An efficient stereological sampling approach for quantitative assessment of nerve regeneration

Aims: The aim of the present study was to find the most efficient sampling strategy for stereological analysis of peripheral nerve, including the number of myelinated nerve fibres, axon cross‐sectional area and myelin sheet thicknesses of nerve fibres. Methods: Two groups of rats underwent experimental resection of the tibial and peroneal nerves. The first group received tibial‐peroneal end to end autograft repair (n = 6). Tibial and peroneal nerves were isolated, transected, and separated 1 cm distal to the trifurcation, where they lay adjacent to each other by a 1‐cm gap, then repaired with an autologous nerve graft taken from the tibial nerve. The proximal stump of the tibial nerve and distal stump of the peroneal nerve were connected to each other by means of the nerve graft. The second group received tibial‐peroneal repair with a flexible collagen tube (n = 6). After 90 days of recovery, animals were sacrificed and nerve segments were removed and sectioned for microscopy. Three different sampling strategies, that is, small, medium and large step sizes were applied to obtain each quantitative parameter. Results: There are no significant differences between these sampling strategies with respect to total number of myelinated nerve fibres, axon cross‐sectional area and myelin sheet thicknesses of nerve fibres. Conclusion: Findings show that one can achieve the desired estimate precisely with a rather large and less time‐consuming sampling approach. In addition, it was observed that the size discrepancy of nerve regeneration can be improved by collagen tube conduit even with a 1‐cm gap.