| Part 1 The changs of bacterial flora in ocular surface of diabetic ratsIn recent years, ocular surface diseases induced by diabetes has attracted wide attention. As an exposed organ, conjunctiva directly links to the skin and the bacteria dwell in it soon after birth. Normal bacterial flora in conjunctiva refers to all aerobic and anaerobic bacteria in the conjunctival sac, which keeps dynamic balance in it. These bacteria are always changes with the personal and environmental factors, and it will cause eye infections when this balance is disturbed.ObjectiveTo observe changes of bacterial flora in conjunctival sac of diabetic rats; To observe changes of microenvironment and susceptibility of diabetic rats.MethodsThirteen SPF SD rats, six weeks old, male and body weight of 240 to 300g, were used in this study and they were fed in SPF animal laboratory of clinical institute in the Sino-Japan hospital. Before the experiments, all animals were examined by slit lamp microscope for making sure that the fundus of each animal was normal. They were divided randomly into diabetic model group (7 animals) and control group (6 animals). For normal control group, the same quantity of sterilized physiological saline was injected intraperitoneally. For diabetic model group,60 mg/kg of STZ was injected intraperitoneally at one time to induce diabetes mellitus type I. Seventy two hours post injection, if glucose concentration measured in vein blood is higher than 16.7mmol/L for 3 days continuously, the rats are thought to be successfully induced as diabetic model. The rats were observed daily for six months and at the sixth month, the swab samples were taken from conjunctival sacs for bacterial isolation, and the conjunctivae were collected for determination of cytokine expressions by reverse transcription polymerase chain reaction (RT-PCR). Statistical software SPSS20.0 was employed to process and analyze the data, with P≤0.05 or less as the significant difference.ResultsIsolation and identification of bacteria from conjunctival sacs demonstrated that Gram-positive bacteria are dominant in both diabetic model group and normal control group. Only two species(Enterococcus faecium and Escherichia coli) shared between the two groups with the most abundant one of Enterococcus faecium. The quantities of isolated common bacteria in model group are higher than the normal group. However, after the development of T1DM, the bacterial flora in the rat bulbar conjunctiva changed considerably, with a reduced complexity evident.ConclusionIn rats with diabetes, the numbers of bacteria have increased and the diversities are higher, indicating the risk of infection.Part 2 Impact of conjunctival blood vessels and tissue in rats with diabetes mellitus (DM) by intervention of Yiqihuoxuetongluo (YQHXTL) formulaeOcular surface in anatomy refers to all mucosal epithelium covering eye surface between the palpebral superior and palpebral inferior, including corneal epithelium and conjunctival epithelium, and the latter covers bulbar conjunctiva, palpebral conjunctiva and conjunctival fornix. Ocular surface epithelium originated from the corresponding stem cells, and the conjunctival epithelium is mainly the stratified squamous cells, with many goblet cells secreting sticky proteins. Ocular surface disease refers to the corneal and conjunctival ocular surface damage in normal structures and functions. Diabetes is a kind of endocrine system diseases, and it is characterized by high blood sugar with a variety of complications caused by metabolic disorder. DM can cause diabetic retinopathy, cataract and refractive error, the corneal lesion and other ocular changes.ObjectiveThis study was to explore the impact of diabetes on conjunctival tissue organization, and blood stream dynamics, and the cytokine and some important proteins expressions by immunohistochemical staining in treatment group by YQHXTL formulae and the control one. This will lay a foundation to clinical application of YQHXTL formulae.MethodsTwenty five SPF SD rats, six weeks old, male and body weight of 240 to 300g, were used in this study and they were fed in SPF animal laboratory of clinical institute in the Sino-Japan hospital. Before the experiments, all animals were examined by slit lamp microscope for making sure that the fundus of each animal was normal. They were divided randomly into diabetic model group without administration of YQHXTL formulae (7 animals) (DMG), diabetic model group with administration of YQHXTL formulae group (6 animals) (DM-YQHXTL), diabetic model group with administration of positive drug of hydroxyl benzene sulfonic acid calcium capsules (6 animals) (DM-pos), and the control group without any induction and treatment (6 animals) (ConG). Except ConG, for animals in other groups,60 mg/kg of STZ was injected intra-peritoneally at one time to induce diabetes mellitus type I. Seventy two hours post injection, if glucose concentration measured in vein blood is higher than 16.7mmol/L for 3 days continuously, the rats are thought to be successfully induced as diabetic model. For groups DM-YQHXTL and DM-pos, the drugs were given immediately after the DM was confirmed in the animals at 10 a.m. each day by intragastric administration. The YQHXTL formulae consists of Radix Astragali preparata, radix ginseng Rubra, Sanchi powder, cassia twig, the root of red-rooted salvia, earthworm, and Poria cocos etc. The concentration of the YQHXTL formulae for administration is lg/ml of solid materials. The DM-pos was given hydroxyl benzene sulfonic acid calcium capsules at a dosage of 250 mg/kg·d.The blood glucose and body weight of the animals was determined regularly every 2-week regularly in the experimental period of 9 months. Before sampling, all the animals were examined by the laser Doppler for measuring blood flow velocity of the upper conjunctival blood vessels. For sampling, the animals were killed by excessive anesthesia using 10% chloral hydrate, immediately after the deep anesthesia of the animals, the bulbar conjunctiva and fornix biopsy were collected for tissue examination by light microscopy and electron microscopy. Immunohistochemical staining was employed to detect expressions of MMP9 (matrix metalloprotein 9), IL-6, IL-12 p40, and TNFa in the conjunctival tissues. Using image analysis software, color intensity of immunohistochemical measurement was obtained for quantitative analysis. Statistical software SPSS20.0 was employed to process and analyze the data, with P≤0.05 or less as the significant difference.Results(1) After the DM model was developed, the body weights of DM rats were significantly reduced during the experimental period comparing with the ConG (P<0.05);(2) The concentration of glucose in blood of the DM rats was significantly higher than ConG (P>0.05) based on the continuous monitoring of blood glucose.(3) Morphological observations showed that in ConG, the conjunctival tissue was integrate; the epithelial cells were complete; and the goblet cells were distributed in abundance in the basal layer of epithelial cells. However, in DMG, disordered arrangement of epithelial cells and inflammatory cells were increased significantly and the conjunctival goblet cells were reduced significantly, mainly in basal epithelial layer. In Con-pos, the disordered arrangement of epithelial cells and inflammatory cells increased; conjunctival goblet cells decreased significantly in basal epithelial layer. In DM-YQHXTL, epithelial cells arranged neatly and conjunctival goblet cells were abundant in the basal epithelial layer.(4) Laser Doppler blood flow diagram demonstrated that the DM modeled animals had slower blood flow velocity than the animals in ConG (P=0.004, P< 0.05) and the DM-YQHXTL (P=0.019, P< 0.05); however, there was no difference between the DM-YQHXTL and ConG (P=0929, P>0.05).(5) MMP9 was expressed significantly higher in conjunctival tissue of the DMG rats than other three groups. AGES was also highly expressed in DMG other than remaining three groups.(6) Electron microscopy confirmed that the inner epithelial cell of conjunctival blood vessels was not intact.Conclusions(1) The SD rats were successfully and reliably induced as diabetic model, which has been successfully applied in study of conjunctival changes in DM rats and also used to evaluate the improvement of conjunctiva-associated DM damages by administering traditional Chinese medicines.(2) The bacterial flora of conjunctival sacs changed in DM rats in both bacterial quantity and species.(3) The conjunctival blood vessels and tissues changed obviously in DM rats.(4) MMP9, a Matrix metallopeptidase 9, and AGEs, advanced glycation end products, are significantly expressed in DM rats compared to the rats without DM.(5) Conjunctival blood flow per unit area in DM rats is slower than those without DM or the ones with treatment by traditional Chinese medicines.(6) YQHXTL formulae are confirmed to be effective in ameliorating conjunctival damages in rats caused by DM. |
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