Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition methods
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Materials and methods
Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; casp1 -/-, n =20) aged 60-90 days were obtained from the Australian Phenomics (Canberra, ACT, Australia) or the Bioresources Facilities (Adelaide, SA, Australia). Genetic caspase-1 deficiency was confirmed by genotyping in experimental mice ( ). Littermates were group housed (Green Line IVC Sealsafe PLUS mouse, Tecniplast, Varese, Italy) in a temperature-specific (22C±1 °C) and light-specific (12 h cycles, lights on at 0700 h) pathogen-free room with water and standard regular chow ad libitum. Animals were assigned and randomize...
Materials and methods
After baseline behavioral testing, animals were submitted to restraint stress for 21 days. Every day, mice were placed in a horizontal resting position inside a well-ventilated (12 holes, 0.5 mm diameter) 50 ml falcon tube at 1000 h and after 4-6 h they were unrestrained.
Pharmacological caspase-1 inhibition with minocycline
The wt animals were treated with either saline (0.2 ml, intraperitoneally, n =27) or minocycline (LKT Laboratories, St Paul, MN, USA; 5 mg kg -1 per day in 10 ml kg -1 saline, intraperitoneally, n =27). Treatment lasted for the same duration of the restraint procedure (21 days).
Chronic restraint stress increases anxiety-like and depressive-like behaviors
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plus maze test, decreasing the time spent in the open arms (F 1, 45 =5.65, P =0.022) and the open to closed arms time ratio (F 1, 45 =4.55, P =0.038, ), as well as in the open field test, increasing the number of defecations (F 1, 45 =12.74, P =0.001, ). Furthermore, restraint decreased body weight gain (F 1, 45 =6.06, P =0.018) and food intake (F 1, 43 =5.75, P =0.021). Nevertheless, restrained mice showed an increase in ratio quotient (F 1, 28 =4.79, P =0.037). Following restraint, no changes were observed in the sucrose preference test (F 1, 33 =0.05, P =0.817, ) or in loco...
Chronic restraint stress affects the gut microbiome
Chronic restraint stress (4-6 h per day for 21 days) affected the gut microbiome compared with nonstressed animals (PERMANOVA P =0.0027, t =2.3492). Although the shallowest level of classification (that is, phylum level) only revealed a nonsignificant trend toward an increased ratio of Firmicutes to Bacteroidetes ( ), the deeper analysis did identify clear differences between the animal groups. In particular, the relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U- test), Bifidobacterium (-4.6%, P =0.0002), Turicibacter (-3.4%, P <0.0007), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-5.8%, P =0.0021) were all reduced in restrained animals, and the relative abundance of the family Lachnospiraceae was increased (+0.3%, P =0.0244). Variance in the relative abundance...
Minocycline affects the gut microbiome
Minocycline treatment (5 mg kg -1 per day for 21 days) also affected microbiota composition compared with saline-treated controls (PERMANOVA P =0.0001, t =3.0947, ) and, interestingly, in a manner very similar to that observed for restrained animals. In particular, minocycline-treated animals were also found to possess lower relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U -test), Bifidobacterium (-5.8%, P <0.001), Turicibacter (-4.2%, P <0.0001), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-7.4%, P =0.003), and significantly high relative abundances of the family Lachnospiraceae (+25.3%, P =0.005) and Ruminococcaceae incertae sedis (+2.4%, P =0.024). Variance in the relative abundance of these taxa accounted for >67% of intergroup variance.
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Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; casp1 -/-, n =20) aged 60-90 days were obtained from the Australian Phenomics (Canberra, ACT, Australia) or the Bioresources Facilities (Adelaide, SA, Australia). Genetic caspase-1 deficiency was confirmed by genotyping in experimental mice ( ). Littermates were group housed (Green Line IVC Sealsafe PLUS mouse, Tecniplast, Varese, Italy) in a temperature-specific (22C±1 °C) and light-specific (12 h cycles, lights on at 0700 h) pathogen-free room with water and standard regular chow ad libitum. Animals were assigned and randomized as described in the. The investigators were not blinded to group assignment. Behavioral phenotyping was performed between 0900 h and 1600 h. Animals were given 30 min of habituation to the behavioral testing room. Tests were performed from the least to the most invasive to minim...
After baseline behavioral testing, animals were submitted to restraint stress for 21 days. Every day, mice were placed in a horizontal resting position inside a well-ventilated (12 holes, 0.5 mm diameter) 50 ml falcon tube at 1000 h and after 4-6 h they were unrestrained.
The wt animals were treated with either saline (0.2 ml, intraperitoneally, n =27) or minocycline (LKT Laboratories, St Paul, MN, USA; 5 mg kg -1 per day in 10 ml kg -1 saline, intraperitoneally, n =27). Treatment lasted for the same duration of the restraint procedure (21 days).
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plus maze test, decreasing the time spent in the open arms (F 1, 45 =5.65, P =0.022) and the open to closed arms time ratio (F 1, 45 =4.55, P =0.038, ), as well as in the open field test, increasing the number of defecations (F 1, 45 =12.74, P =0.001, ). Furthermore, restraint decreased body weight gain (F 1, 45 =6.06, P =0.018) and food intake (F 1, 43 =5.75, P =0.021). Nevertheless, restrained mice showed an increase in ratio quotient (F 1, 28 =4.79, P =0.037). Following restraint, no changes were observed in the sucrose preference test (F 1, 33 =0.05, P =0.817, ) or in locomotor activity in the open field test (F 1, 45 =3.64, P =0.063, ).
Chronic restraint stress (4-6 h per day for 21 days) affected the gut microbiome compared with nonstressed animals (PERMANOVA P =0.0027, t =2.3492). Although the shallowest level of classification (that is, phylum level) only revealed a nonsignificant trend toward an increased ratio of Firmicutes to Bacteroidetes ( ), the deeper analysis did identify clear differences between the animal groups. In particular, the relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U- test), Bifidobacterium (-4.6%, P =0.0002), Turicibacter (-3.4%, P <0.0007), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-5.8%, P =0.0021) were all reduced in restrained animals, and the relative abundance of the family Lachnospiraceae was increased (+0.3%, P =0.0244). Variance in the relative abundance of these taxa accounted for >40% of intergroup variance.
Minocycline treatment (5 mg kg -1 per day for 21 days) also affected microbiota composition compared with saline-treated controls (PERMANOVA P =0.0001, t =3.0947, ) and, interestingly, in a manner very similar to that observed for restrained animals. In particular, minocycline-treated animals were also found to possess lower relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U -test), Bifidobacterium (-5.8%, P <0.001), Turicibacter (-4.2%, P <0.0001), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-7.4%, P =0.003), and significantly high relative abundances of the family Lachnospiraceae (+25.3%, P =0.005) and Ruminococcaceae incertae sedis (+2.4%, P =0.024). Variance in the relative abundance of these taxa accounted for >67% of intergroup variance.
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