Hyperglycemia in global cerebral ischemia and reperfusion: a 31-phosphorous NMR spectroscopy study in rats

31-phosphorous magnetic resonance spectroscopy was used in a rat model of 10 min severe incomplete forebrain ischemia (two-vessel occlusion with hypotension) to assess the effect of hyperglycemia on intracellular pH and high energy phosphates during ischemia and early reperfusion. One group (n = 8) with preischemic hyperglycemia (serum glucose 20 mmol.l-1) showed an increased intracellular acidosis (pH 6.35) during ischemia compared to 6.55 in the normoglycemic control group (n = 7, P less than 0.001), but the recovery of phosphocreatine and ATP in early reperfusion was the same in the two groups. Another group (n = 7) was normoglycemic during ischemia, but received an i.v. bolus of glucose during the first minute of reperfusion. In this group the recovery of intracellular pH in early reperfusion was slower than in the control group (0.034 +/- 0.006 pH units per minute compared to 0.052 +/- 0.11 in the controls, +/- s.d. and P less than 0.01).     

大鼠热缺血肝脏加氧低温灌流31磷-核磁共振频谱与病理的对比研究

目的　观察承受不同程度热缺血的离体大鼠肝脏三磷酸腺苷 (ATP)的再生能力及相应的病理变化 ,从代谢和形态结构两个方面判断其活力。方法　SD大鼠 18只 ,按热缺血时间随机等分成 3组 :A组 ,0min ;B组 ,30min ;C组 ,6 0min。测定加氧低温灌流的离体肝脏31磷 核磁共振频谱 ,同时观察灌流前后肝组织病理变化。结果　灌流 30min时 ,A、B、C 3组大鼠肝脏ATP/亚甲基二磷酸(MDP)平均值为 0 .42 3、0 .387、0 .138(C组有 3例测不到ATP峰值 ) ,A、B两组差异无显著性 (P >0 .0 5 ) ;C组与A、B两组比较差异有非常显著性 (P <0 .0 1)。病理改变 :A组肝细胞轻度浊肿 ;B组还有散在肝细胞空泡变 ;C组 ,特别是测不到ATP的 3例肝脏有大片肝细胞空泡变。肝窦内皮细胞A、B两组存在轻度水肿 ,C组肿胀加重。结论　测定加氧低温灌流离体大鼠肝脏能量代谢能有效鉴别其活力。     

Serum hyaluronic acid for the assessment of graft viability in porcine liver transplantation

For the assessment of graft viability, serum hyaluronic acid (HA) levels during porcine orthotopic liver transplantation were measured in two groups: group 1 (viable:n = 5) in which allografts were transplanted following a minimal cold (4°C) preservation, and group 2 (nonviable:n = 4) in which allografts were transplanted after cold static storage (4°C) for 24 h in University of Wisconsin solution. The changes in the HA levels reached a significant difference between the two groups at 30 min after reperfusion (P < 0.02). In group 1, all animals survived for over 4 days, while all animals in group 2 died within 24 h. The serum HA also demonstrated a significant correlation with prothrombin time, ß-glucuronidase, and aspartate aminotransferase at 120 min after reperfusion. These results suggest that the measurement of serum HA is a potentially effective index for evaluating hepatic allograft viability.     

Investigating viability of intestine using spectroscopy: a pilot study

Background

The differentiation of “viable” from “nonviable” bowel remains a challenge in the treatment of acute mesenteric ischemia. In this study, diffuse reflectance spectroscopy (DRS) was used to investigate the viability of bowel tissue after ischemia and reperfusion in an animal model in vivo and in real time.

Methods

A total of 25 females Sprague–Dawley rats were divided into five groups based on different bowel ischemia times. In each study group for four of them, the superior mesenteric artery was occluded using a vascular clamp for a different period (i.e. 30, 45, 60, and 90 min; n = 5 for each group). Intestinal reperfusion was accomplished by releasing the clamps after the given occlusion period for each group. Spectra were acquired by gently touching the optical fiber probe to the bowel tissue before the induce ischemia, at the end of the induced ischemia, and after the reperfusion. The data acquired before the ischemia were used as a control group. Without occluding the superior mesenteric artery, the spectra were acquired on the bowel with the same time intervals of the experiments were used as a sham group (n = 5). Subsequently, the same bowel segments were sent for histopathologic examination.

Results

Based on the correlation between the spectra acquired from the bowel segments and the results from the histopathologic investigation, DRS is able to differentiate the histopathologic grading that appears when the Chiu/Park score ≥5 (i.e., high-level ischemic injury) than Chiu/Park score <5. Eight out of nine low-level ischemic injury tissue samples were correctly defined using the spectroscopic classification system. All eleven high-level ischemic injury tissues that were histopathologically assigned grade 5 and above were correctly defined using the spectroscopic classification system in the ischemia–reperfusion groups.

Conclusions

DRS could potentially be used intraoperatively for the assessment of bowel viability in real time. These preliminary findings suggest that DRS has the potential to reduce unnecessary resection of viable tissue or insufficient resection of nonviable tissues may reduce the mortality and morbidity rates of intestinal ischemia–reperfusion as acute mesenteric ischemia.     

Minimum sizes for remnant and transplanted livers in rats

Our new technique of hepatectomy in rats, avoiding inferior vena cava constriction, revealed that 5%–10% of the total liver is the minimum required volume for remnant liver, a smaller amount than previously reported. The characteristic histological findings of remnant liver in perioperative death are marked hepatocytosis and hepatocyte apoptosis, and extensive hepatocyte necrosis with sinusoid congestion, all of which are thought to be caused by hyperendotoxemia and mitochondrial energy crisis. Partial liver transplantation in rats with our new technique of hepatectomy in the donor operation showed that a graft of 20% liver was the minimum required volume for successful transplantation, also a smaller amount than previously reported. There was no difference in the rate of increase in wet weight after surgery between 20% remnant liver and 20% transplanted liver. Differences between 10% minimum volume remnant liver and 20% minimum transplanted liver appeared to be caused by ischemia-reperfusion and cold preservation injury to the transplanted liver.     

Usefulness of 31P-MRS as a method of evaluating the viability of preserved and transplanted rat liver

We used phosphorus-31 magnetic resonance spectroscopy (³¹P-MRS) to evaluate the viability of transplanted rat liver. Wistar rats were used as donors and recipients. The donor livers were preserved in saline (group 1), Euro-Collins solution (group 2), or in University of Wisconsin (UW) solution (group 3) for 3 and 6 h in groups 1, 2 and 3 and for 9 h in groups 2 and 3. Thereafter the livers were orthotopically transplanted. ³¹P-MRS spectra were measured after portal reperfusion. Finally, all the recipients were divided into survivors and non-survivors. Survival rates were better in group 3 than in groups 1 and 2. In the 9-h-preserved livers, the livers in group 3 showed a significantly higher β-ATP/Pi ratio than those in group 2. Comparing survivors and non-survivors in the 6-h-preserved livers in group 2, survivors' livers showed significantly higher (3-ATP/Pi ratio than those of non-survivors. We concluded that ³¹P-MRS is a useful method for assessing viability of rat liver grafts.     

The significance of tissue-type plasminogen activator for pretransplant assessment of liver graft viability: analysis of effluent from the graft in rats

We studied the significance of tissue-type plasminogen activator (tPA) on the pretransplant assessment of liver graft viability in rats. The liver grafts were excised from the rats and then divided into two groups. Group 1 consisted of grafts preserved for 4 h in chilled, lactated Ringer's solution (4°C) and group 2 consisted of grafts preserved for 6 h in the same solution. After preservation, the liver grafts were flushed out through the portal vein using 5 ml of chilled, lactated Ringer's solution (4°C). The entire effluent from the hepatic veins was then collected and analyzed for tPA, ammonia, lactate, pyruvate, glutamic oxaloacetic transaminase, and lactate dehydrogenase. The tPA concentration of effluent in group 2 was significantly higher than that in group 1 (0.80±0.23 ng/ml vs 0.42±0.08 ng/ml, P<0.05). The lactate, pyruvate, and ammonia levels in group 2 were also higher than those in group 1 (134±13 mg/dl vs 120±2 mg/dl, 0.34±0.40 mg/dl vs 0.09±0.01 mg/dl, and 183±79 g/dl vs 102±40 g/dl, respectively). However, the discriminative power of tPA was stronger than that of the other parameters. Histological findings revealed a higher number of trypan blue-stained sinusoidal lining cells that were detached and swollen in group 2. We conclude that the amount of tPA in the effluent flushed from the graft can serve as a sensitive and reliable indicator of cold-preserved liver grafts in rats.     

The NMDA antagonist MK-801 improved metabolic recovery after 10 minutes global cerebral ischaemia in rats measured with 31 phosphorous magnetic resonance spectroscopy

Summary The blockade of postsynaptic receptors for excitatory amino acids is a promising new field for the possible treatment of cerebral ischaemia. The most important receptor seems to be the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors and MK-801 is a potent non-competitive antagonist to the NMDA receptor.31P NMR Spectroscopy was used to measure the recovery of intracellular pH and the high energy phosphates Phosphocreatine (PCr) and ATP after ten minutes of temporary global cerebral ischaemia in the rat. Cerebral ischaemia was obtained by combining bilateral carotid ligation and systemic hypotension (2 vessel occlusion model).Two intervention groups with intravenous injection of MK-801 in doses of 0.25mg/kg and 0.5mg/kg 15 minutes before onset of ischaemia were compared to a control group. Both intervention groups showed a more rapid recovery of PCr and ATP than the control group, but there were no significant differences for intracellular pH.     

The effects of brain temperature on temporary global ischaemia in rat brain. A 31-phosphorous NMR spectroscopy study

31-Phosphorus magnetic resonance spectroscopy was used in a rat model of 10 min severe incomplete forebrain ischaemia (2-vessel occlusion with hypotension) to assess the effect of mild brain hypo- and hyperthermia (+/- 2 degrees C) on intracellular pH and high energy phosphates. In three experimental groups intracerebral temperature was maintained at levels of 34, 36 and 38 degrees C during ischaemia and early reperfusion. The steady level of intracellular pH during ischaemia was 6.63, 6.58 and 6.53 in the 34, 36, and 38 degrees C groups, respectively. The rate of initial recovery of intracellular pH in reperfusion was 0.046 +/- 0.012 pH units per min (+/- s.d.) in the 36 degrees C group compared to 0.056 +/- 0.010 (+/- s.d., P less than 0.05) in the 34 degrees C group and 0.032 +/- 0.009 (+/- s.d., P less than 0.01) in the 38 degrees C group. The recovery in early reperfusion of phosphocreatine and ATP was slower in the 38 degrees C group compared to the other groups. The findings were consistent with recent studies, suggesting that even mild hypothermia may afford protection to the ischaemic brain, and furthermore indicate that mild hyperthermia as fever or even subfebricity may be deleterious for the outcome in stroke patients.     

Depletion of glutathione by buthionine sulfoximine decreases random-pattern skin flap viability in rats

BACKGROUND: Glutathione (GSH) is one of the most highly concentrated intracellular antioxidants. Exogenous GSH has been shown to increase random-pattern skin flap survival. However, the effects of endogenous GSH depletion on random-pattern skin flap viability have never been studied. MATERIALS AND METHODS: To evaluate the effects of systemic glutathione depletion on random-pattern skin flap survival in rats, 28 Wistar albino rats were divided into control, sham, and BSO (buthionine sulfoximide, a selective inhibitor for gamma-glutamylcysteine synthetase) groups. Dorsal, cranial-based random-pattern skin-flaps were elevated and the percentage of flap necrosis was measured in all rats at the postoperative day 7. RESULTS: BSO-treated rats showed increased skin flap necrosis when compared with untreated animals (P < 0.001). High-dose BSO treatment group had more clinically evident necrosis than low dose group (P < 0.05). CONCLUSIONS: This study reveals the importance of endogenous GSH for random skin-flap viability.     

Safe use of highly steatotic livers by utilizing a donor/recipient clinical algorithm

The aim of this study was to assess the long‐term safety and clinical outcomes associated with the utilization of highly steatotic donor livers utilizing a specific donor/recipient matching algorithm. This was a prospective, observational, single‐center, 10‐yr follow‐up study. Highly steatotic livers were utilized according to a donor/recipient algorithm that guided the surgeon to use highly steatotic donor organs judiciously in low‐risk recipients. This study initially compared fat assessment based on frozen‐section Ehrlich's hematoxylin and eosin (H&E) to reperfusion biopsy fat assessment and demonstrated that H&E is an insensitive analysis to determine degree of steatosis. Patients were divided into three groups based on donor steatosis (group 1: <30% steatosis, group 2: 30–60% steatosis, group 3: >60% steatosis), and clinical outcomes were assessed. One hundred and sixteen patients were included in the analysis. Patients that received severely steatotic livers (>60% fat) showed increased reperfusion liver injury and delayed return of liver function in the early postoperative period, demonstrated by biochemical markers. However, there were no differences in primary non‐function, postoperative complications, length of stay, and patient and graft survival. Using rigorous donor/recipient matching through a detailed algorithm, these data demonstrate that normal liver allograft outcomes are not superior to those in highly steatotic grafts.     

A validated technique for the analysis of biliary bile acid secretion in donor livers prior to transplantation

Many parameters currently used for the pre-transplant assessment of liver allografts, are not reliable enough in predicting the likelihood of early graft dysfunction or non-function. It is generally accepted that bile secretion is a sign of hepatic function post-transplant and that bile flow shows a close linear relationship to the secretion of bile acids (“apparent choleretic activity”). We have studied bile flow, biliary bile acid concentrations and composition and measured apparent choleretic activity from hepatic bile collected with a new technique under controlled conditions at the time of retrieval from 18 donor livers. More than three samples were collected from each of 13 donors and a total of 65 samples of hepatic bile were analysed. Of these, ten showed typical apparent choleretic activity with a positive slope in the regression line analysis (correlation coefficient of 0.9), validating our collection technique. Received: 1 September 1997 Received after revision: 15 December 1997 Accepted: 9 January 1998     

Intraoperative determination of intestinal viability: a comparison with transserosal pulse oximetry and histopathological examination

BACKGROUND: In this study, we aimed to measure the O2 saturation of ischemic intestinal segments and the relationship between these measures and concomitant pathological gradings. MATERIALS AND METHODS: We used 14 New Zealand White rabbits, anesthetized with a combination of 80 mg/kg (i.m.) Ketamine hydrochloride and 10 mg/kg Acepromazine (i.m.). The superior mesenteric artery (SMA) was explored, and O2 saturation was measured by pulse oximetry 5 cm proximal to the ileocecal valve at the 0th hour. At the same time a 0.5-cm full-thickness wedge biopsy was taken from the same region. Thereafter, the SMA was ligated and the abdomen was closed. All rabbits were undertaken relaparotomy at the 4th hour; O2 saturation was measured by pulse oximetry at 5 cm away from the region of the first biopsy and a 0.5-cm full-thickness wedge biopsy was taken. The abdomen was then closed. The same procedure was performed at the 8th and the 12th hour. Mucosal hemorrhage, transmural congestion, mucosal necrosis, and transmural necrosis were examined in the specimens. RESULTS: Pathologically, transmural necrosis was concomitant with 64% O2 saturation (sensitivity: 100%; specificity: 86%). Mucosal necrosis was concomitant with 76% O2 saturation values (sensitivity: 100%; specificity: 75%). Transmural congestion was concomitant with 81% O2 saturation values (sensitivity: 89%; specificity: 58%). Mucosal hemorrhage was concomitant with 91% O2 saturation (sensitivity: 100%; specificity: 31%). CONCLUSION: O2 saturation measures > 76% may indicate reversible changes as mucosal necrosis, transmural congestion, or mucosal hemorrhage, and O2 saturation measures < 64% may indicate permanent transmural necrosis. As a result, intraoperative evaluation of intestinal viability by pulse oximetry may give us an idea about the degree of pathological changes and subsequently might reduce the number of second-look operations.     

Pattern of ischemia reperfusion injury in a mouse orthotopic liver transplant model

BACKGROUND: The molecular pathways of ischemic injury after liver transplantation are complex and difficult to dissect because of the presence of many variables. Transgenic and genetically deficient strains of mice provide ideal models for the study of the contribution of a single gene product in biological processes in vivo. Although well described in rats, prolonged preservation has not been studied in a mouse model of orthotopic liver transplantation (mOLT). The aim of this study was to establish a model of cold ischemia and reperfusion injury in mOLT and describe the pattern of the regenerative response to various lengths of cold storage. MATERIALS AND METHODS: mOLT was performed using a syngeneic combination. Grafts were preserved at 4 degrees C in University of Wisconsin (Viaspan) solution for increasing periods of cold preservation. After cold storage, the liver grafts were transplanted and recipient survival was monitored. Hepatocellular injury was determined by histology, and the regenerative response was quantitated by interleukin 6 upregulation and DNA replication. RESULTS: Long-term survival was 100%, 100%, 88%, and 0% for cold preservation of 1, 4, 8, and 16 h, respectively. Grafts with short preservation times (1 and 4 h) demonstrated limited injury and a weak regenerative response, with slight IL-6 early upregulation and minimal cell division. Eight hours of cold ischemia resulted in prominent injury and an intense regenerative response accompanied by significant IL-6 upregulation and DNA synthesis. Sixteen hours of storage resulted in all recipients succumbing to liver failure, with histology showing extensive hepatic necrosis. CONCLUSIONS: This study demonstrated the feasibility of using the mOLT model for the study of molecular mechanisms associated with recovery from cold ischemia and reperfusion injury. Increasing lengths of cold ischemia correlate with progressive tissue damage whereas recovery is associated with a regenerative response that correlates with the severity of injury.     

Successful transplantation of a 20% partial liver graft in rats: a technical innovation

BACKGROUND: Graft-size disparity negatively impacts the postoperative outcome in the setting of adult-to-adult living donor liver transplantation. MATERIALS AND METHODS: As a model for partial liver transplantation in rats using a minimized graft volume, we orthotopically transplanted a 20% reduced-size liver with the two-cuff method. Blood chemistry, graft weight restoration, and labeling indices of 5-bromo-2'-deoxyuridine were estimated. RESULTS: We achieved 100% 2-week survival. Hepatic mass was restored rapidly with early normalization of biochemical parameters, although albumin synthesis capacity required more time to recover. CONCLUSION: This technique has great potential for research as well as a therapeutic approach to regeneration and functional recovery of small liver grafts under various posttransplant circumstances.     

血管紧张素转换酶在大鼠原位肝移植缺血再灌注损伤中的作用

目的 通过研究血管紧张素转换酶(ACE)在大鼠原住肝移植(OLT)中的表达情况及其与OLT后缺血再灌注损伤(IRI)的相关性,初步探讨肾素-血管紧张素系统在肝移植缺血再灌注损伤中的可能作用.方法 将OLT大鼠分为无冷保存组(NCP)及冷保存组(CP),假手术组作对照,组织学检查及血ALT检测观察缺血再灌注损伤程度,Real-time PCR,Western blot和免疫组化分别检测ACE的mRNA,蛋白表达及其组织定位,以上指标同时在术后多个时间点作动态观察.结果 OLT后,肝组织ACE在mRNA及蛋白水平均显著高于对照组(P<0.05,P<0.01),且CP组明显高于NCP组(P<0.05);CP组血清ALT水平显著高于NCP组,组织损伤更明显;动态观察显示ACE水平与血清ALT水平及组织损伤程度有很好的相关性.结论 ACE与冷保存导致的OLT后IRI的炎症损伤密切相关,肾素-血管紧张素系统可能在OLT后的IRI中有重要作用.     

Resuscitation of cardiac energy metabolism in the rabbit heart by brief hypothermic reperfusion after preservation studied by 31P NMR spectroscopy

Rabbit hearts were subjected to 24-h cold ischaemic storage (at 0°–2°C in melting ice) after initial flushing with either St Thomas' cardioplegic solution (STS) or modified lactobionate/raffinose solution (LR), and the status of phosphorylated energy metabolites was measured by ³¹phosphorus nuclear magnetic resonance (P NMR) spectroscopy. In both groups signals for ATP and phosphocreatine (PCr) were still detectable by ³¹P NMR after 24 h, and there was significantly more ATP in the LR group (P<0.01). The hearts were then subjected to coronary reperfusion via an aortic cannula using the same storage solution (either STS or LR) at 6°–8°C, which was oxygenated. In both groups PCr recovered within 30 min of cold reperfusion, and by 60 min PCr was significantly higher in the LR group (P<0.001). Also, levels of ATP were maintained at higher values during cold reperfusion in the LR group. These studies suggest two important points: (1) the general supply of phosphorylated high-energy intermediates of hearts during cold ischaemic storage is better preserved using LR, and (2) brief cold reperfusion may be used to restore energy metabolism in hearts before re-implantation.