Evaluating the protective role of ischaemic preconditioning in rat hearts using a stationary small-animal SPECT imager and ^99mTc-glucarate

Abstract

Objective

To examine the protective role of ischaemic preconditioning (IPC) in rat hearts using ^99mTc-glucarate (GLA) and a stationary SPECT imager, FastSPECT.

Methods

Twenty-four rats with 30 min myocardial ischaemia and 150 min reperfusion (IR) were studied as follows. The IPC group (n = 6) underwent IPC (five cycles of 4 min ligation of the left coronary artery and reflow) before IR. The control group (n = 7) was treated by IR without IPC. The SPT group (n = 6) was subjected to IPC and an adenosine antagonist, 8-(p-sulfophenyl)-theophylline (SPT). The vehicle group (n = 5) received IPC and SPT carrier vehicle. GLA was delivered intravenously 30 min post-reperfusion, and 2-h dynamic cardiac images were acquired by FastSPECT.

Results

GLA showed ‘hot-spot’ accumulation in the ischaemic area-at-risk (IAR) and exhibited lower retention (% 5 min peak) in the IPC and vehicle groups (33.8 ± 2.6 vs. 35.7 ± 9.2, P>0.05) than in the control and SPT groups (63.1 ± 5.3 vs. 54.8 ± 4.8, P>0.05). The infarct size (% IAR) was larger in the control and SPT groups (48.2 ± 6.3 vs. 41.7 ± 6.3, P>0.05) than that in the IPC and vehicle groups (21.0 ±1.9 vs. 19.1 ± 4.6, P>0.05). In terms of the ex-vivo IAR-to-normal radioactivity ratio, there was a statistical difference between the control and IPC groups (7.4 ± 0.9 vs. 3.0 ± 0.4), as well as the SPT and vehicle groups (7.4 ± 1.0 vs. 3.4 ± 0.5).

Conclusion

IPC offers cardioprotection and relates to the activation of adenosine receptors in rat hearts. FastSPECT GLA imaging is not only useful in detecting early ischaemia–reperfusion injury, but also valuable in evaluating cardioprotection.

Introduction

Small animals such as rats or mice can provide models of human cardiovascular disease. Using non-invasive molecular imaging techniques, the small-animal heart models have become valuable tools in the cardiovascular sciences. A high-resolution single photon emission computed tomography (SPECT) imager, called microSPECT or small-animal SPECT, plays an important role in basic cardiovascular molecular imaging. Because of the small size and fast beating of the heart, high spatial resolution is critical in imaging rat or mouse heart models. Relative to PET, small-animal SPECT has potential advantages in spatial resolution. Currently, several small-animal SPECT systems, either adapted commercial clinical SPECT imagers or specifically designed smallanimal SPECT imagers, have been used effectively to image rat hearts and collect validated data of myocardial perfusion, necrosis, and apoptosis with ^99mTc-labelled tracers [1–3]. Modern molecular imaging studies often require investigation of washout rates and other kinetic parameters of radiolabelled agents [4,5]. The temporal resolution of a small-animal SPECT imager must be high in order to extract accurate estimates of these parameters. Sensitivity is another critical factor in small-animal imaging for acquiring enough photons for good three-dimensional (3-D) reconstruction within short periods of time, such as a fraction of a heartbeat or a tracer washout time. Accordingly, a stationary SPECT imager with many cameras has a number of unique advantages over other small-animal SPECT systems, such as the high speed, sensitivity, and resolution plus a stationary gantry. FastSPECT, a standard high-resolution SPECT system built at the University of Arizona, is the first stationary SPECT imager in the world and is capable of producing fast tomographic images in rats and mice. The acquisition of tomographic data sets in FastSPECT can be executed with no rotation in either animal or detector for dynamic imaging studies.

Using FastSPECT and well-established rat heart models, we previously explored the property of ^99mTc-labelled glucarate (GLA), a novel infarct-avid imaging agent, to identify early myocardial ischaemia–reperfusion injury. The data from our laboratory and others have demonstrated that GLA can mark non-viable regions by hot-spot imaging in myocardium with acute necroses [1,6–11]. Our experimental studies showed that the severity of myocardial injury induced by different durations of ischaemia following reperfusion in the rat heart models can be assessed using GLA non-invasively and quantitatively. From the imaging point of view, investigators and clinicians need a useful tool, not only for identifying varied injuries and status of the heart disease, but also for determining individualized therapeutic strategies and monitoring the effects of therapeutic interventions.

