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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Anesth Analg. 2021 Mar 1:10.1213/ANE.0000000000005440. doi: 10.1213/ANE.0000000000005440

Dimethyl Fumarate Reduces Oxidative Stress and Pronociceptive Immune Responses in a Murine Model of Complex Regional Pain Syndrome

Tian-zhi Guo a, Xiaoyou Shi b,c, Wenwu Li b,c, Tzuping Wei a, Wade S Kingery a, J David Clark b,c,d
PMCID: PMC8049952  NIHMSID: NIHMS1664171  PMID: 33646995

Abstract

Background:

Complex Regional Pain Syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works though the nuclear factor erythroid 2–related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional and immunological changes measured in a model of CRPS.

Methods:

Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25mg/kg/day PO for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments, and hindlimb weight bearing data were collected. The markers of oxidative stress malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1beta and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wild-type fractured mice to B cell deficient fractured muMT mice helped to assess the pronociceptive activity of humoral factors.

Results:

We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy and the accumulation of IgM in fractured limbs. The sera of fractured vehicle treated but not DMF treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice.

Conclusions:

Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation.

Introduction

The transition of acute to chronic pain after surgery and other forms of trauma is common, results in suffering, causes poor functional recovery and delays return to normal activities. Chronic pain after surgery and trauma is the cause of 15-25% of all pain clinic visits 1. Pain outlasting primary healing after trauma to the extremities is particularly common. For example, more than one-fifth of patients with wrist fractures, ankle fractures or severe hand trauma continue to experience pain and functional loss after more than 1 year 2. Complex Regional Pain Syndrome (CRPS) is a devastating form of chronic pain after trauma to the extremities with 80% of chronic CRPS patients experiencing severe pain and disability 3. Regrettably, efforts to prevent chronic pain including CRPS after surgery and trauma to the extremities have had limited success 4.

Oxidative stress is a well-studied phenomenon occurring after surgery and trauma exacerbated by inadequate perfusion, inflammation, infection and immobilization 5. Strong links exist between limb injury, oxidative stress and chronic pain that involve both the innate and adaptive systems of immunity 6, 7. For example, limb fracture in rats leads to elevated levels of local and systemic pro-nociceptive cytokines including IL-1beta, IL-6 and TNF-alpha 7, 8. In addition, oxidative stress has been linked to autoimmunity, possibly by the generation of immunogenic malondialdehyde (MDA)-protein adducts 9. MDA-protein adducts are found in rodent limb fracture and ischemia models persisting for at least several weeks after injury 6, 7, and are elevated in CRPS patients 10. Recent work in animal models and humans suggests a role for autoimmunity supporting pain in CRPS 11, 12.

Vitamin C and N-acetylcysteine are two agents used with limited success in laboratory and clinical studies to reduce pain and lower the incidence of CRPS by reducing the impact of oxidative stress 6, 7, 13. Dimethylfumarate (DMF), a drug approved by the FDA for the treatment of multiple sclerosis, may hold more promise. First, some effects of DMF are mediated by the Nrf2 antioxidant transcriptional regulator 14. Specific target genes are involved in glutathione synthesis, NADPH regeneration and heme metabolism 15. Second, DMF exerts an anti-inflammatory effect by reducing B cell activity and reducing pro-inflammatory Th1 and Th17 cells while increasing the Th2 subset 16. We hypothesized, therefore, that DMF treated limb fracture animals would demonstrate less nociceptive sensitization along with reduced oxidative stress and immune system activation.

Methods

2.1. Animals

These experiments were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA, USA) and followed the animal subjects guidelines of the International Association for the Study of Pain. The work also conforms to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (https://www.nc3rs.org.uk/arrive-guidelines). Three strains of mice were used in these experiments; 1) muMT mice lacking mature B cells and immunoglobulin, on a C57BL/6J congenic background (#002288, Jackson Laboratory, Bar Harbor, ME), 2) Nrf2 knockout mice on a C57BL/6J congenic background (#017009, Jackson Laboratory), and 3) wildtype C57BL/6J controls (#000664, Jackson Laboratory). Three month old male mice were used in all experiments and were housed 4 per group under pathogen-free conditions with soft bedding and were given food and water ad libitum, with a 12:12 light:dark cycle. During the experimental period the animals were fed Teklad lab rodent diet 2018 (Harlan Laboratories, Indianapolis, IN), which contains 1.0% calcium, 0.7% phosphorus, and 1.5 IU/g vitamin D3, and were kept under standard conditions with a 12-h light-dark cycle. Data collection was conducted blind to group assignment.

2.2. Surgery

The fracture model was performed in mice as previously described 17. Briefly, under isoflurane anesthesia a hemostat was used to make a closed fracture of the right tibia just distal to the middle of the tibia. The hindlimb was then wrapped in casting tape (Delta-Lite, BSN Medical, Hamburg, Germany) so the hip, knee and ankle were all fixed. After fracture and casting, the mice were given subcutaneously 2 days of buprenorphine (0.1 mg/kg) and enrofloxacin (5 mg/kg) as well as 1.0 ml of normal saline. At 3 weeks after surgery the mice were anesthetized with isoflurane and the cast removed. All mice had union at the fracture site by manual inspection.

2.3. Hindpaw nociceptive testing

Mechanical allodynia was assayed using nylon von Frey filaments according to the “up-down” algorithm as previously described 18. The mice were placed on wire mesh platforms in clear cylindrical plastic enclosures 10 cm in diameter and 40 cm in height, and after 15 minutes of acclimation von Frey fibers of sequentially increasing stiffness were applied against the hindpaw plantar skin at approximately midsole, taking care to avoid the tori pads, and pressed upward to cause a slight bend in the fiber and left in place for 5 sec. Withdrawal of or licking the hindpaw after fiber application was scored as a response. When no response was obtained the next stiffest fiber in the series was applied to the same paw; if a response was obtained a less stiff fiber was applied. Testing proceeded in this manner until 4 fibers had been applied after the initial response. Hindpaw testing was performed bilaterally. Estimation of the mechanical withdrawal threshold by data fitting algorithm permitted the use of parametric statistics for analysis 19. These data were analyzed as the difference between the fracture side and the contralateral untreated side, thus a negative value represents a reduction in the fracture hindpaw withdrawal threshold.

An incapacitance device (IITC Life Science, Woodland Hills, CA) was used to measure bilateral hindpaw weight bearing. The mice were manually held in a vertical position over the apparatus with the hindpaws resting on separate metal scale plates and the entire weight of the mouse was supported on the hindpaws. The duration of each measurement was 6s and 6 consecutive measurements were taken at 10s intervals. All 6 readings were averaged to calculate the bilateral hindpaw weight-bearing values. Right hindpaw (fracture side) weight-bearing data were analyzed as a ratio between the right hindpaw weight and the average of right and left hindpaw values ((2R/(R + L)) × 100%), thus a value less than 100% represents a decrease in weight bearing in the fracture limb.

2.4. Hindpaw temperature testing

The temperature of the bilateral hindpaws was measured using a fine wire thermocouple (Omega Engineering, Norwalk, CT) applied to the paw skin, as previously described 20. The investigator held the wire using an insulating Styrofoam block. Three sites were tested over the dorsum of the hindpaw: the space between the first and second metatarsals (medial), the second and third metatarsals (central), and the fourth and fifth metatarsals (lateral). After a site was tested in one hindpaw the same site was immediately tested in the contralateral hindpaw. The testing protocol was medial dorsum right then left, central dorsum right then left, lateral dorsum right then left, medial dorsum left then right, central dorsum left then right, and lateral dorsum left then right. The six measurements for each hindpaw were averaged for the mean temperature. These data were analyzed as the difference between the fracture side and the contralateral untreated side, thus a positive value represents increased temperature in the fracture paw.

2.5. Hind paw thickness testing

A laser sensor technique was used to determine the dorsal-ventral thickness of the bilateral hind paws, as we have previously described 20. For laser measurements each mouse was briefly anesthetized with isoflurane and then held vertically so the hindpaw rested on a table top below the laser. The paw was gently held flat on the table with a small metal rod applied to the top of the ankle joint. Using optical triangulation, a laser with a distance-measuring sensor was used to determine the distance to the tabletop and to the top of the hindpaw at the midpoint of the third metatarsal and the difference was used to calculate the dorsal-ventral paw thickness. The measurement sensor device used in these experiments (Limab, Goteborg, Sweden) has a measurement range of 200 mm with a 0.01 mm resolution. These data were analyzed as the difference between the fracture side and the contralateral untreated side, thus a positive value represents increased paw thickness in the fracture paw.

2.6. Popliteal lymph node dissection and size measurement

The popliteal lymph node is embedded in adipose tissue of the popliteal fossa and is spherical. Mice were euthanized by carbon dioxide asphyxiation followed by cervical dislocation, the hair surrounding the foot-draining popliteal lymph nodes was clipped, the skin over the node was incised, and the subcutaneous tissue, fat, and fascia were carefully dissected under microscope. The lymph node sizes (diameters) were measured in millimeters (mm) using a caliper with the average diameter for each lymph node defined as [(short-axis diameter + long-axis diameter)/2].

2.7. Western blot analysis for IgM deposition in hindpaw skin

The ipsilateral hindpaw skin was harvested from 3 weeks post fracture mice and non-fracture controls and processed for IgM and β-actin western blot analyses as we have previously described 21. IgM/Actin band intensity ratio was calculated to demonstrate IgM levels.

2.8. Enzyme immunoassays for IL-1, IL-6, MDA, and 4-HNE levels in hindpaw skin

The ipsilateral hindpaw skin was harvested from 3 weeks post fracture mice and non-fracture controls and frozen immediately on dry ice. Samples were cut into fine pieces in ice-cold phosphate buffered saline, pH 7.4, containing a cocktail of protease inhibitors (Roche Applied Science, Indianapolis, IN) and followed by homogenization using a Polytron device (Brinkmann Instruments, Westbury, NY). Homogenates were centrifuged at 12,000g for 15 min at 4° C and supernatant fractions were frozen at −80° C until required for ELISA performance. An aliquot was subjected to protein assay (Bio-Rad, Hercules, CA) to normalize mediator levels. Protein levels for the inflammatory cytokines interleukin 1alpha (IL-1) and interleukin 6 (IL-6) using mouse ELISA kits (R&D Systems, Minneapolis, MN), and the lipid peroxidation products malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) protein levels were also determined by using ELISA kits (MyBioSource, San Diego, CA) according to the manufacturer’s instructions. The results of these assays were confirmed by repeating each experiment twice.

2.9. Dimethyl fumarate (DMF) experiments in the fracture mouse CRPS model

These experiments examined the inhibitory effects of DMF treatment on the post fracture nociceptive (hindpaw von Frey allodynia and unweighting), inflammatory (hindpaw warmth and edema), oxidative stress, and adaptive and innate immune responses of wildtype (WT) mice. The right distal tibia was fractured and casted and then the fracture mice were treated daily with either DMF (25 mg/kg/day, PO, MilliporeSigma, Burlington, MA) or vehicle (0.3ml/day of 0.25% methyl cellulose) for 3 weeks, then the cast was removed and the following day the mice underwent repeat testing for nociceptive and inflammatory responses. After testing the mice were euthanized and the bilateral popliteal lymph nodes were collected for diameter measurements and the hindpaw skin and spinal cord were collected for western blot assays for IgM and the hindpaw skin for enzyme immunoassay for the inflammatory cytokines IL-1 and IL-6, and the lipid peroxidation products MDA and 4-HNE.

The inhibitory effects of DMF treatment on the pronociceptive effects of injecting WT fracture mouse serum antibodies into muMT fracture mice lacking B cells and immunoglobulin were also evaluated. The muMT mice first underwent baseline bilateral hindpaw von Frey fiber and weight bearing testing, and then the right tibia was fractured and casted. At 3 weeks post fracture the casts were removed and the muMT fracture mice were tested for hindpaw von Frey allodynia and unweighting, then they were intraperitoneally injected (0.5ml, IP, once) with the serum collected from WT fracture mice that had been treated with either DMF or vehicle for 3 weeks, and then the muMT fracture mice were retested for allodynia and unweighting at 1, 5, 7, 14, and 21 days after injection.

A separate experiment examined whether DMF treatment prevented the development of allodynia and unweighting in muMT fracture mice lacking B cell mediated immune responses. After fracture the muMT mice do develop hindpaw allodynia and unweighting, but to a lesser extent than that observed in WT fracture mice and this attenuated nociceptive sensitization resolves several months before resolution of sensitization in WT fracture mice.12 Mice were fractured and casted and then treated daily with either DMF (25 mg/kg/day, PO) or vehicle for 3 weeks, then the cast was removed. The next day the mice underwent repeat testing for nociceptive responses and were then euthanized.

DMF is an activator of the Nrf2 pathway that regulates innate immune responses and endogenous antioxidant protection. The final set of experiments examined whether the Nrf2 pathway mediates DMF inhibitory effects on post fracture nociceptive sensitization, inflammation, innate immune responses, and oxidative stress. Nrf2 knockout mice underwent right tibia fracture and hindlimb casting. The fracture mice were treated with either DMF (25 mg/kg/day, PO) or vehicle for 3 weeks, then the casts were removed and the next day the mice underwent repeat testing for nociceptive responses and then were euthanized and the hindpaw skin collected for enzyme immunoassay for the inflammatory cytokines IL-1 and IL-6, and the lipid peroxidation products MDA and 4-HNE.

2.10. Study power and statistical analysis

The study was powered for behavioral and biochemical endpoints based on previous experimentation in this model to identify 25% differences in outcomes with 80% power at an alpha of 0.05.

Statistical analysis was performed using a one-way ANOVA or a two-way repeated measures ANOVA with Holm-Sidak multiple comparisons test for post-hoc contrasts as specified in the figure legends. Data are presented as the means ± standard deviation, and differences were considered significant at a P value less than 0.05 (Prism 5, GraphPad Software, San Diego, CA).

Results

3.1. DMF treatment inhibited the post fracture development of hindpaw nociceptive sensitization and warmth

Figure 1 shows that at 3 weeks after tibia fracture and casting the vehicle treated wildtype (WT) mice developed hindpaw von Frey allodynia, unweighting, warmth, and edema. Daily treatment with DMF (25mg/kg/day, PO) for 3 weeks post fracture reduced hindpaw allodynia, unweighting, and eliminated hindpaw warmth in WT fracture mice, but had no effect on hindpaw edema.

Figure 1. Dimethyl fumarate (DMF) treatment inhibited the post fracture (FX) development of ipsilateral hindpaw nociceptive sensitization and warmth in WT mice.

Figure 1.

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At 3 weeks after tibia FX and casting the vehicle (Veh) treated wildtype (WT) C57BL6 mice exhibited unilateral hindpaw von Frey allodynia (A), unweighting (B), warmth (C), and edema (D). Daily treatment with DMF (25mg/kg/day, PO) for 3 weeks reduced hindpaw allodynia, unweighting, and warmth in the FX mice, but had no effect on hindpaw edema. Measurements for A, C, D represent the difference between the FX side and contralateral paw, thus a negative von Frey threshold value represents a decrease in mechanical withdrawal thresholds on the affected side and positive temperature and thickness values represent increased hindpaw warmth and edema on the FX side. Measurements for B represent weight bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. Statistical analysis was performed using a one-way ANOVA, using a Holm-Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 8 per cohort. ### P < 0.001, and #### P < 0.0001 for differences between the vehicle and DMF treatment groups, *P<0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001 for differences from control values. WT: wildtype mice, Control: control mice, no fracture, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: Control vs. FX+Veh, <0.0001 , Control vs. FX+DMF, 0.0011 , FX+Veh vs. FX+DMF, <0.0001 ; Panel B: Control vs. FX+Veh, <0.0001, Control vs. FX+DMF, <0.0001 , FX+Veh vs. FX+DMF, <0.0001; Panel C: Control vs. FX+Veh, 0.0002, FX+Veh vs. FX+DMF, 0.008; Panel D: Control vs. FX+Veh, 0.003, Control vs. FX+DMF, 0.0338.

3.2. DMF treatment slightly inhibited fracture induced popliteal lymphadenopathy and blocked IgM immune complex deposition in the hindpaw skin

At 3 weeks post fracture the diameters of popliteal lymph nodes in the fracture limb were increased 2-fold (in comparison to the contralateral side) in the vehicle treated WT mice (Fig. 2A). Daily treatment with DMF for 3 weeks after fracture slightly inhibited the post fracture increase in popliteal lymph node diameter. Figure 2B illustrates the dramatic increase in IgM deposition in the ipsilateral hindpaw skin of WT fracture mice. Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IgM abundance.

Figure 2. DMF treatment slightly inhibited fracture induced popliteal lymphadenopathy and blocked IgM immune complex deposition in the hindpaw skin.

Figure 2.

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(A) At 3 weeks post fracture the diameters of the popliteal lymph nodes in the fracture limb were increased (in comparison to the contralateral side) in the vehicle treated WT mice (n = 12 per cohort). Daily treatment with DMF for 3 weeks after fracture slightly inhibited the post fracture increase in ipsilateral popliteal lymph node diameter. (B) At 3 weeks post fracture there was increased IgM immune complex deposition in the ipsilateral hindpaw skin WT mice, compared to control nonfracture mouse values, as measured by western blot analysis. Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increased in IgM immune complex deposition in skin and cord (n = 4 per cohort). Statistical analysis was performed using a one-way ANOVA and a Holm-Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD. ## P < 0.01, ####P<0.0001 for differences between the vehicle and DMF treatment groups, **** P < 0.0001 for differences from contralateral or control values. WT: wildtype mice, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle, Ipsi: ipsilateral to fracture, Contra: contralateral to fracture. Exact P values for each panel: Panel A: WT/FX+Veh-Contra vs. WT/FX+Veh-Ipsi, <0.0001, WT/FX+DMF-Contra vs. WT/FX+DMF-Ipsi, <0.0001, WT/FX+Veh-Ipsi vs. WT/FX+DMF-Ipsi, 0.0057; Panel B: WT/Control vs. WT/FX+Veh, <0.0001, WT/FX+Veh vs. WT/FX+DMF, <0.0001.

3.3. DMF treatment blocked the post fracture development of pronociceptive serum antibodies in WT mice

Figure 3 demonstrates that when serum from 3 weeks post fracture WT mice was injected (0.5ml, IP) into 3 weeks post fracture muMT mice lacking immunoglobulin, the mice gradually developed increased hindpaw von Frey allodynia and unweighting over the ensuing week, and consistent with the half-life of IgM, these pronociceptive effects resolved by 2 weeks after injection. Daily treatment with DMF for 3 weeks after fracture in the WT mice, prior to the collection of their serum, inhibited the pronociceptive effects of WT fracture serum injection into the muMT fracture mice. The administration of serum did not change the nociceptive thresholds of non-fracture limbs in this group of muMT mice (Supplemental Figure 1), and previously published results showed that serum transfer does not sensitize uninjured mice 12. Collectively, these results support the hypothesis that fracture induces the production of IgM autoantibodies capable of triggering nociceptive sensitization and that DMF treatment blocks this pronociceptive adaptive immune response to fracture.

Figure 3. DMF treatment prevented the post fracture development of pronociceptive autoantibodies in WT mice.

Figure 3.

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Injecting the serum (0.5ml, IP) from vehicle treated 3-week post fracture WT mice (WT/FX) into 3-week post fracture muMT mice (muMT/FX) caused a gradual increase in fracture hindpaw allodynia (A) and unweighting (B) over the ensuing week, and consistent with the half-life of IgM, these pronociceptive effects resolved by 3 weeks after injection. When the WT/FX mice were treated for 3 weeks with DMF prior to the serum collection, DMF treatment blocked the pronociceptive effects of WT/FX serum injection into the muMT/FX mice. Statistical analysis was performed using a two-way repeated measures ANOVA and a Holm-Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 7 per cohort. #P <0.05, ##P <0.01, ### P < 0.001, #### P < 0.0001 for differences between the vehicle and DMF treatment groups at each time point. FX: fracture, WT: wildtype mice, muMT: mice lacking B cells and immunoglobulin, DMF: dimethyl fumarate, Veh: vehicle, TX: treated. Exact P values for each panel: Panel A: muMT/FX + Veh TX-WT/FX vs. muMT/FX + DMF TX-WT/FX (7 days, 14 days), 0.0004, 0.0271; Panel B: muMT/FX + Veh TX-WT/FX vs. muMT/FX + DMF TX-WT/FX (5 days, 7 days, 14 days), <0.0001, <0.0001, 0.0016.

