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PubMedDate not supplied

Synovial membrane microarthroscopy of the equine midcarpal joint.

OBJECTIVE: To evaluate the value of microarthroscopy in the equine midcarpal joint using the vital stains methylene blue, trypan blue, neutral red, and Janus green B to observe components of the synovial lamina propria, vascular architecture, and synoviocytes. STUDY DESIGN: Experimental. ANIMALS: Ten horses. METHODS: Microarthroscopy of left and right midcarpal joints was performed with and without vital staining of the synovium. Four vital stains (methylene blue, trypan blue, neutral red, and Janus green B) were evaluated, with each stain used in 5 joints. Synovial biopsy specimens were collected from the dorsomedial and dorsolateral aspects of the joint. RESULTS: All dyes were biocompatible. At x 60 without vital staining, synovial surface topography, vascular network, and translucency were observed. Intra-articular vital dyes improved evaluation of synovial surface topography. At x 150 with vital staining, individual synoviocytes were clearly identified with all dyes, except neutral red. Although methylene blue provided the best in vivo microscopic differentiation of the structure of the intima, trypan blue had superior retention in conventionally processed synovial biopsies. CONCLUSIONS: Methylene blue, trypan blue, neutral red, and Janus green B stains can be used safely for microarthroscopy. Good visualization of cells and vascular network can be obtained by microarthroscopy, and microarthroscopic evaluation of the synovium compares favorably with conventional histologic evaluation of biopsy specimens. CLINICAL RELEVANCE: Microarthroscopy may be beneficial in both research and clinical diagnosis of equine articular diseases.

Animals↗
PubMed1979

Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line.

Propagation and plaque assay of human coronavirus prototypes were studied in two human cell lines: a diploid fetal tonsil (FT) and a heteroploid rhabdomyosarcoma (RD) cell lines. Plaques, observed within 2 to 3 days on FT cell monolayers with both 229E and OC43 viruses, appeared as colorless areas after staining with neutral red or crystal violet, whereas neutral red staining was required for visualization of plaques on RD cells. The plating efficiencies were approximately equal between the two cell lines, but virus assay by plaque formation was 15- to 30-fold more efficient than tube dilution assay with 50% endpoints. The discrepancy between 50% endpoint and plaque-forming unit values was striking and appeared to result from the fact that killing of cells (particularly RD cells) by coronaviruses was not accompanied by visible changes in the cells but killing was detected by the failure of infected cells to stain with a vital dye. The latent phase in one-step growth curves was 5 to 6 h for both viruses in either cell line, but the maximum yield of intracellular virus was reached in 18 to 20 h for FT cells and 24 to 28 h for RD cells. Virus release also differed between the two cell lines: in FT cells, the maximum yield of extracellular virus was reached 2 to 3 h later than that of intracellular virus, whereas in RD cells, the difference was 5 h for 229E virus and 10 h for OC43 virus. Although both cell lines appear equally useful for plaque assay, RD cells would be preferred for mass virus propagation because yields (5 X 10(8) plaque-forming units per ml) were 10-fold higher than in FT cells, a finding true for both virus prototypes.

Cell Line↗
PubMed1979

Lysosome stability during lytic infection by simian virus 40.

By 48 h postinfection, 40--80% of SV40-infected CV-1 cells have undergone irreversible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.

Acid Phosphatase↗
PubMed2002

Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro.

BACKGROUND: Previous work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by photodynamic therapy (PDT) using visible light and methylene blue. Antimicrobial PDT (APDT) in vivo would require a therapeutic regimen where bacteria could be killed without damaging adjacent tissue. OBJECTIVES: To study keratinocyte killing in vitro using APDT. METHODS: We used a combination of methylene blue (100 microg mL(-1)) and visible light (42 mW cm(-2)), previously used for microbial killing, to study cytotoxic effects on keratinocytes. Kill rates and subsequent D-values were determined against a human keratinocyte cell line (H103) using trypan blue and neutral red dye viability tests. RESULTS: The kill rates for keratinocytes were exponential over the 90- and 180-min period of the experiment for neutral red and trypan blue, respectively. The corresponding D-values were shown to be 198 and 205 min using trypan blue exclusion and neutral red uptake viability tests, respectively. CONCLUSIONS: The kill rates for keratinocytes were 18-200-fold slower than those previously determined for cutaneous microbial species, suggesting that in vivo, APDT sufficient to reduce microbes by seven log cycles would have little cytotoxic effect on keratinocytes. This approach may offer a safe alternative to conventional antimicrobial treatment.

Bacteria↗
PubMed1994

Reduced surface area in apoptotic rounding of human Chang liver cells from serum deprivation.

BACKGROUND: The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and "condensation of the cytoplasm" in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding. METHODS: Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied 1) the cell cycle from propidium iodide-DNA bindings, 2) uptake of neutral red (NR) dye, a viable cell marker, and 3) cytosolic pH (pHi) modulations from 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH4Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy. RESULTS: Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary Ao (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H+]i/[H+]o gradient promoting proton extrusion. The pHi upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pHi upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells. CONCLUSIONS: Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pHi upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pHi upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death.

Apoptosis↗
PubMed2003

Antiulcerogenic effect and cytotoxic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which, however, is accompanied by toxic effects. On the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin, expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays. To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl, stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC(50)=500 microM in the neutral red uptake and MTT reduction tests and an IC(50)=200 microM in the nucleic acid content test. With regard to hepatocyte viability after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC(50)=10-500 microM in the nucleic acid content and MTT reduction tests and an IC(50)=120 microM in the neutral red uptake test. In another experiment, V79 cells were incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h incubation, semi-synthetic crotonin had an IC(50)=500 microM in the MTT reduction and neutral red uptake tests and an IC(50)=370 microM in nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated hepatocytes were in culture.

Animals↗
PubMed2006

Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil.

To examine the potential of a suite of biomarkers as early warning indicators of environmental pollution, sperm count, neutral red retention time (NRRT) and DNA damage were measured in earthworm Eisenia fetida exposed to increasing concentrations of acetochlor in OECD soil. The neutral red retention time of earthworms coelomocytes was sensitive to acetochlor pollution, and decreased significantly when the concentration was more than 10mgkg(-1) after 30 and 60 days of exposure (P<0.05). The reduced neutral red retention time correlated with the soil acetochlor residual. Sperm count decreased significantly at the concentrations of 40 and 80mgkg(-1) after 15 days of exposure (P<0.05). The DNA damage of earthworms coelomocytes increased significantly after 30 days of exposure at the highest concentration (80mgkg(-1); P<0.05). Earthworms were under physiological stress at field dose of acetochlor (10mgkg(-1)). Higher concentrations of acetochlor caused sperm count decrease and DNA damage of earthworms. Such a suite of biomarkers could serve as indicators of the health of the soil environment and to evaluate the toxicity of acetochlor on earthworms or as a means of monitoring soil acetochlor pollution.

Animals↗
PubMed1981

Interfacial potentials at the disk membranes of isolated intact cattle rod outer segments as a function of the occupation state of the intradiskal cation-exchange binding sites.

Two different methods have been used to determine the interfacial potential at the disk membranes of intact isolated bovine rod outer segments: (1) The photolysis products of rhodopsin are known to be dependent on pH. We have used this property in order to probe the interfacial potential at disk membranes which is considered to change the surface pH at the disk membrane seen by rhodopsin. (2) The pK value of the amphiphilic pH-indicating dye neutral red (uncharged basic form) in water is 6.6, but adsorbed to disk membranes at least 7.8. This makes the distribution of neutral red between disk membranes and bulk water dependent on the interfacial potential at the disk membrane if the pH in the bulk solution is less than 7.8. Both methods yielded comparable results on the influence of ions and ion carriers on the interfacial potential at disk membranes. In particular, we have studied the effect of different occupation states on the internal binding capacity (of rod outer segments) for divalent cations. In the presence of the ionophore A23187, addition of EDTA to a suspension of intact rod outer segments removed all endogenous divalent cations (Schnetkamp, P.P.M. (1979) Biochim. Biophys. Acta 554, 441--459) and resulted in an interfacial pH at the disk membrane surface of about 6.4, whereas the bulk pH was 7.4. Subsequent addition of 2 mM Mn2+ saturated the internal binding capacity and resulted in an apparent shift towards alkaline pH of the surface pH at the disk membrane by 1.0--1.1 pH units. This could indicate a change of the interfacial potential by 60--65 mV. The same change of ionic conditions resulted in a change of the interfacial potential by 72 mV as determined from the partitioning behaviour of neutral red. These results were independent of the presence of H+ ionophores such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and gramicidin. We conclude that the above results can be explained by the presence of fixed net negative charges (charge density: 0.5--1.5 electronic charges/rhodopsin molecule) at the intradiskal membrane surface. That the above charge density can be attributed to the intradiskal membrane surface is inferred from the observation that the presence of A23187 was required for access of divalent cations to the membrane interface involved in both rod outer segments with an intact as well as with a leaky plasma membrane.

Adsorption↗
PubMed1980

[Changes of tissue pH in dog brain during ischemia--alkaline shift (author's transl)].

Brain pH changes during ischemia were observed by the newly established histochemical technique in Dr. Kogure's laboratory (Miami). The technique utilizes intravascular injection of neutral red as a pH indicator. Its use is based on the fact that neutral red is red in acid and yellow in alkaline pH. Thalamic infarction model in dogs was used in this experiment. The methods of producing this model by temporary occlusions of brain arteries have been published elsewhere. The animals were anesthetized with sodium thiopental, immobilized with pancronium bromide, and artificially ventilated with room air. Blood gases and blood pH were adjusted to normal. Before the experimental procedure, 3 ml/kg of a 5% saline suspension of neutral red was injected slowly into the femoral artery. After occlusions of cerebral arteries, the brain was frozen in situ by pouring liquid nitrogen into the bottomless cup. The frozen brain was mounted on a cryostat at -20 degrees C and sliced coronally in 10 micron thickness. Color differences in the serial sliced brain surface were observed. No change in the color was observed in the sham operated brains. Thirty minutes following occlusion, the color of the thalamus is reddish, and it was estimated that this area became acidotic. 1 hour following occlusion, too, the thalamus is acidotic. But 2 hour following occlusion, the color of the thalamus became yellowish, indicative of alkaline shift. From this experimental results, it is considered that the pH of ischemic brain changes dynamically, that is, ischemic brain becomes acidotic in the early stage of ischemia but with the lapse of time alkaline shift occurs.

Animals↗
PubMed1979

Variable effect on peripheral blood leucocytes during JE virus infection of man.

The peripheral blood leucocytes of twenty-four cases of Japanese encephalitis (JE) were studied and the findings were compared with those in twenty-five normal health controls of matching age and sex. In the early phases of the disease marked neutrophil leucocytosis was seen which returned to almost normal levels by the fourth week. Lymphopenia was associated with diminished T lymphocytes but the number of B lymphocytes remained within the normal range. Though the number of T lymphocytes was reduced, their function of leucocyte migration inhibition in the presence of JE virus antigen was significantly higher. The phagocytic activity of the neutrophils, as shown by the uptake of neutral red dye, was diminished but the phagocytic activity of monocytes as shown by the uptake of neutral red dye, was diminished but the phagocytic activity of monocytes as shown by the uptake of neutral red dye or ingestion of latex particles remained unaffected. HI antibodies against JE virus were significantly higher in cases of encephalitis as compared with the control group. Thus, JE virus infection in man has a variable effect on different components of the peripheral blood leucocytes.

Adolescent↗
PubMed1986

In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines.

The cytotoxicity of cadmium toward cultured bluegill fry (BF-2) cells was determined using several assay endpoints. The concentrations of cadmium causing a 50% decrease in colony formation, cell replication, uptake of neutral red, population growth (as determined by protein analysis), and uptake of [3H]uridine and 50% detachment of cells (as determined by protein analysis) were 0.03, 0.04, 0.08, 0.09, 0.12, and 0.21 mM cadmium, respectively. The neutral red assay was used to compare the relative sensitivities of bluegill BF-2 cells and RTG-2 cells, derived from the rainbow trout, toward four metals. The concentrations of cadmium, zinc, copper, and nickel causing a 50% reduction in the uptake of neutral red were 0.08, 0.19, 0.55, and 2.0 mM, respectively, with the BF-2 cells and 0.18, 0.64, 1.45, and greater than 10.0 mM, respectively, with the RTG-2 cells. The RTG-2 cells were less sensitive to the metals, in particular to nickel. The less stringent temperature requirements for growth, their greater sensitivity to pollutants, and their markedly shorter doubling time in vitro make the BF-2 cells the preferable cell line for ecotoxicity screening of aquatic pollutants.

Animals↗
PubMedDate not supplied

A comparative study on the photocytotoxicity of hypericin on A431 cells using three different assays.

BACKGROUND: Three assay models were adopted for assessing the photocytotoxicity of hypericin on A431 cells. The cells were incubated for 1 hour or 24 hours with hypericin to evaluate the importance of the incubation period on the exerted photocytotoxicity. MATERIALS AND METHODS: A neutral red, an antiproliferative and a tetrazolium-reduction (MTT) assay were used for the estimation of cytotoxicity. RESULTS: IC50 values were 296, 321 and > 500 nM after 1 hour, and to 70, 54 and 277 nM after 24 hours, for the neutral red, antiproliferative and MTT assays, respectively. CONCLUSIONS: Our results clearly show that it is imperative to incubate cells for long periods to fully assess the hypericin photocytotoxicity, and that the neutral red assay is as sensitive as the antiproliferative assay but superior to the MTT assay at detecting hypericin cell damage.

Anthracenes↗
PubMed1992

Individual and combined cytotoxic effects of cadmium, copper, and nickel on brown cells of Mercenaria mercenaria.

An assay based on the lysosomal incorporation of neutral red dye by brown cells of Mercenaria mercenaria (Bivalvia) was used to measure the cytotoxicity of Cd2+, Cu2+, and Ni2+ singly and in combination. Cytotoxicity was a linear function of Cd2+ concentration 0.1-1.5 mM and Cu2+ concentration between 10 and 100 microM. Nickel was not cytotoxic at concentrations as high as 10 mM. The presence of Cu2+ lessened the cytotoxic effect of Cd2+. Ni2+ did not affect cytotoxicity in combination with either Cu2+ or Cd2+.Ni2+ inflated estimates of cell survival by the neutral red assay in this study. Cells exposed to Ni2+ yielded measured quantities of neutral red dye in excess of those measured in cells from control treatments.

Animals↗
PubMed1996

Toxicity parameters for cytotoxicity testing of dental materials in two different mammalian cell lines.

The present study compares three specific toxicity parameters for cytotoxicity testing of chemically different dental materials. Two glass ionomer cements, a zinc phosphate cement, and a composite material were used to evaluate the sensitivity of three assays: two viability assays, the MTT assay and the quantifiable neutral red assay, and a proliferation assay based on the determination of the total protein content of a cell culture. The colorimetric assays were carried out using transformed mouse fibroblasts (L-929 cells) and fibroblasts derived from biopsies of normal human gingiva. In most cases, all colorimetric assays detected much weaker cytotoxic responses, if any, in gingival fibroblasts than in L-929 cells. The viability assays indicated cytotoxicity of the extracts to two glass ionomer cements in L-929 cells when the materials were set at 0% relatively humidity for 24 h. The severe cytotoxicity of the zinc-phosphate cement in both viability assays was less influenced by the setting conditions. The cytotoxicity of the composite material was most pronounced in the neutral red assay. In general, both the MTT assay and the neutral red assay were more sensitive than the colorimetric proliferation assay. These assays can be performed very effectively; only few cells are needed for rapid, reliable and inexpensive screening purposes of a large number of samples in a short time. Automated processing with a microplate reader after non-radioactive labeling of the cells and subsequent automated analyses of original data, with no need for sophisticated and expensive equipment, are additional advantages of the systems.

Animals↗
PubMed1982

Effect of nicotine on gastric mucosal blood flow and acid secretion.

The effects of an intravenous infusion of nicotine at a dose of 2 . 5, 5 . 0, 7 . 5, or 10 . 0 micrograms kg-1h-1 on pentagastrin-stimulated gastric mucosal blood flow and acid secretion were investigated in eight healthy male non-smokers. Each dose was tested in two males. Gastric neutral red clearance served as a measure of mucosal blood flow. Nicotine reduced volume secretion, acid secretion, and neutral red clearance in a dose dependent manner. In five healthy male smokers smoking of five cigarettes per two hours induced similar changes to the intravenous infusion of 5 micrograms kg-1h-1 nicotine. As volume secretion was inhibited more than neutral red clearance, it is concluded that nicotine increases blood supply to the gastric mucosa relatively to the reduced gastric secretion. Nicotine is either not associated with the development of peptic ulcers, or it exerts its ulcerogenic action via other mechanisms than change of acid secretion and gastric mucosal blood flow.

Adult↗
PubMed1994

The rates of proton uptake and electron transfer at the reducing side of photosystem II in thylakoids.

Proton and electron transfer at the reducing side of photosystem II of green plants was studied under flashing light, the former at improved time resolution by using Neutral red. The rates of electron transfer within QAFeQB were determined by pump-probe flashes through electrochromic transients. The extent of proton binding was about 1 H+/e-. The rates of proton transfer were proportional to the concentration of Neutral red (collisional transfer), whereas the rates of electron transfer out of QA- and from QAFeQB- to the cytochrome b6f complex were constant. The half-rise times of electron transfer (tau e) and the apparent times of proton binding (tau h) at 30 microM Neutral red were: QA- --> FeIIIQB (tau c < or = 100 microseconds, tau h = 230 microseconds); QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 620 microseconds, tau h = 310 microseconds).

Adaptation, Physiological↗
PubMed1996

Preliminary in vitro cytotoxicity screening of a bead-formed macroporous hydrophilic polymer matrix.

A prescreen of the in vitro cytotoxicity of both the primary fabrication components and potential leachables from a bead-formed macroporous poly(2-hydroxyethyl methacrylate), (pHEMA) matrix has been carried out using INVITTOX Neutral red and Kenacid blue R dye binding methods. Of the eluants obtained from 24, 48, and 72-h incubated beads, only the 72-h eluant produced a greater than 20% (ID20) inhibition of 3T3-L1 cell proliferation with values of 20.98 +/- 2.33% and 21.41 +/- 1.37% inhibition for the Neutral red and Kenacid blue R binding methods, respectively. ID50 values for the fabrication components obtained using the Kenacid blue R method were generally higher than those obtained by the Neutral red assay, although the ranking of the chemicals in terms of their relative cytotoxicities was identical by both methods, i.e. ethylene glycol dimethacrylate > uranyl nitrate > purified HEMA > n-hexane > ethylene glycol (mmol 1(-1)). Whilst extended washing of finished PHEMA beads in water will reduce their acute in vitro cytotoxicity, this will only be achieved with some loss of previously encapsulated water soluble macromolecules.

2,4-Dinitrophenol↗