Comp. Biochem. Physiol. Vol. IOIA, No. 2, pp. 361-361, 1992 0300-9629/92 S5.00 + 0.00 Printed in Great Britain 0 1992 Pergamon Press plc

A TIME COURSE STUDY OF THE ISOMETRIC CONTRACTILE PROPERTIES OF RAT

EXTENSOR DIGITORUM LONGUS MUSCLE INJECTED WITH BUPIVACAINE

J. DAMD ROSENBLATT* Physiological Laboratory, Downing Street, Cambridge CB2 3EG, U.K.

(Received 26 March 1991)

Abe&act-l. The effect of intramuscular injections of the myotoxin bupivacaine on the isometric contractile properties of rat extensor digitorum longus (EDL) muscle was studied.

2. Immediately after injection the EDL muscle did not contract when stimulated. The absolute (mN) and normalized (N/g) peak twitch (P,) and tetanic (P,) tensions gradually returned to normal by 21 days after injection.

3. Thereafter, the injected muscles grew at an accelerated rate and by 80 days were 70% heavier than controls.

4. The hypertrophy was not paralleled by a proportional increase in absolute P, and P,, and consequently the normalized P, and P, were 30% less than controls.

INTRODUCI’ION

Following extensive damage, muscles lose their abil- ity to contract, but regain some degree of function during the subsequent period of regeneration. The contractile properties of regenerating rat skeletal muscles following massive damage have been studied in minced (Carlson and Gutmann, 1972), free and nerve-intact (Carlson et al., 1981) grafts. The recov- ery of the contractile properties of these various graft preparations is always fractional and never returns to normal values.

Injection of myotoxic agents (cardiotoxins and local anaesthetics) is another method used to produce extensive muscle damage. Intramuscular injections of the local anaesthetic bupivacaine induces a rapid muscle necrosis succeeded by a period of rapid regen- eration: 15 min after an injection there is disruption of the plasmalemma, hypercontraction of the myo- fibrils, dilatation of the sarcoplasmic reticulum and pyknosis of the myonuclei (Bradley, 1979). During the next 12 hr there is a loss of the myofilament banding pattern, lysis of the Z-band, rupturing of the myofibrils, fragmentation of the cytoplasm and dis- solution of the plasmalemma (Hall-Craggs, 1974; Nonaka et al., 1983). Macrophages engulf the ne- crotic fibres at 12 hr, infiltrate them by 24 hr and phagocytise the debris over the next 3 days (Nonaka et al., 1983). Myoblasts (of satellite cell origin) are observed at the end of the first day and by the third day have fused to form multinucleate myotubes. Thereafter, growth of the myotubcs into fibres occurs

*Address correspondence to: J. David Rosenblatt, Univer- sity of Ottawa, Department of Physiology, Faculty of Medicine, 451 Smyth Road, Ottawa, Ontario KlH 8M5, Canada. Telephone: (613) 787-6564; Fax: (613) 787-6718.

and by 34 weeks, fibre maturation is complete with apparently total restitution of muscle mass (Jones, 1984), and histochemical and morphometric propcr- ties (Hall-Craggs and Seyan, 1975; Abe et al., 1987). The rapid sequence of events, and the apparent complete recovery, can probably be ascribed to the selective effect of the drug on myofibres and lack of damage to the muscle’s satellite cell population (Hall- Craggs, 1980b), endomysial tubes or basal lamina (Hall-Craggs, 1980a), vascular supply (Grim et al., 1983) and intramuscular nerves (Tomas i Fe& er al., 1989), elements which influence the rate and extent of muscle regeneration (Marechal, 1986); the various graft preparations selectively damage one or more of these important elements. Thus, the specific myotoxic action of bupivacaine provides a unique opportunity for studying regeneration of muscle in an environ- ment conducive to optimal recovery.

Most studies of the effect of intramuscular injec- tions of bupivacaine on muscle and muscle fibre properties have been descriptive in nature, and there are few quantitative data to substantiate the descrip- tive claims. More specifically, the contractile proper- ties have not been examined at all, and the histochemical (Hall-Craggs and Seyan, 1975; Abe et al., 1987), immunohistochemical (Abe et al., 1987) and morphometric (Hall-Craggs, 1974) properties of the fibres have only been described, and then only at a few selected or over a narrow range of times during regeneration. Given the previous, but unquantified, claims that muscles treated with bupivacaine recover rapidly and fully, it is of interest to know whether the contractile properties follow a similar course. The results of such a study would make a significant contribution to our understanding of the effects of bupivacaine on skeletal muscle and establishing the preparation for studies of altered regeneration.

361

362 J. DAVID ROSFJNBLATT

MATERIALS AND METHODS

All procedures were conducted on male Wistar rats (130 f 0.43 g, initial body weight) anaesthetized with pento- barbitone sodium (60mg/kg, i.p.). The right EDL muscle was surgically exposed and injected with 0.6ml of 0.5% bupivacaine hydrochloride (Marcain”, 5.28 mg/ml; Astra Pharmaceuticals Ltd) (Hall-Craggs, 1974). A separate group of animals received no treatment. At l-2 hr (0 days) and 2, 4, 8, 11, 21, 40, 60, 80 and 180 days after injection, in situ measurements of isometric tension development were made from the bupivacaine-injected (BI) and non-injected (NI) EDL muscles with a Sensotec model 31 load cell. Contract- ions were elicited by stimulation of the sciatic nerve with a bipolar electrode. The nerve was stimulated with square wave pulses of 0.2 msec duration. The EDL muscle length was adjusted until the peak isometric twitch force was produced from an applied supramaximal stimulus pulse. This length was held constant and the stimulus strength set at 3-5 times the voltage necessary to produce a maximum isometric twitch. The maximum isometric twitch tension (P,), the twitch time, the time to peak tension and the half-relaxation time were determined from two isometric twitch responses. Peak isometric tension development was measured in response to 1 set trains of stimuli delivered at 10, 20, 30, 40, 50, 75, 100 and 150Hz. The highest force attained during the forcefrequency response was denoted as the maximum isometric tetanic tension (P,). At the completion of the measurements the EDL muscle was removed and weighed. A portion of the whole muscle was then excised and used to determine relative fluid content. Preliminary work demonstrated that bupivacaine caused necrosis of 86% of the fibres in the EDL muscle and that saline injection had no effect, except to produce a transient elevated fluid content at l-2hr. Differences between groups were analysed in a two-way ANOVA (type of injection by duration of recovery) and, where appropriate, post hoc r’-test comparisons of least-squares means.

RESULTS

Both the BI and NI muscles became progressively heavier throughout the study and were similar in weight from l-2 hr to 21 days after injection; at 40 days, however, the EDL muscles in the BI animals were significantly heavier (Table 1) and larger (Fig. 1) than those of controls, and remained heavier and larger thereafter. This difference reached a peak at 80 days, when the mean weight of the BI muscles was 70% greater than that of the NI muscles. The relative water content of the BI muscles l-2 hr to 8 days after injection was significantly greater than that of NI muscles at the same recovery times (Table 1); the

Fig. 1. Whole muscle after 40 days recovery: bupivacaine-in- jetted EDL (right), contralateral control EDL (middle), and non-injected control EDL from separate animal (left). The contralateral muscle was not studied but is presented in this photograph to show that its size was not different from

non-injected control muscle. Minor division = 1 mm.

highest level was measured at 2 days. A gradual reduction occurred thereafter, and by 11 days had returned to normal and remained so for the duration of the study.

Absolute P, (mN) increased progressively through- out the course of the study in both BI and NI muscles. From l-2 hr to 11 days after injection, absolute P, was larger in NI than BI muscles: at l-2 hr, BI muscle produced no force in response to either direct muscle or sciatic nerve stimulation; forces were measurable at 2 days, and gradually returned to normal values by 21 days. BI muscles

Table 1. Wet weight and fluid content of bupivacaine-injected (BI) and non-injected (NI) rat EDL muscles

Wet weight Relative fluid content N (mtx) N (%)

Time ~ tdavs) BI NI BI NI BI NI BI NI SI

0 12 12 6-l* 2 2 12 12 53 f 2 4 12 12 62 + 5 8 12 12 81 +5

11 12 12 105 f 2 21 12 I2 152k8 40 12 12 241 f. 9 60 12 12 313 f 26 80 12 12 395 + 25

180 8 8 400+ 15

51* 1 4 4 64+1 4 4 71+2 4 4 8753 4 4 98+2 4 4

141 * 7 4 4 182f3* 4 4 222 f 18 4 4 232* IO* 4 4 257+13* 8 8

81.7 rt 1.1 83.4 k 0.6 79.6 + 0.6 78.5 f 0.1 71.9 f 0.2 75.1 + 0.8 76.0 + 0.3 75.7 * 0.2 75.2 + 0.3 75.8 + 0.7

76.5 f 1.0. 79.0 f 0.87 76.1 + 1.3’ 77.0 i_ 0.5* 75.8 f 0.6’ - 76.5 k 0.2. 76.2 f 1.2’ - 75.6 + 0.4 -

75.3 * 0.5 74.1*0.5 - 74.8 + 0.4 76.0 + 0.8 -

Values are mean + SEM. *Significant difference between BI and NI or BI and SI at a given time; tsignificantly different from BI and NI,

P < 0.05, two-way ANOVA.

Contractile properties of regenerating muscle 363

Table 2. Absolute and normalized maximum isometric twitch and tetanic tensions of bupivacaine-injected (BI) and non-injected (NI) control rat EDL muscles

Maximum isometric twitch tension Maximum isometric tetanic tension Time N Absolute (mN) Normalized (N/g) Absolute (mN) Normalized (N/g) (days) BI NI BI NI BI NI BI NI BI NI

0 8 8 o+o 294 + IS 0.0 * 0.0 5.0 + 0.2. Ok0 1122 + 32. 0.0 * 0.0 19.2 of: 0.4’ 2 9 10 60+ 15 319 + 18’ 1.3 f 0.2 5.0 + 0.2. 257 + 29 1169 + 43. 4.8 + 0.5 18.2 + 0.6* 4 8 8 180 + 20 339 + 26, 3.0 * 0.2 4.8 f 0.4. 699 + 90 1308+54’ Il.0 + 0.8 18.6 + 0.8* 8 9 8 29Of32 440 * 30. 3.6 f 0.3 5.0 + 0.4. 1127+79 1629 & 80. 13.9 f 0.5 18.5 f 0.8’

I1 8 8 390*31 471 + 36. 3.7 * 0.3 4.8 * 0.4. 1539 f 70 1744*7s 14.6 f 0.7 17.8 f 0.8. 21 8 9 660 + 62 635 & 59 4.4 f 0.3 4.6 f 0.4 2490 f 140 2420 f 130 16.4 f 0.7 17.4 f 0.6 40 8 8 820 + 40 826 + 35 3.3 f 0.2 4.4 f 0.2’ 3210 k 350 3470 * 120 13.1 f 1.4 18.7 + 0.4. 60 8 9 960 + 80 922 rt 34 2.8 k 0.3 4.4 * 0.2. 4530 * 360 3870 + 180. 13.2* 1.0 18.3 f 0.6’ 80 8 11 1076 f 73 1060+47 2.8 f 0.2 4.4 * 0.2. 5130 * 310 4170 f 160. 13.2 It 0.5 17.9+ 1.1’

180 6 6 1420 k 82 1171 * 100’ 3.5 * 0.1 4.5 f 0.4* 4660 k 350 4260 f 250. 11.3*0.5 17.0 * 1.2’

Values are expressed as means f SEM. *Significant difference between BI and NI at a given time, P < 0.05, two-way ANOVA.

continued to produce forces that were similar to the NI muscles until after 80 days, but at 180 days produced greater absolute P, than NI muscles. The P, produced by the NI muscles was linearly related to their mass (combined mean, 4.7 f 0.1, r = 0.92, Pearson product-moment correlation, P < 0.0001, Table 2). Normalized P,(N/g) was greater in NI muscles than BI muscles at all recovery times except 21 days. As with the absolute P,, the restoration of the normalized P, in the BI muscles occurred continuously and by 21 days was 96% of NI muscle. Thereafter, a significant reduction (w 30%) in normalized P, was observed.

Absolute P, followed a course similar to that described for P,, with the exception that BI muscles produced greater forces than NI muscles from 60 days until the end of the study. Normalized P, followed an identical pattern to that described for P,: the mass of, and force produced by, the NI muscles were linearly related (combined mean, 18.1 + 0.2, r = 0.96, Pearson product-moment correlation, P < 0.0001, Table 2); BI muscles had lower normal- ized P,, than controls at all recovery times except at 21 days; the recovery of normalized P, in BI muscles occurred progressively up to 21 days, at which time P, was 94% of control; a lower (N 29%) normalized P, in the BI muscles was recorded thereafter.

The isometric twitch contraction times (twitch time, time to peak tension and half-relaxation time) of the BI and NI muscles were not significantly different (Table 3).

The tensions produced at different stimulation frequencies are illustrated in Figs 2 and 3. Stimulating

at higher frequencies elicited larger absolute forces in both BI and NI muscles at all recovery times. From l-2 hr to 11 days after injection, the force produced by the NI muscles at each stimulation frequency was significantly greater than that produced by BI muscles at the same stimulation frequency, but the difference diminished at each successive recovery time (Fig. 2, top). From 21 to 40 days recovery, the forces in BI and NI muscles at each stimulation frequency were similar. With the lower frequencies of stimu- lation (l&40 Hz), BI and NI muscles continued to produce similar forces up to 80 days; at 180 days, the BI muscles produced significantly larger absolute forces than NI muscles (Fig. 3, top). With the higher frequencies of stimulation (Xl-150 Hz), the BI muscles produced significantly larger forces than NI muscles from 60 days onwards (Fig. 3, top). The normalized tensions produced by BI and NI muscles at each stimulation frequency followed the same pattern as that for normalized P, and P,: NI muscles gave unchanging values for normalized forces at each stimulation frequency, and BI muscles had smaller normalized tensions at all recovery times except for 21 days (Figs 2 and 3, bottom).

When expressed as a percentage of P,, there were no significant differences between BI and NI muscles for any of the forces produced at any of the stimu- lation frequencies. Figure 4 contains the mean force-frequency curves of the NI and BI muscles at all recovery times. The curves overlie each other but there is more dispersion at the lower frequencies; this is due to the lower forces produced by BI muscles at these frequencies at later recovery times.

Table 3. Twitch time, time to peak tension and half-relaxation time of bupivacaine-injected (BI) and non-injected (NI) control rat EDL muscles

Time (davs)

Twitch time Time to peak tension Half-relaxation time N (mW (m+ (mW

BI NI BI NI BI NI BI Nl

0 88 2 9 10 37.2; 2.7 4 8 8 52.6 * 2.8 8 98 56.0 f I .9

11 8 8 58.4 f 2.6

zl 88 8 9 60.1 57.9 f f 4.1 1.6 60 8 9 51.2 f 1.9 80 8 11 58.2 f 2.9

180 6 6 67.5 f 5.2

Values are expressed as means f SEM.

44.4 f 2.6 42.3 * 1.3 48.2 * 3.0 56.1 f 2.2 53.7 f 3.3 61.7 rt 5.0 61.2 f 2.8 52.8 f 2.6 55. I f 2.6 68.3 f 8.1

- 12.4 f 0.8 12.3 f 0.9 12.4 * 0.4 12.3 f 0.6 12.0 f 0.4 13.6 * 0.3 12.6 f 0.6 12.1 *0.3 11.3*0.5 13.0 * 0.7 12.6 & 0.6 11.9*0.6 12.9 f 0.2 11.3*0.3 12.0 f 0.5 11.7f0.4 12.3 f 0.4 15.5: 1.3 16.7; 1.6

- 11.9*0.8 11.0*0.9 12.0 * 0.7 13.9 f 0.7 12.6 f 1.3 14.6 f 0.5 12.5 + 0.6 13.0 f 0.4 12.6 * 0.9 4.6 f 1.0 13.0 f 1.1

11.8 * 0.6 13.3 f 0.3 12.1 f 0.6 11.9*0.3 12.8 f 0.5 12.6 f 0.7 16.0* 1.6 17.7 f 2.9

364 J. DAVID ROSENBLA~

01 0 50 100 150

F=qu=y OIZ)

Fig. 2. Absolute (top) and normalized (bottom) force- frequency relationships for bupivacaine-injected (BI) and non-injected control rat EDL muscle at l-2 hr (0 days) and 2, 4, 8, 11 and 21 days recovery. Plotted values are means f SEM. Forces produced by BI muscles returned to control values by 21 days (P < 0.05, two-way

ANOVA).

DISCUSSION

At l-2 hr after injection, the EDL muscle did not respond to either direct muscle or sciatic nerve stimu- lation. This is likely to be due to the anaesthetic effect that bupivacaine produces by stabilizing the sodium channels of the neurolemma and sarcolemma so reducing their permeability to sodium ions and increasing the membrane excitation thresholds (Seeman, 1972). It is probably not indicative of muscle damage. Although damage to the contractile apparatus occurs in bupivacaine-treated muscles as early as 15 min after injection (Bradley, 1979) it is unlikely that the extent of the damage is sufficient to account for the complete absence of force observed in the present study.

By 2 days after injection, the anaesthetic effect of bupivacaine had worn off and the BI muscles pro- duced very small twitch and tetanic forces. Since at this recovery time fibre necrosis has occurred and regeneration has yet to begin (Nonaka et al., 1983), the small forces measured at 2 days can probably be ascribed to a small number of surviving, viable fibres (14% survival in the present study). This is supported by the finding of an approximately 10% incidence of fibre survival in cross-sections of rat tibialis anterior (Foster and Carlson, 1980) and mouse EDL (Martin and Ontell, 1988) muscles 2-3 days after injection.

0 50 100 150

hwlwacy~)

Fig. 3. Absolute (top) and normalized (bottom) force- frequency relationships for bupivacaine-injected (BI) and non-injected (NI) control rat EDL muscle at 21, 40,60, 80 and 180 days recovery. Plotted values are means f SEM. At 10-40 Hz stimulation, BI muscles produced more absolute force than NI muscles only at 180 days. At 50-150 Hz BI muscles produced significantly larger forces than NI muscles from 60 days onward; the normalixed forces produced by BI muscles at all stimulation frequencies were significantly less than NI muscles at all recovery times except 21 days

(P < 0.05, two-way ANOVA).

The rapid return of the isometric force in BI muscles conlirms the reports of rapid degeneration and regeneration in muscles injected with bupiva- Caine: by 21 days after injection, the isometric twitch and tetanic tensions have returned to control values. This time course is similar to that described for the return of the morphometric (Hall-Craggs, 1974; Sadeh, 1988) and histochemical (Hall-Craggs and Seyan, 1975; Abe et al., 1987) properties of bupiva- Caine-injected rat tibialis anterior muscles.

The wet weight of the BI muscles was the same as the control at all the early recovery times. However, the muscle was also oedematous at these times, which probably compensates for the loss in mass resulting from the dissolution of muscle fibres. It should be noted that the muscle basal lamina, a major com- ponent of the extracellular matrix, is unaffected by bupivacaine (Hall-Craggs, 198Ob), and so even though there is a loss of muscle fibres following bupivacaine injection, the persisting basal membranes maintain the muscle infrastructure. Thus, although muscle wet weight was static during early regener- ation, it may not accurately reflect changes in muscle composition resulting from cellular events occurring within the muscle.

Contractile properties of regenerating muscle 365

0 Y SO 73 100 125 150

Frequew (Ha

Fig. 4. Forccfrequency relationships for bupivacaine-in- jetted and non-injected control rat EDL muscle. Plotted values are means f SEM. The force values (P) are normal- ized for maximum isometric tetanic tension (PA at each stimulation frequency. The greater dispersion at the lower frequencies is caused by the lower forces produced by BI

muscles at later recovery time.

Two significant findings in the present study were the increased mass and decreased normalized isomet- ric tensions of the BI EDL muscles at long recovery times. Both the BI and NI muscles displayed normal growth to 21 days, however, the BI muscles then grew at an accelerated rate until 80 days after injection. Similarly, after returning to control levels at 21 days after injection, values of both normalized P, and P,, declined significantly in BI muscles, and remained lower for the duration of the study; this trend was also observed for the normalized tensions produced by the BI muscles at different stimulation frequencies. The reduction in normalized force is related, at least in part, to the increase in mass. The muscle hypertro- phy was not accompanied by a commensurate in- crease in absolute force production; the BI muscles were heavier from 40 days recovery onwards, but absolute P, was larger only at 180 days after injection, whereas absolute P, was larger from 60 days onward. In addition, with the exception of the forces produced at 180 days recovery, the BI muscles produced larger absolute forces than the control only at stimulation frequencies of 50 Hz or more. Thus, neither the magnitude of absolute P, nor the forces produced at relatively low stimulation frequencies by BI muscles were proportional to its large mass.

