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FK506 and Erectile Function Preservation in the Cavernous Nerve Injury Model: Optimal Dosing and Timing
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ORIGINAL RESEARCH—BASIC SCIENCE FK506 and Erectile Function Preservation in the Cavernous Nerve Injury Model: Optimal Dosing and Timing John P. Mulhall, MD,*† Alexander Müller, MD,*† John F. Donohue, MD,† Dragan Golijanin, MD,† Raanan Tal, MD,† Yemi Akin-Olugbade, MD,* Keith Kobylarz, MS,* Leona Cohen-Gould, PhD,‡ Nelson E. Bennett, MD, and Peter Scardino, MD *Weill Medical College of Cornell University—Department of Urology, New York, NY, USA; †Memorial Sloan-Kettering Cancer Center—Urology, New York, NY, USA; ‡Weill Medical College of Cornell University—Core Facility for Electron Microscopy, New York, NY, USA
John P. Mulhall and Alexander Müller equally contributed to this article. DOI: 10.1111/j.1743-6109.2008.00776.x
ABSTRACT
Introduction. The immunophilin-ligand FK506 has been shown to ameliorate erectile function and preserve cavernous nerve (CN) architecture in short-term-studies using rat models of CN injury. Aim. The aim of this series was to ascertain the optimal dose and timing of FK506 administration in this animal model. Methods. Rats underwent bilateral CN crush and were treated with FK506 at different time points. There were control (C) and sham groups for each time point. Based on preliminary experiments, the CN-crush rats had no treatment (C) or either FK506 1 mg/kg (BL) or 3.2 mg/kg (BH) for 3 days prior to and the day of CN crush (PRE), on the day of and for 3 days following CN crush (POST) and for 3 days pre-, on the day of, and 3 days post-CN crush (PP). Main Outcome Measurements. All animals had measurement of intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratios at 28 days post-CN crush. Structural analysis was conducted in the POST groups. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and immunohistochemically for neural factors (growth associated protein 43 [GAP43], nerve growth factor [NGF], and neural nitric oxide synthase [nNOS]). The CN architecture was examined by transmission electron microscopy (TEM). Results. Sham animals had an ICP/MAP ratio of 70%. Only the BH-POST group revealed an improved ICP/MAP ratio compared with C (50 ⫾ 9% vs. 32 ⫾ 8%, P < 0.01). nNOS staining was significantly restored reaching sham levels in BL-POST and BH-POST groups vs. C (P < 0.05). NGF and GAP43 staining displayed no significant differences between C and treatment groups (P < 0.05). Apoptosis was significantly reduced in BL-POST and BH-POST groups compared with C (16 ⫾ 4%, 21 ⫾ 9%, and 63 ⫾ 7%, P < 0.001). TEM exhibited preservation of CN architecture for BH-POST compared with C. Conclusion. These results suggest that short-term treatment with doses of FK506 higher than previously utilized preserves erectile function in the rat CN-injury model. Pretreatment appears to offer no advantage. However, FK506 administration just prior to CN injury and for a short-time post-injury achieves the best functional and structural preservation outcomes. Mulhall JP, Müller A, Donohue JF, Golijanin D, Tal R, Akin-Olugbade Y, Kobylarz K, Cohen-Gould L, Bennett NE, and Scardino P. FK506 and erectile function preservation in the cavernous nerve injury model: Optimal dosing and timing. J Sex Med 2008;5:1334–1344. Key Words. Immunophilin Ligand; FK506; Animal Model; Cavernous Nerve Injury; Erectile Function Preservation
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FK506 and Erectile Function Preservation Introduction
rat, and furthermore to define the optimal dosing and timing regimen.
P
rostate cancer is the most common malignancy among men in Western society. In the United States alone, 200,000 men will be diagnosed with prostate cancer in 2003. Of these men, approximately 25% will undergo radical prostatectomy (RP) for definitive local therapy [1]. While this procedure is technically successful in longterm disease control, the associated morbidity secondary to urinary and erectile dysfunction (ED) may be burdensome to the patient. Prior to Walsh and Donker’s initial description of the anatomic radical retropubic prostatectomy, ED was an invariable sequela of RP [2]. Since then, numerous centers have reported potency preservation rates in excess of 75% in men undergoing bilateral nerve sparing surgery [3–5]. It is believed that neural regeneration is the mechanism by which erectile function improves over time following RP. While the degree of neural trauma that occurs intraoperatively is a determinant of long-term neural function recovery, biological factors that are involved in neural regeneration are likely important determinants of the completeness of neural recovery. Furthermore, these biological factors are likely a major reason for the interindividual variation in erectile function recuperation after this operation. Neurotrophic factors are molecular signals that promote nerve cell survival and maintain target organ function by facilitating axon regeneration [6]. A variety of nerve growth factors have been implicated in animal studies of penile nerve function [7–10]. Recently, rat models of cavernous nerve (CN) injury have been developed, which have facilitated the study of neuroprotective and neuro-regenerative agents. Immunophilins are molecules that are found in both the immune and neural tissue [11]. The immunophilin ligand FK506 has been found to prevent axonal degeneration and to preserve electrically induced penile erections in the rat [12]. Other non-immunosuppressant neuromodulators have been explored in animal models also [13]. In animal models of stroke and neurodegenerative disease, FK506 has been found to have potent neuroprotective effects. In the rat cavernous injury model, FK506 treatment has been shown to preserve erectile function and CN architecture in the immediate postoperative phase [12,14]. This series of experiments was conducted in an effort to define the impact of FK506 on erectile tissue structure and function in the CN-injured
Methods
Animal Groupings and FK506 Administration Adult male Sprague-Dawley rats weighing initially 250–300 g were used in this study, cared for and housed under strict guidelines established by the Cornell University Institutional Animal Care and Use Committee guidelines. The study was divided into two treatment phases: the first, to define an optimal dosing range (A), and the second, to assess optimal timing (B). Two reference groups were determined: sham (no CN crush, no FK506) and control (bilateral CN crush, no FK506). All animals were randomly distributed into the several groups. In phase I (A), the animals were divided into three FK506 dosing groups: (i) low-dose group (AL)—CN crush, FK506 0.1 mg/kg subcutaneously (sc) daily; (ii) intermediate dose (AM)—CN crush, FK506 0.32 mg/kg sc daily; and (iii) high dose (AH)—CN crush, FK506 1 mg/kg subcutaneously daily. The daily drug administration was commenced at the day of CN crush, right after the nerve injury and the last applications were made 24 hours before functional assessments. Within each group, three different time points were defined, and functional parameters were assessed at 3, 10, and 28 days after CN injury. A corresponding control group (C—CN crush, no treatment) was provided at each time point. At 28 days, only C and AH groups were evaluated. In phase II (B), the animals were divided into control group (C) and two treatment groups: (i) C—CN crush, no treatment; (ii) low dose (BL)—CN crush, FK506 1 mg/kg sc daily; and (iii) high dose (BH)—CN crush, FK506 3.2 mg/kg sc daily. Within each treatment group, three time subgroups were analyzed: (i) initial dose given 3 days prior to and also on the day of CN crush (four doses—PRE); (ii) initial dose given 3 days prior to CN injury, on the day of injury and for 3 days after (seven doses—PP); and (iii) initial dose given on the day of CN crush and for 3 days after CN injury (four doses—POST). In phase II (B), all animals were sacrificed at 28 days after CN crush injury. Overall, 18 groups were formed subdivided in sham group, 10 groups (C-3, -10, and -28; AL-3 and -10; AM-3 and -10; and AH-3, -10, and -28) in phase A and seven groups (C; BL-PRE, -PP, and -POST; and BH-PRE, -PP, and POST) in phase J Sex Med 2008;5:1334–1344
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B, analyzing five animals in each group for a total of 90 animals.
hyde and further processed for the transmission electron microscopy (TEM).
CN Injury For the initial surviving surgery, the animals were anesthetized using 4% isoflurane, and a lower midline incision was made. The sham animals had a laparotomy only. In all other animals, a defined bilateral CN crush injury was induced. Using a Zeiss operating microscope (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA), the major pelvic ganglion (MPG) lying on the dorsal prostate and the CN emanating from the ganglion were identified. Proceeding on both sides, 5 mm distal to the MPG, a #7 Dumont nonserrated hemostat (Fine Science Tools USA Inc, Foster City, CA, USA) was applied to the CN for 30 seconds, was removed for 30 seconds and then reapplied for a further 30 seconds [15].
Structural Assessment At this time, structural analyses have been performed only on BL-POST and BH-POST group specimens (BL-POST and BH-POST) as well as on the two reference groups including sham and control animals. Parrafin microscopy slides have been prepared from 5-mm penile tissue cuts showing the crosssection of the corpora cavernosa from the penile midshaft.
Functional Assessment In phase I (A) either at 3, 10, or 28 days and in phase II (B) at 28 days following bilateral CN crush injury, the functional parameters measuring the intracavernosal pressure (ICP) and the mean arterial blood pressure (MAP) during CN stimulation were assessed. This has been described in detail elsewhere [15]. In brief, the CN was stimulated with a hook electrode placed proximal to the site of nerve crush, using parameters that have been previously outlined (20 Hz, 35-ms delay, duration of 5 ms at 7.5 V), and using a 24-gauge intracorporal needle, the ICP was measured [15]. A carotid artery cannula monitored the systemic blood pressure so that ICP/MAP ratios could be generated. Both nerves were stimulated for 60 seconds each, and the maximal ICP generated with the corresponding MAP (ICP/MAP ratio) was recorded for each side. Tissue Harvesting At the completion of CN stimulation and ICP/ MAP ratio recording, the penis was detached and transected. Using a Zeiss operating microscope, the penis was microdissected so that all extratunical tissue was removed. The whole penis was harvested, and the midshaft segments were used for embedding in paraffin for structural analysis including apoptosis assessment and immunohistochemical staining for neuronal factors (NGF, GAP43, and nNOS). CNs were excised from a point 1 mm proximal to a point 1 mm distal to the point of crush injury initially stored in glutaraldeJ Sex Med 2008;5:1334–1344
Immunohistochemistry (IHC) Secondary antibodies were used at a dilution of 1:2,000 (Vectastain ABC Kit (Avidin Biotinylatedenzyme Complex Kit; Vector Laboratories, Burlingame, CA, USA) [Mouse IgG] cat # PK-6102, Vectastain ABC Kit [Rabbit IgG] cat # PK-6101, and Vectastain ABC Kit [Goat IgG] PK-6105). Immunohistochemical detection was carried out with Ventana Medical Systems’ (Tuscon, AZ, USA) DAB Detection Kit following the manufacturer’s instructions. Nonspecific signal was blocked with blocker D; endogenous peroxidase was inhibited with inhibitor D. All antibodies used were manufactured by Abcam Inc., Cambridge, MA, USA (NGF, ab6198; nNOS, ab1376; and GAP-43, ab11136). The IHC was conducted at the Memorial SloanKettering core facility. Two slides per group from two different animals have been stained for each antibody. During the validation phase of this project, negative controls were utilized to ensure accuracy and reproducibility of antibody staining. Immunohistochemical detection was performed with a Zeiss AxioPlan 2 microscope system (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA) at 100¥ magnification. Images of five nonoverlapping microscope fields of the corpora cavernosa of each slide were captured and stored digitally using Zeiss AxioVision computer software (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA). Image analysis was performed with ImageJ ver.1.33u (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih. gov/ij/, 1997–2004) calculating the mean stained area percentage across the tissue sections. TUNEL Assay Terminal deoxynucleotidyl transferase biotindUTP nick end labeling (TUNEL) assay was used to detect intracavernosal apoptosis. By using in
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situ Cell Death Detection Kit (Roche Applied Science; Indianapolis, IN, USA), apoptotic positive cells appear fluorescent. At least two slides (5-mm tissue cuts from the penile midshaft) representing two different animals from each group were analyzed counting five nonoverlapping intracavernosal spots using 40¥ magnification under a Zeiss Axio Microscope. The percentage of fluorescent positive cells to the total number of cells was recorded as the apoptotic index (AI).
TEM At this time, only CN tissue from sham, control, and BH-POST animals have been evaluated. The nerves were fixed in 4% glutaraldehyde in 0.5 M cacodylate buffer at 4°C for 24 hours. The tissue was fixed in 1% osmium tetroxide, then dehydrated in graded ethanols, infiltrated in graded resins, and finally embedded in fresh 100% resin. Ultrathin sections (70 nm) were cut with a diamond knife and were stained with 5% uranyl acetate and lead citrate. The resultant stained sections were viewed on a Hitachi H-600 transmission electron microscope (Hitachi High Technologies America, Inc, Schaumburg, IL, USA) run at 75 kV. Statistical Analysis Means were calculated for each group and were reported as the mean ⫾ standard deviation. Individual pairwise comparison between groups was analyzed with independent two-tailed Student’s t-tests. Results were considered statistically significant if P < 0.05. Results
All animals including the FK506-treated rats tolerated the initial surgical procedure without any infections or any other potential complications. However, the animals that were treated with more than four doses of FK506 did not gain weight over the course of the study at the same rate as those treated with only four doses or the control animals.
Erectile Hemodynamics Animals in the sham group revealed a mean ICP/ MAP ration of 70 ⫾ 6%. Compared with sham, all C groups (CN crush, no treatment) showed highly significant reduced ICP/MAP ratios of 18 ⫾ 10% at 3 days, 31 ⫾ 13% at 10 days, and 32 ⫾ 8% at 28 days (P < 0.001). In the phase I experiments (A), only the AH group receiving 1 mg/kg FK506 demonstrated with 32 ⫾ 3% significant improve-
Figure 1 Functional data after 3 days (phase A). Functional results reported as the intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratio in % as response of electrical cavernous nerve (CN) stimulation. *P < 0.02; compared with all other groups. AL = FK506 0.1 mg/kg subcutaneously (sc) daily; AM = FK506 0.32 mg/kg sc daily; AH = FK506 1.0 mg/kg sc daily, commencing at the day of CN crush.
ment in ICP/MAP ratio vs. C with 18 ⫾ 10% and only at the 3-day time point (P < 0.02). Figure 1 shows the functional results for the phase I experiments at 3 days. The daily drug administration of the different FK506 dosages demonstrated a trend toward decreasing ICP/MAP rations with increasing dosages at 10 days compared with C (C 31 ⫾ 13%, AL 30 ⫾ 17%, AM 23 ⫾ 10%, P < 0.05; AH 17 ⫾ 6%, P = 0.02 vs. C) and an unchanged ICP/MAP ratio between C and AH at 28 days (32 ⫾ 8% vs. 33 ⫾ 14%, P = 0.9). The functional results of all treatment groups remained significantly lower compared with sham (P < 0.001). Based on the improved functional results after short-term administration of four dosages of FK506 at 1 mg/kg, the second analysis (B) has been initiated aiming an optimal timing and dosing. Figure 2 gives an overview about the functional results of phase (B). In this analysis (B), only the BH-POST group receiving daily 3.2 mg/kg FK506 sc for a total of four dosages revealed with 50 ⫾ 9% significantly improved ICP/MAP ratio compared with C with 32 ⫾ 8% at 28 days after CN injury (P < 0.001). However, BH-POST remained significantly reduced compared with sham with 70 ⫾ 6% (P < 0.01). Within the BH group, the ICP/MAP ration of the BH-POST group was also significantly better compared with BH-PP with 34 ⫾ 6% (P < 0.001). Pretreated animals receiving the higher dosage BH-PRE displayed with 41 ⫾ 11% an improved trend compared with C (P = 0.07). Within the BL group receiving a lower dose of 1 mg/kg FK506, only the BL-POST animals expressed with 43 ⫾ 13% a tendency J Sex Med 2008;5:1334–1344
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Figure 2 Functional data after 28 days (phase B). Reporting mean intracavernosal pressure/mean arterial blood pressure (ICP/MAP) rations in % recorded during electrical cavernous nerve stimulation. *P < 0.001; sham vs. all other groups. #P < 0.001; BH-POST vs. all other groups, except sham, BL-POST, and BH-PRE.
toward improved ICP/MAP values compared with C (P = 0.08).
