Photochemistry and Photobiology, 20**, **: *–*
Hypericin as a Marker for Determination of Myocardial Viability in a Rat Model of Myocardial Infarction Cuihua Jiang1, Yue Li1, Xiao Jiang1, Nan Yao1, Meng Gao1, Xueli Zhang1, Junying Wang1, Xiaoning Wang1, Ziping Sun2, Jian Zhang*1 and Yicheng Ni1,2,3 1
Laboratory of Translational Medicine, Jiangsu Academy of Traditional Chinese Medicine, Nanjing, Jiangsu Province, China The Radiation Medical Institute, Shandong Academy of Medical Sciences, Jinan, Shandong Province, China 3 Theragnostic Laboratory, Department of Imaging & Pathology, Biomedical Sciences Group, KU Leuven, Leuven, Belgium 2
Received 1 April 2013, accepted 19 January 2014, DOI: 10.1111/php.12247
worsen the condition. Such “reperfusion damage” is associated with vascular injury, interstitial hemorrhage, disruption of membranes, release of liposomal enzymes, influx of calcium and cessation of functional activity (4). Instead, these patients should mainly receive symptomatic therapies, or heart transplantation. On the contrary, if there is still much viable myocardium in the infarcted area (e.g. stunned myocardium, myocardial hibernation), aggressive reperfusion measures may achieve functional and morphological recovery. Now, clinically dobutamine stress echocardiography (5), nuclear imaging (6), cardiac magnetic resonance imaging (cMRI) (7) and computed tomography (CT) are the mainstays of viability testing and can provide information on contractile function, cellular metabolism and myocardial fibrosis. However, they do not always permit an optimal differential diagnosis, and in many cases, the true condition tends to be underestimated (8). Hypericin, a photodynamic pigment from St. John’s Wort of the plant genus Hypericum, was shown to inhibit the motility and invasion of human malignant glioma cell in vitro via inhibition of protein kinase C or hexokinase bound to mitochondria (9,10) and it has reached phase II clinical trials as a treatment modality for malignant glioma (11). Meanwhile, hypericin sensitized human tumor cells to ionizing radiation in clinical detection of cancer and antitumoral photodynamic therapy (12–15). In the previous studies, hypericin was proved as a non-porphyrin necrosis-avid agent because of its special affinity for nonviable tissues (16,17). Moreover, hypericin revealed significant potentials as an imaging marker for early assessment of therapeutic response after percutaneous ethanol injection and radiofrequency ablation (18). Radiolabeled hypericin also showed a specific targetability in rodent necrosis models and selective accumulation in tumor necrotic tissues with the necrosis-to-viable ratio up to dozens of times (19–21). In small-molecular tumor necrosis therapy, radiolabeled hypericin combined with the novel vascular targeting agent combretastatin A4 phosphate exerted synergistic targeted theranostic effects (22). In view of these results, we thought that hypericin might hold promise for assessment of myocardial infarction, which has been preliminarily studied in rabbits using radiolabeled hypericin (23), but has never been tested in rats using native hypericin for this purpose. In this study, by utilizing its excellent fluorescent property and necrosis avidity, we qualitatively and quantitatively investigated hypericin in rat models of acute reperfused MI by means
ABSTRACT The aim of this study was to investigate the necrosis-avid agent hypericin as a potential indicator for determination of myocardial infarction (MI). Male Sprague-Dawley rats (n = 30) weighing 350 ± 20 g were subjected to acute reperfused MI. Animals were divided into four groups (n = 6), in which hypericin was intravenously injected at 0, 1, 2 and 5 mg kg 1 respectively. One day after injection, rats were euthanized with their hearts excised for qualitative and quantitative studies by means of microscopic fluorescence examination to decide the dosage of hypericin. Another group was injected with hypericin at the decided dose and evaluated by fluorescence macroscopy in colocalization with triphenyltetrazoliumchloride (TTC) and histomorphology. Infarctto-normal contrast ratio and relative infarct size were quantified. Hypericin-induced red fluorescence was significantly brighter in necrotic than in viable myocardium as proven by a six times higher mean fluorescence density. Mean MI area was 35.66 ± 22.88% by hypericin fluorescence and 32.73 ± 21.98% by TTC staining (R2 = 0.9803). Global MI-volume was 34.56 ± 21.07% by hypericin and 35.11 ± 20.47% by TTC staining (R2 = 0.9933). The results confirm that hypericin specifically labeled necrosis, and enhanced the imaging contrast between the infarcted and normal myocardium, suggesting its potential applications for the assessment of myocardial viability.
INTRODUCTION Myocardial infarction (MI) is a common, frequently occurring disorder with high mortality. The amount of dead tissue after acute MI is the most important prognostic indicator. In patients with coronary heart disease, it is critical to distinguish between reversible and irreversible ischemic myocardium, because timely and accurately identifying the exact viable and salvageable myocardium can be decisive for treatment option and ultimate fate of the patients (1). In an infarcted heart, stunned or hibernating myocardium and necrosis may coexist (2,3). If irreversible MI is already stabilized with little viable myocardium, revascularization interventions either hardly provide any curative benefit or even *Corresponding author email: [email protected] (Jian Zhang) © 2014 The American Society of Photobiology
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of macroscopic and microscopic fluorescence examinations in correlation with histochemical staining and histomorphology.
MATERIALS AND METHODS Rat model of myocardial infarction. Thirty male pathogen-free SpragueDawley rats, weighing 350 20 g, were obtained from the SLAC Laboratory Animal Co., Ltd, (Shanghai, China) to induce ischemic MI. The care and treatment of these rats were in accordance with the NIH publication 85-23 (revised in 1985) on “Principles of laboratory animal care”. Rats were sedated with intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL/100 g, then intubated and artificially ventilated with room air using a rodent ventilator at a respiratory rate of 60–80 strokes/ min, respiratory ratio of 1/1, and tidal volume of 4 mL/100 g during the procedure (Fig. 1). An incision was made along the third and fourth intercostal space on the left chest after skin sterilization. The pericardium was incised as soon as the pleural cavity was opened. Left anterior descending (LAD) coronary artery was located to be 1–2 mm below the junction of the pulmonary conus and the left atrial appendage as previously described (24). At 2 mm depth and 2–3 mm width, a 5.0 silk suture with a curve needle was used to ligate the LAD with a single detachable knot. The chest wall was stitched, with the end of the suture of detachable knot externalized, by layers of 4.0 silk sutures. Forty-five minutes after LAD occlusion, the suture end was pulled to achieve coronary reperfusion in the closed-chest condition while 0.2 mL of 4 mg Xylocaine (AstraZeneca LP, Wilmington, DE) was intravenously infused to avoid arrhythmia. The tracheal catheter was removed after recovery of spontaneous respiration and the rat was given intramuscular injection of penicillin for preventing infection. Hypericin preparation. Hypericin was commercially supplied (Purui Technology Co. Ltd, Chengdu, China) with a purity greater than 98.5%. Before intravenous injection, hypericin was dissolved in a mixture of 25% dimethylsulfoxide, 25% polyethylene glycol 400, 25% propylene glycol and 25% distilled water at a concentration of 1, 2, 5 mg mL 1. The solution was dark red. Study design. As shown in the experimental design (Fig. 2), 24 rats with MI were divided into four groups receiving null, low, medium and high dosage of hypericin intravenously injected via a tail vein at 0, 1, 2, 5 mg kg 1 respectively. Rats were euthanized 24 h after hypericin injection. Hearts were removed and dissected for cryostat sections, which were examined by fluorescence microscopy and hematoxylin and eosin
(H&E) stained histopathology. According to fluorescence intensity of the heart sections and solubility of the solution, we determined the appropriate dose of hypericin. In the further study with another group of six rats, after injection of hypericin at the defined dose, the hearts with MI were removed and dissected for cryostat sections. Then the dissected hearts were qualitatively and quantitatively evaluated by correlating the findings of fluorescence macroscopy and triphenyltetra-zolium chloride (TTC) histochemical staining for the MI size. Fluoromacroscopic and fluoromicroscopic examinations. Twenty-four hours post hypericin injection the animals were sacrificed with an intravenous overdose of 10% chloral hydrate. The rat heart was excised and first photographed under tungsten light and UV366 light to assess gross distribution of fluorescent hypericin in the infarct areas and normal myocardium. Then, the excised hearts were cut into 3 mm-thick blocks perpendicular to the long axis starting from the apex, which were quickly frozen in isopentane cooled to 40°C with dry ice. Cryostat sections of 5 lm slice thickness were made from each heart and detected with fluorescence microscopy (Axioskop 2 plus), which was equipped with a light-sensitive, charge-coupled device digital camera (AxioCam HR, Carl Zeiss, G€ottingen, Germany). The fluorescent microscopy images were acquired from the Zeiss filter set 14 (excitation: BP 510–560 nm, emission: LP 590 nm). Finally, tissue slices were stained with hematoxylin and eosin (H&E) and examined using conventional light microscopy. Histochemical staining. The MI was determined by TTC enzymatic histochemical staining, which is regarded as a gold standard for macroscopic infarct size determination. The 3 mm-thick myocardial tissue blocks were stained with 2% TTC solution and kept for 10 min at 37°C in the dark then they were fixed with 10% neutral buffered formalin and photographed using a digital camera. Quantitative imaging analysis. A clear distinction between necrotic and normal tissue was confirmed based on the H&E-stained histological examination. By means of manually drawing regions of interest in the areas of infarcted and viable myocardium, quantitative results of fluoromicroscopic images were obtained. Fluoromicroscopic images were corrected for background and mean fluorescence densities of different slices were measured with an imaging software system (Image-Pro-Plus, IPP). To evaluate the TTC-stained infarct volume (IV) and fluoromacroscopic volume (FV), we first calculated infarct and ventricular areas, averaged the values from the two sides of each cardiac block, multiplied the slice thickness to obtain the volumes, and finally correlated infarct volumes defined from histochemical and fluoromacroscopic findings.
Figure 1. A modified method for inducing rat models of reperfused MI, (a) The rat was intubated and ventilated, (b) Open-chest exposure of LAD, (c) The LAD was localized and ligated with a curve needle, (d) The chest was closed waiting for reperfusion by pulling the single-nodded suture around the LAD.
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Figure 2. Flow chart of experimental procedure. Note: h: hours; MI: myocardial infarction; n: number of rats; iv: intravenous injection; HE: hematoxylin and eosin; TTC: triphenyltetra-zolium chloride.
Statistics. Numerical results were expressed as meansSD. The statistical analysis was performed by the analysis of variance (one-way ANOVA) followed by Student Newman–Keuls test and linear correlation test. Differences were considered statistically significant at P < 0.05.
RESULTS General conditions Despite relatively small size, surgical operation for creating reperfused MI in rats was successful in this experiment after a beforehand training. Using the transoral method, the rats could be intubated successfully in few minutes. The acute reperfused MI was induced using open-chest coronary occlusion but closechest reperfusion method, which was reported previously only for rabbit experiment (25), but now was reproduced in rats with a success rate above 90% in this study. One rat died during surgery due to overdose of anesthesia and the other two died of acute cardiac arrest. Quantitative outcomes of fluoromicroscopy Fluorescence photomicrography confirmed different fluorescence intensities between cardiac slices of different hypericin dose groups (Fig. 3). During the 24 h after injection, hypericin was predominantly cleared from the normal tissues in contrast to necrotic tissue where hypericin was retained. Fluoromicroscopic images revealed the absence of fluorescence in rats of blank control and a distinct red fluorescence in necrotic myocardial tissue in rats injected with hypericin. Quantitatively, mean fluorescence density ratios (infarct-to-normal) in groups of different intravenous doses of hypericin at 0, 1, 2, 5 mg kg 1 were 1, 2.11 0.25, 6.43 0.96 and 6.40 0.72 respectively. Overall, data indicated four different levels of fluorescence density in necrotic tissue (Table 1). On the basis of these data, we chose 2 mg kg 1 as a diagnostic dose for the next experiment. Macroscopic fluorescence imaging versus TTC staining results of infarct size Altogether 36 pairs of photographs from UV–light fluoromacroscopic images and corresponding digital pictures of TTC-stained heart slices from six rats were taken for analysis. At the border between necrotic and viable tissue, a clear-cut fluorescence signal was seen (Fig. 4). The mean infarct size measured on TTC staining approximated that on fluoromacroscopic images. No significant
Figure 3. Photomicrographs of unstained frozen slides (left), fluorescent photomicrocopy (middle) and corresponding tungsten light photograph of HE stained slides of rat hearts with MI. Rats were sacrificed 24 h after intravenous injection of hypericin at 0 (1st row), 1 (2nd row), 2 (3rd row), and 5 (4th row) mg kg 1 in proper order (a–d). Note: V: viable myocardium; N: necrotic myocardium. Scale bar = 100 lm. Table 1. Ratios of mean fluorescence densities from necrotic (N) over viable (V) myocardium taken from groups of rats intravenously injected with different doses of hypericin. 1
Ratio\Group
0
1 mg kg
N/V ratio (n = 6)
1
2.11 0.25
2 mg kg
1
6.43 0.96
5 mg kg
1
6.40 0.72
N, necrosis; V, viable; n, number of heart slices. Data are presented as mean SD.
differences in infarct size between groups were observed by fluoromacroscopy- and TTC-based measurements. Linear correlation analyses on both slice-by-slice infarct area and heart-by-heart infarct volume showed excellent correspondence between fluoromacroscopy and TTC staining (Figs. 4 and 5). Mean relative
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Figure 4. Macroscopic photographs of 3-mm slices of rat hearts taken under ultraviolet light (middle row) and corresponding photographs of fresh heart slices (upper row), and TTC stained heart slices (lower row) under tungsten light. Rats were sacrificed 24 h after intravenous injection of hypericin at 2 mg kg 1 and the excised heart slices were digitally photographed. Slice-by-slice (both sides) analyses of infarct size were made using IPP imaging software system.
MI-areas were quantified as 35.66 22.88% for fluoromacroscopic images and 32.73 21.98% for TTC staining (R2 = 0.9803). Similarly, global relative MI-volume was 35.11 20.47% on fluoromacroscopic images and 34.56 21.07% on TTC-stained slides (R2 = 0.9933) (Table 2).
DISCUSSION Preclinical models of MI prove to be important in investigating pathophysiological mechanisms and therapeutic strategies. Since
1970s, rat models have been established for experimental cardiac research, because of their physiological features similar to that of humans (26). Furthermore, compared to other animals such as dogs or cats, rat models of MI are with lower costs, easier manipulation, higher survival rate, richer availability and less ethical concerns (27). Such quantifiable rat models have been used to investigate pathophysiology and pharmacology of MI and to test the efficacy of new therapies prior to clinical studies (28). Novel noninvasive imaging techniques such as magnetic resonance imaging, single photon emission computed tomographycomputed tomography (SPECT-CT) and positron emission tomography have been applied for diagnosis of MI and estimation of infarct size in clinical cardiology and preclinical studies (29). The main advantage of using these imagers is the ability to noninvasively and repeatedly measure infarct size in vivo for monitoring evolution of pathophysiology as well as therapeutic response. However, both economical constrains and technical limits would make it a long way to go for these novel technologies to be used as routine laboratory tools. Histochemical TTC analysis is the only widely accepted gold standard for macroscopic identification and quantification of acute MI in preclinical studies. But it is a postmortem technique and not clinically applicable. Furthermore, separated groups of animals are needed in longitudinal studies (30). Therefore, in vivo imaging markers for absolutely nonviable tissue, in contrast to viable myocardium with intact cellular functions of preserved metabolic activity and cell membrane integrity, something similar to ex vivo TTC staining, are eagerly needed for clinical and experimental determination of myocardial viability. Recently, hypericin has been proven to possess an unusual avidity for irreversibly damaged ischemic or necrotic tissues. Van de Putte et al. demonstrated that hypericin was considered to significantly improved the imaging contrast between necrosis and viable tumor or normal liver tissues by means of fluoromacroscopic and fluoromicroscopic examinations (8,22). In this experimental study, we designed a rat model of reperfused MI by ligating and reopening the LAD to evaluate the potential of hypericin for use as a marker of negative viability for possible future studies on MI. As demonstrated in this study, no significant difference between groups was observed for fluoromicroscopy- and TTC-based MI measurements. Both slice-by-slice area and heart-by-heart volume linear correlation analyses showed
Figure 5. Comparisons of infarct sizes between fluoromacroscopic and TTC stained images. Linear regression analysis plots of TTC stained IA and IV versus fluoromacroscopically labeled FA and FV are presented. The correlation coefficients are R2 = 0.9933 and R2 = 0.9803 respectively. Note: LVA: left ventricular area; LVV: left ventricular volume.
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Table 2. Slice-by-slice and global analyses on fluoromacroscopic images and TTC-based measurements of infarct size and the differences between fluoromacroscopic images and TTC staining examined by Student-Newman–Keuls test. Analysis approach
Fluoromacroscopic images
Slice-by-slice (n = 36) Infarct size (% of LVA) Global (n = 6) Infarct size (% of LVV)
FA 35.66 22.88 FV 35.11 20.47
TTC staining
Correlation coefficient*
P†
IA 32.73 21.98 IV 34.56 21.07
0.9803
P > 0.05
0.9933
P > 0.05
FA, fluorescent area; IA, infarct area; FV, fluorescent volume; IV, infarct volume; n, number of slices or hearts. Data are presented as mean SD. *Obtained from linear correlation analysis; †Obtained from the Student-Newman–Keuls test, P < 0.05 indicates a significant difference.
an excellent correspondence between fluoromacroscopic images and TTC-stained specimens with regard to the quantification of infarct size. The results suggest that, complementary to TTC as a marker of positive viability, hypericin offers significant potential in the assessment of infarct size following acute MI. On one hand, it can be used as a virtual “gold standard” to monitor the natural evolution of MI and to evaluate the therapeutic effects of myocardial protection drugs under development. On the other hand, it can be helpful for the determination of myocardial viability of the regions that fail to show ventricular motion or wall thickening, e.g. in scenarios of stunning or hibernation. Similar to the limits of TTC, hypericin is only effective for acute MI but not for early healing MI where present viable granulation tissues that are positively stained by TTC but negatively marked by hypericin. Another crucial issue is how to couple the strong necrosis-avid property of hypericin with any practical imaging modalities. The outstanding fluorescence of native hypericin enables its use for optical imaging, which is limited by light penetration depth and 3D reconstruction for tomography. Hypericin has been radioiodinated for enhancing cardiac SPECT (23), and more efforts are needed for exploring its implementations into other imaging modalities. Hypericin, a potent necrosis-avid agent, features a peculiar affinity for irreversibly damaged ischemic or necrotic tissues, but the mechanism remained unclear. Necrotic cell death is typically associated with loss of membrane integrity, inflammation and activated immune system due to the release of heat shock proteins and degraded cellular peptides (31–33). The behavior of hypericin may specifically associate with degraded proteins or peptides present in the necrotic milieu. In a recent study, a release of hypericin from the lipoprotein complex at some point along its way through the peri-necrotic tumor area and the necrotic tissue debris was discovered, which finding is in line with the idea of a compound-specific mechanism (34) but opposed to the albumin-related trapping mechanism proposed by Hofmann and coworkers (35). Phosphatidylserine (PS) is preferentially distributed in the inner plasma membrane leaflet. When cell death occurs, PS exposes in the outer leaflet of the cell membrane (36). Small-molecular-weight 64Cu-Bis-DOTA conjugation to hypericin had been shown the affinity for injured tissues which might be attributed to the breakdown of the cell membrane and exposure of PS or phosphatidylethanolamine to the radiotracer (37). Hypothetically, the necrotic affinity of hypericin may specifically associate with cell membrane lipids. Further investigations are needed to elucidate the specific interactions between hypericin and the necrotic debris. Therefore, for the time being, all available cardiac imaging modalities should be used to complement to each other. We believe that further research on the necrosis-avid hypericin will
open new opportunities in both clinical and preclinical settings to provide unambiguous diagnoses and optimal therapies, which eventually will benefit cardiac patients. Acknowledgements—This study was partially supported by the grants awarded by the National Natural Science Foundation of China (no. 81071828) and Jiangsu Province Natural Science Foundation (BK2010594). The author Yicheng Ni is currently a Bayer Lecture Chair holder.
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Hypericin as a Marker for Determination of Myocardial Viability in a Rat Model of Myocardial Infarction Cuihua Jiang1, Yue Li1, Xiao Jiang1, Nan Yao1, Meng Gao1, Xueli Zhang1, Junying Wang1, Xiaoning Wang1, Ziping Sun2, Jian Zhang*1 and Yicheng Ni1,2,3 1
Laboratory of Translational Medicine, Jiangsu Academy of Traditional Chinese Medicine, Nanjing, Jiangsu Province, China The Radiation Medical Institute, Shandong Academy of Medical Sciences, Jinan, Shandong Province, China 3 Theragnostic Laboratory, Department of Imaging & Pathology, Biomedical Sciences Group, KU Leuven, Leuven, Belgium 2
Received 1 April 2013, accepted 19 January 2014, DOI: 10.1111/php.12247
worsen the condition. Such “reperfusion damage” is associated with vascular injury, interstitial hemorrhage, disruption of membranes, release of liposomal enzymes, influx of calcium and cessation of functional activity (4). Instead, these patients should mainly receive symptomatic therapies, or heart transplantation. On the contrary, if there is still much viable myocardium in the infarcted area (e.g. stunned myocardium, myocardial hibernation), aggressive reperfusion measures may achieve functional and morphological recovery. Now, clinically dobutamine stress echocardiography (5), nuclear imaging (6), cardiac magnetic resonance imaging (cMRI) (7) and computed tomography (CT) are the mainstays of viability testing and can provide information on contractile function, cellular metabolism and myocardial fibrosis. However, they do not always permit an optimal differential diagnosis, and in many cases, the true condition tends to be underestimated (8). Hypericin, a photodynamic pigment from St. John’s Wort of the plant genus Hypericum, was shown to inhibit the motility and invasion of human malignant glioma cell in vitro via inhibition of protein kinase C or hexokinase bound to mitochondria (9,10) and it has reached phase II clinical trials as a treatment modality for malignant glioma (11). Meanwhile, hypericin sensitized human tumor cells to ionizing radiation in clinical detection of cancer and antitumoral photodynamic therapy (12–15). In the previous studies, hypericin was proved as a non-porphyrin necrosis-avid agent because of its special affinity for nonviable tissues (16,17). Moreover, hypericin revealed significant potentials as an imaging marker for early assessment of therapeutic response after percutaneous ethanol injection and radiofrequency ablation (18). Radiolabeled hypericin also showed a specific targetability in rodent necrosis models and selective accumulation in tumor necrotic tissues with the necrosis-to-viable ratio up to dozens of times (19–21). In small-molecular tumor necrosis therapy, radiolabeled hypericin combined with the novel vascular targeting agent combretastatin A4 phosphate exerted synergistic targeted theranostic effects (22). In view of these results, we thought that hypericin might hold promise for assessment of myocardial infarction, which has been preliminarily studied in rabbits using radiolabeled hypericin (23), but has never been tested in rats using native hypericin for this purpose. In this study, by utilizing its excellent fluorescent property and necrosis avidity, we qualitatively and quantitatively investigated hypericin in rat models of acute reperfused MI by means
ABSTRACT The aim of this study was to investigate the necrosis-avid agent hypericin as a potential indicator for determination of myocardial infarction (MI). Male Sprague-Dawley rats (n = 30) weighing 350 ± 20 g were subjected to acute reperfused MI. Animals were divided into four groups (n = 6), in which hypericin was intravenously injected at 0, 1, 2 and 5 mg kg 1 respectively. One day after injection, rats were euthanized with their hearts excised for qualitative and quantitative studies by means of microscopic fluorescence examination to decide the dosage of hypericin. Another group was injected with hypericin at the decided dose and evaluated by fluorescence macroscopy in colocalization with triphenyltetrazoliumchloride (TTC) and histomorphology. Infarctto-normal contrast ratio and relative infarct size were quantified. Hypericin-induced red fluorescence was significantly brighter in necrotic than in viable myocardium as proven by a six times higher mean fluorescence density. Mean MI area was 35.66 ± 22.88% by hypericin fluorescence and 32.73 ± 21.98% by TTC staining (R2 = 0.9803). Global MI-volume was 34.56 ± 21.07% by hypericin and 35.11 ± 20.47% by TTC staining (R2 = 0.9933). The results confirm that hypericin specifically labeled necrosis, and enhanced the imaging contrast between the infarcted and normal myocardium, suggesting its potential applications for the assessment of myocardial viability.
INTRODUCTION Myocardial infarction (MI) is a common, frequently occurring disorder with high mortality. The amount of dead tissue after acute MI is the most important prognostic indicator. In patients with coronary heart disease, it is critical to distinguish between reversible and irreversible ischemic myocardium, because timely and accurately identifying the exact viable and salvageable myocardium can be decisive for treatment option and ultimate fate of the patients (1). In an infarcted heart, stunned or hibernating myocardium and necrosis may coexist (2,3). If irreversible MI is already stabilized with little viable myocardium, revascularization interventions either hardly provide any curative benefit or even *Corresponding author email: [email protected] (Jian Zhang) © 2014 The American Society of Photobiology
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of macroscopic and microscopic fluorescence examinations in correlation with histochemical staining and histomorphology.
MATERIALS AND METHODS Rat model of myocardial infarction. Thirty male pathogen-free SpragueDawley rats, weighing 350 20 g, were obtained from the SLAC Laboratory Animal Co., Ltd, (Shanghai, China) to induce ischemic MI. The care and treatment of these rats were in accordance with the NIH publication 85-23 (revised in 1985) on “Principles of laboratory animal care”. Rats were sedated with intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL/100 g, then intubated and artificially ventilated with room air using a rodent ventilator at a respiratory rate of 60–80 strokes/ min, respiratory ratio of 1/1, and tidal volume of 4 mL/100 g during the procedure (Fig. 1). An incision was made along the third and fourth intercostal space on the left chest after skin sterilization. The pericardium was incised as soon as the pleural cavity was opened. Left anterior descending (LAD) coronary artery was located to be 1–2 mm below the junction of the pulmonary conus and the left atrial appendage as previously described (24). At 2 mm depth and 2–3 mm width, a 5.0 silk suture with a curve needle was used to ligate the LAD with a single detachable knot. The chest wall was stitched, with the end of the suture of detachable knot externalized, by layers of 4.0 silk sutures. Forty-five minutes after LAD occlusion, the suture end was pulled to achieve coronary reperfusion in the closed-chest condition while 0.2 mL of 4 mg Xylocaine (AstraZeneca LP, Wilmington, DE) was intravenously infused to avoid arrhythmia. The tracheal catheter was removed after recovery of spontaneous respiration and the rat was given intramuscular injection of penicillin for preventing infection. Hypericin preparation. Hypericin was commercially supplied (Purui Technology Co. Ltd, Chengdu, China) with a purity greater than 98.5%. Before intravenous injection, hypericin was dissolved in a mixture of 25% dimethylsulfoxide, 25% polyethylene glycol 400, 25% propylene glycol and 25% distilled water at a concentration of 1, 2, 5 mg mL 1. The solution was dark red. Study design. As shown in the experimental design (Fig. 2), 24 rats with MI were divided into four groups receiving null, low, medium and high dosage of hypericin intravenously injected via a tail vein at 0, 1, 2, 5 mg kg 1 respectively. Rats were euthanized 24 h after hypericin injection. Hearts were removed and dissected for cryostat sections, which were examined by fluorescence microscopy and hematoxylin and eosin
(H&E) stained histopathology. According to fluorescence intensity of the heart sections and solubility of the solution, we determined the appropriate dose of hypericin. In the further study with another group of six rats, after injection of hypericin at the defined dose, the hearts with MI were removed and dissected for cryostat sections. Then the dissected hearts were qualitatively and quantitatively evaluated by correlating the findings of fluorescence macroscopy and triphenyltetra-zolium chloride (TTC) histochemical staining for the MI size. Fluoromacroscopic and fluoromicroscopic examinations. Twenty-four hours post hypericin injection the animals were sacrificed with an intravenous overdose of 10% chloral hydrate. The rat heart was excised and first photographed under tungsten light and UV366 light to assess gross distribution of fluorescent hypericin in the infarct areas and normal myocardium. Then, the excised hearts were cut into 3 mm-thick blocks perpendicular to the long axis starting from the apex, which were quickly frozen in isopentane cooled to 40°C with dry ice. Cryostat sections of 5 lm slice thickness were made from each heart and detected with fluorescence microscopy (Axioskop 2 plus), which was equipped with a light-sensitive, charge-coupled device digital camera (AxioCam HR, Carl Zeiss, G€ottingen, Germany). The fluorescent microscopy images were acquired from the Zeiss filter set 14 (excitation: BP 510–560 nm, emission: LP 590 nm). Finally, tissue slices were stained with hematoxylin and eosin (H&E) and examined using conventional light microscopy. Histochemical staining. The MI was determined by TTC enzymatic histochemical staining, which is regarded as a gold standard for macroscopic infarct size determination. The 3 mm-thick myocardial tissue blocks were stained with 2% TTC solution and kept for 10 min at 37°C in the dark then they were fixed with 10% neutral buffered formalin and photographed using a digital camera. Quantitative imaging analysis. A clear distinction between necrotic and normal tissue was confirmed based on the H&E-stained histological examination. By means of manually drawing regions of interest in the areas of infarcted and viable myocardium, quantitative results of fluoromicroscopic images were obtained. Fluoromicroscopic images were corrected for background and mean fluorescence densities of different slices were measured with an imaging software system (Image-Pro-Plus, IPP). To evaluate the TTC-stained infarct volume (IV) and fluoromacroscopic volume (FV), we first calculated infarct and ventricular areas, averaged the values from the two sides of each cardiac block, multiplied the slice thickness to obtain the volumes, and finally correlated infarct volumes defined from histochemical and fluoromacroscopic findings.
Figure 1. A modified method for inducing rat models of reperfused MI, (a) The rat was intubated and ventilated, (b) Open-chest exposure of LAD, (c) The LAD was localized and ligated with a curve needle, (d) The chest was closed waiting for reperfusion by pulling the single-nodded suture around the LAD.
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Figure 2. Flow chart of experimental procedure. Note: h: hours; MI: myocardial infarction; n: number of rats; iv: intravenous injection; HE: hematoxylin and eosin; TTC: triphenyltetra-zolium chloride.
Statistics. Numerical results were expressed as meansSD. The statistical analysis was performed by the analysis of variance (one-way ANOVA) followed by Student Newman–Keuls test and linear correlation test. Differences were considered statistically significant at P < 0.05.
RESULTS General conditions Despite relatively small size, surgical operation for creating reperfused MI in rats was successful in this experiment after a beforehand training. Using the transoral method, the rats could be intubated successfully in few minutes. The acute reperfused MI was induced using open-chest coronary occlusion but closechest reperfusion method, which was reported previously only for rabbit experiment (25), but now was reproduced in rats with a success rate above 90% in this study. One rat died during surgery due to overdose of anesthesia and the other two died of acute cardiac arrest. Quantitative outcomes of fluoromicroscopy Fluorescence photomicrography confirmed different fluorescence intensities between cardiac slices of different hypericin dose groups (Fig. 3). During the 24 h after injection, hypericin was predominantly cleared from the normal tissues in contrast to necrotic tissue where hypericin was retained. Fluoromicroscopic images revealed the absence of fluorescence in rats of blank control and a distinct red fluorescence in necrotic myocardial tissue in rats injected with hypericin. Quantitatively, mean fluorescence density ratios (infarct-to-normal) in groups of different intravenous doses of hypericin at 0, 1, 2, 5 mg kg 1 were 1, 2.11 0.25, 6.43 0.96 and 6.40 0.72 respectively. Overall, data indicated four different levels of fluorescence density in necrotic tissue (Table 1). On the basis of these data, we chose 2 mg kg 1 as a diagnostic dose for the next experiment. Macroscopic fluorescence imaging versus TTC staining results of infarct size Altogether 36 pairs of photographs from UV–light fluoromacroscopic images and corresponding digital pictures of TTC-stained heart slices from six rats were taken for analysis. At the border between necrotic and viable tissue, a clear-cut fluorescence signal was seen (Fig. 4). The mean infarct size measured on TTC staining approximated that on fluoromacroscopic images. No significant
Figure 3. Photomicrographs of unstained frozen slides (left), fluorescent photomicrocopy (middle) and corresponding tungsten light photograph of HE stained slides of rat hearts with MI. Rats were sacrificed 24 h after intravenous injection of hypericin at 0 (1st row), 1 (2nd row), 2 (3rd row), and 5 (4th row) mg kg 1 in proper order (a–d). Note: V: viable myocardium; N: necrotic myocardium. Scale bar = 100 lm. Table 1. Ratios of mean fluorescence densities from necrotic (N) over viable (V) myocardium taken from groups of rats intravenously injected with different doses of hypericin. 1
Ratio\Group
0
1 mg kg
N/V ratio (n = 6)
1
2.11 0.25
2 mg kg
1
6.43 0.96
5 mg kg
1
6.40 0.72
N, necrosis; V, viable; n, number of heart slices. Data are presented as mean SD.
differences in infarct size between groups were observed by fluoromacroscopy- and TTC-based measurements. Linear correlation analyses on both slice-by-slice infarct area and heart-by-heart infarct volume showed excellent correspondence between fluoromacroscopy and TTC staining (Figs. 4 and 5). Mean relative
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Figure 4. Macroscopic photographs of 3-mm slices of rat hearts taken under ultraviolet light (middle row) and corresponding photographs of fresh heart slices (upper row), and TTC stained heart slices (lower row) under tungsten light. Rats were sacrificed 24 h after intravenous injection of hypericin at 2 mg kg 1 and the excised heart slices were digitally photographed. Slice-by-slice (both sides) analyses of infarct size were made using IPP imaging software system.
MI-areas were quantified as 35.66 22.88% for fluoromacroscopic images and 32.73 21.98% for TTC staining (R2 = 0.9803). Similarly, global relative MI-volume was 35.11 20.47% on fluoromacroscopic images and 34.56 21.07% on TTC-stained slides (R2 = 0.9933) (Table 2).
