Percutaneous electrical nerve field stimulation modulates central pain pathways and attenuates post-inflammatory visceral and somatic hyperalgesia in rats

Neuroscience 356 (2017) 11–21

PERCUTANEOUS ELECTRICAL NERVE FIELD STIMULATION MODULATES CENTRAL PAIN PATHWAYS AND ATTENUATES POST-INFLAMMATORY VISCERAL AND SOMATIC HYPERALGESIA IN RATS REJI BABYGIRIJA, a MANU SOOD, a PRADEEP KANNAMPALLI, b JYOTI N. SENGUPTA a,b AND ADRIAN MIRANDA a*

hyperalgesia. Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Pediatrics, Division of Gastroenterology and Hepatology, Medical College of Wisconsin, Milwaukee, WI, United States

Key words: amygdala, visceral hyperalgesia, colitis, spinal neurons, auricular stimulation.

b

Department of Medicine, Division of Gastroenterology and Hepatology, Medical College of Wisconsin, Milwaukee, WI, United States

INTRODUCTION Recent problems with narcotic dependence and abuse have sparked new ways to think about how to properly manage pain. Improving treatment options and providing alternatives for the treatment of chronic pain in the clinical setting is of critical importance. The challenge primarily lies in avoiding narcotics with a very limited number of treatment options. The development of nonpharmacological or non-addicting approaches to treat or prevent chronic pain is now becoming a major priority. Spinal and deep brain stimulation are an exciting and effective approach that has received much attention (Bittar et al., 2005; Greenwood-Van Meerveld et al., 2005; Lind et al., 2015; Kapural et al., 2016). Unfortunately, due to their invasive nature, they are reserved only for cases of severe, refractory pain. The ability to modulate central pain pathways peripherally, through a noninvasive technique has recently been suggested using the BRIDGEÒ device (Innovative Health Solutions, Versailles, IN, USA), a FDA-cleared, percutaneous, electrical nerve field stimulator (PENFS) developed to alleviate pain. The device uses specific parameters of stimulation with alternating frequencies to target central pathways. While the exact mechanism responsible for the analgesic effects is not known, electrical stimulation of peripheral cranial neurovascular bundles in the external ear are believed to help modulate central pain pathways (Ahmed et al., 1998; Sator-Katzenschlager and Michalek-Sauberer, 2007). The external ear in both rats and humans contains branches of four cranial nerves (V, VII, IX, and X) that have projections to brainstem nuclei, particularly the nucleus tractus solitarius (NTS) (Contreras et al., 1982; Folan-Curran et al., 1994; Folan-Curran and Cooke, 2001; Zhang and Ashwell, 2001). The NTS is known to be a ‘‘relay station” to other brain structures involved in autonomic control and pain including the rostral ventral medulla (RVM), hypothalamus, amygdala and spinal cord (van der Kooy et al.,

Abstract—A non-invasive, auricular percutaneous electrical nerve field stimulation (PENFS) has been suggested to modulate central pain pathways. We investigated the effects of BRIDGEÒ device on the responses of amygdala and lumbar spinal neurons and the development of post-colitis hyperalgesia. Male Sprague–Dawley rats received intracolonic trinitrobenzene sulfonic acid (TNBS) and PENFS on the same day. Control rats had sham devices. The visceromotor response (VMR) to colon distension and paw withdrawal threshold (PWT) was recorded after 7 days. A different group of rats had VMR and PWT at baseline, after TNBS and following PENFS. Extracellular recordings were made from neurons in central nucleus of the amygdala (CeA) or lumbar spinal cord. Baseline firing and responses to compression of the paw were recorded before and after PENFS. Sham-treated rats exhibited a much higher VMR (>30 mmHg) and lower PWT compared to PENFS-treated rats (p < 0.05). PENFS decreased the VMR to colon distension and increased the PWT compared to pre-stimulation (p < 0.05). PENFS resulted in a 57% decrease in spontaneous firing of the CeA neurons (0.59 ± 0.16 vs control: 1.71 ± 0.32 imp/s). Similarly, the response to somatic stimulation was decreased by 56% (3.6 ± 0.52 vs control: 1.71 ± 0.32 imps/s, p < 0.05). Spinal neurons showed a 47% decrease in mean spontaneous firing (4.05 ± 0.65 vs control: 7.7 ± 0.87 imp/s) and response to somatic stimulation (7.62 ± 1.7 vs control: 14.8 ± 2.28 imp/s, p < 0.05). PENFS attenuated baseline firing of CeA and spinal neurons which may account for the modulation of pain responses in this model of post-inflammatory visceral and somatic

*Corresponding author. Address: Division of Gastroenterology and Hepatology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, United States. Fax: +1-414-456-6361. E-mail address: [email protected] (A. Miranda). Abbreviations: CeA, central nucleus of the amygdala; CRD, colorectal distension; NTS, nucleus tractus solitarius; PENFS, percutaneous, electrical nerve field stimulator; PWT, paw withdrawal threshold; RVM, rostral ventral medulla; TNBS, trinitrobenzenesulfonic acid; VMR, visceromotor response. http://dx.doi.org/10.1016/j.neuroscience.2017.05.012 0306-4522/Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved. 11

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1984; Ross et al., 1985; Folan-Curran et al., 1994; Liu et al., 2015). In animals, colonic inflammation has been widely used to investigate the pathogenesis of postinflammatory pain and as a model for irritable bowel syndrome (IBS) since a large number of patients develop IBS following a gastrointestinal infection (Gschossmann et al., 2002, 2004; Kanazawa and Fukudo, 2014; Lo¨we et al., 2016). It is not uncommon to see the presence of both visceral and somatic hypersensitivity in the animal models of colitis and in patients with IBS, suggesting a pathway that involves CNS structures (Zhou et al., 2008; Stabell et al., 2013; Patel et al., 2016). Changes in amygdala connectivity and spinal cord processing have been proposed to play a key role in the development of chronic visceral pain (Wang et al., 2013; Qi et al., 2016). The amygdala is involved in integrating information regarding stress and pain and has been linked to the development of chronic visceral pain in animals and humans (Labus et al., 2009; Johnson et al., 2012; Myers and Greenwood-Van Meerveld, 2012; Rouwette et al., 2012; Wang et al., 2013). Inflammation or pain can cause abnormal activation of the amygdala that could also influence spinal cord processing, since the central nucleus of the amygdala (CeA) projects to brainstem structures and the spinal cord (Burstein and Potrebic, 1993; Saha et al., 2005; Bourbia et al., 2014). Also, primary afferents from the intestine and somatic structures can synapse on the same second-order neurons in the spinal cord (Peles et al., 2004; Lamb et al., 2006). Because of this viscero-somatic convergence, colonic inflammation can influence spinal neurons and higher order structures to produce the phenotype of generalized hyperalgesia (Lamb et al., 2006; Farrell et al., 2016). Overall, however, the exact mechanism leading to postinflammatory hyperalgesia is not known. To date, there are no studies that investigate the effects of PENFS on amygdala and spinal neurons and the development of post-inflammatory visceral and somatic hyperalgesia. The objective of the present study was to use an animal model of experimental colitis to investigate the anti-nociceptive properties of PENFS with the BRIDGE device and to explore a central, neuromodulatory mechanism. We hypothesized that PENFS would modulate the response characteristics of amygdala and spinal neurons and prevent the development of visceral and somatic hyperalgesia.

EXPERIMENTAL PROCEDURES Animals A total of 61 male Sprague–Dawley (SD) rats weighing 250–300 g were used in this study and data were collected from a total of 47 animals. Animals were housed under conditions of controlled temperature (22– 24 °C) and illumination (12-h light cycle starting at 6:00 AM) for at least 7 days before the experiments. Rats were allowed ad libitum access to food and water. All experiments were performed according to the approved protocols and guidelines of the Medical College of Wisconsin and The International Association for the Study of Pain and carried out in accordance with

the National Institute of Health ‘‘Guide for the Care and Use of Laboratory Animals”. All efforts were made to minimize animal suffering and to reduce the number of animal in experiments. Surgical preparation for electrode implantation Adult rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) as previously described (Mickle et al., 2010). A pair of Teflon coated electrodes (Cooner wire, Part#: A5631) were implanted in the abdominal musculature for EMG recordings. The electrodes were externalized subcutaneously and protected using a silastic tube sutured to the dorsal aspect of the neck. All rats received analgesic (carprofen, 5 mg/kg/day, i.m. for 3 days) and antibiotic (enrofloxacin, 2.5 mg/kg/day, i.m. for 3 days) post-operatively. Following surgery, the animals were housed separately and allowed to recover for at least 5 days prior to further interventions. Experimental colitis The rats were fasted for 24 h and then deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A 50% solution of TNBS (0.6 ml of 30 mg/ml TNBS in 50% ethanol) was instilled into the colon using a 7-cmlong oral gavage needle inserted into the descending colon. Rats were placed in the supine position with the lower portion of the body slightly elevated in order to prevent leakage of TNBS. The animals were allowed to recover for 5 days prior to further testing. Measurement of colonic sensitivity Prior to testing the colonic sensitivity, animals were acclimatized to the experimental conditions by placing them inside a plexiglass-restraining tube (Bollman cage) for two hours a day over 3 days. The visceromotor response (VMR) to colorectal distension (CRD) was used as an objective measure of visceral sensation in all groups as previously described (35). Briefly, individual rats were kept in a Bollman cage while a distensible latex balloon (5 cm in length) attached to PE tubing was inserted into the descending colon and rectum. The opposite end was attached to a distension device. EMG recordings quantified contractions of the abdominal musculature in response to graded CRD. Distention pressures (10, 20, 30, 40, 50, and 60 mmHg) were held constant during the 30-second stimulus with a 180-s, inter-stimulus interval. The EMG signal from the external oblique muscle was amplified through a low noise AC differential amplifier (model-3000: A-M Systems, Inc., Sequim, WA. United States) and recorded on-line using the Spike 3/CED 1401 data acquisition program. (CED 1401; Cambridge Electronic Design, Cambridge, UK). Measurement of somatic sensitivity Somatic sensitivity was assessed using the paw withdrawal threshold (PWT). The rats were placed on a screen platform and allowed to acclimate to the environment for 20 min prior to testing. Progressive,

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increasing forces using of Von Frey filaments of various bending forces (100–400 mN) were applied to the planter surface of the left paw for 5 s or until the paw was withdrawn. Two trials were recorded in 5-min intervals. The lowest bending force required to stimulate a withdrawal was recorded as the PWT. Percutaneous, electrical nerve field stimulation (PENFS) For behavioral studies, rats were lightly anesthetized with isoflurane to place the BRIDGE stimulating electrodes in the right ear. The device settings are standard and deliver 3.2 V with a rectangular wave pulse and alternating frequencies (1 and 10 Hz) every 2 s. The positive and negative pulses cycle to prevent nerve saturation and maintain transmission of impulses (Fig. 1). The device consists of a pulse generator (approximately 3.5  1.5  1 cm) that attaches to a harness containing three active wires and 1 ground. The tips of the wires contain an array of four small (2 mm each) titanium needles that create a stimulation field on the tissue. Only one stimulating electrode was inserted with light pressure into pinna on the dorsal side and taped to secure in place. The ground containing only one needle was inserted on the ventral side of the ear. Once connected to the generator to deliver the pulse current, the generator was secured with light adhesive tape to the dorsum in order to allow the animals to move freely within their housing cage during the entire period of stimulation. During the entire period of stimulation, the animals appeared to be in a sleeping position with head tucked in legs and did not show any signs of distress. Animals demonstrated minimal to no attempts to remove the hardware from the ear or back. Experimental protocol Prevention of hyperalgesia. Two groups of rats underwent treatment with intracolonic TNBS. Immediately prior to instilling TNBS, stimulation with the

Fig. 1. Example of one cycle of PENFS with the BRIDGE device. The device delivers 3.2 V in a rectangular wave pulse and is programed to cycle between 1 and 10 Hz every two seconds (positive and negative) before repeating.

