Electrophysiological and Morphological Studies of SOD1 Transgenic Mice: An Animal Model of ALS

Amyotrophic lateral sclerosis (ALS) is a disease where upper and lower motor neurons die, and it is often associated with mutations of superoxide dismutase 1 (SOD1). We have used mouse models to compare physiologic and morphologic characteristics of cervical motor neurons in wild-type and mutant animals. Slices of the cervical spinal cord were prepared from old wild-type and mutant G93A and G85R mice, and intracellular recordings of membrane potential, resistance and responses to application of excitatory neurotransmitters were studied. Some motor neurons were injected with Lucifer Yellow for morphological analysis. There were no significant differences between membrane potential in the SOD1 mutants and aged wild-type mice, but membrane resistance was somewhat higher in the mutant motor neurons. Dendrites of the mutant motor neurons were not responsive to ionophoretic application of excitatory amino acids, although the cell body responded strongly. In Lucifer-filled cells, the dendrites were found to disappear. Mutant motor neurons were sometimes spontaneously active. Responses of mutant motor neurons to perfused glutamate with varying calcium concentrations in the Ringer’s solution were different from those of the wild-type cells.

Physiological and Pharmacological Studies on Cervical Motor Neurons in Slices Prepared from Neonatal and Aged Mice

The effects of age on the physiological properties of cervical motor neurons were examined in slices made from an excised spinal cord graft of ICR mice from the second day after birth to age 350 days. The membrane potential of post-natal day 2 (PD2) to PD350 was about -65 mV and did not change greatly with age, although it was slightly higher at PD2. However, there were significant changes in membrane resistance, which increased with age from about 15 to 30 MΩ. The depolarization induced by the excitatory amino acid agonists, kainic acid, NMDA and AMPA, decreased with aging in spite of the increase in membrane resistance. The motor neurons of the aged mice showed delayed recovery from excitation caused by excitatory amino acid agonists. By injecting Lucifer yellow CH into motor neurons, it was observed that the dendrite trees become thin, and some of the dendrite branches were missing in older animals.

Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

Perinatal Exposure of Bisphenol A Differently Affects Dendritic Spines of Male and Female Grown-Up Adult Hippocampal Neurons

Perinatal exposure to Bisphenol A (BPA) at a very low dose may modulate the development of synapses of the hippocampus during growth to adulthood. Here, we demonstrate that perinatal exposure to 30 μg BPA/kg per mother’s body weight/day significantly altered the dendritic spines of the grownup rat hippocampus. The density of the spine was analyzed by imaging of Lucifer Yellow-injected CA1 glutamatergic neurons in adult hippocampal slices. In offspring 3-month male hippocampus, the total spine density was significantly decreased by BPA exposure from 2.26 spines/μm (control, no BPA exposure) to 1.96 spines/μm (BPA exposure). BPA exposure considerably changed the normal 4-day estrous cycle of offspring 3-month females, resulting in a 4∼5 day estrous cycle with 2-day estrus stages in most of the subjects. In the offspring 3-month female hippocampus, the total spine density was significantly increased by BPA exposure at estrus stage from 2.04 spines/μm (control) to 2.25 spines/μm (BPA exposure). On the other hand, the total spine density at the proestrus stage was moderately decreased from 2.33 spines/μm (control) to 2.19 spines/μm (BPA exposure). Thus, after the perinatal exposure to BPA, the total spine density in males became lower than that in females. Concerning the BPA effect on the morphology of spines, the large-head spine was significantly changed with its significant decrease in males and moderate change in females.

Propionic acid disrupts endocytosis, cell cycle, and cellular respiration in yeast

Objective We previously identified propionic acid as a microbially-produced volatile organic compound with fungicidal activity against several pathogenic fungi. The purpose of this work is to better understand how propionic acid affects fungi by examining some of the effects of this compound on the yeast cell. Results We show that propionic acid causes a dramatic increase in the uptake of lucifer yellow in yeast cells, which is consistent with enhanced endocytosis. Additionally, using a propidium iodide assay, we show that propionic acid treatment causes a significant increase in the proportion of yeast cells in G1 and a significant decrease in the proportion of cells in G2, suggesting that propionic acid causes a cell cycle arrest in yeast. Finally, we show that the reduction of MTT is attenuated in yeast cells treated with propionic acid, indicating that propionic acid disrupts cellular respiration. Understanding the effects of propionic acid on the yeast cell may aid in assessing the broader utility of this compound.

Blood–Brain Barrier Dynamic Device with Uniform Shear Stress Distribution for Microscopy and Permeability Measurements

Neurology has always been one of the therapeutic areas with higher attrition rates. One of the main difficulties is the presence of the blood–brain barrier (BBB) that restricts access to the brain for major drugs. This low success rate has led to an increasing demand for in vitro tools. The shear stress, which positively affects endothelial cell differentiation by mimicking blood flow, is required for a more physiological in vitro BBB model. We created an innovative device specifically designed for cell culture under shear stress to investigate drug permeability. Our dynamic device encompasses two compartments communicating together via a semi-permeable membrane, on which human cerebral microvascular endothelial (hCMEC/D3) cells were seeded. The fluidic controlled environment ensures a laminar and homogenous flow to culture cells for at least seven days. Cell differentiation was characterized by immunodetection of inter-endothelial junctions directly in the device by confocal microscopy. Finally, we performed permeability assay with lucifer yellow in both static and dynamic conditions in parallel. Our dynamic device is suited to the evaluation of barrier function and the study of drug transport across the BBB, but it could also be used with other human cell types to reproduce intestinal or kidney barriers.

Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Zebrafish (Danio rerio) Oatp2b1 as a Functional Ortholog of the Human OATP2B1 Transporter

OATP2B1 belongs to a highly conserved organic anion transporting polypeptide (OATP) family of transporters, involved in the cellular uptake of both endogenous and exogenous compounds. The main substrates of human OATP2B1 include steroid conjugates, bile salts and thyroid hormones, as well as several pharmaceuticals. Human OATP2B1 has orthologous genes in other vertebrate species, including zebrafish (Danio rerio), a widely used model organism in biomedical and environmental research. Our previous studies showed that zebrafish Oatp2b1 was phylogenetically closest to mammalian OATP2B1/Oatp2b1 and that it shares a similar tissue expression pattern. In this study, we aimed at discovering whether zebrafish Oatp2b1 could be a functional ortholog of human and rodent OATP2B1/Oatp2b1. To test this hypothesis, our primary goal was to obtain the first in vitro and in silico insights related to the structure and potential substrate preferences of zebrafish Oatp2b1. We generated cells transiently and stably transfected with zebrafish Oatp2b1 cloned from zebrafish liver, constructed an Oatp2b1 homology model, developed transport activity assays with model fluorescent substrate Lucifer yellow, and finally utilized this assay to analyze the interaction of zebrafish Oatp2b1 with both physiological and xenobiotic substances. Apart from structure similarities, our data revealed the strongest interaction of zebrafish Oatp2b1 with bile acids, steroid sulfates, thyroid hormones and bilirubin, as well as xenobiotics bromosulfophthalein and sulfasalazine, which indicates its functional orthology with human OATP2B1.

Connexin43 is differentially distributed within renal vasculature and mediates profibrotic differentiation in medullary fibroblasts

Connexins (Cxs) form gap junctions for intercellular exchange of inorganic ions and messenger molecules. In the kidney, Cxs play essential roles within its compartments, but data on the precise cellular localization and cell type-related function of their isoforms are scarce. We tested whether Cx43 distribution is restricted to vascular and interstitial cells and whether medullary fibroblasts express Cx43 to coordinate profibrotic signaling. Confocal immunofluorescence techniques, ultrastructural labeling, and functional experiments in cell culture were performed. Cx43 was chiefly expressed in the vasculature but was absent from tubular epithelia. All arterial, arteriolar, and lymphatic endothelia showed continuous Cx43 signal along their borders. In the inner medulla, only the interstitium showed Cx43 signals, which were assigned to fibroblasts and their processes. Cultured Cx43-expressing medullary fibroblasts served to study the role of gap junctions in a profibrotic context. In a dye spreading assay, Cx43-sensitive diffusion of Lucifer yellow was dependent on gap junctional passage. The addition of transforming growth factor-β1 (5 ng/mL for 48 h) activated Cx43 biosynthesis and caused Cx43-sensitive transformation of the fibroblasts into a myofibroblast phenotype. This suggested that Cx43 gap junctional channels enable the coordination of profibrotic signaling between cells of the medullary interstitium. In summary, we demonstrate the presence of Cx43-expressing gap junctions within the two major renal compartments, the vasculature and interstitium. Endothelial Cx43 likely provides functions of an earlier-defined “electrical syncytium” within the vascular wall. Additionally, Cx43 facilitates profibrotic signaling between medullary interstitial fibroblasts.

Endothelial Glycocalyx-Mediated Intercellular Interactions: Mechanisms and Implications for Atherosclerosis and Cancer Metastasis

Purpose The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system. Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Examples of such EC function include interendothelial cell communication via gap junctions and receptor-mediated interactions between endothelial and tumor cells. This review focuses on GCX-dependent regulation of these intercellular interactions in healthy and diseased states. The ultimate goal is to build new knowledge that can be applied to developing GCX regeneration strategies that can control intercellular interaction in order to combat the progression of diseases such as atherosclerosis and cancer. Methods In vitro and in vivo studies were conducted to determine the baseline expression of GCX in physiologically relevant conditions. Chemical and mechanical GCX degradation approaches were employed to degrade the GCX. The impact of intact versus degraded GCX on intercellular interactions was assessed using cytochemistry, histochemistry, a Lucifer yellow dye transfer assay, and confocal, intravital, and scanning electron microscopy techniques. Results Relevant to atherosclerosis, we found that GCX stability determines the expression and functionality of Cx43 in gap junction-mediated EC-to-EC communication. Relevant to cancer metastasis, we found that destabilizing the GCX through either disturbed flow-induced or enzyme induced GCX degradation results in increased E-selectin receptor-mediated EC-tumor cell interactions. Conclusion Our findings lay a foundation for future endothelial GCX-targeted therapy, to control intercellular interactions and limit the progression of atherosclerosis and cancer.