Yutong Liu

A semi-automatic image segmentation method for extraction of brain volume from in vivo mouse head magnetic resonance imaging using Constraint Level Sets

In vivo magnetic resonance imaging (MRI) of mouse brain has been widely used to non-invasively monitor disease progression and/or therapeutic effects in murine models of human neurodegenerative disease. Segmentation of MRI to differentiate brain from non-brain tissue (usually referred to as brain extraction) is required for many MRI data processing and analysis methods, including coregistration, statistical parametric analysis, and mapping to brain atlas and histology. This paper presents a semi-automatic brain extraction technique based on a level set method with the incorporation of user-defined constraints. The constraints are derived from the prior knowledge of brain anatomy by defining brain boundary on orthogonal planes of the MRI. Constraints are incorporated in the level set method by spatially varying the weighting factors of the internal and external forces and modifying the image gradient (edge) map. Both two-dimensional multislice and three-dimensional versions of the brain extraction technique were developed and applied to MRI data with minimal brain/non-brain contrast T(1)-weighted (T(1)-wt) FLASH and maximized contrast T(2)-weighted (T(2)-wt) RARE. Results were evaluated by calculating the overlap measure (OM) between the automatically segmented and manually traced brain volumes. Results demonstrate that this technique accurately extracts the brain volume (mean OM=94%) and consistently outperformed the region growing method applied to the T(2)-wt RARE MRI (mean OM=81%). This method not only successfully extracts the mouse brain in low and high contrast MRI, but can also be used to segment other organs and tissues.

Landmark optimization using local curvature for point-based nonlinear rodent brain image registration

Purpose. To develop a technique to automate landmark selection for point-based interpolating transformations for nonlinear medical image registration. Materials and Methods. Interpolating transformations were calculated from homologous point landmarks on the source (image to be transformed) and target (reference image). Point landmarks are placed at regular intervals on contours of anatomical features, and their positions are optimized along the contour surface by a function composed of curvature similarity and displacements of the homologous landmarks. The method was evaluated in two cases (n = 5 each). In one, MRI was registered to histological sections; in the second, geometric distortions in EPI MRI were corrected. Normalized mutual information and target registration error were calculated to compare the registration accuracy of the automatically and manually generated landmarks. Results. Statistical analyses demonstrated significant improvement (P < 0.05) in registration accuracy by landmark optimization in most data sets and trends towards improvement (P < 0.1) in others as compared to manual landmark selection.

Improved Visualization of Neuronal Injury Following Glial Activation by Manganese Enhanced MRI

Research directed at anatomical, integrative and functional activities of the central nervous system (CNS) can be realized through bioimaging. A wealth of data now demonstrates the utility of magnetic resonance imaging (MRI) towards unraveling complex neural connectivity operative in health and disease. A means to improve MRI sensitivity is through contrast agents and notably manganese (Mn2+). The Mn2+ ions enter neurons through voltage-gated calcium channels and unlike other contrast agents such as gadolinium, iron oxide, iron platinum and imaging proteins, provide unique insights into brain physiology. Nonetheless, a critical question that remains is the brain target cells serving as sources for the signal of Mn2+ enhanced MRI (MEMRI). To this end, we investigated MEMRI’s abilities to detect glial (astrocyte and microglia) and neuronal activation signals following treatment with known inflammatory inducing agents. The idea is to distinguish between gliosis (glial activation) and neuronal injury for the MEMRI signal and as such use the agent as a marker for neural activity in inflammatory and degenerative disease. We now demonstrate that glial inflammation facilitates Mn2+ neuronal ion uptake. Glial Mn2+ content was not linked to its activation. MEMRI performed on mice injected intracranially with lipopolysaccharide was associated with increased neuronal activity. These results support the notion that MEMRI reflects neuronal excitotoxicity and impairment that can occur through a range of insults including neuroinflammation. We conclude that the MEMRI signal enhancement is induced by inflammation stimulating neuronal Mn2+ uptake.

