比格犬阴茎移植模型探讨
摘要
阴茎为男性的外生殖器官,尿道的一部分穿行其内,具有排尿和性交功能。战伤、创伤、肿瘤切除或先天性原因所致阴茎完全或部分缺损,往往使患者丧失了男性的排尿和性交能力,给患者精神和心理上造成巨大的创伤,亦给患者及家庭带来了沉重的压力。因此后期的阴茎康复治疗极为重要,理想的治疗应达到具有良好的外形和感觉、通畅的站立排尿、顺利完成性交三个方面的和谐统一。治疗上目前主要采取阴茎延长术(Penile Lengthening)和阴茎再造术(Penile Reconstruction)来解决患者的站立排尿和性生活问题。阴茎延长术采用切断阴茎浅悬韧带和1/3-2/3阴茎深悬韧带使得阴茎达到延长,据报道两者可分别延长3.2-5.0cm(平均4.1cm)和0.8～1.2cm,由于延长的阴茎长度小于5.0cm,术中容易损伤阴茎背深静脉、背动脉和神经,虽然可以解决站立排尿,但存在阴茎头外观欠佳、长度有限和勃起功能障碍等问题；阴茎再造术自Bagoras于1936年创立腹部皮管转移再造阴茎成功后,目前有数十种手术方法,主要有皮管法和皮瓣法二大类方法,虽然成功的手术可以解决患者站立排尿和性生活,但仍存在需多次手术、外形不够美观和需植入阴茎支撑物等众多缺点。从理论上讲,目前解决阴茎缺损这一世界性难题最理想的方法是通过组织工程和克隆技术重新再造复合组织的阴茎。但组织工程和克隆技术是21世纪基础研究的世纪性课题,在相当长的时间里尚难以过渡到临床。且由于身体其它组织不能替代阴茎独有的可勃起的海绵体组织,因此寻找一种新的方法治疗男性阴茎缺损迫在眉睫。
国外Lee对过去25年来生殖器官移植的实验、动物研究如睾丸移植、卵巢移植、全阴道-尿道-卵巢移植等进行了历史回顾,设想认为如伦理上得到认可,不久的将来同种异体阴茎移植就会变为现实。Koga (?)将成年Brown-Norway大鼠阴茎移植到成年Lewis鼠(完全同种异体)网膜做成的囊腔,分成两组使用免疫抑制药物(FK506)：FK+组、FK-组；FK+组是阴茎移植术后3、5、7、10、14或21天后腹腔内注射FK506,获取移植物,FK-组为不使用FK506,自体阴茎再植作为对照组；所有移植物采用HE染色进行组织学观察；结果剖腹探查发现所移植的阴茎在网膜中生成了圆柱状物质,对照组和FK+组移植物成活率达100%,FK+组移植物在阴茎移植后3天和5天的排斥反应是轻到中度,在7、10、14和21天排斥反应是轻微到消失,FK+组和对照组HE染色的阴茎结构正常,在所有FK-组移植物中可发现大量细胞浸润的排斥反应；Koga认为FK506可成功预防同种异体阴茎移植的排斥反应,同时对于建立能成活的用于阴茎重建的阴茎组织技术具有发展前景。由于鼠非灵长类,对阴茎特殊器官来说与人相差较大,此外由于受伦理道德的影响,很少有人涉足此研究领域,目前国内外均未见有关同种异体阴茎移植的动物实验研究的报导。因此如何寻找一种治疗方法既具有良好的外形和感觉、通畅的站立排尿,又能顺利完成性交,从目前来说同种异体阴茎移植最有可能成为较为理想的治疗方法。
我们选用成年比格犬作为实验对象,试图通过本研究证实阴茎组织同种异体移植后能够存活,恢复排尿功能,建立稳定的手术动物模型,同时初步探讨免疫抑制下阴茎组织镜下病理改变,为进一步过渡到人类同种异体阴茎移植提供强有力的理论依据。
本来研究分以下3部分进行。
第1部分比格犬显微外科技术辅助下阴茎离断再植对照研究
目的：探讨显微外科技术辅助下比格犬阴茎血管、神经吻合成功率及再植阴茎存活率,评估显微外科技术在阴茎再植中的作用。
方法：20例成年比格犬进行阴茎海绵体离断后再植术；随机分成2组,观察组10例采用10倍放大镜显微外科技术辅助手术,对照组10例采用在肉眼直视下手术；手术方式为同一组手术成员采用标准术式,缝合阴茎海绵体、尿道海绵体,两侧阴茎背血管；比较两组手术时间,术中失血量、血管吻合时间、阴茎背血管吻合成功例数、术后远端阴茎颜色、温度、术后远端阴茎坏死例数等各项指标。
