ULTRASTRUCTURAL EVALUATION OF THE RADIOPROTECTIVE EFFECT OF SODIUM SELENITE ON SUBMANDIBULAR GLANDS IN RATS

The aim of this study was to evaluate the radioprotector effect of sodium selenite on the ultrastructure of submandibular glands in rats. Fifty-seven male albino Wistar rats were randomized to 4 groups: control, irradiated, sodium selenite and irradiated/sodium selenite. The animals in the sodium selenite and irradiated/sodium selenite groups received intraperitoneal injections of sodium selenite (0.5 mg/kg body weight) 24 h before irradiation. The animals belonging to the irradiated and irradiated/sodium selenite groups were submitted to 15 Gy of gamma radiation in the head and neck region. The submandibular glands were removed at 4, 8, 12, 24, 48 and 72 h after irradiation. The ionizing radiation induced damage to the secretory cells, especially the serous cells, right from the first period. Vacuolization, lysis of cytoplasmic inclusions and nuclear alterations occurred. The sodium selenite group also presented cellular alterations in the study periods, but with less damage compared to that caused by radiation. There was greater similarity between the irradiated/sodium selenite group and the control group than with the other groups treated in all study periods. Despite the alterations observed in the sodium selenite group, sodium selenite presented a radioprotective action on the secretory cells of submandibular glands.     

Effect of ionizing radiation on rat parotid gland

A common side effect of radiotherapy used in the treatment of oral cancer is the occurrence of structural and physiological alterations of the salivary glands due to exposure to ionizing radiation, as demonstrated by conditions such as decreased salivary flow. The present study evaluated ultrastructural alterations in the parotid glands of rats receiving a fractionated dose (1,500-cGy) of radiation emitted by a Cesium-137 source and rats that were not subjected to ionizing radiation. After sacrifice, the parotid glands were removed and examined by transmission electron microscopy. Damage such as cytoplasmic vacuolization, dilatation of the endoplasmic reticulum and destruction of mitochondria, as well as damage to the cellular membrane of acinar cells, were observed. These findings lead to the conclusion that ionizing radiation promotes alterations in the glandular parenchyma, and that these alterations are directly related to the dose level of absorbed radiation. Certain phenomena that appear in the cytoplasm and nuclear material indicate that ionizing radiation causes acinar cell death (apoptosis).     

60Coγ射线照射大鼠涎腺组织后Mrell蛋白表达的初步研究

目的:检测Mre11基因及其蛋白在放疗后的大鼠腮腺和颌下腺组织中的表达变化。方法:选取近交系雄性Wistar大鼠60只,按单次照射0 Gy,3 Gy、6 Gy、9 Gy、12 Gy、15 Gy剂量分成6组,用60Coγ射线照射大鼠头颈部,2h后收集大鼠两侧腮腺和颌下腺组织。用透射电镜观察涎腺上皮细胞超微结构变化;用RT-PCR检测Mre11的基因表达情况;用免疫组化检测Mre11蛋白表达情况。结果:透射电镜显示:放射后大鼠腮腺腺泡细胞胞质和胞膜均出现不同程度的损坏,随着剂量的增加,这种损坏逐渐加剧,未见到再生、恢复的变化。颌下腺和导管变化不明显。RT-PCR检测Mre11mRNA表达无统计学差异。免疫组化检测Mre11蛋白表达有统计学差异。结论:治疗剂量的60Coγ照射对正常涎腺组织呈不可逆的损伤,损伤的程度具有剂量依赖性;大鼠涎腺细胞Mre11mRNA的表达无剂量依赖性;大鼠涎腺细胞Mre11蛋白的表达与辐射剂量以及组织类型有关,随剂量的增加先增强后减弱。     

Evaluation of radioprotective effect of vitamin E in salivary dysfunction in irradiated rats

