Accelerated stem cell labeling with ferucarbotran and protamine

To develop and characterize a clinically applicable, fast and efficient method for stem cell labeling with ferucarbotran and protamine for depiction with clinical MRI. The hydrodynamic diameter, zeta potential and relaxivities of ferucarbotran and varying concentrations of protamine were measured. Once the optimized ratio was found, human mesenchymal stem cells (MSCs) were labeled at varying incubation times (1–24 h). Viability was assessed via Trypan blue exclusion testing. 150,000 labeled cells in Ficoll solution were imaged with T1-, T2- and T2*-weighted sequences at 3 T, and relaxation rates were calculated. Varying the concentrations of protamine allows for easy modification of the physicochemical properties. Simple incubation with ferucarbotran alone resulted in efficient labeling after 24 h of incubation while assisted labeling with protamine resulted in similar results after only 1 h. Cell viability remained unaffected. R2 and R2* relaxation rates were drastically increased. Electron microscopy confirmed intracellular iron oxide uptake in lysosomes. Relaxation times correlated with results from ICP-AES. Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent

Transient Chemogenetic Inhibition of D1-MSNs in the Dorsal Striatum Enhances Methamphetamine Self-Administration

The dorsal striatum is important for the development of drug addiction; however, the role of dopamine D1 receptor (D1R) expressing medium-sized spiny striatonigral (direct pathway) neurons (D1-MSNs) in regulating excessive methamphetamine intake remains elusive. Here we seek to determine if modulating D1-MSNs in the dorsal striatum alters methamphetamine self-administration in animals that have demonstrated escalation of self-administration. A viral vector-mediated approach was used to induce expression of the inhibitory (Gi coupled-hM4D) or stimulatory (Gs coupled-rM3D) designer receptors exclusively activated by designer drugs (DREADDs) engineered to specifically respond to the exogenous ligand clozapine-N-oxide (CNO) selectively in D1-MSNs in the dorsal striatum. CNO in animals expressing hM4D increased responding for methamphetamine compared to vehicle in a within subject treatment paradigm. CNO in animals that did not express DREADDs (DREADD naïve-CNO) or expressed rM3D did not alter responding for methamphetamine, demonstrating specificity for hM4D-CNO interaction in increasing self-administration. Postmortem tissue analysis reveals that hM4D-CNO animals had reduced Fos immunoreactivity in the dorsal striatum compared to rM3D-CNO animals and DREADD naïve-CNO animals. Cellular mechanisms in the dorsal striatum in hM4D-CNO animals reveal enhanced expression of D1R and Ca2+/calmodulin-dependent kinase II (CaMKII). Conversely, rM3D-CNO animals had enhanced activity of extracellular signal-regulated kinase (Erk1/2) and Akt in the dorsal striatum, supporting rM3D-CNO interaction in these animals compared with drug naïve controls, DREADD naïve-CNO and hM4D-CNO animals. Our studies indicate that transient inhibition of D1-MSNs-mediated strengthening of methamphetamine addiction-like behavior is associated with cellular adaptations that support dysfunctional dopamine signaling in the dorsal striatum

Sex Differences in Context-Driven Reinstatement of Methamphetamine Seeking is Associated with Distinct Neuroadaptations in the Dentate Gyrus

The present study examined differences in operant responses in adult male and female rats during distinct phases of addiction. Males and females demonstrated escalation in methamphetamine (0.05 mg/kg, i.v.) intake with females showing enhanced latency to escalate, and bingeing. Following protracted abstinence, females show reduced responses during extinction, and have greater latency to extinguish compared with males, indicating reduced craving. Females demonstrated lower context-driven reinstatement compared to males, indicating that females have less motivational significance to the context associated with methamphetamine. Whole-cell patch-clamp recordings on dentate gyrus (DG) granule cell neurons (GCNs) were performed in acute brain slices from controls and methamphetamine experienced male and female rats, and neuronal excitability was evaluated from GCNs. Reinstatement of methamphetamine seeking reduced spiking in males, and increased spiking in females compared to controls, demonstrating distinct neuroadaptations in intrinsic excitability of GCNs in males and females. Reduced excitability of GCNs in males was associated with enhanced levels of neural progenitor cells, expression of plasticity-related proteins including CaMKII, and choline acetyltransferase in the DG. Enhanced excitability in females was associated with an increased GluN2A/2B ratio, indicating changes in postsynaptic GluN subunit composition in the DG. Altered intrinsic excitability of GCNs was associated with reduced mossy fiber terminals in the hilus and pyramidal projections, demonstrating compromised neuroplasticity in the DG in both sexes. The alterations in excitability, plasticity-related proteins, and mossy fiber density were correlated with enhanced activation of microglial cells in the hilus, indicating neuroimmune responses in both sexes. Together, the present results indicate sexually dimorphic adaptive biochemical changes in excitatory neurotransmitter systems in the DG and highlight the importance of including sex as a biological variable in exploring neuroplasticity and neuroimmune changes that predict enhanced relapse to methamphetamine-seeking behaviors

Long-acting β2-agonists in asthma: enantioselective safety studies are needed

Long-acting β2-agonists (LABAs) such as formoterol and salmeterol are used for prolonged bronchodilatation in asthma, usually in combination with inhaled corticosteroids (ICSs). Unexplained paradoxical asthma exacerbations and deaths have been associated with LABAs, particularly when used without ICS. LABAs clearly demonstrate effective bronchodilatation and steroid-sparing activity, but long-term treatment can lead to tolerance of their bronchodilator effects. There are also concerns with regard to the effects of LABAs on bronchial hyperresponsiveness (BHR), where long-term use is associated with increased BHR and loss of bronchoprotection. A complicating factor is that formoterol and salmeterol are both chiral compounds, usually administered as 50:50 racemic (rac-) mixtures of two enantiomers. The chiral nature of these compounds has been largely forgotten in the debate regarding LABA safety and effects on BHR, particularly that (S)-enantiomers of β2-agonists may be deleterious to asthma control. LABAs display enantioselective pharmacokinetics and pharmacodynamics. Biological plausibility of the deleterious effects of β2-agonists (S)-enantiomers is provided by in vitro and in vivo studies from the short-acting β2-agonist (SABA) salbutamol. Supportive clinical findings include the fact that patients in emergency departments who demonstrate a blunted response to salbutamol are more likely to benefit from (R)-salbutamol than rac-salbutamol, and resistance to salbutamol appears to be a contributory mechanism in rapid asthma deaths. More effort should therefore be applied to investigating potential enantiospecific effects of LABAs on safety, specifically bronchoprotection. Safety studies directly assessing the effects of LABA (S)-enantiomers on BHR are long overdue