Lower Number Of Apoptotic Cells Research Articles

Beneficial effects of alpha-1 antitrypsin therapy in a mouse model of colitis-associated colon cancer

BackgroundIt is widely accepted that chronic inflammatory bowel diseases significantly higher a risk for colorectal cancer development. Among different types of treatments for patients with colon cancer, novel protein-based therapeutic strategies are considered.AIMTo explore the effect of human plasma alpha-1 antitrypsin (AAT) protein in the chemically induced mouse model of colorectal cancer.MethodsBALB/c mice with azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colorectal cancer (CAC), we intraperitoneally treated with commercial preparation of human plasma AAT (4 mg per mouse). Effects of this therapy were evaluated histologically, and by immunohistochemical and gene expression assays.ResultsWhen compared with non-treated controls, AOM/DSS mice receiving AAT therapy exhibited significantly longer colons, and less anal bleeding. Concurrently, AAT-treated mice had significantly fewer polyps, and lower numbers of large colon tumors. Immunohistochemical examinations of colon tissues showed significantly lower neutrophil counts, more granzyme B-positive but fewer MMP9 (gelatinase B)-positive cancer cells and lower numbers of apoptotic cells in mice receiving AAT therapy. The expression levels of IL4 were significantly higher while TNFA was slightly reduced in tumor tissues of AOM/DSS mice treated with AAT than in AOM/DSS mice.ConclusionHuman AAT is an acute phase protein with a broad-protease inhibitory and immunomodulatory activities used as a therapeutic for emphysema patients with inherited AAT deficiency. Our results are consistent with previous findings and support an idea that AAT alone and/or in combination with available anti-cancer therapies may represent a new personalized approach for patients with colitis-induced colon cancer.

Role of EphB2/ephrin-B1 signalling in the development and progression of obesity-associated colorectal cancer.

Obesity is a major problem worldwide and has been associated with colorectal cancer development, among other diseases. Ephrin receptors and ligands play an important role in the turnover of the intestinal mucosa and intestinal crypt compartmentalization. It has been hypothesised that obesity-induced inflammation affects ephrin signals, leading to carcinogenesis. Therefore, the aim of the present study was to assess the relationship between Eph-ephrin B signalling, obesity and obesity-associated colorectal cancer. An azoxymethane-induced obesity-associated cancer KKAy mouse model developed in our prior study was used. A total of 46 patients with consecutive colorectal cancer and 48 tumours were analysed. Immunohistological analyses were performed in mouse and human samples, and immunoreactive scores (IRS) were determined. KKAy mice were significantly more prone to cancer development compared with control C57/BL mice (2/15 in C57/BL vs. 10/10 in KKAy; P<0.001). TUNEL assay revealed a lower number of apoptotic cells in normal mucosa of KKAy mice (8.8% in C57/BL vs. 3.2% in KKAy; P<0.001) and obese patients (9.2% with BMI <25 vs. 3.6% with BMI ≥25; P=0.021). Immunohistological analysis revealed that ephrin-B1 was downregulated in normal mucosa from KKAy mice and obese patients (IRS, 2.86 with BMI <25 vs. 6.00 with BMI ≥25; P=0.002). Moreover, EphB2 was downregulated in tumours from KKAy mice and obese patients (IRS, 6.58 with BMI <25 vs. 3.83 with BMI ≥25; P<0.001). The distribution of infiltrated macrophages corresponded to the MCP-1 expression pattern in KKAy mice, and the number of macrophages was also significantly higher in those mice (36.3 in C57/BL vs. 120.0 in KKAy; P=0.029). The findings suggested that obesity results in disruption of EphB2/ephrin-B1 signalling, promoting colorectal cancer development and progression.

Preventive Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury after Modelling Stroke in a Rat.

Currently, the main fundamental and clinical interest for stroke therapy is focused on developing a neuroprotective treatment of a penumbra region within the therapeutic window. The development of treatments for ischemic stroke in at-risk patients is of particular interest. Preventive gene therapy may significantly reduce the negative consequences of ischemia-induced brain injury. In the present study, we suggest the approach of preventive gene therapy for stroke. Adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) or gene engineered umbilical cord blood mononuclear cells (UCB-MC) overexpressing recombinant VEGF, GDNF, and NCAM were intrathecally injected before distal occlusion of the middle cerebral artery in rats. Post-ischemic brain recovery was investigated 21 days after stroke modelling. Morphometric and immunofluorescent analysis revealed a reduction of infarction volume accompanied with a lower number of apoptotic cells and decreased expression of Hsp70 in the peri-infarct region in gene-treated animals. The lower immunopositive areas for astrocytes and microglial cells markers, higher number of oligodendrocytes and increased expression of synaptic proteins suggest the inhibition of astrogliosis, supporting the corresponding myelination and functional recovery of neurons in animals receiving preventive gene therapy. In this study, for the first time, we provide evidence of the beneficial effects of preventive triple gene therapy by an adenoviral- or UCB-MC-mediated intrathecal simultaneous delivery combination of vegf165, gdnf, and ncam1 on the preservation and recovery of the brain in rats with subsequent modelling of stroke.

Endoplasmic reticulum stress attenuation promotes bovine oocyte maturation in vitro

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

Effects of Low Frequency-Low Voltage Alternating Electric Current on Apoptosis Progression in Bioelectrical Reactor Biofilm.

Bioelectrochemical systems have undergone several modifications to promote the enzymes or pathways used to reduce the energy required for microbial metabolism. Changes in dominant bacteria, population, and growth rates occur when an electric current is applied intermittently. Applying electricity to bioelectrical reactor (BER) biofilms can either stimulate cells or lead to cell death; therefore, determining the applied voltage range that leads to viable and stimulated bacteria is crucial. We investigated the progression of apoptosis induced by a low frequency-low voltage alternating electric current (AC) in a BER biofilm and found that biofilms on carbon cloth (CC) and stainless steel (SS) 304 electrodes had pHzpc values of 8.67. The pHzpc of the biofilms increased by two compared to that of the inoculant bacteria mass. Furthermore, the Henderson–Hasselbalch equation reveals that the compositions of cell walls of the biofilms that formed on the CC and SS304 electrodes are very similar. In contrast, the CC and SS304 biofilms differ from the inoculant biomass without the influence of an AC field; this indicates that there are differences in the compositions of the cell walls in the present bacteria. Fourier transform infrared spectroscopy was used to compare spectra of the biofilms with that of the inoculation mass, and there were differences in shape and absorbance intensity, indicating variability in the composition, and quantity of each individual biofilm component. In addition, the dehydrogenase activity (DHA) content varied under different applied voltages; the highest DHA was obtained at 8 Vpp. A flow cytometry analysis showed a relatively low number of apoptotic cells (10.93 ± 5.19%) for the AC amplitudes studied. Thus, a low voltage-low frequency AC likely induces significant changes in bacterial metabolic activity but causes no significant change in their viability.

Radical change of apoptotic strategy following irradiation during later period of embryogenesis in medaka (Oryzias latipes)

Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.

Silymarin and celecoxib ameliorate experimental varicocele-induced pathogenesis: evidences for oxidative stress and inflammation inhibition.

The present study was done to investigate the ameliorative effect of silymarin (SMN) and celecoxib (CEL) on varicocele (VCL)-induced detrimental impact in testicular tissue. Mature Wistar rats were divided into control and test groups. Following VCL induction, the animals in test group were subdivided into non-treated VCL-induced, SMN-treated (50mg/kg, orally), CEL-treated (10mg/kg) and SMN + CEL-treated groups. Following 60days, testicular total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GSH-px), total thiol molecules (TTM), mRNA and protein levels of COX2 and mRNA level of iNos were analyzed. Moreover, the germinal cells apoptosis and mRNA damage were examined. Observations revealed that co-administration of SMN and CEL significantly (P < 0.05) up-regulated TAC, SOD, GSH-px and TTM levels and resulted in a remarkable (P < 0.05) reduction in iNos and COX2 expression, NO and MDA contents. The animals in SMN + CEL-treated group exhibited significantly (P < 0.05) lower number of apoptotic cells and cells with mRNA damage per one mm2. The SMN by up-regulating testicular TAC, SOD, GSH-px and TTM levels and the CEL by inhibiting COX2 and iNos expression as well as NO content could fairly ameliorate the VCL-decreased spermatogenesis.

Protective effects of melatonin on the invitro developmental competence of bovine oocytes.

The present study investigated the effects of melatonin on bovine oocyte maturation and subsequent embryonic development invitro. Results showed that the nuclear and cytoplasmic maturation, characterized by first polar body extrusion, normal distribution of cortical granules and mitochondria, as well as increased mitochondrial membrane potential, were significantly improved in 10-9 mol/L melatonin-treated oocytes. Melatonin supplementation reduced intracellular reactive oxygen species level and enhanced glutathione production. Meanwhile, the presence of melatonin (10-9 mol/L) during oocyte maturation resulted in a decreased early apoptotic rate in oocytes. After invitro fertilization, oocytes receiving melatonin supplementation exhibited a significantly higher blastocyst formation rate and yielded a markedly lower number of apoptotic cells. Mechanistic explorations showed that addition of 10-9 mol/L melatonin to invitro maturation media significantly attenuated the transcript level of caspase-3, while the expressions of BCL-2, XIAP, CAT and HSP70 were significantly reinforced in the resultant embryos. Taken together, melatonin ameliorates bovine oocyte maturation potential, and the beneficial effects can affect subsequent embryonic development. The protective role of melatonin may be due to its anti-apoptotic and anti-oxidative activities.

Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts.

Effects of adding different concentrations of melatonin (10-7 , 10-9 and 10-11 M) to maturation (Experiment 1; Control, IVM+10-7 , IVM+10-9 , IVM+10-11 ) and culture media (Experiment 2; Control, IVC+10-7 , IVC+10-9 , IVC+10-11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10-9 M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM+10-9 , IVC+10-9 , IVM/IVC+10-9 ). In Experiment 1, maturated oocytes from Control and IVM+10-9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC+10-7 (43.5%; 56.7%) and IVC+10-9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC+10-9 group compared to Control (3.8±0.6; 3.6% versus 6.1±0.6; 5.3%). In Experiment 3, the IVC+10-9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC+10-9 group (52.6%; 3.0±0.5 versus 46.0%; 5.4±1.0). The IVC+10-9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM+10-9 . Reactive oxygen species production was greater in the IVM/IVC+10-9 treatment group. In conclusion, the 10-9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.

Biodegradable functional polycarbonate micelles for controlled release of amphotericin B.

There is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.

Interleukin-18 Mediates Immune Responses to Campylobacter jejuni Infection in Gnotobiotic Mice.

BackgroundHuman Campylobacter jejuni infections are progressively rising worldwide. Information about the molecular mechanisms underlying campylobacteriosis, however, are limited. In the present study we investigated whether cytokines such as IL-23, IL-22 and IL-18, which share pivotal functions in host immunity, were involved in mediating intestinal and systemic immunopathological responses upon C. jejuni infection.Methodology/Principal FindingsTo assure stable infection, gnotobiotic (i.e. secondary abiotic) IL-23p19-/-, IL-22-/- and IL-18-/- mice were generated by broad-spectrum antibiotic treatment. Following peroral C. jejuni strain 81–176 infection, mice of all genotypes harbored comparably high pathogenic loads in their intestines. As compared to wildtype controls, however, IL-18-/- mice displayed less distinct C. jejuni induced sequelae as indicated by less pronounced large intestinal shrinkage and lower numbers of apoptotic cells in the colonic epithelial layer at day 8 postinfection (p.i.). Furthermore, lower colonic numbers of adaptive immune cells including regulatory T cells and B lymphocytes were accompanied by less distinct secretion of pro-inflammatory cytokines such as TNF and IFN-γ and lower IL-17A mRNA expression levels in colonic ex vivo biopsies of infected IL-18-/- as compared to wildtype mice. Upon C. jejuni infection, colonic IL-23p19 expression was up-regulated in IL-18-/- mice only, whereas IL-22 mRNA levels were lower in uninfected and infected IL-23p19-/- as well as infected IL-18-/- as compared to respective wildtype control mice. Remarkably, not only intestinal, but also systemic infection-induced immune responses were less pronounced in IL-18-/- mice as indicated by lower TNF, IFN-γ and IL-6 serum levels as compared to wildtype mice.Conclusion/SignificanceWe here show for the first time that IL-18 is essentially involved in mediating C. jejuni infection in the gnotobiotic mouse model. Future studies need to further unravel the underlying regulatory mechanisms orchestrating pathogen-host interaction.

Hypocellularity in the Murine Model for Down Syndrome Ts65Dn Is Not Affected by Adult Neurogenesis.

Down syndrome (DS) is caused by the presence of an extra copy of the chromosome 21 and it is the most common aneuploidy producing intellectual disability. Neural mechanisms underlying this alteration may include defects in the formation of neuronal networks, information processing and brain plasticity. The murine model for DS, Ts65Dn, presents reduced adult neurogenesis. This reduction has been suggested to underlie the hypocellularity of the hippocampus as well as the deficit in olfactory learning in the Ts65Dn mice. Similar alterations have also been observed in individuals with DS. To determine whether the impairment in adult neurogenesis is, in fact, responsible for the hypocellularity in the hippocampus and physiology of the olfactory bulb, we have analyzed cell proliferation and neuronal maturation in the two major adult neurogenic niches in the Ts656Dn mice: the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). Additionally, we carried out a study to determine the survival rate and phenotypic fate of newly generated cells in both regions, injecting 5′BrdU and sacrificing the mice 21 days later, and analyzing the number and phenotype of the remaining 5′BrdU-positive cells. We observed a reduction in the number of proliferating (Ki67 positive) cells and immature (doublecortin positive) neurons in the subgranular and SVZ of Ts65Dn mice, but we did not observe changes in the number of surviving cells or in their phenotype. These data correlated with a lower number of apoptotic cells (cleaved caspase 3 positive) in Ts65Dn. We conclude that although adult Ts65Dn mice have a lower number of proliferating cells, it is compensated by a lower level of cell death. This higher survival rate in Ts65Dn produces a final number of mature cells similar to controls. Therefore, the reduction of adult neurogenesis cannot be held responsible for the neuronal hypocellularity in the hippocampus or for the olfactory learning deficit of Ts65Dn mice.

Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells.

The potential application of GPNMB/OA as a therapeutic target for lung cancer will require a greater understanding of the impact of GPNMB/OA ectodomain (ECD) protein shedding into tumor tissues. Thus, in this work we characterized GPNMB/OA expression and extent of shedding of its ECD protein while evaluating the impact on lung cancer progression using three non-small cell lung cancer (NSCLC) cell lines: A549, SK-MES-1 and calu-6. We observed a direct correlation (R2 = 0.89) between GPNMB/OA expression on NSCLC cells and the extent of GPNMB/OA ECD protein shedding. Meanwhile, siRNA-mediated knockdown of GPNMB/OA in cancer cells significantly reduced GPNMB/OA ECD protein shedding, migration, invasion and adhesion to extracellular matrix materials. Also, exogenous treatment of cancer cells (expressing low GPNMB/OA) with recombinant GPNMB/OA protein (rOA) significantly facilitated cell invasion and migration, but the effects of rOA was negated by inclusion of a selective RGD peptide. Further studies in athymic (nu/nu) mice-bearing calu-6 showed that intratumoral supplementation with rOA effectively facilitated in vivo tumor growth as characterized by a high number of proliferating cells (Ki67 staining) coupled with a low number of apoptotic cells. Taken together, our results accentuate the relevance of GPNMB/OA ECD protein shedding to progression of lung cancer. Thus, strategies that suppress GPNMB/OA expression on lung cancer cells as well as negate shedding of GPNMB/OA ECD protein are worthy of consideration in lung cancer therapeutics.

Androgen levels and apoptosis in the testis during postnatal development of finasteride-treated male rat offspring.

The hormone-dependent events that occur throughout the first wave of spermatogenesis, such as the establishment of the number of Sertoli cells (SCs) and spermatogonial stem cells (SSCs) within the seminiferous cords and the setting up of the blood-testis barrier, are important for adult male fertility. Any changes in the T/DHT ratio can result in male subfertility or even infertility. In this study we aimed to evaluate effects of paternal exposure to 5-alpha reductase type 2 inhibitor, finasteride on litter size, androgen levels and germ cell apoptosis in male offspring during postnatal development. The subjects of the study were 7, 14, 21/22, 28, and 90-day-old Wistar male rats (F1:Fin) born from females fertilized by finasteride-treated rats. Offspring born from untreated parental animals were used as a control group (F1:Control). Animals and the collected testes were weighed, blood and intratesticular levels of T and DHT were measured by ELISA, and the apoptotic index of testicular cells was evaluated by TUNEL technique. We observed difficulties in obtaining male newborns from female rats fertilized by finasteride-treated male rats. In the F1:Fin rats, changes in the body and testes weights occurred, and a lower number of apoptotic cells was found during postnatal maturation of the seminiferous epithelium. Changes in androgen concentrations during the first spermatogenesis wave and adult life were also evident. Finasteride treatment of male adult rats may not only cause a decrease in the fertility of parental rats, but also could lead to incorrect, androgen-sensitive course of spermatogenesis in their offspring.

NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation.

Mesenchymal stromal cells (MSCs) are multipotent and can be derived from different adult tissues including fat. Our repeated attempts to produce long-term proliferative cultures of rat abdominal adipose stem cells (aASCs) under normal oxygen concentration (21%) were unsuccessful. We set to examine the events controlling this cytostasis of aASCs and found that it resulted from overproduction of reactive oxygen species (ROS) that led to apoptosis. ROS overproduction in aASCs was accompanied by increased expression of NOX1 but not of NOX2 or NOX4. NOX family members are an important source of intracellular ROS pointing to NOX1 involvement in ROS accumulation. This was verified when aASCs that were grown under 3% oxygen conditions expanded long term, displaying reduced NOX1 expression and decreased ROS accumulation. NOX1 involvement in aASC cytostasis was reaffirmed when cells that were expanded under normoxic conditions in the presence of a specific NOX1 inhibitor, ML171, demonstrated reduced ROS accumulation, reduced apoptosis and long-term expansion. aASC expansion arrest was accompanied also by a weak fat differentiation and migratory potential, which was enhanced by NOX1 inhibition. This suggests an inhibitory role for NOX1-induced ROS overproduction on aASCs, their fat differentiation and migratory potential. In contrast to aASCs, similar cells produced from subcutaneous fat were easily expanded in normoxic cultures, exhibiting low ROS concentrations, a low number of apoptotic cells and improved fat differentiation and migration. Taken together, our results show, for the first time, that NOX1-induced ROS accumulation halts ASC expansion and reduces their differentiation and migratory potential under normoxic conditions. Importantly, this phenotype comprises a tissue-specific signature as it was evident in aASCs but not in subcutaneous ASCs. NOX-induced ROS accumulation and cytokine production by fat are part of the metabolic syndrome. The similarity of this phenomenon to aASC phenotype may indicate that they arise from similar molecular mechanisms.

Synergistic effect of hyperbaric oxygen preconditioning and hydrogen-rich saline in ameliorating rat flap ischemia/reperfusion injury

Aim: This study was conducted to evaluate the synergistic effects of hyperbaric oxygen (HBO) preconditioning and hydrogen-rich saline (HRS) treatment on skin flap survival and apoptosis in a rat ischemia/reperfusion (IR) skin flap model. Methods: Male Sprague-Dawley rats were randomly divided into five groups: one sham surgery group (sham group) and four surgery groups (IR group, HBO group, HRS group, and HBO + HRS group). An extended epigastric adipocutaneous flap (6 cm × 9 cm) was raised over the abdomen in each animal of all five groups. The last four groups underwent 6 h of IR management and were treated, respectively, with normal saline, HBO, HRS (HRS, 0.8 mmol/L), or a combined approach (HBO and HRS). On the 3 rd postoperative day, flap survival rate and perfusion condition, apoptotic index, caspase-3 activity, protein expression of pASK1 and Bcl-2/Bax ratio, and Bcl-2 messenger RNA (mRNA) expression were assessed. Results: Prior studies have shown the protective effects of HBO and HRS, both of which have been associated with an increase in flap survival. Compared to the IR group, the flaps in the HBO, HRS, and HBO + HRS groups showed better perfusion and a larger survival area with a low number of apoptotic cells, low caspase-3 activity and pASK1 expression, and a high Bcl-2/Bax ratio and Bcl-2 mRNA expression. Of these groups, the HBO + HRS group showed the best flap survival. Conclusion: Both HBO and HRS treatments increase the rate of flap survival, while the synergistic application of HBO and HRS showed a higher survival rate as compared to individual treatments of each. The potential regulation of apoptosis with the use of these two modalities may improve skin flap survival.

253 EFFECTS OF ADDITION OF ALPHA-LINOLENIC ACID INTO MATURATION MEDIUM ON IN VITRO DEVELOPMENT AND EXPRESSION OF APOPTOSIS-RELATED GENES OF GOAT EMBRYOS

Fatty acids are important sources of energy for oocytes and embryos. In bovine, an increased level of rumen-inert fatty acids in the diet improved the developmental competence of oocytes to the blastocyst stage and also embryo quality. As described in our previous report, providing appropriate levels of α-linolenic acid (ALA) in maturation medium had beneficial effects on nuclear maturation and embryonic development in the goat. Considering these beneficial effects, here we have conducted experiments to evaluate whether addition of ALA to goat oocyte maturation medium can affect the quality of blastocyst and the transcription of apoptosis-related Bax, Bcl-2, and p53 genes. Ovaries were collected from goats, and cumulus-oocyte complexes (COC) were recovered by the slicing method. The COC were placed in maturation medium supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, oocytes from both the treatment (n = 113) and control (n = 104) groups were subjected to IVF followed by culture in CR1aa medium for 8 days under the conditions stated above. The cleavage and blastocyst rates were recorded at Days 3 and 8 of culture, respectively. To examine the effect of ALA on total cell number and apoptosis of the blastocyst cells, the blastocysts from 50 μM ALA-treated and control oocytes were stained with 4′,6-diamidino-2-phenylindole to count total cell number, and apoptotic cells in these blastocysts were detected with TUNEL assay. Blastocysts derived either from 50 μM ALA-treated oocytes or control oocytes were also evaluated for the expression of Bax, Bcl-2, and p53 genes. The cleavage and blastocyst rates were compared by chi-square analysis. Differentially expressed genes were analysed by 1-way ANOVA. A P-value of less than 0.05 was considered significant. Although cleavage rates after IVF were similar (P &gt; 0.05) between 50 μM ALA-treated (68.1%) and control (55.8%) groups, 50 μM ALA-treated oocytes produced more (25.7%) blastocysts than the control group (13.5%; P &lt; 0.05). Blastocysts derived from oocytes supplemented with 50 μM ALA not only had a greater (P &lt; 0.05) total cell number (115.2), but also a lower (P &lt; 0.05) number of apoptotic cells (3.1) compared with the control blastocysts (110.8 and 4.2, respectively). The relative transcript abundance of Bax and p53 was decreased (P &lt; 0.05) in blastocysts that originated from the 50 μM ALA group compared with control blastocysts. Furthermore, there was an increased (P &lt; 0.05) expression of Bcl-2 transcripts in blastocysts derived from the 50 μM ALA-treated oocytes compared with the control. In conclusion, our findings revealed that ALA-treated medium led to an improvement in blastocyst rate and quality as determined by greater total cell number, lower number of apoptotic cells, and altered expression of apoptosis-related genes.

Neonatal Livers: A Source for the Isolation of Good-Performing Hepatocytes for Cell Transplantation

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (β1-integrin, β-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug-metabolizing capability (cytochrome P450 and UDP-glucuronosyltransferase enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM(+) staining), which could also participate in regeneration of liver parenchyma after transplantation. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human hepatocyte transplantation applicability.

Loss of the mu opioid receptor induces strain-specific alterations in hippocampal neurogenesis and spatial learning

Alterations in hippocampal neurogenesis affect spatial learning, though, the relative contributions of cell proliferation and cell survival on this process are poorly understood. The current study utilized mu opioid receptor (MOR-1) knockout (KO) mice on two background strains, C57BL/6 and 129S6, to assess cell survival as well as determine the impact on spatial learning using the Morris water maze. These experiments were designed to extend prior work showing that both C57BL/6 and 129S6 MOR-1 KO mice have an increased number of proliferating cells in the dentate gyrus (DG) when compared to wild-type (WT) mice. The current study indicates that newly born neurons in the DG of C57BL/6 MOR-1 KO mice exhibit enhanced survival when compared to WT mice, while new neurons in the DG of 129S6 MOR-1 KO mice do not. In addition, C57BL/6 MOR-1 KO mice have a lower number of apoptotic cells in the DG compared to WT mice while, in contrast, 129S6 MOR-1 KO mice have a higher number of apoptotic cells in this region. These alterations collectively contribute to an increase in the granule cell number in the DG of C57BL/6 MOR-1 KO mice, while the total number of granule cells in 129S6 MOR-1 KO mice is unchanged. Thus, although C57BL/6 and 129S6 MOR-1 KO mice both exhibit increased cell proliferation in the DG, the impact of the MOR-1 mutation on cell survival differs between strains. Furthermore, the decrease in DG cell survival displayed by 129S6 MOR-1 KO mice is correlated with functional deficits in spatial learning, suggesting that MOR-1-dependent alterations in the survival of new neurons in the DG, and not MOR-1-dependent changes in proliferation of progenitor cells in the DG, is important for spatial learning.

Galactosylceramide Affects Tumorigenic and Metastatic Properties of Breast Cancer Cells as an Anti-Apoptotic Molecule

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ.