Lipopolysaccharide-Induced Delirium-like Behaviour in a Rat Model of Chronic Cerebral Hypoperfusion Is Associated with Increased Indoleamine 2,3-Dioxygenase Expression and Endotoxin Tolerance

Abstract

Indoleamine 2,3-dioxygenase (IDO) and the tryptophan-kynurenine pathway (TRP-KP) are upregulated in ageing and could be implicated in the pathogenesis of delirium. This study evaluated the role of IDO/KP in lipopolysaccharide (LPS)-induced delirium in an animal model of chronic cerebral hypoperfusion (CCH), a proposed model for delirium. CCH was induced by a permanent bilateral common carotid artery ligation (BCCAL) in Sprague Dawley rats to trigger chronic neuroinflammation-induced neurodegeneration. Eight weeks after permanent BCCAL, the rats were treated with a single systemic LPS. The rats were divided into three groups: (1) post-BCCAL rats treated with intraperitoneal (i.p.) saline, (2) post-BCCAL rats treated with i.p. LPS 100 μg/kg, and (3) sham-operated rats treated with i.p. LPS 100 μg/kg. Each group consisted of 10 male rats. To elucidate the LPS-induced delirium-like behaviour, natural and learned behaviour changes were assessed by a buried food test (BFT), open field test (OFT), and Y-maze test at 0, 24-, 48-, and 72 h after LPS treatment. Serum was collected after each session of behavioural assessment. The rats were euthanised after the last serum collection, and the hippocampi and cerebral cortex were collected. The TRP-KP neuroactive metabolites were measured in both serum and brain tissues using ELISA. Our data show that LPS treatment in CCH rats was associated with acute, transient, and fluctuated deficits in natural and learned behaviour, consistent with features of delirium. These behaviour deficits were mild compared to the sham-operated rats, which exhibited robust behaviour impairments. Additionally, heightened hippocampal IDO expression in the LPS-treated CCH rats was associated with reduced serum KP activity together with a decrease in the hippocampal quinolinic acid (QA) expression compared to the sham-operated rats, suggested for the presence of endotoxin tolerance through the immunomodulatory activity of IDO in the brain. These data provide new insight into the underlying mechanisms of delirium, and future studies should further explore the role of IDO modulation and its therapeutic potential in delirium.

Keywords: delirium; indoleamine 2,3-dioxygenase; kynurenine pathway; neurodegenerative diseases.

Figure 1

Effects of the surgery on behaviour (A). Latency to eat food from the buried food test between the BCCAL (n = 6) and sham-operated rats (n = 6) at four weeks and eight weeks after surgery. (B) Open field test, (B1) total distance travelled, (B2) time spent in the centre, (B3) freezing time, and (B4) latency to the centre between the BCCAL and sham-operated group at four weeks and eight weeks after surgery. (C) Y-maze test, (C1) total number of arms visited, (C2) entries to the novel arm, and (C3) duration in the novel arm between the BCCAL and sham-operated rats at 4 weeks and 8 weeks after surgery. * indicates p < 0.05 versus sham group; ^# indicates p < 0.05 versus week 4 after the surgery. Values are expressed in mean ± standard error of the mean (SEM). Abbreviation: BCCAL: bilateral common carotid artery ligation.

Figure 2

Effects of the surgery on the brain amyloid-β42 (n = 4–6). (A): Amyloid-β42 in the hippocampus; (B): amyloid-β42 in the cerebral cortex. ** indicates p < 0.001 versus the sham-operated rats. Values are expressed in mean ± standard error of the mean (SEM). Abbreviation: BBCAL: bilateral common carotid artery ligation.

Figure 3

Percentage change in weight loss at the baseline (eight weeks after surgery) and 24, 48, and 72 h after LPS administration (n = 8). * indicates p < 0.05 vs. BCCAL-saline-i.p. and ^# indicates p < 0.05 vs. measurement at the previous time point. Values are expressed in mean ± standard error of the mean (SEM). Abbreviations: LPS: lipopolysaccharide; BCCAL: bilateral common carotid artery ligation.

Figure 4

Effects of LPS on the natural and learned behaviours. (A) Latency to eat food from the buried food test, (B) open field test, (B1) total distance travelled, (B2) time spent in the centre, (B3) freezing time, (B4) latency to the centre, (C) Y-maze test, (C1) total number of arms visited, (C2) entries to the novel arm, and (C3) duration in the novel arm by surgical types and LPS administrations at 24, 48, and 72 h post-LPS (n = 10). * indicates p < 0.05, ** indicates p < 0.001 versus BCCAL + saline, and ^# indicates p < 0.05, ^## indicates p < 0.001 versus value at the previous time point. Values are expressed in mean ± standard error of the mean (SEM). Abbreviation: LPS: lipopolysaccharide; BCCAL: bilateral common carotid artery ligation.

Figure 5

Effects of LPS on the TRP-KYN metabolites. (A) TRP levels, (B) KYN levels, (C) QA levels, (D) KYNA levels, (E) KYN/TRP ratio, and (F) KYNA/QA ratio in the serum by surgical types and LPS administrations at the baseline, 24, 48, and 72 h post-LPS (n = 6). * indicates p < 0.05, ** p < 0.001 versus BCCAL + saline, ^# indicates p < 0.05, ^## p < 0.001 versus BCCAL + LPS, ^& indicates p < 0.05, and ^&& indicates p < 0.001 versus value within the same group at the baseline. Values are expressed in mean ± standard error of the mean (SEM). Abbreviations: KYN: kynurenine; TRP: tryptophan; KYNA: kynurenic acid; QA: quinolinic acid, BCCAL: bilateral common carotid artery ligation; LPS: lipopolysaccharide.

Figure 6

The TRP, KYN, and KYN/KYN ratio levels in the hippocampus and cerebral cortex by surgical types and LPS administrations (n = 6). (A) TRP levels in the hippocampus, (B) TRP levels in the cerebral cortex, (C) KYN levels in the hippocampus, (D) KYN levels in the cerebral cortex, (E) KYN/TRP ratio in the hippocampus, (F) KYN/TRP ratio in the cerebral cortex. * indicates p < 0.05 versus BCCAL + saline and ^# indicates p < 0.05 versus BCCAL + LPS. Values are expressed in mean ± standard error of the mean (SEM). Abbreviations: TRP: tryptophan; KYN: kynurenine; HP: hippocampus; CC: cerebral cortex; BCCAL: bilateral common carotid artery ligation; LPS: lipopolysaccharide.

Figure 7

The levels of IDO enzyme in the hippocampus and cerebral cortex by surgical types and LPS administrations (n = 6). (A) IDO levels in the hippocampus, (B) IDO levels in the cerebral cortex. * indicates p < 0.05 versus BCCAL + saline and ^# indicates p < 0.05 versus BCCAL + LPS. Values are expressed in mean ± standard error of the mean (SEM). Abbreviations: IDO: indoleamine 2,3-deoxygenase; HP: hippocampus; CC: cerebral cortex; BCCAL: bilateral common carotid artery ligation; LPS: lipopolysaccharide.

Figure 8

The levels of QA and KYNA in the hippocampus and cerebral cortex by surgical types and LPS administrations (n = 6). (A) QA levels in the hippocampus, (B) QA levels in the cerebral cortex, (C) KYNA levels in the hippocampus, (D) KYNA levels in the cerebral cortex, (E) KYNA/QA ratio in the hippocampus, (F) KYNA/QA ratio in the cerebral cortex. * indicates p < 0.05 versus BCCAL + saline and ^# indicates p < 0.05 versus BCCAL + LPS. Values are expressed in mean ± standard error of the mean (SEM). Abbreviations: QA: quinolinic acid; KYNA: kynurenic acid; HP: hippocampus; CC: cerebral cortex; BCCAL: bilateral common carotid artery ligation; LPS: lipopolysaccharide.

Figure 9

A model describing the role of IDO/KP in the development of delirium during CCH. CCH is associated with increased baseline inflammation with dysregulation of TRP-KP activity and the activation of IDO. Heightened IDO levels during CCH play a crucial role in the development of LPS-induced delirium via immunosuppressive activities and endotoxin tolerance. Arrow () indicates the development of delirium-like behaviour under LPS treatment in the CCH rats. Abbreviations: IDO: indoleamine 2,3-dioxygenase; TRP: tryptophan; KYN: kynurenine; CCH: chronic cerebral hypoperfusion; GCN2: general control nonderepressible 2; eIF-2α: alpha subunit of eukaryotic initiation factor-2; AhR: aryl hydrocarbon receptor; LPS: lipopolysaccharide; QA: quinolinic acid; KYNA: kynurenic acid.

Figure 10

The behavioural assessment schedules in experimental rats. Abbreviations: BCCAL: bilateral common carotid artery ligation; LPS: lipopolysaccharide.