- Email: [email protected]
The Effect of FK1706 on Erectile Function Following Bilateral Cavernous Nerve Crush Injury in a Rat Model
The Effect of FK1706 on Erectile Function Following Bilateral Cavernous Nerve Crush Injury in a Rat Model Narihiko Hayashi, Thomas X. Minor, Rafael Carrion,* Raymond Price,† Lora Nunes and Tom F. Lue‡,§ From the Knuppe Molecular Urology Laboratory, Department of Urology, University of California-San Francisco, San Francisco, California, and Pharmacology Research Laboratory, Astellas Pharmaceuticals, Inc. (RP), Ibaraki, Japan
Purpose: We investigated the neurotrophic effect of FK1706 on erectile recovery following bilateral cavernous nerve crush injury in a rat model. Materials and Methods: A total of 28 male Sprague-Dawley rats were randomly divided into 4 equal groups. Seven animals underwent sham operation and subcutaneous vehicle injection, whereas 21 underwent bilateral cavernous nerve crush injury followed by vehicle injection alone, or by low (0.1 mg/kg) or high (1.0 mg/kg) dose FK1706 treatment. Injections were continued 5 days weekly for 8 weeks. Erectile function was then assessed by cavernous nerve electrostimulation and penile tissue was evaluated immunohistochemically. Results: No erectile dysfunction was identified in the sham treated group (mean maximal intracavernous pressure ⫾ SEM 106.8 ⫾ 6.4 cm H2O), whereas nerve injury significantly decreased ICP to 17.9 ⫾ 7.0 cm H2O. FK1706 facilitated neural and erectile recovery in a concentration dependent manner with a mean ICP in the high dose FK treatment group of 80.1 ⫾ 7.8 cm H2O compared with 44.1 ⫾ 12.9 cm H2O in the low dose group. Similar stepwise findings were observed using mean area under the curve data. Sham treated animals showed regular axon sizes and shapes with homogenous GAP-43 and neurofilament staining, whereas injured axons showed irregular shapes, sizes and staining patterns. FK1706 treatment restored axon shape and staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide phosphate staining and FK1706 treatment showed a nonsignificant trend toward increased staining. Conclusions: Bilateral cavernous nerve crush causes reproducible erectile dysfunction, consistent with prior experiments. High dose subcutaneous FK1706 therapy promotes significant neuroregeneration and erectile function recovery. Key Words: penis; impotence; FK1706; rats, Sprague-Dawley; wounds, nonpenetrating
factors, including growth hormone, vascular endothelial growth factor, brain-derived neurotrophic factor and insulin-like growth factor.2,6 –11 Immunophilin ligands represent a new class of therapeutic agents with the ability to facilitate neuronal recovery.12 One such compound, tacrolimus or FK506, is a clinically used immunosuppressant. FK506 also shows neuroprotective and neurotrophic effects, including stimulation of axonal regrowth and enhancement of functional recovery, in various neurodegenerative disease models.13 FK506 also enhanced erectile recovery in 2 cavernous nerve injury models, including unilateral nerve transection and unilateral transection combined with contralateral ablation.14 Despite the therapeutic potential of FK506 for ameliorating neuronal injury its immunosuppressive activity limits chronic use in patients. However, recently it became clear that nonimmunosuppressive FKBP ligands can still show efficacy in neuronal injury paradigms, suggesting that clinical limitation could be overcome. We tested whether the novel nonimmunosuppressant immunophilin ligand FK170615 could enhance the recovery of erectile function and morphological damage following cavernous nerve crush injury in rats.
adical pelvic surgery, including prostatectomy, cystectomy and proctocolectomy, commonly injure the cavernous nerves carrying parasympathetic innervation to the penis.1,2 Despite nerve sparing procedures stemming from recent advances in pelvic neuroanatomy3,4 the recovery of erectile function is protracted and overall success is disappointing. According to a multicenter outcomes study only 15% of patients achieved erection sufficient for intercourse in the first 6 months following radical prostatectomy. Furthermore, 56% of men who underwent bilateral nerve sparing were still reporting impotence 18 or more months after surgery.2,5 The return of potency after surgical cavernous nerve injury partially depends on axon regeneration in the remaining neural tissue. Using established cavernous nerve injury models in rats we have studied the neuroprotective and neuroregenerative effects of various neurotrophic growth
R
Submitted for publication August 8, 2005. Supported by a grant from Astellas Pharma, Inc., Japan. * Financial interest and/or other relationship with Pfizer. † Financial interest and/or other relationship with Astellas. ‡ Correspondence: University of California-San Francisco, 400 Parnassus Ave., Box 0738, San Francisco, California 94143 (telephone: 415-476-1611; FAX: 415-476-8849; e-mail: [email protected]. edu). § Financial interest and/or other relationship with Genix, Pfizer, Lilly-ICOS, Riant, Astellas, Biopharm and Bayer.
0022-5347/06/1762-0824/0 THE JOURNAL OF UROLOGY® Copyright © 2006 by AMERICAN UROLOGICAL ASSOCIATION
MATERIALS AND METHODS The study included 28 male Sprague-Dawley rats at age 2 months weighing 250 to 350 gm that were randomly divided
824
Vol. 176, 824-829, August 2006 Printed in U.S.A. DOI:10.1016/j.juro.2006.03.071
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
825
into 4 groups of 7 each. Group 1 underwent sham operation, composed of periprostatic dissection and identification of the cavernous nerves bilaterally. The remaining 21 rats in groups 2 to 4 underwent bilateral cavernous nerve crush using a needle driver (2-minute crush per side). Following surgery all injections were continued 5 days weekly for a total of 8 weeks. Groups 1 and 2 received vehicle (1.0 ml/kg 4% HCO-60/EtOH subcutaneously), group 3 received low dose FK1706 (0.1 mg/kg subcutaneously) and group 4 received high dose FK1706 (1 mg/kg subcutaneously). The vehicle was a pharmaceutical detergent that stabilizes FK1706 in solution by micelle formation. Animals were weighed daily and doses were adjusted accordingly. For the surgical procedure animals were anesthetized with a combined intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Isothermia was maintained at 37C by placing the rats on a heated pad. Through a lower midline abdominal incision the prostate gland was exposed, and the cavernous nerves and major pelvic ganglia were identified bilaterally. There was no additional pelvic surgical manipulation in group 1. In groups 2 to 4 the cavernous nerves were isolated and crush injury was applied. Specifically the tips of a surgical needle driver were positioned at a 90-degree angle and the nerves were crushed at a constant 1 click pressure for 2 minutes per side. The abdominal wall was subsequently closed in 2 layers. After 8 weeks of injections erectile function was assessed in all rats by measuring maximal ICP upon direct cavernous electrostimulation. The cavernous nerves were again isolated through a repeat midline abdominal approach. The incision was extended inferior and following ischiocavernous muscle dissection the penile crura were exposed. A 23 gauge butterfly needle was inserted into the penile crus and connected to polyethylene-50 tubing for ICP measurement. A bipolar stainless steel hook electrode, consisting of 2 mm diameter probes separated by 1 mm, attached to a multijointed clamp was used to directly stimulate the cavernous nerves. Monophasic rectangular pulses were generated by a computer with a custom-built constant current amplifier. Stimulus parameters were 1.5 mA, 20 Hz, pulse width 0.2 milliseconds and duration 50 seconds. Each cavernous nerve was stimulated separately and ICP was measured using LabVIEW™ 4.0 software. The mean of the peak (maximal) right and left ICPs was then determined in each rat. In addition, the mean ICP area above resting baseline was calculated from the time of initial stimulation to a point 20 seconds after stimulus termination. These AUC data were calculated from pixels using Image-Pro® Plus software. Mid shaft corporeal penile tissue was harvested for histological studies. To minimize manipulation of the rats we elected to determine systemic blood pressure at the end of the procedure by inserting a butterfly needle into the aorta, as opposed to constant monitoring via a cannula in the carotid artery.
compound and stored at ⫺70C until use. Transverse sections were cut at 4 , adhered to charged slides, air dried for 5 minutes and then rehydrated with 0.05M PBS for 5 minutes Sections were treated with hydrogen peroxide and methanol to quench endogenous peroxidase activity. Decreased NADPH diaphorase staining was performed to determine the expression of NOS containing nerves. For NADPH staining sections were incubated with 0.1 mmol/l NADPH, 0.2 mmol/l nitroblue tetrazolium and 0.2% Triton X-100 (SigmaAldrich, St. Louis, Missouri) in buffer with constant microscopic monitoring for color development. Slides were rinsed in buffer to terminate the reaction when a deep blue stain was detected, signifying NADPH diaphorase positive nerves. After rehydration alternate sections were washed twice in PBS for 5 minutes, followed by 30 minutes of room temperature incubation with 3% serum/PBS/0.3% Triton X-100. After draining excess fluid tissues were incubated overnight at 4C with SMI-31 monoclonal antibody to NF (Sternberger Monoclonals, Lutherville, Maryland) (1:70,000) or monoclonal antibody to GAP-43 (Sigma Chemical, St. Louis, Missouri) (1:8000). Additional sections were prepared without antibody exposure to serve as negative controls. After washing sections were immunostained with the avidin-biotin-peroxidase method (Elite ABC, Vector Laboratories, Burlingame, California) with diaminobenzidine as the chromogen, followed by counterstaining with hematoxylin. Tissues from 5 rats per group were randomly selected for immunohistochemical study. Five randomly selected 200⫻ fields per animal were photographed using a DXM1200 digital still camera with ACT-1 software (Nikon Instruments, Melville, New York). Semi-automated image analysis of representative photos was then performed using a customized algorithm running in Image-Pro™ Plus image software. Briefly, the overall area of interest (total nerve area) was defined manually with subsequent analysis using thresholding for dark staining and exclusion criteria based on object area to select individual objects. Thresholding was automatic for NF and GAP-43 images, while predefined manual thresholding was performed for NOS images due to differences in the distribution of pixel intensity. Object analysis was performed using built-in analysis tools in Image-Pro™ Plus. An object was defined as an area of distinct staining, eg staining surrounded by a nonstained area. Particularly in vehicle sections multiple staining areas could have been present in 1 axon, so that objects were equivalent to axons. The object parameters analyzed were the radius, that is the maximum distance between the object centroid (center) and outline, using the equation, ratio ⫽ maximum radius/minimum radius, the area of the object and integrated optical density, that is the integrated total intensity of all pixels in each object with a higher value indicating a darker object.
Immunohistochemistry and Image Analysis Freshly dissected penile tissue samples were processed and analyzed for NADPH diaphorase, GAP-43 and NF expression. Specifically tissues were first fixed for 4 hours with cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, followed by overnight immersion in buffer containing 30% sucrose for cryoprotection. Tissues were frozen in OCT
Statistical Analysis Data were analyzed using Student’s t test to compare sham and vehicle treated animals or using ANOVA with Dunnett’s post test to compare the vehicle and FK1706 treated groups with significance considered at p ⬍0.05. All results are expressed as the mean ⫾ SEM.
826
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
RESULTS Functional Parameters To evaluate changes in erectile function we measured peak ICP, which correlates with penile rigidity in men, and AUC, which represents erectile duration. Nerve injury induced a significant decrease in mean peak ICP in groups 1 and 2 (fig. 1, A and B). FK1706 treatment dose dependently enhanced peak cavernous pressure (fig. 1, C). Although low dose therapy in group 3 more than doubled peak ICP compared to
TABLE 1. Peak ICP increase in response to electrostimulation 8 weeks after bilateral cavernous nerve crush Group No. 1 2 3 4
Treatment Sham Vehicle Low dose FK1706 (0.1 mg/kg) High dose FK1706 (1.0 mg/kg)
No. Subjects
Mean Cavernous Pressure Increase ⫾ SEM (cm H2O)
6 7 7
106.8 ⫾ 6.4 17.9 ⫾ 7.0 44.1 ⫾ 12.9
7
80.1 ⫾ 7.8
Vehicle vs sham treatment Student’s t test p ⬍0.01 and vs high dose ANOVA/Dunnett’s test p ⬍0.01.
vehicle, a statistically significant improvement was seen only in high dose group 4 (table 1). To determine a secondary functional index of erectile recovery mean AUC data were tabulated in each group. AUC was defined as the ICP area above resting baseline from the start of neurostimulation to a point 20 seconds after stimulus termination (total duration 70 seconds). Consistent with peak ICP values the AUC score in group 2 was significantly lower than the score in group 1 or 4. Again, a concentration dependent response to FK1706 therapy was seen with a larger AUC score in the high vs low dose group (table 2). There was no statistical difference in peak aortic blood pressure or gained weight among the 4 groups (data not shown). Results in 1 sham treated animal are not presented because of instrument error. Immunohistochemistry and Image Analysis To determine whether FK1706 promoted morphological changes we examined the staining patterns of the 3 markers GAP-43, NF and NADPH (fig. 2). GAP-43 and NF stains label living or regenerating axons. Therefore, in these sections we measured the presence of regenerating nerve fibers using 2 main end points, including the shape of stained fibers and the proportion of stained vs unstained axons. We measured the maximum radius to determine differences in fiber shape. Sham treated animals showed similar axon sizes and shapes with homogenous staining, whereas injured axons showed irregular shapes and sizes as well as partial staining with stained areas often forming a donut or target shape (fig. 2). Analysis of the intensity of every object showed that 1) injury decreased the number of axons that were stained, that is it decreased integrated optical density, and 2) FK1706 treatment restored staining toward normal levels, that is mean integrated optical density increased in FK1706 treated sections (fig. 3, A and C). FK1706 treatment
TABLE 2. AUC ICP pixel data in response to electrostimulation 8 weeks after bilateral cavernous nerve crush Group No. 1 2 3 4
FIG. 1. Representative ICP traces (top) in response to electrostimulation (bottom) in sham (A), vehicle (B) and 1 mg/kg FK1706 subcutaneously (C) treated animals.
Treatment Sham Vehicle Low dose FK1706 (0.1 mg/kg) High dose FK1706 (1.0 mg/kg)
No. Subjects
Mean ICP AUC Score ⫾ SEM
6 7 7
3,291.3 ⫾ 482.2 510.8 ⫾ 203.1 1,229.4 ⫾ 430.4
7
2,142.0 ⫾ 326.7
Vehicle vs sham treatment Student’s t test p ⬍0.01 and vs high dose ANOVA/Dunnett’s test p ⬍0.01.
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
827
also induced dose dependent restoration of axon shape (fig. 3, B and D). NADPH diaphorase staining indicates the presence of nNOS containing nerves, which are required for erectile function. For this image analysis we measured the total area of NADPH staining, again by objects. Injury induced a significant decrease in NADPH staining and FK1706 treatment showed a nonsignificant trend toward increased staining (fig. 3, E).
DISCUSSION
FIG. 2. Representative cross-sections of rat penis with NF (A to C), GAP-43 (D to F) and NADPH diaphorase (G to I) staining. Sham, sham treated rats. Vehicle, vehicle treated rats. FK1706, high dose FK1706 treated rats. Scale bar indicates 0.1 mm.
Previously we have reported that several growth factors, including growth hormone, vascular endothelial growth factor, brain-derived neurotrophic factor and insulin-like growth factor, could promote cavernous nerve recovery following injury.6 – 8,16 Groups at our laboratory have used several different models of controlled cavernous nerve trauma in the rat, including freezing, crushing and transection with or without removal of a segment of the contralateral nerve. Although sharp nerve transection with approxi-
FIG. 3. Quantitative immunohistochemistry in 4 images of cavernous nerve per parameter shows integrated optical density (staining intensity measure) and maximum radius for NF (A and B) and GAP-43 (C and D) staining, and mean object area for NADPH staining (E). Analysis was performed as described. Error bars represent SEM. Single asterisk indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.05 vs vehicle. Double asterisks indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.01 vs vehicle. Triple asterisks indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.001 vs vehicle. Pound signs indicate Student’s t test p ⬍0.001 vs sham treatment. Sham, sham treatment. Vehicle, vehicle treatment. FK0.1, low dose 0.1 mg/kg FK1706 treatment. FK1, high dose 1.0 mg/kg FK1706 treatment.
828
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
mation of the cut ends has been used, investigators at our laboratory have noted a large degree of spontaneous erectile function recovery in this model even without treatment (unpublished data). We believe that cryoablation and partial nerve excision represent steadfast methods. However, cavernous nerve injury may be permanent or too severe to allow recovery in a reasonable period regardless of treatment. The major advantages of our current model (bilateral cavernous nerve crush using a dedicated surgical needle driver) are simplicity, reproducibility and consistency. In the current study minimal recovery of erectile function in the control group is similar to that in prior experiments and it confirms the reliability of the previous finding.8 A limitation of this model is that the prostate is not removed and the relationship of this type of nerve injury to the surgical trauma sustained in prostatectomy is unclear. Immunophilin ligands show significant promise as treatment for nerve injury and neurological disease. However, current clinically available immunophilins, eg FK506, are immunosuppressive, thus, restricting their long-term clinical use. The immunosuppressive effects of immunophilins are mediated by binding to FKBP-12 and subsequent calcineurin inhibition.17 However, neither calcineurin inhibition nor binding to FKBP-12 is necessary for the neurotrophic activity of immunophilins, suggesting that nerve regeneration and immunosuppressive properties can