In vivo two-photon imaging of neuronal and brain vascular responses in mice chronically exposed to ethanol.

The effects of ethanol on brain function have been extensively studied using a variety of in vitro and in vivo techniques. For example, electrophysiological studies using brain slices from rodents and non-human primates have demonstrated that acute and chronic exposure to ethanol alters the intrinsic excitability and synaptic signaling of neurons within cortical and sub-cortical areas of the brain. In humans, neuroimaging studies reveal alterations in measures of brain activation and connectivity in subjects with alcohol use disorder. While complementary, these methods are inherently limited due to issues related to either disruption of normal sensory input (in vitro slice studies) or resolution (whole brain imaging). In the present study, we used 2-photon laser scanning microscopy in intact animals to assess the impact of chronic ethanol exposure on sensory-evoked neuronal and vascular responses. Adult male C57BL/6J mice were exposed to four weekly cycles of chronic intermittent ethanol (CIE) exposure, while control mice were exposed to air. After withdrawal (≥72 h), a cranial window was placed over the primary visual cortex (V1), and sensory-evoked responses were monitored using the calcium indicator OGB-1. CIE exposure produced small but significant changes in response amplitude (decrease) and orientation selectivity of V1 neurons (increase). While arteriole diameter did not differ between control and CIE mice under baseline conditions, sensory-evoked dilation was enhanced in vessels from CIE-exposed mice as compared to controls. This was accompanied by a reduced latency in response to stimulation. In separate experiments, pial arteriole diameter was measured in the barrel cortex of control and CIE-exposed mice. Baseline diameter of barrel cortex arterioles was similar between control and CIE-exposed mice, but unlike vessels in V1, sensory-evoked dilation of barrel cortex arterioles was similar between the two groups. Together, the results of these studies suggest that chronic exposure to alcohol induces changes in neurovascular coupling that are region-dependent.

Spermatozoa and seminal plasma induce a greater inflammatory response in the ovine uterus at oestrus than dioestrus.

Leukocyte infiltration and increased synthesis of cytokines in response to insemination is considered to enhance reproductive success. The present study investigated the inflammatory response to whole semen, spermatozoa and seminal plasma, with and without the addition of antibiotics, in the ovine uterus at oestrus and dioestrus. Seminal plasma and spermatozoa both contributed to increased IL-8 secretion (P < 0.01) by endometrial epithelial cells and a concurrent infiltration by neutrophils (P < 0.01). Increased GM-CSF secretion (P < 0.01) occurred in response to whole semen and spermatozoa when antibiotics were not used. Macrophages and eosinophils increased (P < 0.05) in the endometrial stroma when antibiotics were not used, and fewer mast cells were detected in the deep endometrial stroma after treatments containing antibiotics (P < 0.05). Neutrophil and IL-8 responses to insemination were greater at oestrus (P < 0.01) than at dioestrus and the GM-CSF response followed a similar trend. Eosinophil numbers were increased at oestrus (P < 0.01) but minimally affected by insemination. More macrophages were located in the superficial endometrial stroma at oestrus. These results indicate that spermatozoa, seminal plasma and possibly bacteria contribute to the post-insemination inflammatory response, and that leukocytes, GM-CSF and IL-8 secretion in the ovine uterus are influenced by ovarian hormones.

Granulocyte-macrophage colony stimulating factor and interleukin-8 in the reproductive tract of ewes following oestrus and mating.

Cytokines produced in the female reproductive tract after mating may enhance reproductive success. The present study investigated the distribution of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 in tissues and luminal secretions from different sites in the reproductive tract of the ewe following oestrus and after natural mating. Fifteen ewes were mated with a ram for 1 h and their reproductive tracts collected 3, 6, 18, 24 or 48 h later. Another 15 ewes were used as oestrous controls. Luminal secretions and tissue samples were collected from seven sites in each reproductive tract. Secretions were analysed by enzyme-linked immunosorbent assay and tissues were stained immunohistochemically using anti-sheep GM-CSF and anti-sheep IL-8 antibodies. Both cytokines were found in luminal and glandular endometrial epithelium and, to a lesser extent, in cervical epithelium; neither was found in the vaginal epithelium. Twice as many (P < 0.05) luminal samples from mated ewes than non-mated ewes were positive for GM-CSF. The vaginal lumen contained significantly higher (P < 0.01) concentrations of IL-8 compared with other sites, irrespective of mating status. Significant differences (P < 0.05) were found in staining intensity of GM-CSF and IL-8 from different sites. Production of GM-CSF and IL-8 by reproductive tissues is likely to contribute to leucocyte infiltration into the ovine reproductive tract.

Leucocyte population changes in the reproductive tract of the ewe in response to insemination.

Leucocyte changes after insemination may affect conceptus implantation, but information regarding leucocyte populations in the ruminant reproductive tract is limited. The present study investigated changes in leucocyte populations and distribution in the ovine reproductive tract following oestrus and insemination. Fifteen ewes were mated with a ram for 1 h and their reproductive tracts collected 3, 6, 18, 24 or 48 h later. Another 15 ewes were used as oestrus controls. Tissues were collected from 10 sites in each reproductive tract and stained with haematoxylin and eosin, Toluidine blue and immunohistochemically using a monoclonal CD68 antibody. Luminal mucus smears were collected from seven sites and stained with a modified Wright's stain and immunohistochemically. Neutrophils, eosinophils, mast cells and macrophages were identified and quantified, and temporal changes in their distribution within tissues were examined. Neutrophils and macrophages increased significantly (P < 0.05) in posterior cervical and uterine tissues following insemination. In uterine tissues, neutrophils peaked at 6 h after insemination, whereas macrophages peaked at 18-24 h. Mast cells decreased and eosinophils remained constant. Neutrophils increased significantly (P < 0.05) in the cervical and uterine lumen following insemination. In conclusion, leucocyte population changes after insemination vary between different sites in the ovine reproductive tract and may contribute to pregnancy establishment.

Controlled trial of hyperbaric oxygen treatment for alkali corneal burn in the rabbit.

PURPOSE: To evaluate the efficacy of hyperbaric oxygen therapy in the treatment of alkali-induced corneal burns in an animal model. METHODS: Twenty-four rabbits were randomized into a control group (n = 12) and hyperbaric oxygen treatment group (n = 12). After induction of anaesthesia, the alkali burn model was established by application of 1 N sodium hydroxide to one eye of each rabbit. The hyperbaric oxygen treatment group was treated each day for 21 days with hyperbaric oxygen at 2.4 Atmospheres Absolute (ATA) for 1 h. The eyes of the animals were examined daily for 2 weeks and then weekly until the end of the trial. The principal endpoint was that of perforation of the cornea at which time the animals were killed with a lethal dose of either intravenous or intraperitoneal barbiturate and the eyes immediately enucleated and fixed in 10% neutral buffered formalin. All animals in which complete healing took placed were also killed, the eyes removed, fixed and examined histologically. Photographs were taken of the rabbit's eyes at weekly intervals and the area of vascularization and epithelial defects in the hyperbaric and control groups were compared. RESULTS: Equal numbers (seven) of the control and hyperbaric oxygen treated groups had perforated corneas and there was no statistical difference in the mean time to perforation (control 30.1 days; treated 30 days). There was also no statistical difference between the two groups with respect to epithelial defect size. CONCLUSION: Treatment with hyperbaric oxygen for 1 h daily for 21 days had no beneficial effect on alkali-induced corneal burns.