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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma
Accepted Manuscript
Nociceptive and Cognitive Changes in a Murine Model of Polytrauma Peyman Sahbaie , Maral Tajerian , Phillip Yang , Karen Amanda Irvine , Ting-Ting Huang , Jian Luo , Tony Wyss-Coray , J David Clark PII: DOI: Reference:
S1526-5900(18)30313-4 10.1016/j.jpain.2018.06.004 YJPAI 3601
To appear in:
Journal of Pain
Received date: Revised date: Accepted date:
28 October 2017 12 June 2018 19 June 2018
Please cite this article as: Peyman Sahbaie , Maral Tajerian , Phillip Yang , Karen Amanda Irvine , Ting-Ting Huang , Jian Luo , Tony Wyss-Coray , J David Clark , Nociceptive and Cognitive Changes in a Murine Model of Polytrauma, Journal of Pain (2018), doi: 10.1016/j.jpain.2018.06.004
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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma
Peyman Sahbaie a,c, Maral Tajerian a,c, Phillip Yang b,d, Karen Amanda Irvine a,c, Ting-Ting Huang b,d, Jian Luo b,e, Tony Wyss-Coray b,e and J David Clark a,c a
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Department of Anesthesiology, Perioperative and Pain Medicine; Stanford University School of Medicine, Stanford, CA 94305, USA. b Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA. c Anesthesiology Service; Veterans Affairs Palo Alto Health Care System; 3801 Miranda Ave (112-A), Palo Alto, CA 94304, U.S.A. d Geriatrics Research Education and Clinical Center; Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave, Palo Alto, CA 94304, U.S.A. e Center for Tissue Regeneration Repair and Restoration; Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave, Palo Alto, CA 94304, U.S.A.
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Email: Peyman Sahbaie: [email protected] Maral Tajerian: [email protected] Phillip Yang: [email protected] Karen Amanda Irvine: [email protected] Ting-Ting Huang: [email protected] Jian Luo: [email protected] Tony Wyss-Coray: [email protected] J. David Clark: [email protected]
Corresponding Author: Peyman Sahbaie, VA Palo Alto HCS, 3801 Miranda Ave, Palo Alto,
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CA 64304. Phone 650-493-5000 X 65413, Fax: 650-852-3423, email: [email protected].
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Disclosures: This work was supported by the VA Merit Review award 1I01RX001776 and Department of Defense award MR130295 to JDC. The authors have no conflicts of interest to
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declare.
Running Title: Altered descending pain modulation after polytrauma
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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma
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Increased mechanical sensitivity is seen after mild TBI in male and female mice. The combination of TBI and fracture (polytrauma) exacerbates nociceptive changes. Polytrauma enhances risk taking behavior in male but not female mice. Fracture, TBI and polytrauma mice display deficits in recognition working memory. Diffuse noxious inhibitory control is greatly diminished in male and female mice after TBI.
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Abstract
Polytrauma commonly involves concussion (mild traumatic brain injury: mTBI) and peripheral
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trauma including limb fractures. Interactions between mTBI and peripheral injuries are poorly understood, both leading to chronic pain and neurobehavioral impairments. To elucidate these
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interactions, a murine polytrauma model was developed. mTBI alone resulted in similar increased mechanical allodynia in males and females. Female fracture and polytrauma groups
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displayed greater increases in hindpaw tactile hypersensitivity for weeks after injury than respective male groups. Capsaicin evoked spontaneous pain behaviors were greater in fracture and polytrauma females compared to males. The mTBI and polytrauma males displayed significant deficits in spatial working memory. All fracture, mTBI or polytrauma groups had deficits in object recognition memory. Only male mTBI or polytrauma mice showed greater
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agitation and increased risk taking behavior in open field testing as well as zero maze tests. Additionally, impaired diffuse noxious inhibitory control was observed in all mTBI and polytrauma mice. The model presented offers clinically relevant features useful for studying persistent pain, cognitive and other behavioral changes after TBI including polytrauma. A better
reduce persistent pain and disability in these patients.
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understanding of nervous system dysfunction after TBI and polytrauma might help prevent or
Perspective: The polytrauma model presented has relevant features of chronic pain and
neurobehavioral impairments useful for studying mechanisms involved in their development.
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This model may have special value in understanding altered descending pain modulation after TBI and polytrauma.
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Key Words: Traumatic Brain Injury, Fracture, Pain, Memory, Descending Inhibition
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Background With a global incidence estimated to be about 106 per 100,000 population, TBI is a leading cause of trauma-related disability 15. In the US more than 1.7 million injuries occur per year resulting in 250,000 hospitalizations and 52,000 deaths 9. The majority (75-80%) of all TBI cases are,
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however, mild and are accompanied by the rapid resolution of the immediate symptoms
including disorientation, dizziness, tinnitus, nausea and balance problems 24. The incidence of TBI is higher overall in males than females, and is particularly common in the very young,
adolescents and elderly 3. Females have a higher rate of mortality after TBI, but scant attention
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has been given to many other outcomes 8, 31. The economic costs of TBI are immense in terms of medical care and indirect costs including lost wages and productivity. In 2010, total costs in the US were estimated at $76.5 billion 7. TBI is therefore a significant public health problem.
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While the immediate symptoms of mild TBI dissipate rapidly, various long-term sequelae are common. For example, chronic pain is experienced by many with histories of TBI. One recent
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meta-analysis involving data from more than 4200 patients determined the average rate of postTBI pain to be > 50% 28. Surprisingly, the same study showed mild TBI to be associated with
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relatively high (75.3%) rates of pain while moderate to severe injuries associated with much
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lower (32.1%) rates. Although headache is common after TBI 20, the resulting pain can be widespread and may include the back and extremities 11. Quantitative sensory testing of the painful
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limbs of TBI patients has demonstrated mechanical allodynia and other sensory abnormalities consistent with neuroplastic changes in pain processing within the brain and spinal cord 29. Recent studies in human subjects suggest that disrupted descending inhibition of nociceptive signaling pathways may contribute to pain after TBI 6.
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TBI can occur in isolation, but in many cases TBI is accompanied by additional extracranial injuries such as fractures, soft tissue damage and visceral injuries. Motor vehicle accidents, sports-related injuries and military trauma are all settings in which TBI may be a component of the resulting polytrauma. The prevalence of pain in the setting of polytrauma is exceptionally
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high and can exceed 80% 19. In a recent study involving patients with concomitant TBI and
peripheral injury it was noted that worsened functional outcomes were experienced by those with TBI plus a peripheral injury compared with those having TBI alone, but the interaction was more likely in the setting of less severe TBI 26. A recent study using a mouse TBI plus fracture
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multitrauma model identified elevated brain levels of IL-1β a month after injuries leading the investigators to speculate that exacerbated neuroinflammation supported the increased risk taking observed in the animals 41.
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In this report we describe studies using a closed head TBI model with or without tibia fracture to study post-TBI nociceptive sensitization in male and female subjects. We further evaluate the
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role of descending inhibition in these mice.
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Methods Animals, experimental groups and conditions Male and female C57Bl/6J mice 11-12 weeks old obtained from Jackson Laboratories (Bar Harbor, MA) were kept in our facility a minimum of 1 week prior to initiating the experiments.
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All mice were kept under standard conditions with a 12 h light/dark cycle and an ambient
temperature of 22±1°C. Food and water were available ad libitum. Mice were habituated to handling by the experimenters for a few minutes each day for 3 days before initiation of
experiments. Figure 1 provides an outline of the experimental timeline. The following four
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experimental groups were used referring to the use of traumatic brain injury/limb fracture:
sham/sham, TBI/sham, sham/fracture or TBI/fracture (polytrauma). For these experiments, we have calculated group sizes (8-10) to have approximately 80% power to detect 25% changes at
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the p<0.05 level. For behavioral experiments, the experimenters were blind to the identity of treatments but not their experimental condition as fracture and polytrauma mice had obvious gait
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differences after injury. Data analysis was carried out by the experimenters blind to the
Ethics approval
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experimental condition of the animals and identity of treatments.
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All 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
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subjects’ guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Closed-head model of mild traumatic brain injury (mTBI) The closed head mTBI and sham procedures were based on previously described protocols 21, 50. Briefly, a benchmark stereotaxic impactor (MyNeurolab, St. Louis, MO, USA) actuator was
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mounted on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) at a 40° angle with a 5-mm impactor tip. After isoflurane anesthesia induction, mice were placed in a foam mold held in prone position on the stereotaxic frame and maintained under anesthesia for the duration of the procedure. The stereotaxic arm was adjusted so that the head impact was at a
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fixed point relative to the right eye and ear, corresponding to the S1 somatosensory cortex. The impact delivered by the device to the head was 5.8-6.0 m/s with a dwell time of 0.2 s and impact depth of 5 mm. After impact, the mice were recovered from anesthesia on a warming pad prior to returning to their home cages. Skull fractures were not observed in our study similar to
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previously reported studies using comparable head impact forces 21, 50. For sham groups, the above procedure was performed except that the impact device was discharged in the air. Tibial fracture
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Mice underwent right leg distal tibia fracture or sham procedures a day after TBI as previously described 12, 46. Briefly, mice were anesthetized with isoflurane and a closed fracture of the right
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tibia just distal to the midpoint was done using a hemostat. Next, the limb was wrapped in casting tape (Scotchcast™ Plus) so the hip, knee and ankle were all fixed forming a spica around
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the abdomen. The model results in a well characterized transverse bone fracture and soft tissue
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injury and cast immobilization is essential for the extended pain-related phenotype 13. To prevent post injury edema constriction, a window was made in the cast over the dorsal paw and
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ankle. Sham groups underwent the same duration of anesthesia as the fracture mice. Mice were recovered from anesthesia on a warming pad and given 1.5 ml normal saline for hydration. Both injured and sham groups received subcutaneous buprenorphine for two days. Food and water gelpacks were provided on the floor for 72h post procedure. Casts were removed 3 weeks after fracture.
