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PubMed2006

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles.

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.

Animals↗
PubMed1992

In vitro particulate adherence to fibronectin: correlation with in vivo particulate adherence to sites of bladder injury.

This study examined the role of fibronectin in promoting particulate attachment to sites of urothelial injury. Variables influencing adherence of the rat transitional carcinoma cell line 4909 and "non-cellular" styrene-divinylbenzene microspheres to fibronectin were studied in an in vitro system. A soluble synthetic peptide fragment (Gly-Arg-Gly-Asp-Ser [GRGDS]) duplicating the receptor binding domain of fibronectin (RGD) was used to determine whether cell adherence could be inhibited by fibronectin receptor blockade. In vitro findings were correlated with an in vivo assay of both cellular and non-cellular particulate adherence to injured urothelium. Time, plated cell density, substrate concentration, GRGDS concentration, and cell viability, were all found to be significant independent variables influencing in vitro cellular adherence (p less than 0.0001). Receptor blockade with GRGDS significantly decreased in vitro tumor cell adherence to fibronectin. In vitro microsphere binding increased as a direct function of fibronectin concentration but was not time dependent (p less than 0.0001 and p = 0.14 for fibronectin concentration and time respectively). The in vivo adherence of both tumor cells and microspheres was significantly increased in injured bladders compared to controls (p less than 0.01). Receptor blockade with GRGDS failed to inhibit in vivo cell adherence to sites of urothelial injury. Microspheres proved to be competitive inhibitors of cellular adherence in competitive binding assays. In vitro microsphere binding demonstrated a pH dependence with maximal binding at pH 7.2. These data suggest that in vitro tumor cell adherence to fibronectin differs from in vivo tumor cell adherence to sites of urothelial injury. Manipulations which inhibit in vitro adherence, specifically fibronectin receptor blockade and cell death, fail to effect in vivo binding to the extreme that non-cellular particulate appears to bind to the same site, and with similar affinity, as cellular particles.

Animals↗
PubMed2002

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes.

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

Animals↗
PubMed1999

Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium-ionophore activation in the pig.

In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitro-matured porcine oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as handling controls. After subsequent in vitro-culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved with fresh in vitro-capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In Experiment 2, in vitro matured oocytes were injected with freshly ejaculated in vitro-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 microM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Ca-ionophore treatment after injection of spermatozoa was ineffective at 100 microM, where at 50 microM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperm-injected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed.

Animals↗
PubMedDate not supplied

In vitro differentiation and ultrastructure of human extravillous trophoblast (EVT) cells.

Tissue explants of anchoring villi from the first trimester placentae cultured on extracellular matrix (Matrigel) give rise to EVT cells in vitro. This study was designed to address two issues important for further application of the described in vitro model: first, were the observed EVT cells derived by cell proliferation in vitro and second, what is the degree of homology between the in vivo and the in vitro differentiated EVT cells. The cultures (tissue and matrix) were prepared for light and electron microscopic (EM) examinations. Semi-thin sections from Spurr epoxy resin-embedded tissue were used to 'pop-off' the selected area for EM examination. Cell proliferation in vitro was assessed immunohistochemically using proliferative cell nuclear antigen (PCNA) antibodies. Since positive hPL immunostaining has been consistently demonstrated in the invasive subpopulation of EVT cells from placental bed in situ, hPL staining was used as a marker of EVT cell differentiation in vitro. It has been demonstrated that PCNA antibodies immunostained nuclei of cytotrophoblast cells from cell column at the base of the anchoring villi, indicating that these cells expressed proliferative activity in vitro. Cytotrophoblast proliferation resulted in the formation of the flattened zone of cell outgrowths at the tip of anchoring villi. Cells from the distal layer of the cell column detached gradually and migrated into the surrounding matrix. These cells appeared as individual, round-shaped EVT cells with smooth surface cell membrane. Their cytoplasm was rich in glycogen and contained large lipid droplets and flattened cisternae of the RER. Positive PCNA immunostaining, along with the presence of mitotic figures, indicated that EVT cells in vitro retained the ability for cell proliferation. As a result of cell proliferation and migration, the number of EVT cells increased during the culture period of 4 days. EVT cell glycogen content and lipid stores decreased progressively as they migrated into the matrix. Individual EVT cells, as well as EVT cell clusters, became surrounded by the clear zone of digested matrix. Some cells started to express strong positive staining with hPL antibodies as soon as they had migrated outside the villous explant. By day 4 of culture, a small percentage of EVT cells (about 5-10%) ceased to migrate, firmly attached to the substratum and appeared as irregular shaped cells with filopodia-like projections. Their cytoplasm contained dilated cisternae of RER, a small number of glycogen granules and bundles of actin-like filaments located in the cytoplasm inside the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Differentiation↗
PubMed2003

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii).

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

Animals↗
PubMed2006

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy.

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

Animals↗
PubMed1997

Concentration-dependent inhibitory effects of apolipoprotein E on Alzheimer's beta-amyloid fibril formation in vitro.

Recently, many research groups have examined the effect of apolipoprotein E (apoE) on beta-amyloid fibril (betaAf) formation in vitro. However, their data were somewhat controversial and no exact kinetic assessment of the role of apoE has thus far been available. We examined the effect of human apoE on betaAf formation in vitro, starting with various concentrations of freshly prepared beta-amyloid(1-40) (beta1-40) and using fluorescence spectroscopy with thioflavine T. When 50 microM of beta1-40 was incubated with a 1:1000 to 1:100 molar ratio of apoE, a dose-dependent inhibitory effect of apoE was observed. Both the nucleation and extension phases of betaAf formation in vitro were inhibited by apoE. On the other hand, when 300 microM of beta1-40 was incubated with a 1:100 molar ratio of apoE, the inhibitory effect of apoE was completely abolished. We then focused our study on the kinetics of the inhibitory effect of apoE on the extension phase of betaAf formation in vitro, utilizing the recently established first-order kinetic model of betaAf extension in vitro [Naiki, H., & Nakakuki, K. (1996) Lab. Invest. 74, 374-383]. The mathematical treatment of the data suggests that apoE inhibits the extension of betaAf in vitro, by making a complex with beta1-40, thus eliminating free beta1-40 from the reaction mixture. The equilibrium association constant with beta1-40 was practically the same among the three major recombinant apoE isoforms. These results indicate that the effects of apoE on betaAf formation in vitro is differential and could settle some of the controversy about beta-amyloid-apoE interaction in vitro.

Alzheimer Disease↗
PubMed2003

The connection between in vitro water uptake and in vivo skin moisturization.

BACKGROUND/PURPOSE: Humectancy or hygroscopy is the water absorption tendency of a substance from the surroundings. Our interest, from the clinical point of view, consists of correlating this tendency in vitro and its effect in vivo for the development of drugs and formulations for the treatment of dry skin syndrome or diseases accompanied by dry skin. METHOD: In vitro, water absorption was measured using the comparative isopiestic method. This method is based on bringing the vapor of the water to isothermal equilibrium between a reference system and the material to be studied. The in vivo model on guinea-pigs for the dry skin syndrome tested the therapeutic ability of mono-, di- and tri-glycerols to provide moisture to dry skin leading to healing. The moisture content in the stratum corneum was measured with a Corneometer CM 825 PC that measures merely the presence of high dielectric material (humectant or water), whereas the Mexameter MX 16 measures a pathological parameter - the erythema. RESULTS: Adding hydroxyl groups to a consecutive set of polyhydroxyalkanes increases the humectancy of the polyols in vitro. This elevation was found to be linear at low relative humidities (Relative humidity=31.9% and 37 degrees C). In vivo, moisture was returned to normal within a week in all three groups. However, only glycerol managed to abolish the erythema within 7 days. CONCLUSION: A rise in water absorption ability in vitro, at a rate of about 0.25 mol water per hydroxyl group was revealed in a consecutive set of glycerols (mono-, di- and tri-glycerols). One would expect that the better humectant is a material, i.e. in which the higher its physical ability to hold water in vitro the more effective it will be in recovering skin dryness. We have found, however, that glycerol, which has the lowest humectant activity in vitro, from the set of glycerols, di- and tri-glycerol, has been proven to be the best for eliminating the signs of skin dryness. Accordingly, we propose to distinguish between the in vitro humectancy (i.e. the water uptake of a material), and its in vivo moisturizing effect, i.e. its ability to cure skin dryness and erythema. This finding supports our conclusion that the connection between in vitro humectancy and in vivo moisturization is not a simple correlation.

Animals↗
PubMed1980

Cytotoxicity of adriamycin to tumour cells in vivo and in vitro.

Two mouse sarcomas have been used to assess the sensitivity to Adriamycin of tumour cells in vivo and in vitro. Both tumours were tissue-culture adapted so that cell survival could be assayed in vitro after treatment either in vivo or in vitro. For both tumours (WHFIB and CBSAF) cells were highly sensitive when treated in vitro yet very resistant to treatment in vivo, whether assayed by cell survival or regrowth delay. Cells from both tumours treated in vitro with Adriamycin immediately after excision were slightly more resistant than the cells maintained in vitro. However, this was not adequate to explain the marked discrepancy between in vivo resistance and in vitro sensitivity. The discrepancy was not due to a failure of drug delivery. Phase of cell growth was the factor was most significantly affecting chemosensitivity in vitro, plateau-phase cells being much more resistant than log-phase cells. Hypoxia was also an important factor leading to reduced chemosensitivity. Tumour diameter, in the range 2-8 mm, did not appear to be important.

Animals↗
PubMed1985

Assay of anti-cancer drugs in tissue culture: relationship of relapse free interval (RFI) and in vitro chemosensitivity in patients with malignant cerebral glioma.

One hundred and seventeen patients with cerebral glioma (Kernohan grades III and IV) were treated with adjuvant chemotherapy using procarbazine (PCB), CCNU and vincristine (VCR) following whole head irradiation. Cell cultures were prepared from 40 patients in this series and their sensitivity to each cytotoxic drug was assessed in a mictotitration assay with 35 S-methionine incorporation as the end point. Twenty-two of forty (55%) patients responded to PCB and/or CCNU in vitro, and sensitivity to these drugs was linked with increased RFI, whilst sensitivity to VCR was not. The RFI of patients who had responded to PCB or CCNU in vitro was significantly longer than the RFI of patients whose tumours failed to respond in vitro or patients who had not been tested. There was no difference in sex ratio, extent of operation, radiation dose and degree of steroid cover between responders, non-responders and untested groups. Grade III tumours tended to be more sensitive in vitro than grade IV tumours. The age of patients also influenced in vitro chemosensitivity. Patients with chemosensitive tumours in vitro tended to be younger than patients with insensitive tumours in vitro. Further statistical analysis, taking into account these prognostic factors, indicated an association between chemosensitivity in vitro and RFI.

Adult↗
PubMed1999

Role of superoxide dismutase in in vivo and in vitro nitrate tolerance.

We assessed whether pharmacological inhibition of CuZn-superoxide dismutase (SOD) mimics the molecular mechanism of either in vitro or in vivo nitrovasodilator tolerance. In endothelium-intact aortic rings from in vivo tolerant rabbits the GTN- and acetylcholine (ACh)-induced maximal relaxation was attenuated by 36 and 23%, respectively. In vitro treatment of control rings with GTN (1 h 10 microM) similarly attenuated the vasorelaxant response to GTN, but not to ACh. Formation of superoxide radicals (*O2-) in endothelium-intact rings (lucigenin-chemiluminescence) increased 2.5 fold in in vivo tolerance, but significantly decreased in in vitro tolerance. The membrane associated NADH oxidase activity was increased 2.5 fold in homogenates of in vivo tolerant aortae, but was not changed in in vitro tolerant aorta. Conversely, SOD activity and protein expression was halved in in vivo tolerance, but SOD activity was not altered by in vitro tolerance. The *O2- scavenger tiron (10 mM) effectively restored the vasorelaxant response to GTN in in vivo tolerant aortic rings, but not the reduced response to GTN in in vitro tolerant rings. Pretreatment (1 h) of vessels with diethyldithiocarbamate (DETC; 10 mM) attenuated vasorelaxant responses to GTN and ACh, increased vascular *O2- production, and inhibited SOD activity in vessel homogenates to a similar degree as observed in in vivo tolerance. DETC-treatment of in vivo-tolerant vessels induced an additional increase in *O2- production. Increased *O2- production in in vivo nitrate tolerant aorta is associated with activation of vascular NADH oxidase and inactivation of CuZnSOD. Therefore, in vivo tolerance can be mimicked by in vitro inhibition of CuZnSOD, but not by in vitro exposure to GTN, which does not affect vascular *O2- production, NADH oxidase and CuZnSOD.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2002

Stereoselective in vitro degradation pattern of mivacurium in human plasma.

BACKGROUND: Mivacurium is a mixture of three isomers, two of which are rapidly broken down in vivo by plasma cholinesterases. This study investigates the stereospecificity of mivacurium in vitro degradation to determine if it accounts for its in vivo behaviour. METHODS: The in vitro rate of degradation of each isomer of mivacurium and the in vitro rate of formation of their primary (monoesters and alcohols) and secondary (alcohols) metabolites were examined using human plasma from six healthy volunteers. The in vitro rate of degradation of the monoester metabolites was also assessed. All these determinations were made using a stereospecific high-performance liquid chromatography assay. RESULTS: The in vitro rate of disappearance of the two active isomers of mivacurium was very rapid, with mean values for the trans trans and cis trans isomers of 0.803 and 0.921 min(-1) respectively. These values are twofold faster than published in vivo data. The in vitro rate of disappearance was much slower for the cis cis isomer, with a mean value of 0.0106 min(-1). The cis trans isomer was converted exclusively to cis monoester and trans alcohol, while only metabolites in the trans and cis configuration were found for the trans trans and cis cis isomers respectively. Mean in vitro rates of disappearance for the trans and cis monoester were 0.00750 and 0.000633 min(-1) respectively. CONCLUSIONS: The in vitro rates of hydrolysis of the active isomers of mivacurium confirm that plasma cholinesterases play a major role in their in vivo degradation, but that in vivo elimination is slowed by extravascular distribution. Mivacurium hydrolysis is stereoselective, the ester group in the trans configuration being more accessible to enzymatic attack. This stereoselective pattern, along with the relatively slow breakdown of the cis cis isomer, sheds light on the in vivo disposition of the cis alcohol metabolite.

Adult↗
PubMed1993

Effect of pentoxifylline on implantation and post-implantation development of mouse embryos in vitro.

Previous work from our laboratory has revealed that brief exposure of mouse zygotes to 3.6 and 7.2 mM pentoxifylline reduced cell numbers in mouse embryos reaching the blastocyst stage in vitro. Since a decrease in cell numbers may impair further development, the present study aimed to assess the implantation in vitro and the development beyond implantation of these embryos. Blastocysts cultured from the one-cell stage in the presence of 5, 10 and 50 microM pentoxifylline, i.e. concentrations expected in the secretions of the genital tract after oral administration, were shown to develop normally to the egg cylinder stage in vitro. Blastocysts, obtained after culture in vitro of zygotes exposed approximately 29 h after human chorionic gonadotrophin (HCG) for 30 min to 3.6 and 7.2 mM pentoxifylline, i.e. the concentrations used for enhancing sperm function in vitro, could implant but their development to the egg cylinder stage was found to be impaired. The same effect was noticed if blastocysts, grown in vitro from the one-cell stage, were exposed to 3.6 and 7.2 mM pentoxifylline for 30 min in vitro approximately 120 h after HCG. These findings imply that when pentoxifylline is used to enhance sperm function in vitro, it should be washed out of the preparation used for insemination.

Animals↗
PubMed1998

Moments and forces during pedicle screw insertion. In vitro and in vivo measurements.

STUDY DESIGN: Moments and forces during pedicle screw insertion were measured in vivo and in vitro and were correlated to several parameters of the screw-bone interface. OBJECTIVES: To compare the in vitro and in vivo screw insertion loads and to relate these measurements to bone mineral density, pedicle size, and other screw parameters (material, diameter). SUMMARY OF BACKGROUND DATA: The in vitro screw insertion torque has been correlated to the screw pullout forces and the number of cycles to ultimate interface failure. However, there are no comparable in vivo data. METHODS: One hundred three pedicle screws were included in the study, 43 in vivo and 60 in vitro. Duel-energy x-ray absorptiometry boen mineral density data were available for 20 in vivo and 32 in vitro specimens. A custom-made sterilizable six-axis load cell was integrated into a torque wrench, enabling the recording of the applied moments and forces during screw insertion. Statistical analysis was performed to detect differences and correlations. RESULTS: The mean in vivo insertion torque (1.29 Nm) was significantly greater than the in vitro value (0.67 Nm). The linear correlation between insertion torque and bone mineral density was significant for the in vitro data but not for the in vivo data. No correlation was observed between insertion torque and pedicle diameter. Two patterns of torque were observed during the insertion process. CONCLUSIONS: There is a significant difference between the insertion loads measured in vivo and those measured in vitro. Additional research is needed to verify whether this method provides an indication of screw fixation quality.

Absorptiometry, Photon↗
PubMed2001

Is it possible to simulate physiologic loading conditions by applying pure moments? A comparison of in vivo and in vitro load components in an internal fixator.

STUDY DESIGN: Loads acting in an internal fixator measured in vitro under the application of pure moments such as those commonly used for implant testing and basic research were compared with loads measured in 10 patients in vivo. OBJECTIVES: To investigate whether these recommended loading conditions are valid by comparing in vivo measurements and those obtained in an in vitro experiment. SUMMARY OF BACKGROUND DATA: Pure bending moments are often preferred as loading conditions for spinal in vitro testing, either for implant testing or basic research. The advantage of this loading pattern is that the bending moment is uniform along the multisegmental specimen. However, functional loading of the spine by muscles or external loads subjects the spine to a combination of forces and moments. METHODS: In an in vivo experiment, loads acting on an internal spinal fixator in 10 patients were determined before and after anterior interbody fusion during flexion, extension, left and right lateral bending, and left and right axial twisting of the upper body with the patient standing. For comparison, an equivalent in vitro data set was created with 7 human lumbar specimens in which the same type of fixator was used. All specimens were tested under the application of pure bending moments in the three main motion planes in the intact state with fixator, after corpectomy, and with bone graft. RESULTS: Consistent qualitative agreement between in vivo and in vitro measurements for the loads acting in the internal spinal fixator were found for axial rotation and lateral bending. For flexion and extension, reasonable agreement was found only for the intact spines with fixators. After corpectomy and after inserting a bone graft, the median values for axial force and bending moment in the sagittal plane in vitro did not agree with in vivo measurements. An axial preload in the in vitro experiment slightly increased the axial compression force and flexion bending moment in the fixators. CONCLUSIONS: The application of pure moments to intact lumbar spinal specimens in vitro produces forces and moments in implants comparable with loads observed in vivo. During basic research on intact specimens or implant testing involving a removed disc or corpectomy, muscle forces are necessary to simulate realistic conditions.

Humans↗
PubMed1987

Variable expression of the murine natural resistance gene Lsh in different macrophage populations infected in vitro with Leishmania donovani.

Various macrophage populations isolated from mice (including congenic C57BL/10ScSn and B10.LLshr) bearing resistant or susceptible alleles for the natural resistance gene (Lsh) were infected with Leishmania donovani amastigotes in vitro and examined (a) for their ability to support growth of the amastigote population over 7 days of culture in vitro, and (b) for their ability to express Lsh gene controlled resistance and susceptibility in vitro. Resident macrophages from liver (Kupffer cells), spleen and lung, as well as 7-day bone marrow-derived macrophages and bone marrow macrophages obtained after 6 weeks of continuous culture in vitro, all supported growth of the amastigote population. Of these, significant differences in amastigote numbers in macrophages from Lshs and Lshr mice were observed after 48 h of infection in vitro for liver, lung and 7-day bone marrow macrophage populations only. Resident peritoneal macrophages grown in adherent or suspension cultures neither supported growth of the amastigote population nor showed any evidence of Lsh gene expression in vitro. Hence, multiplication of the parasite appeared to be a necessary but not sufficient condition for observation of Lsh gene activity against L. donovani in vitro. Use of tritiated thymidine incorporation and autoradiography to label dividing amastigotes showed equivalent multiplication of the parasite in liver macrophages from Lshs and Lshr mice between 24 h and 48 h after infection in vitro, with a dramatic difference observed thereafter. This was consistent with earlier observations of a 2-3 day delay in expression of Lsh gene controlled resistance in vivo. Comparison with studies using Salmonella typhimurium and Mycobacterium bovis suggests that the gene may be restricted in its action to a particular point in the parasite cell cycle, perhaps at the level of regulating DNA replication.

Animals↗
PubMed1982

Effects of live ischemia on hepatic protein synthesis in vitro and in vivo.

When an in vitro system is used to study the influence of ischemia on hepatic protein synthesis, an important question is whether alterations observed in vitro reflect changes in vivo. In the present study the effects of liver ischemia on protein synthesis were investigated in rats both in vitro and in vivo. Liver ischemia was induced by hepatic artery ligation. Protein synthesis in vitro was determined from leucine incorporation into proteins in liver slices incubated in a medium containing 14C-leucine (0.5 mol/l) and in vivo from leucine incorporation into hepatic proteins after intraportal injection of a tracer dose of 14C-leucine. Leucine incorporation rate in non-ischemic liver was 0.16 mumol.g prot-1.h-1 in vitro and 19.6 mumol.g prot-1.h-1 in vivo. After hepatic artery ligation protein synthesis in vitro was reduced by about 60% and in vivo by about 80%. Thus, the relative changes were of the same magnitude in vitro and in vivo. This indicates that an vitro system can be used to evaluate the effects of liver ischemia on hepatic protein synthesis.

Adenosine Triphosphate↗