Label-free Multiscale Transport Imaging of the Living Cell.

The living cell is characterized by a myriad of parallel intracellular transport processes. Simultaneously capturing their global features across multiple temporal and spatial scales is a nearly unsurmountable task. Here we present a method that enables the microscopic imaging of the entire spectrum of intracellular transport on a broad time scale without the need for prior labeling. We show that from the time-dependent fluctuation of pixel intensity, in either bright-field or phase-contrast microscopic images, a scaling factor can be derived that reflects the local Hurst coefficient (H), the value of which reveals the microscopic mechanisms of intracellular motion. The Hurst coefficient image of the interphase cell displays an unexpected, overwhelming superdiffusion (H > 0.5) in the cytoplasm and subdiffusion (H < 0.5) in the nucleus, and provides unprecedented sensitivity in detecting transport processes associated with the living state.

Sex differences in renin response and changes of capillary diameters after renal ischemia/reperfusion injury.

Activation of the RAS has a crucial role in the progression of ischemia/reperfusion-associated CAD. The regulation of RAS differs in the two genders. However, the extent of gender differences and locations of renin production have not been revealed yet. We investigated in vivo the local renin production in the two genders during ischemia/reperfusion injury. In male and female Wistar rats, renal ischemia was induced followed by a reperfusion period of two, eight, 16, 24, or 48 h. We applied flow cytometry to measure renin content and multiphoton imaging to visualize renin granules and changes of peritubular diameters in vivo during ischemia/reperfusion. Renin content decreased in CD in the first eight h of reperfusion; however, after 16 h, its amount increased. In males, the production of renin was more pronounced, and the duration of vasoconstriction was longer with a subsequent phase of vessel hyperdilation compared to females. Renal ischemia/reperfusion injury induces renin response not only in the JGA, but also in the CD segment. Renin production is more explicit in males than in females which, via increased angiotensin II production, might explain the different dynamism of renal vessel regulation between the two genders.

Visualization of Calcium Dynamics in Kidney Proximal Tubules.

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.

Culturing and Scaling up Stem Cells of Dental Pulp Origin Using Microcarriers.

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Green-Light Activatable, Water-Soluble Red-Shifted Coumarin Photocages.

Easily accessible green-light activatable (>500 nm) photocages based on red-shifted, π-extended coumarin scaffolds are developed with uncaging efficiencies similar to those of recently introduced BODIPY derivatives. The photocages possess increased aqueous solubility, high absorption coefficients within the 450-600 nm range, and exceptionally high two-photon cross sections.

The First Postlesion Minutes: An In Vivo Study of Extravasation and Perivascular Astrocytes Following Cerebral Lesions in Various Experimental Mouse Models.

The immediate alterations following lesions cannot be investigated by using fixed tissues. Here, we employed two-photon microscopy to study the alterations to the permeability of blood-brain barrier and to glio-vascular connections in vivo during the first minutes following cortical lesions in mice. Four models were used: (1) cryogenic lesion, (2) photodisruption using laser pulses, (3) photothrombosis, and (4) bilateral carotid ligation. Sulforhodamine101 was used for supravital labeling of astrocytes and dextran-bound fluorescein isothiocyanate for the assessment of extravasation. Transgenic mice, in which the endothelium and astrocytes expressed a yellow fluorescent protein, were also used. Astrocytic labeling in vivo was verified with postmortem immunostaining against glial fibrillary acidic protein (GFAP). Summary of results: (1) the glio-vascular connections were stable in the intact brain with no sign of spontaneous dynamic attachment/detachment of glial end-feet; (2) only direct vascular damage (photodisruption or cryogenic) resulted in prompt extravasation; (3) even direct damage failed to provoke a prompt astroglial response. In conclusion, the results indicate that a detachment of the astrocytic end-feet does not precede the breakdown of blood-brain barrier following lesions. Whereas vasogenic edema develops immediately after the lesions, this is not the case with cytotoxic edemas. Time-lapse recordings and three-dimensional reconstructions are presented as supplemental materials.

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts.

BACKGROUND: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). METHODS: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. RESULTS: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. CONCLUSIONS: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events.

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.