J Korean Neurosurg Soc.  2024 Jul;67(4):418-430. 10.3340/jkns.2023.0208.

Potential Risk of Choline Alfoscerate on Isoflurane-Induced Toxicity in Primary Human Astrocytes

• Lee HJ
• Cho HR
• Bang M
• Lee YS
• Kim YJ
• Chong K

• KMID: 2556739
• DOI: http://doi.org/10.3340/jkns.2023.0208

Abstract

Objective
: Isoflurane, a widely used common inhalational anesthetic agent, can induce brain toxicity. The challenge lies in protecting neurologically compromised patients from neurotoxic anesthetics. Choline alfoscerate (L-α-Glycerophosphorylcholine, α-GPC) is recognized for its neuroprotective properties against oxidative stress and inflammation, but its optimal therapeutic window and indications are still under investigation. This study explores the impact of α-GPC on human astrocytes, the most abundant cells in the brain that protect against oxidative stress, under isoflurane exposure.
Methods
: This study was designed to examine changes in factors related to isoflurane-induced toxicity following α-GPC administration. Primary human astrocytes were pretreated with varying doses of α-GPC (ranging from 0.1 to 10.0 μM) for 24 hours prior to 2.5% isoflurane exposure. In vitro analysis of cell morphology, water-soluble tetrazolium salt-1 assay, quantitative real-time polymerase chain reaction, proteome profiler array, and transcriptome sequencing were conducted.
Results
: A significant morphological damage to human astrocytes was observed in the group that had been pretreated with 10.0 mM of α-GPC and exposed to 2.5% isoflurane. A decrease in cell viability was identified in the group pretreated with 10.0 μM of α-GPC and exposed to 2.5% isoflurane compared to the group exposed only to 2.5% isoflurane. Quantitative real-time polymerase chain reaction revealed that mRNA expression of heme-oxygenase 1 and hypoxia-inducible factor-1α, which were reduced by isoflurane, was further suppressed by 10.0 μM α-GPC pretreatment. The proteome profiler array demonstrated that α-GPC pretreatment influenced a variety of factors associated with apoptosis induced by oxidative stress. Additionally, transcriptome sequencing identified pathways significantly related to changes in isoflurane-induced toxicity caused by α-GPC pretreatment.
Conclusion
: The findings suggest that α-GPC pretreatment could potentially enhance the vulnerability of primary human astrocytes to isoflurane-induced toxicity by diminishing the expression of antioxidant factors, potentially leading to amplified cell damage.

Keyword

Choline alfoscerate; Glycerylphosphorylcholine; Isoflurane; Toxicity; Astrocytes

Figure

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  Fig. 1. A : Immunocytochemistry images of cultured primary human astrocytes (×100 magnification; green : Alexa Fluor 488-conjugated secondary antibody; blue : 4',6-diamidino-2-phenylindole). Human fibroblasts were used as a positive control for vimentin and a negative control for GFAP. The results indicate that the cultures were enriched with mature astrocytes. B : Morphological alteration of primary human astrocytes by isoflurane exposure in a dose-dependent manner (×100 magnification). The characteristics of morphological damage are manifested through cell shrinkage and diminished intercellular adherence as observed under a light microscope. The numbers depicted in the image denote the concentration of isoflurane. C : Schematic diagram of experimental protocol designed to evaluate the impact of α-GPC on isoflurane-induced toxicity. GFAP : glial fibrillary acidic protein, α-GPC : L-α-Glycerophosphorylcholine, WST : water-soluble tetrazolium salt, qRT-PCR : quantitative real-time polymerase chain reaction.

• 

  Fig. 2. A : WST-1 cell viability assay results of primary human astrocytes subjected to α-GPC and isoflurane. There was no significant difference in cell viability at any dose of treatment with α-GPC for 48 hours compared to control (n=6 for each, left), but isoflurane exposure reduced cell viability in a dose-dependent manner (n=6 for each, right). B : The effect on cell viability by 0.1, 1.0, 10.0 μM of α-GPC pretreatment and 2.5% isoflurane exposure (n=6 for each). After 24 hours of exposure to 2.5% isoflurane, pretreatment with 10.0 μM of α-GPC significantly reduced cell viability by approximately 20% compared to the group not treated with α-GPC (n=6). C : Reanalyzed results considering the individual and combined effects of 10.0 μM of α-GPC and 2.5% isoflurane. Primary human astrocytes, when pretreated with 10.0 μM of α-GPC and subsequently exposed to 2.5% isoflurane, exhibited a 32% reduction in cell viability compared to the non-treated control group. This decrease was notably more substantial than the 14% reduction observed when cells were only treated with 2.5% isoflurane, compared to the non-treated control group. D : Analysis of survivin mRNA expression in each treatment group using quantitative real-time polymerase chain reaction (qRT-PCR). In comparison to the non-treated control group, all groups treated with either 10 μM of α-GPC, 2.5% isoflurane, or a combination of both, demonstrated a statistically significant decrease in survivin expression (n=3 for each). E : Analysis of heme oxygenase-1 (HO-1) mRNA expression in each treatment group using qRT-PCR (n=3 for each). The groups pre-treated with α-GPC exhibited a significant reduction in HO-1 mRNA expression when compared to the control group. F : Analysis of hypoxia-inducible factor-1α (HIF-1α) mRNA expression in each treatment group using qRT-PCR (n=3 for each). In all groups treated with 2.5% isoflurane, the HIF-1α expression significantly decreased by more than 80%. The results are presented as the means±standard deviations. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. A.U. : arbitrary unit, ns : not statistically significant, α-GPC : L-α-Glycerophosphorylcholine, WST-1 : water-soluble tetrazolium salt.

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  Fig. 3. A : Phosphorylation screening analysis using proteome profiler array. The α-GPC pretreatment was identified to decrease the phosphorylation of heat shock protein 60 (HSP60), signal transducer and activator of transcription3 (STAT3), 90 kDa ribosomal S6 kinase 1/2 (RSK1/2), and p53, while increasing the phosphorylation of epidermal growth factor receptor (EGFR). B : One-way hierarchical clustering heatmap that represents non-treated controls and various treated groups. The heatmap was created using the Z-Score of normalized values from 1321 genes that exhibited a fold change greater than 2 and a statistical significance of p<0.05. C : Count of up- and down-regulated genes exhibiting a fold change greater than 2 and statistical significance of p<0.05, according to group comparisons. The number of up- or down-regulated genes increased depending on the administration of 2.5% isoflurane, compared to the variation in gene expression with the administration of 10 μM of α-GPC. D : Gene-set enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database. The comparative analysis between the ‘isoflurane 2.5%’ group and the ‘α-GPC 10.0 μM + isoflurane 2.5%’ group revealed distinct patterns. The most notable finding was the further increase in the gene ratio for ‘metabolic pathways’, which exhibited the highest gene ratio with statistical significance. α-GPC : L-α-Glycerophosphorylcholine, ECM : extracellular matrix.

Reference

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