Effect of Adenosine on Injury Caused by lschemia and Reperfusion in Rats: Functional and Morphologic Study

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Effect of Adenosine on Injury Caused by Ischemia and Reperfusion in

Rats: Functional and Morphologic Study

M.A. Haddad, R. Miranda-Ferreira, N.S.A. Taha, V.C. Maldonado, R.R. Daroz, M.O. Daud, J. Lima Neto,

D.A. Muniz, P.C.S. Silva, H.P. Monteiro, D.J. Fagundes, A. Caricati-Neto, and M.O. Taha

ABSTRACT

To study whether treatment with adenosine (ADO), an agonist of adenosine receptors,

attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rats

with ADO (15 mg/kg or saline solution (SS) intravenously before 60 minutes occlusion of the

superior mesenteric artery (I) and/or 120 minutes after its release (R). After I or I/R, isolated

jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions

stimulated by electrical pulses or KCI with the use of a digital recording system. Thin jejunal

slices were stained with hematoxylin and eosin for optical microscopy. Compared with the

sham group, jejunal contractions were reduced in I

⫹

SS and IR

⫹

SS but similar after treatment

with ADO (I

⫹

ADO and IR

⫹

ADO groups). We concluded that rat jejunal enteric nerves were

damaged in I

⫹

SS and IR

⫹

SS but not in the I

⫹

ADO and IR

⫹

ADO groups. These results

suggested that ADO attenuated intestinal dysfunction due to I and R.

I

ntestinal ischemia (I) and reperfusion (R) injuries con-tribute to the morbidity and mortality of critical illness.1

In the mammalian intestine, I results from local mechanical vascular obstructive or systemic factors, such as hypovolemia, hypotension, hypoxia and/or, sepsis.1_{Both situations can}

pro-duce cellular lesions and death due to oxygen and nutrient deprivation.1_{The blood circulation in R after I also}

contrib-utes to cellular lesions and death owing mainly to lipid peroxidation of cell membranes due to accumulation of free oxygen radicals and other cytotoxic substances.2, 3_{The cellular}

dysfunctions caused by I and R severely compromise motor and secretory functions of the intestines.1–3

Intestinal motility is regulated by several excitatory and inhibitory transmitters released from enteric nerves, includ-ing acetylcholine, neuropeptides, purines (adenosine triphosphate [ATP], nitric oxide (NO), and others.4 _The

actions of these transmitters are highly dependent on the integrity of enteric nerves.5 _{However, intestinal motor}

activity is reduced by IR owing mainly to a loss of enteric nerves structural and functional integrity.5,6_{Several drugs}

have been proposed to attenuate or prevent cellular dys-functions caused by I and R,7,8_{including those that}

inter-fere with adenosine receptors (AR).9 –13

In mammalians, the degradation of ATP produces aden-osine (ADO),9,10_{which is involved in the physiologic}

pro-cess inflammation and platelet aggregation.9,10_{The actions}

of ADO are mediated by 4 distinct subtypes of adenosine

receptors: A1, A2A, A2B, and A3.

9,10 _{Stressed or injured}

tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades, thereby decreasing acti-vation of platelets, leukocytes, and endothelial cells.9,10

In our previous study,14_{ADO improved the function of}

rabbit jejunae undergoing I, but not R. The morphologic study revealed that treatment with ADO after I and R was similar to sham group, suggesting cytoprotection by ADO. To better understand these affects we sought to investigate whether ADO attenuated or prevented intestinal IR injury, by analyzing its effects on the motility and histology of jejunal segments among rats undergoing IR.

MATERIALS AND METHODS

Male Wistar EPM-1 rats (270 –300 g) were anesthetized with ketamine (60 mg/kg) and xylazine (40 mg/kg) intravenously. There-after, they underwent occlusion of the superior mesenteric artery

From the Departments of Pharmacology, Biochemistry, Mor-phology, and Surgery, Federal University of São Paulo (UNIFESP), São Paulo, Brazil; and Medicine School, Federal University of Great Dourados (V.C.M., R.R.D., M.O.D., J.L.N., D.A.M.), Dourados, Brazil.

