Calcium imaging of primary canine sensory neurons: Small‐diameter neurons responsive to pruritogens and algogens

Abstract

IntroductionRodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations.MethodsThis study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin.ResultsThis study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens.ConclusionRatiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

Peripheral mechanisms of arthritic pain: A proposal to leverage large animals for in vitro studies

“…For example, in human DRG neurons, the voltage-gated sodium channel (Na V ) 1.8 blocker A-803467 is much less effective at blocking Na V -mediated currents in human DRG neurons than in rat DRG neurons, suggesting that Na V blockers with efficacy in rodents might not translate to clinical pain relief in human diseases due to different expression levels ( Zhang, et al, 2017 ). The feasibility of obtaining DRG from large animals has been demonstrated in many species including horses ( Russo et al, 2010a ), sheep ( Deng et al, 2018 , Dudek et al, 2017 , Domenico Russo et al, 2010b ), pigs ( Jonas et al, 2015 , Klusch et al, 2018 , Kozłowska et al, 2017 , Obreja et al, 2008 , Sandercock et al, 2019 ) and dogs ( Ganchingco et al, 2019 , Schwarz et al, 2019 ), providing proof-of-concept that DRG neurons from large animals can be utilized as in vitro models for arthritis pain.…”

“…Similar to patch clamp electrophysiology, very few studies have investigated large animal neurons using Ca 2+ imaging in the context of nociception. In vitro Ca 2+ imaging of canine DRG neurons demonstrated their ability to respond to algogens such as, capsaicin and pruritogens such as, histamine ( Ganchingco et al, 2019 ). Similarly a recent in vitro study imaged sheep DRG neurons to show hypoxia and acidosis induced increase in Ca 2+ response ( Ma et al, 2020 ).…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…neurotrophins) [7]. DRG have been isolated from several different mammalian species [8][9][10][11][12], and even human organ donors [13], and studied to better understand sensory neuron biology, including responses to nerve injury and disease [14][15][16], with experiments on mouse ganglia being most common. Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), five or six lumbar (L1 to L5/L6) and four sacral (S1 to S4).…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…neurotrophins) [7]. DRG have been isolated from several different mammalian species [8][9][10][11][12], and even human organ donors [13], and studied to better understand sensory neuron biology, including responses to nerve injury and disease [14][15][16], with experiments on mouse ganglia being most common. Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), ve or six lumbar (L1 to L5/L6) and four sacral (S1 to S4).…”