Acta Dermato-Venereologica 99-12CompleteContent | Page 19

1131 INVESTIGATIVE REPORT Itch-associated Neuropeptides and Their Receptor Expression in Dog Dorsal Root Ganglia and Spinal Cord Joshua J. WHEELER 1,2 , B. Duncan X. LASCELLES 2,3 , Thierry OLIVRY 2,3 and Santosh K. MISHRA 1,2 * 1 Department of Molecular Biomedical Sciences, 2 Comparative Medicine Institute, and 3 Department of Clinical Sciences, North Carolina State University, Raleigh, NC, USA Most canine visits to veterinarians are related to skin diseases with itch being the chief complaint. Histori- cally, several itch-inducing molecules and pathways have been identified in mice, but whether or not these are similar in dogs is not yet known. Herein, we set out to study the expression of pruritogenic neuropeptides, their cognate receptors with a limited functional valida- tion thereof using a multidisciplinary approach. We de- monstrated the expression of somatostatin and other major neuropeptides and receptors in canine dorsal root ganglia neurons. Next, we showed that interleu- kin-31, serotonin, and histamine activate such neu- rons. Furthermore, we demonstrated the physiological release of somatostatin from dog dorsal root ganglia neurons in response to several endogenous itch medi- ators. In summary, our results provide the first evi- dence that dogs use similar pruritogenic pathways to those characterized in mice and we thus identify mul- tiple targets for the future treatment of itch in dogs. Key words: dog; canine atopic dermatitis; somatostatin; NPPB; NMB; GRP. Accepted Aug 22, 2019; E-published Aug 22, 2019 Acta Derm Venereol 2019; 99: 1131–1135. Corr: Santosh K. Mishra, Department of Molecular Biomedical Sciences, North Carolina State University, 1060 William Moore Drive, Office 242, Raleigh, NC 27607, USA. E-mail: [email protected] O utside of preventative health care, dermatological issues account for nearly 20% of all causes for dog veterinary visits; approximately 30% of which are due to pruritus, canine atopic dermatitis being one of the leading causes of such pruritus (1). Research aimed at understanding the etiology of pruritus in dogs has begun with the characterization of receptors for several com- mon pruritic agents found to be expressed in dog dorsal root ganglia (DRG) (2). In mice, a few neuropeptides are released from the DRG into the spinal cord synapses upon a cutaneous pruritic challenge: the natriuretic precursor peptide B (NPPB) (3), somatostatin (SST) (4), neuro- medin B (NMB) (5) and the gastrin-releasing peptide (GRP) (6). However, itch-associated neurotransmitters/ neuropeptides and their respective receptors have not yet been reported in dogs. Here, we used qRT-PCR to determine the mRNA levels of itch-associated neuropeptides and neuropep- tide receptors expressed in dog DRGs and spinal cords. Additionally, we used immunohistochemistry (IHC) to SIGNIFICANCE In the last few years, molecular studies have characterized multiple itch mediators, receptors, and neurotransmitters/ neuropeptides that are required for itch signaling in mice. However, it is unclear if similar signaling components and pathways are involved in the propagation of itch in dogs. Herein, we focus on the physiological expression and func­ tion of itch-associated receptors and neurotransmitters that were first implicated in generating the itch sensation in mice. Our work identifies several new potential targets for antipruritic therapies in dogs. localize the expression of the SST protein in DRGs. Next, we performed calcium imaging on primary cultured dog DRG neurons to identify if they have functional recep- tors for selected itch mediators. Finally, we performed enzyme-linked immunosorbent assay (ELISA) on the supernatants from primary cultured dog DRG neurons stimulated with itch mediators to demonstrate the sub- sequent release of the itch-associated neuropeptide SST. MATERIALS AND METHODS RNA isolation RNA was isolated from fresh-frozen lumbar L4 and L5 DRG from 3 to 5 euthanized Beagles (3 females, 1-year-old; and 2 males, 7-year-old, 27  ±  2.0 kg, all from Marshall Farms). DRG had been flash frozen or placed in RNA later and stored at –80°C until use. RNA was similarly isolated from the lumbar spinal cords from 3 adult beagles. Dogs had no prior history of any known pain or itch conditions. Both DRG and spinal cord tissues were isolated under protocols approved by NC State’s animal care and use policies for different studies. A roughly 2 mm 3 piece was cut from each DRG and transferred to a 10 ml tube containing 3 ml lysis buffer with 1% β-mercaptoethanol from Qiagen’s RNeasy Fibrous Tissue Mini Kit and then homogenized for 30–45 s. For the spinal cord, a length of ~4 mm was transferred to a 10 ml tube containing 3 ml of lysis butter with 1% β-mercaptoethanol from Qiagen’s RNeasy Fibrous Tissue Mini Kit and then homogenized for 30–45 s. Fol- lowing homogenization, RNA was extracted following Qiagen’s protocol without performing a proteinase K digestion. cDNA synthesis To synthesize cDNA 200 ng RNA (in ≤ 8 µl), 2 µl random hexamer primers (Invitrogen, Waltham, MA, USA) and PCR grade water (for a final volume of 10 µl) was heated at 72°C for 3 min. Af- terwards, 4 µl of 5x Reaction Buffer, 2 µl DTT, 2 µl SmartScribe Reverse Transcriptase (Clontech, Mountain View, CA, USA), and 2 µl of a 10 mM dNTP stock (Roche, Branchburg, NJ, USA) were This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2019 Acta Dermato-Venereologica. doi: 10.2340/00015555-3297 Acta Derm Venereol 2019; 99: 1131–1135