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Introduction: In vivo studies using selective, localized opioid antagonist injections or localized opioid receptor deletion have identified that systemic opioids dose-dependently depress respiratory output through effects in multiple respiratory-related brainstem areas. Methods: With approval of the subcommittee on animal studies of the Zablocki VA Medical Center, experiments were performed in 53 decerebrate, vagotomized, mechanically ventilated dogs of either sex during isocapnic hyperoxia. We performed single neuron recordings in the Pontine Respiratory Group (PRG, n = 432) and preBötzinger/Bötzinger complex region (preBötC/BötC, n = 213) before and during intravenous remifentanil infusion (0.1-1 mcg/kg/min) and then until complete recovery of phrenic nerve activity. A generalized linear mixed model was used to determine changes in Fn with remifentanil and the statistical association between remifentanil-induced changes in Fn and changes in inspiratory and expiratory duration and peak phrenic activity. Analysis was controlled via random effects for animal, run, and neuron type. Results: Remifentanil decreased Fn in most neuron subtypes in the preBötC/BötC as well as in inspiratory (I), inspiratory-expiratory, expiratory (E) decrementing and non-respiratory modulated neurons in the PRG. The decrease in PRG inspiratory and non-respiratory modulated neuronal activity was associated with an increase in inspiratory duration. In the preBötC, the decrease in I-decrementing neuron activity was associated with an increase in expiratory and of E-decrementing activity with an increase in inspiratory duration. In contrast, decreased activity of I-augmenting neurons was associated with a decrease in inspiratory duration. Discussion: While statistical associations do not necessarily imply a causal relationship, our data suggest mechanisms for the opioid-induced increase in expiratory duration in the PRG and preBötC/BötC and how inspiratory failure at high opioid doses may result from a decrease in activity and decrease in slope of the pre-inspiratory ramp-like activity in preBötC/BötC pre-inspiratory neurons combined with a depression of preBötC/BötC I-augmenting neurons. Additional studies must clarify whether the observed changes in neuronal activity are due to direct neuronal inhibition or decreased excitatory inputs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, HF, declared a shared affiliation with the authors to the handling editor at the time of review., (Copyright © 2023 Palkovic, Mustapic, Saric, Stuth, Stucke and Zuperku.)

Background: Since nonalcoholic fatty liver disease (NAFLD) has become the leading cause of liver disease in the Western world, clinicians need reliable noninvasive tools for the identification of NAFLD-associated fibrosis. Limited evidence on the performance of the novel shear wave elastography technique Elast-PQ (EPQ) in NAFLD is available., Method: In this prospective, European multinational study we assessed the diagnostic accuracy of EPQ using vibration-controlled transient elastography (VCTE) as a reference standard., Results: Among 353 NAFLD patients, 332 (94.1%) fulfilled reliability criteria of VCTE and EPQ (defined by IQR/median ≤0.3; 41.3% female, mean age: 59 [IQR: 16.5], mean BMI: 29.0 (7.1)). 4/353 (1.1%) and 17/353 (4.8%) had unreliable VCTE and EPQ measurements, respectively. VCTE-based NAFLD fibrosis stages were F0/F1: 222(66.9%), F2: 41 (12.3%), F3: 30 (9.1%), F4: 39 (11.7%). We found a strong correlation (Pearson R=0.87; p<0.0001) and concordance (Lin's concordance correlation coefficient =0.792) of EPQ with VCTE. EPQ was able to identify NAFLD-fibrosis risk with the following EPQ cutoffs: ≥6.5 kPa for significant fibrosis (≥F2) (≥1.47 m/s; sensitivity: 78%; specificity: 95%; AUROC: 0.94), ≥6.9 kPa for advanced fibrosis (≥F3) (≥1.52 m/s; sens.: 88%, spec.: 89%; AUROC: 0.949), and ≥10.4 kPa for cirrhosis (F4) (≥1.86 m/s; sens.: 87%; spec.: 94%; AUROC: 0.949)., Conclusion: The point shear wave elastography technique EPQ shows excellent correlation to and concordance with VCTE. EPQ can reliably exclude NAFLD fibrosis <6.0 kPa (<1.41 m/s) and indicate a high risk of advanced fibrosis ≥10.4 kPa (≥1.86 m/s)., Competing Interests: DB served as a speaker and/or consultant and/or advisory board member for AbbVie and received travel support from AbbVie and Gilead. VM, RM, LM, SM: nothing to declare. DC served as a speaker and/or consultant and/or advisory board member for AbbVie, Gilead, and MSD, and received travel support from AbbVie, MSD, ViiV Healthcare, and Gilead. TB has received travel support from BMS, Gilead, and AbbVie. MM served as a speaker and/or consultant and/or advisory board member for AbbVie, Bristol-Myers Squibb, Gilead, Collective Acumen, and W. L. Gore & Associates and received travel support from AbbVie, Bristol-Myers Squibb, and Gilead. IS served as speaker for AbbVie, BMS, Gilead, Janssen, Echosens, Philips; and received advisory board fees by AbbVie, Merck; Siemens, Canon,Toshiba; and received research support by Philips. GF served as speaker for Canon Medical Systems, Hitachi Ltd., Mindray Medical Systems, and Philips. IG served as speaker for Echosens and Philips. TR served as a speaker and/or consultant and/or advisory board member for AbbVie, Bayer, Boehringer Ingelheim, Gilead, Intercept, MSD, Siemens, and W. L. Gore & Associates and received grants/research support from AbbVie, Boehringer Ingelheim, Gilead, MSD, Philips, and W. L. Gore & Associates as well as travel support from Boehringer Ingelheim and Gilead., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Introduction: Oesophageal varices are routinely diagnosed by esophagogastroduodenoscopy (EGD), and their bleeding has high mortality. We aimed to evaluate diagnostic performance of biochemical tests in comparison to elastography-based approaches, as non-invasive alternatives to EGD, for ruling-out high risk oesophageal varices (HRV)., Material and Methods: Retrospective analysis of patients (N = 861) who underwent liver stiffness measurement (LSM) by transient elastography (TE) in a single centre over 5-year period, with available results of EGD (within 3 months from LSM). Only patients with suspicion of compensated advanced chronic liver disease (cACLD) defined by LSM ≥ 10 kPa were included comprising the final cohort of 73 subjects. Original and expanded Baveno VI criteria (B6C), controlled attenuation parameter (CAP), platelet count (PLT), aspartate aminotransferase to PLT ratio index (APRI), Fibrosis-4 index (FIB4), model for end stage liver disease (MELD) score were evaluated against the results of EGD that served as the reference method., Results: Analysed patients had median age 62 years, 59/73 (0.81) were males, 54/73 (0.74) had alcoholic/non-alcoholic fatty liver disease, and 21/73 (0.29) had HRV. In multivariate logistic regression analysis only LSM and PLT were independently associated with HRV. The best performing tests for ruling-out HRV (% of spared EGD; % of missed HRV) were respectively: LSM < 20 kPa (53.4%; 0%), B6C (38%; 0%), Expanded B6C (47.9%; 4.8%); PLT > 214x10 9 /L (21.9%; 0%); FIB4 ≤ 1.8 (21.4%; 0%), APRI ≤ 0.34 (12.3%; 0%). CAP, MELD = 6 alone or combined with PLT > 150(x10 9 /L) did not show acceptable performance., Conclusion: The best performing biochemical tests for ruling-out HRV in our cohort of patients were PLT and FIB-4, but they were still outperformed by elastography-based approaches., Competing Interests: Potential conflict of interest None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Objective: Liver stiffness measurement (LSM) is a tool used to screen for significant fibrosis and portal hypertension. The aim of this retrospective multicentre study was to develop an easy tool using LSM for clinical outcomes in advanced chronic liver disease (ACLD) patients., Design: This international multicentre cohort study included a derivation ACLD patient cohort with valid two-dimensional shear wave elastography (2D-SWE) results. Clinical and laboratory parameters at baseline and during follow-up were recorded. LSM by transient elastography (TE) was also recorded if available. The primary outcome was overall mortality. The secondary outcome was the development of first/further decompensation., Results: After screening 2148 patients (16 centres), 1827 patients (55 years, 62.4% men) were included in the 2D-SWE cohort, with median liver SWE (L-SWE) 11.8 kPa and a model for end stage liver disease (MELD) score of 8. Combination of MELD score and L-SWE predict independently of mortality (AUC 0.8). L-SWE cut-off at ≥20 kPa combined with MELD ≥10 could stratify the risk of mortality and first/further decompensation in ACLD patients. The 2-year mortality and decompensation rates were 36.9% and 61.8%, respectively, in the 305 (18.3%) high-risk patients (with L-SWE ≥20 kPa and MELD ≥10), while in the 944 (56.6%) low-risk patients, these were 1.1% and 3.5%, respectively. Importantly, this M10LS20 algorithm was validated by TE-based LSM and in an additional cohort of 119 patients with valid point shear SWE-LSM., Conclusion: The M10LS20 algorithm allows risk stratification of patients with ACLD. Patients with L-SWE ≥20 kPa and MELD ≥10 should be followed closely and receive intensified care, while patients with low risk may be managed at longer intervals., Competing Interests: Competing interests: JT has received speaking and/or consulting fees from Gore, Bayer, Alexion, MSD, Gilead, Intercept, Norgine, Grifols, Versantis, and Martin Pharmaceutical. Philip Ferstl received consultancy for SNIPR Biome. Supersonic Imagine supported interaction within the groups, but without specific funding., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We aimed to investigate the diagnostic performance of new 2-D shear-wave elastography (SWE) with propagation maps and attenuation imaging (ATI) for quantification of fibrosis and steatosis in non-alcoholic fatty liver disease (NAFLD). Consecutive patients with NAFLD and healthy volunteers underwent liver stiffness measurement and steatosis quantification by means of vibration-controlled transient elastography coupled with the controlled attenuation parameter as the reference and by 2-D shear-wave elastography (2-D-SWE) with propagation maps and ATI as the investigational methods. We included 232 participants (164 in the NAFLD group and 68 in the healthy control group): 51.7%/49.3% women/men; mean age, 54.2 ± 15.2 y; mean body mass index, 29.4 ± 6.5 kg/m 2 . Significant correlations were found between 2-D-SWE and vibration-controlled transient elastography (r = 0.71, p < 0.0001) and between ATI and the controlled attenuation parameter (r = 0.72, p < 0.0001). NAFLD-specific 2-D-SWE liver stiffness measurement cutoffs were as follows-F ≥ 2: 7.9 kPa (area under the curve [AUC] = 0.91); F ≥ 3: 10 kPa (AUC = 0.92); and F = 4: 11.4 kPa (AUC = 0.95). For steatosis, the best cutoffs by ATI were as follows-S1 = 0.73 dB/cm/MHz (AUC = 0.86); S2 = 0.76 dB/cm/MHz (AUC = 0.86); and S3 = 0.80 dB/cm/MHz (AUC = 0.83). According to Baveno VI criteria, the optimal 2-D-SWE liver stiffness measurement for diagnosing liver cirrhosis is 15.5 kPa (AUC = 0.94), and for ruling out compensated advanced chronic liver disease it is 9.2 kPa (AUC = 0.92). To conclude, 2-D-SWE with propagation maps and ATI is reliable for quantification of liver fibrosis and steatosis in patients with NAFLD., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Aims: To investigate morbidity and mortality in a real-life cohort of patients with type 2 diabetes (T2D) in relation to prevalence and severity of nonalcoholic fatty liver disease (NAFLD)., Methods: Patients with T2D were referred for assessment of liver fibrosis by the FIB-4 test and liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE). Liver steatosis was quantified by the controlled attenuation parameter (CAP). These patients were followed until death or censored date., Results: Among 454 patients (52% males, mean age 62.5 years, BMI 30.9 kg/m 2 ), 82.6% was overweight, 77.8% had fatty liver, and 9.9% and 3.1% had LSM and FIB-4 values suggestive of advanced fibrosis, respectively. During the follow-up period of median 2 years, 106 (23%) patients experienced adverse event (11% cardiovascular) and 17 (3.7%) died, whereas no liver-related morbidity or mortality was observed. Independent predictors of adverse outcomes were age and higher platelet count, while FIB-4, LSM, and CAP were not., Conclusion: In a cohort of T2D patients, no liver-related morbidity or mortality occurred during 2 years. Our patients probably have low real prevalence of advanced fibrosis which is likely overestimated by LSM ≥ 9.6 kPa. Liver fibrosis may be safely reassessed in the 2 years interval in noncirrhotic patients with T2D., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ivica Grgurevic et al.)

Objectives: To investigate diagnostic performance of point shear wave elastography by elastography point quantification (ElastPQ) for non-invasive assessment of liver fibrosis in patients with chronic liver diseases (CLD)., Methods: Liver stiffness measurement (LSM) by transient elastography (TE) and ElastPQ was performed in patients with CLD and healthy volunteers. The stage of liver fibrosis was defined by TE which served as the reference. We compared two methods by using correlation, area under the receiver operating characteristics curve (AUC) analysis, Bland and Altman plot and Passing-Bablok regression., Results: A total of 185 subjects (20 healthy volunteers and 165 patients with CLD (128 non-alcoholic fatty liver disease), 83 (44.9%) females, median age 53 years, BMI 27.3 kg/m 2 ) were evaluated. There were 24.3%, 13.5% and 11.4% patients in ≥ F2, ≥ F3 and F4 stage, respectively. The best performing cutoff LSM values by ElastPQ were 5.5 kPa for F ≥ 2 (AUC = 0.96), 8.1 kPa for F ≥ 3 (AUC = 0.98) and 9.9 kPa for F4 (AUC = 0.98). Mean (SD) difference between TE and ElastPQ measurements was 0.98 (3.27) kPa (95% CI 0.51-1.45, range 4.99-21.60 kPa). Two methods correlated significantly (r = 0.86; p < 0.001), yet Bland and Altman plot demonstrated difference between measurements, especially with TE values > 10 kPa. Passing and Bablok regression analysis yielded significant constant and proportional difference between ElastPQ and TE., Conclusion: ElastPQ is reliable method for assessment of liver fibrosis but LSM values are not interchangeable with TE, especially above 10 kPa. Diagnostic performance of ElastPQ for sub-classification of patients with compensated advanced chronic liver disease should therefore be furtherly investigated., Key Points: • ElastPQ appears to be reliable method for assessment of liver fibrosis, with data presented here mostly applicable to NAFLD. • LSM values produced by TE and ElastPQ are NOT interchangeable-in values < 10 kPa, they are similar, but in values > 10 kPa, they appear to be increasingly and significantly different. • Diagnostic performance of ElastPQ for sub-classification of patients with compensated advanced chronic liver disease should be furtherly investigated.

The aim of the study was to explore (a) prevalence and grade of nonalcoholic fatty liver (NAFL) among outpatients referred for abdominal ultrasound (US) examination and (b) relationship between the presence and severity of liver steatosis and metabolic syndrome (MS). This was a retrospective analysis of patients without history of liver disease examined by abdominal US in the University hospital setting. US was used to detect and semiquantitatively grade (0-3) liver steatosis. Data on patients' age, gender, body mass index (BMI), impaired glucose metabolism (IGM), atherogenic dyslipidaemia (AD), raised blood pressure (RBP), transaminases, and platelet counts were obtained from medical records. MS was defined as having at least 3 of the following components: obesity, IGM, AD, and RBP. Of the 631 patients (median age 60 years, median BMI 27.4 kg/m2, and 57.4% females) 71.5% were overweight and 48.5% had NAFL. In the subgroup of 159 patients with available data on the components of MS, patients with higher US grade of steatosis had significantly higher BMI and increased prevalence of obesity, IGM, AD, RBP, and accordingly more frequently had MS, whereas they did not differ in terms of age and gender. NAFL was independently associated with the risk of having MS in a multivariate model adjusted for age, gender, BMI, and IGM. The grade of liver steatosis did not correlate with the presence of liver fibrosis. We demonstrated worrisome prevalence of obesity and NAFL in the outpatient population from our geographic region. NAFL is independently associated with the risk of having MS implying worse prognosis.

The preBötzinger (preBötC) complex has been suggested as the primary site where systemically administered selective serotonin agonists have been shown to reduce or prevent opioid-induced depression of breathing. However, this hypothesis has not been tested pharmacologically in vivo. This study sought to determine whether 5-HT1A receptors within the preBötC and ventral respiratory column (VRC) mediate the tachypneic response induced by intravenous (IV) (±)-8-Hydroxy-2-diproplyaminotetralin hydrobromide (8-OH-DPAT) in a decerebrated dog model. IV 8-OH-DPAT (19 ± 2 μg/kg) reduced both inspiratory (I) and expiratory (E) durations by ∼ 40%, but had no effect on peak phrenic activity (PPA). Picoejection of 1, 10, and 100 μM 8-OH-DPAT on I and E preBötC neurons produced dose-dependent decreases up to ∼ 40% in peak discharge. Surprisingly, microinjections of 8-OH-DPAT and 5-HT within the VRC from the obex to 9 mm rostral had no effect on timing and PPA. These results suggest that the tachypneic effects of IV 8-OH-DPAT are due to receptors located outside of the areas we studied., (Published by Elsevier B.V.)

Life-threatening side effects such as profound bradypnea or apnea and variable upper airway obstruction limit the use of opioids for analgesia. It is yet unclear which sites containing μ-opioid receptors (μORs) within the intact in vivo mammalian respiratory control network are responsible. The purpose of this study was 1) to define the pontine region in which μOR agonists produce bradypnea and 2) to determine whether antagonism of those μORs reverses bradypnea produced by intravenous remifentanil (remi; 0.1-1.0 μg·kg(-1)·min(-1)). The effects of microinjections of agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO; 100 μM) and antagonist naloxone (NAL; 100 μM) into the dorsal rostral pons on the phrenic neurogram were studied in a decerebrate, vagotomized, ventilated, paralyzed canine preparation during hyperoxia. A 1-mm grid pattern of microinjections was used. The DAMGO-sensitive region extended from 5 to 7 mm lateral of midline and from 0 to 2 mm caudal of the inferior colliculus at a depth of 3-4 mm. During remi-induced bradypnea (~72% reduction in fictive breathing rate) NAL microinjections (~500 nl each) within the region defined by the DAMGO protocol were able to reverse bradypnea by 47% (SD 48.0%) per microinjection, with 13 of 84 microinjections producing complete reversal. Histological examination of fluorescent microsphere injections shows that the sensitive region corresponds to the parabrachial/Kölliker-Fuse complex.

Systemic administration of mu-opioids at clinical doses for analgesia typically slows respiratory rate. Mu-opioid receptors (MORs) on pre-Bötzinger Complex (pre-BötC) respiratory neurons, the putative kernel of respiratory rhythmogenesis, are potential targets. The purpose of this study was to determine the contribution of pre-BötC MORs to the bradypnea produced in vivo by intravenous administration of clinically relevant infusion rates of remifentanil (remi), a short-acting, potent mu-opioid analgesic. In decerebrate dogs, multibarrel micropipettes were used to record pre-BötC neuronal activity and to eject the opioid antagonist naloxone (NAL, 0.5 mM), the glutamate agonist D-homocysteic acid (DLH, 20 mM), or the MOR agonist [D-Ala(2), N-Me-Phe(4), gly-ol(5)]-enkephalin (DAMGO, 100 microM). Inspiratory and expiratory durations (T(I) and T(E)) and peak phrenic nerve activity (PPA) were measured from the phrenic neurogram. The pre-BötC was functionally identified by its rate altering response (typically tachypnea) to DLH microinjection. During intravenous remi-induced bradypnea (approximately 60% decrease in central breathing frequency, f(B)), bilateral injections of NAL in the pre-BötC did not change T(I), T(E), f(B), and PPA. Also, NAL picoejected onto single pre-BötC neurons depressed by intravenous remi had no effect on their discharge. In contrast, approximately 60 microg/kg of intravenous NAL rapidly reversed all remi-induced effects. In a separate group of dogs, microinjections of DAMGO in the pre-BötC increased f(B) by 44%, while subsequent intravenous remi infusion more than offset this DAMGO induced tachypnea. These results indicate that mu-opioids at plasma concentrations that cause profound analgesia produce their bradypneic effect via MORs located outside the pre-BötC region.

