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H VM BI OFLUX
H u m a n & V e t e r in a r y M e dicin e
I n t e r n a t ion a l Jou r n a l of t h e Bioflu x Socie t y
Th e e ffe ct s of ch it osa n a n d ch it in w oun d
dr e ssin gs in h a ir le ss dogs
Tohru Kim ura
Cent er for Ex perim ent al Anim als, Nat ional I nst it ut es of Nat ural Sciences, 38, Nishigo-nak a, My odaij i, Ok azaki, Aichi, 444- 8585, Japan; e- m ail: kim ura@nips. ac. j p
Abst r a ct. The effect s on wou nd healing of chit osan and chit in wound dr essings wer e clinically and hist opat hologically invest igat ed using a split t hickness wound in hair less dogs. Wound dr essings used in t his st udy included exam ined wer e dr ied por cine skin ( DP skin) , cot t on t ype chit osan ( CT chit osan) and chit in fiber dr essing ( chit in FD) . Clinically, t her e wer e no differ ences in wound healing am ong t he sit es t r eat ed wit h chit osan, chit in and t he unt r eat ed cont r ol sit es. I n cont r ast , DP skin and CT chit osan did not pr om ot e wound healing. CT chit osan dr essings induced sever e inflam m at or y r eact ions. I n t he sit es t r eat ed wit h chit in FD, wound healing was delayed. Hist opat hologically, t her e wer e few differ ences in r eepit helializat ion am ong t he sit es t r eat ed wit h chit osan, chit in and t he WD- unt r eat ed sit es. The sit es t r eat ed wit h DP skin r eceived elast ic fiber s fr om DP skin. CT chit osan dr essings had a delet er ious eff ect on wound healing ( r eepit helializat ion and gr anulat ion) . Dysker at osis, congest ion, hem or r hage, cellular infilt r at ion, int r a- and int er cellular edem a wer e seen in t he epider m is. Bleeding, infilt r at ion of inflam m at or y cells and m ast cells, and edem at ous changes wer e seen in t he der m is. I n t he sit es t r eat ed wit h chit in FD, t he invasion of m onon uclear cells, m ast cells and er yt hr ocyt es caused t he r et ar ded developm ent of r eepit helializat ion. I t was concluded t hat chit osan and chit in had few delet er ious ef fect s on wou nd healing wher eas DP skin, CT chit osan and chit in FD induced inflam m at ion in t he wound of hair less dogs. I n addit ion, it was clear t hat t her e ar e species differ ences in t he skin r eact ions t o wound dr essings. These r esult s suggest ed special car e should be t aken in vet er inar y clinical use of hum an m edical wound dr essings.
Ke y W or ds: chit in, chit osan, dr ied por cine skin, hairless dogs, wound dr essings, wound healing.
I n t r odu ct ion. Chit in (β- ( 1- 4) - N- Acet y l- D- Glucosam ine) is a cellulose-lik e biopoly m er w hich is widely dist ribut ed as an im port ant st ruct ural elem ent of t he int egum ent s of art hropods, part icularly crust aceans, m olluscs, unicellular organism s, seaw eeds and fungi. Chit osan, a m ucopoly saccharide having st ruct ural charact erist ics sim ilar t o glycosam ines, is t he alk aline deacet ylat ed product of chit in. These poly saccharides hav e been int roduced int o pharm aceut ical and cosm et ic t echnology , w here t hey can be used as a v ehicle for drugs ( Miy azaki et al 1990; Yoshida 1993) or as a com ponent of m oist urizers ( Muzzarelli 1983) . I n m edicine, t heir reconst ruct ing propert ies are used t o m ak e bandages shaped lik e cont act lenses for dam aged corneas, t o prepare w ound dressings for t he t reat m ent of burns and ulcers, and t o m ak e sut ures ( Muzzarelli 1983; Jay asree et al 1995; Cost ain et al 1997) . Chit osan and chit in are believ ed t o be nont ox ic and ideal biopoly m ers in spit e of lack of adequat e dat a on t heir safet y . How ev er, adv erse react ions of chit osan and chit in hav e begun t o be report ed in cosm et ic and m edical fields ( Cleenewerck et al 1994; Tanak a et al 1997; Pereira et al 1998) .
Recent ly , w e hav e est ablished a colony of hairless hy brids of Mexican hairless dogs ( Kim ura et al 1993) . Hairless dogs hav e been ut ilized for inv est igat ing delay ed cont act derm at it is ( Kim ura & Doi 1994ab) and for ev aluat ing derm at ot oxicit y of agricult ural chem icals ( Kim ura et al 1998) , all-t rans- ret inoic acid ( Kim ura & Doi 1998) , and cort icost eroids ( Kim ura & Doi 1999) .
M a t e r ials a n d Me t h ods
Dogs
Three m ale dogs w ere used in t his st udy . They were N4 hairless hy brids result ing from t he breeding of m ale N3 hairless dogs and fem ale beagle dogs.
Dogs w ere individually housed in st ainless st eel cages ( 90× 90 × 90 cm ) in an anim al room cont rolled at 24± 1° C and 55± 5% relat iv e hum idit y w it h 10 t o 15 ex changes of 100% fresh air/ hour and 12- hour light ( 7AM t o 7PM) , 12- hour dark ( 7PM t o 7AM) cy cle. They w ere fed a com m ercial dry dog food ( Labo D St andard, Nihon Nosan Kogy o Co. , Lt d., Yok oham a, Japan) and w at er ad libit um.
Wound dressing ( WD)
The following reagent s and dressing m at erials w ere t est ed in t his st udy: 1. chit osan ( Wak o Pure Chem ical I ndust ries, Lt d., Osak a, Japan) , 2. chit in ( Sigm a Chem ical Co. , St . Louis, USA) , 3. dried porcine skin ( DP sk in, Alloask D, Taiho Yak uhin Co. , Lt d. , Tok y o, Japan) , 4. cot t on t y pe chit osan m at erials ( CT chit osan) , 5. chit in fiber dressing ( chit in FD) and 6. WD- unt reat ed ( only bandage, cont rol) . CT chit osan w as m edical grade chit osan deriv at iv es m odified int o w at er- soluble m at erials. Six split t hick ness w ounds w ere m ade on t he dorsal sit e of t he anim al.
Procedures
The durat ion of t his ex perim et w as 21 day s following w ound creat ion. The anim als w ere anest het ized wit h k et am ine hy drochloride ( 10 m g/ k g, Ket alar, Sank y o Co. , Lt d., Tok y o, Japan) aft er m edet om idine ( 20 µg/ k g, s. c., Dom it ol, Meij i Seiy ak u Co. , Lt d. , Tok y o, Japan) prem edicat ion. Split t hick ness w ounds ( 2× 2 cm ) w ere m ade on t he dorsal skin of each dog, using st erile surgical blades (Feat her Safet y Razor Co. , Lt d. , Osak a, Japan) . The blade of a scalp w as used t o dissect t he epiderm is and t he upper lay er of t he derm is ov er t he w hole w ound area. Aft er irrigat ing t he w ound w it h st erile physiological saline and 0. 05% chlorhex idine gluconat e ( Hibit ane, Sum it om o Seiy ak u, Co. Lt d., Osak a, Japan) , each reagent or dressing m at erial w as applied t o t he split t hick ness w ound at a rat e of approx im at ely 10 m g/ cm2 ( Fig. 1) . The WD- t reat ed sit es were cov ered wit h t ransparent adhesiv e poly uret hane film ( Tegaderm , 3M Healt h Care, Tok y o, Japan) . Canine prot ect iv e j ack et s and Elizabet han collars (Tsugaw a Yok o Co., Lt d., Toky o, Japan) w ere used t o prev ent t he dogs from dislodging t he dressings or from peeling off. The WD- t reat ed sit es w ere k ept under t he w et env ironm ent during t his st udy. Keeping w ounds m oist allows norm al epiderm al m igrat ion at t he sam e level as t he undam aged epiderm is and wit hout shrink age. The dressings were changed on day 1, 3, 7, 14 and 21 of t he st udy . The adhesiv e poly uret hane film w as also changed on t hese day s.
Figure 1. Diagram of t he dorsal skin sit es ex am ined in a hairless dog. 1 = chit osan; 2 = chit in; 3 = DP skin; 4 = CT chit osan; 5 = chit in FD;
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Clinical ev aluat ions
Clinical observ ations w ere done before t he st udy and daily during t he WD-t reaWD-t m enWD-t . The WD- WD-t reaWD-t ed siWD-t es w ere ev aluaWD-t ed daily WD-t hrough WD-t he WD-t ransparenWD-t adhesiv e film , and clinically observ able changes ( reepit helializat ion, granulat ion, inflam m at ory react ions and ex udat ion) w ere phot ographed.
Hist opat hological ex am inat ion
Tissue specim ens w ere obt ained from bot h t reat ed and unt reat ed sit es of t he dorsal skin of each dog using a 6- m m biopsy punch under general anest hesia wit h m edet om idine on t he day before t he st udy , and at 7, 14 and 21 day s aft er t he st udy st art . Skin specim ens w ere fix ed in 10% neut ral buffered form alin, and 4- µm paraffin sect ions w ere st ained wit h hem at ox ylin and eosin and t oluidine blue, and by v an Gieson’s and Weigert ’s st aining. The t ot al severit y of epiderm al and derm al changes w as graded as negligible (－) , slight ( ± ) , m ild (＋) , m oderat e (＋＋) and m ark ed (＋＋＋) .
Re su lt s
Gross appearances
Cont rols: By 14 t o 21 day s aft er t he beginning of t his st udy, com plet e reepit helializat ion and granulat ion dev eloped in t he WD- unt reat ed sit es. Throughout t his st udy , t here w ere no abnorm al react ions such as w ound infect ion and cont ract ion in t he WD- unt reat ed sit es. Addit ionally , w e could observ e no abnorm al findings in ex udat e form at ion and sk in react ion adj acent t o t he w ound. The w ounds w ere com plet ely reconst ruct ed at t he end of t he st udy .
Chit osan dressings: Ex cept for t he t im e of reepit helializat ion, t here w ere no differences in w ound healing bet w een t he sit es t reat ed wit h chit osan and t he WD-unt reat ed sit es. Aft er 14 day s of t reat m ent w it h chit osan, t he skin show ed reepit helializat ion and becam e pigm ent ed ( Fig. 2) .
Figure 2. Gross appearance of t he sit e aft er 14 day s of t he t reat m ent wit h chit osan. Reepit helializat ion and pigm ent at ion are seen.
Chit in dressings: Alt hough t he sit es t reat ed w it h chit in w ere j udged t o be adequat ely reepit helialised aft er 14 day s of t r eat m ent , chit in subsequent ly caused m oderat e inflam m at ory react ions in t he skin. Ex cept for t his respect , t he process of t he w ound healing in t he sit es t reat ed wit h chit in w as sim ilar t o t hat in t he sit es t reat ed wit h chit osan.
day s aft er beginning t he t reat m ent wit h DP skin. At t he end of t he st udy , w ound healing w ere not observ ed in t he sit e t reat ed wit h DP skin.
Figure 3. Gross appearance of t he sit e aft er 21 day s of t he t reat m ent wit h DP skin. Wound healing is not seen.
Cot t on t y pe chit osan dressings: From t he day aft er t reat m ent wit h CT chit osan, inflam m at ory react ions dev eloped around t he wounds. Alt hough reepit helializat ion w as observ ed by 14 day s aft er beginning t he t reat m ent wit h CT chit osan, eryt hem at ous react ions becam e apparent in t he course of t he t reat m ent wit h CT chit osan ( Fig. 4) . I n addit ion, a large am ount of ex udat e appeared on t he w ound surface for 7 t o 10 day s aft er beginning of t he t reat m ent wit h CT chit osan. CT chit osan did not accelerat e w ound healing in t he skin of hairless dogs. The sit es reepit helialised by applying chit osan, chit in and CT chit osan show ed a num ber of com edones aft er 21 day s of t he t reat m ent .
Figure 4. Gross appearance of t he sit e aft er 14 day s of t he t reat m ent wit h CT chit osan. Sev ere ery t hem at ous react ion is seen.
Chit in fiber t y pe dressings: I n t he sit es t reat ed wit h chit in FD, w ound healing w as apparent ly delay ed in com parison w it h t he sit es t reat ed w it h ot her dressings. During t his t reat m ent , considerable ex udat ion and/ or bleeding from t he w ound surface w ere observ ed. Sk in react ion adj acent t o t he w ound w as m oderat e. At t he end of t he st udy , no w ound healing w as not ed. Form at ion of granulat ion t issue and reepit helializat ion w ere inhibit ed by cov ering wit h chit in FD ( Fig. 5) .
Hist opat hological findings
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Figure 5. Gross appearance of t he sit e aft er 21 day s of t he t reat m ent wit h chit in FD. Wound healing is apparent ly delay ed.
Figure 6. WD- unt reat ed skin at 21 day s aft er beginning of t his st udy. HE. × 175.
Chit osan dressings: Aft er 7 day s of t he t reat m ent w it h chit osan, t hick epiderm is and granulat ion t issues are observ ed in t he split t hick ness w ounds. Punch biopsies rev ealed a progressiv e increase in t he t hick ness of t he epiderm al lay er. During t reat m ent w it h chit osan, derm at ot oxicit y such as necr osis, congest ion and infilt rat ion of inflam m at ory cells w as not found. I n t he course of reepit helializat ion, t here w ere no apparent differences bet ween t he sit es t reat ed wit h chit osan and t he WD- unt reat ed sit es. Chit in dressings: The sit es t reat ed wit h chit in exhibit ed m oderat e congest ion and v asodilat ion wit h inflam m at ory cells in t he derm is aft er 14 day s of t reat m ent . There w ere few differences in reepit helializat ion bet w een t he sit es t reat ed wit h chit in and t he WD-unt reat ed sit es. I n t he sit es t reat ed wit h chit osan and chit in, t he repaired skin of hairless dogs had som e com edo-lik e lesions associat ed w it h occlusion of sebaceous glands by reepit helializat ion.
Figure 7. Dorsal skin aft er 7 day s of t he t reat m ent w it h DP skin. Thin epiderm al layer and m ark ed inflam m at ory cell infilt rat ion
w it h elast ic fibers deriv ed from DP skin. HE. × 175.
Cot t on t y pe chit osan dressings: CT chit osan had delet erious effect s on w ound healing ( reepit helializat ion and form at ion of granulat ion t issue) in hairless dogs. The sit es t reat ed wit h CT chit osan for 7 day s show ed epiderm al degenerat ion such as dy sk erat osis, congest ion, hem orrhage, cellular infilt rat ion, int racellular edem a and int ercellular edem a. I n t he derm is, t here w ere bleeding, infilt rat ion of inflam m at ory cells and edem at ous changes ( Fig. 8) . A num ber of degranulat ed m ast cells w ere observ ed in t he derm is, especially around t he capillary v essels. I n t he derm al- epiderm al j unct ion, disrupt ion of t he basem ent m em brane w as found. Aft er 14 day s of t reat m ent w it h CT chit osan, t he abov e- m ent ioned derm at ological changes becam e apparent . I n som e repaired port ions, so- called “ birds- ey e- st ruct ure” charact erized by py k not ic nuclei and v acuolat ed granular cells w ere observ ed ( Fig. 9) . Aft er 21 day s of t he t reat m ent wit h CT chit osan, t he arrangem ent of t he collagen fibers in t he reepit helialized skin w as sparse and disorderly.
Figure 8. Dorsal skin aft er 7 day s of t he t reat m ent w it h CT chit osan. Epiderm al degenerat ion, bleeding and inflam m at ory cell infilt rat ion
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Figure 9. Dorsal skin aft er 14 day s of t he t reat m ent w it h CT chit osan. “ Birds- ey e- st ruct ure” is seen. HE. × 350.
Chit in fiber t y pe dressings: Aft er 7 day s of t he t reat m ent wit h chit in FD, a large am ount of ex udat e cov ered t he surface of t he w ounds. The inv asiv e st age of fibroblast s, m ononuclear cells, m ast cells and ery t hrocy t es show ed t he ret arded dev elopm ent of reepit helializat ion ( Fig. 10) . Aft er 14 t o 21 day s of t he t reat m ent wit h chit in FD, t here w as m ark ed derm al edem a w hich w as charact erized by hem orrhage and disordered arrangem ent of collagen fibers. Throughout t his st udy , hist opat hologically, w e did not find t hat t he sit es t reat ed wit h w ound dressings w ere in a m ore adv anced st age of reepit helializat ion t han t he undressed w ounds. Hist opat hological findings during t reat m ent wit h w ound dressings are sum m arized in Table 1.
