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