Anesthesia and surgery induce age-dependent changes in behaviors and microbiota

Anesthesia/surgery induced age-dependent changes in behavior in mice

We set out to determine the potential age-dependent changes in behaviors, gut microbiota, brain IL-6 levels, levels of brain synaptic marker, and mitochondrial function in 9 and 18 months old mice. The experimental design was demonstrated in Figure 1. There were no significant differences in blood pressure and heart rate (Supplementary Table 1), or the values of blood gas and electrolyte (Supplementary Table 2) between the 9 or 18 months old mice in the control condition and the 9 or 18 months old mice that received the anesthesia/surgery. These data showed that the anesthesia/surgery in the current studies did not significantly disturb physiology status of the mice.

Figure 1. Experimental design. At the 24 hours before the control condition or anesthesia/surgery, the mice had the behavioral tests. At the 9 hours after the control condition or the anesthesia/surgery, the mice repeated the same behavioral test. The same mice then had Barnes maze training from day 7 to day 10, and Barnes maze testing on day 11 after the control condition or the anesthesia/surgery. The feces of the mice were collected at 24 hours before, 9 hours after and 11 days after the control condition or anesthesia/surgery before the behavioral tests. A different group of mice was used for the collection of the hippocampus at the 9 hours or 11 days after the control condition or the anesthesia/surgery.

The anesthesia/surgery differently induced behavioral changes in buried food test, Y maze test and open field test as compared to the control condition (Figure 2A–2L) between the 9 and 18 months old mice, the composite Z scores of which, obtained from the 9 and 18 months old mice, were summarized and compared (Figure 2M, 2N). Specifically, the anesthesia/surgery caused greater behavioral changes in the 18 months old mice than those in the 9 months old mice (Figure 2O), and the Student’s t-test further demonstrated that the composite Z scores in the 18 months old mice were higher than those in the 9 months old mice (Figure 2P, P = 0.019, n = 10). Note that the mice in the control group did better in some of the behavioral tests at the 9 hours after the control condition than those at the baseline, potentially due to the learning effects of these tests.

Figure 2. Anesthesia/surgery induces age-dependent postoperative delirium-like behaviors in mice. Buried food test in the 9 (A) or 18 (B) months old mice. Y maze test (Entries in novel arm) in the 9 (C) or 18 (D) months old mice. Y maze test (Duration in novel arm) in the 9 (E) or 18 (F) months old mice. Open field test (Freezing time) in the 9 (G) or 18 (H) months old mice. (I) Open field test (Time spent in the center) in the 9 (I) or 18 (J) months old mice. Open field test (Latency to center) in the 9 (K) or 18 (L) months old mice. The composite Z scores quantitatively demonstrate the postoperative delirium-like behavior between the control condition and anesthesia/surgery in 9 (M) or 18 (N) months old mice. The comparison of the qualitative (O) or quantitative (P) changes of the postoperative delirium-like behaviors between the 9 and 18 months old mice. The composite Z scores in the 18 months old mice were higher than those in the 9 months old mice, which demonstrates that the anesthesia/surgery may cause a greater postoperative delirium-like behaviors in the 18 months old mice than those in the 9 months old mice (P). N = 10 in each group. The Student’s t-test was used to analyze the data presented in (A–N). The Mann–Whitney U test was used to analyze the data presented in (P). * = P < 0.05.

Next, we found that the anesthesia/surgery caused significant changes in the latency to identify and enter the escape box, but not speed, as compared to the control condition in the Barnes Maze training in 18, but not 9, months old mice (Figure 3A to 3D). In the Barnes Maze testing, the anesthesia/surgery caused significant changes in the number of wrong holes searched before identifying the escaped box in 18, but not 9 month old mice. The anesthesia/surgery caused significant changes in the latency to identify and enter the escaped box, distance, and the time in target zone in both 9 and 18 months old mice (Figure 3E to 3L). However, the anesthesia/surgery caused greater behavioral changes in the Barnes maze training and testing in18 months old mice than in 9 months old mice (Figure 3M). Specifically, the anesthesia/surgery caused higher composite Z scores, representing the behavioral changes in the Barnes Maze test, in the 18 months old mice than those in the 9 months old mice (Figure 3N, P < 0.0001, n = 10, Student’s t-test). These data demonstrated that the anesthesia/surgery was able to induce age-dependent behavioral changes, e.g., postoperative delirium-like behavior and cognitive impairment, in the mice, with greater changes in the older mice.

Figure 3. Anesthesia/surgery induces age-dependent cognitive impairment in mice. Barnes maze training (latency to identify and enter the escape box) in the 9 (A) or 18 (B) months old mice. Barnes maze training (speed) in the 9 (C) or 18 (D) months old mice. Barnes maze testing (latency to identify and enter the escape box) in the 9 (E) or 18 (F) months old mice. Barnes maze testing (distance) in the 9 (G) or 18 (H) months old mice. Barnes maze testing (wrong holes) in the 9 (I) or 18 (J) months old mice. Barnes maze testing (time in target zone) in the 9 (K) or 18 (L) months old mice. The comparison of the qualitative (M) or quantitative (N) changes of the postoperative delirium-like behaviors between the 9 and 18 months old mice. The anesthesia/surgery causes a greater Barnes maze composite Z scores in the 18 months old mice than those in the 9 months old mice (N). N = 10 in each group. Two-way ANOVA with repeated measurement and post-hoc Bonferroni comparison was used to analyze the data in the (A–D). The Mann–Whitney U test was used to analyze the data presented in (E–L and N). * = P < 0.05.

Anesthesia/surgery induced age-dependent changes in the gut microbiota in mice

Given the findings that the anesthesia/surgery induced age-dependent changes in the behaviors in mice, next, we asked whether the anesthesia/surgery could cause age-dependent changes in gut microbiota in the mice. We found that the anesthesia/surgery induced significant alterations in the component of the gut microbiota in 18, but not 9, months old mice (Figure 4A), and different microbiota diversity between the 9 and 18 months old mice (Figure 4B). The heatmap and taxonomic composition distribution demonstrated that the anesthesia/surgery induced different change rate of reduction in the relative abundance of Lactobacillus (genus level, Figure 4C) and Lactobacillus (species level, Figure 4E). Finally, the quantification showed that the anesthesia/surgery induced a significant change rate of reduction in the relative abundance of Lactobacillus as compared to the control condition in the 18 (F = 29.08, P < 0.001 one- way ANOVA, Figure 4D, genus level; F = 10.89, P = 0.014, one-way ANOVA, Figure 4F, species levels), but not 9, months old mice at 9 hours and 11 days post-anesthesia/surgery. Specifically, the anesthesia/surgery induced greater change rate of reduction in relative abundance of Lactobacillus in the 18 months old mice than that in the 9 months old mice. These data demonstrated that the anesthesia/surgery was able to cause age-dependent changes in the gut microbiota.

Figure 4. Anesthesia/surgery induces age-dependent changes in the gut microbiota. (A) OUT-based PLS-DA analysis showed that the anesthesia/surgery induced significant alterations in the component of the gut microbiota in 18, but not 9, months old mice. Different colors and shapes represented the different groups. (B) The number of community species between the different groups demonstrated that the anesthesia/surgery caused different microbiota diversity between the 9 and 18 months old mice, on the Observed species (top left), chao (top middle), ace (top right), Shannon’s diversity (bottom left), Simpson diversity (bottom middle), and Good’s coverage (bottom right). (C) Heatmap (left panel) indicated the anesthesia/surgery induced different changes in bacterial community structure represented as relative abundance shown in genus level analysis between the 9 and 18 months old mice as compared to the control condition. Anesthesia/surgery caused different profiles in the bacterial community taxonomic composition distribution (right panel) represented as relative abundance shown in genus level between the 9 and 18 months old mice as compared to the control condition. (D) Quantification of the change rate in the relative abundance of gut microbiota at the genus level showed the anesthesia/surgery induced significant change rate of reduction in the relative abundance of Lactobacillus as compared to the control condition at 9 hours and 11 days post-anesthesia/surgery in the 18, but not 9, months old mice (F = 2.01, P = 0.169, one-way ANOVA for the 9 months old mice, F = 29.08, P < 0.001, one-way ANOVA for the 18 months old mice). (E) Heatmap (left panel) indicated the anesthesia/surgery induced different changes in bacterial community structure represented as relative abundance shown in species level analysis between the 9 and 18 months old mice as compared to the control condition. Anesthesia/surgery caused different profiles in the bacterial community taxonomic composition distribution (right panel) represented as relative abundance shown in species level between the 9 and 18 months old mice as compared to the control condition. (F) Quantification of the change rate in the relative abundance of gut microbiota at the species level showed that the anesthesia/surgery induced significant change rate of reduction in the relative abundance of Lactobacillus salivarius as compared to the control condition at 9 hours and 11 days post-anesthesia/surgery in the 18, but not 9, months old mice (F = 3.081, P = 0.122, one-way ANOVA for the 9 months old mice, F = 10.89, P = 0.014, one-way ANOVA for the 18 months old mice). Partial least-squares discriminant analysis = PLS-DA, OUT = operational taxonomic unit. One-way ANOVA and post-hoc Bonferroni comparison was used to analyze the data in the (D–F). * = P < 0.05; ** = P < 0.01.

Anesthesia/surgery induced age-dependent changes in brain IL-6 levels, levels of synaptic marker and mitochondrial function in mice

Consistently, we found that the anesthesia/surgery induced age-dependent increases in brain IL-6 levels in the 9 and 18 months old mice (Figure 5A, F = 8.715, P = 0.008, two-way ANOVA, n = 6). The anesthesia/surgery decreased the brain mitochondrial function as compared to the control condition in either 9 (Figure 5B) or 18 (Figure 5C) months old mice. Moreover, the 18 months old mice had a lower baseline mitochondrial function (Figure 5D) and greater anesthesia/surgery-induced mitochondrial dysfunction (Figure 5E) than the 9 months old mice did, including base respiration rate, ADP respiration rate and maximal respiration rate (data not shown) at 9 hours after the anesthesia/surgery. In addition, the anesthesia/surgery reduced the levels of PSD-95 (Figure 5F and 5G, F = 6.178, P = 0.049, two-way ANOVA, n = 6) and synaptophysin (Figure 5H and 5I, F = 8.061, P = 0.037, two-way ANOVA, n = 6) in the hippocampus of 18 months old mice at 11 days after the anesthesia/surgery. These data demonstrated that the anesthesia/surgery caused an age-dependent increase in pro-inflammatory cytokine IL-6, mitochondrial dysfunction, and synaptic loss in the brain tissues of the mice

Figure 5. Age-dependent postoperative changes in brain levels of IL-6, mitochondria function, and synapse levels. (A). Effects of the anesthesia/surgery on the brain IL-6 levels between the 9 and 18 months old mice. The anesthesia/surgery reduces the mitochondrial function in the brain tissues of the 9 (B) or 18 (C) month old mice as compared to the control condition. The 18 months old mice have lower baseline (D) or greater reduction (E) in mitochondrial function than 9 months old mice have. (F) The effects of the anesthesia/surgery on PSD-95 levels between the brain tissues of the 9 months old mice and the brain tissues of the 18 months old mice. (G) The quantification of the Western blot showing that the anesthesia/surgery decreases hippocampus PSD-95 levels as compared to the control condition in the 18 months old mice but not in the 9 months old mice. (H) The effects of the anesthesia/surgery on synaptophysin levels between the brain tissues of the 9 months old mice and the brain tissues of the 18 months old mice. (I). The quantification of the Western blot showing that the anesthesia/surgery decreases the synaptophysin levels in the hippocampus of the 18 months old mice but not the 9 months old mice. PSD: postsynaptic density; IL-6: interleukin-6. N = 6 in each group. Two-way ANOVA and post-hoc Bonferroni comparison was used to analyze the data in the (A, G and I). * = P < 0.05.

Treatment with Lactobacillus rescued the anesthesia/surgery-induced behavioral and cellular changes in mice

To mechanistically determine the association between the anesthesia/surgery-induced changes in behaviors and gut microbiota, we assessed the effects of treatment with Lactobacillus on the anesthesia/surgery-induced changes. We found that the intragastric administration of Lactobacillus attenuated the anesthesia/surgery-induced increase in latency to eat food in the buried food test (Figure 6A, P = 0.001, Friedman’s test, n = 10 - 12), the freezing time (Figure 6F, P = 0.033, Friedman’s test, n = 10 - 12), and time spent in the center of the open field test (Figure 6H, P = 0.004, Friedman’s test, n = 10 - 12) in the 18 months old mice. Specifically, Figure 6I qualitatively demonstrated that intragastric administration of Lactobacillus attenuated the anesthesia/surgery-induced behavioral changes in the 18 months old mice. Finally, two-way ANOVA showed that there was a significant interaction of group (the control condition versus the anesthesia/surgery) and treatment (saline versus lactobacillus) on the composite Z scores (Figure 6J, F = 4.138, P = 0.049, n = 10 - 12), and the treatment with Lactobacillus attenuated the anesthesia/surgery-induced increase in the composite Z scores (Figure 6J). In addition, the treatment with Lactobacillus attenuated the anesthesia/surgery-induced mitochondrial dysfunction in the hippocampus of the 18 months old mice (Figure 6K). These data further indicated that gut microbiota played a role in the anesthesia/surgery-induced behavioral and cellular changes.

