Glial Glutamate Transporter Modulation Prevents Development of Complete Freund’s Adjuvant-Induced Hyperalgesia and Allodynia in Mice

Glial glutamate transporter (GLT-1) modulation in the hippocampus and anterior cingulate cortex (ACC) is critically involved in nociceptive pain. The objective of the study was to investigate the effects of 3-[[(2-methylphenyl) methyl] thio]-6-(2-pyridinyl)-pyridazine (LDN-212320), a GLT-1 activator, against microglial activation induced by complete Freund’s adjuvant (CFA) in a mouse model of inflammatory pain. Furthermore, the effects of LDN-212320 on the protein expression of glial markers, such as ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation molecule 11b (CD11b), mitogen-activated protein kinases (p38), astroglial GLT-1, and connexin 43 (CX43), were measured in the hippocampus and ACC following CFA injection using the Western blot analysis and immunofluorescence assay. The effects of LDN-212320 on the pro-inflammatory cytokine interleukin-1β (IL-1β) in the hippocampus and ACC were also assessed using an enzyme-linked immunosorbent assay. Pretreatment with LDN-212320 (20 mg/kg) significantly reduced the CFA-induced tactile allodynia and thermal hyperalgesia. The anti-hyperalgesic and anti-allodynic effects of LDN-212320 were reversed by the GLT-1 antagonist DHK (10 mg/kg). Pretreatment with LDN-212320 significantly reduced CFA-induced microglial Iba1, CD11b, and p38 expression in the hippocampus and ACC. LDN-212320 markedly modulated astroglial GLT-1, CX43, and, IL-1β expression in the hippocampus and ACC. Overall, these results suggest that LDN-212320 prevents CFA-induced allodynia and hyperalgesia by upregulating astroglial GLT-1 and CX43 expression and decreasing microglial activation in the hippocampus and ACC. Therefore, LDN-212320 could be developed as a novel therapeutic drug candidate for chronic inflammatory pain.


Introduction
Chronic inflammatory pain is a devastating condition causing enormous physical and emotional burden on millions of people worldwide [1]. "Current analgesics are less effective in relieving chronic pain symptoms and often associated with severe adverse effects including numerous central nervous system related complications, gastrointestinal and renal side effects" [2,3]. Furthermore, the current pain medications have inadequate efficacy due to the limited availability to the site of action [2]. Thus, there is a need to develop effective medications to treat chronic inflammatory pain by targeting novel targets and mechanisms in the central nervous system (CNS).
Emerging evidence indicates that the activation of microglial cells and subsequent release of proalgesic mediators play a critical role in chronic pain facilitation following prior to any behavioral experiments. The mice were housed in standard cages (29 12 cm) with free access to standard laboratory chow and water, under standard lab conditions (22 ± 2 °C, relative humidity 60%), and maintained on a regular 12 h ligh cycle (lights on at 0700 h). All behavioral experiments were conducted between light cycles (09:00-17:00 h). On the day of the behavioral experiments, the mic allowed to habituate to the testing room for at least 30 min before starting the exper All procedures used in this study followed the National Institutes of Health gui for the Care and Use of Laboratory Animals and were approved by the Instit Animal Care and Use Committee at South Dakota State University under ap number 19-040A. The Good Laboratory Practice and ARRIVE guidelines were fol All efforts were made to ensure minimal animal suffering.

Drugs and Treatment
LDN-212320 (Axon Medchem, Reston, VA, USA) was dissolved in normal (0.9% NaCl) with 1% dimethyl sulphoxide (DMSO) and 0.5% Tween-80 (vehicl solution composition for the LDN was selected based on the reported solubili stability of this compound. We have previously reported this composition elsewhe Dihydrokainic acid (DHK) and gabapentin (positive control), purchased from Aldrich (St. Louis, MO, USA), were dissolved in normal saline (0.9% NaCl). All groups received an equal volume of vehicles. All drugs and chemicals were i intraperitoneally (i.p.) at a volume of 10 mL/kg body weight unless otherwise ind The LDN-212320 and DHK injections were administered 24 h and 0.5 h prior behavioral experiments, respectively ( Figure 1). The LDN-212320, DHK, and gaba doses were selected based on previous studies [41][42][43]

CFA-Induced Allodynia and Hyperalgesia
The CFA-induced allodynia and hyperalgesia, an established animal mo chronic inflammatory pain, was used as described previously [44,45]. Briefly, the le paw of each mouse was disinfected with 75% alcohol and injected intraplanar (i.p complete Freund's adjuvant (1 mg/mL, 20 µL). The control animals were injected i.p the same volume of vehicle into the left hind paw. All biochemical experiment

CFA-Induced Allodynia and Hyperalgesia
The CFA-induced allodynia and hyperalgesia, an established animal model for chronic inflammatory pain, was used as described previously [44,45]. Briefly, the left hind paw of each mouse was disinfected with 75% alcohol and injected intraplanar (i.pl.) with complete Freund's adjuvant (1 mg/mL, 20 µL). The control animals were injected i.pl. with the same volume of vehicle into the left hind paw. All biochemical experiments were conducted 7 days after control (vehicle) or CFA i.pl. injection, when symptoms of persistent inflammatory pain were evident.
Tactile allodynia was performed as described previously [46] with minor modifications. Briefly, on days 1, 3, and 7 post i.pl. CFA injection, the mice were placed individually in a plastic cage (45 × 5 × 11 cm) with a wire mesh bottom, which allowed full access to the paws. In addition, behavioral acclimatization was allowed for 30 min. A 50% paw withdrawal threshold (50% PWT) against mechanical stimulation by von Frey filament (Stoelting, Inc., Wood Dale, IL, USA) to the plantar surface of each hind paw was measured using the up-down paradigm [47]. Based on preliminary studies that characterized the threshold stimulus in control animals, the innocuous 0.04 g (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 such as prolonged hind paw withdrawal followed by licking, biting, or scratching were recorded. Mice were tested 3 days before i.pl. injection of the CFA or vehicle to determine baseline thresholds, and then tested at 3 h, 1 day, 3 days and 7 days after i.pl. injection of the vehicle or CFA.
Thermal hyperalgesia was performed as described previously with minor modifications [46,48]. Briefly, the animals were handled twice a day for 3 days prior to the experimental procedures to habituate them to handle stress. The thermal hyperalgesia was measured by paw withdrawal latency from a hot plate using a plantar analgesia apparatus (IITC Life Science Inc., Woodland Hills, CA, USA). To measure the latency time, each mouse was individually placed on a hot plate maintained at 51 ± 0.5 • C in a Plexiglas chamber. The animals' licking, flicking, or jumping was recorded as a positive response. The latency time for each mouse was calculated as a mean of three measurements with a 3 min interval between measurements. A cut-off time of 20 s was selected to prevent tissue damage. The mice were tested 3 days before i.pl. injection of the CFA or vehicle to determine the baseline thresholds, and then at 3 h, 1 day, 3 days, and 7 days after i.pl. injection of the vehicle or CFA.
The raw data from the CFA-induced allodynia and hyperalgesia were converted to the area under the curve (AUC). The AUC depicting the total paw withdrawal threshold versus time was computed using a trapezoidal calculation. The doses of LDN-212320, gabapentin, and DHK were used as described previously [41][42][43].

Immunofluorescence Assay
The immunofluorescence assay was carried out as described previously [26] with minor modifications. Briefly, the mice were euthanized through rapid decapitation and their brains were removed and postfixed in 4% paraformaldehyde fixative overnight at room temperature. The brains were cryoprotected by immersion in 30% sucrose in 0.1 M PBS at 4 • C until the brains sank to the bottom. The brain tissues were embedded with Tissue-Tek OCT (Sakura Finetek USA Inc, Torrance, CA, USA) and sectioned into 15-20-µm-thick sections with a Leica CM1850 cryostat (Leica, Wetzlar, Germany). The sections were blocked with 5% normal goat serum in 0.3% Triton X-100 in 1× PBS for 1 h at room temperature then incubated overnight at 4 • C with anti-p38 (1:100, rabbit polyclonal, Cell signaling technology, Danvers, MA, USA) or CX43 (1:200, rabbit polyclonal, PhosphoSolutions, Aurora, CO, USA). After incubation, the sections were washed with PBS followed by incubation with Alexa Fluor 488 (AF488) conjugated secondary antibodies (1:200; ab 150077, Abcam, Cambridge, MA, USA) for 1 h at room temperature in a dark place. The slides were mounted with mounting medium containing 4 , 6 -diamidino-2-phenylindole (DAPI) for nuclear staining with anti-fade reagent (SouthernBiotech, Birmingham, AL, USA). The stained sections were then examined with an Olympus AX70 Olympus epifluorescence microscope attached to a DP70 Digital Camera (Tokyo, Japan). Image J software (v1.8.0, NIH, Bethesda, MD, USA) was used to quantify the expression of target protein bands using their integrated density.

