Melatonin reduces apoptosis and necrosis induced by ischemia/reperfusion injury of the pancreas

Introduction

The obstruction of blood flow or ischemia is relativelyfrequent in numerous clinical interventions and physio-

pathological processes. Although the recovery of bloodflow is normally followed by the restoration of tissuefunction, the reperfusion process may induce a significant

cell damage [1, 2]. The generation of highly reactive oxygenspecies mostly determine tissue injury during ischemia/reperfusion (IR) syndrome [3, 4]. In particular, the pancreas

is subjected to IR injury during tissue transplantation andother pathological situations characterized by a reductionof abdominal blood flow such as post-traumatic hemor-

rhage, septic syndromes, cardiopathies, vascular dysfunc-tion, embolic-thrombosis processes, and all surgicalinterventions in which a regional arterial bypass or vascularclamping is required.

Different experimental studies have shown that the induc-tion of oxidative stress is the underlying mechanism of IRinjury in pancreas [5–7]. Sanfey et al. [8] showed that the

treatment with enzymatic antioxidants [superoxide dismu-tase (SOD) and catalase (CAT)] reduces the histological signsof tissue damage (glandular edema) induced by IR in

pancreas. The regulation of the extracellular concentration

of nitric oxide [9] or free radicals [10] also reduces pancreatitisrelated to IR. In addition, other experimental studies haverelated the impairment of microcirculation with the severity

of acute pancreatitis induced by IR and toxin [11–15].Fujimoto et al. [16] showed that the induction of pancreatitisby IR is accompanied by the presence of apoptoticmarkers in

the acini.Melatonin (N-acetyl-5-methoxytryptamine), a lipophilic

indole amine derived from tryptophan. Melatonin is apotent antioxidant, well tolerate and without toxicity upon

its administration. Melatonin has a powerful capacity toscavenge free radicals and prevents tissue damage [17–19].In particular, melatonin prevents oxidative stress induced

by I/R in liver [20–22], brain [23, 24], myocardium [25],intestine [26, 27] and kidney [28]. Although, differentstudies described above have attempted to control oxidative

stress during IR-induced pancreatitis [8–10], we could notfind a study describing the cytoprotective effect of melato-nin against oxidative stress, cell death and tissue dysfunc-

tion in pancreas induced by IR.The aim of the study was to evaluate the cytoprotective

properties after the preventive or therapeutic administra-tion of melatonin against cell injury and dysfunction

induced by IR in pancreas.

Abstract: The pancreas is highly susceptible to the oxidative stress induced

by ischemia/reperfusion (IR) injury leading to the generation of acute

pancreatitis. Melatonin has been shown to be useful in the prevention of the

damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney.

The aim of the study was to evaluate the cytoprotective properties of

melatonin against injury induced by IR in pancreas. The obstruction of

gastro-duodenal and inferior splenic arteries induced pancreatic IR in male

Wistar rats. Melatonin was intraperitoneally administered before or/and

after IR injury. The animals were killed at 24 and 48 hr after reperfusion and

there were evaluated parameters of oxidative stress (lipoperoxides,

superoxide dismutase, catalase, glutathione peroxidase and reduced

glutathione), glandular endocrine and exocrine function (lipase, amylase,

insulin) and cell injury (apoptosis and necrosis). The IR induced a marked

enhancement of oxidative stress and impaired pancreatic function. The

histological analysis showed that IR induced acute pancreatitis with the

accumulation of inflammatory infiltrate, disruption of tissue structure, cell

necrosis and hemorrhage. Melatonin administration before or after

pancreatic IR prevented all tissue markers of oxidative stress, biochemical

and histological signs of apoptosis and necrosis, and restored glandular

function. No histological signs of pancreatitis were observed 48 hr after

reperfusion in 80% of the animals treated with melatonin, with only a mild

edematous pancreatitis being observed in the remaining rats. Preventive or

therapeutic administration of melatonin protected against the induction of

oxidative stress and tissue injury, and restored cell function in experimental

pancreatic IR in rats.

Francisco C. Munoz-Casares1,Francisco J. Padillo1, JavierBriceno1, Juan A. Collado2,Juan R. Munoz-Castaneda2,Rosa Ortega3, Adolfo Cruz1,Isaac Tunez4, Pedro Montilla4,Carlos Pera1 and Jordi Muntane2

1Department of General Surgery, 2Research

Unit and 3Department of Pathology, Reina

Sofia University Hospital; 4Department of

Biochemistry and Molecular Biology, School of

Medicine, Cordoba, Spain

Key words: cell death, ischemia-reperfusion,

melatonin, oxidative stress, pancreas

Address reprint requests to Francisco J.

Padillo, Departamento de Cirugıa, Hospital

Universitario Reina Sofıa, Avda. Menendez

Pidal s/n 14004 Cordoba, Espana.

E-mail: med026014@saludalia.com

Received August 25, 2005;

accepted September 28, 2005.

J. Pineal Res. 2006; 40:195–203Doi:10.1111/j.1600-079X.2005.00291.x

� 2006 The AuthorsJournal compilation � 2006 Blackwell Munksgaard

Journal of Pineal Research

195

Methods

Animals

Male Wistar rat (200–350 g) received laboratory food

(Ebro Agrıcolas S.A., Sevilla, Spain) and water ad libitum.The animal room was controlled at constant temperature(22 ± 2�C), humidity and light cycle (08:00–20:00 hr).

Animal care and experimental protocols were in accordancewith the Guide for the care and use of laboratory animalsprepared by the National Academy of Sciences and

published by the National Institute of Health (NIHpublication no. 80-83, revised 1985).

Experimental procedures

Animals (n ¼ 65) were distributed in seven experimentalgroups: (i) control, (ii) sham operated (SO), (iii) sham

operated plus melatonin (SO-MEL), (iv) ischemia/reper-fusion (IR), (v) melatonin administration before IR (MEL-IR), (vi) melatonin administration after IR (IR-MEL), and

(vii) Melatonin administration before and after IR (MEL-IR-MEL). Control animals were not subjected to anysurgical intervention. SO animals were submitted to lapar-

otomy but without blood flow obstruction. IR animals weresubjected to obstruction of gastro-duodenal and inferiorsplenic arteries for 1 hr and subsequent reperfusion.MEL-IR rats received melatonin 30 min before arterial

obstruction. IR-MEL animals received melatonin duringreperfusion. MEL-IR-MEL animals received melatoninboth before and after arterial obstruction. All groups

excluding control animals were killed at 24 hr (five animals)and 48 hr (five animals). There were no differences betweencontrol, SO and SO-MEL groups in relation to all

parameters studied. Thus, only the data from the SO ratsare provided herein.All surgical procedure were carried with the animals

placed on a thermoregulated surface (Harvard Apparatus�

Ltd., Edenbridge, Kent, UK) specially designed for thepurpose and coupled to cold light sources (EuromexIlluminator EK-1; Euromex Microscopes Ltd., Aruhem,

the Netherlands). The animals were anesthetized with asolution including ketamine (Ketolar� 50 mg/mL, 80–85 mg/kg), diazepam (Valium� 10 mg/2 mL, 3–4 mg/kg)

and atropine (Atropina� 1 mg/mL, 0.05–0.06 mg/kg). IRwas carried out by the obstruction of gastro-duodenal andinferior splenic arteries for 1 hr using vascular clamps. The

obstruction induces a marked ischemia in two-thirds of thepancreas while one-third of the pancreas perfused by thesuperior splenic arteries continued to receive blood. Thereperfusion of the pancreas was initiated when the vascular

clamps were removed. After surgical intervention, theabdominal wall was sutured using absorbable material(Polyglactin 910, Vicryl� Ethicon, Johnson and Johnson,

Norderstedt, Germany) and the external skin with nonab-sorbable material (Seda, Mersilk�, Ethicon, Johnson andJohnson). Melatonin (Melatonin; Sigma Co, St Louis, MO,

USA) was administered intraperitoneally at a dose of10 mg/kg using 10% ethanol solution (8 mg/mL). Animalswere killed under anesthesia at 24 or 48 hr after experi-mental intervention removing blood samples and pancreas.

