Login to Access Video or Poster Abstract: MP45-07
Sources of Funding: NIH/NIDDK DK079184.

Introduction

Sonic hedgehog (SHH) protein delivered by nanoparticle based peptide amphiphile (PA) nanofiber hydrogel to the cavernous nerve at the time of crush injury (prostatectomy model), is neuroprotective, promotes cavernous nerve (CN) regeneration, and return of erectile function, in a rat model. Little is known about the mechanism of how SHH promotes CN regeneration. We hypothesize that SHH promotes sprouting of pelvic ganglia and CN neurons that innervate the penis, in order to enhance regeneration. We examine this hypothesis in an in vitro organ culture model.

Methods

The caudal portion of the pelvic ganglia and CN were dissected from adult Sprague Dawley rats (n=47) and were grown on Matrigel in growth factor reduced medium for three to five days. Pelvic ganglia/CN were exposed to Affi-Gel beads containing: 1.) SHH protein, 2.) 5e1 and cyclopamine SHH inhibitors, and 3.) SHH protein delivered by PA. Additional pelvic ganglia/CN tissue underwent CN crush and were exposed to SHH protein or mouse serum albumin protein (control) by PA in vivo for 4 days with an additional 4 days in culture. Sprouting was evaluated for number of sprouts and their length, and by immunohistochemical analysis for sprouting markers (GAP43 and nNOS).

Results

Sprouting of neurons in the pelvic ganglia and CN were increased with SHH treatment. Sprouts were more abundant, longer in length, and had increased branching, in comparison to controls. Sprouting was even further enhanced in CN injured nerves with SHH treatment. Sprouting did not occur in the presence of either SHH inhibitor. The CN had similar sprouting potential at 4 and 9 days after crush injury. SHH induced sprouting even when not delivered to the CN until 4 days after injury. Sprouts continued to grow in organ culture once initiated with SHH PA in vivo. Localization of SHH delivery makes a difference in sprouting potential. Sprouts formed in response to SHH treatment stained strongly for nNOS protein.

Conclusions

SHH PA treatment promotes CN regeneration by enhancing sprouting of pelvic ganglia and CN neurons. Understanding the mechanism of SHH PA action on neuronal tissue is critical for translation to prostatectomy patients and to further enhance regeneration.

Funding

NIH/NIDDK DK079184.