Effect of Ischemic Preconditioning on Injuries Caused by Ischemia
and Reperfusion in Rat Intestine
M.O. Taha, R. Miranda-Ferreira, A.C.R. Chang, A.M. Rodrigues, I.S. Fonseca, L.B. Toral, M.R. Cardoso,
M.J. Simões, I.S. Oliveira-Junior, H.P. Monteiro, D.J. Fagundes, N.S.A. Taha, and A. Caricati-Neto
ABSTRACT
To study whether ischemic preconditioning (IPC) attenuated intestinal dysfunction caused
by ischemia (I) and reperfusion (R), rats were underwent 60 minutes of I which was
produced by occlusion of the superior mesenteric artery, and/or 120 minutes R. The IPC
group had the I procedure previously stimulated for 5 minutes and the R for 10 minutes.
IPC and sham groups were injected with saline solution (SS) via the femoral vein 5 minutes
before the I and R, and for R. After I or I/R, 2-cm jejunal segments were mounted in an
organ bath to study neurogenic contractions stimulated by electrical pulses or KCl using a
digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for
optical microscopy. Compared with the sham group, jejunal contractions were similar in
the IPC
⫹
I and the IPC
⫹
I/R groups, but reduced in the I
⫹
SS and the I/R
⫹
SS groups.
The jejunal enteric nerves were damaged in the I
⫹
SS and the I/R
⫹
SS groups, but not
in the IPC groups. These results suggested that ischemic preconditioning attenuated
intestinal dysfunction caused by I and I/R.
I
NTESTINAL ISCHEMIA (I) and reperfusion (R) in-juries occur in a variety of surgical practices, such as transplantation procedures and critical illnesses, such as neonatal necrotizing enterocolitis.1 Among the internalorgans the intestine is probably the most sensitive to the I/R syndrome.1,2I and R injuries can lead to
hypovole-mia, hypotension, hypoxia, and sepsis,1–3 dramatically
compromising intestinal motor and secretory functions due to cellular lesions caused by deprivation of oxygen and nutrients.1,2In addition, vessels that suffer R after I
can also suffer cellular lesions and death mainly due to lipid peroxidation of cell membranes caused by accumu-lation of free oxygen radicals and other cytotoxic sub-stances.2,3 In response to I, cells undergo changes in
enzyme activities, mitochondrial functions, cytoskeletal structures, membrane transport, and antioxidant de-fenses, which collectively predispose to reoxygenation injury.2,3The morphological and functional injuries after
I and R remain a major challenge associated with high rates of morbidity and mortality both in surgical and trauma patients.2,3
The motor functions of the mammalian intestines are regulated by several excitatory and inhibitory transmit-ters, which are released from enteric nerves, including acetylcholine, 5=-adenosine triphosphate (ATP), nitric
oxide, neuropeptides, and other substances.4The actions
of these transmitters are highly dependent on the integ-rity of the enteric nerves.5 However, intestinal motor
activity is severely compromised in I/R mainly due to the loss of structural and functional integrity of the enteric nerves.5,6
Recent studies have suggested ischemic precondition-ing (IPC) to be a potential therapeutic strategy to improve the tolerance of the small intestine to ischemic and preservation insults. IPC, which was first described by Murry et al,20 refers to a phenomenon in which a
tissue is rendered resistant to the deleterious effects of prolonged I and R by prior brief periods of vascular
From the Departments of Pharmacology, Biochemistry, Mor-phology and Surgery of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
A.C.R.C., A.M.R., I.S.F., L.B.T., and M.R.C. are graduate students of Medicine School of Federal University of Great Dourados (UFGD), Dourados, Brazil.
Supported by São Paulo Research Foundation (FAPESP), Brazil.
Address reprint request 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:[email protected]
0041-1345/12/$–see front matter © 2012 by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.transproceed.2012.07.056 360 Park Avenue South, New York, NY 10010-1710
occlusion.13 However, both in vivo and in vitro models
have shown the cycle of preconditioning to be critical to inducte tolerance to ischemia. Hypoxic periods shorter than 5 minutes or exceeding 15 minutes failed to induce protection.21 Similarly, R periods shorter than 5 or
exceeding 15 minutes had no protective effect.21
Al-though most reports in the literature have used 10 minutes of IPC in rat small bowel I/R injury, the ideal IPC protocol, including the duration of I, is not yet established.22
Wang et al demonstrated that the protective effects of IPC on small intestinal grafts correlated with inhibition of epithelial cell apoptosis.14Other studies have shown
several mediators to play crucial roles in this protective phenomenon, including adenosine,15nitric oxide (NO),16
oxidative stress,17 heme-oxygenase-118 and protein
ki-nase C (PKC).19 Since the molecular mechanisms and
ideal protocol involved in IPC are still not fully known, we sought to study the effects of IPC on the motility and histology of rat jejunal segments undergoing intestinal I and R in rats.
MATERIALS AND METHODS
Male Wistar EPM-1 Rats (270 –300 g) were anesthetized with ketamine (60 mg · kg⫺1) and xylazine (40 mg · kg⫺1) intrave-nously prior to occlusion of the superior mesenteric artery using a metallic clip for 60 minutes (I) followed by removal of the clip with recirculation (R) for 120 minutes. Twelve rats underwent only I (I group) and 12, I and R (I/R group) in addition to a sham group (n⫽6).
Among the I group, six rats were treated only with saline solution 0.9% (SS) (I ⫹ SS) via the femoral vein and six
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 IPC or SS and submitted to I or I/R. *Statistically different than I⫹SS (P⬍.05;n⫽6);
#statistically different than I/R
⫹
underwent IPC (IPC ⫹I) for 5 minutes before I. In the I/R group, six rats were treated with SS and six underwent the IPC (IPC⫹I/R) 10 minutes before R. Both IPC and sham groups were injected with SS via the femoral vein 5 minutes before the I and 10 minutes before the R.
After I or I/R, the rats were sacrificed to retrieve jejunal segments (2 cm) that were isolated, washed, cleared of surrounding tissues, and mounted under 1 g tension at 37°C in an organ bath containing 10 mL of arated nutrient solution of composition (in mmol/L): NaCl 138, KCl 5.7, CaCl21.8, NaH2PO40.36, NaHCO3 15, and dextrose 5.5 (pH 7.4). Using a digital recording system we studied neurogenic contractions induced either by electrical field stimulation (EFS) or by the depolarizing agent KCl (70 mmol/L).6 – 8
EFS (5 and 30 Hz, 1-ms duration, 60 V) was per-formed using platinum electrodes connected to an S88 electrical stimulator (Grass, USA).6 – 8
Responses to EFS and KCl were recorded by force-displace-ment 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 one-way analysis of variance and Student t test.7,8 We also performed histological analyses using optical microscopy of jejunal pieces embedded in paraffin, cut into thin slices, and stained with hematoxylin and eosin.
RESULTS
Figure 1 shows that EFS (5 and 30 Hz) and KCl (70 mmol/L) produced contractile responses in all jejunal segments: sham, I⫹SS, IPC⫹I, I/R⫹SS, IPC⫹I/R. However, the amplitude of these contractions were sim-ilar in IPC⫹I and IPC⫹IR groups, but reduced in I⫹ SS and I/R⫹SS groups compared with the sham group (Fig 1andTable 1).
Histological analysis showed a loss of structural integrity of enteric nerves in the jejunal segments of I/R⫹SS group (Fig 2B), but in I/R⫹HEP (Fig 2C).
DISCUSSION
Our results showed that the amount of and the motility stimulated by transmitters released by enteric nerves were significantly reduced in jejunal segments from rats undergoing intestinal I and R. The deleterious effects of R on the intestine exacerbated the I injury.15Molecular
mechanisms involved in the I/R injury are poorly under-stood, but involve formation of free oxygen radicals (ROS) with consequent oxidative stress, alterations of calcium flux, and activation of phospholipase A2. The
stressed or injured tissues release endogenous adenosine, which blocks potentially destructive inflammatory cas-cades, decreasing activation of platelets, leukocytes, en-dothelial cells, and responses that are mediated mainly by A2Aand A2Bpurinoceptors.
9-12 However, these
dys-functions were reduced or absent among rats subjected to IPC, suggesting that the intestine becomes resistant to the deleterious effects of prolonged I and R following prior exposure to brief periods of vascular occlusion.
The intracellular signaling pathway for IPC in intes-tines undergoing I/R injury has not been completely clarified. Probably more than one pathway acts in se-quence or parallel.23,24 The IPC phenomenon, can be
divided into trigger and mediator phases27: the first
triggering phase involves the release of adenosine,
bra-Table 1. Values of Amplitude of Neurogenic Contractions (Expressed in Grams of Tension) Induced by Electrical Field Stimulation (5 and 30 Hz) or KCl (70 mmol/L) in Jejune of Rats
Treated with IPC or Saline Solution and Submitted to Intestinal I or I/R
Groups 5 Hz 30 Hz KCl
Sham 1.43⫾0.12 2.15⫾0.19 2.25⫾0.18 I⫹SS 0.18⫾0.04* 0.28⫾0.05* 0.41⫾0.10* IPC⫹I 1.01⫾0.12† 1.20
⫾0.19† 1.73
⫾0.25† I/R⫹SS 0.18⫾0.06 0.45⫾0.10 0.61⫾0.17 IPC⫹I/R 1.22⫾0.15‡ 1.27
⫾0.19‡ 1.66
⫾0.23‡
Data corresponding to means⫾standard errors of the means (n⫽6). I, ischemia; R, reperfusion; IPC, ischemic preconditioning.
*Statistically different from sham (P⬍.05). †Statistically different of I⫹SS (P⬍.05). ‡Statistically different of I/R⫹SS (P⬍.05).
dykinin, opioids, and even catecholamines, as well as free radicals and other triggers.26These pathways eventually
activate PKC directly or indirectly, leading to down-stream phosphorylation of endothelial nitric oxide syn-thase and NO formation, which in turn activates soluble guanylyl cyclase (GC) and protein kinase G (PKG). The latter phase initiates opening of mitochondrial KATP
channels27,28and generation of ROS that targets PKC.29
Once this pathway is activated, tissues can be protected for a long time, that is, the heart enters a protected phenotype that persists for an hour or more even after the triggering agonists have been washed away.29
Events in the mediator phase are less well defined. PKC and phospholipase C seem to activate phosphatidyl inositol 3-ki-nase, AKT, nitric oxide syntase, GC and PKG, leading to inhibition of the mitochondrial permeability transition pore (MPTP) that controls the release of calcium and preapoptotic factors into the cytoplasm, thereby improving cytoprotec-tion.28 In addition, downstream PKC activation of
mitogen-activated protein kinase alters, expression25antiapoptotic
pro-tein genes like Bcl2.26
Some authors have demonstrated that PKC32modifies
an A2Bpurinoceptor: endogenous ADO binds to it early
in the R period.30,31 Many downstream signaling events
of A2B purinoceptors recapitulate those in the trigger
pathway. This signaling is thought to ultimately prevent opening of the MPTP.33If the latter are allowed to form,
electrochemical equilibrium between the cytoplasm and mitochondrial matrix abolishes the critical electrochem-ical gradients, resulting in cessation of ATP production and cell necrosis. Thus, a series of signaling steps before and after I lead to blockade of MPTP formation accounts for cytoprotection in IPC.33
In conclusion, we have shown IPC to be an important intestinal protector against lesions caused by I and R that compromised the motility of this organ.
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