ with this method for all samples. Our study shows that Tau protein measurement in the rhinorrhoea fluid may be a relevant new marker for the diagnosis of CSF leakage.


Secondary neoplasias are well known consequences of radiotherapy or chemotherapy for a primary cancer. In this report, we describe two rare secondary neoplasias occurring in the same patient: a meningioma-like intracranial tumor and high-risk myelodysplastic syndrome (MDS) of donor-cells origin, both diagnosed simultaneously, 8 years after an allogeneic hematopoietic stem cell transplantation  for chronic lymphocytic leukemia (CLL). Due to an engraftment failure during the first allo-HSCT of a matched related donor for CLL treatment, the salvage treatment was a second allo-HSCT. At the moment of meningioma-like tumor diagnosis, the patient was pancytopenic due to high-risk MDS, so it was decided to postpone a surgical intervention until hematological improvement. For the high-risk MDS of donor-cells origin the chosen treatment was induction with intensive chemotherapy. Due to refractory disease, the patient was treated with 5-azacitidine and donor-lymphocytes infusion with no response and, finally, a third allo-HSCT of a matched unrelated donor was performed. The patient died 6 months after the third allo-HSCT, in cytogenetic remission but without hematological recovery, due to an intracranial hemorrhage with origin in the meningioma-like tumor. Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) is the only curative approach for the majority of hematooncological diseases. The major limitations of the Allo-HSCT are the short and long-term toxicities related to the procedure. The recent advances in transplantation techniques and supportive care were responsible for a decrease in therapy-related mortality and improvement of overall survival (OS). Increasing the OS leads us to the problematic secondary neoplasias. During the first year after HSCT, apart from relapse, posttransplant lymphoproliferative disorder is the most frequent secondary malignancy with a cumulative incidence between 0,6 and 1,4% in allo-HSCT and extremely rare in autologous HSCT (auto-HSCT). In terms of secondary solid cancers, the cumulative incidence increases over time: 1-2% at 5 years; 2–6% at 10 years, and 3–15% at 15 years. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have an incidence of 5–15% at 5 years after auto-HSCT, but extremely rare after allo-HSCT [A 57-year-old Caucasian male was diagnosed with chronic lymphocytic leukemia (CLL), stage II-B in Rai-Binet System, and unknown cytogenetic risk, in 1998. The CLL was refractory to fludarabine and cyclophosphamide (FC). In September of 2001, an allo-HSCT of a matched related donor (brother) was performed with reduced-intensity conditioning (RIC) of fludarabine and busulfan (FluBu). The acute graft-versus-host disease (aGVHD) prophylaxis was cyclosporine (Csp) and mycophenolate mofetil (MMF). Engraftment failure occurred and a second allo-HSCT of the same donor after RIC with fludarabine and cyclophosphamide plus in vivo lymphodepletion with alemtuzumab was performed. The aGVHD prophylaxis was Csp and MMF again. After successful engraftment and hematological recovery, bone marrow evaluation confirmed complete remission (CR). During the posttransplant follow-up period, neither aGVHD nor chronic GHVD (cGVHD) was observed.In March of 2009, due to headache and behavioural alterations, a cerebral magnetic resonance imaging (MRI) was performed and showed an intracranial extra-axial expansive lesion in the anterior cranial fossa measuring 2,7 × 2,7 × 3,3 cm of transversal, cranial-caudal, and anterior-posterior diameters, respectively, suggestive of olfactory groove meningioma . No cereDuring evaluation for neurosurgery, in May of 2009, after almost 8 years in CR for CLL, the patient presented with pancytopenia. A diagnosis of myelodysplastic syndrome with excess blasts-2 (MDS-EB-2) was made based on a bone marrow smear with dysplastic features, blast count of 11%, and karyotype with monosomy 7 in 14 of 20 metaphases. Chimerism analysis by polymerase chain reaction of short tandem repeats (STR-PCR) showed full-donor chimerism in all lineages, which confirmed the donor-cell origin for the MDS. To investigate occult MDS, the donor bone marrow was evaluated and showed no dysplastic features or cytogenetic abnormalities. The donor is currently free of any hematological disease.7/kg and 1 × 108/kg, respectively, with no response. In January of 2011, a matched unrelated donor (MUD) was identified, which allowed a third allo-HSCT in February of 2011. Prior to the HSCT, the patient was pancytopenic with a bone marrow blast count of 5,5% and a karyotype with monosomy 7. The chosen conditioning regimen was fludarabine and melphalan plus in vivo lymphodepletion with alemtuzumab. The aGVHD prophylaxis was tacrolimus and MMF. In March of 2011, within 1 month of allo-HSCT, the meningioma-like lesion was revaluated by MRI and there was a significant increase in its dimensions , associated with oedema and mass effect . Cytomegalovirus (CMV) reactivation was also diagnosed and daily ganciclovir was started.Neurosurgical intervention was postponed until resolution of the hematological disease. The MDS-EB-2 was resistant to intensive chemotherapy . Between October of 2009 and February of 2010, the patient went on second-line treatment with 5-azacitidine, abandoned at the end of 4 cycles due to absence of hematological and cytogenetical response. Between May and August of 2010, donor lymphocyte infusions (DLI) were performed twice, with CD3 cells