GM1 Ganglioside: In Vivo and In Vitro Trophic Actions on Central Neurotransmitter Systems

Abstract: There is now substantial evidence that GM1 ganglioside is effective in partially correcting the consequences of neuroinjury in a number of in vivo and in vitro model systems. Although the molecular mechanism(s) and the substrates for the neurotrophic activity of the gangliosides are not fully understood, the published experimental work suggests that GM1 has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. This review focuses attention on studies reporting that GM1 restores neuronal integrity and function in the brain of lesioned young as well as aged animals. Critical analysis of these studies can provide guidance for future ganglioside research and may point to novel approaches for treating neuroinjury and a variety of degenerative conditions, including aging.     

Autocrine Hyaluronan Influences Sprouting and Lumen Formation During HUVEC Tubulogenesis In Vitro

Although many studies have focused on a role for hyaluronan (HA) of interstitial extracellular matrix (presumably produced by non-vascular “stromal” cells) in regulating vascular growth, we herein examine the influence of “autocrine HA” produced by vascular endothelial cells themselves on tubulogenesis, using human umbilical vein endothelial cells (HUVECs) in angiogenic and vasculogenic three-dimensional collagen gel cultures. Relative to unstimulated controls, tubulogenic HUVECs upregulated HAS2 mRNA and increased the synthesis of cell-associated HA (but not HA secreted into media). Confocal microscopy/immunofluorescence on cultures fixed with neutral-buffered 10% formalin (NBF) revealed cytoplasmic HAS2 in HUVEC cords and tubes. Cultures fixed with NBF (with cetylpyridinium chloride added to retain HA), stained for HA using “affinity fluorescence” (biotinylated HA-binding protein with streptavidin-fluor), and viewed by confocal microscopy showed HA throughout tube lumens, but little/no HA on the abluminal sides of the tubes or in the surrounding collagen gel. Lumen formation in angiogenic and vasculogenic cultures was strongly suppressed by metabolic inhibitors of HA synthesis (mannose and 4-methylumbelliferone). Hyaluronidase strongly inhibited lumen formation in angiogenic cultures, but not in vasculogenic cultures (where developing lumens are not open to culture medium). Collectively, our results point to a role for autocrine, luminal HA in microvascular sprouting and lumen development. (J Histochem Cytochem 69: 415–428, 2021)     

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.     

Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

Involvement of p38α in Kainate-Induced Seizure and Neuronal Cell Damage

We investigated how p38α mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 α gene (p38α^+/?). Mortality rate and seizure score of p38α^+/? mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38α^+/? mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38α^+/? mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38α ^+/? mice was significantly decreased compared to that of WT mice. These results suggest that p38α signaling pathway plays an important role in epileptic seizure and excitotoxicity.     

Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures

The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the beta-adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the alpha-adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists (phentolamine greater than yohimbine greater than prazosin). alpha 2-Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 greater than NE greater than 6-fluoro-NE greater than epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists with a different order of potency (prazosin greater than phentolamine greater than yohimbine). alpha 1-Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE greater than phenylephrine greater than 6-fluoro-NE greater than methoxamine). In all three tissue preparations, large responses to both alpha 1-receptor activation (increases in inositol phosphate accumulation) and alpha 2-receptor activation (decreases in forskolin-stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (beta, alpha 1, alpha 2) are present in each tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

神经脊来源许旺细胞的体外培养研究

目的:探讨坐骨神经中神经脊来源的许旺细胞所占的比率。方法:将Wnt1-Cre+/-与Rosa-EGFP+/-小鼠杂交,获取Wnt1/EGFP小鼠,其所有起源于神经脊的细胞都有EGFP蛋白表达。取其坐骨神经,经过消化分离纯化,获得许旺细胞。进行抗GFP免疫荧光染色和流式分析的检测。结果:根据P1代许旺细胞的形态学观察,其纯度约为60%。抗GFP免疫荧光染色显示,并非所有的许旺细胞均呈阳性。P3代许旺细胞的纯度约为99%,流式细胞术分析显示约65%左右的GFP阳性率。结论:小鼠坐骨神经中的许旺细胞在体外培养提纯后,神经脊起源的许旺细胞占总许旺细胞的比例约为65%。     

Cell to cell recognition between the ciliatePseudomicrothorax dubius and its food organisms: the role of surface charges

Summary The importance of charged groups during phagocytic recognition of filamentous Cyanobacteria (Oscillatoria formosa andAnabaena spp.) by the stenophagic ciliatePseudomicrothorax dubius has been studied. Anionic and cationic domains are evenly and randomly distributed over the cyanobacterial surface, as demonstrated with scanning electron microscopy following labeling with colloidal gold (–) and colloidal gold coupled with poly-L-lysine (+). The phagocytosis ofOscillatoria was inhibited when filaments were treated with cationic reagents such as poly-L-lysine (pLL), FeCl₃ and carbodiimide. In contrast elimination of cationic charges on theOscillatoria surface by treatment with poly-L-glutamic acid (pLGa) or colloidal gold did not affect phagocytosis. The effects of sequential treatment with pLL and pLGa demonstrated that pLL reduced phagocytosis of pLGa-pretreatedOscillatoria, whereas the pLGa restored phagocytosis of pLL-pretreated filaments. Scanning electron microscopy showed that pLL- or pLGa- treated filaments can still adsorb the oppositely charged colloidal gold particles on their surface. However, the treatment of filaments with pLL followed by pLGa prevented subsequent labeling with gold as well as with pLL-gold particles. Filaments ofAnabaena spp., which are not normally ingested byPseudomicrothorax, were also treated individually or sequentially with pLL and pLGa. None of these treatments, however, provoked phagocytosis ofAnabaena byPseudomicrothorax. We suggest that the surface charge alone does not play a crucial role in phagocytic recognition inPseudomicrothorax and that phagocytosis-specific molecules are implicated.     

Production and Characterization of Single Chain Nimotuzumab: An In Vitro Study

Recombinant expression and EGFR-binding activity assay of single chain nimotuzumab (nimotuzumab scFv) is reported in this study. The scFv was produced in VH-linker-VL format in Origami^? 2(DE3)pLysS bacterial cells and purified using Ni-TNA resin column. 3-D structure prediction using I-TASSER (Iterative Threading Assembly Refinement) server and analyzing the predicted models using YASARA (Yet Another Scientific Artificial Reality Application) viewer revealed that VH and VL domains assemble into a correctly-folded single chain antibody that is able to bind EGFR. The scFv was evaluated in ELISA and western blot tests and proven to be able to bind EGFR-overexpressing cancer cells (A-431 cells in an efficient manner but unable to recognize cancer cells expressing low levels of EGFR (MCF-7 breast cancer cells).     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Changes in the Expression of a Neuronal Surface Protein During Development of Cerebellar Neurones In Vivo and in Culture

The expression of the neurone-specific D2 protein changes both quantitatively and qualitatively during development in vivo and in cultures of cerebellar nerve cells. The total D2 content per unit protein shows a two-fold increase in vivo from birth to postnatal day 6, after which it declines progressively to about 50% of the maximal value. This increase can be accounted for by an immature form of the protein anodic D2 being preferentially expressed at the early stages of cerebellar development. After postnatal day 9 this form gradually switches to a mature form cathodic D2. This switch can be mimicked by neuraminidase treatment, suggesting a developmental loss of sialic acid from the D2 protein. In freshly isolated cells the total D2 content per unit protein is only 30% of that in the corresponding intact tissue from 8-day-old cerebella, but it increases rapidly during the first 8 days of culture to levels similar to those of the equivalent age in vivo. The switch from anodic D2 to cathodic D2 also occurs at a faster rate in culture, probably reflecting the culture conditions that favour differentiation. The changes in the expression of D2 during development of cerebellar nerve cells in culture suggest that anodic D2 is preferentially expressed on nerve cells that are proliferating, migrating, or in the initial stages of differentiation, whereas cathodic D2 is associated with differentiated neurones. The transition between the two forms appears to occur during the formation of interneuronal contacts.     

Photodynamic Therapy in Pythium insidiosum – An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen''s cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.     

魔芋丛生芽诱导成苗及生根技术研究

采用不同激素配方的1-9号培养基,探索了由魔芋丛生芽诱导试管苗的最佳培养基配方。试验结果表明:MS+6BA1.5 mg.L-1+NAA0.5 mg.L-1+IBA1.0 mg.L-1和MS+NAA1.0 mg.L-1+PP3331.0 mg.L-1两种培养基对于诱导出苗及生根效果最好,添加PP333不仅可加快出苗生根,还可促使试管苗矮壮。     

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.