Sprague–Dawley rats (male, 6-week-old, 150~200 g) were obtained from Daehan Bio Link (Chung-buk, Republic of Korea) and allowed to acclimatize to the new environment for 1 week before the experiments. All animals were housed (three rats per cage) in a controlled environment (23±2℃, 50% humidity) under a 12 h-light/dark cycle and given food and water ad libitum. In this study, 124 animals were used for the behavioral test (49), the analysis of gene expression analysis (28) and the immunohistochemical study (46). The animal experiments were approved by the Animal Care and Use Committee of Chungnam National University (approval number: CNUH-019-P0001) and conformed to the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain [16].

In this study, we used a rat model of neuropathic pain induced by SNL, which is a proved model of neuropathic pain in animals [17,18]. Briefly, under isoflurane anesthesia (2% in oxygen; Hana Pharm, Kyung-gi, Republic of Korea), the animals were placed in the prone position, and the left side of the paraspinal muscles were spread wide apart via a 2-cm skin incision. After elimination of the lumbar (L) 6 transverse process to reveal the L4 and L5 vertebrae, the L5 spinal nerve was carefully separated from L4 without injury and ligated three times using 3-0 silk thread (Ethicon, Diegem, Belgium). After ligation, the wound was sutured with silk thread, and the animals were allowed to recover on a warm pad. The sham group received the same surgical procedures as in the SNL group except that the L5 spinal nerve was not ligated.

Pain behavior was evaluated as described previously [16,19,20,21] using von Frey filaments (NC12775-99, Touch Test® Sensory Evaluators, 20-Piece Hand Kit; North Coast Medical & Rehabilitation Products, Gilroy, CA). Briefly, rats were placed on a metal mesh floor covered with clear plastic boxes (18×8×8 cm) and left for 20 minutes to acclimatize to the new environment. Mechanical stimuli were applied with nine different von Frey filaments ranging from 0.6 to 15 g (0.6, 1, 1.4, 2, 4, 6, 8, 10, and 15 g). Stimuli were applied with a von Frey filament in 3~4-second trials, each of which was repeated four times on each hind paw at intervals of approximately 5 minutes. The 2-g filament stimulus was applied first. Then, mechanical threshold was determined with the up-down method [21]. If a positive response occurred, the next smaller von Frey filament was used. If a negative response occurred, the next larger filament was applied. The last filament applied was considered as a threshold of the test.

Intrathecal injection of compounds into the subarachnoid space of the spinal cord was performed as described previously [22]. Briefly, animals were placed in the prone position on an operating table under isoflurane anesthesia. Next, 20 µL solution (PBS or TAP2 [AMALDCFRWGWRMWCSSG, Peptron, Daejeon, Republic of Korea], 25 nmol) was introduced intrathecally into the L5/L6 intervertebral space using a syringe (50 µL; Hamilton, Reno, NV) with a 26.5 G needle. Further, fluorescein isothiocyanate (FITC)-conjugated TAP2 (FITC-TAP2, Peptron) was used to examine the microglial specificity and the duration of action in the spinal dorsal horn of SNL-induced rats. An obvious tail flick was observed when injection was successful.

Total RNA was isolated from BV2 cells or the ipsilateral dorsal horn of the spinal cords (L4/L5 segment, 0.7 cm) using the Hybrid-R kit (GeneAll, Seoul, Republic of Korea) [23] and then transcribed into cDNA using TOPscript RT DryMix (Enzynomics, Daejeon, Republic of Korea). qPCR was performed under the following conditions [24]: 95℃ for 10 minutes, followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minute using the AriaMx Realtime PCR System (Agilent Technologies, Santa Clara, CA). Primer sequences (Cosmogenetech, Seoul, Republic of Korea) used in the qPCR were as follows: mouse GAPDH, forward: 5′-ACC CAG AAG ACT GTG GAT GG -3′, reverse: 5′- CAC ATT GGG GGT AGG AAC AC- -3′; mouse TNF- α, forward: 5′- AGC AAA CCA CCA AGT GGA GGA-3′, reverse: 5′- GCT GGC ACC ACT AGT TGG TTG T -3′; mouse IL-1β, forward: 5′- TTG TGG CTG TGG AGA AGC TGT -3′, reverse: 5′- AAC GTC ACA CAC CAG CAG GTT -3′; mouse IL-6, forward: 5′- TCC ATC CAG TTG CCT TCT TGG -3′, reverse: 5′- CCA CGA TTT CCC AGA GAA CAT G -3′; mouse COX-2, forward: 5′- TGA GTA CCG CAA ACG CTT CT - 3′, reverse: 5′- CTC CCC AAA GAT AGC ATC TGG – 3′ ; mouse iNOS, forward: 5′- GGC AAA CCC AAG GTC TAC GTT – 3′, reverse: 5′- TCG CTC AAG TTC AGC TTG GT – 3′; rat GAPDH, forward: 5′- CTC ATG ACC ACA GTC CAT GC -3′, reverse: 5′- TTC AGC TCT GGG ATG ACC TT -3′; rat TNF-α, forward: 5′- AGA TGT GGA ACT GGC AGA GG -3′, reverse: 5′- CCC ATT TGG GAA CTT CTC CT -3′; rat IL-1β, forward: 5′- CAG CAG CAT CTC GAC AAG AG -3′, reverse: 5′- CAT CAT CCC ACG AGT CAC AG -3′; rat IL-6, forward: 5′- CCG GAG AGG AGA CTT CAC AG -3′, reverse: 5′- ACA GTG CAT CAT CGC TGT TC -3′; rat COX-2 forward: 5′- CAG TAT CAG AAC CGC ATT GCC -3′, reverse: 5′- GAG CAA GTC CGT GTT CAA GGA-3′; rat TLR4 forward: 5′- TGA GCA GTC GTG CTG GTA TC -3′, reverse: 5′- CAG GGC TTT TCT GAG TCG TC -3′. The mRNA levels of each target gene were normalized to that of GAPDH. The fold changes in mRNA levels were calculated using the 2^−ΔΔCt method as described previously [25].

Rats were euthanized with sodium pentobarbital (100 mg/kg, i.p.), perfused with heparinized phosphate-buffered saline (0.5% heparin in PBS, pH 7.4), followed by perfusion with ice-cold 4% paraformaldehyde in PBS for 10 minutes using a peristaltic pump at a rate of 20 mL/min. The lumbar region (L4~L6) of the spinal cord was separated, bathed in the same fixative overnight at 4℃, and then immersed in a series of sucrose solutions in PBS (from 10% to 30%) for cryoprotection [26]. After 2 days, the lumbar segments were cut into transverse sections at a thickness of 30 µm using a cryostat (CM1950; Leica, Wetzlar, Germany) and kept at −20℃ in storage buffer (30% glycerol, 30% ethylene glycol in PBS) [27].

For fluorescence staining, tissues were first blocked with blocking buffer (5% normal serum, 0.3% Triton X-100 in PBS) for 1 hour at room temperature. The spinal tissues were then incubated with a mixture of primary antibodies (iNOS, 1:100 [BD-610329; BD Transduction Laboratories, Franklin Lakes, NJ]; Iba1, 1:400 [019-19741; Wako, Osaka, Japan]); diluted in blocking buffer overnight at 4℃ followed by a mixture of corresponding secondary antibodies conjugated with either FITC or Cy³ (1:400; Jackson ImmunoResearch, West Grove, PA) diluted in the same blocking buffer [28]. The sections were counterstained with DAPI (5 µg/mL in PBS; Thermo Fisher Scientific, Waltham, MA) for 5 minutes and then mounted on glass slides with mounting medium (Biomeda, Foster City, CA). Images were obtained using a laser scanning microscope (TCS SP8; Leica) and used for additional data analysis.

For diaminobenzidine (DAB) staining, the spinal tissues were first exposed to 0.3% H₂O₂ in PBS for 10 minutes to block endogenous peroxidase activity and incubated with the same blocking solution for 1 hour at room temperature. The tissues were then treated with the primary antibody (Iba-1, 1:400; Wako) overnight at 4℃ followed by a biotinylated secondary antibody and the streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). The specimens were visualized with DAB–peroxidase substrate solution (0.05% DAB/0.015% H₂O₂ in PBS) and mounted on glass slides using Polymount (Polysciences, Warrington, PA). Images were obtained by bright field microscopy (ECLIPSE E600 POL; Nikon, Tokyo, Japan).

The intensity of Iba1-immunoreactive (IR), FITC- positive, and DHE-positive cells in the spinal dorsal horn was measured using Image J software (NIH, Bethesda, MD) as described previously [16,29]. In brief, images (3 sections per animal, n = 3~5 animals) were first converted to gray ones. After background subtraction and shading correction, the intensity of rectangular area (650 µm×150 µm) in the layer I–IV of spinal dorsal horn was measured. In addition, the number of FITC-TAP2/Iba1-IR, iNOS-IR, and iNOS/Iba1-IR cells was counted in the layer I–IV of dorsal horn of the spinal cord.

Superoxide anion levels in the spinal cord were examined with dihydroethidium (DHE, Thermo Fisher Scientific), as described previously [30]. Briefly, spinal sections were incubated with DHE (1 µM) solution for 5 min at room temperature and mounted on glass slides. The signals were grabbed with a confocal microscope (Leica Biosystems, Germany), and the fluorescent intensity was quantified with the Image J program.

A rat model of OA pain was established by intraarticular injection of MIA (Sigma, St. Louis, MO) as described previously [31]. Briefly, under isoflurane anesthesia (2% in oxygen mixture; Hana Pharm, Kyung-gi, Republic of Korea), PBS (25 µL) or MIA (2 mg in 25 µL saline) was added to the intraarticular space of the knee joint of the left hind leg through the left patellar tendon using a Hamilton syringe with a 26.5 G needle. Once OA pain was established on day 7 post-MIA injection, PBS or TAP2 (25 nmol) was delivered intrathecally into the rats with the same procedure of SNL model above. The animals then underwent behavioral tests with von Frey filaments on the indicated days to validate the analgesic effect of TAP2 on OA pain. Furthermore, to examine the suppressive effect of TAP2 on spinal microglia in an OA pain model, on day 10 after TAP2 injection, L5 spinal tissues were immunostained with anti-Iba1 antibodies.

The data are expressed as the mean±standard error of the mean (SEM). The significance of differences between groups was compared by one-way or two-way analysis of variance (ANOVA), followed by the appropriate multiple comparison test. Two-group analyses were performed using the two-tailed unpaired Student's t test. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). In all analyses, p<0.05 was taken to indicate statistical significance.

In mouse models of neuropathic pain with spinal nerve transection and chronic construction injury, neuropathic pain is mediated by TLR4 and its downstream molecules [10,11]. However, there have been no reports regarding whether neuropathic pain is also mediated by TLR4 in rats. Therefore, we first examined the expression of TLR4 in rats with neuropathic pain.

To verify the mRNA level of TLR4 in rats with SNL-induced neuropathic pain, we first performed L5 SNL ligation in rats insensitive to von Frey filaments (>10 g, baseline) and confirmed the incidence and continuation of neuropathic pain by von Frey filament test on days 3, 7, and 14 (Fig. 1A). The level of TLR4 transcription in the L4–L5 segments of the ipsilateral spinal dorsal horn was then investigated by qPCR using primers specific for TLR4. The results indicate that the TLR4 mRNA level was upregulated by threefold on day 3 post-ligation in SNL-induced rats and subsequently downregulated (Fig. 1B).

These observations suggest that neuropathic pain may involve TLR4 due to the increased TLR4 expression in the rat model of neuropathic pain with SNL.

Prior to the administration of TAP2 in rats with SNL-induced neuropathic pain, we investigated the anti-inflammatory effects of TAP2 on microglial cells because TLR4 is upregulated on the surface of spinal microglia in animals with neuropathic pain [6]. BV2 cells were pre-treated with TAP2 (10 µM) for 30 min and further incubated with LPS (100 ng/ml) for 3 h. The mRNA levels of the proinflammatory mediators, such as TNF-α, IL-1β, IL-6, COX-2, and iNOS, were markedly decreased by 54–83% in the LPS+TAP2-treated group compared with the LPS-treated group in qPCR analysis (Fig. 2).

These findings indicate that TAP2 has an anti-inflammatory effect on microglia in vitro and is a candidate for the treatment of neuropathic pain via suppression of microglial activation.

Before validating the analgesic effect of TAP2 on neuropathic pain, we investigated whether TAP2 preferentially targets spinal microglia in rats with neuropathic pain because TLR4 expression is markedly upregulated in microglia after surgery [11]. To track the localization of TAP2 in the spinal cord, FITC was conjugated to the N-terminus of TAP2 (FITC-TAP2) and administered intrathecally to rats with SNL-induced neuropathic pain on day 3 after surgery. Immunofluorescent staining indicated that TAP2 was exclusively localized in Iba1-positive microglia of the spinal cord on day 4 post-injection (Fig. 3). Interestingly, the number of TAP2/Iba1-positive cells in the spinal dorsal horn was markedly increased by three-fold on the ipsilateral side compared with the contralateral side (Fig. 3).

