However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant.I The hypocretins (Hcrt), which are also known as orexins, consist of two neuropeptides, orexin-A/hypocretin-1 (Orex-A/Hcrt-1) and orexin-B/hypocretin-2 (Orex-B/Hcrt-2). They originated from the same precursor gene (preprohypocretin) present in a few thousand neurons localized in the perifornical area of the lateral hypothalamus14578More than a decade ago, a dramatic decrease (85–95%) in the number of Hcrt neurons was identified as the cause of human narcolepsy with cataplexy101314125 Radio-Immuno-Assay (RIA) kit available from Phoenix Pharmaceuticals : values below 110 pg/ml (using the Stanford reference sample) are highly specific to NT11Orex-A/Hcrt-1 can be measured in crude CSF using the ISince it involves the use of radioactive materials, RIA requires specific precautions for the use and disposal of these materials. RIAs have many other disadvantages, including possible cross reactivity with matrix constituents, great variability between batches, and low precision and accuracy. To avoid these constraints, new methods of quantification are therefore required for measuring CSF orexin levels. Approaches such as targeted mass spectrometry (MS) and especially multiple reaction monitoring (MRM) using internal standards can be used to develop quantitative assays with a high level of specificity, precision and robustness21The innovative method to quantify Orex-A/Hcrt-1 in human CSF described in the present study is based on the use of liquid chromatography combined with mass spectrometry (LC-MS) in the MRM mode with internal standards to ensure absolute quantification. The performance of the method was tested in terms of specificity, reproducibility, and repeatability, as well as its limit of blank (LOB), of detection (LOD), of quantification (LOQ) and the linearity. A clinical validation was then performed comparing LC-MRM and RIA in a selected population of patients with either normal or hypocretin-deficient conditions.4+ (m/z 891) and [M+5H]5+ (m/z 713), which were selected as precursor ions  at a flow rate of 0.1 mL/min . The totThe analytical precision of the calibration standards (as determined by performing reproducibility tests) ranged from 3 to 14% . In humaIn order to determine the selectivity of the LC-MRM method, MRM chromatograms of three blank CSF samples were compared with that obtained on a blank CSF sample spiked with 50 pg/mL of light orexin-A standard. The fact that no MRM signal was detected in the chromatograms of the blank CSF samples at the orexin-A retention time confirmed the high selectivity of the method.2 > 0.99  and the LOQ was 0.025 pg/mL, based on 3 replicates and 5 measurements. The absence of memory/carry-over effects was confirmed by re-analyzing the calibrator/calibration points without adding SIS at the end of the analytical sequence. The results of LC-MRM quantification were not affected by the dilution of the samples, since the linear regression coefficients consistently showed a high level of linearity (R2 > 0.99). In addition, the fact that the inter-day coefficient of variation was lower than 20% on the 4–20 pg/mL range in the case of all the calibration points shows that the orexin-A standard was highly stable at a temperature of 4 °C.The calibration curves were generated by performing linear regression analysis and plotting the peak area ratios (light orexin-A standard/labeled orexin) versus the concentration ratios . Based o2 > 0.99 . The LOBComparison of the MS peak areas of the SIS spiked either in blank CSF or in H2O/ACN/FA 80/20/0.1, v/v/v revealed a matrix related to ion suppression of 97% of the signal intensity. This difference was mainly due to the previously described high molecular complexity of the CSF matrix in comparison with that of the solvent, which impacted the process of ionizationCSF samples with normal orexin-A levels  and low orexin-A levels  determined using the standard RIA method were reanalyzed using the LC-MRM method. A significant between-group differences in the CSF orexin-A levels were observed with the two methods  . To miniThe reliable innovative quantitative approach developed and validated here for measuring CSF Orexin-A/Hcrt-1 using LC-MRM methods was found to show high specificity, reproducibility, repeatability, linearity and low limits of detection and quantification. This new method could well differentiate two populations of patients with normal and low CSF orexin-A (i.e. patients with NT1) but it generated orexin-A values that were more widely distributed.The demand for means of measuring CSF orexin-A levels in the context of sleep disorders has been increasing since the recent publication of the revised International Classification of Sleep Disorders, in which narcolepsy has been divided into type 1 and 2 depending on CSF hypocretin-1 levels13142526125 RIA is the standard clinical method currently used to measure orexin-A in CSF because this method is sufficiently sensitive to detect this analyte, which is present in humans in very low amounts. However, RIA has some major drawbacks including poor inter-batch intermediate precision, and the need to take highly stringent and costly precautions for the use and disposal of radioactive material. In addition, this method based on immunocompetition is highly susceptible to interference and liable to generate cross-reactions with metabolized fragments, circulating receptors and other analogous endogenous proteins28IOne interesting alternative to immunodetection methods is represented by quantitative mass spectrometry in MRM mode that improves assay specificity. This bioanalytical approach can be used to unambiguously identify the target with a high level of reproducibility and robustnessAs all the patients with NT1 previously tested have been found to show low CSF orexin-A levels <110 pg/ml) due to the loss of approximately 90% of the orexin-expressing neurons, the highly sensitive standard RIA method has been widely used as a diagnostic tool for discriminating between patients with NT1 and other conditions pg/ml du11116In conclusion, the reliable innovative method based on LC-MRM developed and tested here for quantifying CSF orexin-A may therefore well replace RIA in the future. Between-method comparisons on larger populations, including patients with NT2, with other central hypersomnias, with intermediate CSF orexin-A levels based on the RIA findings are needed to replicate and extend our preliminary findings. Further studies are also required to define pathological cutoffs using this alternative method, and to explore the role of orexin-A in the neurobiology of sleep in neurodegenerative disorders.125 RIA; and 2) 22 drug-free patients  with various neurological disorders but not NT1. These patients underwent a lumbar puncture prior to the study and were found to have CSF Hcrt-1 levels >200 pg/ml as determined by performing direct I125 RIA. Demographic, clinical and neurophysiological parameters of these patients are presented in In order to test the MRM method, a pool of CSF samples from patients (n = 20) with various neurological disorders but not NT1 was used (RIA concentration of orexin A>200 pg/mL). CSF was collected in polypropylene tubes under standardized conditions. Each CSF sample was sent to the laboratory within 4 hours of being collected and centrifuged at 1000 g for 10 minutes at 4 °C. CSF was aliquoted into 1.5-mL polypropylene tubes and stored at −80 °C until further analysis. To test the validity of the MRM method the following two clinical population samples were set up: 1) 22 drug-free patients  with typical NT1 defined by the presence of sleepiness, clear-cut cataplexy, human leukocyte antigen (HLA) DQB1*0602 positivity, at least two sleep onset REM periods and a mean sleep latency below 8 min during the multiple sleep latency test. All these patients underwent a lumbar puncture prior to the study and were found to have low CSF Orex-A/Hcrt-1 levels (<110 pg/ml) as determined by performing direct I2) was purchased from Phoenix Scientific and internal orexin standard (pyroEPL*PDCCRQKTCSCRL*YEL*L*HGAGNHAAGIL*T*L-NH2)  Leu* with a purity >97% as assessed by performing RP-HPLC and MS was purchased from Eurogentec . Standard peptides were synthesized in lyophilized form with the same amino acid sequence (33 AA), folding pattern , N-terminal pyroglutamination and C-terminal amidation as the RIA standard . Appropriate dilutions of 200 μg/mL orexin-A stock solutions were made to prepare the calibration curve in the concentration range of 0–200 pg/mL . Samples were spiked with the internal standard at a final concentration of 50 pg/mL.Human orexin-A standard (pyroEPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL-NHstandard . Purchas4OH ref. 338818–100ML) (30/65/5 v/v/v), and 200 μL of water. Bound proteins were eluted in two steps with 50 μL methanol/acidified water with 0.1% TFA (90/10 v/v). The eluted samples were dried on a vacuum concentrator  and stored at −80 °C. Prior to LC-MRM analysis, the samples were resuspended with 20 μL of H2O/ACN/FA (80:20:0.1 v/v/v), vortexed at 1000 rpm for 10 minutes and transferred onto a deactivated glass vial insert .500 μL of CSF (corresponding on average to 350 μg of proteins) were acidified with orthophosphoric acid  . Simultaneously, the Oasis® SPE HLB μElution plate (ref. 186001828BA) was preconditioned with 350 μL of methanol , followed by 350 μL of water. Liquids were handled with a vacuum (Waters manifold for 96-well plates). Acidified samples were loaded onto the plate and washed successively with 200 μL of water, 200 μL methanol/water/ammonium hydroxide  coupled to a 6490 triple quadrupole mass spectrometer with an Electro Spray Ionisation (ESI) source operating in the positive mode and in the MRM mode . Data acquisition and analysis were performed using MassHunter workstation data acquisition and Masshunter Quantitative Analysis (version B07), respectively. Liquid chromatography (LC) separation was performed with a reversed-phase Zorbax 300 SB-C18 column kept at a temperature of 50 °C with a flow rate of 0.1 mL/min. A maximum pressure was set at 400 bars. The mobile phases consisted in (A) 0.1% FA in water containing 3% acetonitrile and (B) 0.1% formic acid in acetonitrile. The solvent gradient was set as follows : 0 min, 15; 0.2 min, 25; 4 min, 38; 4.5 min, 100; 5.5 min, 100; 6 min, 15 min. The ESI was set up using the following settings: capillary voltage 3000 V, gas flow 12 L/min at a temperature of 150 °C, sheath gas flow rate 7 L/min at a temperature of 250 °C, nebulizer at a pressure of 45 psi. Precursor ions were transferred into the first quadrupole with a cell accelerator voltage of 5 V, a high pressure ion funnel RF set at 150 V and a low pressure ion funnel RF set at 110 V. Collision energies were optimized for the peptide transitions of interest . All traCalibration curves were generate over a 0–200 pg/mL range  in water/acetonitrile/formic acid  with unlabeled Orexin-A and spiked with SIS (50 pg/mL). Calibrator standards were vortexed for 10 minutes and transferred to deactivated glass vial inserts prior to (n = 5) LC-MRM analysis. The validity of the method was checked by carrying out linearity tests as defined in the EP17 guidelines published by the Clinical and Laboratory Standards Institute (CLSI)The effects of sample dilution were assessed by analyzing a CSF sample spiked with unlabeled light orexin-A standard (100 pg/mL) and diluted serially 3-, 6-, 9,- and 12-fold with a pool of blank CSF. A 500-μL aliquot of each dilution was taken and spiked with the SIS (50 pg/mL) before preparing the samples and performing LC-MRM measurements. The short-term stability of the analyte was assessed by measuring the calibration standards after storing them in the LC auto-sampler for several hours at 4 °C. The peak area ratios obtained  on orexin-A were analyzed to determine the stability of the analyte.The repeatability of the LC-MS procedure was tested on all the calibration standards by measuring them five times in a row. The Relative Standard Deviation (RSD) and coefficient of variation (CV) were calculated from the area ratios obtained on unlabeled orexin-A/SIS. The repeatability was also determined in CSF samples. Pooled human CSF containing >200 pg/mL of orexin A quantified by RIA was measured five times and individual CSF samples (n = 10) with concentrations of orexin A ranging from 202 pg/mL to 391 pg/mL were measured in duplicate. To assess the reproducibility, calibration standards were prepared independently on three different days and measured five times each, and the inter-day precision was then calculated.To assess the accuracy of the