Alveolar epithelial type II cells (AECIIs) containing lamellar bodies (LBs) are alveolar epithelial stem cells that have important functions in the repair of lung structure and function after lung injury. The ultrastructural changes in AECIIs after high-frequency oscillatory ventilation (HFOV) with a high lung volume strategy or conventional ventilation were evaluated in a newborn piglet model with acute lung injury (ALI). After ALI with saline lavage, newborn piglets were randomly assigned into five study groups (three piglets in each group), namely, control (no mechanical ventilation), conventional ventilation for 24 h, conventional ventilation for 48 h, HFOV for 24 h, and HFOV for 48 h. The lower tissues of the right lung were obtained to observe the AECII ultrastructure. AECIIs with reduced numbers of microvilli, decreased LBs electron density, and vacuole-like LBs deformity were commonly observed in all five groups. Compared with conventional ventilation groups, the decrease in numbers of microvilli and LBs electron density, as well as LBs with vacuole-like appearance and polymorphic deformity, was less severe in HFOV with high lung volume strategy groups. AECIIs were injured during mechanical ventilation. HFOV with a high lung volume strategy resulted in less AECII damage than conventional ventilation.
The use of mechanical ventilation in clinical practice has increased the survival rate of newborns. However, respiratory and neurological complications or sequelae have increased because of ventilator-induced lung injury (VILI), a phenomenon caused or aggravated by mechanical ventilation. Alveolar epithelial type II cells (AECIIs) are alveolar epithelial stem cells targeted in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) or VILI. The structural and functional changes in AECIIs are related to ALI, ARDS, and VILI [
High-frequency oscillatory ventilation (HFOV) is a lung protective ventilation strategy in newborns with ALI or ARDS. However, data on the effect of HFOV on the ultrastructural features of AECIIs are limited. The practical application value of HFOV is still controversial. This study used a newborn piglet model with ALI to study the effect of conventional ventilation and HFOV with a high lung volume strategy on AECIIs. The ultrastructural features of AECIIs after HFOV with a high lung volume strategy in a newborn piglet model with ALI were evaluated.
Fifteen newborn piglets (≤3 days old, weighing 1.0 kg to 1.97 kg) were used. All animal experiments were performed with the approval of the Guangdong Second Provincial People’s Hospital Animal Care and Use Committee (2009-XEK-028).
Piglets were given 10% chloral hydrate (1 mL/kg) orally and placed in a supine position under an infant radiant warmer. The body temperature of the animals was maintained in the range of 38.0°C to 39.5°C. Catheters were inserted in the axillary vein and the femoral artery for injecting medication and fluids, as well as for blood gas analysis and arterial blood pressure monitoring. The maintenance fluids included continuous infusion of 0.9% saline solution containing 5% dextrose (120 mL/kg/d). A dopamine infusion (5
Using the pressure control mode, we set the initial ventilator settings to a positive end-expiratory pressure (PEEP) of 2 cm H2O, a peak inspiratory pressure of 10 cm H2O, an inspiratory-to-expiratory ratio of 1 : 2, and a fraction of inspired oxygen (FiO2) of 0.30. The breathing frequency was set to 25 breaths/min to 30 breaths/min and then adjusted to maintain the pressure of arterial carbon dioxide (PaCO2) in the normal range (35 mm Hg to 45 mm Hg).
ALI was induced by lavaging the whole lung with normal saline. During lavage, all piglets were ventilated by a conventional mechanical ventilator using the pressure control mode. Warmed (37°C) normal saline (35 mL/kg) was instilled into the lung via the endotracheal tube. Saline was allowed to remain in the lung for 10 s before removal. Lung lavage was repeated at 5 min intervals until the pressure of arterial oxygen was below 100 mm Hg for 60 min at the following ventilator settings: peak inspiratory pressure, 24 cm H2O; PEEP, 6 cm H2O; inspiratory-to-expiratory ratio, 1 : 2; FIO2, 1.0; and breathing frequency, 35 breaths/min.
