EXPERIMENTAL EVALUATION OF AUTOLOGOUS GRAFTING USING TUNICA VAGINALIS PROPRIA FOR URINARY BLADDER AUGMENTATION IN DOGS

EXPERIMENTAL EVALUATION OF AUTOLOGOUS GRAFTING USING TUNICA VAGINALIS PROPRIA FOR URINARY BLADDER AUGMENTATION IN DOGS

EL-DAHARAWY, M.H.^* and IBRAHIM A.^**

^*Dept. of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University

^**Dept. of Pathology, Faculty of Veterinary Medicine, Suez Canal University

Email: mohamedeldaharawy@yahoo.com

INTRODUCTION

Augmentation of urinary bladder is a procedure in which a piece of tissue is used to enlarge the bladder capacity, maintain bladder wall integrity, and decrease intravesical pressure. This tissue should be biocompatible and be able to serve as a scaffold for the regeneration of all three layers of the urinary bladder. This process is used in conditions with severe damage or loss of the bladder such as cancer, trauma, infection, inflammation, innervation defects and congenital abnormalities (Parsons and Toozs-Hobson 2005; Eldaharawy et al., 2006 and Wongsetthachai et al., 2011).

Various grafts are used in the reconstruction of urinary bladder. Tunica vaginalis is one of these grafts. Tunica vaginalis is derived from the peritoneum which is composed of mesothelium and connective tissue (Wrobel, 1998). Peritoneal flap has been used successfully as a bladder wall substitute in animal models (Tsuji et al., 1963; Hutschenreiter     et al., 1978 and Sadove et al., 1993). Wongsetthachai    et al. (2011)used autologus tunica vaginalis for bladder substitution in male dogs. On the other hand, Theodorescu et al. (1998); Calado et al. (2005) and Leslie et al. (2009) reported successful urethroplasty with tunica vaginalis in rabbit models. Gajda (2006) used tunica albuginea for ureteral reconstruction in dogs. 

The aim of the current study is to assess the changes of the urinary bladder augmented with tunica vaginalis propria in male dogs, based on clinical findings, cystography and histopathological findings.

MATERIALS and METHODS

Eighteen apparently healthy adult male mongrel dogs 2-3 years old, weighing 12-15 kg were used in this study at Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University. Dogs were assigned into two groups each one consists of 9 individuals. The first one, (control group), was subjected to primary incisional closure of the bladder without wall augmentation. The second group, in which the bladder was reconstructed with a sheet of the tunica vaginalis propria.

Dogs were fasted for 12 hrs before the surgical procedure. Each dog was premedicated with intramuscular injection of chloropromazine hydrochloride in a dose rate of 1 mg/kg, 15 min prior to the induction of general anesthesia (Hall et al., 2001).The surgical site was then clipped, shaved and disinfected with povidone iodine solution (Betadine: Povidone- Iodine U.S.P. 10%, El Nile-Co.). General anesthesia was conducted by intravenous injection of thiopental sodium 2.5% until the main reflexes were abolished. The whole animal, except the sites of operation, was draped with sterile towel. Balanced electrolyte (0.9% Sodium chloride) solution was administered (10 ml/kg/hr) during surgery. Two surgical procedures were performed in each dog. The first procedure was closed orchidectomy. The separated tunica vaginalis was harvested into a circular sheet 3 cm in diameter, and placed in an aseptic petri dish containing lactated Ringer’s solution until used for grafting (Fig. 1). The second surgical procedure was performed to expose the urinary bladder. The bladder was approached through a lower ventral midline incision (Fig. 2). 30% full thickness piece from the dome of the bladder wall was excised. In control group, closure of the bladder without wall augmentation was accomplished with simple continuous sutures pattern using 3-0 polyglactin 910 (Vicryl: Manufactured Johnson & Johnson Jutl). In reconstructed  dogs, a sheet of the prepared tunica vaginalis was sutured to the cut edge of the native bladder using a simple continuous suture pattern using 3-0 polyglactin 910 (Fig.3). The bladder was refilled by saline and small leaks were repaired (Fig.4). Omentum was then placed over the operated area in all dogs and secured to the bladder with a simple interrupted suture of the 3-0 polyglactin 910 material before the abdominal incisions were routinely closed. The linea alba and subcutaneous tissue were closed using polyglactin 910 No. 0 in a simple continuous pattern. Skin edges were sutured using silk No. 0 in a simple interrupted pattern. 

Each animal was injected postoperatively with 1 g of amoxicillin intramuscularly (E.Mox: Egyptian International Pharm. Industries Company, A. R.E) and dipyrone (Analgin 50%: El-Nasr Pharmaceutical Chemicals co., Egypt.) in a dose of 10 mg/kg I.M. for 3 days.

Dogs were monitored for appetite, urine output, urine abnormalities, abdominal distention, abdominal pain and body temperature. The morphologic-functional assessment of the augmented bladder was performed through cystography before operation and at time of the scarification. Cystography was performed by insertion of a catheter gently through the urethra. All urine was aspirated out of the bladder then infused slowly positive contrast medium (tri-iodinated) diluted with saline (1:1) into the bladder until its full capacity. Lateral and ventrodorsal radiographs of the caudal abdomen were taken.

Dogs were euthanized at 4, 8 and 12 weeks postoperatively using an overdose of thiopental sodium solution. The abdominal incision, peritoneal cavity, and all abdominal viscera were evaluated for adhesions and any other post surgical changes. The specimens were collected and fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with haematoxylin & eosin and Masson’s trichrome (Bancroft et al., 1990).

RESULTS

In the control group, six dogs appeared normal with the presence of frequent urination. The other three dogs died 10 days postoperatively. These cases showed inappetence, dullness and increase body temperature. In the augmented group, all cases appeared in good health conditions. Urine output appeared to be normal till the time of sacrificing except two cases died two week postoperatively. These cases suffered from inappetence, dullness, increase body temperature, abdominal distention, abdominal pain, difficult and decreased urine output.

Cystography showed more or less oval urinary bladder preoperatively in all cases. Cystography showed small size bladder in the control group postoperatively. Cystography of augmented group at 4, 8 and 12 weeks postoperatively, showed an elongated, irregular contour of the bladder that was returned nearly to the same size (Figs. 5&6).

Grossly in six dogs of control group, there were adhesions between the bladder wall and omentum. The others three dogs showed wound dehiscence and urine leakage at 10 days postoperatively. In the augmented group at the time of sacrifice, the urinary bladder region showed weak adhesions between bladder wall and omentum (Fig. 7). These could easily be dissected by blunt dissection with gauze in the seven cases. No calcification of wall or stone formation was shown inside the urinary bladder in any of these dogs. The urinary bladder distended normally after filling with saline. There was no leak in any of the operated dogs.A decreased diameter of the graft indicated its shrinkage. It changed into remnant at 12 weeks postoperatively. The demarcation over anastomotic line between bladder wall and the graft was difficult to differentiate and internal surface of the augmented urinary bladder covered with apparently healthy mucosa at 12 weeks postoperatively (Fig.8). The bladder of the two died dogs showed perforation within the graft at two weeks postoperatively.

Microscopically, in the control group, all three layers of normal urinary bladder wall were observed at the closure areas. Plasma cells and lymphocytes were noted in the submucosa and serosa of the closure areas during the experimental periods. In the augmented group at 4 weeks, the two cut borders of the native bladder were joined with fibrous connective tissue containing newly formed blood capillaries (granulation tissue)(Fig. 9). Moderate numbers of lymphocytes and plasma cells were detected within the grafted region (Fig. 10). After 8 weeks, the native and grafted regions were in a good continuity and adhered well with fibrous connective tissue bands extending between both of them admixed with strands of smooth muscle, in addition to some inflammatory cells mostly lymphocytes (Figs. 11-13). 12 weeks postoperatively, the grafted area showed full thickness that was evident by presence of well-organized dense fibrous connective tissue containing minimal inflammatory cells and remnant of suture material. The mucosal surface of

the graft was covered with a mildly hyperplastic urothelium (2-4 layers). Focal aggregation of lymphocytes was noted underneath the transitional epithelium (Figs. 13&14). The tunica musclaris was present connected the graft from both sides with the native bladder which was stained with Masson’s trichrome staining (Fig. 16).

DISCUSSION

Autologous tunica vaginalis grafts have the advantage of easy preparation, no need for preservation, less expense, highly vascularity, and no graft rejection. Use of this kind of the tissue graft is useful for maintaining bladder wall integrity especially in an emergency situation where a massive loss of the bladder wall has occurred (Sadove et al., 1993 and Wongsetthachai et al., 2011).

Our results revealed that six dogs in control group suffered from frequent urination. This symptom may due to bladder inflammation and decrease bladder capacity after partial cystectomy. In the augmented group, bladder began to regain normal capacity at 12 weeks postoperatively. The time is necessary for returning bladder capacity. The cystography and histopathological findings enforced this result. Similar result was obtained by Kropp et al. (1996)whoreported that duration of 14 months is required to regain normal bladder capacity after the resected wall had been substituted with porcine small intestine submucosa. On the other hand, decreased bladder capacitywas reported byWongsetthachai et al. (2011) who mentioned that the shrinkage of the substituted portions of the bladder was resulting from inflammation, fibrosis, dystrophic calcification, and bone metaplasia within the grafted portions.

Three dogs from control group died 10 days postoperatively. The main cause was peritonitis due to urine leakage resulting in wound dehiscence and sepsis. Similar complications were obtained byVilar et al. (2004).

In the augmented group, dogs appeared with normal urine output till the time of sacrificing. Cystography showed integration of the graft into the bladder that was returned nearly to the same size. These results were supported in dogs by Elkoushy (2003); Vilar    et al. (2004) and Eldaharawy et al. (2006). Two dogs from this group, suffered from abdominal distention, abdominal pain, inappetence, increase body temperature, difficult and decreased urine output after two weeks postoperatively. The bladder showed perforation and adhesions between the bladder wall, and omentum. A number of factors may be proposed to be etiologic including chronic urinary tract infection, adhesions and over distention which results in areas of ischemia and contractions. Adhesions result in a fixed configuration that may result in sheering with emptying and filling. These causes were supported by Crane et al. (1991) and Bauer      et al. (1992).

The regeneration time of the bladder wall depended on graft type and the animal species in addition to the extent of the bladder wall defect. Epithelialization of the augmented bladder in the present study completed at 12 weeks. Nearly similar result was mentioned by Wongsetthachai et al. (2011) who observed epitheliazation earlier than the 12 weeks. On the other hand, Hutschenreiter et al. (1978) and Pope et al. (1997) reported that,complete epithelialization was observed at 5 and 3–4 weeks after the use of free peritoneal grafts in rabbits and porcine small intestine submucosa in dogs respectively. Smooth muscle regeneration was present at 8 weeks in this study. This result was agreed by Wongsetthachai et al. (2011)whoshowed smooth muscle regeneration at 6 weeks postoperatively. On contrast, regeneration of smooth muscle after substitution with a free peritoneal graft in rabbits (Hutschenreiter et al., 1978) and a pedicled peritoneal flap in dogs (Tsuji et al., 1963) occurred at 10 and 12 weeks, respectively.

Bone metaplasia is not detected in our study. On contrast, Wongsetthachai et al. (2011) found bone metaplasia at 8 and 10 weeks following bladder wall substitution with tunica vaginalis in male dogs. Bone metaplasia was also reported when the porcine small intestine submucosa was used as a bladder wall substitute in dogs (Pope et al., 1997). Bone metaplasia was not observed when the peritoneum was used as a pedicled flap in dogs (Tsuji et al., 1963) and as a free graft in rabbits (Hutschenreiter et al., 1978). The exact cause of the metaplasia is unknown. Variations in graft preparation and processing may contribute to these contrasting findings.

It could be concluded that the use of tunica vaginalis propria in male dogs has the advantage as a readily available autologous graft for bladder wall reconstruction. It is a simple and less expense method with a reliable regenerative capacity. The disadvantage is that it is only applicable to the male intact dog which is the self donor. Use of tunica vaginalis as an allograft in either sex of dogs requires further investigation and a search for the proper method of the preparation and preservation of the graft.

REFERENCES

Bancroft, J.D.; Stevens, A. and Turner, D.R. (1990): Theory and Practice of histopathological technique 3^rd Ed. Churchill, Livingstone, Edinburgh, London, Melboune and New York.

Bauer, S.B.; Hendren, W.H.; Kozakewich, H.; Maloney, S.; Colodny, A.H.; Mandell, J. and Retik, A.B. (1992):Perforation of the augmented bladder. J Urol. 148: 699-703.

