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NEUROPHARMACOLOGY

Increased Expression of Vanilloid Receptor 1 on Myelinated Primary Afferent Neurons Contributes to the Antihyperalgesic Effect of Capsaicin Cream in Diabetic Neuropathic Pain in Mice

Division of Molecular Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, Nagasaki University Nagasaki, Japan (M.H.R., M.I., H.U.); and Central Research Laboratories, Maruishi Pharmaceutical Co. Ltd., Osaka, Japan (SB)

Received February 25, 2003; accepted April 22, 2003.

	Abstract

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Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) at the peripheral nerve endings. Here, we report that an up-regulation of VR1 expression on myelinated fibers contributes to the antihyperalgesic effect of capsaicin cream in streptozotocin (STZ)-induced diabetic neuropathic pain. Intravenous injection of STZ (200 mg/kg) in mice caused rapid onset of diabetes within 24 h. Thermal and mechanical hyperalgesia developed by 3 days after STZ injection and persisted at all time points tested until 28 days. There was also hyperalgesic response to intraplantar (i.pl.) prostaglandin I₂ (PGI₂) agonist-induced nociception in such mice. Application of capsaicin cream dose dependently reversed the thermal, mechanical, and PGI₂ agonist-induced hyperalgesia observed in the diabetic mice. The i.pl. injection of capsaicin solution (0.4 µg/20 µl) produced nociceptive biting-licking responses in control mice, and these responses were significantly increased in STZ-induced diabetic mice. After neonatal capsaicin-treatment, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses were almost abolished. However, in neonatal capsaicin-treated diabetic mice, the i.pl. capsaicin-induced biting-licking responses reappeared. The i.pl. capsaicin-induced biting-licking responses were blocked by the competitive VR1 antagonist capsazepine. All these results suggest an increase in capsaicin receptor on myelinated fibers due to diabetes. Finally, we confirmed the up-regulation of VR1 expression on myelinated primary afferent neurons of diabetic mice by immunohistochemistry. Together, our results suggest that increased expression of VR1 on myelinated fibers might contribute to the antihyperalgesic effect of topical capsaicin in diabetic neuropathic pain.

The vanilloid receptor 1 (VR1) is a ligand-gated cation channel that can be activated by heat, decreased pH, or exogenous ligand such as capsaicin (Caterina et al., 1997; Tominaga et al., 1998). In addition, VR1 can be activated by endogenous fatty acid-derived mediators such as anandamide and N-arachidonyl-dopamine (Di Marzo et al., 2002). The VR1 protein has attracted tremendous attention because it can serve as a molecular integrator of painful stimuli on the primary sensory neurons. Recent findings also suggest its presence in various brain regions, including hippocampus, hypothalamus, and locus coeruleus (Mezey et al., 2000). VR1 has also been found in the spinal cord postsynaptic neuronal dendrites (Valtschanoff et al., 2001). The functional differences between the VR1 in central nervous system and in the periphery are not yet known. Although neonatal capsaicin treatment kills most VR1-expressing neurons in the sensory ganglia (Jancso et al., 1977), those in the central nervous system are not affected by neonatal capsaicin injection (Mezey et al., 2000). Although poorly known, the neurotoxic effect of capsaicin is reported due to depletion of nerve growth factors (Otten et al., 1983). It has been speculated that neonatal capsaicin treatment does not kill VR1-expressing neurons in the brain because these cells do not depend on any neurotrophic factor for survival that capsaicin may deplete (Mezey et al., 2000). In the periphery, VR1 is mainly expressed on unmyelinated C-fibers with very little presence on the thinly myelinated A-fibers (Caterina et al., 1997). Nevertheless, VR1 has been recognized as a marker of the nociceptive polymodal C-fibers in the sensory ganglia (Caterina et al., 1997).

Topical capsaicin is widely used in the clinic to alleviate various painful conditions, including diabetic neuropathic pain (The Capsaicin Study Group, 1991; Low et al., 1995). Capsaicin stimulates the VR1 and initiates a complex cascade of events, including neuronal excitation and release of proinflammatory mediators as well as desensitization of the receptor (Holzer, 1991; Caterina et al., 1997). The analgesic action of topical capsaicin in painful diseases is believed to occur through desensitization of the capsaicin receptor VR1 (Jancsó and Jancsó, 1949; Holzer, 1991; Szallasi and Blumberg, 1999). Thus, it might be speculated that up-regulated VR1 expression could contribute to neuropathic pain and hyperalgesia. Indeed, recent works indicate the involvement of vanilloid receptors in the development and maintenance of inflammatory and neuropathic pain (Di Marzo et al., 2002). Up-regulation of VR1 has been indicated for the development of nerve injury-induced neuropathic pain in the rats (Hudson et al., 2001). Recently, we have also reported that increased expression of VR1 on myelinated, neonatal capsaicin-insensitive fibers accounts for the antihyperalgesic action of topical capsaicin cream in nerve injury-induced neuropathic pain in mice (Rashid et al., 2003). However, it is not yet known whether an up-regulation of VR1 might contribute to the neuropathic pain in diabetes. Kamei et al. (2001) showed that intrathecal injection of anti-VR1 serum blocked the thermal and mechanical hyperalgesia observed in diabetic mice, suggesting the involvement of this receptor in diabetic neuropathic pain. In the present study, for the first time, we reported an up-regulation of VR1 expression on myelinated primary afferent neurons of STZ-induced diabetic mice. We also showed that this up-regulated VR1 on myelinated fibers might contribute to the antihyperalgesic action of topical capsaicin cream in diabetic neuropathic pain.

