Early alveolar epithelial cell necrosis is a potential driver of COVID-19-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) with COVID-19 is aggravated by hyperinflammatory responses even after the peak of the viral load has passed; however, its underlying mechanisms remain unclear. In the present study, analysis of the alveolar tissue injury markers and epithelial cell death markers in patients with COVID-19 revealed that COVID-19-induced ARDS was characterized by alveolar epithelial necrosis at an early disease stage. Serum levels of HMGB-1, one of the DAMPs released from necrotic cells, were also significantly elevated in these patients. Further analysis using a mouse model mimicking COVID-19-induced ARDS showed that the alveolar epithelial cell necrosis involved two forms of programmed necrosis, namely necroptosis, and pyroptosis. Finally, the neutralization of HMGB-1 attenuated alveolar tissue injury in the mouse model. Collectively, necrosis, including necroptosis and pyroptosis, is the predominant form of alveolar epithelial cell death at an early disease stage and subsequent release of DAMPs is a potential driver of COVID-19-induced ARDS.

The U-shaped association of serum iron level with disease severity in adult hospitalized patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that leads to severe respiratory failure (RF). It is known that host exposure to viral infection triggers an iron-lowering response to mitigate pathogenic load and tissue damage. However, the association between host iron-lowering response and COVID-19 severity is not clear. This two-center observational study of 136 adult hospitalized COVID-19 patients analyzed the association between disease severity and initial serum iron, total iron-binding capacity (TIBC), and transferrin saturation (TSAT) levels. Serum iron levels were significantly lower in patients with mild RF than in the non-RF group; however, there were no significant differences in iron levels between the non-RF and severe RF groups, depicting a U-shaped association between serum iron levels and disease severity. TIBC levels decreased significantly with increasing severity; consequently, TSAT was significantly higher in patients with severe RF than in other patients. Multivariate analysis including only patients with RF adjusted for age and sex demonstrated that higher serum iron and TSAT levels were independently associated with the development of severe RF, indicating that inadequate response to lower serum iron might be an exacerbating factor for COVID-19.

Effects of intraoperative tidal volume on incidence of acute kidney injury after cardiovascular surgery: A retrospective cohort study.

PURPOSE: We performed a retrospective cohort study to evaluate whether intraoperative low tidal volume ventilation reduces the incidence of acute kidney injury (AKI) after cardiovascular surgery. MATERIALS AND METHODS: Records of patients who underwent cardiovascular surgery were reviewed. The primary outcome was AKI diagnosed by changes in serum creatinine values. Intraoperative mean tidal volume relative to predicted body weight (PBW) was calculated. The effects of intraoperative mean tidal volumes on AKI incidence were evaluated. RESULTS: Of 338 examined patients, 105 developed AKI. Among patients who received mean tidal volumes of ≤7, >7 to ≤8, >8 to ≤9, and > 9 mL/kg PBW, the AKI incidence was 12.8% (95% confidence interval [CI]: 6.0-25.1%), 29.9% (95% CI: 22.6-38.4%), 38.7% (95% CI: 30.0-48.2), and 34.5% (95% CI: 23.6-47.3%), respectively. Inverse probability of treatment weighting analysis demonstrated that AKI risk was significantly lower in patients who received ≤7 mL/kg PBW than in those who received >7 mL/kg PBW (odds ratio: 0.14, 95% CI: 0.04-0.46, p = .001). CONCLUSIONS: This study suggests that intraoperative low tidal volume ventilation during cardiovascular surgery is associated with a decreased incidence of postoperative AKI. Lowering tidal volume might be a simple strategy for reducing AKI incidence after cardiovascular surgery.

Necrosis Rather Than Apoptosis is the Dominant form of Alveolar Epithelial Cell Death in Lipopolysaccharide-Induced Experimental Acute Respiratory Distress Syndrome Model.

Alveolar epithelial cell (AEC) death, which is classified as apoptosis or necrosis, plays a critical role in the pathogenesis of acute respiratory distress syndrome (ARDS). In addition to apoptosis, some types of necrosis are known to be molecularly regulated, and both apoptosis and necrosis can be therapeutic targets for diseases. However, the relative contribution of apoptosis and necrosis to AEC death during ARDS has not been elucidated. Here, we evaluated which type of AEC death is dominant and whether regulated necrosis is involved in lipopolysaccharide (LPS)-induced lung injury, an experimental ARDS model. In the bronchoalveolar lavage fluid from the LPS-induced lung injury mice, both the levels of cytokeratin 18-M65 antigen (a marker of total epithelial cell death) and cytokeratin 18-M30 antigen (an epithelial apoptosis marker) were increased. The M30/M65 ratio, which is an indicator of the proportion of apoptosis to total epithelial cell death, was significantly lower than that in healthy controls. In addition, the number of propidium iodide-positive, membrane-disrupted cells was significantly higher than the number of TUNEL-positive apoptotic cells in the lung sections of lung injury mice. Activated neutrophils seemed to mediate AEC death. Finally, we demonstrated that necroptosis, a regulated necrosis pathway, is involved in AEC death during LPS-induced lung injury. These results indicate that necrosis including necroptosis, rather than apoptosis, is the dominant type of AEC death in LPS-induced lung injury. Although further studies investigating human ARDS subjects are necessary, targeting necrosis including its regulated forms might represent a more efficient approach to protecting the alveolar epithelial barrier during ARDS.

Driving-pressure-independent protective effects of open lung approach against experimental acute respiratory distress syndrome.

BACKGROUND: The open lung approach (OLA) reportedly has lung-protective effects against acute respiratory distress syndrome (ARDS). Recently, lowering of the driving pressure (ΔP), rather than improvement in lung aeration per se, has come to be considered as the primary lung-protective mechanism of OLA. However, the driving pressure-independent protective effects of OLA have never been evaluated in experimental studies. We here evaluated whether OLA shows protective effects against experimental ARDS even when the ΔP is not lowered. METHODS: Lipopolysaccharide was intratracheally administered to rats to establish experimental ARDS. After 24 h, rats were mechanically ventilated and randomly allocated to the OLA or control group. In the OLA group, 5 cmH2O positive end-expiratory pressure (PEEP) and recruitment maneuver (RM) were applied. Neither PEEP nor RM was applied to the rats in the control group. Dynamic ΔP was kept at 15 cmH2O in both groups. After 6 h of mechanical ventilation, rats in both groups received RM to inflate reversible atelectasis of the lungs. Arterial blood gas analysis, lung computed tomography, histological evaluation, and comprehensive biochemical analysis were performed. RESULTS: OLA significantly improved lung aeration, arterial oxygenation, and gas exchange. Even after RM in both groups, the differences in these parameters between the two groups persisted, indicating that the atelectasis-induced respiratory dysfunction observed in the control group is not an easily reversible functional problem. Lung histological damage was severe in the dorsal dependent area in both groups, but was attenuated by OLA. White blood cell counts, protein concentrations, and tissue injury markers in the broncho-alveolar lavage fluid (BALF) were higher in the control than in the OLA group. Furthermore, levels of CXCL-7, a platelet-derived chemokine, were higher in the BALF from the control group, indicating that OLA protects the lungs by suppressing platelet activation. CONCLUSIONS: OLA shows protective effects against experimental ARDS, even when the ΔP is not decreased. In addition to reducing ΔP, maintaining lung aeration seems to be important for lung protection in ARDS.

Comparison between High- and Low-Cost Transmission of Tele-Anesthesia in Japan.

Background: We previously reported a tele-anesthesia system that connected Sado General Hospital (SGH) to Yokohama City University Hospital (YCUH) using a dedicated virtual private network (VPN) that guaranteed the quality of service. The study indicated certain unresolved problems, such as the high cost of constantly using a dedicated VPN for tele-anesthesia. In this study, we assessed whether use of a best-effort system affects the safety and cost of tele-anesthesia in a clinical setting. Methods: One hundred patients were enrolled in this study. We provided tele-anesthesia for 65 patients using a guaranteed transmission system (20 Mbit/s; guaranteed, 372,000 JPY per month: 1 JPY = US$0.01) and for 35 patients using a best-effort system (100 Mbit/s; not guaranteed, 25,000 JPY per month). We measured transmission speed and number of commands completed from YCUH to SGH during tele-anesthesia with both transmission systems. Results: In the guaranteed system, anesthesia duration was 5780 min (88.9 min/case) and surgical duration was 3513 min (54.0 min/case). In the best-effort system, anesthesia duration was 3725 min (106.4 min/case) and surgical duration was 2105 min (60.1 min/case). The average transmission speed in the best-effort system was 17.3 ± 3.8 Mbit/s. The system provided an acceptable delay time and frame rate in clinical use. All commands were completed, and no adverse events occurred with both systems. Discussion: In the field of tele-anesthesia, using a best-effort internet VPN system provided equivalent safety and efficacy at a better price as compared to using a guaranteed internet VPN system.