Hypoglossal motoneurons (HMNs) innervate all tongue muscles and are vital for maintenance of upper airway patency during inspiration. The relative contributions of the various synaptic inputs to the spontaneous discharge of HMNs in vivo are incompletely understood, especially at the cellular level. The purpose of this study was to determine the role of endogenously activated GABA(A) and glycine receptors in the control of the inspiratory HMN (IHMN) activity in a decerebrate dog model. Multibarrel micropipettes were used to record extracellular unit activity of individual IHMNs during local antagonism of GABA(A) receptors with bicuculline and picrotoxin or glycine receptors with strychnine. Only bicuculline had a significant effect on peak and average discharge frequency and on the slope of the augmenting neuronal discharge pattern. These parameters were increased by 30 +/- 7% (P < 0.001), 30 +/- 8% (P < 0.001), and 25 +/- 7% (P < 0.001), respectively. The effects of picrotoxin and strychnine on the spontaneous neuronal discharge and its pattern were negligible. Our data suggest that bicuculline-sensitive GABAergic, but not picrotoxin-sensitive GABAergic or glycinergic, inhibitory mechanisms actively attenuate the activity of IHMNs in vagotomized decerebrate dogs during hyperoxic hypercapnia. The pattern of GABAergic attenuation of IHMN discharge is characteristic of gain modulation similar to that in respiratory bulbospinal premotor neurons, but the degree of attenuation ( approximately 25%) is less than that seen in bulbospinal premotor neurons ( approximately 60%). The current studies only assess effects on active neuron discharge and do not resolve whether the lack of effect of picrotoxin and strychnine on IHMNs also extends to the inactive expiratory phase.

Opioids depress the activity of brain stem respiratory-related neurons, but it is not resolved whether the mechanism at clinical concentrations consists of direct neuronal effects or network effects. We performed extracellular recordings of discharge activity of single respiratory neurons in the caudal ventral respiratory group of decerebrate dogs, which were premotor neurons with a likelihood of 90%. We used multibarrel glass microelectrodes, which allowed concomitant highly localized picoejection of opioid receptor agonists or antagonists onto the neuron. Picoejection of the mu receptor agonist [d-Ala(2), N-Me-phe(4), gly-ol(5)]-enkephalin (DAMGO, 1 mM) decreased the peak discharge frequency (mean +/- SD) of expiratory neurons to 68 +/- 22% (n = 12), the delta(1) agonist d-Pen(2,5)-enkephalin (DPDPE, 1 mM) to 95 +/- 11% (n = 15), and delta(2) receptor agonist [d-Ala(2)] deltorphin-II to 86 +/- 17% (1 mM, n = 15). The corresponding values for inspiratory neurons were: 64 +/- 12% (n = 11), 48 +/- 30% (n = 12), and 75 +/- 15% (n = 11), respectively. Naloxone fully reversed these effects. Picoejection of morphine (0.01-1 mM) depressed most neurons in a concentration dependent fashion to maximally 63% (n = 27). Picoejection of remifentanil (240-480 nM) did not cause any significant depression of inspiratory (n = 11) or expiratory neurons (n = 9). 4. Intravenous remifentanil (0.2-0.6 microg.kg(-1).min(-1)) decreased neuronal peak discharge frequency to 60 +/- 12% (inspiratory, n = 7) and 58 +/- 11% (expiratory, n = 11). However, local picoejection of naloxone did not reverse the neuronal depression. Our data suggest that mu, delta(1), and delta(2) receptors are present on canine respiratory premotor neurons. Clinical concentrations of morphine and remifentanil caused no local depression. This lack of effect and the inability of local naloxone to reverse the neuronal depression by intravenous remifentanil suggest that clinical concentrations of opioids produce their depressive effects on mechanisms upstream from respiratory bulbospinal premotor neurons.