Edited by: Liliana Bernardino, University of Beira Interior, Portugal
Reviewed by: Antonio J. Herrera, University of Seville, Spain; Muddanna Sakkattu Rao, Kuwait University, Kuwait
†These authors have contributed equally to this work
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Some recent evidence suggests that microglia activation and inflammatory cytokine production in the hippocampus are associated with the development of pain behavior following peripheral nerve injury. We observed sciatic nerve chronic constriction injury (CCI)-induced inflammation-related gene expression changes that are modulated by minocycline in rat hippocampus. Intra-CA1 administration of minocycline was applied after nerve injury. Genome-wide mRNA expression in the hippocampus was evaluated to monitor the fundamental gene expression levels. We found that minocycline treatment produces a pronounced inhibition of CCI-induced mechanical allodynia. We identified 790 genes differentially expressed in CCI vs. sham rats. Among these changed genes, the 425 differentially expressed genes showed a significantly different effect in CCI vs. minocycline-treated rats. Moreover, 390 transcripts were characterized by an increase in mRNA abundance after nerve injury, and minocycline treatment reduced the level of these changes. Only 35 transcripts were characterized by a decrease in mRNA after nerve injury, and minocycline treatment reversed the decrease in the hippocampus. Noteworthily, cytokine-cytokine receptor interaction and the toll-like receptor signaling pathway are the top two most significantly enriched KEGG (kyoto encyclopedia of genes and genomes) terms in comparing the sham vs. CCI group and CCI vs. minocycline-treated group. Nine kinds of transcription factor gene transcripts (Runx3, Tfec, Pax-1, Batf3, Sp5, Hlx, Nfkbiz, Spil, Fli1) increased in abundance after nerve injury, and minocycline treatment reversed these changes. Afterwards, we selected some genes for further validation by using quantitative PCR: interleukins (Il1β), chemokines (Cxcl13, Cxcl1, Ccl2, Cxcl11, Ccl7, Ccl20), toll-like receptors (Tlr8 and Tlr1), and transcription factors (Runx3, Nfkbiz and Spil). We suggested that the transcriptional changes of these inflammation-related genes are strongly related to the processes of microglia activation underlying neuropathic pain development.
Neuropathic pain is a chronic pain condition that is usually induced by peripheral nerve injury. Recent reports suggest that the inflammation-related cytokines accumulation in dorsal root ganglion, dorsal spinal cord, hippocampus, thalamus, and somatosensoric cortex are paralleled by pain responses in different animal models of neuropathic pain (Al-Amin et al.,
It is clear that many kinds of toll-like receptors (TLRs) are expressed in the hippocampus and act as a type of pattern-recognition receptor that participate in inflammatory responses. TLR1 expression in the hippocampus was increased in the neurons, microglia, and astrocytes in seizure mice (Wang et al.,
Previous studies reveal that chemokine production is enhanced in some neuroimmunological diseases accompanied by pathological pain (Cartier et al.,
It is clear that minocycline is an important modulator of the immune response and easily permeates the blood-brain barrier (Stolp et al.,
In the experiments, adult male Sprague-Dawley (SD) rats (200–220 g) were housed under a 12: 12 h revised light/dark cycle. The protocol was prepared from SD rats in accordance with the National Institutes of Health guidelines in a manner that minimized animal suffering and animal numbers. All experiments involving animals were approved by the Zunyi Medical University Committee on Ethics in the Care and Use of Laboratory Animals.
Rats were anesthetized by pentobarbital sodium (40 mg/kg, i.p.) and mounted in a David Kopf stereotaxic frame (Model 1900, Tujunga, CA, USA) with a flat skull position. An incision was made along the midline and the scalp was retracted. The area surrounding the bregma was cleaned. Stainless steel guide cannulae were unilaterally implanted 1 mm above the CA1 according to rat brain atlases. Two holes were drilled through the skull and two stainless steel needles (28 gauge) were inserted through the holes (A/P-3.3 mm caudal to the bregma, L/R ± 2.0 mm lateral to the midline, D/V2.8 mm ventral to the skull surface) (Paxinos and Watson,
Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and the sciatic nerve (left) was exposed. The left sciatic nerve was exposed and a 15-mm length of sciatic nerve proximal to the sciatic trifurcation was dissected. Four loose ligatures (4.0 braided silk) were made around the sciatic nerve at 1-mm intervals. Sham rats underwent the same procedure but without nerve ligation. After surgery, rats were housed in separate cages (at room temperature for 24 h) to avoid scratching each other (Safakhah et al.,
Mechanical withdrawal threshold (MWT) was recorded to assess the response of the paw to mechanical stimulus. An electronic von Frey plantar aesthesiometer (IITC, Wood Dale, IL, USA) was used (Huang et al.,
Male SD rats were divided into Sham, CCI+0.01M PBS and CCI+ Minocycline groups (
First-strand cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, Beijing, China) according to the standard protocols. Quantitative real-time PCR was carried out using a QuantStudio™ 6 Flex Real-Time System (Applied Biosystems, USA) with UltraSYBR Mixture (CWBIO, Beijing, China). The following PCR amplification program was used: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 50–54°C (changed according to the primer sequences) for 20 s and 72°C for 20 s. A dissociation curve was performed (55–95°C) after the last PCR cycle to ascertain the specificity of the amplification reactions. The abundance of each mRNA was normalized with respect to the endogenous housekeeping gene β-actin, and the relative gene expression levels were determined by the 2−ΔΔCt method.
Microarray experiments were performed to determine gene-expression profiles in rat hippocampus. Based on the differentially expressed gene (DEG) results, the heat maps were constructed using Multiexperiment Viewer (MeV;
The DEGs were ascertained using the DESeq R package (1.10.1) as detailed in a previous study (Wang et al.,
Here, M is the number of genes in the pathway, N is the total number of genes in the genome, m is the number of target gene candidates in M and n is the number of differentially expressed genes. In addition, i = 1, 2, 3, … (M-1) where M represents the number of genes in the pathway. The Fisher's score indicates the ratio of genes (number m) belonging to the functional pathway out of the total differentially expressed genes (number n) (Zhang et al.,
Male SD rats were divided into Sham, Sham+Minocycline, CCI+0.01M PBS and CCI+ Minocycline groups (
Primers used for RT-PCR.
Cxcl13 |
5′-TTTGGTAACCATCTGGCAGTA-3′ | 5′-GCTCGACCTTTATCAATCTAAT-3′ |
Cxcl1 |
5′-TGGCTATGACTTCGGTTTGGGT-3′ | 5′-GGCAGGGATTCACTTCAAGAACA-3′ |
Ccl2 |
5′-GTGCTGAAGTCCTTAGGGTTG-3′ | 5′-GTCGGCTGGAGAACTACAAGA-3′ |
Cxcl11 |
5′-CCAGGCACCTTTGTCCTTTAT-3′ | 5′-GGTTCCAGGCTTCGTTATGTT-3′ |
Ccl7 |
5′-CACCGACTACTGGTGATCTTTC-3′ | 5′-TTCATCCACTTGCTGCTATGT-3′ |
Ccl20 |
5′-GACAAGACCACTGGGACA-3′ | 5′-AGCCTAAGAACCAAGAAG-3′ |
Iba-1 |
5′-CAAGGATTTGCAGGGAGGA-3′ | 5′-CAGCATTCGCTTCAAGGACATA-3′ |
Cd68 |
5′-TCAAACAGGACCGACATCAGA-3′ | 5′- ATTGCTGGAGAAAGAACTATGCT-3′ |
iNOS |
5′-GATGTGCTGCCTCTGGTCCT-3′ | 5′-GAGCTCCTGGAACCACTCGT-3′ |
IL-1β |
5′-CAGCCTTACTGGCCTGCTAC-3′ | 5′-CTGCTACCACGACAGCCATA-3′ |
Tlr8 |
5′-TGCTTCATTTGGGATTTG-3′ | 5′-TGGCATTTACACGCTCAC-3′ |
Tlr1 |
5′-CAGTTTCTGGGATTGAGCGGT-3′ | 5′-TAATGTGCTGAAGACACTTGGGATC-3′ |
Runx3 |
5′-GGCTTTGGTCTGGTCCTCTATC-3′ | 5′-GCAACGCTTCCGCTGTCA-3′ |
Nfkbiz |
5′-CCGTAGAAGTAAGCGAGGTT-3′ | 5′-GAGCATGATCGTGGACAAG-3′ |
Spil |
5′-CAATCTTTGCTCCTCTTT-3′ | 5′-CTACCAATCCTGGCTTCA-3′ |
β-actin |
5′-AGCCATGTACGTAGCCATCC-3′ | 5′-ACCCTCATAGATGGGCACAG-3′ |
All data were presented as mean ± standard deviation (SD.). The behavioral and PCR data were analyzed by one- (compared within the group) or two-way (compared between groups) ANOVA. If significance was established,
To investigate the antinociceptive effect of minocycline on the mechanical nociceptive threshold in neuropathic pain rats, the MWT was recorded on the day before and after surgery (at POD 1, 3, 5, and 7). A total of five doses (1, 2, 5, 10, and 15 μg/μl, twice a day) were administered. We compared the changes of MWT between the different time points (
MWT was determined in different groups. All values represent mean ± SD (
As shown in
To explore the possible role of microglia activation and inflammation within the hippocampus in the development of peripheral neuropathic pain, the DEGs between different groups were identified. According to the results, in the rat hippocampus, there were 790 DEGs between the sham group and the CCI group. Among them, 613 genes were increased and 177 were decreased (as shown in
The DEGs were identified. In
The DEGs were annotated covering molecular biological function, cellular component and biological process. As shown in
GO term classification of increased and decreased genes on DEGs for each pairwise. X axis represents GO term. Y axis represents the number of increased/decreased genes.
Compared with the sham-operated group, the CCI group had 20 differential gene-involved significant pathways. DEGs contained in these pathways (top 14) are shown in
The top 14 most significant KEGG pathways identified with increased and decreased genes among different groups.
Cytokine-cytokine receptor interaction | ||
Toll-like receptor signaling pathway | ||
Phagosome | ||
Fc gamma R-mediated Phagocytosis | ||
TNF signaling pathway | ||
Complement and coagulation cascades | ||
Cell adhesion molecules | ||
Natural killer cell mediated cytotoxicity | ||
NF-κB signaling pathway | ||
Chemokine signaling pathway | ||
Osteoclast differentiation | ||
B cell receptor signaling pathway | ||
Primary immunodeficiency | ||
Platelet activation |
We also noticed that the minocycline-treated group had 20 differential gene-involved significant pathways in comparison with the CCI group. DEGs contained in these pathways (top 14) are also shown in
Cytokines and chemokines were originally identified as essential mediators for inflammatory and immune responses in the formation of neuropathic pain (White and Wilson,
mRNA expression profile of inflammation-related genes among different groups.
Cxcl13 | +7.03 | 7.02E-236 | −8.42 | 2.75 E-207 | −1.39 | 0.13 |
Cxcl1 | +5.44 | 2.07E-31 | −1.87 | 1.75 E-13 | +3.57 | 2.18E-08 |
Ccl2 | +5.35 | 1.35E-48 | −6.65 | 1.77 E-47 | −1.30 | 0.26 |
Cxcl11 | +4.80 | 2.95E-26 | −4.45 | 2.75 E-25 | +0.35 | 0.72 |
Ccl7 | +4.27 | 1.02E-17 | −7.25 | 4.73E-18 | 0 | 0 |
Ccl20 | +3.46 | 0.003003 | −3.44 | 0.0029 | 0.02 | 0.99 |
Ccl3 | +2.40 | 0.000603 | −3.38 | 4.47 E-05 | −0.98 | 0.42 |
Ccl6 | +2.22 | 3.47E-12 | −3.20 | 2.39 E-17 | −0.98 | 0.07 |
Ccl5 | +1.61 | 3.03E-07 | −3.48 | 6.45 E-16 | −1.87 | 0 |
Cxcl16 | +1.27 | 6.02E-31 | −1.88 | 1.70 E-53 | −0.61 | 5.23E-05 |
INos | +5.46 | 2.72E-06 | −5.44 | 2.67 E-06 | 0 | 0 |
Il1β | +4.34 | 9.98E-19 | −3.15 | 4.47E-15 | +1.19 | 0.15 |
Il18rap | +4.13 | 6.00 E-16 | −6.11 | 2.35 E-17 | −1.98 | 0.17 |
Socs3 | +3.67 | 2.94 E-243 | −3.08 | 5.23E-209 | +0.58 | 0 |
C3 | +3.61 | 0 | −3.83 | 0 | −0.22 | 0 |
Tlr8 | +3.49 | 1.05E-11 | −4.20 | 4.72 E-13 | −0.71 | 0.49 |
Ptges | +3.47 | 4.28E-49 | −2.48 | 2.02 E-35 | +0.99 | 0 |
Mt1 | +2.15 | 4.94E-228 | −1.81 | 5.21E-181 | +0.34 | 0 |
Il20rb | +2.08 | 1.32 E-18 | −1.11 | 3.61 E-08 | +0.97 | 0 |
Tlr1 | +1.93 | 1.91E-09 | −2.12 | 1.57 E-10 | −0.18 | 0.66 |
Il21r | +1.92 | 1.10E-18 | −2.01 | 1.21 E-19 | −0.09 | 0.77 |
Il2rb | +1.69 | 2.99 E-06 | −1.78 | 1.14 E-16 | −0.10 | 0.84 |
Tnfrsf1b | +1.66 | 5.44E-32 | −1.20 | 6.16 E-20 | +0.46 | 0.01 |
Hpgds | +1.64 | 1.35E-07 | −1.56 | 3.59 E-07 | +0.08 | 0.84 |
Tlr13 | +1.61 | 8.94E-20 | −1.37 | 1.03 E-15 | +0.24 | 0.27 |
I11r1 | +1.48 | 8.55E-62 | −1.15 | 6.06 E-42 | +0.32 | 0 |
Irf8 | +1.57 | 7.14E-85 | −1.35 | 5.28 E-27 | +0.23 | 0.03 |
Card11 | +1.49 | 1.38E-28 | −1.65 | 9.27 E-33 | −0.16 | 0.36 |
P2ry6 | +1.49 | 4.35E-44 | −1.33 | 8.20 E-37 | +0.17 | 0.21 |
Tlr7 | +1.48 | 7.32E-34 | −1.15 | 4.02 E-23 | +0.33 | 0.02 |
Casp4 | +1.38 | 3.52E-14 | −1.34 | 1.52 E-13 | +0.04 | 0.86 |
Fas | +1.22 | 1.79E-05 | −1.37 | 2.68 E-06 | −0.15 | 0.68 |
Tlr2 | +1.06 | 1.61E-19 | −1.05 | 3.82 E-19 | +0.10 | 0.94 |
Tlr9 | +1.06 | 0.0001 | −1.65 | 8.08 E-08 | −0.60 | 0.10 |
Tifab | +1.01 | 8.91 E-29 | −1.05 | 4.44 E-30 | −0.03 | 0.76 |
Cd68 | +3.45 | 9.91E-95 | −2.90 | 1.19E-80 | +0.54 | 0.05 |
Msr-1 | +2.01 | 7.93E-21 | −1.94 | 7.27 E-20 | 0.07 | 0.82 |
Iba-1 | +1.16 | 3.94E-46 | −1.16 | 1.28E-45 | 0 | 0.98 |
Ox-42 (Cd11b) | +0.72 | 9.34E-30 | −0.55 | 7.70E-19 | +0.17 | 0.01 |
Ptges | +3.47 | 4.28E-49 | −2.48 | 2.02 E-35 | +0.99 | 0.01 |
Mrc1 | +2.85 | 4.43 E-129 | −2.71 | 6.94 E-122 | +0.13 | 0.48 |
Cd86 | +1.46 | 1.41E-07 | −1.86 | 3.20 E-10 | −0.41 | 0.27 |
Tgfβ1 | +1.16 | 2.58 E-49 | −0.96 | 4.06E-36 | +0.20 | 0.03 |
Arg1 | −1.06 | 8.19 E-17 | +0.43 | 0 | −0.62 | 5.18E-08 |
IL4r | +1.03 | 5.18 E-70 | −0.14 | 0 | +0.90 | 3.18E-51 |
Runx3 | +6.39 | 7.42 E-11 | −3.79 | 1.51 E-09 | 0 | 0 |
Tfec | +3.95 | 5.47 E-17 | −5.25 | 7.13 E-19 | −1.30 | 0.26 |
Pax-1 | +3.70 | 0.0009 | −4.68 | 0.0004 | 0 | 0 |
Batf3 | +2.67 | 0.0005 | −1.03 | 4.69 E-08 | −0.11 | 0.62 |
Sp5 | +2.17 | 6.15 E-06 | −1.42 | 0.00076 | +0.76 | 0.20 |
Hlx | +1.58 | 9.94 E-28 | −1.26 | 1.85 E-11 | +0.32 | 0.18 |
Nfkbiz | +1.46 | 9.25E-28 | −2.02 | 1.12 E-42 | −0.56 | 0 |
Spi1 (Pu.1) | +1.33 | 2.57E-34 | −1.19 | 1.12 E-28 | +0.15 | 0.27 |
Fli1 | +1.21 | 3.04 E-35 | −1.11 | 1.33 E-30 | +0.10 | 0.39 |
Lst1 | +2.39 | 0.0001 | −1.29 | 0.01 | +1.10 | 0.14 |
Maff | +1.67 | 1.72 E-09 | −0.45 | 0.04 | +1.23 | 2.50E-05 |
Elf4 | +1.39 | 6.46 E-11 | −0.95 | 1.93E-06 | +0.45 | 0.07 |
Vgl13 | +1.38 | 3.40 E-51 | +0.66 | 2.52E-24 | +2.04 | 2.25E-140 |
Nr2f2 | +1.12 | 2.89 E-144 | +0.59 | 1.82E-71 | +1.72 | 0 |
Cartpt | −2.30 | 2.14 E-70 | +0.17 | 0.23 | −2.13 | 0 |
Six3 | −1.68 | 6.84 E-06 | −0.57 | 0.20 | −2.25 | 1.48E-08 |
Meox1 | −1.64 | 7.13 E-05 | +1.11 | 0.01 | −0.53 | 0.10 |
Tfap-2c | −1.55 | 1.84 E-05 | +0.28 | 0.31 | −1.27 | 0 |
Ebf3 | −1.40 | 2.05 E-05 | −0.44 | 0.22 | −1.84 | 7.32E-08 |
Mkx | −1.12 | 2.27 E-08 | −0.003 | 0.49 | −1.12 | 7.17E-09 |
Mei4 | −1.00 | 2.91 E-05 | +0.09 | 0.4 | −0.91 | 6.09E-05 |
In addition, cytokine signaling-3 (SOCS3) and TLR gene transcripts were upregulated in the hippocampus, and minocycline suppressed the upregulation of SOCSs and Tlr8, Tlr1, Tlr13, Tlr7, Tlr2, and Tlr9 gene transcripts in CCI rats. We found that, after nerve injury, Tlr4 transcripts were only upregulated by <1 fold (Tlr4: fold = 0.56, FDR = 0.0167). The Tnf-α and Nlrp3 transcripts were upregulated by >1 fold (Tnf-α: fold = 1.492, FDR = 0.037; Nlrp3: fold = 1.21, FDR = 4.18E-23) in the hippocampus of CCI rats. Moreover, the upregulated Tnf-α and Nlrp3 gene transcripts were only moderately suppressed by minocycline (Tnf-α: fold = 0.469, FDR = 0.275; Nlrp3: fold = 0.79, FDR = 5.55E-12). Besides, Nlrp1a gene transcripts were only slightly upregulated (Nlrp1a: fold = 0.29, FDR = 0.007). For these reasons, the changes of Tnf-α, Tlr4, and Nlrp gene transcripts are not listed in
More studies have suggested that Cd68, Iba-1 (ionized calcium-binding adaptor molecule-1, involved in microglial motility), Ox-42 (Cd11b, involved in microglial plasticity and motility), Msr-1(macrophage scavenger receptor 1, involved in phagocytosis), and Mhc-II (major histocompatibility complex II) are common markers of microglia activation (Booth and Thomas,
It is clear that microglia/macrophages respond to acute brain injury by becoming activated and developing a pro-inflammatory profile of M1-like or anti-inflammatory profile of M2-like phenotypes (Perego et al.,
It is well known that some transcription factors have been shown to be directly or indirectly associated with the expression of inflammation-related cytokine genes. Compared with the sham group, the upregulated transcription factor genes were maff, Elf4, Nr2f2, Vgl13, Lst1, Runx3, Tfec, Sp5, Nfkbiz, Hlx, Spi1 (Pu.1), Fli1, Batf3, and Pax-1. Among them, we would like to mention that the levels of gene transcripts of Runx3, Tfec, Sp5, Nfkbiz, Hlx, Spi1, Fli1, Batf3, and Pax-1 were largely suppressed by minocycline. It is noteworthy that gene transcripts of Tfec, Nfkbiz, Hlx, Spil, Fli1, and Batf3 and Pax-1 were obviously increased in the CCI group compared with those of sham rats, and the expression of these genes returned to normal level after minocycline administration. On the other hand, as shown in
only upregulated in CCI rats: Maff, Elf4, Nr2f2, Vgl13, Lst1;
only downregulated in CCI rats: Cartpt, Six3, Ap-2c, Ebf3, Mei4;
upregulated in CCI and downregulated by minocycline: Runx3, Tfec, Sp5, Nfkbiz, Hlx, Spi1 (Pu.1), Fli1, Batf3, Pax-1; and
downregulated in CCI and upregulated by minocycline: Meox1.
Many of the genes that were identified by microarray analysis should be subject to validation by RT-PCR. As shown in
RT-PCR showing the expression of Cxcl13, Cxcl1, Ccl2, Cxcl11, Ccl7, Ccl20, Iba-1, CD68, iNOS, IL-1β, TLR8, TLR1, Runx3, Nfkbiz, and Spil mRNA in the rat hippocampus (
We reported here the hippocampal genome-wide transcriptome profiling of rats in neuropathic pain status to elucidate minocycline-mediated analgesic effect at the molecular level. It is well known that the CCI model of neuropathic pain displays some symptoms that are very common in neuropathic pain patients including mechanical and thermal allodynia. Then, we screened the hippocampus of the CCI rats for DEGs.
It has also been proved that minocycline exerts an anti-nociceptive effect in different pain models. Recent studies revealed that the hippocampal CA1 region is more sensitive to ischemic injury and peripheral inflammatory stimulation (Sun et al.,
We observed that minocycline at 1 μg/μl induced significant analgesic effect in comparison to CCI rats. Minocycline at doses of 2 and 5 μg/μl showed better analgesic effects in comparison to minocycline at a dose of 1 μg/μl. Minocycline at a dose of 5 μg/μl showed apparent elevations of the MWT in comparison with minocycline at a dose of 2 μg/μl. On the other hand, minocycline at 10 μg/μl produced a moderate antinociceptive effect in CCI rats. Minocycline at 15 μg/μl produced a slight but significant nociceptive effect. It seems the minimum dose of minocycline at 5 μg/μl shows the maximum analgesic effect. Recently, several studies also showed the negative action of minocycline in animal or cellular models for nervous system disorders. Similarly to what we observed in CCI rats, Matsukawa et al. also support the idea that neuroprotection is dose-dependent, in that only low doses of minocycline inhibit neuronal cell death cascades at the acute stroke phase, whereas high doses exacerbate ischemic injury (Matsukawa et al.,
We found that, in the sham group vs. CCI group and minocycline-treated group vs. CCI group, the top 2 items of KEGG pathway are cytokine-cytokine receptor interaction and TLR pathway, which indicates that minocycline administration can regulate the expression of genes in these two pathways, and reversing these gene expression changes may be considered as one of the important reasons for minocycline-mediated analgesic effect. Nerve damage leads to glial activation and thus facilitates the production and release of pronociceptive factors such as interleukins and chemokines from glial cells. We noticed that, after sciatic nerve injury, IL-1β was the most striking interleukin that increased most seriously in hippocampus of CCI rats. Moreover, the increased gene expression of CXCL13, CXCL1, CCL2, CXCL11, and CCL7 in the rat hippocampus was observed after nerve damage. The increased chemokine expression was obviously suppressed by intra-hippocampal injection of minocycline. It appears that minocycline was able to reduce microglia activity efficiently, which led to the decreased expression of these genes. In addition, the increased expression of interleukins and chemokines should be regulated by some transcription factors. For example, the elevated expression of IL-1β was associated with binding of transcription factor Spil/Pu.1 to IL-1β promoter in activated inflammatory macrophage (Vanoni et al.,
In addition, nerve injury evoked the elevated expression of many different kinds of TLRs (TLR8, TLR1, TLR13, TLR7, TLR2, and TLR9) in the rat hippocampus. After treatment with minocycline, the elevated expression of these TLRs in the hippocampus was significantly lower compared to the CCI group. More recent studies suggest that TLRs play an important role in immune response by producing inflammatory cytokines and chemokines under pathological conditions. For example, TLR1, TLR2, TLR7, and TLR9 activation stimulated the production of IL-1β and MCP-1 in B cells (Agrawal and Gupta,
Some previous studies demonstrate that IκBz can serve as a nuclear inhibitor of NF-κB and is thought to have a key role in inflammatory responses. On the other hand, IκBz is induced quickly in monocytes and macrophages after LPS stimulation (Yamazaki et al.,
In summary, the DEGs were identified, and many inflammation-related genes including TLRs and chemokines were considered as important genes in the formation of neuropathic pain through pathway analysis of microarray data, which may help us to further understand the underlying molecular mechanisms of chronic pain. After the bioinformatics analysis of gene expression profiles, the expression of inflammation-related genes was further identified via the RT-PCR method. Although the results obtained from our experiments indicate that intra-hippocampal injection of minocycline exerts an analgesic effect and many inflammation-related genes may be involved in the formation of neuropathic pain, the study we conducted also has certain limitations that should be considered in future studies. In other words, further studies are required to further explore the roles of these inflammation-related genes in the hippocampus, where it is implicated in the formation of the neuropathic pain.
Publicly available datasets were analyzed in this study. This data can be found here:
The protocol was prepared from SD rats in accordance with the National Institutes of Health guidelines in a manner that minimized animal suffering and animal numbers. All experiments were carried out in accordance with China animal welfare legislation and were approved by the Zunyi Medical University Committee on Ethics in the Care and Use of Laboratory Animals.
JZ, YC, and XL: conceived and designed the experiments. LH, RX, and HT: animal experiment. LH and TX: behavioral assessment of pain. LH, RX, YP, and SC: analyzed the data. JZ, LH, and XL: wrote the paper.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are very grateful to the staff of the Department of Physiology, the Department of Pharmacology, and the Department of Clinical Pharmacotherapeutics of School of Pharmacy in Zunyi Medical University.
The Supplementary Material for this article can be found online at: