Transferase Deoxyuridine Triphosphate Nick End Research Articles

Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis.

Angiotensin II- (Ang II-) induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Choline exerts cardioprotective effects; however, its effects on Ang II-induced cardiomyocyte apoptosis are unclear. In this study, the role and underlying mechanism of choline in regulating Ang II-induced cardiomyocyte apoptosis were investigated using a model of cardiomyocyte apoptosis, which was induced by exposing neonatal rat cardiomyocytes to Ang II (10−6 M, 48 h). Choline promoted heat shock transcription factor 1 (HSF1) nuclear translocation and the intracellular domain of Notch1 (NICD) expression. Consequently, choline attenuated Ang II-induced increases in mitochondrial reactive oxygen species (mtROS) and promotion of proapoptotic protein release from mitochondria, including cytochrome c, Omi/high-temperature requirement protein A2, and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P. The reversion of these events attenuated Ang II-induced increases in cardiomyocyte size and numbers of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive cells, presumably via type 3 muscarinic acetylcholine receptor (M3AChR). Indeed, downregulation of M3AChR or Notch1 blocked choline-mediated upregulation of NICD and nuclear HSF1 expression, as well as inhibited mitochondrial apoptosis pathway and cardiomyocyte apoptosis, indicating that M3AChR and Notch1/HSF1 activation confer the protective effects of choline. In vivo studies were performed in parallel, in which rats were infused with Ang II for 4 weeks to induce cardiac apoptosis. The results showed that choline alleviated cardiac remodeling and apoptosis of Ang II-infused rats in a manner related to activation of the Notch1/HSF1 pathway, consistent with the in vitro findings. Taken together, our results reveal that choline impedes oxidative damage and cardiomyocyte apoptosis by activating M3AChR and Notch1/HSF1 antioxidant signaling, and suggest a novel role for the Notch1/HSF1 signaling pathway in the modulation of cardiomyocyte apoptosis.

Comparison of different energy response for lipolysis using a 1,060-nm laser: An animal study of three pigs.

Non-invasive body-sculpting procedures are becoming increasingly popular. The application of 1,060nm of laser energy transcutaneously to hyperthermically induce the disruption of fat cells in the abdomen is a type of non-invasive procedure. The purpose of this study was to compare the treatment results from two parameters of the same system, each with different energy output levels, in an in vivo porcine model to determine the most effective application. Female pigs (n=3) were used in this study. We examined the effects of the treatment using photography, ultrasonography, gross and microscopic pathology, and histological examination in order to determine the mechanism of action, efficacy, and safety of the procedure. Blood chemistry analysis was performed before each session to check lipid levels and to monitor for any adverse changes in markers that may indicate liver damage. Biopsies were taken and routinely processed with hematoxylin and eosin and Oil Red O stains to examine for tissue damage at baseline and after each treatment. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assays were performed to check for apoptotic-related DNA damage. Ultrasonic imaging of the same area before and after the application of 1,060nm of laser energy at outputs of 0.9 and 1.4W/cm2 showed that the density of the fat layer changed immediately after irradiation due to the transient heat transfer in the fat layer. Preclinical evaluation was performed to obtain comparison data on the safety and efficacy of subcutaneous fat reduction after applying the different energy outputs of 0.9 and 1.4W/cm2 . Based on our findings, we suggest that long-term histologic changes through the use of these devices suggest a comparative effectiveness of the treatment energy.

Ephedrine causes liver toxicity in SD rats via oxidative stress and inflammatory responses.

Ephedrine abuse has spread in many parts of the world and severely threatens human health. The mechanism of ephedrine-induced toxicity still remains unclear. This study was performed to investigate the effects of ephedrine treatment on the liver and explore the underlying mechanisms. Sprague Dawley rats were divided into saline and ephedrine groups. Rats were treated with ephedrine at 20 mg/kg or 40 mg/kg (n = 10) by oral gavage daily for 7 days. Pathological changes were examined by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Enzyme-linked immunosorbent assays were used to measure the liver functional markers, oxidative stress markers, and inflammatory cytokines. Real-time polymerase chain reaction and Western blot were used to measure gene and protein expression, respectively. Our data showed that ephedrine treatment increased hepatocellular cell apoptosis and impaired liver function. Moreover, ephedrine treatment increased oxidative stress and inflammatory responses, which may be due to the increase of transforming growth factor β (TGF-β)/Smad3 expression. Our study demonstrated that short-term treatment of ephedrine caused liver toxicity in rats through regulating TGF-β/Smad pathway.

Holliday junction‑recognition protein modulates apoptosis, cell cycle arrest and reactive oxygen species stress in human renal cell carcinoma.

Holliday junction recognition protein (HJURP) is involved in the regulation of mortality in various cell types, including renal cell carcinoma (RCC) cells. The specific mechanisms by which HJURP regulates RCC cell apoptosis and the cell cycle have not been previously investigated, to the best of our knowledge. In the present study, the expression of HJURP in RCC tissues and adjacent paracancerous renal tissue, as well as in RCC cell lines, was analyzed using reverse transcription‑quantitative PCR and western blot analysis. The A498 RCC cells were transfected with an HJURP overexpression vector, which resulted in reduced proliferation, as demonstrated using immunofluorescence staining, a Cell Counting Kit‑8 assay and a colony formation assay. Flow cytometry and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling assays were used to determine the effect of HJURP on the cell cycle and apoptosis of RCC cells. Proteins associated with the reactive oxygen species (ROS) status were analyzed using western blot analysis. The expression of HJURP was lower in RCC tissues and cells compared with that in the adjacent paracancerous renal tissues and control cells. Furthermore, overexpression of HJURP resulted in a decrease in cell viability and proliferation invitro. Overexpression of HJURP resulted in cell cycle arrest at the G0/G1 phase, cell apoptosis and an increase in ROS stress. In addition, the phosphorylated/total sirtuin 1 (SIRT1) protein ratio was decreased, whereas the expression of peroxisome proliferator‑activated receptor (PPAR)γ was increased in the HJURP‑overexpressing RCC cells. In clinical practice, decreased HJURP expression may be associated with poor prognosis in patients with RCC. These results suggest that HJURP may regulate cell apoptosis and proliferation in RCC cells and this may be mediated by PPARγ/SIRT1. Thus, HJURP may be used as a predictor of prognosis in patients with RCC.

3,4-Methylenedioxymethamphetamine causes retinal damage in C57BL/6J mice.

3,4-Methylenedioxymethamphetamine (MDMA) is a powerfully addictive psychostimulant with pronounced effects on the central nervous system, but the precise mechanism of MDMA-induced toxicity remains unclear, specifically on the retina. This study was performed to investigate the effects of MDMA treatment on the retina and explore the underlying mechanism. C57BL/6J mice were randomly divided into control and MDMA groups. Mice were treated with MDMA at progressively increasing doses (1-6 mg/kg) intraperitoneally 4 times per day. Electroretinography was used to test the retinal function. Pathological changes of the retina were examined by toluidine blue staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Enzyme-linked immunosorbent assays were used to measure the levels of cytokines in the retina. Real-time polymerase chain reaction and Western blot were used to measure gene and protein expression in the retina, respectively. Our study showed that MDMA treatment impaired retinal function and decreased retinal thickness. MDMA treatment also increased transforming growth factor β as well as inflammatory factors in the retina. Moreover, MDMA treatment increased protein expression of matrix metalloproteinases (MMPs) and decreased tight junction protein expression in the retina. Our study indicated that treatment of MDMA caused retinal damage in C57BL/6J mice, associated with an increase of MMPs and a decrease of tight junction proteins.

3:18 PM Abstract No. 229 Antitumor effect of transarterial chemoembolization using doxorubicin-albumin nanoparticle loaded lipid microbubbles combined with ultrasound-targeted activation on VX2 rabbit liver tumors

