Effects of Propionyl-L-Carnitine on Ischemia–Reperfusion Injury in Hamster Cheek Pouch Microcirculation

Background and purpose Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia–reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. Methods The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (VRBC) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2′-7′-dichlorofluorescein (DCF), respectively. Results In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and VRBC decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. Conclusions pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase

Differentiation of Human iPSCs Into Telencephalic Neurons Using 3D Organoids and Monolayer Culture

Human induced pluripotent stem cells (hiPSCs) are emerging as a useful tool for modelling in vitro early brain development and neurological disorders. Molecular mechanisms and cell interactions that regulate the neurodevelopment at early stages remain unclear because of human brain’s complexity and limitations of functional studies. Two major culture methodologies are used to differentiate in vitro hiPSCs into neurons: monolayer (2D) and organoid (3D) cultures. Here we investigate the effect of cell dissociation and the loss of 3D organization during the early differentiation process of neuronal progenitors. Using the same culture media, we first differentiated hiPSCs into neural progenitor cells (NPCs) and then induced their differentiation into neurons in 3 different modalities: 3D undissociated organoids, dissociated NPCs followed by immediate re-aggregation into an organoid, and dissociated NPCs cultured as monolayer. We assessed neuronal differentiation efficiency of each method by immunocytochemistry, qPCR, western blot, and RNA-Seq analysis over a time course. Our data revealed substantial differences in gene and protein expression among the three systems, including genes of the Notch pathway (e.g. NEUROD1, NEUROG2), earliest determinants of cortical region differentiation (e.g. SOX1, FEZF1) as well as later transcriptional regulators that specify cortical neuron subtypes (e.g. TBR1, CTIP2), which were all downregulated in monolayer. Moreover, we found that genes and pathways mediating cell-to-cell interactions (e.g. CNTNs, CAMs) were mostly upregulated in the 3D culture systems, whereas cell-extracellular matrix interaction molecules (e.g. ITG, LAM) were mostly upregulated in 2D, indicating that cell surface molecules may be involved in specification of neuronal cell types. Our results address the methodological question of the appropriateness of a differentiation method for a particular experimental goal, and, beyond that, reveal important early determinants that exert a decisive influence on neuronal differentiation and regional specification of human neural stem cells.

Calcium/Calmodulin-Dependent Kinases in the Hypothalamus, Pituitary, and Pineal Gland: An Overview

: We review the literature on the little-known roles of specific CaMKs in regulating endocrine functions of the pineal gland, the pituitary gland, and the hypothalamus. Melatonin activates hippocampal CaMKII, which then influences dendritogenesis. In the pituitary gland, the signal pathways activated by the CaMK in lower vertebrates, such as fishes, differ from those of mammals. In the teleost anterior pituitary, the activation of CaMKII induces the expression of somatolactin by glucagon b. In rats and humans, CaMKIVs have been associated with gonadotropes and thyrotropes and CaMKII with several types of human tumor cells and with a specific signaling pathway. Neuropeptides such as vasopressin and endothelin are also involved in the CaMKII signaling chain, as is the CaMKIIδ isoform which participates in generating the circadian rhythms of the suprachiasmatic nucleus. What arises from this review is that most of the hypothalamic CaMKs are involved in activities of the endocrine brain. Furthermore, among the CaMKs, type II occurs with the highest frequency followed by CaMKIV and CaMKI

Analysis of calretinin early expression in the rat hippocampus after beta amyloid (1-42) peptide injection

It has already been reported that cannabinoids are neuroprotective agents against excitotoxicity in vitro and increase after acute brain damage in vivo. This background prompted us to study the localization and expression of the calcium -binding protein calretinin in a condition similar to Alzheimer disease and its possible relationship with cannabinoids and their supposed protective role. We carried out quantitative analysis of the transient changes in calretinin expression shown by hybridochemistry within neuronal cell populations in the hippocampus of a beta amyloid-treated rat model of Alzheimer's disease and their correlation with endocannabinoid increase. Calretinin expression increases throughout the first week after cortical amyloid-beta peptide injection, and then decreases towards normal levels in the rat hippocampus during the following weeks, indicating that decreased calretinin gene expression may be associated with either increase of endocannabinoids or VDM11-induced accumulation of endocannabinoids. In contrast, SR1, an antagonist, which limits the cannabinoid effect by selective binding to the cannabinoid receptor CB1, up-regulates calretinin expression with respect to non-treated rats. This could mean that the SR1 endocannabinoid-blocking action through CB1 receptors, that are normally stimulated by endocannabinoids to inhibit calcium increase, might cause a higher calretinin expression. This would allow us to speculate on a possible reverse relationship between endocannabinoid and calretinin levels in the hippocampal calcium-homeostasis balance

Investigation of integrin expression in cancer stem cells isolated from glioblastoma patients

Glioblastoma multiforme (GBM) is the most common primary brain tumor, which despite combined treatment modalities recurs, and is invariably fatal within a year of diagnosis. The recent characterization of stem cell-like populations in this tumor (glioma stem cells, “GSCs”), shown to be responsible for GBM initiation and progression, has prompted the study of new therapies for GBM to specifically target GSCs. Here we have focused our studies on integrins, which are heterodimeric transmembrane surface proteins that, when activated, have been shown to signal through several GBM-relevant pathways, including proliferation, motility, cytoskeleton organization, survival and angiogenesis pathways. Accordingly, these molecules have attracted a lot of interest since potential effects of anti-integrin strategies in GBM therapeutics might be threefold: anti-angiogenesis, anti-invasion and anti-tumor. Here we investigated integrins’ expression in GSCs, isolated and derived from patients’ biopsies and cultured in monolayer and a serum free medium. The expression of different integrins was examined by PCR, FACS and immunocytochemistry. Our results showed that GSCs express αvβ3, αvβ5 and α5β1 integrins, the expression levels being higher expression than tissue control. Our aim is now to modulate integrin signaling by means of downregulation/upregulation of respectively oncogenic/tumor suppressor miRNAs

Differentiation of human iPSCs into telencephalic neurons using 3D organoids and monolayer culture.

Differentiation of Human iPSCs Into Telencephalic Neurons Using 3D Organoids and Monolayer Culture Giovanna GIuseppina Altobelli1,2, Soraya Scuderi2, Gianfilippo Coppola2, Jun Hyan Park3, Vincenzo Cimini1, Flora Maria Vaccarino2,3 1 Department of Advanced Biomedical Sciences, University of Naples “Federico II”, Naples, Italy 2 Child Study Center, Yale University, New Haven, CT, USA. 3 Neuroscience, Yale University, New Haven, CT, USA. Human induced pluripotent stem cells (hiPSCs) are emerging as a useful tool for modelling in vitro early brain development and neurological disorders. Molecular mechanisms and cell interactions that regulate the neurodevelopment at early stages remain unclear because of human brain’s complexity and limitations of functional studies. Two major culture methodologies are used to differentiate in vitro hiPSCs into neurons: monolayer (2D) and organoid (3D) cultures. Here we investigate the effect of cell dissociation and the loss of 3D organization during the early differentiation process of neuronal progenitors. Using the same culture media, we first differentiated hiPSCs into neural progenitor cells (NPCs) and then induced their differentiation into neurons in 3 different modalities: 3D undissociated organoids, dissociated NPCs followed by immediate re-aggregation into an organoid, and dissociated NPCs cultured as monolayer. We assessed neuronal differentiation efficiency of each method by immunocytochemistry, qPCR, western blot, and RNA-Seq analysis over a time course. Our data revealed substantial differences in gene and protein expression among the three systems, including genes of the Notch pathway (e.g. NEUROD1, NEUROG2), earliest determinants of cortical region differentiation (e.g. SOX1, FEZF1) as well as later transcriptional regulators that specify cortical neuron subtypes (e.g. TBR1, CTIP2), which were all downregulated in monolayer. Moreover, we found that genes and pathways mediating cell-to-cell interactions (e.g. CNTNs, CAMs) were mostly upregulated in the 3D culture systems, whereas cell-extracellular matrix interaction molecules (e.g. ITG, LAM) were mostly upregulated in 2D, indicating that cell surface molecules may be involved in specification of neuronal cell types. Our results address the methodological question of the appropriateness of a differentiation method for a particular experimental goal, and, beyond that, reveal important early determinants that exert a decisive influence on neuronal differentiation and regional specification of human neural stem cells

Exercise stimulates angiogenesis and improves beta-adrenergic receptor signaling in the failing heart

Exercise upregulates beta-adrenergic receptor expression and this, in turn, stimulates angiogenesis