Insulin resistance is an important cause of cardiovascular events and cerebral infarction development. We aimed to investigate the association of the triglyceride glucose (TyG) index with atherosclerotic burden and plaques in coronary, intra- and extracranial arteries in participants with non-diabetes, and compared the results with that of the homeostasis model assessment of insulin resistance (HOMA-IR).Participants without diabetes in the PolyvasculaR Evaluation for Cognitive Impairment and vaScular Events (PRECISE) study were included. We categorized participants by tertiles of the TyG index and the concordance/discordance of the TyG index and HOMA-IR. Discordance was defined as a TyG index equal to or greater than the median and HOMA-IR less than the median, or vice versa. The atherosclerosis plaques and burden in coronary, intra- and extracranial arteries were evaluated. The association of HOMA-IR and TyG index with the presence of atherosclerotic plaques and atherosclerotic burden was assessed by binary and ordinal logistic regression models, respectively.Among 2,719 included participants, the average age was 60.9 (± 6.6) years, and 53.0% were female. Both TyG index and HOMA-IR were associated with increased odds of coronary/intra- and extracranial atherosclerotic plaques and burden after adjustment for age, sex, currenting smoking and drinking . However, the association between HOMA-IR and intracranial atherosclerosis was not statistically significant after adjustment for all potential confounders. Discordantly high TyG index with HOMA-IR had a higher odd of extracranial plaque , extracranial atherosclerotic burden , coronary plaque  and segment stenosis score  as compared with concordantly low TyG index with HOMA-IR. The TyG index had a better net reclassification improvement ability than HOMA-IR for atherosclerotic plaques when adding to baseline model.Elevated TyG index was associated with increased odds of atherosclerosis in coronary/intra- and extracranial arteries. Compared with HOMA-IR, the TyG index was more strongly associated with intracranial atherosclerosis. Moreover, discordantly high TyG index with HOMA-IR was also important for atherosclerosis identification.The online version contains supplementary material available at 10.1186/s12933-022-01638-x. Insulin resistance, as a risk factor for metabolic diseases, was involved in the development of cardiovascular disease  and assoWe therefore aimed to investigate the association of the TyG index with comprehensive atherosclerotic burden and plaques in coronary, intra- and extracranial arteries in nondiabetic community-dwelling adults and compared the results with that of HOMA-IR.The PolyvasculaR Evaluation for Cognitive Impairment and vaScular Events (PRECISE) study recruited 3067 participants aged 50 to 75 from a cluster sample of 6 villages and 4 communities in Lishui city between May 2017 and September 2019. Details of the study have been published elsewhere. Partici2)  × fasting glucose [mg/dL]/2)] . Partici2 within and/or adjacent to the coronary artery lumen, which could be clearly distinguished from the vessel lumen and the surrounding pericardial/myocardial tissue  for continuous variables and as the frequency (%) for categorical variables. We used ANOVA or Kruskal-Wallis test for continuous variables and χ2 test or Fisher’s exact for categorical variables. The association of TyG index or HOMA-IR with the presence of atherosclerotic plaques was analyzed by binary logistic regressions and with the atherosclerotic burden by ordinal logistic regression models. The odds ratio (OR) or common odds ratio (cOR) with 95% confidence intervals (CI) were estimated. Logistic regression models were also applied to estimate odds for atherosclerosis associated with concordance/discordance groups. We used 2 models: model 1, unadjusted; model 2, adjusted for age, sex, current smoking, current drinking, BMI, estimated glomerular filtration rate, low-density lipoprotein levels, medical history of transient ischemic attack, stroke, coronary artery disease, hypertension, dyslipidemia, and use of antihypertensive and antiplatelet medications. Restricted cubic splines with 5 knots were constructed to model a non-linear relationship between HOMA-IR or TyG and the presence of atherosclerotic plaques. The area under the receiver operating characteristic curve (AUC), the integrated discrimination improvement (IDI), and the net reclassification improvement (NRI) were determined to assess the predictive performance of insulin resistance index .Two-tailed P value < 0.05 was considered statistically significant, and all analyses were performed using SAS version 9.4 .Among the 3,067 enrolled participants, 307 participants with diabetes, 4 with missing fasting glucose, fasting insulin or triglyceride, and 37 without CTA or MRI examinations were excluded, and a total of 2,719 participants were available for final analysis  had intracranial plaques, 975(35.86%) had extracranial plaques and 1160(42.66%) had coronary plaques. Compared with the first tertile, the third tertile of TyG was associated with increased odds of the presence of intracranial plaques , intracranial atherosclerotic burden , the presence of extracranial plaques , extracranial atherosclerotic burden , the presence of coronary plaques  and segment stenosis score  after adjusting for potential covariates (Table Figure This cross-sectional study showed significant associations between the TyG index and intra- and extracranial, and coronary atherosclerosis after adjustment for confounders. The TyG index was more strongly associated with increased odds for the presence of intracranial atherosclerotic plaques and burden than HOMA-IR after adjustment for potential covariates. Moreover, the TyG index for atherosclerotic plaques had a better improvement ability than HOMA-IR, and the concordance/discordance analysis also reveal extra clinical value of the TyG index.Several studies suggested that TyG index was recognized as a risk factor for cardiovascular disease , 22, 23.Moreover, previous studies have also reported the association between insulin resistance and intra- and extra-cranial arterial disease , 26. TheAlthough the mechanisms of TyG index had superior association than HOMA-IR with atherosclerosis have not yet been fully elucidated, two possible explanations have been proposed. First, the two insulin resistance indexes may reflect different aspects of insulin resistance. The TyG index was thought to reflect the insulin resistance of both the hepatic and peripheral tissues, whereasOur study has several strengths. First, with advanced high-resolution imaging techniques, our study comprehensively assessed the intra-/extra-cranial atherosclerotic plaques and stenosis, and calculated atherosclerosis burden. Second, the concordance/discordance analysis helped us further understood the extra clinical value of the TyG index via the disagreements between the TyG index and HOMA-IR.Some potential limitations also require careful consideration in the present study. First, the cross-sectional data limits the exploration of causality. The longitudinal study of atherosclerosis may better indicate the association between TyG index and progression of atherosclerosis diseases. Second, this study only includes the Chinese population and restricts the generalization of the results. Third, our study population comes from the same neighborhoods, and the inherent selection bias may influence our results to some extent. Despite these limitations, this study provides evidence for the correlation between TyG index and coronary, intracranial or extracranial atherosclerosis. These results need to be further validated in other large-scale populations.Elevated TyG index was associated with increased odds of atherosclerosis in coronary, intra- and extracranial arteries. Compared with HOMA-IR, the TyG index was more strongly associated with intracranial atherosclerosis. Besides, the TyG index could be a more appropriate predictor to identify high-risk atherosclerosis patients.Below is the link to the electronic supplementary material.Supplementary Material 1


The prevalence of males on testosterone therapy (TT) seeking vasectomy reversal (VR) is rising. As medical therapy (MT) to recover spermatogenesis after TT has been previously described, our study’s objective is to present our institution’s management and outcomes of VR in men previously on TT. We performed a retrospective case series of vasectomy patients on TT with subsequent VR by a single microsurgeon between March, 2010 and March, 2022. 14 men undergoing VR during the study period met inclusion criteria. The median age at VR was 43 years with a median obstructive interval of 11 years. Median time from MT to VR was 5 months. Post-operative semen analysis was performed in 10 men and all demonstrated patency. 2 patients had very low sperm counts secondary to continuing TT following VR contrary to medical advice and 5 men with patency achieved pregnancy. Our study noted a high rate of vasovasostomy (VV) (96%) and sustained patency despite a 12-year median obstructive interval. Our findings support favorable outcomes with less stringent VV indications after MT in patients previously on TT that desire VR. The use of MT reduces the recommended wait times for VR after TT discontinuation by more than half. Management and counseling of these patients desiring VR has become progressively more difficult as many are on testosterone therapy (TT). It is well known that exogenous testosterone has contraceptive affects due to its potent inhibitory effects on the hypothalamic–pituitary–gonadal (HPG) axis, resulting in gonadotropin suppression2. This gonadal suppression leads to atrophy of the testicular germinal epithelium and oligo- or azoospermia2. It is also well established that the suppressive effects of TT are dose, route, and duration dependent3. After discontinuation of TT, two-thirds of men will experience return to sperm concentrations of > 20 million/mL within 6 months, while 10% require 1–2 years, and some never have spontaneous return of fertility2.Nearly half a million men of reproductive age undergo vasectomies annually in the United States, at an average age of 31 years old. Of these, approximately 6–10% of men express decisional regret and desire a vasectomy reversal (VR) within 10 years4 and the prevalence of TT use for treatment has increased, on average 15.5% annually, in the last two decades. In men 18–45 years old there has been a four-fold increase in TT compared to older men in which there has only been a three-fold increase5. Despite increasing use of testosterone replacement therapy in men of reproductive age, a clear indication for use is often absent. Previous studies6 have reported that only 4% of men receiving TT for presumed hypogonadism have completed the two pre-TT serum testosterone level assessments recommended by the American Urological Association and Endocrine Society guidelines. Indications reported for starting TT were not standardized and included hypogonadism (48%), fatigue (18%), erectile dysfunction (15%), depression (4%) and psychosexual dysfunction (1%)6.The diagnosis of male hypogonadism and its treatment with TT has become increasingly common. Idiopathic hypogonadism affects a third of men between 45 and 54 years of age. Monitoring gonadotropin levels is also of low utility because they do not correlate with spermatogenesis while on TT7. Thus, the optimal timing of VR following cessation of TT is unknown, with the existing literature recommending waiting 6 months–1 year.Despite the prevalence of VRs and rising incidence of TT, there are currently no guidelines regarding the management of vasectomized patients on TT who present for VR. Medical salvage therapy with clomiphene citrate (CC) or human chorionic gonadotropin (hCG) have been utilized to increase intratesticular testosterone and foster spermatogenesis, but it is a challenge to monitor the effectiveness given that the post-VR anatomy precludes clinically useful semen analysisTo our knowledge, this is the largest case series to date to review the management and post-operative outcomes of VR and pregnancy outcomes after medical therapy in men previously on TT at our institution. As such, the findings of our study will add to the paucity of literature and provide an algorithm to aid providers in evaluating and counseling these complex patients.We conducted a retrospective study of consecutive VR cases performed in a single, large academic urology practice. Our study’s protocol (#00010007) was approved by the OHSU Intitutional Review Board. Because the research is no more than minimal risk to the subjects and does not adversely affect the rights and welfare of the subjects, the need for informed patient consent was waived by the OHSU Institutional Review Board. All experiments were performed in accordance with relevant guidelines and regulations. All microsurgical VRs were performed by a single fellowship-trained microsurgeon (J.C.H) from March 1, 2010 through March 31, 2022. Patients were included in the study if they had a history of TT between time of vasectomy and VR and had complete operative findings documented.Preoperative data, including patient age at time of VR and length of obstructive interval, were abstracted Table . Details8. Intraoperative data including the characteristics of the intravasal fluid  and the methods of reconstruction (VV vs. EV) was obtained. Semen analysis was obtained 4 to 6 months after VR unless pregnancy was already achieved. Phone interviews were conducted in January, 2022 to assess the pregnancy status of couples postoperatively.All patients underwent microsurgical VR with either a 2-layer vasovasostomy (VV) or end-to-side epididymovasostomy (EV) based on intraoperative findingsPresented orally at the American Urological Association Annual Meeting, New Orleans, Louisiana. May 14, 2022.Of all men who underwent VR during the study period, 14 met our study’s inclusion criteria. Median age at VR was 43 years old (IQR 37.8–49) with a median obstructive interval of 11 years (IQR 7.4–14). Prior paternity had been achieved in 13 (93%) men. Median partner age at time of VR consultation was 32 years old (IQR 28.3–36), and median prior TT duration was 24 months (IQR 12–60). The median duration of medical therapy was 5 months (IQR 3.8–6.3) prior to VR and included CC alone in 7 (50%) men, CC with hCG in 5 (36%) men, and no medical therapy in 1 (7%) man.Of the 28 vasa deferens repaired, sperm was noted intraoperatively in 21 (75%) of them. Gross characteristics of the vasal fluid were as follows, in descending order of favorability for pregnancy: clear 32%, opalescent 57%, creamy 7%, and pasty 4%. There was an 86% concordance rate between vasa deferens of the same man. VV was performed in 27 (96%), with VE for the remaining 1 (4%).Patency was defined by the presence of sperm on semen analysis after VR. Of the 14 patients who underwent VR, 2 achieved pregnancy prior to their post-operative semen analysis, so patency was assumed. One patient is hypogonadal on hCG treatment, thus his post-operative semen analysis has been deferred. Of the remaining 11 patients, 8 had post-operative semen analyses within normal limits. Of the 3 patients without normal semen analyses, two resumed their TT postoperatively: 1 had severe oligospermia (< 5 million/mL), and the other had sperm fragments on semen analysis. The third has yet to complete his first post-operative semen analysis.Five men successfully achieved live births, all of whom had post-operative semen analysis results consistent with patency Table . One couOur study found a very high rate of successful VV in men undergoing VR after a short interval of medical therapy after TT, in spite of a long obstructive interval and non-favorable intraoperative findings. This suggests that TT is protective to the vas, and intraoperative decision-making should be modified in this population.6: fourfold in men 18–45 years old compared to threefold in older men5. The prevalence of both vasectomy and vasectomy regret is also high in this group, as 15.9% of American men aged 36–45 years have undergone a vasectomy and 19.6% of vasectomized men in the US desire future children9.The increase in TT among young men will lead to increasing consultations for VR in the setting of prolonged TT. Despite a rise in use, there is a paucity of guidelines or best practice statements to guide the timing of medical and surgical therapies. TT has increased 15% annually10. The gross characteristics, by order of decreasing pregnancy success, are opalescent, creamy, clear, then pasty. In the setting of microscopic identification of motile sperm, intact non-motile sperm, and sperm with short tails, a VV is often performed11. However, in the absence of sperm or the presence of only sperm heads, an EV is completed. The intraoperative findings of our patients on TT did not correlate with their postoperative semen analysis as we would have expected based on the above. Two patients with early pregnancy had clear/clear fluids, which we would usually consider a disadvantageous finding. The other six patients who completed post-operative semen analysis and did not resume TT had excellent semen analysis results with a wide range of intraoperative findings: 7 opalescent, 2 clear, 2 creamy, 1 pasty. The only patient to undergo a VE for pasty fluid had a contralateral VV for opalescent fluid and had an excellent semen analysis.The gross and microscopic intraoperative findings of vasal fluid at the time of VR have been well-described as predictors of reversal success and used to guide surgical decision-making regarding VV or VE12. While IM TT decreases sperm counts in only one month, pellets lead to azoospermia in 2–4 months, suggesting that any form of long-term TT will likely provide vasal protection.The discordance between intraoperative findings and post-operative semen analysis suggests that intraoperative findings following TT should be interpreted differently, with a much higher threshold to pursue EV. TT decreases spermatogenesis, which may impact the male reproductive tract proximal to the vasal ligation . As a result, these tissues may be less chronically dilated, promoting more optimal healing after VR. The protective effect of TT would then be to place the ligated system in a quiescent state, until it is awakened by medical therapy and VR. Routes of TT administration with higher spikes in serum testosterone, such as intramuscular injection (IM), dampen spermatogenesis faster than those with more physiologic levels like pellet implants13. Coward et al. reported a total of 3 VE and 9 VV, with 83% patency and 50% pregnancy13. The medical therapy used in this study was similar, although their workup included Testicular Sperm Aspiration (TESA) for two patients with “uncertain recovery of spermatogenesis based on physical examination and hormone response.” Notably their only patient without a patent semen analysis also underwent bilateral VE.One recent retrospective series focused on 6 men undergoing VR with a history of TT13. Based on our results and the literature described above, we support the algorithm described by Coward et al., and propose a narrower indication for TESA, which should be performed if serum testosterone is persistently low or serum testosterone is appropriate but patients still feel symptoms of low testosterone. Serum levels of 17-hydroxyprogesterone could also be used to evaluate intratesticular testosterone levels in that subgroup14. Serum FSH levels could also be employed at the initial evaluation to assess the current degree of spermatogenesis suppression, with normal levels suggestive of a balanced HPG axis. While this would further shorten the time to reversal, the risk of persistent occult TT-induced azoospermia in the early stages of recovery with normal FSH might alter intraoperative findings and decisions. Thus, 3 months of empiric spermatogenesis-promoting therapy balances optimizing time to conception with surgical outcomes. The decision between VV and VE based on intraoperative findings should favor VV in these men and bilateral VE be avoided as gross vasal fluid findings are not as reliable in this population. Given that two of our patients resumed TT after VR, this suggests the need for further education at every visit regarding the adverse impact of TT on spermatogenesis.Coward et al. recommend an algorithm to manage spermatogenesis salvage therapy when treating men who desire VR after TT that includes discontinuation of TT and initiation of CC with or without hCG for 3 months, after which hormone evaluation  and physical exam should be performedOur findings support favorable outcomes with more liberal VV indications after medical therapy in patients previously on TT that desire VR. The use of medical therapy reduced the recommended wait times for VR after TT discontinuation alone by more than half, and the high rate of VV (96%) and sustained patency despite an 11-year median obstructive interval indicates that VR in this population is very likely to succeed.


The accurate assessment of cerebrospinal fluid opening pressure during spinal puncture provides important medical information in diagnosis, prognosis and therapy of several neurological conditions. However, purpose-specific spinal needle choice is debated. While atraumatic needles are associated with lower incidence of post-puncture headache and re-hospitalisation, some clinicians believe that they lack in accuracy of CSF opening pressure assessment. Our primary objective was to investigate different needle types on correctly assessing CSF opening pressure. We compared typical clinically utilised traumatic (0.9 mm outer diameter) and atraumatic  spinal needles with regards to the assessment of the opening pressure in an experimental spinal puncture model testing experimental and cerebrospinal fluids in predefined pressures. Our goal was to measure the time until indicated pressure levels were correctly shown. Atraumatic needles of at least 0.7 mm diameter had a similar accuracy as traumatic needles without significant differences in time-to-equilibrium. These results were independent of protein and glucose concentration and the presence of haemoglobin. This study demonstrates that atraumatic needles can be used to accurately measure CSF opening pressure. This knowledge might guide clinicians in their choice of needle and help to reduce post-puncture headaches and re-hospitalisation. In general, there are two types of spinal needles: traumatic and atraumatic needles, each in different gauges. It is well known that traumatic spinal needles cause more post-puncture headaches compared to atraumatic needles6.Lumbar puncture (LP) for cerebrospinal fluid (CSF) diagnostics and therapy is a daily routine for neurologists, anaesthetists, neurosurgeons, oncologists, paediatricians, and other medical clinicians worldwide. An additional procedure during LP is the assessment of the lumbar CSF opening pressure7. However, the impact of different pathological conditions such as increased glucose or protein concentrations on assessing CSF opening pressure with traumatic or atraumatic spinal needles remain unknown.Earlier studies that assessed flow rates and CSF opening pressure using different spinal needles showed that both atraumatic and traumatic needles can be used to accurately measure CSF pressure values8. His Quincke needle is formed similar to common medical cannulas as a metal needle with a diagonal sharp cut at its bevelled tip end. On one hand, it is very stable and stiff. On the other hand, as a drawback, its tip may mechanically cut a hole through the dura mater at the injection site during the spinal tap procedure. After discharging the needle, CSF may drain through this leak, causing post-puncture headache syndrome.The lumbar puncture technique was first described in 1891 by the German medical doctor Heinrich Irenäus Quincke (1842–1922)Sprotte needle has a blunt tip designed like the top of a rocket with a cuckoo-hole-like outlet at the side9. Recently, similarly to the Sprotte design, Standl and colleagues introduced the Ballpen needle which comes with a pencil-like tip10. Another type of atraumatic needles is referred to as Whitacre type .In contrast, a modernized alternative needle type was developed 1987 by Günter Sprotte, a contemporary German anaesthetist. The atraumatic Quincke needle with its bevelled tip is commonly referred to as traumatic needle. In contrast, the Sprotte needle and derived replica are characterised by a pencil-like rotationally symmetrical tip and are commonly referred to as atraumatic needle.The classical 3. Lumbar punctures with traumatic needles are associated with a higher rate of post-puncture headache than with atraumatic needles (11% vs. 4.2%) and a higher rate of re-hospitalization after lumbar puncture. Therefore, careful considerations and medical indications for the use of traumatic needles are important to reduce side effects and patient suffering. This study aimed to provide conclusive and practically relevant evidence whether the needle type and size impacts the correct measurement of the CSF pressure during lumbar puncture.A recent meta-analysis covering data from more than 31,000 patients has shown that side effects from spinal tap differ significantly between spinal needle typesTo this end traumatic and atraumatic needles were compared in an experimental model with different liquids compositions and pressures to simulate physiological and pathological CSF constellations.We hypothesized that sufficiently large atraumatic needles have a similar accuracy in assessing CSF opening pressure as traumatic needles.survio.com) and distributed the questionnaire among different hospitals in Hamburg and Northern Germany. The survey consisted of ten questions in a multiple-choice format. The questionnaire focused on the execution of lumbar puncture and the influence of needle types and sizes on CSF pressure assessment. The questions are provided in supplementary Table For an overview of the knowledge about and attitudes towards about CSF pressure measurement technique among medical doctors, we conducted a non-representative online survey ; Becton Dickinson, BD Spinal Needle], and two atraumatic needles [0.7 mm (22G) and 0.45 mm ]. The reason of choice of different diameters was the clinical availability. The main goal of the study was the proof-of-principle whether or not CSF opening pressure can be assessed correctly using atraumatic needles at all. And if so, which minimal diameter should be considered. Therefore, we chose to test the spinal needles that are in use in daily routine, thus methodologically limiting the direct comparison between atraumatic and traumatic needle of the same size. Importantly, all needles were 90 mm long. We measured the pressure with a commercially available instrument .2O); water with normal glucose (0.7 g/L) and normal protein (0.4 g/L); water with increased glucose (7.0 g/L) and normal protein (0.4 g/L); water with normal glucose (0.7 g/L) and increased protein (10 g/L); and water with both increased glucose and protein (7.0 g/L and 10.0 g/L), respectively.We created artificial fluids with different viscosities to imitate clinically common conditions of human CSF by dissolving glucose  and milk powder  in distilled water in different concentrations. We tested five different compositions of artificial ‘CSF’: distilled water  samples of CSF with normal glucose and protein levels from patients with idiopathic intracranial hypertension and (b) CSF with high levels of haemoglobin from patients with subarachnoid haemorrhage, retrieved from external ventricular drains. For human CSF samples, only residuals that were retrieved for therapeutic purposes in routine clinical practice that would have been discarded elsewise were used. The study was registered with the local ethics committee , and written informed consent was obtained from the patients. All probes were anonymized and all methods were performed in accordance with the relevant guidelines.11. We used a multiple linear regression model to test the association between needle type and measured times to reach 80% and 100% of the maximal pressure. Needle size, protein concentration, glucose concentration, and haemoglobin status were used as covariates. Subsequently, we performed ANOVA on the linear model to calculate F statistics.Statistical analysis and visualization were performed within the R environmentForty-four out of 60 participants who were medical professionals of different experience levels and specialties completed the online survey. For details, see Table P = 0.07) differences in terms of the time to reach the peak level between the 20G traumatic  and 22G atraumatic . However, using the small 26G atraumatic needle it took significantly longer to reach the peak pressure  . We found that neither the pressure levels (P = 0.84) nor the glucose concentration (P = 0.96) significantly impacted the time to peak. In contrast, high levels of protein (P = 9.5e−07) prolonged the time to peak across all needles .Subsequently, the effect of the different liquid compositions and pressure levels on the time-to-peak were analysed. Thus, we used water with different milk powder protein concentrations , glucose levels  as well as different pressure levels  and 22G atraumatic  needles (P = 0.92). In contrast, the 26G  significantly (P = 3.1e−13) significantly prolonged the time to peak . Subsequent analysis revealed no significant differences between the 20G traumatic and 22G atraumatic needles across all pressure levels in human CSF with or without haemoglobin .To translate our findings into clinical practice, we compared anonymized CSF samples from patients with idiopathic intracranial hypertension (IIH) and subarachnoid haemorrhages (SAH) in our experimental setup. Similar to the results in our experimental liquid compositions we did not observe significant differences in the time to peak between the 20G traumatic  assesses the same and correct pressure with negligible time delay in the range of a few seconds compared to traumatic needles with an even larger diameter. Thus, atraumatic needles of at least 0.7 mm are suitable to assess the intracranial pressure correctly and safely by lumbar puncture. However, 0.45 mm diameter needles are not suited for this purpose due to the strong time delay, especially with high protein and haemoglobin levels. Our study is limited whilst comparing different needle sizes between traumatic and atraumatic tips. Therefore, it is methodologically not possible to separate our observed effects between the needle size and the tip shape. However, these three needle types were chosen since they are routinely used for spinal taps and our main finding that 0.7 mm diameter atraumatic needle adequately show lumbar pressure without time delay is independent from comparing different needle sizes within the needle shapes. In addition, a possible technical deviation between the indicated Gauge unit of a spinal needle and the real millimetre size cannot be taken into account in this study.3 revealed that atraumatic needles were associated with a decreased rate of rehospitalisation for additional therapies. Importantly, they showed that the safety as well as the efficacy were comparable between traumatic and atraumatic needles.This finding is highly relevant since the usage of traumatic needles is associated with an increased incidence of complications. A recent systematic meta-analysis that included data from more than 30,000 patientsIn conclusion, our data provides evidence that atraumatic needles of at least 22G diameter correctly estimate the CSF opening pressure in comparison to traumatic 20G diameter traumatic needles with no relevant time delay across various conditions. Together with previous studies that have clearly shown the reduced incidence of post-puncture headache and other complications, these data justify the usage of atraumatic needles with at least 22G diameter for lumbar puncture if estimation of CSF opening pressure is required.Supplementary Tables.


Interestingly, the increased expression of the buturylcholinesterase evoked by midazolam was accompanied by a reduced methylation of the BCHE gene and the di-methylation of histone 3 lysine 4 and came along with an increased expression of the lysine specific demethylase KDM1A. Last, inflammatory cytokines were not induced by midazolam. In conclusion, we found a promising mechanistic link between midazolam treatment and delirium, due to a significant disruption in cholinesterase homeostasis. In addition, midazolam seems to provoke profound changes in the epigenetic landscape. Therefore, our results can contribute to a better understanding of the hitherto poorly understood interactions and risk factors of midazolam on delirium.Midazolam is a widely used short-acting benzodiazepine. However, midazolam is also criticized for its deliriogenic potential. Since delirium is associated with a malfunction of the neurotransmitter acetylcholine, midazolam appears to interfere with its proper metabolism, which can be triggered by epigenetic modifications. Consequently, we tested the hypothesis that midazolam indeed changes the expression and activity of cholinergic genes by acetylcholinesterase assay and qPCR. Furthermore, we investigated the occurrence of changes in the epigenetic landscape by methylation specific PCR, ChiP-Assay and histone ELISA. In an  Midazolam is the most abundantly used benzodiazepine in anesthesia and emergency medicine . Due to Currently, it is discussed whether anesthetics cause an alteration of the epigenetic landscape of the cell, which might induce a long-lasting cognitive impairment . One comAlthough there are some theories that could explain the positive correlation between midazolam administration and the high incidence of POD, such as the degree of sedation  and the Currently, a pathogenesis is discussed involving a reduced concentration of the neurotransmitter acetylcholine , neuroinACHE or BCHE gene. However, we speculate that one possible mechanism is the alteration of the expression of cholinergic genes by changing the epigenetic profile of the cells.Recently, we could demonstrate that the GABAergic agent propofol changes the epigenome . In contTherefore, in this study we investigated whether the expression, activity and methylation profile of cholinesterases are changed by midazolam. Furthermore, we study whether midazolam changes the epigenetic landscape of the cell by altering KDM1A expression.2 with 10% fetal calf serum  and 1% penicillin/streptomycin . Cells were maintained every three to four days by adding 5 ml of Trypsin-EDTA 0.25%  after medium removal to dissolve adhesive cells. Furthermore, peripheral blood mononuclear cells (PBMCs) were examined, after the Ethics Committee’s approval , registration at the German Clinical Trials Register  and written informed consent. 80 ml EDTA blood was taken from eight healthy donors  and PBMCs were isolated, using density gradient centrifugation with Ficoll-Paque .Human neuroblastoma cells SH-SY5Y and the glioblastoma cell line U343  were cultured in Dulbecco’s modified Eagle medium  at 37°C and 5% CO2. After RNA isolation and cDNA synthesis of 1 μg RNA using the QuantiTect Reverse Transcription kit , we utilized 2.5 μl of cDNA together with specific primers , following the manufacturer’s instructions. Bisulphite conversion was performed with the EZ DNA methylation-gold kit . All DNA samples were diluted to 10 ng /μl qPCR was performed to detect methylation, as previously described Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Summary: Midazolam is given to patients before surgery. Midazolam is responsible for the delirium experienced by some patients in the days after surgery. Based on previous reports that cholinesterase activity levels are altered in patients who experience delirium, the present work hypothesized that midazolam affects the cholinesterase genes. Stable cell lines and peripheral blood mononuclear cells were assayed for the effect of midazolam on cholinesterase activity and mRNA levels, methylation of cholinesterase genes, demethylation enzyme activity, binding of the BCHE promoter to Histone and neuroinflammation. It was concluded that BCHE gene overexpression may aggravate postoperative delirium.1. Abstract. Please mention that your studies used human SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells.2. Abstract. Please list the methods used in your study.3. You find that midazolam increased the level of BChE activity. However, references 16 and 18 report decreased BChE activity in the blood of patients treated with midazolam, on day 1 after surgery. Perhaps the abstract refers to increased BCHE mRNA rather than BChE activity.4. Page 4 of 20, line 80. Typing error: 2.5% should be 0.25% Trypsin-EDTA5. Page 4 of 20. Did you wash the cells with phosphate buffer saline before adding Trypsin-EDTA? This question is relevant to your assay of AChE activity because fetal bovine serum has substantial AChE activity. If you did not wash the cells, bovine AChE activity may be included in the AChE activity reported for cell lysates.6. Please state the source of midazolam. Which midazolam salt did you use? What solvent was used to dissolve midazolam?7. Page 4 of 20, line 94. Please define the abbreviation BV-2.8. The word “stimulated” is used throughout the text when describing treatment with midazolam. What observation explains your choice of the word “stimulated”. Perhaps the cells proliferated more rapidly in the presence of midazolam?9. Figure 1A. The y axis is labeled AChE/BChE activity, indicating AChE activity was divided by BChE activity. However, the assay measured total AChE and BChE activity because both enzymes hydrolyze the same substrate. Please change the y axis to Cholinesterase activity.10. Figures 1B and 1C. The label on the y axis gives no clue that the assay measures mRNA level. Please modify the label on the y axis to AChE mRNA and BChE mRNA11. The abbreviation ACTB is on the y axis of Figures 1B, 1C, , 2A, 2B, 4A, 4B, 4C, and 4D. Please replace ACTB with a word that is easy to understand. Perhaps the ACTB label is intended to mean that the reported mRNA quantity is relative to the mRNA for beta actin. This should be explained in the figure legend.12. Page 8 of 20, line 178. It is suggested to write “the midazolam antagonist flumazenil reverses midazolam induced effects”13. Figure 2 is missing the letters A and B for panels A and B.14. Figure 2A please change the label on the y axis to AChE mRNA15. Figure 2B please change the label on the y axis to BChE mRNA16. Page 9 of 20. Line 186. Typing error Figure 1A should be Figure 2A.17. Page 9 of 20, lines 194 to 196. Typing error. Figures 2A, 2B, 2C should be Figures 3A, 3B, 3C.18. Figure 3 legend. The words lines and line should be lanes and lane.19. Figure 3C. Please add numbers for DNA sizes. Of special interest are the sizes of the bands in lanes 1 to 7.20. Page 10 of 20, lines 226-227. Typing error. Figures 4A and 4B should be 5A and 5B.21. Conclusion. Line 323. BChE activity is extremely low to almost undetectable in stable cell lines. Your assay measured total cholinesterase activity, which in effect is AChE activity. Please delete the word “activity” in line 323 because BChE activity was not measured.Reviewer #2: This interesting study pursued the epigenetic origin of delirium by focusing on methylation changes in the butyrylcholinesterase(BChE) promoter and seeking the corresponding methylase changes in blood cells from patients treated with Midazolam and in cultured human-originated neuroblastoma cells.The concept is a strength point of this study, but there are weakness points as well, as listed below.1. Why limit the search for methylation but avoid pursuit of microRNAs targeting the cholinesterase mRNA transcripts?2. What is the sex origin of the studied cells and what about the other sex? Pursuing a regulation process in half of humanity seems odd.3. Rather than presenting bar graphs, please shift the graphs into box plots displaying the variability.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  21 Jun 2022Reviewer #1: Summary: Midazolam is given to patients before surgery. Midazolam is responsible for the delirium experienced by some patients in the days after surgery. Based on previous reports that cholinesterase activity levels are altered in patients who experience delirium, the present work hypothesized that midazolam affects the cholinesterase genes. Stable cell lines and peripheral blood mononuclear cells were assayed for the effect of midazolam on cholinesterase activity and mRNA levels, methylation of cholinesterase genes, demethylation enzyme activity, binding of the BCHE promoter to Histone and neuroinflammation. It was concluded that BCHE gene overexpression may aggravate postoperative delirium.In response: We are pleased that you consider our manuscript to address an important topic, and we are happy to discuss the concerns you raised. Please find below a point-by-point reply addressing your remarks.1. Abstract. Please mention that your studies used human SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells.In response: Thank you very much for this helpful comment. We now included into the abstract in lines 20 and 21: In an in-vitro model containing SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells, we found that midazolam altered the activity of acetylcholinesterase /buturylcholinesterase (AChE / BChE).2. Abstract. Please list the methods used in your study.In response: Thank you very much for this helpful comment. We now included into the abstract in lines 18 till 20: Consequently, we tested the hypothesis that midazolam indeed changes the expression and activity of cholinergic genes by acetylcholinesterase assay and qPCR. Furthermore, we investigated the occurrence of changes in the epigenetic landscape by methylation specific PCR, ChiP-Assay and histone ELISA.3. You find that midazolam increased the level of BChE activity. However, references 16 and 18 report decreased BChE activity in the blood of patients treated with midazolam, on day 1 after surgery. Perhaps the abstract refers to increased BCHE mRNA rather than BChE activity.In response: Thank you again for your valuable comment. Indeed, it is described in literature that BChE activity is decreased 48 hours after surgery, while AChE activity is not affected. In our study we could not differentiate between acetylcholesterase (AchE) or butyrylcholinesterase (BChE) activity as both enzymes can hydrolyze acetylcholine. However, we saw that BCHE mRNA expression slightly decreased after midazolam exposure, which was followed by an increase after 24 hours. Hence, this could depict a feedback mechanism for the initial decrease. 4. Page 4 of 20, line 80. Typing error: 2.5% should be 0.25% Trypsin-EDTAIn response: We corrected this typing error.5. Page 4 of 20. Did you wash the cells with phosphate buffer saline before adding Trypsin-EDTA? This question is relevant to your assay of AChE activity because fetal bovine serum has substantial AChE activity. If you did not wash the cells, bovine AChE activity may be included in the AChE activity reported for cell lysates.In response: Thank you very much for this comment. Yes, cell lysate preparation for AChE/BChE activity assay contained a wash step with PBS. We now included in line 111: The proteins were isolated as previously described (20) after washing the cells with PBS.6. Please state the source of midazolam. Which midazolam salt did you use? What solvent was used to dissolve midazolam?In response: We utilized midazolam hydrochloride injection solution from B. Braun, Melsungen. It is dissolved in 10 % HCl and sterile water. We now included in line 94: Briefly, cells were cultured in 6-well