The inflammatory cell product neopterin is elevated in serum before and during delirium. This suggests a role for disordered cell-mediated immunity or oxidative stress. Cerebrospinal fluid (CSF) neopterin levels reflect brain neopterin levels more closely than serum levels. Here we hypothesized that CSF neopterin levels would be higher in delirium.In this prospective cohort study, 139 elderly patients with acute hip fracture were recruited in Oslo and Edinburgh. Delirium was diagnosed with the confusion assessment method performed daily pre-operatively and on the first 5 days post-operatively. Paired CSF and blood samples were collected at the onset of spinal anaesthesia. Neopterin levels were measured using high-performance liquid chromatography.p = 0.003), with highest levels in patients who developed delirium post-operatively. Serum neopterin levels were also higher in delirium . CSF neopterin remained significantly associated with delirium after controlling for relevant risk factors. Higher neopterin levels were associated with poorer outcomes  1 year after surgery (p = 0.02 for CSF and p = 0.03 for serum).Sixty-four (46 %) of 139 hip fracture patients developed delirium perioperatively. CSF neopterin levels were higher in delirium compared to controls (median 29.6 vs 24.7 nmol/mL, This study is the first to examine neopterin in CSF from patients with delirium. Our findings suggest potential roles for activation of cell-mediated immune responses or oxidative stress in the delirium process. High levels of serum or CSF neopterin in hip fracture patients may also be useful in predicting poor outcomes. Delirium is a serious acute neuropsychiatric syndrome comprising acute and fluctuating deterioration in attention, cognition, perception, and level of consciousness . AlthougThere has been much speculation that delirium may involve inflammation. This is because infection is a frequent precipitant of delirium and human studies have found elevated pro-inflammatory cytokines in serum , 7 and cThe pteridine neopterin  was firsIn the central nervous system (CNS), neopterin is produced by microglia . CSF levNeopterin levels have been little studied in delirium. Osse et al. have shown that high pre-operative plasma neopterin levels predict post-operative delirium following cardiac surgery, and high post-operative serum levels were also associated with delirium . High plThis study tested the hypothesis that neopterin is elevated in CSF and in serum in delirium in acute hip fracture patients.Hip fracture patients were recruited from Oslo University Hospital, Norway, between 2009 and 2012, and from the Royal Infirmary of Edinburgh, Scotland, between 2009 and 2011.All patients recruited in Oslo were part of a randomized controlled trial evaluating the effect of an orthogeriatric model where hip fracture patients were treated in an acute geriatric ward . There wThe patients were recruited in the emergency room by a member of the orthopaedic surgical team in Oslo and by a geriatrician (RH) in the Orthopaedic Trauma Unit in Edinburgh. Only patients who had both CSF and a pre-operative serum sample collected after recruitment were included in the present analysis.Patients were screened for delirium once daily with the confusion assessment method (CAM) . All patIn both hospitals, the CAM scores were based on information from nurses, close relatives and hospital records, in combination with a 10- to 30-min interview with the patient. Details of the delirium assessments were previously described . PatientThe Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE), 16 item version, was used as a measure of pre-fracture cognitive status . The IQCVenous blood was collected in 5-mL serum tubes from hip fracture patients pre-operatively. After sampling, tubes were left in the vertical position for 30 min at room temperature for clotting before they were centrifuged. Aliquots of 200–500 μL were then stored at −80 °C in polypropylene tubes. CSF was collected at the onset of spinal anaesthesia before administration of the anaesthetic agent. CSF was collected in polypropylene tubes. The CSF was centrifuged within 4 h, and the supernatant was stored in aliquots of 100–1000 μL at −80 °C.Samples were sent on dry ice to the University Medical Center Rotterdam for analyses. Neopterin in serum and CSF were measured by high-performance liquid chromatography (HPLC) after acid oxidation, as previously described . The anat tests depending on the distribution of the data. In our primary analysis, we compared the levels of neopterin in CSF and serum between patients with and without delirium by a Mann-Whitney U test. In a secondary analysis, we divided the patients into four subgroups based on delirium status and pre-fracture cognitive status :Neither delirium nor chronic cognitive impairmentChronic cognitive impairment, but no deliriumDelirium, but no chronic cognitive impairmentDelirium superimposed on chronic cognitive impairmentCategorical variables were analysed by the Chi-square test. Continuous variables were analysed by Mann-Whitney tests or Prevalent delirium—those who had delirium when CSF was takenIncident delirium—those who were free from delirium when CSF was taken but developed delirium post-operativelySubsyndromal delirium—those who at any point (pre-operatively or post-operatively) experienced some delirious symptoms (defined as at least two positive CAM features) but never fulfilled the criteria for full delirium Never delirium—those who never experienced delirium or subsyndromal deliriumWe also stratified the patients according to delirium status at the time of sampling:The Kruskal-Wallis test was used to assess potential subgroup differences.p value <0.1 in univariate analyses. All analyses were on pooled data from both centres. To assess the possibility that differences in diagnostic judgments between Oslo and Edinburgh could influence delirium rates, ‘centre’ was forced in as a covariate in both regression analyses.To assess the relationship between neopterin and delirium when adjusting for other covariates, we performed logistic regression analyses, one with serum neopterin and one with CSF neopterin as covariates, and both using ‘delirium at any time’ as the outcome variable. The inclusion of other covariates was based on a Because a history of cancer and/or an ongoing infection and/or use of non-steroid anti-inflammatory drugs (NSAIDs)/acetylsalicyclic acid (ASA) might influence neopterin levels, we also ran analyses with patients with any of these excluded. We also assessed the association between neopterin and length of stay and the combined endpoint ‘death/new nursing home admissions’ after 12 months.All statistical analyses were performed using IBM SPSS Statistics version 20.The study was undertaken in accordance with the Declaration of Helsinki. It was approved by the Regional Committee for Ethics in Medical Research in Norway and the Scotland A Regional Ethics Committee. At both sites, informed consent was obtained from the patients or from proxy decision-makers if patients did not have capacity to consent.n = 85; Edinburgh n = 54, Fig. Paired CSF and serum samples were available from 139 hip fracture patients (Oslo p = 0.003) and in serum , Table p = 0.05, Fig. p = 0.009, Fig. Sixty-four (46 %) of the hip fracture patients developed delirium at any time. Compared to patients without delirium, delirious patients had higher levels of neopterin in CSF , followed by those with prevalent delirium (n = 35). The group with subsyndromal delirium (n = 21) had neopterin levels between the levels of patients with no signs of delirium (n = 50) to those with full syndromal delirium . The same was not seen in patients with chronic cognitive impairment .In patients with delirium, there was a significant correlation with delirium severity and neopterin levels in the group without chronic cognitive impairment  and showed a trend in serum (p = 0.08) when adjusting for other risk factors in the logistic regression. The analysis identified two other risk factors for delirium: waiting time for surgery and higher IQCODE  and in CSF . Twelve months after surgery, 32 patients had died and 13 had been newly admitted into a nursing home. When death and new nursing home admissions were combined as ‘poor outcome’, both CSF and serum neopterin levels during hospital stay were significantly higher in patients with this poor outcome compared to those without (CSF: median (IQR) 29.2 (22.8–39.6) nmol/L vs 24.9 (19.4–33.2) nmol/L, p = 0.02; serum: median (IQR) 37.9 (26.8–54.4) nmol/L vs 29.2 (23.1–41.7) nmol/L, p = 0.03).There was a significant correlation between neopterin levels and length of stay in the whole study sample, both in serum  or serum (p = 0.21) between patients who were taking (n = 46) or were not taking NSAIDs/ASA (n = 93). There were, however, trends that patients with a history of cancer and/or an ongoing infection at the time of admission (n = 22) had higher levels of neopterin in CSF (median (IQR) 30.3 (21.3–48.7) nmol/L vs 25.3 (19.8–33.8) nmol/L, p = 0.08) and serum (median (IQR) 49.4 (24.7–69.3) nmol/L vs 29.4 (23.4–41.7) nmol/L, p = 0.01). Accordingly, we carried out subgroup analyses excluding these patients; the association between neopterin and delirium remained strong, both in CSF (median (IQR) 29.3 (22.6–39.6) nmol/L vs 23.2 (18.6–28.5) nmol/L, p = 0.001) and in serum (median (IQR) 37.0 (26.8–48.9) nmol/L vs 26.4 (22.0–33.1) nmol/L, p < 0.001). When regression analyses were performed only with patients without cancer and/or infection, neopterin was significantly associated with delirium both in the models with serum (p = 0.01) and CSF (p = 0.01).There were no significant differences in neopterin levels in CSF (p < 0.001). The median CSF/serum ratio was 0.81, and there was no difference in patients with (median ratio 0.81) and without delirium .There was a significant correlation between neopterin levels in CSF and serum  at 1 year. High neopterin levels, indicating greater immune system activation and oxidative stress, have been shown to be associated with poor outcomes in a range of conditions. High neopterin levels in cancer are associated with poorer survival, and the development of tumour-associated problems , they coThis study has some limitations that should be acknowledged. Due to the cross-sectional design, we are unable to infer causation, although longitudinal CSF studies in delirium are practically and ethically difficult. Due to the nature of the different parent studies at the two sites, the Oslo cohort was frailer than the Edinburgh cohort. It is also difficult to rule out that there might have been some differences in diagnostic judgment between the two centres. The assessment tools for delirium were, however, similar and thorough and both centres used CAM as the final diagnostic tool, and we included study site in the logistic regression analyses to minimize bias.This study is the first to examine neopterin levels in CSF in delirium. We found higher levels in delirium. We also replicated a prior study showing higher serum neopterin in delirium. Our findings suggest potential roles for activation of cell-mediated immune responses and/or oxidative stress in the delirium process. High levels may also be useful in predicting poor outcomes. Future work should examine these results in the context of other CSF biomarkers, including markers of oxidative stress, IFN-γ and other components of the cellular immune response. It would also be useful to examine CSF and serum neopterin levels in delirium in other patient populations, contributing to the overall body of knowledge examining the role of inflammatory processes in delirium.ADL, activities of daily living; APACHE II, The Acute Physiology and Chronic Health Evaluation II; ASA, acetylsalicyclic acid; CAM, confusion assessment method; CNS, central nervous system; CSF, cerebrospinal fluid; DRS, 98, Delirium Rating Scale Revised-98; GTP, guanosine triphosphate; HPLC, high-performance liquid chromatography; IFN-γ, interferon-γ; IQOCDE, The Informant Questionnaire on Cognitive Decline in the Elderly; MDAS, Memorial Delirium Assessment Scale; MMSE, Mini Mental Status Examination; NSAIDs, non-steroid anti-inflammatory drugs


There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis.Patients  undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography – mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels . No such correlations were observed for AEA and leptin.In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior. The endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous agonists of cannabinoid receptors and regulate a multitude of physiological processes including metabolism and food intake –3. It isPrevious studies have demonstrated that fasting is associated with reduced central and peripheral leptin levels in humans and elevated hypothalamic 2-AG levels in rodents , 15–17. Despite the documented opposing roles of endocannabinoids and leptin in the control of feeding and obesity, only one study has examined the interplay between central leptin and endocannabinoid levels in humans . These aThe experiments conducted herein were approved by the Stony Brook University institutional review board and written consent was obtained from each patient.Total knee arthroplasty (TKA) patients n = 35) scheduled for an elective unilateral TKA under spinal anesthesia and a femoral nerve block were enrolled for this study. Patients with documented rheumatoid arthritis, patients scheduled for bilateral TKA, and patients scheduled for a TKA revision were excluded from the current study. Details of the patient population used here are given in [ scheduleImmediately prior to the surgery, at the time of the spinal anesthesia, blood and CSF were collected from fasting (10–18 hours) patients.Blood was drawn from patients into tubes lacking anticoagulant (BD Vacutainer) and the resulting serum samples prepared, flash frozen, and stored at -80°C until use. CSF was collected and subjected to centrifugation at 1800 x g for 15 min at 4°C and the resulting supernatant was flash frozen in liquid nitrogen and stored at -80°C. Leptin levels were quantified using a leptin enzyme-linked immunosorbent assay . The samples were diluted by gender as suggested by the manufacturer. Each sample was assayed in triplicate using the protocol supplied by the manufacturer. The color intensities were read at 450 nm.4-PEA, d2-OEA, d5-2-AG, and 400 pg d4-AEA. Following centrifugation, the organic layer was removed and the sample was extracted again with the same buffer. The organic layer was dried down under argon and resuspended with 120 μl of 2:1 CHCl3:MeOH and 10 μl was injected into a Thermo TSQ Quantum Access Triple Quadropole mass spectrometer in triplicate. Endocannabinoid quantification was performed exactly as described [Serum and CSF (0.5 ml) were mixed with 3.5 ml of 2:1:1 CHCl3:MeOH:Tris  that was spiked with 4 ng described , 36.Zero-order correlation coefficients were determined using the statistical package built into the GraphPad Prism computer program for the Macintosh . First-order Spearman’s ρ values were calculated from the corresponding zero-order (bivariate) values using the formula of Lehmann . MultiplThe characteristics of the collective sample, stratified by gender and diagnosis of diabetes, are shown in 2 values (0.69 and 0.71) with both parameters contributing significantly, and the lowest level of collinearity  was seen with the unlogged values (data not shown).Bivariate correlations were determined between the outcome parameters used in the present study. Before undertaking such correlations, it is important to determine whether the data are normally distributed because a parametric Pearson correlation is not appropriate if this is not the case. We used the D'Agostino & Pearson omnibus normality test and found that the serum 2-AG values were not normally distributed . Consequently, we used non-parametric Spearman’s correlations for the whole dataset. The correlation coefficients for the endocannabinoids and for leptin are shown in There was no significant association between AEA and the corresponding leptin levels. As reported previously for this patient population , serum ATo exclude the possibility that the negative correlation between leptin and 2-AG was a result of an underlying distribution of another significantly associated parameter, higher-order correlations controlling for additional parameters were applied. The first order Spearman’s ρ value for CSF leptin vs. CSF 2-AG controlling for BMI, remained significant . Given that both the CSF leptin and the CSF 2-AG values passed the D'Agostino & Pearson omnibus normality test, it is possible to explore the relationship between these two variables further in multiple regression analyses. The statistical program we used allows different approaches for how the independent variables are presented. We used backward elimination , with CSF 2-AG as the dependent variable and with age, gender, incidence of diabetes, BMI and CSF leptin as the independent variables. Using this approach, the only significant variable remaining in the final model was CSF leptin, and the same was true when forward selection (when variables are added to the model) was tested. Given that the data indicate an association rather than causality, the reverse analysis, i.e. where CSF leptin was the dependent variable, was also investigated. Both methods selected CSF 2-AG and diabetes as significant predictors. The backward elimination analyses are summarized in The endocannabinoid system regulates appetite and metabolism . PreviouLeptin is an adipokine that regulates appetite, metabolism, and inflammation and one of its primary roles is to reduce food intake –44. ObesOur results are in contrast to the study of Jumpertz et al , who didImportantly, the CSF endocannabinoid levels reported herein are in line with the literature . Most ofOne observation at odds with the literature, however, is the finding of a significant correlation between serum and CSF AEA (but not 2-AG) levels. In four separate studies , 56, 61 A second potential factor to consider may be the body weight range of the patient sample. In our dataset, only 2 individuals were classified as normal weight (BMI<25) whilst 9 were overweight (25<BMI<30), 22 were obese (30<BMI<40) and 2 were morbidly obese BMI>40). Thus, our findings relate to an overweight/obese population. This may be of importance for the observed and interpreted results, although the literature is somewhat divergent with respect to the association between circulating endocannabinoid levels and body weight / BMI. Indeed, some studies have reported elevated levels of both AEA and 2-AG in overweight and obese individuals , 22, somIn conclusion, the present study investigated the relationship between obesity factors and peripheral and central endocannabinoids in a population of overweight to obese individuals with severe osteoarthritis. Because endocannabinoid levels are dynamically regulated by feeding and fasting in rodents and humans , 20, 69,S1 TableB in the table refers to the unstandardized coefficients, and these are only shown for analyses with significant ANOVA values.(DOCX)Click here for additional data file.S2 TableB in the table refers to the unstandardized coefficients, and these are only shown for CSF 2-AG, gender and the constant; the other variables did not reach significance in any case (P > 0.3).(DOCX)Click here for additional data file.


Phenothiazinyl and carbazolyl-donor moieties can be covalently coupled to an anthraquinone acceptor unit through an Ugi four-component reaction in a rapid, highly convergent fashion and with moderate to good yields. These novel donor–acceptor dyads are electronically decoupled in the electronic ground state according to UV–vis spectroscopy and cyclic voltammetry. However, in the excited state the inherent donor luminescence is efficiently quenched. Previously performed femtosecond spectroscopic measurements account for a rapid exergonic depopulation of the excited singlet states into a charge-separated state. Calculations of the Gibbs energy of photo-induced electron transfer from readily available UV–vis spectroscopic and cyclovoltammetric data applying the Weller approximation enables a quick evaluation of these novel donor–acceptor dyads. In addition, the X-ray structure of a phenothiazinyl–anthraquinone dyad supports short donor–acceptor distances by an intramolecular π-stacking conformation, an important assumption also implied in the calculations of the Gibbs energies according to the Weller approximation. Th. Th1H anic units , so that8 and 1 as well as the reference systems 2, 3, and 10 were studied by cyclic voltammetry in dichloromethane at room temperature  and 800 mV (8d). The direct comparison of the 3-phenyl derivative 8c with the donor-only reference 10a clearly indicates that the proximity of the electron-withdrawing anthraquinone moiety does not affect the first and second reversible oxidations of the phenothiazinyl moiety in the Do–Acc dyad 8c. The system 8d containing two conjugated phenothiazinyl moieties is particular since the second oxidation wave originates from the electronic coupling within the diphenothiazine unit .–86.1 the8 were studied by absorption and emission spectroscopy (8a–d show very similar absorption characteristics with a major absorption band around 259 nm and a lower intensity band around 325 nm (10a) and anthraquinone-only (3) references phenothiazine as well as anthraquinone absorb in the same region. In the spectrum of the carbazole dyad 8e the carbazole-typical absorption maxima can be found (cf reference systems 3 and 10b), whereas the spectrum of 8f displays just two absorption bands at 255 and 285 nm, originating from the 3-phenylcarbazole moiety.Furthermore, the electronic properties of the donor-anthraquinone dyads troscopy . All phed 325 nm . Accordi8 are expectedly larger than those of the donor-only (10) or anthraquinone-only (3) compounds. Plotting the extinction coefficient against the wavelength it becomes evident that the Do–anthraquinone dyads behave additively with respect to the constituent reference chromophores  and reference solvent used in electrochemistry (εref), and r+ and r− are indicating the effective ionic radii of the donor radical cation and acceptor radical anion, respectively. It is allowed to neglect the forth term, if spectroscopic and electrochemical measurements are performed in the same solvent. Therefore, Where  represents the correction term of the solvent polarity and the effect of the distance of the donor and acceptor moieties according to Where the first two terms indicate the free energy of the charge-separated state calculated from the spectroscopic and electrochemical measurements (in eV) and 8 exergonic Gibbs free energies for the electron transfer are found, both for the simplified free enthalpy ΔGET,simpl, i.e., the first two terms of . Therefore, the extent of the thermodynamically favored charge separation by an intramolecular photo-induced electron transfer (PET), plausibly explaining the observed fluorescence quenching, can be easily determined and compared within the series of related dyad systems  process into a dark, i.e., non-emissive, charge-separated state. The Gibbs free energies of the PET into the charge-separated states are exergonic and can be quickly calculated from absorption and electrochemical data applying the Weller approximation. The concise synthetic concept to donor–acceptor systems is very general, easy to perform and readily expandable to all kinds of functional π-electron systems. The Weller approximation of the Gibbs free energies of the PET allows a semiquantitative evaluation and optimization of photo-induced charge-separation systems. Studies directed towards multicomponent syntheses of more complex light harvesting and charge separation systems are currently underway.4 and potassium hydroxide  were dissolved in methanol and the mixture was stirred for 30 min  in dichloromethane (2 mL) or the neat aldehyde 9  were added to the reaction mixture and the solution was stirred at rt for 1 h, followed by the addition of 1 equiv of acetic acid (6)  and 1 equiv of tert-butyl isocyanide (7)  by syringe. The reaction mixture was stirred overnight at rt. The solvents were removed in vacuo and the crude product was purified by column chromatography on silica gel to give the analytically pure Ugi products 8 and 10.In a 25 mL Schlenk tube 0.50 mmol of the donor hydrochloride File 11H NMR, 13C NMR, UV–vis, fluorescence spectra and cyclic voltammograms of compounds 8 and 10, a summary of the X-ray crystallographic data of S(O)-1, computed xyz-coordinates of the structure 1 and HOMO and LUMO energies.


The radicular groove is a developmental anomaly that predisposes the tooth to periodontal break-down. Sometimes the situation becomes more complicated by pulp necrosis and development of a combined endodontic-periodontal lesion which is a diagnostic and treatment challenge for the clinician. This report presents the successful management of an endodontic-periodontal lesion in a maxillary lateral incisor that has a developmental palatal radicular groove using a combination of nonsurgical endodontic therapy and periodontal regenerative techniques. The combination of nonsurgical endodontic and periodontal regenerative treatment is a predictable method in treating combined endodontic-periodontal lesions caused by palato-gingival groove. Type I; the groove is short but does not extend beyond the coronal third of the root, type II; the groove is long reaching beyond the coronal third of the root but shallow, corresponding to a normal or simple root canal and type III; the groove is long, deep and extends beyond the coronal third of the root, corresponding to a complex root canal system [The palatal radicular  groove is a linear depression or groove that begins at the central fossa of the maxillary incisors, extends over the cingulum and continues specifically to the root surface  and possl system .et al. [Lee et al.  believe et al. . Radiculet al. . Whenevei.e., guided tissue regeneration therapy, intentional replantation [Treatment of the radicular groove, include curettage of the affected periodontal tissues , sauceriantation , 9, 10.Considering the great clinical importance of the palatal radicular groove and its rare occurrence, this paper reports a case involving a primary periodontal lesion with secondary endodontic involvement in a maxillary lateral incisor with palatal radicular groove. A multidisciplinary treatment approach included both endodontic treatment and guided tissue regeneration. A 27-year-old female patient with good general health presented to endodontic department of Dental School, Shahid Sadooghi University of Medical Sciences, Yazd, Iran, with a chief complaint of pus discharge from buccal mucosa adjacent to maxillary left lateral incisor for the past 6 months . The perThe patient did not mention any previous trauma to the maxillary anterior region and had no history of pain in the area. Tooth #10 had an intact crown without caries or fracture. The response to refrigerant spray and Electric pulp tester  was negative while adjacent and opposite teeth responded normally. The mobility of the tooth and its response to percussion and palpation tests were within the normal limits. Periodontal examination revealed a 12-mm pocket in mid palatal region . PurulenBased on history, clinical and radiographic examination, the lesion was provisionally diagnosed as primary periodontal lesion with secondary endodontic involvement according to Simon’s classification  in a maxEndodontic treatment was started under rubber dam isolation. Cleaning and shaping of the canal was carried out using the crown-down technique up to #40 master apical ﬁle using hand K-files . Root canal was irrigated with 1% sodium hypochlorite. The tooth was obturated with gutta-percha  and AH-26 sealer  using lateral condensation technique . One weeAt one-week endodontic follow-up evaluation, the buccal sinus tract and pus drainage from palatal sulcus was eliminated and the sulcus adjacent to the palatal groove could be still probed with gutta-percha cone to a depth of 12 mm. Three-month recall showed no abnormal symptoms while palatal probing depth did not change.Upon further consultation with the periodontist, an exploratory surgery was planned. After flap reflection, a narrow palatal bony defect was noted along the root adjacent to radicular groove. Transillumination revealed no apparent cracks or fractures. The diseased granulation tissue filling the bony defect was curetted to leave the soft tissue more conducive to regeneration and the root surfaces exposed to the defect were planned with an ultrasonic scaler  and hand curettes. Also 3 mm of the root apex was resected in order to eliminate probable extra radicular microbial biofilm. Odontoplasty was performed on the lingual aspect of the root, but the groove was too deep to be totally eliminated. Chemical conditioning of the groove was performed by 10% poly acrylic acid and glass ionomer cement  was applied into the groove . The arewith nonresorbable 3-0 silk suture material . PatientThe healing of the periodontium was uneventful. Sutures were removed 14 days after the surgery. No attempt for probing or deep scaling was made before the 6-months follow-up. A radiography taken 6 months after the surgery revealed partial disappearance of the radiolucency around the lateral incisor due to bone grafting and simultaneous bone regeneration . Also paFollow up after 2 years showed normal periodontal and endodontic condition. Palatal probing depth was 3 mm and periapical radiographies showed bone regeneration and healing of periapical bone defect . PermissRadicular grooves are more common in maxillary lateral incisors. They act as a “plaque trap” and initiating factor in localized gingivitis and periodontitis . Focal aThe prognosis of the affected tooth depends on the location, depth and extension of the groove, severity of the periodontal problem, accessibility of the defect and the type of groove  .In cases with both periodontal and endodontic involvement, like the present case, both forms of intervention are needed. Shallow grooves are located entirely on the crown and might be corrected by odontoplasty and periodontal treatment like curettage of granulation tissue. However radicular grooves associated with severe periodontal breakdown and extensive periapical lesion, need surgical intervention to eradicate inflammatory irritants and eliminating the groove .Several materials such as composite resin, amalgam and mineral trioxide aggregate are utilized to fill the radicular groove . MTA setSeveral materials were utilized to optimize regeneration of the lost periodontal and osseous structures. DFDB allograft was chosen to fill the osseous defect because of its osteoconductivity and ability to be converted into bone . SeveralThe key to achieving long-term success in developmental anomalies is accurate diagnosis. Clinician’s awareness of existence of such a situation may help to avoid misdiagnosis and improper treatment of the tooth.


CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters.To evaluate cerebrospinal fluid , progranulin (GRN), and chromosome 9 open reading frame 72 (C9orf72) mutations), and serum NfL in 118 subjects . In 55 subjects both CSF and serum was determined. In two subjects CSF was available before and after symptom onset (converters). Additionally, NfL levels were correlated with clinical parameters, survival, and regional brain atrophy.In this multicenter case–control study, we investigated CSF NfL levels in patients  were strongly elevated compared with presymptomatic carriers , resulting in a good diagnostic performance to discriminate both groups. Serum NfL correlated highly with CSF NfL  and was similarly elevated in patients. Longitudinal samples in the converters showed a three‐ to fourfold increase in CSF NfL after disease onset. Additionally, NfL levels in patients correlated with disease severity, brain atrophy, annualized brain atrophy rate and survival.CSF has the potential to serve as a biomarker for clinical disease onset and has a prognostic value in genetic FTD.NfL in both serum and  A pathogenic C9orf72 expansion was defined as more than 30 repeats.GRN, only nonsense mutations were included  or ascertained before participation in GENFI (n = 112). Participants were either patients with a mutation, or known presymptomatic carriers, or 50% at‐risk individuals (presymptomatic carriers and controls), or cognitively healthy family members without a mutation (controls). At‐risk individuals are first‐degree relatives of a known carrier of a pathogenic mutation. Genotyping of all participants was performed at local sites and clinical investigators were blinded for the mutation status of at‐risk individuals. At‐risk individuals and control subjects underwent neuropsychological examination. Subjects were categorized as presymptomatic or symptomatic according to criteria at the time of inclusion.n = 102) and converters (n = 4) was defined as the moment of first symptoms noted by a caregiver. In presymptomatic carriers, estimated time from onset was calculated as age at sample collection minus mean familial age at onset, resulting in a negative measure in carriers younger than the estimated onset derived from onset ages in their family.C9orf72, two GRN mutations)For this study, three subject groups were included from 11 centers collaborating in the Genetic FTD Initiative (GENFI)tion n = 3; and  provided written informed consent at enrollment.n = 179) was collected according to standardized local procedures. Serum samples were collected from Dutch participants only (n = 120). Both CSF and serum collection within 1 year were available in 57 out of 61 subjects with both CSF and serum . Longitudinal CSF samples were available in five subjects, including converters; in one converter a third CSF sample was available. T1‐weighted MRI‐images within 6 months of CSF collection were available in 101 subjects and a follow‐up scan in 22 subjects. Detailed data on available biosamples and MRI scans in the three subgroups after exclusion of outliers (see CSF (iers see  are presMeasurements of NfL (in CSF and serum) were performed in one laboratory (of CET respectively JK), blinded to clinical information and mutation status. CSF NfL was measured in duplicates using the enzyme‐linked immunosorbent assay (ELISA) of Uman Diagnostics , according to the manufacturer's instructions over four different batches. Median intra‐assay coefficient of variation (CV) was 0.8% (range 0–66.5%), inter‐assay variability was below 20%. Serum NfL concentrations were measured in duplicate by an earlier described, slightly modified, electrochemiluminescence immunoassay with antibodies identical to those used in the CSF ELISA (Data S1).P < 0.05. NfL values with an intra‐assay CV of >20% (n = 1) and outliers  were excluded. CSF and serum NfL were analyzed using nonparametric tests . Since the data were not normally distributed, and log‐transformation did not normalize the data, square root transformed CSF and serum NfL were used to correct for age in all subjects and disease duration in patients using analysis of covariance (ANCOVA) with post hoc Bonferroni corrections where appropriate. Spearman's correlation coefficient (rs) was used to correlate serum with CSF NfL, NfL levels with clinical measures and CSF NfL with brain volumes, the latter also with correction for gender and study site . Diagnostic performance was assessed by areas under the curve (AUC) with 95% confidence intervals (CIs) obtained by receiver operating characteristic (ROC) analyses, with optimal cut‐off levels at the highest Youden's index (sensitivity + specificity‐1).Statistical analyses were performed in SPSS 21.0 for Windows  and GraphPad Prism 6  applying a significance level of GRN, 29 C9orf72, 19 MAPT), 62 presymptomatic carriers  and 71 controls (Table P < 0.001) and controls (P < 0.001). GRN and C9orf72 patients were older than MAPT patients (P = 0.01 and P = 0.04 respectively). The age at onset in patients was highly variable ranging between 39 and 76 years and several presymptomatic carriers were past their estimated age at onset. However, 50% percent of the patients had an onset between 52 and 62 years; and the age of both converters was close to the estimated onset age. The disease duration in C9orf72 patients was longer than in GRN patients (P = 0.007). The clinical presentation was bvFTD (n = 60), PPA (n = 17), FTD‐ALS (n = 7), predominant memory phenotype (n = 4), mild cognitive impairment (n = 4), progressive supranuclear palsy or corticobasal syndrome (n = 2), and dementia not otherwise specified (n = 7).The total group of 234 subjects consisted of 101 patients . The elevation was confirmed after genetic stratification . Correction for age in all subjects and disease duration in patients on square root transformed CSF NfL yielded similar P‐values as without transformation, except for presymptomatic C9orf72 cases versus C9orf72 patient  were more than eight times higher than in presymptomatic carriers (804 pg/mL) and controls  and controls . Correction for age and disease duration showed similar results, except for the difference between presymptomatic carriers and patients which showed only a trend for the MAPT and C9orf72 mutations  than in presymptomatic carriers . The correlations were strongest in carriers , whereas controls showed only a trend . Sample sets collected on the same day showed slightly, although not significantly, stronger correlations .CSF NfL correlated strongly with serum NfL , but not in patients . In serum, a similar pattern was found . Females and males showed similar NfL levels . CSF NfL levels in patients correlated positively with CDR and CDR‐SB, but not with MMSE or disease duration (Table Age correlated with CSF NfL levels in presymptomatic carriers and controls (P < 0.001) and lower in presymptomatic carriers than in controls (P = 0.04). Cortical volumes were lower in patients than in presymptomatic carriers in all investigated areas, except for occipital , without differences between controls and presymptomatic carriers.Whole‐brain volume as a percentage of TIV was lower in patients than in presymptomatic carriers (n = 28) yielded significant negative correlations for whole brain, frontal cortex, and insular cortex. In presymptomatic carriers, negative correlations were found for whole brain and frontal, temporal, and parietal cortices. Subgroup analyses of scans from presymptomatic and symptomatic carriers combined and a CSF‐MRI interval of 90 days or less, as well as correction for gender and age showed similar patterns, albeit with lower correlation coefficients in the latter. Similar results were obtained after correction for study site. Unexpectedly, a positive correlation between NfL CSF and occipital cortex volume was found in the patient group.CSF NfL in carriers negatively correlated with whole‐brain volume Fig. C and witIn the subgroup of carriers with a follow‐up scan after CSF collection  we found significant correlations between CSF NfL and annualized rate of atrophy for whole brain, frontal, temporal, parietal, cingulate, and insular cortices  and from presymptomatic carriers (AUC 0.97 [0.94–0.99]), with a sensitivity of 84% and specificity of 100% for a cut‐off level of 2165 pg/mL , the median follow‐up of alive patients was 2.8 years . High CSF NfL levels were associated with a poor survival (estimated hazard ratio [HR] of 2.21 (95% CI: 1.30–3.77), P = 0.004, corrected for age and disease duration, Fig. C9orf72 cases, even after correction for ALS . Dividing the cohort into two groups gave similar results; dividing into four groups gave major overlap in CIs. Cox regressions on ‘raw’ CSF NfL confirmed the association with mortality: HR 1.02 for each increase in 1000 pg/mL (P < 0.001). Serum NfL was also associated with survival ; gene‐specific analyses in serum yielded no significant results.The median survival after CSF collection of deceased patients was 3.6 years , with a 5.8‐fold increase (from 9.5 to 55.3 pg/mL) in serum samples available in one converter. A decrease (−48% in 1 year) in CSF NfL of the third relative to the second sample was seen in the symptomatic stage of one converter. Longitudinal CSF samples of one patient and two presymptomatic carriers showed a 0.8–1.5‐fold change ; the CVs of all described longitudinal samples were below 5%.Longitudinal CSF samples of the two GRN, MAPT or C9orf72 mutations showed eight‐fold higher CSF NfL levels in patients than in presymptomatic carriers and controls. CSF NfL discriminated presymptomatic carriers from patients and might be useful to determine conversion. Serum NfL correlated highly with CSF NfL and showed a similar elevation in patients. Additionally, NfL levels in patients correlated with disease severity, brain atrophy, annualized brain atrophy rate, and survival. Hence, NfL in CSF or blood has the potential to serve as a biomarker for clinical disease onset and severity with a prognostic value.The present study on a large cohort of carriers of pathogenic The finding of elevated CSF and serum NfL levels in patients, with a good diagnostic performance to separate them from presymptomatic carriers, confirms the earlier findings in small series of presymptomatic carriers.GRN patients than in MAPT patients are supported by earlier findings of higher CSF NfL in cases with TDP‐43‐pathology than with tau‐pathology,GRN.GRN mutations. The wide range of NfL levels in our C9orf72 patients correlated with the clinical phenotype, with mostly high levels in subjects with concomitant ALS and/or fast progression and low levels in patients with a slow progression. This is in line with high NfL levels in genetic ALS  and sporadic ALS, the latter correlating with a fast progression and DTI abnormalities.The higher NfL levels in The identified correlations of NfL levels with disease severity and survival in genetic FTD patients are also in line with earlier reports in sporadic FTD, AD, and ALS.C9orf72,GRN, showing high NfL levels; however groups were too small for gene‐specific analyses. The correlation between CSF NfL and annualized rate of atrophy in the subset of carriers with two consecutive scans, supports the observed prognostic value of NfL levels in the cross‐sectional analysis. Larger future studies are needed to determine whether gene‐specific rates of atrophy, as found in the study by Whitwell et al.,The negative correlation between CSF NfL and brain and cortical volumes is in line with findings in a cohort of mainly sporadic FTD patients in which a negative correlation with gray and white matter volume was found.GRN mutations, in which a rapid breakdown of the neuroaxonal compartment takes place, instead of a more linear disease progression. Similar dynamics are suggested in ALS.The observed three‐ to fourfold increase in CSF NfL levels within 3 years in our converters and normal levels over the entire presymptomatic phase in our large series of healthy mutation carriers, gives a first indication of the time period in which NfL increases. Although the time to onset is difficult to estimate, due to varying age at onset among families, we showed only a small increase in asymptomatic subjects approaching their estimated onset. The elevation in NfL levels suggests a rather explosive nature of the disease process, at least for Major strengths of our study are the large series of presymptomatic carriers and patients with genetic FTD and the multimodal approach in correlating clinical and imaging data with a fluid biomarker. NfL determinations were performed in one laboratory which excludes an important source of variability.In conclusion, NfL in both CSF and serum is a promising biomarker for disease onset, severity, and survival in genetic FTD. Longitudinal studies are warranted to assess dynamics over time and thereby the usefulness of NfL for clinical trials in FTD.LHM contributed to the study design, in the acquisition and interpretation of data, performed the statistical analysis and drafted the manuscript and figures. CB, JK, and CET contributed to the study design, in the acquisition of data (i.e. NfL determinations), and revised the manuscript. JCvS contributed to the study design and supervision and to the interpretation of data, and drafted the manuscript. All other authors  contributed to the study design, in the acquisition of data and in study coordination, and revised the manuscript.Dr. Cardoso reports grants from UK Medical Research Council (Centres of Excellence in Neurodegeneration grant), from Alzheimer's Research UK, Brain Research Trust, The Wolfson Foundation, during the conduct of the study. Dr. Cash reports grants from UK Medical Research Council (MR/M023664/1), Alzheimer's Research UK (ARUK‐PG2014‐1946), Brain Research Trust, The Wolfson Foundation, Anonymous Charity, during the conduct of the study. Dr. Teunissen reports personal fees from advisory board of Fujirebio and Roche, non‐financial support from research consumables from Euroimmun, IBL, Fujirebio, Invitrogen and Mesoscale Discovery, other from performed contract research for IBL, Shire, Boehringer, Roche and Probiodrug, outside the submitted work. Dr. Benussi reports grants from Ricerca Corrente, Italian Ministry, during the conduct of the study. Dr. Graff reports grants from Swedish Alzheimer foundation, Regional Agreement on Medical Training and Clinical Research (ALF) between Stockholm County Council and Karolinska Institutet, Strategic Research Program in Neuroscience at Karolinska Institutet, Karolinska Institutet's Doctoral Funding, Swedish Medical Research Council, Swedish Brain Power, Swedish Brain Foundation, Old Servants foundation, Gun and Bertil Stohne's foundation, King Gustaf V and Queen Victoria's Foundation of Freemasons, during the conduct of the study; grants from Hoffman La Roche Basel, outside the submitted work. Dr. Dopper reports grants from The Netherlands Organisation for Health Research and Development Memorabel grant 733050103, Netherlands Alzheimer Foundation Memorabel grant 733050103, Dioraphte Foundation grant 09‐02‐03‐00, Association for Frontemporal Dementias Research Grant 2009, The Netherlands Organization for Scientific Research grant HCMI 056‐13‐018, Netherlands Alzheimer Foundation, during the conduct of the study;. Dr. Ghidoni reports grants from Ricerca Corrente, Italian Ministry, during the conduct of the study;. Dr. LE BER reports grants from Program “Investissements d'avenir” ANR‐10‐IAIHU‐06, PHRC FTLD‐exomes, PHRC predict‐PGRN, ANR PrevDemAls, during the conduct of the study. Dr Kuhle's