Hepatic Changes During Various Periods of Reperfusion After Induction of Renal Ischemia in Rats

Background

Recent studies have suggested that ischemic damage to the kidneys causes liver tissue alterations. Thus, the morbidity and mortality in patients with acute renal failure (ARF) may be related to liver complications as well as to renal injury. The aim of the present study was to assess the hepatic changes during various periods of reperfusion after induction of renal ischemia.

Methods

Forty male rats were subjected to either a sham operation or to a 45-minute ischemia followed by 1, 3, 6, or 24 hours of reperfusion. Arterial pressure was continuously monitored. Blood samples were drawn to measure serum creatinine and blood urea nitrogen (BUN).

Results

We evaluated hepatic concentrations of interleukin (IL)-10 and tumor necrosis factor (TNF)-α. Ischemia reperfusion (IR) caused significant reductions in renal function as demonstrated by increased values of serum creatinine and BUN. These rats also showed significant increases in hepatic TNF-α and IL-10 concentrations. The most significant changes among inflammatory factors in the liver were observed at 3 hours of reperfusion: TNF-α, 616 ± 41 vs 215 ± 16, and IL-10, 926 ± 73 vs 125 ± 34, pg/100 mg tissue (P ≤ .05). Twenty-four-hour reperfusion reduced the extent of liver injury.

Discussion

Renal IR affects liver inflammatory status, possibly due to increased renal production or impaired clearance of mediators of tissue injury, namely proinflammatory cytokines. The reduction in liver injury at 24 hours of reperfusion compared with the other groups, suggested activation of late-protective mechanisms. These observations may be important for clinical interventions to reduce the morbidity and mortality of ARF.     

Cytokine Profile in Pleural Fluid and Serum After Lung Transplantation

Background

Lung transplantation is the procedure of choice in several end-stage lung diseases. Despite improvements in surgical techniques and immunosuppression, early postoperative complications occur frequently.

Objective

To evaluate the pleural inflammatory response after surgery.

Patients and Methods

Twenty patients aged 18 to 63 years underwent unilateral or bilateral lung transplantation between August 2006 and March 2008. Proinflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 and vascular endothelial growth factor in pleural fluid and serum were analyzed. For cytokine evaluation, 20-mL samples of pleural fluid and blood (right, left, or both chest cavities) were obtained at 6 hours after surgery and daily until removal of the chest tube or for a maximum of 10 days. Data were analyzed using analysis of variance followed by the Holm-Sidak test.

Results

All effusions were exudates according to Light's criteria. Pleural fluid cytokine concentrations were highest at 6 hours after surgery. Serum concentrations were lower than those in pleural fluid, and IL-1β, IL-6, and IL-8 were undetectable at all time points.

Conclusions

There is a peak concentration of inflammatory cytokines in the first 6 hours after transplantation, probably reflecting the effects of surgical manipulation. The decrease observed from postoperative day 1 and thereafter suggests the action of the immunosuppression agents and a temporal reduction in pleural inflammation.     

Protective role of nitric oxide induced by ischemic preconditioning on cold ischemic-reperfusion injury of rat liver graft

Aims

To investigate the protective role of nitric oxide (NO) induced by ischemic preconditioning (IP) on cold ischemic-reperfusion (IR) injury of rat liver grafts.

Methods

One hundred twenty-eight male Sprague Dawley rats used for orthotopic liver transplantation were randomly divided into four groups (n = 32): administering heparin before ischemic reperfusion (control group); IP with 10-minute ischemia and 10-minute reperfusion before IR (IP group); adenosine before IR (Ade group); and L-NAME (NG-nitro-L-arginine methyl ester) + IP before IR (NAME group). Half of each group were used to investigate 1-week recipient survival rate, and another to obtain blood and hepatic tissue samples after 2-hour reperfusion.

Results

One-week survival bile production, serum NO, and antioxidase activity were higher but serum alanine aminotransferase, tumor necrosis factor-α, and superoxide levels in hepatic tissue were lower in the IP group and Ade group versus the control group or NAME group. Liver sinusoidal endothelial cells in the IP and Ade groups showed less injury than the other groups.

Conclusion

NO induced by IP can improve 1-week survival and rat liver function as well as protect liver sinusoidal endothelial cells.     

Role of Peroxynitrite and Recombinant Human Manganese Superoxide Dismutase in Reducing Ischemia-Reperfusion Renal Tissue Injury

Background

In an acute kidney transplant rejection rat model, we demonstrated that manganese superoxide dismutase (MnSOD) activity was significantly reduced and MnSOD was nitrated by peroxynitrite (ONOO⁻), resulting in tissue injury. We examined whether tissue injury was reduced after external supplementation of recombinant human MnSOD in a rat renal ischemia-reperfusion injury model.

Methods

Male Brown-Norway rats underwent dissection of the right kidney. The animals were divided into 3 groups. The controls had the left renal blood vessels clamped for 90 minutes to induce ischemia, followed by reperfusion for 16 hours. In the intraperitoneal administration group, MnSOD was administered 30 minutes before ischemia and immediately before reperfusion. In the sham group, neither ischemia nor reperfusion was performed. After reperfusion, blood was collected, the left kidney was dissected and renal function and tissue injury were evaluated.

Results

Serum creatinine and K⁺, blood urea nitrogen, and aspartate aminotransferase activity decreased significantly, whereas serum Na⁺ and renal function improved in the MnSOD group compared with the control and sham groups. On hematoxylin and eosin staining, the histological score indicated that acute tubular necrosis was significantly reduced by MnSOD administration. Periodic acid-Schiff staining was absent in the nonadministration group, whereas it persisted in the MnSOD group. In the proximal renal tubules a large proportion of anti-nitrotyrosine staining was present before but absent after MnSOD administration.

Conclusions

MnSOD administration improved renal function and reduced tissue injury. It may also reduce tissue injury in acute kidney transplant rejection and other tissue injuries caused by similar molecular mechanisms.     

Recombinant Human Erythropoietin Pretreatment Attenuates Heart Ischemia-Reperfusion Injury in Rats by Suppressing the Systemic Inflammatory Response

Background

Ischemia-reperfusion (I/R) injury may influence graft function after transplantation. Erythropoietin (EPO) attenuates I/R injury in various animal organs such as intestine, brain, and kidney.

Objective

To evaluate the effects of pretreatment with recombinant human EPO (rhEPO) on I/R-induced heart injury.

Materials and Methods

A rat model of I/R injury was established by ligating the left descending coronary artery for 30 minutes, followed by reperfusion for 4 hours. Fifty Sprague-Dawley rats were divided into 5 groups: sham operation; I/R; I/R+rhEPO, 100 U/kg; I/R+rhEPO, 1000 U/kg; and I/R+rhEPO, 5000 U/kg. Electrocardiograms were assessed continuously to note arrhythmia caused by reperfusion. Serum concentrations of interleukin (IL)-6 and IL-8, and tumor necrosis factor-α were measured at 2 and 4 hours after reperfusion.

Results

The rhEPO-treated animals exhibited dosage-dependent significant reduction in the incidence of ventricular arrhythmia caused by reperfusion, and markedly decreased serum concentrations of IL-6, IL-8, and tumor necrosis factor-α (P < .05) compared with the I/R group (P < .05).

Conclusion

The rhEPO attenuates myocardial I/R injury in rats, at least in part related to inhibition of the system inflammatory response.     

Burn induces a Th-17 inflammatory response at the injury site

Introduction

The high incidence of morbidity and mortality following major burn can be in part attributed to immune dysfunction and wound healing complications. Inflammation plays a major role in the complex process of wound repair. Recently, a novel class of T-helper cells, termed Th-17 cells, has been found to secrete the pro-inflammatory cytokines IL-17 and IL-22. The Th-17 response also involves other cytokines, such as IL-6 and TGF-β, which have been shown to be associated with burn-induced inflammation. Nonetheless, the relationships between the Th-17 response and post-burn inflammation are unknown.

Methods

C57BL/6 male mice (n = 5-6/group) were subjected to a major burn (25% TBSA) or sham procedure. Three hours thereafter, skin samples were collected (uninjured skin and burn skin) and processed for the determination of Th-17 cytokine (IL-6, IL-17, IL-22, IL-23, IL-27, and TGF-β) levels by ELISA.

Results

At 3 h after burn a significant (∼3-fold) increase in tissue levels of IL-17 and IL-22 was observed at the burn site as compared to sham skin. The burn-induced Th-17 response was independent of statistically significant changes in other Th-17 cytokines (i.e., IL-6, IL-23, IL-27 and TGF-β).

Conclusions

These findings indicate the development of a robust Th-17 response at the burn site that may play an important role in subsequent immune and wound healing derangements.     

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecium Infecting Recipients of Solid Organs in the Transplant Surgery Ward in 2005 and 2006

Background

The aim of the investigations was to compare the vancomycin-resistant Enterococcus faecium (VREfm) strains obtained from our patients.

Materials and Methods

Strains were compared using restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE) of bacterial DNA.

Results

VREfm infected 26 liver recipients, 22 kidney recipients, and 9 other surgery or nephrology patients. Only five strains possessed the vanB determinant. The PFGE analysis revealed two large and several small groups of related strains.

Conclusions

The PFGE analysis enabled the investigation of VRE epidemiology among patients after transplantation. Strains with similar patterns most probably originated from one source and clearly suggested an outbreak.     

Mitigation of Cytokine Storm by Intraoperative Use of Renal Replacement Therapy During Combined Liver-Kidney Transplantation

Background

Combined liver-kidney transplantation (CLKT) is a widely used multiorgan transplantation with good graft survival rates. Previous studies have shown beneficial effects of renal replacement therapy in critically ill patients. This observation led us to use intraoperative continuous veno-venous hemofiltration (CVVH) during multiorgan transplantations.

Methods

We analyzed (CRP) inflammatory response parameters of tumor necrosis factor (TNF)α, interleukin(IL)-6, procalcitonin (PCT) and C-reactive protein (CRP) at various stages of the combined transplantations.

Results

All patients survived with well-functioning grafts. Mean ± SD follow-up was 32.8 ± 14.2 months. During the whole operation we used intraoperative CVVH starting at the beginning and continuing in the intensive care unit (ICU) afterward (mean ± SD, 11.2 ± 8.4 hours). Intraoperative TNFα, IL-6, CRP, and PCT were measured before surgery, during hepatectomy in the anhepatic phase, before and after liver reperfusion, exactly before kidney reperfusion, after kidney reperfusion, and upon arrival in the ICU. The wash-out of cytokines together with hemodynamic stability gave optimal circumstances for recovery of the transplanted organs.

Conclusions

CVVH-based therapy offered stable intraoperative parameters, prevention of fluid overload, correction of metabolic disturbances, and wash-out of cytokines, which gave optimal circumstances for recovery of transplanted organs.     

Impact of coculture with ischemic preconditioned hepatocellular carcinoma cell line (Hep-G2) cells on insulin secreting function of rat insulin-secreting cell line (RIN-5F) cells

Introduction

Although Islet cell isolation and culture have been well developed, there has been little progress to prolong transplanted islet sruvival. Hepatic ischemia and insufficient neovascularization of islets are considered to be the barriers to long-term survival, Hepatocytes that survive ischemic injury have been reported to protect themeslves and regenerate using the IL-6 interleukin 6 and STAT3 pathways.

Materials and Methods

The hepatocellular carcinoma (Hep-G2) cell line preconditioned for 0, 2, 4, 6, and 24 hours in a hypoxic chamber, was cocultured with rat insulin-secreting celline (RIN-5F) cells. We measured cell viabilities, insulin secretion, and p-STAT3, IL-6, and NF-κB levels.

Results

Cocultured Hep-G2 and RIN-5F cells aggregated to form spheroids. Viabilities of Hep-G2 cells were no different after various ischemic preconditioning times, but insulin secretion increased in a time-dependent fashion with preconditioning. Western blotting showed p-STAT3, NF-κB, and IL-6 levels to increase with preconditioning time.

Conclusion

The IL-6/STAT3 pathway of Hep-G2 cells after ischemic injury showed beneficial effects on insulin secretion of RIN-5f cells cocultured with themselves.     

Effect of Immunosuppressant Blood Levels on Serum Concentration of Interleukin-17 and -23 in Stable Liver Transplant Recipients

Introduction

T_H17 cells have been recently described to be involved in inflammatory and immune-mediated diseases, but there is no evidence of their role in human liver transplantation. Interleukin (IL)-23 is considered an inducer cytokine, whereas IL-17 is the main cytokine released by T_H17 cells. The aim of our study was to measure the serum levels of IL-17 and IL-23 in stable liver transplant recipients and examine the influence of immunosuppressant concentrations.

Materials and Methods

Serum levels of IL-23 and IL-17 were determined in 38 healthy subjects and 35 stable hepatic transplant recipients who were free of rejection episodes for at least 8 years. The results were analyzed according to the simultaneous blood levels of cyclosporine (n = 20) or tacrolimus (n = 15).

Results

No significant differences were observed in the serum levels of IL-17 and IL-23 between healthy subjects and transplanted patients. In addition, patients with low blood levels of tacrolimus (<6 ng/mL), but not cyclosporine, showed significantly lower serum levels of the 2 cytokines.

Conclusion

These preliminary results suggested a lack of activation of the T_H17 pathway, which was more pronounced among the patient subgroup treated with tacrolimus.     

Cold Ischemia Time Influences Spermatogenesis in a Testicular Ischemia/Reperfusion Injury Model

Objective

To evaluate the influence of cold ischemia time on spermatogenesis in a rabbit model of testicular ischemia-reperfusion (I/R) injury.

Material and Methods

The testicular I/R model was established in 24 male white rabbits. The left testes were preserved using HC-A solution at 0°C to 4°C. Cold ischemia time was 1, 2, 4, and 6 hours. The right testes without vascular occlusion were used as autologous controls. Twenty-four hours after reperfusion, the animals were sacrificed, and samples were obtained at bilateral orchiectomy. Another 8 normal testes were used as normal controls. Testicular tissue Johnsen score, malondialdehyde concentration, and apoptosis index were used to evaluate spermatogenesis.

Results

The Johnsen score decreased and the apoptosis index increased with the duration of cold ischemia time in the I/R groups. The malondialdehyde concentration in the I/R groups was significantly higher than the sham and normal groups, and was highest at 4 hours of cold ischemia time.

Conclusion

Testicular I/R injury is highly related to cold ischemia time. In rabbit models, testis transplantation is best performed within 4 hours of cold ischemia with traditional hypothermic protection.     

Impact of surgery on immunologic function: comparison between minimally invasive techniques and conventional laparotomy for surgical resection of colorectal tumors

Background

Surgical trauma suppresses host immune function, potentially creating an environment vulnerable to tumor cell growth. This study compared immune function after laparoscopy, minilaparotomy, and conventional colorectal tumor resections.

Methods

Seventy-one patients underwent surgery (20 laparoscopy, 21 minilaparotomy, and 30 conventional). Blood samples were taken before surgery and at 3 hours, 24 hours, and 5 days after surgery. White blood cell constitution was determined using monoclonal antibodies. Levels of TH1 cytokines interferon-γ, tumor necrosis factor-α, and interleukin (IL)-2 and TH2 cytokines IL-10, -4, and -6 were measured in plasma and from supernatants of activated peripheral blood mononuclear cells.

Results

At 5 days after surgery, lymphocyte counts remained low in the conventional and minilaparotomy groups (P = .001 and P = .008) but had resolved in laparoscopic patients. Three-hour postoperative serum IL-6 concentrations were lower in laparoscopic than in conventional patients (P = .028). Production of TH1 cytokines 3 hours after surgery were significantly increased in laparoscopic patients (interferon-γ P = .018, tumor necrosis factor-α P = .011, and IL-2 P = .037).

Conclusions

TH1 lymphocyte function is improved transiently and immune homeostasis restored earlier in patients undergoing laparoscopic colorectal cancer resection, which may influence disease recurrence.     

Stem cell factor, interleukin-16, and interleukin-2 receptor alpha are predictive biomarkers for delayed and slow graft function

Introduction

Delayed graft function (DGF) and slow graft function (SGF) due to ischemic and reperfusion injury (IRI) are common complications of deceased donor kidney transplantation. We tested whether a panel of serum and urine cytokines represent early biomarkers for DGF and SGF.

Methods

We collected serum and urine samples from 61 patients 48 hours posttransplantation and used a multiplex enzyme-linked immunosorbent assay (ELISA) technique to measure levels of 23 cytokines. Fourteen patients developed poor graft function (PGF), with 6 having DGF and 8 with SGF.

Results

Area under receiver operation characteristics curve (AUC) demonstrated the following: serum levels of SCF (0.88) and interleukin (IL) 16 (0.74).

Conclusions

This study showed that a select panel of cytokines measured early post kidney transplantation may predict poor graft function.     

Variation in the Levels of Inflammatory Cytokines Depending on Ischemic Time: Effects on Respiratory Variables

Objectives

We sought to evaluate the association between ischemic times, cytokines—interleukin (IL)-6, IL-1b, tumor necrosis factor-alpha, sIL-2r, IL-8, and IL-10—and alterations in gaseous exchange.

Materials and Methods

This prospective study of 42 orthotopic liver transplantation (OLT) recipients examined ischemic times and respiratory variables measured as alterations in intrapulmonary shunt and in the Po₂/Fio₂ ratio. Centrifuged blood samples were frozen at −80°C for storage. The Inmulite-One system (Euro/Dpc, Gwynedd, UK) was used to determine the concentration of cytokines. For statistical analysis, we used the Pearson correlation coefficient.

Results

The average cold ischemic time was 478 minutes (range, 35-929) and warm ischemic time was 69.58 minutes (range, 20-180). The warm ischemic time affected the degree of shunt at the end of the operation (P < .027) and the levels of IL-10 (P < .018) and IL-6 (P < .000). The final degree of shunting and IL-10 (P < .044) showed a correlation. The cold ischemic time affected IL-1 (P < .046) and IL-8 levels (P < .023). The reperfusion syndrome was correlated with the final levels of IL-10 (P < .064) and of IL-8 (P < .066).

Conclusion

Warm and cold ischemic times affect the final cytokine levels and the degree of intrapulmonary shunt.     

Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium

Background

The identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.

Methods

Cystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24 h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.

Results

Treatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.

Conclusion

Our data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not.     

Factors influencing rat survival in a warm renal ischemia model: time to adapt the protocols

Introduction

Survival in warm renal ischemia models is not only dependent on the treatment or surgical technique being evaluated, but also on factors inherent to the model itself. Use of rats of various strains in previous studies makes interstudy comparison difficult when trying to design an appropriate model control that would yield intermediate survival. In this study, impact of rat strain on survival after prolonged warm renal ischemia in the setting of delivery-controlled inhalational anesthesia was evaluated.

Materials and methods

Under general delivery-controlled inhalation anesthesia with isoflurane, Dahl salt-sensitive, Wistar-Furth, Sprague-Dawley, and spontaneously hypertensive rats (n = 66 rats) were subjected to 150 minutes of unilateral renal warm ischemia time, subsequent reperfusion, and contralateral nephrectomy. Animals were followed up for 1 month, after which survivors were euthanized and morphologic changes in kidneys were scored.

Results

Thirty-day survival was: Dahl salt sensitive, 78%; Wistar-Furth, 67%; Sprague-Dawley, 55%; and spontaneously hypertensive rats, 0% (P < .0001). Histologic acute injury scores were higher for non-survivors versus 30-day survivors (P < .0001).

Conclusion

Our data strongly suggest that rat strain is a major factor influencing survival and that strain and warm ischemia time selections must be considered together when designing a model control yielding intermediate survival. Further study is warranted in order to compare the effect of delivery-controlled inhalational versus historical anesthesia methods on animal survival.     

Effects of the anti-ICAM-1 monoclonal antibody, allopurinol, and methylene blue on intestinal reperfusion injury

Purpose:

The aim of this study was to evaluate the effect of allopurinol, methylene blue, and a monoclonal antibody to the adhesion molecule ICAM-1 in intestinal ischemia and reperfusion injury.

Methods:

The rats were divided into 5 groups. CG (n = 8) was untreated controls, SISG (n = 11) received sterile isotonic saline solution, ICAMG (n = 12) received a monoclonal antibody to rat ICAM-1, ALLOG (n = 12) received allopurinol, and MBG (n = 14) received methylene blue. Intestinal ischemia was performed for 60 minutes followed by 60 minutes of reperfusion. The agents were injected 10 minutes before the reperfusion to animals. After 60 minutes of reperfusion, the plasma samples for myeloperoxidase (MPO) activity, tumor necrosis factor alpha (TNF-α) and uric acid levels, and the intestinal biopsies of ileum and jejunum for histopathologic examination were taken.

Results:

The mucosal damage was attenuated, and TNF-α level significantly decreased in ALLOG and ICAMG compared with SISG. The MPO activity was the lowest in ICAMG, and uric acid level was significantly decreased in ALLOG compared with the other groups. Methylene blue decreased TNF-α response to reperfusion injury but significantly increased the grade of the mucosal damage and the MPO activity.

Conclusions:

This study shows that prereperfusion application of allopurinol and monoclonal antibody to the adhesion molecule ICAM-1 may attenuate the damage caused by intestinal ischemia and reperfusion, but the different time-points for application, the effects observed in the different ischemia and reperfusion durations, and the long-term results also should be investigated in the same experimental model before the final conclusion. Methylene blue was not effective to prevent or attenuate the intestinal tissue injury, but because this was the first study examining the effect of methylene blue on intestinal reperfusion injury, further studies with the different doses, ischemic duration, and application times will be needed.     

The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver

Background

Liver ischemia-reperfusion (I-R) injury is a well-known cause of morbidity and mortality following liver surgery and transplantation. Further investigation is warranted to identify measures that reduce the untoward sequelae of liver ischemia.

Methods

Male Sprague-Dawley rats (wild-type) and Zucker rats (with hepatic steatosis) were subjected to 75 minutes of 70% hepatic ischemia and 3 hours of reperfusion. The ischemic periods were based on protocols of either continuous clamping (CC) or ischemic preconditioning (IP). Prior to ischemia induction, rats were pretreated with intravenous methylprednisolone (MP; 2 mg/kg) or normal saline. Warm I-R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum interleukin-6 (IL-6), and hematoxylin and eosin staining.

Results

Histology, serum IL-6, and AST release revealed that MP treatment provided significant protection as compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, livers. The inflammatory response was considerably reduced in MP groups with normal livers but not in steatotic livers. In general, the IP groups showed decreased I-R injury compared to the CC group. However, MP was able to further reduce I-R injury only in normal, not steatotic, livers.

Conclusions

MP attenuated the postischemic and inflammatory response in the normal, and not steatotic, livers. MP pretreatment might be effective in reducing warm I-R injury to livers without steatosis. The mechanism of I-R-related hepatocellular damage in steatotic liver is different than in normal liver.