Asiatic acid in Centella asiatica extract towards morphological development in an intermittent hypoxia intrauterine embryo model and molecular prediction pathway of insulin-like growth factor-1 receptor signalling.

Background: Hypoxia during pregnancy generates oxidative stress that alters the growth and development of the human fetus. Insulin-like growth factor-1 (IGF-1) receptors are essential for normal fetal growth. Asiatic acid in Centella asiatica (CA) has antioxidant properties to prevent growth impairment in hypoxia. Aims: This study aimed to investigate the effect of asiatic acid on the morphological development of an intermittent hypoxia (IH) zebrafish embryo model and analyze molecular docking prediction in IGF-1 receptor (IGF-1R) signaling. Methods: Embryos of zebrafish at 2 hours postfertilization (hpf) were assigned to control negative (C), IH, and combination IH and CA extract groups consisting of 1.25 (IHCA1), 2.5 (IHCA2), and 5 (IHCA3) µg/ml. Hypoxia treatment (conducted 4 hours/day) and CA extract were administered for 3 days (2-72 hpf). The parameters of body length and head length were evaluated at 3, 6, and 9 days postfertilization (dpf). The data were analyzed by a two-way analysis of variance (p < 0.05). Molecular docking was performed to explore the binding affinity of asiatic acid to IGF-1R by Molegro Virtual Docker ver.5 software. Results: The body length and head length of embryos in the IH and treatment groups (IHCA) were shorter than those in the control group at 3 dpf (p < 0.05). However, the body length was more prolonged in the IHCA1 group, but the head length was longer in the IHCA2 group than in the IH group at 6 and 9 dpf. Molecular docking showed the reliable interaction of asiatic acid with IGF-1R signaling in an IH animal model. Conclusion: The administration of CA extract benefits IH through the development and growth of zebrafish embryos at a dose of 2.5-5 µg/ml. Asiatic acid has a binding affinity for IGF-1R signaling.

Hypoxia and platelet lysate sustain differentiation of primary horse articular chondrocytes in xeno-free supplementation culture.

Currently, the main limitation for the use of adult differentiated chondrocytes in cell-based therapy and tissue engineering for the repair of articular cartilage is the difficulty of maintaining their state of differentiation during cell expansion. The adult articular cartilage has no direct blood supply, and local oxygen concentrations range from 5%-10% at the surface near the synovial fluid to less than 1% in the deep layer. Low oxygen tension is currently considered an important environmental condition for chondrocytes, and hypoxia has been explored as a signal potentially promoting differentiation and matrix deposition. In the present study, hypoxia and PL supplementation were studied to maintain differentiation in adult articular chondrocytes. Freshly isolated equine articular chondrocytes were grown in monolayer culture at a low seeding density (condition favoring proliferation and dedifferentiation) and in alginate beads (3D culture condition maintaining chondrocyte differentiation) both in normoxic and hypoxic conditions and in various conditions of supplementation or deprivation (fetal bovine serum [FBS]- and PL-free; 10% FBS; 5% PL; 10% PL). Results demonstrated that hypoxia is a micro-environmental condition that reduces chondrocyte dedifferentiation or maintains differentiation during in vitro expansion, as shown by the sustained expression of differentiation markers (COL2, ACAN, SOX9, HIF1a) and the reduction of dedifferentiation marker expression (COL1, RUNX2). In association with hypoxia, PL supplementation demonstrated a positive effect on chondrocyte differentiation in association with hypoxia. This promising result should be confirmed in other conditions of chondrocyte differentiation before proposing PL as a complete alternative to xenogenic serum for the expansion of articular chondrocytes.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish.

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

Hypoxia induces an increase in intestinal permeability and pulmonary arterial pressures in neonatal Holstein calves despite feeding the flavonoid rutin.

The purposes of this study were to determine whether the naturally occurring flavonoid quercetin, as its glucorhamnoside rutin, reduces intestinal permeability and susceptibility to hypoxia-induced pulmonary hypertension in neonatal Holstein calves. A 2 × 2 between-subjects factorial design was conducted using Holstein steers (n = 16). Factors included oxygen level (simulated altitude of 4,572 m vs. 975 m) and quercetin supplementation as its glucorhamnoside rutin (4 g of quercetin per day vs. 0 g per day). Two days after arrival (d 0 of study) the calves were blocked by body mass into treatment groups, and treatments were initiated. Pulmonary arterial pressure, echocardiography, and serum concentrations of orally administered lactulose (0.45 g/kg) and mannitol (0.15 g/kg) were measured on d 12, 13, and 14, respectively. Calves were euthanized on d 15 and pulmonary tissues collected for semiquantitative scoring of histological lesions. Data were analyzed using linear regression, generalized estimating equations, and 2-sample proportion tests. Hypoxia, but not rutin, was found to be associated with intestinal permeability. The lactulose-mannitol ratio was 0.54 ± 0.13 (standard error) in hypoxic calves and 0.02 ± 0.13 in normoxic controls. Hypoxia increased mean pulmonary arterial pressure. Calves fed rutin under hypoxic conditions tended to have a lower mean pulmonary arterial pressure (59 ± 7 mmHg) than control calves (80 ± 7 mmHg) but similar pressures under normoxic conditions. Paradoxically, however, a greater proportion of calves fed rutin had histological evidence of pulmonary arteriolar medial hypertrophy and adventitial hyperplasia than did controls. In conclusion, the findings of this study indicate that hypoxia increased intestinal permeability in neonatal calves. The flavonoid quercetin, as its glucorhamnoside rutin, had no protective effect on intestinal permeability, and, although it tended to reduce the severity of hypoxia-induced pulmonary hypertension, a greater proportion of calves fed rutin had histological lesions consistent with pulmonary arteriolar remodeling.

Combined effects of salinity, temperature, hypoxia, and Deepwater Horizon oil on Fundulus grandis larvae.

Oil spills have polluted the marine environment for decades and continue to be a major source of polycyclic aromatic hydrocarbons (PAHs) to marine ecosystems around the globe, for example during the 2010 Deepwater Horizon spill. Although the toxicity of PAHs to fish has been well studied, their effects combined with abiotic stressors are poorly understood. The goal of this study was to describe the combined impacts of crude oil and environmental stressors on fish larvae, a sensitive life stage. Gulf killifish (Fundulus grandis) larvae (<24 h post-hatch) were exposed for 48 h to high energy water accommodated fractions (HEWAF; total PAHs 0-125 ppb) of Macondo oil from the Deepwater Horizon spill under different combinations of environmental conditions (dissolved oxygen 2, 6 ppm; temperature 20, 25, 30 °C; salinity 3, 10, 30 ppt). Even under optimal environmental conditions (25 °C, 10 ppt, 6 ppm) larval survival and development were negatively affected by PAHs, starting with the lowest concentration tested (∼15 ppb). Hypoxia and high temperature each increased the adverse effects of HEWAF on development and mortality. In contrast, salinity had little effect on any of the endpoints measured. Importantly, expression of the detoxifying gene cyp1a was highly induced in PAH-exposed larvae under normoxic conditions, but not under hypoxic conditions, potentially explaining the enhanced toxicity observed under hypoxia. This work highlights the importance of considering how suboptimal environmental conditions can exacerbate the effects of pollution on fish early life stages.

Pulmonary hypertensive response of broiler chickens to arginine and guanidinoacetic acid under high-altitude hypoxia.

This study assessed the preventive effects of arginine (ARG) and guanidinoacetic acid (GAA) on the incidence of pulmonary hypertension syndrome (PHS) in broiler chickens. Four isoenergetic and isonitrogenous diets were prepared, including: (i) the control, (ii) the control supplemented with 1 g/kg ARG, (iii) the control supplemented with 1 g/kg GAA, and (iv) the control supplemented with 1.5 g/kg GAA. These diets were fed to broilers (Ross 308) from day 1 to 42 post-hatch. Criteria evaluated in the experiment were growth performance, carcass characteristics, serum and blood variables, lead-II electrocardiogram, and ET-1 and iNOS gene expression in heart and lungs. Mortality from PHS was recorded daily. The results showed that ARG and GAA supplements improved the feed conversion ratio (FCR) compared to the control (P < 0.05). Supplementation of ARG and GAA significantly (P < 0.05) increased serum nitric oxide (NO) concentration. ARG and GAA supplementation significantly reduced the haematocrit value and the heterophil to lymphocyte ratio in the blood. A significant (P < 0.05) decline in S-wave amplitude of the lead-II electrocardiogram, right to total ventricular weight ratio (RV:TV) and ascites mortality was observed by supplementing ARG or 1.5 g/kg GAA. Addition of ARG and GAA supplements did not significantly change ET-1 and iNOS gene expression in the heart and lung relative to the control. In conclusion, GAA supplementation at 1.5 g/kg had a potential to improve growth performance and could prevent PHS.

Controversies related to red blood cell transfusion in critically ill patients.

OBJECTIVE: To review the evolution of and controversies associated with allogenic blood transfusion in critically ill patients. DATA SOURCES: Veterinary and human literature review. HUMAN DATA SYNTHESIS: RBC transfusion practices for ICU patients have come under scrutiny in the last 2 decades. Human trials have demonstrated relative tolerance to severe, euvolemic anemia and a significant outcome advantage following implementation of more restricted transfusion therapy. Investigators question the ability of RBCs stored longer than 2 weeks to improve tissue oxygenation, and theorize that both age and proinflammatory or immunomodulating effects of transfused cells may limit efficacy and contribute to increased patient morbidity and mortality. Also controversial is the ability of pre- and post-storage leukoreduction of RBCs to mitigate adverse transfusion-related events. VETERINARY DATA SYNTHESIS: While there are several studies evaluating the transfusion trigger, the RBC storage lesion and transfusion-related immunomodulation in experimental animal models, there is little research pertaining to clinical veterinary patients. CONCLUSIONS: RBC transfusion is unequivocally indicated for treatment of anemic hypoxia. However, critical hemoglobin or Hct below which all critically ill patients require transfusion has not been established and there are inherent risks associated with allogenic blood transfusion. Clinical trials designed to evaluate the effects of RBC age and leukoreduction on veterinary patient outcome are warranted. Implementation of evidence-based transfusion guidelines and consideration of alternatives to allogenic blood transfusion are advisable.

Molecular cloning, characterization and expression of two tryptophan hydroxylase (TPH-1 and TPH-2) genes in the hypothalamus of Atlantic croaker: down-regulation after chronic exposure to hypoxia.

Recently we discovered that hypoxia causes marked impairment of reproductive neuroendocrine function in Atlantic croaker, a marine teleost, which is due to a decline in hypothalamic serotonergic activity. As a first step in understanding the molecular responses of the hypothalamic serotonergic system to hypoxia, we cloned and characterized the genes for the enzymes regulating the rate-limiting step in serotonin biosynthesis, tryptophan hydroxylase (TPH-1 and TPH-2) in the croaker brain. The full-length croaker TPH-1 and TPH-2 cDNAs contain open reading frames encoding proteins with 479 and 487 amino acids, respectively, which are highly homologous to the TPH-1 (76-93%) and TPH-2 (64-92%) proteins of other vertebrates. Croaker TPH-1 and TPH-2 mRNA expression was detected throughout the brain but was greatest in the hypothalamic region. Both Northern blot analysis and real-time PCR showed that TPH-1 (transcript size approximately 2.1 kb) and TPH-2 ( approximately 1.9 kb) mRNA levels were significantly decreased in the hypothalami of croaker exposed for 2 weeks to hypoxic conditions compared with those in fish exposed to normoxic conditions. Immunohistochemistry of hypothalamic neurons with TPH antibodies showed reduced expression of TPHs in hypoxia-exposed fish compared with normoxic fish. Western blot analysis confirmed that hypoxia caused a marked decline in hypothalamic TPH protein levels, which was associated with decreases in hypothalamic TPH enzyme activity and 5-hydroxytryptophan levels. These results suggest that TPH is a major site of hypoxia-induced down-regulation of serotonergic function in croaker brains. Moreover, they provide the first evidence that hypoxia decreases the expression of TPH transcripts in vertebrate brains.

Pharmacokinetics of once-daily amikacin in healthy foals and therapeutic drug monitoring in hospitalized equine neonates.

The objectives of this study were to investigate the pharmacokinetics of once-daily amikacin in healthy neonates, to determine amikacin concentrations in hospitalized foals, and to determine the minimum inhibitory concentrations (MICs) of amikacin against gram-negative isolates from blood cultures in septic foals. Median half-life, clearance, and volume of distribution of amikacin in healthy 2- to 3-day-old foals after administration of an intravenous bolus of amikacin (25 mg/kg) were 5.07 hours (4.86-5.45 hours), 1.82 mL/min/kg (1.35-1.97 mL/min/kg), and 0.785 L/kg (0.638-0.862 L/kg), respectively. Statistically significant (P <.05) decreases in area under the curve (14% decrease), mean residence time (19% decrease), and C24h plasma amikacin concentrations (29% decrease) occurred between days 2-3 and 10-11. Plasma amikacin concentrations in healthy foals at 0.5 hours (C0.5h) were significantly higher (P = .02) than those of hospitalized foals. Sepsis, prematurity, and hypoxemia did not alter amikacin concentrations. The MIC at which 90% of all gram-negative isolates from equine neonatal blood cultures were inhibited by amikacin was 4 microg/mL, suggesting that amikacin C0.5h of 40 microg/mL should be targeted to achieve a maximum serum concentration to MIC ratio of 10:1. The proportion of foals with C0.5h 40 microg/mL was significantly higher (P < .0001) in hospitalized foals receiving a dose of amikacin at 25 mg/kg (22/24 or 92%) than in foals receiving a dose at 21 mg/kg (9/25 or 36%), whereas no difference was found in the proportion of foals with C24h concentrations > or = 3 microg/mL between the 2 groups. An initial dose at 25 mg/kg is recommended for once-daily amikacin in equine neonates.

Treatment of hypoxemia during xylazine-tiletamine-zolazepam immobilization of wapiti.

Hypoxemia is a commonly observed complication during the chemical immobilization of wild ruminants. If severe and left untreated, it can predispose animals to arrhythmias, organ failure, and capture myopathy. The following prospective study was designed to measure the degree of hypoxemia in wapiti that were immobilized with a combination of xylazine and tiletamine-zolazepam and to assess the response to nasal oxygen therapy. Pulse oximetry and arterial blood gas analysis were used to assess the degree of hypoxemia prior to nasal insufflation of oxygen and to demonstrate any beneficial effects of this intervention. All wapiti exhibited mild to marked hypoxemia (PaO2 = 43 +/- 11.8 mmHg) prior to treatment and showed marked improvement after 5 minutes of nasal insufflation of oxygen at 10 L/min (PaO2 = 207 +/- 60 mmHg). This inexpensive, noninvasive technique has great benefit in treating clinical hypoxemia under field conditions, and we recommend that nasal insufflation of oxygen be implemented during xylazine-tiletamine-zolazepam-induced immobilization of wapiti and other wild ruminants.

Effect of dietary flax oil and hypobaric hypoxia on pulmonary hypertension and haematological variables in broiler chickens.

1. Three experiments were conducted with broiler chickens using hypobaric chambers and control pens, feeding diets containing 25 or 50 g flax oil/kg food or control diets with equivalent amounts of animal/vegetable (A/V) blend oil for 4 weeks. The effect of these diets on haematological variables and the extent of right ventricular hypertrophy (RVH) leading to ascites was determined. 2. Overall growth rate was not consistently affected by dietary treatment, although feeding the 25 g flax oil/kg diet reduced weight gain in week 4 of one experiment. Feeding the 50 g flax oil/kg diet but not the 25 g flax oil/kg diet reduced RVH in birds exposed to hypobaric conditions compared to feeding control diets. 3. Feeding the 50 g flax oil/kg diet under hypobaric conditions reduced the haematocrit and haemoglobin content, increased the erythrocyte deformability and the proportion of unsaturated fatty acids in the erythrocyte membranes, and reduced the whole blood viscosity compared to feeding control diets. These effects were not seen when the 25 g flax oil/kg diet was fed. The ratio of n-3 to n-6 fatty acids in erythrocyte membranes was increased in the 50 g flax oil/kg treatment group compared to controls. 4. Including 50 g flax oil/kg broiler diet reduces RVH in broiler chickens. This may be attributable in part to an increase in erythrocyte deformability from an increased proportion of unsaturated fatty acids in the erythrocyte membranes.

