Induction of intramuscular collateral nerve sprouting by neurally applied colchicine

EXPERIMENTAL

67, 513-523 (1980)

NEUROLOGY

Induction of Intramuscular Collateral Nerve Sprouting by Neurally Applied Colchicine LLOYDGLJTH,SAM

SMITH,EDWARDJ.

DONATI,

AND EDSON X. ALBUQUERQUE’ lleparrments lJnil,ersily

qf‘ Anatomy of Mrrryland

arltf

of‘Phartnacology School of‘ Medicine,

Recei\,ed

September

and E.rperimenral Therapeutics. Baltimore. Matyland 21201 1 I, 1979

The plantaris muscle of the rat is innervated by fibers deriving from spinal nerves L4 and L5. When L4 is transected, the intact residual L5 fibers sprout intramuscular preterminal processes which reinnervate some of the denervated muscle fibers and restore their weight and strength. Experiments by Diamond and his colleagues on cutaneous innervation in salamanders indicated that collateral sprouting can be elicited by applying colchicine to a nerve as well as by transecting it, and it therefore seems that collateral sprouting results from the interruption of axonal transport rather than from nerve degeneration. We tested this hypothesis by either transecting or applying colchicine to spinal nerve L4 in the rat and measuring the isometric strength of contraction of the plantaris 2 weeks later. After transection of L4, electrical stimulation of L4 gave no response whereas stimulation of L5 gave a supranormal isometric tension; after application of colchicine to L4, stimulation of L4 resulted in a normal isometric tension and stimulation of L5 gave a supranormal one. The muscles were examined histologically by combined silver-cholinesterase staining. Colchicine treatment produced abnormalities of innervation pattern characteristic of preterminal collateral sprouting. Because colchicine disrupts axonal transport, we interpret these results to mean that interruption of axonal transport in L4 fibers stimulates intramuscular sprouting of L5 fibers. The data are consistent with Diamond’s hypothesis that nerves possess a propensity for collateral growth which is ordinarily repressed by factors that are dependent on axonal transport. Abbreviation: AChE-acetylcholinesterase. I The authors gratefully acknowledge the expert assistance of Mr. Fred F. Bland III, Ms. Jacqueline G. Krikorian. and Mr. Thomas Samaras. The research was supported by a grant from the Paralyzed Veterans of America and by grants NS-12847 and NS-12063 from the National Institute of Neurological and Communicative Disorders and Stroke.

513 0014-4886/80/030513-11$02.00/0 Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.

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INTRODUCTION The nervous system plays a unique role in integrating the functional activities of all organs and tissues of the body, and it is the specificity of central and peripheral neural connections which, by controlling animal behavior (20), is crucial to evolutionary survival. Consequently, animals have evolved various mechanisms for repairing injury to the central and peripheral nervous systems: (i) Axonal regeneration occurs in all vertebrates after injury to the peripheral nervous system (9) although central neural regeneration is seen only in certain inframammalian species (26). (ii) Neural damage can be repaired by outgrowth from remaining uninjured nerves adjacent to the site of injury. This process is termed collateral sprouting to distinguish it from the regenerative growth of injured fibers. Collateral sprouting has been demonstrated in muscle (5), skin (24), sympathetic ganglia (16), brain (14, 18), and spinal cord (6, 11, 17). When the uninjured fibers form new terminations by this means, the normal specificity of neural connections is compromised. At best, collateral sprouting results in imperfect reflex function, and at worst, the abnormal connections that are formed may produce serious functional abnormalities (15). Even in regenerative repair of injured peripheral nerves, the failure of the regenerating neurites to grow selectively to their original end-organs results in functional deficits (20), but, because these deficits are generally less deleterious than are the effects of denervation, the evolutionary significance of axonal regeneration and collateral sprouting seems indisputable. The nature of the signal by which denervated tissues stimulate sprouting from adjacent uninjured nerves has been examined experimentally. Early investigators, studying collateral sprouting in partially denervated muscle, tested the hypotheses that humoral factors from the denervated muscle fibers, the degenerating nerve fibers, or the proliferating Schwann cells stimulate uninjured fibers to sprout (5). However, no compelling support for these possibilities was obtained, and more recent studies led to quite a different explanation (1,4). In those experiments, one of the peripheral nerves supplying the forelimb of the salamander was either transected to cause degeneration or treated topically with colchicine (to block axonal transport). Physiological measurements showed that both treatments resulted in collateral sprouting from adjacent untreated nerves and, consequently, enlargement of the adjacent sensory fields innervated by these nerve fibers. In the case of the colchicine treatment, both the drug-treated nerve fibers and the adjacent untreated ones were functional, thus showing that collateral sprouting could be induced in the absence of nerve degeneration. Similarly, application of colchicine to the fimbria of the rat hippocampus induced collateral sprouting and increased synaptic density in its

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terminal field, the molecular layer of the dentate gyrus (7). Those experiments suggest that under normal conditions, collateral sprouting and hyperinnervation of peripheral territories is prevented by axonally transported neuronal influences. In the present study, we examined further the role of axonal transport in the regulation of collateral sprouting by determining whether or not neurally applied colchicine would induce collateral sprouting in mammalian muscle as it does in mammalian brain and amphibian skin. The plantaris muscle of the rat is innervated by fibers deriving from spinal nerves L4 and L5. When either of these nerves is transected, intramuscular collateral sprouting of the residual intact fibers occurs (25). In the present study, we either transected or applied colchicine to spinal nerve L4, and 2 weeks later we measured isometric tension after stimulation of L5. We found that after either procedure, stimulation of L5 resulted in greater isometric tension than in control rats. Histological studies indicated that this extension of the peripheral field of L5 nerve fibers resulted from collateral sprouting of their axons. These results support the hypothesis (1, 4) that axonally transported substances regulate the density of innervation of peripheral fields and that collateral sprouting is not triggered by factors from denervated muscle and degenerating nerve. MATERIALS

AND METHODS

Operative Procedztre. Female Wistar rats weighing 190 to 240 g were anesthetized with chloral hydrate (400 mgikg. i.p.). Using aseptic technique, a skin incision was made 1 cm lateral to the dorsal midline and extending from upper lumbar to the middle sacral vertebral levels. The attachments of the abdominal muscles to the ilium and lumbodorsal fascia were severed. By blunt dissection, the fibers of the psoas and iliacus muscles were separated. The abdominal muscles were retracted ventrolaterally and the vertebral muscles dorsomedially. enabling us to expose spinal nerves L4 and L5 from the intervertebral foramina to where they unite and give rise to the sciatic nerve (21, 25). (i) Denervation: L4 was transected just distal to its emergence from the intervertebral foramen. (ii) Colchicine application: Using the procedure of Tiedt et al. (22), L4 was carefully freed from surrounding connective tissue and a 5 x IO-mm rectangle of sterile Parafilm was slipped beneath the nerve to isolate it from the underlying muscle. A small piece of cotton approximately 3x 3 x 1 mm was moistened with 0.05 ml colchicine solution (30, 10, or 5 mM dissolved in sterile isotonic saline solution) and placed on the surface of the nerve. Two additional cotton pledgets of similar size were moistened

