Evaluation of collateral sprouting after end-to-side nerve coaptation using a fluorescent double-labeling technique

Abstract

The mechanism of end-to-side neurorrhaphy is believed to be by collateral sprouting, although evidence for this is lacking. This study validates whether axonal sprouting originates from the donor intact nerve by collateral sprouting with the use of a fluorescent double-labeling technique. End-to-side neurorrhaphy was performed on adult female Sprague-Dawley rats. Eight and 12 months postoperatively, animals were injected with true blue and diamidino yellow into the tibialis anterior and/or gastrocnemius muscles and were transcardially perfused with fixative after 7 days of retrograde transport. The lumbar enlargement and the dorsal root ganglia from L3 to L6 were harvested and serial sectioning and fluorescent microscopy were performed. No double-labeling neurons were observed in control animals, but a group of neurons that were greenish or yellowish in color were seen with single labeling. Double-labeling neurons, however, were seen in animals treated with end-to-side neurorrhaphy whether or not perineurotomy was performed. These results demonstrate that one parent nerve fiber can emanate another axon by collateral sprouting following end-to-side neurorrhaphy. We hypothesize that the causes of collateral sprouting might result from "switching signals" and/or "switching factors."

Re-Innervation of the Bladder through End-to-Side Neurorrhaphy of Autonomic Nerve and Somatic Nerve in Rats

“…One is that the regenerated axons could originate from axons collaterally sprouting from intact donor nerve when double-labeled neurons are detected (Yamauchi et al, 2000), and the other is that newly formed axons also originate from terminal sprouting (Matsuda et al, 2005) because of slight injury to the donor nerve during the nerve suturing process when single-labeled neurons could be observed. The appearance of more double- labeled neurons in group I of our study implied that the main regeneration mechanism was collateral axons sprouting (Zhang et al, 1999).…”

Influence of Breaching the Connective Sheaths of the Donor Nerve on Its Myelinated Sensory Axons and on Their Sprouting into the End-to-Side Coapted Nerve in the Rat

“…51 Similarly, regardless of the way in which the connective tissue sheaths were breached, the majority of other studies demonstrated focal Wallerian degeneration in the donor nerve distal from the site of coaptation at the first few weeks after the end-to-side nerve coaptation, but not 2-3 months later. 32,48,54,57,66,69,70 Accordingly, we found significant focal subepineurial degenerative changes in donor nerves with epiperineurial breaching only at 4 weeks, but not at 8 weeks. As shown in our recent longitudinal study, the differences between the donor and control nerves regarding the number of myelinated axons are not statistically significant beyond 4 months after suturing the end-to-side nerve coaptation without windowing the donor nerve connective tissue.…”

“…51 Accordingly, the number of nerve fibers entering the recipient nerve from the mixed donor nerve was higher after making either an epineurial or perineurial window than after the nerve coaptation with epineurial suturing only, resulting in more effective muscle reinnervation during the first 3 months after end-to-side nerve coaptation. 12,13,29,[48][49][50]54 but not later from 4 up to 8 months after surgery. 16,51,53,64 Interestingly, if epineurial or perineurial windows were enlarged from 1 to 4 or 5 mm, respectively, then, also, the magnitude of axonal ingrowth into the recipient nerve was increased.…”

Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

“…The size of the perineurial windows was increased systematically to demonstrate that there is a linear relationship between the perineurial window size and the number of neurons contributing axons into the recipient denervated nerve stump ( Fig 5 ): few if any neurons regenerated axons through very small perineurial windows and more donor neurons grew axons reliably through larger ones. Sprouting, as defined by the presence of doubly labeled neurons and was reported after end-to-side neurorrhaphy [ 21 ], was a minor contributor of donor axons through the side-to-side cross-bridges to the recipient denervated stump ( Fig 3 ). Most of the donor TIB neurons regenerated their axons across the cross-bridges and into the recipient denervated CP nerve stump as measured and defined by their retrograde labelling from axons within the CP nerve stump and their failure to be backlabelled from the donor TIB nerve 10 mm distal to the last cross-bridge ( Fig 3A ).…”