The authors would like to thank the Buckwalter lab (especially Dr. Todd Peterson) for providing the mouse model and performing the dMCAO and sham surgeries. Additionally, we would like to thank Thomas Liguori from Invicro for his technical assistance with VivoQuant image analysis software, Dr. Tim Doyle, Dr. Laura Pisani, Dr. Frezghi Habte from the SCi3 small animal imaging facility at Stanford for their advice and assistance in developing this imaging protocol, and the Radiochemistry facility (especially Dr. Jun Park) for their help with the synthesis of [11C]DPA-713.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+in+stroke.">The inflammatory response in stroke.</a> J Neuroimmunol. 184 (1-2), 53-68 (2007).</li><li>Brown, R. C., Papadopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+peripheral-type+benzodiazepine+receptor+in+adrenal+and+brain+steroidogenesis.">Role of the peripheral-type benzodiazepine receptor in adrenal and brain steroidogenesis.</a> Int Rev Neurobiol. 46, 117-143 (2001).</li><li>Papadopoulos, V., Lecanu, L., Brown, R. C., Han, Z., Yao, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral-type+benzodiazepine+receptor+in+neurosteroid+biosynthesis,+neuropathology+and+neurological+disorders.">Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders.</a> Neuroscience. 138 (3), 749-756 (2006).</li><li>Scarf, A. M., Kassiou, 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+translocator+protein.">The translocator protein.</a> J Nucl Med. 52 (5), 677-680 (2011).</li><li>Cerami, C., Perani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuroinflammation+in+ischemic+stroke+and+in+the+atherosclerotic+vascular+disease.">Imaging neuroinflammation in ischemic stroke and in the atherosclerotic vascular disease.</a> Curr Vasc Pharmacol. 13 (2), 218-222 (2015).</li><li>Stefaniak, J., O'Brien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neuroinflammation+in+dementia:+a+review.">Imaging of neuroinflammation in dementia: a review.</a> J Neurol Neurosurg Psychiatry. 87 (1), 21-28 (2016).</li><li>Gerhard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TSPO+imaging+in+parkinsonian+disorders.">TSPO imaging in parkinsonian disorders.</a> Clin Transl Imaging. 4, 183-190 (2016).</li><li>Airas, L., Rissanen, E., Rinne, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuroinflammation+in+multiple+sclerosis+using+TSPO-PET.">Imaging neuroinflammation in multiple sclerosis using TSPO-PET.</a> Clin Transl Imaging. 3, 461-473 (2015).</li><li>Fan, J., Lindemann, P., Feuilloley, M. G., Papadopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+evolution+of+the+translocator+protein+(18+kDa).">Structural and functional evolution of the translocator protein (18 kDa).</a> Curr Mol Med. 12 (4), 369-386 (2012).</li><li>James, M. 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=Synthesis+and+in+vivo+evaluation+of+a+novel+peripheral+benzodiazepine+receptor+PET+radioligand.">Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand.</a> Bioorg Med Chem. 13 (22), 6188-6194 (2005).</li><li>Boutin, H., 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=11C-DPA-713:+A+novel+peripheral+benzodiazepine+receptor+PET+ligand+for+in+vivo+imaging+of+neuroinflammation.">11C-DPA-713: A novel peripheral benzodiazepine receptor PET ligand for in vivo imaging of neuroinflammation.</a> J Nucl Med. 48 (4), 573-581 (2007).</li><li>Doyle, K. P., Buckwalter, M. 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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=In+vivo+analysis+of+neuroinflammation+in+the+late+chronic+phase+after+experimental+stroke.">In vivo analysis of neuroinflammation in the late chronic phase after experimental stroke.</a> Neuroscience. 292, 71-80 (2015).</li><li>Rojas, 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=Imaging+brain+inflammation+with+[(11)C]PK11195+by+PET+and+induction+of+the+peripheral-type+benzodiazepine+receptor+after+transient+focal+ischemia+in+rats.">Imaging brain inflammation with [(11)C]PK11195 by PET and induction of the peripheral-type benzodiazepine receptor after transient focal ischemia in rats.</a> J Cereb Blood Flow Metab. 27 (12), 1975-1986 (2007).</li><li>Glazier, S. S., O'Rourke, D. M., Graham, D. I., Welsh, F. 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The authors declare no conflicts of interests.

The presented protocol describes a method for the quantification of neuroinflammation in dMCAO and sham mice using [11C]DPA-713-PET. TSPO-PET is the most widely investigated imaging biomarker for visualizing and measuring neuroinflammation in vivo to date. TSPO expression is upregulated on glia in the brain during inflammation permitting the non-invasive detection and quantification of neuroinflammation. Moreover, it is a highly translatable technique, making it a valuable tool in both clinical and pre-clinical research. This protocol and representative results highlight the suitability of using [11C]DPA-713 PET to detect and monitor neuroinflammatory alterations in stroke and other neurological disorders in vivo.
In this study, dMCAO surgery was carried out using 3-month-old C57BL/6 female mice. This model was chosen as it gives rise to a highly reproducible infarct restricted to the somatosensory cortex, providing a model of permanent focal ischemia with low variability compared to other models of stroke (e.g., middle cerebral arterial occlusion (MCAO) filament method)14. PET imaging of stroke models has the advantage of containing an internal reference region in the brain for each animal using ROIs within the contralateral hemisphere. Since there will be some inflammation that results from the surgery alone, it is important to include mice that underwent sham surgery in the study design, whereby craniotomy and manipulation of meninges without artery occlusion was performed. Craniotomy alone can result in disruption to the underlying neuronal tissue and introduction of pathogens leading to immune responses independent of stroke20. Some inflammation after sham surgery is therefore expected and should be assessed in parallel to dMCAO to exclude the possibility of signal due to surgery alone. To avoid including inflammation resulting from the surgery without stroke in dMCAO cohort analysis, MR imaging must be conducted to confirm successful stroke surgery and infarct development. MRI also provides a structural reference frame, which is essential to accurately draw the infarct and contralateral ROIs. Additionally, accurate image processing including image registration and ROI definition are necessary to ensure reliable quantification.
Additional limitations must be kept in mind when working with C-11 labeled radiotracers for PET and autoradiography studies. It is imperative to consider the short half-life (20.33 min) of C-11, with its use generally restricted to research institutes with on-site cyclotron access. Appropriate radioactivity transportation route, dose administration, and acquisition time-points must be determined in advance with a pre-prepared detailed plan of the workflow of the experiment so that the team can work quickly and efficiently. The design and set-up of this study has been outlined to accommodate imaging of 4 mice simultaneously to increase the data output obtainable when using a C-11 tracer. If possible, it is advisable to have all mice cannulated and in the middle of their CT scan by the time the C-11 tracer arrives at the imaging facility to ensure minimal radiotracer decay prior to injection. This step-by-step protocol is also best carried out by a team containing at least 3 researchers to allow for quick cannulation, dose measurement, tracer injection, PET scanning and brain sectioning prior to significant radioactive decay. It requires two people to conduct the initiation of the PET scan and injection of all 4 mice simultaneously. The reason for beginning the PET acquisition just prior to injection is to ensure the pharmacokinetics and dynamics of tracer distribution in blood and regions of interest are accurately and completely captured. Many steps may require vigorous training and practice to ensure smooth running of the experiment. In particular, this protocol is dependent on successful tail vein cannulation of C57BL/6 mice, which can be difficult due to dark hair present on their tails, and may become more challenging after stroke has occurred or if imaging the same mice at multiple time-points.
Another consideration for PET imaging includes careful recording of radiotracer dose and residual activity measurements, including the exact time of measurement. This is essential for accurate decay correction of the injected dose at the time of the scan and is used to obtain an accurate measurement of tracer uptake (i.e., % ID/g) for each ROI. It is imperative to know the exact amount of radioactivity that was present in each mouse at the time of scanning to ensure accurate image analysis. Therefore, it is advisable to synchronize the clocks on the scanner computer and dose calibrator to avoid error when using short-lived isotopes such as C-11.
Accurate PET image quantification can also be limited by the accuracy of the scanner and set-up. Hence to ensure accurate quantification of PET/CT images, it is important to carry out quality control checks for both the CT and PET components of the scanner. CT quality control checks include X-ray source conditioning, dark/light, and center off set calibrations. These calibrations measure and correct for system noise and must be performed prior to acquisition as recommended by the scanner manufacturer. Calibrations should also be performed for the PET scanner. This typically involves scanning a &#34;standard/ PET phantom&#34; scan, containing a known concentration of radioactivity. When preparing the standard, it is best to use the same radioisotope used in the study, a comparable dose to that administered to a single mouse in a volume similar to the body of a mouse, and the same acquisition parameters as animal imaging. A 20 mL syringe filled with radiotracer diluted in water is used for the standard in this protocol, with the subsequent PET imaging results used to calculate a correction factor based on the actual dose measured by the calibration detector. The correction ratio can be applied to the imaging data acquired in the experiment to ensure accurate quantification of tracer uptake in regions of interest in PET images. This accounts for the positron range of the radionuclide in addition to considering any background activity present on the day of scanning. As the dose calibrator is an integral part of the generation of this correction factor, it is imperative that this equipment is also calibrated regularly according to the manufacturer guidelines.
When conducting ex vivo autoradiography it is important to pick an optimal time-point for euthanasia after injection, to ensure high signal-to-background in region(s) of interest. Thirty minutes post-injection was chosen for [11C]DPA-713 autoradiography using data acquired during dynamic PET imaging -i.e., the in vivo dynamic TACs as a guide, while also considering the short half-life of C-11 and the time involved to section and expose the brain tissue after extraction. Considering this, [11C]DPA-713 autoradiography must be performed on a separate cohort of mice to allow for injection of a higher [11C]DPA-713 dose and a 30 minute time-point for perfusion and euthanasia under anesthesia. Performing a small in vivo PET pilot study with a 3-4 mice prior to conducting ex vivo autoradiography will be helpful for determining the optimal time point for autoradiography. An additional consideration for ex vivo autoradiography is whether to recover the mice after injection or keep them anesthetized until euthanasia. Keeping them anesthetized mimics the conditions of the scan and ensures the radiotracer distribution or excretion kinetics are not altered by recovery. Furthermore, this prevents additional stress on the mice by avoiding recovery and subsequent induction. Finally, a useful addition to the ex vivo protocol would be to assess the regional damage in the brain slices used for autoradiography via immunohistochemical staining (after radioactive decay) to generate a high-resolution image of infarct location and volume.
As there are limitations with the use of a C-11 based tracer, this protocol can easily be modified for use with a F-18 (half-life of 109.77 min) based TSPO tracer, which may be more applicable to locations without an on-site cyclotron. Additionally, this protocol describes the use of a 4-mouse imaging set-up. Although this high throughput method is optimal when using a C-11 tracer, this protocol may also be modified for those using single mouse imaging beds. Careful planning and consistent training in the techniques outlined in this protocol will lead to the generation of a wealth of data using [11C]DPA-713, which can easily be applied to probe the role of neuroinflammation in disease manifestation and progression in other rodent models of neurological disorders. Moreover, this technique could be used to assess the in vivo response to immunomodulatory therapeutics targeted at microglia/macrophages.

Stroke is the fifth leading cause of death and a major cause of disability in the United States1. Ischemic stroke represents an overwhelming majority of these cases (~87%), occurring when there is localized disruption in blood flow to the brain (e.g., by a blood clot or fatty deposit). Oxygen and nutrient supplies to the affected areas are subsequently reduced and a complex pathologic cascade is initiated resulting in neuronal death within the stroke core (infarct) in addition to the surrounding areas. Neuroinflammation is a crucial component in the pathway leading to this damage, with both resident brain immune cells (microglia) and infiltrating peripheral immune cells (neutrophils, T cells, B cells, and monocytes/macrophages) thought to contribute to this destructive cascade2,3. Activated microglia and macrophages are central to this neuroinflammatory response, with reports of both deleterious and beneficial effects following ischemic stroke2. Thus, it is imperative to assess the in vivo contribution of these cells following stroke.
PET is a powerful 3-dimensional molecular imaging technique that enables visualization of biological processes in vivo through the use of specific molecules labeled with positron (&#946;+) emitting radionuclides such as 11C, 13N, 15O and 18F. This non-invasive method has many advantages over ex vivo methods (e.g., immunohistochemistry) as it permits the acquisition of molecular information in real time, in living intact subjects, and allows for longitudinal investigation. PET imaging of TSPO, a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to quantify and track innate immune cell responses within the body, and can be utilized to assess inflammation after stroke and response to therapeutic interventions. TSPO, formerly known as the peripheral-type benzodiazepine receptor, is an 18 kDa protein that is believed to play a role in cholesterol transport and the synthesis of neurosteroids4. Moreover, evidence suggests that TSPO is involved in neuroinflammation and neuronal survival5,6, with reports of increased expression in many neurological disorders involving inflammation including stroke7, dementia8, Parkinson&#39;s disease9 and multiple sclerosis10. TSPO is located on outer mitochondrial membranes and is highly expressed in the periphery, particularly in steroid associated tissues (e.g., glands) and with intermediate levels seen in the heart, kidneys, and lungs10.However in the healthy brain, TSPO levels are low and restricted mainly to glia6,11. Upon neuronal injury, such as that observed in stroke, TSPO levels in the central nervous system (CNS) increase significantly. This observed upregulation of TSPO can be exploited to image neuroinflammation in vivo, with expression levels providing an accurate indicator of inflammation severity. Hence, the goal of this method is to accurately quantify thein vivo contribution of neuroinflammation in a mouse model of ischemic stroke using TSPO-PET.
Multiple TSPO tracers have been developed for PET imaging of neuroinflammation. Here, TSPO-PET imaging is described using [11C]DPA-71312, a promising second generation TSPO tracer, which has shown enhanced signal to noise and lower non-specific binding than the more historically used [11C]PK11195 13. As an example, the dMCAO mouse model of stroke was chosen for this method14. This model involves temporal craniotomy and permanent ligation of the distal middle cerebral artery, resulting in focal ischemia of the somatosensory cortex. This is advantageous in pre-clinical stroke research due the high reproducibility of ischemic damage and low mortality rates associated with this model. To date, TSPO-PET imaging studies have yet to be reported in the dMCAO rodent model. However, previous PET imaging studies using the middle cerebral artery occlusion (MCAO) model, a more severe and variable stroke model, in both mice and rats, have reported TSPO expression to increase from day 3 and peak around day 7 post-stroke15,16,17,18. Hence, we performed PET imaging 6 days post-dMCAO to coincide with elevated TSPO expression. [11C]DPA-713 uptake in the brain was assessed in ipsilateral (infarcted) and contralateral hemispheres. TSPO-PET was combined with structural MRI, allowing for precise delineation of infarct and contralateral regions of interest (ROIs). Here we describe both an atlas-based and an MRI-driven ROI approach to calculate [11C]DPA-713 uptake. Radiotracer uptake in spleen was also assessed to investigate peripheral levels of inflammation between groups. This method has the potential to provide critical insights into the spatiotemporal dynamics and role of specific neuroimmune interactions in stroke and other neurological diseases.

<table border="0" cellpadding="0" cellspacing="0" style="width:589px;" width="589">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Inveon PET/CT scanner</td>
			<td style="width:89px;">Siemens</td>
			<td style="width:119px;">Version 4.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MRI scanner</td>
			<td style="width:89px;">Varian</td>
			<td style="width:119px;"></td>
			<td>7 Telsa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">ParaVision software</td>
			<td style="width:89px;">Bruker</td>
			<td>Version 6.0.1</td>
			<td>MRI operating software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">VivoQuant software</td>
			<td style="width:89px;">InVicro</td>
			<td>Version 2.5</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Inveon Research Workspace software</td>
			<td style="width:89px;">Siemens</td>
			<td>Version 4.2</td>
			<td>Scanner operating software. Includes microQView, the post-processing managing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dose calibrator</td>
			<td style="width:89px;">Capintech</td>
			<td style="width:119px;"></td>
			<td>CRC-15 PET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Typhoon phosphor imager 9410</td>
			<td style="width:89px;">GE Healthcare</td>
			<td style="width:119px;">8149-30-9410</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Butterfly catheters</td>
			<td style="width:89px;">SAI Infusion Technologies</td>
			<td style="width:119px;">BFL-24</td>
			<td>27.5 G needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1 mL syringes</td>
			<td style="width:89px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Insulin syringes</td>
			<td style="width:89px;">BD</td>
			<td align="right" style="width:119px;">329461</td>
			<td>0.5 mL insulin syringes with needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 mL syringe&#160;</td>
			<td style="width:89px;">VWR</td>
			<td style="width:119px;">BD302831</td>
			<td>BD Syringe Slip Tip Graduated</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Tissue glue</td>
			<td style="width:89px;">Santa Cruz Animal Health</td>
			<td style="width:119px;">sc-361931</td>
			<td>3 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Heat lamp</td>
			<td style="width:89px;">Fluker</td>
			<td align="right" style="width:119px;">27002</td>
			<td>5.5&#34; reptile heat lamp with clamp and switch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">0.9% sterile saline</td>
			<td style="width:89px;">Pfizer</td>
			<td style="width:119px;">00409-4888-10</td>
			<td>0.9% sodium chloride for injection, 10 mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Eye lubricant</td>
			<td style="width:89px;">Watson Rugby</td>
			<td style="width:119px;">PV926977</td>
			<td>Artificial Tears Lubricant Eye Ointment, 1/8 oz</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Chux absorbent sheets</td>
			<td style="width:89px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">1420662</td>
			<td>Disposable absorbent padding</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Iris scissors</td>
			<td style="width:89px;">World Precision Instruments</td>
			<td style="width:119px;">503708-12</td>
			<td>11.5cm, Straight, 12-pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Surgical tape</td>
			<td style="width:89px;">3M Durapore</td>
			<td style="width:119px;">1538-0</td>
			<td>1/2&#34;X10 yard roll, silk, hypoallergenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Mouse PET bed</td>
			<td style="width:89px;">In house</td>
			<td style="width:119px;"></td>
			<td>4 mouse PET bed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lighter</td>
			<td style="width:89px;">Bic</td>
			<td style="width:119px;">UDP2WMDC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Isoflurane</td>
			<td style="width:89px;">Henry Schein</td>
			<td style="width:119px;">NDC 11695-6776-2</td>
			<td>Isothesia, inhalation anesthetic, 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oxygen</td>
			<td style="width:89px;">Praxiar</td>
			<td style="width:119px;">UN1072</td>
			<td>Compressed gas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Autoradiography cassette</td>
			<td style="width:89px;">Cole Palmer</td>
			<td style="width:119px;">EW-21700-34</td>
			<td>Aluminum, 8&#34; x 10&#34;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Autoradiography film</td>
			<td style="width:89px;">GE Life Sciences</td>
			<td style="width:119px;">28-9564-78</td>
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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.

Mice underwent MRI to verify successful stroke, and [11C]DPA-713 PET was carried out by scanning 4 mice simultaneously. PET, CT, and MR images were co-registered prior to manually drawing brain ROIs and performing the semi-automated split brain atlas analysis, to investigate tracer uptake in ipsilateral and contralateral regions (Figure 2).
PET/CT images and time activity curves (TACs-radiotracer activity as a function of time) display increased [11C]DPA-713 uptake in the ipsilateral versus contralateral hemispheres (Figure 3A). Quantification of dynamic PET brain images, using summed data from 50-60 min, revealed a significant increase in tracer uptake (% ID/g) in the ipsilateral (infarcted) compared to the contralateral hemisphere in dMCAO, but not in sham mice using the manually drawn ROI approach (Figure 3B). Increased uptake was also observed in the ipsilateral hemisphere between dMCAO and sham mice. No significant differences between ipsilateral and contralateral hemispheres were observed using the atlas approach, likely due to the atlas ROIs being larger than the size of the infarct (usually restricted to the somatosensory cortex), therefore diluting the signal. However, overall increased uptake in dMCAO compared to sham was observed for all ROIs, which aligns with previous reports using MCAO model mice, demonstrating increased TSPO expression in regions outside of the infarct19. Ipsilateral/contralateral ratios were increased in the dMCAO versus sham mice using both approaches; however, this difference was only significant in the cortex using the brain atlas approach due to larger variance in the ROI approach. This may be overcome by increasing the number of mice in each group. Quantification of [11C]DPA-713 uptake in spleen showed no significant differences between groups (Figure 4).
Brain dMCAO mouse PET imaging results were confirmed by ex vivo high resolution digital autoradiography (Figure 5). Increased [11C]DPA-713 uptake was observed in infarcted tissue with negligible signal in surrounding healthy brain tissue. Quantitation of these images revealed ipsilateral to contralateral ratios ranging from 1.4 to 2.09 in dMCAO mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57243/57243fig1.jpg" /> 
Figure 1: PET Scanner and Workspace Set-up. All workspaces were covered in protective absorbent padding to create a sterile environment. (A) After calibrations, a 3D-printed mouse bed, equipped for imaging 4 mice simultaneously was secured in the scanner and nose cones for all 4 mice attached to the anesthesia. (B) Necessary equipment for PET imaging were prepared in advance, including saline-filled 27.5 G catheters, eye lubricant, ethanol swabs, heat lamps, surgical tape, tissue glue, 0.5 mL dose syringes, scissors and a lighter. (C) For radiotracer injection, place saline-flush syringes and scissors at the back of the scanner. <a href="https://cloudflare.jove.com/files/ftp_upload/57243/57243fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57243/57243fig2.jpg" /> 
Figure 2: Ipsilateral/Contralateral ROI and Right/Left-Split Hemisphere Brain Atlas PET Image Analysis Process. Image analysis software was used to determine the tracer uptake in ipsilateral and contralateral regions of interest (ROIs) using manually drawn ROIs and a semi-automated 3D split-brain atlas approach. Automatic 3D PET/CT registration was carried out followed by manual registration of the brain MRI within the corresponding mouse skull defined in the CT image. The 3D ROI tool was used to manually draw ipsilateral (red) and contralateral (green) ROIs using the infarct on the MRI as a reference. For the split-brain approach, the 3D left/right-split mouse brain atlas was loaded and fitted within the skull as defined by the CT image. Brain ROIs used for quantification in this 3D mouse brain atlas included Left Cortex (Dark Grey), Left Hippocampus (Cornflower Blue), Left Striatum (Deep Pink), Right Cortex (Tomato red), Right Hippocampus (Green), and Right Striatium (Cyan). The uptake of [11C]DPA-713 in each region was obtained in nCi/cc and was subsequently converted to %ID/g by normalizing to the decay-corrected dose at time of scanning for each mouse. <a href="https://cloudflare.jove.com/files/ftp_upload/57243/57243fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57243/57243fig3.jpg" /> 
Figure 3: Representative In Vivo [11C]DPA-713 Brain Uptake in DMCAO and Sham Mice. (A) Dynamic PET/CT images and TACs demonstrate increased [11C]DPA-713 uptake in the ipsilateral cortex of mice that underwent DMCAO (n = 3) and a slight increase for sham (n = 3) operated mice, with DMCAO mice demonstrating significantly greater contrast in percentage injected dose between the infarct and contralateral side of the brain (%ID/g). (B) PET quantification (50-60 min summed) revealed significantly increased uptake in the ipsilateral ROI using the ROI approach and in the cortex (Ctx) using the split-brain atlas approach. No significant differences were found in the hippocampus (HC) or striatum (Str). Increased ipsilateral to contralateral ratios were seen using both analysis approaches but was only statistically significant in the Ctx using the brain atlas approach. * (p &#60;0.05), *** (p &#60;0.001) <a href="https://cloudflare.jove.com/files/ftp_upload/57243/57243fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57243/57243fig4v2.jpg" /> 
Figure 4: Representative In Vivo [11C]DPA-713 Spleen Uptake in dMCAO and Sham Mice. (A) [11C]DPA-713 dynamic PET/CT images showing spleen ROIs in dMCAO (n = 3) and sham (n = 3) mice. (B) Quantitative results demonstrate no significant results in spleen uptake between dMCAO and sham mice. <a href="https://cloudflare.jove.com/files/ftp_upload/57243/57243fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57243/57243fig5v2.jpg" /> 
Figure 5: Representative Autoradiography Results. Digital autoradiography images demonstrate increased [11C]DPA-713 uptake in the ipsilateral compared to contralateral hemisphere. <a href="https://cloudflare.jove.com/files/ftp_upload/57243/57243fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke at JoVE.com

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.

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 ...

pet-imaging-neuroinflammation-using-11cdpa-713-mouse-model-ischemic

Research

JoVE Journal

Medicine

12.5K Views.  Stanford University. 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.

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

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 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 ...

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.

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 ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Neuroscience

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Non-Invasive Integrated PAUSAT Whole-Brain Imaging of Ischemic Stroke

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...