JP2023104356A - Method for measuring higher brain functions - Google Patents

Abstract

To provide an objective and low-invasive method for evaluating a subject's higher brain functions.SOLUTION: A method for measuring one or more higher brain functions selected from execution function, attentive function and cognitive flexibility includes the step of measuring, in a biological sample taken from a subject, at least one of the ratios of the numbers of bacterial strains classified as Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces to the total number of bacterial strains.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for measuring higher brain function using the composition of intestinal microflora as an index.

Cognitive functions such as executive function, attention function, and cognitive flexibility are also called higher brain functions, and when these functions are impaired due to aging, head injury, stroke, etc., it is difficult to adapt to daily and social life. may be.
The executive function, which is positioned at the top of the higher brain functions, is also called the executive function, and is defined as the ability to maintain an appropriate posture in order to achieve future goals. It is considered an essential function for normal cognitive function (Non-Patent Document 1). In addition, attention control and cognitive flexibility, which are higher brain functions, play important roles in promoting new behavioral patterns and optimizing behavior in unconventional situations. believed to support such behavior.

It is generally believed that the neural basis that controls these higher brain functions resides in the prefrontal cortex, and therefore the prefrontal cortex is underdeveloped or damaged, resulting in lower higher brain functions. Then, symptoms such as unplanned behavior, inability to prioritize things, haphazard behavior, inability to work efficiently, and inability to start behavior without instructions appear.

Currently, "TMT (Trail Making Test) A and B", which evaluate divided attention and ability to process multiple tasks, have been established as one of the test methods for higher brain function. It is widely used because it can be detected sharply. However, it is easier and more objective because it requires regular examinations by dementia specialists and clinical psychologists and regular hospital visits for patients, and the score is affected by the mood, physical condition, motivation, etc. of the subject. Development of inspection methods and indicators is required. Cerebrospinal fluid examination is considered useful in diagnosing dementia, but its invasiveness is a problem, and it is necessary to develop examination methods and indicators that can be applied to minimally invasive diagnosis.

On the other hand, more than 1000 species and more than 100 trillion bacteria live in the human gastrointestinal tract, forming an intestinal flora. The composition of the intestinal microflora varies from individual to individual due to various factors such as age, dietary habits, disease, microbial infection, stress, and drug administration. Recent studies have revealed that the intestinal microbiota is involved in the pathogenesis of various diseases such as metabolic diseases, immune diseases, and psychiatric diseases, in addition to gastrointestinal diseases. In particular, it has been suggested that the composition of the intestinal flora is closely related to obesity and obesity-induced metabolic diseases.
However, the details of the relationship between the composition of the gut microbiota and higher brain functions have not been elucidated.

Toshiya Fukui, Cognitive Neuroscience (2010) 12: 156-164

The present invention relates to providing an objective, minimally invasive method for assessing higher brain function in a subject.

The present inventors have studied in view of such problems, and found that the ratio of the number of bacterial species classified into a specific genus in the intestinal flora to the total number of bacterial species (genus diversity) and TMT-A and / or TMT It was found that there is a significant correlation or tendency to correlate with the performance time of -B, and that higher brain functions can be measured using the diversity of the specific genus in the subject's biological sample as an index.

That is, the present invention relates to the following 1).
1) genus Bacteroides, genus Ruminococcus, genus Streptococcus, genus Parabacteroides, genus Dorea and genus Actinomyces in biological samples collected from subjects One or more higher brain functions selected from executive function, attention function and cognitive flexibility, including the step of measuring 1 or more of the ratio of the number of bacterial species classified into each genus to the total number of bacterial species Measuring method.

INDUSTRIAL APPLICABILITY According to the method of the present invention, the higher brain function of a subject can be tested objectively and simply in a minimally invasive manner, and early changes in deterioration of higher brain function can be objectively evaluated.