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Mechanical nociceptive assay Mechanical sensitivity was assessed using nylon von Frey filaments (Stoelting Co., IL, USA) according to the “up-down” algorithm developed by Chaplan et al. 5. We have applied this technique previously to detect 50% withdrawal threshold in mice after injury 38, 46. After
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acclimating mice on the wire mesh platform inside plastic enclosures (10cm radius), sequential fibers with increasing stiffness ranging from 0.004 to 1.7 g were applied to the plantar surface of hind limb and left in place 5 sec. 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
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response occurred after application of a fiber then a less stiff fiber was applied, if no response was observed the next stiffest fiber was applied. Mechanical withdrawal threshold was determined by a data fitting algorithm for significance analysis 35.
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Capsaicin-evoked behaviors
This assay was performed in separate cohorts of mice to determine if any differences in latent
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sensitization exist between the treatment groups after recovery 45. The total duration of behaviors (shaking, biting and licking) for 5 min after subcutaneous (s.c.) hind paw capsaicin injection
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(2.0ug/5ul; Sigma–Aldrich, USA) was measured. Control experiments used vehicle (0.25%
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DMSO, 0.25% ethanol, 0.125% Tween-80 in saline) plantar injections. The mice were gently restrained for the plantar injections. The assay was done at 20-22 weeks post TBI and fracture
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trauma when all groups had recovered to baseline mechanical threshold values. Open field (OF), zero maze (ZM) and object memory tests The OF arena (40X40X40 cm) was used to assess locomotion, object location (OLM) and object recognition (ORM) working memory tests. In OF test, total locomotor activity (distance travelled) and time spent in the center 20% of the arena for 10 min was determined. Increased
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time spent in the peripheral zones of a novel environment indicative of thigmotactic behavior was used as an index of anxiety, while time spent in the center 20% used as an index of risk taking behavior. The elevated ZM was used as well, to measure anxiety and risk taking behavior according to
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previously published methods 45. The maze is 24 inches above the floor, has an outer diameter of 24 inches and inner diameter of 20 inches, with two closed (6-inch tall walls) and open
quadrants. Mice were randomly placed facing one of the closed quadrants at the beginning of the 5-min test. Total time spent in open and closed quadrants were recorded. Time spent in the open
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quadrants was compared across groups to measure anxiety and risk taking behavior.
For OLM and ORM experiments, mice were initially habituated to two identical objects after being placed in the middle of the OF arena as previously described 45, 51. Next, during a 5 min
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trial one of the objects was moved to a novel location and exploratory behavior (investigation time) was recorded. Subsequent to a 5 min period in home cages, mice were returned to the arena
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with one of the previous identical objects being replaced with a novel one. The new object had a distinct shape and size difference from the identical object sets, and 5 min recordings were done
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for exploratory behavior towards objects. As mice explore novel locations or objects more than
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familiar ones, time spent exploring the novel compared to familiar was used to assess spatial and non-spatial working memory.
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All recordings from the above experiments were analyzed in real time by TopScan software (Clever System, USA). Diffuse noxious inhibitory control (DNIC) assessment DNIC assessment of post trauma mechanical hypersensitivity was done using a noxious stimulation–induced analgesia protocol modified for mice 18, 33. DNIC was induced by s.c.
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application of capsaicin (7.5 ug/5ul; Sigma-Aldrich, USA) to the right forepaw after brief isoflurane (3%) anesthesia. Control mice received 5ul of vehicle (0.25% DMSO, 0.25% ethanol and 0.125% Tween 80 in saline). The amount of DNIC induced by capsaicin injection was evaluated by hind paw mechanical threshold testing. Separate groups of mice in the Sham/Fx or
hypersensitivity was the same between the two groups.
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TBI/Fx groups were assessed for DNIC at 4 weeks post injury, at which time mechanical
In order to assess DNIC in the TBI alone group, the mice were allowed to recover to baseline mechanical thresholds after TBI (3 weeks). Next, right hind paw Prostaglandin E2 (PGE2)
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injections were given to produce brief hypersensitivity 44. Separate groups of Sham/Sham or TBI/Sham groups were given ipsilateral or contralateral hind paw injection (s.c.) of 100ng/15ul PGE2 (Cayman Chemicals, USA) and tested after 1h for mechanical hypersensitivity. Stock
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PGE2 solutions were made in 100% ethanol and further diluted (1:1000) in 0.9% saline prior to use. Vehicle (15μl) injections contained the same dilution of ethanol in saline. Subsequent to
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mechanical threshold evaluations, mice underwent capsaicin injection in the ipsilateral forepaw, and changes in mechanical withdrawal thresholds were followed.
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Assessment of analgesics on mTBI induced mechanical hypersensitivity
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The effects of anti-inflammatory and anti-neuropathic analgesic agents on TBI induced mechanical hypersensitivity were assessed at the 72h timepoint. Systemic (i.p.) injections of
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carprofen (5mg/kg; Sigma-Aldrich, USA) or gabapentin (50 mg/kg; Sigma-Aldrich, USA) were given to sham or TBI mice and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. The doses of the both drugs were in the high range used by other investigators examining effects in separate models 39, 48. Vehicle controls were employed. Data Analysis
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The data for mechanical sensitivity were analyzed by multiple t-tests with Holm-Sidak method to correct for multiple comparisons (no assumption of same scatter). For these experiments, groups of mice were randomly assigned to treatment groups and followed across days/weeks till recovery. Therefore, measurements for each subject were done for 10 time points for TBI alone
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experiments and 13-14 time points for polytrauma groups. In order to assess overall sex
differences after trauma, repeated two-way ANOVA was used. The data from capsaicin induced spontaneous pain behaviors, open field and zero maze experiments were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Two-way ANOVA
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followed by Tukey’s post-hoc tests (95% confidence interval of differences plus significance: α=0.05) were used to compare groups in the DNIC, analgesic and object memory tests. D'Agostino & Pearson omnibus test showed the data obtained were normally distributed.
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All data are presented as mean ± S.E.M and for all analyses, p<0.05 were taken to be significant.
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Results Mechanical hypersensitivity after mTBI, tibial fracture or combined Injury Male and female mice displayed increased contralateral and ipsilateral (Relative to injury side) hind paw mechanical sensitivity after mTBI, with peak increases lasting for 72h and gradual
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recovery to baseline values by 14 days after injury. TBI generally resulted in increased
contralateral versus ipsilateral limb mechanical hypersensitivity, though significant limb
differences were only seen at some timepoints in female mice between days 1 to 7 post injury (Figure 2 A,B). No significant male vs. female differences for respective contralateral (F9,162 =
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1.64, p= 0.11) or ipsilateral (F9,162 =1.82, p= 0.07) hind paw thresholds after TBI was seen on further analysis.
The mice in the fracture or combined injury polytrauma groups of both sexes displayed increased
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mechanical hypersensitivity for weeks after injury with recovery to baseline values at 17-19 weeks in male cohorts and 19-21 weeks in female ones (Figure 3 A,B). Within group analysis
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revealed that the polytrauma group had significantly greater mechanical hypersensitivity than fracture alone in males across several weeks, no significant differences between the female of
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either injury groups were seen. Further analysis showed significant male vs female mechanical
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hypersensitivity differences after fracture (F12,204 =7.14, p< 0.001) and polytrauma (F12,216 =8.07, p< 0.001), with the female groups demonstrating greater nociceptive sensitivity.
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Capsaicin-induced spontaneous pain behaviors after mTBI, tibial fracture or combined injury In order to detect differences in latent sensitization in the experimental groups, capsaicin evoked behaviors were measured after recovery from mechanical hypersensitivity (20-22 weeks) in male and female mice. Both fracture (males: p< 0.05; females: p< 0.001) and polytrauma groups (males: p< 0.001; females: p< 0.001) displayed increased spontaneous pain behavior after hind paw capsaicin application compared to respective sham groups. For both sexes, combined TBI
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and tibial fracture groups displayed greater capsaicin evoked behaviors than mice with either injury alone (Figure 4 A,B). Female mice displayed greater duration of capsaicin-evoked behaviors than male mice for all injury (TBI: male=37.60 ± 2.50 vs. female=60.85 ± 2.80, p<
vs. female=107.90 ± 4.08, p< 0.001) conditions.
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0.001; Fx: male=47.86 ± 3.61 vs. female=85.24 ± 3.11, p< 0.001 and TBI/Fx: male=76.34 ± 7.10
Working memory deficits after mTBI, tibial fracture or combined injury
Next, we sought to determine if differences in spatial and non-spatial working memory deficits existed between the experimental groups. Previously, we had shown that memory deficits
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accompany pain sensitization after fracture 45. Similar to our previous findings, male fracture mice had deficits in non-spatial (ORM, p= 0.53 novel vs same) but not in spatial (OLM, p< 0.01 novel vs same) working memory. TBI alone and polytrauma male groups displayed deficits in
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OLM as seen by insignificant differences in the time spent on novel vs familiar location (Figure 5 A; TBI: p= 0.98; polytrauma: p= 0.96, novel vs same). Neither sham nor the other injury
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female groups showed significant preference for novel location in the OLM test, therefore it was not possible to assess spatial working memory differences in these groups ((Figure 5 B). Male
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TBI and polytrauma groups displayed deficits in the ORM test, as seen by insignificant
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differences in time spent on novel vs familiar object (Figure 5 C; TBI: p= 0.53; polytrauma: p= 0.95, novel vs same). Contrary to the OLM test performance, the female sham mice displayed
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significant preference for the novel object (ORM, p< 0.05 novel vs same). Neither fracture nor TBI nor polytrauma female mice demonstrated a significant preference for novel object (Figure 5 D; Fracture: p= 0.80; TBI: p= 0.12; polytrauma: p= 0.91, novel vs same). As the sham female mice showed more total combined object exploratory activity in either test than respective males (OLM: males=11.47 ± 2.71 vs. females= 43.14 ± 6.19, p< 0.001; ORM: males=17.29 ± 2.71 vs.
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females= 34.57 ± 6.16. p< 0.05), comparing injury type effects between sexes would be challenging. Evaluation of locomotor activity, anxiety and risk taking behavior after mTBI, tibial fracture or combined injury
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Determination of open field locomotor activity (OF) in the male mice showed increased distance travelled after TBI alone (p< 0.001) or when combined with tibial fracture (p< 0.05) compared to controls (Figure 6 A), indicative of greater agitation in these groups. Female mice after fracture or TBI did not show significant differences in locomotor activity, though the polytrauma mice
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displayed decreases (p< 0.01) in distance travelled compared to controls (Figure 6B). Overall activity in the female groups was higher than in males. Male polytrauma mice spent significantly more time (p< 0.01) in the center 20% of the OF, suggestive of increased risk taking behavior
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and decreased anxiety (Figure 6C). None of the female groups significantly differed in time spent at the center compartment (Figure 6D). Next, we went on to investigate anxiety and risk
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taking behavior in the same groups using the elevated zero maze (ZM) test 34. Male polytrauma mice spent significantly more time (p< 0.01) in the ZM open arms than other male groups
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(Figure 7A), providing corroborating evidence for decreased anxiety and increased risk taking
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behavior. Fracture, TBI and the combination did not alter open arm times in the female groups (Figure 7B).
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Descending noxious inhibitory controls deficits after mTBI, tibial fracture or combined injury Deficient descending modulation of pain (DNIC) has been suggested to worsen some types of pain in TBI patients. In experiments directed at detecting possibly deficient DNIC in TBI and sham injured mice we first allowed TBI animals to recover for three weeks after injuries such that hind paw nociceptive thresholds had returned to baseline (Figure 2). DNIC was then tested
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by sensitizing hind paws with PGE2 followed an hour later by ipsilateral forepaw capsaicin injection to stimulate descending inhibition 33. Figure 8 A, B shows that capsaicin robustly reverses ipsilateral hind paw sensitization in sham injured male and female mice, but has almost no effect in animals 3 weeks after TBI suggesting profound disruption of DNIC by TBI in either
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sex. Further analysis showed differences in response to capsaicin between male and female TBI mice were not statistically different (F1,16 =0.60, p= 0.45). Separate groups of male and female mice were tested for DNIC of the contralateral hind paw. After ipsilateral forepaw capsaicin application, male and female TBI groups exhibited reduced DNIC effects on contralateral paw
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mechanical sensitivity compared to sham groups (Figure 8 C, D).
Additional experiments were undertaken 4 weeks after tibial fracture in mice with or without TBI, when mechanical sensitivity was same between the groups. In these experiments, we
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observed that forepaw capsaicin injection transiently reversed hind paw sensitization in fracture alone groups but that this form of analgesia was very limited in mice that had both tibial fracture
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and TBI (Figure 9 A, B). No significant differences in response to capsaicin were seen between male and female fracture alone groups (F1,16 =0.24, p= 0.63) or combined injury groups (F1,16
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=0.32, p= 0.58). For the both groups, DNIC assessments were done in fractured (Ipsilateral)
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side. As the contralateral paw in either group is not sensitized at 4 weeks, sensitization with PGE2 would be needed for DNIC assessments making interpretation of results difficult.
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Assessment of anti-inflammatory and anti-neuropathic analgesic agents on TBI induced mechanical hypersensitivity. Systemic injections of carprofen (5mg/kg) or gabapentin (50 mg/kg) were given to sham or TBI mice and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. On day 3 following injury, carprofen administration did not attenuate TBI induced hind paw
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sensitization in male or female mice (Figure 10 A, B). Similarly, gabapentin failed to attenuate TBI induced hind paw sensitization in both sexes (Figure 10 C, D). Control experiments using vehicle in sham and TBI groups did not show significant changes in paw withdrawal threshold in either sex (Data not shown). These data suggest the increased mechanical hypersensitivity seen
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after TBI is centrally modulated.
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Discussion Our understanding of the long-term sequalae of Traumatic brain injury (TBI) is presently improving. Cognitive, emotional and sensory symptoms are increasingly recognized in this patient population. Only recently have human and animal studies begun to explore the
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mechanisms supporting high pain prevalence after TBI with very little having been written about the interaction of TBI with peripheral injuries or the impact of sex on pain-related outcomes. In this regard the studies contained in this report are highly novel. Our major findings are: 1) mild TBI causes nociceptive sensitization lasting about two weeks. The resulting sensitization is
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refractory to anti-inflammatory and anti-neuropathic analgesics, being more consistent with central pain syndromes, 2) mild TBI worsens nociceptive sensitization after limb fracture, 3) sex does not impact post-TBI nociceptive sensitization but does affect the sensitization caused by
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limb fracture, 4) descending regulation of nociception is profoundly affected by TBI in both sexes, and 5) both concomitant limb fracture and sex modulate anxiety, risk taking behavior and
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memory changes after TBI. Together these observations provide a basis for exploring the
sequelae.
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molecular, cellular and circuit-level alterations after TBI supporting pain and other long-term
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One aspect of the experimental design adding to the face validity of our mTBI mouse model is the use of closed-head injury in generating mTBI, the predominant form of TBI. Though there
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are many methods used to model brain injury in animals, they most often involve opening or penetration of the skull. Conversely, sports-related concussions or acceleration/deceleration injuries and combat-related exposures to blast waves do not typically cause damage to the skull itself. Previously described TBI injuries used in pain studies including those by our own group have involved the application of a percussive pressure wave to the exposed dura of the animal, or
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the direct impact of an object against the brain 10, 22, 37. The present model first described by Shitaka et al. somewhat more realistically involves a non-penetrating blow against the skull 40. Variants of the model have been used to study repetitive brain injury such as can occur to those playing contact sports 14. In fact, neither we nor others using this closed head TBI model
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identified gross motor or behavioral changes in the TBI mice suggesting that more subtle forms of injury may support pain-related changes in the brain after TBI.
Similar to the other models involving disruption of the skull, we did observe sensitization to noxious mechanical stimuli. For example, Macolino et al. demonstrated sensitization of the
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tissues overlying the skull and the forepaw after cortical impact-induced injury 22. Our group recently focused on hind limb sensitization after TBI induced by lateral fluid percussion 10. The more distal site was assessed to better examine the sensitization of areas well outside of the head
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region, similar to areas affected in patients with TBI including the back and limbs. Different mechanisms may underlie pain and sensitization in the head versus back and limbs as altered
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expression of the neurokinin receptor NK1 has been demonstrated in the trigeminal nucleus after TBI 27, while the enhanced expression of pro-nociceptive BDNF occurs in lumbar spinal cord
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tissue after TBI and associated hind limb sensitization 10.
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It is not uncommon for TBI to occur with other injuries in battlefield, motor vehicle accident, etc. Others have combined TBI with injuries such as long bone fracture to model “polytrauma” . An excellent review has been written detailing the known interactions of TBI and various
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forms of peripheral injury in clinical populations and laboratory models 26. These combined injury models have been used to study bone healing and other consequences of injury, but not persistent pain 23, 47. Most studies have investigated consequences of multiple injuries on serum levels of injury markers and the extent of brain injury within the first several days. One
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exception was a report by Shultz et al. demonstrating in a closed-head TBI plus tibial fracture multitrauma model not only enhanced neuroinflammation and exacerbated changes in ventricular volume, but also increased risk taking behavior in male mice about a month after injury 41. Our own studies add significantly to this literature by showing that polytrauma mice display
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enhanced peripheral nociceptive sensitization for months and have latent sensitization after
recovery. These data are consistent with reports of chronic pain at sites distant from the head in multitrauma TBI patients.
