Variability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4β-hydroxycholesterol (4βOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4βOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range.n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4βOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam.The patients , and hepatic CYP3A4 concentrations , but not with intestinal CYP3A4 concentrations  or intestinal microsomal CYP3A4 activity . 4βOHC concentrations correlated weakly with midazolam absolute bioavailability  and apparent oral clearance , but not with systemic clearance .4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (These findings suggest that 4βOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4βOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs.Clinical.Trials.gov identifier: NCT02386917.The online version contains supplementary material available at 10.1007/s00228-022-03336-9. The cytochrome P450 (CYP) 3A subfamily, consisting mainly of CYP3A4 and the polymorphic CYP3A5 , plays aint), the latter representing metabolic capacity [The current gold standard method to assess in vivo CYP3A4 activity is midazolam, a selective CYP3A4 phenotypic probe drug in vivo with a short half-life –16. Howecapacity –19. Prevcapacity , 21. Thucapacity , 23. Thecapacity –27. The capacity –30. Stilcapacity . Also, tcapacity . The potcapacity –35.Additional investigations are required before 4βOHC can be accepted as a validated biomarker. As part of the primary endpoint in the COCKTAIL study , we eval2 combined with at least one obesity-related comorbidity) scheduled for weight loss treatment with Roux-en-Y-gastric bypass (RYGB) (n = 38) or non-surgical calorie restriction (n = 40), and 18 mainly normal to overweight individuals scheduled for cholecystectomy (BMI 18.5–30 kg/m2). The patients with severe obesity were subjected to a 3-week low-energy diet  before the study investigation, whereas the normal to overweight individuals were not subjected to any defined diet beforehand. Medications, including statins, and/or other substances that are known to alter CYP3A4 activity/midazolam pharmacokinetics were discontinued at least seven half-lives before the investigational day, and none of the patients used any CYP3A4 inducers or time-dependent inhibitors. The study was approved by the Regional Committee for Medical and Health Research Ethics (2013/2379/REK) and complied with the Declaration of Helsinki. All patients signed a written informed consent before study participation.In total, 96 patients from the open-label, three-armed COCKTAIL-study (NCT02386917) were included in the present analysis , 37. Then = 38), and liver biopsies were obtained during cholecystectomy (n = 18), as previously described [Blood samples for determination of plasma 4βOHC concentrations and a 24-h pharmacokinetic investigation using semi-simultaneous oral and intravenous dosing of midazolam were collected from all patients. From 10:00 p.m. before the investigation, patients abstained from food, drink (except water), and drugs. On the investigational day, patients first met for blood sampling before 1.5 mg oral midazolam syrup was administered, followed by 1.0 mg intravenous midazolam 4 h later. Blood samples for determination of midazolam plasma concentrations were collected from a peripheral venous catheter before and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 4.25, 2.5, 5, 5.5, 6, 8, 10, 12, 23, and 24 h following oral dosing. The pharmacokinetic investigation has been described in more detail previously . In vivoescribed , 38.Body composition was measured with Inbody 720, Body Composition Analyzer . The NAFLD liver fat score was calculated as suggested by Kotronen et al. . Standarn = 6) [4βOHC plasma concentrations were determined by a previously described assay  with an n = 6) . All samThe plasma concentrations of midazolam were determined by liquid–liquid sample extraction followed by a previously validated UHPLC-MS/MS method . The samThe UHPLC-MS/MS method was carried out in two labs due to limited capacity. The instrument in lab 1 was a Thermo Fisher Scientific Vanquish HPLC coupled to a Thermo Fisher Accucore (C18 2.1 × 50 mm) analytical column and a Thermo Fisher Scientific Altis QqQ MS. In lab 2, a Waters  Acquity UPLC with a BEH C18 RP-shield  analytical column coupled to a Micromass Quattro micro tandem MS was used. The chromatography was carried out with a flow rate of 0.4 mL/min and 0.2 mL/min and gradient elution for lab 1 and lab 2, respectively. Mobile phase A was 5% acetonitrile in 10 mM ammonium formate, and mobile phase B was 90% acetonitrile and 10% methanol. Positive electrospray ionization and selected reaction monitoring were performed with compound-tuned conditions. For both labs, acquisition was carried out by the MRM transitions m/z 326.1 -> m/z 249.1 and m/z 326.1 -> m/z 291.2 for midazolam, and m/z 332.1 -> m/z 252.1 and m/z 332.1 -> m/z 203.1 for midazolam-d6 in positive mode.Inter-laboratory cross-validation was performed using clinical study samples, and the bias was within ± 12.5%. Calibrators and quality control (QC) samples were prepared in blank plasma and analyzed in each analytical run. Eight calibrators in the range of 0.1 to 20 ng/mL were applied, and back-calculated values of calibrators within 80 to 120% were accepted. LLOQ was 0.1 ng/mL, and the upper limit of quantification (ULOQ) was 20 ng/mL. Samples with midazolam concentrations above ULOQ were diluted in blank plasma and reanalyzed. Dilution integrity with dilution factors of 1/2, 1/5, 1/10, 1/20, and 1/50 was established; mean accuracy ranged from 88.9 to 103.8%, and the imprecision was < 4.5%. Within-series and between-series performance were assessed with the resulting coefficient of variation < 12.3%, and the mean accuracy ranged from 99.3 to 104.3%.The biopsies were transferred into individual cryotubes, snap-frozen in liquid nitrogen directly upon sampling, and stored at − 80 °C until analysis.Proteins were extracted from liver and jejunum biopsies in an SDS-containing (2% w/v) lysis buffer and quantified as previously described . In shorMethods for quantifying CYP3A4 activity in liver and jejunum biopsies have been described previously . In brieCYP3A4 and CYP3A5 variant alleles were analyzed using Taqman-based real-time polymerase chain reaction assays implemented for routine pharmacogenetic analyses at the Center for Psychopharmacology, Diakonhjemmet Hospital, Norway. CYP3A4, the reduced function allele*22 (rs35599367), and CYP3A5, the null allele *3 (rs776746), were included in the analysis. All alleles were in Hardy–Weinberg equilibrium.Km) values were adjusted for the fraction of unbound drug in microsomes, which was predicted from the physicochemical properties of the substrates and microsomal protein concentration using the Simcyp prediction tool (https://members.simcyp.com/account/tools/fumic). The unbound CLint  was calculated from the ratio of maximum velocity (Vmax) to the unbound Km. Due to limited capacity, the hepatic and jejunum microsomal CLint,u were determined in 20 RYGB patients.In vivo absolute bioavailability, apparent oral clearance, and systemic clearance of midazolam from the pharmacokinetic investigation were determined using a previously developed population pharmacokinetic model . Brieflyp value < 0.05 was considered statically significant. Data are presented as median difference ) if not otherwise stated. Demographic data are presented as mean ± standard deviation (SD). All statistical analyses were performed using R for Windows (version 4.1.2) [Neither 4βOHC nor most of the other variables passed the normality tests , and non-parametric statistical tests were therefore used in the statistical analyses. Spearman’s rank-order correlation test was used to describe the rank-based measure of association. Wilcoxon rank-sum test was used to compare patients with obesity with normal to overweight individuals. A n 4.1.2) .CYP3A4 *1/*1), and 10% also expressed functional CYP3A5 (CYP3A5 *1/*3). In line with the inclusion criteria, mean BMI was higher in patients with obesity compared with normal to overweight individuals. Patients with obesity had lower serum total cholesterol compared with the normal to overweight individuals  but higher NAFLD liver fat score  and hs-CRP . Twenty percent of the patients received treatment with cholesterol-lowering drugs (mainly statins) .Patient characteristics at the investigational day are given in Table ρ = 0.30, p = 0.027) and hepatic microsomal CLint,u , but not with systemic midazolam clearance  , but no correlation was observed between systemic midazolam clearance and hepatic microsomal CLint,u , and a positive correlation with apparent oral clearance   Fig. . HepaticIn the patients with obesity, neither 4βOHC concentrations, systemic midazolam clearance, nor absolute bioavailability changed during the 3-week low-energy diet period  nor in patients with obesity (p = 0.33) , whereas in the normal to overweight individuals, the difference did not reach statistical significance (p = 0.12)   and ρ =  − 0.23 (p = 0.027), respectively  Fig. a. Both Nely Fig. b and c. In this study, a comprehensive investigation of 4βOHC as an endogenous biomarker for CYP3A4 activity was performed. To our knowledge, this is the first study where 4βOHC has been compared with three other CYP3A4 metrics: intestinal and hepatic microsomal CYP3A4 activity and protein expression in paired liver and jejunum samples, as well as absolute bioavailability, apparent oral clearance, and systemic clearance of midazolam in vivo. The main findings were that 4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity and hepatic CYP3A4 expression. Equally important, this study also provides evidence of a negligible contribution from intestinal CYP3A4 to the formation of 4βOHC.int,u) supports that 4βOHC concentrations reflect individual CYP3A4 metabolic capacity and provides evidence that 4βOHC is a suitable marker for hepatic CYP3A4 phenotype. However, there was no correlation between 4βOHC concentrations and systemic clearance of midazolam, the gold standard method to assess CYP3A4 activity. The correlation between these two metrics has been investigated in multiple studies previously, with conflicting findings varying from no correlation as in this study to a weak or moderate correlation [int,u) and in vivo systemic midazolam clearance, suggesting that systemic midazolam clearance does not solely reflect CYP3A4 activity in the present population. In contrast to hepatic microsomal CLint,u, in vivo systemic midazolam clearance may also be influenced by differences in hepatic blood flow given its medium to high extraction ratio [The moderate correlation between 4βOHC concentrations and hepatic microsomal activity  was correlated with 4βOHC concentrations. Even though there was a weak, but significant correlation between 4βOHC concentrations and (1) midazolam absolute bioavailability, and (2) apparent oral clearance, but no correlation with systemic clearance, these results indicate that intestinal CYP3A4 has a negligible role in the formation of 4βOHC. There have been some indications of a possible contribution from intestinal CYP3A4 in the formation of 4βOHC [Information regarding the role of intestinal CYP3A4 in 4βOHC formation has been lacking , 53, andof 4βOHC , 54. Howof 4βOHC , which iint,u using midazolam as a probe drug are also reported to be negatively correlated with BMI [2, although the authors suggested that this was due to type 2 diabetes, not obesity [2 and NAFLD [In this study, we also report a negative correlation between BMI and 4βOHC, and that patients with obesity have significantly lower 4βOHC concentrations compared with normal to overweight individuals. Previous studies have also found an inverse correlation between BMI and 4βOHC/C, and body weight and 4βOHC/C , 25. Intwith BMI , 9. Thiswith BMI . The norwith BMI , 51, 56.nd NAFLD , 58, andnd NAFLD . FurtherCYP3A5*1/*3) and non-functional CYP3A5 (CYP3A5*3/*3). Nevertheless, we do not believe that the heterozygote CYP3A5*1 carriers in our study population (~ 10%) have impacted the interpretation of the results in this study to any significant degree. This is supported by the fact that there were no statistically significant differences between the heterozygote CYP3A5*1 carriers and the homozygote CYP3A5*3 carriers in any of the other metrics investigated. Also, there was a tendency of lower 4βOHC concentrations in CYP3A4*22 carriers (reduced function), but the interpretation is challenged by the low number of individuals carrying this allele. Overall, these results are in line with previous studies [Most of the cholesterol formation to 4βOHC is accounted for by CYP3A4 metabolism, whereas the contribution from CYP3A5 is more ambiguous , 60, 61. studies , 25, 50 The major strength of this study includes the comprehensive investigation of three different CYP3A4 metrics in relation to 4βOHC, all obtained from the same individuals at the same time point. The patients with obesity were subjected to a 3-week low-energy diet before the study investigation, which may have influenced the results. However, as neither 4βOHC concentrations, absolute bioavailability, nor systemic clearance of midazolam changed from before to after the diet, a change in the CYP3A4 phenotype during this short period seems unlikely. Nevertheless, in addition to the fact that 20% of the patients used cholesterol-lowering drugs, cholesterol levels also decreased following the diet, which led to a significant increase in 4βOHC/C in the patients with obesity. Several studies report the 4βOHC/C ratio to account for abnormal or changing cholesterol levels. However, the concentration of 4βOHC is less than 0.002% of total cholesterol . We deciIn summary, this comprehensive study showed that 4βOHC plasma concentrations reflect hepatic, but not intestinal CYP3A4 activity well. Hence, 4βOHC may be a valuable biomarker for individualized dosing of intravenously administered CYP3A4 substrate drugs. Additionally, 4βOHC is a valuable supplement to traditional phenotyping with probe drugs such as midazolam given its easy implementation and complementary information when other factors, such as disease state or simultaneous physiological alterations, are expected to influence the pharmacokinetics of CYP3A4 probe drugs.Supplementary file1 (DOCX 1062 KB)Below is the link to the electronic supplementary material.


The scarcity of donor hearts continues to be a challenge in transplants for advanced heart failure patients. With an increasing number of patients on the waiting list for a heart transplant, the discrepancy in the number between donors and recipients is gradually increasing and poses a new challenge that plagues the healthcare systems when it comes to the heart. Several technologies have been developed to expand the donor pool in recent years. One such method is the organ care system (OCS). The standard method of organ preservation is the static cold storage (SCS) method which allows up to four hours of safe preservation of the heart. However, beyond four hours of cold ischemia, the incidence of primary graft dysfunction increases significantly. OCS keeps the heart perfused close to the physiological state beyond the four hours with superior results, which allows us to travel further and longer distances, leading to expansion in the donor pool. In this review, we discuss the OCS system, its advantages, and shortcomings. End-stage organ failure remains a significant cause of mortality around the world. Among many medical breakthroughs in the 20th century, the development of solid organ transplants has been a major advancement . The resOf all the organ failures, heart failure is responsible for the highest number of deaths worldwide . End-staImproving organ preservation techniques can be one of the keys to increasing the donor pool . One of Initial workIn order to solve the drawbacks of cold static preservation, research on the ways for warm preservation was initiated in parallel. Devices used in both the approaches were similar and employed pulsatile warm perfusion of the heart with blood to allow ex-vivo recovery of the hearts with borderline function or hearts following cardiac arrest before implantation. Blood was deemed to be an ideal perfusion solution as blood has an excellent property for oxygen and nutrient delivery. In addition, blood acts as a free radical scavenger and a strong buffer against acidosis and metabolic toxicity. Also, blood protects the function of the endothelium and reduces injury .It was observed for a long time that the heart recovered its function in patients dying from cardiac arrest once the patients were put on cardiopulmonary bypass prior to procurement . Based on this construct, an experimental technique for harvesting donor hearts from pigs dying from cardiac arrest was developed by Fedalen and colleagues in which pigs were paralyzed using a neuromuscular blocker, disconnected from the ventilator, and were allowed to go into cardiac asystole . EnsuingA similar method was utilized during the same period but for a different purpose. Hassanein et al.  placed hHow OCS works (heart from donor's chest to implantation on OCS machine)Recovery of the HeartThe initial step in the establishment of an OCS machine for heart perfusion is the procurement of the heart. The procedure of the heart procurement varies significantly in donation after brain death (DBD) and donation after cardiac death (DCD). Cardiac death patients have a serious enough neurological injury to have a meaningful life but do not fit the criteria for brain death. In the subsequent sections, we will discuss the technique of heart procurement in the DBD and DCD donors and then attaching the heart to the OCS system. Donor After Brain DeathIn using OCS for DBD, after sternotomy and pericardiotomy, the donor's heart is directly assessed, emphasizing the left and right ventricular contractility and the evaluation of coronary arteries by palpation and visual assessment. Then, the aortopulmonary window, superior vena cava, and inferior vena cava are dissected. When the assessment is completed, the OCS machine is prepared by unpacking the OCS disposable module and assembling the OCS machine. The procurement process begins by putting a cardioplegia cannula into the ascending aorta, and a venous drainage cannula is introduced into the right atrium. Once the patient is ready for cross-clamp, a minimum of 1,200 mL of donor blood is drawn from the right atrial cannula in a pre-heparinized bag to prime the OCS machine for the procedure. The superior vena cava is clamped, and the right atrium is vented by sharply cutting the inferior vena cava anteriorly 5 mm above the diaphragm attachment. The left or right superior pulmonary vein is severed to vent the blood returning from the lungs to prevent the left ventricular distension. Then, the ascending aorta is cross-clamped and 2,000 ml of Celsior cardioplegia is administered at a pressure of 150 mmHg in conjunction with topical hypothermia maintained with ice slush in the pericardial cradle. In our institute, we use Celsior. However, it can be different for different institutes. As we cannot monitor the coronary or aortic perfusion pressure, we deliver the cardioplegia with a pressure bag at 150 mmHg so that it is delivered at 60-80 mmHg at the aortic level. The final step in the procurement process is the donor's heart cut at the vena cavae, aorta, pulmonary arteries, and pulmonary veins.Donor After Cardiac DeathFor the donor after cardiac death (DCD), the initial step is to set up all the instruments on a separate table. Then, after injecting 30000 units of heparin, life-sustaining supports are withdrawn. As per our institutional protocol, we wait for a maximum duration of 27 minutes from the time of mean arterial pressure is below 50 mmHg for electromechanical arrest. If the donor does not have electromechanical arrest within this time, we decline the heart. If the electromechanical arrest happens within 27 minutes, and 5 minutes of wait time, the chest is entered quickly within 2-3 minutes, sternal edges are separated with a retractor, pericardium is opened. A large venous cannula inserted is into the right atrium and 1200-1500 mL of donor blood is drawn from the right atrial cannula in a pre-heparinized bag to prime the OCS machine for the procedure. Simultaneously, the OCS machine is prepared by unpacking the OCS module and assembled into the OCS machine, where it will be ready to accept the donor heart when it arrives. After collecting the donor blood, a cardioplegia cannula is inserted high into the ascending aorta. Aorta is cross clamped and 1000 ml of Del Nido cardioplegia is administered at a pressure of 150 mmHg in conjunction with topical hypothermia maintained with ice slush in the pericardial cradle. The procurement process of the donor's heart is similar to DBD procurement; however, it needs more diligence as the heart has not been mobilized earlier and is flaccid after giving cardioplegia, increasing the risk of injury to the superior and inferior vena cava, pulmonary veins, and the main and right pulmonary arteries. Establishment of Myocardial Perfusion on OCS Machine (Mayo Protocol)After the recovery of the heart, it is shifted to the back table. The superior vena cava is closed with a silk tie and the inferior vena cava is closed with a 4-0 polypropylene suture. Closure of the patent foramen ovale is mandatory to make the right heart a closed system for monitoring the coronary blood flow by the pulmonary artery cannula. The aortic and pulmonary cannulae are attached to the corresponding major arteries and securely fixed. The donor's heart is put on the OCS tray with the left ventricle facing anteriorly, ensuring that continuous blood flow through the aortic cannula is maintained during the procedure to de-air the aorta. The left ventricular vent is inserted through the mitral valve and fixed to the left atrial wall with a suture Figure . The pulComparison of OCS to SCSIn the static cold storage (SCS) method, hypothermia reduces cellular metabolism and oxygen consumption to extend the organ preservation time. Once the organ is removed from the donor, the organ is placed on ice and stays in the icebox until implantation of the organ in the recipient -19. HoweThe management of heart transplant recipients has substantially improved over the past few decades, but preservation and optimization of the graft have not received the attention they deserve. OCS can be a game-changer since the longer preservation time not only gives us the flexibility to carry out complex recipient dissections and analyze and manage the donor graft before transplant but it can also increase the pool of possible donors . Listed AdvantagesThere are several advantages of using the OCS system for heart perfusion and storage during transport from the donor hospital to the recipient hospital.1) Cold ischemia time and older age of the donors are major risk factors for morbidity and mortality post-transplant. Increased donor age is associated with a greater risk of coronary artery disease (CAD). Studies have shown that the mortality at one-year post-transplantation doubles when the cold ischemic time is increased from three hours to six hours and halved when it is less than one hour . The OCS2) Post-heart transplant mortality of patients with congenital heart defects is high, especially among those who have undergone previous surgical intervention. It also remains a challenge for the surgeons to do the cardiectomy due to associated complex anatomy and possible severe adhesions from previous surgery and may need complex mediastinal dissection to explant the native heart. Further, many pediatric heart transplants may need reconstruction for complex anatomy after cardiectomy and prior to transplantation. Similarly, patients who have had prior VAD implantation may also benefit from OCS-supported donor hearts due to the anatomical complexity from a previous VAD. The preparation for transplantation can be more challenging and may take longer to do a safe cardiectomy without damaging the other structures.The surgeon always has to wait for the donor heart to land in the vicinity of the recipient hospital before performing any irreversible step of explanting the native heart to avoid catastrophe if any unforeseen event happens. Therefore, organ transport time and time required for complex mediastinal dissection and reconstruction significantly increases the cold ischemia time of the donor's heart even after it reaches the recipient's operating room. The OCS system becomes a savior in these situations, and surgeons can perform more peaceful and conscious mediastinal dissection . Fleck e 3) The heart can be transported long distances with the OCS system, increasing the donor pool. Presently, the heart is procured within a radius of five hundred miles to keep the flight time less than two hours. However, a suitable donor or recipient may be located farther in many instances, so the organ has to be denied. With the OCS system, the heart can be procured and transported for farther distances, increasing the possibility of potentially increasing the donor pool .4) OCS carries the potential for increasing the donor pool by accepting the marginal heart. Presently, hearts with left ventricular hypertrophy, EF (ejection fraction) 40-50%, downtime more than 20 minutes, and donor age >55 years are rejected due to the risk of primary graft dysfunction. However, many of these hearts have the potential to recover completely after improving the coronary perfusion and support on cardiopulmonary bypass for a short duration. The OCS system provides the opportunity to procure these marginal hearts and assess in real-time their function after establishing coronary perfusion. If the heart function improves, these hearts can undergo successful transplantation with a good outcome . In the Organ care system and donor after cardiac deathCirculatory death is an irreversible cessation of all circulatory and respiratory functions . While rLimitationUsing OCS is very expensive; each use will cost around $80,000, not including the additional cost of the hospital stay . AdditioThe OCS holds the potential for increasing the donor pool by increasing the number of DCD hearts, procurement of marginal hearts, and long-distance procurement, which will help distant transplant centers to receive donor hearts. The OCS will help the organ donor pool to expand and increase the number of hearts available for transplantation. However, it needs to be more cost-effective and available for more centers around the country.


Testicular torsion (TT) is an acute inflammatory process leading to male infertility. Today, anti‐inflammatory effects of exosomes derived from blood serum are used in various laboratory procedures. In the present study, the anti‐inflammatory effects of blood‐serum‐derived exosomes in treatment of acute inflammation following TT in mice were evaluated.Eighteen male mice were grouped as healthy control, TT, and TT + exosome. TT was induced surgically, and exosomes were extracted from blood serum and administrated by a single intratesticular injection (10 IU). Malondialdehyde (MDA) and Griess assays were used to evaluate the level of oxidative stress. Sperm indices, testosterone (Tes), and apoptotic gene expression  were also assessed. H&E and immunohistochemistry (IHC) stainings were used for histopathological investigations. Data analysis was applied by SPSS (v.19) software.p < 0.05) in TT group compared with control. Sperm parameters and Tes were significantly increased, and expression of apoptotic genes was significantly reduced in TT + exosome group (p < 0.05).Oxidative stress and apoptotic genes expression were increased significantly (Since the blood‐serum‐derived exosomes have anti‐inflammatory features, the intratesticular application of blood‐serum‐derived exosomes can be used clinically in acute phase of orchitis following TT to inhibit testicular inflammation. Blood‐derived exosomes can act as anti‐inflammatory sources in TT. This study showed that administration of a single dose of exosome immediately after an acute testicular injury can significantly reduce TT complications. Our study showed that exosomes can diminish apoptosis and testicular inflammation and lead to the production of high‐quality sperm. It is suggested that in other experimental studies, the molecular effect of exosomes in the treatment of acute inflammation should be investigated to determine the apparent role of this substance. Thus, the exosome may be suggested as a treatment protocol for the relief of acute inflammation such as TT. For future investigations, it is recommended to use exosomes derived from other body fluids in TT pathologic condition. The most important part of this system is the testicles, which produce sperm and the hormone testosterone. The testicles are located inside the sac and are protected by different layers. The structure of the testicles consists of the intricate tubes that make up sperm. The intermediate layers of the seminiferous tubules are composed of different densities of Leydig cells. Damage to the fallopian tubes leads to the cessation of sperm production, and damage to Leydig cells leads to a decrease in testosterone production and thus the elimination of secondary sexual characteristics in men. Testes are involved in 2 primary physiological processes: spermatogenesis and sexual hormone secretion. Healthy sperms are able to fuse with the egg successfully during the process of fertilization. Thus, the preservation of the healthy status of sperms seems a critical issue. Testes with physiological Tes production and healthy sperm generation are considered normal sexual tissue, whereas inflammation of testis can lead to pathological disruption of sperm production or hormone secretion.TT is a common clinical emergency in men. In TT, the testis rotates around the longitudinal axis of the testicle, leading to artery compression and blood stasis.Exosomes are membrane‐bound vesicles that are present in body fluids. Numerous studies have approved the effect of exosomes on restoration of physiological processes. Today, the majority of the new therapeutic treatments are based on the extraction of exosomes from different body fluids and their applications by different protocols.Since the anti‐inflammatory features of many types of exosomes have been reported in various papers, this animal study was designed to experimentally evaluate the role of serum‐derived exosomes on inhibition of inflammation caused by TT. Various laboratory assays were employed to measure the oxidative levels following TT, such as Griess and MDA. Also, to observe the probable therapeutic effects of exosomes on tissue regeneration, immunohistochemistry (IHC) and routine H&E stainings were used, and gene expression was measured.22.1Following preparation of 18 male adult mice , standard living conditions were provided, including normal environmental temperature, 50%–60% humidity, physiological photocycle, and free access to food and water. All experimental manipulations were conducted in accordance with ethical principles described in an appropriate version of the 1975 Declaration of Helsinki (revised in 2000).2.2Blood serum was used as a source of exosome isolation using the Exosome Purification Kit . This kit contained; 2× Exo‐spin Buffer, 8× Exo‐spin columns with waste collection tubes, and 1× PBS (7 ml) without calcium chloride and magnesium chloride. This kit combines precipitation and size exclusion chromatography (SEC) techniques to inhibit the isolation of non‐exosomal materials. Just prior to exosome purification, the blood samples were centrifuged , and blood serum was used for exosome isolation to preserve the high quality of purified exosomes. For debris elimination, 1 ml of plasm was transferred to a microcentrifuge tube and treated by centrifugation . This protocol was also repeated for the supernatant  to remove probable remaining cell particles. For precipitation of exosome‐containing fractions, the isolated supernatant was transferred to a new microcentrifuge tube and received Exo‐spin Buffer (with a buffer:supernatant ratio of 1:2). This solution was preserved in a refrigerator (4°C) for 60 min (12 000 rpm). The supernatant was removed, and the palette was resuspended in 100 μl of PBS. Exo‐spin column matrix was prepared and re‐equilibrated twice with 250 μl of PBS prior to application. The liquid was allowed to penetrate into the column matrix under gravity, and then the flow‐through buffer was discarded. For purification of exosomes, 100 μl of resuspended exosome‐containing pellet was added to the column (column was located in a collection tube). Flow‐through was discarded, and the column was placed into a new 1.5 ml microcentrifuge collection tube. Then, 180 μl of PBS was added to the column and was allowed to elute. Exo‐spin column was removed from the sample collection tube. Finally, the sample collection tube containing the isolated exosomes was centrifuged  to collect all liquid at the bottom of the tube. Also, no dilution was performed on the resulting product, and a solution containing exosome was injected into the testis (on the involved torsion‐detorsion side) with the volume of 10 IU.2.3For anesthesia induction, 50 IU of ketamin 10 IU/xylazine 90 IU was injected intraperitoneally for in the animals. The left inguinal canal was dissected, and the spermatic cord was exposed. According to the routine procedure of Shahedi et al., the testis was rotated (270°) counterclockwise for 2 h (the anesthetic drug injection was extended every 30 min to continue the anesthesia for 2 h). In this period, the spermatic cord was fixed to the walls of inguinal canal using silk thread (4/0). Thus, blood flow was arrested, and orchitis was induced. Finally, all surgical conditions were restored to their anatomical position, and the walls of inguinal canal were sutured. 2.4n = 6 in each group) including; (1) healthy control , (2) TT , and (3) TT + exosome . Four hours after TT, a single dose of exosome was injected in scrotum (10 IU). In this procedure, the skin of scrotum was elevated and the niddle was inserted subcutaneously. Thus, in this process, the drug was accumulated in subcutaneous tissue surrounding the testis.After 2 weeks of adaption of animals to the new environment, they were categorized into 3 groups . The left testicle was also dissected in 2 identical parts for histopathological  and genetical (frozen in liquid nitrogen) assessments. Also, the tail of the left epididymis was cut in 10 ml of DMEM F12/FBS 5% culture medium for sperm parameter assessments.2.6To evaluate sperm quality, indices of viability, motility, count, and morphology were examined microscopically. Following preparation of cell suspension in previous step, 40 μl of eosin was combined with the same volume of cell suspension. Thus, dead cells were identified, and living sperms were counted using a microscope (40×). According to the Zamir‐Nasta protocol, the normal motility of sperms was assessed, and just the sperms with straight movement were considered to have normal sperm motility. For counting, 400 μl of fixed sperm suspension  was located on a hemocytometer. Also, eosin/nigrosin was used to assess sperm morphology (400×).2.7Concentration of tissue nitric oxide (NO) can be considered as one of the factors expressing tissue damage. NO was measured using an ELISA kit . Following measurement of various dilutions containing NO at 540 nm wavelength, the standard curve was drawn, and the results are shown as optical density (OD).2.8After tissue damage and cell death, the cell membrane lipids are peroxidized, and the resulting fragments are released in the form of thio‐barbituric acid reactive substances (TBARS). Therefore, the TBARS measurement using the MDA kit  can be a criterion for reporting the amount of cellular peroxidation. Following preparation of the associated substances, including lysis buffer, phosphotungstic acid, and BHT (100×), the OD was measured at 532 nm. MDA concentration was measured using the following formula; the amount of MDA (nM)/mg × 4* × D = nM/mg. 4* was the correction for using 200 μl of the 800 μl reaction mix.2.9Tes level is an important index representing the status of Leydig cell damage after TT induction. Also, this index shows the level of repairment following exosome administration. An ELISA kit  was employed to measure the Tes levels in blood serum using the routine procedure of Asghari et al.2.10After fixation of the samples in 10% formalin (for 3 weeks), tissue processing was performed  was applied. Then, tissue sections (5 μm) were prepared using a microtome devise. H&E staining was used for histopathological assessments of slides according to various protocols of deparaffinization, hematoxylin staining, alcoholic‐eosin staining, acid‐alcoholic administration, and xylene‐based clearing. Seminiferous tubule, interstitial tissue, lumen, and spermatogenic cell lines were observed microscopically (40×).2.112O2 (3%). Sodium citrate buffer was used for antigen retrieval . These retrieved slides in a 4°C environment were incubated by mice monoclonal antibodies against Bcl‐2 protein as the primary antibody. After one night, the sections were incubated with biotinylated goat anti‐mouse IgG. Then, they were exposed to 3,3‐diaminobenzidine substrate as chromogen for 8 min. Bounded antibodies appeared in brown.NF‐kB proteins belong to a family of structurally related eukaryotic transcription factors involved in controlling a large number of physiological cellular and organismal processes, such as immune and inflammatory responses. Histological sections were incubated in methanol solution with H2.12CCTCAGCATCTTATCCGAGTGG, R: TGGATGGTGGTACAGTCAGAGC), Caspase‐3 , and BCL‐2  were evaluated. Also, β‐actin  was used as a housekeeping gene. Gene expression was measured by using the 2−ΔΔCt method.Total RNA was extracted from the tissue of the left testis. Then, the quality was examined using a Nanodrop machine at 260/280 nm. Finally, the cDNA was synthesized, and real‐time quantitative PCR was conducted. The primers of related genes were p53  was used for statistical analysis, and the Tukey post hoc test was used to determine the difference between the groups.SPSS software (v. 16) was used for data analysis, and the final graphs were drawn using GraphPad Prism software. Results are expressed mean ± standard deviation (SD), and 33.1p < .05) following TT induction in the TT group compared with the control. These features were alleviated significantly (p < .05) after exosome administration in the TT + exosome group compared with the TT animals  incremental trend in TT groups compared with the control animals. This index was decreased significantly (p < .05) in the TT + exosome group compared with TT animals after exosome injection  compared with control. This androgen was also increased approximately similar to normal level following exosome administration in TT + exosome animals compared with the TT group  in TT animals compared with control. This index was also significantly decreased in the TT + exosome group compared with TT animals (p < .05)  Table .3.5Microscopic examination of H&E‐stained images showed that TT could lead to acute tissue damage to testicular tissue Figure . TT temp3.6p < .05) in TT animals compared with control. Bcl‐2 was decreased significantly (p < .05) in TT animals compared with control. Also, these features (p53 and Caspase‐3) were decreased significantly (p < .05) following exosome administration in the TT + exosome group compared with TT animals. Bcl‐2 was increased significantly (p < .05) following exosome administration in the TT + exosome group compared with the TT group  as well as inhibitors including vildagliptin and sitagliptin, along with nitric oxide synthesis inhibitors. This study was performed on samples with testicular truncation. The results showed that concomitant use of DPP4 inhibitor with nitric oxide synthesis (NOS) inhibitor can reduce the accumulation of oxidative stress and subsequent inflammation. The results of this study clearly show that any factor that reduces the presence of oxidative stress factors can lead to proper cell function.5Blood‐derived exosomes can act as anti‐inflammatory sources in TT. This study showed that administration of a single dose of exosome immediately after acute testicular injury can significantly reduce TT complications. Our study showed that exosomes can diminish apoptosis and testicular inflammation and lead to the production of high‐quality sperm. It is suggested that, in other experimental studies, the molecular effect of exosomes in the treatment of acute inflammation should be investigated to determine the apparent role of this substance. Thus, the exosome may be suggested as a treatment protocol for the relief of acute inflammation such as TT. For future investigations, it is recommended to use exosomes derived from other body fluids in TT pathologic condition.Reza Dadfar conceived and designed the study, supervised the data collection, interpreted the results, and revised the final manuscript. Mona Keivan conducted the data analysis, prepared the tables, and wrote the statistical analysis method and the results. Fatemeh Mansouri Torghabeh and Samira Davoudi conducted the study, collected the data, and organized the dataset. Shima Moradi Maryamneghari assisted in protocol development and drafted the manuscript. All authors approved the final manuscript.The authors declare that they have no competing interests.


Cardiac pulsation was the primary source of pulsatile CSF motion except during deep abdominal breathing, when there was a comparable contribution of respiratory and cardiac 1st harmonic power [0.59 ± 0.78], suggesting respiration can be the primary regulator of CSF depending on the individual differences in breathing techniques. Further work is needed to investigate the impact of sustained training yogic breathing on pulsatile CSF dynamics for CNS health.Cerebrospinal fluid (CSF), a clear fluid bathing the central nervous system (CNS), undergoes pulsatile movements. Together with interstitial fluid, CSF plays a critical role for the removal of waste products from the brain, and maintenance of the CNS health. As such, understanding the mechanisms driving CSF movement is of high scientific and clinical impact. Since pulsatile CSF dynamics is sensitive and synchronous to respiratory movements, we are interested in identifying potential integrative therapies such as yogic breathing to regulate CSF dynamics, which has not been reported before. Here, we investigated the pre-intervention baseline data from our ongoing randomized controlled trial, and examined the impact of four yogic breathing patterns: (i) slow, (ii) deep abdominal, (iii) deep diaphragmatic, and (iv) deep chest breathing with the last three together forming a yogic breathing called three-part breath. We utilized our previously established non-invasive real-time phase contrast magnetic resonance imaging approach using a 3T MRI instrument, computed and tested differences in single voxel CSF velocities  at the level of foramen magnum during spontaneous versus yogic breathing. In examinations of 18 healthy participants  years; age range: 18–61 years), we observed immediate increase in cranially-directed velocities of instantaneous-CSF 16–28% and respiratory-CSF 60–118% during four breathing patterns compared to spontaneous breathing, with the greatest changes during deep abdominal breathing (28%,  CSF movement16 and CSF-ISF exchange19 during wakefulness, sleep and/or anesthesia recently have received particular interest for their implications on pathological states involving CSF. For instance, CSF together with ISF plays an essential role for the removal of solutes and metabolic wastes from the brain interstitium4. This waste removal plays a key role in a number of disease pathologies, such as Alzheimer’s20, which is the most common form of dementia contributing to ~ 60–70% of ~ 50 million dementia cases worldwide21, and is associated with the buildup of amyloid beta peptides23. Understanding the mechanisms driving CSF movement, and interventions that influence and enhance its resultant removal of waste products from the brain is therefore of high scientific and clinical impact.Cerebrospinal fluid (CSF) is one of the two discrete fluid compartments of the brain along with interstitial fluid (ISF), and is crucial for the health of central nervous system (CNS). With the advances in imaging technologies and recent research efforts32 and respiration (0.1–0.3 Hz)38, and is influenced by transient effects such as coughing41, and body posture43. A topic of current interest involving CSF dynamics is identifying the primary regulator(s) of CNS fluids or solute movement within subarachnoid spaces, ventricles, and deep brain parenchyma44. The major drivers of CSF flow implicated in recent studies are: (1) forced inspiration in humans12, (2) cardiac pulsation with some contribution from respiration in humans37, and (3) cardiac pulsation in rodents7. A few earlier studies particularly investigated respiratory-CSF dynamics during normal respiration using echo-planar imaging (EPI) (1.5T MRI)34, normal breathing and breath holding using dynamic EPI (1.5T MRI)45, normal and forced breathing using radial gradient-echo sequence (3T MRI)12. However, these studies measured CSF signal intensities in arbitrary units, and did not measure CSF directionality. A recent real-time multi slice EPI velocity phase contrast MRI (PCMRI) study13 showed directionality and magnitude of respiratory- and cardiac-driven CSF velocities in cm/s during a set of breathing patterns , and breath holding (3T MRI). Recent works have also examined the magnitude, direction, and sensitivity of CSF movement to respiratory performances and locations47.CSF movement is driven by pressure changes in CNS vascular system due to cardiac pulsation (~ 1 Hz)49. In a recent study conducted with subjects sleeping in a magnetic resonance imaging (MRI) scanner, Fultz and colleagues9 demonstrated that CSF flow oscillations during non-rapid eye movement (NREM) sleep were larger (5.52 dB) and slower (0.05 Hz vasomotion) compared to wakefulness (0.25 Hz respiratory), and suggested that changes in pulsatile CSF dynamics during sleep may alter the brain’s waste clearance due to increased mixing and diffusion50.More recently, low-frequency oscillations of CSF  including those during sleep have been of interest51 and removal of solutes54 from the brain is a topic of high clinical impact. Since pulsatile CSF dynamics is sensitive and synchronous to respiratory movements, we are interested in identifying potential integrative therapies such as yogic breathing to regulate CSF dynamics, which has not been reported before. To this end, we designed a study to investigate the impact of yogic breathing on pulsatile CSF dynamics.In short, CSF movement55 encompass a large and diverse group of therapies including yoga, meditation, Tai-Chi, Qi-Gong, and relaxation techniques. Among all, yoga has become one of the most popular integrative and complementary mind–body approaches56 of the 21th century for cultivating overall health and well-being. As a critical component of a traditional yoga practice, yogic breathing  cardiac pulsation on the (magnitude and direction of) instantaneous CSF velocities in absolute units [cm/s], which provides a unique opportunity to study the impact of yogic breathing on pulsatile CSF dynamics. We have utilized this RT-PCMRI in a recent randomized controlled trial (RCT) that aims to investigate effects of two separate 8-week yogic breathing interventions on pulsatile CSF dynamics. While the RCT aims to investigate the long-term impact of yogic breathing among novice practitioners, we herein present the pre-intervention baseline data, prior to randomization, to demonstrate the immediate impact of a set of slow and deep yogic breathing patterns on pulsatile CSF dynamics compared to spontaneous breathing by studying respiratory and cardiac-induced harmonic components of CSF. Briefly, we computed instantaneous-CSF (iCSF) velocities acquired with RT-PCMRI during spontaneous breathing and four yogic breathing practices . We then separated iCSF into three components: respiratory (rCSF), cardiac 1st (c1CSF) and 2nd harmonics (c2CSF), and rigorously tested the differences between spontaneous versus four yogic breathing conditions.We have recently developed a non-invasive real-time phase-contrast MRI (RT-PCMRI) approach65 including in intracranial pressure (ICP) to assess pressure pulsatility, and recently in alligator CSF66 in the spinal canal and the cranial cavity. Further, several studies68 observed changes in CSF between individuals due to age and sex among other factors. Here, we provide cardiac-induced harmonics components of CSF movement as well as associations between demographic covariates  and changes in CSF metrics during spontaneous versus yogic breathing conditions.It is important to keep in mind that most studies of CSF dynamics have investigated the primary component of cardiac pulsation which is usually at the heart rate frequency. Cardiac-induced harmonics have been observed only in a few studies1CSF, and c2CSF, and (2) to quantify the relative contribution of rCSF versus c1CSF and c2CSF during each breathing condition to determine the primary regulator of CSF motion in all breathing conditions.The primary goal of this pre-intervention baseline study is then two-fold: (1) to quantify and compare the immediate impact of four yogic breathing practices versus spontaneous breathing on (magnitude and direction of) velocities of iCSF, rCSF, cThe study was approved by the Institutional Review Board of the Oregon Health & Science University (OHSU), and the full ongoing RCT was registered at the Clinicaltrials.gov (ID # NCT03858309). We received verbal and written informed consent from all study subjects prior to all study procedures. We recruited healthy participants from the Portland metropolitan area using OHSU’s study participation opportunities website, Oregon Center for Clinical and Translational Research Institute (OCTRI) research match for recruitment, flyers throughout the OHSU campus and communities in Portland, and social media (Facebook). We aimed to enroll participants 18 to 65 years of age who were able and available for study activities including undergoing non-invasive MRI scans, had no current or previous regular practice of mind–body therapies focusing on breath awareness and/or training , and were in good health without any history of neurological disorders, sleep disorders, respiratory disorders, problems with heart, circulatory system, and lungs. See Table Each subject’s imaging visit lasted approximately 3-h including study instructions, 1-h MRI scans, and a set of questionnaires . Upon arrival for their imaging visit, we measured each subject’s temperature, blood pressure, and height and weight for body mass index (BMI). We then transitioned subjects to a mock scanner (0T room) for a ~ 30-min instruction for the breathing practices to be performed during the RT-PCMRI scans. The mock scanner room is designed to prepare research study subjects prior to entering the MRI Instrument suite, and is useful to help acclimate subjects to the enclosed space inside an MR Instrument. We first explained and demonstrated each breathing practice, then guided subjects to perform at their own pace first seated on a chair, and then in supine in the mock scanner to mimic the MRI environment.spontaneous breathing (SponB), (2) slow breathing (SlowB), (3) deep abdominal breathing (DAB), (4) deep diaphragmatic breathing (DDB) (5) deep chest breathing (DCB). See Table We instructed subjects to perform the following breathing protocol first in the mock scanner for training purposes, and then in the MRI instrument during the ~ 1-min RT-PCMRI measurements, each to be collected twice with 30–60 s between consecutive measurements in the following order: (1) (SponB),  slow bre— is to make the breath slower and deeper, we included slow and deep breathing practices that were likely to have immediate impact on pulsatile CSF motion, and create larger changes compared to spontaneous breathing in magnitude and frequency of pulsatile CSF motion based on our pilot studies and literature review14. Based on our investigations, we hypothesized slow breathing would create an impact in between spontaneous and deep breathing. Therefore, we utilized slow breathing in between spontaneous and deep breathing measurements. Three-part breath components were utilized in the order they are performed in a traditional yoga practice: abdominal, diaphragm and chest. We utilized this same order for each subject—instead of randomizing—to prevent any “carrying over” effect from slow and deep breathing patterns to spontaneous breathing. This would allow us (i) to compare each subject’s unique spontaneous versus yogic breathing patterns, and corresponding CSF velocity waveforms (ii) to then quantify changes in magnitude and frequency components of CSF for identifying the primary driving force of CSF (respiratory versus cardiac components) during spontaneous versus yogic breathing practices.We chose breathing practices that were easily performed in supine in an MRI instrument without any constraints, and were less likely to cause head motion artifacts. We began with spontaneous breathing to observe each subject’s unique resting-state  breathing patterns, and corresponding instantaneous CSF velocity waveforms. Since a key principle of a regular yogic breathing practice — that have been associated with health-benefits15 provided the influence of deep abdominal versus deep thoracic breathing while noting the two-breathing patterns exerting different muscle groups. During abdominal breathing, the diaphragm is utilized as the inspiratory muscle, and changes in intrathoracic volume and intrathoracic pressure are greater during abdominal versus thoracic breathing. Particularly, more pronounced contracting of the diaphragm during abdominal breathing results in greater opening of costodiaphragmatic recess.While we herein are interested in the impact of different deep breathing practices on CSF velocities — performed as in traditional yogic practices —, it is important to note the physiology of different breathing practices: a recent study69 practices in the Himalayan Tradition, in which yogic breathing is suggested to be performed within each person’s own capacity for safety reasons, with inhale/exhale to be extended and expanded with caution through regular long-term practice. With that goal in mind, for the MRI breathing protocol, we specifically avoided enforcing any specific rate or depth for inhale/exhale other than giving a choice of rate .At the core of yogic breathing practices lies awareness and training of the breath. We designed our RCT yogic breathing interventions from Raja YogaAfter being introduced to the breathing techniques in the mock scanner, we transitioned subjects to a 3T MRI instrument  for baseline data acquisitions using a 64-channel head and neck coil. We positioned subjects in supine, and provided them with (i) a bolster placed under the knees, (ii) foam pads under the elbows, (iii) pads around head and neck for comfort and minimizing motion artifacts, (iv) blankets for warmth, (v) a wireless finger pulse sensor  for pulse data collection, and (vi) a respiration bellow  for respiration data collection during the entire RT-PCMRI data acquisitions. We instructed subjects to lie still in supine during the entire data acquisition.14, consisting of anatomical MRI acquisitions, followed by simultaneous recordings of our previously established RT-PCMRI14 acquisition, respiration and finger pulse acquisitions. Briefly, for consistency across all subjects, we aimed to measure CSF at an angle perpendicular to the spinal cord at the level of the foramen magnum (FM) . To determine the location of FM, we first collected anatomical MR images using a T2-weighted fast spin echo  1200 ms, echo time (TE) 80 ms; Fig. 1); a 3D T1-weighted gradient echo sequence . We then acquired a cardiac gated PCMRI  prior to RT-PCMRI to ensure proper slice location and angle for visibility of CSF pulsations, and that CSF was not obstructed. Upon confirming the pulsatile CSF motion , we then acquired ~ 1-min RT-PCMRI  at the same slice location and angle when subjects performed each of the breathing practices. RT-PCMRI sequence parameters included: velocity encoding value (VENC) 5 cm/s, temporal resolution ~ 55 ms, flip angle 30 degrees, matrix size 78 × 128, field of view (FOV) 196 × 323 mm (in-plane resolution ~ 2.5 × 2.5 mm), EPI factor 7, slice thickness 10 mm, TR 108.88 ms, TE 8.74 ms). RT-PCMRI has previously been described in detail14. During the RT-PCMRI acquisitions, we simultaneously collected respiration and pulse data with a sampling frequency of fs = 400 Hz.We utilized a 1-h data acquisition protocol, similar to our previous work1). In total  each repeated twice) we have acquired 428,820 RT-PCMRI (magnitude and phase) images, and processed the needed 214,410 RT-PCMRI phase images for obtaining CSF velocity time series. We have developed a semi-automated protocol for post-processing all MRI DICOM images, and respiration and pulse data time series using MATLAB software packages .Each RT-PCMRI acquisition series produced 2042 images , which may potentially cause spatial noise due to border zone partial volume effects71. Achieving a high temporal resolution for RT-PCMRI further may reduce the spatial resolution compared to conventional PCMRI, for which we previously developed a correlation mapping technique that allowed us to extract and average only highly correlated CSF voxels for an averaged CSF velocity time series. In this study, we are interested in obtaining and comparing true spatial and temporal CSF velocity values [cm/s] for each breathing practice. Therefore, to capture true spatial peak velocities to our best ability, we utilized a 2-step process to evaluate CSF velocity waveforms at a single voxel9  with our previously developed correlation mapping technique14, and then visually compared the CSF ROI voxels on RT-PCMRI images  with the cardiac-gated PCMRI images  to confirm the location of a single voxel of interest within CSF ROI. We are interested in maximum capacity of (participant) breathing impact on CSF. Anterior CSF velocities were usually larger than posterior velocities across our study population. We selected a single anterior voxel, within the CSF space, with greater velocity, which was usually among the highest correlated voxels (greater than 0.9 correlation coefficient) obtained from the correlation mapping technique. In addition, to confirm deep breathing practices did not cause artifacts in velocity values, potentially by B0 field changes in the head caused by the motion of torso during deep breathing, we computed CSF velocities in a set of voxels within static tissue, and confirmed there were no respiratory or cardiac frequency components  components of CSF signals. We observed  and 2nd harmonics (c2CSF), which were below 4 Hz. To remove higher harmonics and high frequency signals, and investigate only the rCSF, respiration, c1CSF and c2CSF, we then low-pass filtered raw CSF velocity time series . See Fig. 1–5, D1–5.Previous studiesved Fig.  higher osee Fig.  using a 1CSF and c2CSF , using the individual frequency bands , in (i) respiration frequency band of , and (ii) cardiac 1st harmonic frequency band , and (iii) in cardiac 2nd harmonic frequency band  . We repeated the above procedure for each breathing condition of each subject, and obtained time and frequency domain signals for all four distinctive CSF velocity waveforms.To separate and investigate rCSF, cCSF Fig. , we firsCSF Fig. A1, usingnds Fig. A2, in (ind) Fig. A3–5. We 1–5). We confirmed the respiratory component in respiration data, and cardiac (1st and 2nd) harmonic components in pulse data .In parallel, to confirm that estimated frequencies of CSF signals match with the physiological data, we filtered respiratory sensor and pulse sensor data in the same frequency band of respiratory and cardiac components of CSF velocity waveforms. For visualization purposes, we arbitrarily scaled the respiration and pulse data to compare with CSF velocity waveforms Fig. C–D1–5. W 1st and nd harmon1CSF, c2CSF for each subject and breathing condition: (1) peak37 maximum  and peak minimum  during ~ 1-min time series; (2) averaged peak maximum and averaged peak minimum obtained from the average of local peak maximums  above  a threshold set at 97.5th percentile73 ; (3) range32 of peak maximum to peak minimum; (4) range of averaged peak maximum to averaged peak minimum; (5) displacement computed from the integration of the CSF velocity time series [cm/s] and converted to [mm]. Lastly, we computed % change in these metrics, from SponB to SlowB, DAB, DDB, and DCB.We computed the following metrics from time domain CSF velocity waveforms iCSF, rCSF, c74.Note that the traditional method for computing cranially- and caudally-directed velocities are to compute peak maximum and peak minimum values. In addition to peak maximum and minimum, for this study, we also computed averaged peak maximum and averaged minimum values. See Fig. 2). Additionally, to observe individual peak frequency amplitude ratio changes, we computed (3) a peak-to-peak frequency amplitude ratio [r/c1peak] and [r/c2peak] calculated as the ratio of rCSF peak frequency amplitude to the c1CSF, and c2CSF peak frequency amplitudes in the frequency domain .From the instantaneous CSF velocity waveforms, we have computed (1) peak frequencies, and (2) peak frequency amplitudes Fig. A2. Additain Fig. A1–5.Figu2, 1–5), (2) power of rCSF, c1CSF, and c2CSF, and (3) relative contribution37 of the respiration versus cardiac components by defining power ratio14 [r/c1power] and [r/c2power] calculated as the ratio of the power of the rCSF to the power of c1CSF and c2CSF, respectively.To compare the contribution of rCSF, c1CSF, and c2CSF and determine the primary regulatory force(s) for pulsatile CSF, we computed (1) estimated frequency band of rCSF, c1CSF, and c2CSF Figs. A2, 5A1–514 as the integral of the square of the amplitude spectrum over the corresponding frequency band), we used trapezoidal numerical integration in frequency given by Eq. ,  range of iCSF averaged peak maximum and minimum values, (iii) iCSF displacement, (iv) peak frequencies, (v) peak frequency amplitudes, (vi) power values for rCSF, c1CSF, c2CSF, (vii) frequency peak-to-peak amplitude ratios, and (viii) power ratios for a total of 23 metrics  for the following time and frequency domain CSF metrics (i) averaged peak maximum and minimum values for iCSF, rCSF, ccs Table .75, with an overall FDR of 0.05, using the R function “p.adjust”. Unadjusted p values and FDR corrected p values are provided in Table p values mentioned within the text are FDR corrected p values.Independent statistical analysis was conducted by A.H. in R version 4.0.3 . Though data were visually inspected, and extreme values were double checked based on percentiles (with a lower threshold of 5th percentile and a higher threshold of 95th percentile), all values remained in the data to preserve the—diversity of—differences in CSF velocities. Mixed effects linear regression models were built to analyze the associations between each outcome measure and each of the four experimental breathing conditions . Each model included a random subject effect to characterize within-person correlations over repeated measures. Normality assumptions of each model were checked by visual inspection of Q–Q Plots. For multiple comparisons, type I error rate was controlled by using the Benjamini–Yekutieli false detection rate (FDR) procedureIn addition, associations between demographic covariates and outcomes were inspected visually, and tested for significance by Spearman’s rank-order correlation (continuous covariates) or t-test (dichotomous covariates) if a possible association was seen. We reported associations found to be significant in “https://clinicaltrials.gov/ct2/show/NCT03858309). We received verbal and written informed consent from all study subjects prior to all study procedures. All the methods were carried out in accordance with relevant guidelines and regulations.The study was approved by the Institutional Review Board of the Oregon Health & Science University (OHSU). The clinical trial was registered at the ClinicalTrials.gov , and one subject due to inability to follow the MRI breathing protocol. We then utilized 183,780 RT-PCMRI phase images from 18 participants for the final data analyses. See Fig. p value, FDR p values], and (iii) group summary metrics  used for statistical analysis in Table We presented (i) sample datasets from a set of participants to demonstrate the changes in time domain and frequency domain CSF metrics during SponB versus yogic breathing Figs. , 5, 6, , DAB , DDB , and an increase of 60–118% in cranial rCSF velocities during all yogic breathing conditions with statistical significance for DAB  and DDB .For all subjects  and DDB . There were no statistically significant findings for cranial  and caudal c1CSF and c2CSF velocities, as well as iCSF displacement during SponB versus yogic breathing.Caudally-directed velocities (averaged peak maximum) were iCSF [− 2.67 to − 3.03] cm/s, rCSF [− 0.67 to − 1.07] cm/s, cp < 0.05) in estimated rCSF peak frequency (respiration rate) during all yogic breathing conditions with most significance for SlowB  , (2) an increase of 101–234% in rCSF peak frequency amplitude with statistical significance for SlowB , DAB , and DDB , (3) a decrease of 13–21% in c2CSF peak frequency amplitude with statistical significance for SlowB , DAB , and DDB . There were no statistically significant changes in peak frequency for c1CSF and c2CSF , except an increase for DCB; 6%, p = 0.0496, and peak frequency amplitude for c1CSF .When compared to SponB Table , we foun1CSF [r/c1peak] with statistical significance for DAB , and an increase of 166–350% in [r/c2peak] with statistical significance for SlowB , DAB , and DCB .Additionally, we found an increase of 158–359% in peak amplitude ratio or rCSF to cp = 0.0016, Fig. 1), and no statistically significant findings for c1CSF and c2CSF power . We computed relative contribution of rCSF versus c1CSF and c2CSF using the power ratios [r/c1power] and [r/c2power]. Power ratio [r/c1power] for each breathing condition was , , ,  and  demonstrating cardiac 1st as major source of pulsatile CSF motion during SponB. There was an increase of 248–534% in [r/c1power] during yogic breathing compared to SponB, with statistical significance for DAB  when there was a comparable contribution of respiration and cardiac 1st harmonic to pulsatile CSF. For instance, four of the 18 participants .Power ratio , , . . There was also an association between sex and c2CSF frequency peak amplitude for the change score between SponB to DAB (p = 0.009), and SponB to DCB (p = 0.048), with a mean increase in peak amplitude for females and a mean decrease for males.We tested associations between demographic covariates and outcomes during SponB versus yogic breathing. Under the DCB condition, there was a positive correlation between age and change scores (defined as change from the SponB condition) for cranial rCSF velocity , (2) increase of 16–28% in cranially directed iCSF velocities with most statistical significance for DAB , with no significance for DCB, (3) in parallel, an increase of 101–234% in rCSF peak frequency amplitude with most significance for DAB , with no significance for DCB, (4) increase of 187–472% in rCSF power with statistical significance only for DAB , (5) increase of 248–534% in [r/c1power] and 234–589% in r/c2power with statistical significance only for DAB , (6) positive association between age and change scores from SponB to DCB.In short, when compared to SponB, the main results were as follows; there was (1) a statistically significant decrease in respiration rate 18–42% during yogic breathing with most significance for SlowB  respiration rate significantly decreased during slow and deep yogic breathing techniques; (ii) cranial iCSF velocities and in parallel rCSF peak frequency amplitudes increased during yogic breathing with greatest effects in DAB, and with no significant effects for DCB, (iii) cardiac pulsation was the primary driving force for pulsatile CSF during all breathing conditions except DAB when there was a comparable contribution of respiration and cardiac 1st harmonic, and (iv) while there was a comparable contribution of respiration and cardiac 2nd harmonic during SponB, power ratio of respiration to cardiac 2nd harmonic increased during yogic breathing with greatest effects in DAB.76 measuring CSF pressure recordings in response to respiratory changes, coughing and Valsalva maneuver, and non-invasive MRI studies46 investigating respiratory CSF velocities or flow volumes. Briefly, the transmission of venous pressure changes to the collapsible dura through thoracic and epidural veins lining the spine and around the vertebral column causes CSF movement in an ebb-and-flow manner. Lloyd et al.16 recently showed respiratory CSF flow is driven by lumbar and thoracic spinal pressures, and that reduced intrathoracic pressure during inspiration draws venous blood from the lumbar spine and cranium towards the thorax.Using a respiratory bellow, we collected respiration data simultaneously with the RT-PCMRI, thus confirming cranially-directed CSF during inhalation and caudally-directed CSF during exhalation. This result is in agreement with previous studies77. Aktas et al.15 recently demonstrated forced abdominal breathing—compared to forced thoracic breathing—has more pronounced effects on CSF movement within spinal subarachnoid space, resulting in upward net flow during both breathing patterns, whereas low flow rates were identified in the cerebral aqueduct in both breathing patterns. They concluded that abdominal breathing was associated with larger CSF flow due to a more pronounced contraction of the diaphragm compared to thoracic breathing. Furthermore, they suggested that changes in CSF dynamics were due to changes in intrathoracic and intraabdominal pressure being transmitted to the epidural space through the paravertebral venous plexus.Of the four yogic breathing conditions we used in our study , the three of them  significantly increased iCSF velocities, with most pronounced effects observed during DAB with no significant change during DCB. The difference between abdominal and chest (thoracic) breathing we observed is aligned with previous reports indicating abdominal breathing is associated with larger respiratory pressure changes compared to thoracic breathing16.There were no statistically significant changes between SponB and DCB in our study, which may suggest our study population primarily consisted of natural chest breathers although other explanations are possible. While we observed significant increase in cranial directed iCSF velocities during SlowB, DAB, DDB, there were no changes in caudal iCSF velocities suggesting exhalation during spontaneous and yogic breathing in our study population was passive12 presented CSF signal intensities (in arbitrary units) during forced inspiration, and suggested forced inspiration is the major driver of CSF while (ii) Takizawa et al.37 demonstrated velocities of cardiac-driven CSF at cerebral aqueduct were greater than respiratory-driven CSF, while displacement of respiratory-driven CSF was greater than cardiac-driven CSF, (iii) Mestre et al.7 more recently demonstrated cardiac pulsation is the primary regulator of CSF flow through perivascular spaces (PVSs) and is reduced in hypertension, and (iv) Fultz et al. demonstrated CSF flow is driven by vasomotion during sleep.The sources of pulsatile CSF velocity waveforms are cardiac pulsation, respiration and low frequency components such as vasomotion. Several studies recently examined primary regulator(s) of CSF movement and/or flow. For instance, (i) Dreha-Kulaczewski et al.79 investigating ICP measures and a recent study66 investigating CSF dynamics of the American alligator. Studies65 observing cardiac-induced harmonics in ICP interpreted changes in brain pulsatility in the context of system compliance . Young et al.66 more recently (i) studied variations of pulsatile CSF in alligator in the spinal canal and the cranial cavity, (ii) found cardiac-induced harmonics in CSF (not above 3rd order), (iii) hypothesized the absence of higher harmonics could be related to the reptilian meninges and compliance. Taken together, higher harmonics of CSF provide important information for determining the mechanisms regulating CSF dynamics, and need to be investigated in further studies.In our study, we presented respiratory and cardiac components of CSF while separating the cardiac 1st and 2nd harmonic components. During spontaneous breathing, we found that the cardiac 1st harmonic contributed greater power to pulsatile CSF velocities, with comparable contributions by respiration and the cardiac 2nd component. During yogic breathing, (i) the cardiac 1st harmonic contributed greater power to pulsatile CSF velocities, except during DAB, when there was a comparable contribution of respiration and cardiac 1st harmonic effect, and (ii) respiration contributed greater power compared to cardiac 2nd harmonic. During in all breathing conditions, we found a larger frequency amplitude of cardiac 1st versus 2nd harmonic, in agreement with earlier studies39 pointed out cardiac pulsation transmits energy to the CSF, while wave propagation depends on pressure-induced differences in motion. Because venous blood and CSF are in equilibrium across venous membranes, venous changes create larger changes in CSF compared to arterial changes80. This could be the reason why Takizawa et al.37 observed greater cardiac — than respiratory-driven CSF velocities, and greater respiratory — than cardiac-driven CSF displacement.In addition, during yogic breathing compared to SponB, we observed an increase in respiration peak frequency amplitudes. Despite the significant increase in cranially directed iCSF velocities and in parallel in rCSF peak frequency amplitudes during SlowB, DAB, and DDB, cardiac pulsation was still the primary contributor (except during DAB), suggesting that the significant increase in CSF peak velocities or in rCSF peak frequency amplitudes did not necessarily mean that respiration was the major regulator for CSF dynamics. Thus, in future studies, we recommend doing a frequency domain power analysis to determine primary regulator(s) of pulsatile CSF dynamics. For instance, group summary results indicate that yogic breathwork increased both cranial directed CSF velocities and respiratory CSF peak amplitudes. However, only four individual subjects Fig.  had greaOur study participants were naive to mind–body approaches, including breath awareness and breath training. During baseline data collection, most, if not all, of our study participants indicated that they were not familiar with any of the different deep breathing practices in our MRI protocol. Thus, the respiratory dynamics investigated in this baseline dataset provides only the immediate influence of yogic breathing in non-practitioners. In our ongoing interventional RCT study, we hypothesized that respiratory dynamics would be different in advanced practitioners, resulting in larger respiratory dynamics, and thus larger effects on CSF. In the RCT study, we will compare pre- and post-intervention respiratory dynamics utilizing the parameters as described herein to determine whether cardiac pulsation is still the primary driver of CSF post-intervention.68, vascularization81 and/or coupling between arterial inflow and venous outflow82. Based on our tests for associations between covariates , and changes in CSF metrics during SponB versus yogic breathing, we did not observe any significant change except a positive correlation between age and changes in SponB to DCB, in addition to a positive association with age and DCB condition alone. Due to small sample size in our study, we suspect these may be spurious findings. Future studies with larger sample size are needed to explore the associations for these covariates.Differences in CSF dynamics between individuals, and across breathing conditions within individuals, suggest unique bio-individual characteristics of pulsatile CSF dynamics. Previous studies suggested changes in CSF between individuals could be due to age and sex37. We also provided the first comprehensive report studying a higher order (2nd) cardiac harmonic component in human CSF non-invasively during voluntarily controlled breathing conditions. Taken together, our results provide evidence for immediate modulation of pulsatile CSF dynamics with yogic breathing, and the importance of studying CSF dynamics in voluntarily controlled conditions to better understand mechanisms driving CSF.In short, we demonstrated that pulsatile CSF dynamics are highly sensitive and synchronous to respiratory characteristics such as rate, depth and location of respiratory movement, in agreement with previous studies9 conducted neuroimaging in human subjects during sleep by combining blood oxygen level–dependent functional magnetic resonance imaging (BOLD fMRI), electroencephalography (EEG), and CSF flow measurements, and demonstrated (i) CSF flow oscillations during non-rapid eye movement (NREM) sleep were larger and slower (0.05 Hz vasomotion) compared to wakefulness (0.25 Hz respiratory); and (ii) suggested changes in pulsatile CSF dynamics during sleep may alter brain’s waste clearance due to increased mixing and diffusion50. In our study, iCSF velocity waveforms were synchronous to breathing patterns, i.e., slower and larger during slow and deep yogic breathing practices compared to spontaneous breathing. Specifically, we found an increase of 16–28% in cranial iCSF velocities during yogic breathing. Because the entry of CSF along perivascular channels is critical for CSF-ISF exchange in rodents18, and because pulsatile CSF dynamics during sleep potentially may alter9 brain waste clearance due to increased mixing and diffusion, changes in pulsatile CSF dynamics due to yogic breathing could then be investigated for the removal of waste products in future studies.Recently, Fultz and colleagues83 studied drug transport as a function of frequency and magnitude of CSF pulsations during different heart rates and CSF stroke volumes. Both heart rate and CSF stroke volume influenced drug distribution in CSF presenting key factors for interpatient variability in drug distribution. We hypothesize that different breathing rates and CSF velocities via yogic breathing would impact peak concentration of drugs in CSF after injection through mixing and diffusion.In addition, changes in pulsatile CSF dynamics through yogic breathing could be beneficial for investigating intrathecal (IT) drug delivery and factors influencing IT drug transportation. For instance, using medical image-based computational fluid dynamics Hsu et al.84, and other mind–body approaches55 with breathing awareness and/or training, such as mindfulness meditation85, mindfulness based stress reduction87, Tai-Chi88 or Qi-Gong89 in the context of CSF dynamics.Further, our study may shed light on other components of yogaOur study is limited by the small sample size. The parent RCT was designed and powered to detect changes in CSF metrics after two breathing interventions, rather than the pre-intervention baseline analysis, and under the reasonable assumptions  ten subjects per arm would be sufficient to observe the proposed difference in the study arms. Future studies with a larger sample size would allow results to be more generalized across all five breathing patterns and varied demographics. The inherent challenges in MRI acquisition can lead to artifacts in MR images. Measurements with data artifacts were removed from the final analysis. Increased temporal resolution in our RT-PCMRI approach results in reduced spatial resolution, which we believe is handled through rigorous data processing methodology including semi-automated algorithm for extracting CSF signals, visually confirming CSF region of interest, and use of a single voxel approach. This approach eliminated partial volume effects, but limited CSF velocities within one voxel instead of entire cross section of CSF resulting in greater CSF velocities, and increased computational cost. Therefore, future work to develop high spatial and temporal resolution for continuous CSF measurements with analysis within the entire CSF region is needed. Despite these limitations, we have shown that our technique can detect and quantify CSF velocities around the spinal cord. To capture true temporal peak velocities and reduce noise due to transient events, we computed averaged peak velocities, which increased computational cost. We have collected pulse data with a finger pulse sensor, and respiration data with a respiration bellow. Future experimental methodology will include electrocardiography (ECG) measures for investigating the heart rate variability, and potential pressure sensors for measuring intrathoracic and abdominal pressures during yogic breathing techniques. Since our RCT focused on CSF dynamics, we have not investigated arterial and/or venous flow in this study.Future investigations involving larger controlled studies of yogic breathing and/or other mind–body approaches will need to evaluate the effects of training of the breathing techniques on CSF measures. Investigations should: (i) use a larger sample size, (ii) study differences in age, sex, gender, race, activity levels, sleep quality, (iii) evaluate the influence of these covariates on pulsatile CSF magnitude and directionality along the spine and in the cranial cavity, (iv) study the coupling between CSF, arterial and venous flow, (v) utilize ECG, intrathoracic and abdominal pressure measurements in sync with MRI, and (vi) evaluate the effect of breathing induced changes in CSF on the brain’s waste clearance mechanism.To our knowledge, our study is the first report demonstrating the impact of a mind–body approach such as yogic breathing to modulate CSF dynamics, and comparing with spontaneous breathing. We investigated pulsatile CSF velocities during spontaneous versus yogic breathing practices  at the level of foramen magnum using a non-invasive MRI-based quantification in a set of healthy participants without current or previous regular practice of mind–body approaches. With rigorous testing, we demonstrated that the three yogic breathing patterns  immediately increased both cranially directed instantaneous CSF velocities and power of respiratory-driven CSF motion. We observed the most statistically significant effects during deep abdominal breathing. Cardiac pulsation was the primary driver of CSF motion during all breathing conditions except during deep abdominal breathing when there was a comparable contribution of respiration and cardiac 1st harmonic, which suggests respiration can be the primary driver for pulsatile CSF motion depending on individual differences in breathing technique. Further work is needed to investigate the influence of sustained yogic breathing training on pulsatile CSF dynamics for CNS health.Supplementary Information.


The protease BACE1 is a major drug target for Alzheimer’s disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates.To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors.Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal.BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.The online version contains supplementary material available at 10.1186/s13024-023-00596-6. The β-secretase BACE1 (β-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease (AD) –5, thereMultiple BACE1-targeted inhibitors have advanced to phase 2/3 clinical trials for AD and successfully lowered Aβ levels in brain and cerebrospinal fluid (CSF). Yet, most clinical trials were terminated early due to futility and/or the occurrence of side effects, such as non-progressive cognitive worsening and, less frequently, treatment-associated neuropsychiatric findings and sleep disturbances –11. The Identification of BACE1 substrates in vivo is feasible using mouse CSF , 24, becMacaca mulatta, are increasingly used as a preclinical model for the study of CNS pharmacodynamics and pharmacokinetics in the context of neurodegenerative, neuroinflammatory and neurovascular diseases such as AD , p-values < 0.05 are displayed red, * transmembrane regions not annotated at UniPro.Additional file 2: Supplementary Data 1. CSF proteomics of NHP treated with the BACE inhibitor MBI-4.Additional file 3: Supplementary Data 2. CSF proteomics of NHP treated with the BACE inhibitor verubecestat.Additional file 4: Supplementary Fig. S1. CSF proteomics of MBI-4 or vehicle-treated NHP.Additional file 5: Supplementary Fig. S2. Extended plots for NHP CSF proteomics in response to MBI-4 and verubecestat.Additional file 6: Supplementary Fig. S3. Pharmacokinetics and -dynamics of NHP treated with verubecestat.Additional file 7: Supplementary Fig. S4. Full Western blots of sSEZ6 in human CSF after BACE inhibition.Additional file 8: Supplementary Fig. S5. Increased survival of neurons can be blocked by sgp130.


We present the case of a 13-year-old Japanese girl with no previous medical history who presented with a gradually worsening series of orthostatic headaches. We diagnosed spontaneous intracranial hypotension, which worsened by playing the trombone and its Valsalva maneuver effect. She was admitted to our hospital and treated with rest and hydration, and discharged on day 14 without any headaches during the admission. However, her headache recurred when she played trombone, so she stopped it. She now plays percussions, and no headaches recurred for a year. Our case’s headache may have been further exacerbated by cerebrospinal fluid (CSF) leakage due to CSF pressure increase by the Valsalva maneuver while playing the trombone. Intracranial hypotension typically manifests as an orthostatic headache with neck pain, dizziness, and fatigue. Orthostatic headache is affected by posture. The headache continues daily, and for which the medication is less effective. The most frequent underlying factor is cerebrospinal fluid (CSF) leakage. It is hypothesized that dural structural weakness in the dura matter may be responsible for its idiopathic or traumatic tear and consequent leakage . AccordiWe herein describe a 12-year-old girl who presented with an orthostatic headache due to spontaneous intracranial hypotension, which is aggravated by playing the wind instrument trombone. This is one of the rare cases of headache worsened by the Valsalva maneuver due to playing the wind musical instruments .A 13-year-old Japanese girl with no previous medical history presented a gradually worsening series of orthostatic headaches for one month. There was no history of major trauma. Her orthostatic headache has a numerical rating scale of 6/10 without pulsating characteristics. The headache was not present during sitting but occurred some minutes after standing. Especially, she complained that the headache worsened when she played the trombone with both standing and sitting. There was no exacerbation of headaches due to coughing or toileting.The MRI showed partially contrast-enhanced dura matter  via inhibiting aquaporin-4 channels . After aThis is one of the rare cases of headache caused by intracranial hypotension, which was worsened by the Valsalva maneuver due to playing trombone. The expiratory pressure required to play the trombone is up to 65 cmH2O . The comOur case’s headache may have been further exacerbated by CSF leakage due to CSF pressure increase by the Valsalva maneuver while playing the trombone. We suggest that musicians who play wind musical instruments in which high expiratory pressure is required may be at the risk of intracranial hypotension exacerbation and that when such players consult their doctors regarding postural headache, the possibility of spontaneous intracranial hypotension should be considered.The diagnostic criteria for spontaneous intracranial hypotension according to the International Classification of Headache Disorders, third edition (ICHD-3) includes; A) any headache attributed to low CSF pressure or CSF leakage that meets criterion C, B) either or both of the following: low CSF pressure as <60 mm CSF, evidence of CSF leakage on imaging, C) Headache that developed in temporal relation to the low CSF pressure or CSF leakage or that led to its discovery and D) Headache not better accounted for by another ICHD-3 diagnosis  state thData from randomized trials are not available to guide the management of spontaneous intracranial hypotension. Depending on the severity, the first treatment is a short course of conservative treatments over a few days or weeks, including bed rest, high oral fluid intake, caffeine, and an abdominal binder. If these therapies could not improve the symptoms, 1 or 2 epidural blood patches in the lumbar spine are required. A higher volume of blood patches has benefits compared to a lower volume. For patients with persistent symptoms, dynamically computed tomography-myelography or digital-subtraction myelography was used to localize the leak site and directed epidural blood patch, glue injection, or microsurgical repair are considered . Also, iWe experienced the case with an orthostatic headache due to intracranial hypotension exacerbated by the Valsalva maneuver while playing trombone. She did not have any history of major trauma, so we diagnosed spontaneous intracranial hypotension. When we see musicians who play wind musical instruments with orthostatic headaches affected by their postures, the possibility of spontaneous intracranial hypotension should be considered.