Co-induction of cyclooxygenase-2 [correction of cyclooxyenase-2] and early growth response gene (Egr-1) in spinal cord in a clinical model of persistent inflammation and hyperalgesia.

BACKGROUND: This study characterised the effects of persistent peripheral inflammation of the foot on pain and spinal cord expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) and early growth response gene 1 (Egr-1), known markers of neuronal plasticity, in a clinical model of naturally-occurring inflammatory disease and hyperalgesia in sheep ('footrot'), before and after routine treatment (parenteral treatment with antibiotics and antiseptic footbathing). The temporal pattern of expression of COX-1, COX-2 and Egr-1 mRNA and protein were analysed using real-time PCR and Western blotting. RESULTS: Animals affected with persistent peripheral inflammation displayed significant hyperalgesia and lameness (a proxy indicator of spontaneous pain) restricted to the inflamed limb. Hyperalgesia and lameness were significantly attenuated 1 day after treatment, and resolved further by day 7 and day 3, respectively. COX-2 but not COX-1, protein expression was up-regulated in spinal cord from lame animals on day 0, before treatment. Following treatment and attenuation of pain behaviours, levels of COX-2 returned to control levels. Significant induction of Egr-1 mRNA and protein were observed in spinal cord from lame animals. Three days after treatment, levels of Egr-1 mRNA returned to control levels, however, Egr-1 protein remained elevated. CONCLUSION: Elevated levels of spinal COX-2 and Egr-1 protein correlate with the presence of pain and hyperalgesia, and may underlie persistent pain, although a direct causal link has still to be established. Understanding the temporal pattern of expression of key mediators in clinical pain states may lead to better strategies to manage pain.

Validity and Responsiveness of the Generic Health-Related Quality of Life Instrument (VetMetrica™) in Cats With Osteoarthritis. Comparison of Vet and Owner Impressions of Quality of Life Impact.

Validity is not an inherent property of a measurement scale and so evidence for validity relating to its use for particular purposes, with defined populations and in specified contexts must be accumulated. We have published the development of a web-based, generic health-related quality of life instrument (VetMetrica™) to measure the affective impact of chronic disease in cats and provided evidence for its validity in a mixed population of cats, some of which, according to veterinary judgement, were healthy and others of which were suffering from chronic conditions likely to affect their quality of life, often with multiple co-morbidities present. The first aim of the current study was to demonstrate the construct validity of the VetMetrica™ generic instrument when used with cats suffering from osteoarthritis, by testing the hypothesis that the health-related quality of life profile of cats with different severities of osteoarthritis would differ and by demonstrating convergent validity between the health-related quality of life profile scores and independently quantified vet-assessed pain and quality of life impact scores. The latter involved simple correlation analysis and investigation of the relationship between health-related quality of life domain scores and vet-assessed scores, when adjusted for other potential explanatory variables including number of comorbidities and age. Responsiveness-the ability to detect clinically relevant change-is an essential quality for an evaluative instrument and it also provides evidence for "longitudinal validity". Therefore, a second aim of this study was to demonstrate that changes in health-related quality of life domain scores concurred with the clinician's impression of change over time in the health status of cats with osteoarthritis, thus providing evidence for the instrument's responsiveness. Previously, we have reported disagreement between owner and vet impression as to health status in cats in general, but not in relation to any specific disease. Accordingly, the third study aim was to investigate the extent of agreement or disagreement between owner impression of the impact of osteoarthritis on their cats' quality of life and vet impression of such impact. Fifty one percentage of cat owners believed their cats to be perfectly healthy despite a clinician diagnosis of osteoarthritis.

Pre-natal stress amplifies the immediate behavioural responses to acute pain in piglets.

Pre-natal stress (PNS) or undernutrition can have numerous effects on an individual's biology throughout their lifetime. Some of these effects may be adaptive by allowing individuals to tailor their phenotype to environmental conditions. Here we investigated, in the domestic pig Sus scrofa, whether one possible consequence of a predicted adverse environment could be altered pain perception. The behavioural response of piglets to the surgical amputation ('docking') of their tail or a sham procedure was measured for 1 min in piglets born to mothers who either experienced mid-gestation social stress or were left undisturbed throughout pregnancy. A behavioural pain score was found to predict the docked status of piglets with high discriminant accuracy. Piglets exposed to PNS had a significantly higher pain score than controls, and for each litter of tail-docked piglets, the average pain score was correlated with mid-gestation maternal cortisol levels. The data presented here provide evidence that the experience of stress in utero can result in a heightened acute response to injury in early life. Speculatively, this may represent an adaptive alteration occurring as a consequence of a pre-natal 'early warning' of environmental adversity.

The selective metabotropic glutamate receptor 7 allosteric agonist AMN082 inhibits inflammatory pain-induced and incision-induced hypersensitivity in rat.

This study characterized the contribution of metabotropic glutamate receptor 7 (mGlu7 receptor) activation to the development of inflammatory hyperalgesia and allodynia, using a novel, systemically active mGlu7 receptor allosteric agonist, N, N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082). The effects of AMN082 (0.1, 1 or 5 mg/kg, intraperitoneally; 5 or 50 nmol, intrathecally) or diclofenac (5 mg/kg, intraperitoneally) administered 30 min preprocedure or 3 h postprocedure on hindpaw withdrawal latency (in seconds) to thermal stimulation, and response threshold (in grams) to mechanical stimulation, were measured in adult rats (n = 6-8 per group) before and up to 24 h after intradermal injection of carrageenan into the hindpaw or hindpaw incision. Precarrageenan injection of 1 and 5 mg/kg AMN082, but not diclofenac inhibited thermal hyperalgesia, whereas postcarrageenan, both AMN082 and diclofenac attenuated thermal hyperalgesia and allodynia. In the paw incision model, presurgical and postsurgical administration of 1 and 5 mg/kg AMN082 inhibited thermal hyperalgesia, but not allodynia, whereas diclofenac was effective in attenuating both thermal hyperalgesia and allodynia but only when administered postsurgically. Intrathecal injection of AMN082 postcarrageenan and postsurgery also significantly attenuated thermal hyperalgesia. Enhancing endogenous mGlu7 receptor activity inhibits postinjury stimulus-evoked hypersensitivity and may be of therapeutic benefit for the treatment of inflammatory and incision-induced pain.

Development, initial validation and reliability testing of a web-based, generic feline health-related quality-of-life instrument.

OBJECTIVES: The objective of this study was to develop a valid, reliable, web-based generic feline health-related quality-of-life (HRQoL) questionnaire instrument to measure the affective impact of chronic disease. METHODS: A large initial item pool, obtained through interviews with cat owners, was reduced using predetermined criteria, survey scores for relevance and clarity, and the ability of individual items to discriminate between healthy and sick cats when owners completed a prototype questionnaire. Using these data, factor analysis was used to derive a scoring algorithm and provide evidence for factorial validity. Validity was demonstrated further in a field trial using a 'known groups' approach (sick vs healthy cats will have a different HRQoL profile, and the HRQoL profile of cats will deteriorate as comorbidities increase). Test-retest reliability was assessed using intra-class correlation coefficients (ICCs). RESULTS: In total, 165 items were reduced to 20 and, on the basis of a factor analysis that explained 72.3% of the variation in scores input by 71 owners of 30 healthy and 41 sick cats using the prototype, these were allocated to three domains (vitality, comfort and emotional wellbeing [EWB]) with a scoring algorithm derived using item loadings. Subsequently, the owners of 36 healthy and 58 sick cats completed one or two (n = 48) assessments. Median scores (healthy vs sick) for all domains were significantly different ( P <0.001), 78% of cats were correctly classified as healthy or sick and for comorbidities the correlation coefficients were moderate (vitality 0.64; comfort 0.63; EWB 0.50). Test-retest reliability was good (ICC vitality 0.635; comfort 0.716; EWB 0.853). CONCLUSIONS AND RELEVANCE: This study provides initial evidence for the validity and reliability of a novel HRQoL instrument to aid the assessment and management of chronic diseases of cats.

Validation of a structured questionnaire as an instrument to measure chronic pain in dogs on the basis of effects on health-related quality of life.

OBJECTIVE: To validate the use of a novel questionnaire as an instrument for measurement of chronic pain in dogs through its impact on health-related quality of life (HRQL). ANIMALS: 108 dogs with chronic degenerative joint disease and 26 healthy dogs. PROCEDURES: Questionnaire responses were subjected to factor analysis (FA) and questionnaire scores to discriminant analysis to evaluate construct validity. Questionnaire scores were used to explore the potential of this instrument for minimizing respondent bias and for evaluative purposes. RESULTS: FA results revealed a sensible factor structure accounting for 65% of the variance in data, with factors identifiable as domains of HRQL in dogs affected by chronic pain. Further evidence for construct validity was provided when questionnaire scores were used to discriminate, on the basis of 218 questionnaires, between dogs with clinician-awarded pain scores of 0 and dogs with pain scores >or= 1 (88% discrimination, with 95% of no-pain group dogs and 87% of some-pain group dogs correctly categorized). Use of the questionnaire provided minimized respondent bias. CONCLUSIONS AND CLINICAL RELEVANCE: Validation of the questionnaire as an instrument for discriminative and evaluative measurements of orthopedic chronic pain through its impact on HRQL in dogs was provided. Use of the questionnaire, with further testing and refinement, may support improved clinical decision making, facilitate development of evidence-based therapeutic options for chronic diseases, and help veterinarians and owners define humane end points in dogs. IMPACT FOR HUMAN MEDICINE: Information gained here may provide improved measurements of clinical change in animal studies that use dogs with naturally occurring chronic pain to evaluate novel human treatment protocols.

Application of a scaling model to establish and validate an interval level pain scale for assessment of acute pain in dogs.

OBJECTIVE: To establish interval level measurement in a prototype composite measure pain scale (CMPS) for assessment of acute pain in dogs and to investigate the scale's validity. ANIMALS: 20 clinically normal dogs, 20 dogs with medical conditions, and 117 dogs undergoing surgery. PROCEDURE: First, a scaling model was applied to the CMPS descriptors to establish weights for each and create a continuous scale. Subsequently, 5 observers independently used the scale to score signs of pain in 4 groups of dogs (control dogs, dogs with medical conditions, and 40 dogs undergoing soft tissue or orthopedic surgery). Scores from each group and from groups of conditions perceived to cause no, mild, moderate, and severe pain were compared. In addition, the scale was applied to 77 dogs undergoing orthopedic or soft tissue surgery and scores were compared with simultaneously derived numeric rating scale (NRS) scores; comparisons were made between surgical groups and with time after surgery. RESULTS: Calculated scale descriptor weights ranged from -2.0 to 2.0 and were transformed to create a continuous scale from 0 to 10. Median CMPS scores differed significantly among the 4 study groups and among pain severity groups and were typically greater with increasing perceived pain severity. Agreement was determined between CMPS and NRS scores, and there was a significant and expected time effect and difference between the CMPS scores of dogs undergoing orthopedic and soft tissue surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that this interval level measurement scale is a valid measure of acute pain in dogs.

Repeated intradermal injection of low-dose carrageenan induces tachyphylaxis to evoked hyperalgesia.

Changes in the thresholds to noxious mechanical and thermal stimulation were monitored in a group of normal sheep at different times (30 min-24 h) following intradermal injection of either low-dose (0.0625%) carrageenan, a widely used mucopolysaccharide irritant, or saline (0.9%) into the lateral aspect of the fore limb. Thresholds to noxious mechanical stimulation were measured on the ipsilateral limb whereas thresholds to noxious thermal stimulation were measured at a site distant to that of the injection, the ipsilateral ear. During the first 2 h after carrageenan injection the thresholds to noxious thermal stimulation fell, while thresholds to noxious mechanical stimulation were unaltered. The evoked hyperalgesia was reversed by the administration of non-steroidal anti-inflammatory agents. Hyperalgesia was not present 4 h after carrageenan injection in control or treated animals. This experiment was carried out on 3 further occasions and a diminishing response to injection of carrageenan was observed, suggesting development of tachyphylaxis to evoked thermal hyperalgesia.

The effect of abdominal surgery on thresholds to thermal and mechanical stimulation in sheep.

Thresholds to noxious mechanical and thermal stimulation were measured in 6 groups of sheep prior to induction of anaesthesia and subsequently for a period of 2 h in the post-anaesthetic period. Groups 1-4 were anaesthetised using thiopentone and underwent ventral midline laparotomy. Four animals (group 5) underwent anaesthesia but not surgery, and a further 6 sheep (group 6) undergoing surgery were anaesthetised using ketamine. Groups 1-3 were intravenously administered the following drugs intra-operatively: flunixin meglumine, carprofen and buprenorphine, respectively. Groups 4-6 received no additional treatment. Thresholds to the mechanical test were not changed in the post-anaesthetic period for any group. There was a significant reduction in the responses to thermal stimulation after surgery for sheep in group 4 (45 and 60 min), while sheep in group 2 had thresholds to thermal stimulation greater than those recorded in the remaining groups at all time points post-operatively. Responses to thermal stimulation in sheep undergoing anaesthesia but not surgery (group 5) were unaltered during the 2 h recording period after anaesthesia ended. These data indicate that abdominal surgery induces thermal but not mechanical hyperalgesia in sheep, which appears to be centrally mediated. Moreover, the absence of mechanical hyperalgesia raises the possibility that central changes in noxious information processing may not be detected using mechanical stimuli in the same time course as thermal stimuli.

The role of nitric oxide and prostaglandin signaling pathways in spinal nociceptive processing in chronic inflammation.

Both nitric oxide (NO) and prostaglandins (PG) and their associated enzymes nitric oxide synthases (NOS) and cyclooxygenases (COX) (specifically COX-2) have been implicated in the development of hyperalgesia. This study examined the effects of naturally occurring chronic inflammation, chronic mastitis, on spinal nociceptive processing in sheep and focused on potential alterations in spinal PG and NO signaling pathways. Mechanical withdrawal thresholds were significantly lower in animals suffering from chronic inflammation (n=6) compared to control animals (n=6). Hyperalgesia was restricted to the side contralateral to the inflammation (decrease from ipsilateral side: hindlimb 33.2+/-5%, forelimb 19.4+/-5%). Neuronal NOS-immunoreactivity was significantly reduced bilaterally in lumbar and cervical spinal cord throughout laminae I-III (decrease 18.4+/-5% and 16.9+/-4%, respectively) and in lamina X (decrease 29.1+/-6% and 17.1+/-4%, respectively) in mastitic animals relative to control animals. No difference was detected in eNOS or iNOS-immunoreactivity or in NADPH-diaphorase staining, a marker of dynamically active NOS. RT-PCR failed to detect any change in levels of nNOS, eNOS, iNOS, COX-1 or COX-2 mRNAs. However, a marked increase in the PGE receptor, EP(3) (but not EP(2)) mRNA was detected in ipsilateral spinal cord tissue from animals with chronic inflammation. This increase in EP(3) receptor expression indicates that spinal PGs are important in the spinal response to chronic peripheral inflammation. Contralateral mechanical hyperalgesia may not be directly linked to changes in spinal EP(3) receptor mRNA expression, however, the bilateral changes in nNOS suggest that this pathway may contribute to the adaptive behavioural response observed.

Differential expression of central metabotropic glutamate receptor (mGluR) subtypes in a clinical model of post-surgical pain.

Tissue damage during surgery can induce 'central sensitization' and the development of pain and hyperalgesia post-operatively. Metabotropic glutamate receptors (mGluRs) contribute to nociception, inflammatory pain and hyperalgesia. This study characterized the temporal expression of group I (mGluR(1), mGluR(5)) and II (mGluR(2), mGluR(3)) mGluRs in spinal cord following abdominal surgery. Lumbar spinal cord was recovered from adult sheep euthanased 5 h, 1, 2, 3 and 6 days after undergoing a midline laparotomy, and processed for mGluR mRNA (real-time PCR, in situ hybridization) and protein (Western blotting). mGluR(5) mRNA was up-regulated 5 h and 1 day post-surgery in laminae I-II of the spinal cord dorsal horn. mGluR(5) protein was increased 1 day post-surgery. A delayed induction of mGluR(2) and mGluR(3) mRNAs and mGluR(2/3) protein occurred in spinal cord 3 days after surgery. By 6 days, mGluR(2) mRNA levels had returned to normal, however, mGluR(3) mRNA and mGluR(2/3) protein remained elevated. No change was detected in mGluR(1). These results demonstrate that mGluRs are differentially regulated following surgery and support a link between mGluR-mediated activity and post-surgical pain.

Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia.

Evidence from experimental pain research has revealed that metabotropic glutamate receptors (mGluRs) play a pivotal role in nociceptive processing, inflammatory pain and hyperalgesia. The aim of this study was to characterise expression of group I and II mGluRs in spinal cord in a model of naturally occurring persistent inflammation (sheep with unilateral lameness due to inflammation of the digital tissues of the feet, estimated to have been affected by the condition for >2 weeks) and an experimental model of acute inflammation (injection of intradermal carrageenan into lower forelimb in sheep). Animals with unilateral clinical inflammation displayed significant mechanical hyperalgesia on the affected limb. Carrageenan treatment produced significant bilateral limb mechanical hyperalgesia 3 h post-injection. Up-regulation of mGluR(3) and mGluR(5) mRNA was observed in ipsilateral spinal cord recovered from clinically lame animals, restricted to laminae II-V and I-II, respectively. Western blot analyses of protein extracts revealed a bilateral increase in mGluR(2/3) and mGluR(5). No change was detected in spinal cord mGluR(1) or mGluR(2) mRNA. There was no change in mGluR(1,2,3,5) subtype mRNA or proteins in spinal cord recovered from animals 3 h post-carrageenan. These results demonstrate for the first time that mGluR subtypes are differentially expressed in spinal cord dorsal horn in response to persistent inflammation, and suggest that mGluR activity may be involved in mediating altered behaviours associated with clinical inflammatory pain.

Expression of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor in sheep spinal cord.

Consistent with its neuroendocrine role, gonadotropin-releasing hormone (GnRH) is located principally within the hypothalamus, although extra-hypothalamic expression has been reported. The present study characterized the expression of GnRH and GnRH receptor (GnRH-R) in sheep spinal cord using real-time PCR and immunocytochemistry. Both GnRH and GnRH-R mRNA were detected in sheep spinal cord. Expression of GnRH peptide was localized to discrete locations in the spinal cord, including lamina X (the area surrounding the central canal) and motoneurons in the ventral horn. Although there is no known functional role for GnRH in spinal cord, a role as a potential neurotransmitter/neuromodulator is supported by the expression of both GnRH and GnRH-R in this tissue.

Development of a questionnaire to measure the effects of chronic pain on health-related quality of life in dogs.

OBJECTIVE: To develop a reliable, validated questionnaire that can be used for the assessment of chronic pain and its impact on health-related quality of life (HRQL) in dogs. SAMPLE POPULATION: 17 owners of dogs that had chronic pain associated with chronic degenerative joint disease and 165 other dog owners. PROCEDURES: Psychometric methods were used to identify relevant domains, create an item pool, select and validate items, and construct and preliminarily test a structured questionnaire. Relevant domains were identified through semi structured interviews. Descriptor-generating exercises provided the terms owners used to describe these domains and formed an item pool. A selection from this pool was validated and used to construct a questionnaire that underwent preliminary testing. RESULTS: The structured questionnaire contained 109 simple, familiar, descriptive terms associated with good health or chronic pain (most describing subtle aspects of behavior that owners interpreted as expressions of subjective experiences of their dogs) for 13 possible HRQL domains. Each descriptor was associated with a 7-point numeric scale. CONCLUSIONS AND CLINICAL RELEVANCE: The questionnaire was intended to facilitate rapid, sensitive, and accurate rating of a comprehensive range of relevant domains by naïve raters with minimal burden on respondents. The principles underlying the development and design of this structured questionnaire offer a novel approach to the proxy measurement of HRQL and changes in HRQL associated with chronic pain for a range of animal species. IMPACT FOR HUMAN MEDICINE: This novel approach may be applicable to other nonverbal populations (eg, young children or elderly people with cognitive impairment).

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.

Characteristics of the in vitro hypoxic pulmonary vasoconstrictor response in isolated equine and bovine pulmonary arterial rings.

OBJECTIVE: Hypoxaemia accompanies dorsal recumbency in the horse and frequently complicates general anaesthesia. The physiology associated with this phenomenon is poorly understood. One possible cause of poor tolerance to dorsal recumbency is an absent or reduced response to hypoxic pulmonary vasoconstriction (HPV). This study compared the HPV response in isolated pulmonary artery vessels from equivalent regions of equine and bovine lung. ANIMALS: Equine and bovine, in vitro study. MATERIALS AND METHODS: Equine and bovine pulmonary arteries were removed from the lungs of euthanased horses and cattle. Measurements of isometric tension were made on isolated rings of pulmonary vessels at 37 degrees C in a Krebs' saline solution. Hypoxia was induced by bubbling with a nominally 0% O(2) gas mixture. RESULTS: A significant HPV response was observed above a baseline tension induced by phenylephrine (PE; 0.3 microm) or 5-hydroxytryptamine (5-HT; 0.1 microm). The HPV response in equine pulmonary vessels was approximately 33% less than the response observed in equivalent bovine vessels (equine 196 +/- 20%versus bovine 290 +/- 32%; p < 0.05). Removal of the endothelium (by rubbing the luminal surface) significantly reduced but did not abolish the HPV response. Incubation with the nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME; 100 microm), or COX-1/COX-2 inhibitor indomethacin (10 microm) markedly attenuated the HPV response in equine vessels. CONCLUSIONS: These results suggest that a significant HPV response exists in isolated equine pulmonary vessels; a component of this response requires a functional endothelium. Inhibition of cyclooxygenase and NO synthase attenuated the response, suggesting the involvement of a COX product and/or NO in mediating this effect either directly or indirectly. Alternatively, a non-COX related action of the nonsteroidal anti-inflammatory drug, indomethacin, may be involved.

Transient up-regulation of spinal cyclooxygenase-2 and neuronal nitric oxide synthase following surgical inflammation.

BACKGROUND: Surgery induces pain and hyperalgesia postoperatively. The products of cyclooxygenases and nitric oxide synthase (NOS) have been implicated in the development of inflammatory pain and hyperalgesia experimentally, and the use of drugs clinically that modify cyclooxygenase activity has been advocated in the management of perioperative pain. However, regulation of these enzymes following surgery has not been studied. METHODS: Adult female sheep (n = 12) undergoing a midline laparotomy for collection of ova were used in this study. Lumbar and cervical spinal cord tissue was collected from animals euthanized 1 day and 6 or 7 days after surgery and processed for cyclooxygenase (cyclooxygenase-1 and cyclooxygenase-2), neuronal NOS mRNA expression using reverse-transcription polymerase chain reaction and hybridization. Tissues were also processed for NADPH-diaphorase staining and cyclooxygenase-1 and cyclooxygenase-2 protein expression by immunohistochemistry and Western blotting. RESULTS: No alteration in cyclooxygenase-1 or cyclooxygenase-2 mRNA or protein concentrations were detected in spinal cord by reverse-transcription polymerase chain reaction and Western blotting, respectively, at 1 day or 6 or 7 days after surgery. However, using techniques that localize mRNA and protein expression ( hybridization and immunohistochemistry, respectively), increases in cyclooxygenase-2 were identified in lamina V dorsal horn neurons in lumbar spinal cord 1 day after surgery. A significant increase in neuronal NOS mRNA was observed in lumbar spinal cord 1 day after surgery, localized to laminae I-II and lamina V neurons, which returned to baseline concentrations by 6 to 7 days. NADPH-diaphorase staining was significantly increased in laminae I-II in lumbar spinal cord 1 day after surgery but not after 6 to 7 days. CONCLUSIONS: Spinal cyclooxygenase and neuronal NOS pathways are differentially altered following surgical inflammation. The early and transient nature of these changes suggests that these enzymes are implicated in postoperative pain and hypersensitivity.

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis.

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.