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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Lysophosphatidylcholine (LPC) solution preparation
<ol>
	<li>Dissolve 25 mg of LPC powder (see Table of Materials) with 250 &#181;L of chloroform and methanol mixed solution (1:1) to make a 10% LPC solution and transfer it to a 500 &#181;L centrifuge tube. 
	&#160;NOTE: If the LPC is not completely dissolved, place the centrifuge tube in an ultrasonic cleaner and ultrasonicate at 40 kHz for ~1 h to obtain a uniform solution.</li>
	<li>Divide the solution into 3 &#181;L/tube and store at &#8722;80 &#176;C. 
	&#160;NOTE: The solution can be stored for ~2 years.</li>
	<li>Before surgery, dilute the solution (step 1.2) with 27 &#181;L of 0.9% Sodium chloride (NaCl) solution, and keep the solution in a constant temperature water bath at 37 &#176;C. 
	&#160;&#8203;NOTE: Prepare the solution just before starting the injection.</li>
</ol>
2. Surgical preparation
<ol>
	<li>Use a 32 G, 2-inch needle to connect to a 5 &#181;L syringe (see Table of Materials). Ensure that the microliter syringe is unobstructed. Withdraw 5 &#181;L of LPC solution to prepare the injection.</li>
	<li>Anesthetize the mouse in an induction chamber connected to an isoflurane vaporizer with 3% isoflurane mixed with 100% oxygen at a rate of about 0.3 L/min.</li>
	<li>Confirm the depth of anesthesia by the lack of toe-pinch reflex while the breathing is smooth.</li>
	<li>Shave the head of the mouse between the ears using an electric shaver. Apply lubricating eye drops to prevent corneal dryness during the procedure.</li>
	<li>Then, position the mouse in a stereotaxic frame (see Table of Materials) with the dorsal side up, and secure the head with a nose cone and tooth clamp. Maintain anesthesia with 1.2%-1.6% isoflurane through its nose. 
	NOTE: Isoflurane concentration can be adjusted according to the respiratory status of the mice.</li>
</ol>
3. Surgical procedure
NOTE: Animals are placed on a heating pad during all procedures.
<ol>
	<li>Fix the mouse to the stereotaxic apparatus with bilateral ear bars. Ensure that the ear bars are level and the head is horizontal and stable.</li>
	<li>Disinfect the skin on the head by wiping it several times with iodophor, followed by alcohol in a circular motion. Then, a scalpel is used to cut a small incision of about 1.5 cm along the midline of the scalp to expose the skull.</li>
	<li>Wipe the skull with a cotton swab dipped in 1% hydrogen peroxide until the bregma, lambda, and posterior fontanelle are exposed. Place the syringe on the stereotaxic apparatus. 
	&#160;NOTE: Use hydrogen peroxide with care and avoid touching the surrounding tissues.</li>
	<li>Ensure horizontal positioning of the animal&#39;s head (both front and rear and left and right).
	<ol>
		<li>Adjust the Z-axis knob of the stereotaxic frame so that the needle tip and the skull just touch without bending, then measure the Z-axis coordinate. Check the Z coordinates of bregma and posterior fontanelle.</li>
		<li>Adjust the ear bar so that the difference between the Z coordinates of bregma and posterior fontanelle is no more than 0.02 mm. Then, follow the same method to measure the Z coordinates of the corresponding positions on the left and right sides of the midline. Adjust the ear bar to ensure the left and right are at the same level.</li>
	</ol>
	</li>
	<li>Locate the corpus callosum. Set the XYZ origin to bregma. 
	&#160;NOTE: The first injection site is 1.0 mm lateral to the bregma, 2.4 mm deep, and 1.1 mm anterior. The second injection site is 1.0 mm lateral to the bregma, 2.1 mm deep, and 0.6 mm anterior. For example, the coordinate of the bregma is (0,0,0). Measure the Z coordinate of the corresponding location marked as (-1, 1.1, X) and (-1, 0.6, Y). It can be determined the first injection site coordinate of the corpus callosum is (-1, 1.1, &#8722;[X + 2.4]), and the second injection site is (-1, 0.6, &#8722;[Y + 2.1]).</li>
	<li>Once the injection site is determined, make a mark with a sterile marker on the skull and record the coordinates.</li>
	<li>Gently drill the marked site with a skull drill (see Table of Materials). Take care to avoid any bleeding.</li>
	<li>Slowly move the needle to the given coordinates and start the injection. To induce corpus callosum demyelination, inject 2 &#181;L of LPC solution (step 1.2) at each injection site (step 3.5.) at a rate of 0.4 &#181;L/min.</li>
	<li>After injection, keep the needle in each site for an additional 10 min. 
	&#160;&#8203;NOTE: Ensure that the interval of two injections is no more than 20 min.</li>
	<li>Suture the skin with a 4-0 suture and wait for the animal to wake up within 10 min. Administer analgesics postoperatively in accordance with institutional animal care regulations. 
	&#160;NOTE: The recommended time for euthanasia can be determined according to the purpose of the experiment.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>L-&#945;-Lysophosphatidylcholine from egg yolk</td>
			<td>Sigma-Aldrich</td>
			<td>L4129-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>32 gauge needle</td>
			<td>HAMILTON</td>
			<td>7762-05</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l syringe</td>
			<td>HAMILTON</td>
			<td>80014</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed skull drill</td>
			<td>strong,korea</td>
			<td>strong204</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Hager &#38; Meisinger, Germany</td>
			<td>REF 500 104 001 001 005</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrx Animal Aneathesia Ventilator</td>
			<td>MIDMARK</td>
			<td>VMR</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Stereotaxic Instrument for Mouse</td>
			<td>Reward</td>
			<td>68507</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro syringe</td>
			<td>Reward</td>
			<td>KDS LEGATO 130</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETEASY</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>FUSUNPHARMA</td>
			<td>20152021225</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult C57BL/6 male and female mice</td>
			<td>Hunan SJA Laboratory Animal Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a focal demyelination mouse model via a two-point injection of lysophosphatidylcholine

