Brain Kynurenine Pathway and Functional Outcome of Rats Resuscitated From Cardiac Arrest

Abstract

Background Brain injury and neurological deficit are consequences of cardiac arrest (CA), leading to high morbidity and mortality. Peripheral activation of the kynurenine pathway (KP), the main catabolic route of tryptophan metabolized at first into kynurenine, predicts poor neurological outcome in patients resuscitated after out-of-hospital CA. Here, we investigated KP activation in hippocampus and plasma of rats resuscitated from CA, evaluating the effect of KP modulation in preventing CA-induced neurological deficit. Methods and Results Early KP activation was first demonstrated in 28 rats subjected to electrically induced CA followed by cardiopulmonary resuscitation. Hippocampal levels of the neuroactive metabolites kynurenine, 3-hydroxy-anthranilic acid, and kynurenic acid were higher 2 hours after CA, as in plasma. Further, 36 rats were randomized to receive the inhibitor of the first step of KP, 1-methyl-DL-tryptophan, or vehicle, before CA. No differences were observed in hemodynamics and myocardial function. The CA-induced KP activation, sustained up to 96 hours in hippocampus (and plasma) of vehicle-treated rats, was counteracted by the inhibitor as indicated by lower hippocampal (and plasmatic) kynurenine/tryptophan ratio and kynurenine levels. 1-Methyl-DL-tryptophan reduced the CA-induced neurological deficits, with a significant correlation between the neurological score and the individual kynurenine levels, as well as the kynurenine/tryptophan ratio, in plasma and hippocampus. Conclusions These data demonstrate the CA-induced lasting activation of the first step of the KP in hippocampus, showing that this activation was involved in the evolving neurological deficit. The degree of peripheral activation of KP may predict neurological function after CA.

Keywords: cardiac arrest; cardiopulmonary resuscitation; indoleamine 2,3‐deoxygenase; kynurenine pathway; neurological deficit.

Figure 1. Experimental designs.

Experimental designs adopted for (A) aim no. 1 and (B) aim no. 2. *Blood (0.6 mL) drawn for measurement of plasma 1‐methyl‐DL‐tryptophan, kynurenine pathway metabolites, and plasma high‐sensitivity cardiac troponin T. BL=baseline (before cardiac arrest [pre‐CA]). AP indicates arterial pressure; CPR, cardiopulmonary resuscitation; Echo, transthoracic echocardiography; NDS, neurological deficit score; RAP, right atrial pressure; ROSC, return of spontaneous circulation; and VF, ventricular fibrillation.

Figure 2. Indoleamine 2,3‐dioxygenase inhibition counteracts cardiac arrest/cardiopulmonary resuscitation (CA/CPR)–induced kynurenine pathway (KP) activation in plasma.

Plasma levels of (A) tryptophan, (B) kynurenine, and (C) kynurenine/tryptophan ratio 15 minutes before cardiac arrest (pre‐CA; n=13–15), 2 hours post–return of spontaneous circulation (PR; PR 2 hours [n=13–15]), and 96 hours PR (n=9) in vehicle‐ and 1‐methyl‐DL‐tryptophan (1‐DL‐MTRP)–treated rats. Box plots show median, quartile range, and minumim–maximum range. Statistical analysis was performed on absolute values (pre‐CA, PR 2 hours, and PR 96 hours) by ordinary 2‐way ANOVA, followed by Sidak multiple comparisons post hoc test. *P<0.05 and **P<0.01 vs corresponding pre‐CA levels (effect of CA). ⁺ P<0.05 and ⁺⁺ P<0.01 vs time‐matched vehicle‐treated rats (effect of 1‐DL‐MTRP pretreatment).

Figure 3. Indoleamine 2,3‐dioxygenase inhibition counteracts CA/CPR–induced kynurenine pathway activation in hippocampus.

Concentrations of tryptophan, kynurenine, and the kynurenine/tryptophan ratio measured in hippocampus 96 hours post–return of spontaneous circulation (96 hours) in rats treated with vehicle (n=9) or 2 doses of 800 mg/kg 1‐methyl‐DL‐tryptophan (1‐DL‐MTRP) given by gavage 16 and 2 hours before CA (n=9). Since this experiment included 2 independent sessions, each 1 including 3 naive rats, for each session we normalized tryptophan, kynurenine, and the kynurenine/tryptophan ratio in vehicle‐ and 1‐DL‐MTRP–treated rats to the means for the corresponding naive rats (absolute levels are reported in Table S4). Box plots show median, quartile range, and minimum–maximum range. Data were analyzed by Kruskal‐Wallis test followed by Dunn multicomparison test. *P<0.05 and **P<0.01 vs naive. ⁺ P<0.05 vs vehicle time‐matched. Exact P values are reported in the text.

Figure 4. Hippocampal kynurenine pathway metabolites levels positively correlate with those in plasma.

Correlations between hippocampal levels of (A) tryptophan, (B) kynurenine, and (C) the kynurenine/tryptophan ratio 96 hours post–return of spontaneous circulation in vehicle‐ (teal blue circles, n=9) and 1‐methyl‐DL‐tryptophan–treated rats (red circles, n=9). Correlations were calculated by Spearman method: rank correlation coefficient r [95% CI] and P value (2‐tailed) of the correlations are indicated inside the figures. P<0.05 was considered significant.

Figure 5. Time course of the neurological deficit score (NDS) after CA/CPR and correlation with hippocampal and plasmatic levels of kynurenine pathway metabolites.

A, NDS was calculated 24, 48, 72, and 96 hours post–return of spontaneous circulation (PR; PR 24 hours, PR 48 hours, PR 72 hours, and PR 96 hours) in rats treated by gavage 16 and 2 hours before CA with vehicle (n=9) or 1‐methyl‐DL‐tryptophan (1‐DL‐MTRP; 800 mg/kg per dose [n=9]). Box plots show median, quartile range, and minimum–maximum range. Data were analyzed by 2‐way repeated measures ANOVA, followed by Sidak multiple comparisons post hoc test. ⁺ P<0.05 vs time‐matched vehicle‐treated rats. Panels B‐E show correlations between NDS and hippocampal (B and C) and plasmatic (D and E) levels of kynurenine and the kynurenine/tryptophan ratio 96 hours PR in vehicle‐ (teal blue circles) and 1‐DL‐MTRP–treated rats (red circles). Correlations were calculated by Spearman method: rank correlation coefficient r [95% CI] and P value (2‐tailed) of the correlation are indicated inside the figures. P<0.05 was considered significant. Correlations analysis between tryptophan, in plasma and hippocampus, and NDS are reported in Figure S4.