结果：观察组10例血管吻合一次成功例数高达38根(其中静脉20根、动脉18根),而对照组10例血管吻合一次成功例数达22根(其中静脉14根,动脉8根),两组比较有显著性差异(静脉组P=0.020、动脉组P=0.002)；观察组平均手术时间117.4分钟、平均血管吻合时间65分钟、平均失血量85ml,而对照组平均手术时间103.7分钟、平均血管吻合时间52.5分钟、平均失血量68ml,两组各项指标比较有显著性差异(P=0.003、0.002、0.003)。观察组术后阴茎坏死例数为0例,其存活率高达100%,而对照组阴茎坏死例数为3例,其存活率为70%。
结论：显微外科技术辅助阴茎离断再植可明显提高血管吻合成功率,显著改善远端阴茎血流供应,缩短远端阴茎恢复时间,提高远端阴茎存活率。
第2部分比格犬阴茎移植模型建立
目的：探讨成年比格犬作为同种异体阴茎移植动物模型可行性,进一步探讨稳定的阴茎移植动物模型建立条件。
方法：按照实验动物模型建立原则按以下几个方面进行研究。
1、相似性研究：
20例成年比格犬阴茎海绵体解剖,包括阴茎海绵体结构、尿道海绵体结构、主要血供及神经结构、横断面解剖学要点等。10例成年人阴茎海绵体解剖,包括阴茎海绵体结构、尿道海绵体结构、阴茎血管与神经结构、横断面解剖学要点等。通过两者在解剖学上的比较,分析其主要存在共同的特点和相似性,以及主要存在不同点和差异性。
2、重复性与可行性研究：
10对具有亲属关系的成年比格犬作为动物配对实验对象,每对比格犬在显微外科技术辅助下进行交叉阴茎移植,术前常规进行血淋巴细胞毒试验、MP+FK506+MMF免疫诱导,手术方式采用第1部分中所描述之标准方式：吻合两侧阴茎海绵体背深静脉、背动脉和伴行神经,连续缝合阴茎海绵体和尿道海绵体,间断缝合皮下、皮肤恢复阴茎连续性。术后留置10F导尿管引流尿液,术后三天每天给予地塞米松7.5mg/kg静脉滴注给药免疫诱导治疗、三天后改为0.2mg/kg/d肌肉注射给药,另外,联合应用FK506(按1mg/kg/d剂量采用灌胃法给药)+MMF(按20mg/kg/d剂量采用静脉滴注方式给药)免疫抑制维持治疗,同时予以抗生素抗感染、质子泵抑制剂保护胃粘膜、营养支持等综合治疗。术后严密观察并记录移植阴茎的皮肤及阴茎头颜色变化、组织肿胀程度,术后7天拔除导尿管,观察并记录动物每日排尿次数、尿线及通畅情况,术后10天予以膀胱尿道造影检查,术后14天切除移植阴茎进行病理学观察。若观察期间出现移植阴茎头颜色发白、阴茎皮肤颜色发黑、组织肿胀明显,则立即予以切除病理学观察。
3、适用性和可控性研究：
记录20例比格犬阴茎移植手术时间、术中血管吻合时间、术中失血量等指标,与同一组手术人员在显微外科技术辅助下行比格犬阴茎离断再植术10例进行以上各项指标的比较。并与临床上人阴茎再植手术以上相关指标进行比较。分析成年比格犬作为实验对象进行同种异体阴茎移植可操作性差异。
结果：
1、20例比格犬阴茎解剖学观察显示：阴茎头较长,约7cm,内含有软骨组织,在其阴囊起始部可探查到阴茎海绵体组织,阴茎平均直径约2.0cm,具有左右侧阴茎海绵体及尿道海绵体,有致密的白膜分隔,尿道海绵体较为薄弱,尿道直径约12F。阴茎背侧存在对称的阴茎背静脉、动脉及伴行神经束,背静脉平均直径为1.2mm,背动脉平均直径为0.5mm。在阴茎阴囊交界处充分游离阴茎长度可达5cm。10例成人阴茎海绵体解剖观察显示：阴茎平均直径3.5cm,横断面上可见左右海绵体及尿道海绵体,亦有致密白膜包绕并分隔,主要血管存在于阴茎背侧的背深静脉和背动脉,平均直径分别约1.3mmm和0.5mm,同时在血管旁尚可找到多条神经纤维伴行,直径约为0.3mm。两者解剖学上具有高度相似性,相互比较统计学上无显著性差异。
2、所有实验动物比格犬术前配对血淋巴细胞毒试验均为阴性。20例移植阴茎中,2例于术后第1天出现阴茎头颜色苍白、阴茎皮肤颜色苍白、组织肿胀明显,予以切除后送病理检查；3例于术后第3天出现阴茎头坏死脱落、阴茎皮肤颜色瘀黑、组织轻度肿胀,予以切除后送病理检查；3例术后第5天出现阴茎头颜色瘀黑、发干,阴茎皮肤颜色瘀黑,周围组织无明显肿胀；其余12例阴茎头颜色红润,皮肤颜色轻度苍白,周围组织无肿胀,切口愈合良好,于术后第7天拔除导尿管,观察排尿正常,小便呈线状,但存在不同程度的尿末滴沥症状,膀胱尿道造影检查无明显尿道狭窄,于术后第14天切除移植阴茎病理检查。
