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Investigation on the Model of Beagle Dog’s Allogenic Penile Transplantation

Author: ZhaoYongBin
Tutor: HuWeiLie
School: Southern Medical University,
Course: Department of Urology
Keywords: penis anatomy allograft transplantation model pathology
CLC: R699
Type: PhD thesis
Year: 2012
Downloads: 42
Quote: 0
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As the external genitalia of male, penis has both urination and sexual function, through which part of the urethra goes. Complete or partial penis defect caused by war injuries, trauma, tumor or congenital reasons often leads to the loss of micturition and copulation function and would result in huge trauma to the patient emotionally and psychologically. That makes advanced penial rehabilitation extremely important. The ideal treatment should achieve with satisfactory appearance and feeling, unobstructed standing urination, and successful copulation. Penile lengthening and penile reconstruction surgery are used to solve standing urination and sexual problems of the patients. Penis lengthening with cut penis hanging shallow ligament and1/3-2/3penis deep suspensory ligament. It is reported that penis can be extended to3.2~5.0cm (average4.1cm) and0.8~1.2cm respectively through hanging shallow ligament and penis deep suspensory ligament. Due to the extension penis length which is still less than5.0cm, damages of penile deep dorsal vein, dorsal arteries and nerves are usually caused by surgery. Although they could achieve standing urination, but still have significant flaws including poor appearance of the glans, the limited length and erectile dysfunction. Penile reconstruction, originated from Bagoras’successful trying on abdominal skin tube transfer to create a penis in1936, now has developed dozens of surgical procedures, mainly includes the tubed flap method and skin flap method. Although phalloplasty can achieve standing urination and successful copulation, but the disadvantage is obvious that phalloplasty requires multiple surgeries and implantation and always lead to unpleasant appearance. Theoretically, the best way to solve this worldwide problem is re-creating a penis of composite tissue through tissue engineering and cloning technology. However, tissue engineering and cloning technology are still century-old puzzle in the21st century. There is still quite a longtime to go when they can be transited to the clinical. In addition, other tissues of the body are not a substitute for the unique erectile cavernous tissue in penis, so a new approach to the treatment of male penis defect seems to be imminent.Lee made a historical review on genital transplantation experiments, animal studies, including testicular transplantation, ovarian transplantation, and whole vagina-urethra-ovarian transplantation over the past25years, and reached to a suspect that allogeneic penis transplant, if ethical recognized, would become a reality in the near future. Koga transplanted the penis of adult Brown-Norway rat into the capsular space made of Omentum of adult Lewis rat (fully allogeneic). Then the Lewis rats were divided into two groups:FK+group, FK-group. In FK+group, the rats were intraperitoneal injected immunosuppressive drugs (FK506) on the day of3,5,7,10,14or21days after transplantation, while the FK-group did not use FK506, and meanwhile set the rats that received autologous penile replantation as control group.Then the transplants were obtained and all transplants were stained for histological observation. It was found that penis transplanted in the omentum generates a cylindrical material. The graft survival rate in control group and the FK+group were100%. The rejection in FK+group three days and five days after the penis transplant is mild to moderate, and is mild to disappear7,10,14and21days after transplantation. HE staining of the penis structures in FK+group and control group, are normal, but a large number of cellular infiltrations can be found in the FK-group graft indicating transplant rejection. Koga concluded that FK506can successfully prevent rejection in the allogeneic penis transplant, and it indicated prospects for the establishment of the penile tissue technology which can be used in penile reconstruction. Mouse is not primates-animal and its penis is quite different with people. In addition, due to ethical reasons,there are few researches involved in this field, and until now no animal experiments studies had been reported on topic of allogeneic penis transplantation both at home and abroad. Therefore, how to find a cure way not only has satisfactory appearance and feeling, but can also retain unobstructed standing urination and successful copulation, seemed important. At the present stage, allogeneic penile transplantation is most likely to be the ideal treatment.We chose adult beagle dogs as experimental subjects, and tried to confirm that allogeneic transplanted penis can survive, and result in recovery of urinary function. We also try to establish a stable surgical animal model of penile tissue immunosuppressive immune pathological changes, in aim of providing a strong theoretical basis for the further transition to human allogeneic penile transplantation.The article was divided into three parts.Part1A controlled study on microsurgical technique assisted penile replantation of amputated penis in adult Beagle dogsObjection:To explore the success rate of reanastomosis of penile blood vessels and nerve as well as the survival rate of replantation of the penis assisted by microsurgical technique in beagle dogs, and to assess the efficiency of microsurgical techniques in penile replantation.Methods and materials:20adult beagle dogs with amputated penis were randomly divided into2groups, observation group (n=10) and control group (n=10). Penile replantation was carried out under10-time-magnifying microscopy in the observation group, and surgeries were carried out using the naked eye in control group. Surgeries in both groups were