More specifically, p-values of 0.0015 for PBFIV, 0.0183 for TRR, and 0.0048 for L0RR were obtained. The results of this feasibility study indicate the diagnostic potential of the proposed method for the detection of PAD.While being a relatively prevalent condition particularly among aging patients, peripheral arterial disease (PAD) of lower extremities commonly goes undetected or misdiagnosed due to its symptoms being nonspecific. Additionally, progression of PAD in the absence of timely intervention can lead to dire consequences. Therefore, development of non-invasive and affordable diagnostic approaches can be highly beneficial in detection and treatment planning for PAD patients. In this study, we present a contrast-free ultrasound-based quantitative blood flow imaging technique for PAD diagnosis. The method involves monitoring the variations of blood flow in the calf muscle in response to thigh-pressure-cuff-induced occlusion. Four quantitative metrics are introduced for analysis of these variations. These metrics include post-occlusion to baseline flow intensity variation (PBFIV), total response region (TRR), Lag0 response region (L0RR), and Lag4 (and more) response region (L4 + RR). We examine the feasibility of this method through an in vivo study consisting of 14 PAD patients with abnormal ankle-brachial index (ABI) and 8 healthy volunteers. Ultrasound data acquired from 13 legs in the patient group and 13 legs in the healthy group are analyzed. Out of the four utilized metrics, three exhibited significantly different distributions between the two groups ( Lower extremity PAD is the most common form of PAD affecting more than 8.5 million Americans and more than 230 million patients worldwide3. While traditional atherosclerotic risk factors participate in the development of PAD, nicotine addiction with habitual smoking is considered the most prevalent risk for these patients, and diabetes mellitus is likewise an important variable5. An early and the most common clinical presentation of PAD is intermittent claudication, which involves pain in the lower limb  mainly induced by physical activities such as walking and subsiding by rest7. While PAD associated walking impairment adversely influences quality of life for PAD patients, there is an added elevated risk of developing major adverse cardiac events including myocardial infarction, stroke, heart failure and both cardiovascular death and all-cause mortality10. Beyond intermittent claudication and major adverse cardiac events, severe PAD can lead to critical limb ischemia which if untreated, might result in leg amputation and a high mortality rate as a result11. Therefore, early diagnosis of PAD and proper implementation of guideline indorsed risk factor modification is paramount for effective management of these patients12.Peripheral arterial disease (PAD) generally refers to a progressive circulation disorder, characterized by narrowing or occlusion of the peripheral arteries15. For disease screening, symptom evaluation, and periodic disease monitoring, a non-invasive evaluation is more appropriate and involves Doppler assessment and measurement of the ankle brachial index (ABI)16. ABI values in the range of 0.9 to 1.4 are considered as normal18. Values less than 0.9 indicate the presence of arterial disease19 with values below 0.5 consistent with severe PAD, suggestive of critical limb ischemia20. While readily available, easy to perform, and reproducible, the resting ABI assessment may have limited accuracy in conditions such as diabetes mellitus, chronic kidney disease, arterial calcification, isolated iliac arterial disease and in the elderly22. Furthermore, ABI may be insensitive to microvascular impairment, which may coexist with macrovascular disease in PAD patients26. Therefore, normal ABI does not rule out PAD and values greater than 1.4, may indicate high stiffness and poorly compressible arteries 28. As a result, in such cases, the severity of PAD cannot be properly evaluated.Customary clinical diagnostic and monitoring methods for peripheral arterial disease include the use of different imaging modalities such as duplex ultrasonography, computed tomography (CT) and magnetic resonance (MR) angiography32. The impaired microvascular reactivity expresses the imbalance between vasodilators and vasoconstrictors, such as nitric oxide and endothelin, respectively. One study reported the assessment of microvascular reactivity by hyperemic response to contraction and cuff occlusion using BOLD imaging33. Moreover, in response to chronic metabolic demand in PAD, vascular remodeling include increased diameter and density of microcirculations to accommodate the increased blood flow34. Studies suggest that progression of the peripheral arterial disease can lead to disruptions in blood distribution and flow in the muscles of the lower extremity, thus creating the conditions that result in intermittent claudication37. Many of these studies are performed using MR40 and CT-based imaging methodologies42.Arteriolar endothelial function, microvascular regulation and responsiveness to specific stimuli are impaired in patients with PAD which may play a central role in disease development and progression44. Emergence of plane wave ultrasound imaging has shown promise in quantitative high resolution blood flow imaging without the use of contrast agents48. Several studies have examined the practicability of novel ultrasound microvasculature and perfusion imaging strategies for evaluation of blood flow variations in patients with PAD49. While studies using contrast agents have shown promise for assessing slow flow in small vasculature54, there is a need for more accessible and cost-effective methods that do not require contrast agents.In recent years, there has been growing interest in investigating the microvascular flow and muscle perfusion in PAD. However, the use of non-invasive imaging modalities for PAD diagnosis, such as duplex ultrasonography, has been limited to the evaluation of macrovascular blood flow55. We hypothesize that by evaluating the blood flow variations of the lower extremity in response to thigh-cuff occlusion, we can identify PAD-affected legs. In this paper, a novel pressure-based method is proposed for flow imaging that can assess microvascular reactivity using ultrasound. The method measures the changes in microvascular blood flow in the calf muscles in response to a mechanical stimulus, which can be used to assess the function of the microvasculature in the lower extremities. We demonstrate how the measured metrics can provide potential diagnostic information about the conditions of patients in comparison to their ABI evaluations.Monitoring and analysis of the reactive hyperemic response in the lower limb to externally induced occlusion can have diagnostic significance. It is known that a reduction in peak muscle perfusion capacity occurs in response to ischemic provocation tests, such as thigh-cuff occlusion In this section, we present a comparison of the results of our methodology for a 72-year-old female PAD patient clinically diagnosed with moderate PAD in the left leg and the results for the left leg of a healthy 62-year-old female volunteer. Resting ABI values for the left leg of the PAD patient measured at the dorsalis pedis (DP), and the posterior tibial (PT) arteries are reported as 0.62 and 0.61, respectively.Comparative illustration of the results for the aforementioned cases is displayed in Fig. p-value > 0.05) distributional difference between the two groups.A summary of the calculated metric values is presented in Table In this study, we introduced a contrast-free ultrasound imaging framework for evaluation of blood flow variations in response to pressure cuff occlusion in PAD patients as well as healthy subjects. Post-processing and analysis help in visualization and evaluation of flow changes and quantification metrics can have diagnostic potential.56, CT57, and contrast-enhanced ultrasound58 imaging. The potential and significance of the current study lies in its use of simple, inexpensive, and accessible ultrasound imaging without the added burden of injecting contrast agents. This can potentially preclude further discomforts and expenses for the patients, as ultrasound is commonly used for screening purposes.There exist several studies in the literature for monitoring blood flow and perfusion changes in the lower extremity in patients with PAD using different imaging modalities including MRIImplementation of the proposed method on patients with clinically diagnosed PAD  and healthy subjects suggest that there might be observable differences between the hemodynamic response of affected legs in PAD patients to pressure cuff induced occlusion compared to the legs of healthy individuals. Correlation masks presented in Fig. The normalized average Doppler Intensity variations in the Doppler frames leading up to the point of pressure release . This phenomenon has been reported in the literature where PAD subjects generally exhibit a faster perfusion increase as well as a more rapid drop in perfusion post occlusion23.Doppler intensity variations as shown in Fig. 55.The delayed response in the case of PAD patients is also quantified via the percentage of no lag pixels (L0RR) and the pixels with a lag of four frames and more (L4 + RR). This is in keeping with observations in the literature where the time to peak response in PAD patients is reportedly significantly higherThere are some limitations associated with the current study. Firstly, the number of subjects included in the analysis for this study was limited. In the future, we plan to study on a large patient population, PAD patients as well as healthy volunteers, to evaluate the performance of the introduced technique in differentiating between the two groups based on the quantification metrics. We also plan to incorporate exercise as part of our setup to evaluate the combined post-exercise and post-occlusion responses for enhanced differentiation between affected and unaffected lower limbs.To evaluate the efficacy of our methods, imaging studies were conducted for 14 patients with clinical diagnosis of PAD , and 8 healthy volunteers . Age distribution of the patients and healthy subjects was 65.9 ± 16.3 and 64.1 ± 2.8, respectively (mean ± standard deviation). This study was approved by the institutional review board at the Mayo Clinic (IRB#: 19-002559) and was in compliance with the Health Insurance Portability and Accountability Act. All the research was performed in accordance with relevant guidelines/regulations and followed the Declaration of Helsinki. A written IRB approved informed consent form was signed by each participant prior to the study. Patients were recruited based on their clinical ABI examination. Recruitment for the healthy group involved aiming for non-smoker individuals with no history of diabetes or relevant underlying diseases. During the study, both legs of the subjects were scanned. For final data analysis, data from legs of patients with normal ABI, as well as poor data acquisitions were excluded. After exclusions, data from a total of 13 legs with abnormal ABI were compared to data from 13 legs of healthy individuals.Data acquisition was performed using a linear array L11-4v probe (with a center frequency of 7.24 MHz) attached to a Verasonics Vantage 256 ultrasound research system . Ultrafast ultrasound imaging was implemented through coherent compounding of plane wave transmissions at five different insonification angles equally spaced within the [− 5.5° +5.5°] range. Each transmission sequence involved acquiring 500 IQ data frames at a frame rate of 2 kHz and was repeated every 2 s to monitor the flow response over time.With subjects in the supine position, the targeted leg was placed on a stand-alone leg prepper. The ultrasound probe was attached to the subject’s calf muscle for imaging and was secured in place using a multi-joint mechanical arm. A pressure cuff was wrapped around the subject’s thigh and an automatic cuff inflation device  was used for rapid inflation of the cuff. A schematic illustration of the setup is shown in Fig. The 500 IQ data frames acquired during each transmission sequence constitute an ensemble of ultrasound images that was used to generate a Doppler image visualizing blood flow intensities at a given point in time. These frames were first reshaped into a spatiotemporal/Casorati data matrix. Next, a singular value decomposition (SVD) filter was applied to the data to remove the low-rank tissue clutter. In this study, the SVD threshold for the separation of clutter and blood subspaces was chosen empirically and was set to the constant value of 50 for all the Doppler frames . Subsequently, the clutter filtered data frame ensemble underwent a temporal coherent integration to generate the Doppler image.62. In this work, we used a Doppler image generated from an open-air transmission to estimate this profile. Since no significant echo signal was expected in such a transmission, the resulting image would be a good approximation of the TGC-induced noise pattern. Considering the TGC-induced noise was removed after the implementation of SVD, it did not have a direct influence on our choice of the SVD threshold. We then subtracted this noise profile from each of the Doppler images to generate a final flow image. We refer to each of these images as a Doppler frame. Figure The resulting Doppler image was composed of signals generated by blood flow as well as noise. The background noise profile (mainly induced by the time gain compensation (TGC) settings) can be estimated and compensated for, through various meansThe generated Doppler images illustrate the hemodynamic variations in the calf muscle. These variations can be quantified as a function of time, in a time series with a sampling period of 2 s. To monitor the hyperemic response to the pressure induced occlusion, we define a single lag activation step function to represent the onset of the stimulation (pressure release) at the “Deflate” (pressure release) point. We then correlate the Doppler intensity variations from 10 Doppler frames before, up until one frame after the point of pressure release. This results in a correlation value for each pixel in the Doppler image showing its consistency with the expect hyperemic response, together constituting a correlation map after pressure release. Using a threshold on these maps, a binarized image is generated depicting all the pixels that exhibited a compensatory flow response, including muscle perfusion. This threshold was set to 0.5 in this study to strike a balance between the inclusion of slow responding flow elements  and the inclusion of noise. We refer to these binarized images as correlation masks. These correlation masks are utilized to monitor Doppler intensity alterations, by averaging the masked Doppler signal magnitude as a function of Doppler frame sample time for the duration of the study. These temporal intensity profiles can then be used to estimate post-occlusion Doppler intensity variations with respect to the baseline. Therefore, we define a metric called “post-occlusion to baseline flow intensity variation” (PBFIV) as the percentage-wise ratio of the difference between average post-occlusion and average baseline flow intensities over the average baseline intensity:mentclass2pt{minimMoreover, by creating maps of post-occlusion response delay (referred to as lag images) we estimate the lag-specific response regions. Lag0 response region (L0RR) corresponds to the density of pixels exhibiting an immediate increase flow following pressure release, and Lag4 (and more) response region (L4 + RR) represents the pixels for which at least 4 frames are required to manifest a compensatory response (flow increase):mentclass2pt{minimFigure The previously defined metrics were calculated for the two groups . A two-sided Wilcoxon rank sum test was implemented on the distributions of these metrics to evaluate the differences between the two groups and the corresponding p-value was computed for each metric. Differences in metrics with p-values less than 0.05 were considered to be statistically significant. Data processing and statistical analyses were performed in MATLAB R2022b .In this paper, we presented a contrast-free ultrasound imaging technique for monitoring the hemodynamic response to pressure cuff induced occlusion in the lower extremity of PAD patients. Metrics were defined to analyze and quantify the variations in blood flow in a baseline state, and after pressure release. These quantification metrics can be utilized to differentiate between affected and unaffected legs and potentially help with more accurate diagnosis of PAD patients before the need for more costly cross-sectional imaging approaches arises. In our future studies, we plan to recruit larger numbers of patients and perform statistical analyses on the proposed metrics to evaluate their discriminatory potential.


The pre-operative blood basophil count and pre-operative bEBR were higher in AERD patients than in the control group . The results of this study support the hypothesis that polyps removal may contribute to reducing the inflammation and activation of basophils.Aspirin-exacerbated respiratory disease (AERD) is characterized by eosinophilic asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and intolerance to cyclooxygenase-1 inhibitors. Interest is emerging in studying the role of circulating inflammatory cells in CRSwNP pathogenesis and its course, as well as their potential use for a patient-tailored approach. By releasing IL-4, basophils play a crucial role in activating the Th2-mediated response. The main aim of this study was to, first, investigate the level of the pre-operative blood basophils’ values, blood basophil/lymphocyte ratio (bBLR) and blood eosinophil-to-basophil ratio (bEBR) as predictors of recurrent polyps after endoscopic sinus surgery (ESS) in AERD patients. The secondary aim was to compare the blood basophil-related variables of the AERD series (study group) with those of a control group of 95 consecutive cases of histologically non-eosinophilic CRSwNP. The AERD group showed a higher recurrence rate than the control group ( Aspirin-exacerbated respiratory disease (AERD) is an inflammatory condition that consists of eosinophilic asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and respiratory reactions to cyclooxygenase-1 (COX-1) inhibitors. The reported prevalence of AERD in CRSwNP patients ranges between 8.7% and 26% ,4,5. ComA standard histological examination can identify the main inflammatory cells (such as eosinophils and neutrophils) that have been associated with CRSwNP relapse ,10,11,12In 2004, Hartnell et al.  investigIt is unknown which biomarkers effectively predict the recurrence of nasal polyposis in patients with AERD undergoing endoscopic sinus surgery (ESS). The main aim of this study was to, first, investigate the level of blood basophils, bBLR and bEBR as predictors of recurrent polyps after the ESS in AERD patients. The secondary aim was to compare the blood basophil-related variables of the AERD series (study group) with those of a control group of 95 consecutive cases of histologically non-eosinophilic CRSwNP.This retrospective study was conducted in accordance with the principles of the Helsinki Declaration. All patients signed a detailed informed consent form and gave their written permission for clinical case publication. The data were examined in agreement with the Italian privacy and sensitive data laws, as well as the internal regulations of Padova University’s Otolaryngology Section. Furthermore, all enrolled patients signed a form in which they consented “to the use of their clinical data for scientific research purposes in the medical, biomedical and epidemiological fields, also in order to be recalled in the future for follow-up needs”.A consecutive series of 39 adult patients who had undergone surgery for CRSwNP with AERD at the Otolaryngology Section of Padova University between 2009 and 2022 was retrospectively assessed. In contrast, the control group consisted of another consecutive series of 95 CRS cases with a histological diagnosis of non-eosinophilic polyposis who had undergone surgery by the same surgical team in the same period. Patients were excluded from the study in the event of a lack of pre-operatory blood basophils data, a diagnosis of systemic inflammatory disease or autoimmune disease, or acute or chronic infectious conditions other than sinusitis; cancer; blood disorders; a history of systemic corticosteroid use; or chronic kidney disease. Patients whose CRSwNP had already recurred before their blood was sampled post-operatively were also excluded, as their recurrent disease might have influenced their post-ESS eosinophil, basophil and lymphocyte counts.Information about sensitivity to acetylsalicylic acid or other non-steroidal anti-inflammatory drugs (NSAIDs) was obtained from their medical history recorded in Padova University Hospital’s electronic archives . Preoperative blood cell counts and percentages were obtained for each patient. The necessary laboratory tests were performed at least three months after withdrawing oral steroids and one month after withdrawing nasal steroids; they were all processed at the same laboratory  and certified in compliance with ISO standard 15,189. The total and specific IgE for Dermatophagoides pteronyssinus and Dermatophagoides farinae, birch pollen, pellitory, grass mix, cat and dog dander, Alternaria alternata, Aspergillus fumigatus and common ragweed were determined. A diagnosis of asthma was confirmed according to the definition of the Global Initiative on Asthma . The recAfter the ESS, surgical tissue was stained with hematoxylin and eosin to measure the eosinophil count, examining 5 high-power fields (HPFs) (400×) selected from each sample and recording the average number of eosinophils. The eosinophilic histotype corresponded to a mean score of ≥10 eosinophils/HPF . PatientAll patients were treated post-operatively with isotonic saline solution irrigations twice a day (20 mL per irrigation), nasal steroids (fluticasone furoate 110 μg daily (55 μg per nostril) or mometasone furoate 200 μg daily (100 μg per nostril)). Adequate therapy was prescribed for asthmatic and allergic patients. Follow-ups with rigid 0° or 30° endoscopes were scheduled 3, 6 and 12 months after the ESS, and yearly thereafter.9/L), blood basophils percentage, bBLR and bEBR.From a laboratory viewpoint, for all patients considered in the study, the following pre- and postoperative variables were determined and archived: blood basophils count . The results are reported as the median and range for quantitative variables and as the count and percentage for categorical variables.The recurrence-free interval was measured as the time from the ESS to recurrence or the last follow-up evaluation for censored patients.The prognostic role of each blood basophils-related variable on recurrence-free interval for AERD and control patients was assessed with univariate Cox regression.p-value and hazard ratio (HR) with a 95% confidence interval (CI).The plot of the cumulative Martingale residuals against the values of the covariate and Kolmogorov-type supremum test based on a sample of 1000 simulated residual patterns was used to test the proportionality for quantitative covariates. The results are expressed as the The AERD and control groups were compared with the Mann–Whitney test for quantitative variables after having checked the normality with the Shapiro–Wilk test and the Q–Q plot. The chi-square test was applied for categorical variables.p-value < 0.05 was considered indicative of statistical significance.A A total of 39 patients with AERD  and 95 consecutive control cases with non-eosinophilic CRSwNP  were enrolled in the study.The CRSwNP recurrence rate after ESS was significantly higher in the AERD group than in the control group, with a calculated HR of 5.473 (95% CI 2.753–10.881) .9/L, respectively; p-value 0.0364)  .p-value 0.4931).Interestingly, considering the same variable post-operatively, there was no significant difference between the two groups   .p-value 0.6607).Furthermore, this basophil-related variable was not significantly different between the two cohorts after surgery , leading to an accumulation of eosinophils ,42. New The main weaknesses of this study concern the retrospective design and the limited number of patients, in particular those with available post-operative laboratory data. The main strengths lie in the intra-group homogeneity of the series of patients considered since the (i) histopathological analyses were all done by a dedicated head and neck pathologist; (ii) ESS was performed by the same team of surgeons; (iii) the endoscopic follow-up after surgery was conducted by the same team; (iv) recurrent eCRSwNP was always confirmed endoscopically; and, lastly, (v) all blood tests were performed over a long-term post-operative period at the same laboratory.Before surgery, among subjects with CRSwNP, blood basophil counts and bEBR were significantly higher in patients with AERD than in those without AERD. The retrospective nature of the study did not allow for correlating these data with significant blood increases of any cytokines involved. AERD was associated with a higher risk of recurrence of nasal polyps. Among all the CRSwNP cases, polyps removal with ESS may have helped in reducing inflammation and the activation of basophils and eosinophils. The same result is obtained with biological therapy. Therefore, our observations seemed interesting in relation to the routine use of anti-interleukin biologics. Furthermore, a possible synergistic effect of surgery associated with the biologic therapy should be further investigated.


