Remote organ impairments are frequent and increase patient morbidity and mortality after lower limb ischemia-reperfusion (IR). We challenged the hypothesis that lower limb IR might also impair lung, renal, and liver mitochondrial respiration. Two-hour tourniquet-induced ischemia was performed on both hindlimbs, followed by a two-hour reperfusion period in C57BL6 mice. Lungs, liver and kidneys maximal mitochondrial respiration (
Peripheral arterial disease is a frequent pathology and corresponds to a major public health problem. The symptomatology ranges from intermittent claudication to tissue necrosis needing amputation [
Importantly, even subtle muscle damage can lead to significant remote organ injuries, and, thus, improving our knowledge on the mechanisms involved in such lesions appears mandatory. Indeed, lower limb ischemia-reperfusion is well known to result in remote organ injuries, key factors increasing perioperative and long-term morbidities [
Pulmonary injuries have been described after aortic cross-clamping, in humans and in animals, and systemic inflammation has been incriminated along with leukocytes sequestration in lung parenchyma. Histology also showed partial atelectasis with collapsed and pinched alveoli, thicker and felted alveolar walls. Pulmonary damages have been thus largely involved in patient mortality and morbidity through decreased FIO2/PaO2 ratio and impaired oxygenation [
Liver damage related to lower limb ischemia-reperfusion was less demonstrated but, in view of the detoxification role of the liver, it might concentrate inflammatory cells triggered by lower limb IR and should be affected, as previously reported in the setting of gut IR [
Kidneys are particularly concerned in the remote alteration phenomenon after skeletal muscle ischemia-reperfusion injuries. Yassin et al. showed renal dysfunction on a Wistar rat model with bilateral hindlimb IR injuries [
Mitochondria are key factor involved in skeletal muscle alteration during lower limb IR, and, interestingly, skeletal muscle mitochondrial dysfunctions occur early and are reversible after therapy [
Protecting remote organ mitochondria might therefore be a goal since mitochondrial dysfunction might lead to the multiorgan failure syndrome. However, very few data [
The aim of this study was therefore to challenge the hypothesis that lower limb IR results in lung, renal, and/or liver mitochondrial oxidative capacities impairment. We therefore investigated specifically their mitochondrial respiratory chain complexes activities and mitochondrial coupling that might be early markers of remote injuries.
Experiments were performed on 8 to 10 weeks male C57BL6 mice (Depré, France) weighing 20–24 g. Animals were housed in a neutral temperature environment (
Mice were ventilated with gas mixture of 4% isoflurane (AERRANE, BAXTER S.A.S.) and oxygen in a hermetic anaesthetic induction cage and placed on heating blankets (MINERVE, Esternay, France). Spontaneous ventilation was allowed through an oxygen-delivering mask, with different concentrations of isoflurane depending on the surgical stage (2% during painful stimuli and 1% during latent periods).
Then, a tourniquet was placed around each groin without skin incision. The Sham group (Sham group,
After a 2-hour reperfusion period, a midline laparotomy was performed, and both kidneys were dissected from the retroperitoneum and harvested by cutting renal vessels and ureters. The liver was then freed, and large specimens were retrieved from both lobes without cutting the hilum to preserve hepatic vessels and continue the thoracic procedure and harvesting.
A midline sternotomy followed. Pleurae were opened, and both lungs were harvested by cutting pulmonary vessels and main bronchi at the level of the hilum. This stage was performed quickly since mice had spontaneous ventilation that was made impossible when pleurae were opened. Mice were then sacrificed by heart harvesting.
All operations were carried out on ice. A piece of tissue was placed into buffer A containing 50 mM Tris, 1 mM EGTA, 70 mM Sucrose, and 210 mM Mannitol, pH 7.40 at +4°C. Tissue was finely minced with scissors, placed in buffer A, and homogenized with a tissue grinder. Then, the homogenate was centrifuged at 1300 ×g for 3 min, 4°C. The supernatant was centrifuged at 10,000 ×g for 10 min, 4°C, to sediment mitochondria. Finally, the mitochondrial pellet was washed twice and then suspended in 50 mM Tris, 70 mM Sucrose, and 210 mM Mannitol, pH 7.4 at +4°C. Protein content was routinely quantified with a Bradford assay using bovine serum albumin as a standard.
Oxygen consumption was measured polarographically by using a Clark-type electrode (Strathkelvin Instruments, Glasgow, Scotland).
When maximal fibre respiration (
(a) Experimental design. C: control; IR: ischemia-reperfusion. (b) Schematic representation of the mitochondrial respiratory chain with specific substrates and inhibitors. CI: complex I (NADH-CoQ reductase), CII: complex II (succinate-CoQ reductase), CIII: complex III (CoQH2-c reductase), CIV: complex IV (cytochrome c oxidase, COX), and TMPD: N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride. Schematic oxygraph trace showing oxygen consumption by the permeabilized skeletal myofibers, using indicated substrates and inhibitors:
The degree of coupling between oxidation and phosphorylation was inferred fromthe acceptor control ratio (ACR:
Liver and kidneys tissue sections were stained with haematoxylin-eosin, as previously described [
SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Graphics were generated by GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA). Means were compared between groups using a
Measuring oxygen consumption allowed determining the functional oxidative capacity of each organ mitochondrial function and particularly the relative contribution of the respiratory chain complexes I, II, III, and IV to the global mitochondrial respiratory rate.
IR decreased lung maximal oxidative capacity (Figure
Effects of lower limb ischemia/reperfusion on lung mitochondrial respiratory chain complexes activities. (A)
Thus, as inferred from the data obtained with each specific substrate we observed that complex I of the lung mitochondrial respiratory chain was impaired by lower limb IR.
Indeed, lower limb IR caused a slight, not statistically significant, increase in liver mitochondrial respiratory values (Figure
Effects of lower limb ischemia/reperfusion on liver mitochondrial respiratory chain complexes activities. (A)
Thus, lower limb IR failed to alter liver mitochondrial function, and all complexes of the mitochondrial respiratory chain (complexes I, II, III, and IV) showed a similar 9% trend to increase their activities.
Both renal
Effects of lower limb ischemia/reperfusion on kidney mitochondrial respiratory chain complexes activities. (A)
Thus, complexes I, II, and III activities of the mitochondrial renal respiratory chain were enhanced after IR.
Mitochondrial coupling was enhanced in kidney after lower limb ischemia-reperfusion (
Effects of lower limb ischemia/reperfusion on lung, liver, and kidney mitochondrial coupling. ACR: acceptor complex ration (
Liver and kidneys tissue sections, stained with haematoxylin-eosin, demonstrated normal architecture and thus no change was observed after IR (data not shown).
The main results of this study are to demonstrate for the first time that tourniquet-induced bilateral hindlimb ischemia-reperfusion (1) impairs lung maximal oxidative capacity, (2) does not modify liver mitochondrial function, and (3) unexpectedly stimulates kidney mitochondrial respiratory chain complexes activities and coupling in experimental mice.
Lower limb ischemia-reperfusion procedures are routinely performed throughout the world using either artery clamping or tourniquet. Particularly, tourniquet is widely used during orthopaedic surgeries in order to reduce bleeding in the operating zone. These procedures are associated not only with local skeletal muscle mitochondrial dysfunctions but also with remote lesions, generally assessed histologically or at a global, functional level. Since remote lesions largely participate in patient’s morbidity and mortality, a better knowledge of pathophysiological mechanisms involved might be useful to open new therapeutic perspectives. In this view, investigation on remote organ mitochondrial functions might be particularly interesting since mitochondrial dysfunction occurs early in skeletal muscles submitted to IR and is likely to be avoided using adapted strategies of ischemic or pharmacologic conditioning [
Although we showed no lung mitochondrial alteration after lower limb IR secondary to aortic cross-clamping in rats [
Taken together, these data support that circulating factors might be responsible for the reduced mitochondrial respiratory chain complexes observed after lower limb IR. On the other hand, incomplete hypoxia during aortic cross-clamping might be associated with a reduction of the deleterious factors released, as compared to tourniquet-induced complete bilateral hindlimb ischemia [
The circulating factors involved in remote organ damage are not totally known, but reactive oxygen species, cytokines, and complements factors associated with increased circulating leukocytes are likely to play a key role. Accordingly, studies reducing these factors allow organ protection, including lung protection [
Liver mitochondrial function was not impaired in our study. This is consistent with the fact that humans liver dysfunction is observed mainly when lower limb IR is complicated by a shock, linked to multiorgan failure [
Concerning the kidneys, we expected to observe impairments in mitochondrial respiration. Indeed, many experimental and clinical reports demonstrated that kidneys are key targets in the setting of IR. Thus, Miller et al. demonstrated a “relationship between intraoperative leg ischemia and postoperative renal failure, providing epidemiological evidence that the ischemic leg may be an important contributor to rhabdomyolysis-like renal morbidity after thoracoabdominal aortic surgery” [
The kidney functional enhancement observed in this study might correspond to the fact that kidneys had to filter circulating toxic whose numbers highly increase during lower limb IR. Therefore, their energetic capacities needed to be improved. A secondary damage might then occur later, when kidneys mitochondrial defences are overloaded by a higher amount of oxidative stress and inflammatory factors. Accordingly, we did not observe histological change in the kidney early after reperfusion, but other authors demonstrated tubular cell necrosis after 24 h of reperfusion following a 2-hour femoral artery occlusion [
A temporal response to ischemia looking at different time point of reperfusion might be very interesting. This might help discover new pattern of mitochondrial activities in different organs, and, particularly, this might allow determining when and why lung and kidneys functions are impaired after lower limb IR. Further, this will pave the way for new therapeutic approaches. In this view, circulating leukocytes and platelets or released molecules might be analysed by performing cellular depletion or serum transfusion experiments and proteomic assessment of the plasma.
Tourniquet-induced bilateral hindlimb ischemia-reperfusion leads to differential remote organ mitochondrial effects. Liver appeared to be well preserved but lungs were early damaged. Better knowing the pathophysiology of lung injury might help to focus therapeutic approach on lung mitochondrial functions, since they were beneficial for skeletal muscle in lower limb IR settings. Additionally, further kinetic studies will be useful to confirm whether and when kidneys mitochondrial dysfunctions occur and how to reduce these remote organ mitochondrial dysfunctions.
The authors declare that there is no conflict of interests regarding the publication of this paper.