**2. Animal models of SCS for neuropathic pain**

Neuropathic pain is caused by damaged somatosensory neural circuits that have developed into a disease condition as a result of an injury that compromised nerve fibers. Chronic neuropathic pain affects hundreds of millions of people around the world and is one of the main sources of work-related disabilities, contributing to the socioeconomical burden on individuals as well as health systems [10]. SCS is largely indicated for chronic neuropathic pain conditions, thus the development of various animal models that resemble clinical conditions play a critical role in our understanding of the electrophysiological and molecular changes in the establishment and persistence of neuropathic pain.

#### **2.1 SCS in partial nerve injury models**

Prior to the development of the chronic constriction injury (CCI) model, animal models of pain often were lacking in their ability to accurately mimic human peripheral neuropathological conditions or did not reflect conditions involving injuries which spared a portion of a nerve's functioning [11]. The CCI model most commonly used was described by Bennet and Xie in 1988 [11]. The procedure typically involves exposing the common sciatic nerve under anesthesia at the level of the mid-thigh and freeing approximately 7 mm of the sciatic nerve from adhering tissue at a location proximal to the sciatic nerve's trifurcation. Once exposed, four sutures are tied loosely around the sciatic nerve approximately 1-2 mm apart from one another. These ligatures are tied loose enough to just barely constrict the diameter of the sciatic nerve (as observed under 25x-40x magnification), thus preserving partial nerve functioning [11–16]. Constriction of the sciatic nerve was observed histologically beneath all 4 ligature areas as early as one day following the CCI procedure. From days 2-21, adjacent constriction areas tended to progressively merge and were accompanied by thinning of the affected sciatic nerve area. Two to four months after the CCI procedure, thinning of the ligated area was still present, but the swellings typically observed in the area had dissipated.

Importantly, this CCI model has proven to be effective in eliciting a neuropathic pain state as measured by a variety of methods, including von Frey mechanical, chemogenic, and heat and cold thermal stimulation. CCI surgery typically resulted in increased paw sensitivity following von Frey stimulation by Day 2 post-lesion using both the up-down method of analysis [12, 15, 16] and the ascending filament

method of analysis [12–14]. The CCI model is also sensitive to chemogenic pain. Following the application of a noxious substance (a 50% mustard oil solution) to the affected hind paw, CCI rats exhibited exaggerated physical responses and an increase in the amount of time they held their hind paw above the floor of the apparatus [11]. CCI rats also showed hypersensitivity to noxious heat sources, such as radiant beams of light being applied to the affected hind paw [11, 16] and increased allodynic responses to cold, such as a slightly chilled metal floor [11].

Although most applications of the CCI model with SCS have been performed in rats, an ovine model has also been effectively demonstrated [17]. In rats, application of SCS with pulsed signals at 50 Hz frequency, 0.2 ms pulse width (PW), and intensity at 66% of the motor threshold (MT) for 30 minutes [12, 14] decreased pain sensitivity following CCI, but did not return pain levels back to pre-injury control levels. Similar results were obtained when SCS was applied for 180 minutes in rats with CCI lesions using the following SCS parameters: 50 Hz frequency, 0.2 ms PW at 80% of the MT [12]. Lowering the intensity to 20-40% of the MT eliminated the beneficial effect of SCS. Electrodes in these rat studies were aimed at the T11-L1 regions of the spinal cord. In sheep, SCS parameters were set at 40 Hz frequency, 120 μs PW and 0.1 V intensity on a continuous setting for one week with electrodes placed at the L2-L3 region. SCS also attenuated pain responses following CCI in sheep, though also failed to return pain levels back to pre-lesion levels [17]. Most SCS studies following CCI typically utilized conventional (also called tonic) SCS at a stimulating frequency of 50 Hz. However, a more detailed investigation of frequency on treatment outcomes (in which frequency varied from 1 Hz to 150 Hz) found a response curve that suggested that while the GCT could account for a subset of efficacious SCS responses, it is unlikely to be the only mechanism underlying the beneficial effects of SCS [18].