In the present invention, "higher brain function" means one or more cognitive functions selected from executive functions, attentional functions and cognitive flexibility, and includes one or more selected from executive functions and attentional functions. Preferably, and more preferably, it includes at least executive functions.

In the present invention, "executive function" is also referred to as executive function, and is defined as the ability to maintain an appropriate posture to achieve future goals. Specifically, 1) task representation, 2) planning ( planning), 3) execution of tasks, 4) suppression of undesired reactions, and 5) evaluation and correction as necessary (Non-Patent Document 1).

"Attention function" is also known as attention control function, and refers to the function of consciously directing attention to certain important information from a large amount of information (Science Co., Ltd., Tadashi Oyama and Yoshiaki Nakajima, Experimental Psychology). Invitation to School [revised edition], p99). The attention function is mainly composed of three functions of "selective attention", "attention conversion", and "attention division" (Waseda University Clinical Psychology Research, Vol. 13, No. 1, p33). Here, selective attention is the function of directing attention from many stimuli and objects to a specific stimulus or object, and diverting attention is the ability to switch attention from a specific stimulus or object to a specific stimulus or object as needed. It is the ability to pause and switch to other stimuli or objects as appropriate, and splitting attention is the ability to distribute attention to multiple objects simultaneously.

“Cognitive flexibility” refers to the ability to replace maladaptive thoughts and incorporate balanced and adaptive thinking (Yokawa Tokuyoshi and Shoichi Iwasaki (2012) Cognitive Flexibility Scale (CFI) Preparation and Validity of the Japanese Version Proceedings of the 76th Conference of the Japanese Psychological Association, 672).

Higher brain functions (executive function, attention function and cognitive flexibility) in the present invention can be evaluated by TMT (Trail Making Test).
TMT is a method that is often used as a test of higher brain functions, and includes visual attention, visual search, visuomotor coordination, attentional maintenance and selection, visual-motor coordination, speed of information processing, and short-term interference. It is said to reflect higher attentional functions such as memory. In particular, TMT-A is used as an index of attentional function (selectivity of attention), and TMT-B is used as an index of executive function.

In the present invention, "measurement of higher brain function" includes measurement of the state or degree of higher brain function and the presence or absence of executive dysfunction, preferably measurement of the state of higher brain function.
Here, "measurement" can also be interchanged with the terms "detection", "inspection", "determination", "evaluation" or "evaluation aid". In addition, the term "judgment" or "evaluation" used herein does not include judgment or evaluation by a doctor.

In the present invention, "intestinal flora" means an intestinal bacterial population present in the gastrointestinal tract, particularly the large intestine, also called intestinal flora. Intestinal flora can typically be measured as fecal flora.

The method for measuring higher brain function of the present invention is a biological sample collected from a subject. Dorea) and Actinomyces genus, the step of measuring one or more of the ratio of the number of bacterial strains classified into each genus to the total number of bacterial strains.

In the present invention, the subject is not particularly limited. Impossible to prioritize, haphazard behavior, inability to work efficiently, inability to start behavior without instructions, inability to modify work content as needed, etc.), decline in higher brain function. is suspected, and the like.

In the present invention, the biological sample includes a specimen containing intestinal bacteria of a subject, such as stool and intestinal contents. From the viewpoint of ease of collection and reduction of the burden on the subject, it is preferable to use stool.

In the present invention, the ratio of the number of bacterial species classified into a certain genus in the biological sample to the total number of bacterial species is calculated by the following formula: [number of bacterial species classified into a certain genus/total number of bacterial species] × 100 (% ) is preferably used. Such a value corresponds to the ratio of the number of bacterial species classified into the genus in the intestinal flora of the subject from whom the biological sample is derived to the total number of bacterial species, and the number of the genus in the intestinal flora of the subject. It shows diversity (genus diversity). Here, the high diversity of a certain genus indicates that there are many types of bacteria classified into that genus in the intestinal flora. , the intestinal microflora contains a variety of bacterial species classified into a certain genus.

Although the means for measuring the ratio of the number of bacterial species classified into a certain genus in a biological sample to the total number of bacterial species is not particularly limited, it is preferable to measure the number of bacterial species in the biological sample based on the base sequence of the 16S rRNA gene. A method of analyzing species is included.
A method for classifying and identifying bacterial strains in a biological sample based on the nucleotide sequence of the 16S rRNA gene will be described below.

1) Extraction of genomic DNA from biological samples For biological samples collected from subjects, known bacteriolytic enzyme method (BMC Microbiol., 2004, 4:16), bead method (Science, 2008, 320, 1647-1651), etc. After releasing the nucleic acid, known methods known as DNA separation and extraction methods, such as the phenol-chloroform method (Mol. Biol., 1986, 191, 615-624) and the guanidine method (Science, 2005, 308 : 1635-1638) are employed to extract bacterial genomic DNA.

2) Sequencing of 16S rRNA gene in genomic DNA Next, the sequence of the 16S rRNA gene contained in the extracted bacterial genomic DNA is determined. That is, the sequence of the 16S rRNA gene characteristic to each bacterial species is determined, and the structure of the intestinal flora is analyzed based on the sequence data. Therefore, it is necessary to select the region of the 16S rRNA gene to be sequenced so as to reflect the bacterial flora structure. As long as the features of the sequences are reflected, it is preferable to determine and compare the sequences of short regions.
The region of the 16S rRNA gene to be sequenced is amplified by PCR, and the primers in this case are preferably set to regions that are universally conserved among bacterial strains. Examples of such primers include, but are not limited to, those shown in Table 1. Forward and reverse can be used in appropriate combination so that the desired region is amplified (BMC Microbiology, 2012, 12:66).

After purification of the amplified PCR products, sequencing is performed. Any known method can be used for sequencing. For example, rapid sequencing can be achieved by using a next-generation ultra-high-speed sequencing device such as MiSeq (Illumina).
It is known that the 16S rRNA gene of bacteria has a highly variable region (V1 to V9) whose base sequence is not conserved among bacterial strains. Therefore, it is desirable to determine the nucleotide sequence of at least one such region, eg, the region containing V1 and V2, or the region containing V3 and V4. A region containing V1 and V2 can be amplified, for example, with a primer set of 27F and 338R, and a region containing V3 and V4 can be amplified, for example, with a primer set of 338F and 907R.

Analysis of the obtained sequence data can be performed using analysis software such as Qiime (Quantitative Insights Into Microbial Ecology) for the data group of the obtained base sequence. It can be performed according to the system of Database Project (Lan, Y et al, Using the RDP classifier to predict taxonomic novelty and reduce the search space for finding novel organisms. PLoS One 2012. 7:e32491.). It is also possible by homology search using the BLAST algorithm against gene sequence databases such as microorganism identification database (Techno Suruga Labo), NCBI nucleotide database, NCBI 16S microbial rRNA database, Greengenes database, and SILVA (J. Mol. Biol., 1990, 215(3):403-410).
In sequence data analysis, if there is at least one copy of nucleotide sequence data that has at least 97% identity with the nucleotide sequence of a specific bacterial strain, it can be determined that the specific bacterial strain (or related species) exists. can.

As used herein, "at least 97% identity" with respect to nucleotide sequences means identity of 97% or more, preferably 98% or more, more preferably 99% or more, and still more preferably 100%.

As shown in the examples below, the ratio of the number of bacterial species classified to the genus Bacteroides to the total number of bacterial species (that is, the diversity of the genus Bacteroides) is the performance time of TMT, which is widely used as a test of higher brain function. A good correlation was observed. Thus, TMT-A and TMT-B performance times are significantly negatively correlated with Bacteroidetes diversity. Ruminococcus diversity was found to correlate well with TMT performance time. Thus, TMT-B performance time is significantly negatively correlated with Ruminococcus diversity. Diversity in Streptococcus tended to correlate with TMT performance time. That is, the performance times of TMT-A and TMT-B tend to be positively correlated with Streptococcus diversity. The diversity of the genus Parabacteroides correlated well with the duration of TMT. Thus, the performance times of TMT-A and TMT-B are significantly and negatively correlated with the diversity of the genus Parabacteroides. Drea diversity was found to correlate well with TMT performance time. Thus, TMT-B performance time is significantly negatively correlated with Drea diversity. Actinomyces diversity tended to correlate with the duration of TMT. That is, TMT-A performance time tends to be positively correlated with Actinomyces diversity.
In addition, when principal component analysis was performed using the diversity of these six genera and the first principal component was calculated, there was a significant correlation between the first principal component and the performance time of TMT-A and TMT-B, respectively. was accepted. Furthermore, multiple regression analysis was performed using the diversity of these six genera as an explanatory variable and the performance time of TMT-A and TMT-B as objective variables, and a significant regression equation was obtained.
Therefore, the ratio of the number of bacterial species classified into each of the genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces in the biological sample of the subject to the total number of bacterial species is 1 or more By measuring and comparing it with the reference value, it is possible to measure the higher brain function of the subject, specifically one or more selected from executive function, attention function and cognitive flexibility.

The enterobacterial genus that is the target of diversity calculation may be one or more genera selected from the genus Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea, and Actinomyces. , Ruminococcus, Parabacteroides and Dorea are preferred, and one or more genera selected from Bacteroides and Parabacteroides are more preferred. Moreover, from the viewpoint of measurement accuracy, it is more preferable to calculate the diversity for the six genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces. When calculating the diversity of two or more genera, the diversity should be calculated for each genus.

In one example, a subject's executive function is measured. Here, the enterobacterial genus for calculating the diversity is preferably one or more genera selected from the genus Bacteroides, Ruminococcus, Streptococcus, Parabacteroides and Dorea, Bacteroides, Ruminococcus, Parabacteroides More preferred are one or more genera selected from the genera and Dorea. Moreover, from the viewpoint of measurement accuracy, it is more preferable to calculate the diversity for the six genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces. In another example, the subject's attentional function is measured. Here, the enterobacterial genus for calculating the diversity is preferably one or more genera selected from the genus Bacteroides, the genus Streptococcus, the genus Parabacteroides and the genus Actinomyces, and the genus Bacteroides and the genus Parabacteroides 1 The above genera are more preferred. Moreover, from the viewpoint of measurement accuracy, it is more preferable to calculate the diversity for the six genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces. When calculating the diversity of two or more genera, the diversity should be calculated for each genus.

For example, the reference value can be set as follows based on the association between the diversity of the genera and the state of higher brain function.
The state of higher brain function is assessed by TMT. Based on the evaluation results, create a healthy group consisting of subjects judged to have normal higher brain function and a higher brain dysfunction group consisting of subjects judged to have low higher brain function. do. Separately, the diversity of the genus is calculated by the method described above. Then, based on the correlation between the evaluation result of the higher brain function and the diversity of the genus, a reference value suitable for evaluating the state of the higher brain function is determined. Specifically, based on the results of statistical analysis of the diversity of the genera of humans belonging to each group, the numerical range of the diversity of the genera that characterizes each group is determined. This numerical range is determined by setting a certain range above and below the average value of each group. Here, the "fixed range" may be a statistical value such as standard deviation (SD), a 1/2 SD value, a 1/3 SD value, or the like, or may be an arbitrary numerical value set in advance. Alternatively, it can be set within a certain range above and below the median value of each group. Here, the “fixed range” may be the first quartile, the third quartile, or the like, or may be an arbitrary numerical value set in advance.
When calculating the diversity of two or more genera, it is preferable to set a reference value for each genera.
Then, for example, when the diversity of the genus obtained from the subject falls within the range of the diversity of the genus of the higher brain dysfunction group, the subject "has decreased higher brain function." , "higher brain dysfunction" or "high possibility of higher brain dysfunction" can be evaluated.