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with tissue fluid and placed on either side of the colchicine-soaked pledget so as to minimize diffusion of the colchicine into adjacent tissues. The Parafilm was folded over the surface of the pledget and the entire region covered with a moist sterile gauze pad to prevent the tissues from drying. After 45 min, the cotton was removed, and the field thoroughly irrigated with 10 ml sterile isotonic saline solution to remove residual colchicine. In all procedures, the nerve had to be handled as little and as gently as possible, because tugging on it or abrasion from the edges of the piece of Parafilm was found to cause nerve degeneration. Physiological Procedures. Two weeks postoperatively the animals were anesthetized and spinal nerves L4 and L5 exposed widely by removing part of the iliac crest, the paravertebral musculature, and the vertebral transverse process. The nerves were then transected as close as possible to the vertebral column. The gluteal branch of the sciatic nerve was transected at the sciatic notch and the soleus and gastrocnemius tendons were severed at the ankle so as to minimize extraneous movements during electrical stimulation of L4 and L5. The tendon of the plantaris muscle was dissected free from adjacent tissue and tied to a metal hook, then the plant.aris muscle was carefully freed from adjacent connective tissue and muscle while taking care to avoid damage to its nerve or blood vessels. The femur and the foot were clamped rigidly and the tendon of the plantaris attached to a Grass force displacement transducer that was oriented perpendicularly to the leg. The plantaris muscle, which was kept moistened with mineral oil and warm with an incandescent lamp, was stretched to a resting tension of 50 g to ensure that the contractions were virtually isometric. The isometric tension was recorded on a Grass polygraph. Spinal nerves L4 and L5 were insulated from the underlying tissues with a piece of nonabsorbent weighing paper and bathed in a pool of warm mineral oil. For stimulation, the nerves were draped over a pair of platinum electrodes and stimulated separately or jointly about 3 mm proximal to the junction of L4 and L5. Stimuli were delivered via a Grass stimulator and stimulus isolation unit. The nerves were stimulated supramaximally for 0.5 s at 100 Hz with a O.l-ms pulse duration. The voltage required for supramaximal stimulation varied between 3 and 12 V. An interval of at least 60 s between tetani avoided fatiguing the preparation. After completion of the physiological recording, the muscle was removed, weighed, and frozen in liquid nitrogen or in dry ice-isopentane. Frozen sections were cut at 25 mm and stained by Goshgarian’s combined cholinesterase-silver nitrate procedure for neuromuscular junctions (8). Frozen longitudinal sections of spinal nerve L4 were reacted histochemically for acetylcholinesterase activity (in the presence of 10m5 M

INTRAMUSCULAR

COLLATERAL TABLE

I

Effect of Partial Denervation and Colchicine Strength and Weight of Plantaris Muscle Isometric Treatment Unoperated Sham-operated L4 Transection 5 mM Colchicine 10 mM Colchicine

N I1 6 10 5 4

L4 201 k 219 + 0-c 205 t 127 +

18 17 0 14 13

517

SPROUTING

Treatment (Mean -C

on SE)

Tension

(al

L5

L4 + L5

102 i 109-+ 199 ? 235 t 189 +

11 6 24 25 16

281 298 198 332 280

k 2 f f t

19 27 24 16 20

Muscle weight (mgl 1862 174 i 149 -c 1922 184 2

6 11 12” 9 IO

” The weights of the muscles of this group were low because of denervation atrophy. The weights of the contralateral unoperated muscles in these rats averaged 183 t 8 g, thus showing that the rats in all operative groups were comparable in size.

iso-OMPA to inhibit 5 mM colchicine.

nonspecific

activity) 0 to 5 days after application

of

RESULTS Validation of Control Procedures. The physiological data presented in Table 1 show that the exposure of L4 to saline-soaked cotton for 45 min (sham operation) had no effect on neuromuscular function. There was no statistically significant difference in isometric tensions between the shamoperated and the unoperated animals. When spinal nerve L4 was exposed 45 min to cotton soaked in colchicine, the effect was dose-dependent. Exposure to 30 mM colchicine seriously affected neuromuscular function; 2 weeks later electrical stimulation of L4 resulted in no isometric muscle response. When the dose was reduced to 10 mM colchicine, the effect was less severe, but electrical stimulation of L4 2 weeks later still resulted in a significantly reduced isometric tension (127 g instead of 2 19 g). Only after exposure of L4 to a concentration of 5 mM colchicine was there no effect on neuromuscular function. Because in this case the isometric tension elicited by stimulation of L4 was not significantly different from normal, we considered 5 mM colchicine to be the appropriate concentration for this study. Effect of Transection of L4. Two weeks after transection of L4 the isometric tension in response to stimulating L4 and L5 was recorded. At that time, no response of the plantaris muscle was obtained by stimulating L4; however, the response to stimulation of L5 (average 199 g) was nearly

518

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FIG. I. Motor end-plates of plantaris muscle, 14 days after applying 5 mM colchicine to spinal nerve L4. AChE-silver stain. x440.

double that obtained from control preparations (Table 1). This statistically significant increase in isometric tension occurred despite a sizeable loss in muscle weight. Ejfect of 5 VIM Colchicine. A cotton pledget soaked in 5 mM colchitine was applied to the spinal nerve L4 for 45 min, and 2 weeks later we recorded the isometric tension developed by the plantaris in response to nerve stimulation (Table 1). Stimulation of L4 gave an average tension of 205 g, which was not statistically different from that of control preparations. However, stimulation of L5 elicited an average contraction of 235 g, which was nearly double that of the control preparations. When L4 and LS were stimulated simultaneously, the tension developed was slightly but not significantly greater than the control values. Histological Observations. The photomicrographs of Fig. 1 illustrate the common abnormalities of the neuromuscular apparatus that were seen

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-

COLLATERAL

SPROUTING

C -

FIG. 2. Frozen longitudinal sections of spinal nerve L4 stained for acetylcholinesterase control nerve, B-one day after application of 5 mM (AChE) activity, x82. A-normal colchicine showing increased enzyme activity proximal to the site of treatment (left side of figure). C-five days after colchicine treatment showing decreased AChE activity at site of drug application.

after application of 5 mM colchicine to L4. Numerous examples of dually innervated muscle fibers were seen in every specimen examined (Figs. IA-D). In some cases two nerve fibers formed separate but contiguous sole plates on a muscle fiber (Figs. lB, D), and occasionally two wellseparated sole plates were seen on a single muscle fiber (Figs. lA, C). These patterns of innervation were rarely seen in normal muscle. The

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acetylcholinesterase (AChE) activities of a control L4 spinal nerve (Fig. 2A) and of one treated with colchicine 1 or 5 days previously show that the drug treatment caused accumulation of enzyme activity proximally (Fig. 2B) and disappearance of activity at the site of application (Fig. 2C). DISCUSSION Collateral Sprouting after Transecting Spinal Nerve L4. Of the isometric tension generated in the plantaris muscle via indirect stimulation, approximately two-thirds (average 201 g) is furnished by L4 motor units and one-third (102 g) by L5 motor units (Table 1). Thus, an effective partial denervation of this muscle can be achieved by transecting spinal nerve L4. The sequence of events that occurs after this operation was thoroughly summarized by Edds (5). Initially, there is a decrease in isometric strength that is proportional to the loss of the L4 motor units. Within a few days, preterminal axonal sprouts emerge from the residual intact L5 neurites and grow to nearby denervated muscle fibers. As a result of this growth, which is directed toward the denervated sole-plates, functional neuromuscular junctions are soon established between the collateral sprouts of these L5 axons and the muscle fibers that had originally been innervated by L4 neurites. This enlargement of the L5 motor units restores to a considerable degree the weight and strength of the partially denervated muscle (10, 23, 25). In the present study we observed that the average strength of L5 motor units increased from 102 to 199 g within 14 days after transecting L4 (Table 1); this finding is consistent with Edds’s description of the neuromuscular changes evoked by collateral sprouting in partially denervated muscle. The factors initially considered (5) as being responsible for eliciting collateral sprouting in partially denervated muscle included (i) agents released by the denervated muscle fibers; (ii) agents released by the degenerating nerve fibers; and (iii) agents released from the proliferating Schwann cells of the degenerating nerve fibers. Even though more than 30 years have elapsed since the original experiments were performed, we still lack compelling evidence to support any of these possibilities; in fact, the results of the present experiment in which colchicine was applied to spinal nerve L4 render them all unlikely. Physiological Effects of Neurally Applied Colchicine. Colchicine has been widely used experimentally because of its ability to block axonal transport. When applied in high concentration, it produces Wallerian degeneration in peripheral nerves (2, 22), but if the concentration is carefully adjusted, this potentially neurotoxic agent will interrupt axonal

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transport without causing nerve fiber degeneration (2, 22). In our hands, the application of 30 mM colchicine to L4 apparently caused complete degeneration because 2 weeks later, muscular contraction could not be elicited by indirect stimulation. At a concentration of 10 mM colchicine produced degeneration of some, but not all nerve fibers, as the isometric tension was reduced from 219 to 127 g (Table 1). However, if a 5 mM concentration of colchicine was used, the isometric tension recorded 2 weeks later (205 g) was not significantly different from the control value of 219 g (Table 1). Consequently. subsequent interpretations are based on the experiments using the 5 mM concentration of colchicine, because it produced no evidence of nerve degeneration. It is essential to demonstrate that, at 5 mM concentration, the colchicine blocked axonal transport. For this purpose, the endogenous transport of AChE provides a useful marker. When a ligature is placed about a peripheral nerve, AChE accumulates proximally and distally to the ligature, indicating a bidirectional movement of this protein (12, 13). and when two ligatures are placed several millimeters apart, the AChE activity of the intervening segment becomes considerably reduced as a result of the anterograde and retrograde movement of the enzyme toward the ligatures (13). We observed a similar decrease in AChE activity when 5 mM colchicine was applied to a region of peripheral nerve several millimeters long (Fig. 2). This result indicates that the drug had effectively blocked transport of AChE into this region. Colluteral Sprouting lifter Applying Colchicine to Spinal Ner\le L4. When colchicine was applied at 5 mM concentration to L4, the strength of the L4 motor units remained normal (205 g) whereas the strength of the L5 motor units increased from 109 to 235 g (Table 1). Such an enlargement of LS motor units without degeneration of L4 motor units could be accomplished by collateral sprouting only if extensive double innervation of the plantaris muscle fibers had occurred. Our physiological and histological data are indeed consistent with the presence of multiply-innervated muscle fibers: (i) After colchicine treatment, the isometric tension produced by the simultaneous stimulation of L4 and 5 was considerably less (332 g) than was the sum of tensions generated by the stimulation of L4 and L5 separately (440 g). This finding indicates that many of the muscle fibers were jointly innervated by nerve fibers deriving from L4 and L5 spinal nerves. (ii) The silver-cholinesterase histological preparations provided numerous examples of dually innervated muscle fibers (Fig. 2). Such appearances are quite rare in the normal plantaris muscle. Regulation of the Density of Peripheral Inner\wtion. The results of the present experiment lead us to conclude that (i) The functional and struc-

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tural changes observed after colchicine treatment resulted from intramuscular collateral sprouting. (ii) In muscle, as in skin (1) and central nervous system (7), the interruption of axonal transport in a nerve fascicle causes the terminal field of its fibers to be invaded by collateral outgrowths from adjacent unaffected neurites. (iii) The induction of intramuscular collateral sprouting is not dependent on the presence of denervated muscle fibers, degenerated nerve fibers, or proliferated Schwann cells as had been suggested. Intramuscular collateral sprouting has been elicited in several ways: by partial denervation (5), by denervation of the contralateral homologous muscle (19), by prolonged inactivation (3), and by blockade of axonal transport (present study). Consequently, the induction of sprouting cannot be explained solely by products of denervation, loss of neuromuscular function, or development of extrajunctional sensitivity to ACh. Although the mechanism by which the density of the peripheral innervation is regulated remains unknown, the present observations indicate that it must be based on an interaction between factors produced in the periphery and factors transported axonally to or from the periphery, as Diamond (4) suggested. The precise nature of the axonally transported materials, and the mechanisms by which they regulate the density of innervation of peripheral fields, are neurobiological problems of considerable current importance. REFERENCES 1. AGUILAR, C. E., M. A. BISBY, E. COOPER, AND J. DIAMOND. 1973. Evidence that axoplasmic transport of trophic factors is involved in the regulation of peripheral nerve fields in salamanders. J. Physiol. (London) 234: 449-464. 2. ALBUQUERQUE, E. X.. J. E. WARNICK, J. R. TASSE. AND F. M. SANSONE. 1972. Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat. E-1-p.N~rrrol. 37: 607-634. 3. BROWN, M. C., AND R. IRONTON. 1977. Motor neuron sprouting induced by prolonged tetrodotoxin block of nerve action potentials. Natlrre (London) 265: 459-461. 4. DIAMOND, J., E. COOPER, C. TURNER. AND L. MACINTYRE. 1976. Trophic regulation of nerve sprouting. Science 193: 371-377. 5. EDDS. M. V., JR. 1953. Collateral nerve regeneration. Q. Rev. Bid. 28: 260-276. 6. GOLDBERGER. M. E.. AND M. MURRAY. 1974. Restitution of function and collateral sprouting in cat spinal cord-deafferented animal. J. Camp. Neural. 158: 37-54. 7. GOLDOWITZ, D., AND C. W. COTMAN. 1979. Evidence that neurotrophic interactions control synapse formation in the adult rat brain. Brain Res. (in press) 8. GOSHGARIAN, H. G. 1977. A rapid silver impregnation for central and peripheral nerve fibers in paraffin and frozen sections. E.rp. Nefrrol. 57: 296-301. 9. GUTH. L. 1956. Regeneration in the mammalian peripheral nervous system. Physiol. Re~j. 36: 441-478. 10. HINES. H. M., W. H. WEHRMACHER, AND J. D. THOMSON. 1945. Functional changes in nerve and muscle after partial denervation. Am. J. Physiol. 145: 48-53.

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I?. LUBINSKA, L., AND S. NIEMIERKO. 1971. Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves. Brcrin Rcs. 27: 329-342. 13. LUBLNSKA, L.. S. NIEMIERKO. B. ODERFIELD. AND L. SZWARC. 1964. Behaviour of AChE in isolated nerve segments. .I. Nerrrochcnt. 11: 493-502. 14. LYNCH. G.. R. SMITH. AND C. W. COTMAN. 1975. Recovery of function following brain damage: a consideration of some neural mechanisms. In A. TOBIAS AND A. BUERGER. Eds., Thr Nc,rrroph~sio/o~~i~trl Btlxi.s C$ Rehtrhi/irtrri\‘c, ,Mc~c/icirzc~. Charles C Thomas. Springfield, Illinois. IS. MCCOUCH. G. P.. G. M. AUSTIN. C. N. LIU. AND C. Y. LIV. 1958. Sprouting as a cause of spasticity. J. Nertrophy.\iol. 21: 205-216. 16. MURRAY. J. G.. AND J. W. THOMPSON. 1957. The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat. J. Ptvsid. (London~ 135: 133- 162. 17. MURRAY. M., AND M. GOLDBERCER. 1974. Restitution of function and collateral sprouting in cat spinal cord: the partially hemisected animal. J. Camp. Nc~~ro/. 158: 19-36. 18. RAISMAN. G. 1969. Neuronal plasticity in the septal nuclei of the rat. Bruin Rr.5. 50: 241-264. 19. ROTSHENKER, S. 1979. Synapse formation in intact innervated cutaneous-pectoris muscles of the frog following denervation of the opposite muscle. J. Phx.sio/. tlordon) 292: 535-547. 20. SPERRY, R. W. 1945. The problem of central nervous reorganization after nerve regeneration and muscle transposition. Q. Ret,. Bid. 20: 3 I l-269. 21. TIEDT, T. N.. E. X. ALBUQUERQUE, AND L. GUIH. 1977. Degenerating nerve fiber products do not alter physiological properties of adjacent innervated skeletal muscle fibers. Scirncr 198: 839-841. 22. TIEDT, T. N.. P. L. WISLER, AND S. G. YOUNKIN. 1977. Neurotrophic regulation of resting membrane potential and acetylcholine sensitivity in rat extensor digitorum longus muscle. E.rp. Nerrrol. 57: 766-791. 23. VAN HARREVELD. A. 1945. Re-innervation of denervated muscle fibers by adjacent functioning motor units. Am. J. Ptf~.sio/. 144: 477-493. 24. WEDDELL. G.. L. GUTTMANN. AND E. GUTMANN. 1941. The local extension of nerve fibres into denervated areas of skin. J. Nerrrol. Psychicrr. 4: 206-225. 25. WEISS. P.. AND M. V. EDDS. JR. 1946. Spontaneous recovery of muscle following partial denervation. Am. .I. Physiol. 145: 587-607. 26. WINDLE, W. F. 1956. Regeneration of axons in the vertebrate central nervous system, Physid.

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