To explore the antitumor effect of transarterial chemoembolization using Doxorubicin-albumin nanoparticle loaded lipid microbubbles (mbTACE) combined with ultrasound-targeted microbubble activation (US) in rabbit VX2 liver tumor model. VX2 rabbits were assigned to 4 groups (n = 7/group): (i) saline (control); (ii) conventional TACE (cTACE); (iii) mbTACE without ultrasound activation (US-); (iv) mbTACE with ultrasound activation (US+). To assess hepatic damage, serum aspartate transaminase and alanine transaminase levels were measured on baseline and 1,3,and 7 days posttherapy. Changes of tumor volumes were segmented on MRI (baseline, 7 days posttherapy). To assess tumor necrosis, animals were euthanized on day 7, and explanted tumors were stained with hematoxylin and eosin and a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Percentage areas of viable tumors were calculated using digitalized histopathologic specimen images. estimated viable tumor volume was calculated using viable tumor fraction multiplying tumor volume on day 7 MRI. Treated groups show significant tumor suppression effect on MR compared to control group, without significant inter-treatment group difference. Tumor viable fraction and viable volume were 5.34% ± 6.54% and 89.9 mm3 ± 91.73 mm3, 17.28% ± 9.18% and 643.98 mm3 ± 758.15 mm3, 12.37% ± 8.11% and 187.01 mm3 ± 100.79 mm3, and 35.27% ± 6.46% and 1778.167 mm3± 1512.82 mm3in groups US+mbTACE, US-mbTACE, cTACE, and control (P < .001 and P < .001). Liver enzyme levels were elevated after drug delivery but recovered during follow-up. No significant differences were observed between treated groups on days 1, 3, and 7. Treatment with a combination of ultrasound activation and mbTACE showed therapeutic benefit in a liver tumor animal model.

Protective Effect of Stachydrine Against Cerebral Ischemia-Reperfusion Injury by Reducing Inflammation and Apoptosis Through P65 and JAK2/STAT3 Signaling Pathway.

Stachydrine, a constituent of Leonurus japonicus Houtt which also called Japanese motherwort has been shown to improve vascular microcirculation and ameliorate endothelial dysfunction. This study investigated the neuroprotective effect of stachydrine. Male Sprague-Dawley (SD) rats were randomly divided into sham, control, and stachydrine groups. The neurological deficit score was evaluated and the infarct size of the brain was measured using 2,3,5-triphenyltetra-zolium (TTC) chloride staining assay, and the pathological changes in the brain tissues were examined by HE staining. Nissl and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining were performed to assess the numbers of Nissl bodies and the levels of apoptosis in the neurons. The activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) were also measured. The release of inflammatory factors IL-1β and TNF-α were detected by Enzyme-linked immunosorbent assay (ELISA). Compared with the control group, the stachydrine group showed a significant prevention of neurological deficit, as indicated by the reduced infarct volume in the brain. Moreover, the stachydrine treatment reduced the activities of SOD, the levels of MDA and decreased the amount of IL-1β, and TNF-α, indicating that it could function to decrease the level of inflammation, thus reducing brain damage. The ischemic stroke model of PC12 cells was prepared via oxygen-glucose deprivation (OGD) protocol for 6 h. The expression of P65 and JAK2/STAT3 signaling pathway related proteins was measured by western blot. The treatment group was found to have the survival rate of PC12 cells improved and the release of inflammatory factors reduced when compared with the OGD group. This study demonstrated that stachydrine could improve nerve function by inhibiting the phosphorylation of P65/JAK2 and STAT3.

Acute changes in the retina and central retinal artery with methamphetamine

Methamphetamine (METH), an addictive stimulant of neurotransmitters, is associated with cardiovascular and neurological diseases. METH-induced ophthalmic complications are also present but have been insufficiently investigated. The purpose of this study is to investigate the retinal effects of METH. C57BL/6 mice were administrated progressively increasing doses of METH (0–6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Retinal degeneration was examined by morphological changes and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Norepinephrine levels were measured by ELISA, protein expression levels were determined by immunoblot and immunostaining, and gelatinase activity was examined by zymography. The thickness of the retina and the number of nuclei in the inner and outer nuclear layers were decreased by METH. Retinal cell death and astrocyte activation by METH treatment were confirmed by TUNEL assay and glial fibrillary acidic protein expression, respectively. Increased tumor necrosis factor-α protein in the retina and elevated norepinephrine levels in plasma were found in METH-treated mice. Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression level was decreased in the retina and central retinal artery (CRA) by METH treatment, along with the endothelial proteoglycans glypican-1 and syndecan-1. Moreover, a regulator of the extracellular matrix, matrix metalloproteinase-14 (MMP-14) in the retina, and MMP-2 and MMP-9 in plasma, were increased by METH treatment. In conclusion, METH administration is involved in retinal degeneration with a vascular loss of PECAM-1 and the glycocalyx in the CRA and retina, and an increase of MMPs.