Figure 6. Lactobacillus attenuate the anesthesia/surgery-induced changes in the 18 months old mice. Interaction of anesthesia/surgery and the treatment of Lactobacillus on the buried food test (A), Y maze test (duration in novel arm, B), Y maze test (entries in novel arm, C), Y maze test (number of arm visit,D), open field test (total distance, E), open field test (freezing time, F), open field test (latency to center, G), open field test (Time spent in the center, H) in the 18 months old mice. The comparison of the qualitative (I) or quantitative (J) changes of the postoperative delirium-like behavior between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus lactobacillus. The treatment of Lactobacillus attenuated the anesthesia/surgery-induced increase in the composite Z score. (K) The comparison of the brain mitochondrial function between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus lactobacillus. The treatment of Lactobacillus attenuated the anesthesia/surgery-induced mitochondrial dysfunction. n = 10 - 12 in each group of the behavioral studies, and n = 6 in each group of the mitochondrial function studies. Friedman test was used to analyze the data in the (A–C, F and H). Two-way ANOVA and post-hoc Bonferroni comparison was used to analyze the data in the (D, E, G and J). * = P < 0.05.

Probiotic mitigated the anesthesia/surgery-induced behavioral and cellular changes

Probiotic has been shown to improve cognitive function in diabetic rats [38]. We found that intragastric administration of probiotic mitigated the anesthesia/surgery-induced behavioral changes in the 18 months old mice (Figure 7A, P = 0.016, Student’s t-test, Figure 7H, P = 0.0005, Mann-Whitney test, n = 12). Specifically, Student’s t-test demonstrated that the intragastric administration of probiotic decreased the anesthesia/surgery-induced increase in the composite Z scores (Figure 7I, P = 0.022, n = 12, Student’s t-test). The treatment with intragastric administration of probiotic also reduced the anesthesia/surgery-induced increase in IL-6 levels (Figure 7J, Student’s t-test, P = 0.001, n = 4) and mitochondrial dysfunction (Figure 7K) in the hippocampus of the 18 months old mice. These data indicated that probiotic could prevent or treat the anesthesia/surgery-induced behavioral changes and the associated alterations in brain IL-6 levels and mitochondrial function.

Figure 7. Treatment of the postoperative delirium-like behaviors by using probiotic.The comparison between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus probiotic on buried food test (latency to eat food, A), Y maze test (number of arm visit, B), Y maze test (duration in novel arm, C), Y maze test (entries in novel arm, D), open field test (total distance, E), open field test (freezing time, F), open field test (time spent in center, G), and open field test (latency to center, H). (I). The comparison of the Z scores between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus probiotic. There was lower composite Z score in the 18 months old mice received the anesthesia/surgery plus probiotic than that in the 18 months old mice received the anesthesia/surgery plus saline, which suggest that the treatment of probiotic mitigated the anesthesia/surgery-induced postoperative delirium-like behavior in the 18 months old mice. (J) The comparison of the brain IL-6 levels between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus probiotic. (K) The comparison of the brain mitochondrial function between the 18 months old mice received the anesthesia/surgery plus saline and the 18 months old mice received the anesthesia/surgery plus probiotic. IL-6: interleukin-6. n = 12 in each group of the behavioral studies, and n = 6 in each group of the mitochondrial function studies. The Student’s t-test was used to analyze the data presented in (A, B, F and J). The Mann–Whitney U test was used to analyze the data presented in (C–D and G–H). * = P < 0.05.

Mice anesthesia and surgery

The animal protocol was approved by the Massachusetts General Hospital (Boston, Massachusetts) Standing Committee on the Use of Animals in Research and Teaching (Protocol number: 2006N000219). All experiments were performed in accordance with the National Institutes of Health guidelines and regulations. Efforts were made to minimize the number of animals used. We only used female mice in the current studies because our previous studies showed that anesthesia/surgery was able to induce the postoperative delirium-like behavior [37] and postoperative cognitive impairment [57] in the female mice. Mice had 12:12 hours light:dark cycle (lights on 7:00 am) with food and water available ad libitum. The mice were randomly assigned to the anesthesia/surgery group or the control group by weight. Mice in the anesthesia/surgery group had a simple laparotomy under isoflurane anesthesia using the methods described in our previous studies [37, 50, 58]. Specifically, we anesthetized each of the mice using 1.4% isoflurane in 100% oxygen in a transparent acrylic chamber. Fifteen minutes after the induction, we moved the mouse out of the chamber. Isoflurane anesthesia was maintained via a cone device, and one 16-gauge needle was inserted into the cone near the nose of the mouse to monitor the concentration of isoflurane. We made a longitudinal midline incision from the xiphoid to the 0.5-centimeter proximal pubic symphysis on the skin, abdominal muscles, and peritoneum. We then sutured the incision layer by layer with 5-0 Vicryl thread. Some of the mice (e.g., the mice in the studies presented in Figure 6) received the non-absorbable suture, which could cause more inflammation; and some of the mice (e.g., the mice in the studies presented in Figure 7) received the absorbable suture, which could cause lesser inflammation. We applied EMLA cream (2.5% lidocaine and 2.5% prilocaine) to the incision site at the end of the procedure, and then every eight hours for five days or until the euthanasia of the mice, to treat the pain associated with the incision as we did in our previous studies [37, 50, 58–60]. The procedure for each mouse usually lasted about 10 minutes, and we put the mouse back into the anesthesia chamber to continue receiving the rest of the anesthesia consisting of 1.4% isoflurane in 100% oxygen for up to two hours. We used this method because surgery could potentiate the anesthesia neurotoxicity, and such a combination of anesthesia and surgery had been shown to induce postoperative cognitive impairment and postoperative delirium-like behavior [37, 50, 58, 61]. We maintained the rectal temperature of the mice at 37 ± 0.5 °C during the anesthesia/surgery by using DC Temperature Control System (FHC, Bowdoinham, Maine). We returned the mice to their home cage with food and water available ad libitum after recovering from the anesthesia. The mice in the control group also received 100% oxygen for two hours. The blind procedure was not possible in the studies because of the appearance of the abdominal wound in the mice.

