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PubMed2003

A tissue level tolerance criterion for living brain developed with an in vitro model of traumatic mechanical loading.

Traumatic brain injury (TBI) is caused by brain deformations resulting in the pathophysiological activation of cellular cascades which produce delayed cell damage and death. Understanding the consequences of mechanical injuries on living brain tissue continues to be a significant challenge. We have developed a reproducible tissue culture model of TBI which employs organotypic brain slice cultures to study the relationship between mechanical stimuli and the resultant biological response of living brain tissue. The device allows for the independent control of tissue strain (up to 100%) and strain rate (up to 150 s-1) so that tolerance criteria at the tissue level can be developed for the interpretation of computational simulations. The application of texture correlation image analysis algorithms to high speed video of the dynamic deformation allows for the direct calculation of substrate strain and strain rate which was found to be equi-biaxial and independent of radial position. Precisely controlled, mechanical injuries were applied to organotypic hippocampal slice cultures, and resultant cell death was quantified. Cell death was found to be dependent on both strain magnitude and rate and required several days to develop. An immunohistological examination of injured cultures with antibodies to amyloid precursor protein revealed the presence of traumatic axonal injury, suggesting that the model closely replicates in vivo TBI but with advantages gained in vitro. We anticipate that a combined in vitro approach with optical strain mapping will provide a more detailed understanding of the dependence of brain cell injury and death on strain and strain rate.

Journal Article↗
PubMed1990

An experimental in vitro model for evaluating drugs against protoscoleces of Echinococcus granulosus.

Pharmacological studies carried out on protoscoleces in vitro to standardize conditions that would permit a preliminary estimate of the efficacy of drugs with potential activity against Echinococcus granulosus are reported. Media such as PBS and Hanks solution, maintenance temperature, different pH values and concentrations of various solvents have been tested to check the effects on protoscolex survival in tubes in vitro. Mebendazole has been used as the pharmacological standard reference. Changes in the viability of protoscoleces have been used to demonstrate pharmacological activity. Best conditions were obtained employing Hanks solution and propylene glycol at low concentrations. Mebendazole was not completely effective at the concentrations achievable in human therapy. Linear, reproducible results demonstrated that Hanks solution provides an ideal medium for pharmacological studies. Among tested solvents, propylene glycol and dimethyl sulphoxide showed no lethal activity at low concentrations. At concentrations similar to those normally obtained in human sera, mebendazole, as in vivo, demonstrated only partial lethality for protoscoleces. The present study represents a new experimental approach to chemotherapy of hydatid disease.

Animals↗
PubMed1989

[Hormone dependence of malignant ovarian tumors--an in vitro model].

The steroid hormone receptor content of 32 malignant ovarian tumors was compared with the in vitro effectiveness of 4 hydroxytamoxifen (OH-TAM) and medroxy-progesterone acetate (MPA) tested in the Human Tumor Colony Forming Assay (HTCFA). The sensitivity for the receptor determination was 5 fmol/mg cytosol protein. Estrogen receptors (ER) and progesterone receptors (PR) were found in 15 (47%) and 13 (41%) of the tumors respectively. As standard criteria for the HTCFA, a minimum of 30 colonies with a diameter of more than 60 microns and 100 microns was used in the control group. The in-vitro sensitivity of ovarian tumors to OH-TAM and MPA was independent on the ER or PR content, and amounted to 9% for OH-TAM and 6% for MPA. However, all 12 ER-PR-tumors proved resistant to OH-TAM and MPA. 18 ovarian tumors showed a sufficient colony growth, even in the size class exceeding 100 microns. With a minimum colony size of 60 microns and 100 microns, 17% and 33% respectively were sensitive to OH-TAM. A similar effect on the proliferative capacity of the Tumor Colony Forming Units (TCFUs), unrelated to PR, was observed with MPA. Dependent on colony size, we found an increasing sensitivity against MPA from 11% to 22%. The in-vitro effectiveness of both OH-TAM and MPA in the clonogenic assay of malignant ovarian tumors was certainly not as potent as suggested by the results obtained in biochemical steroid hormone receptor analysis. To prove the hormonal response in the HTCFA, it is necessary to determine number and size of the colonies as an expression of their proliferative potential.

Adenocarcinoma↗
PubMed2002

Characteristic distribution of gap junctions in rat lacrimal gland in vivo and reconstruction of a gap junction in an in vitro model.

We investigated localization of the gap junction in rat lacrimal gland in vivo and in vitro using electron microscopy and immunostaining with anti-connexin32 (Cx32) monoclonal antibody (HAM8). In immunofluorescence study of lacrimal gland tissues, Cx32 protein appeared to exist not only at the intercellular borders of acinar cells, but also in the basal regions, where there apparently was no contact with adjacent acinar cells. Thin sectioning and immunoelectron microscopy revealed that lacrimal acinar cells formed autocellular gap junctions (reflexive gap junctions) in the basal regions and intercellular gap junctions with adjacent acinar cell membranes. In immunofluorescence study of primary culture, Cx32 protein was found on the free surfaces of isolated acinar cells at the early stage of culture. With culturing time, cell aggregates were formed. We observed Cx32 immunoreactivity between acinar cells in these aggregates, but not on their free surface. Electron microscopic study confirmed that these aggregates possessed intercellular gap junctions and morphologically differentiated acinar-like structures. However, reflexive gap junctions were not observed in these aggregates. In conclusion, lacrimal acinar cells form intercellular and reflexive gap junctions in vivo. On the other hand, the existence of an acinar-like structure and intercellular gap junctions indicates that acinar cells differentiated in vitro morphologically. The cells may communicate with each other through these junctions, organizing themselves into an acinar cell network in an in vitro situation.

Animals↗
PubMed1996

Comparison of toxicity induced by HT-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model.

HT-2 toxin is a trichothecene mycotoxin occurring naturally in various agricultural products. Many in vitro studies have shown that HT-2 toxin is a major metabolite of the parent compound T-2 toxin. In man as well as in animals both toxins have been shown to cause alimentary intoxications and haematological disorders. Granulomonocytic progenitors (CFU-GM) from human umbilical cord blood and from rat bone marrow were cultured in the presence of HT-2 toxin (10(-7) M to 10(-10) M) for 14 days. The concentration-effect relationship was studied and the IC50 were determined on Days 7, 10 and 14. The IC50 was 1.8 x 10(-9) M, 3.5 x 10(-9) M and 1.8 x 10(-9) M for human cells, and 2 x 10(-9) M, 2.3 x 10(-9) M and 2.2 x 10(-9) M for rat cells. The results have been compared with previous findings for T-2 toxin. Although T-2 and HT-2 toxins had a similar IC50 on human and rat CFU-GM, the expression of their cytotoxicities was different. These findings are relevant to investigation of the cellular targets of T-2 and HT-2 in elucidating the mechanism of trichothecene haematotoxicity.

Animals↗
PubMedDate not supplied

[In vitro model for the analysis of the interaction between Shigella flexneri and the intestinal epithelium].

An in vitro study of the adhesion and invasion of Shigella flexneri was implemented, by means of incubation of laminary cuts of cecal mucosa of Dunkin-Hartley guinea pigs in a suspension of Shigella flexneri, which was isolated from a patient with bacillary dysentery. The laminae were placed in plastic chambers for two hours at 37%C. After this the bacterial suspension was discarded so as to eliminate the bacilli which were not adhered. The epithelium was washed with saline and was processed for analysis with scanning electron microscope. The topology of the mucosa incubated with Shigella flexneri was similar to that of the witnesses. The bacteria which adhered to the mucosa were dispersed individually or in clumps of varied numbers. The main alteration observed upon the epithelial surface were depressions due to a lateral separation of the microvilli which may have originated the endocytic stomas containing bacterias. The results of this study allow the proposition of the use of explants, so as to study the interaction between Shigella flexneri and the intestinal epithelium, with the possibility of modifying different experimental variables.

