A novel in vitro model of ischaemic preconditioning: its effect on cell viability in human skeletal muscle and a possible explanation for the ‘second window of protection’

Background

The ischaemia–reperfusion (IR) injury causes significant morbidity. Ischaemic preconditioning (IPC) is a technique for limiting the effects of the IR injury. Its potential has not yet been harnessed in orthopaedics.

Aims

To establish a novel in vitro IR model using a human skeletal muscle cell line. Secondly, to introduce simulated IPC to the model and examine the effect of this on cell viability.

Methods

A human skeletal muscle cell line was cultured in vitro. Placing the cells in a hypoxic buffer and a closed hypoxic environment simulated ischaemia. Reversing this process simulated reperfusion. IPC was simulated by alternate cycles of ischaemia and reperfusion. Cell viability comparisons were made between control and experimental groups of cells.

Results

A reproducible in vitro IR model was established. The addition of simulated IPC is associated with increased cell death at 12 and 24 h of reperfusion. Significantly greater cell survival is seen in the IPC group when measured at 72 h reperfusion.

Conclusions

We hypothesise that IPC initially decreases cell number. The remaining cells are more robust. This selected cell line then expands over the course of 72 h and displays greater resistance to the IR injury. This theory can help explain delayed preconditioning.     

Anaesthetic-induced myocardial preconditioning: fundamental basis and clinical implications

OBJECTIVE: Volatile halogenated anaesthetics offer a myocardial protection when they are administrated before a myocardial ischaemia. Cellular mechanisms involved in anaesthetic preconditioning are now better understood. The objectives of this review are to understand the anaesthetic-induced preconditioning underlying mechanisms and to know the clinical implications. DATA SOURCES: References were obtained from PubMed data bank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) using the following keywords: volatile anaesthetic, isoflurane, halothane, sevoflurane, desflurane, preconditioning, protection, myocardium. DATA SYNTHESIS: Ischaemic preconditioning (PC) is a myocardial endogenous protection against ischaemia. It has been described as one or several short ischaemia before a sustained ischemia. These short ischaemia trigger a protective signal against this longer ischaemia. An ischemic organ is able to precondition a remote organ. It is possible to replace the short ischaemia by a preadministration of halogenated volatile anaesthetic with the same protective effect, this is called anaesthetic PC (APC). APC and ischaemic PC share similar underlying biochemical mechanisms including protein kinase C, tyrosine kinase activation and mitochondrial and sarcolemnal K(ATP) channels opening. All halogenated anaesthetics can produce an anaesthetic PC effect. Myocardial protection during reperfusion, after the long ischaemia, has been shown by successive short ischaemia or volatile anaesthetic administration, this is called postconditioning. Ischaemic PC has been described in humans in 1993. Clinical studies in human cardiac surgery have shown the possibility of anaesthetic PC with volatile anaesthetics. These studies have shown a decrease of postoperative troponin in patient receiving halogenated anaesthetics.     

Postconditioning: Current Controversies and Clinical Implications

ObjectivesPostconditioning of ischaemic tissue, via mechanical or pharmacological manipulation, offers an exciting avenue towards amelioration of ischaemia–reperfusion injury. Born from the concept of ischaemic preconditioning, postconditioning is advantageous in that prior knowledge of the ischaemic insult is not required, and thus clinical translation may be further reaching. This review explores the current evidence and controversies in both animal and human studies and multiple organ systems.MethodsA Medline search was conducted to identify English-language articles with ‘postconditioning’ as a keyword. Two independent researchers scrutinised the literature search for potentially relevant articles. Reference lists from selected articles were manually searched for further relevant articles.Results and conclusionsPostconditioning has been shown to be successful in reducing ischaemia–reperfusion injury in both animal models and clinical trials. Human studies are presently limited to cardiac studies, but there is scope for research into other organ systems with potential beneficial effects, particularly within the field of vascular surgery where ischaemia–reperfusion occurs by nature of both – the disease and the intervention.     

Preconditioning the human heart.

The phenomenon of ischaemic preconditioning protects the myocardium by limiting infarct size in animal models of ischaemia and reperfusion. Ischaemic preconditioning may be induced by short periods of ischaemia and reperfusion. We investigated whether the human heart can be ischaemically preconditioned during coronary artery bypass grafting (CABG). Patients were enrolled into two separate studies. In the first study myocardial adenosine triphosphate (ATP) was used as the measured endpoint, assayed from myocardial biopsies taken at onset of cardiopulmonary bypass (CPB), at the end of the preconditioning stimulus, and at the end of a 10 min sustained ischaemic insult. In the second study the release of myocardial troponin T was used as the endpoint; taken at pre-CPB, and at 1, 6, 24, and 72 h after CPB. In both studies, patients were randomised into either the preconditioning group or the control group. Preconditioning was induced, after the onset of CPB, with two 3 min periods of crossclamping and an intervening 2 min of reperfusion, followed by 10 min sustained ischaemia. The control group only received 10 min of sustained ischaemia. Ischaemic preconditioning resulted in a slower rate of ATP (mumol/g dry weight) depletion in the preconditioned hearts at the end of the 10 min of sustained ischaemia (preconditioned: 11.5 +/- 0.8 vs control: 7.2 +/- 0.3; P < 0.005). Also, preconditioning resulted in a slower rate of troponin T release which was significantly different at 72 h after CPB in the preconditioned group (0.3 milligram) when compared with the control group (1.4 milligrams; P < 0.05). In addition, more patients in the preconditioned group had troponin T levels lower than 0.5 milligram at 72 h than in the control group (10 vs 3 patients). Both groups of patients received the same number of grafts, and underwent the same length of ischaemia during the procedure. We conclude that in patients undergoing CABG surgery, ischaemic preconditioning may reduce myocardial injury as shown by the favourable changes in myocardial ATP, and serum troponin T levels.     

Ischaemic preconditioning of the brain,mechanisms and applications

Summary Background. The concept of ischaemic preconditioning was introduced in the late 1980s. The concept emerged that a brief subcritical ischaemic challenge could mobilize intrinsic protective mechanisms that increased tolerance against subsequent critical ischaemia. Tissues with a high sensitivity against ischaemia, i.e. myocardium and central nervous system, present the most promising targets for therapeutic application of ischaemic preconditioning. During the last years the mechanisms of neuronal preconditioning were systematically studied and a number of molecular regulation pathways were discovered to participate in preconditioning. The purpose of the present review is to survey the actual knowledge on cerebral preconditioning, and to define the practical impact for neurosurgery. Methods. A systematic medline search for the terms preconditioning and postconditioning was filed. Publications related to the nervous system were selected and analysed. Findings. Preconditioning can be subdivided into early and late mechanisms, depending on whether the effect appears immediately after the nonlethal stress or with a delay of some hours or days. In general early effects can be linked to adaptation of membrane receptors whereas late effects are the result of gene up- or downregulation. Not only subcritical ischaemia can trigger preconditioning but also hypoxia, hyperthermia, isoflurane and other chemical substances. Although a vast amount of knowledge has been accumulated regarding neural preconditioning, it is unknown whether the effects can be potentiated by pharmacological or hypothermic neuroprotection during the critical ischaemia. Furthermore, although the practical importance of these findings is obvious, the resulting protective manipulations have so far not been transferred into clinical neurosurgery. Postconditioning and remote ischaemic preconditioning are additional emerging concepts. Postconditioning with a series of mechanical interruptions of reperfusion can apparently reduce ischaemic damage. Remote ischaemic preconditioning refers to the concept that transient ischaemia for example of a limb can lead to protection of the myocardium and possibly the brain. Conclusion. Possible cumulative neuroprotection by preconditioning and pharmacological protection during critical ischaemia should be studied systematically. Easy to apply methods of preconditioning, such as the application of volatile anaesthetics or erythropoietin some hours or days prior to planned temporary ischaemia, should be introduced into the practice of operative neurosurgery.     

The concept of anaesthetic-induced cardioprotection: mechanisms of action

The mechanisms by which ischaemia reperfusion injury can be influenced have been the subject of extensive research in the last decades. Early restoration of arterial blood flow and surgical measures to improve the ischaemic tolerance of the tissue are the main therapeutic options currently in clinical use. In experimental settings ischaemic preconditioning has been described as protecting the heart, but the practical relevance of interventions by ischaemic preconditioning is strongly limited to these experimental situations. However, ischaemia reperfusion of the heart routinely occurs in a variety of clinical situations, such as during transplantations, coronary artery bypass grafting or vascular surgery. Moreover, ischaemia reperfusion injury occurs without any surgical intervention as a transient myocardial ischaemia during a stressful anaesthetic induction. Besides ischaemic preconditioning, another form of preconditioning was discovered over 10 years ago: the anaesthetic-induced preconditioning. There is increasing evidence that anaesthetic agents can interact with the underlying pathomechanisms of ischaemia reperfusion injury and protect the myocardium by a preconditioning mechanism. Hence, the anaesthetist himself can substantially influence the critical situation of ischaemia reperfusion during the operation by choosing the right anaesthetic. A better understanding of the underlying mechanisms of anaesthetic-induced cardioprotection not only reflects an important increase in scientific knowledge but may also offer the new perspective of using different anaesthetics for targeted intraoperative myocardial protection. There are three time windows when a substance may interact with the ischaemia reperfusion injury process: (1) during ischaemia, (2) after ischaemia (i.e. during reperfusion), and (3) before ischaemia (preconditioning).     

L-Arginine given after ischaemic preconditioning can enhance cardioprotection in isolated rat hearts.

OBJECTIVE: Ischaemic or pharmacological preconditioning with L-arginine has been reported to be insufficient for optimal cardioprotection. The ability of nitric oxide (NO) to enhance ischaemic preconditioning was assessed, and the role of L-arginine-induced ischaemic preconditioning in myocardial protection was determined. METHODS: Isolated rat hearts were prepared and divided into six groups: control hearts (control, n=6) were perfused without global ischaemia at 37 degrees C for 160 min; global ischaemia hearts (GI, n=6) were subjected to ischaemia for 20 min and reperfusion for 120 min; ischaemic preconditioned hearts (IP, n=6) received 2 min of zero-flow global ischaemia followed by 5 min reperfusion, before 20 min of global ischaemia; L-arginine hearts (ARG, n=6) received 1 mmol/l L-arginine for 5 min, before 20 min of global ischaemia; ischaemic preconditioning plus nitro-L-arginine methyl ester hearts (IP+L-NAME, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 3 mmol/l L-NAME in Krebs-Henseleit buffer, before 20 min of global ischaemia; and ischaemic preconditioning plus L-arginine hearts (IP+ARG, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 1 mmol/l L-arginine in Krebs-Henseleit buffer. Haemodynamic parameters and coronary flow were recorded continuously. Nitrites and nitrates (NOx) were measured 5 and 60 min after reperfusion, and infarct size was also determined. RESULTS: In the IP+ARG group, significant amelioration and preservation of left ventricular peak developed pressure and coronary flow was observed compared with the GI, IP, ARG and IP+L-NAME groups. Infarct size in the IP+ARG group was reduced significantly compared with that in the GI, IP, ARG and IP+L-NAME groups. Significant preservation of NOx was observed during reperfusion in the IP+ARG group compared with the GI group. CONCLUSIONS: Inhibition of NO synthase with L-NAME had little impact on ischaemic preconditioning, suggesting that endogenous NO is not a major mediator of ischaemic preconditioning. Nevertheless, enhancement of the effects of ischaemic preconditioning can be achieved with L-arginine, a precursor of NO, improving post-ischaemic functional recovery and infarct size in the isolated rat heart.     

Complex Hepatectomy under Total Vascular Exclusion of the Liver: Impact of Ischemic Preconditioning on Clinical Outcomes

Background

Hepatic inflow clamping during hepatectomy introduces ischemia–reperfusion (I/R) injury, and many authors regard the addition of caval occlusion as adding increased risk. Ischemic preconditioning (IPC) is one of the protective strategies employed to reduce I/R injury in animal experiments and limited clinical series. The aim of the present study was to determine the impact of systematic adoption of IPC in patients undergoing complex hepatectomy under total hepatic vascular exclusion (TVE) based on outcomes review.

Methods

The records of 93 patients who underwent major hepatectomy involving TVE at our center from February 1998 to December 2008 were reviewed. These patients were divided into two groups: group 1 (n = 55, TVE alone) and group 2 (n = 38, TVE with IPC). IPC was performed by portal triad clamping for 10 min followed by 3–5 min of reperfusion prior to TVE and resection.

Results

The two groups were comparable regarding demographics, underlying liver diseases, indications for hepatectomy, duration of TVE, and preoperative liver and kidney function tests. Overall postoperative laboratory results of liver function tests were not significantly different between the two groups. Creatinine levels and prothrombin times were not significantly different between the groups. The use of IPC had no impact on the duration of the operation, blood loss, or hospital stay. The morbidity rates were 37.5 and 34.2 %, respectively.

Conclusions

Our adoption of IPC as a protective strategy against I/R injury under TVE did not affect operative or laboratory parameters and clinical outcomes when compared to continuous clamping for comparable ischemic periods.     

Microvascular preconditioning is not detectable by corrosion casting in the isolated perfused rat heart after 30 minutes of ischaemia

OBJECTIVE: Ischaemic preconditioning protects the myocardium from ischaemic injury and may also protect the vascular endothelium from the deleterious effects of ischaemia and reperfusion. We examined the possibility that ischaemic preconditioning might preserve the integrity of the coronary microcirculation following ischaemia and reperfusion. METHODS: Isolated rat hearts were perfused in Langendorff mode for 30 minutes and then subjected to 30 minutes of global ischaemia with or without ischaemic preconditioning (threexthree minute cycles). Some hearts underwent an additional 60 minutes of reperfusion. At the end of each protocol, microvascular corrosion casts were made by methylmethacrylate injection. RESULTS: Median left ventricular capillary density [interquartile range] after ischaemia was slightly but not significantly better with preconditioning at 6.8 [4.0-14.7]x10(-2) mm3.mg(-1) vs. 5.2 [2.6-7.1]x10(-2) mm3.mg(-1) (p=0.13). After 60 min of reperfusion, capillary density in preconditioned left ventricles was 20.7 [10.7-22.8]x10(-2) mm3.mg(-1) vs. 16.0 [10.2-23.0]x10(-2) mm3.mg(-1) for untreated ventricles (p=0.47). Coronary blood flow and heart rate were unchanged from before ischaemia. CONCLUSIONS: Ischaemia for 30 minutes induced global left ventricular capillary loss which was unmodified by preconditioning. We did not demonstrate vascular preconditioning using this model.     

Remote ischaemic preconditioning does not protect the heart in patients undergoing coronary artery bypass grafting

Remote ischaemic preconditioning (RIPC) gained attention as a possibility to reduce myocardial injury after a subsequent sustained episode of myocardial ischaemia. This prospective randomized study was carried out to assess whether RIPC reduces myocardial injury in coronary artery bypass grafting patients. Eighty patients were assigned to remote preconditioning or control treatment. Ischaemic preconditioning was induced by three 5-min cycles of upper limb ischaemia and reperfusion after anaesthesia induction. Haemodynamic and markers of myocardial damage were analysed preoperatively and over 48 h postoperatively. The cardiac index was higher immediately after remote preconditioning in the main group. There were no differences in other haemodynamic, troponin I and creatine kinase-MB concentrations at any time point between groups. Thus, short-term remote preconditioning improves haemodynamics and does not reduce myocardial injury after coronary artery bypass surgery. Further study of high-risk patients may be needed to fully evaluate the clinical effect of RIPC.     

-Arginine given after ischaemic preconditioning can enhance cardioprotection in isolated rat hearts

Objective: Ischaemic or pharmacological preconditioning with -arginine has been reported to be insufficient for optimal cardioprotection. The ability of nitric oxide (NO) to enhance ischaemic preconditioning was assessed, and the role of -arginine-induced ischaemic preconditioning in myocardial protection was determined. Methods: Isolated rat hearts were prepared and divided into six groups: control hearts (control, n=6) were perfused without global ischaemia at 37°C for 160 min; global ischaemia hearts (GI, n=6) were subjected to ischaemia for 20 min and reperfusion for 120 min; ischaemic preconditioned hearts (IP, n=6) received 2 min of zero-flow global ischaemia followed by 5 min reperfusion, before 20 min of global ischaemia; -arginine hearts (ARG, n=6) received 1 mmol/l -arginine for 5 min, before 20 min of global ischaemia; ischaemic preconditioning plus nitro- -arginine methyl ester hearts (IP+ -NAME, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 3 mmol/l -NAME in Krebs–Henseleit buffer, before 20 min of global ischaemia; and ischaemic preconditioning plus -arginine hearts (IP+ARG, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 1 mmol/l -arginine in Krebs–Henseleit buffer. Haemodynamic parameters and coronary flow were recorded continuously. Nitrites and nitrates (NOx) were measured 5 and 60 min after reperfusion, and infarct size was also determined. Results: In the IP+ARG group, significant amelioration and preservation of left ventricular peak developed pressure and coronary flow was observed compared with the GI, IP, ARG and IP+ -NAME groups. Infarct size in the IP+ARG group was reduced significantly compared with that in the GI, IP, ARG and IP+ -NAME groups. Significant preservation of NOx was observed during reperfusion in the IP+ARG group compared with the GI group. Conclusions: Inhibition of NO synthase with -NAME had little impact on ischaemic preconditioning, suggesting that endogenous NO is not a major mediator of ischaemic preconditioning. Nevertheless, enhancement of the effects of ischaemic preconditioning can be achieved with -arginine, a precursor of NO, improving post-ischaemic functional recovery and infarct size in the isolated rat heart.     

Effects of ischemia and omeprazole preconditioning on functional recovery of isolated rat heart

Objective

The aim of this study was to compare protective effects of ischemic and potential protective effects of pharmacological preconditioning with omeprazole on isolated rat heart subjected to ischemia/reperfusion.

Methods

The hearts of male Wistar albino rats were excised and perfused on a Langendorff apparatus. In control group (CG) after stabilization period, hearts were subjected to global ischemia (perfusion was totally stopped) for 20 minutes and 30 minutes of reperfusion. Hearts of group II (IPC) were submitted to ischemic preconditioning lasting 5 minutes before 20 minutes of ischemia and 30 minutes of reperfusion. In third group (OPC) hearts first underwent preconditioning lasting 5 minutes with 100μM omeprazole, and then submitted 20 minutes of ischemia and 30 minutes of reperfusion.

Results

Administration of omeprazole before ischemia induction had protective effect on myocardium function recovery especially regarding to values of systolic left ventricular pressure and dp/dt max. Also our findings are that values of coronary flow did not change between OPC and IPC groups in last point of reperfusion.

Conclusion

Based on our results it seems that ischemic preconditioning could be used as first window of protection after ischemic injury especially because all investigated parameters showed continuous trend of recovery of myocardial function. On the other hand, preconditioning with omeprazole induced sudden trend of recovery with positive myocardium protection, although less effective than results obtained with ischemic preconditioning not withstand, we must consider that omeprazole may be used in many clinical circumstances where direct coronary clamping for ischemic preconditioning is not possible.     

Sevoflurane induces cardioprotection through reactive oxygen species-mediated upregulation of autophagy in isolated guinea pig hearts

Purpose

Sevoflurane increases reactive oxygen species (ROS), which mediate cardioprotection against myocardial ischemia–reperfusion injury. Emerging evidence suggests that autophagy is involved in cardioprotection. We examined whether reactive oxygen species mediate sevoflurane preconditioning through autophagy.

Methods

Isolated guinea pigs hearts were subjected to 30 min ischemia followed by 120 min reperfusion (control). Anesthetic preconditioning was elicited with 2 % sevoflurane for 10 min before ischemia (SEVO). The ROS-scavenger, N-(2-mercaptopropionyl) glycine (MPG, 1 mmol/l), was administered starting 30 min before ischemia to sevoflurane-treated (SEVO + MPG) or non-sevoflurane-treated (MPG) hearts. Infarct size was determined by triphenyltetrazolium chloride stain. Tissue samples were obtained after reperfusion to determine autophagy-related protein (microtubule-associated protein light chain I and II: LC3-I, -II) and 5′ AMP-activated protein kinase (AMPK) expression using Western blot analysis. Electron microscopy was used to detect autophagosomes.

Results

Infarct size was significantly reduced and there were more abundant autophagosomes in SEVO compared with control. Western blot analysis revealed that the ratio of LC3-II/I and phosphorylation of AMPK were significantly increased in SEVO. These effects were abolished by MPG.

Conclusions

Sevoflurane induces cardioprotection through ROS-mediated upregulation of autophagy.     

The separate and combined influence of antioxidant and anti-inflammatory drugs on the liver during ischaemia-reperfusion of the superior mesenteric artery: An experimental study

Aim-Background

To investigate the influence of N-acetyl-cysteine (NAC) and Atorvastatin, separately and combined, on the liver of rats after inducing ischaemia-reperfusion of the superior mesenteric artery.

Methods

Forty male rats aged 2½ months and weighing 220–250gr were randomly assigned to 5 groups of 8 rats. The rats in the first group were considered as controls. The second group was subjected to ischaemia of the superior mesenteric artery for 45 minutes, followed by reperfusion of the superior mesenteric artery for 3 hours, without any drug administration. In the third group, a single dose of Atorvastatin (10mg/kg) was administered 24 hours before the phase of ischaemia. The fourth group received a single dose of N-acetyl-cysteine (160mg/kg) 24 hours before the phase of ischaemia. The fifth group was given both drugs simultaneously 24 hours before the phase of ischaemia. Three hours after reperfusion of the superior mesenteric artery, the liver was surgically removed and prepared for histological examination.

Results

The histological examination of liver tissue samples revealed diffuse necrosis of hepatocytes in the group without any drug administration prior to ischaemia-reperfusion. The groups of animals that were treated with NAC or Atorvastatin or the combination of both drugs showed quite a different histological picture with focal necrosis of hepatocytes and less degenerative lesions.

Conclusions

This study shows that pretreatment with NAC, Atorvastatin and their combination protects the liver from injury induced by intestinal ischaemia-reperfusion. This is ascribed to the antioxidant and anti-inflammatory effect of the above-mentioned drugs.     

Essential Role for Nuclear Factor κB in Ischemic Preconditioning for Cold Ischemia-Reperfusion Injury of Intestinal Transplantation

Objective

The present study was designed to examine whether ischemic preconditioning could play a protective role on cold ischemia and reperfusion injury associated with intestinal transplantation in rats.

Methods

The 48 male Sprague Dawley (SD) rats were randomly assigned to 2 groups. Ischemic preconditioning was performed in experimental but not control rats by preserving the self donor small bowel in Ringer lactate solution at 4 °C for 3 hours (n = 6), 6 hours (n = 6), 12 hours (n = 6), or 18 hours (n = 6). One hour reperfusion was performed for every rat after orthotopic transplantation of donor small bowel. Small bowel samples were obtained for histological examination and immunohistochemistry analysis of nuclear factor κB (NF-κB) expression.

Result

The small intestinal villus was arranged more regularly in experimental compared with control rats. Ischemia preconditioning also decreased edema in the muscule layer and increased Chiu score in experimental rats. Immunohistochemistry analysis revealed that ischemic preconditioning down-regulated the expression of NF-κB in the epithelia of experimental rats.

Conclusion

Ischemic preconditioning improved intestinal transplantation in rats from cold ischemia and reperfusion injury by down-regulating the expression of NF-κB.     

Atrial natriuretic peptide reduces hepatic ischemia–reperfusion injury in rabbits

Purpose

Atrial natriuretic peptide (ANP) has been known to be protective against hepatic ischemia/reperfusion injury. The purpose of this study was to verify the hypothesis that ANP conserves microvascular circulation and reduces ischemia–reperfusion injury in the in vivo rabbit model.

Methods

With IRB approval, 30 male Japanese white rabbits under pentobarbital anesthesia were studied. These animals were randomly assigned to the following three groups (n = 10 each): control, ANP, and sham group. Animals in the ANP group received continuous infusion of ANP at 0.1 μg/kg/min throughout the study period. Animals in control and ANP groups underwent 90 min of partial hepatic ischemia by clamping the right hepatic artery and portal vein. Descending aortic blood flow (AoF) was monitored with a transit-time ultrasound flowmeter. Hepatic tissue microvascular blood flow (HTBF) at both right (ischemic) and left (nonischemic) lobe was intermittently evaluated with the hydrogen clearance method. After 180 min of reperfusion, hepatic injury was determined with serum AST and ALT. Galactose clearance of reperfused right lobe was also measured as an indicator of hepatic metabolic function. Histopathological change and the number of apoptotic hepatocytes were also evaluated.

Results

Systemic hemodynamic data including mean arterial pressure, heart rate, and AoF did not differ among the three groups during the study period. ANP attenuated ischemia-induced right HTBF decrease. ANP also suppressed histopathological degeneration, apoptosis, and decline in galactose clearance after reperfusion.

Conclusions

ANP attenuated hepatic microvascular dysfunction and hepatocyte injury after reperfusion without significant hemodynamic change.     

Milrinone-induced postconditioning reduces hepatic ischemia-reperfusion injury in rats: the roles of phosphatidylinositol 3-kinase and nitric oxide

Background

Ischemic postconditioning (PostC) protects the liver against ischemia-reperfusion (IR) injury. Milrinone, a phosphodiesterase 3 inhibitor, has been reported to exhibit preconditioning properties against hepatic IR injury; however, its PostC properties remain unknown. This study investigated whether milrinone has PostC properties against hepatic IR injury and the roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS).

Materials and methods

Male Wistar rats were separated into six groups: (1) group S: animals that underwent sham operation without ischemia, (2) group C: ischemia followed by reperfusion with no other intervention, (3) group M: milrinone administered immediately after reperfusion, (4) group MW: wortmannin, a PI3K inhibitor, injected before milrinone administration, (5) group MN: l-NAME, a NOS inhibitor, injected before milrinone administration, and (6) group MD, milrinone administered 30 min after reperfusion. Except for group S, all groups underwent 1 h of warm ischemia of median and left lateral lobes, followed by 5 h of reperfusion. Biochemical liver function analysis and histologic examination were performed.

Results

Serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, histologic damage scores, and apoptotic rate in group M were significantly lower than those in group C. The inhibition of PI3K or NOS prevented this protective effect. Milrinone administered 30 min after reperfusion did not show obvious protective effects.

Conclusions

Milrinone-induced PostC protects against hepatic IR injury when it is administered immediately after reperfusion, and PI3K and NOS may play an important role in this protective effect.     

Ischaemic preconditioning of skeletal muscle. 1. Protection against the structural changes induced by ischaemia/reperfusion injury

Ischaemic preconditioning is a process by which exposure of a tissue to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of a subsequent prolonged ischaemic stress. It has been extensively described in the heart, but few studies have examined the possibility that it can occur in skeletal muscle. We have used a rat model of ischaemia of one limb to examine this possibility. Exposure of the hind limb to a period of ischaemia of five minutes and reperfusion for five minutes significantly protected the tibialis anterior muscle against the structural damage induced by a subsequent period of limb ischaemia for four hours and reperfusion for one hour. This protection was evident on examination of the muscle by both light and electron microscopy. Longer or shorter times of prior ischaemia had no effect.     

Activated protein C attenuates skeletal muscle ischaemia reperfusion injury.

Aims: Pharmacological modulation of skeletal muscle reperfusion injury after an ischaemic insult may improve limb salvage rates and prevent the associated systemic sequelae. Activated Protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties that has been extensively studied in the setting of sepsis. The purpose of our study was to evaluate the effects of APC on skeletal muscle ischaemia reperfusion injury. Methods: Adult male Sprague Dawley rats (n = 24) were randomised into three groups: control group, I/R group treated with normal saline and I/R group treated with APC. Bilateral hind-limb ischaemia was induced by rubber band application proximal to the level of the greater trochanters for two hours. Treatment groups received either normal saline or APC prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Skeletal muscle injury was assessed based on myeloperoxidase content as a measure of neutrophil infiltration and wet to dry ratio as a measure of oedema formation. Histological analysis was also performed on the muscle. Results: APC significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle (Table). These findings were supported by our histological findings. Statistical significance was determined using variance analysis. Conclusion: Activated Protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury.     

Inhalatives Kohlenmonoxid zur Protektion der Lunge während des kardiopulmonalen Bypasses

Introduction

Cardiopulmonary bypass (CPB) causes severe pulmonary and extrapulmonary inflammatory reactions, which may lead to impairment of the lungs as well as disorders of other organs, e.g., the kidneys and liver.

Methods and results

In an animal pig model, we were able to show that normalization of pulmonary perfusion during CPB or pulmonary preconditioning with inhaled carbon monoxide (250 ppm) or both may reduce lung injury induced by CPB. One possible mechanism of carbon monoxide-mediated reduction of inflammation and lung damage may be explained by the induction of a heat shock response. Furthermore, impairment of extrapulmonary organs can be reduced by inhalation of carbon monoxide.

Conclusion

Cardiopulmonary bypass-induced inflammation and the associated tissue damage are caused by ischemia of the lung and the subsequent reperfusion injury. This can be reduced by active pulmonary perfusion and inhalation of carbon monoxide. The combination of active pulmonary perfusion and inhaled carbon monoxide can lead to the reduction of pulmonary damage.