The Effect of Extracellular Glutamate Release on Repetitive Transient Ischemic Injury in Global Ischemia Model

During operations, neurosurgeons usually perform multiple temporary occlusions of parental artery, possibly resulting in the neuronal damage. It is generally thought that neuronal damage by cerebral ischemia is associated with extracellular concentrations of the excitatory amino acids. In this study, we measured the dynamics of extracellular glutamate release in 11 vessel occlusion (VO) model to compare between single occlusion and repeated transient occlusions within short interval. Changes in cerebral blood flow were monitored by laser-Doppler flowmetry simultaneously with cortical glutamate level measured by amperometric biosensor. From real time monitoring of glutamate release in 11 VO model, the change of extracellular glutamate level in repeated transient occlusion group was smaller than that of single occlusion group, and the onset time of glutamate release in the second ischemic episode of repeated occlusion group was delayed compared to the first ischemic episode which was similar to that of single 10 min ischemic episode. These results suggested that repeated transient occlusion induces less glutamate release from neuronal cell than single occlusion, and the delayed onset time of glutamate release is attributed to endogeneous protective mechanism of ischemic tolerance.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670+/-47 nm, n=10) and nucleus (847+/-32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.