Ludovic Ferretti

Effects of Low‐Energy Shockwave Therapy on the Erectile Function and Tissue of a Diabetic Rat Model

Introduction.  Low-energy shockwave therapy (LESWT) has been shown to improve erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). However, the underlying mechanism remains unknown. Aim.  The aim of this study is to investigate whether LESWT can ameliorate DM-associated ED in a rat model and examine the associated changes in the erectile tissues. Methods.  Newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous mesenchymal stem cells (MSCs). Eight weeks later, eight of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group), whereas another eight rats were subject to shockwave (SW) treatment (DM+SW group). Each rat in the DM+SW group received 300 shocks at energy level of 0.1 mJ/mm(2) and frequency of 120/minute. This procedure was repeated three times a week for 2 weeks. Another 2 weeks later, all 24 rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Main Outcome Measures.  Erectile function was measured by ICP. Neuronal nitric oxide synthase (nNOS)-positive nerves and the endothelium were examined by immunofluorescence staining. Smooth muscle and MSCs were examined by phalloidin and EdU staining, respectively. Results.  STZ treatment caused a significant decrease in erectile function and in the number of nNOS-positive nerves and in endothelial and smooth muscle contents. These DM-associated deficits were all partially but significantly reversed by LESWT. MSCs (EdU-positive cells) were significantly more numerous in DM+SW than in DM rats. Conclusion.  LESWT can partially ameliorate DM-associated ED by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis. These beneficial effects appear to be mediated by recruitment of endogenous MSCs. Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, and Lin C-S. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2013;10:738-746.

Both Immediate and Delayed Intracavernous Injection of Autologous Adipose-derived Stromal Vascular Fraction Enhances Recovery of Erectile Function in a Rat Model of Cavernous Nerve Injury

BACKGROUND Intracavernous injection of cultured adipose-derived stem cells (ADSCs) effectively restores erectile function in cavernous nerve (CN)-injured rats when administered at the time of injury. However, culturing exposes ADSCs to the risk of contamination and dedifferentiation. OBJECTIVE Explore the effect of uncultured autologous adipose-derived stromal vascular fraction (SVF) on improving erectile function in a rat model of CN injury when administered at the time of injury or 4 wk after injury. DESIGN, SETTING, AND PARTICIPANTS Eighty-nine male Sprague Dawley rats were randomly divided into four groups. CN injury or sham surgery was performed at the start of the study, and rats were treated with either SVF or vehicle. Functional testing and histologic analysis were performed 12 wk after CN crush or sham surgery. INTERVENTION We used intracavernous injection of saline immediately after CN crush (n=23), intracavernous injection of SVF immediately after CN crush (n=17), intracavernous injection of SVF 4 wk after CN crush (n=23), or sham surgery (n=26). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We studied intracavernous pressure (ICP) response to CN electrostimulation and performed histologic examination of midpenile cross-sections. Data were analyzed using one-way analysis of variance followed by the Tukey-Kramer test. RESULTS AND LIMITATIONS Both immediate and delayed treatment with SVF resulted in a significantly increased ICP-to-mean arterial pressure ratio compared with the vehicle-treated group. Both immediate and delayed treatment with SVF significantly increased expression of neuronal nitric oxide synthase and neurofilament in dorsal penile nerves compared to the vehicle group. Furthermore, the smooth muscle-to-collagen ratio within the corpus cavernosum was significantly improved in both of the SVF groups compared to vehicle-treated rats. The main limitation of the study is the lack of determination of the SVF components. CONCLUSIONS Uncultured autologous SVF injected immediately or 4 wk after CN crush improved erectile function, promoted nerve regeneration, and prevented fibrosis of the corpus cavernosum following CN injury.

Effects of intravenous injection of adipose-derived stem cells in a rat model of radiation therapy-induced erectile dysfunction.

INTRODUCTION Radiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED). AIM To investigate whether injection of adipose-derived stem cells (ADSCs) can ameliorate RT-associated ED. METHODS Thirty male rats were divided into three groups. The control + phosphate-buffered saline (PBS) group received tail-vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail-vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail-vein injection of ADSCs, which were labeled with 5-ethynyl-2-deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining. MAIN OUTCOME MEASURES Erectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification. RESULTS Radiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU-positive cells were visible in MPG. CONCLUSIONS Radiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration.

