Conceived and designed the experiments: LR SU. Performed the experiments: LR AK. Analyzed the data: LR SU. Contributed reagents/materials/analysis tools: LR SU. Wrote the paper: LR SU.
The authors have declared that no competing interests exist.
Mechanical ventilation (MV) of mice is increasingly required in experimental studies, but the conditions that allow stable ventilation of mice over several hours have not yet been fully defined. In addition, most previous studies documented vital parameters and lung mechanics only incompletely. The aim of the present study was to establish experimental conditions that keep these parameters within their physiological range over a period of 6 h. For this purpose, we also examined the effects of frequent short recruitment manoeuvres (RM) in healthy mice.
Mice were ventilated at low tidal volume VT = 8 mL/kg or high tidal volume VT = 16 mL/kg and a positive end-expiratory pressure (PEEP) of 2 or 6 cmH2O. RM were performed every 5 min, 60 min or not at all. Lung mechanics were followed by the forced oscillation technique. Blood pressure (BP), electrocardiogram (ECG), heart frequency (HF), oxygen saturation and body temperature were monitored. Blood gases, neutrophil-recruitment, microvascular permeability and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) and blood serum as well as histopathology of the lung were examined.
MV with repetitive RM every 5 min resulted in stable respiratory mechanics. Ventilation without RM worsened lung mechanics due to alveolar collapse, leading to impaired gas exchange. HF and BP were affected by anaesthesia, but not by ventilation. Microvascular permeability was highest in atelectatic lungs, whereas neutrophil-recruitment and structural changes were strongest in lungs ventilated with high tidal volume. The cytokines IL-6 and KC, but neither TNF nor IP-10, were elevated in the BAL and serum of all ventilated mice and were reduced by recurrent RM. Lung mechanics, oxygenation and pulmonary inflammation were improved by increased PEEP.
Recurrent RM maintain lung mechanics in their physiological range during low tidal volume ventilation of healthy mice by preventing atelectasis and reduce the development of pulmonary inflammation.
Mechanical ventilation (MV) of mice is increasingly used in biomedical research. While the mechanisms of ventilator-induced lung injury (VILI) have been explored intensively
Monitoring of key physiological parameters is standard during mechanical ventilation of humans and should be aimed also in experimental research. These key parameters need to reflect both the pulmonary (e.g. tidal volume, airway pressure) and the cardiovascular (e.g. heart rate, blood pressure) consequences of MV as well as oxygenation and acid-base status. Although MV may affect all these parameters, these entities have rarely been assessed together in the same study in mice (
Reference | Experimental design | Monitored parameters | ||||||
Ventilation | VT [ml/kg] | PEEP [cmH2O] | RM | Lung functions | BGA | BP/cardiac activity | SpO2 | |
|
30 min | 7/10 | 2/0 | * | + | − | −/P | + |
|
60 min | 8 | 6/3 | ** | + | − | − | − |
|
140 min | 30/10 | 2/0 | 2x/min | + | + | −/ECG | − |
|
150 min | 8 | 6/2 | 1x/5 min or 1x/75 min | + | − | −/P | + |
|
4 h | 25/7 | − | − | + | − | −/ECG | − |
|
4 h | 20/6 | 2 | − | − | − | − | − |
|
4 h | 30 | − | − | − | − | − | − |
|
4 h | 20/7 | 0−2 | − | − | − | − | − |
|
4 h | 8 | 4 | − | − | + | +/− | − |
|
5 h | 30/6 | − | − | − | − | −/− | − |
|
5 h | 15/7.5 | 2 | − | − | + | +/+ | − |
|
6 h | 24 | − | − | − | − | +/P | − |
|
6 h | 12 | 6 | * | − | + | +/P | + |
|
4 h/8 h | 20/10 | 2 | 1x/h | − | − | +/− | − |
|
8 h | 12 | 2 | − | − | + | +/− | − |
Present study | 6 h | 16/8 | 2 | 1x/5 min or 1x/60 min or no RM | + | + | +/ECG, P | + |
The table lists those ventilation studies that analyzed lung functions by measurement of lung impedance and studies in which mice were ventilated for at least four hours. VT: tidal volume, PEEP: positive end-expiratory pressure, RM: recruitment manoeuvre, BGA: blood gas analysis, BP: blood pressure, P: pulse, ECG: electrocardiogram, SpO2: pulse oximetry. * One RM at the beginning of ventilation. ** Two RMs at the beginning of ventilation.
Studies on the mechanisms of VILI have identified several beneficial ventilation strategies, among them low tidal volume (VT) ventilation and application of recruitment manoeuvres (RM) as well as high positive end-expiratory pressure (PEEP). Although RM have a sound physiological basis, it remains unclear how they should be applied
The present study had several aims: (1) In order to assess the consequences of MV properly, we established a set-up that permits the ventilation of mice under permanent monitoring of clinically relevant physiological parameters. (2) We used this set of parameters to define ventilatory conditions that guarantee stable lung mechanics, hemodynamics, acid base status and oxygenation over six hours. In particular, we studied the input impedance of the lung at low frequencies to distinguish mechanical properties of conductive airways and the distal lung
Experiments were performed with female C57BL/6 N mice (Charles River, Sulzfeld, Germany) aged 8 to 12 weeks, weighing 20 to 25 grams. The experimental protocols were in accordance with the German animal protection law and approved by regional governmental authorities (Landesamt für Natur, Umwelt und Verbraucherschutz NRW, permission number: AZ 8.87-50.10.35.085).
Mice were initially anaesthetized with an intraperitoneal injection of pentobarbital sodium [75 mg/kg] and fentanyl [40 µg/kg]. Anaesthesia was maintained with pentobarbital sodium [20 mg/kg] via an intraperitoneal catheter every 30 to 60 minutes. Mice were tracheotomized with a 20-gauge cannula and connected to the ventilator. A catheter was inserted into the carotid artery, which allowed blood pressure monitoring and permanent infusion of 0.9% NaCl (200 µL/h) to prevent hypovolaemia and thrombus formation. Pulsoxymetry was performed with a tail clip (MouseOx, STARR Life-Science, Oakmont, PA, USA). Blood pressure and ECG were recorded permanently (PowerLab, ADInstruments, Spenbach, Germany). Heart rate was calculated from the ECG. Body temperature was measured rectally and kept stable between 36.5°C and 37.5°C by a homeothermic blanket (Harvard Apparatus Holliston, MA, USA).
Mice were ventilated for six hours with the flexiVent ventilator (SCIREQ, Montreal, Canada). All mice survived the protocol. Mice were either ventilated with low tidal volume (lowVT) of 8 mL/kg and a frequency of 180 min−1 or high tidal volume (highVT) of 16 mL/kg and a frequency of 90 min−1, so that both groups received the same minute volume. The highVT group was ventilated with 3% CO2 to maintain normocapnia without further decreasing ventilation frequency. The fraction of inspired oxygen (FiO2) was 0.5 in all experiments. A positive end-expiratory pressure (PEEP) of either 2 cmH2O or 6 cmH2O was applied. One recruitment manoeuvre (RM) with 30 cmH2O pressure and six seconds duration was performed after onset of ventilation to open airspaces and standardize lung volume. Resistance, compliance and impedance of the lung were measured by the low-frequency forced oscillation technique every ten minutes.
Two different series of experiments were performed and analyzed separately (summarized in
Mice were ventilated for six hours with low VT = 8 mL/kg or high VT = 16 mL/kg, PEEP = 2 cmH2O and RM every five minutes (RM5), every 60 minutes (RM60) or without RM (noRM). Lung mechanics were measured every ten minutes by the forced oscillation technique. (LowVTRM5: n = 6, highVTRM5: n = 6, lowVTnoRM: n = 5, highVTnoRM: n = 4, lowVTRM60: n = 5). P-values for group comparisons are shown in supplementary
Electrocardiogram (ECG) was recorded permanently.
Arterial blood was analysed after six hours of ventilation.
Cytokines were quantified in blood serum or BAL supernatant with commercial ELISA kits after six hours of ventilation and in unventilated mice. (Unventilated: n = 5, lowVTRM5 n = 6, highVTRM5 n = 6, lowVTnoRM n = 4 in BAL and n = 5 in serum, highVTnoRM n = 4, lowVTRM60 n = 5). * p<0.05, ** p<0.01, *** p<0.001, § *** p<0.001 versus all other groups).
BAL fluid was subjected to cytospin preparation, followed by Diff-Quick staining. From each preparation 400 cells were counted and percentage of neutrophils was calculated. (Unventilated: n = 5, lowVTRM5 n = 5, highVTRM5 n = 5, lowVTnoRM n = 4, highVTnoRM n = 4, lowVTRM60 n = 5). § *** p<0.001 versus all other groups.
