Chick Embryo Partial Ischemia Model: A New Approach to Study Ischemia Ex Vivo

Background: Ischemia is a pathophysiological condition due to blockade in blood supply to a specific tissue thus damaging the physiological activity of the tissue. Different in vivo models are presently available to study ischemia in heart and other tissues. However, no ex vivo ischemia model has been available to date for routine ischemia research and for faster screening of anti-ischemia drugs. In the present study, we took the opportunity to develop an ex vivo model of partial ischemia using the vascular bed of 4th day incubated chick embryo. Methodology/Principal Findings: Ischemia was created in chick embryo by ligating the right vitelline artery using sterile surgical suture. Hypoxia inducible factor- 1 alpha (HIF-1a), creatine phospho kinase-MB and reactive oxygen species in animal tissues and cells were measured to confirm ischemia in chick embryo. Additionally, ranolazine, N-acetyl cysteine and trimetazidine were administered as an anti-ischemic drug to validate the present model. Results from the present study depicted that blocking blood flow elevates HIF-1a, lipid peroxidation, peroxynitrite level in ischemic vessels while ranolazine administration partially attenuates ischemia driven HIF-1a expression. Endothelial cell incubated on ischemic blood vessels elucidated a higher level of HIF-1a expression with time while ranolazine treatment reduced HIF-1a in ischemic cells. Incubation of caprine heart strip on chick embryo ischemia model depicted an elevated creatine phospho kinase-MB activity under ischemic condition while histology of the treated heart sections evoked edema and disruption of myofibril structures. Conclusions/Significance: The present study concluded that chick embryo partial ischemia model can be used as a novel ex vivo model of ischemia. Therefore, the present model can be used parallel with the known in vivo ischemia models in understanding the mechanistic insight of ischemia development and in evaluating the activity of anti-ischemic drug.status: publishe

Thirty days of spaceflight does not alter murine calvariae structure despite increased Sost expression

Previously our laboratory documented increases in calvaria bone volume and thickness in mice exposed to 15 days of spaceflight aboard the NASA Shuttle mission STS-131. However, the tissues were not processed for gene expression studies to determine what bone formation pathways might contribute to these structural adaptations. Therefore, this study was designed to investigate both the structural and molecular changes in mice calvariae after a longer duration of spaceflight. The primary purpose was to determine the calvaria bone volume and thickness of mice exposed to 30 days of spaceflight using micro-computed tomography for comparison with our previous findings. Because sclerostin, the secreted glycoprotein of the Sost gene, is a potent inhibitor of bone formation, our second aim was to quantify Sost mRNA expression using quantitative PCR. Calvariae were obtained from six mice aboard the Russian 30-day Bion-M1 biosatellite and seven ground controls. In mice exposed to 30 days of spaceflight, calvaria bone structure was not significantly different from that of their controls (bone volume was about 5% lower in spaceflight mice, p = 0.534). However, Sost mRNA expression was 16-fold (16.4 ± 0.4, p < 0.001) greater in the spaceflight group than that in the ground control group. Therefore, bone formation may have been suppressed in mice exposed to 30 days of spaceflight. Genetic responsiveness (e.g. sex or strain of animals) or in-flight environmental conditions other than microgravity (e.g. pCO2 levels) may have elicited different bone adaptations in STS-131 and Bion-M1 mice. Although structural results were not significant, this study provides biochemical evidence that calvaria mechanotransduction pathways may be altered during spaceflight, which could reflect vascular and interstitial fluid adaptations in non-weight bearing bones. Future studies are warranted to elucidate the processes that mediate these effects and the factors responsible for discordant calvaria bone adaptations between STS-131 and Bion-M1 mice

Shear stress promotes nitric oxide production in endothelial cells by sub-cellular delocalization of eNOS: A basis for shear stress mediated angiogenesis

This study aims to investigate the role of shear stress in cellular remodeling and angiogenesis with relation to nitric oxide (NO). We observed a 2-fold increase in endothelial cell (EC) migration in relation to actin re-arrangements under 15 dyne/cm2 shear stress. Blocking NO production inhibited the migration and ring formation of ECs by 6-fold and 5-fold, respectively under shear stress. eNOS-siRNA knockdown technique also ascertained a 3-fold reduction in shear stress mediated ring formation. In ovo artery ligation model with a half and complete flow block for 30 min showed a reduction of angiogenesis by 50% and 70%, respectively. External stimulation with NO donor showed a 2-fold recovery in angiogenesis under both half and complete flow block conditions. NO intensity clustering studies by using Diaminofluorescein diacetate (DAF-2DA) probed endothelial monolayer depicted pattern-changes in NO distribution and cluster formation of ECs under shear stress. Immunofluorescence and live cell studies revealed an altered sub-cellular localization pattern of eNOS and phospho-eNOS under shear stress. In conclusion, shear-induced angiogenesis is mediated by nitric oxide dependent EC migration

