Investigative Urology EDITORS

ASSOCIATE EDITORS EDITORIAL BOARD

Helmut Klocker Jack Schalken Bill Watson Georg Bartsch David Neal Karl-Eric Andersson Kazem Azadzoi Olivier Cussenot Christopher Foster Robert Getzenberg Martin Gleave Hans Lilja Marston Linehan Norman Maitland Bruce Malkowicz Joel Nelson John Stein Ulf-Håkan Stenman Christian Stief George N. Thalmann Dan Theodorescu Tapio Visakorpi

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INTERNATIONAL

EDITOR-IN-CHIEF JOHN M. FITZPATRICK

Blackwell Science, LtdOxford, UKBJUBJU International1464-410XBJU InternationalOctober 2004 946 Original Article STEM CELL THERAPY AND NEUROGENIC IMPOTENCE BOCHINSKI et al .

The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury DEREK BOCHINSKI, GUI TING LIN, LORA NUNES, RAFAEL CARRION, NADEEM RAHMAN, CHING SHWUN LIN and TOM F. LUE Department of Urology, University of California, San Francisco, CA, USA Accepted for publication 17 June 2004

OBJECTIVE To isolate embryonic stem cells that have differentiated along the neuronal cell line, and to assess whether injecting these neural stem cells into the corpus cavernosum influences cavernosal nerve regeneration and functional status.

into the major pelvic ganglion (MPG); and nine had bilateral cavernosal nerve crush and injection of NES cells into the corpora cavernosa. Erectile response was assessed by cavernosal nerve electrostimulation at 3 months, and penile tissue samples were evaluated histochemically for nitric oxide synthase (NOS)-containing fibres, tyrosine hydroxylase and neurofilament staining.

MATERIALS AND METHODS RESULTS Embryonic neural stem cells were obtained; 26 male Sprague-Dawley rats were divided into four groups: five had a sham operation; eight (controls) had a bilateral cavernosal nerve crush and injection of culture medium into the corpora cavernosa; four had an injection of neural embryonic stem (NES) cells

INTRODUCTION The cavernosal nerves run posterolateral to the prostate and are vulnerable to injury during radical prostatectomy or cystoprostatectomy. Various techniques have been used to try to decrease the incidence of erectile dysfunction after prostatectomy. Walsh [1] developed a nerve-sparing technique that reduced the incidence of erectile dysfunction after surgery. Despite such advances, erectile dysfunction still affects a significant proportion of patients, and many do not recover their erectile capacity [2]. In many instances during nerve-sparing prostatectomy the cavernosal nerves can be inadvertently damaged by manipulation. The recovery of erectile function may depend on re-sprouting of nerves from the remaining neural tissue [3]. The capacity of the nervous system to regenerate after injury is limited. Embryonic stem cells could potentially be used to promote regeneration by replacing existing damaged nerve cells, or more 904

The groups injected with NES cells into the MPG and corpora cavernosa had significantly higher intracavernosal pressures than the control group. Immunohistochemical staining also revealed differences in the quality of the NOS-containing nerve fibres. Neurofilament

probably by stimulating host factors to promote nerve growth. Various growth factors are thought to be involved in neuronal regeneration after injury; brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, neurotrophin and neurotrophin-4 are all important in neural regeneration after injury [4,5]. The capacity of stem-cell therapy to assist neural regeneration may depend on these particular neurotrophins. The aim of the present study was to isolate stem cells from Sprague-Dawley rats and then to induce them to differentiate along the neural cell pathway. We then assessed the effect of injected neural stem cells (NES cells) on cavernosal nerve regeneration and erectile function in a nerve-crush model, and characterized this regeneration process by immunohistochemical staining. MATERIALS AND METHODS Embryonic stem cells were isolated from blastocysts. Two female Sprague-Dawley rats

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staining was significantly better in the experimental groups injected with NES cells.

CONCLUSION We were able to isolate embryonic stem cells that had differentiated along the neural cell line and, using these NES cells intracavernosally, showed improved erectile function in a rat model of neurogenic impotence.

KEYWORDS Stem cells, neurogenic impotence, erectile dysfunction

were kept in the same cage, with a 12 h/12 h light/dark cycle for 4–5 days, and then a male rat was introduced during the late afternoon. The vaginas of the female rats were checked for a white copulation plug (sperm) every morning. The rat with the white copulation plug was separated to a clean cage and this pregnant rat was killed 3.5 days later. The uterine horns were removed to an autoclaved paper towel, and a syringe filled with 5% newborn calf serum/Dulbecco’s Modified Eagle’s Medium (DMEM) (embryonic stem-cell culture medium) carefully introduced into one end of the horn for 2–3 mm. The uterine lumen was flushed, releasing the embryo into the medium. The plate containing the contents of the uterine horns was put on the stage of a stereoscopic dissection microscope. Blastocysts were captured by a micropipette (100 mm) and transferred to 24-well cellculture dishes (coated with gelatine and each well containing 1 mL of medium). The embryo was incubated at 37 ∞C with 5% CO2 for 4– 6 days. The embryonic stem cells were then extracted by disaggregation of the inner cell mass. The medium was aspirated from the

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STEM CELL THERAPY AND NEUROGENIC IMPOTENCE

well and replaced by 1 mL of PBS. A blunt end of a glass tube was used to push the inner cell mass free from the trophoblast cell layer. The inner cell mass was transferred into a small drop of trypsin/EDTA (50 mL) a 37 ∞C for 5 min. The medium was then naturalized by adding another 50 mL of embryonic stem cell culture medium. The stem cell culture suspension was transferred to the feeder-cell layer embryonic stem cell culture medium. Embryonic stem cells were induced to differentiate into neural cells by transfecting them with BDNF tagged with enhanced green fluorescence protein (EGFP). Embryonic stem cells were plated one day before the transfection experiment and cultured in 0.1 ng/mL solution of leukaemia-inhibiting factor and embryonic stem cell culture medium until there was 60–90% confluence (on the day of transfection). The plates were incubated at 37 ∞C with CO2 for 18–24 h. On the day of transfection, fresh liposome solution was prepared by warming serumfree DMEM to 37 ∞C and adding 10 mL of superfectin to the medium. This was then vortexed gently and the solution placed on ice while mixing with 2–4 mg plasmid pEGFPBDNF DNA. The transfection solution was then incubated at room temperature for 10–30 min, and the medium then removed from the cultured cells. The superfectin-DNAmedia solution was then gently applied to the cells. The plates were incubated at 37 ∞C for 1–4 h in a CO2 incubator. The superfectin/ DNA-containing medium was then removed and the cells washed with medium or 1 ¥ PBS (the media or PBS was warmed to 37 ∞C). We then applied 10 mL of fresh complete growth medium with 0.5 mg/mL G418 (deneticin) and incubated this at 37 ∞C for selection until the positive clone appeared. After the EGFP-BDNF embryonic stem cell line had been constructed the cells were stained by haematoxylin and eosin, and S100. By tagging the cells with EGFP the cells were then assayed by fluorescein isothiocyanate fluorescence (¥100) and appeared to be nerve-like. To assess the implantation of stem cells into the corpus cavernosum and major pelvic ganglion (MPG) after cavernosal nerve injury, 26 male Sprague-Dawley rats (3 months old, 250–300 g) were divided into four groups. At 3 months, the erectile responses were assessed in all rats by electrostimulating the cavernosal nerves and measuring the intracavernosal pressure (ICP). In addition,

