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Results for “Cell Tracking”

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At least 433 records · Page 24Linked to original sources

PubMed2002

Long-term injured purkinje cells are competent for terminal arbor growth, but remain unable to sustain stem axon regeneration.

Long-distance axon regeneration requires the activation of a specific set of neuronal growth-associated genes. Adult Purkinje cells fail to upregulate these molecules in response to axotomy and show extremely weak regenerative properties. Nevertheless, starting from several months after injury, transected Purkinje axons undergo spontaneous sprouting. Here, we asked whether long-term injured Purkinje cells acquire novel intrinsic growth properties that enable them to upregulate growth-associated genes and sustain axon regeneration. To test this hypothesis, we examined axon growth and cell body changes in adult rat Purkinje neurons following axotomy and implantation of embryonic neocortical tissue or Schwann cells into the injury track. Purkinje cells that survived over 6 months after injury/transplantation displayed profuse sprouting in the injured cerebellum and developed extensive networks of terminal branches into embryonic neocortical grafts. In addition, severed Purkinje axons exposed to these transplants 6 months after injury grew faster than their counterparts confronted with the same environment immediately after axotomy. Nevertheless, long-term injured Purkinje cells failed to regenerate stem neurites into Schwann cell grafts, and, under all experimental conditions, they did not upregulate growth-associated molecules, including c-Jun, GAP-43, SNAP-25, and NADPH-diaphorase. These results indicate that the long-term injured Purkinje cells remain unable to activate the gene program required to sustain axon regeneration and their plasticity is restricted to terminal arbor remodeling. We propose that the delayed growth of injured Purkinje cells reflects an adaptive phenomenon by which the severed axon stump develops a new terminal arbor searching for alternative connections with local partners.

Animals↗

PubMed1996

Recruitment of mast cells to muscle after mild damage.

We followed the response of muscle following mild intentional injury to determine a temporal sequence of cellular events involved in muscle repair. We found that intramuscular saline injection induced mild damage to muscle which resulted in the gradual recruitment of mast cells. Around the needle track, mast cells appear around 8 h post-injection. Mast cell accumulation were most dramatic immediately neighboring the posterior tibial vessels supplying the injured muscle. Dystrophin-deficient mdx muscle showed mast cell accumulations 3-fold higher than normal muscle, and this number did not change after saline injection. Additionally, we show that stem cell factor (SCF), a known mast cell chemoattractant, is expressed in both normal and mdx muscle at high levels. This steady-state level did not appear to be influenced by injury or dystrophin status. The implications of these findings are discussed as they relate to the repair of injured muscle and to their possible significance in the pathophysiology of Duchenne muscular dystrophy.

Animals↗

PubMedDate not supplied

Quantifying viral propagation in vitro: toward a method for characterization of complex phenotypes.

For a eukaryotic virus to successfully infect and propagate in cultured cells several events must occur: the virion must identify and bind to its cellular receptor, become internalized, uncoat, synthesize viral proteins, replicate its genome, assemble progeny virions, and exit the host cell. While these events are taking place, intrinsic host defenses activate in order to defeat the virus, e.g., activation of the interferon system, induction of apoptosis, and attempted elicitation of immune responses via chemokine and cytokine production. As a first step in developing an imaging methodology to facilitate direct observation of such complex host/virus dynamics, we have designed an immunofluorescence-based system that extends the traditional plaque assay, permitting simultaneous quantification of the rate of viral spread, as indicated by the presence of a labeled viral protein, and cell death in vitro, as indicated by cell loss. We propose that our propagation and cell death profiles serve as phenotypic read-outs, complementing genetic analysis of viral strains. As our virus/host system we used vesicular stomatitis virus (VSV) propagating in hamster kidney epithelial (BHK-21) and murine astrocytoma (DBT) cell lines. Viral propagation and death profiles were strikingly different in these two cell lines, displaying both very different initial titer and cell age effects. The rate of viral spread and cell death tracked reliably in both cell lines. In BHK-21 cells, the rate of viral propagation, as well as maximal spread, was relatively insensitive to initial titer and was roughly linear over several days. In contrast, viral plaque expansion in DBT cells was contained early in the infections with high titers, while low titer infections spread in a manner similar to the BHK-21 cells. The effect of cell age on infection spread was negligible in BHK-21 cells but not in DBTs. Neither of these effects was clearly observed by plaque assay.

Animals↗

PubMed1999

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.

Animals↗

PubMed2006

Novel insights into the mechanism of action of FTY720 in a transgenic model of allograft rejection: implications for therapy of chronic rejection.

FTY720 is a high-affinity agonist at the sphingosine 1-phosphate receptor 1 that prevents lymphocyte egress from lymphoid tissue and prolongs allograft survival in several animal models of solid organ transplantation. In this study we used a recently developed adoptive transfer model of TCR transgenic T cells to track allospecific CD4+ T cell expansion and trafficking characteristics, cytokine secretion profiles, and surface phenotype in vivo in the setting of FTY720 administration. We report that FTY720 administration had no effect on alloantigen-driven T cell activation, proliferation, acquisition of effector-memory function, or T cell apoptosis. However, FTY720 caused a reversible sequestration of alloantigen-specific effector-memory T cells in regional lymphoid tissue associated with a decrease in T cell infiltration within the allograft and a subsequent prolongation in allograft survival. Furthermore, delayed administration of FTY720 in a cardiac model of chronic allograft rejection attenuated the progression of vasculopathy and tissue fibrosis consistent with the hypothesis that FTY720 interrupts the trafficking of activated effector-memory T cells. These data have important implications for targeting the sphingosine 1-phosphate receptor 1 in solid organ transplantation.

Adoptive Transfer↗

PubMed1986

Complex-spike activity of cerebellar Purkinje cells related to wrist tracking movement in monkey.

Four rhesus monkeys were trained to perform visually guided wrist tracking movements (50). While they performed tasks by wrist flexion or extension from a neutral position, simple-spike (SS) and complex-spike (CS) discharges of a single Purkinje cell (P-cell) were recorded from intermediate and lateral parts of cerebellar hemispheres (lobules IV to VI) ipsilateral to the task-performing wrist. Of approximately 400 P-cells observed, 215 (54%) significantly increased or decreased their SS discharge rate during task performance (task-related P-cells). Of these, 161 were selected for analysis of CS activity; in these P-cells, we could reliably discriminate between CS and background SS by a spike discriminator. The 161 P-cells were further classified into response locked (n = 65) and poorly locked (n = 96) cells according to temporal coupling of the SS frequency modulation to the onset of wrist movements. About 60% of the response-locked P-cells showed a phasic increase (statistical significance level: P less than 0.01) of CS firing rate at the onset of wrist tracking movement. In a few P-cells, a phasic decrease (statistically insignificant) of CS firing rate was observed with the wrist movement. In most P-cells, an increase of CS firing rate was observed with both rapid- and slow-tracking wrist movements. The increase was larger with faster step-tracking movement than with slower ramp-tracking movement. In most P-cells, the CS activity increased with both wrist flexion and extension; in some cells, however, it increased only with either flexion or extension. In most of the response-locked P-cells, the increase of CS firing rate occurred during motor time, i.e., after the onset of the EMG change in prime movers and before the beginning of wrist tracking movement. The increase occurred phasically at the onset and/or at the recovery phase of SS frequency modulation. At neutral wrist position, the maintained frequency of the CS was 0.72 +/- 0.29 CS/s (mean and SD for 161 task-related P-cells). Compared with the frequency at neutral position, the CS frequency did not change tonically during maintained flexed or extended wrist position in any response-locked P-cells. There was no increase of CS firing rate when the monkey returned the handle to center position after completing the tracking task, even in P-cells that had shown a significant increase of CS activity during tracking.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1995

In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles.

Dynamic MRI tracking of rat T-cells in vivo is performed in rat testicles after labeling isolated rat T-cells in vitro with superparamagnetic dextran-coated iron-oxide particles, BMS180549. Tissue inflammation induced by the local injection of the calcium ionophore, A23187, is used to attract labeled T-cells. Gradient-echo MR images of rat testicles show a statistically significant decrease (4%) of the signal intensity in areas of injection of A23187 as early as 30 min after intravenous infusion of 2 x 10(8) labeled T-cells. The signal change reaches its maximum (6-7% decrease) at about 60-120 min after cell infusion. T2-mapping also shows a decrease of T2 in the areas with A23187. Image quantitation, which includes a chemical-shift effect, significantly enhances the sensitivity for detection of superparamagnetically labeled T-cells. Localization of labeled T-cells in rat testicles has been verified by fluorescence microscopy studies of T-cells co-labeled with a lipophilic fluorescent carbocyanine dye, 1,1-dioctadecyl-3,3,3',3'-tetramethyl-lindocarbocyanine perchlorate. These results represent the first successful demonstration of dynamic tracking of specific cells in vivo using MRI.

Animals↗

PubMed2002

Micromechanical mapping of live cells by multiple-particle-tracking microrheology.

