Animal

Adult male Wistar rats (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) weighing 250–285 g were subjected to a light/dark cycle (lights on at 8:00 AM). This study took place in a temperature-controlled environment maintained at 22 ± 1 °C and 70% humidity. Rats had free access to food and water and were housed in Plexiglas cages. For surgical and drug injection procedures, all rats were anesthetized using 2.5% isoflurane inhalation and a sterile formulation. Postoperative care included intraperitoneal administration of cefazolin (170 mg/kg) to prevent infection. All experimental protocols involving animals were approved by the National Sun Yat-sen University Institutional Committee for the Care and Use of Animals (Approval Nos. IACUC-10830 & 11,137) and complied with the guidelines set by the American Physiological Society. In addition, after completing the nociceptive behavior experiments, the rats were humanely euthanized, and their spinal cords were analyzed. Every effort was made to minimize both the number of animals used and their suffering. Only rats showing no signs of hematomas or paralysis were included in the study. This ensured that the observed biological and biochemical effects were not influenced by intrathecal catheterization or manipulation.

Intrathecal catheter implantation and induction of neuropathic pain

We used the catheter implantation technique described by Yaksh and Rudy [44] and previous work [45]. Briefly, a catheter (PE5 tubing: 9 cm; inner diameter: 0.008 inches; outer diameter: 0.014 in) was inserted into the intervertebral space at the lumbar extension, drawn out, and affixed to the cranial surface of the head. Post-surgery, rats were allowed a 5-day recovery period in their home cages. Rats exhibiting severe nerve damage or cerebrospinal fluid hematomas were excluded from the study. After catheterization, rats underwent immediate control or CCI surgery on the right sciatic nerve. All surgeries were performed under anesthesia with 2.5% isoflurane inhalation. CCI was performed on the right sciatic nerve (middle part of the thigh), as described by Bennett and Xie [46] and our previous study [47]. For CCI, a 5 mm segment of the sciatic nerve was dissected and placed into four loosely ligated colons (chronic colon 4–0). A skin incision (at intervals of 1 mm) was made around the sciatic nerve, and the skin incision in each layer was sutured.

Drug administration

LDHA inhibitors, namely FX11 (427,218, EMD Millipore, Temecula, CA, USA) and oxamate (A16532.06, Thermo Fisher Scientific, Heysham, UK), were administered intrathecally using a PE5 catheter. FX11 and oxamate were dissolved in a solution containing 30% (v/v) dimethylsulfoxide (DMSO) (D2650, SIGMA-ALDRICH, St. Louis, MO, USA) and artificial cerebrospinal fluid (aCSF), which includes 2.6 mM K+, 21.0 mM HCO3−, 151.1 mM Na+, 1.3 mM Ca2+, 3.5 mM glucose, 0.9 mM Mg2+, 2, 5 mM HPO42−, and 122.7 mM Cl−, respectively. DMSO was diluted with aCSF.

Experimental Grouping

To investigate the patterns of CCI-induced nociception, we observed several time points during the experiment, including 3, 7, 14, 21, and 28 days (d) post-CCI. For testing of the acute analgesic effects of LDHA inhibitors, rats received intrathecal doses of 0.05, 0.1, 0.25, and 1 µg of FX11 or 1, 5, 10, 50, and 100 µg of oxamate in 10 µl of 30% DMSO post-CCI. Each dosage group included at least six rats.

FX11, a small-molecule LDHA inhibitor, downregulates aerobic glycolysis and lactate production [48] and has shown therapeutic potential in cancer treatment [49,50,51]. Moreover, FX11 attenuates proinflammatory immune cell proliferation in rheumatoid arthritis management [52]. Oxamate, the earliest documented small-molecule LDHA inhibitor discovered in 1959 [53], can induce apoptosis during cancer treatment [54, 55] and reduce serum lactate concentrations while increasing insulin secretion in diabetic mice [56]. However, no recent clinical trials on FX11 or oxamate have been conducted [57, 58]. The maximum daily dose of FX11 is approximately 2 mg/kg in mice with cancer [50] and tuberculosis [59]. In addition, the maximum daily dose of oxamate is approximately 750 mg/kg in mice with cancer [60] and diabetes [56]. Based on the equilibrium dosing conversion, the dosage used in rats is approximately two times less than that in the mice [61]. The systemic-to-intrathecal administration conversion ratio is 100:1 [62]. Collectively, the suggested maximum daily intrathecal doses of FX11 and oxamate in rats are 10 and 3750 µg/kg, respectively. Converting this to hourly intrathecal administration for rats with a body weight of 360 g in the present study, the dosages of FX11 and oxamate are 0.15 and 56.25 µg/h/rat, respectively. In addition, the 1-h 50% effective dose (ED50) for intrathecal FX11 and oxamate in rats with neuropathy were 0.14 and 6.47 µg/rat, respectively. Based on the suggested maximum intrathecal dosage, 1-h ED50, and the dose–response analgesic curve, 0.1 µg/h of FX11 and 5 µg/h of oxamate were selected for intrathecal administration in rats with CCI-induced neuropathy using an osmotic pump. Consequently, the LDHA inhibitors FX11 and oxamate have competitive and therapeutic potential for neuropathic pain management.

For the preventive analgesic effects test, FX11 (0.1 µg/µl/h), oxamate (5 µg/µl/h), or the vehicle (1 µl/h of) were chosen to be continuously infused intrathecally after CCI surgery with an osmotic pump (2001 and 2002, DURECT Corporation, Cupertino, CA, USA) immediately after CCI surgery. Rats were randomly divided into several groups: (I) the control group (rats without CCI surgery except intrathecal vehicle infusion), (II) the 7d group (rats received CCI and intrathecal vehicle infusion for 7 days), (III) the 14d group (rats received CCI and intrathecal vehicle infusion for 14 days), (IV) the 7d + FX11 group (rats received CCI and intrathecal 0.1 µg/µl/h of FX11 infusion for 7 days), (V) the 14d + FX11 group (rats received CCI and 0.1 µg/µl/h of FX11 infusion for 14 days), (VI) the 7d + oxamate group (rats received CCI and 5 µg/µl/h of oxamate infusion for 7 days), and (VII) the 14d + oxamate group (rats received CCI and 5 μg/μl/h of oxamate for 14 days).

Thermal hyperalgesia assessment

The rats were placed in clear plastic cages on elevated glass platforms. We assessed thermal hyperalgesia using an IITC analgesiometer (IITC Inc., Woodland Hills, CA, USA), following the method described by Hargreaves et al. [63]. The medial plantar surface of the right hind paw of rats was exposed to a low-intensity (active intensity = 25) through the glass platform. The mean paw withdrawal latency (PWL; in seconds) was calculated by averaging three positive test responses. Behaviors such as licking or rapid paw withdrawal were considered indicative of nociception. The maximum duration for PWL was set at 30 s.

Mechanical allodynia assessment

The rats were placed in clear plastic cages with raised metal mesh floors to facilitate access to the hind paws. Mechanical allodynia was evaluated using von Frey filaments (Stoelting, Wood Dale, IL, USA), as described by Chaplan et al. [64]. The center of the right hind paw was subjected to a vertical array of von Frey filaments with logarithmically increasing stiffness (0.2–10 g), applied until the filament bent slightly. The average paw withdrawal threshold (PWT; in grams) was determined using the "up-down" method described by Chaplan and was calculated from three positive test thresholds. Quick paw withdrawal or licking was considered a sign of nociception. The maximum threshold for PWT was set at 10 g.

Spinal tissue preparation for western blotting

Rat ipsilateral lumbar spinal cords were collected from the control, 7d, 14d, 7d + FX11, 14d + FX11, 7d + oxamate, and 14d + oxamate groups through exsanguination under isoflurane anesthesia 7 or 14 d post-surgery. Immediately upon collection, the spinal samples were frozen and stored at -80 °C until analysis. The samples were homogenized in ice-cold isotonic homogenizer buffer (20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.32 M sucrose, 0.3 mM fresh phenylmethylsulfonyl fluoride, and 10 mg/mL fresh leupeptin), then centrifuged at 1,000 × g at 4 °C for 30 min to separate supernatant and pellet. The supernatant was mixed with 1% Triton X-100 (v/v), stirred for 30 min at 4 °C, and then centrifuged at 20,000 g (4 °C) for 60 min, yielding the cytosolic and solubilized membrane protein fraction. The pellet was resuspended in hypotonic homogenizer buffer (20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.3 mM fresh phenylmethylsulfonyl fluoride, and 10 mg/mL of fresh leupeptin) to extract nuclear protein fractions [65, 66]. Subsequently, lysates from the ipsilateral lumbar spinal cord were subjected to electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane, following established protocols [67]. The PVDF membrane was blocked with 5% nonfat dry milk in Tris-buffered saline for 2 h at room temperature and then incubated with primary antibodies overnight at 4 °C. We used mouse monoclonal anti-CD86 (Sc-28347, Santa Cruz, Dallas, Texas, USA), rabbit monoclonal anti-LDHA (MBP2-67,483, Novus Biologicals, LLC, Centennial, CO, USA), rabbit polyclonal anti-Malondialdehyde (MDA, ab6463, Abcam, Cambridge, UK), rabbit polyclonal anti-SOD1 (ab13498, Abcam), and rabbit monoclonal anti-pTBK1 (5483, Cell Signaling, Danvers, MA, USA). The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for one hour at 37˚C, and detected using enhanced chemiluminescence (Trident femto Western HRP substrate, GTX14698, GeneTex). Densitometry was utilized to analyze the density of bands relative to the background. HRP-conjugated mouse monoclonal anti-β-actin (12,262, Cell Signaling) and mouse monoclonal anti-PCNA (GB12010, Servicebio, Wuhan, China) served as internal controls. Images were captured using the ChemiDocTM Imaging System (Bio-Rad, Hercules, CA, USA), and relative densitometric quantification was performed using Image Lab 6.1 (Bio-Rad).

Immunofluorescence assay

The immunohistochemistry protocol and image quantification were carried out as described in our previous studies [42, 47, 67]. Briefly, 7 and 14 d post-CCI injury, rats were terminally anesthetized with isoflurane and sacrificed via transcardial perfusion with cold phosphate-buffered saline (PBS, pH 7.4) containing heparin (200 U/mL), followed by 4% paraformaldehyde in PBS. To minimize variations in immunohistochemical procedures, lumbar spinal tissues from various groups were embedded in the same OCT compound (Sakura Finetek, Torrance, CA, USA), sectioned into 20 µm-thick sections using cryostat Microm HM550 (Microm International GmbH, Waldorf, Germany), mounted serially on microscope slides, and processed for immunofluorescence studies. Sections were blocked with a buffer containing 0.2% BSA, 0.3% Triton X-100, 0.05% Tween 20, and 1X PBS for an hour, then incubated overnight at 4 °C with primary antibodies: mouse monoclonal anti-GFAP (MAB3402, Millipore, Burlington, MA, USA), goat polyclonal anti-Iba1 (Ab5076, Abcam), mouse monoclonal anti-CD86 antibody (Sc-28347, Santa Cruz), rabbit monoclonal anti-LDHA (MBP2-67,483, Novus Biologicals, LLC), rabbit polyclonal anti-Ang2 (ab125692, Abcam), rabbit polyclonal anti-CD31 (ab25490, Abcam), rabbit polyclonal anti-HIF-1α (GTX127309, GeneTex, Irvine, CA, USA), rabbit polyclonal anti-SOD1 (ab13498, Abcam), and rabbit monoclonal anti-pTBK1 (5483, Cell Signaling). After incubating in the blocking buffer, the sections were incubated with a mixture of Alexa Fluor 488-conjugated mouse monoclonal anti-NeuN (MAB377X, Millipore), Alexa Fluor 488-conjugated anti-mouse IgG antibody (715–545-151, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Alexa Fluor 488-conjugated anti-rabbit IgG antibody (711–546-152, Jackson ImmunoResearch Laboratories), Alexa Fluor 488-conjugated anti-goat IgG antibody (705–546-147, Jackson ImmunoResearch Laboratories), Cy™ 3-conjugated anti-mouse IgG antibody (715–165-151, Jackson ImmunoResearch Laboratories), Cy™ 3-conjugated anti-rabbit IgG antibody (711–166-152, Jackson ImmunoResearch Laboratories), DAPI (D21490, Invitrogen), or DRAQ5 (ab108410, Abcam) for 40 min at room temperature. Imaging was performed using a Leica DM-6000B fluorescence microscope (Leica, Wetzlar, Germany) equipped with a K5 CMOS camera and Leica Application Suite X 3.8.1.26810 software (Leica Microsystems BV, Rijswijk, The Netherlands). The same exposure times and acquisition parameters were used for all sections on a slide. Fiji 2.14.0, an ImageJ program with a plugin (National Institutes of Health, Bethesda, MD, USA), was utilized for pixel counting and analysis [68]. Immunofluorescence quantification spanned laminae I to III of the ipsilateral spinal cord dorsal horn (SCDH), with data averaged from three rats per group. Confocal images were captured using a Leica TCS SP5-II confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica HyD (hybrid detector). Representative images were taken at 100X, and 400X, and co-localization images at 400X or 630X magnification.

Co-localization of two immunofluorescence signals

Co-localization analysis was performed using Pearson’s correlation coefficient (Francois-Moutal et al., 2015), with quantification based on an intensity correlation coefficient method utilizing Fiji. Pearson’s coefficient values < 0.1 were interpreted as indicating no co-localization, while values > 0.5 suggested near maximal co-localization [69]. To quantify LDHA expression in the nucleus, ImageJ software incorporating the JACoP plugin was used to calculate the Manders coefficient. This coefficient measures the proportion of the fluorescent signal in one channel that overlaps with the signal in another channel and is widely used to assess co-localization between different channels [70]. The total intensity of DAPI in each image was normalized to 100%, and the proportion of DAPI overlapping with the LDHA signal was calculated for each group [71].

Statistical analysis

Data are presented as means ± standard error of the mean (SEMs). Changes in protein levels and immunofluorescence reactivity are reported relative to the control, 7d, and 14d groups. Differences between groups were analyzed using a two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. Statistical significance was set at P < 0.05. All statistical analyses were conducted using SigmaPlot Version 15.0 (Systat Software, Inc., San Jose, CA, USA).