Effect of regulating gut microbiota using probiotics on functional changes in the brain: protocol for a systematic review

Abstract

Introduction

There is a growing number of randomised controlled trials (RCTs) that focus on functional changes in the brain detected by functional MRI (fMRI) and gut microbiota composition changes after using probiotics.

However, the effect of probiotics on functional changes in the brain through gut microbiota remains controversial in existing RCTs. Furthermore, to our knowledge, there is no systematic review to evaluate the effect of probiotics on functional changes in the brain through gut microbiota. Therefore, we aim to summarise literatures evaluating the potential association between probiotics, gut microbiota and functional changes in the brain to elucidate whether probiotics influence gut microbiota and affect functional changes in the brain through gut microbiota.

Methods and analysis

China National Knowledge Infrastructure, Wanfang Data, VIP Databases (the Chongqing VIP Chinese Science and Technology Periodical Database), SinoMed, PubMed, Web of Science, MEDLINE (The National Library of Medicine), EMBASE (Excerpt Medica Database), Scopus, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov will be searched until July 2019. The Grey Literature in Europe (OpenSIGLE) database and Google search engine will also be used. The reference lists of each included study will be reviewed to determine whether there are any further relevant studies. RCTs using probiotics compared with a placebo/control will be included. We will use risk of bias assessment and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to assess the quality of evidence. The results of the systematic review will be synthesised narratively in the domains of the three primary outcome measures: (1) Increased/decreased activity in brain regions or altered functional connectivity (FC) of brain detected by fMRI and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (2) Changes in composition and diversity of the gut microbiota and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (3) Increased/decreased activity in brain regions or altered FC of brain detected by fMRI and the changes in composition or diversity of the gut microbiota after administration of probiotics.

Ethics and dissemination

The results will be disseminated through a peer-reviewed publication. As no private and confidential patient data will be included in the reporting, there are no ethical considerations associated with this protocol.

PROSPERO registration number

CRD42019145114.

Strengths and limitations of this study.

• This is the first systematic review assessing the potential association between probiotics, gut microbiota and functional changes in the brain.

• The study design adheres to all relevant guidelines for systematic reviews and meta-analyses.

• The quality of evidence will be assessed by the Grading of Recommendations Assessment, Development and Evaluation system.

• This systematic review will have inherent limitations related to the included studies such as risk of bias, methodological inconsistencies and incomplete outcome data.

• We will only retrieve data from Chinese and English databases, which may limit available data or result in language bias.

Background

Probiotics are defined as ‘live microorganisms’ that provide health benefits to the host when consumed in adequate amounts.¹ The use of probiotics has emerged as a principal approach to maintain the balance of the human gut microbiota.² Probiotics have a strong ability to modulate the gut microbiota composition in healthy subjects,^{3 4} leading to a significant reduction in several bacterial genera directly involved in the onset of gastrointestinal diseases.³ Furthermore, probiotics regulate the gut microbiota of patients with gastrointestinal diseases and improve clinical symptoms.^{5 6} These findings show that probiotics can be used to treat gastrointestinal diseases by maintaining or changing the gut microbiota.

The gut microbiota includes bacteria, fungi, archaea, protozoa and viruses that interact with the host and each other to affect the host’s physiology and health.⁷ The microbiota has recently emerged as a key player in the gut-brain axis. This interaction between the brain and the microbiota in both animals and humans has led to the recognition of a new term called ‘microbiota-gut-brain axis’.⁸ This interaction is bidirectional, meaning that disturbance in the complex community of microbiota (dysbiosis) can affect the brain and vice versa.⁹ A meta-analysis that was performed on 13 studies up until 2016 has shown that there were significant differences in the levels of Lactobacillus, Bifidobacterium and Faecalibacterium prausnitzii in patients with irritable bowel syndrome (IBS) compared with healthy controls.¹⁰ Alteration in gut microbiota may contribute to pathogenesis of patients with IBS by altering the gut-brain axis.¹¹ Patients with neurological disorders were linked to gastrointestinal dysfunction and changes in gut microbiota.¹² And in another clinical trial, Candida albicans exposure has been found to be associated with gastrointestinal dysfunction and impaired cognitive ability.¹³ The underlying signalling mechanisms of these communicating networks between the gut flora and the gut-brain axis have been of special interest to researchers who are seeking potential therapeutic interventions.⁹

The ingestion of probiotics by healthy humans showed diminished psychological symptoms, including anxiety symptoms,¹⁴ while probiotic intervention significantly ameliorated the severity of depression in patients with major depressive disorder¹⁵ and patients with IBS.¹⁶ The above results show that gut microbiota are related to various diseases, and that modulating gut microbiota through probiotics may offer a potential treatment option. However, most of the psychological assessments in clinical trials^{14 15 17} were based solely on self-reported measures, hence firm conclusions regarding the effects of probiotics cannot be drawn from these findings.

Given the difficulty of studying the brain at the cellular level in humans, neuroimaging has emerged as a tool for increasing our understanding of the brain. Advances in computational biology are beginning to explain how these multifaceted and complex systems interact with each other.^{18 19} Neuroimaging tools as outcome measures are more objective than self-reported measures, which is an advantage over self-reported measures. Functional MRI (fMRI) is able to measure moment-to-moment alterations in the blood oxygen content (the blood oxygen level dependent signal).²⁰ Furthermore, it enables us to identify real time changes in neurological activity and correlate these with changes in behaviour or perception.^21–23 fMRI has been used successfully to identify differences in brain function in gastrointestinal disease states, such as IBS and inflammatory bowel disease, as well as in healthy people before and after chronic ingestion of probiotics.^{20 24 25} fMRI is effective predominantly because of its non-invasiveness, and it can accurately and intuitively observe the location and range of brain function activity from the whole level under physiological conditions.²⁶ Using task-based MRI or resting-state fMRI (rs-fMRI), enables us to examine whether probiotics influence the gut microbiota and affect activity in brain regions or functional connectivity (FC) of brain through the gut microbiota. It helps us better understand the microbiota-gut-brain axis, to potentially help develop therapeutic methods for central nervous system diseases in the future.

Recently, there were randomised controlled trials (RCTs) that focused on increased/decreased activity in brain regions^{24 27 28} or altered FC of brain^{24 29} detected by task-based fMRI or rs-fMRI after using probiotics in healthy subjects. However, some studies have found changes in gut microbiota,^{27 28} behaviour, gastrointestinal/emotional symptoms,²⁷ while others have not.²⁴ Furthermore, to our knowledge, there is no systematic review to evaluate the effect of probiotics on functional changes in the brain through gut microbiota. Therefore, we plan to systematically analyse all published RCTs in the medical literature to assess the effects of probiotics on the gut microbiota and functional changes in the brain. This will improve our understanding of the microbiota-gut-brain axis. Moreover, a deeper understanding of the underlying mechanisms will help refine the clinical use of probiotic supplements in the future.

Objectives

The objective of this systematic review is to summarise and evaluate the literature evaluating the potential association between probiotics, gut microbiota and increased/decreased activity in brain regions or altered FC of brain in healthy subjects after taking probiotics of any type, dosage, time point and duration. To this end, we aim to:

1. Evaluate the relationship between the changes in behaviour, gastrointestinal/emotional symptoms and increased/decreased activity in brain regions or altered FC of brain after probiotics’ administration.

2. Evaluate the relationship between the behaviour, gastrointestinal/emotional symptoms and the changes in composition or diversity of gut microbiota after probiotics’ administration.

3. Evaluate the relationship between increased/decreased activity in brain regions or altered FC of brain and the changes in composition or diversity of gut microbiota after probiotics’ administration.

We aim to determine whether probiotics influence gut microbiota and affect functional changes in the brain through the gut microbiota.

Methods

Retrieval strategy

The following databases will be searched until July 2019 for relevant RCTs: China National Knowledge Infrastructure (CNKI), Wanfang Data, VIP Databases, SinoMed, PubMed, Web of Science, MEDLINE, EMBASE, Scopus, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov. The Grey Literature in Europe (OpenSIGLE) database and Google search engine will also be searched. Furthermore, reference lists of each included study will be reviewed to determine whether there are any further relevant studies. The search terms will include probiotics (eg, ‘probiotics’ or ‘culturelle’ or ‘bifidobacteria’ or ‘lactobailli’ or ‘escherichia coli’ or ‘yoghurt’ or ‘yeast’ or ‘saccharomyces’ or ‘bacillus’ or ‘enterococcus’ or ‘streptococcus’ or ‘fermented milk’ or ‘sour milk ’ or ‘gastrointestinal microbiome’ or ‘gut microbiota’ or ‘gut microbiome’ or ‘gut microflora’ or ‘intestinal microflora’ or ‘ intestinal bacteria’ or ‘intestinal microbiome’ or ‘microbiome’ or ‘microbiota’ or ‘bacteria’ or ‘flora’), fMRI (eg, ‘MRI’ or ‘fMRI’ or ‘functional MRI’ or ‘neuroimaging’ or ‘MRI’ or ‘brain activity’ or ‘MR tomography’ or ‘NMR Imaging’ or ‘MR’ or ‘independent component analysis’ or ‘ functional network connectivity’ or ‘amplitude of low frequency fluctuations’ or ‘regional homogeneity’ or ‘functional connectivity’ or ‘central’ or ‘brain’ or ‘neuro’ or ‘ALFF’ or ‘FALFF’ or ‘REHO’ or ‘ROI ’ or ‘FC’ or ‘ICA’). The following terms will be used in the Chinese database searches: ‘Yi sheng jun’, ‘Chang dao jun qun’, ‘Jun qun’, ‘Nao gong neng cheng xiang’, ‘Gong neng xing ci gong zhen’, ‘He ci gong zhen cheng xiang’ and ‘He ci’. The search strategy for the PubMed database is shown in online supplementary table 1; this strategy will be modified appropriately for other databases.

Inclusion criteria

1. Type of study: randomised clinical controlled trials.

2. Participants: healthy individuals.

3. Interventions: probiotics compared with a placebo/control.

4. Outcome measures: the primary outcome measures for this review will be the assessment of functional changes in the brain, which were measured by fMRI, as they relate to gut microbiota regulated by probiotics. Three different primary outcome measures will be obtained from the following primary research: (1) Increased/decreased activity in brain regions or altered FC of brain detected by fMRI and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (2) Changes in composition and diversity of the gut microbiota and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (3) Increased/decreased activity in brain regions or altered FC of brain detected by fMRI and the changes in composition or diversity of the gut microbiota after administration of probiotics. Secondary outcomes: adverse events and participants’ compliance during the trial.

Exclusion criteria

1. Non-randomised clinical controlled trials.

2. Editorials, literature reviews and meta-analyses.

3. Duplicate publications.

4. Studies without full report of study results or primary data.

Study selection

All the studies retrieved through the search strategy will first be screened based on title and abstract using ENDNOTE (ENDNOTE is a reference management software that helps a lot in collecting or curating research materials, and formatting bibliographies.) which can remove duplicates. Two reviewers will independently screen all titles and abstracts according to the inclusion criteria. Any disagreements will be resolved by discussion or by a third reviewer. Full texts of potentially eligible studies based on title and abstract will be screened for eligibility by two reviewers independently; disagreements will be resolved by discussion or by a third reviewer if necessary. Reasons for study exclusions will be recorded.

Data extraction

Data will be extracted from each eligible study including the following information: authors; year of publication; journal; sample size; demographic characteristics of sample (age, ethnicity, sex); any information (eg, type, duration, dose, time point) we can get on the intervention group and control/placebo group; data analysis strategy used; fMRI data analysis strategy used; outcomes and conclusions.

Quality assessment

Risk of bias assessment: the included studies will be assessed by two independent investigators using the Cochrane Risk of Bias Assessment Tool.³⁰ The assessment will include contents of random sequence generation, allocation concealment, blinding (of participants, personnel and outcome assessors), incomplete outcome data and selective outcome reporting and other sources of bias. The investigators’ judgement will be categorised as ‘Low risk’, ‘High risk’ or ‘Unclear risk’ of bias. The disagreements will be resolved by discussion or a by third analyser if necessary.

Quality of evidence assessment: two independent reviewers will assess overall quality of evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system, examining the quality of the literature in five domains—risk of bias, publication bias, consistency, directness and precision.³¹ This system has four levels of classification (‘high’, ‘moderate’, ‘low’ and ‘very low’) to which each study can be assigned based on different factors. Disagreements will be resolved by discussion or by a third analyser if necessary.

Strategy of data synthesis

The results of the systematic review will be synthesised narratively in the domains of the three primary outcome measures: (1) Increased/decreased activity in brain regions or altered FC of brain and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (2) Changes in composition and diversity of the gut microbiota and their association with changes in behaviour, gastrointestinal/emotional symptoms after using probiotics. (3) Increased/decreased activity in brain regions or altered FC of brain and the changes in composition or diversity of the gut microbiota after administration of probiotics and one secondary outcome measure: the relevant adverse events and participants’ compliance. We will extract the increased/decreased activity in brain regions or altered FC of brain to evaluate whether probiotics are effective in activating/deactivating particular brain regions or are effective in altering FC of brain, where the functional changes in the brain are. Additionally, we will evaluate the relationship between the changes in behaviour, gastrointestinal/emotional symptoms and the increased/decreased activity in brain regions or altered FC of brain after administration of probiotics. If possible from the number of studies available, we will use narrative review to explicate whether a ‘dose-dependent’ or ‘type-dependent’ relationship can be identified between probiotic supplements and functional changes in the brain. We will also evaluate the relationship between the changes in behaviour, gastrointestinal/emotional symptoms and the changes in composition or diversity of the gut microbiota after administration of probiotics, and evaluate the relationship between the increased/decreased activity in brain regions or altered FC of brain and the changes in composition or diversity of the gut microbiota after administration of probiotics. Finally, we will record all the adverse events and participants’ compliance during the trial to assess the safety and popularisation of probiotic supplements, which can be regarded as a new potential therapeutic method for central nervous system diseases in the future. To fully assess for gaps in evidence, we will do the risk of bias assessment, and the strength of evidence for this conclusion will be determined by the items evaluated in the GRADE framework described above. We will highlight areas that need further study and give suggestions for future studies.

Registration and reporting of results

We registered this study in the PROSPERO international prospective register of systematic reviews (PROSPERO is an international database of prospectively registered systematic reviews in health and social care, welfare, public health, education, crime, justice, and international development, where there is a health related outcome.) in July 2019.³² This protocol was drafted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P).³³ The methods and results of the systematic review will be reported in accordance with the PRISMA-P guidelines.³⁴

Patient and public involvement

No patients or public will be involved in this systematic review protocol.

Discussion

According to the Global Burden of Disease Study, 322 million and 264 million people worldwide suffered from depression and anxiety, respectively in 2015. This is an increase of 18.4% and 14.9% over the 2005 figures.³⁵ There were more than 80 million stroke survivors in the world,³⁶ 43.8 million people with dementia,³⁷ 45.9 million patients with an active epilepsy,³⁸ and 6.1 million individuals with Parkinson’s disease.³⁹ Globally, in 2016, neurological disorders were the leading cause of disability (276 million disability-adjusted life years) and the second leading cause of deaths (9 million) in the world.⁴⁰ With the increasing annual growth in industrial production of food containing probiotics worldwide, the interest in elucidating how changes in the gut microbiota and functional changes in brain as a result of ingestion of probiotics can serve as a new way for treating subjects with central nervous system diseases is increasing. In our systematic review, we will synthesise the evidence evaluating the association between probiotic supplements and functional changes in the brain and gut microbiota changes in humans to decipher whether probiotics influence functional changes in the brain and whether the effect of probiotics on functional changes in the brain is associated with changes in gut microbiota. This review provides a new way of thinking about the treatment of central nervous system diseases and health maintenance. To our knowledge, this will be the first systematic review focused on this topic.

Additionally, from the adverse events and participants’ compliance with probiotic supplements, we can accumulate evidence for the popularisation of probiotic therapy, which may be used to treat central system diseases in the future. This can help better understand the clinical application, effectiveness and safety of probiotics.

Furthermore, we will determine gaps or uncertainties in the existing literature, which require further study. However, given the potential implications of such an association, this review will provide a solid foundation for the design and implementation of future studies that can better clarify the relationship between probiotics and functional changes in the brain and gut microbiota and in humans.