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Intrathecal delivery of PDGF produces tactile allodynia through its receptors in spinal microglia
© Masuda et al; licensee BioMed Central Ltd. 2009
Received: 02 April 2009
Accepted: 11 May 2009
Published: 11 May 2009
Neuropathic pain is a debilitating pain condition that occurs after nerve damage. Such pain is considered to be a reflection of the aberrant excitability of dorsal horn neurons. Emerging lines of evidence indicate that spinal microglia play a crucial role in neuronal excitability and the pathogenesis of neuropathic pain, but the mechanisms underlying neuron-microglia communications in the dorsal horn remain to be fully elucidated. A recent study has demonstrated that platelet-derived growth factor (PDGF) expressed in dorsal horn neurons contributes to neuropathic pain after nerve injury, yet how PDGF produces pain hypersensitivity remains unknown. Here we report an involvement of spinal microglia in PDGF-induced tactile allodynia. A single intrathecal delivery of PDGF B-chain homodimer (PDGF-BB) to naive rats produced a robust and long-lasting decrease in paw withdrawal threshold in a dose-dependent manner. Following PDGF administration, the immunofluorescence for phosphorylated PDGF β-receptor (p-PDGFRβ), an activated form, was markedly increased in the spinal dorsal horn. Interestingly, almost all p-PDGFRβ-positive cells were double-labeled with an antibody for the microglia marker OX-42, but not with antibodies for other markers of neurons, astrocytes and oligodendrocytes. PDGF-stimulated microglia in vivo transformed into a modest activated state in terms of their cell number and morphology. Furthermore, PDGF-BB-induced tactile allodynia was prevented by a daily intrathecal administration of minocycline, which is known to inhibit microglia activation. Moreover, in rats with an injury to the fifth lumbar spinal nerve (an animal model of neuropathic pain), the immunofluorescence for p-PDGFRβ was markedly enhanced exclusively in microglia in the ipsilateral dorsal horn. Together, our findings suggest that spinal microglia critically contribute to PDGF-induced tactile allodynia, and it is also assumed that microglial PDGF signaling may have a role in the pathogenesis of neuropathic pain.
Peripheral nerve damage leads to a persistent neuropathic pain state in which innocuous stimuli elicit pain behavior (tactile allodynia) [1–3]. Neuropathic pain might involve aberrant excitability of the nervous system, notably at the levels of the primary sensory ganglia and the dorsal horn of the spinal cord [4–8]. There is a rapidly growing body of evidence indicating that peripheral nerve damage activates glial cells in the dorsal horn and results in changing expression and activity of various molecules [9, 10]. Importantly, pharmacological, molecular and genetic manipulations of the function or expression of glial molecules have been shown to substantially influence nerve injury-induced pain behaviors and hyperexcitability of the dorsal horn pain pathway [11–15]. Therefore, signaling between neurons and glia might critically contribute to the pathologically enhanced pain processing in the dorsal horn that underlies neuropathic pain. However, the mechanisms underlying neuropathic pain caused by neuron-glia communications in the dorsal horn remain to be fully elucidated.
Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) have served as prototypes for growth factor and receptor tyrosine kinase (RTK) function. The biologically active form of PDGF is a disulfide-bonded dimer of A-, B-, C-, or D-polypeptide chains. The PDGF isoforms (PDGF-AA, -AB, -BB, -CC, or -DD) bind two structurally related RTKs (PDGFRα and β). PDGF-AA, -BB, -AB, and -CC bind to PDGFRα, whereas PDGF-BB and -DD bind to PDGFRβ [16–20]. Ligand binding induces receptor dimerization and autophosphorylation, subsequently initiates downstream signaling, and causes cellular responses such as proliferation, differentiation, survival, migration, chemotaxis, and gene expression [21, 22].
Although PDGF signaling is commonly known to have essential roles during development , there is limited evidence for its role in the mature CNS. A recent study has shown that PDGF is expressed in dorsal horn neurons in adult mice, and that intrathecal administration of either a selective inhibitor of PDGFR phosphorylation or an antibody trapping endogenous PDGF suppresses thermal hyperalgesia and tactile allodynia after peripheral nerve injury . Thus, PDGF released from dorsal horn neurons is implicated in neuropathic pain. However, how PDGF produces pain hypersensitivity remains unknown.
To examine whether microglia are involved in PDGF-BB-induced tactile allodynia, we tested the effect of minocycline, which inhibits microglia activation [29, 30], on the decrease in the paw withdrawal threshold after PDGF-BB administration. Daily intrathecal administration of minocycline (100 μg) from one day before PDGF-BB (10 pmol) administration significantly suppressed the decrease in paw withdrawal threshold (P < 0.05, day 3; P < 0.01, other testing days) (Figure 3E). This finding suggests that spinal microglia are involved in PDGF-BB-induced tactile allodynia. The mechanisms underlying the anti-allodynic effect of minocycline remains unclear, but we found that minocycline did not inhibit PDGF-induced PDGFRβ phosphorylation in the dorsal horn (Figure 3F), indicating that minocycline does not directly interrupt the PDGF binding to the PDGFRβ and PDGFRβ dimerization and autophosphorylation. Thus, it is conceivable that minocycline may produce its anti-allodynic effect through inhibiting the downstream consequences of PDGFRβ phosphorylation in microglia including p38 mitogen-activated protein kinase that is an important signaling molecule in tactile allodynia [11, 15] and is also known as one of targets of minocycline [31, 32].
PDGFRs in the CNS have been previously reported to be expressed in O-2A progenitor cells, oligodendrocytes, and neurons [35–38]. In the present study, by showing that acute PDGF stimulation in vivo in adult rats induced PDGFRβ phosphorylation specifically in microglia, in addition to our results in in vitro experiments using cultured microglia, we provide the first evidence that microglia are the predominant cell type expressing functional PDGFRβs in the spinal cord. We further revealed that spinal microglia may mediate tactile allodynia caused by intrathecal administration of PDGF. Recently, Narita et al.  have shown that inhibiting PDGFR phosphorylation results in suppression of tactile allodynia after peripheral nerve injury, implying a crucial role for PDGF signaling in neuropathic pain. Notably, following peripheral nerve injury, a marked enhancement of PDGFRβ phosphorylation in dorsal horn microglia also occurred in a cell type-specific manner, indicating that spinal microglia may be crucial for PDGFR-mediated tactile allodynia under neuropathic pain conditions. It remains unknown how PDGF-stimulated microglia modulate pain processing in the dorsal horn, but we found an increase in the expression of IL-1β mRNA in the dorsal horn after PDGF administration. IL-1β enhances C-fiber-evoked responses in wide-dynamic-range dorsal horn neurons , enhances NMDA receptor-mediated Ca2+ responses , and decreases GABAA receptor-mediated currents . A recent study has also demonstrated a powerful role for this cytokine in excitatory and inhibitory synaptic transmission and an effect of this cytokine on neuronal activity in superficial dorsal horn neurons [42, 43].
Therefore, IL-1β may be a candidate intermediary molecule between PDGF-stimulated microglia and dorsal horn neurons that contributes to central hypersensitization. Further investigation using microglia-specific IL-1β-knockout mice will clarify this issue.
Male Wistar rats (250–280 g, Japan SLC) were used. Rats were housed at a constant temperature of 23 ± 1°C with a 12 h light-dark cycle (light on 8:00 to 20:00) and fed food and water ad libitum. All of the animals used in the present study were obtained, housed, cared for, and used in accordance with the guidelines of Kyushu University.
Rat primary cultured microglia was prepared according to the method described previously . In brief, the mixed glial culture was prepared from neonatal Wistar rats and maintained for 9–15 days in DMEM with 10% FBS. Microglia were obtained as floating cells over the mixed glial culture. The floating cells were collected by gentle shaking and transferred to culture dishes for each experiment.
Under 2% isoflurane anesthesia, rats were implanted with a 32 gauge intrathecal catheter (ReCathCo, Allison Park, PA, USA) in the lumbar enlargement (close to L4-5 segments) for intrathecal drug administration. The catheter placement was verified by the observation of hindlimb paralysis induced by intrathecal administration of lidocaine (2%, 5 μl). Rats that failed to cause paralysis were excluded from the experiments. A recombinant human platelet-derived growth factor, PDGF-BB (0.1, 1 and 10 pmol/10 μl PBS; Millipore Bioscience Research Reagents, Temecula, California, USA), or PBS (10 μl, as a vehicle control) was intrathecally administered in naive rats. AG 17 [100 nmol/10 μl PBS containing dimethylsulfoxide (6%: final concentration); Calbiochem] or PBS containing 6% dimethylsulfoxide (10 μl, as a vehicle control) was intrathecally administered 30 min before PDGF-BB (10 pmol/10 μl PBS) administration. Minocycline (100 μg/10 μl PBS; Sigma) or PBS (10 μl, as a vehicle control) was intrathecally administered once a day from 1 day before PDGF-BB (10 pmol/10 μl PBS) administration. 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, TNP-ATP (30 nmol/10 μl PBS; Sigma), or PBS (10 μl, as a vehicle control) was intrathecally administered on day 7 after PDGF-BB (10 pmol/10 μl PBS) administration.
Neuropathic pain model and Behavioral tests
The left L5 spinal nerve of rats was tightly ligated with 5-0 silk suture and cut just distal to the ligature under 2% isoflurane anesthesia [12, 45]. To assess the level of tactile allodynia, rats were placed individually in a wire mesh cage and habituated for 30–60 min to allow acclimatization to the new environment. From below the mesh floor, calibrated von Frey filaments (0.4–15 g; North Coast Medical, Morgan Hill, California, USA) were applied to the mid-plantar surface of the hindpaw. The 50% paw withdrawal threshold was determined using the up-down method .
The rats used in the experiments were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with ice-cold PBS, followed by ice-cold 4% paraformaldehyde in PBS. The L5 segments of the lumber spinal cord were removed, post-fixed in the same fixative for 4 h at 4°C, and placed in 30% sucrose solution for 24 h at 4°C. Transverse spinal cord sections (30 μm) were sliced by a Leica CM 1850 cryostat and incubated in a blocking solution (3% normal goat serum) for 2 h at room temperature, and then incubated for 48 h at 4°C with the primary antibodies against phospho-PDGF β-receptor (rabbit polyclonal anti-phospho-Tyr1021 of PDGFRβ, 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or cell markers; microglia, OX-42 (mouse monoclonal anti-OX-42, 1:1000, Serotec, Oxford, UK) and ionized calcium-binding adapter molecule-1 (Iba1) (rabbit polyclonal anti-Iba1, 1:2000, Wako, Osaka, Japan); astrocytes, glial fibrillary acidic protein (GFAP) (mouse monoclonal anti-GFAP, 1:2000, Millipore Bioscience Research Reagents); oligodendrocytes, CC-1 (mouse monoclonal anti-APC, 1:500, Millipore Bioscience Research Reagents); neurons, neuronal nuclei (NeuN) (mouse monoclonal anti-NeuN, 1:200, Millipore Bioscience Research Reagents) and microtubule-associated protein-2 (MAP2) (mouse monoclonal anti-MAP2, 1:500, Millipore Bioscience Research Reagents). The sections were then washed and incubated for 3 h at room temperature with the fluorescent conjugated secondary antibodies (goat anti-rabbit IgG-conjugated Alexa Fluor 488 or goat anti-mouse IgG-conjugated Alexa Fluor 546, 1:1000, Invitrogen, Carlsbad, CA, USA). The sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained with a confocal microscope (LSM 5 Pascal; Carl Zeiss, Jena, Germany) and analyzed with Zeiss LSM Image Brower (Carl Zeiss). For quantitative assessment of the immunofluorescence staining, the spinal dorsal horn regions were outlined and the immunofluorescence intensity of the p-PDGFRβ was determined as the average pixel intensity within the field.
Primary microglial cells were seeded on aminopropyltriethoxysilane-coated glass (Matsunami, Osaka, Japan) at 5 × 104 cells/well and incubated for 1 h. After the culture media were replaced with serum-free media, cells were incubated for 2 h and subsequently treated with PBS as a control or 50 ng/ml PDGF-BB for 10 min , and then fixed in 3.7% formaldehyde in PBS for 30 min at 25°C. The cells were permeabilized and blocked by incubating them with blocking solution (3% normal goat serum and 0.3% Triton X-100 in PBS) for 15 min at 25°C, and then incubated overnight at 4°C with the primary antibodies against phospho-PDGF β-receptor (1:400) and OX-42 (1:1000). After washing, the cells were incubated for 1 h with appropriate fluorescent-conjugated secondary antibodies (goat anti-rabbit IgG-conjugated Alexa Fluor 488 or goat anti-mouse IgG-conjugated Alexa Fluor 546, 1:1000) and coverslipped in Vectashield containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained and analyzed as mentioned above.
Real-Time Quantitative RT-PCR
The rats used in the experiments were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with ice-cold PBS. The L5 segments of lumber spinal cord were removed immediately and were subjected to total RNA extraction using Trisure (Bioline, Danwon-Gu, South Korea) according to the protocol of the manufacturer and purified with RNeasy mini plus kit (Qiagen, Valencia, CA, USA). The amount of total RNA was quantified by measuring OD260 using a Nanodrop spectrophotometer (Nanodrop, Wilmington, DE, USA). For reverse transcription with random 6-mer primers, 100 ng of total RNA was transferred to the reaction with Prime Script reverse transcriptase (Takara, Kyoto, Japan). Quantitative PCR was performed with Premix Ex Taq (Takara) using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to protocol of the manufacturer, and the data were analyzed by 7500 System SDS Software 1.3.1 (Applied Biosystems) using the standard curve method. Expression levels were normalized to the values for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The TaqMan probes and primers for interleukin-1β (IL-1β) (Taqman probe, 5'-FAM-TTCTCCACCTCAATGGACAGAACATAAGCCA-TAMRA-3'; forward primer, AAATGCCTCGTGCTGTCTGA; reverse primer, GTCGTTGCTTGTCTCTCCTTGTAC), P2X4 receptor (P2X4R) (Taqman probe, 5'-FAM-AGGAGGAAAACTCCCTCTTCATCATGACCA-TAMRA-3'; forward primer, TGGCGGACTATGTGATTCCA; reverse primer, GGTTCACGGTGACGATCATG), P2X7 receptor (P2X7R) (Taqman probe, 5'-FAM-AAAGCCTTCGGCGTGCGTTTTGA-TAMRA-3'; forward primer, CATGGAAAAGCGGACATTGA; reverse primer, CCAGTGCCAAAAACCAGGAT), P2Y12 receptor (P2Y12R) (Taqman probe, 5'-FAM-CACCAGACCATTTAAAACTTCCAGCCCC-TAMRA-3'; forward primer, TAACCATTGACCGATACCTGAAGA; reverse primer, TTCGCACCCAAAAGATTGC), PDGF receptor α-subtype (PDGFRα) (Taqman probe, 5'-FAM-ATATTCTCCCTTGGTGGCACACCCTACC-TAMRA-3'; forward primer, ACGTCTGGTCTTATGGCGTTCT; reverse primer, CATCCTGTATCCGCTCTTGATCT), and PDGFRβ (Taqman probe, 5'-FAM-AACGACTCACCAGTGCTCAGCTACACAGAC-TAMRA-3'; forward primer, GTCCCATCTGCCCCTGAAA; reverse primer, GGTCTCGGTGAACACAGTTCTTAG), as well as the probe and primer for GAPDH, were obtained from Applied Biosystems.
All data are presented as means ± SEM. The statistical analyses of the results were evaluated by using the Student's t test or two-way repeated measures ANOVA with Bonferroni post tests.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to MT, KI).
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