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Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors.

Willner D, Cohen-Yeshurun A, Avidan A, Ozersky V, Shohami E, Leker RR - PLoS ONE (2014)

Bottom Line: Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.These effects were reversed with the addition of the opioid antagonist naloxone.Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Chronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved.

Methods: Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry.

Results: Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.

Conclusions: Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

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Morphine decreases proliferation of NPCs and induces the apoptotic enzyme active caspase-3 in a dose dependent manner.NPCs were exposed to increasing doses of morphine. This increase caused a decrease in the number of cells expressing BrdU and an increase in the number of apoptotic cells expressing active caspase-3 (1A). Bar graph showing, in a dose-dependent manner, the decrease in BrdU and increase in active caspase-3 expression in NPCs exposed to a single-dose of morphine (***p<0.001 different morphine doses vs. untreated in BrdU and caspase-3; ###p<0.001 0.13 µM and 1.3 µM vs. 13 µM in caspase-3; +++p<0.001 0.13 µM vs. 13 µM in BrdU) (1B).
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pone-0103043-g001: Morphine decreases proliferation of NPCs and induces the apoptotic enzyme active caspase-3 in a dose dependent manner.NPCs were exposed to increasing doses of morphine. This increase caused a decrease in the number of cells expressing BrdU and an increase in the number of apoptotic cells expressing active caspase-3 (1A). Bar graph showing, in a dose-dependent manner, the decrease in BrdU and increase in active caspase-3 expression in NPCs exposed to a single-dose of morphine (***p<0.001 different morphine doses vs. untreated in BrdU and caspase-3; ###p<0.001 0.13 µM and 1.3 µM vs. 13 µM in caspase-3; +++p<0.001 0.13 µM vs. 13 µM in BrdU) (1B).

Mentions: Fetal mouse cortical cells treated with morphine demonstrated a dose dependent decrease in BrdU incorporation compared with the untreated group (58.7±3.3% of the cells in the untreated cultures incorporated BrdU). Of the cells treated with 0.13 µM morphine, 30.2±3.2% were BrdU- positive and 22.6±3.3% and 7.8±1.8% BrdU- positive cells were observed with 1.3 µM and 13 µM morphine, respectively (p<0.001) (Figure 1A, 1B). In parallel, a significant increase in the levels of the pro-apoptotic enzyme active caspase-3 (Figure 1A, 1B) was detected (8.3±1.2% vs. 26.0±2.0%, 33.7±2.1% and 52.0±3.1% for untreated vs. 0.13 µM, 1.3 µM and 13 µM morphine-treated groups, respectively; p<0.001 for all).


Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors.

Willner D, Cohen-Yeshurun A, Avidan A, Ozersky V, Shohami E, Leker RR - PLoS ONE (2014)

Morphine decreases proliferation of NPCs and induces the apoptotic enzyme active caspase-3 in a dose dependent manner.NPCs were exposed to increasing doses of morphine. This increase caused a decrease in the number of cells expressing BrdU and an increase in the number of apoptotic cells expressing active caspase-3 (1A). Bar graph showing, in a dose-dependent manner, the decrease in BrdU and increase in active caspase-3 expression in NPCs exposed to a single-dose of morphine (***p<0.001 different morphine doses vs. untreated in BrdU and caspase-3; ###p<0.001 0.13 µM and 1.3 µM vs. 13 µM in caspase-3; +++p<0.001 0.13 µM vs. 13 µM in BrdU) (1B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4114742&req=5

pone-0103043-g001: Morphine decreases proliferation of NPCs and induces the apoptotic enzyme active caspase-3 in a dose dependent manner.NPCs were exposed to increasing doses of morphine. This increase caused a decrease in the number of cells expressing BrdU and an increase in the number of apoptotic cells expressing active caspase-3 (1A). Bar graph showing, in a dose-dependent manner, the decrease in BrdU and increase in active caspase-3 expression in NPCs exposed to a single-dose of morphine (***p<0.001 different morphine doses vs. untreated in BrdU and caspase-3; ###p<0.001 0.13 µM and 1.3 µM vs. 13 µM in caspase-3; +++p<0.001 0.13 µM vs. 13 µM in BrdU) (1B).
Mentions: Fetal mouse cortical cells treated with morphine demonstrated a dose dependent decrease in BrdU incorporation compared with the untreated group (58.7±3.3% of the cells in the untreated cultures incorporated BrdU). Of the cells treated with 0.13 µM morphine, 30.2±3.2% were BrdU- positive and 22.6±3.3% and 7.8±1.8% BrdU- positive cells were observed with 1.3 µM and 13 µM morphine, respectively (p<0.001) (Figure 1A, 1B). In parallel, a significant increase in the levels of the pro-apoptotic enzyme active caspase-3 (Figure 1A, 1B) was detected (8.3±1.2% vs. 26.0±2.0%, 33.7±2.1% and 52.0±3.1% for untreated vs. 0.13 µM, 1.3 µM and 13 µM morphine-treated groups, respectively; p<0.001 for all).

Bottom Line: Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.These effects were reversed with the addition of the opioid antagonist naloxone.Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Chronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved.

Methods: Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry.

Results: Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.

Conclusions: Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

Show MeSH
Related in: MedlinePlus