Study of parameters in focus simulation functions of virtual slide

As a special function of Virtual Slide (VS) for thick specimens like cytology slides, multilayer (Z-stack) simulated focus and focus fusion were introduced. From the standpoint of surgical pathologist, the optimum parameters for multilayer focus simulation were examined. First, minimal thickness of the layer was checked by measuring thickness of small cells counting the number of the layers that come into focus. Then the optimal number of layers to scan, total thickness, was tried. Small-sized cell nuclei showed around 2μm or less thickness. As minimal thickness of one layer for focus simulation, less than 2 μm is required. Papillary cell mass of urothelial carcinoma, aspiration cytology specimen of breast or thyroid, and uterine cervical smear showed different optimal thickness. Cells piling up more than 4 to 5 layer are difficult to make close up observation. Total 15 (to 30) μm thick scan was enough for most specimens. The “focus fusion” image is single layer image synthesized from multiple layer images. Several layer thicknesses were examined, and there was negligible difference between the focus fusion image synthesized from 0.25 and 1μm thick layers. In the focus fusion image synthesized from 3μm thick layers, some cells not to come into focus. The “focus fusion” seems to contain all the cells in one plane, and easy for screening. To emphasize the existence of myoepithelial cells in fibroadenoma of breast, or to clarify the 3-dimensional tissue structure, multilayer image was better. From our results, 10 layers with 1.5μm thick each provide sufficient information in most specimens

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA