Cerebral expression of neuroglobin and cytoglobin after deep hypothermic circulatory arrest in neonatal piglets.

INTRODUCTION: Deep hypothermic circulatory arrest (DHCA) is used in corrective cardiac surgery for complex congenital heart disease. Endogenous protective mechanisms may be responsible for the prevention of brain damage after hypothermic ischemia. Neuroglobin and cytoglobin are expressed in brain cells and appear to modulate hypoxic-ischemic brain injury. However, their neuroprotective potency is still not understood. Thus the aim of this study was to detect the influence exerted by DHCA on their expression. METHODS: The effects of DHCA were analyzed in a neonatal piglet model with cardiopulmonary bypass, DHCA of 60 and 120 min and subsequent reperfusion of 6h. Complete histological analysis and changes in the mRNA expression of neuroglobin and cytoglobin were measured in the brain. RESULTS: In comparison to animals without DHCA, neuroglobin expression was stable after 60 min DHCA and neuronal cell necrosis in the cortex was mild (< 10 %). Neuroglobin expression was significantly reduced after 120 min DHCA, which was accompanied by substantial neuronal cell necrosis (> 50 %). Cytoglobin expression did not differ significantly between animals with neuronal necrosis vs. sham. CONCLUSION: Constitutive expression levels of neuroglobin may explain the mild neuronal injury after 60 min DHCA. Significant neuronal cell death correlates with reduced neuroglobin expression and might reflect a limited capacity to compensate for ischemic injury. Both respiratory cell proteins may constitute attractive targets for therapeutic modulation of gene regulation, but further studies are necessary.

Large-dose pretreatment with methylprednisolone fails to attenuate neuronal injury after deep hypothermic circulatory arrest in a neonatal piglet model.

Conflicting results have been reported with regard to the neuroprotective effects of steroid treatment with cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). We evaluated the mode and severity of neuronal cell injury in neonatal piglets after prolonged DHCA and the possible neuroprotective effect of systemic pretreatment (>6 h before surgery) with large-dose methylprednisolone (MP). Nineteen neonatal piglets (age, <10 days; weight, 2.1 +/- 0.5 kg) were randomly assigned to 2 groups: 7 animals were pretreated with large-dose systemic MP (30 mg/kg) 24 h before surgery, and 12 animals without pharmacological pretreatment (saline) served as control groups. All animals were connected to full-flow CPB with cooling to 15 degrees C and 120 min of DHCA. After rewarming to 38.5 degrees C with CPB, animals were weaned from CPB and survived 6 h before they were killed, and the brain was prepared for light and electron microscopy, immunohistochemistry, and TUNEL-staining. Quantitative histological studies were performed in hippocampus, cortex, cerebellum, and caudate nucleus. Systemic pretreatment with large-dose MP lead to persistent hyperglycemia but no significant changes of cerebral perfusion. Necrotic and apoptotic neuronal cell death were detected in all analyzed brain regions after 120 min of DHCA. In comparison to the control group, large-dose pretreatment with systemic MP lead to an increase of necrotic neuronal cell death and induced significant neuronal apoptosis in the dentate gyrus of the hippocampus (P = 0.001). In conclusion, systemic pretreatment with large-dose MP fails to attenuate neuronal cell injury after prolonged DHCA and induces regional neuronal apoptosis in the dentate gyrus.

Release patterns of astrocytic and neuronal biochemical markers in serum during and after experimental settings of cardiac surgery.

OBJECTIVE: Brain injury and altered psychomotor development in infants, children and adults after cardiac surgery using cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) is still a matter of concern. Early diagnosis and identification of brain injury that has occurred or is ongoing by measurement of biochemical markers in serum may have diagnostic and prognostic value. The aim of the experimental studies in an animal model was therefore to investigate the release patterns of astroglial and neuronal markers in serum and to determine the morphological and immunohistochemical changes in the brain of animals undergoing similar perfusion conditions of CPB and a period of DHCA. METHODS: Fourteen New Zealand rabbits, (weight, 3.1 +/- 0.25 kg) were anesthetized, intubated and mechanically ventilated. Four animals were sham operated and served as controls. After median sternotomy the animals were connected to CPB by cannulation of the aorta and right atrium. Full flow CPB (200-250 ml/kg/min) was initiated to achieve homogeneous systemic cooling. Circulatory arrest of 60 minutes was induced when rectal and nasopharyngeal temperature of 14 degrees C was achieved. After rewarmed reperfusion and establishment of stable cardiac ejection the animals were weaned from CPB and monitored for 6 hours. Then the animals were killed, the brain was immediately removed and cut in standardized sections. These were fixated, embedded in paraffin and stained for further quantitative histological studies. In the brain astrocyte reactivity for S-100B was assessed immunocytochemically (DPC Immustain Los Angeles, USA). Monoclonal mouse anti-human neurospecific enolase (NSE) antibody was used for the localization of NSE in the fixed and paraffin embedded brain (NSE-DAKO, H14). The concentrations of S-100B protein and neurospecific enolase (NSE) in the serum were analyzed using a commercially available immunoluminometric assay (LIA-mat, Sangtec 100, Byk-Sangtec). Immunospecific monoclonal anti-parvalbumin antibody was used for the detection of parvalbumin in the brain. Serum concentrations of parvalbumin were analyzed using a newly developed ELISA method. RESULTS: In all experimental animals a significant increase of the serum concentration of the astroglial protein S-100B was found immediately after reperfusion and the termination of CPB. In contrast the serum levels of the neuronal proteins parvalbumin and NSE were not increased, but rather decreased. Light microscopy and electron microscopy revealed perivascular astrocytic swelling and minor neuronal cell injury. In comparison to the sham operated animals, increased immunohistochemical staining of S-100B was found. This increased reactivity of S100B antibody was found in the astrocytic processes with immediate connection to the perivascular space and around the perivascular oedema. The immunocytochemical stainings for NSE and parvalbumin in the neuronal cells was not different from that of sham-operated animals and indicated well preserved neurons.