Neural-like cells from adipose-derived stem cells for cavernous nerve injury in rats

Abstract

Although the remaining nerve tissue can regenerate and partly restore erectile function when the cavernous nerve is compressed/severed and function lost, the limited regenerative ability of these nerve tissues often fails to meet clinical needs. Adipose-derived stem cells are easy to obtain and culture, and can differentiate into neural cells. Their proliferation rate is easy to control and they may be used to help restore injured cavernous nerve function. Sprague-Dawley male rats (n = 45) were equally randomized into three groups: fifteen rats as a sham-operated group, fifteen rats as a bilateral nerve crush (BINC) group (with no further intervention), fifteen rats as a BINC with intracavernous injection of one million neural-like cells from adipose-derived stem cells (NAS) (BINC + NAS) group. After 4 weeks, erectile function was assessed by stimulating the cavernous body. The number of myelinated axons in the dorsal cavernous nerve was determined by toluidine blue staining. The area of neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve was measured by immunohistochemical staining. Masson staining was used to analyze the ratio of smooth muscle to collagen in penile tissue. The results demonstrate that maximal intracavernous pressure, the ratio of maximal intracavernous pressure to mean arterial pressure, the numbers of myelinated axons and neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve, and the ratio of smooth muscle to collagen could be increased after cell transplantation. These findings indicate that neural-like cells from adipose-derived stem cells can effectively alleviate cavernous nerve injury and improve erectile function. All animal experiments were approved by the Animal Ethics Committee of Huazhong University of Science and Technology, China (approval No. 2017-1925) on September 15, 2017.

Keywords: adipose-derived neural stem cells; cavernous nerve; cell differentiation; corpus cavernosum; erectile dysfunction; nerve regeneration; neural regeneration; neurons; radical prostatectomy.

Figure 1

Neural differentiation capacity of ADSCs. (A) Primary ADSCs. (B) ADSCs treated with the preinduction medium then cultured in the neurogenic induction medium for 48 hours. (C) Tuj-1 (red) was detected after neural differentiation. (D) GFAP (red) was detected after neural differentiation (original magnification, 100×). ADSCs: Adipose-derived stem cells; GFAP: glial fibrillary acidic protein.

Figure 2

Effects of NAS on ICP and MAP in a rat model of cavernous nerve crush injury at 4 weeks after modeling. (A) Sham-operated group: ICP 109.6 ± 12.8 cmH₂O; (B) BINC group: ICP 40.2 ± 11.2 cmH₂O; (C) BINC + NAS group: ICP 84.5 ± 10.8 cm H₂O. (A–C) The horizontal axis represents the time frequency and voltage at which the cavernous nerve is electrically stimulated. (D) The maximal ICP/MAP ratio. Data are expressed as the mean ± SD (one-way analysis of variance followed by Student-Newman-Keuls test). *P < 0.05, vs. BINC group; #P < 0.05, vs. sham-operated group. NAS: Neural-like cells from adipose-derived stem cells; ICP: intracavernous pressure; MAP: mean arterial pressure; BINC: bilateral cavernous nerve-crush injury.

Figure 3

Effect of NAS on the number of myelinated axons in dorsal nerves of a rat model of cavernous nerve injury. (A–C) Myelinated axons (arrows) in dorsal nerves (original magnification, 1000×). (A) Sham-operated group: Abundant myelinated axons of cavernous nerves with normal morphological appearances; (B) BINC group: the number of myelinated axons decreased substantially and displayed atrophy; (C) BINC + NAS group: a significant increase in the regeneration of well-orientated myelinated axons relative to BINC group; (D) the number of myelinated axons. Data are expressed as the mean ± SD (one-way analysis of variance followed by Student-Newman-Keuls test). *P < 0.05, vs. BINC group; #P < 0.05, vs. sham-operated group. BINC: Bilateral cavernous nerve-crush injury; NAS: neural-like cells from adipose-derived stem cells.

Figure 4

Effect of NAS on the number of nNOS-positive nerve fibers in the penile tissue of a rat model of cavernous nerve injury. (A–C) nNOS (brown-stained fiber) in penile tissue (original magnification, 200×). (A) Sham-operated group: Predominant dark brown staining of nerve fibers; (B) BINC group: a paucity of brown-stained fibers; (C) BINC + NAS group: a significant increase in the number of brown-stained nerve fibers relative to the BINC group; (D) the numbers of nNOS-positive nerve fibers. Data are expressed as the mean ± SD (one-way analysis of variance followed by Student-Newman-Keuls test). *P < 0.05, vs. BINC group; #P < 0.05, vs. sham-operated group. BINC: Bilateral cavernous nerve-crush injury; NAS: neural-like cells from adipose-derived stem cells; nNOS: neuronal nitric oxide synthase.

Figure 5

Effect of NAS on smooth muscle/collagen ratio in penile tissue of a rat model of cavernous nerve injury. (A–C) Smooth muscle (red)/collagen (blue) ratio in penile tissue (original magnification, 100×). (A) Sham-operated group; (B) BINC group: muscle content was decreased and collagen content was increased; (C) BINC + NAS group: muscle content was increased and collagen content was decreased. (D) Ratio of smooth muscle/collagen. Data are expressed as the mean ± SD (one-way analysis of variance followed by Student-Newman-Keuls test). *P < 0.05, vs. BINC group. BINC: Bilateral cavernous nerve-crush injury; NAS: neural-like cells from adipose-derived stem cells.