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Biomedical subjects

G Allan Johnson

Publications and source records attributed to G Allan Johnson.

At least 19 recordsLinked to original sources

Neuroanatomical phenotypes in the reeler mouse.

The reeler mouse (Reln) has been proposed as a neurodevelopmental model for certain neurological and psychiatric conditions and has been studied by qualitative histochemistry and electron microscopy. Using magnetic resonance microscopy (MRM), we have quantitated for the first time the neuromorphology of Reln mice at a resolution of 21.5 microm. The neuroanatomical phenotypes of heterozygous and homozygous mutant Reln mice were compared to those of wild type (WT) littermates using morphometry and texture analysis. The cortical, hippocampal, and cerebellar phenotypes of the heterozygous and homozygous mutant Reln mice were confirmed, and new features were revealed. The Reln(rl/rl) mice possessed a smaller brain, and both Reln(rl/+) and Reln(rl/rl) mice had increased ventricles compared to WT controls. Shape differences were found between WT and Reln(rl/rl) brains, specifically in cerebellum, olfactory bulbs, dorsomedial frontal and parietal cortex, certain regions of temporal and occipital lobes, as well as in the lateral ventricles and ventral hippocampus. These findings suggest that certain brain regions may be more severely impacted by the Reln mutation than others. Gadolinium-based active staining demonstrated that layers of the hippocampus were disorganized in Reln(rl/rl) mice and differences in thickness of these layers were identified between WT and Reln(rl/rl) mice. The intensity distributions characteristic to the dorsal, middle, and ventral hippocampus were altered in the Reln(rl/rl), especially in the ventral hippocampus. These differences were quantified using skewness and modeling the intensity distributions with a Gaussian mixture. Our results suggest that structural features of Reln(rl/rl) brain most closely phenocopy those of patients with Norman-Roberts lissencephaly.

Animals↗

Imaging alveolar-capillary gas transfer using hyperpolarized 129Xe MRI.

Effective pulmonary gas exchange relies on the free diffusion of gases across the thin tissue barrier separating airspace from the capillary red blood cells (RBCs). Pulmonary pathologies, such as inflammation, fibrosis, and edema, which cause an increased blood-gas barrier thickness, impair the efficiency of this exchange. However, definitive assessment of such gas-exchange abnormalities is challenging, because no methods currently exist to directly image the gas transfer process. Here we exploit the solubility and chemical shift of (129)Xe, the magnetic resonance signal of which has been enhanced by 10(5) with hyperpolarization, to differentially image its transfer from the airspaces into the tissue barrier spaces and RBCs in the gas exchange regions of the lung. Based on a simple diffusion model, we estimate that this MR imaging method for measuring (129)Xe alveolar-capillary transfer is sensitive to changes in blood-gas barrier thickness of approximately 5 microm. We validate the successful separation of tissue barrier and RBC images and show the utility of this method in a rat model of pulmonary fibrosis where (129)Xe replenishment of the RBCs is severely impaired in regions of lung injury.

Animals↗

Enhanced T2 contrast for MR histology of the mouse brain.

A 3D Carr-Purcell-Meiboom-Gill (CPMG) sequence was implemented to obtain enhanced T(2) contrast in actively stained (perfusion with fixative and contrast agent) mouse brains at 9.4 T. Short interecho spacing was used to minimize diffusion and susceptibility losses. The sequence produced 16 3D volumes with an interecho spacing of 7 ms for isotropic 43-mu-resolution images of the mouse brains in a scan time of 4 hr. To enhance the signal-to-noise ratio (SNR) and contrast, the multiecho frequency domain image contrast (MEFIC) method was applied, resulting in a composite image with T(2)-weighted contrast. The high SNR and contrast thus achieved revealed aspects of mouse brain morphology, such as multiple cortical layers, groups of thalamic nuclei, layers of the inferior and superior colliculus, and molecular and granular layers of the cerebellum, with a high degree of definition and contrast that was not previously achieved in T(2)-weighted acquisitions at high fields.

Animals↗

Imaging methods for morphological and functional phenotyping of the rodent heart.

Small animal imaging has a critical role in phenotyping, drug discovery, and in providing a basic understanding of mechanisms of disease. Translating imaging methods from humans to small animals is not an easy task. The purpose of this work is to compare two cardiac imaging modalities, i.e., magnetic resonance microscopy (MRM) and microcomputed tomography (CT) for preclinical studies on rodents. We present the two technologies, the parameters that they can measure, the types of alterations that they can detect, and show how these imaging methods compare to techniques available in clinical medicine. While this paper does not refer per se to the cardiac risk assessment for drug or chemical development, we hope that the information will effectively address how MRM and micro-CT might be exploited to measure biomarkers critical for safety assessment.

Animals↗

Three-dimensional imaging of xenograft tumors using optical computed and emission tomography.

The physical basis and preliminary applications of optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) are introduced, as new techniques with potential to provide unique 3D information on a variety of aspects of tumor structure and function. A particular focus here is imaging tumor micro-vasculature, and the spatial distribution of viable tumor cells, although the techniques have the potential for much wider application. The principle attractiveness of optical-CT and optical-ECT are that high resolution (<20 microm) and high contrast co-registered 3D images of structure and function can be acquired for relatively large intact samples. The unique combination of high contrast and resolution offers advantages over micro-CT and micro-MRI, and the lack of requirement for sectioning offers advantages over confocal microscopy, conventional microscopy, and histological sectioning techniques. Optical-CT/ECT are implemented using in-house custom apparatus and a commercial dissecting microscope capable of both transmission and fluorescence imaging. Basic studies to characterize imaging performance are presented. Negligible geometrical distortion and accurate reconstruction of relative attenuation coefficients was observed. Optical-CT and optical-ECT are investigated here by application to high resolution imaging of HCT116 xenograft tumors, about 1 cc in dimension, which were transfected with constitutive red fluorescent protein (RFP). Tumor microvasculature was stained in vivo by tail vein injection of either passive absorbing dyes or active fluorescent markers (FITC conjugated lectin). Prior to imaging, the tumors were removed (ex vivo) and optically cleared in a key process to make the samples amenable to light transmission. The cleared tumors were imaged in three modes (i) optical-CT to image the 3D distribution of microvasculature as indicated by absorbing dye, (ii) optical-ECT using the FITC excitation and emission filter set, to determine microvasculature as indicated by lectin-endothelial binding, and (iii) optical-ECT using the DSRed2 filter set to determine the 3D distribution of viable tumor as indicated by RFP emission. A clear correlation was observed between the independent vasculature imaging modes (i) and (ii) and postimaging histological sections, providing substantial validation of the optical-CT and optical-ECT techniques. Strong correlation was also observed between the RFP imaging of mode iii, and modes i and ii, supporting the intuitive conclusion that well-perfused regions contain significant viable tumor. In summary, optical-CT and optical-ECT, when combined with new optical clearing techniques, represent powerful new imaging modalities with potential for providing unique information on the structure and function of tumors.

Animals↗

Optimized radiographic spectra for small animal digital subtraction angiography.

The increasing use of small animals in basic research has spurred interest in new imaging methodologies. Digital subtraction angiography (DSA) offers a particularly appealing approach to functional imaging in the small animal. This study examines the optimal x-ray, molybdenum (Mo) or tungsten (W) target sources, and technique to produce the highest quality small animal functional subtraction angiograms in terms of contrast and signal-difference-to-noise ratio squared (SdNR2). Two limiting conditions were considered-normalization with respect to dose and normalization against tube loading. Image contrast and SdNR2 were simulated using an established x-ray model. DSA images of live rats were taken at two representative tube potentials for the W and Mo sources. Results show that for small animal DSA, the Mo source provides better contrast. However, with digital detectors, SdNR2 is the more relevant figure of merit. The W source operated at kVps >60 achieved a higher SdNR2. The highest SdNR2 was obtained at voltages above 90 kVp. However, operation at the higher potential results in significantly greater dose and tube load and reduced contrast quantization. A reasonable tradeoff can be achieved at tube potentials at the beginning of the performance plateau, around 70 kVp, where the relative gain in SdNR2 is the greatest.

Algorithms↗

Optimization of multiplanar reformations from isotropic data sets acquired with 16-detector row helical CT scanner.

Institutional review board approval and waiver of consent were obtained for the patient component of this retrospective HIPAA-compliant study. By using an anthropomorphic phantom and metal oxide semiconductor field effect transistor detectors, radiation dose was determined for one eight-detector row and two 16-detector row computed tomographic (CT) protocols. A custom phantom was scanned by using the three protocols to identify isotropy. Contrast-to-noise ratios (CNRs) were determined for the same protocols by using a third phantom. Seven patients had undergone isotropic 16-detector row CT of the abdomen and pelvis. Anonymized coronal reformations at various thicknesses were ranked qualitatively by three radiologists. Effective dose equivalents were similar for the eight- and 16-detector row protocols. When transverse and coronal reformations of data acquired in the custom phantom were compared, coronal reformations obtained with the 16-detector row and 0.625-mm section thickness protocol were found to be nearly identical to the transverse image for all sets of line pairs. CNRs were consistently highest on 5-mm-thick coronal reformations (CNR range, 1.2-3.3). For qualitative assessment, 2- and 3-mm-thick coronal reformations were consistently preferred.

