Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients. To evaluate the potential role of miRs as prognostic molecular markers in ccRCC/TT patients we finally correlated the expression of selected miRs with clinico-pathological features and survival aiming towards a possible clinical use as molecular markers.This study was approved by the local human research ethics committee of the medical faculty of the University of Wuerzburg, Germany (no. 136/08) and was conducted according to the standards set by the declaration of Helsinki; all patients provided written informed consent.We collected paraffin embedded samples of 74 primary tumors of ccRCC patients. After excluding other histological histological subtypes  we used samples of 37 primary tumors of ccRCC/TT patients who consecutively underwent radical surgery at the Department of Urology and Pediatric Urology of the Julius-Maximilians-University Medical Center Würzburg between 1997–2010.th edition) by a uropathologist (AR). The level of tumor thrombus was classified according to the Mayo classification To evaluate the role of the selected miRs in development of venous involvement, a comparison collective of consecutive ccRCC cases without venous invasion (n = 37) was used. All ccRCC samples were paraffin-embedded and areas with >90% cancerous tissue were selected; likewise samples of histologically benign renal tissue were reviewed by one experienced uropathologist (AR) and used non-cancerous renal tissue as controls. ccRCC specimens were staged and graded according to the TNM classification ), abdominal CT and/or abdominal magnetic resonance imaging (MRI) and/or bone scintigraphy. Long-term follow-up data were collected during check-up visits, review of patient records and additional telephone interviews with the urologists of the patients.Total RNA extraction from paraffin-embedded samples was performed using the Recover all Total Nucleic Acid Isolation Kit and the Total RNA Extraction Kit, respectively . RNA concentration and A260/280 ratio were analysed with a Nano Drop ND-100 spectrometer  and RIN (RNA Integrity Numbers) calculated with a Bioanalyzer. RNA samples showing RIN<6.0 were excluded from further analysis. The resulting miR was retained for quantitative Real Time PCR (qRT-PCR). Specific cDNA was synthesized from total RNA with stem-loop reverse transcription primers according to the TaqMan miRassay protocol (PE Applied Biosystems).t for tumor samples and adjacent normal tissue of all miRs were calculated by the comparative Ct method. All samples characterized by expression levels of RNU6B>30 Ct were excluded from further analysis.MiR expression in tissue samples was quantified with TaqManH miRassay kits and the BioRad OPTICON 2, following the manufacturer’s instructions (BioRad). Primers for all miRs were obtained from Applied Biosystems. Cycling conditions were chosen according to manufacturer’s protocols. All reactions were performed in triplicates and samples showing SD>0.5 were excluded. Relative expression values of miRs were normalized to small nuclear RNA (RNU6b) previously described as reference gene. ΔCt) of the different miRs were multiplied by these factors using the formula (4.592× ΔCt miR-21)+(−3.892× ΔCt miR-126)+(−1.938× ΔCt miR-221). A negative factor indicates that higher expression correlates with longer survival, whereas a positive factor correlates with shorter survival. A cut-off for the risk score was again determined by ROC. Differences in mean between miR-expression and clinical parameters were analysed by Student's t-test or ANOVA, respectively.Thresholds for dichotomizing relative expressions of miRs were determined by receiver operating characteristic (ROC) curves  we used the entire collective (n = 106) including 37 cases of ccRCC/TT patients and 37 cases without venous involvement (ccRCC/woTT) as well as 32 cases of non-cancerous renal tissue. We previously excluded all renal cell cancer (RCC) histo-pathological subtypes others than ccRCC from our study collective, as it was shown that different RCC subtypes are characterized by distinct miR expression profiles To prove whether the expression profiles of the selected miRs correctly discriminate ccRCC tissue from non-cancerous tissue, we developed predictive rules using logistic regression. Therefore, we randomly divided the study collective (n = 106) into a learning data set of 58 samples containing 42 ccRCC samples and 16 samples of normal kidney tissue and a test data set containing the remaining 48 samples (32 ccRCC cases and 16 samples from non-cancerous kidney tissue). A combination of five miRs  was determined by the AIC to accurately discriminate between normal kidney and ccRCC samples (ccRCC/TT and ccRCC/woTT together) in the learning data set. The area under the curve characteristics predicted 100% sensitivity and specificity  to non-cancerous renal tissue. As shown in Next, we divided our study group into ccRCC/TT and ccRCC/woTT cases . CompariNext, we evaluated whether the expression of these eight miRs could accurately discriminate ccRCC/TT samples (n = 37) from samples without invasion into the venous system (n = 37). Therefore, we developed a new classification model by logistic regression using a learning data set containing 37 ccRCC samples (19 ccRCC/TT and 18 ccRCC/woTT cases), which was randomly selected from the entire set of our study collective. By linear regression analysis, a combination of miR-21, miR-221 and let-7b was selected. All three together contributed essentially to the predicting model. Using the expression data of these three miRs, our model accurately determined ccRCC/TT samples with high sensitivity and specificity  were significantly regulated in ccRCC/TT. Moreover, using the expression level of three miRs  we successfully identified ccRCC/TT cases with an accuracy of 94%. From these results, we concluded that among the eight selected miRs miR-21, miR-126, miR-221 and let7b might be critically involved in the development of ccRCC with TT. Several studies already gave evidence that all four dysregulated miRs are involved in biological processes controlling malignant transformation and progression of tumor cells by the identification or prediction of various target mRNAs and pathways controlled by these miRs An aggressive surgical approach is the only hope for curing ccRCC patients with any level of TT. Many reports demonstrated that a subgroup of patients with TT can achieve long term survival after aggressive surgical treatment, suggesting that TT invasion in the venous system is not necessarily associated with worse prognosis and aggressiveness of the tumor at the time of surgery. These results match our observation as our ccRCC/TT study collective contains a patient subgroup characterized by low risk for progressive disease. Nonetheless, patients with ccRCC/TT generally characterized as high risk patients having a relative poor prognosis. We observed that a significant proportion of ccRCC/TT patients developed early cancer recurrence while others could be characterized by a long term disease free survival indicating the importance of an additional risk stratification model to accurately select patients who may benefit from early and intensified adjuvant therapy. Therefore, one of the critical issues in treatment of ccRCC/TT patients is the development of an accurate predicting model system in these patients. The impact of a number of prognostic nomograms typically including clinico-pathological variables like TNM staging, tumor grade, performance status and serum blood markers  have been suggested for risk stratification in ccRCC/TT, but so far none of these models achieve the status of an independent, reliable and applicable predictor system in ccRCC/TT In summary, we successfully characterized ccRCC/TT by a distinct miR profile generated by the expression of eight selected oncogenic miRs. The used miR profile is able to accurately classify ccRCC/TT cases. Differential expression of miR-21, miR-221 and let-7b precisely separated ccRCC/TT cases from ccRCC/woTT indicating their possible function in the development of ccRCC/TT. Moreover, a CRS calculated by the expression of three onco-miRs, miR-21, miR-126, and miR-221 was generated, which accurately predicts CSS in the ccRCC/TT collective and in a small independent RCC/TT patient group. Nevertheless, the power of our conclusion may be limited by the relatively small number of ccRCC/TT cases and has to be validated in large ccRCC/TT cohorts. After further evaluation of the reliability and effectiveness of the developed prediction model, we suppose this prognostic molecular marker system will be able to carefully select RCC/TT patients to whom an adjuvant systemic therapy may be advisable.Figure S1Expression of miR-21, miR-210, miR-200c, miR-141, miR-126, let-7b miR-221, and miR-143 in ccRCC. Relative expression (ΔCt levels) of indicated miRs were measured by qRT-PCR and normalized against RNU6B and are shown as Box and Whisker-Plot. Expression of different miRs in ccRCC  are compared with non-cancerous renal tissue . * P<0.05; ** P<0.01; *** P<0.001, Student's t-test.(TIF)Click here for additional data file.Figure S2Kaplan Meier survival analysis for CSS of renal cell cancer with tumor thrombus patients (n = 37) median follow up of the study group was 41.4 months. The 5-yr CSS estimate was 43%. CRD: cancer related death; FU: follow up.(TIF)Click here for additional data file.


As a result of the detailed genetic analyses performed by certain laboratories and of the sophisticated genetic tools they developed, this locus has played a major role in our understanding of piRNA mediated TE repression in animals. Here we review the first discovery of this locus and retrace decades of studies that led to our current understanding of the relationship between genomes and their TE targets.The discovery of transposable elements (TEs) in the 1950s by B. McClintock implied the existence of cellular regulatory systems controlling TE activity. The discovery of  Drosophila melanogaster under the control of a locus called flamenco (flam) was first reported in 1983. Focused on flam, this review retraces the numerous studies that have been performed from its discovery to the understanding of its ability to survey TEs.In the 1950s, Barbara McClintock first discovered transposable elements (TEs) by analyzing genetic stocks of corn that were phenotypically unstable. Her discovery implied that a genetic control exerted by genomes was generally used to regulate TE mobilization. Any loss or decrease of this control would consequently result in severe genetic instabilities due to mobilization of TEs. Just such a genetic instability affecting the genome of ovoD mutation in D. melanogaster . The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. The gain of function ovoD allele results from an extension of the N-terminal region which gives rise to a neomorphic protein that causes female sterility  carrying a y v f mal X-chromosome . In the progeny, reversions of the ovoD mutation generating recessive ovo alleles were frequently observed which allowed fertile daughters to be recovered. Surprisingly, these reversions were also associated with the appearance of mutations in other loci, which could potentially be explained if such crosses were accompanied by the de novo mobilization of TEs. gypsy insertions was observed in the progeny of this cross and that a hot spot for gypsy exists into the ovo locus . Insertions of gypsy into the ovo locus interfere with the coding sequence of the neomorphic allele resulting in a null allele of the gene. Novel gypsy insertions can thus be assayed by the presence of fertile daughters. The gypsy mobilization could then explain both the genetic instability observed in these crosses and the ovoD reversion.In the 1980s, terility . Busson D. melanogaster characterized by an elevated frequency of spontaneous mutations in the germ line up to 10-3 - 10-4. Mutations were recovered in both sexes and displayed the characteristics of being unstable with frequent reversion to wild type or to new mutant states. When analyzing the localization of a battery of TE families, they found that the genomic distribution of P, mdg1, 412 (mdg2), mdg3, and copia did not vary among the individuals of this strain. However, this was not the case for gypsy (mdg4) whose frequency of transposition was high and copy number greatly increased to 30–40 copies.Also, gypsy insertions is high while several other TE families remain stable  of loci regulating their transposition  because it had the ability to make gypsy “dance.” Non-permissive or permissive alleles of flam were defined according to their ability to restrict or allow gypsy mobilization, respectively. A fine-scale analysis of flam genetic characteristics uncovered that: (i) Its control on gypsy activity occurs under a strict maternal effect since transposition is only allowed in the progeny  of homozygous permissive females even if fathers are non-permissive. (ii) The mutant allele present in the MS strains is essentially recessive. (iii) Transposition is largely a premeiotic event. (iv) Although ovoD reversion is primarily controlled by flam, it is influenced by other factors such as age and temperature, reversion being higher in young flies grown at 25°C. (v) The effects of flam on gypsy expression are restricted to the somatic follicle cells that surround the maternal germline  of ZAM or Idefix linked to the LacZ reporter gene. These transgenes provided a convenient read-out for analyzing the control exerted on these elements. Crosses designed to test the influence of the genetic background on these reporter constructs indicated that ZAM and Idefix responded to two types of controls: one restricting their expression to specific somatic cells of the ovaries and the other silencing their expression in the majority of Drosophila lines with only one exception reported in 2003 as being the Rev line  triggering gene silencing and repressing their own transcription  tandem repeats in cription . They ald Idefix . To illud Idefix .gypsy did not contain a single binding region for a putative flam repressor  appeared to be able to target the repression, the only requirement being that gypsy sequences were present within the tested transcript. In addition, gypsy repression was impeded by piwi mutations. Short RNAs from 25 to 27 nucleotides long were also detected. These small RNAs, homologous to sequences within the gypsy 5′ UTR, should be able to guide RNA silencing complexes to gypsy-containing transcripts. In line with growing body of evidence implicating RNA silencing mechanisms in regulating TE activity, these data supported that flam could possibly act through a RNA-dependent mechanism.epressor . They fiflam and COM had been identified proved to be very difficult. Uncertainty in the assembly of repetitive DNA in the early releases of the D. melanogaster genome sequence posed difficulties for heterochromatin studies. As a consequence, flam localized to a sequencing gap in the Release 1 genome sequence  displaying flexibility in its potential to repress different TE families.Cloning of the heterochromatic locus where sequence . The groanalyzed . Robert ccessful . Robert epressed . These fflam region became possible due to improved genome sequence data . The use of flam mutations, P(KG00476) and P(BG02658), indicated that a substantial reduction in piRNAs that uniquely map to flam was observed in mutant flies whereas piRNAs derived from other piRNA clusters were unaffected. This reduction of flam piRNAs was accompanied by a loss of flam transcripts and a high increase of the gypsy retroelement transcription.A significant breakthrough for all RNAs . Their mFrom this piRNA sequencing, ZAM, Idefix, and gypsy had indeed been shown to be active and consequently repressed by flam only in the somatic follicle cells  and showed that its transcriptional activity requires the transcription factor, Cubitus interruptus  was found to be constitutively expressed since it is always present within the processed RNAs. Furthermore, when publicly available RNA-seq libraries were interrogated  are present as a unique copy. This observation supports a key prediction of the transposon trap model that postulates if a TE family is silenced as soon as it inserts flam, it should be present only once in the locus. This study also highlighted the high structural dynamics of this locus because numerous differences resulting from deletions, insertions or duplications were identified between different lines. In addition, sequence analysis of the flam TEs indicated that many of them correspond to TEs that recently inserted the locus. Among them, 12 new TEs were identified. Interestingly, eight of them were found closely related to TEs from D. simulans, D. sechellia, D. yakuba, or D. erecta, consistent with a recent origin from horizontal transfers that occurred between species belonging to the melanogaster subgroup  and Rev (restrictive for gypsy but not ZAM or Idefix). Importantly, a deletion of the region comprised between X:21638001 and 21684449 was found in Rev that encompasses the unique ZAM and Idefix copies present in flam. This observation provides the first evidence that a strict correlation exists between the presence or absence of TE sequences  within flam locus and repression or activity of that particular TE family. These new data highlight how structural variations in piRNA clusters impact the genomic TE distribution across the rest of the genome.To determine what underlies the difference between flam fit with a model of TE invasion and its subsequent genomic control as follows (Figure trans to silence their homologous copies. When this occurs, genomic stability is recovered. Due to their highly repetitive nature, piRNA clusters may subsequently undergo deletion events removing small or large portions of the locus. These deletions can remove TE sequences and may result in sudden bursts of transposition. Thus, periods of stability and instability in global TE dynamics will reflect the mutational events that affect piRNA clusters.Overall, data obtained on s Figure . The besflam locus provides an RNA-mediated adaptive immunity against TE invasions. Interestingly, this system in Drosophila shares striking resemblances with the CRISPR system developed by bacteria to fend off invaders (flam and CRISPR loci, these sequences remaining from invasions are transferred to the progeny in which they constitute genetic marks reflecting environmental changes over time.Conceptually, this dynamics of the invaders . CRISPR flam, it is interesting to measure how far we have gone since that time where heterochromatin was considered as a graveyard for TEs. Today, TEs and piRNA clusters in heterochromatin are thought to play fundamental roles in the organization and stability of genomes. The high structural dynamics of flam and potentially of the other piRNA clusters appears as a formidable evolutionary tool to remodel both euchromatic and heterochromatic regions, or even to play a role in speciation (After 40 years of data obtained on eciation , by its flam lead knowledge of heterochromatin function in the years to come?How far will further work on 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.


G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In  Skeletal muscle atrophy is a devastating condition that is characteristic of neuromuscular diseases such as Duchenne muscular dystrophy (DMD) and many chronic diseases including cancer, heart disease, chronic obstructive pulmonary disease (COPD), sepsis and AIDS  is a cytokine that stimulates haematopoietic stem cell mobilization, myelopoiesis, and particularly the production and activation of neutrophils  on target cells. G-CSFR is highly expressed on haematopoietic stem cells and various myeloid cells . Skeletal muscle samples were obtained under local anaesthesia (1% Xylocaine) from the belly of the tibialis anterior (TA) muscles from 8 to 9 week old C57BL/10 and mdx mice were excised as part of a previously published study  supplemented with 10% (v/v) foetal bovine serum (FBS) . For myotube differentiation experiments, C2C12 were seeded at a density of 150 cells per mm2 and incubated as above. G-CSF was used at concentrations between 0.4 and 100 ng/ml as published previously  were seeded at a density of 50 cells per mm2 in DMEM containing 10% (v/v) FBS and 10% (v/v) conditioned WEHI-3B medium. HEK293T cells (ATCC) were maintained at 37°C, 5% CO2 in high glucose DMEM supplemented with 10% (v/v) FBS (Invitrogen). Human primary muscle cell cultures were established as previously described in our laboratory  FBS or 2% (w/v) BSA, which was replenished every 24 h. After 24, 48, 72, and 96 h C2C12 myoblasts were incubated with 0.1 μg/ml 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI)  for 10 min at room temperature. Images were obtained using the Olympus Fluoview FV10i confocal laser scanning microscope with dedicated software at a 10 x magnification. A minimum of ten images per group were analyzed using ImageJ Software  to quantify the number of nuclei per image.2 for 30 min. Cells were again washed and incubated with anti-BrdU-POD Fab fragments for 30 min at 37°C. After a final series of washes, peroxidase substrate with substrate enhancer was added to the wells and the substrate left to develop in the dark for 5 min before the absorbance was determined at a wavelength of 405 nm.Myoblasts were assayed for DNA synthesis using a 5-bromo-2'-deoxyuridine (BrdU) Labeling and Detection Kit III  according to the manufacturer's protocol. Briefly, following 24 and 48 h of BrdU labeling (10 μ M), myoblasts were fixed in 70% ethanol in 0.5 M HCl at −20°C for 30 min. Following a series of PBS washes containing 10% FBS, cells were incubated with the supplied nucleases at 37°C in the absence of COWhen myoblasts were confluent, DMEM containing 2% HS ± G-CSF was added to induce differentiation. Images were obtained every 24 h for 4 days using an Olympus IX70 microscope , and an attached DS-U3 microscope camera with NIS-Elements imaging software  to assess differentiation. At the same time points RNA was extracted from the cells and RT-PCR was performed on genetic markers of differentiation. The media ± G-CSF was replenished every 24 h throughout the experiment.TRI-Reagent  was used to extract RNA from each cell culture sample or approximately 20–40 mg wet weight of muscle, with RNA concentrations determined using a NanoDrop® ND-1000 spectrophotometer . The RNA was reverse transcribed (RT) to synthesize first strand cDNA using a high-capacity RNA-cDNA reverse transcription kit . Each sample was incubated with one unit of ribonuclease H (RNAse H) (Invitrogen) at 37°C for 30 min following reverse transcription.A mixture containing 0.5 × SYBR® Green PCR Master Mix (Applied Biosystems) and the forward and reverse primers for the gene of interest Table  was addeIn some experiments, the resulting PCR products were separated on a 1.8% agarose gel containing 0.5 × SYBR® Safe DNA Gel Stain (Invitrogen). Images were captured using the Kodak Gel Logic 112 image station  under 302 nm UV light. The cDNA fragments were purified using the QIAquick Gel Extraction Kit  according to the manufacturer's protocol. The purified cDNA from BAF/3[G] and C2C12 myoblasts and C2C12 myotubes was sequenced by the Australian Genome Research Facility .Myoblasts and myotubes were serum starved in DMEM (Invitrogen) for 4 h followed by stimulation with serum free media ± G-CSF. Total protein was extracted after 5, 10, 15, 30, and 90 min and phosphorylation was compared to basal levels following 4 h of serum starvation.Total protein was extracted from muscle tissue and cell culture using 1x radioimmunoprecipitation (RIPA) buffer  with 1 μL/mL protease inhibitor cocktail  and 10 μL/mL Halt Phosphatase Inhibitor Single-Use Cocktail . Samples were centrifuged at 13,000 rpm for 15 min with the protein concentration of the supernatant determined from the development of a standard curve using the bicinchoninic acid (BCA) assay Protein Assay Kit  according to the manufacturer's protocol.Protein deglycosylation was performed using the PNGase F  according to the manufacturer's protocol. In brief, 20 μg total protein was denatured in 1 × glycoprotein denaturing buffer at 100°C for 10 min. Following denaturing 1 × G7 reaction buffer, 2 μl 10% (v/v) NP-40 and 2 μl PNGase F was added to the protein sample. The sample was incubated using a water bath at 37°C for 1 h. The samples were frozen at −80°C for Western blot analysis.Electrophoresis was performed using a 4–12% NuPAGE® Novex Bis-Tris Gel in NuPAGE® SDS MOPS Running Buffer (Invitrogen). Proteins were transferred to a PVDF membrane (Millipore) in a Bjerrum buffer containing 50 mM Tris, 17 mM glycine and 10% (v/v) methanol. The membranes were blocked with 5% BSA in PBS, after which they were incubated overnight at 4°C with anti-G-CSFR antibody (1:1000) , anti-phospho-Akt (Ser473), anti-phospho-STAT3 (Tyr705), anti-phospho-Erk1/2 (Thr202/Tyr204), anti-Akt, anti-STAT3 and and anti-Erk1/2  diluted in PBS containing 5% BSA. Following washing, the membranes were incubated with goat anti-rabbit IgG antibody (1:5000) labeled with an infrared-fluorescent 800 nm dye  in PBS containing 50% Odyssey ® blocking buffer  and 0.01% (w/v) SDS. After washing, the proteins were exposed on an Odyssey ® Infrared Imaging System (LI-COR). Anti-GAPDH antibody (1:5000)  and rabbit anti-mouse IgG antibody labeled with an infrared-fluorescent 680 nm dye  was used as a loading control.A Milliplex assay (Millipore) was used to analyse G-CSF protein expression in the skeletal muscle and plasma samples following the manufacturer's protocol (Millipore). Briefly, the samples and standards were added to a 96 well plate containing premixed beads coated with G-CSF antibody. Following a 30 min incubation plates were washed three times then incubated with pre-mixed detection antibodies. Wells were then washed and incubated with streptavidin-phycoerythrin. After a final series of washes samples were resuspended in 125 μ l of assay buffer. The plate was read on the Bio-Plex Suspension Array System . For skeletal muscle, samples were extracted according to the protein extraction method described above and diluted to 1000 μg/ml prior to the assay. Plasma samples were run undiluted. Intra-assay coefficient of variation (CV%) was 4.2% for the plasma and 7.1% for the diaphragm homogenate.mdx mice were made using an unpaired Student t-test. Significance was set at p < 0.05 for all statistical tests with data represented as mean ± SEM.Statistical analysis was performed using GraphPad Prism 4.1 . Comparisons between wild type control and p < 0.05.For measurements of proliferation a One-Way ANOVA was used to determine the interaction between treatment concentration  at each time point. For BrdU analysis a Two-Way ANOVA was used to determine the interaction between G-CSF concentration  and treatment group . Newman-Keuls Multiple Comparison Test was used to determine differences between groups with significance set at p < 0.05.For measurements of differentiation a One-Way ANOVA was used to determine the interactions of time . Furthermore, a One-Way ANOVA was used to determine the interaction between treatment concentrations  at each day. Newman-Keuls Multiple Comparison Test was used to determine differences between groups with significance set at p < 0.05.For densitometry analysis a One-Way ANOVA was used to determine the interaction of time  at the given concentration of G-CSF treatment. Furthermore, a Two-Way ANOVA was used to determine an interaction between time  and treatment (Control and G-CSF). Newman-Keuls Multiple Comparison Test was used to determine differences between groups with significance set at bone fide sequences, confirming expression of the G-CSFR in myoblasts and myotubes , Western blot analysis confirmed expression of G-CSFR protein in mouse C2C12 and human primary myoblasts and myotubes, as well as in mouse and human skeletal muscle Figure . MultiplMyh7, 4, 2, 1) mRNA levels, known markers of muscle cell differentiation  and day 4 (p < 0.001) when compared to day 0  at day 2  and approximately 80% in the diaphragm muscles (p < 0.001) of 8–9 week old mdx mice compared to littermate controls  and >15-fold (p < 0.001) higher in the TA and diaphragm muscles, respectively, of mdx mice compared with control mice    and diaphragm muscle (p < 0.05) of mdx mice  Figure . However) Figure , consist Figures .To investigate the effects of G-CSF on myoblast proliferation, myoblasts were enumerated by DAPI staining at 24 h intervals over 96 h  continued to increase throughout differentiation. G-CSF had no influence on the mRNA levels of these genes at any time point nor did G-CSF influence the myogenic regulatory factors MyoD and myogenin. This further supports our observation that G-CSF does not increase C2C12 myoblast proliferation or differentiation.The present study demonstrated that G-CSF does not stimulate C2C12 myoblast proliferation in the presence or absence of serum. Importantly, our C2C12 myoblasts doubled approximately every 24 h in serum, and exhibited a block in proliferation without serum, consistent with other studies (Yaffe and Saxel, G-CSF is known to activate Akt, STAT3, and Erk1/2 in cells expressing the G-CSFR (Chakraborty and Tweardy, mdx mice. The relevance of altered G-CSF/G-CSFR in the muscles of mdx mice needs to be investigated to determine a causative, passive role in disease progression or whether G-CSF/G-CSFR is actively signaling to cause reduced G-CSFR levels. However, since G-CSF treatment increases muscle recovery in various rodent models of trauma and/or disease, a reduction in G-CSF within the muscle suggests G-CSF may be protective against damage, or that elevating intramuscular G-CSF promote muscle regeneration. Therefore, G-CSF may have therapeutic potential for managing the pathophysiology of muscular dystrophy.In conclusion, we show that the G-CSFR is expressed not just in myoblasts, but also in differentiated C2C12 and human primary myotubes and mature mouse and human muscles. These findings suggest that the G-CSF ligand may act directly on skeletal muscle via its receptor. We also show that G-CSF and the G-CSFR are reduced in the TA and diaphragm muscles from in vivo. For example G-CSF is known to cooperate with stem cell factor in haematopoiesis (Duarte and Franf, The molecular mechanisms by which G-CSF exerts its effects remain elusive as the current study demonstrates that G-CSF does not increase myoblast proliferation with concentrations ranging from 0.4 to 100 ng/ml. The actions of G-CSF may therefore require the interaction with other cytokines and growth factors 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.


Patients with idiopathic normal pressure hydrocephalus (iNPH) have reduced cerebrospinal fluid (CSF) concentrations of amyloid-β (Aβ) and α- and β-cleaved soluble forms of amyloid precursor protein (sAPPα and sAPPβ). The aims of this study were to examine if changes could also be seen in the CSF for secreted metabolites of APP-like protein 1 (APLP1) and to explore the prognostic value of amyloid-related CSF biomarkers, as well as markers of neuronal injury and astroglial activation, as regards to clinical outcome after shunt surgery.Twenty patients diagnosed with iNPH, 10 improved and 10 unchanged by shunt surgery, and 20 neurologically healthy controls were included. All patients were examined clinically prior to surgery and at 6-month follow-up after surgery using the iNPH scale. Lumbar puncture was performed pre-operatively. CSF samples were analyzed for neurofilament light (NFL), Aβ isoforms Aβ38, Aβ40 and Aβ42, sAPPα, sAPPβ, APLP1 β-derived peptides APL1β25, APL1β 27 and APL1β 28 and YKL40 by immunochemical methods.The concentrations of all soluble forms of APP, all Aβ isoforms and APL1β28 were lower, whilst APL1β25 and APL1β27 were higher in the CSF of iNPH patients compared to controls. There was no difference in biomarker concentrations between patients who improved after surgery and those who remained unchanged.The reduced CSF concentrations of Aβ38, Aβ40, Aβ42, sAPPα and sAPPβ suggest that APP expression could be downregulated in iNPH. In contrast, APLP1 concentration in the CSF seems relatively unchanged. The increase of APL1β25 and APL1β27 in combination with a slight decreased APL1β28 could be caused by more available γ-secretase due to reduced availability of its primary substrate, APP. The data did not support the use of these markers as indicators of shunt responsiveness. Idiopathic normal pressure hydrocephalus (iNPH) is a condition with gait and balance disturbances, cognitive decline and urinary incontinence in combination with enlarged cerebral ventricles , 2. ShunPatients with iNPH exhibit suppressed CSF concentrations of amyloid-β (Aβ) and the precursors soluble amyloid precursor protein α-, and β- , in combination with elevated neurofilament light protein (NFL) –9. HypotAmyloid-like protein 1 (APLP1)-derived peptides are processed by similar enzymatic pathways as APP and share related structural domains and functions –12. APLPThe aim of this study was to examine CSF concentrations of APLP1-derived peptides in iNPH, especially if the APL1β28 form was increased, and to explore the prognostic value of amyloid-related CSF biomarkers. For this purpose, we analyzed the APP-derived peptides sAPPα, sAPPβ, Aβ38, Aβ40 and Aβ42, the APLP1derived peptides APL1β25, APL1β27 and APL1β28 in CSF in 20 patients with iNPH (10 improved and 10 unchanged by shunt operation) and 20 neurologically healthy controls.Ten iNPH patients improved after shunt surgery and 10 non-improved were retrospectively selected. All were diagnosed in accordance with the international guidelines . The patThe improved 10 patients consisted of five men and five women, aged 70.3 ± 3.20 (mean ± SD) and the 10 unimproved patients  consisted of seven men and three women aged 71.6 ± 8 (mean ± SD). The groups did not differ significantly in terms of comorbidities, preoperative MMSE scores, extent of white matter lesions (WML), age or sickness duration. The baseline clinical data of the different groups are outlined in Table All patients were examined clinically prior to surgery and 6 months after by the iNPH scale, composed of items assessing gait, cognition, continence and balance . The extTwenty control subjects were chosen from a population of volunteers who had given consent to CSF sampling prior to knee surgery. The controls had no history of neurological or psychiatric disease, a normal clinical neurological examination and a normal mini-mental state examination score. They consisted of eight men and 12 women aged 71.2 (±6.4). There was no difference in age between the three subcategories .Amyloid β isoforms  were analyzed by electrochemiluminescence assays . The APLP1-derived peptides APL1β25, APL1β27, and APL1β28 were analyzed using a commercial ELISA . The samples were analyzed according to the kit insert with minor modifications. The CSF samples were diluted 1:20 for APL1β25, 1:10 for APL1β27, and 1:5 for APL1β28 by the dilution buffer contained in the kit. All samples were analyzed in duplicate and the CV % for standards and samples was <5 %.NFL was measured by ELISA technology using a commercial kit  with a lower limit of detection of 50 ng/L. For astroglia activation, CSF YKL-40 concentration was measured by solid phase sandwich ELISA  according to the manufacturer’s instructions. All analyses were performed batch-wise on one occasion by board-certified laboratory technicians at the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital, Mölndal, Sweden. Intra-assay coefficients of variation were below 10 %.p = 0.05, if not otherwise stated. No correction for the mass-significance effect was made in order to avoid type II errors. Statistical analyses were made using IBM® SPSS® Statistics for Windows version 21.Non-parametric methods were used for analyses. For comparisons between two groups the Mann–Whitney U test was performed and for comparisons between the three subgroups, the Kruskal–Wallis test was performed. For comparison of two proportions, Fisher’s exact test was used. For association between two independent variables, the Spearman rank order correlation was chosen. The level of significance chosen was CSF concentrations of sAPPα, sAPPβ, Aβ38, Aβ40, Aβ42 and APL1β28 were significantly lower and APL1β25 and 27 significantly higher in iNPH patients compared to healthy controls. Levels of NFL and YKL 40 did not differ between iNPH patients and healthy controls (Table There were no differences in any of the CSF biomarkers between improved and non-improved iNPH patients Table .Table 4LOur data showing substantially reduced CSF concentrations of Aβ38, Aβ40, Aβ42, sAPPα and sAPPβ in patients with iNPH, confirm earlier results , 19–22. This explorative study was in part designed to identify predictive alterations in the amyloid CSF pattern. We included two small groups representing iNPH patients that benefitted the most and those without any response to surgical treatment with shunting, in order to identify possible differences. However, we found no differences in CSF biomarkers between improved and non-improved patients. The low number of cases in each group may have been a limitation in this study. However, if there are subtle variances in CSF amyloid concentrations linked to responsiveness that could be identified in larger sample sets, we believe that these would be of minor clinical importance. Lumbar CSF as opposed to ventricular CSF was analysed to enhance the practical applicability of the generated results. Ventricular CSF might however provide a different pattern as it probably mirrors brain metabolic processes more accurately.The reduced CSF concentrations of Aβ38, Aβ40, Aβ42, sAPPα and sAPPβ in iNPH is consistent with earlier findings , 19–23, CSF APP-derived proteins increase after surgery, more in improved patients than in those that do not improve , 11. EveContrary to the profound alterations in APP metabolites, the APLP1-derived peptides showed only minor changes with a small elevation in APL1β25 and APL1β27 and a slight reduction of APL1β28. APP and APLP1 are processed by the same enzymes, including γ-secretase , 13, 32,There is a difference in APP-metabolite production pattern between iNPH and AD. In iNPH there is a general suppression of APP-metabolites in CSF whereas in AD, there is an isolated Aβ42 reduction, whereas the other Aβ-isoforms are unaffected . MoreoveThis data lends further support to a diagnostic profile in iNPH consisting of a general reduction in CSF concentration of APP-derived proteins. That the amyloid-like proteins behave in a different pattern could support the specificity and importance of the APP-down-regulation in iNPH. The study indicates that the biomarker profile in iNPH is consistent between patients who improve by shunt insertion and those who do not; therefore, our results do not lend support to the idea that these markers can be used for predictive purposes, but rather as an aid in the diagnosis of iNPH. Further studies will be needed to replicate the results and to expand the knowledge on the role of a possible altered amyloid metabolism for the pathogenesis of iNPH and the potential use of markers of amyloid metabolism to identify shunt responders needs to be further elucidated.


Oral mass vaccination (OMV) is considered as an efficient strategy for controlling classical swine fever (CSF) in wild boar. After the completion of vaccination, the presence of antibodies in 6–12 month-old hunted wild boars was expected to reflect a recent CSF circulation. Nevertheless, antibodies could also correspond to the long-lasting of maternal antibodies. This paper relates an experience of surveillance which lasted 4 years after the completion of OMV in a formerly vaccinated area, in north-eastern France (2010–2014). First, we conducted a retrospective analysis of the serological data collected in 6–12 month-old hunted wild boars from 2010 up to 2013, using a spatial Bayesian model accounting for hunting data autocorrelation and heterogeneity. At the level of the whole area, seroprevalence in juvenile boars decreased from 28% in 2010–2011 down to 1% in 2012–2013, but remained locally high (above 5%). The model revealed the existence of one particular seroprevalence hot-spot where a longitudinal survey of marked animals was conducted in 2013–2014, for deciphering the origin of antibodies. Eleven out of 107 captured piglets were seropositive when 3–4 months-old, but their antibody titres progressively decreased until 6–7 months of age. These results suggest piglets were carrying maternal antibodies, few of them carrying maternal antibodies lasting until the hunting season. Our study shows that OMV may generate confusion in the CSF surveillance several years after the completion of vaccination. We recommend using quantitative serological tools, hunting data modelling and capture approaches for better interpreting serological results after vaccination completion. Surveillance perspectives are further discussed.The online version of this article (doi:10.1186/s13567-015-0289-6) contains supplementary material, which is available to authorized users. Sus scrofa) for more than 20 years. Thus, free ranging populations of European wild boar are regarded as potential reservoirs of CSF [Classical Swine Fever (CSF) is one of the diseases entailing strong economic impact on the pig industry in the European Communities . Eradicas of CSF  and theis of CSF –6. The ls of CSF . In facis of CSF . Repeates of CSF  and incrs of CSF  or [6]).s of CSF , which hIn the absence of vaccination, both direct observation of infection (through RT-PCR and virus isolation) and indirect through the detection of antibodies in young wild boars are good indicators of the recent circulation of CSF. Seroprevalence in juveniles from 6 to 12 months-old is particularly useful when using hunting data since the viremia is generally short while wild boar recovering from infection will keep antibodies for life , 9. HoweIn the present study we aimed at improving CSF surveillance in wild boar after the completion of OMV. We focused our study in the Vosges du Nord area, north-eastern France, where two consecutive CSF outbreaks had been previously described during the 1990s and the 2000s  and wher2 comprising 1200 km2 of forests and is uninterrupted with the Palatinate forest through Germany  , 20. TheIn the study area, every wild boar shot or found dead had been examined for serological and virological detection during the 3 years after the completion of vaccination, i.e., from July 2010 up to June 2013 , 24. We Detection of antibodies to CSFV was carried out using commercially available ELISA kits (Idexx CSF Ab) according to the manufacturer’s instructions by two local laboratories. All the sera with positive or doubtful results using ELISA collected from December 2012 up to June 2013 in young hunted wild boars were subsequently analysed using a differential virus neutralization test (VNT) in order to confirm the specificity of the ELISA results and to assess the titre of neutralizing antibodies. Specific neutralizing antibodies (NAb) against the CSFV Bas-Rhin strain versus the Aveyron strain of the border disease (BD) virus  were assayed using the VNT on PK15 cells according to the OIE’s manual of diagnostic tests  by the NUi) accounting for seroprevalence similarities between neighbour municipalities  and a global one (Hi) accounting for seroprevalence unstructured heterogeneity between municipalities. The conservation of the spatial pattern from year–year was also tested by considering the average seroprevalence predicted by the model retained for the previous year as a potential factor of seroprevalence within the model for the current year (Riski). We retained for each year the most parsimonious model having the smaller Deviance Information Criterion  that we call hereafter municipalities. The serological status was likely to be similar between wild boars inhabiting the same municipality by sharing the same antigenic background  and even belonging to the same family groups . The serological status was also likely to be similar between wild boars inhabiting neighbouring municipalities, either because CSF is a contagious disease or because individual wild boars may be shot in a given municipality while the largest part of its home range may be included in a neighbouring one . The occon .nd of July up to the 30th of August 2013. Twelve mobile traps [th June 2013 until the end of August. Each animal was initially identified using numbered ear-tags, and animals captured at the same time and place were marked the same way (colour and shape of tags) as a first indication of the family group. The assignation to family groups was further confirmed using video-cameras on feeding capture sites . The capture area spread over 3000 hectares mainly comprising the municipality of Baerenthal and the neighbour ones Figure . Physicale traps  were depi et al. ). Each wi et al. , 36. An Serological responses were measured in sera (collected with dry tubes centrifuged in the few hours after capture) and virological examinations were performed on whole blood from captured animals (using tubes with EDTA) or spleen in hunted ones. Sample sera were first tested by an ELISA (Idexx CSF Ab) according to the manufacturer’s instructions. Then, positive or doubtful samples were analyzed by differential VNT. Two CSF virus strains were considered here for VNT: the Bas-Rhin strain  and theFrom July 2010 to June 2013, 9331 6–12 month-old hunted wild boars were sampled and exhibited a conclusive serological ELISA result. On average, the seroprevalence dramatically decreased from 27.8% in July–September 2010 (±1.0%) down to 1% in April–June 2013 (±0.8%). However, raw seroprevalence remained high  at the level of some municipalities  were captured once, 25/134 (19%) twice, 11/134 (8%) three times and 4/134 (3%) four times. Video-cameras collected about 39 800 pictures corresponding to 893 wild boar visits of the 12 trapping sites, allowing the identification of 25 social groups: 14 groups comprising adult females and piglets, nine groups comprising only subadults of both sexes, one single adult sow without piglet, and one single adult male. From the 23rd July 2013 up to the 20th December 2014, 51/134 marked animals were found at death time (38%): 47/51 had been shot during hunting, 2/51 had been found sick, and 2/51 had been found dead after a road accident. Dead animals comprised 2/8 adults, 12/19 subadults and 37/104 piglets. The place of death was confirmed for 49/51 individuals: 38/49 in the municipality of capture, 9/49 in a neighbouring municipality  and 2/49 up to 10 km from their captured site.From the 2Metastrogylus) in the lungs , but they were both negative by serology and PCR. The kinetics of antibodies among the twelve seropositive individuals is detailed in Table th November 2013, showed a positive ELISA result at around 6–7 months and a low neutralizing antibody titre (7.5). No other seropositive result was reported in the marked animals until December 2014  piglets belonging to five groups and one adult sow older than 30 months old belonging to the same group as the three seropositive piglets , thus confirming that positive ELISA results were corresponding to antibodies targeting the CSF virus. A study conducted at two different spatial levels  was adopted to clarify the situation.Ui” led to a dramatic decrease in models’ DIC), especially during the two first years. Autocorrelation could correspond to a contagious phenomenon, i.e. the active circulation of CSF virus between municipalities. The capture study showed that a significant proportion of wild boars captured in a given municipality were shot elsewhere (~23%), mainly in the neighbouring municipalities. The low match between municipalities’ boundaries of and wild boar home ranges could also participate in the autocorrelation of seroprevalence. We did not detect an important heterogeneity at a global scale . Nevertheless, some isolated municipalities located in the heart of the forested area, exhibited a higher seroprevalence compared to others and this spatial structure was particularly conserved from 2011–2012 to 2012–2013 . Identifying seroprevalence hot spots was interesting for targeting the surveillance efforts, but could not answer the question of the origin of antibodies in 6–12 month-old wild boars . We thus used a longitudinal approach at a finer scale for clarifying the epidemiological situation.At the inter-municipality level, the hierarchical Bayesian approach was useful before mapping seroprevalence : maps prth month [The capture-mark-recapture performed at the level of the seroprevalence hot spot  showed no virus positive animal. This result could either correspond to CSF eradication or to the low number of sampled wild boar , we thus looked at the semi-quantitative serological results. During July–August 2013, about 10% of the 3–5 month-old piglets, and one out of the seven adult sows above 30 months old  were found seropositive. Seropositive piglets had much lower neutralizing antibody titres than the seropositive adult sow, and they progressively lost their antibodies from July 2013 to November 2013  combining large scale hunting data and local longitudinal survey on marked animals. Due to autocorrelation in the hunting data, a hierarchical Bayesian approach was implemented to decrypt the spatial structure of seroprevalence. The models revealed the presence of localized hot spots of seroprevalence, at the level of some municipalities. However, only the longitudinal survey of marked individuals from the hot spots areas could disentangle the possible sources of antibodies in 6–12 month-old wild boars. In the present case, the progressive disappearance of antibodies in repeatedly captured piglets suggests that hyper-immunized sows having transmitted maternal antibodies to their offspring could explain seropositivity in young wild boars during the 4 years following the completion of OMV. Our approach offers an objective way to interpret surveillance data and adapt the surveillance process after the completion of OMV. This study also pinpoints the difficulty of interpreting seropositivity in young wild boar post-vaccination by simply examining transversal serological results and qualitative serological results. On the contrary to what was initially expected by European experts , 14, theIn that context, we recommend combining different surveillance approaches post-vaccination. First, the passive surveillance of dead or sick animals should be strengthened  since CSF re-emergence is supposed to cause morbidity and mortality in naïve populations . Neverth


Both androgens and estrogens are necessary to ensure proper testisdevelopment and function. Studies on endocrine disruptors have highlightedthe importance of maintaining the balance between androgens and estrogensduring fetal development, when testis is highly sensitive to environmentaldisturbances. This balance is regulated mainly through an enzymatic cascadethat converts irreversibly androgens into estrogens. The most important andregulated component of this cascade is its terminal enzyme: the cytochromep450 19A1 (aromatase hereafter). This study was conducted to improve ourknowledge about its expression during mouse testis development.By RT-PCR and western blotting, we show that full-length aromatase isexpressed as early as 12.5 day post-coitum (dpc) with maximalexpression at 17.5 dpc. Two additional truncated transcripts were alsodetected by RT-PCR. Immunostaining of fetal testis sections and ofgonocyte-enriched cell cultures revealed that aromatase is stronglyexpressed in fetal Leydig cells and at variable levels in gonocytes.Conversely, it was not detected in Sertoli cells.This study shows for the first time that i) aromatase is expressed from theearly stages of fetal testis development, ii) it is expressed in mousegonocytes suggesting that fetal germ cells exert an endocrine function inthis species and that the ratio between estrogens and androgens may behigher inside gonocytes than in the interstitial fluid. Furthermore, weemphasized a species-specific cell localization. Indeed, previous worksfound that in the rat aromatase is expressed both in Sertoli and Leydigcells. We propose to take into account this species difference as a newconcept to better understand the changes in susceptibility to EndocrineDisruptors from one species to another.The online version of this article (doi:10.1186/2051-4190-23-12) contains supplementary material, which is available to authorized users. C57BL/6 mice bred in our animal facility were housed under controlled photoperiodconditions (lights from 08:00 to 20:00 h) with commercial food and tapwater supplied ad libitum, as previously described , 2. The In this paper, we focussed on the ontogenesis of cytochrome P450 aromatase(accession # NP_031836 for protein and NM_007810.3 for mRNA), and named“aromatase” thereafter. To characterize aromatase expression duringmouse testis development, its mRNA level was analyzed by RT-PCR from 12.5 dpc tobirth. Three transcript variants, called T1, T2 and T3, were detected using the TaqMan method which allows thedetection of each isoform using three different internal probes each onespecific for one isoform Figure C.Table 1To determine if aromatase is translated in mouse testis, western blot analysiswas performed using a specific anti-aromatase antibody  Figure D. Two prThese findings are different from those of the only previously published paper onthis topic showing that, in the mouse, aromatase expression starts at 17.5 dpcand reaches the highest level at day 1 post-partum . In our Our findings indicate that different aromatase transcripts are generated in fetalmouse testes. Previous studies in different mammalian species (including themouse) reported that tissue-specific aromatase expression is driven by specificpromoters –6. Each Immunohistochemical analysis of aromatase localization in 17.5 dpc mouse testesusing a specific anti-aromatase antibody  showed astrong staining in Leydig cells. Importantly, there was no detectable stainingin Sertoli cells ; reverse 5′-CACAATAGCACTTTCGTCCA-3′ (located inexon V). Each different exon is highlighted in adifferent color . (DOC 24 KB)Additional file 1:


Both glial fibrillary acidic protein (GFAP) and S100B have been used as markers of astroglial plasticity, particularly in brain injury; however, they do not necessarily change in the same time frame or direction. Herein, we induced a Parkinson's disease (PD) model via a 6-OHDA intrastriatal injection in rats and investigated the changes in GFAP and S100B using ELISA in the substantia nigra (SN), striatum, and cerebrospinal fluid on the 1st, 7th, and 21st days following the injection. The model was validated using measurements of rotational behaviour induced by methylphenidate and tyrosine hydroxylase in the dopaminergic pathway. To our knowledge, this is the first measurement of cerebrospinal fluid S100B and GFAP in the 6-OHDA model of PD. Gliosis (based on a GFAP increase) was identified in the striatum, but not in the SN. We identified a transitory increment of cerebrospinal fluid S100B and GFAP on the 1st and 7th days, respectively. This initial change in cerebrospinal fluid S100B was apparently related to the mechanical lesion. However, the 6-OHDA-induced S100B secretion was confirmed in astrocyte cultures. Current data reinforce the idea that glial changes precede neuronal damage in PD; however, these findings also indicate that caution is necessary regarding the interpretation of data in this PD model. It is caused by loss of catecholaminergic dopamine-producing neurons in thee levels . The inte levels .In addition to the primary characteristic loss of neurons, PD also involves astrocyte dysfunction . Glial cBoth GFAP and S100B have been used as astrocyte markers of brain injury; however, they do not necessarily change in the same time or direction. A better understanding of the glial involvement in dopaminergic degeneration model will allow us to improve our data interpretation regarding PD. Thus, our aim was to investigate the changes in GFAP and S100B in the SN, striatum, and cerebrospinal fluid (CSF) on the 1st, 7th, and 21st days following the striatal injection of 6-OHDA in rats. We observed that glial changes precede neuronal damage, based on the content of tyrosine-hydroxylase (TH), a marker for dopaminergic neurons. Moreover, interestingly, the initial change in CSF S100B was apparently more related to the mechanical lesion of the model than 6-OHDA toxicity.Wistar rats were obtained from our breeding colony  and were maintained under controlled light and environmental conditions (12 h light/12 dark cycle at a constant temperature of 22 ± 1°C) with free access to food and water.All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23) and followed the regulations of the local animal house authorities . in vivo effect of the toxin 6-OHDA, seventy-two adult male rats  were divided into two groups: sham/vehicle (n = 36) and 6-OHDA (n = 36) groups. Behaviour and biochemical analyses were performed on day 1 (n = 12 per group), day 7 (n = 12), or day 21 (n = 12) after surgery. Half of the animals of each group, in each time point, was used for behaviour analysis and immunohistochemistry, and the other half was used for biochemical analysis (after CSF collecting and brain dissection) to evaluate the contents of S100B, GFAP, and TH.To analyse the4, 21.4 mL of propylene glycol, and 5.74 mL ethanol and water, q.s.p. 50 mL) and placed in a stereotaxic apparatus. The animals subsequently received three injections of vehicle  or 6-OHDA  into the right striatum over 20 min. For each injection, 2 μL was infused, 0.5 μL/min, according to the following coordinates: AP: 0 mm, LL: −2.8 mm, and DV: 0 mm from the Bregma; AP: −0.5 mm, LL: −3.9 mm, and DV: −6.0; and AP: −1.2 mm, LL: −4.1 mm, and DV: −6 mm; incisor bar: 0 [μL microsyringe  and an infusion pump  was used. The needle was kept in the brain for 4 min after each infusion to allow drug diffusion. The rats received postoperative care until they were awake and were returned to their home cages.The rats were anesthetised with equithesin   and immeμm) on a cryostat (Leica). The free-floating sections that contained the striatum (between 0 and −1 mm from Bregma [Immunohistochemistry was performed according to Brolese and coworkers . The ratm Bregma ) or SN (m Bregma ) were th pars compacta were isolated. After being removed, striatum was sliced (0.3 mm) using a Mcllwain tissue chopper. SN was not sliced, due to the reduced size. The SN, striatum slices, and CSF were frozen (−80°C) until further analysis.In the second set after surgery, the animals were anesthetised as previously described and then positioned in a stereotaxic holder; the CSF was obtained by cisterna magna puncture. The animals were subsequently sacrificed by decapitation. The brains were removed, placed in cold PBS, and the striatum and ventral midbrain area, which contained the SN, were removed by free-hand dissection. SN was dissected as described by Ding and coworkers . Brieflyμg) of proteins from each sample were boiled in sample buffer  SDS, 5% (w/v) b-mercaptoethanol, 10% (v/v) glycerol, and 0.002% (w/v) bromophenol blue) and electrophoresed in 10% (w/v) SDS-polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane [β-actin band. The bands were quantified using Scion Image software [The brain samples were homogenised and equal amounts (20 membrane . The monsoftware . in vitro effect on secretion and intracellular content of S100B.Primary astrocyte cultures were prepared from eight Wistar rats (1-2 days old) to evaluate 6-OHDA2+- and Mg2+-free balanced salt solution, pH 7.4, which contained (in mM): 137 NaCl, 5.36 KCl, 0.27 Na2HPO4, 1.1 KH2PO4, and 6.1 glucose. The cortices were cleaned of meninges and mechanically dissociated via sequential passage through a Pasteur pipette. After centrifugation at 1400 RPM for 5 min, the pellet was resuspended in DMEM (pH 7.6) supplemented with 8.39 mM HEPES, 23.8 mM NaHCO3, 0.1% amphotericin, 0.032% gentamicin, and 10% FCS. The cultures were maintained in DMEM that contained 10% FCS in 5% CO2/95% air at 37°C, allowed to grow to confluence, and used at 15 days in vitro. The medium was replaced by DMEM with 1% FCS in the presence of 6-OHDA (200 μM) or vehicle for 24 h. A GSH saline solution was used as the vehicle [Astrocyte cultures were prepared as previously described . Fetal c vehicle .μL of sample plus 50 μL of Tris buffer was incubated for 2 h on a microtiter plate previously coated with monoclonal anti-S100B antibody . Polyclonal anti-S100  was incubated for 30 min; peroxidase-conjugated anti-rabbit antibody  was subsequently added for an additional 30 min. The colour reaction with ο-phenylenediamine  was measured at 492 nm. The standard S100B curve ranged from 0.002 to 1 ng/mL.S100B was measured by ELISA as previously described . BrieflyμL samples for 24 h at 4°C. Incubation with a polyclonal anti-GFAP from rabbit  for 1 h was followed by incubation with a secondary antibody conjugated with peroxidase for 1 h at room temperature . A colorimetric reaction with ο-phenylenediamine  was measured at 492 nm. The standard human GFAP  curve ranged from 0.1 to 5 ng/mL.ELISA for GFAP was performed as previously described  by coatiThe protein content was measured by Lowry's method using BSA as the standard .t-test (when two groups were considered) or two-way analysis of variance (ANOVA) followed by Tukey's test, which assumed P < 0.05 as significant.Data are reported as the mean ± standard error and were analysed by Student's F = 32.65, P < 0.001). There was also a time effect  = 4.86, P = 0.031), which was indicated by the increased number of rotations in the 6-OHDA-lesioned rats at day 21 after the lesion compared with days 1  and 7 .To validate the 6-OHDA rat model, we performed a classic behavioural test. An intraperitoneal injection of methylphenidate  induced ipsilateral rotational behaviour in 6-OHDA-lesioned rats as shown in F = 11.19, P = 0.004). There was a time effect  = 7.74, P = 0.004) in 6-OHDA-group, in which the 1 day group had more TH immunocontent than the 7  and 21  day groups.In addition, the striatal slices  and ventF = 5.34, P = 0.034); a reduction in the TH immunocontent in the 6-OHDA-group 21 days after surgery was identified compared with the 6-OHDA-group 1 day after and the sham-group 21 days after surgery . Representative TH-immunohistochemistry of the SN is shown in  pars compacta in the 6-OHDA-group 21 days after surgery.Similarly, in the SN, the 6-OHDA-group was significantly different from the sham-group  = 18.80, P < 0.001). There was also an effect of time  = 5.61, P = 0.007), in which the 6-OHDA group of rats exhibited increased levels of striatal GFAP content 7 and 21 days after surgery compared with the sham-group at the same times ; furthermore, the GFAP content was higher 21 days after the toxin injection . To confirm the changes in GFAP identified in the rats injected with 6-OHDA, we conducted a GFAP immunohistochemistry study in striatum tissue on 7 (data not shown) and 21 days (P > 0.05).The immunocontents of GFAP and S100B were analysed using ELISA in the striatum slices and ventral midbrain area that contained the SN. The values are expressed as the percentage of the contralateral side that did not undergo stereotaxic surgery (assumed to be 100%).  