Maternal stress exposure during pregnancy impairs ultrastructural changes in the white matter and reduces cognitive function in offspring mice

CHENG Yan, GUO Jian-wei?, ZHU Bing, YI Min-min

1.Department of Anesthesiology, Shaanxi Provincial People’s Hospital, Xi'an, Shaanxi, 710068, China

2.Department of Surgery department 1,Shaanxi Provincial People’s Hospital, Xi'an, Shaanxi, 710068, China

Keywords:

ABSTRACT Objective: To explore whether maternal stress exposure during pregnancy impairs cognitive function and white matter ultrastructure in offspring mice.Methods: The parent rats were divided into two groups: pressure exposure group (group PE) and control group (group C),and the positive date of vaginal smear of female SD rats was day 0 of gestation.Female mice in group PE were exposed to binding pressure (3 times/day) on day 14-20 of gestation for 45 min-1 h/time.Behavioral tests (Morris water maze and Y maze) were performed on 1-month-old offspring mice followed by cardiac perfusion of fixed brain specimens and placement in mixed fixative solution.The total volume of white matter, total length and volume of myelinated nerve fibers and total length and volume of myelin sheath were calculated using modern stereoscopic methods, and the inner and outer diameter and inner and outer circumference of the myelin sheath were analyzed.Results: 1) Behavioral tests: compared with the group C,the average latency of the 3th and 4th day in the group PE were significantly prolonged, the percentage of the resting time in the quadrant of the platform and the frequency of acrossing the effective area of platform in the fifth day of space exploration experiment were significantly reduced of Morris water maze test, and visiting distance, duration and numbers in novel arm significantly increased of Y-maze test (P＜0.05).2) Compared with group C, the total volume of white matter, total length of myelinated nerve fibers, total volume of myelinated nerve fibers and myelin sheath in the group PE were significantly reduced (P＜0.05), the inner diameter and outer diameter of myelin sheath decreased significantly (P＜0.05), and the inner perimeter,outer perimeter and inner and outer perimeter differences increased significantly (P＜0.05).3) There was a correlation between behavioral test results and white matter ultrastructure measurement results.Conclusions: Maternal stress exposure during pregnancy could impair the cognitive function and white matter and its ultrastructure in the offspring, and there was a correlation between decreased cognitive function and white matter damages.

1.Introduction

Exposure to maternal mental stress during pregnancy may not only lead to adverse pregnancy outcomes, but may also have serious negative effects on the neural development and cognitive function of offspring.A large number of experiments have proved that pressure exposure during pregnancy can lead to different degrees of damage to the hippocampal structure and function of the offspring, including the reduction of the number of neurons in the hippocampus, the atrophy of the pyramidal neuron top dendrites, the decrease of mitochondrial respiratory enzyme activity and the decrease of mitochondrial Membrane potential[1-7].White Matter (WM) is mainly composed of bundles of myelinated or unmyelinated axons and myelinated Glia.WM accounts for nearly 50% of the total Brain size of human beings.WM is crucial to the transmission of electrical signals across different brain regions, so WM damage can lead to serious neurobehavioral and cognitive disorders[8,9].In recent years, research has found that cognitive function is closely related to the development and integrity of white matter in the brain.The development of white matter and the myelination process of myelinated nerve fibers that occupy the majority of white matter occur in the early stages of life, which coincides with the emergence of cognitive skills and abilities.Therefore, the formation and improvement of myelinated nerve fibers and the maintenance of WM integrity play an important role in the development of cognitive abilities in early life.

This study aims to investigate whether maternal stress exposure during pregnancy impairs cognitive function and brain white matter ultrastructure in offspring rats through a stress constrained SD rat maternal stress exposure model.The aim is to provide a theoretical basis for further clinical intervention and treatment strategies by measuring the behavior and brain white matter and its ultrastructure,It has important theoretical research value.

