Neuronal nitric oxide synthase inhibition reduces neuronal apoptosis after hypothermic circulatory arrest.
BACKGROUND
Neurologic injury, including choreoathetosis and learning and memory deficits, occurs after prolonged hypothermic circulatory arrest (HCA). Apoptosis, or programmed cell death, is a possible cause of the neurologic injury seen after HCA. However, the mechanism of apoptosis is unknown. Hypothermic circulatory arrest causes glutamate excitotoxicity, resulting in increased nitric oxide production. We therefore hypothesized that nitric oxide mediates apoptosis. The purpose of this study was to determine if neuronal nitric oxide synthase inhibition reduces neuronal apoptosis in an established canine model of HCA.


METHODS
Fourteen male hound dogs (weight, 20 to 27 kg) were placed on closed-chest cardiopulmonary bypass, subjected to 2 hours of HCA at 18 degrees C, rewarmed to normothermia, and sacrificed 8 hours after HCA. Group 1 (n = 7) dogs were treated with the neuronal nitric oxide inhibitor 7-nitroindazole, 25 mg/kg intraperitoneally, before arrest and every 2 hours until sacrifice. Group 2 (n = 7) dogs received vehicle only. The brains were analyzed histopathologically. Apoptosis, identified by hematoxylin-eosin staining, was confirmed by DNA terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assay and electron microscopy. Apoptosis was scored by a blinded neuropathologist from 0 (normal) to 100 (severe injury).


RESULTS
Apoptosis occurred early after HCA in select neuronal populations, including the hippocampus, stria terminalis, neocortex, and entorhinal cortex. Apoptotic neurons showed a characteristic shrunken cytoplasm and nuclear chromatin condensation. 7-Nitroindazole significantly inhibited apoptosis (group 1 versus 2: 19.17 +/- 14.39 versus 61.11 +/- 5.41; p < .001).


CONCLUSIONS
Our results provide evidence that apoptosis is associated with the neurologic injury that occurs after HCA and that nitric oxide mediates the apoptosis that occurs after HCA. Strategies for cerebral protection during HCA may include the inhibition of neuronal nitric oxide synthase.
Tseng
E E
EE
Division of Cardiac Surgery, Johns Hopkins Medical Institutions, and Kennedy-Krieger Research Institute, Baltimore, Maryland, USA.
Brock
M V
MV
Lange
M S
MS
Blue
M E
ME
Troncoso
J C
JC
Kwon
C C
CC
Lowenstein
C J
CJ
Johnston
M V
MV
Baumgartner
W A
WA
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001921:Brain / Q000473:pathology; D002315:Cardiopulmonary Bypass; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006324:Heart Arrest, Induced; D007036:Hypothermia, Induced; D007191:Indazoles / Q000494:pharmacology*; D008297:Male; D009474:Neurons / Q000201:enzymology* / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D007191:Indazoles; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C080122:7-nitroindazole