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Brain, Behavior, and Immunity 64 (2017) 59–64

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Short Communication

Constriction of the buccal branch of the facial nerve produces unilateral craniofacial allodynia

http://dx.doi.org/10.1016/j.bbi.2016.12.004 0889-1591/� 2016 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Department of Psychology, Campus Box 345, Univer- sity of Colorado at Boulder, Boulder, CO 80309-0345, USA.

E-mail address: [email protected] (L.R. Watkins). 1 Authors contributed equally to this work. 2 Current address: Department of Critical Care Research, University of Texas MD

Anderson Cancer Center, Houston, USA.

Susannah S. Lewis a,1, Peter M. Grace a,b,1,2, Mark R. Hutchinson b,c, Steven F. Maier a, Linda R. Watkins a,⇑ a Department of Psychology & Neuroscience, University of Colorado, Boulder, USA b School of Medicine, University of Adelaide, Adelaide, Australia c Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, Adelaide, Australia

a r t i c l e i n f o

Article history: Received 27 October 2016 Received in revised form 2 December 2016 Accepted 5 December 2016 Available online 18 December 2016

Keywords: Orofacial Muscle Glia Hyperalgesia Mirror-image pain

a b s t r a c t

Despite pain being a sensory experience, studies of spinal cord ventral root damage have demonstrated that motor neuron injury can induce neuropathic pain. Whether injury of cranial motor nerves can also produce nociceptive hypersensitivity has not been addressed. Herein, we demonstrate that chronic con- striction injury (CCI) of the buccal branch of the facial nerve results in long-lasting, unilateral allodynia in the rat. An anterograde and retrograde tracer (3000 MW tetramethylrhodamine-conjugated dextran) was not transported to the trigeminal ganglion when applied to the injury site, but was transported to the facial nucleus, indicating that this nerve branch is not composed of trigeminal sensory neurons. Finally, intracisterna magna injection of interleukin-1 (IL-1) receptor antagonist reversed allodynia, implicating the pro-inflammatory cytokine IL-1 in the maintenance of neuropathic pain induced by facial nerve CCI. These data extend the prior evidence that selective injury to motor axons can enhance pain to supraspinal circuits by demonstrating that injury of a facial nerve with predominantly motor axons is sufficient for neuropathic pain, and that the resultant pain has a neuroimmune component.

� 2016 Elsevier Inc. All rights reserved.

1. Introduction

Peripheral nerve lesions or disease can initiate neuropathic pain, which is responsible for chronic pain in up to 10% of the gen- eral population (Treede et al., 2008; van Hecke et al., 2014). Due to the fact that pain is a sensory experience, neuropathic pain is fre- quently assumed to only follow damage to sensory neurons. How- ever, recent studies have revealed that selective lesion of spinal motor neurons by L5 ventral root transection induces nociceptive hypersensitivity and microglia activation in the spinal dorsal horn, which are both dependent on tumor necrosis factor (TNF) signaling (Li et al., 2002; Sheth et al., 2002; Xu et al., 2006, 2007). Such neu- roimmune signaling has a well-documented role in the develop- ment of neuropathic pain after injury to mixed (sensory and motor) peripheral nerves (Grace et al., 2014, 2016a). Furthermore, injury of the gastrocnemius-soleus (predominantly motor) nerve results in nociceptive hypersensitivity, and both induces ectopic

activity and amplifies evoked action potentials of sciatic nerve and DRG neurons (Kirillova et al., 2011; Michaelis et al., 2000; Zhou et al., 2010). Thus, injury of spinal motor nerves is sufficient for peripheral neuropathic pain.

To date, several models of craniofacial neuropathic pain have been developed, involving lesions of the sensory infraorbital (Eriksson et al., 2005; Vos et al., 1994), or sensory inferior alveolar nerves (Sugiyama et al., 2013). However, it is not yet known whether injury of cranial motor nerves is sufficient to induce neu- ropathic pain, similar to the spinal system. Uniformity cannot be assumed, given the documented pathophysiological differences between the injured spinal and trigeminal systems. For example, production of spinal dorsal horn interleukin (IL)-6 and sprouting of noradrenergic nerves within the dorsal root ganglia (DRG) occurs after sciatic nerve injury (Latrémolière et al., 2008; McLachlan et al., 1993), but neither occur within the trigeminal ganglia after infraorbital nerve injury (Benoliel et al., 2001; Latrémolière et al., 2008). Furthermore, triptans and calcitonin gene-related peptide (CGRP) receptor antagonists are effective in reversing nociceptive hypersensitivity induced by injury of the infraorbital nerve, but not of the sciatic nerve (Kayser et al., 2002, 2011; Michot et al., 2012, 2015).

Therefore, the goal of this study was to determine whether injury of a motor cranial nerve could produce neuropathic pain.

60 S.S. Lewis et al. / Brain, Behavior, and Immunity 64 (2017) 59–64

The facial nerve (cranial nerve VII) of the rat is an excellent candi- date to address this question, as it is comprised of motor efferent neurons without a significant somatosensory nerve component from the skin (Nerve, 2013), is readily accessible surgically and there is a well-established protocol for demonstrating facial allo- dynia in the rat (Ren, 1999). Given the dimorphic role of pro- inflammatory cytokines in craniofacial and spinal neuropathic pain (Latrémolière et al., 2008), the second goal of this study was to determine whether allodynia induced by facial nerve injury could be attenuated by blocking IL-1 signaling.

Fig. 1. Approximate size and position of the incision with skin retracted. Three chromic gut ligatures are shown in red. The buccal branch of the facial nerve (straight black line) is readily visible upon skin incision. Area for tactile testing is shown in blue square. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2. Methods

2.1. Animals

Adult, male, pathogen-free Sprague-Dawley rats (Harlan Labs, Madison, WI) were used for all experiments. Rats (350–400 g at time of surgery) were housed in temperature (23 ± 3 oC) and light (12 h:12 h light:dark; lights on 0700 h) controlled rooms with water and food given ad libitum. All habituation and behavioral testing procedures were performed during the light phase of the daily cycle. All procedures were approved by the University of Col- orado Boulder Institutional Animal Care and Use Committee. All experimental groups have 6–9 rats per group.

2.2. Facial nerve chronic constriction injury surgery

This novel surgery constricted the buccal branch of the facial nerve. The buccal branch of the facial nerve has the advantage of being readily accessible following a skin incision, allowing for a straightforward surgery with very little damage to tissues sur- rounding the nerve. All surgical instruments were sterilized prior to use and all surgical procedures were conducted under isoflurane anesthesia.

The buccal branch of the facial nerve was aseptically exposed through a 1 cm skin incision. Great care is necessary when shaving the skin, as damage to whiskers alters subsequent behavioral responses. The buccal facial nerve branch is superficial and visible following a skin incision. The incision was made along the line from the corner of the mouth to the ear, about two-thirds of the way to the ear (Fig. 1). Once exposed, the nerve was kept moist with sterile physiological saline drops and only touched with glass instruments to prevent damage through metal instruments. Borosilicate 600 glass pipettes (Fisherbrand, Fisher Scientific, Wal- tham, MA) were molded into a curved ‘L’ shape approximately 8 mm long at the tip and used to gently manipulate the nerve. These steps were undertaken to minimize the variability in nerve damage between rats.

To isolate the nerve, two nicks (each approximately 0.5 mm) were made into the fascia and muscle surrounding the nerve using the tip of a #11 scalpel blade (Havel, Cincinnati, OH, USA). These small incisions were expanded using a pair of shaped glass pipettes in a spreading motion to gently separate the nerve completely from the surrounding fascia and muscle. The spreading motion, rather than additional scalpel incisions, separated the muscle along muscle fibers and minimized damage and bleeding. Care was taken not to stretch the nerve during the separation of the nerve and musculature.