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samples of MPG and penile tissue were collected for NADPH-diaphorase (NADPH-d), tyrosine hydroxylase (TH) and neurofilament staining. All animal experiments in the study were approved by the local ethical committee for animal experimentation (University of California, San Francisco, Institutional and Animal Care Use Committee). Rats were anaesthetized using isoflurane inhalation followed by an intraperitoneal injection with sodium pentobarbital (40 mg/ kg), and kept isothermic using a heating pad (37 ∞C). A lower midline abdominal incision was made after the abdomen was shaved and prepared with an iodine-based solution. The prostate gland was exposed and the cavernosal nerves, tracking posterolaterally, identified and isolated. The MPG was also identified more proximally along the course of the cavernosal nerve. In group 1 (sham surgery, five rats) there was no further surgical manipulation. In the remaining groups the cavernosal nerves were isolated and a crush injury induced using a haemostat clamp for 2 min, after which the control rats (group 2, eight) had stem-cell culture medium injected into the corpora cavernosa, and the abdomen closed. In group 3 (four rats), after bilateral nerve crush, 500 mL (10 000 cells/mL) was injected into the MPG bilaterally using a 30 G needle. In group 4 (nine rats), after bilateral nerve crush, 500 mL (10 000 cells/mL) was injected into the crus of the corpora cavernosa of the rat. After the injection of NES cells in groups 3 and 4 the abdomen was then closed, as in all rats, in two layers. At 3 months erectile function was assessed in all the rats; a repeat midline abdominal incision was made, the cavernosal nerves exposed and isolated, the skin overlying the penis incised and the crura of the penis identified. A 23 G scalp-vein needle filled with 250 U/mL heparin solution was connected to polyethylene-50 tubing and inserted into the right crus body to measure the ICP. A bipolar stainless-steel electrode was used to stimulate the cavernosal nerve directly (probes 2 mm diameter, separated by 1 mm). Monophasic rectangular pulses were generated by a computer with a custom-built constant-current amplifier; the stimulus parameters were 1.5 mA, 20 Hz, pulse width 0.2 ms, and duration 50 s. The ICP was recorded in all rats using a Macintosh computer program with Laboratory VIEW 4.0 software (National Instruments, Austin, TX).

HISTOCHEMISTRY After the functional evaluations a midshaft corporal sample and a sample of the MPG were taken from each rat for immunohistochemical staining for NADPH-d, TH and neurofilaments. For NADPH-d and TH staining, tissues were fixed for 4 h in phosphate buffer containing 0.002% picric acid and 2% formaldehyde, then transferred to 30% sucrose before freezing. Serial cryosections (10 mm) were adhered to charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). After air-drying for 5 min, the sections were incubated with 0.1 mmol/L NADPH, 0.2 mmol/L nitroblue tetrazolium, 0.2% Triton X-100 (SigmaAldrich, St. Louis, MO) in a buffer, with constant microscopic monitoring for colour development. When the medium was deep blue for NADPH-d positive nerves, rinsing in buffer terminated the reaction. For TH staining, endogenous peroxidase activity was quenched by incubating slides in 3% H2O2/ methanol for 10 min. After rinsing, slides were incubated in blocking serum followed by overnight incubation with primary antibody (1 : 100 TH monoclonal antibody, NovoCastra, Belmont, CA). After washing with buffer, sections were immunostained using the avidin-biotin-peroxidase method (Elite ABC, Vector Laboratories, Burlingame, CA), with diaminobenzidine as the chromogen. All sections were photographed using a Nikon DXM 1200 digital still camera attached to a Leica Laborlux microscope, using ACT-1 software (Nikon Instruments Inc., Melville, NY). Some images were analysed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The presence of NADPH-d positive nerves is clear with this stain, seen as a highly localized dense blue region. The staining was assessed by counting the number of NADPH-d-positive nerve fibres in each corpus cavernosum (at ¥ 400; endothelial staining was not included in the count). For neurofilament staining, freshly dissected tissue was fixed for 4 h with cold 2% formaldehyde, 0.2% picric acid in 0.1 mol/ L phosphate buffer, followed by overnight immersion in buffer containing 30% sucrose for cryoprotection. Tissues were frozen in OCT compound (Miles Inc, Elkhart, IN) and stored at - 70 ∞C until use. Sections were cut at 5 mm, adhered to charged slides, air-dried for 5 min, then rehydrated with 0.05 mol/L PBS 905

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FIG. 1. Examples of ICP changes after electrostimulation of the cavernosal nerves at 12 weeks. (a) Group 1; the pressure reaches 100 cmH 2O. (b) Group 2; the pressure reaches only 43 cmH2O. (c) Group 3; the pressure reaches 67 cmH2O. (d) Group 4; the pressure reaches 103 cmH2O. b

ICP, cmH2O

–5 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Stimulation amplitude, mA

Stimulation amplitude, mA

ICP, cmH2O

a 100 90 80 70 60 50 40 30 20 10

0

50

100 90 80 70 60 50 40 30 20 10 –5 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0

50 Time, s

Time, s d

–5 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

ICP, cmH2O Stimulation amplitude, mA

Stimulation amplitude, mA

ICP, cmH2O

c 80 70 60 50 40 30 20 10

0

110 100 90 80 70 60 50 40 30 20 10 –5 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

50

for 5 min. Sections were treated with hydrogen peroxide/methanol to quench endogenous peroxidase activity. After rinsing, sections were washed twice in PBS for 5 min followed by 30 min incubation at room temperature with 3% horse serum/PBS/0.3% Triton X-100. After draining excess fluid sections were incubated overnight at 4 ∞C with SMI-31 monoclonal antibody to neurofilaments (1 : 70 000; Sternberger Monoclonals Inc., Lutherville, MD), or with no antibody, to serve as a negative control. After washing, sections were immunostained with the avidin-biotin-peroxidase method with 906

0

50

Time, s

Time, s

diaminobenzidine as the chromogen, followed by counterstaining with haematoxylin. The results were analysed statistically by comparing the groups using Dunn’s test, with groups considered to be significantly different at P < 0.05. Data are expressed as mean (SD) unless otherwise stated.

RESULTS There were significant differences in mean peak ICPs between group 1, at 96.9 (18.0)

cmH2O; Fig. 1a) and all other groups (P < 0.05). The mean peak ICP in the group 2, at 30.5 (8.3) cmH2O (Fig. 1b) was significantly lower than in both groups 3 and 4 (injected with NES cells), at 55.1 (19.2) and 54.1 (23.8) cmH2O, respectively (P < 0.05; Fig. 1c,d). There was no significant difference in mean peak ICP between groups 3 and 4. Using a neurofilament stain there was a significant difference in the percentage of positive cavernosal nerve staining between groups 3 or 4 and group 2 (Table 1). There was also an increase in neurofilament

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STEM CELL THERAPY AND NEUROGENIC IMPOTENCE

TABLE 1 Neurofilament staining in the MPG, dorsal and distal cavernosal nerves, and NADPH-d staining in the dorsal nerve in the four groups of rats Group Stain 1 Neurofilament Mean (SD) % MPG 3.2 (0.8) Dorsal 18.9 (3.7) Distal 12.5 (4.4) NADPH-d Mean (SD) NADPH-d staining n* 738 (276) TH, %† 0.59 (0.19)

2

3

4

a 0.5 (0.3) 12.4 (2.6) 1.2 (0.7)

1.5 (0.6) 19.7 (2.7) 1.6 (0.9)

3.6 (2.0) 22.0 (5.1) 6.5 (2.5)

255 (106) 0.44 (0.24)

467 (155) 0.42 (0.19)

246 (191) 0.32 (0.23)

MPG; group 1 greater (P < 0.05) than groups 2 and 3 but not group 4; groups 3 and 4 significantly greater than group 2: dorsal nerve; groups 3 and 4 not significantly different from group 1; group 1, 3 and 4 all significantly greater than group 2 (P < 0.05). Distal cavernosal; groups 2, 3 and 4 significantly less than group 1 (P < 0.05); group 3 not significantly different from group 2 (P = 0.4), but group 4 significantly different from group 2 (P < 0.05). *number of nerve fibres staining positive per specimen. Groups 3 ( P = 0.93) and 4 (P = 0.08) not significantly different from group 2; †ratio of NADPH-staining and THstaining fibres; groups 3 ( P = 0.77) and 4 ( P = 0.085) not significantly different from group 2.

staining in the MPG and dorsal nerves of the penis. In this case, group 1 had the best neurofilament staining (Figs 2a-d and 3a-d). Immunohistochemical staining of the corpora cavernosa, dorsal nerve complex and MPG showed a distinct pattern in the specimens. There were significantly fewer NADPH-dpositive nerve fibres in the dorsal nerve of group 2 than in group 1 (P < 0.05; Table 1). With NES treatment the number of NADPH-d positive nerve fibres did not increase significantly, but qualitative assessment of the neuronal fibres in the corpora cavernosa revealed a difference in appearance. After nerve crushing, the microscopic appearance of the nerves in the corpora cavernosa appeared attenuated, thinned and beaded (Fig. 4b). This is in contrast to group 1 (Fig. 4a) and groups 3 and 4 (Fig. 4c,d), where the nerves had more normal morphological features. The findings were similar in the dorsal nerve and MPG. Embryonic stem cells were induced to differentiate into neuronal cells by transfecting them with EGFP-tagged BDNF. The EGFP-tagged cells should have also allowed the NES cells to be detectable under immunofluorescent microscopy after tissue extraction. Interestingly, none of the tissue specimens from the experimental groups showed any evidence of EGFP expression.

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FIG. 2. Neurofilament staining (¥ 200) of MPG; neurofilament is stained brown. (a) Group 1, (b) group 2 (note the relative paucity of neurofilament stain), (c) group 3 and (d) group 4 (note the relative abundance of neurofilament staining).

b

DISCUSSION c

The features of embryonic stem cells that make them special are their ability to renew themselves for long periods through cell division, and that they may be induced to become cells with special functions by altering the physiological or experimental conditions [6]. Embryonic stem cells are unspecialized cells, but these same cells may differentiate and give rise to various cell types. The internal and external signals that trigger stem cell differentiation are complex and not fully understood [7]. Embryonic stem cells are grown first by transferring the inner cell mass into a culture dish; the cells then divide and spread over the inner surface of the dish. To aid in the growth of these cells, the culture dish is usually prepared first with mouse embryonic skin cells, which act as feeder cells. These feeder cells are specifically treated so they will not divide, and the stem cells can then adhere to the feeder cells and grow. The feeder cells also release nutrients into the culture medium. At this point the cultured cells are pluripotent. Various techniques may be used to induce these cells to differentiate along the endodermal, ectodermal or mesodermal cell lines. Stem cells that are directed to differentiate into specific cell types offer the possibility of a renewable

d

source of cells and tissues to replace diseased or injured cells. The mechanism of action of NES cells is still unknown, but stem cells may present a cellular substrate to the lesion site that supports axonal extension. Another possible mechanism contributing to stem cell-induced growth of neurones is the constitutive production of growth factors, such as BDNF, 9 07

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nerve growth factor and neurotrophin-3 [8]. Liu et al. [9] also proposed that stem cells may promote functional recovery after injury by reducing the amount of demyelination. This mechanism is probably less important than the other proposed mechanisms, as functional deficit after injury is usually attributable to loss of axonal connectivity. Klassen et al. [10] reported that neural progenitor cells from humans express cytokines including interleukin-1a, -1b and -6, TGF-b1, TGF-b2 and TNFa. The precise function of these cytokines has not been elucidated, but these signalling molecules are known to be involved in neural development and may play a role in activating quiescent stem cells after nerve injury. Interleukin-1a has been proposed to be involved in neural protection [11] while TNFa has been postulated to be vital for neural proliferation [12]. Similar to humans, in rats NES cells are known to promote the secretion of various cytokines, including TGFb. The expression of cytokines by NES cells highlights the importance of these cells in the interface of inflammation and actual wound repair. Neurofilaments are the intermediate filaments of neurones that add rigidity, tensile strength and possibly intracellular transport guidance to axons and dendrites. Neurofilaments are components of the neuronal cytoskeleton and are thought to be critical in establishing the shape and internal organization of nerve cells [13]. We present immunohistochemical evidence that NES cell therapy enhances the expression of neurofilament in treated rats. The enhanced presence of neurofilament may serve to guide and nurture nerve regeneration after injury. There was also a change in the morphological characteristics of nerve fibres in the corpora cavernosa after NES cell treatment. The NADPH-d staining nerve fibres in the NES cell-treated groups resembled the NADPH-d nerve fibres in the sham group, whereas the control group nerve fibres showed atrophy, attenuation and beading. This preservation of nerve morphology may contribute to the improved erectile function found in groups 3 and 4. Interestingly, in these two groups there was no direct evidence of the presence of stem cells after harvesting the tissue. Initially we had hoped to detect the expression of EGFP in the tissue samples (penis, MPG or cavernosal nerve) under fluorescence microscopy. However, that there was none may reflect further stem cell differentiation without the subsequent expression of EGFP. As discussed previously, stem cells may not require prolonged presence to function; 908

FIG. 3. Neurofilament staining (¥ 200) of the distal cavernosal nerve. (a) Group 1, abundant staining of nerves with neurofilament stain; (b) group 2, a paucity of neurofilament staining, (c) group 3 and (d) group 4; note the abundant neurofilament staining in both.

FIG. 4. NADPH-d/TH staining (¥ 400) of corpora cavernosa; the blue NADPH-d stain represents parasympathetic fibres. (a) Group 1 shows normal the morphological appearance of nerves; (b) group 2 (note the attenuated and beaded appearance of the nerve fibres); (c) group 3 and (d) group 4, relatively normal morphological features of nerve fibres.

a a

b b

c c

d d

their mechanism of action may be through growth factor expression, inhibition of demyelination and as an initial lattice of cellular substrate. The present results show immunohistochemical evidence for nerve preservation after administering NES cells

intracavernosally and locally. We also report functional studies that suggest improved erectile function after NES cell therapy. The greater mean peak ICPs in groups 3 and 4 than in group 2 should indicate improved erectile functioning in these rats.

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CONFLICT OF INTEREST None declared.

REFERENCES 1

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Walsh PC. Radical prostatectomy, preservation of sexual function, cancer control. The controversy. Urol Clin North Am 1987; 14: 663–73 Meuleman EJ, Mulders PF. Erectile function after radical prostatectomy: a review. Eur Urol 2003; 43: 95–102 Carrier S, Zvara P, Nunes L, Kour NW, Rehman J, Lue TF. Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J Urol 1995; 153: 1722–7 Fansa H, Keilhoff G. Factors influencing nerve regeneration. Handchir Mikrochir Plast Chir 2003; 35: 72–82 Sun Y, Jin K, Xie L et al. VEGF-induced neuroprotection, neurogenesis, and

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angiogenesis after focal cerebral ischemia. J Clin Invest 2003; 111: 1843–51 6 Alison MR, Poulsom R, Forbes S, Wright NA. An introduction to stem cells. J Pathol 2002; 197: 419–23 7 Herzog EL, Chai L, Krause DS. Plasticity of marrow derived stem cells. Blood 2003; 102: 3483–93 8 Lu P, Jones LL, Snyder EY, Tuszynski MH. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003; 181: 115–29 9 Liu S, Qu Y, Stewart TJ et al. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci USA 2000; 97: 6126–31 10 Klassen HJ, Imfeld KL, Kirov II et al. Expression of cytokines by multipotent neural progenitor cells. Cytokine 2003; 22: 101–6 11 Whiteley SJ, Klassen H, Coffey PJ, Young MJ. Photoreceptor rescue after

low-dose intravitreal IL-1beta injection in the RCS rat. Exp Eye Res 2001; 73: 557–68 12 Wu JP, Kuo JS, Liu YL, Tzeng SF. Tumor necrosis factor-alpha modulates the proliferation of neural progenitors in the subventricular/ventricular zone of adult rat brain. Neurosci Lett 2000; 292: 203– 6 13 Al-Chalabi A, Miller CC. Neurofilaments and neurological disease. Bioessays 2003; 25: 346–55 Correspondence: Tom F. Lue, Department of Urology, University of California, San Francisco, CA 94143–0738, USA. e-mail: [email protected] Abbreviations: NES, neural embryonic stem; MPG, major pelvic ganglion; BDNF, brainderived neurotrophic factor; EGFP, enhanced green fluorescence protein; TH, tyrosine hydroxylase; DMEM, Dulbecco’s Modified Eagle’s Medium; ICP, intracavernosal pressure.

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