Synopsis

Chronic hepatic encephalopathy(HE) is a well-accepted complication of chronic liver disease(CLD), and finding the right treatment to reduce HE episodes before liver transplant remains a challenge. Both rifaximin and probiotics are currently used to reduce HE symptoms, but their precise effect on brain metabolites have never been studied. Our aims were first to assess in vivo and longitudinally the effect of the combination of probiotics and rifaximin on a rat model of chronic HE; and second to compare the results obtained to groups of non-treated/rifaximin-only treated rats. ¹H-MRS at high field combined with biochemical and behavioral tests were used.

Introduction

Chronic hepatic encephalopathy(HE) is a well-accepted complication of chronic liver disease(CLD). Treatments have focused on reducing plasma ammonium(NH₄⁺) levels, a main toxin implicated in HE pathogenesis. However, finding the right treatment to reduce HE episodes before liver transplant remains a challenge.

The non–absorbable antibiotic rifaximin inhibits the division of colonic bacteria responsible for urea deamination, reducing the production of gut ammonia. In humans, rifaximin has been shown to reduce the frequency of HE-episodes¹. In bile duct ligated(BDL) rats(model of chronic HE^2,3), rifaximin treatment showed negligible effects on neurometabolism⁴, while the exact molecular mechanisms of rifaximin remain unknown. On the other hand, probiotics can change the balance of intestinal bacteria, altering the microbial population by promoting non-ammoniogenic bacteria. Probiotics have been shown to be associated with significant improvement in HE symptoms in humans⁵. In BDL rats⁶ they have shown beneficial effects on brain Gln, osmotic equilibrium and motor activity. Both treatments are currently used to reduce HE symptoms in humans, but their precise effect on brain metabolites have never been studied.

Our aims were: 1) to assess in vivo and longitudinally the effect of the combination of probiotics and rifaximin on a rat model of chronic HE; 2)to compare the results obtained to a group of BDL non-treated rats(n=26)⁷ and BDL rifaximin-only treated rats(n=13)⁴. We used the advantages of ¹H MRS at high field combined with biochemical and behavioral tests.

Methods

Adult Wistar rats(n=6) underwent BDL^2,3. They were scanned before BDL(week0) and at weeks 2,4,6, and 8 after surgery. Probiotic administration(VIVOMIXX® in EU, VISBIOME in USA®, 60 billion bacteria/kg of rat) started two weeks before BDL-surgery until the end of the study. Rifaximin was administered orally twice daily(15.7mg/kg/day) starting 2 weeks after BDL-surgery(‘week2’). Rifaximin was administrated at 8am and 3pm, and probiotic daily at 7pm in order to avoid any direct interaction between both treatments in the gastrointestinal tract.

In vivo ¹H MRS was performed on a 9.4T MRI system(Varian/Magnex Scientific) using a home-built 14mm diameter quadrature ¹H coil as a transceiver. Metabolites evolution was studied using the SPECIAL sequence(TE=2.8ms, TR = 4000ms, 160 averages) in the hippocampus(2x2.8x2mm3) due to its role in memory, which appears to be affected in HE. Field inhomogeneity was corrected using FASTESTMAP(linewidth=9-11Hz). Metabolite concentrations were estimated using LCModel and water as internal reference. Plasma measurements of NH₄⁺, bilirubin, glucose were also performed at week 0,2,4,6 and 8. Open field test was performed at week 4, 6 and 8 to evaluate motor activity as a marker of HE severity⁸.

Results and discussion

All rats displayed the characteristic rise in plasma bilirubin, regardless of treatment group, as well as a similar ammonium increase(Fig.1).

The characteristic pattern of chronic HE is visible in the high quality spectra shown in Fig.2 and observed in the ‘probiotics&rifaximin’ treated group (Fig3,blue): 1) gradual increase of brain Gln as a result of ammonia detoxification by glutamine synthetase enzyme; 2) gradual decrease in the other brain osmolytes(Ins,Tau,tCho) (Ins:-27%,p<0.001); 3) decrease of neurotransmitter Glu; 4)decrease of Cr, accepted for its role in energy metabolism, osmoregulation and potentially, neuroprotective^9,10( ̴̴-12%,p<0.01). No significant variation in Lac, reported to play a major role in the development of brain edema in CLD rats¹¹, were observed.

When comparing the three groups at week 8, both treated groups showed a lower absolute Gln level compared to non-treated group(Fig.4A). However, the relative increase was similar in the three groups(+140-155% compared to week0, p<0.001).

Nevertheless, the synergistic action of probiotics and the antibiotic presented some ameliorations compared to the two other groups(‘non-treated’ and ‘rifaximin-only’, Fig.4). In the ‘probiotic&rifaximin’ group, Asc concentrations remained normal, while they decreased in the two other groups( ̴̴-15%), suggesting that this combination may act by reducing oxidative stress, an important pathogenic factor in HE(Fig.4C). While the correlation between Gln and motor activity was very marked in the non-treated group, there was no significant correlation in the ‘probiotics&rifaximin’ group(Fig.4B), probably as a reduced effect of Gln on motor activity due to treatment. Finally, Tau showed a faster and stronger decrease in both treated groups, suggesting a higher osmotic response(Fig.4D).

Conclusion

Our preliminary results showed that the combination of probiotic VIVOMIXX and rifaximin induced some changes in the neurometabolic profile of rats with CLD(i.e. Asc, Tau). Cross-resistance is a major concern in long-term rifaximin administration¹² and the combination between probiotics and antibiotics is very attractive as both could be used alternately to avoid multiresistant organisms¹³. Further measurements are needed to establish the potential of this combination, to assess the impact of both VIVOMIXX and rifaximin on neurometabolic profile and evaluate its therapeutic efficiency.

Acknowledgements

References