Ischaemic preconditioning (IPC) is a process by which exposure of myocardium to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of subsequent prolonged ischaemic stress [12]. IPC reduces myocardial infarct size [12–14], improves post-ischaemic contractile function [15], and preserves metabolic or energy status of the ischaemic–reperfused myocardium [16]. In rat hearts, it is generally believed that adenosine is a trigger for IPC, as it has been well described in other species [15,17]. However, the exact role and fashion of adenosine in rat hearts with IPC are dissimilar to the experimental results in other animal hearts [18–20]. Typically, a single cycle of short IPC is insufficient to produce adenosine-mediated cardioprotection; the threshold for producing IPC in rat hearts is higher than in other species. A longer duration stimulus or multiple cycles of short occlusion and reperfusion of the coronary artery are often required to trigger the adenosine receptors. Because the in-vivo rat heart model with regional ischaemia–reperfusion has been used in our laboratory to explore strategies of cardioprotection, we set out to clarify whether or not the adenosine A₁ receptor is involved in an IPC protocol with five cycles of 4 min of coronary artery occlusion, each separated by 4 min of reperfusion for cardioprotection in rat hearts. If adenosine is involved in the specific preconditioning protocol, the non-selective adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline should block the IPC cardioprotection and change the kinetic appearance of GLA in the rat hearts.

Consequently, the objective of this study was to determine if dynamic FastSPECT imaging with GLA would provide a unique, noninvasive tool for evaluation of cardioprotection. We examined the protective role of IPC in ischaemic–reperfused rat hearts both non-invasively and quantitatively.

Methods

Preparation of the ischaemic–reperfused rat heart model

Male Sprague–Dawley rats (weighing 250–350 g) were anaesthetized with 1.0–1.5% isoflurane. The model of myocardial ischaemia–reperfusion was developed using the technique described previously [1]. The rat was ventilated using a volume-controlled Inspira Advanced Safety Ventilator (Harvard Apparatus, Holliston, MA) with oxygen. The chest was opened with a left intercostal thoracotomy incision at the 5th intercostal space. A ligature was placed around the left coronary artery (LCA) with a small amount of myocardium. The ligature was pulled tight by passing the suture through a polyethylene tubing and clamping it repeatedly for coronary occlusion. Regional myocardial reperfusion was elicited by releasing the ligature. After ischaemia–reperfusion treatment, the chest of the rat was closed. During the period of surgery, ischaemia–reperfusion treatment, and imaging, the body temperature of the animal was maintained with a pad using recirculated warm water.

Experimental protocols and groups

Group I (IPC, n = 6) animals were preconditioned with a sequence of five cycles of 4 min LCA occlusion and 4 min reflow. Ten minutes later, the rats underwent a treatment of 30 min regional myocardial ischaemia and 150 min reperfusion (IR) by ligating and releasing the LCA.

Group II (control, n = 7) animals received no IPC, but were subjected to equivalent open-chest time as in group I, then underwent the IR treatment as in group I.

Group III (SPT, n = 6) rats were subjected to IPC and IR as in group I. 8-(p-sulfophenyl)-theophylline (SPT; 10 mg · kg⁻¹) was infused intravenously (i.v.) 2 min before and during subsequent 30 min ischaemia, and then the second dose of SPT was given within 30 min reperfusion using a Harvard PHD2000 syringe pump (Harvard Apparatus, Holliston, MA).

Group IV (vehicle, n = 5) rats were treated with IPC as in groups I and III followed by IR. An equivalent volume of SPT carrier vehicle (DMSO/saline) was infused i.v. 2 min before and during 30 min ischaemia and again within 30 min reperfusion as in group III.

Thirty minutes post-reperfusion, GLA was administrated i.v. for 120 min dynamic cardiac FastSPECT imaging in all rats.

Reagents

Glucarate kits were provided by Molecular Targeting Technologies, Inc. (West Chester, Pennsylvania, USA). A vial of glucarate was reconstituted by adding 1.0 ml of ^99mTc as sodium [^99mTc]pertechnetate (1.48 GBq (40 mCi)). The radiochemical purity of GLA was verified by thin-layer chromatography using Gelman instant thin-layer silica gel (ITLC-SG) strips developed in saline and acetone, and exceeded 95% for all experimental injections.

SPT was obtained from Sigma (St Louis, Missouri). A stock solution of SPT was prepared in DMSO and diluted with saline for i.v. administration. The dose and administration schedules were applied according to the methods previously described in the literature and our pilot studies [21–23].

Dynamic high-resolution SPECT imaging

The small-animal SPECT system, FastSPECT, was built in the Radiology Research Laboratory at the University of Arizona based on a stationary SPECT brain imager [24]. The system consists of 24 small modular gamma cameras and a cylindrical aperture with 24 1-mm diameter pinholes. It has the capability of rapid SPECT acquisition without motion of the detector or aperture.

Before the animal was loaded into the FastSPECT system, the jugular vein was catheterized through a surgical procedure. The rat was placed inside the aperture using a translation stage. The animal was positioned so that the heart was localized in the centre of the field of view. Thirty minutes post-reperfusion, GLA (185–222 MBq) was injected i.v. via the jugular vein catheter using the Harvard Apparatus syringe pump, followed by a 0.1 ml saline flush. Beginning immediately upon injection, dynamic cardiac images were acquired every minute for the first 10 min, followed by 5 min acquisition every 15 min until 120 min post-injection. A total of 24 projections were obtained, one from each camera, to generate a data set for tomographic reconstruction.

Image processing

Tomographic reconstructions of FastSPECT data were processed using five iterations of the algebraic reconstruction technique (ART) algorithm. Three-dimensional images were computed to provide images in a 33 × 49 × 49 voxel format and to generate tomographic transaxial, coronal, and sagittal slices with a thickness of 1 pixel (1.0 mm). The maximum threshold was set at approximately 30–35%, and minimum threshold was set at 0% to display radioactive distribution in the myocardium. The oblique re-orientation of the transaxial data was performed by computerized procedures to generate tomographic short-axis (transverse) slices, which cover the entire left ventricle from the base to the apex. Using AMIDE 0.8.15.1 software, 2-D isocontour regions of interest (ROIs) over the ‘hot-spot’ radioactive accumulations were created from all transverse slices on the 120-min images.

The 120-min ROIs were applied to all of the dynamic images from 1 to 120 min for generating averaged hotspot time–activity curves (TACs), which were corrected for ROI size (pixels), radioactive decay, acquisition time and injected dose. The remote normal myocardial time–activity curves were generated using 2-D ROI analysis. Typically, the left ventricular septum was selected as a remote normal zone to establish the ROI. Care was taken to draw an ROI on the normal zone and avoid the ROI overlapping into the blood pool on the early time-point images. To do that, we used the early blood-pool image to outline the left ventricular wall range. The endocardial edges from tomographic 1 min cardiac blood-pool images were co-registered with 120 min images to ensure the ROI establishment off the left ventricular cavity. The percent radioactive retention and washout at each time-point image relative to the initial radioactivity on the 1 min image and 5 min image, as well as the ratios of hotspot activities to remote normal myocardial activities, were calculated subsequently.

Postmortem analysis

At the end of the imaging session, the LCA was re-occluded. Evans blue dye (10%) in 1.0 ml PBS buffer was injected through the femoral vein, allowing the dye to stain the non-ischaemic portion of the heart and determine the ischaemic area-at-risk (IAR). The entire heart was excised, rinsed of excess dye with cold saline solution, and weighed. ^99mTc activity in the heart was measured by a CRC-15W Dose Calibrator/Well Counter (Capintec, Ramsey, New Jersey). Great vessels, atria and right ventricles of the heart were removed. The left ventricle was sectioned into four or five transverse slices, and both sides of all slices were photographed using a digital camera. Triphenyltetrazolium chloride (TTC) staining was used to identify the infarct area. The tissue slices were incubated in a 1% TTC PBS-buffered solution (pH 7.4) at 37°C for 20 min and subsequently fixed in 10% PBS-buffered formalin overnight at 2–8°C. Photographs were taken again of both sides of each TTC-stained tissue slice using the digital camera. The area unstained by Evans blue, as well as the TTC-negative area (white or pale), was quantitatively analysed using the software of SigmaScan (SPSS Science, Chicago, Illinois) in trace-measurement mode. Infarct sizes were calculated as a percentage of total LV mass (% LV) and then normalized by IAR (% IAR).

Non-ischaemic viable (Evans blue and TTC positive) and ischaemic non-viable (Evans blue and TTC negative) segments were dissected from all tissue slices. Theoretically, the remaining tissues would be ischaemic viable. However, TTC staining is not precise enough to delineate myocyte injury in detail. Focal irreversible micro-necroses might be neglected in the tissue with TTC-positive staining. It is difficult to dissect a small piece of tissue from the rat heart into TTC-positive and TTC-negative segments ascertainably. After the non-ischaemic viable and ischaemic non-viable tissues were separated, the remaining tissue segments were characterized as mixed-viability tissues in this study. The dissected tissue samples were weighed and gamma counted by the CRC-15W Calibrator/Well Counter. The ratios of the infarcted tissue and IAR (infarcted and mixed-viability tissues) to remote non-ischaemic viable myocardium were calculated, respectively.

Estimation of hot-spot size on FastSPECT imaging

The hot-spot size was estimated from each transverse slice of 120-min FastSPECT images according to the total number of pixels defined by AMIDE 0.8.15.1 2-D isocontour ROIs analysis over the hot-spot. The 2-D isocontour ROIs were defined so that they encompassed all neighboring values above 55 percentage of the maximum threshold value in the myocardium. After acquiring the number of pixels of the hot-spot on each transverse slice, the total number of pixels in all slices with hot-spots were calculated and defined as the size of the infarct in the whole heart. The hot-spot sizes on 120-min images were determined in pixels for all of the animals in the four groups.

Data analysis

All results were expressed as mean ± SEM. The significance of the quantitative variables was assessed using one-way or two-way ANOVA with subsequent Student–Newman–Keuls post-hoc pairwise tests for comparisons between groups. Comparisons between two variables within a group were made by two-way ANOVA. Probability values less than 0.05 were considered significant. The correlation between GLA hot-spots and infarct size as measured by TTC staining was assessed by linear regression analysis.

Ethics

All experiments were performed in accordance with the Principles of Laboratory Animal Care from the National Institutes of Health (NIH Publication 85-23, revised 1985) and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona.

Results

FastSPECT images of myocardium with ischaemia– reperfusion

Cardiac blood-pool activity was visualized initially within 1–2 min after GLA injection. The wall of the left ventricle was then partly visualized in the stenosis zone of the LCA-supplied area, which exhibited a hot-spot (increased GLA uptake). The hot-spot was unequivocally localized within 10–30 min after intravenous administration. Two hours post-injection, the hot-spot could be well visualized in all tomographic image planes. Similar hotspots were detected in all rat hearts with ischaemia–reperfusion imaged with GLA. The hot-spots were localized on the anterior wall, lateral wall, and apex of the left ventricles. As shown in Fig. 1, gross hot-spot sizes among the hearts could be compared visually on the 3-D reconstructed FastSPECT data set and tomographic slices. The rat heart, which received IPC treatment followed by ischaemia–reperfusion, showed a smaller hotspot on the anterior lateral wall of the left ventricle compared to the larger hot-spot in the heart without IPC treatment.

Fig. 1.

A visual comparison of GLA hot-spot accumulations in two representative ischaemic–reperfused rat hearts treated with the protocol of IPC in the IPC group (left panel) and without cardioprotection in the control group (right panel) 120 min post-injection. The wall of the left ventricle was partly visualized on 3-D representation of a reconstructed FastSPECT data set of the left ventricle (a and e). The hot-spots localized in the lateral wall, anterior wall and apex of the left ventricles. Larger hot-spots were demonstrated on tomographic transaxial, coronal, and sagittal slices (b–d) in the control heart without IPC protection than that in the IPC-protected heart (f–h).

Quantitative analysis of GLA imaging

Myocardial TACs were generated using computerized ROI analysis with background, decay, acquisition time and injected-dose correction. Raw TACs in each group are shown in Fig. 2. The difference observed at each point in time from 5 min to 120 min between the LCA hot-spot and normal zone was significant in the control and SPT groups (P < 0.05), respectively. In the IPC and vehicle groups, the difference between the LCA hot-spot and normal zone started from 10 and 15 min.

Fig. 2.

