Hypoxia Alters miRNAs Levels Involved in Non-Mendelian Inheritance of Autism Spectrum Disorder in Mice

Summary

Here, we present a protocol to induce hypoxia in neonatal mice by delivering them in controlled environments with varying oxygen concentrations. Mouse models subjected to oxygen variations at birth have durably varied the expression of miRNAs involved in the induction of behavioral phenotypic changes such as autism.

Abstract

Hypoxia can occur to varying degrees for different reasons during the perinatal period, affecting development. We aim to examine the consequences of mild to severe neonatal hypoxia in mice, focusing on its potential link to neurodegenerative fetal brain dysfunctions related to autism spectrum disorder (ASD).

We induced hypoxia in neonatal mice by delivering them in controlled environments with varying oxygen concentrations. The results showed elevated levels of HIF-1α (a marker of hypoxia) in the cerebral cortex and hippocampus, as well as increased neurodegeneration in these regions. Moreover, we observed altered expression of ASD-related miRNAs (miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p, and miR-499a-5p) in hypoxic groups, consistent with previous results in human and animal models of ASD. These molecular alterations were accompanied by behavioral changes such as decreased movement, speed, social interaction, and repetitive behaviors, as well as an increased tendency to exhibit corner-seeking behavior in mice subjected to hypoxic conditions. Although behavioral and molecular changes were detectable in the 12% O₂ group (mild hypoxia), the most pronounced changes were observed in the more severe hypoxia groups, at 10% O₂ and 8% O₂.

Overall, the results suggest that neonatal hypoxia can induce lasting molecular changes associated with neurodegenerative diseases with behavioral consequences. Moreover, even in normal birth, exposure to hypoxia during early development can impair memory and learning. The results highlight the need for early detection and intervention in newborns exposed to hypoxia to prevent long-term neurological problems. miRNA levels are regulated during development, and their modifications in tissues, especially in germ cells, constitute a risk factor for the next generation. The analyses provide a basis for studying the regulatory pathways that affect functional and behavioral changes under hypoxia.

Introduction

Defined as a state of oxygen deficiency, hypoxia can elicit unique cellular and molecular responses that are critical to understanding its role in various pathologies. İt can disrupt developmental sequences, leading to molecular, cellular, and neuronal alterations and injuries. Furthermore, early exposure to hypoxia interrupts central nervous system (CNS) development, impairing axonal connectivity and synapse development¹.

Additionally, although hypoxia has been shown to have adverse effects on CNS development, it may also play a critical physiological role under specific conditions, such as during embryogenesis. Normal physiological hypoxia refers to a situation in which oxygen levels are naturally low during embryonic development in a beneficial manner. This process allows embryonic cells to produce energy by promoting anaerobic metabolism, supports the development of the circulatory system by accelerating vascularization, ensures proper development of the heart and bones, and stimulates the migration of neural crest cells². Conversely, hypoxia occurring outside of its physiological context can have adverse effects on development and overall health, including embryonic development, which can lead to prematurity or even embryonic lethality¹. It is, therefore, a major public health problem, as many newborns experience hypoxic conditions during normal delivery, often without immediate recognition by caregivers or medical personnel³. In addition, alternations in neuronal connectivity associated with hypoxia are linked to neurodevelopmental disorders, including autism spectrum disorders (ASD) and intellectual disabilities¹.

From a molecular perspective, hypoxia can influence neurodevelopment by altering the expression of genes that regulate synaptic functions, thereby disrupting neuronal connectivity and contributing to the development of neurodevelopmental disorders⁴.

Clinically, neonatal hypoxia affects CNS development through hormonal and metabolic pathways¹. In response to acute hypoxia, the hypothalamus rapidly releases the corticotropin-releasing hormone and arginine vasopressin, which then triggers adrenocorticotropic hormone release from the anterior pituitary, followed by glucocorticoid production in the fetal adrenal cortex and the degree of this response is correlated with the intensity and duration of hypoxia. This rapid response of the hypothalamic-pituitary-adrenal (HPA) axis to acute stress underscores the critical role of glucocorticoids in limiting the physiological impacts of stress and maintaining fetal homeostasis by influencing associated gene expression, protein synthesis, and cellular metabolism^5,6.

Decreased normal oxygen levels activate various responses to hypoxia, most of which are regulated by transcription factors, Hypoxia-Inducible Factors (HIFs), and cellular oxygen sensors such as prolyl hydroxylases (PHDs)¹. HIF-1, a regulator of O₂ homeostasis as shown in in vitro studies⁷, is a heterodimer composed of HIF-1α and HIF-1β⁸. Under hypoxic conditions, the replacement of the hydrogen atom by a hydroxyl group in the complex is reduced, leading to the accumulation of HIF-1α, which then dimerizes with HIF-1β. As a result, the HIF-1 dimer translocates to the nucleus, where it activates the transcription of hundreds of genes¹.

The oxygen-dependent nature of neurons makes them particularly susceptible to hypoxic stress. Therefore, hypoxia can have a significant impact on brain physiology, with long-term implications on neuronal function, by impairing axonal pathfinding and synapse development⁹. These alterations in neuronal connections can predispose to various neurodevelopmental disorders such as ASDs and intellectual disability^9,10.

Perinatal asphyxia is defined as oxygen deprivation during labor and can occur as a result of perinatal events such as maternal-fetal hemorrhage, umbilical cord compression, placental complications, uterine rupture, shoulder dystocia, or failure of the newborn to take its first breath. This serious event affects approximately four million newborns worldwide each year and results in one million neonatal deaths^11,12,13. Although most infants survive episodes of hypoxia, some patients develop hypoxic-ischemic encephalopathy, which can lead to permanent neurological problems such as seizure disorders, cerebral palsy, cognitive delays, and motor deficits. Additionally, asphyxia and ischemia can cause dysfunction of various organs and systems such as the CNS (28%), cardiovascular system (25%), kidneys (50%), and lungs (23%)¹¹.

In addition, immaturity of the cerebral vasculature system and poor autoregulation are factors predisposing newborns to ischemic risks. In such processes, inflammation and oxidative stress are the main mediators of neonatal hypoxic-ischemic injury and associated cell death mechanisms. Abnormal apoptosis can expose the developing brain to risks of motor and cognitive disorders¹⁴.

Given the complexity of ASD, it is essential to understand the underlying genetics that appear to have a polygenic structure, as well as the environmental factors that determine its development. In particular, the polygenic basis of ASD implies a multifactorial etiology, in which genetic predispositions interact with external factors, including hypoxia. The 60%-92% concordance observed in monozygotic twins highlights the importance of genetic influences, while the discordance indicates that environmental factors also play a role. The prevalence of autism is estimated at 13/10,000, and this rate is believed to have increased in recent years¹⁵. Studies support the hypothesis that exposure to environmental factors during pregnancy may increase the risk of autism, but the results of these studies are often inconsistent due to differences in diagnostic criteria, comparison groups, sample size, and exposure assessment methods^15,16.

Although the distinctive neuropathological findings are unclear, studies reveal microscopic and functional brain abnormalities. These abnormalities suggest that the etiologically relevant period may be in utero and that pathogenesis may begin during the prenatal period¹⁵. Exposures during pregnancy are the main focus of epidemiological research on potential risk factors for autism. In particular, the hypothesis that obstetric complications may increase the risk of ASD is supported by many studies, but the magnitude and consequences of these effects vary across studies¹⁷.

In addition to genetic factors, epigenetic mechanisms play a key role in regulating gene expression under hypoxic conditions. Notably, miRNAs present a key regulatory mechanism in the cellular response to hypoxia, such as their impact on HIF. During the early stages of hypoxia, specific changes in miRNA levels can contribute to the accumulation of HIF-1 and the maintenance of stable levels of HIF-2 and HIF-3¹⁸. Although the expression profile of many miRNAs is altered under hypoxic and ischemia conditions, a limited number of hypoxia-associated miRNAs have been shown to play a functional role and affect HIF expression. This explains why different human tissues differ in hypoxic responses due to tissue-specific miRNA expression¹⁸.

Emerging data also indicates that specific miRNAs have been implicated in ASD pathology. İn our previous work, we demonstrated that miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p, and miR-499a-5p, which are known to regulate cell survival, differentiation, and stress responses in cancer, exhibit aberrant expression in ASD patients, potentially influencing neurodevelopmental outcomes. We then confirmed these findings in blood, hypothalamus, and sperm samples from mouse models of ASD, showing a consistent association of gene regulatory changes with the disease¹⁹. These results indicate that the downregulation of these miRNAs at crucial stages of growth and development may have a significant impact on the development of diseases such as ASD. This further demonstrates the profound influence of neonatal hypoxia on molecular and behavioral characteristics.

In this study, we tested the impact of mild to moderate neonatal hypoxia on the risk of developing autism in adulthood. To do this, neonatal mice were exposed to different levels of hypoxia. Indeed, mice that experienced hypoxia at birth exhibit behavioral changes in adulthood and, more importantly, at the molecular level, alterations associated with ASD. Thus, our results provided valuable information on the long-term effects of hypoxia.

Protocol

All experimental protocols were approved by the Local Animal Experiments Ethics Committee of Erciyes University (HAYDEK) and performed under license number 21/216.

1. Animal subjects

NOTE: This study involved Balb/c mice from the Transgenic Unit of the Betül-Ziya EREN Genome and Stem Cell Center (GENKÖK) at Erciyes University.

1. Use 2-month-old Balb/c mice weighing an average of 18-25 g. Maintain the mice at a temperature of 20 ± 2°C, with a humidity of 52 ± 3% in a 12 h light/dark cycle (6-18 h).
2. Feed the mice with standard mouse pellets, with weekly bedding changes, and allow unlimited access to food and water.
   NOTE: Molecular studies were performed in the Genomics and Hypoxia Laboratories of the GENKÖK and the Histology and Embryology Laboratory of Bozok University. A 'STOP point' indicates where you can pause and preserve your work at the specified temperature.

2. Hypoxia model establishment

1. Design the experiment to include 4 groups, including 3 hypoxic and 1 sham control group.
2. Mate healthy female mice with healthy male mice to form healthy pregnant female mice, which will eventually be classified into the following groups: severe hypoxia (8% O₂) (n = 4), moderate hypoxia (10% O₂) (n = 4), mild hypoxia (12% O₂) (n = 4), and normoxia (21% O₂) as sham control group (n =4).
3. To induce hypoxia, transfer the pregnant mice to the hypoxia laboratory and start monitoring them 72 h before day E21.5.
4. Detect the delivery point of the pregnant mice in the hypoxic groups and immediately place them in hypoxia chambers. Adjust oxygen levels for each chamber as described for each group (shown in step 2.2.).
5. Keep neonates in hypoxic conditions for 5 min only, then immediately transfer them to normal housing conditions.
   NOTE: Keeping subjects in hypoxic conditions for longer periods may result in lethality.
6. Use 2 neonates (randomly chosen) from each group to confirm hypoxia-induced damage and determine sex. Keep the remaining pups next to their mothers in normal housing conditions until the end of the lactation period for additional molecular and behavioral testing for the adulthood phase (See experimental design in Supplementary Figure 1).

3. Sex determination to assess potential differences in hypoxic effects between genders in the neonate stage

1. Weigh each mouse (randomly selected (x2/group)) individually to calculate the required dosage of anesthetics. Administer ketamine at 100 mg/kg and xylazine at 10 mg/kg intraperitoneally.
2. Once the mouse is fully anesthetized, proceed with euthanasia via cervical dislocation and immediately collect tail tissues in sterile labeled 1.5 mL tubes (a STOP point, -80 °C)
3. Immediately collect and transfer all brain tissues to centrifuge tubes containing 10% formaldehyde for further analysis (a STOP point, 25 °C).
4. Prepare tissues for DNA extraction.
   1. Prepare tail samples and thaw at 25 °C (if necessary). Add 300 µL of lysis buffer containing 1 M Tris, 0.5 M EDTA, 4% SDS, dH₂O, and proteinase K to the samples.
5. Homogenize the samples.
   1. Mix samples thoroughly by vortexing and incubate overnight at 55 °C.
   2. The next day, add 100 µL of ammonium acetate and mix briefly by vortexing. Centrifuge at 12,000 g for 20 min at 25 °C
6. Perform phase separation .
   1. Carefully transfer the clear aqueous phase into new sterile, labeled 1.5 mL tubes.
   2. Add 500 µL of isopropanol and incubate for 5 min at 25 °C (a STOP point, -20 °C). Centrifuge at 12,000 g for 10 min at 25 °C
7. Wash the DNA pellet.
   1. After inspecting the pellet at the bottom of the tube, carefully discard the supernatant.
   2. Add 1000 µL of 70% ethanol and gently tap the tube to float the pellet. Centrifuge at 7500 g for 10 min at 25 °C
8. Purify the DNA.
   1. After inspecting the pellet at the bottom of the tube, carefully discard the supernatant.
   2. Use a 10 µL micropipette to remove residual ethanol and allow it to dry completely.
   3. Add 100 µL of Nuclease-Free Water (NFW) to resuspend the pellet. Incubate at +4 °C for 10-15 min.
9. Quantify and store the DNA.
   1. Measure DNA concentration using a spectrophotometer. Store DNA samples at +4 °C until further use. (a STOP point).
10. Perform polymerase chain reaction (PCR).
    1. Design SRY-F, SRY-R, and indirect PAX6-F, PAX6-R primers (see Supplementary Table 1).
    2. Prepare a PCR mix as follows (per sample): 5 µL of PCR buffer (10x), 4 µL of MgCl_2, 4 µL of dNTPs, 0.5 µL of each of the four primers, 31 µL of dH₂O, and 0.5 µL of Taq polymerase.
       NOTE: Taq polymerase should be added last and just before use to ensure optimal amplification.
    3. Prepare the labeled PCR tubes and place them on a +4 °C cooling rack. Distribute 45 µL of the mixture into the tubes.
    4. Gently tap the block to ensure that PCR mixture droplets are at the bottom of the tube.
    5. Add 5 µL of DNA samples and pipette briefly. Vortex briefly and then centrifuge the tubes.
    6. Place the tubes in the PCR machine and set the program to the following conditions: Initially, incubation at 95 °C for 5 min, 40 cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min. After the cycles, a final extension at 72 °C for 10 min, followed by a hold at 40 °C indefinitely.
11. Visualize the result samples using 2% agarose gel electrophoresis.
    NOTE: Samples showing two bands are males, and those with only one band are females.

4. Confirmation of hypoxia damage in neonatal mice

1. Microscopically evaluate brain tissues collected from sacrificed mice to confirm hypoxic damage and examine Immunohistochemically to test HIF-1α.
   1. Fix and paraffin embed the tissue.
      1. Preserve tissues in formaldehyde for 12 h, then wash in water for 6 h.
      2. Fix tissues by dehydrating in ethyl alcohol series for 1 day for each concentration: 70%, 80%, 90%, then 3 h in 100% ethyl alcohol. Place tissues in xylene for 15 min to clear.
      3. Transfer tissues to paraffin series I, II, and III for 1 hour each. After the third hour, orient the brain samples properly and embed them in clean paraffin.
      4. Using a rotary microtome, cut 5 µm thick sections from the paraffin blocks.
   2. Prepare slides.
      1. For hematoxylin and eosin (HE) staining, place sections on plain glass slides.
      2. For immunohistochemistry, place sections on positively charged glass slides.
         NOTE: (a STOP point), slides can be archived at this stage for further evaluation and work.
   3. Perform HE staining.
      1. Immerse the samples in Xylene I solution for 10 min.
      2. Immerse the samples in Xylene II solution for an additional 10 min.
      3. Transfer the samples to a series of decreasing alcohol concentrations, each for 1 min: 100%, 96%, and 80%.
      4. Wash the samples with distilled water for 2 min.
      5. Immerse the samples in hematoxylin for 8 min, then wash them in distilled water for 2 min .
      6. After washing, treat the samples with acidic alcohol for 1 s, followed by a 1-min wash.
      7. Place the samples in ammonia water for 10 s, then repeat the 1-min wash.
      8. Place the samples in 70% alcohol for 30 s.
      9. Immerse the samples in eosin for 10 min.
      10. Gradually transfer the samples by increasing alcohol concentrations for 1 min each: 96%, 96%, and 100%.
      11. Immerse the samples in Xylene I, Xylene II, and Xylene III solutions for 1 min each.
      12. Cover with a coverslip.
   4. Examine using a microscope.
      1. After HE staining, examine the entire brain tissues of each group under a light microscope.
      2. Evaluate hippocampal and cortical areas for neuronal degeneration and pericellular/perivascular edema.
2. Perform HIF-1α staining.
   1. Incubate samples on positively charged slides overnight at 67 °C in an incubator to ensure adequate fixation.
   2. Pass sections through xylene I and xylene II solutions to complete deparaffinization.
   3. Rehydrate sections by placing them sequentially in 100%, 96%, 80%, and 70% ethanol solutions for 5 min each, followed by rinsing in distilled water for 5 min.
   4. Unmask epitopes by microwaving sections in 0.01 M citric acid (pH 6.0) at 665 W for two 5-min cycles.
   5. Cool the sections at room temperature (RT) for 20 min.
   6. Delineate the perimeter of the sections with a hydrophobic pen, then wash the sections 3 times in PBS for 5 min each.
   7. Incubate sections in 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity.
   8. Wash sections again 3 times in PBS for 5 min each time.
   9. To prevent non-specific immunoglobulin binding, incubate sections with a UV-blocking reagent for 7 min.
   10. Incubate sections overnight at 4 °C with the anti-HIF-1α primary antibody (1:200 dilution).
   11. Wash sections 3 times in PBS for 5 min each time.
   12. Apply biotinylated secondary antibody to sections and incubate at RT for 40 min.
   13. Wash sections again 3 times in PBS for 5 min each time.
   14. Apply streptavidin solution to the sections and incubate for 30 min at RT.
   15. Wash the sections 3 times in PBS for 5 min each time.
   16. Develop the staining reaction by applying 3,3'-Diaminobenzidine (DAB) and observing under a light microscope.
   17. Counterstain the sections with modified Gill II hematoxylin solution to stain the nuclei.
   18. Dehydrate sections by passing them through increasing concentrations of ethanol (70%, 80%, 96%, and 100% ethanol).
   19. Clear the sections by incubating them in Xylene I and Xylene II solutions for 10 min each.
   20. Mount sections with a mounting medium and a coverslip.
   21. Examine mounted sections under a light microscope and assess areas of the hippocampus and prefrontal cortex.
   22. Measure immunoreactivity levels using open-source image processing software.