Leslie, B.; Barboza, L.L.; Souza, P.O.; Silva, P.S.; Delcelo, R.; Ortiz, V. and Macedo, A.Jr. (2009): Dorsal tunica vaginalis graft plus onlay preputial island flap urethroplasty: Experimental study in rabbits. Journal of Pediatric Urology. 5: 93-99.

Calado, A.A.; Macedo, A.Jr.; Delcelo, R.; Poli de Figueiredo, L.F.; Ortiz, V. and Srougi, M. (2005): The tunica vaginalis dorsal graft urethroplasty: experimental study in rabbits. J. Urol. 174: 765–770.

Crane, J.M.; Scherz, H.S.; Billman, G.F. and Kaplan, G.W. (1991):Ischemic necrosis: a hypothesis to explain the pathogenesis of spontaneously ruptured enterocystoplasty. J Urol. 146:141-144.

El-Daharawy, M.H.; Shekidef, M.H. and Fetaih, H. (2006): Experimental Evaluation of Colonic Graft De-epithelialization Using Silver Nitrate solution for Bladder Augmentation in Bitches. 4th scientific conference, Faculty of Veterinary Medicine, Suez Canal University, 21st-23rd November, 2006.

Elkoushy, M.A. (2003): Urinary bladder augmentation using new tissue materials. A thesis submitted in partial fulfillment of the requirement of the M.D. degree in urology. Faculty of Medicine, Suez Canal University.

Gajda, M.; Tyloch, J.; Tyloch, F.; Hasse, P.; Sujkowska, R. and Domaniewski, J. (2006): Morphologic changes in the canine urinary tract after ureteral reconstruction with the tunica albuginea (experimental study). International Urology and Nephrology 38: 215–224.

Hall, L.W.; Clarke, K.W. and Trim, C.M. (2001): Principle of sedation, analgesia and premedication in veterinary anaesthesia 10^th Ed. W.B. Saunders P.79.

Hutschenreiter, G.; Rumpelt, H.J.; Klippel, K.F. and Hohenfellner, R. (1978): The free peritoneal transplant as a substitute for the urinary bladder wall. Invest. Urol. 15: 375–379.

Kropp, B.P.; Rippy, M.K.; Badylak, S.F.; Adams, M.C.; Keating, M.A.; Rink, R.C. and Thor, K.B. (1996): Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J. Urol. 155: 2098–2104.

Parsons, M. and Toozs-Hobson, P. (2005): The investigation and management of interstitial cystitis. J. Br. Menopause Soc. 11: 132-139.

Pope, J.C.; Davis, M.M.; Smith, E.R.Jr.; Walsh, M.J.; Ellison, P.K.; Rink, R.C. and Kropp, B.P. (1997): The ontogeny of canine small intestinal submucosa regenerated bladder. J. Urol. 158: 1105–1110.

Sadove, R.C.; Rodgers, J.B.; Fink, B.F. and McRobert, J.W. (1993): Experimental rectus abdominis myocutaneous and rectus abdominis myoperitoneal flaps as urinary bladder wall substitutes in miniature swine. Plast. Reconstr. Surg. 91: 511–521.

Theodorescu, D.; Balcom, A.; Smith, C.R.; McLorie, G.A.; Churchill, B.M. and Khoury, A.E. (1998): Urethral replacement with vascularized tunica vaginalis: defining the optimal form of use. J. Urol. 159: 1708–1711.

Tsuji, I.; Kuroda, K.; Fujieda, J.; Shiraishi, Y.; Kassai, T. and Shida, H. (1963): A clinical and experimental study on cystoplasty not using the intestine. J. Urol. 89: 214–225.

Vilar, F.O.; de Araujo, L.A. and Lima, S.V. (2004):Total bladder replacement with de-epitheliallized ileum. Experimental study in dogs. International Braz J Urol. 30: 237-244.

Wongsetthachai, P.; Pramatwinai, C.; Banlunara, W. and Kalpravidh, M. (2011): Urinary bladder wall substitution using autologous tunica vaginalis in male dogs. Research in Veterinary Science 90: 156–159.

Wrobel, K.H. (1998): Male reproductive system. In: Dellmann, H.D., Eurell, J.A. (Eds.), Textbook of Veterinary Histology, fifth ed. Williams and Wilkins, Philadelphia, pp. 226–227.