Enhancement of glycolysis by inhibition of oxygen-sensing prolyl hydroxylases protects alveolar epithelial cells from acute lung injury.

Cellular bioenergetic failure caused by mitochondrial dysfunction is a key process of alveolar epithelial injury during acute respiratory distress syndrome (ARDS). Prolyl hydroxylases (PHDs) act as cellular oxygen sensors, and their inhibition activates hypoxia-inducible factor (HIF), resulting in enhanced cellular glycolytic activity, which could compensate for impaired mitochondrial function and protect alveolar epithelial cells from ARDS. Here, we evaluated the effects of pharmacological PHD inhibition with dimethyloxalylglycine (DMOG) on alveolar epithelial cell injury using in vitro and in vivo ARDS models. We established an in vitro model of alveolar epithelial injury mimicking ARDS by adding isolated neutrophils and LPS to cultured MLE12 alveolar epithelial cells. DMOG treatment protected MLE12 cells from neutrophil-LPS-induced ATP decline and cell death. Knockdown of HIF-1α or inhibition of glycolysis abolished the protective effect of DMOG, suggesting that it was exerted by HIF-1-dependent enhancement of glycolysis. Additionally, intratracheal DMOG administration to mice protected the alveolar epithelial barrier and improved arterial oxygenation, preventing ATP decline during LPS-induced lung injury. In summary, enhancement of glycolysis by PHD inhibition is a potential therapeutic approach for ARDS, protecting alveolar epithelial cells from bioenergetic failure and cell death.- Tojo, K., Tamada, N., Nagamine, Y., Yazawa, T., Ota, S., Goto, T. Enhancement of glycolysis by inhibition of oxygen-sensing prolyl hydroxylases protects alveolar epithelial cells from acute lung injury. FASEB J. 32, 2258-2268 (2018). www.fasebj.org.

Inhibition of Prolyl Hydroxylase Attenuates Fas Ligand-Induced Apoptosis and Lung Injury in Mice.

Alveolar epithelial injury and increased alveolar permeability are hallmarks of acute respiratory distress syndrome. Apoptosis of lung epithelial cells via the Fas/Fas ligand (FasL) pathway plays a critical role in alveolar epithelial injury. Activation of hypoxia-inducible factor (HIF)-1 by inhibition of prolyl hydroxylase domain proteins (PHDs) is a possible therapeutic approach to attenuate apoptosis and organ injury. Here, we investigated whether treatment with dimethyloxalylglycine (DMOG), an inhibitor of PHDs, could attenuate Fas/FasL-dependent apoptosis in lung epithelial cells and lung injury. DMOG increased HIF-1α protein expression in vitro in MLE-12 cells, a murine alveolar epithelial cell line. Treatment of MLE-12 cells with DMOG significantly suppressed cell surface expression of Fas and attenuated FasL-induced caspase-3 activation and apoptotic cell death. Inhibition of the HIF-1 pathway by echinomycin or small interfering RNA transfection abolished these antiapoptotic effects of DMOG. Moreover, intraperitoneal injection of DMOG in mice increased HIF-1α expression and decreased Fas expression in lung tissues. DMOG treatment significantly attenuated caspase-3 activation, apoptotic cell death in lung tissue, and the increase in alveolar permeability in mice instilled intratracheally with FasL. In addition, inflammatory responses and histopathological changes were also significantly attenuated by DMOG treatment. In conclusion, inhibition of PHDs protects lung epithelial cells from Fas/FasL-dependent apoptosis through HIF-1 activation and attenuates lung injury in mice.

Protective effects of continuous positive airway pressure on a nonventilated lung during one-lung ventilation: A prospective laboratory study in rats.

