Characterization of a novel rabbit model of Peyronie’s disease

Ten adult male, New Zealand white rabbits (Western Oregon Rabbit Co. Philomath, OR USA) weighing 3.5–4 kg were randomized into three experimental cohorts including nonsurgical controls (NSC, N = 3) and those receiving subtunical injections of vehicle (N = 3) consisting of 4 mM hydrochloric acid and 1 mg/ml bovine serum albumin or recombinant human TGF-β1 protein (N = 4). Sample sizes for each group were determined based on our past reported experiments which utilized similar animal replicates per group to determine significance in quantitative assessments [12]. Rabbits were single housed postoperatively, fed with regular rabbit food and allowed free access to water throughout the study. The Institutional Animal Care and Use Committee (IACUC) of University of California, Irvine approved the study protocol (# AUP-20-077) and all experimental procedures adhered to the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals. This study was also conducted in compliance with ARRIVE guidelines (https://arriveguidelines.org).

Surgical procedures

Anesthesia was induced by subcutaneous injection of 35 mg/kg Ketamine (Zoetis Inc, Kalamazoo, MI, USA) and 5 mg/kg Xylazine (MWI, Boise, ID, USA) and maintained by isoflurane via mask inhalation. Animals were placed in a supine position and excess fur was trimmed around the genital area. The surgical field was scrubbed with povidone-iodine solution and 70% ethanol three times and sterilely draped. A 5-0 polyprolene (Ethicon, Somerville, NJ, USA) stay suture was positioned at the tip of the distal penile skin to facilitate surgical manipulations. The skin web located between the penis and anus was divided and a ventral incision was made longitudinally between the penile skin and Buck’s fascia. Subtunical injection (50 µl) of vehicle or TGF-β1 protein (0.5 µg, R&D Systems, Minneapolis, MN) was performed with a 30-gauge needle at a single location in the tunica albuginea ~1.5 cm above the penile radix and between the urethra and lateral neurovascular bundle (Fig. 1). Absorbable 5-0 polyglactin sutures (Covidien, Mansfield, MA, USA) were then utilized to close skin incisions. Rabbits were dressed with Elizabethan collars for 5 days to avoid self-mutilation of the surgical site. For 7 days post-op, animals were evaluated daily to assess any potential surgical complications and then monitored weekly thereafter until scheduled euthanasia at 1 month. Pain management was facilitated by a single subcutaneous injection of Buprenorphine SR (0.12 mg/kg, ZooPharm, Laramie, WY, USA) immediately after the surgery as well as daily subcutaneous injections of Banamine (1 mg/kg, Merck Animal Health, Kenilworth, NJ, USA) for 3 days. Rabbits were also given subcutaneous injection of Enrofloxacin (5 mg/kg, Baytril®100; Bayer Healthcare LLC, KA, United States) prior to surgery and for 3 days postoperatively to prevent bacterial infection.

Fig. 1: Rabbit Peyronie’s disease model.figure 1

Overview of surgical stages of model creation. A Penile skin and Buck’s fascia were incised longitudinally in male rabbits (N = 7). A 30-gauge needle was slightly inserted into the penile tunica albuginea between the urethra and right lateral vascular structures and vehicle and TGF-β1 injections were performed. B Prominent white discoloration with central bullae were observed at injection sites of all study animals.

