Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

This protocol is significant because we develop a clinically relevant model of IVH induced brain injury and hydrocephalus, utilizing hemoglobin injection into the ventricles, which allows for subsequent quantification of ventricle volumes in the application, to testing new therapeutic strategies. The main advantage of this technique is that it allows for the study of the pathology of IVH that is specifically mediated by hemoglobin and iron pathway blood breakdown products. This technique is also easy to use and versatile.

The implications of this technique extend towards developing therapies for post-hemorrhagic hydrocephalus, as it allows for further understanding of IVH pathophysiology and the valuation of clinically relevant treatment strategies for the prevention of neurological sequela following IVH. Demonstrating the procedure will be Sruthi Ramagiri, an Associate Post-doc from the Australia Laboratory. 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 non-rupture 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 8, 000 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-O monofilament suture. For MRI, perform T2 weighted imaging by selecting a T2 weighted fast spin echo sequence.

After setting the MRI parameters as described in the text, click the continue button to start the sequence. For image processing and brain volume analysis, open the native T2 weighted data in the segmentation software. To manually delineate the lateral ventricles, click on the paintbrush mode and select the square brush style, then adjust the brush size to one.

Next, click layout inspector, select axial view and click zoom to fit. Then place the cursor on the image and trace and fill the lateral ventricle space. Finally, click on segmentation in the toolbar, followed by volume and statistics to view the segmented volumes.

Animals that underwent hemoglobin injection developed moderate ventriculomegaly when assessed via MRI. With significantly larger lateral ventricles at 24 hours, and 72 hours post hemoglobin injection, compared to ACSF injected animals. Although the difference in lateral ventricular volume between the two groups of rats was not significant after 38 days, MRI scans demonstrated that 44%of the rats receiving hemoglobin still had unresolved ventriculomegaly.

Moreover, in comparison to ACSF, injection of hemoglobin resulted in a significant decrease in the white matter volume after 38 days. Hemoglobin also promoted an inflammatory response in vivo as evident from the significantly higher level of the pro-inflammatory cytokine tumor necrosis factor alpha after injecting hemoglobin, but not saline or whole blood. Furthermore, immunohistochemical staining of the glial fibrillar acidic protein revealed a significantly higher activation of astrocytes after hemoglobin injection, than after ACSF injection.

The most important thing to remember when attempting this procedure is to set the correct injection coordinates to ensure hemoglobin delivery into the lateral ventricles. Following this procedure, CSF tracers can be delivered into the CSF space to evaluate changes in CSF circulation, and efflux patterns following IVH. This additional method addresses how CSF flow is altered in IVH PHH.