Global Ischemia Activates Nuclear Factor-κB in Forebrain Neurons of Rats by James A. Clemens, Diane T. Stephenson, E. Barry Smalstig, Eric P. Dixon, and Sheila P. Little Stroke Volume 28(5):1073-1081 May 1, 1997 Copyright © American Heart Association, Inc. All rights reserved.

CA1 pyramidal neurons of the hippocampus show histological evidence of PCD. Sections were taken from animals killed at 72 hours after 4-VO. CA1 pyramidal neurons of the hippocampus show histological evidence of PCD. Sections were taken from animals killed at 72 hours after 4-VO. A, Hematoxylin-eosin–stained section through the CA1 layer shows evidence of pyknosis and karyorrhexis of neurons. Some cells show chromosome condensation and fragmentation (arrows). B, TUNEL labeling illustrates DNA fragmentation in CA1 hippocampal pyramidal neurons. Staining is localized to the nucleus. Bar=10 μm. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

Apoptag labeling of hippocampal pyramidal neurons from a rat subjected to 4-VO. Apoptag labeling of hippocampal pyramidal neurons from a rat subjected to 4-VO. A, CA1 neurons show strong nuclear localization of peroxidase reaction product at 72 hours after ischemia. B, CA3 region from the same animal reveals no detectable staining. Sections were lightly counterstained with methyl green. The level of signal shown in B is identical to that observed in the CA1 region of sham-operated controls. Bar=60 μm. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

Immunoreactivity for NF-κB p50 and p65 is detected within hippocampal pyramidal CA1 neurons of the paramedian zone. Immunoreactivity for NF-κB p50 and p65 is detected within hippocampal pyramidal CA1 neurons of the paramedian zone. A, Immunoperoxidase staining with antisera directed to p50 reveals distinctive nuclear staining. B, Nuclear staining is likewise observed, albeit less intensely, in CA1 neurons with antisera directed to p65 (arrows). Bar=10 μm. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

NF-κB p50 and TUNEL are colocalized in the nucleus of CA1 pyramidal neurons. NF-κB p50 and TUNEL are colocalized in the nucleus of CA1 pyramidal neurons. Sections taken from a rat several days after ischemia were immunostained with NF-κB p50 antiserum using a Texas Red–based detection system (red), followed by in situ labeling of DNA fragmentation using a fluoresceinated tag (green) and coverslipping using mounting medium containing the nuclear stain DAPI (blue). A, NF-κB; B, TUNEL; C, DAPI. Arrows highlight individual neurons where NF-κB and TUNEL are colocalized. Bar=10 μm. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

NF-κB p50 immunoreactivity is not detected in intact neurons. NF-κB p50 immunoreactivity is not detected in intact neurons. Nissl staining (A) and the adjacent section stained with anti–NF-κB p50 antiserum (C) in the CA3 region from an animal at 72 hours after 4-VO. Nissl staining (B) and NF-κB p50 staining (D) in the CA1 region are from a sham-operated control animal. Immunostained sections were not counterstained. Bar=20 μm. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

NF-κB is induced in the dorsal hippocampus after ischemia. NF-κB is induced in the dorsal hippocampus after ischemia. Western immunoblots of microdissected homogenates from dorsal hippocampus of animals at 72 hours after ischemia were evaluated in parallel with sham-operated controls. A, Western gel probed with polyclonal anti–NF-κB p50 (Santa Cruz Biotechnology). Lane 1, 4-VO; lane 2, sham; and lane 3, HeLa cell nuclear extracts (H). Bands migrating below 50 kD are nonspecific. B, Western gel immunoblotted with anti–NF-κB p65 (Rockland). Lanes 1 and 2, 4-VO; lanes 3 and 4, sham; and lane 5, HeLa cell nuclear extracts (H). James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

NF-κB DNA binding activity by EMSA in hippocampal nuclear extracts. NF-κB DNA binding activity by EMSA in hippocampal nuclear extracts. Pooled nuclear extracts from either 4-VO (lanes 1 and 3) or sham-operated (lanes 2 and 4) rats were prepared from microdissected dorsal hippocampus. Lanes 1, 2, and 5 were evaluated in the absence of competitor, and lanes 3 and 4 were performed in the presence of competitor. Lanes 1 and 3, 4-VO; lanes 2 and 4, sham; lane 5, HeLa nuclear extracts, which served as a positive control. Free probe can be detected at the bottom of the gel. + indicates presence of cold competitor; −, absence of cold competitor. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

Levels of NF-κB p50 messenger RNA are increased at 72 hours after ischemia. Levels of NF-κB p50 messenger RNA are increased at 72 hours after ischemia. Northern blot analysis of total RNA prepared from sham (A) and ischemic (B) microdissected dorsal hippocampus. Blots were probed with radiolabeled antisense cDNA to NF-κB p50. James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.

Western-EMSA analysis demonstrates the presence of both NF-κB p50 and NF-κB p65 in rat dorsal hippocampus. Western-EMSA analysis demonstrates the presence of both NF-κB p50 and NF-κB p65 in rat dorsal hippocampus. Pooled nuclear extracts were subjected to EMSA, transferred to nitrocellulose, and probed with either p50 (A) or p65 (B) antibodies. Lane 1, 4-VO; lane 2, sham; and lane 3, HeLa nuclear extracts. Identification of p50 and p65 bands was based on molecular weight markers (not shown). James A. Clemens et al. Stroke. 1997;28:1073-1081 Copyright © American Heart Association, Inc. All rights reserved.