DISCUSSION Preclinical models of MI prove to be important in investigating pathophysiological mechanisms and therapeutic strategies. Since
1970s, rat models have been established for experimental cardiac research, because of their physiological features similar to that of humans (26). Furthermore, compared to other animals such as dogs or cats, rat models of MI are with lower costs, easier manipulation, higher survival rate, richer availability and less ethical concerns (27). Such quantifiable rat models have been used to investigate pathophysiology and pharmacology of MI and to test the efficacy of new therapies prior to clinical studies (28). Novel noninvasive imaging techniques such as magnetic resonance imaging, single photon emission computed tomographycomputed tomography (SPECT-CT) and positron emission tomography have been applied for diagnosis of MI and estimation of infarct size in clinical cardiology and preclinical studies (29). The main advantage of using these imagers is the ability to noninvasively and repeatedly measure infarct size in vivo for monitoring evolution of pathophysiology as well as therapeutic response. However, both economical constrains and technical limits would make it a long way to go for these novel technologies to be used as routine laboratory tools. Histochemical TTC analysis is the only widely accepted gold standard for macroscopic identification and quantification of acute MI in preclinical studies. But it is a postmortem technique and not clinically applicable. Furthermore, separated groups of animals are needed in longitudinal studies (30). Therefore, in vivo imaging markers for absolutely nonviable tissue, in contrast to viable myocardium with intact cellular functions of preserved metabolic activity and cell membrane integrity, something similar to ex vivo TTC staining, are eagerly needed for clinical and experimental determination of myocardial viability. Recently, hypericin has been proven to possess an unusual avidity for irreversibly damaged ischemic or necrotic tissues. Van de Putte et al. demonstrated that hypericin was considered to significantly improved the imaging contrast between necrosis and viable tumor or normal liver tissues by means of fluoromacroscopic and fluoromicroscopic examinations (8,22). In this experimental study, we designed a rat model of reperfused MI by ligating and reopening the LAD to evaluate the potential of hypericin for use as a marker of negative viability for possible future studies on MI. As demonstrated in this study, no significant difference between groups was observed for fluoromicroscopy- and TTC-based MI measurements. Both slice-by-slice area and heart-by-heart volume linear correlation analyses showed
Figure 5. Comparisons of infarct sizes between fluoromacroscopic and TTC stained images. Linear regression analysis plots of TTC stained IA and IV versus fluoromacroscopically labeled FA and FV are presented. The correlation coefficients are R2 = 0.9933 and R2 = 0.9803 respectively. Note: LVA: left ventricular area; LVV: left ventricular volume.
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Table 2. Slice-by-slice and global analyses on fluoromacroscopic images and TTC-based measurements of infarct size and the differences between fluoromacroscopic images and TTC staining examined by Student-Newman–Keuls test. Analysis approach
Fluoromacroscopic images
Slice-by-slice (n = 36) Infarct size (% of LVA) Global (n = 6) Infarct size (% of LVV)
FA 35.66 22.88 FV 35.11 20.47
TTC staining
Correlation coefficient*
P†
IA 32.73 21.98 IV 34.56 21.07
0.9803
P > 0.05
0.9933
P > 0.05
FA, fluorescent area; IA, infarct area; FV, fluorescent volume; IV, infarct volume; n, number of slices or hearts. Data are presented as mean SD. *Obtained from linear correlation analysis; †Obtained from the Student-Newman–Keuls test, P < 0.05 indicates a significant difference.
an excellent correspondence between fluoromacroscopic images and TTC-stained specimens with regard to the quantification of infarct size. The results suggest that, complementary to TTC as a marker of positive viability, hypericin offers significant potential in the assessment of infarct size following acute MI. On one hand, it can be used as a virtual “gold standard” to monitor the natural evolution of MI and to evaluate the therapeutic effects of myocardial protection drugs under development. On the other hand, it can be helpful for the determination of myocardial viability of the regions that fail to show ventricular motion or wall thickening, e.g. in scenarios of stunning or hibernation. Similar to the limits of TTC, hypericin is only effective for acute MI but not for early healing MI where present viable granulation tissues that are positively stained by TTC but negatively marked by hypericin. Another crucial issue is how to couple the strong necrosis-avid property of hypericin with any practical imaging modalities. The outstanding fluorescence of native hypericin enables its use for optical imaging, which is limited by light penetration depth and 3D reconstruction for tomography. Hypericin has been radioiodinated for enhancing cardiac SPECT (23), and more efforts are needed for exploring its implementations into other imaging modalities. Hypericin, a potent necrosis-avid agent, features a peculiar affinity for irreversibly damaged ischemic or necrotic tissues, but the mechanism remained unclear. Necrotic cell death is typically associated with loss of membrane integrity, inflammation and activated immune system due to the release of heat shock proteins and degraded cellular peptides (31–33). The behavior of hypericin may specifically associate with degraded proteins or peptides present in the necrotic milieu. In a recent study, a release of hypericin from the lipoprotein complex at some point along its way through the peri-necrotic tumor area and the necrotic tissue debris was discovered, which finding is in line with the idea of a compound-specific mechanism (34) but opposed to the albumin-related trapping mechanism proposed by Hofmann and coworkers (35). Phosphatidylserine (PS) is preferentially distributed in the inner plasma membrane leaflet. When cell death occurs, PS exposes in the outer leaflet of the cell membrane (36). Small-molecular-weight 64Cu-Bis-DOTA conjugation to hypericin had been shown the affinity for injured tissues which might be attributed to the breakdown of the cell membrane and exposure of PS or phosphatidylethanolamine to the radiotracer (37). Hypothetically, the necrotic affinity of hypericin may specifically associate with cell membrane lipids. Further investigations are needed to elucidate the specific interactions between hypericin and the necrotic debris. Therefore, for the time being, all available cardiac imaging modalities should be used to complement to each other. We believe that further research on the necrosis-avid hypericin will
open new opportunities in both clinical and preclinical settings to provide unambiguous diagnoses and optimal therapies, which eventually will benefit cardiac patients. Acknowledgements—This study was partially supported by the grants awarded by the National Natural Science Foundation of China (no. 81071828) and Jiangsu Province Natural Science Foundation (BK2010594). The author Yicheng Ni is currently a Bayer Lecture Chair holder.
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