The Effects of Inverse Ratio Ventilation with PEEP on Respiratory Function and Inflammatory Cytokines in Patients during One-lung Ventilation

The incidence of hypoxemia is reportedly from 5% to 10% during one-lung ventilation (OLV) [1]. High tidal volume and airway pressure during OLV correlated with the development of acute ventilationinduced lung injury in patients undergoing lung resection [2]. Inverse ratio ventilation (IRV) has been proposed for patients with adult respiratory distress syndrome to achieve adequate oxygenation [3,4], due to recruiting atelectatic alveoli [5,6], and it is reported that the optimal inspiratory to expiratory ratio (I:E) is 2:1 when using IRV [7]. It is widely accepted the use of low tidal volume should be accompanied by positive end-expiratory pressure (PEEP), and the previous study reported that PEEP could improve oxygenation [8,9], but it was rarely reported that IRV (I:E = 2:1) applying external PEEP was used in thoracic surgery. The present study tested the hypothesis that IRV (I:E = 2:1) applying external PEEP would improve hypoxemia, promote oxygenation and alleviate inflammation in patients underwent one-lung ventilation for lung lobectomy, is superior to conventional ventilation with PEEP. The primary endpoint was that IRV (I:E = 2:1) applying external PEEP would reduced Pplat. Materials and Methods


Introduction
The incidence of hypoxemia is reportedly from 5% to 10% during one-lung ventilation (OLV) [1]. High tidal volume and airway pressure during OLV correlated with the development of acute ventilationinduced lung injury in patients undergoing lung resection [2]. Inverse ratio ventilation (IRV) has been proposed for patients with adult respiratory distress syndrome to achieve adequate oxygenation [3,4], due to recruiting atelectatic alveoli [5,6], and it is reported that the optimal inspiratory to expiratory ratio (I:E) is 2:1 when using IRV [7]. It is widely accepted the use of low tidal volume should be accompanied by positive end-expiratory pressure (PEEP), and the previous study reported that PEEP could improve oxygenation [8,9], but it was rarely reported that IRV (I:E = 2:1) applying external PEEP was used in thoracic surgery. The present study tested the hypothesis that IRV (I:E = 2:1) applying external PEEP would improve hypoxemia, promote oxygenation and alleviate inflammation in patients underwent one-lung ventilation for lung lobectomy, is superior to conventional ventilation with PEEP. The primary endpoint was that IRV (I:E = 2:1) applying external PEEP would reduced P plat .

Materials and Methods
This study was approved by the Hospital Ethics Committee of Jiaxing maternity and child health care hospital and registered in protocol registration system (registration number: ChiCTR-IOR-15006517). The informed consent was signed before enrollment by the patients. From May 2015 to December 2015, we identified 60 patients undergoing elective lung lobectomy with ASA grade II, age 34-61 years, body mass index (BMI) 21-29 kg/m 2 and minute ventilation volume > 70 L/min to enroll in this study. We excluded the patients that had the history of severe cardiovascular disease, asthma, chronic obstructive pulmonary disease and pneumothorax. There were 38 central type lung cancers and 22 peripheral type lung cancers among. The duration of the operation lasted 86.4-138.2 min. Sixty patients were randomly assigned to either control group (n = 30) or IRV group (n = 30) using computer-generated random number code.
Upon arrival in the operating room, standard monitoring including electrocardiogram, invasive arterial blood pressure (ABP), heart rate (HR), pulse oximetry (SpO 2 ) and central venous pressure (CVP) were applied and venous access was established. Anesthesia was induced with intravenous propofol 2 mg/kg, fentanyl 5 µg/kg and rocuronium 0.6 mg/ kg, then bronchial intubation was performed using direct laryngoscope. Anesthesia was maintained with propofol 6-10 mg.kg -1 .h -1 and remifentanil 0.5-1 ug.kg -1 .min -1 to keep the bispectrail index (BIS) value between 40 to 55 (BIS monitor Model A2000, USA). The lungs were mechanically ventilated with ventilator (DATAX-OHMEDA Aspire anesthesia machine, USA). Muscle relaxation was monitored by the train-of-four (TOF) stimulation on the ulnar nerve (Organon, Type TOF-Watch SX, Holland). Continuous infusion of rocuronium was performed to maintain stable neuromuscular block.
