3-Methyladenine ameliorates surgery-induced anxiety-like behaviors in aged mice by inhibiting autophagy-induced excessive oxidative stress

Postoperative anxiety is a common surgical complication in older patients. Research has recently linked excessive autophagy to several neurological disorders, including anxiety. This study aimed to determine whether 3-Methyladenine (3-MA) administration reduced anxiety-like behaviors in a mouse model following abdominal exploratory laparotomy. An abdominal exploratory laparotomy model of postoperative anxiety was established using male C57BL/6 mice aged 20 months. 3-MA (6, 30, and 150 mg/ml) was administered via intracerebroventricular immediately following surgery. The mice were assessed 14 days after surgery using the marble burying, elevated plus maze tests, and local field potential recording in the amygdala. The levels of expression of phosphorylated-Akt, Beclin-1, LC3B, nuclear factor erythroid 2-related factor 2 (Nrf2)-occupied regions in NeuN-positive cells, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione (GSH) were measured at 24 h after surgery. The injection of 3-MA reversed the increased number of marbles buried, decreased time spent in the open arm, and enhanced θ oscillation power after 14 days of abdominal exploratory laparotomy. In addition, administration of 3-MA reduced the ratio of phosphorylated- to total-Akt, decreased expression in Beclin-1 and LC3B, attenuated MDA levels, and increased the ratio of Nrf2-occupied areas in NeuN-positive cells, SOD activity, and GSH levels under abdominal exploratory laparotomy conditions. 3-MA improved anxiety-like behaviors in aged mice undergoing abdominal exploratory laparotomy by inhibiting excessive autophagy-induced oxidative stress. These results suggest that 3-MA could be an effective treatment for postoperative anxiety.


Introduction
Perioperative stress may influence the hypothalamic neuropeptide system due to its physical, psychological, and emotional impacts (Manou-Stathopoulou et al. 2019). Recently, concerns have been raised about postoperative anxiety in elderly patients (Kagerbauer et al. 2019). This demographic from cortical and thalamic sensory inputs to generate fear and anxiety-related behavioral outputs (Babaev et al. 2018). The amygdala is important for the onset and development of anxiety. Lipopolysaccharide-induced neuroinflammation contributes to anxiety-like behavior by modulating neuronal plasticity in the amygdala (Zheng et al. 2021). Excessive autophagy of mitochondria in the amygdala has been reported to lead to an increase in anxiety-like behavior (Duan et al. 2021). Autophagy regulates cell death or survival based on cell types, environments, and stressors (Parzych and Klionsky 2014). Mild to moderate autophagy can ameliorate oxidative damage via degrading oxidative substances or recycling them (Lu et al. 2021). However, excessive autophagy, which leads to autophagic cell death and excessive oxidative stress, has been identified as a cause of perioperative stress following general anesthesia and surgery (Su et al. 2019). Reportedly, oxidative stress induced by anesthesia and surgery inhibits axonal regeneration and synaptic plasticity (Netto et al. 2018). Recent research suggests that excessive autophagy in the hippocampus leads to progressive neurodegeneration, neuronal loss, and cognitive and memory dysfunction in Alzheimer's disease models in rodents (Bostancıklıoğlu 2019;Guglielmotto et al. 2014;Ułamek-Kozioł et al. 2013). Besides, the inhibition of autophagy has been demonstrated to mitigate cognitive and memory dysfunction (Jung et al. 2020). As far as we know, few studies have investigated the effects of the inhibition of excessive autophagy against postoperative anxiety.
One of the key components of autophagosome formation is the autophagy-specific class III PI3K complex . 3-Methyladenine (3-MA), a dual PI3K and autophagy inhibitor, has a synergistic effect when used with anticancer medicines (Wu et al. 2010). Moreover, autophagy inhibition has been demonstrated to mitigate anxiety disorders and cognitive dysfunction. Until now, whether autophagy inhibition contributes to the alleviation of postoperative anxiety after surgical treatments in aged remains unknown. In the current work, an abdominal exploratory laparotomy model was constructed, and then it was investigated to see if 3-MA administration reduced anxiety-like behaviors in aged mice following surgical treatment. It also investigated how excessive autophagy-induced oxidative stress contributes to postoperative anxiety.

Experimental animals
The adult male C57BL/6 mice aged 20 months were provided by Charles River Co., Ltd. (Beijing, China). All aged mice were housed in a 12-hour cycle of light and darkness with a temperature of 25 °C ± 1 °C and a humidity level of 50-70%. Food and water were available to them at all times.

Grouping
Based on the random number table, mice were divided into one of the five groups listed below: (1) sham; (2) abdominal exploratory laparotomy (AE); (3) AE plus 3-MA-low dose (6 mg/ml, 2 µl) (AE + LM); (4) AE plus 3-MA-medium dose (30 mg/ml, 2 µl) (AE + MM); (5) AE plus 3-MA-high dose (150 mg/ml, 2 µl) (AE + HM) groups. 3-MA administration 312; MedChemExpress, NJ, USA) was carried out immediately following surgery through intracerebroventricular injection (i.c.v.).The dose of 3-MA was verified by a previous study and our preliminary experiment (Wei et al. 2013). Mice were first anesthetized using sevoflurane (induction 7-8%, maintenance 3-4%), then placed on a stereotaxic. After disinfection, a 2 cm incision was made in the scalp from the middle of the parietal skull, and the periosteum peeled off. Next, 2 µl of 3-MA was administered into the left lateral ventricle (0.5 mm behind the anterior fontanelle, 1 mm left to the median, 2.5 mm below the skull surface) at a rate of 0.4 µL/min using a micro infusion pump (TJ-4 A/SL0107-1 A, LongerPump, Baoding, China) immediately after model establishment. To prevent fluid reflux, the microsyringe was left in place for an extra 5 min before being removed. Since 3-MA was dissolved in normal saline, the sham and AE groups of mice received an identical volume of normal saline without 3-MA. Twelve mice in each group were selected to receive electrode implantations in the amygdala 7 days following surgery, and participate in the marble burying, elevated plus maze tests, and local field potential (LFP) recording 14 days post-surgery (n = 12). After 24 h of surgery, twelve mice in each group were sampled to assess superoxide dismutase (SOD) activity, malondialdehyde (MDA), glutathione (GSH) levels, western blot (n = 6), and immunofluorescence (n = 6) assays.

Abdominal exploratory laparotomy
In a prior investigation, an abdominal exploratory laparotomy was done on older mice to induce postoperative stress (Qiu et al. 2016). 7-8% sevoflurane was used for anesthesia induction, and 3-4% sevoflurane was used for maintenance using a face mask. A warming pad kept the mice warm from 37 to 38 °C. The procedure for abdominal exploratory laparotomy was as follows: (1) the abdomen was shaved and cleaned with 75% alcohol; (2) a 1-cm midline incision was made; (3) a sterile probe was used to gently handle the liver, spleen, colon, and stomach (10 min); (4) the small intestine, which measured about 5 cm, was exteriorized out of the peritoneal cavity, wrapped with saline gauze, and then gently kneaded with fingers (20 min); (5) the muscle and skin were stitched together with 6 − 0 Prolene; (6) in order to relieve pain, 0.2% ropivacaine (100 µl) was administered locally at the site of the incision. In the sham group, a skin and muscle incision was sutured under anesthesia. Likewise, 100 µl of 0.2% ropivacaine was used as an incision block.

