Threshold level of NF-kB activation in small bowel ischemic preconditioning procedure

Ischemic preconditioning (IPC), which is obtained by exposure to brief periods of vascular occlusion, improves organ tolerance to prolonged ischemia. The aim of this study was to evaluate the threshold level of NF-kB activation in small intestine during an IPC procedure. Various intestinal IPC were performed on 20 Wistar rats in seven groups: group I (GI, nonpreconditioned); group II (GII, 1-minute ischemia and 1-minute reperfusion); group III (GIII, two cycles of 1-minute ischemia and 1-minute reperfusion); group IV (GIV, 2-minutes ischemia and 2-minutes reperfusion); group V (GV, two cycles of 2-minute ischemia and 2-minute reperfusion); group VI (GVI, 5-minute ischemia and 10-minute reperfusion); group VII (GVII, two cycles of 5-minute ischemia and 10-minute reperfusion). Bowel biopsies were collected after laparotomy (control) as well as at 30, 60, and 120 minutes following IPC. We determined the cytoplasmic and nuclear NF-kB by a chemiluminescence-based ELISA method. Our results showed low, constant NF-kB levels in GI. In the preconditioned groups (GII-GVII), NF-kB was significantly elevated at 30 minutes following IPC (P < .05 vs control). After 1 hour, NF-kB activity decreased to the control level. However, 2 hours after IPC both forms of NF-kB were elevated significantly again, which was independent of the number of IPC cycles (P < .05 vs control). Our experiments revealed that one cycle of 1-minute ischemia and 1-minute reperfusion is a critical threshold level for NF-kB activation during small bowel IPC. Longer and more IPC cycles did not result in further elevation of NF-kB activation.     

Apoptosis is caused by prolonged organ preservation and blocked by apoptosis inhibitor in experimental rat pancreatic grafts

Apoptosis is a major mode of cell death after ischemia/reperfusion injury in several organs. The aims of this study were to evaluate apoptosis induction after different conditions of pancreas preservation and to investigate the impact of a caspase inhibitor on apoptosis induction in pancreatic grafts. METHODS: Inbred male Lewis rats served as donors and recipients. Apoptosis was detected using an in situ cell death detection kit and an apoptotic DNA ladder kit (Roche, Germany). An apoptotic index (AI) was defined as the number of apoptotic cells per field (400x) under a light microscope. The five groups included: group 1 (n = 5), normal pancreata; group 2 (n = 7), pancreata stored in University of Wisconsin (UW) solution (4 degrees C) for 6 hours (hr); group 3 (n = 7), pancreata preserved in UW solution (4 degrees C) for 18 hr; group 4 (n = 7), pancreata preserved in 0.9% saline (4 degrees C) for 6 hours; and group 5 (n = 5), pancreata preserved in 0.9% saline (4 degrees C) for 6 hours with Z-Asp-2,6-dichlorobenzoyloxymethylketone (caspase inhibitor) treatment. The pancreatic grafts in all experimental groups underwent a 2-hr period of reperfusion after transplantation. RESULTS: The results in this study showed that the AI was not significantly increased among pancreatic grafts in group 2 compared to group 1 (P >.05). However, AI in group 3 was significantly higher than that in group 2 (P <.05). The highest AI was observed in group 4. Interestingly, AI in group 5 was significantly lower than that in group 4 (P <.01), and not significantly different from group 1 (P >.05). Apoptotic DNA ladders were detected in the pancreatic grafts in group 2, 3, and 4, but not in group 1 and 5. CONCLUSIONS: Prolonged preservation of pancreata in cold UW solution induces a dramatic increase in apoptotic cell death among the pancreatic grafts following Tx. This observation may well be an important mechanism of graft damage. A caspase inhibitor might be useful to inhibit apoptosis induction in pancreatic grafts.     

Role of NF-kappaB as effector of IPC in donor livers before liver transplantation in rats

The objective of this study was to investigate the effect of ischemic preconditioning (IPC) on NF-kappaB activity during reperfusion early after liver transplantation in rats. METHODS: Male Sprague-Dawley (SD) rats were used as donors and recipients of orthotopic liver transplantations. The donor liver was stored 2 hours in Ringer's solution at 4 degrees C preimplantation. IPC was performed by clamping of the portal vein and hepatic artery of the donor for 10 minutes followed by reperfusion for 10 minutes before harvesting. At 1, 2, 4, and 6 hours after portal vein reperfusion, graft samples were obtained to determine hepatic levels of NF-kappaB activity, tumor necrosis factor (TNF)-alpha and intercellular adhesion molecule (ICAM)-1. Blood samples were obtained to measure serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). RESULTS: After liver transplantation without IPC, serum levels of ALT and LDH increased significantly compared with the sham-operated group. Among the IPC group, serum ALT and LDH decreased significantly. NF-kappaB activity in the graft increased within 6 hours after transplantation. Among the IPC group, NF-kappaB activity was significantly attenuated. Hepatic levels of TNF-alpha and ICAM-1 were significantly elevated in the non-IP group but both were reduced in the IPC group. CONCLUSION: IPC downregulated TNF-alpha and ICAM-1 expression in the graft, most likely through decreased NF-kappaB activation, and attenuated neutrophil infiltration after reperfusion.     

Ischemic pre-conditioning of 5 minutes but not of 10 minutes improves lung function after warm ischemia in a canine model.

BACKGROUND: Protection from reperfusion injury by ischemic pre-conditioning (IPC) before prolonged ischemia has been proven for the heart and the liver. We now assess the efficacy of IPC to protect lungs from reperfusion injury. METHODS: Eighteen foxhounds (25 to 30 kg) were anesthetized, intubated, and ventilated with a fraction of inspired oxygen of 0.3 at a volume-controlled mode to maintain arterial pCO2 of 30 to 40 mm Hg. After left thoracotomy, we performed warm ischemia for 3 hours by clamping the left hilus, and followed with 8 hours of reperfusion (control, n = 6). In the treated groups, IPC was performed either for 5 minutes followed by 15-minute reperfusion (n = 6, IPC-5), or by 2 successive cycles of 10-minute ischemia, followed by 10-minute reperfusion (n = 6, IPC-10) before prior to the 3-hours warm-ischemia period. Pulmonary compliance and gas exchange were determined separately for each lung, and we recorded pulmonary and systemic hemodynamics. We performed bronchoalveolar lavage (BAL) at the end of the experiment and determined total protein concentration as well as tumor necrosis factor alpha (TNF-alpha) mRNA expression in cell-free supernatant and in BAL cells, respectively. We also assessed the wet/dry ratio of the lung. RESULTS: In the controls, on reperfusion, we encountered a progressive deterioration of gas exchange, especially of the reperfused left lung, which we could largely avoid using the IPC-5 protocol. Similarly, pulmonary compliance steadily declined but was much better in the ICP-5 group. Parallel to the improvement of gas exchange and lung mechanics, we found less total alveolar protein content and TNF-alpha mRNA expression in BAL cells in the IPC-5 than in the controls. However, we did not find IPC-10 to be paralleled by a significant improvement of lung function. Neither IPC-5 nor IPC-10 influenced the pulmonary vascular resistance index or the fluid accumulation in the lung. CONCLUSION: The major finding of the present study was that 5 minutes of IPC improved lung function after 3 hours of warm ischemia of the lung.     

缺血预处理对家兔肢体缺血再灌注后肺抗氧化系统的影响

目的：观察肢体缺血预处理对家兔肢体缺血再灌注后肺损伤时肺抗氧化系统的影响并探讨其机制。方法：健康家兔30只，随机分为假手术对照组（SC）、缺血再灌注组（IR）和缺血预处理组（IPC），每组10只。实验结束时，取肺组织测定超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH—PX）、黄嘌呤氧化酶（XOD）活性和丙二醛（MDA）、一氧化氮（N0）含量，同时计算肺系数及进行肺病理学检查。结果：肢体缺血再灌注后肺组织SOD、GSH-PX活性和NO含量显著下降，XOD活性和MDA含量明显增高，肺系数升高，肺组织病变明显，肢体缺血预处理可明显缓解上述变化。结论：家兔肢体缺血再灌注后，肺抗氧化功能下降参与肺脏的损伤过程，肢体缺血预处理对这种肺损伤有明显的保护作用。     

缺血预处理对肢体缺血再灌注损伤的影响

目的　观察缺血预处理 (IPC)对肢体缺血再灌注损伤的影响。方法　选择 2 0例需充气止血带止血进行手术的患者 ,随机分为对照组 (n =10 )和IPC组 (n =10 )。IPC组患者术前应用 3次 5min循环缺血 ,间隔 5min再灌注预处理后在止血带下进行手术 ;对照组直接在止血带下进行手术。在肢体缺血前和再灌注 30min、90min、180min分别取静脉血检测血清肌酸磷酸激酶 (CPK)、谷草转氨酶(AST)、乳酸脱氢酶 (LDH)、丙二醛 (MDA)和过氧化物歧化酶 (SOD)水平。结果　随着肢体缺血再灌注时间的延长 ,血中CPK、AST、LDH、MDA含量逐渐升高 ,而SOD活性逐渐降低。IPC组在缺血前及再灌注同时间 ,血中CPK、AST、LDH、MDA含量低于对照组 (P <0 0 5 ,P <0 0 1) ;而SOD活性高于对照组 (P <0 0 5 ,P <0 0 1)。结论　IPC能有效地减轻肢体缺血再灌注损伤程度 ,减轻脂质过氧化反应 ,提高肢体缺血耐受性     

Pharmacologic stimulation of adenosine A2 receptor supplants ischemic preconditioning in providing ischemic tolerance in rat livers

BACKGROUND: Ischemic preconditioning (IPC) is a promising strategy for conferring ischemic tolerance. We confirmed the acquisition of ischemic tolerance in the liver immediately after IPC and the role of adenosine kinetics in this process. METHODS: Male Lewis rats were used. IPC was administered with a 10-minute ischemia followed by a 10-minute reperfusion. Ischemic tolerance was tested with a 45-minute ischemia. Changes in the adenosine concentrations in liver tissue were evaluated, and the effects of adenosine A1 or A2 receptor agonists or antagonists were examined either in place of or against IPC. RESULTS: The 7-day animal survival was significantly better in the IPC group than in the control group (87% vs 53%; n = 15, P < .05). The release of liver-related enzymes during reperfusion was suppressed better in the IPC group (P < .01). Recovery of adenosine triphosphate levels was faster in the IPC group (P < .01). After IPC, adenosine concentrations in liver tissue immediately increased to 1555 +/- 299 pmol/g wet tissue and were maintained at that level during a subsequent 45-minute ischemia. The ischemic tolerance generated by IPC was mimicked by the administration of adenosine A2 receptor agonist and opposed by adenosine A2 receptor antagonist. CONCLUSIONS: The ischemic tolerance of the liver immediately after IPC can be supplanted by selective pharmacologic stimulation of adenosine A2 receptors.     

Ischemic preconditioning reduces neurologic injury in a rat model of spinal cord ischemia.

BACKGROUND: Ischemic preconditioning (IPC) is an endogenous cellular protective mechanism whereby brief, noninjurious periods of ischemia render a tissue more resistant to a subsequent, more prolonged ischemic insult. We hypothesized that IPC of the spinal cord would reduce neurologic injury after experimental aortic occlusion in rats and that this improved neurologic benefit could be induced acutely after a short reperfusion interval separating the IPC and the ischemic insult. METHODS: Forty male Sprague-Dawley rats under general anesthesia were randomly assigned to one of two groups. The IPC group (n = 20) had 3 minutes of aortic occlusion to induce spinal cord ischemia 30 minutes of reperfusion, and 12 minutes of ischemia, whereas the controls (n = 20) had only 12 minutes of ischemia. Neurologic function was evaluated 24 and 48 hours later. Some animals from these groups were perfusion-fixed for hematoxylin and eosin staining of the spinal cord for histologic evaluation. RESULTS: Survival was significantly better at 48 hours in the IPC group. Sensory and motor neurologic function were significantly different between groups at 24 and 48 hours. Histologic evaluation at 48 hours showed severe neurologic damage in rats with poor neurologic test scores. CONCLUSIONS: Ischemic preconditioning reduces neurologic injury and improves survival in a rat model of spinal cord ischemia. The protective benefit of IPC is acutely invoked after a 30-minute reperfusion interval between the preconditioning and the ischemic event.     

Effect of ischemic preconditioning in whole liver transplantation from deceased donors. A pilot study.

The effect of ischemic preconditioning (IPC) in orthotopic liver transplantation (OLT) has not yet been clarified. We performed a pilot study to evaluate the effects of IPC in OLT by comparing the outcomes of recipients of grafts from deceased donors randomly assigned to receive (IPC+ group, n = 23) or not (IPC- group, n = 24) IPC (10-min ischemia + 15-min reperfusion). In 10 cases in the IPC+ group and in 12 in the IPC- group, the expression of inducible nitric oxide synthase (iNOS), neutrophil infiltration, and hepatocellular apoptosis were tested by immunohistochemistry in prereperfusion and postreperfusion biopsies. Median aspartate aminotransferase (AST) levels were lower in the IPC+ group vs. the IPC- group on postoperative days 1 and 2 (398 vs. 1,234 U/L, P = 0.002; and 283 vs. 685 U/L, P = 0.009). Alanine aminotransferases were lower in the IPC+ vs. the IPC- group on postoperative days 1, 2, and 3 (333 vs. 934 U/L, P = 0.016; 492 vs. 1,040 U/L, P = 0.008; and 386 vs. 735 U/L, P = 0.022). Bilirubin levels and prothrombin activity throughout the first 3 postoperative weeks, incidence of graft nonfunction and graft and patient survival rates were similar between groups. Prereperfusion and postreperfusion immunohistochemical parameters did not differ between groups. iNOS was higher postreperfusion vs. prereperfusion in the IPC- group (P = 0.008). Neutrophil infiltration was higher postreperfusion vs. prereperfusion in both groups (IPC+, P = 0.007; IPC-, P = 0.003). Prereperfusion and postreperfusion apoptosis was minimal in both groups. In conclusion, IPC reduced ischemia/reperfusion injury through a decrease of hepatocellular necrosis, but it showed no clinical benefits.     

Ischemic preconditioning fails to improve microcirculation but increases apoptotic cell death in experimental pancreas transplantation

Brief periods of warm ischemia and subsequent short reperfusion before either long-term cold or warm ischemic insult (ischemic preconditioning, IPC) have proven to ameliorate ischemia/reperfusion (I/R) injury in various organs, such as the liver and lung. The aim of this study was to examine the effect of IPC on pancreatic cell apoptosis and microcirculatory impairments in experimental pancreas transplantation. Male Lewis rats served as donors and recipients of heterotopic syngeneic pancreaticoduodenal transplantation. Recipient animals were divided into two experimental groups: group Tx (n=7) received grafts without IPC, group Tx&IPC received grafts with IPC. Animals that had not undergone transplantation but whose pancreata had been exteriorized served as controls (n=5). All pancreatic grafts were preserved in University of Wisconsin solution for 6 h at 4°C. IPC was induced by interruption of the arterial blood flow for 10 min followed by 10 min of reperfusion. One and two hours after reperfusion, graft microcirculation was assessed by means of intravital microscopy (IVM). Rats were immediately killed after the second measurement and DNA breaks of acinar cells were detected by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end-labelling (TUNEL) assay and gel electrophoresis (laddering). The apoptotic index (AI) was defined as the number of apoptotic cells per high-power field. Analysis of both groups of transplanted grafts showed a significant decrease in functional capillary density (FCD) and a significant increase in leukocyte sticking to postcapillary venules (LAV) at 1 h and 2 h of reperfusion, compared with animals that had not undergone transplantation (P<0.01). In parallel, AI was significantly increased in transplanted grafts compared to the controls (P<0.01). Grafts subjected to IPC showed no significant differences, neither for FCD nor LAV, at both time points if compared with grafts of group Tx. However, IPC resulted in a significant increase in AI (P<0.05). We can conclude that IPC has no effect on pancreatic microcirculation but enhances acinar cell apoptosis in experimental pancreas transplantation. These results indicate that IPC might increase I/R injury after pancreatic cold ischemia.     

Mitochondrial function during ischemic preconditioning

Background. Ischemic preconditioning (IPC) protects the myocardium from ischemia reperfusion injury. The effect of IPC on the mitochondria is not well known. However, one of the mechanisms postulated in IPC (the opening of the mitochondrial K(ATP) channels) is likely to result in changes in mitochondrial function. Therefore, the purpose of this study was to determine the effect of IPC on mitochondrial function during ischemia reperfusion. Methods. Isolated rat hearts (n = 6/group) were subjected to (1) 30 minutes of equilibration, 25 minutes of ischemia, and 30 minutes of reperfusion (RP) (control group) or (2) 10 minutes of equilibration, two-5 minute episodes of IPC (each followed by 5 minutes of re-equilibration), 25 minutes of ischemia, and 30 minutes of RP (IPC group). Left ventricular rate pressure product (RPP) was measured. At end-equilibration (end-EQ) and at end-reperfusion (end-RP) mitochondria were isolated. Mitochondrial respiratory function (state 2, 3, and 4), respiratory control index (RCI), rate of oxidative phosphorylation (ADP/Delta t), and ADP:O ratio were measured by polarography with the use of NADH- or FADH-dependent substrates. Results. IPC improved recovery of RPP at end-RP (72% +/- 5% in IPC vs 30% +/- 4% in control, P <.05). Ischemia reperfusion (IR) decreased state 3, ADP/Delta t, and RCI in both groups compared with end-EQ. IPC improved state 3 (47 +/- 3 in IPC vs 37 +/- 2 ng-atoms O/min/mg protein in control), ADP/Delta t (17 +/- 1 in IPC vs 13 +/- 1 nmol/s/mg protein in control), and RCI (3.7 +/- 0.1 in IPC vs 2.1 +/- 0.2 in control) at end-RP compared with control with the use of NADH-dependent substrate (P <.05 vs control). IPC also improved state 3 (85 +/- 6 in IPC vs 71 +/- 4 ng-atoms O/min/mg protein in control), ADP/Delta t (18 +/- 2 in IPC vs 12 +/- 1 nmol/s/mg protein in control), RCI (2 +/- 0.1 in IPC vs 1.5 +/- 0.1 in control), and ADP:O ratios (1.4 +/- 0.04 in IPC vs 1.7 +/- 0.09 in control) at end-RP compared with control with the use of FADH-dependent substrate (P <.05 vs control). Conclusions. The cardioprotective effects of IPC can be attributed at least in part to the preservation of mitochondrial function during reperfusion.     

不同时限缺血预处理对硬化肝脏保护作用的实验研究

目的: 探讨缺血预处理对硬化肝脏缺血再灌注的作用,并寻找一种有效缺血预处理的时间窗和理想方案. 方法: 将64只雄性、肝硬化SD大鼠随机分为八组,每组八只:假手术组(SO组);缺血再灌注组(I/R组);缺血预处理1、2、3、4、5和6组(IPC1、IPC2、IPC3、IPC4、IPC5和IPC6).以肝组织ATP、ADP、AMP及EC(用高效液相色谱法测定),血清ALT、AST、LDH(用全自动生化仪测定)和肝脏胆汁分泌量来评价肝功能. 结果: 再灌注末,IPC3组、IPC4组、IPC5组ATP含量均明显高于I/R组(P分别为0.01、0.07和0.000);同样,测定EC时发现,IPC3组、IPC4组和IPC5组均明显高于I/R组(P=0.000).与I/R组比较,IPC4组和IPC5组的血清ALT差异有显著性(P分别为0.013和0.000);其血清LDH差异亦有显著性(P=0.023,P=0.000),而血清AST却只有IPC5组显著低于I/R组(P=0.001).IPC3组、IPC4组和IPC5组肝脏的胆汁分泌量明显高于I/R组(P=0.028,P=0.023,P=0.008). 结论: 5~10min予一次或两次缺血预处理,能启动IPC对肝硬化大鼠肝脏I/R损伤的保护作用;10 min的缺血预处理,对肝硬化大鼠肝脏I/R损伤的保护作用最强.     

Adenosine A2A analogue ATL-146e reduces systemic tumor necrosing factor-alpha and spinal cord capillary platelet-endothelial cell adhesion molecule-1 expression after spinal cord ischemia

OBJECTIVE: Inflammation is likely a major contributor to spinal cord reperfusion injury after aortic reconstruction. Systemic 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL-146e), a selective adenosine A(2A) agonist, has been shown to reduce paralysis after spinal cord ischemia. We hypothesized that ATL-146e reduces cytokine production during spinal cord reperfusion, curtailing inflammation and decreasing spinal cord capillary platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression. Study design: New Zealand White rabbits sustained spinal cord ischemia with 45-minute cross-clamping of the infrarenal aorta. One group of animals received intravenous ATL-146e at 0.06 microg/kg/min for 3 hours during reperfusion, beginning after 30 minutes of ischemia. A second group received saline solution vehicle alone for 3 hours, serving as an ischemic control. A third group served as sham-operated animals, undergoing laparotomy with anesthesia. Serum was assayed with enzyme-linked immunosorbent assay for tumor necrosing factor-alpha (TNF-alpha). Animals were allowed to recover for 48 hours and were evaluated for hind-limb motor function with the Tarlov (0 to 5) scoring system. At necropsy, animals from each group yielded spinal cords for immunohistochemical staining for PECAM-1. Data are expressed as mean +/- standard error of the mean, with statistical analysis with Student t test and Kruskal-Wallis nonparametric test. RESULTS: Markedly improved Tarlov scores were seen in rabbits with ATL-146e (P <.001) during spinal cord reperfusion as compared with ischemic control animals. A significant reduction was found in TNF-alpha in the sera of rabbits with ATL-146e infusion (P <.01) as compared with ischemic control animals. Significantly reduced endothelial PECAM-1 staining intensity (P <.05) was seen in microscopic spinal cord sections from rabbits with ATL-146e. CONCLUSION: ATL-146e, an adenosine A(2A) agonist, reduces spinal cord reperfusion injury. The mechanism of the protection may involve a reduction in circulating TNF-alpha during a critical 3-hour reperfusion interval and reduction in spinal cord endothelial PECAM-1 upregulation.     

Evaluation of rapid ischemic preconditioning in a rabbit model of spinal cord ischemia

BACKGROUND: Rapid ischemic preconditioning (IPC) has been shown to reduce cellular injury after subsequent cardiac and cerebral ischemia. However, the data on rapid IPC of the spinal cord is limited. The authors investigated whether pretreatment with sublethal ischemia of spinal cord can attenuate neuronal injury after spinal cord ischemia in rabbits. METHODS: Forty-seven male New Zealand white rabbits were randomly assigned to one of three groups (n = 15 or 16 each). In the IPC(-) group, the infrarenal aorta was occluded for 17 min to produce spinal cord ischemia. In the IPC(+) group, 5 min of aortic occlusion was performed 30 min before 17 min of spinal cord ischemia. In the sham group, the aorta was not occluded. Hind limb motor function was assessed at 3 h, 24 h, 4 days, and 7 days after reperfusion using Tarlov scoring (0 = paraplegia; 4 = normal). Animals were killed for histopathologic evaluation at 24 h or 7 days after reperfusion. The number of normal neurons in the anterior spinal cord (L4-L6) was counted. RESULTS: Neurologic scores were significantly higher in the IPC(+) group than the IPC(-) group at 3 and 24 h after reperfusion (P < 0.05). However, neurologic scores in the IPC(+) group gradually decreased and became similar to those in the IPC(-) group at 4 and 7 days after reperfusion. At 24 h after reperfusion, the numbers of normal neurons were significantly higher in the IPC (+) group than in the IPC(-) group (P < 0.05) and were similar between the IPC(+) and sham groups. At 7 days after reperfusion, there was no difference in the number of normal neurons between the IPC(+) and IPC(-) groups. CONCLUSION: The results indicate that rapid IPC protects the spinal cord against neuronal damage 24 h but not 7 days after reperfusion in a rabbit model of spinal cord ischemia, suggesting that the efficacy of rapid IPC may be transient.     

Evaluation of Rapid Ischemic Preconditioning in a Rabbit Model of Spinal Cord Ischemia

Background: Rapid ischemic preconditioning (IPC) has been shown to reduce cellular injury after subsequent cardiac and cerebral ischemia. However, the data on rapid IPC of the spinal cord is limited. The authors investigated whether pretreatment with sublethal ischemia of spinal cord can attenuate neuronal injury after spinal cord ischemia in rabbits.

Methods: Forty-seven male New Zealand white rabbits were randomly assigned to one of three groups (n = 15 or 16 each). In the IPC(-) group, the infrarenal aorta was occluded for 17 min to produce spinal cord ischemia. In the IPC(+) group, 5 min of aortic occlusion was performed 30 min before 17 min of spinal cord ischemia. In the sham group, the aorta was not occluded. Hind limb motor function was assessed at 3 h, 24 h, 4 days, and 7 days after reperfusion using Tarlov scoring (0 = paraplegia; 4 = normal). Animals were killed for histopathologic evaluation at 24 h or 7 days after reperfusion. The number of normal neurons in the anterior spinal cord (L4-L6) was counted.

Results: Neurologic scores were significantly higher in the IPC(+) group than the IPC(-) group at 3 and 24 h after reperfusion (P < 0.05). However, neurologic scores in the IPC(+) group gradually decreased and became similar to those in the IPC(-) group at 4 and 7 days after reperfusion. At 24 h after reperfusion, the numbers of normal neurons were significantly higher in the IPC (+) group than in the IPC(-) group (P < 0.05) and were similar between the IPC(+) and sham groups. At 7 days after reperfusion, there was no difference in the number of normal neurons between the IPC(+) and IPC(-) groups.     

Liver transplantation in rats using small-for-size grafts: a study of hemodynamic and morphological changes

BACKGROUND: Damage to a small-for-size liver graft after reperfusion is frequently observed but the mechanism of injury remains unclear. HYPOTHESIS: Injury to a small-for-size liver graft is related to the changes of portal pressure and blood flow. MAIN OUTCOME MEASURES: Survival rates, portal hemodynamics, microcirculatory changes, and morphological changes (by light microscopy and electron microscopy). SETTING: A rat model of nonarterialized orthotopic liver transplantation comparing 2 groups of rats transplanted with whole grafts (100% of recipient liver weight) and small-for-size grafts (30% of recipient liver weight). RESULTS: Median survival of the rats with small-for-size grafts was 30 hours (range, 27-37 hours). During the first 15 minutes after reperfusion, mean arterial pressure of the small-for-size graft group was significantly lower than that of the whole graft group (10-minute: 100 vs 132 mm Hg, P =.04; 15-minute: 96 vs 127 mm Hg, P =.04). Portal pressure (in centimeters of water) of the small-for-size graft group was significantly higher in the first 20 minutes after reperfusion than the level before the anhepatic phase (5-minute: 15.1 vs 9.3, P =.02; 10-minute: 16.1 vs 9.3, P =.03; 15-minute, 13.5 vs 9.3, P =.03; 20-minute: 13.4 vs 9.3, P =.03) and was significantly higher than that of the whole graft group in the first 10 minutes after reperfusion (5-minute: 15.1 vs 9.6, P =.02; 10-minute: 16.1 vs 10.3, P =.04). Hepatic microcirculatory blood flow (in milliliters per minute per 100 g) was also significantly higher in the small-for-size graft group during the first 40 minutes after reperfusion (5-minute: 16.3 vs 9.3, P =.02; 10-minute: 14.9 vs 6.6, P =.02; 15-minute: 14.8 vs 5.5, P =.02; 20-minute: 13.1 vs 7.0, P =.02; 30-minute: 13.2 vs 8.8, P =.04; 40-minute: 14.6 vs 7.1, P =.02). Light and electron microscopy showed normal morphological features of whole graft up to 24 hours after reperfusion. The small-for-size graft, however, showed sinusoidal congestion, tremendous swelling of mitochondria of hepatocytes, irregular large gap of sinusoidal lining cells, and collapse of the space of Disse. CONCLUSIONS: In a rat model, the portal hemodynamic changes in small-for-size grafts are transient. Progressive damage of the graft may result from microcirculatory failure due to irreversible endothelial injury after reperfusion.     

Atrial natriuretic peptide protects against ischemia-reperfusion injury in rabbit hearts in vivo

The aim of this study is to investigate whether atrial natriuretic peptide can mimic preconditioning to protect ischemia or reperfusion injury in rabbit hearts. New Zealand white rabbits were randomized into 3 groups: (1) Controls. Hearts received a 60 minute-occlusion of the left anterior descending artery, followed by a 180 minute-reperfusion. (2) Preconditioning. Two 5-minute periods of ischemia separated by a 10-minute reperfusion, followed by a 60-minute ischemia and a 180-minute reperfusion. (3) Atrial natriuretic peptide treatment. Bolus injection of exogenous atrial natriuretic peptide (2.5 microg/kg) given intravenously at 15 minutes prior to 60 minute-ischemia followed by a 180-minute reperfusion. Myocardial necrotic area and area at risk of necrosis were determined by triphenyltetrazolium chloride staining. Ratio of necrotic area to area at risk was 49.95% +/- 1.15%, 7.95% +/- 0.33%, and 8.36% +/- 0.61% in the controls, preconditioning group, and atrial natriuretic peptide group, respectively. Both preconditioning and atrial natriuretic peptide significantly reduced the size of infarct caused by ischemia (preconditioning vs controls, P < .05; atrial natriuretic peptide vs controls, P < .05). Atrial natriuretic peptide can mimic ischemic preconditioning to protect rabbit hearts from prolonged ischemia and reperfusion injury. It may be involved in the cardioprotective mechanisms of preconditioning.     

α7烟碱样乙酰胆碱受体激动剂后处理对大鼠在体心肌缺血/再灌注损伤的影响

目的 观察α7烟碱样乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)激动剂后处理对大鼠在体心肌缺血/再灌注损伤(ischemia reperfusion injurv,IRI)的影响.方法 将40只SD大鼠采用计算机产生随机数法平均分为假手术组(S组)、缺血/再灌注组(IR组)、缺血预处理组(IPC组)和α7nAChR激动剂后处理组(PNU组),每组10只.实验中记录缺血期和再灌注初期心律失常,测定冉灌注30 min和180 min时的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-6(interleukin-6,IL-6)、高迁移率组蛋白1(high mobilitv group box 1 protein,HMGB1)和肌钙蛋白Ⅰ(troponin Ⅰ,TnI)血清浓度,实验结束取心脏,采用伊文思蓝和1%TTC双重染色法测量心肌梗死面积.结果 IR组,IPC组和PNU组的心肌梗死面积值分别为(78.4±16.1)%、(35.3±9.4)%和(60.4±7.0)%,且3组的TnI血清浓度分别为(1.02±0.12)μg/L,(0.25±0.03)μg/L和(0.17±0.04)μg/L与IR组相比,IPC组和PNU组心肌梗死面积(infarct size,IS%)显著减小、TnI血清浓度显著降低,IPC组灌注30min时TNF-α、IL-6血清浓度以及再灌注180 min时TNF-α和HMCB1血清浓度显著降低,PNU组再灌注30 min和180 min时TNF-α、IL-6和再灌注180 min时HMCB1血清浓度显著降低.与IPC组相比,PNU组IS%显著增大,但血清TnI浓度显著降低,冉灌注30 min时TNF-α血清浓度以及再灌注180 min时TNF-α、IL-6和HMCB1血清浓度显著降低,但再灌注30 min时IL-6血清浓度显著升高.结论 在大鼠在体心肌缺血/冉灌注损伤模型,α7nAChR激动剂后处理可通过抑制炎症反应获得心肌保护效应、但其心肌保护效应较缺血预处理弱.     

Cytokines contribute to early hepatic parenchymal injury and microvascular dysfunction after bilateral hindlimb ischemia.

PURPOSE: Hepatic dysfunction may contribute to death from multiple organ dysfunction after abdominal aortic surgery. Several factors are likely responsible, and the purpose of this study was to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are involved in initiating this remote hepatic injury. METHODS: In a normotensive rat model of 4-hour bilateral hindlimb ischemia/reperfusion (I/R), we measured systemic TNF-alpha and IL-1 levels throughout the I/R period. Rats were randomly assigned to either the 3-hour control group, the 3-hour I/R group, or the I/R group with administration of a polyclonal antibody (PAb) to TNF-alpha (I/R + TNF-alpha PAb). Direct evidence of lethal hepatocyte injury through the labeling of nuclei by propidium iodide (per 10(-1)mm(3)) and altered microvascular perfusion were assessed by using intravital microscopy. RESULTS: Systemic TNF-alpha peaked at 83.97 pg/mL (P <.05, n = 5) at 30 minutes of reperfusion and returned to baseline in 60 to 90 minutes. No significant change in systemic IL-1 was detected (P <.05, n = 4). Alanine aminotransferase increased 2.5-fold in the I/R group through 3 hours of reperfusion (P <.05, n = 4), and TNF-alpha PAb did not attenuate this alanine aminotransferase increase (P <.05, n = 6). Lethal hepatocyte injury increased by 8-fold in the I/R group compared with the control group (P <.05, n = 5), whereas TNF-alpha PAb significantly reduced this injury (P <.05, n = 4). No regional differences in injury were noted within the acinus. Total perfusion within the microvascular unit did not drop; however, significant flow heterogeneity was observed. The proportion of continuously perfused sinusoids declined in the I/R group after 3 hours of reperfusion in both periportal (62.0 +/- 2.2, P <.05) and, to a lesser, although significant, degree, in the pericentral regions (73. 2 +/- 1.73, P <.05). CONCLUSION: By scavenging extracellular TNF-alpha with a PAb, we provide direct evidence that TNF-alpha contributes to, but is not solely responsible for, early remote hepatocellular injury and microvascular dysfunction. The administration of TNF-alpha PAb reduced lethal hepatocyte injury in both regions of the acinus and also improved perfusion in the periportal region (76.8 +/- 5.41, P <.05), but not in the pericentral region. This suggests that TNF-alpha released during reperfusion mediates early remote hepatocellular injury and microvascular dysfunction after a remote ischemic insult.