Estrogenic impregnation alters pain expression: analysis through functional neuropeptidomics in a surgical rat model of osteoarthritis.

PURPOSE: Several observational studies suggest that estrogens could bias pain perception. To evaluate the influence of estrogenic impregnation on pain expression, a prospective, randomized, controlled, blinded study was conducted in a Sprague-Dawley rat model of surgically induced osteoarthritis (OA). METHODS: Female rats were ovariectomized and pre-emptive 17ß-estradiol (0.025 mg, 90-day release time) or placebo pellets were installed subcutaneously during the OVX procedures. Thirty-five days after, OA was surgically induced on both 17ß-estradiol (OA-E) and placebo (OA-P) groups. Mechanical hypersensitivity was assessed by static weight-bearing (SWB) and paw withdrawal threshold (PWT) tests. Mass spectrometry coupled with high-performance liquid chromatography (HPLC-MS) was performed to quantify the spinal pronociceptive neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK), somatostatin (SST), and dynorphin-A (Dyn-A). RESULTS: Compared to control, ovariectomized rats presented higher SP (P = 0.009) and CGRP (P = 0.017) concentrations. OA induction increased the spinal level of SP (+ 33%, P < 0.020) and decreased the release of BK (- 20%, (P < 0.037)). The OA-E rats at functional assessment put more % body weight on the affected hind limb than OA-P rats at D7 (P = 0.027) and D56 (P = 0.033), and showed higher PWT at D56 (P = 0.009), suggesting an analgesic and anti-allodynic effect of 17ß-estradiol. Interestingly, the 17ß-estradiol treatment counteracted the increase of spinal concentration of Dyn-A (P < 0.016) and CGRP (P < 0.018). CONCLUSION: These results clearly indicate that 17ß-estradiol interfers with the development of central sensitization and confirm that gender dimorphism should be considered when looking at pain evaluation.

Pain characterization and response to palliative care in dogs with naturally-occurring appendicular osteosarcoma: An open label clinical trial.

This study aimed to characterize bone cancer pain (quantitative sensory testing (QST), stance asymmetry index, actimetry, scores of pain and quality of life (QoL)) in dogs with appendicular osteosarcoma (OSA), and to evaluate a stepwise palliative analgesic treatment. The pain profile of thirteen client-owned dogs with OSA was compared with seven healthy dogs. Dogs with OSA were then enrolled in a prospective, open-label, clinical trial. Outcome measures included: primary and secondary mechanical thresholds (MT), conditioned pain modulation (CPM), stance asymmetry index, actimetry (most and least active periods), visual analog scales and QoL. After baseline assessments, stepwise treatment comprised orally administered cimicoxib (2 mg/kg q 24h), amitriptyline (1-1.5 mg/kg q 24h) and gabapentin (10 mg/kg q 8h); re-evaluations were performed after 14 (D14), 21 (D21) and 28 (D28) days, respectively. Statistics used mixed linear models (α = 5%; one-sided). Centralized nociceptive sensitivity (primary and secondary MT, and dynamic allodynia) was recorded in OSA dogs. Healthy dogs had responsive CPM, but CPM was deficient in OSA dogs. Construct validity was observed for the QST protocol. Asymmetry index was significantly present in OSA dogs. The CPM improved significantly at D14. When compared with baseline (log mean ± SD: 4.1 ± 0.04), most active actimetry significantly improved at D14 (4.3 ± 0.04), D21 and D28 (4.2 ± 0.04 for both). When compared with baseline, least active actimetry significantly decreased after treatment at all time-points indicating improvement in night-time restlessness. No other significant treatment effect was observed. Except for tactile threshold and actimetry, all outcomes worsened when gabapentin was added to cimicoxib-amitriptyline. Dogs with bone cancer are affected by widespread somatosensory sensitivity characterized by peripheral and central sensitization and have a deficient inhibitory system. This severe pain is mostly refractory to palliative analgesic treatment, and the latter was only detected by specific and sensitive outcomes.

Refinement of the Montreal Instrument for Cat Arthritis Testing, for Use by Veterinarians: detection of naturally occurring osteoarthritis in laboratory cats.

Objectives Feline osteoarthritis causes pain and disability. Detection and measurement is challenging, relying heavily on owner report. This study describes refinement of the Montreal Instrument for Cat Arthritis Testing, for Use by Veterinarians. Methods A video analysis of osteoarthritic (n = 6) and non-osteoarthritic (n = 4) cats facilitated expansion of scale items. Three successive therapeutic trials (using gabapentin, tramadol and oral transmucosal meloxicam spray) in laboratory cats with and without natural osteoarthritis (n = 12-20) permitted construct validation (assessments of disease status sensitivity and therapeutic responsiveness) and further scale refinements based on performance. Results Scale osteoarthritic sensitivity improved from phase I to phase III; phase III scale total score ( P = 0.0001) and 4/5 subcategories - body posture ( P = 0.0006), gait ( P = 0.0031), jumping (0.0824) and global distance examination ( P = 0.0001) - detected osteoarthritic cats. Total score inter-rater (intra-class correlation coefficients [ICC] = 0.64-0.75), intra-rater (ICC = 0.90-0.91) and overall internal consistency (Cronbach's alpha = 0.85) reliability were good to excellent. von Frey anesthesiometer-induced paw withdrawal threshold increased with gabapentin in phase I, in osteoarthritic cats ( P <0.001) but not in non-osteoarthritic cats ( P = 0.075). Night-time activity increased during gabapentin treatment. Objective measures also detected tramadol and/or meloxicam treatment effects in osteoarthritic cats in phases II and III. There was some treatment responsiveness: in phase I, 3/10 subcategory scores improved ( P <0.09) in treated osteoarthritic cats; in phase II, 3/8 subcategories improved; and in phase III, 1/5 subcategories improved ( P <0.096). Conclusions and relevance The revised scale detected naturally occurring osteoarthritis, but not treatment effects, in laboratory cats, suggesting future potential for screening of at-risk cats. Further study is needed to confirm reliability, validity (disease sensitivity and treatment responsiveness) and clinical feasibility, as well as cut-off scores for osteoarthritic vs non-osteoarthritic status, in client-owned cats.

Translational pain assessment: could natural animal models be the missing link?

Failure of analgesic drugs in clinical development is common. Along with the current "reproducibility crisis" in pain research, this has led some to question the use of animal models. Experimental models tend to comprise genetically homogeneous groups of young, male rodents in restricted and unvarying environments, and pain-producing assays that may not closely mimic the natural condition of interest. In addition, typical experimental outcome measures using thresholds or latencies for withdrawal may not adequately reflect clinical pain phenomena pertinent to human patients. It has been suggested that naturally occurring disease in veterinary patients may provide more valid models for the study of painful disease. Many painful conditions in animals resemble those in people. Like humans, veterinary patients are genetically diverse, often live to old age, and enjoy a complex environment, often the same as their owners. There is increasing interest in the development and validation of outcome measures for detecting pain in veterinary patients; these include objective (eg, locomotor activity monitoring, kinetic evaluation, quantitative sensory testing, and bioimaging) and subjective (eg, pain scales and quality of life scales) measures. Veterinary subject diversity, pathophysiological similarities to humans, and diverse outcome measures could yield better generalizability of findings and improved translation potential, potentially benefiting both humans and animals. The Comparative Oncology Trial Consortium in dogs has pawed the way for translational research, surmounting the challenges inherent in veterinary clinical trials. This review describes numerous conditions similarly applicable to pain research, with potential mutual benefits for human and veterinary clinicians, and their respective patients.

Evaluation of High Dosages of Oral Meloxicam in American Kestrels ( Falco sparverius ).

To evaluate the toxicity of short-term high doses of meloxicam in American kestrels ( Falco sparverius ), 32 male captive-born, 1- to 4-year-old American kestrels were randomly assigned to 4 groups: 3 groups treated with meloxicam (n = 9 per group) and a control group (n = 5). Meloxicam was administered orally via feeding tube in the proventriculus at 2, 10, and 20 mg/kg every 12 hours for 7 days for the treatment groups, while the control group received saline solution. The birds were evaluated for the presence of clinical signs, abnormalities in the complete blood cell count and in the plasma biochemical panel for the 20-mg/kg group, and gross and histopathologic lesions. No clinical signs or mortality were observed in any group. No significant differences of clinical relevance were found in results of the packed cell volume, total solids, and biochemical panel, and no evidence of renal toxicity was found in the treatment or control groups. A significant correlation was found between hepatic lipidosis and meloxicam dose (P = .02). Two of 9 birds in the 20-mg/kg group developed gastric ulcers, although this result was not significant. None of the birds in the 2- and 10-mg/kg groups had similar lesions. Finally, meloxicam dosages up to 20 mg/kg did not result in nephrotoxicity in American kestrels. Further toxicologic studies to evaluate hepatotoxicity and gastrotoxicity of meloxicam in avian species are needed.

Spinal neuropeptide modulation, functional assessment and cartilage lesions in a monosodium iodoacetate rat model of osteoarthritis.

BACKGROUND AND AIMS: Characterising the temporal evolution of changes observed in pain functional assessment, spinal neuropeptides and cartilage lesions of the joint after chemical osteoarthritis (OA) induction in rats. METHODS AND RESULTS: On day (D) 0, OA was induced by an IA injection of monosodium iodoacetate (MIA). Rats receiving 2mg MIA were temporally assessed at D3, D7, D14 and D21 for the total spinal cord concentration of substance P (SP), calcitonin gene related-peptide (CGRP), bradykinin (BK) and somatostatin (STT), and for severity of cartilage lesions. At D21, the same outcomes were compared with the IA 1mg MIA, IA 2mg MIA associated with punctual IA injection of lidocaine at D7, D14 and D21, sham (sterile saline) and naïve groups. Tactile allodynia was sequentially assessed using a von Frey anaesthesiometer. Non-parametric and mixed models were applied for statistical analysis. Tactile allodynia developed in the 2mg MIA group as soon as D3 and was maintained up to D21. Punctual IA treatment with lidocaine counteracted it at D7 and D14. Compared to naïve, [STT], [BK] and [CGRP] reached a maximum as early as D7, which plateaued up to D21. For [SP], the increase was delayed up to D14 and maintained at D21. No difference in levels of neuropeptides was observed between MIA doses, except for higher [STT] in the 2mg MIA group (P=0.029). Neuropeptides SP and BK were responsive to lidocaine treatment. The increase in severity of cartilage lesions was significant only in the 2mg MIA groups (P=0.01). CONCLUSION: In the MIA OA pain model, neuropeptide modulation appears early, and confirms the central nervous system to be an attractive target for OA pain quantification. The relationship of neuropeptide release with severity of cartilage lesions and functional assessment are promising and need further validation.

Comparison of lidocaine and lidocaine-epinephrine for the paravertebral brachial plexus block in dogs.

OBJECTIVE: To compare the motor and sensory block efficacy and duration of a modified paravertebral brachial plexus block (PBPB) after administration of lidocaine alone (LI) or combined with epinephrine (LE). STUDY DESIGN: Prospective, randomized, blinded, crossover study. ANIMALS: A total of eight healthy female Beagle dogs. METHODS: Under general anesthesia, modified PBPB was performed on the left thoracic limb using neurostimulation and/or ultrasound guidance to administer lidocaine (2 mg kg-1; 0.2 mL kg-1) either alone (treatment LI, n = 10) or with epinephrine (1:100,000; treatment LE, n = 9). Sensory block was evaluated through reaction to a painful mechanical stimulus applied at five sites on the limb. Motor block effect was evaluated according to visual gait assessments and thoracic limb vertical force measurements under dynamic and static conditions. Data were analyzed using repeated-measures generalized estimating equations. All statistical tests were performed two-sided at the α = 0.05 significance threshold. RESULTS: The duration of sensory block did not differ significantly between treatments. Visible gait impairment was more persistent in LE than in LI (118 ± 63 minutes for LI and 163 ± 23 minutes for LE; mean ± standard deviation) (p = 0.027). At nadir value, dynamic peak vertical force was lower in LE than in LI (p = 0.007). For both dynamic and static evaluations, the nadir and the return to baseline force were delayed in LE (return to normal at 180-200 minutes) when compared with LI (130-140 minutes) (p < 0.005). CONCLUSIONS AND CLINICAL RELEVANCE: The addition of epinephrine to lidocaine prolonged the duration and increased the intensity of the regional block, as verified by visual gait assessment and kinetic analysis. No significant difference was noted between treatments regarding sensory blockade. Kinetic analysis could be useful to evaluate regional anesthetic effect in dogs.