method, pooled blank CSF was spiked with 14, 25 and 50 mL of orexin A and all the samples were spiked with SIS (50 pg/mL). Sample preparation and MRM measurement were carried out as described in the case of CSF samples/above. The accuracy was calculated from the measured and theoretical concentrations, using the formula “Accuracy = [/(expected concentration)] ×100”.The selectivity of the method was tested by comparing blank CSF samples and samples of CSF spiked with the orexin-A standard. MRM chromatograms of blank CSF samples were examined to detect any interferences in the retention time window of the orexin-A standard. Carry-over was determined by analyzing blank samples directly after LC-MRM runs with the highest calibration standards.2O/ACN/FA .The occurrence of matrix effects was assessed by comparing the peak areas of the blank CSF spiked with SIS and spiked H125 RIA kits from Phoenix Pharmaceuticals  in line with the manufacturer’s instructions. CSF Orexin-A levels below 110 pg/ml were defined as low, those ranging between 110 and 200 as intermediate, and those over 200 pg/ml as normal. All the values obtained were back-referenced to Stanford reference samples .Orexin-A quantification was performed in duplicate on CSF samples obtained from all the patients using IPercentages were used for categorical variables and medians and ranges for quantitative variables; the distribution of continuous variables was mostly skewed, according to the Shapiro-Wilk test. Mann-Whitney test were used to compare categorical variables between groups. Spearman’s rank correlation coefficient was used to compare two continuous variables. Significance level was set at p < 0.05. The Bland and Altman plotThe study protocol was approved by the local ethical committee (Comité de Protection des Personnes – Ile de France 06). The methods were carried out in accordance with the approved guidelines and regulations. Each participant signed legal consent forms. Informed consent was obtained from all subjects.www.biobanques.eu).The samples/specimens were stored in a registered biological collection (#DC-2008-417) at the Montpellier CHU’s certified NFS 96-900 biobank  in cerebrospinal fluid. Sci. Rep. 6, 25162; doi: 10.1038/srep25162 (2016).


Introduction: To achieve personal goals in exercise task completion, exercisers have to regulate, distribute, and manage their effort. In endurance sports, it has become very commonplace for athletes to consult task-related feedback on external devices to do so. The aim of the present study was to explore the importance of the presence of this information by examining the influence of the absence of commonly available task-related feedback on effort distribution and performance in experienced endurance athletes.Methods: A 20-km cycling time trial was performed. Twenty Participants from a homogenous cyclist population were appointed to a group that did not receive any feedback (NoF), or a group that could consult task-related feedback  continuously during their trial (FF).Results: The distribution of power output (PO) differed between groups. Most evident is the spurt at the end of the trial of FF, which was not incorporated by NoF. Nevertheless, no between-group differences were found in performance time (FF: 28.86 ± 3.68 vs. NoF: 30.95 ± 2.77 min) and mean PO controlled by body mass (FF: 3.61 ± 0.60 vs. NoF: 3.43 ± 0.38 W/kg). Also, no differences in rating of perceived exertion scores were found.Conclusion: The current study provides a first indication that prior knowledge of task demands together with reliance on bodily and environmental information can be sufficient for experienced athletes to come to comparable time trial performances. This questions the necessity of the presence of in-race instantaneous task-related feedback via external devices for maximizing performance. Moreover, it seems that different pacing strategies emerge depending on sources of information available to experienced athletes. The contribution of such feedback has been critically examined in existing research in the area of deception and pacing strategies  than on task-related feedback, and were conservative when setting a pacing strategy  to volitional exhaustion to determine maximal cardiorespiratory values. Furthermore, each participant performed a 20-km cycling time trial as fast as possible while being randomly allocated to an experimental group that received no feedback (NoF) or a control group that was allowed full feedback (FF). Participants did not perform a familiarization trial, as we were interested in imposing a relatively novel task such as cyclists in the Grand Tours are experiencing: each time trial or stage is different, cycled under different conditions. Imposing a familiarized time trial condition in a repeated measures design—instead of a rather novel task in our current design—would compromise ecological validity of the study when interested in road cycling. In addition, we expected that the importance of feedback would be higher in a rather novel task.All participants completed an incremental cycling exercise test  that would elicit ~65–70% of maximal oxygen consumption . HR was recorded every 2 s .The ICET was performed on a cycle ergometer  at a pedal frequency of 80 rpm. After a 10 min warming-up at a work rate of 150 W and 1 min passive rest, the test started on an exercise intensity which was equivalent to 3 W/kgParticipants conducted the trial using their own bike mounted on an ergotrainer . A power meter  was used to record PO, time and covered distance during each trial for subsequent data-analysis. Previous research has shown that this power meter provides valid and reliable PO measurements in laboratory tests , task-related feedback was provided during the entire trial. As a result, they could continuously consult their PO, speed, HR, cadence, covered distance, and time elapsed. Participants appointed to NoF (n = 10) did not receive any feedback during the trial (“blinded”). They only knew they had to cycle 20-km as fast as possible and a stop-sign would be provided when they covered this distance.For participants allocated to FF  and -goal  were the same for both groups. However, whereas NoF-participants were reliant on their own resources  during their trial, participants within FF were able to evaluate their perceived bodily exertion, interim performance, and future task demands via external devices.rel, –], as well as divided by their peak PO established during ICET .To examine the pacing strategy and performance of both groups over the trial, participants' PO-distribution curves were considered. In order to compare the PO-distribution between FF and NoF, the mean PO-distribution curves of both groups over the entire trial were established. To do so, first we normalized the PO-distribution curve of each participant to 1250 data points. This number of data points was based on the completion time in seconds of the fastest participant. Following this, the power data was controlled for body mass differences between participants . In addition to considering PO-data , we were also interested in how the groups relatively distributed their PO over the trial and how the groups' PO was related to the maximal PO-capacity of the participants within the groups. As a consequence, participants' PO throughout the trial was divided by their mean PO over the trial  were used. Furthermore, to consider whether there were differences in PO between and within groups at different intervals within the trial, the PO- and POrel-distributions were divided into 10 equal-sized segments . Also, paired differences between neighboring 10%-segments  were calculated  to examine whether PO-changes over subsequent 10%-segments within the groups differ between the groups. Finally, to consider whether RPE differed between groups, RPE group means were calculated for each time the participants rated their perceived exertion during the trial.To compare overall performance between FF and NoF, calculated group-means of PO and POt-tests were conducted. Repeated measures ANOVA's were used to examine the effects of feedback condition on PO at different parts during the race  and PO-changes over the race . If a main effect for group was found, Bonferroni corrected independent t-tests were performed to consider within which specific segment(s) PO differed between groups. If a main effect for segment was found, Bonferroni corrected paired-samples t-tests were performed to consider which specific neighboring 10%-segments of PO differed from each other within groups.To determine whether there were between-group differences in anthropometric characteristics, and ICET- and overall-performance measures, independent t-tests on mean RPE-scores were performed. As RPE was asked at irregular intervals, no repeated measures ANOVA was applied for the RPE-scores analysis.Finally, to consider differences in perceived exertion between groups, independent Effect sizes were calculated as appropriate. An effect size of 0.2 is considered as small, 0.5 as medium, and >0.8 as large Cohen, . For allThe group characteristics are provided in Table *PPO-boundary per group (dotted lines) is incorporated. The mean PO-distribution curve of FF is usually above or at the 70%*PPO-boundary, whereas the curve of NoF is usually situated at or below the boundary. Nevertheless, the higher mean POICET in FF [0.73 ± 0.06 (–)], compared to NoF [0.68 ± 0.06 (–)], was not significant, but accompanied by a large effect size (Cohen's d = 0.85). Also, differences in mean PT  and mean PO  between groups were not significant, which indicates an absence of performance differences between groups.Figure rel per group. A segment main effect was found for both PO and POrel within FF . Post-hoc comparisons revealed that mean PO in FF was higher in S10, compared to S9, for both PO  and POrel , whereas mean PO in S3 was lower than in S2 for PO  and nearly for POrel . There was no significant main effect for NoF.Figure F(1.74) = 3.97; P = 0.03] and POrel . Post-hoc comparisons revealed that mean PO in S10 was higher in FF, compared to NoF, for POrel  and nearly for PO , whereas mean PO in S5 was higher in NoF for POrel .A group by segment interaction effect was found for both PO  and POrel . Post-hoc comparisons revealed that the mean change in PO was higher in FF, compared to NoF, for both PO  and POrel in CS9 .Table The segment analysis indicates that the PO-distribution of the groups differed from each other. Most evident is the spurt at the end of the trial of FF, which was not incorporated by NoF. In contrast, NoF increased their PO halfway through the trial and FF did not.No differences in perceived exertion scores were found  were compared with a group for whom task-related feedback was provided during the entire trial (FF). The results show no spurt at the end of the trial of NoF, whereas FF incorporated an end spurt. Notwithstanding this and other differences in pacing strategy between groups, no difference in overall performance between groups was found. This supports our hypothesis to find no inferior performance in NoF compared to FF. This finding suggests that in middle distance exercise, experienced athletes do not need task-related feedback from external devices to successfully complete a task of which the demands are known. However, the difference in pacing behavior visible toward the end of the race indicates that task-related feedback influences certain aspects of decision-making regarding how and when to invest the available energy over the race.