Many studies show that ivabradine is effective for stable angina.This meta-analysis was performed to determine the effect of treatment duration and control group type on ivabradine efficacy in stable angina pectoris.Relevant articles in the English language in the PUBMED and EMBASE databases and related websites were identified by using the search terms “ivabradine,” “angina,” “randomized controlled trials,” and “Iva.” The final search date was November 2, 2015.Articles were included if they were published randomized controlled trials that related to ivabradine treatment of stable angina pectoris.Patients with stable angina pectoris were included.The patients were classified according to treatment duration (<3 vs ≥3 months) or type of control group (placebo vs beta-receptor blocker). Angina outcomes were heart rate at rest or peak, exercise duration, and time to angina onset.Seven articles were selected. There were 3747 patients: 2100 and 1647 were in the ivabradine and control groups, respectively. The ivabradine group had significantly longer exercise duration when they had been treated for at least 3 months, but not when treatment time was less than 3 months. Ivabradine significantly improved time to angina onset regardless of treatment duration. Control group type did not influence the effect of exercise duration (significant) or time to angina onset (significant).Compared with beta-blocker and placebo, ivabradine improved exercise duration and time to onset of angina in patients with stable angina. However, its ability to improve exercise duration only became significant after at least 3 months of treatment. Thus, unlike BB, ivabradine slows the heart rate without inhibiting inner heart conduction1 or reducing the left ventricular systolic function.2Coronary heart disease (CHD) is a common and often dangerous condition that is typically initially manifested by angina. Treatments for angina that alleviate its symptoms are needed because it severely limits patient quality of life. An insufficient supply of oxygen forms the pathological–physiological basis of angina pectoris, and the heart rate is one of the determinants of myocardial oxygen consumption. Thus, one treatment strategy for angina pectoris is to slow the heart rate. Some existing drugs that slow heart rate such as beta-blockers (BBs) and nondihydropyridine calcium channel blockers also have adverse effects that prevent their use in the treatment of patients with some other diseases. In recent years, the electrophysiology of sinoatrial node activities has become better understood, which has prompted the development of new drugs to control heart rate. One of these drugs may be ivabradine, whose electrophysiological mode of action has become an area of intense research recently. Ivabradine acts on sinus node cells and specifically inhibits the pacemaker I9 However, since many of these studies assessed clinical ivabradine effectiveness for a short time only, it remains unclear how effective this drug is over longer periods of treatment. The effect of different types of control groups (placebo and BB) on ivabradine efficacy in stable angina also remains unclear. To address these questions, the present meta-analysis of randomized controlled trials (RCTs) on ivabradine treatment for stable angina pectoris was performed.Many studies have demonstrated that ivabradine effectively treats stable angina.Relevant articles in the English language in the PUBMED and EMBASE databases and related websites were identified by using the search terms “ivabradine,” “angina,” “randomized controlled trials,” and “Iva.” There were no limitations on publication date or publication status. The final search date was November 2, 2015.To select the appropriate articles, the following inclusion and exclusion criteria were used. Articles were included if they were published RCTs that related to ivabradine treatment of stable angina pectoris; articles were in English language; the experimental group was treated with ivabradine for stable angina pectoris and the control group was managed by a routine treatment strategy without ivabradine; the curative effect of ivabradine was assessed by measuring the following 4 endpoints: heart rate at rest, heart rate at peak, exercise duration, and time of onset of angina; and the 4 endpoints were expressed as mean ± standard deviation (SD). Studies were excluded if they were not randomized trials or lacked a control group, or if the full text of the article could not be retrieved.9 As a result, the control group data had to be calculated repeatedly when analyzing the data. To make full use of the data, we treated 12 weeks as 3 months.6 To assess the effectiveness of ivabradine in stable angina pectoris, the patients were grouped according to whether they were followed up for less than or at least 3 months. In a separate analysis, the patients were grouped according to whether the control group received a placebo or BB.The data of basic information on the patients Table  and the 10 Furthermore, a 27-item checklist  for transparent reporting of a systematic review were used to be included in the study. The 7 selected RCTs were assessed by 2 reviewers according to the Cochrane Collaboration bias risk tool. Six aspects were mainly involved: random sequence generation, allocation concealment, double blinding, incomplete outcome data, selective reporting, and other biases. All these trails were RCTs obtained by the single-blind method3 or the double-blind method.9The numbers of studies that were screened, assessed for eligibility, and included in the meta-analysis are shown in the flow diagram in Figure RevMan 5.2, which was provided by the Cochrane Collaboration, was used for the meta-analysis. Because all endpoints were continuous variables, the data were expressed as weight mean difference (WMD) and 95% confidence intervals (95% CIs). To improve the analysis of the effect of ivabradine on angina pectoris, the patients were grouped according to follow-up time (<3 months group vs ≥3 months group) and the use of placebo in the control group (placebo group vs BB group). The 4 variables employed in meta-analysis were heart rate at rest, heart rate at peak, exercise duration, and time of onset of angina.P < 0.1) and the I2 statistic were calculated to test the heterogeneity of the studies. I2 < 25% was considered to indicate low heterogeneity; 25% < I2 < 50% was considered to indicate moderate heterogeneity; and when P > 0.1 and I2 > 50%, the heterogeneity was considered to be high.11 When I2 < 50%, the fixed effects Mantel–Haenzel model was used to analyze the data. When I2 > 50%, the random-effects model of DerSimonian and Laird was used. A funnel plot was used to evaluate publication bias.12The Breslow–Day chi-square test , the random-effects model of DerSimonian and Laird was used for data analysis. In patients treated for less than 3 months, the ivabradine and control groups did not differ significantly in terms of heart rate at rest . Five RCTs reported heart rate at rest after at least 3 months of treatment, of which 2 involved 2 experimental groups.9 In the patients treated for at least 3 months, the ivabradine and control groups did not differ significantly in terms of heart rate at rest  . Four RCTs reported heart rate at peak after at least 3 months of treatment, of which 2 involved 2 experimental groups.9 In the patients treated for at least 3 months, the ivabradine and control groups did not differ significantly in terms of heart rate at peak  . Four RCTs reported exercise duration after at least 3 months of treatment, of which 2 involved 2 experimental groups.9 In the patients treated for at least 3 months, the ivabradine and control groups differed significantly in terms of exercise duration  . Three RCTs reported time to onset of angina after at least 3 months of treatment, of which 2 involved 2 experimental groups.9 In the patients treated for at least 3 months, the ivabradine and control groups differed significantly in terms of time to onset of angina  , the random-effects model of DerSimonian and Laird was used for data analysis. The ivabradine-treated and placebo-receiving patients did not differ significantly in terms of heart rate at rest . Two RCTs reporting heart rate at rest used BB-treated controls, of which 2 involved 2 experimental groups.9 The ivabradine and BB-treated groups did not differ significantly in terms of heart rate at rest   Figure A.Heart rate at peak: Only 1 RCT reporting heart rate at peak used placebo-receiving controls. The ivabradine-treated and placebo-receiving patients differed significantly in terms of heart rate at peak . Four RCTs reporting heart rate at peak used BB-treated controls, of which 2 involved 2 experimental groups.9 The ivabradine and BB-treated groups did not differ significantly in terms of heart rate at peak  . Four RCTs reporting exercise duration used BB-treated controls, of which 2 involved 2 experimental groups.9 The ivabradine and BB-treated groups differed significantly in terms of exercise duration  . Two RCTs reporting time to onset of angina used BB-receiving controls, of which 2 involved 2 experimental groups.9 The ivabradine and BB-treated groups differed significantly in terms of time to onset of angina   were not analyzed. In the meta-analysis by Belsey et al,27 only 1 RCT8 reported that ivabradine can improve the result of exercise tolerance test. To further clarify how effective ivabradine is in treating angina pectoris, only relevant RCTs were collected for the meta-analysis described in the present study.Twenty-three RCTs were included in the meta-analysis by Cucherat and Borer,The present meta-analysis showed that the ivabradine-treated and control groups did not differ in terms of heart rate at rest or at peak regardless of whether the treatment was for less than 3 or at least 3 months. The 2 groups also did not differ in terms of exercise duration when they were treated for less than 3 months. However, when treatment was for at least 3 months, the ivabradine-treated group had better exercise duration than the control group. Thus, to improve exercise duration, patients with stable angina should keep taking ivabradine for more than 3 months. The ivabradine-treated group had longer times to onset of angina than the control group regardless of whether the treatment duration was short (less than 3 months) or long (at least 3 months).The present meta-analysis also showed that the ivabradine-treated patients had similar heart rates at rest regardless of whether they were compared with placebo or BB-receiving control groups. However, in terms of heart rate at peak, the ivabradine-treated group had better values than the placebo-receiving group, but not the BB-treated controls. The ivabradine-treated patients had better exercise duration and time to onset of angina than both the placebo and BB-receiving controls.This study had a number of advantageous features. Firstly, the sample size was large (N = 3748). Secondly, a meta-analysis only on the efficacy of ivabradine in stable angina has not yet been published. Thirdly, the study results indicate that further focus should be placed on the effect of ivabradine treatment on angina duration. Fourthly, this meta-analysis assessed both the effect of different treatment durations and the effect of different comparator control groups on the efficacy of ivabradine for treating stable angina pectoris.3 These considerations may affect the accuracy of the present meta-analysis.This study also had several limitations. Firstly, it was based on 7 articles only. Additional well-designed studies are needed to validate the findings of this meta-analysis. Secondly, only 4 indicators of curative effect were analyzed in this study. As a result, the effect of ivabradine on stable angina pectoris was not fully determined. Thirdly, the 7 studies differed in the dose of ivabradine. Fourthly, the studies differed in terms of treatment strategies. For example, in 1 study, ivabradine was only used after treatment with other drugs.A number of issues remain to be addressed by further research. Firstly, although ivabradine therapy effectively treats angina pectoris, can it prevent future heart failure? Secondly, does long-term use of ivabradine have a better prognosis than short-term use? Thirdly, would combination therapies employing ivabradine together with other drugs be more effective in treating angina pectoris than therapy based on ivabradine alone?.Compared with BB and placebo, ivabradine improved the exercise duration and time to onset of angina in patients with stable angina. However, its ability to improve exercise duration only became significant after at least 3 months of treatment.


Functional neuroimaging studies in mild traumatic brain injury (mTBI) have been largely limited to patients with persistent post-concussive symptoms, utilizing images obtained months to years after the actual head trauma. We sought to distinguish acute and delayed effects of mild traumatic brain injury on working memory functional brain activation patterns < 72 hours after mild traumatic brain injury (mTBI) and again one-week later. We hypothesized that clinical and fMRI measures of working memory would be abnormal in symptomatic mTBI patients assessed < 72 hours after injury, with most patients showing clinical recovery  within 1 week after the initial assessment. We also hypothesized that increased memory workload at 1 week following injury would expose different cortical activation patterns in mTBI patients with persistent post-concussive symptoms, compared to those with full clinical recovery. We performed a prospective, cohort study of working memory in emergency department patients with isolated head injury and clinical diagnosis of concussion, compared to control subjects (both uninjured volunteers and emergency department patients with extremity injuries and no head trauma). The primary outcome of cognitive recovery was defined as resolution of reported cognitive impairment and quantified by scoring the subject’s reported cognitive post-concussive symptoms at 1 week. Secondary outcomes included additional post-concussive symptoms and neurocognitive testing results. We enrolled 46 subjects: 27 with mild TBI and 19 controls. The time of initial neuroimaging was 48 (+22 S.D.) hours after injury (time 1). At follow up , 18 of mTBI subjects (64%) reported moderate to complete cognitive recovery, 8 of whom fully recovered between initial and follow-up imaging. fMRI changes from time 1 to time 2 showed an increase in posterior cingulate activation in the mTBI subjects compared to controls. Increases in activation were greater in those mTBI subjects without cognitive recovery. As workload increased in mTBI subjects, activation increased in cortical regions in the right hemisphere. In summary, we found neuroimaging evidence for working memory deficits during the first week following mild traumatic brain injury. Subjects with persistent cognitive symptoms after mTBI had increased requirement for posterior cingulate activation to complete memory tasks at 1 week following a brain injury. These results provide insight into functional activation patterns during initial recovery from mTBI and expose the regional activation networks that may be involved in working memory deficits. Traumatic Brain Injury (TBI) is a significant medical problem worldwide , with aThere is a growing literature on the impact of mTBI on the brain using neuroimaging tools –17. MuchWhile the work that has been conducted to date investigating the consequences of mTBI on brain activation has led to a better understanding of how the brain responds to trauma, only a few studies have studied subjects at time points within the first month following injury, and we are unaware of any studies to date that have enrolled patients at their initial medical contact in the emergency department setting. Moreover, few studies have followed their subjects longitudinally. Therefore, while the extant literature provides important insight about the reorganization of brain function, it is not generalizable to the wider context of acute head injuries, and cannot predict which subjects will recover and which will not.Our objective was to understand the effect of concussive head injury on working memory, at the earliest possible time point after a concussion and in the first week of recovery. We compared patients diagnosed with mTBI in the emergency department to controls using the N-back task for working memory  and sequThis was a prospective cohort study performed in a single tertiary care academic medical center with a level 1 trauma center and 60,000 annual emergency department patient visits. The University of Vermont’s Institutional Review Board (IRB) approved this study and written informed consent was obtained from all subjects prior to enrollment.Research staff screened emergency department patients for the study’s inclusion and exclusion criteria. Eligible patients were those aged 18–60 years old who presented to the emergency department with isolated head injury, and medical diagnosis of a concussion based on standard emergency department criteria of Glasgow Coma Scale (GCS) > 14 at the time of injury. For purposes of the study, we defined an isolated head injury as head trauma with an injury severity score (ISS) for any other organ system <2 . Concussion was defined as a patient with acute head trauma endorsing two or more of the following symptoms associated with their injury: loss of consciousness, persistent headache, blurred vision, confusion, dizziness, memory problems or poor balance. Head CT scans were performed at the discretion of the patient’s physician. Patients were excluded if: 1) they did not exhibit two or more concussive symptoms (above); 2) had a past history of a serious TBI ; 3) they were unable to complete initial MRI within 72 hours of injury; 4) they had a pre-existing neurological disorder; 5) they had a psychiatric condition requiring medical treatment within the past year; 6) any contraindications to MRI scanning. Two control groups were also recruited: normal volunteers without acute injury who responded to flier advertisements and non-head injured patients who presented with an extremity injury to the emergency department within 72 hours of injury. Extremity injury was defined as a non-surgical injury to the arms or legs, including the shoulder and hips, with no head trauma.Initial assessments were completed by structured interviews performed by research staff, from review of emergency department charts, discussion with the patient’s physician, and evaluation of subjects while in the emergency department. Additional details on symptoms were also acquired from questionnaires and computer based assessments. Follow-up assessment was performed by a Research Associate at the time of repeat MRI and ImPACT testing Study data were collected and managed using REDCap electronic data capture tools hosted at UVM .The N-back working memory task was used, at both Time 1 and Time 2; however, at Time 1 only the easiest condition (0-back) was used while at Time 2 three conditions were included . All subjects were trained and checked for proficiency  by research staff prior to completing the tasks in the MRI scanner. The tasks were composed of series of letters that were presented on a projector screen, which the subject could see through a mirror mounted on the MRI’s head coil. Instructions for the tasks were as follows: 0-Back: “please respond when the letter you see matches the letter B (different letters were used at Time 1 and Time 2);” 1-Back: “please respond when the letter you see matches the letter you saw one letter back;” 2-Back: “please respond when the letter you see matches the letter you saw two letters back.” Subjects only responded to the letters when they met the above criteria and did so by pressing a button on a controller with their right index finger. When letters did not meet the above criteria subjects were asked not to respond. Each task lasted for approximately 3 minutes and a block design was used in which there were three blocks of the task (32 sec each) interleaved with blocks of rest (32 sec). The tasks were programed using E-Prime.Brain MRI data was acquired on a Philips Achieva 3.0 Tesla  scanner using an 8-channel head coil. T1-weighted images were acquired using a 3D inversion recovery spoiled gradient echo technique (TE/TR/TI/flip angle = 3.7ms/8.1ms/1008ms/8° with a SENSE factor of 1.5) to generate the Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE) image. A sagittal acquisition matrix of 240x240x160 provided whole-brain coverage with an isotropic 1mm spatial resolution with a scan time of less than 8 minutes. The BOLD signal was captured with a T2* sequence (TE/TR/flip angle = 35ms/2000 ms/90°) with an in-plane resolution of 3 x 3 mm, and a slice thickness of 3.8 mm. For each of the n-back runs, 90 volumes were acquired  to ensure steady-state magnetization).Following the first MRI all subjects completed the Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT) computerized neurocognitive testing battery. The ImPACT test measures attention span, working memory, sustained and selective attention, response variability, non-verbal problem solving and reaction time, each of which is sensitive for mild cognitive impairment. ImPACT also includes a symptom score based on patient self-report. All ImPACT testing was done in a small quiet conference room located just outside of the emergency department. Repeat MRI and ImPACT testing was done 7–10 days following injury. Post-concussive symptoms were obtained as part of the ImPACT test, and include 22 symptoms: headache, nausea, vomiting, balance problems, dizziness, fatigue, trouble falling, sleeping more than usual, drowsiness, sensitivity to light, sensitivity to noise, feeling dazed or stunned, irritability, sadness, nervousness, feeling more emotional than normal, numbness or tingling, feeling slowed down, feeling mentally foggy, difficulty concentrating, difficulty remembering, visual problems. Subjects rated each of these symptoms on a Likert scale ranging from 0  to 6 (the symptom was the worst they had ever experienced). Among these symptoms, we defined cognitive symptoms as difficulty concentrating, difficulty remembering, feeling mentally foggy, feeling slowed down, drowsiness, fatigue and sleeping more than usual as previously defined by factor analysis . The priAll variables were summarized descriptively. The primary outcome of clinical recovery was assessed by determining the number of post-concussive symptoms at time 2. We report the mean number of symptoms for controls and mTBI subjects, with standard deviations, and the estimated magnitude of difference between groups with 95% confidence interval for this difference. We also report the mean and standard deviation of post-concussive symptoms between the subgroups of recovered and non-recovered subjects, along with the difference in means between these subgroups, with the 95% confidence interval. Secondary outcomes of neurocognitive testing results, were reported in the same fashion for both the experimental groups and subgroups. Statistical calculations were performed with SPSS .3 Gaussian smoothing kernel, and scaled to the mean intensity across time-points. The data were then deconvolved, using a boxcar function in which each condition was represented by a regressor . The boxcar function was convolved with a gamma variate hemodynamic response function prior to fitting it in the model. Group-level statistics were performed using ANOVAs. All group-level statistical maps were thresholded using both the alpha level and cluster size (extent of activation). The alpha level was set at p<0.01 and the cluster size was set at 39 contiguous voxels in native space. The results of Monte Carlo simulations (using 3dClustSim) showed that this combination resulted in a corrected alpha level of p<0.05.All images were preprocessed using Analysis of Functional Neuro-Images (AFNI) . The reaParticipants consisted of 27 mild TBI patients, 10 orthopedic controls and 9 healthy controls . Head CTThe number of post-concussive symptoms at time 2 was significantly higher in mTBI subjects compared to controls as expected. In comparison of subjects with cognitive recovery to those with no cognitive recovery (primary outcome), there were no differences in demographics, initial symptoms or neurocognitive measurements  at the initial time point. Because only one subject had an abnormal CT scan, and structural MRI did not reveal additional lesions, none of these clinical measures provided additional predictive power in determining the primary outcome. The secondary outcomes of neurocognitive results at time 2 were similar at both time points for those subjects with and without resolution of cognitive symptoms as shown by the overlapping 95% confidence intervals for each of these measures see .We first compared the two groups of Controls to one another to ensure that there were no differences between these two Control groups. This was done by comparing the 0-back data at Time1 and Time2 (in separate t-tests). No differences were found in either behavioral accuracy or in the imaging data. The RTs for extremity injured controls were faster on the 0-back task at both time points (p < 0.04 and p < 0.03 for Time1 and Time2 respectively); however, because this difference suggested that the information processing of the extremity injured controls was (if anything) more efficient than the other HCs (rather than the reverse), and because their accuracy was comparable, and because their RTs were not different on the 1-back and 2-back tasks, we felt that this difference was likely due merely to random chance rather than to a systematic difference between the groups. Therefore in all subsequent analyses, these two groups of Controls were pooled to form a single Healthy Control (HC) group.For both response time (RT) and accuracy (d’), we conducted two ANOVAs. Because the accuracy data was not normally distributed, it was first log-transformed to assure normality. The first compared 0-back performance across time. This was a 2 X 3, mixed between- and within-subjects ANOVA with time as the within-subjects factor (Time 1 vs. Time 2) and group as the between-subjects factor . For both RT and d’, none of the main effects or interactions were significant see .The next analysis interrogated task difficulty by comparing the three levels of n-back working memory load across the three groups at Time2 see . This waThis 2 X 2 ANOVA, with factors of group (mTBI vs. HC) and time (Time 1 vs. Time 2), was conducted using 3dLME.R, a script distributed with the AFNI suite of image analysis tools that implements the R statistical analysis software. Only the 0-back data was included since only these data were collected at both time points. There was a main effect of Time in several areas including superior frontal gyrus, inferior frontal gyrus, insula and inferior parietal lobule see . In all In the analyses that specifically investigated recovery, we compared those who made a good recovery (8 mTBIs) to those who made minimal or no recovery (10 mTBIs). This 3 X 2 ANOVA, with factors of group  and time (Time 1 vs. Time 2) was similar to the analysis above. As in the previous analysis, only the 0-Back data was analyzed. There was a main effect of Group in both the paracentral lobule and precentral gyrus see , and in We investigated the differential effect of task difficulty (working memory load) across the two Groups (mTBI vs. HC) with a 2 X 3 ANOVA. The factors were group (mTBI vs. HC) and load . Because all three loads were used only at Time 2, only data from this time point was included in this analysis. Of the main effects, only the effect of Load was reliable see . The maiThe above analysis was rerun, but with the mTBI group divided into those who showed good recovery vs. those who did not. The factors were the same, but the factor of group had three levels  rather than two. As in the above analysis, there were no areas that showed a main effect of group. Furthermore, the effect of Load was found in largely the same set of areas as in the above analysis see . No areaIn this study, we set out to better understand the consequences of mild TBI on brain function, focusing on the first week after injury. We scanned individuals with mTBI twice: first within 72 hours of their injury to capture acute changes, and then 7–10 days later to assess recovery. This is the first study to our knowledge to longitudinally investigate working memory within the first week on injury in mTBI patients. We also differentiated between those patients who reported cognitive recovery, and those with persistent cognitive symptoms. Demographics, initial symptoms, neurocognitive measures at time 1, CT scans, and structural MRI, did not provide any predictive power to discriminate the primary outcome of recovery at 1 week. The failure of standard clinical assessment to identify those individuals at risk for post-concussive syndrome highlights the urgency of the current work.While there is a growing body of work investigating working memory in TBI –17, muchWhile the replication of previous results in an acute sample is encouraging, the data from the 0-back task collected within 72 hours of injury is perhaps more important . These dWhile any interpretation of these findings must remain highly speculative, it is possible that hypoactivation of the DN is a signal that the brain has to work harder to accomplish even the relatively simple 0-back task. This interpretation is suggested by work such as Singh and Fawcett , who shoOne seemingly surprising result from the comparison of the 0-back data across time (72 hours after injury vs. 1 week after injury) is that the activation appears to decrease in the DN across time in the HCs and mTBIs who recovered. Given that one might expect the task to become ‘easier’ with practice, one would expect the DN activation to increase. This is because prior research has shown that DN activity decreases as tasks become more difficult  . HoweveOne of the strengths of our study is a highly characterized TBI population. We enrolled subjects in the emergency department and performed structured interviews and neurocognitive testing at the bedside. At initial presentation after head injury, our research staff was able to ascertain reliable information about the event, which is subject to recall bias in other studies that have enrolled patients at later time points. We performed comprehensive clinical evaluations at follow-up to provide characterization of functional recovery in the early phase after mTBI . A second strength is the early neuroimaging obtained by bringing emergency department patients directly to a research magnet. This study design allowed us to obtain a large series of MRIs with corresponding clinical information during the early (<1 week) time period after mTBI. It is only through obtaining data at multiple time points that the mechanisms of functional recovery can be inferred.Our exclusion criteria might be considered less stringent than other studies, because we enrolled all adult emergency department patients with mTBI, only excluding past history of TBI requiring surgical intervention, psychiatric condition within the last year, or active alcohol/ drug intoxication. The less stringent exclusion criteria might also be considered a strength of our study because it makes our results more generalizable to the larger population of emergency department patients with heads injures.Despite our attempts to include a diverse population of mTBI patients, our sample may not represent the whole mild traumatic brain injury population. We did not include children or older adults (our cutoff was 60 years of age). Individuals with alcohol and/or drug intoxication also make up a significant portion of emergency medicine trauma cases, but were not represented in this study.Additionally, we were unable to capture the “true” initial time point after injury, and the timing of the first MRI varied between 11 hours and 72 hours after trauma. Functional recovery after brain injury may begin to occur immediately, and the earliest changes in cortical activation patterns would not be detected with our study design. The injury mechanisms were variable and although we documented the patient’s report of the mechanism, the actual injury parameters  are unknown. Another limitation is that the outcome measure of clinical recovery was based on patient report. This may be problematic, because patients may have deficits that are not apparent to them, but only manifested on neurocognitive testing. In fact, our secondary outcomes of neurocognitive testing did not reveal significant differences in cognitive performance in patients who reported cognitive recovery compared to those who did not. Without pre-injury neurocognitive testing the results of tests obtained after injury can only be compared to standardized databases and tracked over time. However, inasmuch as pre-injury testing was not available on the individuals in this study, we felt that the patient’s report of cognitive improvement and resolution of symptoms was the most valid clinical outcome measure.While the findings reported here are encouraging, they are just the first step towards predicting recovery in mTBI. Few clinics have the facilities to scan every patient who presents with a mTBI, and even fewer have the resources to do so. Future work will be to develop inexpensive, sensitive, diagnostic measures that correlate with changes in brain activation, and that can be easily administered in clinical settings to predict recovery.We describe patterns of brain activation during working memory tasks in the acute and sub-acute phases after mTBI. Normalization of activation patterns in the posterior cingulate cortex, a major node of the default network , corresp


Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.DOI:http://dx.doi.org/10.7554/eLife.10230.001 Stem cells found in adult tissues are vitally important for tissue repair and maintenance. These cells divide in two main ways: equally to create two new stem cells, or unequally to create a stem cell and a cell that can develop into one of the cell types in the tissue. A key challenge for biologists is to understand how these tissue-resident stem cells are activated and organized to regenerate injured or missing tissue.Throughout the life of the axolotl salamander, neural stem cells in the spinal cord occasionally divide to add new nerve cells to the healthy spinal cord. However, the axolotl can also regenerate part of its spinal cord, for example if its tail is lost. Under these conditions, the neural stem cells can convert into a highly regenerative stem cell that can produce all the different cell types needed to regrow the spinal cord.As a stem cell becomes a new cell type, it activates different sets of genes. Therefore, Rodrigo Albors, Tazaki et al. measured gene activity in the neural stem cells involved in axolotl spinal cord regeneration to uncover how these cells develop into a more regenerative form. This revealed that when an axolotl tail is amputated, resident stem cells turn off the genes that are specifically active in neuron-generating cells. In addition, they activate a similar set of genes to that seen in the embryonic cells that form the developing nervous system. These genes speed up cell division and activate an important signaling pathway.This pathway – the Wnt/PCP pathway – fulfils various developmental roles, one being to orient cell divisions, particularly in elongating tissues. In axolotls, this pathway causes the stem cells to divide equally to increase the number of available stem cells, and orients the direction of these divisions to ensure that the regenerating spinal cord elongates correctly. If this pathway is disrupted, the cells return to dividing unequally, generating nerve cells prematurely and halting the growth of the spinal cord. Such insights could help develop methods of repairing damaged nervous tissue in other animals that cannot regenerate to the extent that axolotls can.DOI:http://dx.doi.org/10.7554/eLife.10230.002 An outstanding challenge in biology is to understand how resident stem cells are mobilized to regenerate injured or missing tissues. During regeneration the removal of tissue activates a program that must coordinately control (i) the rate of cell divisions, (ii) the type of daughter cells generated, and (iii) the spatial rearrangements of these cells within the tissue.+ neural stem cells located within 500 μm of the injury is mobilized to regenerate the missing portion of the spinal cord  axis in concert with overall tail regeneration  progressively coalesce to form the neural plate  that then closes into the neural tube  signalling precisely within the cells that will regenerate the new spinal cord. Investigation of this pathway during regeneration revealed that PCP simultaneously controlled posteriorward orientation of cell divisions and the switch from neurogenic divisions to those divisions that expanded the stem cell pool. Together, these findings provide new insights into how molecular cues initiated by injury control the cell biology of neural stem cells to yield complete spinal cord regeneration in the axolotl.p=0.001294 as result of Fisher's exact test on count data) , pre-neural tube (PNT), caudal (CNT) and rostral neural tube (RNT) . To invent data) .10.7554/Egr1 , along with genes related to signaling pathways that during embryonic development promote neurogenesis such as the retinoic acid signaling-readout genes Crabp1 and Rarb  receptor Fgfr2, both linked to neurogenic differentiation during development and as such more highly expressed in the CNT+RNT than in the SZ+PNT  receptor e SZ+PNT , were doe SZ+PNT . Their ae SZ+PNT . These cRasgrp3 and downregulation of the negative regulator Dusp4. It is perhaps surprising that we could not detect transcripts for FGF8, which activates this pathway in the developing spinal cord. However, although other FGFs were not present in the 100-gene list, FGF2 has been shown to be upregulated in the regenerating spinal cord of in another salamander species and thus could be activating MAPK signaling in the context of spinal cord regeneration  signaling is presumably more active in the regenerating spinal cord compared to the uninjured spinal cord, based on the strong upregulation of its transcriptional target gene neration .Wnt5a, Wnt5b, Prickle1 genes, was notable in our dataset. These genes are also highly upregulated in the developmental SZ+PNT sample, consistent with the notion that regenerating neural stem cells in the axolotl dedifferentiate to a SZ+PNT-like state.Although a more extensive analysis of signalling activities during axolotl spinal cord regeneration will require a global genome-wide approach, the activation of non-canonical Wnt/PCP signaling during regeneration, reflected in the strong upregulation of Sox2 did not visibly change during regeneration, the neuronal transcription factors Pax6 and Pax7 and the glial marker Gfap showed indeed lower expression levels in Sox2+ cells at day 1  . The difnal cord . Mitotic mitotic , day 0.  mitotic . These rTo investigate this possibility, we determined the cell cycle parameters of neural stem cells in the uninjured and regenerating spinal cord, in regenerating and non-regenerating populations, at the time of maximum expansion (starting at day 6) . For tha+ cells over time. To label cells undergoing DNA synthesis and their progeny we injected the thymidine analog ethynildeoxyuridine (EdU) daily from day 3 to day 8 after amputation. We then quantified the fraction of EdU+ cells that were positive or not for the neural stem cell marker SOX2 in regenerating and non-regenerating regions of the spinal cord   . These oWnt5a, Wnt5b, and the core PCP gene Prickle1  and the vector of the AP axis of the spinal cord (AP) (SAPProj )  . From throj SAP) . The ProDrosophila have classified PCP phenotypes into two classes. Deletion or overexpression of certain core PCP components such as Prickle randomize cell polarization cell autonomously only in those cells in which Prickle expression levels are deranged  Carrei. Studiesderanged . In contderanged . Similarderanged . We took plasmid  and 3. Qcontrols . HoweverPrickle1 , the lev animals . In contng cells . Prickleed cells . Thus, PVangl2-overexpressing cells as well as their neighbors, thus spreading the disruption of PCP to a larger proportion of spinal cord cells compared to the Prickle1-electroporated tails  cells in control and nal cord . Interesme point . These dVangl2 resulted in premature neurogenic divisions that would gradually reduce the number of proliferative cells during regeneration. To label proliferating cells and their progeny, we injected Vangl2 and control electroporated axolotls with the thymidine analog ethynildeoxyuridine (EdU) daily from day 3 to day 8 and collected the tissue 1 hour later for analysis .Axolotls, later (QIAGEN) to protect their RNA. To generate day 0 samples, 500 μm of spinal cord at the level of the tail myotome 15 from the cloaca were microdissected from uninjured tails. To generate day 1 samples, the 500 μm of spinal cord anterior to the amputation plane, corresponding to the regeneration source zone, were microdissected from 1-day regenerating tails. To generate day 6 samples, the whole regenerated spinal cord was microdissected from 6-days regenerating tails. In every case, 48-50 pieces of spinal cord were pooled for each of three biological replicates. Total RNA was extracted using TRIzol (Life Technologies) and purified using RNeasy MinElute cleanup kit (QIAGEN). RNA quality was assessed using a bioanalyzer (Agilent Technologies) and only samples with a RNA integrity number (RIN) above 9.5 used for NanoString analysis.The tails of uninjured, 1 day, and 6 days regenerating axolotls were collected and submerged in RNARpl4 as housekeeping gene, was designed by the NanoString bioinformatics team. See From the list of genes that undergo major regulatory changes at the onset of neurogenesis in the developing chick spinal cord , we selewww.nanostring.com).NanoString nCounter technology was chosen for gene expression profiling . Brieflyp≥0.05 were considered not significant and blanketed to 1. Counts for genes for which p<0.05 were considered significant and the mean plus three standard deviations of the negative control probes was subtracted to remove the background noise inherent to the method. Count values that fell below 1 were blanketed to 1.NanoString data needs to be normalized before performing differential expression analysis. To correct for technical variation in hybridization and purification efficiency, raw counts were first normalized using the geometric mean of the internal, spike-in positive control probes. Next, to determine the presence or absence of gene expression, a one-tailed Student's t-test of the mean counts of each gene against the mean counts of negative controls was performed. Counts for genes for which Rpl4 to account for input material variation and set ready for differential gene expression analysis.Last, counts were normalized to the housekeeping gene http://www.r-project.org/). Count data were modeled by negative binomial distributions and negative binomial tests were used to determine statistical significance. A gene was considered significantly regulated when p<0.05 (corresponding to a Benjamini-Hochberg adjusted p<0.07)  .Wnt5a (GenBank accession number: Z14047.1), Wnt5b (Z14048.1), Prickle1 (CO780858.1), Pax6 (CO784109.1), Pax7 (AY523019.1), Gfap (CO780148.1), Egr1 (JK976899.1), Cdx4 (CO778950.1), and Greb1 (JK980018.1).Axolotl tails were fixed in fresh MEMFA at 4ºC for 72 hours. 10 μm paraffin sections were used for in situ hybridizations as previously described . RNA proTails were fixed in MEMFA overnight at 4ºC, rinsed in PBS, cryopreserved in 30% sucrose in PBS at 4ºC, and frozen in Tissue-Tek  for cryosectioning. Sections were collected on adhesive slides and dried at room temperature for several hours. Slides were then washed 3 times in PBS/0.3% Triton X-100, and incubated in glycine buffer for 20 minutes. After that, antigen retrieval was carried out boiling the sections in citrate buffer pH 6  for 5 minutes. The sections were allowed to cool down and blocked in blocking buffer (PBS/0.3% Triton X-100/1% goat serum). After 1 hour, primary antibodies were added diluted in blocking buffer and incubated overnight at 4ºC. Several PBS washes later, secondary antibodies were added diluted in blocking buffer and incubated for 2 hours at room temperature. Hoechst (1 mg/ml stock solution diluted 1:500) was added together with the secondary antibodies to stain nuclei. After 3 PBS washes, slides were mounted in 50% glycerol/PBS.For whole-mount immunostaining, the skin and muscles were removed from one side of the tail to expose the spinal cord and facilitate antibody penetration. Washing steps were 1-1.5 hours each and antibody incubations 48 hours. The tails were mounted using plastic paraffin between the slide and coverslip to avoid squeezing the tissue.Antibodies used were: rabbit anti-SOX2 +PCNA+ mitotic cells was annotated. Single optical sections were taken from non-consecutive sections to count the number of SOX2+PCNA+ cells per cross-section. Counts were carried out using Fiji (http://fiji.sc/Fiji)  .http://nbviewer.jupyter.org/gist/fabianrost84/3cc58a27b5688f4e2eba), the number of proliferating cells in a section k from a biological replicate i was estimated by:The length of individual cells in the AP dimension was determined for cells from the uninjured cell population and from regenerating and non-regenerating cell populations 3 days after amputation by counting the number of 1 μm optical sections that encompassed individual nuclei . As the mean cell length was approximately equal for all cell populations  was obtained as the mean mitotic index of the n biological replicates at this time point and the corresponding 1-σ error was extracted by summing the intra- and interexperimental error:The average mitotic index of a specific cell population at a given time  was dissolved at 2.5 mg/ml in PBS. A series of BrdU injections were given intraperitoneally to uninjured and regenerating axolotls, from day 6 after amputation. Adapting the labeling conditions defined by Nowakowski , uninjurAfter several PBS washes and quenching with 0.1 M glycine-Tris (pH 7.4), sections were treated with 2M HCl 15 min at 37ºC to denature DNA. BrdU was detected with a mouse anti-BrdU monoclonal antibody directly coupled to rhodamine (homemade), after overnight incubation at 4ºC. Hoechst was used to label DNA.Every third section was imaged with a Zeiss Axio Observer microscope using a Zeiss Plan-Apochromat 40x 0.95 objective. The fraction of BrdU-labeled cells was calculated as the number of BrdU-labeled cells surrounding the central canal of the spinal cord. Rounded nuclei, characteristic of neurons, were excluded from the analysis. Counts were carried out in Fiji.The modeling framework developed by Lefevre and co-workers  which isR’:Under the assumptions that (i) the length of the cell cycle and the cell cycle phases are constant for all cells, that (ii) divisions occur completely asynchronous and that (iii) all cells are proliferating the model by Lefevre et al. predicts the fraction of BrdU-labeled cells, if r = 1, where t is the time after the start of BrdU injections, CT is the cell cycle length, ST is the length of S-Phase, TG2+M is the combined length of G2 and M-phase and r is the average number of daughter cells remaining proliferating progenitor cells.if i.e., each cell division produces only one proliferating cell and one non-proliferating cell, for the uninjured and non-regenerating cell populations and proliferative cell divisions, i.e., each cell division produces two proliferating cells, for the cell population tissue. Hence, r = 1 for the uninjured and non-regenerating cell populations and r = 2 for the regenerating cell population.We assumed asymmetric neurogenic cell divisions, R with the growth fraction g:As there are also non-proliferating cells, we have to correct the labeling In the case of proliferative cell divisions the growth fraction will shrink over time because the proliferating cells increase and the non-proliferating cells remain constant in number. Hence,t = 0. In the case of neurogenic divisions the growth fraction will be constant, hence g(t) = GF.where GF is the initial growth fraction at SSE) calculated as follows:We estimated the parameters for each cell population by the method of least squares, that is, minimizing the sum of squared errors . This also allowed us to determine the 68% confidence intervals for the fitted parameters based on the parabolic approximation. The BrdU labeling data and the implementation of the parameter estimation can be found in http://nbviewer.jupyter.org/gist/fabianrost84/3cc58a27b5688f4e2eba.where G2+MT = 9 hours. We assumed that cells were homogenously distributed in the cell cycle. Hence, M-phase length is given by MT = mi ∙ cT, where mi is the mitotic index and cT is the cell cycle length. The length of G1-phase can then be calculated as G1T = c - TG2+M - TsT. We estimated the errors of MT and G1T by linear error propagation (see http://nbviewer.jupyter.org/gist/fabianrost84/3cc58a27b5688f4e2eba).Because all mitotic cells were BrdU-labeled 9 hours after the start of the BrdU labeling we estimated the combined length of G2- and M-phase as Prickle1 was found in our EST library (clone BL010D_C10) (Prickle1 open reading frame (ORF) was PCR-amplified with primer pair Prickle_kozak_Nhe1 and Prickle1_T2A_Hd3Pml1. mCherry-NLS  was amplified with the primer pair T2A_Hd3Pml1_Fw and Rb_bGlob_pA_Sac1 using CAGGS-EGFP-T2A-Cherry-NLS (C61) as template. Prickle1-T2A-mCherry-NLS was amplified with Prickle_kozak_Nhe1 and Rb_bGlob_pA_Sac1 and cloned into NheI/ SacI site of CAGGS-EGFP-loxP-Cherry (C36). Full-length axolotl Vangl2 was cloned by PCR from day 6 tail blastema cDNA with primer pairs Vangl2_fullFw1 and Vangl2_fullRv1 and cloned into pCR2.1 (Invitrogen). For CAGGS-Vangl2 construct, Vangl2 ORF was PCR-amplified with primer pairs Vangl2_kozak_Xba1 and Vangl2_stopER1 and cloned into NheI/EcoRI site of CAGGS-mCherry. Primer sequences can be found in 10D_C10) . PrickleElectroporation of the spinal cord was carried out as previously described . BrieflyImages of regenerating tails were acquired on an Olympus SZX16 stereomicroscope using cell^F software. The spinal cord outgrowth was measured from bright field images by drawing a line from the amputation plane to the tip of the regenerating spinal cord using the segmented line tool in Fiji.To analyse the orientation of cell divisions, consecutive 50-μm cross-sections of the posterior-most portion of tails 3-, 6-, and 14-days after tail amputation were collected. Next, slides were washed in PBS, permeabilized with 0.3% PBS/Triton X-100 and nuclei stained with Hoechst overnight at 4ºC. Series of optical sections of late anaphase and telophase cells, in which the position of the mitotic spindle is set , were coSAPProj) was determined as the scalar product between a vector indicating the orientation of the mitotic spindle (S) and a vector indicating the AP axis (AP):The orientation of the mitotic spindle was used as a proxy for the orientation of a cell division. To test whether cell divisions were oriented along the AP axis the projection of the mitotic spindle on the AP axis (S) was calculated from the coordinates of both spindle poles  and B = ):The mitotic spindle poles were deduced from the lack of DNA staining in the pair of sister chromatids . The mitAP) was determined by the two points C =  and D =  located in the first and last optical section of each z-stack, in the middle of the central canal of the spinal cord:and similarly, the vector indicating the AP axis  during the EdU labeling time. For +EdU+/EdU+.Every third cross-section across the entire regenerating spinal cord and at least 10 cross-sections from non-regenerating regions were imaged. Single optical sections were taken from the middle of the tissue sections with a Zeiss C-Apochromat 40x 1.2W objective on a Zeiss LSM 780. For + cells within the regenerating portion of control and Vangl2-electroporated spinal cords. For that, non-consecutive cross-sections spanning the entire regenerate were immunostained for active caspase-3, to detect apoptotic cells, and Tuj1, to delineate the spinal cord tissue. Hoechst was used to label nuclei. Single optical sections were taken with a Zeiss C-Apochromat 40x 1.2W objective on a Zeiss LSM 780. The number of active caspase-3+ nuclei and the number of nuclei per cross-section were counted to calculate the apoptotic index as active caspase 3+/Hoechst+ for each replicate.The fraction of apoptotic cells was determined by counting active caspase-3Prickle1-overexpressing spinal cords were microdissected at day 6 and pooled in each of the three biological replicates per group. Total RNA was extracted using TRIzol (Life Technologies) and purified using RNeasy MinElute cleanup kit (QIAGEN). Power SYBR Green QPCR Master Mix (Life technologies) was used to carry out qPCRs in a LightCycler 480 thermal cycler (Roche). LightCycler 480 software (Roche) was used to analyze relative gene expression. Relative Prickle1 expression was normalized to mRNA levels of the housekeeping gene Rpl4. The primers used were for Prickle1: forward 5'-CACTGGATTTCCGACAACCT-3' and reverse 5'-CGTACCATGGTGTCGACTTG-3', and for Rpl4: forward 5'-CACAAGATGACAAATACAGACCTTG-3' and reverse 5'-CATATGGGTTCAGTTTTACCATGAC-3'.The regenerating portion of control and t-test, and Mann-Whitney U test were carried out using GraphPad Prism 6 . NanoString data analysis was carried out using the DESeq package in R as described above. P values < 0.05 were considered statistically significant. No statistical method was used to determine sample size.The statistical analysis carried out to test the statistical significance of each dataset is indicated in the figure legends. Mean, s.d., s.e.m., Student's  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and two reviewers.Thank you for submitting your work entitled "Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:The authors uncover key molecular mechanisms underlying spinal cord regeneration in the axolotl. The work identifies non-canonical Wnt/PCP signaling as a key player in this process, including transcriptional up-regulation of mediators of this pathway and its requirement for a switch from neuron generating to progenitor generating divisions in the regenerating neural tube. The authors further show that this correlates with the ability of PCP signaling to influence mitotic spindle orientation and so identify a cell biological explanation for this change in cell behaviour. The authors take advantage of some interesting tools; the paper is well written, and the data robust. These findings are interesting and might be important for understanding the spinal cord regeneration mechanisms in general.The authors have tackled the problem of the lack of an axolotl reference genome for RNA seq studies by using recent expression profiling data from the chick embryonic spinal cord to identify gene cohorts likely to be associated with distinct steps in the neural differentiation process. The latter data could be analysed and discussed further in this work, data on lack of cell death following manipulation of PCP signaling should also be provided.Essential revisions:1) In the Introduction, can the authors clarify what is meant by a neural stem cell in this context? They should mention that the axolotl spinal cord produces neurons by asymmetric division of neural progenitors throughout life.2) The comparison with data sets from Olivera-Martinez et al. 2014 should be referred to accurately. These authors pooled in silico, data from SZ and PNT and from CNT and RNT, (rather than SZ and PNT vs NT). Is this the specific comparison used for generating the list of 100 genes?Rarb and Crabp2 levels? This would be consistent with decline in neurogenic genes known to depend on such signaling. It might also relate to the lack of change/detection of FGF signaling – an important role of which in the embryonic spinal cord is to inhibit RA signaling, if RA declines in any case this may obviate the need for increased FGF . Can the authors comment on increased Cdx4, but lack of change of Wnt8a expression? Or the potential roles of other highly upregulated genes such as Epha1 and Greb1? It would also be informative to provide a list of genes that are unchanged in comparison to those in chick PNT.3) It would be helpful to indicate which genes are significantly different from day 0 in 4) Is it possible to determine whether neurons re-enter the cell cycle or if it is just slow cycling neural progenitors that contribute to regeneration? For example, could neurons be labeled with fluorescent protein before amputation? This is not a necessary experiment, but it would be helpful if the authors could clarify the potential origin(s) of regenerating cells.Prickle1 expression in 5) 6) The data showing (quantifying) that lack of cellular blebbing or pyknotic nuclei needs to be provided.7) In the Discussion can the authors comment on whether it is likely that a similar PCP role exists in zebrafish in central canal cells in response to injury and in the mouse central canal? Related to this, it would be helpful to make clear differences that might be due lesion vs amputation conditions.Rarb/Crabp2 expression as this might be expected when neurogenic genes are downregulated. Does this relate to what is happening in adjacent tissues? Which might supply RA or stop doing so?8) Can they also comment on Prickle1 in the regenerative zone leads to the AP division phenotype, yet overexpression of Prickle1 negates the AP oriented divisions. If possible, it would be worth confirming that the expression levels are far higher in the overexpression phenotype relative to the native regeneration induced expression. More directly, shRNA/morpholino experiments would be more helpful to verify the dependence of the AP oriented divisions on Prickle1 overexpression.9) Prickle1 overexpression does not alter cell cycle kinetics reported for the control condition in 10) It is also important to verify (or cite references if previously determined) that 11) Similarly, for Vangl2 it would be important to examine alterations of the cell cycle.The authors state: "Vangl2 has a non-cell autonomous effect, causing polarity defects not only in overexpressing cells but also in their neighbors" The polarity effects here need to be further explained especially in relation to the data presented how these relate to the results presented in 12) It seems possible that the lack of tissue regeneration shown with Vangl2 overexpression could be due to a migration deficit. Experiments should be performed to address this. Essential revisions:1) In the Introduction, can the authors clarify what is meant by a neural stem cell in this context? They should mention that the axolotl spinal cord produces neurons by asymmetric division of neural progenitors throughout life.We thank the reviewer for the tip. We have now included the sentence:“Cells with stem cell features persist throughout life lining the spinal cord central canal of the axolotl. These cells divide occasionally to add neurons to the intact central nervous system of the axolotl, presumably by asymmetric divisions ”.Furthermore, to expand the explanation of the neural stem cell features, we have now included the sentences:“Importantly, lineage tracing of cells lining the central canal showed that at least some cells in the source zone become highly proliferative and multipotent, populating different dorsal-ventral regions of the spinal cord . Furthermore clonal isolates, when transplanted into the regenerating spinal cord contributed to neurons, astrocytes, as well as oligodendrocytes in all regions of the spinal cord, indicating that a single cell can give rise to all cells of the spinal cord .”2) The comparison with data sets from Olivera-Martinez