Browsing by Author "Berry, Raymond"
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Item Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain(2024-03-21) Ampofo, Hannah; Berry, Raymond; Spann, Claire; Liu, Ran; Yang, ShaohuaExpansion Microscopy for Super-Resolution Imaging of the Rodent Brain Hannah Ampofo, Raymond Berry, Claire Spann, Ran Liu, Shaohua Yang Pharmacology and Neuroscience Department, School of Biomedical Sciences, University of North Texas Health Science Center, Texas, United States. Purpose-To establish Expansion microscopy (ExM) for super-resolution imaging of the rodent brain. Background- ExM is a remarkable imaging technology that enables nanoscale resolution in three-dimensional (3-D) imaging of preserved cells and tissues. ExM, which was invented in 2015, physically expands specimens using a hydrogel, allowing high-resolution imaging to be done with conventional diffraction-limited microscopes. The basic idea is to attach anchors to biomolecules or labels chemically and link them to a hydrogel that is uniformly distributed throughout the material. This polymerization technique separates biomolecules while maintaining their spatial organization by enabling isotropic expansion. The procedure is similar to sketching an outline on an inflating object and blowing it up: the ink particles will move apart, but their relative organization remains the same. Traditional optical imaging is unable to resolve nanoscale structures with dimensions smaller than 200–300 nm due to the fundamental physical limitations imposed by diffraction. ExM offers faster imaging speeds as compared to super-resolution methods, and enhanced antibody efficiency due to the decrowding effect generated by expanding biomolecules. The original ExM could resolve the specimen at 70 nm, however, new variants such as iterative ExM, 10X ExM microscopy, and nine-fold microscopy can resolve down to 15 to 30 nm, comparable to super-resolution microscopes. Method- The Paper-MAP version of expansion microscopy, a modified MAP method that allows immunostaining and expansion within two days was employed. The procedure involved staining floating mouse brain sections and incubating with a Paper-MAP cocktail (consisting of TEMED and sodium acrylate) and ammonium persulfate solution. The hydrogel matrix was created in situ through the crosslinking of sodium acrylate and bisacrylamide, forming a dense polyelectrolyte hydrogel. A denaturing solution was used to mechanically homogenize the sample and then expanded using deionized water. The pre and post-expanded sections were imaged using a Zeiss LSM 510 confocal microscope. Results- Following the addition of the monomer solution, the expansion procedure produced a 2 fold increase in size. This was followed by an evident 4 to 5 fold increase after the expansion was completed. We compared the pre-expansion image to the post-expansion image and observed intricate and detailed structures with significantly enhanced resolution that were previously indistinguishable in the pre-expansion section using confocal microscope. Conclusion- Expansion microscopy is a versatile and accessible imaging technique that resulted in significant improvements in imaging the microscopic configuration of the mouse brain. Its broad application offers a powerful tool for biological research in diverse organisms.Item Experimental ischemic stroke induces secondary white matter degeneration and long-term cognitive impairment(2024-03-21) Berry, Raymond; Liu, Ran; Winters, Ali; Spann, Clair; Ampofo, Hannah; Colon-Perez, Luis; Sumien, Nathalie; Yang, Shao-HuaClinical investigations have detected extensive white matter degeneration in individuals affected by ischemic stroke. Nonetheless, current stroke research has primarily concentrated on the infarct and periinfarct penumbra regions. The exploration of white matter degeneration's role after ischemic stroke and its contribution to post-stroke cognitive impairment and dementia (PSCID) has been limited in experimental models. Understanding the impact of white matter degeneration on PSCID in these models could offer valuable insights into potential therapeutic targets and interventions for alleviating cognitive decline following ischemic stroke. In this study, we analyzed the progression of locomotor and cognitive function up to 4 months after inducing ischemic stroke by middle cerebral artery occlusion in young adult rats. Despite evident ongoing locomotor recovery, long-term cognitive and affective impairment persisted after ischemic stroke, as indicated by Morris water maze, elevated plus maze, and open field performance. At 4-month after stroke, multimodal MRI was conducted to assess white matter degeneration. T2-weighted MRI (T2WI) unveiled bilateral cerebroventricular enlargement after ischemic stroke. Fluid Attenuated Inversion Recovery MRI (FLAIR) revealed white matter hyperintensities in the corpus callosum and fornix across bilateral hemispheres. A positive association between the volume of white matter hyperintensities and total cerebroventricular volume was noted in stroke rats. Further evidence of bilateral white matter degeneration was indicated by the reduction of fractional anisotropy (FA) and quantitative anisotropy (QA) in diffusion-weighted MRI (DWI) analysis. FA measures water diffusion directionality; reduced FA implies decreased white matter tract coherence. QA, linked to diffusion directionality, indicates microstructural white matter changes with decreased QA. Reduced FA and QA in DWI MRI suggest brain microstructural integrity changes, involving myelin sheath disruption, axonal damage, or overall white matter deterioration. Additionally, microglia and astrocyte activation were identified in the bilateral corpus callosum after stroke. This inflammatory response indicates the involvement of glial cells in the post-stroke environment, suggesting a complex interplay between structural alterations and neuroinflammatory processes that may contribute to the observed changes in white matter integrity. Understanding these multifaceted mechanisms is crucial for developing targeted interventions aimed at promoting recovery and minimizing long-term neurological consequences following ischemic stroke. The importance of these results is underscored by their potential connection to neurological or neurodegenerative conditions, given that white matter degeneration is commonly noted in diverse neurological disorders, including Alzheimer's disease, multiple sclerosis, and other related conditions. Our study suggests that experimental ischemic stroke induced by MCAO in young rats replicates long-term cognitive impairment and pervasive white matter degeneration observed in ischemic stroke patients. This model provides an invaluable tool for unraveling the mechanisms underlying post-stroke secondary white matter degeneration and its contribution to PSCID. Researchers and clinicians use these metrics to understand and monitor the progression of neurological diseases, potentially aiding in early diagnosis and treatment planning. This research may pave the way for a more comprehensive understanding of the mechanisms underlying post-stroke cognitive impairment and dementia, ultimately leading to improved strategies for patient care and rehabilitation.Item Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes(MDPI, 2023-01-22) Sun, Yuanhong; Winters, Ali; Wang, Linshu; Chaudhari, Kiran; Berry, Raymond; Tang, Christina; Liu, Ran; Yang, ShaohuaAstrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood-brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.Item Tractography as a method for mapping brain connectivity(2024-03-21) Spann, Claire; Yang, Shaohua; Liu, Ran; Berry, Raymond; Ampofo, Hannah; Colon-Perez, LuisPurpose. Mapping the brain and its complex connectivity has proved a challenging feat for neuroscience, though with the development of diffusion tensor imaging and tractography, we are one step closer to understanding brain anatomical connections. This method utilizes diffusion-weighted magnetic resonance imaging, which takes advantage of the Brownian motion of water molecules, to produce a diffusion tensor. In the white matter of the brain, diffusion varies in direction due to cellular membranes and myelin, and the diffusion tensor measures this anisotropic diffusivity to indicate possible tissue orientation. The generalized q-sampling imaging tractography method, developed by Frank Yeh in 2010, uses the diffusion tensor to approximate the course of white matter tracts and can be used to determine the exact location and termination of white matter bundles to assess connectivity between and within different brain regions. Despite limitations that decrease the accuracy of white matter tracking, tractography remains the only method to visualize white matter trajectories in vivo and non-invasively. Though commonly used for human diffusion-weighted images, here we verify tractography as a method to visualize and measure white matter trajectories in the rat brain. Methods. A male 3-month Sprague Dawley rat was used to acquire DWI images that were analyzed using DSI Studio. The DWI was superimposed with the corresponding T2W image and regions of interest (ROIs) were drawn in the corpus callosum and were applied via the built-in Waxholm Space rat atlas. Fiber tracking was seeded from the ROI, and fractional anisotropy, quantitative anisotropy, isotropy, mean diffusivity, axial diffusivity, and radial diffusivity was calculated at each ROI by DSI software. Results. Tractography of the corpus callosum was easily visualized using both drawn and atlas-applied ROIs. Fiber tractography from both ROIs included fibers from the internal and external capsules to ensure the integrity of all corpus callosum fibers. Diffusion metrics were not drastically different between the two seeding methods. Conclusion. This study presents tractography as a tool for visualizing white matter tracts and quantifying different diffusion metrics. Using both hand-drawn regions and regions from the rat atlas, white matter tracts in diseased brains can be compared to controls to measure several aspects of pathology, such as edema, axonal integrity, and axonal density. One application includes the imaging and quantification of both acute and chronic stroke, which exhibit different pathologies that can be visualized and measured with diffusion metrics, allowing for more precise targets of therapy. The use of tractography in adjunct with other established methods can improve the understanding of disease and assist in the development of better treatment.