The study aimed: (i) to characterize reflex responses from peripheral and central chemoreceptors in different age groups of healthy men (<50 years old vs ≥50 years old) and, (ii) to assess, within these groups, whether there is any relationship between ventilatory and hemodynamic responses from chemoreceptors and indices of autonomic nervous system (ANS).Peripheral chemoreflex sensitivity was assessed by the transient hypoxia method and respiratory, heart rate (HR) and blood pressure responses were calculated. Central chemoreflex sensitivity was assessed by the rebreathing method and respiratory response was calculated. ANS was assessed using heart rate variability indices and baroreflex sensitivity (BRS).n = 38, mean age: 32 ± 10 years) and ≥50 years . There were no differences in respiratory response from central and peripheral chemoreceptors between the older and younger groups of healthy males. We found a significantly different pattern of hemodynamic responses from peripheral chemoreceptors between the older and the younger groups. The former expressed attenuated HR acceleration and exaggerated blood pressure increase in response to transient hypoxia. Blunted HR response was related to reduced BRS and sympathovagal imbalance characterized by reduced vagal tone. Blood pressure responses seemed to be independent of sympathovagal balance and BRS.Sixty-seven healthy men were divided into 2 groups: <50 years (Ageing impacts hemodynamic rather than respiratory response from chemoreceptors. Impaired arterial baroreflex and sympathovagal imbalance related to ageing may contribute to decreased heart rate response, but not to increased blood pressure response from peripheral chemoreceptors. Healthy male volunteers, aged between 20 and 80 years participated in the study. Exclusion criteria were: (1) smoking, (2) history of any acute or chronic illness, (3) history of alcohol or drug addiction, and (4) any drug therapy. The study protocol was approved by the local ethics committee. All subjects gave an informed consent. The study was conducted in accordance with the Helsinki Declaration.Prior to the autonomic testing all subjects underwent a physical examination. All subjects were asked to refrain from beverages with caffeine for at least 12 h before the study. Autonomic testing was performed between 9:00 and 11:00 a.m., in the quiet, light-attenuated room with stable ambient temperature ~22 °C.Study protocol consisted of the five stages performed in the following order: (1) familiarization (at least 5 min), (2) resting (15 min), (3) evaluation of peripheral chemoreceptors , (4) resting with controlled breathing (5 min), and (5) evaluation of central chemoreceptors . Stages 1–4 were carried out with subjects lying supine on a bed, attached to the equipment described below . Before the final stage subjects were asked to sit on a chair, the study equipment was replaced  and examination was carried out with subjects sitting in an upright position.5) were measured continuously and non-invasively using a cardiovascular monitor  with continuous, non-invasive, digital recording of finger arterial pressure. ECG wave was recorded using ECG module for Nexfin . Heart rate  was calculated from ECG recording.Hemodynamic parameters: systolic blood pressure , diastolic blood pressure , mean arterial pressure , stroke volume , cardiac output  and systemic vascular resistance  were measured continuously using differential pressure transducer  with 1,000 L/min flowhead . Minute ventilation  was calculated from BR and VT. End-tidal carbon dioxide concentration  was measured continuously using capnograph . Blood oxygen saturation  was measured continuously using pulse oximeter  with probe placed on the subject’s earlobe.Subjects breathed through the oro-nasal face mask  connected with a two-way non-rebreathing T-shape valve . Respiratory parameters: breathing rate  and tidal volume  pure nitrogen (class 5.2), stored in a 10 L gas cylinder, was administrated via the small-diameter tube placed inside a large-diameter breathing tube (ADInstruments) connected directly with a face mask. Nitrogen was administrated with a flow rate > 15 L/s regulated to the nearest 0.1 L using flowmeter. As shown by the previous papers from our lab –23, shorData acquisition device  and laptop  with data acquisition and analysis software  were used for data acquisition. All data were recorded and stored at sampling frequency of 1 kHz (16-bit resolution).T and etCO2 were measured breath-by-breath using gas exchange analyzer . MV was calculated from BR and VT.Subjects breathed through the oro-nasal face mask  with mouthpiece, connected with three-way T-shape inflatable balloon-type valve  with two arms. Lower arm was connected with a reservoir bag pre-filled with 5 L of 100 % oxygen. Upper arm opened outside. Simultaneous closing of upper arm and opening of lower arm created a closed circuit limited to mouthpiece, valve and 5-L bag. BR, VData were stored on a PC computer with data acquisition and analysis software .Resting values of hemodynamic and respiratory parameters were defined as arithmetic averages from 10-min period of an acceptable quality selected from 15-min resting stage recording. The same 10-min period was used for calculation of HRV indices and BRS using the sequence method   and the (i)Time-domain analysis: RMSSD (ms), the square root of the mean of the sum of the squares of differences between adjacent NN intervals; pNN50 (%), number of pairs of adjacent NN intervals differing by more than 50 ms divided by the total number of all NN intervals; RMSSD and pNN50 reflect short-term variability of RR intervals related to parasympathetic tone ;(ii)2), low frequency range (0.04–0.15 Hz) of HRV spectrum; HF (ms2), high frequency range (0.15–0.4 Hz) of HRV spectrum; LF/HF, low frequency HRV to high frequency HRV ratio [Frequency-domain analysis: LF The sequence method: BRS is calculated based on selected sequences of three consecutive heart beats where: (i) increase in RR interval duration by at least 5.0 ms is accompanied by simultaneous increase in SBP by at least 1.0 mmHg or, (ii) decrease in RR duration interval by at least 5.0 ms is accompanied by simultaneous decrease in SBP by at least 1.0 mmHg; BRS is expressed as an average slope of all regression lines linking RR intervals duration to SBP for all sequences selected  ;(ii)The spectral (or α-indices) method: BRS is estimated based on spectral analysis of HRV and blood pressure variability (BPV) and expressed as a ratio of HRV to BPV within two frequency bands: low frequency band (0.04–0.15 Hz)  and high frequency band (0.15–0.40 Hz)  , 25;(iii)The controlled breathing method: based on the synchronization of the imposed breathing rate (6 breaths/min) with rhythmical oscillations in HR and SBP; BRS is calculated by dividing an average amplitude of RR interval duration by an average amplitude of SBP  .Cardiac baroreflex sensitivity (BRS) was evaluated by:The spectral method allows for separate assessment of the sympathetic and parasympathetic contribution to baroreflex regulation of heart rate . The sequence method, however, is considered as a tool for “comprehensive” evaluation of baroreflex modulation of the sinus node, as this modulation is suggested to be wider than that explored by the BRS-αLF and BRS-αHF parameters . The conBRS-CB was estimated based on HR and SBP recording obtained during 5-min of the controlled breathing stage (stage 4), while subjects were asked to adjust the breathing rhythm to the rhythm of the metronome presented on the laptop monitor (6 breaths/min). 200-s period of an acceptable quality was selected from 5 min of HR and SBP recording to calculate BRS-CB.2 exceeded 70 mmHg. Slope of the regression line relating increase in MV to increase in etCO2 during the rebreathing phase was interpreted as a measure of central chemoreflex sensitivity in terms of respiratory response .Central chemoreflex sensitivity was assessed using the rebreathing method . The tes2 with maximal desaturation varying from 65 to 85 % (conditions of short hypoxemic hypoxia). The length of consecutive nitrogen administrations was adjusted ad hoc based on the fall in SpO2 caused by the first 10-s nitrogen administration. Nitrogen administrations were separated at least by 5-min periods of room air breathing. Calculations as follows were performed for each nitrogen administration.Peripheral chemoreflex sensitivity was assessed using transient hypoxia method , 31, 32.Respiratory response 35-s period of recording starting from the end of nitrogen administration were selected. Within this time frame the highest three consecutive MV values and the lowest SpO2 value were isolated. Then, isolated MV values were averaged and plotted against the isolated SpO2 value providing Point 1. Baseline values of MV and SpO2 were defined as arithmetic averages from 90-s period preceding nitrogen administration. Then, baseline MV average was plotted against SpO2 average providing Point 2. Slope of the regression line linking Point 1 and Point 2 were obtained. As the value of the slope was negative, we used its absolute value for subsequent calculations. Arithmetic averages of