Attenuation of Isoflurane-Induced Preconditioning and Reactive Oxygen Species Production in the Senescent Rat Heart

Abstract

BACKGROUND: 

Although attenuation of anesthetic preconditioning in aged ex vivo heart models has been studied extensively, there are no comparable in vivo studies. To extend previous work and to address a possible mechanism underlying age-related differences, we investigated isoflurane-induced preconditioning and reactive oxygen species (ROS) production in the aged rat heart in vivo.

METHODS: 

Male Fisher 344 rats were assigned from their respective age groups (young, 3–5 mo; old, 20–24 mo) to either receive 30 min of 1.0 minimum alveolar concentration isoflurane or to a control group. Rats were subjected to coronary artery occlusion for 30 min followed by 2 h of reperfusion. A fluorescent probe for superoxide anion production (dihydroethidium, 1 mg) was administered in the absence of the isoflurane or just before isoflurane exposure in four additional groups. Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and epifluorescence microscopy, respectively.

RESULTS: 

Isoflurane decreased myocardial infarct size of young rats (26.7% ± 3.0%) compared with young controls (50.9% ± 1.9%; P < 0.001), whereas isoflurane did not significantly affect myocardial infarct size of old rats (39.1% ± 0.9%) compared with old controls (46.5% ± 2.4%; P > 0.05). Isoflurane increased ROS levels in young rats (430.5 ± 95.9 arbitrary units [AU]) compared with young controls (162.7 ± 25.5 AU; P < 0.01). In contrast, no significant changes in ROS levels were observed in old animals (316.4 ± 56.3 AU isoflurane versus 233.8 ± 59.2 AU control).

CONCLUSIONS: 

Reduction in the cardioprotective effects of isoflurane and attenuation of isoflurane-stimulated ROS production were observed in the senescent myocardium in vivo.

In Brief

IMPLICATIONS: This study demonstrates that, in contrast to young hearts, isoflurane-induced preconditioning and enhanced reactive oxygen species production are not observed in the aged rat heart. Attenuation of reactive oxygen species may be a mechanism for the reduced anesthetic preconditioning in the senescent myocardium.

In 1986, Murry et al. first demonstrated the protective effects of ischemic preconditioning (IPC) in the dog myocardium.¹ Since then IPC has been studied extensively in rodent and canine models. In principle, IPC entails a short period of myocardial ischemia and reperfusion that triggers a cascade of intracellular events, thereby creating a memory effect that attenuates future ischemic-reperfusion injury. There are two distinct windows of protection. Early preconditioning is observed for 2 to 3 h after the initial transient ischemic event. Late preconditioning, or the second window, is observed 12–24 h after the initial transient ischemic event and it lasts for up to 72 h.² Certain drugs can confer similar levels of cardioprotection, including those used in anesthesia such as opioids and volatile anesthetics.^3–5 Anesthetic preconditioning (APC) with volatile anesthetics shares similar pathways with IPC, although neither mechanism is fully understood.⁶

Although IPC and APC have been well studied in young adult animals, these cardioprotective effects would most benefit the elderly population who have a higher burden of coronary artery disease than younger patients.^7,8 Clinically, the presence of angina before an acute myocardial infarction is reported to be associated with improved in-hospital outcome in younger patients as compared with elderly patients,⁹ suggesting that older human populations may not benefit from preconditioning. It seems likely that the subtle cellular changes that accompany aging attenuate the adaptive response of the aged heart to stress, and that these changes could potentially impair the efficacy of the preconditioning response. For example, aged mitochondria have been shown to have reduced levels of cardiolipin and reduced cytochrome c oxidase activity, and impaired efficiency of oxidative phosphorylation and antioxidant systems.¹⁰ Indeed, studies of isolated perfused rat hearts have shown attenuation of the cardioprotective response from IPC and APC in the aged myocardium.^11–13 Although transient production of reactive oxygen species (ROS) is necessary for IPC and APC in young animals,^14,15 basal ROS production seems to increase with age¹⁶ and this chronic “oxidative stress” appears to result in mitochondrial damage that could, paradoxically, decrease cardioprotection after IPC or APC.

It has been shown in ex vivo models that myocardial infarct size is increased in the senescent rat myocardium compared with the hearts of younger animals, but whether this is true in whole animal models has not been examined. Furthermore, the effectiveness of APC in aged intact animals has not been investigated. An in vivo model allows for the assessment of other possible modulators of APC including neurohumoral responses that would not be present in ex vivo models. Moreover, once the heart is removed, it may well undergo phenotypic drift. The goal of this study was to determine the effect of age on APC using isoflurane in an in vivo rat model. Since production of ROS is an early trigger for IPC and APC^14,15, we further hypothesized that ROS generation during APC is attenuated in the aged rat heart.

METHODS

All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of SUNY Stony Brook. Furthermore, all procedures conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals.

General Preparation

Male Fischer 344 rats of the following age groups were obtained: 3–5 mo and 20–24 mo. These ages correspond to approximately 20–30 yr and 65–75 human years, respectively.¹⁷ Animals were housed in the Division of Laboratory Animal Resources until the day of study. Anesthesia was induced with an intraperitoneal injection of sodium thiobutabarbital (120–135 mg/kg), with additional maintenance doses given as needed. Rats were tested for the absence of pedal reflexes throughout the experimental protocol to ensure adequate anesthesia. Heparin-filled (10 U/mL) catheters were inserted into the right jugular vein for fluid and drug administration. The right carotid artery was cannulated to measure arterial blood pressure. A tracheotomy was performed and the animals’ lungs ventilated using a Harvard Apparatus model 638 rat ventilator with an air-and-oxygen mixture and 5 cm H₂O of positive end-expiratory pressure. Inspired oxygen concentrations were maintained at 49% and end-tidal carbon dioxide concentration maintained at 35–40 mm Hg by adjusting the respiratory rate or tidal volume throughout the experiment. Arterial blood gas tension and acid–base status were monitored at regular intervals and maintained within a normal range (pH, 7.35–7.45; Paco₂, 30–40 mm Hg; and Pao₂, 90–150 mm Hg). End-tidal concentrations of isoflurane, carbon dioxide and inspired oxygen concentrations were measured using a Poet IQ2 infrared gas analyzer (Criticare Systems Inc., Waukesha, WI). The 1.0 minimum alveolar anesthetic concentration (MAC) value of isoflurane used for rats in the current investigation was 1.4%.¹⁸ Body temperature was maintained at 37.0 ± 0.2°C using a heating pad and radiant warmer.

Surgery Protocol

A left thoracotomy was performed in the fifth intercostal space, and the pericardium was opened. A 6–0 Prolene suture was placed around the proximal left anterior descending coronary artery and vein in the area immediately below the left atrial appendage. The ends of the suture were threaded through a propylene tube to form a snare. Coronary artery occlusion was produced by clamping the snare onto the epicardial surface of the heart with a hemostat and was confirmed by the appearance of epicardial cyanosis. Reperfusion was achieved by loosening the snare and was verified by observing an epicardial hyperemic response. Heart rate and mean arterial blood pressure data were continuously recorded on a polygraph throughout the experiment and stored on a personal computer. At the end of the experiment, the animal was euthanized with an overdose of sodium thiobutabarbital.

Myocardial Infarct Size Experiment

The experimental design was modeled after Ludwig et al.¹⁹ This design is illustrated in Figure 1A. In four separate experimental groups, rats of similar age were randomly assigned to one of two groups: control (ischemia/reperfusion without exposure to isoflurane) or isoflurane 1.0 MAC plus ischemia/reperfusion. A 15 min rest period followed the discontinuation of the volatile anesthetic to allow the end-tidal isoflurane concentration to reach zero. To induce myocardial injury, the rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion.

Figure 1.: (A) The experimental Protocol A used in infarct size experiments. (B) The experimental Protocol B used in measurement of reactive oxygen species. OCC = coronary artery occlusion.

At the end of each experiment, the coronary artery was reoccluded, and patent blue dye was injected IV to stain the normal, nonischemic, region of the heart. The heart was rapidly excised and six 1–2 mm cross-sections of the left ventricle (LV) were obtained using a scalpel. Both surfaces of the six sections were then scanned (Fig. 2) using an Epson 3200 Photo scanner and analyzed using two-dimensional planimetry,²⁰ within Matlab software (Mathworks, Natick, MA) to determine the total weight of the areas at risk (AAR). The ischemic area was reported as AAR divided by the ventricular weight.

Figure 2.: Scanned cross sections of the heart after Patent blue dye injection and 15 min of TTC incubation. TTC = 1% 2,3,5-triphenyltetrazolium chloride.

Within 5 min of preparation, the scanned regions were incubated at 37°C for 15 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to a pH of 7.4. This process left the infarcted area white (Fig. 2). Each slice was weighed, and the slices were then rescanned on both sides. The total area of infarct was determined by two-dimensional planimetry as described above. Infarction size was expressed as the area of infarct divided weight by the total AAR weight and multiplied by 100 to determine the percent infarct (Infarct/AAR).

Measurement of ROS

The second experimental series was conducted to determine whether there was a relationship between the degree of APC effect in the young and old groups and the levels of ROS. ROS were detected using dihydroethidium fluorescence as previously described.¹⁵ Dihydroethidium is oxidized by intracellular ROS (primarily superoxide anion) to produce ethidium that subsequently binds to DNA (Eth-DNA) greatly enhancing its fluorescence.²¹ The fluorescence observed after activation of the Eth-DNA complex is generally stable; thus, an increase in dihydroethidium oxidation to Eth-DNA and the subsequent increase in fluorescence reflect super oxide anion generation during the time interval investigated.

In four additional groups of rats (n = 5 in each group), illustrated in Figure 1B, the ROS probe dihydroethidium (1 mg dissolved in 0.1 mL of dimethyl sulfoxide) was rapidly injected into the right internal jugular vein catheter 30 s before the administration 1.0 MAC (young isoflurane or old isoflurane) or the corresponding time point in control rats not exposed to isoflurane (young control or old control). Isoflurane in both age groups was discontinued after 30 min, and both groups were euthanized after 45 min of “memory time” on the ventilator with a lethal dose of sodium thiobutabarbital. The hearts were rapidly excised, cut transversely below the atrium, embedded in a tissue preservative and frozen in liquid nitrogen. Cryostat sections (10 μm thick) of the ventricle were mounted on standard microscope slides.

The mounted sections were viewed under epifluorescent illumination through a 20 × 0.9 NA plan fluorescence lens using an Olympus IMT2 inverted microscope. A sensitive, high resolution CCD camera was used to acquire the images. At least two slices of each LV were examined with four random fields acquired per section under identical conditions. A background image was also obtained for each specimen. After transferring the images as 12 bit TIFF files, LSR image analysis software was used to assess pixel intensities in each field. The pixel area intensities were background-corrected and then averaged. The average intensities were compared and the significances of the differences among mean intensities determined through an analysis of variance.

Statistical Analysis

Based on previous work,²² we estimated that five animals per group were sufficient to detect a 17% difference with 80% power in infarction size between control and APC groups. Statistical analysis of data within and among groups was performed with two-way analysis of variance followed by Bonferroni post-tests. Statistical significance was determined as P < 0.05. All data are expressed as mean ± sem. Analyses were performed on PRISM statistical software (version 4; GraphPad Software, Inc.).

RESULTS

Twenty-four rats were instrumented to obtain five successful myocardial infarct size measurements per group. Of the 24 animals, two were excluded due to intractable ventricular arrhythmias, one because AAR was <30% and one due to excessive bleeding during instrumentation. For the measurement of ROS, 23 rats were instrumented to obtain 20 successful ROS production experiments, two animals were excluded due to pneumothorax secondary to ventilator malfunction and one for an intractable ventricular arrhythmia after dihydroethidium injection. The age and body weight were the similar in the old isoflurane and old control groups and in the young isoflurane and young control groups (Table 1).

Table 1: Age, Weight, and AAR/Vent for all Groups

Systemic Hemodynamics

No differences in the baseline hemodynamics were observed among experimental groups (Table 2) and no differences were observed in mean arterial blood pressure between the young isoflurane and old isoflurane groups. Isoflurane, however, significantly (P < 0.001) decreased the mean arterial blood pressure in the young isoflurane and old isoflurane groups when compared to their respective control groups. There were no differences in mean arterial blood pressure during the memory period between the young isoflurane and young control groups. A difference in mean arterial blood pressure was observed between the old isoflurane and the old control groups during the memory period.

Table 2: Systemic Hemodynamics

During the second hour of reperfusion, there was a significant difference (P < 0.01) in mean arterial blood pressure in the old control group compared to the old isoflurane group. Decline in the heart rate during coronary artery occlusion and during reperfusion periods was observed in all groups. No statistically significant differences in heart rates were observed among groups.

Myocardial Infarct Size

The AAR/LV values were similar among all groups (Table 1). A 40% AAR/LV was considered an adequate degree of coronary artery occlusion. The Infarct/AAR percentage was similar in old and young control animals (Fig. 3). This showed that an adequate ischemia was induced by coronary artery occlusion. Isoflurane preconditioning significantly reduced (P < 0.001) the Infarct/AAR ratios in the young animals (young isoflurane group [26.7% ± 2.9%] compared with the young control group [50.9% ± 1.9%]). By contrast, there was no significant reduction in Infarct/AAR due to isoflurane preconditioning in older animals (Fig. 3.) (old isoflurane group [39.1% ± 0.9%] versus old control group [46.5% ± 2.4%]).