*PPO-boundary, whereas FF usually exercised above or at the boundary. Although this finding could suggest that NoF might have included some cautiousness within their pacing strategy, between group analyses of overall performance, PO-segments and RPE did not indicate an obvious structural conservativeness in NoF's pacing strategy compared to FF.The lack of performance differences between groups contrasts with the suggestion that cautiousness and a slower speed of completion—designed to reduce errors —is typically observed in performing motor tasks someone is unfamiliar with  strategy is often observed in endurance exercise  and personal goals  and all-out paced  strategy. If this is the case, participants in NoF possibly pursued a particular relatively steady but challenging pace they expected to be sustainable for their estimated durations of the trial  influences effort distribution; which was most obvious during the end phase of the trial. Exercising some cautiousness and (consequently) making situation-based (pre-planned) adjustments to the pacing strategy were proposed as possible consequences of the absence of distance feedback, but our results are not fully conclusive about this. It has already been demonstrated that fit participants with limited specific endurance sports experience were cautious during initial trials (Foster et al., FH and BS designed the study protocol, BS and RP conducted the initial data analysis. All authors contributed to interpretation and conception of the work as well as further development of the proposed ideas. BS wrote a first draft of the work. All authors were involved in further drafting, editing, and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Canine intervertebral disc πherniation causes a naturally-occurring spinal cord injury (SCI) that bears critical similarities to human SCI with respect to both injury pathomechanisms and treatment. As such, it has tremendous potential to enhance our understanding of injury biology and the preclinical evaluation of novel therapies. Currently, there is limited understanding of the role of arachidonic acid metabolites in canine SCI.The CSF concentrations of PLA2 and PGE2 were higher in SCI dogs compared to control dogs , but CSF LCT4 concentration in SCI dogs was significantly lower than that in control dogs (p < 0.0001). Prostaglandin E2 concentration in the CSF was significantly and positively associated with increased severity of SCI at the time of sampling (p = 0.041) and recovery 42 days post-injury (p = 0.006), as measured by ordinal behavioral scores.Arachidonic acid metabolism is altered in dogs with SCI, and these data suggest that these AA metabolites reflect injury severity and recovery, paralleling data from other model systems. Several experimental animal models of spinal cord injury (SCI) have been established, including contusion, laceration, clip compression, and crush in a variety of species –3. TheseCanine intervertebral disc herniation (IVDH) causes a naturally-occurring form of SCI that bears critical similarities to human SCI with respect to both injury pathomechanisms and treatment. The resulting SCI occurs spontaneously, consists of varying components of compression and contusion, and is treated with a combination of decompressive surgery and physical rehabilitation , 6. HistThe arachidonic acid (AA) pathway is a critical mediator of secondary SCI and an attractive target for pharmacologic interventions. Metabolism of AA is ubiquitous; this polyunsaturated fatty acid is released from cell membranes by phospholipase A2 . In rodIn this study, we assessed alterations of AA metabolism after SCI in dogs with IVDH by using enzyme-linked immunosorbent assays (ELISA) to measure CSF concentrations of PLA2, leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). We selected these mediators because they represent critical nodes in the AA pathway that are altered in experimental SCI. Our primary objective in this exploratory study was to determine if there were higher concentrations of AA metabolites in the CSF of dogs with SCI than in healthy control dog CSF. Our secondary objective was to determine if, in dogs with SCI, CSF AA pathway metabolites were correlated with functional deficits at the time of sampling and 42-day post-injury recovery as measured by a validated ordinal score.Sample size was determined a priori and was based on previous studies that examined inflammatory mediators  in the CSF of dogs with IVDH-associated SCIs compared to CSF of healthy control dogs , 25. In A repository of CSF aliquots collected from the cerebellomedullary cistern, stored at −80 °C, and housed at Texas A&M University since December 2009 was screened in February 2014 for samples from dogs with IVDH-associated SCI that met the following inclusion criteria: (1) lesion between T3 and L5 vertebrae; (2) neurologic impairment of <7 days duration; (3) surgical decompression of IVDH with post-operative rehabilitation; and (4) complete medical records including neurologic score at admission and follow-up scoring at day 42 post-surgery. Dogs that were part of on-going clinical trials or had a myelogram performed as part of pre-operative diagnostics were excluded from this study.Healthy control CSF samples were collected from purpose-bred dogs with normal physical and neurologic exams, normal complete blood counts, and normal serum biochemical analysis. All CSF samples from healthy control dogs were collected and stored in the same manner as described for dogs with IVDH-associated SCI and were required to have a normal total nucleated cell count (TNCC) (<5 cells/µL) and total protein concentration (<35 mg/dL).Procedures in dogs with naturally-occurring SCI were performed with owner consent and consisted of standard medical and surgical care. Purpose-bred dogs were obtained and used with approval from the Texas A&M University Animal Care and Use Committee . All studies adhered to the National Institutes of Health Guide for Care and Use of Laboratory Animals.Dogs with SCI underwent complete physical examination, neurologic examination, complete blood count, and serum biochemistry prior to anesthesia. Data including age, gender, duration of SCI, and recent delivery of non-steroidal anti-inflammatory drugs (NSAIDs) or glucocorticoids (GCs) were collected Table . Dogs weFollowing surgery, dogs were recovered and provided intravenous fentanyl citrate  analgesia and bladder evacuation if unable to voluntarily void. Twenty-four hours later, physical rehabilitation consisting of supported overland walking, passive range of motion, and standing strength exercises were initiated. Dogs were released to their owner’s care after pain control was achieved via oral analgesics  and urine could be voluntarily voided or the bladder manually expressed. The owners continued physical rehabilitation exercises for 6 weeks post-operatively.Two separate ordinal SCI scores were used in this study, and were applied at initial evaluation and at a 42-day post-SCI re-evaluation. The modified Frankel score (MFS), and the Texas Spinal Cord Injury Score (TSCIS) have both been validated previously in dogs with IVDH-associated SCI and have been shown to have excellent inter-rater agreement, correlate well with MRI-based measures of SCI, and predict 42-day post-SCI motor outcome . For botThe MFS was used as a coarse ordinal system to stratify injured dogs into groups that parallel those in the American Spinal Cord Injury Association Impairment Scale (ASIA). The MFS consists of six strata where 0 = paraplegic with absent pain sensation from the hindquarters; 1 = paraplegic with pain sensation intact to severe stimuli; 2 = parapaplegic with intact sensation for mild stimuli; 3 = non-ambulatory paraparetic; 4 = ambulatory paraparetic; and 5 = signs consistent with spinal pain only.The TSCIS was developed as a more refined system than the MFS and was used for all analyses that did not require stratification into broad functional categories. With this system, individual limbs are assessed independently and given a score based on sensation, gait, and proprioceptive placing. Sensation was scored as 0 = absent, 1 = sensation present to severe stimuli, but absent for mild stimuli, and 2 = sensation intact. Proprioceptive placing was scored as 0 when absent, 1 when delayed , and 2 when considered normal. For gait assessment, scores ranged from 0 to 6 for each limb as follows: 0 = no voluntary movement present when supported; 1 = intact limb protraction with no ground clearance; 2 = intact limb protraction with inconsistent ground clearance; 3 = intact limb protraction with consistent ground clearance; 4 = ambulatory with moderate paresis/ataxia ; 5 = ambulatory with mild paresis/ataxia ; and 6 = normal gait.Cerebrospinal fluid concentrations of PLA2, LTC4, and PGE2 were measured using commercially available ELISA . The PLA2 ELISA (for the lipoprotein-associated isoform), catalog # MBS015390, was performed following the manufacturer’s protocol, using a standard curve ranging from 800 to 25 ng/mL. The LTC4 and PGE2 ELISAs  were also performed following the manufacturer’s protocol, and the standard curves used were 31.2–1000 pg/mL for LTC4 and 31.25–2000 pg/mL for PGE2. All control CSF samples were run as technical duplicates. A single run was performed on injured dogs samples, because volume available was limited.The primary objective of this study was to determine whether the concentrations of the AA pathway metabolites in CSF are associated with functional recovery status at 42 days. These relationships were explored first by using univariable linear regression with TSCIS at 42 days as the outcome (dependent variable). Multivariable linear regression was then used to examine the effects of the various possible interactions including initial injury severity, time delay between injury and sampling, NSAID and GC administration, through their inclusion as covariates. All analyses were conducted using commercially available software .Secondary objectives of this study were to explore relationships between injury and AA metabolite concentrations. We compared the CSF metabolite concentrations between control and SCI dogs using Mann–Whitney tests. Association between AA metabolite concentration and cell count in the CSF and with SCI severity at presentation were analyzed using linear regression. Figures were generated using GraphPad Prism, version 6.0 .There were 44 dogs in the SCI group Table . The medThe 21 control dogs had a median age of 3 years (range 1–4). The 2 most common breeds were Labrador retriever and beagle , the median red blood cell count (RBC) was 10 cells/µL  and the median total protein concentration was 18 mg/dL (range 9–94 mg/dL). Twelve dogs had pleocytosis (TNCC > 5 cells/μL); of these the median percentage of neutrophils was 49 % (range 0–85 %), monocytes 24.5 % (range 0–100 %), lymphocytes 12 % (range 0–63 %), and eosinophils was 0 % (range 0–3 %). No pleocytosis was detected in control CSF samples. In the control group, the median TNCC was 0 cells/µL (range 0–2 cells/µL), the median RBC was 3 cells/µL (range 0–730 cells/µL) and the median total protein was 26 mg/dL (range 10–35 mg/dL).There was significantly higher CSF PLA2 concentration (p = 0.0370) in dogs with SCI  compared to control dogs , PGE2 and CSF total protein , and PGE2 and CSF RBCs .We explored associations between CSF analytes including TNCC, RBC, total protein concentration, and percentage of leukocytes and AA pathway metabolite concentrations in the CSF of SCI dogs. The only significant associations were between PLA2 and TNCC . Because of the low number of SCI dogs in which PGE2 reached detectable concentrations we examined the sensitivity of this result to the numerous null values by repeating the test but including only the dogs with detectable values; both univariable  and multivariable  analysis revealed a non-significant association, although this may also result from the much reduced power of these tests.Prostaglandin E2 concentration in the CSF was significantly and positively associated with increasing severity of SCI at the time of sampling, as measured by the MFS in univariate and multivariate models Fig. a, b. TheThis study broadly compared measures of AA metabolism in dogs after naturally-occurring SCI with that in healthy control dogs. The concentration of PLA2, which frees AA from phospholipid membranes, was significantly higher in SCI versus control dogs, and had a weak negative association with the total nucleated cell count in the CSF. The CSF concentration of LTC4, a pro-inflammatory leukotriene, was significantly lower in dogs with naturally-occurring SCI compared to control dogs. The CSF concentration of PGE2 was significantly higher in SCI dogs compared to control dogs, and significant relationships existed between CSF PGE2 concentration, initial SCI severity, as well as 42-day post-SCI recovery.Here, we found CSF concentration of lipoprotein-associated PLA2 was significantly higher in SCI dogs compared to control dogs. In studies performed on rodent spinal cord homogenates post-SCI, the secretory isoform of PLA2 increases within hours of injury, is over-expressed for days following SCI, and is negatively correlated with recovery of locomotion . CriticaThe CSF concentration of LTC4 was significantly higher in healthy control dogs compared to those with SCI. This finding is in contrast to data from a guinea pig spinal cord contusion model, which showed increased parenchymal LTC4 10 min after SCI that persisted 60 min post-SCI. Data from dogs with experimental spinal cord contusion likewise showed abrupt, early increases in LTC4 concentration, measured within the CSF . Our resCerebrospinal PGE2 concentration was significantly increased in dogs with SCI compared to healthy control dogs. Additionally, CSF PGE2 concentration was significantly and positively associated with CSF protein concentration and RBC in injured dogs; both these CSF analytes are increased as a result of intrathecal bleeding and blood-spinal cord barrier disruption . FinallyWe assessed three mediators of AA metabolism in the CSF of dogs with SCI and found evidence that PLA2 and PGE2 concentration is altered during injury and PGE2 concentration is associated with SCI severity and recovery. Previous studies in other neurotrauma model systems likewise have suggested that PLA2 and PGE2 exacerbate SCI. Additionally, we found decreased CSF LTC4 concentration in dogs with SCI compared to healthy dogs, which is contrary to what has been described in experimental models. Interspecies differences in inflammatory networks, variability in injury severity and the anatomic level of injury, and assessment of CSF rather than parenchyma may have influenced the results of this study. Future studies using larger populations of injured dogs to confirm findings in this study and more broadly screen AA pathway constituents might provide valuable information, as could quantification of pathway enzyme mRNA.


To study the processes by which donor-conceived children incorporate donor conception into their subjective sense of identity.Cross-sectional.Family homes.Nineteen donor-conceived adolescents.Administration of an interview and questionnaire.The mother-child relationship was assessed through the Friends and Family Interview, a semistructured interview designed to assess adolescents' security of attachment in terms of secure-autonomous, insecure-dismissive, insecure-preoccupied, and insecure-disorganized attachment patterns. The Donor Conception Identity Questionnaire assessed adolescents' thoughts and feelings about donor conception, yielding two factors: [1] curiosity about donor conception and [2] avoidance of donor conception.Statistically significant associations were found between the Curiosity scale and the secure-autonomous and insecure-dismissing attachment ratings. Adolescents with secure-autonomous attachment patterns were more interested in exploring donor conception whereas those with insecure-dismissing patterns were less likely to express curiosity. Insecure-disorganized attachment ratings were statistically significantly correlated with the Avoidance scale, indicating higher levels of negative feelings about donor conception.The results of this study of the influence of parent-child relationships on thoughts and feelings about donor conception in adolescence suggest that the valence of the parent-child relationship influences adolescents' appraisal of their donor conception within the context of their growing sense of identity. Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/slutskyj-integrating-donor-conception-identity/Research on the diverse family forms made possible by donor conception has largely focused on comparisons between family types. These studies have generally shown that donor-conceived families headed by heterosexual couples, lesbian couples, and single heterosexual mothers do not differ in terms of family functioning or child adjustment from heterosexual two-parent families formed without medical assistance Research on the identity formation of adopted adolescents offers a useful starting point for exploring identity development in donor conception Adopted adolescents' curiosity about their family of origin can be hindered by perceived barriers Given the mutual emphasis on narrative development, attachment theory provides a useful theoretical bridge for extending adoption identity theories to donor conception. According to attachment theory As children enter adolescence, increasingly sophisticated metacognitive abilities allow them to positively and negatively evaluate their attachment figures as they simultaneously develop their own perspective on, or narrative about, their attachment to their parent(s) Although potentially beneficial to the process of meaning-making and identity development, engaging with various aspects of donor conception, such as searching for donor relations and initiating conversations about genetic origins, can be perceived as a threatening and intimidating process by both parents and children in donor-conceived families The present study focused on the adolescent children of single mothers and lesbian couples conceived through anonymous sperm donation, as the children in these families are more likely than children in two-parent heterosexual families to have become aware of their donor conception at an early age The participants were recruited through the Donor Sibling Registry, a U.S.-based registry that facilitates contact between same-donor offspring, their parents, and donors. In the first instance, an e-mail giving information about the study and requesting assistance was sent to single mothers and partnered lesbian mothers who [1] were living within the tristate area of New York designated for its accessibility to the researchers, [2] had one or more adolescent children conceived by donor insemination, and [3] had found at least one of their child's donor-siblings. Although 146 e-mails were sent out, it was not possible to determine how many mothers actually received or opened the initial e-mail. As the study was designed primarily as an in-depth qualitative study of adolescents' experiences of contact with their donor siblings (reported elsewhere), the aim was to recruit approximately 20 adolescents. The first 28 mothers to give permission for their contact details to be passed on to the researchers were contacted by one of the authors (J.S. or S.P.) to describe the study and request participation. Twenty-one mothers agreed for their family to take part, representing a participation rate of 78%. Nineteen adolescents conceived by donor insemination took part in this research, representing 90% of those who were approached about the study. Of the two adolescents who did not take part, one was not interested in participating, and one was attending school abroad. As face-to-face interviews were being conducted with donor-conceived adolescents, the study provided an opportunity to assess not only their relationships with donor siblings but also their attachment relationships with parents.The adolescents ranged in age from 12 to 19 years (mean 14.18 ± 2.20 standard deviation). Two sets of two adolescents were from the same family. The mean age of the mothers was 52 years, all were white, and 14 (82%) had a bachelor's degree or higher qualification. The majority  were employed full-time outside of the home. Fifteen (79%) of the adolescents were female, and 4 (21%) were male. All were enrolled in middle school or high school, with the exception of one who had graduated high school. All had been conceived by donor insemination using an anonymous sperm donor. Twelve adolescents (63%) were born to single mothers and 7 (37%) to lesbian couples. When asked when they had been told of their donor conception, 2 (10.5%) adolescents reported they had “always known,” 9 (47.4%) could not recall, and 8 (42.1%) reported they had been told at or before the age of 7. None had been in contact with their donor. However, all had located at least one donor sibling—that is, a half-sibling in another family born from the same donor as themselves.One of two trained researchers (J.S. or S.P.) interviewed the adolescents in their homes. Written, informed consent was obtained from participants aged 18 years and over, and written parental consent and written and verbal assent were obtained from participants under the age of 18 years. Ethics approval for this study was obtained from both the University of Cambridge Psychology Research Ethics Committee and the New School's institutional review board. The researcher administered an audio-recorded, semistructured interview and a questionnaire to the adolescent in a private room. The interviews lasted approximately 1 hour.The adolescents took part in a modified version of the Friends and Family Interview (FFI) . Higher ratings of secure-autonomous attachment were statistically significantly associated with higher levels of Curiosity . The model with insecure-dismissing attachment as the predictor accounted for 25.7% of variance in adolescents' Curiosity . Higher ratings of insecure-dismissing attachment were statistically significantly associated with lower levels of Curiosity .Multiple linear regression was used to further investigate the relationship between secure-autonomous and insecure-dismissing attachment patterns and the adolescents' curiosity regarding their donor conception. The outcome variable was the factor score of Curiosity, and the predictors were the secure-autonomous and insecure-dismissing attachment scale scores. Adolescents' scores on these two attachment patterns were statistically significantly and negatively correlated  into a subjective sense of identity The relationship between attachment and expressed curiosity in donor conception may be understood through the points of convergence between Grotevant and Cooper's This complex balance between autonomy and connectedness is best served by mutual recognition by both parent and child of the need for communication as the family strives to accommodate the ultimately symbiotic but at times conflicting demands of connectedness and autonomy In contrast, adolescents who demonstrated evidence of insecure attachment were more likely to show a preference for avoiding the topic of donor conception altogether. Those who demonstrated insecure-dismissive attachment were least likely to express curiosity. Dismissing attachment patterns in adolescence have been characterized by limited autonomy, less communication with parents The minority of the sample who showed insecure-disorganized attachment patterns were most likely to feel negatively about, and avoid engagement with, the issue of their donor conception. Conclusions about the potential effect of disorganized attachment on donor-conception identity must be drawn carefully given the limited representation of these patterns in this sample. However, it is noteworthy that those adolescents who demonstrated evidence of disorganization, associated with negative psychological outcomes The findings of this study must be interpreted with caution due to several limitations, particularly the small sample drawn from a narrow geographic region. Although the participation rate of families directly contacted by the researchers was high, it is not known how many mothers received or opened the initial e-mail from the Donor Sibling Registry. To the extent that biases were present in the sample, these are likely to reflect a greater engagement with the issue of donor conception and a greater inclination to search for donor siblings than would be found in the general population of donor-conception families. It should also be emphasized that the adolescents in the present study were all aware of their genetic origins. They had all been registered on the Donor Sibling Registry by their parents, most when very young. It is likely that children of parents who had not demonstrated such willingness to embrace the topic of their donor conception would have had a different experience of negotiating this aspect of identity development. Moreover, this study included children of single mothers and lesbian couples only. Curiosity about donor conception has been found to be higher in families where a father is not present An advantage of the study is that it is the first to conduct in-depth interviews with donor-conceived adolescents. This made it possible to assess the adolescents' attachment relationships with their parents. The representation of both insecure and secure attachment patterns and both positive and negative feelings toward donor conception indicate that the sample was diverse enough to reveal associations between attachment security and donor-conception identity. All participants were living at home and thus in environments offering a similar opportunity for conversations with their parents. In spite of the small sample size and need for replication with a more representative sample, meaningful and potentially important associations were identified between attachment security and donor-conception identity, thus shedding light not only on differences in the experiences of donor-conceived adolescents but also on the psychological processes through which these differences arise.Although the study was conducted with adolescents, it was designed to capture parental internal representations formed in childhood as they relate to adolescent identity development. Allen et al.


