We report prospectively recorded observational data from consecutive cases in which the attending pre-hospital critical care anaesthesiologist considered performing pre-hospital advanced airway management but decided to withhold such interventions.st 2011 to October 31st 2012. Included were patients of all ages for whom pre-hospital advanced airway management were considered but not performed. The main objectives were to investigate (1) the pre-hospital critical care anaesthesiologists’ reasons for considering performing pre-hospital advanced airway management in this group of patients (2) the pre-hospital critical care anaesthesiologists’ reasons for not performing pre-hospital advanced airway management (3) the methods used to treat these patients (4) the incidence of complications related to pre-hospital advanced airway management not being performed.Anaesthesiologists from eight pre-hospital critical care teams in the Central Denmark Region  registered data from February 1We registered data from 1081 cases in which the pre-hospital critical care anaesthesiologists’ considered performing pre-hospital advanced airway management. The anaesthesiologists decided to withhold pre-hospital advanced airway management in 32.1% of these cases (n = 347). In 75.1% of these cases (n = 257) pre-hospital advanced airway management were withheld because of the patient’s condition and in 30.8% (n = 107) because of patient co-morbidity. The most frequently used alternative treatment was bag-mask ventilation, used in 82.7% of the cases (n = 287). Immediate complications related to the decision of not performing pre-hospital advanced airway management occurred in 0.6% of the cases (n = 2).We have illustrated the complexity of the critical decision-making associated with pre-hospital advanced airway management. This study is the first to identify the most common reasons why pre-hospital critical care anaesthesiologists sometimes choose to abstain from pre-hospital advanced airway management as well as the alternative treatment methods used. Pre-hospital Advanced Airway Management (PHAAM) continues to be one of the main controversies in pre-hospital critical care and an international group of experts have selected the topic as one of the top five research priorities in pre-hospital care . Sollid When choosing not to perform PHAAM, the pre-hospital critical care provider must have a viable alternative treatment option. We have identified no studies investigating the alternative airway management strategies used to treat these patients.The incidences of complications related to PHAAM are not neglectable even in physician-staffed systems ,11,17,18The main objective was to study the critical decision making process associated with the decision not to perform PHAAM. We did this by investigating:1) the pre-hospital critical care anaesthesiologists’ reasons for considering performing PHAAM in the current population2) the pre-hospital critical care anaesthesiologists’ reasons for not performing PHAAM3) the methods used to treat the patients when PHAAM was not performed4) the incidence of complications related to not performing PHAAM5) the incidence of emergency department endotracheal intubation and difficult emergency department endotracheal intubation in this group of patients.We performed a prospective observational study in which we collected PHAAM-related data from physician-staffed pre-hospital critical care teams according to the consensus-based Utstein-style template proposed by Sollid et al. . We have2 with a population of 1.270.000, and an overall population density of 97.7 inhabitants/km2.The Central Denmark Region covers a mixed urban and rural area of approximately 13000 kmThe EMS in the region is a two-tiered system based on 64 road ambulances staffed by emergency medical technicians (EMTs) supported by ten pre-hospital critical care teams staffed with an anaesthesiologist and a specially trained EMT. Rapid response vehicles deploy nine of the pre-hospital critical care teams; the tenth team staffs a HEMS helicopter. The pre-hospital critical care teams covered by this study employ approximately 90 anaesthesiologists as part-time pre-hospital physicians. There are no full-time pre-hospital critical care physicians in the region – all physicians primarily work in one of the five regional emergency hospitals or at the university hospital. Intensive care medicine is part of the Danish anaesthesiological curriculum. We have described the EMS system in our region in more detail elsewhere .st 2011 and November 1st 2012.We collected data between February 1“Guedel”) airway. We considered bag-mask-ventilation without the use of other adjuncts than an oro-pharyngeal airway not to be advanced airway management. In concordance with Sollid et al [Inclusion criteria: Consecutive patients of all ages in whom the pre-hospital critical care anaesthesiologists considered performing PHAAM but decided against such interventions. Sollid et al.  define aid et al  we definExclusion criteria: Patients in cardiac arrest where no treatment were initiated. Inter-hospital transfers.Primary endpoints were 1) the pre-hospital critical care anesthesiologists’ reasons for considering PHAAM 2) the pre-hospital critical care anesthesiologists’ reasons for not performing PHAAM 3) methods used to treat the patient when withholding PHAAM 4) complications related to not performing PHAAM.Secondary endpoints were 1) Emergency department endotracheal intubation (ED-ETI) 2) difficult ED-ETI.We collected all core data proposed in the consensus-based template by Sollid et al.  and the The indications for performing PHAAM as categorised by Sollid et al.  are: (1)For this study, we categorised the reasons for choosing not to perform PHAAM as1. expected difficult airway management 2. difficult or limited access to the patient3. short transport distance to the emergency department 4. aspects of the patient’s current medical condition5. the patient’s co-morbidity6. physician’s lack of training or experience with the type of patient in question7. lack of proper equipment8. no assistance available9. other.Reason number 4 includes both patients so critically ill or injured that advanced critical care is considered futile and patients so clinically unstable that the attending pre-hospital critical care anaesthesiologist assessed the risks associated with pre-hospital PHAAM to outweigh the potential benefits of a secure airway and controlled ventilation. Number 5 includes patients where co-morbidity renders PHAAM unethical  or terminal cancer) and where palliative care are more appropriate.We analysed the reasons for not performing PHAAM in the following groups of non-cardiac arrest patients: a) trauma b) subarachnoid haemorrhage/stroke c) asthma/COPD and c) cardiac disease. We also analysed the reasons for not performing PHAAM in cardiac arrest patients.The following alternatives to PHAAM were available:without the use of airway manoeuvres (chin lift or jaw trust) or an oro-pharyngeal airway1. spontaneous ventilation (with or without supplementary oxygen) with the use of airway manoeuvres2. spontaneous ventilation (with or without supplementary oxygen) with the use of an oro-pharyngeal airway3. spontaneous ventilation (with or without supplementary oxygen) without the use of an oro-pharyngeal airway4. bag-mask ventilation (BMV) with the use of an oro-pharyngeal airway.5. BMV Possible complications include (as defined in the template ): vomitiPhysio-Control, Redmond, USA).The pre-hospital critical care teams measured oxygen saturation, heart rate and blood pressure by using a LifePak 12 monitor  ED-ETI performed – easy, 1b) ED-ETI performed – difficult, 2) ED-ETI not performed. We defined difficult endotracheal intubation according to the latest version of the ologists  as more We collected data from eight pre-hospital critical care teams, including the HEMS. The anaesthesiologists in the participating teams filled in a registration form containing all the core data recommended by Sollid et al.  as well To reduce the risk of recall bias and selection bias, the primary investigator reviewed the registration forms on a day-to-day basis looking for missing forms or incomplete data sets. We crosschecked the registration forms with the standard pre-hospital records from the pre-hospital critical care teams to ensure the highest possible data coverage. In cases of missing data or inconsistencies, we asked the attending pre-hospital critical care anaesthesiologists to provide additional details for clarification.Stata12 (StataCorpLP). Because of the rigorous crosschecking and day-to-day control, missing data were rare. If the missing data were unobtainable, we performed complete case analyses.We analysed the data in the statistical program No patients had their treatment altered because of the study. All anaesthesiologists participated in the study on a voluntary basis – there were no refusals. The study did not involve any alterations from normal practice and according to Danish law, it did not need the approval of the Regional Ethics Committee, nor did we need the patients’ consent to register and publish the data. The Danish Data Protection Agency approved the study .Figure We display the demographics, co-morbidity data and patient characteristics of the 347 patients in whom the pre-hospital critical care physicians decided against PHAAM as Additional file We present pre-hospital critical care anesthesiologists’ reasons for considering PHAAM in Table solely because of a short distance to the ED in 6.6% (n = 23) of these patients. Of these, 87.0% (n = 20) were endotracheally intubated in the ED.Table Additional file Figure Two (0.6%) of the 347 patients not treated with PHAAM suffered aspiration of gastric content or blood into the lungs. We recorded no other immediate airway complications or complications related to the decision not to perform PHAAM.We display the incidence of ED-ETI according to physicians’ reasons for not performing PHAAM in Table We display the incidence of difficult ED-ETI according to physicians’ reasons for not performing PHAAM in Table The most common reason for withholding PHAAM in our system is patient condition and patient co-morbidity. Unfortunately, our data do not allow us to distinguish between cases where PHAAM were not necessary, cases where PHAAM were considered associated with too high a risks of complications and cases where the attending anaesthesiologist deemed PHAAM futile or unethical. Nevertheless, the current study provides the first important knowledge into why pre-hospital critical care anaesthesiologists sometime withhold PHAAM.2 measurement during CPR to monitor the quality of CPR and to detect return of spontaneous circulation (ROSC) at an early stage [Our current results show that a high percentage of potential PHAAM-patients not treated with PHAAM were in cardiac arrest. It is important to stress that the decision not to perform PHAAM is not the same as deciding not to perform cardio-pulmonary resuscitation (CPR). Ventilating the patient with BMV during CPR may be in accordance with current guidelines . These gly stage . This rely stage . But theIt is noteworthy, that limited access to the patient is a rare reason for withholding PHAAM in our system. In the few case where this played a role in the decision-making, it was typically a cardiac arrest patient lying in a narrow place where PHAAM would be difficult but where BMV was possible.In a small but significant proportion of our patients, PHAAM where not performed because of a short transport distance from the scene to the ED. This might be a sensible solution, especially if there is an increased risk of difficult PHETI or post-RSI complications, or if the pre-hospital critical care anaesthesiologists is occupied performing even more urgent interventions on the patient. On the other hand, even a few minutes transfer from the scene to the ED may prove disastrous if the patient has a severe airway or respiratory problem. Furthermore, even though transfer time itself may be short, patient packaging and loading on