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. 2022 Jan-Dec:18:17448069211060181.
doi: 10.1177/17448069211060181.

Differential target multiplexed spinal cord stimulation programming modulates proteins involved in ion regulation in an animal model of neuropathic pain

Dana M Tilley  1 , David L Cedeño  1   2 , Francesco Vetri  3 , David C Platt  1   2 , Ricardo Vallejo  1   2   3
Affiliations

Differential target multiplexed spinal cord stimulation programming modulates proteins involved in ion regulation in an animal model of neuropathic pain

Dana M Tilley et al. Mol Pain. 2022 Jan-Dec.

Abstract

The effect of spinal cord stimulation (SCS) using differential target multiplexed programming (DTMP) on proteins involved in the regulation of ion transport in spinal cord (SC) tissue of an animal model of neuropathic pain was evaluated in comparison to low rate (LR) SCS. Rats subjected to the spared nerve injury model (SNI) and implanted with a SCS lead were assigned to DTMP or LR and stimulated for 48 h. A No-SCS group received no stimulation, and a Sham group received no SNI or stimulation. Proteins in the dorsal ipsilateral quadrant of the stimulated SC were identified and quantified using mass spectrometry. Proteins significantly modulated by DTMP or LR relative to No-SCS were identified. Bioinformatic tools were used to identify proteins related to ion transport regulation. DTMP modulated a larger number of proteins than LR. More than 40 proteins significantly involved in the regulation of chloride (Cl-), potassium (K+), sodium (Na+), or calcium (Ca2+) ions were identified. SNI affected proteins that promote the increase of intracellular Ca2+, Na+, and K+ and decrease of intracellular Cl-. DTMP modulated proteins involved in glial response to neural injury that affect Ca2+ signaling. DTMP decreased levels of proteins related to Ca2+ transport that may result in the reduction of intracellular Ca2+. Presynaptic proteins involved in GABA vesicle formation and release were upregulated by DTMP. DTMP also upregulated postsynaptic proteins involved with elevated intracellular Cl-, while modulating proteins, expressed by astrocytes, that regulate postsynaptic Cl- inhibition. DTMP downregulated K+ regulatory proteins affected by SNI that affect neuronal depolarization, and upregulated proteins that are associated with a decrease of intracellular neuronal K+ and astrocyte uptake of extracellular K+. DTMP treatment modulated the expression of proteins with the potential to facilitate a reversal of dysregulation of ion transport and signaling associated with a model of neuropathic pain.

Keywords: differential target multiplexed programming; ion transport regulation; neuropathic pain model; proteomics; spinal cord stimulation.

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Conflict of interest statement

Conflict of Interests: D.L.C. and R.V. are paid consultants and advisory board members of Medtronic Inc. They are co-inventors in patents related to differential target multiplexed spinal cord stimulation. Other authors declare no conflicts.

Figures

Figure 1.
Figure 1.
A sample scheme of the proteomics flow for isolation, purification, and quantification (courtesy of Cell Signaling Technology). Left: Trypsinized peptides from each experimental group are isotopically labeled and combined. Center: Labeled peptides are fractioned using high pressure liquid chromatography (HPLC). Right: Each fraction is analyzed in a tandem mass spectrometer (LC/MS/MS) to identify and quantify labeled peptides, which will be differentially assigned to a sample via isotopic shifting in the mass/charge (m/z) according to their respective label.
Figure 2.
Figure 2.
Heat maps illustrating fold changes in expression levels of proteins involved in regulation of K+/Na+ (left), Cl− (center) and Ca2+ (right) by DTMP and LR SCS relative to the pain model (DTMP/SNI and LR/SNI, respectively). The effect of the pain model relative to no injury is also included (SNI/Sham) to compare the effect of SCS treatments. For instance, pain tend to decrease gene expression, while DTMP tends to increase it, thus reversing the effect of pain. * denotes p < 0.05, ** denotes p < 0.001.
Figure 3.
Figure 3.
Illustration of proposed modulation of K+ transport. Proteins modulated by DTMP that are involved in K+ regulation in postsynaptic neurons and surrounding astrocyte cells. K+ leak channels and pumps are important in resetting membrane potential and limiting action potential characteristics. Reduction in K+ efflux, induced by the pain model, promotes depolarization and increased hypersensitivity. DTMP reversed changes in K+ regulation induced by the pain model. Some proteins that further promote K+ regulation, though unaffected by SNI, were also increased following DTMP.
Figure 4.
Figure 4.
Illustration of proposed modulation of Cl− transport. Proteins involved in Cl− regulation that are modulated by DTMP treatment. GABA receptors and the glycine receptors are key proteins allowing Cl− into the cell for inhibitory control and subsequent pain relief. The presynaptic release and recycling of GABA, with the aid of neighboring astrocytes, facilitates continued inhibition and pain control as evidenced by a shift in protein expression that would facilitate increased intracellular Cl−. Regarding proteins involved in GABA signaling and Cl− hyperpolarization, DTMP was able to reverse expression of proteomic changes induced by the pain model, as well as to increase expression of proteins unaffected by this but important to GABA and Cl− signaling pathway.
Figure 5.
Figure 5.
Illustration of proposed modulation of Ca2+ transport. Proteins involved in synaptic Ca2+ concentrations that were modulated by DTMP.
Figure 6.
Figure 6.
A scheme depicting key membrane proteins that are involved with ion flux and that have been modulated by treatment with DTMP.
Figure 7.
Figure 7.
Illustration of proposed regulation of ion transport across neuron, glial, and extracellular space based on proteomic changes due to SNI model, DTMP, or LR. In the uninjured Sham state, extracellular concentrations of Ca2+ and Cl−are elevated with K+ concentration being higher intracellularly in the neuron. The pain model caused a shift in inward Ca2+ currents and inhibiting Cl−influx. This shift in ion regulatory proteins was reversed toward Sham levels in DTMP treated animals with a similar, but less robust, shift due to LR.
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