Appropriate Exercise Level Attenuates Gut Dysbiosis And Valeric Acid Increase To Improve Neuroplasticity And Cognitive Function After Surgery in Mice Part 1

Postoperative cognitive dysfunction (POCD) affects the outcome of millions of patients each year. Aging is a risk factor for POCD. Here, we showed that surgery-induced learning and memory dysfunction in adult mice. 

In modern medicine, surgery is a very common treatment method and is necessary for many diseases. However, some patients will experience postoperative cognitive dysfunction after surgery, which is a troublesome problem for some patients.

Postoperative cognitive dysfunction refers to impairments in memory, attention, and thinking ability that occur after surgery. This condition is rare in younger people but more common in older people. Postoperative cognitive dysfunction does cause some inconvenience to life, but it does not mean that patients need to feel frustrated or disappointed.

Although postoperative cognitive dysfunction will affect a patient's memory to a certain extent, there are many ways to help patients alleviate this condition. First, patients can achieve better results by communicating with their doctors and following their recommendations for treatment. Secondly, patients can improve their cognitive abilities through appropriate exercise, such as reading, writing, listening to music, etc. In addition, normal sleeping and eating habits can also go a long way in restoring memory.

Finally, patients need to maintain a positive attitude and an optimistic attitude. Postoperative cognitive dysfunction is not the end point. As long as the patient persists in treatment and exercises, the patient's memory will gradually recover. Therefore, we should not worry or complain too much, but should strengthen our confidence and believe that we can overcome difficulties and face the future with confidence. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

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Transplantation of feces from surgery mice but not from control mice led to learning and memory impairment in non-surgery mice. Low-intensity exercise improved learning and memory in surgery mice. Exercise attenuated surgery-induced neuroinflammation and decreased gut microbiota diversity. These exercise effects were present in non-exercise mice receiving feces from exercise mice. 

Exercise reduces valeric acid, a gut microbiota product, in the blood. Valeric acid worsened neuroinflammation, learning, and memory in exercise mice with surgery. The downstream effects of exercise included attenuating growth factor decrease, maintaining astrocytes in the A2 phenotypical form possibly via decreasing C3 signaling, and improving neuroplasticity. 

Similar to these results from adult mice, exercise attenuated learning and memory impairment in old mice with surgery. Old mice receiving feces from old exercise mice had better learning and memory than those receiving control old mouse feces. Surgery increased blood valeric acid. Valeric acid blocked exercise effects on learning and memory in old surgery mice. 

Exercise stabilized gut microbiota, reduced neuroinflammation, attenuated growth factor decrease, and preserved neuroplasticity in old mice with surgery. These results provide direct evidence that gut microbiota alteration contributes to POCD development. Valeric acid is a mediator for this effect and a potential target for brain health. Low-intensity exercise stabilizes gut microbiota in the presence of insult, such as surgery.

HIGHLIGHTS

Exercise reduces post-surgery neuroinflammation and impairment of cognition and neuroplasticity.

Exercise decreases gut microbiota changes and valeric acid increases after surgery.

The effects of exercise on surgery-induced changes are transferable by fecal transplantation.

Valeric acid blocks the beneficial effects of exercise.

INTRODUCTION

Postoperative cognitive dysfunction (POCD) affects millions of patients each year in the U.S.A. and is associated with increased mortality and cost of hospitalization [1–4]. Advanced age is an independent risk factor for POCD [1, 2, 5]. Currently, the mechanisms for POCD are not fully understood and effective interventions to reduce POCD have not been identified. Neuroinflammation has been implied in the development of POCD [6, 7]. 

Surgery on peripheral tissues or organs can induce systemic inflammation, which then is transmitted into the brain to cause neuroinflammation to induce POCD [8, 9]. Gut microbiota is involved in regulating inflammation [10]. Interestingly, gut microbiota diversity changes and dysbiosis are associated with learning and memory dysfunction after surgery in animals [11, 12]. 

Pretreated animals with probiotics improved their cognitive functions [13]. However, direct evidence to suggest the involvement of gut microbiota in POCD has not been reported. We and others have shown that environmental enrichment reduces learning and memory impairment after surgery [14, 15]. Environment enrichment may enhance the physical activity of animals. A recent study has shown that gut microbiota is important in determining whether human subjects are responders to exercise to improve glucose homeostasis and insulin sensitivity [16]. 

Exercise has been shown to improve the long-term memory of humans [17]. Better pre-surgery exercise ability is associated with less of a decrease in Mini-Mental State Examination scores after cardiac surgery in humans [18]. An animal study has shown that exercise attenuated the enhanced neuroinflammation and impairment of learning and memory after surgery in low-capacity runner rats. Exercise also improved the diversity of the gut microbiota of these rats. 

Although exercise appears to reduce proinflammatory cytokine production in the brain of high-capacity rats, it does not attenuate surgery-induced learning and memory impairment in these rats [19]. These results suggest that exercise or exercise capacity is associated with better learning and memory. However, it is not clear whether exercise attenuates POCD, especially in old animals, and whether gut microbiota plays a role in the effects of exercise on POCD. 

Based on the above information, we hypothesize that an appropriate level of exercise attenuates neuroinflammation and the impairment of learning and memory after surgery and that these effects are mediated by gut microbiota alterations. To test these hypotheses, we subjected adult and old mice to different levels of exercise. Fecal transplantation was performed to determine the role of gut microbiota in the effects of exercise on POCD development. 

Left carotid artery exposure was chosen to be the surgical procedure because this procedure is a component of carotid endarterectomy that is commonly performed in elderly patients. 

In addition, this procedure shall not affect limb functions, which are needed for learning and memory tests, and organ functions for general health. Our results provide direct evidence for the involvement of gut microbiota in POCD. Valeric acid, a product of gut microbiota, plays a critical role in mediating the effects of gut microbiota on learning and memory impairment after surgery.

RESULTS

Low-intensity exercise prevented surgery-induced cognitive dysfunction, neuroinflammation, and impairment of brain cell generation and dendritic arborization possibly via stabilizing gut microbiota in adult mice
To determine the effect of physical activity on the function and structure of the brain, 9-week-old mice were subjected to forced mouse treadmill running to 35–40% (low intensity), 55–60% (middle intensity), or 75–80% (high intensity) of their maximal exercise capacity, respectively, 5 days a week for 4 weeks before they had left carotid artery exposure for 15 min under isoflurane anesthesia (surgery and anesthesia). 

Mice were assessed by novel object recognition and Barnes maze tests from one week after the surgery. Mice in all groups took less time to find the target box with more training sessions in the Barnes maze test (Fig. 1A). Surgery was a significant factor in affecting the animals to find the target box [F(1, 113) = 7.360, P = 0.008] and low-intensity exercise reversed this surgery effect in the training sessions of the Barnes maze test [F(1, 76) = 5.278, P = 0.024]. 

Mice with surgery took longer than control mice to find the target box one or eight days after the training sessions. This surgery effect was attenuated by low-intensity exercise but not by middle or high-intensity exercise (Fig. 1B). Mice with surgery spent less time with a novel objects than control mice in the novel object recognition test and this decreased time was reversed by low-intensity exercise but not by middle or high-intensity exercise no matter when the test was performed either 30 s or 24 h after the initial exploration with two objects (familiarization phase) (Fig. 1C). 

Various lengths of delay between the familiarization and test phases (from 10 s to 24 h) have been used previously. The memorization after short and long delays may involve the perirhinal cortex and hippocampus, respectively [20]. Our results suggest that surgery induces learning and memory dysfunction and that low-intensity exercise but not middle and high-intensity exercise prevents these surgery effects. 

As an initial step to determine whether gut microbiota played a role in the effects of exercise on learning and memory, mice transplanted with feces from control mice or from mice with low-intensity exercise were subjected to surgery. Transplantation with feces from exercise mice reduced the amount of time to identify the target box in the training sessions [F(1, 26) = 13.071, P = 0.001] and one or eight days after training sessions (Fig. 1D, E). Mice transplanted with feces from exercise mice spent more time with novel objects than mice transplanted with feces from control mice (Fig. 1F). 

These results suggest that gut microbiota mediates the beneficial effects of low-intensity exercise. This level of exercise was used for mice receiving exercise conditioning in the following experiments and was referred to as exercise for simplicity. Exercise modified the diversity of gut microbiota within one sample (α diversity) and the difference in diversity among samples (β diversity) (Fig. 2A, B, and Supplementary Fig. 1a–c). Exercise increased the abundance of some bacteria, such as Bacteroidales and Alistipes. Control mice had abundant lactobacilliaceae and lactobacillus because the linear discriminant analysis (LDA) scores for the comparisons of these bacteria between control and exercise mice were all >3.5 (the direction can be negative or positive) (Supplementary Fig. S1d), a threshold indicating a difference in the abundance of bacteria in samples from different experimental conditions [21]. 

LDA was performed after the difference among samples from different experimental conditions was determined to be significant by the Kruskal–Wallis test by rank per LDA effect size analysis protocol. Mice had a reduced diversity of gut microbiota and the difference in diversity among samples 7 days after surgery. This reduction was attenuated in exercise mice (Fig. 2C–F and Supplementary Fig. S2). Mice receiving fecal transplantation had treatment with a mixture of antibiotics to eliminate their native gut microbiota before the transplantation. This treatment nearly abolished the gut microbiota in these mice because the bacterial DNA in the feces was barely detectable (Supplementary Fig. S3). Mice transplanted with feces from exercise mice also had a reduced difference in diversity among samples (Fig. 2G, H and Supplementary Fig. S4a–c). The gut microbiota of mice transplanted with feces from exercise mice contained an abundant amount of Alistipes. Mice transplanted with feces of control mice contained a large amount of lactobacilliaceae and lactobacillus (Supplementary Fig. S4d). 

These results suggest that exercise changed gut microbiota with increased abundance in phylum Bacteroidetes, such as Bacteroidales and Alistipes, and decreased abundance in phylum Firmicutes, such as lactobacilliaceae and lactobacillus. Exercise also stabilizes gut microbiota after surgery. Consistent with the pattern of microbiota in mice with surgery, mice transplanted with feces of surgery mice had reduced diversity of gut microbiota compared with mice transplanted with feces of control mice. Mice receiving surgery mouse feces had decreased abundance in Bacteroidales and Alistipes compared with mice receiving control mouse feces (Supplementary Fig. S5). These results suggest that altered microbiota in surgery mice is successfully transferred to non-surgery mice. Mice receiving surgery mouse feces took longer than mice receiving control mouse feces to identify the target box in the Barnes maze one day after the training sessions. The mice receiving surgery mouse feces also spent less time with novel objects than mice receiving control mouse feces in the novel object recognition test. However, there was no difference among control mice (naïve mice), mice receiving antibiotics, and mice receiving control mouse feces in the performance of Barnes maze and novel object recognition tests (Supplementary Fig. S6). These results indicate a role of microbiota alteration in learning and memory dysfunction after surgery. 

As an initial step to identify possible molecules downstream of low-intensity exercise and gut microbiota for regulating learning and memory, we performed RNA-seq analysis. Surgery and exercise altered the profiles of mRNA expression in the hippocampus (Fig. 3A, B), a brain region involved in learning and memory [6, 22]. The genes with the most changes by surgery and exercise were displayed in Fig. 3C. Among them, the mRNA of complement 3a receptor 1 (C3ar1) was increased by surgery and this increase was attenuated by exercise based on a two-way analysis of variance with surgery and exercise as two independent factors (Fig. 3C, D). The mRNA expression of C3ar1 after surgery was decreased in mice transplanted with feces from exercise mice (Fig. 3E). 

Consistent with this mRNA result, surgery increased C3ar protein (Fig. 4A, B). C3, a ligand for C3ar [23], was also increased by surgery and this increase was reduced by exercise (Fig. 4C, D). Consistent with the ideas that C3 signaling is important in immunomodulation [24] and that surgery can induce neuroinflammation [8], surgery increased ionized calcium-binding adaptor molecule 1 (Iba-1) and interleukin (IL)−6 expression in the hippocampus and exercise attenuated this increase (Fig. 4A, B, F). However, surgery and exercise did not significantly change the expression of IL-1β (Fig. 4E). These results suggest that surgery induces immune and inflammatory responses and that exercise blocks these responses. Learning and memory often require brain structural modification, such as brain cell genesis and dendritic arborization [14, 22]. Surgery reduced brain cell genesis assessed by 5′-bromo-2′- deoxyuridine (BrdU) incorporation. 

The decreased newly generated cells included glial fibrillary acidic protein (GFAP)- positive cells. Exercise attenuated this surgery effect (Fig. 5A, B). Surgery also reduced intersections among dendritic branches and spine densities. These surgical effects were attenuated by exercise (Fig. 5C, D). These results suggest that surgery impairs dendritic arborization and that exercise blocks this impairment.

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