We are grateful for the support from the SCi3 small-animal imaging facility at Stanford and Dr. Frezghi Habte for his technical assistance with the PET/CT. LC-MS is performed by the core staff at the Stanford University Mass Spectrometry (SUMS) core facility and we appreciate the staff for providing this service. We thank Horizon Therapeutics for very kindly providing the hCD19-mAb and Jodi Karnell in particular for her technical guidance and support. This work was funded by the NIH NINDS (1 R01 NS114220-01A1).

<ol>
	<li>Dobson, R., Giovannoni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis+-+a+review.">Multiple sclerosis - a review.</a> European Journal of Neurology. 26 (1), 27-40 (2019).</li><li>Karussis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+of+multiple+sclerosis+and+the+various+related+demyelinating+syndromes:+A+critical+review.">The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical review.</a> Journal of Autoimmunity. 48-49, 134-142 (2014).</li><li>Hauser, S. 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=B-cell+depletion+with+Rituximab+in+relapsing-remitting+multiple+sclerosis.">B-cell depletion with Rituximab in relapsing-remitting multiple sclerosis.</a> The New England Journal of Medicine. 358 (7), 676-688 (2008).</li><li>Chen, D., Gallagher, S., Monson, N. L., Herbst, R., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inebilizumab,+a+B+cell-depleting+anti-CD19+antibody+for+the+treatment+of+autoimmune+neurological+diseases:+Insights+from+preclinical+studies.">Inebilizumab, a B cell-depleting anti-CD19 antibody for the treatment of autoimmune neurological diseases: Insights from preclinical studies.</a> Journal of Clinical Medicine. 5 (12), 107 (2016).</li><li>Stevens, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+CD19+PET+tracer+for+detecting+B+cells+in+a+mouse+model+of+multiple+sclerosis.">Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis.</a> Journal of Neuroinflammation. 17 (1), 275 (2020).</li><li>Chaney, A. M., Johnson, E. M., Cropper, H. C., James, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+imaging+of+neuroinflammation+using+[11C]DPA-713+in+a+mouse+model+of+ischemic+stroke.">PET imaging of neuroinflammation using [11C]DPA-713 in a mouse model of ischemic stroke.</a> Journal of Visualized Experiments. (136), e57243 (2018).</li><li>Lyons, J. -. A., Ramsbottom, M. J., Cross, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+antigen-specific+antibody+in+experimental+autoimmune+encephalomyelitis+induced+by+recombinant+myelin+oligodendrocyte+glycoprotein.">Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein.</a> European Journal of Immunology. 32 (7), 1905-1913 (2002).</li><li>Haugen, M., Frederiksen, J. L., Degn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cell+follicle-like+structures+in+multiple+sclerosis-With+focus+on+the+role+of+B+cell+activating+factor.">cell follicle-like structures in multiple sclerosis-With focus on the role of B cell activating factor.</a> Journal of Neuroimmunology. 273 (1-2), 1-7 (2014).</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=Imaging+B+cells+in+a+mouse+model+of+multiple+sclerosis+using+64Cu-Rituximab+PET.">Imaging B cells in a mouse model of multiple sclerosis using 64Cu-Rituximab PET.</a> Journal of Nuclear Medicine. 58 (11), 1845-1851 (2017).</li><li>Zeglis, B. M., 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=An+enzyme-mediated+methodology+for+the+site-specific+radiolabeling+of+antibodies+based+on+catalyst-free+click+chemistry.">An enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry.</a> Bioconjugate Chemistry. 24 (6), 1057-1067 (2013).</li><li>Lyons, J. -. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+are+critical+to+induction+of+experimental+allergic+encephalomyelitis+by+protein+but+not+by+a+short+encephalitogenic+peptide.">B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide.</a> European Journal of Immunology. 29 (11), 3432-3439 (1999).</li></ol>

The CD19 antibody was provided by Horizon Therapeutics.

This article describes a streamlined method for imaging human-CD19+ B cells in a mouse model of MS using CD19-PET. Due to the heterogeneous presentation of MS and varying responses to treatments, its management in the clinic can be challenging and new approaches for therapy selection and monitoring are greatly needed. PET imaging could serve as a powerful tool for monitoring disease progression and individual response to B cell-depleting therapy. In addition to MS, CD19-PET imaging could be used to monitor B cell depletion after treatment in subtypes of lymphomas and leukemia or other B cell mediated diseases. This protocol and representative data show the utility of imaging B cells in neurological diseases.
To study human CD19+ B cells in the context of MS, we chose the B cell-dependent MOG1-125 EAE model7. Similar to other EAE models, this model presents with symptoms of progressive paralysis and infiltration of immune cells into the CNS. However, the MOG1-125 model is unique in that it is a B cell-driven model: mice contain varying numbers of B cells in the subarachnoid space in the meninges, brain stem, parenchyma, and ventricles. These lymphocytes can be sparsely scattered throughout these regions and/or form follicle-like structures, which are also observed in humans with MS8,9. In addition to using na&#239;ve mice as controls, a complete Freund&#39;s adjuvant (CFA)-only induction kit may be used (i.e., an identical induction emulsion to what is given to EAE mice sans the MOG protein). In the EAE mouse model, the blood brain barrier (BBB) is dysfunctional and allows larger entities, such as antibodies, to cross. The CD19-mAb radiotracer will only bind and remain in the CNS if B cells are present; the tracer will circulate back into the blood pool if B cells are not present. We have demonstrated this using gamma counting and ex vivo autoradiography of CNS tissues by perfusing before measuring the radioactivity levels in the tissues. We have also demonstrated this in earlier publications reporting the use of mAb-based PET radiotracers (i.e., immunoPET imaging approaches) for detecting B cells in the CNS1,2.
The DOTA chelator was used since it has been used in clinical PET imaging with copper-64 labeled peptides and antibodies, and we aim to translate the hCD19-mAb for clinical imaging of MS patients. DOTA has adequate binding affinity to copper-64 in vivo. The in vivo stability is very important because free 64Cu goes to the liver and can obscure the signal of bound radiotracer; thus, it is important to measure the signal in the liver to calculate the relative signal compared to other organs. Muscle is typically taken as a control tissue, but in the case of EAE, there can be inflammation present in the muscles. The half-life of 64Cu is 12.7 h, which affords ample time for the DOTA-hCD19-mAb to bind to its target while ensuring signal can be measured by PET. When preparing the conjugate, small-scale (75-125 &#181;g) test reactions should be performed to determine the amount of DOTA to add to mAb to produce the desired DOTA/mAb ratio (e.g., a reaction of 6-10-fold excess DOTA-NHS-ester per mol mAb may afford a conjugate of 1-2 DOTA/mAb). The reaction time and temperature (e.g., 2-4 h or overnight at 4 &#176;C or room temperature) also influences the DOTA/mAb ratio and should be optimized. A titration with nonradioactive copper can be performed to calculate the number of DOTAs per mAb; however, we recommend performing MALDI-MS and/or LC-MS for more reliable and accurate results.
The calculated DOTA/mAb ratio is an average value for a particular sample and some variation is expected. For MALDI, several shots are taken per sample for the conjugated and unconjugated mAbs. We then calculate the ratio of conjugated to unconjugated to determine the average number of DOTA/mAb. The DOTA/mAb ratio is important because too many chelators will disrupt antibody binding and too few will lead to inconsistent radiolabeling and low signal. The ratio should be very close between batches of conjugate to maintain consistent signal intensity and binding kinetics; ideally, the same batch of conjugate should be used for all experiments within a particular study. A promising technique to reduce potential effects on immunoreactivity due to possible overconjugation is to use site-specific conjugation10 whereby the chelator conjugation is site-selective on the heavy chain glycans of the antibody, thus guaranteeing the addition of 1 chelator per mAb.
The radiolabeling reaction conditions should be optimized to ensure the highest labeling efficiency and yield since differences in antibody, DOTA/mAb ratio, and 64Cu molar activity, among other conditions, will impact radiolabeling. Using the optimal 64Cu to mAb conjugate ratio may allow the radiotracer to be used without purification, reducing the time required for radiolabeling and loss due to the gravity flow column and radioactive decay. A consistent and reliable molar activity may also be achieved when the same 64Cu to mAb conjugate ratio is used, which is especially important when comparing results across multiple cohorts of mice or imaging studies. The ITLC conditions may also be modified to suit each user. If purification is necessary, an aliquot should be saved for either HPLC and/or UV/Vis spectrophotometry so that the molar activity can be calculated.
It is important to note that using radiolabeled antibodies for imaging can be challenging. It is essential that the antibody used for the radiotracer be biologically inert so as not to have a physiological effect. Moreover, since antibodies have a long blood residence, one must wait long enough for circulation, binding, and clearance of a given mAb to ensure a suitable signal-to-background without compromising image quality. Typically waiting for 20-48 h for a 64Cu-labeled mAb is sufficient but one should image at 2, 4, 6, 12, 24, 48 h post injection when assessing a new mAb PET tracer to determine the best time point for imaging in a given rodent model. The same is true for acquiring ARG images with the highest signal-to-background ratio. The representative images in this protocol were taken at 18-20 h post injection, though other time points may be used depending on the radioisotope used. Different antibodies binding to different epitopes of CD19 will produce varying results and should be rigorously characterized.
When analyzing the spinal cord signal, it is important to position mice on their backs in the scanning bed to reduce movement caused by breathing. Additionally, supine placement can help straighten the spine in mice that have increased spinal curvature due to the progression of EAE disease. Another important aspect to consider when aiming to detect signal in the spine and spinal cord is to avoid injecting MOG1-125 on the flank as the injection sites can bind the tracer due to the associated immune response in those areas. The close proximity of the injection site can interfere with spinal cord analysis; thus, injections in the chest are preferable for the application described herein.
The image analysis techniques used are specific for CNS imaging. A brain atlas tool within the image analysis software affords reproducible and reliable results as long as the registration of PET and CT is accurate. Using the semi-automated 3D brain atlas and adjusting it to fit the skull of each mouse allows for consistent ROIs between animals. Since there is currently no automated or semi-automated approach for analyzing the signal in spinal cords, manual ROIs must be drawn. Notably, when quantifying CD19+ B cells (or any cell type present in both the bone marrow and spinal cord), it is critical to eliminate the signal arising from the spinal column and bone marrow as much as possible. The reason for this is that na&#239;ve mice are known to contain more CD19+ B cells in their bone marrow than EAE mice, in which B cells leave the periphery to infiltrate the CNS5,11. This bone marrow signal can obscure the true signal in the spinal cord.
To delineate true spinal cord signal while minimizing the contribution of signal from the spinal column and bone marrow, Otsu thresholding of the CT image can be used to make an immutable ROI for the spinal column. A separate spinal cord ROI can then be easily drawn within the spinal column. The same technique can also be applied to measure bone marrow in the femur. This is a very useful method to gain insights into tracer binding in the spinal cord. However, due to the relatively low spatial resolution of PET and issues pertaining to the partial volume effect when scanning small anatomical regions of mice, use of additional ex vivo confirmatory techniques (e.g., gamma counting, ARG) enables the validation of radiotracer binding in the spinal cord without the presence of blood, cerebrospinal fluid, or spillover signal from the spinal column.
Signal in the cervical/thoracic spinal cord tends to vary in the EAE mice depending on the severity of disease and how many B cells infiltrate during the adaptive immune response. This variation in the number of B cells that infiltrate, as well as the small amount of B cells in the CNS compared to those in the pelvic/spinal bone marrow of na&#239;ve mice, can make in vivo quantitation of spinal cord tissue challenging in mice. Given the spatial resolution of PET in small animal imaging, signal from the bone marrow can spill over onto the spinal cord signal. Ex vivo biodistribution and autoradiography completed here aid in validating the PET signal of the vertebrae versus spinal cord tissue. Mice are perfused prior to dissection to remove any unbound tracer in the blood pool so that the gamma counting and autoradiography results reflect the tracer that is actually bound in each organ rather than the tracer that is in the blood pool in that organ.
Radiotracers circulate through the blood, and with antibody tracers, specifically, there is often unbound radiotracer present in the blood for weeks after the initial injection. Since we are imaging the brain and spinal cord, which have many blood vessels, it is important to understand what portion of the signal is truly due to tracer binding in brain/tissue of interest versus that present in the blood pool. It is thus necessary to divide the brain signal by signal in the heart/blood pool. In the clinical setting, the same image analysis techniques of Otsu thresholding of vertebrae and ROIs of spinal cord tissues can be used for quantification. Given the larger tissue volumes in humans compared to mice, there should be significantly less impact from partial volume effects, leading to improved accuracy and negating the need for ex vivo techniques to confirm in vivo findings. The use of PET in the clinic will allow clinicians to personalize therapy for each patient depending on their individual B cell burden.
ARG is particularly useful for acquiring high-resolution images to enable more accurate delineation of the spatial location of tracer binding in small regions such as brain stem and cerebellum. The same sections and/or adjacent sections can be saved for immunohistochemical stains to confirm the presence of B cells. We have previously stained CNS tissues with CD45R/B220 (Supplemental Figure S1) to correlate the number of B cells with PET and ARG signal5,9. The staining can then be compared spatially to the ARG results to verify that the radiotracer signal matches the staining pattern. B cells can be present in clusters or diffusely throughout the brain stem; PET sensitivity is sufficiently high to measure the signal, which is encouraging for clinical translation. For spinal cord ARG, removing the spinal cord from the vertebrae ensures that the signal measured is due to tracer binding in the spinal cord tissue rather than the bone marrow and/or blood, which can obscure PET images due to partial volume effects.
Similar to ARG, ex vivo gamma counting enables the quantification of radioactive signal in individual organs. For this particular technique, it is important to measure the wet weight of tissues and ensure they are at the bottom of their respective tubes before placing the tubes in the gamma counter. The tubes must be labeled with the mouse number and tissue, so that the correct tube is used; the tube is then weighed on a calibrated balance and organs are inserted to the nearest tenth of a microgram (0.0001 mg). Some tissues are extremely small and the difference in the tube mass before and after will be in the order of 0.0001 mg. The tissues should be weighed immediately following dissection to prevent loss of moisture, which results in a lower mass. After weighing, the brain and spinal cord tubes should be filled with PBS to prevent from drying before freezing these tissues for ARG.

MS is an immune-mediated neurological disorder; the unique presentation in each patient can make management challenging for both patients and clinicians1. The disease itself is characterized by the presence of demyelinating lesions and immune cell infiltration in the brain and spinal cord, resulting in physical and cognitive impairment2. The traditional paradigm that MS is a T cell-mediated disease was first challenged in a landmark phase II clinical trial of rituximab3, a therapy targeting the CD20+ subset of B cells. Additional B cell therapies have since been developed that target CD194, a pan B cell biomarker that is expressed on a broader range of B cells, which can be both diagnostically and therapeutically advantageous. Moreover, existing methods to assess treatment efficacy (i.e., monitoring the number of relapses and magnetic resonance imaging [MRI] activity) do not afford early measures of response-thus placing patients at significant risk of CNS damage due to suboptimal therapy selection and optimization. Hence, there is a critical need for strategies to monitor specific immune cells, such as CD19+ B cells, in real time in the CNS and periphery of MS patients.
PET imaging is a robust imaging technique that allows in vivo, whole-body visualization of a given target of interest, such as CD19. While blood draws, records of relapse rates, and lesion monitoring via MRI provide snapshots into treatment efficacy, PET imaging can allow researchers and clinicians to monitor effectiveness of a therapy across the entire body. This proactive approach to therapeutic monitoring allows clinicians to assess medication effectiveness in real time, enabling rapid adjustments as needed. Monitoring the location and density of cell populations associated with disease also permits longitudinal assessment of severity using patient-specific anatomic information. It is thus essential to establish reproducible analytical methods to reliably utilize the full potential of PET imaging in clinical and preclinical settings.
This paper describes methods (Figure 1) to perform PET imaging, ex vivo gamma counting, and autoradiography (ARG) of CD19+ B cells with a 64Cu-labeled anti-human CD19 monoclonal antibody (mAb), known as 16C4-TM (64Cu-hCD19-mAb), in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS induced in transgenic mice expressing human CD19 (hCD19) using human recombinant myelin oligodendrocyte glycoprotein 1-125 (MOG1-125). We also provide methods to assess radiotracer binding accurately and reproducibly in the brain and spinal cord, both critical sites of pathogenesis often severely affected in this and other neurodegenerative models. These techniques allow for non-invasive investigation of the role of B cells in disease pathology and have the potential to be clinically translated to assess the efficacy of anti-B cell therapies in MS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL 50 kDa MWCO Centrifugal filter</td>
			<td>MiliporeSigma</td>
			<td>UFC505008</td>
			<td>centrifugal filter</td>
		</tr>
		<tr>
			<td>64Cu-CuCl3</td>
			<td>Washington University in St. Louis; University of Wisonsin, Madison; or another vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AR-2000 Radio-TLC Imaging Scanner</td>
			<td>Eckert &#38; Ziegler</td>
			<td>AR-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoradiography cassette</td>
			<td>Cole Palmer</td>
			<td>EW-21700-34</td>
			<td>Aluminum, 8&#34; x 10&#34;</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>&#160;GE Life Sciences</td>
			<td>28-9564-78</td>
			<td>Storage Phosphor Screen BAS-IP SR 2025 E Super Resolution, 20 x 25 cm, screen only</td>
		</tr>
		<tr>
			<td>Butterfly Needle Catheter</td>
			<td>SAI Infusion Technologies</td>
			<td>BLF-24</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTA-NHS-ester</td>
			<td>Macrocyclics</td>
			<td>B-280</td>
			<td></td>
		</tr>
		<tr>
			<td>EAE Induction Kit</td>
			<td>Hooke Laboratories</td>
			<td>EK-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger Counter</td>
			<td>Ludlum</td>
			<td>14C</td>
			<td></td>
		</tr>
		<tr>
			<td>GNEXT PET/CT Scanner</td>
			<td>Sofie</td>
			<td>GNEXT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hidex Automatic Gamma Counter</td>
			<td>Hidex</td>
			<td>AMG</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Column</td>
			<td>Phenomenex</td>
			<td>&#160;00H-2146-K0</td>
			<td>5 &#956;m SEC-s3000 400 &#197;, 300 x 7.8 mm</td>
		</tr>
		<tr>
			<td>Illustra NAP-5 column</td>
			<td>Cytiva</td>
			<td>17085301</td>
			<td>DNA gravity column</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td></td>
			<td>ARG analysis software</td>
		</tr>
		<tr>
			<td>Low Protein Binding Collection Tubes (1.5 mL)</td>
			<td>Thermo Scientific</td>
			<td>PI90410</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Lite Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840281400</td>
			<td>UV-Vis micro/nano-spectrophotometer</td>
		</tr>
		<tr>
			<td>PCR tubes 0.2 mL, for DNA grade</td>
			<td>Eppendorf</td>
			<td>30124707</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon phosphor imager 9410</td>
			<td>GE Healthcare</td>
			<td>8149-30-9410</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoQuant</td>
			<td>Invicro</td>
			<td>Version 4 Patch 3</td>
			<td>PET Analysis Software; must purchase brain atlas add-on</td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>PI89882</td>
			<td>Desalting column</td>
		</tr>
	</tbody>
</table>

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.

The hCD19-mAb was DOTA-conjugated and radiolabeled with 64Cu as shown in Figure 2. EAE and na&#239;ve mice underwent PET/CT scanning (Figure 3) 18-24 h after injection with 64Cu-DOTA-hCD19-mAb. PET/CT images were co-registered using the PET analysis software, and the CNS tissues were analyzed using manual ROIs or a semi-automated 3D brain atlas. Radiotracer binding in ROIs (Figure 4) was higher in EAE mice than in na&#239;ve mice. Ex vivo gamma counting and ARG showed increased binding in the spinal cord (both lumbar and cervical thoracic segments) and brain (ARG only) of EAE mice compared to na&#239;ve (Figure 5 and Figure 6). Ex vivo gamma counting of perfused mice also showed decreased radiotracer binding in peripheral organs, including spleen, femur, and bone marrow (Figure 5), consistent with B cells leaving the periphery and infiltrating the CNS in this EAE model.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64133/64133fig02.jpg" /> 
Figure 2: Conjugation and radiolabeling scheme for generating 64Cu-labeled human-specific CD19 monoclonal antibody, 16C4-TM mAb (64Cu-DOTA-hCD19-mAb), in addition to quality control data. (A) Reaction of DOTA-NHS-ester with hCD19 monoclonal antibody to produce hCD19-DOTA conjugate (not to scale) and radiolabeling with 64Cu-CuCl3 to produce 64Cu-DOTA-hCD19-mAb.&#160;(B) Representative ITLC chromatograph. The peak at 40-60 cm is the radiolabeled antibody; unbound 64Cu-CuCl3 travels with the mobile phase and would be present from 200 to 240 cm. There is no detectable free 64Cu-CuCl3 in this chromatograph. (C) The quality control specifications of the radiolabeled antibody. Abbreviations: DOTA-NHS ester = 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester; ITLC/HPLC = instant thin layer chromatography/high-performance liquid chromatography; MALDI/LC-MS = matrix-assisted laser desorption/ionization/liquid chromatography-mass spectrometry; CPM = counts per minute. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig02large.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/64133/64133fig03.jpg" /> 
Figure 3: Photographs demonstrating how to secure mice in a 3D-printed bed within the PET scanner to enable high quality imaging of the spinal cord and brain while minimizing motion. (A) 3D printed four-mouse scanner bed (also known as &#34;mouse hotel&#34;) equipped with heating elements and anesthesia tubing. (B) Anesthetized mice in a supine position to maximize straightness of the spine; the bed position of each mouse is recorded. (C) Mice taped securely across their head to minimize motion in the brain and across the belly to minimize motion from breathing, without affecting breathing. (D) Mouse bed positioned within the scanner and taped to the scanning bed. Anesthesia tubing was connected from scanner to bed and isoflurane set to 2%. Mouse breathing was monitored to ensure appropriate isoflurane level before closing the scanner door. Abbreviation: PET = Positron emission tomography. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig03large.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/64133/64133fig04.jpg" /> 
Figure 4: Spinal cord image and brain analysis and results using PET analysis software. (A) i) ROIs (pink and tan) drawn on spine to separate lumbar from the thoracic and cervical vertebrae and prepare image for Otsu thresholding. ii) Spinal vertebrae (turquoise and red) were segmented out using Otsu Thresholding. iii) Vertebrae were then made immutable in the 3D ROI menu, and the spinal cord divided into cervical/thoracic (purple) and lumbar (navy) ROIs. iv) The vertebral ROI was removed, leaving spinal cord ROIs and representative brain atlas applied. (B) Representative analysis of PET results from various CNS regions represented as %ID/g normalized to the ROI of the heart within each animal. PET acquisition was a 10 min static scan via PET/CT imaging. Brain regions quantified using a semi-automated brain atlas approach, shown in panel A. iv) Representative results show either significance or trending toward significant increase in tracer binding in the brain and thoracic spinal cord. Statistics performed using Student&#39;s t-test (*: p &#60; 0.0332). Abbreviations: PET = Positron emission tomography; ROIs = regions of interest; CNS = central nervous system; CT = computed tomography; %ID/g = percent injected dose per gram of tissue; EAE = experimental autoimmune encephalomyelitis. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig04large.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/64133/64133fig05.jpg" /> 
Figure 5: Representative quantification of ex vivo gamma counting in various organs in EAE and na&#239;ve mice expressed as %ID/g. Post-PET scan, mice were perfused with PBS to remove the radiotracer present in the blood, either free or bound to blood-resident CD19+ B cells, and organs quickly dissected and weighed to have an accurate weight of each organ. Tracer binding is significantly decreased in the spleen and bone marrow in EAE mice compared to na&#239;ve ones. Increased radiotracer binding is observed in both the lumbar and cervical/thoracic spinal cord segments of EAE mice. The brain does not show significant increase in radiotracer signal, though it is trending toward significant increase. Statistics performed using Student&#39;s t-test (*: p &#60; 0.0332; ****: p &#60; 0.0001). Abbreviations: PET = Positron emission tomography; %ID/g = percent injected dose per gram of tissue; EAE = experimental autoimmune encephalomyelitis; PBS = phosphate-buffered saline. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64133/64133fig06.jpg" /> 
Figure 6: Ex vivo ARG images depict 64Cu-DOTA-hCD19-mAb binding in sagittal brain sections and whole spinal cords from EAE compared with naive mice. Digital phosphor storage films were scanned using a phosphor imager after being exposed to radioactive tissue samples for approximately 10 half-lives (127 h or 5 days). The resulting images reveal visually higher signal in the brain of EAE mice compared to brain sections from na&#239;ve mice, which is expected due to the regions known to contain B cells in this model5. Specifically, there is increased tracer signal in the brain stem, cerebellum, and ventricles of EAE mouse brain sections. This increase in signal for EAE mouse brain sections mirrors what was found for in the whole-brain PET quantification detailed above. Similarly, there is an increase in radiotracer binding in both the cervical/thoracic and lumbar spinal cord segments compared to na&#239;ve spinal cords, reflecting what was found using ex vivo gamma counting. Abbreviations: PET = Positron emission tomography; EAE = experimental autoimmune encephalomyelitis; Vent = ventricles; Cb = cerebellum; BS = brain stem; TSc = thoracic and cervical spinal cords combined; LSc = lumbar spinal cord. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Staining of CNS tissues of na&#239;ve and EAE mouse brain tissue with CD45R/B220. B cells are observed in brainstem, meninges, and white matter of EAE mice (n = 7 EAE, n = 5 na&#239;ve mice, average four slices per animal). This figure is from 5. Scale bars = 5 mm (low magnification [1x]) in sagittal brain images, 100 &#181;m (high magnification [20x]) in brainstem, meninges, and cerebellar white matter. <a href="https://www.jove.com/files/ftp_upload/64133/Suppl Fig 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography at JoVE.com

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.

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 ...

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1.7K Views.  Stanford University. 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.

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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

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

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

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

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

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, ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...