Name: establishing a mouse model of a pure small fiber neuropathy with the ultrapotent agonist of transient receptor potential vanilloid type 1
Uploaded: 2018-02-13T14:00:00
Description: Watch this Scientific Journal Video about Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1 at JoVE.com

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 ...

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>Chemical reagent</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&#8217;-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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antisera</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antisera</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</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...

Watch this Scientific Journal Video about Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1 at JoVE.com

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 ...

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