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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F2 - Multimodal Study of Ischemic Cardiac Remodeling: Murine 4D Ultrasound and Mass Spectrometry Imaging

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Cardiovascular disease (CVD) is a leading cause of mortality worldwide. In 2016, there were an average of 1000 CVD-related deaths daily in the US, amounting to over 400,000 deaths that year [1]. To prevent, diagnose, and treat CVD, it is crucial to develop methods for understanding and observing the mechanisms of disease initiation and progression. Biomedical imaging has been key in generating physiologic and mechanistic insights, and new imaging technologies and analysis techniques are constantly being developed.

Four-dimensional ultrasound (4DUS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry imaging (MSI) are two methods of spatially observing and analyzing the heart. 4DUS allows in vivo functional metrics to be calculated from the complex shape and movement of the heart, while MALDI MSI permits the spatial study of biological molecules within the cardiac tissue ex vivo. To fully understand alterations in the heart with CVD, we must understand both mechanical and molecular mechanisms. Here, we demonstrate a novel process for studying the murine heart pathophysiologically, coupling 4DUS imaging and analysis with MALDI MSI of lipids in the heart. This methodology is demonstrated with a murine model of myocardial infarction.

Materials and

Methods:  This study utilized 12- to 15-week-old male and female C57Bl6 mice (n=6). The mice were separated into a surgically induced myocardial infarction group (Figure 1A) and a control group. A permanent coronary artery ligation was used to induce acute myocardial infarction in the diseased group [2]. The control group underwent imaging without surgery. 4DUS images of the left ventricle (LV) were acquired using a Vevo3100 ultrasound machine (FUJIFILM VisualSonics, Toronto, Canada, Figure 1B). Images were acquired at days 0, 1, 3, 7, 14, and 28 following surgery. After final imaging, animals were sacrificed, and cardiac tissue was removed and flash-frozen with liquid nitrogen. 4DUS images were analyzed with a custom MATLAB graphical user interface [3]. Analysis results included a computational model of ventricular shape, regional functional metrics, and an estimation of relative infarct size based on surface area strain [4].

Flash-frozen cardiac tissue was sectioned and mounted onto microscope slides (Figure 1C). Sections were collected from base, middle, and apical regions of the heart. The regions chosen were guided and identified by the scaled computational model of the LV developed with the MATLAB interface. Slides were sprayed with dihydroxybenzoic acid matrix and run through the MALDI MSI instrument, Waters Synapt G2-Si, following standard protocol (Waters Corporation, Milford, MA). MSI data was analyzed using HD Imaging software (Waters Corporation, Milford, MA) to identify the intensities and spatial heterogeneities of target test lipids found in previous literature to be associated with myocardial infarction.

Results, Conclusions, and Discussions: Here we have developed a protocol for the pathophysiological study of myocardial infarction by coupling 4DUS image acquisition and analysis with MALDI MSI of lipids in murine cardiac tissue. These two imaging techniques were performed progressively on the same tissue, unveiling physiological and molecular changes. Therefore, changes in left ventricular structure and function could be associated with up- and downregulation of target lipids in the respective region (Figure 2). In the 4DUS image, the green-yellow end of the color spectrum represents tissue with surface area strain magnitude of less than 20%, which corresponds with infarcted tissue [4]. The yellow end of the spectrum represents infarcted regions in the MS image as well, corresponding with lipids known to be upregulated in infarcted tissue [5].

4DUS and MS images and metrics from the diseased group will be quantitatively compared to control group images to verify that disease-related changes can be successfully identified using this protocol. Control group results and comparisons between groups are forthcoming. We hypothesize that disease-associated tissue morphologies and molecular abundances will be evident in disease data when compared to control data.

New imaging methods are essential for studying CVD, consequently improving prevention strategies, diagnoses, treatments, and outcomes. The methodology established here can be implemented in future studies investigating various aspects of cardiovascular health and instances of disease.

[1] Tsao, C et al., Circulation, 145:e153-e639, 2022.
[2] Clark, J et al., Cardiovascular Research, 74:466-470, 2007.
[3] Damen, F et al., Tomography, 3(4):180-187, 2017.
[4] Dann, M et al., AJP HCP, 322:H359–H372, 2022.
[5] Kaya, I et al., J Am Soc for Mass Spec, 31:2133-2142, 2020.

Acknowledgements (Optional):