&#946;-cube equipment (Molecubes NV, Belgium) was supported by the S&#227;o Paulo Research Foundation, FAPESP - Brazil (#2018/15167-1). LES has a PhD student scholarship from FAPESP - Brazil (#2019/15654-2).

All procedures were conducted in accordance with the guidelines of the National Council for the control of Animal Experimentation (CONCEA, Brazil) and were approved by the Ethics Committee for Animal Research of the Medical School of the University of Sao Paulo (CEUA-FMUSP, Brazil - protocol number: 25/15).
NOTE: In this protocol, we show how to induce a lysolecithin rat model of multiple sclerosis and how to acquire and analyze the myelin PET images.
1. Lysolecithin solution preparation
<ol>
	<li>Weigh lysolecithin (L-&#945;-Lysophosphatidylcholine from egg yolk) on an analytical scale using a conic plastic tube (1.5 mL).</li>
	<li>Add sterile saline to the tube to make 1% solution (for example: weigh 1 mg of lysolecithin and dissolve it with 100 &#181;L of saline) and dissolve the lysolecithin by shaking the tube (shaking from side to side and not turning from top to bottom).</li>
	<li>Prepare the solution just before starting the animal model induction (do not stock the final ready solution).</li>
</ol>
2. Lysolecithin rat model - Stereotaxic surgery
<ol>
	<li>Use male Wistar rats weighing between 220 - 270 g. Use gloves and mask during all procedures.</li>
	<li>Induce anesthesia with 5% isoflurane mixed in 100% O2 (1 L/min) using an induction box. Check if the animal is anesthetized by observing absence of movement (only breathing should be observed).</li>
	<li>Place the animal in stereotaxic apparatus on a heating pad. Fix the nose and ears of the animal to the equipment. 
	&#8203;NOTE: Details of how to perform stereotaxic surgery can be found in JoVE Science Education Database. Neuroscience. Rodent Stereotaxic Surgery. JoVE, Cambridge, MA, 2021.
	<ol>
		<li>Monitor the anesthesia for the whole procedure (including surgery and image acquisition). To adjust the isoflurane concentration, observe the animal breathing rate. A fast breathing rate requires higher concentration and a slow breathing rate requires lower anesthetic concentration.</li>
	</ol>
	</li>
	<li>Inject the analgesic (ketoprofen - 5 mg/kg) subcutaneously (dilute the medication to 1 mL in saline, this will help to hydrate the animal).</li>
	<li>Put eye cream in the animal&#39;s eyes to protect against dehydration.</li>
	<li>Use a 0.5% chlorhexidine solution to clean the incision area.</li>
	<li>Inject 100 &#181;L of lidocaine hydrochloride 2% subcutaneously in the region of incision.</li>
	<li>Shave the skull area.</li>
	<li>Use sterile instruments for this procedure.</li>
	<li>Using a scalpel, make an incision of about 2 cm in the skin over the skull.</li>
	<li>Expose the skull with Bulldog clamps.</li>
	<li>Use a swab to clean the skull area with 1% hydrogen peroxidase.</li>
	<li>Locate and mark the bregma (a stereotaxic rat brain atlas can help to identity the Bregma).</li>
	<li>Position the Hamilton syringe (&#181;L neuros syringes) at the bregma.</li>
	<li>Using the bregma and a stereotaxic atlas as reference, position the Hamilton syringe at the following coordinates: Antero-posterior: -0.30 mm, latero-lateral: -3.0 mm, and ventral until it touches skull bone, and mark the skull and note the coordinates orientation on paper.</li>
	<li>Drill the skull at the marked coordinate. Take care with dura mater (damaging dura mater can cause a lot of bleeding).</li>
	<li>Fill the Hamilton syringe with 7 &#181;L of lysolecithin solution (1% in saline). A greater volume can be placed in the syringe, but only 7 &#181;L will be injected (take the bubbles out of the syringe to measure the volume correctly).</li>
	<li>Position the Hamilton syringe at the previous coordinates to start the drug injection (total of 7 &#181;L into 3 different ventral stereotactic coordinates). Considering the previously noted coordinates, lower the Hamilton syringe to ventral coordinate -5.0 mm.</li>
	<li>Very slowly inject 2 &#181;L of lysolecithin solution (1 &#181;L/10 min). Wait 3 min.</li>
	<li>Take the needle up to the next ventral stereotaxic coordinate (-4.2 mm) and slowly inject 2&#181;L of lysolecithin solution (1 &#181;L/ 10 min). Wait 3 min.</li>
	<li>Inject the third ventral coordinate -3.0 mm (3 &#181;L of lysolecithin solution - 1 &#181;L/ 10 min).</li>
	<li>Wait 5 min and then remove the Hamilton syringe from the brain.</li>
	<li>Suture the skin.</li>
	<li>Remove the animal from the stereotactic apparatus and allow the animal to awaken.</li>
	<li>Return the animal to the home cage, keep the animal alone and under supervision to check for signs of discomfort.</li>
	<li>Inject subcutaneous analgesic (ketoprofen - 5 mg/kg) diluted in saline, at 24 h and 48 h after the surgery.</li>
</ol>
3. PET acquisition
<ol>
	<li>Take 7 to 20 MBq of 11C-PIB radioactivity in a syringe (1 mL is the maximum volume allowed for intravenous injection in rats).</li>
	<li>Anesthetize the animal with 5% isoflurane mixed in 100% O2 (1 L/min) using the induction box.</li>
	<li>Inject 11C-PIB (radioactivity defined in item 4.1 above) into the penile vein, or tail vein of the rat (both administration sites are fine, the decision is a personal choice. We only advice not to use a retro-orbital injection since the location is too close to the region of interest (the brain) and can impair image quality. 
	NOTE: Image acquisition of baseline time point is performed before stereotaxic injection and the other 2 time points at 1 week and 4 weeks after surgery.</li>
	<li>Remove the animal from anesthesia to allow the animal to awaken (leave the animal on a warm pad until it is completely awake).</li>
	<li>Return the animal to the home cage.</li>
	<li>Wait 30 min for the next step.</li>
	<li>Anesthetize the rats with 5% isoflurane mixed in 100% O2 (1 L/min) using an induction box.</li>
	<li>Open PET scanner software.</li>
	<li>Select Scan &#62; Pet Ready.</li>
	<li>Complete principal investigator details, Study ID, Series ID, Animal ID, Animal weight (g), and Additional notes. Select Next. 
	NOTE: Use dot (.) for decimal number in the animal weight.</li>
	<li>Edit ROI area for scanning (usually between 3 and 8). This is the area that will be included in the image (areas between the numbers 3 and 8 of the bed cover will appear in the final image).</li>
	<li>Position the anaesthetized rat on a standard rat bed of the PET scanner and turn the anesthesia on (3% isoflurane in 100% O2 is a good start, and then the anesthesia rate should be adjusted as necessary). To adjust the anesthesia, check the monitoring data of the scanner software to verify that the animal is breathing in a constant and slow rhythm. 
	NOTE: Turn on the vacuum pump coupled to the activated carbon filter to collect isoflurane gas excess (if the pump is available).</li>
	<li>Apply eye cream to protect the animal&#39;s eyes.</li>
	<li>Push the animal bed inside the PET scanner.</li>
	<li>Complete Activity details, Activity Calibration Time, Isotope (C-11), and Scan duration (20 minutes). Select Start Scan. 
	NOTE: A Pet Moving Bed message will appear, and the animal bed will move into the equipment and stop at the previously selected ROI position. Time of acquisition will start count down and number of counts per seconds will appear (CPS).</li>
	<li>In the scanner software, navigate to the Monitor tab on the left of the screen, check Change Threshold to Respiratory parameters change (BPM). 
	NOTE: A window will pop up, complete with threshold parameter (500). Select OK.</li>
	<li>Navigate to 0% POWER to change temperature parameters to heat the animal during scan. A window will pop up; complete the parameter (80 - 100). Select OK. 
	NOTE: Choose between 80 and 100% power based on room temperature conditions (the more power, the warmer it will get).</li>
	<li>Navigate back to SCAN tab.</li>
	<li>Navigate to configuration tool (gear icon) and complete Injection Time, Remaining Activity, Remaining Activity Calibration Time. Select Save. 
	NOTE: A window will pop up &#34;Are you sure you want to modify the protocol metadata?&#34;&#62; YES</li>
	<li>Navigate back to the Monitor tab (check the animal&#39;s respiratory parameters, and if necessary, increase or decrease isoflurane concentration - usually 3% in 100% O2 is enough).</li>
	<li>When PET acquisition is finished, Pet Ready message will appear on the Scan tab, check Arrow Out Icon (left of configuration tool) to move out the animal bed.</li>
	<li>Remove the animal from anesthesia and allow to awaken on a warm pad.</li>
	<li>For reconstructing the image, navigate to Reconstruct tab.</li>
	<li>Check plus icon at bottom left of the screen, and select the study file which will be reconstructed. Select Next.</li>
	<li>Navigate to Energy Resolution tool. A window will pop up. Configure the energy peak (keV) to the equipment parameter (based on monthly quality control). Select Close. 
	NOTE: Energy peak can change every month when performing monthly equipment calibration.</li>
	<li>Keep all other parameters as the default (Isometric voxel size: 400 mm; number of iterations: 30; Energy resolution: 30%; Save last iteration only; uncheck Keep binary data) .</li>
	<li>Check Add. 
	&#8203;NOTE: A window will pop up &#34;The reconstruction was added to the queue and will start once the others have finished&#34;. Select OK. A file will appear in the reconstruction list on the Reconstruct tab and the status will appear as waiting or % (progress) or finished.</li>
</ol>
4. Image Analysis
NOTE: Perform image analysis using dedicated image analysis software. In the current protocol the demonstration uses a specific software program, but if it is not available, other options can be used.
<ol>
	<li>Open PMOD &#62; Fuse it.</li>
	<li>Navigate to the Matching tab at the top of the screen.</li>
	<li>Open the Load Input menu in the right middle of the screen and select Autodetect files, select PET file (DICOM, Interfile or NifTI), add to selected, click with Operations, Reorient to Standard Orientation, click Load and Close.</li>
	<li>Verify if the animal Species is correct at the bottom of the screen (RAT).</li>
	<li>Check Crop box at the bottom right of the screen.</li>
	<li>Adjust the yellow box size in the PET image to take the whole brain.</li>
	<li>Click the Rigid button on the right of the screen. At confirmation window click Yes.</li>
	<li>Click on the brain tool at the right middle of the screen to open Reference Atlas. Select Px Rat (W.Schiffer) - T2.</li>
	<li>Select Match Results from the top right tab (Select processing stage tab).</li>
	<li>Click Data reslicing (4thtab at top right menu).</li>
	<li>Align the PET image with reference template using the rotate tool (white icon at center of PET image). Check alignment for 3 anatomical planes.</li>
	<li>Save the co-registration file (right side menu of the screen).</li>
	<li>Select output format (DICOMor NifTI), directory and file prefix. Click Save. 
	NOTE: If co-registered output is saved as NifTi the header image information will be lost.</li>
	<li>Click VOIs menu at the bottom of the screen.</li>
	<li>Navigate to Template &#62; Atlas at the bottom of the screen (below VOIs window).</li>
	<li>Select Px Rat(W.Schiffer) from the drop-down menu. 
	NOTE: If you need to analyze only specific VOIs you can select only the brain areas of interest. If you want all brain areas of the brain atlas, no action is needed.</li>
	<li>Click Outline at the bottom of the screen. 
	NOTE: VOIs of brain areas will appear in VOIs window.</li>
	<li>Manually draw the VOI in the lesion site and contralateral brain hemisphere area (11C-PIB low uptake area and contralateral brain hemisphere, respectively).</li>
	<li>Click in the area of PET image with 11C-PIB low uptake.</li>
	<li>Select New VOI &#62; OK. 
	NOTE: A spreadsheet will appear</li>
	<li>Navigate to SPHERE icon in the middle part of the screen (to the left of the VOIs window) 
	NOTE: A spreadsheet will appear.</li>
	<li>Choose the VOI name: lesion and select Apply. 
	NOTE: A sphere will appear in the co-registered image.</li>
	<li>Align the sphere in the lesion area and adjust the VOI for all anatomical planes.</li>
	<li>Click Close (at the bottom part of the spreadsheet).</li>
	<li>Select the lesion VOI (from VOIs list).</li>
	<li>Navigate to VOI mirroring operations icon (at the top right of the VOIs window) and select Clone and mirror left/right.</li>
	<li>Navigate to Calculate Selected VOI Statistics at the top of the VOIs list window. Navigate to Select statistics to be calculated icon to choose the output data (to the right side of the VOI statistics icon). Usually for myelin content check Statistics for group of VOIs, Average, SD, Min, Max). 
	NOTE: A spreadsheet will appear. The default setting Data Unit is kBq/cc. On the left top of the spreadsheet (VOI Statistics) you can check the Data Unit as SUV (Standardized Uptake Value). If you completed the radiotracer administration and image acquisition details correctly in the scanner software, as described previously, the SUV data will be calculated automatically by PMOD software, if not, you can edit the details.</li>
	<li>Navigate to Copy using system locale number format. 
	NOTE: Verify if all statistics are selected to copy as output (Check icon at the right top of the screen). The data that will be copied depends on the Data Unit selected.</li>
	<li>Paste the output data in a notepad or spreadsheet. 
	NOTE: Take care with software settings for dot and comma symbols between numbers. This can be different between language configurations.</li>
	<li>Save the file with study name and details.</li>
</ol>