A major mechanism for the control of pain-related signaling flowing into the brain from
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periphery is descending regulation. Fibers originating in brainstem send axons to the dorsal horn of the spinal cord where they may positively or negatively regulate signal transmission 30. Such regulation can be demonstrated by applying a noxious stimulus to a site in the periphery while
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following a pain or nociceptive threshold in another area. In animals, this type of regulation is referred to as diffuse noxious inhibitor control (DNIC) while in humans it is referred to as
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conditioned pain modulation (CPM) 49. This name emphasizes the general or “diffuse” nature of the regulation. Laboratory and clinical studies have linked the efficiency of descending
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regulation to chronic pain syndromes such as fibromyalgia, tension headache, neuropathic and
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chronic post-operative pain 33, 43, 49. Importantly, brainstem imaging has recently shown that the extent of injury in the periaqueductal grey matter (PAG) is correlated with pain levels in TBI
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patients 17. Our observations showed that weeks after TBI, mice of either sex had severely reduced DNIC providing a possible basis for the enhanced nociception seen after limb fracture. It should be noted that TBI patients with chronic post-traumatic headache display diminished CPM also 6. Existing therapies such as SNRI drugs might be used to augment DNIC in patients with TBI just as they seem to be useful in controlling pain in patients with other forms of chronic pain
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characterized by dysfunctional DNIC 30. The brainstem which contains major descending regulatory centers including the PAG, locus coeruleus and rostral ventromedial medulla, has been noted to be an area prone to TBI injury 25. Studies directed at understanding the extent of injury to these centers after TBI are warranted.
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Additionally, examining similarities or differences in pain behavior after TBI over other cortical regions besides the somatosensory cortex would complement the findings of our study 16.
The present study has been focused on characterizing a novel polytrauma paradigm and is limited to evaluating evoked tactile sensitivity and pain-related behaviors. Further studies
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examining the extent of spontaneous pain, as well as, additional pharmacological and behavioral testing to distinguish decreased anxiety form increased risk taking behaviors would further strengthen the characterization of this model of TBI and polytrauma.
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Males sustain TBI at a rate far higher than females 3. However, the consequences of those injuries may be different for the sexes. For example, a recent survey of military personnel that
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had suffered mTBI suggested that females experienced higher rates of some sensory changes including sensitivity to light and changes in taste and smell 2. Other analyses have found greater
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mortality after TBI amongst females, although these differences may be less when multiple
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injury severity is taken into account 1. Likewise, functional recovery after TBI does not appear to be strongly linked to sex 4. These data are in contrast to sex related differences in the prevalence
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of various pain syndromes which often show a predilection for females over males 42, including pain after surgical trauma 36. Our data failed to identify sex-linked differences in nociceptive sensitization after mTBI, and no clear difference in the additive effect of TBI to the sensitization observed after tibial fracture. On the other hand, there was a prominent effect of sex on sensitization after tibial fracture alone as reported previously 46. Similar to our previous study,
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male fracture mice had deficits in non-spatial working memory. We went on to evaluate memory and other pain/trauma related behavioral comorbidities in both sexes for interaction of limb trauma and TBI 46. Sex impacted anxiety, risk taking behavior and memory to differing extents in our studies. We did observe both sexes to have deficits in non-spatial working memory after
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limb trauma, TBI or both. These results in combination with the clinical observations suggest that the effects of sex on TBI outcomes might be dependent on the specific outcomes being examined as they may involve different underlying mechanisms. Conclusions
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Nociceptive sensitization in regions distant from the brain can be modeled after closed-head mTBI including sensitization resulting from the combination of mTBI with peripheral injury. This type of model provides a tool critical to studying the very common occurrence of chronic
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pain after TBI including multi- or polytrauma. Our animal data are consistent with clinical reports in suggesting that brain centers involved in descending modulation are involved in
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enhanced pain sensitization after mTBI, while we do not at this point understand the details of these changes. A better understanding of the specific involved brain centers, the modulation of
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their activities and a more detailed understanding of the mechanisms of injury to the brain after
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mTBI might help prevent or reduce chronic pain in this population.
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Abbreviations: mTBI, mild traumatic brain injury; Fx, fracture; ANOVA, analysis of variance; OF, open field; ZM, zero maze; DNIC, diffuse noxious inhibitory control, OLM, object location memory; ORM, object recognition memory; DMSO, dimethyl sulfoxide; PGE2, prostaglandin
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E2; SNRI, serotonin and norepinephrine reuptake inhibitors.
Authors’ contributions: PS participated in the design of the study, performed behavior studies, helped to draft the manuscript and performed the statistical analysis. MT participated in the design of the study, performed the surgeries and helped revise the final manuscript. PY helped
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perform behavior studies and analyzed data, KAI helped perform the surgeries and revised the final manuscript. JL helped develop the animal TBI models and revised the final manuscript. TTH and TWC participated in the design of the study and revised the final manuscript. JDC
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conceived of the study, participated in its design and coordination, and helped draft the
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manuscript. All authors read and approved the final manuscript.
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Acknowledgments: Not applicable
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Figure Legends:
Figure 1. Overview of the experimental timeline. mTBI: Mild traumatic brain injury, Fx:
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Fracture, B: Baseline mechanical assay, DNIC: Diffuse noxious inhibitory controls, OLM: object
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location memory, ORM: object recognition memory and CAP: Capsaicin.
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Figure 2. Mechanical hypersensitivity after mTBI. Male (A) and female (B) mice displayed increased hind paw mechanical sensitivity after TBI. Data were analyzed by multiple t tests
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using the Holm-Sidak method to correct for multiple comparisons. * indicates significant
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difference in comparison with respective limb measurements in sham group. # indicates significant difference between ipsilateral and contralateral limb to the side of injury. Error bars: SEM, n = 8-10/group.
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Figure 3. Mechanical hypersensitivity after peripheral trauma or combined mTBI and peripheral trauma. Male (A) and female (B) mice in the fracture or polytrauma groups displayed mechanical hypersensitivity for weeks after injury with recovery to baseline values at 17-19 weeks in male cohorts and 19-21 weeks in female ones. Male but not the female mice showed
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significant differences between fracture and polytrauma induced mechanical hypersensitivity. * indicates significant difference in comparison with respective sex combined injury group. Error bars: SEM, n = 8-10/group. Data were analyzed by multiple t tests using the Holm-Sidak method
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to correct for multiple comparisons.
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Figure 4. Capsaicin-evoked behaviors after mTBI, peripheral trauma or combined injury. The total duration of behaviors (shaking, biting and licking) in male (A) and female (B) mice for 5 min after hind paw capsaicin (2.0ug/5ul) or vehicle injection was measured. The assay was done at 20-22 weeks post trauma period when all groups had recovered to baseline mechanical
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threshold values. Error bars: SEM, n = 8-10/group, *p<0.05, *** p<0.001 for comparison to respective sham groups and ### p<0.001 for comparison within groups. Data were analyzed by
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one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons.
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Figure 5. Working memory evaluations after mTBI, peripheral trauma or combined injury.
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Object location (OLM) and object recognition (ORM) working memory tests were employed. The interval between acquisition and retrieval was 5 min in either test. (A-C) Male fracture mice displayed no deficits in spatial (OLM) but had deficits in non-spatial (ORM) working memory. The TBI alone and polytrauma male groups displayed significant deficits in both OLM and ORM tests. (B-D) Female mice in either experiential group displayed no significant preference for novel location in the OLM test, but only the female sham mice displayed significant preference
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for the novel object (ORM). All recordings were analyzed in real time by automated software and data obtained were analyzed by two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, *p<0.05, or *** p<0.001 for comparison between novel and familiar
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location/object.
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Figure 6. Locomotor activity and anxiety evaluations after mTBI, peripheral trauma or combined Injury. In the open field test the male (A) mice showed increased distance travelled after TBI or polytrauma compared to controls. Female (B) mice after fracture or TBI did not show significant differences in locomotor activity, though the female polytrauma mice displayed
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decreases in distance travelled compared to controls. Male polytrauma (C) but not the female (D) polytrauma mice spent more time in the center 20% compartment of the arena indicative of
decreased anxiety and increased risk taking behavior. All recordings were analyzed in real time by automated software and data obtained were analyzed by one way ANOVA followed by
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Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, *p<0.05, **p<0.01 or *** p<0.001 for
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Figure 7. Anxiety evaluations after mTBI, peripheral trauma or combined Injury. In the zero maze the male (A) polytrauma mice displayed reduced anxiety as seen by increased time spent in
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the open arms, indicative of increased risk taking behavior. None of the female (B) groups
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showed differences in anxiety levels as measured by time spent in open arms. All recordings were analyzed in real time by automated software and data obtained were analyzed by one way
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Figure 8. Descending noxious inhibitory controls deficits after mTBI. To determine differences in descending pain control inputs, capsaicin (arrow) noxious stimulation–induced analgesia was used. The mTBI male (A, C) and female (B, D) groups show descending pain controls deficits in either hind limbs compared to controls. Hind paw mechanical sensitivity data were analyzed by
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two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, ***
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p<0.001 for comparison between mTBI and sham.
