Penis Allotransplantation Model in the Rat

RESEARCH Penis Allotransplantation Model in the Rat Erhan Sonmez, MD, Serdar Nasir, MD, and Maria Siemionow, MD, PhD, DSc Abstract: In this study we present the first experimental vascularized penile allogenic transplantation model in the rat. In group 1 (n ⫽ 6), the vascular anatomy of the male rat perineal region was determined. In 12 Lewis rats of group 2 (n ⫽ 24), the penile composite graft was transplanted as isotransplant by microvascular anastomosis of the pedicle of the penis to the saphenous vessels and in another 12 Lewis rats of group 2, penile composite graft was transplanted without vascular anastomosis. In group 3 (n ⫽ 12), penis composite graft was transplanted from 6 Lewis-Brown-Norway donors to 6 Lewis recipients under CsA immunosuppression. Direct observation, somatosensory evoked potential test, histologic examination and microangiography were used to assess the viability of the transplants. All vascularized isotransplants and allotransplants survived over 200 days (still under observation) whereas all the nonvascularized grafts were necrosed at 7 days posttransplant. In this study the new penile allograft transplantation model in the rat was developed and a new method of blood supply to a vascularized composite tissue with directly artery-to-corpus spongiosum anastomosis was introduced. Key Words: penile allotransplantation, penis transplantation, penile allograft (Ann Plast Surg 2009;62: 304 –310) T he loss of an extremity or an organ is a devastating injury for any patient. When that injury, such as trauma or resection due to cancer includes the penis, significant psychologic and physical problems are encountered by patients. Penis defects may be caused by traumatic amputations, penile cancer surgeries, congenital anomalies, and complications of surgical manipulations such as circumcision, hypospadias repair, bladder extrophy repair, and so on.1–5 Over the past decades surgical reconstruction of penis has evolved significantly but repairing the penis still remains anatomically, functionally, and aesthetically a great challenge. Current treatment options for penile defects include penile replantation, penile reconstruction, and penile transplantation. Penile replantation is the first choice of treatment following acute amputation injuries. Successful replantation of an amputated penis was first performed by Tuerk et al5 in 1971 and since than many cases have been reported. When the replantation of the amputated part is not possible because of the nature of the injury, ischemia time, or when the amputated part is not available, penile reconstruction or penile transplantation remain the only alternative treatments. Local skin flaps or myocutaneous flaps and free tissue transfers, such as radial forearm flap, superficial inferior epigastric artery flap, superficial circumflex iliac artery flap, and fibula flap are among the flaps most often used for penis reconstruction. UnsatisReceived December 8, 2007, and accepted for publication February 13, 2008. From the Department of Plastic Surgery, The Cleveland Clinic Foundation, Cleveland, OH. Reprints: Maria Siemionow, MD, PhD, DSc, Department of Plastic Surgery, A60, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: siemiom@ccf.org. Copyright © 2009 by Lippincott Williams & Wilkins ISSN: 0148-7043/09/6203-0304 DOI: 10.1097/SAP.0b013e31817dc4d8 304 factory cosmetic and functional results, need for multiple operations, and significant donor site morbidity are the major shortcomings of penile reconstruction. Penile transplantation from cadaveric donors or male-tofemale transsexual surgeries is the other option for the repair of penile defects. The only clinical case of allogenic penile transplantation was recently reported in China. However, in this case the allotransplanted penis was removed at the end of second week posttransplant due to patient’s psychologic problems.6 To the best of our knowledge this is the first report on an experimental model of neurovascular allogenic transplantation of the penis. In this study, we present anatomic basis and microsurgical technique of allogenic transplantation of the penis on neurovascular pedicle. This model may help researchers to study immunologic and functional aspects of this allograft transplant. MATERIALS AND METHODS Fourty-two adult male Lewis rats (LEW, RT1l) and 6 adult male Lewis-Brown-Norway rats (LBN, RT1l⫹n) weighing 200 –300 g were used in this study. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Anesthesia was induced with sodium pentobarbital (50 mg/kg administered intraperitoneally) and supplementary doses of 10 mg/kg per hour were administered as needed. The inguinal regions were shaved and the skin was thoroughly cleansed with povidone-iodine (10%) solution. All the dissections, vascular anastomosis, and nerve repair procedures were performed using an operating microscope (Zeiss OP-MI, 6SD; Carl Zeiss, Gottingen, Germany). Postoperatively, each rat was returned to an individual cage under standard environmental conditions. They were maintained on commercially available balanced food, which was available ad libitum. Postoperative analgesia was provided with acetaminophen (300 mg/kg per oral) for the first 2 days postoperatively. The study was composed of 3 groups: the anatomic study (group 1, n ⫽ 6), isotransplantation (group 2, n ⫽ 24), and allotransplantation groups (group 3, n ⫽ 12). Anatomic Study Group (n ⴝ 6) Male Lewis rats were used for anatomic dissections to determine the vascular anatomy of the male perineal region. All anatomic terminology in this manuscript is based on “Greene’s Anatomy of the Rat.”7 The course of vessels supplying the penis was identified and the feasibility of the transfer of the rat penis as a composite tissue based on the neurovascular pedicle was investigated. Isotransplantation Group (n ⴝ 12) A total of 12 transplantations (6 vascularized and 6 nonvascularized) were performed in this group. The vascularized penile composite grafts were transplanted between 6 Lewis rats (LEW, RT1l 3 LEW, RT1l) by microvascular anastomosis of the vascular pedicle of the penis to the saphenous vessels and coaptation of sensory nerve of the penis to the lateral femoral cutaneous nerve. Between 6 Lewis rats (LEW, RT1l 3 LEW, RT1l) penile composite tissues were transplanted without microvascular anastomosis as a control group, to check if the composite graft will survive without vascular supply. Annals of Plastic Surgery • Volume 62, Number 3, March 2009 Annals of Plastic Surgery • Volume 62, Number 3, March 2009 Penis Allotransplantation Model in the Rat Allotransplantation Group (n ⴝ 6) In this group, penile composite tissue allograft transplantation was performed between 6 Lewis-Brown-Norway donors and 6 Lewis recipients (LBN, RT1l⫹n 3 LEW, RT1l) by microvascular anastomosis of the neurovascular pedicle of the penis to the saphenous vessels and coaptation of donor sensory nerve with lateral femoral cutaneous nerve of the recipient rats. To prevent acute and chronic allograft rejection, Cyclosporine A (16 mg/kg per day, Bedford Laboratories, Bedford, OH) therapy was initiated on the day of transplantation and was tapered to 2 mg/kg per day over a 4-week period and maintained at this level thereafter. Operative Technique for the Donor (Harvesting of the Penile Iso and Allograft) A vertical midline incision was made starting from 0.5 cm above the base of the penis and was followed by circumferential incision around the penis leaving the mucosal preputium of the penis intact and ending at the midline about 0.5 cm inferior to the base of the penis (Fig. 1). Next, superficial external pudental arteries and veins were ligated bilaterally and both of the preputial glands were separated and the suspensory ligament of the penis was incised. The deep external pudendal artery and vein were also ligated bilaterally. The ischiocavernosus and bulbocavernosus muscles were incised and cauterized to relieve the penis. On the dorsum of the penis, 10 mm of dorsal penile vein was dissected and identified with the accompanying dorsal penile nerves bilaterally (Fig. 2). Both of the dorsal penile arteries of the penis were cauterized. Then, the ventral surface of the penis was exposed and 10 mm of corpus spongiosum was dissected and isolated from the penile shaft and transected just distal to the bulb of the penis (Fig. 3). After dissection, corpus spongiosum formed the arterial pedicle, the dorsal penile vein formed the venous pedicle, and the dorsal nerves of the penis formed the neural pedicle. When the isolation of the pedicles was completed both of the crura were ligated and transected proximal to the symphysis pubis, and composite penile graft was created ready for transplantation (Fig. 4). FIGURE 2. View of the isolated dorsal penile vein (DPV), and dorsal penile nerves (DPN) on the milimetric scale under operating microscope. (⫻8, Zeiss OP-MI, 6SD; Carl Zeiss, Goettingen, Germany). Operative Technique for the Recipient To reduce the morbidity and mortality of the recipient rats, instead of performing amputation of the recipient penis we have introduced modification of rerouting of the recipient penis into the scrotum. Otherwise, amputation of the penis causes hemorrhage and FIGURE 3. View of the isolated corpus spongiosum (CS) on the milimetric scale under operating microscope. CC, corpus cavernosum; P, preputium (⫻8, Zeiss OP-MI, 6SD; Carl Zeiss, Goettingen, Germany). FIGURE 1. Skin incisions planned for penile composite tissue harvesting. © 2009 Lippincott Williams & Wilkins urinary tract infections in the recipient rat during the posttransplantation period. The rerouting technique of the penis was performed as follows: a vertical midline incision 2 cm long was made on the anterior surface of the scrotum. Next, a circumferential incision was made leaving a small amount of preputial skin on the penis. The penis was dissected and released from the surrounding tissues and rerouted to the most distal part of the scrotum passing through the testicle. The preputial skin surrounding the glans of the penis was sutured to the scrotal skin after making a small opening (0.5 cm in diameter) within the scrotal skin. After this maneuver, the preputial skin created a hollow tube as a bed for the transplanted penis insertion (Fig. 5). 305 Sonmez et al Annals of Plastic Surgery • Volume 62, Number 3, March 2009 FIGURE 4. The penile composite tissue ex vivo. FIGURE 6. View of the anastomoses on the milimetric scale under operating microscope. SA, saphenous artery; CS, corpus spongiosum; DPV, dorsal penile vein; SV, saphenous vein; DPN, dorsal penile nerve; LFCN, lateral femoral cutaneous nerve (⫻8, Zeiss OP-MI, 6SD; Carl Zeiss, Goettingen, Germany). FIGURE 5. View of the penis (p) that was rerouted to the most distal part of the scrotum passing through the testicle (t). After rerouting of the penis to the scrotum an incision of 2 cm in length was made in the right inguinal region of the rat. The saphenous artery and vein were prepared as recipient vessels and the right femoral cutaneous nerve of the rat was prepared as the recipient afferent nerve. Next, the saphenous vessels were rerouted to the pubic region through a subcutaneous tunnel that was created between the scrotal and inguinal incisions. The saphenous artery of the recipient was anastomosed directly to the corpus spongiosum, and the saphenous vein of the recipient was anastomosed to the dorsal penile vein and finally the right femoral cutaneous nerve of the recipient was coaptated to the right dorsal penile nerve of the transplanted penis by using standard microsurgical techniques under operating microscope (⫻10 magnification) with 10/0 nylon sutures. After completion of microanastomoses the transplanted penis was positioned into the tube formed by the preputial skin of the recipient rat and the preputial skin surrounding the glans of the transplanted penis was attached here using 6/0 absorbable sutures. The inguinal and scrotal incisions were closed using 4/0 absorbable sutures (Figs. 6 – 8). Techniques of Evaluation All allotransplants were evaluated clinically for viability and clinical signs of rejection on a daily basis until 100 days posttransplant. 306 FIGURE 7. Drawing of the design of the transplantation procedure. Somatosensory Evoked Potential Somatosensory Evoked Potential (SSEP) analysis was performed for the evaluation of the sensory recovery of the penile allotransplant, while the rat was under pentobarbital anesthesia. A Bio-Logic A-PAC 486 computer (Bio-Logic Systems Corp, Chicago, IL) was used for testing. Thirty to 1500 Hz was used for bandpass filter settings and the grain setting was placed at 3000. Stimulus duration was set to 200 ␮s with a frequency of 2.7 per second and each response was replicated at least once. A display of © 2009 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 62, Number 3, March 2009 FIGURE 8. View just after the operation. p, penis; pa, penis allotransplant. 100-millisecond window was used. The 2 stimulating electrodes were placed on the glans of the penile allotransplant. The ground electrode was placed to the subcutaneous plane of the tail. Next, a midline scalp incision of 2 cm in length was performed on the sagital suture and the parasagital regions of the parietal bones were exposed. Two burr holes were drilled on both sides of the sagittal suture and the recording electrodes were placed over the parietal cortex through these holes. The cortical responses were recorded and each average consisted of 300 trials. The waveform morphology consisted of a series of negative and positive potentials in the SSEP measurements. An initial negative wave (N1) was followed by a positive waveform (P1) and a second negative waveform (N2) in a charactheristic waveform pattern. Microangiography Microangiography was performed by using the technique described by Rees at al.8 Briefly, intraarterial infusion of lead oxide-gelatin mixture was performed via catheterization of the left femoral artery. Then, the penile allograft specimen was harvested and underwent radiography with a soft x-ray machine (Mammo Diagnost UC, Philips, Hamburg, Germany) at the settings of 22 kV and 5 mAs. Penis Allotransplantation Model in the Rat deep penile artery enters the crura of the corpus collosum of the penis and the artery of the bulb enters the urethral bulb of the penis at the base of the corpus spongiosum of the penis. These 3 arterial systems are connected to each other via small branches along the shaft of the penis but dorsal penile arteries and the artery of the bulb of the penis are mainly connected with major branches at the glans penis. The detailed anatomy of the vascular supply of the penis is reported elsewhere.9 Instead of following the main vascular branches supplying the penis proximally to the internal pudendal vessels, as described before by Akyurek et al10 and Karamursel et al,11 we saw that the rat penis can be successfully arterialized by end-to-end anatomosis of the corpus spongiosum of the penis to the saphenous artery. The average diameter of the corpus spongiosum at the midshaft level measured 1.2 mm and it could be easily anastomosed to the saphenous artery (0.9 mm in diameter). Also, the diameter of the dorsal penile vein at the midshaft level (1.4 mm in diameter) was suitable for anastomosis to the saphenous vein (1.2 mm in diameter). After dissection of the lateral femoral