In conclusion, there was a significant correlation between arteriosclerosis and choroidal vascular changes in DR. We suggest prompt ophthalmic evaluation in patients with systemic arteriosclerosis. If the ophthalmologist notes advanced DR, the patient should be referred to a cardiovascular clinic for detailed evaluation of systemic arteriosclerosis.This study was conducted to assess whether systemic arterial stiffness, indicated by cardio-ankle vascular index (CAVI), is related to changes in the microvasculature of the retina and choroid in diabetes mellitus (DM). This study included 113 patients with a confirmed diagnosis of type-2 DM. Among them, 18 patients did not have diabetic retinopathy (DR), 71 had non-proliferative DR (NPDR), and 24 had proliferative DR (PDR). The mean CAVI was 7.58 ± 1.41 in no DR, 8.72 ± 1.47 in NPDR, and 8.43 ± 1.25 in PDR group. Of the 113 eyes, 42 (37.2%) were classified as abnormal CAVI group (CAVI ≥ 9). This group had significantly higher cardiac autonomic neuropathy risk index score, decreased central choroidal thickness, and decreased choroidal vascularity index (CVI). Deep foveal avascular zone area was higher in the abnormal CAVI group. After adjustment for possible confounding factors, CAVI showed negative correlation with the CVI ( The most important factor determining arterial stiffness is age; the elasticity of the arterial wall decreases with age and arterial stiffness increases. In addition, increased arterial stiffness can occur as a result of elevated blood pressure, smoking, obesity, and other systemic vascular diseases including diabetes mellitus (DM)6.Arterial stiffness is a term used to describe the degree of vascular rigidity due to reduced elasticity of the artery. Many studies have demonstrated the predictive value of arterial stiffness on systemic cardiovascular diseases1, central blood pressure (CBP)7, or cardio-ankle vascular index (CAVI)8. Among these, CAVI is superior to PWV because it is not affected by blood pressure at the time of measurement9. CAVI increases with age. It is high in many arteriosclerotic diseases, such as cerebrovascular diseases, chronic kidney disease, carotid arteriosclerosis, and coronary artery disease; it is also related to many coronary risk factors including smoking, DM, hypertension, and dyslipidemia8.Arterial stiffness, termed arteriosclerosis, can be assessed noninvasively by measuring the pulse wave velocity (PWV)11.Atherosclerosis is a narrowing of the arteries caused by a buildup of plaque, and the term is sometimes used as a specific subtype of arteriosclerosis. Atherosclerosis can be assessed by measuring the ankle-brachial index (ABI) or intima-media thickness of the common carotid artery. Studies on the association between arterial stiffness and atherosclerosis showed conflicting results15. Further, attempts have been made to quantitatively analyze diabetic microvascular changes of the retina and choroid. In patients with DM, increased foveal avascular zone area12, decreased retinal perfusion density14, and decreased choroidal vascularity index (CVI)15 were associated with worsening diabetic retinopathy (DR). Moreover, in patients with DM, these microvascular changes were evident even without DR.Recent advances in multimodal imaging have facilitated many studies on microvascular changes in diabetic retinopathy18. However, these studies evaluated only the presence of DR18 or the grade of severity of DR17. To date, the association between arterial stiffness and diabetic ocular microvascular changes using quantitative vascular parameters has not been explored. Further, the underlying mechanism and the association between large-artery stiffness and diabetic ocular microvascular changes remain unclear.Generally, DR is a metabolic disorder caused by hyperglycemia, and it shares common risk factors with other diabetic microvascular complications including diabetic nephropathy and neuropathy. Some previous studies identified a relationship between diabetic retinopathy and systemic endothelial dysfunction with peripheral arterial stiffnessIn this study, we evaluated the CAVI and the quantitative vascular parameters of the retina and choroid in patients with type 2 DM. The aim of the current study was to assess whether systemic arterial stiffness, as indicated by CAVI, has an association with changes in the microvasculature of the retina and choroid in type 2 DM.P < 0.001), glycated hemoglobin (HbA1c) (P < 0.001), and glycoalbumin level (P = 0.030) differed significantly between each DR group. The mean CAVI was 7.58 ± 1.41 in the no DR, 8.72 ± 1.47 in NPDR, and 8.43 ± 1.25 in the PDR group (P = 0.012). If the ABI was set into ordinal variables , advanced DR severity and abnormal ABI showed a significant correlation . However, the mean ABI was 1.06 ± 0.14 in the no DR, 1.05 ± 0.10 in NPDR, and 1.10 ± 0.10 in the PDR group; the differences were not statistically significant (P = 0.162).In total, 113 eyes of patients with diabetes were included in this study. We have summarized the demographic, ocular, and systemic characteristics of the subjects according to the severity of diabetic retinopathy in Table P = 0.018). We noted decreased central choroidal thickness (P = 0.002) and decreased CVI (P = 0.041) in the abnormal CAVI group. Deep foveal avascular zone (FAZ) area was higher in the abnormal CAVI group . However, retinal microvascular parameters including superficial FAZ area, superficial and deep FAZ circulatory index, and superficial and deep capillary vessel density showed no significant differences between the two CAVI groups . Of the 113 eyes, 34 (30.1%) were classified as abnormally low or high ABI (ABI < 0.9 or ≥1.3). There were no significant differences in terms of ocular microvascular parameters between the two ABI groups .Tables r = −0.276, P = 0.045), and CVI . ABI exhibited no correlation with any ocular microvascular parameters as per Pearson’s correlation analysis .The results of Pearson’s correlation analysis are shown in Table Among the 113 study participants, 6 (5.3%) patients showed asymmetric DR. The degrees of asymmetry were as follows; 3 patients had severe NPDR and PDR, 2 patients had moderate NPDR and severe NPDR, and 1 patient had moderate NPDR and PDR in each of the eyes. Three of these 6 patients were classified as abnormal CAVI group (CAVI ≥ 9). To validate our data, we conducted additional analysis only including 107 participants with symmetric DR; it showed the same results.15. DR remains one of the most important diabetic microvascular complications and is a leading cause of irreversible blindness, highlighting the importance of early detection of progression and close monitoring.In this study, we aimed to assess whether systemic arterial stiffness was associated with the condition of the microvasculature of the retina and choroid in type 2 DM. Higher CAVI  was associated with decreased CVI, and this correlation was statistically significant. CAVI could be used as a surrogate marker for DR progression, given that CVI exhibited some linear correlation with the progression of DR severity21. To the best of our knowledge, this is the first study to investigate the association between CAVI and microvascular changes in diabetes. Our findings revealed that systemic arterial stiffness, as measured by CAVI showed correlation ocular microvasculature changes in type 2 DM. The measurement protocol of CAVI is easy and the reproducibility of CAVI is also acceptable as per a previous report22. Furthermore, CAVI has the advantage that it is not affected by blood pressure (BP) at the time of measurement8. CAVI can be used as a physiologic surrogate marker for monitoring systemic arterial stiffness and the risk of DR progression in type 2 DM.There have been several reports of relationships between systemic arteriosclerosis and ocular diseases, such as branch retinal vein occlusion and age-related macular degeneration23. This increased pressure transmission to the microvessels may be the underlying pathophysiology of the increased risk of DR progression with increased CAVI. Another possible explanation is that, systemic arteriosclerosis and ocular microvasculature changes are both caused by the same underlying pathologic process of DM.The underlying mechanisms of the association between arterial stiffness and ocular microvasculature changes are unclear. Our hypothesis is that, increased pulsatility due to the arterial stiffening affects the retinal and choroidal microvasculatures24. Due to the intrinsic autoregulatory mechanisms, retinal microvessels might be better protected from hypoxic damage compared to the choroidal vessels. Considering autoregulatory disturbances of retinal vessels in diabetic patients25, further studies are required with large-scale data with central retinal artery and vein equivalents, which could provide supportive evidence for our results.These associations were evident in choroidal vascular parameters rather than retinal vascular parameters. One possible mechanism of the lack of correlation between higher CAVI and disturbed retinal microvasculature may be due to the auto-regulation of retinal vessels. As is widely known, there is no neuronal innervation in the retinal vascular beds, and retinal blood flow is regulated by local mechanisms27. In contrast, in a recent study, abnormally low or high ABI was associated with PDR28. Previous studies evaluated only the grade of severity of DR. In our study population, the ABI did not show a correlation with the ocular microvascular parameters; quantitative analysis of the microvascular parameters in our study might provide stronger supportive evidence for our work.In this study, arteriosclerosis was significantly correlated with ocular microvascular changes in diabetes, but atherosclerosis was not. Studies exploring the link between atherosclerosis, as measured by ABI, and DR have produced mixed results. In some reports, DR was associated with an abnormally low ABI (ABI < 0.9), but not with an abnormally high ABI (ABI ≥ 1.3)22. The data from these individuals should be cautiously analyzed while using CAVI due to the possibility of a false low CAVI score. In our study, 5 subjects with low ABI (<0.9) were included. The ABI of these 5 subjects ranged from 0.76 to 0.87, and the CAVI score ranged from 8.3 to 10.2. There was no statistical difference even after excluding these low ABI subjects (data not shown).Subjects with an excessively low ABI (<0.9) may have a severely arteriosclerotic femoral arteryOur study had some limitations. First, systemic arteriosclerosis and DR might share common risk factors and both might be caused by the same underlying pathologic process of DM. This might cause underestimation of the associations between CAVI and ocular microvascular changes. And we did not collect data on the presence of carotid artery disease. Second, due to its retrospective design based on medical records, this study does not show changes of CAVI and ocular microvascular parameters over time, and if unrecognized variables, such as the use of statins or antihypertensive medications within each group, could have affected the results. Therefore, a longitudinal study is needed to confirm the pathophysiological mechanisms linking systemic arterial stiffness and ocular