institution  received in the last 3 years and used exclusively for research support: consulting fees from Novartis, Protagen AG; speaker fees from the Swiss MS Society, Biogen, Novartis, Roche, Genzyme; travel expenses from Merck Serono, Novartis; grants from ECTRIMS Research Fellowship Programme, University of Basel, Swiss MS Society, Swiss National Research Foundation, Bayer (Schweiz) AG, Genzyme, Novartis., outside the submitted work;. Dr. Papma reports grants from The Netherlands Organisation for Health Research and Development Memorabel grant 733050103, Netherlands Alzheimer Foundation Memorabel grant 733050103, during the conduct of the study;. Dr. van Swieten reports grants from The Netherlands Organisation for Health Research and Development Memorabel grant 733050103, Netherlands Alzheimer Foundation Memorabel grant 733050103, during the conduct of the study; grants from FORUM Pharmaceuticals, outside the submitted work;. Dr. Jiskoot reports grants from The Netherlands Organisation for Health Research and Development Memorabel grant 733050103, Netherlands Alzheimer Foundation Memorabel grant 733050103, during the conduct of the study;. Dr. Meeter reports grants from The Netherlands Organisation for Health Research and Development Memorabel grant 733050103, Netherlands Alzheimer Foundation Memorabel grant 733050103, Netherlands Alzheimer Foundation WE.09‐2014‐04, during the conduct of the study;. Dr. Otto reports grants from BMBF : Competence Net Neurodegenerative Dementias (project: FTLDc) and the JPND networks for standardization of biomarkers , during the conduct of the study;. Dr. Laforce reports grants from Novartis, outside the submitted work;. Dr. Rombouts reports grants from The Netherlands Organization for Scientific Research , during the conduct of the study;. Dr. Sánchez‐Valle reports grants from Fundacio La Marató de TV3, grants from National Institute of Health Carlos III (ISCIII) under the aegis of the EU Joint Programme ‐ Neurodegenerative Disease Research (JPND), during the conduct of the study;. Dr. Dick reports grants from UK Medical Research Council (Centres of Excellence in Neurodegeneration grant), Alzheimer's Research UK, Brain Research Trust, The Wolfson Foundation, during the conduct of the study;. Dr. Vandenberghe reports grants from Agency for Innovation by Science and Technology (IWT‐135043), during the conduct of the study;. Dr. Harding reports grants from UK Medical Research Council (Centres of Excellence in Neurodegeneration grant), Alzheimer's Research UK, Brain Research Trust, The Wolfson Foundation, during the conduct of the study;. Dr. Masellis reports grants from Canadian Institutes.Data S1. Methods.Figure S1. Patient numbers per collected material and available MR‐imaging. Displayed numbers are after exclusion of outliers. *Three subjects were excluded from the analysis on the correlation between serum and CSF because the interval between serum and CSF collection was longer than 1 year .Figure S2. NfL levels in presymptomatic carriers and controls. NfL levels in (A) CSF and (C) serum by controls and presymptomatic carriers. Green dashed lines represent the cut‐off line to separate controls from presymptomatic carriers at 1066 pg/mL for CSF  and at 8.3 pg/mL for serum . NfL levels in (B) CSF and (D) serum in controls and presymptomatic carriers specified by genetic group . Significances from the analysis of covariance analyses are displayed (corrected for age). Association between (E) CSF NfL and (F) serum NfL and time from estimated onset in controls (red circles) and presymptomatic carriers . One young individual is omitted from the graphs, but not from the analyses, to prevent disclosure of the genetic status. Presymptomatic carriers with CSF NfL values (n = 9) and serum NfL values (n = 14) of >2SD above the mean of controls were closer to or beyond the estimated onset (CSF mean 1.1 years and serum mean 0.8 years after estimated onset) than the presymptomatic carriers below that cut‐off . In presymptomatic carriers, both CSF and serum NfL significantly correlated with time to onset or estimated onset . Ns, not significant.FigureS3. Square root transformed NfL levels in presymptomatic carriers and patients. Square root of NfL in (A) CSF and (C) serum by controls, presymptomatic carriers and patients. Additionally, square root of NfL levels in (B) CSF and (D) serum specified by genetic group and clinical stage. Significances from the analysis of covariance analyses are displayed . Ns, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.Figure S4. Correlation between CSF NfL and MR‐imaging data. (A) Correlation of whole‐brain volume with CSF NfL in controls (red circles) and presymptomatic carriers . Correlations between CSF NfL and (B) frontal lobe volume and (C) temporal lobes volume in presymptomatic carriers (blue squares) and patients (orange triangles).Figure S5. Longitudinal CSF NfL samples. Longitudinal samples of two converters (green and light blue lines), two presymptomatic carriers (dark blue lines) and one patient (orange line), plotted by time from onset or estimated onset in years.Click here for additional data file.


Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass ( Nodaviridae family, Betanodavirus genus), causative agent of the viral encephalopathy and retinopathy (VER), a disease that alters the brain and retina structure and function  alone (control) or with NNV (107 TCID50/ml) or polyinosinic-polycytidylic acid   for 24 hours at 25°C. After incubation, fragments of tissue were washed with 0.01 M phosphate buffered saline (PBS) and processed for gene analysis as described below.Specimens of European sea bass (n = 6) or gilthead seabream (n = 6) males were completely bled and the testis removed without taking the afferent and efferent blood vessels that are located in the mesentery  connecting the testis to the body wall. The testis were weighed and chopped in 1 mm6 TCID50/fish of NNV since this route of infection has been proven to be the most effective  during 10 min at 52°C. The digoxigenin-11-dUTP incorporated in the PCR products was detected by means of a direct immuncytochemistry method using a specific antiserum anti-digoxigenin-HPR (Roche) in 0.1 M Tri-HCl with 0.1 M NaCl at the optimal dilution of 1:100 for 60 min at room temperature. The peroxidase activity was revealed by incubation with 0.05% 3,3’-diaminobenzidine tetrahydrochloride (DAB) in 0.01 M Tris-HCl with 0.1 M NaCl and 0.05% H2O2 at room temperature for 5 min. Tissue from non-infected fish was used as negative controls. In order to determine the ability of the amplified products to diffuse into the aqueous phase, liquid phase was collected after performing the in situ RT-PCR reactions, run in 1% agarose gel for electrophoresis (Bioline) with 0.5 μg/ml of ethidium bromide (Sigma) and visualized under UV light.Testis sections were used to perform escribed . SectionAfter 15 days of infection, testis fragments from mock- or NNV-infected fish were homogenized in 1 ml of 0.01 M PBS. E-11 cells , derivedin vivo (n = 4–5 fish/group and time) and the in vitro (n = 6 fish/group) experiments, with TRIzol Reagent (Life Technologies) following the manufacturer’s instructions, and quantified with a spectrophotometer (Cecil Instruments Ltd). In the cell culture experiments total RNA was extracted using RNeasy Mini kit (Qiagen) following the manufacturer´s instructions.Total RNA was extracted from testis and brain fragments from both, the Isolated RNA was DNase I treated  and the SuperScript III RNase H−Reverse Transcriptase (Life Technologies) was used to synthesize first strand cDNA with 1 μl of random primers  from 1 μg of total RNA, at 50°C for 60 min.rdrp) and capsid protein (cp) genes of NNV in the testis and brain of in vivo infected gilthead seabream and European sea bass specimens, real-time PCR with TaqMan probe was performed with an ABI PRISM 7500 instrument (Applied Biosystems) using TaqMan® Gene Expression Master Mix (Applied Biosystems). The primers and TaqMan fluorogenic probes are detailed in With the aim of determining the levels of transcription of RNA-dependent RNA polymerase  and the interleukin 6 (il6) and 1 beta (il1b); (ii) specific cellular immune response markers as the beta chain of the T cell receptor (tcrb) and the heavy chain of the immunoglobulin M (igmh); (iii) the sex specific gene double sex-and mab3-related transcription factor 1 (dmrt1); (iv) esteroidogenic enzymes such as gonadal aromatase (cyp19a1a of gilthead seabream or cyp19a1 of European sea bass), neural aromatase (cyp19a2) and steroid 11-β-hydroxylase (cyp11b1); and (v) the estrogen receptors as the estrogen receptor α (era) of gilthead seabream, and the European sea bass estrogen receptor β1 (erb1) and β2 (erb2) were analysed in the testis and brain by real-time PCR using an ABI PRISM 7500 instrument and SYBR® Green PCR Core Reagents (Applied Biosystems) as previously described . 50 μl of extracted sample  were used for each ELISA reaction. The standard, mouse anti-rabbit IgG monoclonal antibody (mAb), and specific anti-steroid antibodies and enzymatic tracers (steroid acetylcholinesterase conjugates) were obtained from Cayman Chemical while the microtiter plates (MaxiSorp) were purchased from Nunc. A standard curve from 6.13 x 10−4 to 5 ng/ml (0.03–250 pg/well), a blank and a non-specific binding control (negative control) was established in all the assays. Standards and extracted serum samples were run in duplicate. The lower limit of detection for European sea bass assays was 24.41 pg/ml and for gilthead seabream assays was 12.21 pg/ml. The intra-assay coefficients of variation  were 9.3 ± 4.3% for E2 and 9.1 ± 3.8% for 11KT assays for serum. Details on cross-reactivity for specific antibodies were provided by the supplier .Serum levels of 17β-estradiol (Esea bass  and adapsea bass . SteroidThe gonads were weighed and the gonadosomatic index (GSI) was calculated as an index of the reproductive stage [100*(MG/MB) (%)], where MG is gonad mass (in grams), and MB is body mass (in grams).All slides were examined with a Nikon eclipse E600 light microscope. The images were obtained with an Olympus SC30 digital camera (Olympus soft imaging solutions GMBH) and Spot 3.3 software (Diagnostic instruments).The genetic nomenclature used in this manuscript follows the guidelines of Zebrafish Nomenclature Committee (ZNC) for fish genes and proteins and the HUGO Gene Nomenclature committee for mammalian genes and proteins.erb1 and erb2 gene expression was determined by means of 2% agarose gel densitometrically scanned using an image analysis using the Gel Logic 100 Imaging System (Kodac) and ImageJ 1.44p software . The data are showed as the mean value ± standard error to the mean (SEM) of the gene expression relative to actb gene expression.The quantification of gilthead seabream All data were analysed by t-Student test to determine statistical differences between infected and control groups, or one-way ANOVA to denote statistical differences at different points in the infected group (P ≤ 0.05). A non-parametric Kruskal–Wallis test, followed by a multiple comparison test, was used when data did not meet parametric assumptions. Statistical analyses were conducted using SPPS 15.0 application. All data are presented as mean ± SEM. Minimum level of significance was fixed in 0.1 . Letters denote statistical differences between different time points (P ≤ 0.05).The experimental infection showed no mortality or disease signs in gilthead seabream specimens, but reached 55% mortality in European sea bass .All the techniques performed to detect NNV were firstly applied to control fish confirming that these fish were free of NNV Figs .in vivo with NNV were determined the transcription levels of the mRNA coding for the two main proteins of the virus, the RdRP and the CP proteins, using a specific probe for each, by TaqMan real-time PCR. Surprisingly, no transcripts of these genes were found in the testis of neither gilthead seabream nor European sea bass, neither in the gilthead seabream brain (data not shown). However, both were detected in the brain of European sea bass after 15 days of infection  genes in both tissues and species upon in vivo infection and in the testis upon in vitro infection  or unchanged   .in vivo infection  and 11β-hydroxilase (cyp11b1), the enzymes involved in E2 and 11KT production, respectively; and several E2 nuclear receptors  were differently regulated. The cyp19a1a expression was up-regulated at 1 day post infection . Interestingly, when the expression pattern of these genes was analysed in the testis upon in vitro challenge with NNV or poly I:C, we found that NNV up-regulated the cyp11b1 and down-regulated the erb1 gene expression, while poly I:C up-regulated the cyp11b1 and down-regulated the cyp19a1a, erb1 and erb2 gene expression.In order to determine whether the NNV infection or the changes in the sex-steroid hormone levels detected in gilthead seabream serum affects the functionality of the testis, we next analysed the expression of some reproduction-related genes in the testis. Firstly, m testis , was slinfection , whilst nchanged . On the nchanged . As the s tissue , and fouat day 7 , whereasin vivo infection with NNV in the testis and the brain. Thus, the expression of cyp11b1 was up-regulated at day 7 and erb1 and erb2 genes at day 1 and 7, respectively, while the three genes were down-regulated at day 15 of infection (cyp19a1 gene expression was undetectable in both control and infected specimens (data not shown). However, in the brain, nodavirus modify the expression pattern of cyp19a2, the neural aromatase, which was down-regulated at day 1 and up-regulated at day 7 post infection , whereas the expression of cyp11b1 gene was down-regulated upon poly I:C exposure. Interestingly, the transcription of the genes coding for both estrogen receptors, erb1 and erb2, was up-regulated upon NNV challenge.Finally, in the testis of European sea bass challenged poly I:C , the cypcp gene using in situ PCR as previously described on grouper embryos [cp RNA on Sertoli cells in the testis of both species, and on tunica albuginea’s and germ cells in the gilthead seabream testis. These results suggest that NNV could be spread into the germ cells of gilthead seabream and within the gonadal fluid of both species. However, as Sertoli cells are intimately associated to germ cells in fish [in situ PCR and immunocytochemistry techniques never showed viral mRNA or protein staining in blood vessels or cells (data not shown).NNV is a single stranded RNA virus which causes VER disease and provokes high mortality rates in several Mediterranean fish species . Europea embryos . Thus, w in fish , the she in fish . In contin vivo infection, IPNV did not trigger an effective immune response, which was triggered upon in vitro infection, so some extragonadal factors might block the immune response in the gonad improving the transmission of the virus [tcrb and igmh genes upon in vivo infection and of il6 and il1b genes upon in vitro infection, whilst in the testis of European sea bass we observed up-regulations of the pro-inflammatory cytokine and igmh gene expressions upon in vivo infection and no changes upon in vitro infection. In fact, the same pattern in the testis expression of others immune-related genes, including antimicrobial peptides (AMPs) [in vivo and in vitro infections [in vivo infection with NNV, the phagocytosis, the cell-mediated cytotoxic activity and the mx transcription started very early upon the infection and stayed at high up to 15 days, while in the European sea bass brain these activities and the mx transcription was up-regulated at day 1 upon infection and quickly decreased to control levels [tcrb and igmh gene expression is up-regulated in the brain of gilthead seabream and down-regulated in the testis, while in the European sea bass only the tcrb is up-regulated in the brain upon 15 days of infection. Regarding the igmh transcription in European sea bass tissues, we observed a decrease at day 1 and an increase at day 15 in the brain, while in the testis this gene expression was up-regulated at day 7.Other viruses have also demonstrated to colonize the testis, such as VHSV and IPNV in the rainbow trout and showed different replicating capacity and in turn they elicited a different immune response . Intereshe virus . NNV, hos (AMPs)  and intes (AMPs) , of bothfections , 48. Intl levels . Furtherl levels  and thisl levels . Similar2 and high levels of testosterone (T) have been related with the progression of vibriosis symptoms [2 serum levels decreased as the infection proceeds only in the European sea bass, which showed mortality during the infection and displayed an earlier but less effective immune response in the brain [2 serum levels increased as the infection progressed. Moreover, in the last years, it has been described in the gilthead seabream that estrogens regulated the inflammatory immune response through endothelial cells and macrophage activation and increased leucocytes recruitment in the testis [in vitro the activity of gilthead seabream phagocytes and their sensitivity to pathogens [2 and T serum levels in the gilthead seabream, increased the complement and peroxidase activities at different time points although unmodified or decreased other humoral immune responses such as anti-protease activity and IgM serum levels [2 serum levels at the beginning of the infection, whilst the serum levels of 11KT were decreased at these time points and increased at day 15. Taking into account that a certain increase in E2 serum levels stimulates the immune response in the gilthead seabream, this data also supports the ability of this species to fight against the NNV infection and overcome the disease.Several pathogen infections change the sex steroid hormone levels of the infected specimens , 32. Cursymptoms . Our dathe brain . In conte testis –54. Furtathogens , 56. On m levels . Our dat2 regulates the inflammatory response as two-edges-sword, triggering stimulation or inhibition of this response depending on several parameters such as the immune stimulus, the tissue involved, the concentration of E2, the expression of estrogen receptors and so on [cyp11b1 and cyp19a1a and cyp19a2) and estrogen receptors  in the testis, the main organ that synthesizes biologically active steroids de novo [in vivo infection and in the testis upon in vitro infection. Our data showed that in the gilthead seabream testis, the expression of the cyp19a1a gene that coded for the aromatase enzyme that transforms T to E2, was increased, coinciding with an increase on the E2 serum level. The expression of this gene was undetected in the European sea bass testis, and therefore the E2 serum levels decreased. However, the sensitivity of the gilthead seabream testis to E2 is decreased as the expression of the era at day 15 and of erb1 and erb2 at day 1 decreased after infection. On the other hand, we analysed the sensitivity of E2 and the local E2 production in the brain as locally produced E2 in the brain regulated important biological functions including reproduction and neuroprotection [era in the brain increased at day 1 and decreased at day 7 upon infection whilst neither erb1 nor erb2 were detected. This is not surprising as, it has been suggested that the expression of er genes is not detected in some areas of the brain probably because its expression is very low [cyp19a2), erb1 and erb2 were differently regulated. Regarding androgens, NNV decreases the 11KT serum levels of gilthead seabream without affecting, in the testis, the expression of the cyp11b1 gene; that coded for the key enzyme in the transformation of T to 11KT in the testis. Although the testis is the main steroidogenic tissue, other tissues can synthesize 11KT or transform T to 11KT [cyp11b1 transcription has been detected in the brain of teleosts [dmrt1 gene expression was slightly decreased upon 15 days of infection. However, the dmrt1 transcription levels were not as low as needed to produce testicular disruption in gilthead seabream males [in vitro infection with NNV are different to those observed in vivo in most of the cases. This suggests that the changes in other tissues due to NNV infection; modifies the gonadal response upon infection.In mammals, End so on . Similarnd so on , 57, 59. de novo , and braotection  and the very low . In cont to 11KT  producinteleosts . Regardiam males . Interesin situ PCR, immunocytochemistry and cell culture. However, the response to the virus in both species is very different. In addition, NNV triggers the inflammatory immune response in the testis of European sea bass whilst seems to be overlooked in the gilthead seabream testis. This also applies to other immune responses, as AMPs or IFN production, previously studied, which could account for the higher presence of NNV in the seabream testis. Furthermore, we had also determined whether NNV is able to modulate the reproductive system to improve its transmission and could demonstrated that NNV alters E2 and 11KT production and the sensitivity of brain and testis to these hormones. Whether this is occurs due to changes in the fish physiological abilities to modulate the immune response or by NNV to improve its ability to replicate and be transmitted is still undetermined and further studies will be needed to understand these mechanisms. Apparently, according to the GSI and dmrt1 expression levels, there is no disruption of testicular functions upon infection, which could favour the shedding and dissemination of the NNV to the water and/or the surrounding animals.In conclusion, we have proved for the first time the ability of NNV to colonise the male testis of gilthead seabream and European sea bass and produce infective particles by means of S1 FileThe liquid phase of each isPCR was run in a 2% agarose gel and showed no amplicons. M, 100-bp ladder; lanes 1–9 correspond to different samples and the bands to primers.(TIF)Click here for additional data file.


Endoscopic repair is considered the treatment of choice in cerebrospinal fluid (CSF) rhinorrhea. The aim of our study was to analyze the etiopathogenesis of CSF rhinorrhea, the outcome of treatment and the causes of failure in a developing-country setting.A retrospective review of patients treated with endoscopic repair for CSF rhinorrhea at a tertiary care hospital in southern India from January 2002 to December 2009 identified 36 patients, the majority of them being women. The defects were closed in three layers using fat, fascia lata and nasal mucosa along with a fibrin sealant in the majority of the patients. Per-operatively, a subarachnoid drain was placed in all patients. Patients were followed up for 1 year. Spontaneous onset of CSF rhinorrhea was noted in 61% of patients. The most common site of leak was found to be the left cribriform plate area. Hence the most common cause of CSF rhinorrhea in our study was spontaneous and the second most common was post-traumatic. Our success rate on the first attempt at endoscopic repair was 100%, with a recurrence rate of 6%. A large defect, failure of localization of the defect, or other co-morbid conditions such as chronic cough may be the most likely causes of recurrence of leak.Accurate localization of the site of lesion using a high-resolution computed tomography (CT) scan with magnetic resonance imaging (MRI) and confirmation of the site of leak by intraoperative Valsalva maneuver along with multilayered closure of the dural defect and post-operative lumbar drain appear to be essential for the successful endoscopic repair of CSF rhinorrhea.  Cerebrospinal fluid (CSF) rhinorrhea has been reported to be spontaneous or secondary to head trauma, surgery, neoplastic invasion, congenital malfor- mations; the most common causes being post-traumatic according to some authors and iatrogenic according to others ,2. The rA retrospective study of patients who presented in our unit with CSF rhinorrhea and underwent endoscopic repair was performed. Etiopathogenesis, outcome of treatment and causes of failure were noted. Our experience is presented in this article. A retrospective inpatient chart audit was performed for all cases of CSF leak repairs carried out in the ear, nose and throat (ENT) unit at Christian Medical College and Vellore Hospital from January 2002 to December 2009. All cases were subjected to a detailed history, regarding the duration of leak, mode of onset, etiology and history of meningitis. Following this, diagnostic nasal endoscopy was performed to confirm the side of leak and to rule out other nasal pathologies. Nasal endoscopy was not useful in identifying the exact site of the leak. Nasal secretion was collected and sent for glucose concentration analysis to explore the likelihood of CSF rhinorrhea. All patients had a high-resolution computed tomography (CT) scan , with adPatients were subjected to hypotensive general anesthesia. A lumbar drain was inserted and kept closed until the site of leak was identified. Fat and fascia lata were then harvested from the lateral side of the thigh. All operations were performed using standard endoscopic sinus surgery equipment without computer-assisted surgical navigation. The middle turbinate was excised and the suspected site of leak in the skull base was denuded of mucosa. The bulla ethmoidalis was opened in cases where the defect was found to be extending into the fovea ethmoidalis. The site of the leak was localized. In a few patients where an active leak was not seen, it was noted by Valsalva maneuver. The margins of the bony defect were then delineated .At this stage, the lumbar drain was opened and fat was then inserted into the defect. The fascia lata was placed over the defect and a prepared fibrin sealant consisting of lyophilized vapor heated sealer human protein concentrate (TISSEEL) was injected onto the edges of the fascia. The mucosa from the middle turbinate was then spread over it, and the remaining fibrin sealant was again injected around the edge of the harvested middle turbinate mucosa. In two patients, a piece of the bone from the excised middle turbinate was used to seal the defect in addition to the fascia. Surgicel was then spread over the fascia/mucosa layer, and finally a Bismuth Iodoform Paraffin Paste pack was placed in position.th and 7th post-operative day, respectively. Thigh sutures were removed on the 10th day. An endoscopic inspection of the operative site with removal of crusts was performed on the same day, leaving the operative site untouched. All patients were followed up for 6–12 months. Postoperatively, patients were advised to adhere to strict bed rest. They were also given TED stockings and subcutaneous low molecular weight heparin for 5 days postoperatively to prevent deep vein thrombosis. Stool softeners were also given.A lumbar drain and nasal pack were removed on the 5A total of 36 patients were identified in whom endoscopic CSF rhinorrhea repair was performed from January 2002 to December 2009. One patient had an additional external approach for frontal sinus CSF leak repair. Most patients were in their 30s, with the youngest being 8 months old and the oldest being 64 years. Twenty-eight of the subjects were women, and eight were men.Fifteen of the 36 patients had a right-sided leak, 18 had a left-sided leak, and three had bilateral leaks. It is also interesting to note that most of the post-traumatic leaks were left-sided. Twenty-two patients had a spontaneous onset of the CSF leak, while nine patients had a post-traumatic leak. Five patients were post-surgical: following functional endoscopic sinus surgery (FESS), excision of astrocytoma, meningocele, arachnoid cyst and hemangiopericytoma maxilla and skull base, respectively . The most common site of leak was the cribriform plate (61%), followed by isolated fovea ethmoidalis, and involvement of both cribriform plate and fovea. One patient had a posterior table of frontal sinus involvement and another had a leak in the lateral recess of the sphenoid sinus .Two patients had recurrent CSF leak 3 months after the initial endoscopic repair (6%). In the first patient who had a recurrence, the size of the bony defect was 3.5 cm with an intervening sliver of bone. In the second patient, although the size of the leak was only 1.5 cm, there was an associated history of persistent cough which required treatment. Both patients had their site of leak identified in the fovea ethmoidalis on revision surgery.CSF rhinorrhea occurs as a consequence of breakdown in the dura, arachnoid and a bony skull base defect, resulting in a fistula between the sub-arachnoid space and the nasal cavity.CSF rhinorrhea may occur directly through the anterior cranial fossa or indirectly from the middle or posterior fossa via the Eustachian tube. The diagnosis and localization of the leak is crucial for surgical management. The endoscopic examination of the nose is invaluable in confirming the site of leak prior to surgery. Preoperative assessment for the site of leak is performed using a high-resolution CT scan of the paranasal sinuses and anterior skull base. This is currently accepted as the imaging technique of choice. Additional MRI (with T2- weighted sequences) is indicated when parenchymal or meningeal herniation is suspected .3 consider intrathecal fluorescein as the most accurate modality for identification of the site of the leak. Intrathecal fluorescein, however, is controversial due to multiple reported complications (Banks ications . Seth inications . Presuttications . In our 7,8 for repair of defects greater than 3-4 cm. Additionally, co-morbid conditions such as chronic cough may contribute to raised intracranial tension and failure of the repair. The size of the defect appears to impact the surgical outcome, with larger defects likely to result in failure of repair and recurrent leaks. Composite osteomucosal or chondromucosal flaps have also been advocatedPresutti, in their 5-year retrospective study of 52 patients with endoscopic closure of CSF leak , had an The most common etiology of CSF rhinorrhea in our study was spontaneous, unlike reports from the West, and the condition is more often seen among women. Better results can be achieved when the following factors are taken into consideration: exact localization of site on MRI/CT prior to surgery, with intraoperative demonstration of the leak by Valsalva maneuver; a three-layer closure technique with fat, fascia, mucosa and fibrin sealant interposition is preferred for small leaks and a meticulous four-layer closure with use of cartilage to give added strength in the repair of large defects.


We compared the changes in cell morphology, total and differential cell counts between cerebrospinal fluid (CSF) samples analyzed within an hour of collection (fresh sample) and after the addition of autologous serum and storage for 24 h (stored sample) in 27 cattle with central nervous system disorders.There was a positive linear correlation between total and differential cell counts in the fresh and the stored samples. Cell morphology was preserved in all stored samples, except for increased vacuolization of mononuclear cells and cleaved nuclei of some small mononuclear cells. In the stored CSF samples, the total nucleated cell count and monocyte percentage were decreased , while the lymphocyte percentage was increased (P = 0.04). Mononuclear pleocytosis diagnosed in 20 fresh samples was cytologically confirmed in 12 of the 20 stored samples. In the remaining eight stored samples, the number of total nucleated cells was within the normal range. Neutrophilic pleocytosis was confirmed in all seven stored samples.The overall agreement rate between cytologic interpretation of the fresh and the stored CSF samples was 70 % (100 % for neutrophilic pleocytosis and 60 % for mononuclear pleocytosis).Adding 11 % of autologous serum to CSF samples might allow delayed analysis with a good agreement rate for CSF cytological interpretation. Caution is nonetheless warranted, as animal age, anamnesis, and neurological presentation need to be considered when interpreting stored CSF without pleocytosis.The online version of this article (doi:10.1186/s12917-015-0502-x) contains supplementary material, which is available to authorized users. Cerebrospinal fluid (CSF), formed by plasma ultrafiltration and active transport mechanism of substances across the blood–brain barrier, circulates in the subarachnoid space of the entire central nervous system (CNS) –5. BesidCFS analysis can be a useful tool in the diagnosis of neurological diseases in living animals –9 and caWith adequate restraint, CSF samples can be safely collected from either the cerebellomedullary cistern or the lumbosacral site in ruminants , 4, 9. BBuilding on the results of this previous work , we eval3 were included in the study.This prospective study was conducted in accordance with current animal welfare regulations . Cattle referred to the Teaching Hospital of the Department of Veterinary Science of Turin  for a neurological problem between March 2009 and March 2013 were selected for the study following physical and neurological examination, the latter by a board-certified neurologist (ADA). Complete blood count (CBC), biochemical profile  and CSF analysis were performed. In selected cases, blood gas analysis, coagulation profile, bacterial culture, synovial fluid analysis, cytologic evaluation, leukocyte immunophenotyping and postmortem examination were additionally performed to establish diagnosis. Only samples with a total cell count >10 cells/mmg for 10 min. CSF was aseptically collected under sedation from the lumbosacral site while the animal was standing, as previously described [Blood samples  were collected from the jugular vein into serum and EDTA vacuum tubes. Serum was separated by centrifugation at 1500 g for 5 min  of 250 μL CSF and stained with May-Grünwald Giemsa. Only samples with at least 10 cells per slide were used for the differential cell count. The percentages of neutrophils, eosinophils and mononuclear cells (lymphocytes and monocytes) were calculated [For the total nucleated cell count, 100 μL CSF was added to 100 μL Turk stain and placed on a Nageotte hemocytometer, and the number of cells in an eight by 10 rectangle was counted. Two slides, one for a differential cell count and one for morphological evaluation, were prepared by cytocentrifugation at 500 lculated . Total p3 and/or the protein content was >400 mg/L. Only samples with total and differential cell counts before and after storage were included.CSF samples were considered abnormal if the total cell count was >10 cells/ mms) was used to test the strength of correlations. Statistical significance was set at P <0.05.Data were analyzed using the R ver. 2. 1.0 freeware statistical software package. The Shapiro-Wilk normality test was used to determine whether the data followed a normal distribution. None of the data was normally distributed. Numerical data are presented as median, minimum and maximum. Not normally distributed data were analyzed using the Wilcoxon rank sum test. Spearman’s rank correlation coefficient . The majority of the animals were female  and beef cattle . Piedmontese breed accounted for 91 % (21/23). Other breeds included Italian Holstein-Friesian (n = 5), Blonde d’Aquitaine (n = 2), and crossbreed (n = 2). A definitive neurological diagnosis was available for all animals. An additional file shows this in more detail on the basis of the VITAMIN D algorithm  [see AddThe most frequent neuroanatomical location of lesions was the forebrain and brainstem (78 %).In all, 27 out of 30 samples met the inclusion criteria. Total protein concentration in the fresh samples was >400 mg/L in 18 animals. Total nucleated cell count was significantly decreased in the stored CSF samples (P = 0.01). Concerning the differential cell count, monocyte and lymphocyte percentages were significantly decreased (P = 0.03) and increased (P = 0.04) in the stored samples, respectively. No significant differences in neutrophil and mononuclear percentages were found between the fresh and the stored samples . There was a positive linear correlation between the total and differential cell counts in the fresh and the stored samples  were the same in both studies. We can only assume that many of the animals with neurological problems had been treated with antibiotics, steroids and non-steroidal anti-inflammatory drugs by the attending veterinarian before being referred. If we had had reliable information about their treatment history, we could have created two groups (treated and non-treated) and then attempted to confirm or reject the over mentioned assumption that previous treatments would potentially affect the degree of cell deterioration. Therefore, the lack of complete anamnestic information is the major limitation of this study.For the purposes of the present study, we used autologous serum rather than other stabilizing agents , as it is easier and more practical to obtain and handle in field conditions.Our findings support the recommendation to analyze CSF within 1 h of collection , 10, 12.


Torsion of the appendix testis is not an uncommon cause of acute hemiscrotum. It is frequently misdiagnosed as acute epididymitis, orchitis, or torsion of testis. Though conservative management is the treatment of choice for this condition, prompt surgical intervention is warranted when testicular torsion is suspected. We report a case of torsion of a large appendix testis misdiagnosed as pyocele. Emergency exploration of it revealed a large appendix testis with torsion and early features of gangrene. After excision of the appendix testis, the wound was closed with an open drain. The patient had an uneventful and smooth postoperative recovery. Torsion of the appendix testis is a common cause of scrotal pain in children. It occurs during the prepubertal years ages 7–14), often precipitated by trauma or exercise [4, often A 13-year-old boy presented to the surgical OPD (outpatient department) with history of a scrotal swelling on right side since 3 years, pain in right hemiscrotum for 10 days, and fever for 3 days. The patient started with a gradually increasing swelling of the right hemiscrotum for 3 years. 10 days before his presentation, he developed pain over the swelling which was dull aching and continuous in nature associated with fever which was mild and continuous. There was no precedent history of any trauma or exercise. On examination the patient had swelling of the right hemiscrotum with edema and erythema of the scrotal skin. Blue dot sign was absent. On palpation local temperature was raised, skin was tender, and testis was not palpable separately. Left side scrotum and testis were normal. On investigation hemoglobin was 12.8 g/dL, total leukocyte count was 12140 per microliter, and other blood parameters were within normal limits. A thick walled septated collection was found in the right tunica vaginalis testis with floating internal debris on ultrasonography . Both teEmergency exploration was done which revealed a large cyst of approximately 5 × 3.5 × 3 cm in size with a stalk which was arising from the upper pole of the testis . TorsionThe appendix of testis otherwise known as hydatid of Morgagni is a remnant of upper portion of the paramesonephric duct (Müllerian duct), whereas portion of the mesonephric duct, cranial to the testis, forms the appendix of epididymis . In 1913Torsion of appendix testis usually presents with sudden onset hemiscrotal pain without any systemic symptoms or urinary complaints. Clinically, the scrotum may be swollen and edematous with tenderness limited to the upper pole of the testis. Presence of a paratesticular nodule along with blue dot sign is pathognomonic for the diagnosis of this condition but this is found only in 21% of cases. Blue dot sign with a normally palpable, nontender testis usually excludes torsion of testis clinically but absence of ipsilateral cremasteric reflex is an indication for strong clinical suspicion of a testicular torsion .Ultrasonography with Doppler done early can be diagnostic. Delay in doing the test frequently leads to misdiagnosis of epididymitis or epididymoorchitis due to increased blood flow to the adjacent epididymis and testis with possibly a reactive hydrocele. An oedematous appendix and head of epididymis sometimes give a “Mickey Mouse” appearance in ultrasonography on transverse lie. Doppler sonography reveals normal flow pattern into the testis, with occasional hypervascularity due to reactive inflammation. Doppler study can achieve a sensitivity of 86% and a specificity of 100% in the diagnosis of testicular torsion . RadionuThe treatment for this condition is basically conservative with rest, observation, analgesics, and scrotal support. Surgical intervention is indicated when the diagnosis of testicular torsion cannot be ruled out and the symptoms are prolonged and do not resolve spontaneously. The gangrenous appendage can be excised easily through a small scrotal incision, resulting in prompt relief of symptoms.Torsion of appendix testis is a common cause of acute scrotal pain, frequently misdiagnosed because of its atypical presentation. The classical finding of torsion of appendix testis with a typical blue dot sign is seen only in few cases. Strong clinical suspicion with a high resolution sonography with Doppler can diagnose this condition but prompt surgical intervention is required in clinically equivocal cases.


CSF abnormalities have been reported in CSF leakage syndrome. However, the mechanism for these CSF changes is actually unknown and they may indicate impaired CSF flow or blood-CSF barrier. Angiopoietin-2 (Ang-2), a protein which is expressed and released by endothelial cells, has been associated with increased vascular permeability. In the assumption that CSF changes are due to an impaired blood-CSF barrier, we hypothesized that subjects with persistent CSF leakage may have increased CSF Ang-2 levels. We enrolled 10 subjects with a clinically definite diagnosis of persisting CSF leakage syndrome and 10 control subjects. In CSF analyses, CSF to serum albumin ratio  was the most frequently increased parameter indicating a disturbed blood-CSF barrier function. Comparison of the mean CSF Ang-2 levels, CSF to serum Ang-2 ratio (QAng-2), and QAng-2/Qalb between the control and CSF leakage patients did not show any significant difference. We suggest that the increase of Qalb results from a low CSF flow. Future studies with phase contrast-MRI in conjunction with CSF analyses before and after epidural blood patch treatment are required to address this question. It would be of particular interest whether Qalb can be used as a marker for successful nontargeted epidural blood patch treatment. Persistent cerebrospinal fluid (CSF) leakage syndrome is a rare cause of chronic headache. The common clinical presentation is orthostatic headache of dull aching nature, which, according to the International Headache Society Classification, occurs or worsens in less than 15 minutes after assuming the upright position and disappears or improves in less than 30 minutes after recumbency . Elevatehttp://www.ihs-classification.org/en/02_klassifikation/03_teil2/07.02.03_nonvascular.html). Beside the clinical symptoms, diagnosis was based on the findings in intrathecal gadolinium- (gd-) enhanced magnet resonance (MR) myelography and/or CSF scintigraphy  subjects with a clinically definite diagnosis of persisting CSF leakage syndrome were enrolled from February 2009 to April 2011 . Exclusion of one outlier  did not result in a significant correlation either .Demographic and clinical characteristics of the enrolled subjects with persistent CSF leakage and those of controls are summarized in Orthostatic headache, which mostly occurred occipitally, was a constant and most reliable symptom in all CSF leakage subjects. Despite the typical orthostatic nature of headache, time from onset of complaints to diagnosis varied considerably . Qalb is a generally accepted measure of the blood-CSF barrier function and was increased in 7 subjects with CSF leakage [CSF analyses revealed lymphocytic pleocytosis in 4 cases with a cell count up to 23 leukocytes per  leakage . In the p = 0.272, U = 35.0; Tables p = 0.405, U = 38.5; Tables 3 of Ang-2 . Normalizing of QAng-2 by dividing by Qalb  did not yield a statistical difference either  and CSF Ang-2 concentrations  with Qalb. CSF IgG, IgA, and IgM concentrations and CSF albumin did not correlate with CSF Ang-2 concentrations or QAng-2 either (data not shown).The mean CSF Ang-2 concentration of all subjects was 0.31 ± SD 0.15 pg/mL without any difference between control and CSF leakage subjects  and QAng-2/Qalb  with CSF lactate concentrations, while there was no significant correlation for CSF Ang-2 with CSF lactate concentration . There were no significant relationships when cell count was correlated with CSF Ang-2 concentrations, QAng-2, or QAng-2/Qalb . Furthermore, we could not find any association of Ang-2 with the obtained clinical or radiologic parameters.We did not detect any difference in mean CSF lactate concentrations between the two cohorts  is the most abundant protein in CSF comprising up to 67% of total CSF proteins and exclusively originates from blood , 17. To Ang-2 forms multimeric structures composed of monomers of 55 kDa and the native form is mainly present as disulfide-linked dimers, but variable oligomeric forms may exist as well . So far,Among other factors, biomechanical stimulation has been shown to be a reliable trigger for the release of Ang-2 . This isAng-2 and related molecules are of high interest for the CSF compartment and may help to elucidate our understanding of the mechanism of the brain barriers and CSF hemodynamics. In this pilot study, we did not find any involvement of Ang-2 in CSF leakage syndrome. There are several limitations inherent in this study. One limitation concerns the exploratory analysis of correlations which are post hoc exploratory analyses that need validation from independent studies. The second limitation is that the control group consisted of subjects who were not healthy. Therefore, a difference in CSF Ang-2 levels between healthy controls and subjects with CSF leakage syndrome cannot be excluded. The control group was deliberately chosen in order to find out whether other conditions, which may lead to an increase of Qalb, will be also accompanied by changes in CSF Ang-2 levels. Unexpectedly, we did not find any difference in CSF Ang-2 levels despite the significantly higher Qalb values and ratios of immunoglobulins in subjects with CSF leakage syndrome suggesting that secretion and elimination of Ang-2 are different from these proteins. Further limitations of the study are the small number of enrolled patients and that we assessed only a single marker of endothelial activation.