Appropriate Exercise Level Attenuates Gut Dysbiosis And Valeric Acid Increase To Improve Neuroplasticity And Cognitive Function After Surgery in Mice Part 3

MATERIALS AND METHODS

The experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA, USA; protocol number: 3114). 

Memory is one of the abilities we often need to use in our daily lives. It can make people smarter, more sensitive, and more conducive to career and interpersonal relationships. Therefore, many people want to improve their memory. There is a relationship between experimental protocols and memory. Let's explore this issue next.

First, experimental protocols can help people improve their memory. In the experiment, people need to follow certain steps to complete the experimental operation. This step-by-step activity can put the human brain in a state of high attention and concentration, thus promoting the performance of brain functions. At the same time, you need to remember some experimental data and experimental steps during the experiment, which will also exercise the brain's memory function. By repeatedly practicing in this way, your memory will gradually improve.

Secondly, the sensory experience during the memory process is also closely related to the design of the experimental plan. In experiments, designers often set certain specific tasks during the experiment, so that participants need to repeat the same operation multiple times, such as the memory and writing of numbers, the memory and recognition of color blocks, etc. This repetitive experience can strengthen people's attention and memory abilities, making it easier for people to process this information into long-term memory.

In addition, in experiments, the use of representative stimulus materials (such as images, sounds, smells, etc.) can also produce good results. Because these stimuli can trigger the brain's excitement and memory processes, thereby improving people's memory ability. At the same time, these stimuli can also be coupled with the environment and situations in life, thereby reducing the occurrence of decoupling and making the information easier to remember and more cohesive.

To sum up, the experimental plan plays a crucial role in improving human memory. By conducting experiments, people can exercise their cognitive abilities, improve their memory levels, and at the same time promote brain function. Therefore, we should continue to learn new experimental methods and participate in more experiments to improve our memory levels and make greater contributions to our careers, relationships, and health. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

Click Know Short-term Memory how to improve

All animal experiments were performed by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80–23) revised in 2011. The sources of key materials were listed in the Key Resources Table in the Supplementary Materials.

Animals and Experimental Design

Eight-week-old male C57BL/6 J mice weighing 19–22 g were housed in a room maintained under constant environmental conditions (temperature 22–24 °C, a 12 h light/dark cycle, and 50 ± 10% humidity) with free access to food and water. 

All of them were allowed to acclimate for one week before experiments. They were randomly assigned to the following groups in the first experiment: (1) control group (not being exposed to anesthesia and surgery), (2) exercise group (exercised at 35–40% maximal capacity, but not being exposed to anesthesia and surgery), (3) surgery group (left carotid artery exposure for 15 min under isoflurane anesthesia for 2 h), and (4) to (6) Exe-l+Sur, Exe-m+Sur and Exe-h+Sur groups: exercised at 35–40%, 55–60% and 75–80% maximal capacity, respectively, and were subjected to anesthesia and surgery. 

The animals were used for learning and memory tests starting 4 days after the surgery (n = 20) or their brains were harvested for biochemical assays at 6 h, 24 h, 48 h, 72 h, 96 h, and 7 days after the surgery (n = 9 or 14), for immunofluorescent staining at 48 h and 19 days after the surgery (n = 6), and for Golgi staining at 19 days after the surgery (n = 8). 

Blood and feces from mice of the first 4 groups were harvested for measuring SCFAs at 7 days after the surgery (n = 7) and for 16 S analyses at 3 and 7 days after the surgery (n = 8) or at the end of 4-week exercise protocol (n = 8) or at the corresponding time in the first 2 groups (n = 16). Mice for tissue harvesting were different cohorts of mice that were used for learning and memory tests. In the second experiment, mice were randomly assigned to (1) the Transcontrol+Sur group and (2) the Trans-exe+Sur group. 

Mice in the first group received 500 μl fecal solution from the control group by gastric gavage and 600 μl by enema once a day for 7 consecutive days after antibiotic treatment to eliminate the native gut microbiota in the recipients. Mice in the second group received 500 μl fecal solution from exercise mice by gastric gavage and 600 μl by enema once a day for 7 consecutive days after the antibiotic treatment. Feces from the recipients were harvested 14 days after fecal transplantation for 16 S analysis. The mice were then subjected to surgery. 

Learning and memory were evaluated from 4 days after surgery (n = 15). The brain was harvested for biochemical studies as in the first experiment (n = 10). In the third experiment, mice were randomly assigned to (1) the Transcontrol group, and (2) the Trans-control+Sur group. Mice in both groups received transplantation of feces from control mice. The second group had surgery 14 days after fecal transplantation. The hippocampus was harvested 2 days after surgery to measure GDNF (n = 10). In the fourth experiment, mice were randomly assigned to (1) the control group (naïve mice that were not exposed to any experimental conditions described in this study), (2) the antibiotic group that received antibiotics for 7 days, and (3) Trans-control group that received transplantation of feces from control mice. 

Learning and memory were assessed 19 days after the completion of fecal transplantation (26 days after the completion of antibiotic treatment in the antibiotic group) (n = 12). In the fifth experiment, mice were randomly assigned to (1) the control group, (2) the Trans-control group that received transplantation of feces from control mice, and (3) the Trans-surgery group that received transplantation of feces from surgery mice. 

Fecal transplantation was performed as described in the second experiment. Feces from the recipients were harvested 14 days after fecal transplantation for 16 S analysis (n = 10). Learning and memory were assessed 4 days after the fecal sample was harvested (n = 17). In the sixth experiment, mice were randomly assigned to (1) surgery plus normal saline group that received 200 µl normal saline (NS) by intraperitoneal injection once a week for 4 weeks and then surgery, (2) exercise plus normal saline plus surgery group that received 200 µl NS by intraperitoneal injection once a week during the 4-week exercise at 35–40% maximal capacity and then surgery, (3) exercise plus valeric acid plus surgery group that received valeric acid (200 mg/kg in 200 µl) by intraperitoneal injection once a week during the 4-week exercise at 35–40% maximal capacity and then surgery. 

The injection was given on the morning of the first exercise day of the week. One additional injection was performed on the surgery day. Behavioral (n = 13) and biochemical (n = 10) outcomes were assessed as in the second experiment. In the seventh experiment, 13-week-old (weighing 23–27 g) male C57BL/ 6 J mice were randomly assigned to (1) the control group, (2) the NS group that received 4 µl NS by intracerebroventricular injections once daily for 4 consecutive days, and (3) valeric acid group that received 4 mg/kg in 4 µl by intracerebroventricular injection once daily for 4 consecutive days. The left and right cerebroventricles were injected on an alternated schedule. Learning and memory were assessed 4 days after the injection (n = 15). 

In the eighth experiment, mice were randomly assigned to (1) surgery plus dimethyl sulfoxide (DMSO) group that received DMSO by intracerebroventricular injection once daily for 4 consecutive days starting on the surgery day, (2) surgery plus C3ar antagonist group that received C3ar antagonist (SB290157) by intracerebroventricular injection once daily for 4 days starting on the surgery day, (3) exercise plus DMSO plus surgery group that received DMSO by intracerebroventricular injection once daily for 4 consecutive days starting on the surgery day after the 4-week exercise at 35–40% maximal capacity, and (4) exercise plus C3ar agonist plus surgery group that received a C3ar agonist by intracerebroventricular injection once daily for 4 consecutive days starting on the surgery day after the 4-week exercise at 35–40% maximal capacity. Behavior tests were started from 4 days after surgery as stated above (n = 15). 

In the ninth experiment, mice were randomly assigned to (1) the control group, (2) the surgery group, (3) the surgery plus GDNF group that received GDNF by intracerebroventricular injection when they had surgery, and (4) surgery plus heat-inactivated GDNF group that received heat-inactivated GDNF by intracerebroventricular injection when they had surgery. Hippocampi were harvested at 48 h after surgery for ELISA study (n = 12). After one week of acclimation, old mice (18-month-old male C57BL/6 J mice) weighing 30–36 g were used in three studies. In the first experiment, old mice were randomly assigned to (1) the control group, (2) the exercise group in which mice had a 4-week exercise at 35–40% maximal capacity, (3) the surgery group, and (4) the Exe+Sur group in which mice had a 4-week exercise at 35–40% maximal capacity before surgery. The animals were used for learning and memory tests starting 4 days after the surgery (n = 12) or their brains were harvested for biochemical assays at 48 h after the surgery (n = 10 or 12), for immunofluorescent staining at 48 h and 19 days after the surgery (n = 6), and for Golgi staining at 19 days after the surgery (n = 8). 

Blood and feces from mice of the 4 groups were harvested for SCFA measurement at 7 days after the surgery (n = 7) and for 16 S analyses at 3 and 7 days after the surgery (n = 8) or at the end of 4-week exercise protocol or at the corresponding time in the first 2 groups (n = 16). Mice for tissue harvesting were different cohorts that were used for learning and memory tests. In the second experiment, old mice were randomly assigned to (1) the Trans-Old Control+Sur group, and (2) the Trans-Old Exe+Sur group. These mice were subjected to surgery 14 days after fecal transplantation as described in the second experiment of young adult mice. Learning and memory were evaluated from 4 days after surgery (n = 10). 

In the third experiment, old mice were randomly assigned to (1) surgery plus NS group; (2) exercise plus NS plus surgery group; and (3) exercise plus valeric acid plus surgery group. These mice received exercise conditioning and valeric acid as described in the sixth experiment of young adult mice. Behavioral outcomes were tested starting 4 days after surgery (n = 11). The sample sizes (n) described above represented the number of animals that were randomized into each group/condition. These numbers of animals were the sum of at least 3 replicates of each experiment. Animals were randomly distributed into groups based on computer-generated randomization tables in each experiment. 

Maximal exercise capacity determination and exercise training

Before surgery, 8-week and 18-month-old mice were exposed to a 4-week treadmill aerobic exercise after a determination of the maximal exercise capacity of individual mice. This determination was performed in a way similar to those described before [47, 48]. Briefly, the mice were acclimated to the treadmill on the first day with initial settings of shock grid at 25 V, 0.3 mA, and 2 Hz and the speed and inclination at zero for 10 min. The treadmill speed was then increased to 10 cm/s with inclination set to 50 for 10 min. Next, the treadmill speed and inclination were increased to 15 cm/s and 100 for 5 min. 

The speed was then increased by 5 cm/s every 5 min to an average maximal speed of 70 cm/s in young mice and 60 cm/s in old mice (around 65–75 cm/s and 55–65 cm/s, respectively), as they reached the criteria for exercise-induced exhaustion. These criteria were: (1) 10 consecutive seconds on the electric grid; (2) spending >50% of the time on the grid; and/or (3) lack of motivation to manual prodding. The mouse was removed immediately from the respective lane once one or more of these criteria were met. On the next day, mice ran on the same treadmill at half of average maximal speed (35 cm/s in young mice or 30 cm/s in old mice) and 100 of inclination until they reached one of the exhaustion criteria again, and the duration of continuous exercise was recorded as the maximal exercise capacity of each mouse. After these two protocols, mice were housed separately for 30 min to avoid noticeable aggressive behavior following exercise. 

After resting for 2 days, mice assigned to exercise training groups were subjected to a 4-week protocol of forced treadmill running at 35–40%, 55–60%, or 75–80% of the maximal capacity, respectively, for 5 days a week. Mice that were shocked for >5% of the total daily exercise time in 2 consecutive days or >3% of the total time in 3 consecutive days would be excluded. 

The non-exercise groups in the same set of experiments as the exercise groups were placed on a non-moving treadmill daily for about 30–40 min. They received 10 shocks for a total of 5 s, the average amount of shocks that mice in an exercise group received each day, during their stay on a non-moving treadmill. If a set of experiments did not have an exercise group, no mice in the set of experiments received shocks.

Antibiotic treatment

To facilitate the colonization of the transplanted microbiota, recipient mice were given an antibiotic treatment once daily for 7 consecutive days to eliminate their original gut microbiota as described by us and others [11, 36]. In brief, after purgation with 10% magnesium sulfate (200 μl/10 g, twice with 3 h intervals) by gastric gavage on the first day, C57BL/6 J mice received amoxicillin/clavulanic acid (200 mg/kg), metronidazole (200 mg/kg) and cefazolin (2 g/kg) by gastric gavage and enema (in 300 μl for gastric gavage and 400 μl NS for enema) in the morning once a day for 7 consecutive days, and amoxicillin/clavulanic acid (20 mg/kg), metronidazole (20 mg/kg) and cefazolin (300 mg/kg) in 200 μl NS daily by intraperitoneal injection for the same 7 days (at 4 pm to 5 pm). To prevent bacterial cross-contamination, mouse handling and daily cage and water bottle changes were performed by technicians wearing a clean gown and gloves in a ventilated hood. Feces from these mice were harvested 24 h after the last treatment for 16 S analyses to determine the effect.

Fecal transplantation

Fresh fecal pellets collected within 30 min after bowel movement from control mice, mice immediately after the completion of the 4-week exercise, or mice 2 to 8 days after surgery were diluted with 100 mg/ml sterile saline. All fecal pellets of 7–10 donors with each experimental condition were mixed and re-suspended together in saline. Briefly, the fecal matter was vortex-mixed for 5 min and then passed through a 70 μm nylon cell strainer to remove undigested food and particulate materials. The recipient mice received 500 μl corresponding fecal solution by gastric gavage in the morning and 600 μl by enema in the afternoon from 24 h after the last dose of antibiotics for 7 consecutive days. Transplanted mice were maintained for 2 weeks after fecal transplantation before they were subjected to fecal collection for 16 S analyses and surgery.

Anesthesia and surgery

The surgery was left carotid artery exposure as we described before [6]. Briefly, mice were anesthetized with 2% isoflurane and kept spontaneous respirations with a facemask supplied with 100% oxygen during the procedure. Rectal temperature was monitored and maintained at 37 °C with the aid of a heating blanket. A 2-cm midline neck incision and soft tissue dissection with 1-cm long common artery exposure were performed without any damage to the vagus nerve after the mouse was anesthetized by isoflurane for at least 20 min. The wound was then irrigated and closed by using a 4–0 surgical suture. The surgical procedure was performed under sterile conditions and lasted around 15 min in young mice and 12 min in old mice. The total duration of general anesthesia was 2 hours. After the surgery, all animals received a subcutaneous injection of 3 mg/kg bupivacaine. No response to toe pinching was observed during the anesthesia.

Intracerebroventricular injections

As described above, some groups of mice received intracerebroventricular injection. Briefly, these mice were placed in a stereotactic head frame in the prone position. The injection site was located as: 1.00 mm mediolateral, −0.3 mm anteroposterior from Bregma, and −2.5 mm dorsoventral depth. Micethe in surgery plus GDNF group received an intracerebroventricular injection of 10 μg/kg recombinant mouse GDNF in 3 μl phosphate-buffered saline (PBS) as described in previous studies [6, 26], and mice in surgery plus heat-inactivated GDNF group received an injection of heat-denatured (5 min at 1000C) GDNF solution. Mice in surgery plus the C3ar antagonist group received 10 μg/kg SB290157 in 3 μl PBS containing 5% DMSO at 0 h, 24 h, 48 h, and 72 h after surgery. Mice in the exercise plus C3ar agonist and surgery group received 1 μg/kg C3ar agonist in 3 μl PBS containing 5% DMSO at the same time as for SB290157. Mice in a valeric acid group of the sixth experiment received 4 mg/kg valeric acid in 4 µl for 4 consecutive days.

Behavioral testing

Learning and memory were evaluated by novel object recognition and the Barnes maze test. All behavioral tests were conducted at 10:00 am−5:00 pm in a sound-isolated room. Mice used in the learning and memory tests were not used for any biochemistry studies to avoid the effects of these tests.

Novel object recognition test

As we and others described before [20, 49, 50], mice were put in an open field chamber for 5 min for habituation 4 days after surgery. The test was performed in the following way. Two of the same objects were placed at adjacent angles of the chamber on the learning day. Mice were put into the chamber with their backs turned toward the objects and allowed to explore the chamber freely for 5 min. The animal was eliminated if the total exploration time on two objects was <5 s. One of the objects was replaced by a novel object 30 seconds or 24 hours later. The mouse was put into the chamber with their backs turned toward the objects and allowed to explore for 5 min. Animal behavior was recorded by ANY-maze behavioral tracking software (Stoelting Co., IL). Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and the memorization ability of the mouse was quantified by discrimination index: DI = T2 / (T1 + T2). The DIs at 30 s and 24 hours after the training reflected the instant and long-term memory, respectively. The field was always provided with even light, and the objects and fields were cleaned with 70% ethanol after each test.

Barnes maze

Seven days after surgery, animals were subjected to the Barnes maze to test their spatial learning and memory as we previously described [6, 49]. Barnes maze is a circular platform with 20 equally spaced holes (SD Instruments, San Diego, CA). One of the holes was connected to a dark chamber that was called a target box. The test started by placing animals in the middle of the Barnes maze. Aversive noise (85 dB) and bright light (200 W) shed on the platform were used to encourage mice to find this box. After training for 4 days, their reference memory was tested on day 5 and day 12. No test was performed during the period from day 5 to day 12. The latency to enter the target box during each trial was recorded by an ANYMaze video tracking system (SD Instruments).

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