Dear Sir,Prenatal torsion of testis is an extremely rare surgical emergency with controversial management guidelines. Controversy exists not only with regard to timing and necessity of exploration but also whether or not to fix the contralateral testis as there is no predisposing anatomical defect. Though not life-threatening, it risks fertility of an otherwise healthy newborn male if overlooked. We describe a case of prenatal testicular torsion and discuss pertinent issues.A term male newborn was found to have edematous and enlarged right hemiscrotum at birth. General condition of neonate was satisfactory with a birth weight of 2.75 kg. Vital signs were within normal range. Systemic examination revealed no abnormality except for the enlarged, edematous and erythematous right hemiscrotum . On palpation, right testis was enlarged, mildly tender, firm in consistency and at a higher position than the left, but with no local rise of temperature. Scrotal transillumination was reduced on the right side. Left testis was in descended position with appropriate size and consistency. Clinical diagnosis of testicular torsion was made. Immediate color doppler ultrasound study of scrotum showed enlarged, heterogeneous right testes, with focal hypoechoic region (suggestive of infarct), no visible internal vascularity, minimal peritesticular fluid and scrotal wall edema; indicating antenatal torsion of the testis. Left testis was found to have normal size, echogenicity and internal vascularity. Since these findings were suggestive of an irreversible ischemia and prolonged duration of torsion, emergent exploration was deferred and decision was taken to manage the case electively.After detailed counseling of parents regarding the objectives and risks of exploration, including the low likelihood of salvaging the affected testis, patient was promptly explored after adequate pre-anaesthetic workup. Surgery under general anaesthesia on 3rd post natal day revealed extravaginal type spermatic cord torsion through two complete turns with gangrenous testes on the affected side. Hence right orchidectomy and left orchidopexy were done using separate scrotal incisions. Histological study of excised testis showed hemorrhagic and gangrenous changes with no viable testicular tissue. Post operative period was uneventful and the neonate is doing well on follow-ups.Prenatal testicular torsion typically presents as a discoloured scrotum seen at birth in an otherwise asymptomatic newborn with decreased scrotal transillumination. Other presentations may be as testicular nubbin, small and hard testis, normal-sized but hard testis or as acute scrotum. Bilateral torsion can occur in up to 20% of affected newborns and is often an asynchronous event [1]. Worryingly, bilateral torsion can present with unilateral signs [2].Doppler ultrasound is an important tool for diagnosing testicular torsion and predicting the viability of testis. Presence of any blood flow suggests incomplete torsion and the potential for salvage. Highest recovery rates have been found in testes with normal, homogeneous echogenicity as compared to no recovery in testes with heterogeneous echogenicity [3].Management of unilateral prenatal torsion is an area of controversy with strategies including observation alone, delayed contralateral orchidopexy and emergent contralateral orchidopexy [1]. Elective orchidectomy with contralateral orchidopexy once anaesthesia risk assessment has been stabilized (within 1-2 days of diagnosis), has been the commonly used and preferred approach [4]. But as bilateral testicular torsion is now being reported with more frequency and in view of recent experiences with lack of significant contralateral symptoms in cases of bilateral asynchronous torsions, many authors now recommend emergent surgical exploration in all cases of prenatal torsions . Studies have shown testicular salvage rate of 5% in cases of prenatal torsion [1]. With this low likelihood of salvage, theoretical risk of contralateral torsion and anorchia must be weighed against the low but real risk of general anesthesia in a healthy newborn.Bilateral torsion is a true emergency and the management is straightforward immediate surgery. Threshold for performing bilateral testicular detorsion and fixation should be low and every effort should be made to leave even necrotic testes in place, as some testicular function may still be possible [5].Prompt recognition and rapid intervention is the key to preserve testicular function in cases of torsion. As salvage rates are poor for prenatally torted testis, prevention of contralateral testicular torsion is imperative. Hence early exploration with contralateral orchidopexy is highly recommended. Doppler ultrasound is useful however any suspicion of bilateral involvement and incomplete torsion should warrant immediate exploration.Source of Support: NilConflict of Interest: None declared


The human motor system permits a wide variety of complex movements. Thereby, the inter-individual variability as well as the biomechanical aspects of the performed movement itself contribute to the challenge of the interpretation of sEMG signals in dynamic contractions. A procedure for the systematic analysis of sEMG recordings during dynamic contraction was introduced, which includes categorization of the data in combination with the analysis of frequency distributions of the sEMG with a probabilistic approach. Using the example of elbow flexion and extension the procedure was evaluated with 10 healthy subjects. The recorded sEMG signals of brachioradialis were categorized into a combination of constant and variable movement factors, which originate from the performed movement. Subsequently, for each combination of movement factors cumulative frequency distributions were computed for each subject separately. Finally, the probability of the difference of muscular activation in varying movement conditions was assessed. The probabilistic approach was compared to a deterministic analysis of the same data. Both approaches observed a significant change of muscular activation of brachioradialis during concentric and eccentric contractions exclusively for flexion and extension angles exceeding 30°. However, with the probabilistic approach additional information on the likelihood that the tested effect occurs can be provided. Especially for movements under uncontrollable boundary conditions, this information to assess the confidence of the detected results is of high relevance. Thus, the procedure provides new insights into the quantification and interpretation of muscular activity. Muscles move us and many scientists move the research on the interpretation of muscular activation. Thus, muscular activation is investigated and described in a wealth of literature to increase the knowledge of physiological movement patterns or pathological movement disorders. But, the human motor system permits a wide variety of complex movements. To meet the challenge of controlling all possible movements, the human motor system exhibits redundant muscular activation strategies ranging from strategies on how to activate one muscle to strategies on activating a consortium of contributing muscles. The study of muscular activation with surface Electromyography (sEMG) provides insights into the contribution of single muscles to the overall performed movement. Like the human motor system itself, the interpretation of muscular activation is complex. There are controversial results from examination of sEMG as well as reviews on the limitation of sEMG. The interpretation of sEMG recordings requires caution . No subject had any known symptoms of neuromuscular disorders, history of orthopedic surgery or any affection of the upper extremities. Subjects avoided strenuous exercises in the day prior to the measurement. All subjects were right handed. All experiments were performed in accordance to the declaration of Helsinki and all subjects gave written informed consent prior to the study.The experimental setup included the measurement of bipolar sEMG and elbow joint angles. sEMG-electrodes, with an inter-electrode distance of 2 cm , rectified and smoothed  in order to compute the envelope of the sEMG signal. Normalization of the envelope was performed with respect to the MVC. For that purpose the mean value of the enveloped sEMG of the recorded three MVC trials was computed. Out of five MVC trials with constant torques the mean of the three MVC sEMG envelopes with the smallest standard deviation is chosen as reference.A decision tree was used to distinguish the present combination of variable movement factors for each sampling point of the recorded sEMG data. When the decision algorithm computes the combination of variable movement parameters, the amplitude of the enveloped and normalized sEMG value is stored with an assignment to this combination Figure .In the example of flexion and extension of the elbow, combinations of the contraction type and flexion and extension angle were assigned to the normalized sEMG value. First, the decision algorithm derived the concentric or eccentric contraction type for the investigated sampling point. Thereby, the contraction types were distinguished in two categories, concentric and eccentric contractions, which were derived from the slope of the flexion and extension course. For the flexing muscles, positive slopes representing flexion of the joint were assigned to concentric contractions, whereas negative slopes representing extension of the joint were assigned to eccentric contractions. Subsequently, the decision algorithm assigned the flexion and extension angle at this sampling point to an angle interval. Here, the decision tree merged the angles into 12 intervals with 10° steps ranging from 0 to 120°, where 0° was full extension.The categorized data allocates the muscular activation to each combination of variable movement factors and was further assessed with a probabilistic approach.The impact of the variable movement factors of flexion and extension angle and contraction type for the combination of constant movement factors was further assessed with the help of a probabilistic approach. All amplitudes of the enveloped and normalized sEMG in one combination of angle category and contraction type were evaluated to derive a relative frequency distribution for each subject separately. From that relative frequency distribution a cumulative frequency can be computed to derive the probability for each possible sEMG value to be within a defined interval Figure .To demonstrate the relevance of assigning possible movement parameters to the sEMG signals, the recorded sEMG signals of brachioradialis of all trials and all subjects were analyzed in three different analyzing scenarios in a deterministic way. For that purpose, for the same data the normalized sEMG envelopes were categorized with three different decision trees to compute mean values ± standard deviations of all trials and all subjects. The results were further analyzed for statistical relevance.The first analyzing scenario A included a decision tree with the contraction type as variable movement parameter only, without any respect to the different elbow flexion and extension angles. Exclusively the contraction type was used to categorize the processed sEMG signal into two contraction types.The second analyzing scenario B included a decision tree with the variable movement parameter flexion and extension angles without any respect to the contraction type. Exclusively the flexion and extension angles were used to categorize the processed sEMG into 12 intervals ranging from 0 to 120°. For the statistical analysis the angle intervals were merged into four intervals with four times 30° steps.The third analyzing scenario C included the described decision tree with the