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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

Abstract

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (µCT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. µCT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Introduction

The pulmonary valve (PV) serves to ensure unidirectional blood flow between the right ventricle and the pulmonary artery. Pulmonary valve malformations are associated with several forms of congenital heart disease. The current treatment for congenital heart valve disease (HVD) is valvular repair or valve replacement, which can necessitate multiple invasive surgeries throughout a patient's lifetime1. It has been widely accepted that the function of the heart valve is derived from its structure, often referred to as the structure-function correlate. More specifically, the geometric and biomechanical properties of the heart dictate its functio....

Protocol

The use of animals in this study was in accordance with Nationwide Children's Hospital institutional animal care and use committee under protocol AR13-00030.

1. Pulmonary valve excision

  1. Autoclave the necessary tools needed for the mouse dissection. This includes fine scissors, micro forceps, micro vascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors.
  2. Acclimate all mice for a minimum of 2 weeks before the operation. Remove C57BL.......

Representative Results

Anastomosis of the pulmonary artery to the pressurization tubing is shown in Figure 1A. Following the application of hydrostatic pressure, the pulmonary trunk distends radially (Figure 1B) indicating that the pulmonary valve leaflets are in a closed configuration. Pulmonary valve conformation was confirmed by µCT. In this case, the leaflets were coapt (closed) and the annulus was circular (Figure 2A). Figur.......

Discussion

Removal of the ventricles serves two purposes. First, exposing the ventricle side to the atmospheric pressure, thereby only needing to apply a transvalvular pressure from the arterial side of the pulmonary valve to close, and second, providing a stable base to prevent twisting of the pulmonary trunk. During pressurization, the pulmonary trunk distends radially and inferiorly, making it prone to twisting, causing the collapse of the pulmonary trunk. Preloading the pulmonary valve with a saline solution offers an additiona.......

Acknowledgements

This work is supported, in part, by R01HL139796 and R01HL128847 grants to CKB and RO1DE028297 and CBET1608058 for DWM.

....

Materials

NameCompanyCatalog NumberComments
25% glutaraldehyde (aq)EMS16210Primary fixative component
0.9% sodium chloride injectionHospira Inc.NDC 0409-4888-10
1 mL syringeBD309659
10 mL syringeBD309604
200 proof ethanolEMS15055
22G needleBD305156
3 mL syringeBD309657
3-way stopcockSmiths Medical ASD, Inc.MX5311L
4% osmium tetroxideEMS19150Staining component
4% paraformaldehyde (aq)EMS157-4-100Primary fixative component
Absorbable hemostatEthicon1961
AcetoneEMS10012
Black polyamide monofilament suture, 10-0AROSurgical instruments CorporationTI38402
Black polyamide monofilament suture, 6-0AROSurgical instruments CorporationSN-1956
C57BL/6 miceJackson Laboratories664Approximately 1 yo
Calcium chlorideSigma-Aldrich10043-52-4
Clamp applying forcepFST00072-14
Cotton tip applicatorsFisher Scientific23-400-118
DPBSGibco14190-144
Dumont #5 forcepFST11251-20
Dumont #5/45 forcepsFST11251-35
Dumont #7 fine forcepFST11274-20
Durcupan ACM resinEMS14040For embedding
Fine scissorFST14028-10
Heliscan microCTThermo Fisher ScientificMicro-CT
Ketamine hydrochloride injectionHospira Inc.NDC 0409-2053
L-aspartic acidSigma-Aldrich56-84-8Staining component
Lead nitrateEMS17900Staining component
low-vacuum backscatter detectorThermo Fisher ScientificVSDBSSEM backscatter detector
Micro-adson forcepFST11018-12
Millex-GP filter, 0.22 um, PES 33mm, non-sterileEMD MilliporeSLGP033NS
Non-woven songesMcKesson Corp.94442000
Potassium hexacyanoferrate(II) trihydrateSigma-Aldrich14459-95-1Staining component
Potassium hydroxideSigma-Aldrich1310-58-3
Pressure monitor lineSmiths Medical ASD, Inc.MX562
Saline solution (sterile 0.9% sodium chloride)Hospira Inc.NDC 0409-0138-22
Size 3 BEEM capsuleEMS69910-01Embedding container
Sodium cacodylate trihydrateSigma-Aldrich6131-99-3Buffer
Solibri retractorsFST17000-04
Sputter, carbon and e-beam coaterLeicaEM ACE600Gold coater
Surgical microscopeLeicaM80
Thiocarbohydrazide (TCH)EMS21900Staining component
Tish needle holder/forcepMicrinsMI1540
TrimmerWahl9854-500
Uranyl acetateEMS22400Staining component
Volumescope scanning electron microscopeThermo Fisher ScientificVOLUMESCOPESEMSerial Block Face Scanning Electron Microscope
Xylazine sterile solutionAkorn Inc.NADA# 139-236

References

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