Finally, 45 and 29 patients from Probio-M8 and placebo groups provided complete fecal and serum samples for further omics analysis, respectively. The results showed that Probio-M8 co-administration conferred added benefits by improving sleep quality, alleviating anxiety, and gastrointestinal symptoms. Metagenomic analysis showed that, after the intervention, there were significantly more species-level genome bins (SGBs) of Bifidobacterium animalis, Ruminococcaceae, and Lachnospira, while less Lactobacillus fermentum and Klebsiella oxytoca in Probio-M8 group (P < 0.05). Interestingly, Lactobacillus fermentum correlated positively with the scores of UPDRS-III, HAMA, HAMD-17, and negatively with MMSE. Klebsiella oxytoca correlated negatively with feces hardness. Moreover, co-administering Probio-M8 increased SGBs involved in tryptophan degradation, gamma-aminobutyric acid, short-chain fatty acids, and secondary bile acid biosynthesis, as well as serum acetic acid and dopamine levels (P < 0.05). Taken together, Probio-M8 synergized with the conventional regimen and strengthened the clinical efficacy in managing PD, accompanied by modifications of the host’s gut microbiome, gut microbial metabolic potential, and serum metabolites.Parkinson’s disease (PD) is mainly managed by pharmacological therapy . However, prolonged use of these drugs would gradually diminish their dopaminergic effect. Gut dysbiosis was observed in some patients with PD, suggesting close association between the gut microbiome and PD. Probiotics modulate the host’s gut microbiota beneficially. A 3-month randomized, double-blind, placebo-controlled clinical trial was conducted to investigate the beneficial effect of probiotic co-administration in patients with PD. Eighty-two PD patients were recruited and randomly divided into probiotic [ It is considered a multifactorial disease that is resulted from both genetic and environmental reasons. Patients with PD are characterized by classic motor symptoms, such as resting tremor, bradykinesia, and rigidity, as well as gastrointestinal (GI) symptoms, including constipation, slower colonic transit time, and small intestinal bacterial overgrowth2. Notably, the GI symptoms of PD patients often precede the motor signs, suggesting an association between gut abnormalities and the onset of PD3.Parkinson’s disease (PD) is a neurodegenerative disease that afflicts seven to ten million people all over the world4. Alterations in the gut microbiota and metabolome have often been observed in patients with PD. For example, the proportion of opportunistic pathogens, such as Anaerococcus, Campylobacter, and Lactobacillus, was found to be elevated, while the polymicrobial cluster of Blautia, Butyricicoccus, Lachnospira, and other short-chain fatty acids (SCFA)-producing bacteria was diminished in patients with PD6. Moreover, the abundance of some gut microbes  was reported to be significantly associated with the severity of PD and its associated symptoms7.The gut has been proposed as the second brain of humans, harboring a dynamic gut microbiome that possesses microbial genomes over 100 times larger than that of the human genome8. Sampson et al.9 found that alpha-synuclein, the most important substance in PD pathology, could be transmitted from the gut to the brain through the vagus nerve causing dyskinesia, confirming an active role of the gut in driving the development and pathogenesis of PD10. Mounting evidence suggests that the gut microbiome has the ability to synthesize and/or regulate various neurochemicals and neurometabolites that may directly or indirectly impact the physiological process of the organism, as well as the onset and development of PD11. Marcus et al. (2021) showed that transplanting microbiota from young donors to aging recipient mice reversed aging-associated impairments in cognitive and behavioral deficits, partly via modulating the production of SCFAs and neurotransmitters12. Additionally, levodopa (the precursor of dopamine), gamma-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT) could be produced by certain microbes in the gut and transmitted to the brain through the systemic circulation, finally relieving neurodegenerative diseases15.The “gut-brain-axis” is a relatively new concept, proposing the existence of bidirectional communication pathways between the intestine and the brain; and misregulation of the communications and interactions between the two body compartments might be related to neurodegenerative diseases including PD18. Therefore, many alternative and/or adjuvant therapies (such as exercise training17 and acupuncture18) have been developed and applied in the management of PD. Owing to the existence of the gut-brain-axis and the close link between gut dysbiosis and pathology of PD, target modulation of the gut microbiome might be an interesting alternative/adjuvant management approach that could alleviate PD.Currently, the main management for PD is administering anti-Parkinson medications, such as Levodopa, Benserazide, Sirelin, and Pramipexole. Pharmacological therapy can improve PD-associated symptoms, but the dopaminergic treatment effect would gradually diminish and larger doses of medication would be needed eventually19. A previous report showed that daily administration of complex probiotics  for 16 weeks conferred protective effects on dopamine neurons and significantly improved motor dyskinesia  in a PD mouse model20. An engineered glucagon-like peptide-1-producing probiotic strain could mitigate neuroinflammation in PD model mice, evidenced by the alleviation of lipopolysaccharide-induced memory impairment and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-related dyskinesia21. Besides murine models, several double-blind randomized placebo-controlled trials (RCTs) have shown that probiotics conferred beneficial effects to patients with PD27, including the relief of GI symptoms . On the other hand, investigations of the effects of probiotics on PD-related motor and non-motor symptoms, as well as inflammation factors, have yielded inconsistent results. One study showed that administering complex probiotics  improved the Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) scores, blood glutathione, high-sensitivity C-reactive protein, malondialdehyde, and insulin of patients with PD24. However, other studies found that the scores of the MDS-UPDRS and Non-Motor Symptom Scale (NMSS), biomarkers of inflammation and oxidative stress28, and fecal calprotectin 25 were not affected by probiotic administration. These reports serve as supporting evidence of the positive impacts of probiotic intake in patients in PD; however, it is likely that factors such as probiotic strain specificity and host conditions would influence the final clinical outcomes. Thus, it would be necessary to perform individual trials to confirm the health-promoting effects of specific probiotics in each case. Moreover, since conventional medications for managing PD may cause significant side effects, it would be of interest to explore the clinical effect of adjuvant probiotic therapy and to find a new candidate strain that synergizes with conventional therapeutics for the management of PD. Few studies have addressed such aspects.Probiotics are “live microorganisms that confer a benefit on the host when administered in adequate amounts”Bifidobacterium animalis subsp. lactis Probio-M8 (Probio-M8) is a novel probiotic strain that was previously isolated from the breast milk of a healthy woman. The administration of Probio-M8 could alleviate cognitive impairment in APP/PS1 transgenic mice, an animal model of Alzheimer’s disease30, and it has also been used as one of the strains in a complex probiotic formulation, which relieved anxiety and stress state of long-term sailors31. Based on these reports, we hypothesized that Probio-M8 was a potential candidate psychobiotic for alleviating neurodegenerative diseases and other neurological conditions. This study aimed to assess the added beneficial effect and mechanism of administering Probio-M8 as adjuvant treatment when given to patients with PD together with a conventional regimen (Benserazide and dopamine agonists) in a double-blind RCT. The clinical outcomes were evaluated by changes in a number of clinical indices, gut microbiome, and serum metabolome of the participants during/after the course of intervention.P = 8.6e-10; placebo group: 14.91 at baseline to 13.85, P < 0.001) and the increase in PDSS scores . Interestingly, improvement in GI-related symptoms was mostly observed in the Probio-M8 group. Marked increases were observed in Bristol scores in Probio-M8 group , though placebo group also showed significant increase from 2.79 at 1 M to 2.91 at 3 M (P = 0.044). Other GI-related parameters, including the times of spontaneous defecations and completed defecation per week, feces hardness, and difficulty in defecation, also improved in greater magnitudes in the Probio-M8 group  but not the placebo group. These results suggested that, compared with a conventional regimen, adjuvant Probio-M8 treatment conferred added clinical effects on improving anxiety, sleep quality, and GI symptoms of PD patients , while the scores only dropped at 3 M (P = 0.037) but not earlier in the placebo group. Besides, the magnitudes of improvement in sleep quality, anxiety state, mental state (measured by MMSE), and depression (measured by HAMD-17) of patients were greater in the Probio-M8 group than in the placebo group  and horizontal (same time point between different groups) comparisons Fig. . MeanwhiB. animalis (SGB_647) was at a very low level across all samples at day 0 . Throughout the intervention period, the abundance of B. animalis remained low in placebo group  but increased significantly in the Probio-M8 group over time . Horizontal comparisons between intervention groups at the same time points also showed consistent and significant increases in the abundance of B. animalis in the Probio-M8 group . Although the standard sequencing depth employed in this study (5.08 ± 1.11 Gbp per sample) like most metagenomic sequencing studies of similar nature was not adequate for strain-level tracking of a target microbe in fecal metagenomic datasets, all participants were requested to refrain from taking major food sources of B. animalis, such as yogurt, fermented milk drinks, probiotic preparations, etc. Thus, the significant increases in SGBs of B. animalis were likely a reflection of amounts of the provided strain (Probio-M8) in patients’ guts. Meanwhile, these results confirmed the participant's adherence during the trial.However, probiotic-driven modulations of fecal microbiota were revealed at the compositional level by a finer taxonomic analysis. The abundance of Butyricimonas sp. CAG:1) in control group at 3 M, while another 19 SGBs showed significant changes in abundance in Probio-M8 group in later time points  distasonis (SGB_536), Mycoplasma sp. CAG:1 (SGB_83), and Evtepia gabavorous (SGB_220) decreased in probiotic group at 1 M and 3 M  oxytoca (SGB_27) and L. fermentum (SGB_127) were diminished. In contrast, the levels of SGBs representing Ruminococcaceae , Lachnospira , and Butyricimonas [Butyricimonas sp. CAG:1(SGB_567)] increased in Probio-M8 group .The multivariable association analysis between gut microbiota and UPDRS, mental metadata , and defecation-related clinical indicators were implemented by MaAsLin2. Our results showed a strong correlation between seven clinical indicators, namely the scores of MMSE, PDSS, HAMA, HAMD, UPDRS Fig. a, b, as Bacteroides (B.) intestinalis (SGB_607), Clostridiales sp. CAG:16 (SGB_255), Clostridium sp. CAG:628 (SGB_100), Firmicutes bacterium AM10 − 47 (SGB_444), Firmicutes bacterium CAG:124 (SGB_261), K. oxytoca (SGB_027), L. fermentum (SGB_127), Roseburia inulinivorans (SGB_368), and uncultured Ruminococcus sp (SGB_384). Of particular interest was L. fermentum (SGB_127) that was diminished in the Probio-M8 group, and it was found to associate with multiple clinical parameters: significant positive correlation with the scores of UPDRS, HAMA, and HAMD-17; significant negative correlation with the scores of MMSE  was another interesting SGB that correlated negatively with two different clinical indexes, i.e., HAMA scores and feces hardness  synthesis, Glutamate synthesis II, Acetate synthesis I, and Glutamate synthesis I were the five metabolic modules having the most coverage across the gut microbiota dataset  was found in the probiotic group than placebo group at 1 M. More obvious changes were detected at 3 M, which were observed in 11 predicted metabolites. At 3 M, the predicted abundances of asymmetrical dimethylarginine (ADMA), bilirubin, cholate, chenodeoxycholate, creatine, erythronic acid, and trimethyllysine  were analyzed.P < 0.05, Fig. P = 0.05, Fig. Seven kinds of SCFAs  and 12 types of neurotransmitters  were detected across samples. The serum concentration of acetic acid was significantly higher in the patients of the probiotic group at 1 M and 3 M , and enhanced the quality of life of patients with PD. The disease improvement was accompanied by changes in the patients’ gut microbiome driven by probiotic application. Probiotic-driven improvement of PD-associated symptoms, such as relief of abdominal pain and bloating, decrease in Bristol scores, increase in the number of spontaneous bowel movements per week, and increases in hs-CRP, Malondialdehyde, and decrease in glutathione and insulin metabolism, have previously been reported34. One possible mechanism of probiotic-driven alleviation of PD-related symptoms was the increase in activity of tyrosine hydroxylase, which is a key factor for L-tyrosine-L-dopa transformation35. Apart from motor symptoms, constipation is prevalent in patients with PD, and it might have occurred 15–24 years before the diagnosis of PD36. Our literature search identified only six studies that investigated the beneficial effect of probiotic administration/ probiotic fermented milk on patients with PD28, and four of which indicated that probiotic consumption increased Bristol scores and improved defecation habits27. As expected, our data showed that administering Probio-M8 also improved stool consistency, the number of spontaneous and completed defecation per week, parameters of difficulty in defecation, incomplete defecation, assisted defecation by hand, times of drain, and hardening of stool, and the scores of PA-CQL.Our study showed that the administration of Parkinson’s drugs (Benserazide and dopamine agonists) along with probiotics significantly improved patients’ UPDRS-III scores. Similar observations were reported in previous works37. Surprisingly, co-administering Probio-M8 for 3 months alleviated anxiety and depression, improved sleep state, and reduced cognitive dysfunction in patients with PD. Adjuvant probiotic treatment has been reported to alleviate symptoms of neurological problems/diseases like anxiety39 and Alzheimer’s disease41 through gut microbiota modulation. Our previous study has demonstrated that probiotic consumption for 12 weeks could alleviate stress-/anxiety-related symptoms42 in stressed adults through probiotic-induced changes in gut microbiota diversity and functional metagenomic potential43, suggesting that the gut microbiota and its environment are important in influencing the development and severity of neurological diseases. The application of probiotics in improving the gut microbiome is thus a promising way to manage problems as such.In addition to constipation-related symptoms, non-motor symptoms, such as depression, anxiety, sleep disorders, and cognitive disorders, also cause significant distress to many patients with PD44 and mild cognitive impairment45. However, a low stool consistency, which is common in patients with PD, has been reported to be associated with a relatively high species richness46. Meanwhile, a previous 2-year trial investigated the gut microbiota of patients with PD and found no significant difference in the Shannon index and beta diversity between different time points47, which is consistent with our results. Thus, probiotic-driven regulation of the gut microbiota did not seem to be obvious on the level of microbiota diversity and structure, but on key species responsible for a specific function in the gut. For example, Bifidobacterium have been considered as pathogens in patients with PD, as they were found to correlate with the pathology of PD47. However, in our study, the relative abundance of B. animalis was found to increase significantly after probiotic intervention, accompanied by the remission of clinical symptoms, suggesting that an increase in B. animalis might mitigate PD-associated symptoms.The diversity and structure of fecal microbiota have not changed significantly during/after the course of intervention. A relatively high gut microbiota diversity is often considered a healthy physiological state, while a lower gut microbiota diversity is generally associated with unhealthy conditions, such as irritable bowel syndrome6. Some taxa in the phylum Firmicutes, such as Butyricimonas and Clostridiam clusters IV and XIV, have been considered as protagonists of carbohydrate fermentation and butyrate production49. Our study found that some SGBs in Firmicutes  increased after probiotic intake. Moreover, SGBs involved in SCFAs  synthesis increased in the Probio-M8 group after intervention. Bacteroides were reported to be GABA-producers, and their relative abundance correlated negatively with the brain signatures associated with depression15. Interestingly, our data showed that more GABA synthesis modules-encoding SGBs were identified in the probiotic group; and more Bacteroides intestinalis was found in the Probio-M8 group, which was associated with increased MMSE scores as well. Our data also showed that more SGBs representing Prevotella and Lactobacillus were found in the placebo group after the course of intervention. Prevotella were found to increase in patients with PD, and they played important roles in the metabolism of mucus layer glycoprotein, the permeability of the intestinal barrier, and inflammation51. Some Lactobacillus could produce specific enzymes that convert levodopa into dopamine before it reaches the brain, thus decreasing the drug efficacy and increasing the required dose for clinical effectiveness52. Our results showed that the abundance of L. fermentum in the placebo group was significantly associated with a less fit mental state  and more serious illness (characterized by higher scores of UPDRS). In addition, some neuroinflammation-associated pathogens decreased after Probio-M8 intervention, such as P. distasonis, Evtepia gabavorous, and, K. oxytoca. The species, P. distasonis, correlated negatively with hippocampal function, while K. oxytoca could induce anxiety and colitis, and the population of apoptotic neuron cells in the brain of mice via production of lipopolysaccharide and enterotoxins55. In our study, K. oxytoca was found to be significantly and negatively associated with feces hardness in patients with PD. Therefore, it was likely that the clinical remissive effect of Probio-M8 was related to the regulation of specific gut microbiota in patients with PD.The gut microbiota of patients with PD was reported to have reduced carbohydrate fermentation and butyrate synthesis capacity and increased proteolytic fermentation compared with healthy individualsP = 0.008) of the probiotic group but not the placebo group (P = 0.999), suggesting a more obvious regulatory effect of Probio-M8 on the gut microbial metabolites. It was previously shown that the alterations in lipid metabolism were related to the carnitine shuttle, sphingolipid metabolism, arachidonic acid metabolism, and fatty acid biosynthesis in patients with PD56. Our study also observed probiotic-driven regulation of lipid metabolism, evidenced by increases in the fecal levels of predicted levels of stearoyl ethanolamide and C20:4 carnitine, as well as a significant increase in the serum acetic acid level. Stearoyl ethanolamide inhibits the conversion of ceramide into sphingosine, and sphingosine has been reported as a neurodegeneration compound in patients with PD57. Moreover, a Caenorhabditis elegans model of PD showed that probiotic administration modified host sphingolipid metabolism and inhibited α-synuclein aggregation58. It has been reported that patients with PD have less carnitine compared with healthy individuals59. Bifidobacterium are acetic acid-producers in the gut60, and it was possible that the high abundance of B. animalis observed during the course of probiotic intervention contributed to the increase in serum acetic acid. Plasma acetic acid concentration was found to be negatively correlated with colonic transit time61, which was consistent with our observation of an increased number of spontaneous defecations per week subject to probiotic treatment.The alterations in the gut microbiota were accompanied by considerable changes in the gut metabolome. Changes in predicted metabolites in each group were calculated, and significant differences were found in the gut metabolites among the three monitored time points  by inhibiting NO synthase64. Faraco et al65. showed that administering l-arginine reversed the cognitive dysfunction of mice by promoting NO production. Cholestenone is the oxidation product of cholesterol, which can be produced by bacteria such as Bacteroides. Up to now, little is known about the effects of cholestenone on health, except that a few studies showed that cholestenone correlated positively with the rapid growth of newborns66. Cholestenone could also be used in treating Helicobacter pylori-infected patients67, and it might affect the function of the immune system68.Another group of microbial neuroactive metabolites, namely amino acids and bile acids, also constitutes information flow along the gut-brain-axisl-phenylalanine (l-Phe), through the conversion into l-tyrosine (Tyr), followed by levodopa (l-dopa), and further converted into dopamine. However, only l-dopa (but not dopamine) could pass through the blood–brain barrier and relieve PD-associated symptoms35. The increased level of serum dopa after one month of Probio-M8 treatment might suggest that more l-dopa entered the circulatory system, suggesting Probio-M8 administration potentially increased the utilization of drugs prescribed to patients with PD and in turn enhanced clinical efficacy. However, such inference needs further experimental verification.Last but not least, we found that the serum level of dopa increased at 1 M in the Probio-M8 group. Dopamine reduction is the main reason for the pathology of PD. Human dopamine is generated from Taken together, a 3-month RCT was conducted in this work to investigate the beneficial effect of adjunctive probiotic treatment in patients with PD. In summary, probiotics Probio-M8 ameliorated the severity of PD and improved the anxiety and depression state of the patients. The potential mechanism of symptom alleviation was via Probio-M8-mediated regulation of the gut microbiota of patients, further regulating the host’s metabolism of lipid, SCFAs, and neurotransmitters, and thus increasing patients’ serum level of dopamine Fig. . Our reshttp://www.chictr.org.cn/; identifier number: ChiCTR1800016977). Informed consent was signed by all PD patients before the trial started.This study complied with the ethical requirements of the Declaration of Helsinki and the regulations of the Good Clinical Practice. This study was approved by the Ethical Committee of Weihai Municipal Hospital (project number 201816). The trial was registered in the Chinese Clinical Trial Registry  patients have clear consciousness and can complete the examination, questionnaire, and medical history collection on their own or with the help of their families; (3) patients who fulfilled functional constipation according to Rome IV criteria, including less than three spontaneous bowel movements per week for the past 3 months with symptom duration of at least 6 months70; (4) patients who agreed to participate in the study and signed informed consent form. Criteria for exclusion: (1) serious cognitive dysfunction that affected written and verbal expression; (2) severe aphasia or dysarthria; (3) mentally unfit; (4) serious physical diseases, including severe abnormal liver or kidney functions; (5) chronic digestive system diseases or tumors ; (6) took immunosuppressive agents for a long time or antibiotics within one month prior to this study; (7) declined to participate.A three-month double-blind RCT was performed and the experimental processes were shown in Fig. n = 50; female to male = 32:18) and probiotic  groups, respectively. The subjects in the Probio-M8 group took two grams of Probio-M8 powder  daily, while the subjects in the placebo group took two grams of placebo powder . The probiotic bacteria and placebo materials were prepared as powder of identical appearance and taste and were provided in individually sealed plastic sachets . Another 18 subjects were further excluded from the study during the course of the intervention, including two in the Probio-M8 group  and 16 in the placebo group . A total of 82subjects completed the trial  were recruited. Thirty-three patients did not meet the inclusion criteria and were thus excluded. The remaining subjects were randomized into Probio-M8 , Hamilton Anxiety Scale (HAMA), and Hamilton Depression Scale-17 (HADM-17) for their mental state72; Parkinson’s Disease Sleep Scale (PDSS) for sleep quality73; Visual Analog Scale (VAS) for the degree of pain74; Activities of Daily Living (ADL) for their daily living ability75; and Patient-Assessment of Constipation Quality of Life (PAC-QCL) for their GI symptoms76 and other constipation-related issues, including Bristol scores, difficulty in defecation, feces hardness, incomplete defecation, assisted defecation by hand, number/week of spontaneous and completed defecation.The treatments continued for 3 months. Clinical improvement was the primary outcome of this study, while changes in the fecal microbiota and serum metabolites were the secondary outcomes. Patients were assessed by Unified PD Rating Scale-III (UPDRS- III) for the overall condition of PDSelf-administered questionnaires for recording clinical symptoms and GI-/constipation-related issues were filled out at the clinic at three time points , the satisfaction of treatment and possibility of continuing medication were quantified and investigated at 3 M and serum samples were collected at the same visit to the clinic . Metagenomic DNA samples with a DNA concentration >20 ng/μL and an optical density ratio (260 to 280 nm) between 1.8 and 2.0 were used for sequencing. Sequencing libraries were generated by NEBNext® Ultra™ DNA Library Prep Kit for Illumina , and index codes were added to attribute sequences to each sample. The built DNA library preparations were sequenced on an Illumina NovaSeq platform to generate paired-end reads .Metagenomic DNA was extracted from the patients’ stool by QIAamp Fast DNA Stool Mini Kit  in accordance with the manufacturer’s instruction; and the quality/integrity/purity of extracted metagenomic DNA and DNA concentration were assessed by 1% agarose gel electrophoresis, Nanodrop spectrophotometer, and the Qubitn = 45 and 29 for Probio-M8 and placebo groups, sampling at 0d, 1M, and 3M, respectively), generating 1.14 Tbp of high-quality paired-end reads (5.14 ± 1.13 Gbp per sample) for gut microbiota analysis. Raw metagenomic reads were quality controlled with the KneadData quality control pipeline , which filtered out low-quality reads (length of reads <60 nt) by Trimmomatic (a flexible trimmer for sequence data generated by Illumina)77. Meanwhile, human contaminating reads were removed by Bowtie2 (v2.3.5.1)78. A total of 1.13 Tbp of clean data , medium-quality , and partial-quality 83. Totally 7538 high-quality genomes , using the parameter “–min-read-percent-identity 0.95–min-covered-fraction 0.4”. The abundance level was expressed in reads per kilobase million (RPKM). Then, the sample diversity was calculated using two R packages (vegan and optparse) based on SGB abundance in RPKM.The abundance of each SGBs was calculated using a normalized method, CoverM  were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthologies (KOs) database to annotate the key gut-brain metabolic modules. The SGBs encoding related modules were identified by Omixer-RPM using the parameter -c 0.6687.A module-based analytical framework described by Valles-Colomerhttps://github.com/lh3/seqtk), and the subsamples reads were compared by the blastx function of DIAMON “-query-cover 90-id 50”. The best hit of each gene was selected for the calculation of the gene abundance profile of each sample. Then the MelonnPan-predict workflow was used to convert gene abundances into a predicted metabolomic table88.Gut metabolites were predicted based on high-quality sequences. One million reads per sample were subsampled using seqtk  was vortex mixed with 100 μL of phosphoric acid solution (36% v/v) for three minutes, followed by adding 150 μL of methyl tert-butyl ether analytical standard (MTBE standard solution). The mixture was vortexed for 3 min again and ultrasonicated for 5 min. After that, the mixture was centrifuged, and the supernatant was collected for GC-MS analysis. An Agilent 7890B GC system coupled to a 7000D GC/MS with a DB-FFAP column  was used to detect the SCFAs and neurotransmitters in the serum samples.P values were generated based on 999 permutations. All graphical presentations were generated under R and Adobe Illustrator (AI) environment.All statistical analyses were performed using the R software (v.4.0.3), and data were expressed as mean ± SD. The Shannon index and principal coordinates analysis (PCoA) were used to assess changes in the microbiota diversity and structure in fecal samples of patients during the course of intervention; the analyses were performed with R package vegan (v.2.5-1) and ggpubr. Analysis of similarities  was used to evaluate differences in gut microbiota communities between Probio-M8 and placebo groups at different time points. Wilcoxon test and t-test were used to evaluate differences in the fecal microbiome and neurometabolites between groups/subgroups. In addition, a multivariable association between clinical metadata and gut microbiota was calculated and visualized by R package MaAsLin2, tidyverse, and ggplot2. Procrustes analysis based on the vegan package was used to determine the similarity between two multivariate axes, and Patients were actively monitored for GI disorders, including common adverse events. The occurrence of any unexpected adverse event was recorded.The primary research question was whether the co-supplementation of the probiotic strain, Probio-M8, offered added beneficial effects when taken as an adjunct to conventional drugs (namely Benserazide and dopamine agonists) for treating PD. This study provided Class I evidence that adjuvant probiotic treatment enhanced the clinical effects  of conventional regimens in the management of PD.Supplementary Information files


Nurse-directed pain protocols for intranasal fentanyl administration are not widely implemented in European (EU) pediatric emergency departments (PED). Barriers include perceived safety concerns for intranasal (IN) fentanyl. The aim of this study is to describe our experience with a nurse-directed triage IN fentanyl protocol with a focus on safety in a tertiary EU PED.We conducted a retrospective analysis of patient records of children aged 0–16 years who received nurse-directed IN fentanyl between January 2019 and December 2021 at the PED of the University Children's Hospital of Bern, Switzerland. Extracted data points included demographics, presenting complaint, pain score, IN fentanyl dosage, concomitant pain medication use, and adverse events.n = 284, 90%). Mild adverse events (vertigo) were reported in two patients (0.6%), without a correlation to concomitant pain medication or protocol violation. The only reported severe adverse event of syncope and hypoxia in a 14-year-old adolescent occurred in a setting where the institutional nurse-directed protocol was violated.A total of 314 patients were identified with ages ranging from 9 months to 15 years. The main indication for nurse-directed fentanyl administration was musculoskeletal pain due to trauma (In accordance with previous studies outside of Europe, our data support the case that when appropriately used, nurse-directed IN fentanyl is a safe potent opioid analgesic for pediatric acute pain management. We strongly encourage the introduction of nurse-directed triage fentanyl protocols Europe-wide in order to provide effective and adequate acute pain management in children. Nurse-directed triage pain protocols  have been shown to improve the time to analgesia in emergency medicine . Nurse-dHowever, as recently shown by a survey study conducted by the Research in European Pediatric Emergency Medicine (REPEM) network, in European (EU) pediatric emergency departments (PEDs), triage nurse-directed pain protocols are still not widely implemented . Indeed,The standard IN dose of 1.5 µg/kg fentanyl has been shown to provide effective analgesia comparable to the analgesic potency of IV morphine , 8. As aThe aim of this retrospective observational study is to describe our experience with a nurse-directed triage IN fentanyl protocol with a focus on safety in a tertiary EU PED.The PED in this study is part of a level 1 pediatric trauma center and university children's hospital with an average of 25,000–30,000 annual presentations of children aged 0–16 years. A nurse-directed triage IN fentanyl protocol has been established since 2013. The protocol allows triage nurses to administer IN fentanyl  in patients older than 12 months without prior physician prescription, when severe pain is present. Pain assessment occurs using one of three different pain score scales dependent on age. These are the “Kindliche Unbehagen und Schmerz Skala—KUSS” (<4 years), the Wong–Baker Faces Pain Scale (4–7 years), and the Visual Analog Scale (>8 years). After IN fentanyl administration, oxygen saturation is monitored continuously for 30 min and patients are clinically reassessed by nurses every 15 min see . The nurWe retrospectively analyzed the electronic medical records of all children <16 years who received IN fentanyl at triage from January 2019 to December 2021. We extracted data pertaining to demographics, presenting complaint, pain score, concurrent pain medication use, effectively administered IN fentanyl dose, and adverse events. Pain scores were corrected to the Visual Analog Scale, with severe pain defined by a score of ≥6. When adverse events were reported in the electronic medical record, a more detailed chart review was completed in order to identify the relationship with fentanyl administration and to assess the severity of the event (according to fentanyl reference safety information provided by the European Medicine Agency). The vital signs monitoring the record of all included patients were manually screened for any atypical observations that may indicate an adverse event, which was not previously captured. A further search for adverse events was conducted by checking the medication administration record for rescue treatments .Continuous variables were described as means ± standard deviations (SD) or median and interquartile range (IQR) and categorical variables as percentages .A total of 314 patients were identified who received nurse-directed IN fentanyl at triage. During the investigated period, our PED reported 76,043 visits. In terms of age, the nurse-directed protocol was violated in two cases, in a 9-month-old infant and an 11-month-old one treated for a scald burn. No adverse events were reported in these patients.Pain in musculoskeletal injury due to trauma was the main indication for nurse-directed IN fentanyl, with isolated injury of the upper extremity being the most reported complaint. Non-trauma-related pain  accounted for only 1% of all treated patients. Interestingly, despite most of the patients receiving IN fentanyl with high pain scores according to the institutional protocol , in 28 patients (8.9%), IN fentanyl was administered with lower pain scores. This phenomenon seemed to occur especially when fracture was clinically suspected in anticipation of an x-ray.n = 117, 37.3%) had already received analgesics in the 2 h prior admission, of which only three patients (1%) had already received intranasal fentanyl by paramedics. None of these had adverse events. No patient had received other opioids before admission. All patients received IN fentanyl within 10 min from the beginning of triage. Dosing errors with doses exceeding >1.7 µg/kg occurred in four patients (1%), with the maximal administered fentanyl dose being 2 µg/kg. Underdosing (<1.3 µg/kg) in relation to the institutional protocol occurred in 13 patients (4%).One-third of all patients  vs. acetaminophen  and metamizol .A total of 152 patients (48.4%) received a non-opioid analgesic in addition to IN fentanyl at triage, with non-steroidal anti-inflammatory drugs (NSAIDs) being the most used comedication (A second dose of fentanyl was administered in 63/314 (20%) of patients during their PED stay.Only three adverse events were reported, one of which was categorized as “severe.” The details of the three patients experiencing adverse events are summarized in 2 not specified). The patient showed rapid recovery, and the administration of naloxone or volume bolus was not needed. Globally seen, this severe adverse event occurred in an atypical setting and the IN fentanyl administration breached the conditions of our institutional nurse-directed pain protocol .One severe adverse event was reported in a 14-year-old, previously healthy patient. IN fentanyl was used to mobilize the patient with severe hip pain out of the parent's car. The patient received two consecutive doses: 120 µg (1.3 µg/kg) and an additional 50 µg (0.6 µg/kg) within 5 min. After a further 10 min, the patient could be mobilized but experienced a syncope and transient desaturation on pulse oximetry requiring short-term oxygen support . A detailed chart review of these patients showed that IN fentanyl was given in anticipation of expected pain during x-ray. Anticipatory pain treatment appears to be an important indication in nurse-directed pain management protocols . In compAlthough nurse-directed pain protocols in general have the potential for providing more effective pain relief in the PED setting and sensitization of caregivers toward adequate pain management for their children, these protocols are still not widely implemented. In a survey among 171 university hospitals and tertiary care centers of 19 EU countries, nurse-directed pain protocols were available only in 53% of all sites and IN fentanyl only in 11 sites (6%) . ImportaIn conclusion, on the basis of our data on nurse-directed IN fentanyl protocol in an EU PED setting, combined with previous evidence on this topic outside Europe, as well as the evidence available on the efficacy and safety of IN fentanyl in general, we encourage the implementation of nurse-directed IN fentanyl pain protocols in EU PED to improve pediatric acute pain management.


The chest pain classifications that are currently in use are based on studies that are several decades old. Various studies have indicated that these classifications are not sufficient for determining the origin of chest pain without additional diagnostic tests or tools. We describe a new chest pain scoring system that examines the relationship between chest pain and ischemic heart disease (IHD). Cross-sectional study conducted in a tertiary-level university hospital and two public hospitals. Chest pain scores were assigned to 484 patients. These patients then underwent a treadmill stress test, followed by myocardial perfusion scintigraphy if necessary. Coronary angiography was then carried out on the patients whose tests had been interpreted as positive for ischemia. Afterwards, the relationship between myocardial ischemia and the test score results was investigated. The median chest pain score was 2 (range: 0-7) among the patients without IHD and 6 (1-8) among those with IHD. The median score of patients with IHD was significantly higher than that of patients without IHD (P = 0.001). Receiver operating characteristic analysis showed that the score had sensitivity of 97% and specificity of 87.5% for detecting IHD. We developed a pre-test chest pain score that uses a digital scoring system to assess whether or not the pain was caused by IHD. This scoring system can be applied easily and swiftly by healthcare professionals and can prevent the confusion that is caused by other classification and scoring systems. Furthermore, lung diseases or peripheral arterial diseases may restrict exercise capacity and the complaints of these patients may very well subside with rest. These disorders may cause increases in symptoms of chest pain during exercise and decreases in discomfort during resting period, thus causing high suspicion of angina pectoris, even though the origin of the symptoms was non-cardiac.In addition to these problems, as mentioned in several studies and as seen in our own clinical experience, the nomenclature of “typical”, “atypical” and “non-cardiac” is often insufficient and may lead to misunderstandings.In this study, our aim was to create a chest pain score that was accurate, swift, more comprehensive and more objective in determining whether or not the chest pain in question was related to IHD.All patients who presented to the cardiology clinics or emergency departments (ER) of Eskisehir State Hospital  or Canakkale Onsekiz Mart University Hospital  complaining of chest pain between June 2017 and February 2018 were included in this study. Patients with prior ST segment elevation myocardial infarction, pregnant patients and individuals under 18 years of age were excluded from the study.After exclusion of any patients who refused to participate in the study and those presenting exclusion criteria, data were collected from the remaining subjects, as our sample of patients. Acardiology specialist applied the chest pain score to all subjects through face-to-face interviews.Implementation of this study was endorsed by our institution’s Internal Review Board .After the patients had been assessed for typical risk factors such as age, gender, diabetes mellitus, hypertension, coronary artery diseases (CAD) and smoking status, the chest pain score developed for this study was applied to them.Data collection was planned before the index test was performed and before the reference standard was implemented. Agroup consisting of 10 experienced cardiology specialists was given the task of creating the scoring system. This group initially determined which cardiac complaints were typical, according to their own clinical experience, and this was followed by meticulous evaluation of the literature on this topic.A total of 10 major questions regarding chest pain properties and the patients’ characteristics were formulated and evaluated by this group of 10 cardiologists. Through this assessment, unnecessary questions were omitted, such that the number of questions was reduced to five. The test consisting of these five questions was then applied to a preliminary group of 20 patients and the questions were then weighted according to the results from this test.The final chest pain scoring system that was proposed in the present study thus consisted of five questions that could only be answered as “yes” or “no”. “No” answers were scored as zero points. For the first and fifth questions, a “yes” answer was scored as 2 points. For the second and fourth questions, a “yes” answer was scored as 1point. The third question consisted of two sub-questions: for each sub-question, a “yes” answer was counted as 1 point, such that the maximum score was 2 points. After the total score from the questions had been obtained, 1 point was added to the score of patients with diabetes and/or those older than 75 years of age .After final scores for the patients evaluated in this study had been obtained, patients who had been found to present a high risk of ischemic heart disease underwent coronary angiography without a stress test. The remaining patients then underwent a treadmill stress test. The treadmill stress test was considered positive for patients who presented 1 mm of horizontal or downsloping ST segment depression in three consecutive leads, or ST segment elevation, or chest pain triggered by the stress test. Myocardial perfusion scintigraphy (MPS) was performed on the patients with suspicious treadmill stress test results. MPS imaging was performed using the imaging protocol developed by the American Society of Nuclear Cardiology.The patients were divided into three groups depending on the results from coronary angiography. Patients with > 50% stenosis in the left main coronary artery (LMCA) or > 70% stenosis in any vessel with a diameter > 2 mm were classified as the “critical coronary artery disease” group. Patients with any lesion not classified as critical or patients with slow coronary flow were classified as the “non-critical coronary artery disease” group. Patients with normal coronary arteries were classified as the “normal coronary arteries” group. The ischemia-positive group included the critical CAD patient group, the non-critical CAD patient group and the group of patients whose non-invasive stress tests were positive. Patients with a positive stress test and normal coronary arteries were included in the ischemia-negative group.The IBM Statistical Package for the Social Sciences (SPSS) software for Windows, version 15.0 , was used for data analysis. The demographic characteristics of the study group were reported using descriptive statistics  and dispersion measurements (standard deviation and minimum-maximum). Initially, the normality of the total scores was tested using the Kolmogorov-Smirnov normality test and graphs. Frequency data were analyzed by using the chi-square test as univariate analysis. Receiver operating characteristic (ROC) analysis was used for calculating the sensitivity, specificity and positive and negative predictive values of cutoff scores from the scale. Median scores were compared using the Mann-Whitney U and Kruskal-Wallis tests. If any comparison yielded a P value less than 0.05, it was considered statistically significant.A total of 484 patients were included in this study. The patients’ mean age  was 52.0 ± 15.0 years (minimum-maximum: 18-84) for the entire study group, while the mean for males was 52.0 ± 14.0 years and the mean for females was 52.0 ± 15.0 years (P = 0.585). The study group consisted of 229 males (47.3%) and 255 females (52.7%).The median chest pain score of those with positive treadmill stress test results was found to be 6, while the median score of the patients with negative results was 1. The scores of those who had positive exercise test results were significantly higher than the scores of those who had negative results (P < 0.001).Regarding MPS results, those with positive results had a median score of 6, while those with negative results had a median score of 3. The chest pain scores of MPS-positive patients were significantly higher than the scores of MPS-negative patients (P < 0.001).Comparison of the chest pain scores of patients with normal carotid arteries and those with critical and non-critical CAD showed that the median score of the critical and non-critical CAD group (median score: 6) was significantly higher than the score of those with normal carotid arteries (median score: 3) (P < 0.001).The median chest pain scores for hypertensive and non-hypertensive patients were 3 and 2, respectively; while the scores for diabetic and non-diabetic patients were also found to be 3 and 2 respectively. Statistical analysis revealed that the scores for hypertensive and diabetic patients were significantly higher than the scores for those without the respective conditions (P < 0.001). Furthermore, it was observed that the chest pain score increased with age and that there was a significant relationship between age and chest pain score (P = 0.001). Regarding sex, the median chest pain scores were 3 and 2 for males and females, respectively. Thechest pain scores of males were significantly higher than those of females. There was no significant relationship between chest pain score and the history of CAD, smoking status, peripheral artery disease or heart failure. The chest pain score was 6 for IHD patientsand 2 for non-IHD patients. The chest pain score for IHD patients was significantly higher (P = 0.001). The relationships between ischemia and chest pain score are shown in The patients were divided into four groups according to chest pain score, presence of IHD and pre-test risk factors for IHD . The firWe also performed ROC curve analysis for each of the questions and for the total score. The results showed that a chest pain score threshold of 4.5 demonstrated sensitivity of 90% and specificity of 95.83% for detecting IHD, while the positive predictive value (PPV) and negative predictive value (NPV) were found to be 84.91% and 97.35%, respectively . The ROC,In this study, we applied a chest pain scoring system consisting of five questions, to patients who had complaints of chest pain, to determine whether or not the chest pain originated from IHD. The study was initially planned because we noticed that the classical classifications for chest pain  were insufficient without additional stress tests.,,,The classical chest pain classification created by Diamond suggests that two positive answers among the three questions that evaluate the association between exercise and pain (except the first question) are enough to determine that the source of pain is myocardial injury. However, in cases of acute coronary syndrome, pain may be triggered without stress or exertion.Another widely accepted chest pain classification score, the WHO-Rose angina questionnaire,Given these facts, we aimed to develop a chest pain scoring system that would inherently solve this problem and could be implemented by all physicians regardless of specialty, and also by other medical staff. The proposed chest pain score is a swift and easy method for determining whether the chest pain in question is caused by myocardial injury, and it can be used in both the ER and the outpatient setting. The scoring system in this study can be used to assess patients’ risk of ischemia and can group patients based on these scores.In this scoring system, we tried to evaluate the association between patients’ complaints and the presence of ischemia, without initially considering risk factors for cardiovascular diseases. In establishing this score, we took into account the four features of chest pain, as described in the European Cardiology Community guidelines: the characteristics of the pain, its location and extent, its duration and its relationship with exercise; while also evaluating other symptoms that accompany chest pain.In the past, various scoring systems for evaluating chest pain were proposed.The present chest pain assessment score is the first, since publication of the chest pain classification of 1983,,,Old age and diabetes are risk factors for IHD but are also misleading factors for physicians because they can affect the anamnesis of patients. Since chest pain and insufficient capacity for exertion can be explained by diabetes and old age, their association with ischemia may be overlooked. Experiencing pain during exertion is a very specific finding in IHD, but this is difficult to detect in elderly patients because they are often not active enough to sense effort-related chest pain. It has been reported in many studies on diabetes and the elderly that pain can be expressed atypically even when it is associated with IHD.The ROC analysis on the scoring system showed that this system has very high sensitivity and specificity for detecting IHD. Although question 4 was not successful on its own, for discriminating whether pain was associated with IHD, it was effective within the overall sensitivity and specificity of the scoring system. Therefore, the question was not omitted.Although men were found to have significantly higher chest pain scores in the current study, we did not attempt to change our scoring system to evaluate differences between the sexes. This was because various studies have shown that there is no significant correlation between sex and the explanations for typical and atypical pain in patients with angina.This study was conducted with 484 patients in two separate centers. This may be considered to be a limitation of the study. Repeating this work with larger populations and different ethnic groups might yield different results. In this study, we used coronary angiography in an attempt to exclude false positive results from non-invasive tests, but the results may still have been impaired by false negative tests. However, we were unable to perform coronary angiography to exclude false negative results due to ethical concerns. There is a need to support this work through using larger populations and multinational studies in which patients are followed for longer periods.We developed a chest pain score that can easily and rapidly be applied by all healthcare workers, and which focuses solely on patients’ chest pain characteristics. The results from ROC analysis indicate that the proposed chest pain scoring system is very successful in identifying patients with pain due to cardiac injury. We believe that this scoring system can be used safely to accurately identify IHD in patients who present chest pain, without the use of non-invasive stress tests.


Visceral hypersensitivity, a fundamental mechanism of chronic visceral pain disorders, can result from both central or peripheral factors, or their combination. As an important regulator of normal gut function, the gut microbiota has been implicated as a key peripheral factor in the pathophysiology of visceral hypersensitivity. Patients with chronic gastrointestinal disorders, such as irritable bowel syndrome, often present with abdominal pain secondary to adverse reactions to dietary components. As both long- and short-term diets are major determinants of gut microbiota configuration that can result in changes in microbial metabolic output, it is becoming increasingly recognized that diet–microbiota interactions play an important role in the genesis of visceral sensitivity. Changes in pain signaling may occur via diet-induced changes in secretion of mediators by both the microbiota and/or host cells. This review will examine the peripheral influence of diet–microbiota interactions underlying increased visceral sensitivity. Diet is a major determinant of gut microbiota composition and function. Additionally, food intake induces both chemical (e.g. release of enteroendocrine mediators) and mechanical stimuli (e.g. bowel distension) within the GI tract capable of activating neural pathways. Yet, despite the multiple effects of diet, processing food within the gut is typically not consciously perceived. However, many patients with chronic GI disorders perceive food as a major trigger of their symptoms. For example, 25–84% of the patients with irritable bowel syndrome (IBS), a disorder characterized by abdominal pain and altered bowel habit in the absence of overt inflammation,9 cite food intolerances unleashing GI symptoms.10–13 While food-induced abdominal pain in GI disorders has traditionally been thought to be secondary to an already hypersensitive GI tract, recent studies suggest that dietary components can stimulate pathways that ultimately lead to visceral hypersensitivity. Since the effects of a particular diet differ from person to person, possibly influenced by a combination of host and microbiome features,14 the gut microbiota is considered an important intermediary between dietary factors and changes in visceral sensitivity. This review will examine the role of diet–microbiota interactions that lead to increased visceral sensitivity.Visceral hypersensitivity, defined as exaggerated pain signaling in response to stimuli within visceral organs such as the gastrointestinal (GI) tract, is a fundamental mechanism of chronic visceral pain disorders.15 These nerves are primarily involved in the transmission of physiological stimuli, such as satiety signaling or ongoing motor activity within the gut.16 Spinal afferent nerves innervate the entire GI tract via both splanchnic and pelvic nerves.17 While subtypes of these afferent nerves may be involved in reflex pathways within the GI tract, other subtypes of spinal afferent nerves are involved in transmission of nociceptive signals to the CNS.19Sensory information from the GI tract to the central nervous system (CNS) follows two main routes: (1) vagal afferent nerves, whose cell bodies lie within the nodose ganglia and (2) spinal afferents, whose cell bodies lie within the dorsal root ganglia (DRG). Vagal afferent nerves are more prevalent in the proximal gut, although studies suggest there is some innervation by vagal afferents in the colon.20 increased synaptic transmission of ascending pathways or decreased inhibition of descending pathways within the spinal cord,21 or changes in processing of sensory input within the brain.22 In this review, however, we will concentrate on the peripheral mechanisms of visceral pain, as the focus will be on diet and microbiota interactions.Visceral hypersensitivity can occur via central or peripheral mechanisms. Central sensitization can occur at different levels within the CNS. This may include increased activation of neurons within the dorsal horn of the spinal cord,et al.23). Visceral hypersensitivity can result when there is an imbalance toward excitatory pathways. This is the case when there is ongoing inflammation, for example, as many inflammatory and immune mediators, acting via G-protein coupled receptors that in turn modulate ion channels, increase excitability of spinal nerves. Consequently, a stimulus (e.g. colonic distension) will now evoke a greater response resulting in either the sensation of abdominal pain following a normally innocuous stimulus  or greater abdominal pain following a painful stimulus . While sensitization of afferent nerves within the gut can be short-lived, there can be long-lasting sensitization following resolution of inflammation. This may be the case for visceral hypersensitivity in patients with post-infectious IBS (PI-IBS)24 or inflammatory bowel disease (IBD) patients who continue to experience chronic abdominal pain, despite achieving endoscopic remission.25 Although several studies have shown evidence of residual low-grade inflammation or immune activation,26 the underlying mechanisms are still to be fully elucidated.Sensitivity of spinal afferent nerves innervating the GI tract is the result of a balance of activation of excitatory and inhibitory pathways  blockade and serine protease inactivation.29 The H1R antagonist ebastine also decreased abdominal pain scores and improved global symptom relief in a small clinical study of IBS patients.30 Additionally, the H1R antagonist and mast cell stabilizer ketotifen has also been shown to improve visceral hypersensitivity in patients with IBS.31 Indeed, histamine release by mast cells, triggered by local immune response to specific food components,32 has been shown to promote visceral hypersensitivity through H1R-mediated TRPV1 sensitiation.32 Other mediators, as well, have been shown to contribute to the development of visceral hypersensitivity.34 A recent study demonstrated that intracolonic infusion of IBS fecal supernatants into mice also increased luminal 5-HT. This increase was due to mast cell secretion of prostaglandin E2 leading to a downregulation of serotonin reuptake transporter on epithelial cells. The 5-HT3 receptor antagonist ondansetron prevented the development of visceral hypersensitivity by IBS fecal supernatants.7 Thus, IBS patients have increased neuroactive mediators within the tissue and lumen that increase pain signaling within the GI tract.There is evidence that patients with IBS have increased excitatory mediators, such as proteases, prostaglandins, histamine and serotonin (5-HT), both within the tissue as well as within the gut lumen, and the continued presence of these mediators may be responsible for ongoing visceral hypersensitivity. Activated mast cells release multiple bioactive mediators, including histamine, prostaglandins and tryptase, and have long been implicated in the pathogenesis of IBS, particularly in the generation of visceral hypersensitivity. Increased number of colonic mast cells, often found in proximity to sensory nerves in IBS patients,10–13 specific dietary components may be one of these drivers. Additionally, the gut microbiota may also contribute to the pool of excitatory mediators, as there is growing evidence that gut bacteria modulate visceral sensitivity via signaling to afferent nerves in the GI tract.Given the heterogenous nature of IBS pathophysiology, drivers of the continued presence of these excitatory mediators may differ in subsets of IBS patients. Since a large proportion of IBS patients report GI symptoms due to food intolerances,36 The gut microbiota evolves during early life until a unique, subject-specific (fingerprint) adult-like community arises, which is highly resilient and relatively stable throughout life.37–41 Inter- and intra-individual variability are very high even among healthy individuals, thus it is difficult to define “the healthy” gut microbiome composition, as this remains a relative state.42 Functional redundancy is a key characteristic of the gut microbiota, with different bacterial consortia performing similar functions in different individuals.43 However, with the recent advances in technology and analyses software, the importance of strain specificity has emerged as well, meaning that not all strains will have the same impact on health or disease.42The highly complex and diverse community of microorganisms, collectively called microbiota , comprising archaea, bacteria, viruses, and eukaryotes is mammals’ closest symbiont.45 while occupying a unique, nutrient rich niche. The central role of the microbiome is highlighted by studies in germ-free, microbiome depleted, or gnotobiotic animals, which demonstrated that gut microbiota is required for normal gut physiology including gut motility,46–49 metabolism, a balanced immune system,50–56 and development of the enteric nervous system (ENS).57 Perception of inflammatory, mechanical and visceral pain is also regulated by the gut microbiota,58–60 however the mechanisms appear to be different depending on the type of pain. While inflammatory and oxaliplatin-induced pain are decreased in absence of bacteria,59 bacterial pathogens can directly activate sensory neurons and induce mechanical and thermal pain.60 Other factors, including sex, may affect the microbial modulation of pain perception as germ-free male but not female mice have increased visceral sensitivity compared to conventionally colonized mice.61The gut microbiota carries out essential functions that the human body is unable to perform,62 proteases,63 and histamine,64 or through stimulating secretion of excitatory mediators from host cells, such as serotonin from enterochromaffin cells.65 Conversely, the microbiota also produces serine proteases that inhibit nociceptive neurons, leading to a decrease in visceral sensitivity.66 Thus, the microbiota is capable of both increasing or reducing visceral pain. It is important to keep in mind that the gut microbiota and the host maintain a bidirectional relationship, such that not only does the gut microbiota influence host physiology but the host also influences microbial structure and metabolism. For example, many neuropeptides, such as substance P, calcitonin-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide, have antimicrobial activity.67 In addition, nociceptors have been recently shown to both sense commensal and dietary cues.68 Moreover, nociceptors can regulate gut microbiota composition, via neuropeptides secretion, which in turn mediate tissue-protective processes in the context of ongoing intestinal inflammation.69 Thus, the balance of excitatory and inhibitory effects on visceral perception will depend on the specific bacterial strains, the intestinal milieu in which the gut microbiota reside , as well as the interaction between the gut microbiota and luminal factors, including those derived from diet.Modulation of visceral sensitivity by the gut microbiota may occur through the activity of mediators that directly excite nociceptive nerves in the GI tract, such as LPS,70 Diet therapies may reduce abdominal pain through multiple mechanisms, including changes in the microbiota. Diet not only impacts microbiome composition, but also influences the microbiome function and metabolite output,72 which in turn may alter nociceptive signaling within the GI tract. Abdominal pain in some IBS patients may also result from local immune activation triggered by dietary antigens, in both the small73 and large intestine.32In gastrointestinal disorders with chronic abdominal pain as the predominant symptom, such as IBS, diet has been regarded among first-line treatments as patients complain of adverse reactions to specific foods.74 the field is fervent with debate on which diet appears to be better.75–77 Studying diet as a therapy is challenging as many patients already use diet modification in an attempt to improve their abdominal pain. For example, a recent study found that IBS patients with worse symptoms, primarily driven by increased abdominal pain and symptom-related negative impact on quality of life, are more likely to adhere to exclusion or restrictive diets.78 These diets, in turn, result in substantial changes to the gut microbiome composition, which might influence IBS symptoms presentation and perception.78 Consequently, this may introduce more heterogeneity into a diet therapy study. Despite these challenges, there has been a significant number of clinical studies investigating diet therapies to treat IBS symptoms. These include traditional dietary advice, a gluten-free diet (GFD), and a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP), all of which have shown varying degrees of effectiveness for improving symptoms.79–84 As the GFD and low FODMAP diets are currently the most commonly employed and investigated, we will focus on these two.While diet has become a common therapy to treat patients with chronic abdominal pain, there are currently no biomarkers associating a subset of patients to a response to a specific diet therapy. And although diet is listed among the first-line treatment options for abdominal pain in IBS,85 however, the proportion of those reactive to gluten protein may be smaller than originally thought.86 Excluding gluten from diet to reduce IBS symptoms has also been questioned due to paucity of randomized, placebo-controlled trials.87 However, a recent study suggested that immune reactivity to gluten in the form of anti-gliadin IgG can be used as a biomarker to identify patients with IBS, mainly with diarrhea, who might benefit from a GFD.80 The underlying immune mechanisms are unclear, as a recent study using confocal laser endomicroscopy (CLE) concluded that more patients responded to a GFD than to a duodenal application of wheat.88 This suggests that some patients, who benefit from a GFD, respond to other components of wheat that do not trigger a response detectable by CLE.88 It is also possible that adverse responses detectable by CLE to wheat do not occur in the duodenum, but in the distal small bowel or even colon, thus explaining discrepant results. Alternatively, it has been proposed that fermentable fructans contained in the wheat, rather than gluten, induce symptoms in IBS patients.89 Reactivity to gluten maybe also be affected by gut microbiota, as several recent studies demonstrated that different bacteria metabolize gluten partially or completely,91 affecting immunogenicity of gluten-derived peptides.Accumulating data suggest that gluten induces symptoms, including abdominal pain, in a subset of IBS patients,93Excessive fermentation of high FODMAP foods leading to gut distension has been proposed as a mechanism contributing to symptom generation in IBS patients. In addition to colonic fermentation, undigested carbohydrates have an osmotic effect increasing water content in the small intestine and colon, that can contribute to gut distension.94 However, a study using magnetic resonance imaging (MRI) showed that IBS patients and healthy controls have similar levels of bowel gas and water content, suggesting that abdominal pain is not primarily driven by greater gut distension in IBS patients but rather by visceral hypersensitivity.95 Another study demonstrated that increased bowel symptoms after lactulose challenge did not correlate with small intestinal water content or bowel distension, although these differed between IBS patients and healthy controls.96 Thus, visceral hypersensitivity is likely a key player, and together with an increase in water content and bowel distension, as well as impaired gas transit and evacuation,95 may generate symptoms such as pain and bloating.97 Although detrimental responses to FODMAPs appear to be driven by peripheral mechanisms, they may also involve central sensitization in response to dietary components as suggested by a recent fMRI study. Following fructan infusion, multiple areas in the brain related to pain, such as the cerebellum, supramarginal gyrus, anterior and midcingulate cortex, insula and thalamus, of IBS patients responded differently compared to healthy controls.98Abdominal pain has been one of the most cited symptoms to improve following reduction in poorly absorbed short-chain carbohydrates, such as FODMAPs.P) due to its pathogenic potential, along with an upregulation in microbial amino acids biosynthesis and carbohydrate metabolism, was suggested to confer the best response to the diet.99 On the other hand, a recent meta-analysis concluded that a low FODMAP diet would be recommendable to patients with high colonic methane and SCFA production.100 Many other studies have pointed toward gut microbiota composition and function as accountable for the diet response, although no clear unifying pattern has been found .Given that the colonic microbiota ferment FODMAPs, microbial profiles have been implicated in visceral hypersensitivity. A specific microbiota, termed a “pathogenic” microbiota .While diet therapies over the past decade have shown efficacy in improving abdominal pain in subsets of IBS patients, mechanistic data supporting their use has been lacking until recently. Clinical trials of diet therapies present with multiple challenges that can weaken their conclusions,32 Therefore, future studies that further phenotype the subgroup of patients to investigate  will lead to better identification of whom is more likely to benefit from a therapy that targets the diet–microbiota interface.A major limitation in studying diet therapy in IBS patients is the heterogeneity within the patient population. Consequently, studying a subgroup of patients only based on the current clinical classification (e.g. IBS diarrhea predominant or IBS constipation predominant) may underestimate a role for a diet–microbiota interaction driving abdominal pain. Furthermore, a subset of patients may have food-induced abdominal pain due to local immune signaling driven by food antigens, rather than microbiota-related mechanisms.105 or epithelial cells106  are necessary, additional translational studies using gut microbiota from the subgroup of interest to colonize germ-free mice to study the effect of a specific diet are ideal to provide mechanistic insight into diet–microbiota interactions in abdominal pain signaling. Alternatively, the microbiota and/or fecal supernatants , before and after the diet, can be used to study pain signaling in a pre-clinical model. This approach allows a parallel comparison of clinical responses and changes in pain signaling in mice induced by the microbiota and/or fecal supernatants. Furthermore, these approaches may elucidate whether microbial mediators directly activate nociceptive nerves and/or indirectly via activation of host immune cellss106 see . In addiDiet therapies may offer relief of chronic abdominal pain to some patients, such as those with IBS. However, the restrictive nature of many diet therapies might not be sustainable in the long-term due to the diet complexity, as well as its potentially negative long-term effects on the microbiome. Only recently have studies begun to elucidate mechanisms of how diet-microbiota the mechanisms of how diet–microbiota interactions may induce visceral hyperalgesia. Therefore, further studies are needed to identify the patients with chronic abdominal pain who would benefit most from a diet directed and/or a microbiome-directed therapy.


Surgical trauma initiates changes in central and peripheral nervous systems that need to be treated therapeutically to facilitate postoperative pain. The quality of postoperative analgesia is expected to affect clinical outcomes positively. Albeit optimal pain relief following cardiac surgery is often complex, researchers have tried to explore several techniques other than conventional ones during the last decade to find a unique analgesic method for postcardiac surgical patients. This study aims to find a unique analgesic approach that maximizes patient satisfaction after off‐pump coronary artery bypass graft (OPCABG) surgery.The current study will compare the analgesic effect of upper thoracic epidural analgesia (TEA) with conventional analgesia after OPCAB graft surgery. For this, we will use a Quasi‐experimental study design. Patients admitted for coronary artery bypass graft (CABG) surgery will be assigned into two groups. The control group  will receive intravenous opioids and nonsteroidal anti‐inflammatory medications, and the study (case) group (TEA) will receive Inj. Bupivacaine 0.25% as an infusion through the epidural catheter. Physiologic parameters like hemodynamic and respiratory variables and pain scores will be recorded in predesigned format periodically.2, PaCO2, PEFR, FEV1). After data collection, the result will be analyzed and published in the public domain and in journals.We expect to analyze a total of 130 consecutive off‐pump CABG surgery patients in Group A  and Group B . Study variables will be the visual analog scale score, hemodynamic parameters (heart rate, mean arterial pressure, and respiratory parameters (respiratory rate, PaOWe expect thoracic epidural analgesia with local anesthetics will be a reliable postoperative analgesic option. The quality of postoperative analgesia positively affects morbidity in cardiac surgery.Off‐pump coronary artery bypass graft (OPCABG) is a commonly practiced procedure for ischemic heart disease worldwide.International Association for the Study of Pain (IASP) defined pain as “An unpleasant sensory and emotional experience associated with or resembling that associated with, actual, or potential tissue damage.”Traditionally opioids, nonsteroidal anti‐inflammatory medications (NSAIDs), or a combination of both, are being used to provide analgesia following cardiac surgery, which is linked to adverse side effects.Despite many new methods, advanced pharmacological analgesic preparations, and proven pain services, postoperative pain control in coronary artery bypass graft (CABG) surgery has remained a challenge. As a general rule, single‐modality therapy is often recommended to treat immediate postoperative pain.This current study will aim to evaluate an effective method of optimum postoperative analgesia using either TEA (Inj. Bupivacaine 0.25% at 3–5 ml/h continuous infusion) or conventional analgesia  after OPCAB surgery. Furthermore, we also aim to evaluate postoperative cardiac and respiratory variables in relation to pain intensity after OPCAB surgery.1.11.1.1There is no significant difference between conventional and upper TEA in pain control after OPCAB surgery.1.1.2Upper TEA with Inj. Bupivacaine will provide better postoperative analgesia than conventional one after OPCAB surgery.1.2Researchers explore significant pain control after cardiac surgery using different methods  of pain services compared to conventional ones. Furthermore, an effective single modality of analgesia has yet not been established.There is seldom a study performed in Bangladesh to explore the role of epidural analgesia after off‐pump CABG. So, the research gap is to find an effective analgesia method after OPCAB surgery using TEA. We characterize the research gap with the PICO framework as follows:Population (P): Patients admitted for scheduled OPCAB surgery.Intervention (I): TEA using Inj. 0.25% Bupivacaine as a continuous infusion pump.Comparison (C): Traditionally used opioids and NSAIDs (Inj. Ketorolac 30 mg iv) analgesics.Outcomes (O): Improved postoperative analgesia and reduced postoperative morbidity.1.3Early extubation and hemodynamic stability are the primary concerns of cardiac anesthesiologists in the modern era of fast‐tracking. The analytical framework Diagram  for this2This Quasi‐experimental Study design will be conducted from February 2021 to July 2022 at the Department of Cardiac Surgery, Bangabandhu Sheikh Mujib Medical University, and Al‐Helal Specialized Heart Hospital, Bangladesh. Patients who will undergo elective off‐pump CABG surgery will be included in the study. The study exclusion criteria are patient refusal or uncooperative patient, abnormal coagulation profile, previous cardiothoracic, cervical, or upper thoracic operation, known case of chronic obstructive pulmonary disease, chronic kidney disease, chronic liver disease malignancy, systemic or local infection, and any spinal abnormality. Also, the patient who converted from OPCABG to On‐pump during the procedure will be excluded from the study.2, partial pressure of carbon dioxide, peak expiratory flow rate, & forced expiratory volume in the 1st second.A total of 130 patients will be evaluated in two groups; Group A (Case 65 patients): off‐pump CABG surgery under general endotracheal anesthesia and will receive postoperative TEA infusion (Inj. Bupivacaine 0.25%); and Group B (Control 65 patients): off‐pump CABG surgery under general endotracheal anesthesia and will receive postoperative conventional analgesia . Study variables will be VAS score, hemodynamic parameters like heart rate, mean arterial pressure, and respiratory parameters  for this null hypothesis test. Therefore, we will need 65 subjects in each group, and the total sample size will be at least 130.In our study, a continuous response variable, pain score by VAS among two groups, TEA and conventional analgesia among OPCAB patients. In a previous study, within each subject group, responses were generally distributed with a 2.3 standard deviation.The above formula used to calculate the sample size is required to compare two groups when the endpoint is quantitative data.2.1Before starting data collection, informed written consent would be taken from all the respondents. The confidentiality of subjects will be protected by offering assurance to respondents that the information they provide will not be revealed and will only be used for study purposes. Patients will have the right to agree, disagree, or leave the study.2.2t‐test will evaluate the continuous data, and categorical data will be evaluated using the χ2 test. A p ≤ 0.05 will be considered statistical significance.Statistical analysis will be conducted using Statistical Package for Social Sciences (SPSS) version 25.0 for windows software. The student's 33.1A VAS is a tool for measuring the pain intensity, which is drawn as 10 cm of a straight horizontal line on a paper, labeled as “no pain at all” and “pain as bad it could be” at the other end3.2The first left internal mammary artery anastomosis to obtuse marginal (OM) was conducted in St Petersburg in 1964 by Kolessov and colleagues without using the heart–lung machine, hence called OPCAB.4Surgical stress, pain, and breathing difficulties contribute to the morbidity and mortality associated with heart surgery. Following cardiac surgery, pain disrupts the humoral and neuroendocrine systems' equilibrium, increasing stress hormone levels and myocardial oxygen demand.Cardiac surgery requires anticoagulants, and it is not widely accepted due to concerns about neuraxial hematoma following anticoagulants.Spinal and epidural instrumentation entails risk, with epidural hematoma formation being the most feared complication.Prospective, randomized research on postoperative pain following coronary artery bypass was conducted in Istanbul to determine the effects of thoracic epidural as an adjunct to general anesthesia. In the early postoperative phase after the CABG, TEA substantially decreased the severity of postoperative pain and the use of pain analgesics.Thoracic epidural anesthesia combined with general anesthesia during OPCAB decreases the need for mechanical ventilation following surgery, decreases the length of stay in the intensive care unit (ICU), and provides better pain control than general anesthesia alone.5Pain after cardiac surgery stimulates a stress response, which has the potential to cause pathophysiological changes in all of the body's major organ systems. Satisfactory postoperative analgesia is associated with a lowered stress response and thus reduces patient discomfort and may reduce morbidity who have undergone OPCAB surgery.In general, single‐modality therapy is the most effective treatment for acute postoperative pain, and it is expected that the TEA (Group A) will be beneficial with significant postoperative pain relief and will show better hemodynamic and respiratory parameters.6Patients' satisfaction with the quality of postoperative analgesia is closely linked to the comparison between predicted and perceived pain. The adequacy of postoperative pain relief remains a concern for patients undergoing OPCAB surgery. As postoperative pain relief is crucial after off‐pump CABG, considering all other modalities, upper TEA may be the choice of postoperative analgesia.Sanjoy Kumar Saha: Conceptualization; data curation; methodology; resources; validation; writing–original draft; writing–review & editing. Redoy Ranjan: Conceptualization; data curation; formal analysis; methodology; resources; writing–original draft; writing–review & editing. Asit Baran Adhikary: Conceptualization; methodology; supervision; validation; writing–review & editing. All authors have read and approved the final version of the manuscript.The authors declare no conflict of interest.Professor Dr. Asit Baran Adhikary affirms that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned  have been explained.