Scaffoldless Tissue Engineering of Stem Cell Derived Cavernous Tissue for Treatment of Erectile Function

INTRODUCTION As one-third of erectile dysfunction (ED) patients do not respond to phosphodiesterase-5 inhibitors, there is great demand for new therapeutic options. Adipose tissue-derived stem cells (ADSCs) represent an ideal source for new ED treatment. AIM To test if ADSCs can be differentiated into smooth muscle cells (SMCs) and endothelial cells (ECs), if these differentiated cells can be used to engineer cavernous tissue, and if this engineered tissue will remain for long time after implantation and integrate into corporal tissue. METHOD Rat ADSCs were isolated and differentiated into SMC and ECs. The differentiated cells were labeled with 5-ethynyl-2-deoxyuridine (EdU) and used to construct cavernous tissue. This engineered tissue was implanted in penises of normal rats. The rats were sacrificed after 1 and 2 months; penis and bone marrow were collected to assess cell survival and inclusion in the penile tissues. MAIN OUTCOME MEASURES The phenotype conversion was checked using morphology, immunocytochemistry (immunohistochemistry [IHC]), and Western blot for SMC and EC markers. The cavernous tissue formation was assessed using rat EC antibody (RECA), calponin, and collagen. The implanted cell survival and incorporation into penis were evaluated with hematoxylin and eosin, Massons trichrome, and IHC (RECA, calponin, and EdU). RESULTS The phenotype conversion was confirmed with positive staining for SMC and EC markers and Western blot. The formed tissue exhibited architecture comparable to penile cavernous tissue with SMC and ECs and extracellular matrix formation. The implanted cells survived in significant numbers in the penis after 1 and 2 months. They showed proof of SMC and EC differentiation and incorporation into penile tissue. CONCLUSIONS The results showed the ability of ADSCs to differentiate into SMC and ECs and form cavernous tissue. The implanted tissue can survive and integrate into the penile tissues. The cavernous tissue made of ADSCs forms new technology for improvement of in vivo stem cell survival and ED treatment.

Tissue Engineering for Penile Surgery: Comparative Study of Noncellular and Cell‐Seeded Synthetic Grafts for Tunica Albuginea Replacement

INTRODUCTION Surgical treatment outcomes in Peyronies disease remain controversial because of high rates of recurrence. AIM The aim of this study was to engineer in vitro a new type of tunica albuginea (TA) autologous graft obtained by culture of autologous fibroblast on a polyglycolic acid (PGA) scaffold. This engineering graft was compared with PGA with morphological and functional outcomes for TA replacement, 4 months after graft upon corpus cavernosum in a rat model. METHODS Thirty-nine Sprague Dawley adult male rats were divided into four groups: (i) control group (C) with resection and resuture of a 5 mm long and 2 mm large piece of original TA; (ii) PGA scaffold group (P) with the same resection of TA and suture of PGA scaffold; (iii) autologous fibroblast-seeded on PGA scaffold graft after resection of the same piece of TA (F + P); and (iv) sham group for functional and histological comparison. MAIN OUTCOME MEASURE The main outcome measure was assessment of graft size variation at 4 months and comparison between the three test groups. The secondary objective is assessment of erectile function by measuring erectile response to cavernous nerve electrical stimulation in each group. RESULTS At 4 months, there was a significant difference in graft area retraction between the groups (P = 0.0081) with higher retraction in P group vs. in C or F + P groups. Erectile response to cavernous nerve stimulation significantly differed between the groups and was sham equivalent to C equivalent to F + P superior to P group. CONCLUSIONS This study provides experimental evidence for the feasibility and the functionality of fibroblast-seeded scaffold compared with acellular graft for TA replacement.

Effect of a Local Delivery of Triiodothyronine (T3) Within Neuroregenerative Guide on Recovery of Erectile Function in a Rat-Model of Cavernous Nerve Injury

INTRODUCTION A promoting effect of thyroid hormones has been established on the maturation of central and peripheral nervous systems. However, effects on autonomic nerves have never been experimentally investigated. AIM To assess the effect of a local treatment combining silicone guides and local administration of Triiodothyronine (T3) on the erectile function and the histological neuroregeneration of crushed cavernous nerves (CNs) in rats. METHODS Forty-five rats were divided into five equal groups: SHAM surgery, guide without crush, crush, crush + guide, crush + guide + T3. All surgical procedures were bilateral. CNs were crushed with microvascular bulldog clamp of 100 g/cm(2). A silicone guide was placed around the nerves. The guides were filled with T3 neuroregenerative solution. MAIN OUTCOME MEASURES Erectile function was assessed 10 weeks post-operatively. Intra-cavernous pressure (ICP) and mean arterial pressure (MAP) were monitored during electrical stimulation of CNs at various frequencies. The main outcome was hardness of erection defined as DeltaICP/MAP. Fluorescent immunohistochemical analysis of CNs was performed to assess regeneration of nerves morphologically. RESULTS Electrophysiological data showed increased recovery of erectile function in the group with guide + T3 neuroregenerative solution compared with the empty guide. Immunohistochemical analysis of cavernous nerves demonstrated in morphology that regenerated axons were straighter in nerves with guide and more regular if guides had been filled with T3. CONCLUSION The use of guides prevented axonal sprouting, facilitated functional neuroregeneration and enabled a local delivery of thyroid hormones. Triiodothyronine improved neuroregeneration and recovery of erectile function after a nerve-sparing-like injury in a rat model.

Testicular-sparing surgery for bilateral or monorchide testicular tumours: a multicenter study of long-term oncological and functional results

To review long‐term oncological and functional outcomes of testicular‐sparing surgery (TSS) in men presenting with bilateral or monorchide testicular tumours at one of five reference centres for testicular neoplasm and infertility.

Efficacy of BloodSTOP iX, surgicel, and gelfoam in rat models of active bleeding from partial nephrectomy and aortic needle injury.

OBJECTIVE To compare the bleeding time using 3 different hemostatic agents in a rat model of partial nephrectomy and aortic needle injury. METHODS Bilateral partial nephrectomy was performed in 20 rats with a total bleeding surface of 1.5 cm(2) without vascular clamping. Surgicel (n = 10) or BloodSTOP iX (n = 10) matrix was applied on each kidney cut surface. Finger pressure was applied to the parenchyma with transparent plastic bubble wrap to allow for visualization of the site and monitor the bleeding time. Pressure was applied until the bleeding stopped and then released to assess the presence of active bleeding for 5 minutes. An additional 24 rats underwent aortic trauma with a 25-gauge needle puncture, and the efficacy of the topical hemostatic agents were compared among Gelfoam (n = 8), Surgicel (n = 8), and BloodSTOP iX (n = 8). RESULTS After partial nephrectomy, the mean bleeding time with BloodSTOP iX and Surgicel treatment was 83.70 ± 13.73 seconds and 168.8 ± 19.41 seconds, respectively, a statistically significant difference (P = .002). After aortic injury, the mean bleeding time was 157.5 ± 31.44 seconds, 187.5 ± 23.20 seconds, and 66.00 ± 13.74 seconds in the Gelfoam, Surgicel, and BloodSTOP groups, respectively, which was statistically significant (P = .004). CONCLUSION The BloodSTOP iX hemostatic surgical matrix was more effective in reducing the bleeding time than Surgicel in a rat model of partial nephrectomy. Similarly, in an aortic needle injury model, BloodSTOP iX achieved hemostasis faster than Gelfoam or Surgicel.

Tunica albuginea allograft: a new model of LaPeyronie's disease with penile curvature and subtunical ossification

The pathophysiology of LaPeyronies disease (PD) is considered to be multifactorial, involving genetic predisposition, trauma, inflammation and altered wound healing. However, these factors have not yet been validated using animal models. In this study, we have presented a new model obtained by tunica albuginea allograft. A total of 40, 16-week-old male rats were used. Of these, 8 rats served as controls and underwent a 10 × 2-mm-wide tunical excision with subsequent autografting, whereas the remaining 32 underwent the same excision with grafting of the defect with another rats tunica. Morphological and functional testing was performed at 1, 3, 7 and 12 weeks after grafting. Intracavernous pressure, the degree of penile curvature and elastic fiber length were evaluated for comparison between the allograft and control groups. The tissues were obtained for histological examination. The penile curvature was significantly greater in the allografted rats as compared with the control rats. The erectile function was maintained in all rats, except in those assessed at 12 weeks. The elastin fiber length was decreased in the allografted tunica as compared to control. SMAD2 expression was detected in the inner part of the allograft, and both collagen-II- and osteocalcin-positive cells were also noted. Tunica albuginea (TA) allograft in rats is an excellent model of PD. The persistence of curvature beyond 12 weeks and the presence of ossification in the inner layer of the TA were similar to those observed in men with PD. Validation studies using this animal model would aid understanding of the PD pathophysiology for effective therapeutic interventions.

Testicular tumours discovered during infertility workup are predominantly benign and could initially be managed by sparing surgery: LAGABRIELLE et al.

To evaluate the pathological features and recurrence of incidental testis tumours treated by partial orchiectomy in a population of infertile men.