Once the nerve was isolated from surrounding muscle and con- nective tissue, three 4-0 chromic gut (Ethicon, Somerville, NJ, USA) ligatures were tied around the nerve with a square knot. Ligatures were tied tightly enough so to not to move along the nerve when gently pushed with forceps, but loose enough not to visibly deform the nerve and spaced approximately 1 mm apart. Again, care was taken not to stretch or deform the nerve during ligation. After

ligation, the chromic gut was cut close to the knot and the skin was then sutured closed with 4-0 silk suture (Ethicon, Somerville, NJ, USA). Sham surgeries were as described above, with the excep- tion that no chromic gut sutures were tied around the isolated nerve.

2.3. von Frey test for tactile sensitivity

Assessment of the development and persistence of tactile allo- dynia was conducted as detailed (Ren, 1999). Briefly, rats were habituated in two 5 min sessions to stand comfortably with their forepaws in a leather glove. This method allows the rats to be com- pletely unrestrained. Calibrated microfilaments (von Frey hairs; Stoelting, Wood Dale, IL, USA) were applied to the hairy skin under the eye by and experimental blind to treatment groups. Microfila- ments were applied in 5 quick up-down applications and the num- ber of brisk head withdrawals or aggravated paw swipes recorded as responses.

Microfilaments ranging logarithmically from 1.2 to 75.86 g were applied starting with a mild stimulus of 3.63 g and increasing or decreasing to find the range from 0 out of 5, to 5 out of 5 responses from the rat. Assessments were made prior to and 3, 7, 10, 14, 21, 28, 35, 42 days following facial nerve CCI or sham sur- gery by an experimenter blind to treatment group. Responses were fitted to a Gaussian integral psychometric function using a maximum-likelihood fitting method as described (Milligan et al., 2000).

2.4. Body weights

Body weights were measured prior to and 3, 7, 10, 14, 21, 28, 35, 42 days following facial nerve CCI or sham surgery by an experi- menter blind to treatment group. Measurements were made between 0900 and 1100 h to reduce variability due to circadian changes.

2.5. Neuronal tracing

Although the majority of the constricted nerve is efferent facial nerve axons, it is possible that there may be a small component of afferent trigeminal axons also mixed within the nerve bundle. In order to determine whether any increase in mechanical sensitivity could be due to damage of intermingled trigeminal afferents in the buccal nerve CCI site, a neuronal tracing study was conducted.

Anterograde and retrograde labeling of the facial and trigeminal brainstem nuclei and trigeminal ganglia with the tracer 3000 MW tetramethylrhodamine-conjugated dextran (Invitrogen, Carlsbad, CA, USA) was used to determine origin/terminus of neurons in

S.S. Lewis et al. / Brain, Behavior, and Immunity 64 (2017) 59–64 61

the constricted region. The nerve was first exposed and isolated identically to that described above. Using a method adapted from May and Hill (2006), the nerve was then transected and parafilm placed under the nerve to isolate it from surrounding tissues. A Q-tip was used to apply DMSO to the cut end of the nerve to increase dextran penetration. Dextran granules were then placed on the nerve, held in place with a small dab of petroleum jelly and the parafilm sealed around the nerve with superglue. This method allowed the dextran to be applied to the nerve for an extended period of time without contaminating nearby tissues, which are innervated by other cranial nerves. By transecting the nerve, all axons in the nerve were exposed to the retrograde tracer.

At 1, 3, 4, 5, 6 or 7 days after dextran placement (n = 2/time- point), rats were deeply anesthetized with sodium pentobarbital (50 mg/kg i.p.) and transcardially perfused, first with a saline flush, and then with 4% paraformaldehyde to fix the tissue. Brains and trigeminal ganglia were harvested and cryoprotected in 30% sucrose. Brains and ganglia were then frozen in dry-ice chilled isopentane and sliced in 50 lm sections in a cryostat. The entire trigeminal ganglion was sectioned, and approximately one out of every 10 sections were stained. Sections were mounted on gelatin coated slides and fluorescence examined immediately on an Olym- pus BX61 fluorescence microscope (Olympus America, Center Val- ley, PA) using Microsuite software (Olympus America).

