
==== Front
Pain Rep
Pain Rep
PAIREP
Painreports
Pain Reports
2471-2531
Wolters Kluwer Philadelphia, PA

PAINREPORTS-D-23-0175
10.1097/PR9.0000000000001179
00008
3
2
Basic Science
Research Paper
Effects of immunosuppression after limb fracture in mice on nociceptive, cognitive, and anxiety-related outcomes
Sahbaie Peyman ab*
Guo Tian-Zhi acwxtguo@yahoo.com

Shi Xiao-you abxyshi@stanford.edu

Kingery Wade S. cwkingery@stanford.edu

Clark J. David abdjclark@stanford.edu

a Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA
b Department of Anesthesia, Perioperative and Pain Medicine, Stanford University School of Medicine, Stanford, CA, USA
c Palo Alto Veterans Institute for Research, Palo Alto, CA, USA
* Corresponding author. Address: VA Palo Alto HCS, 3801 Miranda Ave, Palo Alto, CA 64304. Tel. 650-493-5000 X 65413; fax: 650-852-3423. E-mail address: psahbaie@stanford.edu (P. Sahbaie).
10 2024
10 9 2024
9 5 e117909 11 2023
14 6 2024
20 6 2024
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Reducing adaptive immune response and IgM formation after tibial fracture mitigated nociceptive changes, improved limb weight-bearing, and diminished spontaneous pain in a mouse model.

Supplemental Digital Content is Available in the Text.

Abstract

Introduction:

Chronic pain is a common and problematic consequence of injuries with few proven methods for prevention or treatment. In addition to pain, functional limitations and neuropsychiatric changes such as cognitive impairment and anxiety worsen outcomes.

Objectives:

To determine whether inhibiting activation of the adaptive immune response after limb fracture would reduce pain, functional loss, memory changes, and anxiety.

Methods:

These experiments used a murine tibial fracture/cast immobilization model that develops these adverse outcomes. Adaptive immunity was blocked using the immunosuppressant FK506 beginning at the time of fracture.

Results:

The administration of FK506 reduced mechanical allodynia and hind limb unweighting for weeks after cast removal as well as nonevoked pain measures. Fracture was associated with working memory loss in the Y-maze assay in vehicle- but not FK506-treated mice. Object recognition memory was not improved with FK506 after fracture. Also, vehicle- but not FK506-treated mice developed an anxiety phenotype. Impaired running wheel performance after cast removal over the following 2 weeks was not improved with FK506 administration. In addition, FK506 treatment blocked Immunoglobulin M (IgM) accumulation in the skin of the fractured limbs, and hippocampal enhancement of matrix metalloproteinase-8 expression, a metalloproteinase associated with neuroplastic changes after injuries, was completely blocked.

Conclusion:

Taken together, our results show that blocking the adaptive immune response after limb trauma reduces the severity of nociceptive and biological changes. The same treatment may reduce the adverse consequences of anxiety and memory deficits using some measures, but other measures of memory are not affected, and activity is not enhanced.

Keywords:

Fracture
Pain
Immunity
B cell
Memory
Anxiety
U.S. Department of DefenseW81XWH2010911 J. David ClarkNational Institute of HelathR01NS117340 Wade S. KingeryU.S. Department of Veterans AffairsI01RX001475 Wade S. KingeryOPEN-ACCESSTRUE
SDCT
==== Body
pmc1. Introduction

The chronification of pain after surgery and other forms of trauma is common, results in suffering, poor functional recovery, and delays return to normal activities. Chronic pain after surgery or trauma is the cause of 15% to 25% of all pain clinic visits2,7 and is one of the 7 principal categories of chronic pain in the new (International Classification of Diseases 11th Revision) ICD-11 framework along with cancer-related pain, headache, and other prominent categories.42 Pain after surgery or trauma to the extremities is particularly common and is of high individual and societal impact. For example, more than one-fifth of patients with wrist fractures, ankle fractures, or severe hand trauma continue to experience pain and functional loss >1 year after their injuries.12,31 Even more concerning, greater than 77% of those suffering high-energy lower extremity injuries were found to continue to experience pain several years after trauma.5 Similarly, there is a high incidence of chronic postsurgical pain, ranging from 10% to 85%,21 and both major, eg, joint replacement, and minor, eg, carpal tunnel release, lead to chronic pain in 15% to 30% of patients.4,8 Importantly, chronic postsurgical and traumatic pain increases exposure to opioids; prolonged opioid use after surgery and trauma is a major risk factor for developing opioid use disorder, the condition lying at the heart of the nation's opioid epidemic.20

Although the management of the chronic pain experience itself continues to challenge us, associated consequences and sequelae of pain such as disability and neurocognitive changes including anxiety, depression, and memory problems are also problematic. For example, Workmen's Compensation claims involve disability in association with chronic pain after limb injuries,11,13 and pain interference is highly elevated for up to 7 years after limb-threatening lower extremity injuries.5 Both anxiety and depression are highly associated with chronic pain after limb injuries.5,9 Alterations in cognition have been less well explored in limb injury patients, although studies from those with other forms of chronic pain suggest deficits may be present.27 Whether pain is the direct cause of poor functional, psychological, and cognitive outcomes after injury is unclear. Unfortunately, laboratory and human studies involving injury seldom address whether early pain control reduces the frequency or intensity on nonpain outcomes.

In the laboratory setting deficits in functional capabilities, anxiety-like behaviors and memory tasks have been demonstrated in a model of fracture-related limb trauma persisting for months after the injury,40 and in fact, the effects of limb fracture on physical performance and memory outlast pain sensitization.41 However, we do not know whether controlling nociceptive changes after injury reduces the highly problematic cognitive and mood-related changes. We hypothesized that early and persistent control of pain in the fracture pain model targeting non-neuronal processes outside of the central nervous system (CNS) would result in reduced functional loss, anxiety-like behaviors, and memory loss measured several weeks after injury.

2. Methods

2.1. Animals

All experiments were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA) and followed the animal subjects guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male C57Bl/6J mice were obtained from Jackson Laboratories (#000664; Bar Harbor, MA) and acclimated in the facility a minimum of 1 week before initiating the experiments. All mice were 11 to 12 weeks old at the time of fracture and were kept under pathogen-free conditions with a 12-hour light/dark cycle with food and water available ad libitum. Mice were habituated to handling by the experimenters for a few minutes each day for 3 days before initiation of experiments. Observers were blinded to drug treatment assignments.

2.2. Drugs

FK506 (Tacrolimus; Abcam Inc., Waltham, MA) was used to inhibit T follicular helper cell-dependent stimulation of germinal center B cells.44 FK506 (0.5 mg/kg, i.p.) was given 3 days per week for 6 weeks starting from day 1 after fracture and was dissolved in 5% (dimethylsulfoxide) DMSO, 0.9% NaCl saline solution. Control experimental groups were given 5% DMSO saline solution.

2.3. Tibia fracture

Mice underwent tibia fracture and cast immobilization of the limb (Fx) as previously described.14,33 Briefly, mice were anesthetized with isoflurane, and a closed fracture of the right tibia just distal to the midpoint was performed using a hemostat. Next, the limb was wrapped in casting tape (Delta-Lite; BSN Medical, Hamburg, Germany), so the hip, knee, and ankle were all fixed forming a spica around the abdomen. The model results in a well-characterized transverse bone fracture and soft-tissue injury.17 Control animals received anesthesia without manipulation of the hind limbs. The casts were removed under anesthesia at 3 weeks after fracture. Manual inspection assured union of the fracture.

2.4. Mechanical nociceptive assay

Mechanical sensitivity was assessed using nylon von Frey filaments (Stoelting Co, Wood Dale, IL) according to the “up-down” algorithm developed by Chaplan et al.6 We have applied this technique previously to identify 50% withdrawal threshold in mice after injury.14,40 After acclimation, sequential fibers with increasing stiffness ranging applied to the plantar surface of hind paw and left in place for up to 5 seconds. When 4 fibers had been applied after the first response the testing terminated. Withdrawal of hind paw from the fiber was considered a response. If a response occurred after application of a fiber, then a less stiff fiber was applied, and if no response was observed, the next stiffest fiber was applied. A data fitting algorithm was used to provide an estimation of mechanical withdrawal threshold for parametric significance analysis determination.28

2.5. Hind paw unweighting

To measure hind paw unweighting, an incapacitance device (IITC Inc Life Science, Woodland Hills, CA) was used as we have described previously.18 The mice were held in a near-vertical position over the apparatus within a short restraint cone with the hind paws resting on separate metal scale plates allowing the weight of the mouse to be supported on the hind paws. Hind paw weight-bearing data were analyzed as a ratio between twice the right (fracture limb) hind paw weight and the sum of right (R) and left (L) hind paw values ([2R/(R + L)] × 100%).

2.6. Running wheel exercise assessment

Some experimental mice had ad libitum access to the running wheels 24 h/d, for 7 days at 3 weeks after fracture. A computerized activity wheel (Scurry mouse activity wheel [Model 80820S] and software, Lafayette Instrument, Lafayette, IN) allowed monitoring of the daily distances ran by the mice.

2.7. Conditioned place preference

To assess the effects of FK506 treatment on the spontaneous aversive component of pain associated with limb trauma, a single trail conditioned place preference (CPP) test was used.34 The CPP paradigm reveals the presence or absence of an aversive state associated with spontaneous pain. The CPP experiments were performed using standard conditioning 3-chamber testing enclosures placed inside sound-attenuating cabinets. The 2 side association chambers of CPP box have distinct contextual cues and were used for conditioning sessions. Each experiment started with 3 preconditioning days when the mice had free access to the 3 chambers for 30 minutes of exploration. On preconditioning day 3, mice had free chamber exploration and were video recorded for 15 minutes. Any mouse that spent >80% or <20% of the total experimental time in either of the association compartments was excluded. On day 4, mice received saline injections and were assigned to either one of the association compartments for 50 minutes. Next, mice were placed on the opposite side after 4 hours, immediately after morphine (0.3 mg/kg) administration. On day 5, mice were placed in the middle neutral compartment of the apparatus and were assessed for time spent in outer compartments for 15 minutes. Drug or vehicle injection and chamber assignments were randomized and counterbalanced between groups. TopScan (Clever Sys., Reston, VA) software was used to analyze video recordings for time spent in each association chamber. Difference scores were calculated as (time spent in drug chamber − time spent in vehicle chamber) during the test day. A high score indicates the presence of spontaneous pain shown by preference for the drug side, and a low score indicates absence of a spontaneous pain state.

2.8. Zero-maze and working memory tests

The elevated zero-maze (ZM) was also used to measure risk taking and anxiety according to previously published methods.36 The maze is 24 inches above the floor, has an outer diameter of 24 inches and inner diameter of 20 inches, and 2 closed (6-inch-tall walls) and open quadrants. Mice were randomly placed facing one of the closed quadrants at the beginning of the 5-minute test. Total time spent in open and closed quadrants was recorded. Time spent in the open quadrants was compared across groups to measure anxiety and risk-taking behavior.

Y-maze (YM) testing was used to assess spatial working memory.29 The arena consisted of 3 symmetrical arms (arms A, B, and C) at 120° angles with a dimension of 20 × 8 × 16 cm (L × W × H) for each arm. The mice were placed in the center of the arena, and arm entry was recorded for 10 minutes. Unique triad combination of consecutive arm entries was used as a measurement of spontaneous alternation behavior. Typically, rodents prefer to investigate a new arm of the maze rather than returning to one that was previously visited. The triads of arm entries in which the mouse sequentially visited each possible arm without repeating, ie, ABC, CBA, BCA, BAC, and ACB, was used as a measure of spontaneous alteration. Percentage of alternation was calculated as (number of unique triad combination)/(total number arm entries − 2).

For object recognition memory experiments, mice were initially habituated to 2 identical objects after being placed in the middle of the open field arena, as previously described.36 Subsequently mice were returned to their home cages for 5 minutes. Next, during a 10-minute trial, mice were returned to the arena with one of the previous identical objects being replaced with a novel one, and exploratory behavior (investigation time) was recorded. As mice explore novel objects more than familiar ones, time spent exploring the novel compared with familiar was used to assess spatial working memory. All recordings from the above experiments were analyzed in real time by TopScan software (Version 3.0; Clever Sys.).

2.9. Western blot analysis

The mouse hind paw skin was harvested and processed for detection of IgM deposition as previously described.22 Briefly, tissues were homogenized, and protein concentrations were determined by a Bio-Rad DC protein assay reagent (Bio-Rad, Hercules, CA). After gel electrophoresis (sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS-PAGE]) and transfer onto a polyvinylidene difluoride membrane, blots were blocked and then incubated with primary antibodies against IgM or β-actin (Santa Cruz Biotechnology, Dallas, TX) overnight. Next, the blots were incubated with secondary antibody for 1 hour, and proteins were detected using enhanced chemiluminescence plus chemiluminescence reagent (Thermo Scientific, Waltham, MA). Images were obtained using ChemiDoc MP Image Systems (Bio-Rad) and analyzed by ImageJ.

2.10. Enzyme immunoassay for matrix metalloproteinase-8 and tissue inhibitor of metalloproteinase 2 in the hippocampus

Mouse hippocampus was removed from the isolated brain and frozen immediately on dry ice. All tissues were then processed according to published methods.39 The protein levels of mouse matrix metalloproteinase-8 (MMP8) and tissue inhibitors of metalloproteinase 2 (TIMP2) were measured in duplicate by using mouse MMP8 and TIMP2 ELISA kits (MyBioSource, San Diego, CA) following the manufacturer's instructions.

2.11. Data analysis

A fitting algorithm was applied to mechanical withdrawal response data (von Frey testing) to obtain a 50% mechanical threshold estimation value, thereby permitting the use of parametric statistics for significance analysis determination. Then, data for mechanical sensitivity, as well as, hind limb unweighting were analyzed by 2-way analysis of variance with Tukey correction for multiple comparisons (95% confidence interval of differences plus significance: α = 0.05). Two-way analysis of variance with Sidak post-hoc test was used for determining differences in object recognition memory data. Data from YM, ZM, and CPP as well as protein measurements were analyzed by one-way analysis of variance with the Fisher least significant difference (LSD) test for between-group evaluations. All data are presented as mean ± SEM, and for all analyses, P < 0.05 was taken to be significant. For all experiments, group sizes were calculated to have approximately 80% power to detect 25% changes at the P < 0.05 level.

3. Results

3.1. Continuous treatment with FK506 reduced nociceptive sensitization and improved weight-bearing capacity of the limb after tibia fracture

Figure 1 shows the timeline used for the experiments including the surgeries, drug treatment, and testing. Continuous treatment with systemic FK506 significantly reduced mechanical allodynia (Fig. 2A: F1,66 = 80.04, P < 0.001; significant main effect of treatment) and improved weight-bearing capacity of the affected limb (Fig. 2B: F1,66 = 20.35, P < 0.001; significant main effect of treatment) compared with vehicle treatment in fracture mice for at least 6 weeks after fracture. Systemic FK506 had no effects on either measurement between nonfracture groups.

Figure 1. Overview of the experimental timeline in weeks. B, baseline; CPP, conditioned place preference; FX, fracture; ORM, object recognition memory; YM, Y-maze; von Frey, mechanical sensitivity assay; ZM, zero-maze.

