Pelteobagrusfulvidraco), which provides a promising model for testis development and spermatogenesis. In our previous study, significant differences in morphology and miRNA expression were detected between XY and YY testis of yellow catfish. Here, solexa sequencing technology was further performed to compare mRNA expression between XY and YY testis. Compared with unigenes expressed in XY testis, 1146 and 1235 unigenes have significantly higher and lower expression in YY testis, respectively. 605 differentially expressed unigenes were annotated to 1604 GO terms with 319 and 286 genes having relative higher expression in XY and YY testis. KEGG analysis suggested different levels of PI3K-AKT and G protein-coupled receptor (GPCR) signaling pathways between XY and YY testis. Down-regulation of miR-141/429 in YY testis was speculated to promote testis development and maturation, and several factors in PI3K-AKT and GPCR signaling pathways were found as predicted targets of miR-141/429, several of which were confirmed by dual-luciferase reporter assays. Our study provides a comparative transcriptome analysis between XY and YY testis, and reveals interactions between miRNAs and their target genes that are possibly involved in regulating testis development and spermatogenesis.YY super-males have rarely been detected in nature and only been artificially created in some fish species including tilapia and yellow catfish ( The X latipes ,6, gsdf zonensis , sox3 in dancena , amhy inhatchery , sdY in s mykiss , amhr2 irubripes  and dmrtmilaevis  have beeteleosts –15.Oncorhynchusmykiss) reported obvious differences in gene expression and morphology between XY and YY testis [Oreochromisniloticus)[As an evolutionary link between invertebrates and higher vertebrates, fish species have a very complex sex determination system with XX/XY male heterogametic system as the main sex determination system . Until nY testis ,21. SignY testis . Howeveriloticus). There aIn our previous study, we observed larger spermatogenic cyst and more spermatids in YY super-males than XY males by histological analysis, suggesting a higher degree of testis maturity. Many miRNAs that are potentially involved in testis development and spermatogenesis were identified to be differentially expressed between XY and YY testis . The estde novo assembly, 78148 unigenes were obtained by paired-end method of Trinity and TGICL clustering with mean length of 944bps , SWISS-PROT, KEGG, GO and KOG protein databases by using BLASTX with a cut-off E-value of 1e-5 (Daniorerio while only 776 (3.92%) hits overlapped with Ictalurus punctatus (the close species to Pelteobagrus fulvidraco), perhaps due to the limited amount of genomic data on GeneBank about the species of Siluriformes. In addition, there were a number of unigenes without hitting results, which may contain non-coding fragments or some undetected genes.The unigenes of  of 1e-5 . Finally of 1e-5 , 11591 (vsYY testis) with fold change threshold value = 2 and FDR test (P< 0.05), 4458 unigenes were found expressed with significant difference. Among them 1006 were detected from testis of YY super-male and 1072 were found from testis of XY male. Besides this, 1146 unigenes from YY and 1235 from XY testis were detected with significantly higher expression. The MA scatter plots comparison reveal the differential expressed genes existed between testes of male and super-male in yellow catfish  to determine which kinds of GO term DEGs mainly participated in. Finally, 605 DEGs were annotated to 1604 GO terms  were searched against the Gene Ontology database , PI3K-Akt signaling pathway , phagosome , neuroactive ligand-receptor interaction , cytokine-cytokine receptor interaction , tuberculosis  and focal adhesion . The XY highly expressed unigenes were enriched in pancreatic secretion , protein digestion and absorption  and neuroactive ligand-receptor interaction  .The PI3K-AKT signaling pathway is involved in many fundamental functions including testis development and spermatogenesis, and stimulated by many kind of growth factors that specifically binds to receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR) ,26. As mTo verify the accuracy of the sequencing data, twelve DEGs related to RTK and G protein signaling pathway were arbitrarily selected and validated by quantitative real-Time PCR (qRT-PCR). The twelve DEGs includes four genes  relatively high expressed in XY and eight genes  relatively high expressed in YY testis in the transcriptome data . As in tIn our previous study, relative lower expression of miR-141/429 was observed in YY testis indicating a higher degree of testis maturity than XY testis. A high dose of 17α-ethinylestradiol (EE2) up-regulates the expression of miR-141/429 . Here, wTo determine whether there are direct interactions between miR-141-3p and PI3K-AKT or GPCR signaling pathway, we used dual-luciferase reporter assays to measure the inhibitory effect of this miRNA on Itgb2 and Gria4a. There is one binding site for miR-141-3p in either Itgb2 or Gria4a 3’UTR . We sub-In vertebrates with XY sex-determining system, the expression of sex-determining gene on Y chromosome leads to the development of male phenotypes and testis. Yellow catfish, an important aquaculture fish in China has XY sex-determining system and displays sexual size dimorphism as male grows 2–3 times faster than female. 454 pyrosequencing and illumina sequencing studies have been performed to compare differentially expressed genes and pathways between XX ovary and XY testis and provide a valuable genomic resource for studying fish reproduction, sex determination and differentiation ,28. HoweIn fully mature 18 month-old Nile tilapia, the sperm quality and quantity of XY and YY males were the similar . Howeversyk, prlr and kiss1r, coincides with a higher degree for testis maturity and advantageous spermatogenesis in YY than XY yellow catfish [It is important to know the context under which specific signaling pathways regulate sperm maturation as well as testis development in YY that matures earlier than XY fish. The PI3K-AKT signaling pathway plays essential roles in testis development and spermatogenesis, as loss of p110beta subunit of phosphoinositide 3-OH kinase impaired spermatogenesis and lead to defective fertility . Activat catfish . As one  catfish –34.In mammals, increased expression of miR-141/429 was associated with defects of spermatogenesis –37. HighAll experimental procedures involved in this study were permitted by the Institutional Animal Care and Institute of Huazhong Agricultural University. The total RNAs of XY  and YY  testes were the same biological sample as described before . The RNAP< 0.05 [To detect the differential expression of unigenes, DESeq software package with RPKM (Reads Per Kb Million reads) method was performed to quantify the expression of two expression profiles. The formula applied was  unigene . The Fal unigene . After c[P< 0.05 .-5. The results of BlastX annotation were uploaded on Blast 2 Go to generate Gene Ontology annotations and mapped to the categories of GO database ((geneontology.org/page/download-annotations)[p<0.05). To investigate which GO item and signaling pathways the DEGs participated in, all of the clustered DEGs were mapped back to the GO and KEGG databases. Each enrichment item was corresponded to a specific enrichment score that was calculated by the formula, score value = The unigenes were searched against databases of NCBI nr, SWISS-PROT, TrEMBL, Cdd, pfam and KOG by Blast X with a cut-off E-value of 10eotations). The resnnotation.-ΔΔCt method [Briefly, 1μg of total RNA was reverse transcribed by using PrimeScriptRT reagent Kit (Takara) according to the protocol. The qRT-PCR reaction was performed in a 20μl reaction volume using the Roche LightCycler 480 with SYBR Green PCR master mix(Roche) and three biological replicates were conducted for each reaction. The β-actin was selected as an internal reference to normalize the Ct values of each reaction by using the 2t method . The ANOhttp://genes.mit.edu/GENSCAN.html). Perl scripts of both Target Scan and miRanda were performed for searching the putative targets with default parameters, including context score percentile ≥100 for Target Scanand Max_Energy≤ −20 and for miRandabased on hybrid energy and stability [In our recent study, we found that miR-141-3p and miR-429b-3p have higher expression level in XY than YY testis . To invetability ,45To characterize the interaction between miR-141-3p/-429b-3p and their predicted target genes, the 3’-UTR of selected putative target genes (Itgb2 and Gria4a) were inserted into the pmirGLO expression vector . Hek-293T cells were seeded in 96-well plates and co-transfected with the constructed vectors and microRNA mimics or its control oligonucleotide using DharmaFECT transfection reagent (Dharmacon). 36h post transfection, the dual-luciferase reporter assay system was used to detect reporter (Firefly and Renilla) activity as described . The proS1 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


We investigated plasma levels of M-CSF and conventional tumor markers (HE4 and CA 125) in epithelial ovarian cancer patients as compared to control groups: benign ovarian tumor patients (cysts) and healthy subjects.M-CSF levels were determined by ELISA, HE4 and CA 125 levels - by CMIA method.serous epithelial sub-type of OC. Moreover, CA 125 showed better results of the aforementioned diagnostic criteria than HE4. The combined use of the parameters studied resulted in a further, significant increase in the value of the diagnostic indicators and in the value of the diagnostic power (AUC), especially in the early stages of ovarian cancer.Our results have demonstrated significant differences in the concentration levels of M-CSF, CA 125 and HE4 between the groups of ovarian cancer patients, cysts patients and the healthy controls. In the groups tested M-CSF demonstrated equal to or higher values than both CA 125 and HE4 in diagnostic sensitivity (SE), positive and negative predictive values , and in the area under the ROC curve (AUC), particularly in the group with the serous sub-type of epithelial ovarian cancer and in discriminating between cancer and non-carcinoma lesions, particularly in new diagnostic panels in combination with CA 125 and HE4 for the detection of EOC in the early stages.These findings suggest a high usefulness of M-CSF in diagnosing the The online version of this article (doi:10.1186/s13048-015-0153-3) contains supplementary material, which is available to authorized users. Epithelial ovarian cancer (EOC) remains the most lethal type of all gynecological cancers despite the development of new treatments and therapies. The malignant tumor of the ovaries occurs at all ages with variation in histological sub-type according to age ,2. EstabThe established tumor markers, such as CA 125 (carbohydrate antigen 125) or human epididymis protein 4 (HE4), can be used in the diagnosis and monitoring of epithelial ovarian cancer -8. A numMacrophage-colony stimulating factor is one of the cytokines called hematopoietic growth factors (HGFs). M-CSF regulates the growth, differentiation and functionality of neutrophils or macrophages. Additionally, M-CSF has also been implicated in the pathogenesis of cancer disease ,20. A nuThe aim of this study was to determine plasma levels of macrophage-colony stimulating factor