Ebselen Improves Ischemia-Reperfusion Injury After Rat Lung Transplantation

The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown invivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18h. In nine recipients given 500mg/kg oral ebselen 1h before transplantation, graft PaO2/FiO2 was improved 24h after transplantation, as evidenced with a mean (standard deviation) PaO2 of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p=0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusio

sCR1sLeX reduces lung allograft ischemia-reperfusion injury but does not ameliorate acute rejection

Background: Combined inhibition of complement and leukocyte adhesion by sCR1sLeX reduces lung allograft dysfunction up to 24 h. In the present study its effect on graft function and acute rejection was evaluated up to 5 days after experimental transplantation. Methods: Orthotopic single left lung transplantation was performed in 35 male rats (Brown Norway to Fischer 344) after a total ischemic time of 20 h. Two groups were assessed after 1, 3, and 5 days post-transplant, respectively (n=5 per group and time point): controls vs. recipients which received 10 mg/kg sCR1sLeX 15 min prior to reperfusion. In addition, five animals received 10 mg/kg per day sCR1sLeX for 5 days. For blood gas analysis of the graft, the contralateral lung was occluded for 5 min to assess graft function. Lung grafts were flushed, and histological grading was performed in blinded fashion according to the International Society for Heart and Lung Transplantation criteria. Results: Graft PaO2 in recipients treated with sCR1sLeX was superior on day 1 (383±118 vs. 56±15 mmHg; P≪0.0001) and day 3 (446±48 vs. 231±108 mmHg; P≪0.0001). Five days after transplantation, no difference in PaO2 was found (61±28 vs. 83±31 mmHg; P=0.59). Repeated treatment with sCR1sLeX for 5 days did not improve PaO2 (64±5 mmHg; P=0.65 vs. control; P=0.93 vs. sCR1sLeX). At any time point, there was no difference in the degree of rejection between groups. Conclusions: In this model sCR1sLeX provided marked improvement of graft function up to 3 days, but inhibition of both complement system and selectin dependent leukocyte adhesion failed to protect against acute rejectio

1,25-Dihydroxycholecalciferol with low-calcium diet reduces acute rejection in rat lung allotransplantation

OBJECTIVES The effect of 1,25-dihydroxycholecalciferol (calcitriol, vitamin D3) with a low-calcium diet on the acute lung allograft rejection in a rat unilateral left lung transplantation model was evaluated. METHODS Three transplantation groups were studied (n=5, male Brown-Norway to Fischer F344, 235±15g body weight): calcitriol and low-calcium diet, low-calcium diet and normal diet. Calcitriol (4μg/kg/day) was injected intraperitoneally for 5 days, starting from the day of transplantation. In addition, two non-transplantation groups were compared: (n=3, Brown-Norway) to measure the level of cytokines, and Fischer F344 receiving calcitriol and a low-calcium diet to measure the serum calcium level. The recipients of transplantation were killed on Day 5 post-transplant. The contralateral right main bronchus and the pulmonary artery were occluded for 5min and blood was drawn for the blood gas analysis, and the grafts were assessed for histology (International Society for Heart and Lung Transplantation 1996/rank scale). Lung levels of interleukin (IL)-2, IL-6, IL-12 and tumour necrosis factor-α (TNF-α) were assessed within the calcitriol and low-calcium diet, low-calcium diet and Brown-Norway groups. The serum calcium level was assessed in the Fischer F344 group. An analysis of variance with Tukey's post hoc test was used to compare the arterial blood oxygen pressure and the lung cytokine expression between groups. A non-parametric Kruskal-Wallis test followed by the Siegel and Castellan post hoc test was used to assess the differences between the groups according to the lung graft rejection grading. Student's paired t-test was used to compare the serum calcium level. RESULTS The arterial PaO2 was significantly higher in the calcitriol and the low-calcium diet groups when compared with low-calcium diet or normal diet groups (356±72mmHg; P<0.05 vs other groups). The arterial and bronchial rejection observed in calcitriol and low-calcium diet group was significantly milder than in the low-calcium diet or normal diet groups (A1-2, B1-2; P<0.05 vs other groups). IL-2 and IL-6 levels were significantly higher in low-calcium diet vs calcitriol and low-calcium diet and Brown-Norway groups. IL-12 and TNF-α did not differ among the groups. There was no significant difference in serum calcium level before and after the treatment in the Fischer F344 group. CONCLUSIONS Calcitriol with a low-calcium diet treatment improves lung function, reduces lung allograft acute rejection, decreases IL-2 and IL-6 allograft expression and does not change the serum calcium level significantl

Donor and recipient treatment with the Lazaroid U-74006F do not improve post-transplant lung function in swine

Objective: U-74006F is the only Lazaroid which is currently in clinical use. A number of experimental studies demonstrate that Lazaroids reduce ischemia/reperfusion injury in various organ systems. We evaluated the effect of U-74006F on reperfusion injury in a large animal model of lung allo-transplantation. Methods: Two different treatment modalities were evaluated and compared with corresponding control groups. Unilateral left lung transplantation was performed in 21 weight-matched pigs (24-31 kg). Donor lungs were flushed with 1.5 l cold (1°C) LPD solution and preserved for 20 h. In group I (n=5), donor animals were pretreated with U-74006F (10 mg/kg i.v.) 20 min before harvest. In addition U-74006F was added to the flush solution (10 mg/l). In group III (n=6), the Lazaroid was given to the donor before flush and to the recipient before reperfusion (3 mg/kg i.v.). Group II and IV (n=5) served as control. One hour after reperfusion, the recipient contralateral right pulmonary artery and bronchus were ligated to assess graft function only. Extravascular lung water index (EVLWI), mean pulmonary artery pressure, cardiac output, and gas exchange were assessed during a 5 h observation period. Lipid peroxidation (TBARS) and neutrophil migration (MPO activity) were measured at the end of the assessment in lung allograft tissue. Results: A significant change of TBARS concentration was shown in group III (group III 78.7±4.6 pmol/g vs. group IV 120.8±7.2 pmol/g (P=0.0065) normal lung tissue 41.3±4.2 pmol/g). MPO activity was reduced in group III 3.74±0.25 δOD/mg per min vs. group IV 4.97±0.26 δOD/mg per min (P=0.027), normal lung tissue 1.04±0.27 δOD/mg per min). Pulmonary hemodynamics and gas exchange after reperfusion did not differ between groups. In group I and III, a tendency towards a reduced EVLWI was noted. Conclusion: We conclude that combined treatment of donor and recipient with U-74006F reduces free radical mediated injury in the allograft. However, this intervention did not result in a significant reduction of post-transplant lung edema or improvement of pulmonary hemodynamics. Donor pretreatment alone did not improve lung allograft reperfusion injury. These results indicate that the benefit of U-74006F is too small to consider clinical application in lung transplantatio

Characteristics of inflammatory response and repair after experimental blast lung injury in rats.

BACKGROUND AND OBJECTIVES Blast-induced lung injury is associated with inflammatory, which are characterised by disruption of the alveolar-capillary barrier, haemorrhage, pulmonary infiltrateration causing oedema formation, pro-inflammatory cytokine and chemokine release, and anti-inflammatory counter-regulation. The objective of the current study was to define sequence of such alterations in with establishing blast-induced lung injury in rats using an advanced blast generator. METHODS Rats underwent a standardized blast wave trauma and were euthanised at defined time points. Non-traumatised animals served as sham controls. Obtained samples from bronchoalveolar lavage fluid (BALF) at each time-point were assessed for histology, leukocyte infiltration and cytokine/chemokine profile. RESULTS After blast lung injury, significant haemorrhage and neutrophil infiltration were observed. Similarly, protein accumulation, lactate dehydrogenase activity (LDH), alveolar eicosanoid release, matrix metalloproteinase (MMP)-2 and -9, pro-Inflammatory cytokines, including tumour necrosis factor (TNF) and interleukin (IL) -6 raised up. While declining in the level of anti-inflammatory cytokine IL-10 occurred. Ultimately, pulmonary oedema developed that increased to its maximum level within the first 1.5 h, then recovered within 24 h. CONCLUSION Using a stablished model, can facilitate the study of inflammatory response to blast lung injury. Following the blast injury, alteration in cytokine/chemokine profile and activity of cells in the alveolar space occurs, which eventuates in alveolar epithelial barrier dysfunction and oedema formation. Most of these parameters exhibit time-dependent return to their basal status that is an indication to resilience of lungs to blast-induced lung injury

Strong additive effect of 1,25-dihydroxycholecalciferol and cyclosporine A but not tacrolimus in rat lung allotransplantation

Objectives: 1,25-Dihydroxycholecalciferol (calcitriol, vitamin D3) has immunosuppressive properties. This study evaluates the effect of calcitriol in combination with either cyclosporine A or tacrolimus on acute lung allograft rejection in a rat model of unilateral left lung allotransplantation. Methods: Unilateral left lung transplantation was performed in male rats (Brown-Norway to Fischer F344, 200-250 g body weight). For immunosuppression, the following subtherapeutic doses were used: calcitriol 0.5 μg/kg/day, cyclosporine A 2.5 mg/kg/day i.p., and tacrolimus 40 μg/kg i.m. Five groups (n=5) were analyzed: cyclosporine A; cyclosporine A and calcitriol; calcitriol; tacrolimus and calcitriol; and tacrolimus. The injections were performed for 5 days starting from the day of transplantation. Recipients were sacrificed on day 5 post-transplant. The contralateral right main bronchus and pulmonary artery were occluded for 5 min and blood was drawn for blood gas analysis. The grafts were excised, fixed in formaline and embedded in paraffin. Histological evaluation was done in blinded fashion (ISHLT 1999/rank scale). The mean and standard error of the mean (PaO2) or the median and range (rejection grading) are given. ANOVA followed by planned comparison for the PaO2 and Kruskal-Wallis ANOVA for rejection grading were applied, p<0.05 considered significant. Results: Arterial PaO2 on day 5 was very low in animals treated with subtherapeutic dosages of either cyclosporine A (48±10 mmHg), calcitriol (51±3) or tacrolimus (86±22). Combined treatment with cyclosporine A and calcitriol revealed a significant improvement (248±78; p<0.05 vs. other groups), whereas the combination of tacrolimus with calcitriol did not reveal any benefit (65±9). Rejection grading with these subtherapeutic doses did not show any significant difference between groups. Conclusions: Our data indicate that cyclosporine A, but not tacrolimus, has a strong additive effect with calcitriol on acute rat lung allograft rejectio

Survival and graft function in a large animal lung transplant model after 30 h preservation and substitution of the nitric oxide pathway

Objective: Substitution of the nitric oxide- (NO-) pathway improves early graft function following lung transplantation. We previously demonstrated that 8-Br-cGMP (second messenger of NO) to the flush solution and tetrahydrobiopterin (BH4, coenzyme of NO synthase) given as additive during reperfusion improve post-transplant graft function. In the present study, the combined treatment with 8-Br-cGMP and BH4 was evaluated. Methods: Unilateral left lung transplantation was performed in weight matched outbred pigs (24-31kg). In group I, grafts were preserved for 30h (n=5). 8-Br-cGMP (1mg/kg) was added to the flush solution (Perfadex™, 1.5l, 1°C) and BH4 (10mg/kg/h) was given to the recipient for 5h after reperfusion. In group II, lungs were transplanted after a preservation time of 30h (n=3) and prostaglandin E1 (250g) was given into the pulmonary artery (PA) prior to flush. In all recipients 1h after reperfusion the contralateral right PA and bronchus were ligated to assess graft function only. Survival time after reperfusion, extravascular lung water index (EVLWI), hemodynamic variables, and gas exchange (PaO2) were assessed during a 12h observation period. Results: All recipients in group I survived the 12h assessment, whereas none of the group II animals survived more than 4h after reperfusion with a rapid increase of EVLWI up to 24.8±6.7ml/kg. In contrast, in group I EVLWI reached up to 8.9±1.5ml/kg and returned to nearly normal levels at 12h (6.1±0.8ml/kg). In two animals of group I the gas exchange deteriorated slightly. The other three animals showed normal arterial oxygenation over the entire observation time. Conclusion: Our data indicate that the combined substitution of the NO pathway during preservation and reperfusion reduces ischemia/reperfusion injury substantially and that this treatment even allows lung transplantation after 30h preservation in this mode

Effect of a short period of warm ischemia after cold preservation on reperfusion injury in lung allotransplantation

Objective: A short period of warm ischemia during lung allograft implantation is inevitable. We studied the effect of 2 h of warm ischemia before implantation after 18 h of cold preservation on reperfusion edema and pulmonary hemodynamics in a large animal model. Methods: Left lung transplantation was performed in ten weight-matched pigs (25-31 kg). Donor lungs were flushed with 1.5 l cold (1°C) LPD solution and preserved for 20 h. In Group I (n=5) the grafts were preserved for 20 h at 1°C and topically cooled with ice slush during implantation until reperfusion. In Group II (n=5) lungs were stored at 1°C for 18 h followed by 2 h preservation at room temperature (20°C). Topical cooling was not used during implantation. At 1 h after reperfusion the recipient contralateral right pulmonary artery and bronchus were ligated to assess graft function only. Extravascular lung water index (EVLWI), intrathoracic blood volume (ITBV), mean pulmonary artery pressure (PAP) and cardiac output (CO) were assessed during a 4 h observation period. Quantitative myeloperoxidase (MPO) activity and thiobarbituric acid-reactive substance (TBARS) levels as an indicator for lipid peroxidation were determined in allograft tissue samples taken 5 h after reperfusion. Results: In Group II a tendency to improved pulmonary vascular resistance and cardiac output was noted. Surprisingly, lung edema, assessed by EVLWI, did not increase in animals with warm ischemia. Even a tendency to a reduced EVLWI was noted. However, differences between groups did not reach statistical significance. Gas exchange did not differ statistically significant between groups. Conclusion: Our results indicate that a short period of warm ischemia before reperfusion does not lead to increased pulmonary edema. In animals with a short period of warm ischemia before reperfusion, even a tendency to reduced posttransplant lung reperfusion injury was noted. In this model, topical graft cooling during lung implantation did not improve posttransplant graft functio

Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20ms impulses at 200V/cm) 24h prior to the transplantation. Group A (n=5) received CsA (2.5mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123mmHg, vs 44±8mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II–IIIA). hIL-10 serum levels on day 5 were 14±7pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation

Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20ms impulses at 200V/cm) 24h prior to the transplantation. Group A (n=5) received CsA (2.5mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123mmHg, vs 44±8mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II-IIIA). hIL-10 serum levels on day 5 were 14±7pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantatio