A comparison between argon laser and microsuture anastomosis of the rat uterine horn*

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1 FERTILITY AND STERILITY Copyright,~ 1987 The American Fertility Society Vol. 47, No~ 2, February 1987 Printed in U.SA. A comparison between argon laser and microsuture anastomosis of the rat uterine horn* Douglas R. Lyont Louis A. Vontver, M.D.:j: Dorothy L. Patton, Ph.D.:j: Sheridan A. Halbert, Ph.D.II~ University of Washington, Seattle, Washington For assessment of the use of the argon laser for tubal anastomosis, the uterine horns of 12 Sprague-Dawley rats were surgically divided and then anastomosed, 6 by argon laser photocoagulation and 6 by the conventional technique of microsurgery. After a 4- to 6-week postsurgical period subjects were reexamined. All microsutured anastomoses were fully patent and continuous, with no apparent fibrosis. Four of six laser subjects had complete occlusion; the other two exhibited patencies between 10% and 20% of normal luminal area. Although initially producing satisfactory union, argon laser photocoagulation proved highly tissue traumatic, resulting in poor regeneration of the anastomotic site. Fertil Steril 47:329, 1987 Conventional technique for the repair of tubal occlusion involves excision of the occluded area followed by end-to-end anastomosis. Excision is performed either by microscissors, electrocautery, or the carbon dioxide (C0 2 ) laser.l Anastomosis is conducted under magnification with the application of micro sutures, as characterized by Gomel. 2 Alternative anastomotic techniques include CO 2 laser tissue fusion 3, 4 and the use of fibrin glue. 5 The argon laser photocoagulator is currently under study as a method of tissue reapproxima- Received June 17, 1986; revised and accepted October 29, *Supported in part by the National Institutes of Health grant R01 HD tdepartment of Biology. :l:department of Obstetrics and Gynecology. Department of Biological Structure. IICenter for Bioengineering. ~Reprint requests: Sheridan A. Halbert, Ph.D., Department of Biological Structure, SM-20, University of Washington, Seattle, Washington tion. Previous uses include reparation of the monkey nerve, 6 vascular anastomosis, 7 and treatment of abdominal endometriosis by the laparoscope. 8 When light from the argon laser is directed at a blood-coated surface, a semirigid coagulate forms, binding together underlying tissues. In addition, the coagulate serves to limit light penetration and subsurface heat transfer. If used with a laparoscope, the argon laser offers several advantages for end-to-end anastomosis in the reproductive tract. It provides hemostatic control and eliminates any antigenic response induced by foreign materials (glue, suture, or staples). Potentially, we hoped it would decrease surgical and tissue trauma in addition to eliminating the need for extraabdominal exposure--factors crucial in the prevention of adhesion formation and secondary infection. We investigated the use of argon laser photocoagulation as a method of anastomosis in a rat uterine horn (cornu) model. This method was compared with the conventional technique of microsurgical reconstruction. Lyon et al. Tubal anastomosis with the argon laser 329

2 MATERIALS AND METHODS RATS Female Sprague-Dawley rats, 6 to 8 weeks old, were obtained from Tyler Laboratories, Inc., Seattle, W A. Before experimental procedures all animals were housed and fed in the vivarium facilities of the Department of Animal Medicine at the University of Washington. Subjects were divided into two groups of six rats each. The uterine horns of rats in group I had conventional anastomosis by microsuture, and those in group II were anastomosed by argon laser photocoagulation. LASER A model 171, full-spectrum argon ion laser (Spectra-Physics Corp., Mountain View, CA) was used for anastomotic photocoagulation. Emitted light was focused onto a 660 quartz optical fiber, which terminated into a pencil-like hand piece of 12 divergence. The flux of light energy incident on tissue varied with both the laser output and the fiber-tissue distance. Power densities ranged from to 37.5 W/cm 2 with fiber-tissue distances of 0.34 to 0.22 cm. Figure 2 shows wavelength contribution to total power output. PHOTOCOAGULATION Blood to be used in anastomotic photocoagulation was withdrawn from donor rats, heparinized, and refrigerated at 5 C for 3 to 9 days. Immedi- 1.5 I ~ 1.0 I 0.5 Ii'I III' Ii Wavelength (nm) Figure 2 Absorption spectrum of oxygenated blood. The transmission wavelengths of argon laser light, at top, are listed by percent contribution to total power output. ately before surgery the serum layer was discarded, concentrating the more