Male Sprague–Dawley rats (weighing 200–250 g) were purchased from the Experimental Animal Center of Guangdong Province (production license number: SCXK [Yue] 20180002), and housed in cages under conditions of a temperature of 23±2°C, 55±5% relative humidity, regular 12-h light/dark cycle, and ad libitum availability of food and water. All experimental protocols designed for the diabetes pain model were approved by the Animal Care and Ethics Committee of Sun Yat-Sen University. The experiments were performed following the guidelines of the National Institutes of Health Guidelines for the Care and Use of Experimental Animals. All procedures were designed to minimize animal suffering.
Preparation of diabetic rats
DM was induced among rats as previously described (Tan et al., 2019). Briefly, a single dose of 65 mg/kg streptozotocin (STZ; Sigma, St. Louis, MO, United States) diluted in sodium citrate buffer (0.1 M, pH 4.5) was intraperitoneally administered to rats that had been subjected to an overnight fast. Age-matched rats in the nondiabetic group received the corresponding volume of 0.01 M sodium citrate buffer (pH 4.5). Three days after STZ injection, the blood glucose level was measured from the tail tip using an electronic glucometer (Roche, Fermoy, Ireland), and a diabetic condition was regarded as a blood glucose level ≥16.7 mmol/L. In both DM and nondiabetic rats, body weight and food and water intake were recorded per week.
One week after STZ injection, the DM rats received no treatment (DM-Non group, n = 10), Fer-1 (2 μmol/kg, dissolved in 1% DMSO; Sigma–Aldrich, St Louis, MO, USA; DM-Fer group, n = 10), or a vehicle (1% DMSO; DM-Veh group, n = 10). The Fer-1 or vehicle was injected intraperitoneally once daily for two consecutive weeks (Fig. 1). Rats in the nondiabetic group (Con group, n = 10) received no drugs.
After STZ injection, the behavioral test for paw mechanical withdrawal thresholds (PMWT) was performed weekly among diabetic rats and age-matched nondiabetic controls. The tests were performed between 9:00 and 12:00 am, and on the first day of every week. Before the test, each rat was placed individually on a wire mesh grid and allowed to adapt to the surrounding environment for 30 min. Data from behavioral tests were collected by an independent staff member who was blinded to the group assignment details of the animals. The procedures in the PMWT assessment were carried out as described previously (Guo et al., 2021). In brief, a series of von Frey hair filaments (North Coast Medical, San Jose, CA, USA), with target forces ranging from 1.0 to 15 g, were used to stimulate the plantar surfaces of the rats’ hind-paws in an ascending manner. Each von Frey filament was used five times at 4–6 s intervals. Withdrawal, along with shaking or licking of the paw at least three times over the course of five applications was considered as a positive response. The smallest bending force of the filament eliciting a positive response was expressed as the PMWT.
The iron content was estimated as described in our previous study (Guo et al., 2021). In brief, spinal cord samples were chopped into small masses (0.1–0.2 g) and dried at 60°C for 12 h. Following digestion with 1 ml nitric acid (60%) at 100°C in a water bath for 2 h, the completely dissolved residues were diluted to 10 ml with double distilled water. Atomic iron levels were calculated in terms of micrograms per gram wet weight of tissue, which was assessed by comparing the absorbance assessed via flame atomic absorption spectrophotometry (AA-6800; Shimadzu Corporation, Kyoto, Japan) at 248 nm.
Lipid peroxidation assays
ROS activity was determined based on its reaction with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Chung et al., 2015). In brief, the enlarged lumbar (L4–6) regions of the spinal cords were minced and homogenized in 200 μl phosphate-buffered saline (PBS) at 37°C for 30 min, after which H2DCFDA (Invitrogen, CA, USA) was added to the homogenate. After incubation for 30 min at 37°C, the fluorescence of the reaction medium containing H2DCFDA was assessed and the values of fold changes in each group compared to those in the Con group were calculated.
Also, commercially available kits were applied to assess the concentrations of glutathione peroxidase (GSH-PX, A005-1; Jiancheng Biology, Jiangsu, China), malondialdehyde (MDA, A003-1; Jiancheng Biology), and superoxide dismutase (SOD, A001-1; Jiancheng Biology), according to the manufacturer’s protocols.
Western blot analysis
We performed western blot analyses in line with previously reported protocols (Cheng et al., 2019) with the following primary rabbit antibodies against ACSL4 (1:2000, ab155282; Abcam, Cambridge, United Kingdom), GPX4 (1:2000, ab125066; Abcam), transferrin receptor (TFR; 1:1000, NB200-585; Novus, CA, USA), ferroportin 1 (FPN1; 1:1000, NBP1-21502; Novus), and β-actin (1:2000; 4970; Cell Signaling Technology, NY, USA). Images were obtained using the Tanon 5500 chemiluminescent imaging system (Tanon, Shanghai, China) and analyzed using Image J version 1.52 (National Institutes of Health, Bethesda, Maryland, USA). The optical densities of bands of interest were measured and normalized to those of the β-actin band.
The enlarged lumbar regions of the rat spinal cord samples were sliced into 10 μm transverse sections using a freezing microtome (CM1900; Leica, Munich, Germany). After dewaxing, dehydration, antigen retrieval, and blocking, the spinal cord sections were incubated overnight in a humid chamber at 4℃ with rabbit anti-ACSL4 (1:200, ab155282; Abcam) conjugated with one of the following antibodies: mouse anti-Neuronal nuclei (NeuN, a neuronal marker; 1:400, ab104224; Abcam), mouse anti-glial fibrillary acidic protein (GFAP, an astrocytic marker; 1:300, 3670; Cell Signaling Technology, MA., USA), or mouse anti-ionized calcium-binding adaptor molecule 1 (Iba1, a microglial marker; 1:200, ab15690; Abcam). Then, the sections were rinsed with PBS thrice, followed by incubation with goat anti-mouse (Alexa 594; 1:500, ab150120; Abcam) and goat anti-rabbit (Alexa 488; 1:500, ab150081; Abcam) antibodies and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (2 μg/ml, KGA215-10; KeyGen Biotech Co. Ltd., China). Double-stained sections were observed via fluorescence microscopy (EVOS FL Auto; Thermo Fisher Scientific, MA, USA).
Transmission electron microscopy
A Hitachi HT-7700 transmission electron microscope (Hitachi, Tokyo, Japan) was used to visualize and capture the ultrastructure of mitochondria as described in a previous paper (Guo et al., 2021). Briefly, after fixation with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate at 4°C overnight, the tissues of enlarged spinal lumbar regions were cut into 1 mm3 clumps. Next, the tissue clump cuttings were dehydrated in a graded acetone series, following by embedding in Eponate 812 medium (90529-77-4; Structure Probe, Inc., PA, USA). In every fifth section for each group, we counted the total number of mitochondria, calculated the proportion of mitochondria with abnormal morphologic features, and assessed the mitochondrial planar areas.
Quantitative data, including the results of PMWT, iron content assay, lipid peroxidation assay, Western blot assay, and planar areas of mitochondria, are expressed as the mean ± standard deviation. Two-way analysis of variance with repeated measures was performed to assess intergroup variations followed by Bonferroni post hoc comparisons. All statistical analyses were performed using SPSS version 20.0 (IBM Corp., NY, USA). Analysis items with P values <0.05 were considered statistically significant.