Cell cycle activation contributes to isoflurane-induced neurotoxicity in the developing brain and the protective effect of CR8

Abstract

Aims: It is well established that exposure of common anesthetic isoflurane in early life can induce neuronal apoptosis and long-lasting cognitive deficit, but the underlying mechanisms were not well understood. The cell cycle protein Cyclin B1 plays an important role in the survival of postmitotic neurons. In the present study, we investigated whether cyclin B1-mediated cell cycle activation pathway is a contributing factor in developmental isoflurane neurotoxicity.

Methods: Postnatal day 7 mice were exposed to 1.2% isoflurane for 6 hours. CR8 (a selective inhibitor of cyclin-dependent kinases) was applied before isoflurane treatment. Brain samples were collected 6 hours after discontinuation of isoflurane, for determination of neurodegenerative biomarkers and cell cycle biomarkers.

Results: We found that isoflurane exposure leads to upregulated expression of cell cycle-related biomarkers Cyclin B1, Phospho-CDK1(Thr-161), Phospho-n-myc and downregulated Phospho-CDK1 (Tyr-15). In addition, isoflurane induced increase in Bcl-xL phosphorylation, cytochrome c release, and caspase-3 activation that resulted in neuronal cell death. Systemic administration of CR8 attenuated isoflurane-induced cell cycle activation and neurodegeneration.

Conclusion: These findings suggest the role of cell cycle activation to be a pathophysiological mechanism for isoflurane-induced apoptotic cell death and that treatment with cell cycle inhibitors may provide a possible therapeutic target for prevention of developmental anesthetic neurotoxicity.

Keywords: CR8; apoptosis; cyclin B1; isoflurane.

Figure 1

Isoflurane induces caspase‐3 activation in neonatal mice. (A‐B) Representative immunohistochemistry studies show the expression of cleaved caspase‐3 (brown) positive cells in the brain tissues (scale bar = 500 μm) of postnatal day 7 (P7) mouse pups exposed to either 30% oxygen/air (A) or 1.2% isoflurane (B) for 6 hours. (C) Quantitation of cleaved caspase‐3 immunopositive cells in cerebral cortex and hippocampus demonstrates greater number of cleaved caspase‐3 positive cells in mouse pups receiving isoflurane. (D) Representative immunoblots for cleaved caspase‐3 and the loading control (GAPDH) in animals treated with either 30% oxygen/air or 1.2% isoflurane for 6 hours. (E) Quantitative analysis of western blots shows that cleaved caspase‐3 levels were significantly increased after exposure to isoflurane in the P7 mouse brain. Data presented are mean ± SEM compared with sham (n = 4‐6, ***P < 0.001)

Figure 2

Effects of isoflurane on cell cycle‐related proteins expression. (A) Representative immunoblotting of cell cycle‐related proteins in animals exposed to 1.2% isoflurane for 6 hours. (B‐F) Quantitative analysis of western blots shows the expression of cell cycle‐related proteins cyclin B1(B), CDK1 (C), phospho‐Cdk1(Tyr‐15) (D), phospho‐Cdk1(Thr‐161) (E), and phospho‐n‐myc (F) in the mouse brain after exposure to isoflurane or 30% oxygen/air, normalized to GAPDH levels. Data presented are mean ± SEM compared with sham (n = 4‐6, *P < 0.05, ***P < 0.001)

Figure 3

Systemic administration of CR8 inhibits isoflurane‐induced neuronal cell cycle activation and neuronal apoptosis. (A) Representative immunoblotting of cell cycle and apoptotic proteins in animals exposed to 1.2% isoflurane with or without CR8. (B‐G) Quantitative analysis of western blots shows the effect of CR8 on isoflurane anesthesia induced cyclin B1(B), phospho‐Cdk1(Thr‐161) (C), phospho‐Cdk1(Tyr‐15)(D), cleaved caspase‐3(E), phospho‐Bcl‐x(F), and cytochrome c (G) levels in the mouse brain, normalized to GAPDH levels. Data presented are mean ± SEM (n = 4‐6, *P < 0.05, **P < 0.01, ***P < 0.001 compared with sham; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with Isoflurane)

Figure 4

Isoflurane‐induced cyclin B1 immunostaining positive cells are confined to neurons. Representative immunostaining for cyclin B1 (green), neuronal marker NeuN (red), Merged cyclin B1 and NeuN (yellow) in the brain tissues of postnatal day 7 mouse pups exposed to either 30% oxygen/air (A), 1.2% isoflurane for 6 hours (B), and isoflurane with CR8 treatment (C). The white arrow indicates examples of cyclin B1+/NeuN+/ co‐labeled cells (yellow). (Scale bar = 500 μm)

Figure 5

CR8 inhibits isoflurane‐induced degeneration of neurons in the postnatal mouse brain. (A‐C) Immunofluorescence photomicrographs of Fluoro‐Jade B (green) positive cells in mouse brain exposed to 1.2% isoflurane with or without CR8 (scale bar = 100 μm). (D) Quantitative analysis of neuronal degeneration in the brain as a number of neurons positively stained with Fluoro‐Jade B (degenerating neurons) in sham control, isoflurane, and isoflurane+CR8 groups. Data presented are mean ± SEM (n = 4, ***P < 0.001 compared with sham; ##P < 0.01 compared with Isoflurane)