Aging Decreases Hand Volume Expansion with Water Immersion

Hands may show early signs of aging with altered skin texture, skin permeability and vascular properties. In clinics, a hand volumeter is used to measure swelling of hands due to edema, carpal tunnel syndrome or drug interventions. The hand volume measurements are generally taken without taking age into consideration. We hypothesized that age affects hand volumeter measurements and that the younger age group (≤40 years) records a greater change in hand volume as compared to the older group (&gt;40 years). Four volumetric measurements were taken at 5 min intervals during 20 min of water immersion using a clinically-approved hand volumeter. After 20 min of immersion, the hand volume changes of the younger age group were significantly higher than the older age group (p &lt; 0.001). Specifically, the right-hand volume of the younger age group (≤40 years, n = 30) increased by 4.3 ± 2%, and the left hand increased by 3.4 ± 2.1%. Conversely, the right-hand volume of the older age group (&gt;40 years, n = 10) increased by 2.2 ± 2.0%, and the left hand decreased by 0.6 ± 2.4% after 20 min of water immersion. The data are presented as Mean ± SD. Hand volume changes were not correlated with body mass index (BMI) or gender, and furthermore, neither of these two variables affected the relationship between age and hand volume changes with water immersion. We conclude that the younger age group has a higher increase in hand volume with water immersion as compared to the older age group

Studying the angiogenesis pattern in vessels adjacent to ischemic area.

<p>(A) Angiogenesis pattern of the vessels adjacent to the ischemic area were followed for 3 h. The images of the vessels adjacent to the ischemic area were taken with Nikon Cool Pix camera adapted to a stereo microscope. Images were converted to gray scale and presented. Inset showed the magnified images from ischemia group which revealed formation of new vessels in the area. Yellow arrows showed the formation of new vessels in the area. Images are the representative of 5 individual experiments. (B) Analysis of the images were carried out using Angioquant software. Number of separate vessel complexes was analyzed by the software for different time point. Data presented as fold increase with time. **P<0.001 versus respective time point under non-ischemia. (C) Similarly, the software calculated the length of the vessels in different time of incubation. Data presented as fold increase with time. *P<0.05 and **P<0.001 versus respective time point under non-ischemia. (D) The size of the vessels was calculated by the Angioquant software. Data presented as fold increase with time. **P<0.001 versus respective time point under non-ischemia. (E) The number of junctions in the vessels were calculated by the software and plotted. *P<0.05 and **P<0.001 versus respective time point under non-ischemia.</p

HIF-1α level in ischemic EC.

<p>(A) HIF-1α production was measured in EC treated for different time under ischemia. Cells were grown on collagen coated cover glasses and placed on the top of the ischemic vascular bed of chick embryo. RNA was isolated from the ischemia treated EC using RNA extraction kit. Loading of total RNA for cDNA conversion was normalized by running a gel for total RNA and calculating the band intensities for each set. After amplification, the band intensities from the representative images were calculated and plotted as a graph. Ischemia induced HIF-1α expression in EC in a time dependent manner. **P<0.001 significantly different than 0 min. (n = 5) (B) RL was administered to evaluate its effect on ischemia induced HIF-1α expression by EC. EC grown on collagen coated cover glasses were placed on ischemic chick embryo vascular bed followed by RNA extraction from the EC using RNA extraction kit. Loading of total RNA for cDNA conversion was normalized by running a gel for total RNA and calculating the band intensities for each set. RL attenuated ischemia induced HIF-1α expression by EC. **P<0.001 versus ischemic. (n = 5).</p

Level of different reactive oxygen species and GSH in ischemic EC.

<p>(A) Level of lipid peroxidation in ischemic vascular bed treated EC was quantified using TBA protocol. EC were treated under ischemia for 2 h followed by processing as mentioned in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010524#s4" target="_blank">materials and methods</a>section. Data were normalized by counting the number of cells using hemocytometer. Elevated lipid peroxidation was noted under ischemia treatment. *P = 0.012 versus non-ischemic. (n = 5) (B) Superoxide production by ischemia treated EC was measured using NBT. Cells were treated under ischemia for 2 h followed by incubation with NBT for another 2 h. The formed formazen dye was extracted from the cells as mentioned in methodology. Data were normalized by counting the number of cells using hemocytometer. **P<0.001 versus non-ischemic. (n = 5) (C) Ischemia treated EC were probed with CMFDA to measure the level of GSH in the cells. Cells were treated with ischemic vascular bed driven secondary ischemia for 2 h followed by probing with CMFDA for 30 min. Images were acquired by DP71 camera adapted to an Olympus IX71 microscope. Images are the representative of 5 individual experiments. (D) The green luminosity from the CMFDA probed fluorescent images were measured using Adobe Photoshop version 7.0. Green luminosity of 40 different cells from 5 different sets of experiment were measured and plotted. Ischemia reduced the level of free thiol in EC. **P<0.001 versus non-ischemic. (n = 5).</p

Effect of ischemia on GSH level in vitelline vascular tissue.

<p>(A) Level of GSH was measured using CMFDA fluorescent probe. The ischemic vessels were incubated with CMFDA probe for 30 min. Next, the images were acquired with DP71 CCD camera. Images are the representative of 5individual experiments. (B) The fluorescent intensities of the images were calculated using Adobe Photoshop 7.0 and plotted. Fluorescent intensities were considered as directly proportional to the level of GSH in the vessels. **P<0.001 versus non-ischemic. (n = 5) (C) Ischemic tissues were probed with CMFDA for 30 min. Tissues were then homogenized and centrifuged at 6500 g for 5 min. The supernatants were then read at excitation/emission of 485/515 nm. Values obtained were normalized with the weight of the tissues. **P<0.001 versus non-ischemic. (n = 5).</p