Trapping of Radioactive Microspheres in the Pregnant and Non-Pregnant Rabbit

We injected radioactive microspheres, 15, 25 and 50 μn in diameter in the left ventricle or the lower aorta of rabhits and counted the radioactivity in the lungs to estimate the fraction of microspheres shunted through the systemic circulation. The degree of microsphere shunting was inversely proportional to microsphere diameter. The maximum fraction of 15 μm micro-spheres shunted after an aortic injection was 8.8 % as compared with 6.2 % with 25 μm spheres and 1.2 % with 50 μm spheres. There was no statistically significant change in the fraction of microspheres shunted in pregnancy or with anesthesia. Shunted microspheres may continue to arrive in the lungs for up to 15 min following aortic injection. Microspheres at least 25 μm in diameter should he employed to measure the fractional distribution of cardiac output in rahbits although it may he preferable to use smaller microspheres to measure blood flow within certain organs.     

Shunting of microspheres across the canine coronary circulation.

Coronary shunting of 9 +/-1 micrometer and 25 +/- 5 micrometer radiolabeled microspheres was examined in anesthetized, open-chest dogs, whose left common coronary arteries were perfused at controlled pressures. Shunting was estimated from the difference in radioactivity between perfusion line and coronary sinus blood samples during selective elevations of coronary perfusion pressure (CPP), left ventricular afterload, and inspired oxygen. A linear relationship was found between coronary shunting of 9-micrometer microspheres and CPP over the range 100-200 mmHg. According to regression analysis, percent shunt flow was 4.0% at control CPP (100 mmHg) and 10.0% at CPP of 200 mmHg. No shunting of 25-micrometer microspheres occurred at any CPP. Raising afterload did not affect shunting at control CPP but attenuated the increase in shunting at elevated CPP. Changing inspired gas from room air to 100% oxygen did not influence shunting at control or elevated CPP. Raising CPP to 150 and 200 mmHg also released 2.5% and 5.9% of pretrapped 9-micrometer microspheres, respectively. This study demonstrates that vessels permitting passage of microspheres across coronary circulation are sensitive to elevated perfusion pressure.     

The radioactive microsphere method for the assessment of regional myocardial blood flow after coronary artery occlusion

In this study, we have tried to determine the magnitude of the inaccuracy of the radioactive microsphere method - due to variations in the diameter distribution of the spheres - for measuring regional myocardial blood flow after coronary artery occlusion. In 5 mongrel dogs, three types of 15 mum microspheres, labelled with 125I, 141Ce or 85Sr, were injected simultaneously after the descending branch of the left coronary artery had been ligated. Myocardial samples wert taken from the left ventricle and divided into four groups according to the number of spheres per sample. The radioactivity of the various isotopes per gram tissue was expressed as percentage of their activity per milliliter of the reference sample. The diameter distribution of microspheres, labelled with each of the isotopes, was determined light-microscopically in suspensions belonging to three different batches. The relative error, as determined from the difference in relative radioactivity of the various types of microspheres in the tissue samples, was higher than the theoretical error for each of the number of spheres per sample. It is very likely that this discrepancy is caused by the differences in diameter distribution of the various types of microspheres, resulting in non-random error. The smaller spheres tended to go to low flow areas and the larger ones to high flow areas. Because of the non-randomness, the error due to diameter variations in the spheres can be diminished by randomizing the order of injection of the various isotopes. The present study indicates that the relatively high degree of accuracy of the microsphere method for the determination of blood flow to large parts of the myocardium with an unimpeded coronary circulation, as was described in literature, cannot be extrapolated to the determination of regional myocardial blood flow after coronary artery occlusion, when the combination of small tissue samples, variations in the diameter distribution of the spheres and an unevenly distributed myocardial blood flow unfavourably affect the accuracy of the method.     

Measurement of local blood flow in acute myocardial infarction: loss of 15-micron microspheres during the first hour

Distribution of radiolabelled microspheres is widely utilized for determination of regional blood flow in experimental myocardial infarction studies. The purpose of this investigation was evaluation of the microsphere method during 1 h of regional ischaemia. Special attention was focused upon loss of preocclusion microspheres from ischaemic myocardium; mechanisms for loss and blood flow distribution in non-ischaemic left ventricle. Microspheres (15 micron) were injected into the left atrium in nine pentobarbital anaesthetized cats prior to coronary artery occlusion and again after 1 h of occlusion. Preocclusion blood flow estimates were lower in ischaemic compared with non-ischaemic myocardium (1.36 vs. 1.62 cm3 X min-1 X g-1, P = 0.002), corresponding to 16% apparent loss. In endocardial ischaemic tissue, development of oedema could account for the loss. In epicardial ischaemic tissue, oedema was not present and loss was therefore due to migration of microspheres. Epicardial loss increased in proportion to restoration of left ventricular contractility. There was no evidence for significant microsphere loss through lymphatic pathways. In non-ischaemic left ventricular tissue, myocardial blood flow was evenly distributed from apex to base, and also between endocardial and epicardial layers. This study quantitates an important limitation to measurements of local blood flow in ischaemic myocardium by radiolabelled microspheres.     

Arteriovenous shunting in the lymph node before and after antigenic stimulus.

We have assessed the effects of antigenic stimulation on the regional blood flow of the lymph node. In seventeen New Zealand white rabbits, the regional blood flow to the popliteal lymph nodes was determined using 9 and 15 micron microspheres at 16, 40, and 144 h following antigen administration. The regional blood flow in the resting lymph node measured with 15 micron spheres was 0 . 46 +/- 0 . 07 ml g-1 min-1, with 9 micron spheres 0 . 26 +/- 0 . 03 ml g-1 min-1. There was a significant 'differential flow' (the difference in blood flow between the experimental and control nodes) measured with 15 micron spheres (P less than 0 . 05). This increase in blood flow was larger early (16 h) than late (144 h) following antigen administration (P less than 0 . 01). There was significant (P less than 0 . 05) 'shunt flow' (the difference in blood flow measured with 15 micron and 9 micron spheres) in the lymph node, with more shunting early (16 h) than late (144 h) following antigen administration. No significant shunting was seen in the kidney or thigh muscle. Thus, we have established: (1) a significant increase in blood flow following antigenic stimulus; (2) shunting of 9 micron microspheres in the resting popliteal lymph node; (3) increased shunting following antigenic stimulus; and (4) that the increase in nodal blood flow was, to a large extent, secondary to increased shunt flow. Both the increased blood flow and the shunting shortly after antigenic stimulus may be important in the evolution of the immune response.     

A comparison between direct and indirect measurements of blood flow in the follicular ovary of the rabbit.

A comparison was carried out between direct and indirect measurements of blood flow in follicular ovaries of anaesthetized laparotomized rabbits: Direct measurements by timed quantitative sampling of ovarian venous blood yielded blood flow values 3 times higher than those found with the use of 15 mugm radioactive microspheres. However, when simulating the surgical trauma associated with direct measurements, the microsphere technique gave the same high ovarian flow values, indicating that this type of trauma caused ovarian hyperemia. It is concluded that direct measurements of blood flow, as performed in the present study, are unsuitable for investigations of circulatory events in the ovary. Mechanically induced ovarian vasodilatation is probably a factor which helps to explain the diverging data on blood flow obtained with direct and indirect methods. The radioactive microsphere technique appears to provide a means for reasonably reliable ovarian blood flow measurements in laboratory animals.     

Measurement of local blood flow in acute myocardial infarction: loss of 15-^m microspheres during the first hour

Distribution of radiolabeled microspheres is widely utilized for determination of regional blood flow in experimental myocardial infarction studies. The purpose of this investigation was evaluation of the microsphere method during 1 h of regional ischaemia. Special attention was focused upon loss of preocclusion microspheres from ischaemic myocardium; mechanisms for loss and blood flow distribution in non-ischaemic left ventricle. Microspheres (15 μm) were injected into the left atrium in nine pentobarbital anaesthetized cats prior to coronary artery occlusion and again after 1 h of occlusion. Preocclusion blood flow estimates were lower in ischaemic compared with non-ischaemic myocardium (1.36 us. 1.62 cm³min^-1 g^-1, P = 0.002), corresponding to 16%apparent loss. In endocardial ischaemic tissue, development of oedema could account for the loss. In epicardial ischaemic tissue, oedema was not present and loss was therefore due to migration of microspheres. Epicardial loss increased in proportion to restoration of left ventricular contractility. There was no evidence for significant microsphere loss through lymphatic pathways. In non-ischaemic left ventricular tissue, myocardial blood flow was evenly distributed from apex to base, and also between endocardial and epicardial layers. This study quantitates an important limitation to measurements of local blood flow in ischaemic myocardium by radiolabelled microspheres.     

Estimating perfusion using microCT to locate microspheres

The injection of microspheres into the blood stream has been a common method to measure the spatial distribution of blood flow (perfusion). A technique to conduct this kind of measurement in small animal organs is presented using silver-coated microspheres with a diameter of 16 microm and high-resolution computed tomography (microCT) to detect individual microspheres. Phantom experiments demonstrate the detectability of individual spheres. The distribution of microspheres within a rat heart is given as an example. Using non-destructive, three-dimensional imaging for microsphere detection avoids the cumbersome dissection of the organ into samples or slices and their subsequent registration. The detection of individual spheres allows high-resolution measurements of perfusion and arbitrary definition of regions of interest. These, in turn, allow for accurate statistical analysis of perfusion such as relative dispersion curves.     

Simultaneous estimation of arteriolar, capillary, and shunt blood flow of the gut mucosa

Microspheres of 15 and 9 microns diameter were injected simultaneously into the left ventricle of the dog, and the entrapment of these microspheres in different layers of the gut wall was measured under resting conditions, vasoconstriction, and subsequent vasodilation. Results show that some of the 9-microns spheres passed into the portal blood through all layers of the gut wall, whereas 15-microns spheres were completely entrapped in the tissue. The pattern of entrapment of 15-microns spheres during vasoconstriction and during subsequent vasodilation suggests that these microspheres measure adequately the arteriolar inflow of the muscularis, submucosa, and mucosa (i.e., villus plus crypt) but, due to series arrangement between the arterioles of the villus and the crypt, cannot measure the arteriolar flow of the villus and crypt separately. The entrapment of 9-microns microspheres in the muscularis, submucosa, crypt, and villus that occurred during vasoconstriction did not change during subsequent vasodilation. This suggests that these microspheres became lodged in the precapillary sphincter or capillary and therefore measured the capillary flow of these layers. Accordingly, the difference between the arteriolar (measured by 15 microns) and the capillary (measured by 9 microns) flow of the muscularis, submucosa, and mucosa may provide an estimate of the noncapillary (shunt) flow of these layers.     

Regional Distribution of Blood Flow in Calf Muscles of Rat during Passive Stretch and Sustained Contraction

