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PubMed1996

Comparative study of classical, colorimetric and immunologic staining methods for the assessment of tumor cell viability.

The trypan blue exclusion test, the MTT test and an immunostaining test for apoptosis were performed before and after incubation of SW620 human colonic carcinoma cells with different cytotoxic agents (CTAs) in order to assess tumor cell viability and CTA efficacy in vitro. A modified MTT test using light microscopy was also performed. A good correlation was found between the trypan blue assay and the MTT test, as determined by spectrophotometry. There was no 'overestimation' of cell viability as measured by the trypan blue test. The monitoring of formazan formation by light microscopy was feasible, but not very reliable since it did not show a good correlation with findings determined by spectrophotometry. The apoptosis test failed to show good correlation with other tests. Distilled water had no relevant cytotoxic effect, while chlorhexidine cetrimide (HAC 3.5%), chloramine 0.5% and polyvinylpyrrolidone iodine (PVP-I) > or = 0.05% damaged a large majority of cells. PVP-I at a concentration of > or = 5% was found to be the most effective CTA.

Antineoplastic Agents↗
PubMed2004

Anterior chamber irrigation with an ozonated solution as prophylaxis against infectious endophthalmitis.

PURPOSE: To assess the validity of anterior chamber irrigation with an ozonated solution as prophylaxis against endophthalmitis. SETTING: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan. METHODS: Viability of human corneal endothelium in culture was assessed by the WST-8 assay, lactate dehydrogenase (LDH) release assay, and trypan blue exclusion assay after exposure to a 4 to 40 parts per million (ppm) solution for 10 to 60 seconds. The in vivo effect was observed 1 week after irrigation of a 4 ppm solution in the rabbit anterior chamber by trypan blue exclusion assay. Bactericidal efficacy of the anterior chamber irrigation with the 4 ppm solution was examined by bacterial colony count of the aqueous humor following methicillin-resistant Staphylococcus aureus (MRSA) contaminated intraocular lens implantation in the porcine eye. RESULTS: The WST-8 assay revealed no significant reduction of viability with 10-second exposure to a 4 ppm solution. Lactate dehydrogenase release and trypan blue exclusion assays similarly demonstrated little damage after 60-second exposure to a 4 ppm solution. In the rabbit cornea 1 week after treatment, damage caused by 30-second exposure to a 4 ppm solution was not significant. The MRSA colony count documented almost complete bactericidal action with 5-second exposure to the 4 ppm solution when no ophthalmic viscosurgical device existed in the anterior chamber. CONCLUSION: Limited damage to the corneal endothelium after 10-second exposure and potent bactericidal action with 5-second exposure suggests the validity of anterior chamber irrigation with a 4 ppm ozonated solution as prophylaxis against endophthalmitis.

Animals↗
PubMed2006

Staining ability and biocompatibility of brilliant blue G: preclinical study of brilliant blue G as an adjunct for capsular staining.

OBJECTIVE: To evaluate the effectiveness and biocompatibility of brilliant blue G (BBG) for capsular visualization for continuous curvilinear capsulorrhexis. METHODS: The capsular staining ability of BBG was evaluated at graded concentrations of 10.0, 1.0, 0.5, 0.25, 0.1, and 0.01 mg/mL in enucleated pig's eyes. The biocompatibility of BBG was assessed in rat's eyes for 2 months. The eyes were analyzed using light, fluorescence, transmission electron, and scanning electron microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) was used to detect apoptotic cells, and endothelial cell counts were analyzed using scanning electron microscopy. The results were compared using indocyanine green and trypan blue. RESULTS: The BBG improved capsular visualization, and a complete capsulorrhexis could be performed. In the rat model, no apparent toxic effect was observed using biomicroscopy during 2 months. Histologically, BBG showed satisfactory biocompatibility. Apoptotic cell death of the endothelial cells was detected in only the trypan blue group. In contrast to BBG, indocyanine green and trypan blue showed degeneration of corneal endothelial cells using transmission and scanning electron microscopy. CONCLUSION: The BBG contributed to better capsular visualization and caused no apparent complications to the corneal endothelium.Clinical Relevance The BBG is effective and safe capsular staining for continuous curvilinear capsulorrhexis.

Animals↗
PubMed1992

On the possible mechanisms and predictability of clozapine-induced agranulocytosis.

Studies were conducted on serum removed from 15 patients before, during, and after, clozapine-induced agranulocytosis. Cytotoxic studies were compared with samples taken from patients during treatment with clozapine who did not develop agranulocytosis or treatment controls (TC); additional controls consisted of allogeneic (NC) and autogeneic serum from apparently normal people. The effect of serum on measurable functions of polymorphonuclear neutrophils (PMNs) taken from normal people was tested. Procedures under study included suppression of post-phagocytosis-induced 14CO2-indicated respiratory burst, as well as ejection of trypan blue by test PMNs. PMNs exposed to active agranulocytosis serum plus complement displayed diminished 14CO2 emission during phagocytosis or failed to eject trypan blue. PMNs exposed to serum of TC and NC continued to function normally as regards 14CO2 emission and trypan blue ejection. Five patients studied before the development of agranulocytosis showed suppressed PMN function, which increased to peak value during agranulocytosis and then disappeared within 40 days of recovery. Similar suppression of colony forming units of granulocytes and macrophages (CFU-GM) was found whenever agranulocytosis serum was included in the marrow culture. The cytotoxic material required complement for its full expression, was not dialysable, was neutralised by anti-IgM serum, and was absorbed by test PMNs. Furthermore, solutions of clozapine or 5 of its metabolites offered no similar suppression of PMN function in vitro after incubation in an aqueous medium or with normal serum. These observations favour development of an immunogenic clone in sensitive people during active treatment with clozapine, which eventually leads to precipitous depletion of PMNs and their precursors. The early appearance of this suppressive substance may offer an early warning for development of agranulocytosis.

Agranulocytosis↗
PubMed1999

Retaining ionic concentrations during in vitro storage of tissue for microanalytical studies.

When human or animal tissue is to be investigated by X-ray microanalysis, it is sometimes necessary to store the tissue between removal from the organism and freezing. However, when excised tissue is stored in buffer, the elemental concentrations in the cell may change. In the present study, it was attempted to develop a storage buffer that would retain the cellular elemental concentrations close to their in situ values. To start, the NaCl component in Krebs-Ringer buffer was exchanged for K-gluconate and KCl for NaCl. This buffer was called a '100% high K+ solution'. Starting from this solution, part of the K-gluconate was replaced by an equivalent amount of NaCl. Incubation of excised rat liver (4 degrees C, 4 h) in 85% high K+ solution resulted in retention of cellular Na, K, Ca, S and Mg concentration most closely to the in situ state, whereas cellular Cl was retained best when the tissue was incubated in 75% high K+ solution. For rat submandibular gland, incubation in 80% high K+ solution resulted in optimal retention of cellular Na, K, Ca, P, S and Mg, while Cl was retained best in a 70% high K+ solution. Based on these results, an optimally modified Krebs-Ringer solution for the liver would consist of 119 mM K+, 26 mM Na+ and 45 mM Cl-. An optimally modified Krebs-Ringer bicarbonate solution for the submandibular gland would be composed of 96 mM K+, 53 mM Na+ and 46 mM Cl-. After incubation in the modified solutions (at 4 degrees C), cellular Na, Mg, S, Cl, K and Ca in both tissues were maintained close to the in situ state throughout a 6-h incubation. The cellular P concentration was reduced after incubation for 1 h; thereafter, in the liver cells it remained at this lower level for the rest of the incubation, whereas in the submandibular gland tissue it increased again after 4 h. The increase in cell volume (oedema) was less in tissue stored in the modified solutions, than in the 100% high K+ solutions. Incubation in high Na+ buffers (4 degrees C, 6 h) resulted in a progressive increase in the percentage of cells showing trypan blue uptake. A similar increase in trypan blue uptake was seen in the modified solution, but this increase levelled off after 4 h. After cholinergic stimulation in high Na+ solution (25 degrees C, 1 min), the expected decrease in cellular Cl concentration was seen in submandibular gland cells that had previously been preserved (4 degrees C, 4 h) in the modified solution, but not in those that had been preserved in the 100% high K+ solution.

Animals↗
PubMed1987

Adherence of Salmonella typhimurium to small-intestinal enterocytes of the rat.

The adherence of radiolabeled Salmonella typhimurium to freshly isolated enterocytes of rats was studied. The results established that type 1 fimbriated strains adhered in significantly higher numbers than did related nonfimbriated strains. Adherence was inhibited by D-mannose and methyl alpha-D-mannoside. Results of kinetic studies indicated that adherence was biphasic; the number of bacteria that adhered per enterocyte remained constant for approximately 20 min and then increased rapidly under the assay conditions. The second phase was associated with structural damage to the enterocytes. The addition of chloramphenicol did not prevent the initial attachment of bacteria to enterocytes but did prevent the second phase. Viable and nonviable bacterial cells adhered to enterocytes, but only viable bacteria were destructive. Freshly isolated enterocytes (trypan blue impermeable) and enterocytes stored overnight (trypan blue permeable) were infected by viable S. typhimurium in a similar manner, suggesting that metabolic activity of the host cell was of less consequence than metabolic activity of the bacterial cells. A model for the role of mannose-sensitive fimbriae as a virulence factor is proposed.

Animals↗
PubMed2000

Survey of canine babesiosis in South Africa.

A questionnaire, designed to obtain qualitative information on a number of variables concerning canine babesiosis (biliary fever) in South Africa, was sent to 510 veterinary practices in late 1993. Of the 157 practices that responded, all were presented with cases of babesiosis and most were situated in Gauteng, the Western Cape and KwaZulu-Natal. Apart from the Western Cape, a winter-rainfall region, the prevalence of babesiosis cases in dogs was highest in summer. Most of the respondent practices treated between 1,000 and 5,000 sick dogs that included 100 to 500 babesiosis cases each year. Respondents identified cerebral babesiosis, enterorrhagia, 'red' or haemoconcentrated babesiosis, acute renal failure and pulmonary babesiosis or 'shock lung', amongst others, as the most prevalent forms of complicated ('atypical') babesiosis. Diminazene, imidocarb and trypan blue were the most popular antibabesials. Trypan blue was most often used in shocked patients, whereas diminazene and imidocarb were preferred when there was a high parasitaemia in the absence of shock. At least 19 antibabesial treatment regimens were used in practices. These comprised the use of single doses of antibabesial drugs; split doses with repeat injections, and combined drug variations, some of which are undesirable due to possible sterilisation of Babesia infection or potential toxicity. Side-effects were most commonly associated with imidocarb use. Ninety-six percent of respondents used supportive treatment (e.g. corticosteroids, vitamins and 'liver support') in all cases of babesiosis. The use of blood transfusion as supportive treatment varied according to practice and severity of the case. Most practices never cross-matched blood to be transfused, and transfusion reactions were rare. Diminazene was most frequently incriminated in cases where drug 'resistance' or relapses occurred. Cerebral and 'red' cases resulted in high mortality. Treatment of babesiosis costs the dog-owning public in South Africa more than R20 million each year. Information on the distribution and possible complicating role of Ehrlichia canis was obtained. Development of a vaccine was the first research priority identified.

Animals↗
PubMedDate not supplied

Effects of various drugs on denervation changes in rat muscles.

The effects of drugs, known to affect lysosomal enzyme activities on denervated gastrocnemius muscles of rats were studied. The drugs used were: amiodarone, chloroquine, cortisone, streptomycin and trypan blue. The parameters investigated included changes in fibrillations in electromyographs, morphometric evaluations of muscle atrophy and replacement, and light and electron microscopic analysis of morphological changes and of histochemical acid phosphatase activity. All drug treatments reduced the intensity and frequency of fibrillations. Acid phosphatase spillage was best inhibited by trypan blue. Progressive muscle fiber atrophy with massive aggregation of mitochondria at the cell periphery occurred in denervated fibers. Atrophy was expressed both in loss of muscle weight and in reduction of average muscle fiber diameter. Administration of all the drugs resulted in slowing down of muscle weight loss, particularly 30 days after denervation. All drugs induced slowing down of the loss in average fiber diameter on day 3 after denervation, while on day 10 only cortisone and amiodarone still exerted this effect. A similar transitory slowing of denervation changes was observed in estimations of replacement of muscle by collagen and adipose tissue. Only the effect of trypan blue and amiodarone persisted on day 10. The results indicate that drugs known to inhibit lysosomal enzyme activity may slow down the denervation-derived muscle atrophy to a certain extent, but the effects are transitory.

Acid Phosphatase↗
PubMed1989

Reperfusion injury to endothelial cells following cold ischemic storage of rat livers.

Storage of donor livers in Euro-Collins solution for human transplantation surgery is limited to about 8 hr. Here, tissue damage to isolated rat livers stored under the same conditions as human livers was characterized following reperfusion. The purpose of this work was to determine the importance of nutritional status on injury due to cold storage and reperfusion, to establish whether lethal injury occurs during cold storage or only after reperfusion, and to identify the cell types most vulnerable to damage. Rat livers were perfused with Krebs-Henseleit-bicarbonate buffer, stored 8 to 48 hr in Euro-Collins solution and reperfused with warm, oxygenated Krebs-Henseleit buffer for 15 min. Nuclear trypan blue uptake and lactate dehydrogenase release were used as indices of cell death. After 8 hr of cold storage and reperfusion, little loss of parenchymal or nonparenchymal viability occurred. After 24 or 48 hr, virtually all parenchymal cells remained viable. However, severe damage to nonparenchymal cells was observed, and about 40% of nonparenchymal cells were trypan blue positive. Nutritional status (fed vs. fasted) did not affect the extent of cell damage. Nonparenchymal cell killing was accompanied by cellular rounding, nuclear pyknosis and protrusion of cells into sinusoidal lumens. Scanning electron micrographs demonstrated denudation of the sinusoidal lining. Rounding and pyknosis were not observed in 24-hr-stored livers which were not reperfused, and trypan blue uptake did not occur in stored livers infused with cold, anoxic Euro-Collins solution. Based on cytochemistry and electron microscopy, lethal cell injury occurred predominantly to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Effect of shaking of corneal endothelial preservation.

PURPOSE: To investigate the effect of shaking on corneal endothelial preservation. METHODS: Thirty-six dog corneal buttons were obtained under standard eye bank procedure, after the animals had been sacrificed for cardiovascular experiments in the surgical department. They were equally divided into two groups. In Group I, buttons were put in Dexsol preservative medium and preserved at 4 degrees C. In Group II, buttons were put in Dexsol preservative medium and shaken in a shaking incubator at a speed of 5 rpm. at 4 degrees C for 10 h. After shaking, they were returned to a 4 degrees C refrigerator until examination. Three buttons from each group underwent specular microscopy, scanning electron microscopy (SEM), and alizarin red with trypan blue stain examinations on days 0, 1, 3, 5, 7 and 9, respectively. RESULTS: In Group I, intercellular borders became blurred under specular microscopy, beginning on day 5. However, endothelial cell count did not change significantly until day 9. Intercellular digitations and wrinkling of intercellular borders were seen under alizarin red with trypan blue stain and SEM examination, beginning on day 3. Pleomorphism and ill-defined intercellular junctions were seen under SEM on day 7. There was no obvious denudement of the endothelial sheet, but a few scattered exfoliations were seen in Group I. In Group II, endothelial cell count did not decrease on day 1, but an endothelial image could not be obtained by specular microscopy 3 days after treatment. Alizarin red with trypan blue stain and SEM examination revealed that the endothelial cells were denuded from the Descemet's membrane three days after shaking. Severe stromal edema was seen under SEM five days after shaking. CONCLUSIONS: The results showed that shaking had a detrimental effect on endothelial cell preservation. Vigorous shaking should be avoided during transportation of corneal buttons. It is advisable to perform penetrating keratoplasty as soon as possible upon receiving transported donor corneas.

Animals↗
PubMedDate not supplied

When does the lung die? I. Histochemical evidence of pulmonary viability after "death".

An inadequate number of lung donors for transplantation results in the death of many potential lung recipients awaiting a transplant. Canine experiments in our laboratory have shown effective gas exchange in lungs transplanted from cadaver donors (lungs retrieved after circulatory arrest). The time course of pulmonary cell death after circulatory arrest is unknown. To address this question, we used trypan blue dye exclusion to quantitate lung cell death at postmortem intervals in rats. One hundred ninety Sprague-Dawley rats were killed and separated into four groups: (1) control (n = 10); (2) nonventilated group (n = 60); (3) oxygen-ventilated group (n = 80); and (4) nitrogen-ventilated group (n = 40). At intervals after the animals' deaths, trypan blue was infused into the pulmonary artery followed by fixative, and the left lung was excised. Histologic sections were prepared for each rat lung, and the percentage of nonviable cells was quantified with light microscopy. Control lungs retrieved immediately after death showed little or no uptake of trypan blue dye. In nonventilated rats, 36%, 52%, and 77% of cells were nonviable in lungs retrieved 2, 4, and 12 hours after death, respectively. These results were similar to 34%, 58%, and 71% nonviability at the same intervals in nitrogen-ventilated cadaver rat lungs. Oxygen-ventilated cadaver rats, however, had significantly fewer nonviable lung cells at each time interval: 13%, 10%, and 26%, respectively (p < 0.01). Thus, postmortem mechanical ventilation with oxygen appears to delay lung death in the rat after circulatory arrest. Nonventilated and nitrogen-ventilated cadaver lungs had a similar severity and progression of ischemic injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

A comparison of various methods for the determination of viability of parasitic flagellates.

To find out a simple, rapid, accurate, and reliable viability assay for in vitro anti-protozoa drug testing, a comparison among the various methods for the determination of flagellate viability was made. Micro-colony forming efficiency up to 90% for three different flagellates was obtained by the agar plate method. Macro-colony forming efficiency obtained by the culture tube method was generally lower (20-45%). Viability determined by trypan blue exclusion correlated closely with viability determined by micro-colony count (r = 0.94). The growth of the remaining parasites survived from serum treatment was not inhibited while that from metronidazole pretreatment was inhibited. The standard regrowth assay based on the 48-hr cultivation could be applied only for those survival not inhibited. The inhibitory effect of metronidazole on parasites was further testified by the longer generation time for metronidazole-pretreated flagellates in comparison with that for control (p less than 0.001). If the inhibitory effect of a drug is involved in the mechanisms against parasites, only the trypan blue exclusion and the micro-colony count can give an accurate and reliable viability determination. Moreover, the simple and rapid diagnosis provided by trypan blue exclusion is especially useful for the in vitro anti-protozoa drug susceptibility test.

Animals↗
PubMed2004

The impact of excitotoxic blockade on the evolution of injury following combined mechanical and hypoxic insults in primary rat neuronal culture.

Traumatic brain injury (TBI) involves alterations in neuronal physiology, often complicated by secondary hypoxic or hypotensive events. Excitotoxicity is an important process induced in both TBI and hypoxic or ischemic insults to the brain. We investigated two hypotheses: (1) excitotoxicity is more prominent following combined mechanical and hypoxic injury than either alone; (2) both AMPA and NMDA receptor activation mediate combined mechanical and hypoxic injury. Media in primary mixed neuronal cultures were replaced with conditioned media containing MK801 (NMDA antagonist) and/or NBQX (AMPA/kainate antagonist). Cultures were then subjected to mechanical injury. Afterward, media were exchanged for hypoxic media containing the antagonist, and plates were placed in hypoxia chambers for 7 h. At 24 h following hypoxia, LDH release, trypan blue uptake, and morphologic changes were assessed. Blockade had no effect after mechanical injury. After hypoxia, MK801 and combined MK801/NBQX decreased LDH and trypan blue to control levels. NBQX alone after hypoxia had less impact. After combined mechanical injury and hypoxia, both MK801 and NBQX partially reduced LDH and trypan blue. Combining the antagonists led to reduction to control values for both endpoints. We conclude that excitotoxic processes are more prominent after combined than isolated injuries in neurons and that increased cell death is mediated by both NMDA and AMPA receptor activation following combined injuries.

Animals↗
PubMed1991

Oxidative injury induces influx-dependent changes in intracellular calcium homeostasis.

Understanding of the mechanisms of cell injury and cell death is fundamental to the understanding of both protection against and initiation of cell injury and cell death. We subjected primary cultures of proximal tubular epithelium (PTE) from adult rats to an exogenous oxidative stress, generated by xanthine/xanthine oxidase (X/XOD), and studied its effect on the concentration of cytosolic ionized calcium ([Ca2+]i) by means of digital imaging fluorescence microscopy (DIFM) using a cytosolic calcium probe, fura-2. Exposure to 25 mU/ml X/XOD caused notable increases in [Ca2+]i detectable within 15 sec and increasing to micromolar levels with time. Experiments with Ca(2+)-free medium containing ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) showed that the increase of [Ca2+]i was due to influx from the extracellular space. Smaller and slower increases in [Ca2+]i were seen after exposure to lower concentrations of X/XOD (5 and 10 mU/ml). PTE injury and killing were assessed by measuring the release of cytosolic lactate dehydrogenase (LDH), exclusion of trypan blue, and observation of morphologic changes. Exposures to the 25 mU/ml concentration of X/XOD caused significant LDH release after 2 hr and correlated with trypan blue staining of exposed cells. Again, lesser concentrations of X/XOD resulted in a slower release of smaller amounts of LDH, and thus delayed trypan blue staining. Cytoplasmic bleb formation was seen by phase microscopy within minutes of exposure to 25 mU/ml, followed by cell rounding, retraction, and disintegration. Transmission electron microscopy revealed a progression of changes characteristic of lethal cell injury, beginning with dilatation of the endoplasmic reticulum, detachment of ribosomes, condensation of mitochondria, and chromatin clumping and terminating with mitochondrial swelling and formation of intramitochondrial flocculent densities. These studies clearly show that notable increases of [Ca2+]i precede both sub-lethal and lethal changes in rat PTE. These results indicate that interventions designed to minimize or to accelerate calcium entry could be of importance in cell preservation or cell killing, respectively, and therefore to therapeutic strategies for myocardial infarction, stroke, or shock in the former instance and for cancers in the latter.

Animals↗
PubMed2002

Toxicity of argemone oil: effect on hsp70 expression and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

The effect of argemone oil on hsp70 expression and tissue damage was investigated by studying beta-galactosidase activity, Western blotting and hybridization, and trypan blue staining in the larval tissues of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Different concentrations of argemone oil were mixed with food and third-instar larvae were allowed to feed on them for different time intervals (2, 4, 24, and 48 h). Argemone oil was found to induce hsp70 even in the lowest concentration of the adulterant while maximum tissue damage was observed in the higher two treatment groups. Malpighian tubules and midgut tissue reflected maximum damage as evidenced by both high beta-galactosidase activity and trypan blue staining in these tissues. A prior temperature shock treatment to the larvae was enough to protect the larvae from argemone oil-induced tissue damage as evidenced by little or no trypan blue staining. The present study suggests the cytotoxic potential of argemone oil and further strengthens the evidence for the use of hsp70 as a biomarker in risk assessment.

Animals↗