3、20例阴茎移植手术中,显微外科技术辅助下一次性静脉吻合成功率达95%(38/40),一次性动脉吻合成功率达87.5%(35/40),平均血管吻合时间70.95min,平均手术时间133min,平均失血量135.75m1。10例显微外科技术辅助下阴茎再植手术,一次性静脉吻合成功率达100%(20/20),一次性动脉吻合成功率达90%(18/20),平均血管吻合时间65min,平均手术时间117.4min,平均失血量85m1。12例临床显微外科技术辅助下阴茎离断性再植手术,一次性静脉吻合成功率达100%(24/24),一次性动脉吻合成功率达95.8%(23/24),平均血管吻合时间79min,平均手术时间125min,平均失血量140ml。3组资料采用校正的完全随机单因素方差分析(Welch法)、Games-Howell法以及Fisher's Exact法检验,结果显示比格犬阴茎移植组与人阴茎再植组各项指标相比较差异无统计学意义。
结论：比格犬阴茎海绵体结构与人阴茎海绵体结构具有高度相似性,在阴茎海绵体横断面解剖中其主要解剖结构均相同；显微外科技术辅助下行阴茎再植和同种异体移植手术均获得了成功,其手术操作过程中各项指标与临床中人阴茎再植手术相比较,具有相近的可操作性,重复性和可行性较强。因此,采用成年比格犬作为实验动物建立阴茎移植手术模型切实可行。
第3部分比格犬阴茎移植组织学镜下改变
目的：初步探讨同种异体阴茎移植术后移植阴茎发生排斥反应的主要病理学变化,以及正常免疫抑制状态下移植阴茎病理学特点。
方法：分别截取厚约3mm正常比格犬阴茎海绵体组织,阴茎移植后1天、3天、5天出现坏死的移植阴茎海绵体组织,以及阴茎移植后14天存活的移植阴茎海绵体组织,石蜡包埋,常规切片、HE染色后显微镜下观察。分别记录不同时期阴茎组织病理学改变及其特点。分析并探讨其原因和临床意义。
结果：术后移植阴茎坏死8例中,1例化脓性感染,2例血管栓塞出现干性坏疽,其余5例出现不同程度排斥反应。1例化脓性感染病理表现,肉眼可见阴茎头萎缩,皮肤颜色瘀黑,组织肿胀明显,皮下可见脓性坏死液化渗出,光镜下见皮肤、皮下、阴茎海绵体内大量脓性细胞浸润,间质水肿、退变,海绵体血窦消失、形成脓腔。2例干性坏疽阴茎病理表现,肉眼可见阴茎头颜色瘀黑、发干,皮肤颜色瘀黑,形成硬性痂皮,光镜下皮肤、皮下血管大量血栓形成,海绵体血窦消失,组织退变明显。5例移植阴茎排斥反应病理表现,阴茎头颜色苍白,皮肤颜色苍白或轻度瘀黑,皮下及海绵体肿胀明显,光镜下见组织内炎细胞浸润,小血管闭塞,部分血管内有血栓形成,组织间质水肿、退变明显,海绵体血窦结构破坏。12例移植阴茎存活良好病理表现,肉眼见阴茎头颜色红润,阴茎皮肤颜色稍白,横断面可见皮下组织轻度肿胀,无瘀血及异常渗出,海绵体结构完整；光镜下见皮肤结构良好,皮下组织轻度肿胀,血管内无血栓形成,海绵体血窦结构正常,组织内可见少量炎细胞浸润,未见间质退变坏死。
结论：移植阴茎存活受多方面条件所影响,阴茎移植排斥反应主要病理变现为皮肤、皮下组织间质水肿、退变,海绵体血窦结构紊乱,以及血管闭塞或血栓形成等特点。正常免疫抑制状态下移植阴茎病理学特点为皮肤结构良好,皮下组织轻度肿胀,血管内无血栓形成,海绵体血窦结构正常,组织内可见少量炎细胞浸润,未见间质退变坏死。
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 dogs
Objection: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 model
Objection: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 transplantation
Objection: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.