Animals↗
PubMed1997

Cartilage damage as a result of hemarthrosis in a human in vitro model.

OBJECTIVE: To investigate the direct effect of blood and blood components on human cartilage in vitro. METHODS: Healthy human articular cartilage tissue was obtained post mortem and cultured according to standard procedures. The harmful effects of whole blood and various isolated blood components as well as the reversibility of these effects were assessed by means of proteoglycan synthesis and proteoglycan release. RESULTS: Whole blood anticoagulated with heparin, coagulated blood, mononuclear cells (MNC), erythrocytes [red blood cells (RBC)] and plasma, in this order of potency, decreased proteoglycan synthesis in a dose dependent manner. The effect of the combination of MNC and RBC in concentrations equivalent to those in whole blood was significantly greater than the effect of each of these isolated components alone and did not differ from that of whole blood. Moreover, cartilage exposed for 4 days to this combination exhibited irreversible inhibition of proteoglycan synthesis. The effect was similar to that of whole blood, the opposite of that of the individual components or other combinations. CONCLUSION: Results suggest a direct irreversible harmful effect of whole blood on cartilage, whereby MNC and RBC together are the main factors. Taking into account that the concentration of blood during hemarthrosis approaches 100% and the natural evacuation time of blood from a joint is about 4 days, our results suggest that prompt evacuation of intraarticular blood or prevention of intraarticular bleeding might be crucial in preventing cartilage damage.

Adult↗
PubMed1997

Evaluation of Yucatan micropig skin for use as an in vitro model for skin permeation study.

Skin permeation rates of five non-steroidal anti-inflammatory drugs, three of them lipophilic (ibuprofen, indomethacin and ketoprofen) and two hydrophilic (sodium diclofenac and antipyrine), through Yucatan hairless micropig (YMP) full-thickness skin were determined in vitro using Franz-type diffusion cells, and the usefulness of YMP skin as an animal model skin was investigated. Five-month-old YMP skin showed small variation with respect to drug permeation rates, if the region of skin site was the same. But skin from 6-month-old YMPs showed faster and varied permeation rates due to skin defects. In the case of hydrophilic drugs, no effects on permeation due to the region from which the skin was obtained were observed, although higher permeation rates were observed through the flank than through dorsal skin in the case of ibuprofen and ketoprofen. For YMP skin, the permeation coefficients of drugs were lower than those for hairless rat skin, especially for hydrophilic drugs. Compared with reported human data on permeation coefficients, the permeation coefficients of drugs through YMP skin were 1/2-1/8 of those through human skin, regardless of whether the drugs were hydrophilic or lipophilic. YMP skin could be used as an in vitro animal skin model because of the reproducibility of the permeation rate of drugs through this skin and its similarity to human skin.

Administration, Topical↗
PubMed2002

Calcium-dependent epileptogenesis in an in vitro model of stroke-induced "epilepsy".

PURPOSE: Stroke is the most common cause of acquired epilepsy. The purpose of this investigation was to characterize the role of calcium in the in vitro, glutamate injury-induced epileptogenesis model of stoke-induced epilepsy. METHODS: Fura-2 calcium imaging and whole-cell current clamp electrophysiology techniques were used to measure short-term changes in neuronal free intracellular calcium concentration and long-term alterations in neuronal excitability in response to epileptogenic glutamate injury (20 microM, 10 min) under various extracellular calcium conditions and in the presence of different glutamate-receptor antagonists. RESULTS: Glutamate injury-induced epileptogenesis was associated with prolonged, reversible elevations of free intracellular calcium concentration during and immediately after injury and chronic hyperexcitability manifested as spontaneous recurrent epileptiform discharges for the remaining life of the cultures. Epileptogenic glutamate exposure performed in solutions containing low extracellular calcium, barium substituted for calcium, or N-methyl-d-aspartate (NMDA)-receptor antagonists reduced the duration of intracellular calcium elevation and inhibited epileptogenesis. Antagonism of non-NMDA-receptor subtypes had no effect on glutamate injury-induced calcium changes or the induction epileptogenesis. The duration of the calcium elevation and the total calcium load statistically correlated with the development of epileptogenesis. Comparable elevations in neuronal calcium induced by non-glutamate receptor-mediated pathways did not cause epileptogenesis. CONCLUSIONS: This investigation indicates that the glutamate injury-induced epileptogenesis model of stroke-induced epilepsy is calcium dependent and requires NMDA-receptor activation. Further, these experiments suggest that prolonged, reversible elevations in neuronal free intracellular calcium initiate the long-term plasticity changes that underlie the development of injury-induced epilepsy.

Animals↗
PubMed1997

In vitro model of characterizing the effects of compressive loading on proteoglycans in anatomically intact articular cartilage.

An in vitro method has been developed of cultivating anatomically intact articular cartilage (humeral head of dog) while excluding both bone tissue and other connective tissues with a condom, which controls changes in hydration and minimizes loss of proteoglycans. Compressive loading experiments were carried out with the condom-covered humeral head, to which contact stresses of 2.1 MPa, 3.3 MPa and 6.4 MPa, respectively, were applied with a rubber disc of a simple loading apparatus. The model makes it possible, to compare experimentally loaded regions with experimentally unloaded regions in both the same joint area and the contralateral control joint. Intermittent pressure loading (4-s-on/16-s-off-cycle) during 2 hours of pulse-experiment and 18 hours of chase-experiment resulted in a 40% increase in proteoglycan synthesis rate at 2.1 MPa, an unaltered synthesis rate at 3.3 MPa, and a 45% decrease in proteoglycan synthesis rate at 6.4 MPa, as measured by 35S-sulfate incorporation. No significant increase in degradation rate of proteoglycans was noted during the various loading experiments.

Animals↗
PubMed1984

Induction of acetylcholine receptor-specific suppression. An in vitro model of antigen-specific immunosuppression in myasthenia gravis.

This report describes the in vivo and in vitro induction of murine (AChR)-specific suppressor T cells (Ts) and T cell factors (TsF), and the development of an appropriate assay system for their measurement. The assay described is based on the in vitro Mishell-Dutton culture system. Using this assay, it was shown that the AChR-specific helper cell is an Lyt-2- radiosensitive T cell. Moreover, the proliferating cell measured in the lymphocyte transformation assay was shown to provide AChR-specific T cell help. In vivo induction of Ts cells is achieved by injection of soluble AChR; potent AChR-specific suppression is found in the spleen 1 wk later. In vitro induction of Ts cells involves the primary education of naive splenocytes by culturing them with high concentrations of AChR. Both the in vivo- and in vitro-induced Ts cells were shown to secrete AChR-specific factors that mediate their suppressive effects. The possibility of specifically suppressing the AChR-immune response may be of a particular clinical importance since the AChR is the target autoantigen in the neuromuscular autoimmune disease myasthenia gravis.

Animals↗
PubMed1993

WIF-B cells: an in vitro model for studies of hepatocyte polarity.

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Animals↗
PubMed2000

Use of simple and complex in vitro models for multiparameter characterization of human blood-material/device interactions.

Medical devices, intended for blood contacting applications, undergo extensive in vitro testing followed by animal and clinical feasibility studies. Besides the use of materials known to be intrinsically blood-compatible, the surface of such devices is often modified with a coating in order to improve the performance characteristics during blood exposure. In vitro evaluation of blood-device interactions accompanies the product development cycle from the early design phase using basic material geometries until final finished-product testing. Specific test strategies can vary significantly depending on the end application, the particular study objectives and variables of interest, and cost. To examine the degree to which findings derived from two different in vitro approaches complement one another, this report contrasts findings from a simple multipass loop model with findings from a simulated cardiopulmonary bypass (CPB) model. The loop model consists of tubular test materials, with and without surface modification, formed into valved Chandler loops. The CPB model has an oxygenator with and without surface modification connected to a reservoir and a blood pump. The surface modifications studied in this report are the Carmeda BioActive Surface and Duraflo II heparin coatings. Common blood parameters in the categories of coagulation, platelets, hematology, and immunology were monitored in each model. Ideal models employ the optimal level of complexity to study the design variables of interest and to meet practical cost considerations. In the case of medical device design studies, such models should also be predictive of performance. In the more complex and realistic simulated CPB model, experimental design and cost factors prevented easy/optimum manipulation of critical variables such as blood donor (use of paired samples) and heparin level. Testing in the simpler loop model, on the other hand, readily offered manipulation of these variables, and produced findings which overlapped with observations from the more complex CPB model. Thus, the models described here complimented one another. Moreover, conclusions from consistent findings, such as favorable responses associated with the heparin coatings, between the two models were considered to be more robust.

Biocompatible Materials↗
PubMedDate not supplied

In vivo and in vitro models of medulloblastomas and other primitive neuroectodermal brain tumors of childhood.

Recent advances in understanding the basic biology of the neoplastic cells that populate childhood primitive neuroectodermal tumors (PNET) of the central nervous system (CNS) underline several unique properties of these common pediatric brain neoplasms. For example, studies of posterior fossa cerebellar medulloblastomas (MB), a prototypical group of brain tumors that comprise the largest class of PNET, suggest that the molecular phenotype of subpopulations of neoplastic cells in MB partially recapitulates stages in the acquisition of the neuronal phenotype by normal developing human CNS progenitor cells. However, as reviewed here, it appears that the neoplastic cells in MB exhibit one or more molecular defects in the sequence of normal maturational events that enable CNS progenitor cells to exit the cell cycle, become committed to the neuronal lineage, and undergo terminal differentiation into fully mature, permanently postmitotic CNS neurons. Indeed, since PNET emerge almost exclusively in early childhood, the induction of PNET may result from genetic lesions that arise in developing CNS progenitor cells thereby preventing these neural precursors from executing normal programs of lineage commitment and differentiation in the CNS. Clarification of how lineage commitment and maturation in PNET comprised of neuron-like tumor cells deviate from normal CNS development may clarify how oncogenes and tumor suppressor genes exert their effects in a cell type specific manner at different stages in the normal maturation of CNS cells. Recently, a number of potentially effective in vitro and in vivo model systems of PNET have been developed. Since these model systems could facilitate efforts to elucidate mechanisms of neoplastic transformation and tumor progression in the CNS, we review the potential utility of several recently described in vitro (e.g., MB cell lines) and in vivo (e.g., transgenic mice) experimental systems as models of authentic childhood CNS neoplasms.

Animals↗
PubMed1979

In vitro model for stretch-induced hypertrophy of skeletal muscle.

Mechanical stretch of embryonic chicken skeletal myotubes developed in vitro leads to many of the biochemical changes seen in skeletal muscle hypertrophy. These include increased amino acid accumulation, increased incorporation of amino acids into general cellular proteins and myosin heavy chains, and increased accumulation of total protein and myosin heavy chains. This model system should aid in understanding how the growth rate of skeletal muscle is regulated by its activity.

Amino Acids↗
PubMed2000

Complement activation reaches maximum during equilibrium between antigen and antibody in an in vitro model for thrombolysis with streptokinase.

Treatment with streptokinase (SK) of patients with acute myocardial infarction (AMI) is known to activate the complement system. In a previous investigation we found a great variation in the degree of complement activation taking place during infusion of SK in patients with AMI (range: 0-293% increase in pretreatment complement split product 3d (C3d) levels). In this in vitro study we added SK to serum with different concentrations of SK-antibodies (SK-Ab) and found that both the relative concentrations and the total concentrations of SK and Ab determined the degree of complement activation elicited by SK. This finding was in accordance with the Kendall-Heidelberger equation of antigen-antibody equivalence. Furthermore, we found parallel rises in C3d and C4d, indicating that complement was activated via the classical pathway. A condition for maximal complement activation is antigen-antibody equivalence.

Antigen-Antibody Reactions↗