Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish.

Research has recently demonstrated that larval zebrafish show similar molecular responses to nociception to those of adults. Our study explored whether unprotected larval zebrafish exhibited altered behaviour after exposure to noxious chemicals and screened a range of analgesic drugs to determine their efficacy to reduce these responses. This approach aimed to validate larval zebrafish as a reliable replacement for adults as well as providing a high-throughput means of analysing behavioural responses. Zebrafish at 5 days post-fertilization were exposed to known noxious stimuli: acetic acid (0.01%, 0.1% and 0.25%) and citric acid (0.1%, 1% and 5%). The behavioural response of each was recorded and analysed using novel tracking software that measures time spent active in 25 larvae at one time. Subsequently, the efficacy of aspirin, lidocaine, morphine and flunixin as analgesics after exposure to 0.1% acetic acid was tested. Larvae exposed to 0.1% and 0.25% acetic acid spent less time active, whereas those exposed to 0.01% acetic acid and 0.1-5% citric acid showed an increase in swimming activity. Administration of 2.5 mg l-1 aspirin, 5 mg l-1 lidocaine and 48 mg l-1 morphine prevented the behavioural changes induced by acetic acid. These results suggest that larvae respond to a noxious challenge in a similar way to adult zebrafish and other vertebrates and that the effect of nociception on activity can be ameliorated by using analgesics. Therefore, adopting larval zebrafish could represent a direct replacement of a protected adult fish with a non-protected form in pain- and nociception-related research.

GABAergic regulation of auditory sensory gating in low- and high-anxiety rats submitted to a fear conditioning procedure.

The inferior colliculus (IC) is primarily involved in the processing of acoustic stimuli, being in a position to send auditory information to motor centers that participate in behaviors such as prey catching and predators' avoidance. The role of the central nucleus of the IC (CIC) on fear and anxiety has been suggested on the basis that rats are able to engage in tasks to decrease the aversiveness of CIC stimulation, increased Fos immunolabeling during diverse aversive states and increased CIC auditory evoked potentials (AEP) induced by conditioned fear stimuli. Additionally, it was shown that brainstem AEP, represented by wave V, for which the main generator is the IC, is increased during experimentally-induced anxiety. Rats segregated according to their low or high emotional reactivity have been used as an important tool in the study of fear and anxiety. The IC contains a high density of GABA receptors. Since the efficacy of an anxiolytic compound is a function of the animal's anxiety level, it is possible that GABA-benzodiazepine (Bzp) agents affect LA and HA animals differently. In this study we investigated the GABA-Bzp influence on the modulation of AEP in rats with low- (LA) or high-anxiety (HA) levels, as assessed by the elevated plus-maze test (EPM). GABA-Bzp modulation on the unconditioned AEP response was analyzed by using intra-CIC injections (0.2 µl) of the GABA-Bzp agonists muscimol (121 ng) and diazepam (30 µg), or the GABA inhibitors bicuculline (10 ng) and semicarbazide (7 µg). In a second experiment, we evaluate the effects of contextual aversive conditioning on AEP using foot-shocks as unconditioned stimuli. On the unconditioned fear paradigm GABA inhibition increased AEP in LA rats and decreases this measure in HA counterparts. Muscimol was effective in reducing AEP in both LA and HA rats. Contextual fear stimuli increased the magnitude of AEP. In spite of no effect obtained with diazepam in LA rats the drug inhibited AEP in HA animals. The specificity of the regulatory mechanisms mediated by GABA-Bzp for the ascending neurocircuits responsible for the acquisition of aversive information in LA and HA animals shed light on the processing of sensory information underlying the generation of defensive reactions.

Effects of drugs used in endotoxic shock on oxidative stress and organ damage markers.

The aim of this study was to determine the effects of enrofloxacin (ENR), flunixin meglumine (FM) and dexamethasone (DEX) on antioxidant status and organ damage markers in experimentally-induced endotoxemia. Rats were divided into three groups. To induce endotoxemia, lipopolysaccharide (LPS) was injected into all groups, including the positive control. The two other groups received the following drugs (simultaneously with LPS): ENR + FM + low-dose DEX and ENR + FM + high-dose DEX. After the treatments, blood samples were collected at 0, 1, 2, 4, 6, 8, 12, 24 and 48 h. Oxidative stress parameters were determined by ELISA, while serum organ damage markers were measured by autoanalyser. LSP increased (p < 0.05) malondialdehyde, 13,14-dihydro-15-keto-prostaglandin F(2 alpha) and nitric oxide, while LPS reduced vitamin C. These changes were especially inhibited (p < 0.05) by ENR + FM + high-dose DEX. LPS increased organ damages markers. Cardiac and hepatic damage was not completely inhibited by any treatment, whereas renal damage was inhibited by two treatments. This study suggested that ENR + FM + high-dose DEX is most effective in the LPS-caused oxidative stress and organ damages.

Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows.

Study objectives were to determine whether a nonsteroidal antiinflammatory drug would reduce parturition-induced inflammation and fever and consequently improve appetite, bioenergetic parameters, and production variables in transitioning dairy cows. Multiparous cows (n = 26) were randomly assigned to 1 of 2 treatments beginning at parturition: 1) flunixin meglumine (FM; 2.2 mg/kg of BW; Banamine, 50 mg/mL, Schering-Plough Animal Health, Kenilworth, NJ), or 2) saline (control) at 2.0 mL/45.5 kg of BW. All treatments were administrated i.v. daily for the first 3 d in milk (DIM). Individual milk yield and dry matter intake (DMI) were recorded daily for the first 35 DIM. Rectal temperature was measured daily at 0700 and 1600 h for the first 7 DIM. Milk composition was determined on 2, 7, 14, 21, 28, and 35 DIM and blood plasma was collected on 1, 2, 3, 4, 7, 14, 21, 28, and 35 DIM. Body weight and body condition score were determined on -7, 1, 7, 14, 21, 28, and 35 DIM. Flunixin meglumine treatment slightly increased rectal temperature (38.99 vs. 38.76 degrees C) during the first 7 DIM and reduced overall DMI (22.04 vs. 19.48 kg/d), but there were no treatment differences in overall milk yield (35.2 kg/d), 3.5% fat-corrected milk (37.6 kg/d), energy-corrected milk (37.7 kg/d), DMI (2.97% of BW), or overall energy balance (-2.32 Mcal/d). There were no treatment differences in milk fat (3.91%), protein (3.32%), or lactose (4.57%). Treatment had no effect on plasma glucose (66.5 mg/dL) or nonesterified fatty acids (553 microEq/L), but plasma urea nitrogen tended to be less in FM-treated cows (16.4 vs. 14.5 mg/dL). Daily FM administration to cows for the first 3 d after parturition slightly increased rectal temperatures by 0.23 degrees C, reduced feed intake, and did not improve production or energetic variables during the first 35 DIM in transition dairy cows.

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa.

OBJECTIVE: To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). ANIMALS: 14 adult horses. PROCEDURE: Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. RESULTS: Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.

Levels of progesterone and changes in prostaglandin F(2alpha) release during luteolysis and early pregnancy in llamas and the effect of treatment with flunixin meglumine.

The secretory patterns of progesterone in relation to concentrations of 15-ketodihydro-PGF(2alpha) (PGFM) during the period of luteolysis or of maternal recognition of pregnancy were determined in the blood of llamas mated either with an intact or a vasectomized male. The ability of flunixin meglumine (FM) to postpone luteolysis in non-pregnant llamas was investigated by injecting the drug intravenously every 6 h at a dose of 2.2 mg/kg from days 6 to 12 post-copulation into a group of non-pregnant llamas. A pulsatile pattern of prostaglandin release was recorded during luteolysis in non-pregnant llamas, giving further support to the hypothesis that PGF(2alpha) is the luteolytic agent in llamas. The mean number of peaks per animal rose from 0.3 on day 7 to 3.8 on day 10 and then declined to 1.1 on day 12 with corresponding mean peak amplitude changing from 465 to 1234 and 566 pmol l(-1), respectively. In pregnant llamas, prostaglandin pulsatile release also occurred. The mean number of peaks per animal rose from 0.4 on day 7 to 0.8 on day 10 and then declined to 0.2 on day 11 and 0.6 on day 12, with corresponding mean peak amplitude changing from 494 to 676, 388 and 547 pmol l(-1), respectively. The transient decrease and subsequent recovery in progesterone concentrations was observed to occur in connection with prostaglandin release during early pregnancy. Oestradiol-17beta plasma peak concentrations attained after luteolysis were significantly higher than those recorded in early pregnant animals (around 30 pmol l(-1) and ll pmol l(-1)). Concentrations of PGFM decreased rapidly after the first administration of FM and remained low throughout the first 2 days of treatment. Thereafter, pulsatile release of prostaglandins started, and luteolysis proceeded; but a delay of 1-1.5 days in the progesterone decline was observed. Thus, it might be suggested that a higher dose and/or a more intensive injection schedule is required in llamas than in other ruminants to prevent luteolysis.

Failure of dietary bentonite clay, Silent Herder mineral supplement, or parenteral Banamine to alleviate locoweed toxicosis in rats.

To evaluate treatments purportedly beneficial for livestock grazing locoweeds (LW), growing rats were fed diets containing 10 or 20% whole-plant Oxytropis sericea (LW) with and without Silent Herder mineral mix (1.5% of diet) or bentonite clay (1.5% of diet). Pregnant female rats fed 10% LW were treated i.m. with Banamine (a prostaglandins suppressor) or saline. The LW contained swainsonine (430 micrograms/g DM) and elicited toxicosis within 10 d at intake of 2 mg/kg BW. In Trial 1, 96 immature male Sprague-Dawley rats (BW approximately 100 g) were fed commercial rat feed (CRF) with and without LW, as follows: 100% CRF, free choice; 100% CRF, restricted intake to equal average intake of rats consuming 10 and 20% LW; 90% CRF+10% LW free choice; and 80% CRF+20% LW free choice. Diets with LW contained either no supplement or supplemental mineral mixture (Silent Herder, 1.5% of diet) or added bentonite clay (1.5% of diet). Twelve rats received each of eight dietary regimens through 28 d. Locoweed depressed (P < .05) feed intake and BW gain, increased (P < .05) relative size of liver, kidneys, heart, spleen, and testes, and altered blood serum components (P < .05) indicating toxicosis. Dietary provision of Silent Herder or bentonite failed to benefit rats that ingested approximately 4 or 8 mg of swainsonine/kg BW daily through 28 d. In Trial 2, 68 young adult female Sprague-Dawley rats (approximately 230 g BW) were mated and directly assigned to three diets (100% CRF, free choice, 100% CRF, intake restricted slightly below average intake of diet by rats consuming LW, or 90% CRF+10% LW free choice) and two treatments (i.m. saline or i.m. Banamine at .25 mg/kg BW daily for 10 d) in a 3 x 2 factorial arrangement. Approximately half (31 of 68) of the impregnated rats were killed at d 10, when Banamine was discontinued, but diets were continued until the remaining females gave birth. Ingested LW provided approximately 2 mg swainsonine/kg BW daily and elicited toxicosis in 10 d, but LW failed to affect numbers of live concepti at d 10 (P > .5) or numbers of offspring at parturition (P > .10). Banamine did not alleviate LW toxicosis of dams (P > .10). Provision of Silent Herder or bentonite in the diet or Banamine i.m. had no benefit for rats fed toxic locoweed.

Aflatoxin B1 alters central and systemic tryptophan and tyrosine metabolism: influence of immunomodulatory drugs.

Semi-chronic exposure of ICR male Mice to Aflatoxin B1 (AFB1) in non-toxic doses results in elevated lung tryptophan (TRP) levels without change in serotonin (5-HT) or 5-hydroxyindole-3-acetic acid (5-HIAA) levels. This change is organ specific in that TRP levels are not altered in spleen, duodenum, heart or central nervous system (CNS). Acute (48 hour) flunixin treatment decreases lung TRP levels and reverses the AFB1 mediated increase in lung TRP levels. On the other hand, flunixin treatment decreases CNS TRP levels in control mice but not in AFB1 treated mice. Aflatoxin B1 treated mice have an increase in splenic serotonin (5-HT) content. Acute (48 hour) treatment of mice with E. coli lipopolysaccharide (LPS) also increases splenic 5-HT, and AFB1 treatment followed by LPS have a slightly additive effect on spleen 5-HT content. Treatment of mice with LPS increases heart 5-HT, an effect which is not altered in AFB1 pretreated mice. Both LPS and AFB1 per se increases lung TYR levels although the combination of treatments is not significantly different from the control value. Flunixin treatment increases lung tyrosine (TYR) levels, an effect which is not altered by AFB1 pretreatment. Acute treatment with either LPS or flunixin decreases the CNS TRP/TYR ratio; pretreatment with AFB1 prevents those changes in the CNS TRP/TYR ratio. Central nervous system catecholamines are reduced in AFB1 pretreated mice. However, CNS catecholamine changes in AFB1 treated mice are normalized by vitamin E supplementation during the treatment period.

Castor-oil induced diarrhoea in ponies: a model for acute colitis.

A reproducible, reversible model of colitis induced in ponies by administering castor oil (2.5 ml/kg bodyweight [bwt] per os) was characterised by abdominal pain, fever, watery diarrhoea, dehydration, hypovolaemia, toxaemia, leucopenia, decreased serum Cl, Na and K levels and metabolic acidosis. The signs were most severe between 24 and 48 h post induction, stabilisation was frequently observed after 72 h, although diarrhoea could persist beyond 96 h. Morphological and in vitro transport studies (right ventral colon) were conducted on tissues from animals destroyed at 24, 48 and 72 h. In the caecum and colon, surface epithelial disruption and exfoliation from the basement membrane occurred between 24 and 48 h. Early signs of recovery were evident by replenishment of denuded areas with columnar epithelium at 72 h. The crypt epithelium was unaffected throughout the intestinal tract. In vitro transport studies were consistent with the morphological findings. Decreased Na-Cl absorption and normal Cl secretion indicated an impaired surface epithelium coincident with an undamaged cryptal epithelium. Increased mucosal permeability was demonstrated by high ionic conductance and large unidirectional isotopic fluxes. Tissue conductance improved during in vitro incubation suggesting epithelial repair after removal of castor oil. Changes in the population and proportion of bacteria in the faeces as diarrhoea ensued were confirmed at necropsy with a predominance of E. coli and Enterobacter/Klebsiella sp in the large bowel. The experimental induction of castor oil colitis showed many similarities to intestinal endotoxaemia and the secretory type diarrhoea encountered in naturally occurring acute colitis syndromes in horses. The model could prove applicable in studying the pathophysiological mechanisms precipitating such life-threatening disorders.

Comparison of detomidine, butorphanol, flunixin meglumine and xylazine in clinical cases of equine colic.

Detomidine hydrochloride, butorphanol tartrate, flunixin meglumine and xylazine hydrochloride were evaluated in a blind multi-centre clinical trial in 152 horses with abdominal pain. The drugs were administered as follows: detomidine 20 or 40 micrograms/kg bodyweight (bwt); butorphanol 0.1 mg/kg bwt; flunixin meglumine 1.0 mg/kg bwt; xylazine hydrochloride 0.5 mg/kg bwt. Each centre compared responses to the two doses of detomidine with those to one of the other analgesics. The drugs were administered intravenously (i.v.) after clinical assessment of the degree of sweating, kicking, pawing, head and body movement, attitude, lip curling, stretching to urinate, pulse rate, respiratory rate and rectal temperature. Similar assessments were repeated at 15 min intervals for at least 1 h. The investigators ranked the response to treatment from 'not satisfactory' to 'highly satisfactory'. Significant differences in sweating, kicking, pawing, head and body movement, attitude, pulse rate and respiratory rate were noted between the horses receiving butorphanol and either dose of detomidine. The investigators' subjective evaluation of the analgesic and sedative effects of either dose of detomidine were significantly better than for butorphanol. Analgesia was rated as highly satisfactory or satisfactory in 93.3 per cent and 6.7 per cent of the horses receiving 40 micrograms/kg bwt of detomidine, 73.3 per cent and 26.7 per cent of the horses receiving 20 micrograms/kg bwt of detomidine, and none of the horses receiving butorphanol. There were no differences in the incidence of side effects with the two compounds. Significant differences were noted in kicking, pawing, head and body movement and attitude between the horses receiving flunixin meglumine and either dose of detomidine. Flunixin meglumine provided significantly less analgesia than either dose of detomidine. Analgesia was rated as highly satisfactory or satisfactory in 73.7 per cent and 21.0 per cent of the horses receiving 40 micrograms/kg bwt of detomidine, 42.9 per cent and 21.4 per cent of the horses receiving 20 micrograms/kg bwt of detomidine, and 6.3 per cent and 37.5 per cent of the horses receiving xylazine. Sedation was considered to be at least satisfactory in 84.2 per cent of the horses receiving 40 micrograms/kg of detomidine, 71.5 per cent of the horses receiving 20 micrograms/kg of detomidine and 53.3 per cent of the horses receiving xylazine.

Effects of inhibitors of arachidonic acid metabolism on endotoxin-induced ocular inflammation.

Rabbits were treated with the cyclooxygenase inhibitors indomethacin (2.5 and 10 mg/kg; IP) or flunixin meglumine (2.2 and 8.8 mg/kg; IM), or the dual cyclooxygenase/lipoxygenase inhibitor BW755C (10 and 30 mg/kg; IP) (administered 4 times over 25 hours). One hour following drug administration, 10 ng of E. coli endotoxin was injected into the vitreal chamber and the subsequent inflammatory response was measured 24 h later. Indomethacin was the most effective inhibitor of prostaglandin (PG) E2 and PGF 2 alpha accumulation in the aqueous humor and their ex vivo production by the lens. It was the only agent that decreased iridal hyperemia and protein concentration in the aqueous humor. Flunixin decreased PG production, although generally to a lesser degree than indomethacin. BW755C produced modest inhibition of PG production in the aqueous humor, but did not affect PG production by the lens. The high dose of BW755C also reduced 5-hydroxyeicosatetraenoic acid (5-HETE) levels in the aqueous humor, but did not alter production by the lens. The results indicate that despite drastic reductions in PG levels in the aqueous humor and PG production by the lens, only modest decreases in aqueous humor protein concentration and iridal hyperemia were achieved. Furthermore, 5-HETE does not appear to be important in the modulation of these inflammatory signs, although it is possible that larger decreases in its production could result in more dramatic effects.

Flunixin meglumine: a non-narcotic analgesic.

The N-methyl-d-glucamine salt of flunixin (flunixin meglumine) is a potent non-narcotic analgesic agent after parenteral administration in mice, rats and monkeys. It is significantly more potent than pentazocine, meperidine and codeine in the rat yeast paw test after subcutaneous administration in saline. Activity on intramuscular administration is comparable to that after subcutaneous administration and is enhanced when dissolved in buffered saline as compared to nonbuffered saline. In addition, flunixin meglumine also had oral activity and differs from indomethacin in having more analgesic activity per unit of anti-inflammatory activity. In mice, flunixin meglumine is equipotent to pentazocine and more potent than meperidine and codeine in the abdominal constriction test. In primates, flunixin meglumine at 10 mg/kg i.m., produced a degree of analgesic efficacy comparable to that of a clinically effective dose of morphine (0.3 mg/kg). In contrast to codeine, tolerance to the analgesic action of flunixin meglumine was not observed. Furthermore, flunixin meglumine retained its activity in rats made tolerant to codeine. Unlike narcotics, the analgesic effect of flunixin meglumine is not antagonized by naloxone after acute administration in rats. These results indicate that flunixin meglumine is a parenterally and orally effective analgesic in animals and is unlikely to have narcotic or drug dependence liability.