Neurodegenerative evidence in mice brains with cecal ligation and puncture-induced sepsis: preventive effect of the free radical scavenger edaravone.

Sepsis is a major clinical challenge and septic encephalopathy is its nasty complication. The pathogenesis and underlying mechanisms of septic encephalopathy are not well understood. This study sought to fully characterize sepsis-associated biochemical and histopathological changes in brains of mice after cecal ligation and puncture, regarded as a highly clinically relevant animal model of polymicrobial sepsis. Real-time PCR analysis showed that gene expression levels of proinflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, were significantly up-regulated in brain tissues from septic mice, but to a much lesser extent when compared with those in peripheral tissues such as lungs. Blood-brain barrier (BBB) permeability was significantly increased in septic mice, as determined by the measurement of sodium fluorescein and Evans blue content. Sepsis resulted in increases in NADPH oxidase activity and expression of p47(phox) and p67(phox) and up-regulation of inducible nitric oxide (NO) synthase in brains, indicating that superoxide, produced by NADPH oxidase, reacts with NO to form peroxynitrite, that maybe lead to the loss of BBB integrity. Light and electron microscopic examination of septic mouse brain showed serious neuronal degeneration, as indicated by hyperchromatic, shrunken, pyknotic, and electron-dense neurons. These histopathological changes were prevented by treatment with the free radical scavenger edaravone. Together, these results suggest that sepsis can lead to rapid neurodegenerative changes in brains via free radical species production and possibly subsequent injury to the BBB. We may also provide a potentially useful therapeutic tool for treating septic encephalopathy

Sepsis-induced increase in extravasation of sodium fluorescein (A) and Evans blue (B) in brains.

<p>Tissues were harvested 24 h after surgery. Mean of data from 4–6 animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus sham-operated control.</p

Effect of edaravone treatment on neuronal degeneration in cerebral cortex after sepsis.

<p>In the vehicle-treated CLP septic group, there was serious neuronal degeneration in cerebral cortex (B) compared with sham control (A). In the edaravone-treated CLP septic group, normal-appearing neuronal findings were obtained in cerebral cortex (C). Tissues were harvested 24 h after surgery. Magnified view is shown in each inset. (D) Estimation of densely-stained cells in cerebral cortex. Counts of densely-stained cells were made in the sections at a final magnification of ×100. Mean of data from eight animals in each group is presented, with SEM by vertical lines. **<i>p</i><0.01 versus control. #<i>p</i><0.05 versus CLP treated with vehicle.</p

Sepsis-induced changes in NADPH oxidase activity, NADPH oxidase components p47<i>^phox</i> and p67<i>^phox</i> expression, and oxidative stress parameter in brain tissues.

<p>(A) Time course of changes in NADPH oxidase activity. Mean of data from six animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus control. (B) Western immunoblotting showing up-regulation of p47<i>^phox</i> and p67<i>^phox</i> protein expression after CLP. The result is representative of two independent experiments. (C) The mRNA levels for p47<i>^phox</i> and p67<i>^phox</i> were quantified by real-time PCR. The data are expressed as a fold increase above control (sham operation) normalized GAPDH. Mean of data from six animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus sham-operated control. (D) Levels of 8-OHdG. Mean of data from ten animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05 versus control.</p

Morphological damage of neurons in cerebral cortices from septic mice.

<p>Images from hematoxylin and eosin-stained sections represent cerebral cortices from sham-operated control (A) and from CLP septic mice (B). Tissues were harvested 24 h after surgery. Bottom shows high-magnification images. Arrows indicate densely-stained cells. Shown are representative micrographs from three independent experiments in which the same results were obtained.</p

Sepsis-induced increase in iNOS protein expression in brain tissues.

<p>(A) Time course of changes in cerebral expression of iNOS protein after CLP. β-Actin served as a loading control. The result is representative of two independent experiments. (B) Comparison of iNOS protein expression between sham-operated and CLP septic groups at 24 h after surgery. The summarizing data are presented as iNOS/GAPDH expressed relative to the unoperated control. Mean of data from three animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05.</p

Effect of edaravone treatment on neuronal degeneration in hippocampus after sepsis.

<p>In the vehicle-treated CLP septic group, there was serious neuronal degeneration in hippocampus (B) compared with sham control (A). In the edaravone-treated CLP septic group, normal-appearing neuronal findings were obtained in hippocampus (C). Tissues were harvested 24 h after surgery. Magnified view is shown in each inset. Shown are representative micrographs from two independent experiments in which the same results were obtained.</p

Sepsis-induced up-regulation of proinflammatory cytokine expression in brain and lung tissues.

<p>Tissues were harvested 12 and 24 h after CLP. In (A) and (C), the mRNA levels for TNF-α, IL-1β, and IL-6 were quantified by real-time PCR. The data are expressed as a fold increase above control (sham operation) normalized GAPDH. Mean of data from 3–5 animals in each group is presented, with SEM by vertical lines. *<i>p</i><0.05, **<i>p</i><0.01 versus sham-operated control. (B) Western blots of TNF-α, IL-1β, and IL-6. GAPDH served as loading control. Representative images from two separate experiments are shown.</p

Morphological damage of neurons in hippocampal areas from septic mice.

<p>Images from hematoxylin and eosin-stained sections represent hippocampal areas from sham-operated control (A) and from CLP septic mice (B). Tissues were harvested 24 h after surgery. Bottom shows high-magnification images. Arrows indicate densely-stained cells. Representative micrographs from two independent experiments are shown.</p