Elsevier

Cardiovascular Pathology

Volume 24, Issue 2, March–April 2015, Pages 94-101
Cardiovascular Pathology

Original Article
Effects of isoflurane postconditioning on chronic phase of ischemia–reperfusion heart injury in rats

https://doi.org/10.1016/j.carpath.2014.09.004Get rights and content

Abstract

Introduction

The application of isoflurane in a postconditioning manner, during early reperfusion of ischemic myocardium, reduces the infarct size. Its favorable effect on highly vascularized granulation tissue formation is very important considering the fact that increased genesis of blood vessels in peri-infarct zone reduces the infarct size and improves cardiac function. Taking into consideration the influence of isoflurane on the subacute phase of infarct healing, by using different immunohistochemical markers, we wanted to explore whether isoflurane postconditioning influences the chronic phase of healing.

Methods

The size of infarcted region was measured, and comparisons between isoflurane-treated and control animals were made. Quality of infarcted area was assessed by detecting vascular endothelial growth factor (VEGF), platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) as a marker of angiogenesis, and nestin as a marker of immature progenitor cells, and de novo formed blood vessels (vasculogenesis).

Results

There was no difference between the control and isoflurane-treated groups in VEGF and PECAM-1/CD31 expression. However, a large reduction in infarct size was found (68.1% of control). Also, a marked decrease of nestin expression in immature progenitor cells, along with a marked increase of the same marker in cardiomyocytes, (signs of myocardium regeneration), was found in experimental animals when compared to control animals that did not receive isoflurane treatment.

Conclusions

Based on our results, we can emphasize two morphologically detectable benefits of isoflurane postconditioning: a marked reduction in infarct size along with a more mature-looking infarct area in the chronic phase of infarct healing.

Introduction

Percutaneous coronary intervention and coronary artery bypass grafting (CABG) are therapeutic procedures that can be applied in order to reestablish blood flow in coronary arteries and reduce the size of cardiac infarction [1]. Despite its beneficial effects, reperfusion, paradoxically, increases the intensity of myocardial injury due to the effect on microvascular dysfunction, causing a stronger inflammatory response [2] and initiation of cellular death by apoptosis [3], [4], [5]. These effects inflict an additional damage to the cardiac muscle that is already affected by the ischemic injury. In order to reduce reperfusion injury of myocardium, various mechanical and pharmacological therapeutic procedures are available. They are usually applied immediately before, or at the very start, of the reperfusion and are referred to as postconditioning procedures [6].

In the last 15 years, postconditioning performed by the use of volatile anaesthetics came into use. With this approach, a level of infarct reduction similar to that achieved by ischemic postconditioning could be accomplished, with an added benefit of avoiding the risks of damage caused by balloon inflation in coronary vessels. The positive effect is a result of activation of enzyme paths explained in our last study [7].

It is known that postconditioning by isoflurane decreases the size of myocardial infarction, but most animal studies explored acute effects within a few hours from the beginning of reperfusion [8]. However, the process of myocardial infarction healing continues for several weeks and is divided into three overlapping phases: acute–inflammatory, subacute–proliferative, and chronic–maturation phase [9].

In our previous study, we established that postconditioning by isoflurane reduces the size of the myocardial infarct in the subacute phase of healing (day 4 after reperfusion), wherein a significantly higher number of blood vessels in granulation tissue of rats that were postconditioned by isoflurane were observed [7]. Taking into consideration that several studies indicated that the increased genesis of blood vessels in the peri-infarct zone reduces the infarct size and improves the cardiac function [10], [11], [12], by utilizing several immunohistochemical markers, we wanted to explore whether postconditioning with isoflurane influences the chronic phase of healing.

Vascular endothelial growth factor (VEGF) strongly supports vasculogenesis (“de novo” formation of blood vessels from endothelial progenitors) and angiogenesis (i.e., sprouting and collateral growth of endothelial cells), microvascular permeability, and vasodilatation [13], [14]. It is also important in the process of migration and proliferation of progenitor cells [15]. It has been demonstrated that myocardial reperfusion injury significantly increases the level of VEGF protein and its immune reactivity between days 3 and 7 after reperfusion, while the expression of VEGF significantly decreases on day 21 postinjury [16].

In our previous study, we demonstrated that postconditioning with isoflurane increases the expression of VEGF during the subacute phase (day 4 postinjury) of infarct healing in rats [17]. No data regarding the expression of VEGF and anesthetic postconditioning in the maturation phase of infarct healing can be found. Platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a transmembrane glycoprotein that is expressed on the surface of endothelial cells [18]. It plays an essential role in angiogenesis [19], and its strong expression was found in emerging blood vessels during the healing of ischemic–reperfusion myocardial injury [20].

Nestin is an intermediary filament protein that has been used as a marker of immature progenitor cells capable of developing into different subpopulations of cells important to the process of infarct healing [21]. It is also a marker of proliferating endothelial progenitor cells in human and rat heart damaged by ischemia [22], [23], as well as for “de novo” formed endothelial cells and blood vessels (vasculogenesis). It has been demonstrated that postconditioning by isoflurane increases the number of endothelial progenitor cells and blood vessels (formed by both processes: vasculogenesis and angiogenesis) during the subacute phase of healing [7]. To the best of our knowledge, the influence of postconditioning, using isoflurane, on the chronic phase of myocardial infarct healing has not been investigated until now. Hence, we have used the abovementioned markers to explore the influence of isoflurane postconditioning on the appearance and properties of myocardial infarct 14 days after ischemia–reperfusion injury.

Section snippets

Animal model

All experimental protocols and procedures were approved by the Ethical Committee of the School of Medicine in Split and carried out according to laboratory animal guidelines (European Communities Council Directive of 24 November 1986). Sprague–Dawley female rats (n= 8) weighing 175–225 g were used. They were obtained from the Facility for Experimental Animals, University of Split, Croatia. The rats were housed in individual plastic cages in a temperature-controlled environment, on a 12-h:12-h

General observations

Six animals were excluded due to an incomplete ischemia of the left ventricle. After intraoperative confirmation of appropriate ischemia (pallor affecting the anterior surface of the heart), 8 rats survived until day 14 after reperfusion.

Qualitative differences in the infarcted area

In both the control and isoflurane groups, the infarcted area was clearly visible on objective magnification of × 10 on sections stained with Mallory trichrome staining procedure (Fig. 1A, B). On day 14 after reperfusion, the infarcts were in the chronic phase

Discussion

In this study, using an immunohistochemical approach, we demonstrated the influence of postconditioning with isoflurane on the chronic phase of ischemia–reperfusion injury. In addition to the already observed influence on the subacute phase of healing [7], we found that isoflurane postconditioning causes a decrease of infarct size observed even 14 days after ischemia–reperfusion injury. Although administered only for 15 min, isoflurane caused a long-term effect. We believe this to be an important

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    Funding: This study was funded by the Ministry of Science, Education, and Sports, Republic of Croatia (project nos. 216-2160528-0067 and 021-2160528-0507), whose support is gratefully acknowledged.

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