Studies on the role of NGF in arthritis-induced pain transmission using gait and weight bearing as outcome measures

Pain is one of the most common reasons for seeking healthcare, with approximately forty percent of those suffering from chronic pain having joint pain. Osteoarthritis is the most common cause of joint pain, but currently there are few treatments available. The search for new, effective pain treatment has been mostly unsuccessful, in spite of the discovery of mechanisms that are involved in the transmission of nociceptive signals from the periphery to the central nervous system where pain is experienced. This work focuses on the evaluation of rodent joint pain models, the behavioural manifestations of the injuries, and the possibility to detect treatment effects in these models. Three models have been evaluated in rats; intra-articular injection of carrageenan, Freund´s complete adjuvant (CFA), and monoiodoacetate (MIA) into one hind leg. In mice, two models have been evaluated; intra-articular injection of CFA, and the surgical model of anterior cruciate ligament transection (ACLT). Carrageenan injection resulted in an acute, robust inflammation, CFA injection caused a more long-lasting strong joint inflammation, and MIA injection resulted in an almost complete loss of joint cartilage after a few weeks. The model more resembling osteoarthritis was the surgical model, ACLT, which gave severe cartilage degeneration, osteophytes, and pathophysiological changes in synovia and ligaments. Gait and weight bearing during locomotion have been tested in all models. The degree of weight bearing reduction in the affected limb was largest in the CFA- and carrageenan-induced model, followed by the MIA model and least effect was seen in the ACLT surgical model. Thus the ACLT model was not possible to use for pharmacological evaluation of drugs, whereas carrageenan- and CFA-induced monoarthritis resulted in a big enough difference between animals with monoarthritis and those without, to test drugs commonly used for pain as well as those under investigation for effects on pain. Conventional pain relieving drugs such as non-stereoidal anti-inflammatory drugs (NSAIDs) and opioids were able to normalize effects on weight bearing caused by both the carrageenan- and the CFA-induced monoarthritis, as were treatments based on inhibiting the NGF-TrkA pathway; an anti-NGF antibody and two pan-Trk compounds. However, an antagonist of the TRPV1 receptor lacked effect. We also investigated mice with a mutation in the R100 NGFß gene (hR100E), in comparison with mice possessing a human wild-type NGF (hWT), similar but not exactly like the one found in a hereditary sensory and autonomic neuropathy type V (HSAN V) disorder. This disorder leads to insensitivity to deep pain in homozygous patients, with sensory and autonomic functions remaining almost normal. In mice with the hR100E mutation, we found similar behavioural outcome; normal peripheral sensory functions but less pain-like behaviour when assessing joint pain with gait and weight bearing. In summary, this work shows that in order to detect translatable effects on joint pain, models need to be robust enough, especially for pharmacological testing, but more important, the methods of testing need to be relevant for the study aim

The search for translational pain outcomes to refine analgesic development: Where did we come from and where are we going?

Pain measures traditionally used in rodents record mere reflexes evoked by sensory stimuli; the results thus may not fully reflect the human pain phenotype. Alterations in physical and emotional functioning, pain-depressed behaviors and facial pain expressions were recently proposed as additional pain outcomes to provide a more accurate measure of clinical pain in rodents, and hence to potentially enhance analgesic drug development. We aimed to review how preclinical pain assessment has evolved since the development of the tail flick test in 1941, with a particular focus on a critical analysis of some nonstandard pain outcomes, and a consideration of how sex differences may affect the performance of these pain surrogates. We tracked original research articles in Medline for the following periods: 1973-1977, 1983-1987, 1993-1997, 2003-2007, and 2014-2018. We identified 606 research articles about alternative surrogate pain measures, 473 of which were published between 2014 and 2018. This indicates that preclinical pain assessment is moving toward the use of these measures, which may soon become standard procedures in preclinical pain laboratories.FPU grant from the Spanish Ministry of Education, Culture and SportsSpanish Ministry of Economy and Competitiveness (MINECO, grant SAF2016-80540-R)Ramón Areces FoundationJunta de Andalucía (grant CTS 109)Esteve PharmaceuticalsEuropean Regional Development Fund (ERDF

Gait analysis in rats with single joint inflammation: influence of experimental factors.

