Pharmacological Treatments for Chronic Pain in C57BL/6J Mice: A CFA Model Analysis

Abstract

This study evaluates the efficacy of LDN-212320, Dihydrokianic acid (DHK), and gabapentin in mitigating chronic inflammatory pain induced by Complete Freund's Adjuvant (CFA) in male C57BL/6J mice. Through behavioral tests for allodynia and hyperalgesia, along with biochemical analyses of inflammatory markers, the research aims to contribute to the development of more effective pain management strategies.

Introduction

Chronic inflammatory pain represents a significant challenge in clinical medicine, necessitating ongoing research into novel therapeutic interventions. The study leverages a CFA-induced pain model to simulate chronic inflammatory conditions, assessing the impact of various pharmacological agents on pain perception and biochemical markers of inflammation.

Materials and Methods

Male C57BL/6J mice (weighing 20-30 gm; 7-9 weeks old) were purchased from Jackson laboratories (Bar Harbor, ME, USA). Animals were allowed to acclimatize in quite animal facility for 7 days prior to all behavioral and biochemical experiments. Mice were housed in standard cages (29 × 18 × 12 cm) with free access to food and water, under standard laboratory conditions (22 ± 2 °C, relative humidity 60 %) and maintained on a regular 12 hr light/dark cycle (lights on at 0700 h).

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All of the behavioral and biochemical experiments were conducted between regular light cycle (09:00–17:00 h). On the day of behavioral experiments, mice were allowed to habituate to the testing room for at least 30 minutes before starting the experiments. All procedures used in this study are in compliance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at South Dakota State University.

Good Laboratory Practice and ARRIVE guidelines were followed. All efforts were made to ensure minimal animal suffering.

Drug and Chemicals

LDN-212320 was purchased from Axon Medchem (VA, USA), and Dihydrokianic acid (DHK) and gabapentin were purchased from MCE (MedChem Express, NJ, USA). CFA was purchased from Sigma-Aldrich (St. Louis, MO, USA). LDN-212320 was dissolved in normal saline (0.9% NaCl) having 1% dimethyl sulphoxide (DMSO) and 0.5% tween 80 (vehicle). DHK and gabapentin were dissolved in normal saline (0.9% NaCl). All control animals received an equal volume of vehicle (1% DMSO and 0.5% tween 80 in normal saline (0.9% NaCl) either (intraperitoneal injection as in CFA group) or (intraplantar injection (i.pl) as in control group). All drugs and chemicals were injected intraperitoneally in a volume of 10 ml/kg body weight unless otherwise indicated. The doses used in this study were selected based on previous studies (Rasmussen et al., 2011; Tallarida et al., 2013).

Experiment Timeline

CFA-Induced Allodynia and Hyperalgesia

The CFA-induced allodynia and hyperalgesia, a model for chronic inflammatory pain was used as described previously (Maciel et al., 2013; Wu et al., 2005) with minor modifications. Briefly, mice left hind paw was disinfected with 75% alcohol and intraplantarly injected with Complete Freund’s Adjuvant (1 mg/ml, 20 μl). Control animals were intraplantarly injected with same volume of vehicle into the left hind paw. All biochemical experiments were conducted 7 days after control (vehicle) or CFA intraplantar injection, when the symptoms of persistent inflammatory pain were evident. The raw data from CFA-induced allodynia and hyperalgesia were converted to area under the curve (AUC). The AUC depicting total paw withdrawal threshold versus time was computed by trapezoidal calculation. The doses of LDN-212320, gabapentin and DHK were used as described previously (Rasmussen et al., 2011; Tallarida et al., 2013).

Tactile Allodynia

Tactile allodynia was performed as described previously (Abbas and Rahman, 2016) with minor modifications. Briefly, on day 1, 3, and 7 post CFA intraplantar injection mice were placed individually in a plastic cage (45 × 5 × 11 cm) with a wire mesh bottom which allowed full access to the paws and behavioral acclimatization was allowed for 30 minutes until cage exploration and major grooming activities finished. 50% paw withdrawal threshold (50% PWT) against mechanical stimulation by von Frey filament (Touch-Test TM Sensory Evaluator, North coast Medical, Inc.) to the plantar surface of each hind paw was measured using the up-down paradigm (Chaplan et al., 1994).

Based on preliminary studies that characterized the threshold stimulus in control animals, the innocuous 0.4 mN (#2.44) filament, representing 50% of the threshold force, was used to detect tactile allodynia. The filament was applied to the point of bending six times each to the dorsal surfaces of the left and right hind paws. Positive responses included prolonged hind paw withdrawal followed by licking, biting or scratching were recorded. Mice were tested 3 days before i.pl. injection of CFA or vehicle to determine baseline thresholds, and then tested at 3 hr, 1 day, 3 days and 7 days after i.pl. injection of vehicle or CFA.

Thermal Hyperalgesia

Thermal hyperalgesia was performed as described previously with minor modifications (Abbas and Rahman, 2016; Liao et al., 2017). Briefly, animals were acclimatized by handling twice a day for 3 days prior to experiment to eliminate effects of stress. Thermal hyperalgesia was measured by withdrawal latency period from a hot plate using a plantar analgesia apparatus (IITC Life Science Inc., Woodland Hills, CA). To measure latency time, each mouse was individually placed on hot plate maintained at 51 ± 0.5 °C in Plexiglas chamber. Animals’ licking, flicking or jumping were recorded as positive response. Latency time for each mouse was calculated as a mean of three measurements with interval of 3 minutes between measurements. A maximal of 20 seconds was selected as a cut-off time to prevent tissue damage. Mice were tested 3 days before i.pl. injection of CFA or vehicle to determine baseline thresholds, and then at 3 hr, 1 day, 3 days and 7 days after i.pl. injection of vehicle or CFA.

Western Blot Analysis

Mice were euthanized through rapid decapitation; their anterior cingulate cortex (bregma 1.18 mm) and hippocampi (bregma -1.7 mm) were dissected from 1-mm coronal sections using mouse brain stereotaxic coordinates (Franklin and Paxinos, 2008) and stored at −80 °C until analysis. Western blot analysis was performed as described previously (Xu et al., 2006) with minor modifications. Brain tissue was homogenized in modified RIPA buffer containing Dulbecco’s phosphate-buffered saline (PBS) (pH 7.4), 1 % Igepal CA-630, 0.1 % sodium dodecyl sulfate (SDS), protease inhibitor mix (cOmplete, Mini, Roche, Indianapolis, IN, USA) and phosphatase inhibitor (Catalog #: A32957, ThermoFisher Scientific, MA, USA). The samples were centrifuged (16,000×g, 20 min at 4 °C) and supernatants were collected and stored at −80 °C.

Protein concentration was determined by bicinchoninic acid assay (BCA) (Pierce, Rockford, IL, USA) using albumin as standard. Equal amounts of protein (60 μg) were loaded onto 10 % or 12.5% gels for SDS polyacrylamide gel electrophoresis. Separated proteins were transferred onto nitrocellulose membranes or Polyvinylidene difluoride (PVDF) membrane at 60 V overnight at 4 °C.

Membranes were blocked with 5 % nonfat dry milk in Tris-buffered saline and 0.1 % Tween-20 (TBST) for 1 hr. Then, subsequently incubated overnight at 4 °C with primary antibodies for GLT-1 (1:1000, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, Texas, USA), CD11b (1:1000, rabbit polyclonal, Novus Biologicals, CO, USA), Iba1 (1:500, goat polyclonal, Santa Cruz Biotechnology, Dallas, Texas, USA) or β-actin (1:1000, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, Texas, USA). After incubation, membranes were washed in TBST, followed by incubation with appropriate horseradish peroxide-conjugated secondary antibodies, diluted in blocking buffer at a concentration of 1:5,000. Bound antibodies were detected with ECL Prime reagent (Amersham, Buckinghamshire, UK), and protein quantification was performed using densitometric analysis.

IL-1 estimation by ELISA

Immunoreactive IL-1β protein levels were determined using mouse-specific ELISA kit (Invitrogen). Mice were euthanized through rapid decapitation; their hippocampi and anterior cingulate cortex were harvested and stored at −80 °C until analysis. Tissue samples were placed in sterile PBS containing a protease inhibitor cocktail (cOmplete, Mini, Roche, Indianapolis, IN, USA), homogenized, centrifuged (11000 rpm, 20 min, 4°C), and the supernatant was assayed for IL-1β according to the protocol of the manufacturer. Protein concentrations of all samples were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA) prior to ELISA test, and equivalent amounts of proteins were used for the analyses. Cytokine levels were expressed as pg/mg of tissue.

Immunohistochemistry

Immunohistochemistry was carried as described previously (Alotaibi and Rahman, 2019) with minor modifications. Briefly, mice were euthanized through rapid decapitation; their brains were removed and post-fixed in 4% paraformaldehyde fixative overnight at room temperature. Brains were cryoprotected by immersion in 30% sucrose in 0.1 M PBS at 4°C until brains sank at the bottom. Brain tissues were embedded with Tissue- Tek OCT (Sakura, Finetek, USA) and sectioned into 15-20 μm thick sections with Leica CM1850 cryostat (Leica, Germany). Sections were blocked with 5% goat serum in 0.3% Triton X-100 in 1x PBS for 1 hr at room temperature then incubated overnight at 4°C with CD11b ( 1:100, rabbit polyclonal, Novus Biologicals, CO, USA), anti-p38 (1:100, rabbit polyclonal, Cell signaling technology, MA, USA) or CX43 (1:200, rabbit polyclonal, PhosphoSolutions, CO, USA). After incubation, sections were washed with PBS and followed by an incubation with AF488 or AF647 fluorescence conjugated secondary antibodies (1:200; ab 150077 or ab169348, Abcam, UK) for 1hr at room temperature in dark place.

The slides were mounted with mounting medium containing 4′, 6′-diamidino-2-phenylindole (DAPI) for nuclear staining and anti-fade reagent (SouthernBiotech, Birmingham, AL, USA). The stained sections were then examined with Olympus AX70 Olympus microscope Epi-fluorescence attached with DP70 Digital Camera (Tokyo, Japan). Image J software was used to quantify target protein expressions using integrated density. The hippocampus and ACC coordinates were based on the stereotaxic plates of the atlas of Franklin and Paxinos (Franklin and Paxinos, 2008); anterior-posterior (AP) coordinates referred to bregma, lateral (ML) coordinates to the midsagittal suture line, and ventral (DV) coordinates to the surface of the skull: CA1 ( AP: -1.70 mm; ML: 1.17 mm and DV: 1.34 mm), DG (AP: -1.70 mm; ML: 0.70 mm and DV: 2.04 mm ), and ACC (AP: 0.14 mm; ML: 0.25 mm and DV: 1.00 mm).

Data Analysis and Interpretation

Data analysis employed two-way ANOVA and Tukey’s post-hoc test, facilitating a detailed comparison of treatment effects over time. The area under the curve (AUC) calculations offered a comprehensive overview of behavioral test outcomes.

Tables and Formulas

Table 1: Summary of Drug Efficacy on Behavioral Tests

Formula for AUC Calculation

The AUC, representing the total pain withdrawal threshold over time, was calculated using the trapezoidal rule:

AUC=21∑i=1n−1(xi+1−xi)⋅(yi+1+yi)

Where xi and yi represent the time points and pain thresholds, respectively.

Conclusion

The study underscores the potential of LDN-212320, DHK, and gabapentin as therapeutic agents for chronic inflammatory pain. Through a combination of behavioral and biochemical analyses, it contributes valuable insights into pain management strategies, highlighting the importance of targeted pharmacological interventions in mitigating chronic pain.

This comprehensive approach not only enhances our understanding of pain mechanisms but also opens avenues for the development of more effective and specific treatments for chronic inflammatory conditions.