MicroglialBDNF,PI3K,andp-ERKintheSpinalCordAre

Research ArticleMicroglial BDNF, PI3K, and p-ERK in the Spinal Cord AreSuppressed by Pulsed Radiofrequency onDorsal Root Ganglion toEase SNI-Induced Neuropathic Pain in Rats

Xueru Xu, Shaoxiong Fu, Xiaomei Shi, and Rongguo Liu

Department of Pain Management, Fujian Provincial Hospital, Fujian Key Laboratory of Geriatrics,Provincial Clinic College of Fujian Medical University, Fuzhou, Fujian, China

Correspondence should be addressed to Rongguo Liu; [email protected]

Received 30 November 2018; Revised 7 February 2019; Accepted 28 March 2019; Published 28 April 2019

Guest Editor: Ke Ma

Copyright © 2019 Xueru Xu et al. -is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Pulsed radiofrequency (PRF) on the dorsal root ganglion (DRG) has been applied to alleviate neuropathic paineffectively, yet the mechanisms underlying pain reduction owing to this treatment are not clarified completely. -e activatedmicroglia, brain-derived neurotrophic factor (BDNF), phosphatidylinositol 3-kinase (PI3K), and phosphorylated extracellularsignal-regulated kinase (p-ERK) in the spinal cord were demonstrated to be involved in developing neuropathic pain. Also, it hasbeen just known that PRF on DRG inhibits the microglial activation in nerve injury rats. Here, we aim to investigate whether PRFtreatment could regulate the levels of BDNF, PI3K, and p-ERK in the spinal cord of rats with spared nerve injury (SNI) viasuppressing the spinal microglia activation to ease neuropathic pain. Methods. -e rats with SNI were intrathecally treated withminocycline (specific microglia inhibitor) or same volume of dimethyl sulfoxide once daily, beginning from 1 h before nervetransection to 7 days. PRF was applied adjacent to the L4-L5 DRG of rats with SNI at 45V for 6min on the seventh postoperativeday, whereas the free-PRF rats were treated without PRF.-e withdrawal thresholds were studied, and the spinal levels of ionizedcalcium-binding adapter molecule 1 (Iba1), BDNF, PI3K, and p-ERK were calculated by western blot analysis, reversetranscription-polymerase chain reaction, and immunofluorescence. Results. -e paw withdrawal mechanical threshold and pawwithdrawal thermal latency decreased in the ipsilateral hind paws after SNI, and the spinal levels of Iba1, BDNF, PI3K, and p-ERKincreased on day 21 after SNI compared with baseline (P< 0.01). An intrathecal injection of minocycline led to the reversal of SNI-induced allodynia and increase in levels of Iba1, BDNF, PI3K, and p-ERK.Withdrawal thresholds recovered partially after a singlePRF treatment for 14 days, and SNI-induced microglia hyperactivity, BDNF upregulation, and PI3K and ERK phosphorylation inthe spinal cord reduced on D14 due to the PRF procedure. Conclusion. Microglial BDNF, PI3K, and p-ERK in the spinal cord aresuppressed by the therapy of PRF on DRG to ease SNI-induced neuropathic pain in rats.

1. Introduction

Neuropathic pain is a kind of refractory pain that arises as adirect consequence of a lesion or disease affecting the so-matosensory system [1, 2]. A variety of damages to theperipheral nerves, including diabetes, zoster virus, humanimmunodeficiency virus-acquired immunodeficiency syn-drome, and compression injury, can result in neuropathicpain [3]. Neuropathic pain, characterized by hyperalgesia orallodynia, is associated with central and peripheral sensiti-zation of neurons in the nociceptors [4, 5]. It is hard to treat

due to the complicated etiology and mechanisms, includingseveral neurotransmitter systems, receptors, ionic channels,and cell types [6, 7]. -us, current pharmacotherapy rarelyresolves intractable pain in patients. Pulsed radiofrequency(PRF), a type of electromagnetic stimulation, has beensuccessfully used to treat patients suffering from neuropathicpain [8, 9]. PRF on dorsal root ganglion (DRG) is consideredto be superior to continuous radiofrequency because theelectrode tip temperature of PRF does not exceed 42°Cduring the whole process to avoidmassive tissue destruction.Nowadays, the application of PRF on DRG to treat

HindawiPain Research and ManagementVolume 2019, Article ID 5948686, 15 pageshttps://doi.org/10.1155/2019/5948686

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https://doi.org/10.1155/2019/5948686

neuropathic pain has greatly helped clinicians. However, theanalgesic mechanism of this therapy is not well clarified sofar.

Recently, PRF was administered on DRG in rats withperipheral nerve injury (PNI) to downregulate microglialactivation in the spinal cord and improve pain behaviors[10, 11]. Microglia are the resident macrophages in thecentral nervous system (CNS), and they react to the stimulithat may affect homeostasis and induce pathological alter-ations [12]. As a consequence ofmultiple types of damages inthe nervous system, microglia can transform to reactivestates through a progressive series of cellular and molecularchanges, including morphological hypertrophy, pro-liferation, and expression of various genes [13].-e activatedmicroglial cells play a key role in the peripheral and centralsensitization to develop neuropathic pain conditions [14].-ey secrete brain-derived neurotrophic factor (BDNF),which is a critical microglia-neuron signaling molecule thatgates aberrant nociceptive processing in the spinal cord [15].Many studies support the pronociceptive role of BDNF inpain processes in the peripheral and CNS. Nociceptor-derived BDNF has been shown to be involved in in-flammatory pain and microglial-derived BDNF in neuro-pathic pain [16]. Recently, Liu et al. [17] reported that BDNFparticipated in colitis-induced spinal central sensitization,and the phosphatidylinositol 3-kinase (PI3K)/protein kinaseB pathway mediated BDNF action in the spinal cord.Moreover, the second messengers that PI3K generated couldactivate phosphorylated extracellular signal-regulated kinase(p-ERK) [18, 19]. Inmicroglia, ERK activation occurred afternerve injury, and the inhibition of the activated ERK couldsuppress neuropathic pain development [20]. -e spinalcord, which is the primary integration center of informationand plays a crucial role in central sensitization, was preferredby pain physicians for exerting neuromodulation to relieveneuropathic pain, such as spinal cord stimulation. In pre-cedent different animal models, the microglia, BDNF, PI3K,and p-ERKwere involved in the development of neuropathicpain. However, it remains uncertain that whether the releaseof BDNF, PI3K and p-ERK in the spinal cord regulateschronic pain processing through microglia-dependentmechanism. In addition, whether the application of PRFon DRG for treating neuropathic pain is associated with thedownregulated levels of microglia, BDNF, PI3K, and p-ERKin the spinal cord needs exploration further.

In this study, a microglial inhibitor was intrathecallyadministered to the rats with spared nerve injury (SNI) toaffirm the release of BDNF, PI3K, and p-ERK in the spinalcord via the microglial-dependent mechanism. We alsoinvestigated whether PRF treatment could regulate the levelsof BDNF, PI3K, and p-ERK in the spinal cord of SNI rats viasuppressing the spinal microglia activation to alleviate theneuropathic pain.

2. Materials and Methods

2.1. Animals. Male Sprague–Dawley (SD) rats (4-month-old, 250–280 g) were obtained from the Experimental An-imal Center of Fujian Medical University, Fuzhou. -e

animals were housed under a 12 h light-dark cycle at22°C–24°C with ad libitum access to food and water in thePharmacy College of Fujian Medical University (SPF class).All procedures in this study were approved by the FujianMedical University Experimental Animal Welfare EthicsCommittee (SYXK 2016-0007).

2.2. Treatment Group and Design. Ninety male SD rats wererandomly (according to the method of random numbertable) divided into six groups (n� 15, each): Sham group,SNI group, SNI + PRF group, SNI + free-PRF group, SNIwith minocycline (SNI +M) group, and SNI with dimethylsulfoxide (SNI +DMSO) group.

2.2.1. Effect of Minocycline on Levels of Microglia, BDNF,PI3K, and ERK in the Spinal Cord. -e rats in the followingfour groups (Sham, SNI, SNI +M, and SNI +DMSO) werestudied. All rats (except the Sham group) were subjected toSNI of the right sciatic nerve. -e rats in the SNI +M andSNI +DMSO groups were intrathecally treated with min-ocycline (specific microglia inhibitor) and an equal volumeof DMSO, respectively. Pain behaviors and the levels ofionized calcium-binding adapter molecule 1 (Iba1), BDNF,PI3K, and p-ERK in the spinal cord were assayed andcompared among the four groups.

2.2.2. Effect of PRF on DRG on the Neuropathic Pain andLevels of Microglia, BDNF, PI3K, and ERK in the Spinal Cord.Four groups were observed, including Sham, SNI,SNI + PRF, and SNI + free-PRF. All rats (except those in theSham group) were subjected to SNI of the right sciatic nerve.On postoperative day 7, PRF was applied to the ipsilateralL4-L5 DRG in the SNI + PRF group, and SNI + free-PRFgroup was kept as a control. Pain behaviors and levels ofIba1, BDNF, PI3K, and p-ERK were measured.

-e von Frey behavioral testing for paw withdrawalmechanical threshold (PWMT) and sting thermal imagingfor paw withdrawal thermal latency (PWTL) were per-formed before the operation (D0), on 1st (D01), 3rd (D03),5th (D05), and 7th (D07) postoperative days, and 1st (D1),3rd (D3), 5th (D5), 7th (D7), 9th (D9), 11th (D11), and 14th(D14) days after PRF treatment or completion of intrathecalinjection. -e expression levels of Iba1, BDNF, PI3K, andp-ERK in the spinal cord were measured by western blotanalysis, reverse transcription-polymerase chain reaction(RT-PCR), and immunofluorescence on D14. Western blot,RT-PCR, and immunofluorescence were applied to five ratseach.

2.3. Neuropathic PainModel. PNI was performed accordingto the SNI model as described by Decosterd and Woolf [21].Briefly, the rat’s right common sciatic nerve at the tri-furcation into the tibial, common peroneal, and sural nervesof the rats was exposed under anesthesia. -e tibial andcommon peroneal nerves were transected, leaving the suralnerve intact. -e procedures were performed in the sameway without transecting the nerves in the Sham group.-en,

2 Pain Research and Management

the muscles were massaged back into place, and the incisionwas closed. All operations were performed by the sameresearcher.

2.4. Intrathecal Catheters and Drug Administration. -e ratsreceived intrathecal catheter implantation before SNI. -eywere anesthetized with an intraperitoneal injection of 10%chloral hydrate (300mg/kg). A PE-10 polyethylene catheterwas implanted between the L5 and L6 vertebrae to reach thesubarachnoid space of the spinal cord as described in aprevious study [22]. -e outer part of the catheter wasplugged and fixed onto the skin upon wound closure. -erats showing neurological deficits after the catheter im-plantation were euthanized. Minocycline was dissolved insterile 5% DMSO with 95% saline solution. -e rats wereintrathecally treated with minocycline (40mg/kg, Sigma-Aldrich, St. Louis, MO, USA) or an equivalent volume ofDMSO once daily for 7 days, 1 h before nerve transection.Drugs or vehicles were intrathecally injected through theimplanted catheter in a 10 μl volume of solution followed by10 μl of vehicle for flushing. Each injection lasted for at least5 minutes. After injection, the needle was retained in situ for2 minutes before being withdrawn.

2.5. PRF on DRG. -e rats received PRF treatment on day 7after SNI. -ey were anesthetized using an injection of 10%chloral hydrate (300mg/kg). -e right L4-L5 DRG was ex-posed in the SNI + free-PRF and SNI + PRF groups throughlaminectomy and facetectomy, without injury to the duramater. An RF electrode (type 20G, 5 cm long, 4mm activetip) was placed adjacent to the corresponding DRG via directvisualization by using a radiofrequency device (CosmanMedical, Inc., Burlington, MA, USA)-e motor stimulationtest was used instead of the sensory stimulation test. PRFwaves were applied after carrying out the motor stimulationtest through muscle contraction of the lower extremities.Stimulation parameters of the PRF waves were set as follows:2 bursts/s; duration� 20ms; output voltage� 45V; maxi-mum temperature� 42°C; and the stimulated time� 6min.After the PRF treatment, the RF probe was removed, and themuscles were closed. In the SNI + free-PRF group, theelectrode was put in the same way without any stimuli.

2.6. Behavioral Testing. -e rats were placed in a plasticchamber (20× 25×15 cm3) and habituated for 15min beforethe experiment. PWMT was evaluated using von Frey fila-ments (Stoelting, IL, USA) by the up-down method describedin a previous study [23]. Each filament was applied per-pendicularly to the ipsilateral territory, near the center of thevibrissal pad. Avoiding further contact with the filament,quickly turning head away, scratching the stimulated area, orattacking the filament was considered a positive response. Anallodynic rat was defined as the one with 50% PWT <4.0 g(withdrawal in response to nonnoxious tactile stimulus).

PWTL was tested by measuring the withdrawal responseof the hind paw to heat stimulation using the Plantar TestApparatus (TaiMeng Science and Technology, Chengdu,

China) as described by Hargreaves et al. [24]. -e cutofflatency was 30 s to avoid thermal injury. -e withdrawallatency at each time point was an average of three latenciesseparated by an interval of 5min. -e tests were conductedon the same days as the von Frey test, and both tests wereconducted by the same researcher who was blind to thegroup allocation of the rats.

2.7. Western Blot Analysis. -e spinal cord of the lumbar(L4-L5) ipsilateral quadrant to the lesion was collected,dissected, and homogenized in protein lysis buffer in thepresence of protease inhibitors and incubated on ice for10min. -e samples were centrifuged at 12,000 rpm for15min at 4°C. -e total protein content was determined inthe supernatants using the Bio-Rad DC Protein Assay Kit.Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Millipore,MA, USA). -e membranes were blocked with 5% nonfatmilk at room temperature and incubated overnight at 4°Cwith primary antibodies (rabbit anti-Iba1, ab178680, 1 :1000, Abcam, USA; rabbit anti-BDNF, ab108319, 1 : 1000,Abcam, USA; rabbit anti-p-ERK, #4370, 1 : 1000, CellSignaling Technology, USA; rabbit anti-PI3K, ab40776, 1 :2000, Abcam, USA). -en, the membranes were incubatedwith a horse-radish peroxidase-conjugated secondaryantibody (1 : 5000, -ermo Scientific, USA) at roomtemperature for 2 h. Finally, peroxidase activity was vi-sualized using the ECL Western Blot Detection Kit(Beyotime, China). Western blots were quantitated usingan image analysis system (Bio-Rad, USA). After normal-ization with β-actin, the data were presented as meanpercentages of the ratio of total protein to their respectivesignal intensity levels found in the Sham group animals,indicated as 100%.

2.8.Real-TimeRT-PCR. -e total RNAs were extracted fromthe L4-L5 ipsilateral quadrant spinal cord using TRIzol andreverse transcribed using the High-Capacity cDNA ReverseTranscription Kit (Applied Biosystems, CA, USA). -e real-time PCR was performed using the Power SYBR GreenMaster Mix (Applied Biosystems) according to the manu-facturer’s protocols and analyzed by RT-PCR in a detectionsystem (Applied Biosystems). -e real-time PCR protocolwas as follows: reverse transcriptase was activated and cDNAwas synthesized (50°C for 5min), PCR was activated (95°Cfor 3min), 40 cycles of denaturation were performed (95°Cfor 30 s), and annealing and extension were done for 1min at60°C. At the end of PCR, a melting curve analysis wasperformed by slowly increasing the temperature from 60°Cto 95°C. -e data were analyzed using Software 2.2 using acycle threshold (Ct) value as the readout and relativelynormal levels of β-actin.

-e primers used were as follows:

Iba1: 5′-GCAAGGATTTGCAGGGAGGA-3′ (forward),5′-TGGGATCATCGAGGAAGTGC-3′ (reverse)BDNF: 5′-AATAATGTCTGACCCCAGTGCC-3′ (for-ward), 5′-CTGAGGGAACCCGGTCTCAT-3′ (reverse)

Pain Research and Management 3

PI3K: 5′-ATTTCCAGTGGGTGAGGCAG-3′ (forward),5′-CTCATGGTAGCCGGTGACTC-3′ (reverse)p-ERK: 5′-ATTATGTGCACCGGGACCTG-3′ (forward),5′-TGTCATCCTGGAGGTAGCGA-3′ (reverse)β-Actin: 5′-ACTCTGTGTGGATTGGTGGC-3′(forward),5′-AGAAAGGGTGTAAAACGCAGC-3′(reverse)

