Neurofilament-histomorphometry comparison in the evaluation of unmyelinated axon regeneration following peripheral nerve injury: An alternative to electron microscopy.

BACKGROUND: Currently, assessment of unmyelinated axon regeneration is limited to electron microscopy (EM), which is expensive, time consuming and not universally available. This study presents a protocol to estimate the number of unmyelinated axons in a regenerating peripheral nerve without the need for electron microscopy. NEW METHOD: The common peroneal nerve of Sprague-Dawley rats was transected, repaired and regenerated for 4 weeks. Two distal adjacent segments of the regenerating nerve were then processed for either conventional histomorphometry using toluidine blue or immunolabeling of neurofilament protein. Myelinated axon and total axon counts were obtained, respectively, to generate estimates of unmyelinated axon numbers, which were then compared to unmyelinated axon counts using EM from the same specimens. For comparison, unmyelinated axons were counted in an uninjured rat laryngeal nerve. RESULTS: After 4 weeks of regeneration, the estimated number of regenerating unmyelinated axons was 4044 ± 232 using this technique, representing 81.3% of the total axonal population. By comparison, the proportion of unmyelinated axons in the uninjured laryngeal nerve was 55% of the total axonal population. COMPARISON WITH EXISTING METHOD: These estimates correlate with electron microscopy measurements, both in terms of the proportion of unmyelinated axons and also by linear regression analysis. CONCLUSIONS: The neurofilament staining method correlates with electron microscopy estimates of the same nerve sections. It is useful for the efficient counting of unmyelinated axons in the regenerating peripheral nerve and can be used by laboratories that do not have access to EM facilities.

ErbB2 blockade with Herceptin (trastuzumab) enhances peripheral nerve regeneration after repair of acute or chronic peripheral nerve injury.

OBJECTIVE: Attenuation of the growth supportive environment within the distal nerve stump after delayed peripheral nerve repair profoundly limits nerve regeneration. Levels of the potent Schwann cell mitogen neuregulin and its receptor ErbB2 decline during this period, but the regenerative impact of this change is not completely understood. Herein, the ErbB2 receptor pathway is inhibited with the selective monoclonal antibody Herceptin (trastuzumab) to determine its significance in regulating acute and chronic regeneration in a rat hindlimb. METHODS: The common peroneal nerve of Sprague-Dawley rats was transected and repaired immediately or after 4 months of chronic denervation, followed by administration of Herceptin or saline solution. Regenerated motor and sensory neurons were counted using a retrograde tracer 1, 2, or 4, weeks after repair. Distal myelinated axon outgrowth after 4 weeks was quantified using histomorphometry. Immunofluorescent imaging was used to evaluate Schwann cell proliferation and epidermal growth factor receptor (EGFR) activation in the regenerating nerves. RESULTS: Herceptin administration increased the rate of motor and sensory neuron regeneration and the number of proliferating Schwann cells in the distal stump after the first week. Herceptin also increased the number of myelinated axons that regenerated 4 weeks after immediate and delayed repair. Reduced EGFR activation was observed using immunofluorescent imaging. INTERPRETATION: Inhibition of the ErbB2 receptor with Herceptin unexpectedly enhances nerve regeneration after acute and delayed nerve repair. This finding raises the possibility of using targeted molecular therapies to improve outcomes of peripheral nerve injuries. The mechanism may involve a novel inhibitory association between ErbB2 and EGFR. Ann Neurol 2016;80:112-126.

Side-To-Side Nerve Bridges Support Donor Axon Regeneration Into Chronically Denervated Nerves and Are Associated With Characteristic Changes in Schwann Cell Phenotype.

BACKGROUND: Chronic denervation resulting from long nerve regeneration times and distances contributes greatly to suboptimal outcomes following nerve injuries. Recent studies showed that multiple nerve grafts inserted between an intact donor nerve and a denervated distal recipient nerve stump (termed "side-to-side nerve bridges") enhanced regeneration after delayed nerve repair. OBJECTIVE: To examine the cellular aspects of axon growth across these bridges to explore the "protective" mechanism of donor axons on chronically denervated Schwann cells. METHODS: In Sprague Dawley rats, 3 side-to-side nerve bridges were placed over a 10-mm distance between an intact donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) distal nerve stump. Green fluorescent protein-expressing TIB axons grew across the bridges and were counted in cross section after 4 weeks. Immunofluorescent axons and Schwann cells were imaged over a 4-month period. RESULTS: Denervated Schwann cells dedifferentiated to a proliferative, nonmyelinating phenotype within the bridges and the recipient denervated CP nerve stump. As donor TIB axons grew across the 3 side-to-side nerve bridges and into the denervated CP nerve, the Schwann cells redifferentiated to the myelinating phenotype. Bridge placement led to an increased mass of hind limb anterior compartment muscles after 4 months of denervation compared with muscles whose CP nerve was not "protected" by bridges. CONCLUSION: This study describes patterns of donor axon regeneration and myelination in the denervated recipient nerve stump and supports a mechanism where these donor axons sustain a proregenerative state to prevent deterioration in the face of chronic denervation.

Long-term outcomes following lower extremity sarcoma resection and reconstruction with vascularized fibula flaps in children.

BACKGROUND: Limb salvage operations in patients with bony oncologic defects carry technical challenges and may require long recoveries. This study aimed to evaluate functional outcomes, donor-site morbidity, and complications in lower limb bony oncologic defects reconstructed with vascularized fibula flaps in children. METHODS: The authors performed a retrospective review of consecutive pediatric patients undergoing this procedure between 1994 and 2012. Data on operative details, functional outcomes, and complications were analyzed. A telephone survey was conducted to assess patient satisfaction and quality of life. RESULTS: Eighteen patients who underwent 19 reconstructions were included. Mean age at resection was 10 years (range, 1.5 to 17 years). No patients developed local recurrence, although two patients had metastatic lung nodules resected. All patients were alive at last review, with a mean follow-up of 57 months (range, 10 to 145 months). Flap survival was 95 percent. Median time to bony union was 24 months (range, 9 to 72 months). The fibula flap fracture rate was 52.6 percent. At the end of the study period, 72 percent of patients were fully weight-bearing, all school-age children had returned to full-time school, and 50 percent were involved in sports. Fifty-six percent of patients participated in the follow-up telephone survey; of these, 90 percent expressed satisfaction with the outcome of the surgery. CONCLUSIONS: This study demonstrates that the vascularized fibula flap is an excellent option for reconstruction of lower limb oncologic defects in children. Despite complications, long-term follow-up suggests that most children are able to lead active lifestyles. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.