Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotective after intracerebral hemorrhage.
OBJECTIVE
Tumor necrosis factor-alpha (TNF-alpha) expression is increased in brain after cerebral ischemia, although little is known about its abundance and role in intracerebral hemorrhage (ICH). A TNF-alpha-specific antisense oligodeoxynucleotide (ORF4-PE) was used to study the extent to which TNF-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha during ICH and demonstrate that reducing TNF-alpha expression using antisense oligodeoxynucleotides is neuroprotective.
Mayne
M
M
Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada.
Ni
W
W
Yan
H J
HJ
Xue
M
M
Johnston
J B
JB
Del Bigio
M R
MR
Peeling
J
J
Power
C
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001522:Behavior, Animal / Q000187:drug effects; D002543:Cerebral Hemorrhage / Q000139:chemically induced / Q000188:drug therapy* / Q000473:pathology; D017364:Collagenases; D003342:Corpus Striatum / Q000187:drug effects / Q000473:pathology; D004195:Disease Models, Animal; D004660:Encephalitis / Q000276:immunology / Q000473:pathology; D006493:Heparin; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008279:Magnetic Resonance Imaging; D008297:Male; D008845:Microinjections; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D020556:Neutrophil Infiltration / Q000187:drug effects / Q000276:immunology; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012720:Severity of Illness Index; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
D018696:Neuroprotective Agents; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D006493:Heparin; D017364:Collagenases