Expression Of Cell Death Markers Research Articles

Cellular senescence mediates retinal ganglion cell survival regulation post-optic nerve crush injury.

Traumatic optic neuropathy refers to optic nerve (ON) injury by trauma, including explosion and traffic accident. Retinal ganglion cell (RGC) death is the critical pathological cause of irreversible visual impairment and blindness in ON injury. We previously investigated the patterns of 11 modes of cell death in mouse retina post-ON injury. Here we aimed to identify additional signalling pathways regulating RGC survival in rodents post-ON injury. RNA sequencing analysis identified the upregulation of inflammation and cellular senescence-related genes in retina post-ON injury, which were confirmed by immunoblotting and immunofluorescence analyses. Increased expression of senescence-associated β-galactosidase (SA-βgal) in RGCs and activation of microglia were also found. Transforming growth factor-β receptor type II inhibitor (LY2109761) treatment suppressed p15Ink4b and p21Cip1 protein and SA-βgal expression and promoted RGC survival post-ON injury with decreasing the expression of cell death markers in retina. Consistently, senolytics (dasatinib and quercetin) treatments can promote RGC survival and alleviate the reduction of ganglion cell complex thickness and pattern electroretinography activity post-ON injury with reducing SA-βgal, p15Ink4b, p21Cip1, microglial activation and cell death marker expression. In summary, this study revealed the activation of cellular senescence in rodent retina post-ON injury and contribute to RGC survival regulation. Targeting cellular senescence can promote RGC survival after ON injury, suggesting a potential treatment strategy for traumatic optic neuropathy.

Microglial Activation Modulates Neuroendocrine Secretion During Experimental Sepsis.

Sepsis promotes an inflammatory state in the central nervous system (CNS) that may cause autonomic, cognitive, and endocrine changes. Microglia, a resident immune cell of the CNS, is activated in several brain regions during sepsis, suggesting its participation in the central alterations observed in this disease. In this study, we aimed to investigate the role of microglial activation in the neuroendocrine system functions during systemic inflammation. Wistar rats received an intracerebroventricular injection of the microglial activation inhibitor minocycline (100μg/animal), shortly before sepsis induction by cecal ligation and puncture. At 6 and 24h after surgery, hormonal parameters, central and peripheral inflammation, and markers of apoptosis and synaptic function in the hypothalamus were analyzed. The administration of minocycline decreased the production of inflammatory mediators and the expression of cell death markers, especially in the late phase of sepsis (24h). With respect to the endocrine parameters, microglial inhibition caused a decrease in oxytocin and an increase in corticosterone and vasopressin plasma levels in the early phase of sepsis (6h), while in the late phase, we observed decreased oxytocin and increased ACTH and corticosterone levels compared to septic animals that did not receive minocycline. Prolactin levels were not affected by minocycline administration. The results indicate that microglial activation differentially modulates the secretion of several hormones and that this process is associated with inflammatory mediators produced both centrally and peripherally.

Predictive Nephrotoxicity Profiling of a Novel Antifungal Small Molecule in Comparison to Amphotericin B and Voriconazole.

Background and Purpose Candida albicans is the major fungal species associated with superficial mucosal infections such as oral candidiasis as well as systemic mycoses with high morbidity and mortality. On top of the rising drug resistance, currently available antifungal agents have significant adverse effects. Nephrotoxicity is the major treatment complication associated with antifungal agents.Recently, we discovered a novel antifungal small molecule SM21 with promising antifungal activity. The present study aimed to comparatively evaluate the in vivo and in vitro nephrotoxicity of SM21 comparing with Amphotericin B and voriconazole.Experimental ApproachNephrotoxicity of SM21 and its analogue were comparatively evaluated with Amphotericin B (AmB) and voriconazole. Immortalized human kidney proximal tubule epithelial cells (HK-2) were used for in vitro analysis of nephrotoxicity using cytotoxicity assays and qPCR gene expression analysis (Kim-1/HAVcr-1, CASP3). Sprague Dawley (SD) rat model was used to evaluate the nephrotoxicity in vivo using classical (SCr and BUN) and next-generation kidney injury urinary biomarkers (Kim-1, CLU, ALB, NGAL, β2M, and Cys C) alongside histopathological and immunohistochemical standards.Key ResultsAmB treatment showed a stronger cytotoxic impact on HK-2 viability and gene expression of cell death markers (Kim-1/HAVcr-1, CASP3) compared with SM21 and SM21 analogue in vitro (P < 0.01). In vivo data further demonstrated that SM21 did not significantly increase classical as well as novel nephrotoxic biomarkers, and minimal renal tubular necrosis and abnormalities were observed (15 mg kg−1 BW/day).Conclusions and ImplicationsSM21 had a significantly better safety profile in terms of nephrotoxicity with no major tubular epithelial abnormalities observed in kidney cells and no augmentation of kidney injury biomarkers compared to AmB. Kim-1 and CLU were the most sensitive biomarkers for detection of AmB-induced kidney damage. Future clinical trials should consider inclusion of these novel biomarkers as early indicators of acute kidney injury in antifungal-induced nephrotoxicity.

Expression of Cell Death Markers in Rat Hippocampus after Long-Term Fluoride Intoxication

Expression of Cell Death Markers in Rat Hippocampus after Long-Term Fluoride Intoxication

Cell death in the human infant central nervous system and in sudden infant death syndrome (SIDS).

The brainstem has been a focus of sudden infant death syndrome (SIDS) research with amassing evidence of increased neuronal apoptosis. The present study extends the scope of brain regions examined and determines associations with known SIDS risk factors. Immunohistochemical expression of cell death markers, active caspase-3 and TUNEL, was studied in 37 defined brain regions in infants (aged 1-12 months) who died suddenly and unexpectedly (SUDI). A semi-quantitative mean score of marker expression was derived for each region and scores compared between three SUDI subgroups: explained SUDI (eSUDI; n = 7), SIDS I (n = 8) and SIDS II (n = 13). In eSUDI, active caspase-3 scores were highest in several nuclei of the rostral medulla, and lowest in the hypothalamus and cerebellar grey matter (GM). TUNEL was highest in regions of the hippocampus and basal ganglia, and lowest in the thalamus and cerebellar GM. TUNEL scores were higher in SIDS II compared to eSUDI in the amygdala (p = 0.03) and 5/9 nuclei in the rostral medulla (p = 0.04 - 0.01), and higher in SIDS II compared to SIDS I in the amygdala (p < 0.01), putamen (p = 0.01), lentiform nucleus (p = 0.03) and parietal (p = 0.03) and posterior frontal (p = 0.02) cortex. Active caspase-3 was greater in the hypoglossal nucleus (p = 0.03) of SIDS I compared to eSUDI infants. Co-sleeping, cigarette smoke exposure and the presence of an upper respiratory tract infection in SIDS infants was associated with differences in marker expression. This study affirms the sensitivity of the brainstem medulla to cell death in SIDS, and highlights the amygdala as a new region of interest.

Evaluation of programmed cell death processes on the lens epithelium of older dogs with diabetic and hypermature cataracts

It is well known that posterior capsule opacification (PCO), one of the most common late postoperative complications of cataract surgery, is mainly caused by proliferation and differentiation of remaining lens epithelial cells (LECs) on the posterior lens capsule. Many authors suggest that alterations induced by the pathophysiology of cataracts, its metabolism and the use of 0.1% trypan blue (TB) must cause some degree of cellular damage on these cells, wicht would help to prevent and/or reduce the incidence of PCO after cataract surgery in humans. Therefore, the aim of this study was to evaluate the expression of cell death markers on LECs of older dogs with diabetic and hypermature cataracts, after capsulorhexis, both using 0.1% TB. Twenty samples collected from 13 dogs of different breeds, with ages varying from 8 to 12 years-old, with diabetic and hypermature cataracts, which had been subjected to phacoemulsification surgery (Phaco) using 0.1% TB for staining were studied. Animals were classified as dogs with diabetic (DC) and hypermature cataracts (HC), and expression of molecular markers for apoptosis and autophagy (caspase-3 and beclin-1) on LECs were obtained by immunofluorescence technique. The expression of caspase-3 and beclin-1 was observed in every studied sample and did not differ between groups. In conclusion, our findings suggest that apoptosis and autophagy processes occur to LECs in older dogs presenting diabetic and hypermature cataracts after Phaco utilizing 0.1% TB. Our results may be helpful to future studies of PCO in post-phacoemulsification surgery patients.

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains

Beyond their hemostatic functions, platelets alter their inflammatory response according to the bacterial stimulus. Staphylococcus aureus is associated with exacerbated inflammation and thrombocytopenia, which is associated with poor prognosis during sepsis. Acetylsalicylic acid and statins prevent platelet aggregation and decrease the mortality rate during sepsis. Therefore, we assessed whether these two molecules could reduce in vitro platelet activation and the inflammatory response to S. aureus. Platelets were exposed to clinical strains of S. aureus in the presence or absence of acetylsalicylic acid or fluvastatin. Platelet activation, aggregation, and release of soluble sCD62P, sCD40 Ligand, RANTES and GROα were assessed. Platelet cell death was evaluated by analyzing the mitochondrial membrane potential, phosphatidylserine exposure, platelet microparticle release and caspase-3 activation. All S. aureus strains induced platelet activation but not aggregation and decreased the platelet count, the expression of cell death markers and the release of RANTES and GROα. Acetylsalicylic acid but not fluvastatin limited platelet activation and inflammatory factor release and restored the platelet count by protecting platelets from Staphylococcus-induced expression of cell death markers. This study demonstrates that acetylsalicylic acid limits S. aureus-induced effects on platelets by reducing cell death, revealing new strategies to reduce the platelet contribution to bacteremia-associated inflammation.

In vitro and in vivo analysis of macelignan induced toxicity on heart

Macelignan which isolated from Myristica fragrans Houtt has been suggested that it acts as PPAR α/γ dual agonist for diabetic patients. However, macelignan contains a small amount of safrole considering as carcinogen. Here we investigated that its toxic effect on heart in vitro and in vivo. The systolic blood pressure, pulse and the component of blood serum in spontaneously hypertensive rats (SHR) and control rats were measured. The expression of cell death markers were analyzed by RT‐PCR and immunoblot. We confirmed that PPAR α/γ was expressed on H9c2 cells, neonatal cardiomyocytes and heart. Compared to control, macelignan significantly reduced the beating number and frequency as a concentration dependent manner (p&lt;.05). Cell viability was strikingly decreased at 16 h after treatment. And the expression of autophagic markers as well as apoptotic molecules was maximized at 16 h (p&lt;.05). A significant reduction of the systolic blood pressure was shown in both strains, and pulse was also strikingly decreased after its injection (p&lt;.05). However, the component of serum was not changed by macelignan. Here, we found a cytotoxicity induced by macelignan in vitro and in vivo. Therefore, the use of macelignan as an anti‐cancer or anti‐inflammatory agent should be carefully considered.Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea

Polarized fluid movement and not cell death, creates luminal spaces in adult prostate epithelium

There are two predominant theories for lumen formation in tissue morphogenesis; cavitation driven by cell death, and membrane separation driven by epithelial polarity. To define the mechanism of lumen formation in prostate acini we examined both theories in several cell lines grown in 3D Matrigel culture. Lumen formation occurred early in culture and preceded the expression of cell death markers for apoptosis (active caspase 3) and autophagy (LC-3). Active caspase 3 was expressed by very few cells and inhibition of apoptosis did not suppress lumen formation. Despite LC-3 expression in all cells within a spheroid, this was not associated with cell death. However, expression of the prostate secretory protein coincided with lumen formation and subsequent disruption of polarized fluid movement led to significant inhibition of lumen formation. This work indicates that lumen formation is driven by the polarized movement of fluids and proteins in 3D prostate epithelial models and not by cavitation.

Light and Oxygen Are Not Required for Harpin-induced Cell Death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.