Thus, according to the method of the present invention, the state and degree of higher brain function of a subject can be evaluated non-invasively, simply and accurately.

EXAMPLES Hereinafter, the present invention will be described more specifically by showing examples.

Test Example Correlation analysis between higher brain function evaluated by TMT and diversity of intestinal bacteria [1. Test overview]
(Procedure of test)
Forty males and females aged 65 to 75 (average age 69±4) were subjected to TMT measurement as an evaluation of higher brain function. TMT was measured with the performance time (seconds) of TMT-A and TMT-B set at an upper limit of 300 seconds. In addition, on the day before or on the day of the higher brain function measurement, feces were collected using a stool collection kit (Techno Suruga Labo). DNA extraction and sequence analysis were entrusted to Technosuruga Labo, Inc., and the bacterial strains in feces were identified. Diversity of each genus (genus diversity) was calculated from the identified bacterial species, and the relationship with TMT execution time was analyzed. The genus diversity of intestinal bacteria as used herein refers to the ratio of bacterial species classified into a specific genus to the total number of bacterial species contained in a sample. In other words, a high genus diversity indicates that there are many types of fungal species classified into a certain genus contained in the sample. In other words, it serves as an indicator of the existence of a variety of fungal species classified into that genus.

(DNA extraction)
The collected feces were stored in a preservative solution, transported to Technosuruga Labo, Inc., and subjected to DNA extraction using a DNA purifier (GENE PREP STAR PI-480, Kurashiki Boseki Co., Ltd.).

(sequence analysis)
The 16S rDNA V3-V4 region was subjected to PCR, sequenced by the next-generation sequencer MiSeq (Illumina), and analyzed using Techno Suruga Labo's microorganism identification database.

(Calculation of diversity of each fungal genus)
From the results of sequence analysis, the diversity of each bacterial genus (value indicating the amount of species belonging to the genus, [number of bacterial species within the genus in the sample/total number of bacterial species] × 100 (%)) is calculated and analyzed. Twenty genera with an average diversity exceeding 1% in 40 individuals were included in the correlation analysis.

(data analysis)
Pearson's product-moment correlation analysis, principal component analysis and multiple regression analysis were performed on the measured TMT performance time and the diversity of each fungal genus (SPSS 24.0 was used as statistical analysis software).

[2. result〕
Correlation analysis showed a significant correlation (P <0.05) or a trend towards a correlation (P<0.1) (Table 1). A principal component analysis was performed using the diversity of these six genera to calculate the first principal component. A significant correlation was observed between the calculated first principal component and the duration of TMT (Table 1). Furthermore, multiple regression analysis was performed using the diversity of these six genera as an explanatory variable and the performance time of TMT as an objective variable. As a result, a significant regression equation was obtained. Table 1 shows the adjusted multiple correlation coefficients. Since TMT-A is a test used as an index of attentional function (attentional selectivity) and TMT-B is a test used as an index of executive function, the diversity of these six genera has been shown to be a marker for evaluating higher brain function. rice field.

Claims (5)

1 of the ratio of the number of bacterial species classified into each of the genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces in the biological sample collected from the subject to the total number of bacterial species A method for measuring one or more higher brain functions selected from executive function, attention function and cognitive flexibility, comprising the step of measuring the above. The method according to claim 1, wherein the ratio of the number of bacterial species classified into each of the genera Bacteroides, Ruminococcus, Streptococcus, Parabacteroides, Dorea and Actinomyces to the total number of bacterial species is measured. . 3. The method of claim 1 or 2, wherein the biological sample taken from the subject is a stool sample. 4. The method of any one of claims 1-3, wherein the higher brain functions include at least executive functions. 4. The method of any one of claims 1-3, wherein the higher brain functions include at least attentional functions.