5-HT1F Receptor Agonist Ameliorates Mechanical Allodynia In Neuropathic Pain Via Induction of Mitochondrial Biogenesis and Suppression of Neuroinflammation

doi:10.21203/rs.3.rs-965046/v1

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5-HT_1F Receptor Agonist Ameliorates Mechanical Allodynia In Neuropathic Pain Via Induction of Mitochondrial Biogenesis and Suppression of Neuroinflammation

https://doi.org/10.21203/rs.3.rs-965046/v1

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Background: Neuropathic pain is a devastating disease affecting millions of people worldwide. Serotonin (5-hydroxytryptamine, 5-HT) has long been involved in pain modulation. Several lines of evidence indicate that 5-HT_1F receptor agonists are potent inducers of mitochondrial biogenesis. In this study, we tested the hypothesis that 5-HT_1F receptor agonist ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation.

Methods: Male Sprague-Dawley rats were used to establish neuropathic pain via spared nerve injury (SNI). Paw withdrawal threshold was used to evaluate mechanical allodynia. Real time polymerase chain reaction was used to examine the mitochondrial DNA (mtDNA) copy number. Western blot and immunofluorescence were used to examine the expression of target proteins.

Results: Our results showed that mitochondrial biogenesis was impaired in the spinal cord of SNI rats. Moreover, activation of PGC-1α, the master regulator of mitochondrial biogenesis, attenuated established mechanical allodynia in neuropathic pain rats. Besides, neuronal 5-HT_1F receptor was significantly downregulated in the spinal cord of neuropathic pain rats. Furthermore, selective 5-HT_1F receptor agonist Lasmiditan attenuated established mechanical allodynia in neuropathic pain rats. Finally, Lasmiditan treatment restored mitochondrial biogenesis and suppressed neuroinflammation in the spinal cord of SNI rats.

Conclusion: These results provide the first evidence that Lasmiditan ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation in the spinal cord. Mitochondrial biogenesis inducers may become encouraging therapeutic option for the management of neuropathic pain.

Neurology

Cellular & Molecular Neuroscience

Neuropathic pain

Oxidative stress

Mitochondrial dysfunction

Mitochondrial biogenesis

5-HT1F receptor

PGC-1α

Chronic neuropathic pain is a devastating disease resulting from damage to or dysfunction of the nervous system [1]. It affects millions of people worldwide and severely disrupts their quality of life. Unfortunately, it is refractory to commonly used analgesics such as nonsteroidal anti-inflammatory drugs and opioids [2]. Tricyclic anti-depressants and anti-epileptics also fail to provide sufficient pain relief. Despite rapid advance over the past decades, the specific cellular and molecular mechanisms underlying the development of neuropathic pain remain largely unknown [3, 4]. Therefore, it is urgent to unravel the pathogenesis of neuropathic pain and reveal novel therapeutic targets.

Emerging evidence from our laboratories and others indicate that oxidative stress and mitochondrial dysfunction may be the primary causes of chronic pain [5–7]. Mitochondrial biogenesis, a dynamic process of generating new mitochondria, plays a critical role in regulating mitochondrial homeostasis [8, 9]. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is the master regulator of mitochondrial biogenesis [10, 11]. Once being activated, PGC-1α activates nuclear respiratory factor 1 (NRF1), and subsequently mitochondrial transcription factor A (TFAM). It is reported that impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in neurological diseases such as Alzheimer's disease and spinal cord injury [12, 13]. Moreover, a recent study demonstrates that PGC-1α haploinsufficiency promotes pain chronification after burn injury [14]. However, the role of mitochondrial biogenesis in neuropathic pain remains unknown. Nevertheless, recent evidence indicates that PGC-1α activation exerts neuroprotective effects via suppressing neuroinflammation [15, 16]. Given the vital role of neuroinflammation in chronic pain [17, 18], it is plausible that PGC-1α activation might attenuate pain behaviors in neuropathic pain.

Serotonin (5-hydroxytryptamine, 5-HT), a monoamine widely distributed in the central nervous system, has long been involved in pain modulation [19]. To date, seven different 5-HT receptor families have been identified. Previous studies have demonstrated that several 5-HT receptor subtypes mediate 5-HT-induced antinociception such as 5-HT_5A receptor and 5-HT₇ receptor [20, 21]. However, little is known about the role of 5-HT_1F receptor in neuropathic pain. Although the physiological role of 5-HT_1F is not fully characterized, it is found to be expressed in the spinal cord and various brain regions involved in pain [22, 23]. Moreover, it was reported that LY344864, 5-HT_1F receptor agonist, attenuated formalin-induced inflammatory pain [24]. Currently, Lasmiditan, a highly selective 5-HT_1F receptor agonist, has been approved for the acute treatment of migraine by US Food and Drug Administration [25]. Furthermore, several lines of evidence indicate that 5-HT_1F receptor agonists are potent inducers of mitochondrial biogenesis [26–29]. Given the crucial role of mitochondrial dysfunction in neuropathic pain and the involvement of 5-HT_1F receptor in mitochondrial biogenesis, this study aimed to test the hypothesis that 5-HT_1F receptor agonist ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation. In this study, we used a well-established rat model of neuropathic pain induced by spared nerve injury (SNI) which is characterized by mechanical allodynia similar to neuropathic pain patients.

Animals and Ethical Statement

A total of 496 rats were used in this study. Adult male Sprague-Dawley rats (180-220g) were supplied by Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The rats were housed under controlled conditions (temperature: 22-25℃, relative humidity: 45-65%, and 12-h light to dark cycle, with food and water ad libitum). All experiments were approved by the Experimental Animal Care and Use Committee of Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the ARRIVE Guidelines for Reporting Animal Research [30]. All efforts were made to minimize the number of animals used and their suffering.

Establishment of neuropathic pain model

A rat model of neuropathic pain was induced by SNI as described previously [31, 32]. Briefly, the rats are anaesthetized with 2.5% isoflurane, the right sciatic nerve and its three branches (the common peroneal, tibial, and sural nerves) were exposed. Then, common peroneal and tibial nerves were ligated. And the distal to the ligation was sectioned, cutting 2 to 4 mm of the distal nerve stump. For the sham-surgery rats, the sciatic nerve was exposed without ligation.

Intrathecal catheterization and drug administration

As described previously [33], intrathecal (i.t.) catheters was carried out 5 days prior to the establishment of SNI models. Briefly, the rats were anesthetized using 2.5% isoflurane. Then, the PE10 polyethylene catheters (inner diameter 0.3 mm, outer diameter 0.6 mm) were inserted from L5-L6 spinous processes. The correct position of the catheter was verified by a tail flick response immediately after inserting the catheter and further confirmed using an i.t. injection of 2% lidocaine.

Lasmiditan succinate (Cat# S6489, Selleckchem, Houston, TX, USA), a selective 5-HT_1Freceptor agonist, was dissolved in saline. ZLN005 (Cat# HY-17538, MedChemExpress, Monmouth Junction, NJ, USA), a specific PGC-1α activator, was dissolved in 10% dimethyl sulfoxide (DMSO). SR-18292 (Cat# HY-101491, MedChemExpress), a specific PGC-1α inhibitor, was dissolved in 10% DMSO. In this study, we aimed to investigate a purely spinal mechanism. Therefore, we chose intrathecal injection rather than intraperitoneal or oral injection. The dosages of these drugs were determined by our preliminary experiments. To determine whether a single dose of PGC-1α activator or 5-HT_1F receptor agonist could attenuate established mechanical allodynia in SNI rats, ZLN005 (10, 50, or 100 μg, i.t.) or lasmiditan (50, 100 or 200 μg, i.t.) was given on day 7 after surgery. The behavioral test was conducted before ZLN005 or lasmiditan injection, and 1, 2, 4, 6, and 12 h after the injection. To determine whether repeated injection of a PGC-1α activator or 5-HT_1F receptor agonist could reverse mechanical allodynia in SNI rats, ZLN005 (50 μg, i.t.) or lasmiditan (100 μg, i.t.) was given once daily for five consecutive days starting from day 7. The behavioral test was performed on day 6 and 1 h after ZLN005 injection or 2 h after lasmiditan each day. To determine whether early treatment with a PGC-1α activator or 5-HT_1F receptor agonist could suppress the development of mechanical allodynia in SNI rats, ZLN005 (50 μg, i.t.) or lasmiditan (100 μg, i.t.) was given once daily for seven consecutive days starting from day 1 after the surgery. The behavioral test was conducted before the surgery, and on day 1, 3, 7, 8, 9, 10, 11, 12, 13 and 14 after surgery. To determine whether a PGC-1α inhibitor could reverse the analgesic effects of ZLN005 or lasmiditan, SR-18292 (30 μg, i.t.) was given 30 min before ZLN005 or lasmiditan. The behavioral test was conducted before SR-18292injection, and 1, 2, 4, 6, and 12 h after ZLN005 or lasmiditan injection. To determine whether 5-HT_1F receptor agonist affected the paw withdrawal threshold (PWT) in naïve rats, lasmiditan (100 μg, i.t.) was given once daily for five consecutive days. The behavioral test was performed on 2 h after lasmiditan injection each day. The syringes containing the various drugs were randomly coded by another researcher. The drugs in the syringes were injected into the rats according to the codes.

Behavioral tests

To measure mechanical allodynia, PWT in response to von Frey filament stimuli was assessed as described previously [34]. In brief, the rats were placed in individual pLastic boxes on a metal mesh floor and allowed to habituate for 30 minutes. The von Frey filaments (2, 4, 6, 8, 10, and 15g) were applied for up to 6 seconds per filament to the mid-plantar of the right hind paw. The positive response was defined as the sudden paw withdrawal, licking, and shaking. When a positive response was appeared, the paw was retested after a 5-minute rest, starting with the next descending von Frey filament. When no response occurred, the next higher filament was applied. The lowest amount of force required to elicit a positive response was recorded as the PWT (in grams). The result was shown in Log PWT. All of the behavioral tests were performed by an investigator who was blinded to the experimental design.

Western blotting

The antibody-based procedures complied with the recommendations of the British Journal of Pharmacology [35]. The rats were anesthesia with 2.5% isoflurane. The L4-6 spinal cord was immediately removed and homogenized in an ice-cold mixture of radioimmunoprecipitation assay lysis buffer, phosphatase inhibitor, and phenylmethylsulfonyl fluoride (Boster Biological Technology, Wuhan, Hubei, China), and then centrifuged at 12,000 rpm at 4 ℃ for 30 min. The supernatants were collected and the protein concentration was determined using the Bradford method. The proteins were boiled at 95 °C in a loading buffer for 10 min. Equivalent amounts of samples (30 μg protein) were separated using 10% sodium dodecyl sulfate-polyacrylamide gels electrophoresis and transferred onto polyvinylidene fluoride membranes (Cat# IPVH00010; Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (0.1%) (TBST) for 2 h at room temperature (RT), the membranes were incubated overnight at 4 ℃ with rabbit anti-5-HT_1F receptor antibody (1:500; Cat# DF3499; RRID:AB_2835861; Affinity; OH; USA); rabbit anti-PGC-1α antibody (1:1000; Cat# ab54481; RRID:AB_881987; Abcam, Cambridge, UK); rabbit anti-NRF1 antibody (1:1000; Cat# ab175932; RRID:AB_2629496; Abcam); rabbit anti-TFAM antibody (1:500; Cat# ab131607, RRID:AB_11154693; Abcam); mouse anti-glial fibrillary acidic protein (GFAP, astrocytic marker) antibody (1:500; Cat# 3670; RRID:AB_561049; Cell Signaling Technology, Danvers, MA, USA); goat anti-ionizedcalcium-binding adapter molecule 1(Iba-1, microglial marker) antibody (1:500; Cat# ab5076; RRID:AB_2224402; Abcam); rabbit-anti interleukin-1 beta antibody (IL-1β; 1:1000; Cat# AF4006; RRID:AB_2801567; Affinity); rabbit anti-tumor necrosis factor alpha antibody (TNF-α,1:500; Cat# ab205587; RRID:AB_2889389; abcam); mouse anti-IL-6 antibody (1:500; Cat# A0286; RRID:AB_2757098; ABclonal, Woburn, MA, USA); mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) (1:5000; Cat# AC033; RRID:AB_2769570; ABclonal), respectively. The membranes were then washed in TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:5000; Cat# A21020; RRID:AB_2876889; Abbkine, Wuhan, Hubei, China) , goat anti-mouse secondary antibody (1:5000; Cat# A21010; RRID:AB_2728771; Abbkine) or donkey anti-goat antibody (1:5000; Cat# AS031; RRID:AB_2769846; ABclonal) for 2 h at RT. The bands were finally visualized with SuperLumia ECL Plus HRP Substrate Kit (Cat# K22030; Abbkine) and then detected using a computerized image analysis system (Bio-Rad, ChemiDoc XRS+, USA). The intensity of protein blots was quantified using System with Image Lab software (Bio-Rad Laboratories), normalized to loading control GAPDH antibody and expressed as the fold of control. The blot density of naïve groups was set as 1. The original data was coded by a researcher, the different codes represented different groups, and the data were analyzed by another researcher.

Immunofluorescence

The antibody-based procedures complied with the recommendations of the British Journal of Pharmacology [35]. Under deep anesthesia with 2.5% isoflurane, the rats were perfused intracardially with 0.1 M phosphate buffered saline (PBS), followed by 4% ice-cold paraformaldehyde (PFA) in PBS. Then the L4-6 spinal cord was removed and post-fixed in 4% PFA for 4 h, and subsequently dehydrated in 30% sucrose solution overnight at 4 ℃. The collected spinal cord samples were sectioned to 20 μm thickness in a cryostat (CM1900, Leica, Wetzlar, Germany). For single immunofluorescence: the sections were blocked with 5% goat serum or donkey serum (for detecting the expression of Iba-1) and 0.3% Triton X-100 for 1 h at RT, and then incubated with rabbit anti-5HT_1F receptor antibody (1:50; Cat# DF3499; RRID:AB_2835861; Affinity), mouse anti-GFAP antibody (1:100; Cat# 3670; RRID:AB_561049; Cell Signaling Technology) or goat anti-Iba-1 antibody (1:50; Cat# ab5076; RRID:AB_2224402; Abcam) overnight at 4 ℃. After washing 3 times in PBS, the sections were incubated with Alexa Fluor 594-labeled goat anti-rabbit secondary antibody (1:300; Cat# 111-585-003; RRID:AB_2338059; Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor 488-labeled goat anti-mouse secondary antibody (1:200; Cat# 115-545-003; RRID:AB_2338840; Jackson ImmunoResearch) or FITC-conjugated affinipure donkey anti-goat secondary antibody (1:50; Cat# SA00003-3; RRID:AB_2857365; Proteintech, Chicago, IL, USA) for 2 h at RT. For double immunofluorescence: the sections were blocked with 5% goat serum and 0.3% Triton X-100 for 1 h at RT, and then incubated with a mixture of rabbit anti-5HT_1F receptor antibody (1:50; Cat# DF3499; RRID:AB_2835861; Affinity) and mouse anti-GFAP antibody (1:100; Cat# 3670; RRID:AB_561049; Cell Signaling Technology) or mouse anti-neuronal nuclei antibody (NeuN, neuronal marker) antibody (1:50; Cat# ab104224; RRID:AB_10711040; Abcam) or mouse anti-calcitonin gene-related peptide antibody (CGRP, 1:100; Cat# ab81887, RRID:AB_1658411; Abcam) or FITC conjugated isolectin B4 (IB4, 1:100; Cat# L2140;RRID; AB_2313663; Sigma-Aldrich; St. Louis, Missouri, USA). Then, the sections were incubated with a mixture of secondary antibodies including Alexa Fluor 594-labeled goat anti-rabbit secondary antibody (1:300; Cat# 111-585-003; RRID:AB_2338059; Jackson ImmunoResearch) and Alexa Fluor 488-labeled goat anti-mouse secondary antibody (1:200; Cat# 115-545-003; RRID:AB_2338840; Jackson ImmunoResearch) for 2 h at RT. The sections were blocked with 5% donkey serum and 0.3% Triton X-100 for 1 hour at RT, and then incubated with a mixture of rabbit anti-5HT_1F receptor antibody (1:50; Cat# DF3499; RRID:AB_2835861; Affinity) and goat anti-Iba-1antibody (1:50; Cat# ab5076; RRID:AB_2224402; Abcam). Then, the sections were incubated with a mixture of secondary antibodies including CoraLite 594-conjugated donkey anti-rabbit secondary antibody (1:100; Cat# SA00013-8; RRID:AB_2857367; Proteintech) and FITC-conjugated affinipure donkey anti-goat secondary antibody (1:50; Cat# SA00003-3; RRID:AB_2857365; Proteintech). Images were captured using a fluorescence microscope (BX51, OLYMPUS, Japan). To measure the mean intensity of 5-HT_1F receptor, NeuN, GFAP or Iba-1, three sections from each animal were evaluated. As described previously, the mean fluorescence intensity was calculated using Image J (National Institutes of Health, Bethesda, MD, USA) [36, 37]. The average fluorescence intensity of each pixel was normalized to the background intensity in the same image. To measure the ratio of colocalization in the dorsal horn, the percentage of overlapped pixels (5-HT_1FR⁺/marker⁺) from total marker⁺ pixels were used to represent the degree of colocalization. All images were analyzed by an investigator blinded to the experimental design. The original data was coded by a researcher, the different codes represented different groups, and the data were analyzed by another researcher.

Absolute quantification of mitochondrial DNA (mtDNA) copy number

Under deep anesthesia with 2.5% isoflurane, the rat of L4-6 spinal cord was removed. The DNA isolation of the spinal cord was performed according to the manufacturer’s protocol (DNO7; Aidlab Biotechnologies; Beijing; China) and stored at -20 ℃ until use. The protocols for the absolute quantification of mtDNA copy number were as follows: First, we constructed standard detection pUC57-mtDNA pLasmid, according to the conserved sequence of mtDNA, and then the standard pLasmid was transfected into DH5 α competent cells for further propagation and purification. Second, the concentration and DNA copy number of standard pLasmids were determined according to the follow: copies/μL= (6.02 * 10^23) * (pLasmid concentration ng/μL * 10^-9) / (DNA length * 660). Third, the standard pLasmids (DNA copy number) were diluted with DNase ddH₂O and the range of dilutions were 10^6-10^0 and the diluted pLasmids were used as templates for real time polymerase chain reaction amplification (RT-PCR) (7900HT, Applied Biosystem; USA). Fourth, the standard curve (Y= -0.6388 * X+17.554, X=Ct, Y=log copy number) was made according to the results of RT-PCR. PCR reaction system (20μL) includes 4 μL DNA, 10 μL PCR mix, 1 μL mtDNA primer forward, 1 μL mtDNA primer reverse and 4 μL ddH₂O. The protocols for the PCR were as follows: pre-incubation at 95 °C for 5 min (1 cycle); denaturation at 95 °C for 10 s, annealing and extension at 58 °C for 30 s, 72 ℃ for 15s (repeat denaturation and extension steps for 40 cycles), melting at 95 °C for 15 s, 60 °C for 60 s, (melt curve analysis: 1 cycle). The primers used were as follows: mtDNA forward: 5ʹ- CGCCAGGGTTTTCCCAGTCACGAC- 3ʹ, mtDNA reverse: 5ʹ- AGCGGATAACAATTTCACACAGGA- 3ʹ. Last, the lumbar spinal cord samples were amplified by the same PCR system, and the absolute mtDNA copy number was calculated by standard curve. The original data was coded by a researcher, the different codes represented different groups, and the data were analyzed by another researcher.

Experimental design

All experiments were performed under blinded conditions. The rats were randomly assigned to different groups and allocated to groups using a Research Randomizer (available at https://www.randomizer.org/). Moreover, the sample size of each group was determined according to the Calculators (http://powerandsamplesize.com/Calculators/). The spared nerve injury model results in early (＜24 h), prolonged (＞6 months), robust (all animals are responders) behavioral modifications, moreover, the mechanical (von Frey) sensitivity is increased in the ipsilateral sural and to a lesser extent saphenous territories. For this reason, we chose spared nerve injury as the model of neuropathic pain in this study. There were no excluded animals during the experimental period.

Experiment 1: To explore whether the impaired mitochondrial biogenesis was participated in the development of neuropathic pain, 108 rats were randomly assigned to six groups (n = 18 rats each group). When the final behavioural test was finished (we only showed the behavioral results of 10 rats in each group), the L4-6 vertebra lumbalis were collected for WB, IF, and quantification of mtDNA copy number.

Experiment 2: To determine whether a single dose of PGC-1α activator could attenuate established mechanical allodynia in SNI rats, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 3: To determine whether repeated injection of a PGC-1α activator agonist could reverse mechanical allodynia in SNI rats, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 4: To determine whether early treatment with a PGC-1α activator could suppress the development of mechanical allodynia in SNI rats, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 5: To determine whether a PGC-1α inhibitor could reverse the analgesic effects of a PGC-1α activator, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 6: To determine whether a single dose of a 5-HT1F receptor agonist could attenuate established mechanical allodynia in SNI rats, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 7: To determine whether repeated injection of a 5-HT1F receptor agonist could reverse mechanical allodynia in SNI rats, 72 rats were randomly assigned to four groups (n = 18 rats in each group). When the final behavioural test was finished (we only showed the behavioral results of 10 rats in each group), the L4-6 spinal cord was collected for WB, IF, and quantification of mtDNA copy number.

Experiment 8: To determine whether early treatment with a 5-HT1F receptor agonist could suppress the development of mechanical allodynia in SNI rats, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 9: To determine whether a PGC-1α inhibitor could reverse the analgesic effects of a 5-HT1F receptor agonist, 40 rats were randomly assigned to four groups (n = 10 rats each group).

Experiment 10: To determine whether 5-HT1F receptor agonist affect the paw withdrawal threshold (PWT) in naïve rats, 36 rats were randomly assigned to three groups (n = 12 rats in each group). When the final behavioural test was finished (we only showed the behavioral results of 10 rats in each group), the L4-6 spinal cord was collected for WB and quantification of mtDNA copy number.

Data and Statistical Analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology [38]. Statistical analysis was performed blindly on these independent values. No data points were excluded, and no additional data were subjected to statistical analysis in any experiment. Each animal provided an independent value, and statistical analyses were performed with these numbers. All data are presented as mean ± standard error of the mean (SEM) and analyzed by GraphPad Prism version 6 (Graph Pad Software, San Diego, CA, USA). Data normality was verified with a Shapiro-Wilk's test except for the results of the western blot. Data derived from the Gaussian distribution were further tested for conformity to the ANOVA. Others were log-transformed or tested by non-parametric analysis. For the western blot, normalization was performed to compare the differences among groups, control for unwanted sources of variation, and reveal relevant trends. The protein level was presented as density relative to that of GAPDH. Data were expressed as fold change of sham group. Normalization removed variance from the control values (SEM = 0). Data were subjected to the Kruskal-Wallis test followed by Dunn's post hoc test for non-parametric statistical analysis. In figures, the “fold matched control” was indicated as “relative protein densitometry”. The mtDNA copy number data were analyzed by one-way ANOVA followed by Bonferroni's post hoc test for parametric analysis. The post hoc test was performed only if the F was significant and there was no variance inhomogeneity. The behavioral test was analyzed by two-way, repeated-measures ANOVA (RM ANOVA; treatment group × time) to detect overall differences among treatment groups, followed by Bonferroni's test to detect changes in Log PWT after drug injection over time. Before two-way RM ANOVA, the behavioral test data were analyzed by Mauchly's sphericity test, followed by multivariate ANOVA (MANOVA). When the data did not violate the sphericity assumption (p > 0.05), the MANOVA F value was analyzed by assumed degrees of freedom for sphericity. However, if the sphericity assumption was violated (p < 0.05), the F value was analyzed by Greenhouse–Geisser's corrected degrees of freedom. When the F value was significant, the data were analyzed by Bonferroni's post hoc test. The single immunofluorescent staining were analyzed by one-way ANOVA followed by Bonferroni's post hoc test for parametric analysis. The post hoc test was performed only if the F was significant and there was no variance inhomogeneity. The double immunofluorescent staining was analyzed by an unpaired Student's t test when the data did not violate the sample variance homogeneity assumption (p > 0.05). A p < 0.05 was interpreted as statistically significant for all analyses.

Mechanical allodynia induced by spared nerve injury and mitochondrial biogenesis was impaired in the spinal cord of neuropathic pain rats

In this study, we used a well-established rat model of neuropathic pain induced by SNI. PWT was assessed to evaluate the development of mechanical allodynia at baseline and 1, 3, 7 and 14 days after surgery. As shown in Fig. 1a (group: F (2, 27) = 47.33, p < 0.0001; time: F (4, 108) = 36.07, p < 0.0001; interaction: F (8, 108) = 20.1, p < 0.0001), there is no significant difference regarding the PWT among naïve, sham and SNI rats at baseline. However, the PWT in SNI rats was markedly decreased from day 3 to day 14 (^****p < 0.0001 compared with sham group, n = 10 in each group). In contrast, the PWT in sham rats had no significant change during the observation period. These results indicate that SNI successfully induced the development of mechanical allodynia.

To date, little is known regarding the role of mitochondrial biogenesis in neuropathic pain. We first determined whether mitochondrial biogenesis was impaired in SNI rats. mtDNA copy number and the protein level of PGC-1α, NRF1 and TFAM in the spinal cord were examined. As shown in Fig. 1b, mtDNA copy number was significantly and time dependently decreased in SNI rats, beginning at day 3 after surgery and remain at low level until day 14 (F (5, 30) = 33.81, p < 0.0001; ^**p < 0.01, ^****p < 0.0001 compared with sham group, n = 6 in each group). Similarly, the protein level of PGC‐1α, NRF1 and TFAM were significantly decreased in SNI rats (Fig. 1c: F (5, 30) = 19.54, p < 0.0001; Fig. 1d: F (5, 30) = 40.8, p < 0.0001; Fig. 1e: F (5, 30) = 9.504, p < 0.0001; ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared with sham group, n = 6 in each group). These results suggest that mitochondrial biogenesis was impaired in the spinal cord of SNI rats.

Analgesic effect of PGC-1α activator on mechanical allodynia in neuropathic pain rats

Then, we determined whether activation of PGC-1α, the master regulator of mitochondrial biogenesis, could attenuate mechanical allodynia in neuropathic pain rats. To determine whether a single dose of PGC-1α activator could attenuate established mechanical allodynia in SNI rats, ZLN005 (10, 50, or 100 µg, i.t.) was given on day 7 after surgery. The behavioral test was conducted before ZLN005 injection, and 1, 2, 4, 6, and 12 h after the injection. There is no significant change regarding PWT in SNI rats treated with 10 µg ZLN005 (Fig. 2a: group: F (3, 36) = 4.072, p = 0.0137; time: F (8, 288) = 181.2, p < 0.0001; interaction: F (24, 288) = 5.284, p < 0.0001; p > 0.05 compared with SNI+Vehicle group, n = 10 in each group). However, ZLN005 (50 and 100 µg) significantly increased the PWT in SNI rats, peaking at 1 h, and lasted for at least 2 h (Fig. 2a, ^****p < 0.0001 compared with SNI+Vehicle group, n = 10 in each group). To determine whether repeated injection of a PGC-1α activator could reverse mechanical allodynia in SNI rats, ZLN005 (50 µg, i.t.) was given once daily for five consecutive days starting from day 7. The behavioral test was performed on day 6 and 1 h after ZLN005 injection each day. As shown in Figure 2b (group: F (3, 36) = 58.26, p < 0.0001; time: F (6, 216) = 47.39, p < 0.0001; interaction: F (18, 216) = 12.7, p < 0.0001), repetitive injections of ZLN005 (50 µg, i.t.) considerably reversed established mechanical allodynia in SNI rats (^****p < 0.0001 compared with SNI+Vehicle group, ^####p < 0.0001 compared with Sham+Vehicle group, n = 10 in each group). To determine whether early treatment with a PGC-1α activator could suppress the development of mechanical allodynia in SNI rats, ZLN005 (50 µg, i.t.) was given once daily for seven consecutive days starting from day 1 after the surgery. The behavioral test was conducted before the surgery, and on day 1, 3, 7, 8, 9, 10, 11, 12, 13 and 14 after surgery. As shown in Fig. 2c (group: F (3, 36) = 74.54, p < 0.0001; time: F (10, 360) = 44.01, p < 0.0001; interaction: F (30, 360) = 15.3, p < 0.0001), the PWT was significantly increased from day 3 to day 12 in ZLN005-treated SNI rats compared with vehicle-treated SNI rats (^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared with SNI+Vehicle group, ^####p < 0.0001 compared with Sham+ Vehicle group, n = 10 in each group). To determine whether a PGC-1α inhibitor could reverse the analgesic effects of ZLN005, SR-18292 (30 µg, i.t.) was given 30 min before ZLN005. The behavioral test was conducted before SR-18292injection, and 1, 2, 4, 6, and 12 h after ZLN005 injection. As shown in Fig. 2d (group: F (3, 36) = 3.149, p = 0.0367; time: F (8, 288) = 199.6, p < 0.0001; interaction: F (24, 288) = 7.307, p < 0.0001), the analgesic effects of ZLN005 were reversed by PGC-1α inhibitor SR-18292 (^****p < 0.0001 compared with SNI+Vehicle group, ^###p < 0.001, ^####p < 0.0001 compared with SNI+ZLN005 50 µg+SR-18292 30 µg group, n = 10 in each group). These results indicate that ZLN005 can markedly attenuate established mechanical allodynia in SNI rats, and also delayed the development of mechanical allodynia in SNI rats. Moreover, activation of PGC-1α contributes to the analgesic effects of ZLN005.

Expression and cellular localization of 5-HT_1F receptor in the spinal cord of sham and neuropathic pain rats

To determine the expression and cellular localization of 5-HT_1F receptor in sham and neuropathic pain rats, western blot and immunofluorescence were used. As shown in Fig. 3a-b (Fig. 3b: F (5, 30) = 12.94, p < 0.0001), the expression of 5-HT_1F receptor was significantly decreased in SNI rats, beginning at day 3 after surgery and remain at low level until day 14 (^**p < 0.01 compared with sham group, n = 6 in each group). Consistently, the immunofluorescence results showed decreased expression of 5-HT_1F receptor in the spinal cord dorsal horn of SNI rats (Figs. 3c-i, Fig. 3i: F (5, 30) = 23.15, p < 0.0001; ^****p < 0.0001 compared with sham group, n = 6 in each group). Our double immunofluorescence results showed that 5-HT_1F receptor was mainly colocalized with NeuN (a neuronal marker) in the spinal cord dorsal horn of sham and SNI rats, while the colocalization of 5-HT1F receptor with NeuN was decreased in the SNI rats (Fig. 4, Fig. 4g: t = 2.493, p = 0.0318; Fig. 4h: t = 1.126, p = 0.2864; Fig. 4i: t = 1.414, p = 0.1878; *p < 0.05 compared with sham group, n = 6 in each group). Moreover, 5-HT_1F receptor was also colocalized with CGRP and IB4 in the spinal cord dorsal horn of sham and SNI rats, while the colocalization of 5-HT1F receptor with CGRP was decreased in the SNI rats (Figure 5, Fig. 5e: t = 3.142, p = 0.0105; Fig. 5f: t = 0.79; p = 0.4478; *p < 0.05 compared with sham group, n = 6 in each group).

Analgesic effect of 5-HT_1F receptor agonist on mechanical allodynia in neuropathic pain rats

To determine whether a single dose of 5-HT_1F receptor agonist could attenuate established mechanical allodynia in SNI rats, lasmiditan (50, 100 or 200 µg, i.t.) was given on day 7 after surgery. The behavioral test was conducted before lasmiditan injection, and 1, 2, 4, 6, and 12 h after the injection. There is no significant change regarding PWT in SNI rats treated with 50 µg lasmiditan (Fig. 6a: group: F (3, 36) = 4.795, p = 0.0065; time: F (8, 288) = 120, p < 0.0001; interaction: F (24, 288) = 8.253, p < 0.0001; p > 0.05 compared with SNI+Vehicle group, n = 10 in each group). However, lasmiditan (100 and 200 µg) markedly increased the PWT in SNI rats, beginning at 1 h, peaking at 2 h, and lasted for at least 6 h (Fig. 6a, ^***p < 0.001, ^****p < 0.0001 compared with SNI+Vehicle group, n = 10 in each group). To determine whether repeated injection of a 5-HT_1F receptor agonist could reverse mechanical allodynia in SNI rats, lasmiditan (100 µg, i.t.) was given once daily for five consecutive days starting from day 7. The behavioral test was performed on day 6 and 2 h after lasmiditan injection each day. As shown in Fig. 6b (group: F (3, 36) = 37.3, p < 0.0001; time: F (6, 216) = 25.9, p < 0.0001; interaction: F (18, 216) = 8.494, p < 0.0001), repetitive injections of lasmiditan (100 µg, i.t.) considerably reversed established mechanical allodynia in SNI rats (^****p < 0.0001 compared with SNI+Vehicle group, ^####p < 0.0001 compared with Sham+Vehicle group, n = 10 in each group). To determine whether early treatment with a 5-HT_1F receptor agonist can suppress the development of mechanical allodynia in SNI rats, lasmiditan (100 µg, i.t.) was given once daily for seven consecutive days starting from day 1 after the surgery. The behavioral test was conducted before the surgery, and on day 1, 3, 7, 8, 9, 10, 11, 12, 13 and 14 after surgery. As shown in Fig. 6c (group: F (3, 36) = 43.04, p < 0.0001; time: F (10, 360) = 33.37, p < 0.0001; interaction: F (30, 360) = 12.29, p < 0.0001), the PWT was significantly increased from day 3 to day 10 in lasmiditan-treated SNI rats compared with vehicle-treated SNI rats (^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 compared with SNI+Vehicle group, ^####p < 0.0001 compared with Sham+ Vehicle group, n = 10 in each group). To determine whether a PGC-1α inhibitor could reverse the analgesic effects of lasmiditan, SR-18292 (30 µg, i.t.) was given 30 min before lasmiditan. The behavioral test was conducted before SR-18292 injection, and 1, 2, 4, 6, and 12 h after lasmiditan injection. As shown in Fig. 6d (group: F (3, 36) = 2.99, p = 0.0437; time: F (8, 288) = 160, p < 0.0001; interaction: F (24, 288) = 5.866, p < 0.0001), the analgesic effects of lasmiditan was entirely reversed by PGC-1α inhibitor SR-18292 (^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 compared with SNI+Vehicle group, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 compared with SNI+Las 100 µg+SR-1892 30 µg group, n = 10 in each group). These results indicate that lasmiditan can significantly attenuate established mechanical allodynia in SNI rats, and also delayed the development of mechanical allodynia in SNI rats. Moreover, activation of PGC-1α contributes to the analgesic effects of lasmiditan.

Effect of 5-HT_1F receptor agonist on paw withdrawal threshold and mitochondrial biogenesis in naïve rats

To determine whether 5-HT_1F receptor agonist affect the mtDNA copy number and PWT in naïve rats, lasmiditan (100 µg, i.t.) was given once daily for five consecutive days. The behavioral test was performed on 2 h after lasmiditan injection each day. As shown in Fig. 7a-b (Fig. 7a: F (2, 15) = 0.7022, p = 0.5111; Fig. 7b: group: F (2, 27) = 0.5279, p = 0.5958; time: F (5, 135) = 1.893; p = 0.0996; interaction: F (10, 135) = 0.7842; p = 0.6439), no significant change in the mtDNA copy number and PWT was observed after lasmiditan injection (p > 0.05 compared with Naïve+Vehicle group, n = 10 in each group). To determine whether 5-HT_1F receptor agonist affected mitochondrial biogenesis in naïve rats, lasmiditan (100 µg, i.t.) was given once daily for five consecutive days. Two hours after the final administration, the L4-6 spinal cord was collected to examine mtDNA copy number and the protein level of PGC-1α, NRF1 and TFAM. As shown in Fig. 7c-e (Fig. 7c: F (2, 15) = 1.667, p = 0.2220; Fig. 7d: F (2, 15) = 1.438, p = 0.2683; Fig. 7e: F (2, 15) = 2.101, p = 0.1569), no significant change regarding mtDNA copy number and the protein level of PGC‐1α, NRF1 and TFAM were observed after lasmiditan treatment (p > 0.05 compared with Naïve+Vehicle group, n = 6 in each group). These results suggest that lasmiditan treatment at the current protocol has no impact on paw withdrawal threshold and mitochondrial biogenesis in naïve rats.

Effect of 5-HT_1F receptor agonist on mitochondrial biogenesis in the spinal cord of neuropathic pain rats

To determine whether 5-HT_1F receptor agonist affected mitochondrial biogenesis in SNI rats, lasmiditan (100 µg, i.t.) was given once daily for five consecutive days starting from day 7. Two hours after the final administration, the L4-6 spinal cord was collected. As shown in Fig. 8 (Fig. 8a: F (3, 20) = 5.096, p = 0.0088; Fig. 8b: F (3, 20) = 20.58, p < 0.0001; Fig. 8c: F (3, 20) = 19.21, p < 0.0001; Fig. 8d: F (3, 20) = 34.4; p < 0.0001), mtDNA copy number and the protein level of PGC-1α, NRF1 and TFAM were significantly downregulated in SNI rats, which were reversed by lasmiditan treatment (^*p < 0.05, ^****p < 0.0001 compared with Sham+Vehicle group, ^#p < 0.05, ^####p < 0.0001 compared with SNI+Vehicle group, n = 6 in each group). These results suggest that lasmiditan restored mitochondrial biogenesis in the spinal cord of SNI rats.

Effect of 5-HT_1F receptor agonist on neuroinflammation in the spinal cord of neuropathic pain rats

To determine whether 5-HT_1F receptor agonist affected neuroinflammation in SNI rats, lasmiditan (100 µg, i.t.) was given once daily for five consecutive days starting from day 7. Two hours after the final administration, the L4-6 spinal cord was collected. As shown in Fig. 9a-l (Fig. 9a: F (3, 20) = 44.53, p < 0.0001; Fig. 9b: F (3, 20) = 56.19, p < 0.0001; Fig. 9k: F (3, 20) = 9.339, p = 0.0005; Fig. 9l: F (3, 20) = 42.15, p < 0.0001) the expression of Iba1 and GFAP were significantly increased in the spinal cord of SNI rats, which were markedly suppressed by lasmiditan treatment (^**p < 0.01, ^****p < 0.0001 compared with Sham+Vehicle group, ^##p < 0.01, ^####p < 0.0001 compared with SNI+Vehicle group, n = 6 in each group). Moreover, lasmiditan treatment also inhibited the upregulation of proinflammatory cytokines including IL-1β, IL-6 and TNF-α (Fig. 9m-o, Fig. 9m: F (3, 20) = 20.13, p < 0.0001; Fig. 9n: F (3, 20) = 13.76, p < 0.0001: Fig. 10o: F (3, 20) = 9.059, p = 0.0005, ^*p < 0.05, ^****p < 0.0001 compared with Sham+Vehicle group, ^#p < 0.05, ^####p < 0.0001 compared with SNI+Vehicle group, n = 6 in each group). These results suggest that Lasmiditan suppressed neuroinflammation in the spinal cord of SNI rats.

In this study, we showed that (1) Mitochondrial biogenesis was impaired in the spinal cord of SNI rats; (2) activation of PGC-1α, the master regulator of mitochondrial biogenesis, attenuated established mechanical allodynia, and delayed the onset of mechanical allodynia in SNI rats; (3) neuronal 5-HT_1F receptor was significantly downregulated in the spinal cord of neuropathic pain rats; (4) lasmiditan attenuated established mechanical allodynia, and delayed the onset of mechanical allodynia in SNI rats; (5) lasmiditan treatment restored mitochondrial biogenesis in the spinal cord of SNI rats; (6) lasmiditan treatment attenuated neuroinflammation in the spinal cord of SNI rats. Taken together, these results indicate that 5-HT_1F receptor agonist lasmiditan ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation in the spinal cord.

It is well-established that excessive reactive oxygen species (ROS) accumulation leads to oxidative stress. Our previous study and others have demonstrated that ROS scavengers showed potent analgesic effects against cancer-induced bone pain and neuropathic pain [5, 39]. Nuclear factor erythroid-2-related factor 2 (Nrf2) is the key regulator of endogenous antioxidant defense. Previously, we and others have proved that Nrf2 activator significantly alleviated mechanical allodynia in neuropathic pain [40, 41]. These results indicate that oxidative stress plays a vital role in the development of neuropathic pain. Oxidative phosphorylation at the mitochondria is one of the main sources of ROS [42]. Notably, oxidative stress disrupts mitochondrial respiratory chain, disturbs Ca²⁺ homeostasis, and increases mitochondrial membrane permeability [43]. Therefore, oxidative stress may result in mitochondrial dysfunction, and vice versa. Mitochondrial dysfunction has emerged as a key factor contributing to the development of neuropathic pain [44, 45]. It was reported that impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in neurological diseases such as Alzheimer's disease and spinal cord injury [12, 13]. However, whether mitochondrial biogenesis is impaired in neuropathic pain remains unknown. In this study, we found that mtDNA copy number and the protein level of PGC-1α, NRF1 and TFAM were significantly downregulated in the spinal cord of SNI rats, indicating impaired mitochondrial biogenesis. However, it is worth noting that the mtDNA copy number was progressive decreased following SNI, while the protein level of PGC-1α is relatively stable. This discrepancy might be due to the fact that mitochondrial biogenesis is also regulated by other genes except PGC-1α. Nevertheless, PGC-1α is the master regulator of mitochondrial biogenesis. Therefore, we hypothesized that activation of PGC-1α might attenuate neuropathic pain. Our results showed that a single dose of ZLN005 (a PGC-1α activator) attenuated established mechanical allodynia in SNI rats, which was blocked by PGC-1α inhibitor SR-18292. These results indicate that activation of PGC-1α contributes to the analgesic effects of ZLN005. Besides, repetitive injections of ZLN005 considerably reversed established mechanical allodynia in SNI rats. Moreover, early treatment with ZLN005 for 7 consecutive days from day 1 after surgery delayed the onset of mechanical allodynia in SNI rats. It should be noted that a recent study reported that PGC-1α haploinsufficiency promoted pain chronification after burn injury [14]. Moreover, a very recent study demonstrated that activation of spinal PGC-1α suppressed morphine tolerance by reducing mitochondrial superoxide [46], indicating ameliorative effects of PGC-1α in oxidative stress. Given the pivotal role of oxidative stress in neurological diseases [47], it is plausible that PGC-1α activation attenuates neurological diseases via similar mechanisms. Collectively, our results demonstrated that impaired mitochondrial biogenesis might contribute to the development and maintenance of mechanical allodynia in SNI rats.

Serotonin in the central nervous system has long been considered to play a vital role in the modulation of pain processing [48]. There are accumulating evidence indicating the fundamental role of individual classes of 5-HT receptors in neuropathic pain [49]. For example, intrathecal injection of 5-HT_1A receptor agonist and 5-HT₇ receptor agonist showed potent analgesic effects, respectively [50, 51]. 5-HT_1F receptor is a G-protein coupled receptor subtype whose functions are not fully characterized. Currently, lasmiditan, a highly selective 5-HT_1F receptor agonist, has been approved for the acute treatment of migraine by US Food and Drug Administration [25]. Several lines of evidence indicate that 5-HT_1F receptor agonists are potent inducers of mitochondrial biogenesis [26–29]. However, the role of 5-HT_1F receptor in neuropathic pain remains unclear. In this study, we first examined the expression and cellular localization of 5-HT_1F receptor in the spinal cord. We found that the expression of 5-HT_1F receptor was significantly decreased in SNI rats. Furthermore, our double immunofluorescence results showed that 5-HT_1F receptor was mainly colocalized with neuron in the spinal cord dorsal horn of sham and SNI rats. Our behavioral tests showed that a single dose of lasmiditan attenuated established mechanical allodynia in SNI rats, which was blocked by PGC-1α inhibitor SR-18292. These results indicate that activation of PGC-1α contributes to the analgesic effects of lasmiditan. Moreover, repetitive injections of lasmiditan considerably reversed established mechanical allodynia in SNI rats. Furthermore, early treatment with lasmiditan for 7 consecutive days from day 1 after surgery delayed the onset of mechanical allodynia in SNI rats. Notably, repetitive injections of lasmiditan had no significant effect on the PWT in naïve rats. These results indicate that 5-HT_1F receptor is downregulated in the spinal cord of SNI rats, and lasmiditan possesses potent analgesic effect against SNI-induced neuropathic pain. Importantly, lasmiditan treatment has no significant effect on mitochondrial biogenesis in the spinal cord of naïve rats, but restored mitochondrial biogenesis in the spinal cord of SNI rats. Recently, Simmons et al. found that naïve mice treated with 5HT1F agonist LY344864 (2.0 mg/kg, i.p.) daily for 21 days displayed a 1.4-fold increase in mtDNA content and 1.3-fold increase in PGC-1α mRNA expression [23]. The discrepancy might be explained by difference in drug, route of drug administration, treatment protocol and animal species. Nevertheless, they found that LY344864 promoted recovery from spinal cord injury via induction of mitochondrial biogenesis. These results indicate that 5-HT_1F receptor agonist attenuate neuropathic pain by restore mitochondrial biogenesis in the spinal cord. Moreover, as lasmiditan is orally bioavailable and central nerve system penetrant, with minimal off target adverse effects [25]. A systemic route of administration with lasmiditan may have therapeutic potential for neuropathic pain.

Recent evidence indicates that PGC-1α activation exerts neuroprotective effects via suppressing neuroinflammation in animal models of Parkinson’s disease, depression and brain injury [15, 16, 52]. Accumulating evidence from our lab and others has demonstrated the pivotal role of neuroinflammation in chronic pain [53, 54]. Therefore, we tested the hypothesis that 5-HT_1F receptor agonist could attenuate neuropathic pain via suppression of neuroinflammation. As expected, repeated treatment with lasmiditan significantly inhibited the activation of microglia and astrocyte in the spinal cord. Moreover, lasmiditan also suppressed the upregulation of proinflammatory cytokines including IL-1β, IL-6 and TNF-α. These results indicate that 5-HT_1F receptor agonist attenuate neuropathic pain by suppressing neuroinflammation in the spinal cord. Notably, it was reported that lasmiditan could potentially blocked the release of CGRP and the neurotransmitter glutamate, thus preventing and possibly reversing the development of central sensitization [55]. Therefore, lasmiditan might attenuate neuropathic pain by inhibiting the release of CGRP from primary afferents. Moreover, it has been demonstrated that 5-HT_1F receptor was expressed in rat dorsal root ganglia neurons [56] and activation of peripheral 5-HT_1F receptor had analgesic effects [24], which suggested that a systemic route of administration with lasmiditan may have the potential for greater efficacy as well as clinical relevance.

In summary, this study provided the first evidence that impaired mitochondrial biogenesis in the spinal cord contributes to the development and maintenance of mechanical allodynia in SNI rats, and lasmiditan significantly mitigates established neuropathic pain in SNI rats by induction of mitochondrial biogenesis and suppression of neuroinflammation. Mitochondrial biogenesis inducers may become encouraging therapeutic option for the management of neuropathic pain, although this would require demonstration that the mechanisms detailed here also exist in humans.

DMSO: dimethyl sulfoxide

5-HT: 5-hydroxytryptamine

mtDNA: mitochondrial DNA

NRF1: nuclear respiratory factor 1

Nrf2: Nuclear factor erythroid-2-related factor 2

PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1α

PWT: paw withdrawal threshold

ROS: reactive oxygen species

SNI: spared nerve injury

TFAM: mitochondrial transcription factor A

Acknowledgements

Not applicable

Authors’ contributions

WM and XBT developed the concept of the study; LQZ and YQZ designed the study; JYL, JS and SZ provided experimental support; JYW and SJG analyzed and interpreted the data. LQZ and YQZ wrote the paper.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant Nos.81974170, 82001198, 82071556, and 81873793).

Availability of data and materials

The data and materials supporting the conclusions of this study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All experiments were approved by the Experimental Animal Care and Use Committee of Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the ARRIVE Guidelines for Reporting Animal Research.

Consent for publication

Not applicable

Competing interests

The authors declare no conflict of interests.

Author details

¹Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, People’s Republic of China.

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5-HT_1F Receptor Agonist Ameliorates Mechanical Allodynia In Neuropathic Pain Via Induction of Mitochondrial Biogenesis and Suppression of Neuroinflammation

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