The trachea was intubated using a double-lumen tube (37F for males and 39F for females), the correct position of the double-lumen tube was confirmed using a fiber-optic bronchoscope (Olympus company, Tokyo, Japan) after intubation in both supine and lateral positions. All patients were initially applied with two-lung ventilation. The lungs were initially ventilated using a constant-flow, volume-controlled ventilation mode with an actual tidal volume of 8 mL/kg actual body weight, an inspiratory to expiratory (I:E) ratio of 1:2, a respiratory rate of 12 breaths/min, oxygen flow rate of 1 L/min, and FiO 2 (fraction of inspiratory oxygen) of 1.0 without PEEP. OLV was initiated at the moment of skin incision, and the tube lumen of the non-ventilated lung was opened to room air. During OLV, tidal volume was reduced to 7 mL/kg, and ventilator settings were actual tidal volume (V T ) of 7 mL/kg, respiratory rate of 12 breaths/min, PEEP of 5 cm H 2 O, FiO 2 (fraction of inspiratory oxygen) of 1.0, oxygen flow rate of 1 L/min, I:E of 2:1 (in IRV group) or 1:2 (in control group). At the time of closure of thoracic cavity, two-lung ventilation was started and both lungs were re-expanded by hand bagging in all patients.
Mean airway pressure (P mean ), plateau airway pressure (P plat ), total PEEP (PEEP tot ), dynamic compliance of respiratory system (CL) and end-tidal CO 2 partial pressure (PetCO 2 ) were monitored using a sidestream spirometry device (Anesthesia Monitor D-FPD15-00; GE, Taipei, Taiwan), maintaining PetCO 2 value of 35-45 mmHg. When PetCO 2 > 45 mmHg, tidal volume or breath rate was increased. All the patients were in lateral position with head down 10º during surgery. If SpO 2 was below 92% and lasted 30 seconds during OLV, surgery was temporarily interrupted and double-lung ventilation was alternately applied. The neuromuscular block was reversed with intravenous neostigmine 1 mg and atropine 0.5 mg when TOF > 70%. Fentanyl 1 µg/kg was administered for pain control at 30 min before the end of surgery. Postoperative complications were observed, such as time in Post Anesthesia Care Unit (PACU), time to extubation and incidence of hypoxemia. The Patient was discharged from PACU when the modified Aldrete score was appropriate (score ≥ 9) [10]. Patients were followed up for any complications of lung during their hospital stays. Throughout the perioperative operation, lactated Ringer's solution or hydroxyethyl starch solution was infused at rate of 8-10 ml.kg -1 .h -1 .
Hemodynamic parameters, such as systolic blood pressure (SBP), diastolic blood pressure (DBP), HR and CVP were recorded at 5 min before anesthesia induction (T0), 5 min after bronchial intubation (T1), the initiate of OLV (T2), 45 min of OLV (T3) and the end of surgery (T4), meanwhile, P mean , P plat , PEEP tot and CL were recorded at T1, T2, T3 and T4. Arterial blood gas and mixed venous blood gas were analyzed using a blood gas analyzer (ABL8000A, Denmark) and bronchoalveolar lavage fluid (BALF) of ventilated lung at T1 and T3 point. At T1 and T3 point, we collected bronchoalveolar lavage fluid performed by infilliting 50 mL normal saline in ventilated lung and 20-40% this fluid was recovered with fiber bronchoscope (Olympus company, Tokyo, Japan), then samples were centrifuged at 3,000 rpm for 15 minutes at 4ºC, saved for assay at -70ºC. IL-1β, IL-6 and IL-8 levels were detected by enzµyme-linked immunosorbent assay (ELISA), using the microplate reader (Hyperion MR III, USA). All enzyme-linked immunosorbent assays were performed according to the manufacturers' guidelines.

Statistical analysis
Descriptive statics and compliance with normal distribution was examined with one-sample Kolmogorov-Smirnov test. Between groups Student t test and Mann-Whitney U-test were used. Categorical variables were evaluated with the Chi-square test. Data were presented as mean ± standard deviation. Parametric data were checked for by using t test or categorical data was analyzed by Chi-square test. Significance was considered as P value < 0.05. Data analysis was performed using the statistical software package SPSS 19.0 (SPSS Inc, Chicago, USA). Sample size based on a previous trial test. The main variable in the study, P plat was having a standard deviation of 2.1 from a plot test done on 10 patients. A priori power analysis using two-sided analysis with an α-error of 0.05 and a power of 0.8 showed that 54 patients were needed for this study.

Results
No significant differences in age, body mass index, gender, maximal ventilation volume (MMV), time of OLV and operation time between groups (Table 1) (P > 0.05). At T3, PaO 2 increased significantly in IRV group, there was statistical significance between groups (Table 2) (P < 0.01). Comparison of pH, PaCO 2 , SaO 2 and ScvO 2 , there were no statistical significance in both groups (P > 0.05).
The P mean , PEEP tot and CL were increased significantly in IRV group than those in control group at T1, T2, T3 and T4 (Table 3), there was statistically significant between groups (P < 0.05), but P plat was lower in IRV group (Figure 1), there was statistical significance (P < 0.05). Hemodynamic monitoring reflects the hemodynamic changes in both groups (Figure 2). The comparison of hemodynamic parameters in both groups, there was no statistical significance (P > 0.05). The concentrations of IL-6, IL-8 and IL-1β were lower in IRV group than those in the control group at T3, there was statistically significant between the groups (P < 0.05) ( Table 3). Hypoxemia was 1 case in IRV group and 3 cases in control group during OLV (Table 1), however there was statistical significance between two groups (P < 0.05). There was no significance in the incidence of intraoperative hypoxemia and time in PACU between two groups (P > 0.05) ( Table 1). No postoperative hypoxemia and hypoxemia was observed.

Discussion
One-lung ventilation is a common technique of ventilation in thoracic surgery, which is advantageous to surgical area because of the non-ventilated lung collapse, prevents from the secretions into the healthy lung and protects the healthy lung from pollution. Meanwhile the collapse lung will increase the intrapulmonary shunt, aggravate hypoxia and increase airway pressure of the ventilated lung to result in acute lung injury. High tidal volume and plateau pressure are the major risk factor of acute lung injury, thus the present study compared volume-controlled inverse ratio ventilation (I:E = 2:1) with PEEP to conventional volume-controlled ventilation (I:E = 1:2) with PEEP in patients undergoing one-lung ventilation.
Lower P plat , higher P mean , total PEEP and CL were observed in IRV group compared with the control group. Lower P plat in IRV group was possibly because of the longer inspiratory time or slow inspiratory flow, and higher P mean was achieved by moderate prolongation of I:E ratio [11]. Prolonging inspiratory time resulted in increases in P mean and decreases in P peak or P plat in IRV group. Therefore, when applying low external PEEP, IRV is considered to be superior to conventional ratio ventilation in terms of gas exchange and respiratory mechanics in this study.
The mechanisms of improving oxygenation applying IRV were possibly as following: Firstly, P mean is thought to be a major determinant because it is correlated with mean alveolar pressure and alveolar recruitment [11]. Secondly, increased inspiratory time may have enough time to gas exchange effectively. Lastly, PEEP was an important  Data are mean ± SD, *Means statistically significant (p < 0.05). T1: At 5min after bronchial intubation; T3: At 45 min of one-lung ventilation; ScvO 2 : Oxygen saturation of central venous blood.  factor probably to improve oxygenation, which facilitated the mixing of gas, made collapse alveoli reopen and prevented atelectasis.
IRV would increase the P mean , recruit atelectatic alveoli, reduce intrapulmonary shunt, improve ventilation-perfusion mismatch and decrease dead space ventilation [12]. Prolonged inspiratory time allows enough time to gas exchange. Meanwhile, as expiratory time was shorter, inverse ratio ventilation might lead to air trapping in the lungs with generation of auto-PEEP (internal PEEP). PEEP could increase the mean airway pressure and improve oxygenation. Documents are proved that IRV may lead to intrinsic PEEP [13] and is thought to improve oxygenation and to have advantageous effects on lung mechanics [14]. Higher mean airway pressure was benefit to gas exchange and prevented from lung collapse. PaCO 2 was showed modest decrease in IRV group compared to the control group, there was no significance between two groups. IRV didn't affect elimination of CO 2 [15], which was agreement with the present study that IRV with PEEP didn't prolong the time to extubation and increase the postoperative pulmonary complications.
In our study, there was no significant difference on hemodynamic parameters between two groups, such as: SBP, DBP, HR and CVP. It was suggested that IRV (I:E = 1:2) applying PEEP didn't hamper venous return with possible consequent hemodynamic derangement. It is consistent with the previous statement that increasing the percentage of inspiratory time had no demonstrable changes in hemodynamic during mechanical ventilation [16].
In this experiment, the IL-1β, IL-6 and IL-8 of BALF were increased significantly at 45 min of one-lung ventilation in both groups, and levels of IL-1β, IL-6 and IL-8 in IRV group were significantly lower compared with the control group. One-lung ventilation caused inflammatory response possibly because inflammation factors were activated and released. Inflammatory factors play an important role in the stress response, including IL-1β, IL-6 and IL-8, which are important proinflammatory factor [17]. In the mechanical lung injury, IL-6 is one of the most important inflammatory mediators, its concentration was positively correlated with the degree of lung injury [18]. Neutrophils play a central role in the inflammatory response. Upon infection and/ or tissue damage, the neutrophils must first adhere to endothelium in response to chemokines presented at the endothelial interface and then migrate out of the microvasculature by following other chemokines secreted from nearby macrophages, mast cells, and other serosal cells. The neutrophils would then follow a series of end target chemoattractants such as IL-8 or LTB4 to the final site of infection [19]. In addition, damaged-molecular-patterns (DAMPs) are molecules that have a physiological role inside the cell, but acquire additional functions when they are exposed to the extracellular environment: they alert the body about danger, stimulate an inflammatory response, and finally promote the regeneration process. Beside their passive release by dead cells, some DAMPs can be secreted or exposed by living cells undergoing a life-threatening stress [20]. DAMPs could target the cytokines synthesis during hypoxic condition. They played a role in the inflammatory response, so that the release of the IL-1β, IL-6 and IL-8 would increase in lung tissue. The IL-1β, IL-6 and IL-8 were large released on the condition of hypoxia or lung tension. Studies suggested that inverse ratio ventilation could alleviate the inflammatory response.
The typical forms of lung injury are as following: volutrauma, barotraumas, atelectrauma and biological lung injury [21]. In this study, some degree of inflammation was already present probably due to surgery, trauma, anesthesia and injurious mechanical ventilation, and so on. Inverse ratio ventilation could low the P plat , increase P mean and alleviate the inflammatory response. It maybe alleviate acute lung injury. Using inverse ratio ventilation, we obtained good ventilation and prevented lung injury with PEEP. It is consistent with the protective ventilation strategy.
IRV is different from the normal ratio ventilation: there may be some shortcomings or potential risks, whether will cause respiratory dysfunction or other adverse effects remain to be studied further. Moreover, the other limitation of this study is that anesthesiologist is not blinded to ventilation strategy.
To sum up, IRV applying PEEP can improve hypoxemia, promote oxygenation and increase dynamic compliance of respiratory system. Therefore, when applying low external PEEP, IRV is considered to be superior to conventional ratio ventilation in terms of gas exchange and respiratory mechanics in patients during OLV.