Evaluation of neuronal apoptosis precursors in citosolic, mitochondrial and nuclear fraction of cerebral cortex and hipocamppus in pigs after acute normovolemic hemodilution

Author(s)

Fabrício de Oliveira Frazilio

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2012-01-27

Examining board members

Denise Tabacchi Fantoni; Aline Magalhães Ambrósio; Filomena Regina Barbosa Gomes Galas; Denise Aya Otsuki; Edmar Zanoteli

• Advisor: Denise Tabacchi Fantoni

Abstract

Background: Acute anemia has been associated with neurophysiologic and cognitive dysfunctions in healthy patients. Experimental evidences suggest that hemodilution may increase cerebral lesions, limiting oxygen supply to the brain tissue. Nevertheless, the exact mechanisms through which cerebral lesions occur in anemic patients havent been clearly defined. Therefore, the objective of the present study was to evaluate neuronal apoptosis precursors Bax, Bcl-x in the frontal cortex, caspase 3 and 9 activity in the mitochondrial and cytosolic fractions of the hippocampus, even as DNA fragmentation in the mitochondrial and nuclear fractions of the frontal cortex after acute normovolemic hemodilution. Methods: Twenty four pigs were anesthetized and randomized into 4 groups of 6 animals: sham, acute normovolemic hemodilution (ANH) to reach a hematocrit of 15% (Ht 15%), ANH to reach a hematocrit of 10% (Ht 10%) and hypoxic-hipoxia (HH). ANH was performed with 1ml hydroxyethyl starch 130/0.4 (HES) per ml of blood withdrawn to the desired target hematocrit (10 or 15%). HH consisted of ventilation with low fraction of inspired oxygen (FiO2) of 6% for 60 minutes, serving as a positive control group. Sham animals were not involved in any of these interventions. Pro-apoptotic Bax and anti-apoptotic Bcl-x proteins were evaluated by Western blotting in nuclear and mitochondrial fractions of the frontal cortex and activities of caspases-3 and-9 were evaluated in the mitochondrial and cytosolic fractions of the hippocampus by spectrofluorometry. DNA fragmentation was evaluated by electrophoresis in the mitochondrial and nuclear fraction. Data were compared by analysis of variance (ANOVA) followed by Tukeys test (p<0.05). Results: No statistical significance was found among sham, Ht 15% or Ht 10% groups regarding pro-apoptotic protein Bax, in nuclear or mitochondrial fractions. However, group HH presented significant difference from sham and Ht 15% groups in the nuclear fraction and from all groups in the mitochondrial fraction. No statistical significance was found with Bcl-x. The activities of caspases-3 and-9 in cytosolic and mitochondrial fractions were statisticaly different in group HH when compared with all other groups. No statistical significance was found in relation to DNA fragmentation among sham, Ht 15% or Ht 10%. Conclusion: The evaluation of apoptosis precursors demonstrated that ANH with target hematocrit 15% and 10% did not induce neuronal lesion, suggesting that cerebral oxygenation was preserved (AU)