absolute values of the slopes for all nitrogen administrations was interpreted as a measure of peripheral chemoreflex sensitivity in terms of respiratory response .Hemodynamic responses Calculations of peripheral chemoreflex sensitivity in terms of heart rate response  and systolic blood pressure response  were performed analogously as in the assessment of respiratory response described above with two differences: (i) 55-s period of recording starting from the end of nitrogen administration were selected (instead of 35-s period), (ii) the highest value of HR (for PChS-HR) and the highest value of SBP (for PChS-SBP) were isolated .Detailed description of data processing was presented before .t test was used to test the inter-group differences.Data and statistical analyses were conducted using Statistica 10  and MATLAB . Variables were presented as a mean ± standard deviation  or median with lower and upper quartile (for skewed distributed variables). To normalize the distribution, skewed distributed variables were log transformed ). Unpaired Student P value <0.05 was considered statistically significant.Pearson’s linear correlation coefficient was used to assess the relations between variables. n = 38, mean age: 32 ± 10 years) and ≥50 years .The sixty-seven healthy men who participated in the study were divided into 2 groups: <50 years (The older group (≥50 years) was characterized by reduced SV, CO and elevated SVR as compared to those <50 years. There were no differences between groups in the remaining hemodynamic and all respiratory parameters at rest Table .Table 1CIn the older group, significant reduction in the parasympathetic-related HRV indices  and an increase in sympathetic-related indices  were observed. Table ; Fig. 1.All parameters of BRS, except for BRS-αLE, were significantly lower in men ≥50 years Table ; Fig. 2.There were no differences in respiratory response from central (CChS-Ve) and peripheral chemoreceptors (PChS-Ve) between two groups. However, significant differences were found in hemodynamic responses from peripheral chemoreceptors. The older group reacted to transient hypoxia with a lower heart rate acceleration, but greater systolic blood pressure increase than the younger group Table ; Fig. 3.r = −0.42; p < 0.05) and BRS-αHF . There were no other significant correlations within this group  and respiratory response to hypoxia correlated negatively with LF  and LF/HF . Heart rate response to hypoxia correlated with time-domain HRV indices  and BRS parameters . There were no other significant correlations within this group  no difference in respiratory response from central and peripheral chemoreceptors between the older and younger groups of healthy males, and, (ii) significantly different patterns of hemodynamic responses from peripheral chemoreceptors between the older and the younger groups. The former expressed attenuated heart rate acceleration and exaggerated blood pressure increase in response to transient hypoxia. Blunted heart rate response was related to reduced cardiac baroreflex sensitivity and sympathovagal imbalance characterized by reduced vagal tone. Interestingly, blood pressure response seemed to be independent of sympathovagal balance and baroreflex sensitivity within this group, (iii) additionally we confirmed previous findings that ageing is related to changes in autonomic balance in favour of sympathetic predominance and/or vagal withdrawal.2 sensitivity threshold  was reduced in the subjects aged 65–69 years and 70–74 years as compared with the younger. Thus, one possible explanation of such heterogenic results is that ageing impacts not the sensitivity of this reflex mechanism , but rather the set point around which this mechanism operates . Additional factors may include: relatively small samples studied and combining data from male and female. Taking into account that our paper investigates a sizeable sample of healthy males, we believe that there are no age-related differences in central chemosensitivity  in this population.Previous studies on age-dependent changes in central chemosensitivity provided discordant results. The lack of differences between the younger and the older men in respiratory response from central chemoreceptors found in this study stays in agreement with findings of Chapman and Cherniack , Rubin en = 112 and n = 4 675, respectively) provided, probably, the most relevant attempts to clarify this issue. Garcia-Rio et al. [0.1) instead of minute ventilation to assess ventilatory response from central and peripheral chemoreceptors. P0.1 reflects central inspiratory drive and is believed to be less influenced by age-related changes in lung structure and respiratory mechanics than minute ventilation.Preserved respiratory response from peripheral chemoreceptors in the middle-aged and elderly men as observed in this study, was also described by other authors , 37, 38.o et al.  observedo et al.  respirato et al.  used theData analyzing the possible effects of aging on the hemodynamic component of peripheral chemoreflex are scarce. Blunted hypoxia-induced heart rate acceleration in the elderly was reported by Kronenberg and Drage  and LucyIn the group of men ≥50 years old, robust correlations between heart rate response to hypoxia and HRV parameters  and BRS parameters (positive correlation with BRS-CB and negative correlation with BRS-αLF) were found. It suggests that depressed parasympathetic drive (relations with RMSSD and pNN50), and impaired cardiac baroreflex (relation with BRS-CB), especially overactive sympathetic arm of this response (relation with BRS-αLF) are possibly linked to blunted heart rate response from peripheral chemoreceptors. Both depressed autonomic control of heart function and impaired cardiac baroreflex result in reduced cardiac “plasticity” regarding limited ability of the heart to adjust its function to meet the body’s needs and to react on external stressors properly (e.g. hypoxia).The results of this study seem to support the age-related association between autonomic derangement and deterioration of heart rate response from peripheral chemoreceptors. However, alternative explanations of blunted cardiac response may be considered. Reduced heart rate acceleration in response to hypoxia in the older group may be secondary to exaggerated blood pressure increase with reflex stimulation and response from arterial baroreceptors. However, as baroreflex sensitivity is already deranged in these subjects, this seems unlikely.Secondly, the possible impact of age-related changes in activity of pulmonary stretch receptor reflex should be considered. It was shown recently that pulmonary stretch receptor feedback contributes to inhibition of baroreflex-induced bradycardia in anaesthetized rats . RemarkaFinally, various changes observed in the aging heart  may result in limited ability of the heart to respond to external stimuli  despite Contrary to the results for heart rate response, there were no relationships between systolic blood pressure response to hypoxia and HRV and BRS parameters in the group of men ≥50 years old and only a few in the men <50 years old. It may indicate that age-related changes in blood pressure response to hypoxia are not related to autonomic derangements. Such a result may be surprising as there is an evidence for an anatomical basis for an interaction between peripheral chemoreceptors and arterial baroreceptors . In Interestingly, recent works showed that there is no simple relation between sympathetic outflow to systemic blood vessels (measured by a microneurography technique) and blood pressure at rest . ProbablHeart rate and systolic blood pressure response to hypoxia did not correlate. It stays in line with different pattern of relations with “markers” of autonomic status and regulation found for these responses. A recent study from our laboratory has shown that bilateral carotid body removal in men with systolic heart failure reduced respiratory and systolic blood pressure response to hypoxia, but did not affect cardiac response . Thus, i2 receptors with sympathetic over-activation [As an alternative to the hypothesis of “autonomic derangement”, age-related alterations in blood vessels may promote exaggerated blood pressure increases in response to hypoxia in middle-aged and elderly men. These age-related changes include: stiffening of the blood vessel walls, excessive release of vasoconstrictors with concomitant decline in vasodilator production, and increase in density of αtivation .Some limitations of this study should be emphasized. Clearly, longitudinal observation with intra-individual comparisons would be an ideal design to explore consequences of ageing. To the best of our knowledge, the study of Lhuissier et al.  is the oThe results suggest that the hypothesis of the “autonomic derangement” basis of age-related changes in chemosensitivity may be true for heart rate response from peripheral chemoreceptors, but not for systolic blood pressure response to hypoxia. Moreover, respiratory response from both central and peripheral chemoreceptors seem to be rather independent from sympathovagal balance within the cardiovascular system and barosensitivity.


Epidemiologic evidence, reinforced by clinical and laboratory studies, shows that the rich Western diet is the major underlying cause of death and disability  in Western industrialized societies. The objective of this study is to document the effects that eating a low-fat , high-carbohydrate , moderate-sodium, purely plant-based diet ad libitum for 7 days can have on the biomarkers of cardiovascular disease and type 2 diabetes.Retrospective analysis of measurements of weight, blood pressure, blood sugar, and blood lipids and estimation of cardiovascular disease risk at baseline and day 7 from 1615 participants in a 10-day residential dietary intervention program from 2002 to 2011. Wilcoxon’s signed-rank test was used for testing the significance of changes from baseline.The median  weight loss was 1.4 (1.8) kg (p < .001). The median (IQR) decrease in total cholesterol was 22 (29) mg/dL (p < .001). Even though most antihypertensive and antihyperglycemic medications were reduced or discontinued at baseline, systolic blood pressure decreased by a median (IQR) of 8 (18) mm Hg (p < .001), diastolic blood pressure by a median (IQR) of 4 (10) mm Hg (p < .001), and blood glucose by a median (IQR) of 3 (11) mg/dL (p < .001). For patients whose risk of a cardiovascular event within 10 years was >7.5% at baseline, the risk dropped to 5.5% (>27%) at day 7 (p < .001).A low-fat, starch-based, vegan diet eaten ad libitum for 7 days results in significant favorable changes in commonly tested biomarkers that are used to predict future risks for cardiovascular disease and metabolic diseases. The primary goal of health care should be to decrease all-cause morbidity and mortality. In industrialized societies including those in the United States and Europe, the chief causes of death and disability are noninfectious chronic diseases: atherosclerotic vascular disease, epithelial cell cancers, type 2 diabetes, and autoimmune disorders . The leaThe burden of Western disease can be dramatically reduced by eliminating animal-source foods and vegetable fats from the diet and replacing those foods with low-fat, plant-based foods. When a food rationing system during World War I severely restricted the Danish population’s intake of meats, dairy products, fats, and alcohol but placed no restrictions on such foods as barley, bread, potatoes, and vegetables, Denmark achieved the lowest mortality rate in its history . SimilarA physician can help his or her patients achieve similar benefits by teaching them to eat a low-fat , high-fiber, high-carbohydrate , purely plant-based (vegan) diet –14. The This study is a retrospective analysis of the records of patients who attended a physician-supervised 10-day residential program  from 2002 to 2011. This ongoing program used Internet marketing to attract patients from a wide geographic area to spend 10 days at a hotel in Santa Rosa, California, where they received dietary counseling and were fed a low-fat  diet based on minimally refined plant foods ad libitum to satiety. The educational staff included a medical doctor, a registered dietitian, a psychologist, exercise coaches, and cooking instructors. Patients were also given opportunities for light to moderate exercise. No stress reduction techniques or meditations were included in the program.Upon admission, a standardized questionnaire was used to ask patients if they had any history of hypertension, coronary artery disease, diabetes, hypothyroidism, multiple sclerosis, hypercholesterolemia, or overweight. The patient’s age, sex, and ethnicity were also recorded.A licensed medical doctor saw all patients on at least 3 occasions during the program, and blood pressure was recorded daily. At baseline, the physician took the patient’s history and performed a physical examination. The physician then recommended appropriate changes to each patient’s medications. Medications for hypertension and diabetes were reduced or discontinued at baseline in order to lessen the risk of hypotension and hypoglycemia. Cholesterol-lowering medications (statins) were continued throughout the program for those people taking this class of medication upon entering the program.To facilitate analysis, the following data were recorded according to standardized methods at baseline and on day 7: weight, systolic and diastolic blood pressure, total cholesterol, triglycerides, glucose, blood urea nitrogen, and creatinine. After August 2006, HDL and LDL cholesterol were also measured at baseline and day 7. Body weights were measured with a Detecto® 6800 portable bariatric scale. Blood pressure readings were obtained by a trained operator, manually and/or using an Omron® professional sphygmomanometer. Blood tests were analyzed by using standard medical hospital laboratory procedures.The hotel’s kitchen staff prepared foods according to prescribed guidelines. No animal-derived ingredients  and no isolated vegetable oils  were used. Meals were based around common starches, including wheat flour products, corn, rice, oats, barley, quinoa, potatoes, sweet potatoes, beans, peas, and lentils, with the addition of fresh fruits and non-starchy green, orange, and yellow vegetables. The macronutrient profile was roughly 7% fat, 12% protein, and 81% carbohydrate by calorie. Meals were served buffet-style, and participants were encouraged to eat to the full satisfaction of their appetite.To ensure that the foods would be acceptable to palates accustomed to Western diets, small amounts of simple sugars, salt, and various spices were used in meal preparation. Sugar was included in condiment sauces  and in the low-fat desserts that were served after evening meals. Patients could also add sugar to their cereal in the morning.The kitchen staff used minimal salt, mostly in the form of soy sauce, when preparing the meals. The basic meal plan provided roughly 1000 mg of sodium per day. However, saltshakers were provided at meals, and participants were allowed to use regular table salt ad libitum on the surface of their foods for taste. We conclude that even if a patient added a total of a half-teaspoon of salt per day, the diet would still qualify as low-sodium (approximately 2 g of sodium daily).Data from patients who attended the program from 2002 to 2011 were analyzed in 2014. For patients who attended the program more than once, each visit was regarded as a separate patient record. Records from subjects under the age of 21 were excluded from the analysis. Records with missing data for baseline or day 7 were also excluded.Before August 2006, tests for HDL and LDL cholesterol were not routinely ordered. Only the records that had baseline and day 7 values were included in the analysis of HDL and LDL. The average total cholesterol values and the change in total cholesterol from day 1 to day 7 for the patients with missing HDL and LDL data were compared to the respective values for the patients with complete data on HDL and LDL.The change from baseline in biomarkers was analyzed for risk categories based on the patients’ baseline variables. The cutoffs for the risk categories were arbitrary: total cholesterol <150, 150–179, 180–209, 210–239, or ≥240 mg/dL; triglycerides <150, 150–199, 200–499, or ≥500 mg/dL; glucose <100, 100–126, or >126 mg/dL; systolic blood pressure < 140 or ≥140 mm Hg; diastolic blood pressure <90 or ≥90 mm Hg; creatinine <1, 1–1.4, or >1.4 mg/dL; and blood urea nitrogen <10, 10–20, or >20 mg/dL. Because the patients’ height was not recorded, the patients could not be stratified according to body mass index. Instead, they were stratified according to their weight, as expressed in standard deviations from the mean for his or her sex.Because none of the continuous biomarker variables were normally distributed, Wilcoxon’s signed-rank tests were used to compare baseline values to day 7 values for all biomarker variables. Each comparison was evaluated for the baseline risk categories, as described above. McNemar’s test for paired proportions was conducted to compare the percentage of patients with elevated biomarkers  at baseline whose values fell to normal at day 7 to the percentage of patients with normal biomarkers at baseline whose values were considered elevated at day 7.In 2013, the American College of Cardiology and American Heart Association published guidelines for estimating a patient’s probability of having a cardiac health event  within 10 years . The guiAll statistical analyses were conducted by using SAS 9.3 .A total of 1703 patients attended the program from 2002 to 2011. Of these, 46 patients went through the program more than once. The records for the 12 patients under the age of 21 were excluded. As a result, a total of 1615 patient-records were included in the analyses. There were 1092 records with baseline and day 7 records for HDL and 1091 with baseline and day 7 records for LDL.Table As shown in Table Table The 10-year risk of ASCVD for the full sample  experienced a median decrease of 1 percentage point . However, among patients whose risk was elevated  at baseline, the median decrease in 10-year risk at day 7 was 2.00 percentage points .Table This study documents that a low-fat , high-fiber, high-carbohydrate , vegan diet allows overweight patients to lose weight even if they eat enough food to feel completely satisfied, ad libitum. From eating such a diet for 7 days, the participants lost a median of 1.4 kg (3 pounds) and had significant improvements in blood pressure, blood lipids, and blood sugar. The patients who were the most overweight and had the most unfavorable biomarkers at baseline had the largest favorable responses. The improvements in blood pressure and blood sugar are even more remarkable when one considers that most of the patients discontinued their antihypertensive and antihyperglycemic medication at baseline.This kind of dramatic, early success can be important for maintaining patients’ motivation to adhere to the diet. These rapid results could also motivate physicians to prescribe diet therapy before resorting to the pharmacy for helping their patients. The present study also provides a practical model for health promotion within the healthcare system: an intensive week-long residential educational program. The long-term effectiveness and cost-effectiveness of this approach for managing Western diseases, in comparison with the current approaches that focus on prescription medication and major surgery, deserve further study. We believe that this simple dietary approach can improve patients’ health and ultimately reduce healthcare costs.The diet used in this study differs significantly from some conventional approaches to weight loss. Note, however, that the potential therapeutic value of plant-based diets has been mentioned in the American Diabetes Association’s clinical practice recommendations  as well When conventional dietary approaches fail to reverse type 2 diabetes and cardiovascular disease, prescription medications are used to control the biomarkers of chronic disease. Unfortunately, the drug-induced improvements in biomarkers do not always translate into reduced mortality . ParadoxUncontrolled studies like this one can provide valuable evidence in situations where randomized, blinded clinical trials have not been or cannot be performed . This stPerhaps the biggest limitation of the vegan diet studies comes from the selection of the participants. Subjects who choose to participate in these studies are willing to try a low-fat vegan diet to improve their health and/or appearance. Thus, they may be more likely than the general population to adhere to the diet.In conclusion, in this retrospective study, we observed that an energy-unrestricted low-fat, starch-based, vegan diet consumed in a controlled environment for 7 days resulted in substantial favorable changes in commonly tested biomarkers that are used to predict future risks for cardiovascular disease and metabolic diseases. The results also suggest a model for a cost-effective therapy to offer patients commonly seen in medical practices today.The supporting data from this study will be available in a separate file for publication.


