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. 2013 Aug 29;8(8):e72574.
doi: 10.1371/journal.pone.0072574. eCollection 2013.

Protective effect of surfactant inhalation against warm ischemic injury in an isolated rat lung ventilation model

Akihiro Ohsumi  1 , Fengshi Chen, Jin Sakamoto, Daisuke Nakajima, Masashi Kobayashi, Toru Bando, Hiroshi Date
Affiliations

Protective effect of surfactant inhalation against warm ischemic injury in an isolated rat lung ventilation model

Akihiro Ohsumi et al. PLoS One. .

Abstract

Warm ischemia-reperfusion injury remains a crucial issue in transplantation following the cardiac death of donors. Previously, we showed that surfactant inhalation during warm ischemia mitigated ischemia-reperfusion injury. This study investigated the mechanisms of surfactant inhalation protection of the warm ischemic lung after reoxygenation with ventilation alone. In an isolated rat lung ventilation model, cardiac arrest was induced in the CTRL (control) and SURF (surfactant treatment) groups by ventricular fibrillation. Ventilation was restarted 110 min later; the lungs were flushed, and a heart and lung block was procured. In the SURF group, a natural bovine surfactant (Surfacten®) was inhaled for 3 min at the end of warm ischemia. In the Sham (no ischemia) group, lungs were flushed, procured, and ventilated in the same way. Afterwards, the lungs were ventilated with room air without reperfusion for 60 min. Surfactant inhalation significantly improved dynamic compliance and airway resistance. Moreover, surfactant inhalation significantly decreased inducible nitric oxide synthase and caspase-3 transcript levels, and increased those of Bcl-2 and surfactant protein-C. Immunohistochemically, lungs in the SURF group showed weaker staining for 8-hydroxy-2'-deoxyguanosine, inducible nitric oxide synthase, and apoptosis, and stronger staining for Bcl-2 and surfactant protein-C. Our results indicate that surfactant inhalation in the last phase of warm ischemia mitigated the injury resulting from reoxygenation after warm ischemia. The reduction in oxidative damage and the inhibition of apoptosis might contribute to the protection of the warm ischemic lungs.

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Conflict of interest statement

Competing Interests: These authors have no support or funding to report.

Figures

Figure 1
Figure 1. Physiological lung functions during ventilation.
(A) Airway resistance. (B) Dynamic compliance. **p<0.01 or ***p<0.001 between the Sham (open circles) and CTRL groups (boxes). †p<0.05 or ††p<0.01 or †††p<0.001 between the CTRL and SURF groups (solid circles). §p<0.05 between the Sham and SURF groups. Data are expressed as mean values ± SD (BL = baseline).
Figure 2
Figure 2. Adenine nucleotide levels.
ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; dw, dry weight. Data are shown as mean ± SD.
Figure 3
Figure 3. mRNA expression of cytokines after reperfusion.
(A) iNOS mRNA levels. (B) Caspase-3 mRNA levels. (C) Bcl-2 mRNA levels. (D) Bax mRNA levels. (E) SP-C mRNA levels. All values are expressed as the mean ± SD.
Figure 4
Figure 4. Caspase Activation Assay.
All values are expressed as the mean ± SD.
Figure 5
Figure 5. Immunohistochemical staining and the assessment for oxidative damage (original magnification 400 ×).
(A) Sham group. (B) CTRL group. (C) SURF group. In the 8-OHdG staining, the ratio of 8-OHdG-positive cells was significantly lower in the Sham and SURF groups than that in the CTRL group (D). All values are expressed as the mean ± SD.
Figure 6
Figure 6. Immunohistochemical staining for iNOS (original magnification 400 ×).
(A) Sham group. (B) CTRL group. (C) SURF group. Arrows indicate iNOS positive cells. The ratio of iNOS-positive cells to total cells was significantly lower in the SURF group than in the CTRL group (D). All values are expressed as the mean ± SD.
Figure 7
Figure 7. TUNEL assay (original magnification 400 ×).
(A) Sham group. (B) CTRL group. (C) SURF group. The ratio of apoptotic cells to total cells in the Sham and SURF groups was significantly lower than that in the CTRL group (D). All values are expressed as the mean ± SD.
Figure 8
Figure 8. Immunohistochemical staining for Bcl-2 (original magnification 400 ×).
(A) Sham group. (B) CTRL group. (C) SURF group. Arrows indicate Bcl-2 positive cells. The ratio of Bcl-2-positive cells was significantly higher in the SURF group than that in the CTRL group (D). All values are expressed as the mean ± SD.
Figure 9
Figure 9. Immunohistochemical staining for SP-C (original magnification 400×).
(A) Sham group. (B) CTRL group. (C) SURF group. The amount of SP-C-positive cells in the CTRL and SURF groups was significantly lower than that in the Sham group, however, that in the SURF group was significantly higher than that in the CTRL group (D). All values are expressed as the mean ± SD.
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