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.