Imaging Cerebrospinal Fluid Transport by Contrast-Enhanced Magnetic Resonance Imaging via Intraventricular Infusion in Rodents

Summary

This article presents an intraventricular cannulation and tracer infusion protocol tailored for dynamic contrast-enhanced MRI (DCE-MRI) studies in rodents. The protocol enables continuous tracer delivery during image acquisition, allowing whole-brain mapping of tracer transport and its clearance from the brain within a single, uninterrupted imaging session.

Abstract

Intraventricular (ICV) cannulation enables the delivery of exogenous tracers or therapeutic agents directly into the lateral ventricle, the origin of the cerebrospinal fluid (CSF) circulation pathway. Infused molecules are transported by CSF bulk flow through the ventricular compartments to the subarachnoid space and subsequently enter the brain parenchyma via the perivascular space. However, conventional ICV cannulation methods typically use ferromagnetic cannulas, which are incompatible with MRI studies and therefore require infusion to be performed outside the scanner and removal of the cannula prior to imaging. The time required for animal transport and imaging setup often results in the loss of the initial, and arguably most critical, phase of tracer transport. Here, we describe an MRI-compatible ICV cannulation and infusion protocol for rodents that supports continuous, in-scanner tracer delivery during dynamic MRI acquisition. The infusion line is constructed entirely from non-magnetic materials, enabling uninterrupted image acquisition within a single session. This approach is well-suited for dynamic contrast-enhanced MRI studies of CSF dynamics, glymphatic transport, solute clearance, and intrathecal drug delivery.

Introduction

The brain is one of the most metabolically active and dynamic organs in the body, continuously generating waste products that must be efficiently cleared to maintain normal neural function¹. The accumulation of metabolic wastes is a hallmark of neurodegeneration and brain aging². The glymphatic system plays a central role in this clearance process, facilitating the removal of metabolic by-products and toxic wastes through bulk cerebrospinal fluid (CSF) flow and the exchange of CSF with interstitial fluid (ISF)^3,4,5, a process facilitated by the aquaporin-4 (AQP4) water channels located on the astrocytic endfeet⁶. A substantial understanding of CSF dynamics has been gained by studying the transport of various CSF tracers. Dynamic contrast-enhanced MRI (DCE-MRI) using gadolinium-based contrast agents (GBCAs) is particularly well suited for brain-wide evaluation of tracer transport^7,8. The clinical relevance of this approach has been demonstrated by translational work in humans. Intrathecal administration of GBCAs followed by serial MRI was used to assess CSF dynamics in the human brain⁹. Subsequent work demonstrated brain-wide tracer transport followed by overnight clearance¹⁰. This approach was recently applied to reveal impaired periarterial tracer transport in subjects with reduced intracranial pressure-volume reserve capacity, as well as in patients with idiopathic normal-pressure hydrocephalus¹¹. These studies establish DCE-MRI of CSF tracer transport as a clinically translatable approach and motivate the development of rigorous, reproducible preclinical methods for mechanistic investigation of the regulation of CSF dynamics and waste clearance.

Intra-cisterna magna (ICM) cannulation is commonly used in glymphatic studies for tracer delivery¹². However, only approximately 20% of the GBCAs delivered via this route are transported into the brain parenchyma, with the majority draining directly into the spine or lymphatics⁸. This approach also carries a risk of damaging the cerebellum or brainstem, and securing the cannula for longitudinal studies remains technically challenging. Intraventricular (ICV) cannulation has emerged as a more robust alternative^13,14,15. A recent study demonstrated its reproducibility and key advantages in delivering fluorescent tracers, including stable cannula fixation that allows free animal movement and supports repeated tracer injections in longitudinal studies¹³.

Despite these advantages, existing ICV cannulation protocols are not directly compatible with MRI studies¹⁶. Because ICV cannulation requires penetration of the brain parenchyma, it typically uses ferromagnetic cannulas that give rise to severe susceptibility artifacts in MRI images. As a result, tracer infusion must be performed outside the scanner, with the cannula removed prior to transferring the animal for imaging. A typical workflow involves acquiring a baseline scan, removing the animal for cannulation and tracer infusion, and then repositioning the animal for subsequent dynamic scans. This interrupted workflow precludes continuous, baseline-referenced acquisition and introduces repositioning misalignment that can confound voxel-wise kinetic analysis. More importantly, because the early uptake phase is typically the most dynamic yet transient portion of the tracer kinetics, performing animal transfer and scanner setup during this window results in the loss of the most informative data.

Here, we present a protocol that addresses these limitations by integrating an MRI-compatible ICV infusion line with in-scanner contrast-agent delivery capability that allows continuous dynamic MRI acquisition in rodents. This approach enables quantitative, whole-brain mapping of CSF tracer transport within a single, uninterrupted imaging session. This protocol is well-suited for studying brain-wide CSF dynamics, glymphatic transport, and solute-clearance kinetics. Although described here for adult rats, the protocol can be adapted for mice with appropriate adjustments of cannulation coordinates and infusion parameters¹⁵.

Protocol

The protocol described below was approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University (A-3145-01-2023-0015), in accordance with the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of infusion line

1. Cut a 30-mm segment of copper cannula and a 2-m length of polyethylene (PE) tubing using serrated scissors.
2. Under a microscope, gently flare one end of the PE tubing with a 30 G needle, then insert the copper cannula into the expanded end and secure the junction with cyanoacrylate adhesive. Connect the opposite end of the PE tubing to a 30 G needle.
   NOTE: Copper is highly ductile; take care not to collapse the lumen during cutting. Avoid deforming the cannula or puncturing the tubing during assembly. The length of the PE tubing should allow placement of the syringe pump outside the scanner’s fringe field.
3. Fill the cannula-tubing assembly with degassed tracer solution and connect it to a 50 µL Hamilton syringe. Wrap the syringe tip with polytetrafluoroethylene (PTFE) thread-seal tape to prevent leakage. Use caution to avoid introducing air bubbles during this process.
4. Mount the Hamilton syringe onto the syringe pump and withdraw an additional 30 µL of tracer solution into the syringe.

2. Cannulation procedure

1. Weigh the rat and anesthetize it with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Confirm adequate anesthetic depth by the toe pinch reflex. Supplement with one-third of the initial dose if necessary and recheck before proceeding.
2. Shave the head and neck, remove loose hair, and sterilize exposed skin with 70% ethanol pads.
3. Secure the rat in a stereotaxic frame and place a heating pad covered with absorbent paper beneath the animal. Apply ophthalmic ointment to both eyes to prevent corneal drying.
4. Adjust the nose clamp to level the skull. Make a midline incision and retract the skin with four hemostat clips to expose the bregma and lambda. Apply 3% hydrogen peroxide to the skull to clear residual periosteum and enhance landmark visibility.
   NOTE: Accurate skull leveling is critical for reproducible cannula placement. As a practical quality control measure, compare the mediolateral (ML) and dorsoventral (DV) coordinates at bregma and lambda: if the skull is properly leveled, the difference between the two landmarks should not exceed 0.2 mm in either ML or DV axis.
5. Mount the electric drill with a fine drill bit to the stereotaxic arm and ensure the arm is firmly locked.
6. Position the drill bit at bregma, then move 0.7 mm posterior and 1.4 mm lateral to the right. Lower the drill bit until it contacts the skull surface and record the DV coordinate.
7. Under microscope observation, advance the drill in 0.1 mm increments. Stop immediately upon breach of the skull (typical skull thickness in adult rats is 0.3–0.4 mm).
8. Replace the drill with the pre-loaded cannula on the stereotaxic arm. Advance the infusion pump briefly until fluid just emerges from the cannula tip, confirming an unobstructed fluid path.
9. Ensure the cannula is mounted perpendicular to the skull surface, then align it over the burr hole. Advance the cannula at 0.1 mm/s to a final target depth of 3.5 mm below the dural surface.
10. Apply a drop of tissue adhesive at the cannula-skull interface. Once cured, close the scalp incision with sutures and gently bend the copper cannula parallel to the skull surface to avoid contact with the MRI coil.

3. MRI setup and dynamic acquisition

1. Place the volume transmitter coil at the isocenter of the scanner.
2. Transfer the animal to an MRI-compatible cradle in the prone position. Connect the physiological monitoring system, including the respiratory sensor and rectal temperature probe. Turn on the circulating warm water bath to maintain body temperature at ~37 °C.
3. Maintain proper anesthesia throughout the scanning session using supplemental isoflurane delivered via nose cone. Adjust isoflurane concentration between 0.25% and 1.0% to maintain a respiratory rate of 50–70 breaths/min.
   NOTE: Ketamine-xylazine anesthesia administered before cannulation surgery will provide residual sedation during the initial transfer to the MRI cradle. Introduce isoflurane gradually and monitor respiration rate continuously during the scan.
   CAUTION: Isoflurane is a volatile agent that poses an inhalation hazard; use active scavenging and minimize personnel exposure.
4. Place the infusion pump outside the magnet fringe field, set the infusion rate to 1 µL/min and the infusion duration to 20 min, corresponding to a total infusion volume of 20 µL. Carefully arrange and secure the infusion line along the cradle to prevent tension and displacement.
   CAUTION: Ferromagnetic objects can become dangerous projectiles when placed near an MRI scanner. Keep all MRI-incompatible equipment outside the fringe field.
5. Mount the surface array coil directly over the dorsal skull, centered on the brain. Place a small piece of absorbent paper between the coil and the incision site to absorb any wound seepage.
6. Advance the cradle into the magnet until the brain is at the isocenter. Verify that the infusion line and physiological monitoring leads remain unobstructed.
7. Perform the initial scanner setup and acquire anatomical images to confirm successful cannula placement.
8. After acquiring baseline images, start the infusion pump and the dynamic scan simultaneously, and continue acquisition for a total of 3 h (see Table 1 for acquisition details).

Results

The surgical workstation is configured to allow precise positioning of the drill to target the lateral ventricle (0.7 mm posterior and 1.4 mm lateral to bregma; Figure 1A,D). The infusion line consists of a 30-mm copper cannula, a 2-m segment of PE tubing, and a 30 G needle connected to a Hamilton syringe (Figure 1B). The extended tubing length allows the syringe pump to be placed outside the scanner's fringe field. Following burr-hole drilling,...

Discussion

The protocol described here integrates ICV cannulation with in-scanner infusion and dynamic MRI acquisition, enabling quantitative whole-brain assessment of CSF tracer transport within a single imaging session. The values, as well as the limitations, of ICV infusion are best evaluated in comparison with ICM infusion. ICM infusion avoids trans-parenchymal cannulation and is therefore less invasive. However, because the cisterna magna lies mid-stream in the CSF circulation pathway, ICM infusion, especially at higher infusi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30 G x 1 in. Hypodermic Needle with Plastic Hub	Exel	26439
50 µL, Gastight Syringe, Luer Tip (LT)	Hamilton	80901
Anatomical Narrow Forceps	Fine Science Tools	11003-12
Artificial CSF	TOCRIS	3525
Attane Isofluran 1000 mg/g	ScanVet	55226
Copper Capillary Tube 0.32 mm OD × 0.16 mm ID	Nippon Tokushukan Mfg. Co., Ltd.	C1220-11-109
Copper Clip	IEUYO	FEBA 1801
Cotton Swab	Puritan	868-WCS
Dotarem (gadoterate meglumine) Injection, 0.5 mmol/mL	Guerbet	NDC 67684-2001-1
Drill	Foredom	Model SR
Drill Bit	Fine Science Tools	19007-05
Drill Holder	Stoelting	51630
Dumont Medical Forceps	Fine Science Tools	11252-00, 11253-20
Eye Ointment	Rugby	NDC 0536
E-Z Anesthesia EZ-7000 Classic System	E-Z Systems	EZ-7000
Graefe Forceps	Fine Science Tools	11052-10,11152-10
Heating Pad	Braintree Scientific	5381
High Temperature Cautery Kit	Fine Science Tools	18010-00
Ketaminol Vet 100 mg/mL (ketamine)	Intervet International BV	511519
KWIK-SIL Silicone Adhesive, Low Toxicity, Kit	World Precision Instruments	KWIK-SIL
Needle 5/8 in. single use, sterile, 26 G	BD	305115
Polyethylene Tubing 0.61 mm OD × 0.28 mm ID	Scandidact	SKU252
Rompin Vet 20 mg/mL (xylazine)	KVP Pharma + Veterinär Produkte GmbH	148999
Scalpel Blades	Fine Science Tools	10023-00
Scissors	Fine Science Tools	91460-11, 14007-14
Spatula	Fine Science Tools	10090-13
Stereotaxic Frame	Stoelting	51600U
Surgical Needle Holder	AgnTho’s	12502-12
Syringe Pump	New era pump system	NE-4000
Xylocain 20 mg/mL (lidocaine)	AstraZeneca	158543