5. Behavioral experiments

1. After confirmation of hypoxia, allow the remaining mice to complete the lactation period with their mothers.
2. Perform sex determination on postnatal day 21 based on anogenital distance, then house the animals in different cages based on their sex. When the mice in the hypoxic and sham control groups reach 2 months of age, subject them to behavioral experiments.
3. Before performing behavioral tests, acclimate the subjects to the test environment for 15-30 min before testing.
4. Make sure to disinfect all devices/apparatus between subjects and trials using 70% ethanol.
5. Perform novel object recognition (NOR) test.
   1. Set the camera in a position where subjects can be clearly seen and monitored.
   2. Install appropriate video tracking software.
   3. Prepare the test arena (a 60 cm × 60 × cm × 40 cm box) with two objects no larger than the mouse. Make sure that the two objects are of similar size and shape and easily accessible (10 cm × 10 cm away from the walls and parallel).
   4. Day 1: Familiarization phase:
      1. Place the mouse in the arena with the two identical objects. Allow the mouse to freely explore the objects for a set amount of time (e.g., 10 min).
      2. During this session, observe and record the amount of time the mouse spent with each object. Also, record the number of times the mouse turned toward or interacted with each object
   5. Day 2: Novel object introduction:
      1. Replace one of the familiar objects from day 1 with a novel object (different in appearance, texture, or shape).
      2. Return the mouse to the arena, allowing it to explore freely again for the same amount of time as in the first session.
   6. Record the total time the mouse spent with the familiar object (from Day 1) and the novel object. Record the number of times the mouse turned toward or interacted with each object.
   7. Calculate the Discrimination Index (DI) to assess the mouse's memory and learning using the formula:
      Discrimination Index (DI) = {(Time spent with novel object − Time spent with familiar object)/(Total time spent with both objects)} × 100.
      ​NOTE: A higher DI indicates greater interest in the novel object, suggesting better memory and learning abilities. A DI close to 0 indicates no preference, suggesting either equal interest or a poor memory for the familiar object.
   8. Use the DI to objectively assess the mouse's learning and memory abilities, based on its natural curiosity and ability to recognize and differentiate between familiar and novel objects.
6. Perform tail suspension test (TST)
   1. Install the camera in a position where the mice are clearly visible and can be monitored. Install appropriate video tracking software.
   2. Prepare the experimental setup with space to accommodate 3 mice simultaneously .
   3. Use 25 cm high cardboard panels with 20 cm intervals to isolate the mice so they cannot see each other during the test.
   4. Cut 12 cm long strips of tape and carefully attach them to the tail of each mouse, ensuring that the tail is securely attached without causing damage. Position the tape at the end of the tail (2 cm) so that the mouse can be suspended without injuring itself.
   5. Record the mice for 6 min while they are suspended.
   6. At the end of the duration, stop the recording, remove the mouse, and return it to the housing cage. Analyze the videos to determine the duration of active movement and the duration of immobility.
      NOTE: This will provide insight into the stress response, as immobility often indicates a state of learned helplessness or stress. The degree of immobility is commonly used to measure behavioral despair or stress response in mice, which may be related to the effects of stress or depressive states.
   7. Based on the observed patterns of movement and immobility, assess the emotional state and stress coping mechanisms of mice in response to the forced suspension.
7. Perform marble burying test
   1. Fill an empty cage (20 cm × 35 cm × 15 cm) with a 5 cm height of corn cob. Arrange 20 marbles in 5 rows with 4 marbles each. Ensure the marbles are evenly distributed over the surface.
   2. Place the subject in a corner of the cage, ensuring free access to the entire cage.
   3. Give the mouse 20 min to explore the cage. During this time, the mouse may exhibit some burying behavior, trying to cover the marbles with corn cob.After 20 min, remove the mouse from the cage.
   4. Count the total number of marbles the mouse buried under the corn cob during the 20 min period.Record the total number of marbles buried, as this is the key measure of behavior.
      NOTE: The number of marbles buried is commonly used as an indicator of anxiety , compulsive behavior, or neophobia in rodents. A higher number of buried marbles is often associated with higher levels of anxiety or compulsive tendencies.
   5. Based on the total number of buried marbles, assess anxiety-related behaviors and compulsive tendencies in mice, which can be used to assess the effects of treatments or experimental conditions.
8. Perform social interaction test.
   1. Install the camera in a position where the mice are clearly visible and can be monitored.
   2. Connect the camera to the computer and set up appropriate video tracking software. Set up a rectangular box (60 cm × 40 cm × 25 cm) with two walls (40 cm × 25 cm) with two doors (8 cm) to separate the three chambers.
   3. Place a cage (15 cm × 10 cm) containing a mouse familiar to the subject of the experiment and another cage (15 cm × 10 cm) containing a mouse that has had no previous interaction with the subject of the experiment.
   4. Start recording and place the subject in the middle compartment.
      NOTE: Mice used in this test other than the experimental animals must be of the same sex and healthy.
   5. Monitor the subject for 5 min and record it with the video camera. At the end of the duration, stop the recording, remove the mouse and return it to the housing cage.
   6. Using SIT software, analyze the social interaction time and social novelty tendencies of the mice and compare them with the sham control group.
      NOTE: More time spent on social behaviors (e.g., grooming or sniffing) and less time spent avoiding or displaying aggression can be interpreted as demonstrating high sociability. Avoiding interaction, spending more time in isolation, or displaying increased aggression can suggest anxiety, social withdrawal, or reduced ability to interact socially.
9. Perform open field test.
   1. Position the camera where the mice can be seen clearly and monitored . Connect the camera to the computer and set up suitable video tracking software.
   2. Set up the test arena (a 60 cm × 60 cm × 40 cm box) and divide the box with imaginary lines to create 16 squares.
   3. Start recording and carefully place the mouse in the center of the arena. Observe and record the mouse's behavior for 5 min.
   4. At the end of the duration, stop the recording, remove the mouse, and return it to the housing cage.
   5. Using the tracking software, measure the external and internal behaviors of walking, climbing, escape, grooming, immobility, total crossings, defecation, and urination to assess the subjects' levels of anxiety, locomotor, exploratory, and emotional activity.
10. Perform the Morris water maze (MWM) test.
    1. Prepare the 120 cm diameter and 60 cm deep water maze in a stable position in a behavioral laboratory at 23 ± 1 °C.
    2. Divide the maze into four imaginary quadrants and hang different visual cues (in shape and color) on the center of each quadrant of the maze walls approximately 20 cm from the base. Position a 17 cm long and 10 cm diameter platform 20 cm from the wall.
       NOTE: The maze, cues, and platform should not change position or location so that mice can accurately train their memory.
    3. Fill the maze with 21-26 °C water to 1 cm below the platform and stain it with a black multi-surface acrylic dye.
       NOTE: İncase of using other strains of mice, the water should be colored with a contrast color of the mice to be able to monitor them.
    4. Place the camera in a position that captures the full image and connect it to the MWM software on the computer.
       NOTE: The platform spot should be assigned in the program for the fifth day's trial.
    5. The test lasts for 5 days with four trials each. For the first four days, begin recording and start the trial by carefully placing the subject in a different quadrant each day, with their face directed toward the tank wall.
    6. Assign the platform location in the day 5 test schedule.
       NOTE: This prevents the subjects from forming an associative memory. Careful placement of the subjects in the water and maintaining the correct temperature is essential to avoid adding external stress. Trials last 5 min and end automatically when (i) the platform is reached (ii) the 5-min time limit expires. Over the course of days, subjects should learn the location of the platform and find it in less time.
    7. On the fifth day, remove the platform and carefully place the mice in a square of the maze as described previously.
       NOTE: The trial ends automatically when (i) the mouse finds and steps onto the platform, (ii) the 5-min time limit expires. Healthy subjects should find the location of the previous platform. The software will automatically calculate the time the mouse took to find the platform in each trial and help assess the short- and long-term memory and learning abilities of the subjects.

