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Uterine inflammation is one of the causes of a poor uterine environment. This can result in early embryonic loss in the mare due to an inhibition of or an increased secretion of prostaglandin F2α (PGF2α ). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. Increased secretion or accumulation of PGF2α within the uterus due to uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence prostaglandin production in many species, although the effects on the mare remain unknown. Equine endometrial biopsies were collected and used to establish endometrial epithelial cell and explant cultures to determine the release of PGF2α and PGFM in response to oxytocin stimulation. Endometrial explant cultures were used to determine the inhibitory effects of Atosiban, an oxytocin receptor antagonist, and Indomethacin, a cyclooxygenase –2 inhibitor, on PGF2α secretion. Endometrial explant cultures were challenged with oxytocin (250 nM) and PGF2α concentrations were measured over time. The effects of PUFAs on equine endometrial prostaglandin production were determined using endometrial biopsies harvested on day two of behavioral estrus. Equine endometrial cells were established and shown to replicate in culture and on a basement membrane matrix. Equine endometrial explants stimulated with oxytocin had increased secretion of PGF2α and PGE2 and the secretion of PGF2α was inhibited through an oxytocin receptor antagonist and Cox inhibition. Endometrial explants stimulated with lipopolysaccharide had increased secretion of PGF2α and PGE2, however oxytocin stimulated to a greater extent than LPS. Supplementation with PUFAs, specifically DHA, decreased the secretion of PGF2α and PGE2, however AA and EPA failed to influence this response. Expression of mRNA was not influenced by fatty acid supplementation, however was altered by stimulus. Therefore DHA influences the inflammatory response in vitro through mechanisms other than enzyme expression. Decreased PGF2α production associated with PUFA supplementation in vivo, creates a likely approach for decreasing early embryonic loss associated with post breeding inflammation commonly seen in the equine industry.

The major objective of this project was to examine the potential for mediators of IgE-mediated allergic reactions to alter neural activity. The project was divided into three parts. In Part I, the ability of endogenously released chemical mediators to alter neural activity in vitro was assessed by measuring isometric contractile responses to electrical field stimulation (EFS) (2-128 Hz, 20 V, 0.5 msec. duration) of sensitized rabbit bronchi before and after exposure to the antigen horseradish peroxidase (HRP). Antigen enhanced bronchial responses to EFS with low frequencies: mean log (± S.E.) frequency which produced 20% of maximum response decreased from 1.04 (± 0.05) to 0.90 (± 0.07) Hz (p < 0.05). Responses of unsensitized bronchi were not enhanced by antigen. Chlorpheniramine, an H₁ antagonist, abolished the antigen effect. Antigen did not enhance the responses to exogenous acetylcholine. Hence, the antigen is apparently modulating neural activity and not smooth muscle per se. In Part II, the capacity for histamine to modulate vagally-induced bronchoconstriction in anesthetized, vagotomized, mechanically-ventilated rabbits was examined in vivo. Changes in pulmonary resistance induced by electrically stimulating the cut ends of the vagi (2-32 Hz, 20 V, 0.5 msec. duration) were assessed before and 10 minutes after histamine aerosolization (10 breaths of 10 mg/ml). Histamine inhalation potentiated vagally-induced bronchoconstriction at low frequencies: mean log (± S.E.) frequency producing a 20% change in pulmonary resistance decreased from 0.88 (± 0.09) to 0.56 (± 0.15) (p < 0.05). Chlorpheniramine abolished this effect. In Part III, the dependence on IgE antibodies of the in vitro modulation of neurally-induced contraction of sensitized bronchi was investigated. Rabbits were passively immunized with fractions enriched with HRP-specific IgE, IgG, or IgM antibodies. After 72 hours, rabbits were sacrificed and the responses of bronchi to EFS were assessed before and after antigen challenge. Antigen enhanced the responses to EFS only of bronchi passively sensitized with IgE. Therefore, antigen enhancement of neural activity was dependent on IgE. These studies demonstrate that the interaction between antigen and IgE antibodies can induce the release of chemical mediators which can alter neural activity.

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In these experiments, we primarily investigate the role and source of nerve growth factor (NGF) in peripheral tissues undergoing chronic inflammation. It has been previously determined that there is a significant increase in the levels of NGF following prolonged inflammation of the urinary bladder or the colon, and the first of two projects discussed here mimics this increase with transgenic mice which ectopically produce NGF under control of the smooth muscle alpha-actin promoter. It was determined by this increase that a p75-sensitive increase in sympathetic innervation occurs when an abundance of NGF is produced locally in the descending colon. Sensory innervation in the colon was found to come from two unique populations, one of which increased following heightened NGF levels. The urinary bladders of NGF overexpressing mice were determined to have an increase in sensory axonal density. The second project described here features chemically induced colonic inflammation and observes the nervous and growth factor changes as a response. Transgenic reporter mice are used to observe the cellular source of NGF in the descending colon, which unexpectedly was determined to be Dogiel type II (DgII neurons) based on morphological and chemical characteristics. We report the increase in NGF mRNA and protein observed following a brief 5-day colonic inflammation, and note that there is no increase in axonal density observed. The chemical inflammation did, however, induce an increase in axonal varicosities, used as a measure of axon damage. Finally, a heterozygous null-mutation of NGF was made in a line of transgenic mice to observe changes in local sensory neurons, sympathetic innervation or NGF protein production, but no differences between the heterozygous null mutants and age-matched wild-type siblings were observed. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-09-05 12:08:44.326