Cytotoxic Activity of the Mesoionic Compound MIH 2.4Bl in Breast Cancer Cell Lines.

In this work, we report the synthesis of a new 1,3-thiazolium-5-thiolate derivative of a mesoionic compound (MIH 2.4Bl) and the characterization of its selective cytotoxicity on a panel of breast cancer cells lines. The cytotoxic effect of MIH 2.4Bl on breast cancer cell lines was determined by XTT and crystal violet assays, flow cytometry analysis, electron microscopy characterization, and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) apoptosis assays. As determined using XTT cell growth and survival assays, MIH 2.4Bl exhibited growth inhibition activity on most breast cancer cell lines tested, compared with normal human mammary epithelial cells. Three breast cancer cell lines (MCF-7, T-47D, and ZR-75-1) showed a more potent sensitivity index to growth inhibition by MIH 2.4Bl than the other breast cancer cell lines. Interestingly, these 3 cell lines were derived from tumors of Luminal A origin and have ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) positive expression. Additional analysis of cytotoxicity mediated by MIH 2.4Bl was performed using the MCF-7 cell line. MCF-7 cells displayed both time- and dose-dependent decreases in cell growth and survival, with a maximum cytotoxic effect observed at 72 and 96 hours. The MCF-7 cells were also characterized for cell cycle changes upon treatment with MIH 2.4Bl. Using flow cytometry analysis of cell cycle distribution, a treatment-dependent effect was observed; treatment of cells with MIH 2.4Bl increased the G2/M population to 34.2% compared with 0.1% in untreated (control) cells. Ultrastructural analysis of MFC-7 cells treated with MIH 2.4Bl at 2 different concentrations (37.5 and 75 μM) was performed by transmission electron microscopy. Cells treated with 37.5 μM MIH 2.4Bl showed morphologic changes beginning at 6 hours after treatment, while cells treated with 75 μM showed changes beginning at 3 hours after treatment. These changes were characterized by an alteration of nuclear morphology and mitochondrial degeneration consistent with apoptotic cell death. Results of a TUNEL assay performed on cells treated for 96 hours with MIH 2.4Bl supported the observation of apoptosis. Together, these results suggest that MIH 2.4Bl is a promising candidate for treating breast cancer and support further in vitro and in vivo investigation.

The structural atrophy of the aneurysm wall in secondary expanding aortic aneurysms with endoleak type II

BackgroundAbdominal aortic aneurysm (AAA) has an age-dependent prevalence of 2% to 11% and is a leading cause of death in men aged >65 years if not treated surgically. Today, endovascular aneurysm repair (EVAR) is performed in up to 80% of elective cases and 60% of ruptured cases. Although EVAR improves perioperative, early, and midterm outcomes, it is associated with specific complications, especially endoleaks (ELs). Type II EL occurs in up to 30% of procedures; however, aneurysm sac expansion and rupture are rare, and currently nothing is known about the morphologic changes in this condition. In this study, we investigate the aneurysm wall morphology in secondary expanding human AAA samples after EVAR with persistent type II EL in comparison to nonaneurysmatic control aortic and AAA samples. MethodsSamples were acquired from the aneurysm sac during retroperitoneal feeder vessel ligation in a cohort of 10 patients with secondary expansion after EVAR and type II EL diagnosed by computed tomography and contrast-enhanced ultrasound. Control tissues included 42 AAAs and 13 control aortae published previously. Hematoxylin and eosin staining and immunohistochemistry for CD3/4/31/68 and Ki67 were performed for morphologic analysis. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assays allowed quantification of apoptosis. Reverse transcription-polymerase chain reaction was used to quantify gene expression and Western blot to quantify collagen expression. ResultsSecondary expansion of 33.8% ± 30% during 5 years was seen after EVAR before reoperation. The aneurysm wall after expansion shows significant thinning of the intima-media layer accompanied by a scarcity of cells, with only a little chronic inflammation left compared with AAA samples. Macrophages are seen in abundance, and matrix metalloproteinase expression is significantly upregulated. Relevant apoptosis is not noticed. Fibrous tissue is reduced, and a collagen turnover to different subtypes is noted in comparison to nonaneurysmatic control aorta and AAA. In addition, the transcription factors vascular endothelial growth factor, Kruppel-like factor 4, and BCL2, elevated in AAA, are significantly reduced after secondary expansion. ConclusionsThe aneurysm sac morphology after EVAR with persistent type II EL is characterized by atrophy and proteolysis suggestive of structural weakening. These results should be considered for the follow-up schedule as well as for the potential treatment of this most frequent EVAR complication.

Chlorpromazine protects against acetaminophen-induced liver injury in mice by modulating autophagy and c-Jun N-terminal kinase activation

Background and aimOverdose of acetaminophen (APAP) leads to liver injury, which is one of the most common causes of liver failure in the United States. We previously demonstrated that pharmacological activation of autophagy protects against APAP-induced liver injury in mice via removal of damaged mitochondria and APAP-adducts (APAP-ADs). Using an image-based high-throughput screening for autophagy modulators, we recently identified that chlorpromazine (CPZ), a dopamine inhibitor used for anti-schizophrenia, is a potent autophagy inducer in vitro. Therefore, the aim of the present study is to determine whether CPZ may protect against APAP-induced liver injury via inducing autophagy. MethodsWild type C57BL/6J mice were injected with APAP to induce liver injury. CPZ was administrated either at the same time with APAP (co-treatment) or 2 h later after APAP administration (post-treatment). Hemotoxyline and eosin (H&E) staining of liver histology, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining of necrotic cell death as well as serum levels of alanine aminotransferase (ALT) were used to monitor liver injury. ResultsWe found that CPZ markedly protected against APAP-induced liver injury as demonstrated by decreased serum levels of ALT, liver necrotic areas as well as TUNEL-positive cells in mice that were either co-treated or post-treated with CPZ. Mechanistically, we observed that CPZ increased the number of autolysosomes and decreased APAP-induced c-Jun N-terminal kinase activation without affecting the metabolic activation of APAP. Pharmacological inhibition of autophagy by chloroquine partially weakened the protective effects of CPZ against APAP-induced liver injury. ConclusionsOur results indicate that CPZ ameliorates APAP-induced liver injury partially via activating hepatic autophagy and inhibiting JNK activation.

Ginsenoside Compound K Regulates Amyloid β via the Nrf2/Keap1 Signaling Pathway in Mice with Scopolamine Hydrobromide-Induced Memory Impairments.

The objective of this study was to investigate the neuroprotective and antioxidant effects of ginsenoside compound K (CK) in a model of scopolamine hydrobromide-induced, memory-impaired mice. The role of CK in the regulation of amyloid β (Aβ) and its capacity to activate the Nrf2/Keap1 signaling pathway were also studied due to their translational relevance to Alzheimer's disease. The Morris water maze was used to assess spatial memory functions. Levels of superoxide dismutase, glutathione peroxidase, and malondialdehyde in brain tissues were tested. Cell morphology was detected by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Immunohistochemistry and western blotting were used to determine expression levels of Nrf2/Keap1 signaling pathway-related factors and Aβ. Ginsenoside CK was found to enhance memory function, normalize neuronal morphology, decrease neuronal apoptosis, increase superoxide dismutase and glutathione peroxidase levels, reduce malondialdehyde levels, inhibit Aβ expression, and activate the Nrf2/Keap1 signaling pathway in scopolamine-exposed animals. Based on these results, we conclude that CK may improve memory function in scopolamine-injured mice by regulating Aβ aggregation and promoting the transduction of the Nrf2/Keap1 signaling pathway, thereby reducing oxidative damage to neurons and inhibiting neuronal apoptosis. This study suggests that CK may serve as a future preventative agent or treatment for Alzheimer's disease.

AT1Rknockdown confers cardioprotection against sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway in rats.