culture plates and incubated with 250 ng/ml, 1 µg/ml or 50 µg/ml midazolam  for 2, 4 and 24 h, 10 µg/ml and 50 µg/ml flumazenil or were left unstimulated (control).7. Page 4 of 20, line 94. Please define the abbreviation BV-2.In response: I am sorry for not providing you an abbreviation for this cell line. However, the accession number was now included in the test on line 229: neural glial cells 8. The word “stimulated” is used throughout the text when describing treatment with midazolam. What observation explains your choice of the word “stimulated”. Perhaps the cells proliferated more rapidly in the presence of midazolam?In response: Thank you for this comment. I agree that “stimulate” is not neutral wording but implies a change in the cells biochemical processes. Indeed, we incubated the cells to examine putative stimulation. I changed my language and replaced the word “stimulated” by “incubated” or “treated” throughout the whole manuscript. 9. Figure 1A. The y axis is labeled AChE/BChE activity, indicating AChE activity was divided by BChE activity. However, the assay measured total AChE and BChE activity because both enzymes hydrolyze the same substrate. Please change the y axis to Cholinesterase activity.Figure 1 has been changed. 10. Figures 1B and 1C. The label on the y axis gives no clue that the assay measures mRNA level. Please modify the label on the y axis to AChE mRNA and BChE mRNAFigure 1 has been changed.11. The abbreviation ACTB is on the y axis of Figures 1B, 1C, , 2A, 2B, 4A, 4B, 4C, and 4D. Please replace ACTB with a word that is easy to understand. Perhaps the ACTB label is intended to mean that the reported mRNA quantity is relative to the mRNA for beta actin. This should be explained in the figure legend.The figures were changed and the legends of Figure 2 and 4 were modified. 12. Page 8 of 20, line 178. It is suggested to write “the midazolam antagonist flumazenil reverses midazolam induced effects”This was changed.13. Figure 2 is missing the letters A and B for panels A and B.We changed this. 14. Figure 2A please change the label on the y axis to AChE mRNA15. Figure 2B please change the label on the y axis to BChE mRNAWe changed this. 16. Page 9 of 20. Line 186. Typing error Figure 1A should be Figure 2A.We changed this.17. Page 9 of 20, lines 194 to 196. Typing error. Figures 2A, 2B, 2C should be Figures 3A, 3B, 3C.We changed this.18. Figure 3 legend. The words lines and line should be lanes and lane.We changed this.19. Figure 3C. Please add numbers for DNA sizes. Of special interest are the sizes of the bands in lanes 1 to 7.We added the numbers for DNA size. 20. Page 10 of 20, lines 226-227. Typing error. Figures 4A and 4B should be 5A and 5B.We changed this.21. Conclusion. Line 323. BChE activity is extremely low to almost undetectable in stable cell lines. Your assay measured total cholinesterase activity, which in effect is AChE activity. Please delete the word “activity” in line 323 because BChE activity was not measured.We changed this.Again, thank you very much for carefully reading the manuscript. All points you raised were considered and the errors were corrected. Reviewer #2: This interesting study pursued the epigenetic origin of delirium by focusing on methylation changes in the butyrylcholinesterase(BChE) promoter and seeking the corresponding methylase changes in blood cells from patients treated with Midazolam and in cultured human-originated neuroblastoma cells.The concept is a strength point of this study, but there are weakness points as well, as listed below.In response: We are pleased that you consider our manuscript to address an important topic, and we are happy to discuss the concerns you raised. Please find below a point-by-point reply addressing your remarks.1. Why limit the search for methylation but avoid pursuit of microRNAs targeting the cholinesterase mRNA transcripts?In response: Thank you very much for this outstanding comment. We agree that microRNA has a great potential to regulate cholinesterase mRNA expression. However, the search term “cholinesterase microRNA” yields 70 results in pub med. Hence, we conclude that it depicts a relatively good studied mechanism, while to our knowledge methylation of cholinergic genes was not studied at all. We therefore focused on DNA methylation analysis, but will include studying microRNA in our future studies regarding midazolam and cholinergic activity. 2. What is the sex origin of the studied cells and what about the other sex? Pursuing a regulation process in half of humanity seems odd.In response: Thank you very much for this interesting comment. We mainly studied SH-SY5Y cells, which are from a female origin. In addition, U-343MG cells were studied, which are from a male origin. To completely circumvent gender specific effects, we utilized PBMCs from healthy controls , where no gender dependent effect could be seen. We now included gender distribution into the methods section in line 88 on page 4. 3. Rather than presenting bar graphs, please shift the graphs into box plots displaying the variability.In response: Thank you very much for this outstanding comment. We now changed all graphs. In addition, we have now added new data into figure 3 d, where in the previous version “data not shown” was stated.Attachmentresponse to reviewers.docxSubmitted filename: Click here for additional data file. 24 Jun 2022Midazolam impacts acetyl- and butyrylcholinesterase genes: An epigenetic explanation for postoperative delirium?PONE-D-22-13550R1Dear Dr. Rump,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Israel SilmanAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 29 Jun 2022PONE-D-22-13550R1 Midazolam impacts acetyl- and butyrylcholinesterase genes: An epigenetic explanation for postoperative delirium? Dear Dr. Rump:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Israel Silman Academic EditorPLOS ONE