二胺氧化酶预测迟发性肠坏死的研究

目的研究肠组织及血浆中二胺氧化酶(DAO)在肠缺血时的变化情况及其变化对肠活力的预测价值.方法 32只健康家兔随机分为4组,各选取一段小肠制成完全缺血模型,提取各组缺血肠管组织以及肠系膜静脉血,液体闪烁计数法测定标本中DAO活性变化情况.结果肠缺血时间愈长,缺血肠管肠组织及系膜血中DAO活性下降愈显著(P＜0.05),再灌注24 h后,肠组织及血中DAO活性有不同程度恢复;DAO活性显著变化早于肠管出现的不可逆性改变;肠组织及血浆DAO活性变化呈正相关(r=0.74,P＜0.05).结论肠缺血可导致肠组织及血中DAO活性下降,DAO活性变化先于肠管出现的不可逆改变;DAO活性测定可作为预测迟发性肠坏死的有价值的指标.     

15-F2t-isoprostane在大鼠肠缺血再灌注损伤中的作用

目的 评价异构前列腺素15-F2t-isoprostane在大鼠肠缺血再灌注损伤中的作用.方法 健康雄性SD大鼠32只,体重230 ～ 255 g,采用随机数字表法,将其随机分为4组(n=8):假手术组(S组)、肠缺血再灌注组(I/R组)、血栓烷A2(TXA2)受体拮抗剂SQ-29548组(SQ组)和二甲基亚砜组(DMSO组).采用阻断肠系膜上动脉60 min,再灌注120 min的方法制备肠缺血再灌注损伤模型.SQ组及DMSO组分别于夹闭肠系膜上动脉前30 min腹部皮下注射SQ-29548或二甲基亚砜2μmol/kg.于再灌注120 min时取肠段,观察肠粘膜形态学,并行Chiu评分,取肠粘膜组织,检测髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)、乳酸(LD)含量;采集动脉血样,检测血清二胺氧化酶( DAO)活性及15-F2t-isoprostane、内皮素-1(ET-1)和血栓烷B2(TXB2)浓度.结果 与S组比较,其余各组Chiu评分、DAO活性、15-F2t-isoprostane及TXB2浓度均升高(P＜0.05),SQ组LD和MDA含量、MPO和SOD活性及ET-1浓度差异无统计学意义(P＞0.05);与I/R组比较,SQ组Chiu评分、LD含量、DAO和MPO活性及ET-1浓度降低,SOD活性升高(P＜0.05).结论 15-F2t-isoprostane可通过激活TXA2受体,增加ET-1生成及促进中性粒细胞在肠粘膜聚集参与大鼠肠缺血再灌注损伤过程.     

卡巴胆碱对烧创伤后肠道功能障碍影响的研究

目的观察肠道内给予卡巴胆碱对兔肠部分缺血再灌注(I/R)损伤及重度烧伤患者肠道功能障碍的影响。方法将50只大白兔制成肠部分I/R损伤模型后,随机分为肠部分I/R损伤组(25只)、卡巴胆碱组[25只,于肠系膜上动脉(SMA)阻断后1h肠内注入3g/L卡巴胆碱(3μg/kg)]；另取25只设为假手术组,仅分离SMA,不阻断；取5只作为正常对照组,不致伤,处死后留取标本待测。检测兔SMA阻断前后及肠道内给予卡巴胆碱后肠黏膜的血流量。各致伤组均在处理后2、4、6、8、24、48、72h留取静脉血测定其血浆二胺氧化酶(DAO)活性及D-乳酸和D-木糖含量。并行葡聚糖蓝排出实验,以检测胃肠道吸收功能。同时选择大面积烧伤[烧伤总面积(84±12)%TBSA]患者8例,在患者肠呜音＜2次/min或腹胀明显时,口服1g/L卡巴胆碱(15μg/kg),观察给药后每分钟肠鸣音次数及腹胀情况。结果SMA阻断后肠部分I/R损伤组肠黏膜血流量为(48±6)PU,较正常对照组[(102±5)PU]明显减少,而肠道内注入卡巴胆碱后1h血流量增至(77±3)PU。肠缺血后肠部分I/R损伤组血浆DAO活性及D-乳酸含量开始升高,处理后24h达峰值[(4．63±0．27)U/ml、(7．9±2．4)mg/L],以后逐渐下降,但仍高于正常对照组[(0．89±0．14)U/ml、(2．0±1．1)mg/L,P＜0．05]。卡巴胆碱组的变化基本同肠部分I/R损伤组,但变化幅度较小；而假手术组则无明显变化(P＞0．05)。在给予D-木糖后2h,肠部分I/R损伤组血浆D-木糖含量显著降低,但处理后6h肠部分I/R损伤组及卡巴胆碱组明显升高,以后逐渐下降；假手术组略有波动。SMA处理后2h肠部分I/R损伤组葡聚糖蓝未见排出,处理后6h其运动距离逐渐增加,但处理后24h其运动距离仍明显短于正常值(P＜0．05),48～72h基本恢复正常；卡巴胆碱组注入葡聚糖蓝后即可见其排出,其运动距离明显增加,处理后6h达峰值(43±6)cm,以后逐渐缩短接近正常(28±3)cm。给药前患者肠呜音较弱(1．6±1．1)次/min,给药后10 min明显增强为(6．9±1．7)次/min,30 min时为(8．3±2．4)次/min,给药后1h患者肠鸣音仍较活跃,为(6．1±1．3)次/min。给药后2h患者腹胀明显减轻,其中有6例患者开始排便。结论肠内给予卡巴胆碱可增加兔肠黏膜血流量,改善其肠道运动、吸收、屏障功能；大面积烧伤患者口服卡巴胆碱,可改善其肠道功能障碍。     