Figure 2. Treatment with FK506 reduced nociceptive sensitization and improved weight-bearing capacity of the limb after tibia fracture. Mice underwent distal tibia fracture and 3 weeks of cast immobilization. Ongoing vehicle or FK506 (5 mg/kg; 3 days per week) treatment was initiated the day after fracture. Compared with vehicle-treated fracture mice, FK506-treated mice exhibited reduced ipsilateral hind limb mechanical allodynia (A) and improved hind limb weight-bearing (B), and these effects persisted up to 6 weeks. Data were analyzed by 2-way ANOVA (analysis of variance) followed by Tukey multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant difference from baseline measurements, and ###P < 0.001 indicates significant difference between Fx/vehicle and Fx/drug groups. Error bars: SEM, n = 12/group.

Exercise performance after fracture was assessed using metered running wheels. Both vehicle- and FK506-treated fracture groups displayed decreased running wheel performance during the first 2 weeks after cast removal compared with controls (Figure S1, http://links.lww.com/PR9/A240). No significant differences were seen between vehicle- and FK506-treated fracture groups in wheel running performance. These results suggest that moderate levels of analgesia are insufficient to enhance exercise performance in this model.

3.2. Continuous treatment with FK506 diminished spontaneous pain behavior of mice after tibia fracture

As the results above indicated FK506 treatment had beneficial effects on reducing nociceptive changes and limb weighting impairment after limb trauma, we determined whether these effects extend to nonevoked ongoing pain behaviors. Conditioned place preference was used to determine the presence of an ongoing pain state, and FK506 treatment eliminated place preference behavior suggesting that there was little ongoing pain (Fig. 3: Fx/FK506 vs Fx/vehicle, P < 0.05).

Figure 3. Continuous treatment with FK506 diminished spontaneous pain behavior of mice after tibia fracture. Single trail conditioned place preference (CPP) was used to determine the presence of nonevoked ongoing pain behavior (tonic aversive state) after FK506 or vehicle treatment after fracture (Fx) in mice. FK506 prevented spontaneous pain behavior observed at 6 weeks after injury. Difference scores were calculated as (drug-paired side − vehicle-paired side) time spent on the test day (postconditioning day). Therefore, a higher score represents stronger preference for the drug-paired side where relief of the aversive state was experienced. Conversely, no preference for the drug-paired side indicates lack of evidence for the existence of an aversive state in those animals. Data were analyzed by one-way ANOVA (analysis of variance) followed by Fisher LSD post-hoc tests, *P < 0.05 indicates significant difference between groups. Error bars: SEM, n = 12/group.

3.3. Treatment with FK506 reduced postfracture hind paw skin IgM deposition

Previous work from our laboratory demonstrated that IgM from fracture mice or from patients with chronic pain after limb injuries was pronociceptive in mice when injected systemically, intrathecally, or intraplantarly.15,18 Treatment with FK506, an inhibitor of germinal center B-cell maturation, was previously shown to block IgM formation at 3 weeks after fracture, but it was not known whether this effect could be sustained with continuous FK506 treatment.23 Using western blot analysis, we observed that mice continuously treated with FK506 had significantly reduced hind paw skin IgM deposition compared with vehicle-treated fracture mice 6 weeks after fracture (Fig. 4: P < 0.001).

Figure 4. Treatment with FK506 reduced postfracture skin IgM deposition. Deposition of IgM in hind paw skin of mice after fracture (Fx) and ongoing vehicle or FK506 (5 mg/kg; 3 days per week) treatment. Skin IgM content of the fracture/control limb was quantified using western blot analysis. Data were analyzed by one-way ANOVA (analysis of variance) followed by Fisher LSD post-hoc tests. **P < 0.01 and ***P < 0.001 indicate significant difference between groups. Error bars: SEM, n = 4 mice/group, each sample run in duplicate.

3.4. Anxiety evaluations after FK506 treatment after tibia fracture

Mice exhibit anxiety behavior after limb trauma.40 The elevated ZM test showed that fracture mice spent less time in the open areas of the maze indicating anxiety-like behaviors (Fx/vehicle: 37.81 ± 4.25 vs controls: 63.168 ± 4.09; mean ± SEM, P < 0.01) and (Fx/vehicle: 37.81 ± 4.25 vs FK 506 controls: 55.38 ± 7.31; mean ± SEM, P < 0.05). However, fracture mice treated with FK506 had open arm times that did not differ statistically from FK506-treated controls (Fx/FK506: 49.36 ± 6.59 vs FK 506 controls: 55.38 ± 7.31; mean ± SEM, P = 0.46) or vehicle-treated fracture mice (Fx/FK506: 49.36 ± 6.59 vs Fx/vehicle: 37.81 ± 4.25; mean ± SEM, P = 0.16; Fig. 5).

Figure 5. Anxiety evaluations after FK506 treatment after tibia fracture. Anxiety levels were measured using an elevated zero-maze (ZM) after fracture (Fx) and ongoing vehicle or FK506 (5 mg/kg; 3 days per week) treatment. The elevated ZM has 2 closed and 2 open quadrants. Time spent in the open quadrants was compared across groups to measure anxiety and risk-taking behavior. Higher anxiety levels in mice are expressed as decreased time spent (increased avoidance) of ZM open areas, compared with controls. Data were analyzed by one-way ANOVA (analysis of variance) followed by Fisher LSD post-hoc tests. *P < 0.05 and **P < 0.01 indicate significant difference between groups. Error bars: SEM, n = 12/group.

3.5. Effects of treatment with FK506 on working memory deficits after fracture

It was demonstrated previously that limb fracture in mice caused deficits in spatial and nonspatial working memory.40 Although fracture vehicle-treated mice demonstrated deficits in YM testing in comparison with control animals (mean ± SE of differences: Fx/vehicle = 42.30 ± 16.83 vs control = 58.14 ± 7.61, P < 0.01), FK506-treated fracture mice did not differ from controls (Fx/FK506 = 53.07 ± 14.51 vs control = 58.14 ± 7.61, P = 0.38; Fig. 6A). Novel object recognition was impaired after fracture, and FK506 treatment did not result in improved performance in the object recognition task (Fig. 6B).

Figure 6. Effects of treatment with FK506 on working memory deficits after fracture. Working memory evaluations in mice after fracture (Fx) and ongoing vehicle or FK506 (5 mg/kg; 3 days per week) treatment. Spatial working memory (A) was measured using the Y-maze and nonspatial working memory (B) was measured using object recognition memory (ORM) tests. Data from Y-maze testing were analyzed by one-way ANOVA (analysis of variance) followed by Fisher LSD post-hoc tests and by two-way ANOVA followed by Sidak post-hoc tests for ORM experiments. *P < 0.05 and **P < 0.01 indicate significant difference between groups in (A), and ***P < 0.001 indicates significant time difference between novel and familiar objects (B). Error bars: SEM, n = 12/group.

3.6. Effects of treatment with FK506 on hippocampal matrix metalloproteinase-8 and TIMP2 after fracture

Previous work from our group showed fracture-associated changes in the levels of key hippocampal extracellular matrix components and enzymes, including MMP8 and the endogenous MMP inhibitor tissue inhibitor of metalloproteinase 2 (TIMP2). Elevations in MMP8 levels were further shown to modulate pain, memory, and anxiety in this model.39 In the present experiments, we observed increases in MMP8 after fracture, and that FK506 treatment eliminated the enhanced expression (Fig. 7A: Fx/FK506 vs Fx/vehicle, P < 0.001), but no effects on TIMP2 (Fig. 7B).

Figure 7. Effects of treatment with FK506 on hippocampal MMP8 and tissue inhibitors of metalloproteinase 2 (TIMP2) after fracture. Changes in MMP8 (A) and TIMP-2 (B) protein levels in the C57Bl/6J mice hippocampus after fracture and ongoing vehicle or FK506 (5 mg/kg; 3 days per week) treatment. Both protein levels of the fractured/control limb were quantified using ELISA, and data were analyzed by one-way ANOVA (analysis of variance) followed by Fisher LSD post-hoc tests. ***P < 0.001 and ****P < 0.0001 indicates significant difference between groups. Error bars: SEM, n = 6 mice/group, each sample run in duplicate. MMP8, matrix metalloproteinase 8.

4. Discussion

The transition of postinjury acute pain to a chronic pain state is accompanied by extended suffering as well as the prolongation of disability and the appearance of neuropsychiatric consequences such as mood changes, anxiety, and cognitive difficulties. Consistent with the general estimates of pain after surgical procedures, trauma and surgery to the hands, wrists, and ankles result in pain in approximately 20% of patients one year after their injuries.12 Chronic pain after fracture surgeries is particularly high with one observational study reporting a 57% incidence at 3 months and nearly 43% one year after the procedures.1 The causes of chronic pain after surgery and injuries are under intensive study with nerve injuries, immune system dysfunction, psychological comorbidities, age, genetics, and other factors all possibly playing contributing roles.32 Our studies used a well-validated laboratory model of pain related to orthopedic injury to extend our understanding of the relationship between pain chronification, anxiety, and memory. Our main findings were (1) the immune suppressant FK506 robustly reduced postfracture nociceptive sensitization and hind limb unweighting but not impairment of running wheel performance. These effects were sustained from 3 to 6 weeks after fracture. (2) FK506 also prevented spontaneous pain as measured using the CPP protocol. This is a nonevoked pain measure considered critical to understanding ongoing aversive outcomes of laboratory pain models. (3) Turning to neuropsychiatric outcomes, FK506 treatment prevented the anxiety phenotype observed in vehicle-treated mice (ZM) as well as spatial working memory deficits (YM) but did not change the deficits measured using novel object recognition. (4) Our biochemical studies showed that FK506 treatment prevented hippocampal increases in MMP8 levels, a metalloproteinase involved in neuroplastic changes within the CNS including ones linked to memory.22,40 Together, these results suggest that FK506 treatment is effective in reducing nociceptive changes in the fracture model, but effects on neuropsychiatric outcomes and physical performance are not uniform.

In our studies, we attempted to control post-traumatic nociception by reducing activity of the humoral immune system as this has been shown to support persistent pain after trauma in humans and laboratory animals. Early studies involving the tibial fracture model demonstrated pronociceptive contributions of various cytokines, including IL-1β, IL-6, (tumor necrosis factor) TNFα, and others (reviewed in Ref. 3). However, nociceptive sensitization in the tibial fracture mice long outlives the transient elevations of peripheral nociceptive mediators.45 Activation of humoral immune system, the formation of germinal centers in lymph nodes of the injured limb, and the production of IgM, however, followed a time course more closely matching the months-long period of nociceptive sensitization and pain characteristic of the model.23 Administration of the calcineurin immunosuppressive drug FK506 blocked the formation of germinal centers and autoantibody production.23 The drug works by blocking activation of the transcription factor nuclear factor of activated T cells, a process critical to the function of T follicular helper cells in germinal centers.30 This fairly selective activity of FK506 contrasts with glucocorticoids sometimes used for the treatment of limb pain conditions such as (complex regional pain syndrome) CRPS. Glucocorticoids have broad anti-inflammatory effects resulting from binding to nuclear glucocorticoid receptors.43

Although studies of chronic pain tend to focus on the pain itself, there are important comorbidities that should be taken into consideration. Although the mechanisms are generally poorly described, chronic pain is associated with various types of cognitive difficulties including memory decline and may increase the probability of dementia.19,46 It appears that working memory and long-term memory dysfunction are the most vulnerable in this sense.24 Unclear, however, is whether it is the pain per se that drives the memory problems, or whether inflammation, immobility, stress, or other factors contribute through separate mechanisms. Even so, nociceptive signals reach the hippocampus and related limbic structures through multiple pathways, and the morphology of the hippocampus is altered in those with chronic low back pain and chronic pain involving the limbs.26 Laboratory studies demonstrate neuroinflammation, neuroplasticity, extracellular matrix changes, and impaired neurogenesis in chronic pain models including our tibia fracture model.37,39 Additional evidence links the metalloproteinase MMP8 and its endogenous activity regulator TIMP2 to neoplastic changes in the hippocampus and spinal cord after limb fracture in mice, whereas the downregulation of hippocampal MMP8 in the fracture mice normalized the neuroplastic, extracellular matrix and behavioral changes.38,39 Our findings include the demonstration of elevated MMP8 (but not TIMP2) after tibial fracture, and this increase was prevented by administration of FK506. Despite these effects, memory deficits were not uniformly prevented. It is possible that more profound analgesia would provide better control of changes within the CNS after limb fracture. Alternatively, the non-nociceptive effects of immune system activation after fracture and immobilization may contribute independently to the hippocampal changes and require additional treatment approaches to overcome.

Anxiety is very common in patients with chronic pain affecting more than 30% of these individuals.25 The relationship of pain and anxiety is complex with premorbid anxiety linked to chronic pain after surgery.32 Low back pain is associated with an elevated risk of the development of depression and anxiety, although not all studies demonstrate a causal relationship.10 The neurobiological underpinnings of anxiety are, like other mood and cognitive functions, very complex. However, the hippocampus does have a role. Recent data show that changes in the hippocampus after nerve injury such as reduced neurogenesis are associated with the onset of anxiety-like behaviors in laboratory animals; restoration of neural stem cell populations in the hippocampus by fluoxetine treatment reduced the anxiety-like behaviors without altering pain-related responses.47 In our studies, tibial fracture was seen to be associated with an anxiety-related phenotype in the ZM, whereas fractured animals receiving FK506 were not different from controls. However, more comprehensive testing involving additional paradigms, timepoints, and antinociceptive treatments is required.

Regarding effects on function, FK506 did largely restore fractured hind limb weight-bearing similar to results for anticytokine, anti-NGF (nerve growth factor), and antisympathetic strategies previously tested in this model.14,17,33 However, running wheel performance after fracture was not improved. This may be due to the level of analgesia being inadequate or that atrophy and musculoskeletal changes in the limb because of 3 weeks of casting reduce exercise performance independent of the nociceptive state of the limb.

Despite the many findings presented and discussed, the current work has various limitations. For example, the impact of sex on responses to FK506 was not explored. Our previous work using the same fracture-cast model demonstrated that although male and female animals rely on IgM-mediated mechanisms for postfracture sensitization, females display less dependence on IgM 3 weeks after fracture and have quantitatively different levels of IgM accumulation in skin, sciatic nerve, and spinal cord tissue after fracture.16 Additional studies demonstrate sex-dependent differences in CNS gene expression after fracture and differences in memory and function between the sexes in the fracture model.35,41 Sex-focused studies are, therefore, indicated. In addition, we examined treatment effects beginning close to the time of fracture and extending only 6 weeks. The use of delayed treatment and examination of outcomes at later timepoints would further inform possible translational use of FK506. Finally, it should be recognized that although immunomodulators may have roles in the control of chronic pain, they are not without risks and side effects most notably enhancing susceptibility to infection. Hopefully, future work will identify the minimum extent of immune suppression necessary to reduce chronic pain, memory loss, and anxiety after limb trauma and surgery.

Disclosures

The authors have no conflicts of interest to declare.

This work was supported by Department of Defense award W81XWH2010911 (J.D.C.), R01NS094438 (W.S.K.), R01NS117340 and the VA Merit Review award I01RX001475 (J.D.C.).

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Appendix A. Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PR9/A240.

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painrpts.com).
==== Refs
References

[1] Aulenkamp JL Malewicz NM Brauckhoff JD Zahn PK Ebel M Schnitzler R Clever J Gessmann J Bauer M Meyer-Friessem CH . Chronic pain following fracture-related surgery: posttraumatic rather than postsurgical origin promotes chronification—a prospective observational study with 1-year follow-up. Anesth Analg 2022;134 :974–86.34889805
[2] Barke A Korwisi B Casser HR Fors EA Geber C Schug SA Stubhaug A Ushida T Wetterling T Rief W Treede RD . Pilot field testing of the chronic pain classification for ICD-11: the results of ecological coding. BMC Public Health 2018;18 :1239.30404594
[3] Birklein F Ibrahim A Schlereth T Kingery WS . The rodent tibia fracture model: a critical review and comparison with the complex regional pain syndrome literature. J Pain 2018;19 :1102.e1–19.
[4] Bossmann T Brauner T Wearing S Horstmann T . Predictors of chronic pain following total knee replacement in females and males: an exploratory study. Pain Manag 2017;7 :391–403.28936909
[5] Castillo RC MacKenzie EJ Wegener ST Bosse MJ Group LS . Prevalence of chronic pain seven years following limb threatening lower extremity trauma. PAIN 2006;124 :321–9.16781066
[6] Chaplan SR Bach FW Pogrel JW Chung JM Yaksh TL . Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53 :55–63.7990513
[7] Crombie IK Davies HT Macrae WA . Cut and thrust: antecedent surgery and trauma among patients attending a chronic pain clinic. PAIN 1998;76 :167–71.9696470
[8] Curtin CM Kenney D Suarez P Hentz VR Hernandez-Boussard T Mackey S Carroll IR . A double-blind placebo randomized controlled trial of minocycline to reduce pain after carpal tunnel and trigger finger release. J Hand Surg Am 2017;42 :166–74.28259273
[9] Degen RM MacDermid JC Grewal R Drosdowech DS Faber KJ Athwal GS . Prevalence of symptoms of depression, anxiety, and posttraumatic stress disorder in workers with upper extremity complaints. J Orthop Sports Phys Ther 2016;46 :590–5.27170526
[10] Fernandez M Colodro-Conde L Hartvigsen J Ferreira ML Refshauge KM Pinheiro MB Ordonana JR Ferreira PH . Chronic low back pain and the risk of depression or anxiety symptoms: insights from a longitudinal twin study. Spine J 2017;17 :905–12.28267634
[11] Feuerstein M Miller VL Burrell LM Berger R . Occupational upper extremity disorders in the federal workforce. Prevalence, health care expenditures, and patterns of work disability. J Occup Environ Med 1998;40 :546–55.9636935
[12] Friesgaard KD Gromov K Knudsen LF Brix M Troelsen A Nikolajsen L . Persistent pain is common 1 year after ankle and wrist fracture surgery: a register-based questionnaire study. Br J Anaesth 2016;116 :655–61.27106969
[13] Fulton-Kehoe D Franklin G Weaver M Cheadle A . Years of productivity lost among injured workers in Washington state: modeling disability burden in workers' compensation. Am J Ind Med 2000;37 :656–62.10797509
[14] Guo TZ Offley SC Boyd EA Jacobs CR Kingery WS . Substance P signaling contributes to the vascular and nociceptive abnormalities observed in a tibial fracture rat model of complex regional pain syndrome type I. PAIN 2004;108 :95–107.15109512
[15] Guo TZ Shi X Li WW Wei T Clark JD Kingery WS . Passive transfer autoimmunity in a mouse model of complex regional pain syndrome. PAIN 2017;158 :2410–21.28891866
[16] Guo TZ Shi X Li WW Wei T Clark JD Kingery WS . Sex differences in the temporal development of pronociceptive immune responses in the tibia fracture mouse model. PAIN 2019;160 :2013–027.31033779
[17] Guo TZ Wei T Shi X Li WW Hou S Wang L Tsujikawa K Rice KC Cheng K Clark DJ Kingery WS . Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome. Mol Pain 2012;8 :85.23191958
[18] Guo TZ Wei T Tajerian M Clark JD Birklein F Goebel A Li WW Sahbaie P Escolano FL Herrnberger M Kramer HH Kingery WS . Complex regional pain syndrome patient immunoglobulin M has pronociceptive effects in the skin and spinal cord of tibia fracture mice. PAIN 2020;161 :797–809.31815913
[19] Guo X Hou C Tang P Li R . Chronic pain, analgesics, and cognitive status: a comprehensive mendelian randomization study. Anesth Analg 2023;137 :896–905.37171986
[20] Hah JM Bateman BT Ratliff J Curtin C Sun E . Chronic opioid use after surgery: implications for perioperative management in the face of the opioid epidemic. Anesth Analg 2017;125 :1733–40.29049117
[21] Kehlet H Jensen TS Woolf CJ . Persistent postsurgical pain: risk factors and prevention. Lancet 2006;367 :1618–25.16698416
[22] Li WW Guo TZ Shi X Czirr E Stan T Sahbaie P Wyss-Coray T Kingery WS Clark JD . Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome. PAIN 2014;155 :2377–389.25218828
[23] Li WW Yang Y Shi XY Guo TZ Guang Q Kingery WS Herzenberg LA Clark JD . Germinal center formation, immunoglobulin production and hindlimb nociceptive sensitization after tibia fracture. Brain Behav Immun 2020;88 :725–34.32413559
[24] Mazza S Frot M Rey AE . A comprehensive literature review of chronic pain and memory. Prog Neuropsychopharmacol Biol Psychiatry 2018;87 (pt B ):183–92.28797640
[25] McWilliams LA Cox BJ Enns MW . Mood and anxiety disorders associated with chronic pain: an examination in a nationally representative sample. PAIN 2003;106 :127–33.14581119
[26] Mutso AA Radzicki D Baliki MN Huang L Banisadr G Centeno MV Radulovic J Martina M Miller RJ Apkarian AV . Abnormalities in hippocampal functioning with persistent pain. J Neurosci 2012;32 :5747–56.22539837
[27] Pereira Nery ECH Rocha NP Cruz VT Silva AG . Systematic review and meta-analysis on the association between chronic low back pain and cognitive function. Pain Pract 2023;23 :399–408.36504248
[28] Poree LR Guo TZ Kingery WS Maze M . The analgesic potency of dexmedetomidine is enhanced after nerve injury: a possible role for peripheral alpha2-adrenoceptors. Anesth Analg 1998;87 :941–8.9768799
[29] Prieur EAK Jadavji NM . Assessing spatial working memory using the spontaneous alternation Y-maze test in aged male mice. Bio Protoc 2019;9 :e3162.
[30] Reynolds NJ Al-Daraji WI . Calcineurin inhibitors and sirolimus: mechanisms of action and applications in dermatology. Clin Exp Dermatol 2002;27 :555–61.12464150
[31] Richards T Garvert DW McDade E Carlson E Curtin C . Chronic psychological and functional sequelae after emergent hand surgery. J Hand Surg Am 2011;36 :1663–8.21862240
[32] Rosenberger DC Pogatzki-Zahn EM . Chronic post-surgical pain—update on incidence, risk factors and preventive treatment options. BJA Educ 2022;22 :190–6.35496645
[33] Sahbaie P Li WW Guo TZ Shi XY Kingery WS Clark JD . Autonomic regulation of nociceptive and immunologic changes in a mouse model of complex regional pain syndrome. J Pain 2022;23 :472–86.34699985
[34] Sahbaie P Sun Y Liang DY Shi XY Clark JD . Curcumin treatment attenuates pain and enhances functional recovery after incision. Anesth Analg 2014;118 :1336–44.24755847
[35] Sahbaie P Tajerian M Yang P Irvine KA Huang TT Luo J Wyss-Coray T Clark JD . Nociceptive and cognitive changes in a murine model of polytrauma. J Pain 2018;19 :1392–405.29964216
[36] Shi X Guo TZ Li W Sahbaie P Rice KC Sulima A Clark JD Kingery WS . Exercise reverses nociceptive sensitization, upregulated neuropeptide signaling, inflammatory changes, anxiety, and memory impairment in a mouse tibia fracture model. Anesthesiology 2018;129 :557–75.29994924
[37] Somelar K Jurgenson M Jaako K Anier K Aonurm-Helm A Zvejniece L Zharkovsky A . Development of depression-like behavior and altered hippocampal neurogenesis in a mouse model of chronic neuropathic pain. Brain Res 2021;1758 :147329.33539793
[38] Tajerian M Clark JD . Spinal matrix metalloproteinase 8 regulates pain after peripheral trauma. J Pain Res 2019;12 :1133–8.31118746
[39] Tajerian M Hung V Nguyen H Lee G Joubert LM Malkovskiy AV Zou B Xie S Huang TT Clark JD . The hippocampal extracellular matrix regulates pain and memory after injury. Mol Psychiatry 2018;23 :2302–13.30254235
[40] Tajerian M Leu D Zou Y Sahbaie P Li W Khan H Hsu V Kingery W Huang TT Becerra L Clark JD . Brain neuroplastic changes accompany anxiety and memory deficits in a model of complex regional pain syndrome. Anesthesiology 2014;121 :852–65.25093591
[41] Tajerian M Sahbaie P Sun Y Leu D Yang HY Li W Huang TT Kingery W David CJ . Sex differences in a murine model of complex regional pain syndrome. Neurobiol Learn Mem 2015;123 :100–09.26070658
[42] Treede RD Rief W Barke A Aziz Q Bennett MI Benoliel R Cohen M Evers S Finnerup NB First MB Giamberardino MA Kaasa S Kosek E Lavand'homme P Nicholas M Perrot S Scholz J Schug S Smith BH Svensson P Vlaeyen JW Wang SJ . A classification of chronic pain for ICD-11. PAIN 2015;156 :1003–7.25844555
[43] van der Velden VH . Glucocorticoids: mechanisms of action and anti-inflammatory potential in asthma. Mediators Inflamm 1998;7 :229–37.9792333
[44] Wallin EF Hill DL Linterman MA Wood KJ . The calcineurin inhibitor tacrolimus specifically suppresses human T follicular helper cells. Front Immunol 2018;9 :1184.29904381
[45] Wei T Guo TZ Li WW Kingery WS Clark JD . Acute versus chronic phase mechanisms in a rat model of CRPS. J Neuroinflammation 2016;13 :14.26785976
[46] Whitlock EL Diaz-Ramirez LG Glymour MM Boscardin WJ Covinsky KE Smith AK . Association between persistent pain and memory decline and dementia in a longitudinal cohort of elders. JAMA Intern Med 2017;177 :1146–53.28586818
[47] Zhao Y Zhang L Wang M Yu J Yang J Liu A Yao H Liu X Shen Y Guo B Wang Y Wu S . Anxiety specific response and contribution of active hippocampal neural stem cells to chronic pain through wnt/β-catenin signaling in mice. Front Mol Neurosci 2018;11 :296.30197587
