5. Results
5.2. Effects of ischemia and reperfusion on lungs from WT and Nox2 KO mice
Results 5.2. Effects of ischemia and reperfusion on lungs from WT and Nox2 KO mice
Results 5.2.2. Vascular compliance
Vascular compliance values were not significantly different between nonischemic control lungs and the different experimental groups and were virtually constant throughout the entire experimental period (Table 9).
Table 9. Vascular compliance in WT and Nox2 KO mouse lungs.
Vascular compliance, cm3/mm Hg
Time after onset of reperfusion, min Pre
30 60 90 WT 0.01 (0.002) 0.01 (0.002) 0.01 (0.002) 0.01 (0.002)
Non-ischemic control 0.01 (0.001) 0.01 (0.001) 0.01 (0.001) 0.01 (0.001) WT + SOD 0.01 (0.002) 0.01 (0.002) 0.01 (0.002) 0.01 (0.002) WT + apocynin 0.01 (0.001) 0.01 (0.001) 0.01 (0.001) 0.01 (0.001) Nox2 KO 0.01 (0.001) 0.01 (0.001) 0.01 (0.001) 0.01 (0.001)
Data are presented as mean (SEM) of 4-6 independent experiments. pre – baseline values before starting ischemia; WT – lungs from wild type mice subjected to ischemia/reperfusion; non-ischemic control lungs were continuously perfused and normoxically ventilated; WT+SOD – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with superoxide dismutase (150 U/ml); WT+apocynin – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with apocynin (0.5 mM); Nox2 KO – lungs from Nox2 knock-out mice subjected to ischemia/reperfusion.
Results 5.2.3. Vascular permeability
There were no significant differences in baseline Kfc values among the different experimental groups (Figure 12). In non-ischemic lungs, Kfc values were essentially constant throughout the entire experimental period (Figure 12). Ischemia and reperfusion resulted in increased microvascular permeability in lungs from WT mice compared with time-matched non-ischemic control lungs (Figure 12). Inhibition of NADPH oxidase by apocynin as well as superoxide anion scavenging by SOD significantly attenuated vascular leakage (Figure 12). Similarly, lungs from mice with an NADPH oxidase deficiency (i.e., Nox2 KO mice) were protected against tissue injury (Figure 12).
Time after Onset of Reperfusion (min)
pre 0 30 60 90
Kfc (cm
3 /(s·
mmHg·g·10
4 ))
0 4 8 12 16
WT Control WT+SOD WT+apocynin Nox2 KO Ischemia 90 min
*
*
*
Reperfusion
Figure 12. Vascular permeability in lungs from WT and Nox2 KO mice. Lungs were exposed to anoxic ischemia for 90 min with following reperfusion. Time-matched non-ischemic control lungs from WT mice did not undergo ischemia but were continuously perfused and normoxically ventilated. Kfc values were assessed, as described in Methods.
Ischemia and reperfusion resulted in increased vascular permeability in lungs from WT mice. Pretreatment with apocynin as well as SOD significantly attenuated lung injury. Nox2 KO mouse lungs were protected against injury. Data are presented as mean + SEM of 4-6 independent experiments. *p<0.05 compared with all other groups. Kfc – capillary filtration coefficient; pre – baseline values before starting ischemia; WT – lungs from wild type mice subjected to ischemia/reperfusion; WT+SOD – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with superoxide dismutase (150 U/ml); WT+apocynin – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with apocynin (0.5 mM); Nox2 KO – lungs from Nox2 knock-out mice subjected to ischemia/reperfusion.
Results 5.2.4. Pulmonary edema formation
In non-ischemic lungs, only marginal lung weight gain was observed throughout the entire experimental period (Figure 13). Ischemia and reperfusion resulted in increased fluid accumulation in lungs from WT mice compared with time-matched non-ischemic control lungs (Figure 13). Inhibition of NADPH oxidase by apocynin as well as superoxide anion scavenging by SOD significantly reduced edema (Figure 13). Similarly, lungs from mice with an NADPH oxidase deficiency (i.e., Nox2 KO mice) were protected against tissue injury (Figure 13).
Time after Onset of Reperfusion (min)
pre 0 30 60 90
Lung Weight Gain (g)
0.0 0.2 0.4 0.6
WT Control WT+SOD WT+apocynin Nox2 KO Ischemia 90 min
*
* *
Reperfusion
Figure 13. Lung weight gain in lungs from WT and Nox2 KO mice. Lungs were exposed to anoxic ischemia for 90 min with following reperfusion. Time-matched non-ischemic control lungs from WT mice did not undergo ischemia but were continuously perfused and normoxically ventilated. Lung weight gain was assessed, as described in Methods. Ischemia and reperfusion resulted in increased lung fluid accumulation in lungs from WT mice.
Pretreatment with apocynin as well as SOD significantly attenuated vascular leakage. Nox2 KO mouse lungs were protected against injury. Data are presented as mean + SEM of 4-6 independent experiments. *p<0.05 compared with all other groups. pre – baseline data before starting ischemia; WT – lungs from wild type mice subjected to ischemia/reperfusion; WT+SOD – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with superoxide dismutase (150 U/ml); WT+apocynin – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with apocynin (0.5 mM); Nox2 KO – lungs from Nox2 knock-out mice subjected to ischemia/reperfusion.
Results 5.2.5. Intravascular ROS release
Reperfusion of previously ischemic lungs from WT mice was associated with increased intravascular ROS release as compared with time-matched non-ischemic control lungs (Figure 14). Parallel experiments in the presence of SOD revealed that the majority of ROS release was caused by superoxide (Figure 14). Inhibition of NADPH oxidase by apocynin significantly decreased ROS production (Figure 14). Moreover, in mice lacking Nox2, the catalytic NADPH oxidase subunit, intravascular ROS release was significantly attenuated (Figure 14).
Control WT WT+apocynin Nox2 KO
Signal Intensity Increase Rate (AU/min)
0.0 0.5 1.0 1.5 2.0 2.5
−SOD
+SOD
*
Figure 14. Intravascular ROS release in lungs from WT and Nox2 KO mice. Lungs were exposed to anoxic ischemia for 90 min with following reperfusion. Time-matched non-ischemic control lungs did not undergo ischemia but were continuously perfused. Spin probe CPH was added into perfusate 5 min before reperfusion. Samples from the venous outflow of the isolated lung were taken and measured immediately by ESR spectroscopy. The contribution of superoxide radical was determined in parallel experiments performed in the presence of SOD (150 U/ml) in the buffer fluid. Increased ROS generation upon reperfusion of WT mouse lungs was reduced by SOD and was attenuated in lungs from Nox2 KO mice.
Data are presented as mean + SEM of 4-6 independent experiments. *p<0.05 compared with all other groups. WT – lungs from wild type mice subjected to ischemia/reperfusion; non-isch contr – non-non-ischemic control; WT+apocynin – lungs from wild type mice subjected to ischemia/reperfusion and pretreated with apocynin (0.5 mM); Nox2 KO – lungs from Nox2 knock-out mice subjected to ischemia/reperfusion.
Results 5.2.6. Expression of different NOS isoforms in lungs from WT and Nox2 KO mice Baseline expression of eNOS and nNOS in lungs from Nox2 KO mice was not significantly different from that in lungs from WT mice (Figure 15). No baseline expression of iNOS was detected in lungs from WT and Nox2 KO mice (Figure 15).
eNOS
nNOS β-actin
WT Nox2 KO
A
eNOS nNOS iNOS
NOS/β-actin
0.0 0.4 0.8
1.2 WT
Nox2 KO
B
Figure 15. NOS expression in Nox2-deficiency. (A) Expression of NOS isoenzymes in lungs from WT and Nox2 KO mice. Proteins were extracted from the lungs and subjected to Western blot. (B) The bar graph illustrates the quantification of eNOS/nNOS expression levels normalized to β-actin levels. All data are presented as means + SEM of 4 animals in each group.
Results 5.2.7. NO production in lungs from WT and Nox2 KO mice
The levels of NO metabolites in Nox2 KO mice were not significantly different from those in WT animals (Figure 16).
WT Nox2 KO
N itrite/nitrate ( μ M)
0.0 0.5 1.0 1.5 2.0
Figure 16. NO production in lungs from WT and Nox2 KO mice. All isolated lungs were continuously perfused and normoxically ventilated and perfusate samples were taken after perfusion for 30 min. Accumulation of NO metabolites was determined in perfusate samples by the Griess reaction. There were no significant differences in intravascular NO metabolites accumulation among the groups. Data are presented as mean + SEM of 5-7 independent experiments.
Results 5.3. Effects of lung ischemia and reperfusion in living WT and Nox2 KO