To explore the results of hypertension improvement and renal function preservation after renal artery aneurysm (RAA) repair.This study retrospectively analyzed the change in blood pressure (BP) and renal outcomes of 59 RAA patients throughout either open or endovascular operations and follow-up at a large center. Patients were grouped according to the difference in their BP at the last follow-up vs. their baseline value. Logistic regression was conducted to explore risk factors for perioperative BP relief and long-term hypertension reonset. Previous studies of RAA with records of BP, blood creatinine level, or GFR/eGFR results are reviewed.2. The median follow-up was 854  days. Both open and endovascular techniques significantly relieved hypertension and did not impair renal function much. Lower preoperative systolic BP (SBP) was significantly associated with hypertension relief . Among patients with normal BP after the operation, higher postoperative SBP was significantly associated with new-onset hypertension . Literature review indicated that renal function usually remained normal at follow-up, whereas relief of hypertension varied.Hypertension was observed in 62.7% (37/59) of the patients included. Postoperative BP declined from 132.20 ± 16.46/79.92 ± 9.64 mmHg to 122.41 ± 11.17/71.10 ± 9.82 mmHg, while eGFR changed from 108.17 ± 24.73 to 98.92 ± 23.87 ml/min/1.73 mPatients with lower preoperative SBP were likely to benefit more from the operation, while higher postoperative SBP indicated a higher chance of hypertension reonset. Creatinine level and eGFR generally remained stable regardless of operation type. Renal artery aneurysm (RAA) is a relatively rare condition that entails high risk if ruptured . Around In this study, we retrospectively analyzed the changes in BP and renal outcomes in 59 RAA patients during either open or endovascular operation and throughout follow-up at our institution. Previous studies that had recorded the detailed values of BP, blood creatinine, or glomerular filtration rate (GFR) in patients undergoing RAA open or endovascular operation are reviewed. Multivariable logistic regression was conducted to identify the predictive factors of perioperative BP relief and long-term hypertension reonset.From 2009 to 2021, patients diagnosed with RAA who underwent therapy at the Department of Vascular Surgery, Peking Union Medical College Hospital (PUMCH) were consecutively included in the study. Patients without a detailed record of BP levels were excluded. Computed tomography angiography (CTA), magnetic resonance angiography (MRA), or digital subtraction angiography (DSA) was utilized for RAA diagnosis and evaluation. All participating subjects provided written informed consent, and the study was approved by the institutional review board.Baseline characteristics, perioperative results, and follow-up information were retrospectively collected and analyzed. Indications for intervention included aneurysm diameter >2 cm and symptomatic RAA . Both endovascular and open techniques were applied for treatment.Regarding treatment strategies, coil embolization, as a minimally invasive endovascular procedure with good efficacy and safety, was the first-line treatment if suitable. Stent-assisted coiling (SAC) or simple stent implantation was applied for complicated lesions. For those who were unsuitable for endovascular therapy or too young, open surgery including aneurysmoplasty, resection and primary anastomosis, tailoring, ex vivo repair, and resection and graft reconstruction with either the saphenous vein or a prosthetic graft, was selectively conducted. Detailed procedures can be found in previous literature and in supplemental (1)Always normal patients had normal BP throughout the study.(2)Complete relief of hypertension was defined as SBP and DBP at three or more clinical measurements below 140/90 mmHg without anti-hypertensive drugs.(3)Partial relief was defined as SBP and/or DBP below 140/90 mmHg while taking the same number or reduced number of medications, or a reduction in DBP by at least 15-mm Hg while on the same or reduced number of medications.(4)No relief was defined as no change or inability to meet the above criteria.(5)New onset referred to patients with newly-diagnosed hypertension at follow-up.(6)Relief and new onset was defined as BP relief at discharge but had new-onset hypertension at follow-up.Chronic kidney disease was graded according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative classification  or diastolic BP (DBP) levels of three or more clinical measurements above 140/90 mmHg or taking antihypertensive drugs on admission . BP leveThirty-day and long-term follow-up data were recorded and explored. Technical primary success was defined as the successful introduction and deployment of the device in the absence of surgical conversion or any complication. Color Doppler, CTA, or angiography was applied to evaluate arterial patency, which was defined as evidence of blood flow within the target renal artery or bypass and perfusion in the respective renal parenchyma. Laboratory tests, including creatinine (µmol/L), white blood cell count , hemoglobin , platelet count (PLT 109/L), and D-dimer (mg/L), as well as ultrasounds of the kidney or renal artery, were done at least once preoperatively, postoperatively before discharge, and at three months, six months, and one year or yearly after discharge.Logistic regression of hypertension relief was done between group 2, group 3, group 4, and group 5 patients, who had hypertension before the operation. Patients were divided into complete relief (group 2 and group 5) and incomplete relief (group 3 and group 4). Ordinal regression was first performed between the complete relief group and the incomplete relief group to identify factors with significant associations, which were then incorporated into the multivariate logistic regression model.Logistic regression of new-onset hypertension was run between group 1, group 2, group 5, and group 6 patients who had normal BP after the operation. Patients were divided into new-onset (groups 5 and 6) and no onset (groups 1 and 2) groups. Similar factors as from the ordinal regression model were incorporated.Literature searches in English and Chinese were performed in PubMed, EMBASE and Scopus using the keyword “renal artery aneurysm”. Studies that reported the detailed preoperative, postoperative, follow-up, or changed values of BP, blood creatinine level, or GFR/eGFR level in patients who underwent open or endovascular operation for RAA were included. Single-case reports were excluded. Two authors (SL and FL) reviewed the original articles and identified 10 papers on BP and 16 papers on renal function published from 2001 to 2022.χ2 test or Fisher's exact test, as appropriate. The Wilcoxon test or the Kruskal‒Wallis and Friedman tests were employed for cross- or within-group comparisons, respectively. p < 0.05 was considered significant.Statistical analysis was performed using SPSS 26.0 software . Continuous variables were checked for normality and are reported as median and interquartile range (IQR) or mean ± standard deviation. Categorical variables are presented as frequencies and were compared by the 2.The median follow-up was 64.5 [IQR: 65.2] and 25.1 [IQR: 33.1] months for patients receiving endovascular repair and open surgery, respectively . No signAn average number of types of 0.53 ± 0.63 antiplatelet drugs (aspirin or clopidogrel) were prescribed for patients at the last follow-up . Laboratp < 0.001), prevalence of hypertension history (p < 0.001), preoperative SBP and DBP values (p < 0.001 and =0.005), and postoperative SBP (p = 0.007).All patients were divided into 6 groups according to their BP pattern . Eighteep = 0.047), but no significant difference was observed in the long-term results.BP and renal function alterations were also explored and compared between patients who underwent open or endovascular repair . There wTo further investigate the risk factors for perioperative BP relief and long-term hypertension reonset, logistic regression was conducted in two different sets of groups . Higher The present study demonstrated good results from our twelve-year experience with RAA repair. No significant difference was found in the perioperative laboratory parameters between the open and endovascular groups. Patients receiving open surgery had a transient increase in D-dimer and PLT levels, but their long-term laboratory tests returned to normal. A significantly higher rate of reintervention was observed among patients with new-onset hypertension than for those with other BP statuses. Since RAA patients were generally younger and healthier than other patients in the vascular surgery ward, hypertension improvement and renal function preservation were crucial to reduce morbidity and minimize any potential risk for cardiovascular events in the long run.The mechanism of hypertension in RAA remains unclear. Concurrent RAS or compression might play a part, while microembolization in the distal renal parenchyma and hemodynamic changes from turbulent blood flow within the aneurysm could also contribute . The resConcerning renal function, most RAA patients in this study had normal baseline levels of creatinine and eGFR. During operation, the introduction of nephrotoxic iodinated contrast medium, embolization of a part of the renal parenchyma, failure of artery reconstruction, and clamping of blood supply may all increase the risk of renal impairment . NeverthTo further explore the problem of RAA and BP or renal function, we reviewed the results of BP and indicators of renal function reported in previous studies. Although expertise in RAA repair has accumulated rapidly over the last decade, few studies have reported on the exact values of these parameters. Ten papers that reported the value of BP for RAA were included  3, 7, 8, 19–23. Since controversial results exist, we sought to explore the underlying factors that may contribute to BP alteration using logistic regression models. As for hypertension relief after the operation, we found that patients with lower SBP and higher blood LDL levels preoperatively achieve better outcomes, which could provide predictive value for BP alteration after the operation. Several previous studies concerning BP response after operation for RAS have identified the level of SBP or DBP as a positive predictor of hypertension relief , 31. HowPatients with higher postoperative SBP, though within the normal range, might still be at higher risk of new-onset hypertension. For these patients, renal aneurysm may not be the primary factor causing the hypertension. Thus, the operation could not fully stop the progression of hypertension. A previous study also suggested that a longer duration of hypertension may bring an unfavorable outcome . Since hThis study has some limitations. Besides blood creatinine and eGFR, a more thorough evaluation of renal function, such as urine tests and CTA, was not normally conducted at follow-up, and ambulatory BP monitoring was not done. Subgroup analysis was hampered by the small subject numbers, making it difficult to reach robust conclusions. The activity of the renin-angiotensin system (RAAS) was not routinely tested, so the underlying pathophysiology and etiology of BP change might not be fully explained. More evidence is required to set the optimal RAA diameter threshold for surgical intervention. Nevertheless, through a literature review, we discussed the nature of BP and renal function alteration after RAA operation, which could assist in prognostic prediction for these patients. Larger multicenter studies are necessary to validate our findings.Both open and endovascular techniques for RAA repair demonstrated significant relief of hypertension and little impairment of renal function, yet baseline BP conditions and the extent of hypertension relief or long-term occurrence varied. Patients with lower preoperative SBP were likely to benefit more from the operation, while higher postoperative SBP indicated a higher chance of long-term hypertension reonset. Creatinine level and eGFR generally remained stable regardless of operation type.


Subglottic stenosis, a rare condition of the upper airway, is frequently misdiagnosed as obstructive lung disease. The aim of this study was to investigate whether subglottic stenosis could be identified and distinguished from asthma and chronic obstructive pulmonary disease (COPD) using spirometry or the dyspnea index (DI).The study population included 43 patients with asthma, 31 patients with COPD and 50 patients with subglottic stenosis planned to undergo endoscopic intervention. All patients completed the DI and underwent dynamic spirometry registering both inspiratory and expiratory volumes and flows, including the expiratory disproportion index (EDI), the ratio of forced expiratory volume in 1 s to peak expiratory flow. One-way analysis of variance assessed the discrepancy of the variables among the study groups, and receiver operating curve (ROC) analysis determined the measurement with the best discriminatory power providing a cutoff value, maximizing both sensitivity and specificity.p < 0.001) with a cutoff value of 0.39 , followed by DI  with a cutoff score of > 25 (83% sensitivity and 78% specificity).The only statistically significant variables differing between all three groups were the EDI and the DI. The EDI showed an excellent area under the ROC curve (0.99, In patients with dyspnea of unknown cause, an increase in EDI should arouse a suspicion of extrathoracic airway obstruction, advocating for further evaluation with laryngotracheoscopy.The online version contains supplementary material available at 10.1007/s00405-023-08141-3. Subglottic stenosis (SGS) is a gradual narrowing of the airway caused by chronic inflammation of the tracheal mucosa below the vocal folds, presenting with relatively common symptoms during its early process, such as dyspnea at exertion, wheezing, chronic cough or dysphonia, and stridor in cases of severe obstruction . With an1) to the peak expiratory flow (PEF), when found greater than 0.50 could indicate extrathoracic airway obstruction [Flexible laryngotracheoscopy, although an invasive procedure, is considered the gold-standard examination and is imperative to set the diagnosis of SGS , 5, 7–9.truction –15. Althtruction , 15, andtruction , it is itruction –20. The truction , 22.In summary, the optimal initial investigations to identify patients in need of laryngotracheoscopy presenting with dyspnea refractory to conventional treatment are still not established. Thus, the aim of this study was to identify patterns in spirometry or DI scoring that could distinguish patients with SGS from patients with asthma and COPD, contributing to the correct detection of the disorder without a diagnostic delay.Adult patients with SGS who were scheduled to undergo endoscopic treatment at the Ear Nose and Throat Department at Örebro University Hospital were consecutively included from September 2016 to December 2020. Exclusion criteria were a stricture caused by an external compression of the trachea, malignant tumors, or those with multilevel stenosis further engaging the glottic or supraglottic part of the airway. In the same manner, patients with asthma and COPD referred to the Department of Respiratory Medicine at Örebro University Hospital were consecutively included between 2020 and 2021.1, PEF, PIF, TPF, forced inspiratory volume in 1 s (FIV1), EDI, Forced Vital Capacity (FVC), FEV1/FVC ratio and percent of the predicted FEV1 (FEV1%) and FVC (FVC%) value according to the Global Lung Function Initiative. FVC was replaced with vital capacity when FVC values were missing. The Shapiro‒Wilk test was performed to investigate the normality of the baseline characteristics and all variables. Normally distributed continuous variables are presented using the means and standard deviation (SD), nonnormally distributed variables with the medians and interquartile range (IQR), and categorical variables as the numbers and percentages.All study subjects completed the Swedish version of the DI and underwent spirometry prior to treatment. Spirometry was performed by certified health care professionals from the Department of Clinical Physiology, either at our hospital or at referral hospitals. The following variables were registered: DI score, FEVy-axis) and 1-specificity (placed on the x-axis). The AUC is categorized as excellent (0.90 < AUC < 1.00), good (0.8 < AUC < 0.89), fair (0.70 < AUC < 0.79), poor (0.60 < AUC < 0.69) and failure (0.50 < AUC < 0.59). We further sought to extract a cutoff value, maximizing both sensitivity and specificity for the variables showing at least a good AUC. This value providing balanced sensitivity and specificity is defined as the point on the apex of the ROC curve, being the highest point of the vertical axis and further to the left on the horizontal axis [One-way analysis of variance (ANOVA) including Bonferroni post hoc analysis was used to evaluate the discrepancy of the variables among the three study groups. The diagnostic value of each variable was investigated with receiver operating curve (ROC) analysis by comparing the area under the ROC curve (AUC), which represents the test’s discriminatory power. A ROC curve comprises all different values of each variable according to sensitivity  was used for the statistical analysis. This human study was performed in accordance with the Declaration of Helsinki Guidelines and was approved by the Ethics Review Board in Uppsala, diary number 2016/193. An amendment to include patients with asthma and COPD was approved by the Swedish Ethical Review Authority, diary number 2020-05509. All adult participants provided written informed consent to participate.IBMIn total, 50 patients with SGS, 43 with COPD and 31 with asthma were included. The demographic data of the study population at baseline are listed in Table 1/FVC was significantly reduced in patients with COPD compared with patients with asthma or stenosis. In contrast, PIF, PEF and TPF were all significantly lower in stenosis than in asthma but did not differ from the COPD group.A one-way ANOVA indicated that there was a statistically significant difference in the mean value of all study variables between at least two cohorts; however, the only mean values that differed significantly between both the nonstenosis groups and the stenosis cohort were DI and EDI, as shown in Table The ROC analysis revealed an excellent AUC for EDI (AUC = 0.99), with an optimal cutoff value of greater than 0.39, showing 98% sensitivity and 96% specificity. The DI showed a good AUC = 0.87 and a cutoff score > 25, yet with 83% sensitivity and 78% specificity. ROC analysis with AUC and a visual representation of the ROC curve for each study variable are fully presented in Table 1/FVC ratio.The primary finding of this study suggests that an EDI with a value > 0.39 is the optimal spirometry measurement, showing high sensitivity and specificity for differentiating stenosis from the nonstenosis cohort. Secondary findings are that a DI score > 25 may also contribute to the differentiation of the stenosis against nonstenosis groups and that a reduced PIF or TPF could be used to distinguish stenosis from asthma in patients with a normal FEVShortness of breath not responding to conventional treatment is challenging for every physician. SGS is commonly misinterpreted as “difficult-to-treat asthma” or other causes of lower airway obstruction, such as COPD and bronchitis, resulting in a diagnostic delay because of the rarity of the condition and the overlapping clinical presentation of upper airway obstruction with other causes of dyspnea , 24. It 1 to forced expiratory volume in 0.5 s greater than 1.5 [Other reported potential measurements proposed for diagnosing extrathoracic airway obstruction are the ratio of maximal expiratory flow at 50% of FVC to maximal inspiratory flow at 50% of FVC less than 0.30 or more than 1 , a PIF lthan 1.5 . Neverththan 1.5 .1 were significantly lower in stenosis group and COPD than in asthma, with nonsignificant differences between stenosis and COPD.In COPD, both PEF and PIF may also be reduced, depending on the increased airway resistance that reflects parenchymal inflammation –30. Howe1 in asthmatic patients is often normal in stable situations, is reduced in COPD in moderate to very severe disease  but remains normal in stage 1, corresponding to mild severity. This rationale is also fully in agreement with our findings, where FEV1% was nonsignificantly decreased in stenosis and clearly significantly decreased in COPD compared with the normal mean FEV1% in the asthma group. FEV1 should be normal in upper airway obstruction since it is determined by the status of the small intrathoracic airways, which remain unaffected even in cases of substantial reduction of the tracheal lumen [FEVal lumen .1, provided no parenchymal inflammation, comprises the theoretical background of the EDI: the ratio of FEV1  to PEF (L/s). It was first described by Empey et al. [The unproportioned change of a substantially diminished PEF in relation to a relatively unchanged FEVy et al. , and wasy et al. . These gy et al. , 20, 32.Since it can be easily calculated from data recorded in dynamic spirometry, which is performed at all primary health care centers, we believe it offers a convenient and feasible way to screen patients with suspected SGS, where further assessment with laryngotracheoscopy is needed. In addition, EDI seems to be superior to other spirometry measurements requiring an inspiratory maneuver, such as PIF and TPF, exhibiting excellent specificity and sensitivity. Although DI showed a good AUC, it is practically nonfunctional in distinguishing SGS from nonstenosis considering that the cutoff value of 25 points is quite high, combined with a considerably lower sensitivity and specificity when compared to EDI. In our view, DI could further be used as a complementary measurement for assessing dyspnea in patients already diagnosed with upper airway obstruction, potentially affecting the decision-making and priority of a surgical intervention, as shown by other studies –19.The major strengths of our study are the prospective inclusion of patients with different conditions and the standardized manner in which spirometry was performed in the whole study group. A potential limitation is the dissimilar size of the three cohorts, partly due to an inhibited inclusion rate during the COVID-19 pandemic. However, the data were sufficient to explore a clear pattern of spirometry findings in the respective groups. Finally, it could be presumed that, since the study population of SGS planned to undergo endoscopic intervention, it represented cases of relatively advanced disease suffering from severe dyspnea. However, repeating our analyses with only mild cases of SGS graded as Cotton-Myer 1 or even 2, we found no substantial difference compared to our primary results (data not shown). Subsequently, EDI should be part of the comprehensive work-up during the assessment of dyspnea of unclear cause that is unresponsive to treatment, since it can be useful to identify even mild severity of SGS.An increase in EDI in undiagnosed patients or nonresponders to empirical treatment for exertional breathing difficulties or dyspnea at rest could be a sign of upper airway obstruction and should be further assessed with laryngotracheoscopy.Supplementary file1 (DOCX 15 KB)Supplementary file2 (DOCX 18 KB)Below is the link to the electronic supplementary material.


Kidney transplant patients require careful management of immunosuppression to avoid rejection while minimizing the risk of infection and malignancy for the best long-term outcome. The gold standard for monitoring allograft status and immunosuppression adequacy is a kidney biopsy, but this is invasive and costly. Conventional methods of allograft monitoring, such as serum creatinine level, are non-specific. Although they alert physicians to the need to evaluate graft dysfunction, by the time there is a clinical abnormality, allograft damage may have already occurred. The development of novel and non-invasive methods of evaluating allograft status are important to improving graft outcomes. This review summarizes the available conventional and novel methods for monitoring allograft status after kidney transplant. Novel and less invasive methods include gene expression, cell-free DNA, urinary biomarkers, and the use of artificial intelligence. The optimal method to manage patients after kidney transplant is still being investigated. The development of less invasive methods to assess allograft function has the potential to improve patient outcomes and allow for a more personalized approach to immunosuppression management. HoweverTypically, allograft dysfunction is signaled by a rise in serum creatinine or proteinuria. That said, by the time clinical signs arise, significant damage in the allograft may have already occurred. Protocol biopsy studies have routinely demonstrated the presence of rejection in otherwise clinically stable patients , 7. Immu2The gold standard for diagnosis of allograft dysfunction is the kidney biopsy. The procedure is invasive and not without risk of complications . A study®  is a microarray-based test that uses machine learning to assess the risk of kidney transplant rejection. The test analyzes messenger RNA (mRNA) expression from a biopsy sample to identify patterns associated with rejection  could predict rates of estimated glomerular filtration rate (eGFR) decline over time . The inf3Graft damage and rejection can occur in the absence of an acute rise in serum creatinine. Historically, the only way to do determine changes in allograft status before graft dysfunction would be to perform protocol biopsies. Given the aforementioned risks of the procedure, less than half (46%) of the high-volume transplant centers  in the United States perform protocol biopsies . This ha3.1® Kidney  uses next-generation sequencing and targeted RNA sequencing technology for gene expression profiling to assess immune quiescence . The kSORT looks at relative mRNA expression levels to detect patients who are at higher risk of rejection , which utilizes DNA microarray technology to determine whether a patient’s gene expression is more similar to a reference population with adequate immunosuppression than that with inadequate immunosuppression , 21. A siescence . A studyiescence . Anotherejection . Despiteejection . The kSOejection . AlthougThe use of urinary biomarkers for immune monitoring is an ideal choice because the kidney essentially acts like a “flow cytometer” filtering graft infiltrating cells into the urinary space . Some of®  measures the concentration of adenosine triphosphate (ATP) from CD4+ T-cells after stimulation to monitor the immune response of transplant patients  looks at allo-antigen-specific T-cytotoxic memory cells but has only shown to measure likelihood of TCMR  is being developed to be able to measure the risk of antibody-mediated rejections (ABMR).Tests of lymphocyte function have shown some promise in assessing the overall immunosuppression of a given patient. ImmuKnowpatients . The tespatients . Howeverpatients , 40. Alt of TCMR . A test ®  which analyzes 405 SNPs across all somatic chromosomes, Prospera®  which analyzes over 13,000 SNPs of 4 select chromosomes, and TRAC®  which uses a proprietary number of SNPs  testing. cfDNA is non-encapsulated DNA that can be released after cells have been injured. In solid organ transplantation, dd-cfDNA has been investigated as a potential biomarker for allograft rejection . Single- of SNPs –45. Rece of SNPs . PPV for of SNPs . Allogra of SNPs . The PPVTest combining dd-cfDNA and gene expression have emerged as a solution to improve the diagnostic capabilities. The biggest benefit of these combined tests is the improved PPV. A post-hoc analysis study of 208 patients with biopsy and blood sample results showed that the PPV improved to 81% when both gene expression (TruGraf) and dd-cfDNA (TRAC) were positive (NPV of 88% when both tests were negative) . The stu3.2de novo DSAs (dnDSAs). The presence of DSA has been associated with declined eGFR over time (Donor-specific antibodies (DSAs) are produced by the recipient against the donor’s human leukocyte antigens (HLA) molecules. DSAs can cause ABMR, which is a major cause of graft loss after kidney transplantation . DSAs caver time . DSAs caver time . Around ver time . For exaver time . The risver time . Currentver time . Some evver time . That sa3.3BK polyomavirus is a common DNA virus that infects people of all ages and remains latent in the genitourinary cells . The abbTorque teno virus (TTV) is a non-pathogenic virus that is almost ubiquitous worldwide, with 90% of healthy individuals and up to 100% of transplant recipients infected . TTV is 4Kidney transplant recipients could benefit from improved planning if graft loss can be predicted. As previously stated, histological assessment, gene expression profiling of biopsy, and the presence of DSA have shown potential for prognostication. Proteinuria has also commonly been used to predict poor prognosis . Other p4.1Artificial intelligence (AI) is a rapidly developing field with the potential to revolutionize medicine. One area where AI is being explored is in the prediction of kidney transplant outcome. The iBox (Integrative Box) is an AI-based scoring system, which was developed by analyzing 32 potential prognostic factors in 7,557 kidney transplant recipients . The sco5Improvements in immunosuppressive medications, antimicrobial regiments, and surgical techniques have improved short-term outcomes for kidney transplant recipients over the years. There is still a large gap in our knowledge about how to achieve better long-term outcomes. Personalizing immunosuppressive therapy may be one way to reduce late allograft losses, especially as the organ waitlist continues to grow. Currently, no single non-invasive test is accepted as a reliable alternative to histological assessment. The strong negative predictive values of dd-cfDNA and gene expression have potential to lessen the need for routine biopsies. Monitoring for the presence of DSA and viral infections can be also used as a surrogate marker of over- or under-immunosuppression. In combination with some of the currently available blood biomarker testing, clinicians can potentially identify clinically stable patients at low risk for rejection , clinically stable patients at higher risk for rejection , and those who are over-immunosuppressed (BK viremia) so that adjustments to immunosuppression can be made accordingly and ideally before any graft damage occurs. HH: Writing – original draft, Writing – review & editing. ML: Supervision, Writing – review & editing.


Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure, but its effects in hypertensive diabetes are unknown.1 antagonist valsartan, the ACE inhibitor captopril, valsartan + siRNA or valsartan + captopril for the final 3 weeks. Mean arterial pressure (MAP) was measured via radiotelemetry.To address this, TGR (mRen2)27 rats (angiotensin II‐dependent hypertension model) were made diabetic with streptozotocin over 18 weeks and treated with either vehicle, angiotensinogen siRNA, the ATMAP before treatment was 153 ± 2 mmHg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a change in glomerular filtration rate. All treatments lowered MAP and cardiac hypertrophy, and the largest drop in MAP was observed with siRNA + valsartan. Treatment with siRNA lowered circulating angiotensinogen by >99%, and the lowest circulating angiotensin II and aldosterone levels occurred in the dual treatment groups. Angiotensinogen siRNA did not affect renal angiotensinogen mRNA expression, confirming its liver‐specificity. Furthermore, only siRNA with or without valsartan lowered renal angiotensin I. All treatments lowered renal angiotensin II and the reduction was largest (>95%) in the siRNA + valsartan group. All treatments identically lowered albuminuria, whereas only siRNA with or without valsartan restored podocyte foot processes and reduced glomerulosclerosis.Angiotensinogen siRNA exerts renoprotection in diabetic TGR (mRen2)27 rats and this relies, at least in part, on the suppression of renal angiotensin II formation from liver‐derived angiotensinogen. Clinical trials should now address whether this is also beneficial in human diabetic kidney disease.  MAPmean arterial pressureRASrenin‐angiotensin‐systemTGR (mRen2)27Transgenic rat (mRen2)27Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure in various hypertension models.Angiotensinogen siRNA exerts renoprotection in experimental diabetes by suppressing renal angiotensin formation from hepatic angiotensinogen.Clinical trials should now investigate whether this approach is beneficial in human diabetic kidney disease.1Hypertension is highly prevalent among diabetic patients , such as angiotensin‐converting enzyme inhibitors (ACEi) or 1 receptorAT antagonists . Although these drugs decrease both blood pressure and albuminuria  may be a crucial factor  inhibitor sacubitril to achieve histological improvement on top of blood pressure lowering via RAS‐independent pathways  (mRen2)27 (Ren2 rat). These rats, which overexpress the mouse N‐acetylgalactosamine ‐conjugated small interfering RNA (siRNA) targeting liver angiotensinogen  and an AT1 antagonist . To exclude the possibility of insufficient RAS blockade, we also tested two types of dual RAS blockade, that is, valsartan + captopril and valsartan + angiotensinogen siRNA. We hypothesized that angiotensinogen siRNA, either alone or in combination with valsartan, would lower blood pressure and improve albuminuria to at least the same degree as the classical RAS blockers (or their combination), while simultaneously improving renal microstructural damage, in particular podocyte integrity.In the present study we set out to evaluate this novel treatment tool in the diabetic Ren2 rat, making a comparison versus both an ACE inhibitor , after which the animals were studied for 18 weeks. Following streptozotocin injection, non‐fasting blood glucose and β‐ketone levels were checked every 2 weeks . Only rats with glucose >15 mmol·L−1 were considered diabetic , captopril , valsartan , angiotensinogen siRNA , captopril + valsartan (n = 7), or angiotensinogen siRNA + valsartan (n = 7). The doses of the individual drugs were chosen because of their comparable blood pressure‐lowering effects in previous studies , the isoflurane concentration amounting to 4%–5% in the induction chambers and 2%–3% in the face masks. Under anaesthesia, body temperature was maintained by using a heat therapy pump  connected to a heat pad on which the animals were placed during the entire procedure. For biochemical analysis, we collected 24‐h urine in metabolic cages and blood plasma by venipuncture from the lateral tail vein prior to the induction of diabetes mellitus (baseline) and at weeks 15 and 18 after injecting streptozotocin . GFR measurements were performed at the same 3 time points. At the end of the 3 week‐treatment period, rats were anaesthetized by inhalation of isoflurane and exsanguinated. For angiotensin metabolites quantification 1 ml blood was collected in 10 ml of a 4 mol·L−1 guanidine thiocyanate solution . Animal studies are reported in compliance with the ARRIVE guidelines . In hepatic and renal tissue, angiotensinogen was quantified by western blotting and normalized versus GAPDH as described before , 2/8 (angiotensin II), 8/15 (angiotensin‐(1‐7)), 2/5 (angiotensin‐[1–5]), 2/5 (angiotensin‐[2–8]) and 2/10 (angiotensin‐[3–8]) pg·ml−1 or g tissue, respectively. Urinary albumin was measured with a rat albumin ELISA kit . Plasma N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) was measured with an ELISA . Plasma or serum potassium, sodium, glucose, insulin, creatinine, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urinary sodium, potassium, creatinine and urea (24‐h urine) were measured at the clinical chemistry laboratory of the Erasmus MC.The plasma concentrations of angiotensinogen, renin and total renin (the sum of renin and prorenin) were measured by enzyme‐kinetic assay as described before  and reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit . cDNA amplification was done in duplicate in 40 cycles  followed by a melt curve with a CFX384  using Kapa SYBR® Fast (Kapa Biosystems). Intron‐spanning oligonucleotide primers were designed with NCBI Primer‐BLAST Table . The ΔΔC2.4−1 dissolved in 0.9% saline solution) in the lateral tail vein. A noninvasive clearance–kidney fluorescent detection device and software (Mannheim Pharma & Diagnostics GmbH) was used to generate the elimination kinetics curve of fluorescein isothiocyanate‐sinistrin. GFR was derived from the excretion half‐life (t1/2) of fluorescein isothiocyanate‐sinistrin, using a conversion factor and formula validated for rats  = 31.26 (ml per 100 g body weight)/t1/2 fluorescein isothiocyanate‐sinistrin (min).GFR was determined by transcutaneous measurement of fluorescein isothiocyanate‐labelled sinistrin clearance curves  , 1 (n1), 2 (n2), 3 (n3) and 4 (n4), respectively where (n) represents the number of glomeruli in each category. The glomerulosclerosis index (GSI) was calculated using the formula [(1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)]/(n0 + n1 + n2 + n3 + n4). Tubular atrophy, interstitial fibrosis and tubulointerstitial inflammation were scored in the same kidney section and summed to obtain the tubulointerstitial score (TIS). A score of 0 to 3 indicates <25% of tubulointerstitial tissue damage, a score of 4 to 6 indicates 25%–50% and a score of 7 to 9 indicated >50%. Heart sections, fixed in 4% paraformaldehyde were dehydrated and paraffin‐embedded. Gomori silver staining was applied to deparaffinized left ventricle sections (5 μm) to visualize individual cardiomyocytes. Sirius red staining was used to visualize collagen as a measure of cardiac fibrosis. Cardiomyocyte area and collagen % were quantified using Qwin image analysis software . Only transversally cut cells showing a nucleus were used to determine the cardiomyocyte area.Kidney segments fixed in 4% paraformaldehyde were dehydrated and paraffin embedded. Transversal deparaffinized kidney sections (2 μm) were stained with periodic acid‐Schiff (PAS) and scored semi‐quantitatively in a blinded fashion by a renal pathologist (M. C. C. v. G.) as described previously  and cut in semi‐thin slices (1 μm) to check for tissue representation. After selection, ultra‐thin (60–70 nm) slices were cut and placed on 200 mesh uncoated transmission electron microscopy grids. Contrasting was in an automatic contrasting device for transmission electron microscopy grids . Pictures and interpretation of electron microscopy were obtained and scored in a blinded fashion by a renal pathologist (M. C. C. v. G.).2.7British Journal of Pharmacology on experimental design and analysis in pharmacology , selected post hoc analyses  were performed. Data obtained at multiple points in time were analysed using a repeated‐measures two‐way ANOVA, followed by post hoc correction according to Dunnett or Dunn in case of multiple comparisons, provided there was no significant variance inhomogeneity. Univariate linear associations were assessed by calculation of Pearson's coefficient of correlation. Two‐tailed P < 0.05 was considered statistically significant. Multiple linear regression analysis was performed to identify variables correlating independently. All analyses were performed using Prism, version 8.0.0 .The data and statistical analysis comply with the recommendations of the 2.8Streptozotocin was obtained from Merck Millipore, Amsterdam, the Netherlands. Insulin  immediately prior to treatment was 153 ± 2 mmHg (n = 47). Vehicle did not alter MAP Figure . All tre3.2−1 and plasma renin and prorenin were 41 ± 2 and 507 ± 33 ng angiotensin I per ml·h−1, respectively . Both dual treatments virtually normalized albuminuria. Renal angiotensin II and MAP both correlated strongly with albuminuria  and aspartate aminotransferase (AST), but no treatment subsequently affected these parameters Table .4N‐acetylgalactosamine‐siRNA conjugate silencing the liver expression of angiotensinogen offers renoprotection in diabetic Ren2 rats. This effect involved both blood pressure lowering and angiotensin II suppression in the kidney, given that improved renal histology scores were seen with angiotensinogen siRNA alone and not with valsartan alone or captopril alone, despite the fact that all single treatments lowered blood pressure to the same degree. Moreover, renal angiotensin II levels were an independent determinant of albuminuria on top of blood pressure. In agreement with previous findings, all RAS blocker treatments additionally offered cardioprotection in this model.This study demonstrates that a hepatocyte‐directed The Ren2 rat, by overexpressing mouse (pro)renin, displays angiotensin II‐dependent hypertension  and simultaneously agree with the earlier observation that tissue angiotensin II is largely, if not completely AT1 receptor‐bound  were present in renal tissue, albeit at substantially lower levels than angiotensin I and II. Their levels remained low or became undetectable, during all treatments, with the exception of angiotensin‐(1–7) which rose in parallel with angiotensin I during ACE inhibition. This most likely reflects the shift from angiotensin I‐II conversion by ACE to angiotensin I‐angiotensin‐(1–7) conversion by neutral endopeptidase  over a 6‐month period  small interfering RNA (siRNA), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks. Data are mean±SD of n = 7–9. Numbers in combination with the symbol < denote the lower limit of quantification. *P < 0.05 versus vehicle.Table S3. Main characteristics of diabetic Ren2 rats treated with either vehicle, valsartan, captopril, angiotensinogen (AGT) small interfering RNA (siRNA), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks. Data prior to the induction of diabetes mellitus (DM) and treatment are also provided. Data are mean±SD. #P < 0.05 versus non‐DM; *P < 0.05 versus vehicle.Figure S1. Systolic and diastolic blood pressure  in diabetic Ren2 rats treated with either vehicle, valsartan, captopril, angiotensinogen small interfering RNA (siRNA), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks. Treatment was started 15 weeks after the induction of diabetes. Days −3 to 0 correspond to the period immediately before treatment. Data are mean±SEM of n = 7–9. *P <0 .05 versus vehicle and/or indicated group.Figure S2. Effect of diabetes induction on the levels of angiotensinogen (A), renin, (B) and renin (C) in Ren2 rats after 15 weeks and the relationship between renin (D) or prorenin (E) and angiotensinogen. Data are mean±SEM of n = 47; *P < 0.05.Figure S3. Hepatic (panel A) and renal (panel B) angiotensinogen levels in diabetic Ren2 rats treated with either vehicle, angiotensinogen (AGT) small interfering RNA (siRNA) or AGT siRNA + valsartan. Treatment was started 15 weeks after the induction of diabetes. Panel C shows the immunoblots. Data are mean±SEM of n = 7–9. *P < 0.05 versus vehicle.Figure S4. Renal expression of the angiotensin II type 1a and 1b  receptor (A and B), Neph1 (C), nephrin (D) and podocin (E) in diabetic Ren2 rats treated with either vehicle, valsartan, captopril, angiotensinogen (AGT) small interfering RNA (siRNA), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks. Treatment was started 15 weeks after the induction of diabetes. Data are mean±SEM of n = 7–9. NS, not significant.Click here for additional data file.


Therefore, either position can be used for C-SCI patients as long as MI-E can be performed with manual thrust and sufficient TIV is provided.This prospective pilot study investigated the influence of body position on peak cough flow (PCF) during mechanical insufflation–exsufflation (MI-E) treatment in people with tetraplegia. Fifteen participants with cervical spinal cord injury (C-SCI) were randomized into two groups, which differed in the starting position, that is, the patients were either supine or reclined. Four sessions of MI-E in alternating positions with each session comprising three different maneuvers: five voluntary coughs, five MI-E-assisted coughs, and five MI-E-assisted with manual thrusts were performed with continuous airflow measurement reporting PCF from every cough. PCF was associated with the application maneuvers, total insufflation volume (TIV), and interaction between position and maneuvers but not with the application position. The estimated mean PCF was 1.808, 3.529, and 3.925 L/s when supine and 1.672, 3.598, and 3.909 L/s when reclined from voluntary cough, MI-E, and MI-E with manual thrust, respectively. The estimated PCF change compared to voluntary cough was 1.721  L/s from the combined MI-E and 2.116  L/s from the MI-E with manual thrust, calculated from the linear mixed-model analysis. PCF moderately correlated with TIV (R Additionally, C-SCI results in an impaired ability to cough and breathe deeply, the degree of which depends on the level and severity of the injury2. Various pulmonary hygiene techniques are recommended to support adequate ventilation in C-SCI, including postural drainage, assisted cough, tracheal suctioning, and intrapulmonary percussive ventilation.Respiratory and abdominal muscle weakness due to cervical spinal cord injury (C-SCI) results in a high mortality burden due to respiratory failure, including atelectasis, pneumonia, and ventilator failure5, which indicates an ineffective cough, are candidates for frequent MI-E use. The application of MI-E to such patients improves PCF and reduces the frequency of pneumonia or hospitalization for people with neuromuscular weakness, including C-SCI8. In addition, the results of a study investigating the use of MI-E in C-SCI patients indicated that the patients preferred MI-E over trans-tracheal suction9.As one such airway clearance technique, mechanical insufflation–exsufflation (MI-E) is a noninvasive cough aid that augments in/exsufflation volume and flow by inducing a series of positive and negative pressures followed by a short pause. Gradual application of positive pressures to ensure sufficient inhaled volume to mobilize secretion and induce rapid shifts to negative pressures to produce a high expiratory flow can move secretions toward the mouth, allowing them to be easily removed. Tetraplegic individuals with a reduced peak cough flow (PCF) of less than 4.5–6 L/s (270 L/min)2. A higher forced vital capacity (FVC) has been reported in the supine position than in the sitting position in people with C-SCI, which is the opposite of that in a healthy population10. The diaphragm generates the main force for inspiration, as the intercostal and abdominal muscles are auxiliary. Inspiratory excursion of the diaphragm is better in the supine position in tetraplegic people, as the intra-abdominal contents are displaced and the muscle fibers become longer at the end of expiration when the muscle is in a more effective point of its length-tension curve11. Conversely, expiratory airflow, which is more important in secretion elimination, cannot be further accelerated by increasing muscle effort in the diaphragm. The maximal expiratory airflow is primarily achieved by increasing the operating lung volumes, which is defined as the volume from the end-expiratory lung volume to total lung capacity12. In fact, the forced expiratory volume in 1 s (FEV1) and PCF were reported to be greater in the supine than the sitting position only in people with complete tetraplegia14, and the disparity in pulmonary function becomes negligible when people with both motor complete and incomplete injuries are included in the analysis17.However, body position affects pulmonary function in people with C-SCI differently from that in healthy people or those with other neuromuscular diseases (NMD)12. However, when using MI-E, the insufflation volume may also differ depending on position, as the bowel contents in people with C-SCI can be affected by gravity. Although there were a few reports about preferring the supine position for a better spirometric evaluation assessed during voluntary respiration or cough in C-SCI patients2, no study has been conducted on the effect of posture on expiratory flow generation when using MI-E.MI-E-assisted cough can generate a sufficient insufflation volume more easily than voluntary or manually assisted cough. This is achieved through the application of external positive pressure to compensate for a deficient FVC. Once the maximal insufflation volume is achieved, the individual’s expiratory effort and strength would contribute less to the production of sufficient PCFThis pilot study aimed to investigate how body position influences PCF during MI-E treatment in individuals with C-SCI. Knowledge regarding the differences in PCF depending on body position could suggest proper positioning for MI-E use in individuals with C-SCI in clinical practice.Fifteen participants were recruited and randomized into experimental Group I (n = 8) and Group II (n = 7) Fig. . The mea2O was considered optimal for six participants, +40/−50 cmH2O for six participants, +40/−40 cmH2O for one participant, and +30/−40 cmH2O for two participants. All participants could follow an in/expiratory pressure higher than +30/−30 cmH2O, which was provided as the first pressure during the pre-training session. None of the participants complained of chest–abdominal discomfort, hemodynamic instability, or any of the side effects described in the exclusion criteria for discontinuing MI-E treatment during the study.During the MI-E treatment, the highest pre-determined application pressure of +50/−50 cmHp < 0.001). However, MI-E-assisted cough demonstrated no change in PCF between both applied positions; the reported mean PCF was −3.69 ± 0.48 L/s in the supine and −3.74 ± 0.45 L/s in the reclined position (p = 0.09). During the MI-E combined with manual thrust, the mean PCF was even more similar in both positions as 4.05 ± 0.52 L/s in the supine and 4.04 ± 0.57 L/s in the reclined position (p = 0.52). The median with interquartile range was 1.56[0.62] L/s, 3.78[0.58] L/s, and 4.19[0.74] L/s in the supine position, and 1.33[0.90] L/s, 3.86[0.50] L/s, and 4.23[0.71] L/s in the reclined position, respectively for voluntary cough, MI-E assisted cough, and MI-E combined with manual thrust. A subgroup analysis, which classified the patients into higher cervical SCI  and lower cervical SCI  groups, yielded consistent outcomes. The subgroup analysis demonstrated increasing PCF in the following order: voluntary cough, assistance with MI-E, and MI-E with manual thrust; however, no difference in terms of applied positions was present among both groups.The PCF during voluntary cough significantly differed between the positions with a mean PCF of 1.56 ± 0.51 L/s at supine and 1.36 ± 0.62 L/s at reclined positions, respectively (p = 0.475), treatment day (p = 0.094), and session (p = 0.381), but only TIV was significant (p < 0.001). These results suggested that there was no cumulative effect from consecutive treatment sessions influencing the PCF. Therefore, the final LMM analysis excluded the number of treatment sessions or treatment days as fixed effects and only included the applied position, maneuvers, total insufflation volume (TIV), and the interaction between position and maneuver. The TIV was included because it was significant from the initial analysis.The linear mixed-model (LMM) analysis demonstrated no main effect of assigned group (p < 0.001), maneuver (p < 0.001), and interaction between position and maneuver (p = 0.001); however, the positions did not show a main effect (p = 0.236). The participant-specific random effects were also significant with an estimated intercept of 0.114 (p = 0.005) with a residual of 0.118 (p < 0.001) , showing a supine position preference with a value of 1.59 ± 0.49 L in the supine position and 1.37 ± 0.43 L in the reclined position. During MI-E assisted cough, its positional difference became negligible as mean TIV was 3.14 ± 0.67 L in the supine position and 3.08 ± 0.78 L in the reclined position (p = 0.34), and 3.00 ± 0.73 L in the supine position and 3.04 ± 0.78 L in the reclined position (p = 0.26) during MI-E with manual thrust. The correlation between TIV and PCF was moderate18 of R2 = 0.637 (p < 0.001), demonstrating that a higher TIV was associated with a faster PCF. This correlation effect was also moderate18 in each application position (R2 = 0.624 in the supine position and R2 = 0.648 in the reclined position) , which was smaller than MIC, from smaller insufflation pressure than the pressure generating MIC can reach the fastest PCF. From the pressure–volume-flow relationship, 30–40 mbar (30.6–40.8 cmH2O) generated OIC was suggested to be the candidate for those with NMD to achieve the best PCF. Our study selected the most convenient in/expiratory pressure setting for each participant from a pre-training session, which generated the best PCF from gradually increasing pressure. The most commonly selected pressure settings were +40–50/−50 cmH2O, which was relatively higher than that reported in a previous study19, indicating that MIC or at least submaximal OIC was sufficiently achieved in both positions. The optimal pressure was selected in the supine position as to be higher than the pressure set in the reclined position, because supine position was suitable for generating higher MIP and FVC in C-SCI patients than in the reclined position. This suggests that the patient’s intra-abdominal contents could be well-displaced during insufflation by MI-E, resulting in the best PCF from both positions.A previous study investigating 36 patients with NMD suggested that the best individual PCF could be achieved with intermittent positive pressure breathing-assisted insufflation20, also measured MI-E-assisted cough using a serially connecting flowmeter and reported that MI-E combined with manual thrust showed a synergistic effect to improve PCF only in the reclined position. Compared to MI-E alone in 40 NMD patients with a mean baseline MEP of 25.3 cmH2O, MIP of 19.5 cmH2O, and FVC of 0.67 L, whose inspiratory volume and pressure, that is, lung compliance, were severely decreased compared to our participants, MI-E with manual thrust using ± 40 