¹Department of Urology, The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, Maryland, USA

²Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

³Guilford Pharmaceuticals, Baltimore, Maryland, USA

Correspondence to: A L Burnett, The Johns Hopkins Hospital, 600 North Wolfe Street, Marburg 407, Baltimore, MD 21287-2411, USA. E-mail: aburnett@jhmi.edu

To evaluate whether FK506 and other immunophilin ligands may have potential therapeutic efficacy for erectile function preservation after penile nerve injury, we demonstrated localizations of the immunophilin FK506 binding protein 12 (FKBP 12) in intact and injured rat penile nerves and correlated these findings with localizations of neuronal nitric oxide synthase (nNOS), which neuronally forms nitric oxide for mediation of penile erection, in response to systemically administered FK506. Adult male Sprague-Dawley rats were subjected to unilateral right cavernous nerve forceps crush injury and administered FK506 (1 mg/kg i.p.) or saline at the same time and daily up to 7 days. At 1, 3 and 7 days after injury, bilateral cavernous nerves and major pelvic ganglia were collected for nNOS immunohistochemistry, FKBP 12 immunohistochemistry, and FKBP 12 in situ hybridisation. Protein expressions of nNOS and FKBP 12 were observed in major pelvic ganglion, cavernous nerve and nerve terminals within the rat penis as well as mRNA expression of FKBP 12 observed in the rat major pelvic ganglion neuronal cell bodies to a minimal extent at baseline conditions. After cavernous nerve injury, nNOS immunoreactivity was observed to be slightly diminished in ipsilateral penile nerve structures at only one day following injury while both FKBP 12 protein and mRNA expressions were observed to be increased at each interval of study. FK506 treatment did not affect staining of intact or injured nerves. Our demonstration that FKBP 12 is localized to penile innervation in the rat and becomes upregulated following cavernous nerve crush injury, independent of FK506 treatment, suggests that this immunophilin mediates a neurotrophic mechanism. Whether FK506 affords neuroprotection that preserves penile erection through FKBP 12 upregulation is unclear.

International Journal of Impotence Research (2002) 14, 506-512. doi:10.1038/sj.ijir.3900919

immunophilin; neurotrophic; neuroprotective; nitric oxide

Introduction

Immunophilins constitute a family of cellular proteins that act as receptors for immunosuppressant drugs such as FK506 and cyclosporin A, whose specific immunophilins are FK506 binding proteins (FKBPs) and cyclophilins, respectively.^1,2,3 The immunosuppressive effect resulting from FK506 treatment involves the binding of this drug to FKBP 12 in immune cells.^1,2,3 While FKBP 12 is expressed in immune tissues, it is concentrated in much higher amounts in neuronal structures such as the brain.^4,5 The finding that this immunophilin is so significantly expressed in neuronal structures provided a foundation for subsequent characterizations of immunophilin ligands as neurotrophic and neuroprotective agents.^{6,7,8,9,10,11,12,13} FK506 and other immunophilin ligands have been promoted as having potential therapeutic efficacy for nerve recovery for such clinical presentations as neurodegenerative diseases and nerve injury.^14,15,16,17

We recently demonstrated that FK506 treatment of rats protects penile innervation from degeneration and preserves erectile function in a cavernous nerve crush injury model.¹⁸ This report served as an initial demonstration of the potential therapeutic role of immunophilins for the neuroprotection of focally injured nerves important for penile erection that may occur with radical prostatectomy.¹⁹ As preliminary work in this field, the report focused on a basic physiologic and anatomical description of the effects of FK506 treatment, monitoring intracavernosal pressure responses to cavernous nerve electrical stimulation and nerve morphology by electron microscopic analysis after cavernous nerve injury. The putative molecular mechanism for the FK506 treatment effect in our rat cavernous nerve injury model has not been reported earlier. Herein, we demonstrate FKBP 12 localizations in intact and injured rat penile innervation and correlate these findings with localizations of neuronal nitric oxide synthase (nNOS), which neuronally forms nitric oxide for mediation of penile erection.

Materials and Methods

Experimental treatment

Studies were performed on tissues obtained following experimental treatment as described previously.¹⁸ Briefly, adult male Sprague-Dawley rats (300-450 g, Charles River Laboratories Inc., Wilmington, MA, USA) were anesthetized with pentobarbital sodium (NembutalÒ, Abbott Laboratories, N. Chicago, IL, USA; 50 mg/kg i.p.) for unilateral right cavernous nerve exposure and focal crush with Dumont no. 5 jeweler's forceps three times for 15 s 2 mm distal to the major pelvic ganglion under aseptic conditions. FK506 (PrografÒ, Fujisawa Healthcare Inc., Deerfield, IL, USA; 1 mg/kg i.p.) was administered at the same time as the crush and on successive days, and penile erection and nerve morphology were evaluated 1, 3 or 7 days later. After completion of experimentation, animals were euthanized with pentobarbital sodium (250 mg/kg i.p.), and bilateral cavernous nerves, major pelvic ganglia and penis were collected for further investigation. All experiments were conducted in accordance with the Johns Hopkins University School of Medicine guidelines for care and use of animals.

Immunohistochemistry

Upon removal, tissue specimens were immediately frozen in OCT Compound (Tissue-Tek, Sakura Finetek USA Inc., Torrance, CA, USA). Cryostat sections (6 µm thick) were cut, mounted on gelatin-coated slides and fixed with 4% paraformaldehyde. Sections were processed with primary antibody incubation as described previously,²⁰ using a rabbit polyclonal antibody against human nNOS (amino acids 1419-1433) at a 1:1000 dilution (DiasorinÒ, Stillwater, MN, USA) and rabbit anti-human polyclonal antibody against recombinant FKBP 12 at a 1:500 dilution. The FKBP 12 antibody was raised in New Zealand white rabbits against human recombinant FKBP 12, which was expressed in and purified from E. coli lysates. The immune polyclonal serum was then passed over a Sepharose column with covalently attached FKBP 12 to affect affinity purification. Preabsorption of the antibody with purified FKBP 12 protein abolished the specific binding of the affinity purified antibody. Staining was performed with the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) peroxidase system, with diaminobenzidine (Dako Corporation, Carpinteria, CA, USA) as the chromogen. The omission of primary antibodies provided negative controls. The grade of immunohistochemical staining was assessed by two independent observers.

In situ hybridization

Separate tissues were cryostat sectioned (20 µm thick) for in situ hybridization as described previously.^21,22 Digoxigenin labeled (Dig RNA Labeling Mix, Boehringer Mannheim/Roche Molecular Biochemicals, Indianapolis, IN, USA) RNA oligonucleotide probes (antisense, sense) were prepared from full-length (600 bp) rat FKBP 12 cDNA.²² Alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim/Roche Molecular Biochemicals, Indianapolis, IN, USA) and X-phosphate/nitroblue tetrazolium (Boehringer Mannheim/Roche Molecular Biochemicals, Indianapolis, IN, USA) were used for the detection of signal. Sense probe labeling were used as a negative control. Two independent observers assessed the grade of staining.

Results

nNOS immunohistochemistry

Protein expression of nNOS was observed in major pelvic ganglion, cavernous nerve, and nerve terminals within the rat penis, similar to earlier descriptions²³ (Figure 1). In saline-treated animals, nNOS immunoreactivity was observed to be slightly more faint in the injured right cavernous nerve and major pelvic ganglion compared with that of the contralateral structure at 1 day after right cavernous nerve injury, and otherwise no appreciable differences were observed at 3 and 7 days. In FK506-treated animals, nNOS protein expression was unchanged in the major pelvic ganglion and in the cavernous nerve both ipsilateral and contralateral to the cavernous nerve injury over the entire 7 day time course. The retained nNOS expression in the major pelvic ganglion in distributions ipsilateral to the cavernous nerve following FK506 treatment is consistent with the neuroprotective effect demonstrated functionally by erection monitoring and histologically by electron microscopy.¹⁸ The absence of significantly depleted nNOS protein in the ipsilaterally injured penile nerve distributions with saline treatment may reflect the minor degree of nerve injury and the short time course associated with this experimental model.

