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. 2024 Feb 28;10(5):e26200. doi: 10.1016/j.heliyon.2024.e26200

Prolonged oral intake of green tea polyphenols attenuates delirium-like behaviors in mice induced by anesthesia/surgery

Yao Xue a,1, Yan-Na Zhang a,1, Man Wang b, Hui-Yuan Fu b, Ying-Chao Mao b, Min Hu c, Mei-Tao Sun a, Hong-Gang Guo d, Lin Cao e, Chen-Zhuo Feng a,⁎
PMCID: PMC10943306  PMID: 38495146

Abstract

Postoperative delirium (POD) is a severe postoperative complication characterized by delirium-like symptoms. So far, no effective preventable strategy for POD prevention has been identified. Reports show that the consumption of green tea polyphenols (GTP) is associated with better cognitive function by modulating the composition of gut microbiota. Whether GTP also play a role in alleviating POD through gut microbiota is unknown. Herein, we studied the effect of prolonged (eight weeks) GTP intake on postoperative delirium in C57BL/6 mice with laparotomies under isoflurane anesthesia (anesthesia/surgery). We subsequently investigated anesthesia/surgery caused behavioral changes and increased the expression of malondialdehyde (MAD), an oxidative stress marker, and the activities of superoxide dismutase (SOD), an antioxidant marker, in the mice at 6 h after anesthesia/surgery. However, GTP administration reversed these changes and alleviated anesthesia/surgery-induced decrease in the abundance of gut bacterial genera, Roseburia. Further, fecal microbiota transplant demonstrated that compared with mice in the control group, treatment of C57BL/6 mice with feces from GTP-treated mice had a slight effect on the behavioral changes of mice. These data suggest that daily consumption of GTP could protect against anesthesia/surgery-induced behavioral changes, which is closely associated with gut microbiota modification by GTP.

Keywords: Green tea polyphenols, Postoperative delirium, Oxidative stress, Gut microbiota

Highlights

  • •

    Green tea polyphenols（GTP） alleviates delirium-like behaviors in mice caused by anesthesia/surgery.

  • •

    GTP ameliorates oxidative stress in mice after anesthesia/surgery.

  • •

    GTP might alleviate the delirium-like behaviors by gut microbiota.

  • •

    GTP might be a feasible strategy for managing postoperative impairments.

1. Introduction

Postoperative delirium (POD) is an acute confusional state following surgery, characterized by symptoms resembling delirium [1]. Patients who experience POD may have risk of increasing ICU and hospital length of stay, cognitive dysfunction, and cost [2]. However, the pathophysiological mechanisms underlying POD are still under investigation, and there is currently no effective treatment for it. Researchers have recently confirmed that a relationship between most gut microbiota and the brain's health exists [3]. Altered gut microbiota composition in mice has been associated with delirium-like behaviors after surgery, which can be relieved by taking probiotics [4]. In this paper, we speculated that since diet is an important factor that shapes gut microbiota composition, dietary habits might also affect post-surgical behaviors [5].

Green tea is a healthy beverage that has been popular in China for many years. It contains large amounts of polyphenols with antioxidant and antiinflammation effects [6]. Epidemiological investigation showed that consumption of green tea was associated with better cognitive function and anti-oxidative stress capacity in middle-aged and elderly people [7]. The prevention of cognitive decline is observed in older adults even at very small dosages [8]. This idea is also supported by several animal models of neurological disorders such as Parkinson's disease, Alzheimer's disease, and depression [9]. Catechins are the main active ingredient of GTP, which are mainly composed of (−)-epigallocatechin gallate, (−)-epigallocatechin, (−)-epicatechingallate and (−)-epicatechin. However, the mechanism by which GTP effectively prevent neurological disorders is still controversial, since the majority of dietary polyphenols with higher molecular weight are not absorbed in the small intestine [10].

With the comprehensive research on the microbiota-gut-brain axis in recent years, studies have demonstrated that the major effect of the total intake of polyphenols could be achieved by modulating gut microbiota composition or converting into small molecule metabolites through gut microbiota transformation to enter into the systemic circulation [11]. In this study, we used a mice model to assess whether prolonged consumption of green tea polyphenols alleviates delirium-like behaviors after surgery. Whether green tea polyphenols (GTP) exert their effect by altering the composition of gut microbiota was also investigated.

2. Materials and methods

2.1. Animal and animal treatment

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the USA. The research protocol was approved by the Ethics Committee of Animal Care and Application of Hangzhou medical college, China. Eight-week-old male C57BL/6 mice were purchased from Hangzhou medical college, China, and efforts were made to minimize the number of animals used and their suffering. Mice were housed in a temperature-controlled chamber at 23 ± 1 °C and provided enough food and water during a 12-h light-dark cycle. Mice were then allowed seven days to acclimatize to laboratory conditions before the experiment began. GTP containing >98% pure polyphenols were purchased from Shaanxi Senfo Natural Products Ltd. in Shaanxi Province, China. The mice receiving GTP treatment drank water with 0.05%(w/v) polyphenols for eight weeks according to the previous studies which suggested that 4–8 weeks of treatment of GTP would be enough to induce the change in gut microbiota [12,13]. GTP treatment was applied daily. Based on the daily water consumption, mice ingested ∼100 mg/kg of GTP per day. Mice were initially housed in a group of 2–3 mice per cage. Each mouse was moved to a single cage in case the fight occurred in a cage. Body weight, food intake, and water consumption were recorded once a week. The average food and water intake per mouse per day was calculated on a daily basis by calculating the difference in food and water provided and consumed per cage. We started to collect feces from each group of mice 4 days before the end of the treatment for the subsequent transplantation of fecal microbiota. The feces collected 6 h after anesthesia/surgery were collected for the analysis of bacterial abundance.

2.2. Animal experimental groups

Three sets of experiments were performed in this study. The protocol for each experimental set is presented in Fig. 1, Fig. 4, Fig. 5A.

Fig. 1.

Fig. 1

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Effect of GTP treatment on mice weight, water and diet intake. (A)A flow chart showing the first part of the experiment. (B)Changes in body weight in the mice during GTP treatment. (C)Changes in water consumption in the mice during GTP treatment. (D)Changes dietary intake in the mice during GTP treatment. Data are presented as mean ± S.E.M. (n = 12 for panel B, n = 8 for panel C, D).

Fig. 4.

Fig. 4

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Comparison of SOD and MAD levels in brain tissue of NDW-treated and GTP-treated mice after anesthesia/surgery. (A) A flow chart of the second part of the experiment. (B)Comparison of MAD level in the hippocampus and cortex. (C) Comparison of SOD level in the hippocampus and cortex. Data are presented as mean ± S.E.M. (n = 7). *P≤ 0.05, **P≤ 0.01.

Fig. 5.

Fig. 5

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Effect of GTP treatment on the change of gut microbiota induced by anesthesia/surgery. (A) Relative abundance of Roseburia sp. (B) Relative abundance of Bacteroides sp. (C) Relative abundance of Lactobacillus sp. Data are presented as mean ± S.E.M. (n = 10). *P≤ 0.05.

In the first set of experiments, 56 mice were used to test the behavioral changes. Twenty-eight mice were treated with GTP and then randomly assigned to one of the following groups (n = 14 for each group). One group underwent anesthesia/surgery (GTP+a/s), and the other served as control. Mice in the control group were not exposed to anesthesia or surgery (GTP + con). An equal number of age-matched mice receiving normal drinking water were then divided into two groups: those who underwent surgery/anesthesia (NDW+a/s) and those who did not undergo anesthesia/surgery (NDW + con).

In the second set of experiments (Fig. 4A), 28 mice were used to measure the activity of SOD and the MAD content. Similar to the first set of experiments, the mice were assigned into the following groups: GTP+a/s group, GTP + con group, NDW+a/s group, and NDW + con group. The mice in these experimental groups were sacrificed 6 h after surgery without undergoing a behavioral test.

In the third set of experiments (Fig. 6A), 48 mice were randomly divided into four groups (n = 12 for each group). The first and second groups received fecal suspensions from healthy mice. Then, the first group served as the control group without anesthesia or surgery (FMTN + con), whereas the second group underwent surgery under anesthesia (FMTN+a/s). The third group received the fecal suspensions from GTP-treated mice and was then also divided into two groups: those who underwent (FMTG+a/s) and those who did not (FMTG + con) undergo anesthesia/surgery.

Fig. 6.

Fig. 6

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Effect of FMT on anesthesia/surgery-treated mice with NDW and GTP in Buried food test and Y-maze test. (A) A flow chart showing the third part of the experiment. (B) Comparison of the latency to find food in the buried food test. (C) Comparison of the entries in novel arm in the Y-maze test. (D) Comparison of the time spent in novel arm in the Y-maze test. Data are presented as the mean ± S.E.M. (n = 12). *P≤ 0.05, **P≤ 0.01.

