Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Summary

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Abstract

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(¹⁸F)fluoro-ᴅ-glucose ([¹⁸F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [¹⁸F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

Introduction

Worldwide, stroke is the second leading cause of death and a major cause of disability ¹. The cascade of biochemical and physiological events that occur during and acutely following a stroke event occurs rapidly and with implications for tissue viability and ultimately outcome ². Cerebral hypoxia-ischemia (H-I), which leads to hypoxic-ischemic encephalopathy (HIE), is estimated to affect up to 0.3% and 4% of full-term and preterm births, respectively ^3,4. The mortality rate in infants with HIE is approximately 15% to 20%. In 25% of HIE survivors, permanent complications arise as a result of the injury, including mental retardation, motor deficits, cerebral palsy, and epilepsy ^3,4. Past therapeutic interventions have not proven worthy of adoption as standard of care, and consensus has yet to be reached that the most advanced methods, based on hypothermia, are effectively reducing morbidity ^3,5. Other issues of contention include method of administration of hypothermia and patient selection ⁶. Thus, strategies for neuroprotection and neurorestoration are still a fertile area for research⁷.

Rat models of cerebral H-I have been available since the 1960s, and subsequently were adapted to mice ^8,9. Due to the nature of the model and the location of the ligation, there is inherent variability in the outcome due to difference in collateral flow between animals ¹⁰. As a result, these models tend to be more variable compared to similar models such as middle cerebral artery occlusion (MCAo). Real time measurement of physiological changes has been demonstrated with laser Doppler flowmetry as well as diffusion-weighted MRI ¹¹. The observed intra-animal variability in cerebral flow blood during and immediately after hypoxia, as well as in acute outcomes such as infarct volume and neurological deficit, suggest that simultaneous acquisition and correlation of multimodal data would be beneficial.

Recent advances in simultaneous positron emission tomography (PET) and magnetic resonance imaging (MRI) have allowed for new possibilities in preclinical imaging ^12-14. The potential advantages of these hybrid, combined systems for preclinical applications have been described in the literature ^15,16. While many preclinical questions can be addressed by imaging an individual animal sequentially or by imaging separate animal groups, certain situations – for example, when each instance of an event such as stroke manifests itself uniquely, with rapidly evolving pathophysiology – make it desirable and even necessary to use simultaneous measurement. Functional neuroimaging provides one such example, where simultaneous 2-deoxy-2-(¹⁸F)fluoro-ᴅ-glucose ([¹⁸F]FDG) PET and blood-oxygen-level dependent (BOLD) MRI has recently been demonstrated in rat whisker stimulation studies ¹⁴.

Here, we demonstrate simultaneous PET/MRI imaging during onset of a hypoxic-ischemic stroke in which brain physiology is not at steady state, but instead is rapidly and irreversibly changing during hypoxic challenge. Changes in water diffusion, as measured by MRI and quantified by the apparent diffusion coefficient (ADC) derived from diffusion-weighted imaging (DWI), has been well characterized for stroke in clinical and preclinical data ^17,18. In animal models such as MCAo, diffusion of water in affected brain tissue drops rapidly due to the bioenergetic cascade leading to cytotoxic edema ¹⁸. These acute changes in ADC are also observed in rodent models of cerebral hypoxia-ischemia ^11,19. [¹⁸F]FDG PET imaging has been used in stroke patients to assess changes in local glucose metabolism ²⁰, and a small number of in vivo animal studies have also used [¹⁸F]FDG ²¹, including in the cerebral hypoxia-ischemia model ²². In general, these studies show decreased glucose utilization in ischemic regions, although a study using a model with reperfusion found no correlation of these metabolic changes with later infarction development ²³. This is in contrast to diffusion changes which have been associated with the irreversibly damaged core ²¹. Thus, it is important to be able to obtain the complementary information derived from [¹⁸F]FDG PET and DWI in a simultaneous manner during the evolution of stroke, as this is likely to yield meaningful information about the progression of injury and the impact of therapeutic interventions. The method we describe here is readily amenable to use with a variety of PET tracers and MRI sequences. For instance, [¹⁵O]H₂O PET imaging along with DWI and perfusion-weighted images (PWI) from MRI may be used to further explore the development of the ischemic penumbra and validate current techniques within the stroke imaging field.

Protocol

All animal handling and procedures described herein, and according to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines, were performed in accordance with protocols approved by the Association for Assessment of Accreditation of Laboratory Animal Care (AAALAC) International accredited Institutional Animal Care and Use Committee at the University of California, Davis. Proper surgery should not result in signs of any pain or discomfort in the animal, but proper steps should be taken if these signs are observed, including administration of analgesics or in some cases, euthanasia. The right side of the animals was chosen arbitrarily for the unilateral procedure described.

1. Unilateral Common Carotid Artery (CCA) Ligation

1. Prepare sterile field with sterilized surgical tools and materials positioned conveniently. Ensure heating pad is warmed to 37 °C with temperature probe placed securely on the pad.  Be sure to use a sterile drape to cover the surgical site.  
2. Anesthetize animal (isoflurane, 1-3% in air at 0.5-1 L/min), and place animal in a supine position with the tail facing away. Check anesthetization by pinching the toe - this should elicit no reaction if the animal is properly anesthetized. Apply ophthalmic ointment to the eyes.
3. Apply depilation cream to lower neck to upper chest area using 1-2 cotton swabs. Wait 1-3 min, and then remove hair and cream using wet gauze or alcohol swabs. Swab incision area with Betadine in a circular manner from inside to outside, and then change into sterile surgical gloves.
4. Using surgical scissors, make an incision of around 1 cm along the midline of the lower neck. Carefully separate outer skin from surrounding fascia using surgical scissors.
5. Using two McPherson micro iris suturing forceps, separate the right common carotid artery from fascia, taking care to avoid damaging veins or disturbing the vagus nerve.
6. Using the forceps on the right, exteriorize the right CCA in a stable position. Apply several drops of saline to prevent drying. Pass a suitable length (2-3 cm) of 6-0 silk suture underneath the right CCA, and ligate using a double square knot. Optionally, ligate again using a second length of 6-0 silk suture.
7. Reposition right CCA and clean excess fluid from opening using a sterile sponge tipped swab. Close the incision with 6-0 silk suture. Apply lidocaine topically up to 7 mg/kg.
8. Allow the animal to recover from anesthesia until ambulatory (approximately 30 min) and perform post-surgical monitoring until animal is ready for imaging.

2. Preparation for Imaging: System and Hardware Checks

1. Set up hardware and software for the MRI and PET systems and check their functionality as follows. Ensure all physical connections are secure and software settings are appropriately selected.
   1. Ensure the PET system is at the prescribed operating temperature of 5 °C using the air cooling system.
   2. Mount PET system inside the MRI bore, aligning the PET and MRI field of view (FOV) centers using known axial offsets. Mount the MRI coil inside the bore of the PET system and center the coil with the PET system and MRI magnet centers.
   3. Turn on PET electronics for power and bias voltage (Note: steps will vary by instrument). Perform a quick (5 min) scan using a ⁶⁸Ge cylinder and check the resulting sinogram to ensure all detectors are operational.
   4. Optionally acquire data to be used for a PET/MRI transformation matrix for co-registration purposes: Fill a three-dimensional phantom (e.g., three filled spheres) with 200 µCi of ¹⁸F aqueous solution and acquire for 15 min with PET. Acquire anatomical MRI data: in the Scan Control Window, select the multi-slice multi-echo (MSME) sequence (see Table 1). Repeat for all three major orientations: axial, sagittal, and coronal.
2.  Check the infusion pump settings and operation. Set the pump to 4.44 µl per minute, which in 45 min of constant infusion delivers a total volume of 200 µl, the typical recommended limit for i.v. injection in a 20 g animal.
3. Check the heater operation and confirm that the temperature output is sufficient to keep the animal warm (37 °C). Check that the temperature and respiratory monitoring is operational in preparation for animal placement on the animal bed.
4. Check the operation of the O₂ and N₂ flowmeters (for 0.5 L/min: O₂ at 57.2 mg/min and N₂ at 0.575 g/min) by powering on both with the compressed air source off and O₂ and N₂ sources on. To avoid the risk of damaging the flowmeters, do not turn them on without sufficient input pressure.
5.  Ensure that isoflurane vaporizer is sufficiently filled. Prior to imaging, start isoflurane anesthesia flow at 1-2% and 0.5 to 1 L/min.
6. Prepare animal bed by ensuring that the anesthesia, respiratory pad, and heater systems are positioned securely and functional. For additional PET/MRI co-registration accuracy, fiducial markers (e.g., capillary tubes filled with radiotracer at a similar concentration as injected for imaging) may be attached to the animal bed within the field of view.

3. Imaging workflow

After all necessary equipment checks are completed, proceed to imaging as follows:

1. Anesthetize the animal with isoflurane and insert tail vein catheter (28 G needle, PE-10 tubing less than 5 cm) filled with heparinized saline (0.5 ml heparin, 1,000 USP/ml, in 10 ml saline). Warming the animal and/or tail may improve catheter insertion accuracy. Optionally place a drop of cyanoacrylate adhesive on the site of insertion to secure the IV line.
2. Transfer the animal to the prepared animal bed. Ensure that the animal’s head is secure, with upper incisors secured by the tooth bar and ear bars in place if being used.
3. Apply ophthalmic ointment to eyes to prevent drying. Insert rectal probe thermometer. Ensure that temperature and respiration readings are functional.
4. Draw the radiotracer dose (around 600 µCi in 200 µl) to be injected into heparinized PE-10 tubing of appropriate length – approximately 3 m for PE-10 tubing and a volume of 200 µl. Connect one end of this tubing to the infusion pump syringe, and the other to the tail vein catheter line, taking care not to create punctures in the tubing.
5. Slide the animal bed forward into the bore of the magnet, making sure not to disturb the positioning of the MRI coil and any lines or cables, especially the anesthesia tubing. Ensure that the center of the brain is aligned with the centers of the MRI coil, PET system, and MRI magnet.
6. Perform tuning and matching of the MRI coil by rotating the adjustment knobs on the coil, minimizing impedance (check coil specifications) and frequency (300 MHz for ¹H at 7 Tesla) mismatches by observing the display of the high power preamplifier.
7. (MRI) After tuning and matching, acquire a scout image: select a RARE tripilot sequence and run the sequence from the Scan Control Window. Check positioning of the animal, repeating steps 3.5 and 3.6 as necessary. Reset shims to zero value.
8. (MRI) Acquire a localized, point-resolved spectroscopic scan (PRESS) in a volume within the brain: Run a PRESS sequence (see Table 1)  in a rectangular volume with dimensions 3.9 mm × 6 mm × 9 mm. Check water line width using the CalcLineWidth macro command. If the full width at half-maximum (FWHM) value is acceptable (e.g., 0.2 ppm), continue to step 3.10. If not, proceed to step 3.9.
9. (MRI) Acquire a field map: Run a FieldMap sequence (see Table 1). Use the resulting data for a multi-angle projection shim (MAPSHIM) by running the MAPSHIM macro command and selecting linear and second order (z²) local adjustments.  Repeat step 3.8.
10. (MRI) Position the slice plan for the DWI scan (see Table 1): using the Geometry Editor, ensure that the acquisition FOV is positioned to acquire the desired volume of interest within the brain. If the resulting slice plan is aligned as desired, copy this slice plan in the Scan Control Window for all subsequent DWI scans. Begin acquisition.
11. (PET) With the PET acquisition prepared and ready to begin, start the infusion pump. After the pre-determined delay in which saline from the catheter has been injected, begin the PET acquisition (see Table 1) in order to capture the entry of radiotracer. Monitor the count rate and look for gradual increase in counts indicative of a successful injection.
12. After 10-15 min, initiate the hypoxic challenge concurrent with step 3.12. To initiate hypoxic challenge, turn off medical air flow and immediately power on O₂ and N₂ flowmeters with the predetermined settings to deliver 8% oxygen and 92% nitrogen, and reduce isoflurane to 0.8%. Do not power on flowmeters without input pressure.
13. (MRI) At the same time as step 3.12, begin DWI acquisition prepared in step 3.10 (scan “H1”).
14. (MRI) Begin DWI acquisition (scan “H2”), prepared in step 3.10, immediately after scan H1 is completed. End hypoxic challenge by powering off flowmeters, restoring medical air flow, and returning isoflurane concentration to a suitable value based on physiological monitoring.
15. (MRI) Acquire a post-hypoxia DWI scan prepared in step 3.10. Turn off the infusion pump after this scan has completed.
16. (MRI) Acquire anatomical images in the axial and sagittal planes. In the Scan Control Window – select the MSME sequence (see Table 1). Using the Geometry Editor, ensure that the acquisition FOV covers the brain.
17. Remove animal, return to cage when ambulatory and monitor for signs of morbidity, euthanizing if necessary with administration of CO₂ followed by cervical dislocation as a secondary method.

Results

Figure 1 demonstrates the result of a proper ligation of the common carotid artery, prior to closing the wound with 6-0 silk suture.

In this method, data obtained from imaging is highly dependent upon the temporal arrangement of the experiment, which in turn dictates and is also dictated by experimental limitations including image acquisition schemes and equipment setup. These and other considerations are further explored in the Discussion section. With the protocol described ...

Discussion

Simultaneous anatomic MRI, and dynamic DWI-MRI and [¹⁸F]FDG PET data were successfully acquired from experimental animals during hypoxic challenge following common carotid artery ligation.  This represents a powerful experimental paradigm for multimodal imaging of the rapidly evolving pathophysiology associated with ischemic insults in the brain and could readily be extended to study other PET radiotracers (for example markers of neuroinflammation) and MRI sequences, as well as the impact of interventiona...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Surgery
Surgical scissors	Roboz	RS-5852
Forceps	Roboz	RS-5237
Hartman mosquito forceps	Miltex	7-26
2x McPherson suturing forceps, 8.5 cm	Accurate Surgical & Scientific Instruments	4473	It is useful to reduce the opening width with a band on the forceps used to hold the carotid artery
6-0 silicone coated braided silk suture with 3/8 C-1 needle	Covidien Sofsilk	S-1172
Homeothermic blanket system	Harvard Apparatus	507220F
Super glue	(Generic)
Hypoxia
Flowmeter for O2	Alicat Scientific	MC-500SCCM-D
Flometer for N2	Alicat Scientific	MC-5SLPM-D
O2 meter	MSA	Altair Pro
Imaging
7.05 Tesla MRI System	Bruker	BioSpec	20 cm inner bore diameter with gradient set. Paravision 5.1 software.
Volume Tx/Rx 1H Coil, 35 mm ID	Bruker	T8100
PET system	(In-house)		4x24 LSO-PSAPD detectors, 10x10 LSO array per detector, 1.2 mm crystal pitch and 14 mm depth. 14 x 14 mm PSAPD. FOV: 60x35 mm. 350-650 keV energy window. 16 nsec timing window.
Vessel cannulation Dumont forceps	Roboz	RS-4991
PE-10 polyethylene tubing	BD Intramedic	427401
Infusion pump	Braintree Scientific	BS-300
Animal monitoring & gating equipment	Small Animal Instruments Inc.	Model 1025	Only respiration monitoring used
Animal bed with temperature regulation	(In-house)