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CRITICAL CARE AND TRAUMA

The Influence of Trauma and Ischemia on Carbohydrate Metabolites Monitored in Hamster Flap Tissue

Claudio Contaldo, MD^*, Jan Plock, MD^*, Valentin Djonov, MD, Michael Leunig, MD^*, Andrej Banic, MD, PhD^*, and Dominique Erni, MD^*

*Department of Orthopedic, Plastic and Hand Surgery, Inselspital, University Hospital, CH-3010 Berne, Switzerland; Institute of Anatomy, University of Berne, CH-3011 Berne, Switzerland

Address correspondence to Dominique Erni, MD, Department of Plastic and Reconstructive Surgery, Inselspital, University Hospital, CH-3010 Berne, Switzerland. Address e-mail to dominique.erni{at}insel.ch.

	Abstract

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To monitor hypoperfusion of the peripheral tissues in critical illness caused by injury, we measured the concentrations of glucose, pyruvate, and lactate in traumatized and ischemic hamster flap tissue with the use of microdialysis. The interruption of the anatomic blood supply led to a drastic decrease in microvascular blood flow (laser Doppler flowmetry) and partial tissue oxygen tension (dye fluorescence quenching technique) in the ischemic part of the flap (both P < 0.01). In the traumatized area, blood flow, oxygen tension, and pyruvate were similar to the healthy control tissue throughout the experiments, whereas pyruvate was reduced in the ischemic tissue (P < 0.05 versus baseline and other tissues). Lactate was increased in both parts of the flap (P < 0.01 versus baseline and other groups for ischemic, not significant for traumatized). The sensitivity to detect ischemic hypoxia was 62% for lactate and 93% for lactate/pyruvate ratio (L/P) (P < 0.01). The specificity to discern ischemia-related from trauma-related changes was 71% for lactate and 70% for L/P (not significant). Our results suggest that L/P is more accurate than lactate for monitoring ischemia-related hypoxia after trauma. However, the rate of increased values originating from normally perfused but traumatized tissue was high for both markers.

	Introduction

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Critical illness is characterized by a hypoperfusion of various organs, which may render these tissues hypoxic, thus inducing the production of lactate as the terminal metabolite of anaerobic glycolysis. This scenario may contribute to the development of multiple organ failure (1,2), respiratory complications (2), and subsequent mortality (2,3). Accordingly, it has repeatedly been demonstrated that duration and severity of lactic acidosis correlate well with the outcome of various kinds of critical illness (2–5), and the correction of lactic acidosis has been considered as a main target in the treatment of critically ill patients.

Conversely, the reliability of serum lactate concentration as a marker of ischemic tissue hypoxia, and therefore as a predictor for survival of critically ill patients, may be questioned because enhanced lactate production may also be caused by mechanisms unrelated to tissue-oxygen debt, such as inflammation with (6–8) or without (6,9) infection, and overstimulation of the sympathicoadrenergic drive (10). In such cases, attempts at correction of lactic acidosis may lead to unnecessary use of blood transfusions or inotropic drugs in an effort to improve oxygen supply to tissues that are erroneously supposed to suffer from insufficient oxygen delivery.

As injury is one of the most frequent causes of critical illness, we tested in an experimental model the extent to which the production of lactate in ischemic, hypoxic tissue is influenced by the presence of trauma and, if in such a condition, other markers indicate ischemic hypoxia more accurately. To this end, the previously described hamster skin flap preparation (11–14) was used, which allowed for direct and quantitative measurement of microhemodynamic variables, partial tissue oxygen tension, and the tissue concentrations of carbohydrate energy metabolites simultaneously.

	Methods

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Experiments were performed according to the National Institutes of Health guidelines for the care and use of laboratory animals and with the approval of the local Animal Ethics Committee. Twenty male Syrian golden hamsters (Charles River Laboratories, Sulzfeld, Germany) weighing 78–106 g were included in this study.

A hamster skin flap model was used as previously described in detail (11,13). Anesthesia was induced by pentobarbital injected intraperitoneally (100 mg per kilogram body weight; Nembutal; Abbott Laboratories, Chicago, IL). The carotid artery was cannulated for monitoring arterial blood pressure and for administration of fluid and anesthetics. Catheterization and flap dissection were performed with the aid of an operating microscope at x10 magnification (Wild, Heerbrugg, Switzerland). An island flap measuring 3 x 2 cm was dissected from the shaved and epilated back skin and the entire most superficial layer of the panniculus carnosus muscle was meticulously removed to improve visualization of the microcirculation, thus inducing surgical trauma to this delicate tissue. The flap is perfused by one vascular axis, which bifurcates into two equal-sized branches that nourish two separate vascular territories that are connected to each other by an arterial and venous collateral network. After surgery, the animal was placed on a specially designed Plexiglas stage, and the flap was stretched to its original size with stay sutures.

