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. 2014 Sep 25;9(9):e108366.
doi: 10.1371/journal.pone.0108366. eCollection 2014.

Evaluation of neuronal apoptosis precursors in an experimental model of acute normovolemic hemodilution

Fabrício O Frazilio  1 , Denise Aya Otsuki  1 , Jessica Noel-Morgan  1 , Jessica Ruivo Maximino  2 , Gabriela Pintar Oliveira  2 , Gerson Chadi  2 , Jose Otavio Costa Auler Jr  1 , Denise Tabacchi Fantoni  1
Affiliations

Evaluation of neuronal apoptosis precursors in an experimental model of acute normovolemic hemodilution

Fabrício O Frazilio et al. PLoS One. .

Abstract

Background: The effects of acute anemia on neuronal cells and the safe limits of hematocrit are not well established. The objective of this study was to evaluate neuronal pro- and anti-apoptotic Bax and Bcl-x proteins, caspase-3 and -9 activity, and DNA fragmentation after acute normovolemic hemodilution (ANH).

Methods: Twenty-four pigs were anesthetized and randomized into 4 groups: Sham, ANH to 15% hematocrit (ANH15%), ANH to 10% hematocrit (ANH10%) and hypoxia (Hx). ANH was achieved by simultaneous blood withdrawal and hydroxyethyl starch infusion. Hx consisted of ventilation with a 6% inspired oxygen fraction for 60 minutes. Bax and Bcl-x proteins as well as DNA fragmentation were evaluated in cortical nuclear and mitochondrial fractions. Caspase-3 and -9 activity was evaluated in the cortical mitochondrial and hippocampal cytosolic fractions. The data were compared using analysis of variance followed by Tukey's test (P<0.05).

Results: No changes were observed in Bax protein expression after hemodilution in the ANH15% and ANH10% groups compared to the Sham group. Bax expression in the Hx group was increased in the nuclear and mitochondrial fractions compared to all other groups. No significant difference was observed in Bcl-x expression. Caspase-3 and -9 activity in the cytosolic and mitochondrial fractions was different in the Hx group compared to all other groups. No statistical significance in DNA fragmentation was found among the Sham, ANH15% or ANH10% groups.

Conclusion: ANH to 10 and 15% hematocrit did not induce alterations in apoptosis precursors, suggesting that cerebral oxygenation was preserved during these anemic states.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Western blot analysis.
The relative optical density of the pro-apoptotic protein Bax in the total protein extraction (A) and the neuronal nuclear (B) and mitochondrial (C) fractions from the cerebral frontal cortex of pigs in the sham, 10% hematocrit (Ht10%), 15% hematocrit (Ht15%) and hypoxia groups (n = 7 per group). β-Tubulin III was used as sample loading control. The representative bands of the Bax (23 kDa) and β-Tubulin III (50 kDa) of animals are illustrated. One-way analysis of variance (ANOVA) was applied to compare the groups. Data are presented as the means ± SEM. *p<0.05.
Figure 2
Figure 2. Western blot analysis.
The relative optical density of the anti-apoptotic protein Bcl-x in the total protein extraction (A) and the neuronal nuclear (B) and mitochondrial (C) fractions from the cerebral frontal cortex of pigs in the sham, 10% hematocrit (Ht10%), 15% hematocrit (Ht15%) and hypoxia groups (n = 7 per group). β-Tubulin III was used as sample loading control. The representative bands of the Bcl-x (26 kDa) and β-Tubulin III (50 kDa) of animals are illustrated. One-way analysis of variance (ANOVA) was applied to compare the groups. Data are presented as the means ± SEM.
Figure 3
Figure 3. Western blot analysis.
The relative optical density of NeuN protein 46 kDa (A) and 48 kDa (B) in the total protein extraction from the cerebral frontal cortex of pigs in the sham, 10% hematocrit (Ht10%), 15% hematocrit (Ht15%) and hypoxia groups (n = 7 per group). β-Tubulin III (50 kDa) was used as sample loading control. The representative bands of the NeuN and β-Tubulin III of animals are illustrated. One-way analysis of variance (ANOVA) was applied to compare the groups. Data are presented as the means ± SEM. *p<0.05.
Figure 4
Figure 4. Activity in the cytosolic fraction of caspase 3 and 9 in the cytosolic fraction of the hippocampus (A, B) and the mitochondrial fraction from the cerebral frontal cortex (C, D) of pigs in the sham, 10% hematocrit (Ht10%), 15% hematocrit (Ht15%) and hypoxia groups (n = 7 per group).
Data are presented as the means ± SEM. *p<0.05.
Figure 5
Figure 5. Representative gel electrophoresis and quantitative density images of nuclear (A) and mitochondrial (B) DNA fragmentation in the cerebral frontal cortex of pigs in the sham, 10% hematocrit (Ht10%), 15% hematocrit (Ht15%) and hypoxia groups (n = 4 per group).
Data are presented as the means ± SEM. *p<0.05.
See this image and copyright information in PMC

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