Early phenomena following cryogenic lesion of rat brain : a preliminary study

The cerebrovascular laminin becomes detectable following lesions, whereas the lamina basalis-receptor β-dystroglycan disappears. These alterations may be indirect markers of a glio-vascular detachment which may result in the impairment of blood-brain-barrier. The present study estimates the correlations between the post-lesion exudation and the aforementioned phenomena. Lesions were performed in ketamine-xylazine anaesthesia with a copper rod cooled with dry ice. Immediately, or in 5 or 10 min brains were fixed in buffered 4% paraformaldehyde. Immunohistochemical reactions were performed in floating sections. Exudation was estimated with immunohistochemical detection of plasma-fibronectin and immunoglobulins. Glio-vascular connections were investigated with immunohistochemistry (GFAP, S100, glutamine synthetase), and electron microscopy. Laminin immunoreactivity appeared already at immediate fixation. Exudate was found only around the vessels. β-dystroglycan was still detectable. At five-ten minutes the territory of exudate became confluent and dystroglycan disappeared. Some but not all vessels were free of astrocytes. Electron microscopy demonstrated wide perivascular ’spaces’. ’In vivo’ monitoring was attempted with a multiphoton microscope in the Department of Biophysics of Semmelweis University. Astrocytes were labeled supravitally with sulforhodamine 101 so glio-vascular connections were visible. However, neither in the intact brain nor in 30-min post-lesion period astrocyte motility was observed

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

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

DS_10.1369_0022155418788390 – Supplemental material for The First Postlesion Minutes: An In Vivo Study of Extravasation and Perivascular Astrocytes Following Cerebral Lesions in Various Experimental Mouse Models

<p>Supplemental material, DS_10.1369_0022155418788390 for The First Postlesion Minutes: An In Vivo Study of Extravasation and Perivascular Astrocytes Following Cerebral Lesions in Various Experimental Mouse Models by László Tóth, Dávid Szöllősi, Katalin Kis-Petik, István Adorján, Ferenc Erdélyi and Mihály Kálmán in Journal of Histochemistry & Cytochemistry</p

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

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