MOTOR FUNCTION IN ABBE FLAPS

A Histoehemieal Study of Motor Reinnervation in Transplanted Muscle Tissue of the Lips in Man

By NOEL THOMPSON, F.R.C.S., and A. C. POLLARD, M.A., B.Sc., M.B., B.S. 1

From the Plastic Surgery and Jaw Injury Unit, and the Department of Morbid Anatomy, Stoke Mandeville Hospital, Aylesbury, Bucks.

EVIDENCE is submitted in the following report supporting the concept of motor reinnervation of arterialised transplants of the orbicularis oris muscle in human beings.

Abbe in 1898 first described the operation whereby the tight upper lip, sometimes resulting from the surgical repair of hare lip, may be corrected by the insertion into it of a full-thickness segment of the relatively full lower lip. Such a flap contains all tissues of the lip-skin, orbicularis oris muscle, and oral mucosa, and is transferred in two stages so planned that while an adequate blood supply is preserved throughout, total denervation is inevitable (see Fig. I).

The good function obtained following the use of such flaps has been reported (Ashley, 1955), and recently Gillies and Millard (I957) have recorded their clinical impression that the transposed muscle can become reanimated in its new site. It is indeed not unusual to derive such an impression from the examination of a fully established Abbe flap (see Fig. 2).

Electromyographic investigation has supported the concept of motor reinnervation occurring in similar flaps (Depalma et al., 1958), but yields results difficult of interpretation by reason of the spontaneous action-potentials arising in denervated muscles, electrical interference from the actively contractile muscle in the lateral lip elements, and the effects of tension exerted on the flaP bY l~he latter (Hodes, 1953).

No record has been discovered of any histological evidence relating to possible motor nerve regeneration in Abbe flaps. Such an investigation could not in any case be regarded as simple : experimental muscle denervation in animals has been variously described as ending in complete fibrous replacement of muscle (Tower, 1935), and as preserving throughout the essential histological features of striated muscle despite severe atrophy of the fibres (Sunderland and Ray, 1950), while in human subjects, although shrinkage of muscle fibres is very marked, cross-striation may still be detected even after twenty-four years (Bowden and Gutmann, 1944).

It is uncertain how long motor end-plates persist after denervation of skeletal muscle. Using silver-impregnation methods, in rabbits the end-plate may remain intact though shrunken as long as a year after denervation (Gutmalm and Young, 1944), while in human beings the sarcoplasm of the end-plates progressively disappears after four months, and identification of the end-plate nuclei becomes increasingly difficult after a year (Bowden and Gutmann, 1944).

More recently, histochemical staining methods for locating enzymes have become established for the direct visualisation of the cholinesterase of motor

1 Present appointment : Assistant Pathologist, Courtauld Insti tute of Biochemistry, Middlesex Hospital, London, W.I .

66

MOTOR FUNCTION IN ABBE FLAPS 6 7

end-plates (Gomori, 1948 ; Koelle, 195o; Holt, 1954). Cholinesterase is demonstrable as being so highly concentrated at the level of the neuromuscular junction as to be regarded as capable of hydrolysing the acetylcholine released at each nervous impulse initiating muscular contraction during the brief refractory period (Couteaux, 1955).

No published account has been traced stating the period for which

' V;

L~

FIG. I

Case I. Showing stages in the Abbe flap procedure. A~ Pre-operative state. Showing scarred and retracted upper lip at age 15 years, following

the repair in infancy of congenital bilateral complete lip clefts. B~ C, D~ Aged 16 years : first stage in Abbe flap procedure. B~ Defect in upper lip created by excision of scar tissue. C, Mobilisation of pedicled full-thickness flap from the lower lip. D, Abbe flap, with a single narrow pedicle maintaining an attachment to the lower lip~ and

preserving intact the left inferior labial vessels~ sutured into the upper lip defect. Donor site in lower lip dosed by direct approximation.

E, Abbe flap in si~u two weeks later~ immediately before division of the arterialised pedicle of the flap at the second stage of the Abbe operation.

F~ Two weeks after completion of the second stage. All tissues of the flap are now totally separated from the lower lip.

(By courtesy of Mr J. P. Reidy)

histochemically detectable cholinesterase activity has been found to persist at the motor end-plates of denervated skeletal muscle in human beings.

Experimental studies using histochemical methods, however, indicate that in the rat the cholinesterase content of the end-plate is decreased by about 7 z per cent. at thirty-nine days following motor nerve section (Kupfer, I951), while in the guinea-pig all cholinesterase disappears from the end-plate forty-five days after denervation (Snell and McIntyre, I956). In both mammals cholinesterase activity is found to be concentrated at the motor end-plates following motor nerve regeneration (Sawyer et al., 195o).

It may therefore seem justifiable to regard the presence of normally striated

68 BRITISH JOURNAL OF PLASTIC SURGERY

muscle fibres without gross atrophy, associated with localised areas of cholinesterase activity having the essential configuration of motor end-plates, as very probably indicating the presence of innervated, functional, skeletal muscle elements.

FIG. 2 Case 6. Showing Abbe flap at nine and a half years after operation.

A, Lips at rest. ]3, Lips pursed voluntarily, showing apparent contraction of the Abbe flap, with eversion

of the free border. On palpation between finger and thumb, the flap tissue appeared to become tense.

(By courtesy of Professor T. 2 9. Kilner and M r Rowland Osborne)

The present report is based on a histological and histochemical investigation of muscle biopsies from Abbe flaps in six human beings, taken at intervals of time varying from eleven months to ten years after separation of the flap from the donor lip.

MATERIAL

Six patients, all of whom had received primary operations in infancy for closure of complete bilateral congenital clefts of lip and palate, had in later life each received an Abbe operation, in two stages separated by an interval of two weeks, for the transfer of a full-thickness segment of the lower lip into the upper lip (see Fig. I).

Subsequently, after periods varying from eleven months to ten years, at the time of minor secondary surgical procedures being carried out, biopsies (6 by 4 by 2 mm.) were taken, under vision, from the muscle of the Abbe flap, and also (in all except Case I) from the muscle of the normal lateral upper lip elements ; the latter served as a useful control.

In all cases a preliminary clinical assessment of sensory regeneration in the skin of the flap was made: this disclosed normal or almost normal sensation to

MOTOR FUNCTION IN ABBE FLAPS 6 9

pin-prick and light touch, but a reduction of about 30 to 50 per cent. in two-point tactile discrimination in all cases.

Case I.--B. L., female, aged 26. Abbe flap operation at age 16 (Fig. I) ; biopsy ten years later. Clinical movement in the flap was doubtful.

Case 2.--B. B., female, aged 14. Abbe flap operation at age 13 ; biopsy eleven months later. Clinical movement in the flap was absent.

Case 3.--B. P., male, aged 21. Abbe flap operation at age 19 ; biopsy twenty-one months later. Clinical movement in the flap appeared definite.

Case 4.--D. H., male, aged 20. Abbe flap operation at age 16 ; biopsy three and a half years later. Clinical movement in the flap appeared probable.

Case 5.--A. E. M., male, aged 24. Abbe flap operation at age 17 ; biopsy seven years later. Clinical movement in the flap was absent.

Case 6.--G. F., male, aged 31. Abbe operation at age 21 ; biopsy nine and a half years later. Clinical movement in the flap appeared definite (Fig. 2).

METHODS

Each muscle biopsy was divided into two equal portions on the operating table, and each portion immediately fixed and treated as follows :m

I. After fixation in ice-cold formol-calcium (4 per cent. w/v formaldehyde containing I per cent. w/v calcium chloride) for twenty-four hours, and subsequent treatment with gum-sucrose solution (Holt et al., I96O), frozen sections were cut at 25 to 4 °/z thickness. The sections were stained for cholinesterase to demonstrate the end-plates by a modification of Koelle's method (Gomori, I952), incubation with the substrate being continued until sufficient intensity for photomicrography had been obtained (30 to 6o rain.).

Parallel studies on similar frozen sections, using 5-bromo-4-chloroindoxyl acetate as a substrate (Holt, I958), were also carried out and hmmatoxylin and eosin-stained sections were prepared from the residual formol-calcium fixed material.

2. Following fixation in Bouin's fluid, paraffin sections were cut of thickness 7"5 ~ and 2. 5 /z, and stained for nerve axons using Bodian's silver protargol technique.

RESULTS

The sections stained by KoeUe's technique to demonstrate cholinesterase (Figs. 3, 4, 5, n and B, 6, n and B, 7, n and B, 8) showed that many motor end-plates were visible in both the normal and transplanted muscle, with no obvious numerical reduction in the latter as compared with the control; the marked irregularity of distribution of end-plates throughout both series of sections rendered impossible any precise comparison on this score. The sections stained by the indoxyl acetate method afforded substantially the same appearances as those obtained by the Koelle technique.

7 ° BRITISH JOURNAL OF PLASTIC SURGERY

Given the same incubation time, no difference in staining intensity of the end-plates as a whole was evident in the two series, although at times the end-plates in the transplanted muscle appeared to be a little more diffuse and the sites of activity more fragmentary than in normal control sections ; these appearances were most evident in the youngest transplant, aged I I months (see Fig. 4), but elsewhere did not appear to be correlated with the age of the transplant. Two motor end-plates innervating a single muscle fibre were clearly demonstrable only in Case 4 where the Abbe flap had been completed three and a half years before biopsy ; such an aberration of motor reinnervation is, however, well recognised as occurring in formerly paralysed muscle in animals, as indeed are minor abnormalities in the histological appearances of the end-plates themselves (Gutmann and Young, I944 ; Aitken, I95O ; Hoffman, I95I).

Sections stained by routine hmmatoxylin and eosin methods disclosed that, on the whole, more fibrous and connective tissue was present in the transplanted tissues than in the controls, but the relative proportions of muscle and connective tissue in the biopsies from both regions proved very variable. However, whereas muscle fibres in the normal lip were of fairly uniform diameter, fibres from the transplanted muscle often displayed considerable variation in size, and also in intensity of staining. In some cases, however, the appearances following routine staining of muscle fibres in transplant and control proved virtually indistinguishable, even after ten years (see Fig. 3).

Sections stained by Bodian's technique demonstrated that nerve axons were plentiful throughout the muscle transplants, ramifying parallel to, and over, the muscles fibres. These axons appeared not to branch before terminating on the muscle fibre in a fine arborisation ending in small "but tons " (see Fig. 7c). Although nerve fibres were not shown to enter stained end-plates by the techniques used, it remained probable that the nerve fibres demonstrated were motor in type.

Thus, within the limits of the material examined, and the histochemical and histological methods employed, the muscle of the Abbe flaps appeared almost indistinguishable from the functionally normal muscle of the lip.

DISCUSSION

It is now generally held that in human subjects the muscle cells in free muscle grafts (which from the outset are wholly detached from the donor site) " invariably die, are replaced by fibrous tissue, and fail to resume their normal active function,"

Fig. 3.--Case I. Muscle from transplant, taken ten years after operation. Muscle fibres show essentially normal appearance. Stained hmmatoxylin and eosin, x i5o.

(Section by courtesy of Dr H. ~. Harris)

Fig. 4.--Case 2. Muscle from transplant, taken eleven months after operation. Showing sites of cholinesterase activity at motor end-plates. Stained Koelle's method.

(Not counter-stained.) x 30o.

Fig. 5.--Case 3. A, Muscle from normal upper lip. B, Muscle from transplant, at twenty-one months after operation. Showing sites of cholinesterase activity at motor

end-plates. Stained Koelle's method. (Not counter-stained.) x 3o0.

Fig. 6 .~Case 4. A, Muscle from normal upper lip. B, Muscle from transplant, at three and a half years after operation. Showing sites of cholinesterase activity at motor

end-plates. Stained Koelle's method. (Not counter-stained.) x 3oo.

MOTOR F U N C T I O N IN ABBE FLAPS 71

See opposite page for flegends] FiGs. 3 to 6

72 BRITISH JOURNAL OF PLASTIC SURGERY

FIGS. 7 and 8

Fig. 7A.--Case 5. Muscle from normal upper lip. Fig. 7B.--Case 5. Muscle from transplant, at seven years after operation. Showing sites of cholinesterase activity at motor end-plates. Stained Koelle's method. (Not

counter-stained.) × 30o.

Fig. 7c.--Case 5. Muscle from transplant, at seven years after operation. Stained by Bodian's technique to demonstrate nerve axons. Arrow indicates site of terminal arbor-

isation over a muscle fibre. (Not counter-stained.) × 5o0. Fig. 8.--Case 6. Muscle from transplant, at nine and a half years after operation. Showing sites of cholinesterase activity at motor end-plates. Stained Koelle's method.

(Not cotmter-stained.) x 3o0.

with the possible exception of a few peripherally placed cells able to obtain immediate nourishment from local host tissues (Peer, I955). Muscle deprived of its blood supply will degenerate within a few hours, but muscle fibres permanently deprived of their motor nerve supply only undergo progressive atrophy very much more slowly. Such a denervated muscle may have some function restored at any time before complete atrophy by the ingrowth of motor nerve fibres arising from the original, or some other, motor nerve cells (Ranson and Clark, I959). It is indeed believed that good function may be restored in paralysed human muscles even up to one year, provided the regenerating axons can be directed into the

M O T O R F U N C T I O N IN ABBE FLAPS 73

endoneurial tubes formed at the site of the degenerated motor nerves, to reach the muscle (Sunderland, I95o); however, advancing atrophy and fibrosis of the muscle tend to diminish the degree of functional recovery obtained after the third month (Aird and Naffziger, 1953).

Where the regenerating axon passes along the pathway formerly occupied by a degenerated nerve fibre it penetrates into the old end-plate which has persisted, but after long periods of atrophy the regenerating axon may fail to reach the motor end-plate, and a new end-plate is formed where these fine fibres contact the muscle fibre (Gutmann and Young, I944).

The Abbe flap is at all stages satisfactorily vascularised, initially by the inferior labial artery preserved in its pedicle, and later by the direct vascular anastomoses established between the small vessels of the flap and the host tissues of the upper lip, during the period of two weeks elapsing before the pedicle is severed. The flap, which was largely denervated from the outset, becomes totally so when the pedicle is cut. Any innervation of whatever kind which becomes established in the flap can derive only from the ingrowth of nerve elements into the flap tissue from the normal tissues of the lateral lip elements and columella of the nose. That sensory reinnervation of flaps routinely occurs in this way has been long established (Kredel and Evans, I933), and there exists an appreciable body of evidence to suggest that motor reinnervation can similarly occur from the ingrowth of regenerating motor axons into muscle tissue deprived of its nerve supply.

Thus Heineke (I914) first demonstrated that implantation of the central end of an adjacent severed motor nerve into the denervated limb muscles of a rabbit resulted in the restoration of muscular contractions on faradic stimulation of the implanted nerve. Histological confirmation of motor reinnervation of skeletal muscle under such conditions has been repeatedly obtained in many mammals (Erlacher, I9I 5 a, b ; Steindler, 1916 ; Aitken, I95O ; Hoffman, 195I).

In addition to this " neurotisation" of denervated muscle by the implantation of a healthy peripheral nerve, Erlacher (1915 a) in guinea-pigs claimed successful "muscular neurotisation " of a paralysed muscle by the application to it of a pedicled flap--carrying its own blood and nerve supply--from an adjacent normal muscle ; such a procedure bears an evident basic resemblance to the Abbe flap operation, since each brings into direct contact two fully vascularised muscle elements, only one of which is innervated. An ingrowth of invading nerve fibres from the healthy muscle flap into the paralysed muscle resulted in complete functional recovery of the latter, gross and microscopic appearances becoming normal. The outgrowing motor axons spread most rapidly where they entered the endoneurial tubes of the degenerated nerves, but were also capable of direct spread along the paralysed muscle fibres which they then penetrated to form motor end-plates. Erlacher (I915'b) also reported the successful application of this principle to a human being. A girl aged 7 years, whose tibialis anterior muscle was paralysed by anterior poliomyelitis, was treated by the application to the paralysed muscle of pedicled muscle flaps with intact nerve and blood supply, swung from the adjacent healthy peroneus longus and extensor hallucis longus muscles ; function became restored in the tibialis anterior muscle after eight weeks.

Attempts to reinnervate the facial musculature in human cases of facial nerve paralysis, by establishing contact between the paralysed muscles and pedicled muscle strips transposed from the healthy temporal and masseter muscles have

74 BRITISH JOURNAL OF PLASTIC SURGERY

frequently been made, occasionally with reported success (Sheehan, 1935 ; Owens, 1951 ; Brunner, 1951).

It must appear that the circumstances obtaining in the Abbe flap, where at the time of denervation the paralysed but viable muscle tissue is placed in direct contact with the central end of the axons of divided peripheral motor nerves in the lateral lip elements, must afford optimum conditions for the ingrowth of regenerating motor axons into the muscle of the flap before nerve degeneration in the latter is well established.

Lockhart and Bran& (1938) have suggested that human muscle fibres may usually extend the full length of the muscle they constitute, and although observations on animals do not always support this view (Huber, 1916 ; van Harreveld, I947), it has been suggested by Peer (1955) that the lack of success routinely attending all attempts to transplant striated muscle as a free graft may be in*major degree attributable to the transplant being " largely composed of segments of cells rather than complete cell entities." In the Abbe flap, however, the significance of such an hypothesis, if true, would be greatly reduced if regeneration of damaged muscle fibres occurs--whether by anastomosis between the muscle fibres of the flap and the adjacent normal lip, or by ingrowth of fibres from the latter completely through the transplant. Clark (1946 ) has established that in the skeletal muscle of rabbits a considerable capacity for regeneration exists because of the preservation of the endomysial tubes of connective tissue (which enclosed the original muscle fibres) which then formed guiding pathways for the newly growing fibres budding out from the stumps of the old muscle fibres surviving at the margins of the wound. The same writer (Clark, I958) believes that regeneration of human muscle fibres may similarly occur, provided the lesion is so limited that fibrous replacement of damaged tissue does not obliterate the endomysial framework before the outgrowing new fibres have t ime to arrive there.

Additional evidence suggesting that in Abbe flaps muscle fibre continuity may be restored between transplant and adjacent host muscle is afforded by the following results obtained from animal experiments : first, that muscle regeneration proceeds equally well in normal and in denervated muscle (Saunders and Sissons, I953), and secondly that transected skeletal muscles if sutured immediately result in histologically complete restoration of continuity of their constituent fibres within fifteen days (Gay and Hunt, 1954). The limited biopsy material available unfortunately precluded clarification of this issue in the present investigation, in the context of Abbe flaps.

SUMMARY

Following histological and histochemical investigation of muscle biopsies taken from the Abbe flaps and the normal lateral lip elements of six patients, evidence is submitted to support the concept of motor reinnervation occurring in such flaps. Such evidence isbased chiefly on the demonstration in the flaps of:

I. Normally striated skeletal muscle elements. 2. Motor end-plates exhibiting cholinesterase activity of normal intensity. 3. Nerve axons exhibiting some of the characteristics of motor nerve fibres.

MOTOR FUNCTION IN ABBE FLAPS 75

We are greatly indebted to Dr S. J . Holt of the CourtauM Institute of Biochemistry, Middlesex Hospital, London, IY/.1, for laboratory facilities made available, photomicrography, and much helpful advice; to R. P. GouM of the Department of Anatomy, Middlesex Hospital Medical School, London, W. 1, for the preparation of sections treated by Bodian' s technique; and to plastic surgery colleagues at Stoke Mandeville Hospital, who completed earlier operations on the patients reported above.

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