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Figure 9. Descending noxious inhibitory controls deficits after peripheral trauma or polytrauma. To determine differences in descending pain control inputs, capsaicin noxious stimulation–
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induced analgesia was used. Polytrauma male (A) and female (B) groups displayed deficits in descending pain controls compared to fracture alone. Hind paw mechanical sensitivity data were
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Figure 10. Effects of anti-inflammatory and non-opioid analgesic agents on TBI induced mechanical hypersensitivity. After baseline mechanical sensitivity measurements, Caprofen (5mg/kg, i.p.) or gabapentin (50 mg/kg, i.p.) were given to sham or TBI mice 3 days after injury and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. Neither
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carprofen (A, B) nor gabapentin (C, D) administration (arrows) resulted in significant changes in TBI induced hind paw sensitization in male or female mice. Hind paw mechanical sensitivity data were analyzed by two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n
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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma Peyman Sahbaie , Maral Tajerian , Phillip Yang , Karen Amanda Irvine , Ting-Ting Huang , Jian Luo , Tony Wyss-Coray , J David Clark PII: DOI: Reference:
S1526-5900(18)30313-4 10.1016/j.jpain.2018.06.004 YJPAI 3601
To appear in:
Journal of Pain
Received date: Revised date: Accepted date:
28 October 2017 12 June 2018 19 June 2018
Please cite this article as: Peyman Sahbaie , Maral Tajerian , Phillip Yang , Karen Amanda Irvine , Ting-Ting Huang , Jian Luo , Tony Wyss-Coray , J David Clark , Nociceptive and Cognitive Changes in a Murine Model of Polytrauma, Journal of Pain (2018), doi: 10.1016/j.jpain.2018.06.004
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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma
Peyman Sahbaie a,c, Maral Tajerian a,c, Phillip Yang b,d, Karen Amanda Irvine a,c, Ting-Ting Huang b,d, Jian Luo b,e, Tony Wyss-Coray b,e and J David Clark a,c a
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Department of Anesthesiology, Perioperative and Pain Medicine; Stanford University School of Medicine, Stanford, CA 94305, USA. b Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA. c Anesthesiology Service; Veterans Affairs Palo Alto Health Care System; 3801 Miranda Ave (112-A), Palo Alto, CA 94304, U.S.A. d Geriatrics Research Education and Clinical Center; Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave, Palo Alto, CA 94304, U.S.A. e Center for Tissue Regeneration Repair and Restoration; Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave, Palo Alto, CA 94304, U.S.A.
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Email: Peyman Sahbaie: [email protected] Maral Tajerian: [email protected] Phillip Yang: [email protected] Karen Amanda Irvine: [email protected] Ting-Ting Huang: [email protected] Jian Luo: [email protected] Tony Wyss-Coray: [email protected] J. David Clark: [email protected]
Corresponding Author: Peyman Sahbaie, VA Palo Alto HCS, 3801 Miranda Ave, Palo Alto,
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CA 64304. Phone 650-493-5000 X 65413, Fax: 650-852-3423, email: [email protected].
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Disclosures: This work was supported by the VA Merit Review award 1I01RX001776 and Department of Defense award MR130295 to JDC. The authors have no conflicts of interest to
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declare.
Running Title: Altered descending pain modulation after polytrauma
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Nociceptive and Cognitive Changes in a Murine Model of Polytrauma
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Increased mechanical sensitivity is seen after mild TBI in male and female mice. The combination of TBI and fracture (polytrauma) exacerbates nociceptive changes. Polytrauma enhances risk taking behavior in male but not female mice. Fracture, TBI and polytrauma mice display deficits in recognition working memory. Diffuse noxious inhibitory control is greatly diminished in male and female mice after TBI.
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Abstract
Polytrauma commonly involves concussion (mild traumatic brain injury: mTBI) and peripheral
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trauma including limb fractures. Interactions between mTBI and peripheral injuries are poorly understood, both leading to chronic pain and neurobehavioral impairments. To elucidate these
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interactions, a murine polytrauma model was developed. mTBI alone resulted in similar increased mechanical allodynia in males and females. Female fracture and polytrauma groups
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displayed greater increases in hindpaw tactile hypersensitivity for weeks after injury than respective male groups. Capsaicin evoked spontaneous pain behaviors were greater in fracture and polytrauma females compared to males. The mTBI and polytrauma males displayed significant deficits in spatial working memory. All fracture, mTBI or polytrauma groups had deficits in object recognition memory. Only male mTBI or polytrauma mice showed greater
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agitation and increased risk taking behavior in open field testing as well as zero maze tests. Additionally, impaired diffuse noxious inhibitory control was observed in all mTBI and polytrauma mice. The model presented offers clinically relevant features useful for studying persistent pain, cognitive and other behavioral changes after TBI including polytrauma. A better
reduce persistent pain and disability in these patients.
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understanding of nervous system dysfunction after TBI and polytrauma might help prevent or
Perspective: The polytrauma model presented has relevant features of chronic pain and
neurobehavioral impairments useful for studying mechanisms involved in their development.
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This model may have special value in understanding altered descending pain modulation after TBI and polytrauma.
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Key Words: Traumatic Brain Injury, Fracture, Pain, Memory, Descending Inhibition
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Background With a global incidence estimated to be about 106 per 100,000 population, TBI is a leading cause of trauma-related disability 15. In the US more than 1.7 million injuries occur per year resulting in 250,000 hospitalizations and 52,000 deaths 9. The majority (75-80%) of all TBI cases are,
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however, mild and are accompanied by the rapid resolution of the immediate symptoms
including disorientation, dizziness, tinnitus, nausea and balance problems 24. The incidence of TBI is higher overall in males than females, and is particularly common in the very young,
adolescents and elderly 3. Females have a higher rate of mortality after TBI, but scant attention
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has been given to many other outcomes 8, 31. The economic costs of TBI are immense in terms of medical care and indirect costs including lost wages and productivity. In 2010, total costs in the US were estimated at $76.5 billion 7. TBI is therefore a significant public health problem.
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While the immediate symptoms of mild TBI dissipate rapidly, various long-term sequelae are common. For example, chronic pain is experienced by many with histories of TBI. One recent
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meta-analysis involving data from more than 4200 patients determined the average rate of postTBI pain to be > 50% 28. Surprisingly, the same study showed mild TBI to be associated with
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relatively high (75.3%) rates of pain while moderate to severe injuries associated with much
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lower (32.1%) rates. Although headache is common after TBI 20, the resulting pain can be widespread and may include the back and extremities 11. Quantitative sensory testing of the painful
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limbs of TBI patients has demonstrated mechanical allodynia and other sensory abnormalities consistent with neuroplastic changes in pain processing within the brain and spinal cord 29. Recent studies in human subjects suggest that disrupted descending inhibition of nociceptive signaling pathways may contribute to pain after TBI 6.
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TBI can occur in isolation, but in many cases TBI is accompanied by additional extracranial injuries such as fractures, soft tissue damage and visceral injuries. Motor vehicle accidents, sports-related injuries and military trauma are all settings in which TBI may be a component of the resulting polytrauma. The prevalence of pain in the setting of polytrauma is exceptionally
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high and can exceed 80% 19. In a recent study involving patients with concomitant TBI and
peripheral injury it was noted that worsened functional outcomes were experienced by those with TBI plus a peripheral injury compared with those having TBI alone, but the interaction was more likely in the setting of less severe TBI 26. A recent study using a mouse TBI plus fracture
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multitrauma model identified elevated brain levels of IL-1β a month after injuries leading the investigators to speculate that exacerbated neuroinflammation supported the increased risk taking observed in the animals 41.
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In this report we describe studies using a closed head TBI model with or without tibia fracture to study post-TBI nociceptive sensitization in male and female subjects. We further evaluate the
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role of descending inhibition in these mice.
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Methods Animals, experimental groups and conditions Male and female C57Bl/6J mice 11-12 weeks old obtained from Jackson Laboratories (Bar Harbor, MA) were kept in our facility a minimum of 1 week prior to initiating the experiments.
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All mice were kept under standard conditions with a 12 h light/dark cycle and an ambient
temperature of 22±1°C. Food and water were available ad libitum. Mice were habituated to handling by the experimenters for a few minutes each day for 3 days before initiation of
experiments. Figure 1 provides an outline of the experimental timeline. The following four
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experimental groups were used referring to the use of traumatic brain injury/limb fracture:
sham/sham, TBI/sham, sham/fracture or TBI/fracture (polytrauma). For these experiments, we have calculated group sizes (8-10) to have approximately 80% power to detect 25% changes at
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the p<0.05 level. For behavioral experiments, the experimenters were blind to the identity of treatments but not their experimental condition as fracture and polytrauma mice had obvious gait
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differences after injury. Data analysis was carried out by the experimenters blind to the
Ethics approval
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experimental condition of the animals and identity of treatments.
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All 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
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subjects’ guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Closed-head model of mild traumatic brain injury (mTBI) The closed head mTBI and sham procedures were based on previously described protocols 21, 50. Briefly, a benchmark stereotaxic impactor (MyNeurolab, St. Louis, MO, USA) actuator was
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mounted on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) at a 40° angle with a 5-mm impactor tip. After isoflurane anesthesia induction, mice were placed in a foam mold held in prone position on the stereotaxic frame and maintained under anesthesia for the duration of the procedure. The stereotaxic arm was adjusted so that the head impact was at a
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fixed point relative to the right eye and ear, corresponding to the S1 somatosensory cortex. The impact delivered by the device to the head was 5.8-6.0 m/s with a dwell time of 0.2 s and impact depth of 5 mm. After impact, the mice were recovered from anesthesia on a warming pad prior to returning to their home cages. Skull fractures were not observed in our study similar to
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previously reported studies using comparable head impact forces 21, 50. For sham groups, the above procedure was performed except that the impact device was discharged in the air. Tibial fracture
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Mice underwent right leg distal tibia fracture or sham procedures a day after TBI as previously described 12, 46. Briefly, mice were anesthetized with isoflurane and a closed fracture of the right
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tibia just distal to the midpoint was done using a hemostat. Next, the limb was wrapped in casting tape (Scotchcast™ Plus) so the hip, knee and ankle were all fixed forming a spica around
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the abdomen. The model results in a well characterized transverse bone fracture and soft tissue
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injury and cast immobilization is essential for the extended pain-related phenotype 13. To prevent post injury edema constriction, a window was made in the cast over the dorsal paw and
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ankle. Sham groups underwent the same duration of anesthesia as the fracture mice. Mice were recovered from anesthesia on a warming pad and given 1.5 ml normal saline for hydration. Both injured and sham groups received subcutaneous buprenorphine for two days. Food and water gelpacks were provided on the floor for 72h post procedure. Casts were removed 3 weeks after fracture.
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Mechanical nociceptive assay Mechanical sensitivity was assessed using nylon von Frey filaments (Stoelting Co., IL, USA) according to the “up-down” algorithm developed by Chaplan et al. 5. We have applied this technique previously to detect 50% withdrawal threshold in mice after injury 38, 46. After
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acclimating mice on the wire mesh platform inside plastic enclosures (10cm radius), sequential fibers with increasing stiffness ranging from 0.004 to 1.7 g were applied to the plantar surface of hind limb and left in place 5 sec. 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
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response occurred after application of a fiber then a less stiff fiber was applied, if no response was observed the next stiffest fiber was applied. Mechanical withdrawal threshold was determined by a data fitting algorithm for significance analysis 35.
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Capsaicin-evoked behaviors
This assay was performed in separate cohorts of mice to determine if any differences in latent
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sensitization exist between the treatment groups after recovery 45. The total duration of behaviors (shaking, biting and licking) for 5 min after subcutaneous (s.c.) hind paw capsaicin injection
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(2.0ug/5ul; Sigma–Aldrich, USA) was measured. Control experiments used vehicle (0.25%
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DMSO, 0.25% ethanol, 0.125% Tween-80 in saline) plantar injections. The mice were gently restrained for the plantar injections. The assay was done at 20-22 weeks post TBI and fracture
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trauma when all groups had recovered to baseline mechanical threshold values. Open field (OF), zero maze (ZM) and object memory tests The OF arena (40X40X40 cm) was used to assess locomotion, object location (OLM) and object recognition (ORM) working memory tests. In OF test, total locomotor activity (distance travelled) and time spent in the center 20% of the arena for 10 min was determined. Increased
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time spent in the peripheral zones of a novel environment indicative of thigmotactic behavior was used as an index of anxiety, while time spent in the center 20% used as an index of risk taking behavior. The elevated ZM was used as well, to measure anxiety and risk taking behavior according to
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previously published methods 45. The maze is 24 inches above the floor, has an outer diameter of 24 inches and inner diameter of 20 inches, with two closed (6-inch tall walls) and open
quadrants. Mice were randomly placed facing one of the closed quadrants at the beginning of the 5-min test. Total time spent in open and closed quadrants were recorded. Time spent in the open
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quadrants was compared across groups to measure anxiety and risk taking behavior.
For OLM and ORM experiments, mice were initially habituated to two identical objects after being placed in the middle of the OF arena as previously described 45, 51. Next, during a 5 min
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trial one of the objects was moved to a novel location and exploratory behavior (investigation time) was recorded. Subsequent to a 5 min period in home cages, mice were returned to the arena
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with one of the previous identical objects being replaced with a novel one. The new object had a distinct shape and size difference from the identical object sets, and 5 min recordings were done
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for exploratory behavior towards objects. As mice explore novel locations or objects more than
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familiar ones, time spent exploring the novel compared to familiar was used to assess spatial and non-spatial working memory.
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All recordings from the above experiments were analyzed in real time by TopScan software (Clever System, USA). Diffuse noxious inhibitory control (DNIC) assessment DNIC assessment of post trauma mechanical hypersensitivity was done using a noxious stimulation–induced analgesia protocol modified for mice 18, 33. DNIC was induced by s.c.
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application of capsaicin (7.5 ug/5ul; Sigma-Aldrich, USA) to the right forepaw after brief isoflurane (3%) anesthesia. Control mice received 5ul of vehicle (0.25% DMSO, 0.25% ethanol and 0.125% Tween 80 in saline). The amount of DNIC induced by capsaicin injection was evaluated by hind paw mechanical threshold testing. Separate groups of mice in the Sham/Fx or
hypersensitivity was the same between the two groups.
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TBI/Fx groups were assessed for DNIC at 4 weeks post injury, at which time mechanical
In order to assess DNIC in the TBI alone group, the mice were allowed to recover to baseline mechanical thresholds after TBI (3 weeks). Next, right hind paw Prostaglandin E2 (PGE2)
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injections were given to produce brief hypersensitivity 44. Separate groups of Sham/Sham or TBI/Sham groups were given ipsilateral or contralateral hind paw injection (s.c.) of 100ng/15ul PGE2 (Cayman Chemicals, USA) and tested after 1h for mechanical hypersensitivity. Stock
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PGE2 solutions were made in 100% ethanol and further diluted (1:1000) in 0.9% saline prior to use. Vehicle (15μl) injections contained the same dilution of ethanol in saline. Subsequent to
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mechanical threshold evaluations, mice underwent capsaicin injection in the ipsilateral forepaw, and changes in mechanical withdrawal thresholds were followed.
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Assessment of analgesics on mTBI induced mechanical hypersensitivity
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The effects of anti-inflammatory and anti-neuropathic analgesic agents on TBI induced mechanical hypersensitivity were assessed at the 72h timepoint. Systemic (i.p.) injections of
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carprofen (5mg/kg; Sigma-Aldrich, USA) or gabapentin (50 mg/kg; Sigma-Aldrich, USA) were given to sham or TBI mice and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. The doses of the both drugs were in the high range used by other investigators examining effects in separate models 39, 48. Vehicle controls were employed. Data Analysis
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The data for mechanical sensitivity were analyzed by multiple t-tests with Holm-Sidak method to correct for multiple comparisons (no assumption of same scatter). For these experiments, groups of mice were randomly assigned to treatment groups and followed across days/weeks till recovery. Therefore, measurements for each subject were done for 10 time points for TBI alone
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experiments and 13-14 time points for polytrauma groups. In order to assess overall sex
differences after trauma, repeated two-way ANOVA was used. The data from capsaicin induced spontaneous pain behaviors, open field and zero maze experiments were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Two-way ANOVA
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followed by Tukey’s post-hoc tests (95% confidence interval of differences plus significance: α=0.05) were used to compare groups in the DNIC, analgesic and object memory tests. D'Agostino & Pearson omnibus test showed the data obtained were normally distributed.
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All data are presented as mean ± S.E.M and for all analyses, p<0.05 were taken to be significant.
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Results Mechanical hypersensitivity after mTBI, tibial fracture or combined Injury Male and female mice displayed increased contralateral and ipsilateral (Relative to injury side) hind paw mechanical sensitivity after mTBI, with peak increases lasting for 72h and gradual
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recovery to baseline values by 14 days after injury. TBI generally resulted in increased
contralateral versus ipsilateral limb mechanical hypersensitivity, though significant limb
differences were only seen at some timepoints in female mice between days 1 to 7 post injury (Figure 2 A,B). No significant male vs. female differences for respective contralateral (F9,162 =
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1.64, p= 0.11) or ipsilateral (F9,162 =1.82, p= 0.07) hind paw thresholds after TBI was seen on further analysis.
The mice in the fracture or combined injury polytrauma groups of both sexes displayed increased
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mechanical hypersensitivity for weeks after injury with recovery to baseline values at 17-19 weeks in male cohorts and 19-21 weeks in female ones (Figure 3 A,B). Within group analysis
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revealed that the polytrauma group had significantly greater mechanical hypersensitivity than fracture alone in males across several weeks, no significant differences between the female of
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either injury groups were seen. Further analysis showed significant male vs female mechanical
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hypersensitivity differences after fracture (F12,204 =7.14, p< 0.001) and polytrauma (F12,216 =8.07, p< 0.001), with the female groups demonstrating greater nociceptive sensitivity.
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Capsaicin-induced spontaneous pain behaviors after mTBI, tibial fracture or combined injury In order to detect differences in latent sensitization in the experimental groups, capsaicin evoked behaviors were measured after recovery from mechanical hypersensitivity (20-22 weeks) in male and female mice. Both fracture (males: p< 0.05; females: p< 0.001) and polytrauma groups (males: p< 0.001; females: p< 0.001) displayed increased spontaneous pain behavior after hind paw capsaicin application compared to respective sham groups. For both sexes, combined TBI
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and tibial fracture groups displayed greater capsaicin evoked behaviors than mice with either injury alone (Figure 4 A,B). Female mice displayed greater duration of capsaicin-evoked behaviors than male mice for all injury (TBI: male=37.60 ± 2.50 vs. female=60.85 ± 2.80, p<
vs. female=107.90 ± 4.08, p< 0.001) conditions.
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0.001; Fx: male=47.86 ± 3.61 vs. female=85.24 ± 3.11, p< 0.001 and TBI/Fx: male=76.34 ± 7.10
Working memory deficits after mTBI, tibial fracture or combined injury
Next, we sought to determine if differences in spatial and non-spatial working memory deficits existed between the experimental groups. Previously, we had shown that memory deficits
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accompany pain sensitization after fracture 45. Similar to our previous findings, male fracture mice had deficits in non-spatial (ORM, p= 0.53 novel vs same) but not in spatial (OLM, p< 0.01 novel vs same) working memory. TBI alone and polytrauma male groups displayed deficits in
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OLM as seen by insignificant differences in the time spent on novel vs familiar location (Figure 5 A; TBI: p= 0.98; polytrauma: p= 0.96, novel vs same). Neither sham nor the other injury
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female groups showed significant preference for novel location in the OLM test, therefore it was not possible to assess spatial working memory differences in these groups ((Figure 5 B). Male
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TBI and polytrauma groups displayed deficits in the ORM test, as seen by insignificant
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differences in time spent on novel vs familiar object (Figure 5 C; TBI: p= 0.53; polytrauma: p= 0.95, novel vs same). Contrary to the OLM test performance, the female sham mice displayed
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significant preference for the novel object (ORM, p< 0.05 novel vs same). Neither fracture nor TBI nor polytrauma female mice demonstrated a significant preference for novel object (Figure 5 D; Fracture: p= 0.80; TBI: p= 0.12; polytrauma: p= 0.91, novel vs same). As the sham female mice showed more total combined object exploratory activity in either test than respective males (OLM: males=11.47 ± 2.71 vs. females= 43.14 ± 6.19, p< 0.001; ORM: males=17.29 ± 2.71 vs.
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females= 34.57 ± 6.16. p< 0.05), comparing injury type effects between sexes would be challenging. Evaluation of locomotor activity, anxiety and risk taking behavior after mTBI, tibial fracture or combined injury
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Determination of open field locomotor activity (OF) in the male mice showed increased distance travelled after TBI alone (p< 0.001) or when combined with tibial fracture (p< 0.05) compared to controls (Figure 6 A), indicative of greater agitation in these groups. Female mice after fracture or TBI did not show significant differences in locomotor activity, though the polytrauma mice
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displayed decreases (p< 0.01) in distance travelled compared to controls (Figure 6B). Overall activity in the female groups was higher than in males. Male polytrauma mice spent significantly more time (p< 0.01) in the center 20% of the OF, suggestive of increased risk taking behavior
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and decreased anxiety (Figure 6C). None of the female groups significantly differed in time spent at the center compartment (Figure 6D). Next, we went on to investigate anxiety and risk
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taking behavior in the same groups using the elevated zero maze (ZM) test 34. Male polytrauma mice spent significantly more time (p< 0.01) in the ZM open arms than other male groups
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(Figure 7A), providing corroborating evidence for decreased anxiety and increased risk taking
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behavior. Fracture, TBI and the combination did not alter open arm times in the female groups (Figure 7B).
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Descending noxious inhibitory controls deficits after mTBI, tibial fracture or combined injury Deficient descending modulation of pain (DNIC) has been suggested to worsen some types of pain in TBI patients. In experiments directed at detecting possibly deficient DNIC in TBI and sham injured mice we first allowed TBI animals to recover for three weeks after injuries such that hind paw nociceptive thresholds had returned to baseline (Figure 2). DNIC was then tested
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by sensitizing hind paws with PGE2 followed an hour later by ipsilateral forepaw capsaicin injection to stimulate descending inhibition 33. Figure 8 A, B shows that capsaicin robustly reverses ipsilateral hind paw sensitization in sham injured male and female mice, but has almost no effect in animals 3 weeks after TBI suggesting profound disruption of DNIC by TBI in either
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sex. Further analysis showed differences in response to capsaicin between male and female TBI mice were not statistically different (F1,16 =0.60, p= 0.45). Separate groups of male and female mice were tested for DNIC of the contralateral hind paw. After ipsilateral forepaw capsaicin application, male and female TBI groups exhibited reduced DNIC effects on contralateral paw
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mechanical sensitivity compared to sham groups (Figure 8 C, D).
Additional experiments were undertaken 4 weeks after tibial fracture in mice with or without TBI, when mechanical sensitivity was same between the groups. In these experiments, we
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observed that forepaw capsaicin injection transiently reversed hind paw sensitization in fracture alone groups but that this form of analgesia was very limited in mice that had both tibial fracture
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and TBI (Figure 9 A, B). No significant differences in response to capsaicin were seen between male and female fracture alone groups (F1,16 =0.24, p= 0.63) or combined injury groups (F1,16
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=0.32, p= 0.58). For the both groups, DNIC assessments were done in fractured (Ipsilateral)
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side. As the contralateral paw in either group is not sensitized at 4 weeks, sensitization with PGE2 would be needed for DNIC assessments making interpretation of results difficult.
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Assessment of anti-inflammatory and anti-neuropathic analgesic agents on TBI induced mechanical hypersensitivity. Systemic injections of carprofen (5mg/kg) or gabapentin (50 mg/kg) were given to sham or TBI mice and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. On day 3 following injury, carprofen administration did not attenuate TBI induced hind paw
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sensitization in male or female mice (Figure 10 A, B). Similarly, gabapentin failed to attenuate TBI induced hind paw sensitization in both sexes (Figure 10 C, D). Control experiments using vehicle in sham and TBI groups did not show significant changes in paw withdrawal threshold in either sex (Data not shown). These data suggest the increased mechanical hypersensitivity seen
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after TBI is centrally modulated.
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Discussion Our understanding of the long-term sequalae of Traumatic brain injury (TBI) is presently improving. Cognitive, emotional and sensory symptoms are increasingly recognized in this patient population. Only recently have human and animal studies begun to explore the
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mechanisms supporting high pain prevalence after TBI with very little having been written about the interaction of TBI with peripheral injuries or the impact of sex on pain-related outcomes. In this regard the studies contained in this report are highly novel. Our major findings are: 1) mild TBI causes nociceptive sensitization lasting about two weeks. The resulting sensitization is
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refractory to anti-inflammatory and anti-neuropathic analgesics, being more consistent with central pain syndromes, 2) mild TBI worsens nociceptive sensitization after limb fracture, 3) sex does not impact post-TBI nociceptive sensitization but does affect the sensitization caused by
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limb fracture, 4) descending regulation of nociception is profoundly affected by TBI in both sexes, and 5) both concomitant limb fracture and sex modulate anxiety, risk taking behavior and
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memory changes after TBI. Together these observations provide a basis for exploring the
sequelae.
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molecular, cellular and circuit-level alterations after TBI supporting pain and other long-term
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One aspect of the experimental design adding to the face validity of our mTBI mouse model is the use of closed-head injury in generating mTBI, the predominant form of TBI. Though there
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are many methods used to model brain injury in animals, they most often involve opening or penetration of the skull. Conversely, sports-related concussions or acceleration/deceleration injuries and combat-related exposures to blast waves do not typically cause damage to the skull itself. Previously described TBI injuries used in pain studies including those by our own group have involved the application of a percussive pressure wave to the exposed dura of the animal, or
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the direct impact of an object against the brain 10, 22, 37. The present model first described by Shitaka et al. somewhat more realistically involves a non-penetrating blow against the skull 40. Variants of the model have been used to study repetitive brain injury such as can occur to those playing contact sports 14. In fact, neither we nor others using this closed head TBI model
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identified gross motor or behavioral changes in the TBI mice suggesting that more subtle forms of injury may support pain-related changes in the brain after TBI.
Similar to the other models involving disruption of the skull, we did observe sensitization to noxious mechanical stimuli. For example, Macolino et al. demonstrated sensitization of the
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tissues overlying the skull and the forepaw after cortical impact-induced injury 22. Our group recently focused on hind limb sensitization after TBI induced by lateral fluid percussion 10. The more distal site was assessed to better examine the sensitization of areas well outside of the head
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region, similar to areas affected in patients with TBI including the back and limbs. Different mechanisms may underlie pain and sensitization in the head versus back and limbs as altered
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expression of the neurokinin receptor NK1 has been demonstrated in the trigeminal nucleus after TBI 27, while the enhanced expression of pro-nociceptive BDNF occurs in lumbar spinal cord
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tissue after TBI and associated hind limb sensitization 10.
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It is not uncommon for TBI to occur with other injuries in battlefield, motor vehicle accident, etc. Others have combined TBI with injuries such as long bone fracture to model “polytrauma” . An excellent review has been written detailing the known interactions of TBI and various
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forms of peripheral injury in clinical populations and laboratory models 26. These combined injury models have been used to study bone healing and other consequences of injury, but not persistent pain 23, 47. Most studies have investigated consequences of multiple injuries on serum levels of injury markers and the extent of brain injury within the first several days. One
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exception was a report by Shultz et al. demonstrating in a closed-head TBI plus tibial fracture multitrauma model not only enhanced neuroinflammation and exacerbated changes in ventricular volume, but also increased risk taking behavior in male mice about a month after injury 41. Our own studies add significantly to this literature by showing that polytrauma mice display
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enhanced peripheral nociceptive sensitization for months and have latent sensitization after
recovery. These data are consistent with reports of chronic pain at sites distant from the head in multitrauma TBI patients.
A major mechanism for the control of pain-related signaling flowing into the brain from
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periphery is descending regulation. Fibers originating in brainstem send axons to the dorsal horn of the spinal cord where they may positively or negatively regulate signal transmission 30. Such regulation can be demonstrated by applying a noxious stimulus to a site in the periphery while
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following a pain or nociceptive threshold in another area. In animals, this type of regulation is referred to as diffuse noxious inhibitor control (DNIC) while in humans it is referred to as
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conditioned pain modulation (CPM) 49. This name emphasizes the general or “diffuse” nature of the regulation. Laboratory and clinical studies have linked the efficiency of descending
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regulation to chronic pain syndromes such as fibromyalgia, tension headache, neuropathic and
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chronic post-operative pain 33, 43, 49. Importantly, brainstem imaging has recently shown that the extent of injury in the periaqueductal grey matter (PAG) is correlated with pain levels in TBI
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patients 17. Our observations showed that weeks after TBI, mice of either sex had severely reduced DNIC providing a possible basis for the enhanced nociception seen after limb fracture. It should be noted that TBI patients with chronic post-traumatic headache display diminished CPM also 6. Existing therapies such as SNRI drugs might be used to augment DNIC in patients with TBI just as they seem to be useful in controlling pain in patients with other forms of chronic pain
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characterized by dysfunctional DNIC 30. The brainstem which contains major descending regulatory centers including the PAG, locus coeruleus and rostral ventromedial medulla, has been noted to be an area prone to TBI injury 25. Studies directed at understanding the extent of injury to these centers after TBI are warranted.
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Additionally, examining similarities or differences in pain behavior after TBI over other cortical regions besides the somatosensory cortex would complement the findings of our study 16.
The present study has been focused on characterizing a novel polytrauma paradigm and is limited to evaluating evoked tactile sensitivity and pain-related behaviors. Further studies
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examining the extent of spontaneous pain, as well as, additional pharmacological and behavioral testing to distinguish decreased anxiety form increased risk taking behaviors would further strengthen the characterization of this model of TBI and polytrauma.
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Males sustain TBI at a rate far higher than females 3. However, the consequences of those injuries may be different for the sexes. For example, a recent survey of military personnel that
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had suffered mTBI suggested that females experienced higher rates of some sensory changes including sensitivity to light and changes in taste and smell 2. Other analyses have found greater
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mortality after TBI amongst females, although these differences may be less when multiple
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injury severity is taken into account 1. Likewise, functional recovery after TBI does not appear to be strongly linked to sex 4. These data are in contrast to sex related differences in the prevalence
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of various pain syndromes which often show a predilection for females over males 42, including pain after surgical trauma 36. Our data failed to identify sex-linked differences in nociceptive sensitization after mTBI, and no clear difference in the additive effect of TBI to the sensitization observed after tibial fracture. On the other hand, there was a prominent effect of sex on sensitization after tibial fracture alone as reported previously 46. Similar to our previous study,
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male fracture mice had deficits in non-spatial working memory. We went on to evaluate memory and other pain/trauma related behavioral comorbidities in both sexes for interaction of limb trauma and TBI 46. Sex impacted anxiety, risk taking behavior and memory to differing extents in our studies. We did observe both sexes to have deficits in non-spatial working memory after
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limb trauma, TBI or both. These results in combination with the clinical observations suggest that the effects of sex on TBI outcomes might be dependent on the specific outcomes being examined as they may involve different underlying mechanisms. Conclusions
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Nociceptive sensitization in regions distant from the brain can be modeled after closed-head mTBI including sensitization resulting from the combination of mTBI with peripheral injury. This type of model provides a tool critical to studying the very common occurrence of chronic
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pain after TBI including multi- or polytrauma. Our animal data are consistent with clinical reports in suggesting that brain centers involved in descending modulation are involved in
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enhanced pain sensitization after mTBI, while we do not at this point understand the details of these changes. A better understanding of the specific involved brain centers, the modulation of
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their activities and a more detailed understanding of the mechanisms of injury to the brain after
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mTBI might help prevent or reduce chronic pain in this population.
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Abbreviations: mTBI, mild traumatic brain injury; Fx, fracture; ANOVA, analysis of variance; OF, open field; ZM, zero maze; DNIC, diffuse noxious inhibitory control, OLM, object location memory; ORM, object recognition memory; DMSO, dimethyl sulfoxide; PGE2, prostaglandin
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E2; SNRI, serotonin and norepinephrine reuptake inhibitors.
Authors’ contributions: PS participated in the design of the study, performed behavior studies, helped to draft the manuscript and performed the statistical analysis. MT participated in the design of the study, performed the surgeries and helped revise the final manuscript. PY helped
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perform behavior studies and analyzed data, KAI helped perform the surgeries and revised the final manuscript. JL helped develop the animal TBI models and revised the final manuscript. TTH and TWC participated in the design of the study and revised the final manuscript. JDC
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conceived of the study, participated in its design and coordination, and helped draft the
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manuscript. All authors read and approved the final manuscript.
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Acknowledgments: Not applicable
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Figure Legends:
Figure 1. Overview of the experimental timeline. mTBI: Mild traumatic brain injury, Fx:
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Fracture, B: Baseline mechanical assay, DNIC: Diffuse noxious inhibitory controls, OLM: object
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location memory, ORM: object recognition memory and CAP: Capsaicin.
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Figure 2. Mechanical hypersensitivity after mTBI. Male (A) and female (B) mice displayed increased hind paw mechanical sensitivity after TBI. Data were analyzed by multiple t tests
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using the Holm-Sidak method to correct for multiple comparisons. * indicates significant
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difference in comparison with respective limb measurements in sham group. # indicates significant difference between ipsilateral and contralateral limb to the side of injury. Error bars: SEM, n = 8-10/group.
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Figure 3. Mechanical hypersensitivity after peripheral trauma or combined mTBI and peripheral trauma. Male (A) and female (B) mice in the fracture or polytrauma groups displayed mechanical hypersensitivity for weeks after injury with recovery to baseline values at 17-19 weeks in male cohorts and 19-21 weeks in female ones. Male but not the female mice showed
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significant differences between fracture and polytrauma induced mechanical hypersensitivity. * indicates significant difference in comparison with respective sex combined injury group. Error bars: SEM, n = 8-10/group. Data were analyzed by multiple t tests using the Holm-Sidak method
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Figure 4. Capsaicin-evoked behaviors after mTBI, peripheral trauma or combined injury. The total duration of behaviors (shaking, biting and licking) in male (A) and female (B) mice for 5 min after hind paw capsaicin (2.0ug/5ul) or vehicle injection was measured. The assay was done at 20-22 weeks post trauma period when all groups had recovered to baseline mechanical
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threshold values. Error bars: SEM, n = 8-10/group, *p<0.05, *** p<0.001 for comparison to respective sham groups and ### p<0.001 for comparison within groups. Data were analyzed by
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one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons.
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Figure 5. Working memory evaluations after mTBI, peripheral trauma or combined injury.
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Object location (OLM) and object recognition (ORM) working memory tests were employed. The interval between acquisition and retrieval was 5 min in either test. (A-C) Male fracture mice displayed no deficits in spatial (OLM) but had deficits in non-spatial (ORM) working memory. The TBI alone and polytrauma male groups displayed significant deficits in both OLM and ORM tests. (B-D) Female mice in either experiential group displayed no significant preference for novel location in the OLM test, but only the female sham mice displayed significant preference
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for the novel object (ORM). All recordings were analyzed in real time by automated software and data obtained were analyzed by two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, *p<0.05, or *** p<0.001 for comparison between novel and familiar
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location/object.
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Figure 6. Locomotor activity and anxiety evaluations after mTBI, peripheral trauma or combined Injury. In the open field test the male (A) mice showed increased distance travelled after TBI or polytrauma compared to controls. Female (B) mice after fracture or TBI did not show significant differences in locomotor activity, though the female polytrauma mice displayed
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decreases in distance travelled compared to controls. Male polytrauma (C) but not the female (D) polytrauma mice spent more time in the center 20% compartment of the arena indicative of
decreased anxiety and increased risk taking behavior. All recordings were analyzed in real time by automated software and data obtained were analyzed by one way ANOVA followed by
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Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, *p<0.05, **p<0.01 or *** p<0.001 for
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Figure 7. Anxiety evaluations after mTBI, peripheral trauma or combined Injury. In the zero maze the male (A) polytrauma mice displayed reduced anxiety as seen by increased time spent in
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the open arms, indicative of increased risk taking behavior. None of the female (B) groups
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showed differences in anxiety levels as measured by time spent in open arms. All recordings were analyzed in real time by automated software and data obtained were analyzed by one way
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Figure 8. Descending noxious inhibitory controls deficits after mTBI. To determine differences in descending pain control inputs, capsaicin (arrow) noxious stimulation–induced analgesia was used. The mTBI male (A, C) and female (B, D) groups show descending pain controls deficits in either hind limbs compared to controls. Hind paw mechanical sensitivity data were analyzed by
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two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n = 8-10/group, ***
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p<0.001 for comparison between mTBI and sham.
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Figure 9. Descending noxious inhibitory controls deficits after peripheral trauma or polytrauma. To determine differences in descending pain control inputs, capsaicin noxious stimulation–
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induced analgesia was used. Polytrauma male (A) and female (B) groups displayed deficits in descending pain controls compared to fracture alone. Hind paw mechanical sensitivity data were
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analyzed by two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n = 810/group, *** p<0.001 for comparison between polytrauma and fracture.
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Figure 10. Effects of anti-inflammatory and non-opioid analgesic agents on TBI induced mechanical hypersensitivity. After baseline mechanical sensitivity measurements, Caprofen (5mg/kg, i.p.) or gabapentin (50 mg/kg, i.p.) were given to sham or TBI mice 3 days after injury and mechanical withdrawal thresholds assessed after 90 or 60 minutes respectively. Neither
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carprofen (A, B) nor gabapentin (C, D) administration (arrows) resulted in significant changes in TBI induced hind paw sensitization in male or female mice. Hind paw mechanical sensitivity data were analyzed by two-way ANOVA followed by Tukey’s post-hoc tests. Error bars: SEM, n
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