Next, to determine whether TAP2 mitigates SNL-induced pain in rats, on day 3 after surgery, rats sensitive to von Frey filaments (<4 g) were randomly divided into two groups and treated by intrathecal injection of PBS (20 µL) or TAP2 (25 nmol in 20 µL PBS) (Fig. 4A). The von Frey filament test performed on days 5, 7, 10, 15, 20, and 24 after ligation indicated that the analgesic effect of TAP2 on neuropathic pain was significantly maintained for approximately 2 weeks compared with the control group. Interestingly, the analgesic effect of TAP2 lasted much longer than that of gabapentin (5 mg), which is used clinically for the treatment of neuropathic pain (Fig. 4A).

Microglial activation involved in neuropathic pain is mediated by TLR4 and its downstream signaling pathways [11,32]. Therefore, we examined the reduction in microglial activation induced by TAP2 in SNL-induced rats because TAP2 targets TLR4/MD2 complex expressed on the surface of microglia. On day 4 post-injection of PBS or TAP2, L5 spinal sections were immunostained with anti-Iba1 antibodies. The intensity of Iba1-immunoreactive cells was decreased by 67% in the TAP2-treated group compared with the PBS-treated controls (Fig. 4B).

Furthermore, we investigated the relationship between the reduction of pain and the duration of TAP2 in the spinal cord of SNL-induced rats. Thus, on days 4, 7, 12, 17, and 21 following administration of FITC-TAP2 to SNL-induced rats, the intensity of green fluorescence was measured on the targeted cells of ipsilateral spinal dorsal horn. Interestingly, mechanical allodynia decreased in reverse proportion to the intensity of FITC-TAP2 in the spinal dorsal horn of rats induced by SNL surgery (Fig. 4C).

Taken together, these observations suggest that TAP2 substantially targets microglia in the spinal cord and achieves long-term mitigation of SNL-induced pain in rats by reducing microglial activation.

To understand the mechanism underlying the reduction in neuropathic pain by TAP2, we examined the expression levels of the proinflammatory mediators, such as TNF-α, IL-1β, IL-6, COX-2, and iNOS, because the expression levels of these genes are noticeably upregulated in the spinal dorsal horn of animals with neuropathic pain [3]. On day 4 post-injection, the mRNA levels of TNF-α, IL-1β, and IL-6 in the ipsilateral spinal dorsal horn were significantly decreased by 52–71% in the TAP2-treated group compared with the PBS-treated controls (Fig. 5A). Furthermore, the numbers of iNOS- and iNOS/Iba1-immunopositive cells in the ipsilateral spinal dorsal horn were also markedly decreased by 39% and 46%, respectively, in the TAP2-treated group compared with the controls (Fig. 5B).

As SNL-induced microglial activation enhances the generation of ROS [33], we also examined whether TAP2 inhibits ROS production caused by the blockade of TLR4/MD2 complex and subsequent decrease in microglial activation. When L5 spinal sections were incubated with a ROS marker, dihydroethidium, the level of ROS in the dorsal horn of the spinal cord was significantly decreased by 55% in the TAP2-treated group compared with the PBS-treated controls (Fig. 6).

Together, our findings suggest that the alleviation of neuropathic pain induced by TAP2 may be attributed to decreased levels of proinflammatory molecules and ROS in the spinal dorsal horn.

Finally, to evaluate the therapeutic effect of TAP2 in a physiologically relevant disease model of neuropathic pain, we used an OA model because a subpopulation (~30%) of OA patients show neuropathic-like pain [34,35]. To establish the rat model of OA pain, MIA (2 mg in 25 µL PBS) was administered intra-articularly into the knee of the hind leg, which has been shown to induce OA via gradual death of chondrocytes in the cartilage of the knee joint, mediated by inhibition of ATP production in glycolysis [31]. On day 7 post-MIA injection, PBS or TAP2 (25 nmol in 25 µL PBS) was administered intrathecally to rats with OA pain. The mechanical thresholds were then determined using von Frey filaments on days 1, 2, 3, 5, 7, 10, 14, 17, 19, 21, and 24 post-injection. Similar to its effect on SNL-induced pain, TAP2 significantly improved MIA-induced pain behavior for 19 days compared with PBS control treatment (Fig. 7A).

Further, we investigated whether TAP2 could reudce OA pain by inhibiting microglia activation. When L5 spinal tissues were stained with anti-Iba1 antidodies on day 10 post-TAP2 injection, Iba1-immunoreactivity was decreased by 30% in the TAP2-treated group compared with the PBS-treated controls (Fig. 7B).

These observations indicate that TAP2 causes long-term attenuation of OA pain induced by MIA in rats by prohibiting microglia activation. Therefore, TAP2 is a potential candidate for the treatment of patients with neuropathic pain, including those with OA.