Wound healing requires that wound margins be sufficiently supplied with blood and oxygen. These margins may be at risk after the interruption of their anatomical vascularization resulting from surgery or trauma. This condition may deteriorate further in the course of critical illness. If adequate microcirculation and tissue oxygenation are not maintained, necrosis may occur, thus causing significant morbidity.
A new strategy to protect jeopardized tissues from ischemic and hypoxic damage consists in their preconditioning through exposure to a sublethal degree of environmental stress (1,2). The protective effect of preconditioning is a biphasic phenomenon, with an early phase of 2–3 h and a more efficient and long-lasting late phase that becomes apparent 12–24 h after stress induction (3,4). Preconditioning can be induced by a variety of physical and pharmacological stimuli, the latter being easier to apply (3). Among those, monophosphoryl lipid A (MPL) has gained increasing interest.
MPL is a chemically modified, nontoxic synthetic derivative of endotoxin (5). It has demonstrated a multifaceted therapeutic potential as a vaccine adjuvant (6), an immunomodulator (7), and an inducer of endotoxin hyporesponsiveness (8,9). In addition, it has been successful in protecting the skeletal muscle (10) and myocardium (11–13) against ischemia/reperfusion injury. MPL was well tolerated in healthy volunteers, which emphasizes its possible clinical application (8).
The aim of this study was to test if ischemia-related necrosis of cutaneous wound margins may be reduced in large animals through preconditioning with MPL. Furthermore, we wanted to examine the extent to which circulation and oxygenation are affected in the jeopardized tissue and on a systemic level. For this purpose we chose a porcine flap model, which has been proven useful for investigating these end-points in a variety of previous studies (14,15).
This study was performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the local Animal Ethics Committee of the Canton of Berne, Switzerland. Twenty large white pigs weighing 25–35 kg were included. The number of animals was set according to a statistical power analysis based on previous results with the same model (14,15). The animals were randomly assigned to either the control group (n = 10) or the experimental group (n = 10) receiving MPL. Randomization was performed by the breeder based on the identification number of the animal. The allocation to the groups was blinded for the investigators. All the animals were checked by a certified veterinarian for the presence of diseases or abnormal behavior preoperatively and postoperatively.
Preconditioning was performed 24 h before flap dissection. MPL lyophilisate (Acila AG, Grenchen, Switzerland) was added to 10 mL distilled water, and a homogenous suspension was achieved with the use of ultrasound. The solution was injected into an ear vein at a dosage of 35 μg/kg body weight. The control group received an equal amount of isotonic saline.
General anesthesia was induced with 10 mg/kg body weight ketamine (Narketan®; Parke-Davis, Baar, Switzerland) given IM, followed 10 min later by 0.5 mg/kg body weight midazolam (Dormicum®; Roche, Basel, Switzerland) and 0.05 mg/kg body weight IV atropine (Atropine; Inselspital, Berne, Switzerland) for tracheal intubation. Anesthesia was maintained with isoflurane (Foren®; Abbott, Baar, Switzerland) and 79% nitrous oxide in oxygen. The end-tidal isoflurane concentrations were kept between 0.9% and 1.5% and titrated to achieve stable hemodynamic conditions. The animals were ventilated with a volume-controlled ventilator with a positive end-expiratory pressure of 4–5 cm H2O (Tiberius 19; Drägerwerk AG, Lübeck, Germany). Tidal volume was kept at approximately 10 mL/kg body weight and respiratory rate (13–18 breaths/min) was adjusted to maintain Paco2 constant within a range between 35 and 45 mm Hg. Expired minute volume, tidal volume, respiratory rate, end-expiratory peak and inspiratory pressures, inspired and expired carbon dioxide concentrations, inspired and expired oxygen concentrations, and inspired and end-tidal isoflurane concentrations were monitored continuously throughout the experiment (Hellige SMU 611; Hellige AG, Freiburg, Germany). Ringer’s lactate solution was administered IV at a rate of 10–15 mL · kg−1 · h−1 during surgery and 5 mL · kg−1 · h−1 thereafter. The body and flap temperatures were kept constant with a warming mattress and patient air-warming system (WarmTouch 5700; Mallinckrodt, Hennef, Germany), which was controlled with temperature probes.
Surgery was performed by the first author throughout the series, beginning with the pig in a supine position. A pulmonary artery catheter (thermodilution catheter; Arrow, Reading, PA) was inserted via the right internal jugular vein, and the left common carotid artery was cannulated with a large-bore catheter. The animal was then turned to a prone position, and a rectangular, cranially-based skin flap measuring 14 × 5.5 cm was outlined on each side of the buttocks. This flap design has been shown to produce necrosis of approximately 50% of the flap surface (14). Laser Doppler probes and polarographic microprobes were fixed in each designated flap area. After baseline measurements were taken, the flaps were raised in the epifascial layer and sutured back to their original place.
Mean arterial blood pressure, central venous pressure, mean pulmonary artery pressure, and pulmonary capillary wedge pressure were assessed with a quartz pressure transducer (129A; Hewlett-Packard, Andover, MA), displayed continuously on a multiple-modular monitor (Hellige SMU 611) and recorded (Hellige SMR 821). Heart rate was obtained from the electrocardiograph. Cardiac output was measured by the thermodilution technique and averaged for three consecutive measurements (cardiac output module, Hellige SMU 611) to calculate cardiac index. Central venous blood temperature (°C) was recorded from the thermistor in the pulmonary artery catheter.
Samples for blood gas and lactate analysis were drawn from the carotid artery and jugular vein and analyzed immediately in a temperature-corrected manner (ABL 620; Radiometer, Copenhagen, Denmark). Arterial and venous hemoglobin concentrations and oxygen saturations were measured with an analyzer designed for porcine blood (OSM 3; Radiometer).
Microcirculatory blood flow was measured continuously with a multichannel laser Doppler flowmeter (LDF) system (The Oxford Array; Oxford Optronix Ltd., Oxford, UK). The miniature surface probes (SP 300; Oxford Optronix Ltd.) measured microcirculatory blood flow to a depth of 1 to 2 mm. The laser Doppler unit and probes were calibrated according to the guidelines of the manufacturer. Because of the high intersite variability (15,16), the data are given in percentages of baseline. The probes were sutured at a distance of 2.5 (proximal) and 7.5 cm (distal) from the base of the flap on each side of the buttocks.
Partial tissue oxygen tension was assessed with polarographic microprobes (Revoxode CC1; GMS GmbH, Kiel, Germany) as previously described (15). The probes were inserted subdermally in the vicinity of the LDF probes.
The data for both LDF and tissue oxygen tension were acquired online via a multichannel interface (Mac Paq MP 100; Biopac Systems Inc., Goleta, CA) with acquisition-analysis software (Acqknowledge 3.0; Biopac Systems Inc.) connected to a portable computer (MacIntosh Power Book; Apple Computer Inc., Cupertino, CA). The values represent averages taken over an observation period of 5 min.
The viability of the tissue was examined visually on both the outer and inner surface of the flap (14). Tissue necrosis was characterized by a black or dark brown epidermal surface, a loss of elasticity, and a lack of discernible vasculature on the raw surface. The distribution of tissue necrosis was outlined on a transparent template to assess the percentage of the total flap surface.
Catheterization, probe application, baseline measurements, and flap dissection took approximately 3 h. Before the first postoperative measurements, the animals were left to stabilize for another hour. After completion of the experiment, absorbant adhesive dressings were fixed on all surgical wounds and a transdermal therapeutic system (Durogesic-TTS 50 μg/h; Janssen-Cilag) was applied on the interscapular skin of the animals for postoperative analgesia (17). Thereafter, the animals were tracheally extubated and brought to the veterinary clinic for postoperative care. Throughout the postoperative period, the animals were examined daily for local complications (infection, wound dehiscence, hematoma, seroma), and the dressings were changed if necessary. If any complication occurred or if systemic diseases or abnormal behavior were observed, the animals were immediately anesthetized, euthanized, and excluded from the study. On postoperative day 14, the animals were reanesthetized according to the above descriptions and flap survival was assessed. Thereafter, the animals were euthanized with an IV injection of potassium chloride.
Statistical analysis was performed with the SPSS software package (SPSS™ version 11.0.0; Statistical Package for Social Sciences Inc., Chicago, IL). All data are given as mean ± sd. Differences between groups were assessed by analysis of variance for repeated measurements, using one dependent variable, one grouping factor (control, MPL), and one within-subject factor (time). Differences in flap necrosis between the groups were assessed by Student’s t-test. P < 0.05 was taken to represent statistical significance.
One animal in the control group developed a core temperature exceeding 40°C, which was interpreted as a potential sign of porcine stress syndrome. Consequently, the animal was excluded immediately. The total amount of administered fluid was 2422 ± 151 mL in the control group and 2326 ± 162 mL in the MPL group. All the flaps healed without infection or any other wound healing complications except the expected partial necrosis.
The respiratory variables used to monitor anesthesia are summarized in Table 1. Respiratory rate and inspired end-tidal isoflurane concentrations were gradually increased over time in both groups (P < 0.01). The preconditioned animals received inspired oxygen tensions that were maximally 2.6 mm Hg higher than those given to the control animals (P < 0.05).
During the postoperative observation period, heart rate gradually increased in both groups (P < 0.01; Table 2). The animals receiving MPL showed lower mean arterial blood pressure (not significant), central venous pressure (P < 0.01), and systemic vascular resistance (P < 0.01) than the control group, whereas their cardiac index (P < 0.05) increased. Core temperature gradually increased in both groups; however, it increased to a larger extent in the control animals (P < 0.01 versus baseline for both groups, P < 0.05 between groups).
Although the preconditioned animals revealed higher arterial oxygen tension and oxygenation index (both P < 0.01), arterial oxygen saturation was similar in both groups (Table 3). Lactate concentration gradually increased from 1.02 mmol/L to approximately 1.45 mmol/L in both groups (P < 0.05). However, unlike in the preconditioned animals, pH decreased in the control group over time (P < 0.01). The preconditioned animals showed an increase in oxygen delivery (P < 0.05) and, to a lesser extent, in oxygen consumption (not significant), whereas oxygen extraction ratio was similar in both groups.
Baseline values for LDF ranged from 16 to 54 perfusion units. The values were similar in both groups and all locations. Flap dissection resulted in a decrease in microcirculatory blood flow in the proximal flap skin to 19% ± 12% (n = 10) of the preoperative value in the control group but only to 49% ± 30% (n = 5) in the preconditioned animals (P < 0.01 versus baseline, P < 0.05 between groups) (Fig. 1). In the distal portions of the flap, the flow was similarly reduced to values between 13% and 19% in both groups.
Partial tissue oxygen tension ranged from 41 ± 14 mm Hg to 43 ± 13 mm Hg at baseline. In the proximal flap skin, oxygen tension decreased to values between 2 mm Hg and 2.5 mm Hg in both groups (P < 0.01 versus baseline, not significant between groups) (Fig. 2), whereas oxygen tension in the distal portion of the flap was 0.5 ± 0.5 mm Hg in the control group and 1.3 ± 1.0 mm Hg after preconditioning (P < 0.05 between groups).
Preconditioning diminished flap necrosis from 44% ± 9% of the flap surface (control group) to 35% ± 9% (P < 0.05) (Fig. 3).
The principal finding of this study was a reduction of ischemia-related flap tissue necrosis after preconditioning with MPL, which was paralleled by an improvement in both local and systemic circulation and oxygenation variables.
Preconditioning with MPL caused an increase in cardiac index, which resulted in enhanced systemic oxygen delivery. The benefit to cardiac index was achieved by peripheral vasodilation and improved cardiac performance, as suggested by the decreases in peripheral vascular resistance and central venous pressure. A supportive effect of MPL on systemic hemodynamics has been reported for both swine (18) and rats (19) exposed to endotoxemia. The cardiovascular protection may be attributed to improved response of cardiomyocytes (12,13) and endothelial cells (11) to external stress, as repeatedly documented in hearts subjected to ischemia-reperfusion injury. However, no such effect has been described in the context of prolonged anesthesia. Although isoflurane is considered cardioprotective, it impairs cardiac output at end-tidal isoflurane concentrations larger than 1% in healthy pigs (20,21), which corresponds to the dosages used in the present study. Therefore, our results suggest that MPL may exert a protective effect on the cardiovascular system against volatile anesthetics during prolonged anesthesia.
The initial purpose of this study was to investigate the hypothesized therapeutic benefit of preconditioning with MPL on survival of ischemic flap tissue. Similar to improvement in the systemic variables, improved circulation and oxygenation were also found in the flap tissue. However, there were some discrepancies between the behavior of microcirculation results and tissue oxygen tension in the distal part of the flap, which is where the demarcation of tissue necrosis takes place. In this area, enhanced tissue oxygen tension was obtained in the preconditioned animals, suggesting improved oxygen delivery, whereas LDF was similar in both groups. The most likely explanation is that during the observation period the microcirculation in this area approached the biological zero, a well known phenomenon that is determined by an apparent laser Doppler signal despite absence of flow (16). The improved oxygen tension found in the flap tissue of preconditioned animals may also be related to the markedly higher oxygen tension obtained at the arterial level, which was primarily achieved by an increased oxygenation index, thus suggesting superior alveolar oxygen uptake and, to a much lesser extent, by the inspired oxygen tension that happened to be kept slightly higher in the MPL animals. However, the marked differences in arterial oxygen tension did not translate into arterial oxygen saturation, which was virtually equal in both groups, and which is the factor that determines oxygen delivery.
Both the acute and long-term effects of MPL observed in the current study may be related to an up-regulation of inducible nitric oxide synthase (iNOS), which is the primary mechanism of late preconditioning with MPL (3,12,13). Induction of iNOS results in the production of large amounts of NO in the cardiovascular system for several days, which may protect the system from environmental stress. Besides its hemodynamic effect, increased MPL-induced NOS attenuates neutrophil infiltration (13) and platelet aggregation (22). Activated iNOS has also been reported to enhance vascular endothelial growth factor levels, thus inducing neovascularization (23). All these factors may contribute to diminish ischemia-related flap necrosis. Accordingly, the survival of ischemic skin flaps was reduced by selective iNOS inhibitors and in iNOS knockout mice (23), whereas increased viability was obtained after exogenous administration of the substrate of NOS, l-arginine (24,25)
In conclusion, our results suggest that the development of necrosis in critically ischemic skin flaps may be reduced by preconditioning the organism with a single dose of MPL the day before surgery. The effect may be related to an improvement of the compromised microcirculation and tissue oxygenation. In addition, preconditioning with MPL may stabilize the systemic hemodynamics during prolonged anesthesia with volatile anesthetics.
The authors thank Daniel Mettler, Vet. MD, and Olgica Beslac, veterinary nurse, for technical assistance during surgery (Surgical Research Unit, Inselspital, University Hospital, CH-3010 Berne, Switzerland).
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