Source: Pang, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64059">A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice.</a> J. Vis. Exp. (2022)
This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. ...

Begin with an anesthetized mouse secured in a stereotaxic apparatus with its head shaved.
Disinfect the scalp and make an incision to expose the skull.
Clean the skull to remove the connective tissue layer.
Using the reference point on the skull, mark the target site and drill a hole.
Take a syringe containing a solution of lysophosphatidylcholine, or LPC, fixed on the stereotaxic apparatus.
Determine the coordinates for two injection points in the white matter region of the brain and position the syringe accordingly.
Inject the LPC solution and keep the needle in place for some time after injection at each site.
Suture the incision and let the animal recover.
At the injection site, LPC interacts directly with the myelin, disrupting its structure and causing its breakdown.
This causes local neuronal dysfunction, thus establishing a focal demyelination model.

establishing-focal-demyelination-mouse-model-via-two-point-injection

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

27 Views. Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. (2022) This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflam...

Video: Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine - Concept

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

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

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

Demielinizzazione sperimentale e rimielinizzazione di Murine midollo spinale da iniezione focale Lysolecithin

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

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JoVE Journal

Neuroscienze

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Modello di ratto di demielinizzazione corticale cerebrale diffusa indotta da un'iniezione intracerebrale di citochine pro-infiammatorie

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

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

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

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

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

Imaging tomografico ad emissione di positroni per la misurazione in vivo del contenuto di mielina nel modello di ratto di lisolecitina della sclerosi multipla

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

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Trascrizione

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Demielinizzazione sperimentale e rimielinizzazione di Murine midollo spinale da iniezione focale Lysolecithin

Modello di ratto di demielinizzazione corticale cerebrale diffusa indotta da un'iniezione intracerebrale di citochine pro-infiammatorie

Imaging tomografico ad emissione di positroni per la misurazione in vivo del contenuto di mielina nel modello di ratto di lisolecitina della sclerosi multipla