Dados do Trabalho

Título

PLASMA OF MICE WITH EXPERIMENTAL CEREBRAL MALARIA INCREASES NITRIC OXIDE PRODUCTION IN ISOLATED CEREBRAL ARTERIES

Objetivo (s)

To evaluate the effect of stimulation with plasma from mice with experimental cerebral malaria on the nitric oxide production by isolated cerebral vessels.

Material e Métodos

Nitric oxide (NO) production by isolated cerebral arteries from uninfected mice and mice with experimental cerebral malaria (ECM) was assessed using the 4,5-diaminofluorescein (DAF-2), a fluorescent NO detector. The arteries were incubated at 37°C for 10 minutes with Krebs-HEPES (KH) buffer only (basal production) and KH buffer containing methacholine (MCh, inducer of NO production), L-NAME (Nitric Oxide Synthase inhibitor) or methylene blue (MB, soluble Guanylate Cyclase inhibitor, NOS inhibitor and NO scavenger). Incubations were also performed in neat or diluted control and ECM plasma, in the presence or absence of L-NAME or MB. DAF-2 [5μM] was added to all incubations. After the incubations, the supernatants were collected and deposited on a black 96-well polystyrene plate, and the fluorescence intensity was measured. NO concentrations were calculated as a percentage change from baseline production.

Resultados e Conclusão

Control arteries incubated in KH buffer had a significant increase (17%, p=0.0039) in fluorescence intensity induced by MCh. Incubation with L-NAME decreased the fluorescence intensity levels (-8%, p=0.0017) compared to incubation with only MCh, and MB had a more pronounced effect (-50%, p=0.0262). Neat plasma from ECM mice induced strong production of NO by control vessels (37% increase, p=0.0151), which was not inhibited by L-NAME at 10-3M. Diluted ECM plasma increased production of NO by control (46%, p=0.0007) and ECM vessels (39% increase, p=0.0017) compared to baseline, which was not inhibited by L-NAME at 10-2 M or MB at 10-4M. MB at 10-3 M inhibited NO production by control arteries (73% decrease, p=0.0006) compared to baseline. Only high concentration (1M) L-NAME decreased NO production to levels comparable with the baseline in both control and ECM arteries. The increase in fluorescence intensity induced by MCh and decreased by L-NAME or MB indicate that the ex-vivo assay can specifically detect NO. Plasma from mice with ECM stimulated NO production by the cerebral arteries of control and ECM mice. Confirmation of NO measurements using alternative readouts (e.g., nitrite and cGMP production, functional assays) are necessary to consolidate the findings.

Palavras-chave

Malaria; Cerebral Malaria; Nitric Oxide; Endothelial Dysfunction.

Agradecimentos

CNPq, INCT-NIM, IOC and NIH.