IHC Table 1 gives an overview about the staining results for the antibodies GAP43, NGF, and nNOS, and Figures 3–5 correspond to representative staining pictures for the individual groups (sham, control, BL-POST, and BH-POST). Staining for GAP43 showed a significant lower pattern for sham compared with C and BL-POST (P < 0.05) (Figure 3). Antibody staining for NGF was lowest in sham and significantly different compared with all other groups (P < 0.05). For NGF Table 1 Immunohistochemistry results for GAP43, NGF, and nNOS Mean ⫾ SD
Range
95% CI
1.2 ⫾ 0.8* 2 ⫾ 0.6 2 ⫾ 0.7 1.9 ⫾ 0.8
0.2–2.4 1.2–3.4 1.1–3.6 0.6–3.5
0.7–1.7 1.7–2.3 1.6–2.4 1.4–2.4
NGF
Mean ⫾ SD
Range
95% CI
Sham Control BL-POST BH-POST
1.9 ⫾ 1.7 17.2 ⫾ 10.2‡ 9.7 ⫾ 4.4 6.7 ⫾ 3.0
0.2–4.8 6.0–28.8 5.9–18.2 3.5–11.9
0.8–2.9 8.3–26.1 7.0–12.4 4.9–8.6
nNOS
Mean ⫾ SD
Range
95% CI
3.5–20.2 1.1–5.8 5.8–14.8 6.7–11.9
6.4–11.4 2.2–3.6 5.6–14.1 6.7–10.2
GAP43 Sham Control BL-POST BH-POST
Sham Control BL-POST BH-POST
†
8.9 ⫾ 5.0 2.9 ⫾ 1.4§ 9.8 ⫾ 4.3 8.4 ⫾ 2.0
*P < 0.05; sham showed a significantly reduced GAP43 staining compared with control and BL-POST. † P < 0.05; sham revealed a significant lower NGF staining compared with all other groups. ‡P < 0.05; the control demonstrated a significantly higher NGF staining compared with sham. §P < 0.05; the control showed a significantly lower nNOS staining compared with all other groups. SD = standard deviation, CI = confidence interval.
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staining, the C group displayed the highest staining, which was not statistically different compared with the results for BL-POST and BH-POST (P < 0.05) (Figure 4). Staining for nNOS demonstrated significantly lower values for the C group after untreated CN injury compared with the sham group (P < 0.001) (Figure 5). Both treatment groups BL-POST and BH-POST demonstrated significant restoration of staining pattern, approximating sham levels (P < 0.05).
Apoptosis Assessment Presented in Figures 6 and 7, the TUNEL results exhibited a highly significant increased AI in C (63 ⫾ 7%) compared with sham (11 ⫾ 5%) (P < 0.001). Both treatment groups BL-POST (16 ⫾ 4%) and BH-POST (21 ⫾ 9%) presented a highly significant prevention of cell death compared with control (P < 0.001). BL-POST reached an almost sham level (P = 0.2), but the AI for BH-POST remained statistically higher compared with sham (P = 0.03). TEM The TEM pictures (Figure 8) of the dissected CN of the control showed a reduced number of myelinized nerve fibers compared with sham. In the BH-POST group, an architectural restoration was documented, represented in higher amount of myelinized nerve fibers. Discussion
Despite the advent of nerve-sparing techniques during RP, a significant percentage of men continue to suffer from erectile impairment following this operation. It is estimated that approximately
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Figure 3 GAP43 staining. Representative photomicrographs show GAP43 antibody staining of cavernosal tissue for the sham, control, BLPOST, and BH-POST groups (100¥ magnification).
50,000 men undergo RP in the United States alone each year. ED is a common event after this operation, and the major predictors of erectile function recovery include patient age, preoperative erectile function, erectile hemodynamics, erectile tissue integrity, and nerve-sparing status [4]. The ability to protect the CN architecture and function using pharmacotherapy would thus revolutionize the treatment algorithm for such patients. Immunophilins belong to a family of intracellular proteins called chaperone molecules. These molecules bind and usher messenger molecules that regulate cellular functions. Immunophilins are cytosolic protein receptors for immunosuppressant
drugs like cyclosporin A, rapamycin, and FK506, and these chaperone molecules are often named after the drugs that bind them, e.g. FK-binding proteins (FK-BP12, FK-BP52). These intracellular complexes regulate their physiological role by regulating intracellular Ca2+ efflux controlled by calcineurin. The discovery that immunophilins are 50 times more abundant in brain and nervous system than in the immune system led to research that revealed the important role of immunophilins in neural function [13]. Given the important role of binding proteins in the regulation of cell activity, it is not unexpected that many immunophilin ligands, including FK506, have neuroprotective and neuroregenerative effects [16,17].
Figure 4 NGF staining. Representative photomicrographs show NGF staining of cavernosal tissue for the sham, control, BL-POST, and BHPOST groups (100¥ magnification).
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Figure 5 nNOS staining. Representative photomicrographs show nNOS antibody staining of cavernosal tissue for the sham, control, BLPOST, and BH-POST groups (100¥ magnification).
Figure 6 Apoptotic index (AI). The histogram displays the average AI in % expressing the ration build by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling positive cells divided through the total amount of cells. *P < 0.001; control vs. all other groups. #P = 0.03; sham vs. BH-POST.
Lyons and Steiner showed striking neurotrophic effects of FK506 (also known as tacrolimus, Prograf, Astellas Pharamceuticals, Deerfield, IL, USA) and Rapamycin in PC12 cells and sensory ganglia at sub-nanomolar concentrations [16]. In intact animals, Gold et al. reported enhanced regrowth and functional recovery of crushed sciatic nerves following FK506 treatment [17], and FK506 increased the regeneration of spinal cord axons in a pre-degenerated peripheral nerve autograft [18]. While cross-sectional area of the nerve is augmented by drug treatment, even more pronounced effects are noted in recovery of myelination [19,20] Verkade et al. showed, using
Figure 7 Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. Representative photomicrographs show TUNEL assay for the sham, control, BL-POST, and BH-POST groups. Apoptotic nuclei are fluorescent green compared with blue staining normal cell nuclei (40¥ magnification).
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Figure 8 Transmission electron microscopy (TEM). Cavernous nerve cross-section at the area of the crush injury under the TEM. Reduced number of myelinated nerve fibers in the control compared with sham. BH-POST treatment revealed an architectural restoration compared with the control.
IHC, an increased density of GAP-43 (neuronal growth-associated protein) in regenerating axons comparing with intact myelinated and unmyelinated axon shafts in a model of rat sciatic nerve crush [19]. Similarly, in another study, regenerated axons after rat dorsal root crush injury showed an increased expression of GAP-43 [20]. Our results also showed an increased staining expression for GAP43 in the intracavernosal tissue after FK506 treatment following CN crush compared with sham animals. However, the staining results for both treatment groups were practically the same compared with the control group after CN crush injury without FK506. The therapeutic potential of these neurotrophic drugs is considerable, with multiple targets including injury to CN during radical pelvic surgery. Sezen et al. reported on successful treatment of partial nerve injury in a rat CN model using FK506 [12]. FK506 (1 mg/kg intraperitoneally) preserved erectile function if administered simultaneous to nerve crush, and neuroprotection was documented histologically using optical and electron microscopy, and functionally assessing ICP after CN stimulation, as early as the first day after the treatment. When a longer crush (60 seconds repeated two times) was applied, FK506 (1 mg/kg intraperitoneally) demonstrated a protective effect at 24 and 36 hours compared with normal salinetreated animals, which disappeared 7 days after the surgery.
A more recent study examined the effect of FK506 and GPI-1046 on rats that had undergone CN injury [14]. This study found that animals treated with either FK506 or GPI-1046 had significantly greater recovery of erectile function compared with animals treated with saline. Electrically stimulated maximal ICP and rate of tumescence were better in the treated animals compared with controls. In this study, FK506 appeared to produce better functional results than GPI-1046. These preliminary results suggest an emerging role for direct pharmacologic neuromodulation after CN injury. Currently randomized, placebocontrolled trials assessing GPI-1485 and FK506 have completed enrollment, and data should be available in the near future. In the validation phase of the cavernous injury model [15] and in other experiments, we have shown in the untreated animals that erectile function initially decreases and continues to improve up to the 10-day time point with further improvement out to 28 days. The first phase of our study only found benefit with short-term dosing of 3 days at 1 mg/kg. All other doses (0.1 and 0.32 mg/ kg) did not show any advantage regardless of dose duration. Interestingly, when animals were treated for longer than four doses, they did not gain weight over the course of the study at the same rate as the control animals. It is interesting to note from the Burnett article that toxicity and mortality occurred at dosing schedules of 5 mg/kg for more J Sex Med 2008;5:1334–1344
1342 than a 5-day duration [14]. In the original study from the Burnett group, reported by Sezen et al., the dose of FK506 used was 1 mg/kg, and this was given intraperitoneally [12]. The method of nerve injury was three 15-second crushes of one of the nerves only with the other side used as an internal control. This has implications as it has been shown that unilateral nerve injury causes significant penile morphological changes, and thus the ICP measured following stimulation of the assumed normal nerve may not be maximal because of penile hemodynamic alterations resulting from erectile tissue damage [12,14]. Interestingly, when a more severe injury consisting of two 60-second crushes was applied to the nerve, 1 mg/kg given for 7 days showed no advantage over saline [12]. In the second phase of our study, we examined higher doses given for short periods and whether pretreatment had a role in ameliorating ED following CN crush, as has been suggested for sildenafil [21]. We found that the 1 mg/kg dose resulted in a nonstatistically significant improvement in erectile function when given at the time of injury and for 3 days following injury (BL-POST) with assessment at 28 days. There was no advantage to pretreating or with a combination of both (seven doses). When a higher dose of 3.2 mg/kg was given again following surgery (BH-POST), there was a significant improvement in the ICP/MAP ratio compared with C (P < 0.001). Pretreatment showed a nonsignificant improvement, which may be because of the fact that the last dose was given at the time of the nerve crush and this dose alone may have been sufficient to impart some benefit. Similarly, the combination of pre- and post-treatment (BH-PP) did not demonstrate any difference compared with control, and this may be because of the relatively high dosing over the 7 days, as previously described by Burnett and Becker [14]. Given that the ICP/MAP ratio improvements were most dramatic in the B-POST groups at 28 days, at this time, these groups are the only groups to have undergone detailed structural analyses. TEM (Figure 8) clearly demonstrated the disruption of CN architecture with nerve crush, and the restoration of axon and myelin density in the BH-POST group. This confirms Sezen et al.’s previous findings, although their assessment was for only 7 days compared with 28 days in our study [12]. Assessing IHC for neurotrophic factors, the administration of FK506 at low and high doses, the day of crush and for 3 days afterwards resulted in significant ameliorations in nNOS staining (Table 1). GAP-43 expression was increased miniJ Sex Med 2008;5:1334–1344
Mulhall et al. mally but statistically significantly in the control and in the BL-POST group compared with sham. Nerve crush resulted in a significant increase in NGF staining in the control group, which showed a clear trend toward lower results (although not to sham levels) in the BL-POST and BH-POST groups (P < 0.01). nNOS staining was significantly reduced by nerve crush, supporting the previously described changes identified by Carrier et al. [22], but normalized in the BL-POST and BH-POST groups approximating sham levels. NGF appears to be critical for the survival and maintenance of neurons (neuroprotection), while GAP43 is associated more with nerve growth (neuro-regeneration). Immunocytochemical staining showed that NGF treatment of adult visual cortex selectively increased the level of the phosphorylated form of GAP43, while the total level of GAP43 was not changed [23]. In the future, it may be worth paying closer attention to the phosphorylated form of GAP43. It is possible that the predominant effect of FK506, as dosed in this series of experiments, may be more neuroprotective rather than neuro-regenerative. After bilateral CN crush injury in our rat model, the administration of FK506 was shown to prevent apoptosis in both dosing groups compared with the control animals (P < 0.001). Findings in the literature support our observation of an antiapoptotic effect with FK506 application. Data collected in a rat optic nerve crush model report on a neuroprotection preventing retinal ganglion cells from dying using FK506 administration, which seems to be mediated by interfering with the activation of caspase 9 [24]. In a brain ischemia model in rats, where a 60-minute occlusion of the middle cerebral artery is induced, the administration of FK506 (1 mg/kg) immediately after occlusion ameliorated the degree of cerebral damage by inhibiting both apoptotic and necrotic cell death pathways [25]. Other studies in the literature have reported on the potential benefit of immunophilin ligands, including FK506 and FK1706, in modulating and preserving erectile function after CN injury [6,11– 14,26]. However, the novelty of our series of experiments lies in the fact that we provide new data about optimal dosing and timing of FK506 administration in this model. Conclusions
FK506 reduces the degree of ED following CN injury in the rat model. However, the response is
FK506 and Erectile Function Preservation not dose-dependent, and short-term administration of high-dose FK506 appears to give the optimal results, and pretreatment offers no advantage. The benefits of this pharmacotherapeutic strategy appear to be mediated through reduction in apoptosis and correction of nerve injuryassociated perturbations in neurotrophic factor expression, which may be the reason for the dramatic structural preservation seen in the treatment animals. The future role of FK506 as a pharmacologic neuromodulator in the RP population will be defined by the results of the randomized, placebocontrolled trial, which is ongoing. Corresponding Author: John P. Mulhall, MD, Associate Professor, Director, Sexual Medicine Programs, Departments of Urology, Weill Medical College of Cornell University, and Memorial Sloan Kettering Cancer Center, New York, NY, USA. Tel: 212-7460097 (personal line); 212-746-5653 (assistant); Fax: 212746-0403; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design John P. Mulhall; Dragan Golijanin (b) Acquisition of Data John P. Mulhall; Alexander Müller; John F. Donohue; Yemi Akin-Olugbade; Keith Kobylarz; Nelson E. Bennett (c) Analysis and Interpretation of Data Keith Kobylarz; John P. Mulhall; Alexander Müller; Peter Scardino; Leona Cohen-Gould
Category 2 (a) Drafting the Article John P. Mulhall; Alexander Müller (b) Revising It for Intellectual Content John P. Mulhall; Alexander Müller; Peter Scardino
Category 3 (a) Final Approval of the Completed Article John P. Mulhall; Alexander Müller
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1343 3 Catalona WJ, Carvalhal GF, Mager DE, Smith DS. Potency, continence and complication rates in 1,870 consecutive radical retropubic prostatectomies. J Urol 1999;162:433–8. 4 Rabbani F, Stapleton AM, Kattan MW, Wheeler TM, Scardino PT. Factors predicting recovery of erections after radical prostatectomy. J Urol 2000; 164:1929–34. 5 Walsh PC, Marschke P, Ricker D, Burnett AL. Patient-reported urinary continence and sexual function after anatomic radical prostatectomy. Urology 2000;55:58–61. 6 Burnett AL. Neuroprotection and nerve grafts in the treatment of neurogenic erectile dysfunction. J Urol 2003;170:S31–4. 7 Burgers JK, Nelson RJ, Quinlan DM, Walsh PC. Nerve growth factor, nerve grafts and amniotic membrane grafts restore erectile function in rats. J Urol 1991;146:463–8. 8 Ball RA, Lipton SA, Dreyer EB, Richie JP, Vickers MA. Entubulization repair of severed cavernous nerves in the rat resulting in return of erectile function. J Urol 1992;148:211–5. 9 Te AE, Santarosa RP, Koo HP, Buttyan R, Greene LA, Kaplan SA, Olsson CA, Shabsigh R. Neurotrophic factors in the rat penis. J Urol 1994;152: 2167–72. 10 Dahiya R, Chui R, Perinchery G, Nakajima K, Oh BR, Lue TF. Differential gene expression of growth factors in young and old rat penile tissues is associated with erectile dysfunction. Int J Impot Res 1999;11:201–6. 11 Sezen SF, Hoke A, Burnett AL, Snyder SH. Immunophilin ligand FK506 is neuroprotective for penile innervation. Nat Med 2001;7:1073–4. 12 Sezen SF, Blackshaw S, Steiner JP, Burnett AL. FK506 binding protein 12 is expressed in rat penile innervation and upregulated after cavernous nerve injury. Int J Impot Res 2002;14:506–12. 13 Snyder SH, Sabatini DM, Lai MM, Steiner JP, Hamilton GS, Suzdak PD. Neural actions of immunophilin ligands. Trends Pharmacol Sci 1998;19: 21–6. 14 Burnett AL, Becker RE. Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury. J Urol 2004;171:495– 500. 15 Mullerad M, Donohue JF, Li PS, Scardino PT, Mulhall JP. Functional sequelae of cavernous nerve injury in the rat: Is there model dependency. J Sex Med 2006;3:77–83. 16 Lyons WE, Steiner JP, Snyder SH, Dawson TM. Neuronal regeneration enhances the expression of the immunophilin FKBP-12. J Neurosci 1995;15: 2985–94. 17 Gold BG, Katoh K, Storm-Dickerson T. The immunosuppressant FK506 increases the rate of axonal regeneration in rat sciatic nerve. J Neurosci 1995;15:7509–16. J Sex Med 2008;5:1334–1344
1344 18 Wang MS, Gold BG. FK506 increases the regeneration of spinal cord axons in a predegenerated peripheral nerve autograft. J Spinal Cord Med 1999; 22:287–96. 19 Verkade P, Schrama LH, Verkleij AJ, Gispen WH, Oestreicher AB. Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve. Neuroscience 1997;79:1207– 18. 20 Verkade P, Oestreicher AB, Verkleij AJ, Gispen WH. The increase in B-50/GAP-43 in regenerating rat sciatic nerve occurs predominantly in unmyelinated axon shafts: A quantitative ultrastructural study. J Comp Neurol 1995;356:433–43. 21 Donohue JF, Tal R, Akin-Olugbade Y, Mullerad M, Muller A, Bennett N, Kobylarz K, Paduch DA, Scardino PT, Mulhall JP. Sildenafil and Cavernous Nerve Injury in the Rat: Defining the Optimal Dosing and Timing Regimen. AUA Atlanta 2006, Abstract #993, p. 1205.
