Early nuclear factor-kappaB activation and inducible nitric oxide synthase expression in injured spinal cord neurons correlating with a diffuse reduction of constitutive nitric oxide synthase activity.
OBJECTIVE
Because of toxicity at high concentrations, nitric oxide (NO) contributes to spinal cord injury (SCI) secondary lesions. At low concentrations NO modulates nuclear factor-kappaB (NF-kappaB) activation. The authors investigated the activity of neuronal and endothelial NO synthase (nNOS and eNOS) to determine correlations with NF-kappaB activation and inducible NOS (iNOS) expression soon after SCI.


METHODS
In 48 adult male Wistar rats clip-based (50 g/mm2/10 seconds) SCI was induced, and spinal cords were removed at different intervals for the following evaluations: 1) assaying specific activity of nNOS and eNOS; 2) electrophoresis mobility shift assay for activated NF-kappaB; 3) Northern blotting for iNOS; 4) immunohistochemistry for iNOS and NF-kappaB; and 5) immunofluorescence for iNOS and NF-kappaB. At 15 minutes postinjury, eNOS activity decreased significantly (p < 0.001), as did nNOS activity at 1 hour compared with these levels in control animals and rats killed at 15 and 30 minutes after SCI (p < 0.001). Basal NF-kappaB levels were variable in controls and at 15 and 30 minutes after injury. One hour postinjury, NF-kappaB activation was diffuse. Inducible NOS messenger RNA localized diffusely, peaking 6 hours after injury and remaining stable until 24 hours postinjury. Immunohistochemical analysis showed diffuse iNOS and NF-kappaB staining, especially in neurons inside and around the lesion. Immunofluorescence demonstrated that injured neurons were a source of NF-kappaB and iNOS soon after injury.


CONCLUSIONS
Both nNOS and eNOS exhibited different regulation and roles soon after injury: nNOS correlated with NF-kappaB activation, whereas eNOS may have participated in vascular changes of the injured spinal cord. Neurons seemed to play a pivotal role in modulating and amplifying the inflammatory response in the injured spinal cord.
Miscusi
Massimo
M
Dipartimento di Scienze Neurologiche, Neurochirurgia, Università di Roma La Sapienza, Italia. m.miscusi@libero.it
Ebner
Florian
F
Ceccariglia
Sabrina
S
Menegazzi
Marta
M
Mariotto
Sofia
S
Berra
Luigi
L
Del Fa
Aurora
A
Gangitano
Carlo
C
Lauretti
Liverana
L
Missori
Paolo
P
Delfini
Roberto
R
Suzuki
Hisanori
H
D000818:Animals; D004195:Disease Models, Animal; D008297:Male; D016328:NF-kappa B / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013119:Spinal Cord Injuries / Q000378:metabolism* / Q000473:pathology*; D013904:Thoracic Vertebrae; D013997:Time Factors
D016328:NF-kappa B; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase