Invited Symposium: Hypertension III: Flow-Induced Vascular Remodeling
Materials & Methods
Discussion & Conclusion
Microvascular Adaptations to Reduced Blood Flow: Introduction of a New Model
Contact Person: Donna H. Wang (firstname.lastname@example.org)
Materials and Methods
4-week-old male Wistar rats weighing 112+6 g (mean + SEM) were randomly divided into 3 groups. In group 1, a single cremaster muscle was studied immediately in microcirculatory experiments. The remaining 2 groups were subjected to unilateral orchidectomy and studied 3 weeks later. Group 2 was used for microcirculatory studies of both cremaster muscles, while group 3 was used for blood flow measurements with radioactive microspheres.
Group 2 and group 3 rats were anesthetized, and a unilateral longitudinal incision was made in the abdominal wall lateral and just cephalad to the penis. The testis, epididymis, ductus deferens and the accessory glands were pushed from the scrotum through the inguinal canal into the abdominal cavity. The testicular artery which arises from the internal spermatic artery was ligated proximal to the testis and the testis was surgically removed. The deferential artery and its branch supplying the cremaster muscle, the cremasteric artery, was left intact. The rest of the structure was pushed back into the scrotum and the wound was closed.
BLOOD FLOW MEASURED BY RADIOACTIVE MICROSPHERES:
After the rats were anesthetized, a PE 50 cannula was passed retrogradely through the right carotid into the left ventricle. Radioactively labeled (85 Sr) microspheres were suspended in an isotonic saline solution containing 0.05% Tween 80 to provide a concentration of approximately 2.36 x 106 microspheres/ml. A total volume of 0.2 ml of the sonicated microsphere suspension, containing approximately 4.72 x 105 microspheres, was injected into the left ventricle. Arterial blood was withdrawn into a heparinized syringe at a rate of 0.66 ml/min through PE 50 tubing in the left femoral artery for 2 min. Both cremaster muscles, both kidneys, and the right spinotrapezius muscle were dissected and placed in test tubes for counting in a gamma counter. The flow to the organs was determined from the equation: organ flow = (reference sample flow / activity in reference sample) x activity in organ. The cardiac output was determined by the equation: cardiac output = (reference sample flow / reference sample activity) x total amount of activity injected.
CREMASTER MUSCLE PREPARATION:
Rats were anesthetized and the left carotid artery was cannulated for the measurement of mean blood pressure (MBP) and heart rate (HR). A single cremaster muscle from alternate sides (group 1) or both cremaster muscles (group 2) were surgically prepared for microvascular observation with a technique previously described by Baez (5). The exposed cremaster muscle was spread over a Plexiglas pedestal and continuously superfused at 34+0.5oC at a rate of 2 ml/min with modified Krebs-Henseleit solution, equilibrated with gas mixtures containing 5% CO2 and 95% N2. Warmed water was circulated through the base of the pedestal to help control the temperature. The entire preparation was placed on the stage of a Zeiss microscope, and cremaster arterioles were examined by closed-circuit television microscopy with a Vista Model 308 image shearer.
ADENOSINE CHALLENGE IN CREMASTER ARTERIOLES:
Cremaster arterioles were classified as feeding (1A), arcading (2A), transverse (3A) and precapillary (4A) arterioles on the basis of their branching pattern. Resting inside diameters (IDrest) were measured for all branching orders of arterioles. After topical application of 10-3M adenosine, relaxed inside (IDrelax) and outside diameters (ODrelax) were measured. The number of flowing 4A's on a single 3A were also counted before and after adenosine. Cross-sectional wall area (CSWA) was calculated from the equation: CSWA = p [(ODrelax)2 - (IDrelax)2]/4. Percent arteriolar tone was expressed as 100 [(IDrelax) - (IDrest)]/(IDrelax). Approximately 3-5 2A's, 3A's and 4A's were measured in each animal and a mean value was calculated for each, so that "n" always represents the number of animals.
BLOOD FLOW MEASUREMENTS BY THE DUAL-SLIT TECHNIQUE:
Signals from a dual-fiberoptic photometer system were analyzed with a Princeton Applied Research Model 100A correlator to obtain the time delay between the upstream and downstream signals. Mean velocity (Vm) was calculated using the empirical proportionality factor of 1.6 (6). Arteriolar blood flow was calculated from the mean velocity and the cross-sectional area of the vessel [Flow (ll/sec) = Vm x pr2]. Red cell velocity was measured under resting conditions in every arteriole entering the muscle proximally from outside the field of view, whether they were feeding or arcading arterioles. Total blood flow to the cremaster muscle was obtained by summation of the individual flows and care was taken that flows were summed only once.
The cremaster muscle was perfused with vasodilators and formalin at the individual mean blood pressure of the rat. Cremaster arterioles were then filled with Microfil MV-122. The thickness of the muscle was measured by focusing on the surface of the muscle and the surface of the stage and reading the difference on the calibrated fine focal adjustment scale of the microscope. The weight of the muscle was measured and the volume was calculated assuming a density of 1.06 g/cm3. The area of the muscle was calculated from the volume divided by the thickness. The muscles were then cleared with increasing concentrations of glycerin in water of 50%, 75%, 85% and 100%. Arteriolar density was determined by stereological methods as published by Schmid-Schoenbein et al. (7). The density as length/volume was calculated from, pN/2LT, where N is the number of intersections, L is the total length of the grid with respect to the microcirculation, and T is the thickness of the muscle. The total length of 2A's in each muscle was calculated from the length density times the volume. The distances between 3A branch points along a 2A were measured with a filar eyepiece, and the total number of 3A's in each muscle was then calculated from the total length of 2A's divided by the mean distance between 3A branch points.
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