| Leukopedesis is the most important characteristic change of inflammation. It is a complicated continuous process, including leukocyte margination, adherence, emigration and etc. Leukocytes are recruited to the inflammation focus by chemokines and exert defence function in the local inflammation focus. Leukopedesis process can be observed by in vivo fluorescence microscope, so we can further understand the characteristic of different inflammation and different periods of inflammation. But it is a traumatic observing when observing the position such as meningina and mesentery and not a long-lasting choice. The retinal microcirculation can be observed conveniently and the subjects cannot be hurt, in addition, the retinal microcirculation is similar to cerebral microcirculation, so it can be a window to the study of cerebral microcirculation. For the past few years, scanning laser ophthalmoscopy (SLO) was applied to study the leukokinetics of retinal microcirculation in vivo either in physiological or pathological condition. Several methods have been devised in which the SLO is used to study the cell imaging in vivo. Non-virulence leukocyte tracer technique was studied either in rats or in mice. Several mice was examined either 7 by SLO or Heidelberg Retina Angiograph 2 (HRA2) to observe the retinal gangliocyte apoptosis, It showed that the latter is better in imaging, especially in fluorescence spot imaging. The required condition of this technique is slashing and the reports is few. In order to further explore the pathogenesis of retinal disorder, we have devised this method in which the HRA2 is used to observe the retinal microcirculation and leukocyte movement.Methods and material:Four broods healthy SD rats(ten rats in a brood, body mass 200-300g) without eye disorder and one brood healthy BN rats(six rats in a brood, body mass around 180g) without eye disorder were involved in this study. They all come from the Barrier Shield Animal Laboratory of the Department of Clinical aerospace medicine in the Fourth Military Medical University and were breed in normal temperature of about 25 0C. one brood SD rats need not group and the other broods of rats were all divided into 2 groups averagely in inner brood. One of the group in every brood was used to collect blood from the heart and about 7-10ml blood can be collected from one rat. If one rat cannot be collected enough blood, we can collect blood from several rats one time in order to get sufficient leukocyte to observe. The first brood without grouping was used to observe fundus fluorescein angiography (FFA); the second brood was used to observe the movement of leukocyte which was not activated by ConA in ocular fundus; correspondingly, the third brood was used to observe the movement of leukocyte activated by ConA in ocular fundus; the last brood and BN rats which were exerted fundus laser photocoagulation were also used to observe the movement of leukocyte activated by ConA in ocular fundus. The separating medium is lymphocyte separating medium of rat LTS1083 (Tianjin HaoYang biological product technology limited-liability corporation). HRA2 was produced in German Heidelberg engineering corporation.The rats were anesthetized intraperitoneally with Sumianxin(veterinary surgeon institute of academy of military medical sciences) 0.7ml/kg, its pupils were dilated with compound Tropicamide Eye Drops(Beijing Shuanghe Contemporary Drug Technology limited-liability corporation) 5g/L.The animal and the instrument were adjusted to each other, when the ocular fundus was observed through the HRA2 len, the 100g/L Fluorescein Sodium Injection solution(Guangxi Wuzhou Drug-produced corporation) 0.5ml/kg was injected intravenously in the tail vein immediately and then one eye of the rat was observed and photographed. At every 1-2min interval Tears Naturale gutta (American Alka corporation) was droped to the observed eyeball to avoid cornea drying.Fresh anti-coagulated blood collected from the heart was mixed with Hank's solution in 1:1 proportion, then the mixed liquor was added to isovolumic lymphocyte separating medium of rats gradually to gradient centrifuge, then collect the separated leukocyte and count the cell number, the leukocyte number is 1×109cells/L and the lymphocytes propotion is 60-70%, monocytes propotion is 30-40%. The cells were then suspended in Hank's solution and centrifuged 3 times and then suspended in 5ml RPMI1640 culture fluid. These cells were then co-incubated with CFDA 30min in 37 0C,we can see the cell suspension was dyed with inaurate, then the cell suspension is centrifuged and rinsed 3 times, the last procedure is suspending these cells in 1ml RPMI1640 culture fluid. With the help of cell counting plate, we can count the number of the cell is (5-7)×109cells/L. The leukocyte of another brood of 5 rats was separated in the same way. In order to activate these cells, 10ml cell suspension was co-incubated with 2.5mg/L ConA (Sigma) 30min in 37 0C, then centrifuged and rinsed in RPMI1640 culture fluid 3 times and dyed with CFDA in the same method. These leukocytes were then injected into the circulation of corresponding rat to observe and photograph with HRA2. One of the group of the last brood SD rats and BN rats were exerted fundus laser photocoagulation, the energy of laser beam are 0.3W and 0.08W respectively. The exposure time is 0.1s and the diameter of the light spot is 50μm. The ocular fundus of every rat appeared 52 light spots with the standard of appearing air bubble in slit lamp. The light spot is fourth stage. Then the ocular fundus and the leukocyte movement was observed in the same methods after photocoagulation 2-3 days.Result1 Fundus Fluorescein AngiographyBy this experiment we can clearly see the strike,distribution and morphous of the ocular fundus vessels of rat. The strike and distribution of ocular fundus of normal rat is normal and there's no circuity,exudation and haemorrhage etc. The fourth grade vessels can be observed by this device. Because of the fast velocity and short blood circulation of rat, FFA of rat can hardly be divided into arterial phase,capillary phase and venous phase like human. According to the engorging and visualization of fundus vessel the duration can simply be divided into early phase (1-3s after fluorescein injection, the engorging and visualization of 1st-grade ocular fundus vessels can be seen),metaphase (3s-5min after fluorescein injection, the engorging and visualization of 4th-grade ocular fundus vessels can be seen) and anaphase (5min after fluorescein injection, the angiography can be clearly seen and the background is gradually lightened because of the leakage of fluorescein in the choroid). Owing to the pigment of ocular fundus of BN rats, FFA is more clear and the observing time is longer than 2h.2 The movement of leukocyte in the retinal vesselsThe movement of leukocyte could be clearly seen in the ocular fundus of rat by HRA2, especially conA activated group. Moreover, The leukocyte activated by conA moves more slowly, more leukocyte could be seen in the visual field in the same interval and some ones stay for longer time in visual field. However, the velocity of normal leukocyte is fast and some one cross the vessel like lightning, in addition, the leukocyte number in the visual field is less. Some leukocytes in the small vessels and tissues move slowly, the cell movement trace can be clearly seen, and leukocyte adhension, cross membrane and rolling can also be seen. Laser photocoagulation group and control group has no obvious distinction. The only distinction is more fluorescein leakage in the choroids in the laser photocoagulation group.Conclusion1 The morphous of retinal vessels and leukocyte movement could be clearly observed by HRA2, which could be a valid tool to study the retinal microcirculation and leukocyte movement of rats.2 Compared to SD rats, due to the pigment available , the visual field is more clearly in BN rats.3 After laser photocoagulation, whether leukocyte labeled in vitro chemokine to inflammation focus or not and the observing window time, need further study.
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