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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. &#160;&#160;&#160;
1. Optic Nerve Injection
<ol>
	<li>Anesthetize the animal with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (0.5 mg/kg). 
	NOTE: Following induction of anesthesia, observe the withdrawal reflexes (pinch test, corneal and palpebral reflex, etc.) and their absence as an indication to begin surgery. Continually monitor animals throughout the surgery and administer the additional anesthetic drug (10% of the initial ketamine dose per top-up) if the reflexes are present. Adult rats (&#62;12 weeks) are used in the experiments.</li>
	<li>Shave the skin of the surgical area. Place the animal on a warming pad (37 &#176;C) to maintain body temperature during the surgery. Prepare skin through topical application of povidone-iodine. Apply surgical draping. Apply topical ophthalmic ointment to prevent corneal dryness under general anesthesia. Maintain asepsis by using sterile instruments.</li>
	<li>Make a 1- to 1.5-cm incision in the skin above the orbit of a randomly selected eye. Open the subcutaneous tissue to reach the orbital cavity using fine iris scissors. Open the conjunctiva and anterior Tenon&#39;s capsule under the operating microscope.</li>
	<li>Retract the extraocular muscles and intraorbital lacrimal glands to expose approximately 3 mm length of the optic nerve. Open the dura and arachnoid matter layers around the optic nerve longitudinally using an ophthalmic blade.</li>
	<li>Insert the glass pipette into the optic nerve at a distance of 2 mm posterior to the globe. The glass micropipette is attached to a Hamilton syringe.</li>
	<li>Inject 1% lysolecithin (0.4 - 1.0 &#181;l, with 0.02% Evan&#39;s Blue which doesn&#39;t have effects on myelination) slowly into the nerve approximately over a period of 30 sec.</li>
	<li>Suture the skin incision. Apply antibiotic ointment to prevent infection. Fellow eyes can be served as internal controls for electrophysiology recordings.</li>
	<li>Place the animals on a warming pad to recover from anesthesia.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ketamine 100 mg/ml (Ketamil)</td>
			<td>Troy Laboratories</td>
			<td>AC 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine 1 mg/ml (Domitor)</td>
			<td>Pfizer</td>
			<td>sc-204073</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoeothermic blanket system</td>
			<td>Harvard Apparatus</td>
			<td>NC9203819</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma</td>
			<td>L4129</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#39;s blue</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing local demyelination in the optic nerve using lysolecithin

Source: You, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure of surgically accessing the optic nerve in an anesthetized animal and precisely delivering lysolecithin to induce focal demyelination, creating neuropathological conditions for further study.

Inducing Local Demyelination in the Optic Nerve Using Lysolecithin

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. ...

Begin with an anesthetized rat and make an incision above the eye orbit.
Dissect the subcutaneous tissue to access the orbital cavity, which contains the optic nerve.
Under a microscope, dissect the conjunctiva, followed by the underlying connective tissue.
Then, retract the surrounding muscles and the gland. Cut the protective layers to expose the optic nerve.
The optic nerve consists of lipid-rich myelinated nerve fibers that transmit electrical signals.
Next, take a pre-assembled glass micropipette filled with lysolecithin, a lipid-disrupting agent, and a dye for visual tracking.
Insert the micropipette into the exposed optic nerve and inject the lysolecithin.
Suture the skin incision and apply antibiotic ointment to prevent infection, then allow the rat to recover.
In the optic nerve, injected lysolecithin disrupts the integrity of the myelin sheath surrounding the nerve fibers.
This results in localized demyelination, reducing signal transmission through the optic nerve.

inducing-local-demyelination-in-the-optic-nerve-using-lysolecithin

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Epilepsy and Seizure Disorders

55 Views. Fonte: Tu, Y. et al., Registrazione visiva del potenziale evocato in un modello di ratto di demielinizzazione sperimentale del nervo ottico. J. Vis. Exp. (2015) Questo video dimostra la procedura di accesso chirurgico al nervo ottico in un animale anestetizzato e la somministrazione precisa di lisolecitina per indurre la demielinizzazione focale, creando condizioni neuropatologiche per ulteriori studi. 

Video: Induzione della demielinizzazione locale nel nervo ottico mediante lisolecitina - Concept

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Tecnica mini-invasiva per iniezione in Rat nervo ottico

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Ricerca

JoVE Journal

Neuroscienze

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 ...

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

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

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

Un'iniezione a due punti stabilmente stabilita del modello di demielinizzazione focale indotta da lisofosfatidilcolina nei topi

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

Inducing Local Demyelination in the Optic Nerve Using Lysolecithin

Trascrizione

Inducing Local Demyelination in the Optic Nerve Using Lysolecithin