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BRIDGE was initiated in one group (n = 8), while the other group underwent the same protocol but with an inactive (sham) device (n = 8). The duration of both active stimulation and sham treatment was 4 h per day for five consecutive days. After the fifth day, animals were allowed to rest without stimulation for 2 days and then subjected to standard VMR and PWT testing (day 7 post-TNBS). The same 5-day stimulation protocol with active BRIDGE device was followed in a third group of naı¨ ve (non-TNBS-treated) rats. Treatment of post-inflammatory hyperalgesia. In one group of animals (n = 8), the VMR and PWT were measured to assess visceral and somatic sensitivity sequentially at three different time points: (1) at baseline prior to TNBS injection (2) 7 days after TNBS and (3) after stimulation with the BRIDGE (14 days after TNBS). The stimulation protocol (4 h per day for five consecutive days) started after recording of the second VMR (7 days after TNBS). During all behavioral studies, the investigators were blinded to the active versus sham treatment groups as well as during all VMR and PWT testing. Electrophysiology A group of animals underwent electrophysiology recordings between 14 and 28 days post-TNBS treatment. Surgical preparations for recordings have been previously described (Miranda et al., 2006). Briefly, prior to all electrophysiology recordings, rats were deprived of food, but not water, for 12 h. Rats were anesthetized with an initial dose of urethane (1.5 g/kg, i.p) and maintained by injecting supplemental doses (0.05 mg/kg, i.v.). To monitor blood pressure, the left carotid artery was also cannulated. The body temperature was kept within physiological range (36–37 °C) with an overhead lamp. For spinal recordings, the trachea was intubated and the rats were paralyzed with an initial dose of gallamine triethiodide (10 mg kg, i.v.) and mechanically ventilated with room air (60 strokes min 1). Supplemental doses of gallamine triethiodide (5 mg/kg/h) were given as needed to maintain paralysis. Carbon filament filled glass microelectrodes (0.4–0.8 MX, Kation Scientific, MN, USA) were used for extracellular single-unit recording from the amygdala and spinal cord. Surgery for amygdala recordings. The head of the animal was fixed in place onto a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) and craniotomies were performed. The area for craniotomy was identified from bregma (AP: 2.0 mm, ML: ±4.3 mm and DV: 7.0–8.5 mm) to target the central amygdala (CeA). After removal of the dura membrane, a saline-soaked gelatin sponge (Gelfoam, Pharmacia Upjohn Company, Michigan USA) was placed over the exposed brain. The skin was reflected laterally to make a pool for warm mineral oil (37 °C). The microelectrode was guided to the CeA using a micropositioner. The coordinates positions from bregma (AP 2.0 mm, ML ±4.3 mm and DV 7.0–8.5 mm), was derived from the rat brain atlas (Paxinos and Watson, 2007).

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Surgery for spinal dorsal horn neurons recordings. The rats were placed in a stereotaxic head holder and the lumbar (L1–L4) spinal cord was exposed by laminectomy. After removal of the dura membrane, a 1to 2-cm saline-soaked gelatin sponge (Gelfoam, Pharmacia Upjohn Company, MI, USA) was used to cover the exposed spinal cord segment. The skin was reflected laterally to make a pool for agar solution that was allowed to cool to 38 °C prior to pouring. The agar was allowed to harden and the dorsal surface of the spinal cord was exposed by removing a cubical slice of agar with a scalpel blade. The exposed surface of the spinal cord was covered with warm mineral oil (37 °C). The placement of the electrode for extracellular singleunit recordings was 0.1–0.5 mm lateral from the spinal midline and 0.6–1.3 mm ventral from the dorsal surface (lamina III–X).

Spinal and amygdala recording protocol. The action potentials of amygdala and spinal neurons were amplified through a low-noise AC differential amplifier (model 3000; A-M Systems) and continuously monitored and displayed on an oscilloscope. A dual window discriminator (model DDIS-1; BAK Electronics) was used to discriminate the action potentials and to convert it to rectangular TTL pulse. The frequency of TTL pulses was counted online using the Spike2/CED 1401 data acquisition system (Cambridge Electronic Design, UK). The background and evoked activity of only one neuron was recorded form the right amygdala or lumbar spinal cord in each rat. A neuron was identified by its background activity and responses to brushing, light compression or noxious pinch of the skin in the paw area. For amygdala and spinal recordings, neurons that responded to noxious compression of the paw were selected and recorded. The background activity in the absence of any stimulation was recorded for 5 min. Following the baseline recording, a brief (30-s) compression of the paw was applied twice with the same Von Frey filament (190 mN) 5 min apart to allow the neuron to return to baseline in between compressions. After two compressions were recorded, the BRIDGE device was connected to the ear on the ipsilateral side (same side as recording site) and left undisturbed for 5 min without active stimulation to allow the neuron to return to baseline. The device was then turned on for a total of 15 min after which the baseline firing of the neuron and response to compression of the paw was again recorded using the same protocol. To investigate the effects of PENFS on non-sensitized animals, a separate group of naı¨ ve animals underwent recordings from amygdala following the same protocol. For spinal neuron recordings, the electrical stimulation was given on the contralateral side and the same recording protocol before and after BRIDGE stimulation was followed. Histological verification of the electrode location in the CeA was done post mortem by injecting 0.2 ml of Alcian blue ink and identifying the dye distribution in the

Amygdala. No histological damage except for the electrode trajectory.

was

observed

Data analysis and statistics Statistical analysis was performed using SigmaStat (V2.03, SPSS Inc, Chicago, IL, USA). All results are expressed as mean ± SEM. For PWT, the mean responses at baseline and before and after PENFS were recorded and compared using a one-way analysis of variance (ANOVA). The SRF to graded CRD was constructed to evaluate the changes in visceral sensitivity using the area under the curve (AUC) of the raw EMG amplitude response as a function of pressure for each animal during the 30-sec colon distension (Gschossmann et al., 2002; Miranda et al., 2006). Quantitative comparisons of group mean responses before and after PENFS to respective distension pressures were performed with a two-way repeated measures ANOVA followed by the Holm–Sidak test for multiple comparisons. The data were also subjected to the normality and equal variance test with the Shapiro–Wilk test. A p-value <0.05 was considered to be statistically significant. The electrophysiology data were run through wavemark analysis of the Spike 4 software (Cambridge Electronic Design) to distinguish individual action potentials. The baseline firing of the neurons was calculated as the mean firing frequency during a 60-s resting period and represented as impulses/s. The response of the neurons to paw pinch was measured as action potential counts over 30-s compression of the paw and represented as impulses/s. Baseline firing and response of the neurons to paw pinch were measured as action potential counts over 30-s compression of the paw and were analyzed using a one-way ANOVA and the Mann–Whitney-U test was used to determine the significance among groups. The data were also subjected to the normality and equal variance test with the Shapiro–Wilk test. Values are expressed as mean S.E.M and p-value <0.05 was considered to be statistically significant.

RESULTS Prevention of hyperalgesia In order to investigate whether PENFS can prevent visceral hyperalgesia, the VMR to CRD was measured in a group of rats that received intracolonic TNBS with either active PENFS with the BRIDGE (n = 8) or sham (n = 8). Seven days following intracolonic TNBS, both groups showed an intensity-dependent increase in EMG to graded CRD (10–60 mmHg, 30 s). Animals that received PENFS had a significantly lower VMR to CRD at distension pressures of 40 and 60 mmHg (F5,25 = 13.84, p < 0.001 vs Colitis + Sham, Fig. 2A). CRD 30 mmHg are considered noxious in rats and humans, suggesting that the BRIDGE prevented the development of visceral hyperalgesia in these rats. PWT using von Frey filaments revealed a significant difference in TNBS-treated animals that received active PENFS with the BRIDGE compared to sham device.

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Fig. 2. Summary data of the VMR and PWT of animals after TNBSinduced colitis. These rats received daily (4 h) PENFS with the BRIDGE for 5 days or inactive (sham) device starting the same day of intracolonic TNBS instillation. (A) Rats that received to PENFS had a much lower response to CRD at pressures >30 mmHg compared to those with sham, suggesting the prevention of visceral hyperalgesia. (B) Similarly, rats with PENFS had much higher PWT compared to sham-treated animals, suggesting the prevention of somatic hyperalgesia (*p < 0.05 and **p < 0.001 vs sham).

Following TNBS-induced colitis, the mean PWT with sham stimulation was 160.4 ± 17.87 mN. However, those with colitis that received PENFS with had a mean PWT of 756.9 ± 157.5 (p < 0.001; Fig. 2B), suggesting the prevention of somatic hyperalgesia. In a group of naı¨ ve animals (n = 6), daily stimulation with the

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Fig. 3. Summary data of the VMR and PWT in non-inflamed, naive rats before and after PENFS. These rats received PENFS four hours a day with the BRIDGE device for five days. (A) PENFS had no effect on the VMR to graded CRD. (B) Similarly, PENFS had no effect on the PWT.

BRIDGE device using the same protocol had no effect on visceral or somatic sensitivity (Fig. 3A, B). Treatment of post-inflammatory hyperalgesia In a separate set of experiments, we investigated whether PENFS can reverse post-inflammatory hyperalgesia. In a group of rats (n = 8), the VMR was measured before and 7 days after TNBS-induced colitis. In these rats, VMR was again measured after 5 days of PENFS (14 days after TNBS). Following TNBS, these rats developed a significant increase in the VMR compared to baseline

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pressures of 30 and 40 mmHg (F20,10 = 5.545, p < 0.05 vs Pre-PENFS, Fig. 4A). In the same group of rats, the PWT was measured at baseline, 7 days after TNBS and again after PENFS. The mean PWT at baseline was 810.2 ± 91 mN (n = 8). Following TNBS-induced colitis, the mean PWT was decreased to 181 ± 56 mN. However, following PENFS the threshold for withdrawal significantly increased to 645 ± 98 mN, suggesting that PENFS attenuated the somatic hyperalgesia (p < 0.001; Fig. 4B). Amygdala recordings Extracellular recordings from CeA neurons were made in six rats. A total of six neurons that responded to somatic stimulation (compression of the hind paw) were recorded before and after stimulation with the BRIDGE. All neurons recorded from CeA were nociceptive-specific, since they exhibited responses only to noxious pinch (Randich et al., 1997). Fig. 5A, B shows examples of one CeA neuron before and after PENFS. Prior to stimulation, the mean spontaneous firing of the neurons was 1.71 ± 0.32 imp/s (n = 6). Following compression of the paw (190 mN) these neurons exhibited excitatory responses (3.6 ± 0.52 imp/s, Fig. 5C). There was a 65% decrease in the spontaneous firing of these neurons after 15 min of PENFS (mean: 0.59 ± 0.16 imp/s). Similarly, the response to compression of the paw was decreased by approximately 56% after PENFS (1.6 ± 0.27imp/s, p < 0.05, Fig. 5C). In a separate group of naı¨ ve animals without previously induced colitis (n = 6), PENFS had no effect on baseline neuronal firing or response to compression of the paw (Fig. 6). Spinal recordings

Fig. 4. Summary data of the VMR to graded CRD (10–60 mmh, 30 s) and PWT from rats before intracolonic administration of TNBS (baseline), 7 days after TNBS-induced colitis and after PENFS. (A) The mean analytical data illustrate that in the naı¨ ve, non-inflamed condition, these rats exhibited intensity-dependent increase in EMG activity to graded CRD. Following TNBS-induced colitis, VMR to CRD significantly increased, suggesting the development of visceral hyperalgesia due to colonic inflammation. This increase in VMR was significantly attenuated after PENFS at CRD pressures of 30 and 40 mmHg. (B) Following TNBS, there was a significant decrease in the PWT. However, PENFS significantly increased the PWT, suggesting attenuation of somatic hyperalgesia. (*p < 0.05, ** p < 0.001 vs baseline, #p < 0.05 vs post-colitis).

(non-inflamed condition) response at pressures 30 mmHg (F20,10 = 5.545, p < 0.001 vs Baseline) (Fig. 4A). However, following 5 days of PENFS, there was a significant reduction in the VMR at CRD

A total of five nociceptive specific neurons from the lumbar dorsal horn were recorded from TNBS-induced colitis rats and tested for somatic sensitivity before and after PENFS with the BRIDGE. Fig. 7A, B illustrates examples of one lumbar spinal neuron before and after PENFS. The mean spontaneous firing of the neurons (7.7 ± 0.87 imp/s) increased to 14.8 ± 2.28 imp/s after compression of the paw (Fig. 7C). Following 15 min of PENFS, there was a significant decrease (47%) in the spontaneous firing of the neurons (4.05 ± 0.65 vs control: 7.7 ± 0.87 imp/s, p < 0.05). Similarly, the responses of these neurons to compression of the paw decreased 48% after PENFS (7.62 ± 1.7imp/s, p < 0.05, Fig. 7C).

DISCUSSION In this study, we used a well-established animal model of TNBS-induced colitis to investigate the effects of PENFS with the BRIDGE device on post-inflammatory hyperalgesia. This is the first report demonstrating that the development of post-inflammatory visceral and somatic hyperalgesia can be altered by PENFS. The results also show that the response characteristics of amygdala and lumbar spinal neurons can be modulated by the BRIDGE device.

