Repeated Closed-Head Mild Traumatic Brain Injury Induces Robust Caspase-1 Mediated Inflammation that Contributes to Nociplastic Pain.

doi:10.21203/rs.3.rs-1987260/v1

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Repeated Closed-Head Mild Traumatic Brain Injury Induces Robust Caspase-1 Mediated Inflammation that Contributes to Nociplastic Pain.

https://doi.org/10.21203/rs.3.rs-1987260/v1

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Patients who have experienced concussive mild traumatic brain injuries (mTBIs) suffer from a number of comorbidities including chronic pain. Despite extensive studies investigating the underlining mechanisms of mTBIs-associated chronic pain, the role of inflammation after brain injury, and its contribution towards long-term pain, are still poorly understood. Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes and its effects on TBI-related pain. Utilizing a recently developed transgenic caspase-1 luciferase reporter mouse, we characterized the bioluminescence signal evident in both in vivo and ex vivo tissue following repetitive closed head mTBIs. This allowed us to reveal the spatiotemporal dynamics of caspase-1 activation in individual animals across time. We established that there are significant increases in bioluminescent signal upon protease cleavage in the brain, thorax, abdomen, and paws in vivo which lasted for at least one week after each injury. Enhanced inflammation also was also in ex vivo brain slice preparations following injury event that lasted for at least 3 days. Concurrent with the in vivo detection of bioluminescent signal were persistent decreases in mouse hind paw withdrawal thresholds that lasted for more than two months post-injury. Using a potent small molecule inhibitor of NLRP3 inflammasome-caspase 1 activity, MCC950, we observed reductions in both caspase-1 bioluminescent signal in vivo, caspase-1 p45 expression by immunoblot and increase in hind paw withdrawal thresholds. Overall, these findings suggest that neuroinflammation in the brain following repeated mTBIs is coincidental with a chronic nociplastic pain state, and repeated mTBI associated events can be ameliorated by a highly specific small molecule inhibitor of NLRP3 inflammasome activation.

Caspase-1

mTBI

Neuroinflammation

nociplastic pain

Mild traumatic brain injury (mTBI) is a frequent injury condition observed in both civilian and military populations. The current definition of mTBI, as determined by the International Classification of Diseases (ICD-10), includes a sequela of head injury complex disorders including impaired memory, depression, anxiety and post-traumatic nociplastic pain.¹ Unlike pain from other types of injuries, mTBI-associated nociplastic pain can include both cephalic and somatic symptoms.^1–4 Post-traumatic persistent pain is one of the most common and long-lasting symptoms following mTBI and can be defined as pain symptoms that develop within 7 days after the head trauma and is considered persistent when the condition lasts more than three months. The prevalence rates of persistent nociplastic pain states for up to 3 months range from 30–90% following mTBI, with 50–60% lasting at least one year^5–7. Clinical research also suggests that repeated incidence across time can induce cumulative effects particularly in athletes involved in contact sports^8,9, military personnel^10,11, and the victims of domestic violence.¹¹ Despite extensive research efforts in basic and clinical science to date, the pathophysiology of the persistent chronic pain state due to repetitive mTBI is poorly understood and effective therapeutics are still not available to improve patient outcomes.^{2–4, 12}

The neurological damage responsible for nociplastic pain in mTBI patients is minimal near the time of the injury but is accompanied by acute inflammatory responses which begin within hours of the trauma event. These processes, which are in large part restorative, can become chronic and last for days, weeks, months and even years^13,14. Preclinical studies of generally shorter duration demonstrate that mTBI is accompanied by neuroinflammation, yet it is still unclear what regional and longitudinal profiles of this trauma-induced process contribute to long-term nociplastic pain states. Furthermore, how the development of neuroinflammation progresses over time, in the context of brain-body axis, is not understood. Lack of information regarding longitudinal processes are largely due to technological shortcomings which prevent detection of ongoing mTBI-related real-time neuroinflammatory changes in living animals.

Mechanisms postulated to contribute to both acute and chronic inflammatory conditions include dysregulation of inflammatory responses and systemic release of endogenous mediators acting as danger signals [damage-associated molecular patterns (DAMPs)]. Some of the numerous DAMPs released after trauma trigger the assembly of intracellular multiprotein complexes called inflammasomes predominantly in cells associated with the innate immune response. Inflammasomes are comprised of key cytoplasmic multiprotein complexes of NOD-like receptor (NLR) family members which serve to initiate the assembly of the caspase-1–activating scaffold which effectively cleaves the proinflammatory IL-1 family of cytokines into their bioactive forms, IL-1β and IL-18.^15,16 Accumulating data suggests that the nucleotide-binding oligomerization domain (NOD)-like receptor 3 (NLRP3) is both involved in the generation of tissue damage and immune dysfunction after trauma to the nervous system.¹⁷

Recent studies have changed our understanding of the timing and interactions of the inflammatory processes following the introduction of transgene encoded biosensor into a mouse which is dependent on proteolytic activity of caspase-1 during inflammasome activation. This biosensor rapidly monitors in vivo events from the induction of injury or onset of disease over the course of the animal’s life.^18,19 Using a murine repetitive closed-head mTBI preparation compatible with in vivo imaging²⁰, this caspase-1 reporter mouse model allows for prolonged monitoring of mTBI-associated central and peripheral inflammatory responses. Considering that both a single and repetitive mTBI events can contribute to chronic pain hypersensitivity, we assessed the injury-induced changes in behavioral pain states. Finally, we tested the effects of a potent NLRP3 inflammasome-specific inhibitor MCC950 (CP-456,773, CRID3, Invivogen) on mTBI-induced inflammation and pain behavioral responses.

Animals All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Indiana University School of Medicine, which are in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Wild type C57BL/6 mice at 5–7 weeks old (~ 20–25 g) were randomly divided into sham, injury, and treatment groups for behavioral analysis. Caspase-1 luciferase reporter transgenic mice (with the C57BL/6 background) at 5–7 weeks old were used for in vivo inflammasome imaging. For ex vivo brain slice imaging experiments, Caspase-1 reporter transgenic mice at 5–7 weeks old were divided into multiple groups at different time points following mTBI. Wild type C57BL/6 mice were used for immunohistological assessments.

Thinned-skull cranial window preparation

To create a thinned-skull transparent window for in vivo imaging, mice were anesthetized with 125–250 mg/kg tribromoethanol (intraperitoneally) and fixed on a small animal stereotaxic system. A heating pad set at 37^oC was used to maintain body temperature while under anesthesia. Incisions were made on the scalp to remove a rectangular portion of skin (~ 7x10mm) and expose the underlying skull. The periosteum was removed, and the exposed skull surface was rinsed with sterile saline and dried with a combination of cotton and compressed air. For skull thinning, a sterile IRF 0.6 mm drill bit in a Microtorque Foredom K.1070 drill (Foredom Inc., Bethel, CT, USA) was utilized at ~ 3000–4000 rpm. A rectangular area of (~ 3x4 mm) of skull tissue with respect to the midline was thinned. To thin the bone, the microdrill was used in sweeping motions with the drill bit angled nearly parallel to skull surface; no direct downward pressure was applied. This drilling motion was done uniformly and continuously from anterior to posterior. Thinness of the skull was checked by placing sterile saline over the thinned area and viewed under a dissecting scope. As the transition from the harder thick bone layer to the spongy bone layer (i.e., the “diploe”), drilling speed was reduced to ~ 1000–2000 rpm. Once microvasculature becomes clearing visible, the drilling was stopped. At this time, a sterile dental microblade was used to scrape gently on the thinned skull surface under saline to achieve final thinning and even out the surface area. An ideal optimal thinned skull thickness was about 20–40 µm. Once the thinned skull window was ready, a thin layer of cyanoacrylate glue (C1000, Ted Pella Science, Inc. Redding, CA, USA) was applied using a 1.0 mm glass pipette to cover the thinned-skull area completely. This helped protect the thinned-skull area, slow down bone regrowth, and most importantly, maintain transparency of the thinned-skull area for long-term imaging. Animals were then allowed to recover one week before baseline imaging and mTBI injuries were induced.

mTBI procedure

mTBI was inflicted using a technique described recently^21–23. Briefly, mice were anesthetized with 2–4% isoflurane and heads were stereotactically fixed with heat-pad support below the abdomen. Closed-head mTBI was produced using a control cortical impact (CCI) device with a modified impactor tip via attaching a 3-mm diameter round silicone tip of 1.5 mm thickness on it (Benchmark Stereotaxic Impactor, Leica Impactor One, Meyer Instrument, St. Louis, MO). After the baseline point of the device was set by lowering the tip to the skull surface and the stage position was set to zero, the impactor was retracted, and the impact depth was set. The approximate center of the impact site was 1 mm posterior to the bregma and 0.5 mm lateral from midline on one cortical hemisphere. The skull was struck with the impactor tip at a speed of 4 m/s to a depth of 2 mm. For repeated mTBIs, injury was done once a week for three weeks (total of 3 injuries). All animals survived the injury without any skull fracture or hemorrhage.

Intraocular pressure (IOP) measurements

Intraocular pressure (IOP) changes in the mouse eyes were used to both confirm the severity of the skull impact and verify the absence of skull fracture (Chen et al., 2019). An ICARE TONOLAB (Vantaa, Finland) tonometer was used to measure IOP which is a surrogate of changes in brain pressure. The principle of this device was based on a rebound measuring in which a very light weight probe is used to make momentary contact with the cornea. The IOP measurements were done immediately before and after the injury for up to 30 min (Fig. 1D, n = 10 mice in each group). If the skull bone was fractured, there was an absence of IOP. Alternatively, if there were no changes in IOP, injury to the bone was deemed to be insufficient and the animal was excluded from the study.

MCC950 treatment paradigm

MCC950 (a selective NLRP3 inhibitor) was purchased from Invivogen (San Diego, CA, USA) and dissolved in saline (5 mg/ml). The drug was injected to each mouse at 1 hour and 24 hours after each impact at a dosage of 10 mg/kg as previous reported elsewhere.^24–27 A total of 6 MCC950 injections were done for each animal over the course of each study.

In vivo IVIS SPECTRUM inflammasome bioluminescence imaging

Dynamic bioluminescence images (BLI) were acquired using an IVIS SpectrumCT (Perkin-Elmer Inc. USA) imaging system that could accommodate up to 5 mice per session. Prior to imaging, the skin over the abdomen regions were shaved to minimize light scatter. Anesthetic induction was achieved with 2–4% isoflurane, and animals were administered d-Luciferin (Promega, Madison, WI, USA) at 150 mg/kg via intraperitoneal injection. Mice were immediately transferred to the heated stage (40 ± 1°C) of the IVIS Imager, placed on their abdomen, and sequentially imaged at 2 min intervals for 40 min. At the completion of the sequence, anatomical reference photos were acquired permitting generation of a set of overlaid images. Animals were image at baseline, 24 hours post each injury, 3 days, 1 week, and 1 month after last injury. Identical imaging paradigm was used for mTBI/saline treated and mTBI/MCC950 treated groups.

Brain slice preparations

Caspase-1 reporter mice were anesthetized with 125–250 mg/kg tribromoethanol (intraperitoneally) and decapitated with scissors. After the scalp was removed, the skull was cut along the mid-sagittal line and the two flaps of bone covering both brain hemispheres were removed. The brain was then taken out and immediately placed in an ice-cold 4 ^oC oxygenated sucrose artificial cerebral spinal fluid (s-ACSF) solution (206 mM sucrose, 2 mM KCl, 1 mM MgCl₂, 2 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM D-glucose, 1 mM CaCl₂). After 1–2 mins, the brain was glued onto a cutting stage such that the cortex faced the approaching blade. A piece of agarose gel was used as a cushion against the ventral part of the brain to prevent movement while cutting. Slices of 350 µm thick were cut with a Vibratome (Leica VT1200S; Leica, Nusslock, Germany) while the brain was submerged in a 4 ^oC cutting solution. The slices were then incubated at 32 ^oC for 1 hour in a gridded chamber filled with oxygenated incubating ACSF (124 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM D-glucose, 1 mM CaCl₂). Each slice was kept in a grid of the chamber for up to 4 hours during an experiment.

Ex vivo brain slice IVIS SPECTRUM inflammasome bioluminescence imaging: Imaging paradigm for ex vivo brain slices are identical with in vivo IVIS imaging above. Brain slices were kept in oxygenated gridded chambers (one slice per slot with ~ 1 mL aCSF) at ~ 27–32°C during the duration of imaging. For each imaging session, images were collected at three separate series: first series with only D-Luciferin, second series with Lipopolysaccharides (LPS), and third series with MCC950. Brain slices were imaged at a 2 min interval for a total of 8 minutes for each series. 100 µL of d-Luciferin (20 mg/mL) was added to the chamber one minutes prior to the start of first imaging series to a final concentration of 2 µg/mL, 100 µL of LPS (10 µg/mL; ThermoFisher, Carlsbad, CA, USA) was added to the same chamber up to a final concentration of 1 µg/mL five minutes prior to the second imaging series. Finally, 100 µL of MCC950 (10 mg/mL) was added to the same chamber up to final concentration of 1 µg/mL five minutes prior to the third imaging series.

Immunofluorescence analysis of astrocytes and microglia: To determine potential glial reactivity and neuroinflammation induced by the mTBIs, we used an antibody against ionized calcium binding adaptor protein (IBA1) to label microglia, and an antibody against Glial Fibrillary Acidic Protein (GFAP) to label astrocytes^28,29. C57BL6J (n = 4/group) mice were divided into three groups: sham control, injury 1 day, and injury 3 days. Tissue preparations were done as followed: the mice were deeply anesthetized and perfused transcardially with PBS buffer, followed by 4% paraformaldehyde (PFA) at room temperature. The brains were then dissected and placed in 30% sucrose for 48–72 hours at 4^oC. The tissue was then frozen and sectioned with a cryostat at thickness of ~ 45 µm. Immunofluorescence was performed on free-floating sections by incubating overnight with a primary antibody. For astrogliosis and microglia activation staining, anti-GFAP (mouse; 1:800, Sigma Aldrich G3893) and anti-IBA1 (goat; 1:200, ABCam ab5076) antibodies were followed by secondary antibodies of goat anti-mouse Cy5 (1:500, Jackson Immuno) and donkey anti-goat 488 nm (1:1000, Fischer Scientific). A DNA stain, 4’, 6-diamidino-2-phenylindole (DAPI; 1: 10,000, Fischer Scientific), was added to the solution for a final 5 min as a nuclear counterstain. Finally, fluorescence signals were imaged using a Neurolucida imaging system.

