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 neuropathy12,32,33 and mechanical-induced neuropathy34,35. Thus, the contribution of motor weakness and large-diameter sensory nerve damage could not be completely excluded in the behavioral testing of these neuropathy models. The present protocol describes a new model of small fiber neuropathy in mice, which only affects small-diameter sensory nerves by providing pathological and functional evidence of IENFs degeneration.
RTX is an ultrapotent agonist of TRPV1 and a capsaicin analogue, which may cause loss of peptidergic DRG neurons in culture36 and in vivo systems18,19. Previous studies on RTX and capsaicin have mainly focused on the morphological or functional loss of DRG neuronal cell bodies, which revealed the role of TRPV1 in the thermal transmission response37,38,39. Moreover, a previous study demonstrated systematic high-dose RTX treatment (200 &#181;g/kg) in rats, induced mechanical allodynia and thermal hypoalgesia, possibly due to pathology of large-diameter nerve fibers28. The dose of 200 &#181;g/kg, however, is a lethal dose in mice and this current protocol developed a pure small fiber neuropathy model by reducing the RTX dose (50 &#181;g/kg). This dose of RTX (50 &#181;g/kg) is critical to establishing a pure small fiber neuropathy model, which is superior to that previously reported28, as it spares large fibers18. That is, it only affects small nerve fibers; to wit, only small-diameter neurons were injured, as confirmed by the induction of ATF3 upregulation6,40 on small-diameter DRG neurons and IENFs degeneration6,18,19,41, associated with sensory disorders. These pathological manifestations comprehensively mimic the clinical symptoms of small fiber neuropathy. Moreover, this current model induced the typical neuropathology and neuropathic pain profile of small fiber neuropathy and the effects lasted for 8 weeks post RTX treatment6,18,19. The durations of neuropathology and neuropathic pain were equivalent and could be reversed by promoting the synthesis of nerve growth factor (NGF)18,40,41. Collectively, this protocol both established a pure small fiber neuropathy model and highlighted the possible therapeutic potential of NGF.
Clinically, the gold standard for investigating neuropathies affecting small-diameter nociceptive nerves8,9 is biopsying limb skin for evaluating skin innervation. Our current report applied this technique to the footpad skin of experimental animals to evaluate the skin innervation of a small fiber neuropathy model, which could mimic the pathology of IENFs in the clinic, and also investigated the morphological profiles of DRG sections with the injury marker, ATF3, to reveal the pathological status of neuronal somata. Notably, the spatial distributions of IENFs within the epidermis are highly branching and the counting criteria are the major factor leading to statistical differences among groups. For example, our current protocol counted each IENF with branching points only in the dermis and IENFs with branching points within the epidermis as a single IENF14,18,19. This criterion may have caused a lower density of IENFs in our investigations than in those of other groups. We prepared and processed the skin and DRG sections of experimental animals in a systematic and bulk-evaluation fashion with our current modified protocol. Accordingly, these systematic investigations of IENF degeneration and neuronal injury could avoid the stereological bias of functional and pathological conditions of small-diameter neurons in small fiber neuropathy.
The functional evaluation of small-diameter nerves with behavioral testing, particularly with innoxious von Frey hair filament application, has been traditionally applied to patients&#39; skin for diagnosing the mechanical sensitivity underlying small fiber neuropathy. The observation of mechanical allodynia in experimental animals is challenging due to foot grounding on the metal mesh, which is considered to be exogenous mechanical stimulation, and the animals are highly active during tests. The current protocol optimized a specific sized mesh wire floor (5 mm &#215; 5 mm) in a semi-transparent plastic cage for the environmental adaptation of experimental animals for the behavioral tests. This size of the mesh floor could reduce the exogenous stimulation of foot grounding and avoid foot-dropping.
This RTX mouse model of neuropathy could be applied to different types of small fiber neuropathy, such as diabetes, which is associated with IENF degeneration1,42. However, this model remains limited. For example, the animal model of spinal nerve ligation43 with the characteristics of small fiber neuropathy, referred to as the radiculopathy in the clinic, may also affect the large fibers in the spinal rootlet.

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 (50 &#181;g/kg) was deemed critical and the preliminary study showed that high-dose RTX (200 &#181;g/kg) caused high mouse lethality (Figure 3).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56651/56651fig2.jpg" /> 
Figure 2. Scheme of the mouse model of resiniferatoxin (RTX)-induced small fiber neuropathy. The scheme shows the protocol of established RTX-induced small fiber neuropathy. For systematic assessment, behavior evaluation, and neuropathological examination, it included the hot plate and von Frey tests, and double-labeled immunostaining studies, respectively. <a href="https://cloudflare.jove.com/files/ftp_upload/56651/56651fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56651/56651fig3.jpg" /> 
Figure 3. Dose-effect of resiniferatoxin (RTX) on animal lethality and behavioral dysfunction. (A) Diverse doses of RTX were administered by intraperitoneal (i.p.) injection. The lethality of the dose effect was dose-dependent; for example, a high-dose of RTX (200 &#181;g/kg) caused 100% lethality. (B, C) Thermal latencies and mechanical thresholds were evaluated with the hot plate (B) and von Frey filament tests (C), respectively. A 50 &#181;g/kg dose of RTX induced thermal hypoalgesia and mechanical allodynia compared to the vehicle and the 10 &#181;g/kg-administered group. Open square, vehicle; open circle, 50 &#181;g/kg; open diamond, 10 &#181;g/kg. Dashed line in (B), cutoff time point of hot plate test. *** p &#60; 0.001. <a href="https://cloudflare.jove.com/files/ftp_upload/56651/56651fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Pathologically, there was IENF degeneration and marked ATF3 induction. Double-labeling studies showed that injured neurons were specifically peripherin(+) small-diameter neurons. In contrast, the low-dose of RTX (10 &#181;g/kg) did not establish small fiber neuropathy, including no changes in IENF innervation (Figure 4) and no neuronal injury (ATF3 induction) (Figure 5). Accordingly, this protocol considers the 50 &#181;g/kg dose critical to establishing the mouse model of small fiber neuropathy.
In summary, systematic RTX administration with a 50 &#181;g/kg dose specifically affected small nerve fibers. For instance, it led to neuronal soma injury and peripheral IENF degeneration, which are associated with sensory disorders.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56651/56651fig4.jpg" /> 
Figure 4. Degeneration of intraepidermal nerve fibers (IENFs) in resiniferatoxin (RTX) neuropathy. (A-C) Tissue sections from the footpad skin of mice were immunostained with anti-protein gene product 9.5 (PGP 9.5) antisera in the vehicle (A), 50 &#181;g/kg- (B), and 10 &#181;g/kg-administered (C) groups. PGP 9.5(+) IENFs arise from the subepidermal nerve plexus with a typical varicose appearance. PGP 9.5 (+) IENFs are markedly reduced in the 50 &#181;g/kg, but not in the 10 &#181;g/kg group. (D) IENFs were quantitated according to the immunohistochemical results of A-C. Open square, vehicle; open circle, 50 &#181;g/kg; open diamond, 10 &#181;g/kg. *** p &#60; 0.001 compared to the vehicle group. Scale bar, 50 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56651/56651fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56651/56651fig5.jpg" /> 
Figure 5. Specificity of small-diameter neuron injury in resiniferatoxin (RTX) neuropathy. (A-C) Double-labeling immunofluorescent staining was performed with anti-activating transcription factor-3 (ATF3; A-C, in green) and peripherin (A-C, in red) in the vehicle (A), 50 &#181;g/kg- (B), and 10 &#181;g/kg-administered (C) groups. (D) The diagram indicates the density changes of ATF3(+) neurons. ATF3(+) neurons were increased in the 50 &#181;g/kg, but not in the vehicle and 10 &#181;g/kg groups. Open square, vehicle; open circle, 50 &#181;g/kg; open diamond, 10 &#181;g/kg. *** p &#60; 0.001 compared to the vehicle group. Scale bar, 25 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56651/56651fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Rabies Necropsy Techniques in Large and Small Animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...