No abstract

Objectives To investigated the effect of Granulocyte-colony stimulating factor (G-CSF) on expression of proteins that regulate apoptosis in newborn piglet brain following cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). Methods The newborn piglets were assigned to three groups: 1/DHCA (30 min of DHCA, 1hr of low-flow CPB), 2/DHCA with prior injection of G-CSF (17µg/kg 2hrs before CPB) and 3/sham-operated. After 2hrs 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 one way ANOVA. p<0.05 was considered significant. Results In the frontal cortex, only Fas-L expression was significantly lower in G-CSF group when compared with DHCA group. In the hippocampus, G-CSF increased Bcl-2 (54.3±6.4 vs. 32.3±2.2, p=0.001) and pAkt (141.4±19 vs. 95.9±21.1, p=0.047) when compared to DHCA group. Caspase-3, Bax, Fas, Fas-L, DR6 and pJAK2 levels were unchanged. The Bcl-2/Bax ratio was 0.33 for DHCA and 0.93 for G-CSF groups (p=0.02). In the striatum, when compared to DHCA group, G-CSF group had higher levels of Bcl-2 (50.3±7.4 vs. 31.8±3.8, p=0.01), pAkt (132.7±12.3 vs. 14±1.34, p=2.3×106), pJAK2 (126±17.4 vs.77.9±13.6, p=0.011), and lower Caspase-3 (12.8±5.0 vs. 32.2±11.5, p=0.033), Fas (390±31 vs. 581±74, p=0.038), Fas-L (20.5±11.5 vs. 57.8±15.6, p=0.04) and DR6 (57.4± 4.4 vs. 108.8±13.4, p=0.007). The Bcl-2/Bax ratio was 0.25 for DHCA and 0.44 for G-CSF groups (p=0.046). Conclusions In the piglet model of hypoxic brain injury, G-CSF decreases pro-apoptotic signaling, particularly in the striatum.

No abstract

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 °C, subjected to 30 min of DHCA followed by 1 h of low flow at 20 ml/kg/min, rewarmed to 37 °C (normothermia) or to 33 °C (hypothermia), separated from CPB, and monitored for 6 h. 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 × 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.

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 21% or with 100% oxygen. Following 6-hrs recovery the animals were euthanized, the striatum, frontal cortex, midbrain and hippocampus were dissected out and used to determine levels of Bcl-2, Bax, Caspase-3, p-Akt and p-CREB.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 striatum, was higher with 21% vs.100% group (263±33% vs.197±35%, p<0.05, respectively) but not different in hippocampus. p-Akt expression was higher with 100% vs. 21% oxygen in 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 hippocampus (217±41% vs.132±30%, p<0.01) with not changes in striatum. Much smaller or not significant differences between 100% vs.21% oxygen groups were observed in 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 apneic dependent injury when resuscitation was conducted with 100%, rather than 21%, oxygen enriched gas.