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Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation.

Paulus P, Holfeld J, Urbschat A, Mutlak H, Ockelmann PA, Tacke S, Zacharowski K, Reissig C, Stay D, Scheller B - PLoS ONE (2013)

Bottom Line: Hypoxia induced vasoactive cytokines such as VEGF were reduced.Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1.Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Anesthesiology, Intensive Care Medicine and Pain Therapy, Goethe-University Hospital Frankfurt, Frankfurt am Main, Germany. Patrick.Paulus@kgu.de

ABSTRACT
The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

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Prednisolone preserves the macro- and microstructural aspect of the grafts and reduces reperfusion edema formation following LTX.(A) Image shows explanted lungs 48 hours after LTX. Lungs from prednisolone-treated animals (right picture), Perfadex-treated lungs (middle picture) and sham lungs (left picture) were compared. L = left lobe, R = right lobe. (B) Representative micrographs (H&E staining) showing microstructural alterations and alveolar thickening as edema surrogate by directly comparing shams (left micrographs) with Perfadex-treated animals (middle) and prednisolone-preconditioned lungs (right pictures). (C) Quantification of the alveolar thickening by measuring the percentage of tissue occupying every slide (ratio tissue vs. no tissue, left graph). Values are expressed as % tissue/slide (n=3 random slides per animal). Quantification of the amount of edema (lung water) by evaluating the wet-to-dry ratio (right graph), n=6 per group. Calibration bar for macroscopic pictures (A) represents 1 cm. Calibration bar for H&E micrographs (B) represents 100 µm (200 x magnification, upper micrograph) and 50 µm (400 x magnification, lower micrograph). Significance level was set to P<0.05. P<0.05: *; P<0.01: **, § § and P<0.001: ***. * = significantly different from shams and § = significantly different from Perfadex-group. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
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pone-0073298-g002: Prednisolone preserves the macro- and microstructural aspect of the grafts and reduces reperfusion edema formation following LTX.(A) Image shows explanted lungs 48 hours after LTX. Lungs from prednisolone-treated animals (right picture), Perfadex-treated lungs (middle picture) and sham lungs (left picture) were compared. L = left lobe, R = right lobe. (B) Representative micrographs (H&E staining) showing microstructural alterations and alveolar thickening as edema surrogate by directly comparing shams (left micrographs) with Perfadex-treated animals (middle) and prednisolone-preconditioned lungs (right pictures). (C) Quantification of the alveolar thickening by measuring the percentage of tissue occupying every slide (ratio tissue vs. no tissue, left graph). Values are expressed as % tissue/slide (n=3 random slides per animal). Quantification of the amount of edema (lung water) by evaluating the wet-to-dry ratio (right graph), n=6 per group. Calibration bar for macroscopic pictures (A) represents 1 cm. Calibration bar for H&E micrographs (B) represents 100 µm (200 x magnification, upper micrograph) and 50 µm (400 x magnification, lower micrograph). Significance level was set to P<0.05. P<0.05: *; P<0.01: **, § § and P<0.001: ***. * = significantly different from shams and § = significantly different from Perfadex-group. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.

Mentions: The explanted lungs from the prednisolone group showed a better general macroscopic appearance. Lungs treated with Perfadex only had a dark colored aspect, indicating a higher fluid content. Prednisolone preconditioned lungs were almost identical in their macroscopic aspect to lungs from sham animals (Figure 2, A). Moreover, prednisolone preconditioned lungs had a preserved alveolar appearance and less perivascular or cellular edema 48h after reperfusion compared to the controls (Figure 2, B). For quantification of the edematous changes, the alveolar wall thickening was quantified using an automatized algorithm. Perfadex treated lungs had significantly larger alveoli whereas prednisolone-treated lungs were almost identical to sham lungs. By determining the amount of tissue per picture as described in the materials and methods section, the cellular swelling can be calculated. In Perfadex-treated lungs, 88.26 2.54% of the slide were covered by alveolar tissue, whereas in prednisolone-treated lungs only 54.56 2.54% of the slide consisted of lung tissue (P<0.001*, P<0.01§) (Figure 2, C left graph). For direct evaluation of the lung water as surrogate for edema, the wet-to-dry ratio was calculated. Again, Perfadex-treated 4.991 ± 0.10 (relative values) lungs had significantly increased tissue water than the prednisolone 4.549 ± 0.05 (relative values) preconditioned (P<0.05) and sham 4.034 ± 0.12 (relative values) lungs (P<0.001). Although prednisolone pretreated lungs had less edema than Perfadex treated lungs, their water content was significantly increased when compared to sham animals (P<0.01; Figure 2, C right graph).


Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation.

Paulus P, Holfeld J, Urbschat A, Mutlak H, Ockelmann PA, Tacke S, Zacharowski K, Reissig C, Stay D, Scheller B - PLoS ONE (2013)

Prednisolone preserves the macro- and microstructural aspect of the grafts and reduces reperfusion edema formation following LTX.(A) Image shows explanted lungs 48 hours after LTX. Lungs from prednisolone-treated animals (right picture), Perfadex-treated lungs (middle picture) and sham lungs (left picture) were compared. L = left lobe, R = right lobe. (B) Representative micrographs (H&E staining) showing microstructural alterations and alveolar thickening as edema surrogate by directly comparing shams (left micrographs) with Perfadex-treated animals (middle) and prednisolone-preconditioned lungs (right pictures). (C) Quantification of the alveolar thickening by measuring the percentage of tissue occupying every slide (ratio tissue vs. no tissue, left graph). Values are expressed as % tissue/slide (n=3 random slides per animal). Quantification of the amount of edema (lung water) by evaluating the wet-to-dry ratio (right graph), n=6 per group. Calibration bar for macroscopic pictures (A) represents 1 cm. Calibration bar for H&E micrographs (B) represents 100 µm (200 x magnification, upper micrograph) and 50 µm (400 x magnification, lower micrograph). Significance level was set to P<0.05. P<0.05: *; P<0.01: **, § § and P<0.001: ***. * = significantly different from shams and § = significantly different from Perfadex-group. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
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pone-0073298-g002: Prednisolone preserves the macro- and microstructural aspect of the grafts and reduces reperfusion edema formation following LTX.(A) Image shows explanted lungs 48 hours after LTX. Lungs from prednisolone-treated animals (right picture), Perfadex-treated lungs (middle picture) and sham lungs (left picture) were compared. L = left lobe, R = right lobe. (B) Representative micrographs (H&E staining) showing microstructural alterations and alveolar thickening as edema surrogate by directly comparing shams (left micrographs) with Perfadex-treated animals (middle) and prednisolone-preconditioned lungs (right pictures). (C) Quantification of the alveolar thickening by measuring the percentage of tissue occupying every slide (ratio tissue vs. no tissue, left graph). Values are expressed as % tissue/slide (n=3 random slides per animal). Quantification of the amount of edema (lung water) by evaluating the wet-to-dry ratio (right graph), n=6 per group. Calibration bar for macroscopic pictures (A) represents 1 cm. Calibration bar for H&E micrographs (B) represents 100 µm (200 x magnification, upper micrograph) and 50 µm (400 x magnification, lower micrograph). Significance level was set to P<0.05. P<0.05: *; P<0.01: **, § § and P<0.001: ***. * = significantly different from shams and § = significantly different from Perfadex-group. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
Mentions: The explanted lungs from the prednisolone group showed a better general macroscopic appearance. Lungs treated with Perfadex only had a dark colored aspect, indicating a higher fluid content. Prednisolone preconditioned lungs were almost identical in their macroscopic aspect to lungs from sham animals (Figure 2, A). Moreover, prednisolone preconditioned lungs had a preserved alveolar appearance and less perivascular or cellular edema 48h after reperfusion compared to the controls (Figure 2, B). For quantification of the edematous changes, the alveolar wall thickening was quantified using an automatized algorithm. Perfadex treated lungs had significantly larger alveoli whereas prednisolone-treated lungs were almost identical to sham lungs. By determining the amount of tissue per picture as described in the materials and methods section, the cellular swelling can be calculated. In Perfadex-treated lungs, 88.26 2.54% of the slide were covered by alveolar tissue, whereas in prednisolone-treated lungs only 54.56 2.54% of the slide consisted of lung tissue (P<0.001*, P<0.01§) (Figure 2, C left graph). For direct evaluation of the lung water as surrogate for edema, the wet-to-dry ratio was calculated. Again, Perfadex-treated 4.991 ± 0.10 (relative values) lungs had significantly increased tissue water than the prednisolone 4.549 ± 0.05 (relative values) preconditioned (P<0.05) and sham 4.034 ± 0.12 (relative values) lungs (P<0.001). Although prednisolone pretreated lungs had less edema than Perfadex treated lungs, their water content was significantly increased when compared to sham animals (P<0.01; Figure 2, C right graph).

Bottom Line: Hypoxia induced vasoactive cytokines such as VEGF were reduced.Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1.Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Anesthesiology, Intensive Care Medicine and Pain Therapy, Goethe-University Hospital Frankfurt, Frankfurt am Main, Germany. Patrick.Paulus@kgu.de

ABSTRACT
The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

Show MeSH
Related in: MedlinePlus