Novel application of amino-acid buffered solution for neuroprotection against ischemia/reperfusion injury

Abstract

Ischemic neuron loss contributes to brain dysfunction in patients with cardiac arrest (CA). Histidine-tryptophan-ketoglutarate (HTK) solution is a preservative used during organ transplantation. We tested the potential of HTK to protect neurons from severe hypoxia (SH) following CA. We isolated rat primary cortical neurons and induced SH with or without HTK. Changes in caspase-3, hypoxia-inducible factor 1-alpha (HIF-1α), and nicotinamide adenine dinucleotide phosphate oxidase-4 (NOX4) expression were evaluated at different time points up to 72 h. Using a rat asphyxia model, we induced CA-mediated brain damage and then completed resuscitation. HTK or sterile saline was administered into the left carotid artery. Neurological deficit scoring and mortality were evaluated for 3 days. Then the rats were sacrificed for evaluation of NOX4 and H2O2 levels in blood and brain. In the in vitro study, HTK attenuated SH- and H2O2-mediated cytotoxicity in a volume- and time-dependent manner, associated with persistent HIF-1α expression and reductions in procaspase-3 activation and NOX4 expression. The inhibition of HIF-1α abrogated HTK's effect on NOX4. In the in vivo study, neurological scores were significantly improved by HTK. H2O2 level, NOX4 activity, and NOX4 gene expression were all decreased in the brain specimens of HTK-treated rats. Our results suggest that HTK acts as an effective neuroprotective solution by maintaining elevated HIF-1α level, which was associated with inhibited procaspase-3 activation and decreased NOX4 expression.

Fig 1. Responses of cultured rat cortical neurons during severe hypoxia (SH).

Using different ratios of histidine–tryptophan–ketoglutarate (HTK) solution in volume, the response of cultured rat cortical neurons during SH was evaluated. (A) Lactate dehydrogenase (LDH) released under SH. (B) Cell viability under normoxia, SH and SH + 1/2 HTK, was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Hypoxia-induced factor-1α (HIF-1 α) expression under normoxia, SH, and SH + 1/2 HTK. (D) The ratio of cleaved caspase-3 to total caspase-3 under normoxia and SH (left bar graph). The same ratio with different proportion of HTK. (N = 6 experiments performed at each time point. * p < 0.05 as compared with corresponding controls at the same time points as normoxia in A and C, right bar graph of D, and SH in B, and p < 0.05 in left bar graph of D compared with the untreated SH group at 72 h, # p < 0.05 as compared with normoxia in B and SH in C at the same time points).

Fig 2. The association of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and hypoxia-induced factor 1α (HIF-1α) during severe hypoxia (SH).

Concentration of H₂O₂ release, NOX2 and NOX4 measured during the severe hypoxia (SH) period, with the association hypoxia-induced factor 1α (HIF-1α). (A) The concentration of H₂O₂ released under normoxia, SH and SH + 1/4 HTK (histidine–tryptophan–ketoglutarate). (B) At 72 h, NOX2 and NOX4 expression under normoxia, SH, SH + HTK. (C) Release of lactate dehydrogenase (LDH) in response with HIF-1 antagonist 400083. (D) Expression of NOX4 at 72 h of SH, and the influence of HTK and HIF-1 antagonist 400083. (N = 6 experiments performed at each time point. * p < 0.05 compared to normoxia control, # p < 0.05 compared to SH group, @ p < 0.05 compared to SH + HTK group).

Fig 3. H₂O₂ administration to rat cortical neuron cultures, mimicking oxidative stress during severe hypoxia (SH).

(A) Lactate dehydrogenase (LDH) release using different H₂O₂ concentration. (*p < 0.05 compared to phosphate-buffered saline (PBS) control). For each H₂O₂ concentration, LDH release under the effect of histidine–tryptophan–ketoglutarate solution (HTK) (*p < 0.05 compared to the same H₂O₂ group without HTK). (B) A small amount of viable cells under 800 μM H₂O₂. (C) The PBS + 400 μM H₂O₂ group was used for cell viability test. (*p < 0.05 when compared to the PBS control, and #p < 0.05 when comparing 1/2 or 1/4 HTK with no HTK). (D) The ratio of cleaved caspase-3 to total caspase-3 after H₂O₂ challenge and HTK effect. (*p < 0.05 compared to 400 μM H₂O₂ without HTK). (N = 6 experiments performed at each time point).

Fig 4. Effects of HTK on H₂O₂ scavenge in a neuron-free system.

(A) The representative recordings show luminol-mediated CL counts were dose-dependently increased in response to different concentrations of H₂O₂ after 50 s of background (the first arrow). The PBS, HTK, or recombinant catalase was then treated to examine their antioxidant effects (the second arrow at 200 s). PBS and HTK show similar changes in CL count as first drop and then return (the left and middle panels), and only catalase decreases CL count continuously (the right panel). (B) The area under curve (AUC) after 200 s of recording was calculated and showed that there is no significant difference between PBS or HTK, and only catalase significantly lowers CL counts. Data are expressed as means ± SEMs (n = 6). *P < 0.05 vs. PBS at the same concentration of H₂O₂.

Fig 5. Results of cardiopulmonary resuscitation (CPR) for asphyxial cardiac arrest (aCA).

The CPR time, epinephrine used during CPR, survival rate and neurological deficit (ND) scores in rats, were compared in saline and histidine–tryptophan–ketoglutarate solution (HTK) groups. (A) CPR time (p < 0.05 for the 6’30″ groups (endotracheal tube clamped and asphyxia for 6 min 30 s) compared to the 4’30″ groups (endotracheal tube clamped and asphyxia for 4 min 30s)). (B) The epinephrine dose used for CPR. (C) Survival rate on day 3. (*p < 0.05, HTK vs. saline in the 6′30″ group). (D) Total neurological deficit (ND) scores are shown from day 0 to day 3. Numbers listed beside each icon on the line chart are the survived and observed rats at that time point. Treatment with HTK improved ND scores significantly at day 1 for the 4′30″ group and from day 1 to day 3 for the 6′30″ group. (* p < 0.05 compared with the saline-treated controls with the same duration time of aCA).

Fig 6. Effects of histidine–tryptophan–ketoglutarate solution (HTK) on H₂O₂ production and brain tissue nicotinamide adenine dinucleotide phosphate oxygenase 4 (NOX4) expression.

Changes in H₂O₂ levels in blood (A) and brain cortex (B). Changes in NOX activity (C) and NOX4 mRNA expression (D) in rat brain cortex. (N = 5 experiments for 4′30″ + saline group, N = 6 for 4′30″ + HTK group, N = 4 for 6′30″ + saline and 6′30″ + HTK groups. * p < 0.05 compared with saline-treated controls with the same asphyxial time of CA).