Transferase-mediated dUTP Nick-end Labeling-positive Cells Research Articles

Metformin Attenuates Myocardial Ischemia-Reperfusion Injury through the AMPK-HMGCR-ROS Signaling Axis.

To explore the role and mechanism of metformin (MET) in regulating myocardial injury caused by cardiac ischemia-reperfusion. A rat model of myocardial ischemia-reperfusion injury was established by ligation of the anterior descending branch of the left coronary artery. The myocardial area at risk and the infarction size were measured by Evans blue and 2,3,5‑triphenyltetrazole chloride (TTC) staining, respectively. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining was used to detect apoptosis of cardiomyocytes. The expression of 4‑hydroxynonenal (4‑HNE) was detected by immunohistochemical staining. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and expression of the Adenosine 5'-monophosphate-activated protein kinase (AMPK) - 3‑hydroxy-3‑methylglutaryl-CoA reductase (HMGCR) signaling pathway, respectively. MET treatment decreased the infarct size and the activity of the myocardial enzyme profile, thus demonstrating protection of ischemic myocardium. The number of TUNEL positive cells significantly decreased. Immunohistochemical results showed that MET decreased the expression of 4‑HNE in myocardial tissue and the content of malondialdehyde (MDA) in myocardial cells. Further experimental results showed that MET decreased HMGCR transcription and protein expression, and increased AMPK phosphorylation. In the model of hypoxia and reoxygenation injury of cardiomyocytes, MET increased the viability of cardiomyocytes, decreased the activity of lactic dehydrogenase (LDH), decreased malondialdehyde content and intracellular reactive oxygen species (ROS) concentrations, and regulate the AMPK-HMGCR signaling pathway through coenzyme C (ComC). MET inhibits the expression of HMGCR by activating AMPK, reduces oxidative damage and apoptosis of cardiomyocytes, and alleviates myocardial ischemia-reperfusion injury.

74 Secondary Immunohistochemical Analysis of Viable Cells as Determined by Hematoxylin and Eosin Staining

Abstract Introduction Accurate clinical differentiation of deep partial-thickness (DPT) and full-thickness (FT) wounds is essential in justifying appropriate treatments for heterogeneous burns. Only recently has non-invasive burn depth imaging been ground-truthed with tissue punch biopsies; an endeavor that will no doubt improve AI imaging algorithms in the near future. Prior literature supports unique apoptotic and necrotic properties of DPT and FT wounds, suggesting a use for not only standard stains (H&E), but apoptotic marker staining in assisting clinical diagnosis of burn depth, progression and/or conversion. Methods The aim of this study was to evaluate the presence of apoptotic cells in DPT and FT samples previously diagnosed by a burn biopsy algorithm (BBA-V2) for burn depth and severity by expert review of H&E-stained slides. Eight paired biopsies were collected from DPT and FT wounds in four patients within 72 hours of thermal injury and were immunofluorescently labeled for apoptosis markers by detection of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase-3 (CC-3) staining. Fluorescence intensity in dermal appendages within images was used to quantify signal at 40x magnification for CC-3. % positive TUNEL cells were graded by an investigator blinded to the burn severity. A higher grade indicated higher % positivity. TUNEL grading was evaluated by Chi square test. CC-3 intensity was evaluated by Student’s t-test. Results Across all four patients, FT burns had higher levels of CC-3 in dermal appendages compared to DPT burns (Cy3:DAPI ratios: 1.53 ± 0.28 vs. 0.75 ± 0.13; p=0.1074). Within each patient, all FT biopsy sites had significantly higher levels of CC-3 versus DPT biopsy sites (p< 0.05)(Table). DPT burns had a greater proportion of patients with increasing grades of TUNEL positive cells (DPT: Grade 1: 16%, Grade 2: 36%, Grade 3: 48%; FT: Grade 1: 42.3%, Grade 2: 23.1%, Grade 3: 34.6%, p=0.1178). Conclusions CC-3 and TUNEL levels within dermal appendages align with burn depth classification by H&E staining, wherein appendages graded as necrotic had higher levels of CC-3 and lower levels of TUNEL. Therefore, CC-3 and TUNEL are potential secondary histological markers for burn depth. Further analysis of isolated, individual dermal appendages is underway to identify what additional information this secondary marker may add to the diagnosis of burn depth. Applicability of Research to Practice For the first time there is access to a large tissue repository of biopsies from burn wounds. Although staining from these samples has been used to build diagnosis algorithms with success, much more can be learned from the cellular pathophysiology when secondary analyses are performed. These assays may help us understand the processes of burn conversion.

Liver fibrosis is a common pathological change in the liver of dairy cows with fatty liver

Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3-5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10-14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG <1% wet wt), mild fatty liver (n = 15; 1% ≤ TG <5% wet wt), and moderate fatty liver (n = 5; 5% ≤ TG <10% wet wt). Compared with normal cows, blood concentrations of nonesterified fatty acids and β-hydroxybutyrate, along with alanine aminotransferase and aspartate aminotransferase activities, were greater in the cows with fatty liver (mild and moderate). Hepatic extracellular matrix deposition, as indicated by Picrosirius red staining, was greater in cows with fatty liver than those with normal ones. In addition, we observed an increased proportion of collagen type I fiber in extracellular matrix with increased lipid accumulation in the liver. Compared with normal cows, the area of α-smooth muscle actin (α-SMA)-positive staining along with the mRNA abundance of collagen type I α 1 (COL1A1), ACTA2 (gene encoding α-SMA), and transforming growth factor-β (TGFB) were greater in cows with fatty liver. Compared with normal cows, hepatic contents of malondialdehyde, glutathione disulfide, and 8-isoprostane were greater, whereas total antioxidant capacity, the hepatic content of glutathione, and activities of antioxidant indicators, including superoxide dismutase, glutathione peroxidase, and catalase, were lower in cows with fatty liver. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and abundance of apoptosis-related molecules BAX, CASP3, CASP8, and CASP9 were greater in cows with fatty liver. However, mRNA abundance of the anti-apoptotic gene BCL2 did not differ. The mRNA abundance of pro-inflammatory cytokines including tumor necrosis factor-α (TNFA), interleukin-1β (IL1B), and interleukin-6 (IL6) was greater in the liver of cows with fatty liver. Overall, the present study indicated that fibrosis is a common pathological response to liver damage and is associated with oxidative stress, hepatocyte death, and inflammation.

Alpha-1-Antitrypsin Protects Vascular Grafts of Brain-Dead Rats Against Ischemia/Reperfusion Injury

Endothelial dysfunction is a potential side effect of brain death (BD). Ischemia/reperfusion (IR) injury during heart transplantation may lead to further endothelial damage. Protective effects of alpha-1-antitrypsin (AAT), a human neutrophil serine protease inhibitor, have been demonstrated against IR injury. We hypothesized that AAT protects brain-dead rats' vascular grafts from IR injury. Donor rats were subjected to BD by inflation of a subdural balloon. After 5.5h, aortic rings were immediately mounted in organ baths (BD, n=6 rats) or preserved in saline, supplemented either with vehicle (BD-IR, n=8 rats) or AAT (BD-IR+AAT, n=14 rats) for 24 h. During organ bath experiment, rings from both IR groups were exposed to hypochlorite to simulate warm reperfusion-associated endothelial injury. Endothelial function was measured exvivo. Immunohistochemical staining for caspases was carried out and DNA-strand breaks were evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Data are presented as median (interquartile range). AAT improved IR-induced decreased maximum endothelium-dependent vasorelaxation to acetylcholine in the BD-IR+AAT aortas compared to the BD-IR group (BD: 83 (9-28) % versus BD-IR: 49 (39-60) % versus BD-IR+AAT: 64 (24-42) %, P<0.05). Additionally, an increase in the rings' sensitivity to acetylcholine was noted after AAT (pD2-value: BD-IR+AAT: 7.35 (7.06-7.89) versus BD-IR: 6.96 (6.65-7.21), P<0.05). Caspase-3, -8, -9, and -12 immunoreactivity and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were significantly decreased by AAT. AAT alleviates endothelial dysfunction, prevents increased caspase-3, -8, -9, and -12 levels, and decreases apoptotic DNA breakage due to BD and IR injury. This suggests that AAT treatment may be therapeutically beneficial to reduce IR-induced vascular damage.