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Fig. 6. Shows the summary data of CeA neuron’s mean baseline firing and firing frequency to paw compression in naı¨ ve, non-inflamed rats. These neurons exhibited no difference in baseline firing or response to compression of the paw after PENFS (p > 0.05).

Fig. 5. Shows the response characteristics of CeA neurons before and after PENFS. In panels A and B, the top tracings are neuron’s firing represented as frequency histogram (1-s binwidth), the middle tracings are the neuron’s action potential and the bottom tracings are the duration of mechanical compression of the paw. (A) An example of CeA neuron’s response to paw compression before PENFS. The neuron exhibited an increase in firing frequency during the duration (30 s) of paw compression. (B) Following PENFS, the baseline firing frequency of the neuron and excitatory responses to paw compression were markedly reduced. (C) Shows the mean analytical data of CeA neuron’s responses before and after PENFS. Data show that both resting firing frequencies and excitation to paw compression were significantly reduced after PENFS (*p < 0.05).

The novelty of these results involves neuromodulation of central pain pathways that prevents the development of somatic and visceral hyperalgesia through peripheral, non-invasive electrical stimulation. While the ancient technique of electroacupuncture has been suggested to

improve inflammation and visceral hypersensitivity associated with colitis, the results have been controversial and the technique is much different than that used in this study (Xu et al., 2009; Goes et al., 2014). Electroacupuncture delivers very different electrical current and requires precise placement of needles in specific ‘‘acupoints” in the leg or ear by a trained expert. The percutaneous, titanium needles on the BRIDGE are placed on the ventral and dorsal region of the ear, away from neurovascular bundles to create a field effect in the auricle and not on specific acupoints. It delivers 3.2 V with alternating frequencies of stimulation that is not perceived as painful or noxious by humans or animals. Further, by securing the device with adhesive, this type of treatment is designed to be used in the outpatient setting and does not require more than minimal training to place. Previous reports, including the current study, have demonstrated that central brain structures can be modulated through electrical stimulation of the external ear (Kraus et al., 2013; Frangos et al., 2015). We observed a dampening effect of PENFS on neurons of the CeA with a 65% decrease in the spontaneous firing of the neurons after 15 min of stimulation. Tracing studies have confirmed that the external ear contains branches of cranial nerves that project to the NTS (Contreras et al., 1982; Zhang and Ashwell, 2001). The NTS projects to other brain structures involved in autonomic control and pain, including the RVM, hypothalamus and amygdala (van der Kooy et al., 1984; Ross et al., 1985). The amygdala, in particular, serves as a key limbic structure that is

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Fig. 7. Shows the response characteristics of lumbar spinal neurons before and after PENFS. In panels A and B, the top tracings are neuron’s firing represented as frequency histogram (1-s binwidth), the middle tracings are neuron’s action potential and the bottom tracings are the duration of mechanical compression of the paw. (A) An example of spinal neuron’s response to paw compression before PENFS. The neuron exhibited an increase in firing frequency during the duration (30 s) of paw compression. (B) Following PENFS, the baseline firing frequency of the neuron and excitatory responses to paw compression were markedly reduced. (C) Shows the mean analytical data of spinal neurons responses before and after PENFS. Data show that both resting firing frequencies and excitation to paw compression were significantly inhibited after PENFS (*p < 0.05, ** p < 0.001).

involved in controlling autonomic and visceral responses. The CeA is the output nucleus for major amygdala function and thus has widespread projections to the forebrain and brainstem (Bernard et al., 1996; Davis, 1998;

LeDoux, 2000). A large number of studies have described a key role of the amygdala in modulating chronic pain in animals associated with stress, anxiety and colitis (Davis, 1992; Rosen and Schulkin, 1998; Gao et al., 2004; DeBerry et al., 2015). More specifically, hyperactivity and synaptic plasticity of CeA neurons have been linked to pain-related behaviors (Neugebauer et al., 2004; Ji and Neugebauer, 2007; Fu and Neugebauer, 2008; Fu et al., 2008). For example, the firing rate of CeA neurons in animals with colitis has been shown to be higher than controls, suggesting neuronal excitability (Han and Neugebauer, 2004). In patients with IBS, neuroimaging studies have identified alterations in painrelated signaling and connectivity in the amygdala (Labus et al., 2008, 2014; Qi et al., 2016; Tanaka et al., 2016; Weaver et al., 2016). In the current study, the decrease in amygdala firing during PENFS suggests a possible mechanism to explain the anti-nociceptive effects seen in awake animals. In line with the current study, others have suggested that deactivation of the CeA results in inhibition of nocifensive and affective pain responses, as well as pain-related anxiety-like behavior (Ji et al., 2007; Fu and Neugebauer, 2008). The attenuation of neuronal activity in the amygdala during active inflammation could prevent sensitization and the development of visceral and somatic hyperalgesia since there is substantial convergence of somatosensory and visceral nociceptive input in the CeA (Ji and Neugebauer, 2010). Recent evidence suggests that PENFS with the BRIDGE can rapidly and effectively treat the pain and symptoms associated with opioid withdrawal in humans (Miranda and Taca, 2017). Those findings support to the rapid neuromodulating effects seen in the current study since a common central link between a state of chronic pain and opioid dependence appears to be in the amygdala (Fuchs and See, 2002; Nakagawa et al., 2005; Harris and Aston-Jones, 2007; Elman and Borsook, 2016). This brain region has been associated with the negative emotional state of withdrawal to opioids and drug craving (Koob and Volkow, 2010). Interestingly, our results also suggest that PENFS modulates spinal neurons, which may or may not be related to changes seen in the limbic system. The amygdala modulates lumbosacral spinal neurons that process information of noxious spinal reflexes (Helmstetter and Bellgowan, 1993; Helmstetter et al., 1993; Manning and Mayer, 1995). For example, corticosterone delivered onto the amygdala results in visceral hypersensitivity and an exaggerated response of lumbosacral spinal neurons to mechanical distension of the colon (Greenwood-Van Meerveld et al., 2001; Qin et al., 2003). However, it is possible that the dampening effects of PENFS on spinal neurons as well as the antinociceptive effects are independent of the amygdala. The sensitization of spinal neurons, particularly neurons having viscero-somatic convergence can lead to chronic hyperalgesia in animals and in patients with IBS (Miranda et al., 2004; Peles et al., 2004; Price et al., 2006; Woolf, 2011). The nociceptive drives entering spinal neurons are subject to modulation from pontomedullary nuclei, like PAG and RVM that get input from the NTS (Ross et al.,

R. Babygirija et al. / Neuroscience 356 (2017) 11–21

1985; Chen et al., 1995; Wilder-Smith et al., 2004). Additionally, the hypothalamus helps maintaining homeostasis and changes pain behaviors through its effect on the PAG and RVM (Le Bars et al., 1979; Yarnitsky, 2010; PintoRibeiro et al., 2013). Previous studies have demonstrated that electrical stimulation of the PAG or RVM activates the descending inhibitory pathway to decrease the firing of spinal dorsal horn neurons (Zhuo et al., 2002; Wang et al., 2016). Since we observed a dampening effect of PENFS on lumbar spinal neurons, it can be postulated that four hours of daily stimulation for five days during the critical time of colonic inflammation may prevent the development of spinal sensitization. However, the involvement of other contributing pathways cannot be ignored and deserves further investigation. The ant-inflammatory effects of vagal nerve stimulation (VNS) alone through a decrease in proinflammatory cytokines may have impacted the observed changes in visceral sensitivity. In a model of TNBS-induced colitis, electrical stimulation of the left cervical vagus in freely moving animals (3 h per day for 5 days) has been shown to decrease colonic inflammation (Meregnani et al., 2011). Similarly, in a small subset of patients with inflammatory bowel disease (IBD), VNS has also been shown to improve inflammation (Bonaz et al., 2016). In the present study, we did not examine progressive changes in the degree of colonic inflammation, which is a limitation since this could impact chronic pain behavior. Addressing strategies for pain management has become a major priority given the substantial impact of pain on health, social, and economic welfare. Nonpharmacological treatment or prevention of chronic pain resulting from visceral inflammation, infections or intraabdominal surgeries could have significant clinical implications. Evidence continues to mount for PENFS as a non-invasive, drug-free alternative that influences central pain pathways and improve symptoms of acute and chronic pain.

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(Received 30 December 2016, Accepted 8 May 2017) (Available online 17 May 2017)