Evidence for neutrophil-related acute lung injury after intestinal ischemia-reperfusion

Intestinal ischemia-reperfusion injury is a common and important clinical event associated with the activation of an endogenous inflammatory response. Some of the mediators of this response may be involved in the pathogenesis of multiple organ system failure. The purpose of this study was to determine whether remote organ dysfunction--specifically, acute lung injury--occurs after intestinal ischemia-reperfusion injury. After an ischemia-reperfusion event in rat intestine, whole lungs were obtained for measurement of tissue adenosine triphosphate (ATP) and myeloperoxidase values, and evaluation of histologic condition. In addition, lung microvascular permeability was assessed by determination of the rate at which iodine 125-labeled bovine serum albumin sequestration in the extravascular compartment occurred. Lung tissue ATP levels were no different in sham-operated animals than in those that had undergone 120 minutes of intestinal ischemia. Within 15 minutes of gut reperfusion, however, lung ATP decreased from 3.82 +/- 0.27 to 1.53 +/- 0.90 x 10(-7) moles/50 mg tissue, p less than 0.05. Neutrophil accumulation in the lungs, estimated by tissue myeloperoxidase determination, increased sevenfold (0.13 +/- 0.02 to 0.97 +/- 0.25 units/gm, p less than 0.05) after 120 minutes of ischemia and 15 minutes of reperfusion. Lung microvascular permeability increased threefold after 120 minutes of intestinal ischemia and 120 minutes of reperfusion (0.10 +/- 0.01 vs. 0.35 +/- 0.05 [lung/blood counts per minute], p less than 0.05). Intestinal ischemia followed by reperfusion is associated with acute lung injury characterized by increased microvascular permeability, histologic evidence of alveolar capillary endothelial cell injury, reduced lung tissue ATP levels, and the pulmonary sequestration of neutrophils. These data confirm an acute lung injury associated with intestinal ischemia-reperfusion and suggest a possible pathogenic role for the neutrophil.     

Histamine: a promoter of xanthine oxidase activity in intestinal ischemia/reperfusion.

Xanthine oxidase (XO)-derived oxygen radicals are thought to play an important role in the intestinal injury resulting from ischemia and reperfusion. In vitro data shows enhanced XO activity in the presence of histamine. Histamine is known to be released during intestinal ischemia and reperfusion. The purpose of this study was to evaluate the relationship between histamine and XO in vivo in intestinal ischemia/reperfusion injury. Using an established model of gut ischemia and reperfusion, portal venous plasma was obtained and assayed for histamine levels, XO activity, and xanthine dehydrogenase (XD) activity following injury. Intestinal ischemia for 120 minutes resulted in a 200% increase in plasma histamine levels (263.4 +/- 36.9 nmol/mL control, v 548.7 +/- 35.1 nmol/mL experimental, P less than .05). Reperfusion for 15 minutes resulted in a further increase in plasma histamine (to 658.3 +/- 33.9 nmol/mL), compared with 120 minutes of ischemia alone. No significant change in plasma XO activity resulted after simple ischemia for 120 minutes. However, XO activity doubled within 15 minutes of reperfusion of the ischemic intestine (6.37 +/- 0.53 nmol O2- per milliliter per minute v 3.12 +/- 0.25 nmol O2- per milliliter per minute, P less than .05). Reperfusion for 60 minutes resulted in the maximal observed increase in plasma XO activity (9.49 +/- 0.67 nmol O2- per milliliter per minute). Analysis of XD activity demonstrated no significant decrease compared with controls until 120 minutes of ischemia and 60 minutes of reperfusion (1.62 +/- 0.49 nmol uric acid per milliliter per minute at 60 minutes of reperfusion, versus 5.02 +/- 0.52 nmol uric acid per milliliter per minute control, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Local intravascular coagulation and fibrin deposition on intestinal ischemia-reperfusion in rats

BACKGROUND: This study investigates intravascular coagulation and thrombotic obstruction in the splanchnic vasculature after intestinal ischemia in relation to epithelial integrity and function. METHODS: Intestinal ischemia was induced in rats by superior mesenteric artery occlusion for 20 or 40 minutes. Intestinal injury was assessed by histologic analysis, biochemical markers, and functional studies. During reperfusion, portal and systemic blood samples were collected to analyze activation of coagulation and fibrinolysis. RESULTS: Superior mesenteric artery occlusion resulted in mild to moderate intestinal injury. Twenty and 40 minutes of ischemia and 3 hours of reperfusion resulted in local intestinal thrombin generation and conversion of fibrinogen to fibrin, reflected by 3- and 4-fold increases in thrombin-antithrombin complex levels and a 3-fold elevation of fibrin degradation products (D-dimer), respectively. During reperfusion, after a short-lasting initial activation of local fibrinolysis, plasminogen activator activity was suppressed, as indicated by an approximately 4-fold increase in portal plasma levels of the plasminogen activator inhibitor. D-dimer levels showed that activation of coagulation and depression of fibrinolysis resulted in fibrin formation, which was confirmed to be intravascular fibrin deposition by histologic examination. CONCLUSIONS: Intestinal ischemia-reperfusion results in local intravascular coagulation and fibrin deposition.     

Hypothyroidism protects against free radical damage in ischemic acute renal failure

The effect of hypothyroidism on ischemic acute renal failure was studied in rats. Ten days after thyroidectomy with parathyroid reimplantation, rats underwent right uninephrectomy followed by occlusion of the left renal artery for 60 min. Plasma creatinine was lower in thyroidectomized than control rats 24 hr after ischemia; 1.3 +/- 0.5 vs. 3.2 +/- 0.6 mg%; P less than 0.05. Twenty-four hours after ischemia, inulin clearance was higher in thyroidectomized than control animals (0.40 +/- 0.06 vs. 0.17 +/- 0.03 mliter/min; P less than 0.01), despite an initially lower inulin clearance in thyroidectomized animals (0.81 vs. 1.1 +/- 0.07 mliter/min; P less than 0.05). Administration of the antithyroid drug prophylthiouracil for 14 days also resulted in lower plasma creatinine after ischemia. Kidneys from thyroidectomized animals showed less histologic damage 24 hr after ischemia. Renal cortical content of the lipid peroxidation product malondialdehyde was increased less in thyroidectomy than control kidneys after 60 min ischemia plus 15 min reflow (0.08 +/- 0.02 vs. 0.42 +/- 0.1 nmole/mg protein; P less than 0.005). Renal cortical glutathione content was higher in thyroidectomized animals by approximately 36%, 650 +/- 46 vs. 479 +/- 32 nmole/mg protein (P less than 0.02). In normal rats, glutathione infusion also increased renal cortical glutathione content and resulted in lower plasma creatinine 24 hr after renal artery ischemia. Therefore, hypothyroidism resulted in functional and histologic protection against injury after ischemia. Post-ischemic renal lipid peroxidation was reduced in thyroidectomized animals, perhaps the result of increased scavenging of reactive oxygen species (oxygen free radicals and H2O2) by glutathione.     

Time-dependent impairment of mitochondrial function after storage and transplantation of rabbit kidneys

BACKGROUND: The mitochondrial respiratory chain is implicated as a major target of kidney damage after ischemia-reperfusion. This study measures changes in integrated mitochondrial function and in the activity of enzymes of the respiratory chain after cold storage and transplantation-reperfusion in vivo. METHODS: Mitochondrial oxygen consumption and activities of respiratory chain enzymes and citrate synthase were measured in cortical mitochondria isolated from rabbit kidneys after 1-48 hr of cold ischemia with or without transplantation-reperfusion. RESULTS: State 4 mitochondrial oxygen consumption was significantly increased after 48 hr of ischemia or 24-48 hr of ischemia with transplantation. Prolonged (24 or 48 hr) ischemic storage with and without transplantation caused a significant decrease in state 3 oxygen consumption, as did transplantation after 1, 24, and 48 hr of cold storage. Complex I and complex II-III activity decreased after 24 or 48 hr of ischemia, with transplantation having little additional effect. Complex IV activity was significantly decreased after 48 hr of ischemia, this decrease being exacerbated by transplantation-reperfusion. Complex V activity decreased significantly after 1 hr of ischemia and continued to decrease after 24-48 hr of ischemia. Transplantation after 1-24 hr (but not 48 hr) of ischemia resulted in partial recovery of complex V activity. Citrate synthase activity was decreased significantly only after 48 hr of ischemia and reperfusion, consistent with the loss of mitochondrial membrane integrity seen in electron micrographs of the transplanted 48-hr group. CONCLUSIONS: These data suggest that individual rabbit kidney mitochondrial complexes have different susceptibilities to cold ischemic and reperfusion damage.     

Role of granulocyte-macrophage colony-stimulating factor on apoptosis induced by ischemia-reperfusion in the intestinal epithelium

BACKGROUND: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on ischemia-reperfusion-induced apoptosis in the intestinal epithelium. METHODS: In this study, 50 male Wistar albino rats were used. After midline laparotomy superior mesenteric artery (SMA) was identified only in the sham group, while 60 min of ischemia and 2 h of reperfusion were performed in the control group. In the treatment groups, after 15, 30 and 60 min of ischemia, respectively, 1 microg/kg GM-CSF was administered subcutaneously, followed by 2 h of reperfusion. Malondialdehyde (MDA), campothecin (CAM), an indicator of DNA fragmentation, and histopathology were evaluated in the intestinal mucosa. RESULTS: Tissue MDA levels were found significantly high in all groups at various times of ischemia and 2 h of reperfusion compared with the sham group (p < 0.001). Administration of GM-CSF following 60 min of ischemia caused a significant increase in the MDA levels compared with the control group (6430 +/- 725 vs. 4174 +/- 565 nmol/g protein for jejunum. 7576 +/- 618 vs. 4938 +/- 809 nmol/g protein for ileum, p < 0.05). Intestinal ischemia and reperfusion resulted in a significant increase in tissue CAM levels (p < 0.05). The highest CAM value was found in the group in which 60 min of ischemia and 2 h of reperfusion were performed (50 +/- 3.2 ng/ml for jejunum, 52.8 +/- 2.7 ng/mg for ileum). Compared with the control group, GM-CSF administration following 1 h of ischemia aggravated the tissue injury. CONCLUSIONS: Apoptosis was induced in the small intestine by ischemia-reperfusion. GM-CSF increased the apoptosis of intestinal epithelial cells and exacerbated mucosal injury due to ischemia-reperfusion.     

Serum enzyme levels during intestinal ischemia.

Because the intestinal mucosa is most sensitive to ischemia, serum levels of mucosal enzymes, such as diamine oxidase, may be most likely to indicate intestinal ischemia. Our aim was to compare serum levels of mucosal (diamine oxidase, alkaline phosphatase) and seromuscular (creatinine phosphokinase, lactic dehydrogenase, serum glutamic oxaloacetic transminase) enzymes during intestinal ischemia of varying extent and duration in dogs. Group 1 (n = 6) underwent sham laparotomy. Group 2 (n = 8) had 50% of the small intestine devascularized. Group 3 (n = 8) had the superior mesenteric artery occluded for 2 hours and released. Group 4 (n = 8) had the superior mesenteric artery ligated. Serum samples were obtained before and 2, 4, 8, and 24 hours after operation, and histologic specimens were examined at 4 hours. Creatinine phosphokinase levels became elevated within 4 hours of ischemic injury in group 2 (223 +/- 197 vs. 68 +/- 26, p less than 0.05) and group 4 (212 +/- 136 vs. 76 +/- 29, p less than 0.05). Significant elevation of serum enzymes levels, except diamine oxidase, occurred in groups 2, 3, and 4 at 24 hours, including those with normal histology after temporary superior mesenteric artery occlusion. Thus seromuscular enzymes, particularly creatinine phosphokinase, were more likely to be elevated during intestinal ischemia. Enzyme levels were not influenced by the extent and reversibility of the ischemic injury.     

Serum and intestinal diamine oxidase activity during intestinal adaptation.

Diamine oxidase (DAO) is a cytoplasmic enzyme found primarily in the villus epithelial cells of the small intestine. Serum DAO levels have been evaluated as a potential marker of intestinal disease in a variety of disorders, including gut atrophy, ischemia, and inflammation. In this study serum and tissue DAO levels were evaluated during intestinal adaptation. Twenty dogs were divided into 4 groups: sham laparotomy (n = 5), and 25% (n = 5), 50% (n = 5), and 75% (n = 5) distal enterectomy. Serum DAO activity (basal or postheparin) was measured prior to and 2 days, 4 weeks, 8 weeks, and 12 weeks after operation. Tissue DAO and changes in intestinal length, mucosal protein content, and villus height were measured at sacrifice 12 weeks later. Intestinal remnant length and protein content increased significantly with 50 and 75% resection. Tissue DAO activity was significantly decreased with any enterectomy. Serum postheparin DAO activity was significantly greater than basal at all time points but there was no significant change in either basal or postheparin DAO levels at any time following resection. It is concluded that serum DAO levels are not changed during the early adaptive period following intestinal resection and thus would not be useful as a marker of this process. Tissue DAO levels were diminished during adaptation, suggesting that tissue DAO activity is influenced not only by mucosal mass but by cellular metabolism and the proliferative status of the mucosa.     

Serum and Intestinal Diamine Oxidase Activity During Intestinal Adaptation

Diamine oxidase (DAO) is a cytoplasmic enzyme found primarily in the villus epithelial cells of the small intestine. Serum DAO levels have been evaluated as a potential marker of intestinal disease in a variety of disorders, including gut atrophy, ischemia, and inflammation. In this study serum and tissue DAO levels were evaluated during intestinal adaptation. Twenty dogs were divided into 4 groups: sham laparotomy (n = 5), and 25% (n = 5), 50% (n = 5), and 75% (n = 5) distal enterectomy. Serum DAO activity (basal or postheparin) was measured prior to and 2 days, 4 weeks, 8 weeks, and 12 weeks after operation. Tissue DAO and changes in intestinal length, mucosal protein content, and villus height were measured at sacrifice 12 weeks later. Intestinal remnant length and protein content increased significantly with 50 and 75% resection. Tissue DAO activity was significantly decreased with any enterectomy. Serum postheparin DAO activity was significantly greater than basal at all time points but there was no significant change in either basal or postheparin DAO levels at any time following resection. It is concluded that serum DAO levels are not changed during the early adaptive period following intestinal resection and thus would not be useful as a marker of this process. Tissue DAO levels were diminished during adaptation, suggesting that tissue DAO activity is influenced not only by mucosal mass but by cellular metabolism and the proliferative status of the mucosa.     

The effect of intestinal autotransplantation on serum diamine oxidase activity

Circulating levels of diamine oxidase (DAO), a mucosal enzyme found primarily in the small intestine, have been shown to reflect intestinal mucosal damage in a variety of disease states. Our aim was to assess the usefulness of both basal and postheparin DAO activity as a marker of intestinal allograft rejection by studying the influence of the nonrejection effects of intestinal transplantation on these activities. This separation of the immunological from all other effects of transplantation was achieved by studying 11 dogs who had undergone autotransplantation of the small intestine and 11 unoperated controls. Basal serum DAO activity increased during the first 3 postoperative days following autotransplantation (20.5 +/- 0.7 units/ml on Day 3 versus 6.9 +/- 4.1 units/ml preoperatively, P less than 0.05) but thereafter returned to control levels at 1 month and remained so for more than 18 months. Postheparin DAO activity was similar in both groups with a maximum increase between 15 and 60 min following heparin administration. There was no correlation between maximal DAO activity and time since operation in the transplant group. Intestinal DAO activity was similar to unoperated animals 18 months after autotransplantation. These findings suggest that postheparin serum diamine oxidase activity is not influenced by autotransplantation and thus, is a potential marker of graft rejection following intestinal allotransplantation.     

Intermittent ischemia potentiates intestinal reperfusion injury

We hypothesized that even brief periods of reperfusion interjected between ischemic episodes would increase tissue injury. Studies were performed in a rat small intestine preparation in which metabolic, hemodynamic, and histologic responses to ischemia have been well characterized. Animals were subjected to a total of 30 or 45 minutes of complete intestinal ischemia. Flow interruption was continuous (C, single episode) or intermittent (I, two or three episodes of 15-minute ischemia separated by 5 minutes of reperfusion). In some experiments 5-minute reperfusions were performed with arterial blood depleted of leukocytes (IL). This additional perturbation was included to determine the role of neutrophils that have been strongly implicated in reperfusion injury. In all three protocols histologic sections were obtained after each ischemic insult and after 1 hour of reperfusion with arterial blood. Villous histology was graded in a blinded fashion with 1 = normal and 5 = severe injury. No significant differences were found between groups in immediate postischemic histologies before reperfusion. After 1 hour of reperfusion, intermittent episodes of ischemia were associated with significantly worse histologic injury than that seen with comparable durations of continuous ischemia (30 min: I, 4.4 +/- 0.5 vs C, 2.7 +/- 0.4; 45 min: I, 4.9 +/- 0.2 vs C, 2.8 +/- 0.3). However, if 5-minute reperfusions were with leukopenic blood, this effect was markedly reduced (30 min IL, 3.4 +/- 0.3; 45 min IL, 3.6 +/- 0.2). Even short periods of reperfusion during an ischemic insult greatly increased mucosal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of the rabbit rectus femoris muscle to ischemia and reperfusion.

The rectus femoris muscle of the rabbit is perfused by a single artery and vein and is a valuable new model for study of ischemia-reperfusion injury of skeletal muscle. The consequences of increasing duration of ischemia to the rectus femoris have been examined. Postischemic muscle survival (means +/- SEM), as measured by Nitro blue tetrazolium (NBT) staining 24 hr after ischemia, was 90.5 +/- 1.5% after 2 hr normothermic ischemia, 77.1 +/- 7.7% after 3 hr, 41.8 +/- 7.6% after 3 1/2 hr, and 10.7 +/- 8.7% after 4 hr. Histology confirmed the NBT findings at 24 hr and showed considerable regeneration of muscle fibers 1-2 weeks after injury. The injury caused by 3 1/2 hr normothermic ischemia is the most suitable baseline for study of the effects of pharmacological agents in ischemic muscle injury. Further study of the effects of 3 1/2 hr ischemia by a quantitative Evan's blue method revealed a rapid increase in vascular permeability commencing at the start of reperfusion and lasting for 5-6 hr. Vascular labeling with saccharated ferric oxide showed widespread labeling of venules within the injured muscle and electron microscopic examination showed severe injury to both leaking and nonleaking small blood vessels. However, increased vascular permeability accounted for only a small part of the increase in weight of ischemic muscle.     

Intestinal ischemia-reperfusion injury causes pulmonary endothelial cell ATP depletion.

Intestinal ischemia-reperfusion is a common clinical event associated with both clinical and experimental distant organ injury. In particular, the pulmonary microvasculature appears to be susceptible to injury resulting from systemic inflammatory mediator activation. This study was designed to evaluate the hypothesis that noncellular humoral factors associated with intestinal ischemia-reperfusion result in pulmonary endothelial cell adenosine triphosphate (ATP) depletion. Male Sprague-Dawley rats had intestinal ischemia induced by microvascular clip occlusion of the superior mesenteric artery (SMA) for 120 minutes. Reperfusion resulted from superior mesenteric artery clip removal. After reperfusion for 0, 15, or 30 minutes, plasma samples were obtained from the portal vein. Monolayers of cultured rat pulmonary artery endothelial cells then were incubated with the plasma samples. Adenosine triphosphate levels were determined using a luciferin-luciferase assay. A 51Cr-release assay using labeled endothelial cells was performed under identical conditions to assess cytotoxicity. Potential mechanisms of ATP depletion were evaluated by analysis of cellular energy charge and assessment of microfilament architecture. Endothelial cell ATP levels decreased from 2.23 +/- 0.16 x 10(-11) moles/microgram DNA in sham preparations to 1.23 +/- 0.09 x 10(-11) moles/microgram DNA (p < 0.001) after 4 hours in plasma from animals undergoing 120 minutes of intestinal ischemia. For plasma obtained after 15 minutes of reperfusion, the decrease in cellular ATP concentration persisted (1.23 +/- 0.27 x 10(-11) moles/microgram DNA, p < 0.001 vs. sham). After 30 minutes' reperfusion, cellular ATP levels increased only slightly after the 4-hour incubation (1.39 +/- 0.26 x 10(-11) moles/microgram DNA, p < 0.005 vs. sham). No significant cytotoxic injury occurred in any group when compared with controls. Cellular energy charge was unchanged, and microfilament architecture was preserved. These data confirm the hypothesis that humoral factors, independent of the neutrophil, result in endothelial cell ATP depletion without metabolic inhibition or cell death. Depletion of energy stores by noncellular humoral factors may represent an early event that predisposes the cell to more severe injury by other mediators of the endogenous inflammatory response.     

Glucagon potentiates intestinal reperfusion injury

Vasoactive agents, including glucagon, have been used in treatment of mesenteric ischemia. Such drugs change both intestinal blood flow and metabolism. Since reperfusion injury reflects the metabolic state of an organ as well as the duration and severity of ischemia, we investigated the effect of glucagon in a standard model of intestinal ischemia. Data were generated from denervated isoperfused rat small intestinal preparations (n = 39). Arterial and venous pressures, intestinal blood flow, and oxygen consumption were monitored. Animals were subjected to 15, 30, or 45 minutes of ischemia followed by 1 hour reperfusion. Experiments were performed without drug infusion or during intravenous glucagon administration (0.1, 0.2, or 0.4 micrograms/kg/min). After the rats were killed, histologic sections of intestine were graded 1 through 5 in a blinded fashion with 1 = normal villi and 5 = severe injury. Results (mean +/- SD) were analyzed by analysis of variance (*p less than 0.05). Glucagon at all concentrations increased intestinal blood flow and oxygen consumption before ischemia. For example, with 0.2 micrograms/kg/min glucagon, intestinal blood flow increased from 80.78 +/- 13.5 to 114.79 +/- 21.02 ml/min.100 gm* and oxygen consumption increased from 3.65 +/- 0.73 to 5.73 +/- 1.37 ml/min.100 gm.* Mucosal injury after ischemia reflected duration of ischemia and glucagon infusion rate. At all ischemic intervals, increased glucagon concentrations were associated with greater mucosal injury. In fact the histologic injury with 15 minutes of ischemia + 0.2 microgram/kg/min glucagon (3.04 +/- 0.49) exceeded that of 30 minutes of ischemia (2.87 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of iron in postischemic renal injury in the rat

To determine whether iron participates in free radical-mediated postischemic renal injury and lipid peroxidation, we examined the effects of removal of endogenous iron or provision of exogenous iron following renal ischemia, as well as the effects of renal ischemia and reperfusion on renal venous and urinary "free" iron. Rats underwent 60 minutes of renal ischemia and were studied after either 24 hours (inulin clearance) or 15 minutes (renal malondialdehyde content) of reperfusion. Infusion of the iron chelator deferoxamine (200 mg/kg/hr) during the first 60 minutes of reperfusion resulted in a marked improvement in renal function (inulin clearance: 879 +/- 154 vs. 314 +/- 74 microliter/min; P less than 0.025) and a reduction in lipid peroxidation (renal malondialdehyde: 0.449 +/- 0.06 vs. 0.698 +/- 0.08 mmol/mg prot; P less than 0.05) compared to control animals. Infusion of 50 mg/kg/hr deferoxamine also protected renal function after ischemia (inulin clearance: 624 +/- 116 vs. 285 +/- 90 microliter/min; P less than 0.05) and resulted in less histologic injury. Iron-saturated deferoxamine had no protective effect. Conversely, infusion of the iron complex EDTA-FeCl3 during reperfusion exacerbated postischemic renal dysfunction and lipid peroxidation. Following renal ischemia there was no detectable increase in "free" iron in arterial or renal venous plasma. However, urinary "free" iron increased 10- to 20-fold following reperfusion. Iron chelators which underwent filtration and gained access to this free iron in the urine (free deferoxamine or inulin-conjugated deferoxamine) provided protection, whereas a chelator confined to the vascular space (dextran-conjugated deferoxamine) did not.(ABSTRACT TRUNCATED AT 250 WORDS)