Active Caspase-3 Immunostaining Research Articles

Impact of age on liver damage, inflammation, and molecular signaling pathways in response to femoral fracture and hemorrhage.

Trauma causes disability and mortality globally, leading to fractures and hemorrhagic shock. This can trigger an irregular inflammatory response that damages remote organs, including liver. Aging increases the susceptibility to dysregulated immune responses following trauma, raising the risk of organ damage, infections, and higher morbidity and mortality in elderly patients. This study investigates how aging affects liver inflammation and damage post-trauma. 24 male C57BL/6J mice were randomly divided into four groups. Twelve young (17-26 weeks) and 12 aged (64-72 weeks) mice were included. Mice further underwent either hemorrhagic shock (trauma/hemorrhage, TH), and femoral fracture (osteotomy) with external fixation (Fx) (THFx, n=6) or sham procedures (n=6). After 24 hours, mice were sacrificed. Liver injury and apoptosis were evaluated using hematoxylin-eosin staining and activated caspase-3 immunostaining. CXCL1 and infiltrating polymorphonuclear leukocytes (PMNL) in the liver were assessed by immunostaining, and concentrations of CXCL1, TNF, IL-1β, and IL-10 in the liver tissue were determined by ELISA. Gene expression of Tnf, Cxcl1, Il-1β, and Cxcl2 in the liver tissue was determined by qRT-PCR. Finally, western blot was used to determine protein expression levels of IκBα, Akt, and their phosphorylated forms. THFx caused liver damage and increased presence of active caspase-3-positive cells compared to the corresponding sham group. THFx aged group had more severe liver injury than the young group. CXCL1 and PMNL levels were significantly higher in both aged groups, and THFx caused a greater increase in CXCL and PMNL levels in aged compared to the young group. Pro-inflammatory TNF and IL-1β levels were elevated in aged groups, further intensified by THFx. Anti-inflammatory IL-10 levels were lower in aged groups. Tnf and Cxcl1 gene expression was enhanced in the aged sham group. Phosphorylation ratio of IκBα was significantly increased in the aged sham group versus young sham group. THFx-induced IκBα phosphorylation in the young group was significantly reduced in the aged THFx group. Akt phosphorylation was significantly reduced in the THFx aged group compared to the THFx young group. The findings indicate that aging may lead to increased vulnerability to liver injury and inflammation following trauma due to dysregulated immune responses.

Azithromycin Protects Oligodendrocyte Progenitor Cells against Lipopolysaccharide-Activated Microglia-Induced Damage

Oligodendrocyte progenitor cells (OPC) are the primary cellular targets of brain white matter injury (WMI) in very low-birth weight (VLBW) infants. Microglia plays a significant role in inflammation-induced WMI. Our previous study showed that lipopolysaccharide (LPS)-induced OPC damage is mediated by activated microglia in vitro. We hypothesized that azithromycin (AZ) could protect OPCs against LPS-induced cytotoxicity by blocking microglial activation. Highly enriched primary rat microglia and OPCs were treated with LPS. There were 4 groups: control, LPS + Veh, AZ, and LPS + AZ. Microglia conditioned medium (MCM) was used to determine inflammatory cytokines by enzyme-linked immunosorbent assay or subsequent treatment of OPCs. We found that AZ significantly suppressed TNF-α, IL-1β, and IL-6 in LPS+Veh-treated–microglial MCM and blocked microglial nuclear factor-κB p65 nuclear translocation. AZ prevented LPS-MCM-induced OPC death and improved OPC survival as measured by activated caspase-3 immunostaining and XTT assay, respectively. AZ ameliorated LPS-MCM-induced differentiation arrest and myelin basic protein deficit in oligodendrocytes. Our data suggest that AZ is a potent inhibitor for microglia activation and may hold the therapeutic potential for WMI in VLBW infants.

ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL ALTERATIONS IN HEMOCYTES AND FAT BODY OF THE FLESH FLY (SARCOPHAGA ARGYROSTOMA) LARVAE UPON CANCER CELL LINE INOCULATION

In this study, hemocytes and fat body changes were investigated after activation the immuneresponse of the third instar larvae of Sarcophaga argyrostoma by injecting the larvaewith MDA-MB-231cancer cell lines. To investigate the changes in hemolymph and fat bodytissue, light and transmission electron microscopy (TEM) were used. Apoptosis demonstrationin the larval flesh fly after injection was immunohistochemically tracked using a markerof caspase initiator activity; namely caspase-3 antibody.The results showed remarkable changesin morphology, viability and the spreading ability of immunocompetent hemocytes andfat body cells between normal and immunized S. argyrostoma larvae. In hemocytes, multicellularaggregates were formed to entrap invading cancer cells, in addition to membrane blebbing,cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation.Meanwhile, in fat body cells of injected larvae, depletion of lipid droplets, changes in the appearanceof protein granules and degrade of basement membrane overlying the fat body wereobserved. These features are typical for apoptotic and autophagal cell death which were confirmedby detection of apoptotic fat body cells after cancer cell injection using activatedcaspase-3 immunostaining.

ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL ALTERATIONS IN HEMOCYTES AND FAT BODY OF THE FLESH FLY (SARCOPHAGA ARGYROSTOMA) LARVAE UPON CANCER CELL LINE INOCULATION

In this study, hemocytes and fat body changes were investigated after activation the immuneresponse of the third instar larvae of Sarcophaga argyrostoma by injecting the larvaewith MDA-MB-231cancer cell lines. To investigate the changes in hemolymph and fat bodytissue, light and transmission electron microscopy (TEM) were used. Apoptosis demonstrationin the larval flesh fly after injection was immunohistochemically tracked using a markerof caspase initiator activity; namely caspase-3 antibody.The results showed remarkable changesin morphology, viability and the spreading ability of immunocompetent hemocytes andfat body cells between normal and immunized S. argyrostoma larvae. In hemocytes, multicellularaggregates were formed to entrap invading cancer cells, in addition to membrane blebbing,cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation.Meanwhile, in fat body cells of injected larvae, depletion of lipid droplets, changes in the appearanceof protein granules and degrade of basement membrane overlying the fat body wereobserved. These features are typical for apoptotic and autophagal cell death which were confirmedby detection of apoptotic fat body cells after cancer cell injection using activatedcaspase-3 immunostaining.

Unilateral intranigral administration of \u03b2-sitosterol \u03b2-D-glucoside triggers pathological \u03b1-synuclein spreading and bilateral nigrostriatal dopaminergic neurodegeneration in the rat

The spreading and accumulation of α-synuclein and dopaminergic neurodegeneration, two hallmarks of Parkinson’s disease (PD), have been faithfully reproduced in rodent brains by chronic, oral administration of β-sitosterol β-D-glucoside (BSSG). We investigated whether a single injection of BSSG (6 μg BSSG/μL DMSO) in the left substantia nigra of Wistar rats causes the same effects. Mock DMSO injections and untreated rats formed control groups. We performed immunostainings against the pathological α-synuclein, the dopaminergic marker tyrosine hydroxylase (TH), the neuroskeleton marker β-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using β-galactosidase (β-Gal) assay and active caspase-3 immunostaining, we assessed cell death mechanisms. Golgi-Cox staining was used to measure the density and types of dendritic spines of striatal medium spiny neurons. Motor and non-motor alterations were also evaluated. The study period comprised 15 to 120 days after the lesion. In the injured substantia nigra, BSSG caused a progressive α-synuclein aggregation and dopaminergic neurodegeneration caused by senescence and apoptosis. The α-synuclein immunoreactivity was also present within microglia cells. Decreased density of dopaminergic fibers and dendritic spines also occurred in the striatum. Remarkably, all the histopathological changes also appeared on the contralateral nigrostriatal system, and α-synuclein aggregates were present in other brain regions. Motor and non-motor behavioral alterations were progressive. Our data show that the stereotaxic BSSG administration reproduces PD α-synucleinopathy phenotype in the rat. This approach will aid in identifying the spread mechanism of α-synuclein pathology and validate anti-synucleinopathy therapies.

Mito-tempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity.

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300mg/kg APAP followed by 20mg/kg MT 1.5h or 3h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10mg/kg) 2h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1

In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. Here, we have examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, we examine the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, in the regulation of DEOM histolysis. We find that loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. Herein, we also find that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, our results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death.

Brain-derived neurotrophic factor modulates intestinal barrier by inhibiting intestinal epithelial cells apoptosis in mice.

We aimed to investigate the effects of brain-derived neurotrophic factor (BDNF) on apoptosis of intestinal epithelial cells (IECs) and alterations of intestinal barrier integrity using BDNF knock-out mice model. Colonic tissues from BDNF(+/+) mice and BDNF(+/-) mice were prepared for this study. The integrity of colonic mucosa was evaluated by measuring trans-mucosa electrical resistance and tissue conductance in Ussing chamber. The colonic epithelial structure was analyzed by transmission electron microscopy. Apoptosis involvement was determined with TUNEL staining, active caspase-3 immunostaining and Western blotting for the protein expression of active caspase-3, Bax and Bcl-2. The expression levels and distribution of tight junction proteins were evaluated by immunohistochemistry or Western blots. Compared with BDNF(+/+) mice, BDNF(+/-) mice displayed impaired integrity and ultrastructure alterations in their colonic mucosa, which was characterized by diminished microvilli, mitochondrial swelling and epithelial cells apoptosis. Altered intestinal barrier function was linked to excessive apoptosis of IECs demonstrated by the higher proportion of TUNEL-positive apoptotic cells and enhanced caspase activities in BDNF(+/-) mice. Increased expression of Bax and claudin-2 proteins and reduced Bcl-2 and tight junction proteins (occludin, ZO-1 and claudin-1) expression were also detected in the colonic mucosa of BDNF(+/-) mice. BDNF may play a role in the maintenance of intestinal barrier integrity via its anti-apoptotic properties.

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

ABSTRACTTo investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.

Retinal pigment epithelial cell necroptosis in response to sodium iodate.

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly in developed countries. The late stage of dry AMD, or geographic atrophy (GA), is characterized by extensive retinal pigment epithelium (RPE) degeneration. The underlying molecular mechanism for RPE cell death in GA remains unclear. Our previous study has established that RPE cells die predominantly from necroptosis in response to oxidative stress in vitro. Here, we extend our study and aim to characterize the nature of RPE cell death in response to sodium iodate (NaIO3) in vitro and in a NaIO3-induced retina degeneration mouse model. We found that NaIO3 induces RPE necroptosis in vitro by using a combination of molecular hallmarks. By using TUNEL assays, active caspase-3 and HMGB1 immunostaining, we confirmed that photoreceptor cells die mainly from apoptosis and RPE cells die mainly from necroptosis in response to NaIO3 in vivo. RPE necroptosis in this model is also supported by use of the RIPK1 inhibitor, Necrostatin-1. Furthermore, using novel RIPK3-GFP transgenic mouse lines, we detected RIPK3 aggregation, a hallmark of necroptosis, in the RPE cells in vivo after NaIO3 injection. Our findings suggest the necessity of re-evaluating RPE cell death mechanism in AMD models and have the potential to influence therapeutic development for dry AMD, especially GA.

Kombucha tea prevents obese mice from developing hepatic steatosis and liver damage.

Nonalcoholic fatty liver disease (NAFLD) is associated with the increased accumulation of hepatocellular lipids. Although Kombucha tea (KT) has emerged as a substance protecting the liver from damage, the effects of KT in NAFLD remain unclear. Hence, we investigated whether KT influenced hepatic steatosis. Db/db mice were fed either control or methionine/choline-deficient (MCD) diets for 4 weeks. The MCD diet group was treated with KT or water for 3 weeks. KT treatment alleviated macrovesicular steatosis compared to the MCD-fed group. The levels of triglyceride, ALT, and AST also decreased in the KT+MCD-treated db/db mice. RNA expression in the MCD+KT group showed reduced triglyceride synthesis and uptake of fatty acids. Immunostaining and western blot assays for active caspase-3 demonstrated a lower level of apoptosis in the MCD+KT than in the MCD group. These results demonstrate that KT attenuated lipid accumulation and protected the liver from damage, promoting liver restoration in mice.

Protective effects of melatonin and selenium against apoptosis of olfactory sensory neurons: A rat model study.

Selenium plays a role in the prevention of oxidative damage and has been linked to regulatory functions in cell growth, apoptosis, cell survival, and cytotoxicity. Melatonin has an antioxidant effect, which protects against a number of free radical species. Given its antioxidant properties, melatonin has been widely known to inhibit neuronal apoptosis. We examined the cytoprotective effects of melatonin and selenium in rat olfactory sensory neurons after rhinosinusitis by immunohistochemical evaluation of olfactory bulb mucosa. Rhinosinusitis was induced bilaterally in 24 animals. Twenty-four rats were randomly divided into three equal groups. The melatonin group was treated with intraperitoneal (i.p.) melatonin and ampicillin-sulbactam, the selenium group was treated with i.p. selenium and ampicillin-sulbactam, the antibiotic group was treated with i.p. ampicillin-sulbactam; all three groups were treated for 10 days. After a period of 10 days of treatment, the animals were killed for immunohistochemical analyses. All olfactory bulb mucosae were removed immediately. No histochemical differences were found in the three groups. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells were detected in each group. In the antibiotic group, the appearance of apoptotic cells was higher, whereas the number of apoptotic cells significantly decreased in the melatonin group. When compared with the selenium group, fewer terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells were observed in the melatonin group, which was not significant. In the antibiotic group, the cytoplasmic active caspase-3 and Bax immunostaining in the olfactory epithelium and glandular cells of stroma were higher when compared with the immunostaining in melatonin and selenium groups. Active caspase-3 and Bax immunostaining in the subepithelial stroma was dramatically reduced in the melatonin group. In contrast, the staining intensity and the number of Bcl-2 immunopositive cells were significantly increased in the melatonin group. In the selenium group, Bax and active caspase-3 were moderately immunopositive in the epithelium and subepithelial stroma. However, Bcl-2 immunostaining was more pronounced in the olfactory epithelium and some stromal cells. Our results indicated the possibility that the supplementation of melatonin and selenium, two antioxidant agents for the treatments in the rhinosinusitis rat model, might be reduced or prevent anosmia.

The efficiency of Poly(ADP-Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion.

The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP-ribose) polymerase (PARP) cleavage in testicular torsion-detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4-h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin-eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase-3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4-h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time-dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion-detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.

The effects of α-lipoic acid on immature rats with traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality during childhood. TBI enhances formation of reactive oxygen species that cause neuron damage and apoptosis. α-Lipoic acid (LA) is a free radical scavenger and biological antioxidant. We investigated the effects of LA treatment on the parietal and prefrontal cortex, and on the hippocampal regions of the brain in 7-day-old rat pups that had been subjected to contusion injury. Forty-two male rats were divided randomly into a control group, a TBI group and a TBI + LA treated group. LA was administered 30 min after TBI through an intragastric tube once daily for 2 days. Forty-eight hours after TBI, the animals were sacrificed and tissues were examined for apoptosis and density of neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 immunostaining were used to detect apoptosis. Glutathione peroxidase (GPx), superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels also were measured. Histological evaluation showed that LA treatment significantly reduced TBI-induced neuronal death in the hippocampus, prefrontal and parietal cortex; TUNEL- and caspase-3-positive cells also were decreased in the same regions. In addition, LA administration increased GPx and SOD activity in the prefrontal cortex. It appears that LA may be beneficial for TBI in rats.

Cardiac macrophages and apoptosis after myocardial infarction: effects of central MR blockade.

