The magnitude and duration of the analgesic effect of morphine, butorphanol, and buprenorphine in rats and mice.

This study was designed to determine the magnitude and duration of the analgesic effect of three commonly used opioids: buprenorphine (0.5 mg/kg for rats; 2.0 mg/kg for mice), butorphanol (2.0 mg/kg for rats; 5.0 mg/kg for mice), and morphine (10 mg/kg for rats and mice). We used two standard tests, the hot plate and tail flick assays, to measure opioid analgesia in 62 male, 200 to 300 g Sprague-Dawley rats and 61 male, 25 to 35 g ICR mice. We obtained five baseline measurements then administered the drugs subcutaneously. Morphine gave the highest analgesic effect and was intermediate in duration (2 to 3 h in rats and mice) of analgesia. Butorphanol provided the lowest level of and shortest (1 to 2 h in rats and mice) analgesia. Buprenorphine had an intermediate analgesic effect and the longest duration (6 to 8 h in rats and 3 to 5 h in mice). In light of our results, we recommend the use of morphine (with frequent redosing) for severe pain, butorphanol for mild pain of short duration, and buprenorphine for mild to moderate pain of increased duration. The dosing intervals suggested by our study are 2 to 3 h for morphine in both rats and mice, 1 to 2 h for butorphanol in both rats and mice; and 6 to 8 h in rats and 3 to 5 h in mice for buprenorphine.

Regional changes in forebrain activation during the early and late phase of formalin nociception: analysis using cerebral blood flow in the rat.

This is the first neural imaging study to use regional cerebral blood flow (rCBF) in an animal model to identify the patterns of forebrain nociceptive processing that occur during the early and late phase of the formalin test. We measured normalized rCBF increases by an autoradiographic method using the radiotracer [99mTc]exametazime. Noxious formalin consistently produced detectable, well-localized and typically bilateral increases in rCBF within multiple forebrain structures, as well as the interpeduncular nucleus (Activation Index, AI = 66) and the midbrain periaqueductal gray (AI = 20). Structures showing pain-induced changes in rCBF included several forebrain regions considered part of the limbic system. The hindlimb region of somatosensory cortex was significantly activated (AI = 31), and blood flow increases in VPL (AI = 8.7) and the medial thalamus (AI = 9.0) exhibited a tendency to be greater in the late phase as compared to the early phase of the formalin test. The spatial pattern and intensity of activation varied as a function of the time following the noxious formalin stimulus. The results highlight the important role of the limbic forebrain in the neural mechanisms of prolonged persistent pain and provide evidence for a forebrain network for pain.

Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats.

A series of studies was undertaken to determine whether CO2 can be used as a humane as well as practical agent for euthanasia or anesthesia of rats. Human volunteers rated the degree of discomfort associated with breathing 50 to 100% CO2 mixed with oxygen. Increasing concentrations of CO2 were judged as progressively more noxious, from "highly unpleasant" for 50% CO2 to "painful" for 100% CO2. The practical aspects of anesthesia and euthanasia with 50 to 100% CO2 were studied, using male Sprague Dawley rats. Time to anesthesia and death were inversely related to CO2 concentration, as were the frequency and severity of adverse reactions, including seizures and hemorrhaging from the nose. The severity of edema and hemorrhage, which were observed on histologic examination of the lungs of all rats euthanized with CO2, were greatest in the animals exposed to the lowest concentrations. There were no significant effects of CO2 concentration on time to recumbency or recovery, and there were no significant effects of precharging versus not precharging the chamber on any of the parameters studied. It was concluded that, although CO2 can be used in a humane manner, the concentrations that are least likely to cause pain and distress are associated with the longest times to anesthesia and death, highest incidence of unwanted side effects, and most severe histologic changes in the lungs. Acceptably humane and reasonably practical euthanasia or anesthesia can be achieved using a nonprecharged chamber and a low gas flow rate so that conscious animals are never exposed to CO2 concentrations > 70%.

Evaluation of five agents/methods for anesthesia of neonatal rats.

Although neonatal altricial rodents are frequently used in experimental projects in which they must undergo survival surgical procedures, there are few published guidelines for anesthetizing them. Many of the drugs and methods that are commonly used to anesthetize newborn rodents provide inadequate anesthesia or are associated with problems, such as excessively high mortality. A two-part study was undertaken with the intention of identifying agents or methods that can be used to provide humane, safe, and effective anesthesia for neonatal rats. In part I, 1- to 3-day-old rat pups were anesthetized with methoxyflurane or one of several injectable agents, or by induction of hypothermia (immersion in ice water). Times to loss of the righting reflex, surgical anesthesia (indicated by loss of the pedal reflex and failure to respond to a skin incision), and recovery of the righting reflex were measured, and heart rate, respiratory rate, and oxygen saturation were monitored. Ketamine (100 mg/kg of body weight, i.p.), pentobarbital (30 to 40 mg/kg, i.p.), and fentanyl-droperidol combination (0.16 mg of fentanyl and 8.0 micrograms of droperidol/g, i.p.) proved unsafe (> 50% mortality) and/or ineffective at inducing short-term surgical anesthesia. In contrast, methoxyflurane and hypothermia were safe and effective. On the basis of subjective observations that they struggled and vocalized less, pups placed in protective latex sleeves prior to immersion in ice water appeared to be less distressed than pups immersed unprotected. In part II of the study, it was possible to safely maintain 1- to 3-day-old pups at a surgical plane of anesthesia for 30 min with either methoxyflurane or hypothermia. Supplementation with pentobarbital (20 mg/kg, i.p.) offered no advantages but significantly prolonged time to recovery. Surviving pups in both studies grew at similar rates during the month after testing. It was concluded that methoxyflurane or hypothermia are good choices for short- or long-term anesthesia of neonatal rats, and that use of a protective sleeve appears to reduce distress associated with induction of profound hypothermia.

Cortical potentials evoked by tooth pulp stimulation differentiate between the analgesic and sedative effects of morphine in awake rats.

This study was undertaken to determine whether the cortical potential (CEP) evoked by noxious electrical stimulation of the incisor tooth pulp can be used to measure analgesia in the presence of sedation in the awake rat. Changes in the CEP produced by morphine (5, 10 and 20 mg/kg s.c.), an opioid analgesic with sedative effects, were compared with those produced by droperidol (1.25 mg/kg s.c.), a neuroleptic agent with no analgesic activity. Both drugs had similar small effects on CEP latency. However, whereas morphine produced a dose-related decrease in amplitude and area under the curve, particularly in the earliest component of the CEP, droperidol produced an increase in amplitude and area under the curve. Naloxone (0.5-2 mg/kg s.c.) reversed all effects of morphine. Similar CEPs could be evoked by electrical stimulation of the tooth pulp or surrounding gingiva in lightly anesthetized rats. However, the tooth pulp stimulation-evoked CEP was unchanged after anesthesia of the gingiva with lidocaine, and the gingiva-evoked CEP was unchanged after anesthesia of the tooth pulp. Therefore, stimulation of the rat's incisor can selectively activate intrapulpal fibers, which are sufficient to generate a CEP. This CEP is an indicator of nociception which can be used to distinguish the analgesic effects of drugs such as morphine from their sedative effects.

Sedative efficacy of droperidol and diazepam in the rat.

Droperidol and diazepam were evaluated for sedative properties in 12 male Sprague Dawley rats (Rattus norvegicus). Over a period of several weeks, each rat was treated subcutaneously with 0.5 mg droperidol/kg, 2.0 mg droperidol/kg, 5.0 mg diazepam/kg, 15.0 mg diazepam/kg, and physiologic saline according to a randomized schedule. After each treatment, the animals were evaluated for their response to a series of four common clinical manipulations (tail-vein bleeding, orbital bleeding, teeth clipping, and toenail bleeding) at five time points over the 90 min following the injection. Rats were scored on the basis of their responses to each manipulation. Response to cardiac puncture was assessed once in each animal immediately prior to euthanasia. Histologic lesions associated with subcutaneous and intramuscular administration of these drugs were evaluated in a separate group of animals. Results indicate that both droperidol and diazepam (at either dose) allow easier manipulation for toenail bleeding and teeth clipping when compared with saline control. There was no advantage in using these sedatives for tail-vein bleeding. Orbital bleeding could not be performed humanely with either drug. Diazepam at a dose of 15.0 mg/kg allowed humane cardiac puncture. Subcutaneous injection of diazepam or 2.0 mg droperidol/kg resulted in various degrees of inflammation revealed by histologic examination, although no clinical signs were associated with these lesions. Subcutaneous administration of droperidol at a dose of 0.5 mg/kg is recommended for nonpainful, noninvasive manipulations as it provides adequate sedation for most procedures without inducing the subcutaneous inflammation observed with diazepam or 2.0 mg droperidol/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Spinal antinociception mediated by a cocaine-sensitive dopaminergic supraspinal mechanism.

The role of dopaminergic descending supraspinal processes in mediating the antinociceptive action of cocaine was studied in the rat using a combination of extracellular neuronal recording and behavioral techniques. Neurons in the superficial laminae (I-II) of the spinal dorsal horn with receptive fields on the tail were recorded in anesthetized rats using insulated metal microelectrodes. Stimulation of the receptive field with either high intensity transcutaneous electrical pulses or with an infrared CO2 laser beam produced a biphasic increase in dorsal horn unit discharge. Conduction velocity estimates indicated that the early discharge corresponded to activity in A delta whereas the late response corresponded to activity in C afferent fibers. Cumulative doses of cocaine (0.1-3.1 mg/kg i.v.) inhibited the late response to either electrical or laser stimulation in a dose-related manner. The early response to laser, but not electrical, stimulation was also suppressed by cocaine. Neurons in the spinal dorsal horn with receptive fields on the ipsilateral hindpaw were activated by natural noxious (pinch) or innocuous (tap) somatic stimulation. Cocaine selectively suppressed nociceptively evoked dorsal horn unit discharge. This antinociceptive effect was dose-related (0.3-3.1 mg/kg, i.v.) and antagonized by eticlopride (0.05-0.1 mg/kg, i.v.), a selective D2 dopamine receptor blocker. The same doses of cocaine failed to inhibit the responses of dorsal horn neurons to low threshold innocuous stimulation. Complete thoracic spinal cord transection eliminated the antinociceptive effect of cocaine on dorsal horn neurons and also eliminated the cocaine-induced attenuation of the tail-flick reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatal encephalitis caused by Dactylaria constricta var. gallopava in a snowy owl chick (Nyctea scandiaca).

Dactylaria constricta var. gallopava (Cooke) Salkin et Dixon was found to cause fatal encephalitis in a 28-day-old, captivity-bred snowy owl chick (Nyctea scandiaca). The previously healthy bird suddenly developed ataxia, severe torticollis, and extensor rigidity of the legs. Since the animal did not improve with antibiotic or vitamin-mineral supplement therapy, the chick was euthanized 5 days after the onset of neurologic signs. At necropsy, all tissues except the brain were grossly normal. Cultures inoculated with blood from the brain and heart yielded a dematiaceous mould that subsequently proved to be D. constricta var. gallopava. This is the first report of natural central nervous system infection caused by D. constricta var. gallopava in a snowy owl.

An evaluation of three intravenous anesthetic regimens in New Zealand rabbits.

Intravenous anesthetics can be readily administered to rabbits through the marginal ear vein. In this study, three intravenous anesthetic protocols were evaluated in New Zealand White rabbits. The three anesthetic regimens were: (a) pentobarbital (40 mg/kg); (b) ketamine-xylazine (25-5 mg/kg); (c) midazolam-xylazine-alfentanil (1-1-0.1 mg/kg). The anesthetics were injected slowly over defined time intervals. Reactions to noxious stimuli were determined before and after administration of the anesthetics. Additionally, the effects of the anesthetic agents on the rabbit's cardiopulmonary system were evaluated. Rabbits anesthetized with midazolam-xylazine-alfentanil did not have a pedal withdrawal or ear pinch reflex throughout the testing period. The ketamine-xylazine combination produced a shorter duration of non-responsiveness to noxious stimuli. Rabbits anesthetized with pentobarbital had the greatest variability in response to noxious stimuli. Apnea occurred in at least one rabbit in each group. A side effect unique to the midazolam-xylazine-alfentanil group was the occurrence of opisthotonus or seizure activity during or shortly after the administration of alfentanil. Hypotension, hypercapnia and respiratory acidosis were characteristic of the cardiopulmonary effects of the anesthetics. When choosing an anesthetic regimen for rabbits, intravenous infusion should be considered as an option. Advantages include ease of administration, possibility of redosing as required, and minimal requirements for equipment. Disadvantages of intravenous anesthetic infusion in rabbits include potential for lethal overdose and metabolic alterations after administration.

A risk assessment process for allergic contact sensitization.

This review describes an approach that has been used to assess the skin sensitization risk of new chemicals and product formulations prior to launching the new chemical or product on the market. The risk assessment process utilizes a comparative toxicological approach, in which data on the inherent toxicity of a material, and the exposure to it through manufacturing or consumer use or foreseeable misuse, are integrated and compared with data generated by 'benchmark' materials of similar chemistry or product application, or both. This approach has been valuable in providing an accurate assessment of the skin sensitization potential for a wide range of consumer products and pharmaceuticals, ranging from products with a very transient skin exposure (e.g. some paper products), to cosmetics, to products whose ingredients may be deposited on fabrics and thus result in chronic skin exposure. The risk assessment process described includes both guinea-pig (Buehler) and human skin sensitization test methodologies to evaluate inherent toxicity under relevant epicutaneous exposure conditions. Alternative guinea-pig test methods have been reported to be more sensitive than the Buehler method, particularly those employing intradermal injection of the test material in Freund's complete adjuvant (e.g. maximization test). However, by bypassing the skin barrier at induction, these methods can overstate the sensitization risk of epicutaneous exposure to weak sensitizers (Andersen and Hamann, 1983 and 1984; Matsushita et al., 1975a,b), and can understate the risk to very strong sensitizers possibly through tolerance induction (Buehler, 1985). In addition, materials are tested and classified at concentrations that may not be relevant to anticipated human exposure. The Buehler guinea-pig test data are important in assessing skin sensitization risk in the early phases of product development, where human exposure can be limited, controlled and monitored (e.g. manufacturing employees). The Buehler test can often define consumer skin sensitization risk; however, the ultimate consumer skin safety assessment should in general be developed through a series of controlled human tests; the human repeat insult patch test and, when necessary, the provocative or extended product use tests. Post-market monitoring of skin-related consumer comments is the final phase of the data gathering process. These results can be used to assess further each product and to provide valuable feedback to confirm the validity of the overall risk assessment process. Risk assessment for skin sensitization potential is seldom a simple process.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth of Staphylococcus aureus and synthesis of toxic shock syndrome toxin 1 in different in vitro systems.

Various complex and synthetic bacterial growth media have been used to study the growth of Staphylococcus aureus and the production of toxic shock syndrome toxin 1 (TSST-1) under certain in vitro culture conditions. Because of the biochemical and nutritional differences between these media and human menses, a program was designed to determine the growth and metabolism of S. aureus under conditions that more closely approximate in vivo conditions. Human menses, an artificial menses developed to match human menses, whole human blood, and complex bacterial culture media (with and without added whole human blood) were tested for the ability to support the growth of S. aureus and the production of TSST-1 in vitro. In addition, the impact of other organisms, commonly isolated from the human vagina, on the growth of S. aureus and the production of TSST-1 was evaluated. Results show that the environmental conditions provided by human menses are more closely approximated by the artificial menses and that neither commercial bacterial growth media alone nor complex media plus 25% or 50% human blood provide a menses-like environment for the growth of S. aureus and the production of TSST-1. Furthermore, the addition of a second organism to the S. aureus culture resulted in significant suppression of TSST-1 production, even when in vitro conditions were optimized for the production of TSST-1.

Response of various animal species to experimental infection with different strains of Staphylococcus aureus.

An experimental infection program was conducted in rabbits, pigs, and baboons with toxic shock syndrome (TSS)-associated and non-TSS-associated strains of Staphylococcus aureus to produce an animal model for TSS. TSS-associated strains of S. aureus--whether positive or negative for TSS toxin 1 (TSST-1)--could not be distinguished from non-TSS-associated strains of S. aureus by means of the rabbit whiffle-ball infection model; therefore, limited pilot infection studies were conducted in pigs and baboons. Experimental conditions were optimized in both the pig and the baboon studies to maximize the chance of producing TSS. Pigs infected with TSS-associated S. aureus strain CDC-11 developed some of the clinical signs observed in TSS (fever, hypotension, diarrhea, and vomiting). However, no changes were detected in clinical chemistry or hematology. Baboons infected with S. aureus strain CDC-11 showed only minimal signs of illness, i.e., lethargy, decreased food intake, and loose stools. TSS was not produced in pigs or baboons, even under optimal exposure conditions.

An evaluation of analgesia associated with the immobility response in laboratory rabbits.

The immobility response (IR) was studied in rabbits to evaluate its analgesic properties and reliability as a method of restraint. The participation of the endogenous opioid system in IR was studied indirectly by evaluating the effects of the narcotic antagonist naloxone on this phenomenon. Twenty-four adult New Zealand White rabbits were subjected to six noxious stimuli while restrained by IR and while restrained under control conditions. Testing on each animal was repeated under both conditions following the administration of naloxone. The noxious stimuli consisted of three levels of electric shock (10 volts, 30 volts, and 50 volts) applied to the shaved forearm, and mechanical pressure applied to the pinna, front toe, and hind toe. Withdrawal and changes in blood pressure, heart rate, and respiration were used as indicators of pain perception. Distress associated with noxious electrical and pressure stimulation was significantly reduced by IR, which suggested that the phenomenon does have a significant analgesic component. However, the rabbits showed wide variability in their susceptibility to IR induction, and even animals which did not withdraw in response to noxious stimulation under IR sometimes exhibited physiological changes suggestive of distress. Therefore, IR should not be considered as a reliable or humane alternative to analgesic/anesthetic drugs for laboratory rabbits. Naloxone had little effect on IR or IR-associated analgesia.

Cilia-associated respiratory (CAR) bacillus infection of obese mice.

Cilia-associated respiratory (CAR) bacillus was identified in respiratory tract lesions of obese mice dying of chronic respiratory disease. Neither Mycoplasma pulmonis nor pathogenic bacteria were isolated from cultures of the lesions at necropsy, but there was serologic and histologic evidence of respiratory virus infection. Cranial-ventral areas of lung were firm and demarcated from unaffected lung at gross examination, and representative tissue sank in water. Microscopically, there was suppurative bronchopneumonia with extensive peribronchiole lymphocyte and plasma cell proliferation. The affected bronchiole epithelium was covered with a sheet of slightly basophilic, filamentous, gram negative bacteria. Bronchioles with lesser amounts of lymphocyte accumulations contained lesser amounts of filamentous bacteria. Bronchioles without filamentous bacteria lining the respiratory epithelium lacked peribronchiole lymphocyte accumulations. There was a high correlation between CAR bacillus-positive serology and the identification of diagnostic histologic lesions. CAR bacillus was readily stained using immunohistochemical methods, and the ultrastructural features were similar to that described in rat infections.

Cinnamic aldehyde: a survey of consumer patch-test sensitization.

The potential for cinnamic aldehyde, an important fragrance and flavour ingredient, to induce or to elicit delayed contact hypersensitivity reactions in man was evaluated by analysing patch-test data. Results of studies involving a total of 4117 patch tests on various consumer products and fragrance blends containing cinnamic aldehyde and on the material itself were collected from fragrance and formulator companies. The data indicate that cinnamic aldehyde contained in consumer products and fragrance blends at concentrations up to 6 X 10(-1)%, and patch-tested at concentrations up to 8 X 10(-3)%, has no detectable potential to induce hypersensitivity. Cinnamic aldehyde when tested alone induced a dose-related hypersensitivity response. According to published reports, cinnamic aldehyde elicited positive delayed hypersensitivity responses in dermatitic patients. However, results of the current survey show that when cinnamic aldehyde was tested alone or as part of a mixture in subjects in the general population, no pre-existing hypersensitivity reactions to the fragrance material were observed in any of the 4117 patch tests which constituted the survey. Cinnamic aldehyde at the concentrations contained in consumer products and fragrances, has a very low potential to induce hypersensitivity ('induced' reactions) or to elicit sensitization reactions ('elicited' reactions) in the general population.

Regulation of reaginic antibody production in mice. I. Suppression by antigen of IgE antibody production in vitro.

Regulation of IgE production by antigen in a primed murine splenic lymphocyte culture system was described. Maximum IgE antibody production was found to occur when cells were cultured in the absence of exogenously added antigen. A cells and T lymphocytes did not affect the production of anti-DNP IgE antibody. By using a hapten-carrier antigen system (DNP-EA) for priming mice in vivo, it was found that the production of anti-DNP IgE by spleen cells in vitro was inhibited by hapten when coupled to homologous (EA) or heterologous (BGG) carrier, and was not enhanced or inhibited by homologous carrier. Anti-DNP IgE antibody production by cultures depleted of macrophages or T lymphocytes was found to be as sensitive to the suppressive effects of hapten as was the IgE production by whole spleen cell cultures. Both IgM and IgG secondary anti-DNP PFC responses in vitro were enhanced by the presence of the homologous hapten-carrier or carrier alone. DNP-BGG had no effect on the anti-DNP IgM or IgG PFC responses of the cultures. These data suggest that endogenous production of antibody (IgM or IgG) was not responsible for the observed suppression of the IgE response in vitrol The experimental results presented indicate that the regulation of the IgE production by antigen in the primed mouse splenic lymphocyte cultures was a consequence of the direct interaction of hapten with IgE B cells.

Reaginic antibody production to protein antigens of Escherichia coli and Pseudomonas aeruginosa by mice.

Water-soluble antigens isolated from acetone-dried, gram-negative bacteria elicited reaginic antibody formation in mice. Antibodies specific for Escherichia coli antigens reacted with antigens isolated from several enterobacterial species tested, but not with antigens isolated from Pseudomonas aeruginosa. Reaginic antibodies induced by antigens isolated from a P. aeruginosa strain reacted with antigens isolated from several P.aeruginosa serotypes as well as with a purified protein component of the envelope of P. aeruginosa. The anti-Pseudomonas reagins did not cross-react with enterobacterial antigens. Antigenicity of the bacterial extracts was destroyed by trypsin treatment and reduced by heating, which suggested that the antigens were protein in nature. Whole bacterial cells adsorbed out reaginic antibodies, indicating that the antigens are located at or near the surface of the bacteria.