Protein immunoblotting of caspase-1: Animals were divided into three groups: Sham, mTBIs, and mTBIs with MCC950 treatment. They were subjected to the same 3x mTBI paradigm as described above. MCC950 treatment was performed in the same manner as above, at 1 hour and 24 hours after each injury. At 24 hours after the third injury, animals were euthanized, and the cortical tissues were collected and then rapidly frozen in liquid nitrogen. Immunoblotting was performed the following day after tissue collection. Protein isolation and immunoblotting were perform as previously described^30,31. In brief, cortical brain tissue samples were sonicated in a lysis buffer (RIPA buffer; Alpha Aesar, Catalog #J62524) supplemented with EDTA 0.5 M (Fisher Scientific, Catalog #BP24821), Phenylmethanesulfonyl Fluoride 0.1 M (Sigma, Catalog #93482), and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Catalog #78441). Samples were then cleared by centrifuging (12,000 rpm, 10 minutes, 4°C). Sample concentrations were determined using the Pierce BCA Protein Assay Kit (Fischer Scientific, Catalog #23227) and a FlexStation3 microplate reader (Molecular Devices LLC, San Jose, California). Protein samples were heat-denatured in a Laemmli Buffer (BioRad, Catalog #1610747) supplemented with 2-Mercaptomethanol (Sigma, Catalog #M3148) for 10 min at 95°C and then separated on an SDS-PAGE with 4–20% precast gels (BioRad, Catalog #4561094). The transfer was done on Immuno-Blot PVDF membrane (BioRad, Catalog #1620177). The membranes were blocked for 30 minutes with a TBS-intercept blocking buffer (Li-Cor, Catalog #927-60001) and then incubated with primary antibodies against caspase-1 (Mouse, 1:1000, Adipogen) and β-Actin (Rabbit, 1:1000, Abcam) overnight at 4°C on a rocker. After incubation, membranes were washed 3 times with TBST (150 mM NaCl, 10 mM TRIS-base, 0.1% Tween20, Fischer Scientific), followed by 3 washes with TBS. Membranes were then probed with 800 nm donkey anti-mouse (1:5000, Li-Cor) and 680 nm donkey anti-rabbit (1:5000, Li-Cor) for 2 hours at room temperature on a rocker, and then washed 3 times with TBST and 3 times with TBS. Finally, antibody complexes were detected on LI-COR’s Odyssey CLx (LI-COR Biotechnology, Lincoln, Nebraska) using Image Studio, and the data were analyzed using ImageJ software.

Von Frey pain behavioral assessment: Mechanical hypersensitivity was measured using a mechanical Von Frey aesthesiometer. Calibrated Von Frey filaments measuring 100 µm in diameter and capable of exerting bending forces of 5, 10, 20, 40 mN were applied to the plantar surface of the animal hind paws in succession with the gradually increasing bending force until a paw withdrawal was observed. Then, the corresponding stimulation intensity was recorded. Each stimulus lasted approximately 1 second and had a 10–15 second intermission between applications. Von Frey testing was performed at multiple time points: baseline (before injury), 24 hours after each injury, 3 days, 1 week, 1 month, and 2 months after last injury. The test was done to both the ipsilateral hind paw as well as the contralateral hind paw. The test was conducted by a research analyst who was blinded to treatments and group assignment. An identical behavioral paradigm was used for all mTBI/saline control and mTBI/MCC950 treated groups.

Analysis of IVIS imaging data

For both in vivo animal and ex vivo brain slice images, (to provide visualization, segmentation, and time series quantification from the 10-min scan, BLI and anatomical reference images were imported into LivingImage software (Perkin-Elmer Inc. USA). Pseudo-colored parametric overlays of BLI time-series with anatomical reference images were dynamically constructed for each animal. Using the image of the time series with the peak light emission for each individual animal, ROIs were designated for the region. The extracted time series were then analyzed to determine Total Photon Flux [p/s] of the time course kinetics. Using this information, each BLI time course was integrated to calculate the Area Under the Total Photon Flux Curve (AUC_TPF), which represents the total light emission corresponding to the aerobic metabolism of d-luciferin.

Statistics

Final plots were developed in Microsoft Excel and GraphPad Prism 6 (GraphPad Software, La Jolla, California) and values are reported as mean ± S.E.M. All statistical analyses were done with GraphPad Prism 6 and Jmp Analysis 11 (SAS Institute Inc. 2013. Cary, NC). ANOVA analyses were used for comparing areas under the total photon flux curve for same groups of animals across all time points, areas among control, injury, and treatment groups in vivo (Repeated measures), and among groups of ex vivo brain slices (Repeated measures), comparison of GFAP and IBA1 fluorescence positive cell signals among groups, immunoblotting of caspase-1 among groups (one-way), and Von Frey pain thresholds (two-way). For comparisons that yielded statistical significance, Tukey’s HSD post-hoc analyses were applied for further comparisons between specific groups.

Repetitive mild traumatic brain injury (mTBIs) in mice with a thinned-skull cranial window

Mice were randomly divided into male and female groups (Fig. 1A). To determine the effect of repetitive mTBIs on neuroinflammation in vivo, we subjected mice to a thinned-skull cranial window procedure as described in our previous publications^23,32 (Fig. 1B, C). These thinned-skull cranial windows were minimally invasive and preserved their transparency for months after preparations. Furthermore, the windows retained their integrity despite the physical impact generated following a concussive injury (Fig. 1D). Mice were first subjected to thinned-skull cranial window procedure followed two weeks later to a single mTBI event once a week for three weeks. To confirm that the concussive event did not produce fracture to the skull or cranial window preparation, we measured IOP changes in the mouse eyes before and post-injury. The closed head injury produced a pronounced bilateral increase in intraocular pressure immediately after the impact (19.40 ± 0.25 injury vs. 7.87 ± 0.12 baseline for right eyes and 20.70 ± 0.38 injury vs. 7.83 ± 0.17 baseline for left eyes, p < 0.001, repeated ANOVA; Fig. 1E). This event represented a clinical hallmark of concussion³³ but did not directly affect vasculature in the underlying brain tissue (Fig. 1D). We observed significant increases in astroglyosis as demonstrated by increases in the number of GFAP positive cells in cortical tissues at day 1 and day 3 following the third injury event (94.03 ± 1.4 cells/counted region injury vs. 28.50 ± 1.50 cells/counted region sham at day 1 and 61.00 ± 2.52 cells/counted region injury vs. 25.17 ± 3.51 cells/counted region sham at 3, p < 0.05, one-way ANOVA) (Fig. 1F, G). Image analysis of IBA-1 immunopositive cells yielded evidence of microgliosis as defined by increased numbers of IBA1-immunopositive cells, which was also evident at day 1 and day 3 following third injury event (73.50 ± 7.50 cells/counted region injury vs. 26.13 ± 4.002 cells/counted region sham for day 1 and 112.70 ± 10.71 cells/counted region injury vs. 14.50 ± 3.54 cells/counted region sham for day 3, p < 0.05, one-way ANOVA).(Fig. 1F, G). Therefore, this outcome in the thinned-skull cranial window preparation following repeated impact injury may represent a marker of neuroinflammation events.

3x mTBIs resulted in a robust increase in caspase-1 mediated inflammatory response in multiple body areas lasted at least 1-week post-injury

Detection of inflammation states associated with diseases or injury has historically required tissue removal from sacrificed animals, preventing the real-time dynamic measurement of inflammatory or neuroinflammatory events in individual animals over time. To better understand the spatiotemporal kinetics post-brain trauma, we used a caspase-1 luciferase reporter transgenic mouse model combines with our thinned-skull cranial window preparation, which allowed us to follow the inflammatory dynamics within the same animals over time at many different areas of the body. By utilizing in vivo IVIS imaging, we found significant increase in bioluminescence intensity at 1 day after each injury compared to baseline (Fig. 2, Supplementary Fig. 1). The increase in caspase-1 signals were observed at the brain area (54.73 ± 11.89 male/ 40.74 ± 7.96 female for 1st injury, 28.08 ± 5.34 male/ 29.03 ± 2.78 female for 2nd injury, 60.48 ± 12.21 male/ 61.90 ± 16.93 female for 3rd injury vs. 7.39 ± 1.13 male/ 4.34 ± 0.56 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 2A, B), thoracic area (42.75 ± 10.59 male/ 8.96 ± 2.91 female for 1st injury, 40.44 ± 6.16 male/ 18.65 ± 7.65 female for 2nd injury, 88.33 ± 23.45 male/ 46.21 ± 27.43 female for 3rd injury vs. 0.38 ± 0.14 male/ 0.16 ± 0.1 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 2A, Supplementary Fig. 1A), abdominal area (22.10 ± 6.61 male/ 10.21 ± 4.00 female for 1st injury, 42.30 ± 7.22 male/ 22.70 ± 11.29 female for 2nd injury, 31.95 ± 16.13 male/ 20.48 ± 5.84 female for 3rd injury vs. 0.8 ± 0.57 male/ 0.20 ± 0.16 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 2A, Supplementary Fig. 1B), and paws area (30.00 ± 4.49 male/ 27.78 ± 4.62 female for 1st injury, 39.43 ± 6.22 male/ 19.21 ± 6.18 female for 2nd injury, 34.47 ± 4.5 male/ 31.91 ± 2.82 female for 3rd injury vs. 0.36 ± 0.07 male/ 0.22 ± 0.05 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 2A, C). We observed these enhanced inflammatory signals in both male and female animals.

Furthermore, we continued to monitor inflammatory activity over time after the 3rd mTBI and found remarkably elevated caspase-1-mediated inflammatory response at 1 day that lasted for more than one week after injury (Fig. 3, Supplementary Fig. 2). We observed a signal increase in the brain (176.50 ± 58.79 male/ 127.90 ± 11.35 female at 3 days and 72.01 ± 19.25 male/ 46.66 ± 4.28 female at 1 week vs. 7.39 ± 1.13 male/ 4.34 ± 0.56 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 3A, B) and paws (147.10 ± 19.41 male/ 106.2 ± 12.37 female at 3 days and 39.85 ± 6.78 male/ 35.25 ± 2.24 female at 1 week vs. 0.36 ± 0.07 male/ 0.22 ± 0.05 female of baseline, p < 0.05, all values reported as x 10⁷ p/cm²/sr, repeated ANOVA; Fig. 3A, C) up to 1 week after the third mTBI injury. These changes were observed to be highest at 3 days after injury and were seen in both male and female animals. To confirm that the inflammatory signals were originating from brain tissue, we also performed bioluminescence IVIS imaging on freshly prepared brain slices from thinned skull mice that had been subjected to three mTBIs at 1 day and 3 days after the 3rd injury ex vivo. We found a robust increase in caspase-1 mediated bioluminescence signals in these brain slices for both 1 and 3 days with the greatest increase at 3 days after injuries (7.57 ± 0.93 for 1 day and 9.69 ± 0.83 for 3 days vs. 3.24 ± 0.24 for sham, all values reported as x 10⁴ p/cm²/sr, p < 0.05, one-way ANOVA; Supplementary Fig. 3). We also observed a significant bioluminescence signal increase up to at least 1 month in the abdominal (247.0 ± 97.08 male/ 77.15 ± 25.02 female at 3 days, 24.76 ± 11.01 male/ 20.72 ± 8.38 female at 1 week, and 52.82 ± 9.29 male/ 51.87 ± 8.86 female at 1 month vs. 0.8 ± 0.57 male/ 0.20 ± 0.16 female of baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 3A, Supplementary Fig. 2A) and thorax (156.3 ± 33.05 male/ 83.88 ± 30.18 female at 3 days, 94.25 ± 29.91 male/ 69.38 ± 44.40 female at 1 week, and 36.21 ± 6.27 male/ 37.41 ± 6.79 female at 1 month vs. 0.38 ± 0.14 male/ 0.16 ± 0.1 female of baseline, p < 0.05, all values reported as x 10⁷ p/cm²/sr, repeated ANOVA; Fig. 3A, Supplementary Fig. 2B).

3x mTBIs induced mechanical allodynia for at least 2 months post-injuries

To better understand the degree of how repeated mTBIs affect long-term pain response, we subjected both male and female mice to Von Frey mechanical stimuli. We found a significant decrease in tactile dependent paw withdrawal threshold at 1 day after each injury compared to sham with the greatest loss at 1 day after the 3rd injury (Fig. 4). These mechanically-evoked responses were evident in both the hind paw ipsilateral and contralateral to the injured brain tissue (0.68 ± 0.15 male/ 0.49 ± 0.04 female after 1st injury, 0.55 ± 0.07 male/ 0.47 ± 0.01 female after 2nd injury, 0.62 ± 0.29 male/ 0.20 ± 0.02 female after 3rd injury vs. 1.29 ± 0.08 male/ 1.22 ± 0.1 female after 1st sham preparation, 1.48 ± 0.14 male/ 1.27 ± 0.14 female after 2nd sham preparation, and 1.45 ± 0.13 male/ 1.13 ± 0.21 female after 3rd sham preparation, respectively, p < 0.05, one-way ANOVA, Fig. 4A) and right paws (contralateral to the injured hemisphere) (0.49 ± 0.03 male/ 0.5 ± 0.01 female after 1st injury, 0.5 ± 0.01 male/ 0.43 ± 0.12 female after 2nd injury, 0.40 ± 0.04 male/ 0.07 ± 0.02 female after 3rd injury vs. 1.31 ± 0.11 male/ 1.07 ± 0.04 female after 1st sham preparation, 1.47 ± 0.14 male/ 1.30 ± 0.07 female after 2nd sham preparation, and 1.53 ± 0.1 male/ 1.31 ± 0.1 female after 3rd sham preparation, respectively, p < 0.05, one-way ANOVA; Fig. 4B). We then followed up at multiple time points after the 3rd injury and found this loss in paw withdrawal threshold sustained for at least two months for both left paw (0.61 ± 0.12 male/ 0.20 ± 0.12 female at 3 days, 0.84 ± 0.08 male/ 0.27 ± 0.04 female at 1 week, 1.14 ± 0.05 male/ 0.58 ± 0.15 female at 1 month, and 1.28 ± 0.12 male/ 1.08 ± 0.20 female at 2 month vs. 1.17 ± 0.10 male/ 1.24 ± 0.11 female 3 days sham, 1.37 ± 0.10 male/ 1.09 ± 0.06 female 1 week sham, 1.8 ± 0.12 male/ 1.5 ± 0.19 female 1 month sham, and 1.85 ± 0.14 male/ 1.96 ± 0.08 female 2 months sham, respectively, p < 0.05, one-way ANOVA; Fig. 4A) and right paw (0.60 ± 0.14 male/ 0.28 ± 0.1 female at 3 days, 0.78 ± 0.12 male/ 0.2 ± 0.13 female at 1 week, 0.85 ± 0.16 male/ 0.34 ± 0.1 female at 1 month, and 1.28 ± 0.14 male/ 1.09 ± 0.06 female at 2 month vs. 1.33 ± 0.13 male/ 1.25 ± 0.16 female 3 days sham, 1.31 ± 0.17 male/ 1.26 ± 0.04 female 1 week sham, 1.52 ± 0.14 male/ 1.48 ± 0.18 female 1 month sham, and 1.9 ± 0.05 male/ 1.95 ± 0.07 female 2 months sham, respectively, p < 0.05, one-way ANOVA; Fig. 4B). Interestingly, the heightened mechanical pain sensitivity was more prominent in female animals when compared to males (Fig. 4A, B), especially at 24 hours (0.62 ± 0.29 male vs. 0.20 ± 0.02 female left and 0.40 ± 0.04 male vs. 0.07 ± 0.02 female right), 3 days (0.61 ± 0.12 male vs. 0.20 ± 0.12 female left and 0.60 ± 0.14 male vs. 0.28 ± 0.1 female right), and 1 week (0.84 ± 0.08 male vs. 0.27 ± 0.04 female left and 0.78 ± 0.12 male vs. 0.2 ± 0.13 female right) after the 3rd injury.

NLRP3-specific inhibitor MCC-950 blocked IVIS inflammatory bioluminescence signal increases and attenuated long-term mechanical allodynia observed after 3x mTBIs

First, we directly tested the effect of MCC950, a potent NLRP3-specific inhibitor, on caspase-1-mediated inflammatory response. We treated both male and female ex vivo brain slices of caspase-1 reporter inflammasome mice with LPS to induce acute inflammatory response. As expected, we found significant increase in inflammasome bioluminescence signals, in both male and female mice brain slices, about 10 minutes after LPS treatment compared to prior treatment baseline level (6.60 ± 0.63 male/ 10.99 ± 1.23 female after LPS treatment vs. 0.86 ± 0.13 male/ 1.22 ± 0.17 female baseline, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Supplementary Fig. 4A, B). All treatment was done by adding LPS to the incubation chamber. We then treated these brain slices with MCC950 and found pronounce decrease in response signal levels 10 minutes after treatment in both male and females brain slices compared to LPS treated slices, with more prominent loss in male (2.59 ± 0.37 male/ 5.76 ± 0.5 female LPS + MCC950 treatment vs. 6.60 ± 0.63 male/ 10.99 ± 1.23 female after LPS treatment, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Supplementary Fig. 4A, B). These results indicate the robust inhibitory effects of MCC950 towards NLRP3 inflammasome-mediated caspase-1 activation and its subsequent inflammatory signals.

We next determined if preventing NLRP3 activation would influence mTBI-induced inflammatory responses. In these experiments, we treated mice with MCC950, a potent and NLRP3-specific inhibitor³⁴ intraperitoneally, at 1 hour and 24 hours after each injury (total 6 treatments). MCC950 is known to cross the blood-brain barrier, enabling us to assess its effect on the brain injury-induced peripheral and central caspase-1 mediated inflammatory responses. Using the IVIS imaging, we observed a significant reduction in inflammasome bioluminescence signals at 1 day and 3 days after the 3x injuries in the brain and paws of MCC950 treated compared to saline treated groups for male animals (31.56 ± 9.31 of 1 day and 26.03 ± 7.47 of 3 day with MCC950 vs. 60.48 ± 12.2 of 1 day and 176.5 ± 58.79 of 3 day with saline for male brains, 31.99 ± 5.94 of 3 day with MCC950 vs 147.1 ± 19.41 of 3 day with saline for male paws, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 5A, B, and D) and female animals (35.47 ± 3.57 of 1 day and 25.58 ± 1.89 of 3 day with MCC950 vs. 71.90 ± 16.93 of 1 day and 127.9 ± 11.35 of 3 day with saline for female brains, 9.39 ± 1.82 of 3 day with MCC950 vs. 77.15 ± 25.01 of 3 day with saline for female paws, all values reported as x 10⁷ p/cm²/sr, p < 0.05, repeated ANOVA; Fig. 5A, C, and E). There were also decreases in thoracic and abdominal inflammatory IVIS signals of both male and female MCC950-treated animals at 1- and 3-days post injury compared to vehicle saline treated groups (Supplementary Fig. 5A-D). Immunoblotting, of cortical brain tissue at 24 hours after the third injury, revealed a significant increase in caspase-1 protein level of the ipsilateral cortices after injuries (Fig. 6A, B). This enhancement in caspase-1 expression, however, attenuated remarkably in the group that received MCC950 treatment (Fig. 6A, B). Furthermore, we also assessed the effect of MCC950 on mTBIs-induced mechanical allodynia. With the same treatment paradigm, we injected 3x injured animals with either vehicle (saline) treatment or MCC950 treatment. Through Von Frey testing, we found a pronounced recovery of paws response threshold to tactile stimulus, bilaterally, at 24 hours after each injury in both left paw (1.64 ± 0.21 male/ 1.67 ± 0.11 female after first injury TBI + MCC950, 1.43 ± 0.15 male/ 1.56 ± 0.15 female after the second injury TBI + MCC950, 1.04 ± 0.45 male/ 1.16 ± 0.18 female after the third injury TBI + MCC950 vs. 1.26 ± 0.24 male/ 1.08 ± 0.05 female after first TBI + saline, 0.69 ± 0.17 male/ 0.71 ± 0.1 female after second TBI + saline, and 0.31 ± 0.21 male/ 0.29 ± 0.09 female after TBI + saline, respectively, p < 0.05, one-way ANOVA; Fig. 7A) and right paw (1.68 ± 0.12 male/ 1.40 ± 0.16 female after first injury TBI + MCC950, 1.32 ± 0.21 male/ 1.54 ± 0.10 female after second injury TBI + MCC950, 0.62 ± 0.24 male/ 0.90 ± 0.13 female after third injury TBI + MCC950 vs. 1.04 ± 0.05 male/ 1.27 ± 0.16 female after first TBI + saline, 1.06 ± 0.05 male/ 0.82 ± 0.14 female after second TBI + saline, and 0.28 ± 0.03 male/ 0.40 ± 0.08 female after TBI + saline, respectively, p < 0.05, one-way ANOVA; Fig. 7B). These reduction in pain sensitivity sustained up to one month in both left paw (0.47 ± 0.22 male/ 0.7 ± 0.21 female at 3 days TBI + MCC950, 0.7 ± 0.19 male/ 0.83 ± 0.14 female at 1 week TBI + MCC950, and 0.83 ± 0.2 male/ 1.09 ± 0.13 female at 1 month TBI + MCC950 vs. 0.09 ± 0.01 male/ 0.34 ± 0.13 female 3 days TBI + saline, 0.06 ± 0.02 male/ 0.03 ± 0.02 female 1 week TBI + saline, and 0.21 ± 0.11 male/ 0.36 ± 0.15 female 1 month TBI + saline, respectively, respectively, p < 0.05, one-way ANOVA; Fig. 7A) and right paw (0.88 ± 0.18 male/ 0.83 ± 0.18 female at 3 days TBI + MCC950, 0.75 ± 0.25 male/ 0.85 ± 0.16 female at 1 week TBI + MCC950, and 0.96 ± 0.05 male/ 0.83 ± 0.18 female at 1 month TBI + MCC950 vs. 0.02 ± 0.01 male/ 0.56 ± 0.21 female 3 days TBI + saline, 0.02 ± 0.01 male/ 0.05 ± 0.02 female 1 week TBI + saline, and 0.17 ± 0.088 male/ 0.37 ± 0.15 female 1 month TBI + saline, respectively, p < 0.05, one-way ANOVA; Fig. 7B). However, the pain response threshold of MCC-950 treated injured animals was still trending lower than non-injured sham group for the left paw (0.47 ± 0.22 male/ 0.1 ± 0.21 female at 3 days TBI + MCC950, 0.7 ± 0.19 male/ 0.83 ± 0.14 female at 1 week TBI + MCC950, and 0.83 ± 0.2 male/ 1.09 ± 0.13 female at 1 month TBI + MCC950 vs. 1.17 ± 0.10 male/ 1.24 ± 0.11 female 3 days sham, 1.37 ± 0.10 male/ 1.09 ± 0.06 female 1 week sham, 1.8 ± 0.12 male/ 1.5 ± 0.19 female 1 month sham, respectively) and right paw (0.88 ± 0.18 male/ 0.83 ± 0.18 female at 3 days TBI + MCC950, 0.75 ± 0.25 male/ 0.85 ± 0.16 female at 1 week TBI + MCC950, and 0.96 ± 0.05 male/ 0.83 ± 0.18 female at 1 month TBI + MCC950 vs. 1.33 ± 0.13 male/ 1.25 ± 0.16 female 3 days sham, 1.31 ± 0.17 male/ 1.26 ± 0.04 female 1 week sham, 1.52 ± 0.14 male/ 1.48 ± 0.18 female 1 month sham, respectively). Together, with the trend of recovery with MCC950 treatment, these data provide a therapeutic target and time window opportunity to approach brain injury-induced long-term pain.

This study used a closed head injury mouse model of mTBI which mimics properties of human cephalic traumatic injury. Despite its mild severity, changes in the acute inflammatory responses to repeated trauma play an important role in the development and progression of long term nociplastic pain. In this study, for the first time, we have demonstrated that repeated closed-head mTBI in mice caused significant increases in inflammasome-caspase-1 mediated inflammation, which is coincidental with the development and persistence of long-term nociplastic pain behavior. By blocking the neuroinflammatory signals as demarcated by caspase-1 activation, we were able to diminish both the injury-induced inflammatory responses and stimulus-dependent behavioral changes.

The use of caspase-1 biosensor mice to monitor inflammatory responses in vivo validates the presence of prolonged neuroinflammation following repetitive mTBI. This highly efficient technique provides a reproducible and effective approach to monitoring caspase-1 associated inflammation not only in vivo but also in ex vivo tissue preparations³⁵ and enabled insights into the effects of these inflammatory changes on long-term behavioral deficits. Using this approach, we found repeated 3x mTBIs resulted in a significant increase in caspase-1 mediated inflammation at 24 hours after each injury and persisted for more than 1 week after the last injury, both in the brain and at various areas of the body. Our ex vivo brain slice bioluminescence imaging experiment also confirmed a persistent increase in the brain-derived caspase-1 inflammatory response that lasted to at least three days after the 3rd injury impact. Additionally, cortical caspase-1 levels significantly increased after three injuries. These findings are consistent with previous animal studies that report pronounced and robust increases in caspase-1 activation within the CNS following head trauma³⁶. In keeping with the prediction that neuroinflammatory changes observed with repetitive mTBI, we also observed prolonged alterations in behavioral testing for up to two months after the injuries. The simultaneous monitoring of changes in the brain tissue with increased sensitivity to tactile stimulus provided a global view of NLRP3 inflammasome-mediated caspase-1 activation with the development and progression of nociplastic pain. Though it is unknown to what degree caspase-1 mediated signaling cascade and associated maturation of IL-1β contributes to chronic inflammation states following mTBI, it is entirely possible that other downstream signaling cascades may modulate the persistence of posttraumatic pain states. Indeed, IL-1β exerts its cellular effects by interacting with the cell surface (IL-1R) type I and type II receptors, with most effects mediated by IL-1RI which in turn can elicit activation of p38 MAP³⁷. Previous studies of chronic pain models have also demonstrated that upregulation of glial cell IL-1β production, as a result of NLRP3-Caspase-1 activation, affects several secondary pathways such as inducing Cox-2 activation in neurons³⁸ and promotion of phosphorylation of the NR1 subunit of NMDA receptors (NMDARs)^39,40.

Our behavioral results suggest a greater increase in behavioral responses in females compare to males. This observation may enhance severity of symptoms and longer-term pain catastrophizing in women following traumatic injury and musculoskeletal disorders ^41–46. The preponderance of our imaging and behavioral observations suggests an important role of NLRP3 inflammasome-mediated caspase-1 activation following repetitive mTBI in both the initiating and modulating long-term behavioral sequelae.

Observed signaling activities of the NLRP3 inflammasome mediated caspase-1 activation are indicative of a tightly regimented response to cranial trauma. We hypothesized that blocking the activity of this pathway close to the time of injury could have significant effects on inflammation and ultimately, long-term behavioral changes. Using MCC950, a potent and specific small molecule inhibitor of NLRP3 inflammasome,³⁴ we demonstrated that this small molecule inhibitor effectively diminished caspase-1 activation mediated inflammatory response in multi-body regions of the in vivo animal and in ex vivo brain slice preparations for prolonged periods of time. Similar anti-inflammatory effects in the context of nervous system disease⁴⁶, specifically neurotrauma^47,48 and rodent models of pain^{24–27, 49,50} support these observations. Furthermore, we observed significant increase in paw withdrawal thresholds in injured animals following MCC950 treatments. These observations strongly suggest a caspase-1/NLRP3 pathway as a therapeutic target of injury-induced long-term nociplastic pain behavior. More importantly, the diminished caspase-1 activation observed in our model system and the presumptive reduction of mature IL-1β release perhaps mimics some of the behavioral impacts observed following treatments with the receptor IL-1R1 neutralizing antibody⁵¹ and IL-1β⁵² neutralizing antibody. Suffice to say, the MCC950 treatment paradigm in this study suggests that modulating upstream signaling of the inflammasome-caspase-1 activation pathway is critical to preventing the development of injury-associated changes in nociplastic pain behavior. Taken together, the capase-1 biosensor activation allowed the severity of repetitive mTBI events to be readily monitored in this setting and will yield more precise spatial measurements of the inflammatory changes due to both injury and disease.

Mild head trauma is common among athletes engaged in contact sports, military personnel, and the aging population in the United States. While single mTBIs may not always result in behavioral impairments, repeated mTBIs, can introduce cumulative effects and long term deleterious conditions including cognitive impairment⁵³, psychiatric disease⁵⁴ and posttraumatic chronic pain^13,55. Though some of these conditions are likely associated with white matter tract injury or diffuse axonal injury as observed using neuroradiological techniques, there are often no detectable signs of neuropathology. Therefore, the ability to track, observe and quantify the dynamic progression of neuroinflammation overtime using a bioluminescent reporter mouse that detects caspase-1 activation in combination with a thinned skull cranial window and behavioral assessments, has great experimental advantages as it may provide a drug testing breakthrough for determining the mechanism and efficacy of non-opioid pain medications.

mTBI: mild Traumatic Brain Injury; DAMPs: Damage-Associated Molecular Patterns; NLR: NOD-like Receptors; NLRP3: Nucleotide-binding Oligomerization Domain (NOD)-like receptor 3; CCI: Control Cortical Impact; IOP: Intraocular Pressure; BLI: Bioluminescence Imaging; IVIS: In-Vivo Imaging System; LPS: Lipopolysaccharides; IBA1: Ionized Calcium Binding Adaptor Protein 1; GFAP: Glial Fibrillary Acidic Protein; DAPI: 4’,6-diamidino-2-phenylindole.

