Resistant hypertension (RH) treatment requires an adequate and intense therapeutic approach. However, the results are not always satisfactory despite intensive treatment. Of the different pathophysiological mechanisms involved in the pathogenesis of RH, sympathetic overstimulation and therapies that block the sympathetic system have been widely studied. These approaches, however, are invasive and expensive. Another possible approach is by transcutaneous electrical nerve stimulation (TENS), a noninvasive method that modulates activity by using low-frequency transcutaneous electrical stimulation to inhibit primary afferent pathways. Thus, the current study will evaluate the effect of applying TENS in the cervicothoracic region of subjects with RH and will seek to develop a new low-cost and readily available therapy to treat this group of hypertensive individuals.This is a randomized, single blind (subject), parallel-assignment study controlled with a sham group and including participants aged 40 to 70 years with resistant hypertension. The trial has two arms: the treatment and control (sham group). The treatment group will be submitted to the stimulation procedure (TENS). The sham group will not be submitted to stimulation. The primary outcomes will be a reduction in the peripheral blood pressure and adverse events. The secondary outcomes will be a reduction the central blood pressure. The study will last 30 days. The sample size was calculated assuming an alpha error of 5 % to reject the null hypothesis with a statistical power of 80 %, thereby resulting in 28 participants per group (intervention versus sham).In recent decades, RH has become very common and costly. Adequate control requires several drugs, and in many cases, treatment is not successful. Sympathetic nervous system inhibition by renal denervation and central inhibition have significant effects in reducing BP; however, these treatments are costly and invasive. Another type of sympathetic nervous system inhibition can also be noninvasively achieved by electric current. Therefore, the application of TENS may be a new therapeutic option for treating resistant hypertensive individuals.NCT02365974Clinical Trials The online version of this article (doi:10.1186/s13063-016-1302-8) contains supplementary material, which is available to authorized users. In Brazil, cardiovascular diseases (CVD) accounted for 30 % of deaths in 2008, with hypertension alone affecting more than 36 million people . In the As one of the main risk factors for the development of CVD, hypertension is the starting point of cardiovascular, cerebrovascular, and renal impairment. In most cases, by the time there is a manifestation, this disease, although still manageable, is irreversible. The main risk factors associated with hypertension are smoking, physical inactivity, stress, alcohol consumption, age, obesity, and an unhealthy diet , all of Currently, a broad therapeutic arsenal is available for the treatment of hypertension. Nonetheless, sometimes hypertension becomes difficult to control due to varying situations, thereby characterizing resistant hypertension (RH), which requires a more intensive therapeutic approach. RH is a condition in which the blood pressure (BP) remains above the recommended target even when treated with the maximum tolerated doses of three antihypertensive drugs with synergistic actions, including a diuretic. A condition exists that is known as controlled resistant hypertension, which is defined as high BP that is controlled using four antihypertensive drugs . AlthougThe long-term effect of RH on the vascular system can be catastrophic. Currently, vascular abnormalities are known to exist early in the development of hypertensive diseases and may manifest as severe endothelial dysfunction.Endothelial dysfunction is considered an early marker of vascular complications. It participates in the pathophysiology of atherogenesis, which occurs in one of the first stages of arterial disease , 7 as a Several inflammatory markers, such as proinflammatory cytokines, interleukins , and tumor necrosis factor (TNF-α) related to activated monocytes, have been identified in these situations. Elevated expressions of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells , 15, 16 In animal studies, IL-10 has demonstrated protective roles against the stability and formation of atherosclerotic lesions. Some clinical studies have shown that patients with acute coronary syndrome have reduced IL-10 levels compared to stable patients, raising the possibility that low levels of IL-10 may be related to atherosclerotic plaque instability .Vascular endothelial dysfunction also promotes inflammation by inducing the production of vasoconstrictor agents, adhesion molecules, and growth factors, including angiotensin II (Ang II). Ang II, one of the end products of the renin-angiotensin system (RAS), is actively involved in the pathogenesis/pathophysiology of hypertension. It may be responsible for triggering endothelial dysfunction and vascular inflammation by inducing oxidative stress, a fact that results in the release of inflammatory mediators and in cell growth , 18, 19.The role of Ang II as a proinflammatory mediator of vascular injury is supported by the protective anti-inflammatory effects of RAS inhibitors. Among other effects, RAS inhibitors have properties that result in antithrombotic and fibrinolytic actions. The thrombogenic status mainly results from a high concentration of plasminogen activator inhibitor (PAI-1), which leads to an excessive accumulation of fibrin inside vessels, a situation that contributes to vascular events . Thus, PFibrinogen, an acute phase protein, and cytokines are also directly related to vascular disease . ProspecAs previously mentioned, vascular inflammation and oxidative stress play a crucial role in the pathogenesis of vascular injury mediated by Ang II. Recently, angiotensin-converting enzyme 2 (ACE-2) was identified as having a pleiotropic effect on Ang II, resulting in the neutralization of its proinflammatory and pro-oxidant actions via receptors. Interestingly, the Ang II type-1 receptor blockers may increase ACE-2 expression and activity, thereby reducing cardiovascular and oxidative damage .MMPs are responsible for the degradation of extracellular matrix proteins, breaking them down into their specific peptide bonds, including the cells of the vascular smooth muscle, endothelium, fibroblasts, and inflammatory cells –26. The The aforementioned biochemical changes have a direct relationship with high BP, endothelial dysfunction, and increased vascular stiffness in RH patients, as is demonstrated by reduced flow-mediated vasodilation and increased pulse wave velocity (PWV), which result in higher central BP levels (CBP) .The CBP has a predictive value in the risk stratification of cardiovascular morbidity and mortality as demonstrated by the CAFE  study. This study showed different effects on the CBP of groups treated with amlodipine and atenolol even though reductions in peripheral arterial pressure were similar. This difference has been suggested to be responsible for the favorable cardiovascular outcomes in the amlodipine group of the ASCOT study , which demonstrated the importance of CBP control in relation to peripheral BP and how it can be influenced by certain drug therapies .The CBP is directly influenced by arterial stiffness; the arteries, especially the aorta and carotid, are estimated to stiffen by about 10 to 15 % in men and 5 to 10 % in women each decade . ArteriaThe influence of arterial stiffness on the CBP explains why the pulse wave, generated in every left ventricular ejection period, propagates cyclically throughout the arterial tree to the peripheral arteries. Changes in the impedance and structural or geometric discontinuity of the arterial tree along its path generate reflected waves that move backwards into the ascending aorta and to the heart (backward wave). Therefore, the CBP is the sum of the anterograde and retrograde (reflected wave) components , 43. WavCurrently, nonpharmacological therapeutic methods are being used to treat RH. These are invasive, require long-term follow up to monitor the results and may cause unwanted complications during and after the procedure, besides being expensive. Examples of these methods are denervation or ablation of the renal sympathetic ganglia and chronic carotid baroreflex stimulation . Both thMannheimer et al. demonstrated that low-frequency electrical stimulation applied to the suprathoracic lymph node region produced an anti-ischemic effect by reducing myocardial oxygen demand due to the reduction in the afterload resulting from a reduction in BP . This reGiven the scarcity of publications in the scientific literature, this innovative work will study the effects of the application of TENS in the suprathoracic lymph node region to reduce peripheral BP and CBP in RH patients.Due to the high incidence of cardiovascular morbidity and mortality caused by RH, the introduction of new, more effective, highly specific, and noninvasive therapeutic approaches that are reproducible and less costly from a financial point of view are needed.Does TENS constitute a new therapeutic option in the reduction of peripheral blood pressure of patients with resistant hypertension?Does TENS constitute a new therapeutic option in the reduction of central pressure of resistant hypertensive patients?Does TENS reduce arterial stiffness measurement parameters?Can TENS positively influence metabolic disorders that involve the progression of arterial stiffness?The following research questions will be explored:To evaluate the effectiveness of TENS as antihypertensive therapy in RH patientsTo assess the peripheral and central BP before and after the use of TENSTo evaluate the behavior of arterial stiffness by measuring the AIx and PWV before and after the use of TENSTo observe the biochemical changes, inflammatory markers, and the participation of RAS inhibitors before and after the application of TENSThe research objectives of this study are as follows:The present trial  will be a randomized, open-label, parallel-assignment study controlled with a sham group. All patients who participate in the research will sign informed consent forms. The project design, approved by the Research Ethics Committee of FAMERP, São Paulo, Brazil, which is accredited by the Office of Human Research Protections as an Institutional Review Board  Dermatological abnormalities at the site of the application of TENSUncontrolled diabetes in patients using oral hypoglycemic agents or insulin with glycated Hb ≥ 7.5 %Secondary hypertensionPatients who do not adhere to the lifestyle changes recommended by their physician such as low-sodium diet and weight lossModerate to severe cervical-thoracic scoliosis2Obesity with body mass index (BMI) ≥ 35 kg/mThe exclusion criteria are as follows:A computer-validated software program (Random Allocation) will be used for the random allocation. The study coordinator will organize and number the envelopes, which will be allocated in order of patient enrollment. The professional responsible for the Endo-PAT procedure and evaluation of arterial stiffness parameters will be blinded.TENS will be applied in the cervicothoracic ganglion region in the active comparator group, and the sham group will not be submitted to transcutaneous electrical stimulation. Figure 2), will be calculated. The height will be determined in centimeters using a measuring tape while the individual stands in bare feet. Individuals with a BMI < 25 kg/m2 will be classified as normal, overweight as a BMI between 25 and 30 kg/m2, and obese as a BMI ≥ 30 kg/m2. The waist circumference will be measured using a measuring tape at half the distance between the iliac crest and the lower costal margin. Patients will undergo the following tests before and after the end of the 4-week study period: 24-h ambulatory blood pressure monitoring (ABPM), 24-h dynamic electrocardiography, continuous measurement of beat-to-beat BP, endothelial function measurement (Endo-PAT 2000), and radial artery applanation tonometry (AT). Table Patients will be divided into two groups: treatment and control (sham group). The sham group will be submitted to the procedure to fit the stimulation equipment. However, these patients will not be submitted to stimulation. Both groups will undergo a physical examination at the start and end of the study period to determine anthropometric variables. The BMI, defined as the weight (kg) divided by the patient’s height squared  . Monitornitoring . The folThe SBP and DBP will be obtained by ABPM with the mean values for the 24-h period, daytime, and nighttime being considered for analysis. RH patients with mean BP values of ≥ 130 × 80 mmHg over 24 h, ≥ 135 × 85 mmHg during wakefulness, and ≥ 120 × 70 mmHg when asleep will be considered. PP will be calculated during the periods  using the formula PP = SBP - DBP. The normal nocturnal dip will be defined as a drop >10 % in SBP from wakefulness to the period of sleeping.The assessment of the effects of the inhibition of sympathetic nerve activity on the cardiovascular system will be by 24-h dynamic electrocardiography (Holter System - Cardios-Light/Cardios) in accordance with the Guidelines for the Evaluation and Treatment of Patients with Cardiac Arrhythmias . The HolThe continuous measurement system of beat-to-beat BP  will be used to evaluate the sympathetic activity. Patients will be placed in a quiet room with controlled temperature (22 °C), without having ingested any kind of natural or drug stimulant and without pain or a full bladder , 64. BefThe peripheral endothelial function will be assessed noninvasively by measuring the reactive hyperemia of the vessel using the Endo-PAT system  . PatientA pressure cuff is placed on one arm (study arm), whereas the other serves as a control (control arm). The device, comprising a pneumatic plethysmograph, is placed on the tip of each index finger, and a uniform pressure is applied to the distal digital surface allowing the measurement of changes in the pulse volume. The insufflation pressure of the digital device is electronically set at 10 mmHg below the DAP or at 70 mmHg. After a 10-min regulation period, the BP cuff is inflated to 60 mmHg above systolic pressure or to 200 mmHg for 5 min. The device continues to record for 10 min after the cuff is deflated. The absence of residual pulsatility is monitored in the occlusion period. The hyperemia after the period of occlusion stimulates endothelium-dependent vasodilation, causing an increase in the amplitude of the digital pulse. Changes in the pressure signals are filtered, amplified, recorded electronically, and analyzed using an automated computerized algorithm . The change from the baseline measurement is defined as the reactive hyperemia index (RHI), which will reflect, in part, the vasodilator function of the digital microcirculation. Endothelial dysfunction is defined as a mean value of 2.34 ± 0.33 less two standard deviations of 20 asymptomatic, healthy control subjects with no history of cardiovascular disease and without major risk factors (RHI ≤ 1.68) .The arterial stiffness will also be evaluated by AT of the radial artery, as suggested by taking an average of three measurements after the pressure is stabilized. The HEM9000 AI device  will be used for this exam.The AT examination will be performed in a quiet controlled environment (temperature between 21 °C and 24 °C) after the patient has rested for at least 5 min while sitting with the legs uncrossed, the bladder empty, and away from acute stressors. The patient will not have smoked at all for at least 30 min or ingested alcohol. The OMROM AT equipment uses a radial ultrasonic transducer and cuffs with the correct size for the arm circumference, as recommended by the guidelines to evaluate BP [TENS will be applied in the cervicothoracic ganglion region located between the C7 and T4 vertebral processes for 40 min three times weekly for a total of 4 weeks.Electrostimulators  with two 80-Hz output channels at a pulse width of 150 μs will be used for the application of TENS. The intensity in milliamps (mA) will be adjusted depending on the sensitivity of each individual patient. This electrical stimulus has a high-frequency current that will cause mild local paresthesia without causing pain or unpleasant sensations. The intensity will be increased from zero until the perceived sensation reaches the maximum sensory threshold without pain or discomfort or involuntary muscle contractions. The 5 × 5-cm self-adhesive electrodes  are attached to the patient after marking the correct location using a dermatographic pencil and cleaning the skin. The arrangement thereof will be parallel on each side of the C7 (channel 1) and T4 channel 2) vertebral spinous processes , high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), creatinine, potassium, uric acid, and plasma insulin levels.Blood sugar, TC, HDL-C, and TG levels will be determined by colorimetry after fasting for 12 h using a RXL Dimension device with the Dade Behring reagent. Diagnosis of diabetes mellitus (DM) will be established by patient history, the use of hypoglycemic drugs, and measurement of the serum glucose . The LDLIn order to convert the glucose concentration to mmol/L it is necessary to multiply the value in mg/dL by 0.0555.Measurement of serum creatinine will be performed by a standardized enzyme-kinetic biochemical test and will be considered high when serum creatinine is > 1.4 mg/dL , 72.MDRD (mL/min/1.73 m2) = 186 (serum creatinine)−1.154 × (age)−0.203 ×  × (1.212 if black) [Urinary sodium and potassium levels (mEq/L) will be evaluated by flame photometry. Urinary sodium and potassium levels excretion will be calculated by multiplying the urinary sodium and potassium concentrations in mEq/L by the 12-h urine volume . Microal2 (TXB2); fibrinogen; intercellular adhesion molecule-1 (ICAM-1); plasminogen activator inhibitor (PAI-1); angiotensin converting enzyme (ACE); and angiotensin II (Ang II).Specific biochemical tests will be considered Table : serum aTo evaluate the hsCRP, MMP-9, TNF-α, AS, PRA, TIMPs-1 and −2, nitrites and nitrates, IL-1, IL-6, IL-8, IL-10, IL-18, thromboxane B2, and fibrinogen levels, plasma samples will be obtained after centrifugation at 3500 rpm and 4 °C for 15 min immediately after collection and then stored at −80 °C for later analysis. The IL-1, IL-6, IL-8, IL-10, IL-18, TNF-α, MMP-2 and −9, ACE, and Ang II will be measured using the ELISA technique  according to the manufacturer’s instructions and as described in a previous study . The hsCThe routine PRA assay available today is based on the determination of plasma angiotensin I generated in vitro by the proteolytic action of renin on its substrate: angiotensinogen under predetermined conditions of time and temperature. Blood samples should be collected and maintained with special care: (1) in iced syringes and tubes, (2) using EDTA as a proteolysis inhibitor (angiotensinase), (3) separating the plasma in a refrigerated centrifuge no more than 30 min after each collection, and (4) separating and storing the plasma at −20 °C or less  until the time of the assay. As the PRA levels fluctuate in response to posture, diet, and hydration , reference values exist for the various conditions, but most laboratories require that the patient has fasted and rested lying down for at least 30 min before the collection of baseline samples .A commercial kit  will be used under controlled conditions to measure the total nitrite and nitrate concentrations in the biological fluids and by-products of the production of NO using the NO synthase enzyme. Nitrites and nitrates are stable end products of the NO reaction with molecular oxygen. The proportions of nitrates and nitrites are variable and cannot be accurately determined. Therefore, the best indicator of NO production is the sum of the nitrites and nitrates. The Cayman Chemical kit used to determine nitrites and nitrates provides an accurate and convenient method for measuring the total concentrations through a simple two-step process. This test is based on the determination of nitrite, not of nitrate, using the Griess reaction. The method is very simple, and a large number of samples can be processed in a short time, making it quite suitable for routine application in research laboratories. Plasma samples, collected in an EDTA anticoagulant, are first diluted four times using distilled water and de-proteinized by adding 1/20 volume of zinc sulfate (300 g/L), giving a concentration of 15 g/L. After centrifugation at 10,000 g for 5 min or 1000 g for 15 min at ambient temperature, 100 μL of Griess reagent  is added. After 10 min incubation at room temperature, the sample develops a violet color, the intensity of which is measured in a plate reader using a 540-nm wavelength . Each sa2 plasma levels (metabolite of TXA2) are determined using a commercial enzyme immunoassay kit . This assay uses 500 μL of venous blood plasma and is based on the competition between TXB2 and a TXB2 tracer (TXB2 attached to an acetylcholinesterase molecule) with a limited number of TXB2-specific rabbit antiserum binding sites. The concentration of the tracer is held constant, while the free TXB2 changes. The TXB2 antiserum complex (free tracer) binds to a rabbit monoclonal antibody previously attached to the plate, which is rinsed to remove unwanted reagents, and Ellman’s reagent is added (which contains the substrate for acetylcholinesterase). The product of this enzymatic reaction has a yellow color and absorbs 420-nm wavelength light in a spectrophotometer [Thromboxane B2 will be evaluated in blood samples collected in test tubes containing EDTA. The plasma is separated by centrifugation and stored at −20 °C until testing. The plasma samples are purified using a reversed-phase column , and the TXBotometer , 79.The primary outcome measure will be the change from baseline in the peripheral blood pressure of individuals with resistant hypertension after transcutaneous electrical nervous stimulation.The secondary outcome measures from baseline are the arterial stiffness measurement parameters  of individuals with resistant hypertension after transcutaneous electrical nervous stimulation.Blood pressure (mean of two measurements by an automatic electronic device Omron HEM-711 DLX) and hemodynamic parameters (by Omron HEM 9000 AI device) will be measured in the office at the follow-up visits.The adverse events will be investigated by open questions and by a semistructured questionnaire, which will address general symptoms and the presumed adverse effects of the electrostimulation used in the trial. Laboratory adverse events, such as metabolic changes and inflammatory markers, will be investigated at the final visit of the participants.Participants will be registered with a phone number and address for further contact in case of missing outlined visits.http://www.lee.dante.br/pesquisa/amostragem/amostra.html was used to estimate the sample size. The sample size calculation with a P alpha of 0.05, statistical power of 80 %, standard deviation of 8 mmHg, and maximum acceptable absolute difference of 6 mmHg (diastolic BP), indicated the necessity to study 28 patients per group (intervention versus sham). However, considering a potential 10–15 % dropout or loss to follow-up rate, 32 will be considered for each group. The difference of 5 mmHg (diastolic) has been achieved, on average, in clinical trials that have demonstrated the advantage of a drug over placebo or other nonpharmacological treatment in the prevention of major cardiovascular outcomes.The site t test or the Mann–Whitney test for comparison of continuous variables. The Chi-square test or Fisher’s exact test will be employed to compare categorical variables. The change from baseline to 4-week follow-up in the groups will be evaluated using the paired t test for continuous variables. Pearson’s correlation will be used to assess the relation between BP reduction and treatment (intervention). For all analyses, a two-sided P < 0.05 will be considered statistically significant.All analyses will be performed using SPSS Statistical Software . Continuous variables will be presented as mean ± SD, and categorical variables, as frequencies. Differences between the groups at baseline will be evaluated by an unpaired Over the decades, RH has become very common and costly. Adequate control requires several drugs, and in many cases, treatment is not successful. This is due to target organ damage or even an autonomic imbalance. Studies have shown that sympathetic nervous system inhibition by both renal denervation and by central inhibition have significant effects in reducing BP; however, these treatments are costly and invasive. Moreover, other studies have shown that sympathetic nervous system inhibition can also be noninvasively achieved by electric current. Even so, the effects of electrical stimulation on the metabolic system still need to be elucidated. Additional file The randomization of patients has begun.


Neurodevelopmental disabilities persist in survivors of neonatal hypoxic-ischemic encephalopathy (HIE) despite treatment with therapeutic hypothermia. Cerebrovascular autoregulation, the mechanism that maintains cerebral perfusion during changes in blood pressure, may influence outcomes. Our objective was to describe the relationship between acute autoregulatory vasoreactivity during treatment and neurodevelopmental outcomes at 2 years of age.OPT) at which autoregulatory vasoreactivity is greatest. Cognitive and motor neurodevelopmental evaluations were completed in 19 children at 21–32 months of age. MAPOPT, blood pressure in relation to MAPOPT, blood pressure below gestational age + 5 (ga + 5), and regional cerebral oximetry (rSO2) were compared to the neurodevelopmental outcomes.In a pilot study of 28 neonates with HIE, we measured cerebral autoregulatory vasoreactivity with the hemoglobin volume index (HVx) during therapeutic hypothermia, rewarming, and the first 6 h of normothermia. The HVx, which is derived from near-infrared spectroscopy, was used to identify the individual optimal mean arterial blood pressure  contains supplementary material, which is available to authorized users. Neonatal hypoxic-ischemic encephalopathy (HIE) affects approximately 3 in 1000 births and is the most common cause of perinatal brain injury in full-term neonates . Long-teOPT). The hemoglobin volume index (HVx) monitors autoregulatory vasoreactivity by correlating changes in arterial blood pressure to changes in relative total tissue hemoglobin (rTHb), a surrogate measure of cerebral blood volume obtained by near-infrared spectroscopy (NIRS). HVx is based on the premise that autoregulatory vasodilation and vasoconstriction induce changes in cerebral blood volume that are proportional to changes in rTHb [OPT in neonates with HIE [OPT during rewarming is associated with greater brain injury on MRI in pilot studies of autoregulation during HIE [2) or maintain blood pressures at gestational age in weeks +5 mmHg (ga + 5) to help guide hemodynamic goals in neonates [Dysregulated cerebral blood flow may be a key component in secondary neurologic injury in HIE . Cerebro in rTHb . HVx canwith HIE , 10. We ring HIE , 10. Howneonates .OPT would be associated with neurodevelopmental disabilities; 2) the rSO2 would not be associated with disability; and 3) greater time spent with blood pressure below the ga + 5 would not be associated with disability. We tested each of these hypotheses by comparing autoregulation measurements made during hypothermia, rewarming, and the first 6 h of normothermia to neurodevelopmental outcomes of children 2 years later.The goal of this observational pilot study was to describe the relationship between blood pressure autoregulation during therapeutic hypothermia for treatment of neonatal HIE and cognitive and motor neurodevelopmental outcomes at approximately 2 years of age. We hypothesized that 1) greater blood pressure deviation below MAPThis study was approved by the Johns Hopkins University  Institutional Review Board. Written, informed consent for HVx monitoring was obtained from the neonates’ parents upon admission to the JHU neonatal intensive care unit (NICU) and again before the 2-year neurodevelopmental follow-up evaluations, which took place at the Kennedy Krieger Institute . All neonates who were admitted to the NICU between September 2010 and October 2012 were screened for study eligibility, which was based on the diagnosis of HIE according to criteria used by the NICHD Neonatal Research Network’s clinical trial of hypothermia in neonatal HIE .2, as measured by the NIRS, and blood pressure, as measured by continuous cardio-respiratory monitors, but they were blinded to HVx. Respiratory support parameters, including nasal cannula, high flow nasal cannula, or ventilator support with endotracheal tube were recorded during the rewarming period. Clinical histories and clinical variables were obtained by chart reviews.All clinical care was determined by the treating clinicians and by NICU protocol. Neonates received whole-body hypothermia with a cooling blanket  to a goal rectal temperature of 33.5 ± 0.5 °C for 72 h. They were rewarmed over 6 h  to normothermia (36.5 °C). The clinicians determined the hemodynamic goals, decided when to implement vasoactive or inotropic medications, and selected the sedation regimens. When vasoactive medications were needed, dopamine was initiated followed by dobutamine, epinephrine, or milrinone infusions as necessary. Morphine, fentanyl, or hydromorphone boluses and infusions were used for sedation as necessary. Full montage electroencephalograms (EEGs) were conducted during hypothermia and after rewarming in addition to continuous amplitude-integrated EEG monitoring  during hypothermia, rewarming, and the first 6 h of normothermia. Phenobarbital was administered to treat electrographic or clinical seizures; thereafter, levetiracetam, fosphenytoin, or topiramate was used for persistent seizures. Clinicians could view the rSOAdhesive, neonatal cerebral oximetry probes were placed bilaterally on the neonates’ foreheads and connected to an INVOS 5100 NIRS machine  according to manufacturer guidelines. We synchronously sampled the NIRS signals and arterial blood pressure from the patient hemodynamic monitor at 100 Hz and processed the data with ICM+ software  using a bedside computer. The ICM+ software calculated HVx using a continuous, moving correlation coefficient between MAP and the rTHb  after filtering out high-frequency waves from pulse and respiration , 13. EacOPT in each time period  as the bin that had the most negative HVx when the bar graph exhibited an overall trend of increasing HVx values as MAP deviated from this nadir  We identified the MAPdir Fig. . When a OPT Fig. . These vOPT and analyzed this as a percentage of the autoregulation monitoring period. Second, we determined the maximal blood pressure deviation below or above MAPOPT [OPT and the amount of time spent with blood pressure below MAPOPT. We analyzed time as the absolute duration of autoregulation monitoring to determine the AUC. The AUC (min•mmHg/h) was calculated as time (minutes) spent with blood pressure below MAPOPT and blood pressure deviation (mmHg) below MAPOPT, and then normalized for the duration of monitoring (hours) [2 using the mean between right and left cerebral hemispheres.Blood pressure data were analyzed by three methods within each of the three time periods. First, we calculated the amount of time the neonate spent with blood pressure below, at, or above MAPe MAPOPT , 10. ThiWhen the children were 21–32 months of age, they were evaluated for neurodevelopmental function in a single visit at KKI during a routinely scheduled clinical visit or a one-time research visit. Clinical visits were part of routine and regularly scheduled care in the KKI NICU follow-up clinic and included a neurologic exam, administration of the Capute Scales , 15 compp < 0.05. Neurodevelopmental outcomes were dichotomized into impaired or unimpaired, and the Mullen Early Learning Composite scores were analyzed as a continuous variable. MAPOPT values and the percentage of time spent with blood pressure below MAPOPT during each period  were compared by using Wilcoxon signed rank tests. Blood pressure, MAPOPT, and rSO2 data with respect to neurodevelopmental outcomes were tested separately within each time period. MAPOPT; the percentage of time spent with blood pressure below, at, or above MAPOPT; the maximal blood pressure deviation below or above MAPOPT; AUC; the percentage of time spent with blood pressure below ga + 5; and rSO2 were compared between children with and without impairments by using Mann Whitney rank sum tests. MAPOPT, blood pressure data in relation to MAPOPT and ga + 5, and rSO2 were compared to Mullen scores by using Spearman correlations. Seizure activity and the receipt of a vasopressor  were compared between children with and without impairments by using Fisher exact tests.Data were analyzed with SigmaPlot  and SAS v9.2 . Graphs were generated with GraphPad Prism . We present the data as means with standard deviations (SD) or medians with interquartile ranges (IQR) when appropriate. Differences were considered significant at n = 19; SD = 1.5). During the autoregulation monitoring period, 11 (58 %) neonates had clinical or electrographic seizures that were treated with phenobarbital. Four of these neonates received additional antiepileptic therapy, including levetiracetam, fosphenytoin, lorazepam, or topiramate for persistent seizures. Thirteen (68 %) neonates received vasopressors during HVx monitoring, including 13 with dopamine, four with dobutamine, and one with epinephrine. Morphine infusions were administered to four neonates, and a hydromorphone infusion was given to one neonate. Fourteen neonates were intubated for synchronized intermitted mandatory ventilation (13) or high frequency jet ventilation (1). Seven neonates received nasal continuous positive airway pressure or high-flow nasal cannula respiratory support. During the rewarming period, four intubated neonates had adjustments to their ventilator respiratory rate (range of increase in respiratory rate: 5–14 breaths/min) or peak inspiratory pressure (range of change in peak inspiratory pressure: 1–6 cmH2O). One neonate had inhaled nitric oxide initiated during rewarming. Nine neonates had adjustments made in the inhaled oxygen concentration delivered through nasal cannula (2), high flow nasal cannula (1), or endotracheal tube . No patients received extracorporeal membrane oxygenation (ECMO). Clinical data upon admission to the NICU and physiologic and laboratory data are presented in Tables Twenty-eight neonates with HIE received therapeutic hypothermia and had HVx monitoring. Nineteen of those children participated in neurodevelopmental follow-up examinations at 21–32 months of age. Therefore, data are presented for 19 children . Their mean gestational age was 38.9 weeks . GMFM scores  were similar to those of 2–4-year-old children with cerebral palsy who can walk without assistance  [n = 4; mean = 86.00; SD = 51.61). Overall, 11 (58 %) children had typical performance or mild delays in neurodevelopment based on cognitive performance in the average range and the ability to walk without limitations. These 11 children were coded as having an unimpaired neurodevelopmental outcome for the analysis. Eight (42 %) children had more significant delays based on below-average cognitive performance or the inability to walk without limitations. These eight children were coded as having an impaired neurodevelopmental outcome for the analysis. Seizures or the receipt of a vasopressor were not associated with having an impaired neurodevelopmental outcome .Nineteen children had neurodevelopmental outcomes evaluated at a median age of 25 months . Fifteen children were evaluated in research visits. Because one of those participants did not complete the GMFM, the GMFC level was determined by using the Mullen Gross Motor performance and clinical judgment. Four children had clinical evaluations. Children who participated in the full research battery had a mean performance on the Mullen Early Learning Composite within the normal range (OPT was identified in 15/19 (79 %) neonates during hypothermia, 17/17 (100 %) during rewarming, and 14/16 (88 %) during the first 6 h of normothermia. HVx was monitored for a median of 30.5 h  during hypothermia, 6.5 h  during rewarming, and 6 h  during normothermia. MAPOPT ranged from 35 to 65 mmHg, with the majority of MAPOPT values between 45 and 55 mmHg. Values for MAPOPT were similar during hypothermia, rewarming, and the first 6 h of normothermia . One neonate did not receive HVx monitoring during normothermia because the arterial blood pressure cannula was removed. MAP5 h IQR, 2.4–46.5 OPT was similar between time periods. More specifically, neonates spent a median of 6 %  of the hypothermia period and 41 %  of the rewarming period with blood pressure below MAPOPT (p = 0.119). Neonates spent a median of 31 %  of the normothermia period with blood pressure below MAPOPT .The MAP ranged from 30 to 70 mmHg but remained between 40 and 60 mmHg most of the time  than in those who were unimpaired  and those without impairments during hypothermia  and during normothermia .Values for MAPOPT during the neonatal rewarming period. Children