国外Lee对过去25年来生殖器官移植的实验、动物研究如睾丸移植、卵巢移植、全阴道-尿道-卵巢移植等进行了历史回顾,设想认为如伦理上得到认可,不久的将来同种异体阴茎移植就会变为现实。Koga (?)将成年Brown-Norway大鼠阴茎移植到成年Lewis鼠(完全同种异体)网膜做成的囊腔,分成两组使用免疫抑制药物(FK506)：FK+组、FK-组；FK+组是阴茎移植术后3、5、7、10、14或21天后腹腔内注射FK506,获取移植物,FK-组为不使用FK506,自体阴茎再植作为对照组；所有移植物采用HE染色进行组织学观察；结果剖腹探查发现所移植的阴茎在网膜中生成了圆柱状物质,对照组和FK+组移植物成活率达100%,FK+组移植物在阴茎移植后3天和5天的排斥反应是轻到中度,在7、10、14和21天排斥反应是轻微到消失,FK+组和对照组HE染色的阴茎结构正常,在所有FK-组移植物中可发现大量细胞浸润的排斥反应；Koga认为FK506可成功预防同种异体阴茎移植的排斥反应,同时对于建立能成活的用于阴茎重建的阴茎组织技术具有发展前景。由于鼠非灵长类,对阴茎特殊器官来说与人相差较大,此外由于受伦理道德的影响,很少有人涉足此研究领域,目前国内外均未见有关同种异体阴茎移植的动物实验研究的报导。因此如何寻找一种治疗方法既具有良好的外形和感觉、通畅的站立排尿,又能顺利完成性交,从目前来说同种异体阴茎移植最有可能成为较为理想的治疗方法。
我们选用成年比格犬作为实验对象,试图通过本研究证实阴茎组织同种异体移植后能够存活,恢复排尿功能,建立稳定的手术动物模型,同时初步探讨免疫抑制下阴茎组织镜下病理改变,为进一步过渡到人类同种异体阴茎移植提供强有力的理论依据。
本来研究分以下3部分进行。
第1部分比格犬显微外科技术辅助下阴茎离断再植对照研究
目的：探讨显微外科技术辅助下比格犬阴茎血管、神经吻合成功率及再植阴茎存活率,评估显微外科技术在阴茎再植中的作用。
方法：20例成年比格犬进行阴茎海绵体离断后再植术；随机分成2组,观察组10例采用10倍放大镜显微外科技术辅助手术,对照组10例采用在肉眼直视下手术；手术方式为同一组手术成员采用标准术式,缝合阴茎海绵体、尿道海绵体,两侧阴茎背血管；比较两组手术时间,术中失血量、血管吻合时间、阴茎背血管吻合成功例数、术后远端阴茎颜色、温度、术后远端阴茎坏死例数等各项指标。
结果：观察组10例血管吻合一次成功例数高达38根(其中静脉20根、动脉18根),而对照组10例血管吻合一次成功例数达22根(其中静脉14根,动脉8根),两组比较有显著性差异(静脉组P=0.020、动脉组P=0.002)；观察组平均手术时间117.4分钟、平均血管吻合时间65分钟、平均失血量85ml,而对照组平均手术时间103.7分钟、平均血管吻合时间52.5分钟、平均失血量68ml,两组各项指标比较有显著性差异(P=0.003、0.002、0.003)。观察组术后阴茎坏死例数为0例,其存活率高达100%,而对照组阴茎坏死例数为3例,其存活率为70%。
结论：显微外科技术辅助阴茎离断再植可明显提高血管吻合成功率,显著改善远端阴茎血流供应,缩短远端阴茎恢复时间,提高远端阴茎存活率。
第2部分比格犬阴茎移植模型建立
目的：探讨成年比格犬作为同种异体阴茎移植动物模型可行性,进一步探讨稳定的阴茎移植动物模型建立条件。
方法：按照实验动物模型建立原则按以下几个方面进行研究。
1、相似性研究：
20例成年比格犬阴茎海绵体解剖,包括阴茎海绵体结构、尿道海绵体结构、主要血供及神经结构、横断面解剖学要点等。10例成年人阴茎海绵体解剖,包括阴茎海绵体结构、尿道海绵体结构、阴茎血管与神经结构、横断面解剖学要点等。通过两者在解剖学上的比较,分析其主要存在共同的特点和相似性,以及主要存在不同点和差异性。
2、重复性与可行性研究：
10对具有亲属关系的成年比格犬作为动物配对实验对象,每对比格犬在显微外科技术辅助下进行交叉阴茎移植,术前常规进行血淋巴细胞毒试验、MP+FK506+MMF免疫诱导,手术方式采用第1部分中所描述之标准方式：吻合两侧阴茎海绵体背深静脉、背动脉和伴行神经,连续缝合阴茎海绵体和尿道海绵体,间断缝合皮下、皮肤恢复阴茎连续性。术后留置10F导尿管引流尿液,术后三天每天给予地塞米松7.5mg/kg静脉滴注给药免疫诱导治疗、三天后改为0.2mg/kg/d肌肉注射给药,另外,联合应用FK506(按1mg/kg/d剂量采用灌胃法给药)+MMF(按20mg/kg/d剂量采用静脉滴注方式给药)免疫抑制维持治疗,同时予以抗生素抗感染、质子泵抑制剂保护胃粘膜、营养支持等综合治疗。术后严密观察并记录移植阴茎的皮肤及阴茎头颜色变化、组织肿胀程度,术后7天拔除导尿管,观察并记录动物每日排尿次数、尿线及通畅情况,术后10天予以膀胱尿道造影检查,术后14天切除移植阴茎进行病理学观察。若观察期间出现移植阴茎头颜色发白、阴茎皮肤颜色发黑、组织肿胀明显,则立即予以切除病理学观察。
3、适用性和可控性研究：
记录20例比格犬阴茎移植手术时间、术中血管吻合时间、术中失血量等指标,与同一组手术人员在显微外科技术辅助下行比格犬阴茎离断再植术10例进行以上各项指标的比较。并与临床上人阴茎再植手术以上相关指标进行比较。分析成年比格犬作为实验对象进行同种异体阴茎移植可操作性差异。
结果：
1、20例比格犬阴茎解剖学观察显示：阴茎头较长,约7cm,内含有软骨组织,在其阴囊起始部可探查到阴茎海绵体组织,阴茎平均直径约2.0cm,具有左右侧阴茎海绵体及尿道海绵体,有致密的白膜分隔,尿道海绵体较为薄弱,尿道直径约12F。阴茎背侧存在对称的阴茎背静脉、动脉及伴行神经束,背静脉平均直径为1.2mm,背动脉平均直径为0.5mm。在阴茎阴囊交界处充分游离阴茎长度可达5cm。10例成人阴茎海绵体解剖观察显示：阴茎平均直径3.5cm,横断面上可见左右海绵体及尿道海绵体,亦有致密白膜包绕并分隔,主要血管存在于阴茎背侧的背深静脉和背动脉,平均直径分别约1.3mmm和0.5mm,同时在血管旁尚可找到多条神经纤维伴行,直径约为0.3mm。两者解剖学上具有高度相似性,相互比较统计学上无显著性差异。
2、所有实验动物比格犬术前配对血淋巴细胞毒试验均为阴性。20例移植阴茎中,2例于术后第1天出现阴茎头颜色苍白、阴茎皮肤颜色苍白、组织肿胀明显,予以切除后送病理检查；3例于术后第3天出现阴茎头坏死脱落、阴茎皮肤颜色瘀黑、组织轻度肿胀,予以切除后送病理检查；3例术后第5天出现阴茎头颜色瘀黑、发干,阴茎皮肤颜色瘀黑,周围组织无明显肿胀；其余12例阴茎头颜色红润,皮肤颜色轻度苍白,周围组织无肿胀,切口愈合良好,于术后第7天拔除导尿管,观察排尿正常,小便呈线状,但存在不同程度的尿末滴沥症状,膀胱尿道造影检查无明显尿道狭窄,于术后第14天切除移植阴茎病理检查。
