We investigated the effects of the iron chelator desferrioxamine (DFX) on thrombus formation in the arterioles and venules of the rabbit ear chamber. Thrombi were induced by irradiation with filtered light in combination with a fluorescent dye. The occlusive thrombus formation time was significantly extended by DFX. The mor-phological composition of thrombi in the arterioles and venules was different. In the arterioles, the thrombi consisted of platelet aggregation, but in the venules, platelets and leukocytes accumulated on the endothelium. This suggests that hydroxyl radicals may be important mediators in this model, as DFX is known as a hydroxyl radical scav-enger. Furthermore, the components of thrombi in the arterioles and venules in the skin microvascular system may be different

Hashimoto, Ichiro

Nakanishi, Hideki

Shono, Yoshitaka

Tanaka, Shinji

Tokushima University Institutional Repository

INTRODUCTIONDesferrioxamine (DFX) is an iron chelator usedfor the treatment of acute iron overload states andis known to act as a hydroxyl radical scavenger(1, 2). Recently it has also been reported that DFXprotects the endothelial cells of heart and lung (3, 4)and can also shield skin flaps from necrosis (5, 6).On the other hand, it has been shown that DFXdoes not benefit the endothelium or improve sal-vage of skin necrosis (7). In flap surgery, thrombusformation is one of the causes of flap necrosis. Theeffect of DFX on thrombosis is, however, not clear.In this study, we used the rabbit ear chamber (REC)as a model of skin microcirculation and observedthe thrombus formation resulting from endothelialdamage due to dye/light reaction. The effects ofDFX on this damage were also examined. Sincethis thrombus model is usually used for mesentericvessels, and as there have been no reports onthrombus formation in the skin, the morphologicalfeatures are also described.MATERIALS AND METHODSAnimals/ Rabbit Ear ChamberA transparent round-table chamber (rabbit earchamber : REC) was implanted in the earlobe ofJapanese white rabbits weighing between 2.7 and3.2kg (Fig.1) (8). Five to seven weeks after im-plantation, a complete microvascular network hadformed in the chamber, and arterioles 20-30 µm indiameter and venules 45-55 µm in diameter wereselected for the experiments (Fig.2).The effects of desferrioxamine on thrombus formation ininjured microvessels of the rabbit earIchiro Hashimoto, Hideki Nakanishi, Yoshitaka Shono, and Shinji TanakaDepartment of Plastic and Reconstructive Surgery, University Hospital, The University of TokushimaSchool of Medicine, Tokushima, JapanAbstract: We investigated the effects of the iron chelator desferrioxamine (DFX) onthrombus formation in the arterioles and venules of the rabbit ear chamber. Thrombiwere induced by irradiation with filtered light in combination with a fluorescent dye.The occlusive thrombus formation time was significantly extended by DFX. The mor-phological composition of thrombi in the arterioles and venules was different. In thearterioles, the thrombi consisted of platelet aggregation, but in the venules, plateletsand leukocytes accumulated on the endothelium. This suggests that hydroxyl radicalsmay be important mediators in this model, as DFX is known as a hydroxyl radical scav-enger. Furthermore, the components of thrombi in the arterioles and venules in the skinmicrovascular system may be different. J. Med. Invest. 46 : 200-204, 1999Key words : rabbit ear chamber, desferrioxamine, thrombus formation, microcirculationReceived for publication June 17, 1999 ; accepted July 22, 1999.Address correspondence and reprint requests to IchiroHashimoto, M.D., Department of Plastic and ReconstructiveSurgery, University Hospital, The University of TokushimaSchool of Medicine, Kuramoto-cho, Tokushima 770-8503, Japanand Fax : +81-88-633-7297.Fig.1. Rabbit ear chamber installed in a rabbit earlobe.The Journal of Medical Investigation Vol.46 1999２００Photochemically induced thrombosis modelNo anesthesia was needed for the following studies.Thrombi were produced in the vessels with thedye/light method reported by Sato and Ohshima(9, 10). Briefly, a solution of fluorescein sodium(2.5%, 2 ml/kg ; Wako Pure Chemical Industries,Ltd., Osaka, Japan) was injected through a vein ofthe contralateral ear. After confirming the appear-ance of fluorescence in the microvessels in the REC,filtered light (400-500 nm light passed through afluorescein isothiocyanate excitation filter from ahigh pressure mercury lamp) was irradiated ontothe vessels through an objective lens (x20) (Fig.3).The diameter of the irradiated spot was 130 µm onthe focal plane. The light intensity was controlledat 0.5 mW under the objective lens. Irradiated ves-sels were continuously monitored and recorded withan intravital microscope-television system in allcases and the time course of thrombus formationwas calculated with the aid of these records. In allcases, the intervals“ti”(from irradiation to the onsetof thrombus formation) and“ts”(from irradiation tocomplete obstruction of blood flow) were also cal-culated.Experimental Groups and Administration of DFXThirty rabbits were prepared and divided intothree groups of 10 animals each. In the control group,10cc of saline was injected via the contralateralear 15 minutes before aggregation was induced. Inthe two DFX treatment groups, 100 mg/kg of DFX(Novartis Pharma Co., Tokyo, Japan) and 10 ml ofsaline, or 200 mg/kg of DFX and 10 ml of saline wasinjected in the same way.Statistical AnalysisThe Mann Whitney U-test was used for statisticalanalysis of the results. P values less than 0.05 wereconsidered statistically significant.RESULTSWhen the filtered light was irradiated onto thevessels after confirmation of fluorescence in themicrovessels in the RECs, microthrombi began tostick to the wall, and continued to accumulate onthe inside of the vessels. Eventually the microvesselswere totally occluded by the thrombi and the bloodflow was completely blocked. The composition ofthe thrombi in the arterioles and venules was dif-ferent. Morphologically, the thrombus formationin the arterioles began with platelet aggregation(Fig.4), but in the venules, platelets, erythrocytesand leukocytes started to gather and adhere to theendothelium from the start (Fig.5). This differenceFig.2. A few weeks after installation, a microvascular systemcontaining arterioles and venules is observed in the chamber.Fig. 3. Schematic diagram of the intravital microscope-televisionsystem. Solid line indicates the epi-illumination system to producethrombi. Broken line indicates the transillumination system toobserve the microvascular bed.Fig.4. Thrombus formation in the arteriole. The arrows showaccumulation of platelets on the vessel wall.２０１The Journal of Medical Investigation Vol.46 1999between thrombosis in arterioles and venules wasobserved in all groups.In arterioles and venules, the thrombus forma-tion times, ti and ts, were extended as a result ofDFX (Fig.6). In the arterioles, ti and ts of the controlgroup were 25.2±2.7 (mean±S.D.) and 84.3±4.6sec and those of the DFX 100 mg/kg group were36.5±5.2 (p<0.05) and 95.7±9.2 sec (not signifi-cant). The times of the DFX 200 mg/kg group were45.7±6.0 (p<0.01) and 111.0±8.0 sec (p<0.05). Inthe venules, ti and ts of the control group were25.0±2.0 and 104.9±5.6 sec and those of the DFX100 mg/kg group were 38.3±7.4 (not significant)and 128.3±12.6 sec (not significant). The times ofthe DFX 200 mg/kg group were 43.7±7.6 (p<0.05)and 135.1±11.7 sec (p<0.05).DISCUSSIONDifferent methods to induce thrombosis in vivoare presently available. However, there are fewreports on the application of thrombus models tothe REC. Fleming and Seiler have evaluated theantithrombotic efficacy of some drugs by using alaser-induced thrombosis model in the REC (11,12). In their model using irradiation by ruby laserto vessels, it is thought that the localized thermaleffect on the red cells as a result of absorption of theruby laser evoked the formation of a thrombus,which consisted of platelets accumulated around acore of one or two damaged red cells (13). It is,however, generally accepted that the first step ofthrombogenesis is damage to endothelial cells.Therefore, their model differs from the physiologi-cal mechanism of thrombus formation.We applied the fluorescent dye/light method,which Sato and Ohshima developed for mesentericvessels, to the REC. It has been reported that themechanism of thrombus formation in the in vivomodel of the dye/light reaction does not consist ofa localized thermal effect but of endothelial dam-age due to forms of active oxygen generated withexcited dyes (14, 15). However, details of the mech-anism are not clearly understood at present.Our data showed that DFX lengthened the throm-bus formation time in the arterioles and venules ina dose dependent manner. DFX is known to act asa hydroxyl radical scavenger (1, 2). With almost thesame dye/light method as described here, it hasbeen shown that, in the pial arterioles of the mouse,the hydroxyl radical scavengers dimethyl sulfoxide(DMSO) and glycerol are effective inhibitors ofplatelet aggregation (15). Hydroxyl radicals maytherefore be important mediators of the skin micro-vascular responses in this model. The maximumclinical daily dose of DFX is 80 mg/kg for humans.As no statistically significant difference could beidentified in the DFX 100 mg/kg group, the anti-thrombotic efficacy of this agent may not be verystrong. However it has been reported that a drugmixture containing a small dose of DFX (20 mg/kg)improves experimental flap survival (16, 17). Withthat method, a mixture including a small amount ofeach drug can be effective and DFX can be used ina clinically viable manner.Recently, it has been found that after a prolongedischemic phase, reestablishment of the vascularsupply causes tissue injury which is called ischemiareperfusion injury. In this injury, reactive oxygenFig.5. Thrombus formation in the venule. The arrows showleukocytes in the thrombus.Fig.6. Thrombus formation time. Values show mean±S.E. ■,ti (time when thrombus began to form) ; , ts (time when bloodflow completely stopped) : *p<0.05, **p<0.01 compared withcontrol group.I. Hashimoto et al. Effects of DFX on thrombus formation２０２species such as the superoxide anion radical (O2-),hydrogen peroxide (H2O2), and the hydroxyl radical(OH・) lead to endothelial cell injury which resultsin increased capillary permeability, thrombus forma-tion, and microcirculatory failure. As the thrombiin our model may be due to endothelial injury byhydroxyl radicals, we are convinced that this modelvery closely resembles such clinical conditions (15,18).In this study, it was observed that thrombus for-mation in the arterioles consisted of platelet aggre-gation, but in the venules both platelets and leukocytesgathered and adhered to the endothelium. Whenthe same model as used in this study was appliedto mesenteric vessels, platelet aggregation wasobserved in the venules (9, 10). This difference incomposition of the thrombus between REC andthe mesenteric vessels may be caused by charac-teristics of the organ. In the skin microcirculation,WBC contributed to thrombus formation only inthe venules but not in the arterioles. It is not clearwhether the leukocyte sticking in thrombus iscaused by slow blood flow velocity or by the func-tional features of venous endothelial cells. Furtherstudies are needed to investigate this point.REFERENCES1. Gutteridge JM, Richmond R, Halliwell B :Inhibition of the iron-catalysed formation ofhydroxyl radicals from superoxide and of lipidperoxidation by desferrioxamine. Biochem J184 : 469-472, 19792. Gutteridge JM, Rowley DA, Halliwell B :Superoxide-dependent formation of hydroxylradicals and lipid peroxidation in the presenceof iron salts. Detection of‘catalytic’iron andanti-oxidant activity in extracellular fluids. Bio-chem J 206 : 605-609, 19823. Sellke FW, Shafique T, Ely DL, WeintraubRM : Coronary endothelial injury after cardio-pulmonary bypass and ischemic cardioplegiais mediated by oxygen-derived free radicals.Circulation 88 (part 2) : 395-400, 19934. Pickford MA, Green CJ, Sarathchandra P,Fryer PR : Ultrastructural changes in rat lungsafter 48 h cold storage with and without reper-fusion. Int J Exp Pathol 71 : 513-528, 19905. Green CJ, Dhami L, Prasad S, Healing G,Shurey C : The effect of desferrioxamine onlipid peroxidation and survival of ischaemicisland skin flaps in rats. Br J Plast Surg 42 :565-569, 19896. Angel MF, Mellow CG, Knight KR, O’BrienBM : The effect of deferoxamine on toleranceto secondary ischaemia caused by venousobstruction. Br J Plast Surg 42 : 422-424, 19897. Nishikawa H, Manek S, Fryer PR, Green CJ :Reperfusion injury in rat adipomusculocutaneousflaps : an ultrastructural and microangiographicstudy. Br J Plast Surg 44 : 500-505, 19918. Asano M, Ohkubo C, Sasaki A : A modifiedrabbit ear chamber and an example of itsapplication for intravital-microscopic study onacute effects of topical thermal stimulation. JpnJ Pharmacol 48 : 149-152, 19889. Sato M, Ohshima N : Platelet thrombus inducedin vivo by filtered light and fluorescent dye inmesenteric microvessels of the rat. Throb Res35 : 319-334, 198410. Sato M, Ohshima N : Hemodynamics at stenosesformed by growing platelet thrombi inmesentericmicrovasculature of rat. Microvasc Res 31 : 66-76, 198611. Seiler SM, Buchanan JO, Schumacher WA,Zavoico GB, Gamberdella M, Fleming JS,Meanwell NA : 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl] phenoxy] acetic acid (BMY 42393) : 2).Oral activity and efficacy in animal models ofarterial thrombosis. Throb Res 74 : 125-133,199412. Fleming JS, Buchanan JO, Seiler SM, MeanwellNA : Antithrombotic activity of BMY-43351, anew imidazoquinoline with enhanced aqueoussolubility. Throb Res 63 : 145-155, 199113. Grant L, Becker FF : Mechanisms of inflamma-tion.1. Laser-induced thrombosis, a morphologicanalysis. Proc Soc Exp Biol Med 119 : 1123-1129, 196514. Herrmann KS : Platelet aggregation induced inthe hamster cheek pouch by a photochemicalprocess with excited fluorescein isothiocyanate-dextran. Microvasc Res 26 : 238-249, 198315. Rosenblum WI, El Sabban F:Dimethyl sulfoxide(DMSO) and glycerol, hydroxyl radical scav-engers, impair platelet aggregation within andeliminate the accompanying vasodilation of,injured mouse pial arterioles. Stroke 13 : 35-39, 198216. Lepore DA, Knight KR, Bhattacharya S, Ritz M,Robbins SP, Seig P, Morrison WA, O’BrienBM : Drug mixture which improves survivalof ischemic rabbit epigastric skin flaps. Micro-２０３The Journal of Medical Investigation Vol.46 1999surgery 15 : 685-692, 199417. Kohout M, Lepore DA, Knight KR, Van GJ,O’Brien BM, Stewart AG and Morrison WA :Cool perfusion solutions for skin flaps : a newmixture of pharmacological agents which im-proves skin flap viability. Br J Plast Surg 48 :132-144, 199518. Matsuno H, Uematsu T, Umemura K, TakiguchiY, Asai Y, Muranaka Y, NakashimaM : A simpleand reproducible cerebral thrombosis modelin rats induced by a photochemical reactionand the effect of a plasminogen-plasminogenactivator chimera in this model. J PharmacolToxicol Methods 29 : 165-173, 1993I. Hashimoto et al. Effects of DFX on thrombus formation２０４

The effects of desferrioxamine on thrombus formation in injured microvessels of the rabbit ear

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The effects of desferrioxamine on thrombus formation in injured microvessels of the rabbit ear

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