Imaging Brain Damage Induced by Cerebral Hypoxia-Ischemia in a Mouse Model

Samenvatting

Source: Ouyang, Y., et al. Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia. J. Vis. Exp. (2015)

This video demonstrates a technique to assess cerebral hypoxia injury via simultaneous positron-emission tomography (PET) and magnetic resonance imaging (MRI). An anesthetized mouse undergoes common carotid artery (CCA) ligation, reducing blood flow to one brain hemisphere and predisposing it to hypoxia-induced injury. PET and MRI imaging evaluate glucose metabolism and structural changes during hypoxia. MRI detects restricted water diffusion, while PET reveals reduced glucose uptake, indicating hypoxia-induced cellular stress and metabolic impairment.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 the 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 intravenous (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 the 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 Rapid Imaging with Refocused Echoes (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 diffusion-weighted imaging (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 been 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 the animal, return to the cage when ambulatory and monitor for signs of morbidity, euthanize if necessary with administration of CO2 followed by cervical dislocation as a secondary method.

Table 1. MRI pulse sequence parameters for scans described in protocol, and PET acquisition, histogram, and reconstruction parameters.

Imaging Acquistion Parameters and Hardware Acquisition
Diffusion MRI (EPI-DWI)
Acqusition time	15 min
Matrix size	256 x 64
Slices	10
FOV	30 x 14 x 8 mm
Voxel size	0.117 x 0.219 x 0.8 mm
Effective spectral bandwidth	150 kHz
TE	41 msec
TR	3,000 msec
Averages	6
k-space segments	16
b-values	0, 400, 800 sec/mm2
Anatomical MRI (MSME)
Acquisition time	5 min
Matrix size	256 x 256
Slices	16
FOV	30 x 22 x 12.8 mm
Voxel size	0.117 x 0.086 x 0.8 mm
TE	14 msec
TR	1,000 msec
Averages	1
Repetitions	1
Point-Resolved Spectroscopic Scan (PRESS)
Acquisition time	15 s
Voxel size	3.9 x 6 x 9 mm
TE	20 msec
TR	2,500 msec
Averages	6
FieldMap
Acquisition time	1 min 21 sec
1st TE	1.49 msec
2nd TE	5.49 msec
TR	20 msec
Averages	1
PET Acquisition, Histogram, and Reconstruction Parameters
Tracer	[18F]FDG
Infusion rate	4.44 µL/min
Acquisition time	60 min
Image size per slice	128 x 128
Slices	99
Voxel size	0.4 x 0.4 x 0.6 mm
Dynamic framing	12 x 300 sec
Reconstruction type	OS-MLEM (6 subsets, 6 iterations)

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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, 35mm ID	Bruker	T8100
PET system	(In-house)		4x24 LSO-PSAPD detectors,
10x10 LSO array per detector,
1.2mm crystal pitch and 14mm depth. 14 x 14 mm PSAPD. FOV: 60x35mm. 350-650 keV energy window. 16 ns 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)