Preliminary investigation of the thermal antinociceptive effects of codeine in cats.

OBJECTIVES: The aim of this study was to evaluate the potential thermal antinociceptive effects of oral administration of a single dose of codeine in cats compared with positive (buprenorphine) and negative (saline 0.9%) controls. METHODS: Six adult healthy cats weighing 5.14 ± 0.6 kg were used. Skin temperature and thermal thresholds (TTs) were evaluated using a wireless device (Topcat Metrology) at baseline, 0.5, 1, 3, 6 and 10 h after treatment. In period 1, TTs were evaluated after subcutaneous administration of saline 0.9%. In period 2, cats were administered either oral codeine (10 mg total, 2.0 ± 0.2 mg/kg) or buccal buprenorphine (0.04 mg/kg) in a cross-over, blinded study design. Half of the volume of buprenorphine was administered into each cheek pouch. Δ TT (difference between TTs after and before treatment) was used for data comparison. Mean ± SD data were analyzed using one-way ANOVA followed by Dunnett's or Tukey's test when appropriate (P <0.05). RESULTS: Adverse effects did not occur in any group. Skin temperature was not different between groups nor over time. Temporal changes in TTs were not observed after saline or codeine. Buprenorphine increased Δ TT at 3 h (2.7 ± 3.3°C) when compared with baseline or saline (P <0.05). For buprenorphine, TTs were not >47.6°C at any time point in four cats. The mean highest temperature recorded in the two other cats in that group was 54.5 and 52.8°C at 3 h. CONCLUSIONS AND CLINICAL RELEVANCE: At the dose administered, codeine did not produce thermal antinociception. Mild increases in TT after buccal buprenorphine might be related to the first-pass effect after drug swallowing, drug spillage during administration and/or individual variability. These factors should be taken in to consideration when administering buprenorphine by this route in the clinical setting.

Oxytocin improves the expression of cardiac specific markers in porcine bone marrow stem cells differentiation.

Bone marrow stem cells (BMSCs) treated with 5-azacytidine possess myogenic differentiation potential. Oxytocin (OT) induces cardiomyogenesis in murine embryonic and cardiac stem cells. We attempted to isolate, characterize, and induce OT-mediated cardiomyogenic differentiation of porcine pBMSCs. Cells were treated as: control, OT, and 5-azacytidine groups. During early passages, transcripts of Oct4, GATA4, OT receptor, and phospholamban were expressed. RT-PCR showed upregulation of GATA4 in OT and 5-azacytidine-induced groups. Immunocytochemistry revealed higher expressions of cardiac troponin T and myosin heavy chain in OT than in 5-azacytidine-induced groups (p < 0.01). Western blot analysis showed upregulation of cardiac troponin I in OT-induced pBMSCs (p < 0.01). We infer pBMSCs should be induced during early passages, when expressing transcription factors related to pluripotency and cardiomyogenesis, as well as OT receptor. The more abundant expression of cardiac specific proteins in OT-treated pBMSCs suggests OT could be a more potent cardiomyogenic inducer of pBMSC.

Coxofemoral joint kinematics using video fluoroscopic images of treadmill-walking cats: development of a technique to assess osteoarthritis-associated disability.

The objectives of this pilot study were to develop a video fluoroscopy kinematics method for the assessment of the coxofemoral joint in cats with and without osteoarthritis (OA)-associated disability. Two non-OA cats and four cats affected by coxofemoral OA were evaluated by video fluoroscopy. Video fluoroscopic images of the coxofemoral joints were captured at 120 frames/s using a customized C-arm X-ray system while cats walked freely on a treadmill at 0.4 m/s. The angle patterns over time of the coxofemoral joints were extracted using a graphic user interface following four steps: (i) correction for image distortion; (ii) image denoising and contrast enhancement; (iii) frame-to-frame anatomical marker identification; and (iv) statistical gait analysis. Reliability analysis was performed. The cats with OA presented greater intra-subject stride and gait cycle variability. Three cats with OA presented a left-right asymmetry in the range of movement of the coxofemoral joint angle in the sagittal plane (two with no overlap of the 95% confidence interval, and one with only a slight overlap) consistent with their painful OA joint, and a longer gait cycle duration. Reliability analysis revealed an absolute variation in the coxofemoral joint angle of 2º-6º, indicating that the two-dimensional video fluoroscopy technique provided reliable data. Improvement of this method is recommended: variability would likely be reduced if a larger field of view could be recorded, allowing the identification and tracking of each femoral axis, rather than the trochanter landmarks. The range of movement of the coxofemoral joint has the potential to be an objective marker of OA-associated disability.

Evaluation of osteoarthritis in cats: novel information from a pilot study.

OBJECTIVE: To describe structural changes associated with osteoarthritis (OA) in cats and to quantify OA-associated disability using functional evaluations. STUDY DESIGN: Cross-sectional pilot study with longitudinal data. ANIMALS: Normal cats (n = 2) and coxofemoral joint OA cats (n = 4) were evaluated by physical examination, radiography, and magnetic resonance imaging (MRI). METHODS: Structural changes related to OA were scored using computed radiographs (CR) and MRI. Functional evaluation consisted of podobarometric gait analyses performed using a pressure-sensitive mattress and motor activity assessments using collar-attached, accelerometer-based activity sensors. RESULTS: Structural scores for the coxofemoral joint OA-related lesions were lower in normal cats than OA cats for MRI (P = .07). Use of MRI allowed for whole-organ assessment of the coxofemoral joint. Pelvic limb peak vertical ground reaction force (PVF) was higher in normal cats than OA cats (P = .10). During the night, motor activity was greater in normal cats than OA cats (P = .04). PVF was positively correlated with mean motor activity (Spearman coefficient [Rho] = 0.83, P = .04) and negatively correlated with age and MRI structural score (Rho = -0.93 and -0.79, P < .01 and .06, respectively). CONCLUSIONS: This study provides the first description of OA-related lesions in cats using MRI. Gait analysis and accelerometry should be considered as objective tools to characterize OA-associated disability, although these assessments were weakly correlated with structural changes.

Evaluation of a fracture pain model in domestic pigeons (Columba livia).

OBJECTIVE: To validate a model of postfracture pain in perching birds. ANIMALS: 21 adult domestic pigeons (Columba livia). PROCEDURES: In each bird, a standardized osteotomy of 1 femur was performed and the fracture was immobilized with an intramedullary pin. Degree of postoperative pain was evaluated 6 times/d for 4 days by use of 3 methods: an electronic perch for assessment of weight-bearing load differential of the pelvic limbs, 4 numeric rating pain scales for assessment of pain (all of which involved the observer in the same room as the bird), and analysis of video-recorded (observer absent) partial ethograms for bird activity and posture. Measurements obtained were compared with data collected before the surgery to evaluate the ability of these methods to detect pain. RESULTS: The weight-bearing load differential was a sensitive, specific, reliable, and indirect measure of fracture-associated pain in the model used. Two of 4 tested pain scales (fractured limb position and subjective evaluation of degree of pain) were sensitive and specific for detecting pain and were reliable in a research setting. Interobserver reliability of the 4 pain scales was excellent. Partial ethograms were sensitive for identifying pain-associated behavior in pigeons, particularly during the first 2 days after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: The fracture pain model was reliable and reproducible and may be useful for experimental studies involving postsurgical pain in pigeons. Weight-bearing load differential was the most sensitive and specific means of determining degree of pain in pigeons during the first 4 days after hind limb fracture induction.

Analgesic effects of meloxicam administration on postoperative orthopedic pain in domestic pigeons (Columba livia).

OBJECTIVE: To assess the analgesic effects of 2 doses of meloxicam on the degree of postoperative orthopedic pain in pigeons. ANIMALS: 21 domestic pigeons (Columba livia). PROCEDURES: In each bird, a standardized osteotomy of 1 femur was performed and the fracture was immobilized with an intramedullary pin. Birds were randomly allocated to receive saline (0.9% NaCl) solution (control) or meloxicam (0.5 mg/kg [low dose] or 2.0 mg/kg [high dose]). The first treatment was administered i.m. after surgery was completed. Subsequent treatments were administered p.o. every 12 hours for 9 days. Degree of postoperative pain was assessed for the first 4 days after surgery by use of 3 methods: an electronic perch for assessment of weight-bearing load differential of the pelvic limbs, 4 pain scales, and analysis of video-recorded partial ethograms for bird activity and posture. RESULTS: No significant differences were observed between the control group and the low-dose meloxicam group in any tested variable. The high-dose meloxicam group had a greater degree of weight bearing on the affected limb from the second to the fourth postoperative day as well as lower pain scores for at least the first 2 postoperative days, compared with the other groups. Return to presurgical behavior was achieved faster in pigeons that received high-dose meloxicam than in the other groups. No adverse effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of meloxicam at 0.5 mg/kg appeared ineffective in minimizing postoperative orthopedic pain in pigeons, but the 2.0 mg/kg dose provided quantifiable analgesia that appeared safe in this species in experimental conditions.

Measurements of canine aqueous humor inflammatory mediators and the effect of carprofen following anterior chamber paracentesis.

OBJECTIVES: Phase I: To evaluate levels of prostaglandin E(2) (PGE(2) ), nitrites and nitrates (NO(x) ), tumor necrosis factor-alpha (TNF-α) and expression of inducible cyclo-oxygenase (COX-2), nitric oxide synthase (NOS-2), and matrix metalloproteinases (MMP-3 and -9) in canine aqueous humor following repeated anterior chamber paracenteses (ACP). Phase II: to evaluate the effect of carprofen on PGE(2) , NO(x) , and TNF-α in canine aqueous humor following ACP. ANIMALS STUDIED: Four beagles in phase I and 8 beagles in phase II. PROCEDURES: Phase I: ACP was performed at time (T) 0, 4 and 8 h. Phase II: A randomized, placebo-controlled cross-over design with four dogs per group where carprofen was given 4.4 mg/kg/day on day (D) 1, 2 and 3. ACP was performed at T0 and T1.5 on D3. Statistical analysis was performed with repeated measures anova and post hoc Tukey-Kramer multiple-comparison procedure. In phase II, TNF-α level was analyzed with a Wilcoxon signed-rank test. RESULTS: Phase I: PGE(2) significantly increased (P < 0.0001) to plateau at T4. NO(X) was decreased at T4 (P < 0.06), but increased at T8 (P < 0.0001). COX-2 showed detectable expression only at T8. TNF-α, NOS-2, MMP-3 and -9 were undetectable at all time points. Phase II: At T1.5, PGE(2) was significantly elevated in both groups but was lower in the carprofen group (P = 0.037). NO(x) and TNF-α did not statistically increase in either group. CONCLUSIONS: Following ACP, significant increases in PGE(2) levels confirmed inflammation characterized by a rise of COX-2. The NO(x) pathway took longer to induce as compared with PGE(2) . Carprofen decreased PGE(2) levels and could help control intraocular inflammation.

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model.

INTRODUCTION: The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA). METHODS: Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE(2) and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05. RESULTS: A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE(2) (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone. CONCLUSION: Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Involvement of the nitric oxide-soluble guanylyl cyclase pathway in the oxytocin-mediated differentiation of porcine bone marrow stem cells into cardiomyocytes.

Bone marrow stem cells (BMSCs) express cardiac markers in vitro and in vivo upon induction. Cardiomyogenic differentiation of embryonic stem cells induced by oxytocin (OT) involves the nitric oxide (NO)-soluble guanylyl cyclase (sGC) pathway. Also, OT improved cardiomyogenic differentiation of porcine BMSCs (pBMSCs). Here, we document the role of NO pathway in OT-mediated cardiomyogenic differentiation of pBMSCs obtained from bone marrow aspirates of juvenile pigs. Cells were exposed (OT cells) or not (control cells) to OT, in presence or absence of a NO synthase inhibitor (L-NAME) and a sGC inhibitor (ODQ). Gene (RT-PCR) and protein expression (immunocytochemistry) of NOS was up-regulated after OT induction. Exposure of OT cells to L-NAME, ODQ, or both, leaded to a significant reduction in cardiac troponin I transcripts, and protein (Western Blot) expression. For the latter, ODQ looked more performing in inhibition than L-NAME. Expression of cardiac troponin T and myosin heavy chain (immunocytochemistry) was less abundant in OT cells exposed to inhibitors without apparent synergic effect between L-NAME and ODQ. In control cells, protein expression remained low. Moreover, OT-induced cell proliferation, and this effect was counteracted by NOS/sGC inhibitors. Inhibiting NO production and NO effector, sGC, affected the OT-mediated differentiation of pBMSCs, because abundance of cardiac proteins was reduced to levels similar to those observed in control cells. We propose that following treatment with OT, activation of NO pathway directs pBMSCs to a preferential cardiomyogenic phenotype and stimulates cell proliferation.

Treatment with tiludronic acid helps reduce the development of experimental osteoarthritis lesions in dogs with anterior cruciate ligament transection followed by reconstructive surgery: a 1-year study with quantitative magnetic resonance imaging.

OBJECTIVE: to investigate over a 1-year period in dogs that underwent extracapsular stabilization surgery (ECS) following anterior cruciate ligament (ACL) transection: whether reconstructive surgery could prevent osteoarthritis (OA) progression and whether treatment with the bisphosphonate tiludronic acid (TA) could improve the chronic evolution of OA structural changes. METHODS: ACL transection was performed on dogs on Day 0 and ECS on Day 28. Dogs were randomly divided into 2 groups: 15 received placebo and 16 were treated with TA (2 mg/kg subcutaneous injection) on Days 14, 28, 56, and 84. Magnetic resonance images were acquired on Days -10, 26, 91, 210, and 357, and cartilage volume was quantified. At sacrifice (Day 364), cartilage from femoral condyles and tibial plateaus was macroscopically and histologically evaluated. Expression levels of MMP-1, -3, -13, ADAMTS-4, -5, BMP-2, FGF-2, IGF-1, TGF-ß1, collagen type II, and aggrecan were determined using real-time RT-PCR. RESULTS: the loss of cartilage volume observed after ACL transection stabilized following ECS. Thereafter, a gradual gain occurred, with the cartilage volume loss on the tibial plateaus reduced at Day 91 (p < 0.02) and Day 210 (p < 0.001) in the TA-treated dogs. At sacrifice, TA-treated dogs presented a reduction in the severity of macroscopic (p = 0.03 for plateaus) and histologic (p = 0.07 for plateaus) cartilage lesions, had a better preserved collagen network, and showed decreased MMP-13 (p = 0.04), MMP-1 and MMP-3 levels. CONCLUSION: our findings indicate that in dogs with ACL transection, ECS greatly prevents development of cartilage volume loss. Treatment with TA provided an additional benefit of reducing the development of OA lesions.

Influence of changes in body weight on peak vertical force in osteoarthritic dogs: a possible bias in study outcome.

OBJECTIVE: Force platform gait analysis is a recognized clinical evaluation tool that captures and documents the in vivo pathomechanics of osteoarthritis (OA). In a clinical trial designed to evaluate the impact of 2 specific diets, an increase in body weight (BW) was observed in lame client-owned dogs. Covariance analysis was used to evaluate the interference of BW changes toward the evolution of peak vertical force (PVF) values. These secondary findings are reported in this study. STUDY DESIGN: Prospective study. ANIMALS: Lame dogs (n=26). METHODS: Dogs with radiographic evidence of OA and low PVF values were fed with 2 specific diets for 30 and 60 days. PVF and BW were recorded at baseline, day 30 (D30), and D90. RESULTS: Mean (+/-SD) PVF values (%BW) did not differ significantly over time (D0: 63.9+/-17.2; D30: 65.5+/-17.4; and D90: 66.5+/-20.1). In contrast, BW (kg) was significantly higher at D90 (41.3+/-7.9) when compared with D30 (39.9+/-8.4) and D0 (40.0+/-8.7). Upon covariance analyses, BW changes interfere significantly with PVF values already normalized in %BW (P=.013). Values of PVF adjusted using BW as a covariate were then 63.4+/-17.1 (D0), 65.0+/-17.3 (D30), and 67.6+/-20.5 (D90), whereas D90 was significantly higher than D0. CONCLUSION: These findings highlighted the interference of changes in BW toward locomotor function of OA dogs when using PVF values normalized in %BW. Exacerbation of lameness when a gain in BW occurred was also sustained, raising a possible bias in clinical study outcomes. CLINICAL RELEVANCE: A BW increase in dogs with OA could exacerbate a preexisting lameness and induce a bias in clinical trials.

Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes.

Medetomidine is a potent and selective alpha2-adrenergic agonist. The activation of alpha2-adrenergic receptor mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia. However, our main interest is the sedative effects of medetomidine when used as a premedicant prior surgery in companion animals, especially in dogs. Recently, data suggested that following intravenous infusion at six dosing regiments non-linear pharmacokinetics was observed. Major causes of non-linear pharmacokinetics are the elimination of the drug not following a simple first-order kinetics and/or the elimination half-life changing due to saturation of an enzyme system. The goal of this study was to establish the metabolic stability and determine the metabolic pathway of medetomidine in dog liver microsomes. Consequently, Michaelis-Menten parameters (V(max), K(m)), T(1/2) and CL(i) were determined. The incubations were performed in a microcentrifuge tube and containing various concentrations of medetomidine (10-5000 nM), 1 mg/mL of microsomal proteins suspended in 0.1 M phosphate buffer, pH 7.4. Microsomal suspensions were preincubated with NADPH (1 mM) for 5 min at 37 degrees C prior to fortification with medetomidine. Samples were taken at various time points for kinetic information and the initial velocity (v(i)) was determined after 10 min incubation. The reaction was stopped by the addition of an internal standard solution (100 ng/mL of dextrometorphan in acetone). Medetomidine concentrations were determined using a selective and sensitive HPLC-ESI/MS/MS method. Using non-linear regression, we determined a K(m) value of 577 nM, indicating relatively low threshold enzyme saturation consistent with previous in vivo observation. The metabolic stability was determined at a concentration of 100 nm (<

The use of lingual venous blood to determine the acid-base and blood-gas status of dogs under anesthesia.

OBJECTIVE: To assess the suitability of lingual venous blood (LBG) as an alternative to arterial blood (ABG) samples in determining acid-base balance and blood-gas status in dogs anesthetized for elective procedures and with medetomidine and isoflurane administration under experimental conditions. STUDY DESIGN: Prospective, randomized clinical and experimental study. ANIMALS: Clinical population of 18 ASA I/II dogs for elective surgery and five healthy Beagles (3 females and 2 males) for the experimental study. METHODS: Blood sampling was simultaneously performed at dorsal pedal arterial and lingual venous sites, generating paired data. Two paired samples were collected from each dog in the clinical part and four from each dog in the experimental part (two during isoflurane anesthesia and two during isoflurane plus medetomidine). A modified Bland and Altman method was used to examine data from the clinical part and the experimental data were subjected to a paired sign's test following transformation where appropriate. RESULTS: The pH of LBG overestimated ABG, with limits of agreement of (-0.01, 0.02). The partial pressure of carbon dioxide (PCO(2)) of LBG overestimated ABG by 0.6 mmHg [0.1 kPa], with limits of agreement of (-3.5, 4.6) mmHg [-0.5, 0.6 kPa]. The partial pressure of oxygen (PO(2)) of LBG underestimated ABG by 86.3 mmHg [-11.5 kPa], with limits of agreement of (-199.8, 27.3) mmHg [-26.6, 3.6 kPa]. During medetomidine administration values for PO(2) (p = 0.03) and lactate (p = 0.03) were lower for LBG when compared with ABG. The LBG value of PO(2) was lower (p = 0.03) during medetomidine and isoflurane administration versus isoflurane alone. CONCLUSIONS AND CLINICAL RELEVANCE: The pH and PCO(2) of LBG samples provide clinically acceptable substitutes of ABG samples in the dog population studied. The wider limits of agreement for PO(2) render it less reliable as a substitute for ABG. The difference in PO(2) identified between LBG and ABG during medetomidine administration may not preclude the use of LBG as substitutes for ABG samples.