Jennifer Creed

Brain Oxygenation During Cardiopulmonary Bypass and Circulatory Arrest

Quantitative measurements of oxygen distribution in the microcirculation of the brain cortex of newborn piglets were made during different modes of cardiopulmonary bypass. Three groups of animals, anesthetized and mechanically ventilated, were studied. The first group of animals were maintained on normothermic cardiopulmonary bypass (CPB) at a flow of 100 ml/kg/min, while the second and third groups underwent low flow hypothermic cardiopulmonary bypass (40 ml/kg/min at 18 degrees C) (LFCPB) and deep hypothermic (18 degrees C) circulatory arrest (DHCA), respectively. After bypass, the piglets were monitored for a two hours post-bypass recovery period. CPB caused a decrease in the cortical oxygen from 62 +/- 3 mm Hg to 32 +/- 7 mm Hg at the beginning of bypass and to 36 +/- 5 mm Hg at the end of bypass. During the recovery period, cortical oxygenation steadily decreased, reaching 29 +/- 8 mm Hg at the end of the experiment. With initiation of LFCPB, cortical oxygen decreased to 22 +/- 7 mm Hg. Upon rewarming cortical oxygen increased to 37 +/- 5 mm Hg and then decreased again to about 30 mm Hg at the end of two hours of post-bypass recovery. Similar changes in cortical oxygenation were observed during DHCA. In DHCA cortical oxygen decreased to 19 +/- 4 mm Hg and during rewarming and recovery increased to 35 +/- 6 mm Hg. In conclusion, it has been shown that in newborn piglets recovering from CPB, LFCPB and DHCA, when the blood pressure remained above 55 mm Hg and therefore total blood flow should be well maintained, oxygen pressure in the microvasculature is significantly lower than for pre-bypass. It is suggested that the decreased oxygenation is due to increased heterogeneity in resistance in the microcirculatory units, resulting in broadened distribution of flow rates and oxygen levels.

Effect of perfusion flow rate on tissue oxygenation in newborn piglets during cardiopulmonary bypass.

BACKGROUND Our knowledge of the best perfusion flow rate to use during cardiopulmonary bypass (CPB) in order to maintain tissue oxygenation remains incomplete. The present study examined the effects of perfusion flow rate and patent ductus arteriosus (PDA) during normothermic CPB on oxygenation in several organ tissues of newborn piglets. METHODS The experiments were performed on 12 newborn piglets: 6 with PDA ligation (PDA-L), and 6 without PDA ligation (PDA-NL). CPB was performed through the chest at 37 degrees C. During CPB, the flow rate was changed at 15-minute intervals, ranging from 100 to 250 ml/kg/min. Tissue oxygenation was measured by quenching of phosphorescence. RESULTS For the PDA-L group, oxygen in the brain did not change significantly with changes in flow rate. In contrast, for the PDA-NL group, oxygen was dependent upon the flow rate. Statistically significant decreases in cortical oxygen were observed with flow rates below 175 ml/kg/min. Within the myocardium, liver, and intestine, there were no significant differences in the oxygen levels between the PDA-L and PDA-NL groups. In these tissues, the oxygen decreased significantly as the flow rate decreased below 150 ml/kg/min, 125 ml/kg/min, and 175 ml/kg/min, respectively. Oxygen pressure in skeletal muscle was not dependent on either PDA ligation or flow rate. CONCLUSIONS In newborn piglets undergoing CPB, the presence of a PDA results in reduced tissue oxygenation to the brain but not to other organs. In general, perfusion flow rates of 175 ml/kg/min or greater are required in order to maintain normal oxygenation of all organs except muscle.

Effect of catecholamines on activity of Na+, K+-ATPase in neonatal piglet brain during posthypoxic reoxygenation☆

The present study examined the possible role of dopamine on the response of Na(+), K(+)-ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2-4 days of age and with and without prior treatment with alpha-methyl-p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO(2)) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO(2) was 50+/-3 torr. Following ligation and hemorrhage the PcO(2) decreased to 8+/-0.5 torr. After release of ligation and reperfusion PcO(2) increased to 45+/-4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO(2) between AMT treated and untreated animals. In sham-operated animals striatal Na(+),K(+)-ATPase was 29.1+/-3 micromol/mg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Na(+), K(+)-ATPase in the striatum of newborn piglets.

Cerebral Oxygenation During Repetitive Apnea in Newborn Piglets

This study examined the effect of repetitive apnea on brain oxygen pressure in newborn piglets. Each animal was given 10 episodes of apnea, initiated by disconnecting them from the ventilator and completed by reconnecting them to the ventilation circuit. The apneic episodes were ended 30 sec after the heart rate reached the bradycardic threshold of 60 beats per min. The oxygen pressure in the microvasculature of the cortex was measured by oxygen-dependent quenching of the phosphorescence. In all experiments, the blood pressure, body temperature, and heart rate were continuously monitored. Arterial blood samples were taken throughout the experiment and the blood pH, PaO2 and PaCO2 were measured. During pre-apnea, cortical oxygen was 55.1 +/- 6.4 (SEM, n = 7) mm Hg and decreased during each apnea to 8.1 +/- 2.8 mm Hg. However, the values of cortical oxygen varied during recovery periods. Maximal oxygen levels during recovery from the first two apneic episodes were 76.8 +/- 12 mm Hg and 69.6 +/- 9 mm Hg, respectively, values higher than pre-apnea. Cortical oxygen pressure then progressively decreased following consequent apnea. In conclusion, the data show that repetitive apnea caused a progressive decrease in cortical oxygen levels in the brain of newborn piglets. This deficit in brain oxygenation can be at least partly responsible for the neurological side effects of repetitive apnea.

CREB phosphorylation following hypoxia and ischemia in striatum of newborn piglets: possible role of dopamine.

The goal of the present study was to determine the effects of hypoxia and ischemia and the role of dopamine on phosphorylation of cAMP response element binding protein (CREB) in striatum of newborn piglets. Piglets, with and without prior injection of alpha-methyl-p-tyrosine (AMT), an inhibitor of dopamine (DA) synthesis, were subjected to 1 h of hypoxia (decreased inspired oxygen pressure, FiO2, from 21 to 6%) or 1 h of ischemia (ligation of both carotid arteries and hemorrhage to reduce the systemic arterial pressure to about 40 mmHg), followed by 2 h recovery. Microvascular oxygen pressure in the cortex (pCO2) was measured by quenching of phosphorescence. Extracellular DA was determined by in vivo microdialysis. Striatal levels of phosphorylated CREB (pCREB) and total CREB were determined by Western blots. In sham-operated animals, pCO2 was 49.7 +/- 8.2 mmHg. During hypoxia and ischemia, pCO2 decreased to 6.3 +/- 1.8 mmHg and 10.2 +/- 2.7 mmHg, respectively. There was statistical difference in the level of extracellular DA during hypoxia versus ischemia. At the end of ischemia and hypoxia, the levels of DA were 96 x 10(3) +/- 24 x 10(3)% and 26 x 10(3) +/- 12 x 10(3)% of control, respectively. The pCREB measured after 2 h recovery was not changed after hypoxia but was decreased to 47.8 +/- 24% of control after ischemia. Depletion of endogenous DA abolished the ischemia-induced decrease in pCREB level. Total CREB did not change after either condition. It can be concluded that observed decreases of CREB phosphorylation following ischemia can be at least partially due to the high extracellular DA level.

Brain Injury Following Repetitive Apnea in Newborn Piglets

Repetitive apnea is associated with a significant increase in extracellular dopamine, generation of free radicals as determined by o-tyrosine formation and increase in Fluoro-Jade staining of degenerating neurons. This increase in extracellular dopamine and of hydroxyl radicals in striatum of newborn brain is likely to be at least partly responsible for the neuronal injury and neurological side effects of repetitive apnea.

Monitoring the dynamics of tissue oxygenation in vivo by phosphorescence quenching.

