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The effects of a lethal dose (1380 r) of X-irradiation on plasma and adrenal tissue histamine levels of rats were studied. The plasma histamine response was triphasic (increase at 1-3 hours, decrease at 5 and 9 hours and return to control at 24 hours post-irradiation). The adrenal tissue histamine response was found to be biphasic (decrease at 1 to 9 hours and a return to control level at 24 hours post-irradiation).

Blood flow to the previously active skeletal muscle remains elevated for several hours following an acute bout of aerobic exercise and is dependent on activation of H1 and H2 histamine receptors. Many questions remain unanswered in humans regarding the mechanisms mediating this sustained post-exercise vasodilation and what benefits come of this physiological phenomenon. The studies detailed in this dissertation were designed to examine the upstream mechanisms and explore a potential benefit associated with sustained post-exercise vasodilation. In chapter IV, we examined if oxidative stress is the upstream exercise-related factor mediating sustained post-exercise vasodilation. Intravenously infusing the antioxidant ascorbate blunted sustained post-exercise vasodilation, and this reduction was similar in magnitude to that observed with H1/H2 blockade. However, ascorbate may directly degrade histamine and may also inhibit its formation. Therefore, we conducted a follow-up study to verify the findings in study 1. In this study, we intravenously infused n-acetylcysteine, a potent antioxidant with no known histaminergic interactions. We found that n-acetylcysteine had no effect on sustained post-exercise vasodilation, indicating that exercise-induced oxidative stress is not the exercise related factor mediating sustained post-exercise vasodilation. In chapter V, we attempted to measure interstitial histamine in an effort to demonstrate that exercise induces the local formation of histamine in previously active skeletal muscle. We found that histamine is increased in the interstitial fluid within skeletal muscle during and after exercise. Additionally, we determined that de novo synthesis via histidine decarboxylase contributes to the rise in histamine during and following exercise. We also demonstrated a possible role of mast cells as an additional mechanism augmenting histamine in skeletal muscle. Collectively, these studies demonstrate that histamine is the ligand activating histamine receptors and activation is due to the induction of histidine decarboxylase and mast cell activation. In chapter VI, we attempted to determine if histamine receptor activation contributes to the expression of pro- and anti-angiogenic growth factors during the recovery from exercise. Our preliminary findings indicate that activation of histamine receptors may play a role in the expression of pro-angiogenic growth factors during the recovery from acute aerobic exercise.

Bacterial histamine formation in mackerel and albacore was studied by inducing histamine in the muscles under controlled storage conditions. The optimum temperature for histamine formation was 25°C. The highest level of histamine detected was 283 mg/100 g in the 2-day stored mackerel; and 67.1 mg/100 g in the 6-day stored albacore. To identify the bacteria crucial to histamine formation, histamine formers were isolated using the conventional culture method. Enteric bacteria were most frequently isolated from the fish. Weak histamine formers were found in the gill and skin of fresh fish, and they required the enrichment step. Prolific histamine formers were mostly isolated from the decomposed muscles during storage at 25°C. Morganella morganii was the most prolific histamine former, producing >3,000 ppm in culture broth. M. morganii was the most prevalent histamine former in mackerel. In albacore, however, the most prevalent species was Hafnia alvei, a weak histamine former, resulting in less histamine accumulation than mackerel. Weak histamine formers, identified as natural bacteria in the marine environment, were found in mackerel during storage at 4°C after fish became unsuitable for human consumption. At 0°C, neither histamine-forming bacteria nor histamine was detected in fish. M. morganii formed significant amounts of histamine (>200 mg/100 g) in artificially contaminated fresh and frozen mackerel, albacore, and mahi-mahi when the fish were improperly stored at ambient temperatures (25°C). Growth of M. morganii was controlled by storage of fish at 4°C or below, but histamine formation was controlled only during frozen storage. For rapid detection of M. morganii, a PCR assay was developed by designing 16S rDNA targeted primers. Unique primers found for M. morganii were: the forward primer, 5'-CTCGCACCATCAGATGAACCCATAT-3'; and the reverse primer, 5'-CAAAGCATCTCTGCTAAGTTCTCTGGATG-3'. Nine CFU/ml of M. morganii inoculated in albacore homogenate were detected with a 6 h-enrichment of samples in TSB at 37°C. It would be necessary to monitor the presence of M. morganii in fish during handling and storage due to its high histamine-producing capability and prevent its contamination and proliferation after capture. The PCR assay developed in this study would be helpful to routinely monitor its presence in fish. / Graduation date: 2002

Many factors influence the inflammatory responses in rat trachea, including inflammatory mediators released from nerve fibers, histamine released by mast cells and endotoxin from the cell walls of bacteria. The inflammatory responses include plasma extravasation, subepithelial edema, and hypersecretion of secretory cells. But the mechanism of inflammation mediated by these factors was not completely understood. In the present study, a high dose of histamine was administered intravenously to induce the inflammation in rat airway. India ink was also injected as a tracer to label the leaky blood vessels in different time points. To investigate the serous cell secretion and subepithelial edema formation, the treacheal tissue was processed for histological study. Electron microcopy was carried out to investigate the ultrastructure of serous cells. To investigate the mechanism of histamine effect, the muscarinic receptor antagonist atropine (1 mg/ml/kg) or histamine H1 receptor antagonist mepyramine (10 mg/ml/kg) was injected 15 min before histamine injection. Five minutes after histamine, plasma leakage and serous cell secretion were extensive. The area density of India ink-labeled leaky vessels was 17.24 % ¡Ó 2.03 %. Saline, the vehicle of histamine, produced only a little extravasation. Mepyramine inhibited the histamine-induced plasma extravasation and serous cell degranulation significantly but atropine had no effect. The results suggest that histamine-induced serous cell degranulation is mainly through histamine H1 receptors but not through cholinergic muscarinic receptors in rat trachea.

Inflammatory response in the airway may lead to asthma. Asthma may develop during the childhood in some asthmatic patients. Both environmental and genetic factors may influence the onset and progress of asthma. It is well-known that there may be complex neural innervation and reflex mechanisms between trachea and esophagus. Intraesophgeal infusion of 1N HCl could lead to tracheal inflammation by activating neural reflex pathway and cause tachykinin-like substance to release. In this study, we first sensitized rats with 1ml of OVA-Al［OH］3 mixture containing 200£gg OVA via intraperitoneal injection on days 1, 2, 3 and 11, then perform intraesophageal infusion of ovalbumin to see whether stimulation of esophagus in sensitized rat model could involve inflammatory response in the lower airways. Animals were perfused with saline and fixative at various time points and the esophagus and airway tissues were processed for the subsequent analysis. We observed the extent of plasma leakage and migration of leukocytes in the lower airway. India ink was used to label the leaky blood vessels.The magnitude of plasma leakage was expressed by the area density of India ink-labeled blood vessels. The results showed that the intraesophageal infusion of ovalbumin 75 mg/kg caused an increase in plasma leakage in the lower airways. The plasma leakage peaked at 30 min, the area density of plasma leakage in trachea was 22.43 ¡Ó 3.34¢H; and 20.57 ¡Ó 4.91¢H in right bronchus; 18.47 ¡Ó 5.03¢H in left bronchus and 27.85 ¡Ó 2.71¢H in epiglottis. The extent of leakage gradually diminished 3 hours after ovalbumin infusion. However, a second increased plasma leakage peaked at about 4 hours of ovalbumin infusion. Tissue sections clearly showed degranulation of mast cells in OVA infusion group. Experimental data showed that pretreatment with either bilateral vagotomy, or mepyramine, the histamine H1 receptor antagonist, significantly inhibited the inflammatory response in the lower airways induced by intraesophageal infusion of OVA. In conclusion, there were clearly two phases, early and late phase responses, in inflammatory response in OVA-sensitized rats receiving intra-esophageal OVA challenge. The underlying mechanisms may involve vagal C-fibers and histamine H1 receptors.