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Assessment of the efficacy and safety of vixotrigine in participants with lumbosacral radiculopathy: results of a randomized, placebo-controlled trial

Fiona Forrestal – Biogen

Background and Aims Vixotrigine is a state- and use-dependent voltage-gated sodium channel blocker under investigation for treatment of neuropathic pain. Neuropathic pain associated with lumbosacral radiculopathy (PLSR) is typically due to degenerative changes in the lumbosacral spine compromising L4, L5, and/or S1 nerve roots. No oral medications are approved specifically for PLSR. The safety and efficacy of vixotrigine in this population was assessed in a Phase 2 multicenter trial (NCT02935608; EUDRA CT 2015-004775-78). Methods Double-blind (DB), randomized, placebo-controlled, parallel-group trial in participants with PLSR. Study comprised a 2-week, single-blind, placebo run-in period followed by a 12-week DB period. Participants were eligible for randomization if they had a baseline weekly average pain score ≥4 and ≤9 on the Pain Intensity-Numerical Rating Scale (PI-NRS; range 0–10). Pain scores from the worst affected leg along with low back pain and sleep interference scores were recorded daily in an electronic diary. Baseline was defined as the 7 days prior to the first DB dose. Participants were randomized (1:1:1) to placebo, vixotrigine 200 mg BID, or vixotrigine 350 mg BID. The randomization was stratified by previous back surgery, whether nonsteroidal anti-inflammatory drug use was ongoing at baseline, and geographical region (Eastern versus Western Europe). The primary endpoint was the change from baseline to DB week 12 in the weekly average PI-NRS score. Key secondary endpoints included the percentage of participants with ≥30% and ≥50% reduction from baseline in weekly average PI-NRS scores at DB week 12, and the incidence of adverse events (AEs). Plasma samples for quantification of vixotrigine levels were collected throughout the trial. Results In total, 502 participants enrolled in the study; 425 were randomized, of whom 424 were dosed (placebo [n=142], vixotrigine 200 mg BID [n=140], or vixotrigine 350 mg BID [n=142]). Mean age of randomized participants was 52.7 years, and 63.7% were female. Mean (range) duration of pain was 50.8 months (6–367), and mean baseline pain score was 6.39. There was a reduction in the mean average weekly pain score at DB Week 12 in all treatment groups: placebo (−1.75) and vixotrigine 200 mg BID (−1.50) or vixotrigine 350 mg BID (−1.81), and no difference between the groups was observed (p=0.314, placebo vs 200 mg; p=0.797, placebo vs 350 mg). The percentage of participants with ≥30% (or ≥50%) reduction in weekly average pain scores at DB week 12 were similar in all treatment groups: placebo, 40.1% (22.5%); 200 mg BID, 34.3% (20%); and 350 mg BID, 33.8% (20.4%). Overall, 26.2% of participants receiving vixotrigine experienced at least 1 treatment-emergent AE. The most common ( >2% in any treatment arm) treatment emergent AEs were dizziness (placebo, 0.7%; 200 mg BID, 2.1%; 350 mg BID, 2.1%), headache (placebo, 3.5%; 200 mg BID, 2.1%; 350 mg BID, 2.1%) and alanine aminotransferase increase (placebo, 0%; 200 mg BID, 0.7%; 350 mg BID, 2.8%). Most events were mild; 1 serious AE of dizziness was reported and considered related to the study treatment in the 350 mg BID group, led to withdrawal from the study, and was resolved. The safety profile was similar to that previously reported with no new or unanticipated category of safety concerns noted. Observed mean plasma levels of vixotrigine for the two dosed groups were approximately dose-proportional (1104.21 ng/mL for 200 mg BID and 1807.52 ng/mL for 350 mg BID at week 8 predose). Conclusions The study did not meet any of the efficacy endpoints. Vixotrigine was well-tolerated in this patient population. Conflicts of Interest Fiona Forrestal, Himanshu Naik, Leslie A Shinobu, Romy Christmann, and Wildon Farwell are employees/shareholders of Biogen. Mark Versavel is a paid consultant to Biogen. John Markman has served on 2 advisory boards for Biogen and as a consultant to Abbott, Allergan, Aptinyx, Biogen, Chromocell, Clexio Bioscience, Collegium, Convergence, Depomed, Diacchi Sankyo, Editas Medicine, Eli Lilly, Flexion Therapeutics, Grunenthal, Inspirion, Jansen, Kempharm, Nektar, Novartis, Pfizer, Plasmasurgical, Plasmasurgical, Purdue, Quark, Quartet, Sanofi, Teva, and Trevena. Juan Perez-Cajaraville has received fees for consulting and for lectures, speakers bureaus, expert testimony, employment, and advisory boards from Pfizer, Grunenthal, Mundipharma and St Jude Medical. The terms of this arrangement have been reviewed and approved by NAU in accordance with its policy on objectivity in research. Simon Tate was an employee of Convergence Pharmaceuticals Ltd, a Biogen Company, at the time of the study. Source of Financial Support for the Project Biogen (Cambridge, MA, USA). References NA

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Prokineticin System as a new Target to Counteract Experimental Vincristine Induced Peripheral Neuropathy

Giada Amodeo

Theodora Kalpachidou

Silvia Franchi

Michaela Kress

Giorgia Moschetti, MD – Università degli Studi di Milano

Paola Sacerdote

Background and Aims : Chemotherapy Induced Peripheral Neuropathy (CIPN) is a side effect of several antineoplastic agents. Regardless their mechanism of action, chemotherapeutic agents induce an alteration of neural and glial function, mitotoxicity, cytokines and chemokines release which result in an enhanced nociceptive input. Among chemokines, Prokineticins (PK) have a fundamental role in the development and maintenance of both inflammatory and neuropathic pain [1,2,3]. The aim of our study was to elucidate the role of PK in CIPN induced by Vincristine (VCR), a chemotherapeutic agent commonly used in the clinical practice and related to the development of neuropathy; and whether the block of PK receptors, using an antagonist (PC1), may represent a therapeutic approach.

Methods : CIPN was induced in C57BL/6J male mice by repeated administration of Vincristine (0.1mg/kg once a day for 14 consecutive days). Hypersensitivity was evaluated by measuring mechanical and thermal allodynia as well as thermal hyperalgesia. When hypersensitivity was established (7 days after the 1st VCR injection), PC1 was daily administered (150µg/kg twice a day) until the end of VCR schedule. At day 7, before starting PC1 chronic treatment, and at the end of VCR/PC1 schedule (day 14), Prokineticin2 (PROK2), PK receptors (PK-R1 and PK-R2), cytokines (TNF-α, IL-6, IL-1β and IL.10), CD68, CD11b, TLR4 and ATF3 were evaluated as mRNA (RealTime-qPCR) in dorsal root ganglia and spinal cord. Neurite outgrowth was assessed in DRG cell cultures treated with different nanomolar doses of VCR and PC1 and expression levels of PK system and ATF3 were evaluated by RT-qPCR.

Results : VCR induced a dose-dependent allodynia and hyperalgesia in mice, which was correlated to an upregulation of PK system, the presence of neuroinflammation and an upregulation of ATF3 expression levels, both in peripheral and central nervous system. We found increased expression of CD68, CD11b and TLR4, therefore indicating that activated macrophages/microglia seemed to be key mediators in this pathological condition. PC1 administration was able to counteract hypersensitivity. Its effect could be due to its ability to reduce PROK2 and its receptors levels in nervous tissues and contrast neuroinflammation probably by reducing macrophages activation and migration in DRG. Neurite outgrowth was severely reduced in cultured DRG neurons treated with different nanomolar concentration of Vincristine. Also in vitro PC1 treatment was able to contrast the toxic effect of VCR. The chemotherapeutic agent induced an upregulation of PROK2, PK-R1 and ATF3 expression levels that was instead prevented when the PK antagonist was simultaneously administered with VCR.

Conclusions : PK antagonism may represent a promising pharmacological strategy to handle CIPN.

References : [1] Franchi et al., The prokineticin system: an interface between neural inflammation and pain. Neurol Sci. 2017; 38:27-30.
[2] Maftei et al., Controlling the activation of the Bv8/prokineticin system reduce neuroinflammation and abolishes thermal and tactile hyperalgesia in neuropathic animals. Br J Pharmacol. 2014; 171 (21): 4850-4865.
[3] Castelli et al., Antagonism of the prokineticin system prevents and reverses allodynia and inflammation in a mouse model of diabetes. PLoS One. 2016; 11(1):e0146259.

Conflicts of Interest : No conflicts of interest to declare.

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The EQIPD Framework for Rigour in the Design, Conduct and Analysis of Biomedical Experiments.

Malcolm Macleod, PhD

Tom Van de Casteele

Anton Bespalov

Bruce Altevogt

Esther Schenker

Thomas Steckler

Hanno Wuerbel

Gernot Riedel

Ulrich Dirnagl

Heidrun Potschka

Jan Vollert, PhD – Research Associate, Imperial College London

Kimberley E Wever

Martin C Michel

Andrew SC Rice

Background and Aims : Within the last years, there has been growing awareness of the negative repercussions of inappropriate planning, conduct and reporting of preclinical research. Several initiatives have set the aim of increasing the completeness of reporting of studies, and making their internal validity transparent [Hirst et al., 2014, Percie du Sert et al., 2017, Hooijmans et al., 2014, Simera et al., 2010]. While these initiatives overlap significantly, they differ in detail, and show variance in generalizability or specific challenges for a single field. Consequently, it is challenging for researchers to decide which guidelines to follow, especially at the stage of planning future studies.

Aim of the EQIPD (European Quality in Preclinical Data) framework is to unify current guidance, find a basis in evidence behind their rationale, and prospectively test the newly set framework for feasibility in multi-centre animal experiments.

Methods : We performed a systematic review of existing guidelines on preclinical experimental planning, conduct and reporting. PubMed, Embase and Web of Science were searched systematically to identify guidelines published in English language in peer-reviewed journals before January 2018. Our comprehensive search strategy yielded 13,863 results, of which 62 publications met our predefined inclusion criteria. From these publications, a list of 58 unique items were extracted. In a two-round Delphi process with a subsequent consensus meeting, 33 of these items were included in the final framework. This framework was structured into five major domains.

Results : DOMAIN 1: Answer the following question: Is your experiment testing a predefined scientific hypothesis which is statistically testable (confirmatory research) or is it exploring a space of interesting options to generate hypotheses (exploratory research)?

DOMAIN 2: Whenever and where ever meaningful, possible and feasible, prespecify, document and standardize all methods and analyses before the experiment.

DOMAIN 3: Think about which form of aggregate measures are meaningful for your data and choose appropriate statistical methods.

DOMAIN 4: Whenever and where ever meaningful, possible and feasible, randomize and blind your processes to avoid the introduction of confounding and systematic error.

DOMAIN 5: Not all biases can be avoided, but most can be uncovered: provide full and comprehensive documentation.

Conclusions : The EQIPD framework is currently being tested prospectively for feasibility and limitations in a multi-centre animal study, which will further inform the final version. As many of the suggested methods are coming mainly from expert opinions rather than controlled experiments, we also aim to identify the provenance of diligence – the evidence for an impact of the proposed items, and the history of their application.

References : Du Sert NP, Bamsey I, Bate ST, et al. The Experimental Design Assistant. Nat Methods 2017;14(11):1024–25.
Hirst JA, Howick J, Aronson JK, et al. The need for randomization in animal trials: An overview of systematic reviews. PLoS ONE 2014;9(6):e98856.
Hooijmans CR, Rovers MM, Vries RBM de, et al. SYRCLE's risk of bias tool for animal studies. BMC Med Res Methodol 2014;14:43.
Simera I, Moher D, Hirst A, et al. Transparent and accurate reporting increases reliability, utility, and impact of your research: Reporting guidelines and the EQUATOR Network. BMC Med 2010;8:24.

Conflicts of Interest : No relevant conflicts of interest.

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Symptomatic Profiling of Neuropathic Pain in Different Rat Strains, - an Aid for Improving Translatability?

Axel Hansen – Professor, Department of Veterinary and Animal Sciences, University of

Gordon Munro – Research Scientist, Hoba Therapeutics

Sara Hestehave, DVM – PhD.Student, University of Copenhagen

Klas Abelson – Associate Professor, Dept. of Experimental Medicine, Faculty of Health and Medical Sciences, University of Copenhagen

Tina Brønnum Pedersen – Principal Laboratory Animal Veterinarian, H. Lundbeck A/S

Daniel Andersson – Research Scientist, H. Lundbeck A/S

Josue Castro-Mejía – Post Doc, Department of Food Science, Faculty of Science, University of Copenhagen

Dennis Nielsen – Professor, Department of Food Science, Faculty of Science, University of Copenhagen

Background and Aims : Chronic pain conditions typically involve heterogeneous patient populations characterized by distinct phenotypic sensory profiles that arise as a consequence of functionally diverse disease processes, and genetic and environmental factors. The ongoing failure to develop more efficacious and better tolerated analgesic drugs has led to a re-evaluation of the validity of assays and models used in preclinical drug development. This includes the over-reliance by many investigators on the use of nociceptive withdrawal reflex based assays at the expense of assays incorporating facets of spontaneous pain or sensory loss, and emotional aspects of the pain condition (Rice et al., 2018). Discussions also revolve around choice of pain model in relation to both disease pathology and animal-related factors like gender, age and genotype. E.g. the majority of preclinical pain activities utilizing rats, use outbred Sprague Dawley (SD) or Wistar. Currently, we do not really know if these strains represent the optimal choice for assessing all aspects of pain as a disease entity. Complicating matters further are the unknown effects of environment and gut microbiota on experimental and clinical outcome. In recent years evidence has mounted to show that the mammalian gut microbiome (GM) influences behavior/mood in rodents and likely also in humans. The majority of serotonin is for instance of gut-origin and GM manipulation has in rodents been shown to directly affect behavior. However, whether pain perception is also linked to GM composition is not known. To address some of these fundamentally important issues within the same laboratory setting, we compared behavioral outcomes in a standard neuropathic pain model using four different inbred rat strains (Lewis; LEW, Wistar Kyoto; WKY and two sub-strains of Fischer; F344/ICO and F344/DU) that vary in inherent stress sensitivity and emotionality. We compared pain-outcome via the use of both evoked and functional (gait) assays, explored emotional comorbid burden, and the possible connections between the behavioral outcomes and the GM composition.

Methods : Male subjects of the selected strains (LEW, WKY, SD (Crl:SD), F344/ICO & F344/DU) were exposed to either Spared Nerve Injury (SNI)- or SHAM-surgery, and evaluated for 6 months post-injury on parameters related to pain and anxiety; (i) hindpaw mechanical thresholds (von Frey filaments) measured continuously throughout the study, (ii) CatWalk® gait analysis performed at the end of the study, (iii) anxiety-like behaviors (Elevated Plus Maze (EPM)) each month, and (iv) fecal samples were collected monthly for determination of GM composition using 16S rRNA gene V3-region high throughput amplicon sequencing.

Results : Four out of five of the strains studied variously developed mechanical allodynia as a result of the SNI surgery (F344/ICO=WKY=SD >F344/DU). The two inbred stress-hyperresponsive F344 sub-strains were markedly different, with the F344/DU rats developing less mechanical allodynia from the surgery than the exquisitely sensitive F344/ICO. Surprisingly, the stress-hyporesponsive LEW-strain did not develop any significant effects of surgery, when mechanical sensitivity was compared between SNI and SHAM. On the other hand, when exploring functional gait-adaptations to the neuropathic pain injury by use of CatWalk®, all strains exposed to the nerve injury exhibited similar changes on parameters like dynamic weight bearing and abnormal step-cycles. None of the measured gait parameters indicated that LEW rats should have been less affected by SNI surgery than the other strains. Only one strain (F344/DU) showed changes on the EPM, that could be indicative of increased comorbid anxiety-like behavior related to the neuropathic pain, and the effects were only significantly different from SHAM at month 3 and 4 post surgery. The GMs of all five strains were strain-specific and gradually changed during the study, while still maintaining strain-specific GMs. Interestingly, SNI-surgery induced changes in the GM in only the mechanically insensitive LEW strain, where the microbial composition became significantly different compared with SHAM-injured LEW controls.

Conclusions : Our study showed clear differences in response to neuropathic injury in different rat strains, and even in two sub-strains of the same inbred strain (F344/ICO and /DU). Notably, SNI-induced functional alterations of gait-pattern did not correlate with degree of evoked mechanical allodynia, as shown in the stress-hyporesponsive LEW strain which developed gait-changes in the absence of evoked mechanical allodynia. This sensory phenotype could be explained by (i) neuropathic pain/allodynia not measurable by evoked mechanical stimuli, (ii) sensory loss contributing to the gait-changes, (iii) effects of damaged motor-neurons, or (iv) possible protective effects from the altered GM composition in the SNI-injured LEW rats. Comorbid anxiety-like behavior was only detected in one strain - the moderately pain-sensitive F344/DU – indicating that the development of anxiety-like behavior was not directly correlated to degree of mechanical allodynia following nerve-injury. Thus, different rat (sub)-strains appear to develop distinct sensory and symptomatic profiles associated with gain and/or loss of function after neuropathic injury, and in some strains, there may be an interaction with the gut microbiome.

References : Rice, A, S.C.; Finnerup, N.B.; Kemp, H.I.; Currie, G.L.; Baron, R.:"Sensory profiling in animal models of neuropathic pain: a call for back-translation". Pain 2018.

Conflicts of Interest : Experiments were partially funded by and performed at H. Lundbeck A/S.

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Photobiomodulation inhibits RAGE activation and decreases the expression of NF-kB p65 in streptozotocin-induced type 1 diabetes mellitus

Marucia Chcaur

Igor Rafael Correia Rocha – PhD Student , University of São Paulo

Background and Aims : Type 1 diabetes mellitus has become a largely forgotten disease and still an incurable and untreatable ailment. Painful diabetic neuropathy is one of the most common phenotype of diabetic peripheral neuropathy and the activation of the receptor for advanced glycation end products (RAGE) and the transcriptional factor NF-kB p65 play the central role in initiating and propagating inflammatory responses and thus has been pointed out as one of the signaling pathways responsible for diabetic complications. The present study envisaged the involvement of photobiomodulation in inhibiting RAGE/NF-kB activation and in cytokines modulation.

Methods : Type 1 diabetes mellitus was induced on adult male Wistar rats with an intraperitoneal single dose (85mg/Kg) of streptozotocin. The animals were submitted to 10 sessions of photobiomodulation emitting a wavelength of 904 nm and an output power of 45 mWpk. RAGE, NF-kB, TNF-a, IL-6, IL-1b and IL-10 were analyzed in the rat sciatic nerve using western blot assays.

Results : Diabetic rats showed elevated expression of NF-kB p65, RAGE along with increased levels of TNF-alpha but no changes were observed in IL-6 and IL-1b levels. We observed that photobiomodulation decreased the expression NF-kB p65, RAGE along with increased levels of the anti-inflammatory interleukin IL-10 in diabetic rat sciatic nerve.

Conclusions : It can be concluded that the beneficial effects of photobiomodulation may be attributed to its RAGE/NF-kB inhibitory action as well as cytokines modulatory activity.

References : Diabetes Atlas - International Diabetes Federation.
Lawrence, T., 2009. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb. Perspect. Biol.
Schmidt, A.M., Stern, D.M., 2000. RAGE: A new target for the prevention and treatment of the vascular and inflammatory complications of diabetes. Trends Endocrinol. Metab.

Conflicts of Interest : The authors declare no conflict of interest.

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An adolescent rat model of vincristine-induced peripheral neuropathy

Jamie Renbarger

Ailing Li, PhD – Indiana University Bloomington

Tammy Sajdyk

Yvonne Lai

Andrea Hohmann, PhD

Jonathon Crystal

Background and Aims : Childhood acute lymphoblastic leukemia (ALL) is a significant clinical problem that can be effectively treated with vincristine, a vinca alkaloid-based chemotherapeutic agent. However, due to the increased survival rate, nearly all children receiving vincristine treatment develop dose-limiting sensory, motor and/or autonomic peripheral neuropathy[1,2] We previously reported that pediatric ALL populations are highly vulnerable to developing vincristine-induced peripheral neuropathy (VIPN) [1,3]. The possible long term impact of adolescent vincristine treatment across the lifespan remains incompletely understood. We, consequently, developed an adolescent rodent model of VIPN which can be utilized to study both the mechanisms underlying VIPN and possible long term consequences of vincristine treatment in the developing rat.

Methods : Adolescent rats of both sexes received once daily intraperitoneal injection of vincristine for 15 days during the critical period of adolescence (i.e. postnatal day P35 to P49). Separate groups received saline injections in parallel or were left untreated. Mechanical paw withdrawal thresholds, duration of responding to cold, rotarod descent latency, and grip strength (all paws and forepaws) were assessed before vincristine treatment and once every four days until adulthood P71. Body weight was also monitored throughout the experiment.

Results : Vincristine-treated animals developed hypersensitivity to mechanical and cold stimulation of the plantar hind paw surface. Hypersensitivity to mechanical and cold stimulation developed shortly following the onset of the vincristine dosing regimen and outlasted the period of vincristine treatment for approximately 10 days. However, both mechanical and cold allodynia resolved by P63. No differences in rotarod performance were observed between saline- and vincristine- treated animals. Vincristine-treated animals exhibited delayed increases in grip strength (all paws) compared to the saline-treated group from P54 to P70. No differences were observed in forepaw grip strength between vincristine- and saline-treated groups. A slower onset of weight gain was observed for both males and females in the vincristine-treated groups, with male rats showing a more profound slowing of weight gain.

Conclusions : We developed an adolescent rodent VIPN model that peripheral neuropathy characterized by development of marked sensory abnormalities to mechanical and cold stimulation. Vincristine treatment during the adolescence led to the development of mechanical and cold allodynia which outlasted the period of vincristine treatment but resolved later in adulthood. Vincristine also produced a slowing of gain in grip strength (all paws) that exhibited a delayed onset and persisted into adulthood.

References : 1. Smith E, Li L, Hutchinson R, Ho R, Bryan Burnette W, Wells E, et al. Measuring vincristine-induced peripheral neuropathy in children with acute lymphoblastic leukemia. Cancer Nurs [Internet]. 2013;36:1–26. Available from:
2. Ramchandren S, Leonard M, Mody RJ, Donohue JE, Moyer J, Hutchinson R, et al. Peripheral neuropathy in survivors of childhood acute lymphoblastic leukemia. J Peripher Nerv Syst. 2009;14:184–9.
3. Smith E, Lang L, Ho R, Wells EM, Hutchinson RJ, Skiles J, et al. Patterns and severity of vincristine-induced peripheral neuropathy in children with acute lymphoblastic leukemia during the first year of treatment. Pediatr Blood Cancer [Internet]. 2014;61:S117. Available from:

Conflicts of Interest : Authors declare no conflict of interest.

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Novel telemetric approach to assess the progressing impact of diabetes on the peripheral nervous system

Martin N. Skov, MSc.Eng. Biomed tech – Ph.D. Student, Comparative Medicine Lab, Department of Clinical Medicine, Aarhus University

Michael Pedersen

Background and Aims : Diabetic neuropathy (DN) is a common and sometime a debilitating condition. The prevalence of DN is approximately 30%, and up to 50% will eventually develop neuropathy during the course of their disease. (Maser RE, S. A. (1989).) The disease may affect both large and small fibres and cause sensory and motor disturbances, causing paraesthesia, sensory loss, pain and different degrees of motor weakness. Autonomic nervous system involvement is known, and this condition is associated with an increased mortality and morbidity in patients with diabetes. (Callaghan B. C, C. (june 2012)). To better treat and prevent complications it is of great importance to recognize the sensory and autonomic dysfunction as early as possible. At the moment, it is unclear how and when the autonomic changes develop in relation to the somatosensory signs. It is therefore important to perform longitudinal measurements to determine the progression of autonomic and sensory involvement. However, a single unifying hypothesis for neuropathy pathogenesis has not yet emerged from clinical and experimental animal studies. There is therefore a need for more accurate and regular measurements of the developing of neuropathy of the sensory and autonomic nerves. In humans, such studies may take years to carry out. As alternative, animal-studies using experimental models of diabetic neuropathy represent a way to elucidate the sensory and autonomic changes associated with development of DN.
Aim of the project:
This study addresses the challenges associated with continuous measurements of sensory and motor nerve function, focusing on the sciatic -and tail nerve function in rats, using novel telemetric implant and Qtrac, a threshold tracking software and hardware setup for excitability studies (Hugh Bostock). As an extra validation step in the development process, the median nerve in healthy humans will be tested. Concomitantly, the study aims to reveal the longitudinal denervation associated with the development of diabetes in rat model.

Methods : Methods:
In the roadmap for a fully functional implant, being small enough to be implanted in a rat, the requirement for a small front-end sensor (FES) is a significant part. The FES consists of an amplification- and filtration stage that will not distort the unusual non-linear signal that is received from the nerve and the stimulation artefacts. See the following paragraph Signal shape. The study consists of multiple iterations to determine the best configuration for the amplification, signal-to-noise, filtration, space occupied on the PCB and battery use. The requirements specifications for the implant were determined by looking at already existing implants and by testing.
Signal Shape:
An average signal was found from multiple tests, with an amplitude in the 1-50 mV range and a propagation time of around 4 ms, with a first spike duration-time of 0.5-1 ms. As the signal arrives from an artificial stimulation, with a square wave amplitude of up to 20 V and a duration of 0.5-1 ms, the stimulation artifact becomes an important issue. Due to the short distance between the stimulation and recording sites, only a short time-delay of 1-3 ms between the two signals was observed.
Filtering an amplification:
A traditional way of building a filter for a nonlinear signal is a cascading filter of multiple op-amps. This type of filter was tried out to minimize the 50 Hz noise but was how ever found impossible to work without distorting the signal.
A level shift is chosen to 1.5 V and the battery voltage to 3 V. Thus, the amplification can raise the signal to theoretical 1.5 V and practical 1 V.
To make the implant wireless:
Different SoCs was in the consideration to become the “brain” of the telemetric implant. The choice fell on a PSoC 4 BLE module from Cypress. Since the SoC is pre-built with all the functional blocks needed to build the implant: ADC as interface to the front-end sensor and Bluetooth as a telemetric connection to a computer.

Results : A preliminary study on 8 rats, shows no significant different between, using the standard Qtrac setup and the newly developed FES. As all the data still havened been processed. A finally verdict is still to come. But as off today it seems very promising that the FES can be a new and important tool to the Qtrac setup in animal models.

Conclusions : Multiple different designs were tried out; all with pro and cons. In the end, a new instrumental amplifier (ad8237) with a double clipper input circuit and a DC level shifted output signal, was considered as the best choice. Many methods were tried to lower the amplitude of the square wave to prevent saturation, without distorting the nerve signal. As a square wave consists of all signals in the frequency domain, an amplitude filter was tried. Such filter type was found possible, but due to high component count needed to build, it was not considered of practical use.
A fully functional telemetric prototype is expected to work as a pre-amp in a regular electrophysiology setup within the next 6 months. Due to difficulties in the wireless connectivity to the Qtrac software, it is not expected to work perfect as a telemetric connection without an improvement in the Qtrac software.

References : Maser RE, S. A. (1989). Epidemiological correlates of diabetic neuropathy. Report from Pittsburgh Epidemiology of Diabetes Complications Study. Diabetes.
Callaghan B. C, .. C. (june 2012). Diabetic neuropathy: clinical manifestations and current treatments. The lancet neurology, s. 521-534.
Hugh Bostock. (As the inventer of Qtrac and affiliation to the IDNC)

Conflicts of Interest : No conflicts of interest

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Fluorinated cannabidiol derivative, HUF-101, prevents the development of mechanical and thermal allodynia induced by paclitaxel in mice

Thiago Cunha

Francisco Guimarães

Francisco Gomes – University of Sao Paulo

Alexandre Lopes

Raphael Mechoulam

Miriam Fonseca

Nicole Silva – PhD student, University of Sao Paulo

Background and Aims : Background: Cannabidiol (CBD) is a phytocannabinoid with multiple pharmacological effects and several potential therapeutic properties. Its low oral bioavailability, however, could limit its clinical use. Previous studies indicate that fluorinated CBD compounds could present a higher pharmacological potency. Aim of Investigations:To investigate the antinoceciptive effects of HUF-101, a fluorinated synthetic CBD analog, in Chemotherapy-Induced peripheral Neuropathic Pain model and its possible mechanisms of the action.

Methods : Methods: For the Chemotherapy-induced peripheral neuropathy (CIPN), C57BL/6 mice (20-25-g) received daily injections of HUF-101 (i.p., 1, 3, 10, or 30 mg/kg) or vehicle (VEH) for 7 consecutive days. Paclitaxel (PCX; 8mg/kg) or saline were administered 4 times, every two days (0, 2, 4 and 6), 30min after HUF-101 or VEH injections. Another independent group of animals received injection of PCX (i.p., 8mg/kg) or saline in days 0, 2, 4 and 6. HUF-101 or vehicle were daily administered from 7th to the 20th days. To evaluate the mechanical and thermal allodynia induced by PCX, Von Frey filaments and acetone test were applied to the right plantar surface of the animal. To investigate the possible mechanism of action of HUF-101, the animals were transcardially perfused with PBS and the dorsal root ganglia were removed after the last injection of HUF-101 in the first protocol for further qPCR analysis. Besides that, in vitro HUF-101 effects (0.1, 0.3, 1.0, and 3µM) were evaluated in breast cancer cells culture (4T1) treated with Paclitaxel (3.0 and 10µM) for 24h using the MTT method to evaluate cell viability.

Results : Results: PCX treatment (8 mg/kg) was able to induce mechanical and thermal allodynia in all tested protocols. This effect was prevented by treatment with HUF-101 (1, 3, 10 e 30 mg/kg) for 7 days. However, these same doses were not effective to reverse the already installed allodynia induced by PCX. HUF-101 decreased IBA-1 and TNF-a mRNA relative expression in the dorsal root ganglia when compared to animals that only received PCX. Moreover, HUF-101 did not interfere with the antineoplastic effect of paclitaxel.

Conclusions : Conclusion: These results showed that HUF-101 attenuated persistent pain in the Paclitaxel-induced peripheral neuropathy model, without interfering with its chemotherapeutic effect. Furthermore, they allow us to suggest a possible anti-inflammatory action mediating the HUF-101 effects.However, more experiments are necessary to better understand the mechanism of action of this compound. Thus, HUF-101 could be a therapeutic alternative for the treatment of pain.

