30. Validation of a Novel Transgenic Mouse Model Expressing GFP in Motoneurons for the Investigation of Peripheral Nerve Injury

PURPOSE: Peripheral nerve injury (PNI) is a significant clinical problem, and novel therapies are evidently necessary to optimize functional outcomes. However, the regenerative environment of motor fibers must first be thoroughly elucidated, an effort which is currently limited by inherent drawbacks of conventional visualization techniques. Mnx1 is a homeobox transcription factor that has been established as a spinal motoneuron-specific marker. Here, we propose a transgenic mouse model (Mnx-GFP) in which expression of a ZsGreen1 GFP is driven by the Mnx1 promoter for the specific investigation of motoneuron regeneration. This study aims to confirm an absence of off-target effects of recombination on nerve regeneration, fiber characteristics, and grip strength in male Mnx-GFP mice after median nerve injury. METHODS: 4-week old founder mice (males: heterozygous Mnx1-cre, females: homozygous Rosa expressing ZsGeen1 GFP) were obtained from the Jackson Laboratory and cross-bred in-house, followed by genotyping to confirm recombination. Male transgenic Mnx-GFP (n=2) and wildtype C57BL/6 (n=10) mice underwent surgical transection and immediate repair of the right median nerve. Every 3d following the intervention, unilateral volitional forelimb grip strength was assessed in all mice. On day 27, the median nerve was re-exposed and transected 4mm distal to the original repair site, at which point a 2mm distal segment was collected for histomorphometry. Fluorogold (FG) neurotracer was applied to the nerve stump to label regenerated neurons, followed by collection of DRG and spinal cord tissue 1 week later for immunofluorescent imaging and immunostaining for the neuronal marker Isl1. RESULTS: No significant differences in grip strength (g) were observed between Mnx-GFP and wildtype at baseline (122.7±11.74 vs. 122.8±6.72; p=0.99) or 4-weeks post-injury (31.83±4.41 vs. 39.29±1.45; p=0.071). Retrograde-labelling revealed that motor (274±36 vs. 169±20; p=0.057) and sensory (1160±104 vs. 1072±118; p=0.76) neuron counts were comparable between cohorts. Furthermore, the total pool of Mnx+ motoneurons in the cervical enlargement was observed to include neurons that successfully regenerated to the point of neurotracer application (FG+) as well as those that did not. Interestingly, a considerable number of non-brachial motoneurons were both GFP+ and Isl1+. Histomorphometry of the median nerve revealed no significant difference in myelin thickness (0.56±0.020μm vs. 0.51±0.014μm; p=0.19), axon diameter (1.89±0.040μm vs. 1.81±0.090μm; p=0.75), fiber diameter (3.025±0.025μm vs. 2.84±0.10μm; p=0.44), or g-ratio (0.64±0.025 vs. 0.64±0.012; p>0.99) at the 4-week timepoint. All values are presented as Mnx-GFP vs. C57BL/6. CONCLUSION: Currently, no transgenic models exist to specifically visualize motoneuron regeneration. This data demonstrates that the Mnx-GFP transgene had no measurable impact on regenerative capacity, fiber characteristics, or motor function, supporting this model as a valid tool to study PNI. The use of Mnx-GFP mice alongside more traditional analytical metrics thus has the potential to improve the efficiency and reproducibility of data collected in this field. Further, the Mnx+ motoneuron population was not limited to neurons that successfully regenerated to the site of retrograde-labelling, suggesting a potential utility of this model for assessing regenerative efficacy. Future investigations may aim to examine spinal motor column-specific markers to aid in identifying the cell population of interest within the larger motoneuron pool.