cmH2O pressure generated a PCF of 3.37 L/s, which was better than the value of 2.95 L/s without manual thrust20. Patients with NMD with an impaired VC lower than 40% or baseline PCF of < 3 L/s also showed improved PCF through a combination of MI-E and manual thrust compared to either MI-E alone or manually assisted cough22. The patient satisfaction scale was also reported to be the same between MI-E alone and manual thrust combined with MI-E. Our study investigated C-SCI rather than NMD and reported the same additional benefits of approximately 0.4 L/s from a combination of manual thrust on MI-E. Although the insufflation volume may be sufficient with MI-E alone, further expiratory pressure or force from manual thrust is helpful to generate an even faster PCF.Previous reports have shown an additional benefit of combining manual thrust with MI-E in patients with NMD, including myopathy or motor neuron disease, however, there is very little information regarding MI-E with manual thrust in tetraplegic individuals. Kim et al.22, there is a lack of reports addressing the potential differences in body positioning during MI-E or manual thrust in people with C-SCI. Limited research has provided information about the specific positions used so far. Positional changes in pulmonary function were distinct in tetraplegic patients, as indicated by the results of PFT2. While individuals with NMD typically experience uniform muscle weakening, it is important to highlight that C-SCI patients without ventilator support exhibit a disproportionate diaphragm weakness that is relatively less pronounced compared to the almost complete inactivity of intercostal and abdominal muscles10. The removal of abdominal organ compression in the sitting position pulls the diaphragm downward and makes it flat, which increases the diaphragm’s demand and effort for breathing12. Therefore, the outcomes of the current study should be interpreted within the context of C-SCI patients and cannot be generalized to all individuals with NMD.Although PCF variations caused by the assistance of MI-E or manual thrust have been documented in different populations as mentioned aboveThis study had some limitations. First, we only measured PCF during cough but could not measure the exact amount of eliminated sputum to assess the effectiveness of the procedure in maintaining pulmonary hygiene. Additionally, comparing MI-E-assisted cough at 60° or 90° sitting position can provide more information about the best position for MI-E in people with C-SCI as well. Furthermore, information on long-term pulmonary prognosis, which is affected by the body position applied for MI-E treatment, will further empower future clinical decisions. Last, this pilot study is limited by a small, heterogeneous sample of individuals with varying SCI severities, restricting the generalizability of findings. The estimated sample size for paired t-tests is notably high due to the small mean differences relative to the standard deviations of differences. For instance, n = 557 would be needed for MI-E assisted cough, and n = 80,602 for assessing positional differences during MI-E combined with manual thrust. Therefore, it is imperative to utilize a small sample size but incorporate multiple, repetitive measures using LMM.In conclusion, the PCF during MI-E does not change according to the position, but a beneficial effect of applying MI-E and additional manual thrust could be achieved in both the supine and reclined positions. Generated TIV during cough was important in determining PCF in both positions. Hence, either position can be used as long as MI-E can be performed together with manual thrust and sufficient TIV is provided.5. Participants were excluded if they were tracheostomized or required continuous noninvasive ventilation and oxygen support; had any previous restrictive or interstitial lung disease; had any other contraindications for MI-E use, such as pneumothorax, pneumomediastinum, emphysema, or lung barotrauma within 1 month; had any active communicable respiratory infections; were hemodynamically unstable, i.e., systolic blood pressure < 90 or > 160 mmHg, diastolic blood pressure < 50 or > 110 mmHg, respiratory rate > 33/min, heart rate > 130 bpm, or saturation (SpO2) < 95%23. All participants provided informed consent to participate in this study; then, they underwent PFT in both the supine and sitting positions to evaluate baseline PCF, FVC/FEV1, MEP/MIP, and MIC following instructions of the American Thoracic Society guidelines24 before starting the MI-E treatment sessions. This study was approved and monitored by the Institutional Review Board of the Ethical Committee, in accordance with the Declaration of Helsinki (IRB No. NTRH-20018).This prospective, randomized crossover pilot study recruited C-SCI patients with impaired cough who required further cough augmentation, defined by a PCF < 270 L/min26.Eligible participants were randomized in a 1:1 ratio into groups I or II without stratification using a computer-generated block size of 4. Group I started in the supine position, was later reclined to 40° on the first day, and then the reclined position first followed by the supine position on the next day. Conversely, Group II underwent four sessions of MI-E in the opposite order of the applying position. The angle of 40°-reclining was chosen because previous studies have shown that this angle can achieve a more effective manual thrust combined with MI-E than in a 90°-sitting positionThe study design provides alternating starting positions of either supine or reclined for each day and each group because the cumulative treatment effect can also affect the generated PCF during MI-E. Consecutive MI-E application with eliminated sputum between coughs affects airway resistance and generates better PCF. However, repetitive cough combined with MI-E assistance would result in fatigue because of repeated maximal effort during exsufflation and decreased PCF. Conversely, if more MI-E treatment sessions are completed, more experienced participants can cooperate better with MI-E, which generates a faster PCF. This bias from repetitions of the MI-E application could be avoided by changing the order of the applied posture for every session for all participants.27. A pre-training session was performed in the supine position to determine the personalized pressure setting for each participant. The supine position was chosen for setting the personalized, optimal pressure because a higher FVC was reported in the supine position10. This indicates that a higher insufflation pressure is anticipated to generate larger TIV in the supine position compared to the reclined position. The maximal pressure optimized only in the sitting position might be insufficient to generate enough TIV which can reach MIC in the supine position. By gradually increasing the pressure from 30 to 50 cmH2O in 5 cmH2O increments, the highest pressure that each participant could withstand was measured. If the maximal pressure of ± 50 cmH2O was tolerable, then the pressure at which the fastest PCF could be generated was determined as the applied pressure.MI-E treatment sessions were administered using the CoughAssist E70 , which was set to generate 3 s of insufflation, 2 s of exsufflation, and 2 s of rest for one cough, with a low inhale flow setting and oscillation off to simulate physiological coughing and facilitate better cooperation for higher in-exsufflation pressureAfter determining the optimal MI-E pressure settings, the protocol comprised five consecutive voluntary coughs following 3 s of full inspiration, 2 s of maximal expiration, and rest was initiated. Participants were instructed to cough five times into the facemask while wearing a well-sealed mask (Air Way 96 mm (#4), ACE Medical Co., South Korea) with support from a physiotherapist or caregiver to prevent any air leak. After eliminating the expelled sputum, one set of five MI-E-assisted coughs was provided, followed by another set of five more MI-E-assisted coughs with manual thrusts. The three sets (5 coughs/1 set) of different application maneuvers—voluntary cough, MI-E assist, and MI-E with manual thrust—comprised one session at each position, and these three sets were repeated in the same order at the next assigned position.When performing MI-E combined with manual thrust, the physiotherapist asked the participant to cough at the start of the exsufflation period while delivering manual pressure to the participant’s bilateral costophrenic angle toward the direction of the upper chest simultaneously. An experienced physiotherapist blinded to the analysis of the mean PCF conducted all MI-E treatments and measured the concomitant airflow. A separate researcher blinded to the assigned group and applied position performed all the analyses.28. The total insufflation volume (TIV) was calculated through integration by squaring insufflation time and flow, as follows: volume (L) = time (s) × flow (L/s).The in-expiratory airflow generated in both directions during the MI-E session was measured using a flowmeter  serially connected to one side of a single-use antibacterial filter towards the facemask interface, and the other side to the CoughAssist E70. This flowmeter was formally calibrated and validated annually by IMT Analytics to ensure that the maximal uncertainty error was within 0.75% for all measurements. Expiratory airflow was measured as a negative value; therefore, the lowest value during exsufflation was the PCF, which could be calculated from each cough with continuous airflow measurement2 overall and in each application position. Demographic data comparison between the assigned groups was performed using the Mann–Whitney or Fisher's exact test. Baseline PFT outcomes and measured PCF values were compared between the two applied positions using the Wilcoxon signed-rank test after normative analysis. All analyses were performed using the R software version 4.2.2.The LMM was used to estimate mean PCF, standard error, and confidence interval which described the changes in the PCF according to the position or maneuver. Applied position, maneuvers, TIV, and interaction between maneuver and position were included as fixed effects, and participant-specific random intercepts were included as a random effect to account for repeated measures within participants covering individual, different baseline pulmonary functions. The normality test was confirmed for residuals. The strength of the linear correlation between PCF and TIV was analyzed using Pearson’s correlation analysis after calculating the correlation coefficient RThis study was approved and monitored by the Institutional Review Board of the Ethical Committee, in accordance with the Declaration of Helsinki (IRB No. NTRH-20018).