Adult↗

A liposomal nanoscale contrast agent for preclinical CT in mice.

OBJECTIVE: The goal of this study was to determine if an iodinated, liposomal contrast agent could be used for high-resolution, micro-CT of low-contrast, small-size vessels in a murine model. MATERIALS AND METHODS: A second-generation, liposomal blood pool contrast agent encapsulating a high concentration of iodine (83-105 mg I/mL) was evaluated. A total of five mice weighing between 20 and 28 g were infused with equivalent volume doses (500 microL of contrast agent/25 g of mouse weight) and imaged with our micro-CT system for intervals of up to 240 min postinfusion. The animals were anesthetized, mechanically ventilated, and vital signs monitored allowing for simultaneous cardiac and respiratory gating of image acquisition. RESULTS: Initial enhancement of about 900 H in the aorta was obtained, which decreased to a plateau level of approximately 800 H after 2 hr. Excellent contrast discrimination was shown between the myocardium and cardiac blood pool (650-700 H). No significant nephrogram was identified, indicating the absence of renal clearance of the agent. CONCLUSION: The liposomal-based iodinated contrast agent shows long residence time in the blood pool, very high attenuation within submillimeter vessels, and no significant renal clearance rendering it an effective contrast agent for murine vascular imaging using a micro-CT scanner.

Animals↗

Automated segmentation of neuroanatomical structures in multispectral MR microscopy of the mouse brain.

We present the automated segmentation of magnetic resonance microscopy (MRM) images of the C57BL/6J mouse brain into 21 neuroanatomical structures, including the ventricular system, corpus callosum, hippocampus, caudate putamen, inferior colliculus, internal capsule, globus pallidus, and substantia nigra. The segmentation algorithm operates on multispectral, three-dimensional (3D) MR data acquired at 90-microm isotropic resolution. Probabilistic information used in the segmentation is extracted from training datasets of T2-weighted, proton density-weighted, and diffusion-weighted acquisitions. Spatial information is employed in the form of prior probabilities of occurrence of a structure at a location (location priors) and the pairwise probabilities between structures (contextual priors). Validation using standard morphometry indices shows good consistency between automatically segmented and manually traced data. Results achieved in the mouse brain are comparable with those achieved in human brain studies using similar techniques. The segmentation algorithm shows excellent potential for routine morphological phenotyping of mouse models.

Algorithms↗

Magnetic resonance imaging at microscopic resolution reveals subtle morphological changes in a mouse model of dopaminergic hyperfunction.

Structural abnormalities of the basal ganglia have been documented in several neuropsychiatric conditions associated with dysregulation of the dopamine system. However, the histological nature underlying these changes is largely unknown. Using magnetic resonance imaging at microscopic resolution (MRI, 9.4 T with 43 microm isotropic spatial resolution) and stereological techniques, we have investigated the effect of increased dopamine neurotransmission on brain morphology in mice with elevated extracellular dopamine, the dopamine transporter knockout (DAT-KO) mice. We first demonstrate the usefulness of MRI at microscopic resolution for the accurate identification and measurement of volumes of specific subregions, accounting for less than 0.03% (0.16 mm(3)) of the volume of a mouse brain. Furthermore, the MRI analysis reveals a significantly lower volume (-9%) of the anterior striatum of DAT-KO mice, while the volume of other dopamine-related structures such as the posterior striatum and the substantia nigra pars reticulata is unchanged in comparison to wild type littermates. Stereological analysis performed in the same brains reveals that one important structural factor accounting for this selective change in volume is a reduction of 18% in the absolute number of neuronal cell bodies. The feasibility of assessing accurately small morphological alterations in mouse models, where the molecular and histological pathologies can be easily compared in a controlled manner, provides a paradigm to examine the relevance of selective brain volumetric changes associated with a number of neuropathological conditions.

Animals↗

Ventilation-synchronous magnetic resonance microscopy of pulmonary structure and ventilation in mice.