After lung injury was established, the piglets were randomly assigned into five study groups (with three piglets in each group): control (receiving no ventilation), conventional ventilation for 24 h, conventional ventilation for 48 h, HFOV for 24 h, and HFOV for 48 h. The control piglets were sacrificed by overdosing with 10% potassium chloride under deep anesthesia. In the conventional ventilation group, the piglets were ventilated using a conventional mechanical ventilator (Servo-i) in the pressure control mode at the following ventilator settings: peak inspiratory pressure, 20 cm H2O; PEEP, 4 cm H2O; inspiratory-to-expiratory ratio, 1 : 2; and FIO2, 1.0. The breathing frequency was set to 25 breaths/min to 30 breaths/min and adjusted to maintain PaCO2 in the normal range (35 mm Hg to 45 mm Hg). In the HFOV group, the piglets were placed in HFOV (SLE-5000; Tokibo, Tokyo, Japan) with an oscillatory frequency of 10 Hz, a fractional inspiratory time of 33%, and an FIO2 of 1.0. The mean airway pressure was set to 2 cm H2O higher than that in conventional ventilation, which indicated a high lung volume strategy in HFOV. The amplitude was set to the range of 20 cm H2O to 25 cm H2O and adjusted to maintain PaCO2 in the normal range (35 mm Hg to 45 mm Hg). During ventilation, FIO2 was decreased by 10% every 6 h up to 40%. At the end of ventilation, the piglets were sacrificed by overdosing with 10% potassium chloride under deep anesthesia. The lungs were immediately removed, and samples (1 mm3) were obtained from the lower parts of the right lung and fixed with 2.5% glutaraldehyde.
The samples were prefixed in 2.5% glutaraldehyde, postfixed in 1% osmic acid, and then embedded in Epon 812. Ultrathin sections prepared using an ultramicrotome were stained with uranyl and lead citrate. AECIIs were examined under a transmission electron microscope by two blinded, independent observers.
The ultrastructure of AECIIs showed tight interaction with the basal membrane and AECIs. The nuclei were round and clear, and the chromatin inside each nucleus was homogeneous. Lamellar bodies (LBs) with uniform density and ring-like arrangement were present. Some LBs showed decreased electron density and vacuole-like deformity. Microvilli were displayed distinctly (Figure
Ultrastructural changes of AECIIs in control group (×9700). The nuclei were round and clear. Some LBs were presented as decreased in electron density and vacuole-like deformity; some LBs with density uniformity and ring-like arrangement were shown.
In both the conventional ventilation 24 h and 48 h groups, LBs were arranged around the nucleus in reduced numbers and with decreased electron density. Some AECIIs showed shrunken nuclei containing nonhomogenous, condensed chromatin.
In the conventional ventilation 24 h group (Figure
Ultrastructural changes of AECIIs in conventional ventilation 24 h group (×5800). The secretion of AECII discharged into alveolar space was observed.
At 48 h, some AECIIs were dislodged from the basal membrane (Figure
Ultrastructural changes of AECIIs in conventional ventilation 48 h group (×9700). AECII without nucleus was observed. Microvilli were irregularly arranged.
In the HFOV 24 h group, the juxtaposition of AECIIs to the basal membrane and AECIs was close (Figure
Ultrastructural changes of AECII in HFOV 24 h group (×9700). The juxtaposition of AECII to basal membrane and AECI was close.
Except for small numbers of AECIIs, most of the cells interacted tightly with the basal membrane and AECI under continuous ventilation on HFOV up to 48 h (Figure
Ultrastructural changes of AECIIs in HFOV 48 h group (×5800). The incompletely vacuole-like deformity in LB was presented.
AECIIs cover approximately 4% of the mammalian alveolar surface but constitute 15% of all lung cells as multifunctional cells. AECIIs participate in the defense and pathogenesis of infection. AECIIs also perform various important functions within the lungs, including regulation of surfactant metabolism, ion transport, and alveolar repair in response to injury. Clinically, obtaining lung tissue from neonates on a mechanical ventilator to study AECIIs is difficult. Therefore, we used newborn piglets as models to observe the ultrastructural features of AECIIs in animals with lung injury treated by conventional ventilation or HFOV.
Figure
The results of transmission electron microscopy showed that the juxtaposition of AECIIs in the HFOV 48 h group to the basal membrane and AECIs was closer in ventilated lungs and with the extension of ventilation time. In addition, the electron density of LB in the HFOV 48 h group decreased less than that in the conventional ventilation 48 h group. Lamellar structure was clearly observed in the HFOV 48 h group. This result suggested that HFOV caused less damage on AECIIs than conventional ventilation. Stüber et al. [
AECIIs were injured when newborn piglets were ventilated. AECIIs from piglets under HFOV with a high lung volume strategy showed better structural integrity compared with AECIIs from piglets maintained under conventional ventilation.
The samples of both the control and study groups were small in this study. We concentrated only on the ultrastructural changes in AECIIs following HFOV with a high lung volume strategy or conventional ventilation in a newborn piglet model with ALI. However, our results were all descriptive analysis and lacked quantitative presentation of data. Notwithstanding its limitations, this study is interesting and suggests that AECIIs are injured during mechanical ventilation and that HFOV with a high lung volume strategy results in less AECII damage than conventional ventilation. The sample size and quantitative analysis must be, respectively, increased and broadened to study the possible mechanisms of HFOV to minimize injury.
This study was partly supported by Grant no. 2010B031600258 from the Guangdong Science and Technology Department.