Neuronal apoptosis induced by morphine withdrawal is mediated by the p75 neurotrophin receptor.

Morphine withdrawal evokes neuronal apoptosis through mechanisms that are still under investigation. We have previously shown that morphine withdrawal increases the levels of pro-brain-derived neurotrophic factor (BDNF), a proneurotrophin that promotes neuronal apoptosis through the binding and activation of the pan-neurotrophin receptor p75 (p75NTR). In this work, we sought to examine whether morphine withdrawal increases p75NTR-driven signaling events. We employed a repeated morphine treatment-withdrawal paradigm in order to investigate biochemical and histological indicators of p75NTR-mediated neuronal apoptosis in mice. We found that repeated cycles of spontaneous morphine withdrawal promote an accumulation of p75NTR in hippocampal synapses. At the same time, TrkB, the receptor that is crucial for BDNF-mediated synaptic plasticity in the hippocampus, was decreased, suggesting that withdrawal alters the neurotrophin receptor environment to favor synaptic remodeling and apoptosis. Indeed, we observed evidence of neuronal apoptosis in the hippocampus, including activation of c-Jun N-terminal kinase (JNK) and increased active caspase-3. These effects were not seen in saline or morphine-treated mice which had not undergone withdrawal. To determine whether p75NTR was necessary in promoting these outcomes, we repeated these experiments in p75NTR heterozygous mice. The lack of one p75NTR allele was sufficient to prevent the increases in phosphorylated JNK and active caspase-3. Our results suggest that p75NTR participates in the neurotoxic and proinflammatory state evoked by morphine withdrawal. Because p75NTR activation negatively influences synaptic repair and promotes cell death, preventing opioid withdrawal is crucial for reducing neurotoxic mechanisms accompanying opioid use disorders.

Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation.

Aimed at providing a contribution to the optimization of cryopreservation processes, the present work focuses on the osmotic behavior of human mesenchymal stem cells (hMSCs). Once isolated from the umbilical cord blood (UCB) of three different donors, hMSCs were characterized in terms of size distribution and their osmotic properties suitably evaluated through the exposure to hypertonic and isotonic aqueous solutions at three different temperatures. More specifically, inactive cell volume and cell permeability to water and di-methyl sulfoxide (DMSO) were measured, being cell size determined using impedance measurements under both equilibrium and dynamic conditions. Experimental findings indicate that positive cell volume excursions are limited by the apparent increase of inactive volume, which occurs during both the shrink-swell process following DMSO addition and the subsequent restoration of isotonic conditions in the presence of hypertonic solutions of impermeant or permeant solutes. Based on this evidence, hMSCs must be regarded as imperfect osmometers, and their osmotic behavior described within a scenario no longer compatible with the simple two-parameter model usually utilized in the literature. In this respect, the activation of mechano-sensitive ion-channels seemingly represents a reasonable hypothesis for rationalizing the observed osmotic behavior of hMSCs from UCB.

Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression.

Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission.