Transcriptome profiling reveals activation of inflammation and apoptosis in the neonatal striatum after deep hypothermic circulatory arrest.

OBJECTIVES: Brain injury, leading to long-term neurodevelopmental deficits, is a major complication in neonates undergoing cardiac surgeries. Because the striatum is one of the most vulnerable brain regions, we used mRNA sequencing to unbiasedly identify transcriptional changes in the striatum after cardiopulmonary bypass and associated deep hypothermic circulatory arrest. METHODS: Piglets were subjected to cardiopulmonary bypass with deep hypothermic circulatory arrest at 18°C for 30 minutes and then recovered for 6 hours. mRNA sequencing was performed to compare changes in gene expression between the striatums of sham control and deep hypothermic circulatory arrest brains. RESULTS: We found 124 significantly upregulated genes and 74 significantly downregulated genes in the striatums of the deep hypothermic circulatory arrest group compared with the sham controls. Pathway enrichment analysis demonstrated that inflammation and apoptosis were the strongest pathways activated after surgery. Chemokines CXCL9, CXCL10, and CCL2 were the top upregulated genes with 32.4-fold, 22.2-fold, and 17.6-fold increased expression, respectively, in the deep hypothermic circulatory arrest group compared with sham controls. Concomitantly, genes involved in cell proliferation, cell-cell adhesion, and structural integrity were significantly downregulated in the deep hypothermic circulatory arrest group. Analysis of promoter regions of all upregulated genes revealed over-representation of nuclear factor-kB transcription factor binding sites. CONCLUSIONS: Our study provides a comprehensive view of global transcriptional changes in the striatum after deep hypothermic circulatory arrest and found strong activation of both inflammatory and apoptotic signaling pathways in the deep hypothermic circulatory arrest group. Nuclear factor-kB, a key driver of inflammation, appears to be an upstream regulator of the majority of the upregulated genes; hence, nuclear factor-kB inhibitors could potentially be tested for beneficial effects on neurologic outcome.

Whole Body Periodic Acceleration (pGz) as a non-invasive preconditioning strategy for pediatric cardiac surgery.

We hypothesized that pGz has cardio and neuroprotective effects due to upregulation of pathways which include eNOS, anti-apoptotic, and anti-inflammatory pathways. We analyze protein expression of these pathways in the brain of neonatal piglets, as well as report on the myocardial function after Deep Hypothermic Circulatory Arrest (DHCA) and pGz preconditioning. Animal data affirms both a cardio and neuroprotective role for pGz. These findings suggest that pGz can be a simple, non-invasive cardio and neuroprotective strategy preconditioning strategy in children requiring surgical intervention.

Granulocyte colony-stimulating factor significantly decreases density of hippocampal caspase 3-positive nuclei, thus ameliorating apoptosis-mediated damage, in a model of ischaemic neonatal brain injury.

OBJECTIVES: Ischaemic brain injury is a major complication in patients undergoing surgery for congenital heart disease, with the hippocampus being a particularly vulnerable region. We hypothesized that neuronal injury resulting from cardiopulmonary bypass and associated circulatory arrest is ameliorated by pretreatment with granulocyte colony-stimulating factor (G-CSF), a cytokine and an anti-apoptotic neurotrophic factor. METHODS: In a model of ischaemic brain injury, 4 male newborn piglets were anaesthetized and subjected to deep hypothermic circulatory arrest (DHCA) (cooled to 18°C, DHCA maintained for 60 min, rewarmed and recovered for 8-9 h), while 4 animals received G-CSF (34 µg/kg, intravenously) 2 h prior to the DHCA procedure. At the end of each experiment, the animals were perfused with a fixative, the hippocampus was extracted, cryoprotected, cut and the brain sections were immunoprocessed for activated caspase 3, a pro-apoptotic factor. Immunopositive neuronal nuclei were counted in multiple counting boxes (440 × 330 µm) centred on the CA1 or CA3 hippocampal regions and their mean numbers compared between the different treatment groups and regions. RESULTS: G-CSF pretreatment resulted in significantly lower counts of caspase 3-positive nuclei per counting box in both the CA1 [52.2 ± 9.3 (SD) vs 61.6 ± 8.4, P < 0.001] and CA3 (41.2 ± 6.9 vs 60.4 ± 16.4, P < 0.00002) regions of the hippocampus as compared to DHCA groups. The effects of G-CSF were significant for pyramidal cells of both regions and for interneurons in the CA3 region. CONCLUSIONS: In an animal model of ischaemic brain injury, G-CSF reduces neuronal injury in the hippocampus, thus potentially having beneficial effect on neurologic outcomes.

Granulocyte colony stimulating factor reduces brain injury in a cardiopulmonary bypass-circulatory arrest model of ischemia in a newborn piglet.

Ischemic brain injury continues to be of major concern in patients undergoing cardiopulmonary bypass (CPB) surgery for congenital heart disease. Striatum and hippocampus are particularly vulnerable to injury during these processes. Our hypothesis is that the neuronal injury resulting from CPB and the associated circulatory arrest can be at least partly ameliorated by pre-treatment with granulocyte colony stimulating factor (G-CSF). Fourteen male newborn piglets were assigned to three groups: deep hypothermic circulatory arrest (DHCA), DHCA with G-CSF, and sham-operated. The first two groups were placed on CPB, cooled to 18 °C, subjected to 60 min of DHCA, re-warmed and recovered for 8-9 h. At the end of experiment, the brains were perfused, fixed and cut into 10 µm transverse sections. Apoptotic cells were visualized by in situ DNA fragmentation assay (TUNEL), with the density of injured cells expressed as a mean number ± SD per mm(2). The number of injured cells in the striatum and CA1 and CA3 regions of the hippocampus increased significantly following DHCA. In the striatum, the increase was from 0.46 ± 0.37 to 3.67 ± 1.57 (p = 0.002); in the CA1, from 0.11 ± 0.19 to 5.16 ± 1.57 (p = 0.001), and in the CA3, from 0.28 ± 0.25 to 2.98 ± 1.82 (p = 0.040). Injection of G-CSF prior to bypass significantly reduced the number of injured cells in the striatum and CA1 region, by 51 and 37 %, respectively. In the CA3 region, injured cell density did not differ between the G-CSF and control group. In a model of hypoxic brain insult associated with CPB, G-CSF significantly reduces neuronal injury in brain regions important for cognitive functions, suggesting it can significantly improve neurological outcomes from procedures requiring DHCA.

Effect of granulocyte-colony stimulating factor on expression of selected proteins involved in regulation of apoptosis in the brain of newborn piglets after cardiopulmonary bypass and deep hypothermic circulatory arrest.

OBJECTIVE: The study objective was to investigate the effect of granulocyte-colony stimulating factor on the expression of proteins that regulate apoptosis in newborn piglet brain after cardiopulmonary bypass and deep hypothermic circulatory arrest. METHODS: The newborn piglets were assigned to 3 groups: (1) deep hypothermic circulatory arrest (30 minutes of deep hypothermic circulatory arrest, 1 hour of low-flow cardiopulmonary bypass); (2) deep hypothermic circulatory arrest with prior injection of granulocyte-colony stimulating factor (17 µg/kg 2 hours before cardiopulmonary bypass); and (3) sham-operated. After 2 hours of post-bypass recovery, the frontal cortex, striatum, and hippocampus were dissected. The expression of proteins was measured by gel electrophoresis or protein arrays. Data are presented in arbitrary units. Statistical analysis was performed using 1-way analysis of variance. RESULTS: In the frontal cortex, only Fas ligand expression was significantly lower in the granulocyte-colony stimulating factor group when compared with the deep hypothermic circulatory arrest group. In the hippocampus, granulocyte-colony stimulating factor increased Bcl-2 (54.3 ± 6.4 vs 32.3 ± 2.2, P = .001) and serine/threonine-specific protein kinase (141.4 ± 19 vs 95.9 ± 21.1, P = .047) when compared with deep hypothermic circulatory arrest group. Caspase-3, Bax, Fas, Fas ligand, death receptor 6, and Janus protein tyrosine kinase 2 levels were unchanged. The Bcl-2/Bax ratio was 0.33 for deep hypothermic circulatory arrest group and 0.93 for the granulocyte-colony stimulating factor group (P = .02). In the striatum, when compared with the deep hypothermic circulatory arrest group, the granulocyte-colony stimulating factor group had higher levels of Bcl-2 (50.3 ± 7.4 vs 31.8 ± 3.8, P = .01), serine/threonine-specific protein kinase (132.7 ± 12.3 vs 14 ± 1.34, P = 2.3 × 10(6)), and Janus protein tyrosine kinase 2 (126 ± 17.4 vs 77.9 ± 13.6, P = .011), and lower levels of caspase-3 (12.8 ± 5.0 vs 32.2 ± 11.5, P = .033), Fas (390 ± 31 vs 581 ± 74, P = .038), Fas ligand (20.5 ± 11.5 vs 57.8 ± 15.6, P = .04), and death receptor 6 (57.4 ± 4.4 vs 108.8 ± 13.4, P = .007). The Bcl-2/Bax ratio was 0.25 for deep hypothermic circulatory arrest and 0.44 for the granulocyte-colony stimulating factor groups (P = .046). CONCLUSIONS: In the piglet model of hypoxic brain injury, granulocyte-colony stimulating factor decreases proapoptotic signaling, particularly in the striatum.