combination of two variable movement parameters namely the combination of 12 angle and two contraction type intervals. For statistical analysis angle intervals were merged into four intervals with four times 30° steps.The assignment of probabilities for each possible categorized sEMG value to be within a defined interval enables the test of the impact of movement factors on the muscular activation. Thus, the categorized frequency distributions of the normalized sEMG envelope were used for further analysis. To demonstrate the procedure, the following hypothesis was exemplarily tested.The muscular activation is affected by a combination of contraction type and flexion and extension angle.For the test of the raised hypothesis, the frequency distributions of normalized sEMG envelopes in concentric and eccentric contraction were assessed in the same four flexion and extension angle intervals as performed in analyzing scenario C. For that purpose a probability for each angle interval was computed for each subject as described in the following Figure .interval was defined.For each subject the distance d between the most probable normalized sEMG envelope of the frequency distributions during concentric and eccentric contractions were computed for each angle interval separately. In the middle between the most probable normalized sEMG envelope of the frequency distributions of concentric and eccentric contractions d/2 an analyzing interval Acon  and eccentric Pecc  cumulative frequencies.Subsequently, the probability of the normalized sEMG envelope to occur within the defined analyzing interval was computed for both contraction types separately, as concentric Pcon + ecc  was computed through the multiplication of both cumulative frequencies:The probability that the value of normalized sEMG envelope of concentric and eccentric contractions occurs in both frequency distributions at the defined interval PThe more the frequency distributions of concentric and eccentric contraction apart from each other, the smaller the probability for an overlap of both distributions at the defined interval gets.To compare the probabilistic approach to a deterministic analysis the probabilities for the overlap of concentric and eccentric contractions in all four angle intervals of each subject were computed. Subsequently, the probabilities of each subject in one angle interval were averaged and finally compared to the deterministic outcome of the same categorized data of test scenario C.All statistical analyses were performed with SPSS . The level of statistical significance for all analyses was set to α = 0.05.t-test was used to determine whether there was a statistically significant difference between the categorized normalized sEMG envelopes (dependent variable) during concentric contractions compared to eccentric contractions. Test for normality was performed with Shapiro-Wilk's test.For the categorized data of scenario A a paired-samples Post-hoc analysis with a Bonferroni adjustment was used to compare the change of the normalized sEMG envelope with increasing angles. Normal distribution and sphericity of the categorized data was assessed by Shapiro-Wilk's test and Mauchly's test of sphericity, respectively.For scenario B an One-Way repeated measures ANOVA was conducted to determine whether there were statistically significant differences of the normalized sEMG envelope (dependent variable) over the course of four different angle conditions (within subject factor). A For scenario C a Two-Way repeated measures ANOVA was run to determine the effect of four different angle conditions (within subject factor 1) in two different contraction types (within subject factor 2) on the normalized sEMG envelope (dependent variable). Subsequently, simple main effects on the contraction type were run to assess the difference of normalized sEMG in concentric and eccentric contractions under all angle conditions separately. Again, normal distribution and sphericity of the categorized data was assessed by Shapiro-Wilk's test and Mauchly's test of sphericity, respectively.The described method for the analysis of dynamic sEMG signals assigns the muscular activation to movement parameters originating from the way of the performed movement. Thereby, the results are limited to the constant movement parameters, the constant velocity of 25°/s and the constant external weight of 3.5 kg. Data are mean ± standard deviation, unless otherwise stated.t(9) = 5.256, p = 0.001] with a higher normalized sEMG during concentric contractions (19.14 ± 7.30% MVC) compared to eccentric contractions (13.76 ± 5.62% MVC). The difference of the concentric and eccentric normalized sEMG envelopes of the categorized data was normally distributed, as assessed by Shapiro-Wilk's test (p = 0.703).First, the effect of categorization of the data was tested for the described analyzing scenarios. For the first analyzing scenario A the contraction type was found to take a significant impact on the muscular activation of brachioradialis. A statistically significant difference between normalized sEMG envelope of concentric contractions compared to eccentric contractions were found  with the normalized sEMG envelope increasing from 4.67 ± 2.28% MVC for flexion angles of 0–30° to 23.90 ± 10.55% MVC for flexion angles of 90–120°. Post-hoc analysis with a Bonferroni adjustment revealed that there was a statistically significantly increase from 0–30° to 30–60° (p < 0.0005), from 30–60° to 60–90° (p = 0.005), but not for 60–90° to 90–120° (p = 0.485).For the second analyzing scenario B the flexion and extension angle showed a significant impact on the normalized sEMG envelope. The categorized normalized sEMG envelopes of scenario B were normally distributed at each angle condition, as assessed by Shapiro-Wilk's test (p > 0.05). Additionally, there was sphericity for the interaction term, as assessed by Mauchly's test of sphericity (p > 0.05). A statistically significant interaction between contraction type and angle condition was found . The simple main effects on the contraction type revealed that the normalized sEMG envelope was statistically significantly different for the concentric compared to the eccentric contractions at the angle condition of angles between 30 and 60° , 60–90° , and 90–120° . However, the normalized sEMG envelope was not statistically significantly different for the concentric compared to the eccentric contractions at the angle condition of angles between 0 and 30°  .Analysis of the studentized residuals of the categorized normalized sEMG envelopes of scenario C showed that there was normal distribution, as assessed by the Shapiro-Wilk test . The decreased probabilities represent that the frequency distributions of concentric and eccentric contractions are more likely to differ from each other. For 30–60°, 60–90°, and 90–120° the median of the probability for overlap decreased to 5.77% with a range of 0.01–24.98%, 4.88% with a range of 0.02–24.24%, 5.37% with a range of 0.05–28.82%, respectively . In contrast, the probabilistic parameter for the overlap of both frequency distributions provides additional information on the likelihood that this difference is not present. The decreased probabilities for angles exceeding 30° represent that the frequency distributions of concentric and eccentric contractions are more likely to differ from each other. But at the same time, the value of the probable overlap in a defined interval accounts for the probability of the negative effect, namely that the concentric and eccentric sEMG do not differ from each other. For angles between 30 and 60° the median and range of the probability that both frequency distributions in a defined interval do not differ from each other was computed to 5.77% with a range of 0.01–24.98%. Thereby, in four of the 10 subjects the probability exceeded 20%, meaning that for these subjects the tested hypothesis, with respect to the parameter of overlap, holds true with a probability of less than 80%. With the probabilistic parameter additional information are provided to add a confidence to the obtained results. The ranges of the computed probabilities for the overlap of both frequency distributions reveal that there are inter-individual differences for the activation of brachioradialis in concentric and eccentric contractions along all angle areas.For the comparison of the deterministic approach Figure  to the pFor the deterministic analysis the outcome of the statistical test is to reject or hold the raised hypothesis. In contrast, if the analysis is connected to the test of a specific hypothesis with a probabilistic approach, additional information on the likelihood that the hypothesis holds true or fails are available which adds a confidence to the results. More probabilistic methods could be added to the evaluation of two frequency distributions. Besides the similarity or difference of two frequency distributions one could also assess the probability that one sEMG distribution possess higher sEMG amplitudes than the other.The main potential of the probabilistic approach lies in the fact that each possible value of the normalized sEMG can be assigned to a probability that this value will occur in a specific interval. In combination with parameters, which describe the similarity or difference of two frequency distributions, the information on the likelihood that the analyzed effect occurs or not occurs can be provided. It can be assumed, that inter-individual differences increase with more uncontrollable movement conditions in free movements. A measure on the confidence of the detected results could account for this increasing effect of inter-individual differences.Analyzing sEMG signals during dynamic contractions is challenging. However, the introduced procedure of analyzing categorized data with a probabilistic approach allows to address the challenge of the influence of different movement conditions on the muscular activation and provides additional information on the confidence of the results by adding probabilities to the tested hypothesis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The craniocervical junction (CCJ) is a potential choke point for craniospinal hydrodynamics and may play a causative or contributory role in the pathogenesis and progression of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, MS, and ALS, as well as many other neurological conditions including hydrocephalus, idiopathic intracranial hypertension, migraines, seizures, silent-strokes, affective disorders, schizophrenia, and psychosis. The purpose of this paper is to provide an overview of the critical role of the CCJ in craniospinal hydrodynamics and to stimulate further research that may lead to new approaches for the prevention and treatment of the above neurodegenerative and neurological conditions.  The CCJ comprises the base of the skull, atlas C1), and axis (C2), as well as muscles and connective tissues that connect the skull to the cervical spine. It further includes the dura mater and dentate ligament attachments of the brain and cord to the foramen magnum and upper cervical spine , and axi. MoreoveWhile the exact source, such as the arteries at the base of the brain or choroidal vessels, is still debated, in either case, it is generally accepted that craniospinal hydrodynamics are primarily driven by arterial pulsations –19 and fThere are many different types of malformations and anomalies of the CCJ such as atlantooccipital assimilation, basiocciput hypoplasia, malformations of the condyles, and malformations of the dens . CertainIn addition to blocking CSF flow and causing hydrocephalus, malformations, anomalies, and misalignments of the CCJ can obstruct blood flow through the vertebral arteries –63 and vIn brief, the CCJ is a potential choke point for blood and CSF flow between the cranial vault and spinal canal that can cause faulty craniospinal hydrodynamics and subsequent chronic ischemia, edema, and hydrocephalus. Upright posture and motion of the cervical spine compound the strains and deformation of blood and CSF pathways of the CCJ further contributing to blockage of flow. Faulty craniospinal hydrodynamics have been associated with Chiari malformations –36, cranThe arteries and veins of the CCJ play key roles in craniospinal hydrodynamics. The main arteries that pass through the CCJ include the vertebral and spinal arteries. The venous system of the CCJ includes the (1) dural sinuses of the posterior fossa, (2) suboccipital cavernous sinus, (3) occipital-marginal sinuses, (4) emissary veins, and (5) vertebral veins.The vertebral arteries make up the posterior blood supply routes that provide about twenty percent of blood flow to the brain. The twenty percent of blood flow they supply includes most of the diencephalon  and all of the brainstem, as well as the medial temporal and occipital lobes. The internal carotid arteries are the anterior supply routes and supply about eighty percent of blood flow to the brain including most of the lobes and some of the diencephalon.The circle of Willis encircles the infundibulum located above the sella turcica and links the anterior blood supply routes to the posterior blood supply routes. There are variations in the layout of the circle. In some cases it is incomplete. The design of the circle of Willis affects its ability to supply collateral circulation to the brain. It also affects the direction of arterial flows in the anterior and posterior supply routes under normal and pathological conditions, such as steal syndromes involving the vertebral arteries.The vertebral arteries arise from the proximal segments of the subclavian arteries and are broken down into four segments. The first segment is from the origin at the subclavian artery to where it enters the intervertebral foramen of C6 or C5. The second segment passes through the intervertebral foramen from C6 up to C2. The third segment of the vertebral artery passes through the intervertebral foramen of the CCJ, turns back around the condyles, and then pierces the suboccipital cavernous sinus of the posterior atlantooccipital membrane through which it travels toward the midline of the occiput. As they approach the midline of the occiput the vertebral arteries turn cephalward and pass through the foramen magnum. After passing through the foramen magnum the vertebral arteries penetrate the dura mater and enter the brain. The fourth segment continues from where the vertebral arteries enter the brain up to the pontomedullary junction where they unite to become the basilar artery see .Subclavian steal syndromes occur when the proximal portion of the subclavian artery is obstructed due to stenosis resulting in decreased blood flow and pressure downstream in the vertebral and axillary arteries. This creates a pressure gradient that causes the arm to draw blood from the ipsilateral vertebral artery, which in turn gets its supply from the contralateral vertebral artery via their connection to the basilar artery –161. SubThe ophthalmic arteries are the first branches of the internal carotid artery. Decreased pressure in the circle of Willis relative to arterial pressure in the ophthalmic artery can draw blood away from the eye to the ipsilateral brain. Consequently, relatively lower downstream pressure in the posterior portion of the circle of Willis, basilar, and vertebral arteries, which occurs in a subclavian steal syndrome can result in an ophthalmic steal syndrome and hypoperfusion of the optic nerve head see . HypoperIt is further interesting to note that migraines have been associated with hyperintensity signals on brain scans that indicate ischemic damage. The hyperintensity signals are often found in the distribution of the posterior blood supply, especially the cerebellum , 173. ThIn addition to providing twenty percent of blood flow to the brain, the vertebral arteries provide a significant portion of the blood supply to the cord via the anterior and posterior spinal arteries. The anterior spinal arteries typically arise from the fourth segment of the vertebral arteries after the posterior inferior cerebellar arteries see . The posRecent clinical evidence by Rafael further suggests that ALS may be due to progressive ischemia in the intraparenchymal territory of the anterior-ventral spinal arteries and/or the anterior spinal artery. The anterior-ventral spinal arteries are branches of the inferior thyroid and ascending cervical arteries that stem from the thyrocervical trunk, which arises from the subclavian artery distal to the vertebral artery. The anterior spinal and anterior-ventral spinal arteries supply the ventral aspect of the cord. Rafael found some key common characteristics in ALS patients based on CT and MRI scans of the skull and cervical spine, as well as from observations made during surgery that included (1) anatomical variants of the fourth segment of the vertebral arteries; (2) atherosclerosis of the fourth segment of the vertebral arteries, as well as the basilar artery; (3) atrophy of the pyramids; (4) atrophy of the frontoparietal cortex; (5) questionable zones that appeared to be microinfarcts in the medulla oblongata and cervical cord; (6) exsanguinated short and long circumferential arteries that branch off from the anterior-ventral spinal and/or the anterior spinal arteries; (7) atrophy of small nerve roots in cranial nerves IX, X, and XI; and (8) degeneration of the cervical spine. Based on his clinical and neurosurgical observations, as well as the following omental transplantation for revascularization, Rafael posits that the primary cause in certain cases of olivopontocerebellar atrophy (Parkinson's plus), ALS, and possibly central pontine myelinolysis is ischemic in origin and related to vascular anomalies and atherosclerotic plaques located at the mouths of the collateral branches from the vertebral and basilar arteries , 121.Many authors have also suggested that ALS may be related to trauma, especially head trauma –120. AmoThe third segment of the vertebral artery passes through the suboccipital cavernous sinus  of the CCJ see . The extThe dural sinuses are the large drainage veins of the brain  that can similarly affect blood flow in the vertebral veins and cord. In addition to straining the dura of the CCJ, cervical kyphosis  also causes the internal jugular vein to bend around the transverse process of atlas, reversing its normal course, as well as potentially compressing the vein and increasing resistance to blood flow. In brief, malformations and misalignments of the CCJ may play a role in chronic ischemia and edema, which may in turn lead to neurodegenerative processes and subsequent diseases.CSF is produced in the ventricles. The lateral ventricles are the highest and largest ventricles located in the left and right hemispheres beneath the corpus callosum. CSF flows from the lateral ventricles through the foramen of Monroe into the third ventricle located within the walls of the thalamus of the diencephalon. It flows out of the third ventricle through the cerebral aqueduct of Sylvius in the midbrain and into the fourth ventricle located anterior to the cerebellum and posterior to the pons and medulla. CSF flows out of the fourth ventricle through four openings. The foramen of Magendie, located in the midline of the fourth ventricle, and the two foramens of Luschka, located on the lateral walls, drain into the prepontine cistern and cisterna magna. The central canal of the medulla and cord is connected to the obex of the fourth ventricle. The central canal of the cord is the embryological remnant of the neural tube and homolog of the ventricles and cerebral aqueduct of Sylvius. Although it is often discontiguous in adults, the central canal passes through the center of the lower brainstem and spinal cord and terminates as a cistern in the conus medullaris see .The cisterns are expansions of the subarachnoid space . Trauma can also cause dural tears resulting in CSF leaks and coning. Connective tissue disorders such as Ehlers-Danlos appear to be predisposed to CSF leaks as well. In addition to preventing coning, proper CSF volume is essential to the regulation of intracranial pressure (ICP), which increases exponentially with increases in volume.Chiari malformations are typically attributed to malformations of the brainstem in children in which the lower portion of the cerebellum, called the tonsils, are found to be descended five millimeters or more beneath the level of foramen magnum into the CCJ. In contrast to an oversized cerebellum, research has shown that Chiari malformations are often due to hypoplasia of the posterior fossa resulting in crowding and downward displacement of the cerebellar tonsils –36. SomeAlthough still hotly debated, most of the CSF in spinal cord and cisterns eventually flows up through the subarachnoid space and CCJ toward the top of the brain where it is absorbed back into venous circulation by the arachnoid granulations. The arachnoid granulations are special one-way valves that develop during infancy as a child starts to hold its head up, crawl, and walk. While they can be found in other dural sinuses as well, the bulk of arachnoid granulations are found at the top of the brain in humans and drain into the venous lacunae and superior sagittal sinus . In quadIn addition to flow within the craniospinal compartment, research has further shown that CSF flows along cranial nerve roots, especially the olfactory and optic nerves. In fact, enlargement of the optic nerve sheath is a sign of increased CSF volume and possible hydrocephalus. CSF can also be seen in the trigeminal and other cranial nerves as well. Alternatively CSF can exit the cranial vault and flow through the CCJ into the subarachnoid space of the cord. As in the brain, there is ongoing debate about optional routes for CSF to leave the subarachnoid space of the cord including pathways that follow the nerve roots of the cord similar to cranial nerves in the brain. The bulk of CSF in the subarachnoid space of the cord, however, appears to return to the cranial vault and exit through the arachnoid granulations –204. TheClassic hydrocephalus is an increase in CSF volume coupled with ventriculomegaly. In children, hydrocephalus is typically, but not always, associated with an increase in intracranial pressure (ICP) and head size. For the most part, hydrocephalus is considered a childhood condition. Normal pressure hydrocephalus (NPH) is seen mostly in adults but it is sometimes seen in children as well. NPH is associated with increased CSF volume, ventriculomegaly, and normal or slightly elevated ICP. The size of the head is unaffected in adults. The decrease in compliance of the skull most likely puts a damper on ICP in adults with NPH compared to the highly compliant skulls seen in infants and young children. Despite the development of shunts, decades of research, and recent advances in brain imaging we know very little about the role of faulty craniospinal hydrodynamics in hydrocephalus.hydrocephalus is an active distension of the ventricular system of the brain resulting from inadequate passage of CSF from its point of production within the cerebral ventricles to its point of absorption into the systemic circulation” [According to Rekate a new definition for hydrocephalus is long overdue and so he proposed one as a starting point for discussion in which he maintains that “ulation” , 209. BeOther definitions for hydrocephalus have also been proposed over the years. One definition previously proposed by Raimondi is that hydrocephalus is a pathologic increase in CSF volume independent of hydrostatic or