<ol>
	<li>Oh, J., Vidal-Jordana, A., Montalban, X. <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:+clinical+aspects.">Multiple sclerosis: clinical aspects.</a> Current Opinion in Neurology. 31 (6), 752-759 (2018).</li><li>Sand, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification,+diagnosis,+and+differential+diagnosis+of+multiple+sclerosis.">Classification, diagnosis, and differential diagnosis of multiple sclerosis.</a> Current Opinion in Neurology. 28 (3), 193-205 (2015).</li><li>Thompson, A. 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The biggest advantage of using the lysolecithin model to study multiple sclerosis is the fast timeline for demyelination (about 1 week) and remyelination (about 4 weeks) to occur14. This model can also be induced in mice15, however, induction in rats is more advantageous for in vivo PET imaging due to the larger size of the rat brain compared to mice.
The first step of the induction model is to be extremely cautious. This model was valid...

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/62094">Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis</a>. The citation&#160;was updated.
The citation was updated from:
de Paula Faria, D., Cristiano Real, C., Estessi de Souza, L., Teles Garcez, A., Navarro Marques, F. L., Buchpiguel, C. A. Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis. J. Vis. Exp. (168), e62094, doi:10.3791/62094 (2021).
to:
de Paula Faria, D., Real, C.C., Estessi de Souza, L., Teles Garcez, A., Navarro Marques, F. L., Buchpiguel, C. A. Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis. J. Vis. Exp. (168), e62094, doi:10.3791/62094 (2021).

An erratum was issued for:&#160;<a href="https://jove.unicartagenaproxy.elogim.com/t/62094">Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis</a>. The citation&#160;was updated.

Erratum: 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 that affects the central nervous system, characterized by inflammation, demyelination, and axonal loss1. The prognosis of this disease is variable even with advances in treatment, and it is one of the most common causes of neurological deficits in young people1. The diagnosis of MS is based on the criteria of clinical manifestation and visualization of characteristic lesions by magnetic resonance imaging (MRI)2,3.
Positron emission tomography (PET) can be a useful tool for in vivo monitoring of MS progression and therapeutic effects. The Pittsburgh compound B radiotracer (PIB) labeled with carbon-11 (11C-PIB) is widely used to quantify &#946;-amyloid plaques; however, in the last decade, it has been studied to quantify myelin content and show dynamic demyelination and remyelination4,5,6.
Different amyloid PET tracers (11C-PIB, 18F-florbetaben,18F-florbetapir, 18F-flutemetamol) can be used to quantify myelin and provide important information about disease progression and therapeutic response, allowing identification of demyelination and remyelination processes, without the interference of neuroinflammation, which can occur with conventional magnetic resonance images (MRI)7. Amyloid PET imaging showed decreased tracer uptake in active MS patients compared to non-active patients which could be explained by early white matter damage in the active patients8. Lower amyloid tracer uptake was also associated with cognitive decline in a follow-up study, showing this technique to be a valuable tool for studying the pathophysiology of the disease and clinical outcomes9.
The lysolecithin (LPC) rat model is a chemical induced model of multiple sclerosis, where the injected toxin, LPC, induces a high response of macrophages that results in increased inflammation and, consequently, demyelination10,11. The demyelination is rapidly reversed, in approximately 4 weeks, which makes this a good model for evaluating demyelination and remyelination processes in rodents. This model has already been evaluated using PET imaging, with good results and correlation with post-mortem essays12.
Here we present the protocol for myelin PET imaging with 11C-PIB in the lysolecithin rat model, showing this imaging technique to be a useful tool for in vivo measurement of myelin content.

