Brain cholinergic impairment in liver failure

Abstract

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

Fig. 1

(A) AChE, (B) ChAT and (C) BuChE activity levels in frontal cortex extracts from NC controls (n = 4) and patients with HE (n = 4). (D) Representative profiles of AChE molecular forms (G₄ = tetramers; G₁ + G₂ = monomers + dimers) and the activity of each AChE molecular form are also shown. Values are means ± SEM. *P < 0.05.

Fig. 2

Levels of (A) AChE, (B) ChAT and (C) BuChE in cortical extracts from sham NL controls, BDL rats without and with HD (BDL + HD), HD without liver disease fed with an ammonium-containing diet for 1 week and PCS rats. (D) Plasma and (E) cerebral ammonia levels were also determined for each rat group. Values are means ± SEM. *P < 0.05, significantly different from NL group; there were no statistically significant differences between BDL and BDL + HD groups (at least n = 6 for each group).

Fig. 3

Representative profiles of AChE molecular forms (G₄ = tetramers; G₁ + G₂ = monomers and dimers) and the activity of each molecular AChE form, for sham NL controls, BDL rats and BDL rats with HD (BDL + HD) (n = 6 for each group). Values are means ± SEM. *P < 0.05, significantly different from NL group.

Fig. 4

AChE and ChAT detection in the cerebral cortex of control and experimental rats. (A–I) AChE immunoreactivity in brain frontal cortex in normal (NL) rats (A–C), BDL rats, (D–F) and BDL + HD rats (G–I). The immunopositive staining is mainly localized in the perinuclear area of layer V pyramidal neurons in both control and experimental samples. Inserts show the progressive higher power pictures in the localized areas for each rat model. Scale bar: 250 microns in (A), (D) and (G); 100 microns in (B), (E) and (H); and 30 microns in (C), (F) and (I). (J–O) ChAT immunoreactivity of frontal cortex in normal (NL) rats (J and M), BDL rats (K and N) and BDL + HD rats (L and O). Immunopositive staining is mainly localized in the perinuclear area of layer V pyramidal neurons in control and experimental samples. Inserts show progressive higher power pictures in localized areas for each rat model. Scale bar: 250 microns in (J), (K) and (L); 100 microns in (M), (N) and (O). No significant staining was found in cortical superficial layers in pyramidal cells or interneurons, only apical processes of pyramidal neurons could be followed from layer V. Not staining was observed in the white matter.

Fig. 5

Immunodetection of AChE subunits and detection of AChE and ChAT transcripts in brain cortex from NL and BDL rats. (A) Relative mRNA levels of the transcripts for AChE (T and R; labelled AChE-T and AChE-R in the figure) and ChAT (cholinergic and peripheral; cChAT and pChAT) were analysed by QRT-PCR. Values were calculated using relative standard curves and normalized to GAPDH control from the same cDNA preparations. Specifity of the PCR products was confirmed by dissociation curve analysis. (B) Three major AChE bands of ∼77, 70 and 60 kDa were identified with the antibody E-19 in NL and BDL brain cortical extracts (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing NL and BDL animals. (C) Representative immunoblot of individual G₄ and G₁ + G₂ AChE peak-fractions separated by sucrose gradient centrifugation from NL and BDL brain cortical extracts (equivalent volume of G₄ and G₁ + G₂ AChE peak-fractions were loaded in each lane).

Fig. 6

Levels of ACh in cortical extracts from sham NL controls, and BDL rats without and with HD (BDL + HD). ACh values are uncorrected for probe recovery (32% of recovery) and represent means ± SEM. *P < 0.05, significantly different from NL group; there were no statistically significant differences between BDL and BDL + HD groups (n = 6 for each group).

Fig. 7

Result of the active avoidance test in sham NL controls, BDL rats, BDL rats + HD (BDL + HD), rats fed with a HD without liver disease, and PCS rats. Only cirrhotic BDL and BDL + HD rats showed a decrease in the number of attempts made to avoid the foot shock as compared with NL controls (*P < 0.02). Values are means ± SEM (at least n = 6 for each group).

Fig. 8

Change in levels of brain AChE species in response to rivastigmine or vehicle treatment in NL and BDL rats (A) representative profiles of AChE molecular forms. (B) Immunodetection of AChE variants analysed by western blot with the anti-N-terminal AChE antibody N-19 (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing rivastigmine and vehicle treated animals.

Fig. 9

Effect of the AChE inhibitor rivastigmine on conditioned stimuli response (avoidances) in the active avoidance test (A), the object recognition test (B) and on the rotarod motor coordination test (C), for NL and BDL rats (n = 10 for each subgroup). *P < 0.05 for a difference between NL-vehicle and BDL-vehicle rats; ^†P < 0.05 for a difference between BDL-vehicle and BDL-rivastigmine treated rats. Note that BDL-rivastigmine treated rats show differences in the number of avoidances and in the percentage of time exploring the non-familiar object, as compared with BDL-vehicle rats. No differences in the time on the rotarod were observed between BDL-rivastigmine treated rats and BDL-vehicle rats.