Brain injury after deep hypothermic circulatory arrest (DHCA) is still a matter of concern in corrective cardiac surgery of some complex cardiovascular malformations in neonates (1,2). Understanding the pathophysiological changes, which may be related to brain injury after such a cardiac surgical procedure, is fundamental for the development of pharmacological neuroprotective interventions (3). Because of the contact of the patient’s blood with the large nonphysiological surface of the cardiopulmonary bypass (CPB), a systemic inflammatory response syndrome may develop, which is thought to aggravate ischemia-reperfusion injury in the brain and other organs (4,5). In addition to hypothermia, which plays the major role in the cerebral protection during circulatory arrest, the administration of different medications have been routinely used before and after surgery to achieve organ protection. Methylprednisolone (MP) is one of the frequently used protective drugs in cardiac surgery for the prevention systemic inflammatory response syndrome (1,4–7). Conflicting results, however, have been reported regarding the neuroprotective effects of steroids in several experimental settings (4,8,9). The precise mechanism of neuroprotective action of MP in the brain is still not clearly understood (8). Various neuroprotective mechanisms, such as dermolytic (10) or antioxidant effect (11,12), inhibition of macrophages (13) or the expression of proinflammatory cytokine tumor necrosis factor α (8,14), reduction of the release of excitatory amino acids (15), and improvement of cerebral blood flow (CBF) (9), have been suggested. Several prospective clinical studies have been performed during the last decades for the evaluation of the protective effect of MP using a dose of 30 mg/kg during cardiac surgery with improvement of cardiac performance (4,16) or pulmonary function and survival rate (17) or no clinical improvement (8).
The mode and time of drug administration, including pretreatment, type of ischemic injury, and the evaluated variables in these studies, are heterogeneous and may not necessarily have implications for clinical use of neuroprotective strategies in pediatric cardiac surgery (9,17–19). Thus, an evaluation of the effect of MP pretreatment on neuronal cell changes in the neonatal brain in association with bypass perfusion and DHCA is warranted. Because no significant neuronal cell damage was found in our preliminary experimental studies after 60 min of DHCA (21), prolonged duration of DHCA was chosen in our study to induce significant neuronal cell damage to evaluate any reduction of neuronal cell injury by MP. Similar prolonged DHCA of 120 min has been used in numerous other experimental studies (4,22) to evaluate protective strategies (23). This study was performed to evaluate the pattern of neuronal cell death and a possible improvement by pretreatment with large-dose MP in a neonatal piglet model with prolonged DHCA and subsequent reperfusion.
Nineteen neonatal piglets (age, <10 days; weight, 2.1 ± 0.5 kg body weight [BW]) were included and randomly assigned in this study with approval of the Institution of Animal Care for the State of Berlin (Reg. 0146/98). Seven animals were pretreated with large-dose (30 mg/kg of BW) MP (methylprednisolone-21-hydrogen-succinate; Urbason, Hoechst, Germany), which was administered systemically at 24 h and again at 4 h after surgery (each dose with 30 mg/kg). Twelve animals without pharmacological treatment served as the control group.
Sedation was initiated with IM ketamine 10 mg/kg BW and midazolam 0.5–1 mg/kg BW and was then continued with total IV anesthesia using fentanyl (maintenance dose, 4–10 μg · kg−1 · h−1 and midazolam 5–10 μg · kg−1 · h−1). Muscle relaxation was performed with bolus pancuronium bromide 0.1 mg/kg BW, with a maintenance dose of 0.05 mg/kg every 2–3 h. Continuous invasive arterial blood pressure monitoring and blood gas sampling were achieved with intravascular catheters in the femoral artery and vein and also in the internal jugular vein. Prophylactic antibiotic treatment consisted of IV second generation cephalosporins. During rewarming and in the postoperative phase, dopamine (2–6 μg · kg−1 · min−1) was used for inotropic support in all animals to stabilize cardiac function and to achieve a normal perfusion pressure (24). Furosemide (1–2 mg/kg) was also used to support urine production and normal urine production with 2–4 mL/kg BW per hour.
Nonpulsatile CPB was initiated with a roller pump (Stoeckert, Munich, Germany) by the use of a microporous polypropylene membrane neonatal oxygenator (Safe-Micro, Polystan, Denmark) with a 40-μm arterial filter adapted from our clinical use (Dideco/Sorin, Mirandola, Italy), which had been selected for the size and flow requirements of the animals. The priming volume consisted of 300 mL of fresh homologous blood collected from a donor pig on the day of the study and an electrolyte solution to obtain a minimal target hemoglobin value of approximately 9 mg/dL. NaHCO3 8.4% was added to the prime to achieve a normal pH value of 7.4 ± 0.05. Temperature was kept constant at 38°C ± 0.5°C. After median sternotomy and systemic heparinization (400 IU/kg), a 12–14F venous cannula was inserted into the right atrium via the auricular appendage. After a small incision within the purse string, an arterial cannula (8F) was inserted and secured with the purse-string tourniquet. The activated clotting time was kept at more than 480 s. The animals were connected to the bypass machine and normothermic CPB was established for 20 min at flow rate of 200–250 mL · kg−1 · min−1. Arterial carbon dioxide tension was maintained throughout the hypothermic CPB at 35–45 mm Hg, uncorrected for temperature according to α-stat blood gas management, because of our institutional standard in clinical use (25). With 30 min of systemic cooling, the minimal temperature of 15°C was reached, bypass perfusion was stopped, and DHCA was induced for 120 min. After that, the animals were reperfused and rewarmed for 45 min to achieve a normal rectal temperature for piglets of 38°C ± 0.5°C. The animals were then weaned from bypass and monitored closely for 6 h under general anesthesia with ventilation and stable hemodynamics. Fresh whole blood was given to keep the hemoglobin value within the normal range (9–10 g/dL) during and after CPB according to the preoperative values and as reported for neonatal piglets (24).
Three different fluorescent microspheres (FluoSpheres®, Molecular Probes Inc., Leiden, Holland) were used to evaluate regional brain blood perfusion before and after DHCA. One milliliter of microsphere-solution contains 1.0 × 106 fluorescent polystyrene microspheres with a diameter of 14 μm. The microsphere solution was mixed with 4 mL of aspirated blood and was administered through the aortic cannula within 30 s. A reference blood sample was aspirated from the femoral artery for 2 min with continuous blood flow using an aspiration pump. The hemodynamic variables and bypass flow rate were kept stable and simultaneously documented. After the end of the experiment, tissue samples were collected from representative brain regions according to standardized protocols for determination of regional CBF (rCBF). For homogeneous digestion, the samples were incubated with alkaline sodium hydroxide (NaOH) in 45°C for 48 h. After hydrolysis of the brain tissues, the microspheres were separated from the tissue suspension using a diaphragm-operated vacuum pressure pump (Millipore, Billerica, MA). The diaphragm consisted of a filter device with pore size of 10 μm. The filter devices with the retained microspheres were then dried at 50°C for 15 min. 2-ethoxyethyl-acetate was added as solvent solution to the filter papers to dissolve the different dyes from the microspheres. Extension spectra of dissolved microspheres were then quantified using a luminescence spectrometer.
Cerebral arteriovenous oxygen metabolization was calculated as a difference between the arterial (CaO2) and venous oxygen (CvO2) content according to the formula: avDO2 = CaO2 − CvO2, where CaO2 or CvO2 was calculated from the Po2 with the standardized and common formula, as previously reported (27): (1.34 × Hb × SO2/100) + 0.0031× Pao2 or Pvo2.
At the end of the 6-h postoperative period, the skull was carefully opened, and the brain was immediately removed, cut into coronal sections, and fixed for 48 h in SOMOGYI-solution (28) consisting of paraformaldehyde, glutaraldehyde, and picric acid with neutral pH values (7.4) for subsequent histological, immunohistochemical staining and electron microscopy studies. The specimens for light microscopy were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin. For electron microscopy, the tissue was dehydrated and embedded in araldite. Semi-thin sections were stained with toluidine blue. Apoptotic cell death was either assessed by light microscopy, TUNEL (terminal transferase-mediated dUTP-digoxigenin nick end-labeling; Boehringer, Mannheim, Germany), or electron microscopy.
Necrotic neuronal cell death was identified by eosinophilic cytoplasm and loss of cytoplasmatic structures and nuclear membrane integrity, according to the neuropathological criteria (29,30). Characteristic brain regions, such as the hippocampal regions CA4 and CA1, the cortical cingulate gyrus, the nucleus caudatus, and cerebellum, were studied histologically. For quantification of cell death, 10–40× magnifications and a ZEISS eyepiece graticule with 100 grid squares were used. In every studied brain region, a minimum of 500 cells were evaluated by a blinded neuropathologist, and necrotic neurons were expressed as a percentage of the total counted neuronal cell population. Apoptosis was predominantly found in the dentate gyrus. Apoptotic neuronal cell death was recognized and characterized by a sequence of morphological criteria such as chromatin condensation, nuclear blebbing, and the presence of apoptotic bodies (29,30). Normal and apoptotic cells were identified in hematoxylin and eosin stained paraffin sections, confirmed by semi-thin sections, and also counted in the defined areas of 16 mm (2) using computer-assisted morphometry in the dentate gyrus.
Mann-Whitney and Wilcoxon tests were used and processed with StatView (Cary, NC) and SPSS (version 11.0; SPSS Inc., Chicago, IL) statistical software.
The animals did not differ significantly in arterial blood gases, arterial blood pressure, and hemoglobin, rectal, and nasopharyngeal temperatures before surgery, before the induction of DHCA, and after the end of CPB, as reported in Tables 1 and 2.
The BW before surgery was similar in all groups (2380 ± 150 g) and increased significantly in all groups after the operation. Systemic pretreatment resulted in a slight reduction of postoperative BW gain in the MP-treated group (200 ± 60 g versus 350 ± 65 g; P = 0.08). The brain weight at the end of the experiment was similar in both groups without any statistical difference (34 ± 0.9 g versus 35 ± 1.2 g; not significant).
Blood glucose levels after reperfusion were significantly higher in the MP-treated animals compared with the control group (P < 0.001), as reported in Figure 1. Blood glucose levels after DHCA did not reach baseline values in the MP-treated group but normalized in the group without MP. Insulin treatment of hyperglycemia was not used in any of the animals to keep pharmacological intervention standardized in all groups.
The rCBF in both groups did not differ significantly at baseline after CPB was started. Hypothermic perfusion resulted in a significant decrease of rCBF to nearly 25%–40% of the baseline values in both groups (Fig. 2). However, after reperfusion and rewarming, the percentage recovery of rCBF from baseline in the basal ganglia and parietal and frontal lobe of the brain was slightly higher in the MP-pretreated animals without reaching statistical significance.
Cerebral arteriovenous oxygen content difference (AVDO2) showed a slight increase in the MP-treated group during the early postoperative period, with no statistical difference (P = 0.36). The AVDO2 in both groups was not different at normothermic CPB (control versus MP pretreatment = 5.1 mL/dL versus 5.4 mL/dL) and was significantly reduced during deep hypothermia (1.1 versus 1.0 mL/dL) (P = 0.01). AVDO2-values returned to preoperative values during the postoperative period (between 5 and 6 mL/dL in both groups), without any statistical difference (P = 0.48).
In this neonatal piglet model, the morphological changes after prolonged DHCA consisted of hypoxic necrotic neuronal cell death particularly in the hippocampus in both groups (Fig. 3). No significant reduction of necrotic neuronal cell death was observed in the studied representative regions of the hippocampus CA1-CA4 or the dentate gyrus, cingulate gyrus, and cerebellum after systemic large-dose MP pretreatment. Single-cell apoptosis could be observed in all brain regions investigated in both groups. However, in comparison to the animals without pretreatment, apoptotic neuronal cell death occurred predominantly in the dentate gyrus after systemic MP-pretreatment (P = 0.001) (Fig. 4). The morphological aspects of apoptosis characterized by marginal dark chromatin condensation were confirmed by semithin section (Fig. 5). In situ end labeling of sections of the dentate gyrus in neonatal piglets showed significantly more TUNEL-positive neurons in the MP-pretreated animals (Fig. 4).
Histological evaluation provided no signs of inflammation such as microglial activation or infiltration of the brain parenchyma by hematogenous cells. Global brain edema was manifested predominantly in the astrocytes and perivascular space in all regions of the brain. Light microscopy and electron microscopy showed a marked perivascular swelling of the astrocytic end feet, as reported in our preliminary work (21).
In this neonatal animal model of prolonged DHCA with a reperfusion period of six hours, we did not find any significant neuroprotective effect after systemic pretreatment with large-dose MP. To evaluate attenuation of brain injury after pretreatment with MP, DHCA time was deliberately prolonged to 120 minutes, according to other experimental studies evaluating neuroprotective treatment (4,17,31,32).
