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

This research improves a method to preserve and analyze human resistance arteries, enhancing visualization and molecular insights. It has the potential to advance diagnostics and therapies in cardiovascular medicine. We recently discovered that tetramer hemoglobin composed of both the alpha and beta subunits regulates the release of the vasodilator nitric oxide from the vascular endothelial cells of human arteries.

This technique was pivotal for localizing the alpha and beta globin transcripts to the vascular endothelial cell layer. This protocol effectively addresses the challenge of preserving the structural and molecular architecture of human resistance arteries, facilitating better analysis and understanding of cardiovascular conditions. To begin, obtain the human omental tissues on ice for the procedure.

Cut a piece of the visibly vascularized region of the tissue and transfer it to a Petri dish containing Krebs Henseleit Buffer on ice. Using a dissection microscope, carefully isolate the small resistance arteries of 100 to 300 micrometers in diameter. Clean the arteries from surrounding adipose tissues and remove blood from the lumen without damaging the arteries.

Prepare a culture myograph chamber with two glass cannulas to cannulate and pressurize the dissected arteries. Fill a five-milliliter syringe attached to a clean capillary tubing with fresh cold Krebs Henseleit Buffer and connect the tubing to one end of a cannula. Then fill the glass cannula with the buffer, ensuring no air bubbles are present.

With a wide board transfer pipette, carefully transfer the dissected artery to the myograph chamber. Cannulate the proximal end of the artery by sliding it onto a glass cannula using fine forceps and secure it with two nylon sutures. Place the chamber onto the myograph unit, ensuring it is connected to the pressure controller, and set the initial pressure to 10 millimeters of mercury.

Increase the intraluminal pressure in 10 millimeters of mercury increments every 10 minutes until reaching 60 millimeters of mercury. Use the pressure regulator to maintain consistent pressure throughout the pressurization process. Next, use the micro-positioner to straighten the pressurized artery longitudinally to approximate its physiological length.

Run MyoView, or another suitable software, on the computer monitor to track the inner and outer arterial diameters using digital calipers. Allow the artery to equilibrate for 45 minutes to achieve a stable diameter and ensure that it maintains its physiological dimensions and tone. To test the artery's viability, add a vasoconstrictor or a vasodilator and record the changes in arterial diameter.

Wash the artery with fresh solution to let it restore its resting baseline diameter, and then fix the artery by introducing 10%neutral buffered formalin both into the lumen and around the vessel in the chamber. Incubate overnight. After fixation, rinse the arteries inside and outside with PBS to remove any residual fixative from the artery and chamber at 37 degrees Celsius.

Place the tube of liquefied tissue-stabilizing gel at room temperature for three to five minutes to slightly cool the liquid, and then transfer it to a 40-degrees Celsius heating block to maintain the liquid gel for 10 to 15 minutes. Withdraw the liquid gel using a three-milliliter sterile syringe connected to 10-to 15-inch long capillary tubing. Connect the distal end of the tubing filled with liquid gel to the proximal cannula and gently infuse the gel into the artery lumen.

Immediately replace the PBS in the chamber with an equal volume of liquid gel to fill it to the top. Now, disconnect the myograph unit from the temperature controller and allow the chamber to rest at room temperature for 10 minutes. Using fine forceps, loosen the nylon sutures and gently remove the cannulas from the artery and chamber.

Finally, transfer the entire solidified gel containing the artery from the chamber into a container filled with 70%ethanol. Equilibrated human omental artery in solution was visualized at physiological conditions. Upon exposure to 60 millimolar potassium chloride, viable artery showed a clear constriction followed by a return to baseline diameter after washing.

Hematoxylin and eosin staining of gel-embedded arterial cross-sections showed a well-preserved lumen and clear layering of endothelial, smooth muscle, and adventitial cells. Immunohistochemistry revealed intact expression of CD31 in endothelial cells and alpha-SMA in smooth muscle cells, confirming structural preservation of the vessel wall. In situ hybridization using a probe for Homo sapiens peptidyl-prolyl isomerase B showed bright red punctate signals in smooth muscle cells, indicating preserved mRNA integrity.