KTP-532 laser-assisted microvascular anastomosis (experimental animal study)

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Eur Arch Otorhinolaryngol (2007) 264:823–828 DOI 10.1007/s00405-007-0317-x

HEAD AND NECK

KTP-532 laser-assisted microvascular anastomosis (experimental animal study) Balázs B. Lörincz · Endre Kálmán · Imre Gerlinger

Received: 23 April 2006 / Accepted: 3 April 2007 / Published online: 24 April 2007 © Springer-Verlag 2007

Abstract Former animal studies on laser-assisted microvascular anastomosis performed with CO2-, argon-, diode-, Holmium:YAG- and Nd:YAG-lasers had already proven the stability of the anastomotic sites. Tissue damage remained minimal along the anastomosis, while duration of the surgeries decreased signiWcantly compared to that of traditionally implemented microvascular sutures. In addition to this, foreign body reaction next to end-to-end anastomosis appeared to be minimal due to fewer traditional stitches. This animal study was designed in order to investigate the durability and the histological properties of microvascular anastomosis assisted by KTP-532 laser. Twentyone Wistar albino rats were used: in nine animals the KTPlaser-assisted microvascular anastomosis was carried out on the femoral artery. Those nine animals were divided into three groups and each of them consisted of three rats. The animals in these three groups were sacriWced 4 h, 1 and 4 weeks following the surgery, respectively. In three additional animals laser-assisted microvascular anastomosis was done on the abdominal aorta. Conventional microvascular sutures were carried out on femoral arteries of further

B. B. Lörincz (&) · I. Gerlinger Department of Otolaryngology and Head and Neck Surgery, Medical School, University of Pécs, Munkácsy M. utca 2, 7621 Pécs, Hungary e-mail: [email protected] E. Kálmán Department of Pathology, Medical School, University of Pécs, Szigeti út 12, 7624 Pécs, Hungary Present Address: B. B. Lörincz Department of Otolaryngology and Head and Neck Surgery, Haukeland University Hospital, Jonas Liesvei 65, 5021 Bergen, Norway

nine animals in the control group. The healing process of the femoral arteries is documented with Wgures of histological slides both in the laser-treated and in the conventionally operated group of rats. The KTP-laser-assisted microvascular anastomosis failed on the abdominal aorta, as strong bleedings occurred after the traditional sutures had been taken out. However, the coagulative eVect of the KTP-laser could still be used. The authors share the opinion that the success of the laser-assisted end-to-end microvascular anastomosis does not depend on the wavelength of the applied laser, but can be aVected by both the calibre of the vessel and the intraluminal pressure. Keywords Vascular suture · Microvascular anastomosis · KTP-laser

Introduction Besides pedicled Xaps used for the reconstruction of radical excisions of malignant tumours of the head and neck region, free Xaps fed through microvascular anastomosis are also available for the surgeon [1–5]. Successful application of free Xaps depends on detailed knowledge of the circulation and the vascular network of the transplanted tissues (muscle, skin, bones and nerves), and on a sophisticated surgical technique. In addition to free Xaps involving certain anatomical structures of the lower and upper limbs (radial forearm, lateral upper arm, lateral thigh and Wbular osteocutaneous Xaps), several other donating areas might also be available for free-Xap reconstruction, such as latissimus dorsi Xap and free jejunal tendril. In order to perform a reliable and secure microvascular anastomosis, alternatives to the conventional suture technique are being evaluated. Experiments with glue

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based on cyanoacrylate showed acceptable results with instant tightness [6]. Staples provide a very fast and easy way of vascular wall adaptation, however, they contain some risk for foreign body reaction [7]. An interesting idea emerged during the last decade referring to a possible increase of the quality and stability of the anastomosis, as a result of laserassisted adjustment of vascular walls due to heat-provoked tissue-welding eVect. Several authors performed successful animal studies with stable vascular anastomosis assisted by CO2-, argon-, Nd:YAG-, diode- and Holmium:YAG-lasers with fewer stitches [8–19]. Their experiences showed shorter duration of surgery with minimal tissue-damage and less foreign body reaction in the surrounding area of the anastomosis, compared to traditional sutures. This experimental animal study was designed to investigate the capability of the KTP-532 laser from the point of view of performing stable, durable and reliable microvascular anastomosis within a shorter period of time compared to the traditional and conventional suture-techniques.

Materials and methods The KTP laser device (Orion, LaserScope®, UK) The KTP-532-laser device emits a green-coloured visible light of 532 nm in wavelength [20]. Due to the fact that the KTP-beam can be well absorbed by haemoglobin, it has excellent coagulating properties, but it is also appropriate for incision and can be excellently used for vaporization. The beam can be led to the target area not only with a microscope-mounted micromanipulator, but also with Wber optics led through a hand-held probe called endostat. Animal studies Twenty-one adult Wistar albino rats weighing between 300 and 400 g were prepared for the purposes of the present study. Consent and approval for this investigation were obtained from our institution’s Animal Study Ethical Review Committee. The animals were divided into two main groups. Three animals underwent KTP-laser—(LaserScope Inc., UK)—assisted microvascular anastomosis performed on the abdominal aorta. Nine animals went through KTP-laser-assisted microvascular anastomosis on the femoral artery of the right caudal limb. A third group of nine further animals used as a control group, underwent conventional microvascular sutures on their femoral artery using exclusively 10-0 nylon monoWlament (Ethilon® by Ethicon) sutures (average 6–8 stiches each) guided by an atraumatic needle (Ø 75 m). The animals were placed in a thermostat after the operations. Each rat was anaesthetized by intraperitoneally administered mixture of 0.4 ml diazepam

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(Seduxen® by SanoW-Egis) and 1.6 ml ketamine (Calypsol® by Teva-Biogal). The limbs of the narcotized animals lying on their back were Wxed with strips onto the wooden surgical holdingdevice constructed by the authors particularly for that purpose. Following incisions either on the abdominal wall or on the medial surface of the right caudal thigh, the abdominal aorta and the femoral artery were prepared, respectively. While preparing the femoral artery, its larger branches were ligated with 7-0 sutures (Prolene® by Ethicon) or occasionally cauterised with bipolar diathermy. After positioning the double vascular clip, the capes of the consequently intersected arteries were rinsed with heparinized physiologic saline solution, and the adventitia of the vessels were carefully removed under surgical microscope. Four holding stitches in case of the abdominal aorta, and three for the femoral arteries were necessary to fasten the capes to each other. The stitches were performed with 10-0 nylon monoWlament suture (Ethilon® by Ethicon) guided by an atraumatic needle (Ø 75 m). Following radial Wxation of the fastening stitches, the mural sections between the stitches of the slightly stretched vascular capes of the vessels tightened to each other were treated with KTP-532 laser (LaserScope, UK). Prior to the laser-treatment of the anastomosis, a gout of blood was dropped onto the attached endings of the vessels in order to facilitate the absorption of the laser light. The beam was led towards the targeted area by a handpiece with Wber optics of 200 m in diameter. Using non-contact technique with a beam-diameter calculated to be 0.3 mm, selecting the pulse-mode (0.1 s exposure time at 0.5 s intervals) to 0.2 W, the so-achieved power-density reached the value of 285 W/cm2. Once the completion of the laserassisted microvascular anastomosis had been achieved, the vascular clips were released and the integrity of the anastomosis was conWrmed. Finally, the skin incisions were closed and the animals were placed into thermostat. The clamp-time of all procedures was recorded. Antibiotics were not administered to the animals postoperatively. Three rats of the nine animals that underwent laserassisted femoral artery anastomosis were repeatedly anaesthetized 4 h after the anastomosis had been performed. Another three animals 1 week after the intervention, and the remaining three of those nine rats were narcotized again 4 weeks after the operation. During those second narcotic sessions their vascular system was perfused with a Wxing solution (a mixture of 200 ml of 20% paraformaldehyde, 500 ml of 0.2 mol/l sodium-cacodylate and 300 ml of distilled water adjusted to pH = 7.4 with a few drops of concentrated hydrochloric acid) from a catheter led into the aortic arch through the left cardial ventricle after an incision had been made also in the right auricle to serve as an exit oriWce, in order to Wx the tissues. The nine control

Eur Arch Otorhinolaryngol (2007) 264:823–828

animals were also divided into three groups, each consisting of three rats. The rats of the Wrst control group were narcotized after 4 h. Another three animals were sacriWced after 1 week, while the remaining three control rats were narcotized 4 weeks later. Hereafter the formerly performed anastomosis were rediscovered, the correspondent and already histologically Wxed vessels were cut out, removed and embedded in paraYn. The histological slides of the treated vascular sections were studied under light microscopy in order to Wnd the signs of any thrombotic event, and to check the presence of inXammation in the aVected tissues.

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H.E. staining) revealed damage of the vascular walls, which became amorphous or even acellular along shorter sections (Fig. 1). Nevertheless, the orcein staining revealed that the Wbrous mural structure of the vessels remained unaVected (Fig. 2). As for the control animals, granulocytes could be observed between the sutures (Fig. 3).

Results The durability of the anastomoses At the time of sacriWcing the animals, all nine femoral arteries with laser-assisted microvascular anastomosis had been conducting the blood properly; no alterations referring to eventual hypoxia in the aVected limbs could be observed. As for the nine control animals, each of them showed uneventful recovery without signs of lower limb hypoxia or infection. In three further cases, in which the laser-assisted anastomosis was carried out on the abdominal aorta, it became obvious immediately after the procedures that such a high-pressure anastomosis cannot be safely performed using the laser-beam exclusively, probably due to the deWnitely higher peak pressure of aortic blood. In certain cases, further stitches needed to be placed in order to attach the aortic endings to each other properly, meanwhile the excellent coagulative eVect of the defocused KTP-532 laserbeam could be used to stop bleedings occurring after the vascular clips had been released. The mean clamp-time of the conventionally sutured anastomoses was 19.5 min (SD: 2.88), which was signiWcantly longer than that of the laser-welded anastomoses, i.e. 7.5 min (SD: 2.11).

Fig. 1 “Rolling granulocytes” on the endothelial surface. (Laser group, 4 h postoperatively, H.E. staining, £200 magniWcation)

Fig. 2 Fibrotic network of the vascular wall remained unchanged. (Laser group, 4 h postoperatively, orcein-staining, £400 magniWcation)

Light microscopy studies on KTP-laser assisted anastomosis on the femoral artery Neither intraluminal thrombus nor aneurism was found in any of the femoral arteries having undergone laser assisted microvascular anastomosis. Four hours after the anastomotic sutures had been performed; signs of histological alterations due to early inXammation were detectable around the anastomotic sites. On the aVected endothelial surface, slight precipitations of Wbrin were observed and the granulocytes attached to the endothelial surface showed the so-called “rolling phenomenon”. The surrounding area of the anastomotic sites (with

Fig. 3 Fibrin and granulocytes between the stitches. (Control group, 4 h postoperatively, H.E. staining, £400 magniWcation)

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One week following the vascular interventions, mural granulomatous alterations were detected due to the foreign body reaction caused by the few holding stitches applied. Furthermore, hyperplasia and migration of the endothelial cells could be observed in addition to numerous cellular divisions. In the surrounding area of the anastomotic sites, the mural smooth muscle layer of the treated vessels disappeared in its entire thickness, with the signs of chronic inXammation presenting outside of the vascular wall. Numerous hystiocytes appeared around the sutures (Fig. 4). The orcein staining shows that the vascular Wbers have survived the intervention although they became more elongated, and the diameter of their porotic structure became enlarged (Fig. 5). SigniWcant chronic inXammatory reaction could be observed inside the vascular wall and around the sutures in the control animals (Fig. 6). Four weeks following the surgery, certain signs of Wbrosis were present in the subintimal layer corresponding to the aVected vascular area. Subintimally widely spread transmural scar tissues appeared. Histological signs of inXammatory reaction at the end of the fourth postoperative week were not present anymore (Fig. 7). As also veriWed by the orcein-

Eur Arch Otorhinolaryngol (2007) 264:823–828

Fig. 6 Intense chronic inXammatory reaction around the conventional suture and inside the vascular wall. (Control group, 1 week postoperatively, H.E. staining, £400 magniWcation)

Fig. 7 Subintimal Wbrosis and transmural scar-tissue. No sign of chronic inXammation. Granulomatous foreign body reaction around the holding suture. (Laser group, 4 weeks postoperatively, H.E. staining, £400 magniWcation)

Fig. 4 Endothelial hyperplasia, migration and foreign body granulomatous reaction caused by the holding suture in the vascular wall. The smooth muscle layer of the vascular wall disappeared corresponding to the anastomosis. Signs of chronic inXammation outside the vascular wall. (Laser group, 1 week postoperatively, H.E.-staining, £200 magniWcation)

staining, the inXammatory process calmed down after 4 weeks (Fig. 8). Granulomatous foreign-body reaction could be observed in the vessels of the control group animals caused by the suture material (Fig. 9.). Furthermore, performing a traditional anastomosis took more than 2.5 times longer time than performing the laser-assisted one.

Discussion

Fig. 5 Elongated Wbers of the vascular wall surviving the procedure. (Laser group, 1 week postoperatively, orcein-staining, £400 magniWcation)

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Several authors have published their results with CO2-, argon-, diode-, Holmium:YAG and Nd:YAG-laser-assisted microvascular anastomosis on vessels of diVerent diameters and calibers [8–19, 21]. Recently, Ott reported his favourable experiences using intraluminal laser light source and external solder while creating microvascular anastomoses without holding sutures [16]. Advantages of laser anastomosis are that (1) there is less trauma to the vessels than with conventional suture

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Fig. 8 No sign of inXammation at the cutting zone. (Laser group, 4 weeks postoperatively, orcein-staining, £400 magniWcation)

Fig. 9 Granulomatous foreign body reaction around the conventional suture. (Control group, 4 weeks postoperatively, H.E. staining, £400 magniWcation)

technique; (2) there is a decrease in foreign-body reaction and inXammation; (3) the exposure of suture thread in the vessel lumen is smaller than that in the suture group, with minimal risk of thrombus formation; (4) there is less or no blood leakage after the procedure; (5) ease of technique, shorter learning curve [17]. On the contrary, among the disadvantages of the laser-assisted microvascular anastomosis the following are noted: (1) a certain susceptibility to tension forces; (2) query aneurysm formation [18, 22].

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The evidence for the CO2-laser action mechanism supports the theory that the CO2 laser—due to its longer infrared wavelength—heats the surface water layer on the irradiated tissue, with a subsequent heat conduction to the tissue elements below that surface. The localised heating above 60°C causes denaturation of the protein components. When the laser irradiation is discontinued, the denaturated proteins are being cooled and coagulated. By performing laser assisted microvascular anatomosis, the coagulum to be formed creates a “weld”; a heat-fused bond that holds the two edges together [21]. Vale [11] came to the conclusion based on his animal experiments on rats that in case of CO2-laser assisted microvascular anastomosis an increased Wbrinogen-polymerisation may also occur due to the thermodynamic eVect of the laser, which has a signiWcant impact on the adhesion of the vessels. Visible and nearinfrared lasers transmit their light through all vessel layers. Due to deeper penetrance into the tissues, the action may be a more direct photocoagulation of the proteins, which does not involve heat conduction [9, 10, 12–16, 19, 23]. Using the argon-laser, diVerent authors experienced that the success of anastomotic sutures was still possible around 43– 48°C, but reaching the tissue-temperature of 60°C, the aVected areas were not stable anymore [5, 23]. Schober observed on Nd:YAG-laser-assisted microvascular sutures that the structure of the collagen had been changed around the anastomotic site, so the Wbers ensured perfect closure when sliding into each other from both sides, like the Wngers of a pair of gloves [21]. It is also possible that the structural changes of the mural collagen and the consequent vascular welding process are presented exclusively as a result of the thermodynamic eVect of the laser. By applying a diode laser, the introduction of dyes such as indocyanine green or Xuorescein isothiocyanate, enhances the delivery of the laser energy precisely to the target tissue [13, 14]. Using the holmium-YAG laser, Ott reported good bursting pressures of anastomoses, which appear to be suYcient to withstand psychological conditions [15]. The KTP-laser showed its eVectiveness in coagulating bleedings occurring when the vascular clip had been released; consequently there was no need for further stitches and the surgery took signiWcantly less time. On studying the histological slides, no intraluminal thrombus was detected and the observed signs of inXammation were moderate. Using the KTP-laser with low power settings, only minimal tissue damage could be observed. A particular advantage in our study seemed to be the feature that the green-coloured radiation of the KTP-laser is well absorbed by the blood haemoglobin. This phenomenon was considered when the defocused beam of the KTP-laser was used to stop smaller bleedings: this cannot be achieved with the CO2-laser due to its inappropriate coagulative parameters.

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Another important advantage of the KTP-beam is that it can be led to the target area with Wber optics of 0.2 mm in diameter through a hand-held endostat, so in non-contact mode a 0.2 W laser-beam of approximately 0.3 mm in diameter can be directed onto the target. That high precision cannot be achieved with a microscope-mounted CO2laser. The present study is proposed to verify that the KTP-532 laser is a suitable tool for performing microvascular anastomosis. Although investigation of the adhesive forces was not aimed yet, this question may serve as the basis of further discussions. As a summary of our experiences, it can be concluded that the absorption aVected by the wavelength of diVerent lasers, the chosen parameter settings during the exposure, the thickness and composition of the tissues and the achieved tissue temperature may all have an inXuence on the successful performance of the laser-assisted microvascular anastomotic sutures. The KTP-laser might give a suitable alternative solution for performing microvascular anastomosis between veins and at least between arteries of smaller calibre, but further research needs to be carried out to clarify whether the KTP-laser can give a real alternative for creating microvascular anastomoses in humans, especially in head and neck reconstructive surgery, where the diameter of arteries is often signiWcantly larger. The longterm behaviour of the anastomotic sutures, their solidity, security and the properties of the adhesive forces will serve as the basis for our further experimental studies.

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