DAMAGE OF THE LATISSIMUS DORSI MUSCLE DUE TO VASCULAR DELAY TECHNIQUE AND PRECONDITIONING FOR THE USE AS AN ASSISTING SKELETAL MUSCLE:

FIRST RESULTS OF AN HISTOMORPHOLOGICAL ANALYSIS

L.-P. Kamolz¹, W. Haslik¹, W. Girsch², H. Lanmüller³, M. Rab², R. Koller² and H. Gruber¹

1 Institute of Anatomy, Department III

2 Department for Plastic and Reconstructive Surgery, Clinic for Surgery

3 Department of Biomedical Engineering and Physics

University of Vienna

SUMMARY

In an experimental series in sheep we wanted to produce a Latissimus dorsi muscle (LDM) capable of performing chronic work immediately after the construction of a skeletal muscle ventricle (SMV). The longitudinal division of the LDM into two branches and mobilization of the entire muscle inorder to achieve vascular delay, followed by muscle preconditioning, produced disastrous results.

STATE OF THE ART

According to literature and plastic surgical clinical practice ”vascular delay” is a well known surgical technique to increase the vascularisation of randomized and mobilized tissues. Concerning the power of chronic stimulated skeletal muscles used in cardiomyoplasty or aortomyoplasty preconditioning of the LDM in situ seems to be beneficial. Both described techniques were used to improve the performance of a chronic stimulated left LDM before its transposition around a biological neo-ventricle anastomosed in parallel to the descending aorta.

MATERIAL AND METHODS

Two adult female sheep, weighing 48 and 51 kg, were used for the experiment.

Stimulation device:

All implanted components were designed and manufactured at the Department of Biomedical Engineering and Physics. The nerve pacing leads are made from stainless-steel stranded wire, coiled and embedded in silicon. The battery-powered pulse generator is hermetically sealed in a titanium case. This newly developed pulse generator can be used for activating two skeletal muscles via the motor nerves, using constant-current impulses with a maximum current of 4 mA at a pulse duration of 0,2 to 1 ms.

Surgical procedure:

The sheep were placed in right side position to performed a lateral flank incision on the left side. The left LDM was detached from the thoracic wall, with all perforating vessels from the intercostal vascular bundles being ligated, while the LDM insertion into the humeral bone was kept unaffected. The thoracodorsal nerve was prepared, with the vascular pedicle being carefully preserved. The pulse generator, already connected to the electrode leads, was placed in a subcutaneous pocket at the back of the contralateral side and the electrode leads to the left side were led under the scapula to the nerve. Four ring-shaped electrodes of an inner diameter of 1 mm were sutured to the epineurium of the nerve in helical manner. After the intramuscular vascular architecture of the left LDM had been identified by translumination, the muscle was divided longitudinally from its caudal end up to the entry of the neurovascular bundle in order to create two muscle branches of equal size. The LDM was reattached to the thoracic wall with absorbable sutures in original position. Muscle biopsies were harvested from the cranial, scapular and caudal parts of the LDM. After skin closure the animals were brought in left side position and a lateral flank incision was performed on the right side. The right LDM, especially its orgin at the thoracic wall, was left untouched. Electrode leads were tunneled from the back to the scapular region of the right side and the electrodes were applied to the thoracodorsal nerve as described above. Muscle biopsies were harvested from the cranial, scapular and caudal parts of the LDM and again, the skin closure was made.

Conditioning protocol:

After a delay of 14 days the conditioning program was started. Stimulation was performed via the implanted stimulation device, which was activated and programmed by the external programmer. Rectangular pulses with a pulse width of 600 ms at a pulse frequency of 26 Hz were used for stimulation. The conditioning protocol was set according to the classic stimulation protocol for cardiomyoplasty by Chachques et al., starting with single pulse chronic stimulation and a biweekly increment in the number of pulses to achieve burst stimulation. The rate of muscle contractions was raised from 35 to 50 contractions per minute in the course of the program. "Carousel-stimulation", a special kind of multichannel stimulation using four stimulation electrodes on each nerve, was applied. In the first step of our experimental sequence we could shown that this stimulation technique leads to a less pronounced loss of muscular force after the end of conditioning. Only two electrodes out of four were activated at a distinct point of time. Selection of activated electrodes was changed automatically from burst to burst and repeated cyclically. Four bipolar combinations of electrodes, producing muscle contractions of equal strength, were selected. During the conditioning, the contraction strength of each electrode combination was evaluated by varying the stimulation current between 0 and 4 mA so that maximum tetanic tension was achieved. Both LDM were conditioned simultaneously regardless of the native heart rate. At the end of the conditioning program the construction and implantation of the SMV was planed. According to an unexpected and significant loss of contraction strength the chronic experiment was stopped and therefore no SMV implantation was performed. The two sheep were sacrificed in order to examine the LDM of the left and right side. Both LDMs were completely detached from their origin and insertion. Three biopsies were harvested from the cranial, scapular and caudal part of each muscle.

Histomorphological analysis:

A histomorphological analysis of the right and left LDM was performed. The muscles biopsies were cut into transverse slices with a razor blade. The slices were put on cork discs and immediately snap-frozen at -80°C in isopentane cooled by dry ice and stored at -80°C until use. Serial transverse cryosections of 10 µm thickness from each slice were stained for the following histochemical methods: actomyosin ATPase after alkaline (pH 10,2) and acid (pH 4,3) preincubation according to Guth and Samaha and NADH tetrazolium reductase according to Dubowitz and Pearse. All stained sections were examined by light microscopy at a 100 fold magnification and the resulting random fields were displayed on the monitor of a personal computer by means of a video camera adjusted to the microscope. The histomorphometric measurements and counts were performed with a pen linked to the personal computer using a semi-automatic image-analyzing system. More than 300 muscle fibers per three random fields were counted in each section. After comparison of the serial sections stained for actomyosin ATPase with alkaline (pH 10,2) and acid (pH 4,3) preincubation and for NADH tetrazolium reductase, the muscle fibers were divided into Type I and Type II. The classification into the subtypes of the Type II muscle fibers, i.e.Types IIa and IIb, was performed on sections stained for NADH tetrazolium reductase enzyme activity. The following morphometric parameters were determined for the LDM:

· the percentage of Type I, Type IIa and IIb muscle fibers in relation to the number of muscle fibers counted

· the equivalent diameter of Type I, Type IIa and IIb muscle fibers in µm

· the percentage of perimysial and endomysial connective tissue in relation to the measured area of the section

RESULTS

The conditioning protocol could be performed without any problems for the first four weeks in sheep 1 and for six weeks in sheep 2. When the stimulation frequency was raised from 2 to 3 impulses in sheep 1 and 3 to 5 impulses in sheep 2, the left LDM, which was splited and detached, showed signs of fatigue: contrary to the sudden drop in contraction strength known to occur when unconditioned muscles are strained, a gradual decrease in contraction strength over serveral days took place. The stimulation threshold of the nerve did not change and a rise in the stimulation amplitude did not lead to an increase in muscle force. Considering overstimulation we stopped the conditioning of the left LDM. On the other hand the right muscle showed no signs of fatigue. Conditioning was continued following the original protocol without any delays. After a four week phase for regeneration, conditioning of the left LDM was resumed at lower increments. No more fatigue occurred, contraction strength was visibly reduced, though. According to the significant loss of contraction strength no SMV implantation was performed in both sheep. The examination of the left LDM revealed severe signs of degeneration: in comparison with the right side we found a distinct atrophy of contractile muscle tissue and an strong increase of the perimysial connective tissue. During electrical stimulation no contraction was detected in the caudal part of the left LDM, whereas in the most cranial part muscle-contraction could be observed. Macroscopically the right LDM revealed no signs of degeneration and contractions of all parts of the muscle were detected during electrical stimulation.

Histomorphological analysis:

Morphologically the right LDM showed signs of a completely transformed skeletal muscle. The percentage of the Type I fibers was 100% in the cranial and scapular part of the LDM. The caudal part revealed 96.04% Type I, 1.22% Type IIa and 2.74% Type IIb muscle fibers. The mean diameter of the Typ I fibers was 40.38µm in the cranial and 39.06µm in the scapular part of the LDM. In the caudal part the mean diameter of the Typ I fibers revealed 27.50µm, of the Typ IIa 39.45µm and of the Typ IIb 21.50µm. The percentage of the peri-and endomysial connective tissue was 18.28% in the cranial, 19.63% in the scapular part, and 26.71% in the caudal part of the muscle. We found also a distict fiber transformation in the cranial and scapular part of the left LDM, allthough a high increase in the perimysial-and endomysial connective tissue together with a complete loss of muscle fiber architecture was evident. In the caudal part of the left LDM a histomorphological analysis according to the method decribed above was not possible. Beside the high increase of the connective and fatty tissue the muscle fibers showed typical signs of degeneration or necrosis. In the cranial part we found 93.65% Type I and 2.61% Type IIa muscle fibers. In the scapular part of the left LDM we measured 74.01% Type I and 25.99% Type IIa fibers. The mean diameter of the Typ I fibers revealed 14.52µm in the cranial and 46.47µm in the scapular part of the muscle. The mean diameter of the Typ IIa fibers revealed 23.97µm in the cranial and 33.99µm in the scapular part. The percentage of the peri-and endomysial connective and fatty tissue was 68.94% in the cranial and 47.51% in the scapular part of the left LDM.

DISCUSSION

Chronical FES changes muscle morphology but does not damage the muscle tissue, provided adequate stimulation parameters are applied. This fact has been reported in literature and is also shown in our results of conditioning of the right LDM. It was the increase from about 1 to 2 Hz of chronic stimulation that led to severe damage of the mobilized parts of the muscle. The increment triggered a homogenous degeneration of the muscle fibers. The results of this analysis clearly demonstrate that the combination of ”vascular delay” and preconditioning of a skeletal muscle at the same time before using in cardiomyoplasty or aortomyoplasty is contraproductive for its morphological outcome.

REFERENCES

1. Magovern GJ Sr, Simpson KA. Clinical cardiomyoplasty: review of the ten-year United States experience. Ann Thorac Surg. 1996; 61(1): 413-9

2. Chachques JC, Marino JP, Lajos P, et al. Dynamic cardiomyoplasty: clinical follow-up at 12 years. Eur J Cardio Thorac Surg. 1997; 12 (4) : 560-567

3. Chachques JC, Radermercker M, Tolan MJ, et al. Aortomyoplasty counterpulsation: experimental results and early clinical experience. Ann Thorac Surg. 1996; 61(1): 420-5

4. Salmons S, Jarvis JC. Cardiac assistance from skeletal muscle: a critical appraisal of the various approaches. Br Heart J. 1992; 68(3): 333-8

5. Thomas GA; Isoda S; Hammond RL; et al. Pericardium-lined skeletal muscle ventricles: Up to two years' in-circulation experience Ann Thorac Surg. Dec 1996; 62 (6) : 1698-1706

6. Koller R, Girsch W, Huber L, et al. Influence of different conditioning methods on force and fatigue resistance in chronically stimulated skeletal muscles. Pace. 1996; 19(2): 222-30

7. Barron-DJ; Etherington-PJ; Winlove-CP; Pepper-JR Regional perfusion and oxygenation in the pedicled latissimus dorsi muscle flap: the effect of mobilisation and electrical stimulation. Br-J-Plast-Surg. 1997 Sep; 50(6): 435-42

8. el-Oakley-RM; Jarvis-JC; Barman-D; Greenhalgh-DL; Currie-J; Downham-DY; Salmons-S; Hooper-TL Factors affecting the integrity of latissimus dorsi muscle grafts: implications for cardiac assistance from skeletal muscle. J-Heart-Lung-Transplant. 1995 Mar-Apr; 14(2): 359-65

9. Girsch-W; Koller-R; Lanmuller-H; Rab-M; Avanessian-R; Schima-H; Wolner-E; Seitelberger-R Experimental development of an electrically stimulated biological skeletal muscle ventricle for chronic aortic counterpulsation European-Journal-Of-Cardio-Thoracic-Surgery. Jan 1998; 13 (1) : 78-83

10. Koller R, Girsch W, Liegl C, et al. Long-term results of nervous tissue alterations caused by epineurial electrode application: an experimental study in rat sciatic nerve. Pace. 1992; 15(1): 108-15

11. Mayr W, Bijak M, Girsch W, et al. Multichannel stimulation of phrenic nerves by epineural electrodes. Clinical experience and future developments. ASAIO J. 1993; 39(3): M729-35

12. Guth L, Samaha FJ: Procedure for the histochemical demonstration of actomyosin ATPase. Exp-Neurol. 1970 Aug; 28(2): 365-7

13. Dubowitz, V. Pearse, A.G.E. (1961) Enzymic activity of normal and diseased human muscle: a histochemical study. Journal of Pathology and Bacteriology, 81,365-378.

14. Taylor-GI; Corlett-RJ; Caddy-CM; Zelt-RG An anatomic review of the delay phenomenon: II. Clinical applications. Plast-Reconstr-Surg. 1992 Mar; 89(3): 408-16; discussion 417-8

AUTHOR´S ADDRESS

Lars-Peter Kamolz,

resident at the Institute of Anatomy, Department III

Waehringerstrasse 13, A-1090 Vienna,

e-mail: lars.peter.kamolz@unvie.ac.at