microvascular changes. Third, we did not evaluate the ocular blood flow. The effect of ocular blood flow regulation on DR remains unclear, and retinal capillary vessel density or CVI cannot accurately reflect the ocular blood flow. Fourth, FAZ area and retinal vessel density can be influenced by the spherical equivalent or axial length. We adjusted the spherical equivalent to exclude the confounding effect, but we did not analyze the axial length. Future studies should address these limitations.In summary, systemic arteriosclerosis had a significant correlation with choroidal vascular changes in DR. Further longitudinal studies are necessary to support these findings and correlate them with structural and functional parameters. We suggest prompt ophthalmic evaluation in patients with systemic arteriosclerosis in DR. If the ophthalmologist notes advanced DR, the patient should be referred to a cardiovascular clinic for a detailed evaluation of systemic arteriosclerosis.The study included patients with a confirmed diagnosis of type 2 DM. We recruited all participants between December 2016 and March 2018 at Seoul St. Mary’s Hospital in Korea and conducted a retrospective chart review. This retrospective study adhered to the tenets of the Declaration of Helsinki. Institutional Review Board (IRB)/Ethics Committee approval was obtained from the Catholic University of Korea, which waived the requirement for obtaining informed patient consent because of the retrospective nature of the study. In patients with symmetric DR, only the right eye was included in the analysis. In patients with asymmetric DR, the data of the worse eye was included.30. To minimize the confounding effect of age-related retinal and choroidal thinning, we excluded patients aged over 75 years.Exclusion criteria for the study were as follows: (1) age over 75 years, (2) medication for dyslipidemia started prior to CAVI measurement, (3) diagnosis of cardiovascular disease besides DM, (4) presence of other retinal diseases, including glaucoma, age-related macular degeneration, epiretinal membrane, uveitis, or retinal vein occlusion, (5) clinically significant diabetic macular edema, and (6) eyes with a history of laser treatment, intravitreal injection, or intraocular surgery. Previous studies have demonstrated retinal and choroidal thinning even during the normal aging processPatients were referred to the specialized endocrinology center, and a thorough evaluation for diabetes was performed. In the clinic, all patients were evaluated standardly for cardiovascular diseases (pulse-wave velocity using vascular screening system), peripheral neuropathy test  and autonomic nervous system test. We retrospectively collected these medical records. We recorded the height, body weight, and BMI data. The systolic and diastolic BPs were measured at rest, and the mean arterial pressure was calculated as diastolic BP +1/3 (systolic BP − diastolic BP). Laboratory test data including glycated hemoglobin (HbA1c), fasting blood sugar, eGFR, creatinine, glycoalbumin and lipid profile  were obtained.All subjects underwent ocular examinations including best-corrected visual acuity (BCVA) evaluation , non-contact pneumatic tonometry, slit-lamp biomicroscopy, dilated fundus examination, and optical coherence tomography (OCT). OCT imaging was performed with a swept-source OCT device .31, and classified patients into 3 groups according to the DR severity as follows: no DR, NPDR, or PDR.We defined and graded the severity of the DR according to the modified Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy severity scaleWe obtained retinal and choroidal thicknesses using in-built OCT software. We created thickness maps in accordance with the standard ETDRS subfield, consisting of 3 concentric circular areas with 9 independent sectors, and used the central retinal and choroidal thicknesses of the innermost 1-mm circular area for analysis. The automated segmentation software of the OCT device can identify the outer boundary of the retinal nerve fiber layer (RNFL) and the inner plexiform layer (IPL). For every OCT scan, each segmented layer line can be manually adjusted to avoid possible segmentation errors. The difference between the RNFL and the IPL outer boundary segmentation yields the ganglion cell-IPL (GC-IPL) thickness. In this study, we measured the average macular GC-IPL thickness within an annulus with inner and outer diameters of 1-mm and 3-mm, respectively. We then used the average RNFL thickness at the peripapillary area for analysis.en-face OCT angiography images at the superficial and deep capillary plexuses were used for analysis. Representative OCT images are shown in Fig. We measured the FAZ area, FAZ circulatory index, and vessel density at the superficial and deep retinal capillary plexuses manually using ImageJ software . The 3 × 3 mm sized et al.32. CVI, which represents the choroidal vascular component, was defined as the ratio of the vascular luminal area to the total choroidal area.For quantitative analysis of the choroidal vasculature, we calculated the CVI in the total macular area. We used the segmentation protocol described by Agrawal 33.We assessed arterial stiffness by measuring CAVI. Measurements of CAVI, ABI, and blood pressure were performed using the program embedded in the vascular screening system . Electrocardiography (ECG) and phonocardiography (PCG) were performed, pressure and waveforms of the brachial and ankle arteries were evaluated, and brachial-ankle PWV (baPWV) and subsequently CAVI were calculated automaticallyOcular examination, measurement of CAVI, and the laboratory tests were performed within a 1-month time frame.34. Spectral analysis of heart rate variability (HRV) was performed, and the standard deviation of all normal R-R intervals (SDNN) and the root-mean square of the difference of successive R-R intervals (rMSSD) were evaluated. CAN risk index was assessed with a CAN analyzer . The CAN risk index value was classified as follows: normal <38.4; borderline 38.4–53.8; and abnormal >53.8.We evaluated CAN using Ewing’s traditional five simple tests, namely, changes in the R-R with standing, deep breathing, and the Valsalva maneuver as well as changes in blood pressure in response to sustained handgrip and standing up35. The neurometer generates a constant alternating current stimulus, which is applied to two different test sites, the median + ulnar nerves on the ring finger , and the deep and superficial peroneal nerves on toe 1 . We performed the data analysis using the Neuval® software provided with the device, and used the mean CPT grade for analysis.We evaluated peripheral neuropathy by measuring the current perception threshold (CPT) using a neurometer . The standard method of CPT testing has been described previouslyP < 0.05), the ANOVA was followed by a post-hoc Bonferroni correction. We used the linear-by-linear association test to examine the linear correlation between the advanced DR stage and abnormal ABI. We used Pearson’s correlation test to investigate the associations between CAVI and retinal and choroidal vascular parameters. Statistical significance was assumed if P < 0.05.We expressed categorical data as an absolute number, and continuous data as mean ± standard deviation . We performed statistical analysis using the Statistical Package for the Social Sciences for Windows version 23.0 . The normality of data distribution was confirmed via the Kolmogorov–Smirnov test. Demographic and clinical data of patients were compared by one-way analysis of variance (ANOVA), chi-square, and Fisher’s exact tests. In cases of significant results (


In all, 43 individuals participated in this study. After the index %η was calculated, the accuracy of the index %η in distinguishing healthy subjects and subjects at a high risk of arteriosclerosis was tested via a receiving operating characteristic (ROC) analysis. The calculated %η for the healthy participants and those at a high risk of arteriosclerosis was 13.4 ± 55.1% and −32.7 ± 34.0% (mean ± S.D.), respectively. The area under the ROC curve was 0.77. Thus, it was concluded that the proposed method can be used to evaluate the vascular endothelial function.This paper proposes an algorithm for estimating the arterial viscosity using cuff pressures and pulse waves measured by an automatic oscillometric sphygmomanometer. A change in the arterial viscosity during the enclosed-zone flow-mediated dilation test is calculated as an index for evaluating the vascular endothelial function % One of the main causes of these cardiovascular diseases is arteriosclerosis, which must be diagnosed and treated early as it is a progressive and refractory disease. Vascular endothelial dysfunction is known as an early symptom of arteriosclerosis2. The vascular endothelial function associated with the sympathetic adrenergic system is involved in fine tuning of the blood pressure. Specifically, the activation of the α1 adrenergic receptor and β adrenergic receptor causes vasoconstriction and nitric oxide (NO)-mediated vasodilatation, respectively4. The vascular endothelial function can thus be evaluated via changes in the blood flow rate, vascular diameter or arterial mechanical properties, which are associated with vasoconstriction and vasodilatation.In recent years, cardiovascular diseases such as heart diseases and cerebrovascular diseases have accounted for approximately 25% of the deaths among the Japanese people9. For example, Panza et al. used a plethysmograph to estimate changes in the blood flow rate when a NO agonist or antagonist was administered in an artery10. The flow-mediated dilation (FMD) test11 was then proposed as a clinically practical method that enabled noninvasive evaluation of the vascular endothelial function. In the FMD test, shear stress is applied on the endothelium to induce NO release, and the dilation ratio of the vascular diameter between the pre- and post-cuff occlusion (%FMD) is measured as an index to evaluate the endothelial function. One of the drawbacks of the FMD test is that it requires dexterity because the prolonged stable measurement of the vascular diameter with an ultrasound device is often difficult. To simplify the measurement procedure, a technique applying an oscillometric method12, which is widely used in commercial automatic sphygmomanometers, was proposed14. This technique is referred to as the enclosed-zone flow-mediated dilation (ezFMD) test. In the ezFMD test, a cuff is attached to the upper arm and the cuff pulse wave is measured. The vascular endothelial function is then estimated based on the maximum amplitude change rate between the pre- and post-cuff occlusion of the cuff pulse wave (%ezFMD).Evaluation methods for vascular endothelial function have been extensively studied since the 1980s, and the first attempt involved invasive methods9 because it is a rather direct measure of vasodilation compared to the vascular diameter, which can be affected by changes in the blood pressure. In these studies, several measures such as the pulse wave velocity were used to evaluate the arterial stiffness or viscoelasticity. Our research group has previously proposed a log-linearized peripheral arterial model15, which enabled the estimation of the changes in the arterial viscoelasticity in a beat-to-beat manner during the FMD test16. These recent studies revealed the effectiveness of the use of viscoelasticity in vascular endothelial function evaluation; however, such methods inherit the drawbacks of the FMD test because viscoelasticity estimation in these cases is carried out based on the vascular diameters measured using the ultrasonic devices.In addition, evaluation methods focusing on the arterial mechanical characteristics have been attracted attention since the 2000sTo improve the clinical applicability, this paper presents a concept employing the oscillometric method and an algorithm for estimating the arterial viscosity to assess the vascular endothelial function. The proposed method enables the estimation of the arterial viscosity from only a common oscillometric automatic sphygmomanometer. The algorithm is then tested in terms of its ability of evaluating the vascular endothelial function by measuring the changes in the estimated viscosity induced by reactive hyperaemia.The baseline clinical characteristics of the participants are summarised in Table η was 65.4%, as shown in Fig. η was −57.4%, as shown in Fig. Figure Figure p = 0.87). However, the post-cuff occlusion arterial viscosity measured in the participants at a high risk of arteriosclerosis was significantly lower than the pre-cuff occlusion arterial viscosity . Figure η for the healthy participants and those at a high risk of arteriosclerosis . The change ratio of arterial viscosity %η of the participants at a high risk of arteriosclerosis was significantly lower than that of the healthy participants (p = 0.006).Figure η and the conventional endothelial function evaluation indices %FMD and %ezFMD. Table η and the conventional endothelial function evaluation indices %FMD and %ezFMD were 0.786, 0.695, 0.667, and 0.751, respectively.Figure This paper proposed an estimation method for the vascular viscosity and tested its applicability for endothelial function evaluation. In contrast to the current de facto standard methods such as the FMD and the ezFMD, which evaluate the endothelial function based on changes in vascular diameters, the proposed method uses the vascular viscosity as the arterial mechanical characteristic. The performed ROC analysis showed that the proposed method outperformed the FMD and the ezFMD see Fig. . In addi17. However, the arterial viscosity showed a different trend between the healthy participants and the participants at a high risk of arteriosclerosis. The estimated post-cuff occlusion viscosity did not change significantly compared with the pre-cuff occlusion values in the healthy participants, and the post-cuff occlusion values were decreased compared to the pre-cuff occlusion values for participants at a high risk of arteriosclerosis  to understand the artery behaviors caused by the cuff occlusion. During post-cuff occlusion, the cuff pulse wave and the cuff pulse wave velocity were increased compared with the corresponding pre-cuff occlusion values for both the healthy participant (Sub. 1) and the participant at a high risk of arteriosclerosis (Sub. 30). This indicates that, even for the participant at a high risk of arteriosclerosis, endothelium-derived NO can be detectable, although diminished to underet al.18 where they reported delayed NO effects during reactive hyperaemia in human forearm vessels. Specifically, their finding that the role of NO during the early phase of reactive hyperaemia is minimal can explain that fact that the estimated viscosities in this paper did not change significantly in the first post-occlusion. In addition, modest yet significant effects of NO in the mid-to-late phase can explain the difference between the healthy participant group and the high-risk participant group: The effects of NO in the mid-to-late phase in the healthy participant group counteract the significant decreases in the viscosities after the second pre-cuff occlusions in the high-risk participant group. Further, the nearly constant viscosities observed through the first to the last post-occlusion in the healthy participants and through the second to the last post-cuff occlusions in the high-risk participants could be caused by the effects of the post-cuff occlusions. Because the post-cuff occlusions were applied for every 30 s, and they were conducted based on the protocol of the ezFMD13, the short intervals between the consecutive post-cuff occlusions may impose continuous stimulation to the endothelial function and prevent the viscosities from returning to the normal levels.The transition of the arterial viscosity for each blood pressure measurement also exhibited different trends between the healthy participants and the participants at a high risk of arteriosclerosis. As shown in Fig. et al.19 reported the relationship between the load exerted on the muscle and the lengthening velocity of the muscle, as shown in Fig. The difference between the viscosities estimated from the two subject groups could be attributed to the fact that the vascular endothelial function maintains the vascular viscosity, and vascular dysfunction significantly decreases the arterial viscosity after occlusion. However, this straightforward interpretation does not consider the behaviors of the vascular smooth muscle during vasodilation. Mashima η was significantly different between the healthy subjects and those at a high risk of arteriosclerosis; the change ratio of arterial viscosity %η in the healthy subjects showed a large variation , and 14 subjects at a high risk of arteriosclerosis . Healthy subjects had no history of cardiovascular disease, liver disease, renal disease, autoimmune disease, or malignancy and had no coronary risk factors, including hypertension, dyslipidaemia, diabetes mellitus, and smoking. Subjects at a high risk of arteriosclerosis were defined as those affected by one or more of the following conditions: hypertension, diabetes, and dyslipidaemia. Hypertension was defined as having a systolic blood pressure of ≧140 mmHg or a diastolic blood pressure of ≧90 mmHg measured in a sitting position on at least three different occasions22. Diabetes mellitus was defined according to the guidelines of the American Diabetes Association23. Dyslipidaemia was defined according to the third report of the National Cholesterol Education Program24.A total of 43 adults (average age ± standard deviation: 34.9 ± 20.9 years) participated in the experiment for measuring the proposed change ratio of arterial viscosity %https://upload.umin.ac.jp. Unique identifier. UMIN000004902). Informed consent was obtained from the subjects.This experiment was conducted in accordance with the Declaration of Helsinki and was subject to the approval of the Epidemiological Research Ethics Review Committee of Hiroshima University  widely used in general clinical practice was used to measure the cuff pressure and cuff pulse wave in this experiment. The cuff pressure and cuff pulse wave data were saved on a multimedia card (Transcend) inside the device at a sampling frequency of 125 Hz. The subjects assumed a supine position and were allowed to rest. Their blood pressure was then measured once before the 5-min cuff occlusion and five times after that occlusion. First, to verify the change in arterial viscosity from pre- to post-cuff occlusion, the arterial viscosities 25. Hayashi et al. measured the static arterial diameter invivo when artificial internal pressure was applied to the artery. In the physiological pressure range (60 to 160 mmHg), the results showed that the relationship between the logarithmic value of the intravascular pressure for the reference intravascular pressure and the ratio of the vascular diameter for the reference vascular diameter was linear. Thus, Hayashi et al. proposed the Stiffness Parameter25, which is an estimation index of arterial elasticity and is less susceptible to changes in intravascular pressure. However, it is difficult to express vascular characteristics only using elastic characteristics because invivo experiments have reported arteries to be viscoelastic bodies rather than simple elastic bodies27. To resolve this issue, Hirano et al. proposed the log-linearized peripheral arterial viscoelastic model and evaluated the arterial viscosity considering the nonlinear relationship between the peripheral intravascular pressure and peripheral artery diameter26. The arterial viscoelastic estimation method proposed by Hirano et al. requires the simultaneous measurement of continuous arterial pressures and changes in vascular diameter, which limits the clinical applications of the method. Evaluating the arterial viscosity with an automatic oscillometric sphygmomanometer, which does not require continuous blood pressure measurements, may broaden the clinical applications of this method. This paper proposes an arterial viscoelastic model in which blood pressure is measured by the cuff of an automatic sphygmomanometer wrapped around the upper arm. The proposed model is based on the log-linearized peripheral arterial viscoelastic model proposed by Hirano et al. The log-linearized peripheral arterial viscoelastic model is expressed as follows:t represents time, P(t), ε(t), et al. measured ε(t) ε(t), 28:p(t) applied from inside the vessel on the outside is considered positive. dV(t)/V, dV(t) expresses the change in vascular volume V when p(t) = 0; in an automatic oscillometric sphygmomanometer, slight pressure fluctuations occur in the cuff (cuff volume pulse wave Ppulse(t)) in response to this change in vascular volume dV(t).Conventionally, it is known that the relationship between the vascular diameter and intravascular pressure is nonlinear in humansge 60 to 60 mmHg, 29. The cuff pressure can be considered to act from the outside toward the inside of the arterial wall, as shown in Fig. p(t) acting on the arterial wall is then given as the difference between the intravascular pressure Pb(t) and cuff pressure Pcuff(t):In this study, viscoelastic parameters were estimated using biological signals obtained from the automatic oscillometric sphygmomanometer and Equation . The upp31. The volumetric vascular change dV(t) was then approximated using the following equation because it is proportional to the cuff pulse wave Ppulse(t)13:kr is a positive constant. Considering only the time at which dV(t) = 0 and Pb(t) is equal to or higher than the mean blood pressure, the pressure t is defined as the time at which the cuff pulse wave crosses 0 (Ppulse(t) = 0). Equation  is assumed to be equal to Pb(tj). In this study, Equation post3−5 is the average arterial viscosity value calculated from the 3rd to 5th post-cuff occlusion arterial pressure measurement, and (krη/V)pre is the pre-cuff occlusion arterial viscosity. The post-cuff arterial viscosity is taken as the average of the 3rd to the 5th measurements to avoid a condition in which the blood flow immediately after the cuff occlusion is unstable. In this study, the proposed method was used to distinguish between healthy subjects and subjects at a high risk of arteriosclerosis, and the vascular endothelial function of the subjects was evaluated using the change ratio of arterial viscosity %η.A schematic of the proposed vascular endothelial function evaluation system is shown in Fig.


IBD) is characterized by a low prevalence of traditional risk factors, an increased aortic pulse‐wave velocity (aPWV), and an excess of cardiovascular events. We have previously hypothesized that the cardiovascular risk excess reported in these patients could be explained by chronic inflammation. Here, we tested the hypothesis that chronic inflammation is responsible for the increased aPWV previously reported in IBD patients and that anti‐TNFa  therapy reduce aPWV in these patients.Inflammatory bowel disease . Disease duration (P=0.02) was associated with aortic stiffening as was, in patients with ulcerative colitis, high‐sensitivity C‐reactive protein during follow‐up (P=0.02). All these results were confirmed after adjustment for major confounders. Finally, the duration of anti–TNFa therapy was not associated with the magnitude of the reduction in aPWV at the end of follow‐up (P=0.85).This was a multicenter longitudinal study. We enrolled 334 patients: 82 patients with ulcerative colitis, 85 patients with Crohn disease, and 167 healthy control subjects matched for age, sex, and mean blood pressure, from 3 centers in Europe, and followed them for 4 years . At baseline, aPWV, an established surrogate measure of cardiovascular risk, in patients with IBD. This suggests that effective control of inflammation may reduce cardiovascular risk in these patients.Long‐term anti–TNFa therapy reduces  A long‐term anti–tumor necrosis factor‐alpha (TNFα) therapy in patients with inflammatory bowel disease (IBD) reduces aortic pulse‐wave velocity to a level comparable to that of healthy individuals, whereas high doses of salicylates lead to aortic stiffening.Effective control of inflammation may reduce cardiovascular risk in patients with inflammatory bowel disease as it improves aortic pulse‐wave velocity, an established surrogate measure of cardiovascular risk.Patients with inflammatory bowel disease (IBD) have a low prevalence of traditional risk factors for cardiovascular diseaseIn a meta‐regression of 8 cross‐sectional studiesIn this multicenter longitudinal prospective study, we hypothesized that chronic inflammation is responsible for the increased aPWV previously reported in patients with IBD and that anti‐TNFα drugs could reduce aPWV. Consequently, the aim of the present study was to investigate the role of inflammation and therapy on aPWV in patients with IBD.The data, analytic methods, and study materials will be made available from the corresponding author to other researchers for purposes of reproducing the results or replicating the procedure.This study was a multicenter, open‐label longitudinal prospective study conducted in the Department of Clinical and Experimental Medicine of the University of Catania, Italy; the DIBIMIS of the University of Palermo, Italy; and the Department of Gastroenterology of the Gulhane School of Medicine of Ankara, Turkey.Sequential patients with an established clinical and endoscopic diagnosis of IBD were recruited at our departments. Concomitantly, healthy volunteers matched for age, sex, and mean blood pressure (BP) (ratio of 1 control per IBD patient) were randomly selected from our local community databases and invited to take part in the study. Individuals with previous cardiovascular disease  and those with diabetes mellitus, chronic kidney disease, and infectious and inflammatory disorders other than Crohn disease (CD) and ulcerative colitis (UC) were excluded, as well as those on treatment for hypertension or dyslipidemia. Written informed consent was obtained from each patient included in the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was previously approved by the Ethics Committee on Research on Humans of the University of Catania.All participants were studied in a quiet room with a controlled temperature of 22±1°C after 15 minutes of recumbent rest. In each subject, a noninvasive hemodynamic study was performed by an expert operator blinded to the clinical data and therapy. A second operator, blinded to the hemodynamic examination, collected the clinical data using a standardized questionnaire.®; AtCor Medical, Sydney, Australia) in two centers  using the foot‐to‐foot velocity method, the intersecting tangent algorithm, and the direct distance between the measurement sitesA hemodynamic examination was performed at baseline and at the end of follow‐up. Brachial BP measurements were performed using an oscillometric device . The carotid‐femoral (aortic) pulse wave velocity was measured by a SphygmoCor device =(aPWV at the end of follow up−aPWV at baseline)/follow‐up duration (year).Active disease was defined by Mayo Score ≥2 in subjects with UC and Harvey‐Bradshaw Index ≥5 in subjects with CD.P<0.01) difference in aortic stiffening among groups, hypothesizing that ΔaPWV was −0.03±0.21 m/s per year in patients with IBD treated with anti‐TNFα, 0.20±0.15 m/s per year in those treated with salicylates, 0.02±0.21 m/s per year in those treated with other immunosuppressive drugs, and 0.08±0.09 m/s per year in control subjects, as previously reported in our pilot study.P<0.01) difference in aortic stiffening between groups. These calculations were based on 10 000 Monte Carlo samples.Data were analyzed with IBM SPSS Statistics version 19.0 . Continuous variables are presented as the means (SDs), and categorical variables are presented as percentages. Using ANOVA and the Bonferroni test for multiple comparisons, we determined a sample size adequate to demonstrate that aortic stiffening during follow‐up was lower in patients with IBD treated with anti‐TNFα than in control subjects. The study power (90%) was calculated to detect a significant , where the mean values and SD were calculated in each cohort. Covariates included were age, body mass index, mean BP, heart rate, total cholesterol, high‐density lipoprotein cholesterol, triglycerides, and active disease. Values of these variables at baseline and annual progression were included in the analysis as well as disease duration, immunosuppressive therapy, baseline aPWV, and center of origin. The contribution of these variables to ΔaPWV was first assessed in univariate and then in multivariate analysis adjusted for baseline measurements and the matching variables . A 2‐tailed P<0.05 was considered significant.Clinical and hemodynamic variables were compared using ANOVA for continuous variables with the Bonferroni test for multiple comparisons and the chi‐square test for categorical variables in univariate analyses. Comparisons between measures at baseline and follow‐up were made using repeated‐measures ANOVA. Generalized estimating equations were used to examine correlations of annual progression (taken as the difference between the first and second visits divided by time between visits) in aPWV to conventional risk factors, inflammation, and therapy in patients with IBD. In generalized estimating equations, we used the center‐specific The authors had full access to the data and take full responsibility for its integrity. All authors read and agreed to the manuscript as written.P=0.71). The matching process worked well because age, sex, and mean BP were comparable between groups, as were systolic and diastolic BP. At baseline examination, patients with UC and those with CD had higher aPWV  and heart rate  than did control subjects.The main baseline clinical and demographic data of patients with IBD and matched control subjects are presented in Table Current therapy for the IBD patients included anti‐TNFα therapy , salicylates , and other disease‐modifying drugs . Most patients were taking 2 or more drugs concomitantly. Baseline and follow‐up characteristics of control subjects and patients with IBD sorted for the immunosuppressive therapy at baseline are reported in Table P=0.85). Disease duration, a factor associated with the amount of chronic inflammation over time, was associated with aortic stiffening, whereas an increased baseline aPWV was associated with the largest reduction in aortic stiffness  in 1‐way ANOVA. Moreover, the use of center‐specific z score of ΔaPWV in generalized estimating equations allowed us to reduce further the source of variability of ΔaPWV related to the center of origin  and by 2016/2018 Department Research Plan of University of Catania, Department of Clinical and Experimental Medicine (Project #A).None.


Heart failure with preserved ejection fraction (HFpEF) is a multifactorialsyndrome characterized by a limited exercising capacity. High-intensityinterval training (HIIT) is an emerging strategy for exercise rehabilitationin different settings. In patients with HFpEF, HIIT subacute effects onendothelial function and blood pressure are still unknown.To evaluate the subacute effect of one HIIT session on endothelial functionand blood pressure in patients with HFpEF.Sixteen patients with HFpEF underwent a 36-minute session of HIIT on atreadmill, alternating four minutes of high-intensity intervals with threeminutes of active recovery. Brachial artery diameter, flow-mediateddilation, and blood pressure were assessed immediately before and 30 minutesafter the HIIT session. In all analyses, p <0.05 was consideredstatistically significant.There was an increase in brachial artery diameter  and adecrease in systolic blood pressure . Flow-mediated dilation and diastolic blood pressure  did not change significantly.There were no adverse events throughout the experiment.One single HIIT session promoted an increase in brachial artery diameter andreduction in systolic blood pressure, but it did not change flow-mediateddilation and diastolic blood pressure. Eligibility criteria werepresence of signs and symptoms of heart failure, preserved ejection fraction(> 50%), diastolic dysfunction (left ventricular end-diastolic volume index< 97 mL/m2) with increased filling pressure (E/e' > 8), and inthe case of E/e' < 15, at least one diagnostic criterion for HFpEF, accordingto the abovementioned document. Age between 40-75 years, New York HeartAssociation (NYHA) functional class I to III, and clinical stability underoptimal drug therapy in previous 3 months, was also considered criteria foreligibility. Patients with severe lung disease, moderate-to-severe valvulardisease and peripheral arterial disease were excluded. Similarly, autonomicneuropathy, unstable angina, a history of complex arrhythmias induced by stress,patients with implantable cardiac electronic devices and those with cognitiveand/or limiting musculoskeletal conditions, were excluded.This before-and-after  study was conducted between June 2014and November 2015. Nineteen patients with HFpEF, according to the EuropeanSociety of Cardiology criteria,Firstly, patients underwent a Doppler echocardiography with color flow mapping toconfirm the diagnosis criteria for HFpEF. Then, a maximal cardiopulmonaryexercise testing was performed to assess ventilatory thresholds and peak oxygenconsumption, as well as heart rate response to exercise. Up to 14 days after thecardiopulmonary exercise testing, brachial artery diameter, flow-mediateddilation (FMD) and endothelium-independent dilation were assessed immediatelybefore and 30 minutes after a HIIT session. In the same experimental session, BPand heart rate were measured at two different moments before and after exerciseas described below.Demographic and clinical data were collected on the first day through aquestionnaire and verified in the medical records of each patient.Anthropometric data were collected at the time the questionnaire wascompleted.25,26 Cine loops and static images of 3 consecutive beatswere recorded on standard 2D, M-mode, Doppler and tissue Dopplerechocardiographic views. Left ventricular ejection fraction was calculatedusing the Teichholz formula from the parasternal long-axis view. Forpatients with regional wall motion abnormalities, the Simpson rule was usedto calculate the ejection fraction. Left atrium volume was measured atventricular systole, just before mitral valve opening, and calculated fromapical 4-and 2-chamber views using the biplane method of disks. Leftventricular diastolic function was evaluated with transmitral pulsed Doppler andmitral annulus tissue Doppler velocity .All echocardiographic examinations were performed using equipment Envisor CHD or HD 11  with a standard multifrequency sectorialtransducer by a trained cardiologist. Images were acquired following astandardized protocol, following recommendations present in the currentguidelines.27 BP wasmeasured with a sphygmomanometer  every 3 minutes during the test and also at the physician'sdiscretion. All tests were performed in the morning, with room temperaturebetween 18 and 22ºC and relative humidity around 60%, and they wereconducted always by the same researcher (ADS), a cardiologist with expertisein cardiopulmonary exercise testing, certified by the Department of ExerciseTesting and Cardiovascular Rehabilitation of the Brazilian Society ofCardiology. An individualized ramp protocol was used as described elsewherein this study.28 Testswere considered maximal when the respiratory quotient (R) was equal to orhigher than 1.10.The test was performed on a treadmill , and breath-by-breath expired gas analysis wascarried out using a Cortex Metalyzer 3B system . Heart rate was monitored with a 12-lead electrocardiograph , with electrode placement as described byMason and Likar.BP was measured with a digital device  at fourdifferent points in time: 1) pre-assessment of endothelial function (after 15minutes seating at rest); 2) immediately before HIIT session; 3) 5 minutes afterHIIT session; 4) 30 minutes after HIIT session.Patients were instructed not to do any type of exercise, not to smoke, and not todear or drink any caffeine or alcohol for 24 hours before the evaluation. Theevaluation started after 15 minutes of seated rest in a room with temperaturebetween 18 and 22ºC. Patients stood in the supine position with their left armpositioned comfortably. Noninvasive measurements of endothelial function wereperformed using a two-dimensional Philips EnVisor Ultrasound system  with an electrocardiogram module and a high-frequency (7-12 MHz) vasculartransducer.An image of the brachial artery was obtained 2-5 cm from the antecubital fossa ona longitudinal plane. Artery diameter was manually measured from the anteriorand posterior intimal layer. Visual inspection of single frames was performedand calipers were placed at discrete points along the long axis of the B-modeimage, when means were calculated.After measurements of brachial artery diameter were taken at baseline, asphygmomanometer was inflated on patient's left forearm with 50 mmHg above thesystolic BP, remaining there for 5 minutes. Sixty seconds after deflation of thesphygmomanometer cuff, a new image was recorded synchronized with the R wave ofthe electrocardiogram to identify the artery diameter, enabling FMDmeasurements.After 15 minutes , the artery diameter was measured again.Then, a dose (0.4 mg) of nitroglycerin spray was administered sublingually.After 5 minutes, another image was recorded to measure endothelium-independentdilation. These data were obtained before exercise and 30 minutes after the HIITsession.FMD was expressed as the relative change in brachial artery diameter during thehyperemic phase, as follows: [(post-hyperemic diameter - baseline diameter) /baseline diameter] × 100.15 The session started with an8-minute warm-up at moderate intensity followed by four blocks of 4 minutes eachat 85-95% maximal heart rate, 15 to 17 on Borg rating of perceived exertionscale,29 alternatedwith 3 minutes at 60-70% maximal heart rate, 11 to 13 on Borg scale. It endedwith 3 minutes of cool-down at moderate intensity, totaling 36 minutes. Theheart rate target zone stipulated for each block was based on the maximal heartrate reached at cardiopulmonary exercise testing and was continuously measuredduring training through 12-lead electrocardiographic monitoring .The HIIT session was performed on a treadmill according to the protocolrecommended by the European Society of Cardiology (ESC).2 slope) was described as medianand interquartile range. After meeting the assumptions of normality, the Studentt-test for paired samples was used to compare means of the endothelial functionvariables pre- and post-exercise. Generalized estimating equations (GEE) were used tocompare mean BP and heart rate between four different moments during theexperiment. In all analyses, p <0.05 was considered statisticallysignificant.Data were analyzed using SPSS, version 20.0. Categorical variables are describedas absolute frequencies and percentages. Continuous variables with normaldistribution are described as means and standard deviations. The only variablewithout normal distribution  evaluated by plethysmography up to 30 minutes after exercise.35 Currie et al.20 evaluated coronary artery diseasepatients after one single HIIT session and found an increase in the endothelialfunction after 60 minutes.20 Inother experiment, the same group showed that only individuals with coronary arterydisease with endothelial dysfunction presented augmentation in FMD after 15 minutesof a HIIT session.21Interestingly, as in our experiment, in both studies the brachial artery diameterwas increased.Although there is no previous publication concerning subacute effect of an exercisesession on endothelial function in patients with HFpEF, previous studies haveevaluated patients with heart failure with reduced ejection fraction in a similarcontext.37,38 The hypotensive effect of HIIT is already well establishedin the literature, but prior to this study, BP had not been evaluated in patientswith HFpEF after a session of any type of exercise. In our experiment, we observedan absolute reduction of 12.7 ± 3.8 mmHg in systolic BP 30 minutes after anexercise session. On a chronic basis, this reduction may have clinical relevance,especially in the case of a syndrome whose strict control of BP pressure is crucial.Interestingly, a recent meta-analysis has demonstrated that HIIT performed at least3 times a week for 12 weeks resulted in a significant reduction in systolic BP inoverweigh/obese individuals.19Some evidence points out that exercising performed at submaximal intensities closerto the peak of exercise promotes a greater and longer reduction in BP afterexercising than when exercising less intensively.It is noteworthy that in this subgroup of individuals with HFpEF and reducedfunctional capacity, high-intensity exercising was well tolerated, once appropriateoverload (speed and slope) was individually prescribed, always considering thetarget zones established based on maximal cardiopulmonary exercise test results ofeach individual.Finally, in a condition characterized by exercise limitation, aerobic exercisetraining has a significant role and is indicated for all patients capable ofperforming it. In an acute and subacute setting, HIIT reduced BP and increasedbrachial artery diameter, suggesting that this training modality could be abeneficial alternative for individuals with HFpEF.This was a small, single-center; before-and-after study with HEpEF patients wherethe presence of diabetes, atherosclerosis, gout, and use of tobacco may haveinfluenced the study outcomes. However, these characteristics represent thereality of this complex syndrome which have multiple comorbidities. Weacknowledge that further studies are necessary to evaluate the effect of a HIITsession, especially after one hour, as well as the long-term efficacy of thisexercise strategy as part of a cardiovascular rehabilitation program for thesepatients. Finally, the presence of a control group of matched individualswithout HFpEF could help establishing which responses can be attributed to thesyndrome under study. Likewise, comparing a HIIT session with a continuousmoderate-intensity training session could help establishing the differences inhemodynamic response among these different exercise protocols.One single HIIT session promoted an increase in brachial artery diameter and areduction in systolic BP, and did not change FMD and diastolic BP 30 minutes afterthe exercise session.


Study outcomes included incident clinically significant hyperkalemia  and among patients who experienced incident hyperkalemia, the proportion who had RAASi medication changes . Among 20,184 CKD patients with a median follow-up of 3.9 years, 1,992 (9.9%) patients experienced an episode of hyperkalemia. The overall incidence rate was 3.1 (95% CI: 2.9–3.2) per 100 person-years. Rates progressively increased with worsening eGFR (e.g. 3.5-fold increase in patients with eGFR <15 vs. 45–59 ml/min/1.73m2). Among patients who experienced incident hyperkalemia, 46.6% had changes made to their RAASi treatment regimen following the first occurrence of hyperkalemia (discontinuation: 36.6% and dose reduction: 10.0%). In this analysis of adult RAASi users with CKD, hyperkalemia and subsequent RAASi treatment changes were common. Further assessment of strategies for hyperkalemia management and optimal RAASi use among people with CKD are warranted.Data on hyperkalemia frequency among chronic kidney disease (CKD) patients receiving renin-angiotensin aldosterone system inhibitors (RAASis) and its impact on subsequent RAASi treatment are limited. This population-based cohort study sought to assess the incidence of clinically significant hyperkalemia among adult CKD patients who were prescribed a RAASi and the proportion of patients with RAASi medication change after experiencing incident hyperkalemia. We conducted a retrospective, population-based cohort study (1 January 2013–30 June 2017) using Australian national general practice data from the NPS MedicineWise’s MedicineInsight program. The study included adults aged ≥18 years who received ≥1 RAASi prescription during the study period and had CKD (estimated glomerular filtration rate [eGFR] <60 ml/min/1.73m Blood pressure lowering therapy has shown to prevent the onset of poor cardiovascular outcomes (which remains the leading of cause of death in CKD) and delay the progression of kidney disease  discontinued RAASi treatment and 174 [10.0%] were prescribed a reduced dose of these agents). No significant difference in mean serum potassium levels at the time of incident hyperkalemia according to the type of RAASi medication change  was observed. When assessed according to eGFR category, RAASi medication changes were more prevalent among those with lower levels of eGFR .On univariate analysis, medication change was predicted by healthcare card status , smoking status  and by the presence of comorbid conditions including cardiovascular disease , diabetes  and heart failure  or subgroups of patients with CKD from a broader population. These studies have reported an incidence of hyperkalemia in CKD of 4.1% over 3 years [A systematic review of 39 randomized trials assessing the effects of RAAS inhibitors and other blood pressure lowering agents in CKD reported that the overall incidence of hyperkalemia (serum potassium ≥6 mmol/L) with single or dual RAAS inhibition generally ranged between 1.9 to 6.1% over 3 months to 3.4 years ], Sweden 3 years , 45% ove 3 years  and 7 pe 3 years , respect 3 years  and lack 3 years .The variable rates of hyperkalemia across recent studies may be attributable to differences in study inclusion criteria (broad [e.g. age ≥18 years with blood pressure measurement] or specific [e.g. RAAS inhibitor users]) or definitions of hyperkalemia . Nevertheless, collectively, they suggest that hyperkalemia rates derived from randomized trials may not reflect actual incidence in routine clinical practice  in our study. Similar findings were reported in a recent U.S.-based study [Our study, using a large, national cohort of RAASi users with CKD and a study follow-up of 4.5 years, also shows that hyperkalemia is observed at progressively higher rates among those with poorer levels of kidney function, as well as those with diabetes and heart failure, a finding that is consistent with prior studies reporting increased risk of hyperkalemia among individuals with these conditions. Given that diabetes and heart failure are observed at greater frequency among people with advanced CKD, the complex interplay of these conditions might explain the higher rates of hyperkalemia observed in those with lower levels of eGFR  while another study reported that among patients who received RAASi therapy at maximum dosage, 47% had their dose reduced or therapy discontinued following a hyperkalemia event (defined as ≥5.5 mEq/L) [Reported percentages of RAASi therapy  discontinuation due to hyperkalemia from randomized trials of various patient groups (e.g. hypertension or heart failure) have been relatively low  whil5 mEq/L) . Our resWe observed that the incidence of hyperkalemia was higher among those in whom serum potassium was measured more frequently. Patient data (e.g. serum potassium results) from non-MedicineInsight general practices were not available for analysis and while it is possible that such data may have affected serum potassium testing frequency in the current study, it is reasonable to expect that closer monitoring of potassium levels would lead to greater detection of clinically significant hyperkalemia (and vice versa) which in turn may lead to improved management of RAASi treatment regimens. Indeed, we observed that serum potassium levels at the time of the hyperkalaemia event was the strongest predictor of RAASi medication change. Closer patient monitoring attributed to older age and higher comorbid burden may also explain our findings which showed a lower risk of hyperkalemia with older age, results which are consistent with 3 studies assessing the relationship between antihypertensive agents and the risk of hyperkalemia among individuals with varying degrees of kidney function . Our study was based on a large, nationally representative , population-based cohort of patients with CKD reflecting real-world clinical settings. In addition, we assessed the incidence of hyperkalemia in RAASi users with CKD based on longitudinal data, which allowed for assessments of subsequent medication changes and factors associated with this change. However, our study has some limitations that should be considered. General practices participating in the MedicineInsight program were recruited using non-random sampling. Therefore, while the overall distribution of patients in our study cohort by state and territory was largely consistent with the broader Australian population distribution, sampling differences across Australian regions, states and territories cannot be excluded. There are also some limitations to the data that should be noted including: 1) the potential for incomplete data given that certain variables are not collected within the MedicineInsight program to preserve patient confidentiality, 2) the lack of specific dates pertaining to birth and death (year of birth and death are available) and 3) the potential for duplication of patients across MedicineInsight participating general practices . We defined a hyperkalemia event as potassium >6 mmol/L to avoid spurious elevations of potassium. Nevertheless, we were limited in our ability to confirm actual levels of potassium in those with known cases of spurious potassium elevations.We were also limited in our ability to assess for the use of concomitant medications including those known to increase potassium concentrations such as calcineurin inhibitors, beta-blockers and trimethoprim. We sought to delineate descriptively the relationship between RAASi use, kidney function and study outcomes specifically in a population likely to benefit most from continued RAASi therapy (i.e. CKD). Accordingly, we did not include a “control” group of individuals with normal kidney function and/or receiving other blood pressure lowering medications to whom the risk of study outcomes could be compared against. Finally, we did not have complete information on the receipt of chronic dialysis or kidney transplantation at baseline and the identification of the prevalence of these treatments was limited to relevant text searches of the datasets. Thus, the potential for inclusion of patients who received chronic dialysis or kidney transplantation cannot be excluded.In conclusion, in this study of a large cohort of adults RAASi users with CKD, the incidence of hyperkalemia was frequently observed and rates increased with deteriorating levels of kidney function. RAASi medication change following an episode of hyperkalemia occurred in almost half of patients who experienced hyperkalemia.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.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.


Recently, several new biological therapies targeted IgE and IL‐5 in severe asthma have been developed and approved. In the last few years, there have been some reports on the therapeutic algorithms for severe asthmatic subjects screened by biomarkers. However, these algorithms have one problem. In atopic/eosinophilic overlapping asthmatic subjects, there is no effective answer to the question: “which is the optimal choice between Anti‐IgE and anti‐IL‐5?”We propose a new three‐step algorithm for biological therapy in severe asthma.Step 1 is to divide subjects into four groups by measuring blood eosinophils and FeNO. Step 2 is to divide the subjects further into six groups by atopy test. In the case of elevated blood eosinophils, normal/elevated FeNO, and atopy, we perform a steroid trial in step 3 in order to decide whether to select anti‐IgE or anti‐IL‐5. The steroid trial is to assess the symptoms of asthma, lung function, blood eosinophils, and FeNO before and after 14 days treatment with 0.5 mg/kg oral prednisolone/day. We judge that cases in which blood eosinophils and FeNO decrease together are not “truely steroid resistance.” In such cases, considering the possibility that allergic type inflammation through adaptive immunity is dominant, anti‐IgE is selected when it is difficult to prevent exacerbations by improving environmental factors. Conversely, we consider that cases in which blood eosinophils and/or FeNO do not decrease, are “truely steroid resistance.” In this case, since there is a possibility that non‐allergic type inflammation due to innate immunity, etc. may remain, anti‐IL‐5, which is expected to be effective for steroid‐resistant eosinophilic inflammation, is selected.Our three‐step algorithm including the steroid trial may be applicable to companion diagnostics testing for molecularly targeted therapies in severe asthma. Further validation is required to examine the effectiveness of this algorithm. Asthma is a general, chronic, and heterogeneous disease with different phenotypes. The management of asthma in recent years has become polarized, reflecting the permanence of inhaled corticosteroids (ICS) treatment. Some patients have good control with ICS and/or bronchodilators, while some patients have poor control despite ICS and bronchodilator and/or oral corticosteroid (OCS). Approximately 10% of asthma patients have severe asthma, and they account for 50% of the total asthma‐related costs, which is a major socioeconomic burden.Molecularly targeted treatment of asthma has made remarkable progress in recent years due to advances in clarifying the molecular pathophysiological mechanism and the development and clinical application of therapeutic drugs corresponding to therapeutic target molecules. Many trials have verified a statistically significant and clinically meaningful reduction in daily maintenance OCS use compared with placebo for patients with severe, uncontrolled OCS‐dependent asthma receiving molecularly targeted treatment Recently, there have been some reports on the therapeutic algorithms for severe asthmatic subjects screened by biomarkers −1 is defined as elevated blood eosinophils, and FeNO ≥35 ppb is defined as elevated FeNO. Step 2 is to divide the subjects further into six groups by atopy test. Positive specific IgE to at least one allergen was assumed to confirm the presence of atopy. As a result, in the case of normal blood eosinophils, normal FeNO and non‐atopic, we make an alternative approach except for biological therapy. If the patient has normal blood eosinophils, normal/elevated FeNO, and is atopic, anti‐IgE is the treatment of choice. If blood eosinophils are elevated, FeNO is normal/elevated, and the patient is non‐atopic, anti‐IL‐5 is the treatment of choice. In the case of elevated blood eosinophils, normal/elevated FeNO, and atopy, we perform a steroid trial in step 3 in order to decide whether to select anti‐IgE or anti‐IL‐5.In terms of how to deal with this problem, we propose a new three‐step algorithm for biological therapy in severe asthma Fig.  that we The steroid trial is to assess the symptoms of asthma, lung function, blood eosinophils, and FeNO before and after 14 days treatment with 0.5 mg/kg oral prednisolone/day In fact, we experienced an allergic/eosinophilic overlap asthma case, as shown in Figure There is no direct comparison between anti‐IgE or anti‐IL‐5 in allergic/eosinophilic overlap asthmatics, nor is one planned. Our three‐step algorithm including the steroid trial may be applicable to companion diagnostics testing for molecularly targeted therapies in severe asthma. Further validation is required to examine the effectiveness of this algorithm.Informed consent was obtained from a patient who presented case.KO and KM designed the study and wrote the manuscript. KO and KM contributed to data collection. KO and KM read and approved the final manuscript.The authors declare no conflict of interests.


Severe asthma prevalence is still controversial.5The present study aimed at investigating the prevalence of severe and uncontrolled severe asthma in the general population through the analysis of the electronic database of Veneto region  Drug Regulatory Agency.A 5-years retrospective (2011–2016) cross-sectional analysis was performed, including subjects older than 6. The database provides the information regarding the anti-asthmatic drugs prescriptions and the asthma-related hospitalizations for all the people living in the region and referring to the National Health System (NHS) services.-The whole asthmatic population included subjects with the exemption code (007), which is released by the NHS to asthmatic patients after specialist's certification. Usually, but not always, the certification implies the lung function assessment. Subject with the exemption code, with or without spirometry assessment, were considered. Patients with Chronic Obstructive Pulmonary Disease (COPD), identified by a different exemption code (057), although prescribed with the same drugs (ICS/LABA combination), were excluded. As per Italian national health system regulation, the two exemption codes are mutually exclusive.-Among asthmatic patients, the more severe ones were selected on the basis of anti-asthmatic treatment level in accordance with step 4 and 5 GINA recommendations .-Within the severe asthma population, patients adherent to the treatment were identified. Adherence was considered as a use over 80% of the prescribed treatment within a 12-months period, that is the amount of drugs bought by the patients in one year. Patients who, despite a regular GINA step 4–5 treatment had more than 2 exacerbations/year, needed hospitalization, or used OCS on regular basis for 6 months, were defined as uncontrolled.The ERS/ATS definitionThe database included 4.659.938 subjects . The overall asthma prevalence was 2.2% (103.138 subjects) . A respiAmong adherent patients, 1.163  remained uncontrolled. In this last subgroup, in the whole study time frame (2011–2016) a spirometry was performed in 77% of cases and considering the 12 months prior to the inclusion in the cross-sectional analysis the eosinophil count was available in 0.7% of patients. The mean age of severe uncontrolled asthmatics was 56 years and females were predominant (59%). According to these data, based on prescribed and dispensed asthma medications, the prevalence of severe asthma is 0.07% in the general population and 3.2% among asthmatic patients. Patients uncontrolled despite a regular GINA step 4/5 treatment were 1.2% of asthmatics. In order to obtain a more reliable estimate, only adherent patients were considered for defining severe asthma and severe uncontrolled asthma population.The functional assessment was performed in 27.7% of patients with certified asthma and among patients with uncontrolled severe disease 23% did not perform a spirometry.To the best of our knowledge this is the first investigation on severe and severe uncontrolled asthma prevalence in Italy, reporting data from a general population database. Only patients over 6 years were included, as the prevalence of severe asthma in Youngers is negligible.However, differently from the other reports, our analysis relied on a general population database provided by the regional Drug Regulatory Agency, which can probably be considered more accurate under the epidemiological perspective. Another study performed in The Netherlands estimated that 3.6% of asthmatics is affected by severe refractory disease. The diagnosis of asthma and degree of asthma control were derived from patient-self-completed questionnaires.In accordance with previous studies the lung function assessment was not regularly performed.Some limitations have to be highlighted. First, the database excluded the patients referring to private practice and/or without the exemption code. However, especially in the field of severe asthma, due to the costs of drugs and the complex management it is reasonable to consider that most of patients refer to the NHS services, so that this limitation should not represent a substantial bias. In fact, in the case the patients are followed-up in the context of the NHS, it implies that everything concerning the management of their disease, namely exams and treatments, are accurately registered. Second, the method used for adherence assessment may not exactly reflect the amount of really taken medications. However among the available tool for adherence evaluation, no one is completely free from bias. Furthermore, the applied method can be a way to distinguish true difficult to treat asthma and lack of asthma control due to poor treatment adherence.As severe and severe uncontrolled asthma prevalence assessment is crucial for disease management, prevention strategies and cost/benefit estimations,AV, GS, MC and MA conceived the article structure and drafted the manuscript. LDE analyzed the dataset. All the authors critically revised the manuscript draft and approved the final manuscript.Ethics approval was obtained from the Health Technology Assessment Unit Committee.Not applicable.Data sharing will be provided following a request to the corresponding author.The authors declare that they have no competing interests.Not applicable.


To investigate changes in intraocular pressure (IOP) and axial length (AL) on the ascent to high altitude from Beijing to Lhasa.Twenty volunteers  who had been sent to work in Lhasa, Tibet for more than 6 months were enrolled. One of their journeys from Beijing to Lhasa was chosen as the time for the examination. IOP, AL, corneal curvature (K), and blood pressure (BP) were measured in Beijing  and Lhasa . Their first examination was conducted at least 1 day before arriving in Lhasa and the second examination after they had stayed in Lhasa for 7 days. The data from the highland and lowland examinations were analyzed with a paired-sample T test and Pearson’s correlation coefficient was calculated for the association between IOP and average BP.P<0.05). The mean K was 43.58±2.25 D in Beijing and 43.56±2.21 D in Lhasa and no significant difference was found in this study (P>0.05). A positive correlation between variance of IOP and ACD was found  and no correlation between IOP and average BP was noted.The mean IOP was 12.65±2.34 mmHg in Beijing and 14.85±3.1 mmHg in Lhasa. The mean AL was 24.61±1.50mm in Beijing, and 24.98±1.45 mm in Lhasa. The IOP and AL showed a significant elevation in highland compared with lowland (High altitude may lead to a small but significant change in IOP and axial length. However, the shape of the corneal surface was not influenced by the hypobaric and hypoxic conditions. High altitude has a systemic effect on human beings, including the visual system. But the relationship between IOP and altitude has been inconsistent. Multiple studies have shown varying effects of IOP at high altitudes 2]3]4][3][4]2][[4][3][4][3][4]2]6]..6].China is a vast country, with varying geographic characteristics. Many cities on the Qinghai-Tibetan plateau, such as Lhasa and Shigatse, have an elevation that is over 3600 meters. Every year, many tourists and workers travel between the eastern lowlands and the western highlands of China. A high altitude has diverse systemic and ophthalmic effects on individuals. Ophthalmic effects include changes in IOP7], the , the 7],Lhasa, with an altitude of 3658 m and nearly 1 million population, is one of the highest metropolises in the world. In this study, healthy volunteers from Beijing, a lowland with an elevation of 45 m in eastern China, were enrolled in this study. The IOP and axial length of eye balls were measured in both Beijing and Lhasa, and changes were documented and compared. The aim of this study is to objectively assess human responses to hypoxic and hypobaric stresses.20 volunteers from Beijing who had worked in Lhasa for > 6 months were enrolled between March to July 2017. All subjects were adult Han Chinese, who had a normal slit lamp examination, a best-corrected visual acuity (BCVA) of >20/40 and an IOP < 21 mmHg. Subjects with a history of intraocular surgery and any ocular conditions other than refractive errors were excluded. One of their journeys from Beijing to Lhasa by flight was chosen as the time for the examination. Ophthalmic examination of IOP, biometric parameters, corneal curvature (K), central corneal thickness (CCT) and blood pressure (BP) were measured in Beijing (3 pm-4 pm) 1 day before they arrived in Lhasa. The IOP, biometric parameters, K and blood pressure were measured again in Lhasa (3 pm-4 pm) after the volunteers had stayed in Lhasa for approximately 7 days. Beijing and Lhasa are in the same time zone making the issue of jet lag mute. All measurements were measured at the same time in each city. Both eyes underwent the same ophthalmic examinations and only right eye data was selected for analysis. Values of IOP were measured three times using two identical applanation tonometers , and K was measured three times using two identical autorefractometers  in Beijing and Lhasa. Biometric parameters, including the AL, anterior chamber depth (ACD), lens thickness (LT), and vitreous chamber depth (VCD) of the eye balls, were measured with an A-mode ultrasonic pachymeter in Beijing  and an A-mode ultrasonic pachymeter  in Lhasa. The mean of at least 10 readings were used for each eye. CCT was measured with a noncontact pachymeter  in Beijing only due to limited equipment available in Lhasa. The average BP (ABP) was calculated from diastolic blood pressure (DBP) and systolic blood pressure (SBP) using the formula: average BP = DBP +1/3(SBP-DBP). All examinations were repeated three times in both cities. The means of the measurements were used as the ocular parameters for analysis. Before the study, a consistency check between the two examiners were done at Beijing, and the results were highly coherent.This study was approved by the Medical Ethics Committee of the Tibet Autonomous Region People’s Hospital. All participants gave informed written consent according to the tenets of the Declaration of Helsinki.SPSS  was used for all statistical analyses. Data exhibiting a normal distribution are expressed as the mean±standard deviation. Comparisons were performed between the two measurement using paired Student’s t-test. A value of P<0.05 was considered statistically significant. Pearson’s correlation coefficient was calculated to determine the association between IOP, AL, ACD and average BP.More than 40 volunteers registered for this study but many subjects were excluded due to incomplete data collected during the trips between Beijing and Lhasa. Only 20 volunteers completed the data collection process, including 17 men and 3 women. The mean age was 42.56±5.5 years. The average IOP of the volunteers in Beijing was 12.65±2.34 mmHg and increased to 14.85±3.1 mmHg in Lhasa , with a The ABP was 91.4±9.7 mmHg in Beijing and 105.3±15.9 mmHg in Lhasa. There was no relationship between the variance of IOP and the variance of axial length and the variance of the ABP, except for the variance of ACD . The corThe likely cause of the changes in IOP with variations in altitude is fluctuations in barometric pressure. Three conditions have been used to examine this issue before, flight environment, artificial hypobaric chamber and ascending altitude . The mosThe most common research model was conducted on climbers. Some studies6] showe showe6] In this study, healthy volunteers who lived in the highland city for 6 months were selected. It is presumed that these subjects have high hematocrit levels and other physiological characteristics adjusted to hypobaric and hypoxic conditions. All ophthalmic examinations were performed in indoor clinics. We expect our data is more credible since the subjects were merely affected by altitude.In this study, we found that immigrating from lower land to higher land may cause an increase in IOP, and the variance of IOP was related with the variance in ACD. There may be two reasons to explain the IOP changes.First, increased IOP may be caused by increased systemic blood pressure. Some studies have shown that a 10 mmHg increase in SBP and DBP was associated with 0.2 and 0.4 mmHg increase in IOP, respectively. In thisSecond, the change in CCT may also affect IOP measures. Some research has shown that the CCT increased after exposure to high altitudes19]20].[20].19][.[20].19]Many studies have used a Tono-Pen, which is a handheld electronic instrument with an advanced ergonomic design, to measure IOP in different environments. This deAnother concern about the accommodation of altitude is the change in K and axial length. In this study, we did not find statistically significant changes in K, but we did find changes in AL, which have not been reported to date. It is hard to compare the changes in these ocular parameters at different altitudes because of device limitations. The autofractometer and ultrasonic pachymeters are difficult to carry with mountain climbers. In this study, we chose two ophthalmology clinics in the two cities to implement such examinations. We found that the axial length and anterior chamber length had significant elongation under high-altitude conditions. The elongation of axial length corresponded to an increased diopter of -0.5 to -1D, which could be the reason of blurred vision in such subjects.This transformation of eye shape may be due to a quick adaptation of the eyeball under a lower barometric pressure. The physical characteristics of the eyeball are similar to a latex balloon filled with water. The size of the eyeball would have enlarged a little to maintain a lower pressure because the barometric pressure of Lhasa is only 60% of Beijing. Secretion and drainage of aqueous fluid may achieve a new balance and the decreased pressure difference between the inside and outside of the eyeball would be greater in Lhasa than Beijing. At the same time, we also note that the difference in measurement instruments and examiners may contribute to the prolongation of axial length at Lhasa. One of the examiners might touch the cornea more lightly because of his operating habits.One of the shortcomings of this study is having different examiners at the two examination clinics and this could contribute to examiner bias. Before starting the research, we performed a consistency check between the two examiners. One group of 10 subjects had their IOP tested by the two examiners, and there was no significant difference between the measurements obtained by the two examiners. The difference in IOP measurements between the examiners can be assumed to be very small during this research. Furthermore, the instruments used for measuring keratometry and axial lengths were different between the two clinics. The difference in axial length measurements could be confounded by the different measurement techniques applied by the different examiners. In a similar study, two identical devices were used at two different places, and this was also acceptable considering travel limitations . Some eqIn this study, we concluded that a change in altitude resulted in a statistically significant change in IOP and axial length, which could be due to ocular adaption to hypobaric conditions and may be the reason for blurred sight. We could not identify the underlying mechanism for this phenomenon; however, high blood pressure and elevated episcleral venous pressure may play an important role. Future research with an increased sample size and decreased measurement bias is needed to further validate this.S1 Table(XLSX)Click here for additional data file.