GLA kinetic time–activity curves from normal and ischaemic–reperfused areas in the hearts of all four groups. The curves were corrected by radioactive decay, acquisition time, and injected dose. There was a significant difference between the LCA hot-spot and normal zone at each time point from 5 to 120 min in the control and SPT groups. The difference was significant from 10 min to 120 min in the IPC group, and 15 to 120 min in the vehicle group (P<0.05).

Based on the appearances of the TACs, GLA exhibited slower washout from the LCA hot-spot than the normal myocardial zone. By normalizing the radioactivity at each time point to the initial peak activity at 1 min postinjection, the fractional retention (%) of GLA from the hot-spot was significantly greater than that from the remote viable zone in all hearts. Because the first 1 min image is composed mainly of blood-pool activity, the rapid washout from the normal zone shown on the TACs might represent mostly rapid blood pool clearance. Accordingly, the radioactivity at each time point was furthermore normalized by the activity at 5 min to calculate the percentage of radioactive washout and retention. As shown in Fig. 3, the fractional retention (% 5 min peak) at the conclusion of imaging was still significantly higher in the hot-spot than in the normal zone in all hearts (33.8 ± 2.6 vs. 14.5 ± 1.0 in IPC, P < 0.05; 63.1 ± 5.3 vs. 21.2 ± 2.4 in control, P < 0.05; 54.8 ± 4.8 vs. 18.6 ± 1.7 in SPT, P < 0.05; 35.7 ± 9.2 vs. 15.3 ± 0.9 in vehicle, P < 0.05). In particular, the hot-spot retention in the IPC group was similar to the vehicle group but significantly lower than that in the control and SPT groups (P < 0.05). The increased hot-spot retention in the SPT group was as high as in the control group. The hot-spot retention in the vehicle group was significantly lower than that in the control and SPT groups (P < 0.05).

Fig. 3.

The fractional retention (%) of GLA from the hot-spot and remote viable zone calculated by normalizing the radioactivity at the end of the imaging session to the initial 1 min and 5 min peak activity on FastSPECT images. The retention from the hot-spot was significantly greater than that from the remote viable zone in all hearts. ^#P<0.05 compared to the hot-spot in the control group at 1 min as initial peak; ^&P<0.05 compared to the normal zone in the control group at 5 min as initial peak; *P<0.05 compared to the hot-spot in the control group at 5 min as initial peak; ^@P<0.05 compared to the hot-spot in the vehicle group.

Figure 4 shows that the ratios of the hot-spot to normal myocardial activity increased with time following GLA administration in all hearts. In the control group, the ratio was significantly higher than that in the IPC group beginning at 60 min and also significantly higher than that in the vehicle group beginning at 75 min, but it was not different from the SPT group at any time point. The difference between the SPT group and the vehicle group was significant. The final ratio was 3.6 ± 0.3 in the IPC group, 2.6 ± 0.6 in the vehicle group, 5.2 ± 0.7 in the control group, and 5.4 ± 0.3 in the SPT group.

Fig. 4.

Ratios of the hot-spot to viable myocardial GLA activity over time from the hearts of all four groups. *P<0.05 compared to the control group.

Measurements of myocardial IAR, infarct and hot-spot

The measurements of myocardial IAR, infarct, and hotspot are shown in Table 1. IAR did not differ significantly among experimental groups. Normalized by IAR, the infarct size (% IAR) in the control and SPT groups was significantly larger than that in the IPC and vehicle groups, respectively. No significant difference in infarct size was found between either the hearts treated with IPC versus carrier vehicle or non-IPC control versus SPT blockade. Relative to the control group, there was a significant decrease in the size of hot-spot in the IPC group (44.2%) and vehicle group (53.4%), but not in the SPT group.

Table 1.

Measurements of myocardial ischaemic area at risk (IAR), infarct and GLA hot-spot

	IAR (% LV)	Infarct (% LV)	Infarct (% IAR)	Hot spot (pixels)
IPC (n=6)	49.4 ± 3.9	10.1 ± 0.6*	21.0 ± 2.0*	624.5 ± 74.1*
Control (n=7)	48.8 ± 2.9	23.8 ± 3.7	48.2 ± 6.3	1118.4 ± 102.6
SPT (n=6)	46.2 ± 4.6	18.9 ± 2.9**	41.7 ± 6.3**	1043.9 ± 185.0**
Vehicle (n=5)	47.7 ± 5.2	8.4 ± 1.7*	19.1 ± 4.6*	624.5 ± 74.1*

Mean ± SEM.

