Clinical trials related to investigation of gut-brain axis in relationship with mental health.
A rather new and somewhat unusual concept connects brain functions to gut microbiota. It is called “gut-brain axis” (or “microbiota-gut-brain axis”) and states that probiotics consumption and a healthy gut microbiota positively influence brain functions related to behavior and cognition. Synergistic with a low chronic grade peripheral inflammation, this faulty barrier exposes the aged brain to negative extra-cerebral signals. Given the quasi-constant failure of pharmacological treatments in neurodegenerative diseases, increased interest is directed toward allopathic medicine, including dietary supplements. Interplay between gut microbiota and central nervous system by immune, neural and metabolic pathways is being explored as a possible modulator of cognitive impairment and behavior disorders. In elderly persons, this axis has been reported to be altered, contributing to systemic inflammation and was also indicated as a possible marker for early frailty in younger population. Currently, there are several clinical trials addressing the relationship between gut microbiota and central nervous system psychiatric disorders and at least one directly investigating whether there is a correlation between composition of gut microbiome, permeability of intestinal barrier and systemic inflammation in patients with dementia. This chapter discusses evidence-based data on positive modulation of gut-brain axis to alleviate behavior and cognition alterations in the elderly.
- gut-brain axis
In recent years, great progress has been made in characterizing bidirectional interactions between central nervous system (CNS), enteric nervous system and gastrointestinal tract. Dr Michael Gershon has defined the gut as “the second brain”  for several reasons: the enteric nervous system may function autonomously (it is capable of reflexes in the absence of CNS input), it can communicate with CNS through the parasympathetic (via vagal nerve) and sympathetic (via paravertebral ganglia) nervous system and is susceptible to neurotrophic and neuromodulators signaling.
The term gut-brain axis is often extended to include the role of intestinal flora in the system, in which case the axis is called microbiota-gut-brain axis. The microbiota is not limited to bacteria but also includes protozoa, fungi, nematodes and viruses, so that in the intestinal tract there are over 1000 species of bacteria out of a total of approximately 100 trillion organisms . To maintain the homeostasis of such complex system, permanent correlation between the gut and the brain is essential.
The gastrointestinal microbiota has a symbiotic relationship with enteric cells and contributes to basic physiological processes such as digestion, growth and immune defense. The composition of an individual’s microbiota depends on the mode of delivery at birth, genetic predisposition, age, nutrition, physical activity, environmental factors, stress, infections, other diseases and the use of antibiotics .
Gut microbiota can influence the functioning of the CNS by the ability to synthesize or mimic some molecules such as host-signaling neuroactive molecules, for example, acetylcholine, catecholamine, g-aminobutyric acid (GABA), histamine, melatonin and serotonin . Conversely, the composition of the microbiota is influenced by emotional and psychological stress , resulting, for example, in the decrease in
Studies on GF mice also highlighted another surprising relationship between gut microbiota and aging and in the occurrence of aging-related-diseases. It has been shown that GF mice live significantly longer than normal animals, probably due to the reduction of pathological infections, with a first report back in the mid 1960s , followed by others in mice [22, 23]
We shall further detail some topics related to the gut-brain axis in the elderly, like gut-brain alterations observed in preclinical and clinical studies, some data about neuro-nutraceuticals with a focus on mechanisms and gut-brain axis modulation with a final reference to clinical trials.