3.4. DMF treatment blocked the post fracture development of nociceptive sensitization in muMT mice

Figure 4 illustrates that at 3 weeks post fracture the vehicle treated muMT mice developed hindpaw von Frey allodynia and unweighting, albeit to a lesser degree than observed in 3 weeks post fracture WT mice (Fig.1). Daily DMF treatment for 3 weeks completely blocked the development of hindpaw allodynia and unweighting in muMT fracture mice, indicating that DMF inhibitory effects on fracture induced nociceptive sensitization can’t be entirely attributable to its inhibitory effects on post fracture adaptive immune responses.

Figure 4. DMF treatment blocked the post fracture development of nociceptive sensitization in muMT mice.

Figure 4.

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At 3 weeks post fracture vehicle treated muMT fracture mice (muMT/FX) had developed hindpaw allodynia (A) and unweighting (B), but to a lesser extent than was observed in WT fracture mice (Fig. 1). Daily DMF treatment for 3 weeks completely blocked the post fracture development of hindpaw allodynia and unweighting in the muMT mice. Statistical analysis was performed using a one-way ANOVA and a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 8 per cohort. ### P < 0.001, #### P < 0.0001 for differences between the vehicle and DMF treatment groups, ***P < 0.001, **** P < 0.0001 for differences from control nonfracture values. muMT: mice lacking B cells and immunoglobulin, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: muMT/Control vs. muMT/FX+Veh, 0.0004, muMT/FX+Veh vs. muMT/FX+DMF, 0.0004; Panel B: muMT/Control vs. muMT/FX+Veh, <0.0001, muMT/FX+Veh vs. muMT/FX+DMF, <0.0001.

3.5. DMF treatment blocked the post fracture development of oxidative stress and innate inflammatory responses in WT mice

At 3 weeks post fracture the WT mice developed increased levels of pronociceptive inflammatory cytokines (IL-1, IL-6) and oxidative stress markers (MDA, 4-HNE) in the ipsilateral hindpaw skin (Fig. 5). Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IL-1, IL-6, MDA, and 4-HNE, indicating that DMF treatment had an inhibitory effect on post fracture pronociceptive innate immune responses and oxidative stress.

Figure 5. DMF treatment prevented the post fracture up-regulation of inflammatory cytokines and oxidative stress markers in WT mice.

Figure 5.

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At 3 weeks post fracture vehicle treated WT mice developed increased levels of the inflammatory cytokines (A) interleukin 1β (IL-1) and (B) interleukin 6 (IL-6), and the lipid peroxidation products (C) malondialdehyde (MDA), and (D) 4-hydroxynonenal (4-HNE) in the fracture limb hindpaw skin, relative to control nonfracture mouse values, as measured by enzyme immunoassay. Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IL-1, IL-6, MDA, and 4-HNE. Statistical analysis was performed using a one-way ANOVA and a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 8 per cohort. #P<0.05, ## P < 0.01, ### P < 0.001 for differences between the vehicle and DMF treatment groups, **P < 0.01, ***P < 0.001, ****P <0.0001 for differences from control nonfracture values. WT: wildtype mice, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: WT/Control vs. WT/FX+Veh, <0.0001, WT/Control vs. WT/FX+DMF, 0.0012, WT/FX+Veh vs. WT/FX+DMF, 0.0012; Panel B: WT/Control vs. WT/FX+Veh, 0.0012, WT/FX+Veh vs. WT/FX+DMF, 0.0130; Panel C: WT/Control vs. WT/FX+Veh, 0.0003, WT/FX+Veh vs. WT/FX+DMF, 0.0001; Panel D: WT/Control vs. WT/FX+Veh, 0.0001, WT/FX+Veh vs. WT/FX+DMF, 0.0038.

3.6. DMF treatment blocked the post fracture development of nociceptive sensitization in Nrf2 knockout mice

Figure 6 illustrates that at 3 weeks post fracture the vehicle treated Nrf2 knockout mice developed hindpaw von Frey allodynia, unweighting, warmth, and edema to the same extent observed in 3 weeks post fracture WT mice (Fig.1). Daily DMF treatment for 3 weeks completely blocked the development of hindpaw allodynia, unweighting, and warmth in Nrf2 knockout fracture mice, with no effect on hindpaw edema. These data indicate that DMF inhibitory effects on fracture induced nociceptive sensitization and warmth do not require activation of the Nrf2 pathway.

Figure 6. DMF treatment blocked the post fracture development of nociceptive sensitization in Nrf2 deficient mice.

Figure 6.

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At 3 weeks post fracture vehicle treated Nrf2 knockout mice developed allodynia (A), unweighting (B), warmth (C), and edema (D) to a similar extent as WT fracture mice (Fig.1). Daily treatment with DMF for 3 weeks blocked the development of hindpaw allodynia, unweighting, and warmth in the fracture Nrf2 knockout mice, but had no effect on hindpaw edema. Statistical analysis was performed using a one-way ANOVA, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 7 per cohort. # P < 0.05, #### P < 0.0001 for differences between the vehicle and DMF treatment groups, * P < 0.05, ****P < 0.0001 for differences from control nonfracture values. Nrf2 KO: Nrf2 knockout mice, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0368, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, <0.0001; Panel B: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, <0.0001; Panel C: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, <0.0001; Panel D: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0223, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0223.

3.7. DMF treatment blocked the post fracture development of oxidative stress and innate inflammatory responses

At 3 weeks post fracture the Nrf2 knockout mice developed increased levels of pronociceptive inflammatory cytokines (IL-1, IL-6) and oxidative stress markers (MDA, 4-HNE) in the ipsilateral hindpaw skin (Supplemental Fig. 2). Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IL-1, IL-6, MDA, and 4-HNE, indicating that DMF treatment had an inhibitory effect on post fracture pronociceptive innate immune responses and oxidative stress that does not require activation of the Nrf2 pathway.

Discussion

The pathogenic mechanisms and presentations of pain syndromes initiated by tissue trauma are highly heterogeneous just as the types of surgery and trauma that initiate the syndromes are highly varied. Limb trauma is a common cause of chronic pain and disability with damage to bones, joints, soft tissues and neural structures variably involved 2. One widely recognized pain syndrome linked to limb trauma is Complex Regional Pain Syndrome (CRPS). The model selected for these studies, tibial fracture followed by weeks of cast immobilization, is well-characterized and resembles CRPS in many respects 22. Our main observations were: 1) Post-fracture nociceptive sensitization, limb unweighting and edema were all reduced by the administration of DMF. 2) Fracture with immobilization increases MDA and 4-HNE oxidative markers in the skin of the fractured limbs, and each was reduced by DMF. 3) DMF administration reduced both cytokines that are part of the innate system of immunity and IgM, an effector of the adaptive immune system, in the fractured limb. In fact, DMF prevented fracture animal serum from supporting nociceptive sensitization and limb unweighting in fracture mice lacking the ability to form IgM autoantibodies (muMT mice), and 4) Behavioral, biochemical and immunological effects of DMF were seen in mice lacking a functional Nrf2 gene suggesting that DMF has additional mechanistic targets. These findings are consistent with the hypothesis that oxidative stress contributes to chronic pain after limb trauma via interactions with both the innate and adaptive systems of immunity.

Oxidative stress is common after trauma when oxygen demand exceeds supply. Such settings include those characterized by vascular damage, high tissue interstitial pressures, and constricting immobilizers such as casts. Enhanced demand occurs with extensive tissue damage and infection. Similarly, markers of oxidative stress are found in the serum and saliva of patients with CRPS 23. Oxidative stress has been demonstrated in limb tissues of rodents after fracture 7, or when blood supply is limited by the application of a constricting O-ring 6. The latter model is used as a model of chronic post-ischemic pain. In both types of models, elevations in the products of lipid peroxidation MDA and 4-HNE have been shown. Furthermore, oxidative stress is well-established to activate innate immune cascades including the production of pain-related cytokines 24. Antioxidant agents such as vitamin C, N-acetyl cysteine and TEMPOL (4-hydroxy-2,2,6,6-TetraMethylPiperidine-1-OxyL) reduce nociceptive sensitization, levels of oxidative markers and pro-nociceptive cytokine levels in these models. In our studies, DMF reduced the levels of oxidative markers in the skin of the injured limbs. Likewise, IL-1beta and IL-6 cytokine levels were elevated after fracture and reduced by DMF. The known links between trauma-induced oxidative stress and pain have led to clinical trials of vitamin C where reductions in early postoperative pain 25 and sub-acute pain 26 were demonstrated. Studies examining the effectiveness of perioperative vitamin C to prevent CRPS have reported mixed results 27, 28. It should be noted that the plasma level of vitamin C is tightly regulated, likely limiting the effects of supplemental vitamin C administration on oxidative stress 29.

More broadly, oxidative stress leads to the post-translational modification of proteins that may create neo-epitopes causing autoimmune responses. For example, autoantibodies against MDA-protein adducts were identified in lupus patients 9 a possibility supported by earlier observations of anti-MDA antibodies in lupus-prone MRL/lpr mice 30. A second rheumatologic disease with autoimmune contributions related to oxidative stress is systemic sclerosis. In this condition a complex interplay of vasculopathy, oxidative stress and autoimmunity leads to fibrosis of the skin and organ dysfunction 31. Our previous observations and those of others have demonstrated that autoantibodies may participate in the pathogenesis of CRPS, and that there are likely multiple targets including cell surface receptors and cytoskeletal proteins 11, 12, 32. Our results using DMF demonstrated that this compound could both reduce IgM immune complex deposition in the fracture limb skin and the pronociceptive effects of fracture mouse serum. Previous experiments demonstrated the pro-nociceptive effects of sera from tibia fracture mice and CRPS patients to be mediated by IgM autoantibodies 12.