3、20例阴茎移植手术中,显微外科技术辅助下一次性静脉吻合成功率达95%(38/40),一次性动脉吻合成功率达87.5%(35/40),平均血管吻合时间70.95min,平均手术时间133min,平均失血量135.75m1。10例显微外科技术辅助下阴茎再植手术,一次性静脉吻合成功率达100%(20/20),一次性动脉吻合成功率达90%(18/20),平均血管吻合时间65min,平均手术时间117.4min,平均失血量85m1。12例临床显微外科技术辅助下阴茎离断性再植手术,一次性静脉吻合成功率达100%(24/24),一次性动脉吻合成功率达95.8%(23/24),平均血管吻合时间79min,平均手术时间125min,平均失血量140ml。3组资料采用校正的完全随机单因素方差分析(Welch法)、Games-Howell法以及Fisher's Exact法检验,结果显示比格犬阴茎移植组与人阴茎再植组各项指标相比较差异无统计学意义。
结论：比格犬阴茎海绵体结构与人阴茎海绵体结构具有高度相似性,在阴茎海绵体横断面解剖中其主要解剖结构均相同；显微外科技术辅助下行阴茎再植和同种异体移植手术均获得了成功,其手术操作过程中各项指标与临床中人阴茎再植手术相比较,具有相近的可操作性,重复性和可行性较强。因此,采用成年比格犬作为实验动物建立阴茎移植手术模型切实可行。
第3部分比格犬阴茎移植组织学镜下改变
目的：初步探讨同种异体阴茎移植术后移植阴茎发生排斥反应的主要病理学变化,以及正常免疫抑制状态下移植阴茎病理学特点。
方法：分别截取厚约3mm正常比格犬阴茎海绵体组织,阴茎移植后1天、3天、5天出现坏死的移植阴茎海绵体组织,以及阴茎移植后14天存活的移植阴茎海绵体组织,石蜡包埋,常规切片、HE染色后显微镜下观察。分别记录不同时期阴茎组织病理学改变及其特点。分析并探讨其原因和临床意义。
结果：术后移植阴茎坏死8例中,1例化脓性感染,2例血管栓塞出现干性坏疽,其余5例出现不同程度排斥反应。1例化脓性感染病理表现,肉眼可见阴茎头萎缩,皮肤颜色瘀黑,组织肿胀明显,皮下可见脓性坏死液化渗出,光镜下见皮肤、皮下、阴茎海绵体内大量脓性细胞浸润,间质水肿、退变,海绵体血窦消失、形成脓腔。2例干性坏疽阴茎病理表现,肉眼可见阴茎头颜色瘀黑、发干,皮肤颜色瘀黑,形成硬性痂皮,光镜下皮肤、皮下血管大量血栓形成,海绵体血窦消失,组织退变明显。5例移植阴茎排斥反应病理表现,阴茎头颜色苍白,皮肤颜色苍白或轻度瘀黑,皮下及海绵体肿胀明显,光镜下见组织内炎细胞浸润,小血管闭塞,部分血管内有血栓形成,组织间质水肿、退变明显,海绵体血窦结构破坏。12例移植阴茎存活良好病理表现,肉眼见阴茎头颜色红润,阴茎皮肤颜色稍白,横断面可见皮下组织轻度肿胀,无瘀血及异常渗出,海绵体结构完整；光镜下见皮肤结构良好,皮下组织轻度肿胀,血管内无血栓形成,海绵体血窦结构正常,组织内可见少量炎细胞浸润,未见间质退变坏死。
结论：移植阴茎存活受多方面条件所影响,阴茎移植排斥反应主要病理变现为皮肤、皮下组织间质水肿、退变,海绵体血窦结构紊乱,以及血管闭塞或血栓形成等特点。正常免疫抑制状态下移植阴茎病理学特点为皮肤结构良好,皮下组织轻度肿胀,血管内无血栓形成,海绵体血窦结构正常,组织内可见少量炎细胞浸润,未见间质退变坏死。
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 dogs
Objection: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 model
Objection: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 transplantation
Objection: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.
