High frequency physiologic data are routinely generated for intensive care patients. While massive amounts of data make it difficult for clinicians to extract meaningful signals, these data could provide insight into the state of critically ill patients and guide interventions. We develop uniquely customized computational methods to uncover the causal structure within systemic and brain physiologic measures recorded in a neurological intensive care unit after subarachnoid hemorrhage. While the data have many missing values, poor signal-to-noise ratio, and are composed from a heterogeneous patient population, our advanced imputation and causal inference techniques enable physiologic models to be learned for individuals. Our analyses confirm that complex physiologic relationships including demand and supply of oxygen underlie brain oxygen measurements and that mechanisms for brain swelling early after injury may differ from those that develop in a delayed fashion. These inference methods will enable wider use of ICU data to understand patient physiology. Novel therapeutic interventions are desperately needed for acute brain injuries, which affect large parts of the population worldwide and are associated with poor long-term outcome. IntervenUnderstanding causal inferences amongst biological signals recorded shortly after injury would allow us to better determine the extent to which experimental data replicates the real world scenario. Clinical data are obtained at a time when interventions have the highest chance of changing the disease course but are challenging to analyze due to incomplete data sets, poor signal-to-noise ratios, non-stationarity, hidden dependencies, and correlations on different time scales. Causal inference methods have been successfully applied in neuroscience to underSpontaneous subarachnoid hemorrhage (SAH) is an acute brain injury that frequently leaves patients comatose and can lead to poor outcomes., 9 ComatWe studied all poor-grade aneurysmal SAH-patients in coma (defined as those with a Glasgow Coma Score of 8 or below) admitted to Columbia University Medical Center between June 2006 and October 2013 that underwent invasive brain multimodality monitoring. The diagnosis of SAH was established by computed tomography (CT) or xanthochromia of the cerebrospinal fluid if the CT was negative. Patients were not enrolled if any of the following were met , 11Data were collected as part of an ongoing prospective database approved by the local Institutional Review Board. Columbia University Medical Center IRB (study identification number: AAAL4106) provided approval for all data presented in the current submission. All research activities have been conducted according to the principles expressed in the Declaration of Helsinki and in compliance with all local, state, federal, and IRB mandated policies related to human subjects research. Written consent was obtained when a patient and/or their legally authorized representative, health proxy or surrogate was available to provide written consent. As such the CUMC IRB approved a waiver of documentation of consent as this is a no more than minimal risk observational outcomes study. For patients that we were able to obtain a written signed consent form, the CUMC IRB approved document was presented to the patient and/or their surrogate (as defined below), upon obtaining consent. For patients who were not able to provide a written consent, the CUMC IRB approved a Information Sheet that was provided to the patient. If a surrogate was found then the study team sought written consent, or when the patient was able to provide informed consent, they were presented with the option of continuing participation, not participating in long term follow up but allowing deidentified collection of their hospitalization data, or total removal from the study.TM [20 kDa pores], analyzed for lactate, pyruvate, and glucose using the CMA-600TM, CMA Inc, Stockholm, Sweden; metabolic crisis was defined as lactate pyruvate ratio [LPR] > 40 and brain glucose < 0.7mmol/L), partial brain tissue oxygenation (PbtO2) and brain temperature , regional cerebral blood flow . Physiologic measurements were categorized into normal ranges based on recommendations form the literature or consensus between experienced neurointensevists and neurosurgeons  from General Electric Solar 8000i monitors and inserted into a Microsoft SQL database. Additionally we collected clinical variables including demographics, disease specific variables , laboratory values, neurological examination findings, medications, hospital complications, and three months functional outcome data  as part of an ongoing, prospective observational SAH outcomes study.Physiologic data was acquired using a high-resolution acquisition system  following a previously described approach. We calcuhodology.https://github.com/kleinberg-larb/FLK-NN).[Clinically recorded physiological data may have gaps due to device malfunction or loss of connectivity between the recording and data storage location. After synchronizing the data, and averaging each minute, missing values were imputed using the FLk-NN method (/FLK-NN). This appavg, the causal significance for each relationship , which represents the average impact of the cause on the effect’s value when holding fixed other potential causes. However a threshold must be chosen to determine which values of this measure are statistically significant. Significant relationships for each patient were determined using the causal significance scores and a p-value cutoff of 0.05. Since models were inferred for individuals and each is a separate family for the purposes of hypothesis testing, and the causal significance is only calculated for causes that seem to raise the probability of their effects, there were not a large number of causes tested in each step and thus we did not control for multiple hypothesis testing as we normally would. We explored causal relationships during two predefined time periods or phases: days 0 to 3 after bleed; and days 4 to 7 after bleed.[Instead of testing specific hypotheses, we aim to infer the causal structure between the measured variables in a data driven way. Thus we tested for causal relationships between all measured variables and all other variables at a series of timescales (5 minute windows up to one hour). Because of the heterogeneity of ICU patients and the amount of data collected on each, inference was done for each patient separately (so that 98 separate models were learned). The inference method infers causal relationships and their timing from time-series data, by evaluating how much of an effect’s value a cause explains, relative to other potential causes. This meter bleed., 26We performed k-means clustering to identify specific phenotypes of patients that did have causal relationships identified. Clustering was performed using “kmeans” implemented in R statistical language (v. 3.1.3) using parameters that are known to affect hospital course, such as severity of disease, age, or development of cerebral edema, as well as outcome parameters. The optiWe identified 98 consecutive SAH patients that underwent multimodality monitoring (MMM). All patients were intubated and blood pressure was tightly controlled requiring vasopressors in 91% (N = 89) and antihypertensives in 77% (N = 75) of the cohort . MonitorSystemic physiologic parameters . During ICP. A bidirectional causal relationship involving cerebral perfusion pressure (CPP) and pbtO2 was identified. Also causal effects were seen from pbtO2 and CPP on ICP , ETCO2, and temperature were found to causally affect pbtO2. CPP and pbtO2 had a bidirectional relationship and pbtO2 caused changes in ICP. Higher pbtO2 measures were most frequently linked to normothermia (compared to fever), and higher MAP and CPP measures (Blood flow and water content. BrT caused changes in TW% and rCBF. ETCO2 caused changes in BrT and rCBF caused changes in cardiac index (CI). Increasing TW% was primarily linked to parameters related to volume status . P on ICP . During P on ICP . PbtO2. measures . Blood ftus CVP, .ICP. Again, tightly linked relationships were seen between MAP, ICP, and CPP . 32% (N = 31) of patients developed brain ischemia, also known as delayed cerebral ischemia (DCI).  and CPP . Tempera and CPP . PbtO2. in pbtO2 , while pgenation .Phenotyping. The causal relationships inferred reflect the heterogeneity in the population, with most relationships being found in subsets rather than all patients and some variables having a positive impact in some patients and a negative impact in others. However, we did not identify any patient characteristics that differentiated these groups. To test this, we performed cluster analysis to distribute patients into distinct groups based on parameters that are known to affect hospital course, such as severity of disease, age, or development of cerebral edema, as well as outcome parameters. There was no consistent relationship between any of the clusters (or even averaged close clusters) with causal relationships. The mean distances between clusters representing the patients for any relationship were not smaller than expected from a random distribution.We identified biologically plausible causal relationships using physiologic data routinely collected after brain injury. Combining our causal inference and imputation techniques enabled this real-world data to be used to gain insight into the physiology of patients recovering from hemorrhagic stroke, despite many missing values and a poor signal-to-noise ratio. Interestingly, we confirmed the complex dependencies of partial brain tissue oxygenation (pbtO2) measures primarily on cardiovascular and pressure measures. This complexity illustrates the challenges clinicians face in interpreting this signal at the bedside. Brain waMethodology. One of the biggest challenges in analyzing real-world physiologic data is dealing with variables measured at different timescales and with large gaps between measurements. Many methods exist for imputing missing values, but these ignore the temporal dependencies of these data. Further, missing values for a variable can be dependent on the variable itself  or on other variables (e.g. if monitors are disconnected to perform an intervention) or both. These dependencies also exist across time, so the value of blood flow may depend on temperature with a delay, rather than instantaneously. To address this we developed an imputation method that combines the Fast Fourier Transform with a time-lagged K-Nearest Neighbors method  to account for both types of missingness. This also enables imputation for time instances where all data are missing (e.g. when a device measuring multiple variables is disconnected). The imputation method was validated on a subset of 9 patients from the full dataset. To simulate missingness, patients with more present values were used so that the amount of missingness could be synthetically varied by deleting varying percentages of data (while still having ground truth). In comparisons against leading imputation methods on these data, error rates were the lowest for all amounts of missing data and ranged from a mean absolute error (MAE) of 0.02 for 5% missing to 0.038 for 50% missing. While imCausal inference. Relative consistency of the internal environment known as homeostasis is a fundamental prerequisite for biological functioning and governed by a series of complex interdependent control systems. This biological balance is maintained both by a network of interconnected physiologic properties with bidirectional dependencies between variables  as well as more direct corrective steps . FollowinGiven the significant missing data and noise, even if relationships did have a single discrete lag, we would not observe them as such. Instead, by using time windows (rather than lags) and enabling significance of each causal relationship to be assessed individually (rather than finding a network where one error or omission can have a cascading effect), we were able to efficiently find individualized physiological models. In particular, in preliminary tests of DBNs the methods either overfit to the noise in each patient’s data or make few discoveries, leading to little overlap in both cases.Initial post injury phase. Relationships between physiologic parameters are expected to change over time following acute brain injury particularly in SAH as recovery from the initial injury and development of secondary complications follow a predictable but significantly variable time course. As monitoring only starts following the injury we are unable to provide a pre-injury baseline but most complications such as delayed cerebral ischemia and