The aim of present study was to assess the radioprotective effects of the local application of amifostine to treat acute buccal mucositis in guinea pigs. A total of 32 guinea pigs were randomized into four groups: (Group A) topically administered 50 mg of amifostine plus radiotherapy (RT); (Group B) 100 mg amifostine plus RT; (Group C) normal saline plus RT; and (Group D) normal saline plus sham RT. The opportunity for administration was 15 min before irradiation. When administered, the cotton pieces that had been soaked with 0.5 ml amifostine solution or saline were applied gently on the buccal mucosa of each guinea pig for 30 min. The animals in Groups A, B and C were irradiated individually with a single dose of 30 Gy to the bilateral buccal mucosa. Eight days after irradiation, the animals were scored macroscopically; they were then euthanized, and the buccal mucosal tissues were processed for hematoxylin–eosin staining and ICAM-1 immunohistochemical analysis. In Groups A and B, the mean macroscopic scores were 2.9 ± 0.6 and 2.4 ± 1.1, respectively. There was no significant difference between the two groups (P > 0.05). However, when they were separately compared with Group C (4.4 ± 0.7), a noticeable difference was obtained (P < 0.05). No mucositis was observed in Group D. Comparisons of the expression of ICAM-1 were in agreement with the macroscopic data. Histologically, superficial erosion, exudate and ulcer formation were all observed in the RT groups; only the severity and extent were different. The microscopic observations in the amifostine-treated groups were better than in Group C. The results demonstrated that topical administration of amifostine to the oral mucosa is effective treatment of acute radiation-induced mucositis.
Objective Munc18-1 has an important role in neurotransmitter release, and controls every step in the exocytotic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Munc18 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid (KA) solution into hippocampus of Sprague-Dawley (SD) rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immunochemistry and immunoelectron microscopy with anti-Munc18-1 antibody were used to determine the nuclear localization of Munc18-1. Immunoblotting was used to detect the protein level of Munc18-1. Results The localization of Munc18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunoblotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Munc18-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expression level of Munc18-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relationship between the change of Munc18-1 expression in neuronal nuclei and neuronal over-activation was also tested in primary cultured neurons. After treatment with 50 μmol/L glutamate acid for 3 h, Munc18-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excitatory stimulation can induce the distribution change of Munc18-1 in neuron, which may subsequently modulate neuronal functions in brain.
Objective Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters. Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup of Rasmussen's encephalitis patients. However, the machinery of Munc18 autoimmunity is still elusive. The present study was aimed to investigate Munc18 release from primary cultured neurons, Munc18 distribution on the outer plasma membrane of neurons, and the neurotoxicity of Munc18 antibody. Methods The cerebral cortical neurons from embryonic day 17 SpragueDawley rats were prepared and cultured in neurobasal medium. The proteins in culture medium were precipitated with 10% trichloroacetic acid, and analyzed by immunoblotting. The proteins on neuronal surface were biotinylated with EZ-Link-sulfo-NHS-LC-Biotin, and collected with avidin-conjugated agarose beads followed by immunoblotting analysis. For cell surface immunofluorescent staining, the living neurons were labeled with anti-Munc18 antibody at 4 °C. Neuronal injury was assessed by lactate dehydrogenase(LDH) release. Results Munc18 was detected in culture medium by immunoblotting analysis.After treatment with 50 μmol/L glutamate for 1 h, Munc18 content in medium was increased. Meanwhile, β-actin and syntaxin1were not detected in culture medium, and LDH release was not significantly increased. Moreover, glutamate enhanced Munc18 distribution on outer plasma membrane. Living neuron staining also demonstrated the localization of Munc18 on neuronal surface after glutamate treatment, especially at contacting regions between neurons. Glutamate-induced increase of surface Munc18 distribution was suppressed by NMDA receptor antagonist MK801, but not by AMPA receptor antagonist NBQX. Moreover, compared with c-Fos antibody, Munc18 antibody could induce neuronal injury, when culture medium contained the components of serum. Conclusion A portion of Munc18 can be released from neurons or distributed on neuronal surface, which can be enhanced by glutamate treatment via activation of NMDA receptors. Besides, Munc18 antibody-induced neuronal injury depends on the serum components.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.