Introduction

Owing to the proximity of the cavernous nerves to the capsule of the prostate, neurogenic impotence is still a major problem in pelvic surgery or pelvic cryoablation. The recovery period and severity depends on the degree of damage and the ability of the cavernous nerves to regenerate. The nonadrenergic–noncholinergic nervous system is the principle pathway of penile erection in which its main neurotransmitter is nitric oxide (NO).^{1, 2} Nitric oxide synthase (NOS) is responsible for the production of NO and the primary expression sites for NOS in the penis are in the nerves, endothelium and leukocytes.^{3, 4} Our previous studies in cryoablation of the cavernous nerve in a rat model showed time-related changes in NOS-containing nerve fibers in penile nerves and upregulation of 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 to the regeneration of the cavernous nerve and the effect of growth hormone (GH) enhances the regeneration of NOS-containing penile nerve.^{7, 8} GH's growth-promoting actions are largely mediated by IGF-1, a polypeptide and a member of the insulin gene family. IGF-1 is homologous in sequence and structure to insulin. The blood level of IGF-1 represents primarily hepatic production; however, IGF-1 is produced by most tissues where it is thought to act by a paracrine or autocrine action. The biological effects of IGF-1 are mediated mainly via the activation of the type 1 insulin-like growth factor receptor (IGF-1R). IGF-1R is a tyrosine kinase receptor belonging to the insulin receptor family. The activity of IGF-1 is modulated by its binding proteins (IGFBPs) by affecting IGF-1 turnover and restricting the diffusion of IGF-1.^{9, 10, 11} IGF-1 is known as an important neurotrophic factor in peripheral nerve regeneration.¹² Using rat models for sciatic nerve crush or freezing injury, IGF-1 has shown promising effects in peripheral nerve regeneration.^{13, 14} We previously reported that the cavernous smooth muscle cells, which are supposed to be the most critical element in controlling the erectile function of the penis, secreted IGF-1 and expressed IGF-1R.¹⁵ In this experiment, we studied the effect of IGF-1 and IGFBP on the regeneration of NOS-containing nerve fibers within the corpus cavernosum and the erectile response to cavernous nerve electrostimulation after cryoablation of the cavernous nerve.

Materials and methods

Experimental animals and surgical procedure

In all, 28 male Sprague–Dawley rats (2 months old, 200–250 g weight) were divided into four groups: the sham operation group (n=7); the control group (n=7); treatment group 1 (n=7) (injection of IGF-1); and treatment group 2 (n=7) (injection of IGFBP-3 complex). Each rat was anesthetized using an intraperitoneal injection of sodium pentobarbital (35 mg/kg) after being sedated with methoxyflurane inhalation and placed on a heating pad to maintain its body temperature at 37°C. A lower abdominal midline incision was used and the posterolateral aspect of the prostate gland was explored bilaterally. The major pelvic ganglions and the cavernous nerves were identified and exposed.¹⁶ Surgery was performed under an operating microscope (Olympus, 10–40). All animal experiments in this study were approved by the local ethical committee for animal experimentation (Institutional and Animal Care use Committee)

In the group that underwent a sham operation, the bilateral cavernous nerves were identified only with no further surgical manipulation. In the control and treatment groups, the cavernous nerves were frozen bilaterally for 1 min using a thermocoupler (5 mm in diameter, Omega HH21 hand-held microprocessor 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. 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 probe protruded 2–5 ml outside the tube. The thermocoupler probe, with the centrifuge tube, was always kept in a dry ice thermoflask (Lab-Line Instruments Inc.) to maintain a constant temperature. Using this method, we were able to keep the probe at a constant temperature of -80°C during cryoablation of the nerves. To avoid nerve disruption, saline was used to defrost the tip of the probe before taking it off the nerve. The wound was closed in two layers. The rats were then sent to the animal house after full recovery from anesthesia. In the control group, 2 cm³ of buffer solution were administered intraperitoneally twice a day to the rats for 14 days after cavernous nerve cryoablation. In the IGF-1 treatment group, 2 cm³ of IGF-1 (32 g dissolved in 2 ml of PBS) were administered intraperitoneally twice a day to the rats for 14 days after bilateral cavernous nerve cryoablation. In the IGFBP-3 complex treatment group, 2 cm³ of IGFBP-3 and IGF-1 complex (0.03 ml dissolved in 2 ml of PBS) was administered intraperitoneally once a day for 14 days after cavernous nerve freezing.

Erectile function evaluation and tissue procurement

At 3 months after surgery, rats in each group (weighing 450–550 g) were re-explored to expose and identify the bilateral major pelvic ganglions and cavernous nerves. Direct electrostimulation of the bilateral cavernous nerves was performed before tissue collection. The skin overlying the penis was incised and the right penile crus was exposed by removing part of the overlying ischiocavernous muscle. A 23-gauge needle filled with 250 U/ml heparin solution was connected to PE-50 tubing and was inserted into the right crus for a pressure measurement. A bipolar stainless-steel hood electrode was used to stimulate directly the cavernous nerve (each pole was 0.2 mm in diameter and separated by 1 mm). Monophasic rectangular pulses were generated by a Macintosh computer with a custom-built constant current amplifier. Stimulus parameters were 1.5 mA, frequency 20 Hz, pulse width 0.2 ms, and a duration of 50 s. Intracavernous pressure was measured during nerve electrostimulation and was recorded with a Macintosh computer programmed with Lab VIEW 4.0 software (National Instruments, Austin, Texas, USA). After the functional evaluation was completed, systemic blood pressure was measured by placing the same 23-gauge needle directly into the abdominal aorta. A midshaft penile segment was taken for immunostaining study after the blood pressure measurement was completed.

NADPH diaphorase staining for NOS nerve fibers

Tissue specimens were fixed for 4 h in a cold, freshly prepared solution of 2% formaldehyde, 0.002% picric acid in 0.1 M phosphate buffer, pH 8.0. Tissues were then cryoprotected for 24 h in cold 15% sucrose solution in 0.1 M phosphate buffer, pH 8.0. They were embedded in OCT compound (Tissue-Tek, Miles Laboratory), frozen in liquid nitrogen, and stored at -70°C. Cryostat tissue sections were cut at 7 m and adhered to charged slides, air-dried for 5 min, and hydrated 10 min with 0.1 M PO₄, pH 8.0. Sections were incubated at room temperature with 0.1 NADPH, 0.2 mM nitroblue tetrazolium, and 0.2% Triton X-100 in 0.1 M PO₄, pH 8.0, for 60 min. The reaction was then terminated by washing sections in a buffer. Slides were then coverslipped with buffered glycerin as the mounting medium.¹⁷

The presence of NADPH diaphorase-positive nerves is easily apparent with this stain and is seen as a highly localized, densely blue region. The staining pattern was assessed by counting the number of the NADPH-positive nerve fibers present in four random fields (magnification 400) of each corpus cavernosum (endothelium staining was not included in the count).

Statistical analysis

The data were compared using a Student–Newman–Keul's test (Statview 4.02 software). Values were considered significant at P<0.05. The individual data are expressed as the meanstandard deviation of the mean (s.d.) unless otherwise stated.

Results

Erectile function

The sham group demonstrated the greatest mean maximal intracavernosal pressure (MMIP) of 98.4 cm H₂0. This value was significantly greater than both the control group (MMIP=33.1 cm H₂0) and the IGFBP-3 group (MMIP=54.1). The IGF-1 treatment group (MMIP=80.6 cm H₂0) also demonstrated significantly greater MMIP, after bilateral cavernous nerve stimulation, than both control and IGFBP-3 treatment groups. It should be noted that no significant differences in systemic blood pressure measurements were found between groups. Although no significant differences in MMIP exist between the IGFBP-3 group (Figure 1d) and the control groups (Figure 1b), it appears that the IGFBP-3 complex does stimulate erectile function but does not restore it completely. The pressures in the sham group were relatively higher than the IGF-1 treatment group; however, they were not significantly different (Table 1) (Figure 1a and c).

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.

Full figure and legend (309K)

Table 1 - Maximal intracavernosal pressures in response to the bilateral cavernous nerve stimulation.

Full table

NADPH diaphorase staining for NOS nerve fibers

Histological examination of the midshaft penile tissue revealed a distinct staining pattern on the 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 significantly lower than the sham (111.8) and IGF-1 groups (90.0) (Table 2) (Figure 2a–d).

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 400.

Full figure and legend (242K)

Table 2 - Presence of NADPH diaphorase-positive nerve fibers in the cavernous tissue.

Full table

Discussion

Unlike neurotomy, bilateral cavernous nerve freezing may have substantial recovery of function because the intact neural sheath can provide a conduit for the regeneration of axons to reach the target area. Within 3 months, our control group showed a poor erectile response to nerve electrostimulation and markedly diminished number of NOS-containing nerve fibers as demonstrated by the decrease in NADPH diaphorase staining in the corpus cavernosum. This is consistent with our previous studies.^{5, 6, 18} IGF-1 is a neurotrophic polypeptide that is expressed during nerve injury, and it is believed to play an important role in nerve regeneration.^{9, 19} Although we used an intraperitoneal injection of IGF-1, with its systemic effect (rather than intracorporal injection for its local effect), significant erectile response to nerve electrostimulation, and an increase in the total number of NOS-containing nerve fibers in the corpus cavernosum was clearly shown. We believe that daily injection of IGF-1 can maintain the baseline serum levels and accelerate the cavernous nerve regeneration either as a local or systemic effect. The bioavailability of IGF-1 is modulated in the circulation and extracellular environment by six different IGF binding proteins, the most abundant being IGFBP3. These proteins bind IGF-1 with high affinity²⁰ and typically inhibit its function.²¹ In our experiment, it is possible that daily administration of the IGFBP3 protein caused a decrease in the IGF-1 free form and hence acted to inhibit the regeneration process of the injured nerve. To our knowledge, this is the first report that IGF-1 treatment can influence NOS-containing nerve fibers regeneration in corpus cavernosum and the erectile function after bilateral cavernous nerves freezing. Further study of the optimal dose and duration of IGF-1 treatment and the effect of this neurotrophic factor on cancer growth is necessary before applying it to humans.

Conclusion

Our results showed that IGF-1 administration after freezing of the bilateral cavernous nerves can facilitate the recovery of erectile function and enhance the regeneration of the cavernous nerves.

References

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Acknowledgements

This work was supported financially from the ISSIR (Pfizer) research grant.