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The role of TNF-α in regulating ketamine-induced hippocampal neurotoxicity.

Zheng X, Zhou J, Xia Y - Arch Med Sci (2015)

Bottom Line: In in vivo experiments, genetically silencing TNF-α markedly improved the ketamine-induced memory impairment through Morris water maze test.Our results clearly demonstrated a protective mechanism of down-regulating TNF in ketamine-induced hippocampal neurotoxicity.This study may present a new target for pharmacological intervention to prevent anesthesia-related neurodegeneration in brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia, Zhejiang Hospital, Hangzhou, Zhejiang, China.

ABSTRACT

Introduction: Ketamine is commonly used in pediatric anesthesia but recent studies have shown that it could induce neurotoxicity in the developing brain. The inflammatory cytokine, tumor necrosis factor α (TNF-α) is involved in the pathogenesis of various types of neurodegenerations. In the present study, we examined whether TNF-α may regulate ketamine-induced neurotoxicity in the hippocampus of neonatal mouse.

Material and methods: The in vitro organotypic culture of hippocampal slices was used to investigate the gain-of-function and loss-of-function effect of TNF-α modulation on ketamine-induced hippocampal neurotoxicity. Also, western blotting analysis was used to examine the relative pathways associated with TNF-α modulation. In the in vivo Morris water maze test, TNF-α was genetically silenced to see if memory function was improved after anesthesia-induced memory impairment.

Results: In in vitro experiments, adding TNF-α enhanced (112.99 ±5.4%, p = 0.015), whereas knocking down TNF-α ameliorated (46.8 ±11.6%, p = 0.003) ketamine-induced apoptosis in hippocampal CA1 neurons in the organotypic culture. Western blotting showed that addition of TNF-α reduced (67.1 ±3.7%, p = 0.022), whereas downregulation of TNF-α increased (126.87 ±8.5%, p = 0.004) the phosphorylation of PKC-ERK pathway in ketamine-treated hippocampus. In in vivo experiments, genetically silencing TNF-α markedly improved the ketamine-induced memory impairment through Morris water maze test.

Conclusions: Our results clearly demonstrated a protective mechanism of down-regulating TNF in ketamine-induced hippocampal neurotoxicity. This study may present a new target for pharmacological intervention to prevent anesthesia-related neurodegeneration in brain.

No MeSH data available.


Related in: MedlinePlus

TNF-α regulated ketamine-induced neurotoxicity in vitro. A – The hippocampal cultures were cultured with TNF-α siRNA (100 μM) and nonspecific scrambled siRNA (100 μM) for 24 h. The efficacy of TNF-α siRNA was then verified by western blotting. B – Representative images of TUNEL-positive CA1 neurons of organotypic hippocampal slice cultures that were treated with normal medium (control), 4 h of 0.5 mM ketamine (ketamine) only, 4 h of 0.5 mM ketamine plus 50 ng/ml TNF-α treatment (ketamine + TNF-α), or 4 h of 0.5 mM ketamine plus 100 nM TNF-α SiRNA treatment (ketamine + TNF-α siRNA). C – Quantitative measurements showed that adding TNF-α significantly increased the number of TUNEL-positive CA1 neurons whereas silencing TNF-α significantly reduced the number of TUNEL-positive CA1 neurons. *P < 0.05, as compared to control, ▵p < 0.05, as compared to ketamine treatment (n = 3)
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Figure 0001: TNF-α regulated ketamine-induced neurotoxicity in vitro. A – The hippocampal cultures were cultured with TNF-α siRNA (100 μM) and nonspecific scrambled siRNA (100 μM) for 24 h. The efficacy of TNF-α siRNA was then verified by western blotting. B – Representative images of TUNEL-positive CA1 neurons of organotypic hippocampal slice cultures that were treated with normal medium (control), 4 h of 0.5 mM ketamine (ketamine) only, 4 h of 0.5 mM ketamine plus 50 ng/ml TNF-α treatment (ketamine + TNF-α), or 4 h of 0.5 mM ketamine plus 100 nM TNF-α SiRNA treatment (ketamine + TNF-α siRNA). C – Quantitative measurements showed that adding TNF-α significantly increased the number of TUNEL-positive CA1 neurons whereas silencing TNF-α significantly reduced the number of TUNEL-positive CA1 neurons. *P < 0.05, as compared to control, ▵p < 0.05, as compared to ketamine treatment (n = 3)

Mentions: Ketamine was purchased from Jiangsu Hengrui Medicine Co., Ltd (Jiangsu, China). TNF-α was purchased from Sigma (St. Louis, MO, USA). TNF-α SiRNA was purchased from IDT Inc. (Coralville, IA, USA). To induce neurotoxicity, the hippocampal slices were treated with ketamine (0.5 mM) for 4 h after overnight culture, followed by 3 × 10 min wash at 37°C with 5% CO2. After that, to examine the effect of TNF-α through a gain-of-function paradigm, TNF-α (50 ng/ml) was added into the organotypic hippocampal culture for 24 h. For the loss-of-function paradigm, TNF-α SiRNA (100 nM) was instead introduced into the organotypic hippocampal culture through a gene silencer transfection reagent according to the manufacturer's protocol (GenLentis, CA, USA). The cultures were then maintained for another 24 h before immunohistochemistry and western blotting analysis. The efficacy of TNF-α SiRNA was confirmed by western blotting (Figure 1 A).


