Original article
Functional outcome of autologous anorectal transplantation in an experimental model V. E. Seid1,2 , F. H. F. Galvão1 , A. Vaidya3 , D. R. Waisberg1 , R. J. Cruz Jr4 , E. Chaib1 , S. C. Nahas2 , S. E. A. Araujo2 , L. A. C. D’Albuquerque1 and J. Araki5 1 Laboratory of Liver Transplantation and Experimental Surgery (LIM-37), Division of Liver Transplantation, and 2 Division of Colorectal Surgery, Department of Gastroenterology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 3 Department of Transplant Surgery, Oxford University Hospitals and University of Oxford, Oxford, UK, 4 Intestinal Rehabilitation and Transplant Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA, and 5 Department of Plastic Surgery, University of Tokyo, Tokyo, Japan Correspondence to: Dr J. Araki, Department of Plastic Surgery, University of Tokyo, 7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan (e-mail: [email protected])
Background: Although anorectal transplantation is a challenging procedure, it is a promising option for
patients who have completely lost anorectal function or in whom it failed to develop, as in congenital malformations. The paucity of animal models with which to test functional outcomes was addressed in this study of anorectal manometry in rats. Methods: Wistar rats were assigned randomly to four groups: orthotopic anorectal transplantation, heterotopic transplantation, sham operation, or normal control. Bodyweight and anal pressure were measured immediately before and after operation, and on postoperative days 7 and 14. ANOVA and Tukey’s test were used to compare results for bodyweight, anal manometry and length of procedure. Results: Immediately after the procedure, mean(s.d.) anal pressure in the orthotopic group (n = 13) dropped from 31⋅4(13⋅1) to 1⋅6(13⋅1) cmH2 O (P < 0⋅001 versus both sham operation (n = 13) and normal control (n = 15)), with partial recovery on postoperative day 7 (14⋅9(13⋅9) cmH2 O) (P = 0⋅009 versus normal control) and complete recovery on day 14 (23⋅7(12⋅2) cmH2 O). Heterotopic rats (n = 14) demonstrated partial functional recovery: mean(s.d.) anal pressure was 26⋅9(10⋅9) cmH2 O before operation and 8⋅6(6⋅8) cmH2 O on postoperative day 14 (P < 0⋅001 versus both sham and normal control). Conclusion: Orthotopic anorectal transplantation may result in better functional outcomes than heterotopic procedures.
Surgical relevance Patients with a permanent colostomy have limited continence. Treatment options are available, but anorectal transplantation may offer hope. Some experimental studies have been conducted, but available data are currently insufficient to translate
into a clinical option. This paper details functional outcomes in a rat model of anorectal autotransplantation. It represents a step in the translational research that may lead to restoration of anorectal function in patients who have lost or have failed to develop it.
Paper accepted 28 November 2014 Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9762
Introduction
The technical feasibility of anorectal transplantation in porcine, canine and rat models has been reported1 – 8 , but functional outcomes are unclear. The authors’ group has previously described autologous orthotopic and heterotopic anorectal transplantation rat models5 . The present © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
study tested the potential functional outcome of these procedures using anal manometry in a denervated anorectal autotransplantation rat model. Methods
This study followed the guidelines of the International Council for Laboratory Animal Science and the Brazilian BJS
V. E. Seid, F. H. F. Galvão, A. Vaidya, D. R. Waisberg, R. J. Cruz Jr, E. Chaib et al.
National Council for the Control of Animal Experimentation, and was reviewed and approved by the local institutional ethics committee (CAPPesq no. 0358/09).
Animals Adult male Wistar rats were individually caged in a temperature-controlled room (20–26∘ C) and fasted from solid food for 24 h before the surgical procedure. Ketamine (1 mg/kg) was used for anaesthesia. Following anaesthesia and surgery, animals were observed for 30 min and then placed in individual cages with standard food and water ad libitum. The experimental endpoint was set on postoperative day (POD) 14.
Combined perianal and laparotomy incision, and mobilization of the anorectal segment to the inside of the abdomen. The black arrow indicates the anorectal segment detached from perineum to abdominal cavity
Fig. 1
Surgical procedure For orthotopic and heterotopic procedures, a combined perianal approach and laparotomy was performed through a midline abdominal incision. The rectum, anal sphincter and its adjacent skin were completely disconnected and mobilized inside the abdominal cavity to ensure total denervation of the segment (Fig. 1). The inferior mesenteric artery was dissected down to the aorta under microscopic vision to sever/remove autonomic nerves to the bowel (Fig. 2). The bowel was transected just below the point where the inferior mesenteric vessels meet the intestine, to disrupt intrinsic innervation. With respect to blood supply, care was taken to preserve the branches of the inferior mesenteric artery and vein to and from the detached anorectal segment. The inferior mesenteric artery was clamped for 15 min to simulate transplantation ischaemia–reperfusion (Fig. 3). An end-to-end anastomosis restored intestinal continuity (Fig. 4). The anorectal segment was mobilized back to its original position orthotopically, or positioned heterotopically as a stoma in the lower left abdominal quadrant.
Postoperative assessment
Anorectal graft supplied by the inferior mesenteric artery. The inferior mesenteric artery emerging from the aorta is denoted by the black arrow
Length of the procedure, survival and bodyweight were assessed on POD 2, 7 and 14. Anal manometry was performed immediately before and after surgery, as well as on POD 7 and 14, except in rats undergoing heterotopic transplantation where manometry was performed before surgery and on POD 14 owing to difficulties related to the position of the anus in the abdominal wall.
pressure-sensitive catheter was used. The system was recalibrated before each measurement to maintain a constant hydraulic pressure. The catheter was fixed and anal pressure was recorded for 10 min consecutively under anaesthesia. The software (Proctomaster® 6⋅0; Dynamed) continuously recorded all anal sphincter pressure curves.
Anal manometry
Statistical analysis
Standard equipment (DUAL infusion system; Dynamed, São Paulo, Brazil) connected to a radial four-channel
ANOVA and Tukey’s test were used to analyse length of the procedure, bodyweight and manometry values. To analyse
© 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
www.bjs.co.uk
Fig. 2
BJS
Anorectal transplantation in an experimental model
Table 1
Anal pressures for all procedures at each evaluation point Anal pressure (cmH2 O)
Procedure Orthotopic Heterotopic Sham Normal control
No. of animals
Preop.
Immediately postop.
13 14 13 15
31⋅4(13⋅1) 26⋅9(10⋅9) 34⋅9(14⋅3) 40⋅7(13⋅1)
1⋅6(13⋅1)* – 21⋅6(5⋅4) 40⋅7(13⋅1)
POD 7
POD 14
14⋅9(13⋅9)† 23⋅7(12⋅2) – 8⋅6(6⋅8)* 29⋅9(9⋅9) 33⋅1(10⋅5) 33⋅8(13⋅9) 33⋅5(12⋅2)
Values are mean(s.d.). POD, postoperative day. *P < 0⋅001 versus sham and normal control group; †P = 0⋅009 versus normal control (ANOVA and Tukey’s test).
preoperative and anaesthetic conditions, but had only a laparotomy and a perianal incision. The normal control group had anaesthetic procedures for manometry only.
Length of surgical procedure Clamping of vessels and division of the colon and rectum. The black arrow shows divided anorectal segment; the white arrow indicates the clamp occluding the inferior mesenteric artery
Fig. 3
The mean(s.d.) length of each surgical procedure was 43⋅8(8⋅4) min for orthotopic and 44⋅3(4⋅5) min for heterotopic procedures (P = 0⋅970).
Survival In the orthotopic group, two animals died as a result of technical difficulties (mesenteric thrombosis) on POD 1 and 2, and a further two animals died during the anaesthesia procedure on POD 7, resulting in a total of four deaths in this group. There were five deaths in the heterotopic group, all of which resulted from surgical complications due to bowel obstruction and/or mesenteric thrombosis. No animals in the sham or normal control group died. Final graft aspect after end-to-end colonic anastomosis. The black arrow indicates the inferior mesenteric artery after clamp release; the white arrow shows the colonic anastomosis
Fig. 4
anal pressure, mean(s.d.) pressures (cmH2 O) were obtained from each curve for each animal at each evaluation point. The threshold value for significance was set at P < 0⋅050. All statistical analyses were performed using SPSS® version 18⋅0 (IBM, Armonk, New York, USA).
Weight The bodyweight of rats in the orthotopic and heterotopic groups tended to decrease on POD 2, with progressive regain of weight on POD 7 and 14. In sham and normal control rats, bodyweight increased continuously. ANOVA and Tukey’s test showed no significant differences between groups on any day of evaluation.
Anal manometry Results
Fifty-five rats weighing 230–280 g were divided randomly into the following four groups: orthotopic anorectal transplantation (n = 13), heterotopic transplantation (n = 14), sham operation (n = 13) and normal control (n = 15). Animals in the sham group were exposed to similar © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
The preoperative mean(s.d.) anal pressure for all animals in the study was 33⋅7(12⋅6) (95 per cent c.i. 10⋅8 to 66⋅5) cmH2 O. Mean(s.d.) anal pressures in orthotopic, sham and normal control groups before and immediately after operation, and on POD 7 and 14 are shown in Table 1 and Fig. 5a. ANOVA and Tukey’s test verified that there www.bjs.co.uk
BJS
V. E. Seid, F. H. F. Galvão, A. Vaidya, D. R. Waisberg, R. J. Cruz Jr, E. Chaib et al.
∗
60 Anal pressure (cmH2O)
†
POD 7
POD 14
40 30 20 10 0
a
∗
50
Orthotopic Sham Normal control
Preop.
Immediately after operation
Anal pressure at all evaluation points in orthotopic, sham and normal control groups
60
Orthotopic Sham
Heterotopic Normal control
∗ ∗
Anal pressure (cmH2O)
50 40 30 20 10 0
b
Preop.
POD 14
Comparison with heterotopic group
Mean(s.d.) anal pressures for a orthotopic, sham and normal control groups at each evaluation point, and b all four groups before operation and on postoperative day (POD) 14, to show comparison with heterotopic group. *P < 0⋅001, †P = 0⋅009 (ANOVA and Tukey’s test) Fig. 5
were no significant preoperative differences. Following a significant postoperative drop in anal pressure immediately after operation, pressures had recovered by POD 7 in the sham group but not significantly in the orthotopic group, compared with values in the normal control group. By POD 14, recovery of anal pressure was confirmed in rats in the orthotopic group, with no significant differences compared with sham or normal control animals. The time course of the manometry curves for an animal in the orthotopic group can be seen in Fig. S1 (supporting information). Mean(s.d.) anal pressure for rats in the heterotopic group was 26⋅9(10⋅9) cmH2 O before operation and 8⋅6(6⋅8) cmH2 O on POD 14, when the pressure was significantly lower than that in sham and normal control rats (Table 1 and Fig. 5b). © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
Discussion
In humans, vascularized composite allotransplantation of tissues such as the face9 , hand10 and uterus11 has been proposed to re-establish their function, with promising results. Anorectal transplantation was shown to be technically feasible in pigs and dogs1,4 . However, the function of the anorectal graft has not been assessed in previous studies. Orthotopic rats displayed an almost complete lack of anal pressure immediately after operation, owing to the fact that the pelvic floor muscles were dissected and pudendal nerves cut. In sham rats, a drop in mean anal pressure levels was observed, most likely because the surgical trauma to the abdominal wall affected the natural abdominal pressure, disturbing the evacuation mechanism. There may be close proximity between the donor and recipient pudendal nerves that can result in improved nerve functional regeneration in the orthotopic model. The observation that some function had returned in the orthotopic group by POD 14 inspires rethinking with regard to the mechanism of anorectal reconstitution or readaptation after total nerve disconnection. In fact, the contribution of the pudendal nerve to the innervation of the levator ani muscles, as well as in the evacuatory mechanism, is debatable. Pudendal motor innervation of the external anal sphincter and pudendal sensory innervation of the anal canal seem to be important in faecal continence12 – 14 . However, it remains unclear whether the pudendal afferent nerves contribute to sensation in the distal rectum. Indeed, the distal rectum may receive intramural afferent fibres extending from the upper anal canal, via either the inferior haemorrhoidal nerve or afferent pathways distinct from the pelvic floor mechanoreceptors that travel within the primarily motor branches innervating the levator ani and puborectalis15 – 17 . Furthermore, the contribution of extrasphincteric components (colonic and rectal) is being recognized increasingly15 . There are obvious issues in any comparison of humans and rats. Inferior rectal artery division may cause ischaemia, even with inferior mesenteric preservation18 . Furthermore, complete extrinsic denervation in humans may result in severe faecal incontinence19,20 . Even though translating this approach to humans will be difficult, the authors believe that the present experimental data provide further evidence that it is not impossible. Acknowledgements
The authors thank Laboratory LIM 37 and the statistics service of the Gastroenterology Department, University of São Paulo. Disclosure: The authors declare no conflict of interest. www.bjs.co.uk
BJS
Anorectal transplantation in an experimental model
References 1 O’Bichere A, Shurey S, Sibbons P, Green C, Phillips RK. Experimental model of anorectal transplantation. Br J Surg 2000; 87: 1534–1539. 2 Galvao FH, Seid VE, Waisberg DR, Cruz RJ Jr, Hirano H, Catanozi S et al. An innovative model of autologous anorectal transplantation with pudendal nerve reconstruction. Clinics (Sao Paulo) 2012; 67: 971–972. 3 Araki J, Nishizawa Y, Nakamura T, Sato T, Naito M, Fujii S et al. The development of a canine anorectal autotransplantation model based on blood supply: a preliminary case report. PLoS One 2012; 7: e44310. 4 Araki J, Nishizawa Y, Nakamura T, Sato T, Naito M, Hatayama N et al. Anorectal autotransplantation in a canine model: the first successful report in the short term with the non-laparotomy approach. Sci Rep 2014; 4: 6312. 5 Galvão FH, Seid VE, Nunes dos Santos RM, Kitamura M, de Castro Galvão R, Ambar Pinto R et al. Anorectal transplantation. Tech Coloproctol 2009; 13: 55–59. 6 Galvão FH, Waisberg DR, De Mello Vianna RM, De Castro Galvão R, Seid VE, Andraus W et al. Intestinal transplantation including anorectal segment in the rat. Microsurgery 2012; 32: 77–79. 7 Galvão FH, Waisberg DR, Cruz RJ Jr, Chaib E, Carneiro D’Albuquerque LA. Modified multivisceral transplantation in the rat. Transplantation 2013; 96: e3–e4. 8 Araki J, Mihara M, Narushima M, Iida T, Sato T, Koshima I. Vascularized anal autotransplantation model in rats: preliminary report. Transplant Proc 2011; 43: 3552–3556. 9 Pomahac B, Pribaz J, Eriksson E, Bueno EM, Diaz-Siso JR, Rybicki FJ et al. Three patients with full facial transplantation. N Engl J Med 2012; 366: 715–722. 10 Jones JW, Gruber SA, Barker JH, Breidenbach WC. Successful hand transplantation. One-year follow-up.
11
12
13
14
15
16
17
18
19
20
Louisville Hand Transplant Team. N Engl J Med 2000; 343: 468–473. Brännström M, Johannesson L, Bokström H, Kvarnström N, Mölne J, Dahm-Kähler P et al. Livebirth after uterus transplantation. Lancet 2014; [Epub ahead of print]. Athanasakos EP, Ward HC, Williams NS, Scott SM. Importance of extrasphincteric mechanisms in the pathophysiology of faecal incontinence in adults with a history of anorectal anomaly. Br J Surg 2008; 95: 1394–1400. Duthie HL, Watts JM. Contribution of the external anal sphincter to the pressure zone in the anal canal. Gut 1965; 6: 64–68. Gibbons CP, Trowbridge EA, Bannister JJ, Read NW. Role of anal cushions in maintaining continence. Lancet 1986; 1: 886–888. Kiff ES, Swash M. Slowed conduction in the pudendal nerves in idiopathic (neurogenic) faecal incontinence. Br J Surg 1984; 71: 614–616. Percy JP, Neill ME, Swash M, Parks AG. Electrophysiological study of motor nerve supply of pelvic floor. Lancet 1981; 1: 16–17. Siproudhis L, Bellissant E, Pagenault M, Mendler MH, Allain H, Bretagne JF et al. Fecal incontinence with normal anal canal pressures: where is the pitfall? Am J Gastroenterol 1999; 94: 1556–1563. Vignali A, Gianotti L, Braga M, Radaelli G, Malvezzi L, Di Carlo V. Altered microperfusion at the rectal stump is predictive for rectal anastomotic leak. Dis Colon Rectum 2000; 43: 76–82. McCarthy MJ, Aylott CE, Grevitt MP, Hegarty J. Cauda equina syndrome: factors affecting long-term functional and sphinteric outcome. Spine 2007; 15: 207–216. Benevento BT, Sipski MJ. Neurogenic bladder, neurogenic bowel and sexual dysfunction in people with spinal cord injury. Phys Ther 2002; 82: 601–612.
Supporting information
Additional supporting information may be found in the online version of this article: Fig. S1 Anal manometry pressure curves in a rat subjected to orthotopic anorectal transplantation (TIFF file)
© 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
www.bjs.co.uk
BJS
Functional outcome of autologous anorectal transplantation in an experimental model V. E. Seid1,2 , F. H. F. Galvão1 , A. Vaidya3 , D. R. Waisberg1 , R. J. Cruz Jr4 , E. Chaib1 , S. C. Nahas2 , S. E. A. Araujo2 , L. A. C. D’Albuquerque1 and J. Araki5 1 Laboratory of Liver Transplantation and Experimental Surgery (LIM-37), Division of Liver Transplantation, and 2 Division of Colorectal Surgery, Department of Gastroenterology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 3 Department of Transplant Surgery, Oxford University Hospitals and University of Oxford, Oxford, UK, 4 Intestinal Rehabilitation and Transplant Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA, and 5 Department of Plastic Surgery, University of Tokyo, Tokyo, Japan Correspondence to: Dr J. Araki, Department of Plastic Surgery, University of Tokyo, 7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan (e-mail: [email protected])
Background: Although anorectal transplantation is a challenging procedure, it is a promising option for
patients who have completely lost anorectal function or in whom it failed to develop, as in congenital malformations. The paucity of animal models with which to test functional outcomes was addressed in this study of anorectal manometry in rats. Methods: Wistar rats were assigned randomly to four groups: orthotopic anorectal transplantation, heterotopic transplantation, sham operation, or normal control. Bodyweight and anal pressure were measured immediately before and after operation, and on postoperative days 7 and 14. ANOVA and Tukey’s test were used to compare results for bodyweight, anal manometry and length of procedure. Results: Immediately after the procedure, mean(s.d.) anal pressure in the orthotopic group (n = 13) dropped from 31⋅4(13⋅1) to 1⋅6(13⋅1) cmH2 O (P < 0⋅001 versus both sham operation (n = 13) and normal control (n = 15)), with partial recovery on postoperative day 7 (14⋅9(13⋅9) cmH2 O) (P = 0⋅009 versus normal control) and complete recovery on day 14 (23⋅7(12⋅2) cmH2 O). Heterotopic rats (n = 14) demonstrated partial functional recovery: mean(s.d.) anal pressure was 26⋅9(10⋅9) cmH2 O before operation and 8⋅6(6⋅8) cmH2 O on postoperative day 14 (P < 0⋅001 versus both sham and normal control). Conclusion: Orthotopic anorectal transplantation may result in better functional outcomes than heterotopic procedures.
Surgical relevance Patients with a permanent colostomy have limited continence. Treatment options are available, but anorectal transplantation may offer hope. Some experimental studies have been conducted, but available data are currently insufficient to translate
into a clinical option. This paper details functional outcomes in a rat model of anorectal autotransplantation. It represents a step in the translational research that may lead to restoration of anorectal function in patients who have lost or have failed to develop it.
Paper accepted 28 November 2014 Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9762
Introduction
The technical feasibility of anorectal transplantation in porcine, canine and rat models has been reported1 – 8 , but functional outcomes are unclear. The authors’ group has previously described autologous orthotopic and heterotopic anorectal transplantation rat models5 . The present © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
study tested the potential functional outcome of these procedures using anal manometry in a denervated anorectal autotransplantation rat model. Methods
This study followed the guidelines of the International Council for Laboratory Animal Science and the Brazilian BJS
V. E. Seid, F. H. F. Galvão, A. Vaidya, D. R. Waisberg, R. J. Cruz Jr, E. Chaib et al.
National Council for the Control of Animal Experimentation, and was reviewed and approved by the local institutional ethics committee (CAPPesq no. 0358/09).
Animals Adult male Wistar rats were individually caged in a temperature-controlled room (20–26∘ C) and fasted from solid food for 24 h before the surgical procedure. Ketamine (1 mg/kg) was used for anaesthesia. Following anaesthesia and surgery, animals were observed for 30 min and then placed in individual cages with standard food and water ad libitum. The experimental endpoint was set on postoperative day (POD) 14.
Combined perianal and laparotomy incision, and mobilization of the anorectal segment to the inside of the abdomen. The black arrow indicates the anorectal segment detached from perineum to abdominal cavity
Fig. 1
Surgical procedure For orthotopic and heterotopic procedures, a combined perianal approach and laparotomy was performed through a midline abdominal incision. The rectum, anal sphincter and its adjacent skin were completely disconnected and mobilized inside the abdominal cavity to ensure total denervation of the segment (Fig. 1). The inferior mesenteric artery was dissected down to the aorta under microscopic vision to sever/remove autonomic nerves to the bowel (Fig. 2). The bowel was transected just below the point where the inferior mesenteric vessels meet the intestine, to disrupt intrinsic innervation. With respect to blood supply, care was taken to preserve the branches of the inferior mesenteric artery and vein to and from the detached anorectal segment. The inferior mesenteric artery was clamped for 15 min to simulate transplantation ischaemia–reperfusion (Fig. 3). An end-to-end anastomosis restored intestinal continuity (Fig. 4). The anorectal segment was mobilized back to its original position orthotopically, or positioned heterotopically as a stoma in the lower left abdominal quadrant.
Postoperative assessment
Anorectal graft supplied by the inferior mesenteric artery. The inferior mesenteric artery emerging from the aorta is denoted by the black arrow
Length of the procedure, survival and bodyweight were assessed on POD 2, 7 and 14. Anal manometry was performed immediately before and after surgery, as well as on POD 7 and 14, except in rats undergoing heterotopic transplantation where manometry was performed before surgery and on POD 14 owing to difficulties related to the position of the anus in the abdominal wall.
pressure-sensitive catheter was used. The system was recalibrated before each measurement to maintain a constant hydraulic pressure. The catheter was fixed and anal pressure was recorded for 10 min consecutively under anaesthesia. The software (Proctomaster® 6⋅0; Dynamed) continuously recorded all anal sphincter pressure curves.
Anal manometry
Statistical analysis
Standard equipment (DUAL infusion system; Dynamed, São Paulo, Brazil) connected to a radial four-channel
ANOVA and Tukey’s test were used to analyse length of the procedure, bodyweight and manometry values. To analyse
© 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
www.bjs.co.uk
Fig. 2
BJS
Anorectal transplantation in an experimental model
Table 1
Anal pressures for all procedures at each evaluation point Anal pressure (cmH2 O)
Procedure Orthotopic Heterotopic Sham Normal control
No. of animals
Preop.
Immediately postop.
13 14 13 15
31⋅4(13⋅1) 26⋅9(10⋅9) 34⋅9(14⋅3) 40⋅7(13⋅1)
1⋅6(13⋅1)* – 21⋅6(5⋅4) 40⋅7(13⋅1)
POD 7
POD 14
14⋅9(13⋅9)† 23⋅7(12⋅2) – 8⋅6(6⋅8)* 29⋅9(9⋅9) 33⋅1(10⋅5) 33⋅8(13⋅9) 33⋅5(12⋅2)
Values are mean(s.d.). POD, postoperative day. *P < 0⋅001 versus sham and normal control group; †P = 0⋅009 versus normal control (ANOVA and Tukey’s test).
preoperative and anaesthetic conditions, but had only a laparotomy and a perianal incision. The normal control group had anaesthetic procedures for manometry only.
Length of surgical procedure Clamping of vessels and division of the colon and rectum. The black arrow shows divided anorectal segment; the white arrow indicates the clamp occluding the inferior mesenteric artery
Fig. 3
The mean(s.d.) length of each surgical procedure was 43⋅8(8⋅4) min for orthotopic and 44⋅3(4⋅5) min for heterotopic procedures (P = 0⋅970).
Survival In the orthotopic group, two animals died as a result of technical difficulties (mesenteric thrombosis) on POD 1 and 2, and a further two animals died during the anaesthesia procedure on POD 7, resulting in a total of four deaths in this group. There were five deaths in the heterotopic group, all of which resulted from surgical complications due to bowel obstruction and/or mesenteric thrombosis. No animals in the sham or normal control group died. Final graft aspect after end-to-end colonic anastomosis. The black arrow indicates the inferior mesenteric artery after clamp release; the white arrow shows the colonic anastomosis
Fig. 4
anal pressure, mean(s.d.) pressures (cmH2 O) were obtained from each curve for each animal at each evaluation point. The threshold value for significance was set at P < 0⋅050. All statistical analyses were performed using SPSS® version 18⋅0 (IBM, Armonk, New York, USA).
Weight The bodyweight of rats in the orthotopic and heterotopic groups tended to decrease on POD 2, with progressive regain of weight on POD 7 and 14. In sham and normal control rats, bodyweight increased continuously. ANOVA and Tukey’s test showed no significant differences between groups on any day of evaluation.
Anal manometry Results
Fifty-five rats weighing 230–280 g were divided randomly into the following four groups: orthotopic anorectal transplantation (n = 13), heterotopic transplantation (n = 14), sham operation (n = 13) and normal control (n = 15). Animals in the sham group were exposed to similar © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
The preoperative mean(s.d.) anal pressure for all animals in the study was 33⋅7(12⋅6) (95 per cent c.i. 10⋅8 to 66⋅5) cmH2 O. Mean(s.d.) anal pressures in orthotopic, sham and normal control groups before and immediately after operation, and on POD 7 and 14 are shown in Table 1 and Fig. 5a. ANOVA and Tukey’s test verified that there www.bjs.co.uk
BJS
V. E. Seid, F. H. F. Galvão, A. Vaidya, D. R. Waisberg, R. J. Cruz Jr, E. Chaib et al.
∗
60 Anal pressure (cmH2O)
†
POD 7
POD 14
40 30 20 10 0
a
∗
50
Orthotopic Sham Normal control
Preop.
Immediately after operation
Anal pressure at all evaluation points in orthotopic, sham and normal control groups
60
Orthotopic Sham
Heterotopic Normal control
∗ ∗
Anal pressure (cmH2O)
50 40 30 20 10 0
b
Preop.
POD 14
Comparison with heterotopic group
Mean(s.d.) anal pressures for a orthotopic, sham and normal control groups at each evaluation point, and b all four groups before operation and on postoperative day (POD) 14, to show comparison with heterotopic group. *P < 0⋅001, †P = 0⋅009 (ANOVA and Tukey’s test) Fig. 5
were no significant preoperative differences. Following a significant postoperative drop in anal pressure immediately after operation, pressures had recovered by POD 7 in the sham group but not significantly in the orthotopic group, compared with values in the normal control group. By POD 14, recovery of anal pressure was confirmed in rats in the orthotopic group, with no significant differences compared with sham or normal control animals. The time course of the manometry curves for an animal in the orthotopic group can be seen in Fig. S1 (supporting information). Mean(s.d.) anal pressure for rats in the heterotopic group was 26⋅9(10⋅9) cmH2 O before operation and 8⋅6(6⋅8) cmH2 O on POD 14, when the pressure was significantly lower than that in sham and normal control rats (Table 1 and Fig. 5b). © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
Discussion
In humans, vascularized composite allotransplantation of tissues such as the face9 , hand10 and uterus11 has been proposed to re-establish their function, with promising results. Anorectal transplantation was shown to be technically feasible in pigs and dogs1,4 . However, the function of the anorectal graft has not been assessed in previous studies. Orthotopic rats displayed an almost complete lack of anal pressure immediately after operation, owing to the fact that the pelvic floor muscles were dissected and pudendal nerves cut. In sham rats, a drop in mean anal pressure levels was observed, most likely because the surgical trauma to the abdominal wall affected the natural abdominal pressure, disturbing the evacuation mechanism. There may be close proximity between the donor and recipient pudendal nerves that can result in improved nerve functional regeneration in the orthotopic model. The observation that some function had returned in the orthotopic group by POD 14 inspires rethinking with regard to the mechanism of anorectal reconstitution or readaptation after total nerve disconnection. In fact, the contribution of the pudendal nerve to the innervation of the levator ani muscles, as well as in the evacuatory mechanism, is debatable. Pudendal motor innervation of the external anal sphincter and pudendal sensory innervation of the anal canal seem to be important in faecal continence12 – 14 . However, it remains unclear whether the pudendal afferent nerves contribute to sensation in the distal rectum. Indeed, the distal rectum may receive intramural afferent fibres extending from the upper anal canal, via either the inferior haemorrhoidal nerve or afferent pathways distinct from the pelvic floor mechanoreceptors that travel within the primarily motor branches innervating the levator ani and puborectalis15 – 17 . Furthermore, the contribution of extrasphincteric components (colonic and rectal) is being recognized increasingly15 . There are obvious issues in any comparison of humans and rats. Inferior rectal artery division may cause ischaemia, even with inferior mesenteric preservation18 . Furthermore, complete extrinsic denervation in humans may result in severe faecal incontinence19,20 . Even though translating this approach to humans will be difficult, the authors believe that the present experimental data provide further evidence that it is not impossible. Acknowledgements
The authors thank Laboratory LIM 37 and the statistics service of the Gastroenterology Department, University of São Paulo. Disclosure: The authors declare no conflict of interest. www.bjs.co.uk
BJS
Anorectal transplantation in an experimental model
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Supporting information
Additional supporting information may be found in the online version of this article: Fig. S1 Anal manometry pressure curves in a rat subjected to orthotopic anorectal transplantation (TIFF file)
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