Decreased lung ischemia-reperfusion injury in rats after preoperative administration of cyclosporine and tacrolimus  B. Krishnadasan, MD, B. Naidu, MBBS, M. Rosengart, MD, A.L. Farr, BA, A. Barnes, PharmD, E.D. Verrier, MD, M.S. Mulligan, MD  The Journal of Thoracic and Cardiovascular Surgery  Volume 123, Issue 4, Pages 756-767 (April 2002) DOI: 10.1067/mtc.2002.120351 Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 1 Immunomodulatory effects of CSA and tacrolimus (TAC). With antigen presentation, protein kinase C (PKC) activation leads to production of calcineurin, phophatase increasing production of various transcription factors, including NF-AT, OCT-1, AT-1, and NF-κB. Increase in NF-AT results in transcription of IL-2, powerful T-cell stimulant driving rejection. CSA and tacrolimus block this reaction by binding with cyclophilin and FK-binding protein to inhibit action of calcineurin. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 2 Left lung permeability in control animals. Lung vascular permeability was increased 7-fold in animals that underwent ischemia and reperfusion (P <.001). Animals that underwent thoracotomy alone and ischemia alone also had statistically significant increases in permeability relative to unmanipulated control animals (P <.04; n = 4 in all experimental groups). The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 3 Lung permeability in animals receiving CSA at 1 mg/kg, CSA at 5 mg/kg, and tacrolimus at 0.2 mg/kg. Two groups of positive control animals are shown, one receiving saline solution and one receiving cremaphor vehicle. There was no difference in permeability between these two groups. Animals treated 2 hours into reperfusion (diagonally filled bars) did not demonstrate any significant reduction in lung vascular injury at either dose of CSA or with tacrolimus (n = 6 in all experimental groups). Animals treated at reperfusion (shaded bars) with 5 mg/kg CSA and/or 0.2 mg/kg tacrolimus showed 17% and 18% reductions in lung vascular permeability (asterisk indicates P <.05). Animals treated 6 hours before reperfusion (dotted bars) with CSA at 1 and 5 mg/kg and with tacrolimus at 0.2 mg/kg demonstrated 33%, 54%, and 56% reductions in lung permeability (double asterisk indicates P <.003). The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 4 MPO content in control animals. There was steady increase in MPO contents of left lungs with increasing time of reperfusion. MPO content at 4 hours was statistically increased from unmanipulated control animals (P <.002). Each experimental group included 4 animals. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 5 MPO activity in treated animals. Tissue neutrophil accumulation was modestly decreased in animals receiving CSA at 5 mg/kg 2 hours after reperfusion and just before reperfusion, but decrease was not significant (ns). Animals that received CSA at 5 mg/kg and tacrolimus (TAC) at 0.2 mg/kg 6 hours before experiment demonstrated 38% and 36% decreases in MPO content, respectively. These were statistically significant (P <.008). Each experimental group included 4 animals. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 6 BAL cell count increased significantly in those animals undergoing ischemia followed by reperfusion relative to unmanipulated control animals (P <.005). Small increases in cell count were seen with thoracotomy alone and with ischemia alone, but these increases were not significant. Predominant cell type in animals was alveolar macrophage, and most cells in unperfused lungs were neutrophils. Each experimental group included 6 animals. No relative decrease in alveolar cell count was seen in animals that received CSA at 5 mg/kg after 2 hours of reperfusion or at time of reperfusion. However, significant decrease in alveolar leukocyte accumulation was seen in animals that received CSA at 5 mg/kg and tacrolimus (TAC) at 0.2 mg/kg 6 hours before experiment, resulting in 32% increase in BAL cell counts. Asterisk indicates P <.01. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 7 EMSA for nuclear protein expression of NF-κB. A, Area of interest is boxed. Far left lane shows extract from injured positive control lung that underwent ischemia followed by 4 hours of reperfusion. Second lane shows unmanipulated negative control lung. Third lane shows another positive control for side-by-side comparison. Fourth lane (Cold Competition) shows lung treated with nonradiolabeled NF-κB oligonucleotide that antagonizes radiolabeled oligonucleotide. Band specific for NF-κB drops out at this time. Far right lanes demonstrate absence of NF-κB expression in animals treated with CSA and tacrolimus 6 hours before reperfusion. B, Densitometric results corresponding to lanes on gel. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 8 Ribonuclease protection assay. Four experimental groups are shown: negative control, positive control, and animals treated with 5 mg/kg CSA and 0.2 mg/kg tacrolimus (TAC) 6 hours before reperfusion. Housekeeping genes shown at bottom of gel demonstrate equal loading of RNA in all experimental groups. IFN, Interferon. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 9 Densitometric analyses for ribonuclease protection assay in Figure 8. Illustrated are samples from negative control animals, positive control animals, and animals treated with 5 mg/kg CSA and 0.2 mg/kg tacrolimus (TAC, FK-506) 6 hours before reperfusion. There were generalized decreases in expressions in treatment groups, as seen quantitatively in densitometric analyses. IL-2 (A), TFN-α (B), and interferon γ (C) messenger RNA expressions were decreased in animals treated with CSA and tacrolimus. The Journal of Thoracic and Cardiovascular Surgery 2002 123, 756-767DOI: (10.1067/mtc.2002.120351) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions