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Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain
Pain 122 (2006) 53–62 www.elsevier.com/locate/pain
Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain Hee Kee Kim, Jae Hyo Kim, Xiu Gao, Jun-Li Zhou, Inhyung Lee, Kyungsoon Chung, Jin Mo Chung * Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-1069, USA Received 10 June 2005; received in revised form 22 December 2005; accepted 3 January 2006
Abstract Recent studies suggest that reactive oxygen species (ROS) are critically involved in neuropathic pain. Although vitamin E is a well-known antioxidant, its efficacy on chronic pain is not known. This study investigated the efficacy and mechanisms of vitamin E analgesia in a rat model of neuropathic pain produced by spinal nerve ligation. The effects of vitamin E were investigated using behavioral testing, electrophysiological recording of dorsal horn neurons, and determinations of phosphorylated NMDA receptor subunit 1 (pNR1) levels in the spinal dorsal horn. Results showed that a systemic single injection of a high dose or repetitive daily injections of low doses of vitamin E significantly reduced neuropathic pain behaviors. Vitamin E was also effective in producing analgesia by intrathecal injection, suggesting the importance of spinal mechanisms. In spinal dorsal horn neurons, vitamin E reduced evoked responses to mechanical stimuli as well as the sizes of their receptive fields. In addition, levels of pNR1 in neuropathic rats were also reduced by vitamin E injection. These data suggest that vitamin E produces analgesia in neuropathic rats that is, at least in part, mediated by reducing central sensitization which, in turn, is induced by peripheral nerve injury. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: ROS; Antioxidant; Analgesia; Mechanical allodynia; Free radical scavenger
1. Introduction Nerve injury causes varying degrees of sensory and motor deficits. In some cases, however, it can also trigger severe neuropathic pain which persists for weeks or months (Bonica, 1990). A hallmark of peripheral neuropathic pain is mechanical allodynia or touch-evoked pain. Many patients suffer from neuropathic pain, yet treatment is a challenge because the underlying mechanisms are not clearly understood and existing analgesics, such as non-steroidal anti-inflammatory agents or opiates, have limited efficacy in relieving neuropathic pain. *
Corresponding author. Tel.: +1 409 772 2106; fax: +1 409 772 4687. E-mail address: [email protected] (J.M. Chung).
Recent studies suggest that reactive oxygen species (ROS) are critically involved in the generation of pain in various painful conditions, including neuropathic and inflammatory pain. In the chronic constriction injury model of neuropathic pain, systemic treatment with an antioxidant (Tal, 1996; Khalil et al., 1999; Khalil and Khodr, 2001) reduced hyperalgesia. In the spinal nerve ligation (SNL) model of neuropathic pain in rats, systemic or intrathecal injection of well-known freeradical scavengers, phenyl N-tert-butylnitrone (PBN) or 5,5-dimethyl-pyrroline-N-oxide (DMPO), almost completely alleviated mechanical hypersensitivity for several hours (Kim et al., 2004a). Since PBN was also very effective in producing analgesia by intrathecal injection, it was concluded that PBN-induced analgesia was mediated mainly through spinal mechanisms. In
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.01.013
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inflammatory pain induced by an intradermal injection of carrageenan into the hind paw of the rat, systemic treatment with the superoxide dismutase mimetic, M40403, reduced all measured inflammation parameters including hyperalgesia (Salvemini et al., 1999; Wang et al., 2004). The above evidence suggests that oxidative stress may be an important factor for chronic pain due to various causes. One of the most common antioxidants and most familiar to the general public is vitamin E. However, the analgesic efficacy of vitamin E on persistent pain is not known. In this study, the effects of vitamin E in chronic pain were investigated in SNL neuropathic rats utilizing behavioral, electrophysiological, and molecular techniques. The aims of this study included testing whether vitamin E would: (1) produce analgesia in neuropathic pain, (2) produce this analgesia through spinal mechanisms, (3) reduce responsiveness of dorsal horn neurons to mechanical stimuli in neuropathic rats, and (4) reduce NMDA receptor phosphorylation in dorsal horn neurons. Part of these data have previously been presented in abstract form (Kim et al., 2004b). 2. Materials and methods 2.1. Experimental animals Adult male Sprague–Dawley rats (200–350 g, Harlan Sprague–Dawley Co., Houston, TX) were used for the experiments. Rats were housed under a 12/12 h reversed light–dark cycle (dark cycle: 8:00 A.M.–8:00 P.M.) for at least one week before beginning any experiments. All experiments were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and the animal use protocol was approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee. 2.2. Neuropathic surgery (spinal nerve ligation, SNL) Rats were anesthetized with halothane (3% induction, 2% maintenance) in oxygen, then the L5 spinal nerve was tightly ligated as described before (Kim and Chung, 1992). Briefly, a midline skin incision was made on the back at the lower lumbar region, the paraspinal muscles were retracted, and the left transverse process of the L6 vertebra was removed under the dissection microscope. The left L5 spinal nerve was identified and gently separated from the adjacent L4 spinal nerve; it was then tightly ligated using 6-0 silk thread. The wound was cleaned with saline, closed with wound clips, and rats were returned to their cage after recovering from anesthesia. 2.3. Behavioral testing for the measurement of mechanical thresholds The experimenter who conducted the behavioral tests was blinded to the nature of the experimental manipulation to avoid experimental bias. Behavioral tests were conducted to
measure foot withdrawal thresholds in response to mechanical stimuli applied to the left hind paw. For testing, each animal was placed in a plastic chamber (8.5 · 8.5 · 28 cm) which was placed on top of a metal mesh stand, and mechanical stimuli were applied to the plantar surface of the left hind paw with von Frey monofilaments from underneath. Thresholds were determined by the up-down method (Chaplan et al., 1994; Baik et al., 2003), using a set of von Frey monofilaments [von Frey numbers (log stimulus intensity in mg · 10): 3.65, 3.87, 4.10, 4.31, 4.52, 4.74, 4.92, and 5.16; equivalent to: 0.45, 0.74, 1.26, 2.04, 3.31, 5.50, 8.32, and 14.45 g]. von Frey filaments were applied perpendicularly to the plantar surface at the base of the 3rd or 4th toes, the most sensitive area in SNL, with sufficient force to bend the filament slightly for 2–3 s. An abrupt withdrawal of the foot during stimulation or immediately after stimulus removal was counted as a positive response. A further detailed description of the test has been reported previously (Chaplan et al., 1994; Baik et al., 2003; Kim et al., 2004a). 2.4. Vitamin E injections in different experimental groups Vitamin E, DL-a-tocopherol acetate, and olive oil (a vehicle for dissolving vitamin E) were purchased from Sigma Chemical Company (St. Louis, MO, USA). The olive oil itself contains a small amount of vitamin E. However, the total amount of vitamin E in the largest quantity of olive oil (about 2 ml) used for a single injection is only about 0.2 mg, which is negligible as compared to the amount of vitamin E used in this study. 2.4.1. Systemic injection of vitamin E For systemic treatment, appropriate amounts of vitamin E in olive oil were injected intraperitoneally (i.p.) using a 2 gauge needle. The systemic effects of vitamin E on mechanical thresholds were tested in two different paradigms: immediate effects after a single injection and long-term effects with multiple daily injections. For immediate effects after a single injection, mechanical sensitivity was measured after various doses of vitamin E using the randomized Latin square design (Kirk, 1995) in a group of neuropathic rats. To do this, mechanical thresholds were measured before and 3 days after neuropathic surgery in 8 rats. Beginning the 4th postoperative day, baseline values were taken and rats were randomly divided into 4 groups of 2 rats. Rats in each group received an intraperitoneal injection of one of 3 doses of vitamin E (0.1, 1, or 5 g/kg in 10 ml/kg of olive oil) or vehicle alone (olive oil), and behavioral tests were repeated at 1, 2, 4, 6, and 8 h after the injection. The sessions for injection-testing (with the doses that were not given previously) were repeated at the 6th, 8th, and 10th postoperative days, so that all rats received all 4 compounds (3 doses of vitamin E and vehicle) in a random order by the end of the testing period. For long-term effects with multiple daily injections, 18 neuropathic rats were divided into 3 groups of 6 rats each. Starting on the 3rd postoperative day, rats in each group were given repetitive daily intraperitoneal injections of one of two doses of vitamin E (50 or 100 mg/kg) or olive oil alone (vehicle) for 7 days (until the 9th postoperative day), and mechanical thresholds were measured daily up to 15 postoperative days. Mechanical thresholds were measured just prior to the daily
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injections (approximately 24 h after the previous injections) and continued to be measured at the same time of day even after drug treatment was terminated. 2.4.2. Intrathecal (i.t.) injection of vitamin E For spinal i.t. injections, a catheter was implanted at the same time the spinal nerve ligation was done. While the rat was under halothane anesthesia, the paraspinal muscles were retracted, and the posterior part of the 12th thoracic vertebra was removed to expose the spinal meninges. A catheter (sterilized PE 10 tubing filled with artificial cerebrospinal fluid) was inserted into the subarachnoid space through a small nick in the dura and the tip was placed near the lumbar enlargement of the spinal cord. The remaining part of the catheter was fed subcutaneously to the mid-thoracic level and anchored to muscles by sutures and the tip was exposed and sealed. A total volume of 50 ll of various concentrations of vitamin E dissolved in olive oil was injected. The spread of vitamin E after i.t. injection was estimated by examining stained gross brain and spinal cord structures after injecting a lipid-soluble dye (3.5% Sudan Black B from Sigma, 50 ll in olive oil) intrathecally. 2.5. In vivo electrophysiological recording from dorsal horn neurons – single cell extracellular recording Five to ten days after the L5 spinal nerve ligation, rats were anesthetized with two intraperitoneal injections of 0.6 g/kg urethane given at 10 min intervals and the external jugular vein was cannulated. After a tracheotomy, the animal was paralyzed by an i.v. infusion of pancuronium bromide (1 mg/kg for induction and 0.3 mg/kg/h for maintenance) while being maintained on a ventilator. Endtidal CO2 levels were monitored with a capnometer and maintained between 3.5% and 4.5%. Rectal temperatures were kept at 37 C with a regulated electric blanket. A laminectomy was performed at the 3rd to 6th lumbar spinal segments. A carbon fiber-filled glass microelectrode (impedance: 0.4–0.8 MX at 1 kHz) was advanced into the 4th or 5th lumbar spinal cord segment with a stepping motor to a depth ranging from 500 to 900 lm from the dorsal surface. Single unit activity, evoked by probing the hind paw with a glass rod, was used to identify the dorsal horn cells that have hind paw receptive fields. The receptive fields were mapped by probing with a von Frey filament of 1 g bending force. The size of each receptive field was estimated by a point counting method using a 2.5-mm2 lattice. Evoked activities of these dorsal neurons in response to brush, non-noxious (1 g von Frey filament), and noxious (20 g von Frey filament) pressures, were recorded before and at various times after vitamin E treatment (5 g/kg, i.p.). All stimuli were applied at a rate of once per second for 10 s. Recorded signals were amplified, displayed on a digital oscilloscope, and fed into a window discriminator, whose output is used to compile peristimulus time histograms by a data acquisition system (CED 1401 with Spike 2 software). Evoked responses to each stimulus (brush, 1 g, 20 g) were calculated by subtracting the background from the total activity during the 10-s stimulation period. Evoked responses of all recorded neurons were expressed as spikes per second and displayed as box plots.
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At the end of the recordings, a DC current (2 mA for 20 s) was passed through the recording electrode to produce a microlesion. At the end of the experiment, the spinal cord was taken out and immersed in a 10% formaldehyde fixative solution, sectioned at 40 lm, and then stained with toluidine blue. The lesion indicating recording sites was then reconstructed. 2.6. Immunohistochemical staining for phospho-NR1 Immunohistochemical labeling for phosphorylated NMDA receptor subunit 1 (pNR1) was done. When anesthesia was deep (sodium pentobarbital, 10 mg/kg), the chest was opened and the rat was perfused through the aorta with saline, followed by cold fixative containing 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB), pH 7.2. The L5 spinal cord was removed, post-fixed overnight, and then placed in 30% sucrose until equilibration. The spinal cord was cryosectioned at 16 lm, mounted on gelatin-coated slides, and immunostained for pNR1. The sections were incubated in 5% normal goat serum with 0.3% Triton X-100 in PB for 1 h and then in solution containing rabbit anti-phospho-NR1 (1:500 dilution; Upstate Biotechnology, Lake Placid, NY). The sections were washed and then incubated with rhodamine-conjugated goat anti-rabbit IgG solution (1:200; Chemicon International, CA) for 2 h at room temperature. Sections were then washed, air-dried, and coverslipped with mounting medium. To confirm the specificity of the immunolabeling, some sections were processed as above without the primary antibodies. The sections were examined under a Zeiss epifluorescent light microscope or an Olympus Fluoview confocal microscope. The numbers of pNR1-immunostained dorsal horn neurons were counted from 10 randomly selected sections from each animal by an experimenter who did not know the origin of the tissue. The experimenter counted all neurons showing red fluorescence in the cytoplasm having an unstained nucleus. The density of fluorescence of 413 neurons randomly selected from 9 different sections was measured with an image analysis system (Image Pro-Plus). The density of all but one of 48 labeled cells was more than three standard deviations above the average unlabeled cell density, showing that the labeled and unlabeled cells are clearly separated in almost all cases and so identification of labeled neurons was unambiguous. 2.7. Western blots for phospho-NR1 To examine the changes in total amounts of pNR1 protein in the spinal dorsal horn, a group of 6 SNL rats were used for Western blots. On the 7th postoperative day, rats were deeply anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfused quickly with cold saline. The L4/5 spinal cord segments were removed, divided into left and right halves (ipsiand contralateral sides, respectively), and immediately frozen on dry ice. Spinal cord tissue was homogenized in 300 ll lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 2 ll protease inhibitor cocktail from Sigma, and 4 ll phosphatase inhibitor cocktail from Sigma) and centrifuged at 16,000 · g for 25 min at 4 C. The supernatant was decanted and used for Western blot analyses. The protein
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concentration of the supernatant was measured using a BioRad Protein Assay Kit I (Bio-Rad, Hercules, CA). The supernatant, with equal amounts of 80 lg protein, was then fractionated by 10% (w/v) SDS–PAGE gel and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with immunoaffinity-purified antibody to phospho-NR1 (1:500; Upstate Biotechnology) and monoclonal anti-b-actin antibody (1:50,000, Sigma, St. Louis, MO) for 2 h at room temperature. The blots were washed three times for 20 min each with buffer and then incubated with horseradish peroxidase-conjugated IgG (1:3000, goat anti-rabbit, Upstate Biotechnology; 1:3000, sheep anti-mouse, Amersham Bioscience). The membranes were washed with buffer three times again for 20 min each and enhanced with a chemiluminescence reagent (ECL kit; Amersham Pharmacia Biotech, Arlington Heights, IL). The blots were exposed to autoradiographic film (Eastman Kodak Co., Rochester, NY), the films were scanned into a computer, and the intensities of the immunoreactive bands of interest were quantified and analyzed using Meta Image series software. Densities were calculated based on the formula, Density = log (255/Intensity). The data are expressed by the ratio of density of pNR1 to the density of b-actin. 2.8. Data analysis Behavioral data are presented as means ± standard errors of the mean (SEM) and analyzed using the SAS statistical program. The data were analyzed by two-way repeated-measures ANOVAs with two-repeated factors followed by Duncan’s post hoc test or by two-way repeated-measures ANOVAs with one-repeated factor followed by Duncan’s post hoc test. Electrophysiological dorsal horn activity data were analyzed by two-way non-parametric repeated-measures ANOVAs (Friedman test) followed by Duncan’s post hoc multiple comparison test. For morphological studies, the comparison of the number of dorsal horn neurons expressing phospho-NR1 immunoreactivity and of the gel density in Western blots was done by Student’s t-test between vehicle vs. vitamin E treated neuropathic rats. In all cases, a p value <0.05 was considered to be significant.
3. Results 3.1. Vitamin E reduces pain behaviors in neuropathic pain The effect of vitamin E on pain behaviors, measured by mechanical thresholds, is shown in Fig. 1. The 50% foot withdrawal thresholds (mechanical thresholds) of the rat hind paw are estimated as 5.27 ± 0.0 (mean ± SEM) in normal naive rats. Following L5 spinal nerve ligation, the mechanical thresholds were reduced to 3.64 ± 0.03, thus demonstrating mechanical allodynia as similarly seen in human patients. Single systemic treatment with vitamin E (0.1, 1, or 5 g/kg, i.p. injection) increased mechanical thresholds for 6 h with the peak response at 1 h after vitamin E injection. The mechanical thresholds reached almost the normal level (5.03 ± 0.06) with the highest dose of
vitamin E (5 g/kg) at 1 h after injection (Fig. 1A). The analgesic effect of vitamin E showed graded responses in relation to the amount of injected vitamin E from 0.1 g to 5 g/kg of body weight. Vehicle injection did not change mechanical thresholds throughout the testing period. The mechanical thresholds of neuropathic rats with daily injection of vitamin E are shown in Fig. 1B. Repeated daily treatment with 50 or 100 mg/kg doses of vitamin E induced a gradual increase of mechanical thresholds, thus, the first significant change was detected 3 days after the initiation of treatment and the analgesic effect reached a significantly high plateau (4.43 ± 0.1 with 50 mg/kg and 4.38 ± 0.13 with 100 mg/kg) after 5 treatments. After termination of vitamin E injection, the significant analgesic effect lasted 5 more days and then eventually returned to the previous allodynic condition by the 6th post-treatment day. These data suggest that vitamin E is effective as an analgesic drug in chronic neuropathic pain. 3.2. The spinal cord is an important action site of vitamin E Vitamin E readily crosses the blood–brain barrier (Goss-Sampson and Muller, 1987). Consequently, systemically injected vitamin E may act on multiple sites, including the spinal cord. To determine the importance of the spinal cord as an active site of vitamin E for analgesic effect, the effects of vitamin E in the spinal cord were tested behaviorally and the results are shown in Fig. 1C. When small amounts of vitamin E (3, 10 or 30 mg) were injected intrathecally (i.t.) through implanted catheters, they produced analgesia in a dose-dependent manner and the analgesia lasted approximately 7 h (Fig. 1C). For example, 30 mg vitamin E changed mechanical thresholds from 3.73 ± 0.08 to 4.54 ± 0.09 at 2 h after drug injection and this increase was significantly different from the pre-injection baseline value. Although not shown, a further increase in the injection dose to 50 mg (n = 3) did not cause any further increase in threshold, suggesting the response had reached its maximum level. In general, the magnitude of threshold elevation by i.t. injection was smaller than that after systemic injection of vitamin E. Therefore, multiple sites seem to be involved in mediating the analgesic effect of vitamin E. However, the spinal cord is clearly an important site for vitamin E action. Examination of the spinal cord after an intrathecal injection of Sudan Black B dye (50 ll in olive oil) into the L5 vertebra level in 12 rats showed that the most rostral segments to which the dye spread ranged between the L1 and T7 levels. These data suggest that injected vitamin E stayed mostly within the lumbar enlargement of the spinal cord.
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Fig. 1. The effects of vitamin E on mechanical thresholds in neuropathic rats. (A) Systemic dose–response – SNL produced a significant reduction in mechanical thresholds (L) from the normal presurgery levels (N) in all operated rats (n = 8). Systemic injection (i.p.) of vitamin E, with doses of 0.1, 1, or 5 g/kg, reversed mechanical allodynia in a dose-dependent manner for a period of 6 h. Vehicle treatment (Vehicle) did not affect the mechanical thresholds. Experiments were done by the randomized Latin square design (2 rats/dose/day, 2 day intervals until all doses were tested in each rat). (B) Repeated treatment – daily injection (3–9 post-ligation days, indicated by arrowheads) of vitamin E (50 or 100 mg/kg, i.p.) produced a gradual increase in mechanical thresholds and reached moderately high plateau levels (4.38 ± 0.13, with 100 mg/kg) by the 5th day of treatment in 2 groups of rats (n = 6/group). After the termination of vitamin E treatment, mechanical thresholds maintained moderately high levels for 5 more days and returned to the pretreatment allodynic condition (3.73 ± 0.07) by the 12th post-ligation day. Vehicle treatment (n = 6) did not change the mechanical thresholds throughout the treatment period. (C) Intrathecal dose–response – single intrathecal injection of graded low doses of vitamin E (3, 10, and 30 mg/kg) showed graded analgesic effects on pain behaviors. Vehicle did not influence the mechanical thresholds. Experiments were done by the randomized Latin square design. N, presurgery normal naive rats; L, 3 days after SNL; B, baseline before vitamin E treatment; *, the value is significantly (p < 0.05) different from that of the vehicle control by two-way repeated-measures ANOVAs followed by Duncan’s post hoc tests.
3.3. Vitamin E reduces sensitization of dorsal horn wide-dynamic-range neurons in neuropathic rats The effects of vitamin E on the activities of spinal dorsal horn neurons were examined in normal and neuropathic rats. In particular, activities of single widedynamic-range (WDR) dorsal horn neurons, which receive multi-modal sensory inputs, were recorded by using an in vivo extracellular electrophysiological recording set-up. An example of a WDR neuron recorded from a normal rat is shown in Fig. 2A. This neuron
had a receptive field at the base of the 2nd through 4th toes and responded well to all stimuli. Vitamin E application (5 g/kg, i.p.) did not change these evoked response activities or the size of the receptive field. The neuron was located in the medial part of the lamina IV as indicated in the cross-section of the cord. An example of a WDR neuron recorded from neuropathic rats is shown in Fig. 2B. This neuron showed much higher evoked responses as well as higher spontaneous activities as compared to those in normal rats, thus showing signs of sensitization. This neuron had a
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Fig. 2. Effects of vitamin E on dorsal horn WDR neuron activity. (A) Normal rat – this neuron responded well to all three different stimuli applied to the receptive field. Neither the responses to mechanical stimuli nor the receptive field size changed after vitamin E (5 g/kg, i.p.). The receptive field (mapped with 1 g von Frey) and recording location are shown in the right inset. Br = Brush stimulus. (B) Neuropathic rat (7 day post-ligation) – this neuron responded more vigorously to mechanical stimuli as compared to cells in normal rats. Both responses and receptive field size were greatly reduced after vitamin E injection. (C) Median evoked responses of populations of WDR neurons [10 cells from normal and 14 cells from neuropathic rats] to various mechanical stimuli before and after vitamin E injection. Box plot values: the thick horizontal line within each box is the median value; the upper and lower edges of each box represent the 75th and 25th percentiles, respectively; and the upper and lower whiskers represent the 90th and 10th percentiles, respectively. Asterisks indicate values that are significantly (p < 0.05) different from the pre-injection levels and the number signs (#) indicate values that are significantly (p < 0.05) different from that of the normal group.
receptive field on the plantar surface of 3rd and 4th toes. Systemic injection of vitamin E markedly reduced the responsiveness of this neuron to innocuous stimuli (brush and 1 g von Frey filament), but not to noxious stimuli (20 g von Frey filament). In addition, the size of the receptive field was reduced after vitamin E treatment. Fig. 2C shows a summary of the responses of 10 neurons (one unit/rat) recorded from normal rats and 14 neurons recorded from neuropathic rats. Evoked responses to each stimulus are expressed as spikes per second and displayed as box plots in Fig. 2C. The average sizes of the receptive fields (as mapped with 1 g von Frey filament) of 6 neurons recorded from normal rats were 58.5 ± 13.3 and 56.5 ± 14.2 mm2 before and 1 h after vitamin E injection, respectively. On the other hand, the average receptive field size of 8 neurons recorded from neuropathic rats was 103.9 ± 14.7 mm2 before vitamin E injection. This size was significantly reduced to 77.4 ± 12.3 mm2 1 h after vitamin E injection. Data show that: (1) the responsiveness of WDR neurons to both innocuous and noxious stimuli was higher in neuropathic rats (median value was 30.49 spikes/s to brush; 10.38 spikes/s to 1 g; 33.72 spikes/s to 20 g) compared to that of normal rats (median value
is 13.33 to brush; 5.58–1 g; and 22.35–20 g); (2) vitamin E treatment did not change the responsiveness in normal rats; (3) vitamin E treatment significantly reduced the responsiveness of dorsal horn WDR neurons in neuropathic rats. It is noted, however, that vitamin E reduced activity evoked by weaker stimuli (brush and 1 g von Frey) more than the enhanced responses to stronger stimuli (20 g von Frey). Thus, overall, the average responses of dorsal horn WDR neurons in neuropathic rats returned to the levels of normal rats after vitamin E treatment. Data suggest that spinal dorsal horn WDR neurons were sensitized after nerve injury and then transiently restored to a normal state after vitamin E treatment. 3.4. Vitamin E reduces the phospho-NR1 expression in the dorsal horn neurons Vitamin E reduced the responsiveness of dorsal horn neurons in neuropathic rats, thus suggesting the reduction of central sensitization. To study the potential effects of vitamin E for NMDA receptor phosphorylation, which is a mechanism likely underlying central sensitization, numbers of dorsal horn neurons expressing
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In the ipsilateral dorsal horn, these numbers are higher at 1.95 ± 0.2 and 14.51 ± 1.2 (n = 6) in the superficial and deep layers, respectively. One hour after treatment with vitamin E, numbers of immunoreactive cells on the ipsilateral side were reduced to levels comparable to that of the contralateral side. The number of pNR1 immunoreactive neurons in the contralateral dorsal horn did not change significantly after vitamin E treatment. The total amount of pNR1 protein in the L4/5 spinal cord was also measured by the Western blot method. An example of a Western blot gel for pNR1 and the averaged relative densities of pNR1 are shown in Figs. 3F
pNR1 as well as levels of pNR1 protein in the spinal cord were examined. Examples of pNR1 immunoreactive dorsal horn neurons in a vehicle treated rat (Fig. 3A), 7 days after nerve ligation (Fig. 3B) and 1 h after vitamin E treatment in neuropathic rats (Fig. 3C), are shown in Fig. 3. In the contralateral dorsal horn of the vehicle (olive oil)-treated neuropathic rats, the average (±SEM) number of pNR1 immunoreactive cells per section is 0.52 ± 0.12 (n = 6) in the superficial layers (laminae I–II) and 10.57 ± 1.17 (n = 6) in the deep layers (laminae III–VI) (Figs. 3D and E). These numbers are not significantly different from that of the unoperated normal rats (n = 6, data are not shown).
A
B
C
E
D
F
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G
Fig. 3. Changes in pNR1 protein expression in the L5 spinal cord. Spinal cord tissues were collected from 7 day post-ligation SNL neuropathic rats 1 h after a systemic treatment with either vehicle (SNL + Veh, 10 ml/kg olive oil, i.p.) or vitamin E (SNL + Vit E, 5 g/kg in olive oil). (A–C) Examples of pNR1 immunoreactive neurons in the dorsal horn (lamina V) of neuropathic rats with various treatments: (A) the contralateral side after vehicle; (B) the ipsilateral side after vehicle; and (C) the ipsilateral side after vitamin E treatment. Calibration bar = 50 lm. (D and E) The averaged number of pNR1 immunoreactive neurons found in the superficial (D; laminae I and II) and deep (E; laminae III–VI) layers of the dorsal horn section (16 lm thick). (F) An example of a Western blot gel of pNR1. The averaged values of relative ratio of pNR1 to b-actin from 6 animals are shown in (G). Only the ipsilateral spinal cord showed increased pNR1 levels after SNL + Veh and then returned to the control levels after vitamin E treatment. Data for D, E, and G were collected from the vehicle (open bars, n = 6) and vitamin E (filled bars, n = 6) treated groups. Asterisks indicate values that are significantly different at p < 0.05 compared to the vehicle control group by Student’s t-test.
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and G, respectively. Seven days after L5 spinal nerve ligation, the relative density of pNR1 was significantly increased in the ipsilateral L4/5 spinal cord (Fig. 3G, n = 6) as compared to that of the contralateral side. This increased level was significantly reduced one hour after intraperitoneal injection of vitamin E (5 g/kg). Thus, vitamin E reduced the expression of phospho-NR1 in the spinal cord. These results are consistent with the electrophysiological data showing that vitamin E reduces central sensitization of dorsal horn neurons. 4. Discussion The effect of a common antioxidant, vitamin E, on chronic pain was investigated in the SNL model of neuropathic pain. Vitamin E significantly reduced behavioral signs of mechanical allodynia. The spinal dorsal horn neurons showed reduced responsiveness, reduced receptive field size, and reduced phospho-NR1 expression after vitamin E treatment in neuropathic rats. These data suggest that an important mechanism for vitamin E-induced analgesia is through reduction of central sensitization. Chronic pain is a state where abnormalities are maintained in both the central and peripheral nervous systems (Woolf and Costigan, 1999; Woolf and Salter, 2000). In the spinal cord, dorsal horn neurons become sensitized and respond more vigorously to peripheral input, and they also receive input from expanded cutaneous receptive fields (Cook et al., 1987; Woolf and Thompson, 1991). NMDA receptors mediate central sensitization and many second messengers are involved in the cascade of changes (Woolf and Thompson, 1991; Willis, 1994; Woolf and Costigan, 1999; Woolf and Salter, 2000). In the periphery, abnormal activities are produced and enter the spinal cord to initiate and maintain the central sensitization (Sheen and Chung, 1993; Yoon et al., 1996; Chung and Chung, 2002; Sukhotinsky et al., 2004). Peripheral abnormal activities include ectopic discharges arising from injured afferent neurons (Devor and Seltzer, 1999; Chung and Chung, 2002) and spontaneous activities arising from sensitized intact nociceptors (Ali et al., 1999; Wu et al., 2001a; Shim et al., 2005). Therefore, there are multiple potential sites where vitamin E can exert its effect. The fact that vitamin E was very effective in producing analgesia by intrathecal injection, that it reduced responsiveness and receptive fields of the dorsal horn neurons, and reduced pNR1 expression in the dorsal horn neurons suggests that the spinal cord is a very important site for analgesic actions of vitamin E. Although the results show that the spinal cord is an important site for vitamin E-induced analgesia, other sites, particularly the periphery, need to be considered. Intra-arterial infusion of the free radical donor tert-butyl-hydroperoxide into one extremity of the rat produces
pain in the affected region (Van Der Laan et al., 1998). Increased production of ROS, both at the injury site and in the dorsal root ganglion neurons of the injured nerve, and reduced pain behaviors by nitric oxide synthase inhibitors (Haley et al., 1992; Levy et al., 1999; Khalil and Khodr, 2001) are observed in animal models of neuropathic pain. Thus peripheral ROS are also involved in the generation of pain. The effect of vitamin E on peripheral mechanisms, such as sensitization of intact peripheral nociceptors, needs to be studied to clarify these peripheral mechanisms. Sensitized spinal dorsal horn neurons become normalized after vitamin E treatment in neuropathic rats. This implies that, after spinal nerve injury, excessive oxidants build up in the spinal cord and then sensitize dorsal horn neurons, suggesting an oxidative stress in the spinal cord. Removing excessive oxidants with vitamin E restores the normal physiological condition and thus relieves pain. Oxidative stress is considered an important determinant for neuronal cell death in various neurodegenerative diseases (Olanow, 1992; Balazs and Leon, 1994; Gerlach et al., 1994) and brain injury (Lewen et al., 2000). The majority of these studies show extreme oxidative conditions where irreversible cell death is eminent. When pain is relieved with vitamin E, the neuropathic pain, at least in the early stages, seems to be a temporary dysfunctional state due to mild oxidative stress but not due to permanent cell death. This temporary dysfunctional state may be a window of opportunity to prevent cell death due to prolonged oxidative stress. Regarding the doses of vitamin E, we believe that 5 g/kg body weight is too high for repetitive clinical use and thus only tested this dose in a single injection paradigm. Since the estimated toxic dietary level of vitamin E is 16,000–64,000 IU/kg (equivalent to about 16–64 g/kg) for a bird (Dierenfeld and Traber, 1993), a single dose of 5 g/kg (5000 IU/kg) for the rat seems to be within safe limits. It is difficult to estimate comparable doses of vitamin E between rats and humans considering the difference in relative metabolic rates, which in rats is much higher than in humans. However, the low dose (50 mg/kg) that we used for repeated daily injections for 200 g rats (10 mg per rat) would be equivalent to 3 g for a 60 kg human. This dose is well within the range of clinical use for human patients (2000–5000 IU of vitamin E which represents about 2–5 g) (Sano et al., 1997; Parkinson Study Group, 1998; Graf et al., 2004). It is important to identify the types and sources of oxidants that play a critical role in neuropathic pain. The most likely sources of oxidants in the cord include superoxides generated from mitochondria and nitric oxides generated by nitric oxide synthase (Coyle and Puttfarcken, 1993; Wu et al., 2001b). It is not known which type is more critically involved in neuropathic pain, and it is possible that both types are involved. Because
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vitamin E can react with various reactive oxidants, including peroxyl radicals, peroxynitrite, nitrogen dioxide, and superoxide (van Haaften et al., 2003), our study could not delineate the specific types of oxidants. Since the levels of mitochondrial reactive oxygen species increase in rat neuropathic dorsal horn neurons (Park et al., 2006), superoxides produced by mitochondria may be one of important free radicals for neuropathic pain. A more specific detection method is required in future studies. The slow buildup and decay of analgesia with repetitive injections of low doses of vitamin E suggest the cumulative effects of vitamin E are due to their storage properties in adipose tissue (Butterfield et al., 2002). The preferential location of vitamin E in the membrane fraction of the mitochondria also indicates the importance of antioxidant action in mitochondria (Buttris and Diplock, 1998). Another important question is the mechanism by which the excessive oxidants produce pain. The present study provides evidence that vitamin E desensitizes spinal neurons. Therefore, it is critical to illuminate molecular steps involved in sensitization of the dorsal horn neurons by oxidants. Overall we speculate that oxidants may be an important initiating factor for some or many already known mechanisms, such as the activation of second messenger signaling systems (Ali and Salter, 2001; Zou et al., 2002; Zhang et al., 2003). We used the enhanced expression of pNR1 in the dorsal horn neurons as an indicator of central sensitization in neuropathic rats. This is based on the fact that phosphorylation of NMDA receptors is involved in activation of NMDA receptors (Cerne et al., 1992; Li and Zhuo, 1998; Christie et al., 1999), and the number of spinal neurons expressing pNR1 increases greatly in conditions where central sensitization and persistent pain exist (Zou et al., 2000; Gao et al., 2005). The present study shows that vitamin E reduced pNR1 expression in dorsal horn neurons, supporting the results of the electrophysiological study on neuronal responses. In conclusion, systemic administration of vitamin E produces analgesia in a rat model of neuropathic pain. Further, this analgesia is built up and prolonged with daily repeated administration of small doses of vitamin E. The mechanism of the analgesia, at least in part, is desensitization of dorsal horn neurons in the spinal cord.
Acknowledgments This work was supported by NIH Grants NS 31680, NS 11255, and AT 01474, and a Research Development Grant from the Sealy Memorial Endowment Fund (2547-03). The authors sincerely thank Denise Broker for her excellent service in English editing.
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Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain Hee Kee Kim, Jae Hyo Kim, Xiu Gao, Jun-Li Zhou, Inhyung Lee, Kyungsoon Chung, Jin Mo Chung * Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-1069, USA Received 10 June 2005; received in revised form 22 December 2005; accepted 3 January 2006
Abstract Recent studies suggest that reactive oxygen species (ROS) are critically involved in neuropathic pain. Although vitamin E is a well-known antioxidant, its efficacy on chronic pain is not known. This study investigated the efficacy and mechanisms of vitamin E analgesia in a rat model of neuropathic pain produced by spinal nerve ligation. The effects of vitamin E were investigated using behavioral testing, electrophysiological recording of dorsal horn neurons, and determinations of phosphorylated NMDA receptor subunit 1 (pNR1) levels in the spinal dorsal horn. Results showed that a systemic single injection of a high dose or repetitive daily injections of low doses of vitamin E significantly reduced neuropathic pain behaviors. Vitamin E was also effective in producing analgesia by intrathecal injection, suggesting the importance of spinal mechanisms. In spinal dorsal horn neurons, vitamin E reduced evoked responses to mechanical stimuli as well as the sizes of their receptive fields. In addition, levels of pNR1 in neuropathic rats were also reduced by vitamin E injection. These data suggest that vitamin E produces analgesia in neuropathic rats that is, at least in part, mediated by reducing central sensitization which, in turn, is induced by peripheral nerve injury. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: ROS; Antioxidant; Analgesia; Mechanical allodynia; Free radical scavenger
1. Introduction Nerve injury causes varying degrees of sensory and motor deficits. In some cases, however, it can also trigger severe neuropathic pain which persists for weeks or months (Bonica, 1990). A hallmark of peripheral neuropathic pain is mechanical allodynia or touch-evoked pain. Many patients suffer from neuropathic pain, yet treatment is a challenge because the underlying mechanisms are not clearly understood and existing analgesics, such as non-steroidal anti-inflammatory agents or opiates, have limited efficacy in relieving neuropathic pain. *
Corresponding author. Tel.: +1 409 772 2106; fax: +1 409 772 4687. E-mail address: [email protected] (J.M. Chung).
Recent studies suggest that reactive oxygen species (ROS) are critically involved in the generation of pain in various painful conditions, including neuropathic and inflammatory pain. In the chronic constriction injury model of neuropathic pain, systemic treatment with an antioxidant (Tal, 1996; Khalil et al., 1999; Khalil and Khodr, 2001) reduced hyperalgesia. In the spinal nerve ligation (SNL) model of neuropathic pain in rats, systemic or intrathecal injection of well-known freeradical scavengers, phenyl N-tert-butylnitrone (PBN) or 5,5-dimethyl-pyrroline-N-oxide (DMPO), almost completely alleviated mechanical hypersensitivity for several hours (Kim et al., 2004a). Since PBN was also very effective in producing analgesia by intrathecal injection, it was concluded that PBN-induced analgesia was mediated mainly through spinal mechanisms. In
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.01.013
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inflammatory pain induced by an intradermal injection of carrageenan into the hind paw of the rat, systemic treatment with the superoxide dismutase mimetic, M40403, reduced all measured inflammation parameters including hyperalgesia (Salvemini et al., 1999; Wang et al., 2004). The above evidence suggests that oxidative stress may be an important factor for chronic pain due to various causes. One of the most common antioxidants and most familiar to the general public is vitamin E. However, the analgesic efficacy of vitamin E on persistent pain is not known. In this study, the effects of vitamin E in chronic pain were investigated in SNL neuropathic rats utilizing behavioral, electrophysiological, and molecular techniques. The aims of this study included testing whether vitamin E would: (1) produce analgesia in neuropathic pain, (2) produce this analgesia through spinal mechanisms, (3) reduce responsiveness of dorsal horn neurons to mechanical stimuli in neuropathic rats, and (4) reduce NMDA receptor phosphorylation in dorsal horn neurons. Part of these data have previously been presented in abstract form (Kim et al., 2004b). 2. Materials and methods 2.1. Experimental animals Adult male Sprague–Dawley rats (200–350 g, Harlan Sprague–Dawley Co., Houston, TX) were used for the experiments. Rats were housed under a 12/12 h reversed light–dark cycle (dark cycle: 8:00 A.M.–8:00 P.M.) for at least one week before beginning any experiments. All experiments were carried out in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and the animal use protocol was approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee. 2.2. Neuropathic surgery (spinal nerve ligation, SNL) Rats were anesthetized with halothane (3% induction, 2% maintenance) in oxygen, then the L5 spinal nerve was tightly ligated as described before (Kim and Chung, 1992). Briefly, a midline skin incision was made on the back at the lower lumbar region, the paraspinal muscles were retracted, and the left transverse process of the L6 vertebra was removed under the dissection microscope. The left L5 spinal nerve was identified and gently separated from the adjacent L4 spinal nerve; it was then tightly ligated using 6-0 silk thread. The wound was cleaned with saline, closed with wound clips, and rats were returned to their cage after recovering from anesthesia. 2.3. Behavioral testing for the measurement of mechanical thresholds The experimenter who conducted the behavioral tests was blinded to the nature of the experimental manipulation to avoid experimental bias. Behavioral tests were conducted to
measure foot withdrawal thresholds in response to mechanical stimuli applied to the left hind paw. For testing, each animal was placed in a plastic chamber (8.5 · 8.5 · 28 cm) which was placed on top of a metal mesh stand, and mechanical stimuli were applied to the plantar surface of the left hind paw with von Frey monofilaments from underneath. Thresholds were determined by the up-down method (Chaplan et al., 1994; Baik et al., 2003), using a set of von Frey monofilaments [von Frey numbers (log stimulus intensity in mg · 10): 3.65, 3.87, 4.10, 4.31, 4.52, 4.74, 4.92, and 5.16; equivalent to: 0.45, 0.74, 1.26, 2.04, 3.31, 5.50, 8.32, and 14.45 g]. von Frey filaments were applied perpendicularly to the plantar surface at the base of the 3rd or 4th toes, the most sensitive area in SNL, with sufficient force to bend the filament slightly for 2–3 s. An abrupt withdrawal of the foot during stimulation or immediately after stimulus removal was counted as a positive response. A further detailed description of the test has been reported previously (Chaplan et al., 1994; Baik et al., 2003; Kim et al., 2004a). 2.4. Vitamin E injections in different experimental groups Vitamin E, DL-a-tocopherol acetate, and olive oil (a vehicle for dissolving vitamin E) were purchased from Sigma Chemical Company (St. Louis, MO, USA). The olive oil itself contains a small amount of vitamin E. However, the total amount of vitamin E in the largest quantity of olive oil (about 2 ml) used for a single injection is only about 0.2 mg, which is negligible as compared to the amount of vitamin E used in this study. 2.4.1. Systemic injection of vitamin E For systemic treatment, appropriate amounts of vitamin E in olive oil were injected intraperitoneally (i.p.) using a 2 gauge needle. The systemic effects of vitamin E on mechanical thresholds were tested in two different paradigms: immediate effects after a single injection and long-term effects with multiple daily injections. For immediate effects after a single injection, mechanical sensitivity was measured after various doses of vitamin E using the randomized Latin square design (Kirk, 1995) in a group of neuropathic rats. To do this, mechanical thresholds were measured before and 3 days after neuropathic surgery in 8 rats. Beginning the 4th postoperative day, baseline values were taken and rats were randomly divided into 4 groups of 2 rats. Rats in each group received an intraperitoneal injection of one of 3 doses of vitamin E (0.1, 1, or 5 g/kg in 10 ml/kg of olive oil) or vehicle alone (olive oil), and behavioral tests were repeated at 1, 2, 4, 6, and 8 h after the injection. The sessions for injection-testing (with the doses that were not given previously) were repeated at the 6th, 8th, and 10th postoperative days, so that all rats received all 4 compounds (3 doses of vitamin E and vehicle) in a random order by the end of the testing period. For long-term effects with multiple daily injections, 18 neuropathic rats were divided into 3 groups of 6 rats each. Starting on the 3rd postoperative day, rats in each group were given repetitive daily intraperitoneal injections of one of two doses of vitamin E (50 or 100 mg/kg) or olive oil alone (vehicle) for 7 days (until the 9th postoperative day), and mechanical thresholds were measured daily up to 15 postoperative days. Mechanical thresholds were measured just prior to the daily
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injections (approximately 24 h after the previous injections) and continued to be measured at the same time of day even after drug treatment was terminated. 2.4.2. Intrathecal (i.t.) injection of vitamin E For spinal i.t. injections, a catheter was implanted at the same time the spinal nerve ligation was done. While the rat was under halothane anesthesia, the paraspinal muscles were retracted, and the posterior part of the 12th thoracic vertebra was removed to expose the spinal meninges. A catheter (sterilized PE 10 tubing filled with artificial cerebrospinal fluid) was inserted into the subarachnoid space through a small nick in the dura and the tip was placed near the lumbar enlargement of the spinal cord. The remaining part of the catheter was fed subcutaneously to the mid-thoracic level and anchored to muscles by sutures and the tip was exposed and sealed. A total volume of 50 ll of various concentrations of vitamin E dissolved in olive oil was injected. The spread of vitamin E after i.t. injection was estimated by examining stained gross brain and spinal cord structures after injecting a lipid-soluble dye (3.5% Sudan Black B from Sigma, 50 ll in olive oil) intrathecally. 2.5. In vivo electrophysiological recording from dorsal horn neurons – single cell extracellular recording Five to ten days after the L5 spinal nerve ligation, rats were anesthetized with two intraperitoneal injections of 0.6 g/kg urethane given at 10 min intervals and the external jugular vein was cannulated. After a tracheotomy, the animal was paralyzed by an i.v. infusion of pancuronium bromide (1 mg/kg for induction and 0.3 mg/kg/h for maintenance) while being maintained on a ventilator. Endtidal CO2 levels were monitored with a capnometer and maintained between 3.5% and 4.5%. Rectal temperatures were kept at 37 C with a regulated electric blanket. A laminectomy was performed at the 3rd to 6th lumbar spinal segments. A carbon fiber-filled glass microelectrode (impedance: 0.4–0.8 MX at 1 kHz) was advanced into the 4th or 5th lumbar spinal cord segment with a stepping motor to a depth ranging from 500 to 900 lm from the dorsal surface. Single unit activity, evoked by probing the hind paw with a glass rod, was used to identify the dorsal horn cells that have hind paw receptive fields. The receptive fields were mapped by probing with a von Frey filament of 1 g bending force. The size of each receptive field was estimated by a point counting method using a 2.5-mm2 lattice. Evoked activities of these dorsal neurons in response to brush, non-noxious (1 g von Frey filament), and noxious (20 g von Frey filament) pressures, were recorded before and at various times after vitamin E treatment (5 g/kg, i.p.). All stimuli were applied at a rate of once per second for 10 s. Recorded signals were amplified, displayed on a digital oscilloscope, and fed into a window discriminator, whose output is used to compile peristimulus time histograms by a data acquisition system (CED 1401 with Spike 2 software). Evoked responses to each stimulus (brush, 1 g, 20 g) were calculated by subtracting the background from the total activity during the 10-s stimulation period. Evoked responses of all recorded neurons were expressed as spikes per second and displayed as box plots.
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At the end of the recordings, a DC current (2 mA for 20 s) was passed through the recording electrode to produce a microlesion. At the end of the experiment, the spinal cord was taken out and immersed in a 10% formaldehyde fixative solution, sectioned at 40 lm, and then stained with toluidine blue. The lesion indicating recording sites was then reconstructed. 2.6. Immunohistochemical staining for phospho-NR1 Immunohistochemical labeling for phosphorylated NMDA receptor subunit 1 (pNR1) was done. When anesthesia was deep (sodium pentobarbital, 10 mg/kg), the chest was opened and the rat was perfused through the aorta with saline, followed by cold fixative containing 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB), pH 7.2. The L5 spinal cord was removed, post-fixed overnight, and then placed in 30% sucrose until equilibration. The spinal cord was cryosectioned at 16 lm, mounted on gelatin-coated slides, and immunostained for pNR1. The sections were incubated in 5% normal goat serum with 0.3% Triton X-100 in PB for 1 h and then in solution containing rabbit anti-phospho-NR1 (1:500 dilution; Upstate Biotechnology, Lake Placid, NY). The sections were washed and then incubated with rhodamine-conjugated goat anti-rabbit IgG solution (1:200; Chemicon International, CA) for 2 h at room temperature. Sections were then washed, air-dried, and coverslipped with mounting medium. To confirm the specificity of the immunolabeling, some sections were processed as above without the primary antibodies. The sections were examined under a Zeiss epifluorescent light microscope or an Olympus Fluoview confocal microscope. The numbers of pNR1-immunostained dorsal horn neurons were counted from 10 randomly selected sections from each animal by an experimenter who did not know the origin of the tissue. The experimenter counted all neurons showing red fluorescence in the cytoplasm having an unstained nucleus. The density of fluorescence of 413 neurons randomly selected from 9 different sections was measured with an image analysis system (Image Pro-Plus). The density of all but one of 48 labeled cells was more than three standard deviations above the average unlabeled cell density, showing that the labeled and unlabeled cells are clearly separated in almost all cases and so identification of labeled neurons was unambiguous. 2.7. Western blots for phospho-NR1 To examine the changes in total amounts of pNR1 protein in the spinal dorsal horn, a group of 6 SNL rats were used for Western blots. On the 7th postoperative day, rats were deeply anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and perfused quickly with cold saline. The L4/5 spinal cord segments were removed, divided into left and right halves (ipsiand contralateral sides, respectively), and immediately frozen on dry ice. Spinal cord tissue was homogenized in 300 ll lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 2 ll protease inhibitor cocktail from Sigma, and 4 ll phosphatase inhibitor cocktail from Sigma) and centrifuged at 16,000 · g for 25 min at 4 C. The supernatant was decanted and used for Western blot analyses. The protein
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concentration of the supernatant was measured using a BioRad Protein Assay Kit I (Bio-Rad, Hercules, CA). The supernatant, with equal amounts of 80 lg protein, was then fractionated by 10% (w/v) SDS–PAGE gel and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with immunoaffinity-purified antibody to phospho-NR1 (1:500; Upstate Biotechnology) and monoclonal anti-b-actin antibody (1:50,000, Sigma, St. Louis, MO) for 2 h at room temperature. The blots were washed three times for 20 min each with buffer and then incubated with horseradish peroxidase-conjugated IgG (1:3000, goat anti-rabbit, Upstate Biotechnology; 1:3000, sheep anti-mouse, Amersham Bioscience). The membranes were washed with buffer three times again for 20 min each and enhanced with a chemiluminescence reagent (ECL kit; Amersham Pharmacia Biotech, Arlington Heights, IL). The blots were exposed to autoradiographic film (Eastman Kodak Co., Rochester, NY), the films were scanned into a computer, and the intensities of the immunoreactive bands of interest were quantified and analyzed using Meta Image series software. Densities were calculated based on the formula, Density = log (255/Intensity). The data are expressed by the ratio of density of pNR1 to the density of b-actin. 2.8. Data analysis Behavioral data are presented as means ± standard errors of the mean (SEM) and analyzed using the SAS statistical program. The data were analyzed by two-way repeated-measures ANOVAs with two-repeated factors followed by Duncan’s post hoc test or by two-way repeated-measures ANOVAs with one-repeated factor followed by Duncan’s post hoc test. Electrophysiological dorsal horn activity data were analyzed by two-way non-parametric repeated-measures ANOVAs (Friedman test) followed by Duncan’s post hoc multiple comparison test. For morphological studies, the comparison of the number of dorsal horn neurons expressing phospho-NR1 immunoreactivity and of the gel density in Western blots was done by Student’s t-test between vehicle vs. vitamin E treated neuropathic rats. In all cases, a p value <0.05 was considered to be significant.
3. Results 3.1. Vitamin E reduces pain behaviors in neuropathic pain The effect of vitamin E on pain behaviors, measured by mechanical thresholds, is shown in Fig. 1. The 50% foot withdrawal thresholds (mechanical thresholds) of the rat hind paw are estimated as 5.27 ± 0.0 (mean ± SEM) in normal naive rats. Following L5 spinal nerve ligation, the mechanical thresholds were reduced to 3.64 ± 0.03, thus demonstrating mechanical allodynia as similarly seen in human patients. Single systemic treatment with vitamin E (0.1, 1, or 5 g/kg, i.p. injection) increased mechanical thresholds for 6 h with the peak response at 1 h after vitamin E injection. The mechanical thresholds reached almost the normal level (5.03 ± 0.06) with the highest dose of
vitamin E (5 g/kg) at 1 h after injection (Fig. 1A). The analgesic effect of vitamin E showed graded responses in relation to the amount of injected vitamin E from 0.1 g to 5 g/kg of body weight. Vehicle injection did not change mechanical thresholds throughout the testing period. The mechanical thresholds of neuropathic rats with daily injection of vitamin E are shown in Fig. 1B. Repeated daily treatment with 50 or 100 mg/kg doses of vitamin E induced a gradual increase of mechanical thresholds, thus, the first significant change was detected 3 days after the initiation of treatment and the analgesic effect reached a significantly high plateau (4.43 ± 0.1 with 50 mg/kg and 4.38 ± 0.13 with 100 mg/kg) after 5 treatments. After termination of vitamin E injection, the significant analgesic effect lasted 5 more days and then eventually returned to the previous allodynic condition by the 6th post-treatment day. These data suggest that vitamin E is effective as an analgesic drug in chronic neuropathic pain. 3.2. The spinal cord is an important action site of vitamin E Vitamin E readily crosses the blood–brain barrier (Goss-Sampson and Muller, 1987). Consequently, systemically injected vitamin E may act on multiple sites, including the spinal cord. To determine the importance of the spinal cord as an active site of vitamin E for analgesic effect, the effects of vitamin E in the spinal cord were tested behaviorally and the results are shown in Fig. 1C. When small amounts of vitamin E (3, 10 or 30 mg) were injected intrathecally (i.t.) through implanted catheters, they produced analgesia in a dose-dependent manner and the analgesia lasted approximately 7 h (Fig. 1C). For example, 30 mg vitamin E changed mechanical thresholds from 3.73 ± 0.08 to 4.54 ± 0.09 at 2 h after drug injection and this increase was significantly different from the pre-injection baseline value. Although not shown, a further increase in the injection dose to 50 mg (n = 3) did not cause any further increase in threshold, suggesting the response had reached its maximum level. In general, the magnitude of threshold elevation by i.t. injection was smaller than that after systemic injection of vitamin E. Therefore, multiple sites seem to be involved in mediating the analgesic effect of vitamin E. However, the spinal cord is clearly an important site for vitamin E action. Examination of the spinal cord after an intrathecal injection of Sudan Black B dye (50 ll in olive oil) into the L5 vertebra level in 12 rats showed that the most rostral segments to which the dye spread ranged between the L1 and T7 levels. These data suggest that injected vitamin E stayed mostly within the lumbar enlargement of the spinal cord.
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Fig. 1. The effects of vitamin E on mechanical thresholds in neuropathic rats. (A) Systemic dose–response – SNL produced a significant reduction in mechanical thresholds (L) from the normal presurgery levels (N) in all operated rats (n = 8). Systemic injection (i.p.) of vitamin E, with doses of 0.1, 1, or 5 g/kg, reversed mechanical allodynia in a dose-dependent manner for a period of 6 h. Vehicle treatment (Vehicle) did not affect the mechanical thresholds. Experiments were done by the randomized Latin square design (2 rats/dose/day, 2 day intervals until all doses were tested in each rat). (B) Repeated treatment – daily injection (3–9 post-ligation days, indicated by arrowheads) of vitamin E (50 or 100 mg/kg, i.p.) produced a gradual increase in mechanical thresholds and reached moderately high plateau levels (4.38 ± 0.13, with 100 mg/kg) by the 5th day of treatment in 2 groups of rats (n = 6/group). After the termination of vitamin E treatment, mechanical thresholds maintained moderately high levels for 5 more days and returned to the pretreatment allodynic condition (3.73 ± 0.07) by the 12th post-ligation day. Vehicle treatment (n = 6) did not change the mechanical thresholds throughout the treatment period. (C) Intrathecal dose–response – single intrathecal injection of graded low doses of vitamin E (3, 10, and 30 mg/kg) showed graded analgesic effects on pain behaviors. Vehicle did not influence the mechanical thresholds. Experiments were done by the randomized Latin square design. N, presurgery normal naive rats; L, 3 days after SNL; B, baseline before vitamin E treatment; *, the value is significantly (p < 0.05) different from that of the vehicle control by two-way repeated-measures ANOVAs followed by Duncan’s post hoc tests.
3.3. Vitamin E reduces sensitization of dorsal horn wide-dynamic-range neurons in neuropathic rats The effects of vitamin E on the activities of spinal dorsal horn neurons were examined in normal and neuropathic rats. In particular, activities of single widedynamic-range (WDR) dorsal horn neurons, which receive multi-modal sensory inputs, were recorded by using an in vivo extracellular electrophysiological recording set-up. An example of a WDR neuron recorded from a normal rat is shown in Fig. 2A. This neuron
had a receptive field at the base of the 2nd through 4th toes and responded well to all stimuli. Vitamin E application (5 g/kg, i.p.) did not change these evoked response activities or the size of the receptive field. The neuron was located in the medial part of the lamina IV as indicated in the cross-section of the cord. An example of a WDR neuron recorded from neuropathic rats is shown in Fig. 2B. This neuron showed much higher evoked responses as well as higher spontaneous activities as compared to those in normal rats, thus showing signs of sensitization. This neuron had a
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Fig. 2. Effects of vitamin E on dorsal horn WDR neuron activity. (A) Normal rat – this neuron responded well to all three different stimuli applied to the receptive field. Neither the responses to mechanical stimuli nor the receptive field size changed after vitamin E (5 g/kg, i.p.). The receptive field (mapped with 1 g von Frey) and recording location are shown in the right inset. Br = Brush stimulus. (B) Neuropathic rat (7 day post-ligation) – this neuron responded more vigorously to mechanical stimuli as compared to cells in normal rats. Both responses and receptive field size were greatly reduced after vitamin E injection. (C) Median evoked responses of populations of WDR neurons [10 cells from normal and 14 cells from neuropathic rats] to various mechanical stimuli before and after vitamin E injection. Box plot values: the thick horizontal line within each box is the median value; the upper and lower edges of each box represent the 75th and 25th percentiles, respectively; and the upper and lower whiskers represent the 90th and 10th percentiles, respectively. Asterisks indicate values that are significantly (p < 0.05) different from the pre-injection levels and the number signs (#) indicate values that are significantly (p < 0.05) different from that of the normal group.
receptive field on the plantar surface of 3rd and 4th toes. Systemic injection of vitamin E markedly reduced the responsiveness of this neuron to innocuous stimuli (brush and 1 g von Frey filament), but not to noxious stimuli (20 g von Frey filament). In addition, the size of the receptive field was reduced after vitamin E treatment. Fig. 2C shows a summary of the responses of 10 neurons (one unit/rat) recorded from normal rats and 14 neurons recorded from neuropathic rats. Evoked responses to each stimulus are expressed as spikes per second and displayed as box plots in Fig. 2C. The average sizes of the receptive fields (as mapped with 1 g von Frey filament) of 6 neurons recorded from normal rats were 58.5 ± 13.3 and 56.5 ± 14.2 mm2 before and 1 h after vitamin E injection, respectively. On the other hand, the average receptive field size of 8 neurons recorded from neuropathic rats was 103.9 ± 14.7 mm2 before vitamin E injection. This size was significantly reduced to 77.4 ± 12.3 mm2 1 h after vitamin E injection. Data show that: (1) the responsiveness of WDR neurons to both innocuous and noxious stimuli was higher in neuropathic rats (median value was 30.49 spikes/s to brush; 10.38 spikes/s to 1 g; 33.72 spikes/s to 20 g) compared to that of normal rats (median value
is 13.33 to brush; 5.58–1 g; and 22.35–20 g); (2) vitamin E treatment did not change the responsiveness in normal rats; (3) vitamin E treatment significantly reduced the responsiveness of dorsal horn WDR neurons in neuropathic rats. It is noted, however, that vitamin E reduced activity evoked by weaker stimuli (brush and 1 g von Frey) more than the enhanced responses to stronger stimuli (20 g von Frey). Thus, overall, the average responses of dorsal horn WDR neurons in neuropathic rats returned to the levels of normal rats after vitamin E treatment. Data suggest that spinal dorsal horn WDR neurons were sensitized after nerve injury and then transiently restored to a normal state after vitamin E treatment. 3.4. Vitamin E reduces the phospho-NR1 expression in the dorsal horn neurons Vitamin E reduced the responsiveness of dorsal horn neurons in neuropathic rats, thus suggesting the reduction of central sensitization. To study the potential effects of vitamin E for NMDA receptor phosphorylation, which is a mechanism likely underlying central sensitization, numbers of dorsal horn neurons expressing
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In the ipsilateral dorsal horn, these numbers are higher at 1.95 ± 0.2 and 14.51 ± 1.2 (n = 6) in the superficial and deep layers, respectively. One hour after treatment with vitamin E, numbers of immunoreactive cells on the ipsilateral side were reduced to levels comparable to that of the contralateral side. The number of pNR1 immunoreactive neurons in the contralateral dorsal horn did not change significantly after vitamin E treatment. The total amount of pNR1 protein in the L4/5 spinal cord was also measured by the Western blot method. An example of a Western blot gel for pNR1 and the averaged relative densities of pNR1 are shown in Figs. 3F
pNR1 as well as levels of pNR1 protein in the spinal cord were examined. Examples of pNR1 immunoreactive dorsal horn neurons in a vehicle treated rat (Fig. 3A), 7 days after nerve ligation (Fig. 3B) and 1 h after vitamin E treatment in neuropathic rats (Fig. 3C), are shown in Fig. 3. In the contralateral dorsal horn of the vehicle (olive oil)-treated neuropathic rats, the average (±SEM) number of pNR1 immunoreactive cells per section is 0.52 ± 0.12 (n = 6) in the superficial layers (laminae I–II) and 10.57 ± 1.17 (n = 6) in the deep layers (laminae III–VI) (Figs. 3D and E). These numbers are not significantly different from that of the unoperated normal rats (n = 6, data are not shown).
A
B
C
E
D
F
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G
Fig. 3. Changes in pNR1 protein expression in the L5 spinal cord. Spinal cord tissues were collected from 7 day post-ligation SNL neuropathic rats 1 h after a systemic treatment with either vehicle (SNL + Veh, 10 ml/kg olive oil, i.p.) or vitamin E (SNL + Vit E, 5 g/kg in olive oil). (A–C) Examples of pNR1 immunoreactive neurons in the dorsal horn (lamina V) of neuropathic rats with various treatments: (A) the contralateral side after vehicle; (B) the ipsilateral side after vehicle; and (C) the ipsilateral side after vitamin E treatment. Calibration bar = 50 lm. (D and E) The averaged number of pNR1 immunoreactive neurons found in the superficial (D; laminae I and II) and deep (E; laminae III–VI) layers of the dorsal horn section (16 lm thick). (F) An example of a Western blot gel of pNR1. The averaged values of relative ratio of pNR1 to b-actin from 6 animals are shown in (G). Only the ipsilateral spinal cord showed increased pNR1 levels after SNL + Veh and then returned to the control levels after vitamin E treatment. Data for D, E, and G were collected from the vehicle (open bars, n = 6) and vitamin E (filled bars, n = 6) treated groups. Asterisks indicate values that are significantly different at p < 0.05 compared to the vehicle control group by Student’s t-test.
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and G, respectively. Seven days after L5 spinal nerve ligation, the relative density of pNR1 was significantly increased in the ipsilateral L4/5 spinal cord (Fig. 3G, n = 6) as compared to that of the contralateral side. This increased level was significantly reduced one hour after intraperitoneal injection of vitamin E (5 g/kg). Thus, vitamin E reduced the expression of phospho-NR1 in the spinal cord. These results are consistent with the electrophysiological data showing that vitamin E reduces central sensitization of dorsal horn neurons. 4. Discussion The effect of a common antioxidant, vitamin E, on chronic pain was investigated in the SNL model of neuropathic pain. Vitamin E significantly reduced behavioral signs of mechanical allodynia. The spinal dorsal horn neurons showed reduced responsiveness, reduced receptive field size, and reduced phospho-NR1 expression after vitamin E treatment in neuropathic rats. These data suggest that an important mechanism for vitamin E-induced analgesia is through reduction of central sensitization. Chronic pain is a state where abnormalities are maintained in both the central and peripheral nervous systems (Woolf and Costigan, 1999; Woolf and Salter, 2000). In the spinal cord, dorsal horn neurons become sensitized and respond more vigorously to peripheral input, and they also receive input from expanded cutaneous receptive fields (Cook et al., 1987; Woolf and Thompson, 1991). NMDA receptors mediate central sensitization and many second messengers are involved in the cascade of changes (Woolf and Thompson, 1991; Willis, 1994; Woolf and Costigan, 1999; Woolf and Salter, 2000). In the periphery, abnormal activities are produced and enter the spinal cord to initiate and maintain the central sensitization (Sheen and Chung, 1993; Yoon et al., 1996; Chung and Chung, 2002; Sukhotinsky et al., 2004). Peripheral abnormal activities include ectopic discharges arising from injured afferent neurons (Devor and Seltzer, 1999; Chung and Chung, 2002) and spontaneous activities arising from sensitized intact nociceptors (Ali et al., 1999; Wu et al., 2001a; Shim et al., 2005). Therefore, there are multiple potential sites where vitamin E can exert its effect. The fact that vitamin E was very effective in producing analgesia by intrathecal injection, that it reduced responsiveness and receptive fields of the dorsal horn neurons, and reduced pNR1 expression in the dorsal horn neurons suggests that the spinal cord is a very important site for analgesic actions of vitamin E. Although the results show that the spinal cord is an important site for vitamin E-induced analgesia, other sites, particularly the periphery, need to be considered. Intra-arterial infusion of the free radical donor tert-butyl-hydroperoxide into one extremity of the rat produces
pain in the affected region (Van Der Laan et al., 1998). Increased production of ROS, both at the injury site and in the dorsal root ganglion neurons of the injured nerve, and reduced pain behaviors by nitric oxide synthase inhibitors (Haley et al., 1992; Levy et al., 1999; Khalil and Khodr, 2001) are observed in animal models of neuropathic pain. Thus peripheral ROS are also involved in the generation of pain. The effect of vitamin E on peripheral mechanisms, such as sensitization of intact peripheral nociceptors, needs to be studied to clarify these peripheral mechanisms. Sensitized spinal dorsal horn neurons become normalized after vitamin E treatment in neuropathic rats. This implies that, after spinal nerve injury, excessive oxidants build up in the spinal cord and then sensitize dorsal horn neurons, suggesting an oxidative stress in the spinal cord. Removing excessive oxidants with vitamin E restores the normal physiological condition and thus relieves pain. Oxidative stress is considered an important determinant for neuronal cell death in various neurodegenerative diseases (Olanow, 1992; Balazs and Leon, 1994; Gerlach et al., 1994) and brain injury (Lewen et al., 2000). The majority of these studies show extreme oxidative conditions where irreversible cell death is eminent. When pain is relieved with vitamin E, the neuropathic pain, at least in the early stages, seems to be a temporary dysfunctional state due to mild oxidative stress but not due to permanent cell death. This temporary dysfunctional state may be a window of opportunity to prevent cell death due to prolonged oxidative stress. Regarding the doses of vitamin E, we believe that 5 g/kg body weight is too high for repetitive clinical use and thus only tested this dose in a single injection paradigm. Since the estimated toxic dietary level of vitamin E is 16,000–64,000 IU/kg (equivalent to about 16–64 g/kg) for a bird (Dierenfeld and Traber, 1993), a single dose of 5 g/kg (5000 IU/kg) for the rat seems to be within safe limits. It is difficult to estimate comparable doses of vitamin E between rats and humans considering the difference in relative metabolic rates, which in rats is much higher than in humans. However, the low dose (50 mg/kg) that we used for repeated daily injections for 200 g rats (10 mg per rat) would be equivalent to 3 g for a 60 kg human. This dose is well within the range of clinical use for human patients (2000–5000 IU of vitamin E which represents about 2–5 g) (Sano et al., 1997; Parkinson Study Group, 1998; Graf et al., 2004). It is important to identify the types and sources of oxidants that play a critical role in neuropathic pain. The most likely sources of oxidants in the cord include superoxides generated from mitochondria and nitric oxides generated by nitric oxide synthase (Coyle and Puttfarcken, 1993; Wu et al., 2001b). It is not known which type is more critically involved in neuropathic pain, and it is possible that both types are involved. Because
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vitamin E can react with various reactive oxidants, including peroxyl radicals, peroxynitrite, nitrogen dioxide, and superoxide (van Haaften et al., 2003), our study could not delineate the specific types of oxidants. Since the levels of mitochondrial reactive oxygen species increase in rat neuropathic dorsal horn neurons (Park et al., 2006), superoxides produced by mitochondria may be one of important free radicals for neuropathic pain. A more specific detection method is required in future studies. The slow buildup and decay of analgesia with repetitive injections of low doses of vitamin E suggest the cumulative effects of vitamin E are due to their storage properties in adipose tissue (Butterfield et al., 2002). The preferential location of vitamin E in the membrane fraction of the mitochondria also indicates the importance of antioxidant action in mitochondria (Buttris and Diplock, 1998). Another important question is the mechanism by which the excessive oxidants produce pain. The present study provides evidence that vitamin E desensitizes spinal neurons. Therefore, it is critical to illuminate molecular steps involved in sensitization of the dorsal horn neurons by oxidants. Overall we speculate that oxidants may be an important initiating factor for some or many already known mechanisms, such as the activation of second messenger signaling systems (Ali and Salter, 2001; Zou et al., 2002; Zhang et al., 2003). We used the enhanced expression of pNR1 in the dorsal horn neurons as an indicator of central sensitization in neuropathic rats. This is based on the fact that phosphorylation of NMDA receptors is involved in activation of NMDA receptors (Cerne et al., 1992; Li and Zhuo, 1998; Christie et al., 1999), and the number of spinal neurons expressing pNR1 increases greatly in conditions where central sensitization and persistent pain exist (Zou et al., 2000; Gao et al., 2005). The present study shows that vitamin E reduced pNR1 expression in dorsal horn neurons, supporting the results of the electrophysiological study on neuronal responses. In conclusion, systemic administration of vitamin E produces analgesia in a rat model of neuropathic pain. Further, this analgesia is built up and prolonged with daily repeated administration of small doses of vitamin E. The mechanism of the analgesia, at least in part, is desensitization of dorsal horn neurons in the spinal cord.
Acknowledgments This work was supported by NIH Grants NS 31680, NS 11255, and AT 01474, and a Research Development Grant from the Sealy Memorial Endowment Fund (2547-03). The authors sincerely thank Denise Broker for her excellent service in English editing.
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