photosensitive red blood cells to serve as the coagulation medium. As the packed red blood cell concentrate was applied dropwise from a 1-ml syringe onto the anastomoticjunction, bursts oflaser light were circumferentially applied until a stable anastomotic union was achieved (10 to 15 bursts, 0.8 W of 0.5-second duration). Figure 1 illustrates the procedure. This effect can be explained by the selective absorption of argon light by hemoglobin molecules (Fig. 2). ANESTHESIA Two methods of anesthesia were used: (1) injection of a ketamine and Rompun mixture (Mobay, Animal Health Division, Shawnee, KS) or (2) inhalation gas using 2.5% halothane in oxygen. Figure 1 Schematic of method of argon laser anastomotic photocoagulation of the rat uterine horn. 330 Lyon et ai. Tubal anastomosis with the argon laser SURGICAL PROCEDURE Surgeries were performed with sterile technique. Each rat abdomen was opened with a 2- to 3-cm longitudinal incision. The uterine horns were isolated, and one was randomly selected to receive the experimental procedure; the other served as an internal control. Each experimental horn received identical treatment with regard to irrigation, radiant heat from illumination, and Fertility and Sterility

3 exposure time. At 3-minute intervals tissues were moistened with room temperature saline. The experimental tube was transected, and a 5-mm-long segment was removed. Either the microsuture (group I) or laser (group II) anastomotic technique was then applied. Group I uterine horns were anastomosed conventionally with eight to nine microsutures of 11-0 monofilament nylon (Ethilon, Ethicon Inc., Somerville, NJ). Four were placed in the muscularis, four in the serosa, and one at the mesenteric border. Anastomosis with the argon laser (group II) necessitated the use of an internal supportive catheter (0.64 mm, nonreactive polyethylene) to provide adequate tissue layer and close end-toend approximation. For placement of the catheter, two longitudinal incisions were made in the uterine horn along the antimesenteric border, approximately 1.5 cm to each side of the anastomotic site. After horn transection, a blunt-nosed catheter was inserted through the most cephalad incision past the anastomotic site and out the caudal incision. Anastomosis by photocoagulation followed, and the catheter was removed before closure of the abdominal incision. EVALUATION PROCEDURE After a 4- to 6-week postsurgical period, the rats were reoperated. The uterine horns were examined grossly for any sign of overt anatomic abnormalities, including failure of approximation Clack of end-to-end attachment across the anastomotic site), the presence of a hydrocornu, and adhesions causing significant conformational alteration or stricture. Tissue adhesions were assessed with the aid of a 6-12X binocular dissection microscope. They were quantified as percent of uterine horn surface area demonstrating adhesions within a l-cm band on either side of the anastomotic site. The severity of adhesion on each horn was scored qualitatively with values from 0 to 4. Table 1 describes these values. For assessment of patency, a single longitudinal incision was made through the anastomotic site. Percent of normal patency was calculated by visually comparing the luminal width at the site of anastomosis with the luminal width of undisturbed tissue at each side. When a horn exhibited severe constriction, a dilute solution of methylene blue was injected to establish occlusion before the Table 1. Description of Scores Depicting Adhesion Severity Type o Description No evidence of adhesions 1 Thin and filmy 2 Thin with associated fatty tissue 3 Thick and fatty, with no apparent disruption of normal organ conformation 4 Thick and fatty, with obvious alteration of organ conformation longitudinal incision was made. The sites of anastomosis were sectioned and prepared for light microscopy. Tissues were fixed in methyl Carnoys, dehydrated in a graduated series of ethyl alcohols followed by toluene, and embedded in paraffin. Sections 4 to 6 f.l thick were cut and oriented to view the cornual wall across the site of anastomosis. Tissues were stained with hematoxylin and eosin and Masson trichrome. RESULTS Uterine horn anastomoses were evaluated with regard to attachment, patency, presence of a cornual occlusion, and adhesion formation (Table 2). Tissues were examined for inflammation, continuity, and fibrosis. The uterine horns of all group I rats were 100% patent, with no apparent edema or stenosis. Healing was so complete that the anastomotic sites were indistinguishable from adjoining tissue. Histologic examination showed tissue continuity, no apparent fibrosis, and no sign of inflammation. Thin adhesions with some associated fatty tissue (type 2) were on the average present on 54% of the evaluated surface of the experimental horn, as compared with thin filmy adhesions (type 1) averaging 7.5% of the same area on the control horn; therefore, 46.5% of the evaluated area had adhesions inherent to the microsurgical procedure. Fertilization was attempted in one subject with the subsequent delivery of 13 viable progeny. On reoperation, group II rats showed a moderate to poor quality of union, with stenosis and accumulation of intraluminal fluid associated with cornual occlusion. Patency was observed in only two of six subjects, limited to 10% and 20% of normal luminal area. Hydrocornua, rigid on palpation, were present in the remaining four, indicative of occlusion. Interstitial tissue in the anastomotic site was composed primarily of stringy fibrous tissue. Normal tissue continuity was in- Lyon et al. Tubal anastomosis with the argon laser 331

4 Table 2. Gross Observation of Rat Uterine Horns on Reoperation 4 to 6 Weeks After Surgical Anastomosis Rat no. Attachment Percent patient Hydrouteri Group I (microsuture) 1 Yes 100 No 2 Yes 100 No 3 Yes 100 No 4 Yes 100 No 5 Yes 100 No 6 Yes 100 No Group II (laser photocoagulation) 7 Yes 10 No 8 Yes 0 Yes 9 Yes 0 Yes 10 Yes 0 Yes 11 Yes 0 Yes 12 Yes 20 No Adhesions Experimental Control Quantity Quality Quantity Quality terrupted at all muscular levels and in some cases was completely replaced by a composite of collagen fibers, agglutinated photocoagulate, and adipose tissue. Thick fatty adhesions (type 3) were present across the anastomotic site and averaged 50.8% of the evaluated external area. The control horns were free from adhesion. DISCUSSION Because patency, structural integrity, and presumably function were restored in the uterine horns of all control subjects, conventional microsuture technique proved to be an adequate control model of end-to-end anastomosis. Argon laser photocoagulation was shown to be ineffective, yielding patency in only two of six subjects. At the end of the 4- to 6-week recovery period we found stenosis, occlusion, and hydrocornua, indicative that necrosis and partial coagulate-serosal dehiscence occurred in the recovery interim. Histologic examination showed fibrosis, multilayer discontinuity of the muscularis, and a disproportionate amount of revascularization to each side of the anastomotic junctions. A non suture alternative to argon laser photocoagulation is tissue fusion with the CO 2 laser. Satisfactory anastomosis of the rabbit fallopian tube was accomplished with this technique by Klink et a1. 3 with a power density of 64 W/cm 2. In a similar study, Fayez et au and Choe et al.,4 using 900 and 637 to 796 W/cm 2, respectively, were unsuccessful in attempts at CO 2 laser anastomosis. The success of CO 2 laser function appears to depend on the power density applied; the lesser the value the better the chance of functional repair. In our study we used the argon laser at 27 W/cm 2 with unsuccessful results. This power density is less than half that used by Klink et a1. 3 for successful anastomosis by CO 2 laser fusion. Thus, when comparing CO 2 and argon laser techniques, we are unable to correlate successful anastomoses with power density; instead, the type oflaser radiation seems to determine technique success. The argon and CO 2 lasers differ in their effect on tissue by method of energy transfer. The CO 2 laser emits long wavelength, infrared light (10,640 nm), which is well absorbed by water molecules. The argon laser emits a spectrum of shorter wavelengths (476.5, 488.0, and nm) to which water is relatively transparent. However, these shorter wavelengths are readily absorbed by pigment (e.g., hemoglobin, Fig. 2) or a similar protein complex. This absorption specificity has been reported to be responsible for the selective damage of mitochondria 9 and microvasculature. lo Owing to the spectral dependency of optical absorbance oftissue, the depth of penetration of CO2 and argon laser light differs considerably. Because water is the primary component of tissue, incident CO 2 laser light is rapidly dissipated, whereas argon laser light penetrates to a much greater depth. Bellina et al. ll reported that CO2 laser light is 90% absorbed in less than the first 0.1 cm of fat and that of the argon laser is not diminished until a distance > 1 cm. Because argon laser light penetrates deeper and is selective- 332 Lyon et al. Tubal anastomosis with the argon laser Fertility and Sterility

