This work was supported by grants from the Ministry of Science and Technology (106-2320-B-037-024), Kaohsiung Medical University (KMU-M106028, KMU-S105034) and Aim for the Top Universities Grant (TP105PR15), Kaohsiung Medical University, Taiwan.

All the procedures described are in accordance with ethical guidelines for laboratory animals30, and the protocol has been approved by the Animal Committee of Kaohsiung Medical University, Kaohsiung, Taiwan.
1. Establishment of RTX Neuropathy
CAUTION: RTX is neurotoxic and hazardous. On contact, it acts as an irritant to the eyes, mucous membranes, and upper respiratory tract. Avoid inhalation and wear lab eyeglasses and coats during RTX preparation. Rinse with plenty of water in case of skin contact or after handling.
<ol>
	<li>Add 1 mg of RTX powder to a 200 &#181;L of mixture of equal volume of Tween 80 and absolute ethanol (100 &#181;L for each solvent).</li>
	<li>Aliquot the RTX solution (12 &#181;L/vial) and store at -20 &#176;C, for up to 3 months. This constitutes the RTX stock; discard the remaining RTX solution at expiration.</li>
	<li>Dilute the RTX stock with normal saline to a final volume of 600 &#181;L. The final concentration of the RTX solution should be 0.01%, which equals 1 &#181;g RTX in 10 &#181;L vehicle solution.</li>
	<li>Use 8-week-old adult male ICR mice (35-40 g) as experimental animals and administer a single dose of RTX solution (dose: 200, 50, and 10 &#181;g/kg, respectively) intraperitoneally (i.p.) with a microinjection syringe to the mice.&#160;Mice was anesthetized by inhaler with 5 % isoflurane for deep anesthesia. If mice showed withdrawal action of extremities during RTX injection, mice should take longer inhalation of anesthesia.&#160;&#160; 
	Example: If the mouse weighs 40 g, then it will receive 20 &#181;L of the RTX solution, representing the dose of 50 &#181;g/kg.</li>
	<li>Give one group of mice an equal volume of vehicle (10% Tween 80 and 10% absolute ethanol in saline solution), as a control.</li>
	<li>After RTX injection, return the mice to a plastic cage on a 12-h light/12-h dark cycle and provide food and water ad libitum.</li>
</ol>
2. Evaluation of Neuropathic Behavior
NOTE: Maintain the animals in a comfortable environment (step 1.6) to allow recovery after injection. At day 7 post RTX injection (D7), each animal performs the hot plate and von Frey hair filament tests on the same day to reduce time bias and promote the efficiency of the behavioral tests. Bring the animals to a quiet room that is maintained at a stable humidity (40%) and temperature (27 &#176;C) for optimizing animal acclimatization and reducing environmental effects during the behavioral testing. Do not disturb the animals during test periods; the behavioral tests are scheduled weekly.
<ol>
	<li>Measurement of thermal latencies with the hot plate test
	<ol>
		<li>Place the animal gently on a metal hot plate (27 cm &#215; 29 cm) with a transparent Plexiglas cage (length &#215; width &#215; height: 22 cm &#215; 22 cm &#215; 25 cm; Figure 1A). Set the temperature of the metal hot plate to 52 &#176;C.</li>
		<li>Start measuring the duration of the animal&#39;s thermal latency on the hot plate with the step-on, built-in timer of the hot plate once the animal&#39;s hindpaws touch the hot plate, and observe the responses of the animal&#39;s hindpaws. If the animal shows shaking, licking of hindpaws, or jumping while on the hot plate, remove it and record the duration that the animal remained on the hot plate. This time duration defines the thermal latency of an individual animal. Record the thermal latency to the nearest 0.1 s.</li>
		<li>For each test session, perform three trials with 30-min intervals for response normalization after the last hot plate test. If the animal shows no response on the hot plate, stop the session after 25 s to avoid potential tissue damage.</li>
	</ol>
	</li>
	<li>Measurement of mechanical threshold with the von Frey hair filament test
	<ol>
		<li>Put the animal on the customized metal mesh (mesh size: 5 mm &#215; 5 mm) with a semi-transparent cylinder Plexiglas cage (diameter: 13 cm; height: 12 cm) (Figure 1B) for acclimatization for at least 2 h.</li>
		<li>Apply the different calibers of the von Frey hair filaments to the plantar region of the hindpaw with the up-and-down method31. Start initial application from the middle force of a set of von Frey hair filaments for a duration of filament application of 5-8 s.</li>
		<li>Use a 2 min interval between filament applications to optimize animal normalization. Change the applied filament force based on the animal&#39;s last response. 
		NOTE: A set of von Frey hair filaments consists of 0.064, 0.085, 0.145, 0.32, 0.39, 1.1, and 1.7 g force application. For example, if hindpaw withdrawal occurred with an initial force of 0.32 g, then apply 0.145 g. In the absence of paw withdrawal, a 0.39 g force is then applied. Then four additional filaments of varying forces are applied based on the preceding responses and the mechanical threshold is calculated according to a published formula31.</li>
		<li>For each test session, include the bilateral hindpaws. Perform three trials for each hindpaw. Express the average of these six mechanical thresholds as the mean mechanical threshold (mg) of each animal.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56651/56651fig1.jpg" /> 
Figure 1. Custom-made Plexiglas cage and metal mesh for the evaluation of neuropathic pain in the mouse model of resiniferatoxin (RTX)-induced small fiber neuropathy. (A, B) These graphs show the equipment used to measure (A) the thermal latencies by a metal hot plate (27 cm &#215; 29 cm) with a transparent Plexiglas cage (length &#215; width &#215; height: 22 cm &#215; 22 cm &#215; 25 cm) and evaluate (B) the mechanical threshold by a customized metal mesh (mesh size: 5 mm &#215; 5 mm) with a semi-transparent cylinder Plexiglas cage (diameter: 13 cm; height: 12 cm) in the mice with RTX-induced small fiber neuropathy. <a href="//cloudfront.jove.com/files/ftp_upload/56651/56651fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Skin Biopsy Preparation and Evaluation of IENFs Innervation
<ol>
	<li>After behavior testing, anesthetize the animals with 5% isoflurane and sacrifice the animals by intracardiac perfusion with 0.1 M phosphate buffer (PB) (pH 7.4) followed by 4% paraformaldehyde (4P) in 0.1 M PB.</li>
	<li>Cut the first footpads of the two hindpaws after perfusion and post-fixate them in 4P for another 6 h. Transfer the footpad tissue to 0.1 M PB at 4 &#176;C for long-term storage.</li>
	<li>Cryoprotect footpads with 30% sucrose in PB overnight and cut in a vertical to plantar surface manner in 30-&#181;m thick slices. Label the footpad sections sequentially, and then store in antifreeze at -20 &#176;C. 
	NOTE: The antifreeze composition is as follows: distilled water, ethylene glycerol, glycerol, and 2x PB in a 3:3:3:1 ratio.</li>
	<li>To ensure adequate sampling, select every third section of the footpad.
	<ol>
		<li>Put the chosen footpad sections on coated glass slides and air-dry them.</li>
		<li>Cover a plastic coverplate on the slide and process with standard immunostaining procedures.
		<ol>
			<li>Quench the footpad sections with 1% H2O2 in methanol for 30 min and block with 0.5% nonfat dry milk and 0.1% Triton X-100 in 0.5 M Tris buffer (Tris) for 1 h.</li>
			<li>Incubate footpad sections with antisera against pan axonal marker, PGP 9.5 (raised in rabbit; 1:1,000), overnight at 4 &#176;C.</li>
			<li>Incubate footpad sections with a biotinylated goat anti-rabbit IgG secondary antibody at room temperature (RT) for 1 h, and then incubate with the avidin-biotin complex at RT for 45 min.</li>
			<li>Visualize the reaction product with 0.05% 3,3&#39;-diaminobenzidine (DAB) solution for 45 s. Then wash footpad sections with distilled water and air-dry them for mounting. 
			NOTE: The primary and secondary antisera are diluted with 0.5% nonfat dry milk in 0.5 M Tris.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. DRG Section Preparation and Evaluation of Injured Small-diameter Neurons
<ol>
	<li>Dissect the 4th and 5th lumbar DRG and post-fix for another 2 h.</li>
	<li>Cryoprotect DRG tissues with 30% sucrose in PB overnight and cut to an 8-&#181;m thickness sequentially, place on microscope slides, and label. Store DRG sections in a -80 &#176;C freezer.</li>
	<li>Immunostain DRG sections in 80 &#181;m-intervals to ensure adequate sampling.
	<ol>
		<li>Perform DRG immunostaining procedures as those of the footpad sections, except for the double-labeling immunofluorescent procedures. Alternatively, include ATF3 (raised in rabbit; 1:100), an injury marker, and peripherin (raised in mouse; 1:800), a small-diameter neuronal marker in the primary antisera.</li>
		<li>Incubate DRG sections with the mixture of primary antisera overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate DRG sections with either Texas red or fluorescein isothiocyanate (FITC)-conjugated secondary antisera (1:200), corresponding to the appropriate primary antisera at RT for 1 h, and then mount for quantification.</li>
</ol>

The authors have nothing to disclose

Efficacious therapy of small fiber neuropathy in the clinic is required for promoting the functional recovery and life quality of patients. Currently, there is lack of a therapeutic guide targeting sensory disorder associated with small fiber neuropathy due to lack of comprehensive understanding of the molecular mechanisms underlying small-diameter neuronal injury. Previous models of neuropathy usually affected both large- and small-diameter sensory nerves; for instance, the models of chemotherapy-induced neuropathy<sup ...

Small fiber neuropathy involving neuropathic pain, which is evident by the degeneration of IENFs, is common in various types of conditions, such as DM, and as a result of the neurotoxic effects of chemotherapeutic agents1,2,3,4,5. IENFs are the peripheral terminals of small-diameter neurons located in the dorsal root ganglia (DRG), and are affected in parallel in cases of IENF degeneration6. For example, the altered upstream genetic transcription of neuronal somata has been demonstrated by the upregulation of activating transcription factor-3 (ATF3)6,7. Moreover, the evaluation of IENFs innervation with skin biopsy is useful for the diagnosis of small fiber neuropathy5,8,9. Traditionally, the profiles of the IENFs on the skin biopsy have depended on immunohistochemical demonstration of protein gene product 9.5 (PGP 9.5)1,10,11. Taken together, the pathological profiles of DRG and IENFs reflect the functional condition underlying small fiber neuropathy and may be an indicator for the functional consequences of this type of neuropathy on small-diameter neurons.
Previously, several experimental models have addressed the issue of IENF degeneration in cases of chemotherapy-induced neuropathy12,13 and nerve injury caused by compression or transection14,15,16. These experimental models also affected large-diameter nerves; it was, therefore, not possible to exclude the contribution of affected large-diameter nerves in the observed small fiber neuropathy; for instance, the examination of thermosensation disorder by noxious withdrawal depends on functional motor nerve fibers17,18,19. Thus, establishing a pure small fiber neuropathy model and systematically investigating the pathological status of both neuronal somata and their peripheral cutaneous nerve fibers in small-diameter neurons are necessary and imperative.
RTX is a capsaicin analogue and a potent agonist to transient receptor potential vanilloid receptor 1 (TRPV1), which mediates nociceptive processing20,21,22. Recently, peripheral RTX treatment relieved neurogenic pain23,24,25 and an intraganglionic injection of RTX induced irreversible loss of DRG neurons22. The effect of peripheral RTX administration is dose-dependent20,26,27, which resulted in the transient desensitization or degeneration of IENFs. Intriguingly, systematic high-dose RTX treatment led to neuropathic pain28, a symptom of small fiber neuropathy. These findings suggest that the treatment mode and dose of RTX produce distinct pathological effects and neuronal responses; to wit, peripheral administration prevented pain transmission by local effects29 and affected the neuronal somata that developed neuropathic behavior6. Collectively, these findings indicate that RTX has a multipotency effect and raised the issue whether there is a specific dose of RTX that could systematically affect the peripheral nerves, such as the peripheral IENFs and central neuronal somata. If so, RTX might be a potential agent to specifically affect small-diameter neurons and mimic small fiber neuropathy in the clinic. For example, DM in the clinic is a complicated issue including metabolic disorder and neuropathology of peripheral nerves, which are the main characteristics of small fiber neuropathy. The mechanisms of DM-associated small fiber neuropathy could not exclude the contribution of metabolic disorder that may not be the main agent affecting peripheral nerves. Therefore, DM-associated small fiber neuropathy requires a pure animal model that could exclude the effects of systematic metabolic disorder. This protocol describes the working dose of RTX to develop a typical small fiber neuropathy model, including IENF degeneration and small-diameter neuron injury, as demonstrated by modified immunostaining analysis.

<table><tbody><tr><td>&lt;strong&gt;Chemical reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Resiniferatoxin</td><td>Sigma</td><td>R8756</td><td></td></tr><tr><td>Tween 80</td><td>Sigma</td><td>P1754</td><td></td></tr><tr><td>3,3&amp;rsquo;-diaminobenzidine</td><td>Sigma</td><td>D8001</td><td></td></tr><tr><td>avidin-biotin complex</td><td>Vector</td><td>PK-6100</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antisera&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Peripherin</td><td>Chemicon</td><td>MAB-1527</td><td></td></tr><tr><td>ATF3</td><td>Santa Cruz</td><td>SC-188</td><td></td></tr><tr><td>PGP9.5</td><td>UltraClone</td><td>RA95101</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antisera&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biotinylated goat anti-rabbit IgG</td><td>Vector</td><td>BA-1000</td><td></td></tr><tr><td>Texas Red-conjugated goat anti-mouse</td><td>Jackson ImmunoResearch</td><td>115-075-146</td><td></td></tr><tr><td>Isothiocyanate (FITC)-conjugated donkey anti-rabbit</td><td>Jackson ImmunoResearch</td><td>711-095-152</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hot plate</td><td>IITC</td><td>Model 39</td><td></td></tr><tr><td>von Frey filament</td><td>Somedic Sales AB</td><td>10-600-0001</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shandon coverplate</td><td>Thermo scientific</td><td>72110017</td><td></td></tr><tr><td>Slide rack</td><td>Thermo scientific</td><td>73310017</td><td></td></tr></tbody></table>

establishing a mouse model of a pure small fiber neuropathy with the ultrapotent agonist of transient receptor potential vanilloid type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This protocol describes a novel mouse model of RTX neuropathy, which specifically affects small-diameter neurons, including IENF degeneration, associated with sensory disorders (Figure 2). Following the protocol described herein, animals exhibited thermal hypoalgesia and mechanical allodynia at D7 post RTX injection. To establish this small fiber neuropathy model, three doses of RTX: 200, 50, and 10 &#181;g/kg were administered by the i.p. route. The RTX dose...

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Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

establishing-mouse-model-pure-small-fiber-neuropathy-with-ultrapotent

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.4K Views.  Kaohsiung Medical University. This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 µg/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Video: Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Medicine

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Immunology and Infection

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Establishing the Mouse Model for Trigeminal Neuropathic Pain

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury (DIoN-CCI) model to study trigeminal neuropathic pain in mice. This includes the surgical procedures to perform the chronic constriction injury and the postoperative behavioral tests to evaluate the changes in spontaneous and evoked behavior that are signs of ongoing pain and mechanical allodynia. The methods and behavioral readouts are similar to the infraorbital nerve chronic constriction injury (IoN-CCI) model in rats. However, important changes are necessary for the adaptation of the IoN-CCI model to mice. First, the intra-orbital approach is replaced by a more rostral approach with an incision between the eye and the whisker pad. The IoN is thus ligated distally outside the orbital cavity. Secondly, due to the higher locomotor activity in mice, allowing rats to move freely in small cages is replaced by placing mice in custom-designed and constructed restraining devices. After DIoN ligation, mice exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory.

Author Spotlight: Utilizing Infraorbital Nerve Ligation in Mice for Investigating Trigeminal Neuropathic Pain and Treatment Strategies

Chronic constriction injury of the distal infraorbital nerve in mice induces changes in spontaneous behavior (increased face grooming activity) and nocifensive behavior in response to tactile stimulation (hyperresponsiveness to von Frey hair stimulation) that are signs of ongoing pain and allodynia and serves as a model for trigeminal neuropathic pain.

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury ...

Establishing a Mouse Model of Small Fiber Neuropathy Using Resiniferatoxin

Source: Lee, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56651">Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1.</a>&#160;J. Vis. Exp.&#160;(2018).
This video demonstrates a method for establishing a small fiber neuropathy model in mice using the TRPV1 agonist resiniferatoxin (RTX). It outlines the steps involved in administering RTX to mice and explains how RTX induces neuronal death in small-diameter sensory neurons, thereby establishing the neuropathy model.

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

Neuropathology

Neuromuscular Disorders

Assessing Motor Function in an Experimental Autoimmune Neuritis Mouse Model

Source: Gonsalvez, D. G., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56455">A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments.</a> J. Vis. Exp. (2017).
This video demonstrates the process of assessing motor dysfunction in a mouse model through the administration of Pertussis toxin and an inoculum containing myelin-mimicking peptides and adjuvants, followed by treadmill tests to assess motor function and evaluate the resulting nerve damage due to immune activation and demyelination.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Summary

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A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

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Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Establishing a Mouse Model of Small Fiber Neuropathy Using Resiniferatoxin

Assessing Motor Function in an Experimental Autoimmune Neuritis Mouse Model