operated by the same operator and with the same standard procedures. Several indexes were recorded and analyzed including operative time, intraoperative blood loss, vascular anastomosis, success case of dorsal penile vascular anastomosis, and the distal penile color, temperature and distal penile necrosis after operation.Results:38success cases (including20veins and18arteries) of vascular anastomosis were recorded in observation group, in comparison with22cases (including14veins and8arteries) in control group (vein, p=0.020; artery, p=0.002). The average operative time and average vascular anastomosis time of observation group were, respectively,117.4minutes and65minutes, as compared with103.7minutes and52.5minutes in control group (operative time, p=0.003; vascular anastomosis time, p=0.002). The average blood loss was85ml in observation group,while it is68ml in control group (p=0.003). There was no penile necrosis case with a survival rate of100%in observation group, while there were3cases penile necrosis in control group with a survival rate of70%.Conclusions:Microsurgical technique assisted penile replantation can significantly improve the success rate of vascular anastomosis, improve distal penile blood supply, shorten the distal penis recovery time, and improve the survival of distal penis.Part2Establishment of allogeneic penile transplantation animal modelObjection:To investigate the feasibility to establish an allogeneic penile transplantation animal model in adult beagle dogs. And to explore the modeling conditions of a stable penis transplant animal model.Methods and materials:The research of this part was carried out in accordance with experimental animal models principles as follows.1. Similarity study:20adult beagle dogs’ penis were dissected and several anatomy features including the structure of cavernous body of penis and corpus cavernosum urethrae, the main blood supply and neural structures, and cross-sectional anatomy were all recored. Human adult penis (10cases) was also dissected and anatomy features mentioned above were also recorded. Through the comparison of anatomical features of beagle dogs and human beings, we analyzed its common characteristics and similarities, as well as different points and otherness.2. Repeatability and feasibility studies:10pairs of adult beagle dogs with kinship were selected as experimental subjects, of each pair of beagle dogs received cross-penis transplant with the microsurgical technique. Blood lymphocyte toxicity test and MP+FK506+MMF immune induction were routine carried out preoperative. The surgical procedure has been described in part1:consistent on both sides of penis deep dorsal vein, dorsal artery and accompanying nerve, continuous suture of the corpora cavernous and corpus spongiosum was sutured subcutaneous skin and restore the penis continuity. Indwelling10F catheter drainage of urine, at the first three days after surgery dexamethasone was given7.5mg/kg through intravenous administration in aim of immune induction, three days later it was changed to0.2mg/kg/d intramuscularly. Additionally, FK506(1mg/kg/d, gavage)+MMF (20mg/kg/d, intravenous) were used as maintenance immunosuppressive therapy. At the same time antibiotics, anti-infection, and the proton pump inhibitors, nutritional support were also carried out. After surgery the change in color of skin and the degree of tissue swelling of glans were closely observed. The catheter was removed seven days after surgery, observe and record the animals’ daily frequency of urination, urinary stream and patency. The urethrography check was taken10days after surgery. Transplanted penis was removed for pathology observation14days after transplantation. During observation period If the color of glans of penis become white or penial skin color become black, or significant tissue swelling were observed, the transplanted penis should immediately be removed for pathological observation.3. Applicability and controllability study:Penis transplant operation time, intraoperative vascular anastomosis time, intraoperative blood loss and other indicators of the20beagle dogs were recorded, and all the indicators were compared with that of the dogs which received reanastomosis of amputated penis using microscopy and that of clinical exalted penis replantation surgery. Analysis differences in operability of adult beagle dogs experimental subjects as allogenic penile transplantation model.Results:1.20beagle dogs penis anatomy observation:glans penis of beagle dogs, which contain cartilage tissue, is longer than human beings at a length of about7cm. Corpus cavernosum can be probed at the scrotum beginning part of the penis. The average penis diameter is of about2.0cm. It contains left and right side cavernous body of penis and corpus spongiosum, separated by dense albuginea. The corpus spongiosum is relatively weak, and the urethral diameter is of about12F. The dorsal penis existing symmetrical penis back vein, artery and nerve bundle. The average diameter of dorsal vein and artery is1.2mm and0.5mm, respectively. The penis length could be up to5cm when the root of the penis was sufficiently dissected. Penis anatomical observations of10adult human beings:the average penis diameter is of3.5cm. Cavernous body of penis and corpus spongiosum, which were encircled and separated by dense albuginea, could be seen from the cross section. The major blood vessels, those are deep dorsal vein and dorsal artery, present in the dorsal penile with the average diameter of about1.3mm and0.5mm, respectively. And a number of nerve fibers could be found accompanied by those vessels with a diameter of about0.3mm. The penile anatomy of beagle dogs and human beings has highly similarity, no significant statistical difference was found between them.2. All the results of preoperative paired blood lymphocytotoxicity test of experimental beagle dogs were negative. Of all20penile transplanted beagle dogs,2were found becoming tissue swelling and pale in the glans and the penis skin on the first postoperative day, these two penises were removed for pathology examination. Three cases were found glans necrosis. Penis skin color becoming stasis black and organize mild swelling on postoperative day3. On postoperative day5, another3case were found glans becoming black and dry, penis skin color becoming stasis black without obvious swelling. In the remaining12cases, the glans of penises were red-colored, mild pale-colored penis skin, and the surrounding tissue were not swelling, the incision healed well. On postoperative day7, urethral catheter was withdrew all these12dogs micturated normally with continual urinary stream and varying degrees of terminal dribbling. Urethral stricture was not found by cystography and urethrography. On postoperative day14, all the transplanted penises were removed and sent for pathology examination.3. Of20dogs with penile transplant surgery, one-time vein anastomosis consistent success rate under microsurgical technique was95%(38/40), and one-time artery anastomosis success rate was87.5%(35/40). The average vascular anastomosis time was70.95min. The mean operative time was133min, with an average blood loss of135.75ml. Of10cases of penile replantation surgery assisted by microsurgical techniques, the one-time vein anastomosis success rate was100%(20/20), and one-time artery anastomosis success rate was90%(18/20), with an average vascular anastomosis time of65min and an average operative time of117.4min. The average blood loss was85ml. Of12cases of clinical penis replantation surgery under microsurgical technique, one-time vein and artery anastomosis success rate was100%(24/24) and95.8%(23/24), respectively. The average vascular anastomosis time was79min, and the mean operative time was125min, while the mean blood loss was140ml. Data of these three groups were statistically tested by correction completely randomized single factor analysis of variance (Welch method), Games-Howell test and Fisher’s Exact test, results indicated that no significant statistical differences were found between penis transplantation group of beagle dogs and clinical penile replantation group.Conclusion:Anatomy structure of corpus cavernosum penis in beagle dogs and human has highly similarity to each other. The major anatomical structure in cross-sectional of the penis in beagle dogs and human beings are almost the same. Penile replantation and allograft transplantation assisted by microsurgical technique both achieved success. In comparison with the indicators of clinical human penile replantation surgery, beagle dogs’ surgery model has similar operability, repeatability and strong feasibility. Therefore, it is feasible that setting adult beagle dogs as experimental animals to build penis transplant model.Part3Histological change under microscopy of beagle dogs after allogeneic penile transplantationObjection:To explore the major pathological changes of rejection in allogeneic penile transplantation, and the pathological features of transplant penis in immunosuppressive state.Methods and materials:Dissecting corpus cavernosum penis tissue at the thickness of3mm of normal beagle dogs the necrosis penis on post-transplant day1,3and5, as well as the survival penis transplant after14days, then those samples were embedded in paraffin, routine sectioned, HE staining under the microscope. Penile histopathological changes of different periods were recorded and analyzed to investigate its causes and clinical significance.Results:Of the8cases of necrotic transplant penile postoperative, one was confirmed to be suppurative infections, two cases appeared dry gangrene caused blood clots, the remaining five cases showed varying degrees of rejection. Pathology of the suppurative infections case showed:visible penis head atrophy, skin color stasis black, tissue swelling, subcutaneous purulent necrotic liquefied exudate, a large number of the purulent cell infiltration in the skin, subcutaneous, the corpus cavernosum, interstitial edema, degeneration, cavernous sinusoids disappear, and the formation of abscess. Pathological manifestations of the two cases of dry gangrene penis showed:visible glans penis color becoming stasis black and dry, the black penis skin, formation of hard crusts, the formation of a large number in the skin and hypodermis, vascular thrombosis, cavernous sinusoids disappear, tissue degeneration obvious. Pathological manifestations of five cases of rejection of transplanted penis showed:pale-color glans penis,pale or mild stasis black skin, hypodermis and corpus cavernosum swelling significantly, inflammatory cell infiltration within the organization, occlusion of small blood vessels, thrombosis part of the vessels, interstitial edema, degeneration, cavernous sinuses structural damage. Pathological findings of the12cases of survived penis transplants showed:the ruddy color of the glans penis, little white skin color. Through the cross-section it showed that subcutaneous tissue with mild swelling, bleeding and abnormal exudation, structural integrity of the corpus cavernosum; light microscope, good skin structure, subcutaneous tissue mild swelling, intravascular thrombosis, cavernous sinuses with normal structure, infiltration of inflammatory cells within the organization, no stromal degeneration necrosis.Conclusions:The survival of transplanted penis was influenced by a variety of factors. The major pathological changes of rejection in penis transplant are skin and subcutaneous tissue interstitial edema,degeneration, cavernous sinuses structural disorder, and vascular occlusion or thrombosis. The pathological features of normal immunosuppression penis transplant are normal skin structure,subcutaneous tissue mild oedema, no intravascular thrombosis,normal cavernous sinuses structure, infiltration of inflammatory cells within the organization, no stromal degeneration necrosis.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Urinary tract and male reproductive system surgery
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