Supported by the São Paulo Research Foundation, Brazil. Address reprint requests to Murched Omar Taha, MD, PhD, Professor of Department of Surgery, Federal University of São Paulo, Escola Paulista de Medicina, Rua Botucatu, 740, CEP 04023-900, São Paulo—SP, Brazil. E-mail:taha@uol.com.br

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with a metabolic clip for 60 minutes (I) followed by blood recirculation, for 120 minutes (R) following its removal. Twelve rats underwent only I (I group) and 12 I and R (IR group), in addition to a sham group (n_⫽_6).

In the I group, 6 rats were treated with 0.9% saline solution (SS) and 6 with ADO (15 mg/kg) injected into the femoral vein 5 minutes before I. In the IR group, 6 rats were treated with SS and 6 with ADO (15 mg/kg) injected into the femoral vein 5 minutes before I, 5 minutes before R, and 55 minutes after R. After I or IR, the rats were killed; jejunal segments (2 cm) were isolated, washed, cleared of surrounding tissues, and mounted under 1 g tension at 37°C in an organ bath containing 10 mL of aerated nutrient solution of composition (in mmol/L): NaCl 138, KCl 5.7, CaCl21.8, NaH2PO40.36, NaHCO315, and dextrose 5.5

(pH 7.4). We studied neurogenic contractions induced by electrical field stimulation (EFS) or by the depolarizing agent KCl (70 mmol/L) using a digital recording system.6 – 8

EFS (5 and 30 Hz, 1

ms duration, 60 V) was performed by means of platinum electrodes connected to an electrical stimulator S88 (Grass, USA).6 – 8

Responses to EFS and KCl were recorded by force-displacement transducers connected via a bridge amplifier to an analog/digital recording system (AD Instruments, USA). Data on contractile responses were subjected to statistical analysis using 1-way analysis of variance and Studentt_test.7,8

We also performed histologic analyses of jejunal pieces embed-ded in paraffin, cut into thin slices, and stained with hematoxylin and eosin for optical microscopy.

RESULTS

Figure 1shows that EFS (5 and 30 Hz) or KCl (70 mmol/L)

produced contractile responses in all jejunal segments: sham (Fig 1A), I⫹SS (Fig 1C), I⫹ADO (Fig 1E), IR⫹SS

(Fig 1D), and IR⫹ADO (Fig 1F). However, the amplitude

Fig 1. Typical records of neu-rogenic contractions induced by electrical field stimulation (5 and 30 Hz) or KCl (70 mmol/L) in jejunal segments of rats treated with ADO or SS and submitted to I or I/R. *Statistically different than I⫹SS (P⬍.05;n⫽6); #Statisti-cally different than IR⫹SS (P⬍

.05;n⫽6). ADO, adenosine; SS, saline solution; I, ischemia; R, reperfusion. ns, not significant.

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of these contractions was similar in I⫹ADO and IR ⫹

ADO groups, but reduced in I⫹SS and IR⫹SS compared with the sham group (Fig 1B;Table 1).

Histologic analysis showed a loss of structural integrity of enteric nerves in the jejunal segments of IR⫹ SS group

(Fig 2B), but not in IR⫹ADO (Fig 2C).

DISCUSSION

Our results showed that the motility stimulated by trans-mitters released by and the amount of enteric nerves were significantly reduced in jejunal segments of rats having underwent intestinal I and R. However, these dysfunctions were reduced or absent in segment from hosts treated with ADO, suggesting that activation of ARs by ADO attenu-ated or prevented the motor and neural dysfunctions caused by intestinal I and R.

The deleterious effects of R on intestines exacerbate the I injury.1_{Molecular mechanisms involved in the IR injury}

are poorly understood, but they involve formation of free oxygen radicals and consequent oxidative stress alterations of calcium flux beyond activation of phospholipase A2.