scene and patient handover in the ED may add to the ETI delay.Our 1.8% incidence of PHAAM not performed because the attending pre-hospital critical care anaesthesiologist anticipated a difficult-to-manage airway is in contrast to the approximately 22% incidence of intubations requiring more than one intubation attempt, previously reported from our system . We haveThe fact that the most common method of treating patients in cases where the pre-hospital critical care physician withheld PHAAM were BMV corresponds to the high incidence of cardiac arrest in our material.The risks associated with PHETI, which in our system is comparable to those associated with RSI in British and North-American EDs , must beIt is interesting, that the in-hospital anaesthesiologists in the ED performed ETI in almost 90% of the patients in whom the only reason for not performing PHETI was a short transport distance to the ED. Our data do not allow us to calculate the time delay to ETI in those patients who had their trachea intubated in the ED. We would like to stress that we do not believe that a “maximum acceptable delay” can be defined in these situations. In our opinion, postponing PHAAM solely because of a short distance to the ED is a debateable practice.It is also worth noticing that only one fifth of the patients assessed by the pre-hospital critical care physician as having a potential difficult airway proved to have a difficult ED-ETI. These results supports our previous notion that our system may need additional critical decision-making aids to further improve system performance and patient safety related to PHAAM.The main limitation of the current study is that the attending physicians registered the data. They are therefore subject to registration bias (systematic errors in the registration of data) or recall bias. The high capture rate reduces the risk of selection bias. We tried to reduce the effects of this further as described in the Bias section.We did not introduce a set of criteria for when the attending anaesthesiologists ought to consider performing PHAAM. We wanted to describe our services as they currently are; with no overall guidelines or SOPs for PHAAM. This may very well have influenced the data collected, as the reasons for considering performing PHAAM are more than likely to vary between the pre-hospital critical care anaesthesiologists. However, we feel that this depicts the current situation in our system.As with the other parts of this prospective study , we did Missing data are rare except for the 25% of missing reasons for not performing PHAAM in cardiac arrest cases. We do not know why missing data are more common in this group, but based on the review of the registration forms and the patients’ pre-hospital charts the by far most common reason for not performing PHAAM in this group of patients were the patient’s condition (i.e. the CPR were terminated before the attending anaesthesiologist considered PHAAM to be necessary).It is vitally important to understand that this study does not answer the question whether PHAAM and PHETI should be performed or not. The low incidence of complications associated with not performing PHAAM should not be generalised to the whole populations of pre-hospital critical care patients. Caution is therefor advised when interpreting our results.This was part of a larger study from one homogenous Danish system of anaesthesiologist-staffed pre-hospital critical care teams. This limits the ability to generalise the findings to other systems with different staffing, caseload or case mix. Nevertheless, we believe that our results may be of use to other both physician- and paramedic-staffed pre-hospital services. We also believe that they may be useful when trying to improve patient safety in different pre-hospital systems.Further studies are required to gain more knowledge into the critical decision making process involved in PHAAM and especially to establish whether introducing SOPs and checklists in physician-staffed pre-hospital systems can reduce the incidence of complications and ultimately improve patient safety and outcome . One mayWe have illustrated some of the complexity of the critical decision making process associated with pre-hospital advanced airway management. We have also identified the reasons for not performing pre-hospital advanced airway management and the alternative treatment methods used in our system. Immediate complications related to the decision of not performing pre-hospital advanced airway management were rare.PHAAM: Pre-hospital advanced airway management; EMS: Emergency medical service; HEMS: Helicopter emergency medical service; ETI: Endotracheal intubation; PHETI: Pre-hospital endotracheal intubation; EMT: Emergency medical technicians; RSI: Rapid sequence intubation; ED: Emergency department; GCS: Glasgow coma scale.The authors declare that they have no competing interests.LR and TMH conceived the study. LR, TMH and ET designed the study. LR performed the data collection and –management. All authors contributed to data analysis and -interpretation. LR drafted the manuscript. TMH, HK and ET revised the manuscript critically for important intellectual content. All authors read and approved the final version of the manuscript for publication.Scandinavian Society of Anaesthesiology and Intensive Care Medicine Advanced Educational Programme in Emergency Critical Care.LR is a research fellow at the Norwegian Air Ambulance Foundation, consultant anaesthesiologist at the Viborg Regional Hospital and a pre-hospital critical care physician in the Central Denmark Region. He currently holds the post as Programme Director for the TMH is a consultant anaesthesiologist at the Aarhus University Hospital, Lead Clinician of the Pre-hospital Critical Care Team in Aarhus and a pre-hospital critical care physician in the Central Denmark Region.HK is a consultant anaesthesiologist and professor of emergency medicine at the Aarhus University Hospital.ET is a consultant anaesthesiologist and professor of anaesthesiology at the Aarhus University Hospital.Registration form.Click here for fileDemographic data and patients' characteristics.Click here for fileFurther patients' and treatment details.Click here for file


Analysis of arm-selection patterns in the lesioned animals suggests little influence of the lesion in the ability to predict the reward absence. AcbC lesions did not change exploratory locomotor activity in an open-field test in which there were no rewards. This suggests that the AcbC controls the activity level of planned resting behavior shaped by the 8-arm FFT. Rats receiving training after AcbC lesioning showed a reduction in motivation for reward seeking. Thus, the AcbC also plays important roles not only in controlling the activity level after the last-reward collection but also in motivational learning for setting the activity level of reward-seeking behavior.In recent years, the study of resting state neural activity has received much attention. To better understand the roles of different brain regions in the regulation of behavioral activity in an arousing or a resting period, we developed a novel behavioral paradigm  using the radial 8-arm maze and examined how AcbC lesions affect behavioral execution and learning. Repetitive training on the 8-arm FFT facilitated motivation of normal rats to run quickly to the arm tips and to the center platform before the last-reward collection. Importantly, just after this point and before confirmation of no reward at the next arm traverse, locomotor activity decreased. This indicates that well-trained rats can predict the absence of the reward at the end of food seeking and then start another behavior, namely planned resting. Lesions of the AcbC  The observation that neural activity can increase during awake and intentional resting states has received increasing research attention in recent years, from examination of individual neurons to functional brain imaging to behavior. Examples of intentional resting states at the behavioral level include periods when a subject is not performing a task, times when a subject is in task-residuals A network of brain regions that exhibit increased activity during rest and decreased activity during a given task has been identified. This network has been termed the “default mode network” (DMN) In rats, neural activity is often elevated during a no-task condition or resting state. Taha and Fields, for example, showed that neurons in the nucleus accumbens (Acb) display enhanced activity when the rat fails to exhibit a learned reward-approach behavior in a no-task state. Acb function mainly has been investigated by selective lesion techniques In the present study, we introduced a behavioral task called the 8-arm food foraging task (8-arm FFT), a learning and memory task in which the reward condition changes (from reward-available to reward-unavailable situations). In the 8-arm FFT, animals learn how to predict, without external cues, the reward condition and learn when to voluntarily change their activity level according to changes in the reward condition. The active (reward-available) and resting (reward-unavailable) periods were specifically built into the task to better understand the roles of different brain regions in the regulation of behavioral activities in an arousing or in a resting period. We made selective AcbC lesions in three different conditions. First, we examined how the AcbC contributes to the control of motor activity during active and resting periods by lesioning animals after training on the 8-arm FFT. Second we examined how the AcbC contributes to shaping optimized motor behavior through repetitive training by lesioning the AcbC before training on the 8-arm FFT. Finally, to understand the nature of the behavior inhibited by AcbC activity, we examined the effect of AcbC lesions on general motor activity or simple exploration.This study was carried out in strict accordance with the Guidelines for the Care and Use of Experimental Animals at Saitama University, Japan. All protocols for animal experiments were approved by the Saitama University Institutional Animal Care and Use Committee (permit number: H21-1-16). Subjects were adult male Long-Evans rats  weighing 300–360 g. They were 5–6 months old at the beginning of behavioral testing. All rats were given ad libitum access to water and were handled daily for 10–15 min. Food intake of each rat was restricted: Rats were given daily 12–16 g of solid food pellets  in order to maintain their weight at 85% of their free-feeding weight. This was done in order to motivate the animals to obtain the food reward in the behavioral task.The various experimental groups are explained in detail in the Stereotactic lesions of the AcbC were made using a standard stereotaxic apparatus (David Kopf Instruments). Rats were first injected with pentobarbital sodium  dissolved in saline and then maintained on anesthesia with isoflurane  for the surgical procedure. Bilateral lesions were made by infusing ibotenic acid  through a stainless steel needle (200 µm outer diameter) attached to the stereotaxic device. Coordinates for injection sites were as follows: AP, +1.9 mm rostral to bregma; ML, ±1.7 mm lateral to the midline; V, −6.3 mm ventral to the dura mater. Sham-lesioned rats received injections of the same volume (0.6 µl/hemisphere) of PBS at these coordinates. The needle was left in place for 15–20 min to allow the toxin to diffuse sufficiently within the target sites. Rats were allowed to recover for 7 days following the lesion surgery. For the first 4 days after surgery, rats were given daily i.p. injections of antibiotic solution .An eight-arm radial maze was used for the 8-arm FFT. The maze had an octagonal-shaped center platform (40 cm across), from which eight equally spaced arms radiated outward; each arm measured 50 cm×9 cm. Throughout this report, we refer to the end of the arm farthest away from the maze center as the “tip” and the center platform as the “platform.” A food cup was located at the tip of each arm.3) with black walls and numerous external landmarks . The room ceiling was 180 cm from the maze surface. In preliminary studies, we observed that rats trained in a room with a higher ceiling (250 cm from the maze surface) occasionally showed a random pattern of arm selection after 25 trials . We believe that the higher ceiling induced more anxiety in the rats, causing more re-entry arm errors during the reward-available period. Thus, all of our data in the present study were collected in the lower-ceiling room . Four recessed lights (7 lux at the platform surface) provided indirect illumination, and noise was presented to mask extraneous noise. These environmental conditions ensured that rats reliably performed sequential pattern of arm selection.Eight computer-controlled gates (clear 9×13 cm acryl plates covered with clear blue plastic skirts) situated between the platform and the beginning of the arms controlled entry to the arms. The maze was elevated 40 cm above the floor and was centered in a room  provided indirect illumination. Noise was presented to mask extraneous noise.Rats' movements on the maze were recorded by a CCD camera mounted 150 cm above the maze surface. Video data (440 pixels×440 pixels) were digitized  at a rate of 30 frames/sec and were analyzed in real time with custom software running on a PC. The rat's position (center of the rat's black head) in each frame was determined using graphic analysis functions ; the calculated position for each frame was stored onto a hard disk of the PC, along with the timing of gate-openings and the timing of each reward collection at the food cups. Reward sensors at the arm tips monitored the state of each food cup (filled vs. empty). The computerized tracking system also directed the task schedule for each trial by controlling the arm entry gates (opening/closing). These event signals were also simultaneously recorded onto the disk along with the time-stamps associated with the tracking data.8-arm FFT training: Rats received one trial per day. Prior to training in the 8-arm FFT, however, all animals received a preliminary learning trial, during which sucrose pellets (1811555(5TUT); PMI Certified Rodents TestDiet, Richmond, IN, USA) were liberally placed throughout the surface of each arm, from the beginning of the arms to the tips, and on the platform near the beginning of each arm. In this preliminary learning trial, the rat was first placed on the platform for 2–3 min while all arm gates remained closed. Then, all of the gates opened simultaneously, allowing access to the arms. About 30 min after the start of this preliminary learning trial, we stopped recording the behavior and returned the rat to its home cage.For the 8-arm FFT training, two sucrose pellets were placed in each food cup at the arm tips . As withFor some trials, or portions of trials, we occasionally failed to obtain behavioral data because of an instrument malfunction. These rare trials were counted as having “no data,” and were excluded from analysis.Open-field test: The rat was placed into the center of the arena and allowed to move freely around the open-field and explore the environment. The rat's traveling distance was recorded during a test period of 30 min using the same tracking system that was used in the radial 8-arm FFT. The arena floor was cleaned before the next rat began its open-field test.Analysis of motor activity: For behavioral analyses, we calculated the rats' traveling distance before and after the last-reward collection, the tip-approaching speeds, and the platform-approaching speeds on each arm approach for each trial. We also measured the number of arm re-entry errors for each trial. Traveling distance in a trial was calculated from tracking data, which recorded the rat's maze position every 32 msec. The timing of the last-reward collection was estimated from data obtained from reward sensors. Tip-approaching speed was defined as the rat's mean speed from arm entry to reaching the end of the arm (distance covered divided by time to reach the tip). The calculation started when the rat's head passed over a virtual line at the beginning of the arm (5 cm from the center of the platform) to when it reached the end of the arm and stopped. Platform-approaching speed was defined as the rat's mean speed as it moved away from the end of the arm to the maze center. This calculation started when the rat's head passed over a virtual line at the end  to the point at which the rat's head passed over a virtual line at the platform (5 cm from the border between the arm and the center of the platform). The mean speeds for each rat in five successive trials were analyzed for each arm. These mean speeds were averaged before the last-reward collection in order to compare performance of rats with AcbC lesions and rats with sham lesions during the reward-available period. Tallying the number of re-entry errors in each trial was also done automatically from the same calculation done for the tip-approaching speed.Analysis of prediction of reward absence: We were interested in determining whether rats showed behavioral signs of knowing when the trial was complete, even before they confirmed there would be no more rewards . Selective differences in certain task behavioral parameters on the 8-arm FFT, before and after AcbC lesions, might provide evidence of its role in organizing/regulating motor activity control. Several behavioral parameters were analyzed for this purpose: (1) platform-approaching speed just before (7th platform approach) and after (8th platform approach) the last-reward collection  tipAnalysis of arm-selection patterns: We were interested in determining whether rats displayed a sequential pattern of arm selection, or one that tended to be somewhat random in the 8-arm FFT. Of course for the first arm selection, the pattern of arm selection could not be determined. For the second arm selection, we regarded it as sequential if the rat traversed an arm adjacent to the first one selected, followed by another adjacent arm selection, continuing to the end, selecting each successive adjacent arm  one if at some point in the trial the rat selected and ran down an arm non-adjacent to the prior one selected. In the example shown , the 3rdAnalysis of random pattern of arm selection: To determine a rat's first random arm-selection pattern, we counted the number of first random pattern of arm selections at each arm traverse of the rat in five successive trials.Statistical comparisons: We used one-way ANOVA to examine group differences in traveling speeds (arm tip-approaching and platform-approaching speeds) along the arms in a trial of the 8-arm FFT. Dunn-Sidak post hoc tests were used to determine where any overall speed differences detected in the ANOVA occurred in the sequence of arm visits in the trial. When appropriate, comparisons between rats with AcbC lesions and sham control rats were tested with unpaired t-tests. To test for an occurrence of a random pattern of arm selection on a potential 9th arm selection, we compared the data in the same well-trained rats before and after they received AcbC lesions by using the Chi-square test. To examine the relationship between platform-approaching speed during the reward-available period and locomotor activity (distance traveled) after the last-reward collection, we used correlation analysis. Statistical analyses were performed using GraphPad Prism 6.0 for Mac  and MATLAB .After the end of the behavioral experiments, rats were deeply anesthetized with pentobarbital sodium and perfused transcardially with 0.01 M PBS followed by 10% formaldehyde. The brains were removed and postfixed in 10% formaldehyde and then cryoprotected by placing them in a solution of 30% sucrose in 0.01 M PBS for 48–72 h at 4°C until they equilibrated . Coronal sections (40 µm thick) were cut on a freezing microtome and collected in cold 0.1 M PBS. Sections were mounted onto silane-coated glass slides and stained with cresyl violet . Stained sections containing the anterior end to the posterior end of the AcbC were examined using a light microscope by an investigator who was blind to the behavioral results. Areas of neuronal loss were mapped onto drawings of standardized coronal sections of the rat brain http://rsb.info.nih.gov/ij/) to estimate and compare the extent of the lesioned areas across all rats. Rats with unilateral AcbC lesions did not differ behaviorally from rats with bilateral AcbC lesions. Next, we determined the percentage of the AcbC that was lesioned by using ImageJ to measure the mapped areas of the lesions in the individual drawings of standardized coronal sections at different posteroanterior levels. We chose the area measurement of the largest lesion (either in left or right hemisphere) for the percentage calculations.We used ImageJ software . During this last phase, the rat could freely traverse the platform and arms, if it desired. There were only 8 rewards to be retrieved during a trial, each in a food cup at the arm tip.The horizontal bar of The task also has several critical events. The first important event for the rat—“gate-open” (GO)—is the simultaneous opening of all the gates after the waiting period  often occurred just after the last-reward collection .To examine the contribution of the AcbC on a rat's ability to detect when all rewards are retrieved, we analyzed three parameters that change between the period after the last-reward collection to before the rats reach an empty food cup  at the arm tip. These parameters are (1) platform-approaching speed just before (7th platform approach) and after (8th platform approach) the last-reward collection, (2) tip-approaching speed just before (8th tip approach) and after (9th tip approach) the last-reward collection, and (3) occurrence of a random pattern of arm selection on an attempted 9th arm visit. Although well-trained rats selected each arm sequentially before the last reward was retrieved, a random selection . After the lesion surgery, in AcbC-lesioned rats, it was also 0/30 cases. At the 9th arm selection, 33.3% (10/30) AcbC-lesioned rats showed a random pattern of arm selection. Compared to their performance before lesion surgery, they showed no significant difference . Moreover, there was a significant drop in tip-approaching speed at the 9th approach compared to the 8th approach . In five successive trial sessions in unlesioned rats at the 8th arm selection, 0/30 cases occurred . This was observed in rats trained after they had received AcbC lesions.Use of our 8-arm FFT in the present lesion study revealed the following interesting facts. Well-trained rats showed that they predicted a reward absence just after their last-reward collection and then started the planned resting behavior during the reward-unavailable period. Based on the results of making an AcbC lesion We also examined the influence of AcbC lesions on exploration and habituation in an open-field test . HoweverBased on the results of the present study, we advance a provisional model for the mechanism of activity control mediated by the AcbC . First, It was already reported that some Acb neurons show greater firing levels when animals are in a “no task” state compared to periods when they are performing a task after training has no affect on any aspects of reward-approaching behavior. Even with bilateral AcbC lesions before and after training, arm tip-approaching speed dropped when animals revisited the same, already rewarded arm   see . We alsonovel open field Previous work indicates that lesion of the Acb or AcbC results in hyperactivity in an open-field task The AcbC is strongly involved in activity or learning control of preparatory behaviors that have indirect commitment to reward acquisition before learning impairs instrumental responses Many studies have reported that lesioning the AcbC It has been reported that DA depletion decreases locomotor activity In conclusion, we propose that at least part of the AcbC output activity is utilized to inhibit actions in a situation-dependent manner. As shown here, these actions were redundant for a specific situation. This would contribute to the formation of a temporal gate for a purposeful behavior set.Figure S1Examples of sequential and non-sequential patterns of arm selection. Solid, irregular lines superimposed on schematic of 8-arm maze show movement traces of an example rat's traverses up and down the arms. Lowercase letters at arm tips indicate the order of arm selections. Repetitive training on the 8-arm FFT resulted in rats adopting a sequential pattern of arm selections (A). In this pattern, each successive turn of the rat in the trial is 45°. With a random arm-selection pattern (B), the rat selects a non-adjacent arm at some point(s) in the trial. In this instance, the 3rd visited arm (c) was not adjacent to the 4th visited arm (d). a, 1st selected arm; b, 2nd selected arm; c, 3rd selected arm; d, 4th selected arm; e, 5th selected arm; f, 6th selected arm; g, 7th selected arm; h, 8th selected arm.(TIF)Click here for additional data file.Figure S2Change in the occurrence of random arm-selection patterns before the last-reward collection. To clarify the learning process, we trained 10 adult rats for 50 trials on the 8-arm FFT. Repetitive training on the 8-arm FFT decreased the occurrence of random arm-selection patterns before the last-reward collection . Data represent means ±SEM.(TIF)Click here for additional data file.Figure S3Behavioral performance of well-trained rats assessed in five successive trials before AcbC lesions. Before AcbC- (n = 6) or sham-lesion surgery (n = 6), both groups attained similar performance levels in the 8-arm FFT. Tip-approaching (TA) (A) and platform-approaching (PA) speeds (B), the number of re-entry errors (C), and the traveling distance (D) after the last-reward collection (LRC) of the two groups were not statistically different . Data represent means ±SEM.(TIF)Click here for additional data file.Figure S4Effect of AcbC lesions on re-entry errors before the last-reward collection. We examined the effects of AcbC lesions on well-trained rats  and untrained rats . Lesioning of the AcbC had no effects on re-entry errors before the last-reward collection. Data represent means ±SEM.(TIF)Click here for additional data file.Figure S5Effect of AcbC lesions on tip-approaching speed just before and just after the last-reward collection. We compared the effects of AcbC lesions on the tip-approaching (TA) speed measured between two points: just before (8th arm approach) and just after (9th arm approach) the last-reward collection (LRC). AcbC-lesioned rats that received lesions after training  (A) and others that received lesions before training  (B) showed significant decreases in tip-approaching speed at the 9th arm approach . #P<0.05 compared to TA speed on the 8th arm visit. Error bars denote SEM.(TIF)Click here for additional data file.Figure S6Histological analysis of lesioned groups. Comparison of the percentage of AcbC that was damaged, as assessed in coronal sections, in rats lesioned after training in the 8-arm FFT (dotted line), rats lesioned before training (solid line), and rats lesioned before the open-field test (broken line). There were no significant differences in the extent or distribution of the lesioned areas across the three groups . Error bars denote SEM.(TIF)Click here for additional data file.Figure S7Detailed analysis of running speed of reward-seeking in rats that received AcbC lesion before training. We compared the effects of lesioning the AcbC before training on the tip-approaching (TA) speed and platform-approaching (PA) speed before the last-reward collection (LRC). AcbC-lesioned rats showed no significant differences in tip-approaching speed (A) compared to sham control rats . However, rats receiving AcbC lesions before training showed slower platform-approaching speeds (B) on the 1st arm traverse to the 7th arm traverse than sham controls . Error bars denote SEM.(TIF)Click here for additional data file.Figure S8Quantitation of running speed changes in AcbC-lesioned rats during learning of tip-approaching and platform-approaching before the last-reward collection. As with normal rats, the behavioral performance parameters of rats that received lesions of the AcbC before repetitive training on the 8-arm FFT plateaued before the 30th trial. A: Change in tip-approaching (TA) speed before the last-reward collection (LRC). B: Change in platform-approaching (PA) speed before LRC. Error bars denote SEM.(TIF)Click here for additional data file.Methods S1Supplementary methods.(DOC)Click here for additional data file.


The increase in FA might indicate an increase in motor fiber alignment, myelination, and overall fiber integrity. Crossed and uncrossed fibers from multiple cortical regions might be one reason why the aMF fiber system showed more plastic structural changes that correlate with motor improvements than the CST.Motor impairment after stroke has been related to the structural and functional integrity of corticospinal tracts including multisynaptic motor fibers and tracts such as the cortico-rubral-spinal and the cortico-tegmental-spinal tract. Furthermore, studies have shown that the concurrent use of transcranial direct current stimulation (tDCS) with peripheral sensorimotor activities can improve motor impairment. We examined microstructural effects of concurrent non-invasive bihemispheric stimulation and physical/occupational therapy for 10 days on the structural components of the CST as well as other descending motor tracts which will be referred to here as alternate motor fibers (aMF). In this pilot study, ten chronic patients with a uni-hemispheric stroke underwent Upper-Extremity Fugl-Meyer assessments (UE-FM) and diffusion tensor imaging (DTI) for determining diffusivity measures such as fractional anisotropy (FA) before and after treatment in a section of the CST and aMF that spanned between the lower end of the internal capsule (below each patient’s lesion) and the upper pons region on the affected and unaffected hemisphere. The treated group (tDCS + PT/OT) showed significant increases in the proportional UE-FM scores , while no significant changes were observed in an untreated comparison group. Significant increases in FA  were found in the ipsilesional aMF in the treated group while no significant changes were found in the contralesional aMF, in either CST, or in any tracts in the untreated group. The FA changes in the ipsilesional aMF significantly correlated with the proportional change in the UE-FM ( Motor impairment after stroke has been related to the structural and functional integrity of descending corticospinal tracts Canedo, . BesidesResearch over the last decade has also shown that stroke recovery can be facilitated by targeted non-invasive brain stimulation geared towards enhancing or diminishing local cortical excitability. Techniques such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation  is the most commonly used diffusivity measure. It is a normalized value between 0 and 1 and is a measure of the directional preference of water diffusivity. The higher the FA the more aligned fibers are in a particular voxel. Although voxel-based and deformation-based morphometric studies have shown increases in gray matter signal in training paradigms that spans years  combined with a combination of physical and occupational therapy  consisted of ten stroke patients (≥4 months after their first and only unihemisphere stroke) who participated in an experimental study consisting of a combination of dual transcranial direct current stimulation (tDCS) for 30 min while simultaneously receiving physical/occupational therapy (PT/OT) for 60 min  who were scanned once.All subjects gave their written informed consent following protocol approved by the Committee of Clinical Investigations and Internal Review Board at Beth Israel Deaconess Medical Center.2) using a Phoresor II Auto stimulator . The stimulation consisted of 30 min of 1.5 mA direct current with the anode placed over the ipsilesional motor cortex and the cathode over the contralesional motor cortex. Stimulation sites were identified using the international 10–20 EEG electrode system.The treatment consisted of 10 sessions of tDCS (for 30 min/day) in conjunction with PT/OT (for 60 min/day) over a 2–3 week span. PT and OT techniques included functional motor tasks of the affected arm and hand to promote sensorimotor integration, coordination of movement, and goal-directed activities of practical relevance for the patient. It was left to the physical therapist to determine the motor activities/tasks that would be most helpful for a particular patient. Direct current was delivered through 2 saline-soaked surface gel-sponge electrodes (electrode area was 16.3 cmEach patient underwent the Upper-Extremity Fugl-Meyer Assessment (UE-FM) before and after treatment. The UE-FM assessment is a standardized and validated 30-item motor impairment assessment frequently used in stroke rehabilitation  scanner. The treated group was scanned before and after therapy, while the untreated group was scanned two times with similar time intervals between scans as the treated group. The group of healthy elderly subjects used to generate the canonical CST and aMF were scanned only once.b-value = 1000 s/mm, 1 image with b-value = 0 s/mm). Three patients in each group were acquired using a single-shot, spin-echo, echo-planar imaging sequence .All subjects underwent anatomical scans, which were acquired using high-resolution strongly T1-weighted Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE) sequence (voxel size = 0.93 × 0.93 × 1.5 mm). Two types of DTIs protocols were used. Seven patients in each group (treated and untreated) were acquired using a single-shot, spin-echo, echo-planar imaging sequence .FLIRT and FNIRT), with the aid of lesion masks. The FA image from the second time point was then registered to the normalized FA image from the first time point. All FA images with a left hemisphere lesion were flipped such that all images had the stroke lesion in the right hemisphere. The FA images of the healthy control subjects were normalized directly to the FMRIB standard FA template. BEDPOSTX was conducted on these images in preparation for tractography.For the patients, we registered the FA image of the first time point to the FMRIB standard FA template using linear and non-linear algorithms (z = −24) as a narrow ROI that included only the anterior part of the pons (for the CST) and similarly a single slice ROI was drawn in the posterior part of the pons (for the aMF). Additional ROIs were drawn in the PLIC and the white matter underlying the posterior part of the precentral gyrus. Exclusion ROIs were drawn on the superior and medial cerebellar peduncle to exclude fibers to the cerebellum, as well as the middle sagittal region covering the brain stem and corpus callosum to exclude trans-hemispheric fibers. ProbtrackxTractography of the aMF and CST were outlined in a previous publication  images of each patient were normalized to FSL’s skull-included T1 template. Lesion maps were drawn on the normalized T1-weighted images by an experienced neurologist using T1- and coregistered T2-weighted images as a reference. The lesion maps were then overlapped with the probabilistic CST  is considered to be significant. Mean FA values from tracts that were found to be significantly changed after treatment were correlated with proportional UE-FM change scores.All left-hemisphere lesions were flipped to the right hemisphere for analysis. FA and other diffusivity measures were extracted from the CST and aMF templates on the lesional and contralesional hemisphere in both the treated and untreated groups at both time points. Paired p > 0.1). Otherwise, both groups were well matched with regard to age, time post-stroke-to-enrollment, and between-scan interval. The wCST-LL is a novel measure that combines lesion volume and lesion location by calculating a weighted overlap region between each patient’s lesion and the canonical corticospinal tract as it descends from the cortex through the internal capsule into the brainstem. As one can see from Figure In Figure p < 0.01) were