who developed impairments (n = 8) spent a greater percentage of time with blood pressure below MAPOPT than did children without impairments  had greater maximal blood pressure deviation below MAPOPT . No associations were identified between impairment at 2 years and the percentage of time spent with blood pressure below MAPOPT, maximal blood pressure deviation below MAPOPT, or AUC during hypothermia and normothermia  spent a smaller percentage of the rewarming period with blood pressure above MAPOPT . This association was not present for hypothermia or normothermia . No correlations were identified between the Mullen score and duration of time with blood pressure above or below MAPOPT or blood pressure deviation from MAPOPT during hypothermia or normothermia , the mean rSO2 in any period  was not associated with future impairment or Mullen score (p > 0.10 for all comparisons; Additional file p > 0.10 for all comparisons; Additional file When all children were analyzed (including those with an unidentifiable MAPOPT during rewarming was associated with outcome. Children with impairments at approximately 2 years of age had significantly higher MAPOPT values during the neonatal rewarming period than did children without impairments. Neurodevelopmental impairment in children was associated with more time spent at blood pressure below MAPOPT, having greater maximal blood pressure deviation below MAPOPT, and having greater AUC below MAPOPT during the rewarming period. Children without impairments spent more time with blood pressure above MAPOPT and had greater blood pressure deviation above MAPOPT during rewarming than did those with impairments. Furthermore, higher Mullen scores at 2 years significantly correlated with neonates spending more time with blood pressure above MAPOPT and having greater blood pressure deviation above MAPOPT during rewarming. An association was observed only between neurodevelopmental outcome and blood pressure in relation to MAPOPT during rewarming; no association was apparent between outcome and blood pressure during hypothermia or normothermia. Finally, neither the rSO2 nor time spent with blood pressure below ga + 5 during hypothermia, rewarming, or normothermia was associated with neurodevelopmental outcome. Although a causal relationship between blood pressure autoregulation and neurodevelopmental outcomes cannot be determined in this small, observational pilot study, our findings reveal an association between better neurodevelopmental outcomes and having blood pressures that remain within or above MAPOPT during rewarming. They further suggest that identifying each individual neonate’s MAPOPT with HVx may serve as a better method than rSO2 alone or rules based on gestational age to select blood pressure goals.Several findings relevant to the treatment of neonatal HIE are suggested by this observational pilot study. The range of MAP with optimized cerebrovascular autoregulatory vasoreactivity may be identified by using HVx. Further, deviation from MAPAlthough the overall performance of the 19 children evaluated at 2 years was in the average range, the mean cognitive scores were lower than those in normative samples , 6, and Cerebral NIRS is often used to monitor neonates with HIE during therapeutic hypothermia , 20–23 bOPT at which autoregulation is most functional.We used the autoregulation metric HVx, which incorporates measures of both oxygenated and deoxygenated hemoglobin. Therefore, HVx should be minimally affected by parameters that change tissue oxygen extraction or supply, including temperature and metabolic demand. This method may enable clinicians to develop an individualized approach for neonates with HIE by identifying and aiming for the MAPOPT in neonates by using HVx showed that MAPOPT values vary among individuals. Children who developed impairments had significantly higher MAPOPT during rewarming from hypothermia than did children who did not develop impairments. Intracranial hypertension raises the limits of blood pressure autoregulation [OPT. Identifying MAPOPT would be particularly critical in these neonates to clarify hemodynamic goals that support autoregulation.Our ability to identify MAPgulation . It is pgulation , which cOPT during rewarming. Children with impairments spent more time with blood pressure below MAPOPT, had greater maximal blood pressure deviation below MAPOPT, and had greater AUC below MAPOPT during rewarming than did those without impairments. Greater maximal blood pressure deviation below MAPOPT correlated with a lower Mullen Early Learning Composite score. Likewise, having blood pressure that remained above MAPOPT during rewarming was associated with less impairment. Children without impairments spent a greater proportion of the rewarming period with blood pressure above MAPOPT and had greater blood pressure deviation above MAPOPT than did children with impairments. Moreover, more time with blood pressure above MAPOPT and greater maximal blood pressure deviation above MAPOPT during rewarming correlated with a higher Mullen Early Learning Composite score.We also found an association between neurodevelopmental impairment and blood pressure deviation from MAPOPT during hypothermia or normothermia. The percentages of time that neonates spent with blood pressure below MAPOPT were similar in the hypothermia, rewarming, and normothermia periods. Several possibilities might explain the association between blood pressure deviation from MAPOPT during rewarming and neurodevelopmental outcomes. The inherent risk of secondary neuronal injury may be highest during rewarming. Additionally, severely injured neonates may have less stable cardiovascular regulation and diminished autoregulatory capacity during rewarming. We previously reported that spending more time and having greater blood pressure deviation below MAPOPT during rewarming were associated with greater brain injury on MRI [OPT during rewarming in severely injured neonates. Rewarming itself might adversely affect cerebral blood flow autoregulation and increase the risk of stroke [There were no associations between the 2-year neurodevelopmental outcomes and blood pressure deviation from MAPy on MRI , 10. Thif stroke . Intracrf stroke . Moreovef stroke . Other nf stroke .2, or sedation which might affect cerebral blood flow and potentially confound the interpretation of HVx. Neonates did not have any clinical changes during rewarming that would acutely change their hemoglobin level, such as blood transfusion or hemorrhage. Vasoreactivity is affected by changes in CO2 production [Given the small sample size in this pilot study, we were unable to control for hemoglobin level, PaCOoduction , includioduction , 10, 13.The effects of changing ventilatory support during rewarming on HVx are unclear. Changes in intrathoracic pressure can affect cerebral perfusion pressure  and osci2 and blood pressure based on ga + 5 were not associated with neurodevelopmental outcomes in this study. The “normal” MAP for a neonate is often assumed to be the neonate’s ga + 5 [2 or rules based on gestational age.Regional SOs ga + 5 , a valueOPT might improve outcome, a cause and effect relationship between blood pressure autoregulation and neurodevelopmental outcome cannot yet be determined in this observational pilot study. The risks of raising a neonate’s blood pressure must be considered. While targeting the optimal cerebral perfusion pressure to support autoregulation has not yet been explored in neonates with HIE, it is being evaluated in adult traumatic brain injury [While the data suggest that maintaining a patient’s blood pressure near MAPn injury .OPT during cardiopulmonary bypass [Given the small sample size of this pilot study, we were unable to control for potential confounders such as gender, socioeconomic status and access to therapy services that might affect neurodevelopmental outcome. There are factors in addition to hemodynamic management that may correlate with neurodevelopmental outcomes in HIE, including abnormal EEG and brain imaging  and non-y bypass .OPT varied among individual neonates and was higher during rewarming in children who developed impairments than in those who did not. Blood pressure deviation below MAPOPT during rewarming was associated with greater impairment and lower cognitive scores. Although future studies are warranted, our pilot data suggest that individualizing blood pressure goals based on MAPOPT, especially during the rewarming period, may be superior to rSO2 alone or blood pressure goals based on gestational age to support autoregulation and improve neurodevelopmental outcomes.In this observational pilot study of neonatal HIE and therapeutic hypothermia, we used HVx monitoring to identify associations between cerebrovascular autoregulatory vasoreactivity during the neonatal rewarming period and 2-year neurodevelopmental outcomes. MAP


Asthma is the most common chronic disease of childhood and a leading cause of childhood morbidity. The aim of the current study was to assess the effectiveness of montelukast administered as monotherapy or in combination with current inhaled corticosteroids (ICS) in pediatric patients with uncontrolled asthma as per the Canadian Asthma Consensus Guidelines.Twelve-week, multicentre, open-label, observational study. Primary effectiveness outcome was the proportion of patients achieving asthma control (Asthma Control Questionnaire (ACQ) score ≤0.75) at weeks 4 and 12.A total of 328 patients with uncontrolled asthma (ACQ > 0.75) were enrolled with mean ± SD age of 6.9 ± 3.4 years. Among these, 76 (23.2%) were treated with montelukast monotherapy and 252 (76.8%) with montelukast combined with ICS. By 4 weeks of treatment 61.3% and 52.9% of the patients in the monotherapy and combination group, respectively, achieved asthma control. These proportions increased to 75.0% and 70.9%, respectively, at 12 weeks. Within the monotherapy group, clinically significant improvements in the ACQ score  and the PACQLQ score  were observed. In the combination group, the mean ± SD ACQ score significantly improved from 2.02 ± 0.83 at baseline to 0.90 ± 0.86 at 4 weeks and 0.64 ± 0.86 at 12 weeks (p < 0.001), while the PACQLQ score improved from 4.42 ± 1.35 at baseline to 5.76 ± 1.30 at 4 weeks and 6.21 ± 1.03 at 12 weeks (p < 0.001). After a 12-week montelukast add-on therapy, 22.6% of patients reduced their ICS dosage. Similar results were observed among preschool- and school-aged patients.Montelukast as monotherapy or in combination with ICS represents an effective treatment strategy for achieving asthma control in pediatric patients and improving caregivers’ quality of life.NCT00832455.This study is registered at ClinicalTrial.gov:  Asthma is a chronic inflammatory disorder of the airways with a heterogeneous target age group and an initial diagnosis age of as early as infancy. Its prevalence, especially among children, is increasing worldwide ,2, incluCurrent asthma treatment guidelines recognize the importance of early and aggressive intervention for asthma and recommend low-dose inhaled corticosteroids (ICSs) as first-line treatment in childhood ,5-8. HowLeukotriene receptor antagonists (LTRAs), such as montelukast, provide an alternative treatment for asthma patients who are not controlled or satisfied with ICS therapy ,5-7,12. Although results from controlled randomized clinical trials have provided evidence of the montelukast efficacy in the treatment of asthmatic children ,14, contComité central de l’éthique de la recherche du Ministère de la santé et des services sociaux du Québec, Montréal, Québec), and was conducted in accordance with ICH Good Clinical Practice Guidelines, the World Medical Association Declaration of Helsinki and all applicable local regulations.This was a 12-week, open-label, multicenter, prospective study conducted in 58 Canadian clinics between June 2006 and October 2008. Patients were treated with montelukast sodium for 12 weeks, either as a monotherapy or in combination with their current ICS treatment. Clinical assessments were conducted at baseline, 4 and 12 weeks of treatment at the clinics of their treating physicians. During the course of the study, tapering of ICS dosage was performed at the discretion of the treating physician and on an individual basis when asthma control was achieved. An optional visit after 8 weeks of treatment was performed to determine if an ICS dosage adjustment was necessary and to assess asthma control of patients previously tapered. Parents or legal guardians provided written informed consent prior to the participation of their children in this study. The study was approved by three independent Ethics Review Boards  on an as-needed basis or using an ICS at any dosage. In addition, one of the following conditions had to be satisfied: i) the physician and/or patient was dissatisfied with the current controller therapy; ii) the patient was reluctant to take ICS therapy, or; iii) the patient was insufficiently controlled with the current therapy through the preceding 6 weeks. Finally, eligible patients had to have uncontrolled asthma as per the 2003 Canadian Asthma Consensus Guidelines [Eligible patients were between 2 and 14 years of age and had been diagnosed with asthma for at least 6 months. In order to be included in the study, patients had to have a peak expiratory Flow (PEF) ≥ 80% of the predicted value (applicable only for patients older than 7 years old) and they had to be either currently untreated, using a short-acting β2-agonist (LABA) alone or in a combination product, oral prednisone, regular use of theophylline and/or other asthma medications such as sodium cromoglycate or nedocromil. Patients using an antibiotic for respiratory tract infection at the time of entry into the study or treated with an antibiotic for respiratory tract infection (initiation of antibiotic treatment was permitted during the study) within 30 days were also excluded. A history of cystic fibrosis, immune deficiency requiring specific therapy or any other disease that could influence the evolution of asthma was also a reason for exclusion. Finally, patients with a history of hypersensitivity to any component of montelukast were excluded.Patients were excluded if their asthma symptoms were controlled and if they were treated with montelukast or any of the following treatments at the time of entry into the study: long-acting βConsidering that the primary outcome measure was the proportion of patients achieving asthma control based on the ACQ criteria (ACQ ≤ 0.75), a re-analysis of the data was conducted including only patients with ACQ > 0.75 at baseline the results of which are reported here.2-agonist (SABA) as rescue medication was allowed during the study, but patients were asked to refrain from its utilization for 6 hours prior each study visit.All patients were treated with montelukast sodium  taken once-daily at bedtime as monotherapy or in addition to their current ICS therapy. Patients aged between 6 and 14 years were treated with 5 mg montelukast sodium chewable tablets, while patients between 2 and less than 6 years of age were treated with 4 mg montelukast chewable tablets. The 4 mg granule formulation was also available for the latter age group on demand. The use of a short-acting βThe primary effectiveness outcome measures was the proportion of patients achieving asthma control, defined as a score ≤ 0.75  in the sCompliance with the study medication was assessed by tablet counts, as recorded in the study worksheets. Safety and tolerability were assessed with the incidence of treatment-emergent adverse events, which were coded and reported according to the MedDRA dictionary of terms, version 9.0 .Descriptive statistics were produced for patient demographics and characteristics at baseline. Comparisons between baseline and follow-up visits were performed with the matched Chi-Square test for categorical scales and the paired Student’s t-test for continuous scales. Two-tailed tests were performed using a significance level (α) of 0.05. Subgroup analyses by treatment strategy and stratified analyses for preschool-aged children (less than 6 years old) and school-aged children (6 years of age or older) were performed. There were no imputations for missing data. All analyses were performed using the SPSS version 12.0 for Windows .A total of 420 patients with uncontrolled asthma as per the 2003 Canadian Asthma Consensus Guidelines completed the baseline assessment of whom 92 (21.9%) had an ACQ score of ≤ 0.75 at baseline. Considering that the primary outcome measure was the proportion of patients achieving asthma control based on the ACQ criteria, only the 328 patients with ACQ > 0.75 were included in this analysis. Among these, 320 (97.6%) and 288 (87.8%) patients completed the 4- and 12-week assessment, respectively, while the optional 8-week assessment was performed on 197 (60.1%) patients. There were 40 (12.2%) patients who were discontinued from the study: 10 (3.0%) due to an adverse event, 8 (2.4%) withdrew consent, 9 (2.7%) were lost to follow-up, 4 (1.2%) due to protocol violation, 8 (2.4%) discontinued for other reasons, while the reason of discontinuation was missing for 1 (0.3%) patient.The demographics and baseline characteristics of the study population are summarized in Table Table The mean (SD) ACQ score of the total study sample decreased from 1.94 (0.82) at baseline to 0.85 0.83) at 4 weeks and 0.61 (0.79) at 12 weeks of treatment, representing statistically and clinically significant absolute mean (SD) changes of −1.08 (1.00) and −1.34 (1.03) from baseline to 4 and 12 weeks, respectively (p < 0.001)  PACQLQ score of the total study sample increased from 4.63 (1.36) at baseline to 5.89 1.24) at 4 weeks (mean (SD) change = 1.55 (1.40); P < 0.001) and 6.28 (1.00) at 12 weeks (mean (SD) change = 1.82 (1.30); P < 0.001). Among the patients who adopted the montelukast monotherapy treatment strategy, the mean (SD) PACQLQ score increased from 5.34 (1.14) at baseline to 6.32 (0.89) at 4 weeks and 6.51 (0.85) at 12 weeks. The absolute mean (SD) change in PACQLQ of 0.98 (1.12) at 4 weeks and 1.13 (1.04) at 12 weeks was both clinically (change in PACQLQ > 0.7) and statistically significant (p < 0.001)  reported by 112 (34.1%) patients. Of these, 157 (86.3%) were probably or definitely not related to study medication and 15 led to study drug discontinuation in 12 (3.7%) patients. There were 25 (13.7%) NSAEs possibly, probably or definitely related to montelukast. Of these, the most frequent NSAEs related to montelukast were nightmares and sleep terror (n = 6), abdominal pain (n = 5), insomnia (n = 2) and headache (n = 2). A total of 3 serious adverse events (SAEs) were experienced by 3 patients: 1 asthma episode, 1 bronchitis and 1 pneumonia, none of which were judged to be related to the study medication by the treating physicians.Although results from controlled randomized clinical trials indicate that montelukast is efficacious in the treatment of asthmatic children ,14, contThe results of this 12-week multicenter observational study support the therapeutic effectiveness of montelukast in pediatric patients with uncontrolled asthma, in a clinical setting emulating real-life. Asthma control was achieved by the majority of patients who received montelukast either as monotherapy or in combination with ICS treatment for 12 weeks. Furthermore, clinically and statistically significant decreases in ACQ scores were observed after 4 and 12 weeks of treatment with montelukast mono- and add-on therapies, among both preschool and school-aged patients.Although cross-study comparisons are difficult due to differences in study designs and diversity in efficacy outcomes, the results of the current study are consistent with the efficacy profiles of montelukast in childhood asthma that were previously reported in systematic reviews and randomized clinical trials conducted in preschool ,13,21,22Asthma is the most common chronic disease of childhood and a leading cause of childhood morbidity. In addition to considerably affecting children’s physical, emotional and social lives, uncontrolled asthma also directly correlates with a loss of productivity and quality of life of the children’s caregivers ,32. TherThe results of this study indicate that both asthmatic children and their caregivers can benefit from montelukast therapy since it is an effective treating option enabling asthma control, while significantly improving the caregivers’ quality of life. After 12 weeks of treatment with montelukast administered as monotherapy or in combination with ICS, clinically (mean change of ≥ 0.7 in PACQLQ score) and statistically (p < 0.001) significant improvements in caregivers’ quality of life were observed with mean (SD) changes in PACQLQ score of 1.25 (1.30) and 1.63 (1.32) from baseline, respectively.Furthermore, the vast majority of the patients and physicians were satisfied or very satisfied with montelukast. This high level of satisfaction can be probably attributed to the observed effectiveness of montelukast in controlling asthma symptoms and improving caregivers’ quality of life, the ease of medication administration which enhances treatment compliance, and the safety and tolerability profile of montelukast.et al.[et al.[et al.[The results of previous randomized clinical trials conducted with adult asthmatic patients suggested that montelukast could facilitate a reduction in ICS use ,34. Howeet al. reportedl.[et al. have shol.[et al. reportedOverall, once-daily administration of montelukast for 12 weeks was well tolerated in the context of this study. The observed safety and tolerability results are consistent with the safety profile of montelukast previously reported in asthma childhood -26,37-39Potential limitations of the current study are related to the open-label and single cohort design without a parallel control group. However, since this study emulated the real-world clinical setting, blinding to the treatment used and comparison to a control group were not appropriate. Furthermore, the primary objective of the study was to assess the real-life effectiveness of treatment with montelukast in achieving asthma control and not the comparison of montelukast treatment with alternate treatment strategies. By conducting within- instead of between-group comparison, possible confounding bias related to disease and lifestyle factors that could affect the effectiveness of montelukast were minimized since each patient provided both control (pre-treatment) and on-treatment data. In light of the heterogeneous response documented for both ICS ,40 and lAn important strength of this study is the generalizability of its results to the Canadian target population. Since this study was conducted in a real-life clinical setting, inclusion and exclusion criteria were less selective and therefore more representative of the general population compared to the highly controlled environment of clinical trials. In addition, as recommended by the Global Initiative for Asthma (GINA), the current study focused on asthma control achievement rather than on asthma severity ,5,8. TheIn conclusion, the results of this study indicate that montelukast, administered either as monotherapy or in combination with ICS treatment, is an effective, convenient and well tolerated therapeutic option for the management of asthma in preschool and school aged paediatric patients with uncontrolled asthma symptoms.ICS: Inhaled corticosteroids; LTRAs: Leukotriene receptor antagonists; PEF: Peak expiratory force; SABA: Short-acting β2-agonist; LABA: Long-acting β2-agonist; ACQ: Asthma Control Questionnaire; PACQLQ: Pediatric asthma caregivers quality of life questionnaire; GINA: Global initiative for asthma; SD: Standard deviation; NSAEs: Non-serious adverse events; SAEs: Serious adverse events.DB has received honorariums for consulting (Ad Board) and lecturing (CME) from Abbott, Altana, AstraZenaca, Graceway, GlaxoSmithKline, Merck, Novartis, Nycomed and Pfizer. MD is an employee of Merck Canada Inc. JSS is an employee of JSS Medical Research, the CRO contracted by Merck Canada Inc. to conduct the data management and analysis. AB has received research funding from CIHR, NSERC, AllerGen NCE and unrestricted educational grants from AZ, Graceway, GSK, Merck, Novartis and Nycomed.DB contributed to, interpretation, and the critical revision of the article. MD contributed to study design, interpretation, and the critical revision of the article. JSS contributed to study design, data analysis, interpretation, drafting the manuscript, and the critical revision of the article. AB contributed to data collection, interpretation, and the critical revision of the article. All authors have approved the current version of the manuscript.


PHOX2B gene in both girls. In this report we highlight the use of non-invasive bi-level positive airway pressure ventilators via tracheostomy, overnight end-tidal carbon dioxide trend graphs and outcomes of the patients whose diagnosis and treatment were delayed.Due to the economic downturn in Thailand, two baby girls with congenital central hypoventilation syndrome had to wait for several months to obtain definite diagnosis and long-term mechanical ventilation. Genetic investigation later revealed 20/25 polyalanine expansion of Case 1: A Thai baby girl showed symptoms of apnea and cyanosis from birth and required invasive mechanical ventilation via tracheostomy during sleep. At 5 months, she unfortunately was discharged from the hospital without any ventilatory support due to financial problems. She subsequently developed cor pulmonale, respiratory failure and generalized edema and was referred to us when she was 9-months old. An overnight polysomnogram was consistent with a central hypoventilation disorder, in which the severity of oxygen desaturation and hypercapnia was worsening during non-rapid eye movement compared to rapid eye movement sleep. At 12 months she was allowed to go home with a conventional home ventilator. The ventilator was changed to bi-level positive airway pressure when she was 4-years old. After she received adequate home ventilation, she thrived with normal growth and development.Case 2: A Thai baby girl developed apnea and cyanosis from the age of 5 weeks, requiring ventilatory support (on and off) for 5 months. After being extubated, she had been put on supplemental oxygen via nasal cannula for 2 months. She was then referred to us when she was 7-months old. An overnight end-tidal carbon dioxide trend graph revealed marked hypercapnia without increase in respiratory rate. An overnight polysomnogram was consistent with a central hypoventilation disorder. Since 9 months of age she has been on home bi-level positive airway pressure via tracheostomy without any complications.PHOX2B gene in both girls.Genetic testing confirmed 20/25 polyalanine expansions of Bi-level positive airway pressure, originally designed as a non-invasive ventilator, was found to work effectively and safely, and may be used as an invasive ventilator via tracheostomy in young children with congenital central hypoventilation syndrome. PHOX2B gene is requisite to establish the diagnosis [Congenital central hypoventilation syndrome (CCHS) is a rare disorder most commonly found in young children , charactiagnosis . Since aiagnosis .The levels of financial support for homecare for children on long-term mechanical ventilation are different across the world. In high income countries such as in North America and Europe, ventilators, accessories and home healthcare professionals are generously provided by either a national health system or medical insurance –7. HowevPHOX2B mutation with 20/25 polyalanine expansion. In this report we highlight the use of bi-level positive airway pressure (BPAP) ventilators via tracheostomy, clinical manifestations and complications when home mechanical ventilation could not be initiated, our diagnostic approach when genetic tests were not available and outcomes after the patients received adequate ventilation.Our case studies feature two babies from a low socioeconomic background clinically diagnosed with CCHS. They eventually received home ventilators by donation. Genetic testing later confirmed 1P0 mother, gestational age 38 weeks, birth weight 2500g. The baby was noted to have apnea and cyanosis since birth. At 10-days old, she was intubated due to frequent cyanosis and drowsiness. Six attempts of extubation failed due to apnea, hypopnea, cyanosis and hypercapnia. Tracheostomy was performed at 3 months of age. Ventilatory support was weaned to only at night time.A Thai baby girl was born by caesarian section due to polyhydramnios, to a 38-year-old GDue to financial problems, at 5-months old she unfortunately was discharged from the hospital without any ventilatory support. At home, during sleep she was noted to have intermittent cyanosis which occurred more often and worsened as time went by. The only thing her mother could do was to give tactile stimulation in order to wake her up.At 9-months old, she developed cor pulmonale, respiratory failure, generalized edema and was referred to us in 2009.2) was 95 to 98 % but when asleep SpO2 dropped to 58 to 60 %. Loud pulmonic valve closure (P2) and hepatomegaly were noted. Her hematocrit was 55 %. An electrocardiogram revealed right ventricular hypertrophy with strain pattern and right axis deviation.On admission, a physical examination revealed body weight of 6.8kg (3rd percentile for age), height 67cm (25th percentile for age), head circumference 43cm, respiratory rate (RR) 35, and heart rate (HR) at 90 per minute. When staying awake her oxygen saturation measured by pulse oximetry  1 L per minute (LPM). During sleep her RR was 40 per minute. There were frequent central hypopneas of 30.4 events per hour. Peak end-tidal carbon dioxide (EtCO2) was 76mmHg which seemed to worsen during non-rapid eye movement (REM) compared to REM sleep. The duration that EtCO2 level was greater than 50mmHg was prolonged . Nadir SpO2 was 52 % with prolonged desaturation. There were no significant changes in RR and HR in response to increased carbon dioxide (CO2) and decreased SpO2.An overnight polysomnogram was performed, which revealed that when she was awake her RR was 42 per minute, SpO2O with back up rate 44 per minute. At this setting SpO2 was maintained at 96 to 98% with peak EtCO2 47mmHg. No supplemental O2 was needed.Fortunately, a home ventilator  was donated to her when she was 1-year old. Optimal setting of the ventilator from the titration under polysomnography was as followed: assisted pressure controlled mode, peak pressure 16, positive end-expiratory pressure (PEEP) 3cmHHer mother had been trained at bedside to give care for the tracheostomy, gastrostomy and ventilator for 1 month at our hospital. The patient returned home eventually and was put on a ventilator via tracheostomy only during sleep.At the age of 4 years she grew to be healthy. Her weight, height and development were normal for a 4-year-old child. The first ventilator donated to her started to develop some problems 3 years after use. Fortunately, she received a second one also from a donation. During the 2-month wait for the second ventilator, her mother noted frequent cyanosis during sleep and more agitation during the daytime. Due to a limited budget, a bi-level device  was selected. The setting was adjusted under polysomnography yearly. The ventilatory back up rate was decreased as she grew up.2O, rate 20 per minute. She attended a regular school without any learning problems. Her weight and height were normal for her age. She demonstrated no clinical signs of cor pulmonale. Chest radiography and daytime arterial blood gases without ventilatory support were normal. An echocardiogram revealed improvement in pulmonary hypertension with estimated pulmonary artery pressure of 30 to 35mmHg.At 5 years of age, her BPAP setting was adjusted to inspiratory pressure 17, expiratory pressure 5cmH1P0 mother, gestational age 40 weeks, birth weight 4040g. She was found to have apnea and central cyanosis at 5-weeks old. SpO2 in room air was 80 %. She was then intubated and mechanically ventilated. An echocardiogram revealed dilated right atrium and right to left flow through foramen ovale. Extubation attempts repeatedly failed. After extubation, she could breathe spontaneously at the beginning; however, within 3 to 9 days she yet again developed respiratory failure which necessitated ventilatory support.A Thai baby girl was born by caesarean section to a 29-year-old G2 at the previous hospital revealed pH 7.18, partial pressure of carbon dioxide in arterial blood (PaCO2) 76, partial pressure of oxygen in arterial blood (PaO2) 110mmHg and bicarbonate (HCO3) 28.4mEq/L. The peak PaCO2 was sometimes increased to 200mmHg. She had been intubated in the first and second hospitals on and off for 5 months.During sleep, she was noted to breathe slowly with RR of 18 to 20 per minute and she developed apnea and desaturation. Arterial blood gases on one occasion when she received only supplemental OCCHS was suspected but not confirmed. She had been extubated for 2 months at the second hospital before getting transferred to us when she was 7-months old in 2010. Oxygen 0.5LPM was given to her only during sleep.2 0.5LPM during sleep. We recorded overnight EtCO2 and RR by using BCI Capnocheck®Plus . Microsoft Excel program was used to create a trend graph of EtCO2 and RR monitoring. The EtCO2 was above 50mmHg during most of her sleep time and peak EtCO2 was elevated to 87mmHg. RR did not change in response to elevated EtCO2  was donated to the patient. Her mother was trained by our nurses to be the caregiver. She had been on night time ventilatory support for more than 4 years without readmission. At 4 years of age, the BPAP setting was adjusted to inspiratory pressure 16, expiratory pressure 4cmHBoth patients were seen regularly at the pulmonary clinic. Polysomnographic studies were performed at least once a year to adjust ventilator settings that needed to change as the patients grew older.PHOX2B gene revealed 25 repetitions of polyalanine allele in addition to the normal allele with 20-alanine (GCG) residues, or designated as 20/25, in these two girls. The parents of both girls were found to carry only normal alleles. Primer sequences are as follows: forward 5′-CCAGGTCCCAATCCCAAC-3′ and reverse 5′-GAGCCCAGCCTTGTCCAG-3′.Genetic test using polymerase chain reaction (PCR) followed by direct sequencing of the exon 3 of the PHOX2B mutation. Both have the same mutation of 25 repetitions of polyalanine (20/25 genotype), which is the most common mutation of the PHOX2B gene [We present the case of two girls with CCHS confirmed by 2 which could reduce hypoxia severity. We created overnight EtCO2 and RR monitoring graphs by using Microsoft Excel program. The graphs demonstrated a prolonged period of hypercapnia without any ventilatory response indicating abnormal physiologic control of ventilation. In a place where polysomnography and genetic testing are not available, an overnight EtCO2 and RR trend graph might be another simple method of screening for central hypoventilation.The second patient did not demonstrate signs of cor pulmonale on admission to our hospital probably due to a less severe CCHS or earlier referral, and/or shorter period (only 2 months) of inadequate ventilation during sleep. She also had received night time supplemental OAs CCHS does not resolve spontaneously, nor does it appear to respond to pharmacologic stimulants or improve with advancing age, chronic ventilatory support at home has become a necessity that provides a chance for the patients to leave the hospital and lead a normal life at home with their family . However2  and expiratory positive airway pressure (EPAP). The tidal volume correlates with the difference between the IPAP and the EPAP. Back up rate was also set up in the timed mode of BPAP to guarantee breath delivery especially in small children who had relatively less effort to trigger the ventilator. Heated humidification systems were connected to the ventilator circuits. We attached an exhalation port in the single limb circuit closest to the tracheostomy in order to minimize rebreathing and prevent elevated CO2 Fig.  and 4. A2 administration, suctioning, ventilator management, as well as basic life support. The patients were discharged after they were clinically stable with unchanged ventilator settings. The capabilities of the mothers to handle problems of children with mechanical ventilators were tested and approved by our team. Both children with CCHS who came from low income families have lived happily with normal growth and development. We showed that the mothers, who are not medical professionals, could provide safe ventilator care at home which results in improving the quality of life for the family and decreasing the cost of care, as compared to hospitalization. The members of both families were motivated and they dedicated their time and effort to their children.Since home nursing care is impossible in our country, the mothers were trained to be caregivers . Their ePHOX2B mutation of 25 repetitions of polyalanine was identified. BPAP ventilators were donated to them. The girls’ mothers were trained to be home caregivers. We found that BPAP ventilators were safe to be used via tracheostomy as long-term home mechanical ventilators in young children with CCHS.In conclusion, we reported the cases of two underprivileged girls diagnosed with CCHS. Written informed consents were obtained from the parents of both patients for publication of this case report and accompanying images. A copy of the written consents is available for review by the Editor-in-Chief of this journal.


Intra-uterine growth restriction (IUGR) is present in about 3–10% of live-born newborns and it is as high as 20–30% in developing countries. Since the 1990s, it has been known that abnormalities during foetal growth may result in cardiovascular disease, including hypertension in adulthood.This study evaluated blood pressure parameters (using ambulatory blood pressure monitoring) in children aged six to 10 years old, born as small for gestational age (SGA), and compared them to their healthy peers born as appropriate for gestational age (AGA).p < 0.01). This relationship also occurred in association with the type of IUGR .In the SGA group, an abnormal blood pressure level (prehypertension or hypertension) was present significantly more often than in the AGA group (50 vs 16%, In SGA children, abnormal blood pressure values occurred more frequently than in AGA children. Intra-uterine growth restriction (IUGR) is an important issue for both neonatologists and paediatricians. It occurs in about 3–10% of live-born newborns. The most serious problem of IUGR exists in developing countries where it concerns up to 20–30% of liveborns.1In 1967, the American Academy of Paediatrics introduced nomenclature according to neonatal birth weight as follows: appropriate for gestational age (AGA), located between the 10th and 90th percentile; large for gestational age (LGA), above the 90th percentile; and small for gestational age (SGA), below the 10th percentile.There are two types of IUGR. The first, which accounts for approximately 20–25% of all cases, is called symmetrical IUGR. The disturbances occur in the first or second trimester of pregnancy, during organogenesis. There is a decrease in all dimensions of the foetus’s body and internal organs, usually accompanied by a permanent reduction in growth potential.3 (g/cm3)] in this type is lower than in symmetrical IUGR.3The second type is asymmetrical IUGR, constituting 75–80% of all cases of IUGR. This develops in the late second and third trimester of pregnancy and is the result of abnormal cell growth, rather than their quantity. In this type, infants have a low birth weight while other parameters remain normal . Due to this, Rohrer’s ponderal index  by centiles developed for the Polish population.12A family history of cardiovascular disease was taken, including the occurrence of hypertension. The nutritional status of all the children was assessed from their height, weight and body mass index  during the 24-hour period, (4) SBP load for the 24-hour period , (5) DBP load for the 24-hour period .et al.,According to recommendations developed by Urbina p < 0.05 level.Descriptive statistics were executed by computing the mean and standard deviation (SD) for scale variables, or frequencies for nominal variables. The significance level was computed for the differences between variables in the AGA and SGA groups. To evaluate the differences between the two groups, a parametric t-test and a non-parametric Mann-Whitney test were performed. The normality of scale variables was assessed using the Kolmogorov-Smirnov test. All tests were two sided and performed at the Pearson and Spearman correlation coefficients were computed to evaluate the degree of association between variables either for the control or study group. Statistical analysis was performed using Statistica 10 software  and a dedicated author’s software based on Microsoft SQL Server 2008 database management system.3) and asymmetrical (mean 1.75 ± 0.16 g/cm3) subgroups (p < 0.01).There were no statistically significant differences between the groups in the distributions of gender, age and anthropometric parameters  at the time of the study. Likewise, except for body measurements , Apgar score, and other perinatal parameters  did not differ statistically significantly r = 0.42, r < 0.01). An analysis was performed on the prevalence of risk factors of IUGR in children in the AGA and SGA groups, with an additional division into symmetrical and asymmetrical type.Among the children in the SGA group, the occurrence of one or more risk factors for IUGR was observed significantly more often, regardless of the type of IUGR (p < 0.01), and in the symmetrical subgroup, environmental factors such as smoking (p < 0.05) and alcohol consumption by the pregnant woman (p < 0.05).p < 0.05) but not in SBP .In oscillometric measurements of blood pressure, there was a statistically significant difference between the SGA and AGA groups in DBP . However, abnormal blood pressure (ABPM meeting the criteria for hypertension or prehypertension) was diagnosed significantly more often in the SGA group compared with the AGA group . This relationship also occurred in the asymmetrical  and symmetrical subgroups .Based on ABPM, hypertension was diagnosed in 18% of the children in the SGA group, while it was not found in any child in the AGA group (A significantly higher blood pressure load (both systolic and diastolic) was found in the SGA patients. When comparing symmetrical and asymmetrical subgroups with the AGA group, the values of blood pressure load were also statistically significantly higher p < 0.05), and MAPs during the daytime , compared with those of the AGA group.When analysing ABPM measurements more specifically, we found more significant results. Among children born with features of asymmetrical IUGR, there were higher mean SBPs during the daytime . With oscillometric measurement, DBP was significantly higher in the subgroup below the 5th percentile than in the AGA group  while SBP did not differ significantly.Patients from the SGA group with a birth weight less than the 5th percentile were subjected to a separate analysis. In this group of children, IUGR risk factors also appeared significantly more often than in the AGA group  , as well as a higher mean SBP during the daytime , higher mean DBP during the 24-hour period , and a higher MAP during the daytime  and during the 24-hour period .A significantly higher blood pressure load was also found in this group compared with children from the AGA group . Nevertheless, the combined frequency of hypertension together with prehypertension among the SGA children was compared with the group of children born with a birth weight ≤ 5th percentile and those between the 5th and 10th percentiles. There was no statistically significant difference in instance of abnormal blood pressure values found between these two groups.The analysis showed a statistically significant negative correlation between the occurrence of abnormal blood pressure and birth weight . The difference was not statistically significant. LV mass indices did not correlate significantly with abnormal blood pressure levels.The relationship between blood pressure and other birth parameters, i.e. body length, head circumference and ponderal index, was also examined. A study was conducted on the correlation of family history of hypertension and other cardiovascular diseases with blood pressure values. Factors such as gender, age of the child at the time of the study, and current weight, height and BMI were also analysed. None of these factors correlated significantly with frequency of abnormal blood pressure values.The results of our study indicate that there were significant differences in the incidence of abnormal blood pressure values between children born as SGA and AGA. Our research showed a higher frequency of high blood pressure values diagnosing prehypertension or hypertension in SGA patients, which is consistent with the reports of other investigators.19Incidentally our results correspond with reports of a prospective multi-centre study of 950 American children, of whom 28% were children with IUGR. The authors of that study found that in a group of six-year-old children with IUGR, hypertension occurred in 25% of them, whereas in only 16% of the control group.et al. is relevant. It was based on children 3–6 years old from China and North and South America . The authors found a relationship between higher blood pressure values and lower birth weight.20 There are many other studies in African countries where similar results were observed.Since IUGR affects particularly developing countries, the article by Law In another large, retrospective study, the Collaborative Perinatal Project, analysis of blood pressure in seven-year-old children was based on a single measurement. In that population of more than 2 600 children born with IUGR, the presence of higher blood pressure values was not confirmed compared to children born as eutrophic.et al. differed from ours despite similar methods and a similar age of the study group, although fewer children were included in the study. The authors did not observe a statistically significant higher incidence of abnormal blood pressure values among the IUGR children.It appears therefore that, especially in young children, 24-hour ABPM during ordinary activity of the child is a much more accurate assessment, as was done in our study. The only research available to us that evaluated the association between birth weight and blood pressure assessment using ABPM measurements was in a group of 39 children with IUGR.It has been proven that higher blood pressure values are not only the result of low birth weight but also too rapid weight gain within the first two years of life, which can lead to overweight and obesity.25In our research, there were no significant differences in body weight, height and BMI at the time of the study between the SGA and AGA children. Therefore intra-uterine growth retardation was an independent risk factor for abnormal blood pressure in childhood, and their nutritional status did not matter.In this article, we also distinguished between children with symmetrical and asymmetrical IUGR among the SGA group on the basis of anthropometric measurements at birth, including ponderal index. In children with asymmetrical IUGR, higher daytime and 24-hour SBP as well as daytime MAP were found. This may suggest that this subgroup is more prone to higher blood pressure values in early childhood, due to normal growth potential. However, the size of this subgroup was rather small and this observation needs further investigation.When isolating the subgroup of SGA children according to birth weight (≤ 5th percentile and 5th–10th percentile), there was no significant difference in the incidence of abnormal blood pressure values. It appears that assessing intra-uterine growth restriction only on the basis of birth weight does not reflect the risk of developing hypertension. Some authors tried to find other perinatal parameters describing body proportions that better correlate with metabolic dysfunction and high blood pressure in later life.The major limitation of this work was the relatively small group of patients. However, this was a single-centre study, which could constitute the beginning of a wider research. We intentionally used fewer subjects in the AGA group, so that they could be comparable to the subgroups of SGA children .In children born as SGA, abnormal blood pressure values (prehypertension or hypertension) occurred more frequently than in healthy children. This correlation did not provide a significant relationship with the type of IUGR  or birth weight percentile (< 5th percentile vs 5–10th percentile), which could have been the result of the small number of patients.It seems reasonable therefore that children born with IUGR should remain under paediatric care. In cases of elevated blood pressure values during standard medical examination or any other risk factors of cardiovascular disease, these patients should be directed to a more accurate assessment of blood pressure values using APBM.


EDN1) have been associated with several cardiovascular diseases and may act as genetic prognostic markers. Here, we explored the overall relevance of EDN1 polymorphisms for long-term survival in patients undergoing on-pump cardiac surgery. A prospectively collected cohort of 455 Caucasian patients who underwent cardiac surgery with cardiopulmonary bypass was followed up for 5 years. The obtained genotypes and inferred haplotypes were analyzed for their associations with the five-year mortality rate (primary endpoint). The EDN1 T-1370G and K198N genotype distributions did not deviate from Hardy–Weinberg equilibrium and the major allele frequencies were 83% and 77%, respectively. The cardiovascular risk factors were equally distributed in terms of the different genotypes and haplotypes associated with the two polymorphisms. The five-year mortality rate did not differ among the different EDN1 T-1370G and K198N genotypes and haplotypes. Haplotype analysis revealed that carriers of the G-T (compound EDN1 T-1370G G/K198N T) haplotype had a higher cardiac index than did non-carriers (p = 0.0008); however, this difference did not reach significance after adjusting for multiple testing. The results indicate that common variations in EDN1 do not act as prognostic markers for long-term survival in patients undergoing on-pump cardiac surgery.Genetic variants within the endothelin-1 gene ( EDN1) is localized on chromosome 6, spans 5.5 kb in length, and contains five exons and four introns. EDN1 has been identified as a strong candidate for involvement in cardiovascular diseases, including essential hypertension. Given its vasoconstrictive effects on vessels and hypertrophic impacts on myocardial tissue, the expression of ET-1 has been considered an important risk factor for hypertension[EDN1 is a G-to-T substitution that causes a lysine to asparagine change at codon 198 (K198N); in large populations, this polymorphism was found to interact with the body mass index (BMI) in association with blood pressure[EDN1 T-1370G, is located in the 5' flanking promoter region of the gene and may therefore be involved in its differential transcriptional regulation[EDN1 T-1370G G allele presented with significantly increased left ventricular hypertrophy[Endothelin (ET) is a strongly vasoconstrictive peptide produced by vascular endothelial cells. The thrertension, 5. Moreertension, 8. Furtertension. Additioertension. Additioertension. Based o pressure–13. Acco pressure. Anotheregulation. Carrierpertrophy. This popertrophy.EDN1 T-1370G and K198N in Caucasians, these two polymorphisms were selected for an assessment of their clinical impacts on a cohort of Caucasian patients undergoing on-pump cardiac surgery.Given this background, the potential clinical and functional relevance of EDN1 T-1370G and K198N or their haplotype combinations might impact the long-term (5-year) survival (primary outcome) of patients following cardiac surgery with cardiopulmonary bypass.This study aimed to explore whether The results indicate that common variations in EDN1 do not act as prognostic markers for long-term survival in patients undergoing on-pump cardiac surgery.This study included adult Caucasian patients who were admitted to the University Medical Center Goettingen (UMG) and underwent cardiac surgery with cardiopulmonary bypass from 2006–2007. Patients with known neoplasms were excluded. This study conformed with the ethical principles of the Declaration of Helsinki  and was approved by the University of Goettingen ethics committee in Goettingen, Germany. Written informed consent was obtained either from the patients or their legal representatives.After enrollment, the patients were followed up for 5 years and their deaths were recorded as the primary outcome variable. The cardiac index (CI), systemic (SVRI), and pulmonary vascular resistance (PVRI) indices were calculated from standard formulas as secondary outcome parameters. An intraaortic balloon pump (IABP) and extracorporeal membrane oxygenation (ECMO) requirement were also assessed as secondary endpoints. Similarly, the length of the hospital and intensive care unit (ICU) stay and the in-hospital mortality were also recorded as secondary parameters.aO2/FiO2), positive end-expiratory pressure (PEEP), partial pressure of carbon dioxide (PCO2), arterial pH, lung compliance, pulmonary infiltrates, intraoperative cross-clamp time, cardiopulmonary bypass time, lung injury score, Acute Physiology and Chronic Health Evaluation Score (APACHE II), and Simplified Acute Physiology Score (SAPS II). The hemodynamic measurements included the heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure (PAP), and pulmonary capillary wedge pressure (PCWP). Moreover, catecholamine support and amiodarone, cortisone, nitroglycerin, or vasopressin administrations were recorded. The use of blood cell suspensions and blood products was also recorded over the observation period.Additionally, the postoperative course, including the complete intensive care unit stay, was assessed for relevant pulmonary and hemodynamic parameters. These parameters comprised the oxygenation index (PEDN1 T-1370G (rs1800541) and K198N (rs5370) polymorphisms were genotyped using pre-designed TaqMan genotyping assays according to the manufacturer’s instructions . Twenty percent of the samples were genotyped in duplicate, and complete concordance of the genotyping results was achieved. The DNA sample identities were controlled by sex typing[Using EDTA-treated whole blood that was collected before surgery, genomic DNA was extracted from the peripheral blood lymphocytes using a commercially available kit, as described previously. The EDNEDN1 haplotype estimation was performed with ten independent runs using the seeds 2, 1536, 2936, 3123, 4957, 5283, 6757, 7992, 8633, and 9045. The most likely combinations of individual pairs of haplotypes (best pairs) were used in the further analyses. The minimal probability for the most likely combinations used was 99.5%.EDN1 haplotypes were inferred using the Bayesian statistical-based program PHASE version 2.1, 21. EDNThe statistical analysis was performed using Statistica  or R software . Continuous variables are presented as the mean ± standard deviation, and categorical variables are presented as an absolute number or percentage. Continuous variables were compared using the Kruskal–Wallis test and the F-test. Significance based on contingency tables was calculated using a two-sided Fisher’s exact test or the chi-square test as appropriate. Allele frequencies in the study population were counted and compared to an expected distribution in a normal population according to Hardy–Weinberg equilibrium and were analyzed using the chi-square test. The time-to-event data were compared using the log–rank test from the R survival package. To adjust for possible effects of confounders we used a multivariate Cox regression model with the additional confounder variables Age, SAPS II, Euroscore, APACHE II; the p-value for a significant genotype effect was assessed then using ANOVA. Analysis was performed using the R package survival. A p-value <0.05 was considered a statistically significant difference. The significance levels were adjusted according to the Bonferroni correction for multiple hypothesis testing. A complete list of all tests that were performed and the corresponding test results are provided as EDN1 T-1370G and K198N polymorphisms were successfully genotyped in all subjects. The genotype distributions of EDN1 T-1370G and K198N were 311:130:14 (TT:TG:GG) and 267:170:18 (GG:GT:TT), respectively, which is consistent with Hardy–Weinberg equilibrium . The observed minor allele frequencies of 17.4% (T-1370G) and 22.2% (K198N) were very close to those reported for reference populations like CEU from the HapMap project . The EDN1 haplotype analysis revealed four common haplotypes , long-term follow-up (5 years) data were available for the initial survivors. The overall mortality rate was 25.5% (n = 116), and there were no significant differences in the mortality rate with respect to the genotype or haplotype distributions , Fig 1. EDN1 H2 carriers relative to EDN1 H2 non-carriers . However, these differences did not reach significance after adjusting for multiple testing.An exploratory analysis of the secondary endpoints revealed some statistical trends in several clinical parameters with respect to the genotype and haplotype distributions The mean postoperative ICU and hospital length of stay was also comparable among the genotype carriers. The overall in-hospital mortality rate was 7.9% for all patients included in this study (36 of 455) and did not reach statistical significance when compared with respect to the genotypes and haplotypes. Furthermore, the in-hospital mortality rate remained uniform across urgent and emergency cases, and no significant correlation was observed among the genotypes or haplotypes EDN1 T-1370G and K198N polymorphisms and haplotypes with the long-term mortality rates (5 years) and postoperative clinical courses of patients who underwent cardiac surgery with CPB. The primary endpoint of five-year mortality did not differ among the EDN1 T-1370G and EDN1 K198N genotypes and haplotypes. Furthermore, no significant genotype-based differences were observed in terms of the postoperative in-hospital mortality rate. Of the secondary endpoints, carriers of the EDN1 haplotype H2 (T-1370G G combined with K198N T) were found to have a statistical trend toward an increased cardiac index when compared with the non-carriers.This study examined the associations of the EDN1 T-1370G and K198N genotype distributions among the patients were similar to the database entries for healthy Caucasians and followed Hardy–Weinberg equilibrium. Neither of these genotypes nor the haplotypes was associated with any of the recorded baseline characteristics The EDN1 T-1370G and K198N on the secondary outcome parameter of perioperative and postoperative morbidity. Morbidity was assessed using several relevant clinical measurements with an emphasis on the pulmonary and hemodynamic parameters and the requirement of catecholamine and cardiac organ support In the present study, the complete perioperative course was recorded to explore the clinical impacts of EDN1 H2 carriers when compared the values of EDN1 H2 non-carriers Click here for additional data file.S2 TablesPaO2/FiO2: oxygenation index; PEEP: positive end-expiratory pressure; PCO2: partial pressure of carbon dioxide; APACHE II Score: Acute Physiology and Chronic Health Evaluation Score; SAPS II Score: Simplified Acute Physiology Score; HR: Heart rate; MAP: mean arterial pressure; CVP: central venous pressure; PCWP: pulmonary capillary wedge pressure; PAP: mean pulmonary artery pressure; CI: cardiac index; SVRI: systemic vascular resistance; PVRI: pulmonary vascular resistance; NTG: nitroglycerin; IABP: intraaortic balloon pump; ECMO: extracorporeal membrane oxygenation; ICU: intensive care unit.(DOCX)Click here for additional data file.