Comparison of Different Techniques for Hemostasis in a Rabbit Model of Hypospadias Repair

Comparison of Different Techniques for Hemostasis in a Rabbit Model of Hypospadias Repair Abdol-Mohammad Kajbafzadeh,* Seyedmehdi Payabvash, Seyed Mohammad Tavangar, Amirali Hassanzadeh Salmasi, Zhina Sadeghi, Azadeh Elmi, Farrokh Tirgari and Amirnader Emami Razavi From the Pediatric Urology Research Center, Department of Urology, Children’s Hospital Medical Center (A-MK, SP, AHS, ZS, AE), Department of Pathology, Shariati Hospital (SMT) and Department of Pathology, Cancer Institute of Iran (FT, AER), Tehran University of Medical Sciences, Tehran, Iran

Purpose: We compared the cellular and ultrastructural changes in the urethral wall following application of different hemostasis techniques in a rabbit model of hypospadias surgery. Materials and Methods: Rabbits were allocated into 5 groups. In group 1 animals underwent surgery without application of any hemostasis technique; in group 2 continuous penile tourniquet was applied for 30 minutes; in group 3, 3 intermittent periods of 10-minute penile tourniquet were applied with 3-minute intervals of reperfusion; in group 4 epinephrine was injected to maintain a 30-minute period of hemostasis; and in group 5 epinephrine vehicle (normal saline) was injected during the procedure. Early urothelium ultrastructural damage was studied 1 hour postoperatively with electron microscopy. Apoptotic damage and histopathological changes were determined 48 hours following the procedure. Late onset complications were assessed with retrograde urethrography and evaluation of tissue fibrosis at 8 weeks postoperatively. Results: Electron microscope studies demonstrated urothelium ultrastructural damage in all hemostasis groups compared to controls. However, the changes were most prominent in group 4. The apoptosis index of urethral wall myocytes in groups 1 and 5 was significantly lower compared to other groups. Moreover, the number of apoptotic myocytes in epinephrine injected animals was significantly higher than in the continuous or intermittent tourniquet group as well as the normal saline injected group. At 8 weeks postoperatively collagen deposition in the urethral wall of rabbits in group 4 was higher than that in group 1. Although urethrocutaneous fistula was found in only 1 rabbit in group 4, the difference was not significant. Conclusions: Hemostasis techniques applied for maintaining a bloodless surgical field during hypospadias repair may lead to ischemia/reperfusion tissue damage in the urethral wall. Our findings suggest that epinephrine injection may result in more prominent cellular changes compared to tourniquet techniques. However, further experimental and human studies are required to draw a firm conclusion. Key Words: hypospadias, hemostasis, tourniquets

ypospadias is one of the most common congenital defects of the penis, with anomalous development of the penile urethra.1 Despite considerable progress in the surgical repair of hypospadias, the reconstruction procedure may be associated with complications, including dehiscence, urethrocutaneous fistulas, proximal strictures, diverticula and meatal stenosis.1 Whatever the reconstruction technique, an essential part of the procedure is to maintain a bloodless operative field. Different methods are available for hemostasis, including injection of vasoconstrictive agents, use of bipolar electrocautery and application of a penile tourniquet.2 However, there is not yet consensus among urologists regarding the optimal hemostasis technique.

H

Submitted for publication April 8, 2007. Presented at 17th Annual Congress of European Society of Pediatric Urology, Athens, Greece, April 29, 2006; first place Clinical Poster Presentation Prize. Experimental protocol received local ethics committee approval. * Correspondence and requests for reprints: No. 36, 2nd Floor, 7th St., Saadat-Abad, Ave. Tehran 19987, Iran (telephone: 98-21-22089946; FAX: 98-21-2206-9451; e-mail: [email protected]).

0022-5347/07/1786-2555/0 THE JOURNAL OF UROLOGY® Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION

Redman popularized the application of a rubber band tourniquet in hypospadias surgery beginning in 1986.3 The method is still frequently used for different types of penile surgery. Recently, Crawford et al reported using a Penrose drain as a penile tourniquet during hypospadias repair.4 Some authors advocate injection of epinephrine for maintenance of a bloodless field during hypospadias surgery.5–7 The advantages of epinephrine injection include reduced use of tourniquet, provision of a more selective bloodless field and longer duration of pain control in cases of simultaneous analgesic injection. To date, few comparisons of the different hemostasis techniques applied in hypospadias surgery have been reported in the literature.8 It has been hypothesized that some complications of hypospadias surgery could be the result of ischemic tissue necrosis.2 However, cellular devitalization following temporary penile hemostasis may be due to programmed cell death following reperfusion, comparable to the ischemia/reperfusion phenomenon observed in other tissues. Therefore, accurate evaluation of cellular damage following hemostasis warrants assessment of early ischemic damage and later onset cellular apoptosis. We studied early and late cellular

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Vol. 178, 2555-2560, December 2007 Printed in U.S.A. DOI:10.1016/j.juro.2007.08.051

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injury in the urethral wall tissues following application of different hemostasis methods in a rabbit model of hypospadias repair. MATERIALS AND METHODS Animal Procedures and Sampling A total of 56 white New Zealand rabbits (1,500 to 2,000 gm) were used in this study. Using general anesthesia, the rabbit penis was degloved and the pendulous urethra was exposed (fig. 1). Next, a 1.5 by 0.3 cm segment of ventral wall was excised to create a hypospadias-like defect. A full thickness incision was made on the dorsal distal urethral plate. Then, a 5Fr feeding tube was inserted as the urethral catheter, and both urethral wings were tubularized ventrally using a 7-zero polyglactin suture. The penile skin was approximated by simple interrupted 6-zero polyglactin sutures. The rabbits were randomly allocated into 5 groups, with each containing 12 animals (except group 5, which contained 8). In group 1 animals served as controls and underwent hypospadias surgery without application of any hemostasis technique. However, the procedure was prolonged for 30 minutes to make comparisons between groups more accurate. In tourniquet groups a rubber tourniquet was passed around and both arms of the tourniquet were grasped with a mosquito forceps close to the penile base, producing enough tension to prevent bleeding. In group 2 we applied 30 minutes of continuous penile tourniquet for hemostasis. In group 3 bleeding was prevented by 3, 10-minute intermittent periods of tourniquet application with 3-minute intervals of reperfusion. In group 4 epinephrine (1:100,000 diluted with normal saline) was injected beneath to the urothelium beside the incision line.6 The injection was repeated (if needed) to maintain the hemostasis for 30 minutes (total volume less than 0.3 ml).6 Group 5 contained 8 rabbits as controls for normal saline injection under the urothelium. Urethral samples for electron microscopic evaluation were obtained 1 hour after the procedure from 4 rabbits per study group. At 48 hours postoperatively the entire penile urethra of 4 other rabbits from each group was excised and fixed in formalin for determination of apoptosis index and histopathological study. At 8 weeks postoperatively the remaining 4 rabbits from groups 1 to 4 underwent urethrog-

raphy, followed by excision and fixation of urethral samples for determination of fibrosis. Electron Microscope Study Samples of rabbit urothelium were carefully excised and immersed in phosphate buffered 3% glutaraldehyde. The sections were double stained with lead citrate and 20% uranyl acetate, and were observed and photographed using an electron microscope. A total of 10 representative urothelial cells (per rabbit specimen) were randomly selected and analyzed. The urothelial cells were evaluated for evidence of nuclear changes (chromatin dispersion or clumping), the presence of cytoplasmic changes including macrovacuoles and microvacuoles, the overall shape of the cell (ie shrunken, swollen) and the appearance of RER. The mitochondria were checked for abnormalities in distribution, shape, matrix density, cristae structure and appearance of any abnormal structures. The changes were graded as mild (I), moderate (II) or severe (III), depending on the frequency in each case. In Situ Detection of Apoptosis We used an in situ cell death detection kit for determination of apoptotic nuclei in tissue sections by TUNEL technique. The assays were performed according to manufacturer instructions.9 All TUNEL positive cells fulfilling the morphological criteria for apoptosis were counted. Apoptotic index was calculated as the number of TUNEL positive cells divided by the total number of cells counted in 30 fields at 400⫻ magnification. Radiological Evaluation The rabbits underwent retrograde urethrography at 8 weeks postoperatively. A 5Fr feeding tube was inserted into the meatus and fixed to the penile skin with an adhesive strip. Urethrography was performed after injection of 2 ml isotonic mixture of normal saline and meglumine 76%. Evaluation of Tissue Fibrosis Picrosirius red was used for collagen staining.9 Under fluorescence microscopy collagen fibers exhibit a characteristic red-orange fluorescence, whereas elastin fibers appear as a

FIG. 1. Rabbit model of hypospadias surgery. Penis was held vertically with stay suture through glans. In epinephrine injection group diluted solution was injected beside incision line, just under urothelium (A). In tourniquet groups rubber ring cut from surgical glove was used as rubber band (B).

HEMOSTASIS IN RABBIT MODEL OF HYPOSPADIAS REPAIR strong fluorescent green.10 Image analysis was performed using professional biosoftware. The ratios of positively stained areas to full urethral wall thickness areas were calculated using computerized image analysis.9 For each sample 4 to 5 photomicrographs were used for scoring, and the mean score was assigned as the final value for data analysis. Statistical Analysis Results are expressed as mean ⫾ SD. Statistical evaluation was performed using analysis of variances or chi-square test, where appropriate. A value of p ⬍0.05 was considered statistically significant.

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Ultrastructural changes following penile ischemic hemostasis Grade No. RER changes: 0 I II III No. mitochondrial changes: 0 I II III

Group 1 3 1

Group 2

Group 3

1 3

2 1 1

2 2

1 3

Group 4

Group 5 4

1 3

4

4 1 3

Ultrastructural damage to rough endoplasmic reticulum and mitochondria was graded as none (0), mild (I), moderate (II) or severe (III).

RESULTS Light Microscope Study The degrees of edema and congestion were mild in specimens of groups 1, 4 and 5 vs mild to moderate in samples of groups 2 and 3. Dilated lymphatic channels were observed in some samples of groups 2 and 3 but not in the other 3 groups. The morphology of the urothelium remained normal in all samples, with no sign of inflammation or necrosis. There was no significant difference in histopathological parameters between the study groups. Electron Microscope Study Urothelial cells in the control rabbits from groups 1 and 5 had normal appearing nuclei and cytoplasm without signs of edema or injury (fig. 2, A). Organelles within the cytoplasm, including the mitochondria and RER, also appeared normal. Following application of hemostasis techniques trivial changes were seen in the urothelium nuclei and cytoplasmic macrovacuoles and microvacuoles (data not shown). However, the changes in the mitochondria and RER were prominent (fig. 2, B). Higher grades of swelling and disruption of matrix, cristae and membranes were seen in the urothelial cell mitochondria (p ⬍0.001) and RER (p ⬍0.01) of groups 2, 3 and 4, compared to groups 1 and 5 (see table). Although none of the ultrastructural changes in the study groups was irreversible, changes were most prominent in the epinephrine injection group. Apoptosis The apoptotic cells in the urothelium and submucosal connective tissue were scarce, with no significant differences between the study groups (fig. 3, data not shown). However,

the myocyte apoptosis index in the continuous (6.75 ⫾ 0.94, p ⬍0.01) and intermittent (7.28 ⫾ 0.89, p ⬍0.01) tourniquet groups was higher than in normal controls (3.56 ⫾ 0.29) and normal saline injected controls (3.06 ⫾ 0.76). The number of apoptotic myocytes in the epinephrine hemostasis group (9.81 ⫾ 1.06) was also increased compared to group 1 (p ⬍0.001) and group 5 (p ⬍0.001). The apoptosis index in group 4 was also higher than in group 2 (p ⬍0.01) or group 3 (p ⬍0.05). We found no significant differences in myocyte apoptosis indices between the continuous and intermittent penile tourniquet groups. Retrograde Urethrography Since injection of normal saline in group 5 had no effect on short-term cellular damage compared to normal controls (group 1), we excluded group 5 from long-term studies. During the 8-week followup only 1 rabbit from the epinephrine injected group had a urethrocutaneous fistula. Retrograde urethrography revealed no sign of urethral stricture or extravasation in any other rabbit. Evaluation of Urethral Wall Fibrosis The percentage of collagen fibers in the urethral wall was increased following continuous (25.2% ⫾ 5.6%) and intermittent (28.5% ⫾ 6.9%) tourniquet hemostasis but it was not significantly different from group 1 (20.5% ⫾ 6.4%). However, collagen fiber-to-urethral wall ratio was significantly higher in the epinephrine injection group (36.7 ⫾ 7.4) compared to controls (p ⬍0.05, fig. 4). There was no significant difference between groups 2, 3 and 4.

FIG. 2. Ultrastructural changes in urothelial cells. Normal appearing mitochondria and RER (arrows) in urothelium of rabbit from control group (A). Swollen matrix and cristae, and disruption of membranes in RER and mitochondria (arrows) of urothelial cells in animal from group 4 (B).

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FIG. 3. Apoptotic nuclei were almost absent in urothelium and submucosal connective tissue of urethral wall (A). Number of apoptotic myocytes (arrows) was increased in urethral wall of animals that underwent ischemic hemostasis (B and C).

DISCUSSION The best option for maintenance of effective and temporary hemostasis without permanent tissue injury during hypospadias repair has not yet been determined. However, it is generally accepted that the use of hemostasis methods should be kept to a minimum to avoid ischemic injuries.2 We studied ischemia/reperfusion induced cellular damage following the application of different hemostasis methods. The results demonstrated that all of the aforementioned interventions induced urethral wall tissue damage but the damage was more prominent in the epinephrine injection group compared to the tourniquet groups. Various types of tourniquets have been used in hypospadias surgery through the years. However, there is no consensus regarding the optimal duration of tourniquet hemostasis.8 Redman believes that ischemic periods less than 50 minutes are well tolerated.3 In addition, while some urologists advocate application of a tourniquet during the entire operation,3 others advise intermittent release every 10 minutes.11 Duckett and Keating use a tourniquet intermittently with infiltration of epinephrine/lidocaine into the penile skin.7 There are few reports of tourniquet induced complications following hypospadias repair attributed to the occurrence of reactive hyperemia following tourniquet release.12 Rabinovitch reported penile edema in 4.17% of cases after tourniquet release.13 However, it is generally accepted that complete interruption of the perfusion in the acral areas can be tolerated without damage for at least 1 hour. Moreover, tourniquets have been proved to cause nerve and muscle injury in animal models only when pressures exceed 500 mm Hg.14

A similar controversy exists regarding the optimal epinephrine dose and its use with lidocaine in hypospadias repair. Tissue sloughing is reported following the use of local anesthetic containing 1:20,000 epinephrine. However, different studies have supported the safe application of 1:100,000 to 1:800,000 concentrations of epinephrine.8,14 It was once strictly emphasized that epinephrine containing solutions should not be injected into tissues supplied by end arteries such as in the fingers and toes. However, recent investigations have largely disproved this idea.14 Moreover, there is no known report of any complication following hypospadias surgery attributed to the administration of epinephrine. Application of hemostasis techniques results in temporary tissue ischemia followed by reperfusion, which is similar to cellular damage following ischemia/reperfusion. The underlying mechanism of reperfusion damage is presumably the release of reactive oxygen species from either activated parenchymal cells or invading neutrophils.15 Indeed, high concentrations of reactive oxygen species induce cell death through necrosis, while lower concentrations promote apoptosis. We found no evidence of tissue necrosis following application of different hemostasis methods. However, myocyte apoptosis was significantly increased after ischemia/ reperfusion. The results of electron microscope studies also revealed RER and mitochondrial damage in the hemostasis groups compared to controls, most prominently with epinephrine injection. Early ultrastructural damage was followed by apoptotic cell injury in later hours. The TUNEL assay determines DNA fragmentation, which is a late consequence of apoptotic cell death, with the

HEMOSTASIS IN RABBIT MODEL OF HYPOSPADIAS REPAIR

FIG. 4. Collagen fibers are represented by characteristic fluorescent red-orange color. Collagen fiber–to– urethral wall ratio was significantly lower in control rabbits from group 1 (A), compared to epinephrine injected animals (B).

number of TUNEL positive cells usually peaking 48 hours after reperfusion.15 We also found increased myocyte apoptosis 48 hours after the application of ischemic hemostasis. Interestingly, the myocyte apoptosis index was highest in those animals that received epinephrine injection. The higher myocyte apoptosis indices in group 4 might be due to fairly uncontrolled (and presumably longer) periods of ischemia following epinephrine hemostasis. It is noteworthy that longer ischemic periods lead to higher apoptotic damage.15,16 One may conclude that epinephrine injection cannot maintain the period of ischemia in a manner as strictly controlled as tourniquet hemostasis, leading to unwanted longer duration of ischemia and higher apoptosis indices. However, this phenomenon may also be due to direct proapoptotic effects of epinephrine. Recent studies have shown the cytotoxic effects of epinephrine (1 to 10 ␮M) exposure on myocytes and endothelial cells via activation of the ␤2-adrenergic receptor.17,18 In hypospadias repair surgery solutions with concentrations up to 60 ␮M (1:100,000) are injected locally for hemostasis, which may have potential cytotoxic effects. However, fibroblasts and urothelium seem less sensitive to proapoptotic effects of epinephrine compared to myocytes. It is noteworthy that epinephrine is injected at different sites and doses during hypospadias repair.6 – 8 Moreover, considering the weight of rabbits used in the present study, the actual mg/kg dose of epinephrine used in our animals appears to be different from what is used in children. However, since epinephrine is supposed to have a local effect (and caution should be used to avoid injection into the bloodstream), one may assume that the urethral tissue is exposed

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to similar local concentrations of epinephrine in animals and human subjects. The results following local injection of normal saline in group 5 also confirmed the role of epinephrine in observed cytotoxic effects. Regardless of the triggering factor, the appearance of apoptotic cells is generally associated with later deposition of fibrotic tissue. Clearance of apoptotic debris through phagocytosis stimulates fibrogenesis through generation of transforming growth factor-␤, which is a strong fibrogenic signal.19 Thus, the increase in the number of urethral apoptotic myocytes could have a major role in the induction of fibrosis, since at the end of 8-week followup higher levels of collagen deposition were found in epinephrine injected animals. Although fibrotic changes may contribute to surgical complications such as strictures and meatal stenosis, the present results provide no evidence to support this hypothesis. Moreover, the incidence of major surgical complications was not different between the study groups, although the number of cases was too small to draw a firm conclusion. Interestingly, even meticulous cellular and ultrastructural studies demonstrated no significant differences between continuous and intermittent penile tourniquet application in our series. These data suggest that use of short-term reperfusion intervals does not reduce cellular damage following penile tourniquet hemostasis. However, there is no question that application of shorter periods of ischemic hemostasis will reduce ultimate tissue damage.8 There are also some obvious limitations in our study. First, experimental animal series of hypospadias have the inherent disadvantage of studying events that take place in a normal urethra. Moreover, one cannot necessarily translate the presence of cellular damage to the incidence of surgical complication, since the long-term results of hypospadias repair seem to depend on the severity of the initial abnormality rather than the operative technique.20 Finally, only controlled human studies with long-term followup can definitively show the optimal and safest surgical method. CONCLUSIONS Our results indicate that hemostasis techniques applied for maintenance of a bloodless operative field throughout hypospadias repair induce ultrastructural and cellular damage in the urethral wall. These changes are more prominent following epinephrine hemostasis. Additionally, we found no difference between application of continuous and intermittent penile tourniquets. Since these methods are routinely used during hypospadias repair, they should be applied with more caution, and further evaluation of their safety is needed.

Abbreviations and Acronyms RER ⫽ rough endoplasmic reticulum TUNEL ⫽ terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling

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