Sulforaphane improves syncytiotrophoblast mitochondrial function after in vitro hypoxic and superoxide injury.

INTRODUCTION: Placental mitochondrial dysfunction contributes to the oxidative stress that underlies preeclampsia. Here, we assessed whether sulforaphane (SFN) could improve syncytiotrophoblast mitochondrial function after in vitro hypoxic and superoxide injury. METHODS: Placental cytotrophoblasts were isolated from healthy term placentae (n = 12) and incubated for 48 h in 8% O2 ± 1 µM SFN before acute (4hrs) or chronic (24hrs) hypoxic (1% O2), or superoxide (xanthine/xanthine oxidase) injury. Cytotrophoblasts were also isolated from preeclamptic placentae (n = 5) and cultured in 8% O2 ± 1 µM SFN. Mitochondrial respiration was measured using the Seahorse MitoStress XF assay. Cells were stained with mitotracker red to assess mitochondrial membrane health and mitochondrial gene expression assessed using RT-qPCR. RESULTS: SFN prevented significant reductions in syncytiotrophoblast mitochondrial maximal respiration, spare respiratory capacity, basal respiration and ATP production following acute hypoxia. Chronic hypoxia only reduced maximal and spare respiratory capacity. SFN prevented these negative changes and increased respiration overall. Alternatively, acute superoxide injury significantly increased mitochondrial maximal respiration and spare respiratory capacity. SFN treatment further increased basal respiration following superoxide injury and prevented significant decreases in ATP production and coupling efficiency. In preeclamptic placentae, SFN significantly increased mitochondrial maximal respiration, spare respiratory capacity, basal respiration and ATP production, and decreased proton leak. SFN up-regulated mRNA expression of mitochondrial complexes and corrected an up-regulation in fission gene expression observed after hypoxic-superoxide injury. Finally, preliminary results suggest SFN prevented hypoxia-induced impairment of mitochondrial membrane structure. DISCUSSION: SFN mitigated hypoxia and superoxide induced changes to syncytiotrophoblast mitochondrial function in vitro, and improved mitochondrial respiration in trophoblast cells from preeclamptic placentae.

Description of a method for inducing fetal growth restriction in the spiny mouse.

Intrauterine or fetal growth restriction (IUGR) is a major complication of pregnancy and leads to significant perinatal morbidities and mortality. Typically, induction of IUGR in animals involves the complete occlusion or ablation of vessels to the uterus or placenta, acutely impairing blood flow and fetal growth, usually with high fetal loss. We aimed to produce a model of reduced fetal growth in the spiny mouse with minimal fetal loss. At 27 days gestational age (term is 38-39 days), a piece of silastic tubing was placed around the left uterine artery to prevent the further increase of uterine blood flow with advancing gestation to induce IUGR (occluded). Controls were generated from sham surgeries without placement of the tubing. Dams were humanely euthanized at 37 days gestational age and all fetuses and placentas were weighed and collected. Of the 17 dams that underwent surgery, 15 carried their pregnancies to 37 days gestational age and 95% of fetuses survived to this time. The difference in fetal body weight between occluded and control was ~21% for fetuses in the left uterus side: there were no differences for fetuses in the right uterus side. Offspring from the occluded group had significantly lower brain, liver, lung, kidney and carcass weights compared with shams. Preventing the gestation-related increase of uterine blood flow induced significant growth restriction in the fetal spiny mouse, with minimal fetal loss. This technique could be readily adapted for other small animal.

A review of fundamental principles for animal models of DOHaD research: an Australian perspective.

