Hyperbaric Oxygen Therapy Alleviates Social Behavior Dysfunction and Neuroinflammation in a Mouse Model for Autism Spectrum Disorders

Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder (NDD) characterized by impaired social communication and repetitive behavior, among other symptoms. ASD is highly heritable, with SHANK3 being one of the high-risk genes for ASD. In recent years, knowledge has been growing regarding the neuroplasticity effect induced by hyperbaric oxygen therapy (HBOT) and its potential use for ASD. Here, we characterized the effect of HBOT on a mouse model for ASD with the human genetic condition of InsG3680 mutation in the Shank3 gene. As compared to placebo, HBOT improved social behavior and reduced neuroinflammation in the cortex of the InsG3680(+/+) mice. Specifically, HBOT induced upregulation of Insulin-like growth factor 1 (Igf1) expression levels and reduced the number of Iba1-positive cells in the mouse model for ASD compared to placebo control. Together, our research suggests that HBOT has the potential to improve the clinical outcome of ASD by ameliorating some of the core pathophysiological processes responsible for the development of the disorder.


Introduction
Autism spectrum disorders (ASDs) are neurodevelopmental disorders (NDDs) characterized by impaired social behavior and communication, restricted interests, and repetitive patterns of behavior, among other symptoms [1]. According to the Centers for Disease Control and Prevention, 1 out of 44 children is diagnosed with ASD in the United States of America, making it one of the most common NDDs [2]. Regarding genes known today, up to 25% of ASD cases have a genetic cause [3]. The focus of this study is on the SHANK3 gene, which is one of the high-risk genes for monogenic ASD [1,4]. Micro-deletion and variants of SHANK3 are known to cause Phelan McDermid syndrome (PMS), characterized by autistic-like phenotype, developmental delays, hypotonia, and other symptoms [5].
SHANK3 is a protein that takes part in the postsynaptic density (PSD) of glutamatergic synapses [4]. It is a scaffold protein whose main role is to bind glutamate receptors to the cytoskeleton and therefore is essential for synaptic transmission and integrity [1,4,6]. One of the human mutations found in SHANK3 is an insertion of guanine in position 3680 of the SHANK3 gene [6], which causes a frameshift and the appearance of an early stop

HBOT Improves Social Novelty Preference but Not Anxiety-like Behavior and Motor Coordination in InsG3680 Mouse Model for ASD
The physiological and behavioral improvements demonstrated in the human HBOT study prompted us to study the neurobiological properties of HBOT on a mouse model for ASD that harbors the same insertion mutation in Shank3 as in the human patient. Our motivation was to test whether the improved behavior in the autistic human patient could also be found when proper statistical power is tested, and to study neurobiological properties that can explain the improved behavior reported.
To achieve this, we studied the effects of HBOT (100% oxygen gas, 2 atmospheres absolute [ATA] pressure levels) on InsG3680 (+/+) mice as compared to InsG3680 (+/+) mice in the placebo control group exposed to air (air containing 21% oxygen gas, 1 ATA pressure levels) (Figure 1). All mice survived the procedures with no indications of irregular behavior or discomfort. At the end of the procedure (HBOT or placebo), 90 days-old (P90) mice were tested in behavioral tests that examined social behavior (three-chamber sociability and social novelty test), anxiety-like behavior (open field exploration test and elevated zero maze), and motor coordination (rotarod). Upon completion of the behavioral tests, mice were sacrificed and used for molecular and cellular studies ( Figure 1).

HBOT Improves Social Novelty Preference but Not Anxiety-Like Behavior and Motor Coordination in InsG3680 Mouse Model for ASD
The physiological and behavioral improvements demonstrated in the human HBOT study prompted us to study the neurobiological properties of HBOT on a mouse model for ASD that harbors the same insertion mutation in Shank3 as in the human patient. Our motivation was to test whether the improved behavior in the autistic human patient could also be found when proper statistical power is tested, and to study neurobiological properties that can explain the improved behavior reported.
To achieve this, we studied the effects of HBOT (100% oxygen gas, 2 atmospheres absolute [ATA] pressure levels) on InsG3680 (+/+) mice as compared to InsG3680 (+/+) mice in the placebo control group exposed to air (air containing 21% oxygen gas, 1 ATA pressure levels) (Figure 1). All mice survived the procedures with no indications of irregular behavior or discomfort. At the end of the procedure (HBOT or placebo), 90 days-old (P90) mice were tested in behavioral tests that examined social behavior (three-chamber sociability and social novelty test), anxiety-like behavior (open field exploration test and elevated zero maze), and motor coordination (rotarod). Upon completion of the behavioral tests, mice were sacrificed and used for molecular and cellular studies ( Figure 1).

Figure 1.
Graphical description of experimental procedures. InsG3680 (+/+) and littermates control mice were divided into two groups, HBOT and placebo. After two months of treatment, mice were tested in various behavioral tests, and afterward, InsG3680 (+/+) mice were used for qPCR and immunofluorescence analysis.
Because social behavior deficits are hallmark features of ASD, we studied whether HBOT has the potential to alleviate social behavior dysfunction. In the three-chambers sociability test, both the InsG3680 (+/+) HBOT group and the InsG3680 (+/+) placebo group spent significantly more time with the stranger mouse than with the object, as opposed to previous findings from this mouse model [7] (Figure 2A). Nonetheless, in the social novelty test, the InsG3680 (+/+) HBOT group showed significantly increased exploration time close to the novel mouse than the familiar mouse, whereas the InsG3680 (+/+) placebo group did not show any preference between them, similar to previously reported social novelty deficits [7] ( Figure 2B).
Because elevated anxiety levels are known in ASD compared to typically-developed (TD) individuals, we next sought to characterize the effect of HBOT in ASD on anxietylike behavior. In the open field test, no significant difference in overall exploration 40   . Graphical description of experimental procedures. InsG3680 (+/+) and littermates control mice were divided into two groups, HBOT and placebo. After two months of treatment, mice were tested in various behavioral tests, and afterward, InsG3680 (+/+) mice were used for qPCR and immunofluorescence analysis.
Because social behavior deficits are hallmark features of ASD, we studied whether HBOT has the potential to alleviate social behavior dysfunction. In the three-chambers sociability test, both the InsG3680 (+/+) HBOT group and the InsG3680 (+/+) placebo group spent significantly more time with the stranger mouse than with the object, as opposed to previous findings from this mouse model [7] (Figure 2A). Nonetheless, in the social novelty test, the InsG3680 (+/+) HBOT group showed significantly increased exploration time close to the novel mouse than the familiar mouse, whereas the InsG3680 (+/+) placebo group did not show any preference between them, similar to previously reported social novelty deficits [7] ( Figure 2B).
Because elevated anxiety levels are known in ASD compared to typically-developed (TD) individuals, we next sought to characterize the effect of HBOT in ASD on anxiety-like behavior. In the open field test, no significant difference in overall exploration properties was found, except in the one-time bin (25 min), in which the InsG3680 (+/+) placebo group spent a significantly longer time in the margins compared to the InsG3680 (+/+) HBOT group ( Figure 2C). In the elevated zero maze ( Figure 2D) and the rotarod test ( Figure 2E), no significant difference was found between the InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo group. properties was found, except in the one-time bin (25 min), in which the InsG3680 (+/+) placebo group spent a significantly longer time in the margins compared to the InsG3680 (+/+) HBOT group ( Figure 2C). In the elevated zero maze ( Figure 2D) and the rotarod test (Figure 2E), no significant difference was found between the InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo group. In the rotarod test, no change was found in latency to fall throughout all three trials between HBOT and placebo in both genotypes. However, the WT groups have endured longer on the rotarod compared to InsG3680 (+/+) groups. ns-non significant, * p < 0.05, ** p < 0.01, *** p < 0.0005. Two-tailed ttest (A,B,D). 3-way ANOVA with repeated measures (C,E). Data are shown as mean ± s.e.m.

Social novelty Social interaction A B
Open field Rotarod Elevated zero maze In the rotarod test, no change was found in latency to fall throughout all three trials between HBOT and placebo in both genotypes. However, the WT groups have endured longer on the rotarod compared to InsG3680 (+/+) groups. ns-non significant, * p < 0.05, ** p < 0.01, *** p < 0.0005. Two-tailed t-test (A,B,D). 3-way ANOVA with repeated measures (C,E). Data are shown as mean ± s.e.m.