BACKGROUND: The use of one-lung ventilation (OLV) to facilitate intrathoracic surgery is a cause of lung injury. OBJECTIVE: We hypothesised that application of continuous positive airway pressure (CPAP) to a nonventilated lung during OLV would prevent alveolar hypoxia and blood flow shift from the nonventilated to the ventilated lung, thereby attenuating lung injury. DESIGN: Controlled animal study. SETTINGS: University laboratory. STUDY PARTICIPANTS: Adult male Sprague-Dawley rats (n = 4 to 8 per group, depending on experiments). INTERVENTIONS: Rats were alternately assigned to one of two ventilation protocol groups: control and CPAP groups. Rats received 240 min of OLV followed by 240 min of two-lung reventilation (re-TLV). The nonventilated lungs of rats in the control group were collapsed during OLV whereas rats in the CPAP group received CPAP (5 cmH2O with 100% oxygen) to the nonventilated lungs. MAIN OUTCOME MEASURES: Pulmonary blood flow during OLV was measured by quantification of lung radioactivity after intravenous infusion of indium-labelled macroaggregated albumin. Inflammatory cytokines in the lungs after 240 min of OLV, and after the subsequent 240 min of re-TLV were measured. Additionally, we measured lung wet-to-dry weight ratios after re-TLV. We also measured lung malondialdehyde levels after re-TLV as an indicator of reactive oxygen species produced by reoxygenation. RESULTS: Application of CPAP attenuated the pulmonary blood flow shift from the nonventilated to the ventilated lung. CPAP decreased the levels of IL-6, CXC chemokine ligand-1 and CC chemokine ligand-2 in both lungs after 240 min of OLV. CPAP also decreased CXC chemokine ligand-1 in the nonventilated lung and CC chemokine ligand-2 in both lungs after re-TLV. Moreover, wet-to-dry weight ratios of both lungs were decreased by application of CPAP. However, lung malondialdehyde concentrations were not affected by CPAP. CONCLUSIONS: CPAP applied to the nonventilated lung during OLV suppresses blood flow shift and decreases inflammatory cytokines and water content in both lungs. Application of CPAP may attenuate lung injury during and after OLV.

Adrenaline aggravates lung injury caused by liver ischemia-reperfusion and high-tidal-volume ventilation in rats.

BACKGROUND: We often administer adrenaline to improve hypotension of patients undergoing systemic inflammation that is not treated with volume resuscitation. The effects of adrenaline on injured lungs during shock status have not been elucidated. We previously demonstrated that hepatic ischemia-reperfusion followed by high-tidal-volume ventilation-induced systemic inflammation, hypotension, and lung injury in rats. Using this animal model, we investigated the effects of adrenaline on lung injury and hemodynamics. METHODS: Anesthetized rats were ventilated and underwent hepatic inflow interruption for 15 min twice. After the second liver ischemia-reperfusion, the tidal volume was increased to 24 ml · kg(-1) body weight from 6 ml · kg(-1), and 12 rats in each group were observed for 360 min after reperfusion with or without continuous intravenous adrenaline administration. Extra fluid was administered according to the decline in the arterial blood pressure. RESULTS: Adrenaline administration significantly reduced the volume of intravenous resuscitation fluid. The wet-to-dry weight ratio of the lungs was higher (7.53 ± 0.37 vs. 4.63 ± 0.35, P < 0.001), the partial oxygen pressure in arterial blood was lower (213 ± 48 vs. 411 ± 33, P = 0.004), and the tumor necrosis factor-α concentration in bronchoalveolar lavage (BAL) fluid was higher (10(2.64) ± 10(0.22) vs. 10(1.91) ± 10(0.27), P = 0.015), with adrenaline. Histopathological examinations revealed marked exudation in the alveolar spaces in rats receiving adrenaline. CONCLUSIONS: Continuous administration of adrenaline partially prevented a rapid decline in blood pressure but deteriorated lung injury in a rat model of liver ischemia-reperfusion with high-tidal-volume ventilation. A possibility that adrenaline administration aggravate ventilator-induced lung injury during systemic inflammation should be considered.

Atelectasis causes alveolar hypoxia-induced inflammation during uneven mechanical ventilation in rats.

BACKGROUND: Patients with acute respiratory distress syndrome receiving mechanical ventilation show inhomogeneous lung aeration. Atelectasis during uneven mechanical ventilation leads to alveolar hypoxia and could therefore result in lung inflammation and injury. We aimed to elucidate whether and how atelectasis causes alveolar hypoxia-induced inflammation during uneven mechanical ventilation in an open-chest differential-ventilation rat model. METHODS: We first investigated inflammatory and histological changes in the bilateral lungs of unilaterally ventilated rats, in which the right lung was atelectatic and the left lung was ventilated with high tidal volume (HTV). In the next series, we investigated the effects of normal tidal volume (NTV) ventilation of the right lungs with 60 % O2 or 100 % N2 during HTV ventilation of the left lungs. Then, proinflammatory cytokine secretions were quantified from murine lung epithelial (MLE15) and murine alveolar macrophage (MH-S) cells cultured under a hypoxic condition (5 % O2) mimicking atelectasis. Further, activities of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)-1 were assessed in the nonventilated atelectatic lung and MLE15 cells cultured under the hypoxic condition. Finally, effects of NF-κB inhibition and HIF-1α knockdown on the cytokine secretions from MLE15 cells cultured under the hypoxic condition were assessed. RESULTS: The nonventilated atelectatic lungs showed inflammatory responses and minimal histological changes comparable to those of the HTV-ventilated lungs. NTV ventilation with 60 % O2 attenuated the increase in chemokine (C-X-C motif) ligand (CXCL)-1 secretion and neutrophil accumulation observed in the atelectatic lungs, but that with 100 % N2 did not. MLE15 cells cultured with tumor necrosis factor (TNF)-α under the hypoxic condition showed increased CXCL-1 secretion. NF-κB and HIF-1α were activated in the nonventilated atelectatic lungs and MLE15 cells cultured under the hypoxic condition. NF-κB inhibition abolished the hypoxia-induced increase in CXCL-1 secretion from MLE15 cells, while HIF-1α knockdown augmented it. CONCLUSIONS: Atelectasis causes alveolar hypoxia-induced inflammatory responses including NF-κB-dependent CXCL-1 secretion from lung epithelial cells. HIF-1 activation in lung epithelial cells is an anti-inflammatory response to alveolar hypoxia in atelectatic lungs.

A pilot study of tele-anaesthesia by virtual private network between an island hospital and a mainland hospital in Japan.

We studied the use of tele-anaesthesia between Sado General Hospital (SGH) located on Sado Island and Yokohama City University Hospital (YCUH) located in mainland Japan. The two sites were connected via a virtual private network (VPN). We investigated the relationship between the bandwidth of the VPN and both the frame rate and the delay time of the tele-anaesthesia monitoring system. The tool used for communication between the two hospitals was free videoconferencing software (FaceTime), which can be used over Wi-Fi connections. We also investigated the accuracy of the commands given during teleanaesthesia: any commands from the anaesthetist at the YCUH that were not carried out for any reason, were recorded in the anaesthetic records at the SGH. The original frame rate and data rate at the SGH were 5 fps and approximately 18 Mbit/s, respectively. The frame rate at the transmission speeds of 1, 5 and 20 Mbit/s was 0.6, 1.6 and 5.0 fps, respectively. The corresponding delay time was 12.2, 4.9 and 0.7 s. Twenty-five adult patients were enrolled in the study and tele-anaesthesia was performed. The total duration of anaesthesia was 37 hours. All 888 anaesthetic commands were completed. There were 7 FaceTime disconnections, which lasted for 10 min altogether. Because no commands needed to be given during the FaceTime disconnection, the telephone was not used. The anaesthesia assistance system might form part of the solution to medical resource shortages.

Edaravone prevents lung injury induced by hepatic ischemia-reperfusion.

BACKGROUND: Lung injury is a major clinical concern after hepatic ischemia-reperfusion (I/R), due to the production of reactive oxygen species in the reperfused liver. We investigated the efficacy of edaravone, a potent free-radical scavenger, for attenuating lung injury after hepatic I/R. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were assigned to sham + normal saline (NS), I/R + NS, or I/R + edaravone group. Rats in the I/R groups were subjected to 90 min of partial hepatic I/R. Five minutes before reperfusion, 3 mg/kg edaravone was administered to the I/R + edaravone group. After 6 h of reperfusion, we evaluated lung histopathology and wet-to-dry ratio. We also measured malondialdehyde (MDA), an indicator of oxidative stress, in the liver and the lung, as well as cytokine messenger RNA expressions in the reperfused liver and plasma cytokine concentrations. RESULTS: Histopathology revealed lung damages after 6 h reperfusion of partial ischemic liver. Moreover, a significant increase in lung wet-to-dry ratio was observed. MDA concentration increased in the reperfused liver, but not in the lungs. Edaravone administration attenuated the lung injury and the increase of MDA in the reperfused liver. Edaravone also suppressed the reperfusion-induced increase of interleukin-6 messenger RNA expressions in the liver and plasma interleukin-6 concentrations. CONCLUSIONS: Edaravone administration before reperfusion of the ischemic liver attenuates oxidative stress in the reperfused liver and the subsequent lung injury. Edaravone may be beneficial for preventing lung injury induced by hepatic I/R.

Role of nerve growth factor-tyrosine kinase receptor A signaling in paclitaxel-induced peripheral neuropathy in rats.

The mechanisms underlying paclitaxel-induced peripheral neuropathy remain unknown. Nerve growth factor (NGF) is a representative neurotrophic factor that maintains neuronal function, promotes survival, and mediates neuropathic pain. We investigated expression levels of NGF and its receptors in the dorsal root ganglia (DRG) and spinal dorsal horn (DH) following paclitaxel treatment. Intraperitoneal (I.P.) administration of paclitaxel induced significant mechanical hypersensitivity and cold allodynia in rats, significantly increased the expression of NGF and its receptor tyrosine kinase receptor A (trkA) in the DRG, and increased NGF expression in the DH. In contrast, paclitaxel treatment did not alter the mRNA levels of NGF or its receptors in the DRG, DH, sciatic nerve, or hindpaw skin. Moreover, expression of NEDD4-2, a negative regulator of trkA, was significantly increased in the DRG of paclitaxel-treated rats. Intrathecal (I.T.) administration of the tyrosine kinase receptor inhibitor k252a significantly alleviated mechanical hypersensitivity in paclitaxel-treated rats. Our results suggest that NGF-trkA signaling is involved in mechanical allodynia in paclitaxel-induced neuropathy.

Reevaluation of the effectiveness of ramosetron for preventing postoperative nausea and vomiting: a systematic review and meta-analysis.

BACKGROUND: Ramosetron has been shown to have a very strong effect for preventing postoperative nausea and vomiting (PONV) in previous meta-analyses. However, these previous meta-analyses included a number of studies by Fujii et al. which have now been proven to have been fabricated. In the present meta-analysis, we reevaluated the effectiveness of ramosetron in preventing PONV after excluding Fujii et al.'s randomized controlled trials. METHODS: We searched MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), Embase, and Web of Science. All double-blind randomized controlled trials that tested the efficacy of ramosetron compared with a placebo or other drugs as a control in the prophylaxis of PONV were considered to be eligible. The first postoperative 24 hours were divided into early (0-6 hours) and late (6-24 hours) time periods, and we collected these data separately. RESULTS: A total of 1372 patients were included in the final analysis. Compared with a placebo, ramosetron reduced the incidence of early postoperative nausea (PON) (relative risk [RR] [95% confidence interval] 0.59 [0.47-0.73]: number needed to treat [NNT] [95% confidence interval] 6.0 [4.3-9.7]), late PON (RR 0.65 [0.49-0.85]: NNT 7.2 [4.6-16.6]), early postoperative vomiting (POV) (RR 0.48 [0.31-0.74]: NNT 14.8 [8.3-70.4]), and late POV (RR 0.50 [0.35-0.73]: NNT 12.3 [7.1-47.6]). Compared with ondansetron, ramosetron reduces early POV (RR 0.50 [0.28-0.90]: NNT 24.1 [10.7-98.0]) and late POV (RR 0.53 [0.34-0.81]: NNT 27.2 [12.0-102.0]) but not PON. CONCLUSIONS: Ramosetron has a significant effect for preventing PONV compared with a placebo, but less than that reported in previous analyses. Ramosetron also has statistically significant differences in preventing early and late POV compared with ondansetron, but the clinical significance may be questioned because the NNTs are large.