Measuring oxygen in living tissue: intravascular, interstitial, and "tissue" oxygen measurements.

Oxygen dependent quenching of phosphorescence has been used to measure the oxygen pressure in both the vasculature of the microcirculation and the interstitial spaces of resting muscle tissue. Oxygen sensitive molecules were either dissolved in the blood (intravascular space) or micro-injected into the interstitial space and the distributions, histograms, of the oxygen pressure were measured. The mean oxygen pressures are higher in the blood than in the interstitial space but the oxygen pressures in the lowest 10% of the two spaces were not significantly different, indicating there is minimal (< 1 mm Hg) oxygen gradient between the two spaces in the capillary bed.

Effect of deep hypothermic circulatory arrest followed by low-flow cardiopulmonary bypass on brain metabolism in newborn piglets: comparison of pH-stat and α-stat management.

OBJECTIVE: To compare the effects of pH-stat and α-stat management before deep hypothermic circulatory arrest followed by a period of low-flow (two rates) cardiopulmonary bypass on cortical oxygenation and selected regulatory proteins: Bax, Bcl-2, Caspase-3, and phospho-Akt. DESIGN: Piglets were placed on cardiopulmonary bypass, cooled with pH-stat or α-stat management to 18 °C over 30 mins, subjected to 30-min deep hypothermic circulatory arrest and 1-hr low flow at 20 mL/kg/min (LF-20) or 50 mL/kg/min (LF-50), rewarmed to 37 °C, separated from cardiopulmonary bypass, and recovered for 6 hrs. SUBJECTS: Newborn piglets, 2-5 days old, assigned randomly to experimental groups. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Cortical oxygen was measured by oxygen-dependent quenching of phosphorescence; proteins were measured by Western blots. The means from six experiments ± sem are presented as % of α-stat. Significance was determined by Student's t test. For LF-20, cortical oxygenation was similar for α-stat and pH-stat, whereas for LF-50, it was significantly better using pH-stat. For LF-20, the measured proteins were not different except for Bax in the cortex (214 ± 24%, p = .006) and hippocampus (118 ± 6%, p = .024) and Caspase 3 in striatum (126% ± 7%, p = .019). For LF-50, in pH-stat group: In cortex, Bax and Caspase-3 were lower (72 ± 8%, p = .001 and 72 ± 10%, p = .004, respectively) and pAkt was higher (138 ± 12%, p = .049). In hippocampus, Bcl-2 and Bax were not different but pAkt was higher (212 ± 37%, p = .005) and Caspase 3 was lower (84 ± 4%, p = .018). In striatum, Bax and pAkt did not differ, but Bcl-2 increased (146 ± 11%, p = .001) and Caspase-3 decreased (81 ± 11%, p = .042). CONCLUSIONS: In this deep hypothermic circulatory arrest-LF model, when flow was 20 mL/kg/min, there was little difference between α-stat and pH-stat management. However, for LF-50, pH-stat management resulted in better cortical oxygenation during recovery and Bax, Bcl-2, pAk, and Caspase-3 changes were consistent with lesser activation of proapoptotic signaling with pH-stat than with α-stat.

The effect of hypothermia on neuronal viability following cardiopulmonary bypass and circulatory arrest in newborn piglets.

OBJECTIVE: To determine the effect of recovery with mild hypothermia after cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) on the activity of selected key proteins involved in initiation (Bax, Caspase-3) or inhibition of apoptotic injury (Bcl-2, increased ratio Bcl-2/Bax) in the brain of newborn piglets. METHODS: The piglets were placed on CPB, cooled with pH-stat management to 18 degrees C, subjected to 30 min of DHCA followed by 1h of low flow at 20 ml/kg/min, rewarmed to 37 degrees C (normothermia) or to 33 degrees C (hypothermia), separated from CPB, and monitored for 6h. Expression of above proteins was measured in striatum, hippocampus and frontal cortex by Western blots. The results are mean for six experiments+/-SEM. RESULTS: There were no significant differences in Bcl-2 level between normothermic and hypothermic groups. The Bax levels in normothermic group in cortex, hippocampus and striatum were 94+/-9, 136+/-22 and 125+/-34 and decreased in the hypothermic group to 59+/-17 (p=0.028), 70+/-6 (p=0.002) and 48+/-8 (p=0.01). In cortex, hippocampus and striatum Bcl-2/Bax ratio increased from 1.23, 0.79 and 0.88 in normothermia to 1.96, 1.28 and 2.92 in hypothermia. Expression of Caspase-3 was 245+/-39, 202+/-74 and 244+/-31 in cortex, hippocampus and striatum in the normothermic group and this decreased to 146+/-24 (p=0.018), 44+/-16 (p=7 x 10(-7)) and 81+/-16 (p=0.01) in the hypothermic group. CONCLUSION: In neonatal piglet model of cardiopulmonary bypass with circulatory arrest, mild hypothermia during post bypass recovery provides significant protection from cellular apoptosis, as indicated by lower expression of Bax and Caspase-3 and an increased Bcl-2/Bax ratio. The biggest protection was observed in striatum probably by decreasing of neurotoxicity of striatal dopamine.

Resuscitation with 100%, compared with 21%, oxygen following brief, repeated periods of apnea can protect vulnerable neonatal brain regions from apoptotic injury.

PURPOSE: To determine the effect of repeated intermittent apnea and resuscitation with 100% vs. 21% oxygen enriched gas on levels of key regulatory proteins contributing to cell death (Bax, Caspase-3) or protecting neurons from hypoxic/ischemic injury (Bcl-2, p-Akt, p-CREB). METHODS: The anaesthetized, mechanically ventilated newborn piglets underwent 10 episodes of apnea with resuscitation either with 100% or with 21% oxygen. Following 6h recovery the animals were sacrificed painlessly, the brain dissected out and used to determine levels of Bcl-2, Bax, Caspase-3, p-Akt and p-CREB in the striatum, frontal cortex, midbrain and hippocampus were studied. RESULTS: In hippocampus and striatum, Bcl-2 expression was higher with 100% vs. 21% group (173+/-29% vs. 121+/-31%, p<0.05 and 189+/-10% vs. 117+/-47%, p<0.01, respectively) whereas the Bax expression was lower (88+/-3% vs. 100+/-9%, p<0.05 and 117+/-5% vs. 133+/-10%, p<0.05, respectively). Expression of Caspase-3 in the striatum, was lower with 100% vs. 21% group (197+/-35% vs. 263+/-33%, p<0.05, respectively) but not different in the hippocampus. p-Akt expression was higher with 100% vs. 21% oxygen in the hippocampus and striatum (225+/-44% vs. 108+/-35%, p<0.01 and 215+/-12% vs. 164+/-16%, p<0.01, respectively). The p-CREB expression was higher with 100% vs. 21% oxygen resuscitation in the hippocampus (217+/-41% vs. 132+/-30%, p<0.01) with no changes in striatum. Much smaller or insignificant differences between 100% vs. 21% oxygen groups were observed in the frontal cortex and midbrain, respectively. CONCLUSION: In neonatal piglet model of intermittent apnea, selectively vulnerable regions of brain (striatum and hippocampus) are better protected from apoptotic injury when resuscitation was conducted with 100%, rather than 21%, oxygen.

Response of brain oxygenation and metabolism to deep hypothermic circulatory arrest in newborn piglets: comparison of pH-stat and alpha-stat strategies.

