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 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.

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.

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.

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.

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.

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).

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.

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.

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.

Tissue oxygen tension during regional low-flow perfusion in neonates.

OBJECTIVE: We examined cerebral cortical and peripheral organ tissue Po(2) values in a neonatal piglet model of regional low-flow perfusion. METHODS: Twenty-one neonatal piglets were placed on cardiopulmonary bypass, were cooled to 18 degrees C, then underwent either deep hypothermic circulatory arrest or regional low-flow perfusion at 20 or 40 mL/(kg x min) for 90 minutes. Regional low-flow perfusion was carried out by advancing the aortic cannula into the proximal innominate artery. Tissue mean Po(2) and Po(2) distribution were measured in the cerebral cortex, liver, small bowel, and skeletal muscle through the principle of oxygen-dependent quenching of phosphorescence. Measured quantities were compared by analysis of variance or the Fisher exact test. RESULTS: During regional low-flow perfusion, axillary and femoral arterial pressures, respectively, were 55 +/- 15 and 8 +/- 4 mm Hg at 40 mL/(kg x min) and 37 +/- 10 mm Hg (P =.04) and 17 +/- 5 mm Hg (P =.08) at 20 mL/(kg x min). Venous saturations were 95% +/- 6% at 40 mL/(kg x min) and 84% +/- 6% at 20 mL/(kg x min) (P =.03 at 15, 30, and 45 minutes). Cortical Po(2) was similar to prebypass values during regional low-flow perfusion at 40 mL/(kg x min) (53 +/- 5 mm Hg) but declined during reperfusion and recovery. Cortical Po(2) was lower than before bypass during low-flow perfusion at 20 mL/(kg x min) (38 +/- 7 mm Hg) but increased during reperfusion. Po(2) in liver and bowel was less than 10 mm Hg during low-flow perfusion at both 20 and 40 mL/(kg x min). Fraction of oxygen distribution with Po(2) lower than 15 mm Hg was less during perfusion at 40 mL/(kg x min) than at 20 mL/(kg x min) (P =.001). Three of 6 piglets that received a 40-mL/(kg x min) flow rate had significant upper torso edema, metabolic acidosis, and an unstable recovery period, whereas zero of 6 piglets that received a 20-mL/(kg x min) flow rate did. CONCLUSIONS: In a piglet model, regional low-flow perfusion at 20 mL/(kg x min) resulted in lower cortical tissue oxygenation but better recovery than did perfusion at 40 mL/(kg x min). Neither flow rate adequately oxygenated organs in the lower torso.

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.

Comparison of low-flow cardiopulmonary bypass and circulatory arrest on brain oxygen and metabolism.

BACKGROUND: In the neonatal brain we measured oxygen (Bo(2)), extracellular striatal dopamine (DA), and striatal tissue levels of ortho-tyrosine (o-tyr) during low-flow cardiopulmonary bypass (LFCPB) or deep hypothermic circulatory arrest (DHCA) and the post-bypass recovery period. METHODS: Newborn piglets were assigned to sham (n = 6), LFCPB (n = 8), or DHCA (n = 6) groups. Animals were cooled to 18 degrees C and underwent DHCA or LFCPB (20 mL x kg(-1) x min(-1)) for 90 minutes. The Bo(2) was measured by quenching the phosphorescence, DA by microdialysis, and hydroxyl radicals by o-tyr levels. The results are presented as the mean +/- SD (p < 0.05 was significant). RESULTS: Baseline Bo(2) was between 45 to 60 mm Hg. At the end of LFCPB, Bo(2) was 10.5 +/- 1.2 mm Hg. By 5 and 30 minutes of arrest during DHCA, Bo(2) fell to 4.2 +/- 2.5 mm Hg and 1.4 +/- 0.7 mm Hg, respectively. Compared with control, extracellular DA did not change during LFCPB. During DHCA extracellular levels of DA increased, by 750-fold from baseline at 45 minutes and to a maximum of 53000-fold at 75 minutes. After 2 hours of recovery from DHCA, the o-tyr within the striatum increased about sixfold as compared with control. There was no change in o-tyr measured after LFCPB. CONCLUSIONS: In DHCA, but not LFCPB, levels of DA and o-tyr increased considerably in the striatum of piglets, a finding that may indicate the exhaustion of cellular energy levels and contribute substantially to cellular injury.

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.

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 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.