Increased Bcl-2 Immunoreactivity at 10°C Indicates Additional Protection of Neocortex and Hippocampus during Prolonged Hypothermic Circulatory Arrest (HCA)
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Objective:
We have reported that sensory and motor neocortex and hippocampus are selectively vulnerable to injury in an acute porcine model of HCA at 18°C. Further cooling to 10°C was found to significantly reduce neurological injury during HCA in these regions. The Bcl-2 family of genes participate in pathologic apoptotic cell death. Apoptotic death is preceded by increased Bax and decreased Bcl-2 expression in cells destined to die, while increased Bcl-2 immunoreactivity is observed in cells that survive. The current study assesses the effects of HCA at 18°C on Bcl-2 expression in the porcine brain and determines whether further cooling to 10°C alters the expression of this anti-apoptotic protein.
Methods:
Twelve piglets underwent 75 minutes of HCA after cardiopulmonary bypass at 18°C (n=6) and 10°C (n=6). Four served as normal controls. After 80 minutes of gradual rewarming and reperfusion, treatment animals were sacrificed and brains were perfusion-fixed and cryopreserved. Regional patterns of Bcl-2 protein expression after HCA were characterized using immunohistochemistry. Hematoxylin and eosin histology was used as counterstaining. Bcl-2 positive cells were scored on a scale of 0 to 3 (Grade 0:negative; Grade 1:weakly positive; Grade 2:positive; Grade 3:moderately positive). Comparisons were made using the Mann Whitney U test.
Results:
Positive Bcl-2 immunostaining was observed only in the motor and sensory neocortex and hippocampus. The cerebellum, thalamus and medulla were negative for Bcl-2 immunostaining. Profound cooling to 10°C resulted in increased Bcl-2 immunostaining in the motor and sensory cortex as compared to 18°C. (P<0.05 Mann Whitney U) (Table showing that cerebral protection may be better at profound hypothermia at 10°C compared to18°C during HCA.)
Conclusions:
The motor and sensory neocortex are offered more neural protection during 10°C HCA as indicated by elevated levels of Bcl-2 expression. This is compatible with our findings of selective vulnerability of these regions at 18°C, and neuroprotection during HCA at 10°C as indicated by Tunel assay, strongly suggesting activation of the apoptotic mechanisms at this early stage. Higher levels of Bcl-2 expression in these vulnerable regions during profound hypothermia at 10°C, suggests activation of anti-apoptotic mechanisms and mechanisms of increased cell survival.
Regional Bcl-2 immunostaining | Motor Cortex | Sensory Cortex | Hippocampus |
| HCA 18° | 1.67 + 0.33 | 0.83 + 0.31 | 1 + 0.52 |
| HCA 10° | 2.5 + 0.22* | 1.8 + 0.31* | 1 + 0.45 |
| Controls | 1.8 + 0.25 | 1.8 + 0.63 | 1 + 0.41 |
| *P<0.05, Mann Whitney U (vs 18°C) |
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