Original contribution
Vascular lesions and s-thrombomodulin concentrations from auricular arteries of rabbits infused with microbubble contrast agent and exposed to pulsed ultrasound

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Abstract

Arterial injury resulting from the interaction of contrast agent (CA) with ultrasound (US) was studied in rabbit auricular arteries and assessed by histopathologic evaluation and s-thrombomodulin concentrations. Three sites on each artery were exposed (2.8 MHz, 5-min exposure duration, 10-Hz pulse repetition frequency, 1.4-μs pulse duration) using one of three in situ peak rarefactional pressures (0.85, 3.9 or 9.5 MPa). Saline, saline/CA, and saline/US infusion groups (n = 28) did not have histopathologic damage. The saline/CA/US infusion group (n = 10) at exposure conditions below the FDA mechanical index limit of 1.9 did not have histopathologic damage, whereas the saline/CA/US infusion group (n = 9) at exposure conditions above the FDA limit did have damage (5 of 9 arteries). Lesions were characteristic of acute coagulative necrosis. Mean s-thrombomodulin concentrations, a marker for endothelial cell injury, were highest in rabbits exposed to US at 0.85 and 3.9 MPa, suggesting that vascular injury may be physiological and not accompanied by irreversible cellular injury. (E-mail: [email protected])

Introduction

The American Heart Association reports that coronary heart disease is the single largest killer of Americans and that atherosclerosis is the single largest cause of coronary heart disease (American Heart Association 2005). Atherosclerotic coronary arterial obstruction can cause reduction or obstruction of coronary blood flow, leading to myocardial ischemia and infarction (Schoen 2005). Surviving this disease is dependent on early detection, diagnosis and treatment; thus, an estimated 1.2 million coronary angioplasties and 0.5 million bypass procedures were performed in 2002 in the USA and, in 2005, the direct and indirect costs of coronary heart disease were estimated at $142.1 billion (American Heart Association 2005). Thus, the medical and biomedical engineering communities have recognized that one factor that can contribute to reducing the death rate attributable to coronary heart disease is early detection and diagnosis (Asanuma et al 2004, Bruce et al 2004, Chen et al 2004, Dawson et al 2003, Takeuchi et al 2004, Yip et al 2003, Zhuang et al 2004). To this end, microbubble ultrasound (US) echogenic contrast imaging agents (CAs) were approved by the US Food and Drug Administration (FDA) for use in cardiology in the USA health care market (Al-Mansour et al 2000, Clark and Dittrich 2000; Cohen et al. 1998; Malhotra et al 2000, Mulvagh et al 2000).

Although not currently approved by the FDA for other applications, the ultimate benefit of microbubble US CAs to the patient may lie in their use by physicians as a means of noninvasive assessment of coronary heart disease. By specifically imaging coronary artery and microvascular (capillary) blood flow, myocardial structure, function and perfusion can be assessed and the presence and/or severity of coronary artery occlusion (stenosis) and the severity and extent of myocardial degeneration and replacement fibrosis after myocardial infarction determined. Microbubble US CAs are valuable diagnostic tools for physicians, but their use must be considered in the context of risk-benefit assessment for each patient. The medical significance of and long-term potential benefits from the use of CAs for diagnostic imaging are clear; however, concerns related to their “safe use” have been raised because of reports of CA-induced vascular injury.

Over the last 7 years, a variety of clinical and experimental studies have demonstrated an assortment of structural (vascular alterations) and functional (arrhythmogenesis) changes in the cardiovascular system resulting from the interactions of US with CAs in the vascular system. These vascular alterations included changes in permeability (i.e., leakage of Evans blue dye) and integrity (i.e., petechial hemorrhages), focal endothelial cell and myocyte loss and necrosis, inflammation and replacement fibrosis (Hwang et al 2005, Li et al 2004, Miller and Gies 1998, Miller and Gies 2000, Miller et al 2004, Miller et al 2005, Miller and Quddus 2000, Skyba et al 1998). Arrhythmogenic changes included premature ventricular contractions in healthy adult human beings during triggered second-harmonic imaging of a CA for myocardial perfusion (van Der Wouw et al. 2000); supraventricular tachycardia or nonsustained ventricular tachycardia in patients at risk for syncope, supraventricular tachycardia or ventricular tachycardia after IV administration of perfluorocarbon-exposed sonicated dextrose albumin microbubbles (PESDA), exposure to therapeutic transthoracic low-frequency US (Chapman et al. 2005) and cardiac arrhythmogenesis in a rat model (Zachary et al. 2002). Cavitation has been suggested as the likely mechanism for microbubble-induced premature cardiac contractions (Dalecki et al. 2005).

The results from these studies have been used as indirect evidence to suggest that the interaction of US with CAs in the vascular system causes endothelial cell dysfunction and injury; however, the character and extent of the injury to the vascular endothelium has not been fully described. A lack of a clear understanding of the short- and long-term vascular bioeffects induced by the interaction of US with CAs could impact the regulatory process and impede the introduction of new microbubble US CAs into the health care market for needed medical benefit.

Currently, the medical significance and pathogenesis of such phenomena as applied to CA use in human beings are not clearly understood and, although no serious concerns were raised in the American Institute for Ultrasound in Medicine consensus statements (AIUM 2000a, AIUM 2000b, AIUM 2000c), it was advised that CA exposure conditions that minimize the potential for bioeffect occurrence should be used. However, the consensus statements did not provide guidelines as to what US exposure conditions would minimize the potential for bioeffect occurrence.

The purpose of this study was to: 1. characterize the histopathologic lesions and relative changes in s-thrombomodulin concentrations, a marker for endothelial cell injury, from rabbit auricular arteries (a medium-sized muscular artery model for the coronary artery) after the interaction of pulsed US with CA, and 2. provide, through the cytomorphologic characteristics of the lesions, insight into the mechanism of vascular injury associated with this interaction.

Section snippets

Animals

The experimental protocol was approved by the Institutional Animal Care and Use Committee, University of Illinois, Urbana-Champaign and satisfied all University and United States National Institutes of Health rules for the humane use of laboratory animals. Rabbits were selected as the experimental animal because the anatomic and histologic structures of the auricular artery were reasonable models for the human coronary artery and provided a readily accessible superficial site for insonation of

Pathologic evaluation

The results from all rabbit treatment groups are shown in Table 2 and Fig. 2, Fig. 3, Fig. 4. Of all the rabbits in the negative control and experimental groups, only 5 of 9 rabbits in the highest experimental pressure group (high-pressure positive control group, 9.5 MPa) had histopathologic lesions that allowed them to be scored as damaged auricular arteries. A high-pressure positive control group was included in this study to ensure that auricular arteries could be histopathologically damaged

Discussion and summary

In this study, we have demonstrated three important observational findings. First, the interaction of CA with US at exposure conditions below the FDA MI limit of 1.9 did not cause histopathologic damage of the auricular artery in vivo; however, this interaction can cause histopathologic damage using exposure conditions higher than the FDA limit.

Second, the arterial lesions observed in this study are characteristic of acute coagulative necrosis (necrotic cells) (Kumar et al. 2005) and include:

Acknowledgements

The authors thank the Bioacoustics Research Laboratory and the staff of the Histopathology Laboratory, College of Veterinary Medicine, University of Illinois for technical contributions. This study was supported, in part, by the NIH (grant EB02641m formerly HL58218) awarded to W. D. O’Brien, Jr. and J. F. Zachary.

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