Sedation overuse is frequent and possibly associated with poor outcomes in the intensive care unit (ICU) patients. However, the association of early oversedation with clinical outcomes has not been thoroughly evaluated. The aim of this study was to assess the association of early sedation strategies with outcomes of critically ill adult patients under mechanical ventilation (MV).A secondary analysis of a multicenter prospective cohort conducted in 45 Brazilian ICUs, including adult patients requiring ventilatory support and sedation in the first 48 hours of ICU admissions, was performed. Sedation depth was evaluated after 48 hours of MV. Multivariate analysis was used to identify variables associated with hospital mortality.P = 0.041) and more tracheostomies were performed in the deep sedation group  despite similar PaO2/FiO2 ratios and acute respiratory distress syndrome (ARDS) severity. In a multivariate analysis, age (Odds Ratio (OR) 1.02; 95% confidence interval (CI) 1.00 to 1.03), Charlson Comorbidity Index >2 , Simplified Acute Physiology Score 3 (SAPS 3) score , severe ARDS  and deep sedation  were independently associated with increased hospital mortality.A total of 322 patients were evaluated. Overall, ICU and hospital mortality rates were 30.4% and 38.8%, respectively. Deep sedation was observed in 113 patients (35.1%). Longer duration of ventilatory support was observed (7 (4 to 10) versus 5 (3 to 9) days, Early deep sedation is associated with adverse outcomes and constitutes an independent predictor of hospital mortality in mechanically ventilated patients. Sedation is an important component of care for patients under mechanical ventilation (MV) in the ICU. Significant distress is related to MV itself or to routine procedural interventions  and miniDespite the current recommendations , there iThe aim of this study is thus to describe the association of early sedation strategies (sedation depth and sedative choice) with clinical outcomes of mechanically ventilated adult ICU patients, with hospital mortality as the primary outcome.This study was a secondary analysis of a multicenter prospective cohort conducted in 45 Brazilian ICUs (from the Brazilian Research in Intensive Care Network) from 12 states between 1 June 2011 and 31 July 2011 . AdditioWe excluded patients submitted exclusively to noninvasive MV and those presenting with primary neurological disorders. We also excluded those with missing data regarding sedation depth on the second day of MV. Demographic, clinical and laboratory data were collected during the ICU stay and included cause of respiratory failure, chronic health status, Charlson Comorbidity Index , Simplifr = 0.91, P <0.001) between the GCS and RASS, where GCS = 9 would be equivalent to RASS = -2 [As the primary study was not designed to evaluate sedation strategies , the GlaASS = -2 .Our primary hypothesis was that early sedation strategies, namely sedation depth, could be associated with clinical outcomes including hospital mortality, so we chose to evaluate the sedation level on the second day of MV.The study was approved by the institutional review board of Hospital Sírio-Libanês at the coordinating center  and subsequently by local review boards. Additional file P <0.2 by univariate analysis, or those considered clinically relevant despite P values, were entered into the multivariate analysis to estimate the independent association of each covariate with the dependent variable. Results are presented as the odds ratio with 95% confidence interval. A Kaplan–Meier curve with log-rank test was used to compare patients with light versus deep sedation for hospital mortality (censored at day 30). Two-tailed P <0.05 was considered statistically significant. All statistical tests were carried out using the commercial SPSS19.0 package for Windows .Continuous variables are reported as the median (25 to 75% interquartile range). Univariate and multivariate analyses using a binary logistic regression were used to identify factors associated with the dependent variable . Variables yielding n = 51) were comparable with the included population regarding demographic data, comorbidities, physiologic and disease severity variables and outcomes (data not shown). The main patient characteristics are presented in Table P <0.001), higher SAPS3 and SOFA score (68 (57 to 79) vs. 57 (48 to 67), P < 0.001 and 10 (7 to 12) vs. 8 (5 to 10), P <0.001) and higher percentage of patients with a Charlson Comorbidity Index >2 . Those who died during hospital admission also had a higher frequency of moderate/severe ARDS , need for vasopressors  and renal replacement therapy .From 773 patients derived from the original cohort, a total of 322 patients fulfilled the eligibility criteria Figure . PatientP = 0.033; 8.6% vs. 0.9% for dexmedetomidine, P = 0.005).The most frequently used sedatives were the association of midazolam and fentanyl (39.4%) or of propofol and fentanyl (14%) and the use of fentanyl (12.4%) or midazolam (8.1%) as single drugs. Sedative drug type was also associated with sedation levels: the sole administration of fentanyl and dexmedetomidine was more frequent in lightly sedated patients  (Table P = 0.041) and more tracheostomies were performed in the deep sedation group  despite similar arterial partial pressure of oxygen/fraction of inspired oxygen ratios and ARDS severity. Finally, trends to increased ICU and hospital mortality  were also associated with deep sedation (Table Deep sedation was observed in 113 patients (35.1%) and was associated with a higher overall disease severity, as demonstrated by higher median SAPS 3 (65 (54 to 78) for deep sedation vs. 59 (48 to 70) for light sedation, 1) Table . MoreoveIn a multivariate analysis, age, Charlson Comorbidity Index >2, SAPS3, severe ARDS and deep sedation were independently associated with increased hospital mortality Table .In the present multicenter cohort study we evaluated the association of early sedation strategies with outcomes of adult mechanically ventilated patients in the ICU. Deep sedation in the first 48 hours of MV is a common characteristic, observed in 35.1% of patients. Deep sedation was associated with increased disease severity, longer duration of ventilatory support and higher need for tracheostomy. In a multivariate analysis, deep sedation was an independent factor associated with increased hospital mortality (odds ratio = 2.36).P = 0.033; 8.6% vs. 0.9% for dexmedetomidine, P = 0.005). These findings may be attributed to a rapid onset and offset of action of these drugs and their easiness to titrate, while benzodiazepines are more likely to accumulate, especially during continuous infusion and when associated with other drugs [The more frequently used sedative regimens in our study were the association of midazolam and fentanyl or of propofol and fentanyl. When compared by sedation depth, the administration of fentanyl or dexmedetomidine as single drugs was more frequent in lightly sedated patients  who were sedated and ventilated ≥24 hours [n = 322), and demonstrated that early deep sedation was an independent predictor of hospital mortality.Although important studies were published recently, a systematic review published in 2010 showed that clinical trials on sedation are commonly reported as low quality, mainly due to their design , heterogeneous protocols and the relatively small number of patients in a single center or few centers . These f24 hours . LikewisUnlike the other variables that were also found to be independently associated with mortality in our study, such as ARDS severity, comorbidities and disease severity, sedation depth is a potentially modifiable risk factor for mortality. Implementing sedation protocols to achieve light sedation has been proven feasible and reproducible ,33. ThisFinally, our findings provide validation of the concept that early sedation has a major impact on outcomes. Demonstrating this in a middle-income country in South America is an important step to provide generalizability of previous findings because the main previously published results on this issue are from ANZ ICUs. Several ANZ trials are well known for frequently presenting different results compared with similar interventions in other parts of the world, as demonstrated by studies in the last decade in the critical care field ,36 and er = 0.91, P < 0.001), as described by Ely and colleagues from 1,360 paired observations among 275 adult patients in medical and coronary ICUs [The present study has some limitations that must be considered. First, the sedation level was not assessed using a specific sedation scale. Nonetheless, although originally created to assess the level of consciousness after head injury, it is known that GCS  <9 denotes a comatose state even in nontraumatic settings, such as cerebral depression by pharmacological cause ,41. Moreary ICUs . This stary ICUs ,43, whicAnother limitation is the potential influence of previous neurological status on GCS values. Data regarding GCS on day 1 were collected in patients who were already sedated and under MV. A reported low GCS could therefore be influenced by the baseline neurological status and not only by sedation strategies, because it may also reflect acute neurological conditions associated with the current illness presented by the critically ill, such as encephalopathy. However, this is not an exclusive limitation of the GCS because this may also occur with the more specific sedation assessment scales such as the RASS or Sedation-Agitation Scale (SAS). Moreover, it is possible that certain patients may have required larger sedative doses on day 2 of MV for other reasons  and we did not collect these data. In order to minimize the impact of other conditions in GCS values, we excluded patients with primary or known acute neurological disorders and included only patients under sedation in the present study. Considering the potential confounders mentioned above, the strong association between deep sedation and mortality must be analyzed regarding our interpretation of GCS values mainly as a surrogate of sedation depth.Finally, we have not evaluated the presence of delirium. In mechanically ventilated patients, delirium is an independent predictor of hospital and 6-month mortality . These oEarly deep sedation is associated with worse outcomes and constitutes an independent predictor of hospital mortality in a prospective cohort of mechanically ventilated patients. Randomized clinical trials should be designed to address the impact of light versus deep sedation in the early phase (<48 hours) of MV.• Early deep sedation is associated with longer duration of ventilator support and higher need for tracheostomy in mechanically ventilated patients.• Early sedation depth is independently associated with increased hospital mortality.• Sedation depth is a potentially modifiable risk factor for adverse outcomes; future clinical trials of light sedation with early (<24 hours) randomization of patients should therefore be encouraged.ANZ: Australia and New Zealand; ARDS: acute respiratory distress syndrome; GCS: Glasgow Coma Scale; MV: mechanical ventilation; RASS: Richmond Agitation–Sedation Scale; SAPS3: Simplified Acute Physiology Score 3; SOFA: Sequential Organ Failure Assessment.This study was performed with institutional funds. JIFS received honoraria and unrestricted research grants from Hospira, Inc. São Paulo, Brazil, but this did not interfere with the design, analysis or the elaboration of the manuscript and therefore with adherence to the ethical requirements of the journal. The remaining authors declare that they have no competing interests.LMST participated in study conception, data acquisition, data analysis and interpretation, and drafting of the manuscript. LCPA participated in study conception, data acquisition, data analysis and interpretation, and drafting of the manuscript. MP participated in study conception, data analysis and revising the manuscript for important intellectual content. GS participated in study conception, data acquisition, data analysis and drafting of the manuscript. APNJr participated in data acquisition, drafting of the manuscript and revising the manuscript for important intellectual content. AR-N participated in data acquisition and revising the manuscript for important intellectual content. LT participated in data acquisition and revising the manuscript for important intellectual content. VCdS-D participated in data acquisition and revising the manuscript for important intellectual content. AT participated in data acquisition and revising the manuscript for important intellectual content. TL participated in data acquisition and revising the manuscript for important intellectual content. CP participated in data acquisition and revising the manuscript for important intellectual content. FBC participated in data acquisition and revising the manuscript for important intellectual content. MdOM participated in data acquisition and revising the manuscript for important intellectual content. FPG participated in data acquisition and revising the manuscript for important intellectual content. FRM participated in data acquisition and revising the manuscript for important intellectual content. FD-P participated in data acquisition and revising the manuscript for important intellectual content. AGRdC participated in data acquisition and revising the manuscript for important intellectual content. RBdS participated in data acquisition and revising the manuscript for important intellectual content. PFGMMT participated in data acquisition and revising the manuscript for important intellectual content. MS participated in study conception, data analysis and interpretation, and drafting of the manuscript. JIFS participated in study conception, data analysis and interpretation, and drafting of the manuscript. All authors approved the final copy of the manuscript.Is a table listing the sites, investigators and institutional review boards that participated in the Epidemiology of Respiratory Insufficiency in Critical Care study.Click here for file


Neoadjuvant imatinib for gastrointestinal stromal tumours (GISTs) is increasingly used nowadays. As oesophagectomy is associated with high morbidity and mortality, a preoperative downsizing of an oesophageal GIST to limit the extent of resection would be ideal. Because these tumours are rare and neoadjuvant treatment with imatinib is recent, there is limited literature available regarding neoadjuvant administration of imatinib in patients with oesophageal GISTs. A 50-year-old woman presented with total dysphagia. An upper endoscopy and biopsy revealed a large submucosal KIT-positive GIST obstructing the mid oesophagus. CT confirmed a lesion measuring 99 mm × 50 mm × 104 mm. Because the size and location of the tumour increased the risk of intraoperative rupture, it was decided to administer preoperative imatinib. The patient had an excellent clinical and radiological response. Her dysphagia gradually resolved and the follow-up CT scans of the first 10 months showed a gradually reducing tumour size to 54 mm × 33 mm × 42 mm. The patient underwent an uneventful laparoscopic-assisted Ivor-Lewis oesophagectomy. Postoperatively, the patient continued with adjuvant imatinib. At the last follow-up, 1 year from operation and 38 months from the diagnosis, the patient is disease free. Gastrointestinal Stromal Tumours (GISTs) are mesenchymal neoplasms located primarily in the gastrointestinal (GI) tract and represent 80% of all GI sarcomas . SurgeryA benchmark in dealing with these chemotherapy-insensitive tumours was the introduction of treatment with imatinib and later of sunitinib, two tyrosine-kinase inhibitors , 4. The Although 80% of newly diagnosed cases are resectable at presentation, the high efficacy of imatinib in the metastatic and adjuvant setting quickly made way for its usage also in the neoadjuvant setting –6.The main indications for preoperative imatinib are (1) unresectable or metastatic disease with an aim of tumour downsizing and conversion of patient to candidate for radical resection and 2) resectable metastatic or nonmetastatic disease with an aim of downsizing of disease as well, thus reducing the extent of surgery and postoperative morbidity and mortality . In this second group of patients, with resectable disease at presentation, preoperative administration of imatinib also aims to improve the oncological outcome by reducing the likelihood of intraoperative tumour rupture, which has been identified as being an independent predictor of disease recurrence and is currently included in systems assessing the likelihood of postoperative relapse  resectab.Oesophageal GISTs are very rare, representing less than 2% of GISTs . BecauseWe report a case of large oesophageal GIST in a 50-year-old woman who underwent laparoscopic-assisted Ivor-Lewis oesophagectomy after 26 months of neoadjuvant imatinib therapy.A 50-year-old lady presented to our hospital with a 4-month history of progressive dysphagia. Her symptoms worsened progressively resulting in total dysphagia, pain mainly in the epigastric and lower chest area radiating to her back, and one recent episode of haematemesis. At presentation, she was unable to swallow even her saliva. She had no associated medical comorbidities, except for being an ex-smoker and having a BMI of 41. The physical examination was unremarkable. Standard laboratory test results were within normal range, with haemoglobin of 11 mg/dL. An oesophagogastroscopy revealed a large submucosal obstructing oesophageal mass, partially ulcerated, situated 42 mm above diaphragmatic hiatus, and extending over a distance of at least 10 cm. Biopsy revealed a GIST with diffuse positivity for CD117 and CD34 and a mitotic index of 3/22 hpf . Gene analysis with direct sequencing revealed a 6 bp deletion in exon 11 of the KIT gene [mutation details: c.1670_1675del p.Trp557_Val559delinsPhe (reference cDNA sequence: NM_000222.2)].A computed tomography (CT) scan of chest and abdomen revealed an annular polypoidal mass, 99 mm × 50 mm × 104 mm in diameter in the mid oesophagus, arising 42 mm above the diaphragmatic hiatus and extending cranially over a distance of 10 cm to the subcarinal level . There wThe patient was admitted to our hospital for pain and symptoms of total dysphagia and an 8F nasogastric tube was inserted endoscopically.Her case was further discussed in the sarcoma Multidisciplinary Team (MDT) meeting. Despite the tumour being resectable at presentation, its size and location rendered it to an increased risk of intraoperative rupture. Furthermore, GIST with mutations in exon 11 of KIT is generally associated with sensitivity to imatinib. Therefore, the MDT decided on preoperative administration of imatinib 400 mg daily.She had an excellent clinical response with dysphagia gradually improving, and 12 days later, she was able to tolerate solid food once again.The first follow-up CT scan two months after initiating treatment showed an excellent partial response, with the mass measuring 65 mm × 42 mm × 51 mm and reduction of tumour mass to 27% of the initial volume. New CT three months after showed further response, with mass reduction to 53 mm × 33 mm × 42 mm  revealing an anaerobic threshold of 9.3, the MDT decided the patient should continue treatment with imatinib and further take up an exercise program in an attempt to reduce her risks of perioperative complications.Unfortunately, the next CT scan after 13 months of treatment demonstrated localized but progressive disease (90 mm × 42 mm × 60 mm)  classified the tumour as “high risk” and the decision was taken for further adjuvant imatinib, according to the Scandinavian Sarcoma Group Trial [/50 hpf cAt the last follow-up, 1 year after operation and 38 months from the diagnosis, the patient remained disease free.We present a patient with a large oesophageal GIST who underwent radical surgical resection following neoadjuvant administration of imatinib for 26 months. Despite the fact that the plateau response to imatinib was reached 10 months after initiating treatment, the combination of high operative risk and noncompliance with therapy prolonged the neoadjuvant administration of imatinib. The lack of compliance to daily dosage imatinib resulted in the rapid enlargement of the tumour. The subsequent improved drug compliance resulted in a new response to treatment, with a better radiological response than the one seen on plateau phase before. Finally, the patient underwent an uneventful laparoscopic-assisted Ivor-Lewis oesophagectomy without tumour rupture. Postoperatively, the patient continued imatinib treatment and remained disease free at 1 year follow-up.The discovery that the vast majority of GISTs have characteristic activating mutations in c-KIT (75–80%) or PDGFRA 5–15%) radically changed the management of these tumours by the approval of the use of tyrosine-kinase inhibitors , 15. Ima% radicalAlthough preoperative downsizing of oesophageal GIST in an attempt to reduce the high morbidity/mortality associated with oesophagectomy seems ideal, literature available regarding neoadjuvant administration of imatinib in patients with oesophageal GISTs is very limited as oesophageal GISTs are rare and also neoadjuvant treatment with imatinib is a recent development –12. YanaThe accurate prediction of recurrence is very important for the decision of administration of adjuvant imatinib. The existing risk stratification systems  are based on tumour size, mitotic activity, tumour rupture prior to or at surgery, and tumour location , 13, 17.Regarding oesophageal GISTs, despite the fact that these can be classified according to the risk stratification systems mentioned above, the contribution of oesophageal GISTs to the creation of these systems was very small. This fact raises concerns about the accuracy of these systems regarding the prognosis of patients with oesophageal GISTs. For example, only 8 of the 1625 patients (0.49%) who validated for the creation of Joensuu liner-model had oesophageal GIST .The optimal duration of preoperative imatinib administration is not fully documented. NCCN states that the treatment should be continued until the tumour stops responding to imatinib or if PD appears unresponsive even to increased dose of imatinib . InterruDisease progression during neoadjuvant imatinib has been reported as a rare phenomenon for primary tumours, which has been attributed in part to the emergence of secondary mutations following prolonged treatment , 21. MorThe rarity of oesophageal GISTs explains the lack of clear recommendations regarding their optimal surgical management. As oesophageal segmental and wedge resections are not feasible, the surgical options include the high morbidity/mortality associated oesophagectomy and the much less invasive surgical tumour enucleation , 22–25. In the absence of clear guidelines for the optimal treatment of oesophageal GISTs, preoperative administration of imatinib in an attempt to downsizing, particularly in patients with large tumours, presented as an attractive approach aiming to (1) decrease the risk of intraoperative tumour rupture, thus achieving a better oncological outcome, and (2) reduce the extent of surgery and therefore the risks of perioperative morbidity and mortality. Compliance with drug therapy is essential for the success of this approach.


Volume of blood shifts between trunk and extremities (Vbs) was determined non-invasively as ΔVtr-ΔVb. Three types of breathing were studied: spontaneous (SE), rib cage , and abdominal . During SE and RCE blood was displaced from the extremities into the trunk , while during ABE it was displaced from the trunk to the extremities . At baseline, Vbs swings (maximum to minimum amplitude) were bimodal and averaged 0.13 ± 0.08 L. During exercise, Vbs swings consistently increased . It follows that during leg exercise significant bi-directional blood shifting occurs between the trunk and the extremities. The dynamics and partitioning of these blood shifts strongly depend on the relative predominance of the action of the diaphragm, the rib cage and the abdominal muscles. Depending on the partitioning between respiratory muscles for the act of breathing, the distribution of blood between trunk and extremities can vary by up to 1 L. We conclude that during exercise the abdominal muscles and the diaphragm might play a role of an “auxiliary heart.”Apart from its role as a flow generator for ventilation the diaphragm has a circulatory role. The cyclical abdominal pressure variations from its contractions cause swings in venous return from the splanchnic venous circulation. During exercise the action of the abdominal muscles may enhance this circulatory function of the diaphragm. Eleven healthy subjects  performed plantar flexion exercise at ~4 METs. Changes in body volume (ΔV The cyclical swings in intrathoracic and abdominal pressures caused by breathing influence cardiovascular function at rest and during exercise. The effects on venous return and blood redistribution from and to the limbs have been described qualitatively, both in humans and in animal models  and trunk volume (ΔVtr) were simultaneously tracked using whole body plethysmography (WBP) and opto-electronic plethysmography (OEP). This allowed quantification of blood shifting between trunk and extremities, calculated as the difference between these two simultaneous measurements (Vbs = ΔVtr − ΔVb). We hypothesized that during exercise the three breathing modes would differentially impact on intrathoracic and abdominal pressure variations, and therefore cardiovascular function. In particular, breathing with the diaphragm and the abdominal muscles would primarily act on abdominal pressure, while using the rib cage muscles would primarily act on pleural pressure. We expected that during exercise, compared to resting, the volume of blood displaced in and out the trunk would increase significantly and that the direction and amount of this blood displacement would depend on the relative predominance of the action of diaphragm, rib cage and abdominal muscles during the three breathing modes.We therefore studied 11 healthy subjects during exercise, while breathing in 3 different ways: (a) spontaneously (hereafter referred to as “spontaneous mode”); (b) predominantly using the inspiratory rib cage muscles (“rib cage mode”); and (c) predominantly using the abdominal muscles during expiration and the diaphragm during inspiration . We looked at the impact of these breathing modes during moderate exercise, on blood shifts between the trunk and the extremities. The volume of blood shifting between the trunk and the extremities was continuously measured using double body plethysmography (DBP). Changes in body volume , recruited among laboratory personnel, volunteered for the experiments. Most were experienced in doing complex respiratory maneuvers; all of them were instructed in detail at the beginning of each maneuver. The subjects' characteristics are shown in Table b), resulting from the gas flowing in and out of the lungs, and the simultaneous changes in compression and decompression of thoracic gas , resulting from the lung volume changes plus any blood shifting between the trunk and the extremities (Vbs). By subtracting the two independent measurements of ΔVtr and ΔVb we thus obtained Vbs. ΔVb was measured continuously with the subjects sitting comfortably in the WBP. The flow in and out of the WBP due to changes in body volume was measured by a pneumotachometer mounted on the top of the box, connecting the box's interior with the exterior. From this flow signal ΔVb was obtained by mathematical integration.To quantify blood volume shifting between the trunk and the extremities, we used DBP using a WBP with walls made of transparent cast acrylic sheet (20 mm)  was calculated as the sum of rib cage and abdominal volume (Vrc and Vab respectively). Flow at the mouth was measured using a hot wire anemometer, which measured gas velocity in a tube of known cross-sectional area , connecting the subject to the exterior of the box. Both flow meters were calibrated with a 3 L syringe.ΔVtr, and also to avoid influencing the pneumotachograph placed on the top of the box. Pilot testing in preparation for the experiments showed that this conditioning system allowed keeping temperature quite constant inside the WBP even during exercise, but not relative humidity, which led to integration drift.Temperature and humidity inside the box were continuously controlled with a multi-sensor device  based on a hygroscopic polymer as a humidity sensitive element and a semiconductor as a temperature-sensitive element. Eight Peltier cells  were interfaced with a set of finned heat sinks  and placed inside and outside the box. The air conditioning system was placed at the bottom of the body box out of the line of sight between the OEP cameras and the trunk, to allow the correct measurement of V2 resulting from this procedure was then used.In clinical practice WBP is routinely used for pulmonary function diagnostic tests that last so short that temperature and humidity changes can be neglected. Conversely, if prolonged volume measurements are needed, for example during an exercise test, increases in temperature and humidity, caused by the subject, cause substantial thermal drift , carbon dioxide output (V′CO2) and end-tidal CO2 pressure (PETCO2) were continuously measured on a breath-by-breath basis using a respiratory gas analyzer . O2 and CO2 concentrations at the mouth were measured by paramagnetic and infrared gas analyzers, integral part of the system, calibrated with gases of known composition. The flow meter was calibrated with a 3 L syringe. For each subject, data are presented as the mean of at least 10 breaths collected pre-exercise and the last part of the three exercise bouts.Oxygen uptake , they engaged in 5 min of stepping exercise while breathing spontaneously, followed by 4 min of recovery (no exercise) . Data are expressed as mean ± SD unless otherwise specified.Pre-exercise and exercise phases were analyzed in terms of volume changes and blood shifts. The analysis of V2, reached during exercise between the third and fourth minute while breathing spontaneously, by 3.5 ml kg−1 min−1. The exercise intensity corresponded to an average of 4.3 METs, indicating low to moderate intensity exercise (3–6 METs). Even though the stepping effort was equivalent in the three modes, because of the shorter durations of rib cage and abdominal breathing modes compared to spontaneous mode, steady state gas exchange was not reached in rib cage and abdominal modes (duration of exercise 1 min).The square-wave submaximal stepping exercise was performed at about 30 cycles/min and an average power output of 64 W. Individual metabolic equivalent of task (MET) values were calculated by dividing the V′O2 and V′CO2 compared to pre-exercise condition. V′O2 increased from pre-exercise by 11.1 ± 3.9, 8.5 ± 2.4, and 6.8 ± 2.6 ml kg−1 min−1 , for spontaneous, rib cage and abdominal mode, respectively. V′CO2 increased during exercise (p < 0.001) mostly during spontaneous breathing mode; 12.8 ± 3.6, 7.5 ± 1.4, 6.0 ± 1.9 ml kg−1 min−1 for spontaneous, rib cage and abdominal mode, respectively . PETCO2 during spontaneous breathing was higher compared to pre-exercise, rib cage mode and abdominal mode . The baseline ventilation, PETCO2 and RQ indicated some hyperventilation related to the experimental conditions.Ventilatory parameters for pre-exercise and exercise are shown in Table p < 0.001), during spontaneous, rib cage, and abdominal mode, respectively.Exercise significantly increased ventilation, which passed from 14.4 ± 4.3 L/min at pre-exercise baseline to 31.5 ± 7.8, 42.2 ± 15.1, and 37.7 ± 13 L/min , abdominal volume (Vab), and trunk volume (Vtr), all three obtained with OEP (black lines). Superimposed in red on Vtr is shown WBP-measured body volume (Vb), the difference with Vtr representing blood shifting (Vbs), shown in the bottom panels.Representative traces of spontaneous, rib cage, and abdominal breathing modes during exercise are shown in Figure bs , defined as the difference of end-inspiratory volume and end-expiratory volume, for all breathing modes  as shown in Figure T was reached by an increase of end-inspiratory rib cage volume (VrcEI) and a concomitant decrease in end-expiratory abdominal volume (VabEE). VrcEI increased on average by 0.37 ± 0.28 L (p < 0.01) while VabEE decreased on average by 0.21 ± 0.20 L (p < 0.05) compared with pre-exercise baseline.Figure p < 0.01), mostly due to an increase in end-inspiratory rib cage volume , while end-expiratory abdominal volume was kept nearly constant . No significant differences were found in VcwEE and VcwEI during abdominal exercise. The increase in tidal volume was due to a decrease in VabEE , while VrcEI was kept approximately constant .In rib cage breathing mode chest wall tidal volume significantly increased (rcEI (0.84 ± 0.39 L) during exercise compared to spontaneous and abdominal breathing modes .Rib cage breathing mode significantly increased Vbs swings and variations of average Vbs values (ΔVbs-mean) during spontaneous, rib cage and abdominal breathing modes are presented in Figures bs swings, were calculated as the mean difference between maximum and minimum values for each series of breaths analyzed, for quiet breathing and exercise respectively. During exercise, each subject showed a consistent increase in within-breath Vbs swings compared to pre-exercise . On average, ΔVbs-mean decreased by 0.19 ± 0.31 L and 0.53 ± 0.51 L  respectively during spontaneous and rib cage modes, indicating blood shifts from the extremities into the trunk. Conversely during abdominal exercise ΔVbs-mean tended to increase in average by 0.23 ± 0.16 L (p < 0.01), indicating blood shifting from the trunk to the extremities  while during abdominal mode 0.22 ± 0.20 L of blood  was pushed out of the trunk.Within-breath Vs Figure  and showbs amounted to 134 ml on average, slightly higher as reported before, probably because of slight hyperventilation, while during exercise it increased to about 420 ml, independently of the breathing mode, increasing inferior vena cava venous return from 2.5 L/min at baseline to 11.4 L/min during exercise. It follows that depending on the partitioning between respiratory muscles for the act of breathing, the distribution of blood between trunk and extremities can vary by up to 1 L and that during exercise the abdominal muscles together with the diaphragm might play a role of an “auxiliary heart.”We investigated how changes in breathing mode, during exercise, by acting on intrathoracic and abdominal pressure variations, influence blood shifting between different body compartments, and in particular in and out of the trunk. We expected that during exercise, compared to rest, splanchnic venous blood would be mobilized, that the amount of blood displaced within each breathing cycle would increase, and that the direction and amount of this blood displacement would depend on the relative contributions of the actions of the diaphragm, the rib cage and abdominal muscles. The main results can be summarized as follows: (a) for the first time the extent of blood volume shifting between the trunk and the extremities was non-invasively quantified during a form of leg exercise; (b) compared to pre-exercise baseline conditions, during exercise, while in spontaneous breathing mode, on average 160 ml of blood were pulled into the trunk from the extremities; (c) during abdominal mode, when the subjects breathed predominantly using the diaphragm and the abdominal muscles, about 225 ml was shifted out of the trunk to the extremities; (d) conversely, during rib cage mode, when the subjects voluntary emphasized the use of inspiratory rib cage muscles, about 478 ml was pulled into the trunk from the extremities; (e) at baseline, intra-breath Vbs) shifted from the trunk and presumably the splanchnic venous reservoir, to the extremities. These observations support the notion of a circulatory role for the diaphragm, as also illustrated by the effects of phrenic pacing of the diaphragm on blood shifting reported by Roos et al. , corresponding to blood coming from the splanchnic circulation (presumably mostly from the liver), through the hepatic vein into the inferior vena cava downstream toward the heart.We now quantified the effects of different breathing modes on blood volume redistribution between the trunk and the extremities during physical activity of moderate intensity. Three breathing patterns were chosen deliberately to vary the contribution of different muscles groups, in order to attain various combinations of breath-by-breath changes in intra-thoracic and abdominal pressures. We chose this approach to complement the results from Aliverti et al.  who measbs in two ways. According to Aliverti et al.  and 0.53 ± 0.51 L (p < 