Prolonged stress response induced by chronic stress and corticosterone exposure causes adult neurogenesis inhibition and astrocyte loss in mouse hippocampus

Chronic stress is a pervasive and complex issue that contributes significantly to various mental and physical health disorders. Using the previously established chronic unpredictable stress (CUS) model, which simulates human stress situations, it has been shown that chronic stress induces major depressive disorder (MDD) and memory deficiency. However, this established model is associated with several drawbacks, such as limited research reproducibility and the inability to sustain stress response. To resolve these issues, we developed a new CUS model (CUS + C) that included exogenous corticosterone exposure to induce continuous stress response. Thereafter, we evaluated the effect of this new model on brain health. Thus, we observed that the use of the CUS + C model decreased body and brain weight gain and induced an uncontrolled coat state as well as depressive-like behavior in adult mice. It also impaired learning memory function and cognitive abilities, reduced adult hippocampal neurogenesis as well as the number of hippocampal astrocytes, and downregulated glial fibrillary acidic protein expression in the brains of adult mice. These findings can promote the utilization and validity of the animal stress model and provide new information for the treatment of chronic stress-induced depressive and memory disorders.


Introduction
Stress is a ubiquitous phenomenon that can bring about numerous physiological and psychological changes in human daily life.Organisms undergo stress when they encounter situations that may endanger their lives, and as a result, various physiological processes are initiated in these organisms to maintain survival and homeostasis against stress (Herman, 2013;O'Connor et al., 2021).Even though organisms are capable of stress adaptation, chronic stress may exert detrimental effects on behavioral and neurobiological function (de Kloet et al., 2005).Based on a study on subjects aged 18-23 years, Eganov et al. showed the existence of a significant correlation between mental disorders and socially stressful conditions (Eganov et al., 2020).It has also been confirmed that patients with various mental diseases, such as depression and post-traumatic stress disorder have higher levels of cortisol, a stress hormone, in their hair than normal individuals (Dettenborn et al., 2012;Steudte et al., 2011).Another study showed high levels of cortisol in the hair of young adults who had suffered from unfortunate experiences within the last 3 months (Karlén et al., 2011).These studies suggest that there exists an association between stress and mental disorders.Thus, studies to clarify the effect of stress on brain health and on the treatment of stress-induced mental illnesses are required.
Chronic unpredictable stress (CUS), which was first proposed in 1982 for the systematic exploration of the neurobiological foundation of major depressive disorder (MDD), is one of the most vigorous and variable animal stress models (Katz, 1982).Its utilization has enabled the examination of stress-induced neurobiological modifications in the central nervous system (CNS) as well as the evaluation of the pharmacological efficacy of antidepressants and their potential candidates (Chen et al., 2022;Li et al., 2023;Yalcin et al., 2008;Zhong et al., 2019).Of recent, it was reported that the use of the CUS model reduces neuronal firing rate and elevates inhibitory synaptic input in agouti-expressing neurons of the arcuate nucleus, which is involved in appetite and metabolism regulation as a response to an energy deficiency state (Fang et al., 2021).Additionally, the use of the CUS model hyperactivates lateral habenula neurons projecting on ventral tegmental area neurons leading to depressive-like behavior (Cerniauskas et al., 2019).While the flexibility of this model has facilitated studies on the impact of stress on brain health in organisms, the development of the novel CUS model that can trigger continuous stress response is necessary considering the few inherent limitations of the CUS model, namely: (1) insufficient reproducibility, (2) difficulty maintaining stress response for an extended period, and (3) individual variation of stress perception and susceptibility (Franklin et al., 2012;Markov and Novosadova, 2022;Monteiro et al., 2015).
The hypothalamic-pituitary-adrenal axis is a prominent neuroendocrine pathway that is involved in maintaining homeostasis and managing response to stress in mammals.Corticosterone, a glucocorticoid hormone in rats and mice, is synthesized in the adrenal cortex via stimulation by adrenocorticotropic hormone produced by corticotrophin-releasing hormone and vasopressin in the median eminence (Dallman et al., 1987;Matthews and Challis, 1997).Further, numerous studies in which exogenous corticosterone was administered to cellular and animal models to examine the impact of elevated corticosterone levels on mammalian health have been conducted.These investigations confirmed the wide-range effects of corticosterone on metabolism, immune response, the reproductive system, and the CNS (Kaiser et al., 2015;Koo et al., 2018;Shini and Kaiser, 2009;Zaytsoff et al., 2019).However, the effect of simultaneous exposure to stress and exogenous corticosterone on brain health has not yet been fully investigated.
Therefore, in this study, we established a new CUS model that includes exposure to exogenous corticosterone (CUS+C) and used it to investigate the impact of stress and exogenous corticosterone coexposure on the brain.Our results indicated that the use of the CUS+C model influenced physical conditions and induced behavioral defeat (depression and memory deficit) in adult mice.Its use also inhibited hippocampal neurogenesis, triggered hippocampal astrocytic loss, and downregulated GFAP expression in adult mice brains.Taken together, our CUS+C model represents as a novel model of MDD that induces memory impairment by inhibiting neurogenesis and astrocyte homeostasis in the hippocampus of adult mice.

Animals
Specific pathogen-free adult ICR male mice (age, 4.5 months; weight, 35-40 g) were obtained from Samtaco (Osan, Gyeonggi, Korea) and acclimated in a mouse breeding facility for 2 weeks prior to subjection to stress.Further, the mice were housed under standard conditions as follows: number of mice per cage, 3-4; light-dark cycle, 12-h/12-h; humidity, 55%; temperature, 22 • C. Water and mouse chow diet provided ad libitum (control,n = 20;CUS+C,n = 19).The Institutional Animal Care and Use Committee of Pusan National University (IACUC) approved all the experimental protocols, and all the experiments were conducted in accordance with relevant guidelines and regulations.

Chronic unpredictable stress paradigm with exogenous corticosterone exposure (CUS+C)
The stress paradigm was applied as previously described with minor modifications (Burstein and Doron, 2018;Du Preez et al., 2021;Woodburn et al., 2021).Mice in the CUS group were exposed to diverse stress conditions, including physical restraint, empty cage, tilting cage, cage rotation, rearing in a cage with damp bedding, food and water deprivation, and light/dark cycle disruption for 12 weeks.To induce continuous stress response, mice in the CUS+C group were administered corticosterone solution (C2505, 35 mg/L; Sigma-Aldrich, St. Louis, MO, USA) diluted with 0.45% hydroxypropyl-b-cyclodextrin (H0979, TCI chemicals, Tokyo, Japan) instead of drinking water during the stress exposure period (Choi et al., 2021).The control mice received 0.45% hydroxypropyl-b-cyclodextrin solution as a vehicle.The experimental timeline for CUS+C is shown in Fig S1.

Coat state assessment
The coat state was estimated as previously described (Nollet et al., 2013).Seven mouse body areas (head, neck, back, abdomen, fore and hind paws, and tail) of the coat state were evaluated by an experimenter blinded to experimental groups.The coat score was assigned based on the following criteria: Score 0 = smooth and shiny fur without spiky spots, Score 0.5 = downy fur with some spiky spots, and Score 1 = dirty and unkempt fur with spiky spots.The total coat score was calculated by combining the individual scores of seven body areas.

BruU staining
The brain tissue sections, including hippocampus tissue, were fixed in 4% PFA for 10 min at room temperature (RT).After permeabilization with PBS containing Triton X100, the tissue sections were incubated in 1 M HCl (Daejung Chemicals & Metals Co., Ltd, Gyeonggi-do, Korea) for 30 min at RT.This was followed by neutralization using 0.1 M borate buffer (Sigma-Aldrich) for 15 min, after which the sections were blocked with PBS for 1 h.Then to realize BrdU immunolabeling, the tissue sections were incubated overnight at 4 • C in the appropriate primary BrdU antibodies (cat.no.555627; dilution, 1:1000; Bioscience, Durham, NC, USA).This was followed by incubation in a secondary antibody solution (Alexa Fluor594 goat anti-mouse, cat.no.A11032; dilution, 1:1000; Invitrogen) containing DAPI for 1 h.Finally, the tissue sections were mounted in Fluoro-Gel, and fluorescently labeled cells were visualized via fluorescent microscopy (Thunder Imager 3D Assay; Leica Microsystems GmbH, Wetzlar, Germany).

Western blot analysis
The total protein content of the brain tissue samples was evaluated using a Pro-prep solution (iNtRON, Seoul, Korea) according to the manufacturer's protocol.Briefly, using 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, 70 μg of protein was resolved from the tissue samples and transferred into a polyvinylidene fluoride membrane (Merck Millipore) as previously described (Tran et al., 2020).Next, the membrane was incubated overnight with the primary antibody (GFAP, cat.no.ab53554 dilution, 1:1000; Abcam) and secondary antibody (anti-goat, cat.no.31402; dilution, 1:5000; Invitrogen).Then, to enhance the membranes, we used a chemiluminescence reagent (EMD Millipore Corporation, Burlington, MA, USA), and the optical density of the target band was detected using the Chemi Doc equipment, Lumi-noGraph (Atto Corporation, Tokyo, Japan), and images obtained were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Behavioral analysis
Mice were randomly selected for behavioral testing as previously described (Jung et al., 2017).All the behavioral tests were conducted during the light cycle.On the test days, the mice were transferred to the test room at least 30 min before the test was commenced.Further, the testing was conducted by laboratory technicians blinded to the mouse group information.All the experiments were conducted between 8:00 AM and 6:00 PM, and a rest period of 2 d per week was provided between two consecutive tests.Additionally, all the experimental areas were cleaned using 70% ethanol before and after each test.

Tail suspension test
Each mouse was suspended on the edge of a shelf 50 cm above the surface of a table.Thereafter, the mouse was allowed to move for 6 min while a camera recorded its behavior.The videos obtained were then analyzed using EthoVision XT16 software (Noldus, Leesburg, VA, USA).The duration of immobility over a period of 5 min was recorded.

Forced swimming test
Each mouse was gently placed in a glass cylinder (height, 20; diameter, 15 cm) filled with water (25 ± 2 • C) to a depth of 12 cm, and forced to swim for 5 min.Then, the duration of immobility was recorded using a camera, the videos obtained were analyzed using EthoVision XT16 software (Noldus).

Novel object recognition
First, the mice were placed in an open field arena with two identical objects (2 × 5 × 9 cm) and allowed to freely explore the objects for 10 min.After 6 h, one of the objects was replaced with a novel object having a different shape and color.The mice were then allowed to freely explore the objects for another 10 min.The time spent by the mice interacting with the new and old objects (sniffing or exploring within 2 cm of the object) was recorded.The videos obtained were then analyzed using EthoVision XT16 software (Noldus).

Puzzle box test
To assess mouse memory function and learning ability, we performed the puzzle box test as previously described with minor modifications (Ben Abdallah et al., 2011;O'Connor et al., 2014).The puzzle box arena consisted of two compartments with a bright field area (600 × 280 × 250 mm) and a dark goal box area (150 × 280 × 250 mm) with a narrow underpass (40 × 40 mm).The mice underwent three trials (T) per day for 3 days and were challenged to reach the goal box area by passing a narrow underpass with obstacles that made the task harder every day: Day 1 (T1 -open door underpass; T2, 3 -closed door underpass), Day 2 (T4 -closed door underpass; T5, 6 -underpass filled with sawdust), Day 3 (T7 -underpass filled with sawdust; T8, 9 -underpass blocked with tissue plug).The time limits on T1-7 and T8, 9 were and 240 s, respectively.Each session allowed the assessment of hippocampal-dependent memory ability as follows: T5, 8problem-solving ability; T3, 6, 9 -learning/short-term memory; and T4, 7 -long-term memory (Cowan, 2008;Shepard et al., 2017).The latency time to arrive the goal zone was also noted.

Rotarod test for motor learning ability
Using an accelerating rotarod machine (Scitech Korea Inc., Seoul, Korea) the rotarod test was performed by placing mice on rotating drums (3 cm in diameter).The speed of the rotarod was increased from to 40 rpm within 5 min.Three trials were conducted each day for a period of over 10 d.Further, a recovery time of at least 20 min was allowed between trials.The residence time of the mice on the rotarod machine was also recorded.

Statistical analysis
All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).To compare two independent groups, two-way analysis of variance (ANOVA) and unpaired Student's t-test were performed.Further, significant differences in body weight between groups were determined by performing repeated measures two-way ANOVA.The results were presented as mean ± standard error of the mean, and the p-values for each comparison are provided with the figure legends.Each experiment was performed in a blinded and randomized manner.The animals were randomly assigned to different experimental groups, and data were collected and processed randomly.The allocation and application of stress conditions, and handling of the animals were similar across the study groups.Additionally, all the experiments were independently performed in triplicates.

CUS+C induced changes in adult mice body and brain weights and coat state
Stressful conditions have detrimental effects on the health of organisms and affect their physical, mental, and pathological functioning (Chrousos, 2009).In this study, we first assessed changes in mouse physical condition owing to the CUS+C model.Thus, we observed a significant decrease in body weight in the CUS+C group compared to the control group (Fig. 1A, B).We also observed a significantly smaller brain weight in the CUS+C group than in the control group (Fig. 1C, D).Additionally, the CUS+C group exhibited an irregular and unregulated coat state, showing a score that was significantly higher than that observed for the control group (Fig. 1E, F).These findings indicated that the CUS+C model negatively affected body and brain growth and inhibited self-care behavior in the test adult mice.

CUS+C induced depressive-like behavior and memory dysfunction in adult mice
Forced swimming and tail suspension tests were performed to determine whether the CUS+C model induced depressive-like behavior in mice.Notably, the forced swim test showed a significantly higher immobility time for mice in the CUS+C group than for those in the control group (Fig. 2A).Likewise, in the tail suspension tests, we observed a significantly higher immobility time for the CUS+C mice than for the control mice (Fig. 2B).These results indicated that our CUS+C model possibly induced depressive-like behavior in adult mice similar to the previously established CUS model.
Reportedly, depression is strongly associated with memory impairment (Burt et al., 1995;Dillon and Pizzagalli, 2018).To analyze memory function in each mice group, we performed puzzle box and rotarod tests and thereafter, compared cognitive flexibility and learning ability between groups.The results of our puzzle box test showed that the latency time for reaching the goal box was significantly higher in the CUS+C group than in the control group on T5, 7, 8, and 9 (Fig. 2C).Additionally, in all the trials in the rotarod test, we observed that the CUS+C mice dropped off the rotating drum significantly faster than the control mice (Fig. 2D).Additionally, we performed another test to evaluate differences in recognition memory performance between the control and CUS+C mice.To this end, the mice were exposed to two identical objects for 10 min, and after 6 h, one of the objects was replaced by a novel object.The mice were then allowed to observe the two objects for another 10 min.For the control group, we observed a higher exploration time and proximity to the novel object than to the familiar object; however, for the CUS+C group, there was no significant difference in exploration time and proximity between the two objects (Fig. 2E-G).These results suggested that our CUS+C model negatively affected cognitive function and memory-learning ability as well as motor learning function in adult mice.

CUS+C decreased adult hippocampal neurogenesis in adult mouse brains
Various types of stressors bring about changes in behavioral response to stress and the neuroendocrine system by affecting neurogenesis (Korosi et al., 2012;Levone et al., 2015).Moreover, it is well known that Fig. 2  the hippocampus is a brain region that is vulnerable to stress (McEwen et al., 1992;McEwen and Sapolsky, 1995).We evaluated change of hippocampal neurogenesis owing to the CUS+C model via immunofluorescence using Brdu and Ki-67 antibodies.The number of Ki-67-positive cells in the dentate gyrus of mice in the CUS+C group was remarkably lower than that in the dentate gyrus of mice in the control group (Fig. 3A, B).Similarly, the CUS+C group showed a significantly lower number of BrdU-positive cells in the dentate gyrus than the control group (Fig. 3A, C).These results indicated that our CUS+C model negatively affected progenitor recruitment in the hippocampus of adult mice brain.

CUS+C significantly reduced hippocampal astrocyte number in adult mice brain
An imbalance in brain cellular homeostasis may result in behavioral disruptions, such as depression and memory issues (Duman, 2009;Perry et al., 2019).In this study, we compared the density of brain-derived cells (neurons, astrocytes, and microglia) in the cortical area and hippocampus of mice in the different groups.First, we did not observe any significant difference between the control and CUS+C groups with respect to the number of NeuN-positive cells in the mice brain hippocampal subregion (Fig. S2A-E).Specifically, the number of NeuN-positive cells in the cortical region was the same for both groups (Fig. S2F, G).Similarly, there was no significant difference between the two groups with respect to the number of IBA1-positive cells in the hippocampus and cortical area (Fig. S3A-G).However, GFAP-positive cell number was significantly lower in the hippocampus region of CUS+C mice than in the hippocampus region in the control mice; no such difference was observed in the cortical area (Fig. 4A-E and Fig. S4A, B).Additionally, GFAP protein expression level was significantly reduced in the brain of CUS+C mice compared to its level in the brain of the control mice (Fig. 4F, G).These results suggested that our CUS model induced hippocampal astrocyte loss in adult mice brains.