It is possible that the reduced absolute force pro- duction at lower stimulation frequencies is related to impairment in muscle activation. Since the absolute tension produced at each stimulation frequency is a function of both muscle mass (i.e. physiological cross- sectional area) and degree of activation of the muscle, the effect of activation alone can be approximated by expressing the tensions relative to P,, (Roy et al., 1982). Following this manipulation, differences be- tween the forces produced by NI and BI at the higher frequencies disappeared (Fig. 4). For example, at 80 days recovery, the tension produced by NI muscles

when recruited at a frequency of 75 Hz was 3679 mN, whereas BI muscles produced 4451 mN at the same frequency, a 21% increase. When expressed as a percentage of P,. the tension produced by both NI and BI muscles was 87%; thus the whole of the apparent increase (21%) in absolute force produced by the BI muscle at higher stimulation frequencies may be due to an increase in contractile tissue mass. Therefore, it seems that the larger absolute forces produced by the BI muscles are attributable primarily to its larger muscle mass. The discrepancy between mass and force production, revealed by the reduced normalized tensions, suggests, however, that some of the increase in mass is related to non-contractile elements.

The force produced by a particular normal muscle is proportional to its physiological cross-sectional area (CSA). The mean physiological CSA of muscle is normally estimated by dividing the product of muscle wet mass and the cosine of the angle of pinnation by the product of fibre length and the density of muscle, assumed to be 1.056 g/cm3 (Mendez and Keys, 1960). The fibres of normal rat EDL muscle are approxi- mately the same length and lie at an angle of about 3.5” to the long axis of the muscle, an arrangement that is independent of the age of the animal (Close, 1964) hence the force produced by EDL muscle can also be regarded as being proportional to its mass, which was the indicator of relative force used in the present study. The linear relationship between muscle weight and Pt (r = 0.92) and P, (r = 0.96) in the NI muscles supports the findings of Close (1964) and justifies the use of weight as a measure of relative isometric tension output.

Theoretically, an increase in the mass of a pinnate muscle without a change in its length, or in the length and number of fibres in it, produces an increase in the angle of fibre pinnation (Maxwell et al., 1974), a situation which could occur in animals in which muscle growth occurs independently of skeletal growth (Gollnick et al., 1981). In fact, acute hypertro- phy of fast-twitch plantaris muscle (fibres normally oriented at N 15” angle) in mature rats, produced by the removal of their functional synergist muscles, is accompanied by only a 2” increase in angle of pinna- tion without a change in muscle length or muscle fibre length (Kandarian and White, 1990). Therefore, although the absolute tension produced by a hyper- trophic muscle may increase, an increase in the angle of pinnation would reduce the force applied to the tendons. Whether there was a significant increase in the angle of pinnation in the hypertrophic BI muscles is not deducible from the results. Based on the estimate for muscle physiological CSA, the effect that the small angle of pinnation (3.5”) would have on the force developed by a normal EDL muscle is less than 0.1% (Close, 1964), thus the EDL can almost be considered a parallel-fibred muscle. For argument let us assume the angle of pinnation of the BI muscle increased to lo”, an almost 3-fold change, and greater than any previously reported for enlarged muscles. Further, if we assume a constant fibre length to muscle length ratio, a relationship characteristic of hypertrophic muscles and of normal rat EDL muscles (Elmubarak and Ranatunga, 1984), there would be a 1.5% decrement in specific tension.

366 J. D~vm R~XENBLMT

Unfortunately, there are no data in the literature on the contractile properties of bupivacaine-injected muscles nor their weights after recovery times of longer than 30 days (Jones, 1984) with which to compare the present results. Furthermore, the con- tractile properties of muscles exposed to cardiotoxic snake venoms, many of which, like bupivacaine, have a specific myotoxic effect and cause massive fibre degeneration, have not been measured and so com- parisons with similar models of muscle regeneration can not be made either.

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Acknowledgements-The author wishes to thank the Cambridge Commonwealth (Canadian) Trust (Tidmarsh Scholarship), the Council of the Canadian Centennial Scholarship Fund and the Wellcome Trust for financial support, and Dr R. I. Woods and Dr A. Silver for their . helpful advice and guidance.

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