Purification of neurturin
Recombinant rat neurturin (NTN) was expressed in E. coli as an inclusion body. Cell lysis was performed on a micro-fluidizer, repeated, and inclusion bodies were solubilized in 6 M guanidine-HCL, 0.1 M sodium sulfite, 0.01 M sodium terathionate and 0.02 M Tris pH 8.0 for 4 hours at 25°C. Separation of solubilized inclusion body rat NTN was achieved by centrifugation at 7,000 rpm for 1 hour, which was dialyzed in 4 M guanidine-HCL, 1 mM imidazole, and 0.01 M phosphate (pH 7.2). Unfolded rat NTN was then purified on an affinity nickle charge resin Ni-NTA superflow column (Qiagen Inc, Valencia, California, USA). Solubilized rat NTN was washed with 10× (ten times) column volume of 10 mM imidazole, and eluted with 0.4 M imidazole. Rat NTN fractions were exchanged in pre-refolding buffer containing 4 M urea, 0.1 M phosphate, 10% glycerol, 0.02 M glycine, and 0.02 M Tris pH 8.2. The refolding reaction was carried out by diluting rat NTN 10× in 3 M urea, 15% glycerol, 0.075 M phosphate, 0.3 M NaCL, 0.02 M glycine, 2 mM cysteine, and 0.02 M Tris pH 8.2, which was left incubating at 4°C for 48 hours. Di-filtration was performed and refolded rat NTN was formulated in 0.2 M sodium acetate pH 3.8. Refolded rat NTN was further purified on Toyopearl 650 M-phenyl sepharose HIC media (Tosoh Corp, Tokyo, Japan). Rat NTN was then loaded in 0.2 M sodium acetate and 0.750 M NaCL. A 10× column volume wash was performed in 1 M NaCL, followed by elution of rat NTN in HIC media with 0.2 M sodium acetate. Stripping of unfolded rat NTN and contamination was achieved by adding 25% ETOH to the HIC media. Finally, refolded rat NTN was formulated into 10 mM sodium acetate pH 3.8.
Thirty-two male Sprague-Dawley rats (3 months old, 250–350 g) were randomly divided into four groups, each containing eight animals. Control animals received a sham operation only (identification of the cavernous nerves bilaterally). The remaining 24 animals were divided into 3 treatment cohorts (Groups A, B, and C). Animals in the treatment groups underwent a bilateral cavernous nerve crush injury, followed by direct injection of either albumen (blinded control group), extended release NT-4 or neurturin (dose of 100 ug per animal; microspheres suspended in phosphate buffered solution) to the site of injury. All animal experiments were approved by the local ethical committee for experimentation (University of California, San Francisco, Institutional and Animal Care Use Committee) and complied with National Institutes of Health (NIH) regulations for the care and use of laboratory animals.
Animals were anesthetized for surgical procedures using intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg) and kept isothermic on a heated pad. After the animal was shaved, a lower midline abdominal incision exposed the prostate gland and the cavernous nerves and major pelvic ganglia (MPG) were identified bilaterally. No additional pelvic surgical manipulation was performed in the control group. In groups A, B, and C, the cavernous nerves were carefully isolated and the crush injury induced using a surgical needle driver at a constant 'one-click' pressure for 2 minutes per side. The abdominal wall was subsequently closed in two layers.
At 5 weeks, erectile function was assessed by measuring maximal intracavernous pressure (ICP) upon direct cavernous nerve electrostimulation. The cavernous nerves were isolated via a repeat midline abdominal incision and the crura of the penis was identified. A 23-gauge butterfly needle with 250 U/ml heparin solution was inserted into the penile crus and connected to polyethylene-50 tubing for ICP measurement. A bipolar stainless steel hook electrode (2 mm diameter probes separated by 1 mm) stimulated the cavernous nerves. Monophasic rectangular pulses were generated by a computer with a custom-built constant current amplifier. The stimulus parameters were 1.5 mA, 20 Hz, pulse width 0.2 ms, and duration 50 s. Each cavernous nerve was stimulated separately, ICP measured using LabVIEW 4.0 software (National Instruments, Austin, Texas), and mean maximal right and left ICPs determined for each rat. Systemic blood pressure was measured prior to terminating the procedure using a butterfly needle inserted into the aorta.
The data were first analyzed by non-repeated measures ANOVA with significance considered at p