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PubMed1989

Use of an in vitro model for the assessment of muscle damage from intramuscular injections: in vitro-in vivo correlation and predictability with mixed solvent systems.

The potential of binary mixtures of propylene glycol-water, ethanol-water, and polyethylene glycol 400-water to cause skeletal muscle damage (myotoxicity) following intramuscular injection was examined with an in vitro model using the isolated rat muscle. At moderate concentrations (20-40%, v/v) of the organic cosolvent, the order of myotoxicity was propylene glycol greater than ethanol much greater than polyethylene glycol 400. The in vitro results were then compared with in vivo toxicity in rabbits after injection of normal saline, 40% (v/v) polyethylene glycol 400, 40% (v/v) propylene glycol, indocyanine green in normal saline, and indocyanine green in 40% (v/v) propylene glycol. Employing the area under the creatine kinase activity curve from 0 to 72 hr as the index of skeletal muscle damage, an excellent in vitro-in vivo correlation was observed. The basic myotoxicity relationships obtained from the binary cosolvent systems were then used to examine the myotoxicity of ternary organic cosolvent mixtures. Several mixed solvent systems with the same theoretical molar solubilization power for a model compound, diazepam, were selected to determine (1) if myotoxicity can be reduced by changing the composition of the ternary mixtures and (2) if myotoxicity of the individual components is additive. For the solvent systems containing propylene glycol, ethanol, and water, the total myotoxicity equaled the sum of the individual myotoxicity of each component. In contrast, for the solvent systems containing polyethylene glycol 400, the total myotoxicity was only half of the sum of individual toxicities. These results suggest that polyethylene glycol 400 in mixed cosolvent systems might have a protective effect on the myotoxicity generated by intramuscular injections.

Animals↗

PubMedDate not supplied

Establishment of an in vitro model using NR8383 cells and Mycobacterium bovis Calmette-Guerin that mimics a chronic infection of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis infection affects one-third of the world's population and causes the death of three million people each year. To clarify details of M. tuberculosis survival strategies, it is important to establish a suitable in vitro model that mimics a chronic infection in alveolar macrophages by M. tuberculosis. For this reason, we established a new in vitro model using a rat alveolar macrophage cell line, NR8383. MATERIALS AND METHODS: Basic characteristics, including phagocytotic ability and production of nitrogen oxide and tumor necrosis factor in response to several stimuli, of NR8383 cells were compared with those of primary alveolar macrophages. The course after phagocytosis of live or killed M. bovis bacilli Calmette-Guerin (BCG) was examined over 21 days using NR8383 cells as the host. RESULTS: The characteristics that have been examined to date were nearly the same for both primary alveolar macrophage and NR8383 cells. Live BCG phagocytosed by NR8383 cells had successfully begun to grow in the cells within 7 days, while killed BCG were almost completely destroyed by 21 days. CONCLUSION: BCG-infected NR8383 cells are potentially a suitable in vitro model that mimics a chronic infection with M tuberculosis.

Animals↗

PubMed1976

An organotypic in vitro model system for studying pulmonary surfactant production by type II alveolar pneumonocytes.

This report describes an in vitro model system in which monodisperse fetal rat lung cells reorganize to form alveolar-like structures when cultured on a gelatin sponge matrix. The alveolar-like structures are composed of cells that have morphologic characteristics like those of the type II alveolar pneumonocytes of intact lung. These morphologic characteristics include lightly stained nuclei, microvilli on the apical surface, and osmiophilic lamellar bodies in the cytoplasm. The presence of osmiophilic lamellar bodies and tubular myelin in the lumen of the alveolar-like structures suggests that the cells in these structures are producing pulmonary surfactant. The formation and long-term maintenance of these alveolar-like structures provide a unique in vitro model system for studies of the synthesis, storage, and secretion of pulmonary surfactant.

Animals↗

PubMed1995

Prediction of the percutaneous penetration and metabolism of dodecyl decaethoxylate in rats using in vitro models.

