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

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.

Figure 1

Effect of microglia inhibitors administered 3 h earlier on blood glucose concentration in control and 4-day STZ-diabetic rats. Data are the mean ± S.E.M. of 5 rats in each group. Statistical comparison to control rats is indicated by ***P < 0.001.

Figure 2

Time-course effect of B₁R antagonists administered in the periphery (A) or intrathecally (B) on blood glucose concentration in control and 4-day STZ-diabetic rats. Data are the mean ± S.E.M. of 5 rats in each group. Statistical comparison to control (*) or untreated (0 h) STZ-treated rats (+) is indicated by ⁺ P < 0.05, ***P < 0.001.

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.

Figure 4

Effect of microglia inhibitors administered 3 h earlier on B₁R binding sites in the thoracic spinal cord of control and 4-day STZ-diabetic rats. Shown in (A) are autoradiograms of Control (I), Non-specific (II), STZ (III) and STZ + fluorocitrate (IV), and in (B) are the quantification of specific densities of B₁R binding sites in spinal dorsal horn (Laminae I-III). Data are the mean ± S.E.M. of 5 to 6 rats in each group. Statistical comparison to control rats is indicated by **P < 0.01.

Figure 5

Effect of microglia inhibitors administered 3 h earlier on tail-flick reaction time (MPE %) in control and 4-day STZ-diabetic rats. Shown are the maximal responses measured 1 min after intrathecal injection of either aCSF, 9.6 nmol des-Arg⁹-BK or 6.6 nmol SP. Data are the mean ± S.E.M. of 5 rats in each group. Within groups, statistical comparison to aCSF is indicated by ^††P < 0.01, while statistical comparison to the same agonist in the control group (*) or in STZ + vehicle (+) is indicated by * ⁺ P < 0.05; ^{++ ††}P < 0.01; ***P < 0.001.

Figure 6

Time-course of the inhibitory effect of B₁R antagonists administered in the periphery (A, C) or intrathecally (B, D) on spontaneous paw withdrawal threshold to tactile stimulation (g) in control and 4-day STZ-diabetic rats. All treatments were given at time 0 h. Data are the mean ± S.E.M. of 4 to 12 rats in each group. Statistical comparison to control + vehicle (*) and STZ + vehicle (⁺) is indicated by ⁺⁺P < 0.01; *** ⁺⁺⁺P < 0.001.

Figure 7

Time-course of the inhibitory effect of fluorocitrate (A) and minocycline (B) on spontaneous paw withdrawal threshold to tactile stimulation (g) in control and 4-day STZ-diabetic rats. All treatments were given at time 0 h. Data are the mean ± S.E.M. of 4 rats in each group. Statistical comparison to control + vehicle (*) and STZ + vehicle (⁺) is indicated by *** ⁺⁺⁺P < 0.001.

Figure 8

Time-course of the inhibitory effect of B₁R antagonists administered in the periphery (A, C) or intrathecally (B, D) on paw withdrawal response frequency (%) to cold stimulation in control and 4-day STZ-diabetic rats. All treatments were given at time 0 h. Data represent the mean ± S.E.M. of 4 to 12 rats in each group. Statistical comparison to control + vehicle (*) and STZ + vehicle (⁺) is indicated by ⁺⁺P < 0.01; *** ⁺⁺⁺P < 0.001.

Figure 9

Time-course of the inhibitory effect of fluorocitrate (A) and minocycline (B) on paw withdrawal response frequency (%) to cold stimulation in control and 4-day STZ-diabetic rats. All treatments were given at time 0 h. Data are the mean ± S.E.M. of 4 rats in each group. Statistical comparison to control + vehicle (*) and STZ + vehicle (⁺) is indicated by ⁺⁺P < 0.01; ⁺⁺⁺ ***P < 0.001.