Beta adrenergic receptors are crucial for their role in rhythmic contraction of heart along with their role in the pathological conditions such as tachycardia and high risk of heart failure. Studies report that the levels of beta-1 adrenergic receptor tend to decrease by 50%, whereas, the levels of beta-2 adrenergic receptor remains constant during the risk of heart failure. Beta blockers—the antagonistic molecules for beta-adrenergic receptors, function by slowing the heart rate, which thereby allows the left ventricle to fill completely during tachycardia incidents and hence helps in blood pumping capacity of heart and reducing the risk of heart failure. In the present study, we investigate the potential of glycyrrhizic acid (GA) as a possible principal drug molecule for cardiac arrhythmias owing to its ability to induce reduction in the heart rate and blood pressure. We use in vitro and in silico approach to study GA′s effect on beta adrenergic receptor along with an in vivo study to examine its effect on heart rate and blood pressure. Additionally, we explore GA′s proficiency in eliciting an increase in the plasma levels of vasoactive intestinal peptide, which by dilating the blood vessel consequently, can be a crucial aid during the occurrence of a potential heart attack. Therefore, we propose GA as a potential principal drug molecule via its potential in modulating heart rate and blood pressure. No specific monitoring is needed after single acute overdose”.There exist four subtypes of β-AR—β1-adrenergic receptor (β1-AR), β2-adrenergic receptors (β2-AR), β3-adrenergic receptors (β3-AR) and β4-adrenergic receptors (β4-AR) . Also, βquorice) . It is wquorice) , inflammquorice) ,21,22, bquorice) , diabetequorice) ,25, cancquorice) ,27 and hquorice) ,29,30. Hquorice) . NeverthThe effect of 10 µM GA on the cAMP levels of CHO cells transfected with β-ARs  and stimulated by Nor-epinephrine (0.1 µM) was tested.We did not observe any statistically significant reduction in the cAMP levels in β1-AR. However, a statistically significant drop in the cAMP levels was observed in cells transfected with β2-AR and β3-AR and subsequently stimulated with nor-epinephrine (NE), when compared to β2-AR and β3-AR transfected cells and stimulated with nor-epinephrine (NE) along with 10 µM of GA, suggesting selective antagonistic capability of GA against β2-AR and β3-AR .The DSI small animal telemetry was implanted and the mice were allowed to recover and stabilize the blood pressure and heart rate parameters for at least 10 days post-surgery. GA at a dosage of 10 mg/kg i.p. in PBS (as vehicle) was used for measuring its effect on mice′s blood pressure and heart rate. Wild type mice with stabilized cardiac parameters were treated with GA (10 mg/kg i.p.), an acute single dose, and further monitored in the DSI telemetry system. Followed by the i.p. injection, both the blood pressure and the heart rate dropped significantly. Three to four hours post injection, the blood pressure A as wellThe systolic and the diastolic blood pressure reduced by 28.68% ± 6.48% and 31.56% ± 7.35% and, the heart rate dropped by 27.17% ± 14.73% C, all peGA (10 mg/kg i.p) along with PBS as vehicle was used to treat mice. GA administration was subsequently followed by the measurement of vasoactive intestinal peptide (VIP) plasma levels. We found that i.p. injection of GA instigated a statistically significant but small rise in the VIP levels, after 30 min post i.p. injection when compared to control .t = 1 h [Furthermore, along with VIP, we observed a rise in the secretin levels in mice, peaking at t = 1 h . Interest = 1 h .Upon virtual docking between the 3D structures of GA, NE and alperenolol (retrieved from pubchem database) with β1-AR (PDB ID: 5F8U) and β2-AR (PDB ID: 3NYA), we observed the binding affinity of GA with β1-AR and β2-AR to be −56.27 and −59.51 respectively. Importantly, the binding affinity of GA was found to be higher when compared to the binding score of the control molecules, alperenolol  and NE.Adrenergic receptors are functionally involved in several important physiological functions and are implicated in various cardiovascular disorders particularly heart failure and even aging . ActivatThe adrenergic stimulation by epinephrine and nor-epinephrine  of the hSince GA is capable of modulating heart rate and blood pressure after acute administration via intra peritoneal injection in vivo, hence, it might produce a similar physiological response as that of a β-AR antagonist. β-AR antagonist molecules are well recognized to be an important part of clinical practice  and GA, v/v) CO2 humidified chamber at a temperature of 37 °C. Cell culture was followed by the cAMP assay, wherein, a six well plate with a capacity of 35 mm/well  was plated with 3 × 105 cells and allowed to grow for 24 h before transfection. After 24 h of cell growth, the cells were transfected, wherein, 2 µg of the construct, receptor-pcDNA3.1, was used to transfect the CHO-K1 cells using X-treme GENE HP DNA Transfection Reagent , in a ratio of 1:3 (w/v), for each well. After two days, the transfected cells were incubated with the peptide for 45 min, which was then followed by the measurement of the intracellular levels of cAMP. The assay conducted, utilized the LANCE cAMP assay kit  with Victor x4 multilabel reader (Perkin-Elmer), which followed the manufacturer′s protocol. Also, the CHO-K1 cells transfected with pcDNA3.1 were taken as the negative control. The observed results were reported as the fold change in the intracellular cAMP levels when compared to the basal level (no agonist/antagonist control). Additionally, the cells upon transfection were used for the extraction of total RNA  in order to confirm the receptor′s expression.The cell culture and transfection experiment was carried out as per the standardized protocol in our lab ,52. BrieMale mice of age 2–3 months were used for the experiments. Only 3–4 mice were housed per cage with 12 h light and dark cycle along with free access of food (standard rodent chow) and water with temperature controlled environment at 24 °C. All the animal experiments were carried out according to the guidelines of CULATR . The animals were acclimatized for a week before experiments and then randomized into groups based on their body weight and basal hormonal levels.t = 0, t = 30 min). The blood samples were procured from the tail vain of the mice and were aliquoted in different Eppendorf tubes along with heparin. Further, the serum was separated from the blood samples by centrifuging it for 15 min at 4 °C at 12,000× g. The hormonal levels were estimated using ELISA kit  as explained above [Male WT mice of 2–3 months of age were used for the experiment. After randomization of mice, the mice were grouped according to their basal level of the hormones, measured using ELISA kit provided by Phoenix pharmaceuticals, INC . After grouping the animals, we left them for about two weeks to acclimatize, followed by the drug administration in the animals (GA 10 mg/kg i.p. injection) and blood collection from different animals at different time points (ed above .https://www.datasci.com/products/ implantable-telemetry/mouse-(miniature)/hd-x11). The incision was then closed using the continuous monofilament suture. The animal was administered with buprenorphine (0.3 mg/mL i.p.) for pain relief. Then until the animal recovered from anesthesia they were kept in intensive care unit for about 4–6 h. Afterwards the animals were kept back in temperature controlled 12 h day and night cycle for complete recovery for about 10 days. The blood pressure measurement and the drug administration was started after the animal got fully recovered and the diurnal blood pressure pattern returned back to normal.Mice were first sedated with the help of isoflurane (3%–4%) in 2 L of oxygen flow rate in the induction chamber for 3–5 min. The hair in the neck area were removed using depilatory cream and warm water. Betadine and 70% ethanol were used for sterilizing the area. The animal was then put on a 37 °C temperature controlled pad for operation. The dosage of isoflurane was then changed to 1%–1.5% with 2 L oxygen flow rate. For DSI implantation, approximately 1 cm cut was made longitudinally, separating the neck muscle from the connective and fat tissue, which allowed us to observe clearly the left common carotid artery. One tight suture was made near the head along with the two loose ones in the middle and another at the end of the exposed artery. Afterwards, the artery was opened by piercing with a 90 angled 27 G needle tin followed by the insertion of the pressure probe and further proceeding for 11 mm into the artery from the opening. Then the ECG positive electrode was inserted at the end of the left side of the rib cage, while the other was inserted into the right pectoral region. Further space was created till stomach and then DSI device was implanted (n = 5) were allowed to recover for 10 days and then the blood pressure and heart rate change analysis were done between the days 11–15, after the base line monitoring of the same. First, the normal blood pressure (diastolic and systolic pattern) and heart rate were confirmed and then the drug treatment was done (GA 10 mg/kg i.p. in PBS). We supervised the diastolic and systolic pressure along with the heart rate for next 24 h to check the effect of the drug. PBS was given as vehicle control and the effect was analyzed after the drug responses returned back to normal.After DSI instalment surgery, the mice (WT https://pubchem.ncbi.nlm.nih.gov) in SDF file format and was later converted to PDB file format using open babble software, for the docking stimulation [The in silico virtual docking of the compounds  was performed with their respective receptors with which they are documented to interact with, in order to computationally calculate the binding affinity towards the receptors and also to compare it to their natural ligand molecule and a known drug for the receptor . The PDBmulation . Dockingmulation ,58 and Smulation , and folmulation .p < 0.05) and was performed for in vitro and in vivo assays to compare between two groups. The graphs for in vitro analysis of cAMP results were plotted using GraphPad Prism 7.0 software and all data was expressed as mean + SEM. In vivo treatment groups of hormonal release assay were also analyzed using t-test and were plotted using Microsoft Excel.The results were analyzed by Microsoft Excel, Fold change was calculated as treated/untreated. Significant difference was identified using student “t” test with 95% level of significance (We conclude that GA when administered via intra peritoneal injection, leads to a drop in heart rate and blood pressure in wild type mice model. This physiological effect might be due to GA′s antagonistic activity against beta adrenergic receptors and also possibly via increasing the plasma VIP levels after about 30 min of its injection. The present study, opens up a possibility for further research in GAs action on changes in heart rate and blood pressure.