South paciflc Study Vol.23,No l,2002

Skeletal muscle necrosis in rat induced

by rrmιrFs r sノZαツ
θツ
tridis venom
can be prevented by its serunl proteins

Ⅳlotoo KITANOl*,Ⅳ Iasato HIRANOl,Takashi lsHIHARAl,
Aichi YosHIDA2,shosaku HATTOR13,
Naoko UEDA4,TakahitO CHIJIwA4,Ⅳ lotonori OHN04

Abstract

財 ″θ
″s″
″s′αツ
οツ
プ
″グノ
sの sertlm proteins wcrc fractionatcd by anmnonium sulfatc precipitati
i、e pOrtiOns dcpcnding on the dittcrcnces ofits saturation percentagcs,that
is,0‐20%,20‑30%,30‑409る,
4050%,and 50‑70%Thc cmccts ofthese proteins on 7venOm̲induced rat skeletal musclc damage werein、
cstigatcd、/ith cioscr attention to histopath。
10gical features ofimpainllcnt,necrosis,and regeneration ofmusclc flbers The kllowledgcs which portion of y senlln prOteins is effectivc fOr prevention of local
lcJOns causcd by 7Venom should shed light On the effccJve medicaltreatmcnt afterbi■
cn by γ snakC In
rcrlldC vcnOm together withconscquence,it was fOund thatthe necrotic changc ofthe rats inoculated
η witllthc senlm prOtein fraction ofanlmOnium sulfate saturation percentagc 40‑50%was the smallcst cOmpa
to thOse ofthc rats tested with othcr ttrSertlm protein fractiOns
Key words: r7′772′″ S′77/Sノ αツοツ′′″お (助 ,envenomation,skelctal muscle nccrOsis(町regcncration,myoblast,7scrum protcins,allllnonium sulf・
atc

OneCrOsis),

Introduction
ln conforlnity with recent improvement of the mcdical treatlncnt of ttt777θ
lθ
′
S夕″ZrSソ
οッ″た力s cγ:habu snakc)snakC bites,thc mttOrity Ofrcccnt victirns has bccn savcd/7α
frOm scvere gencralizcd symptoms and dcath. HOwcvcr, local tissue damagc isincvitablc,and in some sevcrc cascs this is responsiblc for sequclac such as a ous
se五slough in the mttor extrcmity and eventually leads tO alllputation(HoMMA andIn the AInaFni iSlands of Kagoshilna Prcfccture and the several islands of Okina、va
Prcfccturc,this accident accounts for abOut 90%Of
γ Snake bitcs.Thcrcfore,it is an
irnpOrtant task to prevent the scvcrc and cxtensive damage of skeletal musclc tissues
caused by myotoxins containcd in 7venOm.
Five phospholipase A2(PLA2)iSOZymcs have becn is01ated from r/venom(TANAKA

ι′α′,1986:LIu

θ′α′,1990;YOsHIzUMI

駆 踏 郷 肱鵠 翌堂
管糊 器

θ′α′,1990;KIHARÀ′α′.,1992):

鋼 蝠 鳴鍬躍 比

called PLA―N(p110.3,neuroto対 c),and tWO
and 10.2, rcspcctivcly. extremcly low lipolytic to egg―yolk cmulsion but strongly

椰y掘鰹F職換都躙 I亭
覆
羅A善
鵞鐵 醜五
牌
EnginccHng,SoJo UniVCrsity*Corresponding Author

South Pacinc study Vo1 23,No l,2002

θ′α′.,1995).BPI and
HARA θ′αノ。
.,1990;Iα
myolytic)(LIu θ′α′,1992;SHIMOHIGAGASHI
2 position of phospholipids in lniccllar state and in
BPII can clcavc arachidonatc at sん…
bilaycr rnembranes with siinilar activity as PLA2.Thus,Inost of PLA2 iSOZymcs can beconsidered to bc rnyotoxins.Thc rnyotoxic action of PLA2S iS nOt neccssarily associatedyolk、vith thcir catalytic activity toward ordinary micenar substrates such as egg¨
emulsion.Thcy are able to disrupt the integrity ofskclctal rnusclc plasma rnembrancs and+and Othcr ions.
possibly causc an increased perlncability to C′
Reccntlェ

NoBllHISA

θ′α′.(1997)reported that thc proteins with binding afflnity toγ VCnOm PLA2S WCre fractionatcd from its scrum on four colums,cach cottugatcd withonc offour PLA2iSOZymes,PLA2,PLA―

B,BPI,and BPH.Five PLA2 inhibitory protcins,

I〜PLI―ヽ;were Obtaincd.PLI― IV and PLI―V is mostly boundtcrllled PLA2 inhibitors,PLI―
B,BPI,alld BPⅡ .
to PLA2,whereas PLI―I has attnity toward basic PLA2S,PLA―
PurincatiOn of PLA2 inhibitors fromγ Seruln by afflnity chromatography is quite
laborous and this lnethod is difflcult to obtain thc inhibitors in a large quantity.For

rSerum proteins were fractionatcd by ammonium sulfate precipi
reasons,η
309る
portions dcpcnding on thc diffcrcnccs of its saturation pcrcentagcs,, 0‑209る, 20…
venom―
induccdrat
30‑40%,40¨50%,and 50‑70%.Thc cffccts ofthcsc protcinsγon、vcrc invcstigated with closcr attcntion to histopathologicalskeletal muscle damage
featurcs of impairlncnt,nccrosis,and regeneration of lnuscle fibcrs.Such studics havcs t e m m e d f r o m o u r r e c e n t d e t a i l c d s t u d i c s uo五
f o ut sh e a ci tn i」o n o f 7 v c n O m a n d i t s
TANO θ′α′.,2001).The
componcnts,some PLA2S,On thc rat skeletal lnuscle flbcrs(●
knowlegdc which portion of 7FSerum protcins is cffcctivc for prcvcntion oflocal lcsions
causcd by[∫ VCnOln should contributc to advanccmcnt ofthe effcctivc lncdical treatmcnt
aftcr bittcn byγ snake.

ⅣIaterials and PIethods
Rαおg in body wcight)ofWiStar/Furth strain
Y o u n g a d u l t f c m a l c a n d m a l c r a t s1(0800 〜
niversity Dcntal School bymaintaincd in Dcpartrnent of(Dral Pathology Kagoshirna l」
θ′α′.,2001)were dividcd into scvcn
sister―brothcr mating(● Iぶ Oθノαノ,1992;KITANO
groups,cach group consisting ёf 6 rats(tOtal;42)(Table,1).
――θ″Cr ル 7降7 VCnOm was collected in Amami Laboratory of ltturiOus Animals,Institutc of
Mcdical Science,Univcrsity ofTokyo and lyophilized aftcr dilution with a small volumc
of、vater. Thc crudc venoln、 vere、vcighcd and dissolvcd in sterilc physiological saline
immediatcly prior to usc.

、
c r i aα″κθ θ′ S ιr ″P r a 彪れsき崎 α″r i a α ″F r α
̀ γ at Amami Laboratory of ltturiOus Animals.Its
Blood of ηrsnake was collectedscruln was prcparcd by rcmoving prccipitated cげ hrocytes and fibrous protcins aftcr
kccping the blood at 49C overllight and stocked at‑8013.At Faculty of Engincc五ng,

20%
S●O UniVersity,ammonium sulfate was added to the serum .At
portionwiseasaturation of arninonium sulfatc thc prccipitatc was collccted by ccntrifugation. Thc

Prevcntion Of βrαッ。ップ
/7dis vcnOm―
induced ryonccrosis by its scrllrn protcins

13

supcmatant waS then brought to 30%ammonium sulfate saturation and thc prccipitate、as conectcd.The samc proccdurc was repeated.Thc protcins cOHcctcd wcrc dissolvcd
in water(or O.05 M Tris―
HCl,pH 8.0),dialyzcd against water,and lyophilizcd.Thc
protcins obtaincd'om 50 mlof 7scrum WCrc O.15g,0.36g,0.35g,1.33g,and O.55g for
O‑20%,20… 30%,30‑40%,40‑50%,and 50‑70%ammonium sulfatc saturation,
respcctively. Each serunl protcin iaction was weighed and dissolvcd in sterile
physiological salincjust bcforc usc.
Expι ri″ι″″′Praιι″ ″s
Thc animal cxperilncnts wcrc conductcd at the Animal Center of Kagoshima
University IDcntal School follo、
ving Guidline ofthc Anilnal Expcrilncnt"ofthis School.
All thc rats of seven groups wcrc ancsthctizcd with pentobarbital sodium(Nembutal:
A b b o t L a b . , U S . A . ) . A m i x t u r e o f 7 F C r u d e V e n O m (μ5g0 c a c h ) a n d C a c h s c r u m p r o t c i niaction(50μg each)in 100
vere ittcctCd into thc antcrior aspect
μl physiological salinc、
ofthc quadriccps fcmoris muscle(QFM)of caCh rat of irst ttve groups(Groups l‑
the ordcr of incrcasing ammonium sulfatc
on satura●pcrccntagc,0〜20%,20〜30%,
30〜
409る, 40〜
509る, and 50〜
70°0(TablC l). The Crude vcnolll(50μg)in 100 μl
vas illlectcd into the rats of the sixth group (Group 6). Sterile
Physiological saline、
physiological salinc alonc was in」 cctcd into thc rats of thc seventh group(Group 7)as a
control Thc anilnals、vere aHowed to suぃ′
ive for a period of48 hours and sacrinced undercthyl ether ancsthesia.Both thc right and left hindlimbs and visccral organs including thebrain wcrc immcrscd for scveral days in 10%buffcrcd formalin(pH 7.4),and prOccssed
lor parafflnic cmbedding Thc scctions of 5〜
6 μ rn thickness wcrc cut and staincd withhcmatoxylin and eosin.
Pathological indings
Thc musclc tissuc ofthe rats of Group 7 ill」
ectcd only with physiological saline had
a typical histology ofskeletal llluscle、vith no abnorlllality in inuscle ibers,nerves,blood
ヽCSSClS,and ibro―
adiposc layers.
In thc rats inoculated with 2γ Crude venom(Group 6),and With a mixturc of thecrudc vcnonl and a scrunl protein fraction of ammoniuln sulfate saturation perccntagcs(Groups l〜 5),no hiStOpathologicaHy signincant differences、 verc dctcctcd among theimale and male rats of cach group,so wc prcfcrrcd to dcscribc hcrc all together(TablCl)rcrtldC vcnom(Group 6)and with a mixture oftllc
In the rats inoculatcd with η
onlycrude venonl and thc scrulln protcin fraction of allallloniulll sulfatc saturation perccntagc
O〜20%(Group l),thcrC Was a widespread musclc nccrosis.Thc myo■brillar matcrials in
necrotic ccns wcrc more amorphous and their distribution within the cenular space wasdense Clampcd masses showcd liqucfactiOn nccrosis(Fig.1)instcad of being morc
homogeneous looking coagulative necrosis 2へ
n abundant innanllnatory inflltratc wasprescnt outsidc thc nccrotic ccH arcas, mainly at thc surrOunding tissuc layers of thc
llccrotic muscle flbcrs. SiFllultancousl)Ъ
rcgcncrativc prolifcration of myoblasts,which、crc characterized by thc prcscnce of scanty basophilic cytoplasnl,was obscⅣ cd in thc
pcriphery of the nccrotic ccH arcas.Thc rcgcncrating cclls wcrc spindlc in shapc、vith avas little hemorrllagc,althoughccntral nucleus,some rcvcaling lnitotic activitics.There、

South Paciflc Study Vol.23,No.1,2002

14edema was considcrably markcd.Fibrosis was not so conspicuous.
Thc sizes of thc necrotic arcas became smaner and slnallcr in accord with thephenomenon that prolifcration of sman regenerating muscle flbers seemed more andmorc rnarked in theく3roups 2,3,4 in thc ordcr ofincrcasing aIIIlmonium sulfate saturation
pcrccntage,20〜
30%,30〜40%,and 40〜50%(Figs.2‐4).HoweVet the rats ofGЮup 5 ofammonium sulfatc satllration pcrccntage
70%showed
50〜
rclativcly greater necroticchangcs than thosc ofGroups 3 or 4.

Tablc l.Histopathologic indings in QFM ofthe rats whcn trcat
protein fractions indicated.
TF venorn and its senll■
7VenOm+Necrosis

lnflanlm atory

Rcgcncrationoflnflltradon

Ofskeletal

Fraction*of(Group l)

40%
30〜

OthersFlbers

…

Ⅲ 「H
・

‖

FF

‖

I‐

2 0 〜3 0 % I 「(Group 2)

Edenlamuscle7Semm
SkCkCtJprotcinmusclc(Group oftissucratS)**

20%*
0〜

Hcmorrhagc

―++

‐丼

I「

―F
―F

―
F FF

¨
―F

―IF

―
―l‐

―I

(Group 3)

40〜
50%

‐
―「F

I‐

IF

‖

(Group 4)

50〜

70%

‐ ‐ ・
「
FI‐

4‐

丼

IF

―

l・

IF
一

(Group 5)
Crudc venom(Group 6)
Salinc only
(Group 7)

I「

丼

¨F
―

IFI

thro壺

bosis

一
¨

* The values of%represcnt thcurn
anlmcl五sulfatc saturati(m rangcs for
量atiOnfractional
Ofthe pscrtlm protcins** Each gЮ up consists of 6 rats(｀
ViStar/Furth strain;F+M)#

The necrodc changcs are composed nlainly of liquefaction necrosis but coagulativc necrosis are alsopresent.

Prevention of r/7aッ
9ν′″″s venom―induced lnyonecrosis by its serum protcins

Fig■

Fig 2

Fig l(lCft):A photomicrograph of a peHphcral area of a largc focus of liqucfaction
nccrosis(N)obscrVCd in thc QFM ofa rat ofGroup 6.Thc necЮsis was induccd by

ηr CrtldC VCnom, inoculated 48 hours beforc.The focus is surrounded by agranulation tissuc (Astc五sks) containing numerous inflammatory ccHs andrcgcncrating lnyoblastic spindlc cclls(HE).

Fig 2(right):A photomicrograph ofa vcry smali necrotic focus(Arrow hcads)inthe QFM
ofa rat of Group 4(HE).

Fig 3

Fig.4

Fig.3(lcft):A larger magnincation ofFig.2.The focusis composcd mainly ofa numerous
inflalnmatory cclls and regenerating myoblastic spindlc cclls.A couplc of nccrotic
musclc■ bcrs(Arrows)arc interlninglcd with them at thc ccntral rcgion ofthc focus.
Fig.4(五ght):A pc五 phcral arca ofthe granulation tissuc of Fig.2.Notc thc charactcristic
myoblastic spindlc cclls(Open circlc).

South Paciflc Study Vo1 23,No l,2002

Discuss10m
Many vcnomous snakcs are rcsistantto their o、vn vcnoms.Thcir natural rcsistancc to
thcir toxins is duc to neutralizing factors in thcir scra.Thcse factors must protcct against
toxicity duc tO accidcntal bitcs by the snake itsclfOr by othcr snakes ofthc same spccics.
Inhibitors of snake venonI PLA2S haVe becn is01atcd from the scra ofvaHous snakes andtheir prilnary structurcs detcllllined. Two kinds of PLA2 inhibitOrs against t
PLA2,namcd PLI―
A and PLI― B,were pu五 icd frOm its serum(INOUE
θ′α′.,1991),and an
inhibitor that ncutralizcs Иgた,s′Юグθ bあ ″力晰 傷 S″′′
Jθ s(mamushi snakc)PLA2 WaS
isoslated from its serum(OHKURA
θ′α′.,1993)CЮ′αルS PLA2 neutralizing factor was
isolatcd from CЮ rr7ル
s″夕′おs s′θ″ ′ θ夕S SCrum(FoRTES― DIAS θ′α′.,1994;PERALESαα′.,1995).
NOBuHISA
θ′α′。(1997)rcported thc is01ation Of flve PLA2 inhibitoぃ/protcins(PLI一I〜V)from
πFSCrum in which PLI― IV and PLI― V arc the samc as PLI― A and PLI― B,

rcspect市
ely.HOwevet the contents ofthc inhibitors
in lγ
serum
scem to bc to a minOr
cxtcnt,so thatthcir scparation from γ scrum is vcry dimcult.Thus,thc cstablishnlcnt ofthc cffcctive mcthod ibr prcparation ofthc scrum fractiOns containing thc inhibitors was
rcquircd.Our prcscnt study has startcd to fractiOnatc■
iseruln protcins to scvcral pOrtions
by prccipitation v/ith increasing conccntration of alllmlonium sulfatc.
It is、
vcll known that the pathological signs charactcristic of bitcSE∫ in humans,
rabbits,and micc arc markcd and cxtcnsivc hcmorrhagc and musclc necrosis(OKONOGI
θ′α′, 1960).PrCViOusly we rcported that Our cxpe五
mcntal rats,howevet failed todcmonstrate thc hcmorrhage as a predominant slgn whcn 7 venOm was inJccted.7
venom did not givc rise to any promincnt local bleeding in the rats, but produccd a
massivc nccrosis in tlle skcletal muscles accompanied by marked edema(KITぶoθ′α′.,2001)Additionally wc repOrtcd that in 48 hours aftcr the ittcctiOn Ofヱ

FVCnOm intO the

QFM of the rats,a widcspread myolytic muscle necrosis was fOund,whic
resulted fronl a dircct venonl action On thc musclc ibers.Muscle nccrosis by thc 10cal
inoculation of Ei VenOm is usuaHy fol10wcd by rapid phagocytosis of dcbris at thc
periphcry ofthc nccrotic area.Thcn thc clcaHng Ofnecrotic rllatc五als by phagocytes、vas
followcd by a rapid regcncration prOccss. Numcrous myoblasts wcrc obscⅣ
ed.「Fhc

exccss and rnarkcd regencration activity ofmyoblasts aftcr rnusclc nccrosis induced by
myotoxins lnight bc ascribcd to thc cOnditions that ncithcr
neⅣblood vessels are
cs normarkedly damagcd by thcsc tOxins,since adcquatc b10od supply and innervaton are thc
essential rcquircmcnts for muscic rcgencration(QuEIROZ

θ′αム,1984;GuITIERREZ

θ′α′,1989).ThCrcfOrc,for analysis ofthcc cffcct市
functions of7sCrum inhibitors againstth
rVCnOm,thc ratis considcred to bc onc ofthc most appropri
myolytic itturicS
η by
uSeflll CXpcrilnental anilnals.
In thc present studン
Ъthe rats ofGroups 3 and 4 inoculated byごfvenOm togethcr、 vith
thc serum protein fractiOns of ammonium sulfatc saturation perccntages 30‑40%a
40‑509る demonstrated the most dOminant changcs in thc foci Of musclc necrosis. Thc
nccrotic foci were lilnited in a narro、
v arca and、verc su■ounded mainly by granulation
tissucs cOntaining numcrous lnacrophages and rcgcncrativc myoblastic ccns.Edcma was
not so marked, cither. These facts suggestcd that thc effcctivc myotOxin inhibitory
protcins wcrc cOntaincd in thc scrum fractiOns prccipitated in thc particular conccntration
rangcs ofalllnlonium sulfatePrevention of rノαッ。ッ
Jガ s venom―
induced myonecrosis by its sertlm protcins
̀″17Conclusivelシ
Ъthe results obtained in thc present study suggest that thc myotoxin
inhibitors inηrserum are effect市e in healing the myotoxic ittuHeS induced by ηrcrude、enom Since application of excess市
c doscs an″ or repetitive application of anti‑7
ヽenom antisera are known to bring about ous
se五 lncdical problems,we certainly expectthat applicati9n OfrFScrum inhibitors in appropriate ways could providc a uscfulthcrapy
ぉrr/snake bites in near futurc.

Acknowledgements
This work、 vas supportcd,in parts,by a Fund for Promotion and Dcvclopment ofArnami lslands sponsored by Kagoshilna Prefecturc and Ministry ofLand,Infrastructurc
and Transport of Japan and by a KagoshiFna University Fund for lmprovcment of
Educatlon and Research Activltles.

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