Selective cerebral perfusion prevents abnormalities in glutamate cycling and neuronal apoptosis in a model of infant deep hypothermic circulatory arrest and reperfusion

Abstract

Deep hypothermic circulatory arrest is often required for the repair of complex congenital cardiac defects in infants. However, deep hypothermic circulatory arrest induces neuroapoptosis associated with later development of neurocognitive abnormalities. Selective cerebral perfusion theoretically provides superior neural protection possibly through modifications in cerebral substrate oxidation and closely integrated glutamate cycling. We tested the hypothesis that selective cerebral perfusion modulates glucose utilization, and ameliorates abnormalities in glutamate flux, which occur in association with neuroapoptosis during deep hypothermic circulatory arrest. Eighteen infant male Yorkshire piglets were assigned randomly to two groups of seven (deep hypothermic circulatory arrest or deep hypothermic circulatory arrest with selective cerebral perfusion for 60 minutes at 18℃) and four control pigs without cardiopulmonary bypass support. Carbon-13-labeled glucose as a metabolic tracer was infused, and gas chromatography-mass spectrometry and nuclear magnetic resonance were used for metabolic analysis in the frontal cortex. Following 2.5 h of cerebral reperfusion, we observed similar cerebral adenosine triphosphate levels, absolute levels of lactate and citric acid cycle intermediates, and carbon-13 enrichment among three groups. However, deep hypothermic circulatory arrest induced significant abnormalities in glutamate cycling resulting in reduced glutamate/glutamine and elevated γ-aminobutyric acid/glutamate along with neuroapoptosis, which were all prevented by selective cerebral perfusion. The data suggest that selective cerebral perfusion prevents these modifications in glutamate/glutamine/γ-aminobutyric acid cycling and protects the cerebral cortex from apoptosis.

Keywords: Energy metabolism; apoptosis; glucose; glutamate; magnetic resonance spectroscopy; neurotransmitters; γ-aminobutyric acid.

Figure 1.

Schematic diagramming central role of glucose in glycolytic, CAC and accessory pathways. Full circle represent ¹³C and empty circle ¹²C. G-6-P: glucose 6-phosphate; CAC: citric acid cycle; PDC: pyruvate decarboxylase; PC: pyruvate carboxylase; α-KG: α-ketoglutarate; OAA: oxaloacetate; GABA: γ-aminobutyric acid; GAD: glutamate decarboxylase; GABAT: GABA aminotransferase.

Figure 2.

Diagram of perfusion protocol (a) and time course of cerebral and somatic regional oxygen saturation ((b), (c)). The two groups differ perfusion protocol after cooling for 45 min. Details are listed in the text. (b) The cerebral regional oxygen saturation (rSO₂) after starting DHCA gradually dropped, while SCP maintained over 80% during DHCA (25 ± 1% in group DHCA vs. 92 ± 2% in group SCP, p < 0.001). (c) The somatic rSO₂ values did not differ at any time points among two groups. CPB: cardiopulmonary bypass; DHCA: deep hypothermic circulatory arrest; SCP: selective cerebral perfusion; Glu: glucose; T0: baseline; T1: at just before starting DHCA; T2: at 60 min of DHCA; T3: completion of rewarming; T4: endpoint. *p < 0.001 vs. DHCA.

Figure 3.

Apoptosis cells and neurons identified by TUNEL staining in representative cerebral cortex sections. ((a), (b)) Detection of apoptosis and quantitation of apoptotic cells (brown/orange stains) from TUNEL staining. TUNEL assay showed no apoptosis in control pigs (Ctrl). Pigs undergoing DHCA had visibly more apoptosis compared with the SCP group, which also exhibited few apoptosis cells. Cleaved caspase-3 immunostaining (brown/orange stains) also showed same results as TUNEL staining. ((c), (d)) Double immunofluorescence staining for TUNEL (green) and the neuronal marker NeuN (red). Nuclei were counterstained with DAPI (blue). TUNEL-positive cells in DHCA group were detected in both neurons and other cells at the similar rate. TUNEL- and NeuN-double positive cells (arrowhead). TUNEL-positive and NeuN-negative cells (arrow). TUNEL, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Scale bar = 100 µm. *p < 0.001.

Figure 4.

Cerebral energy metabolism. (a) Intracellular ATP level and (b) glycogen level were measured by detection assay kits. ATP level was not different among three groups, whereas glycogen level was lower in DHCA group than control and SCP groups. CAC intermediates and entering CAC in the cerebral tissue were measured by GC–MS (C–G). (c) ¹³C-MPE of pyruvate and lactate, derived from exogenous [U-¹³C₆]-glucose. (d) Absolute quantity of pyruvate and lactate, derived from all carbohydrates including endogenous glucose and exogenous [U-¹³C₆]-glucose. (e) Pyruvate decarboxylation (PDC) and pyruvate carboxylation (PC) relative to citrate synthase (CS) flux ratio. (f) Total ¹³C-MPE and (g) absolute quantity of CAC intermediates. These data were not statistically different among three groups. CAC: citric acid cycle; MPE: molar percent enrichment; α-KG: α-ketoglutarate; OAA: oxaloacetate. *p < 0.05.

Figure 5.

Concentrations of amino acid neurotransmitters in the cerebral tissue. Absolute concentrations and ¹³C-labeled metabolite enrichment derived from [U-¹³C₆]-glucose were measured by GC–MS ((a) and (b)) and ¹³C-NMR (typical spectrum in (c) and graphs in (d)), respectively. These data showed that glutamate in DHCA group was lower than in control and SCP groups, while GABA in DHCA group was higher than others. Chemical shifts were as follows: C4 of glutamine, 31.4 ppm; C4 of glutamate, 34.0 ppm; C2 of GABA, 34.9 ppm. GABA: γ-aminobutyric acid; ppm: parts per million. *p < 0.05.