Registration of in vivo MR to histology of rodent brains using blockface imaging

Registration of MRI to histopathological sections can enhance bioimaging validation for use in pathobiologic, diagnostic, and therapeutic evaluations. However, commonly used registration methods fall short of this goal due to tissue shrinkage and tearing after brain extraction and preparation. In attempts to overcome these limitations we developed a software toolbox using 3D blockface imaging as the common space of reference. This toolbox includes a semi-automatic brain extraction technique using constraint level sets (CLS), 3D reconstruction methods for the blockface and MR volume, and a 2D warping technique using thin-plate splines with landmark optimization. Using this toolbox, the rodent brain volume is first extracted from the whole head MRI using CLS. The blockface volume is reconstructed followed by 3D brain MRI registration to the blockface volume to correct the global deformations due to brain extraction and fixation. Finally, registered MRI and histological slices are warped to corresponding blockface images to correct slice specific deformations. The CLS brain extraction technique was validated by comparing manual results showing 94% overlap. The image warping technique was validated by calculating target registration error (TRE). Results showed a registration accuracy of a TRE < 1 pixel. Lastly, the registration method and the software tools developed were used to validate cell migration in murine human immunodeficiency virus type one encephalitis.

Multimodal theranostic nanoformulations permit magnetic resonance bioimaging of antiretroviral drug particle tissue-cell biodistribution

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, “multimodal imaging theranostic nanoprobes” were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution. RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate. CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy.

Manganese-Enhanced Magnetic Resonance Imaging Reflects Brain Pathology During Progressive HIV-1 Infection of Humanized Mice

Progressive human immunodeficiency viral (HIV) infection commonly leads to a constellation of cognitive, motor, and behavioral impairments. These are collectively termed HIV-associated neurocognitive disorders (HAND). While antiretroviral therapy (ART) reduces HAND severity, it does not affect disease prevalence. Despite decades of research, there remain no biomarkers for HAND and all potential comorbid conditions must first be excluded for a diagnosis to be made. To this end, we now report that manganese (Mn2+)-enhanced magnetic resonance imaging (MEMRI) can reflect brain region-specific HIV-1-induced neuropathology in chronically virus-infected NOD/scid-IL-2Rγcnull humanized mice. MEMRI diagnostics mirrors the abilities of Mn2+ to enter and accumulate in affected neurons during disease. T1 relaxivity and its weighted signal intensity are proportional to Mn2+ activities in neurons. In 16-week virus-infected humanized mice, altered MEMRI signal enhancement was easily observed in affected brain regions. These included, but were not limited to, the hippocampus, amygdala, thalamus, globus pallidus, caudoputamen, substantia nigra, and cerebellum. MEMRI signal was coordinated with levels of HIV-1 infection, neuroinflammation (astro- and micro-gliosis), and neuronal injury. MEMRI accurately demonstrates the complexities of HIV-1-associated neuropathology in rodents that reflects, in measure, the clinical manifestations of neuroAIDS as it is seen in a human host.

Precise 3D printing of micro/nanostructures using highly conductive carbon nanotube-thiol-acrylate composites

Two-photon polymerization (TPP) is of increasing interest due to its unique combination of truly three-dimensional (3D) fabrication capability and ultrahigh spatial resolution of ~40 nm. However, the stringent requirements of non-linear resins seriously limit the material functionality of 3D printing via TPP. Precise fabrication of 3D micro/nanostructures with multi-functionalities such as high electrical conductivity and mechanical strength is still a long-standing challenge. In this work, TPP fabrication of arbitrary 3D micro/nanostructures using multi-walled carbon nanotube (MWNT)-thiolacrylate (MTA) composite resins has been developed. Up to 0.2 wt% MWNTs have been incorporated into thiol-acrylate resins to form highly stable and uniform composite photoresists without obvious degradation for one week at room temperature. Various functional 3D micro/nanostructures including woodpiles, micro-coils, spiral-like photonic crystals, suspended micro-bridges, micro-gears and complex micro-cars have been successfully fabricated. The MTA composite resin offers significant enhancements in electrical conductivity and mechanical strength, and on the same time, preserving high optical transmittance and flexibility. Tightly controlled alignment of MWNTs and the strong anisotropy effect were confirmed. Microelectronic devices including capacitors and resistors made of the MTA composite polymer were demonstrated. The 3D micro/nanofabrication using the MTA composite resins enables the precise 3D printing of micro/nanostructures of high electrical conductivity and mechanical strength, which is expected to lead a wide range of device applications, including micro/nano-electromechanical systems (MEMS/NEMS), integrated photonics and 3D electronics.

Mouse brain fixation to preserve In vivo manganese enhancement for ex vivo manganese‐enhanced MRI

To develop a tissue fixation method that preserves in vivo manganese enhancement for ex vivo magnetic resonance imaging (MRI). The needs are clear, as conventional in vivo manganese‐enhanced MRI (MEMRI) applied to live animals is time‐limited, hence limited in spatial resolution and signal‐to‐noise ratio (SNR). Ex vivo applications can achieve superior spatial resolution and SNR through increased signal averaging and optimized radiofrequency coil designs. A tissue fixation method that preserves in vivo Mn2+ enhancement postmortem is necessary for ex vivo MEMRI.

Potential of N-acetylated-para-aminosalicylic acid to accelerate manganese enhancement decline for long-term MEMRI in rodent brain.

BACKGROUND Manganese (Mn(2+))-enhanced MRI (MEMRI) is a valuable imaging tool to study brain structure and function in normal and diseased small animals. The brain retention of Mn(2+) is relatively long with a half-life (t1/2) of 51-74 days causing a slow decline of MRI signal enhancement following Mn(2+) administration. Such slow decline limits using repeated MEMRI to follow the central nervous system longitudinally in weeks or months. This is because residual Mn(2+) from preceding administrations can confound the interpretation of imaging results. We investigated whether the Mn(2+) enhancement decline could be accelerated thus enabling repeated MEMRI, and as a consequence broadens the utility of MEMRI tests. NEW METHODS We investigated whether N-acetyl-para-aminosalicylic acid (AcPAS), a chelator of Mn(2+), could affect the decline of Mn(2+) induced MRI enhancement in brain. RESULTS AND CONCLUSION Two-week treatment with AcPAS (200mg/kg/dose×3 daily) accelerated the decline of Mn(2+) induced enhancement in MRI. In the whole brain on average the enhancement declined from 100% to 17% in AcPAS treated mice, while in PBS controls the decline is from 100% to 27%. We posit that AcPAS could enhance MEMRI utility for evaluating brain biology in small animals. COMPARISON WITH EXISTING METHODS To the best of our knowledge, no method exists to accelerate the decline of the Mn(2+) induced MRI enhancement for repeated MEMRI tests.

An image warping technique for rodent brain MRI-histology registration based on thin-plate splines with landmark optimization

Coregistration of in vivo magnetic resonance imaging (MRI) with histology provides validation of disease biomarker and pathobiology studies. Although thin-plate splines are widely used in such image registration, point landmark selection is error prone and often time-consuming. We present a technique to optimize landmark selection for thin-plate splines and demonstrate its usefulness in warping rodent brain MRI to histological sections. In this technique, contours are drawn on the corresponding MRI slices and images of histological sections. The landmarks are extracted from the contours by equal spacing then optimized by minimizing a cost function consisting of the landmark displacement and contour curvature. The technique was validated using simulation data and brain MRI-histology coregistration in a murine model of HIV-1 encephalitis. Registration error was quantified by calculating target registration error (TRE). The TRE of approximately 8 pixels for 20-80 landmarks without optimization was stable at different landmark numbers. The optimized results were more accurate at low landmark numbers (TRE of approximately 2 pixels for 50 landmarks), while the accuracy decreased (TRE approximately 8 pixels for larger numbers of landmarks (70- 80). The results demonstrated that registration accuracy decreases with the increasing landmark numbers offering more confidence in MRI-histology registration using thin-plate splines.