<table><tbody><tr><td>Analytical Balance</td><td>Marte</td><td>AUWZZOD</td><td>max: 220 g- min: 1 mg</td></tr><tr><td>Anestesia vaporizer</td><td>Nanitech</td><td>15800</td><td></td></tr><tr><td>Beta-cube</td><td>Molecubes</td><td></td><td></td></tr><tr><td>Bulldog clamp</td><td>Stoelting</td><td>5212043P</td><td></td></tr><tr><td>clorexidine</td><td>Rioquimica</td><td></td><td>0.5%/100 mL</td></tr><tr><td>Cotton swabs</td><td>johnson e johnson</td><td></td><td></td></tr><tr><td>Dose calibrator</td><td>Capintech</td><td></td><td></td></tr><tr><td>Drill</td><td>Kinzo powertools</td><td>352901</td><td>Model Q0M-DC3C</td></tr><tr><td>Eppendorf tube</td><td>Eppendorf</td><td>30125150</td><td>1.5 mL</td></tr><tr><td>Eye lubricant</td><td>ADVFARMA</td><td>30049099</td><td>&amp;nbsp;vaseline 15 g (pharmaceutical purity)</td></tr><tr><td>Fine forceps</td><td>Stoelting</td><td>52102-38P</td><td></td></tr><tr><td>Gloves</td><td>Descarpack</td><td>212101</td><td>&amp;nbsp;6.5 size</td></tr><tr><td>Heating pad</td><td>Softhear</td><td></td><td></td></tr><tr><td>Injection Syringe</td><td>Hamilton</td><td>80314</td><td>10&amp;micro;, 32ga, model 701</td></tr><tr><td>Insuline syringe</td><td>BD</td><td>328328</td><td>1 mL insulin syringes with needle</td></tr><tr><td>Isoflurane</td><td>Crist&amp;aacute;lia</td><td>410525</td><td>100 mL , concentration 1 mL/1 mL</td></tr><tr><td>Ketoprofen or other analgesic</td><td>Sanofi</td><td></td><td>100 mg/2 mL</td></tr><tr><td>lidocaine</td><td>Hipolabor</td><td>1.1343.0102.001-5</td><td>2%/20mL</td></tr><tr><td>L-&amp;alpha;-Lysophosphatidylcholine from egg yolk</td><td>Sigma-aldrich</td><td>L-4129</td><td>25 mg - &amp;ge;99%, Type I, powder</td></tr><tr><td>Needle holder</td><td>Stoelting</td><td>5212290P</td><td></td></tr><tr><td>Oxygen</td><td>White Martins</td><td>7782-44-7</td><td>Compressed gas</td></tr><tr><td>PMOD software</td><td>PMOD technologies</td><td>Version 4.1</td><td>module fuse it</td></tr><tr><td>Rat anesthesia mask</td><td>KOPF</td><td>Model 906</td><td></td></tr><tr><td>Saline</td><td>Farmace</td><td>0543325/ 14-8</td><td>0.9% sodium chloride for injection, 10 mL</td></tr><tr><td>Scapel blades</td><td>Stoelting</td><td>52173-10</td><td></td></tr><tr><td>Scapel handles</td><td>Stoelting</td><td>52171P</td><td></td></tr><tr><td>Scissor</td><td>Stoelting</td><td>52136-50P</td><td></td></tr><tr><td>Semi-analytical Balance</td><td>Quimis</td><td>BK-3000</td><td>max:3,100 g; min:0.2 g</td></tr><tr><td>shaver</td><td>Mega profissional</td><td></td><td>AT200 model</td></tr><tr><td>Stereotactic Apparatus</td><td>KOPF</td><td>Nodel 900</td><td></td></tr><tr><td>Universal holder with needle support</td><td>KOPF</td><td>Model 1772-F1</td><td>Hamilton support for 5 and 10 &amp;micro;L</td></tr></tbody></table>

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.

Figure 1&#160;shows illustrative 11C-PIB PET images with myelin changes over time. In the baseline scan, no differences can be seen in myelin content (i.e., no demyelination is present). In the 1-week time-point image, it is possible to see the focal demyelinated lesion (in the right hemisphere) as indicated by the white arrow. Images are presented in the 3 anatomical planes (coronal, axial, and sagittal) and it is possible to identify the demyelinated lesion in all of them. The 1...

Watch this Scientific Journal Video about Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis at JoVE.com

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.

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, ...

positron-emission-tomography-imaging-for-vivo-measuring-myelin

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

4.0K Views.  Universidade de Sao Paulo. 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.

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

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Cancer Research

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Medicine

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Bioengineering

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 ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). Lysophosphatidylcholine (LPC) is an endogenous lysophospholipid associated with inflammation, and it could induce rapid damage with toxicity to myelin lipids, leading to focal demyelination. Here, a detailed protocol is presented for stereotactic two-point LPC injection that could directly cause severe demyelination and replicate the experimental demyelination injury quickly and stably in mice by surgical procedure. Thus, this model is highly relevant to demyelination diseases, especially MS, and it can contribute to the related advancing clinically-relevant research. Also, immunofluorescence and Luxol fast blue staining methods were used to depict the time course of demyelination in the corpus callosum of mice injected with LPC. In addition, the behavioral method was used to evaluate the cognitive function of mice after modeling. Overall, the two-point injection of lysophosphatidylcholine via a stereotaxic frame is a stable and reproducible method to generate a demyelination model in mice for further study.

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

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 ...

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

Summary

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Chapters in this video

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

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

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

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