Percutaneous absorption of a lipophilic surfactant, dodecyl decaethoxylate, can be predicted using in vitro models. In vivo, dermal penetration of dodecyl decaethoxylate was found to be 22.9% in 48 h. All of the absorbed dodecyl decaethoxylate in the rat was metabolised and excreted in expired air as carbon dioxide, or in the urine and faeces. Using rat skin mounted in the unoccluded flow-through diffusion cell with MEM as receptor fluid, in vivo absorption was predicted by the percentage of the applied dose recovered in the stratum corneum, epidermis, dermis and receptor fluid at 24 h (25%). Conversely, the penetration of dodecyl decaethoxylate was over-predicted in the unoccluded static diffusion cell using aqueous ethanol (50% v/v) as the receptor fluid where 49.4% recovered in the receptor fluid at 24 h. In vitro models may be used to predict percutaneous absorption and reduce animal use, provided a suitable receptor fluid is used in which the penetrant is soluble. Dermal metabolism of dodecyl decaethoxylate was low and not considered to influence dermal absorption.

Administration, Topical↗

PubMed1998

Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.

The processes of wound repair were investigated using an in vitro model of mechanical injury on confluent cell monolayers of either human umbilical vein endothelial cells (HUVEC), aortic endothelial (RAEC) or smooth muscle cells (VSMC) of the rat. A mechanical wounder was used to produce 11 parallel (400 microm wide) lesions across the monolayer and the movement of cells into the denuded area was quantified using image analysis. The lesioned area recovered completely in 72h, with proliferation occurring after 24h for endothelial cells and 18h for VSMC, as detected by an increase in cell numbers. The cell migration inhibitor Taxol (1ng/ml) abolished the increase in repair of HUVEC monolayers in the first 24h of repair, while actinomycin D had no effect before 24h but thereafter abolished the further repair which was associated with increased cell numbers. Repair of endothelial cells was accelerated by basic fibroblast growth factor (bFGF), vascular endothelial growth factor or platelet-derived growth factor-BB (PDGF), and in VSMC both bFGF and PDGF increased repair. This simple in vitro model of mechanical injury allows a quantitative study of the repair processes of a previously confluent monolayer and thus is a representation of mechanical damage in vivo.

Journal Article↗

PubMed1992

An in vitro model of rodent nongenotoxic hepatocarcinogenesis.

An in vitro model of liver in which rat hepatocytes are maintained as cocultures with nonparenchymal epithelial cells (NPC) derived from liver has been developed and characterized with respect to maintenance of hepatocyte viability and differentiated function. The system was then evaluated as a model for studying peroxisome proliferator-induced rodent liver nongenotoxic carcinogenesis. Within the coculture model, hepatocyte viability and morphology were maintained for 1 month or more within a system that is both easily accessible for microscopic examination and is free of any additives that may lead to artifacts. Even after 1 month or more, hepatocyte cocultures retained expression of the constitutive liver marker albumin. In addition, they maintained the ability to show induction of the peroxisome proliferator-inducible enzymes peroxisomal bifunctional enzyme (PBE) and cytochrome P450IVA1 in response to the peroxisome proliferator nafenopin. After 4 weeks, NPC cocultures showed a six- and a fourfold induction of PBE and cytochrome P450IVA1 expression, respectively, which compared well with the three- and fivefold induction seen in freshly isolated cells. This was paralleled by an increase in the cytoplasmic volume fraction of peroxisomes averaging eightfold. Interestingly, great heterogeneity was exhibited between adjacent hepatocytes in terms of the degree of peroxisome proliferation, a finding reflected by immunocytochemical staining which indicated heterogeneity in the level of expression of the peroxisome proliferator-inducible enzymes. Other cell lines representing different tissue types, morphologies, and species were also examined for their ability to support hepatocyte survival but were found to be ineffective, with the exception of a bovine corneal endothelial cell line. This line supported hepatocyte survival and maintenance of differentiated function but to a lesser extent than that observed with NPC. Ultrastructural examination of NPC cocultures revealed extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile canalicular structures. There were no junctional complexes between the hepatocytes and the supporting feeder cells with any contact being limited to a close association of the hepatocytes with the extracellular matrix presumably produced by the NPC. The data demonstrate that hepatocytes maintained in vitro within an NPC coculture system retain differentiated function and the ability to respond to the peroxisome proliferator class of nongenotoxic carcinogens. Cocultures will provide us with a model system for the study of changes in hepatocyte growth regulation during rodent liver nongenotoxic carcinogenesis.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2001

A two-compartment in vitro model for studies of modulation of nociceptive transmission.

Here we present a two-compartment in vitro model in which embryonic rat dorsal root ganglia (DRG) neurons are cultured separately from their target dorsal horn neurons. Although separated, synaptic contact can be established between the peripheral and central neurons since the system allows the DRG axons to project into the other compartment, which contains a network of dorsal horn neurons. The efficacy of the model was evaluated by immunocytochemical, calcium imaging and electrophysiological experiments. The results showed that a subpopulation of the DRG neurons had nociceptor characteristics and that these made synaptic contact with the dorsal horn network. Application of current pulses, according to the stimulus paradigm used, evoked action potentials in DRG axons selectively. This in turn gave rise to increased postsynaptic activity in the network of dorsal horn neurons. The model offers a high degree of efficiency since large numbers of DRG axons can be stimulated simultaneously, thus permitting recording of strong output responses from the dorsal horn neurons. This in vitro model provides a means for studying the mechanisms by which modulatory factors, such as immunoregulatory molecules, applied at either the PNS or the CNS level, can affect synaptic activity and nociceptive transmission in single neurons or network of neurons in the dorsal horn.

Action Potentials↗

PubMed1976

An in vitro model for evaluating neural stimulating electrodes.

A model for in vitro evaluation of materials for use as neural stimulation electrodes is developed. Critical areas of concern in developing an in vitro test model discussed include: selection of environment, choice of material, design of stimulating equipment, and analytical procedures used to evaluate materials response. A method of providing quantitative analysis of materials response to stimulation conditions is presented. Evaluation techniques involve the use of scanning electron microscopy, x-ray spectroscopy, atomic absorption spectrometry, and potentiographic and dielectric analysis of the test electrodes. A diagnostics matrix is presented which assigns a scale factor to quantify the relative corrosion response of the candidate materials. The corrosion response of Pt electrodes is evaluated in terms of scale factors assigned through use of the diagnostics matrix.

Corrosion↗

PubMed2005

Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells.

OBJECTIVES: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. DESIGN AND METHODS: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. CONCLUSION: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.

Animals↗

PubMed1994

Primary culture of rat gastric epithelial cells as an in vitro model to evaluate antiulcer agents.

Primary rat gastric cell cultures were investigated as an in vitro model for evaluating antiulcer agents. Following exposure to concentrations of up to 5 mg/mL of an antiulcer agent sucralfate, an aluminum hydroxide complex of sucrose octasulfate, cultured cells were treated with either pH 3.5 medium or 3.5 mM indomethacin. Cytoprotection was evaluated by colony forming efficiency, neutral red uptake, and 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) hydrolysis. By each measure, and depending on damaging agent, 2 and 5 mg/mL sucralfate provided partial (50% of untreated control) to near-complete (90% of untreated control) cytoprotection, respectively. Aluminum hydroxide also provided partial (55% of untreated control) to near-complete (more than 90% of untreated control) cytoprotection at 2 and 5 mg/mL, respectively, for the pH 3.5 medium-induced damage. Over a concentration range of 0.05 to 5 mg/mL, the potassium salt of sucrose octasulfate, KSOS, stimulated cell growth up to 40-60% over untreated controls but had little or no cytoprotective action in the presence of either 3.5 mM indomethacin or pH 3.5 medium. Overall results suggested that sucralfate may have at least two roles in influencing gastric epithelial cell function, cytoprotection and stimulation of cell growth in vitro. These observations serve as a basis for further study of in vitro models in evaluating the cytoprotective activity of antiulcer agents and their respective mechanisms of action.

Acids↗

PubMed1997

[In vitro models for the study of nerve lesions and potential neuroprotective drugs].

INTRODUCTION AND DEVELOPMENT: In order to study the neurotoxic effects of drugs and to search for neuroprotective agents, we need simple models that permit a quick screening of thousands of compounds. In vitro models with cellular cultures fulfill all this criteria and they are very appropriate to study the mechanism of action of drugs. As adult neurons are very difficult to maintain in culture, this kind of studies are carried out with fetal neurons, tumoral cells or chromaffin cells. CONCLUSIONS: This in vitro models have shown the neuroprotective effect of glutamate antagonists, calcium-channel blockers, free radical scavengers and other agents as CDP-choline, that promotes cellular membranes restoration.

Animals↗

PubMed2004

Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury.

We have developed a novel in vitro model of the glial scar that mimics the gradient of proteoglycan found in vivo after spinal cord injury. In this model, regenerated axons from adult sensory neurons that extended deeply into the gradient developed bulbous, vacuolated endings that looked remarkably similar to dystrophic endings formed in vivo. We demonstrate that despite their highly abnormal appearance and stalled forward progress, dystrophic endings are extremely dynamic both in vitro and in vivo after spinal cord injury. Time-lapse movies demonstrated that dystrophic endings continually send out membrane veils and endocytose large membrane vesicles at the leading edge, which were then retrogradely transported to the rear of the "growth cone." This direction of movement is contrary to membrane dynamics that occur during normal neurite outgrowth. As further evidence of this motility, dystrophic endings endocytosed large amounts of dextran both in vitro and in vivo. We now have an in vitro model of the glial scar that may serve as a potent tool for developing and screening potential treatments to help promote regeneration past the lesion in vivo.

Aggrecans↗

PubMed1996

The effects of mechanical compression and hypoxia on nerve root and dorsal root ganglia. An analysis of ectopic firing using an in vitro model.

STUDY DESIGN: This study analyzed in vitro experiments of ectopic firing evoked by mechanical compression or hypoxia of canine lumbar dorsal roots with dorsal root ganglia using an in vitro model. OBJECTIVES: The results were correlated to understand the pathophysiology of radiculopathy, which manifests abnormal sensation and pain. SUMMARY OF BACKGROUND DATA: It has been speculated that blood flow in the nerve root and mechanical compression play major roles in the production of radiculopathy symptoms. However, no precise experimental studies have been conducted on the relationship between these factors and the development of ectopic firing. METHODS: Canine lumbar dorsal roots with dorsal root ganglia were immersed in an oxygenated artificial cerebrospinal fluid, and activity of the nerve root was recorded using bipolar platinum electrodes. Using this model, the effects of quantitative mechanical compression and hypoxia on the ectopic firing were analyzed. RESULTS: When compression was applied, mechanical thresholds for eliciting firing were much lower in dorsal root ganglia than in dorsal roots, and the firing lasted for a longer period in dorsal root ganglia. Under hypoxia, dorsal root ganglia showed firing, and their thresholds from mechanical stimuli decreased significantly. In dorsal roots, impulse propagation was not affected, whereas firing was seen under the hypoxic condition. CONCLUSION: Dorsal root ganglia are highly sensitive to mechanical compression and hypoxia and closely related to abnormal sensations and pain in radiculopathy.

Action Potentials↗

PubMed2000

Mechanism responsible for the formation of focal swellings on injured neuronal processes using a novel in vitro model of axonal injury.

Animals↗

PubMed2001

Studies on the mechanisms responsible for the formation of focal swellings on neuronal processes using a novel in vitro model of axonal injury.

A novel in vitro model of axonal injury using PC12 cells was designed to introduce traumatic alterations on neuronal processes and to identify mechanisms responsible for the formation of focal swellings by observation with phase-contrast and transmission electron microscopes. The injury on the processes was produced by one-dimensional, horizontal oscillation. The fluid shear stress applied by the oscillation did not exceed 380 dyne/cm2. The injured processes showed two forms. One involved an increase in the terminal diameter of the processes and the other entailed beading along the injured portions. Long-term observation of cellular responses to the mechanical insult disclosed that the terminal swelling coincided with the detachment of growth cones from the culture plate. The finding suggests that the detachment of the growth cone destroys the cytoskeletal network, which determines and maintains the cell shape, resulting in spherical deformation of the processes. When the cytoskeletal destruction occurred at non-terminal sites along the processes, spherical deformations developed slowly, and these appeared as beads. The beading also caused the detachment of the growth cones. As the most proximal bead grew, they absorbed the distal segment and their growth cones were pulled proximally with the spreading cytoskeletal destruction. The processes with terminal swellings as well as the bead segments showed regeneration with time evidence of and growth cone formation.

Animals↗

PubMed2006

In vitro models for studying trophoblast transcellular transport.

In vitro models have proven to be effective in studying the placental transporters that play a role in the exchange of nutrients, waste products, and drugs between the maternal and fetal circulations. Although primary cultures of trophoblast cells can be used to perform uptake, efflux, and metabolism studies, only the rodent HRP-1 and the human BeWo cell lines have been shown to form confluent monolayers when grown on semi-permeable membranes. Protocols for the revival, maintenance, passage, and growth of BeWo cells for transporter expression and transcellular transport studies are provided.

Cell Culture Techniques↗

PubMed1991

Drug effects on a novel in vitro model of cartilage breakdown.

A new in vitro model for studying cartilage breakdown has been utilised in this work. Polymorphonuclear neutrophils (PMNs) with phorbol myristate acetate (PMA) were layered onto 2 microns cryostat sections of bovine nasal cartilage. After incubation, the sections were fixed, stained, and the amount of glycosaminoglycan (GAG) contents measured by microdensitometry. PMNs caused GAG loss from sections and this was greatly enhanced when the PMNs were activated by PMA. Various pharmacological agents were then added to the system, namely acetyl salicylic acid, indomethacin, ibuprofen, piroxicam, dexamethasone, D-penicillamine, chloroquine and BN50548. The drugs tested had no direct effect on cartilage matrix, nor did they affect GAG loss from sections treated with non-stimulated PMNs. However, BN50548, a novel protease inhibitor, afforded a dose response protection of cartilage section from GAG loss by PMA stimulated PMNs. This model may prove to be of value in screening novel antiproteases with chondroprotective activity.

Animals↗

PubMed2001

Glutamate injury-induced epileptogenesis in hippocampal neurons: an in vitro model of stroke-induced "epilepsy".

BACKGROUND AND PURPOSE: Stroke is the major cause of acquired epilepsy. The mechanisms of ischemia-induced epileptogenesis are not understood, but glutamate is associated with both ischemia-induced injury and epileptogenesis in several models. The objective of this study was to develop an in vitro model of epileptogenesis induced by glutamate injury in hippocampal neurons as observed during stroke. METHODS: Primary hippocampal cultures were exposed to 5 micromol/L glutamate for various durations. Whole-cell current clamp electrophysiology was used to monitor the acute effects of glutamate on neurons and chronic alterations in neuronal excitability up to 8 days after glutamate exposure. RESULTS: A single, 30-minute, 5-micromol/L glutamate exposure produced a subset of neurons that died and a larger population of injured neurons that survived. Neuronal injury was characterized by prolonged reversible membrane depolarization, loss of synaptic activity, and neuronal swelling. Surviving neurons manifested spontaneous, recurrent, epileptiform discharges in neural networks characterized by paroxysmal depolarizing shifts and high-frequency spike firing that persisted for the life of the culture. CONCLUSIONS: This study demonstrates that glutamate injury produced a permanent epileptiform phenotype expressed as spontaneous, recurrent epileptiform discharges for the life of the hippocampal neuronal culture. These results suggest a novel in vitro model of glutamate injury-induced epileptogenesis that may help elucidate some of the mechanisms that underlie stroke-induced epilepsy.

Action Potentials↗