Kenichi Funamoto

A Novel Microfluidic Platform for High-Resolution Imaging of a Three-Dimensional Cell Culture under a Controlled Hypoxic Environment

Low oxygen tensions experienced in various pathological and physiological conditions are a major stimulus for angiogenesis. Hypoxic conditions play a critical role in regulating cellular behaviour including migration, proliferation and differentiation. This study introduces the use of a microfluidic device that allows for the control of oxygen tension for the study of different three-dimensional (3D) cell cultures for various applications. The device has a central 3D gel region acting as an external cellular matrix, flanked by media channels. On each side, there is a peripheral gas channel through which suitable gas mixtures are supplied to establish a uniform oxygen tension or gradient within the device. The effects of various parameters, such as gas and media flow rates, device thickness, and diffusion coefficients of oxygen were examined using numerical simulations to determine the characteristics of the microfluidic device. A polycarbonate (PC) film with a low oxygen diffusion coefficient was embedded in the device in proximity above the channels to prevent oxygen diffusion from the incubator environment into the polydimethylsiloxane (PDMS) device. The oxygen tension in the device was then validated experimentally using a ruthenium-coated (Ru-coated) oxygen-sensing glass cover slip which confirmed the establishment of low uniform oxygen tensions (<3%) or an oxygen gradient across the gel region. To demonstrate the utility of the microfluidic device for cellular experiments under hypoxic conditions, migratory studies of MDA-MB-231 human breast cancer cells were performed. The microfluidic device allowed for imaging cellular migration with high-resolution, exhibiting an enhanced migration in hypoxia in comparison to normoxia. This microfluidic device presents itself as a promising platform for the investigation of cellular behaviour in a 3D gel scaffold under varying hypoxic conditions.

Local Hemodynamics at the Rupture Point of Cerebral Aneurysms Determined by Computational Fluid Dynamics Analysis

Background: Cerebral aneurysms carry a high risk of rupture and so present a major threat to the patient’s life. Accurate criteria for predicting aneurysm rupture are important for therapeutic decision-making, and some clinical and morphological factors may help to predict the risk for rupture of unruptured aneurysms, such as sex, size and location. Hemodynamic forces are considered to be key in the natural history of cerebral aneurysms, but the effect on aneurysm rupture is uncertain, and whether low or high wall shear stress (WSS) is the most critical in promoting rupture remains extremely controversial. This study investigated the local hemodynamic features at the aneurysm rupture point. Methods: Computational models of 6 ruptured middle cerebral artery aneurysms with intraoperative confirmation of rupture point were constructed from 3-dimensional rotational angiography images. Computational fluid dynamics (CFD) simulations were performed under pulsatile flows using patient-specific inlet flow conditions. Time-averaged WSS (TAWSS) and oscillatory shear index (OSI) were calculated, and compared at the rupture point and at the aneurysm wall without the rupture point. We performed an additional CFD simulation of a bleb-removed model for a peculiar case in which bleb formation could be confirmed by magnetic resonance angiography. Results: All rupture points were located at the body or dome of the aneurysm. The TAWSS at the rupture point was significantly lower than that at the aneurysm wall without the rupture point (1.10 vs. 4.96 Pa, p = 0.031). The OSI at the rupture point tended to be higher than at the aneurysm wall without the rupture point, although the difference was not significant (0.0148 vs. 0.0059, p = 0.156). In a bleb-removed simulation, the TAWSS at the bleb-removed area was 6.31 Pa, which was relatively higher than at the aneurysm wall (1.94 Pa). Conclusion: The hemodynamics of 6 ruptured cerebral aneurysms of the middle cerebral artery were examined using retrospective CFD analysis. We could confirm the rupture points in all cases. With those findings, local hemodynamics of ruptured aneurysms were quanti-tatively investigated. The rupture point is located in a low WSS region of the aneurysm wall. Bleb-removed simulation showed increased WSS of the bleb-removed area, associated with the flow impaction area. Although the number of subjects in this study was relatively small, our findings suggest that the location of the rupture point is related to a low WSS at the aneurysm wall. Further investigations will elucidate the detailed hemodynamic effects on aneurysm rupture.

Fundamental study of ultrasonic-measurement-integrated simulation of real blood flow in the aorta.

Acquisition of detailed information on the velocity and pressure fields of the blood flow is essential to achieve accurate diagnosis or treatment for serious circulatory diseases such as aortic aneurysms. A possible way to obtain such information is integration of numerical simulation and color Doppler ultrasonography in the framework of a flow observer. This methodology, namely, Ultrasonic-Measurement-Integrated (UMI) Simulation, consists of the following processes. At each time step of numerical simulation, the difference between the measurable output signal and the signal indicated by numerical simulation is evaluated. Feedback signals are generated from the difference, and numerical simulation is updated applying the feedback signal to compensate for the difference. This paper deals with a numerical study on the fundamental characteristics of UMI simulation using a simple two-dimensional model problem for the blood flow in an aorta with an aneurysm. The effect of the number of feedback points and the feedback formula are investigated systematically. It is revealed that the result of UMI simulation in the feedback domain rapidly converges to the standard solution, even with usually inevitable incorrect upstream boundary conditions. Finally, an example of UMI simulation with feedback from real color Doppler measurement also shows a good agreement with measurement.

Hemodynamic Analysis of Growing Intracranial Aneurysms Arising from a Posterior Inferior Cerebellar Artery

OBJECTIVE The role of hemodynamics in the growth of intracranial aneurysms is not completely clear. We present a hemodynamic study with two adjacent unruptured aneurysms arising from one parent artery but growing in different ways. This study aimed to investigate whether there were differences in hemodynamic characteristics between the two growing aneurysms. METHODS A 62-year-old female patient presented with six unruptured intracranial aneurysms. Catheter angiography at 6-month intervals revealed that two aneurysms located adjacently at the right posterior inferior cerebellar artery were growing over a 1-year period. Three-dimensional aneurysm geometries were acquired via rotational angiography. Computational fluid dynamic simulations were conducted on the 3D aneurysm geometries under patient-specific pulsatile flow conditions that were measured by magnetic resonance velocimetry. RESULTS The proximal multilobular aneurysm demonstrated high flow and physiological levels of wall shear stress (WSS) in the region of growth, whereas the distal rounded aneurysm had low flow and low WSS in the growing sac. CONCLUSION Growing aneurysms can have heterogeneous hemodynamic and morphologic characteristics and different growing patterns. Growing regions of an aneurysm could be exposed to either high WSS at the inflow zone or low WSS and high oscillatory shear in the aneurysm sac.

Numerical experiment for ultrasonic-measurement-integrated simulation of three-dimensional unsteady blood flow.

Integration of ultrasonic measurement and numerical simulation is a possible way to break through limitations of existing methods for obtaining complete information on hemodynamics. We herein propose Ultrasonic-Measurement-Integrated (UMI) simulation, in which feedback signals based on the optimal estimation of errors in the velocity vector determined by measured and computed Doppler velocities at feedback points are added to the governing equations. With an eye towards practical implementation of UMI simulation with real measurement data, its efficiency for three-dimensional unsteady blood flow analysis and a method for treating low time resolution of ultrasonic measurement were investigated by a numerical experiment dealing with complicated blood flow in an aneurysm. Even when simplified boundary conditions were applied, the UMI simulation reduced the errors of velocity and pressure to 31% and 53% in the feedback domain which covered the aneurysm, respectively. Local maximum wall shear stress was estimated, showing both the proper position and the value with 1% deviance. A properly designed intermittent feedback applied only at the time when measurement data were obtained had the same computational accuracy as feedback applied at every computational time step. Hence, this feedback method is a possible solution to overcome the insufficient time resolution of ultrasonic measurement.

Numerical Validation of MR-Measurement-Integrated Simulation of Blood Flow in a Cerebral Aneurysm

This study proposes magnetic resonance (MR)-measurement-integrated (MR-MI) simulation, in which the difference between the computed velocity field and the phase-contrast MRI measurement data is fed back to the numerical simulation. The computational accuracy and the fundamental characteristics, such as steady characteristics and transient characteristics, of the MR-MI simulation were investigated by a numerical experiment. We dealt with reproduction of three-dimensional steady and unsteady blood flow fields in a realistic cerebral aneurysm developed at a bifurcation. The MR-MI simulation reduced the error derived from the incorrect boundary conditions in the blood flow in the cerebral aneurysm. For the reproduction of steady and unsteady standard solutions, the error of velocity decreased to 13% and to 22% in one cardiac cycle, respectively, compared with the ordinary simulation without feedback. Moreover, the application of feedback shortened the computational convergence, and thus the convergent solution and periodic solution were obtained within less computational time in the MR-MI simulation than that in the ordinary simulation. The dividing flow ratio toward the two outlets after bifurcation was well estimated owing to the improvement of computational accuracy. Furthermore, the MR-MI simulation yielded wall shear stress distribution on the cerebral aneurysm of the standard solution accurately and in detail.

Volumetric and Angiogenic Evaluation of Antitumor Effects with Acoustic Liposome and High-Frequency Ultrasound

Acoustic liposomes (AL) have their inherent echogenicity and can add functionality in serving as drug carriers with tissue specificity. Nonuniform vascular structures and vascular branches/bends are evaluated by imaging the intravascular movement locus of ALs with high-frequency ultrasound (HF-US) imaging. However, the evaluation of antitumor effects on angiogenesis by ALs and HF-US imaging has not been reported. Here, we show that the combination of ALs and an HF-US imaging system is capable of noninvasively evaluating antitumor volumetric and angiogenic effects in preclinical mouse models of various cancers. In this study, the antitumor effects of cisplatin on tumor growth and angiogenesis in mice bearing two different types of tumor cells were assessed. By tracking each AL flowing in the vessel and transferring the images to personal computers, microvessel structures were mapped and reconstructed using the color difference based on SD method. The antitumor effects were confirmed with an in vivo bioluminescence imaging system and immunohistochemical analysis. Our results show that cisplatin inhibits tumor growth by decreasing intratumoral vessel area but does not affect the angiogenesis ratio in the tumor. The vascular occupancy in the outer region of the tumor was larger than that in the inner region; however, both occupancies were similar to those of the control tumor. We propose that this method of mapping microvessels with ALs and an HF-US system can serve as a new molecular imaging method for the assessment of angiogenesis and can be applied to evaluate the antitumor effects by various therapeutic agents.

Reduced Uterine Perfusion Pressure (RUPP) Model of Preeclampsia in Mice

Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as sFlt-1 followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the role of genes on PE using genetically engineered animals has been problematic because it has been difficult to make a useful RUPP model in mice. To establish a RUPP model of PE in mice, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). Consequently, these mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. This novel RUPP model in mice should be useful for investigating the pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Numerical Experiment of Transient and Steady Characteristics of Ultrasonic-Measurement-Integrated Simulation in Three-Dimensional Blood Flow Analysis

In ultrasonic-measurement-integrated (UMI) simulation of blood flows, feedback signals proportional to the difference of velocity vector optimally estimated from Doppler velocities are applied in the feedback domain to reproduce the flow field. In this paper, we investigated the transient and steady characteristics of UMI simulation by numerical experiment. A steady standard numerical solution of a three-dimensional blood flow in an aneurysmal aorta was first defined with realistic boundary conditions. The UMI simulation was performed assuming that the realistic velocity profiles in the upstream and downstream boundaries were unknown but that the Doppler velocities of the standard solution were available in the aneurysmal domain or the feedback domain by virtual color Doppler imaging. The application of feedback in UMI simulation resulted in a computational result approach to the standard solution. As feedback gain increased, the error decreased faster and the steady error became smaller, implying the traceability to the standard solution improves. The positioning of ultrasound probes influenced the result. The height less than or equal to the aneurysm seemed better choice for UMI simulation using one probe. Increasing the velocity information by using multiple probes enhanced the UMI simulation by achieving ten times faster convergence and more reduction of error.

Reproduction of pressure field in ultrasonic-measurement-integrated simulation of blood flow.

Ultrasonic-measurement-integrated (UMI) simulation of blood flow is used to analyze the velocity and pressure fields by applying feedback signals of artificial body forces based on differences of Doppler velocities between ultrasonic measurement and numerical simulation. Previous studies have revealed that UMI simulation accurately reproduces the velocity field of a target blood flow, but that the reproducibility of the pressure field is not necessarily satisfactory. In the present study, the reproduction of the pressure field by UMI simulation was investigated. The effect of feedback on the pressure field was first examined by theoretical analysis, and a pressure compensation method was devised. When the divergence of the feedback force vector was not zero, it influenced the pressure field in the UMI simulation while improving the computational accuracy of the velocity field. Hence, the correct pressure was estimated by adding pressure compensation to remove the deteriorating effect of the feedback. A numerical experiment was conducted dealing with the reproduction of a synthetic three-dimensional steady flow in a thoracic aneurysm to validate results of the theoretical analysis and the proposed pressure compensation method. The ability of the UMI simulation to reproduce the pressure field deteriorated with a large feedback gain. However, by properly compensating the effects of the feedback signals on the pressure, the error in the pressure field was reduced, exhibiting improvement of the computational accuracy. It is thus concluded that the UMI simulation with pressure compensation allows for the reproduction of both velocity and pressure fields of blood flow.