Proof of concept for multiple nerve transfers to a single target muscle
- Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Austria
- Center for Biomedical Research, Medical University of Vienna, Austria
- Department of Electrical Engineering, Chalmers University of Technology, Sweden
- Department of Bioengineering, Imperial College London, United Kingdom
- Department of Clinical and Movement Neuroscience, University College London, London, United Kingdom
- BSICoS Group, IIS Aragón, Universidad de Zaragoza, Spain
- Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Austria
- Karl Landsteiner University of Health Sciences, Department of Plastic, Aesthetic and ReconstructiveSurgery, University Hospital St. Poelten, Austria
Figures
Figure 1
Double retrograde labeling.
(A) The selected donor nerves were both dissected in a right forelimb and placed in a conduit reservoir filled with Fast-Blue (UN) and Fluoro-Ruby (AIN), respectively, for 1 hr. Wet sterile swabs were placed above the surgical site to prevent the tissue from drying and the fluorescent dyes from bleaching. (B) Spinal cord section C8-Th1. Labeled AIN (orange) and UN motoneuron pool (blue). (C) A Kruskal-Wallis H test was conducted to determine if there were differences in labeled motor neuron count between the three groups with different treatment: control (n=7), SNT (n=15), and DNT (n=15). Distributions of motor neuron count were not similar for all groups, as assessed by visual inspection of a boxplot. The mean ranks of motor neuron count were statistically significantly different between groups, χ2(2)=11.147, p=0.004. Subsequently, pairwise comparisons were performed using Dunn’s (1964) procedure with a Bonferroni correction for multiple comparisons. Adjusted p-values are presented. This post hoc analysis revealed statistically significant differences in labeled motor neuron count between the SNT (mean rank=11.90) and DNT (mean rank=24.67; *p=0.004) group, but not between the control group (mean rank=22.07) or any other group combination. DNT, double nerve transfer; GM, grey matter; SNT, single nerve transfer; WM, white matter.
Figure 2 with 1 supplement
Comparison of muscle mass.
Muscle mass after SNT was significantly reduced compared to the untreated muscle mass *p<0.001 while muscle mass following DNT regenerated to 98.83%. Muscle mass following DNT was significantly larger compared to the SNT group **p<0.001. DNT, double nerve transfer; SNT, single nerve transfer.
Figure 2—figure supplement 1
A grouped scatterplot was created to visually assess the linear relationship between treated and untreated muscle mass for each nerve transfer procedure.
Figure 3
Experimental nerve transfer models.
Single-nerve transfer model: The UN (yellow) was transected distally to the palmar cutaneous branch in the forearm and surgically transferred to reinnervate the long head of the biceps (n=30). Multiple-nerve transfer model: Both the UN (yellow) and AIN (green) were redirected to reinnervate the long head of the biceps (n=32). Before both nerve transfer procedures, the originally innervating branch of the MCN was removed. The untreated contralateral biceps muscles served as internal control for both groups. The red lines indicate the level of transection.
Figure 4
Surgical procedure of the ulnar nerve transfer.
(A) Overview of the rats’ supinated right forelimb after the brachial and antebrachial fascia were removed. (B) Two blunt retractors have been placed to pull the flexor carpi ulnaris and the palmaris longus apart, revealing the underlying UN. The yellow line indicates the level of transection to gain sufficient length to reach the biceps’ long head tension-free. To achieve this, the palmar cutaneous branch must be transected, while the dorsal cutaneous branch can be preserved. (C) For better visualization, the brachial fascia was opened above the biceps. A sharp retractor was placed to pull back the pectoral muscles and thus revealed the two biceps heads, which were bluntly separated. In the deep bicipital groove, the MCN and its motor branch to the long head of the biceps were identified. Maximum length of the motor branch to the long head was removed to prevent spontaneous regeneration. (D) Eventually, the UN was rerouted from between the palmaris longus and flexor carpi ulnaris to the long head of the biceps and sutured to the epimysium at the former original motor entry point. This procedure on the one hand spares the denervation of the flexor carpi ulnaris and the flexor digitorum superficialis and the invasive dissection through the cubital tunnel. MCN, musculocutaneous nerve.
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Figure 4—source data 1
Labeled motor neuron count by treatment.
- https://cdn.elifesciences.org/articles/71312/elife-71312-fig4-data1-v2.xlsx
Figure 5
Surgical procedure of the double nerve transfer.
(A) General view of the right supinated forelimb. The proximal hook pulls the pectoral muscles toward proximal for better presentation. (B) The brachial and antebrachial fascia and the motor branch to the pronator teres muscle were removed for better visualization. In the cubital fossa, three branches arise from the median nerve: one muscle branch supplying the pronator teres (resected); one muscle branch supplying the flexor carpi radialis, palmaris longus, and flexor digitorum superficialis; and the AIN supplying pronator quadratus, flexor pollicis longus, and flexor digitorum profundus. After transecting the AIN (yellow line), proximal dissection in an intraneural fashion gains sufficient length to reach the biceps’ motor entry point. (C) Surgical site before wound closure, after both the UN and the AIN were transferred to the physiological motor entry point of the long head of the biceps. FCR, flexor carpi radialis; FDS, flexor digitorum superficialis; PL - palmaris longus.
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Figure 5—source data 1
Muscle mass of treated and untreated sides in [mg] by treatment.
- https://cdn.elifesciences.org/articles/71312/elife-71312-fig5-data1-v2.xlsx
Videos
Video 1
Grooming behaviour.
The grooming behavior of a double nerve transferred animal is provoked by sprinkling 1–3 ml of water or glucose on its snouts and as shown in slow-motion. Notice that the animal can perform a physiological grooming movement with both front paws reaching behind the ears smoothly.
Video 2
Nerve crush of the MCN.
The supinated left forelimb with the exposed biceps muscle and its motor branch is shown. By crushing the MCN repeatedly with increasing pressure from proximal to distal with a micro needle holder, action potentials were elicited toward the biceps’ long head which resulted in muscle fibrillation. MCN, musculocutaneous nerve.
Video 3
Nerve crush of the AIN.
Twelve weeks following DNT, the AIN reinnervating the long head of the biceps was repeatedly crushed with a micro needle holder. This resulted in a macroscopically recognizable muscle response, indicating successful reinnervation. DNT, double nerve transfer.
Video 4
Nerve crush of the UN.
After crushing and neurotomizing the AIN, the UN was crushed. Repeated nerve crushes resulted in adequate muscle fibrillations indicating neuromuscular regeneration.
Tables
Table 1
Overview of qualitative results.
These results provide a detailed overview of the nerve transfer model and evidence of successful reinnervation.
| SNT | DNT | |
|---|---|---|
| Surgery timeBehavior after 12 weeksMacroscopic innervationCrush/neurotomy response | 49±13 minAll max score (n=21)All (n=30)All (n=15) | 78±20 minAll max score (n=30)All (n=32)All (n=17) |
| UN | AIN | |
| Nerve length | 23.08±1.36 mm | 10.50±1.61 mm |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71312/elife-71312-transrepform1-v2.docx
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Source code 1
The Syntax code for SPSS 25 for Mac is divided into analysis for muscle mass on page 1 (ANCOVA) and retrograde labeling analysis (Kruskal Wallis H-Test) on page 2.
The respective headings for the code sections are highlighted using asterisks.
- https://cdn.elifesciences.org/articles/71312/elife-71312-supp1-v2.zip
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Proof of concept for multiple nerve transfers to a single target muscle
eLife 10:e71312.
https://doi.org/10.7554/eLife.71312
