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Letter: Succinylcholine for euthanasia of laboratory animals.
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Carbon dioxide for euthanasia of laboratory animals.
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"Carbon dioxide for euthanasia of laboratory animals".
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Recommendations for euthanasia of experimental animals: Part 2. DGXT of the European Commission.
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[Methods for the euthanasia of experimental animals--the ethics, esthetics and personnel safety].
In this paper there are shown chemical and physical methods of euthanasia of vertebrate animals. All methods are divided into three categories: A) Acceptable methods of euthanasia, B) methods acceptable only for unconscious animals, C) methods that are not acceptable for euthanasia. The acceptability or non-acceptability of the method is determined by ethic or aesthetic conceptions and also by conceptions of the personnel safety and the environment. There is provided a table of acceptable methods of euthanasia. Recommendations of working group of the Federation of Laboratory Animal Science Association (FELASA) were taken into consideration in this paper. The survey can be useful for the experimenters and personnel concerned with laboratory animals.
Effects of systemic glucocorticoid administration on tenocytes.
The effects of systemic administration of glucocorticoids (GC) on Achilles tendons of rats was studied. After the animal euthanasia, Achilles tendons were removed in sterile conditions from Sprague Dawley adult female rats to isolate tenocytes. Animals have been daily treated for 8 weeks with 4 mg/kg methylprednisolone or Sham-treated with saline solution. In vitro, cell proliferation (WST-1), extra-cellular matrix (ECM) synthesis (collagen type I, CICP; proteoglycans, PG; and fibronectin, FBN), and transforming growth factor (TGF-beta1) were evaluated at 3 and 7 days of culture. The effect of glucocorticoids (GC) on tenocytes was evident both at 3 and 7 days of culture, and caused a significant decreases in cell proliferation (P<0.01), CICP (P<0.01) and PG synthesis (P<0.01) as compared to NT tenocytes. In conclusion, GC systemic treatment seems to compromise the normal proliferation rate and synthetic activity of cultured tenocytes. This study was helpful in understanding the fundamental biological processes that occur during corticosteroid systemic administration and tenocyte cultures may be used to further study to improve knowledge of these cell behaviour also in the ambit of tendon tissue-engineered therapies.
Method of euthanasia in food animals must be practical and safe.
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Individual mouse analysis of the cellular immune response to tumor antigens in peripheral blood by intracellular staining for cytokines.
Among the experimental animal models, mice remain the most widely used for the evaluation of immunotherapeutic strategies. Vaccines against parasites and viral antigens are commonly administered to the appropriate mouse strain which also allows testing of the therapeutic effect. Similarly, in mice transgenic for human tumor associated antigens (TAA), cancer vaccines must lead to breakage of immune tolerance to elicit a significant effect on the tumor. However, one of the major drawbacks in the monitoring of cellular immune responses induced by vaccination is that functional immunological assays require suppression of the animals to collect the spleen or lymph nodes for analysis. Here, we report the application of a rapid intracellular staining (ICS) method to quantify antigen-specific T cells responses in small volumes of murine blood. Genetic vaccination with plasmid DNA followed by electroporation (DNA-EP) and the use of adenoviral vectors (Ad) encoding CEA as a model target antigen were applied to different strains of mice. Optimal blood volume, number of lymphocytes, sensitivity and reproducibility of intracellular staining for IFN-gamma were determined both in non-tolerant/wild type mice as well as in tolerant CEA transgenic mice upon restimulation of PBMCs with CEA peptides. Groups of vaccinated mice were then sacrificed and PBMCs and splenocytes from individual animals were compared for intracytoplasmic detection of IFN-gamma and TNF-alpha. A significant correlation was observed between splenic and blood immune responses. Finally, the cellular immune response was followed over time in groups of vaccinated mice. The kinetics of IFN-gamma producing effectors were measured after priming and successive boosting with adenoviral vectors. We show that intracellular staining for mouse PBMCs is a rapid and simple method to measure antigen-specific immune responses. It does not require animal euthanasia and mirrors the response observed in lymphoid organs such as the spleen.
[Osteosarcoma in cats: epidemiological, clinical and radiological findings in 78 animals (1990-1995)].
In this study 78 cats with osteosarcoma were identified from biopsy logs and evaluated retrospectively regarding breed, sex, and age distribution, tumor location, clinical symptoms, radiographic findings, biologic behavior of the neoplasm, and outcome following therapy. There was no significant breed or sex prediffection among the cats. The average age was 10.1 years, with a range of less than one to over 17 years. 36 (46%) of the osteosarcomas were localized in the bones of the limbs, 42 (54%) were found in flat or irregular bones. The hind limbs (25 cases) were affected significantly more often than the front limbs (10 cases). The most prevalent sites were the distal femur, proximal tibia, the humerus and the digits. Four of the tumors occurred after osteosynthesis of a previous fracture. In most of the cats limb tumors were associated with chronic lameness. The skull was the most common site for tumors of the flat and irregular bones (35 cases), which involved the oral cavity in 27 cases. The most common symptoms were dental problems, deformations of the skull, and nasal discharge. The remaining tumors of the flat and irregular bones were located in the pelvis (3), vertebrae (2), scapula (1), and rib (1). Radiographic findings were very variable and ranged from lytic to purely osteoproliferative forms. With tumors of the flat and irregular bones, tumor recurrence was a common cause for euthanasia. Animals with tumors of the limbs had a good prognosis following amputation.
Analysis of the influence of low-power HeNe laser on the healing of skin wounds in diabetic and non-diabetic rats.
PURPOSE: To study the influence of HeNe laser irradiation on the collagen percentage in surgically-induced skin wounds in rats with and without alloxan-induced diabetes, by morphometric analysis of collagen fibers. METHODS: 48 male Wistar rats were used, divided into groups: laser-treated diabetic (group 1); untreated diabetic (group 2); treated non-diabetic (group 3); and untreated non-diabetic (group 4). For groups 1 and 2, diabetes was induced by intravenous injection of alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil; Sigma), into the dorsal vein of the penis, at a rate of 0.1 ml of solution per 100 g of body weight. A wound was made on the back of all the animals. Groups 1 and 3 were treated with HeNe laser (4 J/cm2) for 60 s. One animal from each group was sacrificed on the 3rd, 7th and 14th days after wounding. Samples were taken, embedded in paraffin, stained with hematoxylin-eosin and Masson's trichrome, and morphometrically analyzed using the Imagelab software. The percentages of collagen fibers were determined from the samples from the euthanasia animals. The data were treated statistically using analysis of variance (ANOVA) and the Student t and Kruskal-Wallis tests. The significance level was set at 0.05 or 5%. RESULTS: The results obtained from the samples taken on the third, seventh and fourteenth days after wounding demonstrated that the laser-treated group presented a statistically significant (p<0.05) greater mean quantity of collagen fibers than in the non-treated group, both for diabetic rats (p = 0.0104) and for non-diabetic rats (p = 0.039). CONCLUSION: The low-power laser (632.8 nm) was shown to be capable of influencing the collagen percentage in skin wounds by increasing the mean quantity of collagen fibers, both for the diabetic and for the non-diabetic group.
Inhalation toxicology of decamethylcyclopentasiloxane (D5) following a 3-month nose-only exposure in Fischer 344 rats.
D5 is a low-molecular-weight cyclic siloxane used for industrial and consumer product applications. The objective of the present study was to evaluate the subchronic toxicity of D5 following a 3-month nose-only inhalation exposure. In addition, animals from both sexes of the control and high dose groups were allowed a 4-week recovery period to observe reversibility, persistence, or delayed occurrence of any potential adverse effects. Male and female Fischer 344 rats were exposed for 6 h/day, 5 days/week for 3 months to target concentrations of 0 (30/sex/group), 26 (20/sex/group), 46 (20/sex/group), 86 (20/sex/group), and 224 (30/sex/group) ppm D5. Recovery groups (0 and 224 ppm) comprised 10 rats/sex/group. Body weights and food consumption were monitored at least twice weekly over the course of exposures. Approximately 16 h preceding euthanasia, animals were transferred into metabolism caging for urine collection and were fasted. Rats were anesthetized with pentobarbital and euthanized by exsanguination. Blood was collected for hematological and clinical biochemical analyses. Selected organ weights were measured and a complete set of tissues was taken for histopathological examination. There were several minor changes observed in clinical biochemistry parameters; the most notable was an increase in gamma glutamyl transferase (gamma-GT) in both sexes at the high dose. In females, this effect was dose-related (46-224 ppm) and did not recover upon cessation of exposure. Additionally, there was an decrease in serum lactate dehydrogenase (LDH) observed in females at 86 and 224 ppm which was not resolved during recovery. There was an increase in absolute and/or relative liver weight in rats of both sexes. Taken together, these data suggest that the female rat is more sensitive to the actions of D5 on the liver. Exposure-related increases in absolute and relative lung weights were observed in both sexes at terminal necropsy. This observation was not noted in males in the recovery phase, but was still present in females. Finally, histopathological evidence indicated the primary target organ following D5 inhalation exposure is the lung, with an increase in focal macrophage accumulation and interstitial inflammation in the lungs of male and female rats exposed to 224 ppm D5. This observation did not appear to resolve at the end of a 1-month period of nonexposure. The incidence of these changes was also slightly increased in rats of both sexes exposed to 86 ppm D5. These data suggest that nose-only D5 vapor inhalation provokes minimal changes in the lung which are similar in incidence and severity to spontaneously occurring changes in control animals after nose-only exposures. There were no histopathological findings noted in the livers which support this organ as a target in this study, despite the observed changes in organ weight and in some serum chemistry parameters.
Stimulation of angiogenesis to improve the viability of prefabricated flaps.
The cutaneous area in a prefabricated myocutaneous flap surviving after elevation is dependent on the rate and amount of vascular ingrowth that occurs from the underlying muscle. Two modalities, basic fibroblast growth factor and hyperbaric oxygen, were used separately and together in a prefabricated myocutaneous flap animal model to improve flap survival. The semimembranous muscle, based on the saphenous vessels of 40 female Wistar rats weighing between 250 and 325 grams, was tunneled under the ipsilateral abdominal skin and sutured in place. A 3 x 5-cm silicone sheet was placed beneath the muscle flap, and the ipsilateral epigastric vessels were ligated. Four groups of 10 animals each received one of the following treatment regimes: a 1-ml normal saline infusion into the saphenous arterial pedicle, a 1-ml infusion of basic fibroblast growth factor (1.0 microg/gm of muscle), a 1-ml normal saline infusion and 14 hyperbaric oxygen treatments, or a 1-ml basic fibroblast growth factor infusion and 14 hyperbaric oxygen treatments. After 1 week, the muscle, still based on the saphenous vessels, was elevated with a 3 x 5-cm abdominal skin paddle. The flap was sutured back in place, leaving the silicone sheet intact. The surviving area of each flap was measured 1 week later after it had demarcated into viable and necrotic regions. Laser Doppler skin perfusion measurements were taken before and after flap elevation and before animal euthanasia. Sixteen flaps, 4 in each group, were examined histologically for vascularity by means of hematoxylin and eosin staining. There was a statistically significant increase in flap survival area when either basic fibroblast growth factor or hyperbaric oxygen was used alone. Further improvement was noted with combination therapy. Histology confirmed improved vascularity in the basic fibroblast growth factor and hyperbaric oxygen-treated flaps. This study shows a significant and reliable increase in the area of prefabricated myocutaneous flap survival using either basic fibroblast growth factor or hyperbaric oxygen. There is a further complementary effect when these two modalities are combined, leading to near complete flap survival through improved vascularity.
A reinvestigation of murine cranial suture biology: microcomputed tomography versus histologic technique.
BACKGROUND: Histology remains the standard form to analyze cranial suture in murine models, but this technique provides only limited "snapshots" of the entire suture and requires animal euthanasia with tissue destruction. Because of the bone complex microarchitecture, better methods are required to study the behavior of the cranial suture and its surrounding environment. The authors compared microcomputed tomography and histology as techniques to evaluate murine cranial sutures. METHODS: A total of 360 microcomputed tomography images and 160 to 170 histologic sections were processed from a mouse at postnatal days 22 and 45, respectively. After euthanasia, the posterior frontal and sagittal sutures were imaged with a microcomputed tomography system and subsequently processed for histologic analysis. Quantitative analysis of two-dimensional images was performed to determine the percentage of bone in a 1-mm sample. RESULTS: Quantitative analysis of the percentage of bone within the sutures showed identical patterns by microcomputed tomography and histology techniques. Both methods demonstrated the posterior frontal suture to have heavier fusion patterns in the anterior and endocranial portions, with variable skip areas of complete patency on the endocranial surface, ectocranial surface, or both at day 45. CONCLUSIONS: Cranial suture fusion in the murine model is not an "all-or-none" phenomenon. The posterior frontal suture, previously thought to be completely fused on day 45 by histological analysis, showed variable fusion along the length of the suture by both methods. Quantitative assessment of the percentage of bone within the posterior frontal and sagittal sutures and morphologic assessment of these sutures demonstrated similar findings by both methods. Whereas thorough histologic evaluation of an entire suture would be extremely labor intensive and impractical, these findings help to validate microcomputed tomography as a rapid and reliable method of examining the entire suture in murine models.
Inhalation toxicology of octamethylcyclotetrasiloxane (D4) following a 3-month nose-only exposure in Fischer 344 rats.
Octamethylcyclotetrasiloxane (D4) is a low-molecular-weight cyclic siloxane used primarily in the synthesis of silicone polymers. The objective of the present study was to evaluate the subchronic toxicity of D4 following a 3-month nose-only inhalation exposure. Male and female Fischer 344 rats (20/sex/group) were exposed 6 h/day, 5 days/week for 3 months to vapor concentrations of 0, 35, 122, 488, and 898 ppm D4. Also, an additional 10 per sex in the control and high-exposure groups were allowed a 4-week recovery period to observe reversibility, persistence, or delayed occurrence of any potential adverse effects. Body weights and food consumption were monitored at least twice weekly over the course of exposures. Approximately 18 hours preceding euthanasia, animals were transferred into metabolism cages for urine collection, and were fasted. At necropsy, rats were anesthetized with pentobarbital and euthanized by exsanguination. Blood was collected for hematological and clinical biochemical analyses. Selected organ weights were measured and a complete set of tissues was taken for histopathological examination. A concentration-dependent increase in absolute and relative liver weight (488 to 898 ppm) and a significant decrease in ovarian weight (898 ppm) were observed in female rats. Exposure to D4 via nose-only inhalation (35 to 898 ppm) produced minor alterations in hematological and serum chemistry parameters that were considered either incidental and of little toxicological significance (hematology) or suggestive of metabolic adaptation/alteration (serum chemistry) in response to exposure-related hepatomegaly. There were no histopathological findings noted in the liver. Histopathological evidence indicated the primary target organs following D4 inhalation exposure to be components of the female reproductive tract. Reversible histopathological changes were observed in the ovary (hypoactivity) and vagina (mucification) of female rats in the high-dose group only (898 ppm). Although an increase in the incidence and severity of both macrophage accumulation, interstitial inflammation, and eosinophil infiltration was observed in the lungs of male and female rats exposed to D4, the toxicological significance is uncertain as other inhalation studies at similar concentrations failed to show these effects. In summary, nose-only inhalation of a high concentration of D4 resulted in reversible histopathological changes in the female rat reproductive tract. Lower concentrations did not elicit these same effects.
Humane slaughter of birds.
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