Marble burying test
The marble burying test was used to assess anxiety-like behaviors in aged mice after 14 days of surgical exposure, as described by previous studies (de Brouwer et al. 2019;Jimenez-Gomez et al. 2011). Sixteen vitreous marbles were placed in a typical home cage (35 × 35 × 25 cm 3 ), which was covered with a 5 cm thick layer of clean woodchips. In onefourth of the cage, the marbles were dispersed uniformly (four rows of four), allowing the mice to avoid contact with them. Then the mouse was placed in the area facing the wall where there were no marbles and allowed to interact with the cage. The buried marbles were counted after the mice had been in the cage for 30 min. When at least 90% of a marble's surface was covered in bedding material, the marble was considered to be buried. The number of marbles buried was converted to a percentage for additional statistical analysis.

Elevated plus maze (EPM)
Following 2 h of the marble burying test, anxiety-like behaviors were further evaluated by an EPM test as described in previous studies (Alegre-Zurano et al. 2021; Knight et al. 2021). The movements of mice were captured using a video camera for 5 min after they were placed in the center of an elevated plus maze with four arms, two of which have walls and two of which do not, with walls measuring 40.5 cm in height and arms measuring 51 cm. The time spent in the open arm was analyzed using a computer tracking system obtained from XinRuan Information Technology Co., Ltd. The elevated plus maze was lifted from the ground by 75.5 cm, and 75% alcohol was utilized to block the olfactory cues.

LFP recording
After seven days of surgical exposure, aged mice were fixed on a stereotaxic frame under sevoflurane anesthesia. The bregma and posterior fontanelle were made visible by a longitudinal incision along the middle line of the cranial crest. The right amygdala was recorded following craniotomy and dura removal using an 8-channel linear silicon probe (Cat # KD-MWA, KedouBC, Suzhou, China). The stereotaxic coordinates (AP: -1.7 mm, ML: 3 mm; DV: 4.8 mm) were established using the mouse brain atlas as a guide. After 14 days of surgical exposure, LFPs were recorded while the mice explored the open arm of the elevated plus maze through a NeuroLego amplifier (Jiangsu Brain Medical Technology Co. Ltd, Nanjing, China). The signals were amplified and digitalized at 2 kHz after being filtered using a pass-band of 0.3-300 Hz. A 50 Hz notching filter was applied to the recorded LFPs to eliminate the powerline artifact. The wideband recordings underwent a 1000 Hz downsampling in preparation for LFP analysis. Neuroexplorer (Plexon Inc., Dallas, TX) software was used to analyze the data.

Measurement of total SOD activity, MDA and GSH levels
Mice were given cold heparin saline via the aorta 24 h after surgery under deep sevoflurane anesthesia. The mice were then decapitated, and their cerebral tissues were carefully removed. The cerebral tissues were dissected into coronal slices using a mouse brain slicer at intervals of 2 μm. Amygdala tissue was isolated from the surrounding cortex, according to the mouse brain atlas (Paxinos and Franklin 2001). These tissues were homogenized in 1.15% KCl for MDA measurement, while 0.9% NaCl was used for SOD and GSH analyses. Tissue samples were centrifuged with Yellow Line DI 18 basic. Centrifuge homogenates at a temperature of 4 °C for 15 min at a speed of 15,000 rpm. Before it was tested, the supernatant was collected and refrigerated at -80 °C (Gür et al. 2020). Following the manufacturer's instructions, a SOD assay kit (WST-8 method; Cat # S0103; Beyotime Institute of Biotechnology), an MDA assay kit (Cat # S0131; Beyotime Institute of Biotechnology), and a GSH kit (Cat # A005, Jiancheng, Nanjing, China) were used to measure the total SOD activity, the levels of MDA, and GSH in the amygdala.

Immunofluorescence
Mice were perfused with heparin saline and 4% paraformaldehyde via the aorta after receiving deep anesthesia with sevoflurane following 24 h of surgery. Their brain tissue, the amygdala, was vitrified and then embedded in paraffin after being dried in a succession of ethanol solutions of increasing strength. A 5-µm thick slice of paraffin was then cut and dewaxed. After being rehydrated with alcohol, the slices underwent a 30 min incubation with 0.1% Triton X-100 (Cat # T8200, Solarbio, Bejing, China) and a 1 h sealing procedure with 5% standard bovine serum (Cat # A8010, Solarbio) at a temperature of 25 °C. After rinsing with PBS, monoclonal mouse anti-NeuN (Cat # ab177487, 1:200; Abcam, Cambridge, UK), polyclonal rabbit anti-Nrf2 (Cat # was used to compare the groups, and Turkey's post hoc tests were performed to investigate the groups further. P < 0.05 was considered statistically significant.

The administration of 3-MA reduces anxiety-like behaviors induced by exploratory abdominal laparotomy
Marble burying and EPM tests were typically used to assess the anxiety-like behaviors in aged mice fourteen-day postsurgery ( Fig. 1a-f). According to our findings, old mice that underwent abdominal exploratory laparotomy spent less time in the open arm and buried more marbles than mice who underwent sham surgery (vs. sham, P < 0.0001; Fig. 1c, f). In addition, aged mice in the AE + MM and AE + HM groups spent more time in the open arm compared to the AE group (AE + MM vs. AE, P < 0.0001; AE + HM vs. AE, P < 0.0001; Fig. 1c). However, treatment with a low dose of 3-MA did not affect digging behavior or time spent in the open arm. The speed of mice in the five groups did not differ significantly (Fig. 1d). Additionally, it was revealed that medium (vs. AE, P < 0.0001; Fig. 1f) and high-dose (vs. AE, P < 0.0001; Fig. 1f) doses of 3-MA treatment reduced the digging behaviors following surgical exposure. In conclusion, a higher dose of 3-MA was not superior to a medium dose of 3-MA in reversing the anxiety-like behaviors induced by abdominal exploratory laparotomy. Therefore, the subsequent phase involved additional research on medium-dose.

3-MA administration mitigates θ oscillation power in the amygdala after surgery
The LFPs of aged mice were recorded while they explored an EPM arena attached to the acquisition device. In the frequency ranges of δ (2-6 Hz), θ (6-12 Hz), β (15-30 Hz), and γ (35-70 Hz), spectral analysis of LFPs of mice indicated distinct oscillatory activity patterns (Fig. 2a). When LFP power in different frequency ranges was compared, it became evident that exposure to the open arm caused a significant modification in amygdala activity, which has been suggested to play a crucial role in mediating anxiety behaviors (Fig. 2b). Consistent with these findings, surgerytreated mice, exhibited a substantial increase in LFP power in the theta frequency range in the EPM compared to shamtreated mice (vs. sham, P < 0.0001; Fig. 2c), whereas 3-MA partially rectified these changes in θ oscillation power (vs. AE, P < 0.0001; Fig. 2c). Under oxidative stress condition, an imbalance of excitation/inhibition leads to a significant ab76026; Abcam), and LC3 (Cat # ab192890; Abcam) were used as primary antibodies. Similarly, a secondary antibody was created using Cy3-conjugated goat anti-mouse IgG (Cat # A0521, 1:1000; Beyotime, Shanghai, China) and FITC goat anti-rabbit IgG (Cat # A0562, 1:1000; Beyotime). Following three successive PBS washouts, the latter infiltrated the slices for 1 h. The nuclei were labeled using 4′, 6-diamidino-2-phenylindole (DAPI) (10 µg/ml, Cat # C1002, Beyotime). Imaging was carried out using a fluorescent microscope (Eclipse 50, Nikon, Japan). From each group, six fields with a magnification of 400 in three slices were chosen at random. In order to examine the intensity of LC3B and Nrf2-occupied regions in NeuN-positive cells, Image-Pro plus 6.0 (NIH, Bethesda, MD, USA) was utilized.

Statistical analysis
All mice were randomly allocated to various groups in this study, and none were eliminated. Previous research has demonstrated that 3-MA has a 50 to 60% response rate against neurological impairment (Zhao et al. 2019). A power of 0.05 and 0.8 was obtained overall by predetermining sample sizes (n = 6 for each group). GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) was used to analyze the data presented as mean and standard deviation (SD). One-way analysis of variance (ANOVA)

3-MA administration mitigates oxidative stress in the amygdala after surgery
A growing body of evidence indicates that excessive autophagy is related to oxidative stress, which promotes the development of anxiety (De et al. 2017;Wang et al. 2021). MDA levels in the amygdala region were significantly higher in the AE group following surgery than in the sham group (vs. sham, P < 0.0001; Fig. 4b). However, after administration of 3-MA following surgery, MDA levels reduced considerably (vs. AE, P = 0.0003; Fig. 4b). SOD activity and GSH levels are essential for brain anti-oxidative stress ). According to the study, surgical exposure significantly increased both SOD activity (compared to sham, P = 0.0065; Fig. 4a) and GSH levels (vs. sham, P = 0.0398; Fig. 4c). In contrast, in the AE + MM group as compared to the AE group, 3-MA enhanced SOD activity (vs. AE, P < 0.0001; Fig. 4a) and GSH levels (vs. AE, P < 0.0001; Fig. 4c). alteration in LFP, which may be associated with metabolic disorders of GABA and glutamate (Cuenod et al. 2022). Studies have shown enhanced θ oscillation power in the region of amygdala when mice exhibit anxiety-like behaviors, which is consistent with our findings (Cruces-Solis et al. 2021;Jacinto et al. 2016).

3-MA administration mitigates excessive autophagy in the amygdala after surgery
The ratios of phosphorylated-to-total Akt and Beclin-1 were significantly higher in surgically treated mice than in the sham group after 24 h of surgery (vs. sham, P < 0.0001; Fig. 3a-c). However, 3-MA administration significantly reduced the ratio of phosphorylated-to total-Akt (vs. AE, P < 0.0001; Fig. 3b) and Beclin-1 (vs. AE, P = 0.0001; Fig. 3c) in mice exposed to abdominal exploratory laparotomy plus 3-MA compared to abdominal exploratory laparotomy alone. In addition, immunofluorescent staining of LC3B confirmed that surgical exposure significantly enhanced autophagy activation, confirming that the neuroprotective effect of 3-MA was related to the inhibition of excessive autophagy (vs. sham, P < 0.0001; Fig. 3d, amygdala, triggered excessive autophagy, and elevated oxidative stress. However, therapy with 3-MA reduced anxiety-like behaviors induced by abdominal exploratory laparotomy and prevented excessive autophagy and oxidative stress. Overall, this study revealed that 3-MA might aid in preventing postoperative anxiety by reducing excessive autophagy. According to other research, an abdominal exploratory laparotomy may have caused a considerable decline in cognition, directly related to an inflammatory response and oxidative stress (Muscat et al. 2021;Tang et al. 2022). In the present study, collected data revealed that surgical exposure was related to postoperative anxiety, which is consistent with a previous investigation ). In addition, our research also showed that an abnormal increase of θ oscillation power in the amygdala is related to anxiety-like behaviors in mice (Cruces-Solis et al. 2021). Numerous studies have demonstrated that abnormalities in synaptic inhibition Under oxidative stress, Nrf2, which is believed to be a master switch governing the cellular redox status, translocates from the cytoplasm to the nucleus. Our data revealed that the proportion of NeuN-positive cells occupied by Nrf2 was greater after surgical exposure than after sham therapy (vs. sham, P = 0.0001; Fig. 4d, e), whereas 3-MA further upregulated Nrf2-occupied areas in NeuN-positive cells in mice exposed to both AE and 3-MA (vs. AE, P < 0.0001; Fig. 4d, e).

Discussion
This study demonstrated that abdominal exploratory laparotomy induced anxiety-like behaviors in aged mice, including increased digging behaviors and decreased time spent in the open arm. In addition, abdominal exploratory laparotomy increased the power of θ frequency range in the oxidants such as SOD to MDA was decreased in the model of abdominal exploratory laparotomy, which is consistent with our findings (Wu et al. 2022). These results revealed that abdominal exploratory laparotomy induced anxietylike behaviors in elderly mice, possibly resulting from oxidative stress in the amygdala.
Autophagy refers to a dynamic process of self-digestion that manages the metabolic equilibrium of cells (Glick et al. 2010). Previous research demonstrated that excessive oxidative stress contributes to the pathology of increased protein and lipid oxidation induced by autophagy activation (Luo et al. 2020;Varga et al. 2015). Thus, autophagy is delicately regulated in the brain, and autophagy activity is dynamically balanced in neurological diseases (Cao et al. 2019). Excessive autophagy aggravates oxidative stress and neuronal injuries, thereby worsening neurological outcomes . In this study, we discovered that activated autophagy was characterized by increased expression cause excessive amygdala activity, which may encode the observed excessive avoidance responses (Gründemann et al. 2019). According to reports, oxidative stress, a biological process caused by an imbalance between reactive oxygen species and antioxidants, alters the neuronal circuitry of the amygdala and induces anxiety (Salvi et al. 2020). Reportedly, the molecular basis for neuropathologic alteration in the synapsis is associated with oxidative stress (McCoy et al. 2019;Vollert et al. 2011). Our recent findings revealed that neuropathologic changes, including aggravated oxidative stress in the amygdala, were induced by abdominal exploratory laparotomy. It has been reported that there is a significant decrease of antioxidants, including SOD and GSH, in the brain of aged mice compared with young mice (Al-Amin et al. 2015). Moreover, it was reported that antioxidants, including SOD and GSH, are increased after abdominal exploratory laparotomy, subsequently mitigating oxidative stress. However, the ratio of antioxidants and

Declarations
Competing interests The authors declare that they have no competing interests.

Statement of ethics
All animal studies were carried out under the National Institute of Health's Guideline for the Care and Use of Laboratory Animals. The Animal Review Board also approved the protocols involving animals of the First Hospital of Qinhuangdao (2021Q085).

Consent to participate Not applicable.
Consent to publish Not applicable. of phosphorylated Akt and Beclin-1 and upregulation of LC3B intensity. 3-MA is frequently employed to prevent autophagy by inhibiting class III PI3K. Our data revealed that 3-MA administration after surgical exposure reduced the phosphor-Akt ratio to total Akt, decreased expression of Beclin-1, downregulated LC3B intensity, and reduced oxidative stress in the amygdala. The excessive autophagy induced by oxidative stress is reportedly associated with the development of anxiety in a mouse model of chronic social stress (Duan et al. 2021). In the current study, we also observed that 3-MA ameliorated anxiety-like behaviors following surgery. These findings suggest that postoperative anxiety results from increased autophagy-associated oxidative stress.
There are many flaws in the current study that should be noted. Firstly,we only assessed the effects of 3-MA on postoperative anxiety in male mice; further research should be conducted on female mice. In addition, our data revealed that a single dose mitigated postoperative anxiety-like behaviors in mice and maintained it for 14 days, but the effects of multiple administration against anxiety-like behaviors should be further explored. Besides, further research should focus on postoperative cognitive dysfunction and depression-like behavior in aged mice. Furthermore, it has been reported that the other regions, including hippocampus, bed nucleus stria terminalis (BNST), and nucleus accumbens (NAc), are implicated in the process of anxiety (Jimenez et al. 2018;Salimando et al. 2020;Zhang et al. 2020). The other regions that are associated with anxiety behaviors should be further investigated.
In conclusion, treatment with 3-MA reduces the anxietylike behaviors observed in elderly mice following abdominal exploratory laparotomy. The mechanism involves the suppression of excessive autophagy-induced oxidative stress. Although 3-MA has promising therapeutic potential for treating postoperative anxiety, additional study is required to comprehend the underlying mechanisms fully.
Statements and Declarations. Supplemental Table. Details of statistics.