barometric pressure that can be classified as either intra- or extraparenchymal. Intraparenchymal hydrocephalus is essentially the same as cerebral edema, which is an increase in interstitial fluids in the parenchyma. Extraparenchymal hydrocephalus can be further broken down into intraventricular, cisternal, and subarachnoid space forms .Some authors maintain that hydrocephalus is simply cerebral edema regardless of the cause or location of the excess CSF volume –211. ThiThe generally accepted theory is that, aside from frank blockage, such as stenosis of the cerebral aqueduct, the cause of hydrocephalus is due to insufficient absorption, which occurs primarily by way of the arachnoid granulations, as well as arterial and venous capillary routes. According to Greitz, however, the bulk CSF flow theory fails to explain key issues regarding communicating hydrocephalus, such as what causes the ventricles to enlarge, why CSF pressure remains normal in certain cases, and why some patients improve with shunts or endoscopic third ventriculostomy . Greitz Hydrocephalus in children is typically attributed to blockage of CSF flow somewhere between the ventricles where it is produced and the arachnoid granulations where it is absorbed into venous circulation, which meets the criteria proposed by Rekate's definition. In contrast to children with hydrocephalus, however, blockage of CSF flow is rarely found in adults with normal pressure hydrocephalus (NPH), which is also known as the Hakim-Adams syndrome. Consequently, researchers have long suspected that NPH might be due to insufficient venous drainage of the brain by the dural sinuses , 215–224In 1988 this author proposed that neurodegenerative diseases may be due to obstruction and back pressure in the accessory drainage system of the brain as it passes through the CCJ to connect to the vertebral veins , 21. ObsOne of the primary causes of hydrocephalus due to blockage of CSF flow is stenosis or mass effects causing compression of the cerebral aqueduct that links the third and fourth ventricle. Current research, however, suggests that certain cases of communicating hydrocephalus in children may be due to insufficient venous drainage of the brain. Among other things, researchers now suspect that insufficient venous drainage can cause stenosis of the cerebral aqueduct due to increased CSF volume and pressure in the parenchymal spaces surrounding the midbrain resulting in compression of the aqueduct , 224. InFurther adding to the theory regarding the role of the CCJ in faulty craniospinal hydrodynamics and neurodegenerative diseases, in 2008 Williams proposed a unifying hypothesis for the cause of hydrocephalus, Chiari malformations, syringomyelia, anencephaly, and spina bifida based on dissociation of CSF flow between the cranial vault and spinal canal , 226. TyAmong other things, Williams also suggests that relatively lower spinal canal pressure in females in utero allows the cranial vault to close sooner potentially resulting in hypoplasia of the posterior fossa and disposition to crowding of the brainstem and cerebellum resulting in a Chiari malformation , 226. Thβ-amyloid, tau-protein, and proinflammatory cytokines. Reduction in blood flow in the anterior cerebral arteries decreases blood flow to the frontal regions resulting in local ischemia and possibly clinical signs. Decreased blood flow could also lead to periventricular axonal dysfunction. Prolonged periventricular ischemia would eventually result in myelin degeneration and irreversible axonal loss, possibly explaining why some patients with NPH do not improve after a shunt. The decrease in CSF clearance may also explain the high cooccurrence of Alzheimer-like changes in the cortex of patients with idiopathic NPH. A possible association with Alzheimer's disease, particularly when there is concurrent arterial hypertension and cerebral arteriosclerosis, may explain why many NPH cases remain with severe cognitive and motor deficits after shunting, even when ventricular size decreases postoperatively [Some authors have shown a causal relation between low brain blood flow and symptoms of NPH. They surmise that clinical deterioration is probably due to impaired periventricular blood flow associated with interstitial edema, ependymal disruption, microvascular infarctions, gliosis, and neuronal degeneration. Neuronal injury may result from several factors such as (1) mechanical stretching of the anterior cerebral arteries over the corpus callosum due to ventriculomegaly; (2) periventricular impairment of the blood brain barrier; and (3) reduced CSF turnover with decreased elimination of neurotoxic substances, such as ratively , 71, 73.Among other things, hydrocephalus compresses the cortical bridging veins against the cranial vault, which can further increase venous insufficiency. Compression of the veins and decreased venous outflow also decreases cerebral perfusion pressure and blood flow in the brain , 229. InIn addition to ischemia, edema, structural strains, and pressure waves, hydrocephalus causes sluggish CSF flow. As arteries and veins leave the subarachnoid space the pia mater peels away and follows their course into the parenchyma. This creates a perivascular space (Virchow-Robins space) between the pia mater and tunica adventitia of the blood vessels. CSF flows out of the subarachnoid spaces through the perivascular spaces and into the brain parenchyma where it mixes with interstitial fluids. Interstitial fluids flow out of the parenchyma along perivascular pathways. Since the brain has no lymphatic vessels or glands and it is not connected to the lymphatic system, it instead uses the perivascular spaces to move nutrients in and wastes out. Because of this, the author has also long maintained that the perivascular spaces serve as the lymphatic system of the brain and that sluggish CSF flow may play a role in pathology of neurodegenerative diseases and the increase in the presence of breakdown byproducts, viruses, bacteria, and heavy metals such as aluminum and iron , 234. MoIn this regard, the ventricles are the high side of the CSF pressure gradient. The low side of the CSF pressure gradient is venous pressure in the superior sagittal sinus. Superior sagittal sinus venous pressure and flow is affected by posture, respiration, and movement. It is also affected by downstream pressure in the internal jugular and vertebral veins. Obstruction of flow in the internal jugular veins, such as in Queckenstedt's test, is transmitted to the dural sinuses and increases ICP. Likewise, back pressure against abdominal and thoracic veins, such as in a Valsalva maneuver, is transmitted via the vertebral veins to the dural sinuses, which increases ICP. During upright posture the brain preferentially drains into the vertebral venous plexus (VVP). Blockage of the VVP in the CCJ due to malformations and misalignments may cause chronic venous hypertension, decreased CSF flow, and an increase in CSF volume and pulsatility in the cranial vault.A syrinx, also known as syringomyelia, is enlargement of a portion of the central canal of the cord. It is the homolog of hydrocephalus and sometimes referred to as hydromyelia. The central canal of the cord is typically connected to the obex of the fourth ventricle. In many cases it is not. There are currently three basic theories regarding the cause of syrinx formation and its maintenance. The first theory, called Gardner's theory , 241, isMost syrinxes are found in the cervical and thoracic cord. They are frequently associated with Chiari malformations and attributed to blockage and faulty CSF flow through the CCJ , 246. A Although rare, syrinx and holocord have more recently been associated with previously unsuspected conditions such as (1) a dural arteriovenous fistula at the level of the foramen magnum , (2) cerInterestingly, in 2003 Chang and Nakagawa proposed a hypothesis regarding the pathogenesis of syringomyelia in which the cisterna magna functions as a shock absorber against the pulsatile CSF pressure waves emanating from within the brain . The losFor the most part, the cranial vault is a closed container with little room for expansion or compliance. The spinal canal typically has much greater compliance due to the large lumbar cistern and absence of the cord. CSF volume and pressure in the cranial vault are directly related and pressure increases exponentially with increases in volume. The total volume of the cranial vault is a combination of brain, arterial blood, venous blood, CSF, and ISF. Increases in the volume of blood, CSF, brain, or space occupying lesions, such as tumors, cause an increase in ICP. Arterial volume and pressure in the cranial vault fluctuate causing pulsatile flow and pressure waves in the brain. Arterial flow can be increased or decreased by cerebral autoregulatory circulatory controls located in the cavernous and suboccipital cavernous sinus in order to maintain a relatively stable arterial supply. Stable blood flow is further maintained by proportionate outflows of venous blood and CSF from the cranial vault. A decrease in venous outflow reduces the volume of arterial blood that can enter the brain. It also increases turgor and decreases compliance, which decreases capacity of the veins in the subarachnoid space to buffer incoming arterial pressure fluctuations before they are transmitted to the parenchyma.An increase in brain, blood, or CSF volume in the cranial vault, which occurs in space occupying lesions, hemorrhages, and hydrocephalus, can cause compression of the bridging veins and venous hypertension. Venous hypertension decreases cerebral perfusion pressure and thus arterial inflows. Chronic decreases in arterial flow can lead to oxidative stress, ischemia, and subsequent atrophy resulting in a compensatory increase in CSF volume, which occurs in normal pressure hydrocephalus ex vacuo. Atrophy can also be caused by compression, tension, and shear stresses acting on the brain and blood vessels due to hydrocephalus and increased pulsatility. In either case, enlarged ventricles and cisterns are a sign of hydrocephalus and an increase in CSF volume that can be caused by atrophy or chronic strains and deformation of the brain and brainstem.In brief, craniospinal hydrodynamics are a complex interaction between brain, blood, and CSF in the relatively closed compartments of the cranial vault and spinal canal compounded by cardiac cycles and arterial pulsations that cause continuous fluctuations in blood volume and ICP. Faulty craniospinal hydrodynamics have been associated with hydrocephalus, anomalies of the CCJ, Chiari malformations (cerebellar tonsillar ectopia), craniosynostosis, craniofacial anomalies, and Dandy-Walker complex in children. Faulty craniospinal hydrodynamics may also play a role in neurodegenerative diseases such as Alzheimer's, Parkinson's, multiple sclerosis, dementia, and motor neuron diseases, as well as other neurological conditions including migraines, silent-strokes, seizures, psychosis, schizophrenia, depression, and mania. Manual and surgical methods for correcting obstructions, as well as manipulation of blood and CSF flow, may help to restore or improve faulty craniospinal hydrodynamics in certain cases and decrease the prevalence, progression, and severity of neurodegenerative and other neurological conditions. Further studies using upright and cine MRI coupled with computer modeling are needed to determine the role of malformations and misalignments of the CCJ and spondylosis, stenosis and scoliosis in the lower spine in faulty craniospinal hydrodynamics, and neurodegenerative and neurological conditions, as well as the impact of the manual, mechanical, and surgical correction of structural strains and faulty craniospinal hydrodynamics on patient pathology and symptomatology.