Fluorescent FITC-dextran Research Articles

Abstract 123: Establishing a Zebrafish Model of Ischemic Stroke Induced by Photochemical Thrombosis: a Novel Platform for Translational Researches

Introduction: Ischemic stroke is a leading cause of death and disability worldwide. Animal models of stroke are important for translational researches. We herein developed a novel zebrafish model of ischemic stroke using selective photochemical thrombosis, based on which further pathophysiological studies and pharmacological screening can be done. Method: Using 5 day post-fertilization zebrafish larva, we injected a photosensitizer rose bengal (120 pg) into the cardinal vein and employed a green laser (λ = 532 nm) targeting at selected cerebral arteries to induce photochemical thrombosis. The fluorescent FITC-dextran was used to stain the larval blood. The dynamic cerebral blood flow was continuously monitored. The neuronal death 12 h after arterial occlusion was indicated by acridine orange (AO). A neurological scoring system measuring the movement, coordination, and response to stimuli was established to determine the neurological function at 24 h. Thrombolysis using recombinant tissue plasminogen activator (rt-PA, 300 ng) at 0.5 and 3 h post-occlusion with comparison of the above parameters was used to demonstrate the potential application of this model. Results: Photochemical thrombosis consistently induced occlusion of the selected cerebral arteries. Occlusion at the basilar artery led to high mortality (73.3% at 12 h). All zebrafish survived the occlusion at the 1 st branch of central artery (CtA), but showed significantly increased AO stain and worsened neurological scores than control. Early treatment of rt-PA at 0.5 h post-occlusion resulted in a higher re-canalization rate than that at 3 h (40.0 vs. 26.7%). Moreover, the early treatment group exhibited significantly lower AO stain (42.2 ± 9.4 vs. 102.6 ± 17.4 a.u., P < 0.05) and improved neurological scores (4.2 ± 0.8 vs. 2.7 ± 1.1, P < 0.05). In contrast, no significant improvement was shown in the delayed treatment group even if the occluded artery had been reopened. Conclusion: This zebrafish model of ischemic stroke demonstrates characteristic pathophysiological responses and therapeutic effects seen in clinical stroke. With zebrafish being an increasingly employed model animal, there exist great potentials for this model to be applied in basic researches and translational studies.

Control of Na+ and H+ transports by exocytosis/endocytosis phenomena in a tight epithelium.

The relationship linking Na+ and H+ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution. Na+ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10(-3) M) and amphotericin-B (20 micrograms/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO2 or post NH4 load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H+ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H+ pump insertion. A comparable working hypothesis for the control of Na+ transport is proposed.