0.01), for spontaneous and rib cage exercise, respectively). The stronger contraction of the inspiratory rib cage muscles, by lowering intra-thoracic pressure during inspiration likely resulted in a more pronounced right atrial to inferior vena cava pressure difference, thus facilitating venous return, confirming earlier observations  led to a tidal blood volume of 460 ml, presumably through the hepatic vein, and a global splanchnic output of about ~12 L/min.We acquired esophageal and abdominal pressures in three of our subjects but do not present the results in detail because of the small number of observations and the variability between subjects. But although abdominal and pleural pressure patterns differed between subjects during the various breathing modes, it appeared that the VConversely, when the subjects started to exercise while breathing in rib cage mode, a marked increase in pleural swings was evident, while the abdominal pressure swings were determined by the passive movement of the diaphragm, which transmitted pleural pressure variations to the abdominal content. In this case, as demonstrated by Miller et al. , augmentbs swings during exercise was found during all three breathing modes, suggesting a dynamic impact on venous return through the inferior vena cava to the heart. A “stroke volume” of about 420 ml during all breathing modes was found in agreement of what has been previously published by Aliverti et al. during intermittent expulsive maneuvers  and lungs  and a decrease in intestinal blood flow  moved between the trunk and the extremities. Since we measured the total Vcw using OEP, we could not determine the amount of blood shifted between the rib cage and the abdominal compartments, limiting our assertions on splanchnic blood volume recruitment. Third, in this study we have not investigated the effect of the specific breathing modes at rest in comparison to during exercise in the same subject. Fourth we do not report gastric and esophageal pressures since these were only acquired in 3 subjects and equivocal. Fifth, because of the complexity of thermodynamic control inside the body box exercise duration had to be kept short. Even though our conditioning system was able to keep temperature quite stable during exercise, it did not allow complete control of humidity. We had to develop a mathematical method to correct for this drift. In order to avoid all the possible problems related to the influence of filtering on the signal baseline, we therefore analyzed the blood shifts only in terms of variations of baseline and tidal volumes respectively. Sixth, the respiratory maneuvers were difficult to perform. Several of the subjects had difficulties in performing the complex respiratory maneuvers by changing their breathing modes correctly during exercise. The various attempts were classified according to the variations in compartmental volumes, which led to various numbers of repetitions between breathing modes for some subjects. Finally, because of the complexity of the maneuvers there were missing values precluding the use of simple ANOVA. We therefore chose generalized linear mixed modeling, but in the worst case the proportion of missing values was one third, limiting the robustness of the statistical analysis.When interpreting the results of our study one should keep in mind some limitations. First, because of the complexity of our experimental setup we decided to refrain from measuring cardiac output (heart rate and stroke volume), which precludes any strong conclusions on the effects of the observed blood shifts on cardiac function. Second, what was measured in this study as VIn summary, for the first time the volume of blood redistributed between the trunk and the extremities was quantified non-invasively and continuously during exercise. The amount and the partial redistribution of this blood depended on the activation of rib cage, diaphragm and abdominal muscles on a breath-by-breath basis. In particular we found that during abdominal exercise the action of the diaphragm and abdominal muscles, increasing the abdominal pressure can shift blood from the extremities, while the rib cage inspiratory muscles' action facilitates blood shifting toward the pulmonary circulation. Depending on the partitioning between respiratory muscles for the act of breathing, the distribution of blood between trunk and extremities can vary by up to 1 L. We conclude that during exercise the abdominal muscles assist the diaphragm in its role of an “auxiliary heart.”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.


Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve.A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining.The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group.AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury. The optic nerve derives from the embryonic optic stalks in the optic vesicles located in the diencephalon; therefore, the microenvironment within the optic nerve is the same as within the central nervous system (CNS), which contains astrocytes, oligodendrocytes, microglias. Axons of the RGCs form optic nerve, however, once injury happens the axons will ordinarily fail to regrow through the lesion site while the RGCs will die gradually, leading to permanent vision loss . The faiBasic fibroblast growth factor (bFGF) which is a member of the fibroblast growth factor family plays a critical role in the development of the retina, and has been identified as a potent stimulator of axon growth for developing and regenerating RGCs . SeveralAmniotic epithelial cells (AECs) are differentiated from amnioblasts 8 days after fertilization . It was In the present study we established rat AECs modified with human bFGF gene utilizing lentivirus, and confirmed expression and secretion of bFGF in the genetically engineered AECs culture. Whereafter we transplanted the genetically engineered AECs into the transected optic nerve in rat, observing the effects of the cells on RGCs and the injured optic nerve.Adult Sprague-Dawley rats were used in this study. The animals were housed in seperated cages in a temperature-controlled room with a 12 h light/dark cycle and free access to food and distilled water. All the protocols were approved by the Administrative Committee of Experimental Animal Care and Use of Second Military Medical University, China . The animals had received humane care throughout all the procedures in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health . In addition the ARRIVE  guidelines was followed as well.2.The rat amniotic membrane was collected from deeply anesthetized pregnant rats at 19 days of pregnancy. The collected membrane was then treated with 0.25% trypsin (Gibco) 3 times, each for 20 min. AECs were collected and cultured in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum at 37°C with 5% COA bFGF gene segment (556bp) was synthesized according to human bFGF (hbFGF) cDNA sequences . The restriction enzyme sites: Spe I and Xho I were introduced into the segment. Afterwards, plasmid vector pLenti6/V5-GW/lacZ (Invitrogen) was digested with restriction endonuclease Spe I and Xho I to remove lacZ segment, then linked with bFGF gene segment, constructing plasmid vector pLenti6/V5-GW/hbFGF, which contains blasticidin resistance gene within the sequences.Packaging of recombinant lentivirus was performed utilizing the ViraPower Lentiviral Expression Systems (Invitrogen), and viral supernatant was harvested, storing at −80°C. The titer of the viral supernatant were determined by infecting the human fibrosarcoma cell line HT1080 (Invitrogen) and selected with blasticidin (Invitrogen).6/ml to grow as 85% confluence. Afterwards the medium was replaced by mixed medium containing 500 μl viral supernatant and 10 μg/ml polybrene (Sigma), and one control well that containing normal medium in each plate was set. After incubating for 6h, the infectious medium was removed, and the cells were cultivated with fresh medium for 24h. Then the cells were selected in blasticidin (10 μg/ml) selection medium about 12d till all of the cells in control wells died out, and the survived cells were further cultured.The fourth generation passage rat AECs were seeded into 6-well plates at 1×10After blasticidin selection, total RNA was extracted from AECs/hbFGF of the third passage. Expression of human bFGF mRNA was detected by RT-PCR. The sequences of bFGF primers used were (5’→3’): forward GAGCGACCCTCACATCAA, reverse CGTTTCAGTGCCACATACC. The products (222bp) were determined by agarose gel electrophoresis.Meanwhile, culture medium of the first-passage AECs/hbFGF was collected separately 3, 6, 9 and 12 days after culturing, and was tested for bFGF concentration by ELISA.6/ml, determined by cell counting.10d after blasticidin selection, AECs/hbFGF and the same period subcultured AECs that were not infected by lentivirus were chosen for transplantation. On the day of transplantation, both AECs and AECs/hbFGF that grew as 95% or more confluence were trypsinized and made into single cell suspension. The cell density was 8×10For the purpose of tracing transplanted cells, a certain number of animals (n = 3) in each cell transplantation groups were chosen to receive transplantation of AECs/hbFGF or AECs stained with Hoechst dyes . 24h before transplantation, the cells were cultured in fresh medium with Hoechst (0.5 μg/ml) for 1h, and then were cultured with common medium till being prepared for transplantation as described previously.In the optic nerve transected model, the rat was anesthetized by intraperitoneal injection of 2% pentobarbital sodium (40mg/kg). The optic nerve was exposed through supraorbital approach under microscope, and a clear transverse gap (about 0.5mm) was made 2mm retrobulbarly by sucking the nerve fibers with a micro glass pipette through a small vertical breach on the meningeal sheath, without damaging the vessels . The incNormal group: sham surgery, the optic nerve was exposed without transection (n = 18)Control group: 10μl DMEM/F12 medium was injected into the transverse gap of the optic nerve (n = 21)AECs group: 10μl AECs suspension was injected into the transverse gap of the optic nerve (n = 21)AECs/hbFGF group: 10μl AECs/hbFGF suspension was injected into the transverse gap of the optic nerve (n = 21)Adult male Sprague-Dawley rats weighing 150–180 g were randomized into the following 4 groups and then allocated to the following protocols:Within the AECs group and AECs/hbFGF group, 3 rats were transplanted with cells stained with Hoechst dyes separately.28d after transplantation, the rats in the AECs group, AECs/hbFGF group which were transplanted with cells stained with Hoechst (n = 3) and 3 rats in the control group were sacrificed and fixed by cardiac perfusion with 4% paraformaldehyde. Subsequently, the injured optic nerves were taken out for a 6h post-fixation with 4% paraformaldehyde followed by dehydration with 20% sucrose solution. Afterwards, the optic nerve specimens were made into frozen sections (10 μm) for observation by fluorescent microscope.Some of the rats (n = 6) in each group were chosen to receive RGCs labeling with BDA, which were conjugated with fluorescein (Vector). 26d after surgery, the rats were anesthetized by peritoneal injection of 2% pentobarbital sodium (40mg/kg), and 5μl 10% BDA was injected into vitreous body with a microinjector. 48h later, the rats were sacrificed and fixed by cardiac perfusion with 4% paraformaldehyde, and then the eyeballs were taken for retina flattening to count BDA labeled cells. Each retina was detached from the eyeball, and was cut with scissors into upper, lower, nasal and temporal pieces according to the conjunctival ink mark. Afterwards, the directionally cut retina was flattened on the slide for RGCs counting under fluorescence microscope. High power fields (40×10) were chosen and were photographed at random on upper, lower, nasal and temporal pieces. For each area, 3 sites which were 1/6, 3/6 and 5/6 distance from optic nerve head were chosen, and 2 high power fields were photographed at random. Only cells with an intact membrane profile and plump cytoplasm, as well as bright fluorescence within cytoplasm were included. The number of labeled cells present in the high power fields was counted and analyzed.Meanwhile a same number of rats (n = 6) in each group were sacrificed and the retinas were collected for Nissl staining with toluidine blue. The number of RGCs in which the Nissl’s bodies were stained blue were counted and analyzed as the same method described previously, and only cells with an intact membrane profile and plump cytoplasm, as well as hyperchromatic Nissl’s bodies were included.28d after surgery, the rats (n = 6) were sacrificed and fixed, and the injured optic nerves were collected and embeded in paraffin. Afterwards, serial sections (5μm) were made for immunohistochemical staining. The thin paraffin sections were proceeded to react with primary GAP-43 antibody (1:50) (CALBIOCHEM) and further react with secondary biotinylated IgG (1:200) (CALBIOCHEM). The immunoreactivity was visualized with streptavidin biotin-peroxidase complex method. Then the sections were observed under microscope, and the injured sites in each group were photographed (20×10) to measure the integral optical density value of the immunoreactive substance with Biosens Digital Imaging System (Shanghai Bio-tec).P<0.05 was considered to denote statistical significance.All results were expressed as means ± S.E.M. Differences between groups were determined with one-way ANOVA by SAS software (8.2). A value of The hbFGF gene segment within plasmid vector pLenti6/V5-GW/hbFGF was confirmed by restricting enzyme digestion and DNA sequencing. A bright electrophoresis strip appeared at approximately 550bp . Further5 TU/ml. The fourth generation passage rat AECs infected with the tittered viral supernatant were cultured in blasticidin selection medium till the uninfected AECs of the control well died out . Comparing the differences of bFGF concentration in different time groups and the control group by Dunnett-t Test, it was indicated that bFGF concentrations in different time groups were higher than the control group , and the bFGF concentration in 6, 9, 12d groups were higher than that of the 3 d group   .The optic nerve of rat was exposed surgically and sucked with a micro glass pipette, leaving a 0.5mm clear transverse gap . Since tObservation of optic nerve frozen section showed that 28d after transplantation, both AECs/hbFGF and AECs that stained with Hoechst were still alive within the lesion site. However, the AECs/hbFGF survived more robustly and migrated along with the residual glial cells to a distance of 2–3mm from the transected site .As shown in retina flattening after BDA labeling and Nissl staining respectively, 28d after surgery the RGCs in the normal group were homogeneously labeled with green fluorescein or toluidine blue, and the cell bodies were large and plump. Meanwhile the labeled RGCs in the control group and cell transplantation groups arranged disorderly, as well as large cell bodies could rarely be found .P<0.0001; Nissl staining method: F = 468.42,P<0.0001). Furthermore, RGCs counting in the control group was lower than other groups (P<0.0001), and RGCs counting in the AECs/hbFGF group was higher than that of the AECs group  but was lower than that of the normal group (P<0.0001)  .The protein expression of GAP-43 was not visualized within the optic nerve of the normal group with immunohistochemical staining method 28d after surgery, while the expression of GAP-43 was visualized in other groups . In the P<0.0001). The GAP-43 integral optical density value in the control group was lower than the cell transplantation groups (P<0.0001), and the GAP-43 integral optical density value in the AECs/hbFGF group was higher than that of the AECs group (P = 0.0299) , the result showed that the differences of the GAP-43 integral optical density value among the three groups were statistically significant  .Optic nerve, also known as the second of twelve paired cranial nerves, transmits visual information from retina to brain. Since it is derived from the diencephalon during embryonic development, the optic nerve has generally been considered to be part of the CNS. Furthermore the optic nerve has often been used for investigating the CNS, especially for modeling axonal injury of the brain, due to its intrinsic structure and microenvironment differing from the peripheral nervous system as well as its easily accessible location . The neuGAP-43 is a nervous tissue-specific cytoplasmic protein considered as a neuron specific marker of outgrowing axons, which is expressed at high levels in neuronal growth cones during development and axonal regeneration . GAP-43 Our study demonstrated that AECs/hbFGF could synthesize and secrete bFGF peptide, which could possibly promote AECs to survive and migrate after being transplanted into the injured optic nerve. The reason why we chose current method of damage to the rat optic nerve over the conventional cut with iridectomy scissors or crush with forceps was that it could provide a valid complete transection, while in this extremity no nerve fibers was remained within the gap. Moreover since the meningeal sheath was reserved well and vessels were kept intact, it was possible to inject cell suspension into the transverse gap and keep it. Therefore the effect of transplanted cells could be clearly observed and analyzed. Meanwhile, during the procedures there were displaced amacrine cells (dACs) being labeled by both Nissl staining and BDA staining, but we could possibly distinguish a majority of RGCs with dACs by morphological observation. Generally the cytoplasm of RGC is relatively big with obvious Nissl bodies after staining . So we sIn view of the stem cell characteristics, secretion of several neurotrophic factors and low immunogenicity, as well as avoidance of ethical issues on the cell source, AECs has been considered as one of the promising cell types for transplantation strategies to treat neurological disorders. Okawa et al  proved tIn summary, bFGF gene modified rat AECs could promote RGCs survival and regeneration of axons in the completely injured optic nerve of rat, indicating their potential for cell therapy treating optic nerve injury and related diseases. However, the mechanisms underlying the regeneration process and visual function restoration afterwards needs further investigation.


Although effects of weather changes on human health have been widely reported, there is limited information regarding effects on pregnant women in developing countries.We investigated the association between maternal exposure to ambient temperature and the risk of preterm birth (< 37 weeks of gestation) in Guangzhou, China.We used a Cox proportional hazards model to estimate associations between preterm birth and average temperature during each week of gestation, with weekly temperature modeled as a time-varying exposure during four time windows: 1 week (the last week of the pregnancy), 4 weeks (the last 4 weeks of the pregnancy), late pregnancy , and the entire pregnancy. Information on singleton vaginal birth between 2001 and 2011 was collected. Daily meteorological data during the same period were obtained from the Guangzhou Meteorological Bureau.A total of 838,146 singleton vaginal births were included, among which 47,209 (5.6%) were preterm births. High mean temperatures during the 4 weeks, late pregnancy, and the entire pregnancy time windows were associated with an increased risk of preterm birth. Compared with the median temperature (24.4°C), weekly exposures during the last 4 weeks of the pregnancy to extreme cold  and extreme heat  were associated with 17.9%  and 10.0%  increased risks of preterm birth, respectively. The association between extreme heat and preterm birth was stronger for preterm births during weeks 20–31 and 32–34 than those during weeks 35–36.These findings might have important implications in preventing preterm birth in Guangzhou as well as other areas with similar weather conditions.He JR, Liu Y, Xia XY, Ma WJ, Lin HL, Kan HD, Lu JH, Feng Q, Mo WJ, Wang P, Xia HM, Qiu X, Muglia LJ. 2016. Ambient temperature and the risk of preterm birth in Guangzhou, China (2001–2011). Environ Health Perspect 124:1100–1106; http://dx.doi.org/10.1289/ehp.1509778 Preterm birth, defined as birth < 37 completed weeks of gestation, is a complex syndrome and remains a major global public health problem . It is eSeasonal weather changes have extensive effects on human health outcomes, especially for overall mortality, infectious diseases, and cardiovascular diseases . As a spThe most biologically relevant time period for effects of temperature on preterm birth is not known. Previous studies focused mainly on the short-term exposure, which was defined as a few days or weeks before delivery . BecauseAnimal studies have suggested that high temperature exposure could lead to dehydration, reduce uterine blood flow , and incGuangzhou is the largest metropolis in Southern China with a population of 12.9 million . It has All singleton vaginal births at the gestational age of 20–43 weeks were included in the present study. Because the data were collected by the birth date, short pregnancies from the earliest possible conception dates that terminated before the start date of the study (1 January 2001) were not included in the analysis; similarly, long pregnancies from the latest possible conception dates that terminated after the end date of the study (31 December 2011) were also not included in the analysis . Thus, wThis study was approved by the institutional ethical committee board of Guangzhou Women and Children’s Medical Center. Data used in this study were anonymous, and no individual identifiable information was available for the analysis.Preterm birth was defined as birth < 37 completed weeks of gestation. Gestational age at birth was determined according to an ultrasound examination in the first or second trimester . If the 10; micrograms per cubic meter), sulfur dioxide , and nitrogen dioxide . Air pollution data were measured at a fixed environmental monitoring station located in the center of Guangzhou. The monitoring station providing meteorological and air pollution data follows the quality assurance and quality control programs that were mandated by the Chinese government. These data on temperature and air pollution have been widely used to explore their effects on mortality in Guangzhou  and relative humidity (percent), were collected from the Guangzhou Meteorological Bureau. The meteorological data were recorded at a meteorological station in Guangzhou. Daily values for temperature and relative humidity were calculated by averaging four measurements taken at 6-hr intervals over each 24-hr period. Daily air pollution data from 2006–2010 were obtained from Environmental Monitoring Center, including particulate matter with an aerodynamic diameter ≤ 10 μm  1-week (the weekly mean temperature during week t); b) 4-week ; c) late pregnancy  and d) cumulative   .We modeled time-varying weekly temperatures during each time window as a cubic spline with 3 degrees of freedom (df) to allow a nonlinear relationship with preterm birth, and plotted the resulting hazard ratios (HRs) and 95% confidence intervals (CIs) to show the relationship over the entire temperature distribution. In addition, we calculated specific HRs relative to the local median temperature (24.4°C) for locally extreme cold  and moderate cold , and for locally extreme heat  and moderate heat  . In addiMaternal age was categorized into three groups  and included in the model as a stratified variable , because10, SO2, and NO2 as time-varying covariates, using time windows corresponding to those used for temperature. However, because air pollution exposures might be causal intermediates between temperature and preterm birth, adjustment may block part of the total effects of temperature ] = α + βtTemp,l + NS + NS + γtDOW + δLog(tZ),Log[t represents the day of the observation; tY is the observed number of preterm births on day t; t,lTemp is a matrix representing the two-dimensional relationship of mean temperature and lag days; l denotes the maximum lag days; NS is a natural cubic spline with 5 df for relative humidity; NS is a natural cubic spline with 5 df/year for long-term trend and seasonality; and tDOW is the day of the week . tZ is a daily expected count of preterm births on day t:where jtZ is the number of pregnancies at risk during gestational week j (ranging from 20 through 36 weeks) on day t, and jW is the conditional probability of birth during gestational week j, as proposed by where All statistical analyses were performed using SAS statistical software version 9.3  and the R statistical environment (version 2.15)  with theA total of 1,420,348 singleton births at the gestational age of 20–43 weeks were recorded during the study period. After exclusion of 582,202 cesarean section births (with preterm birth rate of 5.2%), 838,146 singleton vaginal births were included in this study. Among them, 47,209 (5.6%) were preterm births, with a daily average of 11.8 preterm births. The demographic characteristics of the study population are shown in sr = 0.098). The median daily mean temperature and relative humidity were 24.4°C and 74% during the study period. The Spearman’s correlations showed a weak correlation between mean temperature and relative humidity  and 32–34  compared with preterm births during weeks 35–36 , though estimates for later preterm births were more precise, at least in part due to larger numbers of observations  after adjustment for weekly average PM10, SO2, and NO2 concentrations during time windows corresponding to those for temperature were similar to those for overall study population, although HRs were slightly attenuated and less precise after adjustment. However, these differences may be attributable at least partly to the use of a different population (2006–2010 vs. 2001–2011), rather than to effects of adjustment for air pollutants per se .In the sensitivity analyses, we included the month as a spline with 3–6 df, and the effect estimates for temperature remained similar (see Table S1). HRs for temperature and preterm among pregnancies during 2006–2010 . Associations with extreme cold and extreme heat both were strongest for temperature on the same day of preterm birth (lag 0) and null after 4- and 6-day lags, respectively. The association with extreme cold was strongest for the longest cumulative lag (lag 0–27), whereas HRs for extreme heat were > 1 at lag 0–6 but < 1 for longer cumulative lags (see Table S2).In the present study, both low and high temperatures during pregnancy were associated with an increased risk of preterm birth. Extreme cold temperatures were associated with the risk of preterm birth during all time windows examined , and extreme heat was associated with preterm birth risk during all but the shortest time window of exposure. Furthermore, extreme heat was more strongly associated with preterm births during 20–31 and 32–34 weeks of gestation than with preterm births during weeks 35–36.To our knowledge, this study may be the first one to investigate the effects of ambient temperature on the risk of preterm birth in developing countries . Air conThe prevalence of preterm birth among singleton in the study area  was similar to those of previous reports in Guangzhou (5.1% to ~ 6.4% from 2002 to 2012) , Korea , and JapFew studies have explored the effects of ambient temperature on the rate of preterm birth with inconsistent results . Two larThere were several potential reasons for differences between our findings and those of previous studies. First, the range of climatic conditions, socioeconomic level, and population susceptibility may vary across areas. In addition, we speculate that the study design, statistical methods, exposure windows, and sample size are important factors . For exaBiological mechanisms that might contribute to the association between ambient temperature and preterm birth are unclear. Previous studies proposed several hypotheses. High temperature exposure causes dehydration and reduces uterine blood flow in pregnant women, which can induce the uterine contraction and onset of labor . In addiSeveral limitations should be mentioned. First, we used data from one meteorological station to define the exposure for all women in Guangzhou. This could cause exposure misclassification. Second, we were unable to distinguish spontaneous preterm birth from iatrogenic preterm birth, although we restricted the study population to those with vaginal delivery. Exposure to temperature might have different effects on these subtypes of preterm birth. We have no data on the percentages of identification method of gestational age  and their changes during the study period, which might influence the incidence of early preterm birth and late preterm birth. In Guangzhou, it is a routine practice to confirm gestational age using ultrasound examination in the first or second trimester . HoweverExposure to both low and high temperatures during pregnancy was associated with an increased risk of preterm birth in our study population. These findings might have important implications in preventing preterm birth in Guangzhou as well as other areas with similar variations in weather conditions.(1.7 MB) PDFClick here for additional data file.


