Effects of sevoflurane and its metabolite hexafluoroisopropanol on hypoxia/reoxygenation-induced injury and mitochondrial bioenergetics in murine cardiomyocytes.

Background: The volatile anaesthetic sevoflurane protects cardiac tissue from reoxygenation/reperfusion. Mitochondria play an essential role in conditioning. We aimed to investigate how sevoflurane and its primary metabolite hexafluoroisopropanol (HFIP) affect necrosis, apoptosis, and reactive oxygen species formation in cardiomyocytes upon hypoxia/reoxygenation injury. Moreover, we aimed to describe the similarities in the mode of action in a mitochondrial bioenergetics analysis. Methods: Murine cardiomyocytes were exposed to hypoxia (0.2% O2 for 6 h), followed by reoxygenation (air with 5% CO2 for 2 h) in the presence or absence sevoflurane 2.2% or HFIP 4 mM. Lactate dehydrogenase (LDH) release (necrosis), caspase activation (apoptosis), reactive oxygen species, mitochondrial membrane potential, and mitochondrial function (Seahorse XF analyser) were measured. Results: Hypoxia/reoxygenation increased cell death by 44% (+31 to +55%, P<0.001). Reoxygenation in the presence of sevoflurane 2.2% or HFIP 4 mM increased LDH release only by +18% (+6 to +30%) and 20% (+7 to +32%), respectively. Apoptosis and reactive oxygen species formation were attenuated by sevoflurane and HFIP. Mitochondrial bioenergetics analysis of the two substances was profoundly different. Sevoflurane did not influence oxygen consumption rate (OCR) or extracellular acidification rate (ECAR), whereas HFIP reduced OCR and increased ECAR, an effect similar to oligomycin, an adenosine triphosphate (ATP) synthase inhibitor. When blocking the metabolism of sevoflurane into HFIP, protective effects of sevoflurane - but not of HFIP - on LDH release and caspase were mitigated. Conclusion: Together, our data suggest that sevoflurane metabolism into HFIP plays an essential role in cardiomyocyte postconditioning after hypoxia/reoxygenation injury.

Entourage effect for phenolic compounds on production and metabolism of mammary epithelial cells.

Primary culture of mammary epithelial cells (MEC) was exposed to ethyl-acetate, chloroform and hexane extracts of Pistacia lentiscus (lentisk). The hexane extract contained mainly ethyl gallate whereas the chloroform extract contained mainly ethyl-gallate with smaller amount of gallic acid, and the ethyl-acetate extract contained mainly rutin, gallic acid and myricetin. Ethyl acetate extract increased secretion of protein and fat and improved mitochondrial activity. The enhancing effect on protein production was attributed to myricetin, one of the polyphenols in the ethyl-acetate extract whereas gallic acid did not affect protein production or secretion. Interestingly, exposure to the isolated polyphenols did not improve mitochondrial productivity and activity as effectively as exposure to the complete plant extract. The results indicated that polyphenols improve production of milk constituents by MEC, through different modes of action for different polyphenols suggesting an additive or even synergistic effect on production traits of mammary cells.

Carryover effect of atrazine and its metabolite-from treated bovine spermatozoa to the embryo's transcriptome†.

Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 µM) or DACT (1 or 10 µM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV-) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV-) and DACT (AV+, AV-) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV- vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.

Pistacia lentiscus extract enhances mammary epithelial cells' productivity by modulating their oxidative status.

We assessed the potential of phenolic compounds from Pistacia lentiscus (lentisk) to enhance production of milk constituents in bovine mammary epithelial cells (MEC). MEC were exposed to 0 (control), 1 or 10 ppm of polyphenols from lentisk ethanolic extract (PLEE) for 24 h. PLEE were absorbed by the MEC plasma membrane, but also penetrated the cell to accumulate in and around the nucleus. PLEE increased triglyceride content in the cell and its secretion to the medium, and significantly increased intracellular lipid droplet diameter. Compared to control, PLEE increased dose-dependently the lactose synthesis, secretion of whey proteins, and contents of casein. To evaluate mitochondrial activity under pro-oxidant load, MEC were preincubated with PLEE and exposed for 2 h to H2O2. Exposure to H2O2 increased the proportion of cells with impaired mitochondrial membrane potential twofold in controls, but not in PLEE-pre-treated cells. Accordingly, proton leakage was markedly decreased by PLEE, and coupling efficiency between the respiratory chain and ATP production was significantly enhanced. Thus, lentisk polyphenols divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control; alternatively, PLEE enables MEC to maintain energy and oxidative status under extreme metabolic rate required for milk production and secretion, and reduces the limitation on energy required to support production.

Hypoxia sensing by hepatic stellate cells leads to VEGF-dependent angiogenesis and may contribute to accelerated liver regeneration.

Portal vein ligation (PVL) induces liver growth prior to resection. Associating liver partition and portal vein ligation (PVL plus transection=ALPPS) or the addition of the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to PVL both accelerate growth via stabilization of HIF-α subunits. This study aims at clarifying the crosstalk of hepatocytes (HC), hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) in accelerated liver growth. In vivo, liver volume, HC proliferation, vascular density and HSC activation were assessed in PVL, ALPPS, PVL+DMOG and DMOG alone. Proliferation of HC, HSC and LSEC was determined under DMOG in vitro. Conditioned media experiments of DMOG-exposed cells were performed. ALPPS and PVL+DMOG accelerated liver growth and HC proliferation in comparison to PVL. DMOG alone did not induce HC proliferation, but led to increased vascular density, which was also observed in ALPPS and PVL+DMOG. Activated HSC were detected in ALPPS, PVL+DMOG and DMOG, again not in PVL. In vitro, DMOG had no proliferative effect on HC, but conditioned supernatant of DMOG-treated HSC induced VEGF-dependent proliferation of LSEC. Transcriptome analysis confirmed activation of proangiogenic factors in hypoxic HSC. Hypoxia signaling in HSC induces VEGF-dependent angiogenesis. HSC play a crucial role in the cellular crosstalk of rapid liver regeneration.

Sevoflurane sedation attenuates early cerebral oedema formation through stabilisation of the adherens junction protein beta catenin in a model of subarachnoid haemorrhage: A randomised animal study.

BACKGROUND: Severe neurological impairment is a problem after subarachnoid haemorrhage (SAH). Although volatile anaesthetics, such as sevoflurane, have demonstrated protective properties in many organs, their use in cerebral injury is controversial. Cerebral vasodilation may lead to increased intracranial pressure (ICP), but at the same time volatile anaesthetics are known to stabilise the SAH-injured endothelial barrier. OBJECTIVE: To test the effect of sevoflurane on ICP and blood-brain barrier function. DESIGN: Randomised study. PARTICIPANTS: One hundred male Wistar rats included, 96 analysed. INTERVENTIONS: SAH was induced by the endoluminal filament method under ketamine/xylazine anaesthesia. Fifteen minutes after sham surgery or induction of SAH, adult male Wistar rats were randomised to 4 h sedation with either propofol or sevoflurane. MAIN OUTCOME MEASURES: Mean arterial pressure (MAP), ICP, extravasation of water (small), Evan's blue (intermediate) and IgG (large molecule) were measured. Zonula occludens-1 (ZO-1) and beta-catenin (ß-catenin), as important representatives of tight and adherens junction proteins, were determined by western blot. RESULTS: Propofol and sevoflurane sedation did not affect MAP or ICP in SAH animals. Extravasation of small molecules was higher in SAH-propofol compared with SAH-sevoflurane animals (79.1 ±â€Š0.9 vs. 78.0 ±â€Š0.7%, P = 0.04). For intermediate and large molecules, no difference was detected (P = 0.6 and P = 0.2). Both membrane and cytosolic fractions of ZO-1 as well as membrane ß-catenin remained unaffected by the injury and type of sedation. Decreased cytosolic fraction of ß-catenin in propofol-SAH animals (59 ±â€Š15%) was found to reach values of sham animals (100%) in the presence of sevoflurane in SAH animals (89 ±â€Š21%; P = 0.04). CONCLUSION: This experiment demonstrates that low-dose short-term sevoflurane sedation after SAH in vivo did not affect ICP and MAP and at the same time may attenuate early brain oedema formation, potentially by preserving adherens junctions. TRIAL REGISTRATION: No 115/2014 Veterinäramt Zürich.

Mono(2-ethylhexyl) phthalate (MEHP) induces transcriptomic alterations in oocytes and their derived blastocysts.

Mono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. The current study examined whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. Bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A proteomic analysis was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes (Data are available via ProteomeXchange with identifier PXD012092). The study explores, for the first time, the risk associated with exposing oocytes to low concentration (i.e., environmentally relevant concentration) of MEHP to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality.

Sevoflurane Protects Hepatocytes From Ischemic Injury by Reducing Reactive Oxygen Species Signaling of Hepatic Stellate Cells: Translational Findings Based on a Clinical Trial.

BACKGROUND: Randomized controlled trials (RCTs) data demonstrate that sevoflurane postconditioning improves clinical outcomes of liver resection with inflow occlusion, presumably due to hepatocyte protection from ischemic injury. However, mechanisms remain unclear. This study examines liver biopsy samples obtained in an RCT of sevoflurane postconditioning to test the hypothesis that sevoflurane attenuates hepatocyte apoptosis. METHODS: Messenger ribonucleic acid (mRNA) of pro- and antiapoptotic regulators Bax and B-cell lymphoma 2 (Bcl2) was examined in hepatic biopsies obtained during the RCT. Hepatic stellate cells (HSCs) and hepatocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to evaluate the effect of sevoflurane postconditioning on apoptosis. The role of HSC as a potential apoptosis trigger in hepatocytes through the production of reactive oxygen species induced by H/R was explored by transferring supernatants from H/R-exposed HSC to hepatocytes as target cells. RESULTS: In patients of the RCT, the Bax/Bcl2 mRNA ratio in liver tissue was markedly decreased in the sevoflurane arm (25% ± 21% reduction; P = .001). In vitro, H/R increased reactive oxygen species production in HSC by 33% ± 16% (P = .025), while it was abolished in the presence of sevoflurane (P < .001). In hepatocytes, caspase was minimally activated by H/R. However, incubation of hepatocytes with supernatants of HSC, previously exposed to H/R, increased caspase activity by 28% ± 13% (P < .001). When exposed to supernatants from HSC undergoing sevoflurane postconditioning, caspase activation in hepatocytes was reduced by 20% ± 9% (P < .001), similarly to the sevoflurane effect on the BAX/Bcl2 mRNA ratio in the liver samples. CONCLUSIONS: The study shows that sevoflurane postconditioning affects apoptosis of hepatocytes after ischemia-reperfusion injury in patients. It also demonstrates that HSC may be the effector cells of sevoflurane protection.

Aflatoxin B1 impairs sperm quality and fertilization competence.

Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation, fertilization competence and early embryonic development. Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro for 4h with 0, 0.1, 1, 10 and 100µM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes. Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔYm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42h postfertilization, however, the proportion of embryos that developed to blastocysts, 7days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted.

Sevoflurane protects rat brain endothelial barrier structure and function after hypoxia-reoxygenation injury.

BACKGROUND: After cerebral injury blood-brain barrier disruption significantly impairs brain homeostasis. Volatile anesthetics have been shown to be protective in ischemia-reperfusion injury scenarios. Their impact on brain endothelial cells after hypoxia-reoxygenation (H/R) has not yet been studied in detail. METHODS: Rat brain endothelial cells (RBE4) were exposed to severe hypoxia and reoxygenated in air in the presence or absence of sevoflurane. Changes in dextran permeability and architecture of the cellular junctional proteins ZO-1 and ß-catenin were measured. To determine necrosis and apoptosis rate DNA content, LDH release and caspase activity were quantified. The role of vascular endothelial growth factor (VEGF) as an inflammatory mediator increasing vascular permeability was assessed. At the same time, it was evaluated if sevoflurane effects are mediated through VEGF. Results were analyzed by unpaired t-tests or one way-analysis of variance followed by Bonferroni's correction. RESULTS: H/R led to a 172% increase in permeability (p<0.001), cell swelling and qualitatively but not quantitatively modified expression of ZO-1, ß-catenin and F-actin. In the presence of sevoflurane during reoxygenation, barrier function improved by 96% (p = 0.042) in parallel to a decrease of the cell size and less re-arranged junction proteins and F-actin. Sevoflurane-induced improvement of the barrier function could not be explained on the level of necrosis or apoptosis as they remained unchanged independent of the presence or absence of the volatile anesthetic. Increased expression of VEGF after H/R was attenuated by sevoflurane by 34% (p = 0.004). Barrier protection provided by sevoflurane was similar to the application of a blocking VEGF-antibody. Furthermore, the protective effect of sevoflurane was abolished in the presence of recombinant VEGF. CONCLUSIONS: In H/R-induced rat brain endothelial cell injury sevoflurane maintains endothelial barrier function through downregulation of VEGF, which is a key player not only in mediating injury, but also with regard to the protective effect of sevoflurane.

Low level of mono(2-ethylhexyl) phthalate reduces oocyte developmental competence in association with impaired gene expression.

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are reproductive toxicants. However, disruptive effects of MEHP at low concentrations on the oocyte and developing blastocyst are unknown. Previously, we detected low levels of MEHP in follicular fluid aspirated from DEHP-treated cows associated with reduced estradiol levels. Moreover, the MEHP concentrations found were similar to those reported for follicular fluid aspirated from women who have undergone IVF cycles. In the current study, we used an in vitro embryo production model to examine the effect of MEHP at low levels on oocyte developmental competence. We set up several experiments to mimic the follicular fluid content, i.e., low MEHP level and low estradiol. For all experiments, cumulus oocyte complexes (COCs) were aspirated from bovine ovaries, then matured in vitro in standard oocyte maturation medium (OMM) supplemented with: MEHP at a range levels (20-1000nM) or with estradiol at a range levels (0-2000ng/ml). Then, oocytes were fertilized and cultured for an additional 7days to allow blastocyst development. Findings revealed that MEHP at low levels impairs oocyte developmental competence in a dose-dependent manner (P<0.05) and that estradiol by itself does not impair it. Accordingly, in another set of experiments, COCs were matured in vitro with MEHP at two choosen concentrations (20 or 1000nM) with or without estradiol, fertilized and cultured for 7days. Samples of mature oocytes and their derived blastocysts were subjected to quantitative real-time PCR to examine the profiles of selected genes (CYC1, MT-CO1, ATP5B, POU5F1, SOX2 and DNMT3b). Maturation of COCs with MEHP (20 or 1000nM) affected gene expression in the mature oocyte. Maturation of COCs with MEHP (20 or 1000nM) in the absence of estradiol reduced oocyte developmental competence (P<0.05). A differential carryover effect on transcript abundance was recorded in blastocysts developed from MEHP-treated oocytes. In the presence of estradiol, increased expression was recorded for CYC1, ATP5B, SOX2 and DNMT3b. In the absence of estradiol, decreased expression was recorded, with a significant effect for 1000nM MEHP (P<0.05). Taken together, the findings suggest that low levels of phthalate must be taken into consideration in risk assessments.

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality.

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing-thawing.

Comparing the effects of heat stress and mastitis on ovarian function in lactating cows: basic and applied aspects.

Reduced reproductive performance of lactating cows is strongly associated with environmental and pathogenic stressors. This review summarizes the most recent knowledge on the effects of acute or chronic heat stress (HS) and acute or chronic intramammary infection (IMI) on ovarian function. It also offers various approaches for improving the fertility of cows under chronic HS or IMI. Comparing the 2 stressors reveals a few similarities in the mode of alteration in the hypothalamus-pituitary-ovarian axis, in particular, in the follicle and its enclosed oocyte. Both HS and IMI cause a reduction in the preovulatory LH surge, with a pronounced effect in cows with IMI, and consequently, ovulation is being delayed or inhibited. Both stresses induce changes in follicular growth dynamics, reduce follicular steroidogenesis, and disrupt follicular dominance. Unlike their effects on follicular function, the effects of mastitis and HS on corpus luteum (CL) function are debatable. Under chronic summer thermal stress, several, but not all, studies show reduced progesterone secretion by the CL. Subclinical mastitis does not affect CL function, whereas the effect of clinical mastitis is controversial; some show a reduction in progesterone, whereas others do not. Both stresses have been found to impair cytoplasmic and nuclear maturation of oocytes, associated with reduced embryonic development. These findings have provided insights into the mechanism by which HS and IMI compromise fertility, which enable developing new strategies to mitigate these effects. For instance, treatment with GnRH and PGF2α to induce follicular turnover successfully improved conception rate in subpopulations of HS cows during the summer, in particular, primiparous cows and cows with high BCS. The "Ovsynch" program, also based on the use of GnRH and PGF2α, has been shown to improve conception rate of subclinical mastitic cows, most likely due to better synchronization of timing of ovulation with that of AI. Supplementing progesterone after AI improves conception rate of HS cows, particularly those with postpartum uterine disease and low BCS. It should be noted that similarities between the 2 stressors do not necessarily suggest a shared mechanism. Although not clear enough, an additive deleterious effects of HS and IMI on reproduction is suggested.

Inflammatory Kidney and Liver Tissue Response to Different Hydroxyethylstarch (HES) Preparations in a Rat Model of Early Sepsis.

BACKGROUND: Tissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model. MATERIAL AND METHODS: Adult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer's lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants. RESULTS: Cytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression. CONCLUSION: We identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.

Progressive motility - a potential predictive parameter for semen fertilization capacity in bovines.

We examined the association between progressive motility of spermatozoa and in vitro fertilization (IVF) competence of bovine ejaculates. Fresh semen was evaluated using a computerized sperm quality analyzer for bulls using progressive motility as the primary parameter. Ejaculates with high progressive motility (HPM; >81%) were compared with those with low progressive motility (LPM; 0.05). Examination of sperm morphology revealed a higher proportion of spermatozoa with abnormal morphology (P < 0.01) in LPM versus HPM ejaculates, the predominant abnormal feature being a bent tail (P < 0.05). Sperm viability, acrosome integrity and DNA fragmentation did not differ between HPM and LPM samples. Mitochondrial membrane potential was higher (P < 0.01) in HPM versus LPM semen. Zinc concentrations in the seminal plasma correlated with progressive motility (R2 = 0.463, P = 0.03). In addition, representative ejaculates from HPM and LPM groups were cryopreserved in straws and used for IVF. The proportions of embryos cleaved to 2- and 4-cell stages (88.1 ± 1.1 versus 80.5 ± 1.7, P = 0.001) and developed to blastocysts (33.5 ± 1.6 versus 23.5 ± 2.2, P = 0.026) were higher for HPM than LPM semen. The total cell number of embryos and blastocyst apoptotic index did not differ between groups. Although sperm progressive motility is associated with IVF competence, further examination is required to determine whether progressive motility can serve as a predictor of semen fertilization capacity in vivo.

Uptake of ferromagnetic carbon-encapsulated metal nanoparticles in endothelial cells: influence of shear stress and endothelial activation.

AIM: Magnetic field guided drug targeting holds promise for more effective cancer treatment. Intravascular application of magnetic nanoparticles, however, bears the risk of potentially important, yet poorly understood side effects, such as off-target accumulation in endothelial cells. MATERIALS & METHODS: Here, we investigated the influence of shear stress (0-3.22 dyn/cm(2)), exposure time (5-30 min) and endothelial activation on the uptake of ferromagnetic carbon-encapsulated iron carbide nanomagnets into endothelial cells in an in vitro flow cell model. RESULTS: We found that even moderate shear stresses typically encountered in the venous system strongly reduce particle uptake compared with static conditions. Interestingly, a pronounced particle uptake was observed in inflamed endothelial cells. CONCLUSION: This study highlights the importance of relevant exposure scenarios accounting for physiological conditions when studying particle-cell interactions as, for example, shear stress and endothelial activation are major determinants of particle uptake. Such considerations are of particular importance with regard to successful translation of in vitro findings into (pre-)clinical end points.

ACTH administration during formation of preovulatory follicles impairs steroidogenesis and angiogenesis in association with ovulation failure in lactating cows.

Ovulation failure, follicular persistence, and formation of follicular cysts are known to impair dairy cow fertility. Although the underlying mechanism is not entirely clear, stress-induced alteration in adrenal hormone secretion can cause these ovarian pathologies. Six synchronized lactating cows were scanned daily by ultrasound, and plasma samples were taken throughout the estrous cycle. Treatment cows (n = 3) were administered with ACTH analog every 12 h from day 15 to day 21 of the cycle to induce formation of follicular cysts. Ovaries were collected at the slaughterhouse on day 23 of the cycle before appearance of follicular pathologies. Control cows (n = 3) were administered placebo, resynchronized, and administered PGF2α on day 6 of the new cycle to induce development of a preovulatory follicle. Follicular fluid was aspirated from the preovulatory follicles of each group to determine their steroid milieu. Slices were taken from the follicular wall for total messenger (m) RNA isolation and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Administration of ACTH increased (P < 0.02) plasma cortisol concentration and reduced (P < 0.01) milk production. Androstenedione and estradiol concentrations in the follicular fluids were lower (P < 0.05) in ACTH-treated follicles than those in controls. The mRNA expression of luteinizing hormone receptor, 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase (P450arom), and cytochrome P450 17α-hydroxylase (P450c17) were lower (P < 0.02) in the ACTH-treated vs control cows. On the other hand, the expression of steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage did not differ between groups. In addition, mRNA expression of vascular endothelial growth factor (VEGF)120 and VEGF164 was higher (P < 0.01) in control than in ACTH-treated follicles, but that for angiopoietin-1 and 2 did not differ between groups. Findings indicated that ACTH administration throughout preovulatory follicle development alters follicular steroidogenesis in association with impaired angiogenesis. Such alterations might explain, in part, the mechanism underlying ovulation failure and the formation of persistent or cystic follicles under stress.