References : Silva, N.R; Gomes, F.V; Fonseca, M.D; Mechoulam, R; Breuer, A.; Cunha, T.M; Guimarães, F.S. Antinociceptive effects of HUF-101, a fluorinated cannabidiol derivative. Progress in Neuropsychopharmacology & Biological Psychiat, 79, 369-377, 2017.

Breuer, A., Haj, C.G., Fogaça, M. V., Gomes, F. V., Silva, N.R., Pedrazzi, J.F., Bel, E. A Del, Hallak, J.C., Crippa, J. A., Zuardi, A.W., Mechoulam, R., Guimarães, F.S. Fluorinated cannabidiol derivatives: Enhancement of activity in mice models predictive of anxiolytic, antidepressant and antipsychotic effects. PLoS One, 11, 1–19, 2016.

Izzo, A.A.; Borrelli, F.; Capasso, R.; Di Marzo, V.; Mechoulam, R. Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends in Pharmacological Sciences, 10, 515-527, 2009.

Conflicts of Interest : There are not conflicts of interest

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Morphological and Functional Changes in the Nociceptive System in Rat Models of Diabetic Peripheral Neuropathy –Contribution of TRP-channels?

Wolfgang Greffrath

Rolf-Detlef Treede, Dr med – Center of Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Bastian Timo Schlickenrieder, MA – Student, CBTM Mannheim, University Heidelberg

Uta Binzen

Background and Aims : Painful diabetic neuropathy (PDN), as one of the critical complications of diabetes type 1 and type 2, T1DM and T2DM, develops in about one quarter of patients. Diabetic neuropathy is characterized by the coexistence of positive signs like hyperalgesia or allodynia and negative signs like sensory loss. Thus, unfortunately, hyperglycaemic diabetic rodent models may display either hyperalgesic and/or hypoalgesic symptoms, and may also display mixed variable phenotypes. Thus, a plethora of sensory symptoms are observed in PDN varying in quality, intensity, modality and/or with time. We now investigated participation of different nociceptive membrane channels, such as thermo TRPs and purinoreceptors for the development of PDN induced by advanced glycation end products (AGEs) in course of T1DM and T2DM.

Methods : The following diabetes animal models were investigated at different time points. Streptozotocin-injection was used to induce T1DM, i.e. hyperglycaemia with hypoinsulin­aemia, and rats (Wistar) were investigated after 8 or 14 weeks (n = 8, two individual groups); sham-treated animals of the same age served as controls. For T2DM, either Zucker Diabetic Fatty-rats (ZDF; n = 8) or ZSF-1 rats, a hybrid rat of a cross between ZDF rat and Spontaneous Hypertensive Heart Failure rat (n = 20), suffering from hyperglycaemia with hyperinsulin­aemia for 14 weeks or 5-6 months were investigated. The respective normoglycaemic animals served as controls, as well as insulin-resistant Zucker fatty rats (ZF; hyperinsulinaemia with normoglycaemia) and their lean controls.

A novel heat pain test using laser-heat stimulation generated by a diode laser (wavelength 1475 nm) was developed to determine radiant heat pain threshold and used in T1DM (n=16) and T2DM (n=28). Animals were placed either on a quarz glass plate or on the grid of a platform (Ugo Basile) to measure mechanical pain thresholds at the glabrous skin of the paws and, after acclimatization of 10 minutes tested using method of limits: laser pulses were applied with in- and decreasing laser intensities (duration 200 ms), and average of 5 supra- and subthreshold stimuli as indicated by nociceptive behavior (licking, head movement, limb flipping) were calculated. A first test series included STZ rats examined once a week over 8 weeks for heat pain thresholds, only. For comparison, all values were normalized to the individual baseline before STZ injection. ZSF-1 rats suffering from diabetic conditions and respective controls were investigated once after 5-6 months to evaluate differences in laser heat pain thresholds, additionally mechanical pain thresholds were determined with an electronic von Frey.

At the end of the observation period, animals were sacrificed and dorsal root ganglia cells (DRGs) were obtained from all animals. RNA content was screened using microarray analysis (n=24), expression of different nociceptive receptor proteins was investigated in tissue slices using immunohistochemistry (n=18; TRPV1-AB, Alomone; TRPV2-AB/VRL-1-AB by Thermo Scientific, P2X3-AB, Neuromics).

Results : Laser heat pain threshold was significantly lower after 6 month of T2DM (ZSF-1 37.35 ± 1.35 vs. CTR 46.48 ± 3.73 mW; p < 0.01; Student’s T-Test). In contrast, mechanical pain thresholds did not differ significantly in T2DM (ZSF-1 57.15 ± 4.10 vs. CTR 47.72 ± 6.42 g; n.s.). In contrast, only slight changes in heat pain thresholds, if any, were observed during the first 8 weeks of development of T1DM. Repeated laser testing leads to a decrease in heat pain threshold after 8 weeks especially in control animals (Baseline 2.00 vs. CTR 1.884 ± 0.028; log transformation of normalized values mean week 7-8; p< 0.001 students T-test). The T1DM-model displayed only a slight decrease but significantly higher thresholds then their controls(CTR vs. STZ 1.948 ± 0.067; p< 0.05). Microarray analysis displayed no difference in TRPV1- or P2X3 –RNA levels in T2DM for 14 weeks (ZDF). T1DM animals showed a slight downregulation of TRPV1 RNA by 16% (fold change to 0.84 that just hit significance, p=0.05, students T-Test). ZF rats revealed an upregulation of RNA by 22% (p≤0.05; students T-Test). However, expression profiles of TRPV1 were not affected in T1DM or in T2DM nor in the normoglycaemic ZF rats as compared to the corresponding controls (STZ 37.9 ± 3.07% vs. 41.6 ± 3.65% n.s., ZDF 47.6 ± 3.0% vs. 46.5 ± 2.75%, n.s. or ZF rats 36.6 ± 3.13% vs. 37.29 ± 1.8%, n.s.), but P2X3 expressing cells were more present in Pre-diabetic and T1DM and reduced in T2DM (all p< 0.05).

Conclusions : In course of T2DM for 6 month a thermal heat hyperalgesia developed whereas mechanical pain remained unaffected. However, at least after 14 weeks disease duration, no corresponding morphological changes were detected within nociceptive neurons that may explain this pain phenotype making investigations at later disease progression essential.

Only slight morphological and behavioral changes were found after 14 weeks suffering from T1DM (slight heat hypoalgesia and reduction in TRPV1 RNA but not protein). Thus, T1DM may either need longer disease progression and/or may not affect thermo TRP channels as pathophysiological targets.

References : -

Conflicts of Interest : No conflict of interests.

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Metformin prevents the development of Oxaliplatin-Induced Peripheral Neuropathy in a Rat Model

Bruno Nervi

Felipe Court

margarita calvo, MD, MSc, PhD – Associate Professor, Pontificia Universidad Catolica de Chile

Background and Aims :
Colorectal cancer is the third most common cancer worldwide. Chemotherapy is widely used as it increases survival. Oxaliplatin, a platinum derivative, extents the rates of disease free survival by 20%. However, its clinical use commonly induces a disabling form of neuropathy. The rate of patients reporting chronic Oxaliplatin-induced peripheral neuropathy (OIPN) is 64-97% with at least 12% having severe neuropathy. Clinical data indicates that more than 60% of patients reduce or discontinue Oxaliplatin due to this side effect. OIPN can be unpredictable: while symptoms may resolve after chemotherapy is discontinued, they can also continue for years. Various strategies have been proposed to prevent or treat OIPN without success.

In this study we aimed to test if the anti-diabetic drug Metformin can prevent the development of OIPN in a rat model.

Methods :
Sprague Dawley rats (250 grs) were injected intraperitoneally with Oxaliplatin 4 mg/Kg in two consecutive days twice weekly, for 4 weeks. Metformin (250mg/Kg) was injected 6 hours before Oxaliplatin, using the same scheme.

Results : Results
1.Metformin prevents Oxaliplatin-induced intraepidermal fiber loss

2.Metformin pre-treatment prevents OIPN-induced astrocytic activation in the spinal cord

3.Cold, heat and mechanical hypersensitivity seen in OIPN are prevented by pre-treatment with metformin

Behavioural studies: Rats were treated with Oxaliplatin (4mg/Kg) and/or Metformin (250 mg/Kg) as indicated in experimental model (Fig.1A) .A. Cold Hiperalgesia was assayed as the duration of withdrawal response after acetone was applied in plantar surface. Oxaliplatin induced an increase in withdrawal response duration by days 22, 25 and 29 after treatment starting. Metformin inhibited oxaliplatin induced withdrawal response duration increase. B. Mechanical hypersensitivity was assessed using Von Frey filaments. Oxaliplatin induced a decrease in the mechanical withdrawal threshold at days 15, 18 and 25 after starting treatment. Metformin pre treatment inhibited oxaliplatin chemotherapy induced threshold decrease. C. Thermal hypersensitivity was assayed as the withdrawal latency in response to focal temperature increase (Haegreves test). Oxaliplatin induced an increase in withdrawal response latency by days 23, 26 and 30 after starting treatment . Metformin prevented thermal hypersensitivity. (n=10) *p< 0.05, Two Way ANOVA, SEM.

Conclusions :
Metformin is a safe drug widely used as an hypoglycemic that has FDA approval. Here we show that it can prevent Oxaliplatin induced peripheral neuropathy in a rat model of the disease. As OIPN is a common and serious problem that prevents cancer patients from receiving this life-saving drug, this strategy of neuropathy prevention with metformin offers a great opportunity to complete chemotherapy.

References : 1. Kolb, N.A., et al., The Association of Chemotherapy-Induced Peripheral Neuropathy Symptoms and the Risk of Falling. JAMA neurology, 2016. 73: p. 860-866.
2. Balayssac, D., et al., Chemotherapy-induced peripheral neuropathies: from clinical relevance to preclinical evidence. Expert Opinion On Drug Safety, 2011. 10: p. 407-17.
3. Mao-Ying, Q.L., et al., The anti-diabetic drug metformin protects against chemotherapy-induced peripheral neuropathy in a mouse model. PLoS One, 2014. 9(6): p. e100701.

Conflicts of Interest : none

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Combination drug therapy using duloxetine and ambroxol – a potential novel treatment option for oxaliplatin-induced peripheral neuropathy

Sałat Kinga

Anna Furgała, MSc – PhD student in Jagiellonian University , Chair of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University, Medical College

Background and Aims : Background: Peripheral neuropathy is one of the main reasons for the discontinuation of anti-cancer therapy or chemotherapeutic agent dose reduction. Chemotherapy-induced peripheral neuropathy (CIPN) is currently only partially reversible, and in some cases it might be an irreversible condition. The mechanisms responsible for the development of CIPN are not fully understood (Addington and Freimer, 2016).

Duloxetine is one of drugs recommended as the first-line treatment for neuropathic pain accompanying CIPN. Duloxetine is a serotonin and norepinephrine re-uptake inhibitor which leads to increases activity of these neurotransmitters and subsequently reduces the pain perception by modulating nociceptive signals in central nervous system (Roy et al., 2017). Ambroxol, is a voltage-gated sodium channel (Nav) inhibitor with anti-neuropathic pain properties shown previously in a mouse model of oxaliplatin-induced peripheral neuropathy (Furgała et al., 2018).

Methods : Materials and methods: The present study investigated the activity of Duloxetine administered intraperitoneally at two doses: 10 mg/kg and 30 mg/kg and Ambroxol used at doses 90 mg/kg and 150 mg/kg. Both these drugs were used alone or in combination, as single-dose or repeated-dose administered simultaneously or in a time-shifted manner (4h, 6h, 12h between each drug tested). To induce CIPN, oxaliplatin was administered intraperitoneally at a dose of 10 mg/kg , 3 h before pain tests. In order to evaluate thermal and mechanical allodynia, the cold plate test and von Frey test were performed on the day of oxaliplatin administration and then, 7 days later. Additionally, the influence on locomotor activity and motor coordination was also evaluated.

Results : Results: In this experiment duloxetine significantly alleviated both thermal and mechanical allodynia in mice. Chronic administration of duloxetine prevented the development of the late phase of neuropathy. Also Ambroxol reduced both cold and tactile allodynia. Duloxetine and amboxol used as combination drug therapy showed a significant antiallodynic effect which was particularly strong when these drugs were administered 4h and 6h apart.

Conclusions : Conclusions: Taken together, these observations suggest that duloxetine and ambroxol have significant potential for the prevention and treatment of tactile and thermal allodynia caused by chemotherapeutic agents.

References : Addington, J., Freimer, M., 2016. Chemotherapy-induced peripheral neuropathy: an update on the current understanding. F1000Research 5, 1466.
Furgała, A., Fijałkowski, Ł., Nowaczyk, A., Sałat, R., Sałat, K., 2018. Time-shifted co-administration of sub-analgesic doses of ambroxol and pregabalin attenuates oxaliplatin-induced cold allodynia in mice. Biomed. Pharmacother. 106, 930–940.
Roy, M.K., Kuriakose, A.S., Varma, S.K., Jacob, L.A., Beegum, N.J., 2017. A study on comparative efficacy and cost effectiveness of Pregabalin and Duloxetine used in diabetic neuropathic pain. Diabetes Metab. Syndr. Clin. Res. Rev. 11, 31–35.

Conflicts of Interest : None declared

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Efficacy assessment of single oral bolus doses of duloxetine in a rat model of CIPN

Maree T. Smith, PhD – Director, CIPDD, School of Biomedical Sciences, The University of Queensland

Andy Kuo

Janet Nicholson

Laura Corradini

Background and Aims : Patients with chemotherapy induced peripheral neuropathy (CIPN) report one or more sensory symptoms including allodynia (pain evoked by a non-noxious stimulus such as light pressure or touch), hyperalgesia (exaggerated pain response to a noxious stimulus), hypoalgesia (numbness), tingling or electric-shock like shooting pains. CIPN develops in a 'stocking and glove' distribution due to the vulnerability of the long nerves. For the pharmacological treatment of CIPN, the antidepressant, duloxetine, is recommended although it has modest efficacy (1,2). Hence, this study was undertaken to assess the efficacy of single bolus doses of duloxetine in our rat model of cisplatin-induced CIPN as a means to 'back-translate' our model from the clinical setting.

Methods : Ethics approval was from The University of Queensland's Animal Ethics Committee and the experiments were conducted in accordance with the requirements of the National Health and Medical Research Council’s Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male Sprague-Dawley rats (180-200 g) were from the Animal Resources Centre (Perth, WA, Australia) and were housed in groups of two to three per cage in a temperature-controlled room (21°C ± 2°C) with a 12 h/12 h light-dark cycle. Environmental enrichment comprised rat chew sticks, Kimwipes and rodent hutches in all home cages. Rats were acclimatised for at least 3 days before initiation of experimentation. Von Frey paw withdrawal thresholds (PWTs) and paw pressure thresholds (PPTs) were assessed in the bilateral hindpaws prior to the induction of CIPN.

The cisplatin dosing regimen comprised four doses at 3 mg/kg administered at once-weekly intervals. This dosing regimen was initiated once the body weights of animals were in the range, 200-240 g. In brief, rats were administered a subcutaneous injection of 2 mL of sterile saline at ~5 min before each cisplatin injection to prevent renal damage. Rats then received a single intraperitoneal bolus dose of cisplatin on days 0, 7, 14 and 21 to give a cumulative cisplatin dose of 12 mg/kg. Animal body weights, body temperatures, haematocrit levels and urinalysis were recorded during the interval ~ day -2 to day 27. Temporal development of mechanical allodynia and mechanical hyperalgesia in the bilateral hindpaws were documented using von Frey filaments and the Randall Selitto test, respectively. Mechanical allodynia in the bilateral hindpaws was regarded as fully developed if PWTs were ≤6g and mechanical hyperalgesia was regarded as fully developed if PPTs were ≤80g.

Commencing on day 28 post-initiation of the cisplatin dosing regimen, rats with fully developed mechanical hypersensitivity in the bilateral hindpaws were randomized to receive a single oral bolus dose of duloxetine or vehicle in a blinded manner according to a ‘washout’ protocol with at least 2 days between successive doses. Vehicle was 0.5% natrosol with 0.01% Tween 80.

Statistical comparisons between treatment groups were performed using 1-way ANOVA followed by the Dunnett’s test. The statistical significance criterion was P≤0.05.

Results : Animal body weights increased steadily over the course of cisplatin-treatments indicative of good animal health. Body temperatures were maintained within the normal range (37-38oC), haematocrits were within the normal physiological values (34-57%) and urinalysis revealed no sign of renal impairment.

Single oral bolus doses of duloxetine at 3, 10, 30 and 100 mg/kg evoked dose-dependent relief of mechanical hyperalgesia in the bilateral hindpaws. By contrast, the same doses of duloxetine, like vehicle, were inactive for the relief of mechanical allodynia in the bilateral hindpaws.

Conclusions : Our findings show that orally administered bolus doses of duloxetine were efficacious for the relief of mechanical hyperalgesia, but not allodynia, in a rat model of CIPN. This profile is distinct from pregabalin which was efficacious for the relief of both mechanical allodynia and mechanical hyperalgesia in the bilateral hindpaws of the same model.

References : 1. Smith EML, Pang H, Cirrincione C, et al. Effect of duloxetine on pain, function, and quality of life among patients with chemotherapy-induced painful peripheral neuropathy: a randomized clinical trial. JAMA 2013;309(13):1359-1367. doi: 1310.1001/jama.2013.2813
2. Hirayama Y, Ishitani K, Sato Y, et al. Effect of duloxetine in Japanese patients with chemotherapy-induced peripheral neuropathy: a pilot randomized trial. Int J Clin Oncol. 2015;20(5):866-871.

Conflicts of Interest : JRN and LC are paid employees of Boehringer Ingelheim Pharma GmbH & Co. KG

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Chemotherapy treatment reduced ex vivo neurite outgrowth of dorsal root ganglion explants

Justin G. Lees

David Goldstein

Lital Livni – PhD candidate, University of New South Wales, Sydney

Gila Moalem-Taylor

Mallory E. Barkl-Luke

Background and Aims : Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting adverse effect of cancer therapy that results from treatment with neurotoxic agents. One of the hallmarks of CIPN is the degeneration of long peripheral axons required for transmission of sensory information. Although chemotherapy treatment has been shown to inhibit neurite outgrowth from dorsal root ganglion (DRG) neurons in vitro, evidence for this effect in vivo is lacking. Therefore, the aim of this study was to investigate whether chemotherapy treatment in mice alters the capacity for axonal outgrowth from ex vivo cultured DRG explants.

Methods : DRGs were harvested from adult male Balb/cJ mice. DRG explants were either treated in vitro with paclitaxel (PTX) or oxaliplatin (OXA) or were derived from mice treated intraperitoneally with 6 cycles of PTX or 12 cycles of OXA (cumulative dose of 30mg/kg) and dissected at day 30 or 90 following treatment. Ex vivo DRGs were cultured for 72 h before fixation, immunostaining with the neuronal marker anti-BIII tubulin and image acquisition. The images were analysed to assess the extent of axonal outgrowth.

Results : DRG explants isolated at day 30 from mice treated with PTX and OXA showed a significant reduction in neurite outgrowth as compared to DRG explants from control vehicle-treated mice. DRGs that were isolated at day 90 showed recovery of the neurite outgrowth, and no significant differences were detected in comparison to vehicle controls. These results are corroborated with an in vitro model, whereby direct application of both PTX and OXA to DRG explants significantly inhibited axonal outgrowth in a dose-dependent manner.

Conclusions : Our results show that the effect of PTX and OXA on the structural plasticity of DRG is retained ex vivo, for at least 30 days following chemotherapy. Our findings support the use of DRG explants for testing chemotherapy-induced neurotoxicity and screening for potential therapeutic targets or neuroprotective agents.

References : N/A

Conflicts of Interest : None

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Effect of Nerve Resection on Ectopic Neuropathic Discharge

Michael Tal, DMD, MS – Dept. Of Medical Neurobiology, School of Medicine

Background and Aims : Ectopic discharge in damaged afferent axons is a major contributor to chronic neuropathic pain. Clinical experience shows that surgical re-section of nerves (just) proximal to the original injury site is frequently ineffective at eliminating pain for more than a short period and may even exacerbate the pain. The usual explanation is that soon after resection a new neuroma forms, and with it renewed ectopic discharge. We have tested this concept.

Methods : Teased fiber recordings were made of ectopic spontaneous discharge (“ectopia”) originating in the experimental nerve-end neuroma and associated dorsal root ganglia (DRGs) in rats that underwent either a single transection (with ligation) of the sciatic nerve, or two consecutive transections. The intensity of discharge (number of spontaneously active axons per teased microfilament) and the discharge pattern was assessed. The second transection was made 7, 14, 21 or 30 days after the first.

Results : The intensity of ectopia originating in the neuroma following a single cut followed its expected pattern of early intense discharge lasting for up to about 2 weeks followed by decline in the 3rd week to a low, steady level. When neuromas of 14 or 25 days standing were resected and recordings were made 7 days later, activity was low, as expected for neuromas of 21 and 32 days standing. We did not observe resurgence of activity to the high levels typical of 7-day neuromas. Extending the interval between the first and the second cut, or between the second cut and the recording, did not lead to increased firing intensity. Ectopia originating in the DRG also failed to show noticeable resurgence after nerve resection.

Conclusions : Our results do not support the idea that resurgent ectopia follows resection of previously injured peripheral nerves, at least not within the time parameters of our experiment.

References : Non

Conflicts of Interest : No conflict of interest.

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Characterisation of Two Kv1.6 Channel Knock-out Mice: Inclusion of a LacZ (E.coli β-galactosidase) Cassette as a Gene Knock-out Strategy May Yield Confounding Effects Including Primary Afferent Neurotoxicity

Anthony Dickenson

John Dawes – University of Oxford

Andrew Todd

Ryan Patel

Liam Peck – University of Oxford

David L. Bennett, MB, PhD, FRCP – Professor of Neurology and Neurobiology, Neural Injury Group, Nuffield Department of Clinical Neurosciences, University of Oxford

Background and Aims : Kv1.6 is a member of the ‘Shaker­-like’ Kv1 family of voltage-gated potassium channels (VGKCs), which are known to impose a ‘brake’ on neuronal excitation via outward delayed rectifying currents1. Genetic and immune-mediated loss of function in these channels and their interacting complex proteins such as Caspr2 is associated with neuronal hyperexcitability and is implicated in the pathophysiology of neuropathic pain2. Kv1.6 in particular is required for restoration towards normal mechanical sensitivity thresholds after axotomy-induced hypersensitivity3. Here, we describe the behavioural, anatomical and physiological characterisation of two Kv1.6 knock-out mice: one produced by homologous recombination with an E.coli LacZ reporter cassette (Kcna6-tm1), and a second produced via Crispr/Cas9 endonuclease-mediated deletion with no exogenous reporter (Kcna6-em1). Replacement of endogenous alleles with a LacZ selection cassette has historically been a common approach to generate knock-out animals or report gene expression, yet little attention has been paid to downstream consequences of expressing exogenous β-galactosidase.

We aim to assess the contribution of the Kv1.6 subunit to somatosensory and nociceptive function in naïve animals. In doing so we have also identified issues regarding LacZ expression in primary afferent neurons and seek to clarify untoward effects of this selection cassette.

Methods : Kcna6-tm1Lex/Mmucd and Kcna6-em1(IMPC)J/Mmjax mice are both loss of function knock-out models of the Kv1.6 (Kcna6) alpha subunit. Adult (8+wks) male and female mice were used indiscriminately throughout. We identified Kcna6 expression directly using the RNAscope® in situ hybridisation assay (ACD, Inc.) or indirectly using immunohistochemical staining for the Kcna6 surrogate reporter β-galactosidase in Kcna6-tm1 animals, all performed on 4% PFA-fixed tissue. We assessed sensorimotor and pain-related behaviour in both Kcna6-tm1 and -em1 knock-out mice. Calcium imaging experiments were conducted on dissociated adult dorsal root ganglia (DRG) cells after 1 day in vitro. We acquired dorsal horn recordings using an extracellular microelectrode in adult Kcna6-tm1 mice following laminectomy to expose the dorsal lumbar spinal cord under isoflurane-induced anaesthesia.

Results : Kcna6 mRNA is expressed in primary afferent cells of the lumbar DRG, with significantly elevated expression in small neurons (p< 0.05 for < 500µm2 vs. >1000µm2), in particular peptidergic nociceptors expressing calcitonin gene-related peptide (CGRP), a known thermosensitive neuronal population. We also identified expression in satellite glial cells. Kcna6-tm1 homozygotes are significantly hyposensitive to hot plate (50°C and 53°C, p< 0.0001) and Hargreaves (p=0.0001) assays; a similar phenotype was seen in heterozygotes (p=0.0002) on the Hargreaves assay only. Responses to mechanical stimuli were not significantly different; motor function and exploratory behaviour were also normal. Intraepidermal nerve fibre density is comparable across genotypes, and we find that dissociated DRG neurons from wild types and knock-outs alike demonstrate thermosensitivity in the 25-45°C range in vitro. In vivo extracellular electrophysiological recording from wide-dynamic range (WDR) neurons of the dorsal horn reveals significantly reduced activity in response to noxious thermal (48°C) and mechanical (15g von Frey) stimulation, reflecting reduced nociceptive transmission from primary afferents to the spinal cord. Upon anatomical analysis in the superficial dorsal horn, the site of nociceptor termination, we observed large (≤35µm diameter) pathological swelling of CGRP- and IB4-positive terminals in hetero- and homozygous Kcna6-tm1 tissue, which are also demarcated by positive staining for the LacZ product β-galactosidase. EM imaging identified gross accumulation of lipid droplets and autophagosome-like structures in these synapse-forming terminals. However, Kcna6-em1 mice that also lack Kv1.6 but do not express LacZ exhibit no such neurodegeneration.

Conclusions : We reveal expression of Kcna6 in DRG neurons (in particular small-diameter nociceptors) and satellite glia, as well as in spinal cord cell populations. Kcna6-tm1 mice are markedly hyposensitive to noxious heat, despite having normal intraepidermal nerve fibre density and DRG neuron responses to noxious temperatures in vitro. A physiological correlate of the behavioural hyposensitivity was found in reduced WDR responses to noxious stimuli. However, these LacZ-expressing Kcna6 knock-outs exhibit gene-dose dependent degeneration of central nociceptor terminals in the superficial dorsal horn (laminae I and II), which is absent from Kcna6-em1 mice. We reason that the inclusion of a LacZ construct commonly employed in mouse knock-out genetics has neurotoxic effects in IB4 and CGRP nociceptors on a Kcna6-null background, potentially exacerbating or masking the underlying phenotype of the knock-out mouse.

References : 1. Hao, J. et al. Kv1.1 channels act as mechanical brake in the senses of touch and pain. Neuron 77, 899–914 (2013).
2. Dawes, J. M. et al. Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability. Neuron 97, 806–822 (2018).
3. Calvo, M. et al. Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury. Elife 5, 1–26 (2016).

Conflicts of Interest : None

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A spontaneous model of central neuropathic pain – canine syringomyelia

Maria Soendergaard Thoefner, DVM – PhD-fellow, University of Copenhagen, Faculty of Health and Medical Sciences, Department of Veterinary Clinical Sciences

Jens Randel Nyengaard

Mette Berendt

Ole Jannik Bjerrum

Troels Staehelin Jensen

Joergen Steen Agerholm

Background and Aims : Central neuropathic pain (CNeP) occurs spontaneous and with a prevalence of 15% in the canine breed Cavalier King Charles spaniel with cervical syringomyelia and Chiari-like malformation (1). Common clinical signs consistent with CNeP are spontaneous or evoked unilateral scratching directed towards the neck region, avoidance of physical contact and paroxysmal pain manifestations e.g. spontaneous vocalisation when the dog is physical active or when it is touched (2-4). A correlation between clinical signs and magnetic resonance imaging (MRI) findings including syrinx dimension, syrinx:spinal cord ratio and the degree of syrinx asymmetry has been reported (1, 5). We hypothesise that Cavalier King Charles spaniels with syringomyelia-related signs of CNeP represent a spontaneous model of CNeP. As a constituent of the characterisation of this translational model, we aimed to characterise and quantify the neurohistopathological changes in the cervical spinal cord. Additional aims were to investigate the relationship between clinical signs consistent with CNeP and structural damage at the cellular level and to investigate if the spinothalamic tract is involved in CNeP generation in affected dogs.

Methods : Private owned Cavalier King Charles spaniels with a well-characterised pain phenotype and MRI-confirmed syringomyelia (n=8) and asymptomatic controls (n=4) were included. The dogs were donated by their owners after euthanasia. Spinal cord segments C1-C8 were sampled within two hours post mortem, followed by a 48-hour formalin fixation before processing and paraffin embedding. Serial 30 mm sections were stained with haematoxylin-eosin, luxol fast-blue (myelin) and thionine (nuclei) for neurohistopathological characterisation. 10 mm sections were stained with a neurofilament triplet H protein-specific primary antibody (SMI-32) to delineate the dorsal horns’ laminae I-III as a representation of spinothalamic neurons after systematic random sampling. The sampling fraction was 1:2. Volumes of the central canal, syrinx - if present, left and right dorsal horns’ laminae I-III, the remaining left and right grey matter and dorsal, lateral and ventral white matter columns were estimated using the 2D nucleator and the Cavalieri estimator. Data analysis including parametric comparisons (paired, two-tailed t-tests) of estimated volumes were undertaken in SAS Studio 3.71 (SAS Institute Inc.; NC, USA). P < 0.05 was considered significant.

Results : Syringomyelia most frequently occurred within segments C2-C4 in 66% of the cases. Common neurohistopathological findings were: neuronal loss, primarily affecting the dorsal horns, focal white matter spongiosis and grey matter interstitial oedema. In five of eight cases (63%), the cranial origin of the syrinx did not communicate with the central canal. In cases with unilateral clinical signs, prominent lesions in the dorsal root entry zone were seen. The histopathological changes were characterised by deafferentation and reorganization of first-order neurons terminating in deeper grey matter laminae. Comparisons of bilateral volumes of dorsal horn grey matter, ventral horn grey matter, dorsal, lateral and ventral white matter columns were done in cases with unilateral clinical signs. A significant dorsal horn volume loss was found on the affected side to which the symptoms were ascribed compared to the non-affected side (P=0.034). In the spinal cord segment most significantly affected by syringomyelia, the mean reduction in dorsal horn area on the affected side was 56% (range 8% - 95%) compared to the contralateral, non-affected dorsal horn area. The remaining comparisons revealed non-significant differences between affected and non-affected sides. No histopathological abnormalities or volume loss was found in the asymptomatic controls' spinal cords.

Conclusions : Syringomyelia in Cavalier King Charles spaniels with clinical signs of CNeP primarily affected the spinal cord grey matter. The loss of spinothalamic dorsal horn neurons is a neurohistopathological characteristic shared with human syringomyelia patients. Despite localised white matter vacuolisation, a quantifiable spinothalamic fiber damage was absent; the volume of white matter was consistent throughout the spinal cord of affected dogs. The histopathological changes in the dorsal root entry zone have not previously been reported in neither canine nor human CNeP patients. Whether the dorsal root entry zone pathology is a result of spinothalamic neuronal loss or vice versa could not be determined in the present study, since the dorsal roots and ganglions were not systematically sampled.The neurohistopathological findings confirm the back- and forward translational potential of this spontaneous model to fill the gap between induced rodent models and human patients. In addition we have previously presented a randomised controlled trial with strong evidence that pregabalin significantly alleviates clinical signs consistent with syringomyelia-related CNeP. Our results confirm the translational potential, strengthen the applicability of the model and enable the search for novel underlying pain mechanisms and treatment targets. Thus, Cavalier King Charles spaniels with syringomyelia represent a relevant, spontaneous translational CNeP model.

References : 1. Thofner MS, Stougaard CL, Westrup U, Madry AA, Knudsen CS, Berg H, et al. Prevalence and heritability of symptomatic syringomyelia in Cavalier King Charles Spaniels and long-term outcome in symptomatic and asymptomatic littermates. J Vet Intern Med. 2015;29(1):243-50.
2. Rusbridge C, MacSweeny JE, Davies JV, Chandler K, Fitzmaurice SN, Dennis R, et al. Syringohydromyelia in Cavalier King Charles spaniels. Journal of the American Animal Hospital Association. 2000;36(1):34-41.
3. Sanchis-Mora S, Pelligand L, Thomas CL, Volk HA, Abeyesinghe SM, Brodbelt DC, et al. Dogs attending primary-care practice in England with clinical signs suggestive of Chiari-like malformation/syringomyelia. The Veterinary record. 2016;179(17):436.
4. Sparks CR, Cerda-Gonzalez S, Griffith EH, Lascelles BDX, Olby NJ. Questionnaire-based Analysis of Owner-reported Scratching and Pain Signs in Cavalier King Charles Spaniels Screened for Chiari-like Malformation and Syringomyelia. J Vet Intern Med. 2018;32(1):331-9.
5. Rusbridge C, Carruthers H, Dube MP, Holmes M, Jeffery ND. Syringomyelia in cavalier King Charles spaniels: the relationship between syrinx dimensions and pain. The Journal of small animal practice. 2007;48(8):432-6.

Conflicts of Interest : The author declares no conflict of interests

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Candesartan prevents mechanical allodynia induced by vincristine but not mechanical allodynia induced by oxaliplatine in mice

Claire Demiot

Hichem Bouchenaki, PharmD – PhD student, EA6309 Faculties of Medicine and Pharmacy, Limoges

Franck Sturtz

Alexis Desmoulière

Laurence Richard

Flavien Bessaguet

Laurent Magy

Aurore Danigo, PhD – EA6309, Faculties of Medecine and Pharmacy of Limoges

Background and Aims : Neuropathic pain is the major dose-limiting effect of frequently-used chemotherapeutic agents such as vincristine (VCR) or oxaliplatine (OXP). We recently demonstrated that candesartan, an angiotensin II type 1 receptor antagonist, was neuroprotective against resiniferatoxin-induced sensory neuropathy and that this effect is mediated by stimulation of the angiotensin II type 2 receptor (AT2R). Thus, we chose to evaluate the effect of a preventive treatment by candesartan on mouse models of sensory neuropathy induced by VCR or by OXP.

Methods : VCR (100 µg/kg, intraperitoneally (i.p.)) was administered once per day for 7 days and OXP (15mg/kg, i.p.) was administered by a single injection in male Swiss mice. Treatments with candesartan (0.5 mg/kg, i.p.) were started at day 1 before administration of the chemotherapeutic agent, then until day 7. Development of VCR/OXP-induced peripheral neuropathy and effect of treatments were evaluated by functional tests.

Results : Mice treated with VCR showed high mechanical allodynia but no modifications of motor performance or mechanical/thermal nociception. Mice treated with OXP showed mechanical allodynia and cold allodynia/hyperalgesia but no modifications of motor performance. While candesartan totally restores tactile sensitivity in VCR mice, its treatment has no effect on mechanical allodynia in OXP mice. Cold allodynia and cold hyperalgesia were partially prevented by candesartan in OXP mice during the first days following the injection of OXP.

Conclusions : Candesartan prevents mechanical allodynia induced by VCR but not mechanical allodynia induced by OXP. Moreover, candesartan seems to partially prevent cold allodynia and cold hyperalgesia induced by OXP. Our finding encourages evaluation of its therapeutic potential in neuropathies induced by other chemotherapeutic agents. Further investigations of differential mechanisms of chemotherapy-induced neurotoxicity is required for the discovery of new targets to tackle chemotherapy-induced neuropathic pain.

References : Bessaguet F, Danigo A , Bouchenaki H, Duchesne M, Magy L, Richard L, Sturtz F, Desmoulière A, Demiot C. (2018) Neuroprotective effect of angiotensin II type 2 receptor stimulation in vincristine-induced mechanical allodynia. Pain 159(12):2538-2546.

Conflicts of Interest : Authors declare no conflicts of interest

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Diffuse noxious inhibitory controls in healthy dogs and those with naturally-occurring neuropathic pain treated with gabapentin alone or with meloxicam

Guy Beauchamp

Marina Evangelista

Ryota Watanabe

Paulo Steagall

Hélène Ruel, DMV, DES, MSc, DACVIM (Neurology) – PhD candidate, Université de Montréal

Background and Aims : Neuropathic pain is poorly understood and undertreated in companion animals. This study aimed to evaluate the diffuse noxious inhibitory controls (DNIC) in healthy dogs and those with neuropathic pain treated with gabapentin alone (G) or with meloxicam (GM).

Methods : Twenty-nine client-owned dogs with naturally-occurring neuropathic pain were included after magnetic resonance imaging. Dogs were randomly allocated to receive one of two treatments during 21 days: G/placebo/GM or GM/placebo/G. Duration of G, placebo or GM was 7 days. Mechanical nociceptive thresholds (MNT) were measured by applying an increasing pressure on the dorsal aspect of the carpus with an algometer until a behavioral response was observed, or the cut-off reached (20N). A conditioning stimulus was then applied using a blood pressure cuff (200 mmHg for 60 seconds) over the same thoracic limb. DNIC was assessed using the difference between MNT before and three minutes after the conditioning stimulus (delta MNT) at presentation, 7,14 and 21 days after treatment. Data were compared with 15 healthy dogs.1 The percentage of dogs with a sensory profile associated with hyperalgesia (negative values for delta MNT) was calculated.2 Statistical analysis included mixed linear models (p < 0.05).

Results : Delta MNT was greater in healthy than in dogs with neuropathic pain (mean ± SD: 2.1 ± 3.4N and -0.16 ± 3.3N, respectively; p = 0.036), but not significantly different after any treatment [-0.88 ± 2.4N (placebo), 0.74 ± 3.5N (G) and 0.54 ± 2.8N (GM)]. Hyperalgesia was observed in 33.3% in healthy controls, and 61.5% (at presentation), 34.6% (G), 53.8% (GM) and 63% (placebo) in dogs with neuropathic pain.

Conclusions : The magnitude of delta MNT was larger in healthy than in dogs with neuropathic pain, demonstrating a possible DNIC dysfunction in the latter group. Treatment with G re-established DNIC function in dogs with neuropathic pain.

References : 1. Ruel HL, Watanabe R, Evangelista MC, Beauchamps G, Steagall PV. Feasibility and reliability of electrical, mechanical and thermal nociceptive testing and assessment of diffuse noxious inhibitory control in dogs. J Pain Res. 2018 Oct 23;11:2491-2496.
2. Potvin S, Marchand S. Pain facilitation and pain inhibition during conditioned pain modulation in fibromyalgia and in healthy controls. Pain. 2016 Aug;157(8):1704-10.

Conflicts of Interest : Hélène Ruel is the recipient of the Mitacs Accelerate Program which is partly sponsored by Boehringer Ingelheim.

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Two Decades of Diode Laser-Based Selective Assessment of A-delta or C fiber Nociception: Application to Human Neuropathic Pain, Animal Models and Cultured Cells.

Mikhail Nemenov

David Yeomans

Michael Iadarola

Background and Aims : Microneurographic single fiber recording of single unmyelinated (C) or A-delta nociceptive fibers is a highly precise method for investigating pain mechanisms. Despite the clear utility of this approach, because of the complexity and technical difficulty of this method, only a few labs in the world have the capability to perform these studies in humans. In addition, the duration of these studies is usually limited to ~½ hour – which is a narrow time period for a systematic evaluation of the effect of therapeutic interventions or agents. The capacity for selective assessment of C-fiber or A-delta nociceptive activity would be highly desirable considering the differential contribution of the two fiber types to pain and analgesia. However, currently available behavioral tests in rodents, as well as quantitative sensory tests in humans (QST), are not fiber selective and both generally involve simultaneous activation of C- and A-delta fibers in unpredictable proportions. Thus, there is an unmet need for a technically less demanding and highly translatable, means of selective assessment of C- or A-delta nociceptive fibers in animals and humans.

Over the last 20 years, we have performed multiple experiments which demonstrate that diode laser radiation using a high (over 100 C/sec) or low (below 30 C/sec) rate of skin heating to selectively activate A-delta or C fiber thermonociceptors. Here we present a summary of these studies which we have performed in animals, humans, and single neurons using diode laser fiber-specific and selective (DLss) stimulation. The aim of this work was to contribute to our understanding of the mechanisms underlying different pain states, including painful peripheral neuropathies, as well as in the development of novel pain treatments.

Methods : DLss is based on 980 nm wavelength diode laser radiation, which provides relatively homogeneous skin heating that includes epidermis and dermis, which is in contrast to heating achieved with contact thermodes, radiant heat, or CO2laser stimulation (1, 10). DLss C-fiber type laser stimulation is achieved with the following characteristics; duration: 1 to 20 sec, beam diameter: 5 mm. A-delta fiber type stimulation is achieved with the following characteristics: duration: 50 to 200 msec, beam diameter: 1 mm to 2 mm. Activation of nociceptors in in vitrorecordings of TRPV1-expressing HEK cells or DRG neurons was defined as an inward ion current or first reproducible spike response in electrophysiological studies. In animal behavioral studies, nociceptor activation was defined by the latency of evoked muscle twitch or limb withdrawal. In humans, subjective sensation reports, fMRI and MEG imaging, and cortical laser evoked potentials all served to demonstrate nociceptor activation.

Results : In response to C-fiber DLss, volunteers and patients with neuropathic pain uniformly report single modality tonic burning pain – which is characteristic of C-fiber evoked pain. For A-delta DLss, volunteers and patients uniformly report only phasic pricking pain - which is characteristic of A-delta evoked pain. Consistent with these sensory reports, conduction velocity measurements based on sensory testing at two body sites were also attributable to selective activation of A-delta or C-fibers. Patients with painful peripheral neuropathy generally report cutaneous numbness and increased heat pain thresholds when measured using contact thermode-applied heat. However, pain thresholds measured with DLss showed fairly normal C-fiber pain thresholds, but also demonstrated significantly increasedA-delta pain thresholds. Thus, the sensory loss is fiber-type specific. Differential fiber activation is dependent on the slope of the skin heating ramp. Thus, the modality and threshold temperature of activation of A-delta or C-fibers depends on both intensity and stimulus duration, A-delta selective activation occurs with a high intensity ramp with a stimulus duration of between 20 to 300 msec. C-fiber selective activation requires a slower ramp with a stimulus duration of between 1 to 20 sec. In both cases, the threshold surface temperature, was measured using a high speed thermal camera.

Conclusions : Over the past 2 decades, we have developed and rigorously tested diode laser-based protocols for selective activation of A-delta or C-fiber nociceptors. The results of these experiments have shown remarkable consistency across multiple paradigms, including single cell electrophysiology, animal behavioral testing, human volunteers, and neuropathic pain patients (1-18). The studies presented demonstrate the utility of these methods in preclinical evaluation of novel pain treatments as well as clinical evaluation of patients with chemotherapy and diabetic painful neuropathies. The methods presented provide a non-invasive and relativity simple and translatable capability which can provide unique and critical information in our quest to improve neuropathic pain management and evaluate new analgesic agents.

References : 1. Greffrath W., Nemenov M. I., Schwarz S., Baumgärtner U., Vogel H., Arendt-Nielsen L., Treede R. D. Inward currents in primary nociceptive neurons of the rat and pain sensations in humans elicited by infrared diode laser pulses. Pain 2002; 99 (1-2): 145-55
2. Tzabazis, A., Klyukinov, M., Manering, N. A., Nemenov, M. I., Shafer, S. L., and Yeomans, D. C. Differential activation of trigeminal C or Aδ nociceptors by infrared diode laser in rats: behavioral evidence. Brain Res., 1037 (2005) 148-56
3. Jang N., Cooper B.Y., Nemenov M.I. Noninvasive diode laser activation of transient receptor potential proteins and nociceptors. Proc Soc Photo Opt Instrum Eng. 2007 Feb 21; 6428 hpd
4. Veldhuijzen D. S., Nemenov M. I., Keaser M., Zhuo J, Gullapalli R. P., Greenspan J. D. Differential brain activation associated with laser-evoked burning and pricking pain: an event-related fMRI study. Pain, 2009 141 1-2 104-13
5. Cuellar J. M., Manering N. A., Klukinov M., Nemenov M. I., Yeomans D. C. Thermal nociceptive properties of trigeminal afferent neurons in rats. Mol Pain. 2010; 6:39
6. Mitchell K, Bates B. D., Keller J. M., Lopez M., Scholl L., Navarro J., Madian N., Haspel G., Nemenov M. I., Iadarola M. J. Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates. Mol Pain. 2010; 6: 94
7. Tzabazis, A. Z., Klyukinov M., Crottaz-Herbette S., Nemenov M. I., Yeomans D. C. Selective Nociceptor Activation in Volunteers by Infrared Diode Laser. Mol Pain. 2011; 7: 18
8. Lebovitz EE, Keller JM, Kominsky H, Kaszas K, Maric D, Iadarola MJ. Positive allosteric modulation of TRPV1 as a novel analgesic mechanism. Mol Pain. 2012; 8:7
9. Zhang, J., Cavanaugh, D. J., Nemenov, M. I., Basbaum, A. I. The modality-specific contribution of peptidergic and non-peptidergic nociceptors is manifest at the level of dorsal horn nociresponsive neurons. Journal of Physiology –London. 2013; 591 (4): 1097-1110
10. Moeller-Bertram T., Schilling J. M., Backonja M. M., Nemenov M. I. Sensory Small Fiber Function Differentially Assessed with Diode Laser (DL) Quantitative Sensory Testing (QST) in Painful Neuropathy (PN). Pain Medicine 2013; 14 (3): 417-421.
11. Mitchell K., Lebovitz E. E., Keller J. M., Nemenov M.I., Mannes A.J., Iadarola M. Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation of A-delta and C-fibers after TRPV1gene knockout or resiniferatoxin lesion. Pain. 2014 Apr;155(4):733-45.
12. Brown JD, Saeed M, Do L, Braz J, Basbaum AI, Iadarola MJ, Wilson DM, Dillon WP. CT-guided injection of a TRPV1 agonist around dorsal root ganglia decreases pain transmission in swine. Sci Transl Med. 2015 Sep 16;7(305)
13. Blivis D, Haspel G, Mannes PZ, O'Donovan MJ, Iadarola MJ: Identification of a novel
spinal ociceptive-motor gate control circuit in rat following A pain stimuli. Elife May 24;6. pii: e23584, 2017
14. Raithel SJ, Sapio MR, Iadarola MJ, Mannes AJ: Thermal A-δ nociceptors, identifieby transcriptomics, express higher levels of anesthesia-sensitive receptors than thermal C-fibers and are more suppressible by low-dose isoflurane. Anesthesia and Analgesia Anesth Analg. Jul;127(1):263-266, 2018.
16. Raithel SJ, Sapio MR, LaPaglia DM, Iadarola MJ, Mannes AJ: Transcriptional changes in dorsal spinal cord persist after surgical incision despite preemptive analgesia with peripheral resiniferatoxin. Anesthesiology. 128(3):620-635, 2018

Conflicts of Interest : DR. Nemenov is co-owner LasMed LLC

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Decrease of tactile information processing in trigeminal system assessed with Functional Ultrasound co-occur with cool allodynia in oxaliplatin-induced polyneuropathy model

Antti Nurmi

Diana Miszczuk

Nicholas Moore

Artem Shatillo

Anna-Mari Zainana

Background and Aims : Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse effect of cancer chemotherapy with profound impact on quality of life. Symptoms of neuropathy are diverse, ranging from numbness to tingling, trigeminal neuropathy, hypersensitivity to touch and cold temperatures (Authier et al, 2009). Functional ultrasound (fUS) is a novel imaging technique that enables high sensitivity imaging of relative cerebral blood volume (rCBV) with unrivaled temporal and spatial resolution. Here, we validated the novel method to quantify oxaliplatin effect on tactile sensory processing in trigeminal system using fUS. To model platin-based chemotherapy in rodents we validated a sustained chronic model of oxaliplatin-induced polyneuropathy in mice, which suffer from cool/cold allodynia and deficits in trigeminal complex response to somatosensory stimulation.

Methods : Mice (C57BL/6J, male) were treated with oxaliplatin (4.5 mg/kg; i.p.) or vehicle at every 3 - 4 days for a period of 21 days. At the end of oxaliplatin challenge, mice were tested weekly for cool allodynia until 110 days from the model induction. Cool Tail Immersion test was performed by submerging the tail in +15 °C water, and measuring the latency of the tail withdrawal response. Acetone Cooling test was performed similarly during the follow-up period, by measurement of the duration of response to the cooling sensation evoked by acetone droplet applied on the dorsal side of both hind paws. On D22, mice underwent unilateral whiskers mechanical stimulation (4x30s intervals with 1 min of idle time in between). Response to stimulation was assessed with fUS using rCBV signal timeseries analysis from 200px (~2 mm3) ROI placed in the barrel field cortex. Pregabaline, an antiepileptic drug and duloxetine, a selective noradrenaline reuptake inhibitor (SNRI), were tested for their ability to alleviate the cool allodynia symptoms.

Results : Oxaliplatin challenge induced cool allodynia symptoms during and after the induction phase. Cool allodynia was displayed in tail immersion - and acetone tests persistently until 110 days. Pregabaline (20, 40 and 80 mg/kg) and duloxetine (1, 3 and 10 mg/kg) reversed the cool allodynia according to acetone test, while the results of tail immersion test were more variable. Trigeminal system response to somatosensory stimulation using fUS revealed significant reduction of response in oxaliplatin-treated animals compared to controls.

Conclusions : These data demonstrate the utility of oxaliplatin exposure as a model of chemotherapy-induced polyneuropathy, manifested as cool allodynia symptoms and as decreased trigeminal response to tactile sensory stimulation. Moreover, reversion of cool hypersensitivity by duloxetine and pregabaline further proved the utility of this model in preclinical testing of novel therapies for neuropathic symptoms.

References : Authier N, Balayssac D, Marchand F, Ling B, Zangarelli A, Descoeur J, Coudore F, Bourinet E, Eschalier A. (2009) Animal models of chemotherapy-evoked painful peripheral neuropathies. Neurotherapeutics. 6(4):620-9.

Conflicts of Interest : None, all authors are employees of Charles River Discovery Services

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