OBJECTIVETo study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.

METHODSSensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.

RESULTSOne hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50% - 90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or Annexin V-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.

CONCLUSIONSOur study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Annexin A5 ; Apoptosis ; physiology ; Cell Adhesion ; physiology ; Cytological Techniques ; DNA, Single-Stranded ; In Situ Nick-End Labeling ; Oxidative Stress ; physiology ; Sensitivity and Specificity ; Staining and Labeling ; methods

OBJECTIVE: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. METHODS: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). RESULTS: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001). CONCLUSION: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.
Case-Control Studies ; Chromatin ; DNA Fragmentation ; DNA Nucleotidylexotransferase ; DNA ; Humans ; In Situ Nick-End Labeling ; Infertility ; Infertility, Male ; Male ; Male ; Methods ; Reactive Oxygen Species ; Semen ; Sensitivity and Specificity ; Spermatozoa

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.
Adult ; Calibration ; DNA Fragmentation ; Flow Cytometry/instrumentation* ; Humans ; In Situ Nick-End Labeling ; Male ; Observer Variation ; Reference Values ; Reproducibility of Results ; Semen Analysis/methods* ; Sensitivity and Specificity ; Spermatozoa/chemistry*

OBJECTIVE: To observe the changes of DNA fragmentation and its quantity along with the time of injury in nerve cells after brain contusion. METHODS: The model of brain contusion caused by free drop hammer was established. TUNEL and Feulgen's DNA staining conjoined with image analysis technique were used for exploration. RESULTS: With the gradually rising of DNA fragmentation, DNA quantity was declining in the brain tissue after contusion. CONCLUSION TUNEL and Feulgen's DNA staining conjoined with image analysis technique could be utilized in the timing of brain injury and provide a new approach for this issue.
Animals ; Apoptosis ; Brain/pathology* ; Brain Injuries/pathology* ; DNA/analysis* ; DNA Fragmentation ; Disease Models, Animal ; Forensic Pathology ; In Situ Nick-End Labeling/methods* ; Male ; Neurons/pathology* ; Random Allocation ; Rats ; Rats, Wistar ; Staining and Labeling/methods* ; Time Factors

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.
Allografts ; Animals ; Apoptosis ; Brain Death ; China ; Death ; Heart Arrest ; Hepatocytes ; pathology ; ultrastructure ; Humans ; In Situ Nick-End Labeling ; Liver ; pathology ; ultrastructure ; Liver Transplantation ; methods ; Microscopy, Electron ; Organ Preservation ; methods ; Swine ; Tissue Donors ; Tissue and Organ Procurement ; methods

OBJECTIVETo observe the effects of three fluid resuscitation methods on apoptosis of visceral organs in rats with hemorrhagic shock.

METHODSA model of rat with severe hemorrhagic shock and active bleeding was established in 32 SD (Sprague-Dawley) rats. The rats were randomly divided into control group, no fluid resuscitation group (NF group), controlled fluid resuscitation group (NS40 group) and rapid large scale fluid resuscitation group (NS80 group). Each group contained 8 rats. The curative effects were compared. At the same time, the apoptosis in liver, kidney, lung and small intestinal mucosa of survivors after hemorrhage and resuscitation was detected by light microscopy in HE (hematoxylin and eosin) stained tissue sections, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL).

RESULTSThe survival rate of early fluid resuscitation (14/16) was markedly higher than that of NF group (3/8). There was some apoptosis in liver, kidney, lung and small intestinal mucosa of all survivors. Compared with NF and NS40 groups, the apoptosis of liver, kidney and small intestinal mucosa of NS80 group was obviously increased.

CONCLUSIONSAmong three fluid resuscitation methods, controlled fluid resuscitation can obviously improve the early survival rate and the apoptosis of liver, kidney and small intestinal mucosa in rats with severe and uncontrolled hemorrhagic shock, and may benefit improvement of prognosis.

Animals ; Apoptosis ; Blood Pressure ; Flow Cytometry ; Fluid Therapy ; methods ; In Situ Nick-End Labeling ; Intestine, Small ; pathology ; Kidney ; pathology ; Lactic Acid ; blood ; Liver ; pathology ; Lung ; pathology ; Male ; Organ Specificity ; Rats ; Rats, Sprague-Dawley ; Resuscitation ; methods ; Shock, Hemorrhagic ; pathology ; physiopathology ; therapy

PURPOSE: In Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium. METHODS: Argon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy. RESULTS: Corneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy. CONCLUSIONS: Argon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.
Animals ; Apoptosis ; Corneal Diseases/pathology/*surgery ; Disease Models, Animal ; Endothelium, Corneal/*pathology ; In Situ Nick-End Labeling ; Iris/*surgery ; Laser Therapy/*methods ; Lasers, Gas/*therapeutic use ; Ophthalmologic Surgical Procedures/*methods ; Rabbits

PURPOSE: In Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium. METHODS: Argon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy. RESULTS: Corneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy. CONCLUSIONS: Argon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.
Animals ; Apoptosis ; Corneal Diseases/pathology/*surgery ; Disease Models, Animal ; Endothelium, Corneal/*pathology ; In Situ Nick-End Labeling ; Iris/*surgery ; Laser Therapy/*methods ; Lasers, Gas/*therapeutic use ; Ophthalmologic Surgical Procedures/*methods ; Rabbits

OBJECTIVETo investigate the early effects of hypertonic and isotonic saline solutions on apoptosis of intestinal mucosa in rats with hemorrhagic shock.

METHODSA model of rat with severe hemorrhagic shock was established in 21 Sprague-Dawley (SD) rats. The rats were randomly divided into the sham group, normal saline resuscitation (NS) group, and hypertonic saline resuscitation (HTS) group, with 7 in each group. We detected and compared the apoptosis in small intestinal mucosa of rats after hemorrhagic shock and resuscitation by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), FITC (fluorescein-iso-thiocyanate)-Annexin V/PI (propidium iodide) double staining method, and flow cytometry.

RESULTSIn the early stage of hemorrhagic shock and resuscitation, marked apoptosis of small intestinal mucosa in the rats of both NS and HTS groups was observed. The numbers of apoptotic cells in these two groups were significantly greater than that in the sham group (P<0.01). In the HTS group, the apoptic cells significantly decreased, compared with the NS group (P<0.01).

CONCLUSIONIn this rat model of severe hemorrhagic shock, the HTS resuscitation of small volume is more effective than the NS resuscitation in reducing apoptosis of intestinal mucosa in rats, which may improve the prognosis of trauma.

Animals ; Apoptosis ; drug effects ; Disease Models, Animal ; Flow Cytometry ; Fluid Therapy ; methods ; In Situ Nick-End Labeling ; Intestinal Mucosa ; drug effects ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Resuscitation ; methods ; Saline Solution, Hypertonic ; administration & dosage ; Shock, Hemorrhagic ; therapy

We investigated the effect of low dose radiation on diabetes induced suppression of neurogenesis in the hippocampal dentate gyrus of rat. After 0.01 Gy, 0.1 Gy, 1 Gy and 10 Gy radiation was delivered, the dentate gyrus of hippocampus of streptozotocin (STZ)-induced diabetic rats were evaluated using immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU), caspase-3, and terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) staining. The number of BrdU positive cells in the non-diabetic rats, diabetic rats without radiation, diabetic rats with 0.01 Gy radiation, diabetic rats with 0.1 Gy radiation, diabetic rats with 1 Gy radiation and diabetic rats with 10 Gy radiation were 55.4+/-8.5/mm2, 33.3+/-6.4/mm2, 67.7+/-10.5/mm2, 66.6+/-10.0/mm2, 23.5+/-6.3/mm2 and 14.3+/-7.2/mm2, respectively. The number of caspase-3 positive cells was 132.6+/-37.4/mm2, 378.6+/-99.1/mm2, 15.0+/-2.8/mm2, 57.1+/-16.9/mm2, 191.8+/-44.8/mm2 and 450.4+/-58.3/mm2, respectively. The number of TUNEL-positive cells was 24.5+/-2.0/mm2, 21.7+/-4.0/mm2, 20.4+/-2.0/mm2, 18.96+/-2.1/mm2, 58.3+/-7.9/mm2, and 106.0+/-9.8/mm2, respectively. These results suggest low doses of radiation paradoxically improved diabetes induced neuronal cell suppression in the hippocampal dentate gyrus of rat.
Rats, Sprague-Dawley ; Rats ; Radiotherapy/methods ; Neurons/*metabolism ; Male ; In Situ Nick-End Labeling ; Hippocampus/*cytology/metabolism/radiation effects ; Diabetes Mellitus, Experimental/radiotherapy ; Dentate Gyrus/drug effects/*radiation effects ; Cell Proliferation ; Caspase 3/metabolism ; Bromodeoxyuridine/pharmacology ; Apoptosis ; Animals