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, 234 (1), 39-49

Intraspinal Transplantation of GABAergic Neural Progenitors Attenuates Neuropathic Pain in Rats: A Pharmacologic and Neurophysiological Evaluation

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Intraspinal Transplantation of GABAergic Neural Progenitors Attenuates Neuropathic Pain in Rats: A Pharmacologic and Neurophysiological Evaluation

Stanislava Jergova et al. Exp Neurol.

Abstract

Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABA(A) receptor antagonist bicuculline (BIC), GABA(B) receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1-3week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals.

Figures

Figure 1
Figure 1
Responses to mechanical (A), heat (B) and cold (C) stimulation after CCI. Negative difference scores for mechanical and heat stimulation indicate hyperalgesia. Increased responses to cold stimulation indicate cold allodynia. Animals with grafted cells (n=10) showed improvement of pain-related behavior compared to saline injected CCI animals (n=10). Inj= intraspinal injection of cells/saline. **p<0.01, *p<0.05 vs baseline; #p<0.05 vs saline group.M
Figure 2
Figure 2
Electrophysiological recording of spinal neuronal responses (n 8 neurons) after hind paw stimulation. CCI induced enhanced firing of A fibers (A) and C fibers (B, C) compared to pre-injury values. Transplantation of GABAergic NPC reduced wind-up responses (B) and post-discharges of C fibers (C) compared to saline treated animals. Inj= intraspinal injection of cells/saline.*p<0.05 vs baseline; #p<0.05 vs saline group.
Figure 3
Figure 3
The effect of intrathecal injection of drugs on heat hyperalgesia in CCI rats (n=10/group) at 1-2 weeks post grafting. Saline (A) did not show any effect of heat hyperalgesia. Bicuculline (B) and CGP (C) significantly reduced pain threshold in NPC grafted animals compared to baseline values. The opposite effect was observed after SKF (D) injection with enhancement of analgesia in control rats. BL= baseline. *p<0.05 vs baseline; #p<0.05 vs saline group.
Figure 4
Figure 4
The effect of intrathecal injection of drugs on cold allodynia in CCI rats (n=10/group) at 1-2 weeks post grafting. Saline (A) did not change responses to cold stimulation. Bicuculline (B) enhanced cold sensitivity in NPC treated CCI rats. CGP (C) had only mild effect. Analgesic effect of SKF (D) was presented in both CCI groups with significant reduction of sensitivity in saline treated rats. *p<0.05 vs baseline; #p<0.05 vs saline group.
Figure 5
Figure 5
The effect of spinal application of drugs on wind-up response of spinal neurons (n 8 neurons) after peripheral stimulation. Bicuculline (A-C) enhanced wind-up response in NPC treated group (A). The apparent decrease in wind–up potentiation in saline treated group (A) was caused by reduced ratio between initial and final firing of neurons although the overall response was significantly higher in this group (B) compared to NPC group (C). CGP (D-F) enhanced wind-up response in NPC treated animals while overall responses between NPC and saline group were comparable (E, F). *p<0.05 vs baseline; #p<0.05 vs saline group.
Figure 6
Figure 6
The effect of spinal application of drugs on C-fibers post-discharges (n 8 neurons). Increased firing was observed in both groups up to 60 min post bicuculline application (A). Enhanced firing reappeared in saline treated group 120 min post drug application. The effect of CGP (B) was moderate. *p<0.05 vs baseline; #p<0.05 vs saline group.
Figure 7
Figure 7
Photomicrograph showing GABAergic NPC graft in the spinal dorsal horn (A). The graft was identified morphologically based on DAPI (blue) and GABA (red) immunohistochemical staining revealing area with numerous cells nuclei that was not observed at the contralateral side of the spinal cord (B). Grafted cells were able to differentiate into mature GABAergic neurons as showed by overlapping of GABA (red) and NeuN (green) signals (C). The track of the needle in saline injected rats was identified by DAPI staining (D). No GABAergic profiles were found in the saline track (E). For electrophysiological experiment, the recording electrode was placed in the close vicinity of transplanted cells and its position was traced using FastGreen dye (F). Scale bar 50 μm (A,B,D,F), 10 μm (C, E).
Figure 8
Figure 8
Estimation of GABAergic profiles in the spinal dorsal horn laminae of saline-injected or NPC-injected rats. As expected, reduced GABAergic profiles occurred in the ipsilateral spinal cord of saline injected rats compared to contralateral side (*p<0.001, t-test). Intraspinal injection of GABAergic cells enhanced GABAergic immunoreactivity in the ipsilateral spinal cord of the grafted rats compared to saline group (#p<0.001, t-test).

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