Treatment with Lactobacillus in mice

Lactobacillus salivarius (ATCC, Manassas, VA) were cultured in ATCC Medium 78 (ATCC), which consists of 20 g/L Tryptone, 5 g/L trypose, 5 g/L Yeast extract, 200 ml/L Tomato juice, 1g/L liver extract concentrate, 0.05g/L tween 80, 3g/L glucose, and 2 g/L lactose (PH 6.5, 37°C, aerobic environment). Each of the mice received 10⁹ CFU Lactobacillus salivarius (10⁹ CFU in 200 ul of saline) or the same volume of saline once per day for ten days by intragastric administration. Then, each of the mice waited for another ten days for the colonization of the Lactobacillus before receiving the anesthesia/surgery or the control condition.

Treatment with probiotic in mice

The 18 months old mice were randomly divided into two groups: anesthesia/surgery plus saline/corn oil and anesthesia/surgery plus probiotic. The probiotic (Mommy’s Bliss Pharmaceuticals and Pharmapacks) contained Lactobacillus rhamnosus GG. The mice received 10 x 10⁸ CFU of probiotic in 100 ul or the same volume of saline/corn oil once per day by intragastric administration for 20 days before the start of the anesthesia/surgery in the mice.

Harvest of mice brain tissues and collection of mice feces

We harvested mice hippocampus at 9 hours after the anesthesia/surgery for the ELISA studies and the measurement of mitochondrial function by using a Seahorse XFp Extracellular Flux Analyzer, and at 11 days after the anesthesia/surgery for the Western blot analysis. The mice feces were collected at one day before the anesthesia/surgery or control condition, then 9 hours and 11 days after the anesthesia/surgery or the control condition.

DNA extraction and quantification of fecal bacteria

The relative abundance and diversity of the gut bacteria were determined by using the methods described in previous studies [62]. The 16S rRNA gene sequencing and data analysis were performed by BGI America (Cambridge, MA) using the manufacturer’s protocol. Briefly, the fecal samples were collected at the times described in Figure 1. Samples were placed in 1.5 ml tubes, snap-frozen on dry ice, and stored at −80°C until the delivery in dry ice. The 16S rRNA analysis of fecal samples was performed by amplicon-based sequencing, operational taxonomic unit (OTUs) were clustered using USEARCH (7.0.1090) with a 97% threshold. Greengene V201305 was used as a reference database for taxonomic analysis. The MixOmics package for R was used for partial least-squares discriminant analysis (PLS-DA) analysis. OTU rank curse was generated in R (V3.1.1). The tag number of each taxonomic rank (phylum, class, order, family, genus, species) or OTU in different samples were summarized in histograms generated in R (V3.1.1). In order to compare α-diversity, the OTU table was rarified, and four metrics were calculated: Chao1, Observed species, Shannon and Simpson index using MOTHUR ver.1.31.2. The β-diversity analysis was conducted as a heat map of Bray–Curtis diversity (calculated by QIIME ver.1.80) distance by using the heatmap function in the NMF package of R ver. 3.1.1. Partial least squares discrimination analysis (PLS-DA) analysis of the two principal components with the highest contribution was conducted using the mixOmics package of R ver. 3.1.1. Heatmap analysis clustering of the relative abundance of six phylogenetic levels in all samples was conducted using Euclidean and complete functions in the gplots package of R ver. 3.1.1. The change rate of relative abundance of Lactobacillus was calculated by using the raw score of the mouse received anesthesia/surgery to minus the mean of the raw scores of the mice in the control group.

Enzyme-Linked Immunosorbent Assay (ELISA) determination of IL-6

The mouse IL-6 Immunoassay kit (Catalog number: M6000B, R&D Systems, Minneapolis, MN) was used to determine the levels of IL-6 in mouse brain tissues as described by the protocol associated with the immunoassay kit. Briefly, a monoclonal antibody specific for mouse IL-6 was coated onto the microplates. Wells were incubated for two hours at room temperature with test samples (serum) and washed for five times. Then, 100 μL of mouse IL-6 conjugate was added to each well and incubated for another two hours, and the washing was repeated twice. Finally, wells were incubated in 100 μL of substrate solution for 30 minutes and stopped with stop solution (100 μL). Determination of the optical density of each well was set at 450 nm and corrected at 570 nm as performed in our previous studies [50].

Western blot analysis

Western blot analysis was performed using the methods described in our previous studies [63]. Hippocampus tissues were harvested from the mice at 11 days after the anesthesia/surgery or control condition. We used postsynaptic density protein (PSD)-95 antibody (1:1,000, molecular weight of 95 kDa, Cell Signaling, Danvers, MA) to detect PSD-95 level. We used anti-synaptophysin antibody (1:1,000, molecular weight of 37, Abcam, Cambridge, MA) to detect the synaptophysin levels. An β-Actin antibody (1:5,000, molecular weight of 42 kDa, Sigma, St. Louis, MO) was used to detect non-targeted protein β-Actin. β-Actin amount was used to normalize (e.g., determining the ratio of PSD-95 to β-Actin amount) protein levels and control for loading differences in the total protein amount. The quantification of the Western blot was performed as described in previous study [63]. We presented changes in protein levels in the hippocampus of the mice received the anesthesia/surgery as a percentage of those in the mice in the control group. 100% of protein level changes referred to control levels for comparison to experimental conditions. Signal intensity was analyzed using a Bio-Rad (Hercules, CA) image program.

Measurement of mitochondrial function by using a Seahorse XFp Extracellular Flux Analyzer

Mitochondria were isolated from the hippocampus of the mice before the studies of the Seahorse XFp Extracellular Flux Analyzer. Specifically, the harvested hippocampus tissues were homogenized 20 times with a plastic tissue grinder (Thermo Fisher Scientific, Waltham, MA) in an Eppendorf tube with 600 μl of mitochondrial isolation buffer, which consisted of 70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid-free bovine serum albumin (BSA) (pH 7.2 at 37°C). The homogenate was centrifuged at 800 x g at 4 °C for 10 minutes. Then, the supernatant was centrifuged at 8,000 x g at 4 °C for 10 minutes, and the pellets were dissolved in the media without BSA, and protein concentrations were measured by bicinchoninic acid assay. The media was composed of 10 mM sodium succinate, 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.2% (w/v) fatty acid-free BSA (pH 7.2 at 37°C). The remaining supernatant was centrifuged at 8,000 x g at 4 °C for 10 minutes, and the precipitates were dissolved in the media plus BSA. After appropriate dilution with the media plus BSA, the mitochondria were seeded into an XFp cell culture miniplate (Seahorse Bioscience, Billerica, MA) at 30 μg/well. The plate was centrifuged at 2,000 x g at 4 °C for 20 minutes and incubated in the air at 37 °C for 10 minutes. A seahorse cartridge with detection probes (Seahorse Bioscience) was loaded with adenosine 5’-diphosphate (ADP, 2.5 mM), oligomycin (2 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 4 μM), and antimycin A (3 μM) into the injection port A, B, C, and D, respectively. We then transferred the mitochondria and these reagents into the seahorse XFp extracellular flux analyzer. The real-time readings were taken for five stages: stage I, basal level; stage II, addition of ADP to measure ATP production; stage III, addition of oligomycin, a mitochondrial oxidative phosphorylation (OXPHOS) complex 5 inhibitor, to measure protein leakage; stage IV, addition of the mitochondrial OXPHOS complex 4 inhibitor carbonyl cyanide m-chlorophenyl hydrazine to measure maximal respiration; and stage V, addition of the mitochondrial OXPHOS complex 3 inhibitor antimycin A to measure nonmitochondrial respiration. Mitochondrial function was determined by measuring the oxygen consumption rate (OCR) calculated and recorded by the Seahorse XFp software.

Behavior tests

There is no ideal animal model to study delirium. However, as we have shown previously [37], it is possible to identify a delirium-like phenotype in mice by using a battery of tests of natural and learned behaviors. We utilized that battery of tests here in the present studies. As demonstrated in the diagram (Figure 1), all of the mice had multiple behavioral tests in the order of buried food test, then open field test and finally Y maze test at 24 hours before (baseline), and then at 9 hours after the anesthesia/surgery or control condition. We performed the behavioral tests in groups of 3 mice and finished them within 50 minutes, which mimicked the specific features of clinical evaluation of delirium in patients.

Buried food test

The buried food test was performed as described in previous studies [37, 64, 65] with modifications. Specifically, one day before the test, we gave each mouse two pieces of the sweetened cereal. On the test day, we habituated the mice for one hour before the test by placing the home cage with mice in the testing room. Then, the test cage was prepared with clean bedding (3 centimeters high). We buried one sweetened cereal pellet 0.5 centimeter below the surface of bedding so that it was not visible. The location of the food pellet was changed every time in a random fashion. We placed the mouse in the center of the test cage and measured the latency of the mouse to find and eat the food. Latency was defined as the time from when the mouse was placed in the test cage until when the mouse uncovered the food pellet and grasped it in its forepaws and/or teeth. Mice were allowed to consume the pellet they found and were then returned to their home cage. The total observation time was 5 minutes. If the mouse could not find the pellet within 5 minutes, the testing session ended, and the latency was defined as 300 seconds (5 minutes) for that mouse. We emptied the bedding from the test cage and cleaned the cage with a 70% ethanol solution after each test to prevent the transmission of olfactory cues. We also changed gloves after each test.

Open field test

The open field test was performed as described in previous studies with modifications [37, 66, 67]. Specifically, the mouse was gently placed in the center of an open field chamber (40 × 40 × 40 centimeters) under dim light and was allowed to move freely for 5 minutes. The movement parameters of the mouse were monitored and analyzed through a video camera connected to the Any-Maze animal tracking system software (Stoelting Co., Wood Dale, IL). The total distance moved (meters), the time (seconds) spent in the center of the open field, the freezing time (seconds) and the latency to the center (the time in seconds for the mice to reach to the center at the first attempt) of the open field was recorded and analyzed. The floor of the open field was cleaned with 70% ethanol solution between each test.

Y maze test

The Y maze test was performed as described in the previous studies with modifications [37, 68, 69]. Specifically, the Y maze, made of gray polyvinylene, was placed in a quiet and illuminated room. Each maze consisted of three arms (8 × 30 × 15 centimeters, width × length × height), with an angle of 120 degrees between each arm. The three arms included the start arm, in which the mouse starts to explore (always open); the novel arm, which is blocked at the first trial but opened at the second trial; and the other arm (always open). In the experiment, the start arm and other arm were designed randomly to avoid spatial memory error. The Y maze test consisted of 2 trials separated by a two hour inter-trial interval. The first trial (training) occurred at 7 hours after the anesthesia/surgery and was 10 minutes in duration, which allowed the mouse to explore two arms (the start arm and other arm) of the maze with the novel arm being blocked. At approximately 2 hours after the first trial, the second trial (retention) was conducted. For the second trial, the mouse was placed back in the maze in the same start arm with free access to all three arms for 5 minutes. A video camera, which was linked to the Any-Maze animal tracking system software, was installed 60 centimeters above the chamber to monitor and analyze the number of entries and the time spent in each arm. The time spent in and entries into the novel arms indicated the spatial recognition memory (learned behavior). Each of the arms of the Y maze was cleaned with 70% ethanol solution between trials.

Calculation of composite Z-scores

The composite Z-scores to present the postoperative delirium-like behavior of each of the mice were calculated using the methods described in previous studies [37, 70] with modifications. Specifically, the Z score was calculated using the formula described by Moller et al.: Z = [ΔX Anesthesia/surgery − MEAN(ΔX) control]/SD(ΔX) control [70]. In the formula, ΔX control was the change score of mice in the control group at 9 hours after the control condition minus the score at the baseline; ΔX Anesthesia/surgery was the change score of mice in anesthesia/surgery group at 9 hours after the anesthesia/surgery minus the score at baseline; MEAN(ΔX) control was the mean of ΔX control; and SD(ΔX) control was the standard deviation of ΔX control. We also used the method for calculating a composite Z score in patients [71, 72] to determine a composite Z score for each of the mice. Specifically, the composite Z score for the mouse was calculated as the sum of the values of 6 Z scores (latency to eat food, time spent in the center, latency to the center, freezing time, entries in novel arm and duration in novel arm) normalized with the SD for that sum in the controls. Given that the reduction (rather than increase) in time spent in the center, the freezing time (open field test), the reduction in duration and entries in the novel arm (Y maze test) indicate impairment of the behavior, we multiplied the Z score values representing these behaviors by −1 prior to calculating the composite Z score using these values in the mice in either the anesthesia/surgery group or the control group.

Barnes maze

Barnes maze test was performed by using the methods described in other studies [50, 73–76] with modifications. Barnes maze with a circular open platform (about 90 center meter diameter) was located in a quiet area. It had 20 equally spaced holes (one of these holes connects with a small dark recessed chamber called escape box) and was surrounded by a dark curtain with four simple colored-paper shapes (square, circle, triangle, and star) as markers (Stoelting, Wood Dale, IL). A video camera which could capture the entire platform was right above the platform and connected to the Any-Maze animal tracking system software (Stoelting Co., Wood Dale, IL) as described in previous studies [74, 77, 78]. The Barnes maze test in the current studies included Barnes maze training (day 7 to 10 after the anesthesia/surgery) and Barnes maze testing (11 days after the anesthesia/surgery) (Figure 1). The Barnes maze training (day 7 to day 10 after the anesthesia/surgery) consisted of 2 trials (3 minutes each trial and 15 minutes between the trials) for four days. In each trial, the mouse was placed under a bucket in the center of the circular platform for 10 seconds and was allowed to escape under the same stimulation of light and aversive noise. Once reaching the escape box, the mouse was allowed to remain in the escape box for one minute. The mouse was then removed and placed back to the home cage for 15 minutes of rest period before returning for another trial. Between each test, the Barnes maze was cleaned with 70% alcohol solution to avoid olfactory cues. After the training period, the mice had the Barnes maze testing on day 11 after the anesthesia/surgery (Figure 1). All of the mice were habituated to the maze. The mouse was placed in the escape box for two minutes and then placed directly in the hole that led to the escape box for another four minutes. Finally, the mouse was placed under a bucket in the center of the circular platform and motivated to escape under the bright light (200 Watt) and noise (85 decibels) stimulation. The mouse was gently guided to the hole connecting to the escape box when it did not go into the escape box three minutes after the light and noise stimulation. Immediately after the mouse entered the tunnel between the hole and the escape box, the buzzer was turned off. Each mouse was allowed to remain in the escape box for one minute, and then removed and placed back to the home cage. We measured and recorded the latency to identify and enter the escaped box and speed in the Barnes Maze training. In the Barnes Maze testing, we measured and recorded the latency of the mouse to identify and enter the escaped box, distance, numbers of wrong holes searched, and the time in target zone [73–76]. The decreases in escape speed would suggest impairment of locomotor activity. We used the time in the target quadrant rather than the target preference in the current studies according to the previous studies [77, 79]. The composite Z-scores of each of the mice from the Barnes Maze test were calculated also using the methods described in previous studies [37, 70] with modifications. Specifically, the Z score of the mouse to present each of the individual Barnes maze test was obtained by the following steps. First, we used the individual raw score of the behavior following the anesthesia/surgery minus the mean of the raw scores of the behavior following the control condition; second, we divided the calculated change score by SD of the raw scores of the behavior following the control condition to obtain the individual Z score of each Barnes Maze test. We added the individual Z score (total of four) to have a total Z score. Finally, we divided the total Z score of each of the mice by the SD of the total Z score of the mice in the control group to obtain the composite Z score of each of the mice.

Statistics

The data were first examined by Shapiro-Wilk normality test. The data with normal distribution were analyzed by Student’s t-test, one-way ANOVA or two-way ANOVA, and the data with abnormal distribution were analyzed by Mann–Whitney U test or Friedman test. The data were expressed as means ± standard error of the mean (SEM) (e.g., the data of latency to identify and enter the escaped box and the speed in the Barnes Maze training) or means ± standard deviation (SD) (e.g., other data). We used 10–12 mice per group for the behavioral test, we used 6 mice per group for the Western blot, ELISA, and Seahorse XFp Extracellular Flux Analyzer studies. We chose these number of mice per group in the present studies based on the findings from our previous studies [37]. The data without normal distribution were analyzed by Mann-Whitney test, Kruskal-Wallis test or Friedman’s test, and were expressed as median and interquartile range (25% to 75%) There were significant variations in the behavioral data (some of the data were not normally distributed) following the intragastric administration of lactabocillus, probiotic and vehicle; thus, we used log transformation to transform the data (e.g., the data in Figures 6 and 7). The log transformation was performed by using Prism 8 software (Graph Pad Software, Inc, La Jolla, CA). Specifically, in the Barnes maze training, the interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of memory curves (e.g., based on escape latency). Post hoc analysis (Bonferroni comparison) was used to compare the difference in all behavior test parameters for each testing day. Mann-Whitney test was used to compare the difference of latency to identify and enter the escaped box, distance, number of wrong holes searched, and the time in the target zone in the Barnes Maze test. Student’s t-test was used to compare the difference of other studies (e.g., the change rate of relative abundance of lactobacillus). Two way ANOVA or Friedman’s test was used to determine the interaction of treatment (e.g., control versus anesthesia/surgery) and group (vehicle versus Lactobacillus or probiotic) on the changes in the mice. P values less than 0.05 were considered statistically significant. Prism 8 software (Graph Pad Software, Inc) was used to analyze the data.