Expression of Na(+)/K(+)-ATPase alpha subunit isoforms in rat salivary glands: occurrence of sense and antisense RNAs of the alpha3 isoform in the sublingual gland

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.     

The effects of mandibular retractive force on the growing rat mandible

The purpose of this study was to investigate the three-dimensional effects of a mandibular retractive force on the growing rat mandible and mandibular growth after the force was removed. Eighty 4-week-old male Wistar rats served as experimental animals and 100 4-week-old male Wistar rats served as the control group. The experimental rats were divided into four groups. Thirty grams of retractive force was applied to each mandible for 4 weeks. Each group was then killed after 4-, 8-, 10-, 12-, or 16-week periods. At the time of death, tibia, skull, and mandible were removed, measurements of those bone specimens were taken, and histologic examinations were performed. Compared with the control group, the experimental group demonstrated no significant differences in body weight nor in the length of measurements of the tibia, skull, and proximal end of the tibia. The mandibles of the experimental group demonstrated shorter anteroposterior length, higher coronoid processes, and a thicker corpus in the retromolar region and condylar neck. The condylar cartilages of the experimental group showed moderate adaptation in the histologic sections. However, mandibles of the experimental animals did not show any catch-up growth behavior after removal of the force. It was concluded that the mandibular retractive force could produce overall growth retardation and transformation of growing rat mandibles, but the force had no effect on the growth behavior after the appliance was removed.     

骨髓间充质干细胞移植促进大鼠下颌骨牵张成骨的实验研究

目的:探讨大鼠下颌骨牵张间隙内移植自体骨髓间充质干细胞,促进骨痂形成的可行性。方法:选用54只雄性SD大鼠,密度梯度离心法分离骨髓间充质干细胞(MSCs)。所有大鼠行右侧下颌骨牵张;并于术后随机分为试验和对照2组。牵张结束时,实验组牵张间隙内注射自体MSCs;而对照组只注射等量生理盐水。分别于固定期第2、4、8周分3批处死大鼠,进行放射学、组织学观察,并对骨组织形态计量学参数进行统计学分析(t检验)。结果:放射学和组织学观察表明,2组牵张间隙内均形成了骨痂组织,但实验组新生骨组织显著多于对照组。计量学分析也显示,各时间点实验组新生骨量(NBV1和NBV2)和新生骨小梁宽度(TNT)均显著高于对照组(P<0.01)。结论:牵张间隙内行自体骨髓MSCs移植,可有效促进新骨形成,缩短固定期;该方法对众多的颅颌面外科畸形的矫治极具价值。     

茶多酚对复发性阿弗它溃疡大鼠模型外周血T淋巴细胞亚群的影响

目的：探讨细胞免疫与复发性阿弗它溃疡（recurrent aphthous ulcer，RAU）发病机制的关系及茶多酚（tea polyphenols，TP）对RAU的免疫调节作用。方法：用免疫方法建立SD大鼠RAU动物模型，将RAU大鼠随机分成阴性对照组、溃疡糊剂组和3个TP治疗组。用流式细胞仪检测成模前后及用药前后各组大鼠外周血T淋巴细胞亚群CD4^＋、CD8^＋细胞数量及比值。结果：实验大鼠溃疡模型成模后外周血CD4^＋及CD4^＋／CD8^＋较成模前显著降低（P〈0．01），CD8^＋改变无统计学意义（P〉0．05）。茶多酚各治疗组外周血CD4^＋、CD4^＋／CD8^＋回升，与阴性对照组有显著性差异（P〈0．05），茶多酚各治疗组间比较没有显著性差异（P〉0．05）。结论：RAU的发生与细胞免疫功能抑制有关，TP对大鼠RAU具有免疫调节作用，其免疫调节作用与给药方式无关。     

大鼠牙龈组织表达护骨因子和护骨因子配体

目的 观察成年大鼠牙龈组织是否表达护骨因子（Osteoprotegerin,OPG)和护骨因子配体（Osteoprotegerin Ligand,OPGL)，为进一步探讨二者在牙龈组织中表达的生理及病理意义奠定基础。方法 提取成年大鼠牙龈组织总RNA，应用特异引物通过逆转录-聚合酶链式反应（RT-PCR）扩增目的片段，并将其插入pGEM-T载体，测序鉴定。结果 测序表明扩增出的二个目的片段DNA序列与文献报道一致，即为护骨因子和护骨因子配体，护骨因子在大鼠虎龈组织表达水平较高，而护骨因子配体表达水平较低。结论 大鼠虎龈组织表达护骨因子和护骨因子配体，二者表达的相对水平对维持牙槽骨局部骨形成与骨吸收的平衡可能有重要意义。     

Effects of gender on serum biomarkers of systemic inflammation coincident to experimentally-induced periapical lesions

Objective

The literature suggests that females have less adverse effects to infection than males, due to the protective effects of oestrogen. The purpose of our study is to compare the systemic effects of induced periapical lesions between groups of animals with various serum concentrations of oestrogen.

Methods

To induce periapical inflammation, two molar tooth pulps were exposed in ovariectomized (OVX) and normal female (F) and castrated (Cast-M) and normal male (M) Sprague–Dawley rats (Experimental group, E). Sham-operated control animals from each group were also studied (Control group, C). Twenty-eight days later, serum and maxillas were collected. Serum 17β-oestradiol, testosterone, MMP-9, IL-18, IL-6, TNF-α, and IL-1β concentrations were measured by ELISA. Maxillas were cleaned of residual tissue and digital radiographs were made to verify the presence of periapical lesions. Data were compared by factorial ANOVA, post hoc Tukey, and Pearson correlation tests. Groups were considered to be significantly different when p < 0.05.

Results

The serum concentration of IL-18, TNF-α, IL-1-β, IL-6 and MMP-9 was greatest in OVX-E animals, compared to all other groups (p < 0.001). F-E rats had significantly higher serum concentrations of these cytokines, compared to F-C. The fold difference in serum concentration of the biomarkers (between E and C groups) was significantly greater in females than males, even though males had higher baseline concentrations of all these biomarkers.

Conclusion

When females are oestrogen-deficient, their systemic response to periapical lesions is significantly greater than males, suggesting that oestrogen is essential in protecting females from the effects of this type of inflammation.     

大鼠成釉细胞的原代培养和光、电镜观察

目的 通过对大鼠成釉细胞进行分离培养,为在离体状态下研究成釉细胞的发育和影响因素以及与釉质发育的关系奠定实验基础。方法 运用组织细胞分离培养技术分离大鼠的成釉细胞并进行原代培养,在光、电镜下观察其形态结构。结果 成釉细胞成簇生长,胞体多呈柱状,胞浆内细胞器发育完好。结论 原代培养的成釉细胞基本保持其在体时的形态及结构特征,此种方法可以用于成釉细胞的体外研究。     

Tissue oxygen tension and blood-flow changes in rat incisor pulp with graded systemic hyperoxia

The role of oxygen in the regulation of the pulpal microcirculation is unknown. This investigation is aimed to measure tissue oxygen tension and blood-flow changes in the pulp of rat lower incisors during graded systemic hyperoxia, and to determine the response of the pulpal vasculature to various oxygen tensions. Twenty-four Sprague-Dawley rats were anaesthetized and artificially ventilated with the appropriate gas mixture. Recessed oxygen-sensitive microelectrodes were used to measure pulpal tissue oxygen tension via a small access cavity filled with saline on the labial surface of the incisor. A laser Doppler flowmeter was used to record pulpal blood-flow. Inspired oxygen was increased stepwise from 20 to 100% in 20% steps. Systemic blood-gas concentrations were measured at each step. Systemic arterial oxygen tension at 100% oxygen ventilation reached 481.2 +/- 30.7% of the baseline at 20% oxygen breathing (n=21). Pulpal tissue oxygen tension did not change significantly whereas pulpal blood-flow fell dose-dependently to 74.6 +/- 5.0% at 100% oxygen ventilation (n=21). Systemic hyperoxia, therefore, induces a significant reduction in pulpal blood-flow whereas pulpal tissue oxygen tension remains relatively stable, indicating an oxygen-dependent local regulatory mechanism.     

Oxygen distribution and consumption in rat lower incisor pulp

The aim was to determine the oxygen tension (P(O(2))) and rate of oxygen consumption in the pulp. Twelve rats were anaesthetised and artificially ventilated. Under an operating microscope, a recessed oxygen-sensitive microelectrode was inserted into the pulp through a small saline-covered cavity on the labial surface of the lower incisor. P(O(2)) was measured as a function of the transverse distance from the saline medium through to the middle of the pulp. Oxygen profiles were characterised by a decline of oxygen tension outside the pulp in the saline medium and a steeper gradient across the interface, before a localised oxygen consuming region corresponding to the odontoblasts. A plateau with some localised fluctuations was then followed by an increase in oxygen tension in the middle of the pulp. The average oxygen tension in the plateau region was 23.2 mmHg+/-2.1 mmHg (n=12). A mathematical model was used to extract oxygen consumption data from P(O(2)) profiles recorded from non-perfused pulp (created by reducing systemic blood pressure). The analysis revealed that there was a distinct oxygen consumption zone in the outer pulp, which anatomically corresponded to the odontoblast layer. The average oxygen consumption rate of the odontoblasts was 3.2+/-0.2 ml O(2)/min per 100g pulp tissue. The zone of high oxygen consumption was 68.7 micro m+/-6.9 micro m (n=24) thick. It is concluded that pulpal oxygen distribution is heterogeneous and that the odontoblast could be a major oxygen consumer within the rat incisor pulp.     

Cholinergic submandibular effects and muscarinic receptor expression in blood vessels of the rat

In order to functionally characterise the muscarinic vasodilator responses, effects of cholinergic agonists were studied on isolated preparations of the rat submandibular artery and vein and carotid and jugular vessels. Tentatively, a cholinergic regulatory mechanism having different effects on the arterial and venous vessels would enhance vascular fluid recruitment for the secretory response. In vitro functional findings were correlated to the expression and cellular location of the different receptors that were assessed by immunohistochemistry. In order to find in vivo correlates to the in vitro findings, the influence of muscarinic receptors on permeability was studied on the vasculature of the submandibular gland in anaesthetised rats. Staining for muscarinic M1 receptors occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the arterial smooth muscle. In venous endothelium, muscarinic M1 and M4 receptors occurred. In the jugular smooth muscle layer, staining for M1, and possibly also for M3, appeared. Muscarinic agonists caused arteries to relax and veins to contract. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-4)M) markedly reduced the cholinergic-evoked relaxation of pre-contracted carotid arterial preparations. In the presence of 4-DAMP (10(-7)M), the relaxation to cholinergic agonists was inhibited. Pirenzepine (10(-5)M) did not only inhibit the relaxatory effects, but even reversed the effects, while it in the jugular vein abolished the cholinergic effects. The arterial nitric oxide-dependent response to muscarinic receptor stimulation consisted of two parts -- one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only, resulted in cholinergic arterial contraction. Also, the submandibular artery and vein responses to muscarinic receptor stimulation show a resemblance with those of the carotid and jugular vessels, i.e. a pronounced arterial relaxation and a contractile component in the venous response. In vivo examination of submandibular glandular vasculature by studying glandular permeability to Evans blue, confirmed the in vitro observations indicating muscarinic M1 receptors preserving perfusion pressure during the secretory process.