In addition to demonstrating that SCS was effective in decreasing pain sensitivity, the CCI model also has proven valuable in elucidating the biological mechanisms behind the increase in pain following injury and the beneficial effect of SCS. After CCI lesions, rats exhibited an increase in the toll-like receptor 4 and nuclear factor κβ [15, 16], which subsequently could increase the release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. SCS significantly reduced these CCIinduced increases, perhaps by inhibiting the activation of glial cells. Indeed, the co-administration of the microglial inhibitor minocycline was shown to decrease CCI-induced pain and prolonged the effect of SCS [12]. However, this same study also showed an increase in the microglial-reactive marker OX-42 and the astrocyte reactive marker GFAP in the lumbar region of CCI-lesioned rats following SCS treatment. This paradoxical increase may limit the effectiveness of SCS and may partially explain why SCS is not effective in all patients and why pain relief often does not return to baseline levels following SCS. Other studies utilizing SCS in CCI-lesioned rats have identified roles of the adenosine and GABAergic systems in mediating the beneficial effects of SCS. Administration of adenosine or the adenosine A1 receptor agonist R-N6 phenylisopropyladenosine (R-PIA), decreased pain in CCI rats [14]. In addition, giving sub-effective doses of R-PIA to non-responders to SCS during stimulation led to effective pain relief. Zhang et al. [18] also found that administration of bicuculline, a GABAA receptor antagonist, decreased inhibitory responses to SCS.

Although the CCI model has provided significant value in (1) serving as a valid model of neuropathic pain, (2) demonstrating that the use of SCS is effective in reducing neuropathic pain, and (3) advancing our understanding of the biological mechanisms underlying SCS, its use has perhaps been surpassed in recent years by another animal model of partial nerve injury called partial sciatic nerve ligation (PSNL). Most PSNL studies utilize the Seltzer technique [19]. The procedure typically involves exposing the sciatic nerve under anesthesia at high-thigh level. The sciatic nerve is freed from adhering tissue at a location near the trochanter just distal to the point at

#### *Animal Pain Models for Spinal Cord Stimulation DOI: http://dx.doi.org/10.5772/intechopen.96403*

which the posterior biceps semitendinosus nerve branches off the common sciatic nerve. Once the sciatic nerve is properly exposed, a suture with a curved cutting mini needle is inserted into the nerve. Unlike the CCI procedure, which involves a loose ligation of the sciatic nerve, the PSNL procedure involves tightly ligating approximately 1/3 to 1/2 of the dorsal nerve thickness [19, 20]. As with the CCI procedure, the goal is to partially reduce, but not completely block sciatic nerve functioning.

Similar to the CCI model, the PSNL model also successfully induces a neuropathic pain state as measured by a variety of methods. The primary method is to assess mechanical allodynia using von Frey filaments [21–23]. Following PSNL surgery, rats show a decreased paw withdrawal response following mechanical stimulation of the affected paw, as compared to control rats, pre-surgery baseline levels, and paw withdrawal thresholds (PWT) following stimulation of the contralateral, unaffected paw [23, 24]. This increased pain sensitivity usually develops by Day 2 post-PSNL lesion [21] and is still typically observed after two weeks post-PSNL lesion [20, 24, 25]. Meuwissen et al. [26] recorded decreased PWT over 40 days following the PSNL lesion. Although the PSNL technique overall has been successful in inducing a neuropathic pain state, it should be noted that there is a wide range of pain responsiveness across studies, with some studies reporting 100% of subjects exhibiting hyperalgesia with von Frey testing of the affected hind paw [20, 24] and other studies reporting less than 40% of subjects showing hyperalgesia [27, 28]. Increased paw sensitivity to thermal stimuli has also been observed following the presentation of both heat stimuli (e.g., a radiant light beam) and cold stimuli, such as a cold spray directed at the affected paw [19, 29]. However, the degree of thermal sensitivity can vary depending upon the location of the PSNL ligature. Other studies have also confirmed hypersensitivity to pain following PSNL surgery by using a pneumatic pressure device [30] and by observing gait/posture [31]. Overall, the PSNL technique has proven successful at inducing a neuropathic pain state as assessed by numerous methodologies.

The PSNL model has also proven effective at evaluating SCS treatment for chronic neuropathic pain. Both tonic and burst SCS attenuated the pain sensitivity observed in rat PSNL neuropathic pain models [20, 24]. In rats, tonic SCS (50 Hz frequency, 0.2 ms PW, intensity at 66% of the MT) attenuated PSNL-induced hyperalgesia following 30 minutes of stimulation [23, 32, 33] and 60 minutes of stimulation [20, 22, 24]. Though tonic SCS successfully decreased pain sensitivity, SCS treatment typically did not return pain levels in rats back to pre-injury control levels. In a mouse model, SCS treatment following PSNL lesions proved particularly efficacious, with 80% of mice in one study [34] and 100% of mice in another study [25] responding positively to tonic SCS treatment. In this latter study, unlike the typical rat study, the mice returned to baseline levels of paw withdrawal following SCS treatment.

Although most rat studies investigating the effect of SCS following PSNL lesions have utilized conventional stimulation parameters and have shown a significant benefit of tonic SCS treatment, it is clear that not all PSNL rat subjects benefit from SCS treatment, leading researchers to investigate different SCS parameters in hopes of improving efficacy. Some factors that have proved to have significant impact on SCS efficacy following PSNL lesions include: (1) electrode placement, (2) stimulus intensity, (3) timing of treatment, and (4) utilizing a burst (vs tonic) stimulation pattern. Electrodes placed at the T13 level of the spinal cord typically yielded more efficacious treatment outcomes than electrodes placed at the T11 area [28, 31, 35] or L5 and L6 regions [36]. In a study that directly compared the efficacy of electrode location at T13 and T11 [21], the T13 placement yielded significantly better pain relief than placement at T11 with 63% vs 15% improvement, respectively, following 15 minutes of electrical stimulation and 48.5% vs. 18.4% improvement, respectively, following 30 minutes of electrical stimulation. Lowering the intensity to

30-50% of the MT reduced the beneficial effect of tonic SCS [24]. In terms of timing, early SCS treatment given within 24 hours of lesion led to significantly better treatment outcomes than late SCS given 16 days post-lesion [32]. Interestingly, an early round of SCS treatment followed by a subsequent late round of SCS treatment increased the efficacy of the late SCS treatment [37]. Lastly, burst stimulation typically led to similar response rates as tonic stimulation [20, 22, 26]. However, burst stimulation patterns did produce slightly different outcomes. For instance, one study [24] found that tonic SCS was most effective at 66% of the MT, while burst SCS was most effective at 50% of the MT. In addition, burst stimulation took longer following stimulus onset to achieve therapeutic benefits, but the benefits of the burst stimulation lasted longer after the stimulation was turned off [38]. Burst SCS stimulation also led to greater performance than tonic SCS on a mechanical conflict avoidance system (MCAS) task which measured the cognitive-motivational aspects of pain, rather than the more typical mechanical allodynic physical response to pain [26]. These results suggest that although equally efficacious, tonic, and burst SCS stimulation may work, at least in part, by different biological mechanisms.

Given the beneficial effect of SCS treatment following PSNL lesions, many PSNL studies have sought to investigate the biological mechanisms behind it. Many studies have utilized a paradigm in which sub-effective doses of pharmaceutical treatments are given to SCS non-responders to determine if the combination of these treatments can yield beneficial effects. Song et al. [29] showed that subeffective doses of the muscarinic agonist oxotremorine turned SCS non-responders into SCS responders, suggesting that the cholinergic system (particularly M2 and M4 muscarinic receptors) plays an important role in SCS efficacy. A subsequent study by these same authors [39] indicated an important serotonergic role in the pain-relieving effect of SCS, particularly the 5-HT2A and 5-HT4 serotonin receptor subtypes. Similarly, blocking NMDA receptors with sub-effective doses of ketamine followed by 30 minutes of SCS also turned SCS non-responders into SCS responders, indicating that the glutamate system also plays an important role in the beneficial effects of SCS [22]. While blocking the excitatory glutamatergic system likely plays a role in successful SCS treatment, enhancing the inhibitory GABAergic system might also play a significant role in successful SCS treatment [27, 32]. SCS treatment decreased intracellular GABA levels in SCS responders but not SCS nonresponders [32], while increasing extracellular GABA levels in the spinal cord [27]. Lastly, SCS stimulation in PSNL rats also led to an increase in levels of *c-fos*, suggesting immediate early gene modulation may trigger longer term changes which could explain pain relief both during and after SCS stimulation [40]. Overall, PSNL has proven to be a valuable tool in examining the biological mechanisms behind the beneficial treatment effects of SCS.

#### **2.2 SCS in the spared nerve injury model**

The Spared Nerve Injury (SNI) was developed by Decosterd and Woolf in 2000 [41] to evaluate peripheral neuropathic pain in the rat model. The SNI is considered superior to previous denervation and partial denervation models due to its specificity of the affected region, as well as its prompt and long-lasting effect. Unlike other models that were designed to test acute nociceptive pain through behavioral and electrophysiological measurements, denervation and partial denervation models induce sensations such as hypersensitivity that more accurately reflect true clinical chronic pain conditions.

The SNI procedure targets the sciatic nerve at its point of trifurcation (**Figure 1A**) in the hindlimb of the rat. Located directly under the biceps femoris muscle, the nerves are exposed and identified as the tibial, common peroneal, and *Animal Pain Models for Spinal Cord Stimulation DOI: http://dx.doi.org/10.5772/intechopen.96403*

**Figure 1.**

*(A) A scheme depicting the anatomical innervation of the sciatic nerve into the spinal cord of the rat. (B-D) Photographic sequence of the localization, ligation, and sectioning of the tibial and peroneal nerves. (E) Map of nerve coverage to plantar hind paw surfaces.*

sural branches (**Figure 1B**). Distal to the point of trifurcation and in the direction of the terminal end, both the tibial and common peroneal branches are individually ligated with silk sutures (**Figure 1C**). Then, 2–4 mm of nerve is sectioned and removed to ensure a complete disruption of nerve transmission (**Figure 1D**). The incision is then closed, leaving the sural branch fully intact and undisturbed. Minor amendments, such as carefully separating the gluteus superficialis and biceps femoris muscles instead of cutting through to expose the sciatic nerve, were made in some later studies in attempt to reduce unnecessary tissue damage [42]. Hypersensitivity is rapidly established in this model, with behavioral onset occurring at just 24 hours post-induction, lasting no less than 6 months, with peak sensitivity around 2 weeks [41]. The duration of the SNI effectiveness allows for considerable flexibility when considering study design. Per the original study, nonresponders are virtually nonexistent so long as the model is induced correctly.

As shown in **Figure 1E**, the hind paw of the rat is subdivided into three zones which are innervated by the sciatic and saphenous nerves. Transecting two of the three sciatic nerve branches, allows for precise and consistent behavioral testing of the lateral portion of plantar surface, corresponding to the sural nerve. This model permits mechanical and thermal allodynia, as well as thermal hyperalgesia to be evaluated [41] and has been used extensively in basic science studies investigating the use spinal cord stimulation (SCS) for the treatment of neuropathic pain.

In 2015, the SNI-SCS model was taken a step further when Tilley et al. [42] developed a model of continuous stimulation in an awake and freely moving rat, allowing stimulation to be delivered for 72 continuous hours. This method allowed for more clinically relevant testing since human patients receive continuous stimulation. A miniaturized four-electrode cylindrical lead was implanted in the epidural space of the rat and anchored into the musculature in the back (**Figure 2**). The lead exited through the incision and was secured through a custom-made harness and tubing up to a circuit board and stimulator suspended in a swivel so that the full assembly could turn and move with the rat. The rat cage lid was modified to allow free movement of the tubing. In later studies, the lead has been attached to an ethernet port secured to the rat harness with a coiled ethernet cord running to the stimulator connector suspended in the swivel [43]. Mechanical (von Frey filament) and cold thermal (acetone drop) allodynia were tested in this study. SCS was set at 50 Hz frequency, 20 μs PW, and at 70% of the MT. While there was no apparent improvement in cold allodynia following SCS, mechanical allodynia was significantly alleviated 24 and 72 hours after the start of SCS. A follow-up genomic study revealed the biological processes uniquely modulated by the SNI model and SCS in the spinal cord and dorsal root ganglion tissues (**Table 1**) [44]. The primary affected processes in both types of tissues included inflammatory response, ion channel regulation, and immune response.

Following the initial development of the SNI model, Li et al. [35] tested variations in an effort to optimize the model specifically for SCS studies. The study included the original SNI procedure, peroneal axotomy, tibial axotomy, tibial tight ligation (no sectioning), and partial tibial ligation (1/3 to 1/2 of its diameter ligated,

#### **Figure 2.**

*Left: A diagram of the setup for continuous SCS setup reported in references [42, 44]. a: plexiglass lid, b: supporting floor, c: counterweight swivel system, d: connecting board/stimulator (IPG), e: connecting cable, f: harness with lead connector. Right: a lateral x-ray image showing a quadripolar SCS lead placed in the dorsal epidural space of a rat.*

without sectioning). SCS was delivered through an implanted 2 mm disc cathode placed on the dura at the T11 level. The 4 mm anode was placed on the chest wall, subcutaneously. Stimulation parameters were set at 50 Hz frequency, 0.2 ms PW, and amplitude at 90% of the MT. SCS was delivered for a 30-minute duration. Measuring mechanical allodynia via von Frey filament testing, authors found that all variations of the model produced allodynia within one week, lasting for at least 3 weeks. All variations except the partial tibial ligation lasted 7-10 weeks. Paw posture was noted as one difference between models, where peroneal axotomy resulted in an inverted position of the paw which tended to be dragged behind the rat. The other variations presented an eversion posture, with the partial tibial ligation being less prominent. Contrary to the original study, where no non-responders were reported and the contralateral hypersensitivity rate was zero, Li et al. found only 53% of SNI operated rats developed hypersensitivity and 25% had some hypersensitivity in the contralateral paw. Response to SCS showed the SNI group with the smallest responder rate at just 8%, compared to the 40-50% responder rate of the variation groups. The researchers concluded there was an inverse relationship between degree of hypersensitivity and efficacy of SCS, agreeing with previous literature in similar models [45], and that these variations of the SNI may provide better models for use in SCS animal studies. However, it should be mentioned that some later studies report higher responder rates to the original variation of the SNI model and subsequent SCS treatment.

Most recently, Sluka and coworkers [46] evaluated tonic SCS on multiple pain models, including the SNI. Implanting an epidural lead and corresponding neurostimulator, the rats were stimulated for 15 minutes per day at 60 Hz, 0.25 ms PW, and at 90% of the MT. Two weeks after SNI induction, the effect of SCS was evaluated. They found that neuropathic pain was alleviated by tonic SCS, measured by von Frey filaments, with significantly increased withdrawal thresholds. Twentyfour hours after stimulation was turned off, behavioral testing revealed that the effect of SCS was lost. The authors attribute the analgesic effect of tonic SCS on the SNI model to the activation of large Aβ dorsal column axons (supporting the GCT), as well as the electrochemical alteration of cell membranes and the involvement of neurotransmitters, receptors, and glial cells.


#### *Animal Pain Models for Spinal Cord Stimulation DOI: http://dx.doi.org/10.5772/intechopen.96403*

#### **Table 1.**

*Gene ontology biological processes modulated in the ipsilateral dorsal quadrant of the spinal cord (directly under the electrode) and ipsilateral L5 dorsal root ganglion demonstrating molecular changes caused by SCS therapy with the SNI model. Reelevant processes obtained after WGCNA and gene ontology analyses performed on microarray results. Only modules with significant False Discovery Rate (FDR) p-values are shown. Data from reference [44].*

Previously, this group looked at frequency-dependent outcomes of SCS, particularly regarding opioid receptors [47] and glial cell activation with SCS [48]. In the opioid receptor study [47], SNI-induced rats were administered naloxone or naltrindole (both opioid antagonists), or were made morphine tolerant. Rats then received SCS at 4 Hz, 60 Hz, or no SCS daily for 6-hour periods, lasting 4 days for each treatment. Testing for mechanical allodynia, they found naloxone prevented the analgesic effect produced by 4 Hz and 60 Hz stimulation, though a higher dose was required to block the effect of 60 Hz. Interestingly, naltrindole had no effect on 4 Hz SCS, but successfully impeded the effect of 60 Hz SCS. When testing the morphine-tolerant rats, they found that 4 Hz SCS did not have the same analgesic effect that it did in normal rats, while 60 Hz stimulation remained efficacious. This work resulted in the understanding that the frequency of SCS may determine the mechanism by which pain relief is achieved, and in this case, engaging different opioid receptors.

Glial activation, via immunohistochemical staining with known markers (GFAP, MCP-1, and OX-42), was measured in a separate study using 30-minute and 6-hour SCS durations and varying the intensity (as percent of the MT) [48]. Two weeks after the SNI was induced, mechanical hypersensitivity increased, as expected, as well as glial cell activity. The results indicated that withdrawal thresholds were positively correlated with increasing SCS duration (6 h vs 30 min) and by stimulating at higher intensities (90% vs 75% vs 50% MT). Glial cell activation was significantly decreased in both 4 and 60 Hz SCS, delivered for 6 hours at 90% MT.

Additional studies focused on the question of frequency importance in SCS, utilizing the SNI as a pain model. Song et al. investigated conventional 50 Hz (200 μs PW, 80% MT) SCS compared to high frequency (HF) SCS at lower intensity (500, 1000, and 10000 Hz; 24 μs PW, 40–50% MT) [49]. A miniaturized 4-electrode plate lead was implanted into the epidural space of the T13 vertebral level. Performing behavioral testing for mechanical hypersensitivity (von Frey filaments) and thermal hypersensitivity (ethyl chloride spray for cold, modified Hargreaves test for heat), they found no significant difference in the overall analgesic effect of conventional SCS versus SCS at higher frequencies. They did, however, found that conventional SCS had significant effect on increasing the gracile nucleus neuron discharge rate. HF SCS had no effect whatsoever. These results suggest that conventional and HF SCS have different mechanisms of action.

Building on the idea that SCS can be designed to modulate neuron-glial interactions, Vallejo et al. [50] utilized the SNI model with continuous SCS to evaluate a differential target multiplexed programming (DTMP) approach compared to high rate and low rate SCS. The DTMP approach utilizes multiple signals that are intended to target neuron and glial cells differentially. It was found that all SCS treatments resulted in significant reduction of mechanical hypersensitivity, but the DTMP approach provided more significant improvement as well as reduced thermal hypersensitivity (hot/cold plate test) after 48 hours of continuous stimulation. RNA-sequencing was performed to confirm the phenotypes. **Figure 3** provides a heatmap illustrating the significant effect of DTMP on sets of genes (modules) with similar expression patterns obtained through a Weighted Gene Co-expression Network Analysis (WGCNA) showing that the effect of DTMP SCS correlated stronger with the expression patterns of modules in naïve rats, compared to the pattern of untreated animals (No-SCS). In a follow up study, Cedeño et al. [51] demonstrated that the DTMP approach modulated neurons and glial cells (microglia, astrocytes, and oligodendrocytes) in a differential manner by using set of genes that were uniquely expressed (cell-specific transcriptomes) by each of the type of neural cell. The effect of DTMP on each of these cell-specific transcriptomes correlated strongly with the expression pattern of naïve animals, indicating a return of gene expression toward the state of naïve (healthy) animals.

### **2.3 SCS in the spinal nerve ligation model**

The spinal nerve ligation model (SNL) is one of the most popular preclinical models of neuropathic pain due to its reproducibility and lack of autotomy. During the surgery, initially described by Kim and Chung [52], the L5 spinal nerve is ligated with a 6-0 silk suture at a point just distal to the dorsal root ganglion (DRG), and cut distally, after the removal of the paraspinal muscles at the level of the L5 spinous process down to the sacrum, and the removal of the L6 transverse process.

Since the introduction of paresthesia-free stimulation parameters in the clinical setting, questions were raised on the value of the GCT as a practical construction to generate models to optimize SCS parameters. To find answers to some of these questions, Guan and coworkers have used the SNL model [53] to understand the specific

#### *Animal Pain Models for Spinal Cord Stimulation DOI: http://dx.doi.org/10.5772/intechopen.96403*

#### **Figure 3.**

*Heat map of mean module eigengene values for modules with significantly different comparisons (FDR-p < 0.2) between SNI untreated animals (No-SCS) and naïve animals. A total of 23 modules out of the total 39 are affected. Asterisks (\*) indicate significantly different module eigengene values when comparing the SCS treatment to untreated animals (No-SCS). R is the Pearson coefficient for the correlation between eigengene values for naïve and each of the other groups. A negative value indicates an opposite trend. DTMP: differential target multiplexed programming; LR: low rate; HR: high rate; SCS: spinal cord stimulation. Reproduced from reference [50].*

effects of different components of the electrical signals in SCS treatment. Before paresthesia-free SCS, common frequencies used in clinical and animal models ranged from 50 to 60 Hz, since referred to as conventional SCS. Due to the lack of agreement regarding the optimal frequency and stimulation intensity to maximize analgesia, these authors hypothesized that kilohertz-level SCS and conventional 50Hz SCS might differently activate gate-control mechanisms and affect peripheral afferent conduction properties [53].

Using the SNL rodent model of neuropathic pain, the authors evaluated intensity-dependent (20%, 40%, and 80% MT) pain inhibition of SCS at various frequencies (50 Hz, 1 kHz, and 10 kHz) while maintaining the PW constant (24 μs). They further compared the effects of conditioning stimulation of the dorsal column, the primary structure targeted by SCS, at 50 Hz and 1 kHz on the conduction property of afferent Aα/β-fibers and inhibition of dorsal horn wide dynamic range (WDR) neuronal responses.

In their experiments, Guan and coworkers [53] advanced a custom-made quadripolar epidural SCS electrode up to the T10-12 spinal levels, via a small laminectomy at the level of T13. Mechanical hypersensitivity was assessed by determining the PWT using von Frey filaments. To further evaluate clinical conditions in the SNL model, the authors choose stimulation intensities set at either 20%, 40%, or 80% of the MT to test the effect of the described frequencies on pain-like behavior. Stimulation was conducted for 30 mins on days 12, 13, and 14 (week 1) and days 19, 20, and 21 (week 2) post-SNL. Behavioral testing was done at time 0, 15 (within the activation of SCS), 30 (end of stimulation), and 60 min. A cross-over design was implemented to avoid the order effect while switching the different frequencies.

Interestingly, rats exposed to 10 kHz SCS at 80% MT often exhibited signs of discomfort. For comparison, a small number of animals were implanted, but not stimulated, and served as a stimulation sham. When stimulation was applied at 20% MT, the effect was marginal for all the tested frequencies. The average mean PWT across the three treatment days was increased from the pre-stimulation level in all SCS groups but was statistically significantly higher than that of sham stimulation only in the 1 kHz and 10 kHz groups. Notably, there was a trend for SCS induced inhibition to increase gradually from the first to the third treatment in all groups. When using 80% MT the mean PWT was significantly increased from the first day of stimulation in both the 1 kHz and the 10kHz SCS groups. Of notice, the mean PWT in the 1 kHz and 10 kHz were both higher than that of the 50 Hz group on the first day of stimulation. The inhibitory effect of 50 Hz stimulation increased progressively during the second and third days of stimulation. The authors concluded that the SCS analgesia in SNL rats depends on both intensity and frequency, and high-intensity kilohertz level SCS provides earlier inhibition of mechanical hypersensitivity than conventional 50 Hz SCS. These results imply that analgesia from kilohertz and 50 Hz SCS may involve different mechanisms.

In a follow-up report, Guan and coworkers [54] explored how charge delivery affects pain inhibition by different frequencies at intensities that seem to be below the sensory threshold (40% MT), and which component of stimulation runs the therapeutic actions. Epidural electrodes were implanted 5 to 7 days post-SNL, in a similar fashion described by this group previously [53]. Based on the frequency, PW, and intensity, the authors calculated the charge-per-pulse, duty time, and charge-per-second. Then, four patterns of high-dose subthreshold active recharge biphasic signals at different frequencies with similar duty times were produced by adjusting the PW (200 Hz with 1 ms PW, 500 Hz with 0.5 ms PW, 1.2 kHz with 0.2 ms PW, and 10 kHz with 0.024 ms PW). Finally, the authors included one 50 Hz with 0.2 ms PW at subthreshold and a sham (no SCS) group. Because clinical and animal data suggest that subthreshold SCS may have a slower onset, stimulation was carried for 120 mins (one session per day) from days 14 to 17 (week 1). The behavioral response was determined by measuring the PWT 30 mins pre-SCS, at 0, 30, and 60 mins during SCS, and 0, 30, and 60 mins post-SCS to evaluate carry-over effects. In those groups that showed increased tolerance to mechanical hypersensitivity, the peak effect appeared at 60 to 90 mins after initiation of SCS and faded shortly after the stimulation was completed. The onset of significant PWT increase was observed from day one in the 200 Hz and 10 kHz groups and was observed on day two in the 1.2 kHz group. Although 200 Hz SCS had the longest PW, the highest charge-per-pulse and the lowest charge-per-second, and 10 kHz had the shortest PW, the lowest charge-per-pulse, and the highest amplitude and charge-per-second, the two groups provided comparable improvements in PWT. These findings suggest that the efficacy of the inhibitory effect is not correlated to the difference in individual SCS parameters (frequency, PW) but is positively correlated with the electrical dose. Probably, the most interesting finding is that at subthreshold SCS amplitudes, mechanical hypersensitivity was not only inhibited by 10 kHz but also at lower frequencies (200 Hz). The authors concluded that using low-frequency subthreshold SCS and longer PWs, could be a more energy-efficient stimulation paradigm for inhibition of mechanical hypersensitivity when compared with 10 kHz SCS.

### **2.4 SCS in a chemotherapy-induced neuropathic pain model**

The administration of chemotherapy agents for the treatment of cancer often results in the onset of neuropathic pain due to peripheral nerve damage. Recently, Sivanesan et al. [55] reported on the efficacy of SCS in reducing mechanical and thermal hypersensitivity in a rodent model of chemotherapy-induced peripheral neuropathy (CIPN). The chemotherapeutic paclitaxel (PTX), common in the treatment of ovarian, breast, and lung cancers, can induce painful peripheral neuropathy even at therapeutic dosages. Often this pain is severe enough to necessitate a reduced dose of PTX and persists after cessation of the drug in nearly a third of cases. Other chemotherapeutic agents, including platinum-based agents, and proteasome inhibitors like bortezomib, are also known to induce similar neuropathies [56].

Induction of the model began by acclimation of a group of adult male rats that were divided into three groups: (1) SCS + PTX, (2) PTX, or (3) naïve. Behavioral testing consisted of assessments of mechanical hypersensitivity (von Frey filaments) and thermal hypersensitivity (via dry ice application) of the hind paws.

Animals assigned to receive SCS underwent a T13 laminectomy and were implanted with a quadripolar miniaturized lead in the dorsal epidural space corresponding to the T13-L1 spinal cord. Stimulation parameters were set to conventional settings with 50 Hz frequency, 0.2 ms PW, and current intensity at 80% MT. Subsequently, animals in the PTX and SCS + PTX groups were administered 1-2 mg/kg PTX, via intraperitoneal (i.p.) injection, every other day for four days. Naïve animals received i.p. injections of the vehicle used in the PTX groups. SCS was administered daily for 6-8 hours over the course of two weeks. To substantiate whether SCS can avert the development of CIPN, PTX was administered at the same time as the stimulation. Implanted animals that did not receive SCS treatment were included as control animals.

Rats developed hypersensitivity one week after the first administration of PTX that was sustained for 25 days. Interestingly, early administration of SCS attenuated the development of mechanical hypersensitivity associated with neuropathic pain-like behavior induced by administration of PTX (**Figure 4**). SCS did not fully recover to the PWT in naïve animals, but the preemptive effect of SCS is noteworthy. Early application of SCS also prevented the development of cold hypersensitivity. It is also important to note that the analgesic effect of SCS persisted for at least 2 weeks after stopping SCS treatment.

#### **Figure 4.**

*Mechanical hypersensitivity (left) and cold thermal hypersensitivity (right) of animals treated with SCS (SCS + PTX), untreated (PTX) and naïve. The patterned box indicates the time of PTX administration. The black box indicates the time of SCS administration. \* indicates significant differences (p < 0.05) relative to naïve, # indicates significant differences (p < 0.05) relative to PTX + SCS. Values obtained from reference [55].*

L3-L6 spinal cord tissue was harvested 17 days after SCS was applied, and RNA in the samples was sequenced to investigate changes in gene expression and biological processes of treated, untreated, and naïve animals. It was found that some genes associated with mechanosensation, neuroimmune response, and glial activation were affected by the CIPN model. The authors hypothesized that repetitive dosing of SCS increased the expression of genes that enhance adenosine-related activity, which has been shown to enhance pain inhibition by SCS when using the CCI model [14]. The authors also observed that SCS downregulated GABA reuptake-related genes, which is consistent with a previous observation in rats after sciatic nerve injury [57]. They postulated that downregulation of genes such as *Gat3* (a GABA transporter expressed by glial cells) by SCS may increase GABAergic signaling that inhibits neurotransmission in CIPN rats. The GABAergic inhibition of excitatory neurotransmitters may involve then the suppression of calcium influx into presynaptic terminals, which is regulated by astrocytes.