2.9. Immunofluorescence Histochemistry. -e rats wereperfused with 200mL of saline followed by 200mL of 0.1Mphosphate buffer (pH 7.3) containing 4% para-formaldehyde. -e L4-L5 spinal cord was removed, post-fixed in 4% paraformaldehyde for 24 h, and allowed toequilibrate in 30% sucrose in phosphate-buffered saline(PBS) overnight at 4°C. Transverse spinal sections (4–6 μm) were cut using a cryostat and collected in 0.01MPBS, pH 7.3. After washing with PBS, the tissue waspenetrated with 0.3% Triton X-100 and primary antibodiesfor rabbit anti-rat Iba1, BDNF, p-ERK, and PI3K (rabbitanti-Iba1, ab178680, 1 : 100, Abcam, USA; rabbit anti-BDNF, ab108319, 1 : 500, Abcam, USA; rabbit anti-p-ERK, #4370, 1 : 200, Cell Signaling Technology, USA;rabbit anti-PI3K, ab40776, 1 : 200, Abcam, USA). Next, theslides were covered with secondary antibodies containing1 μM 4′-6-diamidino-2-phenylinedole (Sigma, USA).Some sections stained for BDNF, p-ERK, and PI3K weredouble labeled using cell-type-specific Abs for microglia(Iba1). -e nuclei were stained with DAPI (5 μg/mL;Beyotime, USA). Fluorescence signal was detected usinga fluorescence microscope (Olympus, Japan), images werecaptured, and signal co-localization was measured usingMetaMorph (Molecular Devices, USA). -e area fractionwas quantified using Image J software (Rawak Software,Inc., Germany).

3. Statistical Analysis

All data were analyzed using SPSS 20.0 statistical softwarepackage (SPSS Inc., IL, USA) and presented as mean-± standard error of mean (SEM). All data were graphedusing Prism 5.0 (GraphPad, CA, USA). After the data dis-tribution was tested to be normal, behavioral data, westernblot data, and enzyme-linked immunosorbent assay datawere analyzed using a repeated-measures (multiple group-s× time) analysis of variance (ANOVA). Multiple com-parisons were performed using the Bonferroni post hoc testto determine the overall significance. When ANOVAshowed a significant difference, pairwise comparisons be-tween the means were tested using the post hoc Tukeymethod or Fisher’s protected least significant difference(LSD) post hoc test. An alpha value of 0.05 was consideredstatistically significant.

4. Results

4.1. Inhibiting the Spinal Microglia Activation Produced aSignificantly Neuropathic Pain Reduction in SNI Rats.Compared with baseline, the SNI group displayed long-lasting mechanical allodynia (P< 0.01; Figure 1(a)) and

thermal hyperalgesia (P< 0.01; Figure 1(b)) in their ipsi-lateral paws, which reached a peak on the fifth day andmaintained stable withdrawal thresholds until the end ofobservation. No significant changes were found in thecontralateral hind paw in all the groups (P> 0.05;Figures 1(c) and 1(d)), similar to the ipsilateral paw in theSham group (P> 0.05; Figures 1(a) and 1(b)). -e me-chanical allodynia and thermal hyperalgesia were not in-duced during intrathecal injection of minocycline. Althoughthe withdrawal thresholds significantly decreased aftercompleting injections in the SNI +M group compared withthose in the Sham group (P< 0.01; Figures 1(a) and 1(b)),they were still higher than the values in the SNI andSNI +DMSO groups (P< 0.01; Figures 1(a) and 1(b)). Nosignificant differences were found between the SNI andSNI +DMSO groups (P> 0.05; Figures 1(a) and 1(b)).

4.2. Suppression of Microglia Activation Contributes to aRemarkable Reduction of the Expression of Iba1, BDNF, PI3K,and p-ERK in the Spinal Cord. At a higher magnification,almost all BDNF, PI3K, and p-ERK immunofluorescence co-localized with the nuclear marker DAPI (Figures 2(a)–2(c)).-e localization of BDNF, PI3K, and p-ERK inmicroglia wasconfirmed by triple labeling with BDNF/Iba1/DAPI, PI3K/Iba1/DAPI, and p-ERK/Iba1/DAPI (Figures 2(a)–2(c)).Western blot analysis (Figures 3(a)–3(d)), RT-PCR analysis(Figure 4), and immunofluorescence and histochemicalanalysis (Figures 5(a)–5(e)) in the spinal cord demonstratedthat the levels of Iba1, BDNF, PI3K, and p-ERK were low inthe Sham group and increased in the ipsilateral spinal cordin the SNI and SNI +DMSO groups (P< 0.01). -ey de-creased in the SNI +M group compared with the SNI groupbut were still higher than those in the Sham group on D14(P< 0.01).

4.3. PRF Treatment Results in a Significant Neuropathic PainReduction. Indeed, PNI induced long-lasting mechanicalallodynia (P< 0.01; Figure 6(a)) and thermal hyperalgesia(P< 0.01; Figure 6(b)) since the first day after SNI, reacheda peak on the fifth day, and maintained stable withdrawalthresholds until the end of observation compared withthose in the Sham-operated rats. No significant changeswere observed in the contralateral hind paw (P> 0.05;Figures 6(c) and 6(d)) throughout the duration of thestudy. Similar results were obtained on the ipsilateral sidein the Sham group (P> 0.05; Figures 6(a) and 6(b)). In thisstudy, PRF was applied on the L4-L5 DRG in rats with SNIfor 6min on the seventh day after nerve ligation; me-chanical allodynia (P< 0.01; Figure 6(a)) and thermalhyperalgesia (P< 0.01; Figure 6(b)) were partially re-covered in the SNI + PRF group from the first day after asingle application of PRF and maintained throughout aperiod of 14 days, compared with those in the SNI andSNI + free-PRF groups, but could not return to pre-SNIbaseline. In the SNI and SNI + free-PRF groups, the pawwithdrawal threshold and paw withdrawal latency weremaintained at a low level from postlesion 7–21 days.


4.4. PRF Treatment Reduced the Levels of Spinal Iba1, BDNF,PI3K, and p-ERK in SNI Rats. Western blot analysis(Figures 7(a)–7(d)), RT-PCR analysis (Figure 8), and im-muno�uorescent and histochemical analysis (Figures 5(a)–5(d), and 9) in the spinal cord showed that the expressionlevels of Iba1, BDNF, PI3K, and p-ERK in the SNI andSNI + free-PRF groups signi�cantly increased comparedwith those in the Sham group after nerve injury (P< 0.05).�e levels of Iba1, BDNF, PI3K, and p-ERK in the SNI + PRFgroup were downregulated signi�cantly than those in theSNI and SNI + free-PRF groups (P< 0.05) but were still

higher than those in the Sham group after a single appli-cation of PRF on D14 (P< 0.05).

5. Discussion

In the present study, SNI induced a long-lasting increase inmicroglia hyperactivity and BDNF, PI3K, and p-ERKupregulation in the spinal cord and resulted in pain sen-sitization. Minocycline, which was intrathecally injected inthe early phase of pain generation, relieved mechanicalallodynia and thermal hyperalgesia and reversed the

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Figure 1: �erapeutic e�ects of minocycline on neuropathic pain in SNI rats. (a, b) E�ects of minocycline (a speci�c inhibitor of microglialactivation) on mechanical allodynia and thermal hyperalgesia. Reversal of SNI-induced allodynia by intrathecal administration ofminocycline once a day for 7 days (1 h before nerve ligation) in the rats. All rats (except those in the Sham group) were subjected to SNI of theright sciatic nerve. �e rats in the SNI +M and SNI +DMSO groups were intrathecally treated with minocycline and an equal volume ofDMSO, respectively. Each symbol represents mean± SEM; ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group.Repeated-measures (multiple groups× time) ANOVA, n� 15 per group. (c, d) Changes in paw withdrawal threshold and paw withdrawallatency in contralateral hind paw of all groups.


upregulated levels of Iba1, BDNF, PI3K, and p-ERK in thespinal cord after injecting interruption. PRF was applied onthe ipsilateral DRG of the rats on the seventh day after SNI,which also reversed mechanical allodynia and thermalhyperalgesia. �e bene�ts of a single PRF application per-sisted for at least 2 weeks after treating interruption. Just likea speci�c microglia inhibitor, PRF reversed the microglialactivation and expression of BDNF, PI3K, and p-ERK in thespinal cord as well.

5.1. Behavioral Changes Induced by the �erapy of PRF onDRG. Neuropathic pain is a kind of refractory pain. �econventional painkillers, such as opioids and nonsteroidalanti-in�ammatory drugs a�ord poor e£cacy and producemany side e�ects. �erefore, a good e�ective therapy forneuropathic pain is urgently required. PRF has been used to

treat neuropathic pain and has shown satisfactory e£cacywith minimal side e�ects. PRF electromagnetic pulse con-veyed a signal to the target tissue with radiofrequencyelectrodes of 20ms, 500 kHz radiofrequency electromagneticenergy, followed by 480ms interval. �e temperature of thetissue around the tip of the PRF does not exceed 42°C toavoid target tissue and nerve damage. DRG is the oval in-�ation of the dorsal root in the upper region of the in-tervertebral foramen, which contains the �rst-class neuronsof sensory a�erents. DRG has an important role in theprocess of peripheral sensitization. Nociceptive sensoryneurons of DRG are activated by noxious stimuli in theperiphery and transmit information to the CNS. �e acti-vation of immune and immune-like glial cells in the DRGand spinal cord leads to the release of both pro- and anti-in�ammatory cytokines, which are involved in the spinalnociceptive transmission and central sensitization [25, 26].

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Figure 2:�ree immunolabeling of Iba1/DAPI with BDNF (a), PI3K (b), and p-ERK (c). At a higher magni�cation, almost all BDNF, PI3K,and p-ERK immuno�uorescence colocalized with the nuclear marker DAPI. �e localization of BDNF, PI3K, and p-ERK in microglia wascon�rmed by triple labeling with BDNF/Iba1/DAPI, PI3K/Iba1/DAPI, and p-ERK/Iba1/DAPI. Scale bar: 100 μm.


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Figure 3: �e western blot analysis of Iba1 (a), BDNF (b), PI3K (c), and p-ERK (d) proteins in the spinal cord of rats in di�erent groups onD14. Values represented the relative ratio of Iba1, BDNF, PI3K, or p-ERK levels (normalized to β-actin) to that in the Sham rats. Eachsymbol represents mean± SEM. In the Iba1 assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. In the BDNFassay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. In the PI3K assay, ∗∗P< 0.01 against the Sham group and##P< 0.01 against the SNI group. In the p-ERK assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. LSD t test,n� 5 rats per assay.


Because of its active role in the modulation of sensoryprocessing and its anatomic accessibility to clinical in-tervention, DRG has become an excellent clinical target forpain treatment.

-e therapy of PRF on DRG has been demonstrated toimprove pain effectively. For example, Arai reported that thePRF on DRG provided pain relief for patients with in-tractable vertebral metastatic pain [27]. Van Boxem et al.achieved a similar efficacy in chronic intractable lumbosacralradicular pain with PRF therapy on DRG [28]. Here, PRFwas applied on DRG to treat neuropathic pain induced bySNI. As neuropathic pain developed significantly andreached the peak point on day 7 after SNI [29], day 7 wasappointed as the PRF treatment time in the present study.

Various experimental neuropathic pain models have alsobeen shown a pain-relieving effect of PRF on mechanicalhypersensitivity and sometimes on thermal allodynia. -eeffect of PRF on chronic pain was still very discrepant, likelybecause of differences in therapy protocols, various exposuretime, duration of pain, stimulation site of PRF, pain model,and species used. Treatment with 5min PRF stimulation onL5 DRG in the unilateral L5 spinal nerve ligation (SNL)model of rats significantly reduced mechanical hypersen-sitivity and heat analgesia [30]. In the CFA-induced pe-ripheral inflammatory pain, PRF to the L4 anterior primaryramus just close to DRG significantly increased PWMTandPWTL, but PRF to the sciatic nerve in the midthigh justincreased PWTL [31, 32]. However, these studies focused onthe prophylactic effect of PRF rather than on its therapeuticapplication for an established chronic pain syndrome. -eyshowed that PRF relieved, even reversed, mechanical hy-persensitivity and some thermal allodynia. However, thepain-relief effect was seen on mechanical hypersensitivity

only when PRF was applied to established chronic pain[10, 33]. An important finding in the study by Tanaka et al.[34] was that increased exposure time of 2–6min to PRFcurrent showed a significant antiallodynic effect withoutmotor impairment. -erefore, this study applied PRF cur-rent for 6min to the L4-L5 DRG in rats with SNI on theseventh day after nerve ligation. PWMT and PWTL weresignificantly increased following PRF on DRG therapy onday 7 after SNI until day 21. -ese results were consistentwith clinical observations and the findings of Liu et al. [35],further indicating that PRF on DRG was a beneficialtreatment for neuropathic pain. A significant finding in thisstudy was that thermal hyperalgesia was also restrainedwhen PRF was applied for established chronic pain. We alsoobserved that nerve injury-induced mechanical allodyniaand thermal hyperalgesia could be reversed for long-lastingrelief by a single PRF on DRG even after PRF cessation, andthey could not return to pre-SNI baseline. -e exactmechanisms underlying the analgesic effect of PRF onneuropathic pain deserve further study.

5.2. Mechanisms Underlying the Analgesic Effect of PRF

5.2.1. Decreased Microglia Activation. Increasing evidencedemonstrates the pivotal role of spinal microglia in neu-ropathic pain [36, 37]. Following varied types of insults inthe nervous system, including PNI, microglial cells were thefirst to become activated and remained so for several weeks.Microglia transformed to reactive phenotype via displaying aprogressive series of cellular and molecular changes, in-cluding morphological hypertrophy, rapid proliferation,upregulated expression of various genes, and increased

BNDF PI3K p-ERKIba10

1

2

3

ShamSNI

SNI + MSNI + DMSO

&&&&

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tive m

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leve

ls of

Iba1

, BD

NF,

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enta

ge o

f Sha

m

∗∗

∗∗

∗∗

##

ΔΔ

ΔΔ

§§ΔΔ

φφ

ααφφ

φφ

&&ΩΩ

Figure 4: Real-time PCR analysis of Iba1, BDNF, PI3K, and p-ERKmRNA expression in the spinal cord in different groups on D14. Valuesrepresent the relative ratio of Iba1, BDNF, PI3K, and p-ERK mRNA (normalized to GAPDH mRNA) expression to that in the Sham rats.Each symbol represents mean± SEM. In the Iba1 assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. In theBDNF assay, ΔΔP< 0.01 against the Sham group and §§P< 0.01 against the SNI group. In the PI3K assay, &&P< 0.01 against the Sham groupand ΩΩP< 0.01 against the SNI group. In the p-ERK assay, φφP< 0.01 against the Sham group and ααP< 0.01 against the SNI group. LSD ttest, n� 5 rats per assay.


SNI + DMSO SNI + PRF SNI + free-PRF

Sham

Iba1

SNI SNI + M

(a)


BDNF

Sham SNI SNI + M

(b)


PI3K

Sham SNI SNI + M

(c)

Figure 5: Continued.


expression of microglia characteristic markers, such asionized calcium-binding adapter molecule 1 (Iba1). -en,inflammatory cytokines were released by microglia andcontributed to the development of pain hypersensitizationand long-persisting pain [38]. Inhibitors of microglia byintrathecal administration have shown great analgesic effi-cacy in pain models [39, 40], but it was limited to reduce theestablished late-phase pain [41]. In this study, minocyclinewas intrathecally injected to rats with SNI at early stages.-epain was completely inhibited by minocycline during theperiod of drug administration. However, the withdrawalthresholds decreased slightly after treatment interruption,

and the analgesic effect persisted for 21 days. Moreover, thespinal microglia level was restrained. Our finding also in-dicated that microglia inhibition as early as possible couldgain more long-lasting pain relief.

-e therapy of PRF on DRG could induce the changes inthe cell morphology. Ultrastructural changes in the axons,including abnormal membranes and morphology of mito-chondria, and the disruption and disorganization of mi-crofilaments and microtubules were observed in C- and Aδ-fibers on electron microscopy [42]. Recently, it was reportedthat the analgesic effect of PRF might derive from long-termmodulation of cell functions by changing gene expression.


p-ERK

Sham SNI SNI + M

(d)

BDNF PI3K p-ERK Iba10

5

10

15

20

ShamSNI

SNI + MSNI + DMSO

&& &&

Are

a fra

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NF,

PI3

K, p

-ERK

, and

Iba1

(%)

∗∗

∗∗

∗∗

##

ΔΔ

ΔΔ

§§ΔΔ

&&ΩΩ

φφ φφφφαα

(e)

Figure 5: Expression of Iba1 (a), BDNF (b), PI3K (c), and p-ERK (d) in the spinal cord of rats in different groups (immunofluorescence,×100), scale bar� 100 μm. (e) -e intensity of Iba1, BDNF, PI3K, and p-ERK immunofluorescence in the spinal cord in different groups onD14. Each symbol represents mean± SEM. In the BDNF assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. Inthe PI3K assay, ΔΔP< 0.01 against the Sham group and §§P< 0.01 against the SNI group. In the p-ERK assay, &&P< 0.01 against the Shamgroup and ΩΩP< 0.01 against the SNI group. In the Iba1 assay, φφP< 0.01 against the Sham group and ααP< 0.01 against the SNI group. LSDt test, n� 5 rats per assay.


�e expression of pain regulatory genes such as a proin-�ammatory gene returned to baseline values. Numerousreports suggested that microglia in the spinal dorsal hornwere vital in pain facilitation. PRF applied on DRG in a ratmodel of neuropathic pain revealed that the establishedmechanical hypersensitivity reduced, and the activation ofmicroglia in spinal dorsal horn was signi�cantly attenuated[10, 11].�e �ndings of this study were consistent with thoseof previous studies. Mechanical allodynia and thermalhyperalgesia were reversed, accompanied by a signi�cantlydownregulated the expression of Iba1 which was maintainedfor 14 days after a single application of PRF. �is

demonstrated that PRFmight have suppressed the activationof microglia and contributed to the nociceptive relief.

5.2.2. Reversing the Increase of Microglial BDNF, PI3K, andp-ERK in the Spinal Cord of Rats with SNI. Some studieshave shown that neurotrophins, especially BDNF, play animportant role as pain mediators/modulators [43, 44].BDNF is a secreted protein and part of the family of neu-rotrophin family. Neutrophins act on neurons to promotethe survival, growth, and di�erentiation of new neurons andsynapses. However, it has a deleterious e�ect on the spinal

D0 D01 D03 D05 D07 D1 D3 D5 D7 D9 D11 D140

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∗∗

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D0 D01 D03 D05 D07 D1 D3 D5 D7 D9 D11 D14

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)


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##∗∗ ##

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∗∗

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(b)

D0 D01 D03 D05 D07 D1 D3 D5 D7 D9 D11 D140

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5

7

9

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Paw

with

draw

al la

tenc

y (s

)

Time (days)


ShamSNI

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(d)

Figure 6: �erapeutic e�ects of PRF on DRG on neuropathic pain. (a, b) E�ects of PRF on mechanical allodynia and thermal hyperalgesiaapplied to L4-L5 DRG.�e paw withdrawal threshold in response to mechanical hypersensitivity (a) and paw withdrawal latency in responseto thermal hyperalgesia (b) partially recovered from the �rst day after a single application of PRF and maintained throughout a period of 14days. On postoperative day 7, PRF was applied to the ipsilateral L4-L5 DRG in the SNI + PRF group, and SNI + free-PRF group was kept ascontrol. Each symbol represents mean± SEM; ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. Repeated-measures(multiple groups × time) ANOVA, n� 15 per group. (c, d) Changes in paw withdrawal threshold and paw withdrawal latency in con-tralateral hind paw of all groups.


cord following nerve injury. Trang found that P2X4 receptoron the activated microglial surface promoted the synthesisand release of BDNF, which in turn accelerated centralsensitization and maintained neuropathic pain [45].Microglia-derived BDNF is a critical microglia-neuronsignaling molecule that gates aberrant nociceptive pro-cessing in the spinal cord. �e enhanced BDNF, whichelicited nociceptive hypersensitivity, also contributed to theactivation of microglia in the spinal cord in a feedforwardmanner, and the functional inhibition of BDNF signal re-versed allodynia in rats with SNI. �e upregulated expres-sion of BDNF was detected in the spinal cord on day 21 after

SNI in the present study, consistent with the previous re-ports, and further indicated that BDNF was highly involvedin neuropathic pain.

PI3K, a lipid kinase that phosphorylates the D3 positionof phosphatidylinositol lipids to produce PI(3,4,5)P3, acts asa membrane-embedded second messenger. Some progresshas been made about the role of PI3K in the refractory pain.Plantar incision induced a time-dependent activation ofPI3K in the microglia, and the inhibition of PI3K preventedpain behaviors induced by plantar incision [46]. Speci�cinhibitors of PI3K applied before SNI reduced the neuro-pathic pain behaviors induced by L5 SNL [47]. �e PI3K

0

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Sham SNISNI + free-

PRF SNI + PRF


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∗∗

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(d)

Figure 7: (a–d)�ewestern blot analysis of Iba1, BDNF, PI3K, and p-ERK proteins in the spinal cord of rats in di�erent groups onD14. Valuesrepresent the relative ratio of Iba1, BDNF, PI3K, and p-ERK levels (normalized to β-actin) to that in the Sham rats. Each symbol representsmean± SEM. In the Iba1 assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. In the BDNF assay, ∗∗P< 0.01 againstthe Sham group and ##P< 0.01 against the SNI group. In the PI3K assay, ∗P< 0.05 against the Sham group and #P< 0.05 against the SNIgroup. In the p-ERK assay, ∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNI group. LSD t test, n� 5 rats per assay.


signaling pathway was expressed in microglia and partici-pated in bone cancer pain [48].�e treatment of bone cancerpain model in rats with PI3Kcb-speci�c small-interferingRNA resulted in the inhibition of pain-related behavior [49].�e expression of PI3K was assayed in the spinal cord on day21 after SNI in the present study. PI3K was found to besigni�cantly increased on day 21 after SNI. �erefore, thepresent results implied that PI3K also played an importantrole in neuropathic pain.

ERK, a member of the mitogen-activated protein kinasefamily, could transmit a great quantity of extracellular in-formation into intracellular responses. �e ERK signalingpathway in the microglia has been reported to modulatevarious types of pain, and the inhibition of p-ERK couldalleviate the associated pain [50, 51]. �e level of p-ERK inthe spinal cord was found to be signi�cantly upregulated onday 21 after SNI in the present study. �ese results wereconsistent with other reports [52] and suggested that acti-vated ERK contributed to the development of neuropathicpain.

�e microglia, BDNF, PI3K, and p-ERK were con�rmedto be involved in developing neuropathic pain in presentstudy. However, whether the release of BDNF, PI3K, and p-ERK in the spinal cord in rats with SNI to regulate chronicpain processing is through microglia-dependent mechanismremains undetermined. In order to clarify this question,�rstly, we adopted the method of triple labeling with BDNF/Iba1/DAPI, PI3K/Iba1/DAPI and p-ERK/Iba1/DAPI to

prove that the localization of BDNF, PI3K, and p-ERK is inmicroglia. Secondly, minocycline (as microglia inhibitor)was intrathecally injected into rats with SNI at early stages;the increased levels of Iba1, BDNF, PI3K, and p-ERK in thespinal cord were all restrained. Hence, these �ndings in-dicated that BDNF, PI3K, and p-ERK might have beenreleased in the spinal cord in amicroglia-dependent manner.

Although the analgesic e£cacy of PRF on DRG forneuropathic pain was exact, the mechanisms were not fullyunderstood. �e e�ects of PRF might, via electromagnetic�elds, disrupt or somehow modulate pain signal trans-mission and gene expression in the treated sites and CNS.�e suppression of Iba1, BDNF, PI3K, and p-ERK in thespinal cord was important in alleviating neuropathic pain inrats with SNI in the present study, and the release of BDNF,PI3K, and p-ERK in the spinal cord might have occurred in amicroglia-dependent manner. Since the therapy of PRF onDRG in rats with neuropathic pain could induce pain reliefby reducing microglial activation, we deduced that PRFmight regulate the release of BDNF, PI3K, and p-ERK in thespinal cord to relieve neuropathic pain. Just as we assumed,the increased expression of BDNF, PI3K, and p-ERK on day21 was simultaneously downregulated for 6min PRF therapyafter SNI. So, these results revealed that PRF therapy onDRG might attenuate neuropathic pain by reducing therelease of BDNF, PI3K, and p-ERK in the spinal cord inmicroglia-dependent way.

6. Limitations

�is study had some limitations. Firstly, only the short-terme�ectiveness of PRF on DRG was explored. Secondly, in vivo�eld potential recording in PRF-treated rats was absent.

ShamSNI


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, BD

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BNDF PI3K p-ERKIba10

1

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3

&&

&& &&

##ΩΩ

φφ

φφ

φφ

αα

∗∗

∗∗

∗∗

Figure 8: Real-time PCR analysis of Iba1, BDNF, PI3K, and p-ERKmRNA in the spinal cord in di�erent groups on D14. Valuesrepresent the relative ratio of Iba1, BDNF, PI3K, and p-ERKmRNA(normalized to GAPDH mRNA) to that in the Sham rats. Eachsymbol represents mean± SEM. In the Iba1 assay, ∗∗P< 0.01against the Sham group and ##P< 0.01 against the SNI group. Inthe BDNF assay, ΔΔP< 0.01 against the Sham group and §§P< 0.01against the SNI group. In the PI3K assay, &&P< 0.01 against theSham group and ΩΩP< 0.01 against the SNI group. In the p-ERKassay, φφP< 0.01 against the Sham group and ααP< 0.01 against theSNI group. LSD t test, n� 5 rats per assay.

ShamSNI


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&&

&&&&

∗∗∗∗

∗∗

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ΩΩ

φφ φφφφαα

Figure 9: �e intensity of Iba1, BDNF, PI3K, and p-ERK im-muno�uorescence in the spinal cord in di�erent groups on D14.Each symbol represents mean± SEM. In the BDNF assay,∗∗P< 0.01 against the Sham group and ##P< 0.01 against the SNIgroup. In the p-ERK assay, ΔΔP< 0.01 against the Sham groupand §§P< 0.01 against the SNI group. In the PI3K assay, &&P< 0.01against the Sham group and ΩΩP< 0.01 against the SNI group. Inthe Iba1 assay, φφP< 0.01 against the Sham group and ααP< 0.01against the SNI group. LSD t test, n� 5 rats per assay.


-irdly, only one time point was selected to assay the levelsof Iba1, BDNF, PI3K, and p-ERK. Fourthly, the relationshipamong BDNF, PI3K, and p-ERK was not investigated. Moredetailed research about PRF on the DRG to ease the neu-ropathic pain is needed.

7. Conclusions

-e application of PRF on DRG could reverse SNI-inducedneuropathic pain after the treatment period. -e mecha-nisms underlying this treatment might be suppressedmicroglia and downregulated levels of BDNF, PI3K, andp-ERK in the spinal cord in microglia-dependent way. Itsupported PRF treatment as a valuable intervention forchronic neuropathic pain.

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

-e authors declare that they have no conflicts of interest.

Acknowledgments

-e authors would like to acknowledge the financial supportfrom the provincial health system. -is study was supportedby the Project of Medical Innovation of Fujian Province(2016-CX-3 and 2018-CX-6).

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