IL-1β Estimation by ELISA
The IL-1β protein levels were determined using a mouse-specific ELISA kit (Invitrogen, Waltham, MA, USA). The mice were euthanized through rapid decapitation; their hippocampi and anterior cingulate cortex were harvested and stored at −80 • C until the analysis. The tissue samples were placed in sterile PBS containing a protease inhibitor cocktail (cOmplete Mini Roche, Indianapolis, IN, USA), homogenized, and centrifuged (11,000 rpm, 20 min, 4 • C), and the supernatant was assayed for IL-1β according to the manufacturer's protocol. The protein concentrations of all samples were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA) prior to the ELISA test, and equivalent amounts of proteins were used for the analysis. The cytokine levels are expressed as pg/mg of tissue.

Data Analyses
The data were analyzed by a two-way analysis of variance (ANOVA) followed by Tukey's post hoc test to compare the behavioral measures between the experimental groups at different time points using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). The area under the curve (AUC) of the time course was analyzed using a one-way ANOVA followed by Tukey's post hoc test. The biochemical studies were analyzed using a one-way ANOVA followed by Tukey's post hoc test. The data from Western blot studies for GLT-1, CD11b, or Iba1/β-actin expression are presented as % of the control and the results are expressed as means ± S.E.M. The difference between treatments was considered significant at p < 0.05.

Effects of LDN-212320 or Gabapentin on CFA-Induced Tactile Allodynia and Thermal Hyperalgesia
To evaluate the anti-allodynic effects of LDN-212320 or gabapentin on CFA-induced tactile allodynia, we assessed the development of allodynia at 3 h, 1 day, 3 days, and 7 days after unilateral hind paw intraplantar CFA injection. As shown in Figure 2A, the CFA (1 mg/mL, 20 µL) significantly (p < 0.0001) decreased the 50% paw withdrawal threshold at 3 h, 1 day, 3 days, and 7 days compared to the control, indicating the presence of tactile allodynia. Moreover, the pretreatment with LDN-212320 (20 mg/kg) or gabapentin significantly (p < 0.0001) increased the 50% paw withdrawal threshold. The two-way ANOVA revealed that the systemic administration of LDN-212320 (20 mg/kg) significantly attenuated the CFA-induced allodynia at 1 day, 3 days, and 7 days (F 4.80 = 36.89, p < 0.0001), indicating the anti-allodynic effects of LDN-212320 (20 mg/kg) Similarly, the pretreatment with gabapentin (100 mg/kg) significantly (p < 0.0001) increased the 50% paw withdrawal threshold. The two-way ANOVA revealed that the systemic administration of gabapentin (100 mg/kg) significantly attenuated the CFAinduced allodynia at 3 days and 7 days (F 4.20 = 24.53, p < 0.0001) but not at 1 day as observed with the LDN-212320 pretreatment ( Figure 2A). Accordingly, the one way-ANOVA revealed that the overall effects (AUC) of LDN-212320 (20 mg/kg) or gabapentin (100 mg/kg) significantly (p < 0.0001) attenuated the CFA-induced allodynia (F 5.5 = 143.2, p < 0.0001) compared to the control injected with the vehicle, as shown in Figure  2B. Moreover, the pretreatment of LDN-212320 (10 mg/kg) did not significantly increase the 50% paw withdrawal threshold (Figure 2A, B).
To determine the effects of LDN-212320 or gabapentin on CFA-induced thermal hyperalgesia, we evaluated the development of hyperalgesia at 3 h, 1 day, 3 days, and 7 days after intraplantar CFA injection. As shown in Figure 2C, the CFA (1 mg/mL, 20 µL) significantly (p < 0.0001) decreased the latency time on the hot plate as compared to the control, indicating a marked reduction in the response to the heat stimulus. Moreover, the pretreatment of LDN-212320 (20 mg/kg) significantly (p < 0.0001) increased the latency time on the hot plate test. The two-way ANOVA revealed that the systemic administration of LDN-212320 (20 mg/kg) significantly attenuated the CFA-induced thermal hyperalgesia at 3 h, 1 day, 3 days, and 7 days (F 4.16 = 37.84, p < 0.0001), indicating the anti-hyperalgesic effects of LDN-212320 (20 mg/kg) ( Figure 2C). Similarly, the pretreatment of gabapentin (100 mg/kg) significantly (p < 0.0001) increased latency time on the hot plate test. The two-way ANOVA revealed that the systemic administration of gabapentin (100 mg/kg) significantly attenuated the CFA-induced thermal hyperalgesia at 1 day, 3 days, and 7 days (F 4.16 = 49.26, p < 0.0001) ( Figure 2C). The one way-ANOVA revealed that LDN-212320 (20 mg/kg) or gabapentin (100 mg/kg) significantly (p < 0.0001) attenuated the CFA-induced thermal hyperalgesia (F 5.5 = 396.8, p < 0.0001) compared to the control injected with the vehicle, as shown in Figure 2D. However, the pretreatment of LDN-212320 (10 mg/kg) did not cause significantly increased latency times on the hot plate test ( Figure 2C,D).

Effects of LDN-212320 on Microglial Iba1 Expression in the Hippocampus and ACC
To study the effects of LDN-212320 on microglial activation, the Iba1 protein expression in the hippocampus and ACC was quantified via an immunoblot analysis following CFA-induced chronic inflammatory pain. Interestingly, the intraplantar injection of CFA (1 mg/mL, 20 µL) into the hind paw significantly increased the Iba1 expression in the hippocampus (p < 0.01, Figure 4A) and ACC (p < 0.001, Figure 4B). Moreover, the one-way ANOVA showed a main effect of LDN-212320 (20 mg/kg) treatment on Iba1 expression in the hippocampus and ACC (p < 0.01, Figure 4A and p < 0.001 Figure 4B, respectively). Multiple comparisons of the means revealed that the administration of LDN-212320 (10 or 20 mg/kg) significantly decreased the Iba1 expression in the hippocampus (F 3.15 = 25.62, p < 0.01, Figure 4A) and at a higher dose (20 mg/kg) in the ACC (F 3.9 = 120.1, p < 0.0009, Figure 4B) compared to the CFA-injected group. However, the pretreatment with the lower dose of LDN-212320 (10 mg/kg) did not significantly decrease Iba1 expression in the ACC ( Figure 4B). Brain Sci. 2023, 13, x FOR PEER REVIEW 8 of 21

Effects of LDN-212320 on Microglial Iba1 Expression in the Hippocampus and ACC
To study the effects of LDN-212320 on microglial activation, the Iba1 protein expression in the hippocampus and ACC was quantified via an immunoblot analysis following CFA-induced chronic inflammatory pain. Interestingly, the intraplantar injection of CFA (1 mg/mL, 20 µL) into the hind paw significantly increased the Iba1 expression in the hippocampus (p < 0.01, Figure 4A) and ACC (p < 0.001, Figure 4B). Moreover, the one-way ANOVA showed a main effect of LDN-212320 (20 mg/kg) treatment on Iba1 expression in the hippocampus and ACC (p < 0.01, Figure 4A and p < 0.001 Figure 4B, respectively). Multiple comparisons of the means revealed that the administration of LDN-212320 (10 or 20 mg/kg) significantly decreased the Iba1 expression in the hippocampus (F 3.15 = 25.62, p < 0.01, Figure 4A) and at a higher dose (20 mg/kg) in the ACC (F 3.9 = 120.1, p < 0.0009, Figure 4B) compared to the CFA-injected group. However, the pretreatment with the lower dose of LDN-212320 (10 mg/kg) did not significantly decrease Iba1 expression in the ACC ( Figure 4B).

Effects of LDN-212320 on Microglial CD11b Expression in the Hippocampus and ACC
To evaluate the effects of LDN-212320 on microglial CD11b levels in the hippocampus and ACC, we assessed the CD11b expression in the hippocampus or ACC following CFA-induced chronic inflammatory pain. We found that CFA (i.pl.,1 mg/mL, 20 µL) injected into the hind paw significantly increased the CD11b expression in the hippocampus (p < 0.01, Figure 5A) and ACC (p < 0.01, Figure 5B). Moreover, the one-way ANOVA showed a main effect of the LDN-212320 (10 or 20 mg/kg) treatment on the

Effects of LDN-212320 on Microglial CD11b Expression in the Hippocampus and ACC
To evaluate the effects of LDN-212320 on microglial CD11b levels in the hippocampus and ACC, we assessed the CD11b expression in the hippocampus or ACC following CFAinduced chronic inflammatory pain. We found that CFA (i.pl.,1 mg/mL, 20 µL) injected into the hind paw significantly increased the CD11b expression in the hippocampus (p < 0.01, Figure 5A) and ACC (p < 0.01, Figure 5B). Moreover, the one-way ANOVA showed a main effect of the LDN-212320 (10 or 20 mg/kg) treatment on the CD11b expression in the hippocampus (p < 0.05, or p < 0.001, Figure 5A) and LDN-212320 (20 mg/kg) in the ACC (p < 0.01 5B). Multiple comparisons of the means revealed that the administration of LDN-212320 (10 or 20 mg/kg) significantly decreased the CD11b expression in the hippocampus (F 3.12 = 19.08, p < 0.0002, Figure 5A) and achieved the same at 20 mg/kg in the ACC (F 3.9 = 72.17, p < 0.0072, Figure 5B) compared to the CFA-injected group. The pretreatment of LDN-212320 (10 mg/kg) did not significantly decrease the CD11b expression in the ACC ( Figure 5B). cingulate cortex (ACC) (B) on CFA-induced chronic inflammatory pain. Representative Western blot images for Iba1 expression from the hippocampus (A) and ACC (B) (top panel). CFA (i.pl) injection increased the Iba1 expression in the hippocampus (A) and ACC (B) compared to the control. Treatment of LDN-212320 (10 or 20 mg/kg) reversed the CFA-induced increased Iba1 expression in the hippocampus (A) or LDN-212320 (20 mg/kg) in the ACC (B). Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4-6 mice/group). Note: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Effects of LDN-212320 on Microglial CD11b Expression in the Hippocampus and ACC
To evaluate the effects of LDN-212320 on microglial CD11b levels in the hippocampus and ACC, we assessed the CD11b expression in the hippocampus or ACC following CFA-induced chronic inflammatory pain. We found that CFA (i.pl.,1 mg/mL, 20 µL) injected into the hind paw significantly increased the CD11b expression in the hippocampus (p < 0.01, Figure 5A) and ACC (p < 0.01, Figure 5B). Moreover, the one-way ANOVA showed a main effect of the LDN-212320 (10 or 20 mg/kg) treatment on the CD11b expression in the hippocampus (p < 0.05, or p < 0.001, Figure 5A) and LDN-212320 (20 mg/kg) in the ACC (p < 0.01 5B). Multiple comparisons of the means revealed that the administration of LDN-212320 (10 or 20 mg/kg) significantly decreased the CD11b expression in the hippocampus (F 3.12 = 19.08, p < 0.0002, Figure 5A) and achieved the same at 20 mg/kg in the ACC (F 3.9 = 72.17, p < 0.0072, Figure 5B) compared to the CFAinjected group. The pretreatment of LDN-212320 (10 mg/kg) did not significantly decrease the CD11b expression in the ACC ( Figure 5B).

Effects of LDN-212320 on Astroglial GLT-1 Expression in the Hippocampus and ACC
To investigate the effects of LDN-212320 on the astroglial GLT-1 levels in hippocampus and ACC, we quantified the GLT-1 expression in the hippocampus or following CFA-induced chronic inflammatory pain. Interestingly, CFA (i.pl., 1 mg/m µL) injected into the hind paw significantly decreased the GLT-1 expression in bot

Effects of LDN-212320 on Astroglial GLT-1 Expression in the Hippocampus and ACC
To investigate the effects of LDN-212320 on the astroglial GLT-1 levels in the hippocampus and ACC, we quantified the GLT-1 expression in the hippocampus or ACC following CFA-induced chronic inflammatory pain. Interestingly, CFA (i.pl., 1 mg/mL, 20 µL) injected into the hind paw significantly decreased the GLT-1 expression in both the hippocampus and ACC (p < 0.0001, Figure 7A,B, respectively). Moreover, the oneway ANOVA showed a main effect of LDN-212320 (20 mg/kg) treatment on the GLT-1 expression in the hippocampus and ACC (p < 0.0001, Figure 7A,B, respectively). Multiple comparisons of the means revealed that the administration of LDN-212320 (20 mg/kg) significantly increased the GLT-1 expression in the hippocampus (F 3.26 = 29.10, p < 0.0001, Figure 7A) and ACC (F 3.28 = 81.14, p < 0.0001, Figure 7B) compared to the CFA-injected group. Again, the pretreatment with the lower dose of LDN-212320 (10 mg/kg) had no effect and did not significantly increase the GLT-1 expression in the hippocampus or ACC ( Figure 7A,B, respectively).

Effects of LDN-212320 on Astroglial GLT-1 Expression in the Hippocampus and AC
To investigate the effects of LDN-212320 on the astroglial GLT-1 levels hippocampus and ACC, we quantified the GLT-1 expression in the hippocampus following CFA-induced chronic inflammatory pain. Interestingly, CFA (i.pl., 1 mg µL) injected into the hind paw significantly decreased the GLT-1 expression in b hippocampus and ACC (p < 0.0001, Figure 7A,B, respectively). Moreover, the o ANOVA showed a main effect of LDN-212320 (20 mg/kg) treatment on the expression in the hippocampus and ACC (p < 0.0001, Figure 7A,B, respectively). M comparisons of the means revealed that the administration of LDN-212320 (20 significantly increased the GLT-1 expression in the hippocampus (F 3.26 = 29.10, p < Figure 7A) and ACC (F 3.28 = 81.14, p < 0.0001, Figure 7B) compared to the CFAgroup. Again, the pretreatment with the lower dose of LDN-212320 (10 mg/kg) effect and did not significantly increase the GLT-1 expression in the hippocampus ( Figure 7A,B, respectively).

Effects of LDN-212320 on IL-1β Level in the Hippocampus and ACC
To determine the effects of LDN-212320 on proinflammatory mediators produced by glial cells in the hippocampus and ACC, we examined the IL-1β protein expression in these regions following CFA-induced chronic inflammatory pain. The one-way ANOVA indicated that the intraplantar injection of CFA (1 mg/mL, 20 µL) into the hind paw increased the IL-1β levels significantly (p < 0.001) in the hippocampus and ACC ( Figure  9A,B, respectively). Moreover, the one-way ANOVA revealed that the LDN-212320 pretreatment had a significant effect (p < 0.001) on the IL-1β levels in the hippocampus and ACC. A post hoc test for multiple comparisons revealed that the higher dose of LDN-212320 (20 mg/kg) significantly attenuated the CFA-induced increased IL-1β levels in the hippocampus (F 3.12 = 33.82, p < 0.0001), as did both 10 and 20 mg/kg in the ACC (F 3.12

Effects of LDN-212320 on IL-1β Level in the Hippocampus and ACC
To determine the effects of LDN-212320 on proinflammatory mediators produced by glial cells in the hippocampus and ACC, we examined the IL-1β protein expression in these regions following CFA-induced chronic inflammatory pain. The one-way ANOVA indicated that the intraplantar injection of CFA (1 mg/mL, 20 µL) into the hind paw increased the IL-1β levels significantly (p < 0.001) in the hippocampus and ACC ( Figure 9A,B,  respectively). Moreover, the one-way ANOVA revealed that the LDN-212320 pretreatment had a significant effect (p < 0.001) on the IL-1β levels in the hippocampus and ACC. A post hoc test for multiple comparisons revealed that the higher dose of LDN-212320 (20 mg/kg) significantly attenuated the CFA-induced increased IL-1β levels in the hippocampus (F 3.12 = 33.82, p < 0.0001), as did both 10 and 20 mg/kg in the ACC (F 3.12 = 33.10, p < 0.0001) ( Figure 9A,B, respectively).

Effects of LDN-212320 on IL-1β Level in the Hippocampus and ACC
To determine the effects of LDN-212320 on proinflammatory mediators produced by glial cells in the hippocampus and ACC, we examined the IL-1β protein expression in these regions following CFA-induced chronic inflammatory pain. The one-way ANOVA indicated that the intraplantar injection of CFA (1 mg/mL, 20 µL) into the hind paw increased the IL-1β levels significantly (p < 0.001) in the hippocampus and ACC ( Figure  9A,B, respectively). Moreover, the one-way ANOVA revealed that the LDN-212320 pretreatment had a significant effect (p < 0.001) on the IL-1β levels in the hippocampus and ACC. A post hoc test for multiple comparisons revealed that the higher dose of LDN-212320 (20 mg/kg) significantly attenuated the CFA-induced increased IL-1β levels in the hippocampus (F 3.12 = 33.82, p < 0.0001), as did both 10 and 20 mg/kg in the ACC (F 3.12 = 33.10, p < 0.0001) ( Figure 9A,B, respectively).