引文
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[11]龙道畴,陕声国,罗定安,等.阴茎延长术的临床研究.中华整形烧伤外科杂志,1990,6(1)：17-19.
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[13]霍然,李森恺,李养群,等.下腹部轴型皮瓣在阴茎延长术的应用.中华整形外科杂志,2001,17(3)：144-145.
[14]王忠,籁玉莲.阴茎部分切除加阴茎延长术治疗阴茎癌.中华男科学杂志,2005,11(12)：952-953.
[15]李战松.持股下区V-Y成形阴茎延长术(附85例临床报告).中国性科学,2007,16(4)：14-15,17.
[16]潘连军,祝黎洁,王忠,等.连续Z形皮瓣在阴茎延长术中的应用.中华男科学杂志,2008,14(4)：317-320.
[17]丁健,李强,李森恺,等.正常男性的阴茎延长术.中国美容医学,2010,19(6)：805-807.
[18]Bagoras, N.A. Plastic construction of penis capable of accomplishing coirus. Zentralbl. Chir.1936,63:1271.
[19]李文鹏,江华.阴茎再造术的进展.中华男科学杂志,2004,10(12)：937-940.
[20]Chang TS, Hwang WY. Forearm flap in one-stage reconstruction of the penis[J]. Plast Reconstr Surg,1984,74(2):251-258.
[21]Gottlieb LJ, Levine LA. A new design for the radical forearmfree-flap phallic construction[J]. Plast Reconstr Surg,1993,92(2):276-283.
[22]Mutaf M. A new surgical procedure for phallic reconstruction:Istanbul flap[J]. Plast Reconstr Surg,2000,105(4):1361-1370.
[23]杨明勇,李森恺,李养群,等.肩胛皮瓣再造阴茎[J].中华整形外科杂志,2003,19(2)：88-90.
[24]裴国献,顾立强,于立新,等.异体手移植二例报告.中华医学杂志,2000,80：417-421.
[25]Lee,-S, et al. Transplantation of reproductive organs. Microsurgery. 1995,16(4):191-198.
[26]Koga H, Yamata Ka A, Wang K, et al. Experimental allogenic penile transplantation. J Pediatr Surg,2003,38:1802-1805.
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[28]Falke G, Yoo JJ, Kwon TG, et al. Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices [J]. Tissue Eng.2003,9(5):871-879.
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[30]Kropp BP, Cheng EY, Pope JC, et al. Use of small intestinal submucosa for corporal body grafting in cases of severe penile curvature [J]. J Urol,2002,168(4 Pt 2):1742-1745; discussion 1745.
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[13]霍然,李森恺,李养群,等.下腹部轴型皮瓣在阴茎延长术的应用.中华整形外科杂志,2001,17(3)：144-145.
[14]王忠,籁玉莲.阴茎部分切除加阴茎延长术治疗阴茎癌.中华男科学杂志,2005,11(12)：952-953.
[15]李战松.持股下区V-Y成形阴茎延长术(附85例临床报告).中国性科学,2007,16(4)：14-15,17.
[16]潘连军,祝黎洁,王忠,等.连续Z形皮瓣在阴茎延长术中的应用.中华男科学杂志,2008,14(4)：317-320.
[17]丁健,李强,李森恺,等.正常男性的阴茎延长术.中国美容医学,2010,19(6)：805-807.
[18]Bagoras, N.A. Plastic construction of penis capable of accomplishing coirus. Zentralbl. Chir.1936,63:1271.
[19]李文鹏,江华.阴茎再造术的进展.中华男科学杂志,2004,10(12)：937-940.
[20]Chang TS, Hwang WY. Forearm flap in one-stage reconstruction of the penis[J]. Plast Reconstr Surg,1984,74(2):251-258.
[21]Gottlieb LJ, Levine LA. A new design for the radical forearmfree-flap phallic construction[J]. Plast Reconstr Surg,1993,92(2):276-283.
[22]Mutaf M. A new surgical procedure for phallic reconstruction:Istanbul flap[J]. Plast Reconstr Surg,2000,105(4):1361-1370.
[23]杨明勇,李森恺,李养群,等.肩胛皮瓣再造阴茎[J].中华整形外科杂志,2003,19(2)：88-90.
[24]裴国献,顾立强,于立新,等.异体手移植二例报告.中华医学杂志,2000,80：417-421.
[25]Lee,-S, et al. Transplantation of reproductive organs. Microsurgery. 1995,16(4):191-198.
[26]Koga H, Yamata Ka A, Wang K, et al. Experimental allogenic penile transplantation. J Pediatr Surg,2003,38:1802-1805.
[27]Weilie H, Jun L, Lichao Z, et al. A preliminary report of penile transplantation. Eur Urol,2006,50:851-853.
[28]Falke G, Yoo JJ, Kwon TG, et al. Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices [J]. Tissue Eng.2003,9(5):871-879.
[29]Kim BS, Yoo JJ, Atala A. Engineering of human cartilage rods potential application for penile prostheses [J]. J Urol,2002,168(4 Pt2):1794-1797.
[30]Kropp BP, Cheng EY, Pope JC, et al. Use of small intestinal submucosa for corporal body grafting in cases of severe penile curvature [J]. J Urol,2002,168(4 Pt 2):1742-1745; discussion 1745.
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[32]陈实,郭晖.移植病理学.人民卫生出版社,2009.
[33]王慧君,丁彦青,丁云川,等.异体手移植组织病理学研究.中国创伤骨科杂志,2006,2(2)：120-122.
[34]Dubernard JM, Owen E, Herzberg G, et al. Human hand allograft:report on first 6 months. Lancet,1999,353:1315-1320.
[35]Deiler S, Stock W. Allogenic hand transplantation. A case report of first 6 months. Unfallchirurg,1999,102:749-750.
[36]Tobin G, Breidenbach WC, Barker J, et al. Clinical progress of the nation first hand transplant over the first year and a half. Louisville Medicine, 2000,48:57-60.
[37]Lee WP, Mathes DW. Hand transplantation:pertinent data and future outlook. J hand surg.1999,24A:906-913.
[38]Foucher G. Prospect for hand transplantation. Lancet,1999,353:1286-1287.
[39]顾立强.亚太地区断指再植、手指再造与异体手移植治疗进展.中华创伤骨科杂志,2005,7(2)：162-165.
[40]张睿,郑智勇,余英豪.移植肾病理学研究新进展.中国误诊学杂志2010,10(18)：4304-4305.
[41]Solez K, Colvin RB, Racusen LC, et al. Banff 07 Classification of Renal Allograft Pat hology:Updates and Future Directions[J]. American Journal of Transplantation,2008,8 (4):7532760.
[42]邹万忠.我国肾脏病理学的现状和未来[J].中华病理学杂志,2005,34(8)：4962499.
[43]于立新,裴国献,顾立强,等.异体手移植免疫抑制治疗.中国创伤骨科杂志,2000,2(2)：117-119.
[44]Maria S, Frank P, Daniel A, et al. Near-total human face transplantation for a severely disfigured patient in the USA. The World Clinical Medicine. 2009,3(12):1025-1031.
[45]Morris P, Bradley A, Doyal L, et al. Face transplantation:a review of the technical, immunological, psychological and clinical issues with recoomendations for good practice. Transplantation,2007,83:109-28.
[46]Meningaud JP, Paraskevas A, Ingallina F, et al. Face transplant graft procurement:a preclinical and clinical study. Plast Reconstr Surg,2008,122: 1383-89.
[47]Chenggang Y, Yan H. Xudong Z, et al. Some issues in facial transplantation. Am J Transplant,2008,8:2169-72.
[48]田屿嘉雄.实验动物的生物学特性资料[M]TO YO TO:SO FT SCO ENCE, INC.1989.
[49]李常青,黄玲,张奉学,等Beagle犬的心电图研究[J].中国实验动物学 杂志,1999,(4)：225-227.
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[51]刘启德,许庆文,欧卫平,等Beagle犬的生物学特性及在药理毒理试验中的应用.中国新药与临床药理,2000,11(2)：91-94.
[52]刘雯,胡赟,邓锋,等.比格犬下颌骨微种植体的置入部位和方法[J].中国组织工程研究与临床康复,2011,15(41)：7635-7638.
[53]杨卫东,葛久禹,傅尧,等.超声对Beagle犬前牙根管内微生物作用的实验研究.牙体牙髓牙周病学杂志,2007,17(11)：618-621.
[54]陈磊,谢琳.萎缩性牙槽骨实验动物模型的建立.吉林医学,2011,32(6)：1043-1044.