Paired Cisterna Magna Nanoinjection and Laser Speckle Contrast Imaging Assay to Study Cerebral Blood Flow Regulation In Vivo
July 8th, 2025
To study cerebral blood flow regulation in vivo, traditional pharmacological methods are invasive, often damaging the skull, dura, and leptomeninges. In this work, we developed a minimally invasive technique that combines nanoinjector-guided intracisternal magna (ICM) injection with laser speckle contrast imaging, preserving the integrity of the skull and leptomeninges.
Our research aim to understand the mechanism regulating brain vascular reactivity in health and disease. We study host cell type like microglia and exercise work together to maintain vascular function and how this process are disrupted in neurodegenerative disease, especially Alzheimer disease. Using the current protocol, we aim to understand how brain microglial cell regulates both basal cerebral blood flow and neurovascular coupling. In the future on that, we focus on investigating how microglial cell regulates cerebral vascular function under both hemostatic and neurodegenerative conditions.
[Narrator] To begin, weigh six C57BL/6 male mice aged 10 to 14 weeks. After anesthetizing the mice, perform the toe pinch reflex to examine whether the mice are fully anesthetized. Using a cotton swab, apply hair removal cream to the top of the skull and the back of the neck to remove the hair. Apply ophthalmic ointment to the eyes to prevent drying and reapply as needed throughout the procedure. Then place the mouse in the prone position in the stereotaxic frame and ventilate it with medical air. Ensure proper fixation and tilt the head slightly downward to form a 120-degree angle with the body to expose the cisterna magna. Sterilize the surgical area with 70% ethanol before proceeding with the surgery. Under a surgical microscope, locate the occipital crest. Using tweezers, lift the overlying skin and make a one-centimeter midline incision. Use cotton swabs to control any bleeding during the procedure. Then, using forceps, expose the neck muscles and gently separate them along the midline using two curved forceps, one in each hand, carefully pull the muscles aside to expose the cisterna magna. Next, tilt the head downward to form a 150-degree angle with the body for optimal exposure of the cisterna magna. Make a five-millimeter incision along the sagittal suture, covering both the lambda and bregma points. Using two curved forceps, gently pull the skin aside and clean the surface of the skull with saline. Position the mouse under the lens of the laser speckle contrast imaging system. Then apply a one-millimeter thick layer of ultrasound gel on top of the exposed skull to keep the area moist. Turn on the system and it's accompanying software. Use the Auto function to find the correct focus for imaging. Next, secure the pump to the stereotaxic manipulator arm and connect the cable to the controller. Turn on the controller and select the target pump. In the control panel, set direction to infuse. Adjust the speed to two microliters per minute and the volume to 4.5 microliters. After the infusion is complete, remove a glass pipette from the container. using a 34-gauge MicroFil flexible needle, or a similar type of needle, fill the pipette with colorless odorless mineral oil. Then unscrew the pump's collet. Insert the glass pipette and tighten the collet securely. Ensure there are no air bubbles inside the pipette. Now, change the direction setting in the control panel to withdraw. Adjust the speed to two microliters per minute and the volume to 4.5 microliters. Move the stereotaxic arm to gently introduce the pipette into the prepared solution. Use the withdraw function to extract the desired volume. Before injecting vasodilators, adjust the angle of the stereotaxic manipulator arm to 45 degrees in the anterior posterior plane. Under the dissection microscope, expose the cisterna magna using curved tweezers. Use the stereotaxic micromanipulator to move the glass pipette close to the dura of cisterna magna. Once the glass pipette touches the dura, gently maneuver it to pass into the center of the cisterna magna. Then change the direction in the control panel to infuse. Set the injection speed to 300 nanoliters per minute and the volume to 1.5 microliters. Launch the laser speckle contrast imaging software and adjust the focus to achieve the clearest image of the blood vessels. In the control panel, set the frame rate interval to five seconds. Record the baseline cerebral blood flow for approximately 10 minutes, ensuring the baseline remains stable. After 10 minutes of stable baseline recording, initiate the nano injection and note the injection start time in the recording system. Once the injection is complete, mark the finish time, and continue recording for an additional 20 minutes or longer. Injection of DHPG caused a sharp increase in cerebral blood flow, which declined gradually after the injection. Acetylcholine injection resulted in a gradual and sustained increase in cerebral blood flow throughout the 20-minute recording period. Adenosine injection induced a transient increase in cerebral blood flow, which returned to baseline levels within 15 to 20 minutes post injection.
Overview
This study investigates cerebral blood flow regulation in the brain by developing a minimally invasive approach utilizing nanoinjector-guided injections and laser speckle imaging. The research focuses on understanding how microglia and exercise collaborate to maintain vascular functions, particularly in the context of neurodegenerative diseases like Alzheimer's.
Key Study Components
Area of Science
- Neuroscience
- Cerebral Blood Flow Regulation
- Neurodegenerative Diseases
Background
- Understanding brain vascular reactivity is crucial for insights into health and disease.
- Microglia play a key role in regulating vascular function.
- The effects of neurodegenerative conditions on cerebral blood flow are important for developing treatment strategies.
- Traditional methods for studying blood flow are often invasive and damaging.
Purpose of Study
- To investigate how microglial cells modulate cerebral blood flow and neurovascular coupling.
- To develop a technique that allows for the preservation of brain structures during experimentation.
- To understand the interplay between vascular reactivity and neurodegenerative disease mechanisms.
Methods Used
- Minimally invasive technique combining nanoinjector-guided ICM injections and laser speckle contrast imaging.
- Utilization of C57BL/6 male mice as the biological model for testing.
- Steps included anesthetizing mice, hair removal, and sterile surgical procedures for accessing the brain.
- Critical timing for recording baseline cerebral blood flow before and after infusion of vasodilators.
Main Results
- Injection of vasodilators such as DHPG and acetylcholine induced significant increases in cerebral blood flow.
- The response of cerebral blood flow to injections displayed both transient and sustained characteristics depending on the agent used.
- Recording revealed valuable data about the dynamics of cerebral blood flow regulation during the study.
Conclusions
- This study demonstrates a new method for examining cerebral blood flow while maintaining the integrity of brain tissues.
- The work enhances understanding of microglial involvement in vascular function in health and disease contexts.
- These insights may inform future research efforts and therapeutic strategies for neurodegenerative conditions.