be separated. In fact, the novel nonimmunusuppressant immunophilin ligand GPI-1485 is currently in clinical trials for treating post-prostatectomy erectile dysfunction, suggesting that this concept has significant clinical potential. We report that FK1706 dramatically and dose dependently improved erectile function recovery after 8 weeks of therapy. Animals treated with high dose FK1706 demonstrated increased AUC pixel levels above baseline in response to electrostimulation, approaching 65% of the total recorded in the sham treated group. Compared to controls AUC data revealed a quantified index of erectile recovery that was approximately 2 and 4-fold greater in the low and high dose treatment groups, respectively. Treatment groups consisted of only 7 animals each, which may have been under powered to detect significant differences between the vehicle control and low dose groups. NFs maintain and regulate neuronal cytoskeletal plasticity through the regulation of neurite outgrowth, axonal caliber and axonal transport, and they are usually up-regulated during neuronal regeneration. Nerve injury significantly decreased NF staining but staining was restored in FK1706 treated animals, suggesting that our nerve crush model causes significant but reversible damage to penile innervation. GAP-43 is a protein associated with axonal growth and regeneration. Nerve injury decreased GAP-43 staining intensity and FK1706 treatment restored it despite the relatively late time that it was used in this study. These findings suggest that FK1706 has a general effect on nerve regeneration rather than on a specific subpopulation of nerves and in fact several studies of neuroimmunophilin therapy for neurodegenerative diseases failed to demonstrate a relationship between functional recovery and histological evidence.18,19 In our experience NADPH diaphorase stains the same NOS containing nerves as those stained with antinNOS antibody. However, blue staining with NADPH diaphorase staining is easier to detect and, therefore, it is our preferred method. In addition, nNOS recovery in the dorsal
nerve parallels nNOS recovery in the intracavernous nerves but it is much simpler to analyze. In vitro FK1706 was more effective for potentiating nerve growth factor induced neurite outgrowth15 than FK506, suggesting that it has similar in vivo neurotrophic potential. FK1706 showed no neurotrophic effects alone but it significantly enhanced NGF signaling via a common growth factor signaling pathway. Moreover, FK1706 did not dramatically inhibit interleukin-2 production or lymphocyte proliferation, suggesting a lack of immunosuppressive activity.15 The neurotrophic characteristics of immunophilins hold vast potential for the treatment of many neurological conditions, including erectile dysfunction following radical pelvic surgery. Unfortunately the current clinically available immunophilins, such as FK506, cyclosporin A and rapamycin, are restricted in their general use postoperatively and in the long term secondary to their immunosuppressive effects. FK1706, a nonimmunosuppressant derivative of FK506 with significant neurotrophic activity, is an exciting drug that warrants further investigation.
Abbreviations and Acronyms AUC FKBP GAP-43 ICP NADPH
⫽ ⫽ ⫽ ⫽ ⫽
NF nNOS NOS PBS VEGF
⫽ ⫽ ⫽ ⫽ ⫽
area under the curve tacrolimus binding protein growth associated protein-43 intracavernous pressure nicotinamide adenine dinucleotide phosphate neurofilament neuronal NOS nitric oxide synthase phosphate buffered saline vascular endothelial growth factor
REFERENCES 1. 2. 3.
4.
5.
6.
7.
Nehra, A. and Moreland, R. B.: Neurologic erectile dysfunction. Urol Clin North Am, 28: 289, 2001 Lin, C. S. and Lue, T. F.: Growth factor therapy and neuronal nitric oxide synthase. Int J Impot Res, suppl., 16: S38, 2004 Walsh, P. C. and Mostwin, J. L.: Radical prostatectomy and cystoprostatectomy with preservation of potency. Results using a new nerve-sparing technique. Br J Urol, 56: 694, 1984 Paick, J. S., Donatucci, C. F. and Lue, T. F.: Anatomy of cavernous nerves distal to prostate: microdissection study in adult male cadavers. Urology, 42: 145, 1993 Stanford, J. L., Feng, Z., Hamilton, A. S., Gilliland, F. D., Stephenson, R. A., Eley, J. W. et al: Urinary and sexual function after radical prostatectomy for clinically localized prostate cancer: the Prostate Cancer Outcomes Study. JAMA, 283: 354, 2000 Bochinski, D., Hsieh, P. S., Nunes, L., Lin, G. T., Lin, C. S., Spencer, E. M. et al: Effect of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 complex in cavernous nerve cryoablation. Int J Impot Res, 16: 418, 2004 El-Sakka, A. I., Hassan, M. U., Selph, C., Perinchery, G., Dahiya, R. and Lue, T. F.: Effect of cavernous nerve freezing on protein and gene expression of nitric oxide synthase in the rat penis and pelvic ganglia. J Urol, 160: 2245, 1998
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY 8.
9.
10.
11.
12.
13.
Hsieh, P. S., Bochinski, D. J., Lin, G. T., Nunes, L., Lin, C. S. and Lue, T. F.: The effect of vascular endothelial growth factor and brain-derived neurotrophic factor on cavernosal nerve regeneration in a nerve-crush rat model. BJU Int, 92: 470, 2003 Lin, C. S., Lau, A., Tu, R. and Lue, T. F.: Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum. Biochem Biophys Res Commun, 268: 628, 2000 Lin, C. S., Lau, A., Tu, R. and Lue, T. F.: Identification of three alternative first exons and an intronic promoter of human PDE5A gene. Biochem Biophys Res Commun, 268: 596, 2000 Lin, C. S., Ho, H. C., Gholami, S., Chen, K. C., Jad, A. and Lue, T. F.: Gene expression profiling of an arteriogenic impotence model. Biochem Biophys Res Commun, 285: 565, 2001 Wang, M. S. and Gold, B. G.: FK506 increases the regeneration of spinal cord axons in a predegenerated peripheral nerve autograft. J Spinal Cord Med, 22: 287, 1999 Gold, B. G.: Neuroimmunophilin ligands: evaluation of their therapeutic potential for the treatment of neurological disorders. Expert Opin Investig Drugs, 9: 2331, 2000
14.
15.
16.
17.
18.
19.
829
Burnett, A. L. and Becker, R. E.: Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury. J Urol, 171: 495, 2004 Price, R. D., Yamaji, T., Yamamoto, H., Higashi, Y., Hanaoka, K., Yamazaki, S. et al: FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action. Eur J Pharmacol, 509: 11, 2005 Carrier, S., Zvara, P., Nunes, L., Kour, N. W., Rehman, J. and Lue, T. F.: Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J Urol, 153: 1722, 1995 Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J. et al: Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry, 31: 3896, 1992 Poulter, M. O., Payne, K. B. and Steiner, J. P.: Neuroimmunophilins: a novel drug therapy for the reversal of neurodegenerative disease? Neuroscience, 128: 1, 2004 Eberling, J. L., Pivirotto, P., Bringas, J., Steiner, J. P., Kordower, J. H., Chu, Y. et al: The immunophilin ligand GPI1046 does not have neuroregenerative effects in MPTPtreated monkeys. Exp Neurol, 178: 236, 2002
Purpose: We investigated the neurotrophic effect of FK1706 on erectile recovery following bilateral cavernous nerve crush injury in a rat model. Materials and Methods: A total of 28 male Sprague-Dawley rats were randomly divided into 4 equal groups. Seven animals underwent sham operation and subcutaneous vehicle injection, whereas 21 underwent bilateral cavernous nerve crush injury followed by vehicle injection alone, or by low (0.1 mg/kg) or high (1.0 mg/kg) dose FK1706 treatment. Injections were continued 5 days weekly for 8 weeks. Erectile function was then assessed by cavernous nerve electrostimulation and penile tissue was evaluated immunohistochemically. Results: No erectile dysfunction was identified in the sham treated group (mean maximal intracavernous pressure ⫾ SEM 106.8 ⫾ 6.4 cm H2O), whereas nerve injury significantly decreased ICP to 17.9 ⫾ 7.0 cm H2O. FK1706 facilitated neural and erectile recovery in a concentration dependent manner with a mean ICP in the high dose FK treatment group of 80.1 ⫾ 7.8 cm H2O compared with 44.1 ⫾ 12.9 cm H2O in the low dose group. Similar stepwise findings were observed using mean area under the curve data. Sham treated animals showed regular axon sizes and shapes with homogenous GAP-43 and neurofilament staining, whereas injured axons showed irregular shapes, sizes and staining patterns. FK1706 treatment restored axon shape and staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide phosphate staining and FK1706 treatment showed a nonsignificant trend toward increased staining. Conclusions: Bilateral cavernous nerve crush causes reproducible erectile dysfunction, consistent with prior experiments. High dose subcutaneous FK1706 therapy promotes significant neuroregeneration and erectile function recovery. Key Words: penis; impotence; FK1706; rats, Sprague-Dawley; wounds, nonpenetrating
factors, including growth hormone, vascular endothelial growth factor, brain-derived neurotrophic factor and insulin-like growth factor.2,6 –11 Immunophilin ligands represent a new class of therapeutic agents with the ability to facilitate neuronal recovery.12 One such compound, tacrolimus or FK506, is a clinically used immunosuppressant. FK506 also shows neuroprotective and neurotrophic effects, including stimulation of axonal regrowth and enhancement of functional recovery, in various neurodegenerative disease models.13 FK506 also enhanced erectile recovery in 2 cavernous nerve injury models, including unilateral nerve transection and unilateral transection combined with contralateral ablation.14 Despite the therapeutic potential of FK506 for ameliorating neuronal injury its immunosuppressive activity limits chronic use in patients. However, recently it became clear that nonimmunosuppressive FKBP ligands can still show efficacy in neuronal injury paradigms, suggesting that clinical limitation could be overcome. We tested whether the novel nonimmunosuppressant immunophilin ligand FK170615 could enhance the recovery of erectile function and morphological damage following cavernous nerve crush injury in rats.
adical pelvic surgery, including prostatectomy, cystectomy and proctocolectomy, commonly injure the cavernous nerves carrying parasympathetic innervation to the penis.1,2 Despite nerve sparing procedures stemming from recent advances in pelvic neuroanatomy3,4 the recovery of erectile function is protracted and overall success is disappointing. According to a multicenter outcomes study only 15% of patients achieved erection sufficient for intercourse in the first 6 months following radical prostatectomy. Furthermore, 56% of men who underwent bilateral nerve sparing were still reporting impotence 18 or more months after surgery.2,5 The return of potency after surgical cavernous nerve injury partially depends on axon regeneration in the remaining neural tissue. Using established cavernous nerve injury models in rats we have studied the neuroprotective and neuroregenerative effects of various neurotrophic growth
R
Submitted for publication August 8, 2005. Supported by a grant from Astellas Pharma, Inc., Japan. * Financial interest and/or other relationship with Pfizer. † Financial interest and/or other relationship with Astellas. ‡ Correspondence: University of California-San Francisco, 400 Parnassus Ave., Box 0738, San Francisco, California 94143 (telephone: 415-476-1611; FAX: 415-476-8849; e-mail: [email protected]. edu). § Financial interest and/or other relationship with Genix, Pfizer, Lilly-ICOS, Riant, Astellas, Biopharm and Bayer.
0022-5347/06/1762-0824/0 THE JOURNAL OF UROLOGY® Copyright © 2006 by AMERICAN UROLOGICAL ASSOCIATION
MATERIALS AND METHODS The study included 28 male Sprague-Dawley rats at age 2 months weighing 250 to 350 gm that were randomly divided
824
Vol. 176, 824-829, August 2006 Printed in U.S.A. DOI:10.1016/j.juro.2006.03.071
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
825
into 4 groups of 7 each. Group 1 underwent sham operation, composed of periprostatic dissection and identification of the cavernous nerves bilaterally. The remaining 21 rats in groups 2 to 4 underwent bilateral cavernous nerve crush using a needle driver (2-minute crush per side). Following surgery all injections were continued 5 days weekly for a total of 8 weeks. Groups 1 and 2 received vehicle (1.0 ml/kg 4% HCO-60/EtOH subcutaneously), group 3 received low dose FK1706 (0.1 mg/kg subcutaneously) and group 4 received high dose FK1706 (1 mg/kg subcutaneously). The vehicle was a pharmaceutical detergent that stabilizes FK1706 in solution by micelle formation. Animals were weighed daily and doses were adjusted accordingly. For the surgical procedure animals were anesthetized with a combined intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Isothermia was maintained at 37C by placing the rats on a heated pad. Through a lower midline abdominal incision the prostate gland was exposed, and the cavernous nerves and major pelvic ganglia were identified bilaterally. There was no additional pelvic surgical manipulation in group 1. In groups 2 to 4 the cavernous nerves were isolated and crush injury was applied. Specifically the tips of a surgical needle driver were positioned at a 90-degree angle and the nerves were crushed at a constant 1 click pressure for 2 minutes per side. The abdominal wall was subsequently closed in 2 layers. After 8 weeks of injections erectile function was assessed in all rats by measuring maximal ICP upon direct cavernous electrostimulation. The cavernous nerves were again isolated through a repeat midline abdominal approach. The incision was extended inferior and following ischiocavernous muscle dissection the penile crura were exposed. A 23 gauge butterfly needle was inserted into the penile crus and connected to polyethylene-50 tubing for ICP measurement. A bipolar stainless steel hook electrode, consisting of 2 mm diameter probes separated by 1 mm, attached to a multijointed clamp was used to directly stimulate the cavernous nerves. Monophasic rectangular pulses were generated by a computer with a custom-built constant current amplifier. Stimulus parameters were 1.5 mA, 20 Hz, pulse width 0.2 milliseconds and duration 50 seconds. Each cavernous nerve was stimulated separately and ICP was measured using LabVIEW™ 4.0 software. The mean of the peak (maximal) right and left ICPs was then determined in each rat. In addition, the mean ICP area above resting baseline was calculated from the time of initial stimulation to a point 20 seconds after stimulus termination. These AUC data were calculated from pixels using Image-Pro® Plus software. Mid shaft corporeal penile tissue was harvested for histological studies. To minimize manipulation of the rats we elected to determine systemic blood pressure at the end of the procedure by inserting a butterfly needle into the aorta, as opposed to constant monitoring via a cannula in the carotid artery.
compound and stored at ⫺70C until use. Transverse sections were cut at 4 , adhered to charged slides, air dried for 5 minutes and then rehydrated with 0.05M PBS for 5 minutes Sections were treated with hydrogen peroxide and methanol to quench endogenous peroxidase activity. Decreased NADPH diaphorase staining was performed to determine the expression of NOS containing nerves. For NADPH staining sections were incubated with 0.1 mmol/l NADPH, 0.2 mmol/l nitroblue tetrazolium and 0.2% Triton X-100 (SigmaAldrich, St. Louis, Missouri) in buffer with constant microscopic monitoring for color development. Slides were rinsed in buffer to terminate the reaction when a deep blue stain was detected, signifying NADPH diaphorase positive nerves. After rehydration alternate sections were washed twice in PBS for 5 minutes, followed by 30 minutes of room temperature incubation with 3% serum/PBS/0.3% Triton X-100. After draining excess fluid tissues were incubated overnight at 4C with SMI-31 monoclonal antibody to NF (Sternberger Monoclonals, Lutherville, Maryland) (1:70,000) or monoclonal antibody to GAP-43 (Sigma Chemical, St. Louis, Missouri) (1:8000). Additional sections were prepared without antibody exposure to serve as negative controls. After washing sections were immunostained with the avidin-biotin-peroxidase method (Elite ABC, Vector Laboratories, Burlingame, California) with diaminobenzidine as the chromogen, followed by counterstaining with hematoxylin. Tissues from 5 rats per group were randomly selected for immunohistochemical study. Five randomly selected 200⫻ fields per animal were photographed using a DXM1200 digital still camera with ACT-1 software (Nikon Instruments, Melville, New York). Semi-automated image analysis of representative photos was then performed using a customized algorithm running in Image-Pro™ Plus image software. Briefly, the overall area of interest (total nerve area) was defined manually with subsequent analysis using thresholding for dark staining and exclusion criteria based on object area to select individual objects. Thresholding was automatic for NF and GAP-43 images, while predefined manual thresholding was performed for NOS images due to differences in the distribution of pixel intensity. Object analysis was performed using built-in analysis tools in Image-Pro™ Plus. An object was defined as an area of distinct staining, eg staining surrounded by a nonstained area. Particularly in vehicle sections multiple staining areas could have been present in 1 axon, so that objects were equivalent to axons. The object parameters analyzed were the radius, that is the maximum distance between the object centroid (center) and outline, using the equation, ratio ⫽ maximum radius/minimum radius, the area of the object and integrated optical density, that is the integrated total intensity of all pixels in each object with a higher value indicating a darker object.
Immunohistochemistry and Image Analysis Freshly dissected penile tissue samples were processed and analyzed for NADPH diaphorase, GAP-43 and NF expression. Specifically tissues were first fixed for 4 hours with cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, followed by overnight immersion in buffer containing 30% sucrose for cryoprotection. Tissues were frozen in OCT
Statistical Analysis Data were analyzed using Student’s t test to compare sham and vehicle treated animals or using ANOVA with Dunnett’s post test to compare the vehicle and FK1706 treated groups with significance considered at p ⬍0.05. All results are expressed as the mean ⫾ SEM.
826
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
RESULTS Functional Parameters To evaluate changes in erectile function we measured peak ICP, which correlates with penile rigidity in men, and AUC, which represents erectile duration. Nerve injury induced a significant decrease in mean peak ICP in groups 1 and 2 (fig. 1, A and B). FK1706 treatment dose dependently enhanced peak cavernous pressure (fig. 1, C). Although low dose therapy in group 3 more than doubled peak ICP compared to
TABLE 1. Peak ICP increase in response to electrostimulation 8 weeks after bilateral cavernous nerve crush Group No. 1 2 3 4
Treatment Sham Vehicle Low dose FK1706 (0.1 mg/kg) High dose FK1706 (1.0 mg/kg)
No. Subjects
Mean Cavernous Pressure Increase ⫾ SEM (cm H2O)
6 7 7
106.8 ⫾ 6.4 17.9 ⫾ 7.0 44.1 ⫾ 12.9
7
80.1 ⫾ 7.8
Vehicle vs sham treatment Student’s t test p ⬍0.01 and vs high dose ANOVA/Dunnett’s test p ⬍0.01.
vehicle, a statistically significant improvement was seen only in high dose group 4 (table 1). To determine a secondary functional index of erectile recovery mean AUC data were tabulated in each group. AUC was defined as the ICP area above resting baseline from the start of neurostimulation to a point 20 seconds after stimulus termination (total duration 70 seconds). Consistent with peak ICP values the AUC score in group 2 was significantly lower than the score in group 1 or 4. Again, a concentration dependent response to FK1706 therapy was seen with a larger AUC score in the high vs low dose group (table 2). There was no statistical difference in peak aortic blood pressure or gained weight among the 4 groups (data not shown). Results in 1 sham treated animal are not presented because of instrument error. Immunohistochemistry and Image Analysis To determine whether FK1706 promoted morphological changes we examined the staining patterns of the 3 markers GAP-43, NF and NADPH (fig. 2). GAP-43 and NF stains label living or regenerating axons. Therefore, in these sections we measured the presence of regenerating nerve fibers using 2 main end points, including the shape of stained fibers and the proportion of stained vs unstained axons. We measured the maximum radius to determine differences in fiber shape. Sham treated animals showed similar axon sizes and shapes with homogenous staining, whereas injured axons showed irregular shapes and sizes as well as partial staining with stained areas often forming a donut or target shape (fig. 2). Analysis of the intensity of every object showed that 1) injury decreased the number of axons that were stained, that is it decreased integrated optical density, and 2) FK1706 treatment restored staining toward normal levels, that is mean integrated optical density increased in FK1706 treated sections (fig. 3, A and C). FK1706 treatment
TABLE 2. AUC ICP pixel data in response to electrostimulation 8 weeks after bilateral cavernous nerve crush Group No. 1 2 3 4
FIG. 1. Representative ICP traces (top) in response to electrostimulation (bottom) in sham (A), vehicle (B) and 1 mg/kg FK1706 subcutaneously (C) treated animals.
Treatment Sham Vehicle Low dose FK1706 (0.1 mg/kg) High dose FK1706 (1.0 mg/kg)
No. Subjects
Mean ICP AUC Score ⫾ SEM
6 7 7
3,291.3 ⫾ 482.2 510.8 ⫾ 203.1 1,229.4 ⫾ 430.4
7
2,142.0 ⫾ 326.7
Vehicle vs sham treatment Student’s t test p ⬍0.01 and vs high dose ANOVA/Dunnett’s test p ⬍0.01.
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
827
also induced dose dependent restoration of axon shape (fig. 3, B and D). NADPH diaphorase staining indicates the presence of nNOS containing nerves, which are required for erectile function. For this image analysis we measured the total area of NADPH staining, again by objects. Injury induced a significant decrease in NADPH staining and FK1706 treatment showed a nonsignificant trend toward increased staining (fig. 3, E).
DISCUSSION
FIG. 2. Representative cross-sections of rat penis with NF (A to C), GAP-43 (D to F) and NADPH diaphorase (G to I) staining. Sham, sham treated rats. Vehicle, vehicle treated rats. FK1706, high dose FK1706 treated rats. Scale bar indicates 0.1 mm.
Previously we have reported that several growth factors, including growth hormone, vascular endothelial growth factor, brain-derived neurotrophic factor and insulin-like growth factor, could promote cavernous nerve recovery following injury.6 – 8,16 Groups at our laboratory have used several different models of controlled cavernous nerve trauma in the rat, including freezing, crushing and transection with or without removal of a segment of the contralateral nerve. Although sharp nerve transection with approxi-
FIG. 3. Quantitative immunohistochemistry in 4 images of cavernous nerve per parameter shows integrated optical density (staining intensity measure) and maximum radius for NF (A and B) and GAP-43 (C and D) staining, and mean object area for NADPH staining (E). Analysis was performed as described. Error bars represent SEM. Single asterisk indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.05 vs vehicle. Double asterisks indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.01 vs vehicle. Triple asterisks indicate 1-way ANOVA followed by Dunnett’s post test p ⫽ 0.001 vs vehicle. Pound signs indicate Student’s t test p ⬍0.001 vs sham treatment. Sham, sham treatment. Vehicle, vehicle treatment. FK0.1, low dose 0.1 mg/kg FK1706 treatment. FK1, high dose 1.0 mg/kg FK1706 treatment.
828
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY
mation of the cut ends has been used, investigators at our laboratory have noted a large degree of spontaneous erectile function recovery in this model even without treatment (unpublished data). We believe that cryoablation and partial nerve excision represent steadfast methods. However, cavernous nerve injury may be permanent or too severe to allow recovery in a reasonable period regardless of treatment. The major advantages of our current model (bilateral cavernous nerve crush using a dedicated surgical needle driver) are simplicity, reproducibility and consistency. In the current study minimal recovery of erectile function in the control group is similar to that in prior experiments and it confirms the reliability of the previous finding.8 A limitation of this model is that the prostate is not removed and the relationship of this type of nerve injury to the surgical trauma sustained in prostatectomy is unclear. Immunophilin ligands show significant promise as treatment for nerve injury and neurological disease. However, current clinically available immunophilins, eg FK506, are immunosuppressive, thus, restricting their long-term clinical use. The immunosuppressive effects of immunophilins are mediated by binding to FKBP-12 and subsequent calcineurin inhibition.17 However, neither calcineurin inhibition nor binding to FKBP-12 is necessary for the neurotrophic activity of immunophilins, suggesting that nerve regeneration and immunosuppressive properties can be separated. In fact, the novel nonimmunusuppressant immunophilin ligand GPI-1485 is currently in clinical trials for treating post-prostatectomy erectile dysfunction, suggesting that this concept has significant clinical potential. We report that FK1706 dramatically and dose dependently improved erectile function recovery after 8 weeks of therapy. Animals treated with high dose FK1706 demonstrated increased AUC pixel levels above baseline in response to electrostimulation, approaching 65% of the total recorded in the sham treated group. Compared to controls AUC data revealed a quantified index of erectile recovery that was approximately 2 and 4-fold greater in the low and high dose treatment groups, respectively. Treatment groups consisted of only 7 animals each, which may have been under powered to detect significant differences between the vehicle control and low dose groups. NFs maintain and regulate neuronal cytoskeletal plasticity through the regulation of neurite outgrowth, axonal caliber and axonal transport, and they are usually up-regulated during neuronal regeneration. Nerve injury significantly decreased NF staining but staining was restored in FK1706 treated animals, suggesting that our nerve crush model causes significant but reversible damage to penile innervation. GAP-43 is a protein associated with axonal growth and regeneration. Nerve injury decreased GAP-43 staining intensity and FK1706 treatment restored it despite the relatively late time that it was used in this study. These findings suggest that FK1706 has a general effect on nerve regeneration rather than on a specific subpopulation of nerves and in fact several studies of neuroimmunophilin therapy for neurodegenerative diseases failed to demonstrate a relationship between functional recovery and histological evidence.18,19 In our experience NADPH diaphorase stains the same NOS containing nerves as those stained with antinNOS antibody. However, blue staining with NADPH diaphorase staining is easier to detect and, therefore, it is our preferred method. In addition, nNOS recovery in the dorsal
nerve parallels nNOS recovery in the intracavernous nerves but it is much simpler to analyze. In vitro FK1706 was more effective for potentiating nerve growth factor induced neurite outgrowth15 than FK506, suggesting that it has similar in vivo neurotrophic potential. FK1706 showed no neurotrophic effects alone but it significantly enhanced NGF signaling via a common growth factor signaling pathway. Moreover, FK1706 did not dramatically inhibit interleukin-2 production or lymphocyte proliferation, suggesting a lack of immunosuppressive activity.15 The neurotrophic characteristics of immunophilins hold vast potential for the treatment of many neurological conditions, including erectile dysfunction following radical pelvic surgery. Unfortunately the current clinically available immunophilins, such as FK506, cyclosporin A and rapamycin, are restricted in their general use postoperatively and in the long term secondary to their immunosuppressive effects. FK1706, a nonimmunosuppressant derivative of FK506 with significant neurotrophic activity, is an exciting drug that warrants further investigation.
Abbreviations and Acronyms AUC FKBP GAP-43 ICP NADPH
⫽ ⫽ ⫽ ⫽ ⫽
NF nNOS NOS PBS VEGF
⫽ ⫽ ⫽ ⫽ ⫽
area under the curve tacrolimus binding protein growth associated protein-43 intracavernous pressure nicotinamide adenine dinucleotide phosphate neurofilament neuronal NOS nitric oxide synthase phosphate buffered saline vascular endothelial growth factor
REFERENCES 1. 2. 3.
4.
5.
6.
7.
Nehra, A. and Moreland, R. B.: Neurologic erectile dysfunction. Urol Clin North Am, 28: 289, 2001 Lin, C. S. and Lue, T. F.: Growth factor therapy and neuronal nitric oxide synthase. Int J Impot Res, suppl., 16: S38, 2004 Walsh, P. C. and Mostwin, J. L.: Radical prostatectomy and cystoprostatectomy with preservation of potency. Results using a new nerve-sparing technique. Br J Urol, 56: 694, 1984 Paick, J. S., Donatucci, C. F. and Lue, T. F.: Anatomy of cavernous nerves distal to prostate: microdissection study in adult male cadavers. Urology, 42: 145, 1993 Stanford, J. L., Feng, Z., Hamilton, A. S., Gilliland, F. D., Stephenson, R. A., Eley, J. W. et al: Urinary and sexual function after radical prostatectomy for clinically localized prostate cancer: the Prostate Cancer Outcomes Study. JAMA, 283: 354, 2000 Bochinski, D., Hsieh, P. S., Nunes, L., Lin, G. T., Lin, C. S., Spencer, E. M. et al: Effect of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 complex in cavernous nerve cryoablation. Int J Impot Res, 16: 418, 2004 El-Sakka, A. I., Hassan, M. U., Selph, C., Perinchery, G., Dahiya, R. and Lue, T. F.: Effect of cavernous nerve freezing on protein and gene expression of nitric oxide synthase in the rat penis and pelvic ganglia. J Urol, 160: 2245, 1998
FK1706 ERECTILE FUNCTION RECOVERY EFFECT AFTER CAVERNOUS NERVE INJURY 8.
9.
10.
11.
12.
13.
Hsieh, P. S., Bochinski, D. J., Lin, G. T., Nunes, L., Lin, C. S. and Lue, T. F.: The effect of vascular endothelial growth factor and brain-derived neurotrophic factor on cavernosal nerve regeneration in a nerve-crush rat model. BJU Int, 92: 470, 2003 Lin, C. S., Lau, A., Tu, R. and Lue, T. F.: Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum. Biochem Biophys Res Commun, 268: 628, 2000 Lin, C. S., Lau, A., Tu, R. and Lue, T. F.: Identification of three alternative first exons and an intronic promoter of human PDE5A gene. Biochem Biophys Res Commun, 268: 596, 2000 Lin, C. S., Ho, H. C., Gholami, S., Chen, K. C., Jad, A. and Lue, T. F.: Gene expression profiling of an arteriogenic impotence model. Biochem Biophys Res Commun, 285: 565, 2001 Wang, M. S. and Gold, B. G.: FK506 increases the regeneration of spinal cord axons in a predegenerated peripheral nerve autograft. J Spinal Cord Med, 22: 287, 1999 Gold, B. G.: Neuroimmunophilin ligands: evaluation of their therapeutic potential for the treatment of neurological disorders. Expert Opin Investig Drugs, 9: 2331, 2000
14.
15.
16.
17.
18.
19.
829
Burnett, A. L. and Becker, R. E.: Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury. J Urol, 171: 495, 2004 Price, R. D., Yamaji, T., Yamamoto, H., Higashi, Y., Hanaoka, K., Yamazaki, S. et al: FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action. Eur J Pharmacol, 509: 11, 2005 Carrier, S., Zvara, P., Nunes, L., Kour, N. W., Rehman, J. and Lue, T. F.: Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J Urol, 153: 1722, 1995 Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J. et al: Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry, 31: 3896, 1992 Poulter, M. O., Payne, K. B. and Steiner, J. P.: Neuroimmunophilins: a novel drug therapy for the reversal of neurodegenerative disease? Neuroscience, 128: 1, 2004 Eberling, J. L., Pivirotto, P., Bringas, J., Steiner, J. P., Kordower, J. H., Chu, Y. et al: The immunophilin ligand GPI1046 does not have neuroregenerative effects in MPTPtreated monkeys. Exp Neurol, 178: 236, 2002