Pascal Vachon

Gait analysis and pain response of two rodent models of osteoarthritis.

The purpose of this study was to compare the gait parameters recorded on the CatWalk and the mechanical sensitivity with von Frey filaments of two putative models of osteoarthritis over a one month period, and to evaluate the effect of celecoxib on these parameters. Animals underwent either a surgical sectioning of the anterior cruciate ligament with partial medial menisectomy (ACLT+pMMx) to create a joint instability model or received an intra-articular injection of monoiodoacetate (MIA) as a putative inflammatory joint pain model. Animals were assessed for four consecutive weeks and knee joints were then evaluated histologically. Spinal cord lumbar enlargements were harvested for selected neuropeptide analysis (substance P (SP) and calcitonin gene related peptide (CGRP)). With the MIA model, significant changes persisted in selected dynamic gait parameters throughout the study in the injured limb as well as with the von Frey filaments. The ACLT+pMMx model in contrast showed no clear differential response between both hind limb for both gait parameters and pain-related behavior with von Frey filaments occurred only on the last day of the study. Neuropeptide analysis of spinal cord lumbar enlargements revealed a significant increase in CGRP concentration in both models and an increase in SP concentration only in the MIA model. Histological evaluation confirmed the presence of articular cartilage lesions in both models, but they were much more severe in the MIA model. Celecoxib had an effect on all selected gait parameters at the very beginning of the study and had an important alleviating effect on mechanical allodynia. These results suggest that the MIA model may be more appropriate for the evaluation of short term pain studies and that celecoxib may modulate mechanical allodynia through central sensitization mechanisms.

Peripheral nerve injury is associated with chronic, reversible changes in global DNA methylation in the mouse prefrontal cortex.

Changes in brain structure and cortical function are associated with many chronic pain conditions including low back pain and fibromyalgia. The magnitude of these changes correlates with the duration and/or the intensity of chronic pain. Most studies report changes in common areas involved in pain modulation, including the prefrontal cortex (PFC), and pain-related pathological changes in the PFC can be reversed with effective treatment. While the mechanisms underlying these changes are unknown, they must be dynamically regulated. Epigenetic modulation of gene expression in response to experience and environment is reversible and dynamic. Epigenetic modulation by DNA methylation is associated with abnormal behavior and pathological gene expression in the central nervous system. DNA methylation might also be involved in mediating the pathologies associated with chronic pain in the brain. We therefore tested a) whether alterations in DNA methylation are found in the brain long after chronic neuropathic pain is induced in the periphery using the spared nerve injury modal and b) whether these injury-associated changes are reversible by interventions that reverse the pathologies associated with chronic pain. Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by decreased global methylation in the PFC and the amygdala but not in the visual cortex or the thalamus. Environmental enrichment attenuated nerve injury-induced hypersensitivity and reversed the changes in global PFC methylation. Furthermore, global PFC methylation correlated with mechanical and thermal sensitivity in neuropathic mice. In summary, induction of chronic pain by peripheral nerve injury is associated with epigenetic changes in the brain. These changes are detected long after the original injury, at a long distance from the site of injury and are reversible with environmental manipulation. Changes in brain structure and cortical function that are associated with chronic pain conditions may therefore be mediated by epigenetic mechanisms.

Hydroxyl radical production in the cortex and striatum in a rat model of focal cerebral ischemia

BACKGROUND Increases in hydroxyl radical production have been used as evidence of oxidative stress in cerebral ischemia/ reperfusion. Ischemia can also induce increased dopamine release from the striatum that may contribute to hydroxyl radical formation. We have compared hydroxyl radical production in the cortex and striatum as an index of oxidative stress in a rat model of focal cerebral ischemia with cortical infarction. METHODS Using a three vessel occlusion model of focal cerebral ischemia combined with bilateral microdialysis, hydroxylation of 4-hydroxybenzoate (4HB) was continuously monitored in both hemispheres in either the lateral striatum or frontoparietal cortex. The ischemia protocol consisted of one hour equilibration, 30 min of three vessel occlusion, then release of the contralateral common carotid artery (CCA) for 2.5 h. RESULTS Induction of ischemia resulted in a 30-fold increase in dopamine release in the lateral striatum. Compared to the nonischemic striatum, the ratio of the hydroxylation product 3,4-dihydroxybenzoate (34DHB) to 4HB (trapping agent) in the ipsilateral striatum increased significantly 30 min after ischemia induction. In contrast, during the 30 min of three vessel occlusion there was no increase in the ratio in the cortex. Following the release of the contralateral CCA, the ratio from the ischemic cortex increased significantly compared to sham-operated animals. However, under all circumstances, the 34DHB/4HB ratio was greater in the striatum than in the cortex. CONCLUSION The increase in the 34DHB/4HB ratio in the lateral striatum coincides with the increased dopamine release suggesting a role for dopamine oxidation in the increased production of hydroxyl radicals. The significant increase in the ratio from the ischemic cortex compared to that from the sham-operated animals is consistent with increased oxidative stress induced by ischemia. However, the lower 34DHB/4HB ratio in the cortex which does not receive dopaminergic innervation compared to the striatum suggests a different mechanism for hydroxyl radical production. Such an alternate mechanism may represent a more toxic oxidative insult that contributes to infarction.

Pharmacokinetics of vanillin and its effects on mechanical hypersensitivity in a rat model of neuropathic pain.

The analgesic effects of vanillin on neuropathic pain was evaluated using thermal sensitivity and mechanical allodynia using the sciatic nerve constriction model (n = 30 rats). To determine the pharmacokinetics of vanillin, rats (n = 6/administration route) received either 20 or 100 mg/kg of vanillin i.v. and p.o., respectively. For the pharmacodynamic study, baseline levels for hyperalgesia and allodynia were taken for 5 days prior to surgery. Following surgery each group (n = 6 rats/group) received either vanillin (50 mg/kg or 100 mg/kg), morphine (2 mg/kg or 6 mg/kg) or the vehicle only. Pharmacokinetic results following p.o. administrations are Cmax 290.24 ng/mL, Tmax 4 h, relative clearance 62.17 L/h/kg and T1/2 10.3 h. The bioavailability is 7.6%. Mechanical allodynia was decreased on treatment days 1, 2, 3, 5 (p < 0.003) and not on day 4 (p > 0.02) with 50 mg/kg vanillin, whereas at 100 mg/kg p.o. a decrease was noted only on days 7 and 8 (p < 0.003). No effect on hyperalgesia was seen following vanillin administration. In conclusion, vanillin is bioavailable and seems to have an alleviating effect on mechanical allodynia, and not on hyperalgesia, when evaluated with a chronic constriction nerve injury rat model of neuropathic pain. Copyright

Comparison of pharmacokinetics of glucosamine and synovial fluid levels following administration of glucosamine sulphate or glucosamine hydrochloride

OBJECTIVE To compare the pharmacokinetics of glucosamine and the synovial fluid levels attained following treatment with glucosamine sulphate or glucosamine hydrochloride in a large animal model at clinically relevant doses. METHODS Eight adult female horses were used. Crystalline glucosamine sulphate (Dona) or glucosamine hydrochloride was administered at a dose of 20 mg/kg by either intravenous (i.v.) injection or nasogastric (n.g.) intubation. Plasma samples were collected before dosing and at 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid samples were collected from the radiocarpal joints within 48 h before dosing and at 1, 6 and 12 h post-dosing. Glucosamine was assayed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). RESULTS Plasma concentrations reached approximately 50 microg/mL after i.v. injection and approximately 1 microg/mL after n.g. administration of both types of glucosamine. The median oral bioavailability was 9.4% for glucosamine sulphate and 6.1% for glucosamine hydrochloride. Synovial fluid concentrations were significantly higher at 1 and 6 h following oral treatment with glucosamine sulphate compared to glucosamine hydrochloride. Twelve hours following oral administration, glucosamine levels in the plasma and the synovial fluid were still significantly higher than baseline for the glucosamine sulphate preparation, but not for the hydrochloride preparation. CONCLUSION Following oral administration of a clinically recommended dose of glucosamine sulphate (Dona), significantly higher synovial fluid concentrations of glucosamine are attained, when compared to an equivalent dose of glucosamine hydrochloride. Whether this difference is translated into a therapeutic effect on the joint tissues remains to be elucidated.

Adenoviral-Mediated Modulation of Sim1 Expression in the Paraventricular Nucleus Affects Food Intake

Haploinsufficency of Sim1, which codes for a basic helix-loop-helix–PAS (PER-ARNT-SIM) transcription factor, causes hyperphagia in mice and humans, without decrease in energy expenditure. Sim1 is expressed in several areas of the brain, including the developing and postnatal paraventricular nucleus (PVN), a region of the hypothalamus that controls food intake. We have previously found that the number of PVN cells is decreased in Sim1+/− mice, suggesting that their hyperphagia is caused by a developmental mechanism. However, the possibility that Sim1 functions in the postnatal PVN to control food intake cannot be ruled out. To explore this hypothesis, we used adenoviral vectors to modulate Sim1 expression in the postnatal PVN of wild-type mice. Unilateral stereotaxic injection into the PVN of an adenoviral vector producing a short hairpin RNA directed against Sim1 resulted in a significant increase in food intake, which peaked to 22% 6 d after the procedure, compared with the injection of a control virus. In contrast, injection of an adenovirus that expresses Sim1 induced a decrease in food intake that was maximal on the seventh day after the procedure, reaching 20%. The impact of bilateral injections of these vectors into the PVN was not greater than that of unilateral injections. Together, these results strongly suggest that Sim1 functions along a physiological pathway to control food intake.

Joint inflammation increases glucosamine levels attained in synovial fluid following oral administration of glucosamine hydrochloride

OBJECTIVE To compare synovial glucosamine levels in normal and inflamed equine joints following oral glucosamine administration and to determine whether single dose administration alters standard synovial parameters of inflammation. METHODS Eight adult horses were studied. On weeks 1 and 2, all horses received 20mg/kg glucosamine hydrochloride by nasogastric (NG) intubation or intravenous injection. On weeks 3 and 4, 12h after injection of both radiocarpal joints with 0.25 ng Escherichia coli lipopolysaccharide (LPS) to induce inflammation, glucosamine hydrochloride or a placebo was administered by NG intubation. Plasma samples were collected at baseline and 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid (SF) samples were collected within 48 h before dosing and 1, 6 and 12h post-dosing. Glucosamine was analyzed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Clinicopathological evaluation of SF parameters included white blood cell (WBC) count and total protein (TP) analyses. RESULTS No significant differences between groups were observed in SF baseline levels of WBC and TP at any stage of the study. SF WBC and TP significantly increased following IA LPS. The mean (+/-SD) maximal SF glucosamine levels (422.3+/-244.8 ng/mL) were significantly higher (>fourfold) in inflamed joints when compared to healthy joints (92.7+/-34.9 ng/mL). Glucosamine did not have any effect on standard SF parameters of inflammation. CONCLUSION Synovial inflammation leads to significantly higher synovial glucosamine concentrations compared to levels attained in healthy joints following oral administration of glucosamine hydrochloride. Whether these higher levels are translated into a therapeutic effect on the joint tissues remains to be elucidated.

Determination of specific neuropeptides modulation time course in a rat model of osteoarthritis pain by liquid chromatography ion trap mass spectrometry.

Animal models are useful to evaluate pharmacological therapies to alleviate joint pain. The present study characterized central neuropeptides modulation in the monoiodoacetate (MIA) rat model. Animals receiving a single 3mg MIA injection were euthanized at 3, 7, 14, 21 and 28 days post injection. Spinal cords were analyzed by liquid chromatography ion trap mass spectrometry. Up-regulations of the calcitonin gene-related peptide and substance P were observed starting on days 7 and 28 respectively, whereas big dynorphin(₁₋₃₂) content decreased significantly on day 14 in comparison to control animals (P<0.05). Preclinical drug evaluations using this model should be conducted between 7 and 21 days post injection when the lesions resemble most to human osteoarthritis.

Intrathecal Eugenol Administration Alleviates Neuropathic Pain in Male Sprague-Dawley Rats

The main objective of this study was to determine the central effect of eugenol on neuropathic pain when injected intrathecally at the level of the lumbar spinal cord. In a preliminary study the penetrability of eugenol was evaluated in the CNS of rats. Blood, brain and spinal cord samples were collected at selected time points following eugenol administration and concentrations were determined by tandem liquid chromatography‐mass spectrometry. Brain‐to‐plasma and spinal cord‐to‐plasma ratios (3.3 and 6.7, respectively) suggest that eugenol penetrates relatively well the CNS of rats, with a preferential distribution in the spinal cord. Following the induction of neuropathic pain in rats using the sciatic nerve ligation model, intrathecal injections of eugenol were done to evaluate the central effect of eugenol. Treatment with 50 μg of eugenol significantly decreased secondary mechanical allodynia after 15 min, 2 h and 4 h (p < 0.05; <0.005; <0.05, respectively) and improved thermal hyperalgesia after 2 h and 4 h (p < 0.001 and p < 0.05). The results support the hypothesis that eugenol may alleviate neuropathic pain, both allodynia and hyperalgesia, by acting centrally most probably at the level of the dorsal horn of the spinal cord where vanilloid receptors can be found. Copyright

Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry.

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance P, CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 --> 40:60; ACN:0.2% formic acid in water) at a flow rate of 75 microL/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance P, CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 --> [600 + 254], 952 --> [1215 + 963] and 717 --> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance P, CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models.