Myocardial dysfunction is an important manifestation of sepsis. In addition, inactivation of the mitogen-activated protein kinase (MAPK)signaling pathway has been reported to be beneficial in sepsis. The current study used gene expression profiling to demonstrate the overexpression of angiotensin II type 1 receptor (AT1R) and activation of the MAPK signaling pathway in sepsis. In this study, we used a rat model of sepsis established by cecal ligation and puncture to explore the mechanism of AT1R silencing in relation to the MAPK signaling pathway on myocardial injury. Various parameters including blood pressure, heart rate, and cardiac function changes were observed. Enzyme-linked immunosorbent assay was used to measure the concentration of cardiac troponin T (TnT), cardiac troponin I (cTnI), and creatine kinase isoenzyme muscle/brain (CK-MB). Myocardial enzyme, tissue antioxidant capacity, mitochondria swelling, and membrane potential were also detected. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelingstaining was applied to measure cell apoptosis, andmessenger RNAand protein levels of apoptosis-related proteins (Fas ligand [Fasl], B-cell CLL/lymphoma [Bcl-2], p53) were also detected. Initially, sepsis rats exhibited decreased survival rate, but increased ejection fraction (EF), heart rate, and concentrations of TnT, cTnI, and CK-MB. Furthermore, decreased AT1R expression inactivated the MAPK signaling pathway (shown as decreased extracellular signal-regulated kinase and cyclic adenosine 3',5'-monophosphate response element binding protein expression), decreased EF, heart rate, and concentrations of TnT, cTnI, and CK-MB, but increased sepsis rat survival rate. Eventually, decreased AT1R expression inhibited myocardial cell apoptosis (shown as decreased apoptosis rate and p53 and Fasl expression as well as increased Bcl-2 expression). These findings indicated that AT1R silencing plays an inhibitory role in sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway.

Chemoembolization with Vascular Disrupting Agent CKD-516 Dissolved in Ethiodized Oil in Combination with Doxorubicin: A VX2 Tumor Model Study

PurposeTo assess feasibility and efficacy of CKD-516, a vascular disrupting agent, in transarterial chemoembolization in a liver tumor model. Materials and MethodsA VX2 carcinoma strain was implanted in rabbit liver (n = 40) and incubated for 2 weeks. After confirmation of tumor growth using computed tomography, transarterial chemoembolization was performed. CKD-516 was dissolved in ethiodized oil, and animals were allocated to 4 treatment groups (n = 10 in each): group A, ethiodized oil; group B, ethiodized oil/CKD-516; group C, ethiodized oil + doxorubicin; group D, ethiodized oil/CKD-516 + doxorubicin. To assess hepatic damage, serum aspartate transaminase and alanine transaminase levels were measured on day 1, 3, and 7 after delivery. To assess tumor necrosis, animals were euthanized on day 7, and explanted tumors were stained with hematoxylin and eosin and a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Percentage areas of viable tumors were calculated using digitalized histopathologic specimen images. ResultsTumor viability rates were 47.1% ± 11.4%, 27.5% ± 13.6%, 14.4% ± 12.5%, and 0.7% ± 1.0% in groups A, B, C, and D (P < .001). Liver enzyme levels were elevated after drug delivery but recovered during follow-up. Significant between-group differences were observed on days 1, 3, and 7 (aspartate transaminase and alanine transaminase: P = .0135 and P = .0134, P = .0390 and P = .0084, and P = .8260 and P = .0440). ConclusionsTreatment with a combination of CKD-516 and conventional transarterial chemoembolization showed therapeutic benefit in a liver tumor model.

IP051. Atrophy of the Aneurysm Wall: Observations From Secondary Expansion After Endovascular Aneurysm Repair With Type II Endoleak∗

Abdominal aortic aneurysm (AAA) has an age-dependent prevalence of 2% to 11% and is a leading cause of death in men aged >65 years if it is not treated surgically. Endovascular aneurysm repair (EVAR) is performed in up to 80% of elective and 90% of ruptured cases. Although reducing perioperative, early, and midterm outcomes and complications rates, the procedure is associated with specific complications requiring close follow-up, especially endoleaks. Type II endoleaks occur in up to 30% after EVAR; however, aneurysm sac expansion is rare. In this study, we investigate the aneurysm wall morphology in secondary expanding human AAA samples after EVAR due to persistent endoleak type II in comparison to nonaneurysmatic control aortas and AAA samples. Samples were acquired from the aneurysm sac during retroperitoneal ligation of the feeder vessel in a cohort of nine patients. Control tissues included 42 AAAs and 11 control aortas. Hematoxylin and eosin staining and immunohistochemistry for CD3/4/31/68 and Ki67 were performed for morphologic analysis and characterization. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assays allowed quantification of apoptotic cells. Reverse transcription-polymerase chain reaction was used to quantify gene expression and Western blot to quantify proteins. Secondary expansion after EVAR was approximately 17% during 21 ± 12 months before operation. The sac wall after expansion shows significant thinning of the intima-media layer. This is accompanied by a scarcity of cells, with only little chronic inflammation left compared with AAA samples. Macrophages are seen in abundance, and matrix metalloproteinase expression is significantly upregulated. Relevant apoptosis is not noticed. Fibrous tissue is reduced, and a collagen turnover to different subtypes is noted in comparison to nonaneurysmatic control aorta and AAA. This is the first study examining aneurysm sac morphology after EVAR with persistent type II endoleak. Atrophy and proteolysis suggestive of structural weakening are observed and have to be considered indications for follow-up and treatment of this frequent EVAR complication.

Effects of oral cimetidine on the reproductive system of male rats.

Cimetidine is widely used for the treatment of digestive tract ulcers, but it induces testis injury. To explore the mechanisms underlying cimetidine-induced toxicity towards the testis, the effects of oral cimetidine on the reproductive system of male rats were assessed. Cimetidine was orally administered to male rats at 20, 40 or 120 mg/kg/day for 9 weeks. The rats were then euthanized, and serum, testis, epididymis, prostate gland, seminal vesicle, preputial gland, levator ani muscle and sphincter ani samples were collected. Sperm parameters were obtained by computer-assisted sperm analysis. Serum hormone levels were measured by ELISA. Protein expression levels were detected by immunohistochemistry. Apoptosis was assessed with the DeadEnd™ Colorimetric Apoptosis Detection System. The results indicated that the sperm average path velocity, straight line velocity and curvilinear velocity were significantly decreased in the 120 mg/kg cimetidine group compared with the control group, while luteinizing hormone and testosterone levels were significantly higher compared with the control group. Testicular lesions were observed by histopathology in the 120 mg/kg cimetidine group. The amounts of cells positive for cyclooxygenase-2 (COX-2) and nuclear factor κB (NF-κB) were increased in the 120 mg/kg cimetidine group compared with the control group. The amounts of cells positive for iNOS were increased in all cimetidine treatment groups. In addition, apoptotic cells were significantly more abundant in the 120 mg/kg cimetidine group compared with the control group, as indicated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Overall, 9 weeks of oral cimetidine induced pathological changes in the testicles and hormone secretion disorder in rats. COX-2, iNOS and NF-κB upregulation and induction of apoptosis may be associated with the reproductive toxicity caused by cimetidine.

Fluoride-associated ultrastructural changes and apoptosis in human renal tubule: a pilot study.

The susceptibility of the kidneys to fluoride toxicity can largely be attributed to its anatomy and function. As the filtrate moves along the complex tubular structure of each nephron, it is concentrated in the proximal and distal tubules and collecting duct. It has been frequently observed that the children suffering from renal impairments also have some symptoms of dental and skeletal fluorosis. The findings suggest that fluoride somehow interferes with renal anatomy and physiology, which may lead to renal pathogenesis. The aim of this study was to evaluate the fluoride-associated nephrotoxicity. A total of 156 patients with childhood nephrotic syndrome were screened and it was observed that 32 of them had significantly high levels ( p ≤ 0.05) of fluoride in urine (4.01 ± 1.83 ppm) and serum (0.1 ± 0.013 ppm). On the basis of urinary fluoride concentration, patients were divided into two groups, namely group 1 (G-1) ( n = 32) containing normal urine fluoride (0.61 ± 0.17 ppm) and group 2 (G-2) ( n = 32) having high urine fluoride concentration (4.01 ± 1.83 ppm). Age-matched healthy subjects ( n = 33) having normal levels of urinary fluoride (0.56 ± 0.15 ppm) were included in the study as control (group 0 (G-0)). Kidney biopsies were taken from G-1 and G-2 only, who were subjected to ultrastructural (transmission electron microscopy) and apoptotic (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) analysis. Various subcellular ultrastructural changes including nuclear disintegration, chromosome condensation, cytoplasmic ground substance lysis, and endoplasmic reticulum blebbing were observed. Increased levels of apoptosis were observed in high fluoride group (G-2) compared to normal fluoride group (G-1). Various degrees of fluoride-associated damages to the architecture of tubular epithelia, such as cell swelling and lysis, cytoplasmic vacuolation, nuclear condensation, apoptosis, and necrosis, were observed.