Disability and movement-related pain are major symptoms of joint disease, motivating the development of methods to quantify motor behaviour in rodent joint pain models. We used observational scoring and automated methods to compare weight bearing during locomotion and during standing after single joint inflammation induced by Freund's complete adjuvant (0.12-8.0 mg/mL) or carrageenan (0.47-30 mg/mL). Automated gait analysis was based on video capture of prints generated by light projected into the long edge of the floor of a walkway, producing an illuminated image of the contact area of each paw with light intensity reflecting the contact pressure. Weight bearing was calculated as an area-integrated paw pressure, that is, the light intensity of all pixels activated during the contact phase of a paw placement. Automated static weight bearing was measured with the Incapacitance tester. Pharmacological sensitivity of weight-bearing during locomotion was tested in carrageenan-induced monoarthritis by administration of the commonly used analgesics diclofenac, ibuprofen, and naproxen, as well as oxycodone and paracetamol. Observational scoring and automated quantification yielded similar results. We found that the window between control rats and monoarthritic rats was greater during locomotion. The response was more pronounced for inflammation in the ankle as compared to the knee, suggesting a methodological advantage of using this injection site. The effects of both Freund's complete adjuvant and carrageenan were concentration related, but Freund's incomplete adjuvant was found to be as effective as lower, commonly used concentrations of the complete adjuvant. The results show that gait analysis can be an effective method to quantify behavioural effects of single joint inflammation in the rat, sensitive to analgesic treatment

Effect of reference compounds on carrageenan induced monoarthritis.

†<p>LoQ = limit of quantification.</p><p><b>Note:</b> Testing was performed 3 hours after carrageenan injection except for the naproxen experiment when testing took place 5 hours after carrageenan. Oxycodone was given subcutaneously 30 min before test while all other compounds were administered per os 4 hours before testing. Plasma samples were taken from satellite animals (n = 3 per group) at the time of testing or from the tested animals at termination of the experiment corresponding to about 5 hours 20 min after administration of naproxen, 3 hours after oxycodone and 7 hours after ibuprofen, diclofenac and paracetamol. Statistically different from vehicle group:</p>*<p> = <i>p</i><0.05,</p>**<p> = <i>p</i><0.01,</p>***<p> = <i>p</i><0.001, Dunnett's Multiple Comparison test subsequent to 1-way ANOVA.</p

Prints from rats in the PawPrint walkway.

<p>The walking pattern of a normal control rat (upper panel) and a representative monoarthritic rat (lower panel). The inflammatory induction agent was injected into the ankle joint of the left hind paw. The middle trace of each panel shows prints from the left side in red, from the right side in blue. Prints from forepaws are shown in light colour, also seen separately in the lower trace, whereas prints from the hind paws are darker, and shown separately in the upper trace. Each print detected by the PawPrint algorithm is surrounded by a white rectangle, but coloured red for the calculated median print. In places where prints would have been expected in normal gait, but not actually detected, green rectangles occur. When such a “non-print” is chosen as the median print, the rectangle is coloured orange.</p

Paw pressure visual rating scale in rats filmed in observation chambers.

<p>Paw pressure visual rating scale in rats filmed in observation chambers.</p

Injection site of carrageenan plays a role for magnitude of response during locomotion.

<p>Time course of guarding index (upper panel) and regularity index (lower panel) for control rats and rats injected with carrageenan into the ankle or knee joint. Bonferroni's test subsequent to ANOVA: *** = <i>p</i><0.001 compared to the control group at the same time point and †† = <i>p</i><0.01, ††† = <i>p</i><0.001 when comparing ankle to knee injection groups. Data shown as mean and SEM, n = 10 per group.</p

Visual rating scale for weight bearing during locomotion.

<p>Illustration of typical paw prints reflecting the rating scale used. In addition to the visual print pattern, the following criteria were implemented: 0 was assigned to rats where no difference could be detected between the two hind paws, 1.5 required the animals to limp, 2.5 was assigned when each step cycle was complete, i.e. that the affected paw touched the floor at every step, 2.75 when only some of the cycles were complete, and 3 when the paw remained off the floor for the entire crossing.</p