Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems.

Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are useful because they can help us to understand the underlying pathogenetic mechanisms and develop new therapies. The present review article summarizes the results of a consensus conference on the status and future development of experimentation in the field of cardiovascular medicine using murine models of peripheral and myocardial ischemia. The starting point was to recognize the limits of the approach, which mainly derive from species- and disease-related differences in cardiovascular physiology. For instance, the mouse heart beats at a rate 10 times faster than the human heart. Furthermore, healing processes are more rapid in animals, as they rely on mechanisms that may have lost relevance in man. The main objective of the authors was to propose general guidelines, diagnostic end points and relevance to clinical problems.

Extent of intestinal damage in the developing chick embryo after repetitive hypoxia under normoxic or hyperoxic conditions.

BACKGROUND: Episodes of hypoxia and reperfusion play an important role in the development of intestinal damage during perinatal development. The aim of this study was to investigate the histopathology of the intestine in the developing chick embryo after exposure to repetitive hypoxia and recovery under two different conditions: normoxic and hyperoxic (60% O2). METHODS: Chick embryos were exposed to 5 minutes of hypoxia. This was repeated six times with a recovery period of 15 minutes under normoxic conditions (21% O2) for chick embryos in test group 1 (TG1) and under hyperoxic conditions (100% O2) for chick embryos in test group 2 (TG2), from day 11 until day 20. Chick embryos that recovered under hyperoxic conditions (100% O2) were previously incubated under hyperoxic conditions (60% O2 for 24 hours). Histologic evaluation of the ileum was performed at different times after the interventions (2, 4, 8, 16, and 24 hours). RESULTS: In both test groups, only chick embryos older than 19 days showed intestinal damage. Intestinal histology on day 19 showed vasodilation of villus capillaries (10% in TG1 and 15% in TG2), necrosis in the top of the villi (29% in TG1 and 30% in TG2), and necrosis with preservation of base of the crypts (2% in TG1) and transmucosal necrosis (2% in TG2). CONCLUSIONS: Significant histologic changes, compared with the control group, were only found in chick embryos that were studied 2 hours after the interventions. Furthermore, recovery under hyperoxic conditions did not cause more intestinal damage compared with recovery under normoxic conditions.

Hyperoxia and local organ blood flow in the developing chick embryo.

1. Hyperoxia can cause local vasoconstriction in adult animal organs as a protective mechanism against hyperoxia-induced toxicity. It is not known at what time during development this vasoconstrictor capacity is present. Therefore, we measured the cardiac output (CO) distribution in different organs during a period of acute hyperoxia (100 % O2) in the developing chick embryo. 2. Fertile eggs were divided into five incubation time groups (10 and 11, 12 and 13, 14 and 15, 16 and 17, and 18 and 19 days of a normal incubation time of 21 days). Eggs were opened at the air cell and a catheter was inserted into a branch of the chorioallantoic vein for injections of 15 microm fluorescent microspheres during normoxia and at the end of 5 min (test group 1; n = 39) or 20 min (test group 2; n = 21) of hyperoxia exposure (100 % O2). The fraction of CO to an organ was calculated as the fluorescence of the organ sample divided by the sum of the fluorescence of all organs. 3. Only in 18- and 19-day-old embryos did hyperoxia cause a decrease in the fractions of CO to the heart and carcass, and an increase in those to the yolk-sac and chorioallantoic membrane. This response was more pronounced after 20 min (test group 2) than after 5 min (test group 1) of hyperoxia with an additional decrease in the fractions of CO to the brain, intestine and liver (test group 2). 4. These data indicate that local mechanisms for hyperoxia-induced vasoconstriction in the heart, brain, liver, intestine and carcass develop late, during the final 15 % of the incubation period, in the developing chick embryo.

The effect of hyperoxia on embryonic and organ mass in the developing chick embryo.

It is known that hyperoxia stimulates growth late in incubation when the chick embryo outgrows the O2 diffusion capacity. We wondered whether hyperoxia could have an effect in the early period prior to the stage where metabolism exceeds the oxygen diffusion capacity of the eggshell. For this we studied four groups of chicken eggs: control group (CG; n = 100) and three test groups (TGs) exposed during 48 h to 60% O2 on days 10, 14, and 18. In the CG, embryonic and organ mass (brain, heart, lungs, liver and intestine) were measured from day 10 until day 21 of incubation. In the TGs embryonic and organ mass were obtained from 24 h after the start of hyperoxia exposure until the end of incubation. In all TGs the most striking growth rate acceleration was observed in the liver and intestine, maximum growth rate accelerations were respectively, 19 and 42% in TG1, 43 and 173% in TG2 and 39% and 84 in TG3. In contrast, the brain was little affected by the hyperoxia exposure, the maximum growth rate acceleration was 14% in TG2. The results suggest that also in the middle of the incubation period O2 availability can be a limiting factor for growth, before metabolism exceeds the oxygen diffusion capacity of the eggshell.

Induction of antioxidant enzyme activity by hyperoxia (60 % O2) in the developing chick embryo.

1. At premature birth, man and animals are exposed to relatively high oxygen levels, compared with intra-uterine conditions, at a time when their antioxidant enzyme (AOE) system is still immature. Using the chick embryo as a study model, we investigated changes in the AOE system in response to hyperoxia applied at different time points during the incubation period. Relations between hyperoxia and AOE activity were studied in selected organs (brain, heart, liver, intestine and lungs) of developing chick embryos (during the second half of the incubation period). 2. Incubated White Leghorn eggs were divided into four groups: control (n = 100) and three test groups exposed for 48 h to 60 % O2 on day 10 (test group 1, n = 80), day 14 (test group 2, n = 60) and day 18 (test group 3, n = 30). Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) enzyme activities were measured in homogenates of the brain, heart, liver, intestine and lungs. 3. Exposure to hyperoxia at different time points during incubation resulted in a 2- to 10-fold increase in SOD activity in all organs except the brain. Catalase and GPx enzyme activities were only induced in test group 1, 48 h after initiation of hyperoxia. 4. In the developing chick embryo, hyperoxia can produce a temporary induction of AOE activity, which is dependent on the AOE, organ, incubation time and time point of exposure.

Cardiac output distribution in response to hypoxia in the chick embryo in the second half of the incubation time.

1. The fetus develops cardiovascular adaptations to protect vital organs in situations such as hypoxia and asphyxia. These include bradycardia, increased systemic blood pressure and redistribution of the cardiac output. The extent to which they involve maternal or placenta influences is not known. The objective of the present work was to study the cardiac output distribution in response to hypoxia in the chick embryo, which is independent of the mother. 2. Fertilized eggs were studied at three incubation times (10-13 days, 14-16 days and 17-19 days of a normal incubation time of 21 days). Eggs were placed in a Plexiglass box in which the oxygen concentration could be changed. Eggs were opened at the air cell and a chorioallantoic vein was catheterized. Cardiac output distribution was measured with 15 micron fluorescent microspheres injected during normoxia, during the last minute of a 5 min period of hypoxia and after 5 min of subsequent reoxygenation. 3. Hypoxia caused a redistribution of the cardiac output in favour of heart (+17 to +160 % of baseline) and brain (+21 to +57 % of baseline) at the expense of liver (-3 to -65 % of baseline), yolk-sac (-46 to -77 % of baseline) and carcass (-6 to -33 % of baseline). 4. The magnitude of the changes in cardiac output distribution to the heart, brain, liver and carcass in response to hypoxia increased with advancing incubation time. 5. The data demonstrate the development of a protective redistribution of the cardiac output in response to hypoxia in the chick embryo from day 10 of incubation.

Changes in mean chorioallantoic artery blood flow and heart rate produced by hypoxia in the developing chick embryo.

Hypoxia in the mammalian fetus produces cardiovascular changes, such as bradycardia, systemic hypertension, and changes in heart rate variability. This response was studied in 140 chick embryos ranging from stage 34 to stage 42 (d 9-16 of the 21-d incubation), by measuring the changes in mean chorioallantoic artery blood flow (CABF) and heart rate for 5 min in two levels of hypoxia (group 1; n = 90; 100% N2) or (group 2; n = 50; 5% O2). Eggs were opened at the air cell and placed in a small plexiglass holder, which had a continuous gas flow of an O2/N2 mixture (5 L/min), at 38 degrees C and 60% humidity. The chorioallantoic artery was placed in the lumen of a flow probe to measure mean CABF, heart rate, peak flow, and blood flow acceleration. After baseline measurements, the gas mixture was changed to 100% N2 or 5% O2 in N2 for 5 min. Mean CABF and heart rate decreased significantly in both groups (Wilcoxon paired sample test, p < 0.05). This response was more pronounced with the development of the chick embryo. Chorioallantoic artery peak flow (mL/min) and CABF acceleration (mL/s2) increased with incubation time and decreased during periods of hypoxia. During recovery, heart rate returned to baseline levels, whereas mean CABF showed an overshoot. The initial decrease in mean CABF and heart rate was similar in both groups. The cardiovascular response to hypoxia in the chick embryo is similar to the response in the mammalian fetus. The more pronounced response in the more developed chick embryo may represent a maturation of cardiovascular control.

Cardiac output distribution in the chick embryo from stage 36 to 45.

OBJECTIVE: The distribution of cardiac output to different organs is well described in the mammalian fetus. Chick embryos are not often used in perinatal cardiovascular research and therefore it is not known whether they can serve as an animal model for this purpose. In this study we documented cardiac output distribution in chick embryos at increasing incubation time. METHODS: Fertilized eggs from day 10 to 19 with an incubation time of 21 days were studied in 3 increasing incubation time groups (10-13, 14-16 and 17-19 days). For the experiment, the egg was placed in a holder in an incubator. The egg was opened at the air cell and a small vein of the chorioallantoic membrane was catheterized. Twenty thousand fluorescent 15 microns microspheres in 0.2 ml were injected. After 5 min, the embryo was sacrificed and the different organs were dissected and digested for microsphere isolation and subsequent fluorescence analysis. RESULTS: The chorioallantoic membrane, which is the placenta equivalent of the chick embryo, received a relatively large fraction of the combined cardiac output: 52.08% (interquartile range [IQR] 12.67%) on days 10-13 and 40.95% (IQR 27.24%) on days 17-19. Relatively small fractions were distributed: to the heart 2.03% (IQR 1.58) on days 10-13 and 3.18% (IQR 1.95) on days 17-19, and to the brain 3.20% (IQR 1.80) on days 10-13 and 5.02% (IQR 3.39) on days 17-19. As incubation time advanced, the fraction of the combined cardiac output to the chorioallantoic membrane and yolk-sac decreased significantly in favor of the heart and brain. CONCLUSION: This distribution shows great similarity to the one found in the mammalian fetus. The chick embryo is an attractive model for perinatal cardiovascular research.

The chorioallantoic artery blood flow of the chick embryo from stage 34 to 43.

Chorioallantoic artery blood flow and heart rate were studied in the chick embryo from stage 34 until stage 43 (d 9-16 of 21-d incubation). Baseline blood flow profiles of the chorioallantoic artery were measured with a flow probe (Transonic) in 100 chick embryos. The eggs were opened at the air cell and placed in a small plexiglass box with a continuous gas flow of a N2/O2 mixture (5 L/min), at 38 degrees C and 60% humidity. The chorioallantoic artery was localized near the fetal abdomen and placed in the lumen of the Transonic flow probe. The heart rate was derived from the blood flow signal. The mean chorioallantoic artery blood flow rose from 0.35 +/- 0.14 mL/min (mean +/- SD) at stage 34 to 3.13 +/- 1.49 mL/min at stage 43 (R2 = 0.69, p < 0.0001), which correlated with an increase in body weight (1.51 +/- 0.18 g to 15.08 +/- 0.76 g). Heart rate increased from 195 +/- 38 beats/min at stage 34 to 289 +/- 13 beats/min at stage 43 (R2 = 0.38, p < 0.0001). The chorioallantoic artery blood flow, which in avian species correlates with umbilical blood flow in mammals, increased with incubation time as reported in the mammalian fetus. This study shows that the chick embryo could be useful as a preparation for further perinatal cardiovascular research.