Q1, and tissue glucose, Q2 (both µmol). The flows  into the plasma pool (Pool 1) are absorbed glucose entry from dietary sources, labelled glucose infusion, and hepatic glucose production. There is one flow out of Pool 1, glucose uptake by the tissues. Inflows to the tissues pool (Pool 2) are from plasma and glycogenolysis. Outflows from Pool 2 are to plasma, glucose oxidation, and glycogenesis and other metabolism. Application of the model was illustrated using experimental data derived from healthy adult Labrador Retrievers in the fasted and fed  states. In general, model derived estimates of glucose kinetics were representative of normal glucose metabolism, where rates of glucose production and uptake are similar and act to maintain blood glucose concentrations. Furthermore, estimates of within tissue glucose cycling indicated glycogenolysis in fasting and glycogenesis when fed. In the fasted state, model outputs were consistent with those reported in the canine literature derived using a single pool model. A new two-pool model to describe glucose kinetics in the steady state is presented. The pools are plasma glucose,  Isotopic determination of plasma glucose kinetics has been examined in steady and nonsteady state situations and has been resolved using one-, two-, and three-pool models and noncompartmental schemes (reviewed by [Ra) and disappearance (Rd) from the pool are considered the sum of all possible entries and exits into and out of the system. In the steady state, such as fasting, Ra describes hepatic glucose production and Rd denotes whole body tissue uptake (disappearance) based on the assumption that glucose is uniformly distributed in the extracellular fluid space. The single pool model is unable to describe the bidirectional exchange of glucose between plasma and body tissue compartments and the contribution of intracellular glucose recycling. To overcome these limitations and yet preserve the inherent simplicity and mathematical convenience of the single pool model, a new two-pool model is proposed to describe glucose kinetics in the steady state.Diabetes mellitus, a disease characterized by a dysregulation of glucose homeostasis, is increasingly prevalent in companion animals (reviewed by ). Accordiewed by ). For exiewed by  proposediewed by , 5 and fiewed by . Howeveriewed by  has beenRa and oxidation values in the published canine literature derived using a single pool model.The primary aim of this study was to develop a simple two-pool model to describe glucose kinetics in the steady state. The model builds from the existing single pool model by giving explicit representation to the bidirectional exchange of glucose between plasma and tissue compartments and the within tissue cycling of glucose through glycogenesis and subsequent glycogenolysis. The proposed model describes glucose kinetics in the steady state and is solved algebraically. To test its validity, the new model was applied to experimental data derived from our previously published work  and an aQ1, and tissue glucose, Q2 (both µmol). The flows  into the plasma pool (Pool 1) are absorbed glucose entry from dietary sources, F10, labelled glucose infusion, I, and hepatic glucose production, F12. There is one flow out of Pool 1, glucose uptake by the tissues, F21. Inflows to the tissues pool (Pool 2) are F21 and glycogenolysis, FR2. Outflows from Pool 2 are F12, glucose oxidation, F02, and glycogenesis and other metabolism, FR2. The flow F10 is assumed to occur only in the fed state and the flow F12 only in the fasted state. The scheme for labelled glucose assumes no reentry of label into Pool 1 during the infusion period and is shown in q1, µmol), one inflow, namely, the rate of infusion I, and one outflow, namely, labelled glucose uptake by the tissues f21. Sampling, for example, total glucose concentration, G1 (µmol mL−1), and enrichment, e1 , is from the plasma pool.The kinetic scheme for total glucose (labelled plus unlabelled) comprises two pools and seven flows  predominates, and when negative, glycogenolysis predominates. In the fasted state, absorbed glucose entry to the plasma pool from dietary sources is zero and  was calculated according to [V2CO is the volume of CO2 production, e2CO is the isotopic enrichment of expired CO2, e1 is the plasma isotopic enrichment, Ccorr is the bicarbonate correction factor to account for carbon retention, and 6 represents the number of carbon atoms per glucose molecule oxidized.The model was applied to data obtained from two separate experiments (A and B) . As the ll meals . Experimrding to :(7)F02=VRa) (F12) was similar to its rate of disappearance (F21). Within tissue glucose recycling (FR2 − FR2) values were negative, suggesting that tissue glycogenolysis was predominant. Calculated flows in Experiment A derived from extrapolated steady state values were higher than those similarly derived from measured values. Indeed, extrapolated steady state values in Experiment A were similar to those observed in Experiment B where isotopic steady state was achieved. During repeated meal feeding, measured glucose oxidation (F02) and calculated flows  were greater than those observed in fasting, reflecting the increased availability of exogenous glucose. In agreement, within tissue glucose recycling (FR2 − FR2) values were positive, indicating glycogenesis. Glucose oxidation and calculated flows varied considerably within and between experiments. Specifically, oxidation and calculated flows were greater in Experiment B than Experiment A. In Experiment A, oxidation values were lowest when derived from extrapolated steady state enrichments, whereas calculated flows were lowest when derived from measured enrichments.Kinetic measurements and calculated flows derived from Experiment A and Experiment B are presented in I and e1) in turn by 0, ±10, and ±20%. Each calculated flow  was then plotted against the perturbation , and a five-point linear regression of y on x was performed to determine the slope of the line produced. Each average slope was subsequently scaled by its corresponding unperturbed average flow value and multiplied by a hundred to give the scaled slopes dimensions of % change in y per % change in x  model application. Literature Ra values were similar to those in Experiment A derived from extrapolated steady state values and Experiment B, whereas Ra values derived from measured values in Experiment A were lower than experimental and literature values. These findings indicate that Ra is likely to be underestimated if true steady state enrichment is not achieved experimentally. However, extrapolating steady state enrichments yield reasonable estimates of glucose kinetic parameters and can be employed if necessary.In the canine literature, plasma nditions . The undRa from ingested glucose ranged from 35 to 81 μmol/(min·kg) in Experiment A and from 153 to 225 μmol/(min·kg) in Experiment B. In general, these values are comparable to those found by Christopher et al. [μmol/(min·kg). However, direct comparisons are not appropriate, given the differences in experimental methodology. Specifically, our experimental data reflect glucose Ra in canines fed intermittent mixed meals, whereas Christopher et al. [While fasting glucose kinetics has received much attention, no other studies in canines to our knowledge have assessed fed glucose kinetics in the steady state using the repeated meal feeding technique. Fed steady state kinetics has been mimicked by infusing exogenous insulin above basal concentrations under clamped hyperglycemia (and euglycemia)  glucose. Furthermore, calculated flows were higher in Experiment B where dogs were fed more diet than in Experiment A, suggesting that the fed state application of the model is sensitive to changes in dietary derived glucose supply. Together, these findings support the present model as an improved alternative to the existing steady state single pool model.In conclusion, a novel two-pool model to describe glucose kinetics in the steady state was presented. The model was tested using experimental data generated from our laboratory and compared to existing literature values. The model yielded fasting plasma glucose