Assessment of Hippocampal Cell Death and Memory Deficits in Rats Following Neonatal Stress

Objective: Neonatal stress (NS) has harmful effects on the hippocampal neurons of rat neonates. It has been reported to enhance neuronal cell death and impair memory behaviors. The researchers conducted this study in order to assess NS’s effects on hippocampal apoptosis and memory deficits in rat neonates’ hippocampus. Methods: Three groups of male Wistar rat neonates exposed to NS; rat neonates reared with 1 hour neonatal isolation (NI) for 8 consecutive days (P2-P9), rat neonates exposed to febrile seizure (FS) at day 10 (P10) and rat neonates reared with both NI plus FS (NI-FS). Control group was reared normally. Novel object recognition test (NORT) carried out to evaluate the effects of NI, FS and NI-FS on memory deficits. At day 22 (P22), Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) assay was done. Results: NORT demonstrated that rat neonates exposed to NI-FS had short-term and long-term memory deficits (P&lt;0.01 and P&lt;0.001). Rat neonates experienced NI had long-term memory deficits (P&lt;0.05). TUNEL assay results showed that NI-FS increased the count of hippocampal apoptotic neurons in Cornu Ammonis 1 (CA1), Cornu Ammonis 3 (CA3) and dentate gyrus (DG) subfields (P&lt;0.001, P&lt;0.001 and P&lt;0.001). Also, NI increased the count of hippocampal TUNEL positive cells in CA3 and DG subfields (P&lt;0.05 and P&lt;0.01). In addition, FS increased the count of hippocampal apoptotic neurons in DG subfield (P&lt;0.05). Conclusion: The present results showed that NS exerted apoptotic effect and induced memory deficits in the hippocampus of rat neonate.

Epigallocatechin-3-Gallate Attenuates Adriamycin-Induced Focal Segmental Glomerulosclerosis via Suppression of Oxidant Stress and Apoptosis by Targeting Hypoxia-Inducible Factor-1α/ Angiopoietin-Like 4 Pathway

Background: Focal and segmental glomerular sclerosis (FSGS) is a common cause of nephrotic syndrome and end-stage renal disease. It has been reported that overproduction of reactive oxygen species (ROS) and cell apoptosis are associated with the development of FSGS. Epigallocatechin-3-gallate (EGCG) is a bioactive constituent accounting for more than 50% of the total catechins in green tea, which have anti-oxidative and anti-apoptotic effects. Based on this, this study was designed to evaluate the renoprotective effect of EGCG treatment on Adriamycin-induced FSGS. ­Methods: In C57BL/6 mice, Adriamycin nephropathy (AN) was induced by Adriamycin (10 mg/kg body weight, diluted in normal saline) via a tail vein on day 0. Then the mice were given with EGCG (20 mg/kg body weight) or YC-1 (Lificiguat, a specific inhibitor of hypoxia-inducible factor-1α [HIF-1α], 50 mg/kg body weight) or both intraperitoneally. Both the EGCG and YC-1 were given on the day of Adriamycin injection and continued for 6 weeks. The animals were organized into the following 5 groups for the animal experiments: the control group, the AN group, the AN + EGCG group, the AN + YC-1 group and the AN + EGCG + YC-1 group. At 6 weeks, the mice were sacrificed; kidneys and blood samples were collected for further analysis. The HIF-1α and the angiopoietin-like 4 (ANGPTL4) expression were detected by Western blot, real-time PCR, immunohistochemistry or immunofluorescence. Dihydroethidium staining and NADPH oxidase 1 (Nox1) measurement were used to detect ROS production. Terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining and caspase-3 measurement was used to detect cell apoptosis. Results: When the animals were treated with Adriamycin, both the ROS production and TUNEL positive cells increased. Besides, the expression of HIF-1α, ANGPTL4, and caspase-3 were also up-regulated, while EGCG treatment could attenuate these changes. Interestingly, compared with treatment with YC-1 or EGCG alone, more pronounced inhibition of ANGPTL4, caspase-3 and Nox1 were obtained when YC-1 and EGCG were administered simultaneously. Conclusion: EGCG attenuates FSGS through the suppression of Oxidant Stress and apoptosis by targeting the HIF-1α/ANGPTL4 pathway.

