Animals

From the National Research Center in Giza, Egypt, female Wistar albino rats weighing 180–220 g were obtained. One week before the experiment was conducted, the rats were acclimated to standard housing conditions at the Faculty of Pharmacy's Animal Facility in Cairo, Egypt. Moreover, water and a chow diet were provided ad libitum. The adaptation and the investigational periods were under controlled environmental circumstances (25 ± 2 °C; temperature, 60 ± 10%; humidity and 12/12 h light/dark cycle).

Induction of the FM-Like Model

RES (Sigma-Aldrich, Saint Louis, MO, USA) was prepared in 0.5% glacial acetic acid solution. RES was administered subcutaneously in a dose of 1 mg/kg/day in the neck region for three days.

Experimental Design

Thirty female rats were randomly assigned to three groups (n = 10/group) using a random number generator to eliminate allocation bias. The study utilized the G-power calculator version 3.1 (Düsseldorf, Germany) to validate the sample size. The parameters for the power analysis were: an effect size of 0.6, an alpha level of 0.05, and a power of 0.8. The first group was designated the control group and administered a 0.5% acetic acid solution as a vehicle. Group two was administered reserpine at a dose of 1 mg/kg subcutaneously to induce a model similar to FM. Group three was administered reserpine at a dosage of 1 mg/kg subcutaneously for three consecutive days, followed by a single intraperitoneal dose of SURM (Sigma-Aldrich, MO, USA) at a dosage of 100 mg/kg dissolved in sterile water for injection (Kharlamov et al. 2002; Naviaux et al. 2014). The study initially assessed spontaneous pain-related behaviors using the Rat Grimace Scale (RGS) before introducing any stimulus-evoked pain tests. Pain-like behaviors were then evaluated using Von Frey filament, Randall-Selitto test, cold allodynia, hot plate, and the tail immersion tests before and after SURM administration on days 3 and 7, respectively. On day 11, motor abnormalities were evaluated by open-field and rotarod tests. On day 12, depressive-like behavior was measured by a forced swimming test. These tests were conducted in order of least to most stressful, this order was chosen to prevent thermal stimulation from sensitizing animals to subsequent mechanical stimuli, which could lead to mechanical hyperalgesia (Gröne et al. 2012). The tests were conducted in consecutive order, with a 2-h break between each test. Following the conclusion of the behavioral assessment, rats were euthanized by decapitation under the influence of a mild anesthetic consisting of ketamine (50 mg/kg) and xylazine (10 mg/kg) (Khajuria et al. 2012). The initial subset (n = 4) was prepared for histopathological and immunohistochemical examinations by fixing brains and spinal cords in 10% buffered formol saline. For subsequent analysis of the biomarkers using western blot (n = 4) and ELISA (n = 6) techniques, the thalami of the second subset (n = 6) were dissected, flash-frozen in liquid nitrogen, and stored at − 80 °C. Throughout the samples'analysis, the samples'identities were unknown to all inspectors. A different researcher was responsible for coding and decoding the samples. Researchers were blinded to group assignments during behavioral assessments and data analysis to minimize observer bias. The experimental design was illustrated as shown in scheme 1.

Scheme 1

Experimental design representation of timeline for drug administration and behavioral tests

Behavioral TestsRat Grimace Scale

The Grimace scale is a facial expression grading system that has been used to quantify animals'spontaneous suffering. Rats'facial expressions changed significantly and permanently, according to three raters who were blind to the treatment the rats received. Each animal was kept in its own acrylic cage with woodchips for bedding, a 12-h light/dark cycle, a controlled temperature (23 ± 2 °C) and humidity (55 ± 10%), and unlimited access to water and normal laboratory chow. Every test was carried out between 8:00 and 20:00 h of daylight. Then the raters assigned ratings to four action units for every image: changes in the features of the ears and whiskers, nose/cheek flattening, and orbital tightening. In accordance with the rating methodology outlined by Sotocinal et al. (Sotocinal et al. 2011), each action unit was assigned a 3-point rating (0 being not present, 1 being moderately present, and 2 being plainly evident). For each of the four action units, the average ratings were calculated. Additionally, the rat Grimace scale (RGS) score for each image was determined by averaging the three raters. Lastly, each animal's RGS score was determined by averaging the RGS scores of each rat's ten photos(Tanei et al. 2020).

Von Frey Hair Test

Mechanical allodynia was evaluated by observing a notable decrease in withdrawal thresholds during the von Frey hair test. Rats were placed collectively on an elevated mesh platform with 1 cm2 holes within a transparent plastic enclosure, allowing them to acclimate to the testing environment for at least 15 min. Von Frey hairs (obtained from IITC Life Science, Inc.) with pre-calibrated bending forces (measured in grams) were used to deliver precise mechanical stimuli of varying strengths. The test began with the filament exerting the least force, targeting the plantar surface of the left hind paw from beneath the mesh. Each hair was applied to the paw to cause a slight bend and held for 2–5 s. An interval of 6 to 8 s was maintained between each of the five stimulations. A positive reaction was recorded when the paw was quickly withdrawn. The sensitivity threshold of the paw was defined as the lowest force needed to elicit a withdrawal reflex, determined by the filament that led to a rapid paw withdrawal in 3 out of 5 attempts. Any movement associated with walking was not considered a withdrawal response (Deuis et al. 2017).

Tail Immersion Test

The tail-immersion test simulates acute heat-induced pain, assessing spinal thermal sensitivity by immersing a portion of the experimental animal's tail in a hot water bath. Specifically, one-third of the tail was submerged in hot water at 55 °C. The time it took for the rat to retract its tail, with a cut-off time of 15 s, was closely observed and recorded as the tail withdrawal latency (Mokhemer et al. 2023).

Randall-Selitto Test

The Randall-Selitto test assesses the threshold response to mechanical pressure stimulation. It involves applying gradually increasing mechanical force using a Randall-Selitto algesimeter. Each rat was gently restrained during the test, with its left hind paw positioned under a pressure pad. The force applied to the paw, measured in grams, was incrementally raised until the rat withdrew its paw due to intolerance to the pressure. This point marks the Randall-Selitto mechanical threshold. The pressure was promptly released upon paw withdrawal, and the force required to elicit this response was recorded (Santos-Nogueira et al. 2012).

Hot Plate Test

The hot plate test is a methodology employed to evaluate the susceptibleness of rats to heat. Each rat was meticulously positioned on a heated surface for this experiment. Rats initially react to the distress of a thermal stimulus by lapping their feet when exposed to a heated surface. Subsequently, they exhibit pronounced evasive behaviors, such as leaping. The temperature of the heated plate apparatus was 55 ± 1 °C. The time required for the rat to demonstrate rear paw lapping or leaping to evade the distress caused by the heat was recorded as the hot plate withdrawal latency. A maximum time limit of 20 s was enforced to prevent animal tissue damage (Kamel et al. 2022).

Cold Allodynia Test

The cold allodynia test evaluates the sensitivity of rats to cold temperatures. In this test, each rat's hind paw is carefully dipped into a container filled with ice-cold water, kept at a temperature of 4 ± 1 °C. The time taken for the rat to withdraw its paw, known as cold allodynia paw withdrawal latency which was then measured. Each session involved testing only one hind paw at a time, with a maximum duration of 20 s for each immersion to safeguard the animal's well-being. For every rat, two separate measurements were taken for each hind paw, spaced five minutes apart. The cold allodynia paw withdrawal latency was then calculated as the average of these readings from both hind paws. An extended cold allodynia paw withdrawal latency was interpreted as an anti-allodynic effect, indicating reduced sensitivity to the cold, while a shorter paw withdrawal latency suggested more severe allodynia, indicating heightened sensitivity (Kamel et al. 2022).

Forced Swim Test

In the forced swim test, a rat is placed in a water-filled container where it initially attempts to escape but eventually adopts a state of immobility, indicating depressive-like behavior. The test duration is six minutes, consisting of a two-minute pretest and four-minute test periods. During the test, the rat was placed in the water-filled container with no means of escape, and the water temperature was maintained at 23 ± 2 °C. Immobility in the rodent is recognized when it assumes a bent forward position with no movement in its rear legs. Observers unaware of the drug treatment manually record the duration of immobility (Yankelevitch-Yahav et al. 2015; Kamel et al. 2024).

Open Field Test

The open field test is a widely used method for assessing locomotion behaviors. It uses an open square wooden box (80 × 80 × 40 cm) with red walls and a white floor, divided into 16 equal squares by black lines. During the test, a rat is placed in the center of the box's bottom surface, and its movements are tracked and recorded using ANY-Maze video tracking software (Stoelting Co, USA) for 6 min. The parameters evaluated include the time spent in different areas of the open field traveled distance, open field mean speed, open field time immobile, open field latency time, and the number of entries into the central zone. The illumination level is maintained at 150 lx throughout the task (Avila et al. 2010).

Rotarod Test

The rotarod test is utilized to evaluate motor coordination in rats. This involves a cylindrical rod measuring 3 cm in diameter and 120 cm in length, rotating consistently at 20 rpm. Rats must maintain their balance on the rotating rod instead of falling onto a platform beneath it. Prior to the main experimental procedures, each rat undergoes five trial runs. Only animals that successfully remain on the rod for 5 min are selected for the experiment. Following these trials, the probe test was conducted, during which the time it takes for the animal to fall off the rod (fall-off latency) was recorded, with a maximum time limit of 300 s (Avila et al. 2010; Ibrahim et al. 2024).

Histopathological Studies

Brain tissue sections were preserved for 72 h in 10% neutral buffered formalin. Afterward, samples were immersed in paraplast tissue embedding media after being purified in xylene and processed in ethanol of varying grades. Sagittal brain tissue sections, 5 μm in thickness, were subjected to rotatory microtome at the mid-thalamic regions. The sections were then adhered to glass transparencies. As a standard histological examination staining procedure, tissue sections were stained with hematoxylin and eosin (H & E). Subsequently, novice histologists conducted blinded examinations of the sections. They were then evaluated blindly by a skilled histopathologist. According to Culling (2013), all standard procedures for processing samples were performed (C.F. Culling 2013).

Immunohistochemical Analysis

Tissue sections, 4 μm thick and embedded in paraffin, were treated with 3% H2O2 for 20 min following the manufacturer's protocol. After washing, the cells were incubated overnight at 4 °C with the following antibodies: anti-CD163 (Cat # GTX35247, GeneTex, CA, USA); anti-CD86 (Cat # bs- 1035R, Bioss, USA); and anti-CASPASE- 1 antibody (14 F468) [NB100 - 56565—1:100 Novus bio Co.]. The tissues were then treated with diaminobenzidine (DAB) for 10 min after rinsing with PBS, followed by incubation with the secondary antibody HRP EnVision reagent (DAKO) for an additional 20 min. After washing with PBS, the tissues were coverslipped for microscopic examination, counterstained with hematoxylin, dehydrated, and clarified in xylene. The mean optical density of thalamic cellular immunohistochemical expression levels of NLRP3 and caspase- 1, as well as the mean count of CD163 +  +/CD86 +  + reactive microglial cells per field, were determined by scanning at least six representative non-overlapping fields per tissue section of each sample, following the methodology of Mohamed et al. (2023) (Mohamed et al. 2023).Tissue sections were analyzed using Leica application software with a full HD microscopic imaging system (Leica Microsystems GmbH, Germany) operated by an experienced histologist examiner.

Western Blot Analysis of P38 MAPK, Procaspase- 1 and Cleaved Caspase- 1

Following rinsing in PBS, the thalami were lysed utilizing the Ready Prep™ protein extraction reagent manufactured by Bio-Rad Inc. in California, USA. Protein concentration was determined using the Bradford assay. Proteins were separated on 10% SDS-PAGE gels using a Mini-PROTEAN system (Bio-Rad) at 120 V for 90 min. Proteins were transferred onto nitrocellulose membranes using a wet transfer apparatus (Bio-Rad) at 100 V for 1 h. Membranes were blocked in 5% non-fat milk in TBST buffer for 1 h at room temperature. Primary antibodies against Pro-caspase- 1 (Cat # ab179515, Abcam,USA), caspase- 1 (Cat # PA5 - 86,692) and p38 MAPK (Cat # PA1 - 30391), both of which were provided by (Thermo scientific, Rockford, Illinois, USA), were diluted 1:1000 in blocking buffer and incubated with the membranes overnight at 4 °C. After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:5000 dilution) for 1 h at room temperature. Membranes were washed three times with TBST buffer for 10 min each. Proteins were visualized using the ECL detection system (Thermo Fisher Scientific) and imaged using a ChemiDoc imaging system (Bio-Rad). Band intensities were quantified using Image Lab software (Bio-Rad) and normalized to β-actin protein. Protein marker was supplied by FastGene (NIPPON Genetics EUROPE GmbH) to highlight the molecular weight of the detected proteins.

Enzyme-Linked Immunosorbent Assay (ELISA)

Using rat-specific ELISA kits, the following neurotransmitters in the thalamus were determined; dopamine (My BioSource Cat # MBS7214676, San Diego, CA, USA), serotonin (5HT) (My BioSource, Cat # MBS9362408, San Diego, CA, USA), NLRP3 (Cat # MBS2033695, San Diego, CA, USA), NF-κB (Cat # MBS2505513, San Diego, CA, USA), BDNF (Cat # MBS824814, San Diego, CA, USA), IL- 1β (Cat # MBS825017, San Diego, CA, USA), IL- 10 (Cat # MBS2707969, San Diego, CA, USA) and TNF-α (Cat # MBS9362408, San Diego, CA, USA). Abeexa Elisa kits were utilized for assessing P2X4 (Cat # abx537443, Cambridge, UK) andNEK7 (Cat # abx535012, Cambridge, UK). In addition, Life Span BioSciences ELISA kits were used for quantification of P2X7 (Cat # LS-F16541, WA, USA) and gasdermin D (GSDMD) (Cat # LS-F39621, WA, USA). Furthermore, IL- 18 (Elabscience, Cat # E-EL-R0567, Wuhan, China) and IL- 6 (RnDSystems, Cat # R6000B, Minneapolis, MN, USA) were determined referring to the relevant kit's instructions. The results were presented as pg/mg protein for TNF-α, NF-κB, IL- 6, IL- 10, IL- 18, IL- 1β, and BDNF and as ng/mg protein for NLRP3, NEK7, GSDMD, P2X7, P2X4, DA, and 5HT. The total protein concentrations in tissue samples were measured by the BCA kit supplied by G-Bioscience (USA). To determine the level of the protein of interest, the study utilized the kit’s standard curve to calculate concentrations in pg/ml or ng/ml, which were then normalized by dividing by the tissue protein concentration (in mg/ml), yielding results expressed as pg/mg protein or ng/mg protein.