Chronic assessment of cerebral hemodynamics during rat forepaw electrical stimulation using functional ultrasound imaging.

Functional ultrasound imaging is a method recently developed to assess brain activity via hemodynamics in rodents. Doppler ultrasound signals allow the measurement of cerebral blood volume (CBV) and red blood cells' (RBCs') velocity in small vessels. However, this technique originally requires performing a large craniotomy that limits its use to acute experiments only. Moreover, a detailed description of the hemodynamic changes that underlie functional ultrasound imaging has not been described but is essential for a better interpretation of neuroimaging data. To overcome the limitation of the craniotomy, we developed a dedicated thinned skull surgery for chronic imaging. This procedure did not induce brain inflammation nor neuronal death as confirmed by immunostaining. We successfully acquired both high-resolution images of the microvasculature and functional movies of the brain hemodynamics on the same animal at 0, 2, and 7 days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1 mA) ranging from 1 (200 µs) to 25 pulses (5s). Our results indicate that CBV HRF parameters such as the peak amplitude, the time to peak, the full width at half-maximum and the spatial extent of the activated area increase with stimulus duration. Functional ultrasound imaging was sensitive enough to detect hemodynamic responses evoked by only a single pulse stimulus. We also observed that the RBC velocity during activation could be separated in two distinct speed ranges with the fastest velocities located in the upper part of the cortex and slower velocities in deeper layers. For the first time, functional ultrasound imaging demonstrates its potential to image brain activity chronically in small animals and offers new insights into the spatiotemporal evolution of cerebral hemodynamics.

Effects of cell spatial organization and size distribution on ultrasound backscattering.

In ultrasound tissue characterization dealing with cellular aggregates (such as tumors), it can be hypothesized that cell microstructure and spatial distribution dominate the backscatter signal. Effects of spatial organization and size distribution of nuclei in cell aggregates on ultrasound backscatter are examined in this work using 2-D computer simulations. The nuclei embedded in cytoplasm were assumed to be weak scatterers of incident ultrasound waves, and therefore multiple scattering could be neglected. The fluid sphere model was employed to obtain the scattering amplitude for each nucleus and the backscatter echo was generated by summing scattered signals originating from many nuclei. A Monte Carlo algorithm was implemented to generate realizations of cell aggregates. It was found that the integrated backscattering coefficient (IBSC) computed between 10 and 30 MHz increased by about 27 dB for a spatially random distribution of mono-disperse nuclei (radius = 4.5 µm) compared with that of a sample of periodically positioned mono-disperse nuclei. The IBSC also increased by nearly 7 dB (between 10 and 30 MHz) for a spatially random distribution of poly-disperse nuclei (mean radius ± SD = 4.5 ± 1.54 µm) compared with that of a spatially random distribution of mono-disperse nuclei. Two different Gaussian pulses with center frequencies 5 and 25 MHz were employed to study the backscatter envelope statistics. An 80% bandwidth was chosen for each case with approximately 0.32 mm as the full-width at half-maximum (FWHM) for the first pulse and 0.06 mm for the second. The incident beam was approximated as a Gaussian beam (FWHM = 2.11 and 1.05 mm for those pulses, respectively). The backscatter signal envelope histograms generally followed the Rayleigh distribution for mono-disperse and poly-disperse samples. However, for samples with partially ordered nuclei, if the irradiating pulse contained a frequency for which ultrasound wavelength and scatter periodicity became comparable (d ~ λ/2), then the histograms were better fitted by the Nakagami distribution. This study suggests that the shape of an envelope histogram depends upon the periodicity in the spatial organization of scatterers and bandwidth of the ultrasound pulse.

A simulation study on photoacoustic signals from red blood cells.

A two dimensional simulation study was performed to investigate the photoacoustic signal properties of non-aggregated and aggregated erythrocytes. Spatial distributions of non-aggregated blood samples were generated by employing a Monte Carlo method and aggregated blood samples were simulated using a hexagonal packing scheme. For the non-aggregating case photoacoustic signals demonstrated a monotonic rise with hematocrit. For the aggregating case it was found that spectral (<20 MHz) intensity increased (11 dB at 15.6 MHz) when the aggregate size increased. This study strongly suggests that the assessment of erythrocyte aggregation level in human blood might be possible by using a photoacoustic spectroscopic method.

Assessment of accuracy of the structure-factor-size-estimator method in determining red blood cell aggregate size from ultrasound spectral backscatter coefficient.

A computer simulation study to produce ultrasonic backscatter coefficients (BSCs) from red blood cell (RBC) clusters is discussed. The simulation algorithm is suitable for generating non-overlapping, isotropic, and fairly identical RBC clusters. RBCs were stacked following the hexagonal close packing (HCP) structure to form a compact spherical aggregate. Such an aggregate was repeated and placed randomly under non-overlapping condition in the three-dimensional space to mimic an aggregated blood sample. BSCs were computed between 750 KHz and 200 MHz for samples of various cluster sizes at different hematocrits. Magnitudes of BSCs increased with mean aggregate sizes at low frequencies (<20 MHz). The accuracy of the structure-factor-size-estimator (SFSE) method in determining mean aggregate size and packing factor was also examined. A good correlation (R(2) ≥ 0.94) between the mean size of aggregates predicted by the SFSE and true size was found for each hematocrit. This study shows that for spherical aggregates there exists a region for each hematocrit where SFSE works most accurately. Typically, error of SFSE in estimating mean cluster size was <20% for dimensions between 14 and 17 µm at 40% hematocrit. This study suggests that the theoretical framework of SFSE is valid under the assumption of isotropic aggregates.

Monte Carlo study on ultrasound backscattering by three-dimensional distributions of red blood cells.

A Monte Carlo study on ultrasound backscattering by red blood cells (RBCs) is presented for three-dimensional (3D) distributions of particles. The cells were treated as classical spherical particles and accordingly, the Boltzmann distribution was considered to describe probability distribution of energy states of a system composed of such particles. The well-known Metropolis algorithm can generate configurations according to that probability distribution and therefore, was employed in this study to simulate some realizations of both nonaggregating and aggregating RBCs. The study of nonaggregating particles was motivated to compare simulations with existing experimental results and consequently, to validate the model. In the case of aggregating RBCs, the interaction potential between cells was modeled with the Morse potential and the frequency-dependent backscattering coefficient (BSC) was investigated at different hematocrits (H, particle volume fractions). The impact of aggregation potential on the spectral slope (SS) was also evaluated. It is shown that BSC increased as the magnitude of aggregating potential was raised and the effect was more pronounced at higher hematocrits. Moreover, spectral slopes at nonaggregating and low aggregating conditions were found to be around 4, which is consistent with the Rayleigh scattering theory. However, it had diminished significantly, particularly at higher hematocrits as the magnitude of the attractive potential energy was raised. For instance, at H=40% SS dropped from 4.04 for nonaggregating particles to 3.62 at the highest aggregating potential considered in this study. Our results suggest that this 3D model is capable of reflecting the effects of RBC aggregation on BSC and SS.

Gated-SPECT equilibrium radionuclide angiography in right ventricular assessment of patients with repaired tetralogy of Fallot.

BACKGROUND: The long-term prognosis of patients with tetralogy of Fallot (TF) who have undergone repair is determined by right ventricular dilatation resulting from residual pulmonary insufficiency. We have studied the values of right and left ventricle systolic function obtained by gated single photon emission computed tomography (SPECT) equilibrium radionuclide angiography (ERNA) in these patients. METHODS: A study population of 62 patients with surgically repaired TF underwent gated-SPECT ERNA to determine ejection fraction of the right and left ventricle and dimensions of the right ventricle and pulmonary infundibulum. Results were compared with those of a group of 11 patients without heart disease. RESULTS: RVEF (34% vs. 40%, P=0.02) and LVEF (49% vs. 54%, P=0.03) were significantly lower in patients with TF than in the control group. The RVEF and LVEF variation coefficients were 9% and 6.2%, respectively. Volumes of the right ventricle (P=0.003) were significantly greater than those of the control group, although variation coefficients were 15%. CONCLUSIONS: Gated-SPECT ERNA is a non-invasive method of assessing ejection fraction in patients with repaired TF. In these patients, the ejection fraction is decreased in both ventricles, whereas size of the right ventricle is significantly increased. Reproducibility of the RVEF calculation is good, but in the case of volumes it is suboptimal.

Effect of red cell clustering and anisotropy on ultrasound blood backscatter: a Monte Carlo study.

When flowing at a low shear rate, blood appears hyperechogenic on ultrasound B-scans. The formation of red blood cell (RBC) aggregates that also alters blood viscosity is the microscopic mechanism explaining this acoustical phenomenon. In this study, Monte Carlo simulations were performed to predict how RBC clustering increases ultrasound scattering by blood. A bidimensional Gibbs-Markov random point process parameterized by the adhesion energy epsilon and an anisotropy index nu was used to describe RBC positions for a hematocrit H = 40%. The frequency dependence of the backscattering coefficient chi(f) was computed using Born approximation. The backscattering coefficient chi0 at 5 MHz and the spectral slopes n(x) and n(y) (chi alpha f(nx) or f(ny)) measured, respectively, when the insonification is parallel and perpendicular with the RBC cluster axis were then extracted. Under isotropic conditions, chi0 increased up to 7 dB with epsilon and n(x) = n(y) decreased from 4.2 to 3.4. Under anisotropic conditions, the backscattering was stronger perpendicularly to aggregate axis, resulting in n(x) < n(y). The anisotropy in scattering appeared more pronounced when epsilon or nu increased. These two dimensional results generally predict that low-frequency blood backscatter is related to cluster dimension, and higher-frequency properties are affected by finer morphological features as anisotropy. This numerically establishes that ultrasound backscatter spectroscopy on a large frequency range is pertinent to characterize in situ hemorheology.

Assessment of the effect of antiseizure drugs on the labeling process of red blood cells and plasma proteins with technetium-99m.

It is estimated that about 2.5 million people only in the United States are affected by epilepsy. Labelled red blood cells (RBC) and plasma proteins (PP) are used for several evaluations in nuclear medicine and drugs affecting those labelings have previously been described. The aim of this study was to evaluate whether the most popular antiseizure drugs interfere with the 99mTc labeling process of RBC and PP. Heparinized blood withdrawn from Wistar rats was incubated with phenobarbital (0.2, 2, 20, 200, 2,000 microg/ml), phenytoin (0.15, 1.5, 15, 150, 1,500 microg/ml), carbamazepine (0.7, 7, 70 microg/ml), clonazepam (0.5, 5, 50, 500 microg/ml) or valproic acid (0.5, 5, 50, 500 microg/ml) for I hr. Stannous chloride (SnCl2), in two different concentrations (0.012 or 1.2 microg/ml) and 99mTc were added. Plasma and cellular fractions were isolated by centrifugation, soluble and insoluble fractions were separated by trichloroacetic acid precipitation. The percentage of radioactivity was calculated for each fraction. Statistical analysis was performed with ANOVA and Dunnet tests. The analysis of the results has shown that phenobarbital (2,000 microg/ml) and clonazepam (50 microg/ml) significantly have reduced the RBC labeling efficiency when it was used the optimal SnCl2 concentration (1.2 microg/ml) and clonazepam (5, 50 microg/ml) has significantly decreased the PP labeling efficiency with 99mTc. Phenytoin (1,500 microg/ml) has decreased the RBC labeling efficiency when the experiments were carried out with a small SnCl2 concentration (0.012 microg/ml). We can suggest that with this in vitro assay, at the therapeutic level of phenytoin, phenobarbital, carbamazepine and valproic acid will not interfere on the 99mTc labeling process of RBC. Interference is displayed at higher phenobarbital concentrations (2,000 microg/ml). However, humans do not tolerate this concentration. On the other hand, a decreased RBC and PP labeling efficiency with 99mTc may be expected for clonazepam at therapeutic levels.

[99mTc red blood cell scintigraphy for the assessment of active gastrointestinal bleeding]. / Rol de la cintigrafía con glóbulos rojos-Tc-99m en la evaluación de hemorragia digestiva alta.

BACKGROUND: 99mTc red blood cell scintigraphy is a non invasive diagnostic method for low flow gastrointestinal bleeding. AIM: To assess the diagnostic yield of this method in patients admitted with gastrointestinal bleeding in whom upper gastrointestinal endoscopy was negative. PATIENTS AND METHODS: The clinical records of 59 patients, aged 6 to 90 years old (35 male), with active gastrointestinal bleeding subjected to a 99mTc red blood cell scintigraphy were reviewed. All had non diagnostic upper gastrointestinal endoscopic studies, and in 20 a selective arteriography was performed. RESULTS: In 40 patients the scintigraphy was positive for gastrointestinal bleeding, and in 57% of these the exam was positive during the first hour. Fifteen of the 19 patients with a negative scintigraphy did not have an active clinical bleeding at the moment of the examination. In 24 patients, a final etiological diagnosis was reached. In 93% of these patients scintigraphy correctly identified the bleeding site. In one patient with a negative scintigraphy, angiography disclosed a pseudo aneurysm of the splenic artery that was not bleeding actively. CONCLUSIONS: In these patients with negative upper gastrointestinal endoscopy 99mTc red blood cell scintigraphy had a 91% sensitivity for the diagnosis of active gastrointestinal bleeding.

Sonographic assessment of carotid artery stenosis. Comparison of power Doppler imaging and color Doppler flow imaging.

BACKGROUND AND PURPOSE: Power Doppler imaging (PDI) is a new ultrasound technique that, in contrast to color Doppler flow imaging (CDFI), generates intravascular color signals from the reflected echo amplitude depending mainly on the density of red blood cells. We evaluated the diagnostic significance of PDI compared with CDFI for the measurement of carotid stenosis and characterization of plaque surface. METHODS: In 25 internal carotid artery stenoses, reduction of the intrastenotic lumen contrasted by blood density signals and color Doppler signals on longitudinal and transverse views was assessed for correlative evaluation. In addition, the peak systolic flow velocity of the Doppler spectrum was correlated with PDI and CDFI measurements. RESULTS: PDI provided good visualization of the residual lumen in all stenoses, whereas displays on CDFI were inadequate in two calcified plaques. PDI revealed two ulcerative stenoses classified as smooth on CDFI. The correlation between PDI and CDFI was high for measurement of area stenosis (r = .93) and moderate for diameter stenosis (r = .73). Similarly, cross-sectional reduction on both imaging methods correlated more significantly with peak systolic flow velocity than diameter reduction. CONCLUSIONS: This pilot study suggests that PDI provides additional information for luminal measurement and characterization of plaque surface in complicated high-grade carotid stenosis. Because of the absent visualization of hemodynamics, PDI should be used in combination with CDFI.

Radionuclide assessment of penile corporal venous leak using technetium-99m-labeled red blood cells.

In an attempt to evaluate penile corporal venous outflow, a method that utilizes intracorporal injection of Tc-RBC was developed and used in 20 patients with erectile dysfunction. Seven patients showed venous leak and 13 had normal venous outflow. Technetium-labeled RBC corporal clearance in the flaccid state and after intracorporal injection of papaverine (30 mg) and regitine (1 mg) were assessed in sequence by two separate injections of 18.5 MBq of Tc-RBC each. The time for 50% corporal clearance (T50%) was determined from the time activity curves obtained in flaccid state and after intracorporal injection of the vasoactive agent. There were no statistically significant differences in T50% measured in the flaccid state between normal venous outflow (202 +/- 139 sec) and venous leak (92 +/- 35 sec, p = 0.1). However, after intracorporal injection of papaverine and regitine a significant increase in the T50% was noted in normal venous outflow (2892 +/- 1899 sec) as compared to venous leak (213 +/- 123 sec, p less than 0.001). The results suggest that measurement of corporal clearance of Tc-RBC after intracorporal injection of papaverine may be a useful method in detecting venous leak, and could be used as a screening test in patients with erectile dysfunction.

Embolization of platelets after endothelial injury to the aorta in rabbits. Assessment with 111indium-labeled platelets and angiography.

This study exploits the ability of a collateral arterial network to trap platelet aggregates in order to document the frequency of macroembolization in rabbits after endothelial damage. Two weeks after ligation of the right superficial femoral artery, endothelial injury was induced in the distal aorta; within 3 hours the rabbits were studied using either angiography or 111indium-labeled (111In) platelet scintigraphy. Angiography indicated visible aggregates in the thigh region in eight of 19 and arterial occlusion in three of 19 rabbits. The collateral-dependent thigh also showed more 111In-labeled platelet activity than the contralateral side (P less than .001), whether platelets were injected before or 2 hours after injury. Radioactivity in the limbs of rabbits with no injury was distributed symmetrically. Blood pool volume, assessed with technetium-99m-labeled red blood cells, was the same in both thighs, and could not account for these observations. The findings indicate that platelet activation and aggregation after endothelial injury lead to microembolization much more frequently than it leads to macroaggregate formation and visible artery occlusion.