Mariano G. Uberti

Tracking superparamagnetic iron oxide labeled monocytes in brain by high-field magnetic resonance imaging.

Inflammatory cells, most notably mononuclear phagocytes (MP; macrophages and microglia), play a critical role in brain homeostasis, repair and disease. One important event in cellular biodynamics is how MP move in and throughout the nervous system. Prior studies have focused principally on cell migration across the blood–brain barrier during neuroinflammatory processes with little work done on cell movement within the brain. During the past decade our laboratories have studied the role of MP in HIV‐1‐associated dementia (HAD). In HAD MP incite sustained glial inflammatory reactions causing significant neuronal damage. To extend these works we investigated cell movement in brain and its influence for disease in a novel co‐registration system integrating neuropathology with high‐field magnetic resonance imaging (MRI). Human monocytes labeled with superparamagnetic iron oxide particles were injected into the brain of severe combined immunodeficient (SCID) mice. MRI was recorded 1, 7, and 14 days after cell injection. MRI co‐registered with histology verified that the MRI signal modification was due to the labeled cells. MRI showed human monocyte‐derived macrophages along the injection site, the corpus callosum, the ventricular system and in other brain sites. These data support the idea that cell migration can be monitored in vivo and provides an opportunity to assess monocyte mobility in brain and its affects on neurodegenerative processes and notably HAD.

Effects of Transcranial Ultrasound and Intravenous Microbubbles on Blood Brain Barrier Permeability in a Large Animal Model

We sought to determine whether transtemporal-applied 1-MHz ultrasound-induced microbubble destruction may be a safe method of transiently altering blood brain barrier (BBB) permeability for drug delivery in a large animal model. Endothelial cells are an integral component of the BBB but also prevent passage of potentially therapeutic drugs. Ultrasound-mediated destruction (UMD) of microbubbles has been shown to disrupt this barrier in small animals when ultrasound is delivered through bone windows. However, the effects of temporal bone attenuation and scattering in a large animal may limit the clinical application of such a technique. Twenty-four pigs were studied. One-MHz pulsed-wave ultrasound at 2.0 W/cm(2) (20% duty cycle) across the temporal bone was applied for 30 min after intravenous injections of either albumin-coated perfluorocarbon microbubble (PESDA, 8 pigs), lipid-encapsulated perfluorocarbon microbubbles (LEMB, 8 pigs) or ultrasound alone (8 pigs). BBB leak was quantified at 30 and 120 min after insonation using Evans blue. Serial magnetic resonance imaging (MRI) was performed in nine of the pigs (3 for each group) to quantify Gadolinium leak within the parenchyma. Peak negative pressures decreased ten-fold when ultrasound was transmitted across the pig temporal bone. Despite this, spectrophotometric analysis showed that both IV LEMB and PESDA combined with transtemporal ultrasound resulted in a significant increase in Evans blue extravasation across BBB of the treated side at 30 min after insonation (p < 0.001; compared with ultrasound alone) but not at 120 min. There was significant retention of Gadolinium within the insonified parenchyma at 60 and 90 min after insonation, but not at 120 min. Oxygen saturation and arterial pressures were not changed after any microbubble injection. Intravenous microbubbles, combined with transtemporal ultrasound, can transiently increase BBB permeability in a large animal. This induced opening of BBB is reversible and may be a safe noninvasive method of achieving drug or gene delivery across the BBB.

Quantitative 1H magnetic resonance spectroscopic imaging determines therapeutic immunization efficacy in an animal model of Parkinson's disease.

Nigrostriatal degeneration, the pathological hallmark of Parkinsons disease (PD), is mirrored by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. MPTP-treated animals show the common behavioral, motor, and pathological features of human disease. We demonstrated previously that adoptive transfer of Copaxone (Cop-1) immune cells protected the nigrostriatal dopaminergic pathway in MPTP-intoxicated mice. Herein, we evaluated this protection by quantitative proton magnetic resonance spectroscopic imaging (1H MRSI). 1H MRSI performed in MPTP-treated mice demonstrated that N-acetyl aspartate (NAA) was significantly diminished in the substantia nigra pars compacta (SNpc) and striatum, regions most affected in human disease. When the same regions were coregistered with immunohistochemical stains for tyrosine hydroxylase, numbers of neuronal bodies and termini were similarly diminished. MPTP-intoxicated animals that received Cop-1 immune cells showed NAA levels, in the SNpc and striatum, nearly equivalent to PBS-treated animals. Moreover, adoptive transfer of immune cells from ovalbumin-immunized to MPTP-treated mice failed to alter NAA levels or protect dopaminergic neurons and their projections. These results demonstrate that 1H MRSI can evaluate dopaminergic degeneration and its protection by Cop-1 immunization strategies. Most importantly, the results provide a monitoring system to assess therapeutic outcomes for PD.

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.

Coregistration of quantitative proton magnetic resonance spectroscopic imaging with neuropathological and neurophysiological analyses defines the extent of neuronal impairments in murine human immunodeficiency virus type-1 encephalitis

Relatively few immune‐activated and virus‐infected mononuclear phagocytes (MP; perivascular macrophages and microglia) may affect widespread neuronal dysfunction during human immunodeficiency virus type 1 (HIV‐1)‐associated dementia (HAD). Indeed, histopathological evidence of neuronal dropout often belies the extent of cognitive impairment. To define relationships between neuronal function and histopathology, proton magnetic resonance spectroscopic imaging (1H MRSI) and hippocampal long‐term potentiation (LTP) were compared with neuronal and glial immunohistology in a murine model of HIV‐1 encephalitis (HIVE). HIV‐1ADA‐infected human monocyte‐derived macrophages (MDM) were stereotactically injected into the subcortex of severe combined immunodeficient (SCID) mice. Sham‐operated and unmanipulated mice served as controls. Seven days after cell injection, brain histological analyses revealed a focal giant cell encephalitis, with reactive astrocytes, microgliosis, and neuronal dropout. Strikingly, significant reductions in N‐acetyl aspartate concentration ([NAA]) and LTP levels in HIVE mice were in both injected and contralateral hemispheres and in brain subregions, including the hippocampus, where neuropathology was limited or absent. The data support the importance of 1H MRSI as a tool for assessing neuronal function for HAD. The data also demonstrate that a highly focal encephalitis can produce global deficits for neuronal function and metabolism.

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.

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.