6. Confirmation of hypoxia damage for adult mice

1. After behavioral testing is completed, sacrifice the subjects by cervical dislocation.
2. Collect hippocampal tissue and blood samples from 6 mice of each group, including the sham control group, for molecular analysis (section 7).
3. Place the brains of the remaining 4 mice in 10% formaldehyde solution to examine the effects of hypoxia exposure during the neonatal period on adult brain morphology.
4. Use the same technique as previously explained in section 4 to conduct histological analysis of adult mouse brains.

7. Total RNA isolation from adult phase-samples

1. Add 500 µL of trizol to each tissue sample and 500 µL of blood.
2. For brain tissue, homogenize the samples using a syringe.
3. Incubate the mixture at RT for 5 min.
4. Add 200 µL of chloroform to each tube and incubate for an additional 5 min at RT.
5. Centrifuge at 12,000 g for 20 min at +4 °C.
6. Carefully transfer the aqueous phase (RNA) to a new sterile tube.
7. Add isopropanol (1:1 ratio to the aqueous phase) and incubate the mixture overnight. (a STOP point/ -20 °C).
8. The next day, centrifuge at 12,000 g for 10 min at +4 °C.
9. Carefully discard the supernatant and add 1000 µL of 75% ethanol to the pellet.
10. Centrifuge at 7,500 × g for 5 min at +4 °C.
11. Repeat the ethanol wash step twice.
    NOTE: İf the pellet is too small, one wash is sufficient.
12. Discard the supernatant and allow the pellet to air dry at RT.
13. Once completely dry, resuspend the pellet with 50 µL of NFW and incubate on ice at +4 °C for 10-15 min.
14. Measure RNA concentration spectrophotometrically.
15. Store RNA samples at -80 °C until further analysis.

8. Complementary DNA (cDNA) synthesis

1. Working on a cooling +4 °C rack, prepare the RNA samples isolated from blood and brain tissues.
2. Prepare the labeled PCR tubes.
3. Mix the cDNA synthesis kit reagents according to the manufacturer's instructions.
4. Prepare a total of 14 µL of the reaction mixture for each sample and distribute it into the tubes.
5. Add 6 µL of RNA sample to the mix.
6. Perform cDNA synthesis in the PCR device following the temperature and time conditions specified by the manufacturer.
   NOTE: The cDNA synthesized at this stage can be stored at -80 °C until further use.
7. Dilute the synthesized cDNA in a ratio of 1:5.
8. Store the diluted cDNA at -80 °C for future qPCR analysis.

9. Quantitative real-time PCR for miRNA (miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p, miR-499a-5p, and U6 small nuclear RNA as a normalization control) expression

1. Design the primers for each miRNA (see Supplementary Table 2 for sequences).
2. Prepare the PCR reaction mixture according to the manufacturer's instructions.
3. Dispense 14 µL of the reaction mix into each well of a 96-well PCR plate.
4. Add 6 µL of the 1:5 diluted cDNA to each well for a total reaction volume of 20 µL.
5. Perform PCR in duplicate for each sample to minimize variation by following this program: Initial denaturation at 95 °C for 120 s, 45 cycles of denaturation at 95 °C for 10 s, annealing and amplification at 56 °C for 60 s, and melting curve analysis at 60 °C for 15 s, then at 95 °C.
6. Analyze the results using Ct (Cycle threshold) values and normalize the data using the 2^-ΔΔCt method.

10. Statistical analysis

1. .Assess the normality of the data using histograms, Q-Q plots, and the Shapiro-Wilk test to analyze miRNA expression data.
   1. If the data is normally distributed, use One-Way ANOVA.
   2. If the data are not normally distributed, apply the Kruskal-Walli's test , student's t-test, or Mann-Whitney U test.
2. When assessing gender differences within each group, use either the student's t-test or Mann-Whitney U test, depending on the distribution of the data. Statistical significance is determined by a p-value of <0.05.
   NOTE: All analyses should be performed using appropriate statistical software. Here, GraphPad Prism 8 is used.
3. Assess the normality of the data using histograms, Q-Q plots , and the Shapiro-Wilk test to analyze behavioral experiments' results.
   1. If the data are normally distributed, use a Two-Way ANOVA.
   2. For data that are not normally distributed, perform the Kruskal-Walli's test , Student's t-test, or Mann-Whitney U test.
   3. Assess gender differences within each group using the student's t-test or Mann-Whitney U test, depending on the distribution of the data. Again, consider the results statistically significant if the p-value is < 0.05.
4. For immunohistochemical analyses, use Two-Way ANOVA followed by Tukey's post-hoc test to compare female and male groups.
5. To compare female and male groups within each hypoxia condition, use Sidak's post-hoc test. Statistical significance is determined if the p-value is < 0.05.

