Purposeful Misalignment of Severed Nerve Stumps in a Standardized Transection Model Reveals Persistent Functional Deficit With Aberrant Neurofilament Distribution

Abstract

Background: Functional recovery following primary nerve repair of a transected nerve is often poor even with advanced microsurgical techniques. Recently, we developed a novel sciatic nerve transection method where end-to-end apposition of the nerve endings with minimal gap was performed with fibrin glue. We demonstrated that transected nerve repair with gluing results in optimal functional recovery with improved axonal neurofilament distribution profile compared to the end-to-end micro-suture repair. However, the impact of axonal misdirection and misalignment of nerve fascicles remains largely unknown in nerve-injury recovery. We addressed this issue using a novel nerve repair model with gluing.

Methods: In our complete "Flip and Transection with Glue" model, the nerve was "first" transected to 40% of its width from each side and distal stump was transversely flipped, then 20 µL of fibrin glue was applied around the transection site and the central 20% nerve was completely transected before fibrin glue clotting. Mice were followed for 28 days with weekly assessment of sciatic function. Immunohistochemistry analysis of both sciatic nerves was performed for neurofilament distribution and angiogenesis. Tibialis anterior muscles were analyzed for atrophy and histomorphometry.

Results: Functional recovery following misaligned repair remained persistently low throughout the postsurgical period. Immunohistochemistry of nerve sections revealed significantly increased aberrant axonal neurofilaments in injured and distal nerve segments compared to proximal segments. Increased aberrant neurofilament profiles in the injured and distal nerve segments were associated with significantly increased nerve blood-vessel density and branching index than in the proximal segment. Injured limbs had significant muscle atrophy, and muscle fiber distribution showed significantly increased numbers of smaller muscle fibers and decreased numbers of larger muscle fibers.

Conclusions: These findings in a novel nerve transection mouse model with misaligned repair suggest that aberrant neurofilament distributions and axonal misdirections play an important role in functional recovery and muscle atrophy.

FIGURE 1.

Evaluation of whole mount immunostaining of the transected nerve at post-injury day 28 for nerve fiber distribution. For the purpose of evaluation and quantification, imaged whole nerve was divided into three zones: proximal, injury, and distal. (A) Representative compact images of immunofluorescence staining of the whole nerve for NF-H in green for uninjured (top image) and injured (“Flip and Transection with Glue,” FTG; bottom image) nerves. Scale bar, 500 μm; magnification, 5×. (B) Bar graph showing the quantification of misaligned fibers at proximal, injury, and distal zones of transected nerves. Misaligned fibers are shown as the percentage of total number of fibers in each zone. n = 3-4/group, **P<.01, ***P<.001 vs. uninjured, ###P<.001 vs. proximal.

FIGURE 2.

Evaluation of whole mount immunostaining of the transected nerve at post-injury day 28 for angiogenesis. (A) Representative compact images of immunofluorescence staining of the whole nerves for CD31 in purple, uninjured (top image) and injured (Flip and Transection with Glue, FTG; bottom image) nerves. Scale bar, 500 μm; magnification, 5×. (B) Representative images from an uninjured nerve (top images) and injured zone of an injured (FTG) nerve (bottom images) to show CD31-stained blood vessels (left panel) and AngioTool reconstruction results (right panel). AngioTool images clearly depict the blood vessel architecture as red lines and their branching points as blue dots. Bar graph showing the quantification of blood vessels at proximal, injury, and distal zones of transected nerves. (C) Blood vessel density is shown as the percentage of number of blood vessel in total area. (D) Branching index of blood vessels is shown as the number of blood vessel junctions/mm2. n = 3-4/group; **P<.01, ***P<.001 vs. uninjured, ###P<.01 vs. proximal, and $$P<.05 vs. injury. For other details, see Fig. 1.

FIGURE 3.

Effect of misaligned nerve repair on tibialis anterior (TA) muscle histology, muscle mass, and quantitative measurements of the muscle fibers. (A) Representative images of TA muscle cross sections stained with H&E from uninjured and injured (FTG) hind limbs. Scale bar, 50 μm; magnification 20×. (B) TA muscle weight, n = 10/group; (C) Cross-sectional area of TA muscle as μm², n = 3/group; (D) Minimum Feret’s diameter (MFD) of TA muscle as μm, n = 3/group; and (E) quantitative muscle fiber size distribution from the MFD value (μm) in each group as a percentage of total fiber number. n = 3/group. ***P<.001 vs. uninjured limb muscle. For other details, see Fig. 1.

FIGURE 4.

Effect of misaligned repair of transected nerve stumps on the functional recovery (SFI) at post-injury day 7, 14, 21, and 28. n = 5-10 at each time point.