cutaneous nerve (0.7 mm in diameter) it could be easily rerouted to the inguinal region and was suitable for coaptation to the afferent recipient nerve (dorsal penile nerve; 0.6 mm in diameter). The major blood supply to the perineal skin and the preputium of the penis comes from the skin branch of the superficial external pudendal vessels. There is a delicate network of connections between these vessels and the vascular network of the penile body passing from the mucosal preputium.10 Isotransplant Group (Group 2) All vascularized isotransplants survived completely (n ⫽ 6) whereas all nonvascularized isotransplants necrosed at day 3 posttransplant (Fig. 9). There were no mortalities recorded in the vascularized isotransplant group. The minimum follow-up period in the vascularized isotransplant group was 70 days (n ⫽ 1) and the rest of the rats survived up to 100 days posttransplant and presented with completely viable penile graft tissues. Allotransplant Group (Group 3) All allotransplants survived completely (n ⫽ 6) without signs of acute or chronic rejection up to 100 days posttransplant (Figs. 10, 11). Somatosensory evoke potential evaluation tests (SSEP) confirmed that at day 60 posttransplant, stimulation of the glans of the transplanted penis revealed cortical responses recorded in the so- Histology After microangiographic evaluation of the penile allograft specimen, it was paraffin embedded, sectioned, placed on slides, and the slides dried in an oven. Then, the slides were processed with xylene, alcohol, and water for hydration. Next, the slides were stained with hematoxylene and rinsed, then stained with eosin. Again they were processed with water, alcohol, xylene, and finally coverslipped. RESULTS A total of 18 penile transplantations (12 isograft and 6 allograft) were performed. The procedure required an average of 2 hours and the ischemia time was approximately 30 minutes. Anatomic Studies The anatomic dissections in group 1 of the study revealed that the penises of the rats were supplied by 3 branches of the internal pudendal artery. These branches included the dorsal penile arteries, deep artery of the penis, and the artery of the bulb of the penis. The dorsal penile arteries were accompanying the deep dorsal vein of the penis. The average diameters of the dorsal penile artery and deep dorsal penile vein were 0.3 mm and 1.4 mm, respectively.9,10 The © 2009 Lippincott Williams & Wilkins FIGURE 9. Necrosis of the nonvascularized isotransplants on the posttransplant 3rd day. 307 Annals of Plastic Surgery • Volume 62, Number 3, March 2009 Sonmez et al FIGURE 10. Allotransplant on 7th posttransplant day. p, penis; pa, penis allotransplant. FIGURE 11. Allotransplant on 100th posttransplant day. p, penis; pa, penis allotransplant. matosensory cortex of the recipient rat. The mean values of positive (P1, P2) and negative (N1, N2) peaks recorded in SSEP after the onset of the glans stimulation are as follows: P1, –2.8 ⫾ 1.85 uV; P2, –1.5 ⫾ 0.81 uV; N1, 3.0 ⫾ 1.59 uV; N2, 2.6 ⫾ 1.23 uV. The mean values of positive (P1, P2) and negative (N1, N2) latencies were as follows: P1, 28.1 ⫾ 1.54 milliseconds; P2, 27.5 ⫾ 3.2 milliseconds; N1, 18.1 ⫾ 3.4 milliseconds; N2, 30.5 ⫾ 1.87 milliseconds. Moreover, we clinically observed that the rats responded with moving their right leg in response to the physical stimulation of the glans of the transplant. These confirmed the successful afferent innervation of the penile allograft. Microangiographic evaluation of the transplanted penile tissues with lead oxide-gelatin mixture demonstrated well-preserved vascular territories of the transplanted penile allograft at 100 days posttransplant12 (Fig. 12). Histologic evaluation of the transplanted tissues confirmed that there were no signs of rejection at posttransplant day 100 and the tissues were completely viable. No fibrosis, no atherosclerosis, and no infiltration with inflammatory cells were observed. Besides, it was observed on the histologic slides that the artery of the urethral bulb, cavernous structures of the corpus spongiosum, the dorsal penile arteries, and the other small arterial branches were filled with the lead oxide-gelatin mixture, which was infused via intraarterial route (Fig. 13). the end of the second week posttransplant, the medical and ethical debates were raised around the world about justification of this procedure. According to some authors it was an experimental study performed in a human subject and more experimental studies are needed before clinical trials will be initiated.13 For this reason, experimental models are needed to study technical and immunologic aspects of penile allotransplantation. The only experimental allogenic penile allotransplantation model reported in the literature is a nonvascularized transplantation model.14 In this study, the entire penis was transplanted from an adult Brown-Norway rat into a pouch created in the omentum of an adult Lewis rat as a nonvascularized graft. The only way to monitor the transplanted tissue in this model was via laparotomy. After vascularization of the transplant from the omentum in which the penis was covered, authors claimed that it could be transplanted with omentum. However, the isolation of the penile transplant from the omentum was difficult since its vascularization was supplied only by the omentum, not by the penile vascular pedicle, and for this reason experimental and clinical application of this model is not practical. There are 2 replantation models reported in the literature reported by Akyurek et al10 and Karamursel et al11 in 2005. In both of these models, the penile flap was based on the internal pudendal artery. These models can be also used in experimental allogenic penile transplant studies but as authors mentioned, elevation of the penis of the rat based on the internal pudendal artery was a very demanding operation and the free flap transfer of the penis in the rat lasted between 8 to 10 hours.11 When we compare these reports with our model, neither the dissection of the penile dorsal vein and corpus DISCUSSION Following the report of the first clinical penile allotransplantation performed in China, which required allograft amputation at 308 © 2009 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 62, Number 3, March 2009 FIGURE 12. Allotransplant filled with intra arterial lead oxidegelatin mixture (upper), and microangiographic view (lower). SA, saphenous artery; CS, corpus spongiosum; DPV, dorsal penile vein; SV, saphenous vein; DPN, dorsal penile nerve. Penis Allotransplantation Model in the Rat spongiosum nor the microanastomoses performed in our model were considered as demanding operations and the entire transplantation procedure could be accomplished within 2 hours. Because of the short duration of the transplantation, the ischemia time was short (mean 30 minutes) and the morbidity and mortality in the recipient rats was minimal. Besides, there was no need to use old male rats weighing between 400 –500 g and all rats in our study weighed between 250 –300 g.10 The detailed description of the vasculature of the rat penis was reported by Fernandez et al9 in 1991. There are delicate connections between the vascular network of the penile body and the preputial and perineal skin, which were supplied by the superficial external pudendal vessels.10 In our opinion, these connections were not reliable for the vascularization of the entire preputium and the perineal skin. For this reason, we preferred to transplant the penis only with the mucosal portion of the preputium, which can be easily supplied by the branches coming from the vascular network of the penile body. Another advantage of this was that, by decreasing the skin within the allotransplant, we have decreased the antigenic load and immune responsiveness of the allotransplant. It has been documented that skin presents the highest antigenicity within all tissues of composite allografts and is followed by muscle, bone, nerve, tendon, and vessels.15 We were able to achieve 100% survival of penile allografts up to 100 days posttransplant on low dose CsA monotherapy confirming feasibility to transplant this highly vascularized allograft. In our model, we provided arterial blood supply to the penis via saphenous artery-corpus spongiosum end-to-end anastomosis. To the best of our knowledge, this is the first report on arterial blood supplied to a vascularized graft without direct vessel-to-vessel anastomosis. In our model, the saphenous artery was directly anastomosed to the corpus spongiosum, which consists of the artery of the urethral bulb, cavernous sinuses, and the urethra. In the histologic sections taken after lead oxide-gelatin intraarterial infusion, the artery of the urethral bulb and cavernous sinuses were filled with the lead oxide-gelatin mixture whereas the urethra was not at day 100 posttransplant. This confirms the successful supply of the arterial blood to the transplanted penis from the corpus spongiosum, which was patent for more than 3 months after transplantation. This is an important technical finding that can be applied to clinical cases of penile transplantation. CONCLUSIONS In this study, a new penis allograft transplantation model in the rat was developed and feasibility of vascularized penile allotransplantation was confirmed. A new method of blood supply to a vascularized composite tissue with direct artery-to-corpus spongiosum anastomosis was introduced. Long-term survival without signs of rejection was achieved on tapered dose Cyclosporin A monotherapy and viability of allograft was confirmed by microangiography and histologic evaluation. Finally, the sensory function of the penile allograft was confirmed by somatosensory evoked potentials. REFERENCES FIGURE 13. Histologic examination of the penile tissue allotransplant on the 100th posttransplant day. Note the dorsal penile arteries (DPA), artery of the urethral bulb (AUB) and cavernous structures of the corpus spongiosum (arrows) filled with leadoxide (⫻10, hematoxylin and eosin staining). U, urethra; DPN, dorsal penile nerve. © 2009 Lippincott Williams & Wilkins 1. 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Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine. Transplantation. 2004;77:514 –521. © 2009 Lippincott Williams & Wilkins