	Materials and Methods

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Experimental Animals. Male ddY mice were used throughout the experiments. They were housed in the animal facility of the University, which had been maintained at 21 ± 2°C, 55 ± 5% relative humidity, and an automatic 12-h light/dark cycle. The animals received standard laboratory diet (Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water ad libitum. The animals were adapted to the testing environment (maintained at 21 ± 2°C, 55 ± 5% relative humidity, and 12-h light/dark cycle) by keeping them in the testing room 24 h before the experiments. Experiments were performed during the light phase of the cycle (10:00 AM–7:00 PM). All procedures were approved by Nagasaki University Animal Care Committee and complied with the recommendations of the International Association for the Study of Pain (Zimmermann, 1983).

Drugs. The following drugs were purchased: substance P (SP; Peptide Institute, Osaka, Japan), ATP (Nacalai Tesque, Kyoto, Japan), capsaicin (Nacalai Tesque), and capsazepine (CPZ; Sigma-Aldrich, St. Louis, MO). ONO-54918-07 [a stable prostaglandin I₂ (PGI₂) agonist; Iguchi et al., 1989] was a kind gift from Ono Pharmaceutical Co. Ltd. (Osaka, Japan). Capsaicin cream and base cream were prepared at the Central Research Laboratories of the Maruishi Pharmaceutical Co., Ltd. (Osaka, Japan). The capsaicin cream labeled 0.01, 0.025, 0.05, and 0.1% contained 0.1, 0.25, 0.5, and 1 mg of capsaicin in 1 g of hydrophilic cream base, respectively. The base cream contained 18% polyoxy-ethylated castor oil, 17% liquid paraffin, 5% white petroleum jelly, 4% 1-hexadecanol, 0.1% EDTA disodium salt, and 0.75% triethanolamine (Minami et al., 2001). All drugs except capsaicin and capsazepine were dissolved in physiological saline. Capsaicin and capsazepine were dissolved in 10% ethanol, 10% Tween 80, and 80% physiological saline (5 mg/ml stock solution), which were then diluted with physiological saline before injection. This vehicle was found to be innocuous. The cream was applied in a volume of 0.1 ml/10 g and then gently rubbed over the mouse footpad skin 3 h before the behavioral test. The footpad was covered with adhesive tape to prevent the mice from licking up the cream.

Streptozotocin (STZ)-Induced Diabetes. The pancreatic -cell cytotoxic agent STZ is widely used to induce diabetes in rodents. The glucosamine-nitrosourea compound STZ is taken up into the insulin-producing -cells of the islets of Langerhan's via the GLUT-2 glucose transporter. The cytotoxic effect of STZ is mediated through a decrease in NAD levels, and the formation of intracellular free radicals leading to various toxic effects, including DNA-strand breaks (Schnedl et al., 1994). The STZ-induced diabetic rodents are hypoinsulinemic, but generally do not require exogenous insulin treatment to survive. STZ-induced diabetic rodents show common features of human diabetes that include damage to the eye, kidney, blood vessels, and nervous system. Diabetic neuropathic pain occurs mainly due to the damage in the nervous system (Sima and Sugimoto, 1999). In the present study, diabetes was induced in mice by a single i.v. injection of STZ (200 mg/kg; Wako Pure Chemicals, Richmond, VA) as reported previously (Kamei et al., 1991; Rashid and Ueda, 2002). Mice weighing 30 g were injected i.v. with STZ in the tail vein. STZ solution was prepared fresh by dissolving it in saline adjusted to pH 4.5 in 0.1 N citrate buffer. Age-matched nondiabetic control mice were injected with the vehicle alone. Due to frequent urination (polyuria) in the diabetic mice, special care is needed for these animals. The STZ-injected mice were kept in a group of four per cage. The bed of the cage was changed daily and special attention was paid to food and water supplement. The plasma glucose level in the mice was measured using the glucose test kit (Wako Pure Chemicals) in blood samples obtained from tail vein. Only mice with a plasma glucose concentration greater than 300 mg/dl (16.7 mM) were considered as diabetic. All efforts were made to minimize both the sufferings and number of animals used.

Thermal and Mechanical Nociception Tests. In the thermal paw withdrawal test, antinociception or analgesia was measured from the latency to withdrawal evoked by exposing the right hind paw to a thermal stimulus (Hargreaves et al., 1988). Unanesthetized animals were placed in Plexiglas cages on top of a glass sheet and an adaptation period of 1 h was allowed. The thermal stimulus (IITC, Inc., Woodland Hills, CA) was positioned under the glass sheet to focus the projection bulb exactly on the middle of plantar surface of the animals. A mirror attached to the stimulus permitted visualization of the undersurface of the paw. A cut-off time of 20 s was set to prevent tissue damage. The paw pressure test was performed as described previously (Rashid and Ueda, 2002). Briefly, mice were placed into a Plexiglas chamber on a 6 x 6-mm wire mesh grid floor and were allowed to accommodate for a period of 1 h. The mechanical stimulus was then delivered onto the middle of the plantar surface of the right hind paw using a transducer indicator (model 1601; IITC, Inc.). With this apparatus, a control response of 10 g was earlier adjusted for naive mice. A cut-off pressure of 20 g was set to avoid tissue damage.

Algogenic-Induced Nociceptive Flexion (ANF) test. Experiments were performed as described previously (Ueda, 1999; Inoue et al., 2003). Briefly, mice were lightly anesthetized with ether and held in a square-sized cloth sling. The cloth sling had four holes at the corners for hanging the mouse's limbs freely through the holes. After placing the mouse in the sling with four limbs hanging through the holes, two ends of the cloth sling were joined over the flanks of the mouse and the sling was suspended on a metal bar. The mouse's limbs were then tied with soft thread strings. Three limbs were fixed to the floor, whereas the other one (right hind limb) was connected to an isotonic transducer and recorder. A polyethylene cannula (0.61-mm outer diameter) filled with drug solution was connected to a microsyringe and then carefully inserted into the undersurface of the right hind paw. All experiments were started after complete recovery from the light ether anesthesia. Nociceptive flexor responses induced by intraplantar (i.pl.) injection (2 µl) of algogenic substances (SP, ATP, and ONO-54918-07) were evaluated and normalized with control saline response. The flexion responses induced by various algogenics were represented as the percentage of maximal reflex in each mouse as the flexion forces differ from mouse to mouse. The biggest response among the nonspecific flexor responses occurred immediately after cannulation was considered as the maximal reflex. The ANF test has been found to be less stressful and more sensitive than many conventional nociception tests (Inoue et al., 2003).

Capsaicin-Induced Biting and Licking Test. The biting and licking behavior after intraplantar injection of capsaicin solution (0.4 µg/20 µl) was measured as described previously by other investigators (Sakurada et al., 1992). Mice were placed in a Plexiglas cage for an hour to adapt to the environment. Before the test, mice were restrained in hand and gently taken inside a hard paper tube of internal diameter 2.5 cm. The right hind paw was taken out of the tube and capsaicin was injected under the plantar surface of right hind paw in a volume of 20 µl using a 30-gauge needle fitted to a Hamilton microsyringe. Mice were immediately put back to the cage and the time spent on biting and licking of the injected paw was measured with stopwatch for a period of 10 min. In antagonism experiments, mice were treated with 1 nmol of capsazepine in association with capsaicin. Dose of capsazepine has been determined from previous similar reports in mice (Santos and Calixto, 1997). Control animals received 20 µl of the vehicle used to dissolve the drugs.

Neonatal Capsaicin Treatment. For the degeneration of small diameter afferent sensory neurons, capsaicin solution was injected subcutaneously into newborn (P4) ddY mice at a dose of 50 mg/kg (Hiura and Ishizuka, 1989; Inoue et al., 1999). As a control, vehicle (10% ethanol and 10% Tween 80 in physiological saline) was injected. No gross behavioral changes were observed in such treated mice. Induction of diabetes in neonatal capsaicin-treated mice was performed as described in the previous section.

Immunohistochemistry. For immunohistochemical experiments, control mice, diabetic mice (7, 14, 21, and 28 days after STZ injection), neonatal capsaicin-treated control mice, or neonatal capsaicin-treated diabetic mice (7, 14, 21, and 28 days after STZ injection) were used. Mice were deeply anesthetized with sodium pentobarbital (50 mg/kg i.v.) and perfused transcardially with 50 ml of 0.1 M potassium free phosphate-buffered saline (K⁺-free PBS, pH 7.4), followed by 50 ml of 4% paraformaldehyde in K⁺-free PBS. The L4-L5 DRGs were removed, postfixed, and cryoprotected overnight in 25% sucrose in K⁺-free PBS. The DRGs were fast frozen in cryoembedding compound on a mixture of ethanol and dry ice and stored at –80°C until use. The DRGs were cut at 10 µm with a cryostat, thaw-mounted on silane-coated glass slides, and air-dried overnight at RT. For immunolabeling, DRG sections were first washed with K⁺-free PBS three times 5 min each and then incubated with 50% methanol 10 min and 100% methanol 10 min, washed with K⁺-free PBS and incubated with excess blocking buffer containing 2% bovine serum albumin in 2% NaCl, 0.1% Triton X-100 in K⁺-free PBS for 60 min. The sections were then reacted overnight at 4°C with goat polyclonal antibody raised against the C-terminal of vanilloid receptor 1 (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in blocking buffer containing 2% bovine serum albumin in 2% NaCl, 0.1% Triton X-100 in K⁺-free PBS. After three 5-min washes in K⁺-free PBS, the sections were placed in Texas Red-conjugated anti-goat IgG secondary antibody (1:200; Rockland, Gilbertsville, PA) for 60 min at RT. For double immunolabeling, sections were rinsed and first incubated with anti-mouse IgG (1:50; Cappel, Aurora, OH) for 60 min and then reacted with a monoclonal antibody raised against the N52 clone of the Neurofilament 200, a marker of myelinated fibers (Franke et al., 1991) (mouse anti-N52, 1:30,000; Sigma-Aldrich) overnight at 4°C. The sections were then placed in fluorescein isothiocyanate-conjugated anti-mouse IgG (1:200; Cappel) for 60 min at RT. After washing, the sections were coverslipped with Perma Fluor (Thermo Shandon, Pittsburgh, PA) and examined under a fluorescence microscope (Olympus, Tokyo, Japan).

Statistical Analysis. Statistical analysis of the data for the comparisons of the thermal latency or mechanical threshold at different time points after STZ injection in mice were performed by repeated measures analysis of variance (ANOVA) and Bonferroni's post hoc test. Data in the capsaicin sensitivity test for the effects of VR1 antagonist capsazepine were analyzed using a two-way ANOVA and Bonferroni's post hoc test. Statistical analyses of all other data were performed using one-way ANOVA followed by a two-tailed Student's t test. All data were presented as mean ± S.E.M. P values less than 0.05 were considered to indicate statistical significance.

	Results

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Rapid Onset of Diabetes and Thermal and Mechanical Hyperalgesia in Mice by Intravenous Injection of Streptozotocin. Diabetes was induced in mice by i.v. injection of STZ. Intravenous injection of STZ is reported to induce rapid onset of diabetes and hyperalgesia symptoms in rats (Aley and Levine, 2001). In the present study, a series of parameters, including body weights, blood glucose levels, thermal latencies, and mechanical thresholds, were measured at different time points after a single i.v. injection of STZ (200 mg/kg) into the tail vein of mice. A rapid onset of diabetes was observed in the STZ-treated mice within 24 h (blood glucose level, 402.5 ± 22.7 mg/dl). Thermal and mechanical hyperalgesia was detectable by 3 days after STZ administration. Blood glucose levels in the STZ-injected mice were almost similar at all later time points tested (7, 14, 21, and 28 days after STZ injection). Similarly, thermal and mechanical hyperalgesia persisted in the diabetic animals at all these time points (Table 1). The blood glucose level, thermal latency, and mechanical threshold did not differ significantly in the vehicle-treated control mice at all time points tested (data not shown). The rate of increase in the body weight of STZ-treated mice was much slower than the vehicle-treated control mice. The body weight of control nondiabetic mice at 7, 14, 21, and 28 days were 108.8, 123.7, 130.8, and 135.9% of the initial weight, respectively, whereas they were 104.4, 106.4, 111.6, and 107.5% of initial weight, respectively, in case of diabetic mice. The body weight of the STZ-treated mice started to decline at 28 days after STZ injection. In an effort to minimize animal sufferings, we used mice at 7 days post-STZ injection in the following behavioral experiments.

Reversal of Thermal and Mechanical Hyperalgesia in Diabetic Mice by Capsaicin Cream. The effect of the capsaicin cream was evaluated on the thermal and mechanical hyperalgesia observed in diabetic mice. The cream was applied 3 h before examining the thermal latency or pressure threshold where maximal analgesic effect was observed in our previous study (Rashid et al., 2003). As shown in Fig. 1A, application of capsaicin cream onto the footpad of diabetic mice concentration dependently reversed the thermal hyperalgesia from 0.01 to 0.1% concentration in the thermal paw withdrawal test. The cream also concentration dependently reversed the mechanical hyperalgesia in diabetic mice (Fig. 1B). The EC₅₀ values of the capsaicin cream were 0.064 and 0.07% in the thermal and mechanical tests, respectively. Consistent with our previous report (Rashid et al., 2003), capsaicin cream (0.1%) did not significantly change the thermal latency or mechanical threshold in control mice.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Effects of capsaicin cream on the thermal and mechanical hyperalgesia in the STZ-induced diabetic mice. A, concentration-dependent reversal of thermal hyperalgesia in diabetic mice by capsaicin cream with the thermal paw withdrawal test. B, concentration-dependent reversal of mechanical hyperalgesia in diabetic mice by topical application of capsaicin cream with the mechanical paw pressure test. Capsaicin cream labeled 0.01, 0.025, 0.05, and 0.1% or base cream (0%) was applied onto the mouse's footpad 3 h before the test. *, indicates significantly different compared with the base cream (0%)-treated diabetic mice at P < 0.05. Each data point represents mean ± S.E.M. of six to eight separate experiments. The vertical bars represent the standard error of the means. "Control" is the paw withdrawal latency or threshold in vehicle-treated control nondiabetic mice.

Phenotypic Changes in the Peripheral Receptor Ligand-Induced Nociceptive Flexion Responses in Diabetic Mice and Effects of Capsaicin Cream. Using the ANF test in mice, we previously proposed the presence of three distinct types of nociceptors depending on their stimulation by specific receptor ligands. The nociceptors called neonatal capsaicin-sensitive type I were stimulated by i.pl. injection of substance P, bradykinin, and nociceptin/orphanin FQ; the nociceptors called neonatal capsaicin-sensitive type II were stimulated by i.pl. P2X₃ receptor agonists; the nociceptors called neonatal capsaicin-insensitive type III were stimulated by i.pl. PGI₂ agonist ONO-54918-07 (Ueda et al., 2000). Very recently, we reported the peripheral nerve injury-induced phenotypic changes in the above-mentioned three types of nociceptors and the effects of capsaicin cream (Rashid et al., 2003). In the present study, we also found phenotypic changes in these three types of fibers in diabetic mice. As shown in Fig. 2, A and B, SP and ATP produced dose-dependent nociceptive flexion responses in the control nondiabetic and STZ-induced diabetic mice through capsaicin-sensitive type I and type II nociceptive fibers, respectively. There was no significant difference in the responses of SP and ATP in STZ-induced diabetic mice compared with the vehicle-treated control mice. On the other hand, similar to the case with nerve injury model, the capsaicin-insensitive type III fiber-mediated nociceptive responses of the PGI₂ agonist ONO-54918-07 were sensitized in diabetic mice giving nociceptive flexion responses at much lower doses compared with the control mice (Fig. 2C). Application of capsaicin cream concentration dependently blocked the PGI₂ agonist-induced hyperalgesic responses in diabetic mice, suggesting an increase in capsaicin-sensitive sites on neonatal capsaicin-insensitive type III fibers due to diabetes (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Phenotypic changes in the peripheral receptor ligand-induced nociceptive flexion responses in the STZ-induced diabetic mice. A and B, in the ANF test, the flexion responses induced by SP and ATP mediated through neonatal capsaicin-sensitive type I and type II fibers, respectively, remained unchanged in the STZ-induced diabetic mice. It was revealed by no significant difference in the dose-response curves between control nondiabetic and STZ-induced diabetic mice. C, dose-response curves of the neonatal capsaicin-insensitive type III fibers stimulant PGI2 agonist ONO-54918-07 in control nondiabetic and STZ-induced diabetic mice with the ANF test. The dose-response curve for ONO-54918-07 was shifted leftward in diabetic mice giving hyperalgesic responses. The results are represented as the percentage of maximal reflex. Details are described under Materials and Methods. Each data point represents mean ± S.E.M. of six separate experiments. The vertical bars represent the standard error of the means.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Reversal of PGI2 agonist-induced hyperalgesia in diabetic mice by topical application of capsaicin cream. Dose-response curves of the PGI2 agonist ONO-54918-07-induced nociceptive flexion responses in the ANF test in diabetic mice after application of capsaicin cream (0.025, 0.05, and 0.1%) or base cream onto the mouse's footpad 3 h before the test. Prior topical application of capsaicin cream concentration dependently reversed the type III fiber-mediated hyperalgesic responses. The symbols , , , and represent the effects of base cream and 0.025, 0.05, and 0.1% capsaicin cream on the ONO-54918-07-induced responses in diabetic mice, respectively; the symbols and represent the ONO-54918-07-induced responses in control and diabetic mice, respectively. The results are represented as the percentage of maximal reflex. Details are described under Materials and Methods. Each data point represents mean ± S.E.M. of six separate experiments. The vertical bars represent the standard error of the means.

Capsaicin-Induced Pain Sensitivity in STZ-Induced Diabetic Mice. Intraplantar injection capsaicin solution (0.4 µg/20 µl) induced nociceptive biting-licking response in control mice. The i.pl. capsaicin-induced biting-licking responses were significantly increased after STZ treatment (Fig. 4A). The competitive VR1 antagonist capsazepine (1 nmol or 0.377 µg) blocked the capsaicin-induced biting-licking response both in control and diabetic mice (Fig. 4A). After neonatal capsaicin treatment in mice, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses almost completely disappeared (Fig. 4B). However, STZ-induced diabetes in the neonatal capsaicin-treated mice caused reappearance of the i.pl. capsaicin-induced biting-licking behaviors and these newly induced responses were blocked by the VR1 antagonist capsazepine (Fig. 4B). Capsazepine alone did not produce any biting-licking behavior in mice (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Capsaicin-induced biting and licking responses and their blockade by capsazepine in the STZ-treated diabetic mice. A, increase in the i.pl. capsaicin-induced nociceptive biting-licking responses in STZ-treated diabetic mice and their blockade by the competitive VR1 antagonist capsazepine. Capsazepine (1 nmol or 0.377 µg) was injected in association with capsaicin (0.4 µg) in a volume of 20 µl. The symbol * indicates significant difference in the i.pl. capsaicin-induced responses between the control and STZ-treated diabetic mice. #, indicates significant difference in the biting-licking responses between i.pl. capsaicin-treated (Cap) and i.pl. Cap + CPZ treatment group. B, reappearance of capsaicin-induced biting-licking responses in neonatal capsaicin-treated (Neocap) diabetic mice. *, indicates significant difference in the i.pl. capsaicin-induced responses between the Neocap control and Neocap diabetic mice. #, indicates significant difference in the biting-licking responses between i.pl. Cap and i.pl. Cap + CPZ treatment group. Results are represented as the time (seconds) spent in biting and licking of the injected paw for a period of 10 min after i.pl. injection of drug substances. Veh, vehicle-induced response. Each data point represents mean ± S.E.M. of six to eight separate experiments. The vertical bars represent the standard error of the means.

Increased Expression of VR1 on Myelinated, Capsaicin-Insensitive Type III Fibers in the STZ-Induced Diabetic Mice. To confirm our speculation that STZ-induced diabetes in mice caused up-regulation of capsaicin receptors on myelinated, neonatal capsaicin-insensitive type III fibers, immunohistochemical double labeling was performed on DRG neurons with antibody to VR1, the putative capsaicin receptor and antibody to N-52 clone of Neurofilament 200, a marker of the myelinated A-fiber. As shown in Fig. 5A, in DRG of control mice VR1-immunoreactive neurons (red) were not colocalized with N-52-immunoreactive neurons (green), indicating the presence VR1 mostly on unmyelinated C-fibers in naive condition. In the DRG of STZ-induced diabetic mice, there was a visible increase in VR1 expression on myelinated A-fibers, which was revealed by a colocalization of VR1-immunoreactive and N-52-immunoreactive neurons observed as yellow (Fig. 5, B and C). The level of increase in VR1 expression on myelinated fibers was almost similar in the DRGs of diabetic mice at 7, 14, 21, and 28 days after STZ injection (Fig. 5, B, C, and G; 14- and 21-day data are not shown). Moreover, VR1 expression in unmyelinated C-fibers was not significantly increased in the diabetic mice (Fig. 5, B and C). In neonatal capsaicin-treated control mice, the VR1-immunoreactive neurons almost completely disappeared (Fig. 5D). However, in neonatal capsaicin-treated diabetic mice, large numbers of VR1-immunoreactive neurons were observed in the DRGs, which were colocalized with N-52, confirming the up-regulation of VR1 expression on myelinated, neonatal capsaicin-insensitive fibers due to diabetes (Fig. 5, E and F; 14- and 21-day data are not shown). When the numbers of VR1-immunoreactive cells were plotted in a bar graph as percentage of total cells, a significant increase in the numbers of cells that were colocalized with N-52 was observed in the diabetic mice (Fig. 5G).

View larger version (65K): [in this window] [in a new window]   Fig. 5. Up-regulation of VR1 expression on myelinated, neonatal capsaicin-insensitive primary afferent neurons after induction of diabetes in mice. A, VR1 expression in DRG neurons of control nondiabetic mouse. Almost all of the VR1-immunoreactive neurons (red) were not colabeled with A-fiber marker N-52 (green). B and C, VR1 expression in DRG of diabetic mice at 7 and 28 days after STZ injection. In both groups, many VR1-immunoreactive neurons were colocalized with A-fiber marker N-52 (observed as yellow). D, VR1 expression in the DRG of neonatal capsaicin-treated control mouse. Almost complete absence of VR1-immunoreactive neurons indicates loss of VR1-containing primary afferents due to neonatal capsaicin treatment. Most of the DRG neurons were labeled by N-52. E and F, Up-regulation of VR1 expression on myelinated fibers in neonatal capsaicin-treated diabetic mice at day 7 and 28 after STZ injection. In neonatal capsaicin-treated diabetic mice, many VR1-immunoreactive neurons were observed which were colabeled with A-fiber marker N-52 (observed as yellow). G, bar graph showing the percentage of VR1-immunoreactive (VR1-IR) neurons that were colabeled with N-52 in control, diabetic (7 and 28 days after STZ injection), neonatal capsaicin-treated control (Neocap control), and neonatal capsaicin-treated diabetic (Neocap diabetic, 7 and 28 days after STZ injection) mice from three separate experiments. *, P < 0.05. Scale bars, 20 µm.

	Discussion

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In the present report, we attempted to identify whether an up-regulation of VR1 expression on myelinated fibers contributed to the antihyperalgesic effect of capsaicin cream in diabetic neuropathic pain in mice. Intravenous injection of STZ in the tail vein of mice induced a rapid onset of hyperglycemia within 24 h, and significant thermal and mechanical hyperalgesia was detectable by 3 days after STZ injection (Table 1). Our results are consistent with previous reports in rat where i.v. STZ induced hyperglycemia by 24 h and thermal and mechanical hyperalgesia and tactile allodynia within 48 h after injection (Aley and Levine, 2001). The rapid elevation of blood glucose level by i.v. STZ might contribute to the rapid induction of thermal and mechanical hyperalgesia as already suggested in the study of Aley and Levine (2001) where pretreatment with insulin prevented the development of hyperalgesia in STZ-treated rat. The thermal and mechanical hyperalgesia observed in the diabetic mice were concentration dependently reversed by topical application of capsaicin cream onto mouse's footpad (Fig. 1, A and B). Capsaicin, the active ingredient of capsaicin cream, gives its analgesic effect by desensitizing the capsaicin receptor (Jancsó and Jancsó, 1949; Holzer, 1991; Szallasi and Blumberg, 1999). Thus, an up-regulation and/or sensitization of the capsaicin receptor could be speculated in the STZ-induced diabetic mice.

With the ANF test, we recently reported that capsaicin cream could block the nociceptive responses mediated through neonatal capsaicin-sensitive type I and type II, but not neonatal capsaicin-insensitive type III, fibers in naive mice (Rashid et al., 2003). After partial sciatic nerve injury, the type I fiber-mediated responses were lost, type II fiber-mediated responses remained unchanged, and the type III fiber-mediated responses were hypersensitized, and capsaicin cream reversed the type III fiber-mediated hyperalgesia in injured mice (Rashid et al., 2003). In the present study, the substance P-induced nociceptive response, which is mediated through type I fibers, and the ATP-induced nociceptive response, which is mediated through type II fibers, did not differ between the nondiabetic and diabetic mice (Fig. 2, A and B). However, the PGI₂ agonist-induced nociceptive response, which is mediated through type III fibers, was hypersensitized in the diabetic mice compared with the control mice (Fig. 2C). The contrasting phenotypic change in type I responses in partial sciatic nerve-injured and diabetic mice might be due to the intense mechanical injury to the sciatic nerve and consequent drastic changes, including decrease in SP immunoreactivity in DRG and spinal cord with the injury model (Malmberg and Basbaum, 1998; Lee et al., 2001). On the other hand, similar to the case with nerve injury model, capsaicin cream concentration dependently reversed the PGI₂ agonist-induced type III fiber-mediated hyperalgesia in diabetic mice (Fig. 3). Thus, the induction of PGI₂ agonist-induced hyperalgesia in diabetic mice could be due to an up-regulated capsaicin receptor on neonatal capsaicin-insensitive type III fibers. PGI₂ agonist produces nociceptive responses through activation of G_s-coupled prostaglandin I₂ receptor. The hyperalgesic responses of PGI₂ agonist in diabetic mice might be due to a protein kinase A-mediated transactivation of the newly expressed VR1 receptors as reported elsewhere (De Petrocellis et al., 2001).

To further investigate whether capsaicin receptor expression is increased in STZ-induced diabetic mice, we performed tests for capsaicin-induced pain sensitivity in control and diabetic mice. Intraplantar injection capsaicin solution-induced nociceptive biting-licking responses were significantly increased in the diabetic mice, indicating an increase in capsaicin-sensitive sites due to diabetes (Fig. 4A). Moreover, after neonatal capsaicin treatment in mice, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses almost completely disappeared, indicating that capsaicin-induced biting-licking responses in control mice were mainly mediated through the C-fibers (Fig. 4B). This finding is consistent with the fact that capsaicin receptor VR1 is mainly expressed in the polymodal nociceptive C-fibers (Caterina et al., 1997). In the neonatal capsaicin-treated diabetic mice, however, the i.pl. capsaicin-induced biting-licking responses surprisingly reappeared (Fig. 4B). This finding clearly indicates that STZ-induced diabetes in mice caused an up-regulation of the capsaicin receptor on myelinated, neonatal capsaicin-insensitive type III fibers. Furthermore, involvement of capsaicin receptor in the increased i.pl. capsaicin solution-induced responses in the diabetic mice was revealed by the fact that the competitive VR1 antagonist capsazepine completely blocked these responses (Fig. 4, A and B). All these results suggest an increased expression of capsaicin receptor VR1 on previously capsaicin-insensitive type III fibers due to diabetes.

We next confirmed the up-regulation of VR1 expression on myelinated, neonatal capsaicin-insensitive type III fibers due to diabetes by immunohistochemistry. Consistent with our previous report (Rashid et al., 2003), almost all of the VR1-immunoreactive neurons in the DRG of control mice were not colocalized with the A-fiber marker N-52, indicating their presence on unmyelinated C-fibers in naive state. In the STZ-induced diabetic mice, the VR1 expression significantly increased only on the myelinated A-fibers (Fig. 5, B, C, and G). VR1-immunoreactive neurons in the DRGs of neonatal capsaicin treated mice almost completely disappeared, which is consistent with previous reports (Mezey et al., 2000; Rashid et al., 2003). However, STZ-induced diabetes in neonatal capsaicin-treated mice caused an increased expression of VR1, which were colocalized with A-fiber marker N-52 (Fig. 5, E and F). These results confirmed our speculation that capsaicin cream reversed the hyperalgesia in diabetic mice (Figs. 1, A and B, and 3) by desensitizing the newly expressed VR1 receptors mainly located on myelinated, neonatal capsaicin-insensitive type III fibers. Our findings of the up-regulated VR1 expression on myelinated fibers in diabetic mice would be both timely and pertinent in view of the recent indications that endogenous vanilloid receptor agonists such as anandamide N-arachidonoyl-dopamine might play a crucial role in the maintenance of neuropathic pain (Di Marzo et al., 2002). Moreover, phosphorylation of VR1 by protein kinase A and protein kinase C, which are easily produced by proinflammatory mediators such as bradykinin and prostaglandins, has been well known (Premkumar and Ahern, 2000; De Petrocellis et al., 2001). Such phosphorylation increases the probability of channel gating by agonists such as heat, proton and endovanilloids (Vellani et al., 2001). Direct activation of VR1 channel by protein kinase C has also been reported (Premkumar and Ahern, 2000). Thus, the up-regulation of VR1 expression on myelinated fibers may contribute to the altered activities of these fibers as well to the maintenance of peripheral and central sensitization in neuropathy states.

In conclusion, we demonstrate that the thermal, mechanical, and chemical hyperalgesia observed in the STZ-induced diabetic mice might be due to the up-regulation of VR1 expression on neonatal capsaicin-insensitive, myelinated A-fibers. Our results also indicate that this up-regulated VR1 on myelinated fibers may account for the antihyperalgesic action of capsaicin cream in diabetic neuropathic pain.

	Acknowledgements

 
We thank S. Kondo, T. Kawashima, F. Fujiwara, M. Tashiro, and N. Itoh for technical assistance.

	Footnotes

 
This study was supported in part by Special Coordination Funds of the Science and Technology Agency of the Japanese Government, a Research Grant from Environmental Agency, Government of Japan, grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan and a grant for Human Frontier Science Program.

DOI: 10.1124/jpet.103.050948.

ABBREVIATIONS: DRG, dorsal root ganglion; VR1, vanilloid receptor 1; SP, substance P; CPZ, capsazepine; PGI₂, prostaglandin I₂; STZ, streptozotocin; ANF, algogenic-induced nociceptive flexion; i.pl., intraplantar; ONO-54918-07, 15-cis-(4-n-propylcyclohexyl)-16,17,18,19,20-pentanor-9-deoxy-6,9--nitriloprostaglandin F1; PBS, phosphate-buffered saline; RT, room temperature; ANOVA, analysis of variance.

¹ M.H.R. and M.I. contributed equally to this work.

Address correspondence to: Dr. Hiroshi Ueda, Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail: ueda{at}net.nagasaki-u.ac.jp

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