Augmentation of blood flow in delayed random skin flaps in the pig: effect of length of delay period and angiogenesis

Skin capillary blood flow and angiogenesis were studied by radioactive microsphere and morphometry technique, respectively, in delayed random skin flaps in the pig. Skin flaps were delayed for 2, 3, 4, 6, or 14 days. Blood flow was measured 6 hours after complete raising of acute and delayed random skin flaps on the opposite flanks of the same pig. It was observed that the capillary blood flow increased significantly (p less than 0.05) within 2 days of delay compared to the acute skin flaps. This capillary blood flow further increased by about 100 percent between days 2 and 3, started to plateau after day 3, and remained unchanged between days 4 and 14 of delay. This increase in capillary blood flow was mainly in the distal portion of the delayed skin flaps. There was no indication of an increase in the density of arteries in all delay periods studied. Our observations did not support the hypotheses that the delay phenomenon involves angiogenesis or long-term adaptation to ischemia, as have been hypothesized previously. The possible mechanism of delay is discussed.     

Hemodynamics and vascular sensitivity to circulating norepinephrine in normal skin and delayed and acute random skin flaps in the pig

Cutaneous circulation in 4 X 10 cm skin samples and delayed and acute random skin flaps constructed on the flanks of castrated Yorkshire pigs (13.3 +/- 0.7 kg; n = 12) were studied during intravenous infusion (0.5 ml per minute) of 5% dextrose solution (vehicle) and 5% dextrose containing norepinephrine (1 microgram/kg per minute). Total and capillary blood flow and A-V shunt flow were measured by the radioactive microsphere technique 6 hours after the raising of 4 X 10 cm single-pedicle acute and delayed random skin flaps using the technique and calculations published previously. Fluorescein dye test was also performed to assess vascular perfusion. It was observed that the capillary blood flow in the single-pedicle delayed skin flaps was similar to that in the normal skin, and the maintenance of this normal skin blood flow was not due to the closing of A-V shunt flow in the delayed skin flaps. Similarly, the significant (p less than 0.01) decrease in capillary blood flow and distal perfusion in the acute skin flaps compared with the delayed skin flaps was not due to the opening of A-V shunts in the acute skin flaps. There was no evidence to indicate that A-V shunt flow per se was the primary factor for the regulation of capillary blood flow in the acute and delayed skin flaps in the pig. Our data seemed to indicate that tissue ischemia in the distal portion of acute skin flaps was likely the result of vasoconstriction of the small random arteries which supplied blood to arterioles and A-V shunts, and locally released neurohumoral substances may play an important role in the pathogenesis of vascular resistance and ischemia in the acute skin flaps.     

Testicular blood flow and testosterone concentration in spermatic venous blood of anaesthetized rats

Testicular blood flow was measured by a radioactive microsphere technique. A significant correlation, rs equal to 0.82, was found between testicular blood flow and testosterone outflow in the spermatic venous blood, indicating that factors which affect the testicular circulation may influence testicular endocrine function.     

Measurement of carotid body blood flow in cats by use of radioactive microspheres

To resolve the controversy regarding carotid body blood flow, we used the radioactive microsphere technique for determination of tissue blood flow. We also measured the blood flow to several other tissues in the cat. Blood flow experiments were performed on 13 cats that were anesthetized, paralyzed, and mechanically ventilated with air. Different numbers of differently labeled 9-, 15-, and 25-micron microspheres were injected via a catheter into the left atrium. It was determined that one injection of 5 x 10(6) 15-micron microspheres was appropriate for the determination of carotid body blood flow. Flows to the carotid bodies and other organs by use of this protocol were as follows (ml.min-1.100 g-1, means +/- SE): carotid bodies, 1,417 +/- 143; adrenal glands, 406 +/- 89; left kidney, 355 +/- 69; right kidney, 375 +/- 74; heart, 201 +/- 39; liver 81 +/- 14; pancreas, 80 +/- 21; superior cervical ganglia, 62 +/- 9; carotid artery wall, 2.4 +/- 1.1. The blood flow to the carotid bodies was the highest for any organ. This measurement provides new evidence that tissue blood flow to the carotid body is very high. This high flow is consistent with the prompt physiological reflex functions of the carotid body.     

Measurement of renal blood flow in the rat.

The radioactive microsphere technique is a simple method for measurement of RBF and intrarenal blood flow distribution in the rat that does not require surgical manipulation of the kidney or general anesthesia. The results are reproducible and compatible with other established techniques.     

Blood flow in skin wound tissue and systemic hemodynamics as affected by a collagen-dalargin complex

In has been demonstrated that a collagen-dalargin complex increases blood flow in the granulation tissue (microsphere technique) due to the formation of new capillaries and a decrease in the vascular resistance. Dalargin had no effect on the capillary blood flow in the unaffected tissues. It has been concluded that the wound healing effect of dalargin may be related to its involvement in angiogenesis regulation.     

Systemic and regional hemodynamics in conscious rats during 24-hour antiorthostatic posture

The systemic and regional hemodynamics during antiorthostatic hypokinesia were studied in male Wistar rats using the radioactive microsphere technique. The animals were hanged up by the tail with the head tilted down (30 degrees) and were able to exercise using only front limbs. Twenty four hours long exposure to antiorthostasis induced significant changes in systemic hemodynamics as well as in regional blood flow in skeletal muscles, spleen, liver and pancreas. Antiorthostasis induced blood flow changes in lungs, heart and brain were less pronounced.     

Experimental comparison of bone revascularization by musculocutaneous and cutaneous flaps

Revascularization, one of the major components of bone healing, was examined in an experimental model. The radioactive microsphere technique demonstrated that after 4 weeks beneath a musculocutaneous flap, isolated bone segments had significant blood flow, whereas bone beneath a cutaneous flap did not. The muscle flap bone had a blood flow approximately half that of normal control bone. The muscle of the musculocutaneous flap had a blood flow three times that of the skin of the cutaneous flap. The bipedicle cutaneous flap used was designed to have a healthy blood supply, and at 4 weeks it had a blood flow twice that of control skin. Despite this, there was essentially no demonstrable blood flow in the cutaneous flap bone segments at 4 weeks. Only 3 of 17 bone segments underneath cutaneous flaps showed medullary vascularization, whereas 10 of 11 muscle flap bones did. All bone segments underneath muscle flaps showed osteoblasts and osteoclasts at 4 weeks; neither were seen in the cutaneous bone segments. The process of revascularization occurred through an intact cortex and penetrated into the cancellous bone. Because the bone segments were surrounded by an impervious barrier except for one cortical surface, the cellular activity seen is attributed to revascularization by the overlying flap. In this model, a muscle flap was superior to a cutaneous flap in revascularizing isolated bone segments at 4 weeks. This was documented by blood flow measured by the radioactive microsphere technique and by bone histology.     

Measurement of cardiac output in waking rats using a Doppler ultrasonic technic

The ultrasound Doppler technique was used for the determination of cardiac output in conscious Wistar rats. The cuff probes were placed on the ascending aorta and calibrated with the radioactive microsphere technique. The results of experiments show that Doppler ultrasound technique determines the cardiac output with high linearity. The possibility of using both methods in one and the same animal was also confirmed.     

Effect of vascular delay on viability, vasculature, and perfusion of muscle flaps in the rabbit

The delay procedure is known to augment pedicled skin or muscle flap survival. In this study, we set out to investigate the effectiveness of vascular delay in two rabbit muscle flap models. In each of the muscle flap models, a delay procedure was carried out on one side of each rabbit (n = 20), and the contralateral muscle was the control. In the latissimus dorsi flap model, two perforators of the posterior intercostal vessels were ligated. In the biceps femoris flap model, a dominant vascular pedicle from the popliteal artery was ligated. After the 7-day delay period, the bilateral latissimus dorsi flaps (based on the thoracodorsal vessels) and the bilateral biceps femoris flaps (based on the sciatic vessels) were elevated. Animals were divided into three groups: part A, assessment of muscle flap viability at 7 days using the tetrazolium dye staining technique (n = 7); part B, assessment of vascular anatomy using lead oxide injection technique (n = 7); and part C, assessment of total and regional capillary blood flow using the radioactive microsphere technique (n = 6). The results in part A show that the average viable area of the latissimus dorsi flap was 96 +/- 0.4 percent (mean +/- SEM) in the delayed group and 84 +/- 0.7 percent (mean +/- SEM) in the control group (p < 0.05, n = 7), and the mean viable area of the biceps femoris flap was 95 +/- 2 percent in the delayed group and 78 +/- 5 percent in the control group (p < 0.05, n = 7). In part B, it was found that the line of necrosis in the latissimus dorsi flap usually appeared at the junction between the second and third vascular territory in the flap. Necrosis of the biceps femoris flap usually occurred in the third territory, and occasionally in both the second and the third territories. In Part C, total capillary blood flow in delayed flaps (both the latissimus dorsi and biceps femoris) was significantly higher than that in the control flaps (p < 0.05). Increased regional capillary blood flow was found in the middle and distal regions, compared with the control (p < 0.05, n = 6). In conclusion, ligation of either the dominant vascular pedicle in the biceps femoris muscle flap or the nondominant pedicle in the latissimus dorsi muscle flap in a delay procedure 1 week before flap elevation improves capillary blood flow and muscle viability. Vascular delay prevents distal flap necrosis in two rabbit muscle flap models.     

Renal haemodynamics in chronic unilateral ureteral obstruction in the dog

Renal blood flow and its intrarenal distribution have been investigated after 7 and 14 days of unilateral ureteral ligation (UUL) in both the ligated and the undisturbed kidney by the radioactive microsphere technique without opening the abdomen and the obstructed ureter. It has been found that renal blood flow (RBF) decreased to about 26% after 7 days and to about 10% after 14 days of normal controls in the obstructed kidney. There was a pronounced inward shifting of the much reduced RBF. The slight increase in RBF of the undisturbed kidney was not significant and did not compensate the pronounced decrease of RBF in the obstructed one.     

Microsphere maps of regional blood flow and regional ventilation.

Systematically mapped samples cut from lungs previously labeled with intravascular and aerosol microspheres can be used to create high-resolution maps of regional perfusion and regional ventilation. With multiple radioactive or fluorescent microsphere labels available, this methodology can compare regional flow responses to different interventions without partial volume effects or registration errors that complicate interpretation of in vivo imaging measurements. Microsphere blood flow maps examined at different levels of spatial resolution have revealed that regional flow heterogeneity increases progressively down to an acinar level of scale. This pattern of scale-dependent heterogeneity is characteristic of a fractal distribution network, and it suggests that the anatomic configuration of the pulmonary vascular tree is the primary determinant of high-resolution regional flow heterogeneity. At approximately 2-cm(3) resolution, the large-scale gravitational gradients of blood flow per unit weight of alveolar tissue account for <5% of the overall flow heterogeneity. Furthermore, regional blood flow per gram of alveolar tissue remains relatively constant with different body positions, gravitational stresses, and exercise. Regional alveolar ventilation is accurately represented by the deposition of inhaled 1.0-microm fluorescent microsphere aerosols, at least down to the approximately 2-cm(3) level of scale. Analysis of these ventilation maps has revealed the same scale-dependent property of regional alveolar ventilation heterogeneity, with a strong correlation between ventilation and blood flow maintained at all levels of scale. The ventilation-perfusion (VA/Q) distributions obtained from microsphere flow maps of normal animals agree with simultaneously acquired multiple inert-gas elimination technique VA/Q distributions, but they underestimate gas-exchange impairment in diffuse lung injury.     

The blood supply to the abdominal organs of the fetal guinea-pig

Guinea-pigs near term of pregnancy were anaesthetized with diazepam and sodium pentobarbitone. A fetus was exposed and the vitelline artery catheterized to measure blood pressure and heart rate or to render a reference sample of blood for the determination of organ blood flow by the microsphere technique. The radioactive microspheres were injected through a catheter in the right atrium. Mean arterial blood pressure was 4.0 kPa and heart rate was 261 beats min-1. The liver, spleen, pancreas and gut receive most of their blood supply from the same trunk as the vitelline artery. The sample from this vessel was also used to calculate blood flow to the adrenal glands, kidneys, urogenital tract, and placenta, assuming even mixing of microspheres and blood in the abdominal aorta. Umbilical blood flow, corrected to a fetal weight of 100 g, averaged 7.5 ml min-1. The adrenal glands, which are known to increase their cortisol secretion near term, had a very high rate of perfusion. If the microspheres were injected in the umbilical vein, almost all were trapped in the liver, confirming the absence of a ductus venosus in the guinea-pig fetus. Most of these microspheres were found in the quadrate lobe of the liver. Hepatic arterial blood flow was also unequally distributed, with more than two-thirds going to the right lobe of the liver. Although the distribution of portal venous blood flow is not known, it is evident that different areas of the liver are presented with blood of greatly varying oxygen saturation.     

Ovarian capillary blood flow in seasonally anoestrous ewes induced to ovulate by treatment with GnRH

The microsphere technique was used to obtain estimates of ovarian capillary blood flow near ovulation, in 8 seasonally anoestrous ewes, which were induced to ovulate by GnRH therapy. Plasma progesterone concentrations were monitored in jugular blood sampled between Days 4 and 7 after the onset of the preovulatory LH surge. The ewes were then slaughtered. Three of the ewes were treated with a single injection of 20 mg progesterone before GnRH therapy. In these ewes and 1 other, plasma progesterone values increased after ovulation and reached 1.0 ng/ml on Day 7 following the preovulatory LH surge (normal, functional CL), whilst in the other 4 ewes progesterone concentrations increased initially then declined to 0.5 ng/ml by Day 7 (abnormal CL). In the ewes exhibiting normal luteal function, the mean ovarian capillary blood flow was significantly greater (P less than 0.01) than that for ewes having abnormal luteal function. Irrespective of the type of CL produced, capillary blood flow was significantly greater (P less than 0.05) in ovulatory ovaries than in non-ovulatory ovaries. These findings indicate that the rate of capillary blood flow in ovaries near ovulation may be a critical factor in normal development and maturation of preovulatory follicles and function of subsequently formed CL.     

The transient nature of myocardial ischemia during partial occlusion of the coronary artery in waking immobilized rabbits

The radioactive microsphere technique was used to study mechanisms of disappearance of myocardial ischemia during partial occlusion of the left descending anterior coronary artery with implanted device in conscious immobilized rabbits. Microspheres (15 microns, NEN, USA) were injected before occlusion, immediately after ST-segment elevation and after disappearance of ST-segment shift. In ischemic region blood flow dropped by 45% (p less than 0.05) and mean blood pressure decreased by 12% (P less than 0.05) on the 1st minute of coronary occlusion. 8-15 min later ST-segment elevation disappeared and the blood flow in ischemic region became higher than control level (on the average by 35%). It is suggested that ischemia is abolished mainly by dilatation of distal coronary vessels, than by activation of collateral blood flow.     

Use of microspheres in measurement of regional blood flows during +GZ stress

The use of the radiolabeled microsphere technique for the study of the effects of +GZ acceleration on regional blood flow is examined. A theoretical analysis of the limits of this technique in a high acceleration environment is presented. Chronically implanted, electromagnetic, aortic flow probes were used to determine the relationship between aortic blood flow velocity and +GZ acceleration in conscious adult miniature swine. It was found that conscious straining adult miniature swine, with the assistance of an inflated anti-G suit, are able to compensate quite well to acceleration levels less than or equal to +7 GZ. Exposure to +9 GZ often resulted in unstable cardiovascular states involving relative bradycardia, often progressing to asystole, declining aortic blood pressure, and markedly diminished cardiac outputs approaching zero. It was found that, if aortic pressure and heart rate attain a relatively steady state during acceleration, and if heart level mean aortic pressure is greater than or equal to 100 Torr, the application of the microsphere technique during +GZ acceleration is theoretically valid. This hypothesis was tested using the microsphere technique (9.0 +/- 0.8 microns diam) in conscious miniature swine during exposure to +GZ acceleration. It is concluded that within the defined limits the radiolabeled microsphere technique is as accurate for use during acceleration studies as it is for use in routine laboratory studies.     

High spatial resolution measurements of organ blood flow in small laboratory animals

With the use of a newly developed Imaging Cryomicrotome to determine the spatial location of fluorescent microspheres in organs, we validate and report our processing algorithms for measuring regional blood flow in small laboratory animals. Microspheres (15-microm diameter) of four different fluorescent colors and one radioactive label were simultaneously injected into the left ventricle of a pig. The heart and kidneys were dissected, and the numbers of fluorescent and radioactive microspheres were determined in 10 randomly selected pieces. All microsphere counts fell well within the 95% expected confidence limits as determined from the radioactive counts. Fluorescent microspheres (15-microm diameter) of four different colors were also injected into the tail vein of a rat and the left ventricle of a rabbit. After correction for Poisson noise, correlation coefficients between the colors were 0.99 +/- 0.02 (means +/- SD) for the rabbit heart and 0.99 +/- 0.02 for the rat lung. Mathematical dissection algorithms, statistics to analyze the spatial data, and methods to visualize blood flow distributions in small animal organs are presented.     

Fasciocutaneous flaps: an experimental model in the pig

No experimental studies have substantiated the claim that fasciocutaneous flaps are superior to skin flaps. Using fasciocutaneous flaps designed in the pig, both flap survival and blood flow were assessed. The forelimb and hindlimb fasciocutaneous flaps survived to 8.2 +/- 0.3 cm and 7.9 +/- 0.3 cm, respectively, compared with 7.3 +/- 0.3 cm and 6.7 +/- 0.3 cm for the comparable cutaneous flaps, a statistically significant finding (p less than 0.01). Random fasciocutaneous flaps survive 12 to 18 percent longer than skin flaps. Using the radioactive microsphere technique, blood flow was measured after flap elevation, and flap survival was estimated using fluorescein. Again, a significant difference in flap survival was found, but there was no significant difference in measured blood flow. This can be explained by the relatively large interval between blood flow measurements (2 cm) compared with the observed difference in survival length (1.0 +/- 0.3 cm).     

Effect of opioid peptides on regional hemodynamics in waking rats

The hemodynamic effects of intravenously administered relatively selective mu-(DAGO) and delta-(DADL) opioid agonists were investigated in conscious rats. The radioactive microsphere technique was used to measure regional blood flow in 10 zones before and 5 min after bolus injection of each peptide. Both opioid agonists in a dose of 1 mumol/kg produced transient hypotension, bradycardia and apnoea. DADL injection increased blood flow in the adrenals and decreased it in the muscles; vascular resistance spleen. DAGO administration increased blood flow in the adrenals and decreased it in the pancreas and skin, whereas vascular resistance increased in the pancreas and skin and decreased in the adrenals. Naloxone pretreatment diminished regional blood flow responses to DAGO. Regional hemodynamic changes after peptide administration are suggested to be connected with the activation of peripheral opiate receptors. High differentiation of regional vascular responses may be related to heterogeneous distribution of mu- and delta-opiate receptors in the body.