Neuroscience Letters: 6 (1977) 41--46 ~ Elsevier/North-Holland Scientific Piiblishers Ltd.

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LIPOPHILIC NERVE SPROUTING FACTOR(S) ISOLATED FROM DENERVATED MUSCLE

CHARLES D. TWEEDLE and JON J. KABARA

Dept. of Biomechanics, Michigan State University, E. Lansing, Mich. 48824 (U.S.A.)

(Received April 4th, 1977) (Accepted July 7th, 1977)

SUMMARY

This report confirmed earlier studies by other workers which suggested that collateral nerve sprouting in mammalian skeletal muscle is induced by factors released from denervated or atrophic muscle fibers. Crude lipid ex- tracts of denervated but not of normal muscle were found to bring about histological evidence of significantly increased collateral nerve outgrowth when injected into normal muscles. Additional experiments indicated that separated non-polar and polar lipid fractions of muscle were less active. The injection of a chemically pure lipid, monlaurin, also caused significant sprout- ing.

Earlier reports have suggested that lipid extracts of denervated mammahan skeletal muscle [10], or ~f certain other tissues [5], may induce or enhance collateral nerve outgrowth ~n skeletal muscle. We have confirmed and extend- ed these findings by using crude and separated lipid extracts of normal or denervated skeletal muscle injected into normal tongue muscle. Nerve sprout- ing was assayed histochemically. There was significantly more collateral nerve outgrowth in tongues injected with crude lipid extracts from denervated as compared to non-denervated muscle. Additional experiments with fractionated lipid extracts indicated that the polar lipid components from the denervated muscle contained the highest biological activity. These results, combined with lipid analyses of normal and denervated muscles, suggest that increased amounts of specific muscle lipid classes following denervation may cause sprouting from nearby motor nerve endings.

The right sciatic nerves of nine 250-g Sprague-Dawley male rats were severed at the sciatic notch. Ten days later, the denervated gastrocnemius muscles were removed, cleaned of fat, and extracted with chloroform:methanol (2:1) [ 8]. The crude lipid extract was dried under nitrogen at low temperature (15°C), and emulsified in propylene glycol (10 mg total !ipid/ml). The emulsion was stored at- 20°C. A similar procedure was performed on the contralateral

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'control' gastrocnemivs muscles from the same animals. Twenty microliters of the crude lipid suspension from denervated muscle were then injected with a 27-gauge needle into the center of the tongues of five 200-g rats. Two injec- tions were given~ one at tl~e beginning of the experiment and one two weeks later. Nine other animals received similar injections of crude lipid extract from control muscle. Six a n n a l s received injections of the crude lipid extract from control muscle. Six animals received injections of the carrier (propylene

• glycol) alove. Seven ~nimals were left uninjected as additional untouched con- trois. Food(L~b-Blox) and water were given ad libitum.

Twenty-five days after the first injection, the tongue muscles were removed, fixed in formalin and then frozen. The frozen tongues were then split down the middle with a razor blade. Fifty/Jm thick sections were taken from the cut center svxface (usually E~--6 sections were taken). These were stained for endplates according to the combined nerve/cholinesterase method of Gwyn and Heardman [ 4]. Slides were coded by a disinterested party and the bands of intrinsic tongue musculature on the edge of the section were then assayed for percentage of endplates showing evidence of collateral nerve sprouting." Double endplates, branched endings, preterminal, ultraterminal, and nodal sprouts were counted as visual evidence of collateral nerve sprouting [ 1,9]; see Fig. 1. The data were analyzed using non-parametric statistics (Kmskal- Wallace and Mann-Whitney U-tests). The results of the experiment are present- ed in Table 1.

Significant increases in collateral branching were found only in the group injected with crude lipid extracts from denervated muscle. Neither injected

Fig. 1. Photomicrograph showing the appeara~lce of a pre-terminal collateral sprout (arrow) using the combined silve~e.holinesterase stain. × 500.

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TABLE I

HISTOLOGICAL EVALUATION OF COLLATERAL NERVE SPROUTING INDUCED BY CRUDE LIPID EXTRACTS

Treatment

Untouched control 7 Propylene glycol carrier -6 Control muscle extract 5 Devervated muscle extract 9

Number of animals evaluated

Amount of sprouting (see text)

Number of Number Percentage endplates showing showing examined nerve sprouts ~prouts

687 60 8.7 693 49 7.1 712 65 9.1 "843 122 14.5 a

ap < 0.05 as compared to the propylene glycol carrier and control muscle extract.

control muscle lipid extract nor the carrier vehicle alone brought about in- creased collateral outgrowth as compared to normal sprouting frequency.

In a second series of experiments, in order to determine which general iipid class of the denervated muscle extract contained biological activity, the crude lipid extract was separated into polar and non-polar lipid fractions by silica gel colunm chromatography. In these experiments, nine-day denervated gastrocnemius muscle were obtained as previously described. The crude ex- tract was fractionated into non-polar and polar lipids by the method of Lennarz and Talamo [ 7]. Each fraction was taken up in propylene glycol (10 mg total lipid/ml).

Twenty microliters of the crude lipid as well as the non-polar and polar lipid fractions were individually injected into separate rat tongue muscles at the beginning of the experiment and again after two weeks. Collateral sprouting was assayed in the same manner as in our initial experiments. Table II shows the amount of collateral sprouting induced by the lipid fractions as observed 30 days after the first injection.

As in the initial experiment, injection of the crude lipid fraction from denervated muscle elicited the highest amount of collateral sprouting. Other lipid extracts or carrier alone exhibited lower biological activity. Only the groups of animals injected with the crude lipid fraction of denervated muscle d isplayed a significantly increased amount of collateral outgrowth at the 5% confidence level.

The difference in response after the injection of polar lipids extracted from denervated or control muscle was not significant. Since crude but not the separated lipid extracts indicated a significant difference between control and denervated extracts the active principle in the rauscle extract may have solubility characteristics of both non-polar and polar lipids and may be distributed ~u both fractions. Another explanation for seeing differences between crude lipid extracts of control and denervated muscle may be the

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TABL~ II

NERVE SPROUTING vs LIPID FRACTION INJECTED

Muscle lipid extract Number of animals examined

Amoun~ of sprouting (see text)

Number of Number endpl~tes showing examined nerve sprouts

P~rcentage showing sprouts

: ropylene glycol carrier 10 i666 154 9.2 Control muscle extract 4 5-~7 44 8.0 Denervated muscle extract 6 1113 180 16.2 b'c Control muscle non-polar

lipids 5 8~4 95 10.7 Denervated muscle non-

polar lipids 4 890 103 11.6 Control muscle polar lipids 4 710 87 12.2 Denervated muscle polar

lipids 4 635 94 14.8 a

a p < 0.05 as compared to the propylene glycol carrier. b p ,~ 0.005 as compared to the propylene glycol carrier. c p <: 0.02 as coml:~red to control muscle extract.

need for both classes of compounds to be present for full biological potency. Even though the total non-polar lipid extract was not significantly active,

we had reason to believe that the activity of a subfraction could be masked or lost by the procedure used. Initial attempts to isolate a non-polar lipophilic nerve sprouting factor(s) were made by subfractionating the non-polar lipids by ~hin-layer chromatography. The non-polar lipids were chromatographed on silica gel G plates (Analtech, Inc., Newark, Delaware) using heptane:anhydrous ether:petroleum ether:acetic acid (60:20:20:1) as thb developing solvent. The most consistent finding was an increase in lipids migrating between cholesterol and the origin. In our solvent system, mono- and diglycerides standards migrate in this area. Since monoglycerides, but not diglycerides, were previ- ously shown to be active in other biological systems [6], the most active of these monoglycerides, monolaurin, was injected into tongue muscle using the techniques and concentration previously described. Eleven animals were in- jected and, after 25 days, 1728 e.,~dplates were examined. From this series 276 (16%) displayed nerve sprouts (P < 0.005). The nerve sprouting seen in this group was often quite elaborate and bizarre (Fig. 2). Experiments dealing wi~h subfractions of non-polar as well as polar lipids from denervated muscle are being conducted in order to establish a structure-function relationship between lipid classes and nerve sprouting.

Collateral nerve sprouting has been reported in skeletal muscle following partial denervation [ 3 ], after botulinum toxin poisoning [ 11 ], and after blockage of nerve impulse activity with tetrodotoxin [ 2]. Thus, not only can

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Fig. 2. Photomicrograph showing the elaborate and profuse nerve branching that was sometimes found following injection of monolaurin intc, muscle. × 500.

denervation of muscle elicit nerve sprouting, but it also occurs in at least some instances of muscle disuse atrophy. By combining the present results with those of previous studies, it is possible to support a hypothesis that atrophic changes in muscle may lend to the increased production of levels in lipophilic substances capable of diffusing to intact neuromuscular junctions and eliciting collateral nerve outgrowth. Similar substances also may be involved in collateral sprouting found elsewhere in the nervous system. It is suspected that lipophtlic compounds like monolaurin or other surface active lipids may be inducing collateral nerve outgrowth through direct membrane effects. Studies are cur- rently being carried out to further identifT those lipids which are biologically active and to demonstrate their release frozn muscle,

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ACKNOWLEDGEMENTS

S u p p o r t e d b y N S F G r a n t BNS 76-81406 and N I H B R G S funds .

REFERENCES

1 Barker, D. and Ip, M.C, S p r o u ~ and degeneration of m a m ~ motor axons in normal and de-afferented skeletal muscles, M . roy, Soc. B, 163 (1966) 538--554.

2 Brown, M.C. and Ironton, R.,Motor neurone sprouting induced by prolonged tetrodotoxin block of nerve action p o t e n t ~ , Nature (Lond,), 265 (1977) 454--461.

3 Edds, M.V., Collateral nerve regeneration, ~ . Rev. BieL, 28 (!953) 260--276. 4 Gwyn, D.Go and Hesrdman, V., A c h o l i n ~ B i e l c h o ~ v s k y staining method for

msmm-]~n motor endplates, Stain Technol., 40 (1965) 15--18. 5 Hoffman, H., Local re-innervation in partially denervated muscle: a histophysiological

study, Austr. J. exp. Biol. reed. Sci., 28 (1950) 383--397. 6 Kabara, J.J., Lipids as safe and effective antimicrobial qen ts for cosmetics and

pharmaceuticals, Cosmet. Perfum., 90 (1975) 21--25. 7 Lem..arz, W.J. and Talamo, B., The chemical characterization and enzymatic synthesis

of mannolipids in Microeoccw |ysodefttfcw, J. biol. Chem., 241 (1966) 2707--2719. 8 In J.M. Lowenstein (Ed.), Methods in

~ew York, 1969, pp. 245--254. 9 Tuffery, A.R., Growth and depne~t ion of motor endplates in normal cat hindlimb

muscles, J. Anat- (Lond.), I I 0 (1971) 221--247. I0 Van Harreveld, A., On the meehanimt of spontaneous re-innervation in paretic muscle,

Amer. J. Physiol., 150 (194"7) 670--676. I I Watson, W.E., The response of motor neurons to intramuscular injection of botulinum

toxin, J. Physiol. (Lond.). 202 (1969) 611--630.