Increasing use of transgenic animal models for pulmonary disease has raised the need for methods to assess pulmonary structure and function in a physiologically stable mouse. We report here an integrated protocol using magnetic resonance microscopy with gadolinium (Gd)-labeled starburst dendrimer (G6-1B4M-Gd, MW = 192 +/- 1 kDa, R(h) = 5.50 +/- 0.04 nm) and hyperpolarized (3)helium ((3)He) gas to acquire images that demonstrate pulmonary vasculature and ventilated airways in live mice (n = 9). Registered three-dimensional images of (1)H and (3)He were acquired during breath-hold at 2.0 T using radial acquisition (total acquisition time of 38 and 25 min, respectively). The macromolecular Gd-labeled dendrimer (a half-life of approximately 80 min) increased the signal-to-noise by 81 +/- 30% in the left ventricle, 43 +/- 22% in the lung periphery, and -4 +/- 5% in the chest wall, thus increasing the contrast of these structures relative to the less vascular surrounding tissues. A constant-flow ventilator was developed for the mouse to deliver varied gas mixtures of O(2) and N(2) (or (3)He) during imaging. To avoid hypoxemia, instrumental dead space was minimized and corrections were made to tidal volume lost due to gas compression. The stability of the physiologic support was assessed by the lack of spontaneous breathing and maintenance of a constant heart rate. We were able to stabilize the mouse for >8 hr using ventilation of 105 breath/min and approximately 0.2 mL/breath. The feasibility of acquiring both pulmonary vasculature and ventilated airways was demonstrated in the mouse lung with in-plane spatial resolution of 70 x 70 microm(2) and slice thickness of 800 microm.

Anesthesia↗

Effects of breathing and cardiac motion on spatial resolution in the microscopic imaging of rodents.

One can acquire high-resolution pulmonary and cardiac images in live rodents with MR microscopy by synchronizing the image acquisition to the breathing cycle across multiple breaths, and gating to the cardiac cycle. The precision with which one can synchronize image acquisition to the motion defines the ultimate resolution limit that can be attained in such studies. The present work was performed to evaluate how reliably the pulmonary and cardiac structures return to the same position from breath to breath and beat to beat across the prolonged period required for MR microscopy. Radiopaque beads were surgically glued to the abdominal surface of the diaphragm and on the cardiac ventricles of anesthetized, mechanically ventilated rats. We evaluated the range of motion for the beads (relative to a reference vertebral bead) using digital microradiography with two specific biological gating methods: 1) ventilation synchronous acquisition, and 2) both ventilation synchronous and cardiac-gated acquisitions. The standard deviation (SD) of the displacement was < or =100 microm, which is comparable to the resolution limit for in vivo MRI imposed by signal-to-noise ratio (SNR) constraints. With careful control of motion, its impact on resolution can be limited. This work provides the first quantitative measure of the motion-imposed resolution limits for in vivo imaging.

Animals↗

Morphology of the small-animal lung using magnetic resonance microscopy.

Small-animal imaging with magnetic resonance microscopy (MRM) has become an important tool in biomedical research. When MRM is used to image perfusion-fixed and "stained" whole mouse specimens, cardiopulmonary morphology can be visualized, nondestructively, in exquisite detail in all three dimensions. This capability can be a valuable tool for morphologic phenotyping of different mouse strains commonly used in genomics research. When these imaging techniques are combined with specialized methods for biological motion control and animal support, the lungs of the live, small animal can be imaged. Although in vivo imaging may not achieve the high resolution possible with a fixed specimen, dynamic functional studies and survival studies that follow the progression of pulmonary change related to disease or environmental exposure are possible. By combining conventional proton imaging with gas imaging, using hyperpolarized 3He, it is possible to image the tissue and gas compartments of the lung. This capability is illustrated in studies on an emphysema model in rats and on radiation damage of the lung. With further improvements in imaging and animal handling technology, we will be able to image faster and at higher resolutions, making MRM an even more valuable research tool.

Animals↗

Neurotoxicity of carbonyl sulfide in F344 rats following inhalation exposure for up to 12 weeks.

Carbonyl sulfide (COS), a high-priority Clean Air Act chemical, was evaluated for neurotoxicity in short-term studies. F344 rats were exposed to 75-600 ppm COS 6 h per day, 5 days per week for up to 12 weeks. In rats exposed to 500 or 600 ppm for up to 4 days, malacia and microgliosis were detected in numerous neuroanatomical regions of the brain by conventional optical microscopy and magnetic resonance microscopy (MRM). After a 2-week exposure to 400 ppm, rats were evaluated using a functional observational battery. Slight gait abnormality was detected in 50% of the rats and hypotonia was present in all rats exposed to COS. Decreases in motor activity, and forelimb and hindlimb grip strength were also detected. In rats exposed to 400 ppm for 12 weeks, predominant lesions were in the parietal cortex area 1 (necrosis) and posterior colliculus (neuronal loss, microgliosis, hemorrhage), and occasional necrosis was present in the putamen, thalamus, and anterior olivary nucleus. Carbonyl sulfide specifically targeted the auditory system including the olivary nucleus, nucleus of the lateral lemniscus, and posterior colliculus. Consistent with these findings were alterations in the amplitude of the brainstem auditory evoked responses (BAER) for peaks N3, P4, N4, and N5 that represented changes in auditory transmission between the anterior olivary nucleus to the medial geniculate nucleus in animals after exposure for 2 weeks to 400 ppm COS. A concentration-related decrease in cytochrome oxidase activity was detected in the posterior colliculus and parietal cortex of exposed rats as early as 3 weeks. Cytochrome oxidase activity was significantly decreased at COS concentrations that did not cause detectable lesions, suggesting that disruption of the mitochondrial respiratory chain may precede these brain lesions. Our studies demonstrate that this environmental air contaminant has the potential to cause a wide spectrum of brain lesions that are dependent on the degree and duration of exposure.

Air Pollutants↗

Cine magnetic resonance microscopy of the rat heart using cardiorespiratory-synchronous projection reconstruction.

PURPOSE: To tailor a cardiac magnetic resonance (MR) microscopy technique for the rat that combines improvements in pulse sequence design and physiologic control to acquire high-resolution images of cardiac structure and function. MATERIALS AND METHODS: Projection reconstruction (PR) was compared to conventional Cartesian techniques in point-spread function simulations and experimental studies to evaluate its artifact sensitivity. Female Sprague-Dawley rats were imaged at 2.0 T using PR with direct encoding of the free induction decay. Specialized physiologic support and monitoring equipment ensured consistency of biological motion and permitted synchronization of imaging with the cardiac and respiratory cycles. RESULTS: The reduced artifact sensitivity of PR offered improved delineation of cardiac and pulmonary structures. Ventilatory synchronization further increased the signal-to-noise ratio by reducing inter-view variability. High-quality short-axis and long-axis cine images of the rat heart were acquired with 10-msec temporal resolution and microscopic spatial resolution down to 175 microm x 175 microm x 1 mm. CONCLUSION: Integrating careful biological control with an optimized pulse sequence significantly limits both the source and impact of image artifacts. This work represents a novel integration of techniques designed to support measurement of cardiac morphology and function in rodent models of cardiovascular disease.

Animals↗

Measurement of fat/water ratios in rat liver using 3D three-point dixon MRI.

Hepatic steatosis, or fatty liver, is commonly observed during the animal phase of drug safety studies. A noninvasive three-dimensional (3D) three-point Dixon method was used to quantitatively evaluate the fatty livers of rats induced by an experimental microsomal transfer protein (MTP) inhibitor, in an effort to develop a safety biomarker that could be translated to human studies. The method was implemented at 2.0 T for in vivo studies, and at 7.1 T for higher-resolution magnetic resonance (MR) histologic studies. In three separate protocols to study dose response and longitudinal evolution, intrahepatic fatty accumulation was detected by this method and confirmed by chemical and histologic assessments. Consistent with the pathologic changes, the fat/water ratios estimated by the MR technique increased significantly at doses of 1 mg/kg and 100 mg/kg of MTP inhibitor after 14 days of continuous administration. Among the more important findings were: 1). with the 3D three-point Dixon method, in vivo longitudinal studies of liver fat distribution can be conducted at significantly higher resolution than has previously been reported; 2). MR histology allows delineation of distribution at the microscopic scale of 0.0024 mm(3) resolution; and 3). the 3D three-point Dixon technique provides relative estimates of liver fat content and distribution at a high confidence level. This technique will be applicable in future studies in which fatty liver is a potential safety issue.

Adipose Tissue↗

Dynamic lung morphology of methacholine-induced heterogeneous bronchoconstriction.

Hyperpolarized (HP) 3helium (3He) dynamic MRI was used to investigate airway response in rats following intravenous (i.v.) bolus administration of a contractile agent, methacholine (MCh). The method provides direct visualization of the ventilated regions within the lung. Heterogeneous bronchoconstriction following the i.v. MCh injection was evident using this technique. These 3He dynamic lung images revealed that the inspired fresh air was shunted to the less-constricted regions after the MCh challenge in a similar manner as described by Laplace's relationship for the stability between adjacent alveoli. The airways in the more-constricted regions became nearly closed, resulting in air trapping, while the airways in the less-constricted regions remained effectively open, leading to overinflation. These data suggest a lung model of airway constriction partitioned into ventilated and nonventilated regions. These nonventilated regions are heterogeneously distributed in the lung and this distribution cannot be deduced from spirometric measurement of the whole lung. We demonstrate that a combination of functional 3He images and anatomical 1H images provide an effective method to diagnose regional lung abnormalities in rats.

Animals↗