Number Of TUNEL-positive Cells Research Articles

Shear stress inhibits cardiac microvascular endothelial cells apoptosis to protect against myocardial ischemia reperfusion injury via YAP/miR-206/PDCD4 signaling pathway

Cardiac microvascular endothelial cells (CMECs), derived from coronary circulation microvessel, are the main barrier for the exchange of energy and nutrients between myocardium and blood. However, microvascular I/R injury is a severely neglected topic, and few strategies can reverse this pathology. In this study, we investigated the mechanism of shear stress in microvascular I/R injury, and try to elucidate the downstream signaling pathways that inhibit CMECs apoptosis to reduce I/R injury. Our results demonstrated that shear stress inhibited the apoptosis protein, increased PECAM-1 expression and eNOS phosphorylation in hypoxia reoxygenated (H/R) CMECs. The mechanism of shear stress was related to up-regulated expression of YAP, the increased number of YAP entering the nucleus by dephosphorylation, the reduced number of TUNEL positive cells, increased miR-206 and inhibited protein level of PDCD4 in CMECs. However, siRNA-mediated knockdown of YAP abolished the protective effects of shear stress on CMECs apoptosis, similar results obtained from administration with AMO-miR-206, and also prevented PDCD4 (target gene of miR-206) increasing when treatment with both AMO-miR-206 and mimics-miR-206. In vivo, restoring the blood fluid with nitroglycerin (NTG) to mimic in vitro shear stress levels, which subsequently improved cardiac function, reduced infarcted area, lowered microvascular perfusion defects. Functional investigations clearly illustrated that increased the protein expression of PECAM-1 and eNOS phosphorylation, activated YAP, strengthened miR-206 expression, and suppressed PDCD4 expression. In summary, this study confirmed that shear stress reversed CMECs apoptosis, relieved microvascular I/R injury, the mechanism of which involving through YAP/miR-206/PDCD4 signaling pathway to finally suppress myocardial I/R injury.

Histopathological study on neuroapoptotic alterations induced by etomidate in rat hippocampus

In human, there is substantial neurogenesis in the hippocampus that is implicated in memory formation and learning. These new-born neurons can be affected by neuropathological conditions. Anesthesia and surgical procedures are associated with postoperative cognitive changes particularly, impaired memory and learning. Therefore, the aim of this study was to evaluate the possible neurodegenerative effects of etomidate in rat hippocampus. Thirty male Wistar rats weighing 250 ± 30 g were randomly divided into 3 groups: 1) Etomidate group; four times 20 mg intraperitoneal injection with 1-h intervals, 2) Control group; the equal volume of normal saline, and 3) Normal group; without any intervention. 6 h after the last injection, the brains were removed and processed according to routine histological methods. TUNEL assay and toluidine blue staining were performed to evaluate neuro-histopathological changes in different regions of hippocampus. Our results showed that the number of TUNEL positive cells and dark neurons (DNs) in etomidate group were significantly higher in the CA1, CA2, CA3, and dentate gyrus (DG) of hippocampus compared with the control and normal groups (p < 0.05). While, there was no significant difference between the various regions of hippocampus in control and normal groups. Our findings showed that etomidate can increase apoptotic cells and dark neurons induction in different regions of hippocampus mainly in DG.

AM1241 alleviates myocardial ischemia-reperfusion injury in rats by enhancing Pink1/Parkin-mediated autophagy

AimsThe purpose of this study was to reveal the therapeutic efficacy and underlying mechanism of cannabinoid type 2 receptor agonist (AM1241) on myocardial ischemia-reperfusion injury (MIRI) in rats. Main methodsWe established a rat myocardial ischemia/reperfusion (I/R) model and H9c2 hypoxia/reoxygenation (H/R) model. ELISA was used to determine the concentrations of cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in plasma. EB/TTC staining was performed to observe the myocardial infarct size. Besides, the pathological changes of myocardial tissue were identified via H&E staining and Masson's trichrome staining. TUNEL assay was performed to examine myocardial apoptosis. Then, the protein expression of Pink1, Parkin and autophagy-related markers (Beclin-1, P62 and LC3) were detected by Western blot, and autophagy was evaluated by Mitotracker staining. Key findingsThe results of EB/TTC staining, H&E staining, Masson's trichrome staining and cardiac enzymes measuring showed that AM1241 treatment significantly diminished infarct size, the structural abnormalities and the activities of cardiac enzymes (cTnI, CK-MB, AST and LDH). AM1241 also significantly reduced the number of TUNEL-positive cells induced by I/R in a dose-dependent manner. Furthermore, AM1241 activated Pink1/Parkin signaling pathway and upregulated autophagy level. SignificanceAM1241 exerts a protective effect against MIRI in rats by inducing autophagy through the activation of Pink1/Parkin pathway.

The immunoregulatory role of p21 in the development of the temporomandibular joint-osteoarthritis.

ObjectiveWe aimed to identify the immunoregulatory role of the cyclin‐dependent kinase inhibitor p21 in the homeostasis of mandibular condylar cartilage affected by mechanical stress.Materials and methodsTen C57BL/6 wild‐type (WT) and ten p21−/− mice aged 8 weeks were divided into the untreated and treated groups. In the treated groups, mechanical stress was applied to the temporomandibular joint (TMJ) through forced mouth opening for 3 hr/day for 7 days. The dissected TMJs were assessed using micro‐CT, histology, and immunohistochemistry.ResultsTreated p21−/− condyles with mechanical stress revealed more severe subchondral bone destruction, with thinner cartilage layers and smaller proteoglycan area relative to treated WT condyles; untreated WT and p21−/− condyles had smoother surfaces. Immunohistochemistry revealed significant increases in the numbers of caspase‐3, interleukin‐1β, matrix metalloprotease (MMP)‐9, and MMP‐13 positive cells, and few aggrecan positive cells, in treated p21−/− than in treated WT samples. Moreover, the number of TUNEL positive cells markedly increased in p21−/− condyles affected by mechanical stress.ConclusionsOur findings indicate that p21 in chondrocytes contributes to regulate matrix synthesis via the control ofm aggrecan and MMP‐13 expression under mechanical stress. Thus, p21 might regulate the pathogenesis of osteoarthritis in the TMJ.

Protective Effect of Vitamin E on Cadmium-Induced Renal Oxidative Damage and Apoptosis in Rats.

Cadmium (Cd), a widely distributed heavy metal, is extremely toxic to the kidney. Vitamin E (VE) is an important antioxidant in the body. It is known that VE exerts a protective effect on renal oxidative damage caused by Cd, but the effect and mechanism of VE on apoptosis are not fully understood. Thus, we conducted this study to explore the protective effect of VE on Cd-induced renal apoptosis and to elucidate its potential mechanism. Thirty-two 9-week-old male Sprague-Dawley rats were randomly divided into four groups, namely control, VE (100mg/kg VE), Cd (5mg/kg CdCl2), and VE + Cd (100mg/kg VE + 5mg/kg CdCl2), and received intragastric administration of Cd and/or VE for 4 weeks. The results showed that Cd exposure significantly reduced the weight of the body and kidney, elevated the accumulation of Cd in the kidney as well as the levels of BUN and Scr in serum, caused renal histological alterations, decreased the GSH and T-AOC contents and antioxidant enzyme (SOD, CAT, GSH-PX) activities, and increased renal MDA content. And the increased number of TUNEL-positive cells by Cd was accompanied by upregulated mRNA and protein expressions of apoptotic regulatory molecules (Bax, Caspase-3, GRP94, GRP78, Caspase-8) and downregulated Bcl-2 expressions. However, the combined treatment of Cd and VE could restore the above parameters to be close to those in the control rats. In conclusion, VE supplement could alleviate Cd-induced rat renal damage and oxidative stress through enhancing the antioxidant defense system and inhibiting apoptosis of renal cells.

Salvianolate lyophilized injection regulates the autophagy-lysosomal pathway in cerebral ischaemia/reperfusion rats

Ethnopharmacological relevanceActivation of autophagy has been implicated in cerebral ischiemia/reperfusion (I/R) injury. Salvianolate lyophilized injection (SLI) has been widely used in the clinical treatment of cerebrovascular disease in China. Whether SLI has any influence on the activation of autophagy in cerebral I/R injury remains elusive. Aim of the studyThe aim of this study were to assess whether SLI attenuates I/R-induced brain injury and evaluate its associated mechanisms. Materials and methodsFocal cerebral ischaemia was induced by middle cerebral artery occlusion (MCAO). SLI (21 mg/kg) was injected intravenously at the beginning of the reperfusion period and 24 and 48 h after ischaemia. The effects of SLI on brain injury were detected according to infarct volume, neurological score, brain oedema, and HE and TUNEL staining at 72 h post-MCAO. Western blotting was used to detect alterations in the autophagy-relevant proteins LC3, Beclin-1, mTOR, p62, Lamp-1, and CTSD in the ipsilateral cortex at 24 or 72 h post-MCAO. ResultsWe first demonstrated that SLI significantly alleviated the infarct volume, neurological deficits, and brain oedema, and reduced the number of TUNEL-positive cells in rats with cerebral I/R injury. Next, we found that SLI has a bidirectional regulatory effect on autophagy: early-stage (24 h) cerebral ischaemia promotes the activation of autophagy and developmental-stage (72 h) cerebral ischaemia has an inhibitory effect. SLI enhanced I/R-induced autophagy as evidenced by the increased expression level of the autophagy marker protein LC3Ⅱ, as well as the decreased expression of mTOR and the autophagy substrate protein p62, but there was no change in lysosomal activity at 24 h after I/R-induced injury. Moreover, SLI also inhibited excessive activation of autophagy at 72 h after I/R-induced injury, which manifested as downregulating LC3Ⅱ expression, upregulating mTOR and p62 expression, and inhibiting lysosomal activity. ConclusionSLI has a protective effect on cerebral ischaemia/reperfusion injury, which may be mediated by the autophagy-lysosome pathway.

TGF-β1-Mediated Activation of SERPINE1 is Involved in Hemin-Induced Apoptotic and Inflammatory Injury in HT22 Cells.

BackgroundIntracerebral hemorrhage (ICH) is a severe subtype of stroke with high mortality and morbidity. Serpin Family E Member 1 (SERPINE1) has been documented to be upregulated following ICH, however, the participation of SERPINE1 in the development of ICH has never been studied.MethodsHemin was utilized to develop an in vitro model of ICH. Gene levels were evaluated by the use of quantitative reverse transcription polymerase chain reaction, Western blot, as well as enzyme-linked immunoassay assay. The activity of caspase-3 was determined using a commercial kit. Cell viability and apoptosis were assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Terminal deoxynucleotidyl transferase (TdT) d UTP Nick-End Labeling assay.ResultsSERPINE1 was upregulated in hemin-treated HT22 cells. Silencing of SERPINE1 attenuated hemin-induced inhibition of cell viability. Moreover, knockdown of SERPINE1 repressed hemin-induced apoptosis in HT22 cells, as evidenced by the decrease in the number of TUNEL positive cells, caspase-3 activity, and Bax expression, and the increase in Bcl-2 expression. Meanwhile, knockdown of SERPINE1 repressed hemin-induced inflammation in HT22 cells, as indicated by reduced levels of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β, and inducible nitric oxide synthase. We also found that transforming growth factor-beta 1 (TGF-β1) induced SERPINE1 expression in a dose-dependent manner. Besides, SERPINE1 knockdown attenuated the effects of TGF-β1 on hemin-induced neuronal damage.ConclusionTGF-β1-induced SERPINE1 activation exacerbated hemin-induced apoptosis and inflammation in HT22 cells, manifesting a novel mechanism for ICH progression.

NLRP3 Inflammasome: A New Pharmacological Target for Reducing Testicular Damage Associated with Varicocele.

Many bioactive natural compounds are being increasingly used for therapeutics and nutraceutical applications to counteract male infertility, particularly varicocele. The roles of selenium and Polydeoxyribonucleotide (PDRN) were investigated in an experimental model of varicocele, with particular regard to the role of NLRP3 inflammasome. Male rats underwent sham operation and were daily administered with vehicle, seleno-L-methionine (Se), PDRN, and with the association Se-PDRN. Another group of rats were operated for varicocele. After twenty-eight days, sham and varicocele rats were sacrificed and both testes were weighted and analyzed. All the other rats were challenged for one month with the same compounds. In varicocele animals, lower testosterone levels, testes weight, NLRP3 inflammasome, IL-1β and caspase-1 increased gene expression were demonstrated. TUNEL assay showed an increased number of apoptotic cells. Structural and ultrastructural damage to testes was also shown. PDRN alone significantly improved all considered parameters more than Se. The Se-PDRN association significantly improved all morphological parameters, significantly increased testosterone levels, and reduced NLRP3 inflammasome, caspase-1 and IL-1β expression and TUNEL-positive cell numbers. Our results suggest that NLRP3 inflammasome can be considered an interesting target in varicocele and that Se-PDRN may be a new medical approach in support to surgery.

Gemigliptin suppresses salivary dysfunction in streptozotocin-induced diabetic rats

Patients with diabetes commonly experience hyposalivation, which induces discomfort in eating, swallowing, dryness, smell, and speaking, as well as increases the incidence of periodontal disease. Dipeptidyl peptidase-4 (DPP4) inhibitors are frequently used as antidiabetic drugs that lower glucose levels by utilizing similar mechanisms; however, additional protective functions of each gliptin have been discovered. In this study, the protective roles of gemigliptin, a DPP4 inhibitor, against salivary dysfunction under diabetic conditions were investigated. Streptozotocin-induced diabetic rats received gemigliptin 10 mg/kg or 100 mg/kg via oral gavage for 3 weeks. The weights of salivary gland tissues, saliva secretion, and antioxidant capacity in salivary glands were reduced after diabetes induction, but were significantly preserved following gemigliptin treatment. In salivary gland analysis, expression of apoptotic proteins, as well as amylase and aquaporin-5 (AQP5) protein expression, were increased following gemigliptin treatment. Furthermore, the number of TUNEL-positive cells decreased after gemigliptin treatment. Therefore, gemigliptin has protective roles against salivary dysfunction observed in diabetes, mediated via antioxidant, anti-apoptotic, and salivary secretion mechanisms. These results may help in selecting a suitable drug for patients with diabetes experiencing salivary dysfunction.

Cardioprotective Effect of Echinatin Against Ischemia/Reperfusion Injury: Involvement of Hippo/Yes-Associated Protein Signaling.

Background: Echinatin (Ech) has been reported to exert antioxidant and anti-inflammatory activities. In this study, we aimed to characterize the functional role of Ech in myocardial ischemic/reperfusion (MI/R) injury and elucidate its underlying mechanism of action. Method: We established in vivo and in vitro models of MI/R injury to determine the effect of Ech on MI/R injury. Gene expression was examined using quantitative real-time polymerase chain reaction and western blotting. Myocardial infarction was assessed using tetrazolium chloride staining and the degree of myocardial injury was evaluated by measuring lactate dehydrogenase (LDH) and creatine kinase-myocardial band (CK-MB) levels. Cell apoptosis was detected using the terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling (TUNEL) assay. The viability of H9c2 cells was determined using Cell Counting Kit-8 assay. Results: MI/R induced myocardial infarction, which was mitigated by Ech treatment. Moreover, Ech treatment resulted in a marked decline of LDH and CK-MB levels in the serum and myocardium of MI/R rats. Ech treatment also restrained cardiomyocyte apoptosis in vivo and in vitro, as evidenced by reduction in LDH release, the number of TUNEL-positive cells, and caspase-3 activity. Furthermore, Ech administration inhibited MI/R-induced activation of Hippo/Yes-associated protein signaling in vivo and in vitro, as indicated by inhibition of mammalian sterile 20-like protein kinase 1, large tumor suppressor one, and YAP phosphorylation and promotion of YAP nuclear translocation. However, silencing of YAP counteracted the protective effect of Ech on hypoxia/reoxygenation-induced myocardial injury in vitro. Conclusion: Ech exerted its protective effect against MI/R injury at least partially by suppressing the Hippo/YAP signaling pathway, providing novel insights into the remission of MI/R injury.

Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy.

Cyclophosphamide (CP) could cause severe gonadotoxicity via imbalanced activation of primordial follicles through PI3K/AKT/mTOR activation. Whether metformin, a widely prescribed anti-diabetes agent with mTOR inhibitory effect, could preserve ovarian function against CP toxicity is unknown. Female C57BL/6 mice were randomized into seven groups (n = 11), including control, CP-alone, CP + metformin, CP + sirolimus or everolimus, metformin-alone and sirolimus-alone groups. The duration of pharmaceutical treatment was 4 weeks. CP treatment significantly impaired ovarian function and fertility in mice. CP + metformin treatment significantly attenuated the gonadotoxicity comparing to CP-alone treatment (primordial follicle count: 17.6 ± 4.2 versus 10.3 ± 2.7 follicles/high-power field; P = 0.027). CP + metformin treatment also tended to increase antral follicular count (5.4 ± 1.1 versus 2.5 ± 1.6 follicles/section), serum AMH levels (4.6 ± 1.2 versus 2.0 ± 0.8 ng/ml) and the litter size (4.2 ± 1.3 versus 1.5 ± 1.0 mice per pregnancy), compared with CP-alone group. Expression of phospho-mTOR and the number of TUNEL-positive granulosa cells increased after CP treatment and decreased in the CP + metformin groups, suggesting the mTOR inhibitory and anti-apoptotic effects of metformin. In in-vitro granulosa cell experiments, the anti-apoptotic effect of metformin was blocked after inhibiting p53 or p21 function, and the expression of p53 mRNA was blocked with AMPK inhibitor, suggesting that the anti-apoptotic effect was AMPK/p53/p21-mediated. In conclusion, concurrent metformin treatment during CP therapy could significantly preserve ovarian function and fertility and could be a promising novel fertility preserving agent during chemotherapy. The relatively acceptable cost and well-established long-term safety profiles of this old drug might prompt its further clinical application at a faster pace.

23-Hydroxytormentic acid reduces cerebral ischemia/reperfusion damage in rats through anti-apoptotic, antioxidant, and anti-inflammatory mechanisms.

23-Hydroxytormentic acid (23-HTA) is an important herbal medicine purified from immature fruits of African Rubus aceae (Rosaceae). This study was carried out to examine the protection properties and potential mechanisms of 23-HTA against cerebral ischemia/reperfusion (I/R) damage. Rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R) 2/24h. All animals were euthanized 24h after reperfusion. Rats were injected with various concentrations of 23-HTA intraperitoneally. Evaluations of infarct volumes, neurological deficit, and brain water contents were carried out to assess the outcome of 23-HTA treatment. The results showed that 23-HTA reduced infarct volumes, brain water content, and neurological deficit in a dosage-dependent manner. 23-HTA can also significantly reduce the numbers of TUNEL-positive cells, the expression levels of Bax, caspase-3, lipid peroxidation, Sod 1, Sod 2, catalase, and pro-inflammatory cytokines TNF and IL-1β and increase the expression levels of Bcl-2 and p-Akt. 23-HTA has a neuroprotective effect due to its anti-apoptotic, antioxidant, and anti-inflammatory effects.

The Extracts of Angelica sinensis and Cinnamomum cassia from Oriental Medicinal Foods Regulate Inflammatory and Autophagic Pathways against Neural Injury after Ischemic Stroke.

The study indicates inflammation and autophagy are closely related to neural apoptosis in the pathology of ischemic stroke. In the study, we investigate the effects and mechanisms of the extracts of Angelica sinensis and Cinnamomum cassia (AC) from oriental medicinal foods on inflammatory and autophagic pathways in rat permanent middle cerebral artery occlusion model. Three doses of AC extract were, respectively, administered for 7 days. It suggests that AC extract treatment ameliorated scores of motor and sensory functions and ratio of glucose utilization in thalamic lesions in a dose-dependent manner. Expression of Iba1 was decreased and CD206 was increased by immunofluorescence staining, western blotting results showed expressions of TLR4, phosphorylated-IKKβ and IκBα, nuclear P65, NLRP3, ASC, and Caspase-1 were downregulated, and Beclin 1 and LC3 II were upregulated. Low concentrations of TNF-α, IL-1β, and IL-6 were presented by ELISA assay. Additionally, caspase 8 and cleaved caspase-3 expressions and the number of TUNEL positive cells in ipsilateral hemisphere were decreased, while the ratio of Bcl-2/Bax was increased. Simultaneously, in LPS-induced BV2 cells, it showed nuclear P65 translocation and secretion of proinflammatory cytokines were suppressed by AC extract-contained cerebrospinal fluid, and its intervened effects were similar to TLR4 siRNA treatment. Our study demonstrates that AC extract treatment attenuates inflammatory response and elevates autophagy against neural apoptosis, which contributes to the improvement of neurological function poststroke. Therefore, AC extract may be a novel neuroprotective agent by regulation of inflammatory and autophagic pathways for ischemic stroke treatment.

Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein.

The role of cytokines in testicular function under normal conditions has not been completely understood. Here, we evaluated testicular macrophages (TM), steroidogenesis by Leydig cells (LC) and seminiferous tubules integrity in cytokines-deficient rat testes induced by diacerein, an anti-inflammatory drug that inhibits interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). Male rats received daily 100 mg/kg of diacerein (DIAG; n = 8) or saline (CG; n = 8) for 30 days. Serum testosterone (T) levels were measured and the seminiferous tubule (ST) area, epithelial area (EA), frequency of damaged ST and number of Sertoli cells (SC) were evaluated. TUNEL method and immunoreactions for detection of pro-IL-1β, TNF-α, steroidogenic acute regulatory protein (StAR), 17β-hydroxysteroid dehydrogenase (17β-HSD), androgen receptor (AR) and scavenger receptor for hemoglobin-haptoglobin complexes (CD163), a TM marker, were performed. Testicular AR, 17β-HSD and IL-1β levels were detected by Western blot. Data were submitted to Student t test (p ≤ 0.05). In DIAG, T and testicular AR, 17β-HSD and IL-1β levels decreased significantly (p < 0.05). The number of TUNEL-positive interstitial cells increased and LC showed weak StAR, 17β-HSD and AR immunoexpression in association with reduced IL-1β immunoexpression and number of CD163-positive TM in the interstitial tissue from diacerein-treated rats. Numerous damaged ST were found in DIAG, and reduction in the EA were associated with germ cells death. Moreover, the number of SC reduced and weak AR and TNF-α immunoexpression was observed in SC and germ cells, respectively. The cytokines deficiency induced by diacerein impairs TM, LC and spermatogenesis, and points to a role of IL-1β in steroidogenesis under normal conditions. In the ST, the weak AR and TNF-α immunoexpression in SC and germ cells, respectively, reinforces the idea that TNF-α plays a role in the SC androgenic control.

Pretreatment with mGluR2 or mGluR3 Agonists Reduces Apoptosis Induced by Hypoxia-Ischemia in Neonatal Rat Brains.

Hypoxia-ischemia (HI) in an immature brain results in energy depletion and excessive glutamate release resulting in excitotoxicity and oxidative stress. An increase in reactive oxygen species (ROS) production induces apoptotic processes resulting in neuronal death. Activation of group II mGluR was shown to prevent neuronal damage after HI. The application of agonists of mGluR3 (N-acetylaspartylglutamate; NAAG) or mGluR2 (LY379268) inhibits the release of glutamate and reduces neurodegeneration in a neonatal rat model of HI, although the exact mechanism is not fully recognized. In the present study, the effects of NAAG (5 mg/kg) and LY379268 (5 mg/kg) application (24 h or 1 h before experimental birth asphyxia) on apoptotic processes as the potential mechanism of neuroprotection in 7-day-old rats were investigated. Intraperitoneal application of NAAG or LY379268 at either time point before HI significantly reduced the number of TUNEL-positive cells in the CA1 region of the ischemic brain hemisphere. Both agonists reduced expression of the proapoptotic Bax protein and increased expression of Bcl-2. Decreases in HI-induced caspase-9 and caspase-3 activity were also observed. Application of NAAG or LY379268 24 h or 1 h before HI reduced HIF-1α formation likely by reducing ROS levels. It was shown that LY379268 concentration remains at a level that is required for activation of mGluR2 for up to 24 h; however, NAAG is quickly metabolized by glutamate carboxypeptidase II (GCPII) into glutamate and N-acetyl-aspartate. The observed effect of LY379268 application 24 h or 1 h before HI is connected with direct activation of mGluR2 and inhibition of glutamate release. Based on the data presented in this study and on our previous findings, we conclude that the neuroprotective effect of NAAG applied 1 h before HI is most likely the result of a combination of mGluR3 and NMDA receptor activation, whereas the beneficial effects of NAAG pretreatment 24 h before HI can be explained by the activation of NMDA receptors and induction of the antioxidative/antiapoptotic defense system triggered by mild excitotoxicity in neurons. This response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning.

Effects of acute triclosan exposure on gill and liver tissues of zebrafish (Danio rerio)

Triclosan (TCS) is a broad-spectrum antimicrobial agent found in personal care, household, veterinary, medical, and industrial products. TCS has been detected in soil, sediment, and surface water. In the current work, the effects of acute exposure to TCS on gill and liver tissues of zebrafish were evaluated. Adult zebrafish were treated with sublethal concentrations of TCS (34, 85, and 170 μg/L) for 120 h. Gill and liver samples were removed and embedded in paraffin for histopathological investigations. TCS induced aneurysm, capillary dilation, lamellar disorganization, hyperplasia, epithelial lifting, and desquamation in the gills. Liver specimens showed sinusoidal dilation, congestion, vacuolization, hepatocellular degeneration, and necrosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to observe the apoptotic effect of TCS. Accordingly, no apoptotic cells were detected in the specimens of 34 μg/L of TCS exposed gills, while the slides of 85 and 170 μg/L of TCS treated gills showed a high number of TUNEL-positive cells. In the liver, no TUNEL-positive cells were identified in 34 μg/L of TCS treated samples of zebrafish. 85 and 170 μg/L of TCS exposed livers showed few hepatic parenchymal cells undergoing apoptosis.

Memantine treatment exerts an antidepressant-like effect by preventing hippocampal mitochondrial dysfunction and memory impairment via upregulation of CREB/BDNF signaling in the rat model of chronic unpredictable stress-induced depression

Mitochondrial and cognitive dysfunctions have long been associated with major depressive disorders (MDDs). Studies have shown that Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, possesses an antidepressant-like effect. Hence, the NMDA receptor can be a better therapeutic target for MDD. Therefore, the present study was designed to study the impact of Memantine on mitochondrial functional status and depression-like symptoms in the chronic unpredictable stress (CUS) model of depression. CUS for 28 days resulted in depression-like symptoms (as indicated by increased immobility time in the forced swim test) and a decline in the spatial learning and retention memory in the Morris water maze (MWM) test, which was prevented by Memantine (10 mg/kg/day) treatment. We observed elevated plasma corticosterone (CORT) levels, microdialysates glutamate concentration, and synaptosomal calcium (Ca2+) ion levels after 28 days of CUS. Memantine treatment prevented only increased plasma CORT and synaptosomal Ca2+ ion levels. Memantine treatment also restored CUS induced increase in oxidative stress parameters [increased neuronal nitric oxide synthase (nNOS) expression, nitric oxide (NO) levels, lipid peroxidation (LPO) and superoxide dismutase (SOD) activity], decrease in mitochondrial electron transport chain (ETC) enzymes activity and mitochondrial membrane potential (MMP). CUS also reduced the expression of cell survival genes, cyclic-AMP response element-binding protein (CREB), and brain-derived nerve growth factor (BDNF), which was reversed by treatment with Memantine. CUS, however, caused a non-significant decrease in the hippocampal adenosine triphosphate (ATP) levels and a non-significant increase in the expression of pro-apoptotic genes, Caspase 3, and the number of TUNEL positive cells, indicating that hippocampal mitochondrial dysfunction caused due to CUS was not severe enough to affect overall energy production, mitochondrial integrity, and cellular apoptosis status. Thus, Memantine treatment exerts an antidepressant-like effect by preventing CUS induced excitotoxicity, oxidative stress, and enhancing CUS induced decrease in mitochondrial functioning and expression of cell survival genes via upregulation of stress-responsive CREB/BDNF signaling.

An in vitro Model of Human Retinal Detachment Reveals Successive Death Pathway Activations.

Purposewas to create an in vitro model of human retinal detachment (RD) to study the mechanisms of photoreceptor death.MethodsHuman retinas were obtained through eye globe donations for research purposes and cultivated as explants. Cell death was investigated in retinas with (control) and without retinal pigment epithelium (RPE) cells to mimic RD. Tissues were studied at different time points and immunohistological analyses for TUNEL, Cleaved caspase3, AIF, CDK4 and the epigenetic mark H3K27me3 were performed. Human and monkey eye globes with retinal detachment served as controls.ResultsThe number of TUNEL-positive cells, compared between 1 and 7 days, increased with time in both retinas with RPE (from 1.2 ± 0.46 to 8 ± 0.89, n = 4) and without RPE (from 2.6 ± 0.73 to 16.3 ± 1.27, p < 0.014). In the group without RPE, cell death peaked at day 3 (p = 0.014) and was high until day 7. Almost no Cleaved-Caspase3 signal was observed, whereas a transient augmentation at day 3 of AIF-positive cells was observed to be about 10-fold in comparison to the control group (n = 2). Few CDK4-positive cells were found in both groups, but significantly more in the RD group at day 7 (1.8 ± 0.24 vs. 4.7 ± 0.58, p = 0.014). The H3K27me3 mark increased by 7-fold after 5 days in the RD group (p = 0.014) and slightly decreased at day 7 and was also observed to be markedly increased in human and monkey detached retina samples.ConclusionAIF expression coincides with the first peak of cell death, whereas the H3K27me3 mark increases during the cell death plateau, suggesting that photoreceptor death is induced by different successive pathways after RD. This in vitro model should permit the identification of neuroprotective drugs with clinical relevance.

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia.

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

KPNA4 regulated by miR-548b-3p promotes the malignant phenotypes of papillary thyroid cancer

AimKaryopherin α4 (KPNA4, importin α3) has been verified to be an oncogene in many cancers. However, its role in papillary thyroid cancer (PTC), the most frequent endocrine malignancy, is still unclear. Materials and methodsKPNA4 expression was analyzed in PTC tissues and cells. The effects of KPNA4 on the proliferation, invasion, and apoptosis of PTC cells were evaluated after overexpression or downregulation of KPNA4. The influence of KPNA4 on NF-κB activation was evaluated by nuclear NF-κB p65 expression and NF-κB-luciferase reporter assays. Moreover, we also explored whether KPNA4 was regulated by miR-548b-3p. Additionally, the roles of miR-548b-3p and KPNA4 were explored in a xenograft mouse model. Key findingsKPNA4 expression was increased in PTC tissues and cells, and its expression was significantly related to patients' clinicopathologic features and overall survival. Overexpression of KPNA4 significantly promoted PTC cell proliferation and invasion, enhanced nuclear p65 expression and augmented NF-κB luciferase activity. However, KPNA4 silencing showed opposite effects on the above indexes, and induced apoptosis of PTC cells. KPNA4 was a target of miR-548b-3p, which was downregulated in PTC and inhibited proliferation and invasion, but promoted apoptosis of PTC cells. KPNA4 overexpression abrogated the suppression of miR-548b-3p on the malignant phenotypes of PTC cells. Both miR-548b-3p overexpression and KPNA4 downregulation inhibited tumor growth and Ki-67 expression, elevated numbers of Tunel-positive cells, and deceased nuclear p65 expression in mouse tumor tissues. SignificanceKPNA4 was negatively regulated by miR-548b-3p and promoted the development of PTC via activating the NF-κB pathway.