Early Blockade of Matrix Metalloproteinases in Spinal-Cord-Injured Dogs Results in a Long-Term Increase in Bladder Compliance.

Spinal cord injury (SCI) is often accompanied by reduced bladder compliance, which contributes to adverse conditions including urinary tract infections and vesicoureteral reflux. Reduced compliance is, in part, attributed to extensive remodeling of the bladder wall, including the extracellular matrix (ECM). Here, we tested the hypothesis that blockade of matrix metalloproteinases (MMPs), known for their ability to remodel the ECM, improves bladder compliance in dogs with SCI. We first evaluated dogs with naturally occurring SCIs resulting from intervertebral disc herniation (IVDH). After characterizing the natural history of urological recovery by cystometry in healthy dogs (n = 10) and dogs with SCIs (n = 20), we conducted a randomized, double-blinded, placebo-controlled clinical trial in dogs with IVDH-associated SCIs to assess the efficacy of the broad-spectrum MMP inhibitor, GM6001, given within 48 h post-injury. The primary outcomes were bladder compliance, as measured by cystometry, and an ordinal gait score (Texas Spinal Cord Injury Score; TSCIS) at day 42 post-SCI. Dogs (n = 93) were randomized to receive either dimethyl sulfoxide (DMSO) or GM6001+DMSO. There were transient, but significantly (p = 0.023) greater, adverse events (31 of 42; 74%) in the GM6001-treated group relative to vehicle controls (22 of 46; 48%). Whereas there were no differences in TSCIS between treatment groups at day 42 (p = 0.9679), bladder compliance was significantly higher in dogs treated with GM6001+DMSO compared to controls (p = 0.0272). Further studies are needed to determine whether this inhibition results from a direct interaction with the bladder wall or indirectly through neural-based mechanisms.

Comparison of spinal cord contusion and transection: functional and histological changes in the rat urinary bladder.

OBJECTIVE: To compare the effect of complete transection (tSCI) and contusion spinal cord injury (cSCI) on bladder function and bladder wall structure in rats. MATERIALS AND METHODS: A total of 30 female Sprague-Dawley rats were randomly divided into three equal groups: an uninjured control, a cSCI and a tSCI group. The cSCI group underwent spinal cord contusion, while the tSCI group underwent complete spinal cord transection. At 6 weeks post-injury, 24-h metabolic cage measurement and conscious cystometry were performed. RESULTS: Conscious cystometry analysis showed that the cSCI and tSCI groups had significantly larger bladder capacities than the control group. The cSCI group had significantly more non-voiding detrusor contractions than the tSCI group. Both injury groups had more non-voiding contractions compared with the control group. The mean threshold pressure was significantly higher in the tSCI group than in the control and cSCI groups. The number of voids in the tSCI group was lower compared with the control group. Metabolic cage analysis showed that the tSCI group had larger maximum voiding volume as compared with the control and cSCI groups. Vesicular acetylcholine transporter/smooth muscle immunoreactivity was higher in the control than in the cSCI or tSCI rats. The area of calcitonin gene-related peptide staining was smaller in the tSCI group than in the control or cSCI groups. CONCLUSIONS: Spinal cord transection and contusion produce different bladder phenotypes in rat models of SCI. Functional data suggest that the tSCI group has an obstructive high-pressure voiding pattern, while the cSCI group has more uninhibited detrusor contractions.

Transplanted Human Stem Cell-Derived Interneuron Precursors Mitigate Mouse Bladder Dysfunction and Central Neuropathic Pain after Spinal Cord Injury.

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

Efficacy of a metalloproteinase inhibitor in spinal cord injured dogs.

Matrix metalloproteinase-9 is elevated within the acutely injured murine spinal cord and blockade of this early proteolytic activity with GM6001, a broad-spectrum matrix metalloproteinase inhibitor, results in improved recovery after spinal cord injury. As matrix metalloproteinase-9 is likewise acutely elevated in dogs with naturally occurring spinal cord injuries, we evaluated efficacy of GM6001 solubilized in dimethyl sulfoxide in this second species. Safety and pharmacokinetic studies were conducted in naïve dogs. After confirming safety, subsequent pharmacokinetic analyses demonstrated that a 100 mg/kg subcutaneous dose of GM6001 resulted in plasma concentrations that peaked shortly after administration and were sustained for at least 4 days at levels that produced robust in vitro inhibition of matrix metalloproteinase-9. A randomized, blinded, placebo-controlled study was then conducted to assess efficacy of GM6001 given within 48 hours of spinal cord injury. Dogs were enrolled in 3 groups: GM6001 dissolved in dimethyl sulfoxide (n = 35), dimethyl sulfoxide (n = 37), or saline (n = 41). Matrix metalloproteinase activity was increased in the serum of injured dogs and GM6001 reduced this serum protease activity compared to the other two groups. To assess recovery, dogs were a priori stratified into a severely injured group and a mild-to-moderate injured group, using a Modified Frankel Scale. The Texas Spinal Cord Injury Score was then used to assess long-term motor/sensory function. In dogs with severe spinal cord injuries, those treated with saline had a mean motor score of 2 (95% CI 0-4.0) that was significantly (P<0.05; generalized linear model) less than the estimated mean motor score for dogs receiving dimethyl sulfoxide (mean, 5; 95% CI 2.0-8.0) or GM6001 (mean, 5; 95% CI 2.0-8.0). As there was no independent effect of GM6001, we attribute improved neurological outcomes to dimethyl sulfoxide, a pleotropic agent that may target diverse secondary pathogenic events that emerge in the acutely injured cord.

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

The pathophysiology of LaPeyronie's 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 rat's 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.

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.

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.

Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury.

BACKGROUND: Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear. OBJECTIVES: To investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury. DESIGN, SETTING, AND PARTICIPANTS: Sprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest. MEASUREMENTS: IC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG. RESULTS AND LIMITATIONS: IC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. CONCLUSIONS: CN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.

Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord.

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

Cavernous smooth muscle hyperplasia in a rat model of hyperlipidaemia-associated erectile dysfunction.

UNLABELLED: What's known on the subject? and what does the study add? Increased cavernous smooth muscle content has been repeatedly observed in rat models of hyperlipidaemia - associated erectile dysfunction. This study shows that the increased smooth muscle content is due to hyperplasia. OBJECTIVE: • To investigate the structural changes, including possible smooth muscle hyperplasia, in the penis of a hyperlipidaemia-associated erectile dysfunction (ED) animal model. MATERIALS AND METHODS: • Hyperlipidaemia was induced in rats through a high-fat diet. • Penile tissues of normal and hyperlipidaemic rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen, neuronal nitric oxide synthase (nNOS), and collagen type IV (Col-IV) before image and statistical analyses were carried out. • The main outcome measures were the smooth muscle, endothelial, Col-IV and nNOS content of the corpus cavernosum. RESULTS: • Phalloidin intensely stained all smooth muscle in the penis, revealing the circular and longitudinal components of cavernous smooth muscle (CSM). • The CSM content was significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cell numbers in both circular and longitudinal CSM were significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cavernous endothelial content was significantly lower in hyperlipidaemic than in normal rats (P < 0.05). • nNOS-positive nerves within the dorsal nerves, around the dorsal arteries, and in the corpora cavernosa were all significantly lower in the hyperlipidaemic than in the normal rats (P < 0.05). CONCLUSIONS: • Hyperlipidaemia is associated with reduced nNOS-positive nerves, reduced endothelium, and increased CSM in the penis. • The increased CSM is attributable to hyperplasia. • These structural changes may explain why hyperlipidaemic men are more likely to develop ED.

Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes.

OBJECTIVE: To investigate whether fluorochrome-conjugated phalloidin can delineate cavernous smooth muscle (CSM) cells and whether it can be combined with immunofluorescence (IF) staining to quantify erectile dysfunction (ED)-associated changes. METHODS: ED was induced by cavernous nerve crush in rats. Penile tissues of control and ED rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen (RECA), CD31, neuronal nitric oxide synthase (nNOS), and collagen-IV (Col-IV). RESULTS: Phalloidin was able to delineate CSM as composed of a circular and a longitudinal compartment. When combined with IF stain for CD31 or RECA, it helped the identification of the helicine arteries as covered by endothelial cells on both sides of the smooth muscle layer. When combined with IF stain for nNOS, it helped the identification that nNOS-positive nerves were primarily localized within the dorsal nerves and in the adventitia of dorsal arteries. When combined with IF stain for Col-IV, it helped identify that Col-IV was localized around smooth muscles and beneath the endothelium. Phalloidin also facilitated the quantitative analysis of ED-related changes in the penis. In rats with cavernous nerve injury, RECA or Col-IV expression did not change significantly, but CSM and nNOS nerve contents decreased significantly. CONCLUSION: Phalloidin stain improved penile histology, enabling the visualization of the circular and longitudinal compartments in the CSM. It also worked synergistically with IF stain, permitting the visualization of the dual endothelial covering in helicine arteries, and facilitating the quantification of ED-related histologic changes.

Functional, metabolic, and morphologic characteristics of a novel rat model of type 2 diabetes-associated erectile dysfunction.

OBJECTIVES: To conduct a pilot study to investigate functional, metabolic, and penile morphologic changes in a novel model of lean DM2. Erectile dysfunction (ED) is a frequent sequela in patients with type 2 diabetes mellitus (DM2). METHODS: Eight rats received a high-fat diet and 2 weeks later, 2 intraperitoneal injections of streptozotocin (STZ, 30 mg/kg). Five age-matched rats served as controls. Insulin challenge tests were performed at 6 and 12 weeks after induction of DM2. At 12 weeks, erectile function was tested by measurement of intracavernous pressure (ICP) increase upon cavernous nerve stimulation. Penile tissue and serum samples were harvested for histology and biochemistry, respectively. RESULTS: A lean DM2 model was established as demonstrated by decreased insulin resistance, elevated nonfasting plasma glucose levels, hyperlipidemia, and decreased insulin concentration in the absence of obesity. ICP/mean arterial pressure was significantly decreased in DM2 animals (0.29) compared with controls (0.81). Expression of neuronal nitric oxide synthase and rat endothelial cell antigen-1, and the smooth muscle/collagen ratio were significantly decreased in the penis of DM2 animals. CONCLUSIONS: We propose an inexpensive nongenetic animal model of lean DM2-associated ED. Microanatomical changes in the erectile tissue that reflect an advanced stage of the disease were observed.

Cavernous nerve repair with allogenic adipose matrix and autologous adipose-derived stem cells.

OBJECTIVES: To investigate whether adipose-derived matrix seeded with adipose-derived stem cells (ADSC) can facilitate the repair of injured cavernous nerves (CNs). METHODS: Human and rat adipose tissues were decellularized and fabricated into various forms, including adipose tissue-derived acellular matrix thread (ADMT). ADMT seeded with ADSC were transplanted into subcutaneous space and examined for signs of inflammation. ADSC-seeded ADMTs were then used to repair CN injury in rats, followed by assessment of histology and erectile function. RESULTS: Adipose tissue can be fabricated into acellular matrices of various shapes and sizes, including threads and sheets. Seeding of ADMT occurred rapidly: within 24 hours, 55% of the surface was covered with ADSC and within 1 week, 90% was covered. Transplantation of the seeded ADMT into the subcutaneous space of an allogenic host showed no signs of inflammatory reaction. At 3 months after grafting into CN injury rats, approximately twice as many cells were found on seeded ADMT as on unseeded ADMT. The seeded ADMT also had various degrees of S100 and neuronal nitric oxide synthase expression, suggesting CN axonal ingrowth. Rats grafted with seeded ADMT overall had the best erectile function recovery when compared with those grafted with unseeded ADMT and those ungrafted. However, as a result of large variations, the differences did not reach statistic significance (P = .07). CONCLUSIONS: Grafting of ADSC-seeded matrix resulted in a substantial recovery of erectile function and improvement of histology. However, further refinement of the matrix architecture is needed to improve the success rate.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).

OBJECTIVE: To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. MATERIALS AND METHODS: Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of ß-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. RESULTS: MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to ß-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. CONCLUSION: In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.

Pentoxifylline promotes recovery of erectile function in a rat model of postprostatectomy erectile dysfunction.

BACKGROUND: Cavernous nerve (CN) injury during radical prostatectomy (RP) causes CN degeneration and secondary penile fibrosis and smooth muscle cell (SMC) apoptosis. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that further inhibits multiple cytokine pathways involved in nerve degeneration, apoptosis, and fibrosis. OBJECTIVES: To evaluate whether PTX enhances erectile function in a rat model of CN injury. DESIGN, SETTING AND INTERVENTIONS: Forty male Sprague-Dawley rats underwent CN crush injury and were randomized to oral gavage feeding of phosphate-buffered saline (vehicle) or PTX 25, PTX 50, or PTX 100 mg/kg per day. Ten animals underwent sham surgery and received vehicle treatment. Treatment continued for 28 d, followed by a wash-out period of 72 h. An additional eight rats underwent resection of the major pelvic ganglion (MPG) for tissue culture and examination of direct effects of PTX on neurite sprouting. MEASUREMENTS: Intracavernous pressure recording on CN electrostimulation, immunohistologic examination of the penis and the CN distal to the injury site, and length of neurite sprouts in MPG culture. RESULTS: Daily oral gavage feeding of PTX resulted in significant improvement of erectile function compared to vehicle treatment in all treated groups. After treatment with PTX 50 and PTX 100 mg/kg per day, the expression of neuronal nitric oxide synthase in the dorsal penile nerve was significantly higher than in vehicle-treated rats. Furthermore, PTX treatment prevented collagen deposition and SMC loss in the corpus cavernosum. In the CN, signs of Wallerian degeneration were ameliorated by PTX treatment. MPG culture in medium containing PTX resulted in a significant increase of neurite length. CONCLUSIONS: PTX treatment following CN injury in rats improved erectile recovery, enhanced nerve regeneration, and preserved the corpus cavernosum microarchitecture. The clinical availability of this compound merits application in penile rehabilitation studies following RP in the near future.

The effect of adipose-derived stem cells on augmentation ileocystoplasty: A pilot study.

OBJECTIVES: Incorporation of intestinal tissue into urinary tract elicits many metabolic and mechanical complications due to anatomical and physiological differences. Adipose-derived stem cells (ADSCs) improve vascularity and functional outcomes by a paracrine mechanism. In a pilot study we investigated whether ADSCs can survive in the augmented bladder and improve its function. MATERIALS AND METHODS: Autologous ADSCs were harvested from rat paragonadal fat and cultured before injection into a rat model of augmentation ileocystoplasty (study group). Control augmented bladders were injected with cell-free saline. Eight weeks later, rats underwent abdominal ultrasonography for upper tract changes and were examined by conscious cystometry to determine bladder function. After extirpation, augmented bladders were examined using Masson trichrome staining for connective tissue and muscle content, immunohistochemistry for α-smooth muscle actin, and rat endothelial cell antigen staining for endothelial cells. Changes in the extracellular matrix were assessed by determining the elastin content. ADSCs were labelled and tracked by 5-ethynyl-2-deoxyuridine nuclear staining. RESULTS: Abdominal ultrasonography showed better preservation of upper tract function in the ADSC group than in the saline-treated group (P = 0.007). After 2 months there were no differences in the variables assessed by conscious cystometry between the ADSC and saline-treated groups. However, the bladder weight was significantly greater in the ADSC-treated group. On immunohistochemistry, the implanted ADSCs survived up to 8 weeks but did not transdifferentiate into smooth muscle or endothelial cells. CONCLUSION: These results suggested a potential role of ADSCs in modifying the intestinal segment in augmented bladders; this role has to be further elucidated.

Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury.

INTRODUCTION: Erectile dysfunction (ED) remains a major complication after radical prostatectomy. The use of adipose tissue-derived stem cells (ADSCs) has shown promising results for the treatment of ED. However, the mechanisms of action for stem cell therapy remain controversial, with increasing evidence pointing to paracrine pathways. AIM: To determine the effects and to identify the mechanism of action of ADSC and ADSC-derived lysate in a rat model of cavernous nerve (CN) crush injury. METHODS: Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: one group underwent sham operation, while three groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of ADSC, lysate, or vehicle only (injured controls). Erectile function was assessed by CN electrostimulation at 4 weeks. Penile tissue was collected for histology. MAIN OUTCOME MEASURES: Intracavernous pressure increase upon CN stimulation; neuronal nitric oxide synthase (nNOS) content in the dorsal penile nerve; smooth muscle content, collagen content, and number of apoptotic cells in the corpus cavernosum. RESULTS: Both ADSC and lysate treatments resulted in significant recovery of erectile function, as compared with vehicle treatment. nNOS content was preserved in both the ADSC and lysate group, with significantly higher expression compared with vehicle-treated animals. There was significantly less fibrosis and a significant preservation of smooth muscle content in the ADSC and lysate groups compared with injured controls. The observed functional improvement after lysate injection supports the hypothesis that ADSCs act through release of intracellular preformed substances or by active secretion of certain biomolecules. The underlying mechanism of recovery appears to involve neuron preservation and cytoprotection by inhibition of apoptosis. CONCLUSIONS: Penile injection of both ADSC and ADSC-derived lysate can improve recovery of erectile function in a rat model of neurogenic ED.

Erectogenic and neurotrophic effects of icariin, a purified extract of horny goat weed (Epimedium spp.) in vitro and in vivo.

INTRODUCTION: Epimedium species (aka horny goat weed) have been utilized for the treatment of erectile dysfunction in Traditional Chinese Medicine for many years. Icariin (ICA) is the active moiety of Epimedium species. AIM: To evaluate the penile hemodynamic and tissue effects of ICA in cavernous nerve injured rats. We also studied the in vitro effects of ICA on cultured pelvic ganglia. METHODS: Rats were subjected to cavernous nerve injury and subsequently treated for 4 weeks with daily gavage feedings of a placebo solution of normal saline and Dimethyl sulfoxide (DMSO) vs. ICA dissolved in DMSO at doses of 1, 5, and 10 mg/kg. A separate group underwent a single dose of ICA 10 mg/kg 2 hours prior to functional testing. Functional testing with cavernous nerve stimulation and real-time assessment of intracavernous pressure (ICP) was performed at 4 weeks. After functional testing, penile tissue was procured for immunohistochemistry and molecular studies. In separate experiments, pelvic ganglia were excised from healthy rats and cultured in the presence of ICA, sildenafil, or placebo culture media. MAIN OUTCOME MEASURE: Ratio of ICP and area under the curve (AUC) to mean arterial pressure (MAP) during cavernous nerve stimulation of subject rodents. We also assayed tissue expression of neuronal nitric oxide synthase (nNOS), eNOS: endothelial nitric oxide synthase (eNOS), calponin, and apoptosis via immunohistochemistry and Western blot. Serum testosterone and luteinizing hormone (LH) were assayed using enzyme-linked immunosorbant assay (ELISA). Differential length of neurite outgrowth was assessed in cultured pelvic ganglia. RESULTS: Rats treated with low-dose ICA demonstrated significantly higher ICP/MAP and AUC/MAP ratios compared with control and single-dose ICA animals. Immunohistochemistry and Western blot were revealing of significantly greater positivity for nNOS and calponin in penile tissues of all rats treated with ICA. ICA led to significantly greater neurite length in cultured specimens of pelvic ganglia. CONCLUSION: ICA may have neurotrophic effects in addition to known phosphodiesterase type 5 inhibiting effects.

The effect of intracavernous injection of adipose tissue-derived stem cells on hyperlipidemia-associated erectile dysfunction in a rat model.

INTRODUCTION: Hyperlipidemia has been associated with erectile dysfunction (ED) via damage to the cavernous endothelium and nerves. Adipose tissue-derived stem cells (ADSC) have been shown to differentiate into endothelial cells and secrete vasculotrophic and neurotrophic factors. AIM: To assess whether ADSC have therapeutic effects on hyperlipidemia-associated ED. METHODS: Twenty-eight male rats were induced to develop hyperlipidemia with a high-fat diet (hyperlipidemic rats, HR). Ten additional male rats were fed a normal diet to serve as controls (normal rats, NR). Five months later, all rats were subjected to ADSC isolation from paragonadal fat. The cells were cultured for 1 week, labeled with 5-ethynyl-2'-deoxyuridine (EdU), and then injected autologously into the corpus cavernosum of 18 HR. The remaining 10 HR rats were injected with phosphate buffered saline (PBS). At 2 and 14 days post-transplantation, four rats in the HR + ADSC group were sacrificed for tracking of the transplanted cells. At 28 days post-transplantation, all remaining rats were analyzed for serum biochemistry, erectile function, and penile histology. MAIN OUTCOME MEASURES: Erectile function was assessed by intracavernous pressure (ICP) measurement during electrostimulation of the cavernous nerve. Cavernous nerves, endothelium, and smooth muscle were assessed by immunohistochemistry. RESULTS: Serum total cholesterol and low-density lipoprotein levels were significantly higher in HR than in NR. High-density lipoprotein level was significantly lower in HR than in NR. Mean ICP/mean arterial pressure ratio was significantly lower in HR + PBS than in NR + PBS or HR + ADSC. Neuronal nitric oxide synthase (nNOS)-positive nerve fibers and endothelial cells were fewer in HR + PBS than in HR + ADSC. Smooth muscle content was significantly higher in both HR groups than in NR. CONCLUSIONS: Hyperlipidemia is associated with abnormalities in both the nerves and endothelium. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential new therapy for ED.