After myocardial infarction (post-MI), inflammation and apoptosis contribute to progressive cardiac remodeling and dysfunction. Cardiac mineralocorticoid receptor (MR) and β-adrenergic signaling promote apoptosis and inflammation. Post-MI, MR activation in the brain contributes to sympathetic hyperactivity and an increase in cardiac aldosterone. In the present study, we assessed the time course of macrophage infiltration and apoptosis in the heart as detected by both terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and active caspase-3 immunostaining in both myocytes and nonmyocytes, as well as the effects of central MR blockade by intracerebroventricular infusion of eplerenone at 5 μg/day on peak changes in macrophage infiltration and apoptosis post-MI. Macrophage numbers were markedly increased in the infarct and peri-infarct zones and to a minor extent in the noninfarct part of the left ventricle at 10 days post-MI and decreased over the 3-mo study period. Apoptosis of both myocytes and nonmyocytes was clearly apparent in the infarct and peri-infarct areas at 10 days post-MI. For TUNEL, the increases persisted at 4 and 12 wk, but the number of active caspase-3-positive cells markedly decreased. Central MR blockade significantly decreased CD80-positive proinflammatory M1 macrophages and increased CD163-positive anti-inflammatory M2 macrophages in the infarct. Central MR blockade also reduced apoptosis of myocytes by 40-50% in the peri-infarct and to a lesser extent of nonmyocytes in the peri-infarct and infarct zones. These findings indicate that MR activation in the brain enhances apoptosis both in myocytes and nonmyocytes in the peri-infarct and infarct area post-MI and contributes to the inflammatory response.

Rat spontaneous foetal resorption: altered α2-macroglobulin levels and uNK cell number.

During rat pregnancy, some of the foetoplacental units undergo complete spontaneous resorption while the adjacent units remain unaffected. In an attempt to clarify the mechanisms implicated in this spontaneous resorption, implantation units from days 14 and 16 of pregnancy were examined. The number of implantation sites and resorption units was recorded, and uterine paraffin sections were stained with haematoxylin and eosin for the evaluation of tissue morphology. The incidence of resorption was about 9.2 % on day 14 and 8.2 % on day 16. Perforin and active caspase-3 immunostaining were performed for localization and characterization of uterine natural killer (uNK) and apoptotic cells, respectively. The α2-macroglobulin (α2-MG) expression was examined by in situ hybridization, immunohistochemistry and its levels quantified by enzyme-linked immunosorbent assay. A reduction in α2-MG decidual levels in resorpted units was observed when compared to normal implantation units in both days. This potent protease inhibitor is the major product secreted by the mesometrial decidual tissue and may constitute an indicator of maternal tissues remodelling abnormalities. Besides the decreased α2-MG levels, an increase in uNK cell number was found in resorption units. The decreased α2-MG levels may be related to the aberrant control of trophoblast invasion that may activate uNK cells. The elucidation of the mechanisms underlying natural pregnancy loss in rat may contribute for the clarification of the "vanishing twin" phenomenon that occurs in human pregnancy.

Molecular pathways underpinning ethanol-induced neurodegeneration.

While genetics impacts the type and severity of damage following developmental ethanol exposure, little is currently known about the molecular pathways that mediate these effects. Traditionally, research in this area has used a candidate gene approach and evaluated effects on a gene-by-gene basis. Recent studies, however, have begun to use unbiased approaches and genetic reference populations to evaluate the roles of genotype and epigenetic modifications in phenotypic changes following developmental ethanol exposure, similar to studies that evaluated numerous alcohol-related phenotypes in adults. Here, we present work assessing the role of genetics and chromatin-based alterations in mediating ethanol-induced apoptosis in the developing nervous system. Utilizing the expanded family of BXD recombinant inbred mice, animals were exposed to ethanol at postnatal day 7 via subcutaneous injection (5.0 g/kg in 2 doses). Tissue was collected 7 h after the initial ethanol treatment and analyzed by activated caspase-3 immunostaining to visualize dying cells in the cerebral cortex and hippocampus. In parallel, the levels of two histone modifications relevant to apoptosis, γH2AX and H3K14 acetylation, were examined in the cerebral cortex using protein blot analysis. Activated caspase-3 staining identified marked differences in cell death across brain regions between different mouse strains. Genetic analysis of ethanol susceptibility in the hippocampus led to the identification of a quantitative trait locus on chromosome 12, which mediates, at least in part, strain-specific differential vulnerability to ethanol-induced apoptosis. Furthermore, analysis of chromatin modifications in the cerebral cortex revealed a global increase in γH2AX levels following ethanol exposure, but did not show any change in H3K14 acetylation levels. Together, these findings provide new insights into the molecular mechanisms and genetic contributions underlying ethanol-induced neurodegeneration.

N-acetylcysteine counteracts oxidative stress and protects alveolar epithelial cells from lung contusion-induced apoptosis in rats with blunt chest trauma

The aim of this study was to investigate the protective effects of N-acetylcysteine (NAC) on peroxidative and apoptotic changes in the contused lungs of rats following blunt chest trauma. The rats were randomly divided into three groups: control, contusion, and contusion+NAC. All the rats, apart from those in the control group, performed moderate lung contusion. A daily intramuscular NAC injection (150mg/kg) was given immediately following the blunt chest trauma and was continued for two additional days following cessation of the trauma. Samples of lung tissue were taken in order to evaluate the tissue malondialdehyde (MDA) level, histopathology, and epithelial cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and active caspase-3 immunostaining. In addition, we immunohistochemically evaluated the expression of surfactant protein D (SP-D) in the lung tissue. The blunt chest trauma-induced lung contusion resulted in severe histopathological injury, as well as an increase in the MDA level and in the number of cells identified on TUNEL assay together with active caspase-3 positive epithelial cells, but a decrease in the number of SP-D positive alveolar type 2 (AT-2) cells. NAC treatment effectively attenuated histopathologic, peroxidative, and apoptotic changes, as well as reducing alterations in SP-D expression in the lung tissue. These findings indicate that the beneficial effects of NAC administrated following blunt chest trauma is related to the regulation of oxidative stress and apoptosis.

Retinoid Acid Specifies Neuronal Identity through Graded Expression of Ascl1

SummaryCell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors [1]. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals [2, 3], two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively [4–7]. These neurons have different physiological properties, functions, and connectivity [8, 9]. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1 [10], whereas V3 neurons express Sim1 [4]. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.

Ketamine alters the neurogenesis of rat cortical neural stem progenitor cells*

High doses or prolonged exposure to ketamine increase neuronal apoptosis in the developing brain, although effects on neural stem progenitor cells remain unexplored. This study investigated dose- and time-dependent responses to ketamine on cell death and neurogenesis in cultured rat fetal cortical neural stem progenitor cells. Laboratory-based study. University research laboratory. Sprague-Dawley rats. Neural stem progenitor cells were isolated from the cortex of Sprague-Dawley rat fetuses on embryonic day 17. In dose-response experiments, cultured neural stem progenitor cells were exposed to different concentrations of ketamine (0-100 µM) for 24 hrs. In time-course experiments, neural stem progenitor cells cultures were exposed to 10 µM ketamine for different durations (0-48 hrs). Apoptosis and necrosis in neural stem progenitor cells were assessed using activated caspase-3 immunostaining and lactate dehydrogenase assays, respectively. Proliferative changes in neural stem progenitor cells were detected using bromo-deoxyuridine incorporation and Ki67 immunostaining. Neuronal differentiation was assessed using Tuj-1 immunostaining. Cultured neural stem progenitor cells were resistant to apoptosis and necrosis following all concentrations and durations of ketamine exposure tested. Ketamine inhibited proliferation with decreased numbers of bromo-deoxyuridine-positive cells following ketamine exposure to 100 µM for 24 hrs (p<.005) or 10 µM for 48 hrs (p< .01), and reduced numbers of Ki67-positive cells following exposure to ketamine concentration>10 µM for 24 hrs (p<.001) or at 10 µM for 48 hrs (p<.01). Ketamine enhanced neuronal differentiation, with all ketamine concentrations increasing Tuj-1-positive neurons (p<.001) after 24-hrs of exposure. This also occurred with all exposures to 10 µM ketamine for >8 hrs (p<.001). Clinically relevant concentrations of ketamine do not induce cell death in neural stem progenitor cells via apoptosis or necrosis. Ketamine alters the proliferation and increases the neuronal differentiation of neural stem progenitor cells isolated from the rat neocortex. These studies imply that ketamine exposure during fetal or neonatal life may alter neurogenesis and subsequent brain development.