FKBP 12 immunohistochemistry

FKBP 12 protein expression was observed in rat major pelvic ganglion, cavernous nerve, and penile nerve terminals (Figure 2). In both saline-treated and FK506-treated animals, FKBP 12 immunostaining in the major pelvic ganglion was observed to be more intense on the cavernous nerve injury side compared with the contralateral intact side at each interval of study. The observed increase in FKBP 12 immunoreactivity following cavernous nerve injury parallels findings observed in models of facial and sciatic nerve crush,²⁴ suggesting that FKBP 12 is upregulated in response to cavernous nerve injury. The results suggest that FK506 treatment does not significantly alter FKBP 12 protein upregulation following cavernous nerve injury, compared with the effect of saline treatment, at least in the short term, although FK506 does apparently preserve functional erection during this time course.¹⁸

FKBP 12 in situ hybridisation

FKBP 12 mRNA expression was observed in the rat major pelvic ganglion with localizations confined to neuronal cell bodies (Figure 3). Similar to FKBP 12 immunohistochemical results, FKBP 12 mRNA expression was increased in the ipsilateral ganglion associated with cavernous nerve injury compared with the contralateral ganglion in both saline-treated and FK506-treated animals. These data further implicate augmented FKBP 12 expression in the penile innervation in response to cavernous nerve injury.

Discussion

Recent descriptions of immunophilins in neuronal structures have generated great interest in defining their likely roles in neurotrophic mechanisms.^{6,7,8,9,10,11,12,13,14,15,16,17} The prototype immunophilin ligand FK506 has been shown to promote regeneration and functional recovery in in vivo animal models of peripheral nerve injury such as crush injury of sciatic and facial nerves.^8,11,13,24 We recently demonstrated that FK506 exerts potent neuroprotective effects on rat penile innervation and preserves erectile function in a cavernous nerve crush injury model.¹⁸ In instances of sciatic and facial crush nerve injury, FKBP 12 localizations and upregulated expression have been confirmed and linked to nerve regeneration.²⁴ To begin to characterize the mechanism of action of FK506 in penile innervation after cavernous nerve injury, we proceeded to perform localization studies for FKBP 12 in this innervation while correlating changes in expression of nNOS. We also examined FKBP 12 expression in these injured structures with FK506 treatment. Our findings in this study, showing that FKBP 12 is localized to penile innervation in the rat with apparently upregulated expression following cavernous nerve crush injury, independent of FK506 treatment, suggest that this immunophilin mediates a neurotrophic mechanism. Whether the neuroprotective effects of FK506 in our model operate through FKBP 12 upregulation remains unclear.

The in vivo rat model of penile erectile function is an ideal one to establish further a role for immunophilins in functional nerve recovery. This model affords an experimentally advantageous neurophysiological system for purposes of manipulation and quantitation. We have been able to advance earlier-described neurophysiological and morphologic findings after cavernous nerve injury^23,25,26,27 with the addition of a molecular study that characterizes the role of immunophilins in penile nerve function recovery. Findings from this study can also be extrapolated to the human condition such as trauma involving penile innervation following radical prostatectomy.¹⁹ In rats, similar to humans, penile autonomic innervation derives from the major pelvic ganglion, termed pelvic plexus in humans, located bilaterally dorsolateral to the prostate.²⁸ Cavernous nerve fibers arising from this ganglion innervate the erectile tissue of the penis, and its distributions contain nitric oxide synthase (NOS), which constitutively catalyzes the generation of the gaseous molecule nitric oxide, identified to be the principal neuroactive mediator of penile erection.²⁹

Several previous studies have been carried out to investigate nerve injury and regenerative properties in the cavernous nerve, particularly in relation to the production and release of nitric oxide. In rats, unilateral cavernous nerve neurotomy causes depletion of NOS in penile nerves and concomitant decrease in electrically stimulated intracavernosal pressure responses, whereas after 6 months intracavernosal pressure responses are significantly recovered in association with regeneration of NOS-containing nerves.²⁵ Unilateral cavernous nerve freezing has also been shown to damage NOS-containing nerve fibers, with a delayed recovery after treatment.²⁶ More recent investigations have suggested that growth hormone and certain growth factors may serve as neurotrophic agents in the regenerative process following cavernous nerve injury in the rat.^30,31,32,33

Our investigation promoting a neurotrophic role for immunophilins significantly supports an alternative neurotrophic system besides conventionally characterized neurotrophins that may be developed therapeutically for the treatment of nerve injuries and neurological disease. A unique aspect of immunophilin ligands is that they act via specific receptors unlike that of classical neurotrophic mechanisms which may exert diverse and perhaps deleterious effects. Molecular mechanisms for the neurotrophic and neuroprotective actions of immunophilin ligands remain unclear. It is known that the neuroregenerative properties of immunophilin ligands such as FK506 do not involve calcineurin, which is essential for immunosuppression, and non-immunosuppressant FKBP 12 ligands that fail to inhibit calcineurin are neurotrophic.¹⁰ Ongoing characterizations of different FKBP receptor subtypes and other molecular targets subserving the action of immunophilin treatments in the future may shed additional insight into regulatory mechanisms involving immunophilins.

This study focused primarily on evaluating localizations of a major FKBP in the peripheral neuronal pathways relevant to penile erection of the rat. While we did not perform any particular quantification of the upregulation, it is acknowledged that this is a preliminary investigation with results that were likely to be much more demonstrative with a more significant degree of cavernous nerve injury and perhaps long-term assessment. Similarly, the limited alterations in NOS immunoreactivity in penile innervation distinguished from that of previous studies^25,26 may readily be explained by the acute time course used in this investigation. However, our results would indicate that molecular distinctions are less discernible than functional effects under conditions that involve only partial nerve injury.

In summary, we have carried out localization studies for FKBP 12 in correlation with neuronal NOS in the penile innervation of the rat, confirming its expression and upregulation acutely following cavernous nerve crush injury. These results suggest its neurotrophic role, although the consistent level of FKBP 12 upregulation with FK506 treatment does not clarify whether or how it mediates FK506 neuroprotection. Studies are ongoing to investigate additional FKBP receptor subtypes and receptor mechanisms for FK506³⁴ and roles of non-immunosuppressant immunophilin ligands that may serve to preserve penile erection and other nerve-mediated pelvic functions in the face of neurological disease or injury.

Acknowledgements

Supported by National Institute of Health grant DK 02568 (National Institute of Diabetes and Digestive and Kidney Diseases) and National Kidney Foundation-Maryland Research grant.

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Figure 1 nNOS immunohistochemistry of the major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, decreased nNOS immunoreactivity (-ir) is observed in cross sections of the ipsilateral ganglion (R-MPG) compared with the contralateral intact nerve side (L-MPG). In FK506-treated animals, similar nNOS-ir is observed in cross-sections of R-MPG and L-MPG. No primary antibody staining was used as a negative control (scale bar, 15 µm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion.

Figure 2 FKBP 12 immunohistochemistry of major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, increased FKBP 12 immunoreactivity (-ir) is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). In FK506-treated animals, similar FKBP 12-ir is observed in cross-sections of R-MPG and L-MPG. No primary antibody staining was used as a negative control (scale bar, 15 µm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion.

Figure 3 FKBP 12 in situ hybridisation of major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, increased FKBP 12 expression is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). Similarly in FK506-treated animals, increased FKBP 12 expression is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). Sense FKBP 12 cDNA probe was used as a negative control (scale bar, 15 µm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion.