Beneficial Effects of Dichloroacetate in Acute Limb Ischemia, The

Military Medicine, Jun 2007 by Platz, Timothy A, Wilson, Jeffrey S, Kline, Jeffrey A, Rushing, Greg, Et al

Objective: The purpose of this study was to determine the effects of dichloroacetate (DCA) in acute limb ischemia. Methods: Anterior tibialis muscle samples of DCA-treated and control animals (Sprague Dawley rats) were collected and assayed for pyruvate dehydrogenase activity, lactate, adenosine triphosphate, and creatine phosphate using spectrophotometry. A physiograph was used to measure fatigability. In an ischemia/reperfusion model using New Zealand rabbits, serum lactate and end-tidal CO2 were compared. Skeletal muscle was evaluated microscopically for muscle necrosis. Results: DCA administration resulted in a 50% increase in pyruvate dehydrogenase activity (p = 0.025), reversal of the increase in lactate levels seen during acute limb ischemia (p = 0.41), a significant increase in the time to skeletal muscle fatigue (p = 0.05), a trend toward increased adenosine triphosphate (p = 0.07), and a significant increase in creatine phosphate (p

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�C. Spectrophotometric techniques previously described3 were used to assay the muscle samples for PDH activity and lactate levels. These were compared in the DCA-treated and control animals from ischemie and nonischemic limbs. An additional 16 animals underwent acute limb ischemia as described above for 3 hours, at which time the hind limbs were attached to a myograph transducer with a 10-g preload. The gastrocnemius muscle was then stimulated with five pulses per second at 40 V with a 0.2-millisecond duration until fatigue (loss of sustained contraction). The time to muscle fatigue for DCAtreated (n = 8) and control animals (n = 8) was compared in ischemie and nonischemic hind limbs. The details of this model have previously been published.4

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.