Potential mechanism of cell death in the developing rat brain induced by propofol anesthesia

Abstract

Commonly used general anesthetics can have adverse effects on the developing brain by triggering apoptotic neurodegeneration, as has been documented in the rat. The rational of our study was to examine the molecular mechanisms that contribute to the apoptotic action of propofol anesthesia in the brain of 7-day-old (P7) rats. The down-regulation of nerve growth factor (NGF) mRNA and protein expression in the cortex and thalamus at defined time points between 1 and 24h after the propofol treatment, as well as a decrease of phosphorylated Akt were observed. The extrinsic apoptotic pathway was induced by over-expression of tumor necrosis factor (TNF) which led to the activation of caspase-3 in both examined structures. Neurodegeneration was confirmed by Fluoro-Jade B staining. Our findings provide direct experimental evidence that the anesthetic dose (25mg/kg) of propofol induces complex changes that are accompanied by cell death in the cortex and thalamus of the developing rat brain.

Fig. 1

Time-dependent changes in NGF mRNA expression in the cortex (A) and thalamus (B) of P7 rats after the propofol treatment, as revealed by real-time RT-PCR. A significant decrease of NGF mRNA expression was observed in the cortex (*p < 0.05 vs. control).

Fig. 2

Time-dependent changes in mature NGF expression in the cortex (A) and thalamus (B) of P7 rats after the propofol treatment, as revealed by Western blotting. Each graph is accompanied by a representative immunoblot. A significant decrease of mature NGF expression was observed in both structures (*p < 0.05 vs. control).

Fig. 3

Time-dependent changes in phosphorylated Akt expression in the cortex (A) and thalamus (B) of P7 rats after propofol treatment, as revealed by Western blotting. Each graph is accompanied by a representative immunoblot. A significant decrease of phosphorylated Akt expression was observed in both structures (*p < 0.05 vs. control).

Fig. 4

Time-dependent changes in TNFα mRNA and protein expression in the cortex and thalamus of P7 rats after the propofol treatment. A significant increase in TNFα mRNA expression, revealed by RT-PCR, was observed at similar times in the cortex (A) and in the thalamus (B) (*p < 0.05 vs. control). The levels of TNFα protein, as revealed by Western blotting, were significantly increased between 4 and 24 h after the treatment in the cortex (C) and between 2 and 24 h after the treatment in the thalamus (D) (*p < 0.05 vs. control).

Fig. 5

Time-dependent changes in caspase-8 mRNA expression in the cortex (A) and thalamus (B) of P7 rats after the propofol treatment, as revealed by real-time RT-PCR. A significant increase in caspase-8 mRNA expression was detected between 8 and 24 h after the treatment (*p < 0.05 vs. control).

Fig. 6

Time-dependent changes in activated caspase-3 expression in the cortex (A) and thalamus (B) after the propofol treatment, as revealed by Western blotting. The expression of cleaved caspase-3 protein was significantly increased in both structures after the propofol treatment (*p < 0.05 vs. control).

Fig. 7

Fluoro-Jade B staining of frozen brain sections. Controls (saline-injected rats killed 24 h after the injection) had a few Fluoro-Jade B positive cells in the posterior cingulate/retrosplenial cortex (A) and laterodorsal thalamus (B). The densest labeling was found in the posterior cingulate/retrosplenial cortex (C) and the laterodorsal thalamus (D) of animals that were treated with propofol (25 mg/kg) and killed 24 h post-injection. Rats treated with two i.p. doses (0.5 mg/kg) of (+) MK-801 and killed 24 h after the first administration of the drug served as a positive control (E – posterior cingulate/retrosplenial cortex; F – laterodorsal thalamus). Degenerating neurons are marked with arrows and the blood vessels with arrowheads. Normal neurons appear darker than the background. Scale bar – 20 μm.