BDNF-TrkB signaling pathway-mediated microglial activation induces neuronal KCC2 downregulation contributing to dynamic allodynia following spared nerve injury

Abstract

Mechanical allodynia can be evoked by punctate pressure contact with the skin (punctate mechanical allodynia) and dynamic contact stimulation induced by gentle touching of the skin (dynamic mechanical allodynia). Dynamic allodynia is insensitive to morphine treatment and is transmitted through the spinal dorsal horn by a specific neuronal pathway, which is different from that for punctate allodynia, leading to difficulties in clinical treatment. K⁺-Cl^- cotransporter-2 (KCC2) is one of the major determinants of inhibitory efficiency, and the inhibitory system in the spinal cord is important in the regulation of neuropathic pain. The aim of the current study was to determine whether neuronal KCC2 is involved in the induction of dynamic allodynia and to identify underlying spinal mechanisms involved in this process. Dynamic and punctate allodynia were assessed using either von Frey filaments or a paint brush in a spared nerve injury (SNI) mouse model. Our study discovered that the downregulated neuronal membrane KCC2 (mKCC2) in the spinal dorsal horn of SNI mice is closely associated with SNI-induced dynamic allodynia, as the prevention of KCC2 downregulation significantly suppressed the induction of dynamic allodynia. The over activation of microglia in the spinal dorsal horn after SNI was at least one of the triggers in SNI-induced mKCC2 reduction and dynamic allodynia, as these effects were blocked by the inhibition of microglial activation. Finally, the BDNF-TrkB pathway mediated by activated microglial affected SNI-induced dynamic allodynia through neuronal KCC2 downregulation. Overall, our findings revealed that activation of microglia through the BDNF-TrkB pathway affected neuronal KCC2 downregulation, contributing to dynamic allodynia induction in an SNI mouse model.

Keywords: BDNF-TrkB pathway; Dynamic allodynia; KCC2; microglia; spared nerve injury.

Figure 1.

Neuronal membrane KCC2 downregulation in spinal dorsal horn partly mediated SNI-induced dynamic allodynia. (a) Furosemide (30 nmol, i.t.) treatment after SNI surgery prevented the induction of the dynamic allodynia. (WT, n = 8; Sham, n = 6; SNI, n = 5; SNI + Fur, n = 5; red ***p < 0.001, SNI group vs Sham group; blue **p < 0.01, SNI + Fur group vs Sham group; blue ^###p < 0.001, SNI + Fur group vs SNI group, two-way ANOVA followed by Bonferroni post hoc analysis). (b) Treatment with furosemide (30 nmol, i.t.) prevented the induction of punctate allodynia at day 1 after SNI surgery. (WT, n = 8; Sham, n = 6; SNI, n = 5; SNI + Fur, n = 5; red *p < 0.05, **p < 0.01, SNI group vs Sham group; blue *p < 0.05, SNI + Fur group vs Sham group; blue ^###p < 0.001, SNI + Fur group vs SNI group, two-way ANOVA followed by Bonferroni post hoc analysis). (c) Representative immunostaining images of KCC2 (green) and NeuN (red) in the spinal dorsal cord, arrows showing the likely neuron membrane KCC2. Scale bar, 50 μm (left), 5 μm (right). (d) The histograms respectively show the quantification of ratio of immunostaining KCC2 density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (WT, n = 9; Sham, n = 5; SNI, n = 5; SNI + Fur, n = 7; *p < 0.05, SNI group vs Sham group; ^##p < 0.01, SNI + Fur group vs SNI group, unpaired Student’s t test). (e) Western blot of membrane KCC2 in the spinal dorsal cord. (f) The histograms respectively show the relative mKCC2 level on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (WT, n = 4; Sham, n = 4; SNI, n = 5; SNI + Fur, n = 5; **p < 0.01, WT group, SNI group or SNI + Fur group vs Sham group; ^###p < 0.001, SNI + Fur group vs SNI group, unpaired Student’s t test).

Figure 2.

Activation of microglia in spine dorsal horn is involved in SNI induced mKCC2 reduction and dynamic allodynia. (a) Representative immunostaining images of microglia in the spinal dorsal. Scale bar, 100 μm (left), 50 μm (right). (b) Representative immunostaining images of KCC2 (green) and NeuN (red) in the spinal dorsal, arrows showing the likely neuron membrane KCC2. Scale bar, 50 μm (left), 5 μm (right). (c) The histograms respectively show the quantification of ratio of immunostaining density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 5; SNI, n = 5; SNI + Mino; n = 5; SNI + 5BDBD, n = 9, **p < 0.01, SNI group vs Sham group, ***p < 0.001, SNI +Mino group or SNI +5BDBD group vs Sham group; ^#p < 0.05, SNI + 5BDBD group vs SNI group, ^#p < 0.05 SNI + Mino group vs SNI group, unpaired Student’s t test). (d) The histograms respectively show the quantification of ratio of immunostaining KCC2 density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 5; SNI, n = 4; SNI + Mino; n = 5; SNI + 5BDBD, n = 6, *p < 0.05, SNI group vs Sham group; ^##p < 0.01, SNI + 5BDBD group vs SNI group, ^#p < 0.05 SNI + Mino group vs SNI group, unpaired Student’s t test). (e) Western blot of membrane KCC2 in the spinal dorsal cord. (f) The histograms respectively show the relative mKCC2 level on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (SNI, n = 5; SNI + Mino, n = 4; SNI + 5BDBD, n = 4; ^#p < 0.05, SNI + 5BDBD group vs SNI group, ^##p < 0.01, SNI + Mino group vs SNI group, unpaired Student’s t test). (g) Minocycline (30 nmol, i.t.) or 5BDBD (30 nmol, i.t.) treatment after SNI surgery prevented the induction of the dynamic allodynia. (Sham, n = 5; SNI, n = 6; SNI + Mino, n = 6; SNI + 5BDBD, n = 6; red ***p < 0.001, SNI group vs Sham group; blue ***p < 0.001, SNI + Mino group vs Sham group; green ***p <0.001, SNI + 5BDBD group vs Sham group; blue ^###p < 0.001, SNI + Mino group vs SNI group; green ^##p < 0.01, ^###p < 0.001 SNI + 5BDBD group vs SNI group; two-way ANOVA followed by Bonferroni post hoc analysis). (h) Minocycline (30 nmol, i.t.) treatment after SNI surgery had no effect on the punctate allodynia, but 5BDBD (30 nmol, i.t.) treatment after SNI surgery prevented the induction of punctate allodynia at day 1 and day 2. (Sham, n = 5; SNI, n = 6; SNI + Mino, n = 6; SNI + 5BDBD, n = 6; red *p < 0.05, **p < 0.01, SNI group vs Sham group; blue *p < 0.05, **p < 0.01, SNI + Mino group vs Sham group; green *p < 0.05, SNI + 5BDBD group vs Sham group; green ^#p < 0.05, ^###p < 0.001 SNI + 5BDBD group vs SNI group; two-way ANOVA followed by Bonferroni post hoc analysis).

Figure 3.

Activation of spinal dorsal horn microglia triggered BDNF-TrkB pathway induced neuronal KCC2 down regulation contributes to the dynamic allodynia in SNI mice. (a) Representative immunostaining images of microglia in the spinal dorsal. Scale bar, 100 μm (left), 50 μm (right). (b) Representative immunostaining images of KCC2 (green) and NeuN (red) in the spinal dorsal, arrows showing the likely neuron membrane KCC2. Scale bar, 50 μm (left), 5 μm (right). (c) The histograms respectively show the quantification of ratio of immunostaining density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 5; SNI, n = 4; SNI + K252a, n = 9; *p < 0.05, **p < 0.01, SNI group or SNI +K252a group vs Sham group; unpaired Student’s t test). (d) The histograms respectively show the quantification of ratio of immunostaining KCC2 density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 5; SNI, n = 4; SNI + K252a, n = 5; *p < 0.05, SNI group vs Sham group; ^#p < 0.05, SNI + K252a group vs SNI group, unpaired Student’s t test). (e) Western blot of membrane KCC2 in the spinal dorsal cord. (f) The histograms respectively show the relative mKCC2 level on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (WT, n = 5; Sham, n = 6; SNI, n = 8; SNI + K252a, n = 8; **p < 0.01, SNI group vs Sham group, ^##p < 0.01, SNI + K252a group vs SNI group, unpaired Student’s t test). (g) K252a (30 nmol, i.t.) treatment after SNI surgery prevented the induction of the dynamic allodynia. (Sham, n = 7; SNI, n = 7; SNI + K252a, n = 6; red ***p < 0.001, SNI group vs Sham group; blue *p < 0.05, SNI + K252a group vs Sham group; blue ^###p <0.001, SNI + K252a vs SNI group; two-way ANOVA followed by Bonferroni post hoc analysis). (h) K252a (30 nmol, i.t.) treatment after SNI surgery prevented the induction of the punctate allodynia. (Sham, n = 7; SNI, n = 7; SNI + K252a, n = 6; red ***p < 0.001, SNI group vs Sham group; blue ^##p < 0.01, ^###p < 0.001, SNI + K252a group vs SNI group; two-way ANOVA followed by Bonferroni post hoc analysis).

Figure 4.

BDNF treatment reversed anti-glia activation to induce dynamic allodynia through mKCC2 down regulation. (a) Representative immunostaining images of microglia in the spinal dorsal. Scale bar, 100 μm (left), 50 μm (right). (b) Representative immunostaining images of KCC2 (green) and NeuN (red) in the spinal dorsal, arrows showing the likely neuron membrane KCC2. Scale bar, 50 μm (left), 5 μm (right). (c) The histograms respectively show the quantification of ratio of immunostaining density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 5; SNI, n = 4; SNI + Mino, n = 5; SNI + Mino + BDNF, n = 8; ***p < 0.001, SNI group, SNI + Mino group or SNI + Mino + BDNF group vs Sham group; ^##p < 0.01, SNI + Mino group vs SNI group; ^###p < 0.001, SNI + Mino + BDNF group vs SNI group; unpaired Student’s t test). (d) The histograms respectively show the quantification of ratio of immunostaining KCC2 density on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (Sham, n = 6; SNI, n = 9; SNI + Mino, n = 7; SNI + Mino + BDNF, n = 9; *p < 0.05, SNI + Mino + BDNF group vs Sham group, **p < 0.01, SNI group vs Sham group; ^##p < 0.01, SNI + Mino group vs SNI group; &&p < 0.01, SNI + Mino + BDNF group vs SNI + Mino group; unpaired Student’s t test). (e) Western blot of membrane KCC2 in the spinal dorsal cord. (f) The histograms respectively show the relative mKCC2 level on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord after SNI surgery. (SNI + Mino, n = 4; SNI + Mino + BDNF, n = 4; &&&p < 0.001, SNI + Mino + BDNF group vs SNI + Mino group; unpaired Student’s t test). (g) BDNF (3 ng/10 uL, i.t.), once a day for consecutive 2 days after SNI surgery with the Mino (30 nmol, i.t.) or the vehicle co-injected on the 1st day significantly reversed the blockade effect of Mino on SNI-induced dynamic allodynia on both day 1 and day 2. (Sham, n = 9; SNI, n = 8; SNI + Mino, n = 7; SNI + Mino + BDNF, n = 6; red ***p < 0.001, SNI group vs Sham group; blue ***p < 0.001, SNI + Mino group vs Sham group; purple ***p < 0.001, SNI + Mino + BDNF group vs Sham group; blue ^##p < 0.01, ^###p < 0.001, SNI + Mino group vs SNI group; purple ^##p < 0.01, SNI + Mino + BDNF group vs SNI group; purple &P < 0.05, &&&p < 0.001, SNI + Mino + BDNF group vs SNI + Mino group; two-way ANOVA followed by Bonferroni post hoc analysis). (h) BDNF (3 ng/10 uL, i.t.), once a day for consecutive 2 days after SNI surgery with the Mino (30 nmol, i.t.) or the vehicle co[1]injected on the 1st day had no effect on the punctate allodynia. (Sham, n = 9; SNI, n = 8; SNI + Mino, n = 7; SNI + Mino + BDNF, n = 6; red ***p < 0.001, SNI group vs Sham group; blue ***p < 0.001, SNI + Mino group vs Sham group; purple ***p < 0.001, SNI + Mino + BDNF group vs Sham group; two-way ANOVA followed by Bonferroni post hoc analysis).

Figure 5.

The involvement of KCC2 downregulation in BDNF-induced both dynamic and punctate allodynia. (a) Western blot of membrane KCC2 in the spinal dorsal cord. (b) The histograms respectively show the relative mKCC2 level on ipsilateral (ipsi) and contralateral (cont) sides of dorsal spinal cord (Sham, n = 6; Sham + BDNF, n = 6; Sham + BDNF +Fur, n = 6; ***p < 0.001, Sham group vs Sham + BDNF group; ^###P < 0.001, Sham + BDNF group vs Sham + BDNF +Fur group, unpaired Student’s t test). (c) BDNF (3 ng/10 uL, i.t.) with the Fur (30 nmol, i.t.) co-injected on the 1st daysignificantly reversed the BDNF (3 ng/10 uL, i.t.) treatment alone evoked the dynamic allodynia. (Sham, n = 5; Sham + BDNF, n = 6; Sham + BDNF + Fur, n = 7; blue ***p < 0.001, Sham + BDNF vs Sham group; purple ^###P < 0.001, Sham + BDNF + Fur vs Sham + BDNF group, two-way ANOVA followed by Bonferroni post hoc analysis). (d) BDNF (3 ng/10 uL, i.t.) with the Fur (30 nmol, i.t.) co-injected on the 1st day significantly reversed the BDNF (3 ng/10 uL, i.t.) treatment alone evoked the punctate allodynia. (Sham, n = 5; Sham + BDNF, n = 6; Sham + BDNF + Fur, n = 7; blue ***p < 0.001, Sham + BDNF vs Sham group; purple ^#p < 0.05, ^###p < 0.01, ^###p < 0.001, Sham + BDNF + Fur vs Sham + BDNF group, two-way ANOVA followed by Bonferroni post hoc analysis).

Figure 6.

Scematic diagram of KCC2 contributing to dynamic allodynia. SNI activates spinal dorsal horn (SDH) microglia, which leads to the release of BDNF. BDNF downregulates KCC2 in SDH pain transmission neurons by combing with TrkB, causing an increase in intracellular Cl− and leading to a depolarizing shift in the anion reversal potential. The resulting hyperexcitability of pain transmission in neurons contributes to dynamic allodynia.