Insight into the beneficial immunomodulatory mechanism of the sevoflurane metabolite hexafluoro-2-propanol in a rat model of endotoxaemia.

Volatile anaesthetics such as sevoflurane attenuate inflammatory processes, thereby impacting patient outcome significantly. Their inhalative administration is, however, strictly limited to controlled environments such as operating theatres, and thus an intravenously injectable immunomodulatory drug would offer distinct advantages. As protective effects of volatile anaesthetics have been associated with the presence of trifluorinated carbon groups in their basic structure, in this study we investigated the water-soluble sevoflurane metabolite hexafluoro-2-propanol (HFIP) as a potential immunomodulatory drug in a rat model of endotoxic shock. Male Wistar rats were subjected to intravenous lipopolysaccharide (LPS) and thereafter were treated with HFIP. Plasma and tissue inflammatory mediators, neutrophil invasion, tissue damage and haemodynamic stability were the dedicated end-points. In an endotoxin-induced endothelial cell injury model, underlying mechanisms were elucidated using gene expression and gene reporter analyses. HFIP reduced the systemic inflammatory response significantly and decreased endotoxin-induced tissue damage. Additionally, the LPS-provoked drop in blood pressure of animals was resolved by HFIP treatment. Pathway analysis revealed that the observed attenuation of the inflammatory process was associated with reduced nuclear factor kappa B (NF-κΒ) activation and suppression of its dependent transcripts. Taken together, intravenous administration of HFIP exerts promising immunomodulatory effects in endotoxaemic rats. The possibility of intravenous administration would overcome limitations of volatile anaesthetics, and thus HFIP might therefore represent an interesting future drug candidate for states of severe inflammation.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Cellular and molecular mechanisms of heat stress related to bovine ovarian function.

In light of the intensive genetic selection for high milk production and the onset of global warming, it seems that the reduced fertility of lactating cows during the summer will worsen in coming years. Although not entirely clear, the mechanism appears to be multifactorial in nature. It includes alterations in follicular development, depression of follicular dominance, and impairment of steroidogenesis and gonadotropin secretion. Heat-induced perturbations in the physiology of the follicle-enclosed oocyte have also been documented, expressed by impaired cleavage rate and reduced developmental competence. With respect to the oocyte, alterations include an increase in PUFA in the membrane, reactive oxygen species, ceramide formation and caspase activity, and induction of apoptosis via the sphingomyelin and/or mitochondrial pathways. New insight into cellular and molecular alterations has revealed that heat induces perturbations in both nuclear and cytoplasmic maturation events, such as resumption of meiosis, metaphase II plate formation, cytoskeleton rearrangement, and translocation of cortical granules. Alterations in mitochondrial distribution (i.e., low proportion of category I mitochondria) and function (i.e., low membrane potential) have recently been reported for oocytes collected during the summer. These were associated with impaired expression of both nuclear (succinate dehydrogenase subunit [SDHD], adenosine triphosphate [ATP] synthase subunit beta [ATP5B]), mitochondrially NADH dehydrogenase subunit 2 (ND2), and mitochondiral (cytochrome c oxidase subunit II [MT-CO2] and cytochrome b [MT-CYB]) genes that are crucial in the mitochondrial respiratory chain. In addition, season-induced alteration in the stored maternal mRNA has been documented, expressed by reduced transcript levels (oocyte maturation factor MOS [C-MOS], growth differentiation factor 9 [GDF9], POU domain class 5 transcription factor 1 [POU5F1], and glyceraldehyde-3-phosphate dehydrogenase []GAPDH) in metaphase II stage oocytes and embryos before (i.e., 2-, 4-, and 8-cell stages) and after (i.e., 8- to 16-cell stage) embryonic genome activation. Taken together, the findings indicate an association between cellular and molecular modifications and reduced developmental competence during the hot seasons. Such knowledge is essential for the development of new approaches to cope with this unsolved problem.