Tissue oxygen level is a critical determinant of both functionality and viability of cells and tissue in vivo. The oxygen level is highly regulated through complex, multilevel modulation of vascular resistance throughout the vascular tree. We have developed a method for oxygen measurement using oxygen dependent quenching of phosphorescence that is well suited for study of the regulation of tissue oxygenation in vivo. It is a minimally invasive optical method that makes it possible, in real time, to determine either mean oxygen pressure or entire histograms of the distribution of oxygen in the tissue microvasculature. When using near infrared phosphors, the measurements sample the blood volume throughout the tissue between the excitation and collection sites. By measuring phosphorescence lifetimes instead of intensity, interference by other pigments in the tissue that absorb or fluoresce at the measurement wavelengths is avoided. Since the strength of the signal is inversely related to the oxygen pressure, tissue regions with relatively low oxygen (hypoxia) can be readily identified. Calibration of the oxygen dependence of phosphorescence is absolute, eliminating the potential errors due to altered calibration, and the lifetime measurements do not «drift» over time of measurement.

Altered Gene Expression Following Cardiopulmonary Bypass and Circulatory Arrest

This study investigated the effects of normothermic cardiopulmonary bypass (CPB) and circulatory arrest (DHCA) on expression of specific genes in neonatal piglet brain. CPB was performed through the chest at 100 ml/kg/min for 2 hrs at 37 degrees C. In the second group of animals, CPB was begun as described above and then animals were cooled to a nasopharyngeal/brain temperature of 18 degrees C. When the brain temperature reached 18 degrees C, the CPB circuit was turned off. After 60 min of circulatory arrest (DHCA), CPB was resumed at 100 ml/kg/min, and the piglets were rewarmed to a temperature of 36 degrees C. In both groups, the animals remain sedated, paralyzed, mechanically ventilated, and continuously monitored throughout a four hour study period after CPB. Oxygen pressure in the microvasculature of the cortex was measured by oxygen dependent quenching of phosphorescence. The aRNA technique was used to assess mRNA steady-state levels in the brain tissue. Control oxygen pressure (pre-bypass) was 61 +/- 5 Torr and during CPB this decreased to 32 +/- 7 Torr on the beginning of bypass and to 36 +/- 5 Torr at the end of bypass. During the recovery period, cortical oxygenation steadily decreased, reaching 29 +/- 8 Torr at the end of the four hours period. Cortical oxygen decreased during DHCA to near zero and during rewarming and recovery increased to 35 +/- 6 Torr. Measurements of gene expression following CPB revealed significantly increased levels of mRNA for NMDAR1, DARPP-32, CamKII, GluR1, and D1AR. DHCA caused changes similar to those for CPB in levels of mRNA for NMDAR1, DARPP-32, CamKII and GluR1. In contrast, DHCA caused significantly increased levels of mRNA for GluR6 and GABRB1. There was no significant alteration in the level of D1AR following DHCA. The results showed that DHCA caused much larger alterations in gene expression in the critical metabolic signaling pathways tested than did CPB.

Effect of Hypoxia and Ischemia on Expression of Selected Genes in Brain of Newborn Piglets

In the brain, function is acutely dependent on delivery of oxygen at a pressure sufficient to maintain the cellular metabolic status. Interruption of oxygen delivery leads to loss of functional integrity (consciousness) within seconds, with progressive deterioration occurring during periods of oxygen deprivation. This oxygen deprivation can result from either decreased oxygen content in the blood (hypoxic-hypoxia) or low blood flow (ischemia), although these names are frequently used interchangeably in the literature. It has been shown, however, that the physiological response and neuropathology of brain are different for hypoxia than for ischemia. In the present study, we investigated the effects of hypoxic-hypoxia and of ischemic-hypoxia on cortical oxygenation and the expression of specific genes in neonatal piglet brain. Experimental conditions were selected such that the models had similar values for the mean cortical oxygenation. The results show that the changes in expression of the selected genes were significantly greater following ischemic-hypoxia than in hypoxichypoxia.