Figure 10. Dorsal skin aft er 7 day s of t he t reat m ent w it h chit in FD. A large am ount of ex udat e cov ers t he w ound surface. HE. × 175.
D iscussion. The split t hick ness w ounds t reat ed w it h chit osan, chit in and w ound dressings m ade of t hese m at erials w ere m acroscopically and hist opat hologically ex am ined. I n addit ion, w ound healing in t he sit e t reat ed wit h each dressing w as com pared w it h t hat in t he WD- unt reat ed sit e.
Table 1 Hist opat hological findings in t he split t hick ness wounds of hairless dogs
aft er t reat m ent wit h w ound dressings
Findings Chit osan Chit in DP skin
CT chit osan Chit in FD Cont rol
Epiderm is
Reepit helializat ion + + - t o ± ± - +
Dy sk erat osis - - - + + - -
Thickening + + + + - ± t o + + - + +
Ret e ridges + + + + - ± t o + + - + +
I nfilt rat ion of - - + + + + + + + + + -
inflam m at ory cells
Fibroblast s - - - - + + + -
Ery t hrocy t es - - - - + + + -
Mast cells - - - - + + + -
I nt racellular edem a - - - + + - -
I nt ercellular edem a - - - + + - -
Birds- ey e st ruct ures - - - + + - -
Disrupt ion of t he - - - + + + - -
basem ent m em brane
Pigm ent at ion + + - - - +
Derm is
Granulat ion + + + + ± - t o ± - + +
Congest ion ± + + + + + + + + + + ±
Hem orrhage ± + + + + + + + + + + ±
Edem a - - + + + + + + -
Neov ascularizat ion + + + + + + + + +
Vasodilat at ion + t o + + + + + + + + + +
I nfilt rat ion of - t o ± + + + + + + + + + + ± inflam m at ory cells
Degranulat ed m ast cells - t o ± - t o ± - t o ± + + t o + + + + - t o ±
Arrangem ent of + + ± ± ± +
collagen fibers
Sebaceous cy st s + + + - ± - + +
The degr ee of hist opat hological changes: - = negat ive; ± = slight ; + = m ild;
+ + = m oder at e; + + + = m ar ked.
Throughout t his st udy , chit osan did not show any t ox ic effect s in t he t reat ed sit es. Treat m ent wit h t his agent rev ealed no discernible accelerat ing effect s in w ound healing, as com pared wit h non- t reat m ent . I n t he w ound healing process, t here w ere no differences bet w een t he sit es t reat ed wit h chit osan and t he WD- unt reat ed sit es.
DP skin is k now n t o be a st andard w ound dressing used widely in hum an m edicine. I t has been est ablished t hat DP skin is a biodegradable m at erial wit h good biocom pat ibilit y and beneficial biological effect s such as accelerat ion of w ound healing ( Kuroy anagi & Tak ao 1995) . I n cont rast , our result s rev ealed t hat DP skin prov ok ed adv erse effect s t o t he w ounds of hairless dogs. This skin react ion of hairless dogs seem ed t o result from absorpt ion of prot eins ( elast ic fibers) deriv ed from t he porcine skin, i. e. prot eins of different species.
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w ound fluids in hum an beings. Dressing wit h CT chit osan ret ains t he w ound ex udat e t o m aint ain a m oist environm ent at t he w ound surface and im prov es w ound healing ( Biagini et al 1991) . Cont rary t o beneficial result s in hum an clinical report s, t he present st udy rev ealed t hat CT chit osan had clinically and hist opat hologically apparent adv erse effect s on t he split t hick ness w ounds in hairless dogs. Ok am ot o et al ( 1993ab; 1995) report ed ev aluat ion of CT chit osan on open w ound healing in dogs. They described t hat t he ex perim ent al dogs show ed infilt rat ion of inflam m at ory cells and a considerable am ount of ex udat e on t he w ound surface. I n laborat ory anim als, Tanak a et al ( 1997) st udied effect s of CT chit osan on BALB/ c m ice by oral and parent al adm inist rat ion. Their result s show ed t hat CT chit osan did not ev oke t he general and cellular abnorm alit ies. Rao & Sharm a ( 1997) perf orm ed t ox icit y t est s ( acut e sy st em ic t ox icit y t est , py rogen t est , ey e and sk in irrit at ion t est s and int ram uscular t est ) of chit osan using m ice, guinea pigs and rabbit s, and t hey concluded t hat chit osan w as nont ox ic. Clinically and hist opat hologically, t he derm at ot oxicit y of CT chit osan t o hairless dogs w as m ore prom inent t han t hat t o t he ot her laborat ory anim als. These findings rev ealed t hat t here w ere species differences in t he skin react ions t o CT chit osan.
Recent ly , allergic cont act derm at it is due t o a cream w it h chit osan gluconat e has been report ed in hum an beings ( Cleenewerck et al 1994; Pereira et al 1998) . I n t he present st udy , t he skin around t he w ound t reat ed w it h CT chit osan ex hibit ed apparent inflam m at ory react ions. Chit osan deriv at ives are considered t o hav e som e delet erious effect .
I n our prev ious papers ( Kim ura & Doi 1994ab) , w e report ed t hat t he derm is of hairless dogs cont ained fewer m ast cells t han t hat of haired dogs and Beagles. I n t he sit es t reat ed wit h CT chit osan, t here w ere a num ber of m ast cells in t he derm is. The appearance of degranulat ed m ast cells m ay be associat ed wit h irrit at ing pot ent ials of CT chit osan w ound dressings.
Chit in FD is an absorbable m at erial and t his dressing has high air perm eabilit y and w at er absorpt ion abilit y . Several st udies ( Nak aj im a et al 1986; Tanak a et al 1997; Su et al 1997) report ed t ox icologic findings induced by adm inist rat ion of chit osan deriv at ives. Nak aj im a et al ( 1986) report ed t hat an absorbable chit in prov ok ed a great ex t ent of inflam m at ory cell infilt rat ion. I n m ice, Tanak a et al.7 m ent ioned t hat m any hy perplast ic m acrophages w ere observ ed in t he m esent ery and foreign- body giant - cell- t y pe poly k ary ocy t es w ere found in t he spleen. The clinical appearance in t his st udy show ed t hat chit in FD apparent ly inhibit ed w ound healing. I n addit ion, t he hist opat hological findings dem onst rat ed t hat chit in FD caused ret ardat ion of reepit helializat ion and granulat ion in hairless dogs. These result s rev ealed t hat chit in FD w as an unsuit able m at erial for w ound dressings in dogs.
I n conclusion, chit osan and chit in had few delet erious effect s on w ound healing w hereas DP skin and t heir deriv at iv es ( CT chit osan and chit in FD) induced not iceably high derm at ot oxicit y in w ounds of hairless dogs. I n addit ion, it appears t hat t here are species differences in t he skin react ions t o w ound dressing m at erials. These result s suggest ed special care should be t ak en in v et erinary clinical use of hum an m edical w ound dressings.
Con clusion. Chit osan and chit in had few delet erious effect s on w ound healing w hereas DP sk in and t heir deriv at iv es ( CT chit osan and chit in FD) induced not iceably high derm at ot oxicit y in w ounds.
Ack n ow le dge m e n t s. The aut hor declares t hat he is not current ly affiliat ed or sponsored by any organizat ion wit h a direct econom ic int erest in subj ect of t he art icle.
Re fe r en ce s
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Kim ura T., Kurok i K. , Doi K., 1998 Derm at ot ox icit y of agricult ural chem icals in t he dorsal skin of hairless dogs. Toxicologic Pat hology 2 6: 442- 447.
Kim ura T. , Doi K., 1998 Effect s of all- t rans- ret inoic acid on t he dorsal skin of hairless dogs. Toxicologic Pat hology 2 6: 595- 601.
Kim ura T. , Doi K., 1999 Dorsal sk in react ions of hairless dogs t o t opical t reat m ent wit h cort icost eroids. Toxicologic Pat hology 2 7: 528- 535.
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Received: 02 June 2 011. Accept ed: 01 July 201 1. Published on line: 14 July 201 1.
Aut hor : Tohr u Kim ur a, Cent er for Exper im ent al Anim als, Nat ional I nst it ut es of Nat ur al Sciences, 38, Nishigo-naka, Myodaij i, Okazaki, Aichi, 444- 858 5, Japan, em ail: kim ur a@nips. ac. j p