secondary brain swelling occur after the initial four post bleed days. As in prior analyses, ,23 we thSystemic physiology. Causal inferences were identified within cardiovascular  and respiratory parameters . These confirm widely accepted fundamental physiologic principles such as that seen between RR or MV and ETCO2. Our observations confirm that these relationships are robust in the setting of tightly controlled systemic physiology as management for these patients included intubation, respiratory management strategies to keep close ETCO2 goals, tight blood pressure control including vasopressors and antihypertensives as needed, and careful fluid management. These management strategies may in part also explain the identified cardio-respiratory relationships . However, direct vagal nerve mediated cardiorespiratory reflexes or common sympathetic or parasympathetic stimulation may also play a role. As expected, body temperature had a number of systemic, mostly bidirectional, causal links with respiratory  and cardiovascular parameters . Temperature is likely linked to these physiologic parameters via the sympathetic and parasympathetic nervous system but treatment strategies such as targeted temperature management and medications given to counteract shivering are additional factors that may add to these causal links.Invasive multimodality brain monitoring allows insights into dynamic brain physiology in pathologic states such as trauma or stroke. These monitors are increasingly used to directly measure intracranial pressure, brain oxygenation, cerebral blood flow, oxygen extraction, electrical activity, brain temperature, cerebral water content, and metabolites such as glucose, lactate, glycerol, glutamate, and pyruvate., 13, 20 ICP. Confirming the widely accepted observation that MAP changes may directly lead to ICP changes particularly in patients with abnormal vasoreactivity following acute brain injury, a causal inference of MAP on ICP was seen. Similar mechanisms may explain the effect of other cardiovascular parameters on ICP  but sympathetic stimulation or the Cushing’s reflex may also be a common shared link. As CPP is calculated from MAP and ICP, it is not surprising to see several bidirectional relationships between CPP and both MAP and ICP. Note that while CPP = MAP-ICP, we do not necessarily expect relationships between all three in all patients. That is because calculations of CPP reflect MAP and ICP at a single timepoint, and our causal inference method requires that causes must occur prior to their effects (rather than simultaneously), and it is not the case that later values of MAP and ICP must necessarily depend on earlier ones.Respiratory parameters (ETCO2 and RR) directly link to cerebral vascular diameter via PCO2 or pH changes in the blood. Neuronal injury from systemic hypoxia (reflected in oxygen saturation) may lead to brain swelling and raised ICP. Several studies have associated elevated ICP with fever, a relatiPBTO2. Several techniques have been introduced to generate estimates of brain oxygenation at the bedside utilizing either changes in the electrical properties detected at a Clark type electrode as in the current study or direct tissue oximetry using an optical system. PbtO2 is often used as a surrogate for blood flow changes in patients monitored for vasospasm following SAH or to guide blood pressure targets following TBI. We foundBrain water content. Diffuse brain swelling known as global cerebral edema (GCE) after SAH is found in 8% of patients on admission and 12% during hospital course and is associated with poor functional outcome. The etioLimitations. Physiologic relationships may continuously and gradually change making them difficult to detect. Our approach partly handles this change by studying causality in two different time periods separately, but it remains to discover the correct timings in a data driven way and most critically to discover how relationships may change gradually over time. We studied causal relationships that occurred over a time frame of up to one hour after generating one minute averages of each variable. We may therefore have missed faster (<1 minute) or delayed (>1 hour) causal relationships.Causal inference for individuals further requires that variables have variation (so we cannot discover relationships between static factors such as sex and outcomes), and can only infer relationships between the variables that are observed. This limitation is illustrated by the lack of an identified causal relationship between SPO2 and pbtO2, which we would expect and may be caused by the minimal variability of SPO2 values. Additionally, if there is an unmeasured variable that causes two observed ones, we may identify a relationship between them if one follows the other with a consistent time lag. It is further important to realize that we observe the entire network in a situation of stress  and that the detected relationships link upstream and downstream regulatory responses that are intended to restore this equilibrium. A major limitation for studying brain physiology in the pathologic states is the difficulty to differentiate between ongoing pathologic processes and adaptive, potentially protective, responses. This may require interventional trials or moving back and forth between bench and bedside.Challenges of developing a more comprehensive understanding of brain physiology in humans include limitations of the available datasets and prior analytical approaches. Most datasets have a very limited set of physiologic parameters at a low time resolution. The chosen physiologic parameters in this study are only a selection of a convenience sample of variables chosen based on their increasing use in clinical practice. Others such as microdialysis (only collected hourly), imaging , and EEG (adding a large degree of complexity) were not included but may be explored in the future. Cerebral blood flow and TW% in this study were measured using a temperature dissipation methodology and not surprisingly we found that this was dependent on BrT. This likely represents a technique dependent relationship as both variables were measured with the same probe. For the chosen methodology we were required to categorize physiologic measures into ordinal scales of normal and abnormal, as well as if applicable into abnormally high and abnormally low values. These cut points were based on cut points in the literature and clinical judgment if these were not available see . This ne


We conclude that visual structural and functional changes in type 2 diabetes are related to heart dysfunction, when the effects of clinical, demographic and associated risk factors are taken into account, revealing a genuine relation between cardiac and retinal diabetic phenotypes.Cardiovascular disease and diabetes represent a major public health concern. The former is the most frequent cause of death and disability in patients with type 2 diabetes, where left ventricular dysfunction is highly prevalent. Moreover, diabetic retinopathy is becoming a dominant cause of visual impairment and blindness. The complex relation between cardiovascular disease and diabetic retinopathy as a function of ageing, obesity and hypertension remains to be clarified. Here, we investigated such relations in patients with diabetes type 2, in subjects with neither overt heart disease nor advanced proliferative diabetic retinopathy. We studied 47 patients and 50 controls, aged between 45 and 65 years, equally distributed according to gender. From the 36 measures regarding visual structure and function, and the 11 measures concerning left ventricle function, we performed data reduction to obtain eight new derived variables, seven of which related to the eye, adjusted for age, gender, body mass index and high blood pressure using both discriminant analysis (DA) and logistic regression (LR). We found moderate to strong correlation between left ventricle function and the eye constructs: minimum correlation was found for psychophysical motion thresholds , while the maximum correlation was achieved with structural volume density in the neural retina . Controlling the effect of pairwise correlated visual constructs, the parameters that were most correlated to left ventricle function were volume density in retina and thickness of the retinal nerve fiber layers (adjusted multiple R Diabetes is a major cause of morbidity and mortality due to its multiorgan complications –3. In paWith the increase of diabetes in the population, diabetic retinopathy was added to the priority list of preventable visual impairment, since it affects about 4.25% of the world population, and highly contributes to low vision estimates around the world [Moreover, diabetes is related to other major organ complications, in particular nephropathy, which is the leading cause of kidney failure and most importantly cardiac disease . This is due to the fact that type 2 diabetes mellitus is reflected in increased coronary atherosclerosis and left ventricular dysfunction , 6, condMetabolic and physiological risk factors known to be associated with the increase of these deaths are raised blood pressure, being overweight or obese, hyperglycaemia and hyperlipidaemia.According to the Annual Report from a National Observatory of Diabetes from 2012, the prevalence of diabetes in southern European populations can reach 12.7%, although more than half remain undiagnosed. According to that report, attributable deaths due to diabetes were 4.4% of the total deaths, cardiovascular diseases being a major cause of death [Previous studies addressing the relationship between diabetic retinopathy and heart complications have been largely univariate and/or included patients in relatively advanced stages –11. HereOur aim was therefore to establish a relationship between structural alterations in retina, as measured by morphometric analysis, and functional measures, based on motion, colour and/or contrast discrimination with left ventricular alterations measured by cardiovascular magnetic resonance, as a function of other general clinical variables.This study was designed as a pilot, observational and prospective study with one visit where controls and type 2 diabetes mellitus patients performed multimodal imaging examinations, namely, ophthalmological, psychophysical, heart, liver and cerebral imaging. All subjects included in this study were evaluated for eligibility and signed the informed consent, approved by the Ethics Committee from the University of Coimbra. Our institutional ethics committee specifically approved this study.Inclusion criteria were as follows: men and women aged between 40 and 75 years, functionally independent, capable to provide written consent after proper education and discussion with the treating physician and/or the research physician, with type 2 diabetes for the diabetic group and without any type of diabetes for the control group. Exclusion criteria defined for the study were: history of neuropsychiatric, renal, heart, ocular or any other severe non-age disease unrelated to diabetes, pregnancy or lactation. Eligible patients were asked to participate in the study and signed the informed consent form. Then, subjects completed a questionnaire on lifestyle, cardiovascular risk factors and family history of diabetes, current medication, with a particular emphasis in determining the presence or absence of medication for controlling arterial hypertension, physical activity, dietary pattern and quality of life.Afterwards, at the clinic, height, weight, waist perimeter and blood pressure were measured. For the purpose of analysis, height and weight were used to derive body mass index, and waist was discarded as it was collected and registered in few cases. Clinically significant high blood pressure was defined by the current use (presence or absence) of medication for controlling arterial blood pressure.All subjects performed ophthalmic examination including best corrected visual acuity and intraocular pressure measurements, visual psychophysical tests  and multimodal imaging .For the purpose of this particular study, we considered just heart and ophthalmological scanning. Concerning heart imaging, we used True fast imaging with steady state precision (True-FISP) obtained for the heart, performed at 3.0 T . The participants performed the True-FISP imaging in the supine position, with breath-hold at the end of expiration for each image acquisition, in order to discard artefacts due to respiratory motion. Measures were obtained for systolic and diastolic volume , and the correspondent percentage of ejection fraction, weight of the left ventricle , left ventricle peak flow rate , mitral peak E and peak A velocity  