in comparison to plasma levels of HE4 and the established CA 125 tumor marker in epithelial ovarian cancer (EOC) patients in relation to the control groups: patients with a benign ovarian tumor and healthy subjects. Additionally, comparisons between plasma levels of the parameters tested and cancer stage, its histological sub-type and histological type of benign ovarian tumors were performed. Furthermore, the diagnostic criteria  and the receiver-operating characteristic curve (ROC) for the cytokine tested (M-CSF), HE4 and CA 125 alone and in combinations were defined. Moreover, a correlation between the three parameters studied was established.The data obtained may be used in the evaluation of M-CSF usefulness in diagnosing the stages and histological sub-types of ovarian cancer and in discriminating between ovarian cancer and benign ovarian tumors, especially when analysed with HE4 and CA 125.serous and endometrioid sub-types) diagnosed by the Gynecology Group. The control groups comprised 70 benign ovarian tumor patients (cystis serous or cystis endometrioides) and 50 healthy volunteers. All participants enrolled in the study (cancer and control groups) had postmenopausal status at the time of blood collection. Clinical stages and histological classification based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO) were established in all cases. The ovarian cancer patients and the control group (benign lesions) were treated in the Department of Gynecology, Białystok Medical University Teaching Hospital, Poland, between 2006–2012. Epithelial ovarian cancer and benign ovarian tumor histopathology was established in all cases. Patients with renal failure were excluded from the study due to significantly elevated HE4 concentration levels, indistinguishable from ovarian cancer. Written consent including participants’ own statements regarding their medical history  and lifestyle habits including smoking was obtained from all the subjects.Table None of the patients had received chemo- or radiotherapy before blood sample collection. Pretreatment staging procedures included physical and blood examinations, ultrasound scanning and chest X-rays. In addition, CT (computed tomography) scans or MRI (magnetic resonance imaging) were performed where necessary.Healthy patients were recruited from apparently healthy female employees of Białystok Medical University Teaching Hospital between 2006 and 2012. They were not referred from other medical centers. All subjects had undergone annual check-ups . Subjects with a clinical history of prior endometriosis or mild gynecological conditions were excluded. Women included in the control group were volunteers who reported no prior history of gynecological conditions and displayed no visible or perceptible changes in the adnexa. The group were examined by a gynecologist prior to blood collection and an ultrasound examination was performed in every case.The study was approved by the local Ethics Committee of the Medical University in Białystok, numbers: R-I-002/314/2009 and R-I-002/262/2010 and all the patients gave their informed consent for the participation in the study.0 C until assayed. M-CSF was measured with the enzyme-linked immunosorbent assay (ELISA) , according to the manufacturer’s protocols. Duplicate samples were assessed for each patient. The intra-assay coefficient of variation (CV%) of M-CSF - 3.4% at a mean concentration of 227 pg/ml, SD = 7.7. The inter-assay coefficient of variation (CV%) of M-CSF - 3.1% at a mean concentration of 232 pg/ml, SD = 7.3. The assay showed no significant cross-reactivity or interference with numerous human cytokines and other growth factors.Venous blood samples were collected from every patient. Blood was collected into a heparin sodium tube, centrifuged 1000 rpm for 15 min. to obtain plasma samples, and stored at-85Plasma concentrations of HE4 and CA 125 were measured by chemiluminescent microparticle immunoassay (CMIA) . The intra-assay CV for HE4 - 3.7% at a mean concentration of 39.0 pmol/L, SD = 1.4. The inter-assay CV for HE4-2.8% at a mean concentration of 39.0 pmol/L, SD = 1.1. The intra-assay CV for CA 125 is reported to be 2.4% at a mean concentration of 43.5 U/ml, SD = 1.1. The inter-assay CV for CA 125 is reported to be 3.9% at a mean concentration of 43.5 U/ml, SD = 1.7.cut-off points of M-CSF (575.80 pg/ml), HE4 (75.90 pmol/L) and CA 125 (28.40 U/ml) were calculated as 95th percentile from the control group of healthy blood donors.The statistical analysis was performed using the STATISTICA 8.0 PL program. A preliminary statistical analysis (Chi-square test) revealed that the distribution of cytokine and tumor marker levels did not follow normal distribution. Consequently, nonparametric methods were used to compare levels of the parameters tested between the groups of patients. Comparisons between two groups were performed using the Mann–Whitney test, between multiple groups Kruskal-Wallis tests were calculated with post hoc comparisons according to Dwass-Steele-Critchlow-Fligner method. The ROC analyses were utilized in the evaluation of the diagnostic power of tumor markers and the construction of the curves was performed using GraphRoc Program for Windows. Markers were compared by assessing the significance of differences between the areas under their corresponding ROC curves. The Data were presented as median and range. Statistically significant differences were defined as comparisons resulting in p < 0.05. The Spearman rank correlation was used in the correlation analyses.The median of M-CSF levels, similarly to the median levels of the comparative tumor markers HE4 and CA 125 in the total group of OC and in every stage of advancement (I-IV) of cancer disease were significantly higher when compared to the healthy controls   had statistically considerably higher median levels of the parameters researched  than those observed in the group with benign ovarian tumors Table . We alsoIn the case of the total group with benign ovarian tumors, the concentrations of M-CSF and CA 125 were significantly different than in healthy subjects (p < 0.001).serous and endometrioid) and benign tumors control group . It was also observed that the distribution of all the tested parameters among two histological sub-types of epithelial ovarian cancer were significantly different  . Interestingly, the plasma level of HE4 was significantly lower in patients with cystis endometrioides in comparison to the healthy controls (p = 0.042). In addition, we noticed significant differences in the median HE4 (p = 0.046) and CA 125 levels (p = 0.039) when comparing the cystis endometrioides to the cystis serous group  or serous . Moreover, positive correlations were also observed between the M-CSF and CA 125 levels in the healthy control group . The single negative correlation was obtained for the HE4 and CA 125 concentrations  in patients with stage II EOC.The Spearman’s rank correlation was used in the analysis of dependence between the investigated parameters  in every cancer group compared and in every histopathological sub-type of EOC. The combined use of the parameters studied resulted in a decrease in the diagnostic SP Table .serous and for M-CSF in the endometrioid sub-types of EOC.In the total group of EOC patients the PPV had the highest values for HE4 (90%). HE4 reached the highest values of PPV in stage I in contrast to M-CSF, which reached the highest values in stages II-IV. Similarly, the combined use of the studied markers resulted in a decrease in the PPV values Table . Additioserous and CA 125 in the group with endometrioid sub-types of OC . M-CSF also obtained the highest value in stages II-IV with the exception of stage I, where HE4 consistently achieved the best results. Additionally, the NPV values showed a further increase during the combined analysis of the parameters tested Table . The maxOC Table .endometrioid epithelial (0.9129) and M-CSF in serous epithelial sub-types of ovarian cancer (0.9058). The AUCs of M-CSF and the comparative tumor markers were significantly higher compared to AUC = 0.5 in every group of EOC studied .The relationship between the diagnostic sensitivity and specificity was illustrated by the ROC curve. Table serous or endometrioid ovarian carcinoma [The search for an effective screening test for ovarian cancer has been the focus of intensive research efforts. Blood flows into and out of tumors and circulates tumor-specific protein profiles, making serum or plasma the ideal biological media for finding a screening biomarker . The co-arcinoma -8.In this study we investigated the diagnostic usefulness of M-CSF used alone and in combination with HE4 and CA 125 in patients with EOC before surgical intervention or chemo- and/or radiotherapy. Furthermore, we estimated the diagnostic utility of these biomarkers in correlation to the stage and histological type of ovarian cancer.serous, endometrioid, mucinous, clear cell). It is worth noting that Suzuki and colleagues [c-fms, were expressed in gynecological malignancies, and the co-expression of M-CSF and c-fms might be related to progression to the metastatic state [Our results show that M-CSF as well as HE4 and CA 125 plasma levels in the total group of ovarian cancer patients were statistically significantly higher in comparison to the group of healthy controls. Data regarding M-CSF obtained in the present study are also in agreement with our previous studies  and withlleagues  found nolleagues . It shouic state  and is aic state ,38. Furtic state . Our resic state , althougic state  who obseic state ,43. The ic state ,44.et al. [serous, mucinous and endometriotic cysts and also of fibromas, thecoma and benign cystic teratomas were analysed). Suzuki et al. [cystic teratomas were studied. Furthermore, Burke et al. [et al. [In line with our previous report  and otheet al.  discoveri et al.  confirmee et al.  revealed [et al.  observed [et al. . The fin [et al. ,47,48 an [et al. ,36. Sign [et al. ,49,50 we [et al. .et al. [serous epithelial OC displayed significantly different concentrations of all the parameters studied than those with endometrioid epithelial OC. We also demonstrated significant differences in the concentrations of the parameters tested in every group of OC sub-types vs patients with cystis endometrioides and with cystis serous. Comparable results, but only for CA 125, were published by Nolen et al. [cystis endometrioides and cystis serous. These data are at variance with other publications [In contrast to the results published by Suzuki et al.  we obseret al. . Our preet al. ,52,53. Fn et al. , althougications , and mayications ,41,55,56The correlation between M-CSF, HE4 and CA 125 levels was estimated by Spearman’s rank correlation test and most of the measured values tended to increase for every marker. However, the degree of the correlation was not particularly strong (R: 0.32–0.47), and there were some discordant results. This suggests that each marker was elevated concurrently or under different conditions, and these results also support the necessity of combining the three markers. We found similar results in the existing literature ,10,52.endometrioid sub-type). Our results are in agreement with the investigation of Skates et al. [In our analysis of postmenopausal subjects, we found that M-CSF alone provided the highest SE, PPV and NPV,  of any individual