2.Materials and Methods

2.1Animal experiments and their grouping

Parent rats (KM SD rats, purchased from the Animal Experiment Center of Xi'an Jiaotong University) were divided into two groups:pressure restraint exposure group (PE group) and control group (C group), with 15 female and 5 male SD rats in each group.The virgin female SD rats and male SD rats (3:1) were placed overnight for mating.The vaginal smears of female SD rats were examined the next morning, and the date when the smears were confirmed to be positive was the day 0 of the embryo, Then separate the pregnant female SD rats and raise them separately.Exposure to stress restraint: Three times a day on the 14th to 20th day of pregnancy,with a duration of 45 min～1 h per exposure under restraint pressure.Conduct behavioral tests (Morris water maze and Y maze tests) on offspring mice at the age of one month.After the behavioral test,the offspring rats were anesthetized by intraperitoneal injection of 4% Chloral hydrate (300～350 mg/kg, laboratory configuration),and then the brain samples were fixed by cardiac perfusion (normal saline 4% formaldehyde, Xi'an Hite Biotechnology Co., Ltd.).After the perfusion was good, the brain was taken and placed in the previously prepared mixed fixative (2% Paraformaldehyde 2.5%Glutaraldehyde, laboratory configuration) for sample fixation.Embed the fixed specimens under electron microscopy, make electron microscopy sections, and use modern stereological methods to calculate the total volume of white matter, the total length and volume of myelinated nerve fibers, and the total length and volume of myelin sheaths.Calculate and analyze the inner and outer diameters and perimeter of myelin sheaths.

2.2Maternal stress restraint animal model during pregnancy

The restraint device (self-made in the laboratory) is a transparent plastic tube with a diameter of approximately 6.8 cm, with a breathing hole and a closed end, and the length can be adjusted to fit the size of the animal.Pregnant SD rats were exposed to restraint pressure three times a day on the 14th to 20th day of pregnancy,lasting for 45 min to 1 h.The duration of each stress exposure was randomly limited over time periods (08:00 am～11:00 am, 11:00 am～02:00 pm, and 04:00 pm～07:00 pm)[10].

2.3 Behavioral testing of offspring mice

2.3.1 Morris Water Maze test

The water maze instrument of Morris Water Maze Laboratory(Morris Water Maze Analysis System Smart software, China Chengdu Thai Union), the diameter of the pool is about 120 cm,the diameter of the platform is about 8cm, the height is about 15 cm, and the diameter of the effective area of the platform is about 10 cm.The computer automatically tracked and recorded the mice's swimming according to a camera located directly above the center of the pool.Before the experiment, the circular pool was divided into four quadrants, and then a water entry point was determined in each quadrant.Place the platform in the middle of a random quadrant and control the water temperature at (20.0±2.0) ℃.Add titanium dioxide to the water to make it milky white and make the surface of the water about 1cm above the platform so as not to let animals see the underwater platform.The heads of experimental mice were stained with picric acid for camera capture and recording.The test lasted for five days and was divided into two parts: the positioning navigation test (the first four days) and the space exploration test (the last day).The incubation period of each test of mice in the positioning navigation test was recorded (that is, the time from the mouse entering water to the time when all its limbs stood on the platform).If the mouse did not stand on the platform after 90 s, the incubation period was recorded as 90 s.The average incubation period of the mice was calculated for 4 tests per day.The number of times the mice crossed the effective area of the platform and the percentage of time they stayed in the original quadrant of the platform were recorded in the space exploration experiment.[11]

2.3.2 Y-maze test

The Y-shaped maze used in the Y-maze test experiment (Smart software of the Y-maze analysis system, Chengdu, China) is made of gray organic plastic and consists of three arms.The angle between adjacent arms is 120 °, and each arm is 8 cm × 30 cm × 15 cm(width × long × High), the computer can automatically track and record the exploration process of mice based on the camera above the center of the maze; Randomly assign three identical arms: (1)Start arm, mouse begins exploration (always open); (2) The novel arm was blocked during the first experiment but opened during the second experiment; (3) Other arms (always open); Close the novel arm and place the mouse back towards the center of the maze into the starting arm, allowing it to explore directionally in the starting arm and other arms for 15 min; After 1 h of directional exploration,open the novel arm and place the mouse in the starting arm with its back facing the center of the maze, allowing the mouse to freely explore in all three arms for 10 min; All experiments were recorded on a video recorder by using a camera installed above the center of the maze; Subsequently, analyze the video recording and analyze the exploration distance, number of explorations, and exploration time in the novel arm.[12-14]

2.4 Measurement of brain white matter and its ultrastructure by modern stereological methods

1) Calculation of the total volume of the white matter in the Cerebral hemisphere of offspring rats: randomly select several brain slices of offspring rats along the coronal plane, and randomly superimpose the equidistant dot matrix map and slices, as shown in Figure 1A.Count all points Σ PWM overlapping with the white matter under the anatomical microscope (stereoscopic microscope,Nikon, Japan), and calculate the total volume of the white matter VWM (formula: VWM=t × A (p) × ∑ PWM, where t is the thickness of the continuous slices, and a (p) is the area related to each grid point);[13,15-17] 2) Obtaining electron microscope photos: obtain tissue blocks containing white matter from selected brain slices,carry out electron microscope embedding, use transmission electron microscope (H-7650 Transmission electron microscopy, Hitachi,Japan) to observe electron microscope slices, and systematically sample 5 8000× and 10 15000× fields on each slice;[13] 3) Calculate the length density and total length of myelinated fibers in white matter: As shown in Figure 1B, the unbiased counting box is randomly overlaid with an 8000x electron microscope photo to calculate the length density LV (mf/wm) of myelinated fibers in white matter (formula: LV (mf/wm)=2 × ∑ Q (mf)/[a (frame) ×∑Frame], where ∑ Q (mf) is the total number of myelinated fibers in the electron microscope photo obtained by counting the unbiased counting boxes, a (frame) is the area of a single unbiased counting box, and ∑ frame is the total number of counting boxes And the total length L (mf, wm) (formula: L (mf, wm)=LV (mf/wm) ×VWM;[13,15,18] 4) Calculate the volume density and total volume of myelinated fibers in the white matter, as well as the volume density and total volume of the myelin sheath: As shown in Figure 1C, randomly overlay the equidistant lattice plot with an 8000×mm electron microscope photo, and count all the points ∑ P (wm)that fall on the white matter, ∑ P (mf) that fall on the myelin fibers,and ∑ P (ms) that fall on the myelin sheath.Calculate the volume density VV (mf/wm) of myelinated fibers (formula: Vv (mf/wm)=∑P (mf)/∑ P (wm)) The volume density of myelin sheath VV (ms/wm) (formula: Vv (ms/wm)=∑ P (ms)/∑ P (wm)), and the total product of myelin fibers V (mf, wm) (formula: V (mf, wm)=Vv (mf/wm) × VWM and the overall product V (ms, wm) of myelin sheath(formula: V (ms, wm)=Vv (ms/wm) × VWM;[13] 5) Calculate the inner and outer diameters of myelinated fibers: As shown in Figure 1D, randomly overlay the unbiased counting box with a 15000×electron microscope photo to determine the cross-section of the nerve fibers that fall within the unbiased counting box.Estimate the outer diameter of myelinated fibers by measuring the longest contour diameter perpendicular to the longest axis of the axon.Estimate the inner diameter of myelinated fibers by measuring the longest contour diameter perpendicular to the longest axis of the axon.Measure the inner and outer diameters and calculate the ratio and difference between the inner and outer diameters;[13,17,19] 6) Calculate the inner and outer circumference of myelinated fibers:As shown in Figure 1E, the unbiased counting box and equidistant parallel lines are randomly superimposed on a 15000× electron microscope photo to determine the cross-section of the nerve fibers that fall within the unbiased counting box.The inner or outer circumference of the myelinated fibers in the cross-section b (ms) is calculated (formula:b (ms)=π/2) × D ×∑ I.Where d is the vertical distance between equidistant parallel lines, and ∑ I is the total number of points where the equidistant parallel lines intersect with the inner or outer boundary of the myelin sheath that falls within the unbiased counting box, Then calculate the ratio and difference between the inner and outer perimeter.[13,16,19]

2.5 Statistical Analysis

The experimental data was statistically analyzed using SPSS 23.0.Firstly, perform normality tests on each group of data.If normality is met, use parameter tests.If normality is not met, use non parameter tests (rank tests); The parameter test uses independent sample T-test.When P＜0.05, the difference is considered statistically significant.

3.Results

3.1 Body weight analysis of parent and offspring mice during the experiment

The parent rats were selected on the 0, 14, and 20 d of pregnancy,and the offspring rats were selected on the 0, 15, and 30 days of birth for weight statistical analysis.As shown in Figure 2A, the body weights of parental mice on the 0th, 14th, and 20th day of pregnancy were (223.67 ± 10.54) g, (245.87 ± 13.72) g, (251.60 ± 8.86) g in Group C, and (226.33 ± 13.14) g, (242.93 ± 10.16) g, and (254.2± 13.88) g in Group PE, respectively.There was no statistically significant difference between the two groups.As shown in Figure 2B, the body weights of offspring mice on the 0th, 15th, and 30th day of birth were (6.04 ± 0.82) g, (34.88 ± 2.84) g, (73.39 ± 4.62) g in Group C, and (6.05 ± 0.76) g, (39.78 ± 2.78) g, and (75.19 ± 3.59)g in Group PE, respectively.There was no statistically significant difference between the two groups.

Figu 1 White matter and its ultrastructure calculation

Fig 2 Weight comparison between the two groups of parent mice and offspring mice during the experiment(±s D)

3.2 Maternal stress exposure during pregnancy impairs cognitive function in offspring mice

As shown in Figure 3, in the Morris water maze navigation test,the 4-day latency of group C mice was (57.24 ± 12.84) s, (61.09 ±12.24) s, (34.48 ± 4.78) s, (23.65 ± 5.53) s, respectively.The latency of PE group mice was (63.60 ± 8.30) s, (53.55 ± 12.56) s, (65.36 ±6.59) s, and (65.79 ± 6.02) s, respectively.On the third day (F=9.72,t=-25.32, P＜0.05) and the fourth day (F=0.86, t=-34.23, P＜0.05), the incubation period of the PE group mice was significantly prolonged compared to the C group mice.As shown in Figure 4, in the Morris water maze space exploration experiment, the number of times the C group and PE group mice crossed the effective area of the platform was 3.39 ± 1.26 and 0.90 ± 0.82, respectively.The percentage of residence time in the quadrant where the platform was located was 26.48 ± 4.19% and 20.33 ± 2.98%, respectively.Compared with group C mice, the number of times the PE group mice crossed the effective area of the platform (F=21.95, t=10.88, P＜0.05) and the pe-rcentage of residence time in the quadrant where the platform was located were significantly reduced (F=7.60, t=7.87, P＜0.05).As shown in Figure 5, the exploration distances of the Y maze novel arm in group C and PE mice were (43.59 ± 4.52) cm and (111.86 ±18.92) cm, respectively, with exploration times of 24.78 ± 3.85 and 77.69 ± 9.91, and exploration times of 123.89 ± 14.59 and (198.07 ±11.75) s, respectively.Compared with group C mice, the exploration distance (F=86.79, t=-23.76, P＜0.05), exploration frequency(F=54.80, t=-33.57, P＜0.05), and exploration time (F=4.09, t=-26.11, P＜0.05) of the Y maze novel arm in the PE group mice were significantly increased.

Tab 1 Results of stereoscopic analysis of myelin nerve fiber myelin sheath in the white matter of the brain hemispheres between two groups((±S D))

Tab 1 Results of stereoscopic analysis of myelin nerve fiber myelin sheath in the white matter of the brain hemispheres between two groups((±S D))

Note: *P compared with group C

group C group PE Outer diameter(μm) 1.25±0.02 1.18±0.01*Inner diameter(μm) 1.15±0.02 1.08±0.01*Outer diameters- in inner(μm) 0.05±0.01 0.05±0.01 Ratio of inner and outer diameters 0.91±0.02 0.92±0.01 outer perimeter(μm) 20 977.23±125.77 21 241.82±177.47*inner perimeter(μm) 18 602.64±194.72 18 353.87±220.57*Outer - inner perimeter(μm) 2 374.60±225.59 2 887.95±298.40*Ratio of inner and outer perimeter 0.89±0.01 0.86±0.01

Fig 3 Comparison of the time latencies of the Morris water maze positioning navigation test of offspring mice between two groups (±S D)

3.3 Maternal stress exposure during pregnancy impairs the white matter and ultrastructure of offspring mice's brains

Fig 4 Comparison of the target zone frequency and percentage of time in target zone of the Morris water space exploration test of offspring mice between two groups (±s D)

As shown in Figure 6, the total white matter volume of mice in the C and PE groups was (283.2 ± 19.83) mm3(180.69 ± 6.11) mm3,respectively; The total length of myelinated nerve fibers is (10466.87± 831.92) mm and (6091.29 ± 181.08) mm, respectively; The total volume of myelinated nerve fibers was (32079.50 ± 1091.68) mm(330411.31 ± 965.28) mm3, and the total volume of myelin sheath was (8045.91 ± 153.91) mm (36813.07 ± 58.86) mm3, respectively.Compared with group C mice, the total white matter volume(F=51.40, t=32.13, P＜0.05), total length of myelinated nerve fibers(F=75.19, t=33.36, P＜0.05), total volume of myelinated nerve fibers (F=0.27, t=7.56, P＜0.05), and total volume of myelin sheath(F=37.07, t=48.74, P＜0.05) in the PE group mice were significantly reduced.In order to further investigate the specific changes in the myelin sheath of myelinated nerve fibers, the inner and outer diameters and circumference of the myelin sheath were analyzed.As shown in Table 1, compared with group C mice, the inner diameter(F=21.54, t=17.60, P＜0.05) and outer diameter (F=11.20, t=23.22,P＜0.05) of the myelin sheath in PE group mice were significantly reduced, while the inner circumference (F=.39, t=5.62, P＜0.05) The outer circumference (F=13.02, t=-8.12, P＜0.05) and the difference in inner and outer circumference (F=2.87, t=-9.15, P＜0.05)significantly increased.

3.4 Negative correlation between brain white matter ultrastructure and behavioral damage in offspring mice

Fig 5 Y-maze exploration results between two groups (±s D)

As shown in Figure 7, the total volume of white matter, the total length of myelinated nerve fibers, the total volume of myelinated nerve fibers, and the total volume of myelin sheaths were negatively correlated with the latency of the Morris water maze navigation test on day 3 and day 4, the exploration distance, exploration frequency,and exploration time of the Y-maze novel arm, and positively correlated with the percentage of residence time in the quadrant where the Morris water maze spatial exploration test platform is located and the number of effective areas crossing the platform.

4.Discussion

Stress exposure is closely related to brain damage and cognitive dysfunction[4,20].Sapolsky et al.found that stress exposure is involved in the plasticity of the nervous system, especially the Limbic system, which proposed that severe and/or long-term stress weakens the ability of hippocampus dependent explicit learning and the plasticity of its foundation[21].The negative effects of maternal stress exposure during pregnancy on fetal neural development, as well as long-term emotional and behavioral disorders, have been documented in both animals and humans[1].In animal experiments,it was found that the development of Amygdala of offspring mice exposed to pressure during pregnancy was different, indicating that this exposure may be a trigger for fear related behaviors (such as anxiety symptoms) of offspring mice[2]; In the early and late pregnancy of rhesus monkeys, exposure to prenatal pressure can inhibit placental precocity, and Neurogenesis and reduction of hippocampal volume are found in the hippocampus Dentate gyrus[22]; Yang et al.found that pressure exposure during pregnancy may lead to long-term impairment of the enhancement function of hippocampal CA1 region in offspring rats and long-term inhibition,and this change in Synaptic plasticity may be related to the impairment of spatial learning and memory in offspring rats[3].In this study, parental mice were divided into PE group and C group.On the 14th to 20th day of pregnancy, parental female mice in the PE group were exposed to restraint pressure (3 times/day) for 45 min to 1 h/time.Morris water maze and Y maze tests were performed on 1-month-old offspring mice.Morris water maze test is considered the most classic behavioral detection method for animals' spatial reference memory.Y maze task is a dual experiment recognition test used to measure spatial recognition memory.The study found that compared to C group, On the 3rd and 4th day of the Morris water maze navigation test in the PE group mice, the incubation period was significantly prolonged, the percentage of residence time in the quadrant where the spatial exploration test platform was located, and the number of times it crossed the effective area of the platform was significantly reduced.The exploration distance,number of explorations, and exploration time of the Y maze novel arm were significantly increased.The experimental results suggest that maternal mental stress exposure during pregnancy impairs the cognitive function of offspring mice, which is consistent with previous animal research findings.

Human studies have found a correlation between maternal stress exposure during pregnancy and delayed motor and intellectual development, as well as weaker language abilities in children.Huizink et al.found using the Bayley Infant Development Scale that higher daily distress (stress) and anxiety levels of mothers are independent risk factors for intellectual disability in children,and mid pregnancy stress exposure may be one of the determining factors for delayed motor and intellectual development in 8-monthold infants, and may be a risk factor for future developmental problems[23]; In a study of women who became pregnant during severe ice storms in Quebec, Canada, it was found that the objective stress experienced by mothers (including injuries, losses, and changes) was associated with lower scores on the Bayley scale and lower language proficiency in their 2-year-old children[24].In the Scientific control with animals, the stress factors of mental stress exposure during pregnancy introduced at present include constraint, chronic transliteration, immobilization, isolation, social stress, and acute feeling or emotional tension, among which the stress limitation (and constraint) model is the most widely used[10].Therefore, this study applied an animal model of maternal stress restraint during pregnancy and found that maternal stress exposure during pregnancy can significantly damage the cognitive function and learning and memory abilities of offspring mice.The research results are consistent with previous animal studies.If pregnant mice are exposed to severe stress during pregnancy, it may have negative effects on the fetus.Research has shown that severe stress can affect the physiological and behavioral status of pregnant mice,thereby having adverse effects on fetal development.For example,fetal development is limited, possibly due to the production of stress hormones such as cortisol in pregnant mice.High levels of cortisol may cross the placenta and enter the fetal body, affecting its development, which may lead to fetal growth restriction and lower birth weight.[25]Severe stress exposure during pregnancy in pregnant mice refers to the exposure of pregnant mice to high-intensity,long-term stress stimuli during pregnancy.This stress stimulus can include the pressure or Irritability stimulus imposed on pregnant rats under various laboratory conditions, such as electric shock, noise,cold water immersion, etc.These stimuli may cause physiological and behavioral stress reactions in pregnant rats, and thus affect the physiological status and development of pregnant rats and fetuses.[25,26] There was no statistically significant difference in postnatal weight between the two groups of offspring mice in this study, which may be related to exposure intensity and duration.The degree of mental restraint stress exposure during pregnancy in this experiment was much lower than that of severe stress exposure.

After conception, the fetal brain begins to form, and as pregnancy progresses, the fetal WM also gradually changes.WM is a type of tissue in the brain, composed of nerve fiber bundles, primarily responsible for transmitting neural signals.In the third week after conception, neural tubes begin to form, which are the precursors of the fetal central nervous system and ultimately develop into the brain and spinal cord.The basic structure of WM begins to form; From the sixth to the twelfth week after conception, nerve cells begin to migrate from the fetal ventricular region to other regions, which is a Committed step in the formation of cerebral cortex and a Key Stage in the development of WM; After the twelfth week of pregnancy,nerve cells begin to form myelin sheaths, a lipid layer that covers nerve fibers.The myelin sheaths are crucial for nerve signal transmission, enhancing the transmission speed of nerve impulses,which is crucial for the development of WM; In the later stages of fetal development, the connections between nerve cells increase and neurons further differentiate, forming complex neural networks that support various cognitive and motor functions.[27,28]When lipid myelination forms around neuronal axons, its function is to increase the conduction speed of electrical pulses, thereby improving brain connectivity.Myelination rapidly develops throughout early childhood (the first 5 years of life).This ontogenetic pattern is strictly regulated by neural activity and is consistent with the emergence of cognitive skills and abilities.The formation and improvement of myelinated nerve fibers and the maintenance of WM integrity play an important role in the development of cognitive ability in infants and young children[9,29].This study found that compared with Group C, the total volume of white matter, total length and volume of myelinated nerve fibers, and total volume of myelin sheath in mice in the PE group significantly decreased.Further analysis of the inner and outer diameters and circumference of myelinated nerve fibers revealed a significant decrease in the inner and outer diameters of myelin sheath in offspring mice of the PE group, with a significant decrease in inner circumference and an increase in outer circumference.This suggests that early damage to white matter and its ultrastructure may be related to cognitive impairment, Consistent with previous research.This study analyzed the correlation between the behavioral test results of offspring mice and the changes in white matter and its ultrastructure.It was found that the total volume of white matter, the total length of myelinated nerve fibers, the total volume of myelinated nerve fibers, and the total volume of myelin sheath were negatively correlated with the latency period of Morris water maze navigation test on the 3rd and 4th days, the exploration distance, exploration frequency, and exploration time of the novel arm in the Y-maze, There is a positive correlation between the percentage of residence time in the quadrant where the Morris water maze space exploration experimental platform is located and the number of times it crosses the effective area of the platform.Therefore, maternal exposure to mental stress during pregnancy may damage the development, formation, and myelination of white matter in the early stages of life, thereby damaging the white matter and its ultrastructure of offspring mice, and thereby reducing their cognitive function and learning ability.Although previous studies have suggested that the formation, development, and myelination process of white matter in the brain may have an important impact on cognitive function, relevant research has not been conducted from the perspective of white matter ultrastructure.This study uses modern stereological three-dimensional quantitative measurement methods to reveal the relationship between early life white matter damage and cognitive decline, which is innovative and of research value.

In summary, maternal exposure to mental stress during pregnancy can damage the cognitive function, white matter and its ultrastructure of offspring mice, and there is a correlation between cognitive function and white matter.The inherent antioxidant properties in white matter may be relatively low[30,31], while myelin sheaths rich in lipids and proteins can provide rich substrates for peroxidation reactions, leading to the production of a large amount of ROS.Oxidative stress itself may lead to the destruction of membrane integrity, for example, Lipid peroxidation may be related to the loss of phospholipid asymmetry in the synovial membrane[32].The myelin sheath of myelinated nerve fibers is mainly composed of OLs.Some studies have found that oxidative stress induces cell death in OLs and OPC[33], or inhibits the differentiation of oligodendrocyte progenitor cells, which disrupts the maturation of OLs[34].Therefore,oxidative stress may affect the conduction function of WM by disrupting the myelin sheath structure, leading to learning and memory dysfunction.Oxidative stress plays an important role in the impact of white matter damage on cognitive function, but this study did not measure the level of oxidative stress in the brain, and further research can be conducted in the future.

Conflict of Interest

This study does not involve any conflicts of interest.Author’s Contribution

Cheng Yan participated in research design, experimental setup,data collection, data analysis and interpretation, as well as writing this article.Guo Jianwei has made contributions in explaining data, revising manuscripts, and conceptualizing ideas.Zhu Bin participated in completing water maze and Y-maze tests in animal experiments.Yi Minmin participated in the calculation process of the white matter and its ultrastructure in the offspring mouse brain.All authors have approved the final version of the manuscript.