2.6. Drug administration

The effect of proinflammatory cytokines on facial nerve CCI was assessed using interleukin-1 receptor antagonist (IL-1ra, Amgen, Thousand Oaks, CA) administered intracisterna magna (i.c.m.). IL- 1ra or equivolume sterile, endotoxin free saline was administered 21 and 28 days after facial nerve CCI or sham surgery. Mechanical allodynia was assessed 45 min following i.c.m. injection to account for the relatively short cerebrospinal fluid half life of IL-1ra (Milligan et al., 2005).

I.c.m. injections were percutaneously performed as previously described (Frank et al., 2010), using polyethylene-60 (PE60) tubing attached to a 30 gauge 3/800 hypodermic needle. Each rat was briefly anesthetized with isoflurane and a small patch at the nape of the neck was shaved and scrubbed with 70% ethyl alcohol. The rat was then placed in ventral recumbancy on a box with the head positioned beyond the end of the box such that the head bent downward at a 90� angle to the body, allowing easier access to the cisterna magna. The 30 gauge needle was percutaneously inserted into the cisterna magna and a 10 ll injection of either 1 ll of 100 lg IL-1ra plus 8 ll saline vehicle separated by 1 ll air, or 9 ll saline vehicle plus 1 ll air. Injections were given slowly over a 30 s period. A dose of 100 lg IL-1ra was chosen based on prior reports that the same dose intrathecally reversed neuropathic pain induced by sciatic CCI (Grace et al., 2016b) and inflammatory neuropathy (Milligan et al., 2003), and this same dose i.c.m. blocked stress-induced enhancement of pro-inflammatory responses by brain nuclei (Johnson et al., 2004).

2.7. Statistics

Mechanical allodynia was analyzed as the interpolated 50% thresholds (absolute threshold). One-way analysis of variance fol- lowed by the Tukey post hoc test was used to confirm that there were no baseline differences in absolute thresholds between treat- ment groups. Differences between treatment groups were deter- mined using 2-way analysis of variance, followed by the Sidak post hoc test, with a correction for repeated measures for mechan- ical allodynia. P < 0.05 was considered significant, and all data are expressed as mean ± SEM.

3. Results

3.1. Buccal branch CCI produces unilateral craniofacial allodynia

There were no pre-surgical baseline differences between the either surgery group on either side of the face (F3,24 = 0.69, P > 0.05). CCI of the buccal branch of the facial nerve produced sig- nificant orofacial allodynia ipsilateral to the site of injury from day 10 through day 35 after surgery (Fig. 2; Time x Treatment: F7,84 = 3.86, P < 0.01; Time: F7,84 = 4.99, P < 0.001; Treatment: F1,12 = 28.93, P < 0.001). Post hoc tests showed a significant decrease in the CCI group compared to Shams ipsilateral to facial nerve CCI at every time point tested after surgery, until testing was concluded at day 42 (P < 0.05). No significant allodynia devel- oped contralateral to the site of injury (Time x Treatment: F7,96 = 0.53, P = 0.8).

At no point in the six week duration of allodynia was there a significant difference in body weight gain between the facial nerve CCI and sham animals (Treatment: F8,95 = 0.58, P = 1.0, data not shown). No noticeable changes in whisking behavior or eyeblink reflex were subjectively observed following the ligation of the facial nerve.

3.2. No trigeminal afferents were detected at the site of constriction

To test whether injury of a small contingent of sensory nerves in the facial nerve could have accounted for the robust allodynia, trigeminal afferents were labelled with the antero- and retrograde tracer 3kD tetramethylrhodamine-conjugated dextran. This dye has previously produced robust central nervous system cell body labeling of peripheral gustatory sensory nerves (May and Hill, 2006), and tibial and common fibular motor nerves (English et al., 2009). Strong labeling of neurons in the facial nucleus was found 6 days following dextran placement (Fig. 3) with weaker labeling present 5 and 7 days following dextran placement. At no time point (1, 3, 4, 5, 6 or 7 days following dextran placement at the site of transection) was fluorescent labeling detected in the trigeminal ganglion or at any level of the brainstem trigeminal nuclei beyond that seen in an animal without dextran placement. These data indicate that there are no detected trigeminal sensory afferents in the surgical site of the facial nerve.

3.3. IL-1ra reverses established allodynia following facial nerve CCI

Numerous studies have convincingly shown that an increase in neuroinflammation in the dorsal spinal cord importantly con- tributes to allodynia following sciatic CCI (Grace et al., 2014, 2016a). One of the major neuroinflammatory mediators within spinal cord implicated in creating allodynia is following injury to peripheral sensory/motor mixed nerves is IL-1beta (Grace et al., 2014, 2016a). In contrast, IL-1 has never been implicated in allody- nia induced as a consequence of injury to motor axons, either spin- ally or supraspinally. To determine if IL-1 provides a proinflammatory component necessary to maintain the craniofa- cial allodynia seen following facial nerve CCI, tactile sensitivity was assessed 45 min after i.c.m. IL-1ra, in a within-subjects design described above. There were no baseline differences between the sham and CCI group on either side of the face (F3,23 = 1.10, P > 0.05). There was a significant interaction between surgery and drug treatment (Fig. 4; F3,48 = 4.74, P < 0.01), as well as a main effect of treatment (F3,48 = 13.57, P < 0.001), but not of time (F1,48 = 1.42, P = 0.2). Post hoc tests showed that the facial CCI sur- gery produced a robust allodynia prior to the saline and IL-1ra injections on day 21 and 28 post surgery compared to sham treated animals (P < 0.05). The allodynia remained unchanged after

Fig. 2. Chronic constriction injury of the facial nerve leads to the development of tactile allodynia ipsilateral to the surgery. No significant allodynia was found contralateral to injury. Animals with CCI maintained significant allodynia from 10 to 35 days after surgery. Allodynia was no longer significant at 42 days post- surgery. *P < 0.05, **P < 0.01, ***P < 0.001, relative to Sham Ipsilateral. Mean ± SEM are presented, n = 6–99/group.

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an i.c.m. saline injection. However, IL-1ra reversed established craniofacial allodynia, relative to control treatment after facial CCI (P < 0.01), with no significant difference between CCI rats trea- ted with IL-1ra and sham treated animals. IL-1ra had no impact on contralateral mechanical thresholds, which were not altered by facial nerve CCI (data not shown).

4. Discussion

These studies present the first evidence that constriction injury to a cranial nerve with predominantly efferent motor neurons can produce reliable and prolonged tactile allodynia. Notably, con- tralateral allodynia was absent after buccal branch CCI, which con- trasts with that reported for some models of sciatic nerve injury (Grace et al., 2010; Milligan et al., 2003). The allodynia measured in this study was transiently reversed with an intracisterna magna injection of IL-1ra, suggesting a role for central nervous system inflammation in the generation of the allodynia.

To our knowledge, all other craniofacial neuropathic pain mod- els involve damage of sensory nerves (Eriksson et al., 2005;

Fig. 3. Representative micrographs from dextran staining demonstrate that the injury sit the trigeminal nucleus through the hindbrain as well as the trigeminal ganglia were ex fluorescence noted was in the facial nucleus 5, 6 and 7 days following dextran placemen position of the illuminated neurons in the facial nucleus (A, 4�), detailed morphology o

Sugiyama et al., 2013; Vos et al., 1994). The results obtained here demonstrate that injury to the facial nerve, which we show to be devoid of detected trigeminal somatosensory afferents from the skin, is also sufficient to create neuropathic pain. These data paral- lel and importantly extend studies performed in the motor gastrocnemius-soleus nerve (Kirillova et al., 2011; Michaelis et al., 2000; Zhou et al., 2010) and the motor ventral root (Li et al., 2002; Sheth et al., 2002; Xu et al., 2006, 2007), and highlight a common consequence of damage of nerves that innervate mus- cles in the cephalic and spinal systems. Injury of these motor nerves also induces nociceptive hypersensitivity, and spontaneous activity in uninjured DRG sensory neurons (Kirillova et al., 2011; Michaelis et al., 2000; Xu et al., 2006, 2007; Zhou et al., 2010). The facial region below the eye, where hypersensitivity was detected, is innervated by the V2 branch of the trigeminal nerve (Nerve, 2013). The trigeminal and facial nerves are not mixed, but both project to the brainstem. This extra-territorial allodynia may therefore be mediated by central sensitization, rather than by Wallerian degeneration of motor neurons, as occurs in the spinal system (Gaudet et al., 2011; Xu et al., 2006, 2007). Future studies may seek to confirm these results in cephalic nerves com- posed solely of efferent fibers, such as the oculomotor nerve.

Our data also point to the involvement of pro-inflammatory cytokines in neuropathic pain induced by facial nerve CCI. While TNF has previously been implicated in allodynia resultant from injury to motor axons (Li et al., 2002; Sheth et al., 2002; Xu et al., 2006, 2007), no prior study of allodynia in response to motor damage has examined IL-1. Here, IL-1ra reversed allodynia at 21 and 28 days post-surgery, indicating a role for IL-1 in neuropathic pain maintenance, most likely via release within brainstem sites. IL-1 may have a common role in mediating nociceptive hypersen- sitivity after craniofacial and sciatic nerve injury (Grace et al., 2014), unlike IL-6 (Latrémolière et al., 2008). There are several known mechanisms by which IL-1 may increase neuronal excitability in nociceptive pathways (Grace et al., 2014, 2016a), including phosphorylation of postsynaptic NR1 NMDA receptor subunits (Zhang et al., 2008), and down-regulation of both the astrocyte glutamate transporter GLT-1 (Yan et al., 2014) and neu- ronal G protein-coupled receptor kinase 2 (an enzymatic regulator of G protein-coupled receptor homologous desensitization, that protects against overstimulation) (Kleibeuker et al., 2008). IL-1 is elevated in the brainstem and contributes to extra-territorial pain after trigeminal nerve injury (Chai et al., 2012; Takahashi et al., 2011), and this report adds to others demonstrating a causal role for this cytokine in craniofacial neuropathic pain (Won et al.,

e did not contain trigeminal sensory afferents. Brain slices from the caudal portion of amined from 1 to 7 days following dextran placement at the injury site. The only t. Six days was optimal and shown in the above pictures. Micrographs show relative f illuminated neurons (B) and the lack of staining in the trigeminal ganglia (C).

Fig. 4. Intracisterna magna IL-1 receptor antagonist (IL-1ra; 100 lg) significantly attenuated the tactile allodynia that developed following facial nerve constriction. Assessments were made prior to (pre-treatment), and 45 min after administration (post-treatment). No significant change was noted following i.c.m. saline injections. *P < 0.05, **P < 0.01, ***P < 0.001. Mean ± SEM are presented, n = 6–9/group.

S.S. Lewis et al. / Brain, Behavior, and Immunity 64 (2017) 59–64 63

2014). Future studies may investigate whether activated glial cells or recruited immune cells are associated with this nerve injury model, and are responsible for production of IL-1.

In conclusion, this study demonstrates that injury to the facial nerve, which is predominantly composed of motor neurons, is suf- ficient to induce neuropathic pain in rat. This finding is also sup- ported by the clinical literature, as pain is a principal complaint of Bell’s palsy—an idiopathic paralysis of the facial nerve (De Seta et al., 2014). Our data predict that neuroimmune signaling con- tributes to nociceptive hypersensitivity after facial nerve injury, and is a possible therapeutic target for craniofacial neuropathic pain.

Acknowledgments

The authors declare that there are no conflicts of interest. Fund- ing from NIH R01 DE021966. Peter M Grace was a NHMRC (Aus- tralia) CJ Martin Fellow [ID: 1054091] and American Australian Association Sir Keith Murdoch Fellow. Mark R. Hutchinson was a NHMRC (Australia) CJ Martin Fellow (ID 465423; 2007-2010) and an Australian Research Council Research Fellow (DP110100297). The authors are grateful to Drs. Dianna Bartel and Thomas Finger (University of Colorado Denver) for their assistance with the neu- ronal tracing protocol.

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