Consecutive Daily Administration of Intratracheal Surfactant and Human Mesenchymal Stem Cells Attenuates Hyperoxia-induced Lung Injury in Neonatal Rats

Background: Surfactant therapy is a standard of care for preterm infants with respiratory distress and reduces the incidence of death and bronchopulmonary dysplasia in these patients. Mesenchymal stem cells (MSCs) attenuated hyperoxia-induced lung injury. Surfactant reduced the in vitro viability of human MSCs, and the combination therapy of surfactant and MSCs did not have additive effects on hyperoxia-induced lung injury in neonatal rats. The effects of 2 consecutive days of intratracheal administration of surfactant and MSCs on hyperoxia-induced lung injury were undetermined. Methods: Neonatal Sprague Dawley rats were reared in either room air (RA) or hyperoxia (85% O 2 ) from postnatal days 1 to 14. On postnatal day 4, the rats received intratracheal injections of either 20 μL of normal saline (NS) or 20 μL of surfactant. On postnatal day 5, the rats reared in RA received intratracheal NS, and the rats reared in O 2 received intratracheal NS or human MSCs (3 × 10 4 or 3 × 10 5 cells). Six study groups were examined: RA + NS + NS, RA + surfactant + NS, O 2 + NS + NS, O 2 + surfactant + NS, O 2 + surfactant + MSCs (3 × 10 4 cells), and O 2 + surfactant + MSCs (3 × 10 5 cells). The lungs were excised for analysis on postnatal day 14. Results: The rats reared in hyperoxia and treated with NS yielded signicantly higher mean linear intercepts (MLIs) and cytokine levels and signicantly lower vascular endothelial growth factors (VEGFs), platelet-derived growth factor protein expression, and vascular density than did those reared in RA and treated with NS or surfactant. The lowered MLIs and cytokine levels and the increased VEGF expression and vascular density indicated that the surfactant and surfactant + MSCs (3 × 10 4 cells) treatment attenuated hyperoxia-induced lung injury. The surfactant + MSCs (3 × 10 5 cells) group

Results: The rats reared in hyperoxia and treated with NS yielded signi cantly higher mean linear intercepts (MLIs) and cytokine levels and signi cantly lower vascular endothelial growth factors (VEGFs), platelet-derived growth factor protein expression, and vascular density than did those reared in RA and treated with NS or surfactant. The lowered MLIs and cytokine levels and the increased VEGF expression and vascular density indicated that the surfactant and surfactant + MSCs (3 × 10 4 cells) treatment attenuated hyperoxia-induced lung injury. The surfactant + MSCs (3 × 10 5 cells) group exhibited a signi cantly lower MLI and signi cantly higher VEGF expression and vascular density than the surfactant + MSCs (3 × 10 4 cells) group did.
Conclusions: Consecutive daily administration of intratracheal surfactant and MSCs can be an effective regimen for treating hyperoxia-induced lung injury in neonates.

Background
Supraphysiological oxygen is often required to treat newborns with respiratory disorders. However, administering supplemental oxygen to newborn infants with respiratory failure can lead to lung injury.
Term-born rat models are appropriate for studying the effects of hyperoxia on preterm infants with respiratory distress because rats are born at the saccular stage, which is approximately equivalent to a human gestational age of 30 weeks [1]. The prolonged exposure of neonatal rats to hyperoxia results in a decrease in alveolarization and vascularization similar to human bronchopulmonary dysplasia (BPD) [2,3]. The pathogenesis of BPD is multifactorial, and oxygen toxicity plays a crucial role in the process of lung injury leading to BPD [4,5].
Surfactant therapy is a standard of care for preterm infants with respiratory distress syndrome and can reduce the incidence of death and BPD [6]. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have immunomodulatory, anti-in ammatory, and regenerative properties and have been demonstrated to treat hyperoxia-induced lung injury in newborn animals [7][8][9][10][11][12][13][14]. In this study, we demonstrated that the addition of surfactant reduced the in vitro viability of human MSCs through mitochondrial dysfunction and that a combination therapy of surfactant and MSCs had no additive effects on lung development in neonatal rats exposed to hyperoxia [14]. The effects of 2 consecutive days of intratracheal administration of surfactant and MSCs on hyperoxia-induced lung injury were undetermined. We hypothesized that consecutive daily administration of intratracheal surfactant and MSCs improves lung development and that high doses of MSCs enhance this effect on experimental BPD in neonatal rats. The aim of this study was to investigate the effects of an animal-derived surfactant (Survanta) and consecutive daily administration of human MSCs on hyperoxia-induced lung injury in neonatal rats.

Surface tension
Survanta (AbbVie Inc., North Chicago, IL, USA) was prepared through lipid extraction from minced bovine lungs and contained approximately 84% phospholipids, 1% hydrophobic surfactant proteins (SP-B and SP-C), and 6% free fatty acids. The solution was supplied at a 25 mg/mL concentration of phospholipids suspended in a 0.9% sodium chloride solution. The surface tension of Survanta (25 mg/mL), Survanta (12.5 mg/mL), and Survanta (25 mg/mL) and the human MSCs (1.5 ´ 10 6 cells/mL) was determined at a volume of 2 mL by using a surfactometer (Amherst Electronics, Buffalo, NY, USA). Each sample was measured three times.

Isolation of human mesenchymal stem cells
The MSCs were isolated from human umbilical cords, as previously described [12]. The MSCs were to laboratory food and water, kept on a 12:12-h light-dark cycle, and allowed to deliver vaginally at term. Within 12 h of birth, the litters were pooled and randomly redistributed to the newly delivered mothers; the pups were then randomly assigned to room air (RA) or oxygen-enriched atmosphere (85% O 2 ) groups for postnatal days 1-14. The nursing mothers were rotated between the 85% O 2 and the RA groups every 24 h to prevent oxygen toxicity in the mothers and to eliminate differing maternal effects between the groups. An oxygen-rich atmosphere was maintained in a transparent 40 × 50 × 60-cm 3 plexiglass chamber receiving continuous O 2 at 4 L/min. The oxygen concentration inside the hyperoxic plexiglass chamber was continuously monitored using an oxygen sensor (Coy Laboratory Products, Grass Lake, MI, USA). On postnatal day 4, the rats received intratracheal injections of either 20 μL of normal saline (NS) or 20 μL of surfactant (Survanta, AbbVie Inc.), corresponding to approximately 50 mg/kg of phospholipids ( Fig. 1). On postnatal day 5, the rats reared in RA were treated with NS and those reared in The lungs were excised for histological, western blot, and cytokine analyses on postnatal day 14.

Intratracheal administration of surfactant and human MSCs
For intratracheal transplantation, the rats were anesthetized with iso urane and restrained on a board at a xed angle as described by Chen et al. [15]. On postnatal day 4, the rats received intratracheal injections of either 20 μL of normal saline (NS) or 20 μL of surfactant. On postnatal day 5, the rats reared in RA received intratracheal NS, and the rats reared in O 2 received intratracheal NS or human MSCs (3 × 10 4 or 3 × 10 5 cells).

Lung histology
The lungs were placed in 4% paraformaldehyde, washed with phosphate-buffered saline, and then serially dehydrated in increasing concentrations of ethanol before being embedded in para n. To standardize the analyses, lung sections were taken from the right middle lobe. Sections of tissue weighing 5 μm were stained with hematoxylin and eosin, examined using light microscopy, and assessed for lung histology.
The mean linear intercept (MLI), an indicator of the mean alveolar diameter, was assessed in 10 nonoverlapping elds [14].

Immunohistochemistry of lung vascular endothelial growth factor and von Willebrand factor
Immunohistochemical staining was performed on the 5-μm para n sections through immunoperoxidase visualization. After routine depara nization, heat-induced epitope retrieval was performed by immersing the slides in 0.01 M sodium citrate buffer (pH 6.0). To block the endogenous peroxidase activity and the nonspeci c binding of antibodies, the sections were preincubated for 1 h at room temperature in 0. ImmunoResesarch Laboratories Inc., West Grove, PA, USA). After the reagents from an avidin-biotin complex kit (Vector Laboratories, Inc., CA, USA) produced a reaction, the reaction products were visualized with a diaminobenzidine substrate kit (Vector Laboratories Inc.) in accordance with the recommendations of the manufacturer. Pulmonary vessel density was determined by counting the number of vessels with positive vWFs stained in an unbiased manner by using a minimum of four random lung elds at ×400 magni cation [16].

Western blot analysis of growth factors
The lung tissues were homogenized in ice-cold buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, and a protease inhibitor cocktail (complete minitablets; Roche, Mannheim, Germany). The samples were sonicated and then centrifuged at 500 g for 20 min at 4°C to remove cellular debris. Densitometric analysis was performed with AIDA software to measure the intensity of VEGF, PDGF-B, and β-actin bands.

Lung cytokine levels
The lung tissue was homogenized in 1 mL of ice-cold lysis buffer containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.01 M deoxycholic acid, and a complete protease cocktail inhibitor. Cell extracts were centrifuged, and the levels of interleukin (IL)-1β and IL-6 in the supernatants were measured with an enzyme-linked immunosorbent assay kit (Cloud-Clone Corp., Houston, TX, USA).

Statistical analysis
The data are presented as means ± standard deviations. Statistical analyses were performed using oneway ANOVA with the Bonferroni post hoc test for the multiple-group comparisons. The survival rate was evaluated by using the Kaplan-Meier method, and the log-rank test was used for the intergroup comparisons. Differences were considered statistically significant when P < 0.05.

Survival rate
All the rats reared in RA and treated with NS or surfactant survived (Fig. 2). In the O 2 + NS + NS group, one, one, three, and one rats died on postnatal days 5, 9, 10, and 11, respectively. In the O 2 + surfactant +

Body and lung weight and lung-to-body-weight ratio
The body and lung weights and the lung-to-body-weight ratios on postnatal day 14 were comparable among the six study groups (Table 1).

Immunohistochemistry and western blotting of VEGF
The VEGF immunoreactivities were primarily detected in the endothelial cells (Fig. 5a). The rats reared in hyperoxia and treated with NS exhibited signi cantly lower VEGF protein expression than did those reared in RA and treated with NS or surfactant (Fig. 5b). Treatment with surfactant and MSCs (3 × 10 4 or 3 × 10 5 cells) signi cantly augmented the hyperoxia-induced decrease in VEGF protein expression compared with treatment with NS.
Western blot analysis of PDGF Fig. 6 shows the representative western blot of PDGF-A and PDGF-B. The rats reared in hyperoxia and treated with NS exhibited signi cantly lower PDGF-A and PDGF-B protein expression than did those reared in RA and treated with NS or surfactant. Treatment with surfactant and MSCs (3 × 10 5 cells) signi cantly augmented the hyperoxia-induced decrease in the PDGF-A and PDGF-B protein expression compared with treatment with NS.

Lung cytokine levels
The rats reared in hyperoxia and treated with NS yielded signi cantly higher IL-1β and IL-6 levels on postnatal day 14 than did those reared in RA and treated with NS or surfactant on postnatal (Fig. 7). The treatment with surfactant and the treatment with surfactant and MSCs signi cantly diminished the hyperoxia-induced increase in IL-1β and IL-6 levels. Although term-born rats have structurally immature lungs, they are functionally mature and require no surfactant treatment. In one study, neonatal mice exposed to hyperoxia for 4 days exhibited disruptions to type II cell proliferation, which produced pulmonary surfactant [17]. In another study, hyperoxia during the rst 3 days of life induced in ammatory cell in ltration in alveolar spaces and increased the wet-todry lung weight ratio in neonatal Sprague Dawley rats [18]. The results of these studies have suggested that hyperoxia reduces surfactant production and induces lung in ammation in newborn animals. Pulmonary surfactant is a mixture of phospholipids, surfactant-associated proteins, and neutral lipids, which modulate pulmonary in ammation and stabilize the alveoli by reducing surface tension [19]. In our study, the administration of surfactant on postnatal day 4 diminished the hyperoxia-induced increase in MLI and lung cytokines in the neonatal rats. The surfactant treatment did not augment the hyperoxiainduced decrease in pulmonary vascular density. These results support the idea that pulmonary surfactant ful lls an essential role in the lungs for both host defense mechanisms, such as modulating pulmonary in ammation, and for improving alveolarization [14,20].

Discussion
Surfactant therapy has become the standard of care for preterm infants with respiratory distress syndrome and can reduce the combined outcomes of death and BPD [6]. Compared with delayed surfactant treatment, early surfactant treatment was more effective in reducing mortality, air leak, BPD, and BPD or death in preterm infants [21]. Although surfactant has a unique spreading property and can reduce surface tension. The addition of a surfactant reduced the in vitro viability of human MSCs, and the combination therapy of surfactant and MSCs did not exhibit any additional bene ts to lung development in neonatal rats exposed to hyperoxia [14]. For this reason, we administered intratracheal surfactant and MSCs on 2 consecutive days and found that the intratracheal administration of surfactant on postnatal day 4 and MSCs on postnatal day 5 improved alveolarization and angiogenesis in the neonatal rats exposed to hyperoxia. The time interval between the administration of surfactant and the MSCs for achieving optimal therapeutic effects was not determined. Future studies are required to evaluate the effects of different time intervals on hyperoxia-induced lung injury.
In this study, the administration of surfactant and the administration of surfactant with human MSCs to the hyperoxia-exposed rats signi cantly improved lung development in the surviving animals, although the survival rate did not signi cantly improve. The differences in the survival rates between rats treated with surfactant and those treated with surfactant and MSCs were not signi cant on postnatal day 14. The rats reared in hyperoxia and treated with NS exhibited a low survival rate after postnatal day 5. The treatment with surfactant and treatment with surfactant and human MSCs (3 × 10 5 cells) maintained the survival rate from postnatal days 5 to 9. These results suggest that an additional dose of MSCs is required to maintain the survival rate.
In this study, we determined the levels of VEGF, PDGF-A, and PDGF-B expression and elucidated the mechanisms that mediate the MSCs' effects because their mRNA and protein expression decreased in the lungs of newborn animals exposed to 14 days of hyperoxia [22][23][24]. VEGF is a potent endothelial cell mitogen that regulates angiogenesis and alveolar development [25]. PDGF is crucial to alveolarization of normally developing lungs [26]. We demonstrated that the rats reared in hyperoxia and treated with NS exhibited signi cantly lower levels of VEGF, PDGF-A, and PDGF-B protein expression than did those reared in RA and treated with NS or surfactant. Treatment with surfactant and MSCs augmented the hyperoxiainduced decrease in the VEGF, PDGF-A, and PDGF-B protein expression levels. These results suggest that treatment with MSCs enhanced vascular and alveolar development in the neonatal rats through the induction of growth factors.

Conclusions
Consecutive daily administration of intratracheal surfactant and human MSCs likely attenuated hyperoxia-induced defective alveolarization and angiogenesis by increasing VEGF expression. High doses of MSCs enhanced the therapeutic effects more effectively than the low doses of MSCs. Consecutive daily administration of intratracheal surfactant and MSCs can be an effective regimen for treating hyperoxia-induced lung injury in neonates.