Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Secure an anesthetized rat pup on a stereotactic frame. Disinfect the head and incise to expose the skull.

Identify the bregma, an anatomical landmark, to locate the lateral ventricles, cerebrospinal fluid-filled cavities in the brain.

Choroid plexus epithelial cells and ependymal cells line the ventricles, forming a barrier from the periventricular space.

Use a microsyringe to inject hemoglobin into a ventricle, mimicking intraventricular hemorrhage.

Withdraw the needle, close the incision, and allow the pup to recover.

Injected hemoglobin undergoes degradation, releasing heme and generating reactive oxygen species or ROS.

ROS oxidize cell membrane lipids and proteins, causing cell death and disrupting the barrier, which allows heme to enter the periventricular space.

Tissue-resident immune cells recognize free heme as a danger signal and release proinflammatory cytokines to recruit additional immune cells, which damage the neighboring brain tissues.

The loss of periventricular tissue leads to ventricular enlargement, a consequence of intraventricular hemorrhage.

To begin, place the anesthetized rat prone in the stereotactic apparatus, with its nose positioned in the anesthesia adapter. Then, secure the head by tightening the nonrupture ear bars on the external auditory meatus.

To clean the head, first, touch a sterile cotton-tipped applicator soaked in betadine at the center of the head and move outwards to spread the betadine in circles. Then, repeat the process with an applicator soaked in 70% ethanol.

Next, using a scalpel, make a 0.3-centimeter incision vertically down the center of the head, to expose the bregma of the skull. Then, dry the area using a cotton-tipped applicator.

To set up the stereotactic injector, draw the previously prepared hemoglobin solution into a 0.3 or 0.5-milliliter syringe, and place the syringe in the stereotactic injector system.

Next, turn on the stereotactic injector interface, and click on the configuration button to enter the injection volume and rate settings. Click on volume and set the volume at 20,000 nanoliters.

Then, click on infusion rate and set the rate at 8000 nanoliters per minute. Click on the reset position button to exit the configuration. Flush the needle tip by clicking the infuse button, until a small bead of hemoglobin appears at the needle tip.

Then use a cotton-tipped applicator to gently remove the bead. To begin the animal injection, set the bregma as zero on the stereotactic injecting system by adjusting the mediolateral and anteroposterior positions of the syringe.

Then, lower the syringe needle to touch the skull at the bregma gently. After identifying the coordinates of choice, raise the syringe needle one centimeter above the skull to clear the scalp.

Set the mediolateral and anteroposterior coordinates. Then, lower the syringe for the needle to gently touch the skull, and set the dorsoventral coordinate over 30 seconds.

Once the coordinates have been set, begin injection by clicking the RUN button on the stereotactic injector interface. After the injection is finished, leave the needle in place for two minutes to minimize the backflow of the solution.

Next, slowly withdraw the syringe along the dorsoventral coordinate over two minutes until the needle tip is two centimeters above the scalp. Then, rotate the stereotactic injector arm away from the operative field and close the scalp with a 6-0 monofilament suture.