ethidium-homodimer and Necrosis

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg2+, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg2+, ethidium homodimer (5 microM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 microm. Sections were permeabilized with -20 degrees C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.. The Hg2+-treated animals showed a dose-dependent increase in the number of ethidium labeled cells in the proximal tubule, but not in other segments of the nephron. Other results showed that a nephrotoxic dose of gentamicin also caused a significant increase in the number of ethidium labeled cells in the proximal tubule.. These results indicate that this simple and sensitive perfusion technique can be used to evaluate cellular necrosis in the proximal tubule with the three-dimensional cyto-architecture intact.

Topics: Animals; Apoptosis; Cell Count; Cell Survival; Cells, Cultured; Ethidium; Kidney Cortex Necrosis; Kidney Tubules, Proximal; Luminescent Measurements; Male; Microscopy, Fluorescence; Necrosis; Rats; Rats, Sprague-Dawley; Staining and Labeling

Necrosis and apoptosis have been demonstrated in articular cartilage in response to trauma and disease. However, cell death in articular cartilage may also be thought of as a scale of cell death culminating in secondary necrosis with the failure to remove apoptotic cells from the tissue. The in situ detection of cell death is an important technique in studying articular cartilage as it most closely resembles the in vivo situation. The methods described here involve the use of light microscopy and electron microscopy in conjunction with fluorescent and biochemical methods to correctly ascertain the type of cell death that has occurred.

Topics: Animals; Cartilage, Articular; Cattle; Cell Death; Chondrocytes; Cytological Techniques; Ethidium; Fluorescent Dyes; Humans; Male; Microscopy, Electron, Transmission; Necrosis; Osteoarthritis

Coxsackievirus infections may trigger and accelerate pancreatic beta-cell death, leading to type I diabetes. Unrestricted coxsackievirus B5 replication in cultured beta-cells inoculated with high multiplicity leads to rapid lytic cell death. Evidence from other virus-host cell systems indicates that host cell responses to infection may depend on the multiplicity of infection (MOI). Thus, the aim of this study was to compare the mechanisms of beta-cell death during high versus low multiplicity of coxsackievirus B5 infection. Cultures of highly differentiated mouse insulinoma cells and primary adult human islets were infected with coxsackievirus B5 at multiplicities of >1,000 or <0.5 TCID50 per cell. The results of nuclear morphology and viability stainings, TUNEL staining and electrophoretic DNA fragmentation analysis showed high multiplicity infection to predominantly induce necrosis and transient apoptosis. In low multiplicity culture, however, necrosis was only moderately induced and apoptosis increased steadily with time. This was best demonstrated by a tenfold higher apoptosis/necrosis ratio than after high multiplicity inoculation. Expression of gamma-glutamyl cysteine synthetase increased in both infective cultures but the level of intracellular glutathione permanently depleted only at high multiplicity and recovered fully at low multiplicity. Thus, apoptosis represents an important mechanism of beta-cell death after low multiplicity of coxsackievirus B5 infection. This process is associated with maintenance of a physiological intracellular glutathione profile differing dramatically from the high multiplicity infection during which necrosis dominates and intracellular thiol balance deteriorates. These data suggest that the pattern and mechanisms of coxsackievirus B5 infection induced beta-cell death depend on the MOI.

Topics: Animals; Apoptosis; Benzimidazoles; Cell Death; Cell Line, Tumor; Cell Nucleus; Cells, Cultured; DNA Fragmentation; Enterovirus B, Human; Ethidium; Glutamate-Cysteine Ligase; Glutathione; Humans; In Situ Nick-End Labeling; Insulinoma; Islets of Langerhans; Mice; Necrosis; Nitrites

Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.

Topics: Cell Membrane; Cell Survival; Cell Transplantation; Collagenases; Coloring Agents; Ethidium; Fluoresceins; Fluorescent Dyes; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Necrosis; Organic Chemicals; Propidium; Sensitivity and Specificity; Time Factors

To evaluate the potential cytotoxic effects of indocyanine green (ICG) on cultured human retinal pigment epithelium (RPE) and the resultant implications for macular hole surgery.. Human RPE cells were exposed to ICG in concentrations from 0.001 to 5 mg/mL. The exposure duration ranged from 5 minutes to 3 hours. Light microscopy, MTS viability assay, and calcein AM-ethidium homodimer 1 staining were used to evaluate the cytotoxic effects of ICG.. The RPE cells incubated with up to 5 mg/mL of ICG for 5 minutes or less exhibited no morphologic change and no significant decrease in dehydrogenase activity. When RPE cells were exposed to 5 mg/mL of ICG for 10 minutes, 1 mg/mL of ICG for 20 minutes, or 0.01 mg/mL of ICG for 3 hours, cell morphologic features were altered, mitochondrial dehydrogenase activity decreased, and some cells were necrotic.. Indocyanine green caused cytotoxicity in cultured human RPE in a dose- and time-dependent manner. Cell death occurred by necrosis.. Exposure of RPE cells to ICG concentrations up to 5 mg/mL for 5 minutes or less was not injurious; prolonged exposure to a low ICG concentration was toxic. Since ICG may be retained in the vitreous cavity for a lengthy period, thorough washout of ICG during macular hole surgery is required.

Topics: Cell Death; Cells, Cultured; Coloring Agents; Dose-Response Relationship, Drug; Ethidium; Fluoresceins; Humans; Indocyanine Green; Mitochondria; Necrosis; Oxidoreductases; Pigment Epithelium of Eye; Retinal Perforations; Time Factors