The production of antibodies for analytical purposes using invasive procedures on small mammals is common practice in biomedical research. The aim of this study was to develop an efficient method for non-invasive antibody production in the chicken. This thesis presents an alternative method that eliminates the discomfort, pain and distress invoked by traditional immunization procedures on mammals by instead harvesting antibodies (IgY) from the yolk of eggs laid by orally immunized hens. An efficient oral immunization regime was developed by first trying out a suitable non-aggressive oral adjuvant with Bovine Serum Albumine (BSA) as the model antigen. It was found that the pegylated mono/diglyceride RhinoVax® (Softigen®) at a concentration of 20% (v/v) produced a good humoral antibody response in chickens as well as development of IgY antibodies in the egg yolk. The age of the chicken is important in order to have a proper humoral immune response. We found that chicken older than 22 days produced circulating immunospecific anti BSA-antibodies of of IgG, IgM and IgA class when orally immunized with BSA alone, whereas chickens 15 days old only produced IgM and IgA antibodies. This is the first report of oral immunizations with a high dose (250–300mg) of BSA in 20% RhinoVax® consisting of 3 or 5 consecutive daily doses resulting in high concentrations of immunospecific IgY antibodies in the yolk. Using this technique of three consecutive daily doses repeated after 7 weeks and after 18 weeks, a booster effect was induced after the third immunization. This is the first demonstration of a clear anamnestic immune response in orally immunized chickens. The results suggest that it may be possible to further increase the concentration of immunospecific IgY antibodies by modifying the immunization regime. It seems plausible to develop a procedure where the immunogen can be fed to the chickens as in an ordinary egg producing farm thus making antibody production not classified as an animal experiment.

The spinal cord is an important component in the processing and modulation of painful stimuli. Nerve signals from the periphery are relayed and further conducted to the brain (nociception) in the spinal cord, and the most essential modulation of painful information (antinociception) occurs here. Several neurotransmitters are involved in spinal pain modulation, among them acetylcholine. However, the role of acetylcholine has previously been little investigated. In the present thesis, the acetylcholine release in the spinal cord was studied in vivo. By using spinal microdialysis on anaesthetised rats, the effects on the intraspinal acetylcholine release of various receptor ligands and analgesic agents were examined. This, together with pain behavioural tests and in vitro pharmacological assays, was used to evaluate the role of acetylcholine in spinal pain modulation. The four studies in this thesis resulted in the following conclusions: An increased release of spinal acetylcholine is associated with an elevated pain threshold, while a decreased acetylcholine release is associated with hyperalgesia, as seen after systemic treatment with a muscarinic agonist and an antagonist. Lidocaine is a potent analgesic when given systemically. It was found to produce an increase of intraspinal acetylcholine after intravenous injection of analgesic doses. This effect was attenuated after muscarinic, and abolished after nicotinic, receptor blockade. Various a2-adrenergic ligands, associated with nociceptive or antinociceptive effects, were found to affect intraspinal acetylcholine release via action on nicotinic receptors. Finally, the involvement of spinal acetylcholine in the analgesic effects of aspirin and paracetamol was examined. It was found that spinal acetylcholine could participate in the analgesic effects of aspirin, but not of paracetamol. The present thesis provides data that clearly demonstrate a relationship between intraspinal acetylcholine and antinociception, and elucidate interactions between acetylcholine and other mechanisms that mediate antinociception in the spinal cord.

<p>The timing of cell differentiation is important for development and renewal of well functioning organs and tissues. One protein involved in this process is Preadipocyte factor 1 (Pref-1). Most likely, the role of this protein is to maintain cells in an undifferentiated state. </p><p>The work presented in this thesis, has employed the rat as an animal model for the studies of Pref-1. Rat models of obesity (Zucker, ZO) and type II diabetes (Goto-Kakizaki, GK) were used to determine metabolic influence on Pref-1 and adipokine mRNA expression in adipose tissues.</p><p>The Pref-1 cleavage product was purified from rat amniotic fluid and physicochemically characterised. Concentration of Pref-1 in serum, amniotic fluid and urine was determined by ELISA. Soluble Pref-1 and the compartmentalisation of the protein were highly similar to what had previously been demonstrated in mice and humans.</p><p>Immunohistochemistry studies displayed similar staining patterns of Pref-1 in adrenal glands, ovaries and pituitary glands of non-pregnant and pregnant rats. This suggests that pregnancy do not influence the protein expression of Pref-1 in these organs.</p><p>In the GK rats, Pref-1 mRNA was altered and a decrease in the visceral compared to subcutaneous adipose depots was demonstrated, in contrast to the ZO rats. Additionally, adiponectin, leptin, IL-6 and TNF-α mRNA levels were altered in the diabetic strain, indicating that this animal model expresses many of the typical features of type II diabetes.</p><p>In conclusion, the rat is an appropriate model for studies of FA1/Pref-1/dlk. Pref-1 is highly elevated in fetal and maternal serum during pregnancy. However, the expression of Pref-1 in some endocrine tissues did not alter due to pregnancy. The mRNA expression of Pref-1 was altered between adipose depots and demonstrated to be affected by metabolic disturbances in the animals.</p>

The timing of cell differentiation is important for development and renewal of well functioning organs and tissues. One protein involved in this process is Preadipocyte factor 1 (Pref-1). Most likely, the role of this protein is to maintain cells in an undifferentiated state. The work presented in this thesis, has employed the rat as an animal model for the studies of Pref-1. Rat models of obesity (Zucker, ZO) and type II diabetes (Goto-Kakizaki, GK) were used to determine metabolic influence on Pref-1 and adipokine mRNA expression in adipose tissues. The Pref-1 cleavage product was purified from rat amniotic fluid and physicochemically characterised. Concentration of Pref-1 in serum, amniotic fluid and urine was determined by ELISA. Soluble Pref-1 and the compartmentalisation of the protein were highly similar to what had previously been demonstrated in mice and humans. Immunohistochemistry studies displayed similar staining patterns of Pref-1 in adrenal glands, ovaries and pituitary glands of non-pregnant and pregnant rats. This suggests that pregnancy do not influence the protein expression of Pref-1 in these organs. In the GK rats, Pref-1 mRNA was altered and a decrease in the visceral compared to subcutaneous adipose depots was demonstrated, in contrast to the ZO rats. Additionally, adiponectin, leptin, IL-6 and TNF-α mRNA levels were altered in the diabetic strain, indicating that this animal model expresses many of the typical features of type II diabetes. In conclusion, the rat is an appropriate model for studies of FA1/Pref-1/dlk. Pref-1 is highly elevated in fetal and maternal serum during pregnancy. However, the expression of Pref-1 in some endocrine tissues did not alter due to pregnancy. The mRNA expression of Pref-1 was altered between adipose depots and demonstrated to be affected by metabolic disturbances in the animals.