Hypothermic circulatory arrest increases permeability of the blood brain barrier in watershed areas

Abstract

Background: The integrity of the blood brain barrier (BBB) after cardiopulmonary bypass (CPB) with hypothermic circulatory arrest (HCA) is controversial in children. We tested the hypothesis that the BBB is disrupted by HCA.

Methods: Forty-one piglets (mean weight 11 kg) were randomly allocated to acute and survival experiments. Five groups (25 piglets, 5 per group) underwent acute studies: anesthesia alone (control); CPB at 37°C with full-flow (FF); CPB at 25°C with very low flow (LF); HCA at 15°C, and HCA at 25°C. Two groups (16 piglets, 8 per group) underwent survival studies: CPB at 25°C with LF and HCA. In the acute studies, Evans blue dye (EBD) extravasation through the BBB into the brain was measured using two methods: EBD absorbance of homogenized brain, and immunohistochemical localization of EBD-linked albumin for cortex, caudate nucleus, thalamus, hippocampus, and cerebellum. In the survival studies, cerebral histology was assessed with hematoxylin-eosin stain after sacrifice at 4 days after surgery.

Results: The BBB disruption was clearly observed around watershed areas for 25°C HCA compared with other conditions. Microscopic data showed that leakage of EBD in 25°C HCA was more severe than control in all brain areas (p < 0.05), and EBD and albumin were colocalizing. Histologic damage scores were significantly higher in watershed areas with 25°C HCA.

Conclusions: The BBB was impaired around watershed areas by 25°C HCA for 1 hour according to both macroscopic and microscopic data. An increase in permeability of the BBB may be both a sign and a mechanism of brain damage.

Figure 1

Whole brain photographs of sagittal view for 25°C HCA, 15°C HCA and control groups. BBB disruption around watershed area at 25 °C HCA clearly apparent. The leakages of Evans Blue Dye (blue area on 25°C HCA) as a disruption tracer of BBB was not observed under other conditions: 15°C HCA, 25 °C LF, 37 °C FF and control.

Figure 2

Ratio of absorbance of Evans blue dye in cortical and cerebellar tissue vs plasma EBD level three hours after end of bypass. The cortex/plasma ratio was significantly higher for 25°C, HCA than 37°C FF (p = 0.03) and 37 °C OFF (p = 0.005). Cerebellar tissue/plasma ratio was significantly higher for 25°C, HCA than 37 °C OFF (p = 0.005).

Figure 3

Immunohistochemistry in Brain tissue; Extravasation of Evans Blue dye (red) was observed in left photograph. The lectin (green) on endothelial cells was stained as a marker of vascularity. The nucleus (blue) was stained using 4',6'-diamidino-2-phenylindole hydrochloride (DAPI). Leakage of BBB was not found in right photograph.

Figure 4

Blood brain barrier leakage for cortex, caudate nucleus, thalamus, hippocampus and cerebellum using Evans blue dye at three hours after cardiopulmonary bypass. Pixel intensity in 25°C, HCA group was significant higher than control for each brain area (*p<0.05 vs control; one-way ANOVA).

Figure 5

Immunohistochemistry of cortex to demonstrate colocalization of EBD and albumin; (a) Evans Blue dye shows red (b). Albumin is green with FITC-conjugated polyclonal rabbit anti –albumin. (c) Nuclei are blue using 4',6-diamidino-2-phenylindole. (d)