Epidemiology formed the basis of 'the Barker hypothesis', the concept of 'developmental programming' and today's discipline of the Developmental Origins of Health and Disease (DOHaD). Animal experimentation provided proof of the underlying concepts, and continues to generate knowledge of underlying mechanisms. Interventions in humans, based on DOHaD principles, will be informed by experiments in animals. As knowledge in this discipline has accumulated, from studies of humans and other animals, the complexity of interactions between genome, environment and epigenetics, has been revealed. The vast nature of programming stimuli and breadth of effects is becoming known. As a result of our accumulating knowledge we now appreciate the impact of many variables that contribute to programmed outcomes. To guide further animal research in this field, the Australia and New Zealand DOHaD society (ANZ DOHaD) Animals Models of DOHaD Research Working Group convened at the 2nd Annual ANZ DOHaD Congress in Melbourne, Australia in April 2015. This review summarizes the contributions of animal research to the understanding of DOHaD, and makes recommendations for the design and conduct of animal experiments to maximize relevance, reproducibility and translation of knowledge into improving health and well-being.

Maternal creatine in pregnancy: a retrospective cohort study.

OBJECTIVE: To estimate creatine concentrations in maternal plasma and urine, and establish relationships with maternal characteristics, diet and fetal growth. DESIGN: Retrospective cohort study. SETTING: Lyell McEwin Hospital, Adelaide, Australia. POPULATION: A biobank of plasma and urine samples collected at 13, 18, 30 and 36 weeks' gestation from 287 pregnant women from a prospective cohort of asthmatic and non-asthmatic women. METHODS: Creatine was measured by enzymatic analysis. Change in creatine over pregnancy was assessed using the Friedman test. Linear mixed models regression was used to determine associations between maternal factors and diet with creatine across pregnancy and between creatine with indices of fetal growth at birth. MAIN OUTCOME MEASURES: Maternal creatine concentrations, associations between maternal factors and creatine and between creatine and fetal growth parameters. RESULTS: Maternal smoking, body mass index, asthma and socio-economic status were positively and parity negatively associated with maternal plasma and/or urine creatine. Maternal urine creatine concentration was positively associated with birthweight centile and birth length. After adjustment, each µmol/l increase in maternal urinary creatine was associated with a 1.23 (95% CI 0.44-2.02) unit increase in birthweight centile and a 0.11-cm (95% CI 0.03-0.2) increase in birth length. CONCLUSIONS: Maternal factors and fetal growth measures are associated with maternal plasma and urine creatine concentrations. TWEETABLE ABSTRACT: Maternal creatine is altered by pregnancy; fetal growth measures are associated with maternal creatine concentrations.

A randomised controlled trial comparing StarClose and AngioSeal vascular closure devices in a district general hospital--the SCOAST study.

AIMS: AngioSeal and StarClose are vascular closure devices (VCDs) that can be used following cardiac catheterisation via the femoral artery to achieve haemostasis. Both devices have been demonstrated to be superior to conventional manual pressure, which reduce time to haemostasis and time to patient ambulation. We sought to compare these devices in a prospective, randomised trial. METHODS: Patients undergoing elective coronary angiography were randomised to receive either AngioSeal VIP or StarClose VCD with immediate postprocedure mobilisation. Bruising was recorded at 30 min, 60 min and at 1 week postprocedure. Patient satisfaction surveys were taken at 1 h and 1 week postprocedure. Complications for both groups were compared. RESULTS: Four hundred and one patients were included. Arteriotomy closure was achieved in 144 of 208 (69.2%) patients randomised to AngioSeal and 134 of 193 (69.3%) patients randomised to StarClose (p = ns). There was no significant bruising in either group at either 30 or 60 min postprocedure. However, at 1 week, there was significantly more bruising in the AngioSeal group than the StarClose group (63.1 vs. 38.5cm2, p = 0.02). Patient satisfaction and pain perception with the procedure at closure were not significantly different between the groups. Deployment success and instant mobilisation rates were significantly lower for junior, as compared with senior, operators. CONCLUSION: Achievement of haemostasis is similar with both AngioSeal and StarClose. The StarClose vascular closure device results in significantly less bruising at 1 week postprocedure as compared with AngioSeal, with no significant differences in complication rates. Patients' pain perception and satisfaction are similar with both VCDs.

Predicting mortality and rehospitalization in heart failure patients with home monitoring--the Home CARE pilot study.

