186 - Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011
6 – ORIGINAL ARTICLE
Ischemia-Repe usion
E ec s o allopu inol and p econdi ioning on apop osis due o ischemia- epe usion on a
double jejunum-segmen canine model1
E ei os do alopu inol e p econdicionamen o na apop ose de ido a isquemia- epe usão em
duplo segmen o de jejuno em cães
End e B a hI, I en MikoII, No be Neme hIII, Judi Ko acsIV, Ka alin Pe oV, Is an Fu kaVI
1 Resea ch pe o med a he Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e ,
Uni e si y o Deb ecen, Hunga y.
I MD, Assis an Lec u e , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e ,
Uni e si y o Deb ecen, Hunga y. Pe o ming ope a ions and sampling, e alua ing esul s, w i ing pape .
II MD, CSc, PhD, Full P o esso , Head o he Depa men , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical
and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Conduc ing expe imen , e alua ing esul s.
III MD, PhD, Assis an P o esso , Head o Resea ch Labo a o y, Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y,
Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. E alua ing esul s, pe o ming s a is ics, w i ing pape .
IV MD, PhD, Associa e P o esso , Chie Doc o , Depa men o Pa hology, Semmelweis Hospi al, Miskolc, Hunga y. Examina ion o his ological
samples, e alua ing esul s.
V MD, PhD, Assis an P o esso , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e ,
Uni e si y o Deb ecen, Hunga y. Assis ing ope a ions, e alua ing esul s.
VI MD, PhD, DSc, Eme i us P o esso , Head o Mic osu gical Educa ional and T aining Cen e , Depa men o Ope a i e Techniques and Su gical
Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Conduc ing expe imen , e alua ing esul s,
w i ing pape .
ABs RAc
Pu pose: To in es iga e he du a ion o apop osis caused by ischemia- epe usion in he in es ine in a new double jejunum-segmen
model, and o analyze he p o ec i e e ec s o allopu inol o ischemic p econdi ioning (IPC). Me hods: In Expe imen I o ha es ing
he double jejunum-segmen model a e lapa o omy a 30-cm-long jejunum pa was selec ed on mong el dogs (n=24). End- o-end
anas omoses we e pe o med a bo h ends and in he middle o he jejunum pa , c ea ing wo equal segmen s. In one segmen ischemia
was induced by occluding he supplying essels, he o he segmen se ed as con ol. Tissue samples o de ec ing apop osis we e
aken a 30 h minu es, 1s , 2nd, 4 h, 6 h, 8 h, 12 h and 24 h hou s o epe usion. In Expe imen II using he same model he 4-hou
epe usion ime pe iod, allopu inol (50 mg/kg) p e- ea ed and IPC (3 cycles o 5x1) g oups (n=5 pe each) we e also in es iga ed.
Resul s: In Expe imen I he g ea es apop o ic ac i i y was de ec ed a he 4 h and 6 h hou o epe usion (14.2 ± 1.31 and 16.3 ± 1.05
pe isual ield a 40x magni ica ion). In Expe imen II Using he 4-hou epe usion ime pe iod allopu inol p e- ea men inc eased
he apop o ic ac i i y (10.72 ± 0.47 pe 50 in es inal illi) app oxima ely wo- old han he IPC (6.72 ± 0.46 pe 50 in es inal illi) did
(p<0.05). conclusions: Apop o ic ac i i y has a cha ac e is ic ime cu e, eaching he highes alues be ween he 4 h and 6 h hou s a e
30-minu e in es inal ischemia. Ischemic p econdi ioning seemed o be p o ec i e agains he mo phological changes caused by in es inal
ischemia- epe usion.
Key wo ds: Ischemia. Repe usion. Jejunum. Apop osis. Allopu inol. Ischemic P econdi ioning. Dogs.
Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011 - 187
E ec s o allopu inol and p econdi ioning on apop osis due o ischemia- epe usion on a double jejunum-segmen canine model
REsUMO
Obje i o: In es iga a du ação da apop ose causada pela isquemia- epe usão no in es ino em um no o modelo de duplo segmen o de
jejuno e analisa os e ei os p o e o es do alopu inol ou p econdicionamen o isquêmico (IPC). Mé odos: No expe imen o I pa a ob e
o modelo do duplo segmen o de jejuno, após a lapa o omia, uma pa e de 30cm de comp imen o de jejuno oi selecionada em cães
mes iços (n=24). Ana omoses T-T o am ealizadas em ambas as ex emidades no meio do segmen o de jejuno, c iando dois segmen os
iguais. Em um segmen o oi induzida isquemia po oclusão dos asos que o i iga am e o ou o segmen o oi usado como con ole.
Amos as de ecido pa a de ecção da apop ose o am ob idos aos 30 minu os, 1h, 2h, 4h, 6h, 8h, 12h e 24 ho as de epe usão. No
expe imen o II usando o mesmo modelo, no empo de epe usão de 4 ho as, o am in es igados dois ou os g upos (n=5 cada) usando
p econdicionamen o com alopu inol (50 mg/kg) e IPC (3 ciclos de 5x1). Resul ados: No expe imen o I a maio a i idade de apop ose
de ec ada oi às 4h e 6h de epe usão (14,2 ± 1,31 e 16,3 ± 1,05 no campo isual de 40x). No expe imen o II usando o pe íodo de 4ho as
de epe usão o p é- a amen o com alopu inol aumen ou a a i idade apop ó ica (10,72 ± 0,47) ap oximadamen e 2 ezes mais do que
o IPC (6,72 ± 0,46) (p<0,05). conclusões: A a i idade de apop ose em uma cu a ca ac e ís ica, a ingindo maio es alo es en e a 4ª
e a 6ª ho as após 30 minu os de isquemia in es inal. O p econdicionamen o isquêmico pa ece p o ege con a al e ações mo ológicas
causadas pela isquemia- epe usão in es inal.
Desc i o es: Isquemia. Repe usão. Jejuno. Apop ose. Alopu inol. P econdicionamen o isquêmico. Cães.
In oduc ion
Ischemia- epe usion (I/R) inju y o small bowel appea s
in case o small bowel ansplan a ion, hea and ao a su gical
in e en ions, acu e and ch onic mesen e ic a e ial and enous
occlusion, du ing hypo olemic shock, in ense auma, in a ious
o ms o asculi is, and nec o ising en e ocoli is (in childhood)1,2.
Small in es inal mucosa wi h a high egene a i e capaci y
is one o he issues being mos sensi i e o ischemia- epe usion
inju y. Chiu e al.3 e alua ed he ischemia- epe usion in es inal
inju y and classi ied his p ocess in cha ac e is ic his ological
g ades. Simila ly o o he o gans, ee adicals play an impo an
ole in he pa hophysiology o mesen e ial I/R inju y4-6, leading o
di e en ype o cell dea h, such as apop osis o nec osis7,8.
Al hough he pa hophysiology o mesen e ial ischemia-
epe usion inju y has been widely s udied8,9, i s ill in ol es
ques ions wi hou answe . Wha a e he ole and p ocess o
apop osis in he pa hogenesis o I/R inju ies in he in es inal wall?
When does he apop osis become ac i e and in ensi e a e he
dea h signal has been s a ed? When does he mucosal damage
eco e o he no mal unc ion? Fu he ques ions aised whe he
ischemic p econdi ioning o adminis a ion o allopu inol (inhibi o
o xan hine-oxidase) can be e ec i e o keep he no mal apop o ic
le el and mo phological s uc u e. In es iga ing hese ques ions,
we aimed o analyse he du a ion o p esence o apop osis caused
by ischemia- epe usion inju y in he in es inal wall using a double
jejunum-segmen model in he dogs. The second aim was o
in es iga e he e ec s o allopu inol and ischemic p econdi ioning
in he same model.
Me hods
Expe imen al animals
The expe imen s we e pe o med on 39 male and emale
dogs, weigh ing be ween 21-25 kg, wi h he pe mission issued
by he Uni e si y o Deb ecen Commi ee o Animal Resea ch
(UDCAR) (Pe mission N .: 22/1999.).
The animals we e kep in s anda d cages a oom
empe a u e o 18-22ºC, and ed wi h a no mal mixed ood,
wa e ad libi um, and p o iding hem he eedom o mo emen
acco ding o hei demands.
The s udy included wo expe imen s:
Expe imen I. (n=24) a double-jejunum segmen model
has been ca ied ou . A e 30-minu e ischemia he his ological
analyses we e done a he 30 h minu es, 1s , 2nd, 4 h, 6 h, 8 h, 12 h
and 24 h hou s o he epe usion ollowing-up he p esence o
apop osis.
Expe imen II. (n=15) he same model and he same
ischemic ime was used as in Expe imen I, and he his ological
analyses we e done a he 4 h hou o he epe usion compa ing
he e ec s o ischemic p econdi ioning and adminis a ion o
allopu inol.
Expe imen I.
Anes hesia, su gical echniques and expe imen al g oups
Anes hesia was induced and main ained by combined
adminis a ion o SBH-Ke amin (10% ke aminum hyd ochlo icum,
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B a h E e al.
10 mg/kg, i.m.) and P imazin (2% xilazinum hyd ochlo icum, 1
mg/kg, i.m.). The ope a ions we e pe o med in s e ile condi ions
a he expe imen al su gical ope a ing oom o ou depa men .
A e pe o ming midline lapa o omy, a 30-cm long
jejunum pa was selec ed. A bo h ends and in he middle po ion o
he in es inal loop en e o omies we e done blocking he in amu al
colla e al blood low. Th ee end- o-end anas omoses we e
pe o med o main ain he con inui y o he bowel c ea ing wo
segmen s o in es iga ion o he I/R inju y and o con ol (Figu e
1). The essels supplying hese segmen s we e gen ly dissec ed
om he su ounding issues o elimina e connec ions wi h he
sys emic ci cula ion. Then 30-minu e ischemia was induced by
occluding he essels o one segmen (ischemic segmen ) using
an a auma ic ascula clip. The o he segmen did no su e om
ischemia (con ol segmen ).
FIgURE 1 - Jejunum segmen s wi h 3 in es inal anas omoses. A. Con ol
jejunum segmen wi hou clamping. B. Jejunum segmen o ischemia-
epe usion in es iga ions (The a ows show he in es inal anas omosis).
The animals we e di ided in o 8 g oups (n=3 pe each),
and acco dingly, he issue samples we e aken om he in es ine
a he 30 h minu e, 1s , 2nd, 4 h, 6 h, 8 h, 12 h and 24 h hou s o he
epe usion, and h ee pa allel examina ions pe each sample we e
pe o med. Also biopsies we e aken om he con ol jejunum
segmen s (Figu e 2). Du ing he epe usion pe iod he abdominal
ca i y was closed in 2 laye s wi h uning su u e lines in all g oups
excep o he 30 minu es g oup. In his g oup we closed he
abdominal ca i y empo a y wi h pe i onel clamps. The animals
o he 24-hou obse a ion pe iod we e allowed o eco e om
he anes hesia, ecei ed analgesics (Demalgonil®, 1 ml s.c.), and
on he nex day hey we e anes he ized again.
A he end o he epe usion pe iods mac oscopic
ob se a ion o he abdominal ca i y, he egion o he anas omosis
and he lumen o he in es ine a e opening was ca ied ou .
By he end o he expe imen s he eu hanasia we e ca ied
ou by gi ing concen a ed po assium-chlo ide in aca dially unde
gene al anes hesia o all he animals.
FIgURE 2 - The expe imen al p o ocol o in es iga ing apop osis by
di e en du a ion o epe usion a e su ge y and 30-minu e ischemia.
Expe imen II.
Anes hesia, su gical echniques and expe imen al g oups
The anes hesia, ope a i e condi ions we e he same as in
Expe imen I. In all animals he double-jejunum segmen model
has been pe o med as desc ibed abo e.
Ischemia- epe usion (IR) g oup (n=5): 30-minu e
ischemia was induced by a auma ic clamping he essels ha
supply he examined, ischemic segmen .
Ischemic p econdi ioning (IPC) g oup (n=5): p io o
he 30-minu es ischemia p econdi ioning p o ocol was used ha
included h ee imes pe o med 5-minu es ischemic pe iod wi h
1-minu e in e mi en epe usion phases.
Allopu inol p e- ea ed ischemia- epe usion (AP+IR)
g oup (n=5): p io o he 30-minu e ischemia 50 mg/kg allopu inol
(125 mg in 20 ml 0.9 % saline solu ion) was in used in o he
ex e nal jugula ein. Then he ischemia- epe usion was ca ied
ou as in IR g oup.
In hese g oups a 4-hou epe usion pe iod was
allowed, while he abdominal wall was closed empo a y. A he
4 h hou o he epe usion issue samples we e aken om he
ischemic and con ol jejenum segmen s o ligh mic oscopy and
immunohis ochemical s aining. By he end o he expe imen he
eu hanasia was he same as in Expe imen I.
1 hou
30 min.
S
U
R
G
E
R
Y
1.
2.
3.
4.
5.
6.
7.
8.
30 min.
30 min.
30 min.
30 min.
30 min.
30 min.
30 min.
2 hou s
4 hou s
6 hou s
8 hou s
12 hou s
24 hou s
ISCHEMIA REPERFUSION
30 min.
EXPERIMENTAL GROUPS
Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011 - 189
E ec s o allopu inol and p econdi ioning on apop osis due o ischemia- epe usion on a double jejunum-segmen canine model
His ological echniques
Biopsies o he in es inal wall we e ixed in 10 % o malin,
dehyd a ed in a g aded se ies o alcohol, embedded in pa a in,
mic o omed in 5 µm s ep sec ions, and s ained wi h haema oxilin
and eosin (H&E). E alua ion o he ischemia- epe usion
in es inal inju y we e pe o med using he Chiu Sco ing Sys em3:
G ade 0 (G0) – no mal mucosa; G ade 1 (G1) – de elopmen o
subepi helial G uenhagen’s space a he ip o he illus; G ade
2 (G2) – ex ension o he space wi h mode a e epi helial li ing;
G ade 3 (G3) – massi e epi helial li ing wi h a ew denuded illi;
G ade 4 (G4) – denuded illi wi h exposed capilla ies; and G ade
5 – disin eg a ion o lamina p op ia, ulce a ion and hemo hage.
Immunohis ochemical echnique
Apop o ic cells we e iden i ied wi h an in si u apop osis
de ec ion ki using e minal deoxy-nucleo idyl- ans e ase-
media ed, dUTP nick end-labeling (TUNEL) assay isualized
by di ec immunope oxidase eac ion (Apop ag-Ki , Onco ,
Bioma ke ).
In Expe imen I, apop o ic index e e ing o all he
posi i ely s ained nuclei pe isual ield a 40x magni ica ion
was also calcula ed. A leas 10 isual ields pe sec ion we e
in es iga ed in he his ological samples. In Expe imen II, he
a e age numbe o TUNEL posi i e nuclei pe 50 well-o ien ed
illi was calcula ed bo h in con ol and ischemic segmen s o each
g oup.
S a is ical analyses
Because o he small case numbe (3 animals pe ime
poin , 10 isual ields pe each issue sample), in Expe imen I a
limi ed compa ison was made using one way ANOVA on anks
es (Dunn’s me hod). In Expe imen II (5 animals, 50 in es inal
illi om each segmen ) S uden ’s - es o Mann-Whi ney ank
sum es s we e used acco ding o he da a no mali y es s. A p
alue <0.05 was conside ed as s a is ically signi ican .
Resul s
Expe imen I
Mac oscopical obse a ions
Du ing he expe imen he e was nei he mo ali y no
any complica ion in he abdominal ca i y and in he ope a ing
egion. All he in es inal anas omoses unc ioned well.
A e opening he bowel wall we could see ha he e was
a small quan i y o deb is s icked on he in es inal mucosal su ace
a he 30 h minu e, 1s and 2nd hou o epe usion. A he 4 h, 6 h and
8 h hou s o epe usion he in es inal lumen was ull wi h deb is
being and adhe ed o he in es ine wall oo. A he 12 h and 24 h
hou s o epe usion he e was only a small quan i y o deb is.
Mic oscopical in es iga ions
In acco dance wi h he mac oscopical obse a ion, H&E
s aining demons a ed minimal cellula deb is on he in es inal
mucosal su ace a he 30 h minu e, 1s and 2nd hou o epe usion
(Figu e 3). In nume ous illi subepi helial spaces we e obse ed
wi h inc eased cellula i y, mos ly o lymphocy es in he apical
egion. Howe e , he s uc u e o he in es inal wall was well
o ganized (Chiu G2 and G3).
FIgURE 3 - Pic u e o he mucosa a e 30-minu e ischemia and 2-hou
epe usion. A ow shows he la ge amoun o deb is on he mucosa
su ace (H&E s aining, o iginal magni ica ion: 200X).
In he issue samples aken a he 4 h, 6 h and 8 h hou s o
he epe usion he mucosa was cha ac e ized by de elopmen o
subepi helial G uenhagen’s space, usually a he apex o he illus,
o en wi h capilla y conges ion. In ce ain illi, ex ension o he
subepi helial space wi h mode a e li ing o epi helial laye om
he lamina p op ia was also obse ed (G3, G4).
In biopsy ma e ials aken a he 8 h, 12 h and 24 h hou s
o he epe usion he e was no damage o he mucosa, and he
deepe laye s we e well o ganised. De elopmen o subepi helial
G uenhagen’s space was e y a e (G0-1).
In he con ol samples mucosa was no mal wi hou
in lamma o y cells (G0).
190 - Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011
B a h E e al.
Immunohis ochemical in es iga ions
Table 1 shows he numbe and localiza ion o apop o ic
cells (40X magni ica ion).
The con ol specimens showed heal hy s uc u e o
mucous memb ane and o he deepe laye s o he in es inal wall.
The e was no sign o he in lamma ion. The apop o ic ac i i y was
low: 1-2 apop o ic cells pe isual ield we e loca ed in he bases
o he epi helial laye .
The apop o ic ac i i y appea ed a e 30-minu e
epe usion: 3 apop o ic cells pe ield we e de ec ed, bo h in he
basal laye o he mucous memb ane and in he apical egion o
in es inal illi. Also in lamma o y cells, mos ly lymphocy es we e
seen.
A he end o he 1s hou o he epe usion apop o ic
cells bo h in basal and apical a eas we e obse ed. Dominan ly
in e s i ial bu also mucous cells we e in ol ed in he p ocess. The
nume ical alue was 3 apop o ic cells pe isual ield.
In he 2-hou specimens a e age o 6 apop o ic cells we e
seen pe isual ield. In il a ion o in lamma o y cells could be
seen in he apical egion o illi.
ABlE 1 - The numbe and localiza ion o apop o ic
cells in he unc ion o epe usion ime.
means ± S.D.
* p<0.05 s 0 minu es (Con ol)
In he 4 h hou o epe usion showed he in es inal lumen
was hickly co e ed wi h deb is con aining nume ous TUNEL
posi i e cells. Howe e , we p o ed DNA damage in hese cells,
we could no coun hem because he damage migh ha e been
caused by nec osis. The a e age numbe o apop o ic cells pe
isual ield was 14, concen a ed in he apical egion o he illi.
Howe e , hey also appea ed in he basal egion, in he c yp s, and
e en in he deepe laye s o he in es inal wall (Figu e 4).
The apop o ic ac i i y inc eased p og essi ely in he
in es iga ed specimens eaching i s peak a e 4-6 hou s o
epe usion. In he 6 h hou o epe usion we ound he highes
numbe o apop o ic cells: 16 TUNEL posi i e cells we e seen pe
ields.
FIgURE 4 - Loca ion o he TUNEL posi i e cells in he in es inal
mucous memb ane a e 30-minu e ischemia and 4-hou epe usion. The
a ow shows a TUNEL posi i e cell (Apop ag Ki , o iginal magni ica ion:
100X).
In he 8 h hou o epe usion he numbe o apop o ic
cells (placed bo h apically and basally) d opped o 4 pe isual
ield. Howe e , in he unica p op ia he e was an ex ensi e
in lamma o y cell in il a ion.
In he 12- and 24-hou specimens he numbe o apop o ic
cells was jus like in he con ol specimens.
Expe imen II
Mac oscopical obse a ions
Simila ly o he ele an esul s (4-hou ) o Expe imen
I we obse ed signi ican amoun o cellula deb is s icked on
he in es inal mucosal su ace in each g oup. In AP+IR g oup he
Du a ion o he
epe usion
he numbe o he
UNEl posi i e apop o ic
cells / isual ield
a 40x magni ica ion
localiza ion o he
apop o ic cells in he
in es inal illi
0 minu e (Con ol) 0.7 ± 0.57 basal and apical egion
30 minu es 2.96 ± 0.61 * basal and apical egion
1 hou 3 ± 0.74 * basal and apical egion
2 hou s 6 ± 1.17 * apical egion
4 hou s 14.2 ± 1.31 *
a he ip o he illi,
basal egion and in he
c yp s
6 hou s 16.3 ± 1.05 *
a he ip o he illi,
basal egion and in he
c yp s
8 hou s 3.83 ± 0.79 * basal and apical egion
12 hou s 0.8 ± 0.61 basal and apical egion
24 hou s 0.83 ± 0.59 Basal and apical egion

Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011 - 191
E ec s o allopu inol and p econdi ioning on apop osis due o ischemia- epe usion on a double jejunum-segmen canine model
amoun o deb is seemed o be la ge .
Mic oscopical in es iga ions
H&E s aining demons a ed he cellula deb is on he
mucosal su ace in all IR, IPC and AP+IR g oups, howe e , he
his ological g ade did no di e be ween he g oups. Each g oup
p esen ed mucosal changes anging om comple ely no mal
apical illi o ex ension o he space wi h mode a e epi helial
li ing (Chiu G0-G2).
Immunohis ochemical in es iga ions
Table 2 shows he numbe o apop o ic cells in con ol
and ischemic jejunum-segmen s o IR, AP+IR and IPC g oups.
In each g oup he e was signi ican di e ence be ween
con ol and ischemic segmen (IR: p<0.001; IPC: p=0.008; AP+IR:
p=0.008). The highes apop o ic ac i i y was obse ed in AP+IR
g oup, p esen ig almos wo o ou - old di e ence compa ed o
he IPC o IR g oup, espec i ely (p<0.001). Apop osis was ound
o be mos in ensi e a he ip o he illi, howe e i was p esen
in he deepe laye s o he in es inal wall, oo.
ABlE 2 - The numbe o apop o ic cells in mucosal
illi o con ol and ischemic jejunum-segmen s o IR, AP+IR and
IPC g oups.
means ± S.D.
IR = ischemia- epe usion; IPC = ischemic p econdi ioning;
AP+IR = allopu inol p e- ea ed ischemia - epe usion
* p<0.05 s IR g oup, # s Con ol segmen
Discussion
The inju ies caused by he ischemia- epe usion
o igina e om many ac o s, such as eac i e oxygen in e medie s,
leukocy e adhesion molecules, ni ogen oxide10. The main sou ce
o he ee adicals in he in es inal issue is he xan hine-oxidase/
dehyd ogenase sys em and he ac i a ed polymo phonuclea
leukocy es. Du ing ischemia he xan hine-dehyd ogenase
con e s o xan hine-oxidase. This p ocess is e y as in in es inal
issue; wi hin one minu e o mesen e ic ischemia i ans o ms
comple ely10-12. In he hypoxic cell he xan hine-dehyd ogenase
enzyme - i s physiological elec onaccep o is NAD+ - u ns in o
oxida ed o m, o which he molecula oxygen is he elec on
accep o . A he same ime du ing anoxia he ATP molecule is
dephospho ila ing in he cell, hus he concen a ion o AMP is
inc easing. The e o e in he cells su e ing om hypoxia bo h
he xan hine-oxidase and one o i s subs a e a e p esen . Du ing
epe usion- eoxygena ion he o he subs a e, he molecula
oxygen appea s and he xan hine-oxidase s a s o educe he
molecula oxygen in o supe oxide adical and hyd ogenpe oxide12.
Bu ke e al.13 sugges ed ha oxida i e s ess induces apop osis,
and many o i s inhibi o s ha e an ioxidan ac i i ies o enhance
cellula an ioxidan de enses.
The in es ine con ains he highes quan i y o hese
enzymes in he body10. G ange e al.12 demons a ed ha he
xan hine-oxidase is p esen ed in he highes quan i y and ac i i y
in he mucous memb ane and in he apical egion o he illi,
he e o e he damage caused by he ee adicals a e mo e in ensi e
in his a ea. These indings may explain, why he his ological slides
showed so in ensi e damage in he mucous memb ane and why
he numbe o apop o ic cells a he 4 h and 6 h hou o epe usion
was so high.
I was supposed ha he xan hine-oxidase inhibi o
allopu inol may educe he jeopa dizing e ec s o ischemia-
epe usion14. In ou p e ious models we success ully used he
50 mg/kg dosage o allopu inol o diminish he hemo heological
changes (impai ing o ed blood cell de o mabili y) o hind limb
o enal ischemia- epe usion inju y15,16. In cu en s udy i was
also expec ed ha he numbe o apop o ic cells oge he wi h he
nec o ic ones dec eases13. In e es ingly, in his jejunum-segmen
model he allopu inol p e- ea ed g oup exp essed he highes
le el o apop o ic ac i i y, and hus, he a io be ween nec osis
and apop osis signi ican ly al e ed in his g oup. Since signi ican
oxida i e s ess is known o induce nec osis, i is supposed ha
he e ec o allopu inol migh di ec he dominan cell dea h ype
om nec osis o apop osis by dec easing he le el o eac i e
oxygen species, and a he inc easing he numbe o apop o ic
cells. Howe e , he e ec o allopu inol is dose-dependen and
supposedly he way o adminis a ion would ha e an in luence on
he esul s, oo17,18.
Du ing ischemia and epe usion bo h nec osis and
apop osis may appea depending on he du a ion and ex en o he
ischemia. While nec osis is a passi e p ocess, he p og ammed
Expe imen al
g oups con ol
segmen Ischemic
segmen
IR 0.16 ± 0.09 2.66 ± 0.82 #
IPC 0.14 ± 0.05 6.72 ± 0.46 # *
AP+IR 0.12 ± 0.04 10.72 ± 0.47 # *
192 - Ac a Ci ú gica B asilei a - Vol. 26 (3) 2011
B a h E e al.
cell dea h, he apop osis is ei he an ac i e, gene- egula ed
p ocess o ac i a ion induced. The mo phology o apop o ic
p ocess caused by ischemia- epe usion inju y in he in es ine was
i s ly published by Shah e al.8: single cell dele ion, ounded up
apop o ic cells, loose con ac wi h neighbou s and condensa ion
o sh inkage, compac ion o ch oma in o ming apop o ic bodies,
memb ane blebbing wi hou loss o in eg i y, in ac lysosomes,
phagocy osis by neighbou ing cells o mac ophages, and no
in lamma o y esponse occu ing.
Nico e a e al.19 conside ed he ATP le el as a
de e mina i e ac o in e e ence o he cells diminishing ei he in
apop o ic o nec o ic way. I he in acellula ATP le el was low he
signal s imula ing apop osis could cause nec osis. The in acellula
ene gy le el in luences wha ypes o cell-dea h would e en ua e.
The condi ions de e ming apop osis/nec osis a e he ollowings.
Apop osis: ATP sho age o 25-50%, inc eased le el o Ca2+ 200-
400 nM, mode a e ee- adical o ma ion, ac i a ion o caspase-
sys em is necessa y o he p ocess. Nec osis: ATP sho age o
70-100%, inc eased le el o Ca2+ >1μM, la ge amoun o ee-
adical o ma ion, Bcl-2 ac i a ion- ee adical p oduc ion, Bcl-2
inhibi ion, no ac i a ion o caspase-sys em a e equi ed/necessa y
o he p ocess20.
Biochemical e en s o apop osis induced by pa hological
o physiological s imuli a e igh ly egula ed p ocesses by syn he ic
and ac i a ion s eps, ecqui ing ene gy, mac omolecula syn hesis
and “de no o” gene ansc ip ion. One impo an cha ac e is ic o
apop osis appea s o be DNA agmen a ion20-22.
The DNA agmen s a e o med unde he in luence o
endonuclease ac i a ion, an enzyme deg ading he DNA a he
in e nucleosomal linke egions22.
The enzymes ha ca alyse his agmen a ion a e
usually non-lysosomal nuclea endonucleases. In ce ain cells
hey a e ac i a ed by Ca2+ and Mg2+ and inhibi ed by Zn2+. In
he mechanism o apop osis ac i a ion o issue ansglu aminase,
which appea s o p oduce c osslinking o p o eins has possible
ole o o m apop o ic bodies20.
In ou model he 30-minu e ischemia caused
mo phological changes and inju ies cha ac e is ically s a ed
a in es inal illi and sp eaded ou down he c yp s. The e was
minimal deb is s icked on he mucosal su ace om 30 minu es
ill 2 hou s o epe usion.
I is also well known ha apop osis is a p ocess which
demands ac i e p o ein syn hesis ha needs ime23. This could be
he eason why he numbe o apop o ic cells was low a he 30 h
minu e, 1s and 2nd hou o epe usion, and inc eased a e wa ds
peaking be ween he 4 h and 6 h hou s o he epe usion. The
numbe o apop o ic cells was low a he 8 h, 12 h and 24 h hou o
he epe usion. I is sugges ed ha he p ocedu e was inished,
hus u he in es iga ions o e hese pe iods o epe usion ime
a e no app op ia e.
This was he eason why in Expe imen II we choosed
a 4-hou epe usion pe iod o in es iga e he p o ec i e e ec o
allopu inol o ischemia p econdi ioning. Al hough bo h p ocedu e
showed simila cellula deb is on in es inal illi (Chiu G ade 0-2),
he allopu inol p e- ea men caused highe apop o ic ac i i y as
discussed abo e. Howe e , ischemic p econdi ioning esul ed in
lowe numbe o TUNEL-posi i e cells compa ed o allopu inol
p e- ea men g oup.
Ischemic p econdi ioning by exposing a issue o a b ie
pe iod o ischemia ollowed by epe usion allowes he abili y o
he issue o su i e he sus ained ischemia24-26. In expe imen al
models o mesen e ial ischemia, bowel-au o ansplan a ion o
jejunal laps ischemic p econdi ioning was shown o educe local
acidosis, neu ophil ec ui men as well as oxida i e s ess by
complex pa homechanisms26-29. Conce ning he e ec i e pe iods
o ime, Fe encz e al.27 ound ha he nuclea ac o κB- ela ed
adap i e mechanisms we e ini ia ed wi hin 1 hou o ischemic
p econdi ioning and peaking a 3 hou s in a canine model o small-
bowel au o ansplan a ion. This ime pe iod i well wi h ou
obse a ions.
Howe e , he signi icance o apop osis in in es inal
ischemia- epe usion s ill need u he s udies o cla i y he
balance o disbalance be ween apop o ic and nec o ic e en s, and
hei p e en ion o ea men .
conclusions
Expe imen I showed ha apop o ic ac i i y has a
cha ac e is ic ime cu e, eaching he highes alues be ween
he 4 h and 6 h hou s a e 30-minu e in es inal ischemia. Thus his
pe iod seems o be he mos sui able ime o in es iga ing he
p ocess o apop osis. Apop osis was ound o be he mos in ensi e
a he peak o in es inal illi. In Expe imen II he e ec o
adminis a ion o allopu inol was con adic o y, bu he ischemic
p econdi ioning (3 x 5 minu es) seems o be sui able p o ocoll
o diminish mo phological changes du ing ischemia- epe usion
inju y in his canine model.
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co espondence:
End e B a h M.D.
Depa men o Ope a i e Techniques and Su gical Resea ch
Ins i u e o Su ge y, Medical and Heal h Science Cen e
Uni e si y o Deb ecen
H-4012 Deb ecen, POB. 21 Hunga y
Phone: +36-52-416-915
[email p o ec ed]
Con lic o in e es : none
Financial sou ce: none
Recei ed: No embe 10, 2010
Re iew: Janua y 14, 2011
Accep ed: Feb ua y 15, 2011