Discussion
Considerable progress has been made regarding the exploration of the neurobiological factors that contribute to the behavioral symptoms associated with psychiatric disorders.Research based on several animal stress models spanning a period of 60 years has provided evidence that stress can modify the behavior, physiology, and neuropsychiatry of organisms (Patchev and Patchev, 2006).Among the diverse animal stress models reported so far, CUS as a model of MDD has provided insights into the impact of stressful stimuli on neuropsychiatric functioning based on the partial simulation of pathological phenomena and atypical behavior owing to stress.Nonetheless, recent studies have indicated that there has been no substantial progress in the investigation of new MDD treatment approaches using animal stress models.Moreover, the CUS model is limited in terms of reliability and reproducibility (Antoniuk et al., 2019;Planchez et al., 2019), and the threshold for stress response that translates physiological effects into harmful consequences varies according to an individual's neuroendocrine and genetic factors (Franklin et al., 2012).These previous reports suggest the need for the development of a novel CUS model that can deliver robust and sustained stress responses in experimental animals to enhance the reproducibility of research associated with stress and MDD.Therefore, in this study, we developed the CUS+C model, a new CUS paradigm, that combines diverse stressful conditions and exogenous corticosterone exposure, and thereafter, evaluated its effects on mouse brain homeostasis.
Patients with depression often face more challenges with self-care than their non-depressed counterparts owing to their feelings of hopelessness and lethargy (Ludman et al., 2013).Further decrease in body weight gain is a distinctive feature of the effects of chronic stress and major depression in humans (Nollet et al., 2013).It has also been shown that the degeneration of the mouse coat state and a depressive-like behavior are representative physical and behavioral hallmarks, respectively, in stress-susceptible animals (Frisbee et al., 2015;Teng et al., 2021).In a previous study, the subjection of 7-week-old young adult mice to CUS induced body weight loss and worsened mouse coat state (Surget et al., 2009).Another study showed that corticosterone administration to C57BL/6 N mice worsened coat condition and depressive-like behavior (Sturm et al., 2015).In this study, the CUS+C model disrupted normal body and brain weight gain and worsened coat state as well as depressive-like behavior in adult male mice.These results indicated that the CUS+C model negatively influenced physical condition, brain health, and self-care capacity in adult mice; thus, can be employed as a stress-related animal model for MDD.
There is a significant relationship between depression and memory deficits.Individuals with depression display memory impairment as well as a propensity for remembering more unfortunate events than healthy individuals (Dillon and Pizzagalli, 2018).It has also been observed that memory impairment may intensify depressive symptoms more severely (Sumner et al., 2010).Studies involving experimental animals have shown that stress impairs memory function and cognitive flexibility (Stevenson et al., 2009;Zhang et al., 2017).Therefore, based on these reports, we investigated whether the CUS+C model could induce memory disability by performing various behavior tests to evaluate memory function.Our results revealed that CUS+C impaired cognitive, learning as well as motor learning memory.According to a previous study, predictable chronic mild stress (stress exposure at a fixed time) elevates learning and memory abilities as well as hippocampal neurogenesis (Parihar et al., 2011).Another study revealed that rats exposed to predictable chronic mild stress conditions (e.g., 5 min restraint stress for a period of 28 d) show resistance to depressive and anxiety behaviors induced by CUS (Suo et al., 2013).These results suggest that the induction of adverse behavioral effects by stress necessitates persistent stress response that prevents adjustment by animals.Thus, incorporating exogenous corticosterone exposure into the general CUS model may help prevent stress adaptation by keeping the mice in a consistent stress response state.
In the dentate gyrus, granule cell neurogenesis continues for an extended period, from the embryonic stage to adulthood (Gould and Tanapat, 1999).It has also been observed that in the dentate gyrus in adults, new neurons contribute to functional circuit maturation by providing considerable neuroplasticity in the hippocampal circuit (Song et al., 2012).Tomohisa et al. suggested that adult neurogenesis of the dentate gyrus is highly regulated by environmental factors, implying that stressful events may influence this process (Toda et al., 2019).Moreover, it has been observed that the process of neurogenesis in the hippocampus of adults is related to cognitive function and memory performance (Deng et al., 2010).Adult rats exposed to early life stress have also been shown to exhibit a decrease in hippocampal neurogenesis and the deterioration of spatial learning ability (Oomen et al., 2010).Our findings are consistent with these reports and suggest that the inhibition of adult hippocampal neurogenesis by the CUS+C model may contribute to cognitive dysfunction and memory impairment.
Astrocytes, the most abundant glial cells in the CNS, display a complex and multifaceted morphology with protoplasmic and fibrous subtypes.They have also been identified as crucial players in diverse neurologic functions.For example, they (1) contribute to the integrity of the blood-brain barrier and fluid balance, (2) regulate synaptic transmission and plasticity by participating in neurotransmitter cycling and clearance; (3) ensure immune regulation by releasing pro-and antiinflammatory factors; and (4) protect neurons from oxidative stress and neurotoxicity (Haim and Rowitch, 2017;Khakh and Sofroniew, 2015;Sofroniew, 2020).In this study, the CUS+C model induced astrocytic loss in the hippocampal regions of adult mice but did not bring about neuronal and microglial loss.Additionally, the CUS+C model downregulated the protein expression level of GFAP, a typical astrocyte marker, in mice brains.Although studies with results consistent with those of this study have been reported, our findings are debatable because other studies have suggested that stress exposure increases astrocytic density and alters their morphology in the mouse hippocampus (Du Preez et al., 2021;Virmani et al., 2021).Previous studies have also shown that the activation of glucocorticoid signaling by corticosterone hinders astrocyte proliferation as well as the expression of the astrocytic growth factor gene in the hippocampus (Yu et al., 2011;Zhang et al., 2015).Therefore, we reasoned that the observed hippocampal astrocyte depletion induced by the CUS+C model in our study could be attributed more to the biological effects of exogenous corticosterone exposure than to stress conditions.Additionally, studies on the role of astrocytes in the hippocampus have shown that hippocampal astrocytes play an important role in memory formation and maintenance H.S. Shin et al. (Kol et al., 2020;Zhou et al., 2021).Taken together, our results indicated that a decline in hippocampal astrocyte number owing to the CUS+C model possibly induced memory dysfunction in adult mice.
The novelty of our study is that we uncovered the impacts of sustained stress response on the brain of adult mice by incorporating exogenous corticosterone exposure in the established CUS model.To the best of our knowledge, this is the first report on the combined effects of CUS conditions and exposure to exogenous corticosterone.Further, using the CUS+C model, we observed a decrease in hippocampal neurogenesis and hippocampal astrocyte number in adult mice.However, to expand understanding regarding the CUS+C model, in future, it would be necessary to explore the mechanism by which the model suppresses hippocampal cell proliferation and leads to astrocytic cell loss in the hippocampus.In conclusion, the CUS+C model may contribute to enhancing comprehension regarding the pathogenesis of MDD and memory disorder caused by chronic stress and provide new insights into MDD and stress-induced memory defect therapy.

Conclusions
In this study, our results showed that subjecting adult mice to the CUS+C model resulted in diminished body and brain growth, degeneration of the coat state, and depressive-like behavior.Additionally, the use of this model disrupted learning memory, motor learning memory, and cognitive ability in adult mice while also inhibiting hippocampal neurogenesis and loss of hippocampal astrocytes.This study uncovered the collaborative effects of diverse stress conditions and exogenous corticosterone exposure on brain health.

Fig. 1 .
Fig. 1.CUS+C phenotype.(A) Representative images of the bodies of mice in the control and CUS+C groups.Scale bar = 10 mm.(B) Body weight changes for control and CUS+C mice during the CUS+C period.[Time effect: F = 12.62, P < 0.0001, Group effect: F = 3.776, P = 0.0643, Time/Group effect: F = 2.527, P = 0.0049; n = 13 and 12 mice in the control and CUS+C groups, respectively; repeated measures two-way ANOVA].(C) Representative images of the brains of control and CUS+C mice.Scale bar = 3 mm.(D) Brain weight.[t 18 = 3.969, P = 0.0009; n = 11 and 9 mice for the control and CUS+C groups, respectively; two-tailed Student's t-test].(E) Representative images of the coat state of control and CUS+C mice.Scale bar = 10 mm.(F) Mouse coat state score.[t 9 = 10.98,P <0.0001; n = 6 and 5 mice in the control and CUS+C groups, respectively; two-tailed Student's t-test].Data are presented as mean ± standard error of the mean.** P <0.01, *** P <0.001 , **** P <0.0001 vs. control.

Fig. 3 .
Fig. 3. CUS+C impairs neural progenitor proliferation in the dentate gyrus of adult mice brain.(A) Hippocampal coronal sections of the brains of mice in the control and CUS+C groups were stained with Ki-67 and BrdU antibodies.Scale bar = 200 μm.(B, C) Quantifications of stained cells as described in (A).[Ki-67: t 18 = 2.733, P = 0.0137; BrdU: t 18 = 2.155, P = 0.045; n = 10 of dentate gyrus images from 4 mice brains for control and CUS+C groups respectively; two-tailed Student's t-test].Data are presented as mean ± standard error of the mean.*P <0.05 vs. control.