9 –11

The stressed or injured tissues release endogenous ADO, which blocks potentially destructive inflammatory cascades and decreases platelet, leukocyte, and endothelial cell

acti-vation as well as responses mediated by A2A and A2B

receptors.9,10

Recently, Hart et al12 _{showed increased intestinal IR}

injury, compared with wild-type mice, among transgenic mice with selective knockout of A2B receptors, suggesting

their protective role in IR injury. They also showed that selective inhibition of A2B receptors in wild-type mice

increased intestinal inflammation and injury during IR. In addition, treatment with an agonist of A2Breceptors (BAY

60 – 6583) protected wild-type mice from intestinal injury, inflammation, and premeability dysfunction; in contrast, their therapeutic effects were abolished following targeted gene deletion of A2Breceptors.

Similarly, Haskó et al13_{showed that pre- and}

posttreat-ment with a selective agonist of A2Areceptors (CGS-21680)

protected lungs against lesions caused by I. ADO reduced the expression of adhesion molecules and the release of proinflammatory mediators, eg, reactive oxygen species, elastase, and tumor necrosis factor alpha.14_{Synthetic A}

2A

and A2Bselective agonists are currently undergoing

preclin-ical testing for the treatment of IR injury and other pathologic conditions, including allergen-induced inflam-mation, autoimmune diseases, and sepsis.14 _{It is possible}

that these mechanisms may be involved in the protective effects of ADO against cellular lesions caused by I and R, as observed in present study.

Some authors15–17 _{have demonstrated that many A} 2B

purinoceptors cause downstream signaling that ultimately prevents opening of mitochondrial permeability transition pores (MPTP).17_{When MPTP are allowed to open,}

elec-trochemical equilibrium between the cytoplasm and mito-chondrial matrix abolishes the critical electrochemical gra-dients, resulting in cessation of ATP production and cell necrosis. Therefore, when ADO activates A2Breceptors, a

series of signaling steps before ischemia lead to blockade of MPTP formation accounting for tissue cytoprotection.17,18

A previous study by our group show ADO to improve the functional response of rabbit jejunae after I but not after R. The results suggested that the lesions caused by R were

Table 1. Values of Amplitude of Neurogenic Contractions (Expressed in Grams of Tension) Induced by EFS (5 and 30 Hz) or KCl (70 mmol/L) in Jejunae of Rats Treated with ADO or SS

and Submitted to Intestinal I or IR

Group 5 Hz 30 Hz KCl

Sham 1.95⫾0.19 2.15⫾0.19 2.25⫾0.18 I⫹SS 0.19⫾0.04a _0.29

⫾0.05a _0.41 ⫾0.10a I⫹ADO 0.25⫾0.04ns _0.48

⫾0.05b _0.67 ⫾0.05b I/R⫹SS 0.18⫾0.06 0.45⫾0.10 0.61⫾0.17 I/R⫹ADO 0.61⫾0.08c _0.97

⫾0.09c _1.16 ⫾0.12c

Data corresponding to mean⫾SEM (n⫽6).

a_{Statistically different from Sham (}

P⬍.05).

b_{Statistically different from I}

⫹SS (P⬍.05).

c_{Statistically different from IR}

⫹SS (P⬍.05).

Fig 2. Histologic aspects of jejunae of sham rat (A) (without ischemia and/or reperfusion) and rats treated with saline solution (B) of adenosine (C) and submitted to intestinal ischemia and/or reperfusion. The images show the longitudinal muscle (ME), circular muscle (MI), and enteric nerves (arrows). (Hematoxylin and eosin method,⫻400).

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more pronounced that those caused by I, perhaps because of the long time that blood was stopped in the vessel during this process.19_{In that work, we did not observe functional}

or morphologic improvement after I, in contrast with the present work. This difference may have been due to the rabbits used in the previous study more sensitive to the effects of ischemia than rats.20

In conclusion, treatment with ADO attenuated small bowel motor, and neural dysfunctions caused by I and R in rats.

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