The authors would like to acknowledge the Center for Molecular and Genomic Imaging at UC Davis and the Biomedical Imaging Department at Genentech. This work was supported by a National Institutes of Health Bioengineering Research Partnership grant number R01 EB00993.

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
<ol>
	<li>Prepare sterile field with sterilized surgical tools and materials positioned conveniently. Ensure heating pad is warmed to 37 &#176;C with temperature probe placed securely on the pad. &#160;Be sure to use a sterile&#160;drape to cover the surgical site. &#160;</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Allow the animal to recover from anesthesia until ambulatory (approximately 30 min) and perform post-surgical monitoring until animal is ready for imaging.</li>
</ol>
2. Preparation for Imaging: System and Hardware Checks
<ol>
	<li>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.
	<ol>
		<li>Ensure the PET system is at the prescribed operating temperature of 5 &#176;C using the air cooling system.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;Ci of 18F aqueous solution and acquire for 15 min&#160;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.</li>
	</ol>
	</li>
	<li>&#160;Check the infusion pump settings and operation. Set the pump to 4.44 &#181;l per minute, which in 45 min&#160;of constant infusion delivers a total volume of 200 &#181;l, the typical recommended limit for i.v. injection in a 20 g animal.</li>
	<li>Check the heater operation and confirm that the temperature output is sufficient to keep the animal warm (37 &#176;C). Check that the temperature and respiratory monitoring is operational in preparation for animal placement on the animal bed.</li>
	<li>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.</li>
	<li>&#160;Ensure that isoflurane vaporizer is sufficiently filled. Prior to imaging, start isoflurane anesthesia flow at 1-2% and 0.5 to 1 L/min.</li>
	<li>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.</li>
</ol>
3. Imaging workflow
After all necessary equipment checks are completed, proceed to imaging as follows:
<ol>
	<li>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.</li>
	<li>Transfer the animal to the prepared animal bed. Ensure that the animal&#8217;s head is secure, with upper incisors secured by the tooth bar and ear bars in place if being used.</li>
	<li>Apply ophthalmic ointment to eyes to prevent drying. Insert rectal probe thermometer. Ensure that temperature and respiration readings are functional.</li>
	<li>Draw the radiotracer dose (around 600 &#181;Ci in 200 &#181;l) to be injected into heparinized PE-10 tubing of appropriate length &#8211; approximately 3 m for PE-10 tubing and a volume of 200 &#181;l. 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>(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.</li>
	<li>(MRI) Acquire a localized, point-resolved spectroscopic scan (PRESS) in a volume within the brain: Run a PRESS sequence (see Table 1)&#160; in a rectangular volume with dimensions 3.9 mm &#215; 6 mm&#160;&#215; 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.</li>
	<li>(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.&#160; Repeat step 3.8.</li>
	<li>(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.</li>
	<li>(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.</li>
	<li>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.</li>
	<li>(MRI) At the same time as step 3.12, begin DWI acquisition prepared in step 3.10 (scan &#8220;H1&#8221;).</li>
	<li>(MRI) Begin DWI acquisition (scan &#8220;H2&#8221;), 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.</li>
	<li>(MRI) Acquire a post-hypoxia DWI scan prepared in step 3.10. Turn off the infusion pump after this scan has completed.</li>
	<li>(MRI) Acquire anatomical images in the axial and sagittal planes. In the Scan Control Window &#8211; select the MSME sequence (see Table 1). Using the Geometry Editor, ensure that the acquisition FOV covers the brain.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Donnan, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Lancet. 371, 1614-1623 (2008).</li><li>Turner, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+cerebral+ischemia+and+the+quest+for+neuroprotection+navigating+past+failure+to+future+success+A+review.">The science of cerebral ischemia and the quest for neuroprotection navigating past failure to future success A review.</a> Journal of Neurosurgery. 118, 1072-1085 (2013).</li><li>Vannucci, R. C., Perlman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventions+for+perinatal+hypoxic+ischemic+encephalopathy.">Interventions for perinatal hypoxic ischemic encephalopathy.</a> Pediatrics. 100, 1004-1014 (1997).</li><li>Chicha, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+brain+repair+in+neonatal+hypoxia&#8211;ischemia.">Stem cells for brain repair in neonatal hypoxia&#8211;ischemia.</a> Childs Nervous System. 30, 37-46 (2014).</li><li>Barks, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+controversies+in+hypothermic+neuroprotection.">Current controversies in hypothermic neuroprotection.</a> Seminars in Fetal and Neonatal. 13 (1), 30-34 (2008).</li><li>Jantzie, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+ischemic+stroke+a+hypoxic+ischemic+injury+to+the+developing+brain.">Neonatal ischemic stroke a hypoxic ischemic injury to the developing brain.</a> Future Neurology. 3, 99-102 (2008).</li><li>James, A., Patel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic+ischaemic+encephalopathy.">Hypoxic ischaemic encephalopathy.</a> Paediatrics and Child Health. 24 (9), (2014).</li><li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxic+ischemic+encephalopathy+in+rats.">Anoxic ischemic encephalopathy in rats.</a> The American Journal of Pathology. 36 (1), (1960).</li><li>Vannucci, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+stroke+in+the+female+diabetic+db+db+mouse.">Experimental stroke in the female diabetic db db mouse.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 21, 52-60 (2001).</li><li>Sheldon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+related+brain+injury+in+neonatal+mice+subjected+to+hypoxia+ischemia.">Strain related brain injury in neonatal mice subjected to hypoxia ischemia.</a> Brain Research. 810, 114-122 (1998).</li><li>Adhami, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia+hypoxia+induces+intravascular+coagulation+and+autophagy.">Cerebral ischemia hypoxia induces intravascular coagulation and autophagy.</a> American Journal of Pathology. 169 (2), 566-583 (2006).</li><li>Catana, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+vivo+positron+emission+tomography+and+magnetic+resonance+imaging.">Simultaneous in vivo positron emission tomography and magnetic resonance imaging.</a> Proceedings of the National Academy of Sciences. 105, 3705-3710 (2008).</li><li>Judenhofer, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+PET+MRI+a+new+approach+for+functional+and+morphological+imaging.">Simultaneous PET MRI a new approach for functional and morphological imaging.</a> Nature Medicine. 14, 459-465 (2008).</li><li>Wehrl, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+PET+MRI+reveals+brain+function+in+activated+and+resting+state+on+metabolic+hemodynamic+and+multiple+temporal+scales.">Simultaneous PET MRI reveals brain function in activated and resting state on metabolic hemodynamic and multiple temporal scales.</a> Nature Medicine. 19, 1184-1189 (2013).</li><li>Judenhofer, M. S., Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+for+preclinical+PET+MRI.">Applications for preclinical PET MRI.</a> Seminars in Nuclear Medicine. 43 (1), 19-29 (2013).</li><li>Wehrl, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+Translational+PET/MR+Imaging.">Preclinical and Translational PET/MR Imaging.</a> Journal of Nuclear Medicine. 55, 11S-18S (2014).</li><li>Heiland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+Perfusion+Weighted+MR+Imaging+in+Acute+Stroke+Principles+Methods+and+Applications.">Diffusion and Perfusion Weighted MR Imaging in Acute Stroke Principles Methods and Applications.</a> Imaging Decisions MRI. 7, 4-12 (2003).</li><li>Loubinoux, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+vasogenic+edema+and+cytotoxic+edema+assessed+by+quantitative+diffusion+and+T2+magnetic+resonance+imaging.">Spreading of vasogenic edema and cytotoxic edema assessed by quantitative diffusion and T2 magnetic resonance imaging.</a> Stroke. 28, 419-427 (1997).</li><li>Ouyang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+2+[18F]fluoroacetate+kinetics+in+rodent+models+of+cerebral+hypoxia&#8211;ischemia.">Evaluation of 2 [18F]fluoroacetate kinetics in rodent models of cerebral hypoxia&#8211;ischemia.