Discussion
In the present study, the major findings are that the GLT1 activator LDN-212320 significantly attenuated allodynia and hyperalgesia associated with a CFA-induced model of chronic inflammatory pain. We found that the anti-allodynic and anti-hyperalgesic effects of LDN-212320 were prevented by pretreatment with DHK, a GLT-1 antagonist. In addition, LDN-212320 significantly reversed CFA-induced increases in microglial activation, decreased astroglial GLT-1 and CX43 expression, and increased the inflammatory mediator IL-1β's levels in the hippocampus and ACC. Thus, the present study reveals novel mechanisms of LDN-212320 in the hippocampus and ACC associated with pain modulation in chronic inflammatory pain. To the best of our knowledge, we show here for the first time the cellular mechanism associated with GLT-1 modulation in chronic inflammatory pain induced by CFA.
Our study indicated that LDN-212320 inhibits microglial activation associated with chronic inflammatory pain induced by CFA. In accordance with these observations, previous reports found that suppressing microglial activation delayed the development of allodynia in a chronic pain model [6]. In addition, systemic treatment with minocycline, a microglial inhibitor, attenuated hyperalgesia, and allodynia in a rat model of neuropathic pain [11]. Importantly, we have demonstrated that intraplantar CFA administration increased the expression of the microglial markers Iba1 and CD11b and microglial-induced proinflammatory cytokine production in the hippocampus and ACC. These findings are consistent with previous studies showing that peripheral inflammation induces microglial activation and proinflammatory cytokine release in forebrain regions [51,52]. Previous reports also indicated that microglial activation plays an important role in pain facilitation induced by peripheral tissue injury [53][54][55]. It is noteworthy to mention that pharmacological approaches targeting microglial activation prevented microglial responses and subsequently attenuated chronic pain associated with peripheral tissue injury [56,57]. In fact, microglial activation induces a range of pronociceptive molecules that may potentiate pain transmission [58,59]. For example, p38 is predominantly expressed in the spinal microglia after peripheral nerve injury [60,61]. In addition, the activation of microglial cells following peripheral nerve injury facilitates persistent pain via p38 and IL-1β mediation [62,63]. It is worth mentioning that increased glutamate activity may induce microglial p38 activation to promote injury [64,65], which is in line with our observations. Moreover, our findings demonstrate that p38 upregulation is consistent with the activation of microglia within the hippocampus and ACC. This notion is also supported by previous reports regarding the p38-dependent activation of microglia and associated pain facilitation [8,66].
Consistent with our previous report on formalin-induced nociceptive pain [26], we have demonstrated that GLT-1 modulation inhibited microglial activation and reduced CFA-induced hyperalgesia and allodynia. These results are also consistent with previous findings showing that systemic treatment with ceftriaxone, a known GLT-1 activator, protected rats against cerebral ischemic injury by preventing microglial activation in the striatum [67]. Moreover, previous studies have shown that GLT-1 modulation prevents chronic pain [68,69]. For example, systemic treatment with ceftriaxone attenuated allodynia and hyperalgesia in animal models of chronic pain [37,70]. Furthermore, ceftriaxone treatment significantly reversed GLT-1 downregulation in a chronic pain model [69,71], which is consistent with our findings.
In this study, we found that astroglial CX43 is downregulated in the hippocampus and ACC following peripheral tissue injury. This is consistent with a recent report suggesting that decreased astroglial CX43 expression might facilitate pain transmission by enhancing algesic targets following nerve injury [72]. Interestingly, increasing spinal astroglial CX43 expression to normal levels inhibited mechanical hypersensitivity during chronic pain [39]. Furthermore, astroglial CX43 was shown to play a major role as a mediator of injury-dependent downregulation of GLT-1 in the hippocampus [73,74]. These observations support the notion that reduced GLT-1 expression could be partially attributed to astroglial CX43 downregulation following peripheral tissue injury. These results, in addition to our findings provide strong evidence that GLT-1 modulation by LDN-212320 in the hippocampus and ACC is important in attenuating chronic inflammatory pain.
Previous studies reported that increased glutamate accumulation activates glutamate ionotropic receptors leading to increased IL-1β synthesis and expression in microglial cells in the rat brain [75], supporting the notion that increased glutamate may be in part responsible for microglial activation in our model. Indeed, it was found that the application of glutamate in the rodent cerebral cortex sensitized microglial cells to produce inflammatory mediators [76], giving functional relevance to our observations. Importantly, it was shown that IL-1β significantly influenced the glutamatergic release in the CNS following peripheral tissue injury [77][78][79]. For example, IL-1β was found to increase the physiological function of the glutamate N-methyl-D-aspartate (NMDA) receptor by increasing the frequency of NMDA receptor channel opening [80] and directly enhancing the activity of hippocampal neuronal NMDA receptors [81]. In addition, IL-1β was shown to facilitate inflammatory pain by enhancing the phosphorylation of NMDA receptors [77]. Several studies have suggested that IL-1β might modulate presynaptic glutamatergic release via signaling pathways involving increased Ca 2+ influx in the hippocampus [82][83][84]. Moreover, IL-1β was shown to decrease the expression of the astroglial GLT-1 leading to impaired glutamate transport [85]. Consistent with these reports, we have shown that the CFA-induced increase in IL-1β release could decrease the GLT-1 expression in the hippocampus and ACC, and this downregulation was reversed by LDN-212320. It is important to note that in the present study we focused on IL-1β mainly due to its critical involvement in the glutamatergic system, as noted previously, and for its role as a key mediator of neuroinflammatory responses. However, future studies are important to determine the effects of other proinflammatory cytokines on glutamatergic neurotransmission in chronic inflammatory pain.
It is well documented that glutamate transporters are also expressed in peripheral tissues [86]. Given this, LDN-212320 might have additional effects by targeting glutamate transporters expressed in these tissues. However, we have shown that the systemic treatment of LDN-212320 does not produce any noticeable change to the expression of peripheral inflammation markers [26], suggesting an insignificant role. However, it is possible that acute treatment in a formalin-induced nociceptive model could limit any observable effects of LDN-212320 in peripheral tissues. Therefore, future studies are necessary to examine the effects of LDN-212320 on the peripheral glutamate transporter system.
It is widely believed that the hippocampus and ACC play an important role in pain perception and modulation [87,88]. Pertinent to this study, peripheral nerve injury induces robust glutamate release in the hippocampus and ACC [89,90]. Furthermore, previous studies demonstrated that increased glutamatergic neurotransmission modulates microglial activation directly through glutamate receptors or indirectly via extracellular adenosine triphosphate signaling in response to glutamate release [91,92]. More importantly, increased extracellular glutamate release following nerve injury stimulates glutamate receptors, which in turn activate microglia to induce the release of cytokines such as IL-1β in the hippocampus [93], which could be relevant to peripheral nerve injury. Similarly, a previous study has shown that accumulated glutamate leads to microglial activation through the release of proinflammatory mediators [92]. It is of interest to state that microglia-neuronastroglia crosstalk is evident in most peripheral injury models [4]. Importantly, it was found that microglial activation could modulate glutamate neurotransmission in the CNS following central insults [94]. For example, CX3C chemokine ligand 1 (CX3C1), acting on microglia, was shown to indirectly modify GLT-1 expression on astroglia via the release of soluble factors such as the adenosine A1 receptor [95]. Most interestingly, it was found that gabapentin reduces microglial activation through the blockage of CX3C1 signaling following CFA-induced peripheral inflammation [96], suggesting the crucial involvement of GLT-1 and CX3C1 in the crosstalk between microglia and astroglia. Furthermore, several studies support the notion that GLT-1 activity is greatly involved in glutamate receptor activation [97]. For instance, GLT-1 upregulation is considered the limiting step for the induction of long-term depression in the hippocampal synapse by modulating metabotropic glutamate receptor activation [98]. In accordance with these reports and from our observations, we propose that GLT-1 modulation controls microglial activation through mediators or the direct modulation of the glutamatergic system in microglial cells. Moreover, a previous report has shown that gabapentin decreased microglial activation in rodents with chronic pain [99]. Interestingly, other reports showed that reduced gabapentin effectiveness was associated with decreased GLT-1 expression [41], suggesting that gabapentin's effect is GLT-1-dependent, which is similar to our findings with LDN-212320. However, future studies are required to confirm this notion in the CFA-induced chronic inflammatory pain model.
Previous evidence suggested that the excessive accumulation of glutamate may be responsible for central sensitization in chronic pain [100,101]. Glutamate accumulation may occur due to increased presynaptic release through increased neuronal activity [102,103] or impaired glutamate uptake caused by GLT-1 downregulation during peripheral tissue injury [26,104]. It is worth mentioning that the glutamate transporter competes with glutamate receptors for glutamate binding [97], suggesting that glutamate transporters critically modulate glutamate receptor activity and function. In accordance with this, we have shown that the removal of glutamate from the synapses through GLT-1 activation prevents microglial activation. Similarly, earlier data reported that the systemic administration of MK-801, an NMDA receptor antagonist, prevents microglial activation in the hippocampus [105][106][107].
We explicitly observed that glutamate transporter modulation in the hippocampus and ACC prevented microglial activation and extended our previous findings with LDN-212320 on formalin-induced nociceptive behavior in mice [26]. Our data demonstrate that LDN-212320 significantly attenuated microglial activation associated with CFA-induced chronic inflammatory pain, likely through the regulation of GLT-1 in the hippocampus and ACC. However, additional studies are necessary to confirm these observations by using site-specific intracranial injections of LDN-212320 in these brain regions. Future studies are warranted to elucidate the role of astroglial GLT-1 modulation using site-specific routes such as intracranial or intranasal administration. Intranasal administration can be preferred since it influences drug delivery to the brain. Additional studies should focus on lipid-based nanoformulations for an improved pharmacokinetic profile.

Conclusions
Our novel findings provide strong evidence that LDN-212320 prevents CFA-induced allodynia and hyperalgesia. This effect is likely mediated by reversing CFA-induced microglial activation and upregulating astroglial GLT-1 and CX43 expression in the hippocampus and ACC in a mouse model of inflammatory pain. Together, these findings give compelling support to the idea that LDN-212320 could be developed as a novel therapeutic drug candidate for chronic inflammatory pain.
It is noteworthy to mention that we have studied the effects of the astroglial GLT-1 modulation in the hippocampus and ACC in male mice. However, astroglial GLT-1 regulation in female mice is critical in acute and chronic inflammatory pain. Therefore, future studies are needed to examine the astroglial GLT-1 modulation in female mice for acute and chronic inflammatory pain.
Author Contributions: G.A. conducted the experiments, analyzed the results, and drafted the manuscript. S.R. conceived and designed the study, reviewed, and edited the manuscript, and helped with and secured funding to conduct the experiments. A.K., P.J.R. and K.L. helped to review and edit the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All data included in this study are available upon request from the corresponding authors.