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Key role for spinal dorsal horn microglial kinin B1 receptor in early diabetic pain neuropathy.

Talbot S, Chahmi E, Dias JP, Couture R - J Neuroinflammation (2010)

Bottom Line: Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1.Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations.The enhanced mRNA expression (B1R, IL-1beta, TNF-alpha, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Université de Montréal, C,P, 6128, Succursale Downtown, Montréal, Québec, H3C 3J7, Canada.

ABSTRACT

Background: The pro-nociceptive kinin B1 receptor (B1R) is upregulated on sensory C-fibres, astrocytes and microglia in the spinal cord of streptozotocin (STZ)-diabetic rat. This study aims at defining the role of microglial kinin B1R in diabetic pain neuropathy.

Methods: Sprague-Dawley rats were made diabetic with STZ (65 mg/kg, i.p.), and 4 days later, two specific inhibitors of microglial cells (fluorocitrate, 1 nmol, i.t.; minocycline, 10 mg/kg, i.p.) were administered to assess the impact on thermal hyperalgesia, allodynia and mRNA expression (qRT-PCR) of B1R and pro-inflammatory markers. Spinal B1R binding sites ((125I)-HPP-desArg10-Hoe 140) were also measured by quantitative autoradiography. Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1. Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations.

Results: STZ-diabetic rats displayed significant tactile and cold allodynia compared with control rats. Intrathecal or peripheral blockade of B1R or inhibition of microglia reversed time-dependently tactile and cold allodynia in diabetic rats without affecting basal values in control rats. Microglia inhibition also abolished thermal hyperalgesia and the enhanced allodynia induced by intrathecal des-Arg9-BK without affecting hyperglycemia in STZ rats. The enhanced mRNA expression (B1R, IL-1beta, TNF-alpha, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%.

Conclusion: The upregulation of kinin B1R in spinal dorsal horn microglia by pro-inflammatory cytokines is proposed as a crucial mechanism in early pain neuropathy in STZ-diabetic rats.

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Effect of microglia inhibitors administered 3 h earlier on Iba-1 microglial immunoreactivity in the spinal dorsal horn of control and 4-day STZ-diabetic rats. Shown are (A) confocal microscopy pictures of microglial cells, and (B) the quantification of the mean pixel energy of Iba-1 (in arbitrary unit, AU). Scale bar = 100 μm. Data are the mean ± S.E.M of 4 pictures per rat from 4 rats in each group. Background mean energy was subtracted for each picture. Statistical comparison to control (*) or STZ rats treated with vehicle (+) is indicated by *** +++ P < 0.001.
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Figure 3: Effect of microglia inhibitors administered 3 h earlier on Iba-1 microglial immunoreactivity in the spinal dorsal horn of control and 4-day STZ-diabetic rats. Shown are (A) confocal microscopy pictures of microglial cells, and (B) the quantification of the mean pixel energy of Iba-1 (in arbitrary unit, AU). Scale bar = 100 μm. Data are the mean ± S.E.M of 4 pictures per rat from 4 rats in each group. Background mean energy was subtracted for each picture. Statistical comparison to control (*) or STZ rats treated with vehicle (+) is indicated by *** +++ P < 0.001.

Mentions: A quantitative immunolabelling with a specific immunomarker of microglia Iba-1 was employed to validate the use of minocycline and fluorocitrate as inhibitors of microglia activity. As shown in Figure 3-A, immunoreactivity to Iba-1 was much more striking in the spinal dorsal horn of STZ-diabetic rats than in matched control spinal dorsal horn. Immunoreactive microglial cells in STZ spinal cord were more numerous, thicker and displayed higher mean pixel energy than microglia of control spinal dorsal horn. Importantly, treatment with fluorocitrate or minocycline reversed and normalized the enhanced Iba-1 immunoreactivity in STZ spinal cord microglia (Figure 3-B). The same treatment with fluorocitrate or minocycline had no effect on the mean pixel energy of Iba-1 in control spinal dorsal horn.


Key role for spinal dorsal horn microglial kinin B1 receptor in early diabetic pain neuropathy.

Talbot S, Chahmi E, Dias JP, Couture R - J Neuroinflammation (2010)

Effect of microglia inhibitors administered 3 h earlier on Iba-1 microglial immunoreactivity in the spinal dorsal horn of control and 4-day STZ-diabetic rats. Shown are (A) confocal microscopy pictures of microglial cells, and (B) the quantification of the mean pixel energy of Iba-1 (in arbitrary unit, AU). Scale bar = 100 μm. Data are the mean ± S.E.M of 4 pictures per rat from 4 rats in each group. Background mean energy was subtracted for each picture. Statistical comparison to control (*) or STZ rats treated with vehicle (+) is indicated by *** +++ P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913947&req=5

Figure 3: Effect of microglia inhibitors administered 3 h earlier on Iba-1 microglial immunoreactivity in the spinal dorsal horn of control and 4-day STZ-diabetic rats. Shown are (A) confocal microscopy pictures of microglial cells, and (B) the quantification of the mean pixel energy of Iba-1 (in arbitrary unit, AU). Scale bar = 100 μm. Data are the mean ± S.E.M of 4 pictures per rat from 4 rats in each group. Background mean energy was subtracted for each picture. Statistical comparison to control (*) or STZ rats treated with vehicle (+) is indicated by *** +++ P < 0.001.
Mentions: A quantitative immunolabelling with a specific immunomarker of microglia Iba-1 was employed to validate the use of minocycline and fluorocitrate as inhibitors of microglia activity. As shown in Figure 3-A, immunoreactivity to Iba-1 was much more striking in the spinal dorsal horn of STZ-diabetic rats than in matched control spinal dorsal horn. Immunoreactive microglial cells in STZ spinal cord were more numerous, thicker and displayed higher mean pixel energy than microglia of control spinal dorsal horn. Importantly, treatment with fluorocitrate or minocycline reversed and normalized the enhanced Iba-1 immunoreactivity in STZ spinal cord microglia (Figure 3-B). The same treatment with fluorocitrate or minocycline had no effect on the mean pixel energy of Iba-1 in control spinal dorsal horn.

Bottom Line: Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1.Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations.The enhanced mRNA expression (B1R, IL-1beta, TNF-alpha, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Université de Montréal, C,P, 6128, Succursale Downtown, Montréal, Québec, H3C 3J7, Canada.

ABSTRACT

Background: The pro-nociceptive kinin B1 receptor (B1R) is upregulated on sensory C-fibres, astrocytes and microglia in the spinal cord of streptozotocin (STZ)-diabetic rat. This study aims at defining the role of microglial kinin B1R in diabetic pain neuropathy.

Methods: Sprague-Dawley rats were made diabetic with STZ (65 mg/kg, i.p.), and 4 days later, two specific inhibitors of microglial cells (fluorocitrate, 1 nmol, i.t.; minocycline, 10 mg/kg, i.p.) were administered to assess the impact on thermal hyperalgesia, allodynia and mRNA expression (qRT-PCR) of B1R and pro-inflammatory markers. Spinal B1R binding sites ((125I)-HPP-desArg10-Hoe 140) were also measured by quantitative autoradiography. Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1. Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations.

Results: STZ-diabetic rats displayed significant tactile and cold allodynia compared with control rats. Intrathecal or peripheral blockade of B1R or inhibition of microglia reversed time-dependently tactile and cold allodynia in diabetic rats without affecting basal values in control rats. Microglia inhibition also abolished thermal hyperalgesia and the enhanced allodynia induced by intrathecal des-Arg9-BK without affecting hyperglycemia in STZ rats. The enhanced mRNA expression (B1R, IL-1beta, TNF-alpha, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%.

Conclusion: The upregulation of kinin B1R in spinal dorsal horn microglia by pro-inflammatory cytokines is proposed as a crucial mechanism in early pain neuropathy in STZ-diabetic rats.

Show MeSH
Related in: MedlinePlus