Representative lung sections stained with hematoxylin and eosin from:
Scoring criteria were: neutrophils in the alveolar or interstitial space, alveolar septal thickening, alveolar congestion and formation of hyaline membranes. Scores from 0 to 4 were given according to the number of fulfilled criteria. (Unventilated: n = 5, lowVTRM5 n = 5, highVTRM5 n = 5, lowVTnoRM n = 4, highVTnoRM n = 4, lowVTRM60 n = 5). ** p<0.01, *** p<0.001.
Mice were ventilated for six hours with low VT = 8 mL/kg, PEEP = 6 cmH2O and RM every five minutes (RM5), every 60 minutes (RM60) or without RM (noRM). Lung mechanics were measured with the forced oscillation technique every ten minutes. (n = 4 in all groups). Please see supplementary
Arterial blood was analysed after six hours of ventilation and the pO2/FiO2 ratio was calculated. (n = 4 in all groups). ** p<0.01, *** p<0.001.
Cytokines levels after six hours of ventilation were measured in blood serum or BAL supernatant by ELISA. (n = 4 in all groups). * p<0.05, ** p<0.01.
Leukocytes were counted after cytospin preparation of BAL fluid. From each preparation 400 cells were counted and percentage of neutrophils was calculated. (n = 4 in all groups). * p<0.05.
Mice received 1 mg bovine serum albumin (BSA) intravenously 90 min before exsanguination for analysis of microvascular permeability. Mice were sacrificed by exsanguination via the carotid artery. Blood samples from ventilated mice were analysed for pO2, pCO2, pH, HCO3− and standard base excess (SBE) by blood gas analysis (ABL700, Radiometer, Copenhagen, Denmark). Blood gas analysis from anaesthetized control mice was not representative due to reduced breathing activity.
First experimental series | Second experimental series | |||||||
LowVT RM5 | HighVT RM5 | LowVT noRM | HighVT noRM | LowVT RM60 | PEEP6 _RM5 | PEEP6 _noRM | PEEP6 _RM60 | |
|
8 | 16 | 8 | 16 | 8 | 8 | 8 | 8 |
|
180 | 90 | 180 | 90 | 180 | 180 | 180 | 180 |
|
2 | 2 | 2 | 2 | 2 | 6 | 6 | 6 |
|
12 | 12 | - | - | 1 | 12 | - | 1 |
|
0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
|
0 | 3 | 0 | 3 | 0 | 0 | 0 | 0 |
VT: tidal volume, f: breathing frequency, PEEP: positive end-expiratory pressure, RM: recruitment manoeuvre of 1 s and 30 cmH2O pressure, FiO2: fraction of inspired oxygen, lowVT: low tidal volume, highVT: high tidal volume, RM5: one recruitment manoeuvre every five minutes, noRM: no recruitment manoeuvres, RM60: one recruitment manoeuvre every 60 minutes, PEEP6: PEEP = 6 cmH2O.
After thoracotomy lungs were perfused free of blood with ice cold phosphate buffered saline. Bronchoalveolar lavage of the right lung was performed by instilling two times 300 µL NaCl via tracheal tubing. About 500 µL BAL fluid was retrieved from each mouse. BAL fluid was centrifuged and the supernatant was frozen for quantification of proteins. The pellet was transferred to a cytospin preparation, followed by a modified Giemsa stain (Diff-Quick; Medion Diagnostics, Düdingen, CH) and differential leukocyte count. Cytokine levels in serum and BAL fluid were quantified with commercial enzyme-linked immunosorbent assays (ELISA) (R+D Systems, Abingdon; UK). BSA was quantified in blood serum and BAL fluid by ELISA (Bethyl Laboratories, Montgomery, USA). The ratio of BSA in serum and BAL fluid was calculated to determine microvascular permeability. Additionally, total protein levels were measured using a DC protein assay (BioRad, Hercules, CA, USA).
The left lung was filled with 4% formalin for fixation and embedded in paraffin for histopathological examination. Sections of 3 µm thickness were stained with hematoxilin and eosin (HE). Histopathology was evaluated in a blinded manner. The following scoring system based on four criteria was used for each histological section: neutrophils in the alveolar or interstitial space, alveolar septal thickening, alveolar congestion and formation of hyaline membranes. Each criterion scored one point, if present, thus scores ranged from 0 (none criterion found) to 4 (all four criteria observed). Nine to ten sections per lung were evaluated and the mean was calculated for every lung and every group.
Lung mechanics were analysed with the mixed model procedure followed by correction for false discovery rate (FDR). To evaluate lung mechanics from the groups lowVTRM60 and PEEP6_RM60 in a linear model, one time point every 60 minutes before and one after RM were analyzed separately, resulting in two separate linear slopes (lowVTRM60a and PEEP6_RM60a: before RM, lowVTRM60b and PEEP6_RM60b: after RM). BoxCox transformation was performed to achieve homoscedasticity and normal distribution, when necessary. Analyses of parametric data were carried out with One-Way Analysis of Variance and FDR correction. Non-parametric data were analysed with the Kruskall-Wallis test followed by Dunn's post-test. Data in figures are shown as mean ± standard error of the mean (SEM). P-values<0.05 were considered as significant. Statistical analyses were carried out with JMP 7 or SAS 9.1 software (SAS Institute Inc., Cary, NC, USA).
The first series of experiments was performed at a PEEP = 2 cmH2O, a PEEP level that is commonly used in animal studies that aim to study VILI (see
These findings indicate that ventilation without RM leads to impaired lung functions, which stabilize within two hours, though at a low level. We therefore examined the effect of repetitive RM (30 cmH2O) every 5 min. In both the lowVTRM5 and the highVTRM5 group, lung mechanics stayed in a physiological range and remained unchanged during the whole experiment. Depending on the different tidal volumes, C and H differed reciprocally between low- and highVT groups (p<0.05). The stability of the lung mechanics demonstrates that application of deep inflations of 1 s duration and 30 cmH2O are sufficient to prevent the deterioration of lung functions in healthy mouse lungs ventilated with 2 cmH2O PEEP and moderate tidal volumes.
Further, we examined whether it would suffice to apply RM only every 60 min instead of every 5 min. This was studied in the low VT group (lowVTRM60) only. In this group, respiratory conditions were instable. R and C worsened comparable to the lowVTnoRM group, but improved significantly after each RM. These alterations were reflected in changes in G and H. However, even though each RM was beneficial, after six hours tissue elastance had increased permanently, indicating that one deep inflation per hour is not sufficient to maintain lung volume at base line values. This is illustrated by the finding that H (p<0.001), R, C, G (all p<0.01) and Raw (p<0.05) were significantly different between the lowVTRM60 and the lowVTRM5 group.
Anaesthesia with pentobarbital sodium resulted in reduced heart frequency (300–400 min−1) and mean blood pressure (50–60 mmHg), compared to average data of unsedated mice. ECG (data not shown), HF and BP remained unchanged throughout ventilation and were not significantly different between the groups (
Blood gas analyses revealed that infusion of saline and application of 3% CO2 in the high VT groups were adequate to keep the acid-base status stable and that ventilation resulted in normocapnia in the lowVTRM5 and highVTRM5 group, with pCO2 levels ranging from 35 to 40 mmHg (
LowVTRM5 | HighVTRM5 | LowVTnoRM | HighVTnoRM | LowVTRM60 | |
|
283.5±13.4 | 287.7±12.7 | 108.6±13.6 | 261.8±17.8 | 205.5±72.4 |
|
38.4±3.9 | 34.9±4.0 | 58.3±7.61 | 40.6±15.1 | 50.1±9.4 |
|
7.33±0.03 | 7.36±0.04 | 7.22±0.03 | 7.32±0.08 | 7.28±0.05 |
|
19.5±1.6 | 19.2±1.6 | 23.6±2.3 | 19.7±3.4 | 21.8±1.9 |
|
− 6.0±1.6 | − 5.5±1.7 | − 3.5±2.1 | − 5.4±2.3 | − 3.5±1.6 |
Blood gas analyses from arterial blood after six hours of mechanical ventilation with PEEP = 2 cmH2O. Data are shown as mean ± standard deviation. SBE: standard base excess, lowVT: low tidal volume, highVT: high tidal volume, RM5: one recruitment manoeuvre every five minutes, noRM: no recruitment manoeuvres, RM60: one recruitment manoeuvre every 60 minutes.