Investigations were performed using an intravital microscope (Axioplan 1; Zeiss, Jena, Germany). Microscopic images were captured by a television camera (Intensified CCD camera; Kappa Messtechnik GmbH, Gleichen, Germany), recorded on video (50 Hz; Panasonic, Osaka, Japan) and displayed on a television screen (Trinitron PVM-1454QM; Sony, Tokyo, Japan). The preparation was observed visually with a x40 objective, resulting in a total optical magnification of x909 on the video monitor. The arterioles were classified according to physiologic and anatomic features into A1 to A4. Microvascular diameter was measured off-line. For the A1 arterioles, this required transillumination with a green filter to obtain a well-defined image of the width of the erythrocyte column.

We used combined bare fiber probes (Oxy Lite probes; Oxford Optronix, Oxford, UK) to continuously measure tissue oxygen tension, temperature, and microvascular blood flow. Microvascular blood flow was measured with 2 230-µm fibers. The sensitive tip of the oxygen probe (100 µm diameter) consisted of Ruthenium-III-(tris)-chloride, which measured Po₂ by fluorescence quenching of the dye. A T-type thermocouple was attached to the probe, which was coated with a biocompatible sleeve of polyurethane. According to the manufacturer, the bare fiber probe provides resolutions of 0.1 mm Hg and 0.1°C for partial oxygen tension and temperature, respectively. The data on blood flow were displayed in arbitrary perfusion units and further processed into percentages of the baseline.

The interstitial concentrations of glucose, pyruvate, and lactate were assessed by microdialysis as previously described (15,16). The system used in our study included microprobes (CMA/20; CMA Microdialysis AB, Stockholm, Sweden) carrying a microcapillary that was perfused by a microinjection pump (CMA/100). The molecular cut-off of the membrane was 20,000 Dalton. The outlet tube was connected to a refrigerated fraction collector (CMA/200 F), where the dialysates were collected in microvials, stored at 4°C, and further processed for laboratory analysis (CMA 600). The microcapillary was continuously perfused with isotonic Ringer’s solution at a flow rate of 0.75 µL/min, which resulted in a time delay from the membrane to the microvial of 7 min. The sampling time was set at 60 min. Before each usage, the probes were prepared according to the guidelines of the supplier. This included flushing and equilibration of the probes, and establishing the relative recovery of the carbohydrate metabolites by placing the probes in an Eppendorf tube containing a standard solution (Calibrator A; CMA Microdialysis AB) with known concentrations of glucose (5.55 mmol/L), lactate (2.5 mmol/L), and pyruvate (250 µmol/L).

The data collected at the three last time points were pooled to calculate the sensitivity and specificity of a variable for detecting ischemia. The result was considered positive when it was beyond the 95% confidence interval for control tissue. The sensitivity was reflected by the percentage of positive results per total number of measurements for the ischemic tissue. Specificity was determined by the percentage of positive results obtained in the ischemic tissue per sum of positive results in traumatized and ischemic tissue.

The terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to detect cell death (17). The flap harvested at the end of the experiment was cut into slices of 3 mm thickness that were transferred to gelatinized micro-slides and air-dried overnight at 37°C. The sections were dewaxed in xylene (3 changes), rehydrated in ethanol, and rinsed in Tris-buffered saline (50 mM Tris/NaCl, pH 7.4, containing 100 mM sodium chloride [2 changes]) and then incubated in 20 µg/mL proteinase K for 15 min at room temperature. Endogenous peroxidase activity was suppressed by treatment with 0.3% hydrogen peroxide for 10 min. The sections were then incubated with terminal deoxynucleotidyl transferase enzyme for 60 min at 37°C followed by peroxidase-conjugated anti-digoxigenin antibody for 30 min at room temperature. The reaction was visualized by diaminobenzidine substrate for 8 min at room temperature. Finally, the sections were washed three times with Tris-buffered saline, counter-stained with hematoxilin, and mounted by using Aquatex (Merck, Darmstadt, Germany) for light microscopy examination.

The animals were kept under light anesthesia with a continuous infusion of 50 mg/mL pentobarbital given at a rate of approximately 0.5 mg/min/kg body weight throughout the experiment. After surgery, the depth of anesthesia was considered adequate when the animals showed a noxious reflex to pinching of the hind paw but no nonaversive reflexes (palpebral, corneal, and jaw reflex) (11). A heating pad and a room temperature of 28°C were used to prevent the animals from hypothermia, which was verified with a microthermometer placed on the abdominal skin.

Microdialysis and Oxy Lite probes were inserted subcutaneously in the middle of each vascular territory of the flap and in the intact dorsal skin (control). The collection of the microdialysates started after a period of 30 min had elapsed for stabilization. One hour later, the baseline values were obtained. Thereafter, one of the two branches of the vascular blood supply to the flap was transected to render the corresponding vascular territory ischemic. This tissue was merely perfused via a collateral vasculature connecting the two vascular beds. Care was taken that all measurements were obtained in areas supplied by vascular networks that did not contain any collateral vessels.

Exclusion criteria were abnormalities of the vascular anatomy, insufficient optical clarity, and mean arterial blood pressure (MAP) <60 mm Hg. The animals were killed with an overdose of pentobarbital at the end of the experiment.

The InStat version 3 program (Graph Pad Software, San Diego, CA) was used for statistical analysis. The data were presented as mean ± sd, and the time-related differences between repeat measurements and the differences between the tissues were assessed by the paired and unpaired analysis of variance, respectively. All tests were followed by the Bonferroni posttest. A value of P < 0.05 was taken to represent statistical significance. The statistical differences for sensitivity and specificity were assessed by the Fisher’s exact test.

	Results

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Two animals did not fulfill the inclusion criteria and were excluded from this study. MAP gradually declined from 91 ± 6 mm Hg to 83 ± 11 mm Hg during the experiment (not significant).

The behavior of microvascular diameters is presented in (Figure 1. The diameter in the artery feeding the flap tended to decrease from 105 ± 15 µm to 97 ± 8 µm over time (not significant). A different pattern was found in the arterioles in each part of the flap: the arterioles in the tissue of mere trauma showed continuous slight vasoconstriction (P < 0.01 for A1), whereas the opposite occurred in the ischemic arterioles, which tended to dilate over time (P < 0.01 between tissues).

View larger version (37K): [in this window] [in a new window]   Figure 1. Microvascular diameter in feeding flap artery (A0) and arterioles in traumatized and ischemic parts of the flap after inducing ischemia. A1–A4 = first to fourth order arterioles. Data represent means ± sd *P < 0.05 and **P < 0.01 versus baseline. P < 0.05 and P < 0.01 versus traumatized tissue.

The baseline values for microvascular blood flow ranged between 20 PU and 78 PU, and were similar in all tissues. The values remained virtually unchanged in the control skin and traumatized tissue, whereas ischemia reduced blood flow to approximately 10% of baseline (P < 0.01) (Fig. 2).

View larger version (41K): [in this window] [in a new window]   Figure 2. Microvascular blood flow (laser Doppler flowmetry) in normal control skin, and in traumatized and ischemic flap tissue before and after inducing ischemia. The data are given as percentage of baseline and represent means ± sd. **P < 0.01 versus baseline, control and traumatized tissue.

The mean baseline tissue Po₂ values ranged from 24.4 mm Hg to 26.7 mm Hg in traumatized and ischemic flap tissue as well as in control skin (not significant) (Fig. 3). After induction of ischemia, the corresponding tissue Po₂ was diminished to a mean value of 10.2 ± 3.7 mm Hg (P < 0.01).

View larger version (42K): [in this window] [in a new window]   Figure 3. Partial oxygen tension in normal control tissue, and in traumatized and ischemic flap tissue before and after inducing ischemia. Data represent means ± sd. **P < 0.01 versus baseline, control and traumatized skin.

At baseline, glucose values were 3.5 ± 0.4 mmol/L in the control tissue and 2.4 ± 1.2 mmol/L and 2.4 ± 1.0 mmol/L in the 2 parts of the flap (not significant) (Fig. 4). The values remained virtually unchanged in the control skin over time, whereas they gradually decreased in the flap tissue, reaching 1.7 ± 1.0 mmol/L in the traumatized tissue and 0.7 ± 0.5 mmol/L in the ischemic tissue at 4 h, after which they leveled out (P < 0.01 versus baseline and between all tissues). The mean pyruvate values were between 86 and 93 µmol/L in the different tissues at baseline. No major changes were seen in either control tissue or traumatized tissue over time, whereas the values decreased to 63 ± 30 µmol/L in the ischemic tissue (P < 0.05 versus baseline and other tissues). The mean baseline lactate was 1.5 ± 0.2 mmol/L in the control skin. The values tended to be higher in the traumatized (1.9 ± 0.7 mmol/L) and in the ischemic (2 ± 0.7 mmol/L, both not significant versus control) tissues. The values remained close to baseline in the control skin and in the traumatized flap tissue thereafter, unlike in the ischemic tissue, where they gradually increased to 2.8 ± 0.9 mmol/L (P < 0.01 versus baseline and other tissues).

View larger version (42K): [in this window] [in a new window]   Figure 4. Glucose, lactate and lactate/pyruvate (L/P) ratio in normal control tissue, and in traumatized and ischemic flap tissue before and after inducing ischemia. Data represent means ± sd. *P < 0.05 and **P < 0.01 versus baseline; oP < 0.05 and ooP < 0.05 versus control skin; P < 0.05 and P < 0.01 versus traumatized tissue.

The sensitivity to detect ischemia was highest for glucose (100%) and lactate to pyruvate ratio (L/P) (93%), whereas it was significantly less for lactate (62%, P < 0.01 versus other variables) (Table 1). The specificity to differentiate between the effects of ischemia and trauma was 71% for lactate and 70% for L/P ratio but only 53% for glucose (P < 0.05 versus other variables).

The TUNEL assay revealed an abundance of positively stained cells in the ischemic tissue, which were evenly distributed in all layers, whereas in the traumatized tissue, positive staining was restricted to the degenerating corneal layers of the epidermis (Fig. 5). These findings were characteristic in all specimens but were not further quantified.

View larger version (130K): [in this window] [in a new window]   Figure 5. TUNEL assay of dying cells in traumatized (a and c) and ischemic (b and d) flap tissue 6 h after induction of ischemia. Please note the massive accumulation of apoptotic cells (black nucleus) in the ischemic flap tissue. In the traumatized tissue, apoptotic cells are restricted to the degenerating corneal layers of the epidermis (arrowheads), whereas dying cells were scattered throughout all layers of the ischemic flap tissue (arrows).

	Discussion

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Our model provided the unique opportunity to monitor microhemodynamics, partial oxygen tension, and carbohydrate metabolites quantitatively and simultaneously in healthy, traumatized, and ischemic tissue. The present flap model has previously been proven to be useful to study the behavior of microhemodynamics and oxygenation in ischemic tissue and to test the efficacy of therapeutic manipulations focused on the reduction of hypoxia (11–14). Direct and quantitative assessment of the microhemodynamics was made possible by the use of intravital microscopy, which requires thin and transparent skin, as found in small rodents. The areas of healthy, traumatized, and ischemic tissues were clearly defined, thus ensuring reproducible data. Surgical trauma was applied during the dissection of this delicate tissue. The interruption of the anatomic blood supply led to an abrupt and drastic decrease in both microvascular blood flow and partial tissue oxygen tension in the ischemic tissue, thus suggesting a decrease in oxygen delivery. Although some microcirculatory blood flow and tissue oxygenation were still maintained, the severity of the subsequent oxygen debt was confirmed by the massive accumulation of dying cells in the ischemic tissue, whereas in the normally perfused, traumatized part of the flap, some cell death was restricted to the corneal layer, as reported for healthy skin (18).

The principal finding of this study was that L/P ratio was significantly more sensitive to detect ischemic hypoxia than lactate, whereas both markers showed a relatively frequent rate of trauma-related changes (approximately 30%), thus reducing their specificity.

Traumatization resulted in a slight lactate increase and a pronounced decrease in glucose. In this tissue, both microcirculatory blood flow and partial tissue oxygen tension were similar to the control tissue at all time points, which suggests that the glucose reduction was not supply-dependent but related to an accelerated oxidative energy metabolism resulting in lactate increase, a constellation that may be ascribed to inflammation, as reported previously (6,9). Because pyruvate remained virtually normal, the increased lactate was paralleled by an increased L/P ratio, which was substantial enough to keep the specificity of this variable to discern ischemia-related from trauma-related changes relatively low (70%), as low as the specificity for lactate. Furthermore, it may be assumed that the specificity for both markers could be even less if traumatization were more severe.

The induction of ischemia led to an additional increase in lactate and to a reduction of pyruvate, both signaling accelerated anaerobic glycolysis. The pyruvate reduction was responsible for rendering L/P ratio more sensitive than lactate to indicate ischemic hypoxia. Furthermore, the lactate increase may have been blunted by the ischemia-related decrease in glucose supply, as suggested by the decrease in glucose. However, the lactate increase appeared to be severe enough to induce vasodilation in the terminal arterioles, which have been reported to be the most susceptible for metabolic vasoregulation (19).

The observed pattern of carbohydrate metabolites cannot necessarily be extrapolated to systemic values obtained in the circulating blood because of the contribution of other tissues, limited washout from the ischemic tissues, and hepatic clearance. Furthermore, our findings would still have to be confirmed in human tissue.

In conclusion, our results suggest that the L/P ratio is more appropriate than lactate to monitor ischemic hypoxia in the presence of traumatized tissues. However, both markers yielded a relatively low specificity to discern ischemia-related changes from trauma-related changes. Provided our data can be extrapolated to clinical conditions, they suggest that monitoring L/P instead of lactate may reduce the probability of overlooking ischemic hypoxia in underperfused lesser organs in traumatized patients, whereas the risk of diagnosing ischemic hypoxia erroneously may be relatively high for both markers.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Contaldo, C. Articles by Erni, D. Search for Related Content PubMed PubMed Citation Articles by Contaldo, C. Articles by Erni, D. Related Collections Critical Care Resuscitation