Bioluminescence Tomography Guided Small-Animal Radiation Therapy and Tumor Response Assessment

The image guided small animal arc radiation treatment platform has adopted onboard cone beam computed tomography and bioluminescence tomography (BLT). We used BLT to guide irradiation delivery and quantitatively assess irradiation-induced tumor response. BLT was first validated on a tissue-simulating phantom, where the internal chemiluminescent liquid had a constant volume while its luminescence intensity gradually decayed. Then, invivo experiments were performed on BALB/c mice orthotopically inoculated with 4T1 breast carcinoma cells expressing luciferase. Animals either received radiation treatment (radiation therapy [RT] group, n=9) or did not (control group, n=9). BLT was used to guide delivery of a single-fraction 5-Gy radiation dose to the tumor and to evaluate the treatment response. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was used to evaluate irradiation-induced DNA damage and cell apoptosis. Phantom results showed that BLT not only recovered the constant target volume with <2% deviation but also accurately monitored the decay of the chemiluminescent molecules. For the RT group of animals, there was significant reduction in both the BLT-based tumor volume (21%±10%, P=.001) and bioluminescence intensity (48%±17%, P=.0008). For the control group, a significant increase was detected in the BLT tumor volume (35%±12%, P<.0001) but not the BLT bioluminescence intensity (P=.4). There was a significant difference in the BLT tumor volume between the RT and control groups 7days after irradiation (P=.03). Regression analysis suggests a strong correlation between the BLT and cone beam computed tomography tumor volume (R2=0.93). Analysis using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining showed a significant difference in tumor cell apoptosis between the RT and control groups (20.6%±2.9% and 3.2%±1.7%, respectively; P<.05). BLT onboard the image guided small animal arc radiation treatment platformcan be used to accurately guide irradiation delivery and to quantitatively assess treatment response by simultaneously monitoring tumor volume and cancer cell population.

Expression of diaphragmatic myostatin and correlation with apoptosis in rats with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and loss of lung function. The present study aimed to investigate the diaphragmatic protein expression of myostatin and its correlation with apoptosis in a rat model of CPOD. Sprague Dawley rats were randomly divided into a control group and a COPD group, the latter of which were exposed to cigarette smoke to build a rat model of COPD. The validity of the COPD model was evaluated by assessment of lung function and histopathological analysis. Diaphragmatic myostatin expression and apoptosis were measured by western blot and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, respectively. The rat model of COPD was efficiently established by cigarette smoke exposure. Diaphragmatic myostatin expression and apoptotic index in COPD rats were obviously increased as compared with that in the control animals. A positive correlation between diaphragmatic myostatin expression and apoptotic index was identified (r=0.857). Diaphragmatic myostatin overexpression in rats with COPD may promote diaphragmatic apoptosis and atrophy, leading to diaphragm weakness and respiratory muscle dysfunction, which is involved in the pathology of COPD.

Apoptosis in the cochlear nucleus and inferior colliculus upon repeated noise exposure.

The time course of apoptosis and the corresponding neuronal loss was previously shown in central auditory pathway of mice after a single noise exposure. However, repeated acoustic exposure is a major risk factor for noise-induced hearing loss. The present study investigated apoptosis by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay after a second noise trauma in the ventral and dorsal cochlear nucleus and central nucleus of the inferior colliculus. Mice [Naval Medical Research Institute (NMRI) strain] were noise exposed [115 dB sound pressure level, 5–20 kHz, 3 h) at day 0. A double group received the identical noise exposure a second time at day 7 post-exposure and apoptosis was either analyzed immediately (7-day group-double) or 1 week later (14-day group-double). Corresponding single exposure groups were chosen as controls.No differences in TUNEL were seen between 7-day or 14-day single and double-trauma groups. Interestingly, independent of the second noise exposure, apoptosis increased significantly in the 14-day groups compared to the 7-day groups in all investigated areas.It seems that the first noise trauma has a long-lasting effect on apoptotic mechanisms in the central auditory pathway that were not largely influenced by a second trauma. Homeostatic mechanisms induced by the first trauma might protect the central auditory pathway from further damage during a specific time slot. These results might help to understand the underlying mechanisms of different psychoacoustic phenomena in noise-induced hearing loss.