PURPOSE: The purpose of this study was to use novel transgenic mouse models - Thy1-YFP(16), Myo-GDNF and Thy1-YFP(16)xMyoGDNF mice, to assess (1) the degree of motor axonal sprouting induced by Botulinum Toxin A (BTxA) poisoning and (2) the degree to which glial cell line-derived neurotrophic factor (GDNF) accelerates axonal sprouting post-BTxA injection and recovery of neuromuscular function.
METHODS: 96 mice were divided into four experimental groups: (A) Wild-type (WT) CF1 mice, (B) Myo-GDNF mice -- which over-express glial cell line-derived neurotrophic factor (GDNF) in its musculature, (C) Thy1-YFP(16) - which constitutively express YFP in its peripheral nerves and (D) Thy1-YFP(16)xMyo-GDNF mice - which constitutively express YFP in its peripheral nerves and over-expresses GDNF in its musculature. All groups underwent exposure of the right gastrocnemius muscle with subsequent BTxA injection. These animals served as their own controls by undergoing contralateral gastrocnemius muscle exposure and injection with vehicle solution (0.1% BSA). Groups C and D were analyzed by live fluorescent imaging obtaining high resolution, digital images of the motor end-plate regions at 7, 21, and 35 days post-BTxA injection. Outcome measures included both the number and length of axonal sprouts in the toxin injected and contralateral control side. On post-operative day 42, the Thy1-YFP(16) and the Thy1-YFP(16)xMyo-GDNF mice were sacrificed and underwent trans-cardiac perfusion with subsequent gastrocnemius muscle harvest for confocal microscopy analysis. Groups A and B underwent similar right gastrocnemius exposure with BTxA injection and contralateral vehicle injection. At post-operative days 0, 7, 14, 21, 28, and 35, six animals from groups A and B were randomly selected to undergo walking track analysis using the Sciatic function index (SFI), and muscle force testing to attain peak tetanic forces. These animals then underwent bilateral gastrocnemius muscle harvest for inter-group comparison by confocal microscopy.
RESULTS: At the 7, 21, and 35 day time points post-BTxA injection the Thy1-YFP(16)xMyo-GDNF mice exhibited increased motor axonal sprouting as compared to the Thy1-YFP(16) mice. Additionally, confocal imaging and muscle force testing showed (1) earlier return of neuromuscular recovery and (2) higher peak tetanic forces in the Myo-GDNF mice compared to WT CF1 mice controls.
CONCLUSION: GDNF, a potent motor neurotrophic factor, was seen to augment and accelerate recovery from Botulinum Toxin A poisoning in a transgenic mouse gastrocnemius model. Mice constitutively expressing GDNF (Thy1-YFP(16)xMyo-GDNF) show (1) evidence of increased axonal sprouting via fluorescence live-imaging versus non-Myo-GDNF controls(Thy1-YFP(16) mice),(2) increased axonal sprout length and (3)increased number of sprouts per motor end-plate. Additionally, Myo-GDNF mice exhibit earlier neuromuscular recovery with higher peak tetanic force generation compared to CF1 controls. This data directly applies to the future treatment paradigm of muscular paralysis secondary to unintended BTxA spread. As horizon treatment modalities could include alteration of local muscle GDNF levels by direct muscular injection of GDNF, therein reducing the amount of unintended muscular paralysis time.