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First published online August 6, 2004
Journal of Experimental Biology 207, 3213-3220 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01034
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Chaperones, protein aggregation, and brain protection from hypoxic/ischemic injury

Rona G. Giffard1,2,*, Lijun Xu1, Heng Zhao2, Whitney Carrico1, Yibing Ouyang1, Yanli Qiao1,2, Robert Sapolsky3, Gary Steinberg2, Bingren Hu4 and Midori A. Yenari2

1 Department of Anesthesia, Stanford University, Stanford, CA 94305, USA
2 Department of Neurosurgery, Stanford University, Stanford, CA 94305, USA
3 Department of Biology, Stanford University, Stanford, CA 94305, USA
4 Cerebral Vascular Disease Research Center, University of Miami School of Medicine, Miami, Florida 33136, USA



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  		Fig. 1. Ubiquitin staining patterns in CA1 neurons, 24 h following 8 min of dense forebrain ischemia. Ubiquitin staining is color-coded green, ß-gal staining is color-coded red. (A) A sham control animal not subjected to ischemia was examined 24 h later. A diffuse pattern in the processes (arrowheads) with strong nuclear staining (arrow) is observed. (B) In an animal subjected to dense forebrain ischemia, the pattern has changed to a patchy pattern in processes (arrowheads) with little nuclear staining (arrow). (C) An animal subjected to ischemia following injection with Herpes vector encoding only ß-gal and then colabeled for ß-gal to identify a vector targeted cell shows the same pattern in the targeted neuron as in B and in neighboring untargeted neurons with loss of nuclear staining (arrow). (D) An animal injected with Hsp-72 vector shows a relatively maintained pattern of ubiquitin staining in the neuron that is overexpressing Hsp72; note colocalization of ubiquitin and ß-gal staining in the nucleus (arrow) identified by the yellow color.

 


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  		Fig. 2. The pattern of ubiquitin immunostaining in astrocytes changes after glucose deprivation (GD). Astrocyte cultures were subjected to sham wash (A,B) or allowed to recover 16 h after an 8 h GD insult (C–I). Uninjured cells show diffuse immunoreactivity with greater nuclear staining compared to cytoplasmic (A,B). After GD there is loss of nuclear staining and the appearance of aggregates throughout the cytoplasm, varying from coarse to fine. Scale bar, 10 µm.

 


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  		Fig. 3. Hdj-2 reduces aggregation caused by GD. Cultures were subjected to 8 h GD and 16 h recovery. (A) A cell overexpressing Hdj-2. It retains a diffuse staining pattern with darker nuclear staining even after GD. (B) A cell expressing ß-galactosidase as a control. There are densely staining patches throughout the cytoplasm. Scale bar, 10 µm.

 


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  		Fig. 4. Geldanamycin (GA) blocks astrocyte death induced by glucose deprivation (GD). Sister early cultures of astrocytes subjected to 24 h GD without (A,B) or after pretreatment with GA (C,D). The same microscope field of each culture after 24 h GD was photographed using phase contrast optics (A,C) or by fluorescence microscopy (B,D) after staining with propidium iodide (PI) and Hoechst dye. The insert in B shows two of the apoptotic PI-staining nuclei from the lower part of the field, at higher magnification so the apoptotic bodies can be easily seen. (E) The extent of apoptotic and necrotic cell death in early cultures was determined by nuclear morphology after GD and PI plus Hoechst staining. At least 100 cells per culture were counted, with 4–8 cultures per condition. Values are means ± S.E.M. {dagger}P<0.05 compared to GD without GA treatment (Control). (F) Quantitation of cell death in mature cultures after 24 h GD by lactate dehydrogenase (LDH) release. GA was present beginning 8 or 4 h prior to GD or first added at the beginning of GD (During); No Tx indicates GD without GA treatment. Pretreatment for 8 or 4 h was most effective, but treatment only during GD was still able to reduce injury in older cultures. *P<0.05 compared to GD with GA (No Tx) condition. N=8–12 cultures per condition.

 

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© The Company of Biologists Ltd 2004