The pancreas was used for histological analysis, and for the

measurement of antioxidant status, lipoperoxides andapoptotic markers. The blood-derived serum was frozenand further used for the measurement of glucose, insulin,

amylase and lipase.

Measurement of lipid peroxidation and antioxidantstatus

The presence of lipid peroxides (malondialdehyde +4-hydroxynonenal) in tissue was determined using a com-

mercial assay (LPO-586; Byoxitech, OXIS International,Portland, OR, USA). The pancreas was homogenized in20 mm Tris-HCl pH 7.4 and centrifuged at 10,000 g during

15 min at 4�C. The assay is based on the reaction of achromogenic reagent, N-methyl-2phenylindole, with lipop-eroxides at 45�C. The results were expressed in relation tothe protein content.

The measurement of reduced glutathione (GSH) wasdetermined using a commercial assay GSH-400 (ByoxitechS.A). Briefly, all mercaptans present in the sample react

with 4-chloro-1-methyl-7-trifluromethyl-quinolinium meth-ylsulfate in a first chemical reaction. The second stepconsisted of the b-elimination reaction under alkaline

conditions of the product from the first reaction into achromogenic thione-derived product.Glutathione peroxidase (GSH-Px) was measured accord-

ing to the method described by Flohe and Gunzler [29].CAT was determined following the method described byAebi [30]. SOD was assessed according to the methodpublished by Sun et al. [31].

Serological parameters measuring the function ofpancreas

The function of the pancreas was assessed by the measure-ment of serological concentration of glucose, insulin, lipase

and amylase. The concentration of glucose and amylasewas determined using an autoanalyzer Cobas Integra 400(Roche� Diagnostics, Indianapolis, IN, USA). The pan-creatic lipase was quantified using a commercial colorimet-

ric enzymatic test (LIPASE EE500; Linear Chemicals S.L.,BBI Diagnostics, West Bridgewater, MA, USA). Theinsulin was measured by radioimmunoassay using a com-

mercial assay (INSULIN-CT; CIS Bio International, OrisGroup, Gif/Yvette, France).

Measurement of apoptosis in pancreas

The evaluation of DNA fragmentation and caspase-3-

associated activity was used as index of apoptosis inpancreas [32, 33]. DNA fragmentation was assessed inpancreas (20 mg) homogenized (Ultra-turrax T25; Jankeand Kunkel IKA Laboratory, Stafen, Germany) in lysis

solution [1% SDS, 10 mm Tris-HCl, 50 mm ethylenediam-inetetraacetic acid (EDTA)], pH 7,4. Samples were incuba-ted with RNAse (1 mg/mL) (Sigma) for 2 hr at 37�C and

proteinase K (1 mg/mL) (Sigma) for 45 min at 48�C. DNAwas obtained by phenol:choloform:isoamyl alcohol 25:24:1(Sigma) extraction and precipitated with isopropanol (v/v)

and 0.5 m NaCl for 12 hr at )20�C. DNA was recovered bycentrifugation at 15,000 g for 20 min, and the precipitated

Munoz-Casares et al.

196

was washed with 70% ethanol, dried, and re-suspended inTris-EDTA buffer (10 mm Tris, 50 mm EDTA), pH 7.4.Samples (250 lg DNA) were applied and analyzed on 2%

agarose gel with ethidium bromide (0.5 lg/mL). In order tocarry out a statistical analysis of the results, it wasdetermined the absorbance of each band of the ladderobserved in agarose gel.

The evaluation of caspase-3-associated activity wasassessed in pancreas (20 mg) homogenized (Ultra-turraxT25) in lyses solution (50 mm Tris-HCl pH 7.5, 2 mm

EDTA, 100 mm NaCl, 1% nonidet NP-40, 1 mm phenyl-methylsulfonyl fluoride, 20 lg/mL aprotinin, 20 lg/mLleupeptin and 20 lg/mL pepstatin A) at 4�C for 10 min,

transferred to Eppendorf tubes and centrifuged at 20,800 gat 4�C for 5 min. The caspase-3-like activity in the cellextract (25 lg) was measured by colorimetric assay usingthe peptide-based substrate ac-N-acetyl-Asp-Glu-Val-Asp-

p-nitoanilide (AcDEVD-pNA) (Bachem AG, Bubendorf,Switzerland). The increase in absorbance of enzymaticallyreleased pNA was measured at 405 nm for 10 min in a

DU� 640 Spectrophotometer (Beckman Coulter, Inc.,Fullerton, CA, USA).

Histological examination

The injury of the pancreas was evaluated by histological

examination of tissue sections (5 lm) fixed with 4%paraformaldehyde solution pH 7.0 and embedded inparaffin. After removing paraffin with xylol, the sectionswere stained with hematoxylin-eosin or periodide-acid

Schiff [34]. The histological observation was carried outusing an optical microscope (Nikon�. Biophot, Tokyo,Japan) by different independent anatomopathologist. The

expert graded the severity of the injury based on theprevious experimental studies [10, 11, 35] (Table 1).

Statistical analysis

All results were expressed as mean ± standard error. Ifdata followed a normal distribution, the differences

between groups were assessed by the one-way analysis ofvariance (ANOVA) using the multiple comparison Tukeytest for independent samples. In order to compare paired

samples using the time of killing after IR as a factor, datawere submitted to multiple mean ANOVA using DMS testas post hoc analysis. When normal distribution was not

observed, data was submitted to post hoc Kruskal–Wallisand Mann–Whitney tests for independent samples, andpost hoc Friedman and Wilcoxon tests for paired samples.

For the measurement of the percentage of histologicalinjury between samples a chi-squared test (v2) was used foranalysis in a data matrix structure. The v2 analysis with

Yates correction was used in the case of matrix 2 · 2. Whenthe expected frequency was lower than 5 Fisher test wasapplied.The statistical significance was set at P < 0.05 and

indicated using superscript symbols in the figures.

Results

Different parameters related to lipoperoxidation and anti-oxidants (GSH, SOD, CAT and GSH-Px) have been

assessed in pancreas from IR rats treated or not treatedwith melatonin (Fig. 1). IR increased significantly thegeneration of lipoperoxides in pancreas in comparison withthe SO group (P < 0.001). This exacerbation of tissue

oxidative injury was correlated with a depletion ofantioxidant status (GSH, SOD, CAT and GSH-Px) inpancreas. All groups treated with melatonin showed a

marked reduction of lipoperoxides and a recovery ofantioxidant values of the IR animals.Ischemia/reperfusion induced a significant reduction of

insulin concentration in serum at 24 and 48 hr after injury(P < 0.001) (Fig. 2A). Nevertheless, this effect was notaccompanied by any changes on glucose concentration

between SO and IR groups (144 ± 11.2 versus 178 ± 54.9at 24 hr and 210 ± 49.7 versus 189 ± 45.0 at 48 hr,respectively). The administration of melatonin restoredthe level of insulin in IR animals reaching the control values

at 48 hr in MEL-IR-MEL animals (Fig. 2A).The level of amylase and lipase in serum increased

rapidly in rats subjected to IR compared with SO group at

24 hr (P ¼ 0.032 and P < 0.001, respectively) and 48 hr(P ¼ 0.009 and P < 0.001, respectively) (Fig. 2B,C). Mela-tonin administration reduced significantly lipase in all IR

group at 24 and 48 hr (P < 0.001) (Fig. 2C). Nevertheless,melatonin was only able to diminish significantly theconcentration of amylase at 48 hr in IR rats treated withmelatonin (P < 0.01) (Fig. 2B).

The fragmentation of DNA and caspase-3 activity wereused as a markers of apoptosis in pancreas (Figs 3 and 4).A relevant increase on DNA fragmentation in pancreas was

only observed 24 hr after IR (Fig. 3A). This observationwas confirmed with the measurement of absorbance ofethidium bromide band in agarose gel (Fig. 3B). Caspase-3

activity also raised 24 hr after IR in pancreas homogenate(Fig. 4). Melatonin prevented the increase of DNA frag-mentation and caspase-3 activity in pancreas from

IR-subjected rats (Figs 3 and 4).

Table 1. Degree of histological injuryaccording to the presence of edema,inflammatory infiltrates necrosis andhemorrhage

Tissue injury EdemaInflammatoryinfiltrates Necrosis Hemorrhage

LEVEL 0No or mild pancreatitis No/Minimum No/Minimum No No

LEVEL 1Moderate pancreatitis Relevant Relevant No No

LEVEL 2Acute pancreatitis Intense Intense Yes Yes

Melatonin prevents injury to pancreas

197

The histological analysis showed that the acini were wellconserved, defined and without edema or inflammatoryinfiltrate in pancreas from SO rats (Fig. 5A–D). Weobserved that 24 hr IR induced acute edematous pancre-

atitis, marked interacinar edema, vascular congestion and

an interstitial inflammatory infiltrate (Fig. 5B). At 48 hr,the majority of pancreas (80%) showed an exacerbation ofacute pancreatitis with an important accumulation ofinflammatory infiltrates in the injured area that included

an extensive cell necrosis with disruption of the acini and

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µmol/mg

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***

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***

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**

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Fig. 1. Effect of melatonin on lipoperox-ides, reduced glutathione (GSH), super-oxide dismutase (SOD), catalase (CAT)and glutathione peroxidase (GSH-Px)levels in pancreas subjected to ischemia/reperfusion (IR). All groups were com-pared statistically with the IR group.*P < 0.05, **P < 0.01, ***P < 0.001.

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***

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6000

5000

4000

3000

2000

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***

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***

***

**

Fig. 2. Effect of melatonin on insulin,amylase and lipase serum concentrationafter the induction of ischemia/reper-fusion (IR) in pancreas. All groups werecompared statistically with the IR group.*P < 0.05, **P < 0.01, ***P < 0.001.

Munoz-Casares et al.

198

lobule structures, as well as the presence of variablehemorrhage (Fig. 5C).The comparative histological analysis showed that

although the administration of a single dose of melatoninbefore or after IR did not prevent the induction of acuteedematous pancreatitis at 24 hr IR, the antioxidant wasable to prevent any further evolution to grave acute

pancreatitis at 48 hr (Fig. 5D). In addition, a remarkablecytoprotective effect of melatonin was observed in MEL-IR-MEL animals. In this group, a normal histological or

minimum interacinar edema (60%) and acute edematouspancreatitis (40%) were observed at 24 hr after IR.Melatonin cytoprotection was even more pronounced at

48 hr after IR when 80% of pancreas showed normalhistology (Fig. 6).

Discussion

During the last decade, melatonin has been intensivelyinvestigated for its potent antioxidant properties, broad

absorption and low side effects. These properties suggestthat melatonin may be useful for treating all oxidativestress-related dysfunctions including IR syndrome [36,

37]. Numerous recent evidence suggests that the genera-tion of free radicals mediates the injury in differenttissues, such as pancreas, induced during IR syndrome

[38–40]. Nevertheless, in spite of the antioxidant proper-ties demonstrated by melatonin, the studies involving itsuse for the treatment of oxidative stress-induced damagein pancreas is limited [41–44]. In this sense, only the

study conducted by Jaworek et al. [45] has shown thatthe administration of melatonin reduces lipid peroxida-tion and pancreatic edema, and improves interleukin-10,

tumor necrosis factor-a and amylase concentration inserum during IR. We found no study that correlates theinduction of oxidative stress, lipid peroxidation, cell

death, histological disturbances and cell dysfunctioninduced by IR in pancreas. In addition, the study ofthe protective effects of melatonin against IR has aremarkable therapeutic interest for the treatment of

patients suffering from blood flow obstruction. Thepancreas is subjected to IR injury during tissue trans-plantation and other numerous pathological situations

characterized by a reduction in abdominal blood flowsuch as post-traumatic hemorrhage, septic syndromes,cardiopathies, vascular dysfunction, embolic-thrombosis

processes, and all surgical interventions in which regionalarterial bypass or vascular clamp are required.In the present study, IR induced an intense oxidative

stress characterized by an increase of lipid peroxides anddepletion of enzymatic (CAT, SOD and GSH-Px) and non-enzymatic (GSH) antioxidants in pancreas. The treatmentof melatonin exerted a powerful antioxidant effect with a

reduction of free radical-derived products,MDA + 4HNE, and recovery of antioxidant status inpancreas subjected to IR. Melatonin restored CAT and

GSH-Px activities in pancreas to levels similar to thosefound in the SO group. Although, the administration ofmelatonin before IR exerted the highest cytoprotective

effect against injured pancreas, the therapeutic administra-tion also exerted a remarkable positive effect on tissue

A

B

SO

IR

MEL-IR

MEL-IR-MEL

Arbitraryunits

Base pairs

1230

1033

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298234

Groups 1 2 3 4 5 6 7 8 9 10 11 12 13

1400

1200

1000

800

600

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200

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***

48 hr

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*** *

**

**

Fig. 3. Effect of melatonin on DNA fragmentation in pancreasfrom animals subjected to ischemia/reperfusion (IR). (A) Repre-sentative image of DNA fragmentation in agarose gel. Groups are:(1) Control, (2) sham operated (SO) (24 hr), (3) sham operated plusmelatonin (SO-MEL) (24 hr) (4) SO (48 hr), (5) SO-MEL (48 hr),(6) ischemia/reperfusion (IR) (24 hr), (7) melatonin administrationbefore IR (MEL-IR) (24 hr), (8) melatonin administration after IR(IR-MEL) (24 hr), (9) MEL-IR-MEL (24 hr), (10) IR (48 hr), (11)MEL-IR (48 hr), (12) IR-MEL (48 hr), (13) MEL-IR-MEL(48 hr). In (B) statistical analysis of the absorbance of each band ofthe ladder corresponding to DNA fragmentation from all groupscompared with IR animals. *P < 0.05, **P < 0.01,***P < 0.001.

SO

IR

MEL-IR

MEL-IR-MEL

Abs/(h × mg protein)

4

3.5

3

2.5

2

1.5

1

0.5

024 hr 48 hr

**

IR-MEL

**

**

***

Fig. 4. Effect of melatonin on the caspase-3 activity in pancreasfrom animals subjected to ischemia/reperfusion (IR). All groupswere compared statistically with the IR group. *P < 0.05,**P < 0.01, ***P < 0.001.

Melatonin prevents injury to pancreas

199

oxidative stress and cell dysfunction in pancreas induced byIR.As observed by others [10], our study also showed that

IR syndrome in pancreas reduced insulin concentration in

blood. The administration of melatonin before or after IRimproved insulin concentration in serum. Double admin-

istration of melatonin fully recovered basal insulin con-centration 48 hr after IR. In our study, we could notobserve any correlation between insulin and glucoseconcentration in plasma. It is possible that the ischemia

for 1 hr was not sufficient to alter glucose concentration inserum.

A B

C

E

D

Fig. 5. (A) Histological analysis of pancreas from control rats. Pancreatic acini are well conserved, defined, without edema or inflammatoryinfiltrate. A normal islet is present. (B) Photomicrography of a pancreas subjected to ischemia/reperfusion injury for 24 hr. Acute edematouspancreatitis characterized by an inflammatory infiltrate and interacinar edema are apparent. The acini structure is well conserved withoutnecrosis or hemorrhage. (C) Histological appearance of a pancreas subjected to ischemia/reperfusion injury for 48 hr. Acute necroticpancreatitis with hemorrhage characterized by the presence of extensive inflammatory infiltrate with parenchymal necrosis, as well asnecrosis of adipocytes (D) in lobules and acini are apparent. (E) Photomicrography of a pancreas subjected to ischemia/reperfusion injury(48 hr) with administration of melatonin before and after ischemia. The section shows normal histology similar to that observed in controlanimals. All sections are stained with hematoxylin-eosin. Magnification 40·.

Munoz-Casares et al.

200

In concordance with others [7, 16], we have shown asignificant rise in amylase and lipase concentration in serumafter IR. Melatonin abolished the increase of both enzymesafter IR especially when the antioxidant was administered

before ischemia.Our study also elucidated the relationship between the

cytoprotective effect of melatonin against oxidative stress

and the recovery of glandular dysfunction and histologicaldamage induced by IR in the pancreas. Herein, weinvestigated the association between the alteration of

endocrine and exocrine pancreatic function and the pres-ence of cell death markers, such as apoptosis and necrosis,in pancreas after IR treated or untreated with melatonin.

Fujimoto et al. [16] detected apoptosis in pancreatic acini24 hr after IR injury. We have observed that IR increasedDNA fragmentation and caspase-3 activity in pancreas at24 hr. Melatonin prevents free radical induced apoptosis in

rat brain astrocytes [46]. The administration of melatoninbefore or after IR was able to prevent apoptosis inpancreas. We did not observe any signs of apoptosis at

48 hr after IR. Sandoval et al. [47] have suggested that theaccumulation of inflammatory infiltrate in the pancreas atthe last stage of IR may shift cell death from apoptosis to

necrosis. In agreement with these studies [16, 47], thereduction of apoptosis should be associated with anincrease of pancreatic cell necrosis. In this sense, a detailedhistological analysis of pancreas submitted to IR showed

the presence of acute pancreatitis. Several authors havenoted an increase of edema, accumulation of inflammatoryinfiltrate in lobules and acini, and the presence of cell

necrosis and hemorrhage in relation to the intensity of theinsult [16, 39, 48]. Our study has demonstrated the

presence of acute pancreatitis and the development ofedematous pancreatitis in all samples at 24 hr IR tonecrotic-hemorrhage pancreatitis in the 80% of organs at48 hr. This exacerbation of the tissue damage may explain

the lack of apoptotic markers at 48 hr after IR. Weconsider relevant the observation that melatonin, althoughthe presence of acute edematous pancreatitis in all samples

was maintained at 24 hr, prevented the induction ofnecrotic-hemorrhage pancreatitis at 48 hr after IR. Inaddition, the administration of melatonin before and after

IR reduced the number of samples evaluated at 24 hr withacute edematous pancreatitis to 40% while the remainingpancreas (60%) showed normal histology. At 48 hr, no

case of necrotic-hemorrhage pancreatitis was apparent,and 80% of the samples showed normal histology. Overall,this data clearly show that melatonin reduced all bio-chemical and histological markers of apoptosis and

necrosis induced by IR in pancreas.In conclusion, the administration of melatonin before or

after IR reduced oxidative stress, antioxidant depletion, cell

death and restored the pancreatic histological structure andfunction disrupted by the insult. Our study documents thatmelatonin administration may also have a positive impact

on surgical situations in which an ischemic process hasalready occurred and the restoration of blood flow is stillpossible.

Acknowledgments

This study was supported by the Programa de Promocion

de la Investigacion en Salud del Ministerio de Sanidad yConsumo (FIS 02/0155, RTIC C03/03, G03/156).

MEL-IR

IR-MEL MEL-IR-MEL

100% *

*

**

80

60

40

20

024 hr 48 hr

IR

100%

80

60

40

20

024 hr 48 hr

100%

80

60

40

20

0

24 hr 48 hr

100%

80

60

40

20

0

24 hr

No pancreatitis

Edematous pancreatitis

Necrotic pancreatitis

48 hrFig. 6. Comparative figures in relation tothe presence of edematous or necroticpancreatitis in rats subjected to ischemia/reperfusion (IR). All groups were com-pared statistically with the IR group.*P < 0.05, **P < 0.01.

Melatonin prevents injury to pancreas

201

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