Optogenetic Inhibition of Insular Cortex Glutamatergic Neurons Regulates Trigeminal Neuropathic Pain

Introduction and Background

Trigeminal neuropathic pain (TNP) is a severe facial disorder characterized by rapid and intense stabbing pain attacks that spread along the cutaneous segments of the trigeminal nerve. TNP occurs almost twice as frequently in women as in men. While traditional surgical procedures such as rhizotomy or microvascular decompression are effective for most typical TNP patients, surgical outcomes are often unsatisfactory for atypical TNP patients, with some patients continuing to experience symptoms or side effects. Therefore, there is an urgent need to develop new analgesic interventions through in-depth research on different brain regions.

The insular cortex (IC) plays a key role in several sensory and cognitive processes, including social interaction, learning and memory, emotional expression, taste, and anxiety. The IC is divided into three subregions: the anterior insula, the dysgranular insula, and the agranular insula. Previous studies have shown that the dysgranular insular cortex (DPIC) plays an important role in pain processing.

Paper Source

This study was conducted by Jaisan Islam, Elina KC, Soochong Kim, Moon Young Chung, Ki Seok Park, Hyong Kyu Kim, and Young Seok Park from various institutions in South Korea, including Chungbuk National University, Soonchunhyang University, and Eulji University. The paper was published online in the journal Neuromolecular Medicine on September 12, 2023.

Research Methods

Experimental Procedure

The study used 40 eight-week-old female Sprague-Dawley rats, constructing a TNP model using the chronic constriction injury of infraorbital nerve (CCI-ION) model. The rats were randomly divided into TNP, sham surgery, and control groups.

CCI-ION Surgery

CCI-ION group rats (n=16) underwent surgery on the left infraorbital nerve (ION), which included exposing the ION and ligating it with silk thread. Sham surgery group rats (n=16) underwent a similar procedure but without ION ligation.

Optogenetic Virus Stereotaxic Injection

After stereotaxic injection of optogenetic virus (AAV2-CAMKIIa-eNpHR3.0-EYFP) or control virus (AAV2-CAMKIIa-EYFP), optical fibers for light stimulation were implanted in the rats. The injection site was in the contralateral DPIC on the side opposite to CCI-ION. Behavioral observations were conducted daily after surgery for each group.

Behavioral Analysis

This included air-puff test, cold hyperalgesia test, mechanical allodynia test, and elevated plus maze test. Behavioral tests were conducted before CCI-ION surgery and on days 7, 14, 21, and 28 post-surgery.

In Vitro Single Neuron Electrophysiological Recording under DPICG Optogenetic Inhibition

Yellow laser light stimulation was used on DPICG, and in vitro single neuron electrophysiological recording techniques were used to observe neural activity in the trigeminal nucleus caudalis (TNc) and ventral posteromedial thalamus (VPM) contralateral to the CCA-ION side. Analysis results included overall neural firing rate and burst firing rate, as well as the effects of optogenetic inhibition on these firing rates.

Immunofluorescence and Histological Examination

Immediately after behavioral testing and electrophysiological recording, cardiac perfusion was performed. Rat brain tissues were fixed in 4% paraformaldehyde, followed by sectioning and immunofluorescence staining to observe the expression of c-Fos, pERK, and CREB-positive neurons.

Data Analysis

Data analysis was performed using GraphPad Prism software, with results expressed as mean ± standard deviation (SD). Data were compared using analysis of variance (ANOVA) or t-tests, with p<0.05 considered statistically significant.

Research Results

Behavioral Test Results

Air-Puff Test

TNP group rats showed significant mechanical allodynia, with their air-puff tolerance level decreasing from 22.71 ± 2.49 psi to 13.98 ± 2.32 psi over 4 weeks.

Cold Hyperalgesia Test

The cold hyperalgesia test showed that the cold sensitivity score of TNP group rats increased from 16.875 ± 2.65 to 23.208 ± 2.63.

Mechanical Allodynia Test

In the mechanical allodynia test, TNP group rats showed significantly increased sensitivity to mechanical stimulation, with their threshold decreasing from 12.652 ± 1.92 g to 7.24 ± 2.06 g.

Elevated Plus Maze Test

TNP group rats exhibited significant anxiety-like behavior, with both time spent and entries into open arms significantly reduced, from 0.45 ± 0.033% and 0.47 ± 0.066% to 0.29 ± 0.037% and 0.31 ± 0.052%, respectively.

Effects of DPICG Optogenetic Inhibition on TNc and VPM Neural Firing

Optogenetic inhibition of DPICG significantly reduced the firing rate of TNc neurons in TNP group rats from 26.94 ± 6.11 spikes/s to 20.87 ± 3.49 spikes/s, while also significantly reducing the burst firing rate.

Similarly, in VPM neural activity, optogenetic inhibition reduced the firing rate from 45.57 ± 3.90 spikes/s to 36.88 ± 2.17 spikes/s, while the burst firing rate decreased from 0.281 ± 0.0253 to 0.234 ± 0.0178.

Immunofluorescence Results

After optogenetic inhibition, c-fos expression in DPIC and TNc was significantly reduced, indicating decreased neural activity. Additionally, the expression of post-trauma creb and perk-positive neurons was also significantly reduced, further validating the analgesic effect of DPICG inhibition on TNP.

Conclusion

This study found that optogenetic inhibition of DPICG can effectively improve behavioral responses to TNP, reduce neural activity in TNc and VPM, thereby producing significant analgesic effects. The results indicate that DPICG plays an important role in the neural circuitry of TNP, and inhibiting its overactive state can modulate pain processing pathways, ultimately producing analgesic effects. This provides an effective experimental method for further studying the molecular mechanisms of DPIC in TNP and also provides a theoretical basis for developing new analgesic therapies using optogenetic methods.

Research Significance

This study verified the importance of DPICG in TNP regulation and proposed a new method of producing analgesic effects through optogenetic inhibition of DPICG. This not only provides new ideas for the treatment of TNP but also demonstrates the potential of optogenetic technology in pain research. The results may provide a foundation for developing new analgesic therapies targeting TNP, with important theoretical and practical value.