Antisense Oligodeoxynucleotide Inhibition of Tumor Necrosis Factor-α Expression Is Neuroprotective After Intracerebral Hemorrhage

Abstract

Background and Purpose —Tumor necrosis factor-α (TNF-α) expression is increased in brain after cerebral ischemia, although little is known about its abundance and role in intracerebral hemorrhage (ICH). A TNF-α–specific antisense oligodeoxynucleotide (ORF4-PE) was used to study the extent to which TNF-α expression influenced neurobehavioral outcomes and brain damage in a collagenase-induced ICH model in rat. Methods —Male Sprague-Dawley rats were anesthetized, and ICH was induced by intrastriatal administration of heparin and collagenase. Immediately before or 3 hours after ICH induction, ORF4-PE was administered directly into the site of ICH. TNF-α mRNA and protein levels were measured by reverse transcriptase–polymerase chain reaction and immunoblot analyses. Cell death was measured by terminal deoxynucleotidyl transferase–mediated uridine 5′triphosphate-biotin nick end labeling (TUNEL). Neurobehavioral deficits were measured for 4 weeks after ICH. Results —ICH induction (n=6) elevated TNF-α mRNA and protein levels ( P <0.01) at 24 hours after the onset of injury compared with sham controls (n=6). Immunohistochemical labeling indicated that ICH was accompanied by elevated expression of TNF-α in neutrophils, macrophages, and microglia. Administration of ORF4-PE (2.0 nmol) directly into striatal parenchyma, 15 minutes before (n=4) or 3 hours after (n=6) ICH, decreased levels of TNF-α mRNA ( P <0.001) and protein ( P <0.01) in the brain tissue surrounding the hematoma compared with animals treated with saline alone (n=6). Mean±SEM striatal cell death (cells per high-powered field) was also reduced in animals receiving ORF4-PE (34.1±5.0) compared with the saline-treated ICH group (80.3±7.50) ( P <0.001). ORF4-PE treatment improved neurobehavioral deficits observed at 24 hours ( P <0.001) after induction of ICH (n=6) compared with the untreated ICH group (n=6). This improvement was maintained at 28 days after hemorrhage induction ( P <0.001). Conclusions —These results indicate a pathogenic role for TNF-α during ICH and demonstrate that reducing TNF-α expression using antisense oligodeoxynucleotides is neuroprotective.