2009 - Volume 36 - Issue 4

309-320
Social Rank Influences the Distribution of Blood Leukocyte Subsets in Female Growing Pigs
by Bodil Margrethe Hjarvard, Ole Næsbye Larsen, Helle Risdahl Juul-Madsen, Erik Jørgensen & Karin Hjelholt Jensen

The effect of high (DOM) and low (SUB) social rank on blood immune variables was examined in female growing pigs. Pigs were mixed with unfamiliar pigs at 9 weeks of age and kept in stable groups of 4 pigs for 5 weeks. Social rank was determined using a feeding competition test. SUB pigs showed reduced growth as compared to DOM pigs confirming their lower social status. Blood was sampled for immunological assessments immediately before grouping the pigs and again after the 5 weeks of social housing. White Blood Cell (WBC) counts, percentage of CD4 positive cells (CD4+), percentage of CD8 positive cells (CD8+), percentage of swine leukocyte antigen II (SLAII) carrying cells, LPS-stimulated Toll-like Receptor 4 (TLR4) expression, and LPS-stimulated tumor necrosis factor-alpha (TNF-alpha) responsiveness were determined at both times. IgG and IgM concentrations were measured following the 5 weeks of social housing only. From the WBC counts it was found that the percentage of neutrophils was higher in SUB pigs and the neutrophil to lymphocyte ratio was higher in DOM pigs. The percentage of CD4+ cells decreased with time in both DOM and SUB pigs, but only significantly in SUB pigs. The percentage of CD8+ cells was higher in SUB pigs than in DOM pigs and decreased with time in both DOM and SUB pigs. In addition, SUB pigs had a higher ex vivo TNF-alpha responsiveness as compared to DOM pigs. Both the percentage of SLAII carrying cells and LPS-stimulated TLR4 expression increased with time, but here no significant effect of social rank was found. In addition, neither IgG nor IgM concentrations showed any relationship with social rank. The findings indicate that social rank influences the distribution of blood leukocyte subsets in female growing pigs, suggesting that the pig would be a good model for investigating the effects of long-term immunomodulation on health.

325-342
Effects of Antirheumatic Drugs on the Development of Experimental AA Amyloidosis in C57BL/6 Mice
by L. Leonaviciene, D. Povilenaite, R. Bradunaite, D. Vaitkiene & A. Venalis

Because there is no known specific effective therapy for secondary amyloidosis at the present time, the aim of this study was to determine whether antirheumatic drugs inhibit the development of experimental AA amyloidosis, induced in a C57BL/6 mice by injections of casein and fibrin. Monotherapy with sulfasalazine (SSL) and diclofenac (D) and combined treatment with diclofenac and prednisolone (D/P) by using prophylactic and therapeutic treatment protocols were investigated. The drugs were administered through intragastric gavage 5 times a week for 5 or 6 weeks in the following doses: D - 1 mg/kg, P - 10 mg/kg, and SSL - 100 mg/kg. Histopathological examination of splenic, kidney and hepatic tissues of mice was performed. The amount of amyloid was assessed semi-quantitatively by polarizing microscopy after Congo Red staining. Our study indicated that no positive effect from prophylactic treatment with D could be seen on amyloid deposition in investigated organs. Prophylactic combined treatment with D/P resulted in significant improvement of disease symptoms and markedly reduced amyloid deposits in the spleen, kidneys, and liver (P< 0.02-0.001). SSL therapy alone has been more successful in the prophylactic treatment of experimental amyloidosis: the decrease of amyloid deposits was statistically significant in all investigated organs (P< 0.04 – 0.001) and the most suppression of amyloid formation in the kidneys and liver was observed (P< 0.004-0.001). In therapeutic treatment of experimental amyloidosis, combined treatment with D/P showed the most inhibition of amyloid formation in the internal organs (P < 0.006 – 0.001). The highest suppression (by 86.7%; P < 0.001) of amyloid deposits was observed in the liver. Treatment of mice with D alone produced a significant reduction in amyloid deposition only in the liver (P < 0.03) and with SSL – only in the spleen (P < 0.03). These findings suggest that D/P and SSL at relevant doses suppress amyloidogenesis and this suppression is possibly related to the anti-inflammatory effect of antirheumatic drugs. Although these drugs cannot completely inhibit the disease in this model, a possibility remains that they may be clinically useful in rheumatic diseases associated with the formation of amyloidogenic derivatives.

347-351
Technical Report: Replacement of Vertebrates by Locust in Student Laboratory Exercises
by Alaburda A., Kaminskas O. & Ruksenas O

The student laboratory exercises are a very important part in the curriculum of physiology, neurophysiology and biophysics. Knowledge from lectures and books should be supported with some practical experience based on work with live subjects. But this raises a very important question – is the use of animals for teaching purposes acceptable? This is a controversial question for a long time in physiology teaching and has numerous arguments for and against. With respect to efficiency and quality of teaching it is essential. However, is this enough to justify the use of animals? Traditionally laboratory exercises are performed on frogs or rodents. Despites obvious advantages this has serious disadvantages, namely relatively high costs related to animal facility and care of the animals. Applying the 3R‘s principle, we replaced vertebrates by invertebrates and introduced laboratory exercises based on recording of action potentials from wing stretch receptors of the locust (Robertson, 1992). Locusts are cheap to buy (approx. 2 locust for 1 euro) and easy to care for. Preparation of locusts for experiment is very simple. However, in this laboratory exercise students can learn such basic concepts like generation of action potential, natural variability of recorded signals in a live system, adaptation and principles of coding in the nervous system. Apart from this, modification of the experimental setup enabled us to investigate physiological concepts like neural coding in more natural conditions.

355-361
Elimination of P. Aeruginosa in Mice by Treatment with Chlorine, and the use of Microbiological and PCR Analyses
by E. Mahabir, D. Bulian, S. Bensch, J. Schmidt

Unwanted micro-organisms in mice can jeopardize experimental protocols and should be eliminated if a colony becomes infected. The opportunistic bacterium Pseudomonas aeruginosa has been shown to cause infection in mice. In order to eliminate this bacterium from a specific-pathogen-free full barrier mouse area at our animal facility we treated mice with 7 ppm chlorine for 7 weeks and then with 10 ppm chlorine in the drinking water. The P. aeruginosa status of mice was examined by agar culture and PCR. The results show that 6 months after commencement of the treatment the colony was free of P. aeruginosa.

363-368
A Simple and Reliable Method of Endotracheal Intubation in Mice: Advantages of Exposing the Trachea
by Akihiko Ikeda, Shonosuke Matsushita & Yuzuru Sakakibara

Mice are popular models in experimental studies because they can be genetically modified and have a short gestation period. In long-term studies requiring recovery from anesthesia or repeated measurements of pulmonary function, endotracheal intubation is advantageous. However, this is difficult because of the small anatomic structures involved. Various methods have been previously reported, the majority of them calling for expensive devices or techniques which require special training. Therefore, we have created a simple method of endotracheal intubation in mice using a light-emitting diode (LED) light, a metal laryngoscope, a 22-gauge plastic cannula and a stylet made of a 0.3mm piano wire, which can all be easily prepared. Transillumination after exposure of the trachea makes it possible to illuminate the oropharynx with a lowpriced LED light and a metal laryngoscope provides good visualization of the tracheal opening. With direct vision, a 22-gauge plastic cannula can easily be inserted into the trachea. The custom-made stylet is suitably flexible for performing tracheal intubation without tissue injury. In a series of 42 mice, the success rate with the procedure was 97.6% (n=41). Only in one case was it necessary for the procedure to be repeated. There were no airway complications. Though exposure of the trachea is invasive, it does have advantages. We conclude that this method is simple, safe and inexpensive, and could be used by any researchers interested in it, no matter how small their budget.