Fluorescent Microsphere Method Research Articles

Quantification of leptomeningeal collateral blood flow in hypertensive rats during ischemic stroke.

There is increasing evidence that poor leptomeningeal collateral blood flow in hypertensive animals is due to increased vascular myogenic tone, indicating that therapies to enhance collateral blood flow during ischemic stroke may be particularly effective. To develop such therapies, we need a greater understanding of the factors that regulate collateral blood flow in the setting of hypertension. Therefore, we aimed to quantify blood flow velocity, diameter and absolute blood flow in individual collateral vessels in an ischemic stroke model in spontaneously hypertensive rats (SHRs) and determine which factors had the greatest influence on blood flow. We quantified collateral flow velocity and vessel diameter and calculated absolute collateral blood flow in SHRs (n = 5) during 70 min of middle cerebral artery occlusion (MCAO), using a fluorescent microsphere method. Average collateral blood flow significantly increased post-occlusion relative to baseline (pre-MCAO: 16.8 ± 7.1nL/min vs. post-MCAO: 146.4 ± 37.7nL/min, p = 0.02). Within animal linear regression analysis showed a strong positive correlation between changes in collateral blood flow versus changes in collateral diameter during stroke (r = 0.7-0.99, p = 0.3-0.002). In contrast, collateral blood flow was only weakly correlated with collateral blood flow velocity during stroke (r = -0.03-0.97, p = 0.9-0.1). Collateral blood flow and velocity significantly increased post-occlusion. Collateral flow was strongly influenced by vessel diameter, likely because of marked baseline vasoconstriction of collaterals which is flow-limiting.

Serum level of eight cytokines in Han Chinese patients with systemic lupus erythematosus using multiplex fluorescent microsphere method.

Aim of the studyTo investigate the role of 8 cytokines and their correlation with clinical characteristics in systemic lupus erythematosus (SLE) in Han Chinese population by detecting their serum levels using the multiplex fluorescent microsphere method.Material and methodsSerum was separated from 79 patients with SLE and 40 healthy controls. The serum cytokine detection was conducted according to the instruction of MILLIPLEX MAP human cytokine detection kit on the Luminex liquid phase array platform with 0.01 pg/ml detectable level. The 8 cytokines were interferon α2 (IFN-α2), IFN-γ, interleukin 6 (IL-6), IL-8, IL-10, IFN-γ-inducible protein 10 (IP-10), tumor necrosis factor α (TNF-α) and IL-17. Variable data were in skewed distribution and were expressed with median (P25, P75). Mann-Whitney analysis was used in statistical analysis.ResultsAt the baseline level without any stimulus, the level of IP-10 expression was the highest among the 8 cytokines and the second highest was IL-8. The level of IL-17 was too low to be detected. The level of 7 cytokines was higher in SLE patients than in healthy controls (p < 0.01). The level of dsDNA antibody, C3, CRP, ESR and anti-nucleosome antibody was correlated with IL-10. Proteinuria was not correlated with any cytokine.ConclusionsEight cytokines were measured in our study, while not all of them were detected. The most important finding was the usefulness of IL-10 as a disease activity biomarker for Han Chinese patient with SLE. None of cytokines reflected kidney injury.

Local cooling reduces regional bone blood flow

Local cooling is very common after bone and joint surgery. Therefore the knowledge of bone blood flow during local cooling is of substantial interest. Previous studies revealed that hypothermia leads to vasoconstriction followed by decreased blood flow levels. The aim of this study was to characterize if local cooling is capable of inducing reduced blood flow in bone tissue using a stepwise-reduced temperature protocol in experimental rabbits. To examine bone blood flow we utilized the fluorescent microsphere (FM) method. In New Zealand white rabbits one randomly chosen hind limb was cooled stepwise from 32 to 2°C, whereas the contra lateral hind limb served as control. Injection of microspheres was performed after stabilization of bone and muscle temperature at each temperature level. Bones were removed, dissected and fluorescence intensity was determined to calculate blood flow values. We found that blood flow of all cooled regions decreased relative to the applied external temperature. At maximum cooling blood flow was almost completely disrupted, indicating local cooling as powerful regulatory mechanism for regional bone blood flow (RBBF). Postoperative cooling therefore may lead to strongly decreased bone blood flow values. As a result external cooling has capacity to both diminish bone healing and reduce bleeding complications.

Influence of castration on bladder blood flow and function during the rapid phase of androgen deprivation

Objective. This study examined the effects of androgen deprivation on bladder blood flow (BBF) and bladder function during the acute phase in castrated rats. Material and methods. Nine-week-old male Wistar rats were divided into six groups as follows: 24 h post-sham-operation (24hPS), no operation (control), 24 h post-castration (24hPC), 48 h post-castration (48hPC), 7 days post-castration (7dPC) and 12 weeks post-castration (12wPC). BBF was measured in the 24hPS, control, 24hPC, 48hPC, 7dPC and 12wPC groups, and prostate blood flow was measured in the control, 24hPC, 48hPC and 7dPC groups using laser Doppler methods. In select groups, BBF was measured using the fluorescent microsphere method. Bladder function was tested in the 24hPS, control, 24hPC and 12wPC groups. The bladder was irrigated with saline and 0.25% acetic acid. Maximum voiding pressure and voiding intervals were measured. Results. BBF significantly increased within 24 h after castration (p < 0.001); these changes did not persist beyond 24 h. However, prostate blood flow decreased significantly within 24 h after castration (p < 0.001). Shortening of the voiding interval upon acetic acid stimulation was significantly suppressed in group 24hPC compared to the control group (p < 0.001). The maximum voiding pressure did not significantly change in the 24hPS, control, 24hPC and 12wPC groups. Conclusions. During the acute phase of androgen deprivation following castration, BBF significantly increased and the bladder became receptive to stimulation. This temporary increase may be because of a decrease in the prostate blood flow, indicating that androgens do not directly affect the BBF.

Experimental model of laparoscopic gastric ischemic preconditioning prior to transhiatal esophagectomy

Cervical esophagogastric anastomotic disruption following transhiatal esophagectomy (THE) is a significant problem. Gastric tip ischemia is a primary cause of anastomotic failure. We examined gastric tip blood flow when laparoscopic "ischemic preconditioning" was attempted by selectively ligating the short gastric (SG) vessels or both the left and short gastric (LG/SG) vessels prior to THE. Seventeen (25 kg) mongrel dogs underwent laparoscopy followed 3 weeks later by THE. Three groups were studied: control group = laparoscopy only, no preconditioning (n = 6); SG group = laparoscopic ligation of SG vessels only (n = 5); and LG/SG group = laparoscopic ligation of LG and SG vessels (n = 6). Tissue blood flow was assessed using the fluorescent microsphere method. The initial microsphere injections occurred prior to pneumoperitoneum and upon completion of the laparoscopy. At the second operation, transhiatal esophagectomy was performed and microsphere blood flow assessment occurred after induction of anesthesia, after mobilization of the stomach, and after completion of the cervical esophagogastric anastomosis. The animals were euthanized and regional gastric tissue was analyzed for microsphere estimates of blood flow. Differences in blood flow were evaluated using Student's t test. The mean baseline gastric blood flow was 0.58 ml/min/g. After THE, the proximal gastric blood flow fell to 16% of baseline in control and 22% in SG, but was reduced to only 60% of baseline in LG/SG. This relative preservation of blood flow among the LG/SG group approached significance compared with the laparoscopy-only (control) group (P = 0.07). Ligation of SG vessels alone provided no preservation of proximal gastric blood flow following THE. Preoperative "ischemic preconditioning" through ligation of both the short and left gastric vessels may achieve preservation of blood flow to the gastric tip. Preconditioning during laparoscopic staging of esophageal carcinoma may be considered to reduce anastomotic complications following esophagectomy.

Development of a fluorescent microsphere technique for rapid histological determination of cerebral blood flow

The purpose of this study was to develop a more efficient fluorescent microsphere method to facilitate the rapid use of the histological technique and to enable its use in large tissue regions. Using fluorescent plate/slide imaging technology and automated detection and analysis software, we were able to rapidly image, detect, and count 3 separate microsphere colors in 200 μm thick tissue sections from piglet brain. In resting newborn piglets ( n = 6) on isoflurane anesthesia, we measured a median total cerebral blood flow (CBF) of 105 ml/min/100 g (range 27–206 ml/min/100 g). Compared with other FM analysis methods, our method reduces the time required to determine blood flow, improves accuracy in lipid-rich tissues and large tissue regions and, unlike the radiolabeled microsphere method, can be combined with histological analysis.

Diffuse optical monitoring of hemodynamic changes in piglet brain with closed head injury

We used a nonimpact inertial rotational model of a closed head injury in neonatal piglets to simulate the conditions following traumatic brain injury in infants. Diffuse optical techniques, including diffuse reflectance spectroscopy and diffuse correlation spectroscopy (DCS), were used to measure cerebral blood oxygenation and blood flow continuously and noninvasively before injury and up to 6 h after the injury. The DCS measurements of relative cerebral blood flow were validated against the fluorescent microsphere method. A strong linear correlation was observed between the two techniques (R=0.89, p<0.00001). Injury-induced cerebral hemodynamic changes were quantified, and significant changes were found in oxy- and deoxy-hemoglobin concentrations, total hemoglobin concentration, blood oxygen saturation, and cerebral blood flow after the injury. The diffuse optical measurements were robust and also correlated well with recordings of vital physiological parameters over the 6-h monitoring period, such as mean arterial blood pressure, arterial oxygen saturation, and heart rate. Finally, the diffuse optical techniques demonstrated sensitivity to dynamic physiological events, such as apnea, cardiac arrest, and hypertonic saline infusion. In total, the investigation corraborates potential of the optical methods for bedside monitoring of pediatric and adult human patients in the neurointensive care unit.

Fluorescent Cardiac Imaging

The purpose of the present study was to examine whether the effect of coronary stenoses of variable severity on myocardial perfusion can be quantitatively assessed in vivo by analysis of fluorescent cardiac imaging (FCI) compared with the gold standard, the fluorescent microsphere method. FCI is a novel technology to visualize coronary vessels and myocardial perfusion intraoperatively using the indocyanine green dye with an infrared-sensitive imaging device. Graded stenoses and total vessel occlusion of the left anterior descending coronary artery were created in 11 open-chest pigs. Stenoses were graded to reduce resting left anterior descending coronary artery flow by 25%, 50%, 75%, and 100% of baseline flow measured by transit-time flowmeter. FCI images were analyzed with a digital image processing system. The impairment of myocardial perfusion was quantified by background-subtracted peak fluorescence intensity and slope of fluorescence intensity obtained with FCI and compared with myocardial blood flow assessed by fluorescent microsphere. All stenoses resulted in an impairment of myocardial perfusion visualized by FCI. Occlusion of the left anterior descending coronary artery resulted in a total perfusion defect (no fluorescence intensity) of the corresponding anterior myocardial wall. During graded stenosis and total vessel occlusion, normalized background-subtracted peak fluorescence intensity and slope of fluorescence intensity decreased significantly (P<0.0001). Both background-subtracted peak fluorescence intensity (r=0.92, P<0.0001) and slope of fluorescence intensity (r=0.93, P<0.0001) analyzed by FCI demonstrated good linear correlation with fluorescent microsphere-derived myocardial blood flow. The impairment of myocardial perfusion in response to increased coronary stenosis severity and total vessel occlusion can be quantitatively assessed by FCI and correlates well with results obtained by fluorescent microsphere.

Continuous inhalation of carbon monoxide induces right ventricle ischemia and dysfunction in rats with hypoxic pulmonary hypertension

We aimed to investigate the toxicity of carbon monoxide (CO) in rats with right ventricle (RV) remodeling induced by hypoxic pulmonary hypertension (PHT). A group of Wistar rats was exposed to 3-wk hypobaric hypoxia (H). A second group was exposed to 50 ppm CO for 1 wk (CO). A third group was exposed to chronic hypoxia including 50 ppm CO during the third week (H+CO). These groups were compared with controls. RV and left ventricle (LV) functions were assessed by echocardiography and transparietal catheterization. Ventricular perfusion was estimated with the fluorescent microsphere method. Results were confirmed by histology. PHT induced RV hypertrophy and function enhancement. In the H group, RV shortening fraction (RVSF; 71 +/- 12% vs. 41 +/- 2%) and RV end-systolic pressure (RVESP; 54 +/- 6 vs. 19 +/- 2 mmHg) were increased compared with controls. Moreover, myocardial perfusion was increased in the RV (36 +/- 2% vs. 22 +/- 2%) and decreased in the LV (64 +/- 3% vs. 78 +/- 2%). In the H+CO group, RVSF (45 +/- 3% vs. 71 +/- 12%) and RVESP (38 +/- 3 vs. 54 +/- 6 mmHg) were decreased compared with the H group. RV perfusion was decreased in the H+CO group compared with the H group (21 +/- 5% vs. 36 +/- 2%), and LV perfusion was increased (79 +/- 5% vs. 64 +/- 3%). PHT and RV hypertrophy were still present in the H+CO group, and fibroses localized in the RV were detected. Similar lesions were observed in an additional group exposed simultaneously to hypoxia and 50 ppm CO over 3 wk. We demonstrated that rats with established PHT were more sensitive to CO, which dramatically alters the RV adaptive response to PHT, leading to ischemic lesions.

In vivo suppression of TRPM4 compromises autoregulation of cerebral blood flow

The transient receptor potential channel TRPM4 is a key component of the cerebral artery myogenic response and as such should play a central role in cerebral blood flow (CBF) autoregulation. In this study, we tested the hypothesis that in vivo suppression of cerebrovascular TRPM4 channel expression compromises the maintenance of constant CBF in the face of rising mean arterial pressure (MAP). In vivo suppression of cerebrovascular TRPM4 expression was achieved by infusing sense (S) or antisense (AS) oligodeoxynucleotides (ODNs) into the cerebral spinal fluid of 350g Sprague-Dawley rats at 280μg·day−1 for 7 days using an osmotic pump that discharged into the lateral cerebral ventricle. ODNs tagged with a fluorescent label could be visualized in confocal images of cerebral arterial smooth muscle cells isolated from arteries of treated rats indicating that the ODNs were delivered to the target tissue. Semi quantitative RT-PCR indicated that the message for TRPM4 was decreased in the arteries following AS-treatment. Our hypothesis was directly tested by measuring CBF in TRPM4 S- and AS-treated rats using the fluorescent microsphere method. CBF was not different between S and AS-treated rats at a MAP of 110±5 mmHg (69±4 vs. 90±19 mL·100g−1·min−1 S vs. AS, n=5). Venous infusion of noradrenaline (35μg·min−1) increased MAP to 185±10mmHg causing CBF to be increased significantly more in AS vs. S-treated animals (295±42 vs. 190±27 mL·100g−1·min−1, n=5). Thus, in vivo suppression of TRPM4 compromised CBF autoregulation at high MAP. This suggests that TRPM4 and myogenic constriction are important for protecting the brain from overperfusion when MAP is elevated. (Supported by AHA #0525983T and NIH #HL58231)

Fluorescent Microspheres Are Reliable for Serial Bone Blood Flow Measurements

The fluorescent microsphere method is one of the current techniques to determine regional blood flow in various organs. The purpose of this study was to examine the suitability of fluorescent microspheres for serial measurement of regional bone blood flow. Six anesthetized female New Zealand rabbits received five left ventricular injections of fluorescent microspheres in 20-minute intervals. To test the precision of the measurement two types of fluorescent microspheres were injected simultaneously at the first and last injections. Blood flow was calculated in the kidneys, lungs, brain, femurs, and tibias after measuring the fluorescence intensity in each reference blood and tissue sample. Comparison of blood-flow values obtained by simultaneously injected microspheres showed an excellent correlation and a minimal percentage difference at the first and last injections, indicating valid measurements of regional bone blood flow. No significant differences were observed when comparing blood flow in the corresponding regions of bones on the right side and left side. Mean blood flow in the femur and tibia significantly increased at the fourth injection whereas flow distribution within the femur and tibia essentially remained unchanged throughout the experiment. Comparison of blood flow values obtained by simultaneously injected microspheres showed moderate agreement for the kidneys and lungs at the last injections. Because this finding might be attributable to disturbances of microcirculation caused by accumulation of spheres in high-flow organs, the increase in regional bone blood flow observed in our experiments has to be interpreted carefully. This study showed that bone blood flow can be determined reliably in anesthetized rabbits by as many as three serial injections of fluorescent microspheres.

Fluorescent microsphere method is suitable for chronic bone blood flow measurement: a long-term study after meniscectomy in rabbits.

The fluorescent microsphere (FM) method is considered a reliable technique to determine regional bone blood flow (RBBF) in acute experiments. In this study, we verified the accuracy and validity of this technique for measurement of RBBF in a long-term experiment and examined RBBF after meniscectomy. Twenty-four anesthetized female New Zealand white rabbits (3 groups, each n = 8) received consecutive left ventricular injections of FM in defined time intervals after meniscectomy: group 1 from preoperation to 3 wk postoperation; group 2 from 3 to 7 wk postoperation; and group 3 from 7 to 11 wk postoperation. To test the precision of the FM method, two FM species were injected simultaneously at the first and last measurement. After the experiment, humeri, femora, tibiae, and reference organs (kidney, lung, brain) were removed and dissected according to standardized protocols. Fluorescence was determined in each reference blood and tissue sample, and blood flow values were calculated. Blood flow in kidney, lung, and brain revealed no significant difference between right and left side and remained unchanged during the observation period, thus excluding errors due to shunting and dislodging of spheres in our experiments. Comparison of relative bone blood flow values obtained by simultaneously injected FM showed an excellent correlation at the first and last injection, indicating valid RBBF measurements in long-term experiments. We found a significant increase in RBBF 3 wk after meniscectomy in the right tibial condyles compared with the nonoperated left side. Similar changes were found in the femoral condyles. RBBF in other regions of tibia, femur, and humerus revealed no significant differences between right- and left-sided bone samples of the same region. Our results demonstrate that the FM method is valid for measuring RBBF in long-term experiments. In addition, we were able to demonstrate that meniscectomy leads to an increase in RBBF in the tibial condyles at a very early stage. This increase might be caused by stress-induced alterations of the subchondral bone.

Determination of Regional Bone Blood Flow by Means of Fluorescent Microspheres Using an Automated Sample-Processing Procedure

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 × 10⁶ spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 ± 1.1 and 8.5 ± 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 ± 1.1 to 6.0 ± 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 ± 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF.

Endothelin-1 elevates regional cerebral perfusion during prolonged ventricular fibrillation cardiac arrest in pigs

Since adrenaline (epinephrine) also has negative effects during and after cardiopulmonary resuscitation (CPR) a non-adrenergic vasoconstrictor like endothelin might be an alternative to increase vital organ blood flow. We studied the effect of different doses of endothelin-1 compared with adrenaline on the ability to resuscitate, cerebral and myocardial blood flow (MBF) in a closed chest cardiac arrest pig model. After 5 min of ventricular fibrillation, CPR with a ventilator and a mechanical compression device was started. At 10 min, 31 pigs were randomized to receive a single dose of endothelin-1 50, 100 or 200 μg or repeated doses of adrenaline 0.04 mg kg −1 or saline every 3 min. After 25 min, the pigs were defibrillated to achieve restoration of spontaneous circulation. Blood flow was measured with the fluorescent microsphere method. In animals receiving endothelin-1 50, 100 and 200 μg the cerebral blood flow (CBF) increased from median 28 (25th; 75th quartile: 16; 40), 32 (15; 48) and 17 (4; 65) to 36 (31; 54), 47 (39; 57) and 63 (35; 83) ml min −1 per 100 g, respectively, 6 min after drug administration ( P<0.05 endothelin-1 50 μg vs. Control, P<0.01 endothelin-1 100 and 200 μg vs. Control). At the same time CBF decreased in the control and adrenaline group from 36 (21; 41) and 39 (15; 50) to 12 (2; 25) and 24 (15; 26) ml min −1 per 100 g, respectively, ( P<0.05 adrenaline vs. endothelin-1 200 μg). There was no difference in MBF between the treatment groups despite a higher coronary perfusion pressure (CoPP) in the endothelin-1 groups. Restoration of spontaneous circulation could be only achieved in the endothelin-1 50 μg (3 of 7; 43%) and 100 μg (5 of 7; 71%) group. This study suggests that endothelin-1 enhances CBF during CPR better than adrenaline and increases resuscitation success.

Role of microvascular rarefaction in the increased arterial pressure in mice lacking for the endothelial nitric oxide synthase gene (eNOS3pt-/- ).

Mechanisms involved in hypertension in homozygous mice for the defective endothelial nitric oxide synthase gene (eNOS-/-) have not been fully elucidated. As NO is a potent vasodilator agent and possibly promotes angiogenesis, we investigated whether vasoconstriction and/or microvascular rarefaction could explain hypertension in these mice. Immunohistochemistry with mouse monoclonal smooth muscle alpha-actin antibody was used to detect arterioles, and quantification of arteriolar density was performed in the left ventricle and in the gracilis muscle of 12-week-old male eNOS+/+ and eNOS-/- mice. Haemodynamic parameters - mean arterial pressure (MAP), cardiac index (CI), total peripheral résistance (TPR), myocardial blood flow, muscular blood flow and corresponding resistances - were measured or calculated using the fluorescent microsphere method in basal conditions and after infusion of sodium nitroprusside (SNP) (5 to 150 microg/kg per min) in eNOS-/- mice, compared with eNOS+/+ mice. We evidenced a significant decrease in arteriolar density in the heart (-16%, P < 0.02) and in the gracilis muscle (-22%, P < 0.05) in eNOS-/- mice. In basal conditions, eNOS-/- mice developed significant hypertension (MAP = 127 +/- 14 versus 77 +/- 14 mmHg, P < 0.001) associated with decreased CI (-29%, P < 0.001) and increased TPR (+ 125%, P < 0.001). Coronary and gracilis muscular resistances were increased (by 75 and 89% respectively, P < 0.001) compared with eNOS+/+ mice, whereas myocardial and skeletal muscle tissue blood flows were not affected. After SNP administration (10 microg/kg per min), a dose that did not significantly modify haemodynamic parameters in eNOS+/+ mice, MAP, TPR and regional resistances were normalized in eNOS-/- mice, showing that vasodilation may correct hypertension in eNOS-/- mice. However, under maximal vasodilating conditions, TPR and regional resistances remained significantly higher in eNOS-/- mice than those of eNOS+/+ mice. Anatomical and functional results show that both vasoconstriction and arteriolar rarefaction are involved in hypertension of eNOS-/- mice. Indeed, under maximal vasodilation, arterial pressure and TPR remained significantly higher in eNOS-/- mice than in eNOS+/+ mice, evidencing a major role of microvascular rarefaction in this model of hypertension.

Validation of automated spectrofluroimetry for measurement of regional organ perfusion using fluorescent microspheres

The fluorescent microsphere (FM) method for determination of regional organ blood flow is labor intensive, requiring processing of 100 s to 1000 s of samples per organ. To save time and reduce handling errors, we developed an automated fluorescence analysis system by interfacing a commercially available spectrofluorimeter and sample delivery unit to a PC, operated by a Windows95®-based program, WINFAC. WINFAC allows versatile analysis setup and instrument control for fluorescent intensity acquisition at fixed wavelengths or by synchronous scanning. Data is presented on screen, in real-time, and stored in text format. Reference analyses are performed at specified intervals and the coefficient of variation is continuously updated to monitor instrument performance. The automated system was validated against radioactive microspheres (RM) for lung perfusion in a pig and sheep and evaluated for analysis reproducibility. Fluorescent and radioactive microspheres were delivered by simultaneous injection into a femoral vein. Lungs were excised, flushed, dried at total lung capacity, and cubed into ≈2cm 3 pieces ( n=833 and 1560, pig and sheep, respectively). Radioactive count rates were determined for each lung piece (corrected for decay, background and spillover). Fluorescent dyes were extracted in solvent and intensities were determined at fixed wavelengths, using the automated spectrofluorimeter (corrected for background and spillover). Multi-color reference solutions were measured every 50 samples to monitor instrument reproducibility. Blood flow estimates for each piece determined by RM and FM methods were highly correlated: R 2=0.98±0.017, Slopes=1.00±0.007 and Intercepts=0.00±0.006 (mean±SD). The CV of repeat reference analyses was 0.71%±0.16, a 30% to 50% reduction relative to manual analysis. Automated spectrofluorimetry reduces measurement errors and is a reliable and time saving advancement. With this technology, use of FM to measure regional lung perfusion approaches the ease and accuracy of the RM method.

A new sample-processing unit for the fluorescent microsphere method.

The use of fluorescent-labeled microspheres (FM) for measurement of regional blood flow is an attractive alternative to the use of radioactive-labeled microspheres. In the FM method the FM have to be completely recovered from the tissue samples in a time- and labor-intensive process. For this reason, a considerable loss of FM is possible. The aim of this study was to develop a filtration device that allows the tissue sample to remain in a single container throughout the procedure to make the process easier and to avoid the loss of FM. The core of the sample-processing unit (SPU) is a single-tube filtration device with a polyamide wire mesh. The protocol for processing tissue from different organs (heart, kidney, liver, spleen, intestine, muscle, bone, lung, brain) was modified and thus shortened significantly. Furthermore, the SPU allows direct filtration of the blood reference sample without previous digestion. Different experiments showed that the SPU in combination with the new protocol excludes the loss of 15-micrometers FM. The modifications of the whole procedure render it faster and highly standardized.

Cerebral blood flow determinations using fluorescent microspheres: variations on the sedimentation method validated

We validate a modification of the sedimentation method for measuring fluorescent microspheres (FM) that improves the determination of regional cerebral blood flow (rCBF). Our FM method for rCBF determination is compared to the radioactive microspheres (RM) method for rCBF measurement by simultaneous injection of one radioactive and two fluorescent labeled doses, at two separate time points, into the left ventricle of a pig. The pig was killed, the brain and spinal cord removed, and divided into 92 pieces averaging 0.83 g. Our modifications to FM analysis by sedimentation includes: 2 instead of 1 week of autolysis, pellet washing with 1% Triton X-100 instead of 0.25% Tween 80, phosphate buffer addition during rinse, fluorescent dye extraction using 2-ethoxyethylacetate instead of 2-(2-ethoxyethoxy)ethyl acetate and polypropylene instead of glass tubes. Comparing rCBF using Sc46 RM, to yellow-green and orange FM, yielded mean differences of 0.026 and 0.021 ml/min per piece, respectively. Sn 113 RM compared to blue-green and scarlet FM gave mean differences of −0.010 and 0.137 ml/min per piece, respectively. All RM–FM differences, except those for scarlet FM, are within acceptable limits. This assay provides a reliable method for determining rCBF.

Use of an automated fluorescent microsphere method to measure regional blood flow in the fetal lamb

We have developed a method for measuring regional blood flow by means of fluorescent microspheres in all organs and tissues of the fetal lamb, including brain, heart, lung, liver, gut, spleen, kidney, adrenal, brown fat, skin, muscle, bone, and placenta. Five different fluorescent-labeled microspheres were used: blue (B), yellow-green (Y), orange (O), red (R), and crimson (C). An automated, 96-well microplate fluorescent reader (bottom reading) was chosen for the assay because of the rapidity and high throughput that it offers. Tissue samples were digested by 4 M ethanolic KOH. The sedimentation method and dye extraction with Cellosolve acetate, as previously reported by others, were used for the sample processing. The bones were crushed and allowed to directly soak in Cellosolve acetate to extract the dye. The relationship between microsphere number and fluorescent intensity was linear over a broad range of microsphere numbers (80-20,000/mL). The coefficients of variation of within-run and between-run precision were 3.39 +/- 1.10% and 4.54 +/- 1.10%, respectively. Recovery of microspheres from tissues and blood averaged 94.3 +/- 2.5% and was not dependent on microsphere number. The spillover of the fluorescent signals into adjacent colors was 4.0 +/- 0.1% for O to Y, 8.1 +/- 0.4% for O to R, and 9.1 +/- 0.5% for R to C, and these values were constant over a wide range in concentrations of the microsphere pairs. No evidence was obtained for quenching of the emission of one fluorophore via photon absorption by another fluorophore. The measurements of regional blood flow obtained with fluorescent microspheres in three chronically instrumented fetal lambs at approximately 140 days gestation were similar to the flow estimates obtained using radioactive microspheres in four other fetal lambs at the same gestational age. The fluorescent method is thus a viable alternative to the radioactive technique for the measurement of regional blood flow to all fetal organs and tissues, particularly when an automated fluorescent microplate reader is employed to reduce analysis time.

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