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Item Geometric Navigation of Axons in a Cerebral Pathway: Comparing dMRI with Tract Tracing and Immunohistochemistry(Oxford University Press, 2018-04-01) Mortazavi, Farzad; Oblak, Adrian L.; Morrison, Will Z.; Schmahmann, Jeremy D.; Stanley, H. Eugene; Wedeen, Van J.; Rosene, Douglas L.; Pathology and Laboratory Medicine, School of MedicineBrain fiber pathways are presumed to follow smooth curves but recent high angular resolution diffusion MRI (dMRI) suggests that instead they follow 3 primary axes often nearly orthogonal. To investigate this, we analyzed axon pathways under monkey primary motor cortex with (1) dMRI tractography, (2) axon tract tracing, and (3) axon immunohistochemistry. dMRI tractography shows the predicted crossings of axons in mediolateral and dorsoventral orientations and does not show axon turns in this region. Axons labeled with tract tracer in the motor cortex dispersed in the centrum semiovale by microscopically sharp axonal turns and/or branches (radii ≤15 µm) into 2 sharply defined orientations, mediolateral and dorsoventral. Nearby sections processed with SMI-32 antibody to label projection axons and SMI-312 antibody to label all axons revealed axon distributions parallel to the tracer axons. All 3 histological methods confirmed preponderant axon distributions parallel with dMRI axes with few axons (<20%) following smooth curves or diagonal orientations. These findings indicate that axons navigate deep white matter via microscopic sharp turns and branches between primary axes. They support dMRI observations of primary fiber axes, as well as the prediction that fiber crossings include navigational events not yet directly resolved by dMRI. New methods will be needed to incorporate coherent microscopic navigation into dMRI of connectivity.Item Hydrodynamic delivery for the study, treatment and prevention of acute kidney injury(2014-07-07) Corridon, Peter R.; Atkinson, Simon; Basile, David P.; Bacallao, Robert L.; Dunn, Kenneth William; Gattone II, Vincent H.Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.Item In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging(2014-02-25) Freeman, Kim Renee; Rubart-von der Lohe, Michael; Atkinson, Simon; Hurley, Thomas D., 1961-; Gattone II, Vincent H.The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing.Item Large-scale, three-dimensional tissue cytometry of the human kidney: a complete and accessible pipeline(Elsevier, 2021) Ferkowicz, Michael J.; Winfree, Seth; Sabo, Angela R.; Kamocka, Malgorzata M.; Khochare, Suraj; Barwinska, Daria; Eadon, Michael T.; Cheng, Ying-Hua; Phillips, Carrie L.; Sutton, Timothy A.; Kelly, Katherine J.; Dagher, Pierre C.; El-Achkar, Tarek M.; Dunn, Kenneth W.; Kidney Precision Medicine Project; Anatomy, Cell Biology and Physiology, School of MedicineThe advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell's physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.Item Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics(American Physical Society, 2020) Tavakoli, Meysam; Jazani, Sina; Sgouralis, Ioannis; Shafraz, Omer M.; Sivasankar, Sanjeevi; Donaphon, Bryan; Levitus, Marcia; Pressé, Steve; Physics, School of ScienceFluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.Item Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues(Springer Nature, 2019-04-04) Chen, Yi; Shen, Qi; White, Sharla L.; Gokmen-Polar, Yesim; Badve, Sunil; Goodman, Laurie J.; Pathology and Laboratory Medicine, School of MedicineThe tumor microenvironment can be spatially heterogenous, which makes it challenging to fully characterize with standard 2D histology-based methods. In this study, we determined the feasibility of a CLARITY tissue-processing approach to analyze biopsies from breast cancer patients. Formalin-fixed human breast cancer core-needle biopsy specimens, were embedded, lipid-cleared, and multiplexed immunostained to identify key biomarkers (pan-cytokeratin, Ki67, CD3). Confocal microscopy was then used to image the specimens after refractive index matching. These data sets were then quantitatively compared to conventional slide-based FFPE histology. Using CLARITY, the gross and cellular morphology of the tissues were well preserved, and high optical transparency was achieved, with the exception of fibrotic regions. Specific staining of various cellular and nuclear markers was achieved using optimized antibody conditions. Manually determined composite Ki67 scores from the CLARITY datasets agreed with histology results. However, the CLARITY datasets (3D) revealed variation in the intra-tumoral Ki67 expression that was not evident in individual FFPE sections (2D). We further demonstrated that archived FFPE clinical specimens can be CLARITY-processed, immunostained, and imaged. In short, CLARITY-processed specimens may enable a more accurate, unbiased analysis of tumor samples in comparison to conventional slide-based histology, thus allowing for improved visualization of intra-tumoral heterogeneity.Item Vector Competence of Thrips Species to Transmit Soybean Vein Necrosis Virus(Frontiers, 2019-03-19) Han, Jinlong; Nalam, Vamsi J.; Yu, I-Chen; Nachappa, Punya; Anatomy and Cell Biology, IU School of MedicineSoybean vein necrosis virus (SVNV) is a newly discovered species of tospovirus infecting soybean plants that is transmitted by the primary vector, soybean thrips (Neohydatothrips variabilis), and two additional secondary vectors, tobacco thrips (Frankliniella fusca) and eastern flower thrips (F. tritici). This study was undertaken to elucidate the association between virus acquisition [6, 12, 24, and 48 h acquisition access period (AAP)] and transmission efficiency [12, 24, and 48 h inoculation access period (IAP)] in the primary vector, N. variabilis, and to examine the mechanisms of vector competence by analyzing the effect of AAP (6, 12, and 24 h) on virus infection in various tissues. In addition, we examined virus infection in tissues of the two secondary vectors. We found a significant effect of virus acquisition on transmission efficiency, transmission rate post 6 and 48 h AAP was significantly lower than 12 and 24 h AAP. Our analysis did not reveal a correlation between virus transmission rate and virus RNA in corresponding N. variabilis adults. On the contrary, N. variabilis adults harboring higher accumulation of the virus (>104) resulted in lower transmission rates. Analysis of SVNV infection in the tissues revealed the presence of the virus in the foregut, midgut (region 1, 2, and 3), tubular salivary glands and principal salivary glands (PSG) of adults of all three vector species, however, the frequency of infected tissues was highest in N. variabilis followed by F. fusca and F. tritici. The frequency of SVNV infection in individual tissues specifically the salivary glands was lowest after 6 h AAP compared to 12 and 24 h AAP. This finding is in agreement with the transmission assays, where significantly lower virus transmission rate was observed post 6 h AAP. In addition, N. variabilis adults with high PSG infection (12 and 24 h AAP) were likely to have high percentage of foregut and midgut region 2 infection. Overall, results from the transmission assays and immunolabeling experiments suggest that shorter AAP results in reduced virus infection in the various tissues especially PSG, which are important determinants of vector competence in SVNV-thrips interaction.Item Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis(Optical Society of America, 2017-07-17) Höök, Peter; Brito-Robinson, Teresa; Kim, Oleg; Narciso, Cody; Goodson, Holly V.; Weisel, John W.; Alber, Mark S.; Zartman, Jeremiah J.; Medicine, School of MedicineA technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions.