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.

INTRODUCTION

Traumatic peripheral nerve injury represents a major clinical problem, and it is increasingly common in combat-related extremity injuries.^1,2 Traumatic peripheral nerve injury occurs along a spectrum from injuries in which some axonal continuity is maintained to complete nerve transection, and combat-injury-induced neurotrauma significantly contributes to the morbidity and mortality in military casualties.^3–5 It is estimated that roughly 3% of all trauma patients have peripheral nerve injuries and more than 50,000 peripheral nerve repair procedures are performed annually in the United States alone.^6,7 The current treatment of choice for nerve transection injury is advanced microsurgical end-to-end repair with tensionless epineurial sutures or autologous nerve grafting if end-to-end anastomosis is not possible.^8,9 Despite these highly advanced microsurgical and reconstructive techniques, the functional recovery after peripheral nerve repair is poor.^10,11

Although the optimal functional recovery requires regenerating axons to cross the injury site (nerve bridge) and innervate the target organ, axonal misdirection, inadequate axonal regeneration, delayed muscle reinnervation, and muscle atrophy all contribute to the poor functional outcome and therapeutic failure after nerve repair.^12–17 It has been shown that functional outcomes following nerve repair depend on the accuracy of fascicular apposition.^18,19 Proper orientation of transected nerve stumps and their fascicles is thus crucial in nerve repair because aberrant growth of regenerating axons into inappropriate endoneurial tubes leads to poor functional recovery even with optimal axonal regeneration. Although it is difficult to investigate the pathophysiology of poor functional outcomes in humans with axonal misdirection or misalignment of nerve fascicles, this was rarely addressed in animal model of nerve transection with rigor and quantitative analysis of neurofilaments, neural revascularization, and neurogenic muscle atrophy.²⁰ We developed a novel standardized peripheral nerve transection technique, stepwise cut and fibrin glue (STG), in mice using fibrin glue for modeling peripheral nerve transection injury with reproducible gap distance between the severed nerve ends.²¹ Fibrin glue is widely used in laboratory research and clinical nerve repair scenarios.^22,23 Fibrin glue is easy to use and causes less local inflammation and scarring.²⁴ This new method demonstrated a close resemblance to the pathophysiological characteristics of nerve transection with gold-standard epineural suturing, and there was no negative impact of fibrin glue on nerve regeneration. Our findings with STG are consistent with others where sciatic nerve transection repair in mice with fibrin glue is reported to enhance axonal elongation compared to sutures during early nerve regeneration.²⁴ Our novel STG technique provides distinct advantages when compared with gold standard end-to-end neurorrhaphy, and these could be highly significant clinically, including: (i) standardized gap distance without suturing, (ii) simplicity and reproducibility with faster procedure time, and (iii) lack of confounding factors associated with surgeon manipulation.²¹ This model can be easily reproduced by any lab, and the data generated by this method would significantly contribute to better understand the nerve pathophysiology and molecular mechanisms of nerve regeneration with long-term follow-up.

In this study, we used our novel nerve transection and repair method with gluing to investigate the impact of misalignment of nerve fascicles in nerve-injury-induced neuro-vascular changes, muscle atrophy, and functional recovery. We hypothesized that the repair of a transected nerve with purposefully and precisely misaligned nerve stumps even with rigorous prevention of stump retraction would result in an increased number of misdirected nerve fibers and poor functional recovery. The preliminary findings of this study were presented as an abstract at 2019 Military Health System Research Symposium.²⁵

MATERIALS AND METHODS

Animals

The experimental design and animal protocols were approved by the Institutional Animal Care and Use Committee at Penn State University College of Medicine. Ten-week-old male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) weighing 20 to 25 g were used in this study. The mice were housed at the animal facility and the experimental animals were handled according to the Institutional Animal Care and Use Committee guidelines for the care and use of laboratory animals.

Mouse Models of Peripheral Nerve Transection Injury

Briefly, the mice were anesthetized with intraperitoneal ketamine (60 mg/kg) and xylazine (4 mg/kg) anesthesia, the right hind limb and lower back were shaved, washed with 70% ethanol, and prepped with povidone iodine. A 2-cm-long skin incision was made on the extended posterior right hind limb to carefully expose the right sciatic nerve through trans-gluteal approach under an operating microscope by a trained microsurgeon. Extreme care was taken to avoid any iatrogenic mechanical damage to the sciatic nerve.

In our recently established novel “Stepwise Transection with Glue (STG)” method (Appendix), to prevent the gap formation, the nerve was incompletely cut to 80% of its width in one attempt at a location 2 mm proximal to the trifurcation of sciatic nerve using fine microscissor (Integra Miltex, Plainsboro, NJ).²¹ Then, 20 µL of fibrin glue was applied to the laceration site and the remaining 20% of the nerve was completely transected before the complete clotting of the glue. This method not only effectively minimized gap formation caused by elastic retraction of the cut nerve ends, but it was also free from additional injury and manipulation caused by suturing. In our complete “Flip and Transection with Glue” (FTG) model (Appendix), the right sciatic nerve was “first” transected to 40% of its width from each side and the distal stump was transversely flipped, and then 20 µL of fibrin glue was applied around the transection site and the central 20% nerve was completely transected before complete clotting of fibrin glue. Complete transection was confirmed under direct microscopic evaluation. The skin was closed with surgical staples and postoperative slow-release buprenorphine (0.05 mg/kg) was given subcutaneously to all animals as an analgesic. Animals were monitored on the warming pad until active and then returned to the animal facility under the supervision of the attending veterinarian. Mice were followed for 28 days with weekly assessment of sciatic function index (SFI). The surgical staples were removed on post-surgery day 14, and sciatic nerves and tibialis anterior (TA) muscles were harvested for histomorphological and cellular analysis on post-surgery day 28. FTG was performed on the right sciatic nerve and thus the right sciatic nerve is termed as “FTG” or injured. The left sciatic nerve had no injury and it is termed as “uninjured” or healthy nerve. Similarly, muscles from right hind limb supplied by the injured right sciatic nerve are termed “FTG,” and muscles from left hind limb supplied by the uninjured left sciatic nerve are termed “uninjured.”

SFI as Determined by Walking Track Analysis

Walking track analysis was performed as previously described to evaluate the direct in vivo global motor functional recovery.²⁶ Briefly, mice were trained to walk freely along a 77 cm by 7 cm corridor lined with white paper, and individual footprints of the hind limbs were obtained by painting each foot with ink. At least three measurable footprints for each hind limb were obtained. Sciatic function index was calculated using three parameters of footprints [(1) toe spread (TS, first through fifth toes), (2) total print length (PL), and (3) intermediate toe spread (IT, second, third, and fourth toes)] and the following formula: SFI = −38.3 [(EPL − NPL)/NPL] + 109.5 [(ETS − NTS)/NTS] + 13.3 [(EIT − NIT)/NIT]—8.8, where E is the experimental paw and N is the normal paw. In general, an index of 0 to −10 indicates normal function and an index of −100 represents complete loss of function.

Whole Mount Immunostaining of Nerves

The whole mount nerve immunostaining was performed as described by Dun XP.²⁰ Briefly, after SFI analysis on post-surgery day 28, nerves were collected and fixed for 5 hours in 4% paraformaldehyde at 4°C. Grossly, the nerves were found in well-connected condition and in good continuity with a bulge at the repair site, and there was no dehiscence after 28 days post-surgery. Nerves were washed 3 times for 10 minutes each with phosphate buffered saline (PBS) with 1% Triton X-100 (Sigma X100) (PTX) and incubated in blocking solution (10% normal goat serum (Jackson Immunoresearch, 005-000-121) in 5% BSA PTX) overnight at 4°C. On the next day, nerves were transferred into primary antibodies in 5% BSA PTX and incubated for 72 hours at 4°C with gentle rocking. Primary antibodies were neurofilament heavy chain (NF-H) (1:1,000, Novus biologicals, NB300-135), P-zero Myelin protein (MP0) (1:500, Aves Labs, PZ0), and CD31 (1:100, BD Pharmingen, 553370). Nerves were then washed with PTX for 4 hours at 4°C, with a change of PTX every 1 hour. After PTX washes, nerves were incubated with Alexa Fluor 488, 594, and 647-conjugated secondary antibodies (1:500, Invitrogen) for 48 hours at 4°C with gentle rocking. Nerves were washed in PTX 3 times for 15 minutes each, followed by 4-hour washing in PTX with PTX change at every hour. Nerves were then washed overnight without changing PTX at 4°C. Next day, nerves were washed with PBS (3 times for 10 minutes each) for the removal of triton and cleared sequentially in 25%, 50% glycerol (Sigma, G6279) in PBS for 6 and 12 hours, respectively, for each glycerol concentration. Following clearing, nerves were mounted in SlowFade™ Gold Antifade Mountant with DAPI (Invitrogen, S36939) or without DAPI (Invitrogen, S36937). Stained whole nerves were imaged using ZEISS Axio Observer 7 equipped with an Apotome.2 (Carl Zeiss Microscopy GmbH, Jena, Germany). Tiling and Z-stack functions both were employed to image whole nerve. Maximum Intensity projection was used to pull the data from all Z-stacks and represented as 2D image.

Quantitative Analysis of Nerve Fibers and Blood Vessels

At least three uninjured left sciatic and injured (FTG) right sciatic nerves were imaged as paired manner with an apotome 2, which can capture optical sections of tissue. These 3D images were then projected as a 2D image using Zeiss microscopy software. Maximum Intensity projected 2D images were used for the quantification of horizontal and vertical fibers. Nerve images were captured at different depths using Z-stack imaging. Data from different depths (3D data) was pulled together in 2D images using maximum intensity projection. Use of maximum intensity projected 2D image nullifies possibility of recounting same fiber at different depths. The bulging in the nerve was the transection site with a nerve bridge, and we termed this as the injury zone.^20,27 For the purpose of quantification, the imaged whole nerve was divided into three zones: proximal, injury, and distal. All nerve regions immediately before and after the bulge were proximal and distal zones, respectively. Each zone was then divided into grid using Image J. In the nerve, fibers are arranged parallel to the proximal-distal axis. Any deviation from this is considered aberrant.

The number of horizontal and vertical fibers was calculated in every grid manually. The division of each zone into many grids was performed to ease manual counting. The percentage of horizontal and vertical fibers was then calculated from the data. Three nerve sections were analyzed per group. Directional Image analysis (degree of orientation) of nerve fibers was done using Orientation J plugin from Image J (National Institutes of Health, Bethesda, MD).²⁸ AngioTool version 0.6a (02.18.14) was used to quantitate angiogenesis in distinct nerve zones like proximal, injury, and distal. Increased vessel density and branching index indicates angiogenesis. AngioTool is an open source windows executable software which provides automated measures of vessel area and number of junctions.²⁹

Hematoxylin and Eosin Staining and Histomorphometric Analysis of Muscles

After functional analysis on post-surgery day 28, TA muscles of contralateral and injured hind limbs were harvested from surrounding tissues and weighed. Hematoxylin and eosin (H&E) staining and histomorphometric analysis of TA muscles were performed as described previously.²⁶ Briefly, muscles were washed in chilled PBS and immediately transferred to 30% sucrose and kept at 4°C until tissue sank to bottom. Muscles were embedded in optimal cutting temperature (OCT) tissue freezing medium (Sakura Torrance, USA) in dry ice isopentane (2-methyl butane) slurry. Three paired TA muscles from the uninjured (healthy) left hind limbs and from injured (FTG) right hind limbs were used for cryosectioning. Three 10-μm thick sections from each muscle were cut at −21°C using a Cryostat (Microm HM505e) and collected on superfrost plus microscopic slides (Fisherbrand, Cat # 12-550-15). Slides were then stained for H&E and imaged on Olympus BX53 microscope. Quantitative analysis for cross-sectional area (CSA), minimum Feret’s diameter (MFD), and fiber distribution of muscle fiber was done with Image J software on three random microscopic fields from each muscle.

Data Analysis

The number of animals (n) needed is based on the conservative use of animals for the least sensitive data type, and is determined based on a desired power level greater than 80% and a significance value of <.05. This results in n = 9-10 animals per group for in vivo functional analysis (SFI), with fewer animals needed for in vitro studies (n = 3-4). The probability that an animal dies of causes unrelated to the experiment in our longest group is approximately 8% based on previous experience. We therefore arrive at a preliminary calculation of 10 animals in this study. All results are presented as means ± SEM and all experiments were repeated at least three times. Data were analyzed using GraphPad PRISM 7 (GraphPad Software, San Diego, CA, USA). Statistical analyses were performed using either paired Student t-test, or one- or two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. A P value of <.05 was considered to be statistically significant.

RESULTS

Misaligned Nerve Repair and Axonal Regeneration

Despite optimal axonal regeneration after microsurgical repair, misdirection of the regenerating axons is considered one of the major critical factors in poor functional outcome after nerve injury and repair.^12,13,17 To evaluate the distribution, direction, and alignment of regenerating nerve filaments, immunofluorescence staining of the whole nerve was performed for NF-H and MP0. At post-injury day 28, we found all nerves in good continuity with a bulge near the transection site and there was no dehiscence with any repair method. Figure 1A (lower image) outlines the zoning of the transected nerve as described in the Method. Uninjured nerve displays normal nerve architecture with uniform NF-H (Fig. 1A, top image) staining over the entire length, and unidirectional nerve fibers are compactly packed and parallelly aligned. Neurofilaments which run parallel to proximal-distal axis are vertical filaments. In the injured nerve (Fig. 1A, lower image, FTG), axons from proximal stump tried to grow towards the distal end through the repair site and led to a bulging at the nerve bridge. NF-H intensity in the proximal, injury, and distal zones were more pronounced compared to uninjured nerve probably because of increased number of misdirected nerve fibers. To evaluate the extent of misdirection, we quantified nerve fiber alignment in different zones. Figure 1B shows that the number of misaligned nerve fibers (“as a percentage of total fibers”) were significantly higher in all three zones of injured (FTG) nerves as compared to the uninjured nerve (1.57 ± 0.61, n = 4), and they were mostly distributed in the injured (54.66 ± 4.75, n = 3) and distal (44.37 ± 4.61, n = 3) zones than in the proximal zone (17.05 ± 3.61, n = 3). Here it is noteworthy that transected nerves with STG model had markedly reduced number of misaligned nerve fibers at all zones when compared to this FTG model.²¹

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.

Misaligned Nerve Repair and Neural Angiogenesis

Angiogenesis is reported to play an important role in axonal sprouting, regeneration, and reinnervation following nerve transection injuries.^20,30 Using the endothelial cell marker CD31, we examined new blood vessel formation at post-injury day 28, and Fig. 2A displays the immunofluorescence staining of the whole nerves with CD31. In the uninjured nerves, immunofluorescence staining was uniform over the entire length. In contrast, CD31 staining intensity in the injury and distal zones of injured (FTG) nerves was more pronounced compared to the proximal zone. Figure 2B shows the representative CD31-stained blood vessels (left) and AngioTool result (right) images of uninjured and injured (FTG) nerves. AngioTool images depict the blood vessel architecture (red lines) and their branching points (blue dots). Quantification of blood vessel density (“as % vessels/total area,” Fig. 2C) and branching index (“as junctions/mm,”² Fig. 2D) revealed that blood vessel density and branching index in the proximal zone (17.68 ± 2.16 and 96.76 ± 16.38, n = 3) of the injured (FTG) nerves were not significantly different from the uninjured nerves (14.11 ± 0.94 and 58.82 ± 6.70, n = 4), but they were significantly higher in the injury (39.23 ± 2.58 and 655.88 ± 79.99, n = 3) and distal (26.18 ± 3.68 and 340.45 ± 97.61, n = 3) zones as compared to proximal zone of the injured nerve, and these findings were consistent with increased number of misdirected nerve fibers in Fig. 1B.

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.

Misaligned Nerve Repair and Muscle Atrophy

Denervation causes rapid muscle atrophy within weeks of the injury.^15,16,31 To evaluate muscle atrophy: First, we checked the changes in muscle mass at post-injury day 28 and then we performed histological and histomorphometric analysis of the muscles. Figure 3A displays representative H&E images of frozen transverse sections of TA muscle from different groups. Uninjured TA muscles show normal muscle architecture with a tightly packed homogeneous polygonal shaped muscle fiber distribution, peripherally placed nuclei, minimal intramyofiber spacing, and little cellular infiltration. Compared to muscles in uninjured limbs, transverse sections of muscles from the injured (FTG) limbs showed increased intermyofiber spacing and cellular infiltration.

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 3B shows that the gross muscle weight (mg) in injured right limb was markedly reduced (32.42 ± 0.86, n = 10) compared to contralateral healthy left limb (56.29 ± 0.83, n = 10). Quantitative histomorphometric analysis of muscle (n = 3 muscles/group) revealed significantly decreased CSA (1,301 ± 13.69 µm², n = 965 muscle fibers; Fig. 3C) and MFD (34.58 ± 0.21 µm; n = 965 muscle fibers, Fig. 3D) in injured (FTG) limb compared to uninjured limb (CSA, 2,103 ± 27.34 µm²; MFD, 43.99 ± 0.32 µm; n = 958 muscle fibers). We also analyzed the muscle fiber distribution (Fig. 3E) based on the MFD data which revealed that ~66% fibers in the uninjured group were distributed evenly in a range 35-55µm, other 13% had higher MFD (55-70µm) and only ∼20% fibers were smaller (<35 µm). From the 20% smaller fibers, 15% were within MFD of 30-35 µm. In the injured (FTG) limb, the ratio of muscle fiber within 35-55 µm and <35 µm ranges were 46% and 53%, respectively. Histograms for the muscle fibers in injured (FTG) limb thus revealed a left shift in fiber distribution with a predominance of smaller fibers.

Misaligned Nerve Repair and Functional Recovery

To evaluate how the changes in regenerating axons, blood vessels, and muscles are associated with the functional recovery, SFI was determined weekly for 4 weeks (Fig. 4). We had 10 mice in this study, but only 5 mice were able to walk on post-injury day 28 and other 5 mice had no readable footprints from the injured limb for analysis. The SFI remained markedly and persistently low throughout the entire period of experiment, and SFI value on day 7, 14, 21, and 28 was −66.26 ± 3.56 (n = 10), −58.90 ± 4.34 (n = 10), −70.30 ± 3.77 (n = 10), and −65.82 ± 7.87 (n = 5), respectively. In contrast, we observed that all 10 mice with STG model (personal communication) were able to walk on post-injury day 28 day, and SFI value on day 7, 14, 21, and 28 was −61.94 ± 3.34, −66.03 ± 3.56, −58.93 ± 4.69, and −54.55 ± 4.23, respectively.

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.

DISCUSSION

The main finding of this study is that misaligned nerve repair results persistently poor functional recovery with significantly increased number of aberrant axonal neurofilaments in the injured and distal nerve segments than in the proximal nerve segment. Increased aberrant neurofilament profiles in the injured and distal nerve segments were associated with significantly increased neural blood vessel density and branching index. Muscle fiber distribution in the atrophied muscle demonstrated a significantly increased number of smaller muscle fibers and decreased number of larger muscle fibers. These findings in a novel nerve transection mouse model with purposefully misaligned repair suggest that aberrant neurofilament distributions and axonal misdirections play an important role in post-injury functional recovery.

Although a complex multicellular event takes place after nerve transection, the therapeutic failure with nerve repair often results from misdirection of regenerating axons to functionally inappropriate end organs.^{12,13,17,32,33} In mouse sciatic nerve transection injury, whole mount imaging demonstrated an extensive axon guidance defect around the nerve bridge with axons growing along the outside of both the proximal and distal nerve ends.²⁰ Consistent with the negative impact of misdirected nerve fibers in nerve regeneration and functional recovery, qualitative and quantitative analysis of transected nerves in our recent work demonstrated that closer approximation and minimal manipulation of the severed nerve ends are crucial to limit misdirectional axonal regrowth.^12,13,17,21 We observed the lowest number of misaligned nerve fibers in the proximal, injury and distal zones of regenerating axons in our optimally aligned STG model compared to a less-aligned simple nerve transection model.²¹ In this study, we further demonstrate that purposeful misalignment of the transected nerve stumps leads to increased propensity of misdirectional axonal growth in all zones compared to our findings with STG model. These findings confirm that irrespective of closer approximation of severed nerve ends, proper orientation of transected nerve stumps and their fascicles play a crucial role in reducing nerve fiber misalignment during nerve regeneration. This role is assumed, but rarely measured with rigor. Importantly, these findings have tremendous implications in postsurgical recovery process of nerve gap and grafting because of the complexity associated with multiple bridge formation at the both ends of a nerve graft.

Angiogenesis following an injury plays an important role in nerve regeneration because Schwann cells use the newly formed blood vessels as tracks to guide the regeneration of axon with the right direction across the nerve bridge.^33,34 Recently, whole mount imaging of mouse sciatic nerve demonstrated CD31-positive new blood vessel formation in the proximal and distal stumps of the transected nerve.²⁰ Consistent with this imaging study, we confirmed neural angiogenesis in our novel STG method by quantitative analysis and demonstrated a significantly increased blood vessel formation in all zones of the transected nerve and new blood vessels were optimally maintained in the proximal and distal zones because of optimal alignment of the longitudinal blood vessels in the epineurium.²¹ However, the increased blood vessel density and branching index in the injured (FTG) nerves were seen in the injury and distal zones but not in the proximal zone of misaligned nerve indicating a maladaptive pattern of neural angiogenesis. This is because of the loss of aligned epineurial longitudinal blood vessels in flipped distal nerve stumps, often assumed but rarely quantified in traumatic peripheral nerve injury.

Denervation of the muscle leads to rapid muscle-fiber atrophy, and it is characterized by reduced muscle mass, decreased CSA, increased connective tissue, increased fibrosis, and altered muscle fiber distribution.^15,16,31,34 Muscle wet weight is widely used to evaluate muscle innervation.^16,35 However, reduced muscle mass and CSA with FTG method were comparable to our recent findings with STG method.²¹ These findings thus indicate that the alignment of the nerve fibers is not the critical factor when it comes to preventing some features of muscle atrophy. Although these findings are interesting, future studies with long-term follow-up for 12 weeks would be required to completely address the mechanisms of muscle atrophy in relation to morphologically intact regenerating nerves, changes of neuromuscular junctions, and nerve conduction.

CONCLUSIONS

Misaligned repair of the transected nerve stumps differentially affects the architectural structure of nerves and neural microvessels as compared to our novel STG model.²¹ Poor SFI recovery with misaligned FTG method indicates that proper alignment of the transected nerve stumps and subsequent coordinated favorable neurovascular and muscular processes are crucial for post-injury functional recovery. We thus provide direct quantitative evidence that misalignment of the transected nerve stumps can lead to increased misdirectional axonal growth, and it is the proper alignment of the nerve stumps that plays the key role in supporting a favorable microenvironment for directional neurovascular and axonal growth into the distal stump for better functional recovery.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at Military Medicine online.

FUNDING

This work was supported by grants from the National Institutes of Health (K08 AR060164-01A) and the Department of Defense (W81XWH‐16‐1‐0725) to John C. Elfar in addition to the institutional support from Pennsylvania State University Medical Center.