Introduction

Acute limb ischemia represents a significant source of morbidity and mortality annually. Mortality rates as high as 25% and limb loss rates as high as 20% in survivors have been reported,1 with presumed higher rates in military combat environments secondary to significant delays in access to tertiary care and revascularization. In an effort to decrease the known morbidity and mortality associated with acute limb ischemia, investigations of the potential benefits of dichloroacetate (DCA), a potent blocker of pyruvate dehydrogenase (PDH) kinase (PDHK), have been undertaken. Under aerobic conditions, PDH catalyzes decarboxylation of pyruvate produced during glycolysis, producing acetate and CO2. Within the mitochondria, this provides substrate for the TCA cycle and subsequent adenosine triphosphate (ATP) production.

During ischemia, PDH activity is inhibited by PDH-K, thus shifting the metabolism of pyruvate away from acetyl CoA production in favor of lactate. This results in skeletal muscle accumulation of lactic acid and subsequent tissue injury. DCA maintains PDH activity even under anaerobic conditions by directly inhibiting PDH-K activity2 in cardiac myocytes. We theorized that DCA administration during acute limb ischemia would similarly increase skeletal muscle PDH activity which would in turn decrease tissue lactate levels, increase time to skeletal muscle fatigue, and increase skeletal muscle ATP production and creatine phosphate (CrP) stores. In addition, it is theorized that serum lactate levels, end-tidal CO2, and skeletal muscle tissue necrosis would decrease significantly after ischemia/reperfusion with DCA administration.

Materials and Methods

All procedures and protocols were approved by an institutional animal care and use committee. Thirty-two Sprague-Dawley rats were anesthetized with 30 mg/kg ketamine and 6 mg/kg xylazine subcutaneously. Venous access was then obtained via cutdown placement of an indwelling catheter in the right internal jugular vein. Using a retroperitoneal approach, the right common iliac arteries were exposed and ligated to produce hindlimb ischemia. After 2 hours, DCA (15 mg/100 g body weight) was administered via the venous access to 16 of the animals, while an additional 16 animals received an equivalent volume of normal saline. After an additional 1 hour of hind-limb ischemia, the tibialis anterior muscles were surgically exposed in both the ischemie and nonischemic hind limb. Skeletal muscle tissue was excised and freeze-clamped in liquid nitrogen and stored at -70

In a separate protocol, an additional 44 adult male rats underwent surgical ligation of iliac artery under anesthesia described above. After 2 hours, DCA (15 mg/100 g body weight) or an equivalent volume of saline (control) was administered intravenously. At 3 hours (n = 22) and 6 hours (n = 22) of hind limb ischemia, muscle samples from the anterior tibialis were harvested, freeze-clamped, and stored at -70°C. Skeletal muscle ATP and CrP levels were measured using spectrophotometry. Results were compared from ischemie and contralateral nonischemic limbs for both DCA and control animals.

In a third protocol, 36 female New Zealand rabbits were anesthetized with ketamine, xylazine, and inhaled anesthesia. Animals were intubated and monitored continuously for oxygen saturation via pulse oximetry and end-tidal CO2 through capnography. Animals had their common iliac (high ligation, n = 18) or common femoral (low ligation, n = 18) arteries surgically exposed and then ligated. Mer 2 hours of ischemia, animals received 15 mg/100 g of DCA or equivalent normal saline (controls) through venous access. After an additional 2 hours of ischemia, the ligatures were removed; 15 minutes after reperfusion, end-tidal CO2 was recorded and blood was drawn from the ear vein and assayed via spectrophotometry for lactate concentration. The surgical wounds were closed and pulses were assessed by continuous wave Doppler for 1 hour and then at 6, 12, 24, 36, and 48 hours and categorized for limb use. At 48 hours, the tibialis muscle was sampled and cross-sections were taken and stained with hematoxylin and eosin; using light microscopy, sections were evaluated for percentage of muscle necrosis. Significant necrosis was defined as > 10% total volume of necrosis for evaluated sections. The details of this model have previously been published.5 Please refer to Figure 1 for summary of the protocols performed.

Data were expressed as mean ± SEM. Skeletal muscle PDH activity (µmol/min/g dry weight), lactate (µmol/g), ATP (µmol/ g), CrP (µmolmol/g), and fatigue time (minutes) were compared between control and DCA-treated animals and ischemie and nonischemic limbs by use of the Student t test. For the reperfusion model, blood samples were assayed using spectrophotometric techniques for lactate concentration (mmol/L). Comparison was made between high and low ligation and by the Student f test. Changes in end-tidal CO2 (percentage gas concentration) were compared between DCA- and saline-treated animals by the Student t test. Comparisons were similarly made between treated and control animals for recovery of pulses and limb use. Cross-sections were evaluated microscopically for degree of necrosis with 10% necrosis selected as the percentage to quantify favorable versus unfavorable (lowest amount of necrosis easily quantified under simple light microscopy).

Results

In nonischemic hind limbs, DCA administration resulted in a similar measured PDH activity of 13.2 ±1.3 compared to 9.6 ± 1.1 µmol/min/g dry weight controls (p = 0.13). However, in ischemie hind limbs, skeletal muscle PDH activity was significantly higher (p = 0.025) in DCA-treated animals (19.6 ± 1.6 µmol/min/g dry weight) than in controls (13.1 ± 1.3) (Fig. 2). With DCA treatment, there was no significant difference between lactate levels measured in ischemie limbs (83.3 ± 37.3 jumol/g) compared to the contralateral nonischemic limb (75 ± 41.5 µmol/g) (p = 0.41), indicating a similar level of acidosis. In the absence of DCA, the muscle lactate level of control animals was significantly higher in ischemie (121.9 ± 55.7 µmol/g) compared to nonischemic limbs (53.3 ± 23.8 µmol/g) (p < 0.005) (Fig. 3).

Acute limb ischemia results in a significant increase in tissue lactate (p = 0.005). This effect was blocked by DCA administration (p = 0.41). Ischemia significantly decreased the time to muscle fatigue in both DCA-treated animals (p = 0.002) and saline-treated control animals (p = 0.001). DCA treatment improved muscle function in ischemie hind limbs. After 3 minutes of ischemie time, the gastrocnemius muscle fatigue time of DCA-treated animals (2.6 ± 0.3 minutes) was significantly increased (p < 0.05) compared to controls (2.0 ± 0.6 minutes). Muscle fatigue time in nonischemic limbs was not significantly (p = 0.51) different between DCA-treated (3.3 ± 0.5 minutes) and control (3.1 ± 0.6) animals. At 3 hours of ischemia, DCA-treated animals showed no significant difference in skeletal muscle ATP or CrP. After 6 hours of ischemia, CrP levels in the DCA-treated ischemie hind limbs showed a significant increase from ischemie placebo hind limbs (p < 0.02) (Fig. 4) and a trend toward an increase in ATP levels compared to placebo (p < 0.08) (Fig. 5).

In the ischemia/reperfusion model, higher serum lactate levels were seen after high compared to low ligation in control animals (3.2 versus 1.2 mmol/L, p = 0.01). DCA treatment significantly reduced serum lactate levels measured 15 minutes after reperfusion in both high (0.27 ± 0.16 mmol/L versus 3.2 ± 1.51 mmol/L, p < 0.001) and low (0.4 ± 0.5 mmol/L versus 1.2 ± 0.58 mmol/L, p = 0.01) (Fig. 6). DCA-treated animals had significantly (p < 0.001) less rise (1.2 ± 2.3) in end-tidal percentage (38.3-39.4%) CO215 minutes after reperfusion than did control animals (8.2 ± 3.1,36.8-46.8%) (Fig. 7). All animals had loss of Doppler-detected pulses after ligation and full recovery of normal pulses and spontaneous limb use after ligature removal by 24 hours. There was no significant difference in the rate of recovery of function at 6 and 12 hours, with 11 of 18 control animals having spontaneous limb use by 6 hours compared with 10 of 17 DCA-treated animals, and 16 of 18 control animals having spontaneous limb use by 12 hours, compared with 16 of 17 in the DCA-treated animals.

In high-ligation animals, ischemia resulted in significant necrosis in 50% of sections in controls, but no DCA-treated animals had >10% necrosis. In low-ligation animals, there was no significant difference in proportion between control and DCA-treated animals. When data from the high- and low-ligation animais were pooled, there was still a significant difference in the proportion of animals having <10% area necrosis (Figs. 8 and 9).

Discussion

In the experimental model of acute limb ischemia, the ligation of the iliac artery for 3 hours produced significant decreases in PDH activity and an increase in tissue lactate levels. Treatment with DCA, a pyruvate molecule with two chloride ions attached to the methyl group that inactivates PDH-K, appeared to provide metabolic protection to the skeletal muscle. DCA directly inhibits PDH-K activity and allows PDH to remain active in skeletal muscle even under ischemie conditions. This allows PDH to continue to decarboxylate pyruvate and provide substrate for aerobic metabolism and inhibit lactic acid accumulation. Treatment with DCA increased PDH activity and decreased tissue lactate levels.

Although limb ischemia did not change muscle lactate levels in the DCA-treated group, control animals demonstrated significantly higher lactate levels in ischemie versus nonischemic limbs, validating this model of acute limb ischemia. This also demonstrates the protective effect of DCA in muscle metabolism during ischemia. The difference in lactate levels in DCA-treated and control ischemie limbs was not statistically significant (p = 0.09), which may represent inadequate ischemie time or a type II statistical error. It may be appropriate in future studies to lengthen the ischemie time to appreciate statistical significance. In addition, physiologic studies of muscle function showed an increase in time to muscle fatigue with the use of DCA. It was also concluded that DCA treatment increased time to muscle fatigue after 2 hours of ischemia. This increase in muscle function is thought to be due to a more favorable pH and possibly higher levels of high-energy phosphate molecules such as CrP or ATP.

During short periods (minutes) of ischemia, energy stores in the form of CrP, which is hydrolyzed by CrP kinase to convert ADP to ATP, are adequate to meet the metabolic demands of the cell, although accumulation of lactate levels will still occur. An additional objective of this study was to evaluate the effects of DCA on skeletal muscle ATP and CrP levels after acute ischemia. During hypoxia, energy stores become depleted resulting in physiologic failure and cell death. Minimizing the time between the onset of muscle ischemia and reperfusion remains the best way to minimize tissue damage and irreversible necrosis. DCA administration resulted in increased levels of ATP after acute limb ischemia when compared to controls. Even though no significant difference in skeletal muscle ATP levels was found at 3 (no difference) and 6 (p < 0.08) hours of ischemie time, a definite trend toward increased ATP levels is recognized. It is postulated that if a similar study was performed with longer times to ischemia, a statistically significant difference would be seen when comparing ischemie limbs in those treated with DCA versus controls; this work is currently underway in our laboratory.

However, a significant difference was seen with CrP levels when comparing DCA versus control animals in ischemie limbs. It was shown at the 6-hour ischemia time point that CrP levels were significantly higher (p < 0.02) in the DCA-treated animals. Thus, it is concluded that with DCA treatment in acute limb ischemia, a physiologic protective effect is provided to skeletal muscle tissue. The depletion of CrP stores in control animals represents the pathway responsible for maintenance of adequate ATP levels and may partially explain the failure in our study to show significant difference in ATP levels. As CrP stores are completely depleted, a rapid decrease in available ATP would be expected at longer ischemia times.

In the rabbit model of ischemia and reperfusion, DCA administration resulted in significantly lower serum lactate levels and a corresponding lower rise in end-tidal CO2 after reperfusion. This is consistent with the previous study findings demonstrating significant increases in PDH activity and decreased lactate levels in ischemie skeletal muscle when DCA was administered. The benefit of DCA in acute limb ischemia is further supported by histologie studies showing a significant decrease in the percentage of animals having >10% skeletal muscle necrosis after 4 hours of limb ischemia when DCA was administered. DCA has shown functional improvement in animal models during myocardial ischemia6,7 and in this study for hind-limb ischemia. It seems likely that decreased levels of lactate seen as a result of increased PDH activity after DCA administration would have a benefit during reperfusion as well.

Prevention of postreperfusion metabolic acidosis should have definite clinical benefits with prevention of organ dysfunction and may be reduced by limiting anaerobic metabolism during acute ischemia. Direct evaluation of this postreperfusion organ dysfunction and the possible benefits of DCA administration, especially in lung and kidney tissue, is an area of current study in our laboratory. The reduction in significant muscle necrosis after DCA treatment suggests a muscle protective effect that is more complex than just maintaining an optimal pH for cellular function. With administration of DCA in acute limb ischemia, PDH-K is inhibited (maintaining PDH activity) thus allowing for continuous substrate to be provided for the tricarboxylic acid cycle for production of ATP. It is known that mitochondria can produce ATP at extremely low oxygen tension if adequate substrate is provided. Being able to provide substrate during acute ischemia with the use of DCA should allow at least limited ATP production within the cell which would extend the ischemie time available before irreversible cell loss and decrease injury seen after reperfusion.

Although DCA administration has not shown clear benefit in subsequent clinical trials in the treatment of acute myocardial ischemie syndromes,8,9 it was found to be safe for administration for humans, with little or no side effects, in doses found to be effective in maintaining PDH activity in animal and organ culture models of ischemia. The application of DCA in the treatment of skeletal muscle ischemia had not been previously evaluated in human or animal models. Even although there has been no clinical benefit demonstrated in myocardial infarction in humans with the use of DCA, we theorize that in skeletal muscle, which has less flow-dependent oxygen extraction and better tolerates intermittent (hours) periods of ischemia, DCA administration may demonstrate better efficacy. In addition, it is proposed that DCA administration in acute ischemia would decrease mortality and morbidity in terms of limb loss and functional deficits, specifically in rural and military warfare settings where the time between onset of ischemia and access to surgical repair and reperfusion may be quite long.

Conclusions

In several animal models of acute limb ischemia and ischemia reperfusion, DCA administration maintains skeletal muscle PDH activity, decreases both tissue and serum lactate levels, increases time to muscle fatigue, and decreases postreperfusion end-tidal CO2. Skeletal muscle CrP stores are significantly preserved with DCA administration with a trend toward higher ATP levels. Additionally, histologie studies demonstrate lower amounts of significant muscle necrosis after ischemia and reperfusion when DCA is administered. Further studies to evaluate the physiologic benefits of DCA in limb ischemia including human studies to evaluate its potential benefit in terms of decreased limb loss, morbidity, and mortality are warranted.

© 2007 Association of Military Surgeons of the United States Provided by ProQuest LLC. All Rights Reserved.