Prior to implantation, right ventricular dysfunction (RVD),ejection fraction < 25% and use of high doses of diuretics (HDD) increased therisk of cardiac death and Tx by 3.9-, 4.8-, and 5.9-fold, respectively. In thefirst year after CRT, RVD, HDD and hospitalization due to congestive heart failureincreased the risk of death at hazard ratios of 3.5, 5.3, and 12.5, respectively.In the second year after CRT, RVD and FC III/IV were significant risk factors ofmortality in the multivariate Cox model. The accuracy rates of the models were84.6% at preimplantation, 93% in the first year after CRT, and 90.5% in the secondyear after CRT. The models were validated by bootstrapping.We developed predictive models of cardiac death and Tx at different stages of CRTbased on the analysis of simple and easily obtainable clinical andechocardiographic variables. The models showed good accuracy and adjustment, werevalidated internally, and are useful in the selection, monitoring and counselingof patients indicated for CRT. Thus,multifactorial indexes or scores need to be developed to more accurately identifysurvival predictors and treatment responders4. Such indexes shouldinvolve variables related to mortality reduction, with high rates of sensitivity andspecificity.However, 30%-40% of CRT cases do not achieve favorable outcomes, which means that thesepatients undergo surgery with high risks and costs but with no clinical, hemodynamic, orsurvival benefitsThis work aimed to develop predictive models for the combined endpoint of cardiac deathand transplantation (Tx) at different stages of CRT.This prospective observational study evaluated 116 patients with multisite pacemakersimplanted consecutively in a tertiary university hospital between January 2008 and March2013 , who hadOf the 147 patients who underwent implantation during the study period, only 116 wereincluded in the study for the following reasons: 4 had an EF >35%, 3 had totalatrioventricular block, 2 had primary valvular heart disease, 2 had pacemaker infection,7 had incomplete data, 4 had loss of capture in the left ventricle electrode, 2 did notundergo complete follow-up, 1 had severe comorbidity, 5 were hospitalized for class IVCHF at the time of inclusion, and 1 died of premature respiratory infection.The electrodes of the right ventricle were positioned preferentially in the apicalregion (84%). The models used in 92, 12, 10, and 2 patients were from St. Jude Medical,Biotronik, Medtronic, and Guidant, respectively. Patients with concomitant indicationfor an implantable cardioverter-defibrillator (CRT-D group) (54% of the 116 patients)were also included in this study. This indication was for primary prevention in 47patients and for secondary prevention in 16 patients.st-degreeatrioventricular block; QRS duration; QRS narrowing after implantation; R wave in the V1lead in patients with LBBB; and QRS axis in the frontal plane after implantation. Theechocardiographic variables were: left ventricular (LV) diastolic and systolicdiameters; EF computed using Simpson’s method; degree of diastolic dysfunction (DD) fromI to IV; degree of mitral regurgitation from I to III; right ventricular dysfunction(RVD); and dyssynchrony.Assessments were performed in the preimplantation period , at 1 yearafter implantation , and at 2 years after implantation according to a fixed protocol. We analyzed 12 clinical, 8 electrocardiographic, and 7echocardiographic variables. The clinical variables were age, sex, body mass index,cardiac cachexia, FC, etiology of cardiomyopathy, cardiac vein where the electrode waspositioned in the left ventricle, serum creatinine level, systolic and diastolic bloodpressures, use of high-dose loop diuretics (≥ 80 mg/day of furosemide), andhospitalization due to heart failure. The electrocardiographic variables were: atrialfibrillation; LBBB or RBBB; previous cardiac pacemaker; 1A 12-lead surface electrocardiogram was recorded at the speed of 25 mm/s and amplitudeof 10 mm/mV. The longest duration of the QRS measured in one of the leads of the frontalor horizontal plane, which was the lead with the highest value and thus allowed forbetter evaluation, was taken into account. Cardiac mortality was defined for deaths ofend-stage CHF or for sudden death.6. Three experienced physicians performed theechocardiographic examinations, 72% of which were performed by the same specialist.The examinations were performed using the GE Vivid 7 Ultrasound System .The echocardiographic guidelines for the analysis of various echocardiographicparameters were followed, as well as the guidelines for dyssynchrony for the analysisof such parameters5. Right ventricular function was analyzedqualitatively, differentiated between the presence and absence of any degree ofdysfunction5.The systolic function analysis of the cardiac chambers was performed using Simpson’smethod in the two-dimensional mode. Ventricular diameters were obtained on M-modeechocardiography, according to the standard guideline7.Diastolic dysfunction analysis was conducted by assessing mitral flow , tissue Doppler images, and flow propagation speed oncolor M-mode. Results were used to classify DD into four grades 5. In this practical context, the Coanda effect was interpreted as amoderate reflux when restricted to the atrial sidewall and accentuated when itstretched through the upper pole of the left atrium.The degree of mitral regurgitation was assessed as the percentage of the left atriumfilling using color Doppler echocardiography. The percentage was less than 20% inmild reflux, and between 20% and 40% in moderate reflux; values above thesepercentages indicated severe refluxAll patients provided informed consent, and the ethics committee of the hospitalapproved the study, whose protocol conforms to the ethical guidelines of thedeclaration of Helsinki.t test was used to compare the distribution ofapproximately normal, continuous variables, and the Wilcoxon/Mann-WhitneyU test was used for the comparisons of continuous variableswithout normal distribution. Distributions were considered significantly different ifp < 0.05.The categorical variables were presented as frequencies and percentages, whereas thecontinuous variables were presented as means and standard deviations, or medians. Thecategorical variables were compared using the McNemar, Stuart-Maxwell, or chi-squaretest. The Student The univariate relationship between the clinical, electrocardiographic, andechocardiographic variables and the combined endpoint of cardiac mortality and Tx wasevaluated by using the Kaplan-Meier survival curve, log-rank test, and Cox regressionanalysis. Some continuous variables were assessed to determine a cutoff value.Cox multiple regression models were developed in the following analysis times toassess the independent contribution of each of the significant variables in the Coxunivariate model: preimplantation (time 1), first year after CRT (time 2), and secondyear after CRT (time 3). Variables with p < 10% were considered potentialconfounders. Each of the variables was included in the multivariate model accordingto hazard descending order and was excluded when p ≥ 5%. After obtaining thefinal model, the previously excluded variables were included again in the model andtested individually using the same criteria.8 as an independent variable to measure risk, andcardiac death/Tx as the dependent variable. The accuracy of the models was testedwith the receiver-operating characteristic (ROC) curve, along with its sensitivityand specificity. Models were prepared by dividing the hazard scores into riskcategories according to the number of variables present and classified as low (classA), medium (class B), and high risk (class C).We conducted logistic regression analyses by using hazardKaplan-Meier survival curves were elaborated individually for the independentvariables and risk classes, and compared using the log-rank test.For the proposed models, all the variables were tested for compliance with theproportional hazards assumptions by performing the Schoenfeld test and a visualanalysis of the Schoenfeld residuals against the time of deaths or censorship. Foreach model, the effect of each observation on the estimated parameters was analyzed.To achieve this, after the deletion of an observation, the model was estimated againand the new estimates were compared to the previous ones. Values should not changemuch or the model may be too sensitive to a particular observation.To obtain the bootstrap confidence intervals, the original data were sampled 10,000times to obtain 10,000 pseudo-samples of size 60. Then, for each pseudo-sample, thehazard ratios of the three models were estimated. These estimated hazard rates weresorted, and the 95% confidence interval was reported.The data were analyzed by using Stata/SE version 12.1  and the "R" software .During the study, 29 deaths were recorded, representing a total mortality rate of 25%during the follow-up period of 34.09 ± 17.9 months. Cardiac mortality/Txaccounted for 16.3% (19 patients) of the cases. Six patients underwent Tx during thestudy period, 5 for refractory CHF and 1 for recurrent arrhythmic storm. Three Txpatients died prematurely due to disease severity at the time of Tx. No sudden deathoccurred in the CRT-D group, but sudden death occurred in 3 patients in the CRT-P(pacemaker without defibrillator) group. In the CRT-D group, 6 patients with fastventricular tachycardia or fibrillation were treated with effective shock. The baselinecharacteristics of the patients and the comparison of the results of the variablesduring the assessment period are shown in No significant statistical evidence showed that the assumption of proportional hazardswas violated. The effect of each observation on the estimated parameters for each modelwas analyzed. The data obtained do not suggest influential observations. Bootstrappingconfidence intervals for a 95% level of significance were obtained and confirmed thestatistical significance of the estimated hazard ratios. These results did not