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Method Article

In Vitro Intraluminal Gel Infusion: An Advanced Approach for Microscopic Analysis of Human Resistance Arteries

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DOI:

10.3791/68773

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August 19th, 2025

 ,  ,  ,  , 
1Physiology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, 2Surgical Oncology Program, Center for Cancer Research, National Cancer Institute, 3Infectious Disease Pathogenesis Section, National Institute of Allergy and Infectious Diseases

In This Article

Summary

Here, we present a protocol for preserving human arterial architecture by infusing tissue-stabilizing gel into the vessel lumen before sectioning for molecular or histopathological analysis.

Abstract

Resistance arteries, which include small arteries and arterioles, play essential roles in regulating blood pressure and tissue perfusion. Dysfunction in these arteries can lead to various cardiovascular conditions such as hypertension, atherosclerosis, and heart failure, as well as neurovascular conditions. The examination of human resistance arteries is crucial for understanding cardiovascular disease mechanisms and developing targeted therapeutic strategies. This study presents an innovative method for preparing isolated human resistance arteries by infusing a tissue-stabilizing gel into the lumen, preserving their native architecture and cellular integrity. Human omental tissues were obtained from patients undergoing abdominal surgeries, and small resistance arteries (100-300 µm in diameter) were isolated by careful micro-dissection. The arteries were then cannulated, pressurized in a culture myograph chamber, and fixed with 10% neutral buffered formalin. A tissue-stabilizing gel was infused into the lumen of the cannulated artery, which was allowed to solidify to preserve the three-dimensional structure. Histological, immunohistochemical, and gene expression analyses were performed to assess the preservation of the arteries. Histological sections revealed the well-preserved structural integrity and natural architecture of the arteries, maintaining well-defined endothelial and smooth muscle layers. Immunohistochemical staining showed distinct localization of markers such as CD31 and α-smooth muscle actin. In situ hybridization revealed specific gene expression patterns, providing insights into the molecular mechanisms. This advanced method of intraluminal gel infusion offers significant advantages, enabling advanced imaging and comprehensive analysis of arterial structure, remodeling, and cellular interactions in both healthy and diseased states. This method has the potential to improve clinical diagnostics and therapeutic strategies for vascular diseases, thereby providing a valuable tool for advancing vascular biology research.

Introduction

Resistance arteries regulate blood flow and pressure and undergo significant changes in conditions like hypertension, diabetes, and atherosclerosis1,2,3,4,5. Examining human resistance arteries is essential for understanding cardiovascular pathologies and developing targeted therapies6,7,8,9,10. Traditional methods for investigating blood vessels have often led to artifacts and loss of three-dimensional integrity10,11,12,13,14,15. To address these limitations, we present an innovative method for preparing isolated human resistance arteries by infusing a gel into the lumen, thereby preserving their approximate ex vivo and/or in vivo structure.

This intraluminal gel infusion technique maintains the natural architecture of the arteries, allowing for a more accurate representation of their physiological state. By stabilizing the vessel structure, this method facilitates detailed examination of arterial remodeling, cellular structure, microdomains, hetero-cellular junctions, and gene/protein expression across different cell layers. This technique is particularly advantageous for studying cell-type-specific components in both healthy and diseased states.

This technique has been successfully applied in our recent study16, demonstrating its effectiveness in preserving the structural and molecular integrity of human resistance arteries, facilitating advanced imaging and comprehensive analysis. The success of this application inspired us to document this detailed protocol for others to use.

In this method paper, we describe the protocol for preparing isolated human resistance arteries and infusing them with a biocompatible tissue-stabilizing gel (Tissue Guard Gel). Here, we describe the experimental method and illustrate its applications for the assessment of arterial structure and the investigation of gene and protein expression patterns in different cell layers. By providing a comprehensive and reproducible technique for the preparation of human resistance arteries, we aim to advance cardiovascular biology research, improve the reproducibility of results, and contribute to novel diagnostic and therapeutic approaches for cardiovascular diseases.

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Protocol

Before conducting the following experiments, ensure that the use of human tissues and the following procedures are approved by the Institutional Review Board (IRB). The collection of omental tissue from patients undergoing clinically indicated abdominal operations at the National Institutes of Health (NIH) Clinical Center was conducted with the approval of the NIH Institutional Review Board (NCT01915225). All participants provided written informed consent.

1. Materials and equipment

  1. Refer to the Table of Materials for the information on all reagents and materials required for this protocol. For additional details, the manuals and websites associated with the respective vendors can be consulted as needed.

2. Collection of human omental tissues

  1. Collect human omental tissues from the surgical team.
  2. Immediately place the tissues into a pre-chilled specimen container containing cold Krebs Henseleit (KH) buffer (composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES, 2.0 mM CaCl2, and 11.1 mM glucose) on ice.
  3. Ensure the tissues are fully submerged in the buffer and then securely close the lid.
  4. Transport the tissues in the closed container on ice to the laboratory within 30 min of collection to ensure tissue viability.
    NOTE: Make sure to adjust pH to 7.4 using NaOH during preparation of the buffer.

3. Microdissection of human small resistance arteries

  1. Upon arrival at the laboratory, rinse the omental tissues with cold KH buffer to remove excess blood.
  2. Select a piece of the more vascularized region of the tissue for dissection in cold buffer in a petri dish on ice.
  3. Under a dissection microscope, identify small resistance arteries (100-300 µm in diameter) based on their location and branching pattern.
  4. Carefully isolate the identified arteries from the surrounding adipose and connective tissues. Using fine forceps and micro-scissors, cut and remove the surrounding tissues without damaging the artery.
  5. Keep the isolated arteries in a cold buffer throughout the dissection procedure to maintain viability and prevent any enzymatic degradation.
    NOTE: Before dissection, ensure the dissection area is clean. Verify that the microscope objective scale and/or camera software are properly calibrated to accurately measure the diameter at different magnifications. Usually, the veins are clearly visible, and the arteries are located right next to or around the veins. The walls of arteries are thicker than those of veins. When isolated from surrounding tissue, arteries maintain their structure as intact vessel tubes. In contrast, veins have thinner walls, often appear brighter due to the blood in their lumen, and tend to be more fragile once separated from the tissue.

4. Preparation of the culture myograph chamber

  1. Prepare a culture myograph chamber with two glass cannulas for the cannulation and pressurization of the dissected arteries.
  2. Prepare a 5 mL syringe with clean capillary tubing and fill with fresh KH buffer. Connect the tubing to one end of the cannula and fill with the KH, leaving no air bubbles.
  3. Fill the chamber with cold buffer and place it on ice to maintain a low temperature.
    NOTE: A custom-made small dish with two cannulas, designed to mimic the myograph chamber, can be used.

5. Cannulation and pressurization of arteries

  1. Carefully transfer the dissected artery to the myograph chamber using fine forceps or a transfer pipette with a diameter larger than that of the artery.
  2. Prepare four open knots with the nylon suture (11/0 suture, 7 ply) and put two of them onto each cannula before cannulating the artery.
  3. Cannulate the proximal end of the artery by sliding it onto a glass cannula using fine forceps and secure it with the two nylon sutures.
  4. Use the KH buffer in the syringe to gently flush the cannulated artery to remove any remaining blood.
  5. Cannulate the distal end of the artery and secure it similarly.
  6. Place the chamber onto the myograph unit that is connected to the pressure and temperature controller.
  7. Set the temperature to 37 °C.
  8. Set the initial intraluminal pressure to 10 mmHg.
  9. Increase the intraluminal pressure by 10 mmHg increments every 10 min until reaching 60 mmHg. Ensure that the pressure is consistently maintained using the pressure regulator connected to the culture myograph system.
  10. Use the micro-positioner to longitudinally straighten the pressurized artery as needed to bring the length approximately to its physiological level.
  11. Run MyoVIEW or other suitable software on the computer monitor to track the inner and outer arterial diameters with digital calipers.
  12. Allow the artery to equilibrate for 45 min to achieve a stable diameter, ensuring it maintains its physiological dimensions and tone.
  13. Test the viability of the artery by using a vasoconstrictor (e.g., 60 mM KCl or Phenylephrine) or vasodilator (e.g., Acetylcholine) and record the changes in the arterial diameter.
    NOTE: Applying excessive pressure with a syringe can damage the artery, so it is important to exercise caution when flushing it. Use a minimal amount of buffer and proceed gently. Alternatively, the arterial blood can be flushed out by using the perfusion buffer while pressurizing the artery. Arteries can be pressurized at 80 mmHg, and equilibration time can be up to 1 h.

6. Artery fixation

  1. Following equilibration, fix the arteries luminally and abluminally overnight with 10% neutral buffered formalin. Maintain a constant pressure of 60 mmHg to ensure the fixation process preserves the structural integrity of the arterial wall and cellular components.
  2. After fixation, rinse the arteries luminally and abluminally with PBS to remove any residual fixative from the artery and the chamber.
    NOTE: Depending on the study objective and the region of interest for analysis, the mounted vessel can be fixed in the presence of pharmacological agents such as a vasodilator or vasoconstrictor. At this stage, the artery can be stored in PBS at 4 °C for up to 48 h. 4% Paraformaldehyde can also be used to fix the arteries.

7. Preparation of tissue-stabilizing gel

  1. Liquefy the tissue-stabilizing gel in its tube by heating it to 60 ± 5 °C before use.
  2. Place the tube at room temperature (RT) for 3-5 min for the liquid gel to lower the temperature, and then place it on a heating block at 40 °C to maintain the liquid gel for 10-15 min.
  3. Use a 3-mL sterile syringe connected to a 10-15 inch-long capillary tubing to withdraw the liquid gel. Proceed immediately to the infusion steps below.
    NOTE: The gel can be liquefied using different methods. It is essential to follow the manufacturer's instructions.

8. Intraluminal gel infusion and embedding

  1. Keep the fixed arteries cannulated in the chamber with PBS at 37 °C.
  2. Connect the distal end of the capillary tubing filled with liquid gel to the proximal cannula.
  3. To avoid air bubbles going through the lumen while infusing the gel, take the proximal end of the artery off the first cannula to let any air bubbles out and allow the gel to fill the cannula.
  4. Mount the artery back onto the cannula and gently infuse the gel into the artery lumen until it comes out of the distal cannula.
  5. Immediately replace the PBS in the chamber with an equal volume of liquid gel to fill the chamber to the top level.
  6. Allow the chamber with the arteries to rest at RT by disconnecting the unit from the temperature controller for around 10 min to allow the gel to solidify inside the lumen as well as outside the lumen in the chamber. This ensures that the artery is fully embedded in the gel, preserving its three-dimensional structure.
  7. Using fine forceps, loosen the nylon sutures and gently pull out the cannulas from the chamber.
  8. Transfer the entire solidified gel containing the artery from the chamber into a container filled with 70% ethanol. The ethanol serves as a holding solution to preserve the sample until further processing.
    NOTE: A three-way valve can facilitate removing air bubbles before infusing the gel. Solidification of the gel can be confirmed by gently touching the gel in the chamber using a clean pipette tip.

9. Histopathology

  1. Cut the artery transversely while still in the gel and place these samples in the tissue processing machine for processing according to the manufacturer's instructions.
  2. Subsequently, embed the tissue to make a paraffin block. Ensure the samples are embedded "on-end" so that cross sections are facing down on the block.
  3. Cut axial sections at 5 µm thickness using a microtome. Transfer the cut sections onto adhesive slides.
  4. Bake the sections at 60 °C for 1 h, then dewax with xylene, followed by rehydration with graded alcohol washes.
  5. Stain the sections with hematoxylin and eosin (HE) for routine histopathology and differentiate in 1% acid alcohol. Examine the sections under light microscopy using an appropriate microscope and acquire photographs using a compatible camera.

10. Immunohistochemistry

  1. Achieve staining using an automated system with the polymer refine detection system according to the manufacturer's protocol.
  2. Dewax the tissue sections at 72 °C for 30 s with a dewaxing solution, then rehydrate the sections with graded alcohol washes and wash solution.
  3. Perform heat-induced epitope retrieval by heating the sections to 100 °C for 20 min in epitope retrieval solution 1 and apply a peroxide block for 5 min to quench endogenous peroxidase activity.
  4. Apply primary antibodies (e.g., anti-CD31 or anti-smooth muscle actin) at appropriate concentrations diluted in background reducing antibody diluent. Incubate the sections with a horseradish peroxidase polymer for 8 min and colorize the sections with a chromogen solution for 10 min. Counterstain the slides with hematoxylin for 5 min.

11. RNAScope chromogenic in situ hybridization

  1. Perform staining using an automated system with an appropriate kit and the polymer refine red detection kit according to the manufacturer's protocol.
  2. Bake the sections for 30 min at 60 °C, then dewax the sections for 30 s in a dewax solution heated to 72 °C.
  3. Rehydrate the tissues with absolute ethanol washes and wash solution.
  4. Treat the sections with epitope retrieval solution 2 for 15 min at 95 °C and incubate the sections with an enzyme solution for 15 min at 40 °C.
  5. Hybridize the samples with the appropriate RNAScope probes for the target genes.
  6. Use a medium-expressing housekeeping gene probe (e.g., peptidylprolyl isomerase B) to confirm adequate RNA abundance and integrity.

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Results

Human omental tissues were obtained from patients undergoing clinically-indicated abdominal surgeries. Small resistance arteries (100-300 µm in diameter) were meticulously isolated from the surrounding adipose and connective tissues under a dissection microscope (Figure 1A-C). The isolated arteries were cleaned, successfully cannulated, and mounted onto the glass cannulas in the culture myograph chamber for pressurization (Figure 1D

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Discussion

Advancements in medical imaging, surgical techniques, and research methodologies have improved our understanding of 3D vascular architecture, revolutionizing diagnostics, surgical planning, and research17,18,19,20. Despite these innovations, preserving the 3D structure of isolated human resistance arteries remains challenging due to their delicate and intricate nature, which complicates isolati...

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Disclosures

NIAID GenAI was used to generate an initial outline and draft of the manuscript, which was thoroughly written, reviewed, and edited by the human authors to ensure accuracy and coherence.

Acknowledgements

This research was funded by the Division of Intramural Research in the National Institute of Allergy and Infectious Diseases, project number AI001150 to HCA. We thank our former lab member, Dr. Steven Brooks, as well as our current lab members, Mohamed Ibrahim, Mary Jackson, and Jian Wu from Malaria Functional Genomics Section, for their assistance in capturing images during the process.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1% Focus Acid AlcoholStatLab SL89-1
10% NBSLeica Biosystems3800598
4K Microscopy HDMI CameraHitech Instruments, Inc.HT-XP4K8MA
Antibody Diluent- Background ReducingDakoS3022
Automated Tissue Processor Leica BiosystemsHistoCore PEGASUS/Cat# 14048858005
Bond Dewax SolutionLeica BiosystemsAR9222
Bond Epitope Retrieval Solution 1Leica BiosystemsAR9961
Bond Epitope Retrieval Solution 2Leica BiosystemsAR9640
Bond Polymer Refine DetectionLeica BiosystemsDS9800
Bond Polymer Refine Red DetectionLeica BiosystemsDS9390
Bond Wash Solution (10x)Leica BiosystemsAR9590
Cannula Pack (glass), 80–100 μmDMTProduct Code: 300407
CD31Abcamab28364
Dissection Dishes, Large (pyrex glass), 93 mmLiving Systems InstrumentationSKU: DD-90
Dissection Dishes, SmallLiving Systems InstrumentationSKU: DD-50
Dissection MicroscopeHitech Instruments, Inc.ZEISS SteREO Discovery.V8 
Dumont #5 ForcepsFine Science ToolsItem No. 11295-10
Dumont #5SF ForcepsFine Science ToolsItem No. 11252-00
EcoMountBioCare MedicalEM897L
EosinIHC WorldIW-3100B
HematoxylinBioCare MedicalCathe-MM
Iamging cameraOlyumpusDP28
Imaging MicroscopeOlyumpusBX51
Krebs Henseleit Buffer (with 2 mM Calcium Chloride)Boston BioProducts, Inc.SKU: C-10625N-200mL
Krebs-Henseleit Buffer (without Calcium)Boston BioProducts, Inc.SKU: C-9906W-200mL
Mounting MediaThermoFisher Scientific4111
Myograph chamber and UnitDMT202/204CM
PBS (phosphate buffered saline)Thermofisher Scientific10010023
PPIB ACDBio313908
RNAscope 2.5 LS Reagent Kit-REDACDBio322150
StainerLeicaLeica Bond RX automated system
Stainless steel dissection pinsLiving Systems InstrumentationSKU: PIN-0.1MM | PIN-0.2MM | PIN-#3 | PIN-VP-1
Surgipath ParaplastLeica Biosystems39601006
Tissue Guard GelStatLab TissueGuard/SKU: TG12
Vannas Spring Scissors - 2 mm Cutting EdgeFine Science ToolsItem No. 15000-03
Vannas Spring Scissors - 2.5 mm Cutting EdgeFine Science ToolsItem No. 15000-08
Vannas Spring Scissors - 3 mm Cutting EdgeFine Science ToolsItem No. 15000-00
α-SMAAbcamab5694

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Human Artery AnalysisMicrodissection TechniqueCulture Myograph ChamberArterial PressurizationImmunohistochemical StainingIn Situ HybridizationEndothelial Cell MarkersSmooth Muscle Cells
In Vitro Intraluminal Gel Infusion: An Advanced Approach for Microscopic Analysis of Human Resistance Arteries
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