biomarker tested . We demonstrated that the utilization of a combination of all the markers under investigation consistently provided the highest sensitivity for the detection of every stage, and particularly early stages, of EOC with the AUC value of 0.9069–0.9894, which was superior to the value of either marker used alone. Furthermore, we observed that the areas under the curve for the cytokine tested and the comparative markers were statistically significantly larger compared to AUC = 0,5 - borderline of diagnostic usefulness of the test. Test data published by Zhang et al. [The AUC numerically describes the overall performance of a marker, with the AUC of 1 indicating a perfect SE and SP. We observed that M-CSF had the highest, comparable to conventional markers, diagnostic power in the groups with stages I-III of OC  and in the patients with g et al.  are at vg et al. ,52,56,62g et al. , the resserous sub-type of EOC, particularly in combination with HE4 and CA 125 as a new diagnostic panel. The utilization of the tested parameters in combination may also measurably improve the OC diagnosis in the early stages. Additional studies with larger, early-stage carcinoma patient groups may be needed to further strengthen the statistical power of the evidence.In conclusion, challenges regarding the early diagnosis of epithelial ovarian cancer remain. Although hundreds of biomarkers have been identified as being associated with this common type of cancer, their efficacy in the early diagnosis of the disease has not yet been established. To our knowledge, this is the first study in which plasma levels of M-CSF, HE4 and CA 125 were simultaneously analysed in a relatively large group of untreated patients with primary malignant and nonmalignant ovarian tumors. While each marker has its own strengths and weaknesses, CA 125 still remains better than HE4 in the diagnostics of EOC patients. However, M-CSF obtained even better results than CA 125. Our findings suggest a highly potential role for M-CSF as a tumor marker in the diagnosis of the


The presence of ethanol in all samples and the wide range of concentrations clearly indicated contamination of the samples of some sort, which affected the 1H-NMR spectra quality and the interpretation. To determine where in the sampling protocol the ethanol contamination occurs, we performed a CSF sampling protocol simulation with 0.9 % NaCl  instead of CSF and detected ethanol in all simulation samples. Ethanol diffusion through air during sampling and preparation stages appeared the only logical explanation. With a bench study, we showed that ethanol easily diffuses into ex vivo CSF samples via air transmission. Ethanol originated from routinely used skin disinfectants containing ethanol and from laboratory procedures. Ethanol affected the CSF sample matrix at concentrations above ~9.4 mM and obscured a significant part of the 1H-NMR spectrum. CSF sample preparation for 1H-NMR-based metabolomics analyses should therefore be carried out in a well-ventilated atmosphere with laminar flow, and use of ethanol should be avoided.Standardization of body fluid sampling, processing and storage procedures is pivotal to ensure data quality in metabolomics studies. Yet, despite strict adherence to standard sampling guidelines, we detected variable levels of ethanol in the The online version of this article (doi:10.1007/s00216-015-8663-9) contains supplementary material, which is available to authorized users. Other studies reporting the presence of ethanol in CSF have interpreted this as either a contaminant [Cerebrospinal fluid (CSF) is generally believed to reflect brain physiology and is therefore frequently used to assess biomarkers for brain disorders –3. CSF staminant , 6 or dutaminant , 8.In the present study, we set out to identify the origin of ethanol contamination and to assess the effect it has on the sample matrix.1H-NMR-based metabolomics (method 3) . Following centrifugation, the supernatant was transferred to a 15-mL polypropylene falcon tube and divided in 0.5-mL aliquots, placed on dry ice within 30 min of sampling and transferred to −80 °C for storage within 60 min of sampling. In sampling methods 1 and 2, cold ethanol was added to the CSF during the sample handling to denature proteins and to be able to thaw the samples before measurements at lower temperatures. Because this was a likely source of ethanol contamination, we also analysed three CSF samples obtained for clinical diagnostic purposes to check whether ethanol is also present in these samples. Disinfection of the skin with chlorhexidine (5 g/L)/denatured ethanol 70 %, sample collection and sample processing of these clinical samples were similar to the research samples with the exception that ethanol was not used anywhere in the handling of these samples. For additional details on sampling and processing of the research and clinical CSF samples, see the The cohort in which we initially discovered ethanol contamination consisted of human CSF samples that were collected for research purposes, namely to investigate the pathophysiology of migraine. The study protocol was approved by the Medical Ethical Committee of Leiden University Medical Center (LUMC), and all subjects gave written informed consent prior to collection. For disinfection of the skin, chlorhexidine (5 g/L)/denatured ethanol 70 %  was used. CSF samples were taken via lumbar puncture. Three different sample handling protocols were used simultaneously for the preparation of samples for mass spectrometry (MS)-based metabolomics methods 1 and 2) and for  and 2 an1H-NMR-based metabolomics (method 3). In methods 1 and 2, one pipette was used throughout the entire sampling protocol, including pipetting of ethanol. In method 3, a different pipette was used which had never been exposed to ethanol; only these samples were measured with 1H-NMR as described below.To determine the origin of ethanol during our sample processing procedure, we performed a CSF sampling protocol simulation. A 0.9 % NaCl solution  was used instead of CSF in an exact simulation of the CSF sampling protocol see Fig. . Differe1H-NMR analysis. The second cryotube was removed after 30 min. This process was repeated without the beaker in the same well-ventilated work space. All samples were then prepared for and analysed by quantitative 1H-NMR spectroscopy as described below.To test how fast ethanol can diffuse into CSF via air, we created a work area simulation. Ethanol  was placed into 2 × 1.8-mL cryotubes . An up-turned 500-mL glass beaker was placed over two open cryotubes containing 1 mL of saline and one open cryotube containing ethanol see Fig. . After 51H-NMR spectrum is affected. Two hundred microlitres of CSF was aliquoted into 10 × 1.5-mL eppendorf tubes containing ethanol , decreasing in concentration in regular stepped increments so that the final ethanol concentrations were 9.1 × 10−3–500.0 mM. Samples were prepared for 1H-NMR analysis as described below.A serial dilution was made to test at which level of ethanol contamination the CSF matrix and thereby the whole 3. Following thorough mixing by repeated inversion, 190 μL of the sample was transferred into 3-mm NMR tubes in a cooled rack at 6 °C.In 1.5-mL eppendorf tubes that were placed on ice, 225 μL of CSF, 0.9 % NaCl, or CSF/ethanol was added to 25 μL of pH 7.0 phosphate buffer (50 mM) in D2O containing 4 mM of sodium 3-trimethylsilyltetradeuteriopropionate (TSP) and 2.0 mM NaN1H-NMR data were obtained using a Bruker 600 MHz AVANCE II spectrometer equipped with a 5-mm TCI cryoprobe and a z-gradient system; a Bruker SampleJet sample changer system was used for sample transfer; samples were kept at 6 °C while queued for acquisition. One-dimensional (1D) 1H-NMR spectra were recorded at 300.0 K using the first increment of a NOESY pulse sequence [sequence  with presequence . The dursequence  on the lsequence .Quantification of ethanol peaks was carried out by iterative line fitting in the frequency domain .1H-NMR spectra of not only all research CSF samples but also in three clinical CSF samples where ethanol is only used during disinfection and not during sample processing  disinfection of the skin with chlorhexidine/70 % ethanol prior to lumbar puncture and (ii) use of ethanol in the same room as where the CSF samples were prepared for 1H-NMR spectra. The two signal groups of ethanol at 1.18 and 3.66 ppm can mask metabolites that lie in the same chemical shift region of 1H-NMR spectra. The methylene quartet at 3.66 ppm will itself be masked, as it lies in the heavily crowded glucose region of the spectrum. The methyl signal of the small metabolite 3-hydroxybutyric acid, however, normally occurs in the same region of the CSF spectrum as the ethanol methyl triplet (1.18 ppm). In the presence of ethanol, the detection and subsequent quantification of this metabolite are heavily compromised.The most important effect of ethanol contamination is on the interpretation of the 1H-NMR spectra prior to multivariate analysis; 2) spectrum alignment might be required prior to analysis in cases of severe matrix distortion; 3) processing of CSF samples for 1H-NMR analysis in an ethanol-free zone or well-ventilated area such as a laminar flow cabinet; 4) use of non-ethanol-containing disinfectant instead of chlorhexidine/ethanol.There are several solutions to minimize the risk of ethanol contamination and to mitigate its effects on metabolomics analysis, as follows: 1) ethanol regions can sometimes be removed from We believe that the recommendations resulting from this study should be considered when collecting CSF for biomarker research in brain disorders.ESM 1(PDF 18 kb)


Increased α-synuclein oligomers in asymptomatic leucine-rich repeat kinase 2 mutation carriers showed a sensitivity of 63.0% and a specificity of 74.0%, with an area under the curve of 0.66, and a sensitivity of 65.0% and a specificity of 83.0%, with an area under the curve of 0.74 for sPD cases. An inverse correlation between CSF levels of α- synuclein oligomers and disease severity and duration was observed. Our study suggests that quantification of α-synuclein oligomers in CSF has potential value as a tool for PD diagnosis and presymptomatic screening of high-risk individuals.Mutations in the leucine-rich repeat kinase 2 gene are the most common cause of autosomal dominant Parkinson’s disease (PD). To assess the cerebrospinal fluid (CSF) levels of α-synuclein oligomers in symptomatic and asymptomatic leucine-rich repeat kinase 2 mutation carriers, we used enzyme-linked immunosorbent assays (ELISA) to investigate total and oligomeric forms of α-synuclein in CSF samples. The CSF samples were collected from 33 Norwegian individuals with leucine-rich repeat kinase 2 mutations: 13 patients were clinically diagnosed with PD and 20 patients were healthy, asymptomatic leucine-rich repeat kinase 2 mutation carriers. We also included 35 patients with sporadic PD (sPD) and 42 age-matched healthy controls. Levels of CSF α-synuclein oligomers were significantly elevated in healthy asymptomatic individuals carrying leucine-rich repeat kinase 2 mutations ( SNCA) were the first genetic factors to be linked to familial PD ; Spillantini et al., in vitro and in vivo, whereas amyloid fibrils may not be directly toxic  is the most common age-related movement disorder and the second most common neurodegenerative disorder after Alzheimer’s disease. The earliest clinical features of PD are typically retrospective and not specific, and they may include depression, hyposmia, constipation, and sleep disorders. At least 70% of the neurons in the substantia nigra (SN) are lost prior to the appearance of any major motor symptoms  score  and a superdex 200 column at 4°C. Concentrated 0.5 ml of CSF were loaded onto the column and eluted with PBS (pH 7.4) at a flow rate of 0.1 ml/min (0.5 ml/fraction). The elution of α-syn was monitored at absorbance wavelengths of 215 nm. Fractions of 1 ml were collected, concentrated to 100 μl using a speed vac, and analyzed by the western blotting for the presence of α-syn. To determine the elution time of monomeric and oligomeric α-syn, molecular weight standards , fresh α-syn solution and aged α-syn solution were co-injected into the column and eluted at the same conditions mentioned above.The CSF fractions were separated on NuPAGE Bis–Tris 4–12%, 1 mm gels  and then transferred to nitrocellulose membranes (0.45 μm) at 30 V, 125 mA for 45 min . Membranes were boiled for 5 min in PBS then blocked for 1 h with 5% marvel dried skimmed milk and dissolved in PBS–Tween 20 (0.05%) (PBST). The membranes were probed overnight at 4°C anti-α-syn (211) mouse monoclonal antibody to α-syn (aa 121–125). The membranes were washed several times with PBST followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse , for 60 min. The membranes were again washed several times with PBST. The protein bands were visualized using ECL reagents  as described by the manufacturer.A sandwich enzyme-linked immunosorbent assay (ELISA) methods were employed to measure total or oligomeric α-syn levels in CSF samples as described previously  was analyzed to assess the most appropriate cut-off values for the level of CSF α-syn oligomer and the oligomers/total-α-syn ratio in the CSF to distinguish between groups. All analyses were conducted using GraphPad Prism software .Differences between groups were compared using a Mann-Whitney U test. Significance was defined as LRRK2 families were investigated in the present study. Thirteen individuals with LRRK2 point mutations had developed symptomatic PD, including 11 males who were carrying the most common LRRK2 point mutation, G2019S, and two females who were carrying a different LRRK2 point mutation, N1437H. The 13 individuals had a mean age of 64.0 years ± 13.3 years. In contrast, 20 individuals were healthy asymptomatic LRRK2 mutation carriers [G2019S (n = 16) and N1437H (n = 4)]. These 20 individuals had a mean age of 55.4 years ± 15 years. None of the healthy asymptomatic LRRK2 mutation carriers (LRRK2-H) had any complaints of a movement disorder. Some were receiving medication for diabetes mellitus, mild hypertension, and other minor health problems. In addition, 35 patients with sPD and 42 age-matched healthy controls were also included in this study. No significant difference was noticed in disease duration between symptomatic PD patients with LRRK2 mutations (LRRK2-PD) and sPD patients. Moreover, there was no difference between the groups with regard to CSF levels of leukocytes or total protein, albumin, and glucose levels, including plasma glucose levels. Controling for age and gender did not significantly alter the results in any case. A summary of the patient population employed in the present study and the respective demographic details are shown in Table In total, 33 Norwegian individuals from 12 different To measure the total α-syn (t-α-syn) in CSF samples, we recently optimized our original α-syn ELISA protocol using a chemiluminescence-based read-out arm for HRP-labeled antibody detection , LRRK2-PD , and LRRK2-H  than in age-matched controls  .As illustrated in Figure After measuring the total α-syn in CSF samples from our cohort, we then measured the levels of CSF α-syn oligomers (o-α-syn) in the same samples. For such measurements, we used an ELISA system for α-syn oligomers that specifically detects soluble oligomers without detecting monomeric forms of α-syn  relative to the healthy age-matched control group  . Significantly higher levels of CSF o-α-syn (approximately 2-fold higher) were also detected in LRRK2-H  relative to the control group  . Unexpectedly, LRRK2-PD patients  did not show any significant difference in the level of o-α-syn in the CSF relative to the healthy control group , possibly because of the low number of individuals in this group (n = 13 vs. n = 42). Furthermore, we observed a significant inverse correlation  between the severity of the disease and the level of o-α-syn in the CSF of sPD patients using H&Y grading  between H&Y grading and the levels of o-α-syn in the CSF of LRRK2-PD patients was observed , but CSF o-α-syn did not correlate with disease duration in LRRK2-PD group. Whereas, we observed significant negative correlation between CSF o-α-syn levels and disease duration when the sPD and LRRK2-PD groups were combined together . In contrast, CSF o-α-syn levels did not correlate with UPDRS scores within any of the groups (data not shown). In addition, no correlation was observed between the level of o-α-syn in the CSF and the age of the patients (data not shown).A scatter plot representing the CSF levels of o-α-syn for each patient in the four groups is shown in Figure n = 35)  and LRRK2-H group   than in the healthy control group . No significant difference in the ratio of o-α-syn to t-α-syn was found in the LRRK2-PD relative to controls  , possibly because of the small number of individuals in this group (n = 13 vs. n = 42).The measurement of both total and oligomeric α-syn in RLU/s allowed us to calculate the ratio of o-α-syn to t-α-syn (o-α-syn/t-α-syn ratio [%]) in the CSF of each patient, as shown in Figure Figure Currently the diagnosis of PD is based mainly on clinical symptoms. However, differential diagnosis from other parkinsonisms can be difficult and can lead to misdiagnosis. To date, no simple laboratory biomarker is available to detect individuals at risk for PD before most of their dopaminergic neurons have been lost.LRRK2 mutations are an excellent population for validating surrogate biomarkers for early stages of PD. A recent study reported a lack of a statistically significant relationship between PET scan evidence of lost striatal dopaminergic function and the levels of DJ-1 and t-α-syn in CSF from LRRK-H or LRRK-PD cases . However, we noticed in this group that patients with mild PD (H&Y grade ≤2) showed high levels of CSF o-α-syn similar to those of sPD patients. Conversely, patients with higher H&Y grades (>2) had lower levels of α-syn oligomers, which supports the hypothesis that at the early stages of the disease, high levels of toxic o-α-syn accumulate in the brain. In support of this notion, we also found that levels of CSF o-α-syn were inversely correlated with disease duration and H&Y grade in LRRK2-PD and sPD cases, which confirms that CSF levels of o-α-syn decrease with increasing PD severity (Table Numerous studies have suggested that neuronal cell death may result from the formation of oligomeric species of α-syn in the brain (Irvine et al., s Figure , which ss Figure , which cs Figure . UnexpecUnified Parkinson’s Disease Rating Scale is the most commonly used clinical scale to provide an efficient and flexible assessment of motor performance in PD patients and to monitor the degree of resultant disability. However, thus far, no strong linear relationship has been established between UPDRS scores and the progressive nigrostriatal degeneration in PD, which may underlie the absence of a correlation between CSF α-syn levels and UPDRS scores. Cerebrospinal fluid biomarkers mirror changes within the brain as an entire unit, whereas UPDRS scores primarily reflect changes in the nigrostriatal dopaminergic system. In addition, compensatory responses in PD may further confound the correlation between CSF biomarkers and the severity of PD motor symptoms (Shi et al., LRRK2 point mutation, G2019S, initiates and enhances the formation of α-syn aggregates (Lin et al., LRRK2-related PD cases are pathologically and clinically undistinguishable from sPD, LRRK2 mutation carriers exhibit considerable clinical and pathological variability (Wider et al., It has been recently reported that the most common In conclusion, our current pilot study suggests for the first time that quantification of o-α-syn in CSF has strong potential value as a tool for PD diagnosis and presymptomatic screening of high-risk individuals who are good candidates for clinical trials. However, large-scale, prospective, and well-controlled studies, especially those that include subjects at genetic risk, are necessary to validate the use of CSF o-α-syn as a biomarker for PD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Information processing in the striatum requires the postsynaptic integration of glutamatergic and dopaminergic signals, which are then relayed to the output nuclei of the basal ganglia to influence behavior. Although cellularly homogeneous in appearance, the striatum contains several rare interneuron populations which tightly modulate striatal function. Of these, cholinergic interneurons (CINs) have been recently shown to play a critical role in the control of reward-related learning; however how the striatal cholinergic network is functionally organized at the mesoscopic level and the way this organization influences striatal function remains poorly understood. Here, we systematically mapped and digitally reconstructed the entire ensemble of CINs in the mouse striatum and quantitatively assessed differences in densities, spatial arrangement and neuropil content across striatal functional territories. This approach demonstrated that the rostral portion of the striatum contained a higher concentration of CINs than the caudal striatum and that the cholinergic content in the core of the ventral striatum was significantly lower than in the rest of the regions. Additionally, statistical comparison of spatial point patterns in the striatal cholinergic ensemble revealed that only a minor portion of CINs (17%) aggregated into cluster and that they were predominantly organized in a random fashion. Furthermore, we used a fluorescence reporter to estimate the activity of over two thousand CINs in naïve mice and found that there was a decreasing gradient of CIN overall function along the dorsomedial-to-ventrolateral axis, which appeared to be independent of their propensity to aggregate within the striatum. Altogether this work suggests that the regulation of striatal function by acetylcholine across the striatum is highly heterogeneous, and that signals originating in external afferent systems may be principally determining the function of CINs in the striatum. The striatum is a large subcortical structure involved in major cognitive and sensorimotor functions, which is regarded as the primary input to the basal ganglia network . Lackinghttps://dx.doi.org/10.6084/m9.figshare.3420961.v1).The authors confirm that all data underlying the findings are fully available without restriction. All data files are available from the data repository figshare . All experimental procedures were approved by the University of Queensland Animal Ethics Committee , and were conducted in accordance with the Animal Care and Protection Regulation  and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes .This research has been performed in agreement with the ChATBAC-EGFP transgenic mice (http://jaxmice.jax.org/strain/007902.html) were used in this study [ad libitum and were kept with their littermates until used for the experimental procedures described below. Experiments were carried out during day time.Four two-month-old male wild-type C57Bl/6 mice and four two-month-old male homozygous is study ,16. AnimNaïve mice were taken from their home cage and rapidly anaesthetized through exposure to isoflurane (4% in air) in a sealed chamber followed by an injection of sodium pentobarbital (350 mg/kg i.p.), and transcardial perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5) using an air pressure system (constant flow of 15 ml/min). Vibratome-cut free-floating coronal sections were processed for immunofluorescence staining as described previously . BrieflyChATBAC-EGFP mouse and imaged them using a Metafer slide scanning system (MetaSystems). Images were processed using ImageJ2/Fiji . The outer boundary of the right striatum and its anatomical subdivisions , shell (ShVS) and core (CoVS) of the ventral striatum, dorsomedial (pDMS) and dorsolateral (pDLS) areas of the posterior striatum) were manually drawn in accordance with a mouse brain atlas [2) and Cartesian coordinates of the points defining each region’s edge were obtained. Images were then thresholded based on EGFP intensity levels and individual CIN somata (EGFP positive) were automatically detected using the “Analyze Particle” command. A table containing the Cartesian coordinates  of the centroid was obtained.We obtained 102 serial sections  from a in atlas , and the2) of CINs was calculated for each striatal territory. The same data files were used to assess CIN spatial organization using the DBSCAN  clustering algorithm in MATLAB. For all analyzed sections, we set the parameters for DBSCAN algorithm considering a point to be included within a cluster when it had at least six neighboring points located within a circle of 150 μm radius. Finally, we calculated the nearest neighbor distance of CINs using a customized triangulation-based algorithm in MATLAB. Finally, a custom written program in MATLAB [K-function [K-function from simulated point maps (500 simulations/section) containing the same number of CINs at a given area for each striatal section analyzed [L-function was used to test whether the distribution of points within a concentric circle of radius r obeys complete spatial randomness, in which case L(r)–r will be zero. If at some radius the value of L(r)–r is positive, it means that more points have been encircled at that radius than would have been expected for a random distribution, i.e. the points are clustered. Cluster maps were generated from Ripley’s K-function in which each dot corresponds to one CIN, which was pseudo-colored according to its individual L(r) value calculated for a 200-μm radius. We chose r = 200 μm to represent clustering in each section because at that radius, the L(r) value calculated from the Ripley’s K-function peaked in most of our samples.The spatial distribution of all detected CINs was digitally reconstructed using a customized program in MATLAB (The MathWorks). The total count and density  for the digitization and analysis of striatal cholinergic global neuronal branching. High magnification images were acquired with a LSM 510 META confocal laser-scanning microscope (Carl Zeiss). Serial optical sections along the z axis were obtained from each striatal subregion (3 z-stacks per subregion composed of 70–100 images). Analysis of global spatial complexity of the striatal cholinergic network was performed using NeuronStudio [We used 20 μm-thick sections from three onStudio . Data reFive sections  from four C57Bl/6 mice were co-stained with antibodies against ChAT and p-rpS6 and imaged using the Metafer slide scanning system. The location of 2,107 CINs was recorded as described above and a region of interest (ROI) of the contour of individual somata was obtained and overlaid onto the p-rpS6 image to measure the fluorescence intensity  for individual neurons.a priori alpha level was set at p < .05. One-way analysis of variance (ANOVA) was conducted followed by Tukey’s (HSD) multiple comparisons test when the variances were equal across groups . However, when the assumption of homogeneity of variances was violated , a Welch’s ANOVA correction was applied together with the Games-Howell post hoc test. To measure the relationship between p-rpS6 intensity and distance to the most dorsomedial point, Pearson’s correlation coefficient (r) and its significant level were calculated.Data were analyzed using SPSS Statistics software . The ChATBAC-EGFP mouse, and mapped the position of 9,732 fluorescently labeled CINs (p < .001), and in ventral regions, the average in the ShVS was also twofold higher than in the CoVS (p = .039) (p < .05).To analyze the spatial organization of the striatal cholinergic system, we digitally reconstructed 102 serial coronal sections spanning the rostrocaudal extent of the right striatum from a led CINs . We nextled CINs . The rosled CINs . When cl = .039) . OverallF = 10.475, p < .001) (p < .001), pDMS (p < .001) and pDLS (p = .004). The aDS also contained significantly greater concentration of CIN when compared to the pDMS (p = .012), which suggests a rostrocaudal gradient of CIN density. Furthermore, within the ShVS, there was a gradual two-fold increase of density along the rostrocaudal axis (from 20 to 47 CINs/mm2), whereas in the CoVS we observed a two-fold decrease density from anterior to most posterior levels (from 29 to 12 CINs/mm2)  . We obseINs/mm2) , inset. F = 24.957, p < .001) (p < .001). Next, we considered whether CINs form spatially organized clusters, as other interneuronal populations do in the mouse neocortex and the spinal cord [p < .05 vs CoVS, pDMS and pDLS). There was not significant difference in the propensity of clustering between the other striatal sectors  . In linenal cord . We firs sectors .K-function [In order to determine if these spatial arrangements were observed more often than expected from complete randomness, we used a cluster-analysis method based on Ripley’s function ,21 and cChATBAC-EGFP mice. We reconstructed the cholinergic multineuron network contained in a ~100 μm3 box of the different striatal sectors (3 volumes/region) with similar neuronal density and assessed their spatial complexity through automated quantitative methods  = 6.676, p < .001). The total content of cholinergic neuropil in the CoVS was significantly shorter compared to the aDS (p = .001), the ShVS (p = .002) and the pDLS (p = .007), but not the pDMS (p = .170)  = 2.163, p = .111)  . Further = .111) . These dCholinergic interneurons  typically display a tonic pattern of firing that is maintained even if all their synaptic inputs are blocked ,17. ReceF = 139.023, p < .001) (p < .001). We also found that the degree of rpS6 phosphorylation in the ShVS was significantly lower than in the CoVS (p < .001). In regions of the posterior striatum, CINs in the pDMS displayed higher levels of p-rpS6 signal compared to those located in the pDLS (p < .001). These findings reveal that CINs residing in different striatal subdivisions display particular levels of intrinsic cellular activity in naïve animals, which raises the possibility that functional cholinergic gradients may exist throughout the striatal tissue in basal conditions.Interestingly, we found that CIN p-rpS6 fluorescence intensity varied significantly among striatal subdivisions ( < .001) . In the r = -.439; B 1.1 mm: r = -.371; B 0.62 mm: r = -.390; B 0.14 mm: r = -.386; B -0.22 mm: r = -.396; p < .0001 for all levels analyzed). Taken together, these results suggest that the topographical organization of excitatory cortical and thalamic synaptic inputs to the striatum, and probably others, might play a role in the region-specific differences observed in striatal CIN intrinsic activity in naïve animals.It is well known that cortical and thalamic projections to the striatum distribute along a dorsomedial-to-ventrolateral zone, forming a gradient of excitatory inputs ,27. TherIn this study, we employed a quantitative imaging approach to analyze the intrinsic architecture of the entire CIN population in a fully reconstructed mouse striatum and to assess overall CIN activity levels throughout this brain region. Expanding previous observations in rodents and primates with greater detail and accuracy, we found that the rostral striatum contained a higher concentration of CINs than the caudal striatum, a gradient that has also been observed in the rat striatum –31. The Although it is generally assumed that CINs are dispersed in a random manner throughout the striatum, the novel visualization and analytical tools employed in the current study enabled us to determine this observation quantitatively. The different spatial point pattern statistics that we applied to our neuronal dataset revealed that the proportion of detected CINs arranged into cluster was low (17%), but there was however significant clustering of CINs along the rostrocaudal extent of the striatum, particulary in the anterior dorsal striatum and the shell of the ventral striatum. Interestingly, the observation that these clusters were confined to several consecutive sections in the two-dimensional space raises the possibility that CINs may establish local functional ensembles, or microcircuits, within the striatal network. Indeed, it is likely that future neuroanatomic studies addressing the three-dimensional organization of this interneuronal population using non-sectioned tissue of the whole striatum, will find a higher degree of clustering than what we could observe here ,38. The Of particular interest are our findings in naïve animals that baseline intrinsic activity of CINs varied depending on their location within the striatal tissue. CINs have been extensively studied electrophysiologically and can be reliably identified based on their distinctive morphological features and membrane properties ,8. HowevIt is well known that the function of CINs is largely influenced by excitatory thalamic and cortical inputs, whose glutamatergic terminals establish decreasing density gradients along the dorsomedial-to-ventrolateral and the medial oblique axes, respectively ,28,41. IAn additional level of complexity regarding the functional organization of the striatum is its segregation into patch (striosome) and matrix compartments . This clThe functional investigation of neuronal circuits and systems is a fast-growing area in neuroscience, and it requires detailed information on the spatial organization and connectivity of full neuronal networks within the brain. In this study we have undertaken an imaging approach that provides an exhaustive representation of the distribution and baseline activity of a large number of cholinergic interneurons throughout the striatum of the mouse, thus expanding our understanding of the striatal cholinergic network, a critical system within the basal ganglia. The comprehensive data generated from this and other neuroanatomical studies, combined with genetic approaches for specific neuronal activity monitoring and manipulation, should contribute to generate a realistic functional connectome of the striatum, which will help deepen our knowledge of basal ganglia function.


Cerebrospinal fluid (CSF) leak is the most commonly encountered perioperative complication in transsphenoidal surgery for pituitary lesions. Direct closure with a combination of autologous fat, local bone, and/or synthetic grafts remains the standard of care for leaks encountered at the time of surgery as well as postoperatively. The development of the vascularized nasoseptal flap as a closure technique has increased the surgeon’s capacity to correct even larger openings in the dura of the sella as well as widely exposed anterior skull base defects. Yet these advances in the technical nuances for management of post-transsphenoidal CSF leak are useless without the ability to recognize a CSF leak by physical examination, clinical history, biochemical testing, or radiographic assessment. Here, we report a case of a patient who developed a CSF leak 28 years after transsphenoidal surgery, precipitated by a robotic-assisted hysterectomy during which increased intra-abdominal pressure and steep Trendelenberg positioning were both factors. Given the remote nature of the patient’s transsphenoidal surgery and relative paucity of data regarding such a complication, the condition went unrecognized for several months. We review the available literature regarding risk and pathophysiology of CSF leak following abdominal surgery and propose the need for increased vigilance in identification of such occurrences with the increasing acceptance and popularity of minimally invasive abdominal and pelvic surgeries as standards in the field. Interestingly, the CSF leak developed in the immediate postoperative period following the robotic procedure, separated by 28 years from the initial TSS, prompting our review of potential pathophysiological mechanisms by which such a CSF leak could occur.The patient is a woman who underwent a sublabial transsphenoidal approach for resection of a pituitary adenoma at the age of 30. An autologous abdominal fat graft was applied at the sellar floor secondary to intraoperative observation of a CSF leak. She tolerated the procedure well and had an uneventful postoperative course. The pathology of the lesion was consistent with a nonfunctional pituitary macroadenoma. At the age of 58, the patient underwent a robotic-assisted total hysterectomy and oophorectomy due to persistent menorrhagia. During the procedure, the operative table was placed in extreme Trendelenberg position, noted to be approximately 45° from the floor . In the 13The causes of CSF fistulae of the anterior skull base are varied, ranging from infection to tumor invasion and, quite commonly, surgical/intraoperative leak. The majority of the latter are immediately recognized by the surgical team and repaired via autologous fat graft, artificial dural substitute, vascularized nasoseptal flap, or any combination of the aforementioned. More insidious CSF fistulae caused by infection or tumor are also managed in the same manner, usually using an endoscopic endonasal approach for identification and repair, albeit more delayed in initial diagnosis than a witnessed intraoperative CSF leak. Adjuncts to treatment such as intrathecal injection of fluorescent dyes or placement of a lumbar drain aid in both identification and repair of occult CSF leaks where defects in the anterior skull base cannot be clearly seen on radiographic imaging.15 Unlike postsurgical CSF leaks, the treatment of traumatic CSF leaks associated with anterior skull base fractures can range from immediate surgical repair to conservative management, as the rate of spontaneous resolution can be up to 60% of cases with conservative management including placement of a lumbar drain for temporary CSF diversion.16 Larger defects in the skull base consistently require surgical repair, using an endoscopic or open approach.18Traumatic injury to the anterior skull base resulting in CSF rhinorrhea complicates approximately 2% to 3% of all head trauma cases.1921 Unfortunately, the resultant dependencies of the head and neck often manifest in postoperative facial edema, with the risk being higher in cases with longer operative times.23 Similarly, the risk of increased intraocular pressure (IOP) makes such positioning, and the procedures requiring such position, a relative contraindication for patients with previously diagnosed glaucoma.24 Studies of the direct measurement of IOP under steep Trendelenberg and pneumoperitoneum have shown a reversible increase in IOP, with operating room table angulation having a greater impact on the degree of IOP increase than the degree of pneumoperitoneum.25 Although IOP is not a reliable predictor of intracranial pressure (ICP), the venous hypertension caused by the head down position leading to increased IOP also serves to increase the intracranial pressure.27 Such increases in ICP have a clear association with the development of a spontaneous CSF fistula as well as compromise of a preexisting skull base defect.28The advancement of minimally invasive approaches to intra-abdominal and pelvic surgeries has been dependent on the development of improved endoscopic tools as well as optimization of patient preparation, including anesthetics and positioning. While pneumoperitoneum creates the necessary working space for the placement of surgical instrumentation, patient positioning allows for passive manipulation of viscera out of the surgical field, often requiring extreme angulation of the operating table. Steep Trendelenberg, usually defined as a 45° angle from the horizontal plane, is considered a standard position for many robotic and laparoscopic surgeries, including minimally invasive gynecologic and urologic procedures, such as hysterectomies and prostatectomies.26 Intraoperative measurements of ICP in multisystem trauma patients undergoing laparoscopic procedures without significant Trendelenberg can result in an increase in ICP ranging from 9 to 60 mm Hg within 10 minutes.29 In patients with ventriculoperitoneal shunts for hydrocephalus who undergo a subsequent laproscopic procedure, evaluation of the ICP revealed an increase during maximal insufflation. Recommendations from this work include that special attention be given to shunted patients with an intraperitoneal catheter during laparoscopic surgery, although several groups have shown such use of laparoscopy without neurological injury.3033 Additional data from large animal studies have confirmed the relationship between increased abdominal pressure and the resulting increase in intracranial pressure during pneumoperitoneum.3436Even in the absence of steep Trendelenberg, pneumoperitoneum can cause an independent increase in intracranial pressure. In cases of intra-abdominal hypertension (IAH), ICP has been found to be elevated.38 The normal mucosal regrowth that occurs following such repairs is believed to create permanent barrier across the defect, hence the importance of the presence of healthy mucosa within the sinuses during the healing process.40 Hadad et al41 described the early use of the local nasoseptal flap to cover defects in the skull base and prevent CSF fistula progression. In this setting, the vascular supply from the tissue pedicle alleviates need for mucosal in-growth across a defect. Yet, even in the setting of a vascularized flap closure, a repair of the CSF fistula may fail, usually due to persistently increased intracranial pressure, requiring the need for CSF shunting.42 No clinical data exist regarding transient increases in ICP and recurrence of CSF leak. Yet, despite this fact, prolonged increases in ICP, that is, over 1 to 3 hours, as would be expected during steep Trendelenberg and pneumoperitoneum, could reasonably result in adequate disruption of a “healed” CSF fistula with resultant postoperative CSF rhinorrhea as demonstrated by the current clinical case.Improvements in graft material and the development of dural sealants have advanced the ability to definitively repair a CSF fistula, regardless of its etiology. Several retrospective studies have determined that repairs with such material remain viable indefinitely.4345 Both steep Trendelenberg and pneumoperitoneum play pivotal roles in the technical execution of such procedures, with the additional risk of a transient increase in intracranial pressure, which, in the absence of a preexistent skull base defect, may not be clinically significant. Patients with known surgical histories including transsphenoidal and anterior skull base procedures should be considered at relatively increased risk for developing a postoperative CSF fistula following the application of steep Trendelenberg and/or pneumoperitoneum. In this patient population, reduction in Trendelenberg angle, minimization of pneumoperitoneum, or exploration of alternative surgical approaches may be of benefit to the patient.Laparoscopic and robotic-assisted abdominal and pelvic surgeries have been shown to be safe and effective approaches for abdominal and pelvic surgeries based on both perioperative and short-term outcomes, although long-term outcome studies are still pending.


Cerebrospinal fluid (CSF) biomarkers, although of established utility in the diagnostic evaluation of Alzheimer’s disease (AD), are known to be sensitive to variation based on pre-analytical sample processing. We assessed whether gravity droplet collection versus syringe aspiration was another factor influencing CSF biomarker analyte concentrations and reproducibility.Standardized lumbar puncture using small calibre atraumatic spinal needles and CSF collection using gravity fed collection followed by syringe aspirated extraction was performed in a sample of elderly individuals participating in a large long-term observational research trial. Analyte assay concentrations were compared.p = 0.10; t-tau 83.9 (46.6) vs 84.7 (47.4) p = 0.49; p-tau 43.5 (22.8) vs 40.0 (17.7), p = 0.05). The mean duration of collection was 10.9 minutes for gravity collection and <1 minute for aspiration.For the 44 total paired samples of gravity collection and aspiration, reproducibility was high for biomarker CSF analyte assay concentrations (concordance correlation [95%CI]: beta-amyloid1-42 (Aβ42) 0.83 [0.71 - 0.90]), t-tau 0.99 [0.98 - 0.99], and phosphorylated tau (p-tau) 0.82 [95 % CI 0.71 - 0.89]) and Bonferroni corrected paired sample t-tests showed no significant differences (group means (SD): Aβ42 366.5 (86.8) vs 354.3 (82.6), Our results demonstrate that aspiration of CSF is comparable to gravity droplet collection for AD biomarker analyses but could considerably accelerate throughput and improve the procedural tolerability for assessment of CSF biomarkers. In 2011, proceedings from the National Institute on Aging - Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease (AD) recommended the addition of biomarkers as an important component of diagnosis .The CSF in established AD is characterized by a decrease in Aβ42 and increases in total tau (t-tau) and tau phosphorylated at threonine 181 (p-tau). The diagnostic performance of these CSF biomarkers to discriminate AD from non-demented older individuals is high, with sensitivity and specificity figures of 80 %–90 % . AdverseConsiderable variability in absolute concentrations of AD biomarkers has been reported by different centers using the same assay, leading to different cutoff values . A qualiA key issue which was not considered in the ABSI statement was the methodology for CSF collection. During LP CSF can be collected by allowing it to drip into the collecting tube (gravity drip) or by aspiration with a syringe (syringe aspiration). Proponents of gravity drip maintain that when a syringe is used to aspirate CSF, the extra surface area of the syringe (even when it is polypropylene) may adsorb analytes and thus influence assay results, while others believe that if a suitable polypropylene syringe is used, the resulting assays for the biomarkers of AD will not be affected. Consequently, some investigators use gravity flow in the belief that this will minimize this effect . GravityTo investigate potential effects of collection methodology we compared gravity drip collection (directly from the needle) with negative pressure aspiration using a polypropylene syringe when assaying AD CSF biomarker concentrations, during the same lumbar puncture for each subject. All other pre-analytical and analytical aspects of the procedures were identical. We hypothesized that analyte concentrations would be in agreement between techniques.A total of 54 participants , eight with mild cognitive impairment (MCI) and eight with AD) from the Australian Imaging Biomarker and Lifestyle (AIBL) study of aging were evaluated using standardized LP. Description of the psychometric test battery and characterization of AIBL participants at baseline has been reported previously . All protemena.com) spinal needle micro-tip (22/27G x 103 mm) (CAT 21922–27). If there was difficulty using this fine needle, a RapID set pencil point spinal needle, 25G  was used. The procedure was constrained by the AIBL CSF collection protocol which required gravity drip collection, so all aspiration samples for this study were taken after gravity drip collection. Up to 6 mLs of CSF was initially aspirated for routine microbiological and biochemical assessment, as well as concurrent studies, after which 8 mLs of CSF was collected by gravity flow into a 15 mL polypropylene tube (Greneir Bio-One188271), and placed immediately onto wet ice. At the completion of the gravity collection, a polypropylene syringe  was then used to aspirate 2 mL of CSF, which was then transferred to a polypropylene tube on ice. Samples were processed within one hour and kept at 4 °C during transport to the laboratory. The CSF was centrifuged  and the supernatant transferred to a new polypropylene tube and gently inverted. The CSF was then aliquoted in 300 μL volumes into Nunc cryobank polypropylene tubes (NUN374088). Samples were transferred to dry ice immediately, and then into liquid nitrogen vapor tanks within one hour and only thawed once immediately before analysis. All participants were contacted by telephone the following day, and also provided a phone number if there were any concerns such as headache or backache.CSF was collected by LP in the morning from overnight fasted participants using protocols aligned with the ABSI . All LPs2O or supplied diluents according to the kit instructions. The filter plate was washed once using 225 μL/well of 1x wash buffer and vacuum aspirated immediately before use. A total of 100 μL of bead suspension  was added to each well and the plate vacuum aspirated. Then 25 μL of the conjugate working solution was added to each well. A total of 75 μL of standards, kit and pooled CSF controls and samples were added to the plate in duplicate. A buffer blank was also included. The filter plate was sealed, the bottom of the plate was dabbed dry and the plate was wrapped in aluminum foil and incubated on an orbital plate shaker at room temperature (21–23 °C). The plate was incubated overnight (at least 14 hours). On the second day, the remaining reagents were prepared at room temperature. The filter plate was aspirated and then washed with 225 μL of 1x wash buffer, three times. A total of 100 μL of diluted detection reagent was then added to each well and the plate re-covered in foil for one hour on an orbital plate shaker. The filter plate was aspirated and washed with 225 μL of 1x wash buffer, three times. Then, 100 μL of read solution was added to each well and the plate was finally incubated on an orbital plate shaker for five minutes covered in foil, at room temperature. The sample concentrations were then quantified using a Bio-Rad Bioplex 200 instrument  using 5PL logistic regression. The data were fitted to calibration curves constructed with the median fluorescence values for each replicate of the standards. Concentrations were determined by sigmoidal curve fitting. Data from duplicate sample measurements for each individual’s CSF sample that had a percentage coefficient of variance (%CV) above 20 % (as recommended by the manufacturer) were discarded and the samples were reanalyzed. For all plates, the internal standard control duplicate analyses were within the accepted %CV.All samples were measured in duplicate using the AlzBio3 xMAP assay  according to the included protocol for human CSF Aβ42, t-tau and p-tau. A total of four assay kits were used to collect the data spread over several days, and all steps were conducted by the same operator blinded to participant and CSF collection method, using multichannel pipettes and a manual wash/vacuum manifold. The same kit batch-number was used for all assay plates used in the study and the participant samples were distributed randomly on the analysis plates. Briefly, all reagents, standards, controls and samples were brought to room temperature and vortex-mixed immediately before the assay. Coated beads were vortexed for three minutes in a sonicating water bath. All working solutions were diluted in Milli-Q Hp-value assessment, with test results compared against 0.0167 (0.05/3). Statistical analyses for concordance and sample mean differences were performed on the original set of 35 samples where the %CV was less than 20 %, as well as the complete set of 44 samples to determine whether there were any comparable differences between the original assay set and the repeated samples. Linear models between CSF collection methods were compared using analysis of variance. Demographic characteristics were assessed using the independent samples t-test (age), Chi square analyses (APOEε4 and sex), and Kruskal Wallis tests  score and Mini–Mental State Examination (MMSE) score). All statistical analyses were conducted using the R software version 3.0.2.Biomarker agreement was assessed using the Concordance Correlation test, with a correlation coefficient close to 1 representing perfect repeatability. Bland-Altman plot analysis was conducted to assess agreement between methods . To detep = 0.40) or in the distribution of males and females between clinical classifications (p = 0.41). There were more APOEε4 carriers in the AD group; however, this was not statistically significant (p = 0.20). As expected, the CDR scale was significantly higher and the MMSE score significantly lower in the MCI and AD groups (p < 0.0001). The mean inter-assay %CV based on the lowest concentration included standards was 11.6 % for Aβ42, 7.26 % t-tau, and 11.29 % for p-tau.Of the 54 participant samples, 19 failed quality control for one or more of the multiplexed analytes, with %CV greater than 20 %. Of these 19, nine samples were re-run and performed with < 20 % %CVs and were included in the final analysis using a total of 44 participant samples. Demographic characteristics of the participants are shown in Table Using the complete sample (n = 44), concordance correlations between CSF collection methods showed strong reproducibility for Aβ42, t-tau, and p-tau . Reducing the sample to only those with a < 20 % %CV in the original assay (n = 35) performed similarly compared to that of the complete sample . Bland-Altman plots also displayed good agreement between the two collection methods Fig. , i. Tablp = 0.10; t-tau: gravity: 83.9 (46.6) vs aspiration: 84.7 (47.4), p = 0.49; and p-tau gravity: 43.5 (22.8) vs aspiration: 40.0 (17.7), p = 0.05). Analyses of the reduced set (N = 35) found similar results for each of the analytes (Aβ: gravity: 373.4 (88.0) vs aspiration: 366.5 (84.8) p = 0.35; t-tau: gravity: 82.8 (48.5) vs aspiration: 82.3 (48.7), p = 0.64; and p-tau gravity: 41.9 (22.7) vs aspiration: 38.5 (18.6), p = 0.07). Assessment of intra-classification means showed only small changes in biomarker level between gravity and aspiration collection methods; t-tests were not performed due to low sample size  in mean Aβ42, t-tau, and p-tau levels between gravity-fed and aspiration collection methods (Aβ: gravity: 366.5 (86.8) vs aspiration: 354.3 (82.6) ze Table . Gravityze Table .th and 90th percentiles . Using this method, only one participant was classified differently between collection methods (with a 6 % difference between collection methods).We also classified each participant using the standardized cut-off scores of our laboratory  and dichotomized using the 10In four of the 54 participants, it proved difficult to find the CSF with the fine #27 gauge needles and the 25 gauge pencil point needle was successfully employed. Of the 54 subjects, two complained of mild headache when questioned the following day by telephone follow-up. Both headaches resolved quickly with mild analgesics . A further participant complained of nagging back pain, but had suffered back pain for many years and the pain following the LP was not clearly different to pre-existing pain. The duration for CSF extraction was a mean of 10 range 5–23) minutes. A total of 16 mLs was taken from all participants except one in whom only 11 mL was extracted because of slow gravity drip (22 minutes) during which the participant became tired  due to increased assay variation, causing a reduced agreement statistic as compared with t-tau. Analyses of the three biomarkers via paired samples t-tests showed no significant difference between collection methods; however, there was a trend towards a difference in p-tau levels (largely driven by the AD group) prior to adjustment for multiple comparisons. We included MCI and AD controls to address the issue of varying HC levels of AD pathology potentially influencing the dynamic levels of each specific analyte or the way they behave Importantly, all CSF samples were taken sequentially from the same LP and all analyzed simultaneously. The CSF samples were thus not subjected to any variation in pre-analytical handling other than the method of collection and, furthermore, were not exposed to technical variations, which may have contributed to previously seen variation observed between laboratories, underscoring the accuracy of the results.The method of CSF collection is an important step in the pre-analytical handling of CSF samples. While some investigators routinely use gravity, others use aspiration. Gravity drip has the drawback of unpredictable variation of collection times and may potentially take considerably longer than aspiration, thereby reducing feasibility in busy clinics. In dementia evaluation settings, the longer duration of CSF acquisition can be a particular problem for a patient with memory impairment or dementia since repeated reassurance and explanation may be required. Our results demonstrate that syringe aspiration does not have a significant effect on analyte concentrations and, therefore, should be acceptable and allow predictable and more rapid CSF collection.In our study, post lumbar puncture headaches using the #27 and #25 gauge needles occurred in 2/54 procedures (4 %) of patients consistent with previous reports when using small caliber needles . This isReasons for a preference of gravity flow collection over aspiration in some centers may be historical. A belief that aspiration may cause radicular pain by traumatizing local nerve roots  appears To our knowledge, this is the first report of concordance of CSF biomarkers when CSF is collected either by standard gravity drip or syringe aspiration. In particular, more rapid collection by aspiration suggests that wider adoption of aspiration is feasible and may become the preferred means of CSF collection for the detection of AD CSF profiles. Shorter duration should help facilitate acceptance both by patients undergoing the procedure and also staff conducting the LP. Indeed, the implications of reducing the time of the extraction of the CSF from around ten minutes for gravity feed to approximately one minute for aspiration, may allow the procedure to be suitable for mass screening as advocated by Herskovits .These results also indicate that the use of an extra polypropylene contact step (in the syringe) used in the aspiration technique made no significant difference to the CSF biomarker concentrations, despite prior concerns . With thA limitation to the study was that all aspirations were performed subsequent to gravity drip collection. This was because the protocol for the AIBL study demanded that samples be taken by gravity drip, and, therefore, it was only after the required samples had been obtained for AIBL that we were able to take the aspiration sample. Although we consider it unlikely that reversing the CSF collection technique order will significantly influence analyte concentrations, formal assessment of this issue could be the subject of future studies.A further limitation in this study was the relatively small sample size, with varying proportions of variation per analyte, and insufficient power to evaluate analyte concordance within clinical classifications. Further studies with larger samples sizes are required to evaluate whether clinical classification influences correlations between methods.The results of this study indicate that there is no significant difference in CSF AD biomarker concentrations for Aβ42, p-tau, and total tau between gravity collection and polypropylene syringe aspiration, yet aspiration is much quicker. We conclude that aspiration should be considered as the favored collection technique for CSF AD biomarker evaluation as this technique becomes more widely adopted throughout the world.


Background: Serum troponin elevation, characteristic of ischemic myocardial injury, has been observed in some acute ischemic stroke (AIS) patients. Its cause and significance are still controversial. The purpose of this study is to find determinants of troponin elevation and its relationship with stroke severity and location.Methods: Between January 2013 and August 2013, 114 consecutive AIS patients confirmed by diffusion-weighted magnetic resonance imaging were recruited in this study. Serum troponin T level was measured as part of routine laboratory testing on admission. Ten lead standard electrocardiogram (ECG) was performed and stoke severity was assessed based on National Institutes of Health Stroke Scale (NIHSS).Results: Troponin T was elevated in 20 (17.6%) of 114 patients. Patients with elevated troponin were more likely to have higher age, higher serum creatinine and ischemic ECG changes. Troponin levels were higher in patients with more severe stroke measured by NIHSS [7.96 (6.49-9.78) vs. 13.59 (10.28-18.00)]. There was no association between troponin and locations of stroke and atrial fibrillation. There were 6 (5%) patients with elevated troponin in the presence of normal creatinine and ECG.Conclusion: Stroke severity, not its location, was associated with higher troponin levels. Abnormal troponin levels are more likely, but not exclusively, to be due to cardiac and renal causes than cerebral ones. Previous studies have shown that troponin is elevated in 10-30% of acute stroke patients.4-6 This rise can be due to concomitant coronary artery disease and myocardial infarction (MI), congestive heart failure, renal insufficiency or direct neurogenic myocardial injury.7,8 Some researchers have found association between troponin level and location and size of infarction, severity of stroke [measured by National Institutes of Health Stroke Scale (NIHSS)], ischemic electrocardiogram (ECG) changes and increased mortality.9-12 The purpose of this study is to investigate the relationship between cardiac troponin T and severity and location of a stroke.Troponin is a sensitive marker of myocardial injury.Subjects with a diagnosis of acute ischemic stroke (AIS) presenting to Shariati Hospital Tehran, Iran from January 2013 to August 2013 were enrolled. Stroke patients were diagnosed according to World Health Organization definition  and were diagnosed as AIS if their brain computed tomography (CT) scan was normal or showed acute ischemic changes. Ischemic stroke was confirmed by showing diffusion restriction on diffusion-weighted imaging magnetic resonance imaging (MRI) using Siemens Magnetom Avanto 1.5 Tesla . When MRI could not be performed (e.g. cardiac pacemaker), acute stroke was confirmed by showing new hypodensity on repeat brain CT scan 4 days later. Stoke severity was assessed based on NIHSS.Serum troponin T was measured as part of routine laboratory testing on admission. Levels of Troponin T were measured by Elecsys and cobas e analyzer (Roche diagnostics) and was considered abnormal if it was ≥ 24 ng/l. Ten lead standard ECG was performed and repeated every hour if there was any sign of ischemic changes .Descriptive statistics was shown as mean. P-P plot  and Kolmogorov–Smirnov test were used to assess normal distribution violation of variables. T-test and Kruskal–Wallis test, when non-parametric test had to be used, were used to compare groups in their troponin levels. Spearman correlation coefficient was used to assess the correlation between NIHSS and troponin level. Univariate general linear model was used to control for confounding variables in assessment of the association between stroke severity and troponin level. P < 0.500 was considered as significant. All analyses were done using SPSS software .A total of 120 AIS patients were enrolled. After review of patients’ records, 6 of them were excluded because their troponin levels were not measured. Compared with included patients, they were non-significantly younger (59.67 ± 17.33 vs. 66.34 ± 14.98), more masculine (83.3 vs. 55.3%), and had non-significant less severe strokes (6.17 ± 5.04 vs. 8.18 ± 6.59).Mean of serum troponin was 22.61 ± 43.63. It was abnormal in 20 (17.5%) patients. Although dichotomizing NIHSS did not yield significant association between this variable and serum troponin, Four stroke variables which had P < 0.200 in univariate analyses were entered in a multivariate analysis after exclusion of subjects who had either abnormal creatinine levels or ischemic ECG changes. Only NIHSS remained a significant predictor of troponin levels. After adjustment by age and sex, NIHSS was still a significant predictor (P = 0.002). However, the effect of NIHSS on troponin level was too small in a way that mean of troponin among stroke patients with NIHSS < 10 was 7.96 [95% confidence interval (CI) = 6.49-9.78] compared with 13.59 (95% CI = 10.28-18.00) in patients with NIHSS ≥ 10. And, among subjects with ECG ischemic signs, NIHSS was not a significant predictor of troponin .There were 78 AIS subjects without any evidence of renal impairment or ECG ischemic signs. 8 of them (10%) had abnormal troponin levels; 6 of them were older than 70 and 4 had NIHSS more than 9. All 8 subjects had at least one of these predictors.13 meanwhile, IHD is the second most common cause of death after stroke.14,15 While stroke and IHD share the same risk factors , it is not unexpected to see both diseases in one patient.16 Many studies have shown elevated serum troponin in significant proportion of acute stroke patients (11-36.4%).4,17-19Stroke is the second-fourth most common cause of death, after ischemic heart disease (IHD);20-23 In our study, this association was found just between troponin T and age, renal impairment, ECG changes and stroke severity (i.e. NIHSS). 17.5% of acute stroke patients in our study had elevated serum troponin level which was congruent with previous studies. Associations, causes and value of troponin rise have been the core of many studies. It has been demonstrated that troponin elevation could be associated with higher age, more severe stroke, larger stroke, specific stroke locations, renal dysfunction, previous IHD, ECG changes and poor outcome.We did not measure infarction size, and outcome determination was not in design of our study. Renal impairment and ischemic ECG changes were greater determinant than NIHSS; in a way that in patients with high creatinine or ischemic ECG changes, effect of NIHSS on troponin was not significant. However in patients without ischemic ECG or renal problem, higher troponin was seen in patients with more severe stroke. This effect was independent and could not be explained by other factors.24 Therefore, insula and brainstem were center of attention of many researchers.25 Some studies have shown that troponin level is higher in the right insular, brainstem or middle cerebral artery territory infarction.5,10,26,27 Nevertheless, our findings along with the study of Barber and Morton. did not corroborate those results, and we found no correlation between stroke location and troponin level.28It has been hypothesized that damage to centers regulating autonomic function can cause autonomic dysregulation resulting in sympathetic overflow and neurogenic myocardial injury.7 and Scheitz et al.29 proposed that the most likely causes of increased troponin in AIS patients are silent acute MI before stroke, heart failure and renal insufficiency. Our data also show that most troponin elevations in AIS patients occurred in the context of renal or cardiac dysfunction and 3 (2.5%) of the patients had concomitant acute MI. However, there were 8 patients with elevated troponin (mean 43.75) at the presence of normal ECG and renal function. In these patients, troponin elevation could not be attributed to renal or cardiac problem, and neurogenic cardiac injury could be suspected. Nonetheless, we did not go through those cases and coronary status was not evaluated to be sure of the absence of coronary artery disease as predisposing factor for troponin elevation.Apart from associations of it, the possible causes of troponin elevation in AIS have been investigated previously. Jensen et al.According to our findings, although there are some AIS patients with possible neurogenic myocardial injury, it is prudent to be vigilant in those with high troponin and perform appropriate cardiac and renal evaluation.This was the first Troponin-Stroke relationship study in Iranian population. The number of patients (sample volume) was limited to 114, which made specific location stroke groups small, and might have made probable associations statistically insignificant. As mentioned, we did not evaluate cardiac structural (echocardiography) and coronary status; therefore the number of real neurogenic myocardial injury patients might have been over- or underestimated.Troponin T elevation in AIS patients was associated with higher age, creatinine, ECG changes and severity of stroke, but location of stroke was not a determinant factor. Cardiac and renal impairment were the cause of troponin elevation in the majority of patients; however, there are some patients with possible neurogenic myocardial injury.The authors declare no conflict of interest in this study.