Pain modulation effect on motor cortex after optogenetic stimulation in shPKCγ knockdown dorsal root ganglion-compressed Sprague-Dawley rat model

Abstract

Neuropathic pain can be generated by chronic compression of dorsal root ganglion (CCD). Stimulation of primary motor cortex can disrupt the nociceptive sensory signal at dorsal root ganglion level and reduce pain behaviors. But the mechanism behind it is still implicit. Protein kinase C gamma is known as an essential enzyme for the development of neuropathic pain, and specific inhibitor of protein kinase C gamma can disrupt the sensory signal and reduce pain behaviors. Optogenetic stimulation has been emerged as a new and promising conducive method for refractory neuropathic pain. The aim of this study was to provide evidence whether optical stimulation of primary motor cortex can modulate chronic neuropathic pain in CCD rat model. Animals were randomly divided into CCD group, sham group, and control group. Dorsal root ganglion-compressed neuropathic pain model was established in animals, and knocking down of protein kinase C gamma was also accomplished. Pain behavioral scores were significantly improved in the short hairpin Protein Kinase C gamma knockdown CCD animals during optic stimulation. Ventral posterolateral thalamic firing inhibition was also observed during light stimulation on motor cortex in CCD animal. We assessed alteration of pain behaviors in pre-light off, stimulation-light on, and post-light off state. In vivo extracellular recording of the ventral posterolateral thalamus, viral expression in the primary motor cortex, and protein kinase C gamma expression in dorsal root ganglion were investigated. So, optical cortico-thalamic inhibition by motor cortex stimulation can improve neuropathic pain behaviors in CCD animal, and knocking down of protein kinase C gamma plays a conducive role in the process. This study provides feasibility for in vivo optogenetic stimulation on primary motor cortex of dorsal root ganglion-initiated neuropathic pain.

Keywords: Optogenetics; dorsal root ganglia; motor cortex; neuropathic pain; protein kinase C gamma; thalamus.

Figure 1.

Experimental animal model and timeline: (A) experimental timeline, (B) schematic diagram of experimental animal model and optogenetic virus injection site, and (C) stainless steel rod insertion at L4 and L5 intervertebral foramen to induce DRG compression. DRG: dorsal root ganglion; CCD: chronic compression of DRG; PKCγ: protein kinase C gamma.

Figure 2.

Optogenetic stimulation procedure: (A) complete setup of optic stimulation, (B) optic fiber implantation position, and (C) stimulation giving by blue laser on primary motor cortex area.

Figure 3.

Behavioral pain responses to mechanical and thermal tests of CCD group and sham group. (A) Paw withdrawal latency in response to mechanical pain with a plantar aesthesiometer of ipsilateral and contralateral hind paw in CCD group. (B) Ipsilateral hind paw withdrawal latency between CCD group and sham group. (C) Paw withdrawal threshold of ipsilateral and contralateral hind paw in CCD group. (D) Ipsilateral hind paw withdrawal threshold between CCD group and sham group. (E) Hind paw thermal latency in hot plate test between DRG-compressed group and sham group. **p < 0.01, ****p < 0.0001 significant difference between the indicated values (ANOVA). DRG: dorsal root ganglion.

Figure 4.

Alterations of hyperalgesia in animal groups with blue laser stimulation. (A) Paw withdrawal latency scores, (B) paw withdrawal threshold scores, and (C) thermal latency scores of all eight animal groups. Only CCD-Opto-shPKCγ group (a) and CCD-Opto-shPKCsafe group (e) exhibited significant changes of behavioral scores with blue light “ON” state. In other animal groups, blue laser stimulation “ON” state did not have any significant alterations in behavioral response. *p < 0.05; **p < 0.01, ***p < 0.001 significant difference between the indicated values (ANOVA). CCD: chronic compression of dorsal root ganglion; PKCγ: protein kinase C gamma.

Figure 5.

Electrophysiology results of thalamic output by optic stimulation in motor cortex. (A) Evoked firing rates in the neurons of VPL thalamus of CCD animals compared to sham-operated animals. Unpaired t-test was used to compare between sham group and CCD group. (B) Burst firing rates of CCD-grouped animals following optical stimulation. Significant change was seen in only CCD-Opto-shPKCγ group. Burst rates decreased in CCD-Opto-shPKCsafe group also, but it was not significant. (C, D) In vivo recording of CCD-Opto-shPKCγ-grouped animals (C) and CCD-Opto-shPKCsafe-grouped animals (D) from the VPL thalamus. Firing output (spikes/s) declines under blue laser stimulation, which is higher in the pre- and post-light states. (E, F) No changes of firing rates in CCD-Null-shPKCγ and CCD-Null-shPKCsafe group, respectively. Two-way ANOVA test was used to compare neuronal activity according to the optical stimulation. *p < 0.05; **p < 0.01; ***p<0.001 significant difference between the indicated values (ANOVA). (G) Average action potential waveform of VPL neuron of CCD-Opto-shPKCγ-grouped animal. (H) Average action potential waveform of CCD-Opto-shPKCsafe-grouped animal. (G, H) In both cases, amplitude got decreased during optical stimulation on motor cortex. (I, J) Perievent raster histogram of responses of CCD-Opto-shPKCγ-grouped animal’s VPL neurons. (K, L) Raster plot responses of CCD-Opto-shPKCsafe-grouped animal’s VPL neurons. (I, K) Increased firing response when there is no optical stimulation present. (J, L) During optical stimulation, VPL firing response got decreased. Bin size = 50 ms. CCD: chronic compression of dorsal root ganglion; PKCγ: protein kinase C gamma.

Figure 5.

Electrophysiology results of thalamic output by optic stimulation in motor cortex. (A) Evoked firing rates in the neurons of VPL thalamus of CCD animals compared to sham-operated animals. Unpaired t-test was used to compare between sham group and CCD group. (B) Burst firing rates of CCD-grouped animals following optical stimulation. Significant change was seen in only CCD-Opto-shPKCγ group. Burst rates decreased in CCD-Opto-shPKCsafe group also, but it was not significant. (C, D) In vivo recording of CCD-Opto-shPKCγ-grouped animals (C) and CCD-Opto-shPKCsafe-grouped animals (D) from the VPL thalamus. Firing output (spikes/s) declines under blue laser stimulation, which is higher in the pre- and post-light states. (E, F) No changes of firing rates in CCD-Null-shPKCγ and CCD-Null-shPKCsafe group, respectively. Two-way ANOVA test was used to compare neuronal activity according to the optical stimulation. *p < 0.05; **p < 0.01; ***p<0.001 significant difference between the indicated values (ANOVA). (G) Average action potential waveform of VPL neuron of CCD-Opto-shPKCγ-grouped animal. (H) Average action potential waveform of CCD-Opto-shPKCsafe-grouped animal. (G, H) In both cases, amplitude got decreased during optical stimulation on motor cortex. (I, J) Perievent raster histogram of responses of CCD-Opto-shPKCγ-grouped animal’s VPL neurons. (K, L) Raster plot responses of CCD-Opto-shPKCsafe-grouped animal’s VPL neurons. (I, K) Increased firing response when there is no optical stimulation present. (J, L) During optical stimulation, VPL firing response got decreased. Bin size = 50 ms. CCD: chronic compression of dorsal root ganglion; PKCγ: protein kinase C gamma.

Figure 6.

Immunofluorescence results confirmed viral expression in primary motor cortex area (A–L). The low-magnification figures (A to C) and higher magnification figures (D to F) showed the optogenetic virus-infected and DAPI-stained neurons in the motor cortex region. (G to I) and (J to L), respectively, showed the low-magnification and higher magnification of histological section of null virus-infected and DAPI-stained neurons in the motor cortex region. (A, D, G, J) EYFP, (B, E, H, K) DAPI, and (C, F, I, L) Merge. (A, B, C, G, H, I) Scale bar = 200 µm. (D, E, F, J, K, L) Scale bar = 100 µm.

Figure 7.

Immunohistochemistry results of DRG cells of CCD animals. (A, B) DRG cells of shPKCγ-injected animals showing no bindings with anti-PKCγ antibody as shPKCγ inhibits the activation of PKCγ within DRG cells. (C, D) DRG cells of shPKCsafe-injected animals showing bindings (arrow bars) anti-PKCγ antibody with activated PKCγ within DRG cells. (A, C) Scale bar = 20 µm. (B, D) Scale bar = 50 µm.