Sara Adães

Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.

UNLABELLED Although there are several reports on pain behavioral tests in rat models of knee osteoarthritis (OA), most of them focus on the paw. The aim of this study was to investigate pain-related behaviors on the affected knee joint, the primary source of nociception, in animals with mono-iodoacetate-induced OA, using the knee-bend (which provides information on movement pain) and pin-prick tests, and to evaluate nociception elicited by walking using the CatWalk test. The von Frey and Randall-Selitto tests applied to the paw allowed us to compare our study results with previous studies. A further aim was to compare the behavioral nociceptive responses of the most used doses of mono-iodoacetate, 2 and 3 mg. Knee-bend score of OA animals was higher than those of control animals throughout the study (P < .05). At every time point, the ipsilateral hind-paw load of OA rats, as measured by the CatWalk test, was lower than that of control rats (P < .05), and paw withdraw threshold to von Frey filaments was also decreased (P < .01). No changes were observed in pin-prick and Randall-Selitto tests. Results obtained with the 2 doses of mono-iodoacetate were similar. The knee-bend and CatWalk tests are effective for evaluating movement-related nociception, a hallmark of clinical OA, which was present throughout the experimental period. PERSPECTIVE Behavioral characterization of models of OA pain is important and useful for use in future studies to test pharmacological treatments. Furthermore, it is important to find methods that correlate better with the human symptoms of OA.

Increased Endothelial Apoptotic Cell Density in Human Diabetic Erectile Tissue—Comparison with Clinical Data

INTRODUCTION Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. AIM To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patients clinical data and erection status. METHODS Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). MAIN OUTCOME MEASURES Establish an index of ACD and correlate those results with patient clinical data. RESULTS Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. CONCLUSIONS We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.

Phenotypic alterations of neurons that innervate osteoarthritic joints in rats

OBJECTIVE Pain is a prominent feature of osteoarthritis (OA). To further understand the primary mechanisms of nociception in OA, we studied the expression of the phenotype markers calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200) in sensory neurons innervating the OA knee joint in rats. METHODS OA was induced in rats by intraarticular injection of 2 mg of mono-iodoacetate (MIA) into the knee. Neurons innervating the joint were identified by retrograde labeling with fluorogold in dorsal root ganglia (DRG) and colocalized with neurochemical markers by immunofluorescence. The total number of DRG cells was determined by stereologic methods in Nissl-stained sections. RESULTS A 37% decrease in the number of fluorogold-backlabeled cells was observed in rats with OA when compared with control rats, even though no decrease in the total number of cells was observed. However, an increase in the number of medium/large cell bodies and a decrease in the number of the smallest cells were observed, suggesting the occurrence of perikarya hypertrophy. The percentage of CGRP-positive cells increased significantly, predominantly in medium/large cells, suggesting the occurrence of a phenotypic switch. Colocalization of CGRP and NF200 revealed no significant changes in the percentage of double-labeled cells, but an increase in the number of medium/large double-labeled cells was observed. No differences in the expression of either IB4 or NF200 were observed in fluorogold-backlabeled cells. CONCLUSION These results indicate that MIA-induced OA causes an up-regulation of CGRP in different subpopulations of primary afferent neurons in DRG due to a phenotypic switch and/or cell hypertrophy which may be functionally relevant in terms of the onset of pain in this pathologic condition.

Dose-dependent expression of neuronal injury markers during experimental osteoarthritis induced by monoiodoacetate in the rat

BackgroundIt was recently reported that the mono-iodoacetate (MIA) experimental model of osteoarthritis (OA) courses with changes of neurons innervating the affected joints that are commonly interpreted as a neuronal response to axonal injury. To better characterize these changes, we evaluated the expression of two markers of neuronal damage, ATF-3 and NPY, and the growth associated protein GAP-43, in primary afferent neurons of OA animals injected with three different doses of MIA (0.3, 1 or 2 mg). Measurements were performed at days 3, 7, 14, 21 and 31 post-MIA injection.ResultsOA animals showed the characteristic histopathological changes of the joints and the accompanying nociceptive behaviour, evaluated by the Knee-Bed and CatWalk tests. An increase of ATF-3 expression was detected in the DRG of OA animals as early as 3 days after the injection of 1 or 2 mg of MIA and 7 days after the injection of 0.3 mg. NPY expression was increased in animals injected with 1 or 2 mg of MIA, at day 3 or in all time-points, respectively. From day 7 onwards there was a massive increase of GAP-43 expression in ATF-3 cells.ConclusionsThe expression of the neuronal injury markers ATF-3 and NPY as well as an up-regulation of GAP-43 expression, indicative of peripheral fibre regeneration, suggests that axonal injury and a regeneration response may be happening in this model of OA. This opens new perspectives in the unravelling of the physiopathology of the human disease.

Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis

IntroductionAnimal models currently used in osteoarthritis-associated pain research inadequately reproduce the initiating events and structural pathology of human osteoarthritis. Conversely, intra-articular injection of collagenase is a structurally relevant model, as it induces articular degeneration both by digesting collagen from cartilage and by causing articular instability, thereby reproducing some of the main events associated with osteoarthritis onset and development. Here, we evaluated if the intra-articular injection of collagenase can be an alternative model to study nociception associated with osteoarthritis.MethodsOsteoarthritis was induced by two intra-articular injections of either 250 U or 500 U of collagenase into the left knee joint of adult male Wistar rats. A six weeks time-course assessment of movement- and loading-induced nociception was performed by the Knee-Bend and CatWalk tests. The effect of morphine, lidocaine and diclofenac on nociceptive behaviour was evaluated in animals injected with 500 U of collagenase. Joint histopathology was scored for both doses throughout time. The expression of transient receptor potential vanilloid 1 (TRPV1) in ipsilateral dorsal root ganglia (DRG) was evaluated.ResultsAn increase in nociceptive behaviour associated with movement and loading of affected joints was observed after intra-articular collagenase injection. With the 500 U dose of collagenase, there was a significant correlation between the behavioural and the histopathological osteoarthritis-like structural changes developed after six weeks. One week after injection of 500 U collagenase, swelling of the injected knee and inflammation of the synovial membrane were also observed, indicating the occurrence of an early inflammatory reaction. Behavioural changes induced by the 500 U dose of collagenase were overall effectively reversed by morphine and lidocaine. Diclofenac was effective one week after injection. TRPV1 expression increased six weeks after 500 U collagenase injection.ConclusionWe conclude that the intra-articular injection of 500 U collagenase in the knee of rats can be an alternative model for the study of nociception associated with osteoarthritis, since it induces significant nociceptive alterations associated with relevant osteoarthritis-like joint structural changes.

Injury of primary afferent neurons may contribute to osteoarthritis induced pain: an experimental study using the collagenase model in rats

OBJECTIVE Pain exacerbated by movement and loading on the joint is the major symptom of osteoarthritis (OA), but the mechanisms of chronic pain in this pathology are still poorly understood. Using the intra-articular (i.a.) injection of collagenase in the knee of rats as a model of OA, we aimed at evaluating whether injury of sensory neurons may contribute to the development of OA-associated nociception. DESIGN OA was induced by i.a. injection of collagenase into the left knee joint of adult male Wistar rats. Histopathological changes and movement and loading-induced nociception were assessed for 6 weeks. A time-course analysis of the expression of the neuronal injury markers activating transcription factor-3 (ATF-3) and neuropeptide Y (NPY) and of the neuropeptide SP in the dorsal root ganglion (DRG) was performed. Gabapentins effect on nociception was evaluated, as well as the expression of the α2δ-1 voltage-gated calcium channel subunit. RESULTS Collagenase induced the development of OA-like histopathological changes and of movement-induced nociception. Altered expression of ATF-3, NPY and SP was observed in the DRG, correlating with the degree of articular degeneration after 6 weeks of disease progression. Repeated administration of gabapentin reversed the nociceptive responses 6 weeks after the induction of OA. α2δ-1 was upregulated in the DRG. CONCLUSION By inducing nociceptive behaviours associated with relevant joint structural changes, the i.a. injection of collagenase presents itself as a pertinent model for the study of OA pain. The findings in this study support the hypothesis that injury of sensory neurons innervating OA joints may be a significant element in the mechanisms of OA-associated pain.

Muscarinic M3 facilitation of acetylcholine release from rat myenteric neurons depends on adenosine outflow leading to activation of excitatory A2A receptors

Abstract  Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A2A receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle–myenteric plexus of the rat ileum. Blockade of the muscarinic M2 receptor with 11‐[[2‐1[(diethylamino) methyl‐1‐piperidinyl]‐ acetyl]]‐5,11‐dihydro‐6H‐pyrido [2,3‐b][1,4] benzodiazepine‐6‐one (AF‐DX 116), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) and atropine facilitated [3H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M3 receptors activation, as predicted by blockade with J104129 (a selective M3 antagonist), 4‐DAMP and atropine, whereas the selective M2 antagonist, AF‐DX 116, was without of effect. Blockade of A2A receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto‐5′‐nucleotidase with concanavalin A, all attenuated release inhibition caused by 4‐DAMP. J104129 and 4‐DAMP, but not AF‐DX 116, decreased (∼60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 μmol L−1) facilitated the release of [3H]ACh (34 ± 4%, n = 5) and adenosine (57 ± 3%, n = 6) from stimulated myenteric neurons. 4‐DAMP, dipyridamole and concanavalin A prevented oxotremorine‐induced facilitation. ZM 241385 blocked oxotremorine facilitation of [3H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M2 and facilitatory M3 autoreceptors. While the M2 inhibition is prevalent during brief stimulation periods, muscarinic M3 facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A2A receptors.

Glial activation in the collagenase model of nociception associated with osteoarthritis

Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3–L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

530 EFFECT OF ANALGESIC DRUGS IN EXPERIMENTAL OSTEOARTHRITIS EVALUATED BY TWO CLINICALLY RELEVANT BEHAVIOURAL TESTS

Background and Aims: Recognition of the necessity for new strategies in the management of pain has led to the development of innovative drugs with favorable side-effect. Nowadays antiinflammatory drugs are being discovered and developed based on their effects on signal transduction. MCS-18 is a novel natural product isolated and purified from Helleborus purpurascens. Its local analgesic and anti-inflammatory efficacy has been confirmed by a rapid and long-lasting effect in eliminating pain and increasing mobility in patients with shoulder periarthritis. By focusing on MCS-18 mechanisms of action, our study aimed to investigate the analgesic and anti-inflammatory properties of this product. Methods: The isolated and highly purified MCS-18 product was tested by using a complex schedule including: effect on nitric oxide (NO) production (Griess method); effect upon production and release of free oxygen radicals (FOR) in human neutrophils and murine macrophages activated at TLRs level (chemiluminometry); action on TRPV1 pain receptors (capsaicin-induced eye wipe test); TLR-2 antagonist action on human monocytes/macrophages (flowcytometry). Results: Our experimental data demonstrate that MCS-18 inhibits NO can decrease the pain sensation after TRPV1 receptors activation and cancels FOR-release by human activated neutrophils and mouse macrophages. MCS-18 also acts on human monocytes/macrophages as a TLR-2 antagonist. Conclusions: The results obtained reveal that the novel natural product MCS-18 can down-regulate inflammatory pain, has antiinflammatory capacities and might be a potential TLR-2 antagonist.

130 INCREASED EXPRESSION OF NEURONAL INJURY MARKERS IN THE DRG OF OSTEOARTHRITIC RATS

Background & Aims: Pain is the most prominent but least well-studied feature of osteoarthritis (OA). Retrograde labelling of neurons innervating the OA joint decreases 40% at 31 days of disease progression, with the total number of DRG cells remaining unaltered, but increasing the number of medium-large cells. Therefore, we hypothesized that neuronal damage might be occurring during OA, and to test this hypothesis we evaluated the expression of ATF-3 and NPY (known to be increased by neuronal damage) in DRG neurons. Since ATF-3 has also been associated with the regeneration of injured cells, we also evaluated its colocalization with the regeneration marker GAP-43. Methods: All procedures were performed according to the ethical guidelines for the study of experimental pain in conscious animals. OA was induced by injection of 2mg of mono-iodoacetate in the knee joint of adult Wistar rats. Animals were sacrificed at 3, 7 and 14 days post-injection. L3-L5 DRGs were used for immunohistochemistry for NPY, ATF-3 and GAP-43. Results: An increase in the number of ATF-3-positive cells was observed 3 days after the induction of OA. Such increase diminished over time, but the percentage of ATF-3 cells positive for GAP43 increased at days 7 and 14. NPY expression showed a similar pattern as ATF-3 expression. Conclusion: The increased ATF-3 and NPY expression suggests that damage in DRG neurons innervating the OA joint may be occurring. The increased co-localization of ATF-3 and GAP-43 over time indicates that neuronal regeneration may be taking place as a response to neuronal damage.