The regional distribution and number of microspheres in the calf muscles of rat has been studied during isometric sustained contraction and in stretched uncontracted muscles in situ. Carbonized microspheres, 15 ± 5 μm, were injected into the aortic arch and muscle blood flow arrested 6 sec later. The calf muscles were freeze sectioned (12 slices of 40μm) and the microspheres counted microscopically. The microsphere concentration in the gastrocnemius and plantaris muscles during rest was 4.6 ± 1.6 spheres/mm³ (mean ± S.E.). One min after a standardized exercise programme the sphere concentration was increased to 20.5 ± 3.9 spheres/mm³. At increasing force of contraction following the standard exercise programme, the microsphere concentration fell from 11.6 ± 2.5 at 25% of maximal force of contraction (MFC) to 2.2 ± 0.6 spheres/mm³ at 100% MFC (5.6 kg/cm²). Corresponding measurements in stretched, uncontracted muscles showed a similar fall in microsphere concentration when stretch was increased from 50 to 175% MFC. The ratio between microsphere concentration in the central inner zone and in the peripheral outer zone was slightly higher than unity (1.08-1.16) in muscles at rest and at light postexercise hyperemia. At 75 and 100% MFC the ratio was 0.76 ± 0.07 and 0.57 ± 0.13, significantly lower than unity. Stretching of the uncontracted muscle group to 175% MFC reduced the ratio towards zero. The greater reduction in blood flow to the inner central zone of contracted calf muscles shown by microsphere distribution was confirmed by measurement of ¹²⁵I-antipyrine distribution. These results show an increased resistance against blood flow during active contraction or stretching of the calf muscles, most pronounced in the central inner zone at high tensions.     

Regional distribution of blood flow in calf muscles of rat during passive stretch and sustained contraction.

The regional distribution and number of microspheres in the calf muscles of rat has been studied during isometric sustained contraction and in stretched uncontracted muscles in situ. Carbonized microspheres, 15 +/- 5 mum, were injected into the aortic arch and muscle blood flow arrested 6 sec later. The calf muscles were freeze sectioned (12 slices of 40 mum) and the microspheres counted microscopically. The microsphere concentration in the gastrocnemius and plantaris muscles during rest was 4.6 +/- 1.6 spheres/mm3 (mean +/- S.E.). One min after a standardized exercise programme the sphere concentration was increased to 20.5 +/- 3.9 spheres/mm3. At increasing force of contraction following the standard exercise programme, the microsphere concentration fell from 11.6 +/- 2.5 at 25% of maximal force of contraction (MFC) to 2.2 +/- 0.6 spheres/mm3 at 100% MFC (5.6 kg/cm2). Corresponding measurements in stretched, uncontracted muscles showed a similar fall in microsphere concentration in the central inner zone and in the peripheral outer zone was slightly higher than unity (1.08-1.16) in muscles at rest and at light postexercise hyperemia. At 75 and 100% MFC the ratio was 0.76 +/- 0.07 and 0.57 +/- 0.13, significantly lower than unity. Stretching of the uncontracted muscle group to 175% MFC reduced the ratio towards zero. The greater reduction in blood flow to the inner central zone of contracted calf muscles shown by microsphere distribution was confirmed by measurement of 125 I-antipyrine distribution. These results show an increased resistance against blood flow during active contraction or stretching of the calf muscles, most pronounced in the central inner zone at high tensions.     

Retention and biodistribution of microspheres injected into ischemic myocardium

Intramyocardial injection of therapeutic agents may enhance heart repair after infarction. Incomplete retention of intramyocardial injections has been reported, but modes of loss are undefined. We determined the fate of neutron-activated microspheres injected into acutely ischemic rat myocardium using saline, Pluronic F127, or Matrigel as vehicle. Twenty minutes after injection in saline, 63% +/- 12% of 10-mum microspheres was retained in the heart. Similar retention was observed after 6 days. Injection site leakage accounted for 14% +/- 5% of the microspheres, whereas exit via coronary veins resulted in 11.2% +/- 9.5% collecting in the lungs. Microspheres distribution to other organs was minimal. Retention of 40-mum microspheres was similar to that observed with the 10-mum microspheres. Pluronic F127 and Matrigel reduced immediate leakage to 4% +/- 1% and 2% +/- 1%, respectively. Surprisingly, microsphere retention in the heart was not improved at 20 min using either gelling vehicle, suggesting that leakage occurs over a prolonged period. Thus, most injected particles are retained in the ischemic rat heart following direct injection, but significant fractions are lost from the injection site and through coronary veins. Gelling agents reduced short-term leakage, but failed to enhance longer-term retention. Hydrogels with stiffer mechanical properties might enhance retention and reduce variability.     

The microsphere method for measuring low blood flows: theory and computer simulations applied to findings in the rat cochlea

When the microsphere method of blood flow measurement is used in small organs, the number of spheres actually recovered may be small. This introduces errors which, however, are not necessarily important as compared with the biological variation. A mathematical model was constructed which allowed estimation of the extra uncertainty caused by the low number of spheres. It was applied to an experimental material of over 100 measurements of cochlear blood flow in the rat, where only about 60 spheres were recovered per cochlea. It was concluded that this paucity of spheres introduced only a small to moderate extra error. Simple approximate formulae were derived allowing easy estimation of the uncertainty caused by low number of spheres under different conditions. In general, there appear to be many situations where fewer microspheres than are commonly used would suffice.     

Influence of coronary venous retroinfusion and vasodilatation on regional myocardial blood flow measurement with microspheres. An analysis of ‘microsphere loss’ from ischaemic and reperfused porcine hearts

The influence of coronary venous retroinfusion and a vasoselective calcium antagonist felodipine on the microsphere loss in a porcine model of myocardial ischaemia and reperfusion was studied. Sixteen open-chest pigs underwent 45 min of myocardial ischaemia induced by occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Either felodipine (felo-retro group, 7 nmol kg^-1, n= 6) or the corresponding amount of vehicle (vehicle-retro group, n= 5) was infused retrogradely into the coronary veins over 30 min, starting 5 min before reperfusion. In a third group, the same amount of felodipine was administered intravenously (felo-iv group, n= 5). Myocardial regional blood flow was measured with radiolabelled microspheres (ø= 15 μm) injected before ischaemia to investigate a possible loss during ischaemia. In the felo-retro group, the apparent blood flow in the ischaemic areas, expressed as a percentage of the corresponding values in the non-ischaemic areas (%-flow), were 73±15, 73±11 and 75±19 in the subendocardial, midmyocardial and subepicardial layers, respectively. The corresponding percentage flows were 64±11, 70±11 and 62±9 in the vehicle-retro group and 75±18, 77±15 and 76±11 in the felo-iv group. The differences between the groups were not satistically significant. It is concluded that in this open-chest preparation microsphere loss observed in the ischaemic and reperfused myocardium is not increased by coronary venous retroinfusion or by a concomitantly administered vasodilative agent like felodipine.     

A method to improve the size uniformity of microspheres

When microspheres are used to measure blood flow, the amount that may be injected is limited by side effects, primarily a rise in arterial pressure. It is therefore desirable that the proportion of spheres that actually get stuck in the capillaries is as large as possible. Too small spheres, which pass through the capillaries, as well as too large spheres, which may be held up in the arterioles, should be avoided. This is especially the case if blood flow is measured in small tissue segments, since the spheres found may be so few that the statistical variability of the result becomes embarrassing. From this point of view, commercially available microspheres, in particular the more inexpensive varieties, tend to have too wide size ranges. Also, in developing automatic methods for sphere counting, uniform sphere size may be advantageous.     

Use of microspheres to measure small intestinal villus blood flow in the dog.

The accuracy of using 7- to 10-micron microspheres to measure blood flow to dog small intestinal villi was studied. These spheres appear to pass unimpeded through the afferent arterioles of the villus and lodge at the villus tip because 1) virtually all villus spheres were located at the tip, 2) flow determined by visually counting spheres did not differ significantly from flow determined from radioactivity of the sheared-off villus layer, 3) the size distribution of spheres in the villus and subvillus layers was virtually identical to that administered, indicating no premature impaction of the larger spheres, and 4) spheres lodging in the submucosa during vasoconstriction did not subsequently migrate to the villi during vasodilatation. Studies with 25-micron spheres indicated that 28% of the 7- to 10-micron spheres shunted through vessels greater than 10 micron. Double isotope studies showed that lodges spheres do not migrate and that the injection of 2--4 X 10(6) spheres does not alter villus flow. Thus, 7- to 10-micron microspheres pass to and lodge in villus vessels in proportion to capillary flow and should provide an accurate estimate of villus nutrient blood flow.     

Poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for cartilage tissue engineering

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.     

Effects of volume expansion and vasodilators in acute pericardial tamponade

Pharmacologic therapy may be useful as emergency treatment for acute pericardial tamponade while preparing for pericardiocentesis. We studied the effects of volume expansion and vasodilators in acute pericardial tamponade in spontaneously breathing mongrel dogs. Hemodynamic parameters and blood flow (15-micrometer microspheres) to brain, heart, kidney, colon, stomach, liver, and temporalis muscle were measured. Arterial pressure and cardiac output rose, and blood flow to all major organs, except stomach, increased after volume expansion (n = 17) by infusion of 20 ml/kg of dextran after tamponade. With nitroprusside (8-75 microgram/min) after volume expansion (n = 5), ventricular filling pressures were reduced to control levels, whereas the favorable effects of volume expansion on cardiac output, blood pressure, and kidney and heart blood flow were maintained. Hydralazine alone or hydralazine plus volume expansion was not as effective as the combination of nitroprusside and volume expansion. Thus, volume expansion alone or the combination of volume expansion and nitroprusside infusion should be beneficial as emergency therapy while preparing for pericardiocentesis in patients with acute pericardial tamponade.     

Blood flow distribution in the renal portal system of the intact hen. A study of a venous system using microspheres

The blood flow in the renal portal system of intact hens was characterized by determining the fractional distribution of 15 μm and 50 μm microspheres in this system after injection into a leg vein. Validation tests showed that only 50 μm spheres gave a reliable estimation of this distribution. The blood flow in the coccy-geomesenteric vein was directed towards the liver in nearly all cases. On the average 44, 47 and 8% of the portal blood from the external iliac vein perfused the ipsilateral kidney, the liver and the lungs, respectively. However, the distribution of portal blood to these organs varied considerably between individuals and changed appreciably within 35 min in half the animals studied. The reason for these variations is not clear. The portal blood from the right and left external iliac veins was asymmetrically distributed in most animals illustrating the importance of local factors in its regulation. Variation was great in the regional distribution of portal blood within the kidney possibly due to local vasoconstriction of portal vessels. Further information e.g. on the regulation of renal portal blood flow is needed to explain its physiological significance. Microspheres provide a convenient method for such studies. A combination of microsphere and Sperber techniques allows us to determine the renal excretion efficiency of a given substance.     

A comparison of the effects of ischemia and of selective occlusion of capillaries, pre-capillaries and terminal arterioles on coronary reperfusion

To study the 'no-reflow' phenomenon is ischemic myocardium, the effects of ischemia and selective embolic blockade of capillaries, precapillaries and terminal arterioles were compared in isolated rat hearts. Hearts received oxygenated Krebs-Henseleit buffer for 10 min via an aortic cannula, and then coronary perfusion was stopped. The pattern and extent of reperfusion after 15-90 min of global ischemia and after the injection of 9, 15 or 55 mu diameter microspheres were determined from the distribution of injected 6.7% fluorescein isothiocyanate-dextran in frozen transverse sections of the ventricles. Following ischemia, progressively larger subendocardial regions surrounding the left ventricle could not be reperfused. In contrast, embolic occlusion of capillaries, precapillaries or terminal arterioles caused a transmural reduction in perfusion and a fine linear or herringbone pattern of fluorescence. Sixty min of ischemia followed by microsphere injection had no effect on the subendocardial zone of no-reflow but much reduced the intensity of fluorescence elsewhere. Thus thrombosis, erythrocyte plugging and occlusion of capillaries, precapillaries or terminal arterioles are unlikely to be primary causes of the reperfusion defect which develops in ischemic myocardium.