Cavernosography and cavernosometry

Cavernosographic and cavernosometric evaluations were performed 1 month following subtunical injections in experimental animals to assess papaverine-induced, erectile function using previously reported protocols [12, 15]. Rabbits were supine positioned under general anesthesia described above and a stay suture was placed at the tip of glans. The penile skin was degloved and a 22-gauge IV catheter was inserted into the right cavernous body below the subtunical injection site at the level of penile radix. For cavernosography, contrast medium (Omnipaque 300; GE Healthcare Inc., Marlborough, MA, USA) diluted with 1:1 saline was infused into the cavernous body through the IV catheter. Serial X-ray images were acquired in the anterior/posterior and lateral directions by using a C-arm fluoroscope (BV Pulsera; Philips, Eindhoven, Netherlands) while the penis was at its maximal erection point during contrast infusion. For cavernosometry, a second 22-gauge IV catheter was inserted into the left cavernous body adjacent to the penile radix. A urodynamics system (Goby CT; Laborie, ON, Canada) was connected to the right catheter for continuous recording of intracorporal pressures (ICP). Baseline ICP levels were recorded and then heparinized saline (10 U/ml) was infused (1 ml/min) into the corporal bodies in combination with the vasodilator, Papaverine-HCl (15 mg/ml, Sigma-Aldrich, Inc; MO, USA) to induce penile erection. Maximum ICP values were acquired and maintained for a period of 10 min with saline infusion. Photomicrographs of penile erections were captured at maximum ICP levels. Rabbits in both experimental groups as well as NSC were euthanized by intravenous injection of 0.2 ml/kg pentobarbital sodium and phenytoin sodium euthanasia solution (Euthasol; Virbac AH, Westlake, TX, USA) and tissues were collected for histological, immunohistochemical (IHC), and histomorphometric analyses.

Histological, immunohistochemical, and histomorphometric analyses

Penile tissues harvested from NSC as well as vehicle and TGF-β1-treated cohorts following 1 month post-op were excised for routine histological procedures following animal harvest. Tissue specimens were fixed in 10% neutral-buffered formalin for 12 h, dehydrated in graded alcohols, and paraffin embedded using standard protocols. Five micron sections were stained with Masson’s trichrome (MTS) and picrosirius red (PSR) to visualize total collagen content as previously described [16, 17]. Parallel sections in all groups were also evaluated for the presence of elastin fibers in control tissues and PD plaques using a commercially available, Verhoeff Van Gieson (VVG) staining kit (ab150667, Abcam, Cambridge, MA, USA). IHC evaluations were performed on tissue sections following antigen retrieval (10 mM sodium citrate buffer, pH 6.0) and incubation in phosphate-buffered saline with 0.3% Triton X-100, 5% fetal bovine serum, and 1% bovine serum albumin for 1 h at room temperature. Sections were then independently stained with primary antibodies including anti-collagen type I (ab34710, 1:200 dilution, Abcam), anti-collagen type III (ab7778, 1:50 dilution, Abcam), anti-MMP1 (10371-2-AP, 1:200 dilution, Proteintech, Rosemont, IL, USA), anti-MMP2 (436000, 1:150 dilution, Invitrogen, Waltham, MA), anti-MMP9 (AV33090, 1:200 dilution, Sigma-Aldrich), anti-MMP12 (22989-1-AP, 1:100 dilution, Proteintech), anti-TIMP1 (BS-0415R, 1:200 dilution, Bioss, Woburn, MA, USA), and anti-elastase 2B (orb183368, 1:100 dilution, Biorbyt, St Louis, MO, USA). Samples were then incubated with species-matched, horseradish peroxidase (HRP)-conjugated secondary antibodies and 3,3’Diaminobenzidine (DAB) substrate followed by hematoxylin counterstain. Sample visualization was performed with a Zeiss Axio Imager M2 model (Carl Zeiss MicroImaging, Thornwood, NY) and representative fields were captured with Zen software (version 3.1). Parallel specimens stained with secondary antibodies alone served as negative controls and led to no detectable background signal. For histomorphometric evaluations, quantitation of selective marker expression was calculated across 2–3 independent global sections per group replicate and displayed as a proportion of stained area per total field area examined utilizing ImageJ software. Analyses were performed with nonblinded observers.

Statistics

Statistical analyses of quantitative data between groups was performed using the Mann–Whitney U test considering a value of p ≤ 0.05 as significant. Quantitative data were represented as mean ± standard deviation (SD).

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