&#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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+multiple+sclerosis:+2017+revisions+of+the+McDonald+criteria.">Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria.</a> Lancet Neurology. 17 (2), 162-173 (2018).</li><li>Veronese, 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=Quantification+of+C-11+PIB+PET+for+imaging+myelin+in+the+human+brain:+a+test-retest+reproducibility+study+in+high-resolution+research+tomography.">Quantification of C-11 PIB PET for imaging myelin in the human brain: a test-retest reproducibility study in high-resolution research tomography.</a> Journal of Cerebral Blood Flow and Metabolism. 35 (11), 1771-1782 (2015).</li><li>Carvalho, R. H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-11+PIB+PET+imaging+can+detect+white+and+grey+matter+demyelination+in+a+non-human+primate+model+of+progressive+multiple+sclerosis.">C-11 PIB PET imaging can detect white and grey matter demyelination in a non-human primate model of progressive multiple sclerosis.</a> Multiple Sclerosis and Related Disorders. 35, 108-115 (2019).</li><li>Stankoff, B., 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+central+nervous+system+myelin+by+positron+emission+tomography+in+multiple+sclerosis+using+[methyl-(1)(1)C]-2-(4'-methylaminophenyl)-+6-hydroxybenzothiazole.">Imaging central nervous system myelin by positron emission tomography in multiple sclerosis using [methyl-(1)(1)C]-2-(4'-methylaminophenyl)- 6-hydroxybenzothiazole.</a> Annals of Neurology. 69 (4), 673-680 (2011).</li><li>Faria, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+positron+emission+tomography+(PET)+imaging+in+multiple+sclerosis.">Myelin positron emission tomography (PET) imaging in multiple sclerosis.</a> Neural Regeneration Research. 15 (10), 1842-1843 (2020).</li><li>Pietroboni, A. 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=Amyloid+PET+as+a+marker+of+normal-appearing+white+matter+early+damage+in+multiple+sclerosis:+correlation+with+CSF+-amyloid+levels+and+brain+volumes.">Amyloid PET as a marker of normal-appearing white matter early damage in multiple sclerosis: correlation with CSF -amyloid levels and brain volumes.</a> European Journal of Nuclear Medicine and Molecular Imaging. 46 (2), 280-287 (2019).</li><li>Pytel, V., 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=Amyloid+PET+findings+in+multiple+sclerosis+are+associated+with+cognitive+decline+at+18+months.">Amyloid PET findings in multiple sclerosis are associated with cognitive decline at 18 months.</a> Multiple Sclerosis and Related Disorders. 39, (2020).</li><li>Faria, D. d. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+imaging+of+glucose+metabolism,+neuroinflammation+and+demyelination+in+the+lysolecithin+rat+model+for+multiple+sclerosis.">PET imaging of glucose metabolism, neuroinflammation and demyelination in the lysolecithin rat model for multiple sclerosis.</a> Multiple Sclerosis Journal. 20 (11), 1443-1452 (2014).</li><li>Rinaldi, 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=Galectin-1+circumvents+lysolecithin-induced+demyelination+through+the+modulation+of+microglial+polarization/phagocytosis+and+oligodendroglial+differentiation.">Galectin-1 circumvents lysolecithin-induced demyelination through the modulation of microglial polarization/phagocytosis and oligodendroglial differentiation.</a> Neurobiology of Disease. 96, 127-143 (2016).</li><li>Faria, D. d. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+imaging+of+focal+demyelination+and+remyelination+in+a+rat+model+of+multiple+sclerosis+comparison+of+[C-11]MeDAS,+[C-11]CIC+and+[C-11]PIB.">PET imaging of focal demyelination and remyelination in a rat model of multiple sclerosis comparison of [C-11]MeDAS, [C-11]CIC and [C-11]PIB.</a> European Journal of Nuclear Medicine and Molecular Imaging. 41 (5), 995-1003 (2014).</li><li>Faria, D. d. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+imaging+of+focal+demyelination+and+remyelination+in+a+rat+model+of+multiple+sclerosis:+comparison+of+[11C]MeDAS,+[11C]CIC+and+[11C]PIB.">PET imaging of focal demyelination and remyelination in a rat model of multiple sclerosis: comparison of [11C]MeDAS, [11C]CIC and [11C]PIB.</a> European Journal of Nuclear Medicine and Molecular Imaging. 41 (5), 995-1003 (2014).</li><li>vander Star, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+and+In+Vivo+Models+of+Multiple+Sclerosis.">In Vitro and In Vivo Models of Multiple Sclerosis.</a> CNS &amp; Neurological Disorders-Drug Targets. 11 (5), 570-588 (2012).</li><li>Najm, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-based+modulation+of+endogenous+stem+cells+promotes+functional+remyelination+in+vivo.">Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.</a> Nature. 522 (7555), 216 (2015).</li></ol>

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://www.jove.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://www.jove.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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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>&#160;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&#181;, 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&#225;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-&#945;-Lysophosphatidylcholine from egg yolk</td>
			<td>Sigma-aldrich</td>
			<td>L-4129</td>
			<td>25 mg - &#8805;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 &#181;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...

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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

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

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 ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

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 ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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 ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...