Reducing tissue injury in acute ischemic stroke by targeting NF-κB with transcription factor decoys.
Background: The transcription factor, NF-κB, plays a pivotal role in the inflammatory cascade. Transcription factor decoys (TFDs)are phosphorothioated oligonucleotides that bind activated NF-κB and prevent the transcription of inflammatory genes. We have shown that TFDs can be delivered into brain endothelial cells in vitro using mannitol and that an NF-κB TFD can block the upregulation of the ICAM-1 gene(Stroke 2000;31:1179–86). Objectives:1.) To determine if intra-arterial mannitol can deliver TFDs into endothelial cells in vivo 2.) To determine if an NF-κB decoy delivered with intra-arterial mannitol reduces cerebral infarct size. Methods: The temporary middle cerebral artery suture occlusion (2 hrs) model was performed in male Wistar rats. Intra-arterial mannitol was used to transfect phosphorothiaoted oligonucleotide decoys into brain endothelial cells. Decoy was detected using fluoroscein-tagged TFDs with immunohistochemical amplification. Infarct size was measured at 48 hours by TTC staining. The NF-κB decoy was a 38 mer with 3 NF-κB binding sites. A scrambled decoy (38 mer with same base composition but random sequence) was used as control. Results: With intra-arterial mannitol, the TFD was visualized in cerebral vessels in the injected hemisphere only. Double-labeling studies using a lectin (Griffonia simplicifolia) to label endothelial cells indicated that much of this labeling was in endothelial cells. Without mannitol or with a cationic liposome preparation, no decoy could be visualized. Animals treated with NF-κB decoy (at reperfusion) had significantly smaller infarcts than control animals (saline treatment)p=.036 Fishers PLSD [Control (N=6) 33.8 ± 1.1 (mean + SE) % of hemisphere vs NF-κB decoy with mannitol (N=4) 25.9 ± 3.4; mannitol alone (N=3)33.8 ± 4.3 , scrambled decoy with mannitol (N=3)32.3 ± 2.0]Conclusions: Intra-arterial mannitol permits the delivery of phosphorothioated oligonuclotides into brain endothelial cells in vivo. An NF-κB decoy delivered with intra-arterial mannitol may decrease infarct size. This approach may have clinical application as an adjunctive treatment to intra-arterial tPA