HBOT Increases the Expression Level of Insulin-like Growth Factor 1 (Igf1) and Hif1a and Decreases Neuroinflammation in the Brain of InsG3680 Mouse Model for ASD
To seek neurobiological properties that could support the improved brain perfusion in the human patient and the improved behavior in our mouse model, we examined whether HBOT affects hypoxia and neuroinflammation properties. The indications of a hypoxic state and neuroinflammation in ASD [15,17,22] support the potential beneficial outcomes of HBOT as a potential treatment for the InsG3680 mouse model.
To examine whether HBOT impacted hypoxia, we examined protein levels of Hif1a. This protein is known to be upregulated during either hypoxia or hyperoxia and to function as a transcription factor to induce brain recovery. We found an increased intensity of Hif1a in the motor cortex of the InsG3680 (+/+) HBOT group compared to InsG3680 (+/+) placebo group ( Figure 3A). This may indicate a recovery process of the tissue since Hif1a regulates angiogenesis processes and may alleviate neuroinflammation.
To characterize neuroinflammation properties, we examined the number of Iba1positive cells and the expression level of key neuroinflammation-related transcripts in the whole cortex of InsG3680 (+/+) HBOT compared to InsG3680 (+/+) placebo. We found a reduced number of Iba1-positive cells in the motor cortex of the InsG3680 (+/+) HBOT group compared to InsG3680 (+/+) placebo group ( Figure 3B,C). This result may imply a reduction in neuroinflammation following HBOT.
Since previous studies showed that IGF1 administration is effective in ameliorating ASD deficits [38,39] and could lead to a reduction in chronic neuroinflammation, we wanted to determine whether HBOT affects Igf1 transcript expression level. We found upregulation in the expression level of Igf1 in the whole cortex of the P120 InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo ( Figure 3D). Interestingly, we found a significant negative correlation between the number of Iba1-positive cells and Igf1 expression levels in both InsG3680 (+/+) HBOT and InsG3680 (+/+) placebo ( Figure 3E). No significant difference was found in the expression level of CD11b ( Figure 3F) and CD68 ( Figure 3G), which are key neuroinflammation-related genes, as measured in the whole cortex of the P120 InsG3680 (+/+) HBOT group compared to InsG3680 (+/+) placebo group. upregulation in the expression level of Igf1 in the whole cortex of the P120 InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo ( Figure 3D). Interestingly, we found a significant negative correlation between the number of Iba1-positive cells and Igf1 expression levels in both InsG3680 (+/+) HBOT and InsG3680 (+/+) placebo ( Figure 3E). No significant difference was found in the expression level of CD11b ( Figure 3F) and CD68 ( Figure 3G), which are key neuroinflammation-related genes, as measured in the whole cortex of the P120 InsG3680 (+/+) HBOT group compared to InsG3680 (+/+) placebo group.

Discussion
In this study, we demonstrate for the first time that the core social behavioral deficit in ASD can be partially reversed by HBOT in a mouse model for ASD with Shank3 gene mutation as in the human condition. As such, this study has the potential for translational and medical implications. It enables us to gain molecular and cellular mechanistic explanations that link behavioral improvement and brain changes induced by HBOT.
We found that HBOT improves the social behavior of adult InsG3680 (+/+) mice compared to InsG3680 (+/+) placebo control group mice, indicating that HBOT is effective in adults and not necessarily in the early postnatal developmental stage only. Specifically, HBOT improved social novelty preference, a subtype of social behavior tested in the three chambers social interaction test. The improved preference of a novel mouse compared to a familiar mouse in the social novelty test could also indicate improved cognition and memory. It may demonstrate that InsG3680 (+/+) treated mice remember the familiar mouse better than the InsG3680 (+/+) placebo group [40]. Nevertheless, it is important to note that the results in the three chambers social interaction test do not replicate the social preference deficit previously characterized in InsG3680 (+/+) mice compared to controls [7]. The lack of social deficit in our study might result from the repeated exposure of test mice to other test mice, coming from different cages and litters, daily, throughout the 2 months of the experiment. Also, the lack of improvement in anxiety-like behavior following HBOT might be related to a critical age at which the treatment should be given to reduce anxiety-like behavior to normal levels.
In search of molecular and cellular evidence that will explain how HBOT affected neurobiological properties, we characterized parameters related to the hyperoxic-hypoxic paradox [28]. Along the course of HBOT, the oxygen levels fluctuated, altering from normal (normoxia) to high (hyperoxia) oxygen levels. Those repeated fluctuations generate a new setting point where hyperoxia is interpreted as the new baseline and normoxia is interpreted as hypoxia with increased expression of HIF-1α. HIF-1α is upregulated and enters the nucleus to join an active complex with HIF-1β during hypoxia, while in normoxia, most HIF-1α is degraded in the cytoplasm [28,41]. A higher expression level of HIF-1α induces the expression of genes responsible for tissue regeneration, angiogenesis mediators such as VEGF, mitochondria metabolism, and anti-inflammatory processes [28]. In this study, we found increased expression of Hif-1α in the brain of the ASD mouse model following HBOT as compared to the InsG3680 (+/+) placebo group. To determine whether HBOT ameliorated the brain's neuroinflammation, we studied the properties of microglia, resident macrophages of the brain. We found a reduced number of microglia (Iba1-positive cells) in the InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo group. Previous studies indicated elevated immune response in the brain of autistic patients [42,43]; therefore, the reduced number of microglia due to HBOT may improve neural circuit activity [12] and reduce neuroinflammation in the brain of the mouse model for ASD. Nevertheless, microglia number does not necessarily represent the functionality of these cells. Hence, to gain more insight into the microglial properties on the molecular level, we studied the Igf1 mRNA expression level following HBOT. IGF1 is a neurotrophic factor essential for central nervous system development [44][45][46][47], and microglia are an important source of IGF1. We measured an upregulation in the expression level of Igf1 transcript in the whole cortex of the P120 InsG3680 (+/+) HBOT group compared to the InsG3680 (+/+) placebo group.
Previous studies found significantly reduced levels of IGF1 in the cerebrospinal fluid (CSF) of autistic children and infants compared to TD controls [48,49]. Of clinical relevancy, two studies in human patients with PMS showed improved social behavior, motor skills, and other behavioral symptoms following IGF1 administration [38,39]. In neurons derived from induced pluripotent stem cells (iPSCs) from ASD patients with SHANK3 microdeletion, IGF1 rescued synaptic transmission deficits in excitatory neurons [50]. Similarly, injection of IGF1 to Shank3 deficient mice rescued abnormal motor skills and reversed major deficits related to excitatory synapse signaling [51]. Based on the above, IGF1 is a potential treatment for both syndromic and non-syndromic types of ASD [46,[52][53][54]. Therefore, the upregulation of the Igf1 expression level we measured following HBOT may lead to a cascading effect [55][56][57] that may improve brain functionality and behavioral outcomes.
Interestingly, when testing for a correlation between the number of Iba1-positive cells and the expression level of Igf1, we found a significant negative correlation between the two parameters. This correlation suggests that higher levels of Igf1 are correlated with a potential reduction in neuroinflammation following HBOT and that Igf1 may induce tissue repair, as was also found in previous studies [58][59][60]. Nonetheless, the results of our research show only a correlative and not a causative relationship.
Overall, our data suggest that HBOT is an effective treatment in the ASD genetic condition tested, reduces neuroinflammation, increases Igf1 expression, and improves social behavior in adult mice.
The molecular and cellular effects we characterized following HBOT were examined 1 month after the HBOT session, suggesting a long-term effect of HBOT. Moreover, while HBOT was mainly studied in acute neurological conditions or chronic conditions in adulthood, our study shows the efficacy of HBOT in treating a genetic condition associated with chronic NDD starting from embryogenesis.
This research gives hope for the potential new biological intervention for a subtype of ASD, and future research should better dissect its long-term clinical effect. Looking forward, the use of treatments for ASD that are specifically proven to be effective on a specific genetic condition rather than on the phenotype of behavior hopefully marks the future of effective treatments for ASD. Since other subtypes on the ASD spectrum have overlapping pathophysiology, such as hypoperfusion, this study may be relevant to these other conditions as well.

Animal Work Statement
Terminology: InsG3680 (+/−) -mice that are heterozygous to the mutation. InsG3680 (+/+) -mice that are homozygous to the mutation. WT-mice without the mutation on both chromosomes. Breeding: To test the efficacy of HBOT on the InsG3680 mutation, InsG3680 (+/−) mice were crossed with InsG3680 (+/−) (Het-Het mating). InsG3680 and WT mice are with a C57 B6/S129 Sv mixed background. The resulting mice are either homozygous to the mutation (referred here as InsG3680 (+/+) ), heterozygous to the mutation (referred here as InsG3680 (+/−) ), or do not have the mutation (referred here as WT). The mutation occurs in all the cells of the body.
Housing: Mice of the same gender were contained in cages with 2-4 littermates with random genotypes. The environmental conditions were stable with 20-24 • C under a 12 h light/dark cycle (lights at 07:00 until 19:00), with food and water available at all times. All experimental procedures conformed to the guidelines of the Institutional Animal Care and Use Committee of Tel Aviv University, Tel Aviv, Israel. All efforts were made to minimize animal pain and suffering and the number of animals used.

