Ischemic preconditioning improves oxygenation of exercising muscle in vivo

BACKGROUND: Ischemic preconditioning (IP) improves tissue tolerance to prolonged ischemia. In this study, we investigated the functional effect of IP on skeletal muscle of rat hind limb by means of near-infrared spectroscopy (NIRS) and by measuring myeloperoxidase (MPO) activity. MATERIALS AND METHODS: Adult male Sprague Dawley rats were divided into four separate protocol groups according to different preparations prior to 2 h of global ischemia: a group of ischemic reperfusion without any preparation (I/R), ischemic reperfusion with ischemic preconditioning (IP+IR), ischemic reperfusion with adenosine infusion (ADO+I/R), and sham operation. Ischemia and ischemic preconditioning were induced by clamping infrarenal abdominal aorta and left common iliac artery. For each rat, an exercise test of gastrocnemius muscles was performed by stimulating sciatic nerve before and after global ischemia while performing NIRS. MPO activity of ischemic muscles was also measured. RESULTS: Half-resaturation time after exercise and MPO activity were significantly improved in IP+IR and ADO+I/R groups. Difference of oxyhemoglobin during exercise was also improved in the IP+IR group. CONCLUSION: This study has demonstrated that IP provides the protective effect on in vivo skeletal muscle oxygenation during exercise.     

缺血预处理减轻骨骼肌缺血再灌注损伤

目的 观察缺血预处理对骨骼肌缺血再灌注损伤的保护作用。方法 选择２４只健康兔,随机等分为实验组和对照组。实验组先进行缺血预处理,再持续阻断后肢血流４ｈ;对照组直接阻断后肢血流４ｈ,制作骨骼肌缺血再灌注损伤模型。测定再灌注期血清中肌酸磷酸激酶（ＣＰＫ）和天门冬氨酸氨基转移酶（ＡＳＴ）,镜下观察骨骼肌变化。结果 实验组血清中ＣＰＫ和ＡＳＴ的含量均明显低于对照组（Ｐ〈０．０５）。实验组骨骼肌线粒体空泡变     

Remote preconditioning improves hepatic oxygenation after ischaemia reperfusion injury

Hepatic ischaemia reperfusion injury (IRI) lowers hepatic oxygenation and induces tissue acidosis. Remote ischaemic preconditioning (RIPC) reduces hepatic IRI through increased hepatic blood flow but its effect on hepatic oxygenation and acidosis is not known. This study investigates these effects through near infrared spectroscopy (NIRS). Twenty-four NZ rabbits were grouped into four: sham, RIPC, IRI alone, RIPC + IRI. RIPC was induced through three cycles of 10 min ischaemia and reperfusion to the limb. Total hepatic ischaemia was produced by complete portal inflow occlusion for 25 min. Serum transaminases, bicarbonate and hepatic venous nitrite/nitrate (NO(x) ) levels were measured 2 h postreperfusion. Hepatic oxygenation was monitored with NIRS. At 2 h post reperfusion, IRI alone resulted in reduced mitochondrial oxygenation (CytOx CuA Redox), serum bicarbonate, hepatic venous NO(x) with an increase in serum transaminases and hepatic deoxyhaemoglobin levels. RIPC before IRI caused significant improvement in mitochondrial oxygenation (P = 0.01), increased serum bicarbonate (P = 0.02), hepatic venous NO(x) (P = 0.025) with a decrease in serum transaminases (P = 0.04) and hepatic deoxyhaemoglobin levels (P = 0.03). There was a positive correlation (P = 0.02) between hepatic venous NO(x) levels and mitochondrial oxygenation. RIPC before IRI improves hepatic mitochondrial oxygenation and reduces acidosis and currently undergoing clinical study.     

Ischemic preconditioning of skeletal muscle: duration of late-phase protection

BACKGROUND: The time course of the late phase of ischemic preconditioning (IPC) was determined in latissimus dorsi muscle (LDM) flaps using viability and function as the endpoints. MATERIALS AND METHODS: LDM flaps from Sprague-Dawley rats were allocated into 6 groups. LDMs were preconditioned with 2 30-minute periods of ischemia separated by 10 minutes of reperfusion and subjected to a 4-hour ischemic insult after 24, 48, 72, and 96 hours from IPC. LDMs were evaluated for percent necrosis and muscle contractile function and compared with controls. RESULTS: The late phase of IPC provides significant protection against necrosis up to 72 hours. Conversely, when the end point used was muscle contractile function, the protection only lasted 48 hours. CONCLUSION: The time course of late-phase protection in skeletal muscle is 2-3 days. Late phase IPC appears to protect muscle flaps during the most critical time period following elevation.     

Ischemic preconditioning of skeletal muscle mitigates remote injury and mortality

BACKGROUND: Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality. METHODS: Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Student's t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio. RESULTS: Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59). CONCLUSIONS: We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.     

Ischaemic preconditioning improves microvascular perfusion and oxygenation following reperfusion injury of the intestine

BACKGROUND: Ischaemia-reperfusion (IR) injury of the intestine occurs commonly during abdominal surgery. Ischaemic preconditioning (IPC) provides a way of protecting the organ from damage inflicted by IR. This study was designed to evaluate the beneficial effect of IPC, focusing on the intestinal microcirculation and oxygenation in intestinal IR injury. METHODS: Rats were allocated to three groups. Animals in the IR and IPC groups underwent 30 min of intestinal ischaemia followed by 2 h of reperfusion. In the IPC group this was preceded by 10 min of ischaemia and 10 min of reperfusion. Animals in the third group underwent laparotomy but no vascular occlusion. Intestinal microvascular perfusion, oxygenation and portal venous blood flow (PVF) were monitored continuously. At the end of the reperfusion period, blood samples were obtained for measurement of lactate dehydrogenase (LDH) and biopsies of ileum for histological evaluation. RESULTS:: IPC improved intestinal microvascular perfusion and tissue oxygenation significantly at the end of the reperfusion period (P < 0.001). PVF improved significantly in the IPC compared with the IR group (P = 0.005). The serum LDH concentration was significantly lower in the IPC than the IR group (mean(s.e.m.) 667.1(86.8) versus 1973.8(306.5) U/l; P < 0.001) Histological examination showed that ileal mucosa was significantly less injured in the IPC group. CONCLUSIONS: This study demonstrated that IPC improves intestinal microvascular perfusion and oxygenation.     

Ischemic preconditioning: lack of delayed protection against skeletal muscle ischemia-reperfusion

We investigated the ability of ischemic preconditioning to induce expression of heat shock protein 70 (Hsp 70) and/or to increase muscle survival after ischemia-reperfusion in the rat hind limb. Ischemic preconditioning regimens tested were; 1 x 5 min of ischemia, 4 x 5 min of ischemia interrupted by 10 min of reperfusion, 1 x 10 min of ischemia or 2 x 10 min of ischemia interrupted by 15 min of reperfusion. Western blot analysis revealed only a modest induction of Hsp 70 at 24 h after preconditioning using the latter two protocols of 1 x 10 min of ischemia or 2 x 10 min. Used at 24 h prior to prolonged ischemia, neither protocol improved muscle survival measured at 24 h after reperfusion. In conclusion, ischemic preconditioning did not produce delayed protection from ischemia-reperfusion in this model and the study suggests that ischemic preconditioning is not a useful protective strategy against skeletal muscle necrosis in the long-term.     

Ischemic preconditioning improves rat kidney graft function after severe ischemia/reperfusion injury

Ischemic preconditioning (IP) has been shown to ameliorate renal ischemia reperfusion injury. Using a rat kidney transplantation model we determined if IP improves graft function after prolonged cold storage. MATERIALS AND METHODS: Syngeneic rat kidneys were divided into two groups. Prior to 42 hours of cold storage in UW and transplantation, one group (n = 10) received IP (15 minutes of warm ischemia/10 minutes of reperfusion), whereas another group (n = 10) received no treatment. Early graft function and 1-week recipient survival were assessed. RESULTS: Recipient survival was not significantly different between groups [70% (IP) vs 40% (non-IP); P = .28]. IP treatment led to a quicker recovery of renal function. On PODs 3 and 6, serum creatinine levels in the IP group were significantly lower compared with the untreated group. In conclusion, one cycle of IP (15/10) accelerates recovery of renal graft function after severe ischemia reperfusion injury. This simple treatment modality may improve outcomes of renal transplants with prolonged cold storage.     

Hypercapnia improves tissue oxygenation

BACKGROUND: Wound infections are common, serious, surgical complications. Oxidative killing by neutrophils is the primary defense against surgical pathogens and increasing intraoperative tissue oxygen tension markedly reduces the risk of such infections. Since hypercapnia improves cardiac output and peripheral tissue perfusion, we tested the hypothesis that peripheral tissue oxygenation increases as a function of arterial carbon dioxide tension (PaCO(2)) in anesthetized humans. METHODS: General anesthesia was induced with propofol and maintained with sevoflurane in 30% oxygen in 10 healthy volunteers. Subcutaneous tissue oxygen tension (PsqO(2)) was recorded from a subcutaneous tonometer. An oximeter probe on the upper arm measured muscle oxygen saturation. Cardiac output was monitored noninvasively. PaCO(2) was adjusted to 20, 30, 40, 50, or 60 mmHg in random order with each concentration being maintained for 45 min.(2) (2) RESULTS: Increasing PaCO(2) linearly increased cardiac index and PsqO(2) : PsqO(2) = 35.42 + 0.77 (PaCO(2)), < 0.001. CONCLUSIONS: The observed difference in PsqO(2) is clinically important because previous work suggests that comparable increases in tissue oxygenation reduced the risk of surgical infection from -8% to 2 to 3%. We conclude that mild intraoperative hypercapnia increased peripheral tissue oxygenation in healthy human subjects, which may improve resistance to surgical wound infections.     

缺血后处理和预处理对大鼠骨骼肌缺血-再灌注损伤的作用

目的 探讨缺血后处理( IPost)和缺血预处理(IPC)对大鼠骨骼肌缺血再灌注(IR)损伤的影响.方法 将40只大鼠随机分成缺血再灌注组(A组)、缺血后处理组(B组)、缺血预处理组(C组)、缺血预处理加缺血后处理组(D组)以及对照组(E组),采用切断患肢全部皮肤、肌肉和神经,保留患肢股动、静脉的动物模型,通过夹闭和开放股动、静脉造成骨骼肌缺血再灌注损伤,通过测定骨骼肌缺血4h、再灌注1h后血清丙二醛(MDA)和骨骼肌髓过氧化物酶(MPO),以及再灌注6h后骨骼肌的坏死程度来观察缺血后处理.缺血预处理及缺血预处理加缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响.结果 B组、C组和D组再灌注1 h MDA和MPO水平以及再灌注6h骨骼肌坏死程度均低于A组(P＜ 0.05),但是高于E组(P＜0.05);B组和D组再灌注1 h MDA和MPO水平以及再灌注6h骨骼肌坏死程度基本相同(P＞0.05);B组和D组再灌注1 h MDA和MPO水平低于C组(P＜0.05),但再灌注6h骨骼肌坏死程度基本相同(P＞0.05).结论 应用缺血后处理和缺血预处理对大鼠骨骼肌缺血再灌注损伤有一定的保护效果,联合应用缺血后处理和缺血预处理,对骨骼肌缺血再灌注损伤的保护作用并没有明显增强.     

Protective effect of preconditioning and adenosine pretreatment in experimental skeletal muscle reperfusion injury.

BACKGROUND: Prolonged ischaemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue injury. Ischaemic preconditioning (IPC), achieved by repeated brief periods of I/R before prolonged ischaemia or adenosine pretreatment, can prevent I/R injury in cardiac muscle. The aim of this study was to ascertain in a rodent model if damage to skeletal muscle due to global hindlimb tourniquet-induced I/R could be similarly attenuated. METHODS: Anaesthetized rats were randomized (n = 6-10 per group) to five groups: sham-operated controls; I/R (4 h of ischaemia, 2 h of reperfusion); IPC (three cycles of 10 min of ischaemia/10 min of reperfusion) alone; IPC immediately preceding I/R; or adenosine 1000 microg/kg immediately before I/R. At the end of reperfusion, biopsies were taken from the left gastrocnemius muscle for measurement of myeloperoxidase (MPO) and reduced glutathione (GSH). Before ischaemia and at the end of reperfusion, blood samples were taken for measurement of nitric oxide metabolites, tumour necrosis factor (TNF) alpha and macrophage inflammatory protein (MIP) 2. RESULTS: IPC before I/R resulted in lower levels of MPO (P < 0.001) and TNF-alpha (P = 0.004), and higher levels of GSH (P < 0.001) and nitric oxide metabolites (P = 0.002) than I/R alone. Adenosine had effects comparable to IPC pretreatment (P < 0.001 for MPO, P = 0.002 for GSH, P = 0.02 for nitric oxide metabolites and P = 0.001 for TNF-alpha). There was no difference in the blood pressure or the MIP-2 concentration among the groups. CONCLUSION: IPC or pretreatment with adenosine ameliorates the I/R injury of skeletal muscle.     

Sympathetic block significantly improves reperfusion in skeletal muscle following prolonged use of tourniquet

The effects of guanethidine sympathetic nerve blocks on reperfusion of skeletal muscle was studied in rats. After 3 hours of ischaemia reperfusion was significantly better in animals that had received guanethidine.     

Erythropoietin improves functional and histological recovery of traumatized skeletal muscle tissue

Apart from its hematopoietic effect, erythropoietin (EPO) is known as pleiotropic cytokine with anti‐inflammatory and anti‐apoptotic properties. Here, we evaluated for the first time the EPO‐dependent regeneration capacity in an in vivo rat model of skeletal muscle trauma. A myoblast cell line was used to study the effect of EPO on serum deprivation‐induced cell apoptosis in vitro. A crush injury was performed to the left soleus muscle in 80 rats treated with either EPO or saline. Muscle recovery was assessed by analysis of contraction capacities. Intravital microscopy, BrdU/laminin double immunohistochemistry and cleaved caspase‐3 immunohistochemistry of muscle tissue on days 1, 7, 14, and 42 posttrauma served for assessment of local microcirculation, tissue integrity, and cell proliferation. Serum deprivation‐induced myoblast apoptosis of 23.9 ± 1.5% was reduced by EPO to 17.2 ± 0.8%. Contraction force analysis in the EPO‐treated animals revealed significantly improved muscle strength with 10–20% higher values of twitch and tetanic forces over the 42‐day observation period. EPO‐treated muscle tissue displayed improved functional capillary density as well as reduced leukocytic response and consecutively macromolecular leakage over day 14. Concomitantly, muscle histology showed significantly increased numbers of BrdU‐positive satellite cells and interstitial cells as well as slightly lower counts of cleaved caspase‐3‐positive interstitial cells. EPO results in faster and better regeneration of skeletal muscle tissue after severe trauma and goes along with improved microcirculation. Thus, EPO, a compound established as clinically safe, may represent a promising therapeutic option to optimize the posttraumatic course of muscle tissue healing. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Protective effect of ischaemic preconditioning against ischaemia-induced reperfusion injury of skeletal muscle: how many preconditioning cycles are appropriate?

We investigated the efficacy of ischaemic preconditioning (IPC), consisting of repeated brief episodes of vascular occlusion followed by reperfusion, as protection against ischaemia-reperfusion injury of skeletal muscle, using a rat amputation-like model. Wistar rats underwent temporary amputation at the level of the femur, excluding the femoral vessels. The femoral artery and vein were clamped for 4h, using a micro-clamp, in the groups exposed to ischaemia. The rats were randomly divided into eight groups: a control (C) group (n = 7) with non-amputated and non-ischaemic hind limbs; a sham control (SC) group (n = 7) with amputated but non-ischaemic hind limbs; an ischaemia-reperfusion (IR) group (n = 7) with amputated and ischaemic hind limbs; and five IPC groups (n = 7 in each) with hind limbs that were subjected to 4h of ischaemia after one to five cycles of brief ischaemia and reperfusion for 10 min each, respectively. All rats were sacrificed 24h after reperfusion. The viability of the anterior tibial muscles was evaluated using nitroblue tetrazolium staining. The total viable area ratio (T-VAR) of the muscle tissue was calculated in each animal as follows: T-VAR\total viable area/total slice areae 100%. The T-VAR values of the eight groups were as follows: C group, 100% +/- 0%; SC group, 100% +/- 0%; IR group, 73.5% +/- 1.7%; IPC1 group, 79.4% +/- 6.5%; IPC2 group, 70.5% +/- 6.2%; IPC3 group, 90.6% +/- 2.8%; IPC4 group, 90.0% +/- 1.6%; and IPC5 group, 87.8% +/- 1.8%. The T-VARs in the IPC3, IPC4 and IPC5 groups were significantly higher (alpha < 0.01) than those in the IR group. In contrast, there were no significant differences between the T-VARs of the IPC1 and IPC2 groups and those of the IR group. In conclusion, three to five cycles of IPC could protect skeletal muscle against ischaemia. 2002 The British Association of Plastic Surgeons.     

Ischemic preconditioning improves mitochondrial tolerance to experimental calcium overload

BACKGROUND: Ca(2+) overload leads to mitochondrial uncoupling, decreased ATP synthesis, and myocardial dysfunction. Pharmacologically opening of mitochondrial K(ATP) channels decreases mitochondrial Ca(2+) uptake, improving mitochondrial function during Ca(2+) overload. Ischemic preconditioning (IPC), by activating mitochondrial K(ATP) channels, may attenuate mitochondrial Ca(2+) overload and improve mitochondrial function during reperfusion. The purpose of these experiments was to study the effect of IPC (1) on mitochondrial function and (2) on mitochondrial tolerance to experimental Ca(2+) overload. METHODS: Rat hearts (n = 6/group) were subjected to (a) 30 min of equilibration, 25 min of ischemia, and 30 min of reperfusion (Control) or (b) two 5-min episodes of ischemic preconditioning, 25 min of ischemia, and 30 min of reperfusion (IPC). Developed pressure (DP) was measured. Heart mitochondria were isolated at end-Equilibration (end-EQ) and at end-Reperfusion (end-RP). Mitochondrial respiratory function (state 2, oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI, state 3/state 4); rate of oxidative phosphorylation (ADP/Deltat), and ADP:O ratio) was measured with polarography using alpha-ketoglutarate as a substrate in the presence of different Ca(2+) concentrations (0 to 5 x 10(-7) M) to simulate Ca(2+) overload. RESULTS: IPC improved DP at end-RP. IPC did not improve preischemic mitochondrial respiratory function or preischemic mitochondrial response to Ca(2+) loading. IPC improved state 3, ADP/Deltat, and RCI during RP. Low Ca(2+) levels (0.5 and 1 x 10(-7) M) stimulated mitochondrial function in both groups predominantly in IPC. The Control group showed evidence of mitochondrial uncoupling at lower Ca(2+) concentrations (1 x 10(-7) M). IPC preserved state 3 at high Ca(2+) concentrations. CONCLUSIONS: The cardioprotective effect of IPC results, in part, from preserving mitochondrial function during reperfusion and increasing mitochondrial tolerance to Ca(2+) loading at end-RP. Activation of mitochondrial K(ATP) channels by IPC and their improvement in Ca(2+) homeostasis during RP may be the mechanism underlying this protection.