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University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Reanimation, University Hospital of Strasbourg, France
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Physiology, University Hospital of Strasbourg, France
Corresponding author. Department of Vascular Surgery and Kidney Transplantation, University Hospital of Strasbourg, 1 place de l’hôpital, 67091, Strasbourg Cedex, France.
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Vascular Surgery and Kidney Transplantation, University Hospital of Strasbourg, France
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Anaesthesiology, Critical Care and Peri-operative Medicine, University Hospital of Strasbourg, France
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Physiology, University Hospital of Strasbourg, France
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Vascular Surgery and Kidney Transplantation, University Hospital of Strasbourg, France
University of Strasbourg, FMTS, Research Unit 3072, Mitochondria, Oxidative Stress and Muscular Protection, Strasbourg, FranceDepartment of Physiology, University Hospital of Strasbourg, France
The aim of this study was to investigate whether remote ischaemic per-conditioning might protect skeletal muscle during lower limb ischaemia–reperfusion (IR).
Methods
Twenty-three male C57BL/6 mice were randomised into three groups: sham group (n = 7), IR group (unilateral tourniquet induced three hours of ischaemia followed by 24 hours of reperfusion, n = 8), and remote ischaemic per-conditioning group (RIPerC) (three cycles of 10 minute IR episodes on the non-ischaemic contralateral hindlimb, n = 8). Oxygraphy, spectrofluorometry, and electron paramagnetic resonance spectroscopy were performed in order to determine mitochondrial respiratory chain complexes activities, mitochondrial calcium retention capacity (CRC) and reactive oxygen species (ROS) production in skeletal muscle.
Results
IR impaired mitochondrial respiration (3.66 ± 0.98 vs. 7.31 ± 0. 54 μmol/min/g in ischaemic and sham muscles, p = .009 and p = .003 respectively) and tended to impair CRC (2.53 ± 0.32 vs. 3.64 ± 0.66 μmol/mg in ischaemic and sham muscles respectively, p = .066). IR did not modify ROS production (0.082 ± 0.004 vs. 0.070 ± 0.004 μmol/min/mg in ischaemic and sham muscles respectively, p = .74). RIPerC failed to restore mitochondrial respiration (3.82 ± 0.40 vs. 3.66 ± 0.98 μmol/min/g in ischaemic muscles from the RIPerC group and the IR group respectively, p = .45) and CRC (2.76 ± 0.3 vs. 2.53 ± 0.32 μmol/mg in ischaemic muscles from the RIPerC group and the IR group respectively, p = .25). RIPerC even impaired contralateral limb mitochondrial respiration (3.85 ± 0.34 vs. 7.31 ± 0. 54 μmol/min/g in contralateral muscles and sham muscles respectively, –47.3%, p = .009).
Conclusion
RIPerC failed to protect ischaemic muscles and induced deleterious effects on the contralateral non-ischaemic muscles. These data do not support the concept of RIPerC.
Ischaemic conditioning strategies could provide tremendous potential in vascular surgery and improve patient outcomes by protecting tissues suffering from ischaemia–reperfusion injury. However, pre-conditioning and post-conditioning strategies do not fully translate into the clinical setting: pre-conditioning is limited by the failure to predict the onset of ischaemia while post-conditioning might be deleterious. This study investigated a per-conditioning protocol, having the advantage of being applied after the onset of ischaemia, during the surgical procedure itself.
Introduction
Skeletal muscle ischaemia–reperfusion (IR) impairs the entire local muscle environment (endothelial and muscle cells, vessels, and nerves) via complex processes, leading to loss of muscle function and even failure of remote organs, contributing to the peri-operative morbidity and mortality of vascular surgery procedures.
2017 ESC guidelines on the diagnosis and treatment of peripheral arterial diseases, in collaboration with the European Society for Vascular Surgery (ESVS).
In parallel, xanthine dehydrogenase, which is found in the microvascular endothelial cells of skeletal muscle, is converted to xanthine oxidase. After two hours of ischaemia, adenosine diphosphate is catabolised into hypoxanthine and xanthine.
During reperfusion, hypoxanthine is converted to uric acid by xanthine oxidase, producing a massive burst of reactive oxygen species (ROS), to mitochondrial respiratory chain dysfunction, resulting in further energy depletion, membrane potential depolarisation, mitochondrial swelling, apoptosis, and necrosis.
Vascular surgery procedures often lead to IR situations, due to necessary arterial clamping. IR can therefore lead to the development of compartment syndrome and skeletal muscle damage due to apoptosis and necrosis, resulting in limb amputation in severe cases. Ischaemic conditioning strategies could provide tremendous potential in vascular surgery and might improve outcomes following IR.
Pre- and post-ischaemic conditioning, defined as brief exposure to IR performed before ischaemia or at the onset of reperfusion respectively have been mainly investigated.
However, these strategies did not fully translate into the clinical setting. Indeed, pre-conditioning is limited by the failure to predict the onset of ischaemia except in elective vascular surgical settings, while post-conditioning may further increase injury by enhancing mitochondrial dysfunction and oxidative stress.
Moreover, caution should be exercised because local conditioning strategies require repeated vascular clamping in an already calcified and diseased vascular zone.
Ischaemic per-conditioning has therefore been proposed as an alternative since the IR stimulus can be applied after the onset of ischaemia and during the surgical procedure itself. Moreover, remote conditioning can be applied at a distant site, without further impairing the vessels involved in the ischaemic zone. It consists of several IR short lasting cycles performed in a remote non-ischaemic zone during the time of sustained ischaemia of the target organ to be protected. Remote ischaemia is generally achieved non-invasively by placing a cuff around the proximal part of a limb, although any tissue can be used for the induction of ischaemia. Remote ischaemic per-conditioning (RIPerC) has demonstrated experimental and clinical protective effects in cardiac and cerebral IR.
Effect of in-hospital remote ischemic perconditioning on brain infarction growth and clinical outcomes in patients with acute ischemic stroke: the RESCUE BRAIN randomized clinical trial.
Experimental RIPerC protective effects have also been demonstrated in liver or kidney IR, but little is known about the potential protective effects of RIPerC on skeletal muscle during lower limb IR.
Therefore the aim of this study was to determine whether RIPerC might protect skeletal muscle from IR induced mitochondrial dysfunction.
Materials and methods
Animals
Twenty-three male C57BL/6 mice weighting 20 – 25 g were handled in accordance with the Helsinki Declaration for Humane Treatment of Animals during Experimentation and 3Rs Principles (Replacement, Reduction, Refinement). Mice were chosen rather than rats in order to perform future studies on knockout (KO) mice (easier to obtain than KO rats). The research protocol was approved by the ethics committee on animal research (APAFIS#22119-2019092416585166v5). Animals were housed in a thermally neutral environment of 22 ± 2°C on a 12 hour day/night cycle and were provided food and water ad libitum.
Experimental design
The number of mice included in the present study was based on studies on ischaemia reperfusion previously performed, showing that eight mice were sufficient in order to demonstrate the deleterious effects of IR damage compared with control animals.
It was hypothesised that the same number of mice would be sufficient to demonstrate the potential protective effects of RIPerC. Mice were therefore randomised into three groups: sham group (n = 7), IR group (n = 8), and RIPerC group (n = 8). Mice were placed in a hermetic cage for induction of anaesthesia and ventilated with a mixture of 4% isoflurane (CSP, Cournon, France) and oxygen. After induction, mice were placed on supine position heating blankets (Minerve Esternay, France) with a pre-selected 37°C temperature maintained during the entire procedure. Spontaneous ventilation was allowed through an oxygen delivering mask, with concentration of isoflurane maintained at 1.5%.
The sham group was maintained under anaesthesia for three hours and mice were sacrificed 24 hours later. The right hindlimb of the IR group was submitted to three hours of ischaemia induced by tourniquet applied to the root, the left hindlimb serving as a control, as previously reported.
Ischaemia was characterised by cyanosis and coldness of the limb, and confirmed by lactatemia. After three hours of ischaemia, the tourniquet was removed, tramadol (20 mg/kg) was injected intraperitoneally and mice were sacrificed after 24 hours of reperfusion.
In the RIPerC group, mice underwent the same right hindlimb ischaemia protocol for three hours. Two hours after the onset of ischaemia, RIPerC was applied on the contralateral non ischaemic left hindlimb, consisting of three cycles of 10 minutes of ischaemia followed by 10 minutes of reperfusion. The tourniquet was then removed, tramadol (20 mg/kg) was injected intraperitoneally, and mice were sacrificed after 24 hours of reperfusion (Fig. 1).
Figure 1Mice were randomly allocated to sham group, ischaemia–reperfusion (IR) group and remote ischaemic per-conditioning (RIPerC) groups and submitted to three hours of anaesthesia. In the IR and RIPerC groups, the right hindlimbs were submitted to three hours of ischaemia followed by 24 hours of reperfusion. In the RIPerC group, after 120 minutes of ischaemia, ischaemic per-conditioning was applied (three sequences of 10 minutes ischaemia followed by 10 minutes of reperfusion) on the contralateral hindlimb.
The study of mitochondrial complex activity was measured by oxygraphy through oxygen consumption in skinned muscle fibres, using a Clark type electrode (Strathkelvin Instruments, Glasgow, UK), assessing the functional oxidative capacity of the skeletal muscle (Table 1). Substrates were added in order to activate or inhibit the different complexes of the respiratory chain. Oxidative phosphorylation through Complex I (OXPHOS CI) was stimulated by the addition of adenosine diphosphate (2 mM). Amytal (0.02 mM) was added to inhibit Complex I and succinate (25 mM) was then added to activate Complex II (OXPHOS CII). The results were expressed as μmol/min/g dry weight.
Table 1Principle of each method used for the experimental study of ischaemic per-conditioning for protection of lower limb skeletal muscle ischaemia–reperfusion injury in mice
Method
Principle
Mitochondrial complex activity (oxygraphy)
Measurement of oxygen consumption in skinned muscular fibres in order to determine the functional oxidative capacity of the skeletal muscle in its cellular environment Cells are permeabilised using saponin and are provided with specific substrates to measure complex mediated respiratory activities
Calcium retention capacity (spectrofluorometry)
The mitochondrial transition pore opening and its inhibition are evaluated by determining the calcium retention capacity of mitochondria to calcium GreenTM-5N, a low affinity membrane impermeable dye that exhibits an increase in fluorescence emission intensity on binding to calcium
Reactive oxygen species (electron paramagnetic resonance)
Measurement of the production of reactive oxygen species using a molecular probe that readily reacts with ROS to produce stable nitroxide radicals. The amount of nitroxide radicals can be quantitatively measured par electron paramagnetic resonance and directly reflects the amount of ROS (through the intensity of the signal)
Calcium retention capacity represents the high cut off amount of calcium that determines the opening of the mitochondrial transition pore (mPTP), which allows the release of calcium that will in turn lead to apoptosis (Table 1). Calcium pulses (5 μM) were applied to skinned gastrocnemius muscle fibres. Each pulse was recorded as a peak of extramitochondrial calcium concentration using the fluorescent probe calcium green (1 μM; 500/530 nm). Calcium is then very rapidly taken up by the mitochondria. Abrupt increase in extramitochondrial calcium concentration indicated a massive release of calcium by mitochondria due to mPTP opening.
Detection of reactive oxygen species in muscle with electron paramagnetic resonance
Muscles were fragmented in 1 mm3 fragments and incubated with a 200 μM molecular probe (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrolidine, CMH), which was oxidised in the presence of unpaired electrons of ROS. The amount of oxidised probe was measured through the intensity of the resonance signal (Table 1).
Statistical analysis
Results were compared using one way analysis of variance followed by the Newman Keulspost test. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, USA). Results are expressed as mean ± standard error of the mean (SEM). Results with p < .050 were considered statistically significant.
Results
Effects of hindlimb ischaemia reperfusion
IR impaired mitochondrial respiration: OXPHOS CI activity in ischaemic muscles was 3.66 ± 0.98 μmol/min/g compared with 5.74 ± 0.77 μmol/min/g in contralateral non-ischaemic muscles (–36.2%, p = .009) and 7.31 ± 0. 54 μmol/min/g in sham muscles (–49.9%, p = .003) (Fig. 2A). No significant difference was noted for OXPHOS CII: 4.31 ± 0.86 μmol/min/g in ischaemic muscles, 5.26 ± 0.75 μmol/min/g in contralateral non-ischaemic muscles and 5.82 ± 0.53 μmol/min/g in sham muscles (p = .43 and p = .39 respectively, Fig. 2B).
Figure 2Effects of ischaemia–reperfusion (IR) and remote ischaemic per-conditioning (RIPerC) on mitochondrial respiration (O2 consumption) in ischaemic (dark red and blue bars) and contralateral non-ischaemic (light red and blue bars) skeletal muscle in mice indicated by (A) oxidative phosphorylation with substrates for mitochondrial complexes I (OXPHOS CI) and (B) OXPHOS CII activities presented as mean ± standard error of mean.
IR tended to impair CRC: CRC was 2.53 ± 0.32 μmol/mg in ischaemic muscles compared with 3.73 ± 0.39 μmol/mg in contralateral muscles in the IR group (–32.2%, p = .054) and 3.64 ± 0.66 μmol/mg in sham muscles (–30.5%, p = .066) but no statistical significance was reached (Fig. 3).
Figure 3Effects of ischaemia–reperfusion (IR) and remote ischaemic per-conditioning (RIPerC) on ischaemic (dark red and blue bars) and contralateral non-ischaemic (light red and blue bars) skeletal muscle calcium retention capacity in mice presented as mean ± standard error of mean.
IR did not modify ROS production in the skeletal muscle: ROS production was 0.082 ± 0.004 μmol/min/mg in ischaemic muscles compared with 0.070 ± 0.004 μmol/min/mg in contralateral non-ischaemic muscles (+17.1%, p = .065) and 0.087 ± 0.012 μmol/min/mg in sham muscles (–5.7%, p = .74) (Fig. 4).
Figure 4Effects of ischaemia–reperfusion (IR) and remote ischaemic per-conditioning (RIPerC) on reactive oxygen species (ROS) production in ischaemic (dark red and blue bars) and contralateral non-ischaemic (light red and blue bars) skeletal muscle in mice presented as mean ± standard error of mean.
RIPerC failed to restore mitochondrial respiration: OXPHOS CI activity in ischaemic muscles from RIPerC group did not differ compared with ischaemic muscles from IR group (3.82 ± 0.40 μmol/min/g vs. 3.66 ± 0.98 μmol/min/g respectively, p = .45) (Fig. 2A).
OXPHOS CI activity of non-ischaemic muscles in the RIPerC group was even impaired (–47.3%) compared with the muscles from sham group (3.85 ± 0.34 μmol/min/g vs. 7.31 ± 0. 54 μmol/min/g respectively, p = .009).
No significant difference was noted for OXPHOS CII: OXPHOS CII activity in ischaemic muscles from RIPerC group did not differ compared with ischaemic muscles from IR group (4.56 ± 0.48 μmol/min/g vs. 4.31 ± 0.86 μmol/min/g respectively, p = .54). OXPHOS CII activity in ischaemic muscles from RIPerC group did not differ compared with the sham group (4.56 ± 0.48 μmol/min/g vs. 5.82 ± 0.53 μmol/min/g (p = .23, Fig. 2B).
RIPerC had no effect on CRC: CRC in ischaemic muscles from the RIPerC group did not differ compared with ischaemic muscles from the IR group (2.76 ± 0.3 μmol/mg vs. 2.53 ± 0.32 μmol/mg respectively, p = .63) and compared with the sham group (3.64 ± 0.66 μmol/mg, p = .25) (Fig. 3).
In the RIPerC group, ROS production tended to increase in the ischaemic and in the contralateral hindlimbs compared with the sham and IR groups without reaching statistical significance (0.102 ± 0.012 and 0.101 ± 0.009 in ischaemic and contralateral hindlimbs in the RIPerC group, vs. 0.087 ± 0.012 μmol/min/mg in sham muscles) (p = .06, Fig. 4).
Discussion
The main results of this study are that the RIPerC protocol failed to restore IR induced skeletal muscle mitochondrial dysfunction and even led to mitochondrial dysfunction in the non-ischaemic contralateral hindlimb, highlighting the deleterious effects of RIPerC.
It is well described that IR induced mitochondrial dysfunction is an early marker of skeletal muscle impairment.
The results confirm these data since IR impaired mitochondrial respiration and tended to decrease CRC. Indeed, mitochondrial dysfunction was mainly characterised by a decreased oxidative phosphorylation through Complex I and remained present for 24 hours after reperfusion. This is consistent with Wang et al., who found mitochondrial membrane potential impairment after 24 hours of reperfusion.
Accordingly, in this study, the CRC modifications supported that mPTP opening tended to occur earlier after the calcium load. Similarly, in previous studies, the RNA level of the BaX/BCl2 ratio was increased and apoptosis was probably activated in association with mPTP opening after reperfusion.
The results thus confirm that mitochondrial dysfunction is a key factor in IR induced skeletal muscle impairment.
Decreasing the degree of IR injury has therefore great practical significance, especially in the vascular surgery field. Conditioning strategies have been developed, based on the idea that brief episodes of IR can provide protection from cellular injury.
It has later been described that ischaemia at a site distant to the organ to protect may confer the same cytoprotection through circulating or neural mediators released from distant organs exposed to ischaemia. Remote ischaemic conditioning strategies were therefore developed and remote pre-conditioning as well as remote post-conditioning protocols have been investigated, but these strategies could not fully translate into the clinical setting because pre-conditioning was limited by the failure to predict the onset of ischaemia except in elective vascular surgical settings, while post-conditioning was found to increase ischaemia reperfusion injury.
RIPerC was first proposed in 2007 by Schmidt et al as an easy to use procedure that could be applied in every elective and unscheduled situation whenever a greater danger of developing IR injury exists.
Intermittent peripheral tissue ischemia during coronary ischemia reduces myocardial infarction through a K-ATP dependent mechanism: first demonstration of remote ischemic perconditioning.
RIPerC has more beneficial features from a practical point of view. The essence of this strategy is that the brief, remote ischaemic cycles are applied after the induction of target organ ischaemia, but before the onset of reperfusion.
This technique could become easily feasible in clinical settings, because it could be of interest in the setting of acute ischaemia, for example embolic situations, aneurysm surgery needing arterial clamping, or vascular trauma. Accordingly, if protective, RIPerC could be the most promising protective strategy among conditioning procedures and could be applied in situations where perfusion cannot be restored within two hours. However, there is scant literature and experimental results are disparate, due to the heterogeneity of the protocols, in terms of animals, IR situations studied, outcomes measured, and the RIPerC protocol itself. For instance, Keskin et al. observed that RIPerC reduced histopathological damages in musculocutaneous flaps in rats
Local and systemic coagulation marker response to musculocutaneous flap ischemia-reperfusion injury and remote ischemic conditioning: an experimental study in a porcine model.
In the present study, RIPerC failed to protect skeletal muscle mitochondria from IR injury. It might be assumed that the RIPerC protocol used in this study was not adapted for muscle protection, as all organs do probably not need the same number or intensity of IR cycles to be protected. Moreover, duration of ischaemia, as well as duration of reperfusion might be confounding factors. Different durations may yield different results. In fact, there is a substantial heterogeneity in the current literature and no study has currently addressed the optimal site and duration of the RIPerC stimulus, or the optimal number of repetitions. Moreover, the temporal aspect of RIPerC effectiveness has not been addressed.
In this study, RIPerC even enhanced the deleterious effects of unilateral hindlimb IR by increasing the mitochondrial dysfunction in the non-ischaemic contralateral skeletal muscle. These results support the hypothesis that the RIPerC was not an optimal protocol. These deleterious effects on the contralateral non-ischaemic limb also support the hypothesis that circulating factors might be involved. These circulating factors (IR induced damage associated molecular patterns) are not totally known, but reactive oxygen species, cytokines, and complement factors associated with increased circulating leucocytes probably may play a key role.
RIPerC tended to increase ROS production, in both the ischaemic and contralateral limb. It is well described that ROS are a double edged sword: small amounts of ROS are beneficial by activating the antioxidant defence network, while excessive ROS production can be harmful.
This might participate in the RIPerC harmful effects observed in this study. The main question still remains: how is it possible to produce the specific beneficial level of ROS in order to protect the target organ? Remote ischaemic conditioning strategies may still be promising in vascular surgery and RIPerC would be the ideal strategy, because it can be applied in every elective as well as unscheduled situation, whenever a greater danger of developing IR injury exists, after the onset of ischaemia and during the surgical procedure. However, future studies are needed in order to define the optimal RIPerC protocol and the mechanisms through which protection might be exerted.
Remote ischemic perconditioning – a simple, low-risk method to decrease ischemic reperfusion injury: models, protocols and mechanistic background. A review.
The mechanisms of signal transduction from remote tissue to the target organ might be the same as those involved in ischaemic pre-conditioning (which has been studied more extensively) and might invoke neurogenic, humoral, and systemic pathways.
However, further studies are needed to confirm this hypothesis and to identify the ideal RIPerC protocol that could activate these protective pathways.
This study suffers from several limitations. First, whether the RIPerC protocol itself is the optimal RIPerC modality is unclear. One might assume that the contralateral limb was not the optimal site for remote conditioning, as well as the duration of stimulus and the number of repetitions. The temporal aspect is also un unresolved issue because the ideal time between the onset of ischaemia and the RIPerC is not defined. On the other hand, the underlying mechanisms have not been investigated.
In conclusion, per-conditioning could be the ideal conditioning protocol to translate into the clinical setting: it could be applied after the onset of ischaemia, during the surgical procedure itself. However, in the experimental study, RIPerC failed to protect skeletal muscle from IR injury and even appeared to be deleterious in the contralateral non-ischaemic muscle. Future studies are needed in order to identify a protocol that might offer protection and the underlying mechanisms.
References
Aboyans V.
Ricco J.B.
Bartelink M.E.L.
Björck M.
Brodmann M.
Cohnert T.
et al.
2017 ESC guidelines on the diagnosis and treatment of peripheral arterial diseases, in collaboration with the European Society for Vascular Surgery (ESVS).
Effect of in-hospital remote ischemic perconditioning on brain infarction growth and clinical outcomes in patients with acute ischemic stroke: the RESCUE BRAIN randomized clinical trial.
Intermittent peripheral tissue ischemia during coronary ischemia reduces myocardial infarction through a K-ATP dependent mechanism: first demonstration of remote ischemic perconditioning.
Local and systemic coagulation marker response to musculocutaneous flap ischemia-reperfusion injury and remote ischemic conditioning: an experimental study in a porcine model.
Remote ischemic perconditioning – a simple, low-risk method to decrease ischemic reperfusion injury: models, protocols and mechanistic background. A review.
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