The increasing worldwide prevalence of heart failure is associated with numerous and protracted hospital admissions. The multidisciplinary team approach together with telemonitoring aims at reducing the number of rehospitalizations, length of hospital stay, and mortality rates. Novel cardiac resynchronization therapy (CRT) devices have a Home Monitoring capability, offering wireless, everyday transfer of the essential status and therapy data to the attending physician. The transmitted data include potential predictors of death or hospitalization, such as the onset of atrial and ventricular arrhythmias, duration of physical activity, mean heart rates over 24 h and at rest, percentage of CRT delivered, and lead impedances. We present here interim results of the prospective, longitudinal, multicenter Home CARE Phase 0 study, conducted in 123 patients (age: 67+/-9 years, 83% male) with clinical indication for CRT. Twenty-nine patients (24%) received a CRT pacemaker, 52 (42%) a prophylactic implantable cardioverter defibrillator (ICD), and 42 (34%) had other ICD indications. All devices have an integrated Home Monitoring feature. In a mean (interim) follow-up period of 3 months (9194 observational days), 11 unplanned rehospitalizations of cardiovascular etiology and 9 deaths occurred. In 70% of the rehospitalization events, the retrospective analysis of transmitted data via Home Monitoring revealed an increase in mean heart rate at rest and in mean heart rate over 24 h within 7 days preceding hospitalization. A decrease in the percentage of CRT was observed in 43% and a reduction in the patients' daily activity in 30% of rehospitalized patients. These interim findings suggest that Home Monitoring data may predict events leading to hospitalization and encourage further research.

Role of resynchronisation therapy and implantable cardioverter defibrillators in heart failure.

The worldwide prevalence of heart failure is increasing in part because of an aging population. In the developed world, heart failure affects 1%-2% of the general population, accounting for 5% of adult hospital admissions. There is now convincing evidence supporting the beneficial effects of cardiac resynchronisation therapy for the treatment of heart failure. Numerous observational studies, as well as a series of randomised controlled trials, have shown the safety, efficacy, and long term benefits for patients with chronic systolic heart failure who have broad QRS complexes and refractory symptoms despite optimal medical therapy. These studies have consistently found statistically significant improvements in quality of life, New York Heart Association functional class, exercise tolerance, and left ventricular reverse remodelling. Recent evidence suggests that the benefit may at least in part be because of a reduction in mechanical dysynchrony.

The role of cardiac resynchronization therapy in heart failure.

The worldwide prevalence of heart failure is increasing in part due to an ageing population. In the developed world, heart failure affects 1-2% of the general population, accounting for 5% of adult hospital admissions. There is now convincing evidence supporting the beneficial effects of cardiac resynchronization therapy for the treatment of heart failure. Numerous observational studies, as well as a series of randomised controlled trials, have demonstrated the safety, efficacy, and long-term benefits for patients with chronic systolic heart failure who have broad QRS complexes and refractory symptoms despite optimal medical therapy. These studies have consistently demonstrated statistically significant improvements in quality of life, NYHA functional class, exercise tolerance, and left ventricular reverse remodeling. Recent evidence suggests that the benefit may at least in part be due to a reduction in mechanical dyssynchrony.

Biventricular pacing in heart failure.

Optimal pharmacological therapy for heart failure improves patients' prognosis and symptoms. Despite this, the long-term prognosis for these patients is very poor and symptoms are debilitating. Biventricular pacing, or resynchronization therapy, should be considered for patients who remain symptomatic despite optimal therapy and have evidence of dyssynchrony.

Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/reperfusion injury.

The cardioprotective efficacy of the pyrazolinone-piperidine dipeptide growth hormone secretagogue (GHS) CP-424,391 was studied in an in vivo rabbit model of ischemia and reperfusion. CP-424,391 was administered at 25 mg/kg p.o. x 7 days. Ischemia was induced by left coronary artery occlusion for 30 min, after which the heart was reperfused for 2 h. At the end of reperfusion, animals were euthanized and the infarct size was determined. The area at risk of infarct was not different between the control (45.8+/-3.7%, n=6) and CP-424,391-treated groups (36.9+/-4.3%, n=11). The infarct size of the control animals was 49.5+/-7.1% and was significantly (P<0.05) lower in the CP-424,391-treated group (infarct size=17.3+/-3.0). There was a trend, albeit not significant, for the left ventricular function to recover to a greater extent in CP-424,391-treated rabbits. Thus, the treatment of rabbits for 7 days with CP-424,391 was cardioprotective against ischemia/reperfusion injury.

A novel sodium-hydrogen exchanger isoform-1 inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo.

The cardioprotective efficacy of zoniporide (CP-597,396), a novel, potent, and selective inhibitor of the sodium-hydrogen exchanger isoform 1 (NHE-1), was evaluated both in vitro and in vivo using rabbit models of myocardial ischemia-reperfusion injury. In these models, myocardial injury was elicited with 30 min of regional ischemia and 120 min of reperfusion. Zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) of 0.25 nM) in the isolated heart (Langendorff) and reduced infarct size by 83% (50 nM). This compound was 2.5- to 20-fold more potent than either eniporide or cariporide (EC(50) of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide (58% reduction in infarct size). In open-chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) of 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (maximum inhibition 93%). Furthermore, zoniporide did not cause any in vivo hemodynamic (mean arterial pressure, heart rate, rate pressure product) changes. Zoniporide represents a novel class of potent NHE-1 inhibitors with potential utility for providing clinical cardioprotection.

Polychlorinated biphenyls and organochlorine pesticides in EELS (Anguilla anguilla L.) from Welsh rivers.

Organochlorine concentrations in the muscle tissues of eels at 41 freshwater sites across Wales were surveyed in 1993. Positive but weak relationships were detected between eel age or length and the concentration of some organochlorines. Isomers of HCH were below detection at most sites. Dieldrin was widespread at 10-100 microg kt(-1) wet weight, despite its ban in sheep dip in 1989. Total DDT residue concentrations averaged 73 microg kg(-1). Total PCB burdens expressed as Arochlor 1260, were > 50 microg kg(-1) at 76% of sites and > 100 microg kg(-1) at 46% of sites. The range of concentrations was comparable with other UK data. Rural sites had relatively low levels of PCBs, highest contamination occurring in the lower reaches of industrialised catchments. The inferences for environmental effects are discussed.

CP-71,362: a pentapeptide renin inhibitor selective for the canine enzyme.

Most renin inhibitors are primate-specific. In the present paper, we describe the effects of CP-71,362, a pentapeptide which preferentially inhibits canine (and to a lesser extent, rat) plasma renin. Vs. the canine enzyme, its affinity (IC50 = 3.3 x 10(-12) M) is 1000x greater than for rat renin (IC50 = 3.3 x 10(-9) M), and 1000x greater than for human (IC50 = 2.3 x 10(-8) M), cynomolgus monkey (IC50 = 1.6 x 10(-8) M), or guinea pig (IC50 = 5.2 x 10(-8) M) enzyme. In anesthetized, sodium-depleted dogs, intravenous infusion of CP-71,362 (ED50 = 1.1 micrograms/kg/min) resulted in dose-dependent decreases (up to -35 mm Hg) in mean arterial pressure (MAP). The maximum fall in MAP was equivalent to that produced by i.v. captopril (5 mg/kg). Similar falls in MAP were observed in conscious sodium-depleted SHR (ED50 = 5 micrograms/kg/min). Via bolus injection, the action of CP-71,362 was relatively brief in dog, guinea pig, and SHR. We conclude that CP-71,362 is a potent canine/rat renin inhibitor and causes profound MAP lowering in these species.

Vasoconstrictor action of angiotensin I-convertase and the synthetic substrate (Pro11,D-Ala12)-angiotensin I.

A chymase (also referred to as angiotensin I-convertase) specific for the conversion of angiotensin (Ang) I to Ang II has been identified in human heart. This serine protease is also present in dog and marmoset vasculature. We examined the vasoconstrictor effects of Ang II putatively generated from an angiotensin-converting enzyme (ACE)-resistant convertase synthetic substrate (SUB) in vivo and in vitro. In marmosets, SUB (7 to 700 micrograms/kg i.v.) or Ang I (0.1 to 30 micrograms/kg) caused similar dose-dependent increases in mean arterial pressure (10 to 100 mm Hg) and decreases in heart rate. Pressor effects of SUB were slightly attenuated at low (but not high) doses by captopril (CAP, 1 mg/kg i.v.) and blocked by losartan (5 mg/kg i.v.); in contrast Ang I pressor effects were substantially blocked by both. In isolated canine superior mesenteric artery, Ang I-induced contraction was eliminated by losartan and reduced but not eliminated by 10 mumol/L CAP. When combined with the serine protease inhibitor chymostatin, CAP eliminated Ang I-induced contraction, but chymostatin alone had no effect. SUB-induced contraction was not blocked by CAP but was equally blocked by chymostatin (25 mumol/L) alone or by the combination of CAP (10 mumol/L) and chymostatin (25 mumol/L); losartan (10 mumol/L) eliminated SUB-induced responses. Previous studies have suggested that Ang I-convertase is important for production of Ang II in the heart. Our results are consistent with a potential role for Ang I-convertase in the production of Ang II in the vasculature, resulting in Ang II-mediated vasoconstriction.

Diagnostic imaging of human neuroblastoma with radiolabeled antibody.

In a previous study, the authors showed that iodine-131 labeled monoclonal antibody (Mab 3F8) could be used to image human neuroblastoma xenografts in mice with excellent tumor-to-tissue ratios. In this study they report their experience with six patients scanned with radiolabeled 3F8. There was strong accumulation of the labeled antibody in viable tumor, but no significant uptake was noted in normal brain, liver, spleen, or adrenal glands. Tumor-to-nontumor activity ratios varied but were approximately 10:1-20:1. This ratio yields good contrast for visualization. Time-activity curves show that radioactivity levels in normal tissue have a half-time of about 40 hours, whereas tumor tissues show a half-time of about 60 hours. Significant gastric secretion of free iodine demonstrated that the Mab was being deiodinated. Calculated radiation doses indicate that tumors receive at least ten times the dose to other tissues. The results indicate that Mab 3F8 has clinical potential for both imaging and therapy of human neuroblastomas.

Complete tumor ablation with iodine 131-radiolabeled disialoganglioside GD2-specific monoclonal antibody against human neuroblastoma xenografted in nude mice.

The antibody 3F8, an IgG3 murine monoclonal antibody (MoAb) against disialoganglioside GD2, could target iodine-131 (131I) to established subcutaneous human neuroblastoma (NB) xenografts in BALB/c nude mice. 131I-radiolabeled MoAb (0.125-1 mCi) was injected iv. Tumor radioactivity over time was calculated from scintigraphy, and radiation dose to individual tumors was calculated. Tumor shrinkage occurred only with 131I-labeled 3F8, but not with nonradioactive 3F8 or radiolabeled irrelevant antibody. While the tumor of the control mice enlarged by tenfold, the treated tumor showed over 95% shrinkage by 12 days. Both the rate of shrinkage and duration of tumor response were dose dependent. Calculated doses of more than 10,000 rad could be achieved. Only those tumors that received more than 4,200 rad were completely ablated without recurrence. Recurrent tumors were not antigen negative or radioresistant. These results confirmed the prediction based on imaging studies that human NB xenografts could be effectively eradicated with the use of 131I-labeled MoAb 3F8 with tolerable toxicities.

Electrical stunning and stillness of lambs.

