Effect of Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor Binding Protein-3 Complex in Cavernous Nerve Cryoablation

Effect of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 complex in cavernous nerve cryoablation

D Bochinski1, P-S Hsieh1, L Nunes1, G-T Lin1, C-S Lin1, EM Spencer2 and TF Lue1*

1Department of Urology, School of Medicine, University of California, San Francisco, California, USA; and 2Department of Medicine, Davies Medical Centre, San Francisco, California, USA

The purpose of this work was to study the effect of insulin-like growth factor 1 (IGF-1) and its binding protein (IGFBP-3) on the recovery of erectile function in a rat model for neurogenic impotence. In all, 28 male Spraque–Dawley rats were divided into four groups: seven underwent a sham operation; seven underwent bilateral cavernous nerve freezing (control group); seven underwent bilateral cavernous nerve freezing followed by intraperitoneal injection of IGF-1; and seven underwent bilateral cavernous nerve freezing followed by intraperitoneal injection of IGFBP- 3. Erectile response was assessed by cavernous nerve electrostimulation at 3 months, and samples of penile tissue were evaluated histochemically for nitric oxide synthase (NOS)-containing fibers. In the sham and IGF-1 group, there were significantly higher maximal intracavernous pressures compared to the IGFBP-3 complex and the control group. Correspondingly in the cavernosum, there were significantly more NOS-containing nerve fibers in the sham and IGF-1 groups. In conclusion, administration of IGF-1 can facilitate the regeneration of NOS-containing nerve fibers in penile tissue and enhance the recovery of erectile function after bilateral cavernous nerve cryoablation. The reverse effect was noted with the IGFBP-3 complex injection. International Journal of Impotence Research (2004) 16, 418–423. doi:10.1038/sj.ijir.3901190 Published online 19 February 2004

Keywords: IGF-1; IGFBP-3; nerve freeze; regeneration

Introduction insulin-like growth factor 1 (IGF-1) messenger RNA expression in the penile tissue.5,6 After surgical neurotomy, partial recovery of erectile function due Owing to the proximity of the cavernous nerves to the to the regeneration of the cavernous nerve and the capsule of the prostate, neurogenic impotence is still effect of growth hormone (GH) enhances the regene- a major problem in pelvic surgery or pelvic cryoabla- ration of NOS-containing penile nerve.7,8 GH’s tion. The recovery period and severity depends on growth-promoting actions are largely mediated by the degree of damage and the ability of the cavernous IGF-1, a polypeptide and a member of the insulin nerves to regenerate. The nonadrenergic–noncholi- gene family. IGF-1 is homologous in sequence and nergic nervous system is the principle pathway of structure to insulin. The blood level of IGF-1 penile erection in which its main neurotransmitter is represents primarily hepatic production; however, 1,2 nitric oxide (NO). Nitric oxide synthase (NOS) is IGF-1 is produced by most tissues where it is thought responsible for the production of NO and the primary to act by a paracrine or autocrine action. The expression sites for NOS in the penis are in the biological effects of IGF-1 are mediated mainly via 3,4 nerves, endothelium and leukocytes. Our previous the activation of the type 1 insulin-like growth factor studies in cryoablation of the cavernous nerve in a rat receptor (IGF-1R). IGF-1R is a tyrosine kinase receptor model showed time-related changes in NOS-contain- belonging to the insulin receptor family. The activity ing nerve fibers in penile nerves and upregulation of of IGF-1 is modulated by its binding proteins (IGFBPs) by affecting IGF-1 turnover and restricting the diffusion of IGF-1.9–11 IGF-1 is known as an important neurotrophic factor in peripheral nerve *Correspondence: TF Lue, MD, Department of Urology, 12 University of California, San Francisco, CA 94143-0738, regeneration. Using rat models for sciatic nerve crush or freezing injury, IGF-1 has shown promising USA. 13,14 E-mail: [email protected] effects in peripheral nerve regeneration. We Received 22 July 2003; revised 6 November 2003; accepted previously reported that the cavernous smooth 17 November 2003 muscle cells, which are supposed to be the most IGF-1 and IGFBP-3 and cavernous nerve cryoablation D Bochinski et al 419 critical element in controlling the erectile function of solved in 2 ml of PBS) were administered intraperi- the penis, secreted IGF-1 and expressed IGF-1R.15 In toneally twice a day to the rats for 14 days after this experiment, we studied the effect of IGF-1 and bilateral cavernous nerve cryoablation. In the IGFBP on the regeneration of NOS-containing nerve IGFBP-3 complex treatment group, 2 cm3 of IGFBP- fibers within the corpus cavernosum and the erectile 3 and IGF-1 complex (0.03 ml dissolved in 2 ml of response to cavernous nerve electrostimulation after PBS) was administered intraperitoneally once a day cryoablation of the cavernous nerve. for 14 days after cavernous nerve freezing.

Erectile function evaluation and tissue procurement Materials and methods

At 3 months after surgery, rats in each group Experimental animals and surgical procedure (weighing 450–550 g) were re-explored to expose and identify the bilateral major pelvic ganglions and cavernous nerves. Direct electrostimulation of the In all, 28 male Sprague–Dawley rats (2 months old, bilateral cavernous nerves was performed before 200–250 g weight) were divided into four groups: the tissue collection. The skin overlying the penis was sham operation group (n ¼ 7); the control group incised and the right penile crus was exposed by (n ¼ 7); treatment group 1 (n ¼ 7) (injection of IGF-1); removing part of the overlying ischiocavernous and treatment group 2 (n ¼ 7) (injection of IGFBP-3 muscle. A 23-gauge needle filled with 250 U/ml complex). Each rat was anesthetized using an in- heparin solution was connected to PE-50 tubing and traperitoneal injection of sodium pentobarbital was inserted into the right crus for a pressure (35 mg/kg) after being sedated with methoxyflurane measurement. A bipolar stainless-steel hood elec- inhalation and placed on a heating pad to maintain its trode was used to stimulate directly the cavernous body temperature at 371C. A lower abdominal midline nerve (each pole was 0.2 mm in diameter and incision was used and the posterolateral aspect of the separated by 1 mm). Monophasic rectangular pulses prostate gland was explored bilaterally. The major were generated by a Macintosh computer with a pelvic ganglions and the cavernous nerves were custom-built constant current amplifier. Stimulus identified and exposed.16 Surgery was performed parameters were 1.5 mA, frequency 20 Hz, pulse under an operating microscope (Olympus, Â 10–40). width 0.2 ms, and a duration of 50 s. Intracavernous All animal experiments in this study were approved pressure was measured during nerve electrostimula- by the local ethical committee for animal experimen- tion and was recorded with a Macintosh computer tation (Institutional and Animal Care use Committee) programmed with Lab VIEW 4.0 software (National In the group that underwent a sham operation, the Instruments, Austin, Texas, USA). After the func- bilateral cavernous nerves were identified only with tional evaluation was completed, systemic blood no further surgical manipulation. In the control and pressure was measured by placing the same 23-gauge treatment groups, the cavernous nerves were frozen needle directly into the abdominal aorta. A midshaft bilaterally for 1 min using a thermocoupler (5 mm in penile segment was taken for immunostaining study diameter, Omega HH21 hand-held microprocessor after the blood pressure measurement was completed. digital thermometer) as a probe. We used a 15 ml disposable centrifuge tube with a hole at the bottom that comfortably fit the probe of the thermocoupler. NADPH diaphorase staining for NOS nerve fibers The tube was filled with finely ground dry ice as a freezing material and the thermocoupler was passed through the center of the tube until the tip of the Tissue specimens were fixed for 4 h in a cold, freshly probe protruded 2–5 ml outside the tube. The prepared solution of 2% formaldehyde, 0.002% thermocoupler probe, with the centrifuge tube, was picric acid in 0.1 M phosphate buffer, pH 8.0. Tissues always kept in a dry ice thermoflask (Lab-Line were then cryoprotected for 24 h in cold 15% sucrose Instruments Inc.) to maintain a constant tempera- solution in 0.1 M phosphate buffer, pH 8.0. They ture. Using this method, we were able to keep the were embedded in OCT compound (Tissue-Tek, probe at a constant temperature of À801C during Miles Laboratory), frozen in liquid nitrogen, and cryoablation of the nerves. To avoid nerve disrup- stored at À701C. Cryostat tissue sections were cut at tion, saline was used to defrost the tip of the probe 7 mm and adhered to charged slides, air-dried for before taking it off the nerve. The wound was closed 5 min, and hydrated 10 min with 0.1 M PO4, pH 8.0. in two layers. The rats were then sent to the animal Sections were incubated at room temperature with house after full recovery from anesthesia. In the 0.1 NADPH, 0.2 mM nitroblue tetrazolium, and 0.2% 3 control group, 2 cm of buffer solution were admi- Triton X-100 in 0.1 M PO4, pH 8.0, for 60 min. The nistered intraperitoneally twice a day to the rats for reaction was then terminated by washing sections in 14 days after cavernous nerve cryoablation. In the a buffer. Slides were then coverslipped with buffered IGF-1 treatment group, 2 cm3 of IGF-1 (32 mg dis- glycerin as the mounting medium.17

International Journal of Impotence Research IGF-1 and IGFBP-3 and cavernous nerve cryoablation D Bochinski et al 420 The presence of NADPH diaphorase-positive considered significant at Po0.05. The individual nerves is easily apparent with this stain and is seen data are expressed as the mean7standard deviation as a highly localized, densely blue region. The of the mean (s.d.) unless otherwise stated. staining pattern was assessed by counting the number of the NADPH-positive nerve fibers present in four random fields (magnification Â 400) of each Results corpus cavernosum (endothelium staining was not included in the count). Erectile function

Statistical analysis The sham group demonstrated the greatest mean maximal intracavernosal pressure (MMIP) of 98.4 cm H20. This value was significantly greater The data were compared using a Student–Newman– than both the control group (MMIP ¼ 33.1 cm H20) Keul’s test (Statview 4.02 software). Values were and the IGFBP-3 group (MMIP ¼ 54.1). The IGF-1

Figure 1 Electrostimulation of the cavernous nerve: (a) sham operation (representative sample); (b) control group (3 months after cavernous nerve freeze) (representative sample); (c) IGF-1 group (3 months after cavernous nerve freeze followed by IGF-1 injection) (representative sample); and (d) IGFBP-3 group (3 months after cavernous nerve freeze followed by IGFBP-3 injection) (representative sample). Note higher maximal intracavernosal pressure in the IGF-1 than in the control and IGFBP-3 group.

International Journal of Impotence Research IGF-1 and IGFBP-3 and cavernous nerve cryoablation D Bochinski et al 421 treatment group (MMIP ¼ 80.6 cm H20) also demon- significant differences in systemic blood pressure strated significantly greater MMIP, after bilateral measurements were found between groups. cavernous nerve stimulation, than both control and Although no significant differences in MMIP exist IGFBP-3 treatment groups. It should be noted that no between the IGFBP-3 group (Figure 1d) and the

Table 1 Maximal intracavernosal pressures in response to the Table 2 Presence of NADPH diaphorase-positive nerve fibers in bilateral cavernous nerve stimulation the cavernous tissue

Groups Sham Control IGF1 IGFBP-3 Groups Sham Control IGF1 IGFBP-3 (n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 7) (n ¼ 7)

98.4721.7a 33.1713.6b 80.6728.9a 54.1720b 111.8738.6a 25.0712.3b 90.0745.0a 26.0716.5b

7 All values are expressed as cm H20; mean s.d. Note:No Values represent mean number of NADPH-positive nerve significant differences in maximal intracavernosal pressures was fibers7s.d. in four random fields. Note: No significant difference found between the control group and IGFBP-3 group (P40.05). in number of NADPH diaphorase-positive nerve fibers was found a Mean values that are significantly different from control group between control group and IGFBP-3 group (P40.05). a (Po0.05). Values that are significantly different than control group b Mean values that are significantly different from sham group (Po0.05). b (Po0.05). Values that are significantly different than sham group (Po0.05).

Figure 2 NADPH diaphorase staining of cavernous tissue (nerve): (a) sham operation; (b) control group; (c) IGF-1 group; and (d) IGFBP-3 group. Note greater number of positive nerve fibers in the cavernous tissue (nerve) of the IGF-1 group than in the control and IGFBP-3 group. Magnification Â 400.

International Journal of Impotence Research IGF-1 and IGFBP-3 and cavernous nerve cryoablation D Bochinski et al 422 control groups (Figure 1b), it appears that the freezing. Further study of the optimal dose and IGFBP-3 complex does stimulate erectile function duration of IGF-1 treatment and the effect of this but does not restore it completely. The pressures in neurotrophic factor on cancer growth is necessary the sham group were relatively higher than the IGF- before applying it to humans. 1 treatment group; however, they were not significantly different (Table 1) (Figure 1a and c). Conclusion NADPH diaphorase staining for NOS nerve fibers Our results showed that IGF-1 administration after freezing of the bilateral cavernous nerves can Histological examination of the midshaft penile facilitate the recovery of erectile function and tissue revealed a distinct staining pattern on the enhance the regeneration of the cavernous nerves. corpus cavernosum. The number of NADPH diaphorase-positive nerve fibers in the trabecular smooth muscle (in four random fields) of the control group (25.0) and IGFBP-3 complex group were Acknowledgements significantly lower than the sham (111.8) and IGF- 1 groups (90.0) (Table 2) (Figure 2a–d). This work was supported financially from the ISSIR (Pfizer) research grant. Discussion References Unlike neurotomy, bilateral cavernous nerve freezing may have substantial recovery of function 1 Burnett AL et al. Nitric oxide: a physiologic mediator of penile because the intact neural sheath can provide a erection. Science 1992; 257: 401. conduit for the regeneration of axons to reach the 2 Kim N et al. A nitric oxide-like factor mediates nonadrener- target area. Within 3 months, our control group gic–noncholinergic neurogenic relaxation of penile corpus showed a poor erectile response to nerve electro- cavernosum smooth muscle. J Clin Invest 1991; 88: 112. 3 Sullivan ME et al. 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