Erectile Function Restoration After Repair Of Cavernous Nerves Injury By Adipose Derived Stem Cells In Rats

Part I Culture, Identification and multilineage differentiation potential of adipose derived stem cells in vitroObjective It is reported that adipose derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro, which provide foundation to restore ED caused by carvernous nerve (CN) injury.Methods Adipose tissue from SD rat abdomen and caputepididymidis was digested with collagenase type Ⅰ, followed by filter and centrifugation. The isolated adi- pose stromal cells were cultured in dishes. ADSCs markers were measured by flow cytometry.Cells at third passage were used for in vitro differentiation. Adipogenic and neuronal differentiation was induced by incubation of the ADSCs with different ind-uction media. Two methods were used to induce ADSCs into neuron-like cells. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor (EGF), basic fibroblast growth factor(bFGF), and brain derived neurotrophic factor (BDNF) for 3 days, then with the neurogenic induction medium contain-ning isobutylmethylxanthine (IBMX), indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. Oil red O staining was carried out to identify adipose cells, immuneofluorescence and western blot test protein of GFAP and beta tubulin Ⅲ expressioned by nerve cells.Results Flow cytometry demonstrated that the ADSCs expressed CD44 and CD90, but CD45 and CD34 is very little, the percentage were 96.0%,98.5%,0.6%, 0.5%. ADSCs differentiated into other mesodermal cells including adipocytes, chondrocytes, and osteocytes. These cells were induced to differentiate into adipose cells as evidenced by adipocytes morphology and the lipid droplet was dyed red by oil red O dyeing. These cells were induced to differentiate into neuron-like cells as evidenced by neuronal morphology and the presence of neuronal markers including GFAP and β-tubulin Ⅲ. The induction rate of method 1 and method 2 respectively for 74±3.3%、65±2.1% and 51±1.2%、41±1.1%. The number of positive cells exhibited significant difference after the induction (p<0.05).Conclusions ADSCs can be sucessfuly obtained from a small amount fat tissue and developed in culture. It suggested that ADSCs are able to differentiate into neural-like cells in vitro. It is high rate and short time of ADSCs neural differentiation on method 1. The administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro. ADSCs may be an alternative source of stem cell therapy for neuropathic ED.Part II Research of adipose derived stem cells on cavernous nerve regeneration in a rat modelObjective It is reported that nerve-sparing radical prostatectomy is the treatment of choice for localized prostate carcinoma insexually active men. Postoperative erectile dysfunction (ED) frequently occurs in patients who received surgical or radiation therapy for their prostate. In many cases, the cavernous nerves (CNs) may have been inadvertently damaged by manipulation during nerve-sparing prostatectomy. The aim of this study was to evaluate whether the intracavernous injection of ADSCs had neuromodulatory effects on the regeneration of CN and also to evaluate the recovery of erectile function after bilateral CN-crush injury in a rat model.Methods Thirty Sprague-Dawley male rats were randomly divided into three equal groups:one group underwent sham operation, while two groups underwent bil-ateral CN crush. Crush-injury group was treated at the time of injurywith intracaver-nous injection of ADSCs, or injured control group with no further intervention. Ere-ctile function was assessed by CN electrostimulation after 3 months. Crushed nerves were collected for Toluidine Blue Staining. Penile tissues were collected for Masson’s Trichrome Staining. Western blot test protein of nNOS expressioned by penile tissues.Results Three months after surgery, in the group that underwent bilateral nerve crushing with no further intervention, the functional evaluation showed a lower mean maximal intracavernous pressure (ICP)(38.6±12.4) and maximal ICP per mean arterial pressure (MAP)(0.26±0.03) with CN stimulation than those in the shamgroup. In the group with an immediate intracavernous injection of ADSCs, the mean maximal ICP (90.1±12.8) and maximal ICP/MAP(0.44±0.05)were significantly higher than those in the injured control group(p<0.05). The group treated with ADSCs showed a statistically significant increase in the number of myelinated axons compared with the injured control group(92.5±18 versus 58±20,p<0.05). On the Masson’s trichrome staining, the muscle content was decreased and collagen content was increased in the injured control group as compared with the ADSCs treated group(5.4±1.1 versus 9.2±2.8,p<0.05).Conclusions These results show that the intracavernous injection of ADSCs to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that penile injection of ADSCs can improve recovery of erectile function in a rat model of neurogenic EDPart Ⅲ Research of erectile dysfunction estoration by adipose derived stem cells combined with autologous vein graft in ratsObjective Cavernous nerve (CN) injury is the main cause of erectile dysfunction (ED) following radical prostatectomy. The recovery of erectile function following this procedure remains challenging. Here, we investigated the ability of adipose derived stem cells (ADSCs) combined with autologous vein graft to improve erectile function in a ratmodel of bilateral long CN resection.Methods Sprague-Dawley rats (n=36) were randomized into four groups:A underwent sham operation group;B ADSCs combined with autologous vein graft treat group, C PBS combined with autologous vein graft treat group; D underwent bilateral CN resection group(0.8 cm). Erectile function assessed after 3 months by measuring intracavernosal pressure. Penile tissues were collected for histology. Penile tissues were collected for Masson’s Trichrome Staining. Immunohistochemical staining of nNOS-positive fibers expressioned by penile tissues. The anatomy of transplanted veins under microscopy.Results Erectile function assessed after 3 months by measuring intracavernosal pressure demonstrated significant recovery in erectile function in ADSCs combined with autologous vein graft treat group than PBS combined with autologous vein graft treat group and bilateral CN resection group(p<0.05). Immunohistochemical staining showed that the nNOS-positive fibers was significantly larger in Group B than in Group D(p<0.05). On the Masson’s trichrome staining, ADSCs combined with autologous vein graft treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. The group treated with ADSCs showed a statistically significant increase on the smooth muscle/collagen ratio compared with bilateral CN resection group (8.5±2.1 versus 4.6±1.2,p<0.05).The newly formed nerve tissue was seen in ADSCs combined with autologous vein graft treat group.Conclusions ADSCs combined with autologous vein graft can improve regenerating CN tissue and restoring autonomic erectile function after long bilateral CN resection (0.8 cm) in rats.