A Contrast in Pathogenic Responses between C57BL/6J and BALB/cJ Mice Using a Model of Retinal Injury

Ischemia is associated with the pathogenesis of retinal disease, including diabetic retinopathy and glaucoma. As a result, the retinal ischemia/reperfusion injury model has been used to study neurovascular changes. Historically, murine models of retinal disease are established in C57BL/6J (B6) mice, which have been described as type 1-dominant responders. In bacterial keratitis models, B6 mice are susceptible, whereas BALB/cJ (BALB/c; type 2-dominant) mice exhibit a resistant phenotype. As such, we questioned whether the type 1/type 2 paradigm could be extrapolated to events associated with retinal pathogenesis. The current study compares the retinal response of B6 with BALB/c mice to investigate strain-specific differences. Retinas were collected at 2 and 10 days after ischemia/reperfusion injury to examine differences in neurovascular degeneration, leukostasis, oxidative stress, glial activation, and select inflammatory mediators. Although both strains showed signs of retinal injury, significantly more damage was observed in B6 mice. Retinal thickness was reduced and vascular damage was more severe in B6 mice. Exacerbated response to injury in B6 versus BALB/c retinas was furthersupported by increased leukostasis, inflammatory mediators, reactive oxygen species, and lipidperoxidation. In addition, more terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and increased glial activation were detected in B6 mice. These data indicate that B6 and BALB/c retinas differentially respond to injury, which has broader implications regarding the development and study of retinal diseases.

New generation of drug delivery systems based on ginsenoside Rh2-, Lysine- and Arginine-treated highly porous graphene for improving anticancer activity

In this study, Rh2–treated graphene oxide (GO-Rh2), lysine-treated highly porous graphene (Gr-Lys), arginine-treated Gr (Gr-Arg), Rh2–treated Gr-Lys (Gr-Lys-Rh2) and Rh2–treated Gr-Arg (Gr-Arg-Rh2) were synthesized. MTT assay was used for evaluation of cytotoxicity of samples on ovarian cancer (OVCAR3), breast cancer (MDA-MB), Human melanoma (A375) and human mesenchymal stem cells (MSCs) cell lines. The percentage of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The hemolysis and blood coagulation activity of nanostructures were performed. Interestingly, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2 were more active against cancer cell lines in comparison with their cytotoxic activity against normal cell lines (MSCs) with IC50 values higher than 100 μg/ml. The results of TUNEL assay indicates a significant increase in the rates of TUNEL positive cells by increasing the concentrations of nanomaterials. Results were also shown that aggregation and changes of RBCs morphology were occurred in the presence of GO, GO-Rh2, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2. Note that all the samples had effect on blood coagulation system, especially on PTT. All nanostrucure act as antitumor drug so that binding of drugs to a nostructures is irresolvable and the whole structure enter to the cell as a drug.

Deficiency in cold-inducible RNA-binding protein attenuates acute respiratory distress syndrome induced by intestinal ischemia-reperfusion

Intestinal ischemia-reperfusion can occur in shock and mesenteric occlusive diseases, causing significant morbidity and mortality. Aside from local injury, intestinal ischemia-reperfusion can result in remote organ damage, particularly in the lungs. Cold-inducible RNA-binding protein (CIRP) was identified as a novel inflammatory mediator. We hypothesized that a deficiency in CIRP would protect the lungs during intestinal ischemia-reperfusion injury. Intestinal ischemia was induced in adult male C57BL/6 wild-type and CIRP knock-out (CIRP-/-) mice via clamping of the superior mesenteric artery for 60minutes. Reperfusion was allowed for 4hours or 20hours, and blood, gut, and lung tissues were harvested for various analyses. After intestinal ischemia-reperfusion, the elevated levels of serum lactate dehydrogenase and inflammatory cytokine interleukin-6 were reduced by 68% and 98%, respectively, at 20hours after ischemia-reperfusion in CIRP-/- mice compared with the wild-type mice. In the gut, mRNA levels of inflammatory cytokine interleukin-6 were reduced by 67% at 4hours after ischemia-reperfusion in CIRP-/- mice. In the lungs, inflammatory cytokine interleukin-6 protein and myeloperoxidase activity were reduced by 78% and 26% at 20hours and 4hours after ischemia-reperfusion, respectively, in CIRP-/- mice. Finally, the elevated lung caspase-3 was significantly decreased by 55%, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells decreased by 91%, and lung injury score decreased by 37% in CIRP-/- mice at 20hours after ischemia-reperfusion. Increased levels of proinflammatory cytokines, myeloperoxidase, and apoptosis are the hallmarks of acute respiratory distress syndrome. We noticed after intestinal ischemia-reperfusion the proinflammatory milieu in lungs was elevated significantly, while the CIRP-/- mice had significantly decreased levels of proinflammatory cytokine, myeloperoxidase, and apoptotic cells leading to decreased lung injury. These findings strongly established a causal link between CIRP and acute respiratory distress syndrome during intestinal ischemia-reperfusion injuries. Targeting CIRP may therefore be beneficial for treatment of intestinal ischemia-reperfusion-associated acute respiratory distress syndrome acute respiratory distress syndrome.

Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

PurposeTo investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface.MethodsLEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2’7’-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed.ResultsTBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group.ConclusionsOverexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

The Effect of Everolimus on Scar Formation in Glaucoma Filtering Surgery in a Rabbit Model

ABSTRACTPurpose: To investigate the efficiency of everolimus on the prevention of postoperative scar in a rabbit model of glaucoma filtering surgery in comparison with mitomycin-C (MMC).Materials and Methods: Thirty New Zealand albino rabbits were randomly assigned into 3 groups, each including ten rabbits: an everolimus group (Group 1), a MMC group (Group 2), and a sham group (Group 3). A limbal-based trabeculectomy was performed on the right eyes of all the rabbits. For 28 days following surgery, the eyes were evaluated in terms of intraocular pressure (IOP), morphological and biomicroscopic changes, and complications in the bleb. On the 28th day, four eyes randomized from each group were enucleated and histologically and immunohistochemically analyzed. Transforming growth factor-β1 (TGF-β1), metalloproteinase (MMP-2, MMP-9), and proliferative cell nuclear antigen (PCNA) expressions in each group were evaluated. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was used for apoptosis.Results: Bleb survival was statistically significantly longer for a period in Group 1 compared to Group 2. When postoperative IOPs of three groups were measured, it was seen that there is significant IOP reduction in all three groups. However, there were increases in the mean IOP values beginning from the 5th day in Group 2 and from the 3rd day in Group 3 while in Group 1 mean IOP values began to increase beginning from 10th day and the mean IOP values in Group 1 remained at a lower level in comparison to the other groups for 28 days (p < 0.05). The expressions of TGF-β1, MMP-2, MMP-9, and PCNA were reduced in Group 1 compared to other groups. TUNEL positive apoptotic cells were significantly increased in Group 1 compared to other groups (p < 0.05).Conclusion: Everolimus appears to suppress the proliferation of fibroblast and thus may provide an effective treatment strategy in glaucoma filtering surgery.

PD35-01 RENAL ARTERIAL INFUSION OF ADIPOSE TISSUE-DERIVED STROMAL VASCULAR FRACTION PROTECTS THE RENAL FUNCTION AGAINST ACUTE KIDNEY INJURY INDUCED BY ISCHEMIA-REPERFUSION INJURY

You have accessJournal of UrologyStem Cell Research1 Apr 2016PD35-01 RENAL ARTERIAL INFUSION OF ADIPOSE TISSUE-DERIVED STROMAL VASCULAR FRACTION PROTECTS THE RENAL FUNCTION AGAINST ACUTE KIDNEY INJURY INDUCED BY ISCHEMIA-REPERFUSION INJURY Myoung Jin Jang, Bo Hyun Kim, Chunwoo Lee, Dalsan You, In Gab Jeong, and Chung-Soo Kim Myoung Jin JangMyoung Jin Jang More articles by this author , Bo Hyun KimBo Hyun Kim More articles by this author , Chunwoo LeeChunwoo Lee More articles by this author , Dalsan YouDalsan You More articles by this author , In Gab JeongIn Gab Jeong More articles by this author , and Chung-Soo KimChung-Soo Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1073AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) is a major challenge in critical care medicine, with high rates of in-hospital morbidity and mortality. Preclinical studies suggested that administered cultured stem cells protected kidney function against AKI induced by IRI. Due to several concerns regarding the implantation of cultured stem cells into humans, however, recent clinical studies have focused on the use of stromal vascular fraction (SVF) in treating various diseases. The purpose of this study was to determine the therapeutic effects of adipose tissue-derived SVF and the optimal route for SVF delivery in a rat model of AKI induced by IRI. METHODS Fifty male Sprague-Dawley rats were randomly divided into 5 groups (10 animals per group): sham, nephrectomy control, IRI control, intra-renal arterial SVF infusion, and subcapsular SVF injection. To induce AKI induced by IRI, the left renal artery was clamped with a nontraumatic vascular clamp for 40 minutes, and the right kidney was removed. Serum creatinine and glomerular filtration rate were evaluated 1 day prior to IRI, and 2, 4, 7, 14 days after IRI. For histomorphometric studies, the kidney was removed 2 weeks after IRI. RESULTS Intra-renal arterial infusion of SVF significantly reduced the extent of elevation in serum creatinine compared with the IRI control group 4 days after IRI. The glomerular filtration rate of intra-renal arterial SVF infusion group was maintained at a level similar to those of the sham and nephrectomy control groups 14 days after IRI. Masson's trichrome stain for the degree of fibrosis showed that intra-renal arterial infusion of SVF was significantly less fibrosis than that observed in the IRI control group (P<0.001). Staining for alpha-smooth muscle actin showed that the number of small vessels was noticeably higher in the intra-renal arterial SVF infusion and subcapsular SVF injection groups than in the IRI control group (P<0.001). The number of terminal transferase-mediated dUTP nick-end labeling-positive cells increased after IRI, suggesting apoptosis or necrosis had occurred. Terminal transferase-mediated dUTP nick-end labeling assay showed significantly decreased apoptosis in both the intra-renal arterial SVF infusion and subcapsular SVF injection groups compared to the IRI control group (P<0.001). A greater increase in glutathione reductase and glutathione peroxidase was observed in both the intra-renal arterial SVF infusion and subcapsular SVF injection groups than in the IRI control group (P<0.001). These findings further suggest that anti-oxidative responses were elicited by IRI and SVF treatment contributed to further anti-inflammatory and anti-oxidative effects after IRI in this study. CONCLUSIONS Our study showed that renal function is effectively rescued from AKI induced by IRI through administration of SVF in a rat model and intra-renal arterial infusion of SVF was more effective than subcapsular injection. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e844 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Myoung Jin Jang More articles by this author Bo Hyun Kim More articles by this author Chunwoo Lee More articles by this author Dalsan You More articles by this author In Gab Jeong More articles by this author Chung-Soo Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats

Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis.

Puerarin attenuates the inflammatory response and apoptosis in LPS-stimulated cardiomyocytes.

Patients with septic shock suffer from high mortality rates, particularly when complicated by severe myocardial depression which is characterized by hypotension and a reduction in cardiac output. Inflammation is an important factor involved in the early stages of sepsis. The aim of the present study was to investigate the effect of the Chinese herbal compound puerarin (1, 5, 10, 20 and 40 µM) on cardiomyocyte inflammatory response in a sepsis model using H9c2 cardiomyocytes stimulated with 1 µg/ml lipopolysaccharide (LPS). The mRNA expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-β were evaluated using reverse transcription-quantitative polymerase chain reaction. In addition, the protein expression levels of various factors were determined using western blot analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to evaluate the apoptosis rates in the various groups, and immunocytochemical analysis was employed to determine the effect of puerarin on the nuclear translocation of p65 protein. The present study demonstrated that LPS stimulation increased IL-1β and TNF-α mRNA expression levels, as compared with the controls (P<0.05). Following treatment with various concentrations of puerarin, the expression levels of IL-1β and TNF-α were markedly blunted, particularly in the LPS + 40 µM puerarin group (P<0.05 vs. the LPS group). Furthermore, puerarin administration significantly inhibited LPS-induced apoptosis in H9c2 cardiomyocytes, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (TUNEL positive cells: LPS + 40 µM puerarin group, 5.5% vs. LPS group, 10.5%; P<0.01). In addition, puerarin significantly decreased LPS-induced phosphorylated nuclear factor (p-NF)-κB p65 and Bax expression levels, and increased the expression levels of Bcl-2, as compared with the LPS group (P<0.05). These data indicated that puerarin may serve as a valuable protective agent against cardiovascular inflammatory diseases.

Increased O-GlcNAcylation of NF-κB Enhances Retinal Ganglion Cell Death in Streptozotocin-induced Diabetic Retinopathy

Purpose: Hyperglycemia results in increased flux through the hexoxamine biosynthetic pathway. We examined whether hyperglycemia increases O-GlcNAcylation in the diabetic retina and whether elevated O-GlcNAcylation of nuclear factor (NF)-κB increases apoptosis of retinal ganglion cells (RGCs) in diabetic retinopathy (DR).Materials and methods: Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin. All mice were killed 2 months after injections and expression levels of O-GlcNAcylated proteins, O-linked N-acetylglucosamine transferase (OGT), β-d-N-acetylglucosaminidase and NF-κB, and the extent of RGC death were examined. Immunoprecipitations were performed to investigate whether O-GlcNAcylation of NF-κB led to its activation and RGC death in DR.Results: The expression levels of O-GlcNAcylated proteins and OGT were markedly higher in diabetic retinas than in control retinas. OGT colocalized with NeuN, a RGC-specific marker, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in the ganglion cell layer of diabetic retinas. The p65 subunit of NF-κB was O-GlcNAcylated and the level of O-GlcNAcylated p65 was higher in diabetic retinas than in control retinas.Conclusion: The present data suggest that hyperglycemia increases O-GlcNAcylation in DR and that O-GlcNAcylation of the p65 subunit of NF-κB is involved in hyperglycemia-induced NF-κB activation and RGC death in DR.

Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway.

Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui (GV20) acupoint for 30 minutes at 1 mA and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α (AMPKα) and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation.

Effect of cholecystokinin on learning and memory, neuronal proliferation and apoptosis in the rat hippocampus.

Background:Cholecystokinin (CCK) has roles in learning and memory, but the cellular mechanism is poorly understood. This study investigated the effect of CCK on spatial learning and memory, neuronal proliferation and apoptosis in the hippocampus in rats.Materials and Methods:Experimental groups were control and CCK. The rats received CKK octapeptide sulfated (CCK-8S, 1.6 μg/kg, i.p.) for 14 days. Spatial learning and memory were tested by Morris water maze and finally immunohistochemical study was performed; neurogenesis by Ki-67 method and apoptosis by Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) assay in hippocampal dentate gyrus (DG).Results:Cholecystokinin increased Ki-67 positive cells and reduced TUNEL positive cells in the granular layer of hippocampal DG. CCK failed to have a significant effect on spatial learning and memory.Conclusion:Results indicate neuroprotective and proliferative effects of CCK in the hippocampus; however, other factors are probably involved until the newly born neurons achieve necessary integrity for behavioral changes.

Therapeutic effects of CCAAT/enhancer-binding protein α gene on liver fibrosis in mice

Objective To investigate the difference of CCAAT/enhancer-binding protein α (C/EBP-α) gene induced apoptosis between hepatocytes and hepatic stellate cells (HSC) in mice with liver fibrosis.Methods Sixty BALB/c mice were evenly divided into normal group,model group,treatment group,blank control group and negative control group,12 mice in each group.Except the mice of normal control group,the mice of other groups were treated with intraperitoneal injection of CCl4 to establish liver fibrosis mice model.Mice of treatment group,blank control group and negative control group were administrated with C/EBP-α carried adenovirus (Ad-C/EBP-α),phosphate buffered solution and empty vector of adenovirus (Ad-EGFP) respectively through tail vein for the first week.The expression of C/EBP-α and α-smooth muscle actin (α-SMA) was detected by immunohistochemistry method.Sinusoidal endothelial structure of peri-portal regions and far from portal regions was observed by transmission electron microscope (TEM).Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was applied to detect apoptosis of cells in liver tissue.The degree of liver fibrosis in mice was determined with sirius red staining and hydroxyproline content measurement.Single factor variance analysis was performed for comparison among multiple groups,and t test was used for comparison between two groups.Results C/EBP-α was expressed in nucleus of hepatocyte in normal control group mice.The expression decreased in model group,blank control group and negative control group.However,the expression of C/EBP-α of treatment group increased,and mainly expressed in cells located in perisinusoidal and perivascular.Hepatic sinusoids was distorted,blood vessel wall thickened.Hepatocyte degeneration and lots of lipid droplets was found in model group,blank control group and negative control group.The thicken degree of endothelial layer of blood vessel of treatment group was lower than that of model group.The percentage of sirius red positive cells of normal group,model group,treatment group,blank control group and negative control group was (0.10±0.03)％,(5.81±0.32)％,(2.32±0.45)％,(6.34± 0.81)％ and (6.10± 0.92)％,respectively; content of hydroxyproline was (0.07±0.00) μg/mg,(0.69 ± 0.10) μg/mg,(0.19±0.06) μg/mg,(0.56±0.03) μg/mg and (0.64±0.08) μg/mg,respectively; the percentage of α-SMA positive cells was (0.50 ±0.03)％,(5.30 ± 0.52)％,(2.15 ± 0.29)％,(5.53 ± 0.43) ％ and (5.42 ± 0.25) ％,respectively; the number of TUNEL positive cells was (0.25 ± 0.08),(0.15±0.02),(7.10±1.53),(0.13±0.03) and (0.18±0.07),respectively.The differences between the groups were statistically significant (F=113.74,148.29,292.43 and 140.25,all P＜0.05).The difference between normal group and model group,between model group and treatment group,between treatment group and blank control group,between treatment group and negative control group were statistically significant (tarirus positive cell =-52.54,-16.20,-10.60 and-7.99,thydroxyproline content =-168.00,11.53,11.07 and 12.54,ta SMA pusitive cells-24.77,-13.82,15.94 and 18.37,tTUNEL positive cells =3.26,-11.91,-11.95 and-11.88,all P＜ 0.05),there was no statistically significant difference between model group and blank control group,between model group and negative control group (both P＞0.05).TUNEL positive cells mainly located in perisinusoidal and perivascular of liver in mice,which was consistent with the distribution of α-SMA-positive cells.Conclusion C/EBP-α could effectively relieve CCl4 induced liver fibrosis in mice mainly through inducing HSC apoptosis,however no apoptosis effect on hepatocytes. Key words: Liver cirrhosis; Hepatic stellate cells; CCAAT-enhancer-binding proteins

SY07-2 * ROLE OF OXIDATIVE STRESS IN METHAMPHETAMINE-INDUCED DOPAMINERGIC TOXICITY MEDIATED BY PROTEIN KINASE C

This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδexpression. Go6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδknockout (−/−) mice. MA-induced oxidative stress was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ(−/−) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter, and vesicular monoamine transporter 2 were not significantly observed in rottlerin-treated or PKCδ(−/−) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity.

Upregulation of myeloid cell leukemia-1 potentially modulates beclin-1-dependent autophagy in ischemic stroke in rats

BackgroundThe mechanisms that underlie autophagy in cerebral ischemia remain poorly defined. Myeloid cell leukemia-1 (Mcl1), an anti-apoptotic member of the Bcl-2 family of proteins, regulates the balance between autophagy and apoptosis. However, little is known regarding its expression profile and contribution to cell fate in the brain following ischemic stroke.ResultsIn this study, we investigated the expression profile and cellular distribution of Mcl1 in brains from transient middle cerebral artery occlusion (MCAO) model rats. Brain slices from sham-operated control rats showed minimal immunoreactivity for Mcl1. Mcl1 was mainly produced in neurons. Immunoreactivity for Mcl1 increased as early as 4 hours after MCAO, peaked at 24 hours, and then declined, but still remained high, at 72 hours. Mcl1 positive cells never colocalized with either cleaved caspase-3 or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. Both microtubule-associated protein 1 light chain 3 (LC3) and beclin-1 were evident in ischemic brain between 4 and 72 hours after MCAO. Most cells with strong LC3 staining were also labeled with beclin-1. Beclin-1 did colocalize with caspase-3 or Mcl1. Beclin-1/caspase-3 positive cells displayed the characteristic features of apoptosis including cell shrinkage and pyknotic nuclei, whereas beclin-1/Mcl1 positive cells had normal morphology. Pretreatment with 3-methyladenine attenuated autophagy without affecting the level of Mcl1 protein.ConclusionsThese findings demonstrate that the expression of Mcl1 is involved in the survival of neuronal cells. In addition, the coexpression of Mcl1 with beclin-1 may attenuate beclin-1-dependent autophagy during ischemic stroke in rats.