lucifer-yellow and Hypoxia-Ischemia--Brain

Astrocytes play a vital role in the brain; their structural integrity and sustained function are essential for neuronal viability, especially after injury or insult. In this study, we have examined the response of astrocytes to hypoxia/ischemia (H/I), employing multiple methods (immunohistochemistry, iontophoretic cell injection, Golgi-Kopsch staining, and D-aspartate uptake) in a neonatal pig model of H/I. We have identified morphological changes in cortical gray matter astrocytes in response to H/I. Initial astrocytic changes were evident as early as 8 h post-insult, before histological evidence for neuronal damage. By 72 h post-insult, astrocytes exhibited significantly fewer processes that were shorter, thicker, and had abnormal terminal swellings, compared with astrocytes from control brains that exhibited a complex structure with multiple fine branching processes. Quantification and image analysis of astrocytes at 72 h post-insult revealed significant decreases in the average astrocyte size, from 686 microm(2) in controls to 401 microm(2) in H/I brains. Sholl analysis revealed a significant decrease (>60%) in the complexity of astrocyte branching between 5 and 20 microm from the cell body. D-Aspartate uptake studies revealed that the H/I insult resulted in impaired astrocyte function, with significantly reduced clearance of the glutamate analog, D-aspartate. These results suggest that astrocytes may be involved in the pathophysiological events of H/I brain damage at a far earlier time point than first thought. Developing therapies that prevent or reverse these astrocytic changes may potentially improve neuronal survival and thus might be a useful strategy to minimize brain damage after an H/I insult.

Topics: Animals; Animals, Newborn; Astrocytes; Brain; Cell Size; D-Aspartic Acid; Female; Glial Fibrillary Acidic Protein; Glutamic Acid; Hypoxia-Ischemia, Brain; Immunohistochemistry; Isoquinolines; Male; Nerve Fibers, Unmyelinated; Swine; Time Factors

An acute reduction in cell membrane permeability could provide an effective strategy to prolong anoxic survival. A previous study has shown that in the western painted turtle whole-cell neuronal conductance (G(w)) decreases during anoxia, which may be mediated by the activation of adenosine A(1) receptors and calcium. Reduction in G(w) is thought to be the result of ion channel closure, but closure of gap junctions could also be responsible for this phenomenon. In our study, antibody staining of connexin 32 and 43 (Cx32 and Cx43) suggested the presence of gap junctional components in the turtle cortex. To examine if gap junctions were involved in the previously measured anoxic decrease in G(w), neuronal connectivity was assessed through the measurement of whole-cell capacitance (C(w)). Turtle cortical sheets were perfused with normoxic (95%O(2)/5%CO(2)), anoxic (95%N(2)/5%CO(2)), high calcium (4 mM) and adenosine (200 microm) artificial cerebral spinal fluid (aCSF). No significant change in C(w) was observed under any of the above conditions. However, during hypo-osmotic aCSF perfusion C(w) decreased significantly, with the lowest value of 50+/-10.4 pF (P<0.05) occurring at 30 min. To visualize changes in gap junction permeability lucifer yellow was loaded into turtle neurons during normoxic, anoxic, 0 calcium, hypo-osmotic, cold shock, (+)-isoproterenol, nitric oxide donor S-nitoso-acetyl penicillamine, and 8-bromo-guanosine 3',5'-cyclic monophosphate aCSF perfusion. Dye propagation was only observed in 3 of 20 cold shock experiments (4 degrees C). We conclude that gap junctions are not involved in the acute reduction in G(w) previously observed during anoxia and that our results support the hypothesis that ion channel arrest is involved.

Topics: Animals; Cell Communication; Connexin 43; Connexins; Electric Capacitance; Electric Conductivity; Female; Fluorescent Dyes; Gap Junction beta-1 Protein; Gap Junctions; Hypoxia-Ischemia, Brain; Isoquinolines; Neurons; Patch-Clamp Techniques; Turtles