This paper introduces the method of live-cell multiple-particle-tracking microrheology (MPTM), which quantifies the local mechanical properties of living cells by monitoring the Brownian motion of individual microinjected fluorescent particles. Particle tracking of carboxylated microspheres imbedded in the cytoplasm produce spatial distributions of cytoplasmic compliances and frequency-dependent viscoelastic moduli. Swiss 3T3 fibroblasts are found to behave like a stiff elastic material when subjected to high rates of deformations and like a soft liquid at low rates of deformations. By analyzing the relative contributions of the subcellular compliances to the mean compliance, we find that the cytoplasm is much more mechanically heterogeneous than reconstituted actin filament networks. Carboxylated microspheres embedded in cytoplasm through endocytosis and amine-modified polystyrene microspheres, which are microinjected or endocytosed, often show directed motion and strong nonspecific interactions with cytoplasmic proteins, which prevents computation of local moduli from the microsphere displacements. Using MPTM, we investigate the mechanical function of alpha-actinin in non-muscle cells: alpha-actinin-microinjected cells are stiffer and yet mechanically more heterogeneous than control cells, in agreement with models of reconstituted cross-linked actin filament networks. MPTM is a new type of functional microscopy that can test the local, rate-dependent mechanical and ultrastructural properties of living cells.

3T3 Cells↗

PubMed1994

Tracking of murine spleen cells in vivo: detection of PKH26-labeled cells in the pancreas of non-obese diabetic (NOD) mice.

Flow cytometry was used to track the in vivo migration of PKH26-labeled donor spleen cells from diabetic NOD mice that were injected into non-diabetic recipient NOD mice. Flow cytometric analysis of recipient mouse tissues revealed that the donor cells were present in the peripheral blood, spleen and lymph nodes 24 h following injection and could still be detected after 28 days. PKH26(+) cells were also detectable in the pancreas 7 days after injection. Phenotypic analysis of the PKH26(+) cells that migrated into these target organs and tissues showed that the major cell population detected was Thy1.2(+) T-lymphocytes, predominantly the Thy1.2(+)/L3T4(+) subpopulation, but Thy1.2(+)/Lyt2(+) cells as well as B220(+) cells (B lymphocytes) were also present.

Animals↗

PubMed1996

A new plasma-based method to promote cell adhesion on micrometric tracks on polystyrene substrates.

A new procedure has been developed in order to obtain heterogeneous polymer surfaces for the promotion of cell adhesion. For this purpose, a microelectronic photosensitive resin was spin coated on polystyrene (PS) substrates. The resin was then submitted to UV light irradiation through a mask and partially developed. The sample was further submitted to a plasma oxygen discharge prior to dissolution of the remaining resin. The characterization by time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle (DCA) allowed us to conclude that hydrophilic paths were created on the more hydrophobic PS substrate together with the complete removal of the resin. In order to optimize cell adhesion contrast, the modified surfaces were then conditioned with a solution containing both a surfactant (pluronic F68) and a protein. Two different proteins were tested (collagen I and fibronectin). PC12 cell cultures on those conditioned surfaces showed that cell adhesion occurs only on the hydrophilic tracks. ToF SIMS spectra and images recorded on those substrates revealed the presence of the proteins only in the hydrophilic tracks. In the same time, the surfactant is suspected to adsorb mainly on the hydrophobic areas of the samples.

Adsorption↗

PubMed1994

Double-strand breaks from 125I incorporated in the DNA and cell death.

Track structure calculations of the local energy deposition by electrons emitted during the decay of 125I are used to demonstrate that the range of high energy deposition is small (< 10 nm) and restricted to the DNA and its immediate environment. An experiment in which 125I is incorporated into the DNA of synchronized CHO cells during a pulse and decays are allowed to accumulate a given time after the incorporation is described. Here it is shown that damage from 125I decays in newly replicated DNA (cells frozen for decay accumulation within 1 h after labelling) are relatively non-toxic whereas decays in mature DNA (cells frozen 5 h after labelling) are highly lethal. It is suggested that during DNA maturation the labelled DNA becomes associated with (or reorganized into) a radiosensitive nuclear structure and that damage to this structure is the primary cause of radiation-induced cell death.

Animals↗

PubMed1999

Novel cellular tracks of migrating Dictyostelium cells.

After Dictyostelium cells were settled on a coverslip and allowed to migrate freely on the surface, they were stained with fluorescently labeled Concanavalin A. Tracks with distinct patterns that consist of dots and short fibers were observed behind the cells. In this study, we refer to these tracks as "cellular tracks", CTs for short. We characterized the biological effect of CTs on cell behavior and development. CTs decreased the strength of cell-substratum adhesion, increased the velocity of cell migration, but did not affect growth of cells. CTs also promoted cell aggregation. When pre-aggregation cells touched the CTs of other cells, they avoided or orthogonally crossed them, but did not migrate along them. These observations suggest that the CTs of pre-aggregation cells prompts cells to disperse uniformly on substratum and may enable cells to sense cell density. On the other hand, when aggregation-competent cells touched the CTs of other aggregation-competent cells, a half of them migrated along the CTs. Pre-aggregation cells did not migrate along the CTs of aggregation-competent cells. The CTs of aggregation-competent cells may help the cells to aggregate toward the aggregation center.

Animals↗

PubMed2002

NK cell receptors of the orangutan (Pongo pygmaeus): a pivotal species for tracking the coevolution of killer cell Ig-like receptors with MHC-C.

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif.

Alleles↗

PubMed2002

In vivo magnetic resonance tracking of magnetically labeled cells after transplantation.

During the last few years, the therapeutic use of stem and progenitor cells as a substitute for malfunctioning endogenous cell populations has received considerable attention. Unlike their current use in animal models, the introduction of therapeutic cells in patients will require techniques that can monitor their tissue biodistribution noninvasively. Among the different imaging modalities, magnetic resonance (MR) imaging offers both near-cellular (i.e., 25- to 50-mu) resolution and whole-body imaging capability. In order to be visualized, cells must be labeled with an intracellular tracer molecule that can be detected by MR imaging. Methods have now been developed that make it possible to incorporate sufficient amounts of superparamagnetic iron oxide into cells, enabling their detection in vivo using MR imaging. This is illustrated for (neural stem cell-derived) magnetically labeled oligodendroglial progenitors, transplanted in the central nervous system of dysmyelinated rats. Cells can be followed in vivo for at least 6 weeks after transplantation, with a good histopathologic correlation including the formation of myelin. Now that MR tracking of magnetically labeled cells appears feasible, it is anticipated that this technique may ultimately become an important tool for monitoring the efficacy of clinical (stem) cell transplantation protocols.

Animals↗

PubMed1987

Closed loop television tracking of beating heart cells in vitro.

The motion of beating heart cells in vitro has been used as a sensitive indicator of the presence of drugs. The combination of closed loop television video tracking and latex microspheres placed on the cells as markers results in an improved measurement of cell motion. Latex microspheres, 5.2 micrometers, are allowed to settle onto beating myocardial heart cells in vitro, where they move in concert with the cell motion. These microspheres, when viewed by televised transmission microscopy with normal optics, present high contrast circular targets that are excellent for closed loop television video tracking techniques. With video tracking, an analog voltage is generated proportional to the horizontal center of intensity of the target being tracked. This signal represents the location of a video demodulator gate that is maintained centered over the video target by feedback control.

Animals↗

PubMed2006

Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting.

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cgamma1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig gamma1 constant region gene segment (Cgamma1). In these mice, Cre-mediated recombination at the fas, Igbeta, IgH, and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM(+) B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cgamma1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C(H) gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cgamma1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo. To expedite the genetic analysis of GC B cells, we have established Cgamma1-cre F(1) embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

Alleles↗

PubMed2001

Effect of glucose on antipsychotic drug-induced changes in dopamine neuronal activity.

The effect of glucose on antipsychotic drug-induced changes in the spontaneous activity of rat midbrain dopaminergic neurons was tested with the cells-per-track extracellular electrophysiology method. After daily s.c. treatment with vehicle, haloperidol (0.5 mg/kg), or clozapine (20 mg/kg) for 21 days, rats were anesthetized and recordings performed on one side of the brain. Then, glucose (250 mg/kg, i.v.) was administered and recordings were made from the contralateral midbrain. Glucose significantly reduced the number of spontaneously active A9 and A10 dopaminergic cells per track in control rats, but significantly attenuated the chronic haloperidol- and clozapine-induced reductions in dopaminergic cells per track. These results suggest that caloric intake may influence antipsychotic drug-induced changes in the population activity of midbrain dopaminergic neurons.

Animals↗

PubMed2005

Adult neural stem cell therapy: expansion in vitro, tracking in vivo and clinical transplantation.

Neural stem cells (NSCs) are present not only in the developing nervous systems, but also in the adult human central nervous system (CNS). It is long thought that the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus are the main sources of human adult NSCs, which are considered to be a reservoir of new neural cells. Recently adult NSCs with potential neural capacity have been isolated from white matter and inferior prefrontal subcortex in the human brain. Rapid advances in the stem cell biology have raised appealing possibilities of replacing damaged or lost neural cells by transplantation of in vitro-expanded stem cells and/or their neuronal progeny. However, sources of stem cells, large scale expansion, control of the differentiations, and tracking in vivo represent formidable challenges. In this paper we review the characteristics of the adult human NSCs, their potentiality in terms of proliferation and differentiation capabilities, as well as their large scale expansion for clinical needs. This review focuses on the major advances in brain stem cell-based therapy from the clinical perspective, and summarizes our work in clinical phase I-II trials with autologuous transplantation of adult NSCs for patients with open brain trauma. It also describes multiple approaches to monitor adult human NSCs labeled superparamagnetic nanoparticles after transplantation and explores the intriguing possibility of stem cell transplantation.

Animals↗