Results

In this study, pregnant Balb/c mice were exposed to hypoxic conditions of 8% O₂ (severe), 10% O₂ (moderate), and 12% O₂ (mild) during delivery, and the newborn mice were maintained under these hypoxic conditions for 5 min to establish models of mild, moderate and severe neonatal hypoxia. The group designated as the sham control group was subjected to normoxic (21% O₂) birth conditions then was allowed to live under standard care conditions. This helps isolate the true ...

Discussion

Perinatal hypoxia, a major neonatal health problem, can cause molecular, cellular, and neuronal changes by disrupting critical developmental processes and has been associated with various neurodevelopmental disorders, including autism spectrum disorder (ASD)¹. While severe hypoxia has been widely studied for its immediate effects, the long-term consequences of mild to moderate hypoxia during the perinatal period remain less well understood. Our study aimed to fill this gap by examining the neurobe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(-20 °C) Freezer	Siemens	KD86NAWE0N
(+4 °C) Block	Antagen Bioscience	https://antagen.net/product/384-well-pcr-plate-rack-cat-rkp384/
(+4 °C) Refrigerator	Siemens	KD86NAWE0N
(-20 °C) Block	Cleaver Sientific	https://www.cleaverscientific.com/electrophoresis-products/coolcube-storage-rack/
Absolute Ethanol	Merck	1070172511
Ammonium Acetate	Sigma	238074-500G
Analytical Balance	Adam Equipment	LAB214i
Chloroform	Merck	1024451000
Citric Acid	Merck	100241
Computer	HP	https://www.hp.com/us-en/shop/pdp/hp-all-in-one-24-cr1000t-238-9p2v3av-1
EDTA	Sigma	E9884-1KG
Entellan	Merck	107961
Eosin	Merck	109844
Ependorf (1.5 mL)	?solab	078.03.021
Ethovision System	Noldus	https://noldus.com/ethovision-xt
Etuv	NUVE	EN 400/500 N/A
Falcon Tubes (15 mL)	Isolab	078.02.001
Formaldehyde	Merck	104002
Heat Camera	Samsung	SC2000
Hematoxylin	Merck	105175
HIF-1α Antibody	Bioss	bs-20398R
Hypoxia Chambers	Biospherix	A-Chamber
Isoproponal	Merck	1096342511
Light Cycler 480 II	Roche	5015278001
Marble Burying Test's Cage	Vishnu	https://guide.labanimal.com/product/standard-housing-stainless-steel
Microcentrifuge Tubes (1.5 mL)	Greiner	616201
Micropipette Tips (100 µL)	Greiner	774353
Micropipette Tips (1000 µL)	Greiner	778353
Micropipettes (10 µL)	Gilson	FD10001
Micropipettes (100 µL)	Gilson	FD10004
Micropipettes (1000 µL)	Gilson	FD10003
Micropipettes Tips (10 µL)	Greiner	772353
Micropipettes(2 µL)	Gilson	GFAM00064
Microscopy	Olympus	Bx53
miRCURY LNA RT Kit (cDNA Synthesis Kit)	Qiagen	339340
miRCURY LNA SYBR Green PCR Kit	Qiagen	339346
Multiwell plates	Roche	4729692001
Nano Spectrophotometer	Shimadzu	C101-E112
Novel Object Recogniton Test's Arena	Global Biotech	137
Open Field Test's Arena	Global Biotech	117
Oxycycler	Biospherix	A42OC
Paraffin	Leica	39602012
PBS Tablet	Sigma	P4417-50TAB
PCR Device	Sensoquest	T3-0140
Plate Spin	Peqlab	91-PSPIN-P
Prism	GraphPad	https://www.graphpad.com/
Proteinase K	Sigma	P6556-10MG
QIAzol Lysis Reagent Isolation Reagent	Qiagen	79306
SDS	Sigma	8170342500
Seconder Antibody	Abcam	A0545-1ML
Social Interaction Test's Three chambers	Global Biotech	118
Spin	Gilson	PMC880
Syringe (2 mL)	Baybi	https://beybi.com.tr/urunler/medikal-urunler/medikal-sarf-malzemeleri/parcali-enjektorler
Tail Suspension Test's Setup	Self Made, you can find similar at Harvard Apparatus	Self Made, you can find similar at (https://www.harvardapparatus.com/tail-suspension-test.html)
Thermomixer	Eppendorf	#5355
Trizma base	Sigma	1503-1KG
Vortex	Heidolph	541-10000-00-1
Xylenes	Isolab	9,90,01,92,501