The Effect Of VEGF Gene Transfer To The Penis On A Rat Model Of Erectile Dysfunction

Objective Erectile dysfunction (ED) is a common disease that is estimated to affect 10-30 million men in American, 100 million in the world, and 50-60% of these individuals may have a vascular component of their problem. Atherosclerosis and diabetes may induce ED by impairing penile blood flow and vascular function. ED is often associated with problems in vascular perfusion to the erectile components of the penis. Despite some advances in oral medicine (such Viagra) and percutaneous revascularization techniques, therapeutic options for patients with arteriogenic ED are limited. In this study, we try to test the hypothesis that intracavernous injection of pcDNA-VEGF₁₆₅ plasmid restores erectile function in an arteriogenic ED rat model and to look for a new way to treatment ED. Materials and Methods 1. Purification of pcDNA-3.0 and pcDNA-VEGF₁₆₅ plasmid and Constructed the retroviral vector expression plasmid. The pcDNA-3.0 and pcDNA-VEGF₁₆₅ plasmid was purified by ultracentrifugation. Primers were synthesize base on the sequence of the human VEGF₁₆₅cDNA, and readjusted the direction of restriction endonucleases sites (BamH â… and EcoR â… ) on the both ends of VEGF₁₆₅cDNA, were 5'-CTCGAATTCACCATGAACTTTCTGCTGTC-3'(sense) and 5'-CTCGGATCCTCACCGCCTCGGCTTGTC-3'(antisense), the whole sequence of VEGF₁₆₅ were amplified by PCR from pcDNA-VEGF₁₆₅. The VEGF₁₆₅ sequence and retroviral vector pLXSN plasmid were digested by restriction enzymes BamHâ… and EcoRâ… , and ligated by T4 ligase, so the retroviral vector expression plasmid containing VEGF₁₆₅cDNA sequence was constructed. Its sequence was analyzed and named pLXSN-VEGF₁₆₅. 2. Established arteriogenic ED rats model and preparation of plasmids to transfect to the corporal smooth muscle in vivo. A total of 32 three months old male Wistar rats were used in these studies. The bilateral internal iliac arteries of rat were ligated. Minutes later, intracavernous injection of 200μl phosphate buffered saline (PBS) containing 20% sucrose (Group 1, n=8), 100μg pcDNA-3.0 in 200μl PBS containing 20% sucrose (Group 2, n=8), 100μg pcDNA-VEGF₁₆₅ in 200μl PBS containing 20% sucrose (Group 3, n=8), sham operated rats (Group 4, n=8). ALL four groups'animals were maintained separately for four weeks. 3. Apomorphine (APO) study. The rat was placed in the test cage and acclimated to the test environment 10 minutes. Light was dimmed for videomonitoring. A predetermined dose of apomorphine hydrochloride (100μg/kg) and vehicle (ascorbic acid. 0.5mg/kg,dissolved in normal saline), made fresh daily, was then delivered subcutaneously (5ml/kg) into the loose skin at the back of the rat's neck. During a 30-min period recorded on a video recorder, the number of yawns and erections was counted. 4. Electrostimulation of cavernous nerve and recording of intracavernous pressure (ICP). After the rats were anesthetized with 35mg/kg pentobarbital intraperitoneally, a midline abdominal incision was made, the cavernous nerve was exposed and electrostimulated. Stimulus parameters were 1.0 mA, frequency 20 Hz, pulse width 0.2 milliseconds and duration 1 min. A 23-gauge needle was inserted into the right penile crura, and a catheter was inserted into the internal carotid artery, both connected to a computer. Intracavernous and blood pressures were measure and recorded with a computer. 5. Electron microscopy study. After the neurostimulation, the penis was harvested. A part of penile samples were immersion fixed in osmium tetroxide, dehydrated in graded ethanol and embedded. Thin sections (approximately 900?) were cut, mounted on copper grids and stained with Uranyl acetate. Ultrastructural examination was performed with a transmission electron microscope. 6. Immunohistochemistry of VEGF and Fâ…§factor. A part of penile samples were fixed, sectioned and rehydrated. The sections were incubated in order with engogenous peroxidase blocking solution, non-immunone serum, first antibody to VEGF or Fâ…§factor, biotinylate second antibody, streptavidin peroxidase, and then examined...