Effect of dietary flax oil and hypobaric hypoxia on right ventricular hypertrophy and ascites in broiler chickens.

1. The effect of dietary flax oil on growth rate, blood haemoglobin content, mortality and incidence of pulmonary hypertension and ascites in broilers at ambient pressure and at reduced atmospheric pressure was examined. 2. Birds were housed either in hypobaric chambers simulating 1000, 1500 or 2200 m altitude or in pens at ambient atmospheric pressure and fed on diets containing 100 g/kg added fat as either an animal/vegetable (A/V) blend or flax oil. 3. Birds raised under hypobaric conditions had a decreased growth rate and increased mortality, blood haemoglobin content, and incidence of pulmonary hypertension and ascites compared to the groups at normal atmospheric pressure. 4. Broilers fed on the diet containing flax oil showed no difference in growth rate or blood haemoglobin content compared to birds fed on the A/V fat diet raised at the same altitude. 5. Inclusion of flax oil in the diet decreased mortality and the incidence of ascites at 2200 m and pulmonary hypertension at 1500 m. 6. Flax oil may be an effective method of reducing ascites and pulmonary hypertension in broilers without affecting performance.

Hypoxia in neonatal calves: effect on intestinal transport of immunoglobulins.

The effect of reduced oxygen tension in arterial blood (hypoxic hypoxia) for 24 h postpartum on the absorption of colostral Ig was studied in 12 neonatal calves. During this period, inspired air for hypoxic calves contained 10.5% O2, whereas that for normoxic calves contained 21% O2. After 24 h, inspired air for all calves contained 21% O2. Three dietary regimens also were imposed during the initial 24-h period: colostrum at 0 and 12 h, whole milk at 0 and 12 h, or fasting. Colostrum was fed to all calves at 24, 36, and 48 h. During the initial 24-h period, means for arterial partial pressure of oxygen were 26 and 73 mm Hg for hypoxic and normoxic calves, respectively. In those fed colostrum at 0 and 12 h, hypoxia extended the period of Ig absorption from 20 to 40.5 h. In calves fed whole milk or those that were fasted, no such effect was noted. These observations suggest that the effect of hypoxia on the cessation of Ig absorption by the small intestine is mediated through a secondary and as yet undetermined mechanism.

Effects of aspirin on xylazine-induced hypoxaemia in sheep.

Aspirin (10 mg kg-1) administered intravenously to conscious sheep four hours before intravenous xylazine injection (50 micrograms kg-1), failed to abolish or attenuate the hypoxaemic effect of xylazine in this species. Serum thromboxane levels measured in one animal revealed that aspirin administered in this way reduced serum thromboxane levels by 95 per cent. Xylazine (3 x 10(-5) M--4 x 10(-3) M) failed to induce platelet aggregation in vitro. It appears that the mechanism whereby xylazine causes arterial hypoxaemia in sheep does not involve a cyclo-oxygenase-dependent aggregation of platelets.

Metabolic consequences of induced hypoxia in newborn lambs.

Metabolic and nutritional consequences of making newborn lambs breath a 5% oxygen, 95% nitrogen atmosphere during six hours after birth were studied. Blood samples were collected at the end of the treatment and two hours after every meal given 6 h 30, 12 h 30, 18 h 30 and 24 h 30 after birth. Rectal temperature, blood pH, packed cell volume and plasmatic glucose, lactate, triglycerides, non-esterified fatty acids, inorganic phosphorus, uric acid and iron were measured. Comparison with control lambs established that all these parameters were strongly altered except packed cell volume and non-esterified fatty acids disturbed to a lesser extent. Nevertheless, in the last sample, taken after 26 h 30 of aerial life, no significant differences remained detectable, concerning the parameters quoted above. The exception was triglyceridemia, which was still higher in treated lambs, and could be due to lack of utilization of alimentary lipids.