Abstract title: Disease Relevance In Vitro? Comparison of Excitability in DRG Neurons from Neuropathic and Inflammatory Disease Models
Poster number: PTH241
Presentation time: Thursday, September 13, 3:15 – 4:15 PM
Authors: Paul Karila1, Diana Nascimento2, Resti Rudjito2, Alex Bersellini Farinotti2, Susanne Lardell1, Camilla I Svensson2
Affiliations: 1Cellectricon AB, Mölndal, Sweden; 2Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
Introduction: The aetiologies of inflammatory pain and neuropathic pain are fundamentally different. In dorsal root ganglion (DRG) neurons, differential expression of certain ion channels, neuropeptides and markers of nerve stress indicate mechanistic differences in the pain pathophysiology. However, after prolonged peripheral inflammation a number of changes in the DRG neurons resemble changes observed after nerve injury, suggesting that pain of inflammatory origin may evolve into a condition that resembles neuropathic pain. In our previous work we characterized the neurochemical profile of DRG neurons in mice from an arthritis model that show this “shift” in pain mechanism over time.
The aim of the current study was to explore if changes in neuronal excitability induced by nerve injury or long-term inflammation is still present after establishment of primary cell cultures.
Methods: BALB/c female mice were subjected to a spared nerve injury (SNI, model of neuropathic pain) or collagen antibody-induced arthritis (CAIA, model of long-lasting joint inflammation with a neuropathic component). SNI was induced by the ligation and transection (around 3mm distal from the node) of both tibial and common peroneal branches of the sciatic nerve, while the sural nerve was kept intact, and CAIA by intravenous injection of a cocktail of anti-collagen type II antibodies followed by lipopolysaccharide (LPS) 5 days later. Mechanical hypersensitivity was assessed by von Frey filaments and the degree of joint inflammation by visually inspecting the joints and scoring the number of swollen digits and ankle joints. Lumbar DRG neurons (L1-L6) were collected 6 days after SNI and 50 days after induction of CAIA and primary DRG neuronal cell cultures were prepared in 384 well plates. Day 2 after plating the cells were loaded with the calcium indicator Calcium 5 (Molecular Devices) and transient calcium elevations were recorded as fluorescence intensity changes in neurons during simultaneous electric field stimulation. The excitability responses in DRG neurons from the respective animal models were compared to DRGs from untreated or sham operated control animals.
Results: All animal work was carried out in accordance with our local ethical committee and IASP guidelines. Mechanical hypersensitivity was observed from day 1-6 in the SNI model and day 3-50 in the CAIA model. A robust joint inflammation was noted from day 7-30 in the CAIA model and even though the inflammation resolved, pain-like behavior persisted for at least an additional 20 days. There was a clear difference between the contra- and ipsilateral DRGs from the SNI model. On average (N=6 animals and 18 wells/condition) the excitability response (measured as a ratio of the fluorescence intensity) increased from 1.2 in DRG neurons from the contralateral side to 1.4 on the ipsilateral side. This corresponds to a 100% increase in the excitability response. A difference in neuronal excitability in ipsilateral DRG neurons from mice subjected to the SNI model compared to control mice was also observed. Data from experiments in which the neuronal cultures were established from DRGs collected 50 days after induction of CAIA will also be presented.
Conclusions: It is critical to advance our understanding of mechanisms underlying chronic pain. Here, using a high capacity in vitro system, we demonstrate that primary neuronal cultures established from mice subjected to nerve injury retain differences in excitability in culture. Further work is warranted in order to determine how long the condition-induced changes are retained in culture, and to what extent the changes are comparable to the in vivo situation. It is intriguing, however, that these types of neuronal model systems may provide an in vitro platform for studies of how an underlying condition changes the excitatory properties of sensory neurons and offer opportunities to explore how such changes can be modulated. Thus, this finding may open new avenues for utilizing in vitro models to advance our understanding of pain pathophysiology.
Acknowledgments/Disclosures: Grant support was received from Swedish Research Council, Knut and Alice Wallenberg Foundation, Family Lundblad Foundation. SL and PK are employees of Cellectricon AB.