Date of Award
Master of Science (MS)
Douglas I. Johnson
Mastitis, the inflammation of the mammary gland by bacterial infection, is one of the costliest diseases to the dairy industry primarily due to a loss in milk production. The aim of this study was to investigate the mechanisms underlying reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces cell apoptosis, oxidative stress and increases hypoxia while inhibiting milk gene expression in the mammary gland. To test this hypothesis, mice were bred to pregnancy, and 3 days post-partum the left and right sides of the 4th pair of mammary glands were alternately injected with either the endotoxin liposaccharide (LPS, E. coli 055:B5, 100 ul of 0.2 mg/ml) or sterile PBS through the teat meatus. At 10.5 and 22.5 h post-injection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the fourth glands were individually collected (n=8 pairs) and analyzed for hypoxia, gene expression and oxidative stress. LPS treatment induced mammary gland inflammation as shown by increases in inflammatory cytokine expression (P < 0.001) and neutrophil recruitment at 12 and 24 h. LPS promoted cell apoptosis in a transient manner; an abundance of cleaved caspase 3 was evident only at 12 h after LPS challenge (P = 0.02). Increased H2O2 content was seen at 12 h (P < 0.001) but decreased dramatically after 24 h of LPS treatment (P < 0.001). Total antioxidative capacity tended to decrease at both 12 and 24 h (P = 0.067 and 0.061, respectively). In agreement with these findings, LPS activated Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidative signaling in the mammary gland, demonstrated by increased expression of its target gene Nqo1 at 12 h (P = 0.05) and xCT at 24 h (P = 0.076). Hypoxyprobe staining, indicative of hypoxia, was greater in the alveoli of PBS-treated glands than LPS-treated glands at both 12 and 24 h. This suggests oxygen tension rises in response to LPS treatment. Conversely, milk expression genes, β-casein gene (CSN2) and α-lactalbumin (LALBA), were inhibited by LPS treatment across time. Expression of α-S1 casein (CSN1S1) mRNA increased with LPS treatment at 24 h, but protein expression was reduced at this same time point (P < 0.05). In summary, intramammary LPS challenge incurs inflammation, augments cell apoptosis, induces oxidative stress and activation of the Nrf2 antioxidation pathway, increases oxygen tension, and inhibits milk protein expression in the mammary gland. This study provides functional insight into mechanisms of reduced milk production during mastitis and provides possible approaches to combat reduction in milk production, such as enhancing the Nrf2-antioxidative signaling pathway and reducing inhibition of milk protein expression.
Number of Pages
Spitzer, Alexander Jonathan, "Endotoxin Increases Oxidative Stress And Oxygen Tension While Reducing Milk Protein Gene Expression In The Mammary Gland" (2019). Graduate College Dissertations and Theses. 1123.