5 ly absorbed by critical organelles and tissues, it has a much greater noxious potential than the CO 2 laser. The damage resulting from our use of argon laser photocoagulation was likely of two causes. First, thermal energy transferred to the photocoagulate during irradiation dissipated to the underlying serosa, causing protein denaturation and membrane decomposition. Subsequent necrosis of the serosal layer prevented the tissuecoagulate attachment needed to maintain the close end-to-end approximation critical for proper tissue regeneration. Second, absorption of excess or misdirected light by tissue pigments caused damage to blood vessels and intracellular organelles adjacent to the anastomotic junction. Tissue necrosis was thus promoted indirectly by loss of vascular circulation and directly by cellular damage, preventing the normal course of repair. The poor performance of the argon laser, when used to anastomose vascularized tissue, suggests that it is inappropriate for tubal anastomosis. However, in addition to its use in tissue reattachment, the unique ability of argon laser light to pass through tissue and be selectively absorbed by pigment makes it well suited for use as a lytic or hemostatic technique when a restriction of vascular flow is required. Irrespective of application, it is imperative to minimize exposure and damage to nontarget tissue. Four modifications of our technique that could substantially reduce such deleterious effects on nontarget tissues are: (1) Creation of a condensed coagulate that will more completely absorb incident light. A higher concentration of the hemoglobin tetramer in the coagulate could be obtained by either extraction or centrifugation. (2) Decrease in beam spot size. Accomplished by reduction of fiber optic diameter or lensing, this would improve directional accuracy and minimize scatter. (3) Reduction of pulse duration. Shortening the duration of each pulse would lower peak temperatures. (4) Exclusion of noxious light frequencies. Use of prism dispersion or direct filtration may enable elimination of those frequencies that damage intracellular organelles. In conclusion, the unmodified argon laser photocoagulator proved to be highly tissue destructive, performing poorly as an anastomotic tech- nique when compared with the conventional technique of microsurgery. This can be attributed to thermal damage of tissue adjacent to the coagulate mass, coagulation within the cornual angioarchitecture, and damage to intracellular organelles. Because. of tissue transparency and organelle and vascular sensitivity, caution should be exercised when the argon laser is used as a lysis or hemostatic tool. Technique refinement and a thorough understanding of the argon laser's biologic significance will define its appropriate place in the surgical field. Acknowledgments. We thank Patricia Cosgrove and Catherine Gemora for their aid and assistance in histologic preparation and surgery. The argon laser was donated by the Spectra Physics Corporation, Mountain View, California. Microsuture was graciously provided by Nancy Mandoli, Ethicon Inc., Somerville, New Jersey. Illustrations were by Jan Hamanishi. REFERENCES 1. Fayez JA, McComb JS, Harper MA: Comparison of tubal surgery with the CO 2 laser and the unipolar microelectrode. Fertil Steril 40:476, Gomel V: Training in microsurgery. In Microsurgery in Female Infertility. Boston, Little, Brown & Co., 1983, p Klink F, Grosspietzsch R, Von Klitzing L, Endell W, Husstedt W, Oberheuser F: Animal in vivo studies and in vitro experiments with human tubes for end-to-end anastomotic operation by a CO 2 laser technique. Fertil Steril 30:100, Choe JK, Dawood MY, Bardawil WA, Andrews AH: Clinical and histologic evaluation of laser reanastomosis of the uterine tube. Fertil Steril 41:754, Scheidel PH, Wallwiener DR, Wiedemann RA, Hepp HK: Experimental anastomosis of the rabbit fallopian tube using fibrin glue. Fertil Steril 38:471, Almquist EE: Evaluation of the use of the argon laser in repairing rat and primate nerves. Unpublished data 7. Krueger RR: Argon laser coagulation of blood for peripheral nerve and blood vessel repair. Master of Science in Engineering Theses, University of Washington, Keye WR Jr, Matson GA, Dixon J: The use of the argon laser in the treatment of experimental endometriosis. Fertil Steril 39:26, Gamaley NF, Rounds DE: Alteration of mitochondria in myocardial cells induced by microbeam argon laser. In Hemic Cells in Vitro, Edited by P Farnes. Baltimore, Williams & Wilkins, 1969, p Landthaler M, Haina D, Waidelich W, Braun-Falco 0: A three-year experience with the argon laser in dermatotherapy. J Dermatol Surg Oncol 10:456, Bellina JH, Ross LF, Holmquist N, Voros JI, Moorehead ME: Linear and nonlinear effect of the argon laser on a fallopian tube animal model. Lasers Surg Med 2:343, 1983 Lyon et al. Tubal anastomosis with the argon laser 333