doses of 1 × 10s effect . In May In June of 2011, the patient complained of headaches again. The meningioma-like lesion was reassessed by MRI, which showed an increase in size , in relation to intratumor hemorrhage, worsening of oedema, and mass effect . The neuIn terms of etiology for donor-cell MDS/leukemia, the possible explanations are divided into two groups: donor-cell factors and host factors . For donThe first in vivo reports of donor-cell MDS/leukemia were made by cytogenetic evaluation of sex-mismatched allo-HSCT, showing abnormalities in a karyotype of different sex . A few yThe treatment of donor-cell MDS/leukemia is also subject of debate. In a multicentre retrospective study, from 14 patients with donor-cell leukemia, 9 were treated with intensive chemotherapy, and CR was achieved in 6  patients . Only onSecondary brain tumors are rare events after bone marrow transplantation . Meningi


The neuropathology of Alzheimer's disease (AD) is characterized by the widespread accumulation of neuritic plaques and neurofibrillary tangles composed of deposits of beta-amyloid peptide (Aβ) and abnormally phosphorylated tau protein (phospho-tau) respectively. Considerable effort has been expended to identify methods to retard the deposition of these proteins or to enhance their clearance. It is strikingly surprising that until now, very few researchers have attempted to remove these proteins using mechanical procedures.In this article, we start by showing the rationale of mechanical dilution of cerebrospinal fluid (CSF) as a therapeutic approach in AD. Then, we present models of implantable systems allowing mechanical dilution of CSF by means of CSF replacement and CSF filtration (liquorpheresis). We conclude that even though this approach seems simplistic, it is feasible and deserves exploration. The neuropathology of AD is characterized by the widespread accumulation of neuritic plaques and neurofibrillary tangles composed of deposits of beta-amyloid peptide (Aβ) and abnormally phosphorylated tau protein (phospho-tau) respectively Figure . Aβ accu Considerable effort has been expended to identify methods to retard the deposition of these proteins or to enhance their clearance . PerhapsIt is strikingly surprising that until now, very few researchers have attempted to remove these proteins using mechanical procedures. There are a good number of studies where AD CSF biomarkers were correlated with prognosis of long-term cognitive outcomes in patients with idiopathic normal pressure hydrocephalus (iNPH) after shunting treatment. It is important to note that in these studies, AD comorbidity could not be excluded before shunting. Indeed, cortical biopsy findings indicate that normal pressure hydrocephalus (NPH) is at present a heterogeneous syndrome and has notable overlapping with AD -11. In mThere is a theory where AD is proposed to be directly caused by a CSF circulatory failure -13; the Putting aside the theory conceiving AD as a CSF circulatory failure, there is no doubt Aβ and phospho-tau play a central role in the pathogenesis of AD, and removing them would be a major step towards the cure of the disease. All previous studies used the classical one-way ventriculo-peritoneal shunt, where very limited clearance of CSF peptides takes place by a diluting effect. We propose a new approach for mechanical clearance of Aβ and phospho-tau by diluting their concentration in the CSF continuously. This can be achieved by using implantable systems for CSF replacement or CSF filtration.The CSF volume, estimated to be about 150 ml in adults, is distributed between 125 ml in cranial and spinal subarachnoid spaces and 25 ml in the ventricles, but with marked interindividual variations. CSF production is around 500 ml per day and it is renewed about four times every 24 hours , 15. As Some implantable systems for CSF replacement and CSF filtration (liquorpheresis) have been described . The cenSeveral different types of filters for Aβ and tau can be developed in the future; from mechanical filters, similar to those used in plasmapheresis, to "immunotechnological filters", based on antibodies against Aβ and tau able to catch these proteins from the CSF Figure . BiologiThese systems allow the replacement of “dirty” CSF (with Aβ and phospho-tau) with “clean” CSF (without Aβ and phospho-tau) several times a day. With any of these systems, the CSF concentration of Aβ and phospho-tau should decrease sharply. This decrease is supposed to have a “clearance effect” over the ISF concentration of Aβ and phospho-tau Table  1], pre, pre1], We cannot compute the extent of this “clearance” effect” because there are some variables we still do not know about. For instance, we do not know how such systems would affect the production of CSF. Anyway, the dilutional effect would be particularly driven by the renewal rate and how the turnover is programmed.We show the rationale for mechanical dilution of CSF as a therapeutic approach in AD. Mechanical dilution of Aβ and phospho-tau at early stages of the disease may ease clearance of these peptides in the interstitial fluid. Thus, we can prevent the deposition of these peptides in the brain tissue. We also review some innovative models of implantable systems for CSF replacement and liquorpheresis.The idea of treating AD by simply diluting "dirty" CSF with "clean" CSF may seem far-fetched at first. However, from a theoretical point of view, this seems a completely feasible approach that deserves exploration. Product development and preclinical studies should be addressed to verify the usefulness of these systems.


Transplantation of testicular cells and tissues has been studied for the investigation of immunology of the testis, which is an immunologically privileged organ. However, reports of transplant of the testis at organ level have been extremely limited because of technical difficulties of the orthotopic testis transplantation (OTT) in experimental animals. In the present study, we developed a new and simple model of the heterotopic testis transplantation (HTT), which is donor testis transplantation into the cervical region of recipients, in a syngeneic model in rats [donor Lewis (LEW) graft to LEW recipient]. The duration of HTT was significantly shorter and success rate higher than that of OTT. To histologically evaluate HTT, the local immune responses were compared among the syngeneic model, an acute rejection allogeneic model [donor Augustus Copenhagen Irish (ACI) graft to LEW recipient] and a chronic rejection allogeneic model (donor F344 graft to LEW recipient) at postoperative day 3. We found that allogeneic ACI grafts resulted in mild and not severe orchitic lesions, whereas immune responses of allogeneic F344 grafts seemed intact and were not significantly different from those of syngeneic LEW grafts. These results suggest that our new operative procedure will be useful in future for the investigation of the testicular immunology. The testis is an immunologically unique site because new differentiation autoantigens-bearing spermatids and spermatozoa that appear in the seminiferous tubules long after the establishment of immune tolerance survive with no rejection by self-immunity under healthy conditions , 2. It bWith regard to whole testis transplantation, Lee et al. were the first to formulate a model for orthotopic testis transplantation (OTT), which is characterized by an end-to-side anastomosis between the graft’s great arteriovenous segments with testicular arteriovenous vessels and the recipient’s great arteriovenous vessels that is subsequently retracted into the scrotum . SeveralMale inbred Lewis rats (LEW/SsN Slc) at 10 weeks of age were used as the donors (n = 19), recipients (n = 31), and positive and negative controls (n = 24). Male inbred Fischer 344 rats  and Augustus Copenhagen Irish rats  at 10 weeks of age were used as donors in the allogeneic models. The animals were purchased from SLC  and kept in the Laboratory Animal Center of Tokyo Medical University for 2 weeks prior to the experiments. Animals were maintained at 22°C–24°C and 50%–60% relative humidity with a 12 h light–dark cycle. This study was approved by the Tokyo Medical University Animal Committee . All surgeries were performed under deep anesthesia using isoflurane, and all efforts were made to minimize suffering. Finally, rats were euthanized under general anesthesia with an isoflurane overdose (5%), which was maintained for 1 min after cessation of breathing and exsanguination during organ procurement.The operative time and success rate of the transplants between the HTT and OTT groups were evaluated. Vasectomy (Vx), ischemia (IS), and cryptorchidism (CR) groups were generated as positive controls for the consideration of possible effects of vas deferens occlusion, blood flow disturbance, and elevated temperature, respectively, during HTT. Therefore, this study was performed using the following six groups: the sham group as a negative control (n = 6), HTT group (n = 12), OTT group (n = 7), Vx group (n = 6), IS group (n = 6), and CR group (n = 6). For the OTT group, we used examples from Lee’s transplantation model where transplanted testes were retracted into the scrotal sac . In the We compared the local immune response in the transplanted testes in the syngeneic model (donor LEW graft to LEW recipient) with two allogeneic models (donor ACI graft to LEW recipient and donor F344 graft to LEW recipient) at postoperative day 3 after HTT. LEW rats receiving syngeneic grafts served as controls (LEW group). There are several reports that evaluate allograft rejection in various organs using LEW rats as recipients and ACI and F344 rats as donors , 20. MHCSurgical procedures were performed under 20–30× magnification using a desktop operating microscope . The microsurgical instruments for this study included straight super-grip forceps, micro-hemostat clamps, and micro-dissecting scissors. All intraoperative rats were placed on a heating plate  to account for decreased body temperature caused by anesthesia. All HTT, OTT, Vx, IS, CR, and sham operations were performed by two researchers who are experts at the experimental transplantation of hearts and kidneys in rodents.For HTT, the animals were anesthetized using isoflurane . The abdomen was shaved, swabbed with 70% alcohol, and opened through a long midline incision. Rats in which the right testicular artery arose from the abdominal aorta (AA) and the ipsilateral testicular vein arose from the inferior vena cava (IVC) were considered as suitable donors. IVC was ligated just above the entrance of the right testicular vein and transected 1 cm below the entrance of the right testicular vein. Similarly, AA was ligated just above the entrance of the right testicular artery and transected 1 cm below the entrance of the right testicular artery. The right vas deferens was transected near the bladder Figs  and 2A. For OTT, the procedure was based on the model established by Lee and Goldstein , 10. BriThe procedure for HTT was based on the heterotopic heart transplantation method used by Heron et al. with some modifications . The aniThe procedure for OTT was based on the methods used by Lee and Goldstein , 10. BriFor HTT, the donor testis with the epididymis and a part of the vas deferens was transplanted into the neck of the recipient. AA and IVC with the testicular vessels of the donor were overlaid at the end of CCA and EJV with the cuff of the recipients and then fixed to each cuff with a circular ligature of 7–0 silk . The claThe procedure for OTT was based on the methods used by Lee and Goldstein , 10. BriThe subcutaneous temperature in the scrotum, neck, and abdominal cavity of the recipients was measured using a thermometer  immediately before the removal of transplanted organs. Three temperature probes were inserted into each region under anesthesia. Temperatures were measured when the rectal temperature reached 36.5–37.0°C after regaining consciousness.The isolated testes were fixed in Bouin’s solution and then embedded in plastic  without cutting the organs to prevent artificial damage to the testicular tissue. Sections (3–4-μm-thick) were obtained at 15–20-μm intervals, stained with Gill No. 3 hematoxylin and 2% eosin Y, and observed using a light microscope . The degree of spermatogenic disturbance was determined using Johnsen’s scoring system, which ranged from a score of 1 (no cells in the seminiferous tubules) to 10 (complete spermatogenesis) .2O2 as the chromogen. As a semi-quantitative analysis, the number of seminiferous tubules with Ki67-positive cells per 100 round and oval-shaped seminiferous tubules was determined in each testis. The number of infiltrating CD4+ and CD8+ lymphocytes per mm2 of testicular interstitium was evaluated.The isolated testes were frozen in liquid nitrogen and stored at −80°C until use. Cryosections (5 μm) were fixed in 1% glutaraldehyde for 10 min and then digested in pepsin solution (0.006% in 0.01 N HCl) for 10 min at 37°C. Samples were treated with 4 N HCl for 30 min at room temperature and then neutralized with borate buffer . The samples were incubated with primary antibodies against rabbit anti-Ki67 , mouse anti-rat CD4 , and CD8 . Next, the samples were incubated with an alkaline phosphatase-labeled goat anti-rat IgG (Sigma-Aldrich). Immunoreactive cells were visualized using a Vectastain ABC Kit (Vector Laboratories) with 0.05% 3,3′-diaminobenzidine  and 0.01% HTotal RNA was isolated from the transplanted testicular tissues using a TRIzol RNA extraction kit  and then reverse-transcribed into cDNA using a High-Capacity cDNA Archive Kit  according to the manufacturer’s instructions. The quantification of cDNA was performed using SYBR Premix Ex Taq II  and the Thermal Cycler Dice Real-Time System TP800 (TaKaRa). Glyceraldehyde-3-phosphate (GAPDH) was used as a housekeeping gene to normalize mRNA levels. The relative expression of real-time PCR products was determined using the ΔΔCt method to compare the mRNA expression of the target gene and GAPDH. Primers used in this study are listed in Values are presented as the mean ± standard deviations. The significance of differences was determined using either a paired unmatched t-test or an analysis of variance (ANOVA) with Tukey–Kramer post-hoc tests using Prism 6.0d for Macintosh . All statistical tests were performed as two-sided analyses, and P < 0.05 was used as the threshold for significance.Histological examination of the arterioles inside the testis revealed that the sham, HTT, OTT, Vx, and CR groups were normal, including numerous red blood cells. However, inside the arterioles of the IS group, red blood cells were hardly observed and only eosinophilic materials were observed .The operative time and success rate were compared between the HTT and OTT groups . HTT wasThe histological appearance of the testes and the epididymides in HTT and OTT models were similar to those in the sham group. Most seminiferous tubules appeared normal and showed active spermatogenesis, whereas some seminiferous tubules exhibited disorganized spermatogenesis . In the Immunohistochemically, Ki67-positive germ cells (spermatogonia) were observed along the basement membrane of the seminiferous tubules . In the + T cells and CD8+ T cells were detected  prominently increased in the grafts of the ACI group compared with those of the LEW group . The expThis study was performed to assess testis transplantation into a heterotopic area  with blood vessel anastomosis in rats using a new HTT technique. Compared with the OTT model, the HTT model had a significantly shorter operative time and improved success rate. Furthermore, the testes transplanted by HTT appeared histologically intact, as in the sham group, in the syngeneic model (donor LEW graft to LEW recipient). In contrast, transplanted testes in the acute rejection model (donor ACI graft to LEW recipient) exhibited significant inflammatory changes compared with those in the chronic rejection model (donor F344 graft to LEW recipient) and syngeneic model.The operative time for the OTT model was previously reported as 3–5 h –12. SimiThe following three issues must be considered in HTT. First, the effect of the testicular temperature at the cervical lesion should be assessed. It is well known that the process of spermatogenesis is highly sensitive to variations in the environment, particularly fluctuations in temperature. One of the most widely used models to investigate the effect of temperature on the testis is the experimental cryptorchid model. In previous reports, testicular weight decreased 4 to 14 days after the experimental induction of cryptorchidism , 27. In in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive (measurement of cell death) germ cells and the volume and density of intravascular polymorphonuclear leukocytes increased compared with those in naïve testis [The second issue relates to blood flow disturbance caused by folding of the long and thin testicular arteriovenous vessels into the cervical subcutaneous region. Testicular torsion results in obvious spermatogenic disturbances because the blood supply to the testis is cut-off by the twisting of the testis . Severale testis , 25, 29.The third issue is the effect of the draining of the donor vas deferens to the outside of the recipient body for HTT. There is a possibility that the distal tip of the vas deferens is dried and occluded. A previous study described epididymal distension resulting from raised intraluminal pressure through the occlusion of spermatozoa in the early phases of a vasectomy . In the + and CD8+ lymphocytes around arterioles, and increased mRNA expression levels of IFN-γ, IL-1β, IL-10, caspase 3, and caspase 8 were found in the transplanted testes of ACI group but not in those of F344 group. This suggests that the testicular arterioles play little role in immune privilege and become targets for acute allograft rejection under the experimental condition of the present study. There remains a possibility that testicular arterioles equipped with endothelial barrier antigens relatively protect the testicular parenchyma from immune attack compared with those in other transplanted organs. To verify whether the severity of rejection of transplanted testis is milder than that of other transplanted organs in ACI group, we are currently comparing local immune responses in the transplanted testes with those in other transplants, such as the heart and kidney from postoperative day 1 to postoperative day 60. In this long-term evaluation, we will also observe chronic rejection model in F344 group to compare with the syngeneic model.In general, the testis is an immunologically suppressed organ, i.e., an immunologically hospitable site in which allogeneic tissues, such as the thyroid gland and pancreatic islets, show long-term survival –35. The Chemotherapy or radiation therapy for malignant cancers often injure the spermatogenesis of young patients. In particular, prepubertal patients may suffer from permanent infertility because mature spermatozoa in semen cannot be cryopreserved prior to therapy. Thus, transplantation of children’s testes and epididymides to their fathers or brothers before receiving medical therapy may be helpful for obtaining mature spermatozoa from the transplanted testes at a future date. It is also possible that the transplanted testes in recipients are transplanted back into donors after medical treatment. Therefore, the establishment of the HTT model may contribute to this unmet clinical need.We developed a new practical method for the transplantation of the testis with the epididymis in rats. HTT is technically more simple and reproducible than OTT. This newly developed operative procedure could be useful for the further investigation of testicular immunology.S1 Table(XLSX)Click here for additional data file.S1 VideoThis video illustrates each step of the HTT surgical procedure at sextuple speed with a running time of 3 min.(MP4)Click here for additional data file.


The study highlights the shedding pattern of Senecavirus A (SVA) during an outbreak of vesicular disease in a sow farm from the South-central Minnesota, USA. In this study, 34 individual, mixed parity sows with clinical signs of vesicular lesions and 30 individual piglets from 15 individual litters from sows with vesicular lesions were conveniently selected for individual, longitudinal sampling. Serum, tonsil, rectal, and vesicular swabs were collected on day1 post outbreak, and then again at 1, 2, 3, 4, 6, and 9 weeks post outbreak. Samples were tested at the University of Minnesota Veterinary Diagnostic Laboratory for SVA via Real Time Polymerase Chain Reaction (RT-PCR)In sows, vesicular lesions had the highest concentration of SVA, but had the shortest duration of detection lasting only 2 weeks. Viremia was detected for 1 week post outbreak, and quickly declined thereafter. SVA was detected at approximately the same frequency for both tonsil and rectal swabs with the highest percentage of SVA positive samples detected in the first 6 weeks post outbreak. In suckling piglets, viremia quickly declined 1 week post outbreak and was prevalent in low levels during the first week after weaning (4 weeks post outbreak) and was also detected in piglets that were co-mingled from a SVA negative sow farm. Similar to sows, SVA detection on rectal and tonsil swabs in piglets lasted approximately 6 weeks post outbreak.The study illustrates the variation of SVA shedding patterns in different sample types over a 9 week period in sows and piglets, and suggests the potential for viral spread between piglets at weaning. Pircornaviridae and has been associated with idiopathic vesicular lesions on the snout and coronary bands in swine [Senecavirus A (SVA) or commonly known as Seneca Valley Virus belongs to the family in swine , 8. In 2in swine , 11, 12.in swine , 8, 10. in swine . VesiculIn early September of 2015, a sow farm in South-central Minnesota, US, exhibited the sudden and dramatic onset of severe vesicular lesions in nearly 80% of all sows on the site. Due to the relative paucity of information regarding this virus in the field, the objectives of this study were to assess the shedding patterns of SVA in a population of sows and suckling piglets, and assess the potential spread of SVA in weaned pigs. Not only will this information provide valuable insight into the infection dynamics of SVA within a population, but it may also aid in future FADs investigations, as well as assisting in the development of better control and elimination measures of SVA in sow herds.A farm in South central Minnesota, US, containing two geographically different sow sites ), a FADs investigation was initiated and samples were collected by the state veterinary office and sent to the National Veterinary Services Laboratories (NVSL), the Foreign Animal Disease Diagnostic Laboratory at Plum Island Animal Disease Center and the University of Minnesota Veterinary Diagnostic Laboratory (UMNVDL). At NVSL, the samples were tested for Foot-and-Mouth Disease virus (FMDv), SVA, and Swine Vesicular Disease virus (SVDv) by real time RT-PCR. At the UMNVDL, the samples were tested for FMDv according the United States Department of Agriculture Standard Operation Procedure (SOP) titled, “Extraction of Total RNA Using a MagMax-96 Total RNA Isolation Kit for the Detection of Classical Swine Fever and Foot-and-Mouth Disease Viruses” (SOP-PVS-0004.1), which is publically available from the NVSL upon request. The detection of SVA occurred by real time Reverse Transcription Polymerase Chain Reaction (RT-PCR) using AgPath-ID One-Step RT-PCR Reagents (Thermo), forward (5′- TCTCTTGCCCTAACACTGGGG-3′) and reverse primers (5′- CTTGCCTCTAAGGACCACCACA-3′) and probe (5′- TGGCCCAAA/ZEN/GTCTCACCACTATGATCAATG-3′) with the following thermal cycling parameters: reverse transcription, 10 min at 45 °C; Taq activation, 10 min at 95 °C; followed by 40 cycles of 15 s at 95 °C, and 45 s at 60 °C.On the second day post visualization of vesicular disease day 1 PO), 34 individual, mixed parity sows in gestation from site 1 were conveniently selected based on the presence of vesicular lesions on their snouts and feet. In addition, 30 mixed gender, neonatal piglets from 15-mixed parity litters showing similar clinical signs were conveniently selected and tagged at site 1 between approximately 12 and 24 h of age. Tonsil, rectal, vesicular swabs (when vesicles were present), and serum samples were collected on day 1 and then 1, 2, 3, 4, 6, and 9 weeks PO from both sows and piglets. Last samples were collected at 9 weeks PO and did not occur at weeks 5, 7, and 8 due to concerns of increased stress of the pigs due to sampling. Vesicular swab samples were not collected from the piglets since they lacked clinical vesicular lesions. At week 3 PO (at weaning), the 30 piglets from site 1 were transported to site 6 and were comingled with 30 individually identified mixed gender pigs from site 2 and sampled at 4, 6, and 9 weeks PO . Day −1 PO, lameness and decreased feed intake was more prevalent among the rest of the sows in the herd. Vesicular lesions were observed on day 0 PO, consisting of un-ruptured vesicles on the snouts and feet of approximately 5–10% of the animals on site 1, and the state veterinary office was notified. By day 2 PO, nearly 80% of the animals in the herd had detectable vesicular lesions (ruptured and un-ruptured) on the snouts and feet. Lameness, lethargy, and decreased feed intake were the main clinical concerns during this time; however, by 1 week PO, there was noticeable improvement in the clinical signs within the population. At no time was fever a major concern within the herd. In farrowing, there was an increase in pre-weaning mortality from an average of 12% to a high of 22% during the first 7 days PO, and returned to average levels by 2 weeks PO. The majority of the piglet mortality was associated with dehydration due to poor milk production by the sow, and piglets being laid on, potentially due to sow lameness. No other clinical signs were detected in piglets in farrowing, and there were no detectable signs at the nursery barns at any point during or after the outbreak.The samples collected by the state veterinary office at site 1 on the day 0 PO were negative for SVDv at NVSL while the NVSL and UMNVDL had the