et al. 2014 should be referred to accurately. These authors pooled in silico, data from SZ and PNT and from CNT and RNT, (rather than SZ and PNT vs NT). Is this the specific comparison used for generating the list of 100 genes?Indeed, we generated the 100-gene list from the pooled SZ+PNT versus CNT+RNT comparison. We have now referred to this accurately throughout the text.3) It would be helpful to indicate which genes are significantly different from day 0 in Genes significantly different from day 0 are already highlighted in the heat map , as we eThe data presented in this figure is not dealt with in any detail in the Results or Discussion, which focus largely on PCP signaling. Can the authors provide a more detailed analysis of their data and its comparison with the chick data? For example, what differences are there between the major signaling pathways active in the PNT vs the regenerating axolotl?We agree that the description of this very interesting dataset had been a bit sparse. We have now expanded the section describing the genes found in this dataset in the Results, subheading “Neural stem cells in the injured axolotl spinal cord reactivate molecular programs associated with embryonic neuroepithelial cells”.It appears that retinoid signaling, which is low in SZ and PNT is also declining in the regenerating spinal cord, based on readout of retinoid activity, Rarb and Crabp2 levels? This would be consistent with decline in neurogenic genes known to depend on such signaling. It might also relate to the lack of change/detection of FGF signaling – an important role of which in the embryonic spinal cord is to inhibit RA signaling, if RA declines in any case this may obviate the need for increased FGF .Rarb, Crabp1 and Meis2 suggests that retinoid signalling declines during regeneration. We have now commented on this change in the Results (from paragraph three). However, regarding FGF signaling, it is important to bear in mind that we only looked at the expression changes of a limited, 100-gene set. Thus, although our data show that Fgf8 is not expressed, we cannot rule out that other FGF ligands such as FGF2 are responsible for the decline of RA signaling or the activation of the MAPK pathway. Zhang and colleagues actually showed that FGF2 is upregulated in the regenerating spinal cord in newts (PMID:10985857). Interestingly, they also showed dynamic changes in the expression of FGF receptors in the regenerating spinal cord. Consistent with our gene expression analysis in axolotl, FGFR2 is downregulated in the early stages of newt spinal cord regeneration –while FGFR1 is upregulated (PMID: 12379240).That is a very interesting observation from the reviewers. Indeed, the downregulation of RA target genes Can the authors comment on increased Cdx4, but lack of change of Wnt8a expression?Cdx4 could be triggered by Wnt ligands not included in our 100-gene set such as Wnt3a . Another possibility is that FGF/MAPK signaling is regulating Cdx4 expression (PMID: 16982047).As discussed above, the upregulation of Or the potential roles of other highly upregulated genes such as Epha1 and Greb1?Greb1 has been associated with estrogen-induced cell proliferation and cancer progression (PMID: 15986123) and Epha1 was first isolated from cancer cell lines (PMID: 2825356). It is thus likely that both contribute to the conversion to the more proliferative state of regenerating neural stem cells in axolotl. We have now included a few notes about these highly upregulated genes in the Results (paragraphs two and four).It would also be informative to provide a list of genes that are unchanged in comparison to those in chick PNT.This information can be also found in 4) Is it possible to determine whether neurons re-enter the cell cycle or if it is just slow cycling neural progenitors that contribute to regeneration? For example, could neurons be labeled with fluorescent protein before amputation? This is not a necessary experiment, but it would be helpful if the authors could clarify the potential origin(s) of regenerating cells.+ neural stem cell population (PMID: 17507409 and 12557212). However, we have added a small note in the Introduction to make this point clear (paragraph two).The contribution of neurons to the regenerating spinal cord has already been addressed by Zhang and colleagues (PMID: 12557212). They retro-labeled motor neurons using fluorescent dextran before amputation and followed them during regeneration to find that some neurons move into the regenerating spinal cord, but do not dedifferentiate or re-enter the cell cycle. It is well accepted that the new spinal cord stems mainly from the SOX25) Prickle1 expression in Prickle1 and Wnt5a are consistently upregulated in the blastema/mesenchymal cells surrounding the spinal cord. Based on previously literature it is likely that PCP also directs polarized growth/morphogenesis in the mesenchymal component of developing and regenerating tissues. Sugiura and colleagues showed in Xenopus laevis that provision of Xwnt-5a in lateral sites of injured tadpole tails can induce regeneration of a complete ectopic tails containing both spinal cord and mesodermal tissues (PMID 18977433). In addition, Gros and colleagues (PMID 21055947) showed a role for wnt5 signalling in controlling cell-oriented behaviors of limb bud mesenchyme leading to directional during limb bud outgrowth. These observations indeed indicate that non-canonical Wnt signalling acts also in mesenchymal tissues to direct oriented outgrowth of vertebrate appendages.Indeed, 6) The data showing (quantifying) that lack of cellular blebbing or pyknotic nuclei needs to be provided.We thank the reviewers for the suggestions. We have now quantified the fraction of apoptotic cells in control and Vangl2-overexpressing spinal cords and show this in 7) In the Discussion can the authors comment on whether it is likely that a similar PCP role exists in zebrafish in central canal cells in response to injury and in the mouse central canal? Related to this, it would be helpful to make clear differences that might be due lesion vs amputation conditions.We have now added an extensive section at the end of the Discussion to reflect on the function of Wnt5 in developmental and regenerative contexts. The work in development clearly shows that Wnt5 expression is associated with outgrowths of both epithelium and mesenchyme-containing structures. The functional analysis of Wnt5 in different regeneration systems has not yielded as uniform of a conclusion. While in amphibians Wnt5 is clearly associated with tail outgrowth (for example this study and PMID: 18977433), in the zebrafish, fin activation of Wnt5 supressed regeneration (PMID: 17185322). It is unclear if this latter observation is due to the global activation of Wnt5 signalling, which would derange PCP making regeneration less directed, or whether, as hypothesize as the authors, this reflects an antagonism between canonical and non-canonical Wnt pathways in fin cells. To our knowledge, the role of Wnt/PCP signalling after spinal cord injury has not been studied in zebrafish or mouse, but would clearly be important for comparative purposes.8) Can they also comment on Rarb/Crabp2 expression as this might be expected when neurogenic genes are downregulated. Does this relate to what is happening in adjacent tissues? Which might supply RA or stop doing so?We now provide a discussion of this expression in the Results as mentioned above, and discuss this issue in the Discussion, starting in paragraph five. Indeed, it is likely that non-spinal cord cell sources provide retinoic acid signalling dynamically during the course of regeneration and, given the similarities between the developmental and regeneration situations, that at one point, RA is a pro-neurogenic factor during regeneration. In another project in the laboratory we are starting to study these issues with respect to myotome regeneration, and therefore eventually we will link the differentiation of myotomal tissues with the onset of neurogenesis in the spinal cord. The focus of the current manuscript is primarily the early switch from the uninjured state to the PNT-like state. Taking all into consideration, an experimental link between RA from non-spinal cord sources and neurogenesis will be taken up in a future manuscript.9)  Prickle1 in the regenerative zone leads to the AP division phenotype, yet overexpression of Prickle1 negates the AP oriented divisions. If possible, it would be worth confirming that the expression levels are far higher in the overexpression phenotype relative to the native regeneration induced expression.Prickle1 in control and Prickle1-overexpressing spinal cord and added this in We thank the reviewers for pointing out this difficulty of understanding the rational to this experiment. To address this issue, we have now inserted background information with references that validates the use of overexpression to disrupt PCP . We have also quantified the expression levels of More directly, shRNA/morpholino experiments would be more helpful to verify the dependence of the AP oriented divisions on Prickle1 overexpression.Prickle1 5’ sequence and splicing-disrupting morpholinos are not realistic due to lack of genome sequence. Our laboratory is currently working on other (CRISPR/Cas9) knockdown strategies for the spinal cord, but validation of these technical developments will take time beyond the revision scope of this manuscript.Unfortunately GeneTools were unable to design an acceptable morpholino against the axolotl 10) It is also important to verify (or cite references if previously determined) that Prickle1 overexpression does not alter cell cycle kinetics reported for the control condition in Prickle1-overexpressing cells, and found that it is indistinguishable from mCherry-only-transfected cells. The data is in Prickle1 construct.We have now addressed this issue by assessing the rate of EdU incorporation in control and 11) Similarly, for Vangl2 it would be important to examine alterations of the cell cycle.In The authors state: "Vangl2 has a non-cell autonomous effect, causing polarity defects not only in overexpressing cells but also in their neighbors" The polarity effects here need to be further explained especially in relation to the data presented how these relate to the results presented in We have now included more extensive background information on the PCP system and the different classes of phenotypes (autonomous versus non-autonomous) that are expected based on which core component is overexpressed .12) It seems possible that the lack of tissue regeneration shown with Vangl2 overexpression could be due to a migration deficit. Experiments should be performed to address this.In the lab, we are currently performing two other projects that impinge on this question. First we are performing live time-lapse imaging of regeneration. We observe that the major time of what could be termed collective cell migration occurs at 48 hours, which is the phase before the onset of the Vangl2 phenotypes. At this stage, Vangl2 overexpressing spinal cords extend rather normally. In a second project in the laboratory, we have followed labeled cells or small clusters during spinal cord regeneration quantitatively based on their initial position behind the amputation plane. This data, along with some of the data presented in the current manuscript, are being used to construct a quantitative model of regeneration. The results so far indicate that migration plays a minor role in spinal cord regeneration, and that growth can be explained largely by proliferation and some slight “pushing” from more anterior cells, due to their slow expansion. This is an extensive project and will not be submitted prior to the current manuscript. We would prefer to keep that work separate, but are happy to provide the data for perusal when requested.


Pck1 gene) is the rate-limiting enzyme in gluconeogenesis and is largely responsible for the maintenance of glucose homeostasis during fasting in fruit-eating bats. To test whether Pck1 has experienced adaptive evolution in frugivorous bats, we obtained Pck1 coding sequence from 20 species of bats, including five Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our molecular evolutionary analyses of these sequences revealed that Pck1 was under purifying selection in both Old World and New World fruit bats with no evidence of positive selection detected in either ancestral branch leading to fruit bats. Interestingly, however, six specific amino acid substitutions were detected on the ancestral lineage of OWFBs. In addition, we found considerable evidence for parallel evolution, at the amino acid level, between the PEPCK1 sequences of Old World fruit bats and New World fruit bats. Test for parallel evolution showed that four parallel substitutions  were driven by natural selection. Our study provides evidence that Pck1 underwent parallel evolution between Old World and New World fruit bats, two lineages of mammals that feed on a carbohydrate-rich diet and experience regular periods of fasting as part of their life cycle.Bats are an ideal mammalian group for exploring adaptations to fasting due to their large variety of diets and because fasting is a regular part of their life cycle. Mammals fed on a carbohydrate-rich diet experience a rapid decrease in blood glucose levels during a fast, thus, the development of mechanisms to resist the consequences of regular fasts, experienced on a daily basis, must have been crucial in the evolution of frugivorous bats. Phosphoenolpyruvate carboxykinase 1 (PEPCK1, encoded by the  Pck1 gene), which is located in the cytosol, and phosphoenolpyruvate carboxykinase 2 , which is restricted to the mitochondria  (A. lituratus and L. yerbabuenae), grouped together to comprise the suborder Yangochiroptera (BPP = 1.00)  . The two = 1.00) .Pck1 in OWFBs and NWFBs, we set either the ancestral branch to OWFBs or NWFBs as the foreground branch and performed two-ratio model tests on these branches. Our results showed that the two-ratio model with the ancestral branch to OWFBs as the foreground was a better fit than the one-ratio model (null hypothesis)  (N/dS) on the ancestral branch leading to OWFBs that was higher than on the background . These results indicate that a change in selection pressure on the Pck1 gene occurred on the OWFB lineage. However, the two-ratio model test with the ancestral branch for NWFBs as the foreground was not significantly different from the one-ratio model   , which iPck1 gene occurred in OWFBs, and that this change mainly effected the rate of nonsynonymous substitutions   , and the fit of branch-site model A was better than the M1a model  . However; test2) . Since tPck1 codons, we calculated the values of ω among OWFBs, NWFBs, and insectivorous bats using SWAAP 1.0.2. The results of this analysis showed that the estimated ω values for OWFBs are much larger than those of insectivorous bats, with a maximal ω value of 1.9 observed on the nucleotide binding domain 1 , suggesting that this amino acid substitution likely occurred in parallel by chance  on the ancestral branch of OWFBs is higher than for the remaining branches of the species tree, and that the rate of nonsynonymous substitutions in the Pck1 gene of OWFBs is significantly higher than that of insectivorous bats. Although there was a change in the selection pressure acting on the Pck1 gene on the ancestral branch of NWFBs, the omega value is still quite low indicating that Pck1 is still under purifying selection.In this study, we examined the molecular evolution of the glycemia ,25. By cpck1 gene, as the result of the explicit test of positive selection (test2) was negative. Test1, by itself, is unable to distinguish between relaxation of constraints and positive selection [Pck1 experienced relaxed selection in Old World fruit bats is probably low as PEPCK1 has a very important function across mammals [Pck1 genes we found that the crucial residues in the active site loops, including the R-loop, P-loop and Ω-loop, are completely conserved. These results indicated that PEPCK1 retains an important role in glucose homeostasis in frugivore bats [Pck1 in mice leads to hypoglycemia and death [Pck1 has experienced strong purifying selection not only in fruit bats, but also in all other mammalian species examined. Although the results are congruent with the fact that the PEPCK1protein is very critical for hepatic glycometabolism, there are two possible explanations that may explain the failure to detect positive selection. First, OWFBs diverged from the other Yinpterochiroptera lineages nearly 60 million years ago [Although the results of test1 of the branch-site model A test revealed 13 amino acid replacement with elevated omega value, we are unable to rule out the possibility that these were due to a relaxation of the selection acting on the election . However mammals . Even be mammals . From ouore bats . Previound death , supportears ago , and preears ago . Second,ears ago –61. In aResults from the ancestral reconstruction of the amino acid sequences using the maximum parsimony method with Mesquite 2.74  identifiPck1 gene may have two explanations. First, OWFBs evolved their carbohydrate-rich diet earlier than the NWFBs  [In contrast to OWFBs, we did not find any specific amino acid replacements in the NWFBs. It is interesting to note that other recent molecular evolution studies focusing on diet-related genes in bats also found similar patterns –65. For ctively) , thus hactively) , change Pck1 gene of OWFBs and NWFBs, this does not mean that this gene did not undergo adaptive evolution. Parallel evolution between OWFBs and NWFBs is also a strong indicator of adaptive evolution [Pck1 genes in OWFBs and NWFBs is molecular adaption to the regular fasting by these fruit bats. It is widely accepted that the hepatic gluconeogenesis plays a critical role in the maintenance of glucose homeostasis during fasting [Pck1 in OWFBs and NWFBs might relate to the evolution of adaptive mechanisms for fasting resistance in fruit bats. Here we found four credible parallel evolution sites  in PEPCK1. All of these sites are located near the nucleotide binding domain, which might influence the binding of GTP to PEPCK1 and thus enzymatic activity [Although no evidence for positive selection was found in the volution . Previouvolution ,68. A po fasting ,8, and i fasting . PEPCK1, fasting . Thus, iactivity  Fig. 5)Pck1 geneactivity .Pck1 gene, which plays an important role in the maintenance of glucose homeostasis during fasting, has undergone parallel evolution between the OWFBs and NWFBs, which provides evidence for adaptive evolution in both lineages, although our evidence for NWFBs is limited to only two species of Phyllostomid bats. Greater sampling of NWFBs is needed to confirm this finding. Apart from gluconeogenesis, PEPCK1 is also the rate-limiting enzyme in glyceroneogenesis, which is responsible for recycling free fatty acid back to triglyceride [Pck1 gene between OWFBs and NWFBs, which could possibly be explained by its function in glyceroneogenesis. However, additional in-depth studies are needed to address this issue. We speculate that additional deeper studies will reveal that numerous other genes related to carbohydrate metabolism will be found to have experienced adaptive evolution in OWFBs and NWFBs.In conclusion, our results show that the lyceride . Chroniclyceride , therefolyceride . Here weS1 FigFour unique enzymes required in gluconeogenesis are shown in the rectangles. All of the remaining steps are catalyzed by glycolytic enzymes.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


The red cells transfusion is a mainstay in the treatment of anemic patients. These blood transfusions are not without risks. The risk-benefit profile for red cell transfusions to treat anaemia is uncertain, but they may contribute to adverse patient outcomes in some situations. The ability of a patient to tolerate anaemia depends on their clinical condition and the presence of any significant co-morbidity; maintenance of circulating volume is of paramount importance. There is no universal transfusion trigger. Advances in the development and validation of physiological, accessible, practical and reliable markers to guide therapy are expected. To improve patients' outcomes, further study is required to more fully explore the risk of anemia, optimal hemoglobin level, and the risk and efficacy of RBC transfusion. Future clinical investigations with high priority should determine the efficacy of transfusion in those classified as uncertain scenarios. In the absence of data, it is prudent that transfusion is administered with caution in these clinical scenarios. Red blood cell (RBC) transfusions are a mainstay in the treatment of anemic patients, making it the most common medical procedure in hospitalized patientsMost RBC transfusions (RBCT) are prescribed for patients with relatively low levels of hemoglobin (Hb) and only in controlled situations. The underlying thinking is that the transfusion will increase oxygen transport and therefore decrease deficiencies thus "relieving" tissue hypoxia. However, this hypothetical benefit of RBC transfusions has not been unequivocally demonstrated.An inadequate supply of oxygen to tissues can lead to multiple organ failure and increased morbidity and mortality. These deleterious effects appear only with very low Hb levels when compensatory mechanisms do not work properly or are insufficient; however, that level is not exactly known.The risks and complications of RBCT include: the transmission of infectious diseases, immune suppression, acute respiratory distress syndrome, circulatory overload and errors in administration. With these potential or actual adverse effects taken together with the large variability in observed prescriptions, it has been determined that when faced with "liberal" or "defensive" traditional transfusion criteria, more restrictive transfusion practices are adopted along with an enhanced usage of alternative treatments. However, we must be cautious because this trend can lead us to expose patients unnecessarily to risks of anemia and hypoxia by not administering transfusions.This makes it essential to specify an appropriate risk/benefit ratio for the transfusion. This is because it is also not permissible to subject the patient to an intervention whose effectiveness has not been documented in terms of reduced mortality or morbidity.In the 1988 Consensus Conference of the NIH  the lack of justification for "classic" transfusion practice was highlighted and it was concluded that "the available evidence does not support the use of a single criterion for transfusion, such as Hb<100 g/L. No single measure can replace good clinical judgment as the basis for decision making concerning RBCT".Weiskopf asserts that although millions of units of red blood cells are transfused, the biological effectiveness of this measure has not been demonstrated in prospective, controlled studies, nor do other criteria exist by which one could judge the effectiveness of the transfusion of concentrated red blood cellsAfter almost a century of the clinical use of RBCT, the continued use of this form of therapy is now being questioned. This is not necessarily due to adverse effects, but to the lack of studies that document its effectiveness.A recent systematic review of the literature considered the results of 45 cohort studies with 272,596 patients in critical condition and revealed that in 42 of the 45 studies transfusion represented greater risk than benefit to patients and in the transfused cohort it presented a higher rate of adverse events than it did to those not receiving transfusions---.Similarly, in clinical randomized controlled trials (RCTs) evaluating the efficacy/ effectiveness of RBCTThese findings oblige a more judicious use among the many patients who are routinely transfused. A greater effort should be made to understand that there is a transition point at which the physiological mechanisms fail to compensate for the decreased oxygen supply associated with anemia, in which case transfusion favors the results.2 (oxygen delivery) to a level of 7.3 mL/kg/minIt has been noticed that in young, alert, healthy adults when subjected to an isovolemichemodilution with a decreased hemoglobin concentration of 12.5 g/dL to 4.8 g/dL that an increased heart rate, systolic volume index and the cardiac index is produced with no systemic evidence of hypoxic changes despite a reduction in the DO2 could be 3.8 mL/kg/ minA study conducted with septic patients suggests that the critical DO2 with anemia does not always translate into decreased VO2 (oxygen consumption) due to the intervention of compensatory physiological mechanisms, such as the increase in cardiac output and tissue O2 extraction. The VO2 is, in turn, balanced by the ability of peripheral tissues to modify oxygen extraction (EO2) in hypoxemic states thus altering micro-vascular blood flow and maintaining a stable tissue pO2The decrease in DO2 with RBCT have been interpreted as a lack in transfusion effectiveness and are attributed to the loss of 2,3-diphosphoglycerate and nitric oxide during storage or to increased viscosity,The studies that reveal a lack of increased tissue OCardiac function determines the clinical tolerance limit to anemia in any patient. Oxygen delivery to the myocardium increases only by means of improving the blood flow in the coronary arteriesThe consequent reduction of coronary flow can thus increase the pressure at the end of the diastole, to produce changes in the ECG and subsequent symptomatic ischemiaSome studies in humans have thrown light on the limits of physiological compensation of anemia. The critical Hb at rest has been estimated at approximately 20-25% of normal Hb2 in this patient under anesthesia was 4.9 mL O2/kg/min for a VO2 of 2.4 mL O2/kg/min and death occurred at aHb level of 4.0 g/dL. The dissociation curve of oxyhemoglobin, after correction for changes in the pH and PCO2, deviates to the right with a hematocrit of 8%. This indicates a decrease in oxygen affinity of hemoglobin as a compensatory mechanism to facilitate the delivery of O2 to peripheral tissues in extreme anemiaThe first report was documented in an 84 year old Jehovah's Witness who refused transfusion and died after surgery with aHb of 1.6 g/dL. The critical DO2 varies between 3.8-4.5 mL O2/kg/min. For a VO2 of 2.4 mL O2/kg/min, the critical EO2 was approximately 60%Furthermore, in critically ill patients sedated after cessation of life support, the DO2 varies with different metabolic requirements, subsequent studies were carried out taking measurements during states of consciousness in which the VO2 is higher. It has been shown that healthy humans at rest are able to tolerate acute isovolemichemodilution with a Hb of 5 g/dL,Considering that the critical DO2 despite the decrease in DO2 during progressive isovolemichemodilution with 5% albumin and/or autologous plasma to a level of Hb 5 g/dLIn 32 conscious individuals at rest, no significant change was produced any in the concentration of lactate or VO2 of 3.4 mL O2/kg/min, the critical DO2 during acute hemodilution with 5% albumin and autologous plasma was less than 7.3 mL O2/kg/min and 4.8 g/dL of HbIn another study with conscious young healthy volunteers it was found that for a VO,However, in the presence of coronary artery disease, the Hb threshold may increase. In animal models it has been reported that Hb threshold may be at a level of 7-7.5 g/dL in the presence of coronary artery stenosis with limited tolerance to isovolemic hemodilution2 can be reversed and corrected with RBCT by increasing the Hb to 1.9 g/dLContractile dysfunction induced by ischemia and compromise in the delivery and consumption of OThe results of these and other experimental studies, however, are not easily extrapolated to clinical situations for patients with co-morbidities and changes in the balance of supply and demand of oxygen. , Currently, "the transfusion threshold" is based on predetermined values for Hb concentrations that are derived from a few randomized clinically controlled trials, various observational cohort studies, or from the opinion of experts 2) reaches a maximum at a 30% Hct (Hb 100 g/L), and it decreases as hemodilution and hemoconcentration progress. In diverse experimental animal models it has been shown that TO2 and survival are optimal at an Hct between 30 to 40%.Two concepts form the basis for the use of the Hb concentration as a determinant of RBCT: the optimal level of Hb and the minimum acceptable level of Hb. The optimal level of Hb is the concentration of Hb at which organic functionality is maximal. Studies carried out on individuals undergoing acute normovolemichemodilution, found that oxygen transport  can be a useful but nonspecific parameter for determining the presence of tissue hypoxia, especially in the post-operative setting of certain surgeries. After coronary artery bypass surgery, the Pa-vCO2 is influenced by the metabolic rate, body temperature (possibly due to the release of CO2 during re-warming the patient) and decreased pulmonary elimination of CO2. In these circumstances, patients with abnormally elevated Pa-vCO2 show a greater incidence of post-operative complications from tissue hypoxia The arterio-venous difference in the partial pressure of CO2) is the relationship between the consumption and delivery of O2 (VO2/DO2). It is expressed in percentages and indicates the percentage of O2 provided that has been utilized. Normal CEO2 is 25%. Some studies of normovolemic anemia performed with different animals have focused on the study of CEO2 as a good indicator of when to perform the T. Its application to daily clinical practice is lacking.The oxygen extraction quotient  and it is very difficult to know the time at which each patient needs to increase their transport capacity. This is especially so when you consider that their oxygen needs pre-operatively, during the intervention, and post-operatively are quite different.Although the only actual reason for establishing an indication for RBCT is for maintaining the O2 with Hb levels around 50 g/L, this same number should be applicable to individuals ASA I anaesthetized in which O2 consumption is lower than while at rest. Overall, general anesthesia with neuromuscular blockage and mechanical ventilation decreases oxygen consumption by 20-40%, which can make the critical TO2 descend to a lower level than in the conscious individualOnce anesthetized, the patient is in a state of minimal metabolic demand. If young, healthy individuals get adequate TO2 demands increase during the post-anesthetic recovery and VO2 multiplies by a factor of 2-3, depending on the type of anesthesia used, the aggressiveness of the surgery, the degree of post-operative analgesia in the presence of pain and characteristics of the patient. Again, this situation, which is generally well-tolerated by young and healthy patients with active compensating mechanisms for anemia, may be post-operatively complicated by a compromised cardiovascular function, or among patients with sepsis, especially if they are elderly.A recent review2 and cardiac reserve, the critical level of Hb has an individual value as there is no general threshold for RBCT. Given the evidence that myocardial ischemia is a critical factor in the patient's ability to tolerate anemia, no patient over forty years of age with Hb<100 g/L should undergo elective surgery without previously ruling out myocardial ischemia.In contrast, OThe effectiveness of the RBCT can only be established by results from well-designed randomized clinical trials. Until now the clinical trials conducted to compare two transfusion strategies  in different types of patients have not shown significant differences in terms of morbidity, mortality and functional status of patients, with the possible exception of those with AMI or unstable angina.As previously noted, at the 1988 Consensus Conference organized by the U.S. National Institute of Health the threshold concentration of hemoglobin (Hb) from consensus was set at 70 g/L. It was emphasized that there was a direct call to establish needs and clinical symptoms as the basis for the transfusion decision, and not base the decision solely on Hb concentrations. That is, to transfuse if Hb<70 g/L, to individualize the decision for Hb levels of 70-100 g/L and not to transfuse if Hb>100 g/L. Since then, several guidelines have been published, the result of other Consensus Conferences along the same lines; that is, the reasons for the use of "restrictive" transfusion criteria prevailed over those more "liberal".In the field of intensive care, in a multi-centric, randomized, prospective study, Transfusion Requirements in Critical Care (TRICC), the mortality rate in critically ill patients undergoing a "restrictive" RBCC protocol (Hb<70 g/L to keep it between 70 and 90 g/L) was compared with "liberal" criteria However, according to the subsequent analysis of the TRICC study datap= 0.03).In subjects with ischemic heart disease, mortality was greater in the restrictive group than in the liberal group ; whereas in patients without ischemic heart disease, mortality was lower in the restrictive group than in the liberal group  , recently published are as followsRecommendation 1: The AABB recommends adhering to a restrictive transfusion strategy (7-8 g/dL) in hospitalized, stable patients .Recommendation 2: The AABB suggests adhering to a restrictive strategy in hospitalized patients with a preexisting cardiovascular disease and consider transfusion in patients with symptoms or a hemoglobin of 8 g/dL or less .Recommendation 3: The AABB cannot make a recommendation for or against the use of a liberal transfusion threshold or a restrictive one, for hospitalized patients, hemodynamically stable with acute coronary syndrome .Recommendation 4: The AABB suggests that transfusion decisions be based on the symptoms, as well as on hemoglobin concentrations .Reed blood cells transfuctions is widely used in the treatment of anemia, although adequate thresholds for its use remain controversial. Although therapeutic modalities should be subject to a rigorous evaluation of its efficacy and safety prior to use in clinical practice, RBCT has not been subjected to a similar examination. Besides the already known complications from transfusions, numerous studies indicate that RBCT may be associated with unfavorable outcomes.The few conclusive results and non-controversial data have not overcome the difficulties that have prevented previous attempts to establish a policy or guide for RBCT. Despite these limitations and the lack of definitive answers, doctors often have no other choice but the consensus guidelines-.In building a "standard of care", consensus guidelines and reviews often do not represent the actual current clinical practice, and several examples illustrate its ineffectiveness in modifying clinical practiceAlmost two thirds of physicians report regularly transfusing RBC in probably unnecessary situations,