^*P<0.05 compared to control;

^**P<0.05 compared to vehicle.

The hot-spot sizes on FastSPECT images matched consistently with the unstained areas on TTC staining. There was a significant correlation between the TTC infarct measurements and GLA FastSPECT imaging (r² = 0.775, P < 0.001).

Distribution of GLA in the hearts with ischaemia– reperfusion

The whole heart radioactivity (%ID/g) in the IPC (0.15 ± 0.03) and vehicle (0.14 ± 0.02) groups was significantly lower that in the control group (0.35 ± 0.04, P < 0.05), as well as in the SPT group (0.31 ± 0.06, P < 0.05), respectively. No statistical difference in the whole-heart radioactivity between the IPC group versus the vehicle group, as well as the control group versus the SPT group, was found (P > 0.05). Table 2 shows the ex-vivo gamma-counting results in dissected left ventricular myocardium. The area with negative TTC staining was considered as infarct tissue. In terms of the ratio of infarct/non-ischaemic viable myocardium, the control and SPT groups demonstrated a higher trend than the IPC and vehicle groups, but this did not reach statistical significance. The distribution of GLA in the IAR, including infarct zone and mixed-viability zone, was significantly higher than that in the remote viable tissues. Specifically, the ratio of IAR/non-ischaemic viable myocardium in the control group was higher than that in the IPC and vehicle groups, but not in the SPT group. As expected, the difference between the SPT group and vehicle group was significant.

Table 2.

Ex-vivo gamma counting results (ratios)

	Infarct/NIV	MV/NIV	IAR/NIV
IPC (n=6)	6.19 ± 1.45	1.66 ± 0.19*	3.04 ± 0.41*
Control (n=7)	9.62 ± 1.32	3.36 ± 0.53	7.37 ± 0.98**
SPT (n=6)	10.45 ± 1.92	4.87 ± 0.86***	7.37 ± 1.40**
Vehicle (n=5)	6.56 ± 1.11	2.38 ± 0.22	3.36 ± 0.48*

Mean ± SEM.

^*P<0.05 compared to Control;

^**P<0.05 compared to vehicle.

MV, mixed-viability; NIV, non-ischaemic viable; IAR, ischaemic area at risk (infarct and mixed-viability).

Discussion

Ischaemic heart disease is the single leading cause of death in the United States, accounting for one of every five deaths. It is also the leading cause of congestive heart failure. Reperfusion is regarded as the primary means of salvaging myocardium in patients with evolving infarction. However, even after blood flow is restored, reperfusion injury with myocyte death may occur after a critical period of coronary occlusion [25,26]. A variety of therapeutic strategies to prevent myocardial ischaemia– reperfusion injury are being developed, but prospective trials in human subjects require large numbers of patients over durations. Currently, most existing cardioprotective research data are based on animal heart models. A noninvasive approach to assess in-vivo pathophysiological progression in the animal models can bridge the knowledge gap in clinical translation of experimental interventions. In this present study, we were seeking to demonstrate the concept of applying stationary SPECT imaging techniques for non-invasive assessment of cardioprotection.

The time course of IPC protection consists of an initial early phase (2–3 h) and second late phase (12–24 h) [27]. The early phase is an immediate response caused by activation of membrane receptors and downstream kinases. The late phase is a sustained window of protection that lasts at least 72 h and requires synthesis of new proteins following genetic reprogramming of myocytes. The early-phase and late-phase cardioprotection may share certain triggers or mediators. Identified triggers to initiate ischaemic preconditioning include adenosine, bradykinin and catecholamines [28].

Adenosine has been proposed as an important mediator of IPC because it has the potential to exert cardioprotection during all three windows of cardioprotection, including preconditioning, ischaemia and reperfusion [29]. Adenosine mediates its cardioprotection via a cardiac potassium-activated ATP (K-ATP) channel-linked mechanism [28,30]. Although there are still controversies on how adenosine receptors (A₁, A_2A, A_2B and A₃) mediate the cardioprotection, it is generally accepted that the A₁ adenosine receptor is the primary receptor. Activation of the adenosine A₁ receptor plays a major role in most animal species tested, including rabbits, dogs, pigs and humans. In rat hearts, adenosine involvement in preconditioning has been positively reported in several isolated global ischaemia models and intact regional ischaemia models. However, the duration of preconditioning and endpoints to assess myocardial damage in the various studies have produced conflicting results regarding the involvement of adenosine in rat hearts [31].

In the present study, the role of IPC in cardioprotection via adenosine receptor activation was well demonstrated in rat heart models. Because of the higher threshold for producing IPC, we chose a slightly longer protocol of five cycles of 4 min of coronary artery occlusion. A significant tolerance to myocardial ischaemia–reperfusion injury was induced with the IPC protocol. Ischaemic–reperfused treatments in the two models with and without IPC produced an average infarct size (% IAR) of 21.0 ± 2.0 and 48.2 ± 6.3 with significant difference.

In terms of adenosine receptor blockade, we adopted a protocol similar to that reported in an isolated rat heart model by de Jonge et al. [21]. SPT was administrated by continuous infusion 2 min before and during subsequent 30 min ischaemia and 30 min reperfusion before GLA administration. As a result, SPT attenuated the benefits of IPC cardioprotection in the present study. This result is controversial to literature data by Ganote and Armstrong [20], in which it was concluded that adenosine does not involve preconditioning of rat hearts because adenosine receptor antagonists could not attenuate the cardioprotective effects of IPC. The higher interstitial adenosine in rat hearts than that in other species might be one of the reasons that SPT could not abolish the cardioprotection of IPC in the literature [16,21]. The short half-life of SPT (about 10 min) might be another reason for the failure to abolish the protection when it was intravenously injected as in the study reported by Li and Kloner [23]. Using alternative experimental protocols and administration of the adenosine agonist or antagonist, several studies in the literature have shown similar results as in this study. Headrick [16] used a cardiac microdialysis technique and demonstrated that IPC resulted in an interstitial adenosine increase in the rat hearts, which required more adenosine receptor antagonist to attenuate the cardioprotective effects. Peart and Gross [32] administered an adenosine kinase inhibitor, 5-iodotubercidin, to elevate adenosine concentrations and found that the elevated adenosine concentrations might activate adenosine receptors and lead to pronounced cardioprotection in an open-chest rat heart model with infarction.

GLA is a specific necrotic marker in the very early stages of myocyte injury, but it is not an apoptotic marker [33]. The uptake of GLA is associated with disruption of the myocyte and nuclear membranes, allowing free intracellular diffusion and electrochemical binding of the negatively charged glucarate complex to positively charged histones. This uptake mechanism is driven by the avidity of glucarate to the nuclear protein, mitochondrial and cytoplasmic proteins with a positive charge.

In the current study, the cardioprotection via the IPC pathway was assessed non-invasively by following observations relating to GLA necrosis-targeting properties. First, a smaller size of GLA hot-spot was found as a direct definition of the reduced injury extension by effective IPC protection in comparison with a larger hot-spot on constant myocardial ischaemic area-at-risk in the hearts without IPC treatment. The size of the hot-spot represents a good agreement with the infarct measurements on biochemical staining. Second, a faster kinetic washout of GLA and subsequent lower radioactive retention reflect the effectiveness of IPC against ischaemia–reperfusion injury. An accelerated washout of GLA from the regional ischaemic–reperfused myocardium was found in the animals with IPC. The ischaemic–reperfused hearts without IPC protection showed a slower radioactive washout, which resulted in a significant increase in the retention of GLA. When the cardio-protection of IPC was abolished by SPT treatment, the GLA retention resumed to the level in the hearts with no IPC protection. Third, the intensity of GLA distribution determined by FastSPECT imaging in the ischaemic–reperfused myocardium represents the severity of ischaemia–reperfusion injury, which can be evaluated by calculating the ratio of hot-spot to normal zone radioactivity in vivo. The ratios in the hearts with IPC treatment tend towards a plateau about 1 h after radiotracer injection. The ratios in the ischaemic– reperfused hearts with no IPC protection increased progressively following GLA administration and exhibited significant difference beginning at 60 min post-injection in comparison with that in the hearts with IPC. This result may imply that GLA is preferentially retained in ongoing injured myocardium after onset of ischaemia–reperfusion without sufficient cardioprotection. The exvivo measurements of GLA distribution in the ischaemic–reperfused hearts with and without IPC confirmed the in-vivo observations regarding GLA hot-spot uptake. Although there was no statistical difference in the measured ex-vivo radioactivity from dissected TTC-negative tissues for the control group versus the IPC group, as well as the SPT group versus the vehicle group, the overall radioactivity in the unprotected hearts was significantly higher than that in the IPC-protected hearts. More specifically, in terms of GLA distribution, there was a significant difference between the protected and unprotected hearts over the entire ischaemic area-at-risk. Scattered myocardial infarcts or micronecroses, which were ignored by TTC staining, might contribute to the increase of GLA uptake in the ischaemic area-at-risk determined by negative Evans blue and positive TTC staining.