2.3. Anesthesia and surgery

To obtain mice model with delirium-like behaviors, laparotomies were performed under isoflurane anesthesia as described in our previous study [14]. The surgery was performed between 7:00 a.m. to 9:00 a.m. Mice were anesthetized by inhalation of 1.5% isoflurane in 100% oxygen gas for 20 min in a transparent chamber. Once mice lost the righting reflex, they were immediately moved out of the chamber and placed on a nebulizer mask to maintain their unconscious state (1.5% isoflurane in 100% oxygen) during laparotomy.

Before the surgery, the skin in the surgery site was shaved and disinfected with povidone-iodine. Then, a 1.5 cm abdominal midline incision was made, and a sterile cotton swab was inserted into the abdominal cavity to manipulate the viscera and musculature for 2 min. The small intestine was then exteriorized from the abdominal cavity, rubbed gently between the surgeon's thumb and forefinger for 1.5 min, and then returned to the abdominal cavity. After that, the incision site was sutured with 5-0 silk surgical sutures. To relieve pain, 5% lidocaine cream was applied to the wound at the end of the surgery and every 8 h for at least two days after surgery. The surgery lasted approximately 20 min, after which the mice were returned to the anesthesia chamber for 2 h of continuous anesthesia dosage. The mice were returned to their home cages once they recovered from the anesthesia effects. The control group of mice did not undergo surgery or receive anesthesia.

2.4. Behavior tests

All behavioral tests were performed in a soundproof room during the light phase of the light/dark cycle. The behavioral changes of the mice following anesthesia/surgery were assessed using a buried food test, open field test, and Y maze test as previously described [15]. The behaviors in the Y maze and open field test were collected and analyzed with the SMART video tracking system version 3.0. To assess the behavioral performance at baseline, mice were subjected to behavioral tests 24 h before anesthesia/surgery and 6 h after anesthesia/surgery.

2.5. Buried food test

We used a modified version of the buried food test that was described in previous studies [15,16]. Mice were given a piece of sweetened cereal two days before the test and were food-restricted to maintain 90% of their original body weight. Each mouse was tested only once on the experiment day. During the test, the mice were placed in the center of the test cage filled with 3 cm of new bedding and a piece of cereal buried 1 cm below the surface. The location of the cereal was chosen randomly within the cage. The latency of locating buried food for each mouse was recorded from when it was placed in the cage to when it located the cereal. If a mouse could not find the cereal ring within 5 min, the test was stopped and a latency of 300 s was recorded for that mouse. After each trial, the test cage was cleaned with 70% ethanol to eliminate possible olfactory clues.

2.6. Y maze test

The Y maze consisted of three symmetrical arms with a 120° angle between each arm. These arms were labeled as "starting arm," "novel arm," and "other arm." The test was conducted with two trials, with a 1-h interval between each trial. During the first training trial, the novel arm was blocked with a baffle, and the mouse was allowed to explore only two arms (the starting arm and the other arm) for 10 min. During the second trial, the baffle was removed, and the mouse was placed back in the starting arm, and allowed to explore all three arms for 5 min. The time spent on the novel arm and the number of entries into it were recorded and analyzed. The maze was cleaned with 70% ethanol after each trial.

2.7. Open field test

The open field test was performed as previously described [17]. The apparatus for the test was made of grey acrylic panels (50 cm × 50 cm, arm height 30 cm) and was divided equally into 16 small squares. The four central squares were defined as the central area, and the remaining outer squares were defined as the peripheral area. Individual mice were placed in one corner of the open field and allowed to explore for 5 min. The path of each mouse was recorded, and the total distance covered, time spent in the center, freezing time, and latency to the center area were analyzed. The apparatus was cleaned with 70% ethanol after each mouse was tested.

2.8. Calculation of composite Z scores

The composite Z score for each mouse was calculated using methods routinely used to study postoperative delirium in mice [15]. The postoperative data were subtracted from the pre-operative data. The results were then divided by the corresponding Standard Deviation（SD）generated by the control group. The composite Z score for each mouse was calculated as the sum of the following six Z scores divided by the SD of the sum of the Z scores in the control group: latency to find food (buried food test), the time spent in the center (open field test), latency to the center area (open field test), the freezing time (open field test), the entries in the novel arm (Y maze test) and the time spent in the novel arm (Y maze test). Reduction of time spent in the center, freezing time, entries in the novel arm, and time spent in the novel arm indicated impaired behaviors. We multiplied the Z scores by −1 to obtain positive Z scores and the final composite compound Z scores that increase with impairment severity.

2.9. Tissue collection and measurement of MAD and SOD

Mice were anesthetized with isoflurane 6 h after anesthesia/surgery, then perfused with 0.9% saline via the heart, and the brains were immediately removed and placed on ice. Next, the cortex and hippocampus were removed and weighed to obtain tissue weights. The samples were immediately put into liquid nitrogen for temporary storage and moved to a −80 °C freezer for long-term storage. Afterward, the MAD and SOD content in the brain tissues were measured. The levels of MAD and SOD in brain tissues were determined using the MAD Assay Kit (Beyotime Biotechnology, Shanghai, China) and SOD Assay Kit (Beyotime Biotechnology, Shanghai, China), respectively, according to the manufacturer's instructions.

2.10. Bacterial quantification by real-time qPCR [[18], [19], [20], [21]]

Bacterial DNA was isolated from the collected fecal samples using the MolPure® Stool DNA Kit kit (Yeasen Biotechnology, Shanghai) according to the manufacturer's instruction. The DNA quality and quantity were assessed using a Nanodrop spectrophotometer. Quantitative PCR reaction was carried out in SYBR Green Mix. The sequences of primers used for detecting the bacteria have been validated in previous studies and are shown in Table 1 qPCR with primers detecting all bacteria was also carried out for each sample to normalize the differences in starting samples. The relative abundance of Bacteroides sp., Lactobacillus sp. and Roseburia sp. in each study group was calculated by against the NDW + con group using the comparative threshold cycle method.

Table 1.

Primers used in qPCR.

Eubacteria [16] Forward: 5′-CGGYCCAGACTCCTACGGG-3′(Y is C or T)
Reverse: 5′-TTACCGCGGCTGCTGGCAC-3′
Bacteroides sp. [17] Forward: 5′-GAGAGGAAGGTCCCCCAC-3′
Reverse: 5′-CGCTACTTGGCTGGTTCAG-3′
Lactobacillus sp. [18] Forward: 5′-CACCGCTACACATGGAG-3′
Reverse: 5′-AGCAGTAGGGAATCTTCCA-3′
Roseburia sp. [19] Forward: 5′-AAATACCCGTGGTGTTACCG-3′
Reverse: 5′-GTGTCTCCCTCTGTAAAGTCA-3′
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2.11. Fecal microbiota transplant

Pseudo-germ-free mice were prepared before FMT, as previously described [22]. Briefly, four-month-old male C57BL/6 mice were given drinking water with the following antibiotics: 1 mg/ml of ampicillin, 0.5 mg/ml of vancomycin, 1 mg/ml neomycin and 250 mg/ml of metronidazole (Sigma-Aldrich, St. Louis, MO, USA) daily for two weeks. The feces from the mice with or without GTP treatment were collected with sterilized filter paper and stored in a -80°C freezer until transplant. The fecal microbiota suspension was prepared by diluting 1 g of fecal in 10 ml saline, which was then centrifuged at 800g for 3 min. Each mouse was given 0.2 ml of the suspension by gavage for 14 consecutive days.

2.12. Statistical analysis

The normality of the data was tested using the Shapiro-Wilk normality test. The differences between two groups were assessed with Student's t-test if data were normally distributed or the Mann-Whitney U test if the data were non-normally distributed. The effects of interaction between GTP treatment and anesthesia/surgery were assessed using two-way ANOVA, followed by Turkey post hoc test for normally distributed data or by Kruskal-Wallis test followed by the Mann-Whitney U test for non-normally distributed data. Two-way repeated-measure ANOVA with Bonferroni post hoc tests were used to compare diet intake, water consumption, and body weight among groups. Continuous normally distributed data were expressed as mean ± S.E.M. Data were analyzed using Prism 9 software (GraphPad Software, Inc, La Jolla, CA). P ≤ 0.05 was considered statistically significant.

3. Results

3.1. Treatment with GTP ameliorated behavioral changes in mice in the buried food test caused by anesthesia/surgery

According to the guide of dose conversion between human and mouse [23], mice were treated with a small amount of GTP, equivalent to human consumption of less than 1g of GTP per day. Prolonged treatment of GTP did not affect the mice weight (Fig. 1B, F8,414 = 0.422, p = 0.908), average water consumption (Fig. 1C, F8,126 = 0.818, p = 0.588), and average diet intake (Fig. 1D, F8,126 = 0.761, p = 0.637).

The buried food test is a common behavior test to show olfactory ability. It also requires hippocampal function to finish the test [24]. In this study, we found that at 6 h after anesthesia/surgery, the latency to find food was significantly longer in the NDW+a/s group than in the NDW + con group (Fig. 2A, p = 0.005, Mann-Whitney U test). However, the change was not observed between the GTP + con group and the GTP+a/s group (Fig. 2A, p = 0.265, Mann-Whitney U test). Two-way ANOVA showed a significant interaction between anesthesia/surgery and GTP (Fig. 2A, F1,52 = 4.035, p = 0.0498) and a significant main effect of anesthesia/surgery (F1,52 = 10.32, p = 0.002) with no effect of GTP (F1,52 = 1.139, p = 0.291).

Fig. 2.

Fig. 2

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Performance of GTP-treated mice in Buried food test Y-maze test and Open field test after anesthesia/surgery. (A) Comparison of the latency to find food in the buried food test. (B) Comparison of the entries in novel arm in the Y-maze test. (C) Comparison of the time spent in novel arm in the Y-maze test. (D) Comparison of the time spent in the center in the open field test. (E) Comparison of the freezing time in the open field test. (F) Comparison of the latency to the center in the open field test. Data are presented as mean ± S.E.M. (n = 14). *P≤ 0.05, **P≤ 0.01.

3.2. Treatment with GTP ameliorated behavioral changes in mice in the Y-maze test caused by anesthesia/surgery

We assessed the effects of GTP on short-term spatial memory impairments caused by anesthesia/surgery using the Y-maze test. The entry times in the novel arm were significantly lower in the NDW+a/s group compared with the NDW + con group (Fig. 2B, p = 0.046, Mann-Whitney U test), as shown in Fig. 2B. The decrease in the time spent in the novel arm was not statically significant, however a downward trend was observed in the NDW + con group (Fig. 2C, p = 0.077, Mann-Whitney U test). In contrast, the mice treated with GTP exhibited better performance on short-term spatial memory after anesthesia/surgery since there was no difference between the GTP + con group and GTP+a/s group on the percent entries into the novel arm (Fig. 2B, p = 0.370, Mann-Whitney U test) or the percent time spent in the novel arm (Fig. 2C, p = 0.972, Mann-Whitney U test). Two-way ANOVA revealed no interaction between anesthesia/surgery and GTP (Fig. 2B, F1,52 = 0.584, p = 0.448; Fig. 2C, F1,52 = 0.729, p = 0.397), and nonsignificant effect of anesthesia/surgery (Fig. 2B, F1,52 = 2.536, p = 0.117; Fig. 2C, F1,52 = 0.461, p = 0.500) or GTP (Fig. 2B, F1,52 = 1.249, p = 0.269; Fig. 2C, F1,52 = 0.245, p = 0.623) was observed.

3.3. Treatment with GTP ameliorated behavioral changes in mice in the open field test caused by anesthesia/surgery

Compared with the control group, the mice fed with a normal diet spent less time in the center of the open field box after anesthesia/surgery (Fig. 2D, p = 0.016, Mann-Whitney U test), while those treated with GTP behaved similarly to the control group (Fig. 2D, p = 0.401, Mann-Whitney U test). Two-way ANOVA revealed no interaction between anesthesia/surgery and GTP (Fig. 2D, F1,52 = 0.425, p = 0.518), and nonsignificant effect of anesthesia/surgery (F1,52 = 2.478, p = 0.122) or GTP (F1,52 = 0.549, p = 0.462) was observed. The mice fed with a normal diet tended to spent less time freezing after anesthesia/surgery, but the differences were not statistically significant (Fig. 2E, p = 0.068, Student's t-test). The mice treated with the GTP did not show such a significant trend (Fig. 2E, p = 0.260, Student's t-test). The latency of mice to reach the center was unaltered in the mice after anesthesia/surgery with or without GTP treatment (Fig. 2F, normal diet: P = 0.352, Mann-Whitney U test; Fig. 2F, GTP treatment: p = 0.834, Student's t-test).

A Composite Z score was used for assessing the delirium-like characteristic in the animal model. This method has been successfully used in previous studies [15,25]. The values of composite Z scores of mice (Fig. 3A and B) with normal diet and mice treated with GTP were compared.

Fig. 3.

Fig. 3

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Summary of composite Z scores after anesthesia/surgery in GTP-treated mice in the first part of the experiment. (A) The values of composite Z scores in the mice fed a normal diet. (B) The values of composite Z scores in the mice with GTP treatment. (C) Comparison of the Composite Z scores. Data are presented as mean ± S.E.M. (n = 14). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P≤ 0.0001.

Two-way ANOVA showed a significant interaction between anesthesia/surgery and GTP on the composite Z scores (Fig. 3C, F1,52 = 4.643, p = 0.036) with significant main effects of anesthesia/surgery (F1,52 = 25.12, p < 0.0001) and GTP (F1,52 = 6.639, p = 0.013). This result suggested that GTP treatment prevented postoperative cognitive impairment caused by anesthesia/surgery.

3.4. Treatment with GTP ameliorated oxidative stress in mice caused by anesthesia/surgery

Oxidative stress in the mice brains has been implicated in behavioral changes caused by anesthesia/surgery [15]. Although the buried-food test and the open-field test are the most reliable olfaction and emotion assays in rodents so far, they were reported to be closely associated with other cognitive domains [24,26,27]. Thus, we analyzed the level of oxidative stress marker MAD and the activity of antioxidant marker SOD in hippocampal and cortical tissues from each group of mice to evaluate the effect of GTP treatment on the oxidative stress in mice undergoing anesthesia/surgery. It was revealed that after anesthesia/surgery, the MAD activity was higher in the hippocampus (Fig. 4B, p = 0.001, Mann-Whitney U test) and cerebral cortex (p = 0.001, Student's t-test) of mice in the NDW+a/s group than in the NDW + con group. However, those differences did not appear in the mice with GTP treatment (Fig. 4B, hippocampus; p = 0.128, Mann-Whitney U test; cortex: p = 0.086, Student's t-test). Two-way ANOVA showed a significant interaction between anesthesia/surgery and GTP on MAD activity in the cortex (Fig. 4B, F1,24 = 4.707, p = 0.040) but insignificant in the hippocampus (F1,24 = 1.672, p = 0.208). A Significant main effect of anesthesia/surgery or GTP was found in both the cortex (anesthesia/surgery: F1,24 = 20.54, p = 0.0001; GTP: F1,24 = 12.82, p = 0.002) and hippocampus (anesthesia/surgery: F1,24 = 22.28, p < 0.0001; GTP: F1,24 = 6.146, p = 0.01).

Furthermore, the effect of GTP on SOD activity was assessed, and it was found that anesthesia/surgery enhanced SOD activity resulting from adaptive response to oxidative stress induced in the hippocampus (Fig. 4C, p = 0.041, Student's t-test) and cortex (p = 0.009, Student's t-test) of mice fed with a normal diet. However, this increase in SOD activity was not observed in GTP-treated mice after anesthesia/surgery (Fig. 4C, hippocampus: p = 0.519, Student's t-test; cortex: p = 0.623, Student's t-test). The interaction between anesthesia/surgery and GTP on SOD activity among the four groups was statically significant in the cortex (Fig. 4C, F1,24 = 4.324, p = 0.048) but not in the hippocampus (F1,24 = 0.625, p = 0.437). The main effect of anesthesia/surgery had greater significance in the cortex (F1,24 = 7.383, p = 0.012) than in the hippocampus (F1,24 = 3.499, p = 0.074). The main effect of GTP did not reach significance in either cortex (F1,24 = 0.035, p = 0.853) or hippocampus (F1,24 = 0.426, p = 0.520).

3.5. Treatment with GTP alleviated the decrease of the abundance of Roseburia sp. in mice gut caused by anesthesia/surgery

Reports suggest that the change in gut microbiota may contribute to the oxidative stress-related cognitive deficits induced by anesthesia/surgery [22]. GTP which is already known for its antioxidant activity has been reported in reducing gut microbiota dysbiosis [13]. Thus, we hypothesized that the gut microbiota modified by GTP plays an important role in the behavioral changes induced by anesthesia/surgery. To test this hypothesis, we examined the abundance of Lactobacillus sp., Roseburia sp., and Bacteroidetes sp. which have been reported to be involved in the function of GTP in the mouse fecal samples. We found that anesthesia/surgery induced a significant decrease in Roseburia sp. in mice fed with a normal diet, which was not observed in mice with GTP treatment (Fig. 5A, normal diet p = 0.045, Mann-Whitney U test; GTP treatment: p = 0.678, Student's t-test). The interaction between anesthesia/surgery and GTP on Roseburia sp. was not statistically significant (F1,36 = 0.437, p = 0.513). Also, a nonsignificant effect of anesthesia/surgery (F1,36 = 0.894, p = 0.351) or GTP (F1,36 = 2.733, p = 0.107) was observed. The abundance of Lactobacillus sp. and Bacteroidetes sp. was not altered after anesthesia/surgery in mice, regardless of GTP treatment. (Fig. 5B, normal diet: p = 0.427, Mann-Whitney U test; GTP treatment: p = 0.850, Mann-Whitney U test. Fig. 5C, normal diet: p = 0.678, Mann-Whitney U test; GTP treatment: p = 0.910, Mann-Whitney U test).

3.6. FMT from mice treated with GTP ameliorated behavioral changes in mice caused by anesthesia/surgery

We performed fecal microbiota transplantation in pseudo-germ-free mice. Fecal microbiota extracted from mice that received normal or GTP-treated diet were orally administrated to pseudo-germ-free mice for 14 consecutive days before anesthesia/surgery. It was found that time spent to find buried food was longer in the buried food test, FMTN+a/s group compared with the FMTN + con group (Fig. 6B, p = 0.001, Student's t-test). The percent of novel arm entries (Fig. 6C, p = 0.026, Student's t-test) and the proportion of time spent in the novel arm (Fig. 6D, p = 0.004, Mann-Whitney U test) in the Y maze test were less in FMTN+a/s group than in FMTN + con group. The FMTN+a/s group spent less time in the central area (Fig. 7A, p = 0.023, Student's t-test) and freezing (Fig. 7B, p = 0.012, Student's t-test) after anesthesia/surgery in open field test than in FMTN + con group. The latency of mice to reach the center was unaltered after anesthesia/surgery whether or not gut microbiota were modified by GTP (Fig. 7C, no GTP-modified gut microbiota: p = 0.364, Student's t-test; GTP-modified gut microbiota: p = 0.893, Student's t-test). The changes in each of those group parameters were relatively small in the FMTG + con group compared with the FMTG+a/s group (Fig. 6B-D，Fig. 7A–C). The values of composite Z scores in pseudo-germ-free mice with FMT from normal diet-fed mice and in pseudo-germ-free mice with FMT from GTP-treated mice (Fig. 7D and E) were compared. Although the two-way ANOVA analysis revealed no interaction between anesthesia/surgery and gut microbiota in composite Z value, we found that treatment with GTP-modified gut microbiota was consistent with treatment with GTP in preventing behavioral changes caused by anesthesia/surgery (Fig. 7F, F1,44 = 2.703, p = 0.107). The main effect of anesthesia/surgery (F1,44 = 28.88, p < 0.0001) was significant, while the main effect of GTP (F1,44 = 0.713, p = 0.403) was not.

Fig. 7.

Fig. 7

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The effect of FMT on anesthesia/surgery induced mice treated with NDW and GTP in Open field test and the composite Z values of mice in the third part of the experiment. (A) Comparison of the time spent in the center in the open field test. (B) Comparison of the freezing time in the open field test. (C) Comparison of the latency to the center in the open field test. (D) The values of composite Z scores in pseudo-germ-free mice with FMT from normal diet-fed mice. (E) The values of composite Z scores in pseudo-germ-free mice with FMT from GTP-treated mice. (F) Comparison of the Composite Z scores. Data are presented as the mean ± S.E.M. (n = 12). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P≤ 0.0001.

4. Discussion

Animal models are crucial tools for developing preventative treatments in preclinical POD research. In the present study, we assessed the effect of prolonged GTP treatment on behavioral changes and oxidative stress in the animal model before and after anesthesia/surgery. The results of the open field test, Y-maze test, and buried food test showed that GTP treatment ameliorated the anesthesia/surgery-induced behavioral changes in mice and increased the activities of MAD and SOD. Furthermore, our experiment on fecal microbiota transplantation confirmed that gut microbiota played an important role in attenuating the behavioral changes induced by anesthesia/surgery.

The most common cause of acute brain dysfunction in POD is anesthesia/surgery. The symptoms of POD occur in different cognitive domains, but only a few of these symptoms have been validated in animal models [[28], [29], [30]]. Herein, we used data from the mice in behavioral tests, which not only depend upon spontaneous behavior but also cognitive function including attention, organized thinking, and consciousness, to assess behavioral changes induced by anesthesia/surgery. These changes are similar to those in humans experiencing delirium. Our study found that approximately 71% of mice developed POD, which is consistent with a previous study [15] and suggests that this animal model can be used to study POD in the future.

Previous studies suggest that oxidative stress is a primary cause of behavioral change induced by anesthesia/surgery in young mice [15,31]. MAD data is preferred in determining oxidative stress over ROS data because of the challenges posed by ROS instability and its extremely reactive nature. Compared with ROS, MAD is a stable end product of lipid peroxidation that is accompanied by ROS expression. Our results revealed an increase in MAD expression in the mice brain after anesthesia/surgery, suggesting a strong correlation between oxidative stress and behavioral changes after anesthesia/surgery, consistent with a previous study [31]. We also assessed the change in SOD, another oxidative stress marker. The high SOD activity in the brains of mice after anesthesia/surgery indicated that the mice developed physiological adaption to oxidative stress since the symptoms presented in our mouse model were acute and reversible [15]. However, our results were contradictory to a previous study [31]. This could be due to the fluctuation of SOD activity over time, and differences in the time points of sample collection.

GTP possess strong antioxidant properties and has been proven effective in mitigate the negative impact of oxidative stress in neurological diseases such as Parkinson's disease, Alzheimer's disease, depression, and other age-related ailments [9]. However, information on the effects of GTP on POD is scanty. In this research, we preferred prolonged rather than short-term GTP treatment before anesthesia/surgery because short-term treatment with GTP (data not shown) had no effect on the mice. GTP treatment reduced behavioral changes induced by anesthesia/surgery and also modulated oxidative stress, implied by low MAD and SOD contents. These results suggested that GTP could potentially reduce the likelihood of POD. Given that GTP is the most abundant compound in green tea that can be taken on a daily basis, it is a feasible strategy for managing postoperative impairments.

The underlying mechanism for the neuroprotective potential of GTP against neurotoxicity remains unknown, although GTP exhibits strong radical scavenging activities, which can effectively inhibit excessive ROS production and protect cells against oxidative stress damage [32]. Numerous studies regarding the potential health benefits of GTP on the brain have been attributed to the interplay between GTP and gut microbiota for several years [33]. Our data which FMT from mice treated with GTP ameliorated behavioral changes in mice caused by anesthesia/surgery suggests that gut microbiota might mediate the benefit of GTP. Additionally, studies have reported a linkage between gut microbiota and POD development [4,22,34], which is also supported by our data.

Our study had some limitations. First, our study only provided some preliminary data on the effect of GTP in POD. Our study observed GTP treatment prevented the decrease of the abundance of Roseburia sp. after anesthesia/surgery, which suggested that GTP supplement might prevent anesthesia/surgery-induced gut microbiota dysbiosis. However, more bacteria or SCFA should be investigated to uncover the mechanism by which GTP affects behavioral changes induced by anesthesia/surgery. Second, GTP contains multiple active ingredients, while we have no evidence about which components of GTP may be more effective. Finally, considering that POD is more common in elderly patients, whether the effect of GTP observed in young mice is also present in aged mice should be clarified in future studies.

Funding

This work was supported by the Zhejiang Provincial Natural Science Foundation of China [LY22H090011], Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents [2018] and Zhejiang Provincial Public Welfare Research Program [LGD22C040024].

CRediT authorship contribution statement

Yao Xue: Writing – review & editing, Writing – original draft, Formal analysis, Data curation, Conceptualization. Yan-Na Zhang: Writing – review & editing, Formal analysis, Data curation. Man Wang: Writing – review & editing, Formal analysis, Data curation. Hui-Yuan Fu: Writing – review & editing, Formal analysis, Data curation. Ying-Chao Mao: Writing – review & editing, Formal analysis, Data curation. Min Hu: Writing – review & editing, Formal analysis, Data curation. Mei-Tao Sun: Writing – review & editing, Software, Resources. Hong-Gang Guo: Writing – review & editing, Software, Resources. Lin Cao: Writing – review & editing, Conceptualization. Chen-Zhuo Feng: Writing – review & editing, Writing – original draft, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to thank all the people who participated in the project, especially Xiao-Li Ji, for her valuable time and energy in helping us in the experiment of surgery.

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