as well as the mitral E/A ratio, mitral deceleration time  and minimum and maximum left atrium volume .Optical Coherence Tomography (OCT) became an integral part of retinal clinical practice due to its detailed visualization of the retina morphology , 13. OCT2).Computerized psychophysical tests from the multifunctional module of the threshold of visual discrimination measure the ability of subjects to detect movement , achromatic contrast  and chromatic contrast according to Protan, Deutan and Tritan axes, corresponding to distinct cone populations . All the tests used lateral, randomly moving pairs of dots, one being the reference point within each meridian used. Peripheral presented stimuli were of short duration, between 400 and 900 milliseconds, and also of short dimension and reduced spatial amplitude . Periphery distances to the fixation point were of 7.5 visual degrees if the selected meridian was the 0° or 90°, or of 10 or 15 visual degrees on meridians 45° and 135°, respectively. Central fixation was controlled by an eye-tracker device, and that information was used in real time to validate the trial. If there was no central fixation, the trial would be successively repeated until validation. Response to each trial was given after a sound stimulus, which occurred at the end of vertical fixation. Properties as screen background point size and central cross remained constant for all the tests , and the only property (dependent variable) that changed was the one being analysed at each case. These tests returned a threshold that represents the minimal difference between the properties being analysed to the asymptotic value at chance level. The screen background was achromatic (grey), and the luminance used had a sufficient magnitude to guarantee that the test occurred in conditions of photopic response , fulfilling the inclusion/exclusion criteria for this study were included in the analysis.Ninety seven subjects  aged between 45 and 65 years old (54.67 ± 0.65 years) with mean body mass index of 27.09 ± 0.40 kg/mThese subjects were classified according to the absence or presence of type 2 diabetes, according to the general gold standard for diagnosis. Thus, 50 subjects were controls for this disease (51.55%), and the remaining 47 subjects were type 2 diabetics (48%), with non-proliferative diabetic retinopathy. None of the subjects had chronic eye or heart disease; nevertheless, 41 of the 97 subjects (42.27%) were undergoing therapy for hypertension.th and 75th percentiles. Normality was assessed through the Kolmogorov-Smirnov test in order to decide whether to use Pearson or Spearman rank-order correlation coefficient, or to decide between Student’s t test and Mann-Whitney’s test for independent univariate comparisons.All qualitative data are expressed in terms of counts and observed percentages. For all quantitative data we present its range, mean and standard error of the mean, and also its median values and the 25As there were 50 variables in the analysis, for less than 100 participants, and multiple comparisons problem would arise between correlated variables, new (derived) variables were generated using two methods applied in this context to perform data reduction: discriminant analysis and logistic regression, considering the presence of type 2 diabetes mellitus as dependent variable. In this particular case, both were used in order to perform a variable reduction, generating an overall measure for each set of variables. The motive for using two different methods was for confirmatory reasons. This is because some the assumptions of discriminant analysis might not hold , 17. TheWe took into account that diabetes and cardiovascular disease prevalence are dependent on age, gender, body mass index and the presence of arterial hypertension. Therefore, this set of variables was forced to enter in all the variable reduction that was performed.1 to f8, described below), summarizing, with correction, structural integrity of the retina (1 and 2), visual (3–7) and cardiac function (8) for 97 participants instead of 50 variables for 97 participants, each one of them corrected for age, gender, body mass index and diagnosed hypertension. These are depicted as follows, where non-identified abbreviations are the individual measurements described for each construct within the Protocol sub-section:Variable reduction with these methods permitted us to obtain new constructs related to each set of structural and functional assessment , and work with 8 variables OCT VS = f2OCT RNFL = f3SPEED = f4ACHROM = f5CHROM-P = f6CHROM-D = f7CHROM-T = f8True-FISP = fj with j from 1 to 8, was obtained either by Discriminant Analysis (functions DA1 to DA8) and by Logistic regression (functions LR1 to LR8).For the true FISP construct (variable 8), and in spite of having other variables such as the ejection fraction and the ratio between Peak E and Peak A, they were not considered as they are function, respectively, of systolic and diastolic volume (EF (%) = 100-SV/DV*100), and values for peak E and peak A (EA = peak E/peak A). Each function fj is the number of variables used for each construct.Logistic regression delivers a probability, for each one of the eight constructs, of a participant to have type 2 diabetes, given the set of independent characteristics involved, and may be written as j is the number of variables used for each construct. The right probability of significance for the chi-square distribution of the squared Mahanabolis distances (D2) [DSj), and the centroid  obtained and given by each one of the eight linear discriminant functions defined as ces (D2)  between Enter method, in order to force models to use age, gender, body mass index and presence of hypertension, and also all the variables involved in each construct, unless measured variables were linear combinations of others which would create multicollinearity and over-fitting.Both discriminant analysis and logistic regression procedures were performed using the Therefore, each construct represented the conditional probability of a participant to have type 2 diabetes, given a set of independent measures, and it would be a measure of positive correlation with type 2 diabetes given a specific set of variables.2 and Hosmer and Lemeshow test for logistic regression were presented, in order to validate models and measure their fitness.Receiver Operating characteristic curves were obtained in order to validate these constructs, since they may only measure positive correlation between the presence of type 2 diabetes and each construct if they discriminate between groups, that is, if they may be used as classifiers. Also canonical correlation and Wilk’s statistic for discriminant analysis and Nagelkerke’s RFinally, correlation between eye-related constructs and left ventricle impairment construct, were obtained. Afterwards, stepwise multiple linear regression using true-FISP as dependent variable, and eye-related constructs as independent was performed in order to eliminate multicollinearity between eye-related derived variables. This procedure enabled the identification of correlation, moderated by the probability of the presence of type 2 diabetes, between the heart-related construct and a linear combination of eye-related constructs, using age, gender, body mass index and blood pressure as covariates.Statistical analysis was performed using SPSS®, version 22, and all statistical tests were evaluated at a 5% significance level. Confidence intervals (95%) are presented for area under the ROC curve.The sample was gender-matched but, as expected, type 2 diabetes participants presented higher prevalence or diagnosed hypertension , since 62 than controls. However, note that both groups presented mean values for body mass index within overweight limits. Moreover, note that derived variables were corrected for gender, blood pressure, and age and body mass index.Controls were also younger and presented lower body mass index than type 2 diabetics ; in factConcerning eye related variables measured with OCT, we only found statistically significant differences between the two types of participants on the temporal-inferior region of the Retinal Nerve Fiber Layer , where type 2 diabetic participants presented thinner retinal nerve fiber layers. There was also a tendency for a thinning on the retinal nerve fiber layer in the nonproliferative type 2 diabetic group on the nasal superior region of the dominant eye . These results might be explained by age differences, thus the need for multivariate analysis. Further analysis of univariate results is presented in We found that diabetic participants presented lower motion or movement detection discrimination under meridians 0° and 135°  than controls . ConcernType 2 diabetic patients seemed to show lower Systolic and Diastolic volume , despite no statistically significant differences being found in the percent of Eject Fraction . Although Peak E of controls and type 2 diabetics was similar , diabetic participants presented statistically significant higher values of Peak A  and, thus significantly lower values on the ratio between peaks E and A , as presented in The other variables did not show significant statistical differences between groups . This result is a measure of accuracy of each construct.2 is obtained for the same construct (R2 = 0.536). Both of those measures showed to be, at least, reasonable, and the obtained correspondent values for the all the other derived variables were higher.Wilk’s Lamda test  indicate2. All the other constructs presented higher area under the ROC curve, whichever the method applied.Besides, the areas under the ROC curve presented excellent discrimination between type 2 diabetics and controls, being able to separate groups with an accuracy of, at least, 84.4% (p < 0.001) with DA and 84.5% (p < 0.001) with LR methods . These vTherefore, these eight constructs might be considered as validated to be used as new variables, and we may be able to trace the type 2 diabetes profile based on each construct, according to the obtained coefficients for each method, presented on In fact, DA and LR models are quite concordant about signals of their coefficients, which are translated in an increase higher risk for the presence of type 2 diabetes whenever they are positive. Dissimilarities are observed for the Outer Inferior region of volume scan density, as referred before, and for variables gender and thickness on the nasal region for the RNFL construct, and for variables Mitral Deceleration Time and maximum volume obtained for Left Atrium in what concerns the True-FISP construct.In fact, DA and LR use different weights for each variable within a set , but botWhichever the method used, the new eye-related constructs obtained, adjusted for gender, age, blood pressure and body mass index appeared to be strongly correlated with the new left ventricle related construct, also adjusted for the same set of variables, as we may observe in However, these variables are, obviously, correlated between themselves thus stepwise multiple regression may identify which is the minimum set of eye-related constructs that are statistically correlated with heart-related construct, all adjusted for gender, age, body mass index and blood pressure. Both models presented acceptable Durbin-Watson statistic , showing that residuals from this regression are serially uncorrelated.2 of 0.730 , Retinal Nerve Fibre Layer (p = 0.014) and Achromatic contrast (p = 0.020) constructs explain 81.9% of the variability obtained in True FISP construct, while the Logistic Regression method only considers the same first two variables in the model, VS (p < 0.001) and RNFL (p = 0.001), with an adjusted Rof 0.730 . HoweverIn fact, correlation between the identified non-collinear sets of eye-related constructs measuring positive correlation to the presence of type 2 diabetes and the heart-related variables moderating the presence of type 2 diabetes present excellent correlation, as observed in table above  and beloTherefore, the loss of density in the neuro-retina and increased thickness of the retinal nerve fiber layers, naturally related to diabetic retinopathy and its impact on visual dysfunction, is correlated to left ventricle dysfunction in this type of patients. Such relation is described on The main finding of this study was that early diabetic retinopathy and asymptomatic heart function are related, even when a large number of covariates are taken into account  axis where we found significant statistical differences in contrast sensitivity whichever the meridian used, as presented before in Concerning heart related variables, namely left ventricle impairment as measured by cardiovascular