Name: positron emission tomography imaging for in vivo measuring of myelin content in the lysolecithin rat model of multiple sclerosis
Uploaded: 2021-02-28T14:30:00
Description: 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

&#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 ...

<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...

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, ...

This protocol has the name of monitoring in-vivo myelin chains by PET imaging with PIB labeled with carbonyl in the lysolecithin rat model of multiple sclerosis. This imaging technique has the ability of allowing in-vivo images of positions in different points. Getting point touch of the data of mining chains over time.In this video, We show how to induce the animal model by stereotactic injection and how to acquire and analyze. The mining PIP images. Take out needed material for the lysolecithin solution preparation Take the lysolecithin solution weight in an electronic balance using a glass tube take the glass tube out from the electric balance open the cell line and take the right volume for prepare 1%lysolecithin solution.Put the cell line in the plastic tube close the plastic tube and shake it using your fingers. Prepare for the surgery with the animal in the right position inject Put the eye cream the animal eyes are protecting against the hydration. use a 5%codoxygen solution for cleaning the incision area of the surgery inject lidocaine where the incision will be made.Shave the hair in the heads of their skull make an incision of about two centimeters expose the scope era with udak clamps clean area with a swab and mark the bregma position the hemato syringe on the bregma injection will be made in this three marked areas through in the right coordinate. Take the hemato needle through the drute area Go to the ventrical coordinate for start the injection, Finishing the surgery, Suture the incision. Use an appropriate place for the tracer injection.The tracer must be injected intravenously, and it can be done either, In the penile vein or in the tail vein. Image acquisition of baseline time point is performed before stereotactic surgery. And the other two time points one week and four weeks after the stereotactic surgery.Open the PET scanner software and chose the field of view to be scanned during image acquisition. Position the animal in the bed. Be sure that the animal is straight.Take the eye cream to protect the animal eyes. Cover the animal bed. Push the animal bed inside the pet scanner and start acquisition.Check the animal griefing until the end of the PET scan. When one day image acquisition is finished. Take animal out of the bed.Open remote software and choose Fuse It'model select image bio at and open with operations. Use a cylinder orientation and load the image. Go to matching and open the PET image.Adjust color scale to make the brain visible. Press crop'to crop the image and adjust the window size to cover the whole brain. It's parameters are on the bottom right side of the screen press blue shift'Be sure that the animal is positioned right in the software.Select the correct MRI template. if you need it, you can adjust the coverage restriction between PET and MRI template. Press device'button.Select the right brain template. Then you can look at different brain regions available and you can select or unselect all of them. Go to outline guides, and they will be all selected.In the top button, you can select any parameter you want to appear in this statistic data. You can touch the statistic button and then you can choose between KilobeckerCSS or SUV. Copy to clipboard and close.Your data can be pasted in an Excel sheet. For quantifying the and delusion Go to the new board, select these few and apply. You can create a new void in the contralateral side.And then go to the statistic button S2B4. It is possible to see the lesion one week after injection and the lesions covered four weeks after injection. Significant difference are seen between cell lining one week and four.Following this protocol, you'll get the three steps of taking the region of interest, being the injection site and that the principle area to be analyzed. As a large student of protocol, thus taking lesion presents the mining chains over time, where the mining nation is represented by decreased trace uptake and reimagination trace uptake. It's important to emphasize that the laser listing injection must be very slow, avoiding tissue damaging and causing only mining chains which will be detect by peep PIP imaging.

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

Research

JoVE Journal

Neuroscience

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 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 ...

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 ...