An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.
The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables.
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.
1. Prior to the Experiment
2. Day of the Experiment
3. Microvessel Harvesting
4. Cannulating the Vessel
5. Representative Results
Figure 1A. This figure presents the basic microvessel station setup. A = television for viewing vessel; B = digital calipers; C= syringe for pushing superfusate through inflow pipette as necessary; D = hydrostatic column for changing perfusate inflow pressure; E = microscope; F = microvessel chamber; G = superfusate reservoir; H = pressure monitor (multiple can be added as necessary); I = microvessel chamber drain pump for superfusate; J = equilibration gas mixture tank; K = waste contained (for superfusate); L = circulating water heater; M = anti-vibration/floating table.
Figure 1B. This figure presents a closer view of the microvessel chamber setup. In this figure, E = microscope; F = microvessel chamber; H = pressure monitor (while we show only one for simplicity, multiple can be added to different sites as necessary); I = microvessel chamber drain pump; N = tubing attached to the very beginning of the perfusate inflow pipette (a continuation from ‘D’ in Figure 1A); O = tubing attached to the perfusate outflow pipette.
Figure 1C. This figure presents the closest view of the microvessel chamber. In this figure, N = the perfusate line (i.e., tubing attached directly to the perfusate inflow pipette); O = tubing attached directly to the perfusate outflow pipette; P = the horizontal and vertical location controls for the inflow pipette; Q = the superfusate drain for the water bath (connected to ‘I’ in Figures 1A and 1B); R = the inflow for the water bath coming from the reservoir (connected to ‘G’ in Figure 1A).
Figure 2. This figure presents a schematic representation of the flow of fluids and gases in the system. In this figure, A = PSS superfusate line inflow; B = PSS perfusate line to inflow pipette; C = PSS superfusate drain from bath to waste container; D = PSS perfusate outflow from outflow pipette; E = heated water input into chamber jacket; F = heated water output from chamber jacket.
Figure 3. This figure presents individual tie loops made from ophthalmic suture (viewed through a dissecting microscope with 10x eyepieces and a 4.5x adjustable zoom magnification; Panel A) that are used in our preparation and the storage of multiple tie loops from 8-0 and 10-0 suture on reversed tape around a microscope slide which is stored in a covered Petri dish (to avoid losing them; Panel B).
Figure 4. This figure provides an image (viewed through a standard dissecting microscope) of the gracilis muscle resistance arteriole prior to surgical isolation and removal to allow for proper spatial orientation.
Figure 5. This figure provides a representative image of a cannulated microvessel. Panel A shows the entire length of the vessel, with both inflow (A) and outflow (B) pipettes as well as tie loops (C) shown. Panel B presents an image at a high magnification, clearly showing the determination of the arteriolar inner diameter with the digital calipers (in this case the diameter is 112 μm).
Figure 6. This figure presents representative images of a cannulated microvessel following equilibration. The top image is of a vessel under control, unstimulated conditions at the equilibration pressure, the middle image is of the same vessel following dilation to challenge with acetylcholine (10-6 M in the bath), and the bottom image is of that vessel following constriction to challenge with phenylephrine (10-7 M in the bath).
Figure 7. This figure summarizes the responses of an isolated microvessel in response to challenge with: hypoxia (Panel A, a reduction in perfusate and superfusate PO2 in our system from ~135 mmHg to ~40 mmHg), increasing concentrations of acetylcholine (Panel B), changes in intravascular pressure under control conditions and following incubation of the vessel with a calcium-free PSS (Panel C).
The protocol presented describes the isolation, removal and double cannulation of a skeletal muscle microvessel, although this general technique can be readily applied to most tissues. For the current manuscript, the term “arteriole” has been used by the authors to describe a resistance vessel ranging between 70-120 μm in diameter under resting active tone, which is also a major contributor to the regulation of perfusion resistance to an organ or tissue.
With some modifications, this system can be fitted to multiple applications for the specific investigator and readily allows for the incorporation of additional techniques to provide more in depth experimentation (e.g., use of transmembrane electrodes for determination of membrane potential17). A critical step in the basic preparation is always insuring that the inflow and outflow pipettes that are used to cannulate the vessel are well matched, remain clear, and allow flow and pressure to be maintained in a facile manner. When the pipettes become clogged with minute pieces of debris, it may be necessary to break off very small pieces of the tips to restore flow. If the investigator is careful, pipettes can be reused multiple times until they reach a point where they either cannot be unclogged or the diameters sufficiently large that cannulation of the vessel becomes extremely difficult. However, with mastery of this surgical and experimental preparation, careful attention to detail, and the appropriate modifications to suit specific conditions and requirements, investigators can readily evaluate pathways determining vascular reactivity in response to a myriad of physiological and pharmacological challenges. This preparation will also allow the investigator to perform basic analyses on alterations to vascular wall mechanics under conditions of no active tone14. This is indeed a very powerful and adaptable technique and it is one that can readily be used for applications spanning the range of detailed mechanistic study to high-throughput screenings of more basic responses.
The authors have nothing to disclose.
This work was supported by the American Heart Association (EIA 0740129N) and NIH T32 HL90610.
Reagents and Equipment | Company | Comments/Catalogue # |
Vessel Chamber | Custom | Dave Eick (MCW) |
Heated Circulating Water Bath | PolyScience and Haake | Haake DC 10 |
Pipets | Frederick Haer & Co. | Capillary Tubing 2.0 mm OD x 1.0 mm ID (27-33-1) |
Pressure Monitor | World Precision Instruments | |
Water Jacketed Reservoir | Custom | |
External Light Source | World Precision Instruments | Novaflex |
Pipet Puller | MicroData Instruments | PMP102 Micropipet Puller |
Full complement of surgical tools | Fine Science Tools | Dumont |
Ultra Fine Forceps | Fine Science Tools | Inox #5 |
Silk Suture Thread | Ethilon | #10-0 or 9-0 |
Stereo Microscope | Olympus | Olympus SZ-11 |
Analog Video Calipers | Boeckeler | Via Controller (Via-100) |
High Resolution Analog Camera | Panasonic | GP-MF 602 |
Oxygen Tank | Regional | 21% balance nitrogen and 5% CO2 balance nitrogen |
Tubing | Tygon | |
Drain Pump | Cole Parmer Instrument Co. | |
Modified Rat PSS | See recipe below | |
Van Breemen’s Relaxant PSS | See recipe below |
Table 1. A list of the major components of isolated microvessel station setup presented in the Figures.
Modified Rat PSS Recipe | To make two liters of PSS | 20X Salt Stock (2L) | 20X Buffer Stock (2L) |
NaCl | 278.0 g | ||
KCl | 14.0 g | ||
MgSO4-7H2O | 11.5 g | ||
CaCl2-H2O | 9.4 g | ||
NaHCO3 | 80.8 g | ||
EDTA | 0.4 g | ||
NaH2PO4 | 0.28 g | ||
Glucose | 1.98 g | ||
20x Salt Stock | 100 mL | ||
20x Buffer Stock | 100 mL | ||
Distilled Water | 1800 mL |
Table 2. Recipe for standard physiological salt solution (PSS) used in the isolated microvessel protocols.
Comments on Recipe: Make 2 L of Salt Stock and 2 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final PSS.
Van Breemen’s Relaxant PSS | To make 2 liters of PSS | 20X Salt Stock (1L) | 20X Buffer Stock (1L) |
NaCl | 107.4 g | ||
KCl | 7.0 g | ||
MgSO4-7H2O | 5.76 g | ||
MgCl2-6H2O | 81.32 g | ||
NaHCO3 | 40.4 g | ||
EDTA | 0.2 g | ||
EGTA | 15.22 | ||
NaH2PO4 | 0.28 g | ||
Glucose | 1.98 g | ||
20x Salt Stock | 100 mL | ||
20x Buffer Stock | 100 mL | ||
Distilled Water | 1800 mL |
Table 3. Recipe for Van Breemen’s relaxant physiological salt solution (PSS) used in the isolated microvessel protocols under conditions of zero active tone.
Comments on Recipe: Make 1 L of Salt Stock and 1 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final relaxant PSS.