The present study examined the effect of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP) on rabbit skeletal muscle flap survival after warm ischaemia.

Two muscle flap models, rectus femoris pedicle flap and latissimus dorsi free flap, were subjected to a total ischaemia of 4 hours at 37°C and 20"C, respectively. Immediately prior to revascularisation, the muscles were infused with either Hanks' balanced salt solution (BSS) or Hanks' BSS containing 200 pmol PEP and 6.6 pmol ATP. Quantification of muscle damage was deter- mined by measuring the plasma levels of creatinine kinase (CK), lactate de- hydrogenase (LDH), lactate, potassium, and phosphate at 0, 2, 24, and 96 hours after revascularisation. Infusion of PEP/ATP compared with Hanks' BSS alone significantly decreased the efflux of CK in both rectus femoris (P < 0.025) and latissimus dorsi muscles (P < 0.05) and of LDH in the rectus femoris muscle (P < 0.01). No significant changes were observed, however, for the plasma levels of lactate, potassium, and phosphate. From this study it was concluded that PEP and ATP partially protect skeletal muscle from is- chaemia and reperfusion injury.

MICROSURGERY 10:8-14 1989

EFFECT OF PHOSPHOENOLPYRUVATE AND ADENOSINE TRIPHOSPHATE ON RABBIT SKELETAL MUSCLE AFTER ISCHAEMIA: PRELIMINARY BIOCHEMICAL STUDY

RAKESH KUMAR KHAZANCHI, M.D., KENNETH R. KNIGHT, Ph.D., MICHAEL F. ANGEL, M.D., W. CHRISTOPHER PEDERSON, M.D., SERENA A. COE, B.Sc., and BERNARD McC. O'BRIEN, M.D.

W i t h the widespread application of microsurgical tech- niques, replantation of limbs and composite tissue transfers have become common practice. Despite the advances in these techniques, success can be limited by the effect of ischaemia on involved tissues. The sensitivity of tissues to ischaemia lies partly with the metabolic activity of the tis- sues, with skeletal muscle being very sensitive to ischaemia compared with skin and cartilage.

Depletion of high-energy phosphate stores in ischaemic muscle is an important factor in determining whether or not necrosis will occur. Levels of adenosine triphosphate (ATP) and creatine phosphate have been known for years to de- crease rapidly in ischaemic skeletal muscle.' In an attempt

~ ~ ~ ~~

From the Microsurgery Research Centre, St. Vincent's Hospital, Fitzroy 3065, Victoria, Australia.

Acknowledgments: The authors wish io thank the staff of the Experimental Surgical and Medical Research Unit of St. Vincent's Hospital for their invalu- able surgical assistance. This work was supported by grants from the National Health and Medical Research Council (Australia).

Address reprint requests to Dr. K. R. Knight, Microsurgery Research Centre, St. Vincent's Hospital, Victoria Parade, Fitzroy 3065, Victoria, Australia.

Received for publication March 25, 1988; revision accepted October 24, 1988.

0 1989 Alan R. Liss, Inc.

to replenish energy stores and thus increase tolerance to ischaemia, ATP and phosphoenolypyruvate (PEP) have been successfully administered experimentally, resulting in increased cardiac and smooth muscle viability during ischaemia."-" To date, no work has been done on their potential application to skeletal muscle. Thus, this experi- ment investigated the ability of PEP and ATP to lessen skeletal muscular damage during periods of complete is- chaemia using two muscle models. The latissimus dorsi muscle in the rabbit is a long, thin muscle; it was used as a free tissue transfer in this study. The rectus femoris muscle, which is more compact and dense than the former, was used as a pedicle flap.

METHODS AND MATERIALS Fifty adult outbred New Zealand white rabbits (2.2-2.7

kg) were used. They were anaesthetised with phenobarbi- tone and maintained with halothane, nitrous oxide, and ox- ygen. The rabbits were divided into six groups.

Animal Groups Group 1 (n=10) (rectus control). In this group, the rectus femoris muscle of the rabbit was used. The anatomy of this muscle flap has been previously d e ~ c r i b e d . ~ The muscle was raised on its pedicle, which then was clamped with two soft microvascular clamps, one for the artery and

PEP and ATP in lschaemic Muscle 9

Femoral A & V JL Lu-/ Femoral A & v

Figure 1 . Schematic representation of the infusion of the rectus fem- oris muscle flap prior to the release of the microvascular clamp. Note that a Gilson pump was used to control the rate of infusion.

the other for the vein, for 4 hours. The venous clamp was removed and the flap infused for 15 minutes by means of a Gilson infusion pump with 2 ml of filter-sterilised Hanks’ BSS, through a cannula introduced into the femoral artery distal to the rectus femoris muscle branch (Fig. 1). Re- vascularisation then was restored by release of the arterial clamp. The muscle flap was secured to the fascia by a single 310 suture and the wound closed.

Peripheral blood samples ( 3 ml) were taken from the ear vein just before inducing the anaesthesia immediately after arterial clamp release, then at 2, 24, and 96 hours after revascularisation. Two ml of blood was placed in a lithium heparin tube and 1 ml in a fluoride oxalate tube, the latter for lactate assay only. Creatine kinase (CK), lactate dehy- drogenase (LDH), potassium, lactate, and phosphate levels were determined as described elsewhere.8 The rabbits were finally sacrificed at 96 hours.

Group 2 (n=10) (rectus PEP). This procedure was as for Group 1, but with 200 kmol PEP and 6.6 kmol ATP in 2 ml Hanks’ BSS, filter-sterilised, and infused over the 15- minute period immediately prior to revascularisation.

Group 3 (n = 10) (latissimus control). Under sterile conditions, the latissimus dorsi muscle was elevated on its vascular pedicle. The anatomy of this flap has been previ- ously described.’ One third of the muscle was removed along with its vascular pedicle and left in a moist swab in a sealed sterile container at room temperature (20°C) for 4 hours. Toward the end of this ischaemic time, the muscle was revascularised by anastomosis of the femoral vessels in the groin. The venous clamp was released and the muscle

Figure 2. Schematic diagram showing the infusion of the latissimus muscle flap prior to release of the microvascular clamp. The sites of the microvascular anastomoses are indicated.

infused as for Group 1 with 2 ml of Hanks’ BSS via the thoracodorsal artery, distal to the point where it gives the branch to latissimus dorsi muscle (Fig. 2). After removing the arterial clamp and inspecting the anastomoses, the wound was closed. Blood samples were taken as in Group 1.

Group 4 (n = 10) (latissimus PEP). This was the same as Group 3 , except with 200 kmol of PEP and 6.6 kmol of ATP in Hanks’ BSS as the infusate prior to revascularisation.

Group 5 (n = 5). This group acted as a nonischaemic, sur- gery-only control to Groups 1 and 2. The rectus femoris muscle was elevated and replaced with its pedicle attached without subjecting it to ischaemia. Blood samples were col- lected at the same time periods as in previous groups.

Group 6 (n=5). This group acted as a surgery-only con- trol to Groups 3 and 4. The latissimus dorsi muscle was removed as in Group 3 . Four hours later the femoral vessels were ligated and divided. However, the removed muscle was not revascularised and was discarded. Blood samples were collected as in previous groups.

One problem encountered in this project was the pre- mature death of three rabbits using the rectus femoris mus- cle and of seven rabbits using the latissimus dorsi muscle. An additional 10 rabbits were used to make up for those lost. The premature deaths probably were due to the surgi- cal anaesthesia, compounded by the frequent withdrawal of blood. Consequently, for some rabbits, blood samples were not obtained for all time points. Furthermore, the amount of blood drawn on a few occasions was insufficient to assay

10 Khazanchi et al.

TABLE 1. Creatine Kinase (Units per Litre): Release Into Blood After Muscle Ischaemia.

Postop

Time Preop 0 hr 2 hr 24 hr 96 hr

Rectus femoris muscle No ischaemia lschaemia + Hanks' BSS lschaemia + PEPiATP t-test (pooled data)

Latissimus dorsi muscle No ischaemia lschaemia + Hanks' BSS lschaemia + PEPiATP t-test (Dooled data)

ND 251 7 2 627 599 ? 63 2824241 3 732-C 133 2524k228

NS

860 2 227 14502 173 902k 101 1618k357 684282 1 293 2 229

NS

31 50281 2 802722192' 53392412"

NS

291 25625 6830? 3461 4626? 1 01 5

NS

13,00822068 62,67721 1,639*" 33,609&4646"*

P < 0.025

83222 2576 63,717*14,798*** 25,040 t 4324***

P < 0.05

931 k237 10,666+3970"

5373?2193* NS

843 5291 7262219

1052-C 176 NS

Resulfs of assays on blood samples collected from the rabbif ear vein at the times indicated. Mean _t SEM is reported. There were five rabbits in each control (no ischaemia) group and 70 rabbits in each ischaemia group. insufficient blood was collected for a full analysis in some rabbits. Statistical analysis comparing the PEWATP infusion group with the Hanks' BSS infusion group is reported. NS, not significant, i.e., P > 0.05. ND, not determined. Statistical significance by the pooled t-test comparing each ischaemiahnfused group with the no ischaemia group is shown as follows: ' P < 0.05; " P < 0.01; "'P < 0.001; all others, NS.

for all parameters. Priority in these cases was given to the assay of CK. The results were expressed as mean k SEM, and values compared by the Student's t-test.

Materials

ATP, disodium salt, was purchased from Sigma Chem- ical Company, St. Louis, MO and PEP, monosodium salt, from Boehringer Mannheim, Mannheim, West Germany. Hanks' BSS was purchased from Commonwealth Serum Laboratories, Melbourne, Australia.

RESULTS Creatine Kinase

The creatine kinase levels had risen above their basal levels at the time the flap was revascularised in all groups (Table 1, Fig. 3). They showed a further increase 2 hours after revascularisation. Maximal levels were seen 24 hours after revascularisation. At this point, levels in PEP/ATP- BSS-treated groups (Groups 2 and 4) were significantly lower than those in Hanks' BSS-treated groups (Groups 1 and 3) in both the rectus femoris model ( P < 0.025) and the latissimus dorsi model (P < 0.05). Note that the levels were higher in the ischaemic rectus femoris model in which is- chaemia was at 37"C, as compared with corresponding groups in latissimus dorsi model in which ischaemia was at 20°C. A similar trend in the levels was also seen at 2 hours after revascularisation, although the differences were not statistically significant. Also note that removal of a muscle flap alone without ischaemia (Groups 5 and 6) causes a rise in CK levels, which was highest at 24 hours, but this was significantly lower than the corresponding ischaemic mus- cle groups.

Lactate Dehydrogenase

Any samples with haemolysis had a significant effect on LDH levels and were excluded from the results (Table 2, Fig. 4). As with CK, LDH levels increased in all groups, with maximal release of LDH occurring at 24 hours. There was a significantly lower release of LDH at 24 hours in the PEP/ATP-treated group (Group 2) compared with the Hanks' BSS-treated group in the rectus model (Group I ) (P < 0.01). Although a similar decrease was evident at the same time for the latissimus dorsi model (Groups 3 and 4), the higher standard deviations in the values resulted in this not being significant (NS). LDH levels were significantly higher in ischaemia compared with nonischaemic muscle groups (Groups 5 and 6) at several postischaemic times, notably at 24 hours.

Lactate, Potassium, and Phosphate

Lactate levels increased maximally at 2 hours in all ischaemic muscle groups (Groups 1-4) (Table 4). These increases (Groups 1 and 2) were significant at P < 0.05 in the nonischaemic group (Group 5 ) for the rectus femoris model at 2 hours postoperatively, and for the Hanks'-in- fused ischaemic group (Group 1) at 24 hours postopera- tively. PEP/ATP infusion did not produce statistically sig- nificant changes compared with Hanks'-only infusion. No significant changes were observed in potassium levels (Table 3) nor in the phosphate levels (not shown).

DISCUSSION

Although skin, bone, and tendon may tolerate relatively long periods, skeletal muscle is very sensitive to ischaemia. This is of clinical significance in major limb replantations and in compromised free muscle or musculocutaneous

RECTUS FEMORIS

I p( 0.025 \

PEP and ATP in lschaemic Muscle 11

KINASE -

LATISSIMUS DORSI

P < 0.05 m Pro.op. 0 2 24 Praop. 0 2 24 86

n m (hrs.) TIME (hrs.)

Figure 3. Time course showing the plasma levels of creatine kinase in units/litre for the two muscle flap models: rectus femoris and la- tissimus dorsi. The symbolic representation of the groups is as fol- lows: control, surgery only with no ischaemia (----); 4 hours is- chaemia with Hanks' BSS infusion I-); 4 hours ischaemia with

PEP/ATP in Hanks' BSS infusion (. . . . ). Error bars indicate the standard error of the mean. Student's t-test has been used to corn- pare the statistical differences between the PEP/ATP infused group and the Hanks' BSS infused group at 24 hours postischaemia.

Table 2. Lactate Dehydrogenase (Units per Litre): Release Into Blood After Muscle Ischaernia.

POStOD

Time Preop 0 hr 2 hr 24 hr 96 hr Rectus femoris muscle

No ischaemia 302246 10682 182 7482154 10232201 227231 lschaemia + Hanks' BSS 341 230 6412107 15202269' 3061 2473"' 51 1 +94** lschaernia + PEPlATP 233k19 590284 10712183 1621 222V 446274** t-test (pooled data) NS NS P < 0.01 NS

286283 No ischaemia 302k46 5852143 7802277 685k241 lschaernia + Hanks' BSS 243217 447296 8442246 24602941I 454 k 94' lschaemia + PEP/ATP 288229 488244 1082+ 182 1131 2259 3482115 t-test (pooled data) NS NS NS NS

Statistical significance of the PEPIATP-treated ischaemic group compared with the Hanks' BSS-treated ischaemic group is shown in the table. Statistical significance of each ischaemiahfused group versus no ischaemia group is indicated by asterisks as described for Table 1.

Latissirnus dorsi muscle

transfers. Larsson and Lewis" studied the reflow pattern in human skeletal muscle after tourniquet ischaemia and found the existence of a no-reflow phenomenon. The exact cause of this no-reflow phenomenon is unknown. Strock and Majno" found many structural and functional alterations in the microcirculation, which they suggested resulted in a

no-reflow phenomenon in ischaemic muscles in rat hind limbs. Gidlof et found that this was due partially to progressive endothelial swelling.

One potential factor in skeletal muscle necrosis from ischaemia is the depletion of high-energy phosphate stores. Levels of ATP have been shown to decrease rapidly in

12 Khazanchi et al.

LACTATE DEHYDROGENASE

3750

3500

3250

3000

2750

J 2500

2250 \

2000

1750

1500

1250

RECTUS FEMORIS

T LATISSIMUS DORSl

NS

Pro-op. 0 2 24 96 TIME (hrs.)

Figure 4. Time course of the plasma levels of lactate dehydroge- nase in unitshtre for the two muscle flap models. The symbolic

representations are as described for Figure 3. NS, not statistically significant.

Table 3. Potassium (Millimoles per Litre): Release Into Blood After Muscle Ischaemia.

POStOD

Time Preoo 0 hr 2 hr 24 hr 96 hr

Rectus femoris muscle No ischaemia ND 4.3k0.2 3.6k0.4 3.ako.3 4.320.3 lschaemia + Hanks' BSS 4.1 20.3 3.6k0.4 5.520.8 4.520.4 3.8k0.3 lschaemia k PEPiATP 3.920.3 3.7k0.3 4.1 20.2 3.9k0.2 3.520.2

No ischaemia 3.720.4 4.2k0.3 4.5k0.5 4.1 20.3 4.2k0.3

lschaemia + PEPiATP 3.7k0.2 3.6k0.4 4.6k0.6 4.0k0.2 3.9k0.2

Latissimus dorsi muscle

lschaemia + Hanks' BSS 4.4k0.4 3.720.5 3.820.2 5.0k0.9 3.820.3

Details are the same as given in Table 1. There were no sfatisfica//y significant differences between any groups at any one postoperative time.

ischaemic skeletal m ~ s c l e . ~ In cardiac muscle, ATP stores decrease further with reperfusion. l 3 The combination of these two effects leads to markedly decreased levels of ATP with reperfusion of devascularised muscle. As a terminal event in ischaemia, both skeletal and cardiac muscle un- dergo rigor, which is related to decreased levels of ATP and is partially reversible by the addition of ATP. l4 Further- more, ATP levels in endothelial cells also decrease during ischaemic periods, which can lead to irreversible spasm in microvessels and could be a factor in no-reflow phenomenon.6 This spasm cannot be remedied by direct

replacement with exogenous ATP3*6 because of its larger molecular size (molecular weight 578 vs. 208 for PEP) and greater polarity, although some authors dispute this. l5 PEP, on the other hand, without question can cross the cell membrane6 and as an intermediate of glycolysis it is con- verted to enolpyruvate by pyruvate kinase. With the transfer of the high-energy phosphate group to ADP, intracellular ATP is replenished. In addition, ATP causes transient phos- phorylation of plasma membrane and further facilitates the influx of PEP into the cells.16

Working with a rat heart model, Hultman3 showed that

PEP and ATP in lschaemic Muscle 13

Table 4. Lactate (Millimoles per Litre): Release Into Blood After Muscle Ischaemia.

Postop

Time Preop 0 hr 2 hr 24 hr 96 hr

Rectus femoris muscle No ischaemia ND 4.220.8 4.1k1.3 3.4k0.3 3.2k0.8 lschaemia + Hanks’ BSS 4.9k0.9 5.020.9 7.9k1.2’ 6.7?1.4’ 4.1 k0.4 lschaemia + PEP/ATP 4.3-CO.6 4.920.5 8.021 .o* 4.720.7 3.6k0.6

No ischaemia 4.2k0.5 4.320.5 4.1 20.9 3.7k0.5 4.8k0.6 lschaemia + Hanks’ BSS 4.3?0.5 4.2k0.6 5.320.7 5.121.0 4.3k0.5 lschaemia + PEP/ATP 4.2-CO.3 4.4k0.5 5.1 20.6 4.320.7 4.0k0.4

Latissimus dorsi muscle

~~

There were no statistical/y significant digerences (NS) between ischaemia + Hanks’ BSS and ischaemia f PEP/ATP groups at any measured postoperative time. The asterisks indicate statistically significant differences between each ischaemialperfused group and the respective no ischaemia group at P < 0.05 (pooled t-test).

supplementation with PEP and ATP during reperfusion sig- nificantly decreased efflux of CK into the circulating blood, resulting in increased energy content and improved left ven- tricular performance.

It has been shown previously that CK and LDH activity in the postischaemic period is a reliable index of the extent of ischaemic damage. ” In the present experiment, infusion of two different types of ischaemic muscle flaps with PEP and ATP prior to revascularisation resulted in a decrease in efflux of CK and LDH into the blood stream when compared with similar muscles treated with Hanks’ BSS. The levels at 24 hours after revascularisation were significantly lower for CK in both the models. For LDH the difference was sig- nificant in the rectus model but not in latissimus dorsi model, which suggests that PEP and ATP infusion in ischaemic skeletal muscle prior to reperfusion offers some protection against damage caused by ischaemia and reperfusion.

Potassium and lactate levels showed no significant vari- ations. It has been shown previously that ischaemia causes a rise in potassium levels, to peak at 8 hours after revascu- larisation, a time that was not examined in these experi- ments.

The optimal method and time of PEP/ATP administra- tion has yet to be determined. If the increase in ATP levels proves to be the crucial factor in ultimate muscle-flap sur- vival after ischaemia, local infusion of PEP/ATP is there- fore likely to be superior to parenteral administration. A comparison of these two alternatives was not made here. Furthermore, ischaemic, noninfused flaps were not tested in these experiments. Flushing the acidic metabolites with Hanks’ BSS may offer some short-term advantage over non- infused flaps,5 although, in replanted limbs18 and skin flaps, 19**0 the infusion of osmotically balanced salt solu- tions after ischaemia and prior to revascularisation has sometimes been shown to be detrimental to ultimate flap survival. The importance of these factors on muscle-flap survival remains to be investigated.

Parameters such as muscle blood flow and histologic analysis would greatly aid in assessing the effect of this

treatment regimen on ischaemic muscle. In addition, the present study was concerned solely with the effect of PEP/ ATP on muscle survival. We had difficulty in accurately assessing the survival or failure of the muscle flap by gross examination, as there appeared to be partial areas of sur- vival in most cases. Subsequent pilot studies suggest that the labelling of muscle with nitroblue tetrazolium dye2’ and of the blood vessels with India ink infusion would allow an accurate assessment of the viable areas of muscle. These results will be the subject of a future publication. Clinically, it is important for a transferred muscle, such as the gracilis transfer for facial paralysis, to be viable and functional; therefore, the effect of PEP/ATP on the functional state of previously ischaemic muscle is another important area for future investigation. If this treatment is found to be bene- ficial, it would have a substantial role in the management of major limb replants and microvascular muscle transfers.

CONCLUSIONS

Infusion of ischaemic skeletal muscles with PEP and ATP immediately prior to revascularisation in two different muscle models partially protects the muscle against is- chaemic damage, as is apparent from biochemical studies of the release of CK and LDH into the circulating blood.

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14 Khazanchi et al.

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