The role of TNF-α in regulating ketamine-induced hippocampal neurotoxicity.

Zheng X, Zhou J, Xia Y - Arch Med Sci (2015)

TNF-α regulated ketamine-induced neurotoxicity in vitro. A – The hippocampal cultures were cultured with TNF-α siRNA (100 μM) and nonspecific scrambled siRNA (100 μM) for 24 h. The efficacy of TNF-α siRNA was then verified by western blotting. B – Representative images of TUNEL-positive CA1 neurons of organotypic hippocampal slice cultures that were treated with normal medium (control), 4 h of 0.5 mM ketamine (ketamine) only, 4 h of 0.5 mM ketamine plus 50 ng/ml TNF-α treatment (ketamine + TNF-α), or 4 h of 0.5 mM ketamine plus 100 nM TNF-α SiRNA treatment (ketamine + TNF-α siRNA). C – Quantitative measurements showed that adding TNF-α significantly increased the number of TUNEL-positive CA1 neurons whereas silencing TNF-α significantly reduced the number of TUNEL-positive CA1 neurons. *P < 0.05, as compared to control, ▵p < 0.05, as compared to ketamine treatment (n = 3)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697060&req=5

Figure 0001: TNF-α regulated ketamine-induced neurotoxicity in vitro. A – The hippocampal cultures were cultured with TNF-α siRNA (100 μM) and nonspecific scrambled siRNA (100 μM) for 24 h. The efficacy of TNF-α siRNA was then verified by western blotting. B – Representative images of TUNEL-positive CA1 neurons of organotypic hippocampal slice cultures that were treated with normal medium (control), 4 h of 0.5 mM ketamine (ketamine) only, 4 h of 0.5 mM ketamine plus 50 ng/ml TNF-α treatment (ketamine + TNF-α), or 4 h of 0.5 mM ketamine plus 100 nM TNF-α SiRNA treatment (ketamine + TNF-α siRNA). C – Quantitative measurements showed that adding TNF-α significantly increased the number of TUNEL-positive CA1 neurons whereas silencing TNF-α significantly reduced the number of TUNEL-positive CA1 neurons. *P < 0.05, as compared to control, ▵p < 0.05, as compared to ketamine treatment (n = 3)
Mentions: Ketamine was purchased from Jiangsu Hengrui Medicine Co., Ltd (Jiangsu, China). TNF-α was purchased from Sigma (St. Louis, MO, USA). TNF-α SiRNA was purchased from IDT Inc. (Coralville, IA, USA). To induce neurotoxicity, the hippocampal slices were treated with ketamine (0.5 mM) for 4 h after overnight culture, followed by 3 × 10 min wash at 37°C with 5% CO2. After that, to examine the effect of TNF-α through a gain-of-function paradigm, TNF-α (50 ng/ml) was added into the organotypic hippocampal culture for 24 h. For the loss-of-function paradigm, TNF-α SiRNA (100 nM) was instead introduced into the organotypic hippocampal culture through a gene silencer transfection reagent according to the manufacturer's protocol (GenLentis, CA, USA). The cultures were then maintained for another 24 h before immunohistochemistry and western blotting analysis. The efficacy of TNF-α SiRNA was confirmed by western blotting (Figure 1 A).

Bottom Line: In in vivo experiments, genetically silencing TNF-α markedly improved the ketamine-induced memory impairment through Morris water maze test.Our results clearly demonstrated a protective mechanism of down-regulating TNF in ketamine-induced hippocampal neurotoxicity.This study may present a new target for pharmacological intervention to prevent anesthesia-related neurodegeneration in brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia, Zhejiang Hospital, Hangzhou, Zhejiang, China.

ABSTRACT

Introduction: Ketamine is commonly used in pediatric anesthesia but recent studies have shown that it could induce neurotoxicity in the developing brain. The inflammatory cytokine, tumor necrosis factor α (TNF-α) is involved in the pathogenesis of various types of neurodegenerations. In the present study, we examined whether TNF-α may regulate ketamine-induced neurotoxicity in the hippocampus of neonatal mouse.

Material and methods: The in vitro organotypic culture of hippocampal slices was used to investigate the gain-of-function and loss-of-function effect of TNF-α modulation on ketamine-induced hippocampal neurotoxicity. Also, western blotting analysis was used to examine the relative pathways associated with TNF-α modulation. In the in vivo Morris water maze test, TNF-α was genetically silenced to see if memory function was improved after anesthesia-induced memory impairment.

Results: In in vitro experiments, adding TNF-α enhanced (112.99 ±5.4%, p = 0.015), whereas knocking down TNF-α ameliorated (46.8 ±11.6%, p = 0.003) ketamine-induced apoptosis in hippocampal CA1 neurons in the organotypic culture. Western blotting showed that addition of TNF-α reduced (67.1 ±3.7%, p = 0.022), whereas downregulation of TNF-α increased (126.87 ±8.5%, p = 0.004) the phosphorylation of PKC-ERK pathway in ketamine-treated hippocampus. In in vivo experiments, genetically silencing TNF-α markedly improved the ketamine-induced memory impairment through Morris water maze test.

Conclusions: Our results clearly demonstrated a protective mechanism of down-regulating TNF in ketamine-induced hippocampal neurotoxicity. This study may present a new target for pharmacological intervention to prevent anesthesia-related neurodegeneration in brain.

No MeSH data available.


Related in: MedlinePlus