The aim of this study was to evaluate the radioprotective effect of vitamin E in salivary gland function, as well as analyse the total protein concentration. For this purpose 90 male rats were used and randomly divided into five experimental groups: control (I), in which animals received olive oil solution but were not irradiated; irradiated-olive oil (II), in which animals received olive oil solution and were irradiated with a single exposure dose of 15 Gy of gamma rays to the head and neck region; irradiated (III), in which animals were only irradiated with a single exposure dose of 15 Gy of gamma rays; vitamin E (IV), in which animals received alpha tocopherol acetate solution but were not irradiated; irradiated-vitamin E (V), in which animals received alpha tocopherol acetate solution before irradiation with a single exposure dose of 15 Gy gamma rays. The animals were sacrificed 4, 8 h and 30 days after the irradiation procedure. No differences were observed in salivary volumes between the groups at 4 and 8 h. At 30 days, the salivary volume in the animals pertaining to the irradiated-olive oil group was significantly reduced in relation to the control group. The only irradiated group (III) presented significantly diminished salivary volume. In the salivary composition, no significant differences were observed in the total protein content between the groups studied. It was concluded that radiation had no effect on the total protein content and that vitamin E protected the salivary function 30 days after irradiation. Thus, vitamin E can be considered as a potential radioprotective substance.     

分次照射豚鼠腮腺早期放射性损伤的病理学观察

目的观察豚鼠在常规分割局部照射头颈部后腮腺早期放射损伤的病理变化。方法成年雄性豚鼠67只分别于照射剂量0,10,20,30,40,50Gy和50Gy后60天随机分为A～G7个组,给予常规分割γ射线局部照射头颈部,观察各组体重、腮腺重量、组织形态学改变、残存腮腺面积和炎症细胞计数。结果豚鼠体重在照射初期出现短暂体重下降之后逐渐增长。A～F组腮腺重量无显著差异（F=0.997,P=0.43）,G组腺重较其他组显著增加（P〈0.05）;照后腮腺面积随照射剂量的增加而呈递减趋势,并出现不同程度的腺体结构改变和炎症细胞浸润,F、G组炎症细胞计数显著高于A～E组（P〈0.05）,F组与G组相当（t=0.17,P=0.87）。结论照射剂量与腮腺早期放射性损伤的程度呈正相关,放射诱导的炎症反应可能参与其中。     

Rad50在放射后大鼠涎腺中的表达

目的:检测Rad50在放射后大鼠涎腺组织中的表达水平。方法:选用60只Wistar大白鼠,随机分成6组,每组10只。分为对照组(未照射)、3 Gy组、6 Gy组、9 Gy组、12 Gy组、15 Gy组。放射组大白鼠用60Co一次性照射后2 h内处死,立即取出其腮腺、下颌下腺组织备用。用电镜观察放射后各组涎腺组织超微结构的变化,用反转录聚合酶链反应(RT-PCR)检测Rad50基因表达水平的变化,用免疫组织化学法观察其蛋白表达水平的变化。结果:各放射组涎腺组织中Rad50的表达均高于对照组(P<0.05),但不同放射剂量的各放射组之间Rad50的表达无明显差异(P>0.05)。透射电镜下可见大鼠腮腺、下颌下腺细胞超微结构随着放射剂量的增加,发生较明显改变。结论:放射可引起涎腺组织Rad50表达水平增高,但其表达水平并未随放射剂量的增加而增高,提示Rad50在涎腺放射损伤修复中的表达水平有限,这可能是涎腺放射敏感性机制之一。     

Structural and functional injury in minipig salivary glands following fractionated exposure to 70 Gy of ionizing radiation: an animal model for human radiation-induced salivary gland injury

This study explored the feasibility of developing an animal model for radiation-induced salivary gland injury with a radiation protocol identical to current clinical practice. Three male Hanford minipigs were subjected to fractionated daily irradiation with a total dose of 70 Gy; structural and functional measures were compared with those of a control group of minipigs. We found that irradiated submandibular and parotid glands were one-third to one-half the gross size of control glands. Whereas no pathologic changes were noted in control glands, irradiated glands consistently demonstrated significant parenchymal loss with extensive acinar atrophy and interstitial fibrosis, enlarged nuclei in remaining acinar cells, and ductal dilatation and proliferation. Stimulated salivary flow was reduced by 81% in irradiated animals compared with preirradiation flow (P <.001); salivary flow in the control group increased by 30% during the same period (P <.001). The observed radiation-induced structural and functional salivary gland changes are comparable in every respect to those observed following irradiation of human salivary glands.     

Oxidative stress in alcohol-induced rat parotid sialadenosis

This study evaluated the effect of chronic ethanol consumption on the oxidative status of rat parotid and submandibular glands. To identify the endogenous response to ethanol ingestion, the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined. In addition, the antioxidant alpha-tocopherol was supplied to the animals in order to estimate its action in ethanol-associated glandular damage. The thiobarbituric acid reactive substances (TBARS), and the protein carbonyl (PC) content, both markers of cellular oxidative stress on lipid and protein structures, respectively, were recorded. Animals subjected to alcohol ingestion showed a low body growth rate with concomitant enlargement of absolute and relative parotid wet weight, compared with pair-fed calorie-controlled rats. Parotid glands of ethanol-treated animals showed increased SOD and GPx activity, and alpha-tocopherol was able to reduce their activities to the control levels. TBARS and PC were enhanced after chronic ethanol treatment in rat parotids. Supplemental alpha-tocopherol suppressed the oxidative ethanol-induced damage in lipid without affecting induced protein oxidation. Submandibular glands revealed no alterations in the weight, enzymatic and oxidative parameters tested due to ethanol and/or alpha-tocopherol ingestion. These findings indicate the involvement of oxidative stress in parotid gland sialadenosis due to ethanol consumption and the capability of alpha-tocopherol to halt lipid damage, although this low-molecular antioxidant compound leads to neither increased glandular weight nor protein oxidation in ethanol-induced parotid alterations.     

Early immunohistochemical and functional markers indicating radiation damage of the parotid gland

In order to evaluate the correlation between functional impairment and changes in the expression pattern of immunohistochemical antibodies in the early phase of radiation-induced dysfunction of salivary glands, eight rabbits were scintigraphically examined prior to and 24 h after irradiation with 15 Gy. The parotid glands were studied using HE-staining, Ki-67, -smooth muscle actin (ASMA) and tenascin-C antibodies at every scintigraphic examination. The results demonstrated a significant alteration in the ^99mTc-pertechnetate uptake in all irradiated glands. HE-staining showed no relevant impairment of salivary gland tissue in this early phase. Immunohistochemically, we observed a marked re-distribution of ASMA and tenascin-C as well as a reduction of the proliferating rate of acinar cells. This immunohistochemical change correlated with the functional impairment manifested scintigraphically. This study proves the possibility to assess disorders of salivary gland function with immunohistological antibodies as early as 24 h after irradiation and yields the prerequisites to prove the effects of radioprotective agents on salivary gland tissues.This study was financially supported by the German Research Foundation (DFG)     

The effect of X-ray irradiation on the function and saliva composition of rat parotid and submandibular/ sublingual glands

Radiation therapy to the head and neck area frequently causes severe salivary gland dysfunction and xerostomia. Morphological studies of irradiated salivary glands have suggested that the submandibular/sublingual gland may be less radiosensitive than the parotid gland. The purpose of this study was to evaluate the effect of radiation on major salivary gland functions in rats with radiation-induced xerostomia. The effect of salivary gland irradiation on salivary function was examined in specific pathogen-free Sprague-Dawley rats. The animals were irradiated with a single exposure of either 22 Gy or 32 Gy. Stimulated saliva excretion time was measured for the parotid and submandibular/ sublingual glands, and the total protein in saliva was analysed. Our results showed that the saliva flow rate and protein concentration of parotid saliva were significantly reduced in the 32 Gy-irradiated rats.     

Effect of ionizing radiation on sympathetic nerve function in rat parotid glands

Ionizing radiation (IR) irreversibly damages salivary glands. The pathologic mechanism is unknown. Previously we reported that parotid serous acinar cells may not be the primary site of damage by IR. The purpose of this study was to determine if IR alters sympathetic nerve function in rat parotid glands. Male adult rats received a single dose of radiation (20 Gy) to the head and neck. Three days after IR, parotid saliva secretion induced by norepinephrine (NE) was completely blocked. Catecholamine uptake and metabolism were studied by injecting [3H] dopamine ([3H]DA) into irradiated rats, as a bolus. After 60 min, animals were sacrificed and the parotid gland, submandibular gland, and left ventricle removed. Tissue contents of [3H]DA and [3H]NE, identified by HPLC, were unaffected by IR. The results indicate that IR abolishes acinar responsiveness to NE without affecting parotid sympathetic nerve function.     

血管内皮生长因子对小型猪腮腺放射性损伤影响的实验研究

目的：应用血管内皮生长因子（VEGF）对放疗后腮腺微血管内皮损伤进行保护,改善微血管内皮细胞功能,评价其对腮腺放疗后早期腺体及血管变化的影响。方法：将9只实验用小型猪分为三组（n=3）。空白对照组,单纯放疗组逆行注射4ml生理盐水至腮腺导管,VEGF给药放疗组通过腮腺导管逆行注射1μg（4ml）重组人VEGF165蛋白至腮腺。11天后三组动物双侧腮腺进行放疗,空白对照组0Gy/每侧,VEGF＋放疗组及单纯放疗组20Gy/每侧。放疗后4小时取腮腺标本行HE及免疫组化染色,观察腮腺早期组织学与微血管密度变化。结果：三组小型猪腮腺CD31阳性染色颗粒数有显著差异（F=47.727,P〈0.01）;VEGF给药放疗组较单纯放疗组CD31染色颗粒密度明显增高。空白对照组与两放疗组比较AQP1阳性染色颗粒数有统计学意义（P〈0.05）;VEGF＋放疗组与单纯放疗组比较无统计学意义（P〉0.05）。结论：放疗后4小时腮腺组织CD31、AQP1表达明显下降。放疗前导管逆行注射VEGF可使放疗后腮腺组织内微血管密度升高。     

小型猪腮腺放射损伤唾液流率和血常规及生化动态观察

目的 :动态观察小型猪腮腺放射损伤前后腮腺流率、血常规及血生化变化 ,为建立小型猪腮腺放射损伤动物模型 ,及基因治疗涎腺放射损伤奠定基础。方法 :试验组 8只小型猪 ,1 5Gy单侧腮腺放射损伤 ,对侧腮腺做对照 ,2只 0Gy放射做空白对照。放疗前、放疗后 4周、8周和 1 6周 ,用吸盘法收集唾液 ,前腔静脉取血 ,观察唾液流率、血常规和血生化改变。结果 :放疗后 8周唾液流率下降 ,1 6周唾液流率下降明显并与放疗前有显著性差别。随着放射后的时间延长 ,白细胞计数下降越来越明显 ,第 8周和 1 6周与放疗前有显著性差别。红细胞在放疗后第 8周及第 1 6周稍有增加但没有显著性差别 ,血小板在 4周下降 ,第 8周及第 1 6周增高超出放射前水平。乳酸脱氢酶血清含量放疗后呈持续下降趋势 ,与放疗前有显著性差别(P <0 0 5 )。血清淀粉酶在放疗后 1周增高 ,第 4周显著下降 ,与放疗前相比差别有显著性 ,随后缓慢回升。结论 :1 5Gy放疗1 6周时放疗侧腮腺唾液流率明显下降 ,血液常规白细胞计数和血清乳酸脱氢酶、淀粉酶改变     

Radioprotection of the murine submandibular gland by isoproterenol: autoradiography study with <Superscript>3</Superscript>H-leucine

Irradiation of the salivary glands results in the generation of free radicals from metal ions present in the secretory granules of acinar cells, a process that is believed to exacerbate radiation damage to the salivary glands. We therefore conducted a comparative investigation of radiation damage to the acinar cells of murine submaxillary glands in which granule secretion had been induced, and used autoradiography to visualize the pathological changes. Male BALB/c mice, at 8 weeks of age, were divided into four groups: a no-isoproterenol (IPR) and no-irradiation group (group I), a no-IPR, irradiated group (group II), an IPR, no-irradiation group (group III), and an IPR, irradiated group (group IV). Intraperitoneal injections of IPR were used, and 3h later, the submaxillary region was irradiated with X-rays at a dose of 10Gy. Three days after the irradiation, ³H-leucine was administered, and submaxillary glands were removed at predetermined times. Thin sections were prepared, and light- and electron-microscope autoradiography was performed. The number of reduced silver particles per unit acinar cell area was determined by light-microscopic autoradiography, and the proportion of reduced silver particles in the rough endoplasmic reticulum, the Golgi apparatus, and secretion granules was determined by electron-microscopic autoradiography. The result indicated that the effects of the radiation on the secretory potential of the submaxillary glands were diminished in acinar cells with a higher secretory granule content.     

Tea polyphenols protect against irradiation-induced injury in submandibular glands' cells: a preliminary study

Aim

To study the protective effect of tea polyphenols (TPs) on submandibular glands affected by radiation injury.

Methods

Sixty rats were randomly divided into radiation group (R-group, N = 30) and TP-pre-treated-radiation group (TPR-group, N = 30). The rats were intragastrically administered with TP or normal sodium from 14 days before radiation, continuously daily, until the experiment. All the rats in both groups were irradiated with a single exposure dose of 15 Gy gamma rays that were delivered to the head and neck areas. Ten rats of each group were anatomised on the 3rd, 6th and 30th day after irradiation, respectively. The submandibular glands of the rats were removed for the study. The morphologic changes of the submandibular glands were observed by transmission electron microscopy (TEM). The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labelling (TUNEL) method was used to detect apoptosis of the submandibular glands’ cells.

Results

Electron microscope observation of the submandibular glands showed that the lesions of the TPR-group were mild. Change in apoptosis of the cells was not obvious compared with the R-group. The cell apotosis was typical after irradiation in the R-group. Apoptosis index that was detected in the cells of submandibular glands of the TPR-group was statistically significantly decreased compared with the R-group (P < 0.01) on the 3rd, 6th and 30th day after irradiation.

Conclusion

TP could protect submandibular glands from radiation injuries, and the protection mechanism may be realised by anti-apoptosis.     

大鼠头颈部60Co照射后腮腺组织病理变化及临床意义

目的研究60Co照射对腮腺组织的影响及临床意义。方法模拟临床头颈放射治疗,用60Co对40只正常成年SD大鼠头颈部按剂量归一的方法进行照射。照射后分别于第5、10、20、30、60天取腮腺组织作常规病理切片观察。结果60Co照射后,随着时间的推移,大鼠腮腺腺泡细胞胞质肿胀、空泡变性和坏死明显加重,间质血管充血、肿胀及变性逐渐加剧,未见到再生、恢复的变化。结论治疗剂量的60Co照射对正常腮腺组织有损伤,主要作用于腺泡细胞和间质血管,损伤的程度具有时间依赖性,呈不可逆的损伤。     

SSB1在腮腺放射性损伤修复过程中的表达变化

目的：研究正常大鼠腮腺及放射后大鼠腮腺组织单链DNA结合蛋白（ single?strand DNA?binding protein 1，SSB1）的表达及变化规律，探讨SSB1与涎腺放射损伤修复的关系。方法：取近交系成年Wistar大鼠40只，一次给予6 Gy剂量照射头颈部，于放射后每小时随机处死5只动物取腮腺组织，共形成8个实验组，另取5只大鼠作为对照组。应用免疫组织化学与RT?qPCR的方法检测SSB1在大鼠腮腺组织中表达情况。结果：对照组大鼠腮腺组织有一定量的内源性SSB1表达，不同腮腺组织之间SSB1含量不同，导管＞腺泡＞结缔组织；放射后，大鼠腮腺组织中SSB1的表达量随着时间变化呈先增加（P＜0?05）后减弱趋势，并在后期表达量降至接近正常水平。结论：辐射能诱导大鼠腮腺SSB1表达量增加，可能参与了大鼠腮腺放射性损伤修复过程。     

The effects of heparan sulphate mimetic RGTA-OTR4120 on irradiated murine salivary glands

J Oral Pathol Med (2012) 41 : 477–483 Background: This study focuses on the potential of ReGeneraTing Agent OTR4120 (RGTA‐OTR4120) to treat radiation‐induced damage of salivary glands. RGTAs are biopolymers designed to mimic the effects of heparan sulphate, thereby stimulation tissue repair and regeneration. Methods: C3H mice were irradiated with a single dose of 15 Gy in the head and neck region. RGTA‐OTR4120 was injected 24 h after radiotherapy, followed by weekly injections. At 2, 6 and 10 weeks after radiotherapy, salivary flow rates were measured and animals were sacrificed to obtain parotid and submandibular glands for histology. Periodic acid Schiff stain was performed to visualize mucins that are produced by acinar cells. Amylase and total protein content were measured in saliva samples. Results: Salivary flow rates were increased at 2 weeks, but not at 6 and 10 weeks after radiotherapy with RGTA‐OTR4120 administration, compared to irradiated controls. Two and 10 weeks after radiotherapy, the mucin production activity of acinar cells was increased under influence of RGTA administration. RGTA‐OTR4120 did not influence amylase or total protein secretion. Conclusion: RGTA‐OTR4120 administration has a positive effect on salivary flow rates in irradiated mice on the short term. The effect was absent 10 weeks after radiotherapy, while at that time point, mucin producing activity of acinar cells was elevated by RGTA‐OTR4120 administration. Given these results and the advantages of RGTA use in irradiated patients, further investigation on the potential of this drug to treat radiation‐induced salivary gland damage, alone or in combination with other drugs, such as amifostine, is suggested.     

Absorbed Radiation Doses During Tomographic Examinations in Dental Implant Planning: A Study in Humans

Objective: The aim of this human study was to evaluate the radiation doses in the buccal cavity and face, during panoramic, spiral conventional tomography, and helicoidal computerized tomography exams. Material and Methods: Lithium fluoride TL detectors (TLD‐100) were placed on the skin at anatomic points such as parotid glands, submandibular glands, thyroid glands, and crystalline to assess the skin entrance dose in 19 patients who were to undergo dental implant surgery. Results: In the panoramic exam, maximum doses were observed near the parotid glands at 1.57 (±18%) mGy on the right and 1.89 (±18%) mGy on the left. In the spiral conventional tomography exam, the maximum dose was 4.41 (±21%) mGy near the right and left parotid glands, whereas near the right or left submandibular glands, the maximum doses reached 40.7 (±18%) mGy. In the helicoidal computerized tomography for mandibular and maxilla exams, the maximum dose was 40.9 (±15%) mGy near the parotid glands and 41.0 (±18%) mGy near the submandibular glands. Near the thyroid and eye lens, doses were lower than 0.23 (±21%) in all exams. Conclusion: Regardless of the exam target area, the submandibular and parotid glands represented the most irradiated organs. This data suggests that efforts should be made by professionals to improve and optimize methods in order to reduce doses without losing the information necessary for treatment planning.     

电离辐射对大鼠腮腺旁细胞分泌途径损伤及紧密连接蛋白claudin-4的影响

目的: 探讨电离辐射对大鼠腮腺旁细胞分泌功能损伤及紧密连接 (tight junction,TJ) 蛋白claudin-4的影响与潜在作用机制。方法: 24只8周龄雄性Wistar大鼠,随机分为对照组（6只）和照射组（照射后1周组、4周组、12周组,每组6只）。照射组一次性20 Gy射线局部照射实验侧腮腺区。采用Schirmer实验,检测各组腺体唾液静息分泌量;H-E染色,光镜下观察腺体组织病理变化;透射电镜观察TJ超微结构改变;免疫荧光染色和蛋白质印迹检测毒蕈碱型乙酰胆碱受体（M受体）亚型M3、水通道蛋白5（AQP5）及claudin-4的蛋白表达。采用SPSS 23.0软件包对数据进行统计学分析。结果: 照射后1、4、12周,腺体静息分泌量较对照组显著减少（P<0.05）;12周较1周、4周时减少更显著（P<0.05）。组织学观察可见,照射后腺体早期间质血管扩张、充血,后期腺泡细胞计数显著减少（P<0.05）;TJ结构模糊、电子密度降低、宽度减少（P<0.05）。免疫荧光染色和蛋白质印迹检测结果显示,M3受体及AQP5蛋白在照射后1、4、12周表达显著下调,而claudin-4蛋白表达显著升高。结论: 电离辐射后腮腺旁细胞分泌功能降低,TJ结构改变,claudin-4表达上调,可能参与腺体分泌功能损伤。