rejectthe adjustment of the model with the proposed variables .Of the 27 variables analyzed during the first study period (preimplantation), 13 weresignificant in the Cox univariate regression model. In the Cox multivariate model,RVD, EF <25%, and use of high-dose diuretics (HDD) were independently associatedwith increased cardiac mortality/Tx, with hazard ratios of 3.9, 4.8, and 5.9,respectively .Significant variables in the multivariate model were also significant in theKaplan-Meier model when compared using the log-rank test. The analysis of the modelby using the ROC curve showed an area under the curve (AUC) of 0.81, with asensitivity of 61.1%, a specificity of 89.5%, and an accuracy of 84.6% .From the combinations of these variables, we developed a model with three classes asfollows: class A (low risk for cardiac death/Tx) was the absence of the variables orthe presence of only one of the significant variables in the multivariate analysis,implying a 30-month cardiac event-free rate of 93%. The combination of two (class B)and three variables (class C) resulted in 30-month cardiac event-free rates of 61%and 0%, respectively.During time 2 (first year after CTR), 13 variables were significant in the Coxunivariate regression model. In the Cox multivariate model, RVD, use of HDD, andhospitalization due to CHF were independently related to increased cardiacmortality/Tx rate, with hazard ratios of 3.5, 5.3, and 12.5, respectively.The significant variables in the multivariate model were also significant in theKaplan-Meier model, when compared by using the log-rank test. The analysis of themodel by using the ROC curve showed an AUC of 0.910, with a sensitivity of 76.4%, aspecificity of 96.3%, and an accuracy of 93% .From the combinations of these variables, we were able to construct a model withthree classes . Class AHospitalizations due to CHF, use of HDD, FC, DD, RVD, EF < 30%, Chagas disease,and systolic blood pressure < 110 mmHg were significant in the univariate Coxregression model in the second year after CRT.In the multivariate Cox model, RVD and FC III/IV were independently related toincreased cardiac mortality/Tx rate, with hazard ratios of 7.7 and 12.0,respectively. The significant variables in the multivariate model were alsoseparately significant in the Kaplan-Meier model when compared using the log-ranktest (p < 0.001). The analysis of the model using the ROC curve showed an AUC of0.789, with a sensitivity of 40%, a specificity of 98.4%, and an accuracy of 90.5%.From the combination of these variables, we were able to construct a model with threeclasses. Class A means low risk of cardiac death/Tx, composed by the absence of thetwo significant variables in the multivariate analysis, implying a 30-month cardiacevent-free rate of 97.5%. The presence of the combination of two (class B) and threevariables (class C) resulted in 30-month cardiac event-free rates of 83.1% and 38.5%,respectively.In the present study, we developed three predictive models for the risk of cardiac deathand Tx at different stages of CRT. To our knowledge, this is the first study tosequentially and prospectively analyze predictive variables in the same population andat different stages of development  and to develop risk models for cardiac death/Tx. Themodels identified simple variables that, when present, were associated with a high riskfor cardiac death/Tx.9, the mortality was 30% inthe group without intervention, compared with 20% in the group with CRT, during a29.4-month follow-up. In the COMPANION study10, the mortality rate was 21% (131/617) in the CRT group, comparedwith 25% (77/308) in the control group, during a 24-month follow-up. Therefore, ourtotal mortality data are within the range described by large-scale studies. In ourstudy, we analyzed the combined endpoints of cardiac mortality and Tx, aiming atidentifying more-specific variables related to CRT results11.The total mortality rate was 25% (29/116) at 34 ± 17 months. In the CARE-HFstudy12, QRSwidth13, QRS narrowing14, presence of dyssynchrony15, female sex16, type of bundle branch block17, LV diameter18, the aortic velocity time integral13, and DD19.Several studies have evaluated predictors of response or death in different populationsand with different response criteria, and the results were inconsistent. However,several publications identified the following predictors of response: dilatedcardiomyopathy20 showed that the regression of the right ventricleoccurred later (15 months), whereas Leong et al21 identified the right ventricle as an independent predictor ofmortality. Therefore, patients with RVD should not be excluded from the indication forCRT, although they represent a subgroup at higher risk of cardiac death or Tx afterCRT22. The importance of theright ventricle in CRT has been demonstrated in other recent studies, but not in theelaboration of risk models for different evolution stages24.The patients with RVD (20.9% of the group) had worse evolution in all the analysistimes. However, we noticed that 6 patients with good outcomes had regression of thealterations in the right ventricle. The study by Praus et al25. Inthe multivariate analysis, Chagas disease did not remain as an independent variable,probably because 41% of the patients had RVD, a variable that was significant at all theanalysis times. Therefore, the relevance of RVD was not exclusively related to Chagasdisease, as 19 patients had RVD due to other etiologies.Thirteen patients (11.2%) had Chagas disease, 5 of whom had RVD. Chagas cardiomyopathywas related to increased mortality in the survival curve, similar to another study thatrelated it with worse outcome26, in a subanalysisof the REVERSE study, have shown that a basal EF <30%, compared with values between30% and 40%, was positively related to survival. Meanwhile, Kronborg et al27 showed that a basal EF < 22.5%determined an increased mortality after CRT.A preimplantation EF <25% identified a subgroup with the highest risk for cardiacdeath. Linde et al10.The hospitalizations for heart failure proved to be an independent variable in relationto the prediction of cardiac mortality/Tx in the first year after CRT. The studyrepresents, to our knowledge, the first time this variable was included as independentin the analysis of mortality risk in the first year after CRT and not as part of theoutcome combined with death. Hospitalization due to CHF is a well-defined risk factorfor cardiomyopathy, with a reduction in the incidence of these events after CRTdemonstrated in several studies28 reported an association between chronic non-use of diuretics andresponse to CRT. Meanwhile, Cleland et al29 observed that the use of HDD was related to a worse prognosis onlyin the univariate analysis. We believe that the description of this variable as anindependent value of cardiac death in two periods of the CRT analysis in our study is anoriginal observation.Another easily obtainable clinical variable that showed significant value in thepreimplantation period and first year after CRT was the use of high-dose loop diuretics(furosemide ≥ 80 mg/day). Van Boven et al30, including the following four independent variables:LV end-diastolic diameter (LVEDD) > 65 mm, non-LBBB morphology, creatinine level> 1.5, and non-use of beta-blockers. In our study, LVEDD and creatinine level weresignificant only in the univariate analyses. Hospitalization due to CHF, use of HDD, andRVD, some independent variables in our work, were not included in the previousstudy.A clinical prediction rule to identify patients at heightened risk for early demiseafter CRT has been recently elaborated31.The models used in this study showed good accuracy,ranging from 84.6% to 93%, and can be used in three different stages of CRT, which isanother original contribution of our work. At the usual significance levels, the modelwas validated internally and did not reveal lack of adjustment or exaggeratedsensitivity to the data.We achieved a significant improvement in the specificity of the predictive models ofmortality or response after CRT, reaching 96% in the first year after CTR and 98% in thesecond year after CRT, when compared with the specificity of 22%-70% of previouslydescribed models in relation to total and cardiac mortality. These results are inaccordance with the target outcomes of CRT in the treatment of patients with severeillnesses, with high costs and risks in the procedureWe believe that the study contributes to and advances the search for better criteria forprognostic evaluation, with the composition of simple multifactorial indexes and withthe inclusion of easily obtainable variables that are used in clinical practice. Themodels will be useful in the selection, monitoring, and counseling of patients indicatedfor CRT.Analyses of intraobserver and interobserver variabilities of echocardiographic andelectrocardiographic parameters were not performed. The patients did not undergooptimization of the atrioventricular interval after surgery. The models created werenot validated externally, although they were validated internally. This study is alsolimited by the small number of patients, the large number of excluded patients, andthe fact that it was conducted at a single center. The RV function was analyzedqualitatively due to the absence of correlation between the RV measures and theprognosis at the beginning of the study. These results must be considered within thestudy population, who had 59.4% of dilated cardiomyopathy, 11.2% of Chagascardiomyopathy, 12% of patients with RBBB and 16.3% of patients with prior cardiacpacemaker. Future larger prospective studies will help validate the importantvariables related to cardiac death or Tx after CRT.We developed predictive models of cardiac death or Tx at different stages of CRT basedon the analysis of simple and easily obtainable clinical and echocardiographicvariables. The models showed good accuracy and adjustment, were validated internally,and are useful in the selection, monitoring, and counseling of patients indicated forCRT.


Dysregulation of the autonomic nervous system is frequent in subjects with cardiovascular disease. The contribution of different forms of renovascular hypertension and the mechanisms contributing to autonomic dysfunction in hypertension are incompletely understood. Here, murine models of renovascular hypertension with preserved  and reduced  kidney mass were studied with regard to autonomic nervous system regulation  and baroreflex sensitivity of heart rate by spontaneous, concomitant changes of systolic blood pressure and pulse interval. Involvement of the renin-angiotensin system and the rho-kinase pathway were determined by application of inhibitors.C57BL6N mice (6 to 11) with reduced kidney mass (1K1C) or with preserved kidney mass (2K1C) developed a similar degree of hypertension. In comparison to control mice, both models presented with a significantly increased sympathetic tone and lower baroreflex sensitivity of heart rate. However, only 2K1C animals had a lower parasympathetic tone, whereas urinary norepinephrine excretion was reduced in the 1K1C model. Rho kinase inhibition given to a subset of 1K1C and 2K1C animals improved baroreflex sensitivity of heart rate selectively in the 1K1C model. Rho kinase inhibition had no additional effects on autonomic nervous system in either model of renovascular hypertension and did not change the blood pressure. Blockade of AT1 receptors  normalized the sympathetic tone, decreased resting heart rate, improved baroreflex sensitivity of heart rate and parasympathetic tone.Regardless of residual renal mass, blood pressure and sympathetic tone are increased, whereas baroreflex sensitivity is depressed in murine models of renovascular hypertension. Reduced norepinephrine excretion and/or degradation might contribute to sympathoactivation in renovascular hypertension with reduced renal mass (1K1C). Overall, the study helps to direct research to optimize medical therapy of hypertension. Nephrogenic arterial hypertension comprising renovascular and renoparenchymal aetiologies is increasingly prevalent . HyperteThe autonomic nervous system is such a potential target. Baroreflex function is attenuated in renovascular disease, regardless of residual kidney mass . On the The pathomechanism of sympathoactivation in renovascular hypertension is unclear. In experimental renovascular hypertension with preserved kidney mass (2K1C), the renin-angiotensin-aldosterone system (RAAS) is found to be activated , and cenThe intracellular Rho A/Rho kinase system emerges as a novel target for the treatment of cardiovascular disease . Rho A, Established Rho A/ROCK inhibitors are Fasudil but also statins, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase inhibitors. Statins inhibit the formation of geranyl-geranylpyrophosphate, a prerequisite for RhoA membrane anchoring ,17. We hIn the present study, we hypothesize that sympathoactivation is more pronounced in renovascular hypertension with preserved (2K1C) versus reduced (1K1C) kidney mass when using heart-rate independent surrogates of sympathetic tone. Sham surgery animals and irbesartan (Irb)-treated 2K1C animals were used as control groups. The AT1-receptor blocker treatment was used as a positive control for its sympathoinhibitory actions -24. SecoMale C57BL/6 N mice  were housed in individual cages in a separate room under standard conditions , standard chow and drinking water ad libitum. Care was provided daily at the same time, body weight was taken weekly. All animal procedures and experiments adhered to the APS’s Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training. During surgeries, inhalational anaesthesia using a precision vaporizer with isoflurane , and subcutaneous (SC) fentanyl (0.06 mg/kg) were used. Following surgeries, pain-relief medication buprenorphine (0.3 mg/kg SC), and antibiotic prophylaxis with ampicillin (50 mg/kg SC) were administered. After observing the animals for 4–6 weeks, mice were sacrificed , and heart weight (absolute and relative to body weight) was determined. Ethical approval was obtained from local animal-care officials and the supervising federal authority .Aortic blood pressure of unrestrained, conscious mice was monitored by telemetry  attached to a femoral-artery catheter. For catheter placement, a 15 mm skin incision was made, and the left femoral artery and vein were separated using a non-serrated fine-tip forceps . The left femoral artery was tied off  caudally of the superficial epigastric and superficial circumflex iliac artery. A second tie was placed 10 to 12 mm cranially and kept under tension to stop perfusion. A 90°-bent 26 gauge injection needle serving as a catheter introducer was inserted into the left common femoral artery right above the distal tie. The telemetry catheter was inserted and advanced to the proximal tie. After releasing the proximal tie temporarily, the catheter was further advanced into the lower aorta  and secured by two knots. After freeing a subcutaneous pouch on the right flank, the transmitter unit connected to the intra-arterial catheter was inserted and 0.05 – 0.1 g gentamicin solution (3 mg/g) was applied before skin suture . Blood pressure readings were transmitted to a receiver placed below the mouse cage, digitized with a sampling rate of 1000 Hz and stored and analyzed on a workstation in a separate room. Systolic and diastolic blood pressures, pulse pressure and pulse intervals (defined as consecutive dP/dt) were extracted from aortic blood pressure waveforms using ART 4.2 Gold software . One week later, the mice were randomized in a 1:2 fashion to sham surgery  or unilateral renal-artery stenosis, i.e. the 2K1C model of hypertension with preserved kidney mass. There, a U-shaped metal clip  was implanted around the right renal artery as reported previously . One wee2 and ms2) for a given spectral bandwidth. The cumulative intensity of the low-frequency band (0.15-0.6 Hz) of power spectrum of systolic blood pressure (LF-SBP) was regarded as a quantitative measure of sympathetic tone, whereas the cumulative intensity of the high-frequency band (2.5-5.0 Hz) of power spectrum of heart rate (HF-HRV) was considered as a quantitative measure of parasympathetic tone [After a two-week recovery period, a one-hour baseline recording was taken in the morning. Thereafter, intraperitoneal injections of atropine-methyl nitrate   or metoptic tone ,27.Power spectrum (high-frequency band: 1.0-5 Hz) of systolic blood pressure (HF-SBP) ,29 and pIn addition, changes in resting heart rate after administration of metoprolol or atropine were determined. An overnight recovery was required after injection of either substance.http://www.haraldstauss.com/HemoLab/HemoLab.php). Concomitant changes of systolic blood pressure (of at least 15 mmHg) and pulse intervals of at least 4 consecutive heart beats were correlated. For individual baroreflex curves, a correlation coefficient of at least 0.9 was mandated for analysis. In addition, a time delay of 0 seconds was chosen for analysis of concomitant blood-pressure and pulse-interval changes according to a previous study with murine models [Baroreflex sensitivity was determined by the sequence technique  of concoe models . The aveMice were placed in metabolic cages (Tecniplast) for 24-hour urine collection. Urine was collected over 24 hours in a vial containing 30 μl HCl (0.5 mol/l), stored at −20°C. For analysis urinary norepinephrine, dopamine and epinephrine were determined by a radioimmunoassay method .(Irb) was dissolved in water  and given to five 2K1C mice orally ad libitum. Drug uptake was recorded by weighing drinking bottles every 48 h. Likewise, the Rho-kinase inhibitor SAR407899A was given to 2K1C and 1K1C mice with the drinking water . The actual drug uptake is shown in Table The AT1 receptor blocker irbesartan Results are given as means ± one standard deviation. For inter-group comparisons with equal variances, one-way ANOVA/Newman-Keul post-hoc test or one- or two-tailed student’s t-test were used, where appropriate. If the normality test failed, nonparametric tests  were used, where appropriate. A p < 0.05 was considered significant. Asterisks highlight significances . Statistical analysis was carried out with Graphpad .Pulse pressure, systolic and diastolic blood pressure were significantly elevated in both models 2K1C, 1K1C) of renovascular hypertension as compared to controls  or metoprolol (MET) Table . HoweverIn comparison to controls, baroreflex sensitivity of heart rate was significantly attenuated in both models of renovascular hypertension. Beta-1-adrenergic blockade with MET did not alter this difference Figure A-B. FollIn comparison to controls, both models of renovascular hypertension  presented with an elevated sympathetic nervous system tone as determined by power spectral analysis of systolic blood pressure. In 2K1C as well as in 1K1C animals, sympathoactivation persisted after ATR application Figure A-B. In tParasympathetic tone, as determined by the cumulative intensity of HF-HRV, was significantly attenuated in both models of renovascular hypertension. Beta-adrenergic blockade with MET confirmed the significant attenuation of parasympathetic tone in 2K1C animals. An AT1-receptor blockade with Irb prevented the significant attenuation of parasympathetic tone in comparison to controls  associates with a lower parasympathetic tone when compared to normal controls, which is reversed, at least partly, by AT1 receptor blocker treatment.Urinary norepinephrine excretion was significantly reduced in renovascular hypertension with reduced kidney mass (1K1C) when compared to normal controls , AT1-receptor blockade improved baroreflex sensitivity of heart rate. These data are supported by observations in humans with “essential” hypertension . In addiConcerning the parasympathetic tone, different results have been obtained for the renovascular hypertension models with and without reduced kidney mass  as well. 2K1C animals showed a significantly depressed parasympathetic tone which persisted after beta-adrenergic blockade. As novel findings, AT1-receptor blockade with Irb significantly increased parasympathetic tone, decreased resting heart rate, and restored baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass (2K1C). Baroreflex and heart-rate data are in line with published evidence from experimental renoparenchymal hypertension . Given tAs another main result, both models of renovascular arterial hypertension exhibited a state of sympathoactivation as detected by power spectral analysis of blood pressure. Thus, previous results obtained with selective blockade (atropine and propranolol) experiments on heart rate were confirmed ,9. For rAs main results, sympathetic tone  was found to be increased, whereas baroreflex sensitivity of heart rate was depressed in models of renovascular hypertension, irrespective of residual renal mass. Differential results relate to parasympathetic tone (with or without beta 1-adrenergic blockade) that was depressed in the 2K1C model only. In addition, left ventricular hypertrophy was present in experimental renovascular hypertension with reduced renal mass (1K1C) only. Renal norepinephrine excretion was reduced in the 1K1C model exclusively. Hypothetically, renal reduced catecholamine excretion and/or impaired renal catecholamine degradation may be considered as mechanisms of sympathoactivation in the 1K1C model. Finally, Rho-kinase inhibition improved baroreflex function solely in experimental renovascular hypertension with reduced renal mass (1K1C), whereas AT1 blockade improved baroreflex sensitivity of heart rate in renovascular hypertension with preserved kidney mass (2K1C). Taken together, Rho-kinase inhibition might be an additive strategy to improve survival in renovascular hypertension with reduced renal mass, whereas low-dose AT1 blockade might be a therapeutic choice in all other cases of renovascular hypertension.The authors declare that they have no competing interests.RUP made substantial contributions in study conception and design and the acquisition of data. He performed experiments and drafted the manuscript. SB and OJ collected data, performed experiments and provided substantial input in data interpretation and analysis. RPB conceived the study, contributed to study design and was involved in drafting the manuscript. All authors gave final approval to the version to be published.


The value of noninvasive monitoring peripheral perfusion to predict outcome remains to be established in septic patients after resuscitation. Moreover, the prognostic value of PI has not been investigated in septic patients.The peripheral perfusion index (PI) is a noninvasive numerical value of peripheral perfusion, and the transcutaneous oxygen challenge test (OCT) is defined as the degree of transcutaneous partial pressure of oxygen  - (baseline PtcO2)), and the oxygen challenge index ((10min-OCT)/(PaO2 on inspired 1.0 oxygen - baseline PaO2)) during the OCT.Forty-six septic patients, who were receiving PiCCO-Plus cardiac output monitoring, were included in the study group. Twenty stable postoperative patients were studied as a control group. All the patients inspired 1.0 of FiO2, 10min-OCT and oxygen challenge index (OCI) in all the patients. The control group had a higher baseline PtcO2, 10min-OCT and PI than the septic shock group. In the sepsis group, the macro hemodynamic parameters and ScvO2 showed no differences between survivors and nonsurvivors. The nonsurvivors had a significantly lower PI, 10min-OCT and OCI, and higher arterial lactate level. The PI, 10min-OCT and OCI predicted the ICU mortality with an accuracy that was similar to arterial lactate level. A PI <0.2 and a 10min-OCT <66mmHg were related to poor outcome after resuscitation.The PI was significantly correlated with baseline PtcOThe PI and OCT are predictive of mortality for septic patients after resuscitation. Further investigations are required to determine whether the correction of an impaired level of peripheral perfusion may improve the outcome of septic shock patients. Recent reports using the noninvasive PtcO2 have observed a relationship between low oxygen challenge test values to mortality and new organ failure [Blood flow is diverted from the peripheral tissue to vital organs during circulation shock. It is assumed that peripheral tissue is the first tissue bed to sacrifice in shock and the last to be reperfused in resuscitation ,2. Tissuod gases . Becausepatients -8. In ad failure ,10.However, septic shock, in contrast to other forms of shock associated with peripheral vasodilation, is known as 'warm shock'. During septic shock resuscitation, perfusion assessment is difficult and usually a complex determination. The value of peripheral tissue perfusion is controversial in systemic sepsis . To the This study has been partially presented in abstract form .The Institutional Research and Ethics Committee of the Peking Union Medical College Hospital approved this study for human subjects. Informed consent was obtained from all patients or next-of-kin before data were included into the study.2) >80%; were pregnant; aged <18 years; were not expected to live; were brain dead; due to the decision to withhold or withdraw treatments. Septic shock was defined as severe sepsis with sepsis-induced hypotension persisting, despite adequate fluid resuscitation, and requiring the administration of vasopressors [When the research team was available, all adult patients within 48 h after the onset of severe sepsis or septic shock sequentially admitted to the Department of Critical Care Medicine of Peking Union Medical College Hospital, who required PiCCO-Plus cardiac output monitoring and mechanical ventilation for resuscitation, were eligible for the study. The attending intensivists decided PiCCO catheter placement according the severity of the patient's condition after the early hemodynamic support. The local reference standard of PiCCO catheter insert is: (1) tissue perfusion or organ function has not improved after early goal-directed therapy ; (2) accpressors .Twenty stable patients during the first 24 h of the Department of Critical Care Medicine admission who were ventilated and sedated, without infection, and admitted for postoperative monitoring were measured and considered to be the control group.2) was at 70% or more [5; after the macro hemodynamic variables were optimized, if the lactate was not decreasing, sources for the elevation of lactate were sought, and/or maneuvers to lower oxygen consumption were instituted . The intensivists were blinded for the results of the peripheral perfusion variables.All the patients needed mechanical ventilation and sedatives. All septic shock patients received broad-spectrum antibiotic coverage. The local hemodynamic support algorithm of septic shock patients: the early goal-directed therapy was as follows: central venous pressure was 8 to 12 mmHg; mean arterial pressure was above 65 mmHg; urine output was above 0.5 ml/kg of body weight , and the central venous oxygen saturation  ; SequentSimultaneous basic blood gases from the arterial, central venous catheters were obtained . Blood gas samples were taken anerobically in 3 ml heparinized syringes  and analyzed on blood gas bedside machines  or . The thermodilution cardiac output was measured by injecting 15 ml of 0.9% saline at 0°C for the PiCCO-Plus . Three cardiac outputs, which were within 10% of each other, were obtained and averaged. These global hemodynamic variables were recorded simultaneously.2 and PtcCO2 were recorded. The OCTs were performed (FiO2 was increased to 1.0 for 10 min), and the new PtcO2 value was recorded every 1 min. The values for PaO2 were examined on inspired of 1.0 FiO2. PI and pulse oxygen saturation percentage (SpO2) were measured in the index finger by using the IntelliVue MP70 monitor . The MP70 system calculates the PI as the ratio between the pulsatile component and the nonpulsatile component of the light reaching the light-sensitive cell of the pulse oximetry probe. The ambient temperature of the room was kept constant at approximately 23 to 25°C (climate controlled) during all phases of testing.Transcutaneous sensors  having previous validation studies were calibrated to known oxygen and carbon dioxide levels according to the manufacturer's guidelines. The sensors were attached to the patients' upper torso below the clavicle in nonbony areas, and skin was prepared with alcohol and shaved when necessary. Areas of injury such as hematomas were avoided. With a total of 15 min equilibration time, heating of the probe to 44°C required 5 min. Baseline PtcO2 index is baseline PtcO2/baseline PaO2; 10min-OCT is (PtcO2 after 10 minutes on inspired 1.0 oxygen) - (baseline PtcO2); the OCI is (10min-OCT)/((PaO2 on inspired 1.0 oxygen) - (baseline PaO2)); survival is leaving the ICU alive for that admission.The PtcOP <0.05 was required to exclude the null hypothesis. The areas under the ROC curves were compared using a Hanley-McNeil test [A descriptive analysis was performed. All data were expressed as mean ± standard deviation, unless otherwise specified. A Mann-Whitney test was used to compare groups on continuous variables, and chi-square and Fisher's exact tests were used to compare categorical variables. Comparisons of two continuous variables were performed using a linear regression. Discrimination of values was assessed with the receiver operating characteristic analysis. All comparisons were two-tailed, and eil test . The sta2 (ScvO2 <60%). Twenty-six of the 46 septic shock patients were also engaged in a previous reported study [From July 2009 to May 2012, a total of 66 patients were enrolled. Fifty-five septic shock patients were screened for the study, and nine septic shock patients were not included in the analysis because of the failure of the correction of ScvOed study . The med2, PtcCO2, and arterial lactate level between the control patients and the septic patients , MAP, 10min-OCT and PI than the septic group , PtcO2 index and ScvO2 showed no differences between survivors and nonsurvivors. Statistically significant variables between the survivors and the nonsurvivors included arterial lactate level , PI , 10min-OCT  and OCI .Global, metabolic and peripheral perfusion variables after resuscitation of the septic patients according to survival are shown in Table 2 ≥70% after resuscitation, and the mortality was 9/26 (35%) in this subgroup. Statistically significant variables between the survivors and the nonsurvivors included arterial lactate level , PI .There were 26 patients in septic group with ScvO2, PtcO2 index and ScvO2. In contrast, the PI was correlated with baseline PtcO2 , 10min-OCT , OCI , lactate . There is a significantly positive relationship between PaCO2 and PtcCO2 .In the whole group, the PI was not correlated with the baseline PtcCO2 (P(v-a)CO2) and ScvO2. In these PtcO2-related parameters, the 10min-OCT and OCI were significant predictors of lethal outcome, but not baseline PtcO2 and PtcO2 index. For predicting ICU mortality, a threshold of 10min-OCT at 66 mmHg was associated with a sensitivity of 65% and a specificity of 96.2%, and a cutoff of OCI at 0.55 was associated with a sensitivity of 60% and a specificity of 88.5%. The ROC curves of baseline PtcO2, PtcO2 index, 10min-OCT and OCI are shown in Figure 2 and Pv-aCO2 are shown in Figure The cutoff value and areas under the receiver operating characteristic (ROC) curves for the related variables used for predicting ICU mortality are shown in Table The main finding of our study was that lower peripheral perfusion and higher metabolic variables are associated with increased ICU mortality in septic patients after resuscitation. The PI, 10min-OCT and OCI predicted the ICU mortality with an accuracy that was similar to arterial lactate level. Thus, peripheral perfusion monitoring appears as a simple but powerful tool to assess global resuscitation status and outcome.As we know, this is the first study to explore the relationship between PI and ICU mortality in septic patients after resuscitation. We found that the cutoff of the PI value was ≤0.2 for predicting ICU mortality, resulting in a sensitivity of 65% and a specificity of 92.3%. The PI, which was defined as the ratio of the pulsatile to nonpulsatile component of the pulse oximetry plethysmograph, is used as a simple and accurate indication of changes in digital blood flow. It has been suggested as a reliable and early indicator of regional block success, and known to increase due to the effect of autonomic blockade during spinal anesthesia ,19,20. SThe change of the finger PI resulted from the blood volume pulsations, the dispensability of the vascular wall and the intravascular pulse pressure, which can be complicated in critically ill adult patients ,23. Ther2-related parameters in critically ill patients. The PI was significantly correlated with baseline PtcO2, 10min-OCT and OCI. We concluded that all these parameters are dependent on peripheral blood flow, and reflect peripheral tissue perfusion.The impact of possible therapy aimed at increasing the PI was not explored in current study. There is a need of further investigations to test such hypothesis. In our study, we investigated the relationship between PI and PtcO2/PtcCO2 is the direct measurement of the local partial pressure of oxygen/carbon dioxide, achieved by placing a Clark polarographic oxygen/carbon dioxide electrode on the skin surface. When the circulation is normal, the PtcO2 reflects PaO2, but in low flow state, it always underestimates PaO2. Shoemaker et al. attempted the translation of PtcO2 values to cardiac hemodynamics, when they explored the dependence of PtcO2 on PaO2 using a PtcO2 index (PtcO2/PaO2) to assess the severity of low flow shock [2 index below 0.7 has been associated with hemodynamic failure in adult patients [2 index has been used to estimate the adequacy of cardiac output and peripheral blood flow. The OCT is another method to determine the situation of circulation using the noninvasive PtcO2 technique. In nonshock states, PtcO2 correlates with PaO2 to strongly increase response to the FiO2. When peripheral perfusion severely decreases, the PtcO2 values will deviate from their relationship with the arterial tensions and lack of response to a FiO2 of 1.0. In our previous study, we showed that an OCT could improve the diagnostic accuracy of the PtcO2 in estimating the cardiac index (CI) value, and a cutoff 10min-OCT value of 53 mmHg has a good positive predictive value and positive likelihood ratio for predicting low CI value [PtcOow shock . A PtcO2patients ,26. The CI value .2, PaO2 (on FiO2 1.0), 10min-OCT than the septic shock group. Therefore, we speculated the control group had a better lung function. However, there were no difference of baseline PaO2 and PaO2 (on FiO2 1.0) between the survivors and nonsurvivors. Hence, PtcO2 showing a poor response of FiO2 1.0 in the nonsurvivor was not due to the lung function, but due to a poor peripheral tissue flow. Moreover, the 10min-OCT and OCI as a predictor of lethal outcome were more sensitive and specific than baseline PtcO2 and PtcO2 index in the patients with septic shock. For predicting ICU mortality, a threshold of 10min-OCT at 66 mmHg was associated with a sensitivity of 65% and a specificity of 96.2%, and a cutoff of the OCI at 0.55 was associated with a sensitivity of 60% and a specificity of 88.5%. In addition, there is another hypothesis for the OCT to determine tissue perfusion. If there is a cellular oxygen deficit, then the additional dissolved oxygen after the oxygen challenge would be utilized, resulting in a minimum rise in PtcO2. The OCT as a method to assess tissue perfusion mainly described in adult patients by using intramuscular PO2, as in the previous study [et al. reported an incremental change of less than 21 mmHg in PtcO2 in response to a 1.0 FiO2 has been associated with mortality reports during 5min-OCT [2 value at 10 minutes always being higher than at 5 minutes [2 on OCT is not well established in critically ill adult patients. Shah et al. suggested that the ideal time for the OCT is at least 10 minutes in the evaluation of patients with hypoxic wounds [2, and the regulation of skin microcirculation during increasing normobaric oxygen is complex [We mainly explored the correlation between the outcome and the OCT in this study. We found that the control group had a higher baseline PaOus study ,28. Yu e minutes . The idec wounds . Further complex . Therefo2; (2) the peripheral perfusion monitors may provide information on internal organ perfusion with the assumption that the peripheral tissue is the last to perfuse during shock resuscitation; (3) the techniques are noninvasive, and the equipment is readily available and inexpensive. Its importance will be affirmed if peripheral perfusion monitoring during shock resuscitation becomes routine, and if the critical values defined in this paper are validated by further study.There are several advantages of the PI and OCT in comparison with the current global endpoints of resuscitation: (1) the peripheral perfusion indicators may provide a specific endpoint of resuscitation rather than the average survival values of ScvO2) has been applied to estimate regional perfusion by near-infrared spectroscopy (NIRS), and the changes of StO2 during a vascular occlusion test (VOT) have been used as a marker of microvascular reactivity [2, and that peripheral circulation influences StO2 resting values and the StO2 reoxygenation rate [2 could be used to evaluate microvascular reactive hyperemia as during a VOT [2, PI, and PtcO2) indicators are used to assess regional perfusion and reactive hyperemia with close values.In recent years, tissue oxygen saturation  after resuscitation, and these patients had high lactate and poor peripheral perfusion. We acknowledge that the using of ScvO2 <60% to reveal the imbalance between oxygen delivery and oxygen consumption is relatively arbitrary. According to early goal-directed therapy, ScvO2 ≥70% is used to reflect the balance between oxygen delivery and oxygen consumption. Therefore, we analyzed a subgroup of septic shock patients with ScvO2 ≥70%, and we found these peripheral indicators still work. This revealed that peripheral perfusion parameters may provide a specific endpoint of resuscitation after ScvO2 ≥70%.Several limitations should be acknowledged. First, the study period may be considered not long enough to evaluate other relevant clinical outcomes and the selected time points are relatively arbitrary. A small number of subjects at a single time point is an important issue and our findings should be confirmed in a larger multicenter study before being translated into regular clinical practice. Second, one can argue that the assessment of these objective parameters requires stability of a number of factors that might affect tissue perfusion and oxygenation. To reduce those uncertainty factors, we strictly stabilized the related parameters . Third was our failure to directly determine skin microcirculation, local blood flow and subjective peripheral tissue perfusion indicators in this study. Fourth, one can argue that this chest skin circulation is not representative of other site circulations. Recently, some studies have been done to explore the value of evaluating peripheral tissue perfusion of other sites in septic shock patients, such as thenar, deltoid, masseter, and ear sites ,41. PeriThe PI and OCT are predictive of mortality for septic patients after resuscitation, comparable to arterial blood lactate. Further investigations are required to determine whether the correction of an impaired peripheral tissue value may improve the outcome of these patients.• The PI and OCT are predictive of mortality for septic patients after resuscitation, comparable to arterial blood lactate.• For predicting ICU mortality, the cutoff of the PI value was ≤0.2 resulting in a sensitivity of 65% and a specificity of 92.3%, and a threshold of 10min-OCT at 66 mmHg was associated with a sensitivity of 65% and a specificity of 96.2%.• The peripheral perfusion indicators may provide a specific endpoint of resuscitation.2: fraction of inspired oxygen concentration; GEDVI: global-heart end-diastolic volume index; ICU: intensive care unit; MAP: mean arterial pressure; NE: norepinephrine; NIRS: near-infrared spectroscopy; OCI: oxygen challenge index; OCT: oxygen challenge test; PaCO2: arterial pressure of carbon dioxide; PaO2: arterial partial pressure of oxygen; PEEP: positive end-expiratory pressure; PI: peripheral perfusion index; PtcCO2: transcutaneous partial pressure of carbon dioxide; PtcO2: transcutaneous partial pressure of oxygen; Pv-aCO2: difference between central venous and arterial PCO2; ROC: receiver operating characteristic; ScvO2: central venous oxygen saturation; SpO2: pulse oxygen saturation percentage; SOFA: Sequential Organ Failure Assessment; StO2: tissue oxygen saturation; SVRI: systemic vascular resistance index; VOT: vascular occlusion test; WBC: white blood cell count; 10min-OCT: 10 minute oxygen challenge test value.APACHE: Acute Physiology and Chronic Health Evaluation; CI: cardiac index; CVP: central venous pressure; FiOThe authors declare that they have no competing interests.DL conceived and designed the study, interpreted data and helped draft the manuscript. HWH participated in the study conception and design, recruited patients, collected data, performed the statistical analysis, interpreted the data and drafted the manuscript. YL and XTW participated in patient recruitment, data collection, technical support and contributed to the critical review of the manuscript. All of the authors read and approved the final manuscript.