Figure 3.: Histogram with error bars of myocardial infarct size expressed as a percentage of the left ventricular area at risk in preconditioning vs control; *P < 0.001. ISO = isoflurane.

ROS Production

Representative images of ventricular slices acquired by epifluorescence microscopy are shown in Figure 4. Bright areas represent areas of high ROS production. Higher levels of fluorescence were observed in the young isoflurane group (430.5 ± 95.9 fluorescence intensity units) compared with the young control group (162.7 ± 25.5 fluorescence intensity units). ROS levels in the old isoflurane group, however, were not significantly different than the old control group (316.4 ± 56.3 vs 233.8 ± 59.2 fluorescence intensity units, respectively) (Fig. 5).

Figure 4.: Representative epifluoresence photomicrographs of reactive oxygen species (ROS) dihydroethidium dye in 10 μm thick sections of ventricular myocardium. (A) Young control. (B) Young isoflurane. (C) Old control. (D) Old isoflurane.

Figure 5.: Histogram depicting intensity of Ethidium-DNA fluorescence under epifluorescent illumination in young and old experimental models. *P < 0.001 compared to control within the same time period. AU = arbitrary units. ISO = isoflurane.

DISCUSSION

Our study compared the in vivo effects of isoflurane-induced preconditioning and ROS production in young and old rat hearts. We found an approximately 50% reduction in myocardial infarction size in young animals receiving isoflurane preconditioning compared with the young control group. In contrast, there was no reduction in myocardial infarction size in the old animals exposed to isoflurane compared with their respective control group. In the second part of our study, we found that ROS production in young rat hearts after exposure to isoflurane was increased by 265% compared with young control animals; whereas, ROS levels were not significantly elevated in old rats after exposure to isoflurane despite the apparently higher ROS levels in old control animals.

Our results using a whole animal model extend the findings of other studies using an ex vivo heart model to demonstrate that the response to IPC and APC in the aged myocardium is attenuated compared with younger animals.^10–12 One study showed an approximately 50% decrease in myocardial infarct size in young animals whereas showing no benefit in older animals during APC.¹⁷ The degree of protection observed was similar to that demonstrated in ex vivo studies.

In our study, isoflurane-induced ROS response in younger animals was less than observed in the rabbit model reported by Tanaka et al.¹⁵ One possibility is that levels of xanthine oxidase are higher in rats compared to rabbits resulting in higher baseline levels of ROS.²³ This, and the already reported elevation in oxygen-derived free radial production compared to other species,²⁴ could explain the reduced isoflurane-induced ROS elevation observed in our rat model.

The attenuation of APC in the aged myocardium may be attributed to multiple factors. We specifically examined whether ROS could be a source for this attenuation since it is theorized that volatile anesthetics, by generating small amounts of ROS in the mitochondria, trigger preconditioning pathways resulting in myocardial protection.²⁵ Aged mitochondria have been shown to have impaired efficiency of oxidative phosphorylation and antioxidant capacity and multiple studies have confirmed the reduced effectiveness of the aged mitochondria stress responses that accompany ischemia.^26,27 Hence, it is plausible that a defect in the mitochondria of the senescent myocardium accounts for the lack of isoflurane-induced myocardial protection.

ROS have been shown to be an early trigger for IPC and APC, whereas high levels, as seen in reperfusion, may lead to myocardial stunning, infarction, and apoptosis.^28–30 It is possible that isoflurane-induced mitochondrial ROS production in the aged myocardium is not sufficient to trigger preconditioning. Alternatively, the cellular mechanism required to trigger APC may be desensitized or defective. In the former case, stress/survival pathways may already be maximally activated, therefore, protection against further damage may not be possible through this mechanism.

A possible limitation of our study is that the same dose of isoflurane was used in both age groups even though the MAC value declines with age. Nonetheless, the anesthetic concentration we used was within previously studied ranges for APC. For example, as little as 0.25 MAC of isoflurane delivered 30 min before an ischemic insult triggers a myocardial preconditioning response, and this response increases with increased anesthetic concentration equivalent to 1.25 MAC.³¹

In conclusion, our findings compliment the results from prior studies in ex vivo models of APC confirming in an in vivo model that APC is limited in aged myocardium. Attenuation of an increase in ROS production in response to isoflurane might explain the attenuated cardioprotective effects of the volatile anesthetic in the senescent myocardium.