Browsing by Subject "Multiphoton microscopy"

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    Intravital imaging of real-time endogenous actin dysregulation in proximal and distal tubules at the onset of severe ischemia-reperfusion injury
    (Springer Nature, 2021-04-15) Corridon, Peter R.; Karam, Shurooq H.; Khraibi, Ali A.; Khan, Anousha A.; Alhashmi, Mohamed A.; Medicine, School of Medicine
    Severe renal ischemia-reperfusion injury (IRI) can lead to acute and chronic kidney dysfunction. Cytoskeletal modifications are among the main effects of this condition. The majority of studies that have contributed to the current understanding of IRI have relied on histological analyses using exogenous probes after the fact. Here we report the successful real-time visualization of actin cytoskeletal alterations in live proximal and distal tubules that arise at the onset of severe IRI. To achieve this, we induced fluorescent actin expression in these segments in rats with hydrodynamic gene delivery (HGD). Using intravital two-photon microscopy we then tracked and quantified endogenous actin dysregulation that occurred by subjecting these animals to 60 min of bilateral renal ischemia. Rapid (by 1-h post-reperfusion) and significant (up to 50%) declines in actin content were observed. The decline in fluorescence within proximal tubules was significantly greater than that observed in distal tubules. Actin-based fluorescence was not recovered during the measurement period extending 24 h post-reperfusion. Such injury decimated the renal architecture, in particular, actin brush borders, and hampered the reabsorptive and filtrative capacities of these tubular compartments. Thus, for the first time, we show that the combination of HGD and intravital microscopy can serve as an experimental tool to better understand how IRI modifies the cytoskeleton in vivo and provide an extension to current histopathological techniques.
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    Letter to the editor: "Quantifying albumin permeability with multiphoton microscopy: why the difference?"
    (APS, 2014-05-01) Sandoval, Ruben M.; Molitoris, Bruce A.; Department of Medicine, IU School of Medicine
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    Mitochondrial depolarization and repolarization in the early stages of acetaminophen hepatotoxicity in mice
    (Elsevier, 2020-06) Dunn, Kenneth W.; Martinez, Michelle M.; Wang, Zemin; Mang, Henry E.; Clendenon, Sherry G.; Sluka, James P.; Glazier, James A.; Klaunig, James E.; Medicine, School of Medicine
    Mitochondrial injury and depolarization are primary events in acetaminophen hepatotoxicity. Previous studies have shown that restoration of mitochondrial function in surviving hepatocytes, which is critical to recovery, is at least partially accomplished via biogenesis of new mitochondria. However, other studies indicate that mitochondria also have the potential to spontaneously repolarize. Although repolarization was previously observed only at a sub-hepatotoxic dose of acetaminophen, we postulated that mitochondrial repolarization in hepatocytes outside the centrilobular regions of necrosis might contribute to recovery of mitochondrial function following acetaminophen-induced injury. Our studies utilized longitudinal intravital microscopy of millimeter-scale regions of the mouse liver to characterize the spatio-temporal relationship between mitochondrial polarization and necrosis early in acetaminophen-induced liver injury. Treatment of male C57BL/6J mice with a single intraperitoneal 250 mg/kg dose of acetaminophen resulted in hepatotoxicity that was apparent histologically within 2 h of treatment, leading to 20 and 60-fold increases in serum aspartate aminotransferase and alanine aminotransferase, respectively, within 6 h. Intravital microscopy of the livers of mice injected with rhodamine123, TexasRed-dextran, propidium iodide and Hoechst 33342 detected centrilobular foci of necrosis within extended regions of mitochondrial depolarization within 2 h of acetaminophen treatment. Although regions of necrosis were more apparent 6 h after acetaminophen treatment, the vast majority of hepatocytes with depolarized mitochondria did not progress to necrosis, but rather recovered mitochondrial polarization within 6 h. Recovery of mitochondrial function following acetaminophen hepatotoxicity thus involves not only biogenesis of new mitochondria, but also repolarization of existing mitochondria. These studies also revealed a spatial distribution of necrosis and mitochondrial depolarization whose single-cell granularity is inconsistent with the hypothesis that communication between neighboring cells plays an important role in the propagation of necrosis during the early stages of APAP hepatotoxicity. Small islands of healthy, intact cells were frequently found surrounded by necrotic cells, and small islands of necrotic cells were frequently found surrounded by healthy, intact cells. Time-series studies demonstrated that these "islands", consisting in some cases of single cells, are persistent; over a period of hours, injury does not spread from individual necrotic cells to their neighbors.