The therapeutic agent used in our experiments, DMF, has powerful anti-oxidant properties in many experimental systems, and is FDA-approved for the treatment of multiple sclerosis. Classically, DMF is felt to work through the activation of the Nrf2 transcriptional regulator linked to the antioxidant response element (ARE) 33. Affected genes include ones involved in glutathione regeneration, reactive oxygen species detoxification, NADPH regeneration and heme metabolism 15. One rodent neuropathic model study using DMF has shown that the analgesic properties of DMF rely on the presence of the Nrf2 gene 34, and the testing of Nrf2 activators as treatments for CRPS has been advocated 35. The present results, however, demonstrated that Nrf2 deficient mice exhibited very similar analgesic, anti-oxidative and anti-inflammatory response when compared to wild-type mice. In this respect, our results are compatible with the observations of Schulze-Topphoff et al. who showed that the actions of DMF to reduce innate and adaptive immune responses in a model of multiple sclerosis (MS) did not depend on the mice having a functional Nrf2 gene 36. Additional analyses of the circulating cells of MS patients indicated that DMF treatment reduced T follicular helper cell numbers 37, a cell type critical in lymph node germinal center formation. Furthermore, DMF is able to reduce cytokine production by targeting NF-κB rather than Nrf2 38.

This study does have important limitations. Although carefully validated, our rodent model of CRPS does not perhaps recapitulate all aspects of the human pain syndrome, particularly as seen in chronic CRPS sufferers. Also, we did not explore DMF effects on mice with established sensitization. In addition, only male mice were used in this study although we know the timing of activation of the innate and adaptive immune systems differ somewhat between sexes in this model 39. Still, our data interpreted in the context of existing literature on chronic pain after trauma, including surgery, suggests that therapeutic approaches designed to reduce oxidative stress and activation of the innate as well as adaptive systems of immunity may provide long-term benefit to patients.

Supplementary Material

Supplemental Data File (.doc, .tif, .pdf, etc., Published Online Only)_1

Supplemental Figure 1. Wild-type fracture mouse serum had pronociceptive effects in muMT fracture mice were observed only in the ipsilateral fracture limb, not in the contralateral intact limb. This figure presents the time course of the raw von Frey fiber withdrawal threshold data in the ipsilateral and contralateral hindpaws that were presented as side-to-side difference scores in Figure 3A of this paper. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited ipsilateral hindpaw von Frey allodynia (A) with no changes in the contralateral hindpaw thresholds (B). Wild-type fracture mice (WT/FX) were treated for 3 weeks with DMF (25mg/kg/day, PO) or vehicle prior to the serum collection for injection into muMT 3 weeks post fracture mice (muMT/FX). After intraperitoneal injection of vehicle treated WT/FX mouse serum (0.5ml, I.P), the muMT/FX mice gradually developed increased allodynia in the ipsilateral hindpaw over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 3 weeks post-injection. The pronociceptive effects of the vehicle treated WT/FX serum were restricted to the FX limb. No pronociceptive effects were observed after intraperitoneal injection of DMF treated WT/FX serum in muMT/FX mice. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Holm-Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 7 per cohort. # P < 0.05, and ## P < 0.01 for differences between the vehicle and DMF treatment groups at each time point. FX: fracture, WT: wildtype mice, muMT: mice lacking B cells and immunoglobulin, DMF: dimethyl fumarate, Veh: vehicle, TX: treated. Exact P values for each panel: Panel A: muMT/FX + Veh TX-WT/FX vs. muMT/FX + DMF TX-WT/FX serum (7 days, 14 days), 0.0078, 0.0477.

Supplemental Data File (.doc, .tif, .pdf, etc., Published Online Only)_2

Supplemental Figure 2. DMF treatment prevented the post fracture up-regulation of inflammatory cytokines and oxidative stress markers in Nrf2 deficient mice. At 3 weeks post fracture vehicle treated Nrf2 deficient mice developed increased levels of the inflammatory cytokines (A) interleukin 1β (IL-1) and (B) interleukin 6 (IL-6), and the lipid peroxidation products (C) malondialdehyde (MDA), and (D) 4-hydroxynonenal (4-HNE) in the fracture limb hindpaw skin, relative to control nonfracture mouse values, as measured by enzyme immunoassay. Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IL-1, IL-6, MDA, and 4-HNE, similar to its effects in WT fracture mice. Statistical analysis was performed using a one-way ANOVA and a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 8 per cohort. #P<0.05, ## P < 0.01, ### P < 0.001, ####P <0.0001 for differences between the vehicle and DMF treatment groups, *P < 0.05, **P < 0.01, *** P < 0.001, ****P <0.0001 for differences from control nonfracture values. Nrf2 KO: Nrf2 knockout mice, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, <0.0001; Panel B: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0099, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0013; Panel C: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0194, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0097; Panel D: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, 0.0002, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0163, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0487.

Key Points.

Question:

Does dimethylfumarate (DMF) reduce evidence of oxidative stress, immune system activation, nociceptive sensitization and functional impairment in a rodent model of complex regional pain syndrome (CRPS)?

Findings:

We found that DMF strongly reduced the biochemical, immunological and behavioral consequences of tibial fracture in male mice.

Meaning:

These data suggest that DMF and similar dugs may have a role in controlling the pain and pathophysiological changes that accompany CRPS and chronic pain after limb trauma or surgery.

Acknowledgments

Disclosure of funding: This study was supported by the National Institutes of Health grants NS117340 and NS094438, the Department of Veterans Affairs, Rehabilitation Research and Development Merit grant RX001475 and the Department of Defense grant CP190028. The authors do not have financial or other relationships that might lead to conflict of interest.

Glossary

4-HNE

4-Hydroxynonenal

ANOVA

nalysis of variance

CRPS

Complex Regional Pain Syndrome

DMF

Dimethyl fumarate

ELISA

Enzyme-linked immunosorbent assay

FDA

Food and Drug Administration

MDA

Malondialdehyde

MS

Multiple sclerosis

NADPH

Nicotinamide adenine dinucleotide phosphate

Nrf2

Nuclear factor erythroid 2–related factor 2

TEMPOL

4-hydroxy-2,2,6,6-TetraMethylPiperidine-1-OxyL

WT

Wild-type

Footnotes

Conflicts of interest: None

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data File (.doc, .tif, .pdf, etc., Published Online Only)_1

Supplemental Figure 1. Wild-type fracture mouse serum had pronociceptive effects in muMT fracture mice were observed only in the ipsilateral fracture limb, not in the contralateral intact limb. This figure presents the time course of the raw von Frey fiber withdrawal threshold data in the ipsilateral and contralateral hindpaws that were presented as side-to-side difference scores in Figure 3A of this paper. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited ipsilateral hindpaw von Frey allodynia (A) with no changes in the contralateral hindpaw thresholds (B). Wild-type fracture mice (WT/FX) were treated for 3 weeks with DMF (25mg/kg/day, PO) or vehicle prior to the serum collection for injection into muMT 3 weeks post fracture mice (muMT/FX). After intraperitoneal injection of vehicle treated WT/FX mouse serum (0.5ml, I.P), the muMT/FX mice gradually developed increased allodynia in the ipsilateral hindpaw over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 3 weeks post-injection. The pronociceptive effects of the vehicle treated WT/FX serum were restricted to the FX limb. No pronociceptive effects were observed after intraperitoneal injection of DMF treated WT/FX serum in muMT/FX mice. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Holm-Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 7 per cohort. # P < 0.05, and ## P < 0.01 for differences between the vehicle and DMF treatment groups at each time point. FX: fracture, WT: wildtype mice, muMT: mice lacking B cells and immunoglobulin, DMF: dimethyl fumarate, Veh: vehicle, TX: treated. Exact P values for each panel: Panel A: muMT/FX + Veh TX-WT/FX vs. muMT/FX + DMF TX-WT/FX serum (7 days, 14 days), 0.0078, 0.0477.

NIHMS1664171-supplement-Supplemental_Data_File___doc___tif___pdf__etc___Published_Online_Only__1.tif (244KB, tif)
Supplemental Data File (.doc, .tif, .pdf, etc., Published Online Only)_2

Supplemental Figure 2. DMF treatment prevented the post fracture up-regulation of inflammatory cytokines and oxidative stress markers in Nrf2 deficient mice. At 3 weeks post fracture vehicle treated Nrf2 deficient mice developed increased levels of the inflammatory cytokines (A) interleukin 1β (IL-1) and (B) interleukin 6 (IL-6), and the lipid peroxidation products (C) malondialdehyde (MDA), and (D) 4-hydroxynonenal (4-HNE) in the fracture limb hindpaw skin, relative to control nonfracture mouse values, as measured by enzyme immunoassay. Daily treatment with DMF for 3 weeks after fracture blocked the post fracture increase in IL-1, IL-6, MDA, and 4-HNE, similar to its effects in WT fracture mice. Statistical analysis was performed using a one-way ANOVA and a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SD, n = 8 per cohort. #P<0.05, ## P < 0.01, ### P < 0.001, ####P <0.0001 for differences between the vehicle and DMF treatment groups, *P < 0.05, **P < 0.01, *** P < 0.001, ****P <0.0001 for differences from control nonfracture values. Nrf2 KO: Nrf2 knockout mice, FX: fracture, DMF: dimethyl fumarate, Veh: vehicle. Exact P values for each panel: Panel A: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, <0.0001; Panel B: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0099, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0013; Panel C: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, <0.0001, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0194, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0097; Panel D: Nrf2 KO/Control vs. Nrf2 KO/FX+Veh, 0.0002, Nrf2 KO/Control vs. Nrf2 KO/FX+DMF, 0.0163, Nrf2 KO/FX+Veh vs. Nrf2 KO/FX+DMF, 0.0487.

NIHMS1664171-supplement-Supplemental_Data_File___doc___tif___pdf__etc___Published_Online_Only__2.tif (436.3KB, tif)

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