Transcriptome Analysis of Gene Expression Provides New Insights into the Effect of Mild Therapeutic Hypothermia on Primary Human Cortical Astrocytes Cultured under Hypoxia

Abstract

Hypothermia is increasingly used as a therapeutic measure to treat brain injury. However, the cellular mechanisms underpinning its actions are complex and are not yet fully elucidated. Astrocytes are the most abundant cell type in the brain and are likely to play a critical role. In this study, transcriptional changes and the protein expression profile of human primary cortical astrocytes cultured under hypoxic conditions for 6 h were investigated. Cells were treated either with or without a mild hypothermic intervention 2 h post-insult to mimic the treatment of patients following traumatic brain injury (TBI) and/or stroke. Using human gene expression microarrays, 411 differentially expressed genes were identified following hypothermic treatment of astrocytes following a 2 h hypoxic insult. KEGG pathway analysis indicated that these genes were mainly enriched in the Wnt and p53 signaling pathways, which were inhibited following hypothermic intervention. The expression levels of 168 genes involved in Wnt signaling were validated by quantitative real-time-PCR (qPCR). Among these genes, 10 were up-regulated and 32 were down-regulated with the remainder unchanged. Two of the differentially expressed genes (DEGs), p38 and JNK, were selected for validation at the protein level using cell based ELISA. Hypothermic intervention significantly down-regulated total protein levels for the gene products of p38 and JNK. Moreover, hypothermia significantly up-regulated the phosphorylated (activated) forms of JNK protein, while downregulating phosphorylation of p38 protein. Within the p53 signaling pathway, 35 human apoptosis-related proteins closely associated with Wnt signaling were investigated using a Proteome Profiling Array. Hypothermic intervention significantly down-regulated 18 proteins, while upregulating one protein, survivin. Hypothermia is a complex intervention; this study provides the first detailed longitudinal investigation at the transcript and protein expression levels of the molecular effects of therapeutic hypothermic intervention on hypoxic human primary cortical astrocytes. The identified genes and proteins are targets for detailed functional studies, which may help to develop new treatments for brain injury based on an in-depth mechanistic understanding of the astrocytic response to hypoxia and/or hypothermia.

Infusion of Plasma from Exercised Mice Ameliorates Cognitive Dysfunction by Increasing Hippocampal Neuroplasticity and Mitochondrial Functions in 3xTg-AD Mice

“…Increasing evidence suggests that AD pathogenesis is not restricted to the neuronal compartment. Glial cells, particularly reactive astrocytes and activated microglia, appear to play critical and interactive roles in neurodegeneration [22,23]. Accumulating evidence of their role in brain energy metabolism and reduced oxygen supply to the brain clearly point to their critical involvement in the prevention, initiation, and progression of neurodegenerative diseases, including AD [10,24].…”

IL-6 Trans-Signaling in the Brain Influences the Metabolic Phenotype of the 3xTg-AD Mouse Model of Alzheimer’s Disease

“…Glial cells and neuroinflammation may have a critical role in the prevention, but also initiation and progression of AD [54] interfering in multiple brain functions as energy metabolism [3]. They are involved in the maintenance of the CNS homeostasis and protection from various stresses (i.e., hypoxia) by providing metabolic and structural support [55]. Regarding the role of IL-6 trans-signaling, previous studies demonstrated decreased neuroinflammation, including IL-6 production, in response to peripheral LPS if sgp130 is administered icv [56]; and using the same GFAP-sgp130Fc, a prominent down-regulation of the neuroinflammation caused by the transgenic expression of IL-6 was observed [24].…”

Whole Blood Gene Expression Reveals Specific Transcriptome Changes in Neonatal Encephalopathy

“…Therefore, we acknowledge that hypothermia may have contributed to the gene expression patterns. Although we cannot completely exclude that some of the differentially expressed genes were stimulated by hypothermia, preclinical evidence has shown that a reduction by 4–10°C for over 6 h was required to influence gene expression in animal models [28-32]. Moreover, none of the hypothermia-inducible genes were found to be significantly differently expressed between neonatal encephalopathy and control babies [33].…”