Ischemia and reperfusion-reoxygenation (I/R) induces cellular and subcellular changes characterized by impaired tissue oxygenation, decreased intracellular oxygen levels, interstitial edema, cellular swelling, alterations in high-energy phosphate (HEP) metabolism, and mitochondrial dysfunction (1–4). The pathophysiology of I/R becomes clinically apparent in different time intervals after surgery or intervention (3–6). Microsurgical skeletal muscle transplants may be particularly vulnerable to I/R because of prolonged ischemia during reconstructive surgery (3–7). Treatment aims to save initially viable tissue before it is compromised in secondary hypoxia or ischemia by decreased tissue oxygenation and disturbed cellular metabolism (5, 8). Although the ischemic muscle tissue is a target of therapy, the pathophysiology of tissue oxygenation and oxygen metabolism is of laboratory and clinical importance (3–5). Furthermore, monitoring tissue oxygenation will provide accurate values for evaluating emerging therapies in acute I/R-associated events. In previous studies, we found that the recovery muscle energetics and viability are directly related to oxygen availability at microvascular and cellular/mitochondrial level (8, 9). Local hypothermia and pharmacological interventions have been used to attenuate the I/R damage to tissue (4, 6, 10, 11). Pretreatment strategies such as cyclosporine (cyclosporin A, CsA) may be effective during ischemia and protective in the perioperative and postoperative period when reperfusion injury processes are the greatest (4, 6, 12–17). Suppression of the mitochondrial permeability transition (MPT) with CsA, a specific inhibitor of the MPT pore, may provide protection against such I/R-related injury and irreversible tissue damage (6, 12–17).
Consequently, we studied the effects of CsA on tissue oxygenation and mitochondrial cytochrome oxidase redox (CytOx) state to test the hypothesis that the pretreatment with single-dose CsA prevents alterations and improves tissue oxygen levels (PtO2) and mitochondrial CytOx in skeletal muscle I/R.
MATERIALS AND METHODS
Animals used in this study were handled in accordance with institutional and National Institutes of Health animal care and use guidelines. All animals received human care in accordance with the Principles of Laboratory Animal Care for the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1985).
Male New Zealand white rabbits (Charles River Laboratories GmbH, Sulzfeld, Germany) were housed in a climate-controlled room (18°C–20°C, a humidity of 50%–60%, and a day-night rhythm of 12 h) and provided with standard rabbit pellets and water ad libitum. General anesthesia and surgical technique under aseptic conditions followed protocols as described previously (16,17). Briefly, in all animals, anesthesia was induced with intramuscular injection of ketamine (Ketanest, 50 mg kg/body weight) and xylazine (Rompun, 5 mg/kg body weight). After orotracheal intubation (Portex tube; Smiths Medical International, Keene, NH), mechanical ventilation providing the inspiratory oxygen fraction (FIO2) (Servo 900; Siemens, Bromma, Sweden) was performed. Refinements in mechanical ventilation were designed to preserve a pH of 7.35 to 7.45 and normocarbia (arterial carbon dioxide partial pressure [PaCO2] between 38 and 45 mmHg) using continuous end-tidal CO2 monitoring and arterial blood gas analysis. Pulse oximeter was used to monitor the heart rate and the level of oxygenation. After the placement of a central venous line, anesthesia was maintained by continuous intravenous infusion of fentanyl (100 μg · kg−1 · h−1) and midazolam (0.2 mg · kg−1 · h−1). A femoral artery catheter was placed for monitoring systemic arterial blood pressure and sampling blood for blood gas analysis. The animals were maintained at normothermia (38°C–39°C) using an air warming system (WarmTouch; Tyco Healthcare UK Ltd., Gosport, UK), and warmed physiologic saline was given at 5 mL · kg−1 · h−1 intravenously to replace insensible fluid loss.
The rabbits (n = 20, 2.5 ± 0.5 kg) were divided into two groups. The animals were randomized to receive a 60-mg/kg intravenous single bolus of CsA (cyclosporin A, Sandimmun; Novartis Pharma GmbH, Nuernberg, Germany) (CsA group, n = 10) or physiologic saline (control group, n = 10) at 10 min before ischemia onset. After preparation of the vascular pedicle, ischemia was induced by clamping of the thoracodorsal artery and vein for 4 h, followed by 2 h of reperfusion. A small tissue oxygen (PtO2) microcatheter probe (CC1, Licox; Integra Neurosciences, Andover, UK) was inserted in the muscle tissue (distal part) and used to measure tissue oxygen simultaneously with mitochondrial CytOx state by noninvasive optical near-infrared (NIR) spectroscopic measurement (Fig. 1). During each experimental period, venous and arterial blood samples were obtained for blood gases and hemoglobin saturation to achieve stable experimental conditions.
A circumferential skin incision was made to expose the latissimus dorsi (LD) muscle. After dissection of the myocutaneous LD muscle flap, the dominant vascular pedicle was prepared, the muscle mobilized, and the thoracodorsal nerve was transected. The exposed LD muscle was reimplanted to the site using standard microsurgical techniques, and the overlying skin was closed with Prolene sutures. A small opening access was used for clamping of the vascular pedicle to induce muscle ischemia.
Tissue oxygen (PO2) measurement
After surgical preparation and dissection, a small and highly flexible oxygen microcatheter probe (CC1, 500-μm diameter, Licox; Integra Neurosciences) was inserted into the muscle tissue (distal part of the LD muscle) using a guide cannula and connected to a digital monitor (Licox CMP; Integra Neurosciences). The microcatheter sensor utilizes the Clark-type polarographic electrode technique, providing a real-time quantitative local oxygen tension (PtO2) measurement in the muscle tissue. An integrated temperature sensor compensates the values for regional muscle tissue temperature, providing an accurate assessment of oxygen levels.
NIR optical spectroscopy
Mitochondrial cytochrome oxidase redox state was measured with a research-based modified NIR spectroscopy system (Critikon; Johnson and Johnson Medical, Berks, UK). The used miniaturized sensor consists of a laser emitter, two light detectors, and a light-emitting diode for compensating differences in coupling. A full spectral charge-coupled device spectrometer system (wavelength range λ = 720–920 nm) was used. The changes in chromospheres concentration of mitochondrial CytOx were converted to units of concentration (i.e., μmol/L of tissue). The noninvasive NIR spectroscopic probe was positioned adjacent to the tissue oxygen microcatheter sensor on the surface of the prepared LD muscle (Fig. 1) and fixed by sutures. The differential path length has been determined specifically for the rabbit LD muscle and was used in all animals for quantitative measurements. All PtO2 and CytOx data were measured continuously with 5-s resolution and averaged in 2-min blocks, processed, and stored on a computer system for subsequent analysis.
31P magnetic resonance spectroscopy
Data were acquired at high-field Bruker magnetic resonance spectrometer (4.7 T, BioSpec; Bruker BioSpin GmbH, Rheinstetten, Germany) using a surface coil (10/18 mm) placed directly on the muscle adjacent to the NIR optical probe. Shimming for optimizing magnetic field homogeneity and pulse power adjusting for deep tissue acquisition of 31P spectra were performed. The positioning of the coil over the muscle was confirmed by T1-weighed 1H images in the axial plane. The pulse width that optimized the phosphocreatine (PCr) signal was chosen for each study. Spectra (repetition time = 5 s, four averages) were measured at baseline, before ischemia, end of ischemia, and end of reperfusion. All spectra were computed and analyzed using a peak fitting routine with least-squares program. The calculated levels of PCr and adenosine triphosphate (ATP) determined from the spectra peak intensity were normalized to 100% by using as reference the average value obtained during the baseline measurements.
The extent of skeletal muscle edema was measured in tissue samples by the wet-to-dry weight technique. Immediately after harvesting, muscle tissue specimens were blotted, weighed, and placed in a drying oven at 60°C until a constant weight was obtained. Samples were then weighed again, and wet-to-dry weight ratio was estimated.
Mitochondrial viability index
Mitochondrial viability was assessed by the reduction of tetrazolium salt to water-insoluble colored formazan crystals by electron carriers and oxidative enzymes in the mitochondria of the tissue specimens. At the end of the experiment, the LD muscles were harvested, and the tissue was cut into pieces to increase surface area and tissue uptake of the tetrazolium salt. Each piece was weighed and placed in a small tube supplemented with 300 μL of 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (Sigma, St Louis, Mo). The samples were then incubated for 3 h at 37°C in the dark on a rotating mixer. Subsequently, they were removed, washed with distilled water, and blotted dry. The water-insoluble formazan salt was extracted in 3 mL of 2-propanol for 6 h at 37°C in the dark on a rotating mixer. Two-hundred-microliter aliquots were removed, and the absorbance was determined at 570 nm using a microplate reader. The tissue samples were then dried at 90°C for 24 h. The mitochondrial viability index was expressed as percentage of OD570 relative to dry tissue weight in the muscle (percentage viability index of fresh and nonischemic sample considered as 100% viability).
The contralateral LD muscle served as nonischemic control and was also instrumented except that no ischemia was applied. After finishing the experimental protocol, the animals were killed with an overdose sodium pentobarbital (200 mg/kg intravenously administered) under anesthesia.
Data were analyzed statistically using the SigmaStat (version 2.03) statistical software (SPSS, Inc, San Rafael, Calif). The calculated sample size to achieve sufficient power was n = 7 for each group. The following tests were carried out: Kolmogorov-Smirnov to assess the normality of variable distribution, descriptive analysis of data with values reported as means ± SD, repeated two-way analysis of variance for comparisons between baseline and post–ischemia-reperfusion values, one-way analysis of variance for comparison of groups at baseline, and χ2 and Goodman tests to compare proportions. Percent changes (Δ) of all variables were calculated. The Pearson or Spearman coefficient of correlation was used to assess correlation between values. P < 0.05 was considered significant.
There were no significant differences among the two groups in baseline hemodynamic and blood gas data (Table 1). Mean arterial pressure and tissue PtO2 tended to increase after intravenous application of CsA in the CsA group, but were not significantly changed.
Baseline values of tissue PtO2 were comparable between the groups and show no significant difference (Fig. 2). After vascular clamping and induction of ischemia, PtO2 declined rapidly in the first 10 min, and then the decrease slowed, and reached their lowest levels at end of ischemia (Fig. 2). The tissue PtO2 values in the CsA group tended to be higher than the corresponding values in the control group during ischemia and were significantly different at 5 and 15 min of ischemia (Fig. 2). During ischemia, tissue PtO2 decreased precipitously followed by a decrease in mitochondrial CytOx in all LD muscles (Figs. 2 and 3). After ischemia, tissue PtO2 recovered with an overshoot and remained significantly low in the control group (Fig. 2). In the CsA group, the tissue PtO2 levels reach nearly baseline after 1 h of reperfusion (Fig. 2). During reperfusion and reoxygenation, tissue PtO2 values were significantly different in both groups (P < 0.05; Fig. 2). At the end of reperfusion, recovery of tissue PtO2 was significantly improved in the CsA group compared with the control group (CsA group: 31.5 ± 4.6 mmHg vs. control group: 13.9 ± 3.1 mmHg; P < 0.05) (Fig. 2).
During ischemia, the decline of mitochondrial CytOx and the recovery during reperfusion were significantly different in the CsA group versus control (P < 0.05; Fig. 3). A significant difference in mitochondrial CytOx values between the two groups was found, and the difference progressively increased during last time interval of ischemia and during reperfusion (P < 0.01; Fig. 3). During reperfusion and reoxygenation, the recovery of mitochondrial CytOx in the CsA group was very rapid with evident two-step phases (Fig. 3). Near-baseline CytOx values were reestablished at 60 min of reperfusion (Fig. 3). In contrast, the recovery of mitochondrial CytOx during reperfusion was much lower and significantly different (P < 0.01) in the control group compared with the CsA group (Fig. 3). The mitochondrial CytOx values at the end of reperfusion were significantly different in the groups (CsA group: +0.2 ± 0.04 μmol/L compared with the control group: −1.7 ± 0.08 μmol/L, P < 0.01; Fig. 3).
There were no significant differences in the mean PCr or ATP between the control group and the CsA group before ischemia (Figs. 4 and 5). Ischemia caused a decline in HEPs to levels that were higher in the CsA group than in the control group at the end of ischemia period (Figs. 4 and 5). Muscle PCr was significantly better preserved during ischemia in the CsA group compared with the control group (P < 0.01; Fig. 4). The ATP levels were also significantly higher at the end of ischemia (P < 0.05; Fig. 5). By the end of postischemic reperfusion, the mean percent PCr (P < 0.01) and ATP (P < 0.05) were significantly improved in the CsA group than in controls (Figs. 4 and 5).
Comparison of the mitochondrial viability index showed significant differences between the CsA group and the control group (76.9% ± 6.8% vs. 51.4% ± 5.6%, P < 0.01) (Fig. 6). Mitochondrial viability index results confirmed better mitochondrial preservation in the CsA group (Fig. 6), and the values correlated with tissue oxygen levels (PtO2) (r = 0.85) and mitochondrial CytOx (r = 0.92).
Muscle ischemia followed by reperfusion resulted in tissue edema as indicated by a significantly increased wet-to-dry weight ratio (control group: 5.6 ± 0.5 vs. nonischemic control: 3.26 ± 0.2, P < 0.01) (Fig. 7). After ischemia and 2-h reperfusion, the CsA group developed decreased edema with significantly different wet-to-dry weight ratio compared with the control group (P < 0.05) (Fig. 7).
Light microscopy confirmed better morphological preservation in the CsA group (Fig. 8) and revealed marked morphological changes, necrosis, and edema in the muscle specimen of the control group animals.
After ischemia-reperfusion, recovery of tissue oxygenation and cellular/mitochondrial oxygen metabolism are key determinants for oxidative processes to generate HEP (PCr, ATP) and determine tissue vitality (1–4). In this study, the tissue PtO2 levels were significantly higher after I/R in the CsA group in comparison to controls (Fig. 2). The tissue oxygen and mitochondrial CytOx data are consistent with the findings in the mitochondrial viability index and tissue edema. Pretreatment with a single CsA bolus markedly improved microvascular tissue oxygenation and intracellular/mitochondrial oxidative processes in line with the recovery of HEP (PCr and ATP levels).
Oxidative ATP synthesis is a function of three factors: mitochondrial morphology and function, oxygen supply, and the metabolism. We found that tissue reoxygenation is significantly improved in the CsA group compared with control group and may result from excessive oxygen demand or inadequate microvascular tissue perfusion during reperfusion (Fig. 2). It has been reported that significant vasoconstriction and vasospasm in arterioles (particularly in the terminal arterioles) occur after I/R (8, 18, 19). Cellular oxygen delivery in the postischemic tissue could be further compromised by alterations to oxygen diffusion (due to changes in arteriole diameter, interstitial edema formation, perivascular edema, capillary recruitment, and endothelial swelling), which can critically decrease the oxygen diffusion distance and reduce oxygen flux, determined by the rate of mitochondrial respiration (5). The effects of I/R on tissue PtO2 have been documented in experimental and clinical settings (5, 8, 9, 20–22). We obtained tissue PtO2 values during baseline that are comparable to in vivo mitochondrial oxygen tension values reported as ∼30 to 40 mmHg (22).
The measurement of mitochondrial CytOx using optimized NIR spectroscopy provides in situ measurement of mitochondrial oxidative capacity (24, 25). The time course of CytOx response during I/R in the control group was significantly different from that with CsA pretreatment (Fig. 3). These findings favor the protective effects of CsA and related alterations of MPT on mitochondrial function by conditioned suppression during ischemia and accelerated recovery during reperfusion-reoxygenation (26–30). Mitochondrial swelling is a well-documented ultrastructural change occurring after I/R and may be the response for uncoupling or inhibition of oxidative phosphorylation (31, 32). A subcritical decreased intracellular energy state promotes necrosis (5, 7, 28). Cyclosporine pretreatment blocks MPT pore opening and may prevent the energy-depleting effects of I/R injury with preservation of oxidative phosphorylation and ATP synthesis (12–17, 26–30, 33). Inhibition of MPT preserves the hydrogen ion gradient required for oxidative phosphorylation and ATP synthesis and mitochondrial morphology and function (12–17, 26–30, 33). Furthermore, increased ATP availability may also improve cellular calcium handling and mitochondrial performance (26, 28).
Protective effects on mitochondria and tissue including were observed in experimental investigations with different CsA doses (11–17, 28, 33). The reported dose of CsA in a previous rabbit myocardial I/R study was 25 mg/kg (16) and 50 mg/kg in a brain damage model (12). We used the single higher-dose bolus to achieve maximal load on the ischemic tissue with an effective dose of CsA.
Cyclosporine as a strong immunosuppressive drug is widely used clinically, and its adverse effects are well known (34, 35). Extensive research concerning the long-time use of CsA in organ transplantation revealed some adverse effects, which may include hypertension, reduction of kidney and liver function, paresthesia, tremor, gum hypertrophy or bleeding gums, increased risk of bacterial, and fungal or viral infections (34, 35). There are very few experiences about the adverse effects in the single-dose CsA treatment (11–17, 28, 33). Further investigations are needed to determine the optimal timing of application, routes (systemic or local administration), dose regimen, and common adverse effects.
We can only speculate about the significance of the results of this study and their practical clinical implications. Restoration of blood flow and reoxygenation of the tissue are the main steps to prevent or slow down ischemic injury processes. However, reperfusion can cause further impairment and may exacerbate ischemia-related injury, especially in longer periods of transient ischemia. Under certain circumstances, however, ischemia and reperfusion probably cause persistent tissue injury. The possibility of a pharmacological preprocedural and/or periprocedural protective intervention to attenuate the effects of I/R may be beneficial in different medical fields (transplantation, cardiac and vascular surgery, invasive cardiology, invasive neurology, neurosurgery, reconstructive surgery, vascular medicine, intensive care medicine, trauma) and is supported by recent investigations, particularly in the treatment of myocardial ischemia-reperfusion (6, 13, 14, 16, 36).
The results from this study support our hypothesis that single bolus CsA pretreatment prevents alterations and improves tissue oxygenation and mitochondrial oxidation in skeletal muscle I/R.
CsA — cyclosporine
CytOx — cytochrome oxidase redox
I/R — ischemia and reperfusion-reoxygenation
LD — latissimus dorsi
HEP — high-energy phosphates
Pto2 — oxygen tension
PCr — phosphocreatine
ATP — adenosine triphosphate
MPT — mitochondrial permeability transition
NIR — near-infrared
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Latissimus dorsi muscle; mitochondrial oxidation; oxygen tension; cyclosporine; ischemia-reperfusion; near-infrared spectroscopy; rabbits