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PubMed1999

Neuroprotective effects of riluzole: an electrophysiological and histological analysis in an in vitro model of ischemia.

The protective effects of riluzole against the neuronal damage caused by O2 and glucose deprivation (ischemia) was investigated in rat cortical slices by recording electrophysiologically the cortico-cortical field potential and by evaluating histologically the severity of neuronal death. Five minutes of ischemia determined an irreversible depression of the amplitude of the field potential. In addition, this insult caused a clear enhancement of the number of death cells that were specifically colored with trypan blue (a vital colorant which stains altered cells). We found that riluzole, which by itself depressed the synaptic transmission, neuroprotected when perfused 15-20 min before and during ischemia. In fact, due to the treatment with riluzole, the ischemia-induced irreversible depression of the field potential recovered and less cells were stained with trypan blue. These findings demonstrate that riluzole prevents neuronal death in an in vitro model of ischemia and suggest a therapeutic use of this drug in order to reduce the pathophysiological outcomes of stroke.

Animals↗
PubMed2004

Fucoidan modulates the effect of transforming growth factor (TGF)-beta1 on fibroblast proliferation and wound repopulation in in vitro models of dermal wound repair.

Aberrant wound healing, either causing scarring or chronic wounds, is a significant cause of morbidity. There is therefore, considerable interest in agents which can modulate certain aspects of the wound healing process. Fucoidans, sulphated polyfucose polysaccharides which may be extracted from Fucus spp., have been shown to modulate the effects of a variety of growth factors through mechanisms thought to be similar to the action of heparin. We investigated the interaction between two commercial preparations of fucoidan and transforming growth factor (TGF)-beta(1). These preparations of fucoidan, as well as heparin, inhibited fibroblast proliferation at concentrations from 0.01 to 100 mg/ml. The anti-proliferative effects of 1 ng/ml TGF-beta(1) on dermal fibroblasts were abrogated by fucoidan preparation F7 when used at concentrations over 1 mg/ml. In a three dimensional in vitro model of wound repair, the fibroblast populated collagen lattice or "dermal equivalent", TGF-beta(1) reduced the rate of fibroblast repopulation of a wound defect created by punch biopsy. Addition of fucoidan to the model in the presence of TGF-beta(1) increased the rate of fibroblast repopulation of the wound and at 10 mg/ml of fucoidan the number of cells which had migrated into the wounded defect was similar to that of control cultures. These data suggest that fucoidan has properties which may be beneficial in the treatment of wound healing.

Cell Proliferation↗
PubMed2000

A comparison of the erosive potential of different beverages in primary and permanent teeth using an in vitro model.

The aim of this study was to compare the erosive potential of different beverages and foodstuffs in primary and permanent teeth. Sixty primary and 60 permanent human teeth were immersed for 3 min in the solution under study (5 teeth per treatment group). Surface microhardness was measured before and after exposure. Initial (baseline) surface microhardness was lower for primary teeth than for permanent teeth. In both primary and permanent teeth, Sprite showed the highest decrease in surface microhardness, whereas yogurt showed an increase in surface microhardness in the primary teeth. Overall decrease was 27.2 +/- 17.5 KHN (mean +/- SD) for primary and 25.9 +/- 15.6 KHN for permanent teeth. The comparison of the erosive susceptibility in this in vitro model showed that primary teeth were not more susceptible to erosion compared to permanent teeth.

Beverages↗
PubMed2003

The DNA synthesome: its potential as a novel in vitro model system for studying S-phase specific anticancer agents.

A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.

Antineoplastic Agents↗
PubMed2003

The EOL-1 cell line as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.

We recently identified the chimeric kinase FIP1L1-platelet-derived growth factor receptor alpha (PDGFRalpha) as a cause of the hypereosinophilic syndrome and of chronic eosinophilic leukemia. To investigate the role of FIP1L1-PDGFRA in the pathogenesis of acute leukemia, we screened 87 leukemia cell lines for the presence of FIP1L1-PDGFRA. One cell line, EOL-1, expressed the FIP1L1-PDGFRA fusion. Three structurally divergent kinase inhibitors--imatinib (STI-571), PKC412, and SU5614--inhibited the growth of EOL-1 cells. These results indicate that the fusion of FIP1L1 to PDGFRA occurs rarely in leukemia cell lines, but they identify EOL-1 as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and for the analysis of small molecule inhibitors of FIP1L1-PDGFRalpha.

Apoptosis↗
PubMed1989

Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization.

Stromal cells derived from proliferative or secretory human endometria, cultured in the absence of steroid hormones, grew as monolayers that showed only occasional areas of immunoreactive fibronectin and did not produce detectable levels of prolactin (PRL) or laminin. Treatment with physiological doses of estradiol and progesterone induced PRL production and stimulated cell proliferation, resulting in multilayering with an increase of the saturation density. Electron microscopy showed the development of gap junctions, whereas immunofluorescence revealed a dense pericellular matrix containing fibronectin and laminin. These findings show that human endometrial stromal cells in culture respond to physiological doses of ovarian hormones with ultrastructural, proliferative, and biochemical changes that are characteristic of decidualization in vivo. This culture system thus provides an in vitro model for human decidualization.

Cell Division↗
PubMed1989

Human fetal lens epithelial cells in culture: an in vitro model for the study of crystallin expression and lens differentiation.

We have cultured and passaged human fetal lens epithelial cells. Cultured cells exhibited hexagonal, cuboidal shape typical of epithelial cells. Unlike previous observations made with cultured mammalian lens epithelial cells, indirect immunofluorescence and temporal analysis of 35S-labeled proteins demonstrated undiminished levels of alpha B-crystallin in primary, secondary, and tertiary cultures. Among the alpha-crystallins only alpha B synthesis was detected. Two dimensional gel electrophoresis indicated the presence of alpha B2 and no alpha B1. beta B2-crystallin, a fiber cell specific protein hardly detectable in primary cultures, increased significantly upon passaging. Human fetal lens epithelial cell cultures, described in this report, thus present a useful in vitro model for the study of lens epithelial cell differentiation and its pathological manifestations.

Biomarkers↗
PubMed1994

An "in vitro" model for combined functional and morphological investigations on the rat pituitary gland.

We have developed an alternative model for combined "in vitro" functional and morphological studies on the rat pituitary gland. To test its use for biochemical investigations we studied some aspects of the secretion in pituitary thyrotrophs under basal conditions and after stimulation with TRH. After stabilisation of basal TSH release, the stimulation with TRH significantly increased the TSH secretion. To test the usefulness of our model for morphological investigations the tissue was processed for conventional and immunocytochemical light and electron microscopy at the end of the incubation. Different types of pituitary cell resulted to be well preserved. Our model allows combined biochemical and morphological studies on the pituitary gland and thus reduces the number of animal groups, eliminates the extrapolation of results from different groups and replaces the "in vivo" by an "in vitro" experiment.

Animal Testing Alternatives↗
PubMed1987

Round window membrane permeability. An in vitro model.

The round window membrane is regarded to be the main route for passage of potentially ototoxic substances from the middle ear cavity into the inner ear. This is of clinical importance in acute otitis media and chronic otitis media, where sensorineural hearing impairments sometimes develop. The accuracy and reliability of an in vitro round window membrane permeability model was studied. The round window membrane of the mongolian gerbil was resected, together with its bone niche. The preparation was mounted between two glass chambers, representing the middle ear cavity and the perilymphatic space respectively. Passage through the round window membrane did not occur within 3 h for low-density lipoprotein with a molecular weight of 2,300 kD and high-density lipoprotein with a molecular weight of 115-350 kD. Horseradish peroxidase, with a molecular weight of 40 kD, passed the round window membrane at a mean rate of 6 micrograms/h when the concentration of the protein was 10 g/l in the middle ear chamber. The present in vitro model is considered to be free of leakage artifacts between the chambers. Passage rates for different substances through the round window membrane can be calculated under controlled conditions using this type of in vitro model.

Animals↗
PubMed1996

Characterization of a new in vitro model for studies of reepithelialization in human partial thickness wounds.

Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5-7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this human in vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement to in vivo studies on epidermal healing.

Basement Membrane↗
PubMed2005

In vitro model of normal, immortalized ovarian surface epithelial and ovarian cancer cells for chemoprevention of ovarian cancer.

BACKGROUND: Epithelial ovarian cancer has the highest mortality rate among the gynecologic cancers. The synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR), has been used in the chemoprevention of ovarian cancer. However, the effectiveness of its application for different populations has been questioned because of the genetic differences among normal, high risk, and women with cancer. OBJECTIVE: To explore the similarities and the differences in 4-HPR effects on different ovarian epithelial cells which mimic different populations of women, normal ovarian surface epithelium to represent the normal population of women, immortalized ovarian surface epithelium to represent premalignant changes, and cells derived from ovarian cancer cells to represent malignant changes were used as in vitro models. METHODS: Normal ovarian surface epithelial cells, immortalized ovarian surface epithelial cells, and ovarian cancer cells were incubated for different intervals with increasing concentrations of 4-HPR. Growth inhibition, the fraction of apoptotic cells, the expression of apoptosis-related genes, including p53, p16, p21, and caspase-3, and mitochondrial permeability transition were measured before and after 4-HPR treatment. RESULTS: Treatment with 4-HPR produced growth inhibition and apoptosis in a dose-dependent manner for all 3 cell types. 4-HPR produced the strongest activation of the p53 pathway in normal ovarian epithelial (NOE) cells, while it caused the largest increase in MPT in the cancer cells, suggesting a different mechanism for growth inhibition and/or apoptosis in these cell lines. 4-HPR, at a concentration of 10 muM, had a maximal effect on caspase-3 activity at 72 h in normal cells and at 48 h in immortalized and cancer cells, although the effects were modest. CONCLUSIONS: Normal ovarian surface epithelial cells, immortalized ovarian surface epithelial cells, and ovarian cancer cells showed a differential response to 4-HPR. Although the same endpoints of growth inhibition and apoptosis induction were present in response to 4-HPR, these endpoints may be regulated through different pathways. IMPLICATIONS: Clinical trials with higher concentrations of 4-HPR should prove beneficial.

