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Poster 10
Scientific Poster Session sites is unknown, and 8 AQPs are reported to be expressed in various tissues of the GI tract, including AQPs 1, 3, 4, 5, 7, 8, 9, and 10. While the recently identified AQP10 is known to be expressed in keratinized gingiva, the specific combinations of AQP expression found in DF and OKCs are not previously reported for skin, suggesting the DF tissues may be developmentally unique. AQP3 is functionally heterogeneous and possesses water and solute permeation mechanisms. The differential expression of AQP3 may be important in OKC development and may help understand their etiology. References Wang W, Hart PS, Piesco NP, et al: Aquaporin expressions in developing human teeth and selected orofacial tissues. Calcif Tissue Int 1, 2003 Horster M: Embryonic epithelial membrane transporters. Am J Physiol 279:F982, 2000
POSTER 10 Immunolocalization of Opioid Receptors in Primate Trigeminal Ganglion Stephen B. Milam, DDS, PhD, University of Texas Health Science Center, Dept. of OMS, San Antonio, TX (Tobler J; Cui Y; Zardeneta G) Opioids have been administered peripherally to regulate pain and neurogenic inflammation, presumably by action via opioid receptors expressed by primary trigeminal ganglion neurons. However, to date, opioid receptor expression by trigeminal ganglion neurons has not been fully characterized. The aim of this study was to determine the percentage of neurons expressing either of the opioid receptors (MOR, KOR or DOR) in trigeminal ganglia (tgg). Methods: Fresh trigeminal ganglia were harvested from baboons (P. cynecephalus) and either frozen in OCT or used to establish primary neuronal cultures for 7 days. Cross-section slices (20 micromolar) from OCT embedded tgg were used for immunohistochemistry using specific antibodies against the opioids (Biodesign). Species-specific secondary antibodies tagged with a peroxidase were employed and the standard DAB colorimetric substrate used. Percentage of neurons staining positive for these receptors were counted independently by 2 examiners using light microscopy. Dispersed tgg were grown in media supplemented with nerve growth factor (10 nanomolar) containing mitotic inhibitors to establish primary neuron cultures. Maximum neuronal outgrowth was observed after 7days in culture. These cultured tgg neurons were used to verify opioid receptor expression using standard western blot analyses. Neurons were lysed in an SDS-containing solution, and analyzed by SDS-PAGE followed by western blot using the same antibody scheme as above. Opioid receptors were visualized by ECL. 86
Results: The average percentage of tgg neurons containing MOR, KOR, or DOR were found to be 34%, 27%, and 24%, respectively. Cultured tgg neurons were found to express all 3 of the opioid receptors, although the percentage of cultured tgg neurons expressing these was not determined by this method. Conclusion: A significant percentage of neurons (24% to 34%) from adult primate trigeminal ganglion were found to express MOR, KOR, or DOR in vivo. These opiate receptors also were identified in tgg cultures by western blot. This study provides evidence that MOR, KOR, and DOR are expressed by tgg neurons. Future studies will be required to characterize specific trigeminal ganglion neural responses to peripherally-administered opioid receptor subtype-selective opioid agonists. References Hargreaves KM, Keatin K, Cathers S, et al: Analgesic effects of morphine after PDL injection in endodontic patients. J Dent Res 70: 445, 1991 List T, Tegelberg A, Haraldson T, et al: Intra-articular morphine as analgesic in temporomandibular joint arthralgia/osteoarthritis. Pain 94: 275, 2001 Funding Source: UTHSCSA PREF award.
POSTER 11 Effects of Radiation on Primary Human Oral Mucosal Keratinocytes Takayoshi Tobita, DDS, PhD, University of Michigan, Dept. of OMS, Ann Arbor, MI 48109-0018 (Izumi K; Feinberg SE) Purpose: Oral mucositis is a serious side effect for patients undergoing radiotherapy for head and neck tumors. It consists of 4 stages: 1) an initial inflammatory/ vascular phase, 2) an epithelial phase, 3) an ulcerative/ bacteriological phase, and 4) a healing phase. This investigation is a preliminary report in the development of an in vitro model of radiation-induced oral mucositis. Our objective was the evaluation of the primary effects of gamma radiation, in vitro, on a monolayer of oral keratinocytes. Materials and Methods: Oral keratinocytes were obtained from surgically discarded oral mucosa (6 samples: 2 male and 4 female) and cultured in chemically defined serum free medium (EpiLife, Cascade Biologics, Portland, OR). When oral keratinocytes reached 60% to 70% confluency, they were irradiated with 0, 0.5, 1, 3, 5, and 8 Gy by a cobalt 60 source. Colony-forming efficiency (CFE) for cell proliferation was determined by plating irradiated cells in a 60-mm culture dish at a density of 25 cells/cm2. Surviving fraction (SF) of irradiated cells was evaluated 12 days postirradiation by fixing and staining cell colonies with 0.2% Crystal Violet. Colonies, which were larger than 16 cells, were counted and calculated AAOMS • 2003
POSTER 10 Immunolocalization of Opioid Receptors in Primate Trigeminal Ganglion Stephen B. Milam, DDS, PhD, University of Texas Health Science Center, Dept. of OMS, San Antonio, TX (Tobler J; Cui Y; Zardeneta G) Opioids have been administered peripherally to regulate pain and neurogenic inflammation, presumably by action via opioid receptors expressed by primary trigeminal ganglion neurons. However, to date, opioid receptor expression by trigeminal ganglion neurons has not been fully characterized. The aim of this study was to determine the percentage of neurons expressing either of the opioid receptors (MOR, KOR or DOR) in trigeminal ganglia (tgg). Methods: Fresh trigeminal ganglia were harvested from baboons (P. cynecephalus) and either frozen in OCT or used to establish primary neuronal cultures for 7 days. Cross-section slices (20 micromolar) from OCT embedded tgg were used for immunohistochemistry using specific antibodies against the opioids (Biodesign). Species-specific secondary antibodies tagged with a peroxidase were employed and the standard DAB colorimetric substrate used. Percentage of neurons staining positive for these receptors were counted independently by 2 examiners using light microscopy. Dispersed tgg were grown in media supplemented with nerve growth factor (10 nanomolar) containing mitotic inhibitors to establish primary neuron cultures. Maximum neuronal outgrowth was observed after 7days in culture. These cultured tgg neurons were used to verify opioid receptor expression using standard western blot analyses. Neurons were lysed in an SDS-containing solution, and analyzed by SDS-PAGE followed by western blot using the same antibody scheme as above. Opioid receptors were visualized by ECL. 86
Results: The average percentage of tgg neurons containing MOR, KOR, or DOR were found to be 34%, 27%, and 24%, respectively. Cultured tgg neurons were found to express all 3 of the opioid receptors, although the percentage of cultured tgg neurons expressing these was not determined by this method. Conclusion: A significant percentage of neurons (24% to 34%) from adult primate trigeminal ganglion were found to express MOR, KOR, or DOR in vivo. These opiate receptors also were identified in tgg cultures by western blot. This study provides evidence that MOR, KOR, and DOR are expressed by tgg neurons. Future studies will be required to characterize specific trigeminal ganglion neural responses to peripherally-administered opioid receptor subtype-selective opioid agonists. References Hargreaves KM, Keatin K, Cathers S, et al: Analgesic effects of morphine after PDL injection in endodontic patients. J Dent Res 70: 445, 1991 List T, Tegelberg A, Haraldson T, et al: Intra-articular morphine as analgesic in temporomandibular joint arthralgia/osteoarthritis. Pain 94: 275, 2001 Funding Source: UTHSCSA PREF award.
POSTER 11 Effects of Radiation on Primary Human Oral Mucosal Keratinocytes Takayoshi Tobita, DDS, PhD, University of Michigan, Dept. of OMS, Ann Arbor, MI 48109-0018 (Izumi K; Feinberg SE) Purpose: Oral mucositis is a serious side effect for patients undergoing radiotherapy for head and neck tumors. It consists of 4 stages: 1) an initial inflammatory/ vascular phase, 2) an epithelial phase, 3) an ulcerative/ bacteriological phase, and 4) a healing phase. This investigation is a preliminary report in the development of an in vitro model of radiation-induced oral mucositis. Our objective was the evaluation of the primary effects of gamma radiation, in vitro, on a monolayer of oral keratinocytes. Materials and Methods: Oral keratinocytes were obtained from surgically discarded oral mucosa (6 samples: 2 male and 4 female) and cultured in chemically defined serum free medium (EpiLife, Cascade Biologics, Portland, OR). When oral keratinocytes reached 60% to 70% confluency, they were irradiated with 0, 0.5, 1, 3, 5, and 8 Gy by a cobalt 60 source. Colony-forming efficiency (CFE) for cell proliferation was determined by plating irradiated cells in a 60-mm culture dish at a density of 25 cells/cm2. Surviving fraction (SF) of irradiated cells was evaluated 12 days postirradiation by fixing and staining cell colonies with 0.2% Crystal Violet. Colonies, which were larger than 16 cells, were counted and calculated AAOMS • 2003