21 days . However 21 days  (two-wayP > 0.05) or the SN (P > 0.05).In contrast to GFAP, no changes were identified in the S100B content in the striatum   = 18.33, P = 0.002). There was a time effect  = 36.37, P < 0.001); that is, we observed an increase on the 7th day after surgery; however, this change was transient because it was not observed on the 21st day .In addition to the tissue changes of astrocyte markers, we evaluated their content in the CSF. The CSF content of GFAP, which was evaluated by ELISA, was not altered in the sham-group at 1 to 21 days after stereotaxic surgery . HoweverF = 10.415, P < 0.001) in the 6-OHDA and sham-groups. We observed a clear increment in both groups on 1 day after stereotaxic surgery . No significant changes were identified 7 or 21 days after surgery in the 6-OHDA and sham-groups.The CSF S100B content is shown in  per se induced S100B secretion. Therefore, we exposed astrocyte cultures to 6-OHDA and evaluated S100B secretion. The 6-OHDA induced an increase in S100B secretion at 1 and 24 h , without affecting the intracellular content of S100B during this time .Considering that the mechanical injury (by the needle) in this model caused an increase in CSF S100B, it was not possible to determine if 6-OHDAand 24 h   in the 6-OHDA model of PD. CSF GFAP increments have been clinically identified in some conditions of acute brain injury, such as stroke, as well as chronic conditions, such as Alzheimer's disease or vascular dementia. However, no changes were identified in PD patients in two previous studies , 27. HerIn two investigations with PD patients did not identify changes in CSF S100B , 28; howt-test, P < 0.05). The increment on CSF S100B reflects astrocyte secretion in response to an acute brain lesion. This secretion is not restricted to the striatum, and the secreted amount is frequently small compared with the intracellular content, which explains why extracellular and intracellular changes are not necessarily associated [μM, 6-OHDA was able to induce S100B secretion at 1 and 24 h, without significant changes in the intracellular content.This increase in S100B at 1 day was observed in the 6-OHDA and sham-groups. This increment is likely due to the mechanical lesion induced by the needle. Striatal astroglial activation (based on GFAP immunoreactivity) induced by a needle lesion has been described . It is psociated . To deteIt is important to note some limitations of this work. First, we chose the 6-OHDA model of PD; however, we are aware that it is necessary to confirm these data regarding glial parameters in other models. Second, a mechanical lesion induced by the needle masked the lesion induced by 6-OHDA. In future studies in this PD model, the measurement of CSF S100B between 2 and 5 days would help to identify a specific increment of CSF S100B induced by 6-OHDA. Third, this study focused on two classical markers of astroglial activation. However, it is important to consider the astroglial heterogeneity and that other astrocyte markers also deserve attention, such as Aquaporin-4  or Glt-1In summary, we evaluated two markers of astroglial plasticity in a PD model in rats obtained with intrastriatal administration of 6-OHDA. The current data reinforce several ideas regarding PD; however, the findings also indicate several limitations of the model used for this disorder. Glial activation  precedes neuronal damage; however, the initial change in CSF S100B in this PD model was related to a mechanical lesion, which masks 6-OHDA glial toxicity. The 6-OHDA glial toxicity was confirmed by increased S100B secretion in astrocyte cultures.


Neurological outcomes of preterm infants with post-hemorrhagic hydrocephalus (PHH) remain among the worst in infancy, yet there remain few instruments to inform the treatment of PHH. We previously observed PHH-associated elevations in cerebrospinal fluid (CSF) amyloid precursor protein (APP), neural cell adhesion molecule-L1 (L1CAM), neural cell adhesion molecule-1 (NCAM-1), and other protein mediators of neurodevelopment.The objective of this study was to examine the association of CSF APP, L1CAM, and NCAM-1 with ventricular size as an early step toward developing CSF markers of PHH.CSF levels of APP, L1CAM, NCAM-1, and total protein (TP) were measured in 12 preterm infants undergoing PHH treatment. Ventricular size was determined using cranial ultrasounds. The relationships between CSF APP, L1CAM, and NCAM-1, occipitofrontal circumference (OFC), volume of CSF removed, and ventricular size were examined using correlation and regression analyses.CSF levels of APP, L1CAM, and NCAM-1 but not TP paralleled treatment-related changes in ventricular size. CSF APP demonstrated the strongest association with ventricular size, estimated by frontal-occipital horn ratio (FOR) , followed by NCAM-1  and L1CAM . TP was not correlated with FOR .Herein, we report the novel observation that CSF APP shows a robust association with ventricular size in preterm infants treated for PHH. The results from this study suggest that CSF APP and related proteins at once hold promise as biomarkers of PHH and provide insight into the neurological consequences of PHH in the preterm infant. Intraventricular hemorrhage (IVH) is the most common, severe neurological complication of preterm birth, occurring in roughly 25% of very low birth weight infants. Post-heDespite the profound morbidity associated with PHH, there remain few clinical, radiographic, or laboratory parameters to guide treatment for PHH. Physical signs such as occipitofrontal circumference , splaying of the cranial sutures, and tenseness of the anterior fontanel are imprecise measures, and changes in vital signs such as apnea or bradycardia occur only late in the disease course. Imaging-based measures of ventricular size are frequently used for individualized treatment; yet ventricular size and/or morphology may be affected by IVH, hypoxia-ischemia, white matter injury, and impaired brain development—all of which are common among preterm infants. Thus, tUsing tandem multi-affinity immunodepletion quantitative nano-LC-MS proteomics, we previously observed alterations in the abundance of key protein mediators of neurodevelopment in the CSF of a different group of infants with PHH  prior toApproval from the Washington University Human Research Protection Office (WU-HRPO) was acquired prior to initiation of this study. Informed consent was obtained from subjects’ parent(s) or guardian(s) in all cases. As enrollment, consent, and/or sample acquisition were frequently performed while the mother was receiving medical care at another facility or was unable to travel to St. Louis Children’s Hospital, and as the study posed no more than minimal risk to participants, verbal consent was deemed sufficient by the WU-HRPO (#201101887). In the case of control subjects, written informed consent was obtained . Consent, verbal or written, was documented in a regulatory binder for each participant.0/7–416/7 weeks PMA), the requirement for continued CSF removal was assessed, and a determination was made if the infant required a permanent ventriculoperitoneal shunt (VPS).As part of routine clinical care, all preterm infants ≤34 weeks estimated post-menstrual age (PMA) admitted to the St. Louis Children’s Hospital Neonatal Intensive Care Unit (NICU) underwent routine head ultrasound (US) examinations between 0–5 days of age to screen for IVH. Those found to have IVH then had weekly ultrasounds to monitor for PHH, and their OFC was recorded daily. Infants who demonstrated a frontal-occipital horn ratio (FOR) ≥0.55 aEthics Statement above), was obtained in all cases; however, only the 12 subjects included herein had ≥5 ‘paired’ CSF samples and ultrasound examinations, where both the CSF sample and the ultrasound were acquired within a 24 window, an a priori criterion for enrollment in the current study. Eleven of the 12 subjects required a VPS; the remaining subject was transitioned to palliative care status due to cardiopulmonary disease and expired prior to TEA. In those who underwent VPS, the final CSF sample was obtained at the time of shunt implantation. None of the subjects enrolled in the study developed CSF, RES, or VPS infections. In order to account for differences in clinical risk severity uniformly across the cohort, subjects were scored using the Clinical Risk Index for Babies II (CRIB II)[The current study spanned 2.5yrs, during which time 25 infants underwent implantation of a RES for PHH. Consent, verbal or written (see (CRIB II) taking i(CRIB II). Length (CRIB II).Cranial ultrasound examinations (US) examinations were obtained in infants with PHH at least 1–2 times per week using a Zonarez.one UltraSmartCart diagnostic ultrasound system . Ventricular size was measured on each US using two previously reported methods: ventricular index  and fronCSF samples were obtained either directly from the operating room at the time of initial ventricular cannulation (during reservoir implantation) or at the NICU bedside at the time of percutaneous ventricular or RES taps. Final CSF samples were collected from the operating room during VPS surgery. In all cases, CSF was acquired under sterile conditions and only as clinically indicated. Once acquired from the operating room or NICU, each CSF sample was transported to the Washington University Neonatal CSF Repository on ice and then centrifuged at 2500 rpm for 6 minutes. The supernatant was then aliquotted into microcentrifuge tubes (500μl) and stored at -80°C until experimental analysis.Cord blood was collected at term delivery from healthy newborn infants (in evacuated tubes with anticoagulant), plasma separated within 2 hours, aliquotted and stored at -80°C until analysis.Enzyme-Linked ImmunoSorbent Assays (ELISAs) were used to examine the CSF concentrations of APP, L1CAM, and NCAM-1 throughout the PHH treatment interval. Sandwich DuoSet ELISA development systems  were used to measure APP and NCAM-1, as previously described . BrieflyThe Pierce Bicinchoninic Acid (BCA) protein assay kit  was used to determine the total concentration of protein in each CSF sample. Briefly, 25μl of CSF and BSA standards were placed into the microplate wells (in duplicates); 200μl of the working reagent was then added and the plate was incubated for 30min at 37°C. The plate was cooled to room temperature and the absorbance was measured at 562nm on a plate reader. Total protein concentrations were determined using a four parameter logistic standard curve.Data were expressed using mean ± standard deviation. Associations between continuous factors were assessed using the Pearson correlation coefficient. Rates of linear change over time for markers and other measures were calculated using standard linear regression with the marker/factor as outcome and timepoint as predictor. All data analyses were performed using SAS 9.2.Twelve prematurely born subjects  with gestational age of 25.7±2.5 weeks and birthweight of 865±361 grams were included. Characteristics of the cohort are shown in Variability was observed in the total protein (TP) in the CSF of the 12 PHH subjects, both in terms of absolute levels and dynamic trends in concentration (increases/decreases) throughout the PHH treatment interval . InitialIn order to evaluate the relationship between candidate CSF biomarkers and ventricular size, two commonly referenced ultrasound-based ventricular size parameters were assessed for strength of association with CSF APP, L1CAM, and NCAM-1 levels. Specifically, ventricular index  and FOR were analyzed. FOR, which is ratiometric and takes into account the bilateral frontal and occipital horns of the lateral ventricles and the brain size, consistently showed a more robust relationship to normalized CSF levels of APP, L1CAM, and NCAM-1 than VI . Thus, FAs detailed above, CSF was removed via RES taps as clinically indicated for the treatment of each infant. To evaluate the relationship between CSF APP, L1CAM, and NCAM-1 levels and ventricular size, the levels of each protein were measured in CSF samples obtained within 24h of each cranial US. Common neurosurgical practice is to taper or stop RES taps prior to VPS implantation in order to: 1) assess the need for permanent VPS after temporizing PHH treatment with RES taps ; and 2) facilitate the function of the VPS, if one is required (to “prime” the VPS). As shown in In order to quantitate the strength of association between normalized CSF APP, L1CAM, and NCAM-1 with FOR, the rate of linear change in each CSF protein was plotted against the rate of linear change in FOR for each subject . While tIn addition to examining the relationship between APP, L1CAM, and NCAM-1 levels and ventricular size in individual infants in repeated CSF samples over time, we also examined the CSF levels of these proteins across all subjects at the time of the initial CSF sample . After normalizing for TP, APP, L1CAM, and NCAM-1 levels, measured in CSF obtained at the time of initiation of PHH treatment, were not consistently associated with FOR . Similarly, no clear association was observed between initial APP, L1CAM, or NCAM-1 levels and length of time (in days) after IVH diagnosis .As IVH is a critical factor in the pathogenesis of PHH, it is important to exclude blood itself as a major potential source for CSF APP, L1CAM, and NCAM-1. To this end, we examined the concentration of APP, L1CAM, and NCAM-1 in the plasma of age-matched infants without PHH or other known neurological injury. In this cohort, plasma APP = 126±51ng/ml, plasma L1CAM = 13±5ng/ml, and plasma NCAM-1 = 442±102ng/ml. For comparison, the concentrations of APP, L1CAM, and NCAM-1 measured in the initial CSF samples obtained in the 12 PHH subjects included in this study were 3194±1952ng/ml, 771±472ng/ml, and 483±183ng/ml, respectively. In contrast to NCAM-1 (p = 0.53), APP and L1CAM demonstrated significantly higher levels in PHH CSF than in the plasma of age-matched infants without PHH or other neurological injury .The current study was directed at evaluating the relationship between CSF levels of APP, L1CAM, and NCAM-1, with ventricular size. Our previous work suggested that these and other specific protein mediators of neurodevelopment exhibited increased levels in the CSF of infants with untreated PHH, but they decreased to control levels after the initiation of neurosurgical treatment. Using tThe finding that CSF proteins, particularly APP or others with involvement in neurodevelopmental processes may be associated with ventricular size lends support to the notion that these proteins may represent candidate markers of PHH and its associated neurological disability. At present, crude physical findings and semi-quantitative parameters of ventricular size, often obtained in the setting of abnormal ventricular morphology  are used to direct treatment for PHH. Thus, there is significant momentum to develop novel candidate PHH biomarker to complement existing physical and image-based parameters to aid in the diagnosis and management of PHH.The mechanism by which PHH results in increases in the CSF levels of APP, L1CAM, NCAM-1, and perhaps other proteins remains unclear. While it would seem possible that the initial IVH and blood products that remain in the CSF could result in elevations in these CSF proteins , the data reported herein do not support this explanation. Using age-matched control plasma samples, we found that APP and L1CAM levels are 25 fold and 59 fold (respectively) higher in PHH CSF than control plasma, making it unlikely that blood products alone could be responsible .In the current study, CSF APP tracked ventricular size closely, even demonstrating increases when ventricular size increased. These findings also argue against blood products as the origin of these CSF protein levels; rather, they raise the possibility that secondary axonal and/or neuronal injury may be contributing to elevations. Amyloid precursor protein is known to be released by axonal injury or stretch; thus itIn the setting of hydrocephalus, increased ventricular size may be associated with increased intracranial pressure (ICP). While it is of interest to determine the relationship of these candidates CSF biomarkers to ICP, this presents several challenges in the study of human preterm infants with PHH. First, the validity of ICP measurements in preterm infants with patent cranial sutures is unclear, since the open sutures likely attenuate increases in ICP. For this reason OFC is often used as a surrogate for ICP, but this measurement is subject to measurement error under the best of conditions. Further, treatment of PHH with RES is dominated by intermittent CSF removal, leading to fluctuations in ICP, rather than the more continuous CSF diversion provided through ventriculo-subgaleal shunts or VPS; however, by their very nature (risk of infection), routine CSF access is not practical with these devices. Further, ICP in the preterm infant with PHH may be affected by a number of other factors independent of CSF removal, including IV sedation, anesthesia (particularly at surgery), and wide variations in ventilator support, all of which would be expected to influence ICP. Finally and most importantly, bedside percutaneous RES taps are often accompanied by crying and movement by the infant, which inevitably transiently increases ICP during the tap, complicating the use of traditional manometers and the interpretation of their values. However, we were able to record ICP (measured as opening pressure on manometry after insertion of the needle into the RES) in two infants with serial RES sampling. While preliminary, ICP and normalized APP did demonstrate an association in these two infants . Currently, we are investigating CSF APP, L1CAM, and other proteins in hydrocephalus of varying etiologies that affect older children and adolescents, where many of these issues will be mitigated, permitting a clearer assessment of the relationship between candidate CSF markers, ICP, and ventricular size. However, it is likely that animal models will ultimately be needed to fully resolve this issue.The findings of this study are subject to a number of limitations. The data were derived from a small number of preterm infants (n = 12), with inherent variability in medical co-morbidities and clinical care. In addition, 11 of the 12 subjects required a permanent VPS, while the final subject expired from non-neurological causes before reaching TEA, the point at which VPS is considered. Since only 70–80% of patients typically require VPS,36, it iIn the current study, we report the novel observation that the CSF level of APP is associated with ventricular size in preterm infants undergoing treatment for PHH. The results from this study suggest that specific CSF proteins, such as APP, when paired with physical parameters and traditional imaging-based measures of ventricular size, may provide additional information regarding disease status and therapeutic efficacy. Future work will be directed at evaluating the role of CSF APP and related proteins as candidate markers of PHH and at investigating the pathophysiological implications and developmental consequences of these CSF protein changes.S1 FigTotal CSF protein concentrations (μg/mL) (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S2 FigCSF Amyloid Precursor Protein concentrations (ng/mL) (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S3 FigCSF L1CAM concentrations (ng/mL) (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S4 FigCSF NCAM-1 concentrations (ng/mL) (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S5 FigNormalized CSF Amyloid Precursor Protein levels (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S6 FigNormalized CSF L1CAM levels (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.S7 FigNormalized CSF NCAM-1 levels (blue left y-axis) and FOR measurements (red right y-axis) plotted versus relative time of reservoir surgery in all timepoints used for all twelve subjects.(TIF)Click here for additional data file.


Low CSF pressure headache is a clinical syndrome that identifies patients who were previously headache-free and who develop a persistent headache over 24 h, or just a few days. The diagnostic feature of low CSF pressure headache is that of worsening when upright and significant improvement on lying flat. Conservative treatment options include bed rest, abdominal binder, hydration, caffeine, and corticosteroids. If conservative treatment fails, epidural blood patching is often the next option.Since the accumulation of CSF leakage spreads widely, the injection site for the epidural blood patch may be difficult to choose in the management of low CSF pressure headache. We prefer the distal approach for the epidural blood patch which will be demonstrated in the following case.A 31-year-old man spontaneously developed orthostatic headaches for 2 weeks. The pachymeningeal enhancement of brain is noted with venous sinus engorgement in the images of brain MRI. Spinal MRI demonstrated CSF signals along bilateral C5-6, C6-7, C7-T1, T1-2, T8-9, T9-10, T10-11, and T11-12 neural foramens with epidural fluid accumulation from C7 to T12 compatible with CSF leakage. Conservative treatment with bed-rest, intravenous fluid infusion of 3000 ml QD, nonsteroidal anti-inflammatory drugs, and caffeine failed. An epidural blood patch of 6mL through T10-11 interspinous space resulted in immediate headache relief.Some patients have spontaneous low CSF pressure headache, most often due to a cryptic CSF leak. Epidural blood patch at distal part relieves headache by immediately compressing the dural sac and raising intrathecal pressure.No conflict of interest.