Acknowledgments

Data presented herein were obtained at In-Vivo Imaging Core Facility of the Radiology and Imaging Science Center, which is a Simon Cancer Center core research facility of Indiana University School of Medicine. The facility is funded through user fees and generous financial support of the Indiana University School of Medicine and the Indiana Clinical and Translational Sciences Institute (CTSI).

Authors’ contributions

TN and FW conceptualized the study and designed experiments. TN, NN, AC, MJ, SS carried out experiments. TN analyzed the data. TN wrote the manuscript, with guidance of JS, ST, EC, AGO, and FAW. All authors read and approved the final manuscript.

Fundings

This study is supported by funding from the Indiana Clinical and Translational Sciences Institute which is funded in part by UL1TR002529 and by Award Number TL1TR002531, National Institutes of Health (NIH R01 NS102415) and Congressionally directed Medical Research Program (W81XWH-18-1-0433).

Availability of data and materials

All data and materials are available from the corresponding author upon request.

Ethics approval and consent to participate

All animal work was performed in compliance with the Institutional Animal Care and use Committee (IACUC) at Indiana University School of Medicine (protocol number 20036).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

Author details

¹Department of Anesthesia, IU School of Medicine, Indianapolis, IN, USA. ²Stark Neurosciences Research Institute, IU School of Medicine, Indianapolis, IN, USA. ³Department of Anatomy, Cellular Biology, & Physiology, IU School of Medicine, Indianapolis, IN, USA. ⁴Department of Biomedical Engineering, IUPUI, Indianapolis, IN, USA. ⁵Department of Psychological Sciences, Purdue University, West Lafayette, IN, USA. ⁶Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA. ⁷Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA.

Smith-Seemiller, L., Fow, N. R., Kant, R. & Franzen, M. D. Presence of post-concussion syndrome symptoms in patients with chronic pain vs mild traumatic brain injury. Brain Inj 17, 199–206, doi:10.1080/0269905021000030823 (2003).
Naugle, K. M. et al. Physical activity behavior in the first month after mild traumatic brain injury is associated with physiological and psychological risk factors for chronic pain. Pain Rep 6, e969, doi:10.1097/PR9.0000000000000969 (2021).
Naugle, K. M. et al. The role of deficient pain modulatory systems in the development of persistent post-traumatic headaches following mild traumatic brain injury: an exploratory longitudinal study. J Headache Pain 21, 138, doi:10.1186/s10194-020-01207-1 (2020).
Carey, C., Saxe, J., White, F. A. & Naugle, K. M. An Exploratory Study of Endogenous Pain Modulatory Function in Patients Following Mild Traumatic Brain Injury. Pain Med 20, 2198–2207, doi:10.1093/pm/pnz058 (2019).
Nampiaparampil, D. E. Prevalence of chronic pain after traumatic brain injury: a systematic review. JAMA 300, 711–719, doi:10.1001/jama.300.6.711 (2008).
Lucas, S., Hoffman, J. M., Bell, K. R. & Dikmen, S. A prospective study of prevalence and characterization of headache following mild traumatic brain injury. Cephalalgia 34, 93–102, doi:10.1177/0333102413499645 (2014).
Hoffman, J. M., Lucas, S., Dikmen, S. & Temkin, N. Clinical Perspectives on Headache After Traumatic Brain Injury. PM R 12, 967–974, doi:10.1002/pmrj.12338 (2020).
Semple, B. D. et al. Interleukin-1 Receptor in Seizure Susceptibility after Traumatic Injury to the Pediatric Brain. J Neurosci 37, 7864–7877, doi:10.1523/JNEUROSCI.0982-17.2017 (2017).
DeKosky S.T., I. M. D., Gandy S.. Traumatic Brain Injury-–Football, Warfare, and Long-Term Effects. New England Journal of Medicine 363, doi:doi: 10.1056/NEJMp1007051. (2010).
Ojo J.O., M. B., Algamal M., Leary P., Lynch C., Abdullah L., Evans J., Mullan M., Bachmeier C., Stewart W., et al. Chronic Repetitive Mild Traumatic Brain Injury Results in Reduced Cerebral Blood Flow, Axonal Injury, Gliosis, and Increased T-Tau and Tau Oligomers. Journal of Neuropathological Epxerimental Neurology 75, 636–655 (2016).
Zieman G., B. A., Cárdenas J.F. Traumatic Brain Injury in Domestic Violence Victims: A Retrospective Study at the Barrow Neurological Institute. Journal of Neurotrauma 34, 876–880 (2017).
Diaz-Arrastia, R. et al. Pharmacotherapy of traumatic brain injury: state of the science and the road forward: report of the Department of Defense Neurotrauma Pharmacology Workgroup. J Neurotrauma 31, 135–158, doi:10.1089/neu.2013.3019 (2014).
Niu X., B. L., Sun Y., Wang Y., Bai G., Yin B., Wang S., Gan S., Jia X., Liu H. Mild traumatic brain injury is associated with effect of inflammation on structural changes of default mode network in those developing chronic pain. Journal of Headache Pain 21, 135 (2020).
Chaban V., C. G. J. B., Skandsen T., Islam R., Einarsen C.E., Vik A., Damås J.K., Mollnes T.E., Håberg A.K., Pischke S.E.. Systemic Inflammation Persists the First Year after Mild Traumatic Brain Injury: Results from the Prospective Trondheim Mild Traumatic Brain Injury Study. Journal of Neurotrauma 37, 2120–2130 (2020).
Liu, H. D. et al. Expression of the NLRP3 inflammasome in cerebral cortex after traumatic brain injury in a rat model. Neurochem Res 38, 2072–2083, doi:10.1007/s11064-013-1115-z (2013).
Ting, J. P., Willingham, S. B. & Bergstralh, D. T. NLRs at the intersection of cell death and immunity. Nat Rev Immunol 8, 372–379, doi:10.1038/nri2296 (2008).
O’Brien W., P. L. e. a. The NLRP3 inflammasome in traumatic brain injury: potential as a biomarker and therapeutic target. journal of Neuroinflammation 17 (2020).
Talley, S. et al. A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo. J Immunol 203, 2497–2507, doi:10.4049/jimmunol.1900619 (2019).
Talley, S. et al. DSS-induced inflammation in the colon drives a proinflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod. J Neuroinflammation 18, 263, doi:10.1186/s12974-021-02317-6 (2021).
Nguyen, T., Al-Juboori, M. H., Walerstein, J., Xiong, W. & Jin, X. Impaired Glutamate Receptor Function Underlies Early Activity Loss of Ipsilesional Motor Cortex after Closed-Head Mild Traumatic Brain Injury. J Neurotrauma 38, 2018–2029, doi:10.1089/neu.2020.7225 (2021).
Jennifer A. Creed, A. M. D., Douglas P. Fox, Alan R. Tessler, Ramesh Raghupathi. Concussive Brain Trauma in the Mouse Results in Acute Cognitive Deficits and Sustained Impairment of Axonal Function. Journal of Neurotrauma 28, 547–563 (2011).
Han X., C. Z., Ping X., Song L., Ma C., Ruan Y., Jin X. In vivo two-photon imaging reveals acute cerebral vascular spasm and microthrombosis after mild traumatic brain injury in mice. frontiers in Neuroscience (2020).
Nguyen T., A. J. M., Walerstein J., Xiong W., Jin X. Impaired glutamate receptor function underlies early activity loss of ipsilesional motor cortex following closed-head mild traumatic brain injury. Journal of Neurotrauma (2020).
Chen SP., Z. Y., et al. Pharmacological inhibition of the NLRP3 inflammasome as a potential target for cancer-induced bone pain.. Pharmacological Res. 147 (2019).
Zhang LG., C. J., et al. Effect of alcohol on chronic pelvic pain and prostatic inflammation in a mouse model of experimental autoimmune prostatitis. Prostate 79, 1439–1449 (2019).
Khan N, K. A., Brockman DA., Cooper MA., Smith MT. Pharmacological inhibition of the NLRP3 inflammasome as a potential target for multiple sclerosis induced central neuropathic pain. Inflammaopharmacology 26, 77–86 (2017).
He W., L. T., et al. Microglial NLRP3 inflammasome activation mediates IL-1β release and contributes to central sensitization in a recurrent nitroglycerin-induced migraine model. journal of Neuroinflammation 16, 78 (2019).
Imai Y., K. S. Intracellular signaling in M-CSF-induced microglia activation: role of Iba1. Glia 40, 164–174 (2002).
Liedtke W., E. W., Bieri PL., Chiu FC., Cowan NJ., Kucherlapati R., Raine CS. GFAP is necessary for integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron 17, 607–615 (1996).
Due M., P. J., Zhen L., Walls M., Allette Y., White F., Shi R. Acrolein involvement in sensory and behavioral hypersensitivity following spinal cord injury in the rat. Journal of Neurochemistry 128, 776–786 (2014).
Talley S, V. R., Anderson L, Cannon AR, Choudhry MA, Campbell EM. DSS-induced inflammation in the colon drives a proinflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod. journal of Neuroinflammation 18, 163 (2021).
Silasi G., X. D., Vanni M., Chen A, Murphy T. Intact skull chronic windows for mesoscopic wide-field imaging in awake mice. Journal of Neuroscience Methods 267, 141–149 (2016).
Haider M.N, L. J. J., Aronoff N. et al. Intracranial pressure changes after mild traumatic brain injury: a systematic review. Brain Injury 32, 809–815 (2018).
Coll, R. C. et al. MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat Chem Biol 15, 556–559, doi:10.1038/s41589-019-0277-7 (2019).
Talley S., K. O., Winek M., Paik W., Cannon A., Alonzo F., Choudhry M., Knight K., Campbell E. A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo. Journal of Immunology 203, 2497–2507 (2019).
Abdul-Muneer PM., L. M., Conte A., Santhakumar V., Pfister B. High Ca2 + Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death. Molecular Neurobiology 54, 3964–3975 (2017).
Asih PR., P. E., Stefanoska K., Tan ARP.. Ahel HI., Ittner A. unctions of p38 MAP Kinases in the Central Nervous System. Frontiers in Molecular Neuroscience 13, doi:10.3389/fnmol.2020.570586 (2020).
Samad TA, M. K., Sapirstein A, Billet S, Allchorne A, Poole S, Bonventre JV, Woolf CJ.. Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity. Nature 401, 471–475 (2001).
Guo W., W. H., Watanabe M., et al. Glial–Cytokine–Neuronal Interactions Underlying the Mechanisms of Persistent Pain. Journal of Neuroscience 27, 6006–6018 (2007).
Zhang R., L. A., Liu B. et al. IL-1ra alleviates inflammatory hyperalgesia through preventing phosphorylation of NMDA receptor NR-1 subunit in rats. Pain 135, 232–239 (2008).
Madsen TE, M. S., Zhai W, et al.. Gender Differences in Pain Experience and Treatment after Motor Vehicle Collisions: A Secondary Analysis of the CRASH Study. Clinical Therapeutics 40, 204–213 (2018).
Wolfe F, W. B., Perrot S, Rasker JJ, Hauser W. Fibromyalgia diagnosis and biased assessment: Sex, prevalence and bias. Plos One 13 (2018).
Elsamadicy AA, Y. S., Sergesketter AR, Ashraf B, Charalambous L, Kemeny H, Ejikeme T, Ren X, Pagadala P, Parente B, Xie J, Lad SP. Prevalence and Cost Analysis of Complex Regional Pain Syndrome (CRPS): A Role for Neuromodulation. Neuromodulation 21, 423–430 (2018).
Meucci RD, F. A., Faria NM. Prevalence of chronic low back pain: systematic review. Revista de saude publica 49 (2015).
Cimas M, A. A., Sanz B, Agullo-Tomas MS, Escobar A, Forjaz MJ. Chronic musculoskeletal pain in European older adults: Cross-national and gender differences. Eur J Pain 22, 333–345 (2018).
Wijnhoven HA, d. V. H., Picavet HS. Prevalence of musculoskeletal disorders is systematically higher in women than in men. The Clinical Journal of Pain 22, 717–724 (2006).
Jiao J, Z. G., Wang Y., Ren P., Wu M. MCC950, a Selective Inhibitor of NLRP3 Inflammasome, Reduces the Inflammatory Response and Improves Neurological Outcomes in Mice Model of Spinal Cord Injury. Frontiers in Molecular Biosciences (2020).
Ismael S., N. S., Ishrat T. MCC950, the Selective Inhibitor of Nucleotide Oligomerization Domain-Like Receptor Protein-3 Inflammasome, Protects Mice against Traumatic Brain Injury. Journal of Neurotrauma 35, 1294–1303 (2018).
Tang B., L. D., Chen L., Liu Y. NLRP3 inflammasome inhibitor MCC950 attenuates primary dysmenorrhea in mice via the NF-kB/Cox-2/PG pathway. Journal of Inflammation 17 (2020).
Dolunay A., S. S., et al. Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB, Caspase-1/11, ASC, NOX, and NOS Isoforms. Inflammation 40 (2017).
Schafers M, B. J., Neukirchen S, Maziniak M, Sommer C. Combined epineural therapy with neutralizing antibodies to tumor necrosis factor-alpha and interleukin-1 receptor has an additive effect in reducing neuropathic pain in mice. Neuroscience Letters 310, 113–116 (2001).
Kawasaki Y, X. Z., Wang X, Park JY, Zhuang ZY, Tan PH, Gao YJ, Roy K, Corfas G, Lo EH, Ji RR. Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nature Medicine 14, 331–336 (2008).
Guskiewicz K.M., M. S. W., Bailes J., McCrea M., Cantu R.C., Randolph C., Jordan B.D. Association between recurrent concussion and late-life cognitive impairment in retired professional football players. Neurosurgery 57, 719–726 (2005).
Lippa S.M., L. R. T., French L.M., Iverson G.L.. Performance Validity, Neurocognitive Disorder, and Post-concussion Symptom Reporting in Service Members with a History of Mild Traumatic Brain Injury. Arch Clin Neuropsychology 33, 606–618 (2018).
Khokhar B.R., L. M. A., Walker W.C. Post-mTBI Pain Interference in a U.S. Military Population: A Chronic Effects of Neurotrauma Consortium Study. Mil Med. 186, e293-e299 (2021).

No competing interests reported.

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Repeated Closed-Head Mild Traumatic Brain Injury Induces Robust Caspase-1 Mediated Inflammation that Contributes to Nociplastic Pain.

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