magnetic resonance, it is known that left ventricular diastolic function is highly prevalent in type 2 diabetes , 24 and 2. The worst fit was obtained for chromatic vision along the Protan axis, whichever the statistical method used, but presenting good accuracy (minimum AUC between 0.844 and 0.845) and moderate to strong correlation between variables and the new defined construct, as it may be observed in For each set of variables, we always found some dependency between type 2 diabetes and visual or heart dysfunction, which was in general moderate when assessed at an univariate level. New constructs obtained for each set of variables, adjusted for age, gender, blood pressure and body mass index, using either discriminant analysis and or logistic regression, were able to classify and discriminate between groups, as presented in On the other hand, cardiovascular variables as assessed by MR imaging and corrected for age, gender, blood pressure and body mass index, acting as covariates, were also associated with type 2 diabetes presence.Those new derived eye-related variables were revealed to be strongly correlated to the cardiovascular construct, as presented on Simple partial correlations would be useful to ascertain which eye-related measures are associated with cardiovascular function in type 2 diabetes mellitus; however, it is difficult to control for the effect of six variables. An alternative method, which also assumes normality but is more robust to its absence, since it uses the F distribution, is multiple regression, where we may find which variables within the set of eye-related variables are most correlated to the heart-related construct, moderated by the presence of type 2 diabetes since every constructs were built in order to give the probability that a subject is a type 2 diabetic.Therefore, by adjusting variables to age, gender, body mass index and blood pressure, we found that there exists a strong correlation between cardiovascular impairment and the thickness of the retinal nerve fiber layer and increase of volume scan density in retina, moderated by the presence of type 2 diabetes, as presented in Therefore, visual dysfunction due to loss of density in the neuro-retina and increased thickness of the retinal nerve fiber layers, related to type 2 diabetes, is correlated to left ventricle dysfunction moderated by the presence of type 2 diabetes. Moreover, type 2 diabetes may either be accurately predicted through visual or by the cardiac phenotype.We conclude that the cardiac and retinal diabetic phenotypes are related even when an array of clinical and demographic variables, and associated risk factors are taken into account. This means that a genuine mechanistic relation exists between diabetic retinopathy and cardiovascular complications.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Asthma is characterized by airway inflammation which can be reversible.Investigation the importance of eosinophils, ECP and IL-5 in asthmatics versus patients with obstructive bronchitis and healthy subjects. We investigated the values before and after the treatment in asthmatics.We studied 77 subjects divided in three groups as follows: 1) asthma patients; 2) patients with obstructive bronchitis and 3) control group of healthy subjects. In all the subjects there were determined: Total number of eosinophils (Eo), eosinophilic cationic protein (ECP), Interleukin 5 (IL-5) and allergy tests.The total number of eosinophils was significantly increased in the group of asthma patients versus second and third group. We found that the presence of ECP demonstrate an ongoing inflammation, with or without clinical symptoms of asthma patients. There was significant difference between the values of ECP of asthma patients versus second and third group. Our results have shown that IL-5 was significantly increased versus second group and controls (p < 0.01). We also found the decrease of the values of inflammatory markers after the treatment with corticosteroids.Eosinophils, ECP and IL-5 could be useful markers for selecting allergic patients and could be the monitors of treatment effects. The allergy is called an epidemic of the twentieth century. The allergic diseases are among the most common diseases in European countries and cover 15-30% of the  population. Asthma represents a possible disease of the entire human population and it is a rising illness. Worldwide about 2% of the total population suffers from asthma. Industrially developed countries: 2-10%; USA: approximately 18 million asthmatics, out of which about 4,8 are children; Russia: approximately 20 million inhabitants suffer from asthma; Republic of Macedonia: about 5% from the population (above 100 000).The morbidity and the mortality caused by asthma continue to grow, even in the present, in times of advanced scientific researches and modern therapeutic treatment -5.Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation. Both symptoms and airflow limitation characteristically vary over time and in intensity. These variations are often triggered by factors such as exercise, allergen or irritant exposure, change in weather, or viral respiratory infections. Symptoms and airflow limitation may resolve spontaneously or in response to medication, and may sometimes be absent for weeks or months at a time. On the other hand, patients can experience episodic flare-ups (exacerbations) of asthma that may be life-threatening and carry a significant burden to patients and the community .The most common asthmatics triggers are as follows: allergic ; environmental factors ; professional irritants; respiratory infections; food and food additives; medicines ; hyperventilation and physical effort; vasculitis; parasitic infections and some other less expressed triggers.There is also a genetic predisposition defined as atopy. If there is not atopic parent 15% of their children could be affected. If there is one atopic parent 40-50% of their children could be affected. If both parents are atopic 75% of their children could be affected.Based on the ethiology asthma could be , 4, 5: aPulmonary inflammation is characterized by: oedema, mucosal and vascular permeability, mucus secretion, reduction in mucociliary clearance, epithelial damage, subepithelial fibrosis, increased neuron sensitivity and cells infiltration.regulators and effectors of the immune function. After chronic exposure to antigen, two subpopulations of T-cells may be identified as: T-helper1 (Th1) and T-helper2 (Th2). There is common participation with: B-lymphocyte cells, mast cells, eosinophils and macrophages. The atopic allergy is type-2 (Th2) hypersensitivity to antigen with genetic origin and environmental impact.T-cells are central to the immune response as Cytokines are low-molecular proteins which are produced by almost all eukaryotic cells and they are acting through specific “cell-surface” receptors. Cytokines have multiple activities, presented in the cytokine cascade. We can present the immunological reaction and cytokine cascade on The inflammatory cells and various mediators cause damage of the airway (respiratory) epithelium, inducing bronchial hyperreactivity  7]..7].The bronchial obstruction is manifested in three stages :1)early stage) occurs 10-15 minutes after a spasmogenic contact with trigger stimulus. The stage is an early reaction with short term, with increased resistance in airways and it is reversible.Bronchoconstriction (2)Second stage is bronchial obstruction with an inflammatory oedema of the bronchi, on which a mucus secret is complemented (related). It occurs 5-8 hours after the trigger contact; lasts a few days and it is sensitive to therapy with corticosteroids.3)Subacute or chronic inflammation is severe inflammation expressed with infiltration and eosinophilic invasion.Eosinophils are significant effector cells, involved into the late and chronic stage of the allergic inflammatory response. The tissue eosinophilia is mainly a result of the cured IL-5 of the allergen specific T-Ly -11.According to literature, the values of ECP are , 12: higInterleukin 5 (IL-5) has the central part in the maturation of eosinophils (Eo), with their release, activation, extension of survival and degranulation , 11.The aim of our study was to show the importance of inflammation as key of asthma; to determine some parameters as markers of the inflammatory process; to show the significance of Eo and ECP, while measuring the level of a specific type of inflammation; to make an investigation of ECP as a marker of antigen exposition; to make an investigation if the value of ECP could be an indication of the effect of the treatment and to determine the significance of IL-5 in the inflammatory chain and the evolution of asthma.We have investigated 77 subjects at the age among 23 and 79 years old. At each subject, there were complete examinations made for proper diagnosis and categorization.st group: 44 patients with asthma (15 with intrinsic asthma and 29 with extrinsic asthma);1nd group: 21 patients with chronic obstructive bronchitis;2rd group: 12 healthy subjects.3The classification was according to the actual version of the GINA guidelines   and actuIn all subjects we were investigated: total number of eosinophils (Eo); eosinophilic cationic protein (ECP); Interleukin 5 (IL-5) and allergy skin tests.The results were statistically analysed according to the ANOVA test, Kruskal-Wallis test, Wilcoxon and Man Whithey test. The significances value were taken p<0.05 and a highly significant p < 0.01.In our study there is significant deference between the average values of Eo in asthmatics versus average values of Eo in patient with obstructive bronchitis and healthy controls .Total number of eosinophils was significantly increased in patient with intrinsic versus extrinsic asthma .Average values of ECP were significantly increased in asthmatics versus ECP in patient with obstructive bronchitis and group of healthy controls .Values of ECP were significantly increased in patients with intrinsic versus extrinsic asthma .Average values of IL-5 were significantly increased in asthmatics versus IL-5 in the group of patients with obstructive bronchitis and group of healthy controls .Values of IL-5 were significantly increased in patients with intrinsic versus extrinsic asthma .The values of ECP in patients with asthma were significantly decreased after treatment with corticosteroids .The values of IL-5 in patients with asthma were significantly decreased after treatment with corticosteroids .Activated Eo in asthmatics delivered granulated proteins, like ECP detected in bronchial biopsies, BAL, sputum and blood  . AccordiWe noticed significant correlation between the values of ECP and skin allergy tests as it is known in literature. Our results did not show significant correlation between ECP, FEV1 and symptom score. According Ronchi serum ECP values were increased in asthmatics versus healthy controls, but there was not the correlation with clinical symptoms and pulmonary function . In our There is significant deference between the values of IL-5 in asthmatics versus group of patients with bronchitis and healthy controls. IL-5 suggests inflammation in asthma , 18. TheThe values of ECP and IL-5 have tendetion of decrease after corticosteroid therapy. In literture there are several studies according which anty-inflammatory therapy decrease the values of the markers as ECP and IL-5.In conclusion, the investigations that were made, confirm the concept that asthma is a chronic inflammatory disease. Determining and measurements of the markers of inflammation contributes to the diagnosis of asthma, monitoring the course of disease and contribute in the proper treatment of the asthmatic patients.There is significant difference of the values of the eosinophils in asthmatics versus patients with bronchitis and healthy subjects. Total number of eosinophils versus patients with obstructive bronchitis is not different as in healthy controls.There are significantly higher values of ECP in asthmatics versus patients with obstructive bronchitis and healthy subjects. Correlation between ECP and FEV1 and ECP and symptom score were not found. ECP could be higher in non-asthmatic atopic patients like patients with rhinitis, egzema, urticaria. There is significance in average values of IL-5 in asthmatics versus values of IL-5 in patients with obstructive bronchitis and healthy subjects. We found positive correlation between Eo and ECP and Eo, ECP and IL-5 with allergy tests. Values of ECP and IL-5 decreased after therapy with corticosteroids.Values of markers of inflammation have their importance together with other tests ad criteria for proper diagnosis and treatment of asthma.


Introduction. The prognostic value of age, physical status, and duration of surgery on perioperative course has been extensively studied. However, the impact of deep hypnotic time  has not been well evaluated. Methods. We designed an observational study to clarify the relative influence of deep hypnotic time (DHT) on outcome. Eligible participants were mentally stable patients over 18 years old scheduled for elective major abdominal surgery. In total, 248 patients enrolled. Data were analyzed using Fisher's exact test and multiple logistic regression. Results. Five variables  showed statistically significant association with complications, when examined independently. However, when all variables were examined together in a multiple logistic regression model, age and comorbidity were no longer associated with outcome. DHT, hypotension, and duration of surgery were significant predictors of “complications,” and “hypotension” was a significant predictor of prolonged hospital stay (P < 0.001).  Conclusion. Deep hypnotic time emerged as a new factor associated with outcome, and its impact compared to other factors such as age, surgery duration, hypotension, and comorbidity is redefined. Monitoring and managing depth of anesthesia during surgery are important and should be part of careful operation planning. The role of physical status and age on patient outcome after major surgery has been thoroughly studied . It is wIn 2005, an innovative study by Monk et al. attempted to identify new variables such as duration of deep hypnosis defined as low Bispectral Index values and duration of intraoperative hypotension as potential risk factors for long-term (one-year) postoperative mortality . These aIn a previous study, we demonstrated that persistent hypotension during surgery is a significant risk factor for postoperative complications . In addiFollowing approval by the Institution Ethics Committee, we conducted this prospective observational study at a tertiary care university hospital. The study included patients scheduled for major elective abdominal surgery (expected duration > 2 hours) over a two-year period. Inclusion criteria were mentally healthy patients who had given written informed consent and elective major abdominal surgery. Exclusion criteria were pregnancy, inability to comprehend or follow orders, history of psychiatric disease or mental instability, and use of drugs that could influence the central nervous system. Demographic and clinical data for every patient were entered in a secure electronic database. Variables recorded included age, gender, marital status, educational level, alcohol and tobacco use, body mass index (BMI), and patient physical status characteristics. Physical status was evaluated using three widely used methods: the New York Heart Association (NYHA) Functional Class, the ASA Physical Status, and the Charlson Comorbidity Index in order to establish comorbidity scores on admission to hospital. All data were collected by trained physicians and were reviewed for reliability and completeness immediately after collection.In the operating room, monitoring included five lead ECG and continuous blood pressure monitoring using a radial arterial line. Before induction of anesthesia, a low thoracic (T9–T12) epidural catheter was inserted and was used for perioperative analgesia, with incremental administration of 0.5–1.5 mL/dermatome (depending on patient age) of a preservative-free solution containing ropivacaine 3.75 mg/mL and fentanyl 2 mcg/mL. Anesthesia was induced with 100 mcg fentanyl, 1-2 mg midazolam, and 1.5–2 mg/kg propofol and was maintained with 1.1–1.2 MAC sevoflurane. Cisatracurium was used to facilitate tracheal intubation and maintain neuromuscular blockade. A Bispectral Index monitor was used to measure depth of anesthesia . In the recovery room, a programmable infusion pump was used to administer a mixture of ropivacaine 1 mg/mL and fentanyl 2 mcg/mL for patient-controlled epidural analgesia, using the following settings: basal infusion 7 mL/hour, bolus 3 mL, and lockout time 30 minutes. The postoperative analgesic protocol also included intravenous paracetamol 600 mg every 6 hours and intravenous parecoxib 40 mg twice a day.Perioperative data, including surgery type and duration, anesthetic drugs used , hemodynamic variables, fluid and blood product administration, and urine output were recorded on a standardized data collection sheet. BIS values were automatically transferred to an electronic database and were then transferred to the main database. Recording was meticulously inspected for artifacts, such as high BIS values with muscle activity, and all artifacts or obviously erroneous readings were corrected manually. The summation of time with BIS values lower than 40 was recorded as total DHT in minutes. We chose to use BIS < 40 as cutoff value for DHT, because 40 has been used as cutoff value in clinical studies, including the B-Aware trial, has been shown to be associated with morbidity and mortality , and is Patients were assigned to two groups based on intraoperative DHT: (a) patients with DHT = 0 and (b) patients with DHT > 0. Hypotension was defined as mean arterial pressure (MAP) < 60 mm Hg or as MAP < 70 if MAP had decreased by more than 30% below baseline . The sumPatients were evaluated daily until death or hospital discharge. Postoperative data were recorded on standardized data sheets and included pain assessed with visual analog scale, additional analgesic requirements, patient sedation, nausea or vomiting, itching, mobilization, oral intake of liquids or food, epidural catheter removal, medications administered or skipped, sleep disturbances, and complications. All patients were assessed by interview, clinical examination, medical record review, and consultation with their primary in-hospital healthcare provider. For the purposes of this study, complications were defined as the need for any acute medical intervention or treatment. Patients were divided into two groups depending on whether or not they had postoperative complications. The scope of complications was not restricted to major complications but also included lesser forms of morbidity which can influence length of hospital stay and healthcare cost.For the purposes of data analysis, we treated gender as a qualitative characteristic and defined clear cutoff points for all other variables that were analyzed. The cutoff point for age was 65 years, since 65 is the retirement age in most Western societies and also because morbidity and mortality increase with age > 65 , 16. WitP < 0.05.Statistical analysis was performed using the SPSS version 17.0 statistical software package . For the purposes of this analysis, patients were divided into two groups for each independent variable  using clear cutoff points, as described above. Two variables  were assessed as dependent variables. Categorical variables were compared between groups using Pearson's chi square or Fisher's exact test. Multivariate logistic regression was then used to analyze all variables together and adjust the specific risk that each independent variable attributed to the dependent variable. In a univariate basis, the (unadjusted) risk from any independent variable could be attributable to some other independent variable, if an interaction existed. Therefore, all variables  were analyzed together using logistic regression in order to improve prediction accuracy for the dependent variable. The Hosmer-Lemeshow goodness of fit test was used to assess how well each logistic regression model fits the data. In addition, collinearity diagnostics  were used in an attempt to assess if collinearity between predictor variables was a concern for the regression models. Observed associations are expressed as adjusted odds ratio (OR) with 95% confidence intervals (CI). An effect was considered significant when In the 2-year study period 248 consecutive patients  enrolled. Demographic and clinical data are summarized in Tables Five variables  showed significant association with “complications” by Fisher's exact test . Of all The five variables found to be significantly associated with “complications” were analyzed further, using two multivariate logistic regression models.P > 0.05 , thus confirming that observed and expected rates were well calibrated and the models were well adjusted. When we corrected (adjusted) the influence of each factor by entering all variables into the multivariate logistic regression model , thereby implying that the variance of the dependent variable “complications” could be attributed to factors such as surgery duration, hypotension, or DHT. The risk of complications increased 4.170-fold  if a patient was under deep anesthesia, increased 2.946-fold  with lengthy surgery, and increased 4.670-fold  with intraoperative hypotension. A higher percentage of patients under deep anesthesia developed complications (56%) compared to patients not under deep anesthesia (5.2%). Furthermore, 36% of patients with any complication stayed longer in hospital compared to those who had none (0.8%).In the first model, “complications” was the dependent variable, whereas in the second model “hospital stay” was the dependent variable. In both models the Hosmer and Lemeshow test for goodness of fit showed on model , the varP < 0.001). However, when the variable “complications” was excluded from the model, because of concerns about multicollinearity, “hypotension” was the only significant predictive variable (P < 0.001) increased risk of prolonged hospital stay.In the second model, “hospital stay” was analyzed as dependent variable. When the variable “complications” was entered in the analysis as independent variable together with all other variables, it was the only variable that remained significant  profile. Inflammatory response plays a major role in tissue repair and patient healing and can lead to immune system suppression, hypoperfusion, coagulopathy, and complications such as infection, ischemia, multiple organ failure, sepsis, or death –24. It hPulmonary and gastrointestinal dysfunction rather than cardiac complications were the most important complications in our study , whereasLimitations of this study include study design, as observational studies are generally considered less rigorous than prospective clinical trials. In addition, it is possible that our sample may not be representative of the population and the sample size may not be large enough to show significant effects . We atteIn conclusion, our study suggests that complications are common after elective major abdominal surgery, are related to underlying pathology and surgical factors, and are influenced by anesthetic management. Persistent low BIS values and prolonged hypotension are associated with bad outcomes but may be masked by confounding factors such as age or physical status. Considering the limitations of our study, well-designed prospective randomized trials are needed to better evaluate the impact of DHT and determine whether maintaining a lighter state of anesthesia can improve outcome.


Studies suggest that statins have pleiotropic effects, such as reduction in bloodpressure, and improvement in endothelial function and vascular stiffness.To analyze if prior statin use influences the effect ofrenin-angiotensin-aldosterone system inhibitors on blood pressure, endothelialfunction, and vascular stiffness.Patients with diabetes and hypertension with office systolic blood pressure≥ 130 mmHg and/or diastolic blood pressure ≥ 80 mmHg had theirantihypertensive medications replaced by amlodipine during 6 weeks. They were thenrandomized to either benazepril or losartan for 12 additional weeks whilecontinuing on amlodipine. Blood pressure (assessed with ambulatory blood pressuremonitoring), endothelial function , andvascular stiffness (pulse wave velocity) were evaluated before and after thecombined treatment. In this study, a post hoc analysis was performed to comparepatients who were or were not on statins .The SU group presented a greater reduction in the 24-hour systolic blood pressure, and in the brachial artery flow-mediateddilation  when compared with the NSU group . There was nostatistically significant difference in pulse wave velocity .Combined use of statins, amlodipine, and renin-angiotensin-aldosterone systeminhibitors improves the antihypertensive response and endothelial function inpatients with hypertension and diabetes. Studies show that these drugs havepleiotropic effects, such as improvement in endothelial vasodilation response, increasein nitric oxide bioavailability, and reduction in endothelin levels5.In addition, this effect was greater in patients undergoing pharmacological treatmentfor hypertension, suggesting a potential synergistic mechanism between theantihypertensive drugs and the statins.One of the pleiotropic effects of the statins is the reduction in blood pressure (BP). Arecent meta-analysis that included more than 20 thousand patients showed a reduction of2.6 mmHg in systolic BP with statinsThe aim of this study was to analyze whether the use of statin would influence theeffect of renin-angiotensin-aldosterone system (RAAS) inhibitors on BP, endothelialfunction, and vascular stiffness in a population of patients with hypertension and type2 diabetes mellitus (T2DM).Universidade do Estado do Riode Janeiro (UERJ). The inclusion criteria were a previous diagnosis ofhypertension and T2DM, age between 40 and 70 years, and systolic BP ≥ 130 mmHgand/or diastolic BP ≥ 80 mmHg. The exclusion criteria included resistanthypertension, insulin use, stages 4 and 5 chronic kidney disease, previous history ofmyocardial infarction and/or stroke, stages C and D heart failure, atrialfibrillation, symptomatic peripheral arterial disease, retinopathy with reducedvisual acuity, nephrotic syndrome, and symptomatic diabetic neuropathy. The study wasapproved by the Research Ethics Committee of Hospital Universitário PedroErnesto (UERJ) under the number 01539612.6.0000.5259. All participantsread and signed a free and informed consent form. The study is registered atClinicalTrials.gov under the number NCT01603940.Patients selected for this project had hypertension and T2DM and were followed up asoutpatients in the internal medicine service at The study was an open randomized clinical trial, with two active treatment groups andwithout use of placebo. At visit 1, patients underwent a clinical evaluation and hadtheir antihypertensive medications replaced by amlodipine 5 mg/day. Visit 2 wasconducted 6 weeks later and included clinical and laboratory evaluation, ambulatoryBP monitoring (ABPM), and assessment of endothelial function and vascular stiffness.During this visit, the patients were randomized to benazepril 10 mg/day or losartan50 mg/day while continuing on amlodipine. Two reevaluations (visits 3 and 4) wereperformed at intervals of 4 weeks for adjustment of BP, which was aimed at values< 130 x 80 mmHg. The antihypertensive medications in the study could have theirdoses doubled (benazepril 20 mg and losartan 100 mg), and the addition ofhydrochlorothiazide 25 mg/day was allowed on visits 3 and 4. The dose of amlodipinewas maintained at 5 mg/day. At the end of 12 weeks of treatment with RAAS inhibitors,the final visit (visit 5) was conducted, and the assessments performed on visit 2were repeated. The dose of the T2DM medication was maintained constant. Adherence totreatment was assessed by a comparison between actual and expected tablet intake. Weevaluated the use of antihypertensive drugs, statins, acetylsalicylic acid, and oralhypoglycemic agents. Patients were considered as having good adherence when theactual intake was equal to or greater than 80% of the expected intake.On visit 1, patients were classified as users or non-users of statin . Throughout the 18 weeks of the study, the statin wasmaintained in those who were already taking it, and was not allowed to be started inthose who were not taking it. The statin used in the study was simvastatin at a doseof 20 mg/day. All patients in the SU group were taking a statin for more than 12weeks as prescribed by the physician assistant.All tests were conducted in the morning after a 12-hour fast. Patients wereinstructed to not smoke, consume caffeine, or practice physical activity within 24hours of the tests. The rooms were air-conditioned and had the temperature set toaround 23 ± 2ºC and the air relative humidity between 50 and 70%. The patientswere instructed to take the antihypertensive medication 1 to 2 hours prior to theevaluation.For office BP measurements, the patients sat down and rested for 10 minutes. We useda semiautomatic, calibrated equipment, model HEM-705CP , with the cuff adjusted to the arm circumference. Three measurements wereobtained in each arm, and their respective mean values were calculated. The highestmean value was used for analysis of the data.ABPM was assessed with SpaceLabs 90207 . The evaluation wasscheduled to start between 8 and 9 a.m. and last for at least 24 hours. The BP wasmeasured every 20 minutes between 6 a.m. and 11 p.m., and every 30 minutes between 11p.m. and 6 a.m. The test was considered satisfactory when at least 70% of thereadings were valid, with a minimum of 16 readings during the daytime and 8 readingsduring sleep and no more than two hours without measurements. We considered the sleeptime as that reported by the patient in the activities diary. Nocturnal BP reductionwas calculated as [(awake mean BP - sleep mean BP) /awake mean BP] x 100.-1.154*Age-0.203. C-reactive protein was determined by nephelometry . To measure microalbuminuria, we used the albuminuria/creatinuria ratiodetermined in a sample of first-morning urine.Venous blood samples were collected after a 12-hour fast for biochemical tests. Whentriglyceride levels were < 400 mg/dl, we calculated the LDL-cholesterol fractionwith the Friedewald formula. Glomerular filtration rate (eGFR) was estimated with theMDRD formula: eGFR = 186*Creatinine6. The examiner was blinded to thepatients’ treatment. We used the ultrasound equipment Vivid 3  with a 10 MHz high-resolution linear transducer. The patientwas placed in the supine position, with the right arm abducted. After locating thebrachial artery, the transducer was placed on the anteromedial surface of theright arm, perpendicular to the axis of the arm, 2 to 5 cm above the antecubitalfossa, on the topography of the brachial artery. The measurements were obtained atthe end of the diastole (R wave on the electrocardiogram). Ischemia was induced byinflation of the cuff 50 mmHg above the systolic BP during 5 minutes. The largestdiameter of the artery was recorded 30, 60, and 90 seconds after deflation of thecuff. FMD was calculated as the variation of the largest diameter of the artery inrelation to its baseline measurement.The test was conducted according to the latest guidelines on the method7. The distances between the pulses were obtained directlywith a tape measure. The carotid-femoral distance was multiplied by 0.8, and thisvalue was used by the equipment for the calculation. Two PWV measurements werecarried out, and when the difference between them was greater than 0.5 m/s, athird measurement was obtained. The average of these measurements was used for theanalysis.Pulse waves were obtained with the equipment Complior SP . The transducers were positioned on the right carotid, right radial(crPWV), and right femoral (cfPWV) arteries8. This software calculated thecentral systolic BP, central pulse pressure (PP), augmentation pressure (AP), andaugmentation index (AIx). Two measurements were obtained, and when the differencebetween them was greater than 10%, a third measurement was performed. The averageof these measurements was used for the analysis.Pulse waves at the right radial artery were obtained with a tonometer  calibrated according to the pressure in thebrachial artery. Analysis of this arterial wave by applanation tonometry wasperformed to derive central aortic pressures and other hemodynamic parametersusing the system SphygmoCor 9. Thestatistical analysis was performed using a significance level of 5% and the softwareStatistica 12 .The data are presented as median (interquartile range) for numeric variables anddistribution of frequencies and proportions for categorical variables. TheMann-Whitney test and Fisher's exact test were used to compare numerical andcategorical variables, respectively. The Wilcoxon test was used for paired comparisonof variables. Based on a maximum type I error of 0.05 and a standard deviation of3.5%, a sample size of 14 patients in each group would have 80% power to find adifference ≥ 4% in FMD. The calculation of the sample size used the expectedFMD difference between the benazepril and losartan groups according to the design ofthe original studyAccording to the inclusion criteria, 47 patients were selected, and 30 were randomizedto one of the treatment groups . OnvisiIn the analysis of the office BP in the SU group, there was a non-significantdecrease in diastolic (8.0 mmHg or 9% reduction) and systolic BP (11.6 mmHg or 8%reduction), although no statistical significance was observed . In the There was a similar percentage of patients using ACEI (54 vs 41%) and ARB (46 vs 59%)in the SU and SNU groups, and a similar proportion of patients who required dosageadjustment of these medications  and/or association ofhydrochlorothiazide for BP control .The SU group presented a greater FMD response and a higher reduction in systolicaortic BP when compared with the NSU group . There w13. The present study showed thatsimvastatin improves endothelial function and increases the antihypertensive effect ofACEI or ARB in association with amlodipine in patients with hypertension anddiabetes.The most recent guidelines recommend that patients with T2DM should use RAAS inhibitors(ACEI or ARB) and statins14. In addition, a synergism is alsospeculated between statins and RAAS inhibitors through reduced expression of type 1angiotensin receptors and blockade of intracellular pathways associated with angiotensinII action, both caused by statins15.Many of these benefiting mechanisms of the statins are independent of the reduction inLDL-cholesterol. In the present study, the reduction in BP and improvement inendothelial function occurred despite statistically similar LDL-cholesterol values inthe SU and NSU groups.The antihypertensive mechanisms of the statins are probably due to an increase in nitricoxide bioavailability with an increase in endothelium-dependent vasodilation response,and a reduction in endothelin-1 concentration and free radicals formation5 observed a mean systolic BP reduction of 2.6 mmHg in patients onstatins, even in those without a diagnosis of hypertension5. In hypertensive patients, the systolic BP may reduce upto 5.8 mmHg5. In diabetics, the studyestimated a reduction of 6.5 mmHg in the systolic BP and 4.0 mmHg in the diastolicBP5. Another meta-analysis byStrazzullo et al. also examined the antihypertensive effect of statins and observed anaverage reduction of 4.0 mmHg in the systolic and 1.2 mmHg in the diastolic BP16. Strazzullo et al16 observed in a subgroup of diabeticpatients a reduction of 3.7 mmHg in the systolic and 0.8 mmHg in the diastolicBP16. None of these studiesinvestigated the association of statins with ACEI or ARB.In a recent meta-analysis, Briasoulis et al.17 studied theinteraction between ACEI and statins and observed that the group receiving a combinationof both showed greater BP reduction when compared with the group receiving ACEI alone17.However, Mancia et al18 observed noreduction in BP with the association of pravastatin and fosinopril. Koh et al19 evaluated the combination of losartanand ramipril with simvastatin and did not observe greater BP reduction with thisassociation20. In the present study, the association of benazepril orlosartan in patients already receiving simvastatin and amlodipine promoted greaterreduction in systolic and diastolic BP, both in casual measurements