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Intrathecal delivery of PDGF produces tactile allodynia through its receptors in spinal microglia.

Masuda J, Tsuda M, Tozaki-Saitoh H, Inoue K - Mol Pain (2009)

Bottom Line: Interestingly, almost all p-PDGFR beta-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. junyamsd@kyudai.jp

ABSTRACT
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 beta-receptor (p-PDGFR beta), an activated form, was markedly increased in the spinal dorsal horn. Interestingly, almost all p-PDGFR beta-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-PDGFR beta 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.

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PDGF-BB phosphorylates its receptors in spinalmicroglia. (A) The immunoreactivity of phosphorylated PDGFRβ protein was detected by a specific antibody for p-PDGFRβ 30 min after intrathecal administration of vehicle or PDGF-BB (10 pmol) in the L5 spinal dorsal horn. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of vehicle treated rats and PDGF-BB treated rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 5). ***P < 0.001 vs vehicle by Student's t-test. (C) Double immunofluorescence labeling of the dorsal horn 30 min after intrathecal PDGF-BB administration with p-PDGFRβ (green) and cell markers (red); OX-42, a microglia marker; GFAP, an astrocytes marker; CC1, an oligodendrocytes marker; NeuN and MAP2, neurons markers. Scale bars, 20 μm. (D) PDGFRα (116 bp) and PDGFRβ (145 bp) mRNA expression in primary microglia by RT-PCR analysis. Spinal cord, cerebral cortex, and spleen are positive controls. (E) Triple immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red) and DAPI (blue), a nuclear marker, in primary microglia treated with PBS as a control or PDGF-BB (50 ng/ml) for 10 min. Scale bar, 20 μm.
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Figure 2: PDGF-BB phosphorylates its receptors in spinalmicroglia. (A) The immunoreactivity of phosphorylated PDGFRβ protein was detected by a specific antibody for p-PDGFRβ 30 min after intrathecal administration of vehicle or PDGF-BB (10 pmol) in the L5 spinal dorsal horn. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of vehicle treated rats and PDGF-BB treated rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 5). ***P < 0.001 vs vehicle by Student's t-test. (C) Double immunofluorescence labeling of the dorsal horn 30 min after intrathecal PDGF-BB administration with p-PDGFRβ (green) and cell markers (red); OX-42, a microglia marker; GFAP, an astrocytes marker; CC1, an oligodendrocytes marker; NeuN and MAP2, neurons markers. Scale bars, 20 μm. (D) PDGFRα (116 bp) and PDGFRβ (145 bp) mRNA expression in primary microglia by RT-PCR analysis. Spinal cord, cerebral cortex, and spleen are positive controls. (E) Triple immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red) and DAPI (blue), a nuclear marker, in primary microglia treated with PBS as a control or PDGF-BB (50 ng/ml) for 10 min. Scale bar, 20 μm.

Mentions: To identify the cell types on which intrathecally delivered PDGF-BB acts, we performed immunohistochemical experiments using an anti-phospho-Tyr1021 PDGFRβ antibody (p-PDGFRβ) that recognizes activated receptors [27]. The immunofluorescence for p-PDGFRβ in the dorsal horns of vehicle-treated rats remained at low levels, but was markedly increased 30 min after intrathecal PDGF-BB (10 pmol) administration (P < 0.001) (Figure 2A, B). By double-staining with cell type-specific markers, we found that almost all p-PDGFRβ-positive cells were double-labeled with OX-42 (a marker of microglia), but not with GFAP (an astrocyte marker), CC1 (an oligodendrocyte marker), MAP2 or NeuN (neuronal markers) (Figure 2C). These results indicate that activation of PDGFRβ evoked by intrathecally delivered PDGF-BB occurs specifically in microglia. Consistently, both PDGFRα and PDGFRβ mRNAs were detected in primary cultured microglia and in the spinal cord as well as positive control tissues (cerebral cortex and spleen) (Figure 2D). Furthermore, applying PDGF-BB (50 ng/ml) to primary cultured microglial cells enhanced the immunofluorescence for p-PDGFRβ (Figure 2E).


Intrathecal delivery of PDGF produces tactile allodynia through its receptors in spinal microglia.

Masuda J, Tsuda M, Tozaki-Saitoh H, Inoue K - Mol Pain (2009)

PDGF-BB phosphorylates its receptors in spinalmicroglia. (A) The immunoreactivity of phosphorylated PDGFRβ protein was detected by a specific antibody for p-PDGFRβ 30 min after intrathecal administration of vehicle or PDGF-BB (10 pmol) in the L5 spinal dorsal horn. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of vehicle treated rats and PDGF-BB treated rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 5). ***P < 0.001 vs vehicle by Student's t-test. (C) Double immunofluorescence labeling of the dorsal horn 30 min after intrathecal PDGF-BB administration with p-PDGFRβ (green) and cell markers (red); OX-42, a microglia marker; GFAP, an astrocytes marker; CC1, an oligodendrocytes marker; NeuN and MAP2, neurons markers. Scale bars, 20 μm. (D) PDGFRα (116 bp) and PDGFRβ (145 bp) mRNA expression in primary microglia by RT-PCR analysis. Spinal cord, cerebral cortex, and spleen are positive controls. (E) Triple immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red) and DAPI (blue), a nuclear marker, in primary microglia treated with PBS as a control or PDGF-BB (50 ng/ml) for 10 min. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: PDGF-BB phosphorylates its receptors in spinalmicroglia. (A) The immunoreactivity of phosphorylated PDGFRβ protein was detected by a specific antibody for p-PDGFRβ 30 min after intrathecal administration of vehicle or PDGF-BB (10 pmol) in the L5 spinal dorsal horn. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of vehicle treated rats and PDGF-BB treated rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 5). ***P < 0.001 vs vehicle by Student's t-test. (C) Double immunofluorescence labeling of the dorsal horn 30 min after intrathecal PDGF-BB administration with p-PDGFRβ (green) and cell markers (red); OX-42, a microglia marker; GFAP, an astrocytes marker; CC1, an oligodendrocytes marker; NeuN and MAP2, neurons markers. Scale bars, 20 μm. (D) PDGFRα (116 bp) and PDGFRβ (145 bp) mRNA expression in primary microglia by RT-PCR analysis. Spinal cord, cerebral cortex, and spleen are positive controls. (E) Triple immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red) and DAPI (blue), a nuclear marker, in primary microglia treated with PBS as a control or PDGF-BB (50 ng/ml) for 10 min. Scale bar, 20 μm.
Mentions: To identify the cell types on which intrathecally delivered PDGF-BB acts, we performed immunohistochemical experiments using an anti-phospho-Tyr1021 PDGFRβ antibody (p-PDGFRβ) that recognizes activated receptors [27]. The immunofluorescence for p-PDGFRβ in the dorsal horns of vehicle-treated rats remained at low levels, but was markedly increased 30 min after intrathecal PDGF-BB (10 pmol) administration (P < 0.001) (Figure 2A, B). By double-staining with cell type-specific markers, we found that almost all p-PDGFRβ-positive cells were double-labeled with OX-42 (a marker of microglia), but not with GFAP (an astrocyte marker), CC1 (an oligodendrocyte marker), MAP2 or NeuN (neuronal markers) (Figure 2C). These results indicate that activation of PDGFRβ evoked by intrathecally delivered PDGF-BB occurs specifically in microglia. Consistently, both PDGFRα and PDGFRβ mRNAs were detected in primary cultured microglia and in the spinal cord as well as positive control tissues (cerebral cortex and spleen) (Figure 2D). Furthermore, applying PDGF-BB (50 ng/ml) to primary cultured microglial cells enhanced the immunofluorescence for p-PDGFRβ (Figure 2E).

Bottom Line: Interestingly, almost all p-PDGFR beta-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. junyamsd@kyudai.jp

ABSTRACT
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 beta-receptor (p-PDGFR beta), an activated form, was markedly increased in the spinal dorsal horn. Interestingly, almost all p-PDGFR beta-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-PDGFR beta 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.

Show MeSH
Related in: MedlinePlus