Anticarcinogenic Agents↗
PubMedDate not supplied

In vivo-in vitro model to assess chloroquine activity in monkeys.

The bioassay technique and high-performance liquid chromatography (HPLC) method were used to establish chloroquine (CQ) concentration equivalents in serum samples collected from Aotus and Saimiri monkeys after administration of CQ. The results indicate some differences in CQ metabolism between the two simian species. They also indicate a strong positive relationship (rs = 0.96) between data obtained by the bioassay and HPLC technique. The findings suggest that the use of the bioassay in these small primates, using only a fraction of the serum required for HPLC analysis, should provide a useful mean for obtaining preliminary information about the degree and duration of serum antimalarial activity of promising experimental drugs. Apart from reducing the number of monkeys required for drug evaluation, this in vivo-in vitro model should also decrease the overall cost and duration of developing new antimalarial agents.

Animals↗
PubMed2002

A new in vitro model to study endothelial injury.

BACKGROUND: Endothelial dysfunction or "endothelialitis" is a prominent feature in several disease states ranging from atherosclerosis to transplant rejection. This dysfunction is also caused by drugs such as cyclosporin A (CyA) and leads to allograft vasculopathy and eventual graft loss. Despite the frequency and importance of this injury, there is no model to study the morphological effects of endothelial injury and dysfunction in vitro. METHODS: We utilized a model in which mouse endothelial cells (SVEC 4-10) can be induced to form capillary tubes by culturing on a laminin-rich matrix (Matrigel). In this morphological model of endothelial cell function, we studied the effect of varying doses of CyA on two parameters of tube formation: initiation of tube formation and disruption of mature capillary tubes. As a positive control we used IFN-gamma, which inhibited capillary tube formation. We developed this assay in 96-well culture plates to test several samples simultaneously. RESULTS: The assay could be adapted to a 96-well format by optimizing the cell density. Endothelial dysfunction was seen when the endothelial cells were incubated with cyclosporin A, which affected both morphological parameters of tube formation. At higher doses (2-20 microg/ml) CyA both inhibited capillary tube formation and disrupted mature capillary tubes. At lower doses CyA only inhibited the initiation of tube formation; it did not disrupt mature capillary tubes. IL-2 (400-1000 pg/ml) and IFN-gamma (10-400 pg/ml) inhibited initiation of tube formation but did not disrupt mature capillary tubes. None of these agents, including high doses of CyA, impaired endothelial cell viability. CONCLUSION: CyA-induced endothelial dysfunction can be modeled in vitro by this novel morphological assay of capillary tube formation. This assay can discern mild and severe degrees of endothelial dysfunction. The different effects of low and high levels of CyA on capillary tube formation imply that similar dysfunction in vivo may be responsible for allograft vasculopathy caused by CyA. This novel model can also be utilized to study other forms of vasculitis.

Animals↗
PubMed1988

Interaction between lymphocytes and cultured high endothelial cells: an in vitro model of lymphocyte migration across high endothelial venule endothelium.

The interaction between lymphocytes and cultured high endothelial venule endothelium has been studied using light and electron microscopy. High endothelial cells (HEC) in monolayer culture bound lymphocytes 15-200-fold more efficiently than aortic endothelium, aortic fibroblasts or serum-coated glass. Light microscopy of lymphocytes bound to HEC showed two populations. Type I lymphocytes were phase-light, round and revealed no intracellular detail. Type II lymphocytes were phase-dark and flattened. The nucleus and cytoplasm of type II cells were clearly visible under high power light microscopy. The relative positions of these two populations were determined by electron microscopy. Type I lymphocytes were attached to the surface of HEC and type II lymphocytes were flattened underneath. The transition from type I to type II was accompanied by a loss of surface microvilli and a redistribution of intracellular organelles. This suggested that lymphocytes actively migrated across the HEC layer in this assay. The relationship between migrated lymphocytes and total adherent cells was determined by analysis of surface phenotype. Lymphocytes did not adhere randomly from the cell population plated. After 60 min there was an enrichment for B over T lymphocytes and the adherent T cell population was itself enriched for CD8+ cells over CD4+ cells. Migrated cells were, however, a random subpopulation of lymphocytes which adhered to HEC. This is clear evidence that migration was preceded by specific binding to HEC. Lymphocyte adhesion was independent of viable HEC showing that it was a passive event on the part of the endothelium. Lymphocyte migration was, however, completely dependent on viable high endothelium. We conclude that cultured HEC provide a biologically relevant, 3-dimensional matrix which supports the specific adhesion of lymphocytes and actively promotes lymphocyte migration. These observations suggest to us that cultured high endothelium provides a novel in vitro model for the study of lymphocyte migration into lymph nodes from the blood.

Animals↗
PubMedDate not supplied

Effects of T-2 mycotoxin on gastrointestinal tissues: a review of in vivo and in vitro models.

T-2 mycotoxin, a trichothecene, is the principal toxic component of Fusarium sp. Agricultural products and food are frequently contaminated with this toxin. Various animal models have been used to determine its metabolic fate, rate of excretion, and distribution. A modulation effect on cell-mediated immunity and alterations in gastrointestinal propulsion have been demonstrated. The toxin has been shown to produce some similar pathologic alterations in various animal species studied. The consistent alteration appears to mainly affect mitotic cells of the gastrointestinal tract and the lymphoid system. A host of bioassay systems are now being used as alternative methods to the use of animals for testing of the mycotoxin. These tests may accurately assess and define the role of the subject-toxin interactions following consumption of T-2 mycotoxin contaminated food sources. T-2 mycotoxin, as observed above with in vivo and in vitro models, promotes a chemically-induced change in structure and function of affected gastrointestinal cells from a transient and reversible aberration in a single enzymatic reaction to cell death. Regardless of the end point measured, the toxic response brought about in cells appears to involve the interactions of virtually all subcellular processes--membrane transport and permeability, chemical metabolism, DNA function, and energy production/expenditure--as cells attempt to maintain their functional integrity while disposing of the toxicant. The variation in the quality of the toxic response with dose suggests that more cellular processes are perturbed as the chemical dose is increased.

Animals↗
PubMed2003

Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model.

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

Barrett Esophagus↗
PubMed2004

Evaluation of magnetic navigation in an in vitro model of uterine artery embolization.

PURPOSE: To compare steering of a novel magnetic guide wire with a standard 0.014-inch guide wire within a vascular phantom. MATERIALS AND METHODS: The magnetic guiding system (MGS) was composed of two permanent magnets on each long side of the fluoroscopy table generating a 0.1-T magnetic field, and a C-arm angiography system. The magnetic field was created according to vectors drawn onto two radiographic projections. Consequently, the tip of the intravascular guide wire containing a permanent magnet was deflected parallel to the magnetic field. Ninety-six catheterizations were performed in water-filled polyvinyl chloride tubes imitating the arterial tree of a female pelvis. This vascular phantom resembled a total of 12 uterine arteries with three different calibers (inner diameters: 1.1 mm, 1.7 mm, and 4.2 mm). Fluoroscopy and procedure times were measured to compare magnetic-assisted and conventional catheterization. RESULTS: Catheterization to every predefined target was successful for all attempts with both guiding techniques. The fluoroscopy time during magnetic navigation was significantly shorter in vessels of all three sizes compared with conventional navigation (means of 19.6 sec, 5.9 sec, and 4.8 sec vs. 48.8 sec, 49.8 sec, and 32.7 sec for small, medium, and large vessels, respectively; P < .05). Procedure times with use of the magnetic guide wire (149.6 sec, 52.1 sec, and 39.9 sec) were not significantly different than those with conventional navigation (60.4 sec, 68.6 sec, and 47.7 sec). CONCLUSIONS: The MGS enables exact endovascular navigation with shorter fluoroscopy time in an in vitro model. The MGS may offer opportunities to reduce x-ray exposure to patients and staff.

Catheterization↗
PubMed1991

The effects of estrogens on cartilage degradation using in vivo and in vitro models.

We have investigated the effects of estradiol on cartilage breakdown. The model of arthritis used involved the subcutaneous implantation of rat femoral head cartilage (FHCs) into the dorsal region of female mice. The FHCs had been previously wrapped in sterile 5 mg cotton to provoke the growth of granulomatous tissue adjacent to cartilage. Animals were dosed daily, a day after implantation, for 14 days with either 17 beta-estradiol or tamoxifen at different doses. The FHCs were removed and analysed for glycosaminoglycan (GAG) content. The results demonstrated that estradiol accelerated cartilage breakdown in a dose dependent manner, an effect which is blocked by tamoxifen. In addition, estradiol was found to increase cartilage degradation in an in vitro model using FHCs. This effect was enhanced by the addition of fibroblasts.

Animals↗