Multimodal Study of Murine Cardiovascular Remodeling: Four-Dimensional Ultrasound and Mass Spectrometry Imaging

Summary

Here, we describe a protocol to use in vivo four-dimensional ultrasound imaging and ex vivo mass spectrometry imaging to assess biomechanical and biomolecular alterations in the murine cardiovascular system. This technique is applied to analyze cardiac remodeling in surgically induced myocardial infarction and vascular changes in aging animals.

Abstract

Cardiovascular disease (CVD) is the leading cause of death in the United States. Damage in the cardiovascular system can be due to environmental exposure, trauma, drug toxicity, or numerous other factors. As a result, cardiac tissue and vasculature undergo structural changes and display diminished function. The damage and the resulting remodeling can be detected and quantified with ultrasound (US) imaging at the organ level and mass spectrometry imaging (MSI) at the molecular level. This manuscript describes an innovative methodology for studying murine cardiac pathophysiology, coupling in vivo four-dimensional (4D) ultrasound imaging and analysis with ex vivo matrix-assisted laser desorption/ionization (MADLI) MSI of the heart. 4D ultrasound can provide dynamic volumetric measurements, including radial displacement, surface area strain, and longitudinal strain throughout an entire cardiac cycle. In the vasculature, MSI and ultrasound are used to assess vessel wall compositions, hemodynamics, and vessel wall dynamics. The methodology can be tailored to study a myriad of CV diseases by adjusting functional metrics of interest and/or varying MALDI MSI protocol to target specific molecules. MALDI MSI can be used to study lipids, small metabolites, peptides, and glycans. This protocol outlines the use of MALDI MSI for untargeted lipidomic analysis and the use of ultrasound imaging for cardiovascular hemodynamics and biomechanics.

Introduction

Cardiovascular disease (CVD) is a leading cause of mortality worldwide¹. Prevention and treatment of CVD require an in-depth understanding of molecular adaptations to biomechanical forces and the resulting changes in mechanical properties. Throughout the entire cardiovascular system, biomechanical forces play an important role in the function and structure of the tissue². The mechanical properties of cardiovascular (CV) tissue are influenced by these forces, making them indicators of health and disease^3,4,5....

Protocol

The described animal experiments are carried out with the University of Tennessee, Knoxville Institutional Animal Care and Use Committee approval.

1. Ultrasound imaging¹³

1. Setup and animal preparation
   1. Prior to starting the experiment, check the isoflurane anesthesia level and refill if necessary. Check oxygen tank levels before turning on the gas. Weigh the charcoal and record it on the container.
   2. Set up the imaging spa.......

Discussion

US imaging can be operator-dependent, but the use of anatomical landmarks and adequate training can limit user bias. 2D ultrasound is particularly susceptible to inter-user variability because views are angle-dependent, whereas 4DUS is less susceptible as acquisition encompasses the entire volume and is angle-independent. It was also determined that image reproducibility is easier to achieve because of the adjustable animal platform and transducer holder. US data collection should ideally be conducted by the same researc.......

Materials

Name	Company	Catalog Number	Comments
2,5-dihydroxybenzoic acid (DHB)	Supelco,  >99.0% (HPLC)	85707-10MG-F	DHB matrix substance for MALDI-MS; https://www.sigmaaldrich.com/US/en/product/sigma/85707?cm_sp=Insite-_-wimsShippingEmailRecs_wims EmailAPI_wimsGruCrossEntropy-_-wimsEmailAPI10-3
9-aminoacridine (9AA)	Supelco,  ≥99.5% (HPLC)	92817-1G	9-Aminoacridine matrix substance for MALDI-MS; https://www.sigmaaldrich.com/US/en/product/sial/92817?srsltid=AfmBOooiQjQ4pWv_XxITkU 4Lkm0UnHXKekGS_ dFl7V40V9QLWoPpNLoc
Aquasonic Ultrasound Gel	Parker Laboratories	Parker 01-02	Ultrasound Gel; https://www.parkerlabs.com/products/aquasonic-100-ultrasound-transmission-gel/
Benchtop Dewar Flasks	ThermoScientific	4150-2000	Container for liquid nitrogen; https://www.thermofisher.com/order/catalog/product/4150-4000?gclid=Cj0KCQjwpvK4BhDUARIsA DHt9sQVc2f-NxN04Nb5Mv F6TZ7GLHWWDEeqDYmEvtKJSQ YHDeVgZ9qylvYaAs27EALw_wcB &source=google_shopping&ISO_ CODE=us&LANG_CODE=en&ef_id =Cj0KCQjwpvK4BhDUARIsADHt9 sQVc2f-NxN04Nb5MvF6TZ7GLHWWDE eqDYmEvtKJSQYHDeVgZ9qylvYa As27EALw_wcB:G:s&s_kwcid=AL!3652 !3!716188292869!!!g!2366243726129 !!21787513085!171591181194&ev_chn =shop&cid=0se_gaw_30092024_ PBYTXL&source=google_shopping &ISO_CODE=us&LANG_CODE= en&gad_source=1
Cryostat	Leica Biosystems	CM Series	https://www.leicabiosystems.com/us/histology-equipment/cryostats/
Dessicator	VWR	89054-052	https://us.vwr.com/store/product/9104882/desiccator-plastic-ace-glass-incorporated
Epredia MX35 Premier Disposable Low-profile Microtome Blades	Fisher Scientific	3052835	Cryostat blade; https://www.fishersci.com/shop/products/mx35-premier-disposable-low-profile-microtome-blades/3052835
Falcon 15 mL Conical Centrifuge Tubes	Fisher Scientific	14-959-53A	Conical Tubes; https://www.fishersci.com/shop/products/falcon-15ml-conical-centrifuge-tubes-5/1495953A?gclid=Cj0KCQjwpvK4BhDUARIsA DHt9sSBcy5n-lhShligJUOX5KKVGn0bt87 8AB2_muOD2PPTue1phpZgeqwa AqgiEALw_wcB&ef_id=Cj0KCQjw pvK4BhDUARIsADHt9sSBcy5n-lhS hligJUOX5KKVGn0bt878AB2_muO D2PPTue1phpZgeqwaAqgiEALw_ wcB:G:s&ppc_id=PLA_goog_20861 45674_81843405034_1495953A__ 386247001345_165426395473886 37329&ev_chn=shop&s_kwcid=AL!4428!3 !386247001345!!!g!856907751004!& gad_source=1
Flex-Tubes Microcentrifuge Tubes	Eppendorf	EP022364120	Centrifuge tubes; https://www.sigmaaldrich.com/US/en/product/sigma/ep022364120?utm_source=google%2Cgoogle&utm _medium=organicshopping%2Ccpc&utm _campaign=21043330280&utm_ content=&gclid=Cj0KCQjwpv K4BhDUARIsADHt9sTHRD35k CHPtfI2A41axodnMVr6a1eBKk zM4bSUYQAyfEKo3UgTAEQa Ap7wEALw_wcB
Gas Nitrogen	Airgas	N/A
Glass microscope slides	Electron Microscopy Sciences	71873-02	https://www.emsdiasum.com/positive-charge-microscope-slides
Liquid Nitrogen	Airgas	N/A
Mass Spectrometer	Waters	Synapt G2-Si	https://www.waters.com/waters/en_US/SYNAPT-G2-Si-Mass-Spectrometry/nav.htm?locale=en_mkcid=1000251Foodety%3C/a%3E&cid=134740653&bcid= 134528734
Matrix Sprayer	HTX Technologies	M3+	https://www.htximaging.com/htx-m3-sprayer
Methanol (HPLC), >99.9%	Fisher Chemical	A4524	Methanol; https://www.fishersci.com/shop/products/methanol-hplc-fisher-chemical-9/A4524?crossRef=A4524#?keyword=A4524
Preclinical Ultrasound System	FUJIFILM VisualSonics	Vevo 3100	https://www.visualsonics.com/product/imaging-systems/vevo-3100; Vevo F2 has replaced the Vevo 3100 in production. System includes isoflurane vaporizer and induction box.
Reynolds Wrap	N/A	N/A	Aluminum foil
Signagel  Electrode Gel	Parker Laboratories	Parker 15-60	Electrode Conducting Gel; https://www.parkerlabs.com/products/signagel-electrode-gel/
Sterile Lubricating Jelly	Medline	MDS032273Z	Lubricating Gel; https://www.medline.com/ce/product/Sterile-Lubricating-Jelly/Lubricating-Jelly/Z05-PF03664?sku=MDPMDS032273H
Surgical instruments: scissors, forceps/tweezers, sutures	Fine Science Tools	11252-00, 11050-10, 14016-14, 14084-08, 15000-08	info@finescience.com
Surgical Sponges 200 Pack –Gauze Pads Non sterile -First Aid Wound Care Dressing Sponge –Woven Medical Nonstick, Non Adherent Mesh Scrubbing	Medpride	B08RZGQ5GW	Gauze; https://www.amazon.com/Medpride-Surgical-Sponges-200-Pack/dp/B08RZGQ5GW/ref=asc_df_B08RZGQ5GW/?tag=hyprod-20&linkCode=df0&h vadid=693270340506&hvpos= &hvnetw=g&hvrand=960915122 2290977669&hvpone=&hvptwo= &hvqmt=&hvdev=c&hvdvcmdl=& hvlocint=&hvlocphy=9192978&hv targid=pla-1245491514869&psc= 1&mcid=33f4d647c88630c79116 888d565a63b0
Tissue-Plus O.C.T. Compound	Fisher Scientific	23-730-571	OCT; https://www.fishersci.com/shop/products/tissue-plus-o-c-t-compound-2/23730571
Wood Handled Cotton Swabs and Applicators	Fisherbrand	22-363-160	Cotton swab; https://www.fishersci.com/shop/products/wood-handled-cotton-swabs-applicators-8/p-7146852