Pharmacological inhibition of MKP-1 exacerbates central post-stroke pain and neuroinflammation in mice

Thalamic hemorrhage induces persistent mechanical and thermal hypersensitivity in mice

To establish a CPSP model, we induced thalamic hemorrhage (TH) via stereotactic collagenase injection into the ventral posterolateral nucleus of C57BL/6J mice. Behavioral testing at multiple post-surgery time points revealed sensory abnormalities.

Mechanical sensitivity assessed with von Frey filaments showed increased withdrawal frequencies in TH mice beginning on day 1 post-injury and persisting throughout the 21-day observation period (Figure 1B and C). Sham-operated mice exhibited stable baseline responses across all time points.

Thermal nociception evaluated by paw withdrawal latency to radiant heat revealed significantly decreased latencies in TH mice compared to sham controls from day 1 through day 21 post-injury (Figure 1D and E). These data indicate that thalamic hemorrhage produces persistent mechanical allodynia and thermal hyperalgesia in mice, confirming successful CPSP model establishment.

Thalamic hemorrhage produces anxiety-like behaviors without affecting locomotor activity

Because CPSP patients frequently experience affective disturbances, we examined anxiety-related behaviors using multiple paradigms. In the open field test, total distance traveled did not differ significantly between groups (Figure 2B), indicating preserved motor function after TH. However, TH mice showed reduced distance traveled in the center area (Figure 2C) and decreased time in the center zone compared to sham controls (Figure 2D), consistent with increased anxiety-like behavior.

In the light-dark box test, TH mice entered the light compartment less frequently (Figure 2F) and spent significantly less time in the light area (Figure 2G) relative to sham mice. Similarly, elevated plus maze testing revealed fewer open arm entries (Figure 2I) and reduced open arm exploration time (Figure 2J) in TH mice. These collective findings demonstrate that thalamic hemorrhage induces significant anxiety-like behavioral alterations without affecting general locomotor activity.

MKP-1 expression increases in microglia following thalamic hemorrhage

To determine whether MKP-1 participates in CPSP pathogenesis, we examined thalamic MKP-1 expression after TH. Western blot analysis revealed significantly increased MKP-1 protein levels at 4 and 14 days post-TH compared to sham controls (Figure 3A and B). Immunofluorescence staining confirmed elevated MKP-1 expression in the peri-lesional thalamic region. Double-labeling demonstrated strong MKP-1 colocalization with the microglial marker Iba1 (Figure 3C), with quantitative analysis confirming that most MKP-1-positive cells were microglia (Figure 3D). These results indicate that MKP-1 upregulation after thalamic hemorrhage occurs predominantly in thalamic microglia.

MKP-1 inhibition exacerbates neurological deficits and sensory hypersensitivity

To investigate MKP-1 function in CPSP, TH mice received the MKP-1 inhibitor BCI215. Neurological deficit scores increased significantly in TH mice compared to sham controls during the first two post-injury weeks (Figure 4A). BCI215 administration further aggravated neurological deficits in TH mice, particularly at days 4, 7, and 14 post-injury.

Behavioral testing revealed that MKP-1 inhibition significantly worsened mechanical hypersensitivity. BCI-treated TH mice showed higher withdrawal frequencies to von Frey stimulation compared to vehicle-treated TH mice (Figure 4B and C). Thermal nociception testing demonstrated that BCI administration further decreased withdrawal latency following TH (Figure 4D and E), indicating enhanced thermal hyperalgesia. These findings suggest that MKP-1 inhibition exacerbates sensory hypersensitivity and neurological impairment after thalamic hemorrhage.

MKP-1 inhibition worsens anxiety-like behaviors after thalamic hemorrhage

We next assessed whether MKP-1 inhibition affects affective behavioral responses associated with CPSP. Open field testing revealed no significant effects of TH or BCI treatment on overall locomotor activity (Figure 5B). TH mice showed reduced center distance and decreased center time compared to sham controls (Figure 5C and D), and BCI administration further reduced these parameters in TH mice.

Light-dark box testing showed that TH mice entered the light compartment less frequently and spent less time in the light area compared to sham mice (Figure 5F and G). BCI treatment further enhanced these behavioral alterations. Elevated plus maze testing revealed fewer open arm entries and reduced open arm exploration time in TH mice relative to sham controls (Figure 5I and J), and MKP-1 inhibition significantly aggravated these anxiety-like behaviors. These results indicate that MKP-1 inhibition exacerbates affective behavioral disturbances following thalamic hemorrhage.

MKP-1 inhibition enhances neuronal damage following thalamic hemorrhage

To evaluate pathological changes induced by MKP-1 inhibition, we performed histological analyses of thalamic tissue. Compared to sham animals, TH mice exhibited neuronal damage and increased apoptotic cell death in the peri-lesional thalamic region. BCI215 treatment significantly aggravated these pathological alterations. Quantitative analysis revealed more apoptotic cells in the TH+BCI group compared to TH mice without BCI treatment (Figure 6C), suggesting that MKP-1 inhibition enhances neuronal injury following thalamic hemorrhage.

MKP-1 inhibition enhances inflammatory signaling through p38 mapk pathway in microglia

To explore molecular mechanisms underlying MKP-1-mediated effects, we examined inflammatory signaling pathways in BV2 microglial cells. Western blot analysis demonstrated that MKP-1 inhibition significantly increased p38 MAPK phosphorylation, accompanied by elevated expression of inflammatory mediators (Figure 7). These findings suggest that MKP-1 negatively regulates inflammatory signaling in microglia at least partially through modulation of the p38 MAPK pathway.

Transcriptomic analysis identifies mkp-1-associated inflammatory and synaptic pathways after thalamic hemorrhage

To identify molecular pathways potentially influenced by MKP-1-mediated regulation, we performed transcriptomic analysis of thalamic tissues following TH. Among differentially expressed genes (Figure 8A and B), we observed significant enrichment in inflammatory signaling pathways (consistent with our MKP-1 findings) and, unexpectedly, in synaptic transmission-related pathways (Figure 8C). Notably, several inflammation-associated genes (including Il1b, Tnf, Ccl2) showed reciprocal expression patterns to MKP-1, while synaptic genes (Grin2a, Gria2) exhibited alterations potentially relevant to pain circuit remodeling (Figure 8D). These transcriptional changes provide candidate pathways for future investigation of MKP-1's broader effects on thalamic circuitry following stroke.

Data limitations and perspectives

Several limitations warrant consideration. First, we acknowledge that our conclusions regarding MKP-1 function are derived primarily from loss-of-pharmacological-function experiments using BCI215, which has reported activity against both MKP-1 and MKP-3. ³⁹ While our in vitro data in BV2 microglia and the consistent exacerbation of phenotypes upon BCI215 treatment support a protective role for the MKP-1/MKP-3 axis, we recognize that this pharmacological evidence does not substitute for genetic validation (e.g., microglia-specific MKP-1 conditional knockout) or gain-of-function experiments (e.g., MKP-1 overexpression). The present study should therefore be viewed as providing foundational pharmacological evidence that identifies this phosphatase axis as a candidate target for CPSP, rather than as definitive proof of MKP-1’s cell-autonomous causal role. Future studies incorporating MKP-1 overexpression and/or targeted gene deletion will be necessary to establish causality. Second, the transcriptomic changes we observed identify candidate pathways for future investigation, but functional validation of specific genes (including MerTK and synaptic signaling molecules) requires additional study. Third, we examined only male mice; given evidence for sex differences in neuroinflammatory responses and pain processing, future studies should include both sexes.

Despite these limitations, our findings identify MKP-1 as an endogenous regulator restraining neuroinflammation after thalamic injury. Given the limited therapeutic options currently available for CPSP, understanding endogenous anti-inflammatory mechanisms may provide new intervention strategies. Our results suggest that modulating MKP-1 signaling could represent a potential approach for alleviating post-stroke neuropathic pain.

Animals

Adult male C57BL/6J mice (8–10 weeks old, 22–25 g) were obtained from an accredited laboratory animal center. Animals were housed under controlled environmental conditions (12 h light/dark cycle, temperature 22 ± 2 °C, relative humidity 50–60%) with food and water available ad libitum. All experimental procedures were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the Animal Ethics Committee of the affiliated institution.

Thalamic hemorrhage model

Central post-stroke pain was induced by stereotactic injection to generate a thalamic hemorrhage model as previously described with minor modifications. ⁸ Briefly, mice were anesthetized with isoflurane and placed in a stereotactic frame. A small burr hole was drilled in the skull, and 0.075 U collagenase type VII (Sigma-Aldrich) in 0.5 μL sterile saline was injected into the ventral posterolateral nucleus of the thalamus (coordinates: 1.8 mm posterior to bregma, 1.5 mm lateral to midline, 3.5 mm depth from dura) using a microsyringe at 0.1 μL/min. After injection, the needle remained in place for 5 min to prevent reflux before slow withdrawal. Sham animals underwent identical surgical procedures without collagenase injections. Animals recovered on a heating pad before returning to home cages.

Drug administration

BCI215 (MedChemExpress), an inhibitor of MKP-1 phosphatase activity, was dissolved in vehicle solution (10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered intraperitoneally at 5 mg/kg every 48 h beginning immediately after surgery. Control animals received equivalent volume of vehicle solution.

Mechanical sensitivity test

Mechanical sensitivity was assessed using von Frey filaments (Aesthesio Precision Tactile Sensory Evaluator, DanMic Global). Mice were placed in individual testing chambers on a perforated metal floor and allowed to acclimate for 15 min. Filaments with forces of 0.07 g and 0.4 g were applied to the plantar surface of the contralateral hind paw (ipsilateral to the thalamic hemorrhage) for 10 consecutive trials per filament. Paw withdrawal frequency was calculated as (number of withdrawals/10) × 100%.

Neurological deficit scoring

Neurological deficits were evaluated using the Bederson score with minor modifications. ⁴⁰ Briefly, the following criteria were used: 0 = no observable deficit; 1 = forelimb flexion (mild); 2 = decreased resistance to lateral push (modest); 3 = circling to the contralateral side of the stroke hemisphere (severe); and 4 = no spontaneous walking with depressed level of consciousness (most severe). Assessments were performed by an investigator blinded to the experimental groups at 1, 4, 7, 14, and 21 days post-surgery.

Thermal nociception test

Thermal nociception was evaluated using a Hargreaves apparatus (IITC Life Science). Mice were placed on a transparent glass platform and allowed to acclimate for 30 min. A radiant heat stimulus was applied to the plantar surface of the hind paw, and withdrawal latency was recorded. Cutoff time of 20 s prevented tissue injury.

Open field test

Open field test assessed locomotor activity and anxiety-like behavior. Mice were placed in the center of an open field arena (40 × 40 × 40 cm) and allowed to explore freely for 30 min. Animal movement was recorded using an automated video tracking system (EthoVision XT, Noldus). Total distance traveled, distance in the center zone (20 × 20 cm), and time spent in the center area were quantified.

Light-dark box test

Anxiety-like behavior was further evaluated using the light-dark box test. The apparatus consisted of two compartments: a brightly illuminated chamber (30 × 20 × 25 cm, 400 lux) and a dark enclosed chamber (30 × 20 × 25 cm, <5 lux) connected by a small opening. Mice were initially placed in the dark compartment and allowed to freely explore both chambers for 10 min. Number of entries into the light compartment and time spent in the light area were recorded.

Elevated plus maze test

Elevated plus maze test assessed anxiety-like behavior. The maze consisted of two open arms (30 × 5 cm) and two closed arms (30 × 5 × 15 cm) elevated 50 cm above the floor. Mice were placed at the center facing an open arm and allowed to explore freely for 10 min. Number of entries into open arms and time spent in open arms were recorded using an automated tracking system.

Immunofluorescence staining

Mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were collected, post-fixed overnight at 4 °C, and cryoprotected in 30% sucrose. Coronal brain sections (30 μm) were prepared using a cryostat. Sections were blocked with 5% normal donkey serum in PBS containing 0.3% Triton X-100 for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies: rabbit anti-MKP-1 (1:200, Santa Cruz Biotechnology, sc-1102) and goat anti-Iba1 (1:500, Abcam, ab5076). After washing, sections were incubated with appropriate fluorescent secondary antibodies (Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 donkey anti-goat; 1:500, Invitrogen) for 2 h at room temperature. Images were captured using a confocal microscope (Zeiss LSM 880). Quantitative analysis was performed using ImageJ software.

Western blot analysis

Thalamic tissues were homogenized in RIPA lysis buffer (Beyotime) containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined using BCA assay (Thermo Scientific). Equal protein amounts (30 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies: rabbit anti-MKP-1 (1:500, Santa Cruz Biotechnology), rabbit anti-phospho-p38 (1:1000, Cell Signaling Technology, #4511), rabbit anti-total p38 (1:1000, Cell Signaling Technology, #8690), and mouse anti-β-actin (1:5000, Proteintech, 66009-1-Ig). After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Millipore) and quantified using ImageJ software.

Cell culture and in vitro experiments

BV2 microglial cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin at 37 °C with 5% CO₂. Cells were treated with BCI215 (10 μM) or vehicle for 24 h. After treatment, cells were harvested for protein analysis by western blot.

Transcriptomic analysis

Total RNA was extracted from thalamic tissues using TRIzol reagent (Invitrogen). RNA quality was assessed by Bioanalyzer 2100 (Agilent). RNA sequencing was performed on an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) were identified using DESeq2 with criteria |fold change| > 1.5 and adjusted p < 0.05. Functional enrichment analyses, including Gene Ontology (GO) and KEGG pathway analysis, were performed using clusterProfiler.

Statistical analysis

All data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9.0. Comparisons between two groups were performed using two-tailed Student's t-test. Multiple group comparisons were analyzed using one-way or two-way ANOVA followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.