Total protein levels were increased in all ventilated mice, compared to unventilated controls. Protein levels were highest in the groups without RM (
Interleukin-6 (IL-6) and Keratinocyte-derived chemokine (KC) were elevated in the BAL fluid and blood serum from all ventilated mice (
The only cell types found in BAL fluid of unventilated control mice were monocytes and macrophages. In contrast, all samples from ventilated mice also contained neutrophils (
Histopathological samples revealed that no severe lung injury was induced by the ventilation strategies applied (
In the second series of experiments the ventilation strategies RM5, noRM and RM60 were applied to mice ventilated with low VT and a PEEP of 6cmH2O, to find out whether the impairment of lung mechanics during ventilation with noRM and RM60 could be prevented by a higher PEEP (
One aim of the second series of experiments was to investigate whether a higher PEEP can prevent the worsening of gas exchange in mice ventilated at a PEEP = 2 cmH2O and RM60 or noRM. The pO2/FiO2 ratio was around 500 mmHg in the PEEP6_RM60 group and around 400 mmHg in the PEEP6_noRM group (
In the second set of experiments IL-6 and KC levels in the BAL fluid were highest in the PEEP6_noRM group, followed by the PEEP6_RM5 group (
In the second part of the study, in which all mice were ventilated with low VT, neutrophil numbers were clearly highest in the group PEEP = 6_noRM. Numbers of neutrophils were lower in the BAL fluid from PEEP6_RM5 mice and lowest in the PEEP6_RM60 group (
There is an increasing demand for mechanical ventilation of mice, not only in pulmonary research, but also in many other areas such as neurology
The effects of repetitive RM are not well established neither in experimental animals nor in men, one reason being that trials are difficult to compare
We demonstrated that application of deep inflations of 1 s duration and 30 cmH2O peak pressure in five minute intervals was sufficient to keep respiratory mechanics stable and thereby lung volume constant in low and high VT ventilation, without increasing PEEP over 2 cmH2O (
In order to keep blood gases stable, the low and high VT groups had to be ventilated with different respiratory rates and, based on other studies
A pressure of 30 cmH2O for each RM was necessary to maintain lung volume stable. In accordance with previous studies
In fact, lung volume declined only to a certain threshold and a plateau phase was reached in all mice ventilated without RM. This supports the hypothesis that depending on the pressure applied to the lung some populations of alveoli are open and others closed
Further evidence for the formation of atelectasis was given by blood gas analysis, which revealed impaired gas exchange in lowVTnoRM and lowVTRM60 mice (
Nonetheless, protein leakage, indicating increased microvascular permeability, another hallmark of VILI, was highest in those low VT groups that developed atelectasis due to lacking adequate RM (noRM, RM60) (
The present study demonstrates that repetitive recruitment manoeuvres are not only beneficial for lung functions, gas exchange and barrier function, but also reduce the liberation of pro-inflammatory cytokines (
This study aimed to define settings for ventilation under stable conditions and therefore a second set of experiments was performed with a PEEP of 6 cmH2O, to address the question whether a higher PEEP could prevent the severe impairment of lung functions and the effects on oxygenation and inflammation, which were observed in the lowVTnoRM and lowVTRM60 groups ventilated with a PEEP of 2 cmH2O. Lung mechanics and blood gas results revealed that a higher PEEP, even when combined with RM60, was not sufficient to prevent formation of atelectasis. C decreased and R, G and H increased strongly during the first 180 min of the experiments in the PEEP6_noRM group before parameters reached a plateau. A previous study demonstrated that an increase in PEEP from 2 cmH2O to 6 cmH2O alone did not prevent an increase in Raw, G and H during 150 min of ventilation, but was more effective during pressure controlled RM
It is well known that PEEP plays a critical role in preventing/reducing lung injury
We compared lung mechanics during ventilation with and without RM in healthy mice over six hours during close monitoring of many clinically relevant parameters including lung mechanics, cardiovascular functions and gas exchange. Stable cardiovascular conditions resulted amongst others from appropriate fluid support, emphasizing the need for careful monitoring and stabilisation of vital parameters in animal studies.
The present study shows that protective non-injurious ventilation requires the application of frequent non-injurious recruitment manoeuvres and low VT. A ventilation pattern including deep inflations is closer to the variable breathing pattern of spontaneously breathing subjects than monotonic MV without variation in VT or breathing frequency, supporting the finding that variable or ‘noisy’ ventilation improves protective ventilation in the porcine and human lung
Furthermore, our data indicate that a PEEP level of 6 cmH2O has protective effects and is therefore advisable in studies that require MV. A low PEEP of 2 cmH2O combined with recurrent RM is less protective, but still suffices to gain stable respiratory conditions and may be preferable in models that aim to investigate VILI.
We conclude that ventilation with low VT, recurrent RM and sizable PEEP is the most protective ventilation strategy for healthy mice.
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