Unveiling Nerve Match in Free Gracilis Muscle Transplantation: A Comprehensive Anatomical Investigation

doi:10.21203/rs.3.rs-3874753/v1

Objective: To evaluate the compatibility between common donor nerves such as the accessory nerve, phrenic nerve, and intercostal nerves in terms of cross-sectional area and the number of myelinated nerve fibers, compared with the obturator nerve's anterior branch to the gracilis muscle.

Methods: Six fresh frozen cadavers were dissected to expose the accessory nerve, phrenic nerve, intercostal nerves, and the anterior branch of the obturator nerve to the gracilis muscle. Measurements of cross-sectional area, and the number of myelinated nerve fibers were taken.

Results: The number of myelinated nerve fibers in the gracilis muscle nerve was 2870.56 ± 1216.99, with a cross-sectional area of 0.56 ± 0.24 mm²; the accessory nerve had 2210.55 ± 1461.82 myelinated nerve fibers with a cross-sectional area of 0.43 ± 0.28, giving a fiber ratio of 0.77 compared to the gracilis muscle nerve; the phrenic nerve had 2845.65 ± 765.40 myelinated nerve fibers with a cross-sectional area of 0.55 ± 0.15, giving a fiber ratio of 0.99. The detailed counts of myelinated nerve fibers, cross-sectional areas, and their ratios compared to the gracilis muscle nerve for each intercostal nerve are presented in Table-1 and Table-2.

Conclusion：

This study focuses on optimal nerve matching in functional muscle transplantation for brachial plexus injury reconstruction using FMT. Findings indicate a 0.7 ratio as optimal when using three intercostal nerves, compared to 0.77 and 0.99 for SAN and phrenic nerve matches, respectively.

Nerve match

accessory nerve

phrenic nerve

intercostal nerve

gracilis muscle nerve.

Brachial plexus injuries, particularly total brachial plexus injuries, are highly debilitating conditions. The lack of effective treatment methods remains a global challenge. Currently, the main treatments include nerve transfer and functional muscle transplantation. However, for unsatisfactory upper limb functional recovery following nerve transfer surgery and in cases of chronic brachial plexus injuries, functional muscle transplantation may be the only surgical option[1].

Yang et al. [2] achieved favorable outcomes by combining multiple nerve transfers with free gracilis muscle transplantation in the treatment of brachial plexus injuries.

The gracilis muscle is favored for functional free muscle transplantation due to its consistent anatomical structure in terms of arteries, veins, and nerves, which are easily identifiable. The vascular pedicle and nerve can be harvested at a considerable length. The muscle's shape is similar to the biceps brachii, and it possesses a long tendon that can be extended to the dorsal side of the forearm, connecting to the extensor tendons of the thumb and other four fingers. Additionally, the superficial location of the gracilis muscle and the presence of numerous synergistic muscles mean that its removal has minimal impact on lower limb function. Hence, the gracilis muscle presents unique advantages as a donor site for functional free muscle transplantation, and its clinical application is increasingly widespread[3].

Despite the use of free gracilis muscle transplantation for reconstructing upper limb functions following total brachial plexus injuries, there remain significant controversies. Terzis and colleagues reported suboptimal muscle strength (averaging 2.25 ± 0.6) when using a gracilis muscle innervated by three intercostal nerves, thus not preferring it as their primary choice for transplantation[1]. Chuang [4] documented satisfactory outcomes using the gracilis muscle solely for elbow flexion reconstruction. However, there are doubts about the feasibility of using a single free gracilis muscle transplant to simultaneously reconstruct two functions, such as shoulder abduction with elbow flexion, or elbow flexion with finger flexion.

The essence of free gracilis muscle transplantation is the anastomosis of nerves and vessels in free tissue transplantation.Several factors influence the effectiveness of free gracilis muscle transplantation. These include reducing the distance and time for nerve reinnervation, avoiding the use of grafts, tension-free nerve suturing, preventing misdirected axonal regrowth, and particularly, the optimal compatibility between donor and recipient nerves, which is a crucial factor for functional recovery of the transplanted gracilis muscle. All intraplexal and extraplexal nerves can serve as donor nerves for the gracilis muscle[5]. However, for patients with total brachial plexus injuries, the sources of donor nerves are limited. According to literature, the accessory nerve[6], intercostal nerves[7], phrenic nerve[3], and the healthy contralateral C7 nerve [2]are the primary donor nerves for gracilis muscle free transplantation in patients with total brachial plexus injuries.

Therefore, this study utilizes fresh cadavers to conduct a match analysis, including the count of myelinated nerve fibers and cross-sectional area, between donor nerves such as the accessory nerve, phrenic nerve, and intercostal nerves, and the anterior branch of the obturator nerve innervating the gracilis muscle.

1. Materials:

Six fresh frozen cadavers were utilized for this research. The cadavers were positioned supine, and anatomical dissections simulating surgical procedures were performed by the same operator, cutting the nerves at the plane of neural anastomosis. Specimens from both the left and right sides were dissected and harvested: the accessory nerve, phrenic nerve, intercostal nerves (3–6), and the anterior branch of the obturator nerve to the gracilis muscle.

2. Nerve Match Study:

0.5 cm segment of each nerve was fixed in 10% formalin, dehydrated, and embedded in paraffin for sectioning, with sections approximately 5 µm thick. Nerve samples were stained using Sihler's chromalum staining. Photographs of the sections were taken using a microscope with a scale (Olympus BX51, Olympus Corporation, Japan). The cross-sectional area of the nerve fibers was measured using Photoshop software, and the number of myelinated nerve fibers was calculated with computer assistance.

3. Statistical Analysis:

Statistical analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The results are presented as mean ± SD. The correlation between the ratio of donor nerve to gracilis muscle nerve and the muscle strength reported in the literature review was analyzed using linear regression analysis to fit the model. The dependent variable in this analysis was muscle strength outcomes, and the independent variable was the nerve specimen count ratio.

A total of 12 sides from 6 specimens were analyzed. The gracilis muscle nerve had an average of 2870.56 ± 1216.99 myelinated nerve fibers, with a cross-sectional area of 0.56 ± 0.24 mm². The accessory nerve had 2210.55 ± 1461.82 myelinated nerve fibers, with a cross-sectional area of 0.43 ± 0.28, resulting in a myelinated nerve fiber ratio of 0.77 compared to the gracilis muscle nerve. The phrenic nerve had 2845.65 ± 765.40 myelinated nerve fibers, with a cross-sectional area of 0.55 ± 0.15, yielding a ratio of 0.99. For the intercostal nerves, which were harvested as mixed branches, the count of motor nerve fibers was calculated based on the proportion reported by Samardzic et al.[8], where 30% of the total nerve fibers in each intercostal nerve are motor fibers. Detailed counts of myelinated nerve fibers, cross-sectional areas, and their ratios compared to the gracilis muscle nerve for each intercostal nerve are presented in Table-1 and Table-2.

Appropriate nerve matching is a crucial criterion in nerve suturing, yet the optimal ratio of myelinated nerve fibers and cross-sectional area between donor and recipient nerves has been less studied, particularly in functional muscle transplantation. Schreiber et al., through anatomical studies and literature reviews, reported that for C5-C6 brachial plexus injuries, using the ulnar nerve fascicle to repair the biceps brachii branch (Oberlin procedure) for elbow flexion reconstruction, the myelinated nerve fiber ratio between the donor and recipient nerves should exceed 0.7:1 to ensure functional recovery[9]. Oberlin and colleagues noted that when using ulnar nerve fascicle transfer to reconstruct elbow flexion in C5-C6 brachial plexus injuries, the cross-sectional area of the recipient nerve is approximately 7–17% of that of the donor nerve[10]. Furthermore, studies indicate that even under optimal suture and regeneration conditions, at least 25% of nerve fibers are lost at the anastomosis site. Therefore, this factor should be considered when selecting the number of myelinated nerve fibers from the donor nerve during nerve anastomosis.

Gutowski et al. reported the count of myelinated nerve fibers in the accessory nerve to be 1700[11]; Bhandari et al. found it to be 1671[12]; Pruksakorn et al. reported 1603 myelinated nerve fibers in the accessory nerve[13]; while Vathana et al. noted a count of 1054[14]. These results show considerable variability. In our study, the accessory nerve had 2210.55 ± 1461.82 myelinated nerve fibers, with a cross-sectional area of 0.43 ± 0.28 mm². When compared to the nerve innervating the gracilis muscle, the ratio was found to be 0.77, similar to the 0.7 reported by Schreiber et al. Among patients using the accessory nerve as the donor nerve, 85% achieved good to excellent elbow flexion strength and range of motion[2]. Given the simplicity of the accessory nerve's anatomy, absence of significant donor site complications post-harvesting, sufficient length for harvesting, rapid recovery, and a ratio greater than 0.7, we recommend the accessory nerve as the donor nerve for free gracilis muscle transplantation in reconstructing upper limb function following total brachial plexus injuries.

Wang et al. reported the count of motor fibers in the phrenic nerve ranging from 3078 ± 684 to 4794 ± 638[15]. In our study, the phrenic nerve had 2845.65 ± 765.40 myelinated nerve fibers, with a cross-sectional area of 0.55 ± 0.15 mm², resulting in a ratio of 0.99 when compared to the nerve innervating the gracilis muscle. This indicates that the phrenic nerve can also serve as an effective alternative donor nerve for free gracilis muscle transplantation. However, the use of the phrenic nerve as a donor nerve might impact respiratory function in elderly patients, which is a factor that surgeons need to consider.

This study also explores the match between the intercostal nerves and the anterior branch of the obturator nerve to the gracilis muscle. Samardzic et al. in 1986 reported that the 2nd to 4th intercostal nerves have 520–720 myelinated motor nerve fibers[8]; Malungpaishrope et al. reported the myelinated nerve fiber counts of the 3rd, 4th, and 5th intercostal nerves as 742, 830, and 1353 respectively, with cross-sectional areas of 77747 ± 10737, 95150 ± 15515, 160202 ± 8505 um²[16].

Coulet [17] et al. reported on seven patients with total brachial plexus injuries who underwent free gracilis muscle transplantation using the 3rd, 4th, and 5th intercostal nerves as donor nerves, achieving an average muscle strength of 2.9 ± 1.3. In a large series reported by Kovachevich[18]et al., where intercostal nerves were used as donor nerves for gracilis muscle transplantation, the 2nd, 3rd, 4th, 5th, and 6th intercostal nerves were all viable options. However, the use primarily focused on the 3rd to 6th intercostal nerves, with the 2nd to 4th intercostal nerves accounting for 94% of the cases.

Chuang[7]suggests that using two intercostal nerves as donor nerves for gracilis muscle transplantation may prolong the time of functional recovery and reduce the effectiveness of functional restoration. Therefore, he recommends using three intercostal nerves as donor nerves. Terzis et al. reported better outcomes using three intercostal nerves as donor nerves for latissimus dorsi muscle transplantation to reconstruct elbow flexion, compared to using two intercostal nerves [1]. Hattori[19] et al. reported on the use of the 5th and 6th intercostal nerves as secondary donor nerves for gracilis muscle transplantation to reconstruct finger flexion. Barrie[6]et al. reported on using the 3rd and 4th, 4th and 5th, or 5th and 6th intercostal nerves as primary or secondary donor nerves for gracilis muscle transplantation to restore upper limb functions after total brachial plexus injuries. In our study, the motor fiber count of intercostal nerves ranged from 615.72 to 721.46, with a total cross-sectional area of 0.40–0.47 mm². When using two intercostal nerves as donor nerves, the ratio is relatively low, less than 0.5, whereas using three intercostal nerves, the ratio can reach 0.7.

We also conducted a literature analysis, due to inconsistencies in muscle strength evaluation standards across various studies, we chose BMRC ≥ 4 as our inclusion criterion, as it is a widely accepted standard. Moreover, our analysis was limited to the correlation between myelinated nerve fiber count and muscle strength. Factors such as the time from injury to surgery, the distance of nerve regeneration, and the general condition of the patient were not included, potentially causing some variability in the results. We lacked data on the cross-sectional area and myelinated nerve fiber count when using the contralateral lateral pectoral nerve and ulnar nerve as donor nerves, as reported in the literature, preventing further analysis. Some limitations existed in the analysis of the correlation between the ratio of donor nerve to gracilis muscle nerve and muscle strength from the literature review. For instance, some articles did not clearly describe the donor nerves, preventing data extraction and reducing the analyzable sample size. This led to a statistically non-significant result (P = 0.230), even though the nerve count could explain 59.3% of the variability in muscle strength outcomes. Considering that the ratio of donor nerve to gracilis muscle nerve can reach 0.7 when using three intercostal nerves, and 0.77 and 0.99 respectively when using the accessory nerve and phrenic nerve, all above 0.7, we recommend that the ratio of myelinated nerve fibers between the donor and recipient nerves should at least reach 0.7 when selecting a donor nerve. However, the optimal ratio and the effective range of this ratio for donor and recipient nerves require further research (Tables 3 and 4).

This study delves into the match between donor and recipient nerves in functional muscle transplantation, combining anatomical research with literature review findings to conduct relevant analyses and preliminarily establish matching principles. The ration for SAN and phrenic nerve in donor-recipient nerve match in reconstruction of brachial plexus injury by FMT were 0.77 and 0.99, respectively; In contrast, employing three intercostal nerves achieves a ratio of 0.7. Consequently, the study proposes 0.7 as the optimal ratio for this type of surgical nerve matching.

Ethical Approval

Not applicable

Funding

This manuscript was partly supported by Natural Science Foundation of Guangdong Province (2023A1515011724) and Featured Clinical Technique of Guangzhou（C3230111）：Precise repair of brachial plexus injury.

Availability of data and materials

All data generated or analyzed during this study are presented within the article.

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Table1 Histological Results of Donor Nerves（N=12）
	Area（mm²）		Myelinated Nerve Fibers
	Mean	Standard Deviation	Mean	Standard Deviation
GN	0.56	0.24	2870.56	1216.99
SAN	0.43	0.28	2210.55	1461.82
PN	0.55	0.15	2845.65	765.40
ICN3	0.44	0.14	686.05^*	209.35^*
ICN4	0.47	0.13	721.46^*	205.27^*
ICN5	0.40	0.19	615.72^*	300.57^*
ICN6	0.45	0.19	703.68^*	300.75^*
GN= gracilis nerve SAN=spinal accessory nerve PN=phrenic nerve ICN=intercostal nerve *=motor myelinated axons

Table 2 The Ratio of Myelinated Nerve Fibers between Donor Nerves and Nerve of the Gracilis
SAN/GN	0.77
PN/GN	0.99
ICN3+4/GN	0.49
ICN4+5/GN	0.47
ICN5+6/GN	0.46
ICN3+4+5/GN	0.70
ICN4+5+6/GN	0.71
GN= gracilis nerve SAN=spinal accessory nerve PN=phrenic nerve ICN=intercostal nerve

Table 3 Literature Review on the Reconstruction of Brachial Plexus Injuries Using Gracilis Muscle Transplantation with Different Donor Nerves

Nerve	Number^#	MRC≥4 Number	MRC≥4 （%）	References
SAN	1	1	100.00	[20]
SAN	2	2	100.00	[21]
SAN	5	5	100.00	[22]
SAN	2	2	100.00	[23]
SAN	14	10	71.43	[24]
SAN	3	1	33.33	[25]
SAN	6	0	0.00	[1]
SAN	9	5	55.56	[6]
SAN	40	34	85.00	[2]
Total	82	60	73.17
PN	1	0	0.00	[1]
PN	2	2	100.00	[2]
Total	3	2	66.67
3/4 ICN	4	0	0.00	[24]
3/4 ICN	1	1	100.00	[25]
3/4 ICN	6	4	66.67	[6]
Total	11	5	45.45
5/6 ICN	7	2	28.57	[24]
Total	7	2	28.57
3/4/5 ICN	7	3	42.86	[17]
3/4/5 ICN	4	2	50.00	[26]
Total	11	5	45.45
Ulnar Nerve Connected to CC7	1	1	100.00	[27]
Ulnar Nerve Connected to CC7	1	1	100.00	[28]
Ulnar Nerve Connected to CC7	17	10	58.82	[29]
Total	19	12	63.16
Contralateral Lateral Pectoral Nerve	12	7	58.33	[30]
Total	12	7	58.33

# Total Number of Patients or Number of Sides

Table 4 The Correlation between the Ratio of Donor Nerve to Gracilis Muscle Nerve and Muscle Strength in Literature Review

No competing interests reported.

Unveiling Nerve Match in Free Gracilis Muscle Transplantation: A Comprehensive Anatomical Investigation

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1