BACKGROUND: To determine the effect of pH-stat as compared with alpha-stat management on brain oxygenation, level of striatal extracellular dopamine, phosphorylation, and levels of protein kinase B (Akt) and cyclic adenosine 3', 5'-monophosphate response element-binding protein (CREB), and levels of extracellular signal-regulated kinase (ERK)1/2, Bcl-2, and Bax in a piglet model of deep hypothermic circulatory arrest (DHCA). METHODS: The piglets were placed on cardiopulmonary bypass (CPB), cooled with pH-stat or alpha-stat to 18 degrees C, subjected to 90 minutes of DHCA, rewarmed, weaned from CPB, and maintained for two hours recovery. The cortical oxygen was measured by: quenching of phosphorescence; dopamine by microdialysis; phosphorylation of CREB (p-CREB), ERK (p-ERK) 1/2, Akt (p-Akt), and level of Bcl-2, Bax by Western blots. RESULTS: Oxygen pressure histograms for the microvasculature of the cortex show substantially higher oxygen levels during cooling and during the oxygen depletion period after cardiac arrest (up to 15 minutes) when using pH-stat compared with alpha-stat management. Significant increases in dopamine occurred at 45 minutes and 60 minutes of DHCA in the alpha-stat and pH-stat groups, respectively. The p-CREB and p-Akt in the pH-stat group were significantly higher than in the alpha-stat group (140 +/- 9%, p < 0.05 and 125 +/- 6%, p < 0.05, respectively). There was no significant difference in p-ERK1/2 and Bax. The Bcl-2 increased in the pH-stat group to 121 +/- 4% (p < 0.05) compared with the alpha-stat group. The ratio Bcl-2:Bax increased in the pH-stat group compared with the alpha-stat group. CONCLUSIONS: The increase in p-CREB, p-Akt, Bcl-2, Bcl-2/Bax, and delay in increase of dopamine indicated that pH-stat, in the piglet model, prolongs "safe" time of DHCA and provides some brain protection against ischemic injury.

Brain oxygen and metabolism is dependent on the rate of low-flow cardiopulmonary bypass following circulatory arrest in newborn piglets.

OBJECTIVE: To determine the optimum rate of low-flow hypothermic cardiopulmonary bypass (LF), following circulatory arrest (DHCA) on brain oxygenation (bO(2)), extracellular dopamine (DA), phosphorylation of select neuroregulatory proteins responsible for neuronal injury, and survival following ischemic brain injury: CREB, Erk1/2, Akt, Bcl-2, and Bax. METHODS: The piglets were placed on cardiopulmonary bypass (CPB) and cooled to 18 degrees C. They were then subjected to 30 min of DHCA followed by 1h of LF at 20, 50, or 80 ml/(kg/min), rewarmed, separated from CPB, and maintained for 2h. The bO(2) was measured by quenching of phosphorescence; DA by microdialysis; phosphorylation of CREB, ERK1/2, Akt, Bcl-2, and Bax by Western blots. The results are means+/-SD for seven experiments. RESULTS: Pre-bypass bO(2) was 47.4+/-4.2 mmHg and decreased to 1.9+/-0.8 mmHg during DHCA. At the end of LF at 20, 50, and 80 ml/(kg/min), bO(2) was 11.8+/-1.6, 26+/-1.8, and 33.9+/-2.6 mmHg, respectively. The DA increased 510-fold relative to control (p<0.001) by 15 min of LF-20 with maximum increase occurring at 45 min. With LF-50, increase in DA was not statistically significant and no increase was observed when LF-80 was used. Bcl-2 immunoreactivity increased after LF-50 and LF-80 (140+/-14.5%, p<0.05 and 202+/-34%, p<0.05, respectively). Neither flow increased Bax immunoreactivity. The ratio of Bcl-2/Bax, pCREB, pAkt, pErk increased significantly with increasing the flow rate of LF. CONCLUSIONS: The protective effect of LF following DHCA on brain metabolism is dependent on the flow rate. Flow-dependent increase in pCREB, pErk1/2, pAkt, increase in Bcl-2/Bax, and decrease in DA indicated that to minimize DHCA-dependent neuronal injury, LF flow should be above 50 ml/(kg/min).

Regulation of brain cell death and survival after cardiopulmonary bypass.

BACKGROUND: This study investigated the effect of low flow cardiopulmonary bypass, circulatory arrest, and selective cerebral perfusion on expression and phosphorylation of selected regulators of cell death and survival in striatum of newborn piglets. METHODS: Animals were assigned to sham operation and three experimental groups. The experimental groups were placed on bypass, cooled to 18 degrees C, and subjected to 90 minutes of deep hypothermic circulatory arrest (DHCA), low-flow cardiopulmonary bypass (LFCPB) at mL/(kg x min), or selective cerebral perfusion (SCP) at 20 mL/(kg x min), followed by rewarming and 2 hours of recovery. The oxygen pressure in the microcirculation of the cortex was measured by quenching of phosphorescence. Levels of phosphorylated and total protein were determined by Western blot analysis. RESULTS: Control oxygen pressure was 55 +/- 9 mm Hg and decreased during DHCA, LFCPB, and SCP to 1.1 +/- 0.6 mm Hg, 9.8 +/- 2.3 mm Hg, and 9.3 +/- 1.9 mm Hg, respectively (p < 0.001). After DHCA, N-terminal of Bcl-2-associated X protein (N-Bax) levels increased (295% +/- 15%, p < 0.01), B-cell leukemia protein (Bcl-2) levels decreased (31% +/- 9%, p < 0.01), and phosphorylation level of protein kinase B (pAkt) and extracellular signal-regulated kinase 1/2 (pERK1/2) did not change. After LFCPB and SCP, N-Bax and Bcl-2 levels were unchanged, pAkt levels increased (367% +/- 122%, p < 0.05 and 337% +/- 47%, p < 0.01, respectively), pERK1 (484% +/- 70% and 501% +/- 255%, respectively; p < 0.01) and pERK2 (569% +/- 128%; p < 0.001 and 494% +/- 162%; p < 0.05, respectively) levels increased, and total ERK2 levels also increased (279% +/- 90% and 153% +/- 44%, respectively, p < 0.05). CONCLUSIONS: Stable levels of Bcl-2 and Bax and the increases in pAkt and pERK1/2 after LFCPB and SCP are likely indicators of improved chances for cell survival.

Brain oxygen and metabolism during circulatory arrest with intermittent brief periods of low-flow cardiopulmonary bypass in newborn piglets.

OBJECTIVE: We performed this study to determine whether brief intermittent periods of low-flow cardiopulmonary bypass during deep hypothermic circulatory arrest would improve cortical metabolic status and prolong the "safe" time of deep hypothermic circulatory arrest. METHODS: After a 2-hour baseline, newborn piglets were placed on cardiopulmonary bypass and cooled to 18 degrees C. The animals were then subjected to 80 minutes of deep hypothermic circulatory arrest interrupted by 5-minute periods of low-flow cardiopulmonary bypass at either 20 mL x kg(-1) x min(-1) (LF-20) or 80 mL x kg(-1) x min(-1) (LF-80) during 20, 40, 60, and 80 minutes of deep hypothermic circulatory arrest. All animals were rewarmed, separated from cardiopulmonary bypass, and maintained for 2 hours (recovery). The oxygen pressure in the cerebral cortex was measured by the quenching of phosphorescence. The extracellular dopamine level in the striatum was determined by microdialysis. Results are means +/- SD. RESULTS: Prebypass oxygen pressure in the cerebral cortex was 65 +/- 7 mm Hg. During the first 20 minutes of deep hypothermic circulatory arrest, cortical oxygen pressure decreased to 1.3 +/- 0.4 mm Hg. Four successive intermittent periods of LF-20 increased cortical oxygen pressure to 6.9 +/- 1.2 mm Hg, 6.6 +/- 1.9 mm Hg, 5.3 +/- 1.6 mm Hg, and 3.1 +/- 1.2 mm Hg. During the intermittent periods of LF-80, cortical oxygen pressure increased to 21.1 +/- 5.3 mm Hg, 20.6 +/- 3.7 mm Hg, 19.5 +/- 3.95 mm Hg, and 20.8 +/- 5.5 mm Hg. A significant increase in extracellular dopamine occurred after 45 minutes of deep hypothermic circulatory arrest alone, whereas in the groups of LF-20 and LF-80, the increase in dopamine did not occur until 52.5 and 60 minutes of deep hypothermic circulatory arrest, respectively. CONCLUSIONS: The protective effect of intermittent periods of low-flow cardiopulmonary bypass during deep hypothermic circulatory arrest is dependent on the flow rate. We observed that a flow rate of 80 mL x kg(-1) x min(-1) improved brain oxygenation and prevented an increase in extracellular dopamine release.

Brain oxygenation and metabolism during selective cerebral perfusion in neonates.

OBJECTIVE: To investigate the possible neuroprotective effects of selective cerebral perfusion (SCP) during deep hypothermic circulatory arrest on brain oxygenation and metabolism in newborn piglets. METHODS: Newborn piglets 2-4 days of age, anesthetized and mechanically ventilated, were used for the study. The animals were placed on cardiopulmonary bypass, cooled to 18 degrees C and put on SCP (20 ml/(kg min)) for 90 min. After rewarming, the animals were monitored through 2h of recovery. Oxygen pressure in the microvasculature of the cortex was measured by oxygen-dependent quenching of phosphorescence. The extracellular level of dopamine in striatum was measured by microdialysis and hydroxyl radicals by ortho-tyrosine levels. Levels of phosphorylated cAMP response element binding protein (pCREB) in striatal tissue were measured by Western blots using antibodies specific for phosphorylated CREB. The results are presented as mean+/-SD (p<0.05 was significant). RESULTS: Pre-bypass cortical oxygen pressure was 48.9+/-11.3 mmHg and during the first 5 min of SCP, the peak of the histogram, corrected to 18 degrees C, decreased to 11.2+/-3.8 mmHg (p<0.001) and stayed near that value to the end of bypass. The mean value for the peak of the histograms measured at the end of SCP was 8+/-3 mmHg (p<0.001). SCP completely prevented the deep hypothermic circulatory arrest-dependent increase in extracellular dopamine and hydroxyl radicals. After SCP, there was a statistically significant increase in pCREB immunoreactivity (534+/-60%) compared to the sham-operated group (100+/-63%, p<0.005). Measurements of total CREB showed that SCP did induce a statistically significant increase in CREB as compared to sham-operated animals (168+/-31%, p<0.05). CONCLUSION: SCP, as compared to DHCA, improved cortical oxygenation and prevented increases in the extracellular dopamine and hydroxyl radicals. The increase in pCREB in the striatum following SCP may contribute to improved cellular recovery after this procedure.

Brain oxygenation and metabolism during repetitive apnea with resuscitation of 21% and 100% oxygen in newborn piglets.

The oxygen distribution in the microcirculation of the piglet's brain and striatal extracellular dopamine were determined during repetitive apnea and resuscitation with 21% or 100% oxygen. Pre-apnea cortical oxygen was 49.5+/-10.4 mm Hg and during each apnea decreased to 8+/-0.9 mm Hg. After ten apneic episodes followed by resuscitation with 21% or 100% oxygen, 7.48+/-1.6% or 2.6+/-0.5% of the tissue volume was below 10 mm Hg, respectively. Extracellular dopamine increased progressively with an increase in the number of apneas with resuscitation of 21% oxygen and at the end of ten apneic episodes it was up to 59,500+/-11,320% of control. There was no increase in extracellular dopamine during apnea resuscitated with 100% oxygen. Repetitive apnea caused progressive increase in fraction of hypoxic brain tissue in newborn. The magnitude of the increase is dependent on whether the animals were resuscitated with room air or 100% oxygen.

Circulatory arrest and low-flow cardiopulmonary bypass alter CREB phosphorylation in piglet brain.

BACKGROUND: The purpose of this study was to determine the effects of low-flow cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest followed by postbypass recovery on the phosphorylation state of transcription factor, cyclic adenosine 3', 5'-monophosphate response element-binding protein (CREB), in the striatum of neonatal brain. METHODS: Neonatal piglets (1.4 to 2.5 kg) anesthetized with isoflurane and fentanyl were put on CPB. The animals were cooled to 18 degrees C during a 20-minute period. The CPB circuit flow was then either reduced to 20 mL.kg(-1).min(-1) for 90 minutes (low-flow CPB) or turned off for 90 minutes (deep hypothermic circulatory arrest), following with a gradual increase in the flow and rewarming during a 30-minute period and a 2-hour recovery. At the end of the recovery period, the animals were rapidly euthanized, and the striata were removed and frozen for immunochemical analysis by Western blot technique using antibodies against phosphorylated and total CREB. The results are presented as mean +/- standard deviation (p < 0.05 was significant). RESULTS: Deep hypothermic circulatory arrest did not result in alteration in either the level of CREB or its degree of phosphorylation in the piglet striatum whereas after low-flow CPB, CREB phosphorylation was significantly increased (p < 0.005) and there was also an increase in CREB expression (p < 0.01). CONCLUSIONS: This study indicates that at 2 hours of recovery, low-flow CPB but not deep hypothermic circulatory arrest causes an increase in CREB phosphorylation and expression. Future studies will determine the degree to which the increase in CREB phosphorylation correlates with cell survival and neuronal injury after CPB.

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.

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.