Sonic hedgehog regulation of cavernous nerve regeneration and neurite formation in aged pelvic plexus

Abstract

Introduction: Erectile dysfunction (ED) is a significant health concern that greatly impacts quality of life, and is common in men as they age, impacting 52% of men between the ages of 40 and 70. A significant underlying cause of ED development is injury to the cavernous nerve (CN), a peripheral nerve that innervates the penis. CN injury also occurs in up to 82% of prostatectomy patients. We recently showed that Sonic hedgehog (SHH) protein delivered by peptide amphiphile (PA) nanofiber hydrogel to the CN and penis of a prostatectomy model of CN injury, is neuroprotective, accelerates CN regeneration, improves erectile function ~60%, preserves penile smooth muscle 56% and suppresses collagen deposition 30%. This regenerative potential is substantial in an adult prostatectomy model (P120). However prostatectomy patients are typically older (61.5 ± 9.6 years) and our models should mimic patient conditions more effectively when considering translation. In this study we examine regenerative potential in an aged prostatectomy model (P200-329).

Methods: The caudal portion of the pelvic ganglia (MPG) and CN were dissected from adult (n = 11), and aged (n = 13) Sprague Dawley rats, and were grown in organ culture 3 days. Uninjured and 2 day CN crushed MPG/CN were exposed to Affi-Gel beads containing SHH protein, PBS (control), or 5e1 SHH inhibitor. Neurites were quantified by counting the number of growth cones normalized by tissue perimeter (mm) and immunohistochemistry for SHH, patched1 (PTCH1), smoothened (SMO), GLI1-3, and GAP43 were performed.

Results: SHH treatment increased neurites 3.5-fold, in uninjured adult, and 5.7-fold in aged rats. Two days after CN crush, SHH treatment increased neurites 1.8-fold in adult rats and 2.5-fold in aged rats. SHH inhibition inhibited neurite formation in uninjured MPG/CN but not in 2 day CN crushed MPG/CN. PTCH1 and SMO (SHH receptors), and SHH transcriptional activators/repressors, GLI1-3, were abundant in aged MPG/CN with unaltered localization. ROCK1 was induced with SHH treatment.

Conclusions: Reintroduction of SHH protein in an aged prostatectomy model is even more effective in promoting neurite formation/CN regeneration than in the adult. The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. These studies are significant because most prostatectomy patients are not young and healthy, as with adult rats, so the aged prostatectomy model will more accurately simulate ED patient response. Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients.

Keywords: Aging; Cavernous nerve regeneration; Neurite; Prostatectomy; Sonic hedgehog.

Figure 1:

(A) Photos of the pelvic plexus from adult (P115–120) and aged (P200–329) Sprague Dawley rats. The CN is flat and thin in the adult rat with a small, clearly defined MPG. In the aged rat, the CN is thicker and rounder in appearance, with a larger, less well-defined MPG. The vascular supply does not appear diminished with age. (B) Magnified view of MPG treated with SHH protein, shows growing neurites with clearly visible growth cones at the tips and elongating fibers. (C) Neurites were confirmed with GAP43 (growth cones marker). 100–800X magnification.

Figure 2:

Uninjured adult and aged MPG were grown in organ culture for 3 days with PBS (control) or SHH protein and neurites were quantified (A and B). The number of neurites/mm increased 3.5 fold with SHH treatment in adult rats (249%, p=0.0001). The number of neurites/mm increased 5.7-fold with SHH treatment in aged rats (468%, p=0.013). There was no difference in neurite formation in adult and aged rats treated with PBS (control, p=0.085). SHH treatment was 2.7-fold less in aged rats in comparison to SHH treated adult rats (63%, p=0.001). 800X magnification.

Figure 3:

Adult and aged MPG that underwent CN crush and were isolated after two days were grown in organ culture for 3 days with PBS (control) or SHH protein, and neurites were quantified (A and B). The number of neurites/mm increased 1.8 fold with SHH treatment in adult rats (82%, p=0.044). The number of neurites/mm increased 2.5-fold with SHH treatment in aged rats (150%, p=0.030). There was no difference in neurite formation in adult and aged rats treated with PBS (control, p=0.298) or with SHH protein (p=0.197). Red line indicates enlarged region. 800X magnification.

Figure 4:

Uninjured (A) and 2 day CN crushed (B) aged MPG/CN were grown in organ culture for three days with Affi-Gel beads delivering 5E1 SHH inhibitor. SHH inhibition of uninjured MPG/CN had neurite formation in only one small region away from the Affi-Gel beads containing SHH inhibitor (A). In MPG that underwent CN crush and then were cultured after 2 days, SHH inhibition did not decrease neurite formation (B). 800X magnification.

Figure 5:

MPG from aged Sprague Dawley rats that underwent CN crush/MPG tension injury were dissected after two days and grown in organ culture for three days with PBS (A) or SHH protein (B). Aged rat MPG responded to CN injury with neurite formation in the CN, and also in the pelvic nerve and hypogastric nerve, which innervate the bladder (A). Aged pelvic plexus were responsive to SHH treatment (B). 800X magnification.

Figure 6:

IHC analysis was performed in uninjured adult and aged rat MPG/CN that was grown in organ culture for 3 days. (A) In aged rats, nNOS was less abundant by visual observation in MPG neurons that innervate the penis. The SHH receptors, PTCH1 and SMO, were identified in adult and aged rat MPG neurons that innervate the penis, with no apparent difference with age. (B) PTCH1, the binding part of the SHHH receptor, was identified in growing neurites, as confirmed with dual staining for β–III tublin. Arrows indicate protein. 100–1000X magnification.

Figure 7:

IHC analysis was performed on uninjured adult and aged rat MPG/CN that was grown in organ culture for 3 days and were assayed for GLI1–3. All three GLI isoforms were identified in glial cells surrounding MPG neurons of adult and aged rats. Arrows indicate GLI1–3 proteins. 1000X magnification.

Figure 8:

IHC analysis was performed on uninjured MPG/CN from aged rats that were treated with PBS or SHH protein. ROCK2 was abundant in uninjured MPG/CN treated with PBS and SHH (A). ROCK1 was not identified in uninjured MPG/CN (A), however ROCK1 was induced in response to SHH treatment (A). ROCK2 was localized in MPG neurons, while ROCK1 was present in associated glial cells (B). 50–400X magnification.