FastSPECT is able to perform dynamic SPECT imaging for small animals such as rats and mice. A rapid sequence of 3-D images can be acquired in the rat heart model to study the kinetic washout of radiopharmaceuticals. Thus, the myocardial GLA radioactivity could be quantified, and the myocardial washout of GLA was determined effectively in the present study. Dynamic GLA Fast SPECT imaging takes advantage of the high spatial and temporal resolution of modern imaging techniques and provides an ideal investigational tool to evaluate various interventional protocols, such as ischaemic and pharmaceutical preconditioning. It provides investigators with a unique tool to assess myocytic injury, differentiate reversible and irreversible myocardial damage, and evaluate various cardioprotective strategies. Without sacrificing animals, myocardial injury and salvaged amounts can be determined by quantifying the size of hot-spots, measuring the accumulation of GLA, and calculating the kinetic washout of GLA. The ongoing cardioprotection can be examined in the early or delayed phase, depending on the experimental procedures.

Although an advanced stationary SPECT system was applied, there were several limitations in this study partly because of the small rat heart and lack of techniques. The low level of GLA accumulation in normal myocardium makes it difficult to calculate the percent of infarct over the whole left ventricle in hearts with small infarcts. Instead of calculating the percent infarct of the left ventricle, we quantified the number of pixels of the hot-spot on the tomographic images of GLA. One pixel equals 1 × 1 mm in the reconstructed images in our SPECT system; the size of the hot-spot could be estimated. It is also challenging to control the errors in the resulting hot-spot quantification because it is difficult to obtain ‘ground truth’ from which to find the boundary and segment of the image into infarct and non-infarct regions. Because there is essentially little or no GLA uptake in normal myocardium, the ROI location must be carefully selected to avoid the ROI on the early time-point images not overlapping into the ventricular chamber and being contaminated by the blood pool. Small changes in the ROI location could result in significant differences in the radioactive ratios of ischaemic zone to normal zone. In the current study, we applied tomographic blood-pool images to ensure the ROI establishment off the left ventricular cavity. The quantification of radioactive hot-spots in rats with smaller infarcts might be facilitated by the use of dual-isotope SPECT imaging with GLA and a perfusion tracer like ²⁰¹Tl. A dual-modality small-animal SPECT/CT system may be useful to identify the endocardial edges from X-ray CT by administration of contrast agents. Using the physically co-registered SPECT/CT images, the total radiotracer activity in the myocardium may be determined more accurately. We did not correct for radioactive scatter because the scatter influence from adjacent non-cardiac structures is minimal in FastSPECT. Gating SPECT imaging provides a valuable adjunct in enhancing the assessment of infarct avid imaging. However, gating imaging acquisition is impractical in FastSPECT, but is available in a new system, FastSPECT II, also built in our laboratory.

Conclusion

Significant tolerance to myocardial ischaemia–reperfusion injury, as assessed by biochemical assay and non-invasive infarct-avid imaging, was induced with an ischaemic preconditioning protocol in the rat model. The adenosine antagonist 8-(p-sulfophenyl)-theophylline could block the cardioprotection of ischaemic preconditioning. Thus, the preconditioning cardioprotection is in direct relationship with the activation of adenosine receptors in rat hearts. ^99mTc-glucarate FastSPECT imaging is not only useful in detecting early ischaemia–reperfusion injury, but is valuable in evaluating the effects of cardioprotective treatments. Quantitative analysis of dynamic images with ^99mTc-glucarate provides a unique tool for evaluation of cardioprotection. High-resolution imaging studies in small-animal models in cardiology permit longitudinal studies in the same animals. Such imaging studies, once validated, can yield data that will serve as biomarkers for effects of drug intervention.