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 34 (5), 836-844 (2014).</li><li>Kuhl, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+stroke+on+local+cerebral+metabolism+and+perfusion+mapping+by+emission+computed+tomography+of+18FDG+and+13NH3.">Effects of stroke on local cerebral metabolism and perfusion mapping by emission computed tomography of 18FDG and 13NH3.</a> Annals of Neurology. 8, 47-60 (1980).</li><li>Planas, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+Brain+Imaging+in+Small+Animal+Stroke+Models+MRI+and+PET.">Noninvasive Brain Imaging in Small Animal Stroke Models MRI and PET.</a> Neuromethods. 47, 139-165 (2010).</li><li>Marik, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+of+glial+metabolism+using+2-18F-fluoroacetate.">PET of glial metabolism using 2-18F-fluoroacetate.</a> Journal of Nuclear Medicine. 50 (6), 982-990 (2009).</li><li>Mart&#237;n, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depressed+glucose+consumption+at+reperfusion+following+brain+ischemia+does+not+correlate+with+mitochondrial+dysfunction+and+development+of+infarction:+an+in+vivo+positron+emission+tomography+study.">Depressed glucose consumption at reperfusion following brain ischemia does not correlate with mitochondrial dysfunction and development of infarction: an in vivo positron emission tomography study.</a> Current Neurovascular Research. 6, 82-88 (2009).</li><li>Carson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+physiological+measurements+using+constant+infusion.">PET physiological measurements using constant infusion.</a> Nuclear Medicine and Biology. 27, 657-660 (2000).</li><li>Greve, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BOLD+effect.">The BOLD effect.</a> Methods in Molecular Biology. 771, 153-159 (2011).</li><li>Flores, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+anesthetic+agent+and+carrier+gas+on+blood+glucose+and+tissue+uptake+in+mice+undergoing+dynamic+FDG-PET+imaging+sevoflurane+and+isoflurane+compared+in+air+and+in+oxygen.">The effects of anesthetic agent and carrier gas on blood glucose and tissue uptake in mice undergoing dynamic FDG-PET imaging sevoflurane and isoflurane compared in air and in oxygen.</a> Molecular Imaging and Biology. 10, 192-200 (2008).</li><li>Delso, G., Ziegler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+MRI+system+design.">PET MRI system design.</a> European Journal of Nuclear Medicine and Molecular Imaging. 36, 86-92 (2009).</li></ol>

JM and SW are employees of Genentech.

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.&#160; 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...

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 &#8211; for example, when each instance of an event such as stroke manifests itself uniquely, with rapidly evolving pathophysiology &#8211; make it desirable and even necessary to use simultaneous measurement. Functional neuroimaging provides one such example, where simultaneous 2-deoxy-2-(18F)fluoro-&#7429;-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.

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

simultaneous pet/mri imaging during mouse cerebral hypoxia-ischemia

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-&#7429;-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.

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 ...

Watch this Scientific Journal Video about Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia at JoVE.com

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

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.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

simultaneous-petmri-imaging-during-mouse-cerebral-hypoxia-ischemia

Research

JoVE Journal

Medicine

12.2K Views.  University of California, Davis.  De hier gepresenteerde methode gebruikt simultane positron emissie tomografie en magnetische resonantie beeldvorming. In de cerebrale hypoxie-ischemie model, dynamische veranderingen in diffusie en glucosemetabolisme optreden tijdens en na beschadiging. De evoluerende en reproduceerbare schade in dit model vereist gelijktijdige aankoop als zinvolle multimodale beeldvorming gegevens moeten worden verworven. 

Video: Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Cancer Research

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Biology

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Samenvatting

Meer video's verkennen