The stillness obtained with head-to-back stunning results from the current depolarizing spinal neurones. The same effect can be achieved by sequentially applying a head stun, followed by a current through the heart to stop it and a current down the spinal cord to abolish movement. The level of speckle bruising for this sequential stun is lower than that associated with head-to-back stunning and is similar to that of head-to-foreleg stunning. After head-only stunning, current passed continuously down the spinal cord through a good contact at voltages around 40 V gave adequate stillness, which improved when the voltage was raised to 80 V. With a frequency of 14 Hz, 40 V presented no hazard to workers. After head-only stunning, a brief application of current down the entire spinal cord for 2-4 s at 110 V or greater also effectively suppressed movement. With legs as contact points voltages as high as 400 V were necessary. For effective animal stillness good electrode contact is necessary and electrodes should be placed as close to the spinal cord as possible. With a continuous current flowing the oesophagus remained closed until muscle exhaustion occurred; even with a brief current down the spinal cord, the oesophagus remained closed for at least 30 s. Low voltage stimulation in the form of a continuous current cannot replace high voltage stimulation, but can supplement it, and therefore could be incorporated into the electrical stimulation process. Head-only stunning, followed by a cut through the major blood vessels of the neck, is acceptable for Halal slaughter and is now in use commercially in New Zealand. Subsequent movement is suppressed by a spinal discharge at 400 V using leg contacts.

Differential effects of electrical stunning on the early post-mortem glycolysis in sheep.

Electrical stunning methods have been examined to determine the effect on post-mortem glycolysis in various muscles of sheep. Although there was no effect on the ultimate pH achieved in any of the muscles, there were marked differences in the pH drop which occurred during stunning. When curare was used to block neuromuscular transmission much of the effect of stunning on glycolysis was removed. In the absence of neuromuscular blockage, head-only stunning produced the least drop in initial pH and the head-to-back and head-to-foreleg methods the greatest drop, especially in the anterior portions of the LD. Gash cutting without prior stunning produced marked drops in pH in most muscles. Prolonged (120s) head-to-back stunning gave more substantial drops in pH in the LD, BF, SM and ST muscles than did a shorter stun (1·4s). The prolonged stunning did not produce a change in rate of pH fall. Electrical stunning cannot be considered as a substitute for effective electrical stimulation as a means of hastening glycolysis to avoid cold shortening.

Responses of different types of ox muscle to electrical stimulation.

The effects of electrical stimulation on fall in pH upon stimulation (ΔpH), rate of pH fall (dpH/dt at 35°C, cold shortening and muscle ultrastructure were investigated for the Cutaneus trunci (predominantly fast-twitch glycolytic fibres), the Masseter and Diaphragm (predominantly slow-twitch oxidative fibres) and the Sternomandibularis and Longissimus dorsi (bot fast- and slow-twitch fibres) of the ox. The Masseter and Diaphragm showed a small ΔpH and no increase in dpH/dt upon stimulation. Stimulation produced supercontracture but no tearing of the fibres throughout all of the Masseter. Stimulation of the Cutaneus trunci resulted in a significantly increased ΔpH and dpH/dt, loss of glycogen, mitochondrial swelling but no gross sarcomere changes. The Longissimus dorsi and Sternomandibularis had a moderate ΔpH and an increase in dpH/dt intermediate between that of the Masseter and the Cutaneus trunci. The Longissimus dorsi showed supercontracture, but the Sternomandibularis did not. Cold shortening responses at 2°C and 0°C were virtually unaffected by stimulation, being greatest in the Masseter and Diaphragm and least in the Cutaneus trunci. All muscles showed significantly greater shortenings at 0°C than 2°C. Stimulation of the Cutaneus trunci did not affect the tenderness of the cooked meat, but the toughness increased dramatically in cold-shortened Cutaneus muscle, regardless of stimulation. The Cutaneus trunci least requires stimulation as it does not cold shorten appreciably and therefore early rigor would confer no advantage. The Masseter and Diaphragm have a fast dpH/dt and therefore would enter rigor early. Mixed muscles apparently have the combined, least desirable characteristics of the muscle fibre types-i.e. their rate of rigor development is slow and they cold shorten. Electrical stimulation confers a significant advantage by mitigating these mixed-muscle characteristics in carcass muscles.