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PubMed1979

Electrical measurements of complement-mediated membrane damage in cultured nerve and muscle cells.

Cultured neuroblastoma-glioma hybrid cells and primary mouse and rat muscle cells were studied by using intracellular microelectrodes to monitor membrane electrical potential and resistance changes during complement-mediated lysis. The cell membrane was TNP modified under mild conditions and subsequently coated with rabbit IgG anti-TNP, with no electrical changes observed. However, upon addition of guinea pig C the membrane potential dropped from approximately -50 mv to less than -12 mv within a few minutes, with parallel decreases in electrical resistance. Ten to 60 min after these electrical changes the cells became stainable with trypan blue. No electrical changes or trypan blue staining was observed in the absence of antibody or with heat-inactivated C. With more dilute C so that only a fraction of cells became trypan blue positive, all cells nevertheless showed the membrane electrical changes; surviving cells recovered their original membrane properties within 1 hr. Thus the C-mediated damage to the membrane measured electrically is not in itself sufficient to ensure the subsequent death of the cell. The early electrical changes observed appear to be comparable to increases in 86Rb efflux measured by others.

Animals↗
PubMed1990

[The influence of macrophages on the leukemogenic activity of Rauscher murine leukemia virus].

A single application of trypan blue 3 to 24 hours before or simultaneous with inoculation of the Rauscher leukemia virus (RLV) resulted in enhancement of leukemogenesis with an especially high viremia. Repeated administration of trypan blue after RLV infection resulted in reduced spleen weight. Viremia, however, was also increased compared with controls (no application of trypan blue), but in a lower rate than in mice treated with the virus plus trypan blue. Since trypan blue is known as an inhibitor of the functions of macrophages the results point at an important role of this cell type in preventing infection with this oncogenic retrovirus. In this regard there are differences in the role of macrophages in human immunodeficiency virus (HIV) infections.

Animals↗
PubMed1984

Phagocyte C3-mediated attachment and internalization: flow cytometric studies using a fluorescence quenching technique.

The dynamics of phagocyte C3-mediated attachment and internalization of fluorescein-isothiocyanate (FITC)-labelled zymosan particles was studied by a flow cytometric (FCM) fluorescence quenching technique, using trypan blue as quenching agent. Trypan blue effectively quenched the fluorescence of extracellular, i.e. free and phagocyte-attached, zymosan particles, but did not influence on the fluorescence of particles internalized by phagocytes. During phagocytosis, an average of 2 C3-coated zymosan particles were simultaneously attached to the phagocyte surface, and the number of attached particles could not be increased by increasing the zymosan to leukocyte ratio, the concentration of C3, the incubation time, or by inhibiting internalization by Cytochalasin B. Phagocyte C3-mediated internalization of zymosan particles was dependent on the concentration of complement, and in the presence of sufficient amounts of C3, internalization continued until saturation was reached at 11 particles per phagocyte.

Complement C3↗
PubMed1988

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism.

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Adenine Nucleotides↗
PubMedDate not supplied

Staining of intraocular lenses with various dyes: a study of digital image analysis.

PURPOSE: To evaluate the interaction of various intraocular lens (IOL) materials with commonly used dyes. MATERIALS AND METHODS: One IOL of each of the five groups (polymethylmethacrylate, silicone, three-piece hydrophobic acrylic, single-piece hydrophobic acrylic and single-piece hydrophilic acrylic) was bathed in trypan blue 0.1%, fluorescein sodium 2% and indocyanine green (ICG) 0.5% for 15 min. Digital photographs of the IOLs were obtained prior to bathing and after the 15-min bath in each dye. The same IOLs were immersed in the same dye for another 15 min and digital images were reobtained to depict the 30-min dye uptake. New IOLs from the group that exhibited visible colour change after the 15-min bath were then bathed in twofold dilution and if there was still visible dye uptake, fourfold dilution was performed, repeating the 15-min bath and digital photography. The images were then processed using Adobe Photoshop 5.1 to get mean luminosity and red-green-blue values. These values were compared between the groups and the undyed control. Also, dye washout was observed in serum-containing vials. RESULTS: None of the polymethylmethacrylate, silicone and hydrophobic acrylic IOLs were stained with the dyes used. The only IOL material that changed colour was hydrophilic acrylic and did so with all dyes. The most marked colour change was with fluorescein sodium 2%, the least was with trypan blue 0.05%. Twofold dilution of trypan blue and fourfold dilutions of fluorescein sodium and ICG still stained the hydrophilic acrylic IOL; however, fourfold dilution of trypan blue did not cause a significant colour change. Trypan blue washed out within 6 h, while the IOL stained with ICG remained stained for longer than 24 h. COMMENT: Hydrophilic acrylic IOLs should be used with caution together with dyes since this material demonstrates marked dye uptake and washout may also take some time.

Coloring Agents↗
PubMed1986

Kinetics of cell death induced in 10T1/2 cells by methyl methanesulfonate and the effects of extracellular calcium on cell death.

The cytotoxic effect of methyl methanesulfonate (MMS) on C3H 10T1/2 cells is characterized by a complex pattern of changes in the permeability of the cell membrane to trypan blue and, therefore, presumably to extracellular calcium. 10T1/2 cells are temporarily, and reversibly, permeable to trypan blue during the initial 30 minutes following exposure to MMS when incubated in the presence of extracellular calcium. By 90-120 minutes after the exposure, the MMS treated cells have restored control of the membrane permeability, and for the next 6-7 hours they exhibit a level of trypan blue uptake comparable to that observed in the untreated cohort population. Between 9 and 15 hours after exposure to MMS the fraction of the population permeable to trypan blue increases rapidly, ultimately approximating the level of cell killing measured concurrently in a colony formation assay. Transient culture in calcium-free medium immediately after exposure to MMS does not protect 10T1/2 cells from cytotoxicity, but incubation in the calcium-free medium does prevent the initial transient episode of permeability to trypan blue observed when the 10T1/2 cells are incubated in calcium-containing medium. These observations suggest that MMS-induced cytotoxicity results from a complex course of events, possibly from damage to an intracellular target rather than from damage to the plasma membrane.

Animals↗
PubMedDate not supplied

Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: protection by allopurinol.

Trypan blue uptake and lactate dehydrogenase release were measured as indices of irreversible cell damage in isolated, perfused rat livers during low-flow hypoxia. In livers from fasted rats perfused in the anterograde direction, trypan blue uptake took place beginning at about 45 min of hypoxia. Cells which took up trypan blue first were located in narrow bands at the border between anoxic pericentral areas and normoxic periportal regions of the liver lobule. After longer periods of hypoxia, trypan blue uptake progressed towards the central vein until after 120 min virtually all cells in the pericentral regions were stained. Under these conditions, cells in periportal regions were spared. In perfusions in the retrograde direction, cell death began in midzonal regions and spread towards the portal vein. Release of lactate dehydrogenase into the effluent paralleled trypan blue uptake, beginning at about 40 min of low-flow hypoxia and peaking at 80 min. In contrast to livers from fasted rats, trypan blue was not taken up, and lactate dehydrogenase was not released in livers from fed rats exposed to low-flow hypoxia for as long as 120 min. To test the hypothesis that xanthine oxidase-mediated oxygen-free radical formation was involved in cell injury at the border between anoxic and normoxic regions (anoxic edge), allopurinol, an inhibitor of xanthine oxidase, was studied. Allopurinol (0.2 to 5 mM) delayed the release of lactate dehydrogenase during low-flow hypoxia in a dose-dependent fashion (e.g., 5 mM allopurinol delayed hypoxia-induced lactate dehydrogenase release by about 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol↗
PubMed1995

The mechanism of action of the two-layer cold storage method in canine pancreas preservation--protection of pancreatic microvascular endothelium.

We have demonstrated that oxygenation of a pancreas during preservation by a two-layer method leads continued ATP production to maintain cellular integrity and produces an extended period of preserved pancreatic viability. The aim of this study is to examine the effect of ATP vs. oxygenation per se on viability of nonparenchymal cell (vascular endothelium), using 2.4 dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. Mongrel dogs of both sexes, weighing 12-18kg were used. Under general anesthesia, a left lobectomy of the pancreas was performed. The segmental pancreas graft was autotransplanted immediately (group 1; control) or after 48-hour preservation (group 2; simple cold storage method with Euro-Collins' solution [EC], group 3; two-layer cold storage method using EC, group 4; two-layer cold storage method using EC with 0.2 mM DNP), and the remainder of the pancreas was excised at the time of autotransplantation. Graft viability was judged from graft survival after transplantation. A K-value of intravenous glucose tolerance test more than 1.0 at 2 weeks after transplantation was considered graft survival. Tissue concentration of ATP was determined after preservation using high-performance liquid chromatography. Viability of vascular endothelium was examined using trypan-blue perfusion/fixation test after preservation. Nuclear staining by trypan blue in eosin-counterstaining sections was indicative of loss of cell viability. Pancreatic tissue perfusions, which reflect pancreatic microcirculation, were also measured using H2 clearance technique after 30 to 240 min of reperfusion. Graft survival rates in groups 1, 2, 3 and 4 were 5/5, 0/4, 4/4 and 0/3 respectively. ATP tissue concentration was significantly higher in group 3 compared with group 2 (7.91 +/- 1.21 [n = 4] vs. 1.21 +/- 0.31 [n = 4] mumol/g dry weight, p < 0.01). DNP caused a significant decrease in tissue ATP in group 4 (0.61 +/- 0.07 [n = 3] vs. 7.91 +/- 1.21 [n = 4] mumol/g dry weight, p < 0.01). The percentage of nuclear trypan blue uptake of nonparenchymal cells in group 3 was significantly lower than group 2 (11.29 +/- 3.71 [n = 3] vs. 26.41 +/- 1.66 [n = 3] %, p < 0.01), and DNP (group 4) increased trypan blue uptake (30.10 +/- 4.08 [n = 3] vs. 11.29 +/- 3.71 [n = 3] %, p < 0.01). Tissue perfusions after 2hr-reperfusion in group 3 were significantly higher than group 2 (68.64 +/- 8.62 [n = 5] vs. 45.56 +/- 12.84 [n = 5] ml/min/100g, p < 0.01). Moreover, DNP (group 4) caused a significant decrease in pancreatic tissue perfusions (28.84 +/- 9.09 [n = 5] vs. 68.64 +/- 8.62 [n = 5] ml/min/100g. p < 0.001). It was clear that the two-layer method (group 3) protected microvascular endothelium against cold ischemic damage and inhibition of ATP production using DNP (group 4) caused endothelial damage, microcirculatory disturbance after reperfusion and consequently loss of graft viability. We conclude that microvascular endothelium of the pancreas graft is protected against cold ischemic injury by maintaining ATP tissue levels during preservation by the two-layer method. This is one of the mechanisms of action of the two-layer method in successful pancreas preservation.

Adenosine Triphosphate↗
PubMed1992

Characterization of the oxidative stress initiated in cultured human keratinocytes by treatment with peroxides.

Cultured human keratinocytes were treated with H2O2, Fe++/Fe , H2O2 + Fe++/Fe , t-butylhydroperoxide (tBHP), or cumene hydroperoxide (CHP). Fe++ +/- Fe was without effect on cell viability. Neither CHP, tBHP, nor H2O2 at 200 microM led to alteration of trypan blue exclusion, but with 700 microM CHP or tBHP there was uptake of trypan blue after 20 min and lysis of cells beginning at 4 h of treatment. Lysis occurred even if the organic hydroperoxide was removed from the media after 1 h. Treatment with 700 microM H2O2 resulted in half of the cells becoming permeable to trypan blue by 60 min, but > 80% of the cells remained intact and functional, and eventually recovered their impermeability to trypan blue. No concentration of H2O2, tBHP, or CHP produced significant thiobarbituric acid (TBA)-reactive material, and Fe++/Fe , H2O2 + Fe++/Fe , and CHP + Fe++/Fe led to the formation of only small amounts of TBA-reactive material. This was attributed to a lack of polyunsaturated lipid in cells cultured in synthetic media. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is sensitive to oxidative damage and thus was used as an indicator of oxidative stress along with the ratio of reduced/oxidized glutathione (GSH/GSSG). Using these two criteria, we found that CHP or tBHP treatment led to an oxidative stress that was more protracted as compared with the effect of H2O2. The organic peroxides also led to depletion of total glutathione, an effect not found with H2O2. It was also found that H2O2 was more rapidly metabolized than the organic peroxides. In summary, cultured human keratinocytes treated with peroxides underwent a number of changes, which included inactivation of GAPDH, a decrease in the ratio GSH/GSSG, and a loss of trypan blue exclusion. However, as long as the duration of this oxidative stress was short, these changes were reversible and the cells survived. Prolonged oxidative stress led to irreversible damage and cell death. H2O2 was rapidly metabolized and relatively well tolerated by keratinocytes. On the other hand, organic hydroperoxides were metabolized more slowly and were lethal at sub-millimolar concentrations. The relative toxicity of organic hydroperoxides is hypothesized to be related to their non-polar nature.

Cell Survival↗
PubMed1989

Adenine uptake by rat connective tissue cells with special reference to macrophage precursors.

After 3 daily injections of [14C]adenine ([14C]A) (1 microCi/g body weight each) into young adult rats, the large majority of typical, spindle-shaped fibroblasts in the connective tissues of most organs showed almost no labeling, with the exception of a few weakly labeled ones. In contrast, in the interstitial connective tissues of pancreas and heart, unexpectedly numerous mesenchymal cells were found to exhibit especially strong labeling with [14C]A even after RNase treatment. Such cells had a large, round or ovoid, euchromatic nucleus and showed the blast-like appearance. The term mesenchymal cells was used to denote a special type of connective tissue cells that retain the potentiality of embryonic mesenchymal cells. Double labeling experiments using [14C]A and trypan blue by 7 daily injections (1 microCi/g body weight of [14C]A and 3 ml/rat of 0.5% aqueous solution of trypan blue each) disclosed that the mesenchymal cells exhibiting heavy [14C]A labeling did not ingest trypan blue at all. It was further revealed that, in addition to the mesenchymal cells, macrophages showing a remarkable trypan blue uptake were also labeled with [14C]A, though to a lesser extent than were the former cells. Moreover, transitional forms between the mesenchymal cells and macrophages were occasionally observed. The transitional forms were characterized by having the capacities not only for a fairly high rate of DNA synthesis but also of ingesting trypan blue, though to a slight extent. On the basis of the above findings, it can be stated that the mesenchymal cells showing heavy labeling with [14C]A are most likely the precursor cells of macrophages.

Adenine↗
PubMed1997

Effect of perfusion pH on rat lung viability of non-heart beating donors.

OBJECTIVE: Non-heart beating donors could be an important source of lungs for transplantation. In prior experiments, trypan blue exclusion was used to assess the percentage of viable lung cells after different intervals following circulatory arrest. In this study, we assessed the importance of the trypan blue perfusate pH because in liver preservation studies, pH of the perfusate appears to be very important with the presence of a 'pH paradox'. METHODS: Forty Sprague-Dawley rats (n = 5 each group) were sacrificed, left at room temperature, and ventilated with 100% O2 (tidal volume 0.01 cc/g, rate 30/min) for 4 or 8 h after death. Lungs were then flushed with 150 cc of trypan blue in Krebs-Heinseleit buffer via the pulmonary artery, then with 150 cc of fixative (2%) paraformaldehyde + 2% gluteraldehyde). The pH of the trypan blue solution was varied from 6.5 to 7.8. Lungs were sectioned, and cell viability was quantified based on exclusion of trypan blue. RESULTS: Longer cadaver time was associated with less viability, but pH had no effect on viability. The results were similar in groups ventilated for the same period of time and then flushed with solutions at different pH (4 h, viability 80-84%; 8 h, viability 68-72%). Over the range investigated, the pH of the trypan blue solution had no impact on lung viability in non-heart beating donors and the lungs did not show the 'pH paradox'. CONCLUSION: Lung tissue is not susceptible to the 'pH paradox' observed in liver preservation studies. Because more than 80% of lung cells are still viable 4 h after death if the lungs are ventilated with 100% O2 during the cadaveric period, retrieval and transplantation of cadaveric lungs may be feasible and might alleviate the paucity of lung donors.

Animals↗
PubMed1989

In vivo and in vitro staining of acidophilic neurons as indicative of cell death following kainic acid-induced lesions in rat brain.

An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6h after kainic acid injection. This time was approximately 2-3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.

Animals↗
PubMed1994

Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes.

A number of reports have been published describing phagocytosis assays for flow cytometric analysis. In some of these, the fluorescence quenching technique has been used to discriminate between adherent and ingested particles. In this report, we have evaluated the efficacy of a quantitative fluorescence quenching technique with crystal violet and trypan blue for application in a phagocytosis assay with polymorphonuclear leukocytes and sensitized sheep red blood cells. We set the requirements to a high quenching efficiency of the fluorescence of extracellularly bound particles and no intracellular quenching. The latter was determined using polymorphonuclear leukocytes stained with the fluorescent nuclear dye hydroethidine. We observed that both trypan blue and crystal violet efficiently quench the fluorescence of PKH26 (a red fluorescent membrane-associated dye) erythrocytes but that only crystal violet quenches intracellular fluorescence. In testing trypan blue and crystal violet from different manufacturers, there was no real difference between different brands of crystal violet, but only the trypan blue from Merck turned out to be an efficient quencher, whereas the other brands of trypan blue showed low quenching efficiency. Trypan blue at a concentration of 25-50 micrograms/ml proved to be a good quencher of the fluorescent erythrocytes and exerted minimal side effects: over 90% quenching of the erythrocytes, no intracellular quenching, moderate increase in autofluorescence of the polymorphonuclear leukocytes, and no cell loss.

Animals↗
PubMed2004

Anterior capsule staining for capsulorhexis in cases of white cataract: comparative clinical study.

PURPOSE: To compare the safety and efficacy of trypan blue 0.1%, gentian violet 0.001%, indocyanine green 0.5% (ICG), fluorescein 2%, and the patient's autologous blood for anterior capsule staining in cases of white cataract. SETTING: Rajendra Prasad Center for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India. METHODS: Fifty eyes of 50 patients with age-related white cataract had anterior capsule staining with trypan blue, ICG, or gentian violet under an air bubble or subcapsularly with fluorescein or autologous blood followed by phacoemulsification with foldable intraocular lens implantation. Each stain was used in 10 eyes. The ease of creating a continuous curvilinear capsulorhexis (CCC) and the complications during the surgery were noted. Postoperative examinations at 6 hours, 1 day, 1 week, and 1 month included slitlamp microscopy, uncorrected visual acuity, and best corrected visual acuity (BCVA). The staining patterns on the anterior capsule, side port, corneal tunnel, and anterior cortex were assessed intraoperatively and within 6 hours and at 1 day. The intraocular pressure (IOP) was assessed at 1 day; pachymetry, at 1 day and 1 month; and the endothelial cell count, at 1 month. RESULTS: The surgeon had best visualization during the anterior capsulorhexis with trypan blue, ICG, and gentian violet, and a complete CCC was achieved in all eyes in the 3 groups. Two eyes each in the fluorescein and autologous blood groups had extension of the CCC so that the capsulorhexis was complete but not curvilinear. Anterior capsule fibrosis was detected with trypan blue (1 eye) and ICG (2 eyes). The anterior vitreous was stained with fluorescein in 2 eyes. All eyes achieved a BCVA of 20/30 or better from 1 week postoperatively to the last follow-up. The side port and corneal tunnel were stained most intensely with gentian violet followed by trypan blue and ICG and less intensely with fluorescein and autologous blood. The IOP, pachymetry, and endothelial cell loss were comparable between the stains. CONCLUSION: Although trypan blue, ICG, gentian violet, fluorescein, and autologous blood were safely used to stain the anterior capsule for phacoemulsification in eyes with white cataract, trypan blue, ICG, and gentian violet were more effective in staining the capsule.

Anterior Eye Segment↗
PubMedDate not supplied

Development of an intact blood-brain barrier in brain tissue transplants is dependent on the site of transplantation.

Transplantation of fetal septal forebrain tissue was performed to the anterior chamber of the eye, or intracranially to the rostral hippocampal formation in rats, to evaluate the impact of transplantation site on the development of an intact blood-brain barrier (BBB). The tissue was studied at 1,2,3, and 4 wk following transplantation by means of intravenous injection of Trypan blue, which is a vital stain not normally penetrating the BBB, as well as with an antibody specifically directed against the rat BBB, SM171. In the intraocular septal transplants, there was a significant leakage of Trypan blue 1 wk postgrafting, associated with a few laminin-immunoreactive blood vessels that did not contain any SM17I-immunoreactivity. However, at 2 wk postgrafting, the intraocular grafts exhibited an extensive plexus of thin-walled blood vessels expressing SMI71 immunoreactivity and no Trypan blue leakage. Thus, it appeared that a BBB had developed to some degree by 2 wk postgrafting in oculo. In the intracranial grafts, on the other hand, Trypan blue leakage could be seen as long as 3 wk postgrafting, and a dense plexus of blood vessels with SMI71 immunoreactivity was first seen at 4 wk postgrafting. Thus, the development of Trypan blue impermeability was delayed with 1 to 2 wk in the intracranial versus the intraocular grafts. Control experiments using psychological stress in adult rats as a means to transiently disrupt the BBB revealed that an increase in Trypan blue leakage correlated well with the disappearance of SMI71 immunoreactivity. Taken together, these studies demonstrate that the site of transplantation can influence the development of an intact BBB in neural tissue grafts.

Animals↗
PubMed1993

Rat pleural mesothelial cells show damage after exposure to external but not internal cigarette smoke.

