Name: Hypoxia Alters miRNAs Levels Involved in Non-Mendelian Inheritance of Autism Spectrum Disorder in Mice (Video) | JoVE Journal
Uploaded: 2025-07-11T13:20:00.000+00:00
Duration: 9 min 13 s
Description: Discover how neonatal hypoxia alters brain development and behavior in mice, linking oxygen deprivation to autism spectrum disorder through microRNA biomarkers and behavioral testing.

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This study was funded by the Scientific and Technological Research Council of Turkey (T&#220;B&#304;TAK), and the authors thank Celalettin G&#246;kta&#351; (Transgenic animal facility) and the biologist Selda Ta&#351;demir (Behavioral laboratory).

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<li>Adachi, K., Umezaki, H., Kaushal, K. M., Ducsay, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+hypoxia+alters+ovine+fetal+endocrine+and+physiological+responses+to+hypotension%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Long-term hypoxia alters ovine fetal endocrine and physiological responses to hypotension.</a> Am J Physiol Regul Integr Comp Physiol. 287 (1), R209-R217 (2004).</li>
<li>Imamura, T., Umezaki, H., Kaushal, K. M., Ducsay, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+hypoxia+alters+endocrine+and+physiologic+responses+to+umbilical+cord+occlusion+in+the+ovine+fetus%5BTitle%5D)+AND+%22J+Soc+Gynecol+Investig%22%5BJournal%5D)">Long-term hypoxia alters endocrine and physiologic responses to umbilical cord occlusion in the ovine fetus.</a> J Soc Gynecol Investig. 11 (3), 131-140 (2004).</li>
<li>Fajersztajn, L., Veras, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia%3A+from+placental+development+to+fetal+programming%5BTitle%5D)+AND+%22Birth+Defects+Res%22%5BJournal%5D)">Hypoxia: from placental development to fetal programming.</a> Birth Defects Res. 109 (17), 1377-1385 (2017).</li>
<li>Ruas, J. L., Poellinger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia-dependent+activation+of+HIF+into+a+transcriptional+regulator%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Hypoxia-dependent activation of HIF into a transcriptional regulator.</a> Semin Cell Dev Biol. 16 (4-5), 514-522 (2005).</li>
<li>Mukandala, G., Tynan, R., Lanigan, S., O'Connor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+hypoxia+and+inflammation+on+synaptic+signaling+in+the+CNS%5BTitle%5D)+AND+%22Brain+Sci%22%5BJournal%5D)">The effects of hypoxia and inflammation on synaptic signaling in the CNS.</a> Brain Sci. 6 (1), 6(2016).</li>
<li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+networks+associated+with+non-syndromic+intellectual+disability%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">Gene networks associated with non-syndromic intellectual disability.</a> J Neurogenet. 32 (1), 6-14 (2018).</li>
<li>Fattuoni, C., Palmas, F., Noto, A., Fanos, V., Barberini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perinatal+asphyxia%3A+a+review+from+a+metabolomics+perspective%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Perinatal asphyxia: a review from a metabolomics perspective.</a> Molecules. 20 (4), 7000-7016 (2015).</li>
<li>Locci, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+perinatal+asphyxia+by+metabolomics%5BTitle%5D)+AND+%22Metabolites%22%5BJournal%5D)">Exploring perinatal asphyxia by metabolomics.</a> Metabolites. 10 (4), 141(2020).</li>
<li>De Haan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+and+cognitive-behavioural+development+after+asphyxia+at+term+birth%5BTitle%5D)+AND+%22Dev+Sci%22%5BJournal%5D)">Brain and cognitive-behavioural development after asphyxia at term birth.</a> Dev Sci. 9 (4), 350-358 (2006).</li>
<li>Armstrong-Wells, J., Bernard, T. J., Boada, R., Manco-Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurocognitive+outcomes+following+neonatal+encephalopathy%5BTitle%5D)+AND+%22NeuroRehabilitation%22%5BJournal%5D)">Neurocognitive outcomes following neonatal encephalopathy.</a> NeuroRehabilitation. 26 (1), 27-33 (2010).</li>
<li>Gardener, H., Spiegelman, D., Buka, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+risk+factors+for+autism%3A+comprehensive+meta-analysis%5BTitle%5D)+AND+%22Br+J+Psychiatry%22%5BJournal%5D)">Prenatal risk factors for autism: comprehensive meta-analysis.</a> Br J Psychiatry. 195 (1), 7-14 (2009).</li>
<li>Hicks, S. D., Middleton, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+review+of+microRNA+expression+patterns+in+autism+spectrum+disorder%5BTitle%5D)+AND+%22Front+Psychiatry%22%5BJournal%5D)">A comparative review of microRNA expression patterns in autism spectrum disorder.</a> Front Psychiatry. 7, 176(2016).</li>
<li>Cassidy, F. C., Charalambous, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+imprinting%2C+growth+and+maternal-fetal+interactions%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">Genomic imprinting, growth and maternal-fetal interactions.</a> J Exp Biol. 221 (Suppl_1), jeb164517(2018).</li>
<li>Serocki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miRNAs+regulate+the+HIF+switch+during+hypoxia%3A+a+novel+therapeutic+target%5BTitle%5D)+AND+%22Angiogenesis%22%5BJournal%5D)">miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target.</a> Angiogenesis. 21 (2), 183-202 (2018).</li>
<li>Ozkul, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+heritable+profile+of+six+miRNAs+in+autistic+patients+and+mouse+models%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A heritable profile of six miRNAs in autistic patients and mouse models.</a> Sci Rep. 10 (1), 9011(2020).</li>
<li>Siniscalco, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autism+spectrum+disorders%3A+is+mesenchymal+stem+cell+personalized+therapy+the+future%5BTitle%5D)+AND+%22J+Biomed+Biotechnol%22%5BJournal%5D)">Autism spectrum disorders: is mesenchymal stem cell personalized therapy the future.</a> J Biomed Biotechnol. 2012, 480289(2012).</li>
</ol>

The authors declare no conflict of interest.

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...

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. &#304;t 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&#160;(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 metabolism5,6.
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-1&#945; and HIF-1&#946;8. Under hypoxic conditions, the replacement of the hydrogen atom by a hydroxyl group in the complex is reduced, leading to the accumulation of HIF-1&#945;, which then dimerizes with HIF-1&#946;. 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 disability9,10.
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 deaths11,12,13. 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 methods15,16.
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. &#304;n 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.

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hypoxia alters mirnas levels involved in non-mendelian inheritance of autism spectrum disorder in mice

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

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-1&#945; (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.

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 ...

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.

Hypoxia can occur to varying degrees for different reasons during the perinatal period, affecting development. We aim to examine the consequences of mild ...

We studied how different levels of neonatal hypoxia affect brain development, behavior, and ASD related molecular changes in mice, emphasizing the severity dependent effects and need for early intervention. Research studies link neonatal hypoxia to autism-related brain changes. Focus is shifting to repurpose drugs targeting neuroinflammation and neuro-immune dysfunction to prevent neuronal damage and long-term cognitive decline.We used hypoxia chambers, qPCR, behavioral tracking, and microscope to track molecular and structural changes in neonatal brains. A key challenge is translating molecular changes like microRNA dysregulation into behavior due to ASD's complexity. Limitations include species differences, lack of sex-specific analysis, and difficult controlling hypoxia therapy.Our study shows that even mild neonatal hypoxia causes lasting behavioral and molecular changes in mice, be those dependent effects and potential microRNA biomarkers, emphasizing early detection and integration. To begin, design the experiment to include four groups made of three hypoxic groups and one sham control group. Mate healthy female mice with healthy male mice to generate pregnant female mice.Transfer the pregnant mice to the hypoxia laboratory 72 hours before embryonic day 21.5 and begin monitoring them. Place the mothers into hypoxia chambers at the time of delivery. Adjust the oxygen levels in each chamber according to the specifications for each group.Select two neonates from each group to confirm hypoxia induced damage and determine sex. To perform the novel object recognition test, place a two-month-old mouse in an arena with two identical objects, allowing it to explore freely. Record the number of times the mouse turned toward or interacted with each object.The next day, replace one of the familiar objects with a novel object. Return the mouse to the arena and allow it to explore for another 10 minutes. To perform the tail suspension test, install the camera to clearly monitor the mice.Then cut 12 centimeter long strips of tape and attach them to the end of each mouse's tail, positioning the tape two centimeters from the tip to suspend the mouse safely. Record the mice for six minutes while suspended. After the test, stop the recording, remove the mice and return them to their housing cages.For the marble burying test, fill an empty cage with a five centimeter layer of corn cob bedding. Arrange 20 marbles in five rows of four on the bedding surface. Place the subject mouse in a corner of the cage, allowing free access to the entire area.Allow the mouse to explore for 20 minutes during which it may bury marbles. After 20 minutes, remove the mouse and count the number of marbles buried under the bedding. Next, perform the social interaction test.Install the camera to clearly monitor the mice and connect it to the computer with appropriate video tracking software. Set up a rectangular box with two walls containing two doors to create three chambers. Place a cage containing a mouse that is familiar with the experimental setup in one chamber and another cage with a mouse that had no previous experience in another chamber.Start recording and place the subject mouse in the middle compartment, allowing it to explore for five minutes. Return the mouse to its housing cage after the session. To perform the open field test, position the camera and connect it to the computer.Then set up the test arena and divide it with imaginary lines to create 16 squares. Start recording and carefully place the mouse in the center of the arena, observing its behavior for five minutes. After the session, stop the recording, remove the mouse, and return it to its housing cage.Use the tracking software to measure behaviors to assess anxiety levels, locomotor activity, exploratory behavior and emotional responses. To perform the Morris Water Maze test, prepare a water maze in a stable position within a behavioral laboratory. Hang distinct visual cues at the center of four imaginary quadrants of the maze wall, approximately 20 centimeters from the base.Next, position a platform measuring 17 centimeters in length and 10 centimeters in diameter about 20 centimeters from the wall. Fill the maze with water at a temperature between 21 and 26 degrees Celsius to a level one centimeter below the platform. Stain it with a black multi-surface acrylic dye.Place the camera to capture the full maze. Connect it to the Morris Water Maze software on the computer. Begin recording and place the subject mouse in a different quadrant for the first four days, with its face directed toward the tank wall.Assign the platform location in the day-five test schedule. On the fifth day, remove the platform and carefully place the mice in a square of the maze as demonstrated. In the novel object recognition test, mice exposed to 8%10%and 12%hypoxic oxygen conditions during birth showed significantly greater exploration of the novel object.Total distance traveled was significantly reduced in the 8%10%and 12%oxygen groups compared to the 21%oxygen group with a corresponding significant reduction in velocity. Discrimination Index scores were significantly higher in the 10%and 12%oxygen groups, indicating increased novelty preference. The percentage of novel object discovery was significantly reduced in the 10%and 12%oxygen groups.Marble burying behavior was significantly reduced in the 8%10%and 12%oxygen groups, with the most pronounced decrease observed in the 10%oxygen group. In the SIT, male mice of the 21%group showed a significantly greater preference for the cage. Distance traveled significantly decreased in the 10%and 12%oxygen groups.Only the female mice of the 21%group traveled more than males. Movement speed was significantly lower in the 10%and 12%oxygen groups. Female mice in the 21%oxygen group moved significantly faster than males.In the Morris Water Maze test, interest in the platform decreased from day one to day five in all groups, indicating memory decline. In the open field test, all groups spent more time in the peripheral area than in the central area, suggesting anxiety-related behavior. Significant sex-based differences were observed across all groups, though no differences were found between sexes within individual groups.All oxygen groups showed increased total movements.

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Research

JoVE Journal

Behavior

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