2.1 Animal model
SOD1G93A transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Transgenic mice were crossed with wild type (WT) B6SJL/F1 females to produce hemizygotes and maintain. In accordance with previous reports, PCR was used for genotyping mice. All experiments were performed using mice that were matched on age and sex, and matching WT littermates served as control. The mice were kept in a twelve-hour light and dark cycle at constant room temperature (22 ± 2°C), and they were fed autoclaved food, sterile water, and filtered air. All animal procedures were performed in accordance with the Regulations for the Administration of Laboratory Animals set forth by the Ministry of Science and Technology of the People's Republic of China. All experiments were approved by the Research Ethics Committee of the Second Hospital of Hebei Medical University (Shijiazhuang, Hebei, P.R. China, Approval No. 2022AE269).
2.2 Administration of probiotics
The treatment group received the bifidobacterium triple live bacteria capsule (LBE), which was purchased from Shanghai Xinyi Pharmaceutical Co. Ltd., China. LBE (1 capsule =210mg) were suspended in physiological saline to prepare live bacterial suspensions. Each mouse of the treatment group received daily 300 µl of live bacterial suspensions by oral gavage from postnatal day 60 until the experimental endpoint. SOD1G93A mice was randomly divided into two groups including LBE -and vehicle-treatment groups. Age- and gender-matched WT littermate mice served as controls and were also randomly divided into two groups including LBE -and vehicle-treatment groups. In total 148 animals were used for this research. Fifteen to eighteen female and male mice in each group were arbitrarily set as sample size to evaluate the motor function, disease attack and survival time. Other female mice were killed at 40, 90 and 120 days for histological analysis or Western blotting analysis and detection of intestinal microbiota and metabolites. The experimental time line is described in Figure 1.
2.3 Assessment of behavior in mice
Behavior in SOD1G93A mice was evaluated by weights, step lengths, performance on accelerating rotarod test and neurological scoring [34, 35], Weights assessment were performed twice a week from the 60 days of age, and evaluation of step lengths, performance on accelerating rotarod test and neurological scoring once a week began at 80 days of age and twice a week from the 120 days of age until the endpoint. All behavioral experiments were performed between 7 and 9 pm.
Rotarod locomotor test: The rotarod locomotor test was used to measure motor coordination and balance. The mice were placed on a rotating rod at an accelerating speed (from 2 rpm to 30 rpm over a three-minute period) in a rotating rod apparatus (Ugo Basile, Italy), which automatically captured the latency for each mouse to fall off the rotating rod. Prior to the formal locomotor tests, mice are usually trained to become familiar with rotating rod apparatus. Before any formal test, an adaptation test was conducted to allow mice to adapt to the new situation. Three duplicates of each test were conducted with 30 min of rest between the test, and the longest time was recorded.
Neurological scoring: Based on the ALS Therapy Development Institute's criteria [36], the mice was scored: Score of 0: when the mice was suspended by the tail, the hindlimb presented full extension, and the mice can maintain the posture for at least 2 seconds. Score of 1: when the mice was suspended by the tail, their hindlimbs present abnormal splays, e.g., collapse, partially collapse towards the lateral midline, tremble and retract or clasp of hind legs. Score of 2: toes curled downward at least twice during a 90 cm walk or any part of the foot dragged along the cage floor/table when the mouse was allowed to walk. Score of 3: rigid paralysis or minimal joint movement in its hindlimbs. Score of 4: the mice was incapable of righting themselves within 30 seconds on their side.
The onset of disease and survival: When the mice could not insist on 3 min in three rotarod locomotor tests and/or reached the score of 1 in neurological scoring, the mice were defined as disease onset. From the age of 120 days, mice were monitored daily. The end stage of SOD1G93A mice was defined when they reached a neurological score of 4 and/or when more than 15% of their body weight was lost.
2.4 Immunofluorescence
The mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and euthanized with pentobarbital sodium (150 mg/kg, i.p.), and were transcardially perfused with ice-cold phosphate-buffered saline (PBS, PH7.4). Then the lumbar vertebrae (L4-L5), ileum and colon (1 cm to ileocecal junction) were taken and were fixed in paraformaldehyde for 12h in low-temperature conditions. a solution of 30% sucrose was used for dehydration. After that, these tissues were embedded in O.C.T medium and were cut at 10 μm with a cryostat microtome. Before immunofluorescence staining, the frozen sections were placed at room temperature for 20 minutes, then they were rinsed with 1×PBS 3 times for 5 minutes each time followed by using PBS-0.3% Triton X-100 to break for 15min and wash with 1×PBS 3 times for 5 minutes each time. After this, the sections were blocked in 10% donkey serum in PBS for 1h at room temperature, and then they were incubated with anti-Neun (1: 600, CST, # 24307 s), SOD1 (1:200, Abcam, ab16831), IBA1 (1: 500, Abcam, ab178847), casepase3(1:200, Affinity, AF7022), GFAP (1:200, Cell Signaling, # 3670), Claudin1 (1:200, proteintech, 13050-1-AP), PGP9.5 (1:400, HUABIO, ET1703-22), TNF-α (1: 200, Santa Cruz Biotechnology, sc-52746) overnight with shaking. Following washes with 1×PBS, 3 times for 20 minutes each time. the slices were incubated with goat anti-rabbit second antibody labeled with Alexa Fluor 594 (1:1000, Invitrogen, A11037) and goat anti-mouse second antibody labeled with Alexa Fluor 488 (1:1000, Invitrogen, A11029) at room temperature for 1 hour and then were washed three times with 1× PBS for 20 min each. The nucleus was stained with DAPI Fluoromount-G (Southern Biotech). Finally, the sections were observed by fluorescence confocal microscope (LSM900, ZEISS, Germany).
For whole-mount immunofluorescence staining, ileum and colon (1 cm to ileocecal junction) were taken, washed, flattened and fixed in paraformaldehyde for 12h in low-temperature conditions. then the mucosa layer was gently scraped with a scalpel, and the obtained intestinal tissues were cutted into the square (0.5 cm × 0.5 cm). These tissues were rinsed with 1×PBS 3 times for 10 minutes each time followed by using PBS-0.3% Triton X-100 to break for 60min and washed with 1×PBS 3 times for 10 minutes each time. Afterward, these tissues were blocked in 10% donkey serum in PBS at 4 °C for 48 hours with shaking. and then they were incubated with anti-PGP9.5 (1: 600, CST, # 24307 s), GFAP (1:200, Cell Signaling, # 3670), TNF-α (1: 200, Santa Cruz Biotechnology, sc-52746) at 4 °C for 72 hours with shaking. Following washes with 1×PBS, 12 times for 60 minutes each time. the slices were incubated with goat anti-rabbit second antibody labeled with Alexa Fluor 594 (1:1000, Invitrogen, A11037) and goat anti-mouse second antibody labeled with Alexa Fluor 488 (1:1000, Invitrogen, A11029) at 4 °C for 48 hours with shaking, and then were washed with 1× PBS, 12 times for 60 minutes each time. Finally, the sections were observed by fluorescence confocal microscope (LSM900, ZEISS, Germany).
2.5 Western blot analysis
A section of the spinal cord 1cm in length (at T9 ~ T11) and ileal and colonic tissues 1 cm in length (1 cm to ileocecal junction) were collected. These tissues were ground with a full-automatic grinding instrument (JXFSTPRP-CL, shanghai Jing Xin) in lysis buffer to extract total protein. After centrifugation at 10,000g for 10 min at 4°C, the supernatant was collected. and then the protein content of the supernatant was determined by using a protein assay kit. Equal amounts of total protein (50 μg) were separated by using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat skim milk in TBST (0.05% Tween-20 in TBS) for 1h at room temperature, and then they were incubated with antibodies against TNFα (1: 200, Santa Cruz Biotechnology, sc-52746), GFAP (1:200, Cell Signaling, # 3670), SOD1 (1:200, Abcam, ab16831), Claudin1 (1:500, Invitrogen, 37-4900), Occludin (1:100, Invitrogen, 71-1500) at 4 °C overnight with shaking. After washing 3 times with TBST, appropriate horseradish peroxidase-conjugated secondary antibodies were used as secondary antibodies and incubated for 1 hour at room temperature. GAPDH (1:5000, proteintech, 1049-1-AP) was used as an internal control. The bands were visualized by using enhanced chemiluminescence, and images were acquired with Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA). The relative band intensities were quantified by using ImageJ.
2.6 16sRNA sequencing
Ceca and cecal contents were collected from each mouse and flash frozen in liquid nitrogen for storage at −80 °C. To analyze the composition of cecal microbiota in different groups, 16S rRNA sequencing was performed in Shanghai Zhongke New Life Biotechnology Co. The fecal genomic DNA extraction was performed with the fecal microbiota DNA extraction kit (DP712) using a magnetic bead-based method. PCR was performed with Phusion® High-Fidelity PCR Master Mix with GC Buffer and high-fidelity enzyme (New England Biolabs). The DNA samples were used for library construction using the TruSeq® DNA PCR-Free Sample Preparation Kit. All PCR amplification of microbial samples was analyzed at the Illumina NovaSeq6000 platform. The Clean reads of all samples were Clustered into OTUs using Uparse (http://drive5.com/uparse/) at the 97% identity threshold. Then, species annotation was performed on the OTUs representative sequence using the Silva database (https://www.arb-silva.de/). Community diversity (α-diversity) was calculated based on the chao1 and ace indexes. The variation between the experimental groups (β-diversity) was presented with principal coordinate analysis (PCoA) plots.
2.7 Determination of serum SCFAs concentrations
After animals have fasted for 3 h, blood samples were collected from the eye by coagulation-promoting tubes and were centrifuged (at speed of 3000 rpm for 10 min) to obtain serum. The serum was stored at −80°C. Determination of serum SCFAs concentrations was performed in Shanghai Zhongke New Life Biotechnology Co. ADB-Wax column (30 m×0.25 mm ID×0.25 μm; Agilent Technologies Inc.) was used for sample separation. MS analysis was performed using a gas chromatograph/mass spectrometer detector (Agilent 7890A/5975C, GC/MS, USA). The retention time and peak area of chromatography were extracted using MSD ChemStation software, and the content of short-chain fatty acids in the sample was calculated by drawing a standard curve.
2.8 Statistical analysis
The statistical analysis was performed using SPSS software (SPSS version 21.0). All data are expressed as mean ± SEM. Differences between two groups were assessed using the t-test. Differences among more than two groups were assessed using repeated-measures analysis of variance (ANOVA). Values of p < 0.05 were considered statistically significant. Significance being defined as *P < 0.05, **P < 0.01, ***P <0.001. The probability of survival was calculated using the Kaplan–Meier method, and statistical analysis was performed using a log-rank test.