Experimental nerve transfer model in the neonatal rat

Abstract

Clinically, peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries. Most surgical concepts, however, have investigated nerve reconstructions in adult animal models. The immature neuromuscular system reacts differently to the effects of nerve lesion and surgery and is poorly investigated due to the lack of reliable experimental models. Here, we describe an experimental forelimb model in the neonatal rat, to study these effects on both the peripheral and central nervous systems. Within 24 hours after birth, three groups were prepared: In the nerve transfer group, a lesion of the musculocutaneous nerve was reconstructed by selectively transferring the ulnar nerve. In the negative control group, the musculocutaneous nerve was divided and not reconstructed and in the positive control group, a sham surgery was performed. The animal´s ability to adapt to nerve lesions and progressive improvement over time were depict by the Bertelli test, which observes the development of grooming. Twelve weeks postoperatively, animals were fully matured and the nerve transfer successfully reinnervated their target muscles, which was indicated by muscle force, muscle weight, and cross sectional area evaluation. On the contrary, no spontaneous regeneration was found in the negative control group. In the positive control group, reference values were established. Retrograde labeling indicated that the motoneuron pool of the ulnar nerve was reduced following nerve transfer. Due to this post-axotomy motoneuron death, a diminished amount of motoneurons reinnervated the biceps muscle in the nerve transfer group, when compared to the native motoneuron pool of the musculocutaneous nerve. These findings indicate that the immature neuromuscular system behaves profoundly different than similar lesions in adult rats and explains reduced muscle force. Ultimately, pathophysiologic adaptations are inevitable. The maturing neuromuscular system, however, utilizes neonatal capacity of regeneration and seizes a variety of compensation mechanism to restore a functional extremity. The above described neonatal rat model demonstrates a constant anatomy, suitable for nerve transfers and allows all standard neuromuscular analyses. Hence, detailed investigations on the pathophysiological changes and subsequent effects of trauma on the various levels within the neuromuscular system as well as neural reorganization of the neonatal rat may be elucidated. This study was approved by the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Research and Science (BMWF-66.009/0187-WF/V/3b/2015) on March 20, 2015.

Keywords: brachial plexus birth injury; experimental rat model; extremity reconstruction; methodological paper; neonatal rat; nerve reconstruction; nerve regeneration; nerve transfer; neural plasticity; peripheral nerve surgery.

Figure 1

Experimental design. (A) Positive control group with sham surgery (exposure of anatomical situs). (B) Negative control group with a dissected musculocutaneous nerve and no reconstruction of elbow flexion. (C) Nerve transfer group with a nerve transfer from the ulnar nerve to the musculocutaneous nerve. 1: Ulnar nerve; 2: musculocutaneous nerve; 3: biceps muscle (3¹: long head, 3²: short head); 4: nerve transfer.

Figure 2

Surgical anatomy in the neonatal rat. (A) Operation situs before the nerve transfer. Especially note the highly plastic and indiscriminate tissue. (B) Selective nerve transfer: The ulnar donor nerve is transferred to the musculocutaneous nerve to restore elbow flexion. Neural confusion is provoked to evaluate neural plasticity. (C) As suturing was not feasible, the nerve endings were connected with a 2-component fibrin adhesive. (D) Nerve transfer after 12 weeks. 1: Ulnar nerve; 2: musculocutaneous nerve (2¹ proximal, 2² distal); 3: pectoral muscles; 4: brachial artery; 5: biceps muscle; 6: fibrin glue; 7: median nerve; 8: nerve transfer.

Figure 3

Nerve transfer surgery in the neonatal rat. (A) Male neonatal rat before surgery at the age of 20 hours. Note the transparent skin and the still undeveloped anatomical structures (hands, eyes and ears). (B) Rat at the age of 1 week after nerve transfer surgery. Especially note the healed, infraclavicular incision and the paralysis of the right extremity at that time.

Figure 4

Regenerative process in the Bertelli test. At the age of 2 weeks, neonates do not achieve full score, which is indicated by the sham group. Within the first four weeks after birth, the grooming response fully develops. The negative control (n = 5) and nerve transfer group (n = 10) were initially unable to flex their elbow sufficiently. In the nerve transfer group, this deficit fully recovered over the following weeks. Even though biceps function was not restored in the negative control group, they achieved a satisfying grooming response. The trend of the positive control group (n = 10) depicts the development of grooming within the first weeks after birth in neonatal rats. The Y-axis indicates functional regeneration of global shoulder and elbow funciton, assessed by the Bertelli test (Bertelli and Mira, 1993). Here, the grooming response is differentiated from grade 1 (no movement or mouth), to grade 2 (region below the eye), grade 3 (eye), grade 4 (front of the ears), and grade 5 (behind the ears). Values are presented as mean and standard deviation. * Significant difference between nerve transfer and positive control group (P < 0.001). ** Significant difference between nerve transfer and negative control group (P < 0.001). One-way analysis of variance with post hoc Tukey's test was used.

Figure 5

Functional muscle testing and muscle analyses. Following completed nerve regeneration, functional muscle testing and evaluation of the biceps's long head demonstrated successful reinnervation in all nerve transfer animals (n = 10). Comparing the nerve transfer to the negative control group (n = 5), a statistically significant difference was found in maximum tetanic muscle force, muscle weight and cross sectional area analyses (independent samples t-test, **P < 0.001). Especially note that in the negative control group, no muscle contraction was excitable to any extent. However, the nerve transfer group did not completely achieve the values in the sham group (independent samples t-test, *P < 0.001). A high Pearson correlation coefficient was found between cross sectional muscle area and muscle weight (r = 0.96, P > 0.01)), but also cross sectional muscle area and maximum tetanic muscle force (r = 0.94, P > 0.01). Values are presented as mean and standard deviation.

Figure 6

Histological muscle assessment. To examine the effects of muscle de- and reinnervation, cross-sectional-areas of biceps muscle were evaluated. Especially note the typical signs of muscular denervation in the negative control group: endo- and perimysial fibrosis, fatty degeneration, decreased myofiber diameter and number. The biceps muscle was reinnervated in the nerve transfer group. However, it remains histologically altered. The enlarged details of each group on the right side are marked in the respective muscle on the left side by squares. (10 μm cross-sectional muscle samples, Masson-Goldner-Trichrome staining, transmitted-light microscopy with TissueFAXS imaging system, 10× magnification). By applying a Masson-Goldner-Trichrome staining eutrophic muscle fibers can be distinguished from muscle fibrosis. Apart from the fibrosis grade, these results do not allow more precise conclusions on the changes on a muscular level.

Figure 7

Motoneuron analysis of the spinal cord. (A) The musculocutaneous and ulnar nerve were retrograde labeled and natively contained 330.4 ± 36.75 (n = 5) and 278.8 ± 19.89 (n = 5) motoneurons, respectively. After transferring the ulnar nerve within 24 hours after birth, due to post-axotomy motoneuron death, only 77.56 ± 14.96 (n = 9) motoneurons survive for 12 weeks. Values are presented as mean and standard deviation (independent samples t-test, *P < 0.001, **P < 0.001). (B) Spinal cord photographed at the level of C5 following retrograde labeling. First, the retrograde dye (Fluoro-Ruby) is transported to the spinal cord. Consecutively, the corresponding motoneurons of an individual peripheral nerve fluoresce and can be detected as shown above. Here, the musculocutaneous nerve's motor neuron column of a sham rat is depicted and shows a coherent motoneuron pool in the dorsolateral region of the ventral horn (longitudinal section, confocal microscope, 10× magnification). (C, D) Semi-thin cross sections of the ulnar nerve harvested in pilot animals. The native ulnar nerve structure (C) can be compared to the selectively transferred ulnar nerve, taken directly at the coaptation site (D). Especially note the morphologic differences. Following nerve transfer, multiple pathologies, like thinly myelinated and dystrophic axons, but also small diameter axons, are found (staining with Osmiumtetroxid and 1,4-phenylendiamin, optical microscope, 60× magnification).