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Mulhall et al. 22 Carrier S, Zvara P, Nunes L, Kour NW, Rehman J, Lue TF. Regeneration of nitric oxide synthasecontaining nerves after cavernous nerve neurotomy in the rat. J Urol 1995;153:1722–7. 23 Liu Y, Meiri KF, Cynader MS, Gu Q. Nerve growth factor induced modification of presynaptic elements in adult visual cortex in vivo. Brain Res 1996; 732:36–42. 24 Grosskreutz CL, Hanninen VA, Pantcheva MB, Huang W, Poulin NR, Dobbberfuhl AP. FK506 blocks activation of the intrinsic caspase cascade after optic nerve crush. Exp Eye Res 2005;80:681–6. 25 Furuichi Y, Noto T, Li JY, Oku T, Ishiye M, Moriguchi A, Armaori I, Matsuoka N, Mutoh S, Yanagihara T. Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats. Brain Res 2004;1014:120–30. 26 Bella AJ, Hayashi N, Carrione RE, Price R, Lue TF. FK1706 enhances the recovery of erectile function following bilateral cavernous nerve crush injury in the rat. J Sex Med 2007;4:341–6; discussion 346–7.
ORIGINAL RESEARCH—BASIC SCIENCE FK506 and Erectile Function Preservation in the Cavernous Nerve Injury Model: Optimal Dosing and Timing John P. Mulhall, MD,*† Alexander Müller, MD,*† John F. Donohue, MD,† Dragan Golijanin, MD,† Raanan Tal, MD,† Yemi Akin-Olugbade, MD,* Keith Kobylarz, MS,* Leona Cohen-Gould, PhD,‡ Nelson E. Bennett, MD, and Peter Scardino, MD *Weill Medical College of Cornell University—Department of Urology, New York, NY, USA; †Memorial Sloan-Kettering Cancer Center—Urology, New York, NY, USA; ‡Weill Medical College of Cornell University—Core Facility for Electron Microscopy, New York, NY, USA
John P. Mulhall and Alexander Müller equally contributed to this article. DOI: 10.1111/j.1743-6109.2008.00776.x
ABSTRACT
Introduction. The immunophilin-ligand FK506 has been shown to ameliorate erectile function and preserve cavernous nerve (CN) architecture in short-term-studies using rat models of CN injury. Aim. The aim of this series was to ascertain the optimal dose and timing of FK506 administration in this animal model. Methods. Rats underwent bilateral CN crush and were treated with FK506 at different time points. There were control (C) and sham groups for each time point. Based on preliminary experiments, the CN-crush rats had no treatment (C) or either FK506 1 mg/kg (BL) or 3.2 mg/kg (BH) for 3 days prior to and the day of CN crush (PRE), on the day of and for 3 days following CN crush (POST) and for 3 days pre-, on the day of, and 3 days post-CN crush (PP). Main Outcome Measurements. All animals had measurement of intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratios at 28 days post-CN crush. Structural analysis was conducted in the POST groups. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and immunohistochemically for neural factors (growth associated protein 43 [GAP43], nerve growth factor [NGF], and neural nitric oxide synthase [nNOS]). The CN architecture was examined by transmission electron microscopy (TEM). Results. Sham animals had an ICP/MAP ratio of 70%. Only the BH-POST group revealed an improved ICP/MAP ratio compared with C (50 ⫾ 9% vs. 32 ⫾ 8%, P < 0.01). nNOS staining was significantly restored reaching sham levels in BL-POST and BH-POST groups vs. C (P < 0.05). NGF and GAP43 staining displayed no significant differences between C and treatment groups (P < 0.05). Apoptosis was significantly reduced in BL-POST and BH-POST groups compared with C (16 ⫾ 4%, 21 ⫾ 9%, and 63 ⫾ 7%, P < 0.001). TEM exhibited preservation of CN architecture for BH-POST compared with C. Conclusion. These results suggest that short-term treatment with doses of FK506 higher than previously utilized preserves erectile function in the rat CN-injury model. Pretreatment appears to offer no advantage. However, FK506 administration just prior to CN injury and for a short-time post-injury achieves the best functional and structural preservation outcomes. Mulhall JP, Müller A, Donohue JF, Golijanin D, Tal R, Akin-Olugbade Y, Kobylarz K, Cohen-Gould L, Bennett NE, and Scardino P. FK506 and erectile function preservation in the cavernous nerve injury model: Optimal dosing and timing. J Sex Med 2008;5:1334–1344. Key Words. Immunophilin Ligand; FK506; Animal Model; Cavernous Nerve Injury; Erectile Function Preservation
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FK506 and Erectile Function Preservation Introduction
rat, and furthermore to define the optimal dosing and timing regimen.
P
rostate cancer is the most common malignancy among men in Western society. In the United States alone, 200,000 men will be diagnosed with prostate cancer in 2003. Of these men, approximately 25% will undergo radical prostatectomy (RP) for definitive local therapy [1]. While this procedure is technically successful in longterm disease control, the associated morbidity secondary to urinary and erectile dysfunction (ED) may be burdensome to the patient. Prior to Walsh and Donker’s initial description of the anatomic radical retropubic prostatectomy, ED was an invariable sequela of RP [2]. Since then, numerous centers have reported potency preservation rates in excess of 75% in men undergoing bilateral nerve sparing surgery [3–5]. It is believed that neural regeneration is the mechanism by which erectile function improves over time following RP. While the degree of neural trauma that occurs intraoperatively is a determinant of long-term neural function recovery, biological factors that are involved in neural regeneration are likely important determinants of the completeness of neural recovery. Furthermore, these biological factors are likely a major reason for the interindividual variation in erectile function recuperation after this operation. Neurotrophic factors are molecular signals that promote nerve cell survival and maintain target organ function by facilitating axon regeneration [6]. A variety of nerve growth factors have been implicated in animal studies of penile nerve function [7–10]. Recently, rat models of cavernous nerve (CN) injury have been developed, which have facilitated the study of neuroprotective and neuro-regenerative agents. Immunophilins are molecules that are found in both the immune and neural tissue [11]. The immunophilin ligand FK506 has been found to prevent axonal degeneration and to preserve electrically induced penile erections in the rat [12]. Other non-immunosuppressant neuromodulators have been explored in animal models also [13]. In animal models of stroke and neurodegenerative disease, FK506 has been found to have potent neuroprotective effects. In the rat cavernous injury model, FK506 treatment has been shown to preserve erectile function and CN architecture in the immediate postoperative phase [12,14]. This series of experiments was conducted in an effort to define the impact of FK506 on erectile tissue structure and function in the CN-injured
Methods
Animal Groupings and FK506 Administration Adult male Sprague-Dawley rats weighing initially 250–300 g were used in this study, cared for and housed under strict guidelines established by the Cornell University Institutional Animal Care and Use Committee guidelines. The study was divided into two treatment phases: the first, to define an optimal dosing range (A), and the second, to assess optimal timing (B). Two reference groups were determined: sham (no CN crush, no FK506) and control (bilateral CN crush, no FK506). All animals were randomly distributed into the several groups. In phase I (A), the animals were divided into three FK506 dosing groups: (i) low-dose group (AL)—CN crush, FK506 0.1 mg/kg subcutaneously (sc) daily; (ii) intermediate dose (AM)—CN crush, FK506 0.32 mg/kg sc daily; and (iii) high dose (AH)—CN crush, FK506 1 mg/kg subcutaneously daily. The daily drug administration was commenced at the day of CN crush, right after the nerve injury and the last applications were made 24 hours before functional assessments. Within each group, three different time points were defined, and functional parameters were assessed at 3, 10, and 28 days after CN injury. A corresponding control group (C—CN crush, no treatment) was provided at each time point. At 28 days, only C and AH groups were evaluated. In phase II (B), the animals were divided into control group (C) and two treatment groups: (i) C—CN crush, no treatment; (ii) low dose (BL)—CN crush, FK506 1 mg/kg sc daily; and (iii) high dose (BH)—CN crush, FK506 3.2 mg/kg sc daily. Within each treatment group, three time subgroups were analyzed: (i) initial dose given 3 days prior to and also on the day of CN crush (four doses—PRE); (ii) initial dose given 3 days prior to CN injury, on the day of injury and for 3 days after (seven doses—PP); and (iii) initial dose given on the day of CN crush and for 3 days after CN injury (four doses—POST). In phase II (B), all animals were sacrificed at 28 days after CN crush injury. Overall, 18 groups were formed subdivided in sham group, 10 groups (C-3, -10, and -28; AL-3 and -10; AM-3 and -10; and AH-3, -10, and -28) in phase A and seven groups (C; BL-PRE, -PP, and -POST; and BH-PRE, -PP, and POST) in phase J Sex Med 2008;5:1334–1344
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B, analyzing five animals in each group for a total of 90 animals.
hyde and further processed for the transmission electron microscopy (TEM).
CN Injury For the initial surviving surgery, the animals were anesthetized using 4% isoflurane, and a lower midline incision was made. The sham animals had a laparotomy only. In all other animals, a defined bilateral CN crush injury was induced. Using a Zeiss operating microscope (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA), the major pelvic ganglion (MPG) lying on the dorsal prostate and the CN emanating from the ganglion were identified. Proceeding on both sides, 5 mm distal to the MPG, a #7 Dumont nonserrated hemostat (Fine Science Tools USA Inc, Foster City, CA, USA) was applied to the CN for 30 seconds, was removed for 30 seconds and then reapplied for a further 30 seconds [15].
Structural Assessment At this time, structural analyses have been performed only on BL-POST and BH-POST group specimens (BL-POST and BH-POST) as well as on the two reference groups including sham and control animals. Parrafin microscopy slides have been prepared from 5-mm penile tissue cuts showing the crosssection of the corpora cavernosa from the penile midshaft.
Functional Assessment In phase I (A) either at 3, 10, or 28 days and in phase II (B) at 28 days following bilateral CN crush injury, the functional parameters measuring the intracavernosal pressure (ICP) and the mean arterial blood pressure (MAP) during CN stimulation were assessed. This has been described in detail elsewhere [15]. In brief, the CN was stimulated with a hook electrode placed proximal to the site of nerve crush, using parameters that have been previously outlined (20 Hz, 35-ms delay, duration of 5 ms at 7.5 V), and using a 24-gauge intracorporal needle, the ICP was measured [15]. A carotid artery cannula monitored the systemic blood pressure so that ICP/MAP ratios could be generated. Both nerves were stimulated for 60 seconds each, and the maximal ICP generated with the corresponding MAP (ICP/MAP ratio) was recorded for each side. Tissue Harvesting At the completion of CN stimulation and ICP/ MAP ratio recording, the penis was detached and transected. Using a Zeiss operating microscope, the penis was microdissected so that all extratunical tissue was removed. The whole penis was harvested, and the midshaft segments were used for embedding in paraffin for structural analysis including apoptosis assessment and immunohistochemical staining for neuronal factors (NGF, GAP43, and nNOS). CNs were excised from a point 1 mm proximal to a point 1 mm distal to the point of crush injury initially stored in glutaraldeJ Sex Med 2008;5:1334–1344
Immunohistochemistry (IHC) Secondary antibodies were used at a dilution of 1:2,000 (Vectastain ABC Kit (Avidin Biotinylatedenzyme Complex Kit; Vector Laboratories, Burlingame, CA, USA) [Mouse IgG] cat # PK-6102, Vectastain ABC Kit [Rabbit IgG] cat # PK-6101, and Vectastain ABC Kit [Goat IgG] PK-6105). Immunohistochemical detection was carried out with Ventana Medical Systems’ (Tuscon, AZ, USA) DAB Detection Kit following the manufacturer’s instructions. Nonspecific signal was blocked with blocker D; endogenous peroxidase was inhibited with inhibitor D. All antibodies used were manufactured by Abcam Inc., Cambridge, MA, USA (NGF, ab6198; nNOS, ab1376; and GAP-43, ab11136). The IHC was conducted at the Memorial SloanKettering core facility. Two slides per group from two different animals have been stained for each antibody. During the validation phase of this project, negative controls were utilized to ensure accuracy and reproducibility of antibody staining. Immunohistochemical detection was performed with a Zeiss AxioPlan 2 microscope system (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA) at 100¥ magnification. Images of five nonoverlapping microscope fields of the corpora cavernosa of each slide were captured and stored digitally using Zeiss AxioVision computer software (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA). Image analysis was performed with ImageJ ver.1.33u (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih. gov/ij/, 1997–2004) calculating the mean stained area percentage across the tissue sections. TUNEL Assay Terminal deoxynucleotidyl transferase biotindUTP nick end labeling (TUNEL) assay was used to detect intracavernosal apoptosis. By using in
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situ Cell Death Detection Kit (Roche Applied Science; Indianapolis, IN, USA), apoptotic positive cells appear fluorescent. At least two slides (5-mm tissue cuts from the penile midshaft) representing two different animals from each group were analyzed counting five nonoverlapping intracavernosal spots using 40¥ magnification under a Zeiss Axio Microscope. The percentage of fluorescent positive cells to the total number of cells was recorded as the apoptotic index (AI).
TEM At this time, only CN tissue from sham, control, and BH-POST animals have been evaluated. The nerves were fixed in 4% glutaraldehyde in 0.5 M cacodylate buffer at 4°C for 24 hours. The tissue was fixed in 1% osmium tetroxide, then dehydrated in graded ethanols, infiltrated in graded resins, and finally embedded in fresh 100% resin. Ultrathin sections (70 nm) were cut with a diamond knife and were stained with 5% uranyl acetate and lead citrate. The resultant stained sections were viewed on a Hitachi H-600 transmission electron microscope (Hitachi High Technologies America, Inc, Schaumburg, IL, USA) run at 75 kV. Statistical Analysis Means were calculated for each group and were reported as the mean ⫾ standard deviation. Individual pairwise comparison between groups was analyzed with independent two-tailed Student’s t-tests. Results were considered statistically significant if P < 0.05. Results
All animals including the FK506-treated rats tolerated the initial surgical procedure without any infections or any other potential complications. However, the animals that were treated with more than four doses of FK506 did not gain weight over the course of the study at the same rate as those treated with only four doses or the control animals.
Erectile Hemodynamics Animals in the sham group revealed a mean ICP/ MAP ration of 70 ⫾ 6%. Compared with sham, all C groups (CN crush, no treatment) showed highly significant reduced ICP/MAP ratios of 18 ⫾ 10% at 3 days, 31 ⫾ 13% at 10 days, and 32 ⫾ 8% at 28 days (P < 0.001). In the phase I experiments (A), only the AH group receiving 1 mg/kg FK506 demonstrated with 32 ⫾ 3% significant improve-
Figure 1 Functional data after 3 days (phase A). Functional results reported as the intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratio in % as response of electrical cavernous nerve (CN) stimulation. *P < 0.02; compared with all other groups. AL = FK506 0.1 mg/kg subcutaneously (sc) daily; AM = FK506 0.32 mg/kg sc daily; AH = FK506 1.0 mg/kg sc daily, commencing at the day of CN crush.
ment in ICP/MAP ratio vs. C with 18 ⫾ 10% and only at the 3-day time point (P < 0.02). Figure 1 shows the functional results for the phase I experiments at 3 days. The daily drug administration of the different FK506 dosages demonstrated a trend toward decreasing ICP/MAP rations with increasing dosages at 10 days compared with C (C 31 ⫾ 13%, AL 30 ⫾ 17%, AM 23 ⫾ 10%, P < 0.05; AH 17 ⫾ 6%, P = 0.02 vs. C) and an unchanged ICP/MAP ratio between C and AH at 28 days (32 ⫾ 8% vs. 33 ⫾ 14%, P = 0.9). The functional results of all treatment groups remained significantly lower compared with sham (P < 0.001). Based on the improved functional results after short-term administration of four dosages of FK506 at 1 mg/kg, the second analysis (B) has been initiated aiming an optimal timing and dosing. Figure 2 gives an overview about the functional results of phase (B). In this analysis (B), only the BH-POST group receiving daily 3.2 mg/kg FK506 sc for a total of four dosages revealed with 50 ⫾ 9% significantly improved ICP/MAP ratio compared with C with 32 ⫾ 8% at 28 days after CN injury (P < 0.001). However, BH-POST remained significantly reduced compared with sham with 70 ⫾ 6% (P < 0.01). Within the BH group, the ICP/MAP ration of the BH-POST group was also significantly better compared with BH-PP with 34 ⫾ 6% (P < 0.001). Pretreated animals receiving the higher dosage BH-PRE displayed with 41 ⫾ 11% an improved trend compared with C (P = 0.07). Within the BL group receiving a lower dose of 1 mg/kg FK506, only the BL-POST animals expressed with 43 ⫾ 13% a tendency J Sex Med 2008;5:1334–1344
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Figure 2 Functional data after 28 days (phase B). Reporting mean intracavernosal pressure/mean arterial blood pressure (ICP/MAP) rations in % recorded during electrical cavernous nerve stimulation. *P < 0.001; sham vs. all other groups. #P < 0.001; BH-POST vs. all other groups, except sham, BL-POST, and BH-PRE.
toward improved ICP/MAP values compared with C (P = 0.08).
IHC Table 1 gives an overview about the staining results for the antibodies GAP43, NGF, and nNOS, and Figures 3–5 correspond to representative staining pictures for the individual groups (sham, control, BL-POST, and BH-POST). Staining for GAP43 showed a significant lower pattern for sham compared with C and BL-POST (P < 0.05) (Figure 3). Antibody staining for NGF was lowest in sham and significantly different compared with all other groups (P < 0.05). For NGF Table 1 Immunohistochemistry results for GAP43, NGF, and nNOS Mean ⫾ SD
Range
95% CI
1.2 ⫾ 0.8* 2 ⫾ 0.6 2 ⫾ 0.7 1.9 ⫾ 0.8
0.2–2.4 1.2–3.4 1.1–3.6 0.6–3.5
0.7–1.7 1.7–2.3 1.6–2.4 1.4–2.4
NGF
Mean ⫾ SD
Range
95% CI
Sham Control BL-POST BH-POST
1.9 ⫾ 1.7 17.2 ⫾ 10.2‡ 9.7 ⫾ 4.4 6.7 ⫾ 3.0
0.2–4.8 6.0–28.8 5.9–18.2 3.5–11.9
0.8–2.9 8.3–26.1 7.0–12.4 4.9–8.6
nNOS
Mean ⫾ SD
Range
95% CI
3.5–20.2 1.1–5.8 5.8–14.8 6.7–11.9
6.4–11.4 2.2–3.6 5.6–14.1 6.7–10.2
GAP43 Sham Control BL-POST BH-POST
Sham Control BL-POST BH-POST
†
8.9 ⫾ 5.0 2.9 ⫾ 1.4§ 9.8 ⫾ 4.3 8.4 ⫾ 2.0
*P < 0.05; sham showed a significantly reduced GAP43 staining compared with control and BL-POST. † P < 0.05; sham revealed a significant lower NGF staining compared with all other groups. ‡P < 0.05; the control demonstrated a significantly higher NGF staining compared with sham. §P < 0.05; the control showed a significantly lower nNOS staining compared with all other groups. SD = standard deviation, CI = confidence interval.
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staining, the C group displayed the highest staining, which was not statistically different compared with the results for BL-POST and BH-POST (P < 0.05) (Figure 4). Staining for nNOS demonstrated significantly lower values for the C group after untreated CN injury compared with the sham group (P < 0.001) (Figure 5). Both treatment groups BL-POST and BH-POST demonstrated significant restoration of staining pattern, approximating sham levels (P < 0.05).
Apoptosis Assessment Presented in Figures 6 and 7, the TUNEL results exhibited a highly significant increased AI in C (63 ⫾ 7%) compared with sham (11 ⫾ 5%) (P < 0.001). Both treatment groups BL-POST (16 ⫾ 4%) and BH-POST (21 ⫾ 9%) presented a highly significant prevention of cell death compared with control (P < 0.001). BL-POST reached an almost sham level (P = 0.2), but the AI for BH-POST remained statistically higher compared with sham (P = 0.03). TEM The TEM pictures (Figure 8) of the dissected CN of the control showed a reduced number of myelinized nerve fibers compared with sham. In the BH-POST group, an architectural restoration was documented, represented in higher amount of myelinized nerve fibers. Discussion
Despite the advent of nerve-sparing techniques during RP, a significant percentage of men continue to suffer from erectile impairment following this operation. It is estimated that approximately
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Figure 3 GAP43 staining. Representative photomicrographs show GAP43 antibody staining of cavernosal tissue for the sham, control, BLPOST, and BH-POST groups (100¥ magnification).
50,000 men undergo RP in the United States alone each year. ED is a common event after this operation, and the major predictors of erectile function recovery include patient age, preoperative erectile function, erectile hemodynamics, erectile tissue integrity, and nerve-sparing status [4]. The ability to protect the CN architecture and function using pharmacotherapy would thus revolutionize the treatment algorithm for such patients. Immunophilins belong to a family of intracellular proteins called chaperone molecules. These molecules bind and usher messenger molecules that regulate cellular functions. Immunophilins are cytosolic protein receptors for immunosuppressant
drugs like cyclosporin A, rapamycin, and FK506, and these chaperone molecules are often named after the drugs that bind them, e.g. FK-binding proteins (FK-BP12, FK-BP52). These intracellular complexes regulate their physiological role by regulating intracellular Ca2+ efflux controlled by calcineurin. The discovery that immunophilins are 50 times more abundant in brain and nervous system than in the immune system led to research that revealed the important role of immunophilins in neural function [13]. Given the important role of binding proteins in the regulation of cell activity, it is not unexpected that many immunophilin ligands, including FK506, have neuroprotective and neuroregenerative effects [16,17].
Figure 4 NGF staining. Representative photomicrographs show NGF staining of cavernosal tissue for the sham, control, BL-POST, and BHPOST groups (100¥ magnification).
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Figure 5 nNOS staining. Representative photomicrographs show nNOS antibody staining of cavernosal tissue for the sham, control, BLPOST, and BH-POST groups (100¥ magnification).
Figure 6 Apoptotic index (AI). The histogram displays the average AI in % expressing the ration build by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling positive cells divided through the total amount of cells. *P < 0.001; control vs. all other groups. #P = 0.03; sham vs. BH-POST.
Lyons and Steiner showed striking neurotrophic effects of FK506 (also known as tacrolimus, Prograf, Astellas Pharamceuticals, Deerfield, IL, USA) and Rapamycin in PC12 cells and sensory ganglia at sub-nanomolar concentrations [16]. In intact animals, Gold et al. reported enhanced regrowth and functional recovery of crushed sciatic nerves following FK506 treatment [17], and FK506 increased the regeneration of spinal cord axons in a pre-degenerated peripheral nerve autograft [18]. While cross-sectional area of the nerve is augmented by drug treatment, even more pronounced effects are noted in recovery of myelination [19,20] Verkade et al. showed, using
Figure 7 Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. Representative photomicrographs show TUNEL assay for the sham, control, BL-POST, and BH-POST groups. Apoptotic nuclei are fluorescent green compared with blue staining normal cell nuclei (40¥ magnification).
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Figure 8 Transmission electron microscopy (TEM). Cavernous nerve cross-section at the area of the crush injury under the TEM. Reduced number of myelinated nerve fibers in the control compared with sham. BH-POST treatment revealed an architectural restoration compared with the control.
IHC, an increased density of GAP-43 (neuronal growth-associated protein) in regenerating axons comparing with intact myelinated and unmyelinated axon shafts in a model of rat sciatic nerve crush [19]. Similarly, in another study, regenerated axons after rat dorsal root crush injury showed an increased expression of GAP-43 [20]. Our results also showed an increased staining expression for GAP43 in the intracavernosal tissue after FK506 treatment following CN crush compared with sham animals. However, the staining results for both treatment groups were practically the same compared with the control group after CN crush injury without FK506. The therapeutic potential of these neurotrophic drugs is considerable, with multiple targets including injury to CN during radical pelvic surgery. Sezen et al. reported on successful treatment of partial nerve injury in a rat CN model using FK506 [12]. FK506 (1 mg/kg intraperitoneally) preserved erectile function if administered simultaneous to nerve crush, and neuroprotection was documented histologically using optical and electron microscopy, and functionally assessing ICP after CN stimulation, as early as the first day after the treatment. When a longer crush (60 seconds repeated two times) was applied, FK506 (1 mg/kg intraperitoneally) demonstrated a protective effect at 24 and 36 hours compared with normal salinetreated animals, which disappeared 7 days after the surgery.
A more recent study examined the effect of FK506 and GPI-1046 on rats that had undergone CN injury [14]. This study found that animals treated with either FK506 or GPI-1046 had significantly greater recovery of erectile function compared with animals treated with saline. Electrically stimulated maximal ICP and rate of tumescence were better in the treated animals compared with controls. In this study, FK506 appeared to produce better functional results than GPI-1046. These preliminary results suggest an emerging role for direct pharmacologic neuromodulation after CN injury. Currently randomized, placebocontrolled trials assessing GPI-1485 and FK506 have completed enrollment, and data should be available in the near future. In the validation phase of the cavernous injury model [15] and in other experiments, we have shown in the untreated animals that erectile function initially decreases and continues to improve up to the 10-day time point with further improvement out to 28 days. The first phase of our study only found benefit with short-term dosing of 3 days at 1 mg/kg. All other doses (0.1 and 0.32 mg/ kg) did not show any advantage regardless of dose duration. Interestingly, when animals were treated for longer than four doses, they did not gain weight over the course of the study at the same rate as the control animals. It is interesting to note from the Burnett article that toxicity and mortality occurred at dosing schedules of 5 mg/kg for more J Sex Med 2008;5:1334–1344
1342 than a 5-day duration [14]. In the original study from the Burnett group, reported by Sezen et al., the dose of FK506 used was 1 mg/kg, and this was given intraperitoneally [12]. The method of nerve injury was three 15-second crushes of one of the nerves only with the other side used as an internal control. This has implications as it has been shown that unilateral nerve injury causes significant penile morphological changes, and thus the ICP measured following stimulation of the assumed normal nerve may not be maximal because of penile hemodynamic alterations resulting from erectile tissue damage [12,14]. Interestingly, when a more severe injury consisting of two 60-second crushes was applied to the nerve, 1 mg/kg given for 7 days showed no advantage over saline [12]. In the second phase of our study, we examined higher doses given for short periods and whether pretreatment had a role in ameliorating ED following CN crush, as has been suggested for sildenafil [21]. We found that the 1 mg/kg dose resulted in a nonstatistically significant improvement in erectile function when given at the time of injury and for 3 days following injury (BL-POST) with assessment at 28 days. There was no advantage to pretreating or with a combination of both (seven doses). When a higher dose of 3.2 mg/kg was given again following surgery (BH-POST), there was a significant improvement in the ICP/MAP ratio compared with C (P < 0.001). Pretreatment showed a nonsignificant improvement, which may be because of the fact that the last dose was given at the time of the nerve crush and this dose alone may have been sufficient to impart some benefit. Similarly, the combination of pre- and post-treatment (BH-PP) did not demonstrate any difference compared with control, and this may be because of the relatively high dosing over the 7 days, as previously described by Burnett and Becker [14]. Given that the ICP/MAP ratio improvements were most dramatic in the B-POST groups at 28 days, at this time, these groups are the only groups to have undergone detailed structural analyses. TEM (Figure 8) clearly demonstrated the disruption of CN architecture with nerve crush, and the restoration of axon and myelin density in the BH-POST group. This confirms Sezen et al.’s previous findings, although their assessment was for only 7 days compared with 28 days in our study [12]. Assessing IHC for neurotrophic factors, the administration of FK506 at low and high doses, the day of crush and for 3 days afterwards resulted in significant ameliorations in nNOS staining (Table 1). GAP-43 expression was increased miniJ Sex Med 2008;5:1334–1344
Mulhall et al. mally but statistically significantly in the control and in the BL-POST group compared with sham. Nerve crush resulted in a significant increase in NGF staining in the control group, which showed a clear trend toward lower results (although not to sham levels) in the BL-POST and BH-POST groups (P < 0.01). nNOS staining was significantly reduced by nerve crush, supporting the previously described changes identified by Carrier et al. [22], but normalized in the BL-POST and BH-POST groups approximating sham levels. NGF appears to be critical for the survival and maintenance of neurons (neuroprotection), while GAP43 is associated more with nerve growth (neuro-regeneration). Immunocytochemical staining showed that NGF treatment of adult visual cortex selectively increased the level of the phosphorylated form of GAP43, while the total level of GAP43 was not changed [23]. In the future, it may be worth paying closer attention to the phosphorylated form of GAP43. It is possible that the predominant effect of FK506, as dosed in this series of experiments, may be more neuroprotective rather than neuro-regenerative. After bilateral CN crush injury in our rat model, the administration of FK506 was shown to prevent apoptosis in both dosing groups compared with the control animals (P < 0.001). Findings in the literature support our observation of an antiapoptotic effect with FK506 application. Data collected in a rat optic nerve crush model report on a neuroprotection preventing retinal ganglion cells from dying using FK506 administration, which seems to be mediated by interfering with the activation of caspase 9 [24]. In a brain ischemia model in rats, where a 60-minute occlusion of the middle cerebral artery is induced, the administration of FK506 (1 mg/kg) immediately after occlusion ameliorated the degree of cerebral damage by inhibiting both apoptotic and necrotic cell death pathways [25]. Other studies in the literature have reported on the potential benefit of immunophilin ligands, including FK506 and FK1706, in modulating and preserving erectile function after CN injury [6,11– 14,26]. However, the novelty of our series of experiments lies in the fact that we provide new data about optimal dosing and timing of FK506 administration in this model. Conclusions
FK506 reduces the degree of ED following CN injury in the rat model. However, the response is
FK506 and Erectile Function Preservation not dose-dependent, and short-term administration of high-dose FK506 appears to give the optimal results, and pretreatment offers no advantage. The benefits of this pharmacotherapeutic strategy appear to be mediated through reduction in apoptosis and correction of nerve injuryassociated perturbations in neurotrophic factor expression, which may be the reason for the dramatic structural preservation seen in the treatment animals. The future role of FK506 as a pharmacologic neuromodulator in the RP population will be defined by the results of the randomized, placebocontrolled trial, which is ongoing. Corresponding Author: John P. Mulhall, MD, Associate Professor, Director, Sexual Medicine Programs, Departments of Urology, Weill Medical College of Cornell University, and Memorial Sloan Kettering Cancer Center, New York, NY, USA. Tel: 212-7460097 (personal line); 212-746-5653 (assistant); Fax: 212746-0403; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design John P. Mulhall; Dragan Golijanin (b) Acquisition of Data John P. Mulhall; Alexander Müller; John F. Donohue; Yemi Akin-Olugbade; Keith Kobylarz; Nelson E. Bennett (c) Analysis and Interpretation of Data Keith Kobylarz; John P. Mulhall; Alexander Müller; Peter Scardino; Leona Cohen-Gould
Category 2 (a) Drafting the Article John P. Mulhall; Alexander Müller (b) Revising It for Intellectual Content John P. Mulhall; Alexander Müller; Peter Scardino
Category 3 (a) Final Approval of the Completed Article John P. Mulhall; Alexander Müller
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