only found in the ipsilesional aMF in the treated group while no significant changes  were found in the contralesional aMF, CST in either hemisphere, and in the untreated group . The increase in FA in the ipsilesional aMF was mainly due to an increase in axial diffusivity , although this did not become statistically significant (p > 0.1). We did not detect any significant changes or even any non-significant trends in radial diffusivity (RD) or in MD in any of the tracts (p > 0.2).The mean FA changes in the CST and aMF on the lesional as well as contralesional side were calculated. Significant increases in FA .The FA changes in this portion of the aMF were also significantly correlated Figure  with proIn this study we found a significant increase in FA in the portion of the ipsilesional aMF that spans between the inferior internal capsule and the upper pons. This increase in FA correlated with the proportional change in UE-FM comparing pre- with post-intervention assessments. The most significant and most consistent FA change across subjects was seen in the portion of the aMF in the mid to upper pons region and in the white matter in the vicinity of the red nucleus. None of the other tracts showed any consistent or significant changes across subjects in the treated group. No significant changes were seen in the control group.The red nucleus and its surrounding white matter has been shown to have alterations of FA values when compared to normal elderly controls in a cross-sectional analysis of chronic stroke patients  in 7 of 10 patients in each of the two groups. This sequence was chosen to minimize movement artifacts in our group of moderate to severely impaired patients. However, this diffusion sequence had non-isotropic voxels and 25 diffusion directions. Acquiring higher resolution images with isotropic voxels could improve the accuracy of parameter estimation in the DTI analysis. Nonetheless, any systematic errors associated with our DTI sequence should be equally evident across all tracts and both groups. Second, this pilot study has a relatively small sample of experimentally treated patients which might limit generalizability of our results. However, our interpretation correlating rubro-spinal tract microstructural changes with motor recovery may be extrapolated to other stroke patients and other tracts. Third, we cannot determine whether the FA effects in the aMF are due to tDCS alone, physical/occupational therapy alone, or a combination of both as we suggest in this paper. Control groups of chronic stroke patients in previous studies who received any kind of peripheral sensorimotor activities to stimulate recovery, showed relatively little change (e.g., only 3.2% in UE-FM in the group who received sham-tDCS and PT/OT in Lindenberg et al.  and it wIn summary, we interpret the observed FA changes in the pons and in particularly in the vicinity of the ipsilesional red nucleus in the experimentally treated chronic stroke patients as a result of plastic remodeling of a polysynaptic tract that receives more widespread cortical input than the CST and has crossed and uncrossed fibers as has been shown in animal and human studies. The strong correlation of local FA values with UE-FM scores suggests that the observed diffusivity alterations are functionally meaningful. We hypothesize that the cortico-rubral and cortico-tegmental tracts which are making up the aMF could be particularly sensitive and responsive to experimental treatments that modulate cortical excitability in both sides of the brain involving primary and non-primary motor regions and could play a more prominent role in post-stroke recovery than has been assumed so far.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.


In Western countries, better knowledge about patient-related determinants of treatment adherence (medication and lifestyle) is needed to improve treatment adherence and outcomes among hypertensive ethnic minority patients of African descent.To identify patient-related determinants of adherence to lifestyle and medication recommendations among hypertensive African Surinamese and Ghanaian patients with suboptimal treatment results (SBP≥140) living in the Netherlands and how culturally appropriate hypertension education (CAHE) influenced those determinants.This study analysed data of 139 patients who participated in the CAHE trial. Univariate logistic regression analysis was used to measure the association between patient-related determinants  and treatment adherence. We also tested whether CAHE influenced the determinants.Medication self-efficacy and social support were associated with medication adherence at baseline. At six months, more medication self-efficacy and fewer concerns about medication use were associated with improved medication adherence. Self-efficacy was also associated with adherence to lifestyle recommendations at baseline. CAHE influenced patients’ illness perceptions by creating more understanding of hypertension, its chronic character, and more concerns about the associated risks.In this high-risk population, health care providers can support medication adherence by paying attention to patients’ medication self-efficacy, the concerns they may have about medication use and patients’ perceptions on hypertension. The CAHE intervention improved patients’ perception on hypertension. Hypertension is a major risk factor for cardiovascular disease and especially stroke.  To reducIn Western countries, hypertension is more common among ethnic minority groups of African descent than among whites. –6 AgyemaSince studies in several countries have shown that adherence to hypertension treatment recommendations is generally lower among disadvantaged populations, such as ethnic minority groups, it has been hypothesised that patients who belong to these groups may benefit from culturally tailored educational approaches. –17 Additthe consequence, timeline, personal-control, treatment-control and identity) and medications are predictive of adherence to medication and lifestyle recommendations. [Research based on models of health behaviour has provided evidence that adherence to treatment is a self-regulatory process and that patients’ ideas about their illness  Socio-demographic data were obtained at the start of the intervention. The research assistants who performed the baseline and follow-up assessments received a specific training for the purpose of this study and were blinded to the study conditions. [This study analyses data that were collected among participants who completed the CAHE study. Full details of the design and methods of this cluster-randomised trial and the content of CAHE intervention have been published previously. , 25 BrieFig 1)For this paper we performed a stepwise analysis of 1) the association between background characteristics and patient-related determinants of adherence and self-reported adherence to medication and lifestyle recommendations at baseline, 2) the association between changes in patient-related determinants of adherence and changes in self-reported adherence of medication and lifestyle recommendations at six months and 3) the influence of the CAHE intervention on changes in patient-related determinants of adherence. . [MMAS-8).  This scaMMAS-8). , 35–37 TSelf-reported adherence to lifestyle recommendations was assessed with a three-item scale derived from the Morisky scale. [Results of a psychometric analysis of these outcome measures are shown in Tables D and E in S1 File).y scale.  This scaTable B in S1 File)Self-reported data on age, gender, educational level, financial status, duration of hypertension, and years living in the Netherlands were collected at the start of the study, to describe background characteristics of the study sample. (Table A in S1 File):The following five measures were used to obtain information on potential patient-related determinants of adherence (Perceptions of hypertension were assessed with the Brief Illness Perceptions Questionnaire (IPQ-brief). The validity and the reliability of this questionnaire is well established The IPQ-brief asks patients to provide information about 8 dimensions of illness (hypertension) that can be answered on a 5-point Likert scale. Five dimensions are related to cognitive illness representations , two dimensions are related to emotional representations (respondents’ emotions and concerns about the illness) and one dimension is related to illness comprehensibility (coherence). [erence).  SeparateMedication self-efficacy was assessed with the Medication Adherence Self-Efficacy Scale (MASES-R). This scale shows good internal consistency, predictive validity, and convergent validity in African-American patients with hypertension. [tension.  The patitension.  Scores wBeliefs about medication were assessed with the ten-item Beliefs about Medicines Questionnaire (BMQ). [e (BMQ).  This inse (BMQ). , 42 The Social Support patients experienced when dealing with their condition was assessed using the validated 12-item Duke Social Support Scale (DUSOCS). [The amount of DUSOCS).  As demonDUSOCS).  DUSOCS iSatisfaction with care was measured using five items of the Consumer Quality Index-Diabetes [Diabetes  and fiveDiabetes  Similar Diabetes , 47 The Diabetes  A higherData were analysed using SPSS version 19 . The distributions of continuous data were graphically assessed. Means and standard deviations were reported for normally distributed data and medians and quartiles for non-normal distributed data.Univariate logistic regression analysis was conducted to assess the association between each determinant of adherence and each outcome variable (adherence to medication or lifestyle recommendations). Since the number of participants in the study was low, relative to the number of measured determinants,  a p-valuTo assess the influence of the CAHE intervention on the determinants of adherence we compared the change in the determinants between the intervention and the control group. Scores were compared using a chi-square test for categorical data and an independent t-test for normally distributed continuous data.http://www.controlled-trials.com/ISRCTN35675524). In accordance with the Declaration of Helsinki, written informed consent was obtained from all patients.The study protocols were approved by the medical ethics committee of the Academic Medical Center in Amsterdam (protocol ID MEC 09/070 # 09.17.0725) and CCMO (NL27507.018.09). This trial is registered at the ISRCTN Register under registration number ISRCTN35675524 (Table C in S1 File)Patients’ background characteristics at baseline are shown in Table B in p<0.001 and p = 0.026, resp.). Patients who were adherent to medications experienced less social support from non-family members as compared to non-adherent patients (p = 0.046). . Additionally, older patients were more likely to show an improvement in medication adherence than younger patients (p = 0.033). Patients who were more concerned about their medication use at baseline (BMQ-concern) were less likely to report an improvement in medication adherence. In this group the reported level of medication adherence remained low or decreased (p = 0.04). There were no significant associations between changes in determinants of adherence and changes in adherence to lifestyle recommendations.Fig 1). We found that medication self-efficacy and social support were the main determinants of adherence to lifestyle and medication recommendations at baseline. An increase in self-efficacy and a decrease in concern about medication use during the course of the clinical trial, led to an improvement in medication adherence. We found that the CAHE intervention did not significantly influence the aforementioned determinants of adherence. However, CAHE did improve patients’ illness perceptions, in terms of a better understanding of hypertension, more concerns about the associated risks and more awareness of the chronic character of hypertension.This study examined the pathway from patient education (CAHE) to adherence via patient-related determinants of adherence, among treated hypertensive patients of African descent from Dutch general practices with suboptimal treatment control To our knowledge this is the first study that examines patient-related determinants of hypertension treatment adherence among ethnic minority patients of African descent with uncontrolled blood pressure outside the US. In the CAHE study self-report instruments for assessing adherence to medications and lifestyle recommendations have been used. The self-reported format to acquire data has general advantages and disadvantages.  To obtailidated.  However,The used Morisky scale for assessing lifestyle adherence is of a complex nature since it summarizes multiple items of lifestyle adherence . Preferably the association of each item with the possible determinants would be tested individually on each item, which is unfeasible due to the limited population size.Since this study is a secondary analysis, some measurements and instruments are not the best imaginable fit to obtain the answers to our research questions. Considering ‘medication adherence’ the actual medication intake would be a preferred measure, and also for ‘lifestyle adherence’ a more objective measurement would be preferred.The self-efficacy reported for both lifestyle and mediation adherence is measured with the MASES-R. Since MASES-R is tailored to measure medication adherence, we can’t state that CAHE is not associated with lifestyle adherence self-efficacy. A possible explanation that CAHE did not affect medication-self efficacy is the limited room for improvement, as the level of medication self-efficacy was already very high at the start of the intervention .The BMQ used to measure beliefs about medication was shown to be insufficient to capture cultural variations in medication beliefs.  As CAHE We have chosen the MMAS-8 for measuring medication adherence because the instrument has been well validated and had reported good predictive power. , 35–37 HDue to the relatively low numbers of participants we were forced to reduce the number of determinants that could be taken into consideration within our analysis. By excluding some of the determinants based on univariate relations, we also excluded interaction terms involving these determinants.The development of more effective methods for addressing treatment adherence among hypertensive patients of African descent is a priority for research and practice in Western countries. , 60 Our Our study deepens the understanding of the pathway from patient education (CAHE) to adherence to lifestyle and medication recommendations for hypertension in ethnic minority patients of African descent with uncontrolled blood pressure. Firstly, more medication self-efficacy and less social support from people outside the family are associated with higher adherence to medication and lifestyle recommendations. Secondly, improvement in medication self-efficacy and decrease in concerns about medication is associated with increased medication adherence. Thirdly, the CAHE intervention appears to have a beneficial effect on patients’ illness perceptions, by contributing to a better understanding of hypertension and concerns about the associated risks, but not on patients’ medication self-efficacy or their concerns about medication.S1 File(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Background. Despite aggressive intensive medical management acute liver failure (ALF) may require high-urgency liver transplantation (LTx). Available prognostic scores do not apply for all patients; reliable tools to identify individuals in need of LTx are highly required. The liver maximum function capacity test (LiMAx) might represent an appropriate option. Referring to a case of ALF after Amanita phalloides-intoxication the potential of the LiMAx-test in this setting is discussed. Presentation of Case. LiMAx was performed in a 27-year-old patient prior to and after high-urgency LTx. In accordance with clinical appearance of hepatic encephalopathy, coagulopathy, and acute kidney failure, the LiMAx-test constituted a fulminant course of ALF with hardly any detectable metabolic activity. Following LTx with a marginal donor organ (95% hepatosteatosis), uptake of liver function was demonstrated by postoperative increase of the LiMAx-value. The patient was discharged from hospital on postoperative day 26. Discussion. ALF often is associated with a critical state of the patient that requires almost immediate decision-making regarding further therapy. Application of a noninvasive liver function test might help to determine the prognosis of ALF and support decision-making for or against LTx as well as acceptance of a critical donor organ in case of a critically ill patient. Amanita phalloides toxin induced ALF first promising data has been reported using the LiMAx-test [Acute liver failure (ALF) represents a potentially fatal complication of severe hepatic illness due to various reasons. Many patients suffering from ALF profit from aggressive intensive medical management; nonetheless, salvage of a substantial proportion of affected individuals still relies on orthotopic liver transplantation (LTx) , 2. SincMAx-test . As thisAmanita phalloides is a wild mushroom that can cause severe liver damage in man  and intravenous silibinin (20 mg/kg/body weight) was initiated immediately. Due to significant rise of transaminases (at norm on admission) and development of coagulopathy .We report on a healthy 27-year-old male patient who received HU-LTx due to ALF following Figures  therapy  Figures  but stilDespite continuation of the aforementioned therapy the patient's condition worsened continuously with requirement for intensive care treatment: transaminases further increased, coagulopathy worsened dramatically Figures , and relOn day 6 after toxin ingestion the patient received the first offer for LTx. The sighted deceased donor was a 59-year-old obese (body mass index of 28) female who suffered from a subarachnoidal bleeding due to a ruptured cerebral aneurysm. The donor liver was deemed steatotic by macroscopic appearance with confirmation by histology (60% micro- and 35% macrosteatosis). Since no other organ was available and the patient's condition was most critical, the organ eventually was accepted for transplantation following extensive interdisciplinary discussion.13C-labeled CO2 using the “Fast Liver Investigation Package” (FLIP) according to the manufacturer's instructions. By this means, onset of fulminant ALF was confirmed, as hardly any liver metabolism could be detected  by intravenously injecting a body weight-adapted dose of methacetin (2 mg/kg body weight) followed by detection of the exhaled detected .In accordance with this finding severe derangement of blood coagulation was found performing rotational thromboelastometry (ROTEM) according to the manufacturer's instructions  and interpreted referring to published reference ranges  Figure . Amanita phalloides toxin  steatotic organ of the aforementioned donor and a cold ischemic time of 7 h 37 min. LTx was performed with end-to-end vascular and biliary anastomoses. The warm ischemic time was 47 min. Due to diffuse bleeding the liver was packed and a planned second look was performed after 36 h. Triple immunosuppression (tacrolimus/mycophenolate mofetil/steroids) with anti-CD25 induction-therapy was provided. Histopathological work-up of the recipient's own liver demonstrated the severe liver damage caused by Figures .Despite known hepatosteatosis of 95% the graft showed an acceptable initial function. Only on postoperative day (POD) 1 mass substitution of blood products was performed . Thereafter no further substitution of blood products apart from Antithrombin III was necessary. The LiMAx-test and ROTEM analysis were repeated on POD 3 and POD 10, respectively, demonstrating continuous recovery of liver function in the course  decreased continuously and the patient was free of catecholamines on POD 6. He quickly was weaned from the respirator (POD 6). Kidney function likewise rapidly improved following LTx with limited diuresis starting on POD 6 and cessation of hemodialysis on POD 9. Following bacteremia with detection of Staphylococcus sciuri but good response to an antibiotic treatment with Vancomycin (in the course switch to Ciprofloxacin), admission to the surgical ward eventually was performed on POD 11. The patient was discharged from hospital in good condition on POD 26.Following the second look operation on POD 2, the requirement for catecholamines , or cryptogenic liver failure likewise provided very promising results: whereas all biochemical parameters  were statistically indistinct, the liver maximum functional capacity test turned out to be of prognostic relevance with full recovery after ALF for a LiMAx value >38 μg/kg/h [Primarily, the LiMAx-test has been developed for effective preoperative surgical risk evaluation to prevent major complications or even liver failure-related death. It has proven reliable in the assessment of liver function  and dete μg/kg/h .2 and acetaminophen. The latter is a well-known hepatotoxin that may lead to ALF itself when being overdosed [Despite the great potential for the management of ALF and being a noninvasive breath test, the major hypothetical threat of the LiMAx-test in this setting also needs to be discussed: the intravenously applied substrate methacetin is metabolized by the liver to COverdosed . The thrverdosed . Whetherverdosed  and thusReferring to the present case another issue related to transplantation can be discussed: following listing of a patient for HU-LTx it might be of great benefit to further define the time frame in which transplantation should be performed or, in other words, providing further evidence whether there is a need to accept a suboptimal organ to save the patient's life is highly desirable. In case of the young man listed for HU-LTx, we confirmed that ALF has taken a fulminant course applying the LiMAx-test (hardly any metabolic activity detectable). Within 48 h after listing of the patient a first offer for transplantation was obtained, which is in the usual range . Neverth Amanita phalloides-intoxication—often is associated with a critical state of the patient that requires almost immediate decision-making regarding further therapy. Application of a noninvasive liver function test might help to define the prognosis of ALF [In conclusion, ALF—especially followings of ALF , 24, sup


Since the Belgian elderly population is expanding at a very high rate, it is important for public health care systems to better understand ageing-related syndromes and diseases, such as sarcopenia. This condition is characterized by a progressive loss of skeletal muscle mass and its action generating capacity . Therefore it also implies risk of adverse outcomes like fall-related injury and impaired quality of life. Furthermore, it is well known that both muscle mass and muscle function are under strong genetic determination. However, most previous studies only considered individual associations of one specific gene variant with muscular phenotypes, whereas it is likely several related polymorphisms that combine to influence muscular characteristics. The data of the current study were assessed within the framework of the first (2002-2004) and third (2012-2015) generation Flemish Policy Research Centre Sport. A total of 579 healthy, Flemish Caucasian adults  were measured during a follow-up period of ten years. Genomic DNA was extracted and 384 gene variants were genotyped. Force and velocity characteristics of the knee extensors were measured using the Biodex Medical System 3® dynamometer. Furthermore, four genetic predisposition score (GPS) models were designed: a total GPS , a data-driven GPS , a weighted GPS (weighting is done based on the proportion of the variance explained by those variants with significant b-coefficients in stepwise regression) and an elastic-net GPS . Afterwards, these GPS models will be compared and used to predict genetic predisposition towards muscle mass and muscle function and its loss by ageing via linear regression analysis.


We assessed the Chinese version of the Drug Abuse Screening Test (DAST-10) for identifying illicit drug use during pregnancy among Chinese population. Chinese pregnant women attending their first antenatal visit or their first unbooked visit to the maternity ward were recruited during a 4-month study period in 2011. The participants completed self-administered questionnaires on demographic information, a single question on illicit drug use during pregnancy and the DAST-10. Urine samples screened positive by the urine Point-of-Care Test were confirmed by gas chromatography-mass spectrometry. DAST-10 performance was compared with three different gold standards: urinalysis, self-reported drug use, and evidence of drug use by urinalysis or self-report. 1214 Chinese pregnant women participated in the study and 1085 complete DAST-10 forms were collected. Women who had used illicit drugs had significantly different DAST-10 scores than those who had not. The sensitivity of DAST-10 for identify illicit drug use in pregnant women ranged from 79.2% to 33.3% and specificity ranged from 67.7% to 99.7% using cut-off scores from ≥1 to ≥3. The ~80% sensitivity of DAST-10 using a cut-off score of ≥1 should be sufficient for screening of illicit drug use in Chinese pregnant women, but validation tests for drug use are needed. Illicit drug use during pregnancy causes significant adverse obstetric and fetal outcomes, and is becoming an increasing major public health concern worldwide123456812In light of the alarming rise in illicit drug use in Chinese populations and lack of effective screening tools, we validated the Chinese version of Drug Abuse Screening Test (DAST-10) to detect illicit drug use among low risk Chinese pregnant women. We studied the psychometric properties of DAST-10 and examined its use in identifying illicit drug use during pregnancy and in assessing the degree of related problems.A total of 1483 Chinese pregnant women from Hong Kong were recruited during the 4-month study period, 1416 were attending their first antenatal visit and 67 were visiting the maternity ward for the first time. Overall, 1214 pregnant women participated in this study (81.9% response rate) and 1214 DAST-10 (Chinese version) screening forms were collected. Demographic characteristics are shown in Among the 1085 women with completed DAST-10 forms, 3 urine samples were lost or spilt before transfer to laboratory for test. 13 urine samples were confirmed to be positive for one or more of the illicit drugs tested. Among the 1069 urine samples which screened negative by the POCT cards, 42 urine samples were randomly selected as negative controls for confirmatory GC-MS, which were all confirmed to be negative for any drugs.In the anonymous questionnaires collected, one woman did not answer the single question on illicit drug use during current pregnancy, 14 women admitted to a history of illicit drug use during pregnancy and 1070 women denied a history of illicit drug use during pregnancy. A total of 24 pregnant women had either positive urinalysis for drug use or self-reported a history of illicit drug use.2 (45) = 1472.30 (P < 0.001) indicated the correlations between items were sufficiently large for PCA. Eigenvalues were calculated for each component in the data. Three components had eigenvalues over Kaiser’s criterion of 1 and in combination could explain 50.2% of the variance. A principal component analysis (PCA) was conducted on the 10 items of the DAST-10 using oblique rotation (direct oblimin). The Kaiser-Meyer-Olkin measure (KMO = 0.688) verified the sampling adequacy for the analysis. Barlett’s test of sphericity XThe DAST-10 scores of pregnant women were significantly different between those who had and those who had not used illicit drugs.Using urinalysis as a measure of illicit drug use (Measure 1), 13 pregnant women had positive urinalysis with a median DAST score of 1 (interquartile range 0 to 2), whereas 1069 women had negative urinalysis with a median DAST score of 0 . There was significant correlation between DAST-10 scores and the results of urinalysis .Using the self-reported single question as a measure of illicit drug use (Measure 2), 14 pregnant women self-reported illicit drug use during pregnancy with a median DAST score of 3 (interquartile range 1 to 5), whereas 1070 women reported no illicit drug use during pregnancy with a median DAST score of 0 . There was significant correlation between DAST-10 scores and the self-reported drug use .Using urinalysis-or-self-reported illicit drug use during pregnancy (Measure 3) as measure of illicit drug use, 24 pregnant women showed evidence of illicit drug use with a median DAST score of 2 (interquartile range 1to 3), whereas 1061 women had no history of illicit drug use with a median DAST score of 0 . There was significant correlation between DAST-10 scores and evidence of drug use by Measure 3 .The DAST-10 scores also significantly correlated with past history of illicit drug use before pregnancy  and with current cigarette smoking .The DAST-10 (Chinese version) had high face validity. Among the pregnant women with evidence of illicit drug use during pregnancy, those who denied current illicit drug use during pregnancy had significantly lower DAST scores  compared to those who admitted current illicit drug use. The distribution of participants in the five categories of DAST-10 scores according to the degree of problems Illicit drug use is an important public health problem worldwide. In Hong Kong, a survey showed that 1.6% of female students had history of illicit drug use82021192021Therefore, the use of bioanalytical measures as the gold standard for studying the validity of the screening tool would be more objectiveHowever, using just either one of the methods  as gold standards has limitations. In fact, the two methods are complementary to each other. Bioanalysis alone is not an ideal measure of illicit drug use. It may fail to detect drug use as it can only detect drug levels above a specific cut-off level within a specific period of time after drug use1410Our study on DAST-10 (Chinese version) was performed on more than 1200 low risk pregnant women in a general antenatal clinic setting in a Chinese population, which was one of the largest study to date, compared to previous studies conducted on vulnerable populations 10The internal consistency in our study was 0.64, which was low compared to other studies20Among 1483 invited participants, total 1214 questionnaires were collected (response rate 81.9%). As participation was anonymous and volunteer, the data and background information of the 269 non-participants was lacking. And because our study was anonymous in its design, test-retest reliability could not be performed. The information of gestational age of women at the time of study was not included in the questionnaire, and the recall time period was not available for analysis. Nevertheless, the majority (99.9%) of the respondent were recruited from out-patient clinic at their first antenatal visits. In our antenatal clinic, around 70–80% of pregnant women were less than 20 weeks of gestation at their first antenatal visits. We also included the unbooked pregnant women attending the maternity ward for the first time because those with illicit drugs may have had no previous antenatal careIn light of the alarming rise in illicit drug use in Chinese populations and lack of effective screening tools, our study is the first to validate in the literature the Chinese version of DAST-10 to detect illicit drug use among low risk Chinese pregnant women. As it has not ever been applied to any other independent groups  of Chinese population, the comparison of our results with these groups is not possible. However the results in our study in assessing the DAST-10 (Chinese version) for identifying illicit drug use among Chinese pregnant women were comparable to the previous studies on validation of DAST-10 (English version) on detection of drug use among pregnant women in other population10132021The DAST was developed and validated by Dr. Harvey A. Skinner at the Addiction Research Foundation, Toronto, Canada . The DAST is copyrighted by Dr. Harvey A. Skinner, although it has been made available for non-profit research, clinical, and training purposes. Permission for the use of DAST-10 in this study was obtained from the developer. The DAST-10 is a 10-item, yes/no, self-report instrument that has been shortened from the original 28-item DASTThe Chinese version of the DAST-10  was firsIn the questionnaire, they were also asked directly whether they were currently using illicit drugs during their pregnancy, using the question FirstSignTMOne Step Multi Drug Screen (MDS-10) Cards and FirstSignTMOne-Step Drug of Abuse Test Multi-drug  were used to test the collected urine samples for the presence of the following drugs: Amphetamine (AMP), Barbiturate (BAR), Benzodiazepine (BZD), Cocaine (COC), Ketamine (KET), Methylenedioxymethamphetamine (MDMA/Ecstasy), Methadone (MTD), Methamphetamine (MAMP), Opiates (OPI), and Cannabinoids (THC). These drugs are the most commonly used drugs among adult drug users in Hong KongThe The POCT immunoassay urine tests are sensitive27The study was conducted in two major public hospitals (each with approximately 5000 deliveries per year) in Hong Kong, Kwong Wah Hospital from May 2011 to June 2011 and Princess Margaret Hospital from September 2011 to October 2011. Local Chinese pregnant women newly attending the antenatal clinics and local unbooked Chinese pregnant women presenting to the maternity ward for the first time were recruited for the study. Pregnant women who were not Hong Kong residents were excluded from the study.After routine urine testing for albumin and glucose, the pregnant women were asked to provide their urine samples. The urine sample, questionnaire with direct questions on illicit drug use during pregnancy, and the self-administered DAST-10 (Chinese version) screening form from the same pregnant woman were anonymously labeled with the same unique number. The DAST-10 scores were calculated from the DAST-10 screening forms. The urine samples collected were tested by POCT and were performed by a trained staff who was blinded from the results of the DAST-10 screening and the results of the single question on self-reported drug use. Urine samples screened positive by the POCT were sent to a laboratory for confirmatory testing by gas chromatography-mass spectrometry. The laboratory staff performing the confirmatory tests was also blinded from the urine POCT results and all other results from DAST-10 or the self-report drug use.Different measures were used as the gold standards for detection of illicit drug use and to assess the performance of DAST-10 in identifying illicit drug use during pregnancy. (Measure 1) Urinalysis: positive urinalysis results were urine samples that tested positive by the POCT assay confirmed by GC-MS, whereas negative urinalysis results were urine samples that tested negative by the POCT assay or tested negative by GC-MS. (Measure 2) Self-reported drug use: positive results were those pregnant women who self-reported illicit drug use during pregnancy, whereas negative results were those who reported no illicit drug use during pregnancy. (Measure 3) Evidence of drug use by urinalysis or self-report: positive results were those who were positive for either the urinalysis or the questionnaire result, whereas negative results were those who were negative for both the urinalysis and questionnaire result.Details of the study were explained to the pregnant women by the nurses in the antenatal clinic or maternity ward, and the women were given an information sheet about the study. The pregnant women were free to choose whether to leave the urine sample or to fill in the anonymous questionnaires and DAST-10 screening form. Informed consent was obtained from all participants. Methods in the study were carried out in accordance with the approved guidelines of the Clinical Research Ethics Committee of the Hong Kong Hospital Authority. Ethics approval was obtained from the Clinical Research Ethics Committee (Kowloon West Cluster) of the Hong Kong Hospital Authority.Mann–Whitney U test was used to compare the difference of total DAST-10 scores between participants with and without illicit drug use for each of the measures. Spearman’s rho correlation coefficients were obtained from bivariate correlation tests. Receiver Operating Characteristic (ROC) curve data, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and overall accuracy with different cut-off scores of DAST-10 were calculated. Sensitivity refers to the true positive rate . Specificity refers to the true negative rate . PPV is defined as the proportion of true cases that were correctly identified by DAST-10 . NPV is defined as the proportion of pregnant women with negative result for illicit drug use who were correctly identified by DAST-10 (the proportion of women scored lower than the cut-off score of DAST having negative results for illicit drug). Overall accuracy is defined as the number of true positives and true negatives among the total number of women undergoing the tests. For the ROC curves for DAST-10, test sensitivity vs. (1 – specificity) for each cut-off point was plotted. The area under the curve (AUROC) was calculated as a measure of overall test performance, with a score of 1.0 representing a perfect test and a score of 0.5 a test that provided no information.How to cite this article: Lam, L. P. et al. Validation of the Drug Abuse Screening Test (DAST-10): A study on illicit drug use among Chinese pregnant women. Sci. Rep.5, 11420; doi: 10.1038/srep11420 (2015).


With the effectiveness of TIL therapy already well established in multiple Phase II studies in melanoma, there is a push to translate it to other malignancies such as ovarian cancer (OvCa) [Immunotherapy has become an effective cancer therapy, particularly in the case of checkpoint blockade and adoptive T cell therapy (ACT). ACT exploits the presence of tumor-infiltrating lymphocytes (TIL) by exponentially expanding their numbers r (OvCa) .The presence of TIL is correlated with greatly increased survival in OvCa ,3 sugges+ TIL outgrowth as well as favored the expansion of NK cells. Importantly, success rate of TIL growth was increased from 40% to 90% for cultures grown without and with anti-41BB respectively. It was established next that the CD3+ TIL initially grown with anti-41BB could be rapidly expanded at least 1000 fold over two weeks. Finally, the rapidly expanded T cells exhibited anti-tumor capabilities in the context of re-directed killing assays.Extensive flow cytometry analysis detected a robust, activated T cell infiltrate that can be grown from OvCa samples obtained pre- and post-chemotherapy. The addition of an agonistic anti-41BB antibody to the cultures preferentially increased CD8in vitro and in vivo experiments. However, the initial data suggest that TIL therapy for OvCa could be a viable therapeutic option in the future.In conclusion, further flow cytometry analysis to identify other agonistic and inhibitory targets is needed along with additional


Results indicated that phenolic release was mainly achieved during the gastric phase (recovery 59.38%–97.04%), with a slight further release during intestinal digestion. Dialysis experiments showed that dialysable phenolics were 64.11% and 22.93%–26.05% of their native concentrations, respectively, for gallic acid/simple gallate esters and ellagitanins/ellagic acid, in contrast to 20.67% and 28.37%–55.35% for the same groups in the non-dialyzed part of the intestinal media. Investigation of human gut microbiota metabolites of Padma Hepaten and pure ChETs  established the formation of bioactive urolithins . The fact of urolithin formation during microbial transformation from ChETs and ChET-containing plant material was revealed for the first time. Evaluation of the protective effect of ChETs colonic metabolites and urolithins on tert-butyl hydroperoxide (t-BHP)-induced oxidative injury in cultured rat primary hepatocytes demonstrated their significant reversion of the t-BHP-induced cell cytotoxicity, malonic dialdehyde production and lactate dehydrogenase leakage. The most potent compound was urolithin C with close values of hepatoprotection to gallic acid. The data obtained indicate that in the case of Padma Hepaten, we speculate that urolithins have the potential to play a role in the hepatic prevention against oxidative damage.Chebulic ellagitannins (ChET) are plant-derived polyphenols containing chebulic acid subunits, possessing a wide spectrum of biological activities that might contribute to health benefits in humans. The herbal formulation Padma Hepaten containing ChETs as the main phenolics, is used as a hepatoprotective remedy. In the present study, an  In our research, the protective effect of urolithins correlated with the number of hydroxyl groups with a higher value for urolithin C having one o-di-hydroxy- and one mono-hydroxy-substituted phenolic rings and lower value for urolithin B having only one mono-hydroxy-substituted phenolic ring. These data are in good agreement with the results of the previous studies used 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical (ABTS+) [The results of  (ABTS+) , ferric  (ABTS+) . Considein vivo study in a liver fibrosis mouse model, where Padma Hepaten significantly attenuated hepatic fibrosis.Based on the data obtained during the present studies, we propose the following interpretation of hepatoprotective effectiveness of Padma Hepaten. This formulation is a source of two groups of phenolic compounds with high bioaccessible values such as gallic acid and its derivatives and with low bioaccessibility as ellagitannins (both chebulic and non-chebulic groups) and ellagic acid. Both groups characterized by different ways of digestion of human organism. An uptake of gallic acid from the gastrointestinal tract into the inner media of organism started at 1–4 h after taking the medicine. Ellagic acid derivatives cannot transfer to target organ in the native form by any mechanisms that is why they undergo structural changes caused formation of urolithins. The duration of this phase may reach two to four days. Despite the different rates of assimilation of gallic acid and urolithins as a result of a periodic application of Padma Hepaten a constant uptake of bioactive compounds to the target organ should be supposed. These data support the results of an t-BHP-induced oxidative injury demonstrate the benefits of application of the herbal formula Padma Hepaten as a perspective hepatoprotective preparation.In this study, we investigated the componential profile of the herbal formula Padma Hepaten which is rich of chebulic ellagitannins as well as non-chebulic ellagitannins, gallic acid, simple gallate esters, and ellagic acid. Investigated compounds characterized by the various rates of bioaccessibility in the simulated gastrointestinal digestion experiments with the maximal values for the gallic acid and simple gallate esters and the lowest values for chebulic ellagitannins. Despite this fact, our data firstly showed that the chebulic ellagitannins transformed into bioaccessible colonic metabolites urolithins by gut microbiota. The high potential of urolithins to defend the hepatocytes against