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Effects of ketamine on neurogenesis, extracellular matrix homeostasis and proliferation in hypoxia-exposed HT22 murine hippocampal neurons.

ABSTRACT: Ketamine is a widely used drug in pediatric anesthesia, and both neurotoxic and neuroprotective effects have been associated with its use. There are only a few studies to date which have examined the effects of ketamine on neurons under hypoxic conditions, which may lead to severe brain damage and poor neurocognitive outcomes in neonates. In the present study, the effects of ketamine on cellular pathways associated with neurogenesis, extracellular matrix homeostasis and proliferation were examined in vitro in hypoxia-exposed neurons. Differentiated HT22 murine hippocampal neurons were treated with 1, 10 and 20 µM ketamine and cultured under hypoxic or normoxic conditions for 24 h followed by quantitative PCR analysis of relevant candidate genes. Ketamine treatment did not exert any notable effects on the mRNA expression levels of markers of neurogenesis (neuronal growth factor and syndecan 1), extracellular matrix homeostasis (matrix-metalloproteinase 2 and 9, tenascin C and tenascin R) or proliferation markers (Ki67 and proliferating cell nuclear antigen) compared with the respective untreated controls. However, there was a tendency towards downregulation of multiple cellular markers under hypoxic conditions and simultaneous ketamine treatment. No dose-dependent association was found in the ketamine treated groups for genetic markers of neurogenesis, extracellular matrix homeostasis or proliferation. Based on the results, ketamine may have increased the vulnerability of hippocampal neurons in vitro to hypoxia, independent of the dose. The results of the present study contribute to the ongoing discussion on the safety concerns around ketamine use in pediatric clinical practice from a laboratory perspective.

SUBMITTER: Pichl T

PROVIDER: S-EPMC7403805 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Effects of ketamine on neurogenesis, extracellular matrix homeostasis and proliferation in hypoxia-exposed HT22 murine hippocampal neurons.

Pichl Thomas T Keller Titus T Hünseler Christoph C Roth Bernhard B Janoschek Ruth R Appel Sarah S Hucklenbruch-Rother Eva E

Biomedical reports 20200717 4

Ketamine is a widely used drug in pediatric anesthesia, and both neurotoxic and neuroprotective effects have been associated with its use. There are only a few studies to date which have examined the effects of ketamine on neurons under hypoxic conditions, which may lead to severe brain damage and poor neurocognitive outcomes in neonates. In the present study, the effects of ketamine on cellular pathways associated with neurogenesis, extracellular matrix homeostasis and proliferation were examin ...[more]

PMID: 32765862

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Project description:BACKGROUND:Epigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression. METHODS:HT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus. RESULTS:Methylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p?=?0.023). CONCLUSIONS:We demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.

Project description:Mood instability, a subjective emotional state defined as rapid mood oscillations of up and down, is a symptom that occurs in several psychiatric disorders, particularly major depressive disorder and bipolar disorder. Heat shock protein A12A (HSPA12A) shows decreased expression in the brains of schizophrenia patients. However, the causal effects of HSPA12A in any psychiatric disorders are completely unknown. To investigate whether HSPA12A affects mood stability, Hspa12a-knockout mice (Hspa12a-/-) and wild-type (WT) littermates were subjected to tests of open field, forced swimming, elevated plus maze, and sucrose preference. Cerebral lactate levels were measured in cerebrospinal fluid (CSF). Adult hippocampal neurogenesis (AHN) was assessed by BrdU labeling. We found that acute mood stress increased hippocampal HSPA12A expression and CSF lactate levels in mice. However, Hspa12a-/- mice exhibited behaviors of mood instability (anhedonia, lower locomotor activity, antidepression, and anxiety), which were accompanied by impaired AHN, decreased CSF lactate levels, and downregulated hippocampal glycolytic enzyme expression. By contrast, HSPA12A overexpression increased lactate production and glycolytic enzyme expression of primary hippocampal neurons. Intriguingly, lactate administration alleviated the mood instability and AHN impairment in Hspa12a-/- mice. Further analyses revealed that HSPA12A was necessary for sustaining cerebral lactate homeostasis, which could be mediated by inhibiting GSK3β in hippocampal neurons, to maintain AHN and mood stabilization. Taken together, HSPA12A is defined as a novel regulator of mood stability and exerts therapeutic potential for mood disorder. Our findings establish a framework for determining mood disorder and AHN relevance of cerebral lactate homeostasis. HSPA12A is a novel mood stabilizer through inhibiting GSK3β in hippocampal neurons, thereby sustaining glycolysis-generated lactate to maintain cerebral lactate homeostasis, which ultimately leading to maintenance of hippocampal neurogenesis and mood stabilization.

Dataset Information

Effects of ketamine on neurogenesis, extracellular matrix homeostasis and proliferation in hypoxia-exposed HT22 murine hippocampal neurons.

Publications

Effects of ketamine on neurogenesis, extracellular matrix homeostasis and proliferation in hypoxia-exposed HT22 murine hippocampal neurons.

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