Local Delivery of Supplemental Agrin at the time of Injury Prevents Motor Endplate Degradation
Henry Hoang, B.S Cand.1, Winnie A. Palispis, M.D2, Jennifer Uong, B.S Cand.1, Justin P. Chan, B.A1, Ranjan Gupta, M.D2.
1University of California at Irvine, Irvine, CA, USA, 2University of California at Irvine, Orange, CA, USA.
PURPOSE: Traumatic nerve injuries often result in loss of function despite optimal surgical management. One reason for poor recovery is the degradation of the neuromuscular junction(NMJ) after injury. Our lab has previously shown the novel finding that preservation of the NMJ after nerve injury actually improves functional recovery following reinnervation. Developmental neurobiology literature suggest that Agrin, a proteoglycan involved in synaptogenesis, may play a critical role in NMJ preservation. Our experiments with a murine model demonstrated that genetic deletion of MMP-3, a matrix-metalloproteinase that degrades agrin on a homeostatic basis, allowed sufficient agrin levels to remain in denervated motor endplates. Moreover, our lab further demonstrated that denervated motor endplates with sufficient agrin levels have superior morphometric qualities and are highly conducive to promoting successful reinnervation. Here, we sought to determine if direct local delivery of agrin after a traumatic nerve injury might preserve the motor endplates.
METHODS: As previously described, a denervation model was created in 6 week old WT and agrin deficient C57BL/6 murine strains by excising 10 mm right sciatic nerve segment from the mid-thigh of the mice and suturing the proximal nerve stump to the gluteal muscle with 9-0 suture so as to prevent regeneration. Agrin deficient mice were either injected with supplemental agrin or PBS as a control at the site of injury. The downstream denervated and contralateral control soleus, plantaris and gastrocnemius muscles were harvested for immunohistochemistry, cryo-sections with H&E staining, and quantitative western blots at the 1,2,4,8 and 16 week timepoints.
RESULTS: Fluorescence confocal imaging of harvested soleus muscles revealed that agrin supplemented animals retained superior motor endplate morphology over control animals up to the 16 week timepoint (Fig.1). The average surface area of agrin supplemented denervated endplates were significantly greater than control endplates in all analyzed timepoints (Fig.2).
CONCLUSION: These experiments demonstrated that supplemental agrin delivered locally at the site of injury is effective in preserving motor endplates in denervated mice hindlimbs. Consistent with our work that showed the knockdown of MMP3 allowed agrin to remain at the NMJ and thereby preserved the motor endplates so as to improve function recovery, these current experiments support this strategy and role of agrin in maintaining the motor endplates after traumatic nerve injury. These data support our strategy of developing therapeutic targets that will delay or prevent NMJ degradation so as to prolong the window of opportunity for surgical intervention and maximize the opportunity for functional recovery.
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