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>
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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 border="0" cellpadding="0" cellspacing="0">
	<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 &#38; 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></td>
			<td></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></td>
			<td></td>
			<td></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, 35mm 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, 
			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.</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 &#38; 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. 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.

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

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...