With our preliminary experimental model, we described neuropathology after 60 minutes of DHCA and six hours subsequent reperfusion, with major changes on the astroglial and endothelial level, without detecting significant neuronal cell pathology (21,33). Clinically, it is recommended to keep DHCA times less than 60 minutes (34,35). Nevertheless, longer periods of DHCA have been reported in the literature, e.g., during corrective surgery of hypoplastic heart syndrome (35). It was confusing that systemic pretreatment with MP was not associated with any improvement in cerebral perfusion and oxygenation, as has been reported (9). Improvement of regional brain perfusion may indicate attenuation of brain injury after DHCA. However, changes of brain perfusion within the normal range in animals undergoing DHCA may not necessarily indicate significant reduction of ischemic neuronal cell death (9). Astroglial cell injury and perivascular edema has been found to precede neuronal injury after 60 minutes of DHCA (33). Systemic pretreatment of MP did not significantly reduce the postoperative BW gain in comparison to that of the control group, and brain weight at the end of the experiment was similar in both groups. Although quantitative evaluation of the water content may provide better information on the severity of brain edema, histological and ultrastructural studies showed no significant obvious improvement of perivascular or neuronal edema in the pretreated group.
Significant neuronal cell death was expected after prolongation of DHCA. The mode of neuronal cell injury in this study was similar to that reported in other experimental piglet studies with DHCA (36,37). But the quantitative regional neuropathological assessment revealed no reduction of necrotic neuronal cell death in the systemically pretreated animals. Similar to the findings of other experimental studies, the hippocampal regions CA4 and CA1 have been shown to be the most sensitive brain regions to ischemia and suitable for the assessment of pharmacological protective intervention (37,38).
However, MP induced significant apoptotic neuronal cell death, which was predominantly found in the dentate gyrus of the hippocampus and cortex. However, the occurrence of apoptosis seems to be regional and not global (30,36,37). Ischemia and reperfusion have been found to induce both necrotic and apoptotic neuronal cell death with variations according to mode of ischemic injury, neuronal cell type, and regional distributions (22,36,37). Apoptosis has been found more frequently in immature brain regions, which may explain the selective vulnerability of the dentate gyrus to apoptotic cell death after ischemia (36,39). In some physiological and pathophysiological settings (40–42), steroid treatment itself has been found to induce apoptotic cell death in different cell types, including the brain (43,44). But the precise molecular mechanism by which MP induces apoptotic cell death in the hippocampus is still unknown. One explanation for the induction of apoptosis may be derived from a study (45) of the rat hippocampus, where activation of the glucocorticoid receptor increased the ratio of the proapoptotic molecule Bax relative to the antiapoptotic molecule BcL-xL. In addition, hyperglycemia, induced by MP, may also have contributed to an increased number of apoptotic and necrotic cells in the systemically pretreated animals (46–50).
Because of differences in the clinical and experimental setting, time of MP application, and mode and duration of ischemia, comparative conclusions concerning the neuroprotective advantage of MP are difficult (9,12,43,44,51). Where other studies have suggested a protective effect, in contrast to our findings, one should cautiously consider the conclusion of a protective effect of MP in neonates and infants undergoing such extreme physiological changes from DHCA (9).
The limitations of our study were that the concentration of steroids in the serum and at the cerebral level were not measured, which may limit the interpretation of the protective effect of MP regarding the mode of application and cerebral permeation. Also, TUNEL-positive neurons may not necessarily always indicate apoptotic cell death because DNA fragmentation has also been reported in neurons undergoing stages of necrosis (29,30,52). Untreated persistent hyperglycemia in the MP group may additionally have worsened the cerebral pathology after DHCA, but it was not considered to be controlled according to our protocol. Additional studies should evaluate the independent role of hyperglycemia regarding aggravation of necrosis and apoptosis. Additional biochemical, molecular, and ultrastructural studies are required, including treatment of metabolic side effects of MP treatment.
In conclusion, a different effect of steroids could be suspected in the neonatal age, especially in the context of ischemia and reperfusion, and the use of steroids has to be reconsidered in critically ill neonates and infants suffering hypoxic-ischemic events (49). This statement is emphasized by a recent meta-analysis concerning postnatal steroid treatment in preterm infants because of bronchopulmonary dysplasia, in which an altered neurodevelopmental outcome was described (53,54).
We would like to thank Anne Gale of Deutsches Herzzentrum Berlin for editorial assistance.
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