Biomedical subjects

Cheng-Hung Chuang

Publications and source records attributed to Cheng-Hung Chuang.

3 recordsLinked to original sources

PubMed2004

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay.

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.

Animals↗

PubMed2004

A downstream algorithm based on extended gradient vector flow field for object segmentation.

For object segmentation, traditional snake algorithms often require human interaction; region growing methods are considerably dependent on the selected homogeneity criterion and initial seeds; watershed algorithms, however, have the drawback of over segmentation. A new downstream algorithm based on a proposed extended gradient vector flow (E-GVF) field model is presented in this paper for multiobject segmentation. The proposed flow field, on one hand, diffuses and propagates gradients near object boundaries to provide an effective guiding force and, on the other hand, presents a higher resolution of direction than traditional GVF field. The downstream process starts with a set of seeds scored and selected by considering local gradient direction information around each pixel. This step is automatic and requires no human interaction, making our algorithm more suitable for practical applications. Experiments show that our algorithm is noise resistant and has the advantage of segmenting objects that are separated from the background, while ignoring the internal structures of them. We have tested the proposed algorithm with several realistic images (e.g., medical and complex background images) and gained good results.

Algorithms↗

PubMed2002

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues.

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 units/g liver at 37 degrees C for 20 min) for isolation of intact cells for subsequent comet assay We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation (-85 degrees C for 72 h). To minimize this damage, minced liver (1.0 g) and kidney (0.5 g) tissues were added to 20 ml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO (-85 degrees C for 72 h and 3 weeks), and to a slightly lesser extent with glycerol (72 h), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150units/g liver and 300units/g kidney for 10 min produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at - 85 degrees C for 72 h. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.

Animals↗