in the office and in24-hour measurements.Spósito et al21 recently published ameta-analysis on the effects of statins in brachial artery FMD in patients with T2DM.The final result showed an improvement of 0.94%  inabsolute FMD values. Koh et al19conducted a study in which patients were randomized to three groups: ramipril,simvastatin, or a combination of both. Their cohort, comprised of 76% of hypertensivepatients, included individuals with a mean age of 60 years, T2DM, and LDL-cholesterol> 100 mg/dL. The authors observed that the combined use of statins and ACEI promoteda greater increase in FMD than each medication alone. In contrast, no improvement in FMDwas observed by Van Venrooij et al22with atorvastatin, and Beishizen et al. with cerivastatin and simvastatin23. Tan et al24, in apopulation with T2DM and dyslipidemia, observed an improvement in FMD with atorvastatinwhen compared with placebo. In the present study, which assessed a population ofdiabetic patients with hypertension, only those already using simvastatin had astatistically significant improvement in brachial artery FMD when benazepril or losartanwere associated.Zhang et al25 evaluated the use ofsimvastatin in patients with hypertension and dyslipidemia and observed improvements inPWV, aortic BP, and AIx. However, no improvement in aortic pressure was observed withatorvastatin by William et al26 inhypertensive patients and Fasset et al27 in patients with chronic renal failure27. Raisone etal28 were one of the few toobserve worsening in PWV with atorvastatin. In contrast, Pirro et al29 and Maki-Petaja et al30 observed improvement in PWV withstatins. So far, there are no studies on the effects of statins on aortic pressure andPWV in diabetic patients with hypertension. The present study showed a reduction incentral aortic pressure, in parallel with reductions in office BP and ABPM insimvastatin users, although no statistically significant changes were observed in AIxand PWV.Kanaki et alpost hoc analysis, thegroups were not randomized according to statin use. In addition, approximately half ofthe study sample had no prior use of statin. Although it was not possible to evaluatethe reasons, the group without prior use of simvastatin could have pre-selected patientswith poor adherence to antihypertensive drugs before recruitment for the study. Thesepatients could have BP levels above the recommended target, higher prevalence ofvascular lesions, and as a result, inadequate response to antihypertensive drugs orlower improvement in endothelial function with the treatment when compared with thegroup of statin users. There was also a trend towards more male patients in the group ofsimvastatin users. We believe that this fact had little relevance, because despitehaving a higher cardiovascular risk, this group had the greatest benefit in BPreduction. The open use of the medication is also a limiting factor. However, theresults were reinforced by the BP reduction in the ABPM and in central aorticmeasurements, since these methods are known to be minimally influenced by the placeboeffect.The study has some limitations. Since this is a Prior use of statin increases the antihypertensive effect and improves the endothelialfunction in hypertensive patients with T2DM treated with amlodipine associated with anRAAS inhibitor. Randomized studies with larger samples and 2x2 design are needed toevaluate the interaction between statins and RAAS inhibitors.


Blood samples were taken at a first follow-up 6 years after the stroke. At a second follow-up, additional arterial events were registered for 140 patients, new blood samples were taken, and measurements of cIMT and blood pressure (BP) were performed. Unadjusted logistic regression analysis showed that cIMT ≥1 mm was associated with age, male gender, additional arterial events, BP, cholesterol, sedimentation rate, haemoglobin, triglycerides, creatinine, glycolysed haemoglobin (HbA1c) and FcγRIIIB-NaII/NaII. Adjusted backward stepwise logistic regression showed significance for age (odds ratio (OR) 1.13, 95 % confidence interval (CI) 1.04 to1.23, p = 0.003), male gender , HbA1c  and FcγRIIIB-NaII/NaII . In this long-term follow-up study of patients with ischemic stroke at a young age, FcγRIIIB-NaII/NaII was identified as a possible contributing factor for cIMT ≥1 mm together with known risk factors, such as age, male gender, systolic BP, additional arterial events and HbA1c.Lifestyle risk factors, inflammation and genetics play a role in the development of atherosclerosis. We therefore studied Fc gamma receptor (FcγR) polymorphisms, interleukin (IL)-10 polymorphisms and other biomarkers related to carotid intima-media thickness (cIMT) in patients with ischemic stroke at a young age. Patients were evaluated 12 years after stroke occurrence. Patients ( Atherosclerosis is an inflammatory disease with both lifestyle and genetic risk factors. Receptors for the Fc portion of immunoglobulin G (IgG), known as FcγRs, are the binding link between humoral and cellular immunologic reactions. Endothelial cells express FcγRs and pro-inflammatory mediators, such as immune complexes and C-reactive protein, that can activate FcγR-dependent pathways that lead to oxidative burst, degranulation, phagocytosis, cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC) . The FcγInterleukin (IL)-10 is another important immune regulator that may be important in the pathogenesis of atherosclerosis . Low levWe have previously reported data from 232 patients who had suffered acute arterial ischemic stroke at ages between 15 and 49 years and found a 10-fold higher mortality rate and a 5-fold increased vascular morbidity rate among long-term survivors compared with controls . After aChlamydia pneumoniae antibodies. For multivariate analysis, we related these parameters and other relevant clinical information, such as age, gender, blood pressure, number of smoking years and additional arterial events other than the index stroke to cIMT.In the present study, we analysed these retrospectively selected young stroke patients with the following biomarkers: C-reactive protein (CRP), homocysteine, cholesterol and triglycerides, sedimentation rate (SR), haemoglobin (Hb), glycolysed haemoglobin (HbA1c), creatinine, leukocytes, thrombocytes, and FcγR polymorphisms, IL-10 polymorphisms and We identified 232 patients of less than 50 years of age with first-ever ischemic stroke between 1988 and 1997 from five acute care hospitals in Hordaland County in western Norway . The meaFcγR polymorphisms were analysed as previously described . In brieDNA was extracted from whole-blood samples and examined for IL-10 polymorphisms at positions −1082 (G/A), −819 (T/C) and −592 (A/C) using PCR and gene sequencing as previously described . The IL-Right- and left-sided cIMT examination of near and far walls of the distal common carotid artery (CCA), bifurcation (BIF) and the proximal internal carotid artery (ICA) were performed at four predefined angles. CCA, BIF and ICA vessel segments were identified by using the tip of the flow divider as an internal landmark. Segments had a length of 10 mm, and the mean value was measured. A total of 5944 segmental mean IMT or plaque values were obtained. The maximum value of 48 possible individual cIMT measurements was used as outcome variable. cIMT results were used to divide subjects into two cIMT categories: <1.0 and ≥1.0 mm. Our 140 patients had a mean age of 53 years, the oldest patients were 65 years old at that final clinical follow-up, and total maximum IMT of 1.0 mm was chosen as measure of dichotomisation due to literature , 23. MetC. pneumoniae antibodies. p values were calculated from the likelihood ratio test. Multiple logistic regression analysis was done using the dichotomised cIMT results as outcome in a backward stepwise selection process retaining variables significant at the 5 % level in the final model. Multiple linear regression analysis was done using continuous maximum cIMT results as outcome in a backward stepwise selection process retaining variables significant at the 5 % level in the final model. The analysis was performed using the Statistical Package for Social Sciences (SPSS) 20. p values <0.05 were considered significant.Biomarkers were compared between the groups with cIMT <1 mm and cIMT ≥1 mm. Age, additional arterial events, systolic and diastolic BP, number of smoking years, SR, HbA1c, creatinine, Hb, leukocytes, thrombocytes and triglycerides were used as results from the second follow-up. CRP, homocysteine, total cholesterol, LDL cholesterol and HDL cholesterol were used as results from the first and second follow-up. Categorical variables were analysed with 3×2 chi-square test and Fisher’s exact test, and Wilcoxon–Mann–Whitney test was used for continuous variables. Unadjusted logistic regression analysis of the dichotomised cIMT was used to evaluate the potential relationships with FcγR polymorphisms, IL-10 haplotypes with ATA/ATA as reference variable and with C. pneumoniae antibodies, sedimentation rate and C-reactive protein were not associated with cIMT ≥1 mm.Table cIMT ≥1 mm was associated with total cholesterol, LDL cholesterol and homocysteine at the first, but not at the second, follow-up Table . HaemoglThis is the first clinical study of long-term survivors of ischemic stroke at a young age to evaluate follow-up blood samples and carotid IMT measurements. The mean observation time was nearly 12 years, and the cIMT measurements also included plaques, as we measured at standardised sites in order to perform a staging of the carotid artery wall disease.C. pneumoniae antibodies and cIMT. The association between C. pneumoniae antibodies and cardiovascular disease is unclear [We have studied immunological and non-immunological risk factors for cIMT as parameters for carotid atherosclerosis. Our results showed no association with  unclear . FurtherWe also studied the FcγR and IL10 genotypes as they have been linked to atherosclerosis. The frequencies of the FcγR and IL-10 genotypes among our patients were comparable to 272 healthy controls previously reported from western Norway , 26.We found a significant association between FcγRIIIB NaII/NaII and cIMT ≥1 mm. However, linear regression analysis showed non-significant results for continuous maximum cIMT values. Others have reported that soluble FcγRIIIA is associated with maximum cIMT , that thThe present study is the first to show a possible association with the low-affinity FcγRIIIB receptor and atherosclerosis as measured by cIMT ≥1 mm. FcγRIIIB, as well as FcγRIIA and FcγRIIIA, activates immune functions such as phagocytosis, ADCC and release of inflammatory mediators and superoxide radicals . FcγRIIIStimulation of activating FcγRs on macrophages has been found to induce an anti-inflammatory immune response with increased IL-10 production . It has We have also studied the polymorphisms of IL-10 since 50–70 % of the variance in IL-10 levels is genetically determined . HoweverWe found that HbA1c, SR, haemoglobin and creatinine were risk factors in the unadjusted logistic regression analysis, but only HbA1c showed significance in the final adjusted logistic and linear regression analysis. Elevated blood glucose is a known risk factor for endothelial dysfunction and early atherosclerosis , and theThe strength of our study is that we have a very well characterised patient population and clinically related immunological and non-immunological parameters were available for analysis. The frequencies of FcγR and IL-10 polymorphisms in our patients are similar to those observed in population studies performed from the same area , 26. We The weakness of our study is that patients were included retrospectively and selection was affected by death and a 23 % dropout rate of long-term survivors. The 23 % dropout rate at our last clinical follow-up, without any missing data concerning the dead-alive status, is, however, comparable to that of other clinical studies with follow-ups after 10 years .Differences occurred after logistic and linear regression analysis concerning significancies related to cIMT in this study. However, age and HbA1c were in both analyses related to cIMT, and these parameters are well-known risk factors for increased cIMT and atherosclerosis. Male gender and increased BP are also well-known risk factors for atherosclerosis and additional arterial events, even if the results by the two types of regression analysis were slightly different. A new finding in this study is that FcγRIIIB-NAII/NAII appeared as a significant factor for increased cIMT ≥1 mm. However, further prospective studies with higher sample sizes are necessary to confirm these results.In conclusion, we found that in patients with ischemic stroke at a young age, FcγRIIIB-NAII/NAII may be a contributing factor for increased cIMT together with known risk factors, such as age, male gender, systolic BP and increased HbA1c.


Atherosclerotic plaques with a low content of connective tissue proteins are believed to have an increased risk of rupture and to give rise to clinical events. The aim of the present study was to investigate if the content of elastin, collagen and of the matrix metalloproteinase (MMP) −1, −3, −9 and −12 in plaques removed at surgery can be associated with the occurrence of ipsilateral symptoms.The atherosclerotic plaques of 221 patients undergoing carotid endarterectomy were analyzed and their composition was related to the incidence of preoperative, intraoperative and postoperative neurological events.Elastin, collagen and MMP-12 contents were lower in males and diabetic patients. Elastin (P .010), MMP-3 (P .008) and MMP-9 (P < .0001) were lower, while MMP-1 (P .004) and MMP-9 (P .002) were higher in plaques of patients with preoperative symptoms, even after correction for the time between the occurrence of symptoms and surgery. Elastin and MMP-12 decreased  while MMP-1  and MMP-9 (r = .21 P <.0001) increased with age. After a mean follow-up time of 39.6 ± 16.6 months, 7.7% of patients had suffered one or multiple ipsilateral neurological events. Patients with plaque elastin levels lower than the median (52 mg/g) had increased post-operative incidence of ipsilateral stroke (P for trend 0.009 using Log Rank Chi-square test). This finding was confirmed when controlling for age, gender, hypertension, diabetes, smoking, pre-operative symptoms and statin usage in a Cox Proportional Hazard model .These observations support the concept that elastin may be important for plaque stability, and suggest that a low plaque content of elastin is associated with a higher risk for ipsilateral stroke. Several randomized trials –3 have cAn unstable plaque phenotype can, in theory, result in the occurrence of intraoperative symptoms as caused through mechanical stress by vessel manipulation during CEA. Furthermore, the extension of the atherosclerotic lesions in diseased arteries is beyond the carotid bifurcation. Therefore, we theorized that reduced plaque levels of the components of the ECM could be associated with an increased risk of related events.In order to prove this hypothesis, the elastin, collagen and MMPs content of human carotid plaques harvested during CEA was analyzed and correlated with the incidence of early and late neurological events, taking account of comorbid conditions and the patients´ neurologic presentation.Two hundred-twenty-one patients aged 69.7 ± 8.4 years who underwent CEA between October 2005 and October 2009 at our Vascular Department were included in this study. Written informed consent was given by each patient, and the study was approved by the local ethical committee (Decision numbers 472/2005 and 16209/2012).amaurosis fugax (AF), transient ischemic attacks (TIAs) or stroke in the six months prior to surgery.The degree of carotid artery stenosis was preoperatively assessed with ultrasound based on flow velocities as previously validated.  IndicatiInformation about comorbidities and past medical history was obtained through standardized preoperative interviews and review of the medical records. Comorbidity factors such as hypertension, diabetes, peripheral artery disease (PAD), and smoking, as well as the body mass index (BMI), serum levels of triglycerides (TG), low-density lipoproteins (LDL) and high-density lipoproteins (HDL) were recorded. Intraoperative and perioperative events were analyzed from reviewing medical charts. The Swedish national inpatient health register was analyzed in order to identify postoperative neurologic events, with corresponding International Classification of Diseases, Tenth Revision (ICD-10) codes G45 and G46 from October 2005 to December 2010. This is a nationwide validated register in which more than 99% of all somatic (including surgery) and psychiatric hospital discharges are registered. [All neurological ipsilateral events  were identified as described above and registered and analyzed singularly. Events occurring in the first 24 hours postoperatively were considered as procedure-related and assumed as intraoperative for analysis.Patients suffering more than one episode of the same event  were classified as suffering multiple events. In these cases only the first chronological event was taken into account in the survival analysis.The plaques were processed as described previously. –11 BriefTo measure elastin, a Fastin Elastin assay was used . Plaque homogenate (30μl) was mixed with cooled Elastin Precipitating Reagent (1000μl), incubated and centrifuged . Supernatants were discarded, and Fastin Dye Reagent and ammonium sulfate (1000 μl+100μl) were added. The samples were mixed, centrifuged , supernatants were discarded and Fastin Dissociation Reagent (1000μl) was added. The samples were measured on a Tecan Elisa plate reader (492 nm).When measuring collagen, Sircol soluble collagen assay was used . Samples (5μl) were added to 995 μl of Sircol Dye reagent, mixed, incubated (30 min) and then centrifuged . This assay detects acid-soluble and pepsin-soluble collagens types I to V. The supernatants were discarded and alkali reagent (1000μl) was added. The samples were measured (absorbance 540nm) on a Tecan Elisa plate reader.MMPs −1, −3 and −9) were analyzed in homogenate supernatants (25 μl) using Mesoscale human MMP ultrasensitive kit , according to the manufacture´s instructions.MMP-12 levels were analyzed by the Proximity Extension Assay (PEA) technique using the Proseek Multiplex CVD96x96 reagents kit  as previously described. [Results were normalized to the wet weight of the plaque. Continuous variables are presented as mean ± standard deviation (SD) when not stated otherwise, while categorical variables are presented as percentages. Pearson´s Chi-square test was used for categorical variables. Student’s t-test and Pearson´s correlation were used for continuous variables whenever normally distributed, while a Mann-Whitney U test and Spearman´s rank correlation were used for non-normally distributed variables. Elastin and MMPs values were revealed to be non-normally distributed while collagen values were normally distributed. Simple and multiple linear regressions were used to explore the relationship between two or more variables. Freedom from postoperative events was calculated by life-tables according to Kaplan-Meier survival analysis. Correction for age, gender, hypertension, diabetes, smoking, the occurrence of pre-operative symptoms and statin usage was carried out through Cox regression analysis. A P-value of < .050 was considered statistically significant. Statistical analysis was performed using SPSS 20.0 .Two hundred-twenty-one plaques removed by CEA were analyzed for total elastin and collagen content. Of these, two hundred-nine plaques were analyzed for MMP-1, 3- and 9 while only two hundred-one plaques for MMP-12 contents, due to lack of specimen material. Fifty-five % (n = 121) of the plaques were associated with preoperative symptoms, and among those, 7% (n = 15) had more than one preoperative neurological event. There were no differences in risk factor distribution between patients with and without preoperative symptoms.There was a positive correlation between the plaque contents of elastin and collagen  and MMP-12 (r = −.288 P <.0001) content of the plaques decreased while the MMP-1  and MMP-9  content increased with age. Significantly lower levels of elastin, collagen and MMP-12 were found in patients with diabetes as compared with non- diabetics. Current smokers have lower levels of MMP-9 compared to no- or past-smokers. Furthermore, when analyzing time interval between symptoms and CEA (22.2 ± 28 days), plaque content of collagen was significantly lower if the surgical treatment was performed in the first month after symptom onset (P = .007). There were no similar association for the elastin and MMPs content.Two patients (0.9%) suffered an intraoperative (i.e. in the first 24 hours post-CEA) ipsilateral stroke. After a mean follow-up time of 39.6 ± 16.4 months, 7.7% of patients (n = 17) had suffered one or multiple postoperative ipsilateral events . Only one of these patients had a restenosis that needed treatment.vs 68.5 ± 54.5 mg/g, P .033), whereas no significant differences were observed for collagen and MMPs  also has impact on the reliability of the present findings. It would be reasonable to assume that there would be an inverse association between MMP-12 and the plaque elastin content. Instead, we unexpectedly observed a positive correlation. However, it should be kept in mind that elastin turnover involves a number of other factor including expression of growth factors, MMP inhibitors as well as number and types of cells in the plaques. We have previously found a positive association between the plaque contents of platelet-derived growth factor and elastin suggesting that may have an important role.  AccordinThe finding that patients with a low plaque content of elastin have an increased risk of developing an ipsilateral stroke during the follow-up could not be confirmed when excluding those five patients who suffered an ipsilateral stroke perioperatively, i.e. between 24 and 72 hours after CEA. However, this could suggest the instability of a plaque with a lower content of elastin, both in the short and the long term.In conclusion, this study supports the concept that elastin seems to play a key role in plaque stabilization and destabilization. The need for closer follow-up and more aggressive secondary prevention of patients with reduced elastin content in harvested carotid plaques should be further investigated.