Basic and Clinical July 2016. Volume 7. Number 3 The Role of C Fibers in Spinal Microglia Induction and Possible Relation with TRPV3 Expression During Chronic CrossMark Inflammatory Arthritis in Rats Sasan Gazerani 1, Jalal Zaringhalam 1,2*, Homa Manaheji 1,2, Sahar Golabi 1 1. Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2. Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Citation: Gazerani, S., Zaringhalam, J., Manaheji, H., & Golabi, S. (2016). The role of C fibers in spinal microglia induction and possible relation with TRPV3 expression during chronic inflammatory Arthritis in rats. Basic and Clinical Neuroscience, 7(4), 231-240. http://dx.doi.org/10.15412/J.BCN.03070308 : http://dx.doi.org/10.15412/J.BCN.03070308 A B S T R A C T Article info: Introduction: Stimulation of peptidergic fibers activates microglia in the dorsal horn. Microglia Received: 02 March 2015 activation causes fractalkine (FKN) release, a neuron-glia signal, which enhances pain. The First Revision: 28 March 2015 transient vanilloid receptor 1 (TRPV1) mediates the release of neuropeptides, which can Accepted: 27 August 2015 subsequently activate glia. TRPV1 and TRPV2 are generally expressed on C and Aδ fibers, respectively. Expression of both proteins is upregulated during inflammation, but expression of TRPV3 after induction of inflammation is unclear. Methods: Adult male Wistar rats were used in all experiments. Arthritis was induced in them by single subcutaneous injection of complete Freund’s adjuvant (CFA) in their right hindpaws. Resiniferatoxin (RTX) was used to eliminate peptidergic fibers. We examined the relation between FKN and TRPV3 expression by administration of anti-FKN antibody. Results: Our study findings indicated that 1) spinal TRPV3 was mostly expressed on nonpeptidergic fibers, 2) expression of spinal TRPV3 increased following inflammation, 3) elimination of peptidergic fibers decreased spinal TRPV3 expression, 4) alteration of hyperalgesia was compatible with TRPV3 changes in RTX-treated rat, and 5) anti-FKN antibody reduced spinal TRPV3 expression. Discussion: It seems that the hyperalgesia variation during different phases of CFA-induced Key Words: arthritis correlates with spinal TRPV3 expression variation on peptidergic fibers. Moreover, Hyperalgesia, TRPV3, spinal microglial activation during CFA inflammation is involved in TRPV3 expression changes Microglia, Fractalkine via FKN signaling. 1. Introduction Gonzalez-Ros, & Ferrer-Montiel, 2012; Fein, 2012). The inflammatory mediators activate the ends of pain nerve pro- ain is described as "an unpleasant" sensory cesses. Excitation of pain receptors stimulates the specialized and emotional felling accompanied with nerves, such as C and Aδ fibers that carry pain impulses to different aspects of tissue injuries (Fernan- the spinal cord (Omoigui, 2007). C and Aδ fibers are essential P dez-Carvajal, Fernandez-Ballester, Devesa, for determining painful stimuli and launching pain sensation * Corresponding Author: Jalal Zaringhalam, MD, PhD Address: Department of Physiology, Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel: +98 (912) 516 8313 Fax: +98 (21) 22439971 E-mail: [email protected] 231 (Chen & Pan, 2006). Some authors suggested that sharp pain facial nerve axotomy, inflammatory condition, and viral in- is transmitted by Aδ fibers that carry information from dam- fections (Patro, Nagayach, & Patro, 2010). Activated glia re- aged thermal and mechanical nociceptors, and it is followed leases various substances such as proinflammatory cytokines by a more prolonged aching pain which is transmitted by C and other mediators, which can enhance pain transmission fibers that carry signals from polymodal nociceptors Kandel,( (Shan et al., 2007). Schwartz, Jessel, Siegelbaum, & Hudspeth, 2013). Studies also indicated that proinflammatory agents such The nociceptors are equipped with a wide range of recep- as nerve growth factor (NGF), bradykinin, and chemokines tors that render them sensitive to thermal, mechanical, and lead to TRPV1 sensitization (Palazzo et al., 2010). Fractal- some chemical stimuli. Transient receptor potential (TRP) kine (FKN) is a chemokine expressed on the extracellular channels are temperature sensitive and when activated, depo- surface of spinal neurons and sensory afferents (Milligan et larize nociceptor terminals. Thermo TRP channels in mam- al., 2004). It is a critical mediator of spinal neuron-microglial mals are TRPV, TRPM, and TRPA subfamilies. Expression communication in chronic pain. Previous studies indicated of TRP receptors on related nerve fibers vary during different that membrane bound FKN is located on neurons in the dor- physiological and pathological situations. Transient vanil- sal horn of the spinal cord (Staniland et al., 2010), whereas loid receptor 1 (TRPV1) receptors are activated by capsa- its receptor is exclusive and primarily expressed by microglia icin, heat, and low pH (Spicarova & Palecek, 2008; Brandt (Milligan et al., 2004; Staniland et al., 2010). FKN protein et al., 2012). Mice deficient of TRPV1 shows impaired pain increases in (CX3CR1) KO mice following inflammation, response to heat (Chen & Pan, 2006). TRPV1 receptors are and loss of FKN/receptor attenuates hyperalgesia (Staniland predominantly expressed in small sensory C fibers and to a et al., 2010). lesser extent in Aδ fibers (Brandt et al., 2012). TRPV1 ex- pression increases in all 3 types of DRG (dorsal root gan- Altogether, we aimed to determine the role of C fibers in glion) neurons after CFA injection (Yu et al., 2008). hyperalgesia, spinal microglia induction, and its possible re- lation with TRPV3 expression during chronic inflammatory In rat dorsal root ganglia, TRPV2 is expressed primarily in arthritis in male rats. myelinated afferents, most likely in Aδ nociceptors (Shimo- sato et al., 2005). TRPV2 has a higher temperature activation 2. Material and Methods threshold, above 52°C (Sachdeva, Pandit, & Bafna, 2012). Moreover, previous studies indicated that TRPV2 immuno- 2.1. Laboratory animals reactivity in DRG neurons is augmented 2 days after CFA- induced inflammation Fernandez-Carvajal,( et al., 2012). In this study adult male Wistar rats are used in weighing TRPV3 has a unique heat threshold. It is activated at innocu- 180-220 gr. They were protected in Plexiglas cages under ous temperatures with an activation threshold around 33°C hygienic and standard condition such as 12/12-hour light/ to 35°C and exhibits increasing responses at higher noxious dark cycle and food and water ad libitum. All experiments temperatures. The responsiveness of TRPV3 receptors con- achieved the local and international standards for research tinues to increase as the temperature rises in noxious range. on experimental animals and pain induction (Zimmermann, These receptors can also sensitize, suggesting that TRPV3 1983). receptors may contribute in pain sensitization (Sachdeva, Pandit, & Bafna, 2012). Although some studies demonstrat- 2.2. Induction of adjuvant arthritis ed that acute noxious heat thermo-sensation is diminished According to our previous studies we induced adjuvant in TRPV3−/− mice, there is no evidence about the role of arthritis by injection of CFA. Briefly, we injected subcuta- TRPV3 receptors in hyperalgesia changes after inflammatory neously 100μL of CFA (sigma, St. Louis, MO/USA) into insult (Fernandez-Carvajal, et al., 2012). the rats’ right hindpaws. It is clear that, CFA is heat killed Mycobacterium tuberculosis suspended in sterile mineral More studies about pathological pain have primarily fo- oil (10mg/mL). In control group we had tried only 100μL of cused around neuronal mechanisms involved in pathological mineral oil into same paw. Ipsilateral inflammation was seen pain; however, neighboring astrocytes and microglia have on first day following CFA injection. According to our previ- been recognized as a powerful modulator of pain too (Mil- ous studies, first week after CFA injection considered as “in- ligan, Watkins, 2009). Microglia are further activated after flammatory phase” and the next two week named as arthritic various insults such as nerve injury, by displaying morpho- phase (Tekieh et al., 2011). Then, all assessments were done logical changes and upregulation of microglia markers such on days 0, 7, 14, and 21 after induction of arthritis as Iba1 (Ji, Berta, & Nedergaard, 2013). The expression of Iba1 is also upregulated in activated microglia following 232 Zaringhalam, J., et al. (2016). Spinal microglia induction and possible relation with TRPV3 expression. Basic and Clinical Neuroscience, 7(4), 231-240. Basic and Clinical July 2016. Volume 7. Number 3 2.3. Thermal hyperalgesia assessment binant and natural rat fractalkine. According to the paper, we administered 1μg/d of anti-fractalkine antibody for 7 days be- In order to evaluate paw withdrawal latency (PWL) fol- ginning on day 11 postinduction (Clark, Grist, Al-Kashi, Per- lowing painful heat as a thermal hyperalgesia indicator, retti, & Malcangio, 2012). Antibody was dissolved in sterile we used plantar test according to our previous study (Ugo phosphate-buffer (PBS) for intrathecal injection, and control Basile, Verse, Italy) (Zaringhalam, Tekieh, Manaheji, Akh- animals received PBS. This solution was prepared fresh at tari, 2013). Briefly, 10 to 15 minutes prior to experiment, rats most 30 minutes prior to injection.