Assessment of in vivo and post-mortem mechanical behavior of brain tissue using magnetic resonance elastography

The knowledge of in vivo brain tissue mechanical properties is essential in several biomedical engineering fields, such as injury biomechanics and neurosurgery simulation. Almost all existing available data have been obtained in vitro by invasive experimental protocols. However, the difference between in vivo and post-mortem mechanical properties remains poorly known, essentially due to the lack of a common method that could measure them both in vivo and ex vivo. In this study, we report the use of magnetic resonance elastography (MRE) for the non-invasive assessment of in vivo brain tissue viscoelastic properties and for the investigation of their evolution after the death. Experiments were performed on seven adult male rats. Shear storage and loss moduli were measured in vivo, just after death and at post-mortem time of approximately 24h. A significant increase in shear storage modulus G(') of approximately 100% was found to occur just after death (p=0.002), whereas no significant difference was found between in vivoG(') and G(') at 24h post-mortem time. No significant difference was found between shear loss modulus G(')in vivo and just after death, whereas a decrease of about 50% was found to occur after 24h (p=0.02). These results illustrate the ability of MRE to investigate some of the critical soft tissue biomechanics-related issues, as it can be used as a non-invasive tool for measuring soft tissue viscoelastic properties.     

Mechanical properties of porcine brain tissue in vivo and ex vivo estimated by MR elastography

The mechanical properties of brain tissue in vivo determine the response of the brain to rapid skull acceleration. These properties are thus of great interest to the developers of mathematical models of traumatic brain injury (TBI) or neurosurgical simulations. Animal models provide valuable insight that can improve TBI modeling. In this study we compare estimates of mechanical properties of the Yucatan mini-pig brain in vivo and ex vivo using magnetic resonance elastography (MRE) at multiple frequencies. MRE allows estimations of properties in soft tissue, either in vivo or ex vivo, by imaging harmonic shear wave propagation. Most direct measurements of brain mechanical properties have been performed using samples of brain tissue ex vivo. It has been observed that direct estimates of brain mechanical properties depend on the frequency and amplitude of loading, as well as the time post-mortem and condition of the sample. Using MRE in the same animals at overlapping frequencies, we observe that porcine brain tissue in vivo appears stiffer than porcine brain tissue samples ex vivo at frequencies of 100 Hz and 125 Hz, but measurements show closer agreement at lower frequencies.     

Measurement of the dynamic shear modulus of mouse brain tissue in vivo by magnetic resonance elastography

In this study, the magnetic resonance (MR) elastography technique was used to estimate the dynamic shear modulus of mouse brain tissue in vivo. The technique allows visualization and measurement of mechanical shear waves excited by lateral vibration of the skull. Quantitative measurements of displacement in three dimensions during vibration at 1200 Hz were obtained by applying oscillatory magnetic field gradients at the same frequency during a MR imaging sequence. Contrast in the resulting phase images of the mouse brain is proportional to displacement. To obtain estimates of shear modulus, measured displacement fields were fitted to the shear wave equation. Validation of the procedure was performed on gel characterized by independent rheometry tests and on data from finite element simulations. Brain tissue is, in reality, viscoelastic and nonlinear. The current estimates of dynamic shear modulus are strictly relevant only to small oscillations at a specific frequency, but these estimates may be obtained at high frequencies (and thus high deformation rates), noninvasively throughout the brain. These data complement measurements of nonlinear viscoelastic properties obtained by others at slower rates, either ex vivo or invasively.     

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade¹. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development². The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues³. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner⁴. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques⁵. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map⁶. The resulting measurements at each voxel have been shown to strongly correlate (R²>0.9914) with data collected using dynamic mechanical analysis⁷. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.     

生物组织弹性成像方法及其新技术进展

弹性是一种描述物质物理意义的重要参数,在描述物质在热力学和动力学的变化过程中有着重要的意义。在医学上,弹性的变化往往和病变联系在一起。然而,绝大多数生物组织在他们的力学特性上所表现出的复杂性并不是弹性模量一项参数就可以完全表述的,在对于他们的粘弹性表征和流变学行为的描述中,粘滞性往往和弹性一样的重要。现在被广泛用来对生物组织机械特性表征的成像技术是弹性成像,其基本原理是给组织施加一个激励,组织会产生一个响应,而该响应的分布结合技术的处理方法,可以反映出其弹性模量等力学属性的差异。本文介绍了生物组织常见的弹性成像方法:超声弹性成像,磁共振弹性成像以及光学相干弹性成像;详细阐述了新发展起来的技术-光声弹性成像和光声粘弹成像,并讨论分析其应用前景。     

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis

The biomechanical functions of articular cartilage are governed largely by the composition and density of its specialized extracellular matrix. Relationships between matrix density and functional indices such as mechanical properties or interstitial solute diffusivities have been previously explored. However, direct correlations between mechanical properties and solute transport parameters have received less attention, despite potential application of this information for cartilage functional assessment both in vivo and in vitro. The objective of this study was therefore to examine relationships among solute diffusivities, mechanical properties, and matrix density of compressed articular cartilage. Matrix density varied due to natural variation among explants and due to applied static compression. Matrix density of statically compressed cartilage explants was characterized by glycoaminoglycan (GAG) weight fraction and fluid volume fraction, while diffusion coefficients of a wide range of solutes were measured to characterize the transport environment. Explant mechanical properties were characterized by a non-linear Young's modulus (axial stress-strain ratio) and a non-linear Poisson's ratio (radial-to-axial strain ratio). Solute diffusivities were consistently correlated with Young's modulus, as well as with explant GAG weight and fluid volume fractions. Therefore, in vitro mechanical tests may provide a means of assessing transport environments in cartilage-like materials, while in vivo measurements of solute transport (for example with magnetic resonance imaging) may be a useful complement in identifying localized differences in matrix density and mechanical properties.     

Anisotropic mechanical properties of magnetically aligned fibrin gels measured by magnetic resonance elastography

The anisotropic mechanical properties of magnetically aligned fibrin gels were measured by magnetic resonance elastography (MRE) and by a standard mechanical test: unconfined compression. Soft anisotropic biomaterials are notoriously difficult to characterize, especially in vivo. MRE is well-suited for efficient, non-invasive, and non-destructive assessment of shear modulus. Direction-dependent differences in shear modulus were found to be statistically significant for gels polymerized at magnetic fields of 11.7 and 4.7 T compared to control gels. Mechanical anisotropy was greater in the gels polymerized at the higher magnetic field. These observations were consistent with results from unconfined compression tests. Analysis of confocal microscopy images of gels showed measurable alignment of fibrils in gels polymerized at 11.7 T. This study provides direct, quantitative measurements of the anisotropy in mechanical properties that accompanies fibril alignment in fibrin gels.     

Wideband MRE and static mechanical indentation of human liver specimen: Sensitivity of viscoelastic constants to the alteration of tissue structure in hepatic fibrosis

Despite the success of elastography in grading hepatic fibrosis by stiffness related noninvasive markers the relationship between viscoelastic constants in the liver and tissue structure remains unclear. We therefore studied the mechanical properties of 16 human liver specimens with different degrees of fibrosis, inflammation and steatosis by wideband magnetic resonance elastography (MRE) and static indentation experiments providing the specimens? static Young?s modulus (E), dynamic storage modulus (G′) and dynamic loss modulus (G″). A frequency-independent shear modulus μ and a powerlaw exponent α were obtained by fitting G′ and G″ using the two-parameter sprinpot model. The mechanical parameters were compared to the specimens? histology derived parameters such as degree of Fibrosis (F), inflammation score and fat score, amount of hydroxyproline (HYP) used for quantification of collagen, blood markers and presurgery in vivo function tests.     

P-31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid-N₂-frozen guinea pig brains and their organophosphate profiles examined by P-31 nuclear magnetic resonance (NMR) spectroscopy. Thirty-two phosphorus-containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P-31 NMR is its pronounced ribose 5-phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide-induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α-glycerolphosphate and inorganic orthophosphate contents relative to control. P-31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P-31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5-phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3-phosphorylethanolamine (0.3δ); glycerol 3-phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri-(β-ATP) and di-(β-ADP) phosphate ionized end-groups (−6.2δ); α-ATP, α-ADP, and nicotinamide adenine dinucleotides esterified end-groups (−11.1δ); uridine diphosphohexose, hexose esterified end-groups (−13.0δ); and β-ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P-31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P-31 spectrum.     

Magnetic resonance imaging of glutamate

Glutamate, a major neurotransmitter in the brain, shows a pH- and concentration-dependent chemical exchange saturation transfer effect (GluCEST) between its amine group and bulk water, with potential for in vivo imaging by nuclear magnetic resonance. GluCEST asymmetry is observed ～3 p.p.m. downfield from bulk water. Middle cerebral artery occlusion in the rat brain resulted in an ～100% elevation of GluCEST in the ipsilateral side compared with the contralateral side, predominantly owing to pH changes. In a rat brain tumor model with blood-brain barrier disruption, intravenous glutamate injection resulted in a clear elevation of GluCEST and a similar increase in the proton magnetic resonance spectroscopy signal of glutamate. GluCEST maps from healthy human brain were also obtained. These results demonstrate the feasibility of using GluCEST for mapping relative changes in glutamate concentration, as well as pH, in vivo. Contributions from other brain metabolites to the GluCEST effect are also discussed.     

In Vivo Measurements of Ethanol Concentration in Rabbit Brain by 1H Magnetic Resonance Spectroscopy

In vivo 1H magnetic resonance spectroscopy was used to measure the cerebral ethanol concentration in the rabbit after both intraarterial and intragastric administration. There was good agreement between cerebral and blood ethanol concentrations at all times after administration by either route. Cerebral ethanol levels, measured using in vivo 1H spectroscopy, agreed well with those measured in perchloric acid extracts of brain, analyzed by both high-resolution 1H spectroscopy and gas chromatography. Ethanol may be useful as an indicator to measure cerebral blood flow by 1H spectroscopy and chemical shift-selective magnetic resonance imaging.     

Measuring the Characteristic Topography of Brain Stiffness with Magnetic Resonance Elastography

Purpose

To develop a reliable magnetic resonance elastography (MRE)-based method for measuring regional brain stiffness.

Methods

First, simulation studies were used to demonstrate how stiffness measurements can be biased by changes in brain morphometry, such as those due to atrophy. Adaptive postprocessing methods were created that significantly reduce the spatial extent of edge artifacts and eliminate atrophy-related bias. Second, a pipeline for regional brain stiffness measurement was developed and evaluated for test-retest reliability in 10 healthy control subjects.

Results

This technique indicates high test-retest repeatability with a typical coefficient of variation of less than 1% for global brain stiffness and less than 2% for the lobes of the brain and the cerebellum. Furthermore, this study reveals that the brain possesses a characteristic topography of mechanical properties, and also that lobar stiffness measurements tend to correlate with one another within an individual.

Conclusion

The methods presented in this work are resistant to noise- and edge-related biases that are common in the field of brain MRE, demonstrate high test-retest reliability, and provide independent regional stiffness measurements. This pipeline will allow future investigations to measure changes to the brain’s mechanical properties and how they relate to the characteristic topographies that are typical of many neurologic diseases.     

Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements

Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations in in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue.     

Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria

Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.     

The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo

The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.     

Mechanical properties of brain tissue in-vivo: experiment and computer simulation

Realistic computer simulation of neurosurgical procedures requires incorporation of the mechanical properties of brain tissue in the mathematical model. Possible applications of computer simulation of neurosurgery include non-rigid registration, virtual reality training and operation planning systems and robotic devices to perform minimally invasive brain surgery. A number of constitutive models of brain tissue, both single-phase and bi-phasic, have been proposed in recent years. The major deficiency of most of them, however, is the fact that they were identified using experimental data obtained in vitro and there is no certainty whether they can be applied in the realistic in vivo setting. In this paper we attempt to show that previously proposed by us hyper-viscoelastic constitutive model of brain tissue can be applied to simulating surgical procedures. An in vivo indentation experiment is described. The force-displacement curve for the loading speed typical for surgical procedures is concave upward containing no linear portion from which a meaningful elastic modulus might be determined. In order to properly analyse experimental data, a three-dimensional, non-linear finite element model of the brain was developed. Magnetic resonance imaging techniques were used to obtain geometric information needed for the model. The shape of the force-displacement curve obtained using the numerical solution was very similar to the experimental one. The predicted forces were about 31% lower than those recorded during the experiment. Having in mind that the coefficients in the model had been identified based on experimental data obtained in vitro, and large variability of mechanical properties of biological tissues, such agreement can be considered as very good. By appropriately increasing material parameters describing instantaneous stiffness of the tissue one is able, without changing the structure of the model, to reproduce experimental curve almost perfectly. Numerical studies showed also that the linear, viscoelastic model of brain tissue is not appropriate for the modelling brain tissue deformation even for moderate strains.     

Contribution of magnetic resonance imaging to the knowledge of CNS malformations related to chromosomal aberrations

Summary Twelve patients presenting with various clinicopathological syndromes related to chromosomal diseases have been evaluated using magnetic resonance imaging. They include patients with trisomy 21, trisomy 18, trisomy 13, 4p-syndrome, 5p-syndrome, and 7p-syndrome. In all these patients karyotype studies were performed demonstrating the chromosomal aberrations. All patients were examined using magnetic resonance imaging to evaluate the head and neck malformations which may be specifically associated with their chromosomal anomaly. We were particularly interested in brain abnormalities and the morphological findings correlated with some pathologic anatomical findings. A review of the literature on neuropathological data is reported and compared with the in vivo anatomical results obtained using this highly anatomical non-ionising and non-invasive investigative procedure. Particular interest is paid to trisomy 21 in which all recognizable stereotyped morphological skull and brain malformations are depicted with magnetic resonance and some other malformations demonstrated such as the excessive forward bending and ascension of the brainstem which correlated well with a simian cephalic organization.     

Reliability and Validity of Quantifying Absolute Muscle Hardness Using Ultrasound Elastography

Muscle hardness is a mechanical property that represents transverse muscle stiffness. A quantitative method that uses ultrasound elastography for quantifying absolute human muscle hardness has been previously devised; however, its reliability and validity have not been completely verified. This study aimed to verify the reliability and validity of this quantitative method. The Young’s moduli of seven tissue-mimicking materials (in vitro; Young’s modulus range, 20–80 kPa; increments of 10 kPa) and the human medial gastrocnemius muscle (in vivo) were quantified using ultrasound elastography. On the basis of the strain/Young’s modulus ratio of two reference materials, one hard and one soft (Young’s moduli of 7 and 30 kPa, respectively), the Young’s moduli of the tissue-mimicking materials and medial gastrocnemius muscle were calculated. The intra- and inter-investigator reliability of the method was confirmed on the basis of acceptably low coefficient of variations (≤6.9%) and substantially high intraclass correlation coefficients (≥0.77) obtained from all measurements. The correlation coefficient between the Young’s moduli of the tissue-mimicking materials obtained using a mechanical method and ultrasound elastography was 0.996, which was equivalent to values previously obtained using magnetic resonance elastography. The Young’s moduli of the medial gastrocnemius muscle obtained using ultrasound elastography were within the range of values previously obtained using magnetic resonance elastography. The reliability and validity of the quantitative method for measuring absolute muscle hardness using ultrasound elastography were thus verified.     

Are in vivo and in situ brain tissues mechanically similar?

Brain tissue mechanical properties have been well-characterized in vitro, and were found to be inhomogeneous, nonlinear anisotropic and influenced by neurological development and postmortem time interval prior to testing. However, brain in vivo is a vascularized tissue, and there is a paucity of information regarding the effect of perfusion on brain mechanical properties. Furthermore, mechanical properties are often extracted from preconditioned tissue, and it remains unclear if these properties are representative of non-preconditioned tissue. We present non-preconditioned (NPC) and preconditioned (PC) relaxation responses of porcine brain (N = 10) obtained in vivo, in situ and in vitro, at anterior, mid and posterior regions of the cerebral cortex during 4mm indentations at either 3 or 1 mm/s. Material property characteristics showed no dependency on the site tested, thus revealing that cortical gray matter on the parietal and frontal lobes can be considered homogenous. In most cases, preconditioning decreased the shear moduli, with a more pronounced effect in the dead (in situ and in vitro) brain. For most conditions, it was found that only the long-term time constant of relaxation (tau > 20 s) significantly decreased from in vivo to in situ modes (p < 0.02), and perfusion had no effect on any other property. These findings support the concept that perfusion does not affect the stiffness of living cortical tissue.     

Shear wave induced resonance elastography of soft heterogeneous media

In the context of ultrasound dynamic elastography imaging and characterization of venous thrombosis, we propose a method to induce mechanical resonance of confined soft heterogeneities embedded in homogenous media. Resonances are produced by the interaction of horizontally polarized shear (SH) waves with the mechanical heterogeneity. Due to such resonance phenomenon, which amplifies displacements up to 10 times compared to non-resonant condition, displacement images of the underlying structures are greatly contrasted allowing direct segmentation of the heterogeneity and a more precise measurement of displacements since the signal-to-noise ratio is enhanced. Coupled to an analytical model of wave scattering, the feasibility of shear wave induced resonance (SWIR) elastography to characterize the viscoelasticity of a mimicked venous thrombosis is demonstrated (with a maximum variability of 3% and 11% for elasticity and viscosity, respectively). More generally, the proposed method has the potential to characterize the viscoelastic properties of a variety of soft biological and industrial materials.