same results for FMDv and SVA . Subsequent, SVA testing only occurred at the UMNVDL through the study.Sows displayed vesicular lesions on the snouts at day 0 PO, and SVA was present in 33 out of 34 (97%) of the vesicle swabs, with an average cycle threshold (Ct) value of 16 (range = 12–19). At 1 week PO, SVA was present in 34 out of 34 (100%) of the lesion swabs. At 2 weeks PO, most lesions had healed (only few skin tags and ruptured vesicles remained in the population), and SVA detection decreased to 12 out of 34 samples (35%). By 3 weeks PO, all of the vesicular lesions had healed, and no additional samples were collected.Generally, viremia was detected up to 1 week PO in sows. A single positive sample was identified at 3 and 6 weeks PO Fig. . At the Tonsil swabs yielded the highest percentage of SVA detection in site 1 sows (32 out 34 (94%)) and piglets (25 out of 30 (83%))Fig. . In sowsn = 3); however, a single piglet from site 1 was still shedding SVA at 9 weeks PO.The peak of SVA detection from rectal swabs in sows (31 out of 34 samples (91%)) occurred at day 1 PO and continued to steadily decrease and was not detected at 9 weeks PO Fig. . In siteThe oral fluid and serum samples were positive for SVA from site 5, but negative from site 3 and 4. SVA was detected in 9 out of 10 (90%) of the serum samples , and the single oral fluid sample had a , despite the fact that there were no obvious detectable clinical signs in site 5. Of importance is that site 5 appears to have received SVA positive weaned pigs from site 1 before the detection of clinical signs of decrease in feed intake and vesicular lesions.The study assessed the shedding pattern of SVA in sows and piglets during an outbreak on a farm in the US and investigated the spread of SVA between pigs during the post weaning period. In addition, the study suggests the spread of SVA to distant pig sites via piglets before the onset of vesicular lesions.Fresh vesicular lesions on the sows had the greatest amounts of SVA; however, the lesions were only present for a very short period of time (2 weeks). This is important because state and federal veterinarians conducting the FAD investigations are trained to identify vesicles and collect their fluid. However, these data suggest a lower rate of detection of SVA in ruptured vesicles and dried skin tags (that are present after the first week of the outbreak) when compared to tonsil and rectal swabs, especially later in the outbreak when vesicles were no longer present. In sows, the detection of SVA in tonsil and rectal swabs was greater than 90% at 0 week PO and remained as high as 50% through 5 weeks PO, these sample types should be collected and submitted, in addition to vesicular lesion swabs and fluid (if present), as part of FAD investigations for the detection of SVA. In addition, the detection of SVA in oral fluid samples collected from site 5 and the high percentage of positive SVA tonsillar swabs suggests they may be valuable tools for the detection of SVA in pigs.Surprisingly, SVA was detected in serum and oral fluids from site 5, which had received weaned pigs approximately −2 weeks PO, before the detection of clinical signs at site 1. Furthermore, obviously detectable clinical signs were lacking in piglets at site 5, indicating the potential to spread SVA without realization through weaned pig movements. This is a major concern for epidemiologists as well as state and federal veterinarians because the prevalence of SVA within the US swine herd could be greatly underestimated. Therefore, as part of any epidemiological investigation of SVA outbreaks (particularly on sow farms), a social network analysis, which details all pig movements to the level of the specific trailers and trucks used during the previous month should be conducted to identify the potential scope of virus spread.Commingling of piglets at weaning is common in the US swine industry. When the piglets were commingled at week 4 PO (approximately 1 week after weaning), 12 of the 30 (40%) of the piglets sampled from sites 1 and 2 were viremic. Interestingly, site 1 and 2 piglets had the same level SVA shedding in the tonsil and rectal swabs (2 out of 30 (7%), and 3 out of 30 (10%), respectively) at 6 weeks PO. These results suggest the potential spread of SVA to site 2 piglets, and SVA may be transmitted during commingling of piglets. Therefore, caution should be exercised when considering the movement of weaned pigs during an outbreak, which includes avoiding the comingling of piglets from different sow farms.While SVA was detected in site 2 piglets, the levels of virus were minimal in the tonsil and rectal swabs potentially implying some underlying immunity to SVA in site 2 piglets that limited viral infection and shedding. While site 2 tested negative by PCR, indicating that the herd was not infected at that time, a proper immunological assay, which may help elucidate if the herd had been infected in the past and has some level of immunity, is lacking at the time of writing this manuscript. With that in mind, immunological assays including several different ELISA targets are currently under development at several US veterinary diagnostic laboratories . In addiOur results suggest that SVA can be detected in a population of sows in tonsil and rectal swabs for up to 9 weeks; however, the viability of SVA at 9 weeks is currently unknown. Future studies should be developed to discern the viability of the virus from these samples, especially in the later stages of the outbreak. This information would be beneficial to veterinarians working on eliminating SVA from affected sow farms since it would dictate the duration of sow herd closure  in order to elimination the virus from that population. Additionally, as part of any plan to eliminate SVA from sow farms, achieving rapid and homogeneous exposure of all animals is crucial. Therefore, studies are needed to identify the most efficient route of SVA exposure to eliminate the virus from infected sow herds.In conclusion, this study highlights the detection of SVA in vesicular lesions, serum, tonsil and rectal swabs and suggests the spread of SVA between piglets during the post weaning period. In addition, this study suggests that tonsil and rectal swabs, and oral fluids may be useful for monitoring populations SVA positive animals over time.


To better understand shape memory materials and self-healing materials, a new series of liquid-crystalline shape memory polyurethane (LC-SMPU) composites, named SMPU-OOBAm, were successfully prepared by incorporating 4-octyldecyloxybenzoic acid (OOBA) into the PEG-based SMPU. The effect of OOBA on the structure, morphology, and properties of the material has been carefully investigated. The results demonstrate that SMPU-OOBAm has liquid crystalline properties, triple-shape memory properties, and self-healing properties. The incorporated OOBA promotes the crystallizability of both soft and hard segments of SMPU, and the crystallization rate of the hard segment of SMPU decreases when the OOBA-content increases. Additionally, the SMPU-OOBAm forms a two-phase separated structure (SMPU phase and OOBA phase), and it shows two-step modulus changes upon heating. Therefore, the SMPU-OOBAm exhibits triple-shape memory behavior, and the shape recovery ratio decreases with an increase in the OOBA content. Finally, SMPU-OOBAm exhibits self-healing properties. The new mechanism can be ascribed to the heating-induced “bleeding” of OOBA in the liquid crystalline state and the subsequent re-crystallization upon cooling. This successful combination of liquid crystalline properties, triple-shape memory properties, and self-healing properties make the SMPU-OOBAm composites ideal for many promising applications in smart optical devices, smart electronic devices, and smart sensors. Stimuli-responsive polymers play an important role in many applications, such as diagnostics, smart optical systems, and tissue engineering, as well as in micro-electromechanical systems, biosensors, textiles, and coatings ,2. ShapeTo date, numerous SMPs based on various structures have been developed, including polyurethane, polystyrene copolymer, and epoxy-based polymers. Among them, the thermal-induced shape memory polyurethane (SMPU) has been mostly used in practice because of its competitive mechanical and shape memory properties, e.g., high specific strength, high parameters of shape fixity and shape recovery, and a transition temperature that can be approximately tuned to room and body temperature ,9. A varn-hexadecyloxybenzoic acid (HOBA) [As important functional fillers, liquid crystals (LC) are widely used to fabricate various liquid-crystalline polymer composites because they combine the basic properties of conventional polymers with those of LC ,19,20,21d (HOBA) ,25. Our d (HOBA) . It was Therefore, a new kind of LC-SMPCs, termed SMPU-OOBAm, were prepared with PEG-based SMPU containing about 40 wt % hard segment as the polymer matrix, and OOBA mesogen as the LC fillers. This study investigates the effect of OOBA on the structure, morphology, and properties of the SMPU-OOBAm. Notably different from previous systems, the present LC-SMPCs show a successful combination of the liquid crystalline properties, triple-shape memory effects, and self-healing properties. In particular, the self-healing mechanism may be notably different from the common self-healing materials. Thus, this study may be useful in order to fundamentally examine the multi-functional properties, multi-responsiveness, and efficient actuating method of SMPs and polymer composites.n = 6000, PEG6000), was dried before usage at 80 °C under 0.1–0.2 MPa for 6 h. 4,4-Diphenylmethane diisocyanate , 1,4-butanediol , 4-octocyloxy benzoic acid  and dimethylformamide  were purchased from Aladdin Chemical Reagent Co., Ltd.  and were used without further purification.Extra pure grade polyethylene glycol  spectra were scanned from smooth polymer films with a thickness of 0.2 mm using a Nicolet 6700 FT-IR spectrometer  by the FT-IR attenuated total reflection (ATR) method. Ten scans at 4 cmThe surface morphology of the samples was examined using scanning electron microscopy , 20 kV, equipped with an energy dispersive X-ray detector for elemental composition. Prior to scanning, the samples were coated with a thin layer of gold. Fraction morphology was scanned after the samples were fractured with liquid nitrogen.X-ray diffraction (XRD) experiments were performed on a BRUKER AXS D8 Advance diffractometer  with a 40 kV FL tube as the X-ray source (Cu Kα) and a Lynxeye-Xe detector. The applied voltage is 40 kV. Film samples were used in this experiment.2 flow.Thermo Gravimetric Analyzer (TGA) spectra were recorded on a computer-controlled TG Q50 system  after drying at 100 °C, under the following operational conditions: heating rate 10 °C/min, temperature range 100–600 °C, sample weight about 5.0 mg, using the film sample in platinum crucibles, 60 mL/min NDifferential scanning calorimetry (DSC) measurements were carried out with a TA Q200  instrument having nitrogen as the purged gas. Indium and zinc standards were used for calibration. Both heating scan rate and cooling. The scan rate is 10 °C/min. The second heating curves were used for analysis.Dynamic mechanical analysis (DMA) curves of the samples were determined using a Perkin-Elmer DMA  at 1 Hz and at a heating rate of 2 °C/min. The change in modulus and the recovery stress were measured with a DMS module and TMA module, respectively.Mesophases were identified, and the phase transition temperatures were determined using a Zeiss-Axioscope polarised optical microscope (POM)  equipped with a Linkam-THMS-600 variable-temperature stage at a scan rate of 2 °C/min. The samples were heated from 25 to 160 °C and cooled from 160 to 25 °C.Thermally induced triple-shape memory behaviours were examined via thermo-mechanical analysis using a TA Instruments DMA Q800 system  with tension clamps in controlled-force mode. Samples were dried at 100 °C for 24 h and cut into rectangular pieces of 10 mm × 2.0 mm × 0.5 mm./min) to a designated crystallization temperature (Tc) and held at this temperature until the end of the exothermic crystallization. The heat flow during the isothermal crystallization process was recorded as a function of time. Tc was chosen as 137 °C in this experiment. The amounts of heat generated during the development of the crystal phase were recorded and analysed according to the typical equation used for evaluating the degree of relative crystallinity (Xt):t0 and t = ∞ are the times at which the sample reached isothermal conditions  and the time at which the dominant sharp exothermic peak ends, respectively. H is the enthalpy of crystallization at time t. After isothermal crystallization, the sample was heated to 200 °C, and the crystal melting temperature (Tm), indicated by the maximum of the endothermic peak, was recorded. Isothermal crystallization experiments were performed with a TA-Q200 DSC instrument  using nitrogen as the purge gas, according to the procedure described in the literature. The sample (4–6 mg) was initially heated to 200 °C at a rate of 10 °C/min and held for 5 min to remove the thermal history of the crystallizable phase. Subsequently, the sample was rapidly cooled  can be converted into the following form:X(t))} against log(t) should yield a straight line. By fitting the lines, n and log(K) can be calculated from the slope and intercept, respectively.A plot of log{−ln(1 − t(0.5)) is defined as the time at which the crystallinity is equal to 50%, and is related to the Avrami parameter K as determined with the following expression:Additionally, the half-crystallization time , the N–H stretching vibration shifts to a low wavenumber, which implies the formation of a hydrogen bond between the OOBA and the hard segment of SMPU. Additionally, P4 shows a new frequency at approximately 1685 cm−1, which can be ascribed to the C=O stretching vibration of OOBA. The frequency at approximately 1166 cm−1 also likely results from the C–O stretching vibration of OOBA. These results suggest that OOBA has successfully incorporated into SMPU and has formed an SMPU-OOBAm composite. −1 obviously increases from P1 to P5, which confirms the increased OOBA content. In the previous literature, the LC phase was lost when LC mesogens containing pyridine moieties were attached to the SMPU containing carboxyl groups through hydrogen bonding between pyridine and COOH [and COOH . HoweverThe surface morphology of the SMPU-OOBAm composites was investigated systematically via SEM and XRD. As shown in α and β crystalline phases) at low temperature. Therefore, this new peak may be ascribed to the second crystal type (β phase) of OOBA. Thus, the crystallizability of the first crystal type (α phase) of OOBA is restrained, and OOBA subsequently tends to form a second β phase when the OOBA content increases. This phenomenon can also be reflected in the thermal properties and phase transitions.Wide-angle X-ray diffraction (WAXD) experiments were used to further study the morphology of the SMPU-OOBAm composites. As shown in The thermal properties of the SMPU-OOBAm composites were investigated by TGA and DSC. The TG curves show that all samples appear to decompose in two stages, which corresponds to the decomposition of the soft (at 200–300 °C) and hard segments (at 350–450 °C) of the SMPU matrix see a. The TGα and β crystalline phases, respectively. The β crystal-melting transition also indicates that OOBA enters the smectic phase. The third peak at 107 °C indicates a smectic-nematic phase transition, and the fourth peak at 148 °C suggests a phase transition from the nematic phase to the isotropic phase. When OOBA is incorporated into the SMPU matrix, the two crystal-melting transitions in the composites  at 49 °C and 74 °C can be attributed to the crystal-melting of the soft segment of SMPU and the α phase of OOBA, respectively. Meanwhile, a new weak exothermic peak which can be attributed to the β crystal-melting transition appears at 89 °C. When the molar ratio of OOBA/MDI is lower than 0.4 (m ≤ 0.4), the β crystal-melting transitions cannot be detected on the second DSC heating curve  and time are shown in X(t))} on log(t). It was found that log{−ln(1 − X(t))} is linearly related to log(t), but the curve slightly deviates during the later period of crystallization. This deviation implies the existence of a secondary crystallization, which should be attributed to the phase transformation from sphero-crystal to lamella-crystal. Finally, based on the original data and fitting a straight line, the kinetic parameters n, K, and t1/2 of the isothermal crystallization were easily calculated. The results are summarized in n value of all samples is ~1.3, which further indicates that the crystallization mechanism of the hard segment of SMPU in all composites is the nucleated mechanism. The crystallization rate decreases because the K value decreases when the OOBA-content increases. One possible reason for this is that the increased OOBA content might affect the aggregation of the hard segment of SMPU, though they provide more crystal seeds.For understanding the crystallization behaviors of the hard phase in the SMPU-OOBAm composites, the isothermal crystallization kinetics were investigated using DSC analysis. Detailed calculations are provided in E’g) below −30 °C, whereas the storage modulus gradually decreases because of the glass transition of the soft segment of SMPU. The first significant storage modulus occurs when the temperature increases to 46 °C, because of the crystal-melting of the soft segment of SMPU. Thereafter, SMPU enters the rubber state. The DMA curves demonstrate that the rubber modulus at 46~60 °C in the SMPU-OOBAm composites tends to be less than those of SMPU. This result again confirms that the OOBA crystals reinforce the crystallinity of the soft segment of SMPU, and act as crystal fillers in the rubber state. The second storage modulus change occurs above 77 °C, particularly in samples with higher OOBA-content. This modulus change should be ascribed to the crystal melting transition of OOBA because it exactly corresponds to the Tm of OOBA. This observation is also notably consistent with the DSC and POM results. Thus, OOBA is another isolated phase that controls the storage modulus. The OOBA isolated phase provides a second reversible phase to the shape fixation and shape recovery. Therefore, SMPU-OOBAm composites may have a triple-shape memory effect.Dynamical mechanical properties can also be used to investigate phase transitions by characterizing the changes of the storage modulus. The liquid-crystalline properties and the phase transition behaviors of SMPU-OOBAm composites were investigated by POM. As we known, OOBA exhibits two crystalline forms, followed by smectic and nematic LC phases upon heating. Typical POM textures of OOBA upon heating are shown in For the SMPU-OOBAm composites, sample P5 has a bright abundant crystalline texture at 28 °C a becauseAdditionally, a comparison study of the POM textures of the composites is shown in Triple-shape memory behaviours of SMPU-OOBAm composites were examined in a thermo-mechanical analysis using a TA Instruments DMA800 with tension clamps in controlled-force mode. In this experiment, the rectangular samples were first elongated at 100 °C, and then fixed at 80 °C, while the second elongation and fixation were carried out at 80 °C and 0 °C, respectively. Thereafter, the two step shape recovery is tested each for 40 min at 80 °C and 100 °C, respectively. The strain deformation, strain fixation, and strain recovery process were recorded for analysis. The SMPU-OOBAm composites were also found to show self-healing properties in this work. As shown in This paper describes a new type of LC-SMPU composites, which were prepared with PEG-based SMPU and OOBA mesogens. The effect of OOBA on the structure, morphology, and properties of the material was carefully investigated. The results demonstrate that the LC-SMPU composites have liquid crystalline properties, triple-shape memory properties, and self-healing properties. The incorporated OOBA promotes the crystallizability of both the soft and hard segments of SMPU. However, the isothermal crystallization kinetics revealed that the crystallization rate of the hard segment decreases when the OOBA-content increases. Additionally, the SEM confirmed that the SMPU-OOBAm composites form a two-phase separated structure, which was comprised of an SMPU phase and an OOBA phase. DMA further reveals that the composites show two-step modulus changes upon heating. Therefore, the SMPU-OOBAm composites exhibit triple-shape memory behaviour, but the shape recovery ratio decreases with the increase in OOBA-content. Because of the crystalline behaviours and LC properties of OOBA that are maintained in all SMPU-OOBAm composites, the SMPU-OOBAm composites have good self-healing properties, due to the heating-induced “bleeding” of OOBA in the liquid crystalline state and its subsequent crystallization upon cooling. Therefore, the SMPU-OOBAm composites have promising applications in smart optical devices, smart electronic devices, and smart sensors.