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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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Immunofluorescence of phosphorylated PDGF β-receptors and expression of PDGF receptor mRNAs in rats after nerve injury. (A) The immunoreactivity for p-PDGFRβ was detected in the L5 spinal dorsal horn 4 days after nerve injury. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of contralateral side (contra) and ipsilateral side (ipsi) of nerve injured rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 3). **P < 0.01 vs contra by Student's t-test. (C) Double immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red), a microglia marker. Scale bars, 20 μm. (D) Total RNA extracted from the L5 spinal cords of naive rats and peripheral nerve injured rats was subjected to quantitative analysis of PDGFR mRNA expression by real-time RT-PCR. Data are means ± SEM of the percentage over the naive value (ipsilateral side/contralateral side, n = 5).
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Figure 5: Immunofluorescence of phosphorylated PDGF β-receptors and expression of PDGF receptor mRNAs in rats after nerve injury. (A) The immunoreactivity for p-PDGFRβ was detected in the L5 spinal dorsal horn 4 days after nerve injury. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of contralateral side (contra) and ipsilateral side (ipsi) of nerve injured rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 3). **P < 0.01 vs contra by Student's t-test. (C) Double immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red), a microglia marker. Scale bars, 20 μm. (D) Total RNA extracted from the L5 spinal cords of naive rats and peripheral nerve injured rats was subjected to quantitative analysis of PDGFR mRNA expression by real-time RT-PCR. Data are means ± SEM of the percentage over the naive value (ipsilateral side/contralateral side, n = 5).

Mentions: Activation of PDGFRs in the spinal cord is implicated in tactile allodynia after peripheral nerve injury [24]. Thus, we determined the type of cells in which PDGFRβ activation occurs under a neuropathic pain condition. In contrast to the contralateral dorsal horn, where p-PDGFRβ immunofluorescence was low, we observed strong p-PDGFRβ immunofluorescence in the dorsal horn ipsilateral to the nerve injury; the level of p-PDGFRβ immunofluorescence in individual cells in this region was also much higher than that in individual cells in the dorsal horn contralateral to the nerve injury (P < 0.01) (Figure 5A, B). Furthermore, almost all p-PDGFRβ-positive cells were also labeled for the microglia marker OX-42 (Figure 5C). These results indicate that PDGFRβ activation in the dorsal horn occurs exclusively in microglia after nerve injury. How PDGFR activity is enhanced remains unclear, but we examined the time course for changes in the expression levels of PDGFR mRNAs after nerve injury and found no significant change during the period from 1 day to 14 days post-nerve injury (Figure 5D). It is thus possible that the enhanced PDGFR activity might be due to an increase in the level of endogenous PDGF within the dorsal horn after nerve injury, as suggested by a previous study [24]. Consistently, low levels of PDGFRβ phosphorylation in the dorsal horns of normal rats (Figure 2A) and in the contralateral dorsal horns of nerve-injured rats (Figure 5A) were observed. In the adult spinal cord, PDGF has been shown to be expressed in dorsal horn neurons [24]. It is thus assumed that PDGF might be a candidate for signaling molecules between neurons and microglia, thereby producing tactile allodynia, although further investigations are needed to determine the pattern and change in the expression of endogenous PDGFRβ ligands in the dorsal horn after nerve injury.


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)

Immunofluorescence of phosphorylated PDGF β-receptors and expression of PDGF receptor mRNAs in rats after nerve injury. (A) The immunoreactivity for p-PDGFRβ was detected in the L5 spinal dorsal horn 4 days after nerve injury. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of contralateral side (contra) and ipsilateral side (ipsi) of nerve injured rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 3). **P < 0.01 vs contra by Student's t-test. (C) Double immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red), a microglia marker. Scale bars, 20 μm. (D) Total RNA extracted from the L5 spinal cords of naive rats and peripheral nerve injured rats was subjected to quantitative analysis of PDGFR mRNA expression by real-time RT-PCR. Data are means ± SEM of the percentage over the naive value (ipsilateral side/contralateral side, n = 5).
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Figure 5: Immunofluorescence of phosphorylated PDGF β-receptors and expression of PDGF receptor mRNAs in rats after nerve injury. (A) The immunoreactivity for p-PDGFRβ was detected in the L5 spinal dorsal horn 4 days after nerve injury. Scale bar, 200 μm. (B) The intensity of p-PDGFRβ immunofluorescence was quantified in the dorsal horn region of contralateral side (contra) and ipsilateral side (ipsi) of nerve injured rats. Data represent the means ± SEM of the immunofluorescence intensity (n = 3). **P < 0.01 vs contra by Student's t-test. (C) Double immunofluorescence labeling of p-PDGFRβ (green) with OX-42 (red), a microglia marker. Scale bars, 20 μm. (D) Total RNA extracted from the L5 spinal cords of naive rats and peripheral nerve injured rats was subjected to quantitative analysis of PDGFR mRNA expression by real-time RT-PCR. Data are means ± SEM of the percentage over the naive value (ipsilateral side/contralateral side, n = 5).
Mentions: Activation of PDGFRs in the spinal cord is implicated in tactile allodynia after peripheral nerve injury [24]. Thus, we determined the type of cells in which PDGFRβ activation occurs under a neuropathic pain condition. In contrast to the contralateral dorsal horn, where p-PDGFRβ immunofluorescence was low, we observed strong p-PDGFRβ immunofluorescence in the dorsal horn ipsilateral to the nerve injury; the level of p-PDGFRβ immunofluorescence in individual cells in this region was also much higher than that in individual cells in the dorsal horn contralateral to the nerve injury (P < 0.01) (Figure 5A, B). Furthermore, almost all p-PDGFRβ-positive cells were also labeled for the microglia marker OX-42 (Figure 5C). These results indicate that PDGFRβ activation in the dorsal horn occurs exclusively in microglia after nerve injury. How PDGFR activity is enhanced remains unclear, but we examined the time course for changes in the expression levels of PDGFR mRNAs after nerve injury and found no significant change during the period from 1 day to 14 days post-nerve injury (Figure 5D). It is thus possible that the enhanced PDGFR activity might be due to an increase in the level of endogenous PDGF within the dorsal horn after nerve injury, as suggested by a previous study [24]. Consistently, low levels of PDGFRβ phosphorylation in the dorsal horns of normal rats (Figure 2A) and in the contralateral dorsal horns of nerve-injured rats (Figure 5A) were observed. In the adult spinal cord, PDGF has been shown to be expressed in dorsal horn neurons [24]. It is thus assumed that PDGF might be a candidate for signaling molecules between neurons and microglia, thereby producing tactile allodynia, although further investigations are needed to determine the pattern and change in the expression of endogenous PDGFRβ ligands in the dorsal horn after nerve injury.

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