Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

Diabetic polyneuropathy is the most common acquired diffuse disorder of the peripheral nervous system. It is generally assumed that insulin benefits human and experimental diabetic neuropathy indirectly by lowering glucose levels. Insulin also provides potent direct support of neurons and axons, and there is a possibility that abnormalities in direct insulin signaling on peripheral neurons relate to the development of this disorder. Here we report that direct neuronal (intrathecal) delivery of low doses of insulin (0.1-0.2 IU daily), insufficient to reduce glycemia or equimolar IGF-I but not intrathecal saline or subcutaneous insulin, improved and reversed slowing of motor and sensory conduction velocity in rats rendered diabetic using streptozotocin. Moreover, insulin and IGF-I similarly reversed atrophy in myelinated sensory axons in the sural nerve. That intrathecal insulin had the capability of signaling sensory neurons was confirmed by observing that fluorescein isothiocyanate-labeled insulin given intrathecally accessed and labeled individual lumbar dorsal root ganglion neurons. Moreover, we confirmed that such neurons express the insulin receptor, as previously suggested by Sugimoto et al. Finally, we sequestered intrathecal insulin in nondiabetic rats using an antiinsulin antibody. Conduction slowing and axonal atrophy resembling the changes in diabetes were generated by anti-insulin but not by an anti-rat albumin antibody infusion. Defective direct signaling of insulin on peripheral neurons through routes that include the cerebrospinal fluid may relate to the development of diabetic peripheral neuropathy.

OBJECTIVE-Heightened expression of the receptor for advanced glycation end products (RAGE) contributes to development of systemic diabetic complications, but its contribution to diabetic neuropathy is uncertain. We studied experimental diabetic neuropathy and its relationship with RAGE expression using streptozotocin-induced diabetic mice including a RAGE Ϫ/Ϫ cohort exposed to long-term diabetes compared with littermates without diabetes.RESEARCH DESIGN AND METHODS-Structural indexes of neuropathy were addressed with serial (1, 3, 5, and 9 months of experimental diabetes) electrophysiological and quantitative morphometric analysis of dorsal root ganglia (DRG), peripheral nerve, and epidermal innervation. RAGE protein and mRNA levels in DRG, peripheral nerve, and epidermal terminals were assessed in WT and RAGE Ϫ/Ϫ mice, with and without diabetes. The correlation of RAGE activation with nuclear factor (NF)-B and protein kinase C ␤II (PKC␤II) protein and mRNA expression was also determined. RESULTS-Diabetic peripheral epidermal axons, sural axons,Schwann cells, and sensory neurons within ganglia developed dramatic and cumulative rises in RAGE mRNA and protein along with progressive electrophysiological and structural abnormalities. RAGE Ϫ/Ϫ mice had attenuated structural features of neuropathy after 5 months of diabetes. RAGE-mediated signaling pathway activation for NF-B and PKC␤II pathways was most evident among Schwann cells in the DRG and peripheral nerve.CONCLUSIONS-In a long-term model of experimental diabetes resembling human diabetic peripheral neuropathy, RAGE expression in the peripheral nervous system rises cumulatively and relates to progressive pathological changes. Mice lacking RAGE have attenuated features of neuropathy and limited activation of potentially detrimental signaling pathways. Diabetes

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Proteinase-activated receptor-1 (PAR-1) is activated by thrombin and can be selectively activated by synthetic peptides (PAR-1-activating peptide: PAR-1-AP) corresponding to the receptor's tethered ligand. PAR-1 being expressed by a erent neurons, we investigated the e ects of PAR-1 agonists on nociceptive responses to mechanical and thermal noxious stimuli. Intraplantar injection of selective PAR-1-AP increased nociceptive threshold and withdrawal latency, leading to mechanical and thermal analgesia, while control peptide had no e ect. Intraplantar injection of thrombin also showed analgesic properties in response to mechanical, but not to thermal stimulus. Co-injection of PAR-1-AP with carrageenan signi®cantly reduced carrageenan-induced mechanical and thermal hyperalgesia, while thrombin reduced carrageenan-induced mechanical but not thermal hyperalgesia. The fact that thrombin is not a selective agonist for PAR-1 may explain the di erent e ects of thrombin and PAR-1-AP. These results identi®ed analgesic properties for selective PAR-1 agonists that can modulate nociceptive response to noxious stimuli in normal and in¯ammatory conditions.

OBJECTIVE-Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats.RESEARCH DESIGN AND METHODS-We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons.RESULTS-ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor ␤-subunit (IR␤), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IR␤ protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the ␣2␦1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-B (NF-B; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-B, and RAGE protein.CONCLUSIONS-ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.

Aims/hypothesis: The support of distal regenerating axons and epidermal nerve fibres through growth factor delivery may depend on the site of delivery. While low-dose systemic insulin provides trophic support for regenerating axons or axons from diabetic animals, its potential action upon the most distal neurites within the epidermis is unknown. In diabetic neuropathy, distal loss of axons is an important clinical and pathological feature. We hypothesised that insulin and IGF-1 delivered intrathecally could support the most distal epidermal nerve fibres. Materials and methods: As insulin and IGF-1 receptors are present upon sensory ganglion perikarya, we studied the impact of intrathecal delivery of low-dose insulin and equimolar IGF-1 on the density of epidermal axons expressing protein gene product 9.5 in experimental diabetic rats. After 2 months of diabetes induced by streptozotocin injection, intrathecal delivery of low-dose insulin or IGF-1 or saline was provided for 1 month, with comparison to compatible doses of subcutaneous insulin delivery. Results: Diabetes, in itself, was associated with a decline in epidermal nerve fibre density. Delivery of both intrathecal IGF-1 and insulin was associated with significant improvement in epidermal fibre density (greatest with IGF-1) and length relative to placebo. Conclusions/ interpretation: Central intrathecal delivery of IGF-1 and insulin offers remote support for epidermal nerve fibres, subjected to 'dying-back' in early diabetic polyneuropathy.

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