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

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.