Peripheral nerve injuries

Adam Harding The use of thy-1-YFP-H mice in analysing peripheral nerve regeneration. Introduction: Peripheral nerve injuries (PNIs) may occur during surgical procedures, including those in the orofacial region; for example, during 3rd molar extraction there is a 0.5% risk of a nerve injury occurring with permanent sensory disturbance1. Regeneration is an intrinsic property of the peripheral nervous system (PNS) and if injury is mild nerves may regenerate fully without intervention or require surgical repair. However, when surgical intervention is required, the level of functional recovery rarely reaches the level prior to the injury and patients may develop neuropathic pain2. In 2000, Feng et al3. generated a series of mouse strains using a modified thy-1 gene insert which enables the expression yellow fluorescent protein (YFP) within nerve cells. The pattern of labelled nerve cells varied greatly between strains, however, the pattern within each strain was found to be stable. Of the strains generated, the thy-1-YFP-H strain, has low levels of YFP expression in peripheral nerves which is fairly equally distributed among nerve type and size4. These properties enable realistic, representational visual analysis of nerve regeneration; high levels of YFP expression would obscure many axons from view and be unsuitable for analysis. Recently we have used this strain in a number of studies on peripheral nerve regeneration, the results of two are discussed here as examples of the potential of the thy-1-YFP-H strain for use in nerve regeneration studies. One study assessed the effect of mannose-6-phosphate (M6P) on nerve regeneration. M6P is a potential anti-scarring agent which inhibits the activation of transforming growth factor β a cytokine linked to scar formation5. Previous studies have demonstrated that reducing scarring can improve nerve regeneration6. The second was a pilot study assessing the performance of hollow poly(ethylene-glycol) (PEG) nerve guides versus nerve graft repair in order to create a baseline result for future work on nerve guide conduits (NGCs). Currently nerve grafts are the most reliable form of repair following nerve injuries where tissue has been lost, however, they have drawbacks including: unpredictable level of recovery, sacrifice of healthy donor nerves, and potential neuropathic pain development. In future, NGCs could provide better prospects but currently only six hollow NGCs are FDA/EU approved7. Materials and Methods: 1

Adam Harding Anti-Scarring Agents Surgical Setup: The common fibular (CF) nerve in the right leg of a thy-1-YFP-H mouse was exposed under anaesthesia and freed from surrounding tissues. A gap of 2.5-3.0mm was created between the sectioned nerve endings and then repaired, using a graft taken from the CF nerve of a wild type (WT) littermate that had been harvested and soaked in a solution of 600nm M6P or PBS for 30 minutes prior to use. The repair was secured using fibrin glue. Following surgery, mice were allowed to recover for a period of two-weeks. Following recovery animals were anaesthetised and the CF nerve was re-exposed and fixed in situ using 4% paraformaldehyde. The fixed nerve was sectioned proximally and distally to the repair site and mounted on a microscope slide using Vectashield®. Artificial Nerve Guide Surgical Setup: For graft repairs, both WT and YFP mice were simultaneously anaesthetised and the WT graft was transferred to the YFP mouse immediately following sectioning. For NGC repairs, nerve endings were placed approximately 0.5mm inside a hollow PEG conduit. Following a 3-week recovery period nerves were harvested as described above. Image Acquisition and Analysis: Image Pro-Plus was used to obtain confocal-like images of harvested nerves, which were used to create a montage, using Adobe Photoshop software, that provided a 3D image of the entire injured region. Analysis was conducted to determine: axon sprouting levels along the length of the repair, number of unique axons crossing the repair, and disruption of axons across the initial 1.5mm of the repair [fig.1]. Results were analysed statistically using Graphpad Prism software: 2way ANOVA with Bonferroni post-tests for sprouting index results; 2-tailed t-test for all other results.

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Figure 1: Axon Sprouting - Lines were drawn across the image at 0.5mm intervals starting from the point at which normal morphology ended and finishing at a point where axons had exited the graft (3.5mm in anti-scarring study; 4.0mm in NGC study). Axons were counted at each of the 0.5mm intervals and compared to the number prior to the first interval to give a percentage sprouting index for each interval along the repair. Unique Axons Crossing the Repair: To determine the number of functionally unique axons regenerating successfully, axons were traced back from the final interval to the start to see how many unique axons had reached the distal nerve ending. Axon disruption was measured by tracing axons back from the 1.5mm interval to the start and then measuring their lengths.

Results: Anti-Scarring Agents: Axons in M6P treated grafts had a similar sprouting profile across the length of the repair to those in vehicle treated grafts [fig.2] and also had a similar number of unique axons cross the repair and reach the distal nerve stump [fig.3]. On average, axons in M6P treated grafts crossed the initial 1.5mm of the repair using a significantly shorter route than those in vehicle treated grafts [fig.4].

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Figure 2: No significant differences between M6P and vehicle treated grafts were found at any 0.5mm interval across the repair.

Figure 3: No significant differences in the percentage of unique axons regenerating across the repair were found between M6P and vehicle treated grafts.

Figure 4: Axons in M6P treated grafts took a significantly shorter route across the initial 1.5mm of the repair than those in vehicle treated grafts. M6P: 13.55% (SEM = 1.17); Vehicle: 20.51% (2.36); p