The combination of cigarette smoke and high-level occupational asbestos exposure produces a synergistic increase in the incidence of lung cancer; however, smoking does not affect the incidence of mesothelioma. Here we present the results of tests of two theories that have been proposed to explain this phenomenon; namely, that pleural mesothelial cells are resistant to cigarette smoke-induced damage and that the pleural connective tissue acts as a barrier that prevents smoke from reaching the mesothelial cells. To test these hypotheses, excised whole rat lung preparations were exposed to either internal (intratracheal) or external (pleural surface) smoke. For comparison, additional excised lung preparations were exposed to solutions of hydrogen peroxide either externally or intratracheally. Mesothelial cells exposed to external smoke showed widespread, dose-dependent uptake of Trypan blue. Mesothelial cells did not take up Trypan blue after exposure to internal smoke. Bronchial epithelial cells exposed to internal smoke did show uptake, but to a lesser degree than externally exposed mesothelial cells. Examination by scanning and transmission electron microscopy showed that internal smoke did not affect mesothelial cell ultrastructure, whereas external smoke produced obvious mesothelial cell damage and mesothelial cell detachment. Catalase and deferoxamine, scavengers of active oxygen species, provided protection against smoke-induced mesothelial cell injury, but inactivated catalase did not. External hydrogen peroxide produced a very similar, dose-dependent pattern of Trypan blue uptake and ultrastructural changes. Intratracheal hydrogen peroxide also damaged mesothelial cells, but the extent of damage was always less than with comparable concentrations of external hydrogen peroxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae.

All tissues of larval Drosophila melanogaster express Hsp70, the major heat-shock protein of this species, after both mild (36 degrees C) and severe (38.5 degrees C) heat shock. We used Hsp70-specific immunofluorescence to compare the rate and intensity of Hsp70 expression in various tissues after these two heat-shock treatments, and to compare this with related differences in the intensity of Trypan Blue staining shown by the tissues. Trypan Blue is a marker of tissue damage. Hsp70 was rarely detectable before heat shock. Brain, salivary glands, imaginal disks and hindgut expressed Hsp70 within the first hour of heat shock, whereas gut tissues, fat body and Malpighian tubules did not express Hsp70 until 4-21 h after heat shock. Differences in Hsp70 expression between tissues were more pronounced at the higher heat-shock temperature. Tissues that expressed Hsp70 slowly stained most intensely with Trypan Blue. Gut stained especially intensely, which suggests that its sensitivity to heat shock may limit larval thermotolerance. These patterns further suggest that some cells respond primarily to damage caused by heat shock rather than to elevated temperature per se and/or that Hsp70 expression is itself damaged by heat and requires time for recovery in some tissues.

Animals↗
PubMed1988

Irreversible injury of isolated adult rat myocytes. Osmotic fragility during metabolic inhibition.

Isolated myocytes can be established as a valid model for studying changes in cytoskeletal proteins during the development of irreversible injury only if isolated cells develop lesions similar to those that occur during irreversible injury to intact hearts, specifically osmotic fragility and subsarcolemmal blebs. In the first experiment, isolated cells were irreversibly injured by metabolic inhibition with 5 mM Iodoacetic acid (IAA) and 6 mM amobarbital (Amy). Osmotic fragility of control and injured cells was determined by comparing the rates of development of trypan blue permeability during 60 minutes of isotonic or hypotonic (50% reduction in osmolality) incubations. Cell morphology was monitored by light and electron microscopy. Control cells remained elongated and excluded trypan blue. Metabolically inhibited cells rapidly contracted to a nearly square shape. The inhibited squared cells initially excluded trypan blue, but during 60 minutes of incubation became permeable to trypan blue. Cells in hypotonic buffer developed blue staining at a more rapid rate than cells in isotonic buffer, indicating increased osmotic fragility. In a second experiment, control and inhibited cells were first incubated for 25 minutes in isotonic buffer and then in either isotonic or hypotonic buffer. In this experiment, inhibited cells also developed more extensive and rapid permeability increases when transferred to the hypotonic buffer than cells maintained in the isotonic buffer. In both experiments, increased permeability of cells to trypan blue was accompanied by formation of subsarcolemmal blebs along the lateral cell border and at the intercalated disks. The results show that metabolically inhibited, isolated myocytes do exhibit morphologic lesions and increased osmotic fragility properties similar to those reported during anoxic or ischemic injury to intact hearts. Therefore, isolated myocytes may be a useful model with which to study cytoskeletal-sarcolemmal membrane changes during development of irreversible injury.

Animals↗