pyrophosphate and Ischemia

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Diphosphates; Ischemia; Kidney Cortex; Magnesium; Oxidative Phosphorylation; Rats; Rats, Inbred Strains

Increased vascular permeability is an early and sensitive indicator of ischemic muscle injury, occurring before significant histologic or radionuclide changes are evident. We investigated the effect of iloprost, a stable prostacyclin analog, on microvascular permeability in a rat striated muscle model. In six control and six experimental animals the cremaster muscle was dissected, placed in a closed-flow acrylic chamber, and suffused with a bicarbonate buffer solution. Dextran labeled with fluorescein was injected intravenously as a macromolecular tracer, and microvascular permeability was determined on the basis of clearance of the fluorescent tracer. Two hours of ischemia were followed by 2 hours of reperfusion. In the experimental group iloprost (0.5 microgram/kg/min) was given in a continuous intravenous infusion. Microvascular permeability increased significantly during reperfusion in both control and experimental animals (p less than 0.0001). Treatment with iloprost, however, significantly attenuated this response compared to the control group, 4.8 +/- 0.3 versus 7.3 +/- 0.5 microliters/gm/min, respectively (p less than 0.0001). Iloprost decreases the rise in vascular permeability after ischemia and reperfusion. Experimental clinical use of iloprost under controlled conditions in the treatment of patients with acute skeletal muscle ischemia appears justified.

Topics: Animals; Capillary Permeability; Diphosphates; Iloprost; Ischemia; Male; Microcirculation; Muscles; Radionuclide Imaging; Rats; Rats, Inbred WF; Reperfusion; Technetium; Technetium Tc 99m Pyrophosphate; Time Factors

Limiting the rate of reperfusion blood flow following prolonged ischemia in skeletal muscle has been shown beneficial. However, the persistence of this benefit with reinstitution of normal blood flow remains undefined. We investigated the role of temporary limited reperfusion on ischemia-reperfusion injury in an isolated gracilis muscle model in six anesthetized dogs. Both gracilis muscles were subjected to 6 hr of ischemia followed by 2 hr of reperfusion. Reperfusion blood flow was limited for the first hour in one gracilis muscle to its preischemic rate followed by a second hour of normal reperfusion (LR/NR). The contralateral muscle underwent 2 hr of normal reperfusion (NR/NR). Muscle injury was quantified by technetium-99m pyrophosphate (TcPyp) uptake and by histochemical staining using triphenyltetrazolium chloride (TTC) with planimetry of the infarct size. Capillary permeability was evaluated by muscle weight gain. Results are reported as the mean +/- SEM: [table: see text] These data demonstrate a sustained benefit from temporary limited reperfusion. This methodology should be considered in the surgical management of the acutely ischemic limb.

Topics: Animals; Blood Flow Velocity; Blood Pressure; Diphosphates; Dogs; Female; Histocytochemistry; Ischemia; Kinetics; Male; Muscles; Organ Size; Reperfusion Injury; Technetium; Technetium Tc 99m Pyrophosphate; Tetrazolium Salts; Time Factors

Skeletal muscle necrosis will result from prolonged periods of ischemia. The purpose of this study was to develop a method to estimate the extent of necrosis using nitroblue tetrazolium staining and technetium scanning. The bilateral canine gracilis muscle preparation with total vascular isolation was exposed to 4 hr of complete normothermic ischemia followed by reperfusion. After 45 hr of reperfusion 99mTc pyrophosphate (PYP) was injected and 3 hr later the muscles were harvested, cut into six slices, and stained with nitroblue tetrazolium. Biopsies were taken from tetrazolium-positive and -negative areas for electron microscopy to confirm the ability of the stain to distinguish viable from necrotic muscle. Computerized planimetry of the staining pattern was used to estimate the extent of necrosis as a percentage of the total muscle. Electron microscopy confirmed the validity of nitroblue tetrazolium to discriminate between viable and necrotic skeletal muscle in this experimental model. After 4 hr of ischemia the percentage necrosis was 30.2 +/- 6.1% (mean +/- SEM, n = 12), there was no difference in the extent of necrosis in left vs right paired muscles, using tetrazolium staining or technetium PYP uptake. There was a statistically significant correlation between the percentage necrosis and the density of 99mTc PYP uptake per muscle (r = 0.83, P less than 0.001) and per slice (r = 0.94, P less than 0.001). This study demonstrates the ability of tetrazolium staining to accurately differentiate between viable and necrotic skeletal muscle and provides a reproducible method for estimating the extent of necrosis in the gracilis muscle model.

Topics: Animals; Diphosphates; Dogs; In Vitro Techniques; Ischemia; Muscles; Necrosis; Nitroblue Tetrazolium; Radionuclide Imaging; Staining and Labeling; Technetium; Technetium Tc 99m Pyrophosphate

The study of ischemia and reperfusion injury in the extremity has been hampered by lack of an accurate method of measuring skeletal muscle injury. We used a bilateral isolated in vivo canine gracilis muscle model in 15 anesthetized dogs. The experimental muscles had 4, 6, or 8 hours of ischemia and 1 hour of reperfusion. The contralateral gracilis muscle served as a control. Technetium 99m pyrophosphate (99mTc-PYP), an agent which localizes in injured muscle cells, was used to quantitate canine skeletal muscle damage. After 6 hours of ischemia and 1 hour of reperfusion, there was a significant increase of 215% of 99mTc-PYP uptake in the experimental vs the control muscle. Experimental muscle uptake was 8% greater than control after 4 hours and 405% more after 8 hours of ischemia and reperfusion. Segmental distribution of 99mTc-PYP uptake showed localization to be greatest in the middle of the muscle at the entry site of the gracilis artery. Electron microscopic evaluation also documented this area to have undergone the most severe injury. Distal portions of the muscle did not show increased damage. Our results show that 99mTc-PYP effectively quantitates skeletal muscle ischemia and reperfusion injury. The pattern of 99mTc-PYP uptake suggests that considerable injury is caused during reperfusion.

Topics: Animals; Diphosphates; Dogs; Female; Ischemia; Male; Microscopy, Electron; Muscles; Organ Size; Perfusion; Radionuclide Imaging; Regional Blood Flow; Technetium; Technetium Tc 99m Pyrophosphate; Time Factors

The management of an acutely ischemic extremity requires knowledge of the response of skeletal muscle (the largest component of the lower limb) to prolonged periods of complete normothermic ischemia. We have used the canine gracilis muscle model to evaluate the extent and distribution of ischemic necrosis after 3 and 5 hours of ischemia and 48 hours of reperfusion. Each muscle was cut into six slices, and the extent and distribution of postischemic necrosis identified by means of nitroblue tetrazolium staining and 99mTc pyrophosphate uptake. After 3, 4, and 5 hours of ischemia the extent of necrosis was 2.0% +/- 0.9%, 30.3% +/- 6.0%, and 90.1% +/- 3.5% (mean +/- SEM), respectively. A statistically significant correlation exists between the extent of necrosis and the uptake of 99mTc pyrophosphate uptake per gram of tissue (y = 1574.9x - 8.4, r = 0.84, p less than 0.001). Most necrosis was centrally located and found in the thickest portion of the muscle. We conclude that there is a graded response in the extent of skeletal muscle necrosis related to the length of ischemic stress rather than an "all-or-none" phenomenon. This central distribution of necrosis makes the usual external evaluation of ischemic damage clinically unreliable. In addition, since there was no enveloping fascia in this model, a compartment release alone may not prevent the development of skeletal muscle necrosis. This knowledge of the response of skeletal muscle to ischemia may lead to an improved clinical approach to an extremity suffering a prolonged ischemic insult.

Topics: Animals; Diphosphates; Dogs; Ischemia; Muscles; Necrosis; Nitroblue Tetrazolium; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Time Factors; Ultrasonography; Vascular Patency

Topics: Adult; Arterial Occlusive Diseases; Diphosphates; Humans; Ischemia; Leg; Male; Middle Aged; Radioisotopes; Radionuclide Imaging; Rhabdomyolysis; Technetium; Technetium Tc 99m Pyrophosphate; Thallium

Renal ischemia injury was induced by transient 30-min occlusion of the main artery in five rabbits and by transient 60-min occlusion in four rabbits. The vessels to the contralateral kidneys were ligated in all nine animals and in six additional control rabbits. Renal blood flow was restored immediately following the transient ischemia. Twenty-four hours later, 99mTc-pyrophosphate was injected and renal uptake was monitored for 100 s and 10-min intervals for 90 min following injection. At 90 min postinjection, the animals were killed and the percent injected dose remaining in the kidneys was calculated for three animals in each group. At 60 min postinjection, in vivo activity in the 30-min ischemic kidneys was 2.3 times greater than that of controls, and activity in the 60-min ischemic kidneys 4.6 times greater than that of controls. These results suggest that 99mTc-pyrophosphate scanning may be useful in assessing slight ischemic damage to the kidney.

Topics: Animals; Diphosphates; Ischemia; Kidney; Kidney Tubular Necrosis, Acute; Rabbits; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Time Factors