Omega-conotoxin MVIIA reduces neuropathic pain after spinal cord injury by inhibiting N-type voltage-dependent calcium channels on spinal dorsal horn

Abstract

Spinal cord injury (SCI) leads to the development of neuropathic pain. Although a multitude of pathological processes contribute to SCI-induced pain, excessive intracellular calcium accumulation and voltage-gated calcium-channel upregulation play critical roles in SCI-induced pain. However, the role of calcium-channel blockers in SCI-induced pain is unknown. Omega-conotoxin MVIIA (MVIIA) is a calcium-channel blocker that selectively inhibits N-type voltage-dependent calcium channels and demonstrates neuroprotective effects. Therefore, we investigated spinal analgesic actions and cellular mechanisms underlying the analgesic effects of MVIIA in SCI. We used SCI-induced pain model rats and conducted behavioral tests, immunohistochemical analyses, and electrophysiological experiments (in vitro whole-cell patch-clamp recording and in vivo extracellular recording). A behavior study suggested intrathecal MVIIA administration in the acute phase after SCI induced analgesia for mechanical allodynia. Immunohistochemical experiments and in vivo extracellular recordings suggested that MVIIA induces analgesia in SCI-induced pain by directly inhibiting neuronal activity in the superficial spinal dorsal horn. In vitro whole-cell patch-clamp recording showed that MVIIA inhibits presynaptic N-type voltage-dependent calcium channels expressed on primary afferent Aδ-and C-fiber terminals and suppresses the presynaptic glutamate release from substantia gelatinosa in the spinal dorsal horn. In conclusion, MVIIA administration in the acute phase after SCI may induce analgesia in SCI-induced pain by inhibiting N-type voltage-dependent calcium channels on Aδ-and C-fiber terminals in the spinal dorsal horn, resulting in decreased neuronal excitability enhanced by SCI-induced pain.

Keywords: N-type voltagedependent calcium channels; neuropathic pain; omega-conotoxin MVIIA; spinal cord injury; spinal dorsal horn.

Figure 1

MVIIA induces analgesia improvement of mechanical allodynia and locomotor function in spinal cord injury (SCI). Rats with spinal contusion injury received a vehicle via i.t. injection 4 h after surgery (SCI rats) and received MVIIA (200 pmol) via i.t. injection 4 h after surgery (SCI + MVIIA rats). Closed circles represent SCI rats, and open circles represent SCI + MVIIA rats. (A) Fourteen days after contusion injury (D14), SCI + MVIIA rats revealed a significantly increased mechanical threshold for paw withdrawal compared with SCI rats (n = 12/SCI rats, n = 8/SCI + MVIIA rats). Data are presented as mean ± standard deviation (SD), ^**p < 0.01, unpaired t-test. (B) Over the observational period of 14 days, the Basso, Beattie, and Bresnahan (BBB) score gradually increased in both SCI and SCI + MVIIA rats; however, the BBB score for SCI + MVIIA rats was significantly higher at most points than that for SCI rats (n = 12/SCI rats, n = 8/SCI + MVIIA rats). Data are presented as mean ± SD, ^*p < 0.05, ^**p < 0.01, one-way repeated-measures analysis of variance.

Figure 2

MVIIA suppresses phosphorylated extracellular signal-regulated kinase (pERK) in the spinal dorsal horn in spinal cord injury (SCI). Representative images of pERK-positive neurons in the spinal dorsal horn slices. Images corresponding to (A) naïve rats (n = 6) and (B) rats with spinal contusion injury receiving a vehicle by i.t. injection 4 h after surgery (n = 7). (C) Images corresponding to rats with spinal contusion injury receiving MVIIA (200 pmol) via i.t. injection 4 h after surgery (SCI + MVIIA rats, n = 11). (D) The graph represents the number of pERK-positive neurons in each slice of the spinal dorsal horn (laminae I–II). Data are presented as mean ± standard deviation, ^**p < 0.01, one-way analysis of variance followed by Bonferroni post-hoc comparison.

Figure 3

MVIIA suppresses both spontaneous and stimulus-evoked firing on the spinal dorsal horn in spinal cord injury (SCI). In vivo extracellular recordings of the superficial spinal dorsal horn. Rats with spinal contusion injury received a vehicle via i.t. injection 4 h after surgery (SCI rats) and received MVIIA (200 pmol) via i.t. injection 4 h after surgery (SCI + MVIIA rats). (A) Spontaneous firing of the superficial spinal dorsal horn in SCI + MVIIA rats (n = 20) was significantly lower than that in in SCI rats (n = 18). (B) Similarly, stimulus-evoked firing in the superficial spinal dorsal horn in SCI + MVIIA rats (n = 20) was significantly lower than that in SCI rats (n = 18). Data are presented as mean ± standard deviation, ^**p < 0.01, unpaired t-test. vFF = von Frey Filament.

Figure 4

MVIIA inhibits the presynaptic excitatory interneurons of substantia gelatinosa neurons in the spinal dorsal horn in spinal cord injury (SCI). In vitro patch-clamp recordings of substantia gelatinosa neurons in the spinal dorsal of SCI rats with spinal contusion injury. (A) Miniature excitatory post-synaptic currents (mEPSCs) were isolated by adding tetrodotoxin (TTX, 0.5 μM) and direct application of MVIIA to the spinal cord (1 μM, 30 s) in SCI rats. (B) Direct application of MVIIA to the spinal cord did not change the mean mEPSCs amplitude (n = 16; paired t-test) or affect the cumulative distribution of mEPSCs amplitudes (Kolmogorov–Smirnov test). (C) MVIIA significantly decreased the mean mEPSCs frequency (n = 16; paired t-test), and the cumulative inter-event interval distribution showed a significant rightward shift (Kolmogorov–Smirnov test). Data are presented as mean ± standard deviation, ^**p < 0.01. TTX = tetrodotoxin.

Figure 5

MVIIA does not affect the inhibitory interneurons of substantia gelatinosa neurons in the spinal dorsal horn in spinal cord injury (SCI). In vitro patch-clamp recordings of substantia gelatinosa neurons in the spinal dorsal of SCI rats with spinal contusion injury. Miniature inhibitory post-synaptic currents (mIPSCs) were isolated by adding tetrodotoxin (TTX, 0.5 μM) and direct application of MVIIA to the spinal cord (1 μM, 30 s) in SCI rats. The direct application of MVIIA to the spinal cord did not change the mean mIPSCs amplitude (n = 7; paired t-test) or frequency (n = 7; paired t-test). Data are presented as mean ± standard deviation. TTX = tetrodotoxin.

Figure 6

MVIIA inhibits the amplitude of monosynaptic glutamatergic evoked-excitatory post-synaptic currents (EPSCs) expressed on primary afferent Aδ-and C-fibers of substantia gelatinosa neurons in the spinal dorsal horn in spinal cord injury (SCI). In vitro patch-clamp recordings of substantia gelatinosa neurons in the spinal dorsal of SCI rats with spinal contusion injury. Direct application of MVIIA (1 μM, 30 s) decreased both the amplitudes of monosynaptic Aδ-fiber-evoked EPSCs (n = 15; paired t-test) and those of monosynaptic C-fiber-evoked EPSCs (n = 11; paired t-test). Data are presented as mean ± standard deviation, ^**p < 0.01.

Figure 7

MVIIA does not affect the miniature excitatory post-synaptic currents (mEPSCs) frequency and amplitudes of monosynaptic glutamatergic evoked EPSCs expressed on primary afferent Aδ-and C-fibers of substantia gelatinosa neurons in the spinal dorsal horn in spinal cord injury (SCI) under Ca²⁺ free circumstances. In vitro patch-clamp recordings of substantia gelatinosa neurons in the spinal dorsal of spinal cord injury rats with spinal contusion injury. (A) In Ca²⁺ free Krebs, direct application of MVIIA (1 μM, 30 s) does not change the mean mEPSCs amplitude (n = 10; paired t-test) or frequency (n = 10; paired t-test). Data are presented as mean ± standard deviation. TTX = tetrodotoxin. (B) In Ca²⁺ free Krebs, direct application of MVIIA (1 μM, 30 s) does not change the amplitudes of monosynaptic Aδ-fiber-evoked EPSCs (n = 10; paired t-test) or those of monosynaptic C-fiber-evoked EPSCs (n = 8; paired t-test). Data are presented as mean ± standard deviation.

Figure 8

Mechanism of analgesic action of MVIIA in the spinal dorsal horn in spinal cord injury (SCI). MVIIA acts on N-type voltage-dependent calcium channels on both Aδ-and C-fiber terminals in the spinal dorsal horn to inhibit the conduction of pain evoked by stimulation and decrease glutamate release. These mechanisms suggest that administration of MVIIA in the acute phase after SCI may contribute to the analgesic effect, inhibiting spinal neuronal excitability enhanced by SCI-induced pain.