Specialized microdomains which have cholesterol-rich membrane regions contain transient receptor potential vanilloid subtype 1 (TRPV1) are involved in pain development. Our previous studies have demonstrated that the depletion of prostatic acid phosphatase (PAP) – a membrane-bound ectonucleotidase ­– and disordered adenosine signaling reduce the antinociceptive effect. The role of membrane integrity in the PAP-mediated antinociceptive effect in small-fiber neuropathy remains unclear, especially with respect to whether TRPV1 and PAP are colocalized in the same microdomain which is responsible for PAP-mediated antinociception. Immunohistochemistry was conducted on the dorsal root ganglion to identify the membrane compositions, and pharmacological interventions were conducted using methyl-β-cyclodextrin (MβC) – a membrane integrity disruptor that works by depleting cholesterol – in pure small-fiber neuropathy with resiniferatoxin (RTX). Immunohistochemical evidence indicated that TRPV1 and PAP were highly colocalized with flotillin 1 (66.7%±9.7%) and flotillin 2 (73.7%±6.0%), which reside in part in the microdomain. MβC mildly depleted PAP, which maintained the ability to hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and delayed the development of mechanical allodynia. MβC treatment had no role in thermal transduction and neuronal injury following RTX neuropathy. In summary, this study demonstrated the following: (1) membrane cholesterol depletion preserves PAP-mediated antinociception through PI(4,5)P2 hydrolysis and (2) pain hypersensitivity that develops after TRPV1(+) neuron depletion-mediated neurodegeneration following RTX neuropathy is attributable to the downregulation of PAP analgesic signaling.

Neurogenic inflammation is an onset characteristic of small fiber neuropathy (SFN), which is attributed to neuropathic manifestations. Tumor necrosis factor-α (TNFα) is a cytokine that mainly mediates neurogenic inflammation through the ligand receptor TNF receptor 1 (TNFR1), and targeting TNFα/TNFR1 signaling is a direction toward treating inflammatory diseases and injury-induced neuropathy. However, the relationships between TNFα/TNFR1 signaling and Ret signaling, which mediates pain hypersensitivity, remains elusive. This study used resiniferatoxin (RTX), an ultrapotent analog of capsaicin, to generate a mouse model of SFN, leading to marked hindpaw edema (p = 0.013) and parallel the release of TNFα (p = 0.014), which was associated with the upregulation of Ret(+) neurons (p = 0.0043) and partial depletion of TNFR1 caused by colocalization with TRPV1 depleted by RTX. Pharmacological intervention of TNFα with etanercept (Enbrel, Wyeth), a clinical application of TNFα blockers, relieved neurogenic inflammation and caused a reduction in hindpaw thickness (p = 0.03) and TNFα releases (p = 0.01), which were determined to be associated with the normalization of mechanical allodynia (p = 0.22). The extraction of either TNFR1(+) or Ret(+) neurons from total of TNFR1(+):Ret(+) neurons indicated that TNFR1(-)/Ret(+) neurons correlated with the mechanical threshold in an antiparallel fashion (r = -0.84, p < 0.0001) but had no relationship with thermal latencies. This study confirmed that TNFα rather than TNFα mediated neuropathic manifestation through the Ret receptor, specifically mechanical allodynia in RTX neuropathy.

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 µg/kg, respectively) and concluded that a critical dose of RTX (50 µg/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.