2. Evidence-based data of gut-brain axis alteration in aged laboratory animals
Aging is (so far) an irreversible process which impacts on all cell populations, with several common denominators such as genomic instability, epigenetic alterations and oxidative stress . It equally affects gut lining and brain cells, but it also changes the gut microbiota composition [27, 28] which in turn is associated with behavioral and physiological modifications. An anxious behavioral phenotype was observed while transferring fecal microbiota between two strains of mice . Also, fecal microbiota transplantation from depressed patients to microbiota-depleted rats may have the potential to provoke behavioral and physiological features specific to depression, including anhedonia, anxiety-like behaviors, as well as modifications in tryptophan metabolism, suggesting that gut microbiota could be an important player in neurobehavioral changes in the rat . Reduced anxiety and depression-related comportment in mice were connected to dysregulated GABA signaling by metabolizing dietary glutamate through certain strains of
3. Evidence of gut-brain axis alteration in old patients
Each human organism hosts a widely diverse population of microbes that form a “metaorganism” (comprising 10 bacterial cells for every one of our own) . This metaorganism, which is now studied thoroughly in major scientific endeavors such as Human Microbiome Project , impacts on its host as much as the human host changes it back throughout different life stages. Microbial diversity and intestinal microflora stability decrease with age [27, 28] and are accompanied by a reduction in brain function and a decrease in cognitive abilities [34, 35]. Furthermore, alteration of gut microbiota homeostasis, through dietary or environmental factors, can lead to an imbalance between symbionts and pathobionts, resulting in reduced intestinal barrier function such as a dysbiosis state can be related to subsequent metabolic and inflammatory disorders, visceral pain and even alterations in brain functioning . Other studies have shown that while the microbial composition of the gut changes with age, it is not a linear process. The two dominant phyla in younger persons,
Gut microbiota and extreme longevity are the main topics of several interesting studies. There are evidence that extreme aging might be supported by subdominant gut microbiota species, along with pro-inflammatory species and health-associated taxa . Another study was focused on long-lived Chinese people and gut microbiota, with a special reference to the above-mentioned study. Significant differences in community membership and structures between the Italian and Chinese long-living groups were observed, attributed to many factors such as geography, genetics, diets and DNA-extraction methods .
These findings emphasize the importance of sampling a healthy population, both in terms of age, geographic and cultural traditions in order to identify features of gut microbiomes signatures related to age .
Age-associated alterations in gut microbiota are associated with subsequent decline with age in the functionality of the immune system (immunosenescence) and a chronic low-grade inflammatory status, partially due to increased intestinal permeability and increased colonic cytokine expression (inflammaging) . The inflammaging process can undermine the homeostatic equilibrium and accelerate the changes in gut microbiota structure and composition that could be related to the progression of diseases and frailty in the elderly population .
Mabbott et al. in 2015  provided an important advance in our understanding on the effects of aging on intestinal permeability and innate mucosal immune responsiveness in elderly patients. Using human terminal ileal biopsy tissues, they demonstrated that intestinal permeability to solutes, but not macromolecules, was significantly increased in the intestines of elderly humans. Tran and Greenwood-Van Meerveld in 2013  showed for the first time a pivotal contributing factor to geriatric vulnerability to gastrointestinal dysfunction may increase colonic permeability via age-associated remodeling of intestinal epithelial tight junction proteins. On the opposite side is a recent report by Valentini et al. that found no correlation between aging and increased intestinal permeability (or altered intestinal barrier), but mentioned low-grade inflammation as a possible favoring factor .
Age-related physiological modifications in the gastrointestinal tract undoubtedly affect the gut microbiota and high susceptibility to infections becomes an inevitable consequence. The gut microbiota homeostasis seem to play a crucial role for the gut well-being during the aging process and, in this perspective, dietary control of the gut microbiota of the elderly could be an important target for preserving a healthy gut microbiota community .
4. Neuro-nutraceuticals: definition, examples and mechanisms of action
Nutraceuticals are defined as “
However, the evaluation of nutraceutical efficacy and safety represents a big challenge due to the complex chemical compositions and multiple mode of actions . Starting from chemical classification of bioactive compounds , several classes have been repeatedly associated with beneficial neurological effects.
A special class of food derivatives or food ingredients includes prebiotics and probiotics.
Both types of nutraceuticals exert positive effects beyond local, intestinal environment. Prebiotics were demonstrated to exhibit triglycerides and cholesterol-lowering effects , with beneficial effect on blood-brain barrier integrity and brain lipid metabolism. Among many local effects, it is worth mentioning that, related to gut-brain axis, probiotics modify the gut microbiota, which is the main determinant of tryptophan levels, the serotonin precursor, hence the documented effect of these food supplements as anxiety alleviators.
Throughout the next sections, some evidence of modulation of gut-brain axis with nutraceuticals in the elderly will be discussed, in relationship with experimental results from preclinical and clinical studies.
5. Modulation of gut-brain axis with nutraceuticals in the elderly: evidence-based benefits in animal models
Diet and altered gut physiology, together with age-related changes in intestinal microbiome and a weakened immune system [74, 75] result in low-grade inflammation in the gut, that is associated with systemic release of pro-inflammatory metabolites. Finally, the inflammatory process is related to glial cells activation, neuroinflammation and to cognitive decline in the elderly [37, 41].
A useful model for the study of gut-brain axis is the germ-free (GF) mouse. GF mice have socially impaired behavior  and present an exaggerated stress response  that can be directly correlated with changes occurring in different regions of the brain , depressed neurogenesis  and prefrontal cortical hypermyelination . However, preclinical studies reveal that oral administration of probiotics and/or nutraceuticals is sufficient to reduce anxiety-like and depressive-like behaviors and to induce changes in brain chemistry [10, 79]. For example, the impaired microglial function in GF animals was rescued by the oral treatment with short-chain fatty acids . Also, Distrutti et al. , showed that a
The gut microbiome is also essential to the bioavailability of
Since short-chain fatty acids (SCFA) seem to play an important role in shaping the gut microbiota metabolism, it was necessary to study the effect of omega-3 polyunsaturated acids (n-3 PUFA) on cognitive decline in aging. Animal studies have shed light on their neuroprotective roles through pathways of synaptic plasticity, neuroinflammation and oxidative stress, evidencing also positive correlations between peripheral n-3 PUFA levels and regional gray matter . Supplementation of n-3 PUFA increased hippocampal neurogenesis and the fatty acid docosahexaenoic acid (DHA—an intermediate molecule in the metabolism of eicosapentaenoic acid) was able to down regulate microglial activation . Furthermore, age-related decline in c-Fos expression, that reflects neuronal response to extracellular signals such as growth factors and is triggered during action potentials, is attenuated by n-3 PUFA diet . The protective role of n-3 PUFA supplementation in counteracting cognitive decline, emotional dysfunctions and brain atrophy is governed by antioxidant and anti-inflammatory mechanisms [86, 89]. Some evidence in animal models suggested that long-term consumption of fish oil (rich in n-3 PUFA) may predispose the brain to lipid oxidation. The enrichment of fish oil with quercetin significantly attenuated behavioral impairments, restored the ROT-induced oxidative markers, depleted dopamine levels in striatum and reduced mitochondrial dysfunction, offering a higher neuroprotection in animal model .
In conclusion, preclinical studies support the rationale of further clinical testing of nutritional strategies to improve aged brain function, including the use of bioactive compounds with antioxidant, anti-inflammatory or neuroprotective properties in the diet of the elderly.
6. Neuro-nutraceuticals/gut-brain axis targeting in clinical trials
The direct modulation of gut microbiome by targeted dietary and probiotic uptake seem to have a positive impact in treating particular age-related disorders and represent a promising therapeutic option for the aging process itself . Well supported evidence came from findings from ELDERMET, a research consortium (http://eldermet.ucc.ie) which studied and characterized the gut microbiota in an Irish elderly population anti its relationship to mental health in aging . As a consequence of a growing body of evidence regarding a positive correlation between the health of gut microbiota and brain health, funding for clinical research is also spent for study of modulation of gut-brain axis with impact on healthy aging and mental health.
Clinical trials targeting not only the cross-talk between the gut microbiota and the central nervous system focus on its association with various neuropsychiatric and neurodegenerative disorders (anxiety, depression, dementia, etc.), but also on its involvement in the regulation of hunger/satiety and in pathologies affecting the digestive tract (Table 1). The therapeutic solutions investigated these clinical trials involve the use of prebiotics and probiotics, dietary changes and fecal microbiota transplant.
|No.||Clinical trial||Main objective||Status||Reference|
|1||Microbiome and the gut-brain axis||To examine the relationship between digestive tract microbes and mental health||Completed||https://clinicaltrials.gov/ct2/show/NCT02693327|
|2||Microbiome and dementia||To study the role of gut microbiome composition and gut permeability in the progression of dementia||Recruiting||https://clinicaltrials.gov/ct2/show/NCT03167983|
|3||Full4Health: understanding food-gut-brain axis across the life course||To examine how dietary manipulation influences the relationship between hunger/satiety/food preference and gut hormones, neural activation and energy metabolism||Completed||https://clinicaltrials.gov/ct2/show/NCT01597024|
|4||Oral fecal transplant in cirrhosis||To evaluate the safety and tolerability of oral fecal transplant in patients with cirrhosis and hepatic encephalopathy||Recruiting||https://clinicaltrials.gov/ct2/show/NCT03152188|
|5||Reduced appetite in Crohn’s disease: the role of the brain in the control of food intake||To investigate the changes in activity in areas of the brain that control food intake||Recruiting||https://clinicaltrials.gov/ct2/show/NCT02772458|
|6||FMT treating constipation patients with depression and/or anxiety symptoms - clinical efficacy and potential mechanisms||To observe the clinical efficiency of fecal microbiota transplant and the role of the gut microbiome in treating patients with constipation, depression and/or anxiety||Recruiting||https://clinicaltrials.gov/ct2/show/NCT03233100|
|7||Prebiotic intake reduces the waking cortisol response|
and alters emotional bias in healthy volunteers
|To investigate the effects of two prebiotics on the secretion of cortisol and emotional response in healthy individuals||Completed|||
|8||A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome||To study how the administration of ||Completed|||
|9||Assessment of psychotropic-like properties of a probiotic formulation (||To study the effects of probiotic formulations on stress, anxiety, depression and coping strategies in healthy volunteers||Completed|||
|10||A randomized controlled trial to test the effect of multispecies probiotics on cognitive reactivity to sad mood||To evaluate whether a multispecies probiotic formulation can reduce cognitive reactivity in non-depressed individuals||Completed|||
|11||Metabolic effect of new foods through gut-brain axis||To measure the effectiveness of coffee melanoidins, bread melanoidins, beta-glucans and a ||Recruiting||https://clinicaltrialbase.com/study/NCT01851304/|
|12||FMT for patients with IBS with fecal and mucosal microbiota assessment||To assess the efficiency of fecal microbiota transplant in treating irritable bowel syndrome||Recruiting||https://clinicaltrialbase.com/study/NCT03125564/|
|13||Effects of green-MED diet via the gut-fat-brain axis||To investigate the effect of a Mediterranean diet low in processed meat on the gut microbiota and age-related declines (changes in adiposity, cognitive function and cardiometabolic risk)||Recruiting||https://clinicaltrialbase.com/study/NCT03020186/|
|14||The clinical research on the relationship between circadian rhythm and gut microbiota in TBI patients||To examine whether the gut microbiota is involved in the modulation of sleep disorders in patients with brain traumatic injury||Recruiting||https://clinicaltrialbase.com/study/NCT02849028/|
Reports of already completed trials support the findings of preclinical trials, demonstrating that modulation of gut microbiota results in anxiolytic effect without a direct intervention on neurotransmitter circuitries. For example, the reported results of Schmidt et al. , after completion of trial, supported previous evidence that fructooligosaccharides, or Bimuno®-galactooligosaccharides supplementation lowered neuroendocrine stress response, measured by cortisol awakening response. Rao et al.  reported that supplementation with specific lactic acid probiotic bacteria for 8 weeks improved the anxiety scores of patients with chronic fatigue syndrome. A complex report by Messaoudi et al. , involving both laboratory rats as well as healthy human volunteers showed that supplementation with a combination of
Gut-brain axis is a bidirectional chemical network sending information between the intestine and the brain via soluble, chemical messages as well as nervous sympathetic and parasympathetic inputs. A third player with a determinant role in the health of both systems is the gut microbiota. Aging affects all players of the microbiota-gut-brain axis, leading to modified composition of bacterial taxa, chronic low-grade inflammation, modified intestinal metabolism, resulting in diminished availability of neurotransmitters or neurotransmitter precursors and short-chain fatty acids. Along with synaptic impairment, oxidative stress and neuroinflammation, aging leads to behavior alterations, anxiety and cognitive impairment. Interventions on gut-brain axis with nutraceuticals (food or food-derived products that have putative beneficial effects) alleviate some age-associated behavioral and cognitive alterations, as repeatedly demonstrated in animal models. Recently, clinical trials have begun to explore the beneficial effect of gut-brain axis modulation, in mood or cognition-associated disorders. Upon reports of positive results, gut-brain axis emerges as an important, easy to access target for promoting a healthy brain aging.
This work has been supported by National Authority for Scientific Research and Innovation (ANCSI), Program POC Axis 1 Action 1.2.3. Grant ID P_40_197, Contract No. 52/05.09.2016.