Ian F. Gibson

Effects of NG-substituted analogues of L-arginine on NANC relaxation of the rat anococcygeus and bovine retractor penis muscles and the bovine penile artery

1 The effects of two inhibitors of nitric oxide synthase, NG‐monomethyl l‐arginine (l‐NMMA) and NG‐nitro l‐arginine (l‐NOARG), were examined on non‐adrenergic non‐cholinergic (NANC) inhibitory transmission in the rat anococcygeus, bovine retractor penis (BRP) and bovine penile artery. 2 In the rat anococcygeus, l‐NMMA (10–1000 μm) produced a concentration‐dependent augmentation of guanethidine (30 μm)‐induced tone and inhibited NANC relaxation at all frequencies tested (0.1–20 Hz): the maximum inhibition obtained was 56 ± 6% (n = 6). l‐NOARG (0.3–30 μm) also augmented tone and inhibited NANC relaxation in a concentration‐dependent manner, but unlike l‐NMMA the maximum inhibition was 100%. 3 In the BRP, l‐NMMA (10–100 μm) had no effect on tone or NANC‐induced relaxation, but at 1000 μm tone was increased and NANC relaxation inhibited by 25 ± 7% (n = 6). l‐NOARG (0.3–30 μm) produced a concentration‐dependent increase in tone and inhibition of NANC relaxation. As in the rat anococcygeus, inhibition of NANC relaxation was complete. 4 The effects of l‐NMMA and l‐NOARG were stereospecific since d‐NMMA (10–1000 μm) and d‐NOARG (1–1000 μm) had no effect on tone or NANC relaxation of the rat anococcygeus or BRP. 5 l‐Arginine (10–300 μm) had no effect by itself on NANC‐induced relaxation of the rat anococcygeus or BRP. It did, however, reverse the ability of l‐NMMA (10–1000 μm) to augment tone and inhibit NANC relaxation in the rat anococcygeus and BRP. The actions of low concentrations l‐NOARG (0.3–10 μm) were also reversed by l‐arginine (300 μm), but those of higher concentrations were not. d‐Arginine (1000 μm) had no effect on the ability of l‐NMMA or l‐NOARG to augment tone and inhibit NANC relaxation in the anococcygeus and BRP. 6 On the bovine penile artery, both l‐NMMA (100μm) and l‐NOARG (30 μm) augmented the tone induced by guanethidine (30 μm) and 5‐hydroxytryptamine (0.2 μm) in an endothelium‐dependent manner. l‐NMMA had no effect on NANC‐induced relaxation, but inhibited acetylcholine‐induced endothelium‐dependent relaxation. l‐NOARG abolished NANC relaxation at all frequencies tested and inhibited acetylcholine‐induced relaxation. d‐NOARG (30 μm) had no effect on NANC or acetylcholine‐induced relaxation. 7 The ability of l‐NOARG to abolish NANC‐induced relaxation in the rat anococcygeus, BRP and bovine penile artery suggests that the l‐arginine‐nitric oxide pathway mediates neurotransmission in all three tissues. The effectiveness of l‐NMMA in blocking NANC relaxation in the rat anococcygeus but not the BRP and bovine penile artery suggests a species difference in the neuronal nitric oxide synthase. The neuronal and endothelial nitric oxide synthases in the penile artery also appear to differ.

Pharmacokinetics and tissue disposition of danofloxacin in sheep.

The plasma pharmacokinetics of danofloxacin administered at 1.25 mg kg−1 body weight by the intravenous and intramuscular routes were determined in sheep. Tissue distribution was also determined following administration by the intramuscular route at 1.25 mg kg−1 body weight. Danofloxacin had a large volume of distribution at steady state (V   dss) of 2.76±0.16 h (mean±S.E.M.) L kg−1, an elimination half‐life (t1/2β) of 3.35±0.23 h, and a body clearance (C1) of 0.63±0.04 L kg−1 h−1. Following intramuscular administration it achieved a maximum concentration (Cmax) of 0.32±0.02 μg mL−1 at 1.23±0.34 h (tmax) and had a mean residence time (MRT) of 5.45±0.19 h. Danofloxacin had an absolute bioavailability (F) of 95.71±4.41% and a mean absorption time (MAT) of 0.81±0.20 h following intramuscular administration. Mean plasma concentrations of >0.06 μg mL−1 were maintained for more than 8 h following intravenous and intramuscular administration. Following intramuscular administration highest concentrations were measured in plasma (0.43±0.04 μg mL−1), lung (1.51±0.18 μg g−1), and interdigital skin (0.64±0.18 μg g−1) at 1 h, duodenal contents (0.81±0.40 μg mL−1), lymph nodes (4.61±0.35 μg g−1), and brain (0.06±0.00 μg mL−1) at 2 h, jejunal (10.50±4.31 μg mL−1) and ileal (5.25±1.67 μg mL−1) contents at 4 h, and colonic contents (8.94±0.65 μg mL−1) at 8 h.

EFFECTS OF METABOLIC INHIBITORS ON ENDOTHELIUM-DEPENDENT AND ENDOTHELIUM-INDEPENDENT VASODILATATION OF RAT AND RABBIT AORTA

1 Basal release of endothelium‐derived relaxing factor (EDRF) rendered endothelium‐containing rings of rat aorta 4.7 fold less sensitive to the contractile actions of phenylephrine and depressed the maximum response when compared with endothelium‐denuded rings. The responsiveness and maximum response to phenylephrine was, however, similar in rings of rabbit aorta with or without endothelium. 2 Rotenone (1 nm‐0.1 μm), an inhibitor of oxidative phosphorylation, induced a profound, irreversible blockade of phenylephrine‐induced tone in endothelium‐containing and endothelium‐denuded rings of rat aorta, but induced only slight inhibition of tone in rings of rabbit aorta. 3 2‐Deoxy glucose (10 mm), an inhibitor of glycolysis, had no effect on phenylephrine‐induced contraction in endothelium‐denuded rings of rat aorta, but inhibited reversibly the endothelium‐dependent depression of contraction in endothelium containing rings. 2‐Deoxy glucose had no effect on phenylephrine‐induced contraction in rings of rabbit aorta with or without endothelium. 4 Rotenone (0.1 μm) inhibited acetylcholine‐induced, endothelium‐dependent relaxation of phenylephrine‐contracted rings or rat and rabbit aorta. In endothelium‐denuded rings of rat aorta, relaxation induced by glyceryl trinitrate of isoprenaline was also inhibited, but relaxation induced by 8‐bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCl‐contracted, endothelium‐denuded rings of rat aorta was also unaffected. 5 2‐Deoxy glucose (10 mm) inhibited acetylcholine‐induced, endothelium‐dependent relaxation of phenylephrine‐contracted rings of rat and rabbit aorta. In endothelium‐denuded rings of rat aorta, relaxation induced by glyceryl trinitrate and by isoprenaline was also inhibited, but relaxation induced by 8‐bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCl‐contracted, endothelium‐denuded rings of rat aorta was also unaffected. 6 These data suggest that in rabbit and in rat aorta, rotenone inhibits acetylcholine‐induced relaxation by inhibiting EDRF production, and by depressing smooth muscle sensitivity to EDRF, respectively. They further suggest that 2‐deoxy glucose inhibits acetylcholine‐induced relaxation in both tissues by depressing the sensitivity to EDRF, probably as a result of reduced synthesis of cyclic GMP. The additional possibility that 2‐deoxy glucose inhibits EDRF production warrants further investigation. 7 The blockade by 2‐deoxy glucose of the endothelium‐dependent depression of phenylephrine‐induced tone in rat aorta probably reflects blockade of the actions of spontaneously released EDRF.

Effects of hypoxia and metabolic inhibitors on production of prostacyclin and endothelium-derived relaxing factor by pig aortic endothelial cells.

1 The content of adenosine triphosphate (ATP) and basal and bradykinin‐stimulated production of prostacyclin and endothelium‐derived relaxing factor (EDRF) was measured in primary cultures of porcine aortic endothelial cells under normoxic (14.4% O2) and hypoxic (2.8% O2) conditions, and following treatment with rotenone and 2‐deoxy glucose, which inhibit oxidative and glycolytic metabolism, respectively. 2 ATP content and basal and bradykinin‐stimulated production of prostacyclin were similar under normoxic and hypoxic conditions. EDRF production, assessed as endothelial guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) content, was also similar under both conditions. 3 Treatment with rotenone (0.3 μm) had no effect on ATP content or basal or bradykinin‐stimulated production of prostacyclin or of EDRF, measured as endothelial cyclic GMP content. Elevation of cyclic GMP content by atriopeptin II was also unaffected. 4 Treatment with 2‐deoxy glucose (20 mm) in glucose‐free Krebs solution lowered ATP content, reduced bradykinin‐stimulated production of prostacyclin and abolished the bradykinin‐stimulated elevation of cyclic GMP content. Resting production of prostacyclin was unaffected but basal content of cyclic GMP was lowered in some experiments. Elevation of cyclic GMP content by atriopeptin II was abolished. 5 Combined treatment with rotenone (0.3 μm) and 2‐deoxy glucose (20 mm) lowered ATP content more than with 2‐deoxy glucose alone. Basal production of prostacyclin rose slightly and bradykinin‐stimulated production was powerfully inhibited. Basal content of cyclic GMP was unaffected, but bradykinin‐stimulated production was abolished. Elevation of cyclic GMP by atriopeptin II was also abolished. 6 Cascade bioassay experiments using endothelium‐denuded rings of rabbit aorta as a detector system confirmed that bradykinin‐stimulated production of EDRF was blocked by 2‐deoxy glucose, but not by rotenone. 7 These data indicate that porcine aortic endothelial cells in culture operate under mainly glycolytic metabolism and this probably explains why production of prostacyclin and EDRF is unaffected under hypoxic conditions. They also indicate that glycolytic metabolism is required for agonist‐stimulated production of prostacyclin and EDRF by these cells.

Actions and interactions of NG‐substituted analogues of l‐arginine on NANC neurotransmission in the bovine retractor penis and rat anococcygeus muscles

1 The effects and interactions of a series of NG‐substituted analogues of l‐arginine known to inhibit nitric oxide synthase were examined on non‐adrenergic, non‐cholinergic (NANC) neurotransmission in the bovine retractor penis (BRP) and rat anococcygeus muscles. 2 Treatment of BRP muscle strips with either NG‐nitro l‐arginine (l‐NOARG: 0.1–10 μm) or NG‐nitro l‐arginine meythl ester (l‐NAME; 0.1–100 μm) produced a concentration‐dependent blockade of NANC relaxation: blockade was complete at the highest concentration of each. 3 Pretreatment with l‐arginine (1–10 mm) had no effect on NANC relaxation by itself, but inhibited, in a concentration‐dependent manner, the subsequent ability of both l‐NOARG (0.1–300 μm) and l‐NAME (0.1–1 mm) to produce blockade. l‐Arginine (1–10 mm) reversed established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm) or l‐NAME (1 μm), but had little effect on maximal blockade induced by these agents. 4 In contrast to l‐NOARG and l‐NAME, NG‐monomethyl l‐arginine (l‐NMMA; 1 μm‐1 mm) had no effect by itself on NANC relaxation of the BRP. l‐NMMA (0.1–1 mm) did, however, like l‐arginine, inhibit, in a concentration‐dependent manner, the subsequent ability of both l‐NOARG (0.1–1 mm) and l‐NAME (0.1–3 mm) to produce blockade, but was more potent. As with l‐arginine, l‐NMMA (0.1–1 mm) reversed established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm) or l‐NAME (1 μm), but had little effect on maximal blockade induced by these agents. 5 In contrast to the effects on BRP, treatment of rat anococcygeus muscle with either l‐NOARG (0.1–10 μm) or l‐NMMA (1–100 μm) produced concentration‐dependent inhibition of NANC relaxation: the maximal inhibition induced by l‐NOARG and l‐NMMA was 100% and 40.1 ± 5.9% (n = 8), respectively. l‐Arginine (1–10 mm) reversed established submaximal inhibition of NANC relaxation induced by l‐NOARG (1 μm), had little effect on maximal blockade by this agent, and reversed maximal blockade induced by l‐NMMA (100 μm). 6 In the presence of partial blockade of NANC relaxation on rat anococcygeus by a maximal concentration of l‐NMMA (100 μm), subsequent blockade by l‐NOARG (0.1–100 μm) was inhibited. l‐NMMA (100 μm) produced a partial reversal of established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm), but had little effect on maximal blockade induced by this agent. 7 These findings suggest a complex series of interactions between l‐arginine and certain of its NG‐substituted analogues that are commonly used to inhibit nitric oxide synthase. The most striking new finding is that l‐NMMA does not block NANC relaxation in the BRP, but acts with greater potency than the endogenous substrate, l‐arginine, to inhibit the blockade induced by l‐NOARG or l‐NAME. Even on rat anococcygeus where l‐NMMA acts as a partial blocker of NANC relaxation, further blockade by l‐NOARG is inhibited.

The impact of prenatal stress on basal nociception and evoked responses to tail-docking and inflammatory challenge in juvenile pigs

The consequences of tail-docking (at 2-4 days) and prenatal stress (maternal social stress during the 2nd third of pregnancy) on baseline nociceptive thresholds and responses to acute inflammatory challenge were investigated in juvenile pigs in two studies. Nociceptive thresholds were assessed on the tail root and on the hind foot using noxious mechanical and cold stimulation before and after acute inflammatory challenge by intradermal injection of 30 μg capsaicin (study 1) or 3% carrageenan (study 2) into the tail root. Four groups of 8 (study 1, n=14-16 pigs/treatment) or 5 (study 2, n=6 pigs/treatment/sex) week-old pigs were exposed to the main factors: maternal stress and treatment (docked vs. intact tails). In study 1, tail docking did not significantly alter thresholds to noxious mechanical stimulation, whilst prenatally stressed pigs had significantly higher baseline thresholds to noxious mechanical stimulation on the tail root and on the hind foot than unstressed pigs, whether tail-docked or intact. Capsaicin injection induced localised mechanical allodynia around the tail root in all treatment groups, but had no effect on noxious plantar mechanical responses; however prenatally stressed offspring exhibited significantly attenuated response thresholds to capsaicin compared to controls. In study 2 tail docking did not alter thresholds to either mechanical or noxious cold stimulation. Baseline response durations to noxious cold stimulation of the tail root were significantly shorter in both sexes of prenatally stressed pigs, whilst male but not female prenatally stressed pigs exhibited significantly higher baseline thresholds to mechanical stimulation than controls, although results in female pigs tended towards significance. Carrageenan injection into the tail root induced localised mechanical and cold allodynia in all treatment groups, effects that were attenuated in prenatally stressed pigs. Collectively, these findings indicate that prenatal stress can induce long-term alterations in nociceptive responses, manifest as a reduced sensitivity to noxious mechanical and cold stimulation and evoked inflammatory allodynia. Neonatal tail-docking does not lead to long-term alterations in nociception in pigs.

Development of a mechanical stimulator and force measurement system for the assessment of nociceptive thresholds in pigs.

A mechanical stimulator and force measurement system was developed to quantify withdrawal thresholds to noxious mechanical stimulation of the foot in young pigs. The device and associated PC software have design and control features not previously used in other mechanical stimulators. The device, capable of delivering stimulus rates between 2 and 17 mm/s, maximum force 27 N, was validated in a cross-over study on 8 juvenile pigs (6-8 weeks of age) to check the repeatability and reliability of force threshold measurement and assess its ability to measure changes in force threshold following an inflammatory challenge. Threshold force measurements were obtained over several time periods before and after the pigs received a 0.25 ml subcutaneous injection of 3% carrageenan in 0.01 M phosphate buffered saline (PBS) or PBS in the hind foot. Consistent withdrawal thresholds were measured in injected (ipsilateral) and contralateral feet, 24 h and 30 min prior to injection (mean 8.4; 95% CI 7.1-9.7 N). Carrageenan injection, but not PBS injection, induced a significant decrease in withdrawal thresholds 90 min after injection (4.6+/-0.9 N) which remained reduced for 6h after injection. The testing system provided reliable and reproducible measurements of foot withdrawal thresholds to noxious mechanical force in young pigs (weight range 32-39 kg), and was capable of detecting and monitoring changes in threshold sensitivity following the induction of acute local inflammation in the foot. The system is suitable for studying nociceptive mechanisms in pigs.

Neutering affects urinary bladder function by different mechanisms in male and female dogs

Acquired urinary incontinence is a significant, incurable problem, prevalent in neutered bitches but rarely seen in entire bitches or males. Decreased urethral closure pressure has been proposed as a causative factor for altered detrusor contractility in the neutered bitch. In post menopausal women, acquired urinary incontinence is associated with acquired urinary incontinence and changes in collagen deposition within the bladder wall. The aim of this study was to determine effects of neutering on smooth muscle in the canine urinary bladder. Tissue bath studies were used to assess contractile function and morphometric analysis to determine percentage of collagen in the bladder wall from male and female, neutered and entire canines. Maximal response to both carbachol and neurogenic stimulation was significantly lower in bladder strips from neutered animals of both sexes. Sensitivity to carbachol was also significantly reduced by neutering in both sexes. The percentage of collagen was significantly higher in the bladder wall from neutered vs. entire females, which were similar to that of both neutered and entire males. Neutering a canine decreases urinary bladder responsiveness to muscarinic stimulation in vitro, in both sexes, but only increases the percentage of collagen in the bladder wall in females. While increased percentage collagen may predispose bitches to acquired urinary incontinence, the sex difference in this parameter indicates that more than one mechanism underlies the changes in bladder responsiveness seen following neutering. This alternative effect of neutering may be in the muscarinic receptor effector pathway and act as a therapeutic target for acquired urinary incontinence treatment.

248 DEVELOPMENT OF QUANTITATIVE MECHANICAL SENSORY TESTING APPROACHES FOR PAIN ASSESSMENT IN PIGS

248 Citation: European Journal of Pain, Volume 13, Supplement 1, September 2009, Page S79 DEVELOPMENT OF QUANTITATIVE MECHANICAL SENSORY TESTING APPROACHES FOR PAIN ASSESSMENT IN PIGS D. Sandercock, I. Gibson, H. Brash, M. Scott, A. Nolan 1 University of Glasgow, Division of Cell Sciences, Faculty of Veterinary Medicine, Glasgow, United Kingdom 2 University of Edinburgh, Department of Hepatology, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom 3 University of Glasgow, Department of Statistics, Glasgow, United Kingdom Background and Aims: Obtaining accurate and repeatable measurements of sensory thresholds in freely behaving animals presents a considerable challenge. The aim of these studies was to develop methods of quantitative sensory testing (QST) in juvenile pigs and evaluate two models of acute inflammatory pain. Methods: Two QST approaches were developed to detect allodynia and hyperalgesia; (1) von Frey filaments for mechanical force threshold testing around the tail root, and (2) noxious mechanical stimulation of the plantar surface of the foot pad. Assessment protocols were developed in three groups of 8 juvenile pigs (7-9 weeks). Pigs were habituated to the investigators, apparatus and procedures before testing. Response thresholds and behaviours were measured before, and up to 24 h after inflamogen injection. Inflamation was induced by injection of capsaicin (10-100 μg) or carrageenan (3%) into the tail root or hind foot pad. Data analysed using GLM repeated measures ANOVA. Results: Inflamogen injection significantly (p < 0.05) reduced mechanical force thresholds in both tests and behavioural signs of mechanical allodynia and hyperalgesia were observed (tail flicking and foot withdrawal). Maximum reductions in force thresholds were measured 30 min after capsaicin and 4 h after carrageenan injection. Conclusions: Quantification of thresholds using the two approaches reported provides reliable data. Capsaicin and carrageenan induced inflammation and hypersensitivity was measurable in pigs, and its onset and duration was consistent with laboratory species. These approaches will be used in future studies to investigate the effects of neonatal tail-docking on nociceptive processing in pigs. Supported by BBSRC