HBOT
Treatment was performed on 1-month-old mice for 40 sessions for 2 months, 5 days a week, 1 h a day. A total of 30 mice were divided into 4 groups: InsG3680 (+/+) HBOT, InsG3680 (+/+) placebo, WT HBOT, and WT placebo. The HBOT group received 100% oxygen gas in 2 ATA pressure levels, while the placebo group received air (containing 21% oxygen) with 1 ATA pressure levels. The HBOT chamber was filled with 100% oxygen prior to compression for 5 min to enrich the oxygen in the chamber. Compression and decompression were carried out gradually to reduce the risks of changing pressures.

Behavioral Studies
Behavioral tests were performed and analyzed with the experimenter blinded to the type of treatment and genotype. Mice in the experiment went through habituation in the test room for 1 h before all tests. Each group of test mice was used for 4 behavioral tests and had at least 3 days between tests. The mice were tested at the age of 3 months (2 months after the start of the treatment).

Social Preference Test
To perform the test, C57-black mice were ordered from the Jackson Laboratory with similar ages and body weights. Habituation for the stranger mice in the social preference test was performed by placing the mice inside an inverted wire cup for 30 min, 2 sessions per day for 3 consecutive days prior to the test. The test apparatus (65 cm long × 44 cm wide × 30 cm high) was divided into three sections (left, right-21 × 44 cm each, and center-21 × 22 cm), which were connected via a lever-operated door that was opened 5 cm from the floor. The object, stranger, and familiar mice were placed inside an inverted wire cup (10 cm high, diameter of bottom 10 cm, bars with 0.8 cm spacing), and on top of it, a weighted cup was placed to block the test mice from climbing over it. Following each trial, the wire cup was cleaned with ethanol and water. The test was comprised of three phases (15 min each): habituation, sociability, and social novelty. In the habituation phase, the test mouse was placed in the center and explored the environment of all three sections with the inverted wire cups empty. Next, in the sociability test, the tested mouse was placed in the center section with the doors closed while placing a stranger mouse and an object inside the wire cups of the left and right sections; Afterwards, the doors were lifted to allow the mouse to explore. Finally, in the social novelty test, the mouse was placed again in the center section with the doors closed while placing a novel mouse instead of the object, and afterward, the doors were lifted to allow the mouse to explore. The place of the object and stranger mouse were switched between trials to exclude place preference. Each of the stimulation mice were used only twice a day. Following the test, the experimenter analyzed the time spent with the object compared to a stranger mouse and the time spent with a familiar mouse compared to a novel mouse, blinded to treatment type, using the EthoVision XT 14.0.1326 software (Noldus Information Technology BV, Wageningen, The Netherlands).

Open Field Exploration Test
Mice were placed in the center of a Plexiglas box (40 cm long × 40 cm wide × 30 cm high) for 1 h (one mouse in each box). Motor activity and exploration were videotaped and were measured by the time spent in the margins of the box while the experimenter was blind to the treatment type.

Elevated Zero Maze
The maze contains 2 sections-open and closed arms (Height 60 cm). Mice were placed in the closed arms of the zero maze and explored for 5 min. Movement between the sections was videotaped and was measured by the time spent in the open arms, while the experimenter was blind to the type of treatment.

Rotarod
Mice were placed on the apparatus of an accelerating rotarod, and motor coordination was measured by latency to fall. Each subject was tested three times with a time interval of 30 min between each test.

Brain Tissue Extraction
After treatment and behavioral experiments, male mice were deeply anesthetized with isoflurane. Consciousness was tested via a response to a pinch on the foot, and tissue samples from one ear were taken for genetic verification. Then, mice were perfused with 15 mL of ice-cold PBS solution, followed by brain dissection. The brain was separated into two hemispheres. One was kept in 4% paraformaldehyde (PFA) diluted in PBS for 4 consecutive days, and one was taken to a petri dish with PBS for dissection. The Cortex was separated from other brain tissue using cleaned surgical tools and a stereomicroscope (OLYMPUS, Kyoto, Japan). Cortices were placed in 200 µL of RNA-later solution (Invitrogen by Rhenium, Modi'in, Israel) and preserved at 4 • C for 24 h. RNA-later was taken out of the tubes, and samples were frozen at −80 • C. All tools and equipment were sterilized with ethanol and sprayed with RNAse inhibitor (RNase-ExitusPlus, Biological Industries, Israel). Then, samples were centrifuged for 20 min at 4 • C, 800× g (13,800 rpm) (Eppendorf Centrifuge 5430R, Eppendorf by Lumitron, Petah Tikva, Israel). The homogenate was separated into 3 layers-protein, DNA, and RNA, which was the top layer. The clear RNA layer was transferred to a new tube and was diluted with a proportion of 1:1 with isopropanol (Bio-Lab Ltd., Jerusalem, Israel). The tubes were manually shaken, incubated for 5 min at RT and centrifuged for 15 min at 4 • C, 800× g (13,800 rpm). After the centrifuge, the RNA was precipitated in the bottom of the tubs, isopropanol was taken out, and the remaining pellet was washed with 1 mL of 80% ethanol (Sigma-Aldrich, Rehovot, Israel) diluted in DEPC-treated water (Biological Industries, Kibbutz Beit-Haemek, Israel), followed by centrifugation for 15 min at 4 • C at 13,800 rpm.
This was repeated twice, while the ethanol was replaced the second time. All the ethanol was removed, and the tubes were placed upside down and opened on Kim wipes to let the remaining ethanol aspartate for approximately 30 min. 35 µL of DEPC-treated water was added to each sample, and then the sample was heated for 5 min at 60 • C. Finally, samples were pipetted to create homogenous concentration, measured using the Thermo Scientific NanoDrop One device (Thermo Fisher Scientific, USA), and kept frozen at −80 • C.  [61]. To normalize each of the samples, the mRNA of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was also measured. Results are shown as fold change (FC) relative to the control group (InsG3680 (+/+) placebo).
Primers were programmed in the lab and ordered from Hy Laboratories Ltd. (Rehovot, Israel). The final dilution of the primers was done with 10 mM in DEPC-treated water (see detailed sequence in Table 1).

Immunofluorescence Staining
Brains were extracted as described above and then sectioned at a 100 µm thickness using a vibratome (Leica Biosystems, Deer Park, IL, USA). Sections were stained using the free-floating method as follows: from each mouse, a section from the motor cortex was chosen (approximately bregma 0.5 mm, according to the mouse atlas) and was washed 3 times in 1 mL PBS for 5 min each. Then, sections were permeabilized with 1.2% Triton X-100 in PBS for 15 min. Following permeabilization, sections were washed in 1 mL PBS for 5 minutes each and blocked with 5% normal goat serum (NGS), 2% bovine serum albumin (BSA), and 0.2% Triton X-100 in PBS for 1 h. Afterward, sections were placed in a 96-well plate with 250 µL of primary antibodies diluted in blocking buffer (described above) overnight at 4 • C. The next morning, sections were washed in 1 mL PBS, once for 5 min and twice for 15 min each. Slices were then incubated with secondary antibodies conjugated with Alexa Fluor 488, 555, and 647 (1:1000; catalog nos. A11001, A21424; Invitrogen, Waltham, MA, USA) diluted in blocking buffer for 1 h. sections were washed in 1 mL PBS, once for 5 min and twice for 15 min each. Finally, for the mounting process on glass slides, VECTASHIELD Hardset Antifade Mounting Medium with DAPI (catalog nos. H-1500-10, Vector Laboratories, Newark, USA) was used. Images were captured using a light microscope (IX-83, Olympus, Tokyo, Japan) with the experimenter blind to the treatment type. For quantification of cellular properties in the motor cortex, images were taken with ×10 or ×20 magnification according to the type of staining and analysis. Cell number and intensity were quantified manually using the ImageJ program.

Statistical Analysis
Data are presented as the mean ± standard error of the mean (s.e.m.), as calculated by GraphPad Prism 9.4.1 for Windows (GraphPad Software, San Diego, CA, USA) p-values were calculated using Student's t-test, 3-way repeated measures ANOVA, Kolmogorov-Smirnov and Pearson's correlation coefficient, with p < 0.05 considered significant (* p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001). The normality of distributions and equality of variances were checked and addressed accordingly using the appropriate statistical analysis. Outliers were determined via the extreme studentized deviate (ESD) method (p < 0.05).