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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 inhibits neutrophil invasion after LTX.(A) Specific neutrophil staining (arrows, micrographs) and quantification (left graph). Data are expressed as number of neutrophils/field. (B) Measurement of tissue protein expression of neutrophil chemoattractants in transplanted lungs by protein array. Data are expressed in mean pixel densities. (C) Evaluation of MMP-2 mRNA expression (measured by RT-PCR, data are expressed as % of Perfadex group, Perfadex group has been set at 100%) and its Inhibitor TIMP-1 by protein analysis (protein array). (D) Representative protein array pictures. Calibration bar for micrographs (A) represents 100 µm (200 x magnification, upper micrographs) and 50 µm (400 x magnification, lower micrographs). Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
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pone-0073298-g004: Prednisolone inhibits neutrophil invasion after LTX.(A) Specific neutrophil staining (arrows, micrographs) and quantification (left graph). Data are expressed as number of neutrophils/field. (B) Measurement of tissue protein expression of neutrophil chemoattractants in transplanted lungs by protein array. Data are expressed in mean pixel densities. (C) Evaluation of MMP-2 mRNA expression (measured by RT-PCR, data are expressed as % of Perfadex group, Perfadex group has been set at 100%) and its Inhibitor TIMP-1 by protein analysis (protein array). (D) Representative protein array pictures. Calibration bar for micrographs (A) represents 100 µm (200 x magnification, upper micrographs) and 50 µm (400 x magnification, lower micrographs). Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.

Mentions: Rats receiving lungs that had been treated with Perfadex only had significantly more neutrophil cells 46.88 ± 2.47 compared to prednisolone treated 5.12 ± 0.74 or sham lung cells 5.25 ± 1.04 (P<0.001, Figure 4 A). Prednisolone protects from hypoxia-induced inflammation by downregulating tissue expression of specific neutrophil chemoattractants such as CINC-1 (854.0 ± 74.43 mean pixel density), CINC-2α/β (283.5 ± 38.40 mean pixel density) and LIX (1300 ± 54.64 mean pixel density) vs. Perfadex (2134 ± 99.54, P<0.01; 621.5 ± 28.82, P<0.01 resp. 2309 ± 80.91, P<0.01 mean pixel density). Regarding LIX expression, prednisolone (1300 ± 54.64 mean pixel density) treatment resulted in lower levels compared to sham (2379 ± 28.8 mean pixel density, P<0.01) animals (Figure 4 B). Neutrophil invasiveness, which was quantified by MMP-2, was significantly lower in prednisolone-pretreated lungs than in Perfadex (101.9 ± 31.92%) treated ones (P<0.01). However, levels in prednisolone-treated lungs (25.38 ± 4.61%) were still significantly higher than in sham animals (3.37 ± 0.50%; P<0.001, Figure 4C). In contrast, the natural inhibitor of MMP-2, TIMP-1, was significantly upregulated in prednisolone-pretreated (4642 ± 57.36, mean pixel density) lungs compared to Perfadex (3285 ± 37.44, mean pixel density) treated lungs, whereas both interventional groups had significantly higher levels compared to sham (593.4 ± 46.56, mean pixel density) animals (P<0.001, Figure 4C).


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 inhibits neutrophil invasion after LTX.(A) Specific neutrophil staining (arrows, micrographs) and quantification (left graph). Data are expressed as number of neutrophils/field. (B) Measurement of tissue protein expression of neutrophil chemoattractants in transplanted lungs by protein array. Data are expressed in mean pixel densities. (C) Evaluation of MMP-2 mRNA expression (measured by RT-PCR, data are expressed as % of Perfadex group, Perfadex group has been set at 100%) and its Inhibitor TIMP-1 by protein analysis (protein array). (D) Representative protein array pictures. Calibration bar for micrographs (A) represents 100 µm (200 x magnification, upper micrographs) and 50 µm (400 x magnification, lower micrographs). Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
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Related In: Results  -  Collection

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pone-0073298-g004: Prednisolone inhibits neutrophil invasion after LTX.(A) Specific neutrophil staining (arrows, micrographs) and quantification (left graph). Data are expressed as number of neutrophils/field. (B) Measurement of tissue protein expression of neutrophil chemoattractants in transplanted lungs by protein array. Data are expressed in mean pixel densities. (C) Evaluation of MMP-2 mRNA expression (measured by RT-PCR, data are expressed as % of Perfadex group, Perfadex group has been set at 100%) and its Inhibitor TIMP-1 by protein analysis (protein array). (D) Representative protein array pictures. Calibration bar for micrographs (A) represents 100 µm (200 x magnification, upper micrographs) and 50 µm (400 x magnification, lower micrographs). Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.
Mentions: Rats receiving lungs that had been treated with Perfadex only had significantly more neutrophil cells 46.88 ± 2.47 compared to prednisolone treated 5.12 ± 0.74 or sham lung cells 5.25 ± 1.04 (P<0.001, Figure 4 A). Prednisolone protects from hypoxia-induced inflammation by downregulating tissue expression of specific neutrophil chemoattractants such as CINC-1 (854.0 ± 74.43 mean pixel density), CINC-2α/β (283.5 ± 38.40 mean pixel density) and LIX (1300 ± 54.64 mean pixel density) vs. Perfadex (2134 ± 99.54, P<0.01; 621.5 ± 28.82, P<0.01 resp. 2309 ± 80.91, P<0.01 mean pixel density). Regarding LIX expression, prednisolone (1300 ± 54.64 mean pixel density) treatment resulted in lower levels compared to sham (2379 ± 28.8 mean pixel density, P<0.01) animals (Figure 4 B). Neutrophil invasiveness, which was quantified by MMP-2, was significantly lower in prednisolone-pretreated lungs than in Perfadex (101.9 ± 31.92%) treated ones (P<0.01). However, levels in prednisolone-treated lungs (25.38 ± 4.61%) were still significantly higher than in sham animals (3.37 ± 0.50%; P<0.001, Figure 4C). In contrast, the natural inhibitor of MMP-2, TIMP-1, was significantly upregulated in prednisolone-pretreated (4642 ± 57.36, mean pixel density) lungs compared to Perfadex (3285 ± 37.44, mean pixel density) treated lungs, whereas both interventional groups had significantly higher levels compared to sham (593.4 ± 46.56, mean pixel density) animals (P<0.001, Figure 4C).

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