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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Engineering

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

Published: August 5th, 2021

DOI:

10.3791/62581

1Center for Electron Microscopy and Analysis, The Department of Materials Science and Engineering, Ohio State University, 2Center for Regenerative Medicine, Nationwide Children’s Hospital, 3Thermo Fisher Scientific

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.

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.

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....

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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.......

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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.......

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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.......

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

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Name Company Catalog Number Comments
25% glutaraldehyde (aq) EMS 16210 Primary fixative component
0.9% sodium chloride injection Hospira Inc. NDC 0409-4888-10
1 mL syringe BD 309659
10 mL syringe BD 309604
200 proof ethanol EMS 15055
22G needle BD 305156
3 mL syringe BD 309657
3-way stopcock Smiths Medical ASD, Inc. MX5311L
4% osmium tetroxide EMS 19150 Staining component
4% paraformaldehyde (aq) EMS 157-4-100 Primary fixative component
Absorbable hemostat Ethicon 1961
Acetone EMS 10012
Black polyamide monofilament suture, 10-0 AROSurgical instruments Corporation TI38402
Black polyamide monofilament suture, 6-0 AROSurgical instruments Corporation SN-1956
C57BL/6 mice Jackson Laboratories 664 Approximately 1 yo
Calcium chloride Sigma-Aldrich 10043-52-4
Clamp applying forcep FST 00072-14
Cotton tip applicators Fisher Scientific 23-400-118
DPBS Gibco 14190-144
Dumont #5 forcep FST 11251-20
Dumont #5/45 forceps FST 11251-35
Dumont #7 fine forcep FST 11274-20
Durcupan ACM resin EMS 14040 For embedding
Fine scissor FST 14028-10
Heliscan microCT Thermo Fisher Scientific Micro-CT
Ketamine hydrochloride injection Hospira Inc. NDC 0409-2053
L-aspartic acid Sigma-Aldrich 56-84-8 Staining component
Lead nitrate EMS 17900 Staining component
low-vacuum backscatter detector Thermo Fisher Scientific VSDBS SEM backscatter detector
Micro-adson forcep FST 11018-12
Millex-GP filter, 0.22 um, PES 33mm, non-sterile EMD Millipore SLGP033NS
Non-woven songes McKesson Corp. 94442000
Potassium hexacyanoferrate(II) trihydrate Sigma-Aldrich 14459-95-1 Staining component
Potassium hydroxide Sigma-Aldrich 1310-58-3
Pressure monitor line Smiths Medical ASD, Inc. MX562
Saline solution (sterile 0.9% sodium chloride) Hospira Inc. NDC 0409-0138-22
Size 3 BEEM capsule EMS 69910-01 Embedding container
Sodium cacodylate trihydrate Sigma-Aldrich 6131-99-3 Buffer
Solibri retractors FST 17000-04
Sputter, carbon and e-beam coater Leica EM ACE600 Gold coater
Surgical microscope Leica M80
Thiocarbohydrazide (TCH) EMS 21900 Staining component
Tish needle holder/forcep Micrins MI1540
Trimmer Wahl 9854-500
Uranyl acetate EMS 22400 Staining component
Volumescope scanning electron microscope Thermo Fisher Scientific VOLUMESCOPESEM Serial Block Face Scanning Electron Microscope
Xylazine sterile solution Akorn Inc. NADA# 139-236

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